Practical Vaginal Microecology————————————The Legacy of Outstanding Chinese Culture
First Edition, 2025EDITED BYwww.mospbs.comPractical Vaginal MicroecologyYongmei Jiang , PhDDirector of the Department of Laboratory MedicineWest China Second University Hospital, Sichuan UniversitySichuan, China.Zhengqiang Hu , MDChief TechnologistWest China Second University Hospital, Sichuan UniversitySichuan, China.
CONTENTSEDITORIAL BOARD, ⅠTRANSLATING COMMITTEE, ⅡABOUT THE AUTHOR, ⅢPREFACE, Ⅳ1. Overview of the Vaginal Microecology, 12. Vaginal Microecological Evaluation System, 153. Lactic Acid-Producing Bacteria, 334. Bacterial Vaginitis, 495. Vulvovaginal Candidiasis, 636. Trichomoniasis Vaginalis, 787. Aerobic Vaginitis, 1048. Vulvovaginitis in Young Girls, 1299. Atrophic Vaginitis, 14810. Cytolytic Vaginitis, 16611. Mixed Vaginitis, 17212. Vaginal Microbiota Inhibition, 18013. Vaginal Microecological Treatment, 19614. Operation Specification and Quality Control of Vaginal Microecological Examinations, 22715. Interpretation of the Vaginal Microecology Report, 25116. Progress in Vaginal Microecological Diagnosis--Automated Detection and Artificial Intelligence, 268AFTERWORD
EDITORIAL BOARDYongmei JiangWest China Second University Hospital, Sichuan UniversitySichuan, China.Zhengqiang HuWest China Second University Hospital, Sichuan UniversitySichuan, China.Ying Wang West China Second University Hospital, Sichuan UniversitySichuan, China.Wenjie ZhouWest China Second University Hospital, Sichuan UniversitySichuan, China.Yuanting TangWest China Second University Hospital, Sichuan UniversitySichuan, China.Jing ZhangWest China Second University Hospital, Sichuan UniversitySichuan, China.Xin-ai YueWest China Second University Hospital, Sichuan UniversitySichuan, China.Meng ChenWest China Second University Hospital, Sichuan UniversitySichuan, China.Chief EditorMembers of Editorial BoardHongwei LiuWest China Second University Hospital, Sichuan UniversitySichuan, China.Fan YuWest China Second University Hospital, Sichuan UniversitySichuan, China.Xiaojuan LiuWest China Second University Hospital, Sichuan UniversitySichuan, China.Subeditor
TRANSLATING COMMITTEEZhengqiang HuWest China Second University Hospital, Sichuan UniversitySichuan, China.Chief TranslatorYongmei JiangWest China Second University Hospital, Sichuan UniversitySichuan, China.Translation InstructorMembers of Translating CommitteeXin-ai YueWest China Second University Hospital, Sichuan UniversitySichuan, China.Tingting ZhuWest China Second University Hospital, Sichuan UniversitySichuan, China.Yiduo ZhangWest China Second University Hospital, Sichuan UniversitySichuan, China.Yunxia Li363 HospitalSichuan, China.
ABOUT THE AUTHOR Dr. Yongmei Jiang is a director technician and a doctoral supervisor. She is also the director of the Department of Laboratory Medicine, West China Second University Hospital, Sichuan University; The 11th academic and technical leaders of Sichuan province; The health family planning talents of Sichuan province; The Women's and children's clinical inspection quality control center director of Sichuan province; The deputy director of the 11th Laboratory professional committee of Sichuan Provincial Medical Association; The deputy director of the fifth committee of the physicians association first branch of blood transfusion; The deputy director of of the Laboratory Medicine Special Committee of Sichuan Rehabilitation Medical Association. The director of the Inspection Professional Committee of Sichuan Sex Society.
Mr. Zhengqiang Hu is a Master and a director technician of the West China Second University Hospital, Sichuan University. He is also an academic andtechnical leader of the 12th batch of Health and Family Planning Commissionof Sichuan Province; A committee member of Reproductive InspectionCommittee of Sichuan Sexual Society; A New Era "Health Guard" of SichuanProvince; A medical appraisal expert of Chengdu Medical Association; A projectevaluation expert of Sichuan Science and Technology Department; A Scienceand Technology evaluation expert of Chengdu Science and Technology Bureau.A review expert of the Journal of "Modern Clinical Medicine"; A Sichuan Province special care medical and health service expert.
PREFACEFemale's vagina is a unique and dynamic changing microecological system. There are a variety of normal microbial communities, which constitute a mutual restriction and coordination between the host and the environment, and plays an important role in maintaining a healthy vaginal microecological environment. With the application of non-culture methods in the study of vaginal microecosystem, people have a more new understanding of the vaginal microbiota.As a part of the human microecosystem, the vaginal microecosystem is closely related to the pathogenesis of reproductive tract infections.Vaginal microecological evaluation provides a detection means for the analysis and understanding of vaginal microecological changes, including morphological diagnosis and functional diagnosis. Among them, morphological diagnosis includes the evaluation of bacterial flora, the detection of pathogenic microorganisms and the assessment of disease status; functional diagnosis includes the assessment of the function of vaginal Lactobaccilus species, the detection of microbial metabolites and inflammatory reactions of the body.Vaginal microecological examination can not only accurately and quickly diagnose various single vaginal inflammation and mixed infections, reduce the rate of missed diagnosis and misdiagnosis, but also evaluate the prognosis, and also have guiding significance for applying vaginal microecological regulators after treatment. Finally, a good quality control is needed in the whole process of vaginal microecological examination.The "Practical Vaginal Microecology" has accumulated the Laboratory and Clinical Team of the West China Second University hospital, Sichuan University for nearly 20 years of clinical experiences, and combines the vaginal microecological evaluation and the latest progress of clinical diagnosis and treatment. It is a rare and excellent book to promote the diagnosis and treatment specification and key technology of female reproductive tract infection, training primary doctors and examiners and inspection personnel technical level and service ability. I hope the publication of this book can play a certain role in helping Chinese obstetrician, gynecologists and clinical examiners in the diagnosis and treatment of female reproductive tract infections. The writing of
this book has been strongly supported by the Infectious Diseases Cooperative Group of Obstetrics and Gynecology Branch of Chinese Medical Association and Professor Mingyuan Li of West China School of Basic and Legal Medicine, Sichuan University. I would like to thank them for their hard guidance and dedication in the field of vaginal microecology and medical microbiology over the years.This book was translated by our Translating Committee on the basis of the original book "Practical Vaginal Microecology". In the process of translation, the translators found the deficiencies of chapter 3 and Chapter 7 of the original book, and made some beneficial modifications and supplements (for example, "7.7.2 Gram staining of smear combined with clinical features of AV combination diagnostic criteria" was added in of Chapter 7 ). This makes the book more substantial and complete. Due to the limited level of editors and the short time, there are some shortcomings in the book. Welcome obstetricians and laboratory colleagues to criticize and correct this book.Yongmei Jiang, Zhengqiang HuTranslator: Zhengqiang Hu, January, 2025
C H A P T E R - 1Overview of the Vaginal MicroecologyZhengqiang Hu, Yongmei Jiang, Hongwei LiuTranslator: Zhengqiang HuMicrorobes that often live in the body surface and the cavity connected to the outside world, commonly known as normal microbiota or normal flora. It is necessary for the human body, and it is usually beneficial and harmless. A healthy adult has about 1×1013 human body cells, and the total number of normal microorganisms as high as 1×1014, mainly distributed in the skin and oral cavity, digestive tract, respiratory tract, urinary tract and reproductive tract and other viscera mucosal surface. These microorganisms like the "guard" of these open cavity and skin, resist the invasion of exogenous pathogenic microorganisms. In the long-term evolution process of the human body, through mutual adaptation and natural selection between species, different species, normal flora and hosts, and the environment are in a dynamic balance, forming an interdependent and mutually restricted human microecosystem. The female lower reproductive tract is an important part of the human microecosystem, and the microecosystem of female reproductive tract is composed of normal vaginal anatomy, endocrine regulatory system, the local vaginal immune system, and various vaginal microbiota. Various vaginal microbiomes influence and interact with each other, forming the microecological environment of the vagina.1.1 Normal Vaginal AnatomyVolvo-vaginal anatomy is the first line of defense against exogenous genital tract infection. The labium majus on both sides of the vulva naturally is closed and cover the vaginal and urethral entrance. The anterior and posterior walls of the vagina are close to a potential lumen in the natural state. Histologically, the vaginal epithelial lining is covered by intact stratified squamous epithelial cells. Squamous epithelial cells in childbearing age can proliferate and thicken with the rise of estrogen levels in the body, and also fall off periodically with the cycle change of estrogen. The vaginal epithelial cells of healthy women of childbearing age are rich in glycogen. Through the action of Lactobaccilus
2 Chapter 1species, glycogen decomposition produces lactic acid, maintaining the acidic environment of the vagina, and the pH value is maintained between 3.8-4.5. There are no secretory glands in the vagina, and their secretions come from the mucus secreted by the cervix, the vestibular glands, the paraurethral glands, the endometrium and fallopian tubes, and the exudates discharged from the submucosa. The vaginal environment of healthy women of childbearing age is acidic, and the cervical mucus plug is alkaline, so the vagina prevents the upward of pathogenic microorganisms, and the cervix limits the upward of pathogenic microorganisms, so it maintains the cleanliness of the upper part of the cervix, and the upward infection is not easy to occur in the natural state.Although the female pelvic cavity is physically connected with the outside world, it is often "sterile" due to layers of defense factors.The entry of any microorganism may be pathogenic, causing the infection of the upper genital tract or pelvic cavity. More than 90% of pelvic infections are caused by the upward infection of the lower reproductive tract pathogens, and the vagina is the first pass of opening to the outside world, but also the first pass to prevent the female reproductive tract infection; the cervix is the "throat" leading to the upper reproductive tract, it is the last pass to protect the female reproductive tract from being infected.In recent years through 16S rDNA gene amplification sequencing analysis, real-time quantitative PCR and microbial traditional culture method research, broke the "pelvic and on the reproductive tract for sterile environment" of traditional cognition, found that normal female pelvic and reproductive tract also exist microorganisms, reveals the vaginal tract to the cervical canal, uterine cavity, fallopian tube, until the pelvic physical structure and microflora structure has a certain continuity.Although the female pelvic cavity is physically connected with the outside world, it is often "sterile" due to layers of defense factors. The entry of any microorganism may be pathogenic, causing the infection of the upper genital tract or pelvic cavity. More than 90% of pelvic infections are caused by upward infection of lower reproductive tract pathogens, and the vagina is the first pass of opening to the outside world, but also the first pass to prevent female reproductive tract infection. The cervix is the "throat" leading to the upper reproductive tract, and it is the last pass to protect the female reproductive tract from being infected. In recent years, through 16S rDNA gene amplification and sequencing analysis, real-time quantitative PCR and traditional microbial culture method, the traditional cognition that "pelvic and upper reproductive tract is sterile environment" has been broken. We found that microorganisms also exist
Overview of the Vaginal Microecology 3in the pelvic cavity and the upper reproductive tract of normal women, which revealed the continuity of the physical structure and microbiota structure of the reproductive tract from the vagina to the cervical canal, from the uterine cavity and the oviduct to the pelvic cavity.1.2 Endocrine Regulatory SystemThe microbiota in the vagina are diverse, both antagonistic and symbiotic. Influenced by various factors in vitro and outside, it participates in the formation of a microecosystem with complex structure and function.The vaginal microbiota of normal women of childbearing age takes Lactobaccilus species as the dominant bacteria. The normally functioning vaginal microecosystem is an important gateway to maintain female reproductive health. However, the vagina is an open cavity, which directly connects with the outside world, vulnerable to the influence of various internal and external factors, leading to the imbalance of vaginal microecology, and then causing infection and other diseases.The internal cause is mainly closely related to the sex hormone level in different reproductive endocrine states of women.1.2.1 Role of Estrogen Progesterone Levels on Vaginal MicroecologyEstrogen and progesterone are the sex hormone of women, which plays an important role in maintaining the body function of women, and is also closely related to whether the vaginal microecology is in a balanced state. Estrogen promotes hyperplasia and maturation of the vaginal epithelium and keratinizates the superficial epithelial cells. The mucosal layer is thickened, thickening of the vaginal wall and increasing the nonspecific immune function of the vagina. At the same time, estrogen maintains the muscle function of the pelvic floor, making the anterior and posterior walls of the vagina close, and plays an important role as an anatomical barrier to maintaining the microecological environment of the vagina. Estrogen makes epithelial cells rich in glycogen, lactobacilli decompose glycogen to form lactic acid, hydrogen peroxide, etc., maintaining the acidic environment of the vagina, and inhabiting the reproduction of pathogenic microorganisms. Progesterone, on the other hand, can accelerate the loss of vaginal epithelial cells, reduce the number of lactobacilli in the vagina and vaginal cleanliness. The combined action of estrogen and progesterone makes the vaginal epithelial cells in a dynamic renewal process.
4 Chapter 11.2.2 Characteristics of Vaginal Microecology in Female Reproductive Endocrine States of Different AgesYoung Girl PeriodIn this period, the hypothalamus-pituitary-ovary (H-P-O) axis function has not been initiated, and the reproductive organs are not mature. The estrogen and progesterone in the body are at low levels, and the vaginal epithelium is at low estrogen levels. The compound squamous epithelial cells of the vagina have low hyperplasia, thin epithelium, and low glycogen content in the epithelial cells. The dominant bacteria are not Lactobaccilus species, which cannot maintain the acidic environment of the vagina. The mucus layer on the vaginal surface of young girl period is thin, and other pathogenic microorganisms grow more than reproductive age period. Common pathogens are Staphylococcus, Streptococcus and Escherichia coli.，prone to bacterial vaginosis (BV), but less Candida infection. Fortunately, this period of the outside interference to the vagina is less, and the existence of hymen in the vaginal anatomy of young girls also plays a certain role in protecting the vaginal environment, so the infection of young girls is not common.Reproductive Age Period This period is the period of vigorous female H-P-O axis function, and its main characteristic is the maturity of ovarian function and periodic ovulation. The level of sex hormone changes periodically with the follicle growth and development, maturation, ovulation, luteal formation, and luteal degeneration in the ovary. The definition of normal vaginal microecosystem is more applicable to women of childbearing age. Vigorous ovarian function and periodic estrogen progesterone are important factors to maintain normal vaginal microecosystem.The status of vaginal microecosystem also varies during different periods of the menstrual cycle of women of childbearing age. Menstruation is the result of luteal atrophy and reduced sex hormone levels, which is the lowest stage of sex hormone levels throughout the menstrual cycle. At the same time, due to menstrual interference, vaginal microecological state will occur transient imbalance, the Dominant of Lactobaccilus species is not obvious. With the development of follicles and the increase of estrogen levels, the vaginal microecological environment gradually changes to the normal（See Chapter 2, 2.3, The Significance of Vaginal Microecological Evaluation）. The vaginal pH value also varies in different periods, with ovulation period was lower than with follicular period, and the pH value during pregnancy is lower most.
Overview of the Vaginal Microecology 5The vaginal microecological state before and after menstruation is relatively poor, and some vaginal infections are more common, such as trichomoniasis vaginalis（TV), BV, etc., all of which are associated with low estrogen levels. During luteal stage, estrogen and progesterone level is high, which makes Candida species suitable for growth in acidic environment more easy to reproduce, so vulvovaginal candidiasis（VVC) is more likely to attack before menstruation. Patients with menstrual disorders caused by H-P-O axis dysfunction are likely to cause the imbalance of vaginal microecosystem due to abnormal sex hormone levels and long-term vaginal bleeding.Pregnant PeriodEstrogen levels increase during pregnant period, and glycogen increases in vaginal epithelial cells. Lactobacilli decomposes glycogen into lactic acid, and pH decreases, which is conducive to the growth of Candida species. At the same time, high levels of progesterone in pregnancy can reduce the number of vaginal lactobacilli and cleanliness, vaginal microbial reproduction, cause flora imbalance in the vagina, and immune tolerance of pregnancy, prone to vaginal infection, especially VVC. There are limited medication options for vaginal infections during pregnancy, coupled with immune factors leading to poor treatment effects.Puerperal State PeriodBefore ovarian function recovery during postpartum, estrogen at low levels, combined with the amniotic fluid flushing, postpartum lochia is not net, pelvic floor muscle relaxation, antibacterial drugs using multiple factors, such as vaginal micro ecological barrier become fragile, domestic studies found that only 13% for dominant bacteria, Gardnerella and bacteroids, staining variable bending bacteria, vaginal flora disorders, easy to cause a variety of vaginal infectious diseases, postpartum vaginal microflora normal less than 10%, Nugent score one third shows BV, the rest for BV intermediate state.Before the recovery of postpartum ovarian function, estrogen is at a low level, coupled with the amniotic fluid washing in childbirth, postpartum lochia dripping, pelvic floor muscle relaxation, antimicrobial use and other multiple factors, the vaginal microecological barrier becomes fragile. Domestic studies found that in only 13% of this population, Lactobacillus species were dominant bacteria, Gardnerella, bacteroids and indefinite staining campylobacter species increased significantly. Vaginal flora are imbalance, easy to cause various vaginal infectious diseases. Less than 10% of the postpartum vaginal microecological
6 Chapter 1flora is normal, one third of the Nugent scoring shows BV, and the rest are intermediate in BV.Late-Menopause Period Menopause is due to the ovarian reserve gradually reduced with age, the final ovarian follicles natural depletion, no follicle growth and estrogen sample, menstruation permanent stop. Losing of estrogen support, vagina gradually atrophy. Epithelial layer is decreased and become thinning. Glycogen, vaginal estrogen and glycogen gradually decrease, leading to the pH value increasing of the vagina, unable to maintain the vaginal acid environment, aerobic bacteria including Staphylococcus aureus, group B streptococcus and Escherichia. coli can gradually increase with age, part of the elderly women in a state of vaginal flora inhibition. Geriatric vaginitis is associated with reduced estrogen levels and vaginal microecological imbalance. There is reactive Lactococcus growth in some postmenopausal women, making the formation of a new microecological balance in the vagina.1.3 Vaginal Local Immune SystemThe local immune system of the female genital tract also belongs to the mucosal immune system, which is characterized by the large amount of secretary immuglobulin A (sIgA) on the mucosal surface, which plays the role of local immune defense. In addition, it also contains a small number of T and B lymphocytes, macrophages and other immune cells to maintain the physiological level of immune activities and protect the female reproductive tract from the invasion of pathogenic microorganisms and other antigens. Among them, vaginal mucosa response to microorganisms is the main immune response, which can be divided into non-specific immunity and specific immunity.1.3.1 Nonspecific Immunity Non-specific vaginal immunity against infection mainly includes barrier structure, phagocytes, and cytokines. Vaginal Mucosal Barrier Structure a. Mechanical blocking effect: The vaginal mucosa is compound squamous epithelial cells, and the complete vaginal mucosa can effectively prevent bacteria, fungi and other microorganisms from invading the body. b. Weak acid environment: The optimal acidity of the growth of most pathogenic microorganisms is pH7.2~7.6. The weak acid environment in
Overview of the Vaginal Microecology 7the vagina during childbearing age is not conducive to the settlement and reproduction of pathogenic microorganisms. c. Antagonism of the normal microflora: The presence of a large number of lactobacilli in the vagina will consume a large amount of glycogen, reducing the nutrients of other microorganisms and inhibiting the growth. Biofilms formed by a large number of lactobacilli can protect the host vaginal wall from other microorganisms. Phagocytes Phagocytes include neutrophils and mononuclear phagocytes. When the pathogen breaks through the mucosal barrier, the phagocytes escape from the capillaries and accumulate to the pathogen site to exert their phagocytic effects. Under normal circumstances, pathogens can be phagocytosis elimination, only virulent, a large number of pathogens can enter the human blood or other organs, and then by the blood and the phagocytes of other organs continue to phagocytosis killing effect.Cytokines When the outside microbes are invaded, reproductive tract epithelial cells will secrete killer cytokines and anti-microbial peptides, such as the tumor necrosis factor-α (TNF-α), interleukins-1 (IL-1), IL-6, IL-8, human definsin (HD, a class of cationic polypeptides rich in disulfide bonds, which is an important regulatory molecule in the human immune system), lysozyme (widely found in a variety of tissues in the human body, which is an alkaline enzyme that hydrolyzes mucopolysaccharides in pathogenic bacteria, antibacterial, anti-inflammatory, antiviral effects, etc.), calferin (A calponin derived from neutrophils and macrophages, whose expression is tissue-or cell-specific, and can be used as markers of acute inflammatory cell activation), histone-rich (Small molecule cationic peptides rich in histidine), etc., while promoting the establishment of local-specific cellular immunity. Studies found that the vaginal HD-5 level was significantly increased in different vaginal infection states, which also indicates that the innate immune factor HD-5 is involved in the pathogenesis of lower genital tract infection. Studies have also found that the mannose-bound lectin (MBL) exists in the female vagina, which can combine with the cue cell surface covering bacteria and fungi, and may play an important role in the local immune defense of the female genital tract. After binding to pathogens, MBL can bind to the phagocyte lectin receptor to initiate the phagocytic process. Recent studies have confirmed
8 Chapter 1that the concentration of local vaginal MBL is elevated at VVC and may be involved in the body's immune defense response and contribute to the clearance of infectious microorganisms. Primary human vaginal epithelial cells have been successfully cultured by tissue block culture method, and therefore, it is known that human vaginal epithelial cells have the effect of congenital anti-Candida albicans. When C. albicans infected human vaginal epithelial cells, the secretion of TNF-α and IL-10 are both increased, and the secretion of IL-2 and IL-8 are both no change.1.3.2 Specific Immunity Specific immunity to vaginal infection includes both cellular and humoral immunity, which is mediated by T and B lymphocytes, respectively. Cellular Immunity Cellular immunity is the result of multiple cellular interactions between immune cells leading to the release of multiple cytokines. Th1 cytokines include IL-2, interferon-γ (IFN-γ), IL- 12 and TNF-β, which mediate cellular immunity by promoting the activation and proliferation of natural killer cells (NK) and macrophages. Th 2 cytokines include IL- 4, IL-5, IL- 6, IL- 10 and IL- 13, whose main function is to stimulate the proliferation of B lymphocytes and produce antibodies to mediate the humoral immune response.Th1 and Th 2 are cross-regulate, and they inhibit each other. General studies indicate that the vaginal infection is related to the cell-mediated immune status. The results showed that the levels of IFN- γ, IL-5, IL-2, IL- 13 and IL- 8 were increased or decreased to different degrees, indicating that the local cellular immune function of the vagina also changes under vaginal infection. Humoral ImmunityHumoral immunity is an important part of the immune defense system, which plays an important role in the defense against pathogenic microorganisms. Under normal circumstances, IgG, IgM, IgA and IgE can be detected in the vagina, and they are generally at normal levels, especially sIgA. Several studies in China and abroad have shown that sIgA and IgG in the vaginal infection group are significantly higher than that in the normal control group. It is speculated that humoral immunity may have a certain role in anti-infection, but its exact role and mechanism need further research. SIgA is a major antibody present in the vaginal discharge and is a line of defense against bacterial and viral invasion.
Overview of the Vaginal Microecology 9The female reproductive tract contains rich plasma cells and produces a large amount of sIgA, which plays an immune role in the infection of the reproductive tract mucosa. Vaginal local immunity has been researched for more than ten years, the researchers in China also have their own vaginal primary cell culture patent technology and mature research methods. However, because of its research method is so multifarious, its sampling is so difficult, its research cost is too high, the field of vaginal ecological research is not extensive, especially in all kinds of vaginal diseases of immune mechanism and immune factor changes very little. We look forward to greater progress in the research of vaginal local immunity, and to provide more and better theoretical basis for the diagnosis and treatment of clinical genital tract infectious diseases.1.4 The Vaginal MicrobiotaIn normal conditions, multiple microorganisms are present in the vagina. At present, more than 200 kinds of microorganisms have been isolated in vaginal samples, among which the most important and the largest number are lactobaccili. It has been determined that the microbiota colonized in normal vagina is mainly composed of bacteria, including gram-positive aerobic bacteria or facultative anaerobic bacteria, such as Lactobacillus species, Corynebacterium, non-hemololytic streptococcus, Enterococcus and Staphylococcus epidermidis; gram-negative aerobic bacteria or facultative anaerobic bacteria such as Escherichia coli, Gardnerella vaginalis; anaerobic bacteria including Clostridium, Peptotreptococcus, Bacteroid and Fusobacterium, etc.. Under the normal status, the ratio of anaerobes and aerobic bacteria in the vagina is 5:1~10:1, and the two are in a dynamic equilibrium state. In addition, the normal vagina also colonizes Mobiluncus species, fungi, parasites, chlamydia, mycoplasma, viruses and other microbial groups. With the change of age, pregnancy and so on, the succession process of different microbial populations occurs currently. Under normal circumstances, Lactobuccilus species is the vaginal dominant bacteria. It is isolated in the vaginal discharge samples of healthy women and the isolation rate is up to 50%~80%. Lactobacillus is gram-positive large bacterium, without budding, elongated bent or ball rod, rod, single, double or chain, unpowered, microaerobic or facultatively anaerobic, but grow better in the anaerobic environment. The optimal growth temperature of lactobacilli is 35℃ to 38℃. There are 107 to 108 Colony-Forming Units (CFUs) in per gram of vaginal discharge sample. Current studies have shown that more than 120
10 Chapter 1species of Lactobacillus can be isolated in the vagina of healthy women, and the most common Lactobacillus species in the vagina are Lactobacillus acidophilus (L. acidophilus), L. salivarius and L. fermentans. The main H2O2 producers are L. friillius, L. gluthrii, L. Janensis and L. acidophilus.The normal presence of Lactobuccilus species in the vagina plays a key role in maintaining the normal vaginal flora. Glycogen in vaginal squamous epithelial cells is decomposed into lactic acid by Lactobuccilus, making the local vagina form a weak acidic environment (pH≤4.5, mostly between 3.8 and 4.5), which can inhibit the overgrowth of other parasitic bacteria and pathogenic bacteria. In addition, Lactobuccilus prevents pathogenic microorganisms from adhering to vaginal epithelial cells through substitution and competitive exclusion mechanisms. At the same time, the secretion of hydrogen peroxide, bacteriocin, bacteroids and biosurfactants can inhibit the growth of pathogenic microorganisms, so as to maintain the balance of vaginal microecological environment.The types of microorganisms detected in vaginal discharge are closely related to the testing methods. The number of strains that can be detected is determined by the traditional culture methods with the selection of medium appropriate or not. This methods will lose part of strains that are not suitable for the growth of the culture condition, and cannot fully reflect the composition of the vaginal microbiota. Therefore, the study of the vaginal microbiota is no longer limited to the traditional bacterial culture method. In recent years, the rapid development of molecular biology detection methods can detect almost all the microbial species we have found in the vagina. The high-throughput sequencing technology associated with metagenomics has greatly promoted the deep study of the microbiome. From the gene level, high-throughput sequencing can comprehensive, fast and efficient to obtain microbial classification information of all the vaginal microenvironment, and the application of the technology promote the vaginal microbiome in the scale of laboratory research, so that the study obtained a large number of accurate and fine data, thus become an important means of detection of vaginal microbiome. 1.5 Factors Affecting the Vaginal Microecosystem1.5.1 Interactions Between the Host tothe Flora and the Flora to the Flora The host to the flora and the flora to the flora are restricted, interactive and interdependent, or symbiotic or antagonistic, which are located in the microecological environment of the vagina, and maintain a coordinated and
Overview of the Vaginal Microecology 11balanced state. This balance maintains the normal physiological function of the human vagina. No matter which kind of microorganisms grows too much, or which function is too strong, local environment dysfunction or vaginal inflammation can be caused. In addition, the changes in estrogen levels, menstruation, pregnancy, childbirth, the occurrence of sexual activities, local medication, surgery (vagina, cervix, uterine cavity) and other factors, will also cause a series of changes in the vaginal microbiome. The fluctuation of estrogen levels in the physiological range is conducive to the change of the microbiome and the adaptation of the host to the environment. Once beyond its scope, it can cause the occurrence of inflammation and other diseases. With the increase of age, the decrease of estrogen levels, the thinning of vaginal epithelium, the emergence of aging, the decrease of vaginal lactobacilli’s nutrition leads to the decrease of lactobacilli’s quantity. Its isolation rate is constantly reduced, leading to the gradual increase of vaginal pH and the destruction of the acidic environment of the vagina. After the start of menstruation, the number of live bacteria decreased, about 100 times less than before the next menstruation. Sexual activities and local medication and other factors can cause the change of the local pH value of the vagina, resulting in the decline of vaginal resistance and disease. With age, the obligate anaerobes in the vagina remained the same, while Candida albican, Corynebacterium and Lactobacillus species decreased with age, on the contrary, group B streptococcus, Staphylococcus aureus and Escherichia coli in the vagina increased with age. New research found that due to the decrease of lactobacilli in menopausal women, some people will have reactive growth of lactococcus, they will produce acid, hydrogen peroxide and bacteriocin, not only to keep the weak acidic vaginal environment, achieving the purpose of inhibiting other pathogenic microorganisms growth, but also to make the vaginal micro ecological environment to achieve a new balance. The aerobic bacteria and anaerobic bacteria in the vagina restrict each other, interact and depend on each other, and are in a dynamic state of balance. All kinds of microbiome whether too much or too little will have adverse effects on the human body.1.5.2 Vaginal DoucheWhether the vagina should be flushed is a more controversial topic. Vaginal douche is a method of cleaning the vagina using a solution, which can be dated back to centuries. The purpose of initiation and maintenance are complex, including hygiene requirements, disease prevention, treatment,
12 Chapter 1contraception, and enhanced sexual experience. Among them, the exaggeration and mistransmission of vaginal douche benefits is also an important reason for its popularity. Although the prohibition of vaginal washing during pregnancy is widely recognized, there is still no consensus on whether it is conducive to personal hygiene or relieves vaginal inflammation. Many literature have reported the consequences against reproductive health, such as the exact relationship with the occurrence of bacterial vaginosis and acute pelvic inflammatory disease. The reason is that vaginal douche can lead to the destruction of the acidic vaginal environment, vaginal pH increase (nearly neutral), the normal bacteria are inhibited, anaerobic bacteria or other pathogenic bacteria excessive breeding. At the same time, vaginal douche may lead to micro-damage to the vaginal epithelium, breaking its complete physical barrier. The possibility of bacterial vaginosis, aerobic vaginitis, cervicitis, or pelvic inflammatory disease is greatly increased. Although some studies have shown the negative side of frequent douche, they have been reported in considerable literature, such as vaginal douche to reduce the risk of HIV infection through sexual transmission. Different types of vaginal douche solutions have different antibacterial effects. Three rinse solutions containing vinegar may be more beneficial than those of four antimicrobial agents. Therefore, the behavior of vaginal douche, under what conditions or environment, what ways and solutions to take, cause harmful results or get beneficial help, all need to be more scientific and appropriate assessment. Conclusion: Under normal circumstances, continuous and frequent vaginal douche is not advocated. In some vaginal infectious diseases, the removal of large amounts of vaginal discharge containing pathogens has certain therapeutic benefits; for certain diseases (such as sexually transmitted diseases), vaginal douche may reduce the risk of infection.Vaginal Microbial Electrical Potential Bacteria must be able to adhere to the vaginal wall to maintain long and stable residence in the vagina, and function by interacting with the host cell or the bacterial surface to the epithelial surface. The normal flora binds with the vaginal epithelial cell receptors through its own adhesion mechanism, grows on the mucosal surface of the vaginal wall, and participates in material metabolism and nutritional transformation. As an antigen, it stimulates the host to produce antibodies and enhance its immunity. Its metabolites can also constitute a biological barrier to prevent the invasion of foreign bacteria, and play an important role in maintaining the stability of the vaginal environment. The
Overview of the Vaginal Microecology 13biopotential of the vagina is affected by estrogen, and the acidic environment of the vagina is also beneficial to reduce the negative charge on the cell surface and remove the sugar group covering on the surface of the receptor, exposing the receptor and contributing to bacterial adhesion.1.5.3 Other FactorsAge, menstruation, sexual behavior, contraceptive methods, surgical procedures, the use of antibiotics and immunosuppressants can all affect the ecological balance of the vaginal microbiota. AgeAt birth, lactobacilli is dominant due to the acquisition of estrogen from the mother, but with the disappearance of maternal estrogen, various cocci and Escherichia coli gradually replaced Lactobacillus species as the dominant flora. In childhood, the vaginal flora includes skin parasites and intestinal flora. After menarche, the vaginal pH value drops, the vaginal acidic environment gradually forms, and the microflora begins to be dominated by lactobacilli. After menopause, estrogen drops, the vaginal epithelium begins to gradually atrophy, the pH value rises, and lactobacilli gradually drops.MenstruationDuring menstruation, the level of hormones and glycogen changes, while menstruation changes the vaginal pH value. The role of protective flora dependent on the acidic environment of the vagina will decline, and some pathogenic microorganisms can take advantage of it. There are some relevant studies to explore the solutions to the problems of women in this special period.The Use of Antimicrobial Drugs and Immunosuppressants The use of antimicrobial drugs and immunosuppressants will affect the vaginal microecological environment by enhancing or weakening the selective advantages of some special vaginal flora. Sexual Behavior Sexual behavior changes the external environment associated with the vagina, leading to changes in the local vaginal micro-environment. Semen is alkaline, and frequent sexual behavior may affect the pH value of the vagina. While unprotected sex also has an effect on vaginal Lactobuccilus, causing not only the loss of lactobacilli but also raising the levels of Escherichia coli and facultative anaerobes.
14 Chapter 1Contraception Contraception generally has a protective effect on vaginal microecosystem. Effective contraception reduces unwanted pregnancy, reduces uterine operation, and then has a promotion effect on reproductive health. Condoms are the only contraceptive method that is considered to prevent sexually transmitted diseases, which is beneficial to the maintenance of vaginal microecosystem. The most commonly used IUD in Chinese women has no obvious impact on the vaginal microecosystem, but it has some interference with the vaginal microenvironment during the perioperative period. In the form of contraception, the negative effect on the vaginal microenvironment is chemical sperkiller, which interferes with the vaginal microecosystem and may cause damage to the vaginal cervical epithelial cells. It is not recommended for people at high risk of sexually transmitted diseases.References[1] Qingping Liao. Female Vaginal Microecological Map．Beijing：The People's Health Publishing House, 2014 (Chinese)[2] Hongwei Liu. Vaginal microecological changes in different reproductive endocrine states．Journal of Practical Obstetrics and Gynecology，2018，34(10): 732-734 (Chinese)[3] Qingping Liao. Female vaginal microecology and it’s Evaluation. Journal of Practical Obstetrics and Gynecology, 2010, 26 (2)：81-83 (Chinese)
C H A P T E R - 2Vaginal Microecological Evaluation SystemFan Yu, Xiaojuan LiuTranslator: Zhengqiang Hu2. 2Since the 1950s, there are conducted researches on the imbalance of vaginal flora and flora adjustment in China, starting from the related research of vaginal flora and vaginitis to the relationship between vaginal flora and pregnancy outcomes and the link between vaginal flora and cervical cancer. With the deepening of the research on vaginal microecology, the concept of evaluating the vaginal microecological environment from the holistic and balanced view has gradually been recognized and accepted by people. However, the non-standard research methods and inconsistent evaluation standards have always limited the application of vaginal microecology in clinical work. This situation was not improved until the establishment of a rapid clinical evaluation system for vaginal microecosystem. Through the description of the vaginal flora density, flora diversity, dominant bacteria, inflammatory reaction and the morphology of pathogenic bacteria , combined with vaginal pH, H2O2, leukocyte esterase, sialidase and other vaginal microbial metabolites and enzyme activity functional test indicators, both are complementary, a comprehensive evaluation of vaginal microecological environment is conducted, as shown in figure 2-1. Because the sensitivity and specificity of functional test methods can not be balanced at present, and the product performance of different manufacturers is different, it is particularly necessary to point out that if the morphological detection and functional detection results are inconsistent, morphological detection is still taken as the main reference indicators. The expert consensus of this comprehensive evaluation system in clinical work has laid a strong foundation for vaginal microecological regulation. This chapter will introduce the clinical vaginal microecological evaluation system in detail.2.1 Morphological ExaminationMorphological examination of vaginal microecosystem mainly involves: • Microscopic examination of vaginal discharge by gram staining, which was
16 Chapter 2to observation flora density, flora diversity, dominant bacteria (gram positive coccus, gram negative bacteria, gram negative vibrio, etc.), the body inflammatory reactivity (white blood cells, septic leukocytes, etc.), epithelial cells and their change, pathogenic microorganisms (such as Trichomonas vaginalis, yeast-like fungal spores, yeasts with budding, yeast-like fungal pseudohyphae, etc.), gram positive bacteria, gram negative bacteria, etc., Various scores (such as Nugent score, AV score, etc.). • b. A semi-quantitative analysis of the morphology, which was to assist in the clinical evaluation of female vaginal microecological status. The standardized sample pre-analysis process is the prerequisite for the objective and correct evaluation of the above indicators. For the relevant contents, see Chapter 14 of this book "Operation Speciffcation and Quality Control of Vaginal Microecological Examinations". It will not be repeated in this chapter, but only explains the evaluation criteria of each indicators.2.1.1 Vaginal Flora Density Vaginal flora density is the distribution of bacteria, the density of the arrangement, combined with the size of the vaginal sample, which can reflect the total amount of microflora in the microecological area. The classification standard is: Grade I (1+): Observed under oil lens (10×100 times), the average number of bacteria per oil field is 1~9, see Figure 2-1a; Grade (2+): The average number of bacteria per oil field is 10 to 99, see Figure 2-1b; Grade (3+): the average number of bacteria per oil field was 100 or more; Observed under high power field (10×40 times), bacteria are full of view, see Figure 2-1c; Grade (4+): Under oil lens and high power lens observation, bacteria appear to aggregate or densely cover mucosal epithelial cells, see Figure 2-1d. The reporting method of the density is detailed in Table 2-1.Figure 2-1 Classification of vaginal flora densitya Grade Ⅰ b Grade Ⅱ
Vaginal Microecological Evaluation System 17Figure 2-1 Classification of vaginal flora density2.1.2 Vaginal Flora DiversityVaginal flora diversity is the number of all bacterial species in the vaginal smear, which was distinguished by positive or negative, different size, morphology and arrangement of gram staining. The classification criteria is: Grade I (1+): to identify 1-3 bacteria species, as shown in Figure 2-2a; Grade (2+): to identify 4~6 bacteria species, as shown in Figure 2-2b; Grade (3+): to identify 7~9 bacteria species, as shown in Figure 2-2c; Grade (4+): to identify 10 or more bacteria species, as shown in Figure 2-2d. The flora diversity is reported in Table 2-1..Figure 2-2 Classification of the vaginal flora diversityc Grade Ⅲ d Grade Ⅳa Grade Ⅰ b Grade Ⅱc Grade Ⅲ d Grade Ⅳ
18 Chapter 2Table 2-1 The Reporting method of flora density and flora diversityObserve under microscope (bacteria numbers)Reporting of vagi-nal flora densityObserve under microscope (bacteria species)Reporting of vaginal flora diversityabsent absent absent absent0 ~ 9/oil field 1+ 1 ~ 3 1+10 ~ 99/oil field 2+ 4 ~ 6 2+≥100/oil field 3+ 7 ~ 9 3+Gather into groups 4+ ≥10 4+2.1.3 Dominant Bacteria Dominant bacteria is the bacteria with the largest biomass or population density in the flora can, to a large extent, affect the function of the entire flora and have the greatest impact on the physiological pathology of the host. 1. Gram-positive bacilli: gram stain positive, no spores, elongated bending or ball rod, rod, single or double chain, no power, most of which are Lactobacillus species. 2. Gram-positive cocci or gram-positive vibrio: gram stain positive, no spores, elongated bending or ball, can be arranged into a chain, no power. The common bacterium is streptococcus. 3. Gram-negative short bacteria or gram-negative vibrio: negative or indefinite gram staining, no spores, short rod or rod shape, morphology is smaller than Lactobacillus species. Common bacteria are: (a) Gardnerella vaginalis: Gram-negative short bacillus or Gram-negative small proteobacterium; (b) Prevotella: Gram-negative bacilli; (c) Acetobacter: curved Gram-negative bacillus, Gram-stained variant, curved, curved microbacillus.2.1.4 Lactobacillary GradeThe Lactobacillary grade was based on the relationship between lactobacilli. and other flora. Grade Ⅰ: many of lactobacilli, no other flora; Grade Ⅱa: mixed flora, but mainly is lactobacilli; Grade Ⅱb: mixed flora, but the proportion of lactobacilli as significantly decreased, other bacteria are dominant; Grade Ⅲ: lactobacilli seriously reduced or missing, other flora are overgrowth. Studies have shown that under the condition of normal vaginal microecosystem, the
Vaginal Microecological Evaluation System 19Lactobacillary grade is good, Grade Ⅰ ~ Ⅱa accounts for more than 90%, and grade Ⅱb ~ Ⅲ proportion is very small, which was showing that lactobacilli plays a very important role in maintaining the balance of vaginal microecosystem.2.1.5 Leukocytes CountingLeukocytes counting mainly reflects the pathogenicity of the flora or the inflammatory response of the body to the flora. Generally according to 10×40 times microscopic white blood cells, body inflammatory reactivity is reflected. Leukocytes counting of vaginal discharge in trichomonas vaginitis, aerobic vaginitis, cervicitis and pelvic inflammation often rise. It is generally believed that the leukocyte count of vaginal discharge > 10 / high power field indicates the possibility of the above inflammation, and careful identification of the cause should be needed.2.1.6 The Vaginal Epithelial Cell Changes Vaginal epithelial cells attach to the surface layer of the vaginal lumen, forming the vaginal epithelial tissue, without glands, many of rampant wrinkles, with greater extension. Epithelial cells are divided into bottom layer, middle layer and surface layer. Their growth has a great relationship with the influence of estrogen, so the proportion of women is different in different periods of life, women of childbearing age, epithelial cells will undergo periodic changes in different periods of the menstrual cycle. Glycogen secreted by vaginal epithelial cells is converted into lactic acid under the action of vaginal Lactobacillus species, which plays an important role in maintaining the stable vaginal environment and the microecological balance of the vagina. Vaginal epithelial cell changes include:Clue Cells Clue cells are squamous epithelial cells infected by anaerobic bacteria such as Gardnerella, Prevotella, and kinetobacter. Its morphology has been changed, such as irregular edges, rough and fuzzy edges, which is a morphological manifestation of the adhesion of a large number of anaerobic bacteria on the epithelial cells of the vagina. It is the only specific and sensitive indicator in the diagnosis of bacterial vaginosis (BV). In the Amsel diagnostic criteria of BV, positive clue cells (Clue cells account for more than 20% of all epithelial cells) is a necessary diagnostic criteria. Combined with the increased pH value of vaginal samples, the accurate diagnosis of BV sensitivity is 83 to 93%, and the specificity is 82 to 94%.
20 Chapter 2Parabasal EpitheliocytesThe size of parabasal epitheliocytes is about 15~20μm, round, oval and shaped, and have a high nucleoplasm. The higher proportion of parabasal epitheliocytes in all epithelial cells, the greater likelihood of an aerobic vaginitis (AV), see Figures 2-4.Cell Lysis Women of reproductive age suffer from the lysis and breakdown of vaginal epithelial cells due to overgrowth of lactobacilli. Through microscopic observation of vaginal discharge, a large number of lactobacilli, naked nucleus, cell lysis and epithelial cell debris can be found, commonly found in cytotic vaginosis (CV).2.1.7 Vaginal Cleanliness The vaginal cleanliness is one of the important bases for the diagnosis of vaginal inflammation in women. It is a comprehensive judgment indicator through the combination of vaginal pathogens, miscellaneous bacteria, epithelial cells, leukocytes and lactobacilli, which reflects the vaginal cleaning status and is divided into grade I~IV. The classification standard of vaginal cleanliness was provided in the fourth edition of "Clinical Laboratory Operation Procedures of China" in Table 2-2. However, in practice, the authors found that various components in vaginal discharge often intersect when compared with the classification standard, which means that the standard is relatively simple. It is impossible to comprehensively and reasonably solve the various situations faced in the actual testing process of clinical samples, resulting in the laboratory operators being hesitant and unable to accurately grade. Therefore, after communication with clinicians, the author's laboratory has formulated a more comprehensive cleanliness classification standard, as shown in Table 2-3 for details.Table 2-2 Classification standard for vaginal cleanlinessGrade of Vaginal cleanlinessLactobacillus speciesMiscellaneous bacteriaEpithelial cellsLeukocytes (HPF: high power field)Grade I >30 / oil field <1 / oil field Full of field 0-4 / HPFGrade Ⅱ 5-30 / oil field 1-4 / oil field 1/2 field 5-15 / HPF
Vaginal Microecological Evaluation System 21Grade of Vaginal cleanlinessLactobacillus speciesMiscellaneous bacteriaEpithelial cellsLeukocytes (HPF: high power field)Grade Ⅲ 1-4 / oil field 5-30 / oil field <1/2 field 16-30 / HPFGrade Ⅳ <1 / oil field >30 / oil field absent >30 / HPFNote: The standard was provided in the fourth edition of "Clinical Laboratory Operation Procedures of China"2.1.8 Pathogenic Microorganisms Pathogenic microorganisms refer to the microorganisms that can cause different vaginal infectious diseases. The common ones in vaginal discharge are: Yeast-Like FungusYeast-like fungus is a unicellular fungus that reproduces through the extended bud that does not leave the mother cell is called pseudohyphae, while budding from pseudohyphae can produce yest budding. The yeast-like spores are Candida yeast, Gram-positive fungal microorganisms, 5~8 times the volume of ordinary gram-positive coccus. It is normal vaginal opportunistic pathogens, playing a role in asymptomatic residence and transmission. When the body immunity decline or combined with other diseases, yest phase turning into hyphal phase, symptoms. The finding of spores alone indicates that Candida species is in a carrier state and cannot diagnose vulvovaginal candidiasis (VVC) originally. The occurrence of yeast pseudohyphe and yeasts with buddings indicates that Candida species has the ability of infection and invasion. Pseudohyphe is gram-positive large bacteria, different length, 5~8 times that of ordinary gram-positive bacteria. Either of the two findings(yeast pseudohyphe and yeasts with budding) indicates the existence of VVC, combined with clinical symptoms can diagnose simple or complex VVC. Based on the above reasons, spores, yeasts with budding or pseudohyphae should be reported instead of only yeast-like bacteria. Trichomonas VaginalisTrichomonas vaginalis is a flagellates in the urogenital tract, which is 2~3 times larger than white blood cells, pear-shaped, round, oval or irregular, particles with uniform thickness in the cytoplasm. Its nucleus is small, oval, 1/3 at the end of the worm, extended from the axial column to the worm. Four anterior flagella can be clearly observed, occasionally, a posterior flagellum is visible. There is no Trichomonas in the normal vagina, and the parasites is mainly parasitic in
22 Chapter 2Table 2-3 Reference opinions on common judgment mode of vaginal cleanliness in Department of Laboratory Medicine , West China Second University Hospital, Sichuan UniversityVaginal cleanlinessEpithelial cellsLeukocytesLactobacillus speciesFungusTrichomonas vaginalisG- diplococ-cusRatio of clue cellsMiscellane-ous bacteriaYest sporesYests with buddingYest pseudo-hyphaGrade I1/2 or full of field≤5/HPF＞30/oil fieldAbsentAbsentAbsentAbsentAbsentAbsentAbsentGrade Ⅱ1/2 or full of field≤15/HPF1~30/oil fieldAbsentAbsentAbsentAbsentAbsentAbsentAbsentGrade Ⅱ1/2 or full of field5~15/HPF＞30/oil fieldAbsentAbsentAbsentAbsentAbsentAbsentAbsent or present Grade Ⅱ1/2 or full of field≤15/HPF≥1/oil fieldAbsent or present AbsentAbsentAbsentAbsentAbsentpresent Grade Ⅱ1/2 or full of field≤15/HPF≥1/oil fieldpresent AbsentAbsentAbsentAbsentAbsentAbsent or present Grade Ⅱ1/2 or full of field≤15/HPF≥1/oil fieldAbsentAbsentAbsentAbsentAbsent1~20%present Grade Ⅱ1/2 or full of field16~30/HPF≥5/oil fieldAbsentAbsentAbsentAbsentAbsentAbsentAbsentGrade Ⅲ1/2 or full of field16~30/HPF<5/oil fieldAbsentAbsentAbsentAbsentAbsentAbsentAbsent or present
Vaginal Microecological Evaluation System 23Vaginal cleanlinessEpithelial cellsLeukocytesLactobacillus speciesFungusTrichomonas vaginalisG- diplococ-cusRatio of clue cellsMiscellane-ous bacteriaYest sporesYests with buddingYest pseudo-hyphaGrade Ⅲ≤1/2field＞30/HPF≥1/oil fieldAbsentAbsentAbsentAbsentAbsentAbsentAbsent or present Grade Ⅲ≤1/2fieldAny range≥1/oil fieldAbsentAbsentAbsentAbsentAbsent>20%present Grade Ⅲ≤1/2fieldAny range≥1/oil fieldpresent present Absent or present Absent or present AbsentAny rangeAbsent or present Grade Ⅲ≤1/2fieldAny rangeAny rangeAbsent or present Absent or present Absent or present present AbsentAny rangeAbsent or present Grade Ⅲ≤1/2field0~30/HPF<1/oil fieldpresent present Absent or present Absent or present AbsentAny rangeAbsent or present Grade Ⅲ≤1/2field0~30/HPF<1/oil fieldAbsent or present Absent or present Absent or present Absent or present Present inside and outside of leukocytesAny rangeAbsent or present Grade IV≤1/2field＞30/HPF<1/oil fieldAbsent or present Absent or present Absent or present Absent or present Absent or present inside and outside of leukocytesAny rangeAbsent or presentNote: It is recommended not to report vaginal cleanliness in children under 14 years old due to the differences in vaginal flora from women of childbearing age.
24 Chapter 2the vagina and urethra of women, which can cause trichomoniasis vaginalis and urethritis. After sampling, Trichomonas can be seen in wet tablets in a short time and at suitable temperature.Gram Negative (G-) Diplococcus Under microscopic observation of vaginal discharge, G- diplococcus are the pathogen of gonorrhea infection. The common infection site in women is cervix, but it is visible in vaginal discharge. They are stained gram-negative and nephroform a double arrangement. If intracellular inspection, it is gathered in groups. If only extracellular inspection, it is suspicious and must be cultured for identification. When acute gonococcal infection occurs in the cervix or vagina, G-diplococcus can be found both inside and outside of white blood cells. The microscopic epithelial cells are few, with a large number of inflammatory cells.Gardnerella Vaginalis Gardnerella vaginalis is found in the vagina of all BV patients, but also in 50% of the healthy population. Its specific morphology is gram-negative or stain-indefinite bacteria or variant coccal-like microbacillus. It is about 0.4~2um size, circular ends, polymorphism, amastigote capsule and spores. Large numbers of Gardnerella attached to squamous epithelial cells are often seen to form clue cells.Other BacteriaOther bacteria includes some common Gram-positive cocci causing aerobic vaginitis (AV): Staphylococcus aureus, coagulase-negative staphylococcus (such as Staphylococcus epidermidis), Group B streptococcus (Streptococcus agalactiae) and Enterococcus faecalis; Gram-negative bacteria are mainly Escherichia coli. 2.1.9 Nugent Score by Gram Staining Nugent score by Gram staining is a common laboratory method for more accurate diagnosis of BV. The score includes Lactobacillus score, Gardnerella and Prevotella species score and Mobiluncus score, and the sum of the three score is the total Nugent score. The Nugent score 0 to 3 indicates normal, 4 to 6 indicates intermediate BV; ≥7 indicates BV. Specific scoring criteria are shown in Table 2-4.
Vaginal Microecological Evaluation System 25Table 2-4 The Nugent scoring system for vaginal discharge smearsNugent scoreLactobacillus scoreGardnerella and Prevotella sp. scoreMobiluncus score0 >30 / oil field 0 01 5-30 / oil field <1 / oil field 0-4 / oil field2 1-4 / oil field 1-4 / oil field >4 / oil field3 <1 / oil field 5-30 / oil field -4 0 >30 / oil field -2.1.10 Aerobic Vaginitis ScoringAerobic vaginitis (AV) is a common genital tract inflammatory disease in clinical gynecology, and there is no standardized and recognized diagnostic standard for AV. The clinical diagnosis currently mainly depends on Tempera diagnostic criteria and Donders scoring method. Tempera Clinical diagnostic criteria include the following four: a. Vaginal discharge odor and yellow, but negative amine test; b. pH value increased to > 5.0; c. There are large number of white blood cells under microscopic observation; d. Lactobacillary grade are grade IIb or grade III. The Donders scoring method based on phase contrast microscope observation of wet smear is the mainstream method for laboratory diagnosis of AV. The score includes five aspects: Lactobacillary grade, the proportion of white blood cells containing toxic particles, and the proportion of parabasal epitheliocytes (PBC). The sum of the five scores is the total Donders score. AV can be diagnosed when the score is ≥3, but need to combine with the clinical features. In recent years, experts in China have proposed a modified AV diagnostic criteria based on gram stain smear combined with clinical features.The two specific diagnostic criteria are shown in Table 7-1 and 7-2 of Chapter 7. Examples of the morphology of white blood cells containing toxic particles and PBC are shown in Figure 2-3 and Figure 2-4, respectively.
26 Chapter 2Figure 2-3 l Leukocytes containing toxic granule Notes: The arrow shows the white blood cells containing toxic particles, and also shows the swelling of white blood cell body and the increase of cell nucleus. More particles of different sizes are found in the cytoplasm of the fine tuning lens.Figure 2-4 Parabasal epitheliocytesNote: The arrow shows parabasal epitheliocytes, which are about 15~20 μm in size, round, oval and boat shape, and relatively high nucleoplasm.
Vaginal Microecological Evaluation System 272.1.11 Vaginal Flora Imbalance Vaginal flora imbalance mainly includes three conditions, which may have no symptoms of vaginal infection: • Flora inhibition: The flora in per volume of the vaginal samples were significantly reduced, specifically as no dominant bacteria, density and diversity are ≤ grade I or completely without lactobacilli, common in atrophic vaginitis. • Overgrowth of flora: Gram-positive bacilli with a morphology similar to lactobacilli were as the dominant bacteria. the flora density and flora diversity are grade Ⅲ-Ⅳ , which is common in cytolytic vaginosis (CV). • Dominant bacteria abnormality: mainly refers to the dominant bacteria is not Lactobacillus species, but Gram-positive microbacilli, gram-positive cocci, gram-negative bacilli and other bacteria.2.2 Functional Tests of Vaginal FloraIn the clinical evaluation of vaginal microecosystem, in addition to morphological examination, different microorganisms in the vagina can produce different metabolites as well as different activities of enzymes. Therefore, according to the different microbial metabolites and enzyme activity to set up different markers of function indicators, it is not only necessary to conduct a comprehensive and objective evaluation of the vaginal microecological environment, but also to find the causes of some patients that cannot be tracked by morphological examination, and even to make up for the omission and misexamination of morphological results. At present, more and more clinical workers and researchers have begun to gradually apply combined enzymatic profiles or metabolites to analyze the status of vaginal microecosystem. In addition to vaginal pH value, these enzyme profiles and metabolites include hydrogen peroxide (H2O2), sialidase (SNa), leukocyte esterase (LE), β-glucuronidase (GUS), coagulase, β-N-acetylglucosaminosidase (NAG), prolyl aminopeptidase(PAP), oxidase(OX) and lactic acid(LA). This section will explain the principles and significance of some common functional tests.2.2.1 pH Value DetectionAcid-base indicator method generally requires precision pH test paper (3.8-5.4) to test the pH value of vaginal discharge. When using the pH test paper, it is best to contact the vaginal discharge directly with the pH test paper, or use a dry cotton swab sampling to smear on the pH test paper. Many manufacturers
28 Chapter 2have developed manual, semi-automatic and automation products, which are generally sampling vaginal discharge with dilutions before detection. The indicator substrate on the pH paper pad will show the corresponding color change at different acid and base values. The manufacturer has made the diluted pH correction before this kind of product goes to market to avoid affecting the test results. When Lactobacillus species is the dominant bacterium in the vaginal microecosystem, it uses the glycogen in the vaginal epithelial cells to produce lactic acid, which makes the normal vagina has a weak acidic environment, and the pH value is between 3.8 and 4.5. Generally speaking, if the Lactobacillary grade is between grade I and IIa, the average value of vaginal pH is within the normal range; if the Lactobacillary grade is grade IIb or III, the average value of vaginal pH increases, indicateing that Lactobacillus species is very important in maintaining the vaginal pH value. 2.2.2 H2O2 Detection The metabolites of Lactobacillus species and Lactococcus include lactein, H2O2, and lactic acid. The concentration of H2O2 is positively correlated with the number of H2O2-producing Lactobacillus species or Lactococcus, and the normal function of Lactobacillus species can also be determined according to the concentration of H2O2. Most vaginal Lactobacillus species have the ability to release H2O2.The produced H2O2 has inhibitory or toxic effects on neighboring bacteria, fungi and viruses. Especially in the presence of peroxidase and halide, the bactericidal capacity of H2O2 is greatly enhanced. In the presence of peroxidase, H2O2 decomposition to produce oxygen, which can change the color of the chromogenic substrate, and the color depth is proportional to the H2O2 concentration. When the number or function of vaginal lactobacilli is decreased, the H2O2 concentration is < 2μmol/L, and the test hole does not change color, which indicates that H2O2 is positive. H2O2 positive represents decreased number of Lactobacillus species and Lactococcus or low H2O2 function. 2.2.3 Lactic Acid Detection Lactic acid (LA) is one of the main metabolites of Lactobacillus species and Lactococcus. After puberty with female estrogen levels rise, glycogen content on the vaginal wall and epithelial cell number also gradually increased. Glycogen decomposition of LA and glucose, maintaining the healthy women’s low vaginal pH value. In vitro studies, it was found that the growth of lactobacilli acidification can inhibit pathogen proliferation, such as Candida, Escherichia
Vaginal Microecological Evaluation System 29coli and Gardnerilla vaginitis. LA produce pyruvate and H2O2 under the action of lactate oxidase. After peroxidase, in the presence of tetramethylbenzidine, the color of H2O2 depth is proportional to the concentration of LA. In addition, Lactobacillus species, streptococcus, enterococcus can also produce LA, and the detection rate is high. So theoretically speaking, using LA to detect lactobacilli in vaginal discharge, the probability of false positive is large, so we can not only use LA to detect the clinical significance of Lactobacillus species. 2.2.4 Leukocyte Esterase (LE) Detection LE is directly proportional to the number of destroyed leukocytes, which indirectly reflects the proliferation level of pathogenic microorganisms. But specifically, LE is only found in neutrophils, not in lymphocytes and monocytes.Thus LE positive indicates that a large number of multinucleated leukocytes are destroyed in the vaginal discharge so as to release this enzyme. The vaginal mucosa is damaged and there is an inflammatory reaction happened. LE can hydrolyze 5-bromine-4-chloride-3-hydroacetate, releasing colorless bromodo group. In special substances after the reaction discoloration, its color depth is proportional to the activity of LE. The positive results often indicate that there may be all kinds of genital tract inflammation, such as cervicitis, pelvic inflammation, vaginitis and so on. 2.2.5 Sialidase Detection BV is a severe microecological imbalance of the vagina. When the pathogenic bacteria of BV are present (such as Gardnerella vaginalis, Prevotella species, Peptostreptococcus, etc.), the activity of sialidase increases. So, positive sialidase reaction is a relatively specific indicator of BV. Although it can not be used for diagnosis, but can be used for clinical screening. Sialidase can hydrolyze cresol blue-N-acetylneuraminoside sodium salt to produce sialic acid and chromogroup, which develops color under alkaline conditions, and its color depth is proportional to the content of sialidase. 2.2.6 Prolyl Aminopeptidase (PAP) Detection PAP is a non-specific indicator. PAP is positive when infected with some Gardnerella vaginalis, Mobiluncus and Candida albicans. The reaction principle is that proline aminopeptidase hydrolyzes the substrate L-proline p-nitroaniline and releases colored substances. Its color depth is proportional to the activity of PAP.
30 Chapter 22.2.7 β-N-Acetylglucosaminosidase (NAG) Detection NAG in normal female vagina should be negative. When NAG is positive, combined with pH≥4.8, it often indicates trichomonas vaginalis or anaerobic infection. When NAG is positive combined with pH≤4.6, it indicates Candida infection or its previous infection. NAG can react with opposite (adjacent) nitrobenzene-N-acetyl-β-D-glucosaminside, and the released opposite (or adjacent) nitrobenzene shows color under alkaline conditions, and its color depth is proportional to the content of NAG. However, clinical patients often suffer from vaginitis mixed infection, so in addition to the pH value, NAG and morphological results are also very important. 2.2.8 β-Glucuronidase (GUS) Detection GUS is an acidic lysosomal enzyme that hydrolyzes protestations--the main component of the basement membrane, dedicating damage to the vaginal membrane. It is mostly synthesized by aerobic bacteria, such as group B streptococcus and Escherichia coli, which is beneficial to understand the reproduction and invasion of AV pathogens. The detection principle is that β-glucuronidase can hydrolyze 5-bromine-4-chloride-3-glucuronide, releasing colorless bromodo group. After the reaction of color, its color is proportional to the activity of β- glucuronidase.2.2.9 Coagulase Detection Coagulase is a plasma coagulase, which is mainly produced by staphylococcus aureus. It indicates the proliferation of vaginal aerobe, prone to cause AV and vaginal microecological imbalance. 2.2.10 Oxidase (OX) Detection The positive of OX has a certain indication on the gonococcal infection, which must be confirmed in combination with the morphological results. In the presence of oxidase, oxygen oxidizes the intracellular prototype cytochrome C to oxidized cytochrome C, the substrate tetraethylp-phenylenediamine hydrochloride under the action of oxidative cytochrome C, its color depth is proportional to the oxidase activity. 2.2.11 Limitations of Functional Detection The functional detection indicators are more and more, but not the sensitivity
Vaginal Microecological Evaluation System 31and specificity of each indicator are enough to make it a diagnostic indicators. Moreover, the test results are relative to the reagent quality and manufacturer’s manufacturing level, which also makes the conclusions different and even more controversial in various research and clinical applications. At present, functional indicators mostly exist as an auxiliary diagnostic role for morphological examination.2.3 The Significance of Vaginal Microecological EvaluationThe normal vaginal microecological status is generally defined as: the vaginal flora density is grade II~III, the vaginal flora diversity is grade II~III, the dominant bacteria are Lactobacillus species, vaginal pH is 3.8~4.5, normal function of lactobacilli (negative H2O2 test results), and negative enzyme indicators such as LE. When any of the vaginal flora density, vaginal flora diversity, dominant bacteria, white blood cell count of vaginal discharge, pH value and Lactobacillus function is abnormal, it can be diagnosed as vaginal microecological imbalance.Vaginal microecological evaluation system can not only diagnose clinical common types of vaginal infection, but also can diagnose some asymptomatic, or only "vulva pruritus, increased vaginal discharge", especially unexplained vaginitis with only abnormal cleanliness and no specific pathogens. Such patients often due to the special pathogenic microorganisms cannot be found by the traditional morphology routine examination of vaginal discharge and are difficult to diagnose. Finally, they are relied on vaginal microecological evaluation system to improve the clinical diagnosis rate. Vaginal microecosystem is complex and changeable, which is affected by many factors. Various factors restrict and regulate each other. Researches believe that most of the microecological disorder is temporary, the body resistance can return to normal. The treatment principle of vaginitis has been transformed from the traditional sterilization concept to the concept of antibacterial-repair-restore microecosystem. The factors that affect the balance of vaginal microecosystem are worth continuous exploration by researchers. We use morphological examination and functional indicators better together, so as to more comprehensively evaluate the changing characteristics of vaginal microecosystem, timely correction of vaginal flora disorders, so that female reproductive tract infection can be more effectively prevented and treated.
32 Chapter 2References[1] Infectious Diseases Cooperative Group of the Obstetrics and Gynecology Branch of the Chinese Medical Association. Expert consensus on the clinical application of vaginal microecological evaluation. Chinese Journal of Obstetrics and Gynecology, 2016,51 (10): 721-723.[2] Hongli Wang. Experimental diagnostics. Beijing: People's Health Publishing House, 2001.[3] Department of Medical Administration, Ministry of Health, PRC. National Operating Procedures for Clinical Laboratory. Beijing: People's Health Publishing House, 2015.[4] Mengting Dong, Chen Wang, Huiyang Li, et al. Expert recommendation for the combined diagnostic criteria of aerobic vaginitis based on Gram stained smear combined with clinical features. Chinese Journal of Practical Gynecology and Obstetrics, 2021, 37(3): 327335. DOI: 10.19538/j. fk2021030115.
C H A P T E R - 3Lactic Acid-Producing BacteriaJing Zhang Translator: Yunxia Li3. 1Vaginal symbiosis is characterized by the presence of a beneficial lactic acid-producing microbiota, mainly from the Lactobacillus species. The vagina of healthy women and the microbial community therein constitute a balanced microecosystem, with lactobacilli being the main flora of the vagina of healthy women of reproductive age. It was early believed that the main Lactobacillus species in the vagina were Lactobacillus acidophilus (L.acidophilus) and L. fermentans. Up to now, there are about 120 Lactobacillus species reported, among which four are mainly dominant flora: L.crispatus, L.gasseri, L.iners and L.jensenii. The colonization of lactobacilli plays an important role to maintain healthy vaginal micro-environment and antagonistic pathogenic invasion. The decrease of flora diversity and quantity of vaginal lactobacilli may be associated with diseases, such as bacterial vaginosis (BV), fungal infection, sexually transmitted diseases (i.e. Acquired Immunodeficiency Syndromes), urinary tract infection, etc.. In addition, studies showed that vagina that lacks of lactobacilli is correlate to premature of pregnant women. Cacoethic vaginal microecosystem may be associated with cervical persistent high-risk type HPV infection, and increase the incidence of cervical cancer. 3.1 Vaginal Microenvironment and its MicrofloraNormally, the vaginal flora is generally distributed in the vaginal wall, posterior vaginal fornix, cervix and external cervical opening. Human microbiome-related studies indicate that the vaginal microecosystem has a relatively simple structure and low diversity. Before menarche in women, the species of vaginal flora are generally unstable and similar to the Lactobacillus species of skin and intestinal, but the dominant bacteria in the vagina after menarche are relatively constant, and the Lactobacillus species are the most common, and relatively stable. At present, more than 120 kinds of Lactobacillus species have been found. In the reproductive stage, due to the role of estrogen and progesterone, lactobacilli
34 Chapter 3will become advantageous. Estrogen will promote the proliferation of vaginal epithelial cells and the synthesis of glycogen, and progesterone will stimulate the dissolution of epithelial cells, promoting the release of glycogen.Lactobacilli and some other bacteria digest these glycogen, turning it into glucose and maltose and further into lactate, maintaining vaginal pH at 3.8 to 4.5. Currently, vaginal Lactobacillus species of healthy women generally include Lactobacillus iners (L. iners), L. crispatus, L. gasseri, L. jensenii. A subset of healthy or no clinical women whose vaginal dominant bacteria are not Lactobacillus species, but provotella, Gardnerella, Atopobium vaginae and Megacococcus. This view was confirmed by microscopic examination, bacterial culture and molecular biological research methods. There was clear evidence that the same female individual has changes in the vaginal microflora during different periods of the menstrual cycle or after sexual behavior. At a certain point in the menstrual cycle, healthy women will have a decline of dominant lactobacilli and an increase in the number of other bacteria. Clinical examination found that the Nugent score increases, but patients did not appear clinical symptoms of bacterial vaginosis (BV). In women after menopause, especially those with dry vaginal atrophy, there will be a decrease of lactobacilli, mainly L. crispatus, and microscopic examination will find the inhibition of bacterial flora.Newborn female baby of vaginal delivery did not have vaginal microbes at birth, but high levels of estrogen were obtained from the mother, and the vaginal microbiota similar to their mother appeared after about 2 to 3 weeks..Changes in the composition of the vaginal flora are driven by dramatic changes in estrogen during a woman's lifetime. In early childhood, the vaginal acidity is neutral or slightly alkaline.With the increase of estrogen levels during puberty, the amount of glycogen stored in the vaginal epithelium increases, prompting the final dominance of lactobacilli. The vaginal microenvironment returned to childhood until menopause. Vaginal microbes are a dynamically changing microecosystem, whose presence and quantity are controlled by estrogen, and the main influencing factors are age, menstrual cycle, sexual behavior and ethnicity. In short, the vaginal microenvironment controls the types of microorganisms, whereas the vaginal microbes also control the vaginal microenvironment.3.2 Lactococcus SpeciesIn addition to the presence of Lactobacillus species in the vagina of healthy women, Lactococcus species is present in the vagina of healthy women. In menopausal women, due to the decrease of lactobacilli, some people will also
Lactic Acid-Producing Bacteria 35have a reactive growth of Lactococcus species. These cocci play a very important role in maintaining the process of balancing the vaginal flora, including: a. Metabolizing lactic acid, thus maintaining the acidic vaginal environment and inhibiting the overgrowth of pathogenic bacteria; b. Producing bacteriocins, which have selective antibacterial effect: no inhibitory effect on themselves and lactobacilli, but strong inhibitory effect on the pathogenic bacteria, such as Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Candida albicans and other pathogenic bacteria; c. Some lactococci produce hydrogen peroxide (H2O2) in the metabolic process, which has a strong inhibitory effect on pathogenic bacteria. Lactococcus species includes Enterococcus faecalis, Strong Enterococcus, Enterococcus faecium and Streptococcus bovis in Streptococcus species. These lactococci have a synergistic effect with lactobacilli, which can enhance the inhibition ability of pathogenic bacteria and expand the scope of bacteriostasis. They are a group of probiotics that maintain the balance of normal vaginal flora. At the same time, many lactococci can produce metabolites such as lactic acid, bacteriocin and H2O2, and have selective antibacterial effect, namely only effect on pathogenic bacteria, not on itself and probiotics such as lactobacilli. So, these cocci plays an important role in maintaining the ecological balance: a. can be used for prevention and treatment of gynecological diseases caused by the flora imbalance; b. can also use them to develop a variety of microecological agents for clinical use. 3.3 Lactobacillus SpeciesThe Lactobacillus species is a group of Gram-positive bacteria belonging to the kingdom of bacterial, phyla of Firmicutes, class of Bacillus, order of Lactobacillus, family of Lactobaceae, along with the Lactobacillus species and Peelococcus. There are currently 185 species and subspecies within this genus, named for the large amounts of lactic acid produced by fermented sugars. Common strains include Lactobacillus acidophilus (L. acidophilus), L.alimentarius, L. animalis, L. brevis, L. casei, L. catenaformis, L. divergens, L. fermentans, L. gasseri, L. plantarm, L. minutis, and L. vaginalis.The bodies of Lactobacillus species are long and elongated, sometimes curved and shorter, often present as corynebacterium species, with a rounded tip that can form chains, similar to streptococcus, do not produce spores, and are usually unflagellated. Culture characteristics: facultative anaerobic, sometimes slightly aerobic, 5% CO2 can promote growth, some bacteria need anaerobic isolation.
36 Chapter 3Lactobacillus species is an extreme caustic bacterium that requires nutrient-rich medium for growth; It is most suitable for growth at pH 5.5~6.2, but can still grow at pH 3.0~4.5 and generally does not produce any pigment. Biochemical characteristics: fermented carbohydrates, isotype fermented glucose; most of the end products are lactic acid, allotype fermentation produces lactic acid, carbon dioxide, ethanol and (or) acetic acid; no reducing nitrate, no liquefied gelatin, do not produce indole or hydrogen sulfide, touch enzyme and oxidase are negative.3.3.1 Overview the Study History of Vaginal Lactobacillus Species The first article on the study of vaginal microbes was published by Doderlein et al in 1894 when the discovery of Gram-positive bacilli was exactly members of the Lactobacillus species that we are now known about. Early culture-based studies suggested that the main vaginal Lactobacillus species were L. acidophilus and L. fermentans, and more different normal or pathogenic bacteria were discovered in several decades. With the development of anaerobic culture techniques and molecular biology techniques without culture, about 120 species of vaginal Lactobacillus have been discovered so far.In recent years, the high-throughput sequencing technology of 16S rDNA is increasingly mature, which enables us to have a deeper understanding of the diversity and abundance of the vaginal microbiota (VMB). In 2011, according to the community status type of VMB (community state type, CST), Ravel et al divided VMB into 5 types: CST I type (dominant group is L. crispatus), CSTⅡ type (dominant group is L. gasseri ), CSTⅢ type (dominant group is L. iners ), CST Ⅳ type (dominant group is anaerobic bacteria , no or low levels of Lactobacillus species, Where Ⅳ-A takes mainly the Anaerococcus, Peptoniphilus, P. bivia et al, Ⅳ-B takes mainly the Atopobium vaginae and Megacococcus) and CST-V type (dominant group is L. jensenii). The composition and abundance of VMB vary in different individuals or individuals during different periods. The reasons for this change are not only individual hormone levels, immune system and behavior, but also ethnic factors.3.3.2 Classification of Vaginal Lactobacillus Species L. crispatus L. crispatus was first discovered by Brygoo et al in 1953. L. crispatus is a slender rod, slightly bent chain arrangement, amastigotes and no spores; it can ferment mannose, galactose, glucose, maltose, melbiose, raffinose, ribose, sucrose,
Lactic Acid-Producing Bacteria 37trehalose, fructose, and not liquefied gelatin. L. crispatus is one of the dominant Lactobacillus species that persist in the vaginal microenvironment for a long time, and most of its characteristics are similar to the other three major vaginal Lactobacillus species, but it has a strong antibacterial effect. L. crispatus can produce large amounts of H2O2, which is beneficial to maintaining low vaginal pH value and limiting pathogen colonization. The L. crispatus genome contains the gassericin A operon, which is a gene related to bacteriocin production. ST1 strain of L. crispatus exhibited a strong protein-dependent adhesion effect on vaginal epithelial cells, thereby inhibiting the adhesion of pathogenic Escherichia coli. In addition, L. crispatus also has probiotics, for example, L. crispatus CTV-05 can be used in the treatment of urinary tract infections and Bacterial vaginosis (BV). L. crispatus DSM20584 can produce positively charged S-lamin to bind the negatively charged heavy metal contaminant arsenic. L. gasseri L. gasseri was discovered by Eckha R, Dlaue R et al in the early 1970s. From seven strains classified as L. acidophilus, using the DNA-DNA hybridization technique, L. gasseri was distinguished as new Lactobacillus species by the differences of electrophoretic mobility and cell wall structure of L-lactic dehydrogenase. L. gasseri is slender rod, most single or chain arrangement, amastigotes, usually fermentation glucose, fructose, sucrose, cellobiose and salicyside, do not ferment mannitol, pentose, rhamnose, inositol and sorbitol, do not produce indole, hydrogen sulfide, lipase, lecithase; urease negative, not liquefied milk or gelatin. L. gasseri has a specific adaptation to human mucosa, tolerate low pH environment, resist bile salts, produce bacteriocin, and can meet the selection criteria of many probiotics. Its probiotic effects include maintaining the balance of vaginal microenvironment, alleviating Helicobacter pylori infection and improving diarrhea. L. inersL. iners was discovered by Enevold falsen et al in 1999. L. iners strain is rod-shaped, often single scattered, pairs or in short chains arrangement; decomposing glucose for the production of L(+) -lactic acid, no gas production. Some strains can decompose maltose to get lactic acid; do not decompose L-arabinose, D-alglycosyl, cyclodextrin, lactose, mannitol, songpond, melbiose, amylopectin, raffinose, sorbitol, sucrose, D-xylose; α -glucosidase. Leucine arylamidase and phosphoramidase are positive. Alkaline phosphatase, arginine hydrolase, α-galactosidase, β-galactosidase, β-glucosidase, α-mannosidase, β-mannosidase, lipase C4, trypsin, and urease are negative.
38 Chapter 3L. iners is found in the latest of the four major kinds of Lactobacillus species, and later than most of the vaginal Lactobacillus species. In addition to the commonality of Lactobacillus species, it also has some special characteristics: L. iners can not grow on MRS and Rogosa media, and needs to be cultured on blood plates, which is the main reason why it has been ignored and not been detected. Recent studies have shown that it is not only the main vaginal Lactobacillus species, but also a high abundance. L. iners is associated with BV: it persisted in the vagina, including in the microenvironment of BV. Studies have shown that L. iners has different expression profiles in healthy women and BV women. More than 10% of the genomic components were expressed in BV and associated with metabolism in the BV environment. The genome of L. iners is able to express a mannitol-dependent cytolysin (inerolysin, INY), similar to the cytolysin of Streptococcus intermedius and Gardnerella vaginalis. After colonization of L. iners on the vaginal epithelial cells, it can activate signaling pathways associated with BV, leading to upregulated expression of transcription factors and pro-inflammatory cytokines. L. iners is associated with preterm birth. Studies have found that if L. iners is the dominant species of vaginal microbiota in early pregnancy, it is more likely to cause prematurity than other species of vaginal Lactobacillus, which may be related to the weak H2O2 production capacity of L. iners and its correlation with BV. L. jenseniiL. jensenii was discovered by Gasser et al in 1970 in human samples. There were seven Lactobacillus strains whose phenotypes were very similar to L. leichmanii, but with different electrophoretic mobility and DNA base GC content. L. jensenii fermented glucose to produce D-lactic acid. It can also ferment cellobiose, sucrose, maltoamygdalin, chestnut and salicin. Some strains can ferment galactose, melbiose, raffinose, can not ferment lactose, pine triose, mannitol, sorbitol, gum aldose and xylose. L. jensenii has a strong H2O2 production capacity, and the possible relevant factor for its adhesion to vaginal epithelial cells is carbohydrates, while L.jensenii may be a glycoprotein.3.3.3 The Role of Vaginal Lactobacillus Species Lactic acid-producing bacteria is the general term of bacteria that produce lactic acid by carbohydrate fermentation. At present, there are many species of vaginal Lactic acid-producing bacteria, such as Lactobacillus, Enterococcus, Streptococcus, etc., among which Lactobacillus is the main species. As early
Lactic Acid-Producing Bacteria 39as 1892, German scholars described the vaginal flora, and found Gram-positive bacilli in vaginal discharge. On this basis, the isolated Gram-positive bacilli were classified as L. acidophilus.Lactic acid-producingThere is glycogen in the vaginal epithelial cells, which is used by lactobacilli to produce lactic acid and maintain the vaginal pH value at 3.8 to 4.5. Previous studies have shown that the acidic environment is not conducive to the growth of viruses such as HIV, Rotavirus, Influenza virus, pathogenic microorganisms such as Trichomonas vaginalis and most bacteria. However, the microorganisms in the vagina, such as yeast and enterococci, can resist acid and resist the effects of hydrogen peroxide (H2O2), and the mechanism may be related to its cell wall structure and biofilm formation. H2O2-producing H2O2 is an oxidant with toxic effects on most anaerobic microorganisms. Vaginal Lactobacillus species can be divided into two types of production with or without H2O2. The majority of H2O2-producing Lactobacillus species are L. James, L. gasseri, and L. curvililius. Studies have found that H2O2 producers provide protection against vaginal infection. On the one hand, H2O2-producing lactobacilli and the host interact with each other. On the other hand, it is found that H2O2-producing lactobacilli can stimulate the vaginal epithelial cells to secrete antibacterial substances and increase the activity of synthesized defense protective factors, such as lysozyme, lactoferrin, and epithelial metabolites. Lactobacilli, especially H2O2-producing lactobacilli, has a certain defensive effect in preventing the development of BV and reducing the risk of preterm birth and chorioamnionitis in pregnant women. This Lactobacillus species was found to be severely deficient in women with BV, especially in pregnant women. Bacteriocins and Bacteriocoids-Producing Bacteriocins and bacteriocoids refer to substances with similar antimicrobial activity. Bacteriocins refer to the proteins secreted by bacteria that can inhibit and kill similar strains. Bacteriocoids refer to the polypeptides or proteins that are synthesized by ribosomes to inhibit and kill bacteria in the metabolic process. Bacteriocins, bacteroids can not only inhibit pathogenic bacteria such as Gardnerella, Pseudomonas aeruginosa, but also inhibit the growth of normal vaginal flora. Several studies have shown that the bacteriocins and bacteroids produced by lactobacilli have the ability to inhibit the growth of pathogenic bacteria, such as Gardenella vaginalis, Escherichia coli, etc.. But the activity of
40 Chapter 3some bacteriocins also inhibit the growth of normal lactobacilli, which may also be the reason why lactobacilli sometimes decreases spontaneously. Competitive Adhesion and Stimulating Local Immunity Several studies indicate that the lactobacilli can attach to epithelial cells and form biofilm, so as to play the role of resisting pathogens. At the same time, estrogen, vaginal pH value, glycogen content can affect the adhesion ability of lactobacilli. The occupancy effect against vaginal epithelial cells during Candida infection was observed by electron microscopic scanning. In addition, lactobacilli also has the ability to stimulate local immunity, which has a certain impact on the regulation of non-specific immunity or specific immunity. Nutritional CompetitionGlycogen secreted by vaginal epithelial cells is the main nutrient source of vaginal microorganisms, and the limited survival resources make a fierce struggle between bacteria. A large number of lactobacilli colonized in the vagina are in a dominant state in nutrient competition, leading to the failure of other pathogens to obtain sufficient nutrients, which is not conducive to their large proliferation. Vaginal lactobacilli regulates the symbiotic proportion of the normal microflora in the vagina in multiple levels, and maintains the balance of the vaginal microecosystem. Because of these characteristics of lactobacilli, people put forward the vaginal ecological therapy, by supplementing the vaginal physiological lactobacilli to inhibit the growth of a variety of pathogens, improve the internal environment of the vagina, restore the vaginal ecological balance, fundamentally avoid the use of antibiotic resistance, double infection, allergic reaction and the side effects of the body.3.3.4 Functional Substances by which Vaginal Lactobacilli Exert Immunity RolesAs the main component of vaginal flora, lactobacilli can inhibit the growth of miscellaneous bacteria by directly combining H2O2 and combining with other peroxides/halides, maintain vaginal homeostasis, and achieve the purpose of preventing infection. In addition, lactobacilli can also produce immune effects on the vaginal microenvironment. The specific material component involved in immunomodulation is still being explored. Some researchers believe that the cell wall component peptidoglycan (PGN) may be a functional substance for lactobacilli to exert immunity.PGN, a conserved structural component of the bacterial cell wall, is a
Lactic Acid-Producing Bacteria 41molecular structure unique to bacteria and is not present in eukaryotes. It is currently believed that peptidoglycan may have various functions, including anti-infection, immune regulation, anti-tumor, etc.. Studies indicated that PGN can activate the human immune system through the proinflammatory cytokines: interleukin-12 (IL-12), IL-8, tumor necrosis factor-α (TNF-α), etc.). PGN stimulates an inflammatory response and may be related to the Toll-like receptor (TLR) signaling pathway. Through the TLR 2 pathway, PGN induces inflammatory responses and promotes the expression of inflammatory cytokines by inhibiting MyD 88. In addition, transcriptional activation of certain cytokines and chemokine genes with TLR2 and PGN can cause activation of NF-κB, and other related signal transduction pathways may also be activated, such as studies found that PGN also stimulates PGlyRP3 and has an anti-inflammatory effect. Extraction with cell wall of lactobacilli and treatment with LPS separately could induce NOD2 expression in cells and activated nuclear translocation of the NF-κB pathway and nuclear translocation of PPARct. However, Xiyi Ma et al found that PGN of lactobacilli can activate peritoneal macrophages, NK cells to produce IFN-γ, TNF-α and NOD2, which may induce some of its antitumor effects.3.3.5 Vaginal Lactobacillus with Health Vagina is the only channel connecting the female reproductive organs with the outside world, and it is also an important part of the microecology of the entire human reproductive system and the distributions area of microorganisms. The bacteria exist almost all of the vaginal lumen, and generally Lactobacillus species is the dominant bacteria in the vaginal flora. Lactobacilli are important for the maintenance of vaginal pH value in a normal acidic state, so as to inhibit the growth and reproduction of pathogenic bacteria and maintain a healthy microecosystem in the vagina. Once the dominant position disappears, it will lead to the overgrowth of other bacteria, break the balance of the normal vaginal microecology, and lead to the female vaginal microecological disorder. At the same time, the vaginal microecosystem will be affected by various factors in vivo and in vitro, such as individual growth and development and fluctuations of hormone levels in vivo, personal hygiene habits and systemic and local application of antibiotics, living environment and living standards, etc.. As the normal dominant flora of the vagina, there is no doubt about the "probiotics" for maintaining a healthy microenvironment and resisting pathogen colonization, such as antagonizing the growth of BV related bacteria (such as
42 Chapter 3Gardnerella vaginalis). L.acidophilus, L.crispatus, L.gasseri, L.plantarum, L.rhamnosus can be used as probiotic preparations. Studies have shown that probiotics of Lactobacillus species have a certain role in the treatment of BV. However, vaginal lactobacilli also has a "pathogenic" side, with L.iners associated with BV and preterm birth. In addition, not all women take lactobacilli as the dominant vaginal flora, and being diagnosed as asymptomatic BV is not necessarily abnormal, but the individual does not take lactobacilli as the main flora. For example, the average vaginal pH value of the black ethnic is higher than that of the white ethnic, but the former also has a higher diversity. 3.3.6 Vaginal Lactobacillus Species and Vaginal Diseases Bacterial Vaginosis (BV) BV is one of the most common vaginal infectious diseases of women of childbearing age. The main cause of the disease is long-term use of large doses of immunosuppressants, antibiotics, hormones, radiation, incorrect vaginal douche or frequent sexual intercourse, etc., which make vaginal lactobacilli decreased, anaerobic bacteria proliferate, and the normal flora out of balance, resulting in a vaginal mixed infection. Volvovaginal Candidiasis (VVC) VVC previously called fungal vaginitis or candida vulvovaginitis, is a common vaginal inflammatory disease in women of childbearing age. Common pathogens include Candida albicans (C. albicans) species and non-albicans Candida species. When vaginal glycogen increases, acidity rises, or when the body resistance decreases, C. albicans will reproduce. When vaginal C. albicans reach a certain amount, the human body will become sick. During pregnancy, the glycogen content of vaginal epithelial cells increases, the vaginal acidity increases, coupled with the renal sugar threshold of pregnant women reduced, nutritional glucuria often happened, the sugar content in urine is elevated, and the growth and reproduction of C. albicans is promoted. Long-term application of broad-spectrum antibacterial drugs and adrenocorticoids can lead to the imbalance of vaginal flora, change the mutual restriction relationship between microorganisms in the vagina, and reduce the ability to resist infection, thus making the number of Candida species relatively increase. Medical reports in China and abroad report that VVC is mostly seen in local immunity decline (such as pregnant women, diabetic patients and people receiving corticosteroid hormone therapy), vaginal flora imbalance caused by long-term application of
Lactic Acid-Producing Bacteria 43broad-spectrum antibiotics, local humidity, etc.. Trichomonas Vaginitis (TV) TV is a vaginal infectious disease caused by trichhomonas vaginalis. Trichomonas vaginalis reproduces in pH 5.5 to 6.0, inhibiting growth below pH 5.0 or above pH 7.5. It is also easy to survive and spread after leaving the human body. Trichomonas vaginalis exists in the vagina for a long time, consuming or phagocyting glycogen in vaginal epithelial cells , hindering lactate production. Trichomonas vaginalis not only destroys the weak acidic environment of the vagina, but also may cause abnormal development of vaginal cells and induce pre-cancer lesions. In some patients because Trichomonas vaginalis can swallow sperm, it can cause infertility, affect the quality of life, and cause psychological burden.Senile Vaginitis (SV)SV also known as atrophic vaginitis, is a kind of vaginal allergy or inflammation caused by thinning and atrophy of vaginal tissue and reduced lubrication of the vaginal wall, which is usually caused by reduced estrogen and is common in postmenopausal women. Those who can cause a decrease of estrogen levels in the patient may also have the disease, such as artificial menopause (oophorectomy, chemotherapy, radiation, etc.) or some women during childbirth or lactation. The treatment principle of SV is to inhibit bacterial growth, supplement estrogen (systemic or local), and enhance vaginal resistance. But the long-term use of systemic estrogen therapy has increased the level of estrogen in the blood, which may increase the incidence of endometrial cancer and breast cancer, so many elderly patients have great concerns about this treatment. Aerobic Vaginitis ( AV) AV mainly caused by aerobic bacteria infection. In normal vagina of women of childbearing age, Lactobacillus species which produces hydrogen peroxide is dominant flora. When AV occurs, the lactobacilli producing hydrogen peroxide in vagina decreases or disappears, and other aerobic bacteria such as group B streptococcus, staphylococcus, Escherichia coli and enterococcus, etc. increase, and vaginal mucosal inflammatory changes occur. Factors contributing to changes in vaginal flora of AV remain unknown and may be related to estrogen deficiency, vaginal colonization of intestinal bacteria, and abnormal immunomodulatory mechanisms in the vagina. Streptococcus spp., Staphylococcus aureus, coagulase-negative Staphylococcus were identified as the most common Gram-positive pathogen in women with
44 Chapter 3AV with a prevalence of 58%, 41.7% and 37.4%, respectively, while E. coli is the most common Gram-negative pathogen with a prevalence of 23% in symptomatic women. Observation of vaginal discharge smear in women with AV, lactobacilli deficiency, coccus or crude bacilli, parabasal epithelial cells and leukocytes can be found. The presence of these aerobic bacteria in the vagina may be endogenous, or caused by fecal contamination. The treatment of AV is currently mainly for antibacterial drugs against aerobic bacteria, such as aminoglycoside suppositories. The available data indicate that the prevalence of AV cannot be ignored and the possibility that AV is prevalent is considerable. Due to the lack of understanding and examination methods, many women with AV are not treated or treated as BV or unexplained vaginitis by experience. It is important to emphasize that AV is an independent presence, and antimicrobial therapy against BV or other common vaginitis may not have any effect or little effect on AV, and therefore, the treatment of AV must be based on an antimicrobial susceptibility pattern. Cytosolytic Vaginosis (CV) CV, also known as lactobacilli overgrowth syndrome or Doderlein's cytolysis, is a vaginal disease caused by the overgrowth of lactobacilli in the rupture of vaginal squamous epithelial cells, hence cytolytic vaginosis. There are about 80 kinds of Lactobacillus species in the vagina of patients with CV. When the conditions that limit the growth of lactobacilli are reduced, such as long-term use of antifungal drugs, acidic liquid vaginal douche, etc., they will overgrow and reproduce, resulting in a large number of lactobacilli to decompose vaginal epithelial cells, leading to cell lysis. Women of childbearing age have vigorous sample of estrogen, and the vaginal mucosal epithelial cells are rich in glycogen. When the cells fall off or disintegrate, the interaction of enzymes and bacteria forms a low pH environment, which contributes to the growth of lactobacilli. Diabetic patients contain a large number of lactobacilli because of high blood sugar, which may also cause CV. This symptom is more obvious in the luteal phase. Studies have shown that the number of lactobacilli colonized has increased significantly during the luteal phase. Some patients with CV show symptoms of VVC, but these symptoms are not sensitive to antifungal drugs, and these patients may suffer from CV.
Lactic Acid-Producing Bacteria 453.3.7 Discussion of the Mechanism of Lactobacilli Treatment of Vaginal Diseases In women of childbearing age, Lactobacillus species is the dominant bacteria of vaginal flora, which can decompose glycogen to produce lactic acid, hydrogen peroxide, and a variety of anti-microbial factors. Acidic environment produced by lactobacilli is beneficial to reduce the negative charge on the cell surface and remove the sugar base covering the surface of the receptor. This removing exposes the receptor and contributes to bacteria adhesion to antimicrobial factors produced in Lactobacillus metabolism, also contributes to inhibit the growth of other bacteria, thus can adjust the symbiotic proportion of normal vaginal flora from multiple levels and maintain the balance of the vaginal microecosystem. Two major communities, L. curvillius and L. iners, are closely related to the normal flora. L. crispatus-dominated flora is associated with low pH value, but L. iners is required to maintain low pH value when L. curvillius is absent. Four communities are associated with BV. Metronidazole treatment reduced the microbial diversity and disrupted the microorganisms associated with BV, but almost rarely led to the establishment of a microbial community dominated by Lactobacillus species. Active Lactobacillus species helps to maintain the natural and healthy balance of vaginal flora, which is particularly important in pregnant women, because the abnormal of vaginal flora, such as BV and AV, are important factors in preterm birth and perinatal complications.L. iners may have specialized mechanisms adapted to the vaginal environment, such as the iron-sulfur cluster assembly system and some unique σ factors used to regulate gene transcription to adapt to fluctuations in the vaginal environment. In addition, an isotype of cholesterol-binding cytolytic enzyme that is potentially highly expressed in L. iners can also promote the adhesion of host cells or acts as a defense mechanism against other microbes. L. iners is widely present in the vagina of healthy women and those with BV or who have undergone antibacterial treatment, indicating that it is an important vaginal flora. The ability of pathogenic microorganisms to adhere to each other and to the vaginal mucosa is the key to their infectious process. Probiotics can successfully prevent repeated infection, mainly manifested in their strong adhesion to the pathogens, which can bind the pathogens to the coagulation group. If the adhesion of lactobacilli to the pathogen exceeds the adhesion between the pathogen, it is a destructive process. This process enables dense pathogen biofilm penetration also through the production of biosurfactants, but then lactobacilli accumulates into multiple layers to form the structure of the pathogen, so that
46 Chapter 3the antibacterial molecules of lactobacilli can destroy the biofilm and reduce the viability of the pathogen. Studies shows that Candida infection induces a proinflammatory response in vaginal epithelial cells, and estrogen and lactobacilli inhibit the expression of NF-κ B-related inflammatory genes. The probiotic lactobacilli can induce the expression of interleukin-1α (IL-1α) and interleukin-1β (IL-1β) through selective signal transduction pathways (e.g., MAPK/AP-1). A selective signaling mechanism for activating cytokine production in repair epithelial cells by lactobacilli may be one mechanism by which probiotics regulate VVC incidence. Scholars used gelatin vaginal capsule of L. curvililius strain (CTV-05) in healthy women and found a high colonization rate of this bacteria on the first day and many days later. Lactobacilli colonization in the vagina can interfere with the adhesion and invasion of pathogens to the vagina, and change the microecosystem to promote the function of vaginal biological barrier . 3.3.8 Lactobacillus Preparations for the Treatment of Vaginal Diseases According to the properties of vaginal discharge and laboratory testing methods, the selection of the corresponding antimicrobial drugs is the main treatment of all kinds of vaginal inflammation. However, a large number of relevant studies have shown that the use of antimicrobial drugs is easy to cause pathogenic microorganisms to develop drug resistance and recurrence after drug withdrawal, leading to difficulties in clinical treatment. Therefore, there is an urgent need for an effective and non-recurrent treatment. Based on the pathogenesis of vaginitis, vaginitis is accompanied by dysbiosis of microbial flora, so only from the root of the concept of vaginal microecology can we find an effective treatment. Therefore, starting from the vaginal microbial flora, starting from the concept of restoring the vaginal microecological environment, the treatment of female reproductive tract infection has become the main treatment method. Larsen, a scholar from Demoin University, proposed the treatment view of "Fight while adjusting", that is, antibiotics are used to reduce the load of vaginal pathogens, and also use Lactobacillus preparations for conditioning. The history of using live Lactobacillus preparations for the treatment of bacterial vaginosis dates back to the 1980s. With the deepening of vaginal microbial flora research, it is found that Lactobacillus species has different strains, and each strain may have different biological activities. In recent years, through the adoption of high-throughput sequencing technology of vaginal microbial flora of ribosome 16S r DNA gene sequence analysis, the researchers found that vaginal microbial flora
Lactic Acid-Producing Bacteria 47of healthy women has a certain ethnic difference. About 90 percent of Asian women and Caucasian white people are of a single species type, that is to say, the dominant microbial flora is lactobacilli. The study found that the vaginal microbiota of these women was more stable with dominant bacteria such as L. curvililus or L. jensenii, and less likely to develop microbial disorder and vaginal diseases. Women whose vaginal dominant bacteria were L. iners and L. gasseri or lactobacillus deficiency had a significantly increased risk of microbial disorder and vaginal diseases. Therefore, it may be an effective way to prevent and correct the vaginal microecological disorders to isolate and directly supplement the vagina and restore the vaginal microecological environment with lactobacilli as the dominant bacteria as soon as possible. Therefore, isolating Lactobacillus strains from healthy women and directly supplementing them to the vagina of patients with vaginal microecological disorders, and restoring the vaginal microecological environment with Lactobacillus as the dominant bacteria as soon as possible, may be an effective way to prevent and correct the vaginal microecological disorders.There are not many microecological agents used for the treatment of vaginal diseases, the most common are live bacterial preparations of Lactobacillus. The main ingredient of a Lactobacillus preparation that has been used in clinical practice in China is L. delbrueckii DM8909 in the vagina of healthy women. It is reported that its short-term efficacy is significant and can effectively improve the microecological environment in the vagina. Moreover, studies have shown that L. delbrueckii can tightly adhere to the vaginal epithelial cells and produce sufficient amount of lactic acid and H2O2, reduce vaginal pH value, maintain the vaginal acidic environment, change the proportion of vaginal aerobes and anaerobes, restore the balance of the vaginal environment, and fundamentally treat BV.In addition, lactobacilli vaginal capsules is a microecological preparation made from live Streptococcus intestinalis, which can decompose carbohydrates to produce lactic acid and increase the acidity of vagina, so as to be used for the treatment of vaginal diseases caused by microbial disorders. Conventional therapy with lactobacilli vaginal capsule can better treat BV, TV, and VVC. At present, L. crispatus preparation has entered the clinical trial stage, and there are lactobacilli and estrstriol vaginal preparation abroad, looking forward to a better preparation (See Chapter 13: Vaginal Microecological Treatments).
48 Chapter 3References[1] Haiyan Xiong, Yining He, Youjie Lin, et al. The differences of the main populations of vaginal Lactobacillus species related to health. Shanghai Preventive Medicine, 2017,29 (8): 643-647(Chinese).[2] Karimi S, Jonsson H, Torbj rm L, et al. Lactobacillus reuteri strains protect epithelial barrier integrity of IPEC-J2 monolayers from the detrimental effect of enterotoxigenic Escherichia coli. Physiol Rep, 2018, 6(2): e13514.[3] Tada H, Masaki C, Tsuka S, et al. The effects of Lactobacillus re-uteri probiotics combined with azithromycin on peri-implantitis: Arandomized placebo-controlled study. J Prosthodont Res, 2018, 62(1) :89-96.[4] Jung YJ, Choi YJ, An SJ, et al. Tannerella forsythia Gro EL induces inflammatory bone resorption and synergizes with IL-17. Mol Oral Microbiol, 2017, 32(4): 301-313.
C H A P T E R - 4Bacterial VaginitisXin-ai YueTranslator: Xin-ai Yue4. 4Bacterial vaginosis (BV) is the most common condition causing abnormal vaginal discharge in women of reproductive age. It is associated with a variety of gynecological and obstetric diseases, as well as neonatal illnesses, and can also affect women's mental health. The pathogenesis of BV remains unclear, but the early stages of the disease are characterized by a lack of lactobacilli and the overgrowth of anaerobic and facultative anaerobic bacteria. In recent years, increasing evidence has shown that biofilm formation occurs on both the surface of epithelial cells in BV patients and on the surface of Gardnerella vaginalis (G. vaginalis) in the vagina. The formation of biofilms is closely related to the pathogenesis of BV. About 50% of BV patients have no typical clinical symptoms, and more than 50% of patients r1elapse with BV after initial successful treatment. Therefore, active treatment and follow-up are particularly important.BV can occur in women of all ages and was once referred to as non-specific vaginitis. In 1955, American scientists Gardner and Dukes first introduced the concept of BV, calling it Haemophilus vaginalis, mainly because Haemophilus vaginalis was believed to be the primary pathogen responsible for BV at the time. Later, it was discovered that the microorganism called H. vaginalis, did not belong to the Haemophilus genus, and it was subsequently renamed Gardnerella vaginalis. Therefore, BV was initially referred to as Gardnerella vaginitis. It was not until 1984, when it was found that BV patients have vaginal dysbiosis, with a lack of lactobacilli and an overgrowth of anaerobic and facultative anaerobic bacteria, that the condition was formally named BV.In BV, the acid-producing lactobacilli are absent, and anaerobic bacteria, primarily Gardnerella vaginalis, proliferate. The main clinical symptoms are increased vaginal discharge and an unpleasant vaginal odor. Many gynecological and obstetric conditions are associated with BV, including infertility, miscarriage, preterm birth, premature rupture of membranes, chorioamnionitis, post-surgical
50 Chapter 4infections in gynecology and obstetrics, and neonatal infections. In addition, BV is linked to several sexually transmitted infections, such as HIV, gonorrhea, chlamydia, trichomoniasis, and herpes simplex virus infections.4.1 Epidemiology of Bacterial VaginosisCurrently, epidemiological data worldwide indicates that BV may be a sexually transmitted infection, with an incubation period of about 4 days, accounting for approximately 22% to 50% of vulvovaginal infections. The incidence of BV varies significantly between countries and regions, ranging from 20% to 60%. Sub-Saharan Africa has the highest global incidence, reaching 58.3%, while Australia, New Zealand, and Western European countries have a much lower rate of 4.7%. Other regions, such as South Asia, Southeast Asia, Latin America, and the United States, report intermediate incidence rates of 20% to 30%. The reasons for these variations between countries and regions are not yet clear; they may be related to cultural and environmental factors, as well as differences in diagnostic methods and clinical criteria. Additionally, about 50% of BV patients are asymptomatic, which may lead to discrepancies in reported incidence rates. Furthermore, BV significantly affects the mental health and quality of life of patients. A quality of life (QOL) survey found that the impact is mainly due to concerns about vaginal odor, feelings of low self-esteem, depression, social withdrawal, and a decline in overall quality of life. Given the high incidence of BV, its complications, and the psychological distress it causes women, it has become a global public health issue.4.2 Vaginal Microbiota and Common Pathogenic MicroorganismsThe vaginal environment of healthy women contains a variety of microorganisms that mutually constrain and depend on each other, forming an ecosystem that maintains the stability of the vaginal environment. The collection of various microorganisms in the vagina is referred to as the vaginal microbiota (VMB). The dynamic balance system formed between different species of microorganisms, the normal microbiota and the host, as well as the normal microbiota, the host, and the environment, is known as the vaginal microecology.4.2.1 Vaginal Microbiota ChangesIn general, throughout a woman's life, the vaginal microbiota changes in response to hormonal fluctuations. From birth, a newborn's vagina is devoid of microorganisms. Approximately 7 to 8 hours after birth, microorganisms begin
Bacterial Vaginitis 51to appear in the infant’s vagina, primarily Staphylococcus, Enterococcus, and Corynebacterium species. At this time, maternal estrogen levels in the infant's blood remain relatively high, which stimulates an increase in glycogen content in the vaginal epithelial cells, providing an environment conducive to the colonization of lactobacilli. Within 2 to 3 days, lactobacilli temporarily become the dominant species. However, as the infant’s adrenal glands and ovaries are underdeveloped, and with the decline of maternal estrogen, glycogen in the vaginal epithelium disappears. As a result, the amount of lactobacilli in the newborn female’s vaginal microbiota decreases, and eventually, coagulase-negative Staphylococcus, Streptococcus, Escherichia coli, and other intestinal bacteria become the dominant microbiota. The vaginal environment also gradually shifts from acidic to neutral or weakly alkaline. With the onset of puberty and the arrival of menarche, the glycogen content in the vaginal mucosal epithelial cells increases, and the number of lactobacilli increases rapidly. This leads to the formation of a relatively stable, normal vaginal microbiota.The vaginal microbiota consists of various anaerobic and aerobic bacteria, with a ratio of approximately 5:1 to 10:1. Each milliliter of vaginal sample contains about 109 colony-forming units (CFU). In most healthy women, the common bacteria in the vaginal microbiota are primarily species of Lactobacillus. In addition to Lactobacillus species, other bacteria such as Staphylococcus, Escherichia coli, Bacillus, Group B Streptococcus, Enterococcus faecalis, Mycoplasma, Gastric cocci, and Corynebacterium are also present.4.2.2 Common Pathogenic MicroorganismsWhen BV occurs, lactobacilli in the patient's vagina are reduced or disappear, and other anaerobic bacteria proliferate in large numbers. These anaerobes are closely associated with the onset and development of BV. In recent years, with the advancement of detection methods, an increasing number of anaerobic bacteria have been identified. Routine culturing of vaginal microbiota from BV patients has revealed a series of typical anaerobic bacteria, including Gardnerella vaginalis (G.vaginalis), Atopobium vaginae, Mobiluncus mulieris, Prevotella bivia, Fusobacterium nucleatum, Ureaplasma urealyticum, and Mycoplasma hominis. With the advancement of molecular diagnostic technologies, even more BV-associated anaerobes have been discovered, such as Eggerthella, Megasphaera, Leptotrichia, Dialister, Bifidobacterium, Arthrobacter, Caulobacter, and Butyrivibrio. Later, as part of the Human Vaginal Microbiome Project, new microorganisms were found within the Clostridiales order, and
52 Chapter 4these microorganisms were named BV-related bacteria (BVAB), namely BVAB1, BVAB2, and BVAB3. In summary, a wide variety of microorganisms are associated with the occurrence of BV. Studies have found that, in addition to the dominant G.vaginalis, other BV-associated microbial communities exhibit certain variations depending on ethnicity or region. In the Americas, anaerobic bacteria such as Cocci species, BVAB1, and BVAB3 are predominant in the vaginas of BV patients, while in Europe, Mycoplasma hominis and Mobiluncus species are more common in BV patients. 4.3 Etiology and PathogenesisCurrently, research on BV has surpassed 60 years, but its exact etiology remains unclear. However, it is well-established that vaginal lactobacilli play a crucial role in maintaining the vaginal microecological balance. This is because lactobacilli produce lactic acid, hydrogen peroxide (H₂O₂), and bacteriocins during their metabolic processes, which effectively prevent other pathogenic microorganisms from colonizing the vaginal walls. Lactic acid and hydrogen peroxide acidify the vaginal environment, maintaining the vaginal pH between 3.8 and 4.5. Additionally, lactobacilli enhance the barrier defense of vaginal epithelial cells and stimulate the host’s innate immune response. In BV patients, under certain factors, lactobacilli are often absent, while anaerobes proliferate excessively. The ratio of anaerobes to aerobes can increase from the normal 5:1 to 100:1 or even 1000:1. At this point, the vaginal pH typically rises above 4.5. Therefore, factors that disrupt the normal vaginal microbiota, affect the vaginal microecological environment, and raise the vaginal pH are all potential causes of BV. Common influencing factors include endocrine changes due to menstruation, pregnancy, and menopause, multiple sexual partners, overuse of antibiotics, vaginal douching, and unprotected sexual intercourse. These factors not only lead to BV but can also cause aerobic vaginitis (AV) and other sexually transmitted infections (STIs). In recent years, environmental factors and dietary habits, such as poverty, low vitamin D intake, and prolonged psychological stress, have also been considered contributing factors to the development of BV.G.vaginalis has long been considered the primary pathogenic microorganism responsible for the development of BV, as it is found in the vaginal samples of approximately 95%–100% of BV patients. In vitro studies have also shown that G.vaginalis causes more damage to the host compared to other BV-related bacteria. G.vaginalis adheres extensively to vaginal epithelial cells and releases a protein toxin called Vaginolysin, which is a cholesterol-dependent toxin. This
Bacterial Vaginitis 53toxin can lyse red blood cells and vaginal epithelial cells, disrupting the vaginal mucosal barrier and disturbing the vaginal environment. As a result, the normal vaginal microbiota, which is dominated by lactobacilli, is replaced by G.vaginalis and other anaerobic bacteria.In recent years, some researchers have found that G.vaginalis can also be detected in the vaginal samples of women who have never been sexually active or who have a normal vaginal microbiota. This indicates that the colonization of G.vaginalis in the vaginal mucosa does not necessarily lead to the development of BV. Therefore, the presence of G.vaginalis alone is not a sufficient condition for the occurrence and progression of BV.Similarly, the pathogenesis of BV remains unclear, and the main point of contention is whether BV is caused by a single primary pathogen or by the combined action of multiple pathogenic microorganisms. These microorganisms are typically transmitted through sexual activity. However, regardless of the controversy, it is undeniable that the change in the vaginal microbiota associated with BV is characterized by a reduction or disappearance of lactobacilli and a significant proliferation of other anaerobes or facultative anaerobes. This change is referred to as a shift in vaginal flora. The exact causes of this shift remain unclear, but it could be triggered by the colonization of a new pathogen, changes in lifestyle, or other factors. Additionally, the formation of biofilms (BF) has been widely recognized as playing a crucial role in the development and progression of BV. Currently, the following mechanisms of BV pathogenesis are widely accepted:Vaginal Microecological ImbalanceThe vaginal microbiota of healthy women consists of more than 200 species, including Lactobacillus, Bifidobacterium, Bacteroides, Enterococcus, Staphylococcus epidermidis, Streptococcus, Bacillus, Escherichia coli, Gardnerella, and others, as well as viruses, protozoa, mycoplasma, Candida albicans, and more. These microorganisms primarily inhabit the mucosal surfaces of the vaginal side walls and the anterior and posterior walls. They interact with and constrain each other, maintaining the balance of the vaginal microecology. This balance undergoes changes with age, menstrual cycles, pregnancy, and other factors, leading to a dynamic process of microbial evolution. When conditions arise that disrupt the vaginal microecological balance—such as changes in hormonal levels, alterations in vaginal epithelial glycogen content, or a weakened immune system—BV can develop.
54 Chapter 4Microbial InfectionAs early as 1955, Gardner and Duke proposed that BV was caused by G.vaginalis infection. By 2004, Ferris and others indicateed that Atopobium vaginae was also associated with BV. Although the exact pathogenesis of BV is still not fully understood, the microbial infection hypothesis remains one of the key areas of research. In 2005, Fredricks and colleagues used PCR to detect bacteria in the vagina and found a significant difference in the bacterial detection rates between BV patients and non-BV patients.Immunodeficiency A 2007 report indicated that mutations in the exon 54 codon of the mannose-binding lectin 2 (MBL2) gene are more common in patients with recurrent BV. Although further evidence is still lacking, an increasing number of studies indicate that individuals with compromised immune systems are more prone to developing BV compared to healthy women.Biofilm FormationBiofilm is a concept introduced in recent years, typically produced by bacteria themselves and adhering to their own surfaces. The main components of biofilms are polysaccharide matrices, fibrin, lipoproteins, and other substances, which help tightly connect bacteria, ultimately forming a membranous structure covering their surfaces. Biofilm is a form of life that evolves to adapt to the growth environment, in contrast to the planktonic form, the other mode of existence of microorganisms. In fact, biofilms can form on any biological or non-biological surface, including medical devices. Due to their antagonistic effect against antimicrobial agents and their resistance to the host's immune system, biofilms play a significant role in the development and progression of diseases. Increasing evidence confirms that biofilms form on the surface of vaginal epithelial cells and G. vaginalis in BV patients, which may be one of the main reasons for the chronic and recurrent nature of BV.4.4 Clinical Manifestations and Laboratory Examinations4.4.1 Clinical ManifestationsThe most common clinical manifestation of BV is an increase in vaginal discharge with a fishy odor. About 30% of patients present primarily with increased discharge, while approximately half of the patients show no clinical symptoms. A small number of patients may experience vaginal itching or pain, and in such cases, differentiation from vulvovaginal candidiasis should be
Bacterial Vaginitis 55considered. During physical examination, a homogeneous, thin vaginal discharge with an odor is typically observed, but there is no significant erythema or inflammatory reaction in the vulvar and vaginal mucosa.4.4.2 Laboratory ExaminationsVaginal Discharge Smear This method is the most commonly used for diagnosing gynecological vaginal microbial infections. After preparing a smear, it can be examined using either a wet mount or Gram stain. The presence of clue cells (with a proportion >20%) on microscopy is helpful for diagnosing BV (Figures 4-1, 4-2). Additionally, there is an inverse correlation between vaginal cleanliness and the detection rate of BV.Whiff Test When 10% KOH is added to vaginal discharge and a distinctive, foul fishy or ammonia-like odor is released, it is referred to as a positive amine test. The presence of an ammonia odor has high diagnostic value for BV, but the sensitivity of this test is low. A negative amine test does not rule out BV. This test is the most specific but the least sensitive indicator for diagnosing BV.Vaginal pH The normal vaginal pH is between 3.8 and 4.5. A pH greater than 4.5 is the most sensitive indicator for diagnosing BV, but it has lower specificity. A small sample is collected from the vaginal sidewall and then applied to pH test strips to measure the vaginal pH. Semen, cervical mucus, menstrual blood, and other substances in the vagina can raise the pH of the discharge.Vaginal Discharge-Related Biochemical Markers In general, BV patients test positive for hydrogen peroxide and sialidase, while leukocyte esterase is typically negative in simple BV cases.G. Vaginalis Culture of Vaginal Discharge This method has a sensitivity of 92%, but a specificity of only 69%. G. vaginalis can be isolated from the vaginal discharge of 40% to 50% of healthy women. Therefore, this method has limited diagnostic value for BV and is not recommended as a diagnostic approach for BV.DNA Probe for VPIII Type Microbial Identification The DNA probe for identifying VPIII-type microorganisms is currently one of the newer methods for diagnosing BV. However, due to its longer turnaround time and high cost, it is not easily adopted by clinicians.
56 Chapter 44.5 Diagnosis of Bacteria VaginosisApproximately half of patients with BV exhibit no clinical symptoms, making diagnosis challenging and often difficult to distinguish from other forms of vaginitis. Currently, there are two main diagnostic criteria for BV: the Amsel criteria, based on clinical symptoms, and the Nugent score and Hay/Ison criteria, which rely on laboratory examinations.Figure 4-1: Vaginal microbiota and microscopic appearance of vaginal discharge in healthy women and BV patientsNotes: Figure A: Normal vaginal mucosa with a small amount of discharge. Figure B: Mi-croscopic appearance of normal vaginal discharge using the wet mount method, with lacto-bacilli as the dominant bacteria and no inflammatory cells. Figure C: Vaginal mucosa of a BV patient with a larger amount of homogeneous discharge, containing bubbles. Figure D: Microscopic appearance of BV patient vaginal discharge using the wet mount method, with no Lactobacillus and the formation of typical clue cells. Figure source: Dan L. Longo, M.D. Bac-terial Vaginosis and Desquamative Inflammatory Vaginitis.. N Engl J Med 2018;379:2246-54.4.5.1 The Amsel CriteriaThe Amsel method remains the most commonly used approach for diagnosing
Bacterial Vaginitis 57BV, combining clinical presentation with laboratory examination. This method is relatively simple and convenient to perform and has high specificity. However, it is easily influenced by various factors, such as sample collection and transport and the operator's experience, which results in a lower sensitivity compared to other standards.a. Thin, homogeneous vaginal discharge b. pH > 4.5 c. Positive amine test d. Positive clue cells A diagnosis of BV can be made if three out of these four criteria are met, with criterion 4 being mandatory.4.5.2 Nugent ScoringThe Nugent scoring is a 10-point scoring system based entirely on the results of Gram-stained vaginal smear microscopy. It was first proposed by Robert Nugent in the 1990s and is considered the gold standard for laboratory diagnosis of BV according to the CDC guidelines in the United States. It has high sensitivity; however, its limitation lies in its inability to clearly define the clinical significance of intermediate microbiota.In this method, the sample is Gram-stained and observed under oil immersion to evaluate bacterial morphology. A semi-quantitative assessment is made based on the presence of Gram-positive large rods (primarily Lactobacillus species), Gram-positive small rods, Gram-negative bacilli (mainly G. vaginalis and Prevotella species), and Gram-negative curved rods (primarily Mobiluncus species). The scores for each bacterial morphology are then summed to calculate the total score. For specific scoring criteria, refer to Chapter 2, "Vaginal Microecological Evaluation System," Table 2-4 of this book. A total score greater than 7 points is used to diagnose BV.4.5.3 Hay/Ison CriteriaThis standard combines vaginal discharge examination, Gram staining, and pH measurement and is currently one of the more commonly used diagnostic criteria for BV in Europe. One of the main advantages of this method is that it specifically identifies non-BV-related bacteria observed under microscopy. Compared to the Nugent score, this standard provides a more comprehensive bacterial classification description, includes imbalances of other clinically
58 Chapter 4common microbiota such as aerobic bacteria, and simplifies the quantification and disease severity assessment process of the Nugent score, saving doctors time and effort. However, its diagnostic efficacy is comparable to that of the Nugent score. The specific grading criteria are as follows (see Figure 4-2):• Grade 0: Unrelated to BV; only epithelial cells present, no lactobacilli, indicateing recent use of antibiotics.• Grade I: Normal flora (predominantly lactobacilli).• Grade II: Intermediate flora (reduced lactobacilli with other bacterial species present).• Grade III: Abnormal flora (few or no lactobacilli, with a significant increase in other bacteria and clue cells).• Grade IV: Unrelated to BV; only Gram-positive cocci present, no lactobacilli.BV can be diagnosed under the following conditions: microscopy results show Grade III; or microscopy results show Grade II with a vaginal pH > 4.5.4.6 Therapy of Bacteria VaginosisMany patients with BV have subtle or mild symptoms, or they may only experience self-limiting intermittent foul-smelling discharge. Additionally, with the widespread availability of over-the-counter vaginal medications or washes, many patients begin treatment in the early stages without proper guidance, which is one of the reasons for recurrent BV.For patients with early mild BV, treatment focuses on simple prevention of worsening, which includes avoiding excessive vaginal douching or the use of aromatic vaginal washes. It is also recommended for patients to use condoms.For patients diagnosed with BV for the first time, simple preventive measures are ineffective, and pharmacological treatment is required. The treatment of BV often involves the use of antibiotics targeting anaerobic bacteria, such as metronidazole and clindamycin. In recent years, because BV is associated with vaginal dysbiosis, the use of probiotics has been indicateed as part of the treatment, such as lactobacilli preparations. Metronidazole is an ideal treatment because it inhibits the growth of anaerobic bacteria while having minimal impact on lactobacilli.Indications for treating uncomplicated BV include:a. Symptomatic patients b. Before gynecological and obstetric surgeriesCommonly used medications and dosages are as follows:
Bacterial Vaginitis 59Figure 4-2 Microscopic morphology of bacterial vaginosis (Gram stain, 1000×)N o t e s : F i g u re A : F l o r a d e n s i t y 4 + , f l o r a d i v e r s i t y 3 + , d o m i n a n t b a c t e -r ia : Gram-nega t i ve shor t rods , pa thogen: Tr ichomonas ( - ) , f unga l in f ec-tion: spores (-), germinated spores (-), hyphae (-), Nugent score: 9, AV score: 2. Figure B: Flora density 4+, flora diversity 4+, dominant bacteria: Gram-neg-a t i v e s h o r t ro d s , p a t h o g e n : Tr i c h o m o n a s ( - ) , f u n g a l i n f e c t i o n : s p o re s ( - ) , g e r m i n a t e d s p o re s ( - ) , h y p h a e ( - ) , N u g e n t s c o re : 9 , AV s c o re : 3 . Figure C (Mixed infections of BV and AV): Flora density 4+, flora diversity 4+, dom-inant bacteria: Gram-positive short rods, pathogen: Trichomonas (-), fungal in-fection: spores (-), germinated spores (-), hyphae (-), Nugent score: 8, AV score: 6. Figure D: Flora density 3+, flora diversity 2+, dominant bacteria: Gram-positive curved rods, pathogen: Trichomonas (-), fungal infection: spores (-), germinated spores (-), hyphae (-), Nugent score: 7, AV score: 2.a. Systemic medication: • Metronidazole 400 mg, orally, twice daily for 7 days; • Tinidazole 2 g, orally, once daily for 5 days; • Ornidazole 1 g, orally, once daily for 5 days;
60 Chapter 4• Clindamycin 300 mg, orally, twice daily for 5 days.b. Topical medication: • 0.75% Metronidazole gel 5 g, once daily for vaginal use, for 5 days; • Metronidazole vaginal suppositories (tablets) 200 mg, once daily for vaginal use, for 5–7 days; • 2% Clindamycin cream 5 g, once nightly for vaginal use, for 7 days; • Clindamycin vaginal suppositories 100 mg, for vaginal use at bedtime, for 3 days.c. Probiotics: Preparations that restore the normal vaginal microbiota can be used.d. Treatment precautions: During treatment with nitroimidazole drugs, alcohol should be avoided for 24 hours after taking metronidazole and for 72 hours after taking tinidazole to prevent a disulfiram-like reaction. Clindamycin vaginal suppositories (within 72 hours of use) or clindamycin cream (within 5 days of use) may weaken the protective effect of latex condoms due to their oily base. It is recommended that patients avoid sexual intercourse during treatment.4.7 Recurrent Bacterial VginosisMore than 50% of patients experience a recurrence after initial successful treatment. Recurrent bacterial vaginosis (RBV) does not have a precise definition, but it is generally considered to be the occurrence of three or more episodes of BV within 12 months, or the recurrence of BV within three months after treatment. Currently, it is believed that RBV is due to the reactivation of vaginal-associated microorganisms, rather than reinfection. Most treatments for BV have a cure rate of 80%-90%, but 15%-30% of cases relapse within three months after treatment.The causes of RBV are still debated. Commonly proposed factors include: reactivation of vaginal-associated microorganisms, rather than reinfection; ineffective treatment of BV; inability to effectively restore a lactobacilli-dominated vaginal microbiota; and infections from sexual partners. Epidemiological data from both domestic and international studies show that the occurrence of initial BV is often associated with new sexual partners, while RBV is more common in women with stable sexual partners and regular sexual activity.The treatment of RBV is challenging, and there is currently no optimal management plan. Generally, the treatment regimen used prior to recurrence may
Bacterial Vaginitis 61be continued, or an alternative recommended treatment may be tried. In parallel with treatment, it is important to actively identify and address high-risk factors for BV, exclude other infections that may be mixed with BV, and restore the vaginal microbial balance. Current treatment strategies that can be considered include: intensified treatment, maintenance therapy, combination therapy, or microbiota-based therapy.a. Intensified treatment• 400 mg Metronidazole, orally, twice daily for 14 consecutive days; • 0.75% Fucidin vaginal gel 5 g, once nightly for 10 consecutive days.b. Maintenance treatmentAfter intensified treatment: • 0.75% Metronidazole vaginal gel 5 g, twice weekly as intermittent therapy for 4 to 6 months; • Alternatively, 2.0 g Metronidazole, orally, once monthly.c. Combination treatment During the intensified treatment phase, both oral and vaginal Metronidazole should be used simultaneously, followed by continuation of the maintenance treatment plan.d. Microbiota-based treatmentCertain benefits in preventing BV recurrence can be received from this therapy.e. Precautions• Identify and address high-risk factors for BV; • Exclude any possible mixed infections and provide appropriate treatment; • Restore the vaginal microbial balance.4.8 Follow-Up and Management of Sexual PartnersIn general, routine follow-up is not necessary after symptoms disappear. However, because BV tends to recur, patients with persistent or recurrent symptoms should be advised to seek medical attention promptly if symptoms return. For patients with recurrence, different treatment plans can be chosen, but local treatment remains the primary approach. If possible, vaginal microbiota testing can be performed after treatment to assess the recovery of the vaginal flora and the treatment's effectiveness. Routine treatment of sexual partners is generally not recommended, but recent literature has indicated that microorganisms associated with BV can be isolated from the male genital
62 Chapter 4tract. Therefore, providing treatment to sexual partners may help reduce the occurrence of RBV.References[1] Chinese Society of Obstetrics and Gynecology, Infectious Diseases Collaboration Group. Guidelines for the Diagnosis and Treatment of Bacterial Vaginosis (2021 Revised Edition). Chinese Journal of Obstetrics and Gynecology, 2021, 56(1): 3-6.[2] Society of Obstetricians and Gynaecologists of Canada(SOGC). Guideline of Clinical Practice: Screening and Management of Bacterial Vaginosis in Pregnancy. J Obstet Gynaecol Can 2017,39(8):e 184-e191[3] Amsel R, Torten PA, Spiegel CA, et al. Nonpecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. Am J Med, 1983,74:14-22[4] Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol, 1991,29:297-301
C H A P T E R - 5Vulvovaginal CandidiasisXin-ai YueTranslator: Xin-ai Yue5. 5Vulvovaginal candidiasis (VVC), formerly known as yeast vaginitis or vulvovaginal candidiasis, is a common inflammatory condition of the vulva and vagina. The primary pathogen responsible for VVC is Candida albicans(C.albicans), a species of the Candida genus. In recent years, non-albicans Candida (NAC), particularly Candida glabrata (C. glabrata), has become increasingly recognized as a cause of VVC. C. albicans typically colonizes the vagina in its yeast form. When the balance between the vaginal environment and C. albicans colonization is disrupted, the yeast form can transform into the hyphal form, leading to the development of VVC.5.1 Epidemiology of Vulvovaginal Candidiasis The primary at-risk population for VVC is women of reproductive age, with a high incidence rate. VVC is considered the second most common cause of vulvovaginal inflammation after bacterial vaginosis BV. Approximately 70-75% of women will experience at least one episode of VVC in their lifetime, and over 50% of infected individuals will suffer from recurrent episodes, with 5-10% eventually developing recurrent vulvovaginal candidiasis (RVVC). The incidence of VVC varies greatly across different countries and regions. Reports indicate that African countries have the highest incidence of VVC, approximately 50%, while European countries have the lowest, around 15%. The incidence in women of reproductive age is significantly higher than in postmenopausal women.VVC is generally associated with factors such as the overuse of antibiotics, poor lifestyle habits, oral contraceptive use, pregnancy, and diabetes. The high global incidence of VVC has made it a significant public health issue. VVC severely affects the physical and mental health of affected women, causing pain, discomfort, anxiety, and low self-esteem, all of which negatively impact their work and emotional well-being. VVC is also associated with various gynecological conditions, such as pelvic inflammatory disease, infertility, ectopic
64 Chapter 5pregnancy, spontaneous abortion, and menstrual disorders. Therefore, active prevention, early diagnosis, and treatment of VVC are essential.5.2 Pathogens and Triggering FactorsCandida species are widely distributed in nature and can be found in humans, livestock, wild animals, and even in many hospital environments. Candida albicans, which exists in its yeast form, is a part of the normal human microbiota and commonly colonizes the mucosal surfaces of the reproductive tract, urinary tract, respiratory tract, and gastrointestinal tract. It is also frequently found on the surface of the mouth, nails, hair, and skin. Candida species are opportunistic pathogens, maintaining a balanced relationship with the host under the influence of vaginal lactobacilli and the host's immune system. However, when changes occur in the vaginal or vulvar environment that disrupt this balance, it can lead to the development of VVC.5.2.1 PathogensThe most common pathogen causing VVC is Candida albicans, accounting for approximately 80-90% of cases. A minority of VVC cases are caused by non-Candida albicans (NCAC) species, such as Candida glabrata (C. glabrata), Candida tropicalis (C.tropicalis), Candida parapsilosis (C.parapsilosis), and Candida krusei (C.krusei). In recent years, there has been an increasing trend in VVC cases caused by Candida glabrata. Additionally, about 1-10% of VVC cases are caused by two or more Candida species.An acidic environment is favorable for the growth of Candida. During infection, the vaginal pH typically ranges from 4.0 to 4.7, usually <4.5. C.albicans is a dimorphic fungus, existing in both a yeast form and a hyphal form. The yeast form, consisting of spores and blastospores, plays a role in asymptomatic colonization and transmission. The hyphal form, which involves the elongation of blastospores into pseudohyphae, enhances the fungus's ability to invade tissues. C.albicans is highly sensitive to heat and is killed after being exposed to 60°C for one hour. However, it is more resistant to dryness, sunlight, ultraviolet radiation, and chemical inhibitors.10-20% of non-pregnant women and 30% of pregnant women have Candida spores colonizing the vagina, but in very low quantities and in the yeast form, which does not cause any clinical symptoms. Only when the host's conditions change or external environmental factors stimulate the overgrowth of Candida in its yeast form, converting to the hyphal form, does it lead to vaginal mucosal
Vulvovaginal Candidiasis 65inflammation, resulting in epithelial cell damage and the production of inflammatory mediators, which causes a range of clinical symptoms.With the widespread use of antifungal medications in recent years, the incidence of VVC caused by C.albicans has decreased, while the incidence of VVC caused by C.glabrata has increased. This trend is attributed to the stronger antifungal resistance of C.glabrata compared to C.albicans. This resistance is also one of the reasons for the higher incidence of RVVC.5.2.2 Common Triggers and Pathophysiology There are many factors that can trigger VVC, and it is generally believed that both host factors and behavioral factors contribute to the onset and development of VVC. Host Factors include: Pregnancy and hormone replacement therapy, Use of corticosteroids or immunosuppressive medications, Diabetes, Extensive use of broad-spectrum antibiotics, Genetic factors ; Behavioral Factors include: Oral contraceptive use, Intrauterine device (IUD) placement, Wearing tight, non-breathable clothing, Unprotected sexual intercourse. Pregnancy and Hormone Replacement Therapy The incidence of VVC in pregnant women is significantly higher than in non-pregnant women, closely related to the hormonal changes during pregnancy. Additionally, the colonization rate of C.albicans in the vagina of pregnant women is also higher compared to non-pregnant women. VVC is often recurrent throughout pregnancy, particularly in the late stages when estrogen levels peak, leading to the highest occurrence of VVC. VVC is also common in the luteal phase of the menstrual cycle in non-pregnant women, as estrogen and progesterone levels are highest at this time. As a result, the incidence of VVC is very low in prepubertal girls and postmenopausal women, where estrogen levels are much lower.Hormone replacement therapy (HRT) is a treatment option used by some postmenopausal women to mitigate the effects of declining estrogen levels, such as osteoporosis, diabetes, cardiovascular disease, and neurodegenerative disorders. However, HRT can increase the risk of VVC in postmenopausal women because it alters their hormonal balance.The two main sex hormones involved in VVC pathogenesis are estrogen and progesterone. Estrogen, primarily estradiol (E2), is mainly produced by the ovaries and placenta and can increase up to 30 times during pregnancy. Estrogen plays a crucial role in the development of secondary sexual characteristics, uterine growth, and the protection of vaginal mucosa.
66 Chapter 5Progesterone, primarily produced by the corpus luteum, placenta, and adrenal cortex, increases during the luteal phase of the menstrual cycle and can rise more than 10 times during pregnancy.The increase in sex hormones during pregnancy can lead to higher glycogen content in vaginal epithelial cells, enhancing the adhesion and affinity of Candida to these cells. Adhesion is the first step in Candida invasion of the host and is a hallmark of its pathogenicity. Once Candida invades the host, it triggers complement activation and releases a series of chemokines and inflammatory mediators, which initiate specific immune responses and delayed hypersensitivity reactions. These reactions lead to the release of inflammatory mediators from monocytes, causing local tissue damage. Additionally, glycogen provides essential nutrients for Candida growth. Enzymes released by Candida can accelerate tissue damage, further facilitating its invasion of the host.Diabetes The incidence of VVC in diabetic patients ranges from 32% to 67.5%, which is significantly higher than the 11% to 23% incidence in non-diabetic patients. Additionally, the rate of Candida colonization in the vagina and the occurrence of RVVC are both higher in diabetic patients compared to non-diabetic individuals. Studies have shown that the most common pathogens in diabetic patients with VVC are NCAC, particularly C.glabrata. NCAC is less sensitive to conventional antifungal medications, which contributes to the higher incidence of RVVC in these patients.The pathophysiology of VVC in diabetic patients is related to impaired immune function and increased glycogen content in vaginal tissues, which enhances Candida adhesion to the vaginal epithelial cells. The elevated glycogen levels also provide nutrients that promote Candida growth and facilitate adhesion between Candida cells. Whether in premenopausal, postmenopausal, or pregnant diabetic women, the host defense mechanisms are often compromised, leading to a decrease in the random migration, chemotaxis, phagocytosis, and microbial killing capabilities of neutrophils. While the exact mechanisms are still unclear, these changes make diabetic patients more susceptible to Candida infections.Clinically, the use of corticosteroids is also a contributing factor to the development of VVC. In addition to the hyperglycemia associated with diabetes, corticosteroids themselves can suppress the immune response, making patients not only more susceptible to VVC but also prone to fungal infections at other sites.
Vulvovaginal Candidiasis 67Immunosuppressants The stability of the vaginal environment plays a crucial role in inhibiting the growth of Candida, but this balance can coexist with Candida colonization for long periods. When the immune function of the vaginal environment is suppressed, Candida can proliferate excessively, leading to the development of VVC. Conditions that impair the immune system, such as HIV infection, chemotherapy, corticosteroid treatment, organ transplantation, cancer, diabetes, tuberculosis, and other chronic diseases, can disrupt the vaginal environment and decrease immune function, making it easier for Candida to grow and cause infection. Corticosteroids are also a contributing factor to VVC. In addition to the elevated glucose levels in the patient's body, corticosteroids can suppress the immune response, which increases the susceptibility to Candida infections not only in the vagina but also in other parts of the body. Therefore, patients treated with corticosteroids are at higher risk for Candida infections in multiple sites.Antibiotics Long-term use of antibiotics can inhibit the growth of lactobacilli, which play a protective role against Candida by suppressing its growth. The inhibitory effects of lactobacilli primarily manifest in their competition with Candida for nutrients and binding sites on the vaginal epithelial cells. Lactobacilli have a stronger affinity for the vaginal epithelial cells compared to Candida. Additionally, lactobacilli secrete surfactants that can inhibit the adhesion of Candida. Substances such as stearic acid produced by lactobacilli can also prevent Candida from transitioning from the yeast form to the hyphal form.Genetic Susceptibility Genetic susceptibility to VVC has gradually gained attention. Some studies have found that African American women have fewer lactobacilli in their vaginal microbiota, which may contribute to their higher incidence of VVC. Additionally, it has been observed that a higher proportion of women with VVC have the Lewis non-secretor blood type.Oral Contraceptives Women who use oral contraceptives containing steroid hormones for extended periods are more prone to developing RVVC, similar to the effects of hormone replacement therapy.Intrauterine Device (Iud) The IUD is an economical and effective form of contraception, but it also
68 Chapter 5increases the risk of infections in women. This is possibly due to the easier formation of a biofilm on the surface of the IUD, which in turn promotes the adhesion and growth of Candida species.Other Factors Other contributing factors include vaginal douching, the use of feminine hygiene products, wearing tight and non-breathable clothing, obesity, chemical exposure, local allergies, and unprotected sexual behavior.VVC is generally an endogenous infection. Candida species, as opportunistic pathogens, can colonize not only the vagina but also the oral cavity and gastrointestinal tract. Under suitable conditions, these sites can become sources of infection, and the infections can spread between these areas. A small percentage of patients may acquire the infection through sexual contact or indirectly through contaminated clothing.5.3 Clinical Manifestations and Classification5.3.1 Clinical ManifestationsThe clinical manifestations of VVC vary greatly among individuals. Some patients may be completely asymptomatic, while others experience severe symptoms, sometimes in conjunction with other types of vaginitis, such as bacterial vaginosis, trichomoniasis, and gonorrhea. Typical symptoms of VVC include vulvar itching, burning pain, and in severe cases, significant discomfort that disrupts daily life. Some patients may also experience urinary frequency, dysuria, and dyspareunia. Other possible symptoms include erythema, swelling, and fissures on the vulva or vagina, with or without increased vaginal discharge, which is often characteristic in classic cases of VVC.Vulvar Itching Vulvar itching may or may not be accompanied by pain or a burning sensation. It typically begins in the labia minora and can spread to the entire vulva and vagina. The severity of itching often correlates with the intensity of erythema, though some patients show minimal visible changes.Abnormal Vaginal Discharge Patients with typical VVC often have notable changes in discharge, which may appear white, thick, and curd-like, resembling cottage cheese, or it can be thin and watery. Typical discharge may be white and clumpy due to the presence of epithelial cells, hyphae, yeast, and pseudohyphae. In some cases, the discharge
Vulvovaginal Candidiasis 69may be watery or purulent. However, discharge abnormalities are not necessarily a primary symptom. At the onset, discharge might be minimal but may increase over time. Some patients present with discharge that appears relatively normal in consistency, volume, color, and odor.Erythema Erythema is the most common sign of VVC, usually localized to the mucosal surface between the labia minora. With vulvitis, well-defined erythematous patches appear on the vulva, often surrounded by smaller satellite lesions. In some cases, scratch marks or fissures on the vulvar skin are visible. If vaginitis is present, the vaginal mucosa may exhibit edema and erythema.Vaginal Mucosal Inflammation Inflammation of the vaginal mucosa may present as varying degrees of edema and hyperemia, sometimes extending to the external cervical os. Membrane-like samples, often white but occasionally yellow, may adhere to the vaginal walls. These samples are often tightly attached, making them difficult to remove. When removed, they reveal an underlying erythematous mucosal surface. During acute episodes, the eroded and superficially ulcerated areas may be found beneath the white patches.5.3.2 Classification and ScoringThe classification of VVC plays an important role in selecting the appropriate treatment plan. In 1998, the CDC in the United States categorized VVC based on its epidemiology, clinical presentation, microbiological findings, host factors, and treatment outcomes into uncomplicated VVC and complicated VVC. About 80% of cases are classified as uncomplicated VVC, while approximately 10% to 20% are complicated VVC. See Table 5-1 for details. According to the severity of the patient's symptoms, the Infection Collaboration Group of the Obstetrics and Gynecology Branch of the Chinese Medical Association proposed a scoring system for VVC:• Score <7: Mild to moderate VVC• Score ≥7: Severe VVC, with significant symptoms and damage to the vulvar or vaginal mucosa.Based on Table 5-2 for details, RVVC is defined as having symptomatic VVC episodes four or more times within one year.
70 Chapter 5Table 5-1 Clinical classification of VVCUncomplicated VVC Complicated VVCFrequency of OccurrenceSporadic VVC RVVCClinical Manifestations Mild to moderate SevereFungal species C. albicans.Non-Candida albicans species.Host condition Healthy host.Special hosts, such as: pregnancy, uncontrolled diabetes, immunosuppression, etc.Treatment outcome Good PoorTable 5-2 VVC scoring criteria0 1 2 3Itching NoneOccasional, can be ignoredNoticeablePersistent, causing restlessnessPain None Mild Moderate SevereCongestion, EdemaNone Mild Moderate SevereScratches, Fissures, ErosionsNone - - PresentDischarge AmountNone in-creasedSlightly more than normalAbundant, no overflow Abundant, with overflowNote: The total VVC score is the sum of the scores for each clinical symptom.
Vulvovaginal Candidiasis 715.4 Laboratory ExaminationsLaboratory examinations include vaginal discharge smear microscopy and fungal culture.5.4.1 Smear MicroscopySmear microscopy of vaginal discharge remains the primary method for diagnosing VVC. Direct wet mount microscopy or Gram staining after fixation can be used. Currently, many primary hospitals in China use the wet mount method, and some hospitals, where conditions permit, send both dry and wet smears for examination. When using the wet mount method, 10% KOH should be used instead of saline, as it is easier to identify Candida species. The dry smear method, after Gram staining, allows for easy identification of all three forms (spores, yeast buddings, and pseudohyphae).In most asymptomatic cases, C.albicans is present in the yeast form, so a diagnosis can only be made when pseudohyphae are present. The positive rate for wet mount examination with 10% KOH is 70-80%, while after Gram staining, the positive rate can reach more than 80%. Therefore, Gram staining is more commonly recommended in clinical practice (see Figures 5-1, 5-2, 5-3).Figure 5-1 Pseudohyphae and yeast buddings of Candida (Wet Mount Method)Note: Figure source: Vulvovaginal candidosis, Jack D Sobel, Lancet 2007; 369: 1961–715.4.2 Fungal Culture Not all cases of VVC require fungal culture; it is generally only considered for refractory VVC, suspected drug resistance, or when a non-Candida albicans species is suspected as the cause of VVC.Currently, there are traditional culture methods using solid media, which are A (100X) B (400X)
72 Chapter 5Figure 5-2 Candida spores and Pseudohyphae (Gram Staining, 1000×)Notes: The white arrow points to Candida spores, and the red arrow points to Candida Pseudo-hyphae. Figure 5-2A: Flora density 2+, flora diversity 2+, dominant bacteria: Gram-positive bacilli, pathogen: Trichomonas infection (-), fungal infection: Spores (+); yeast buddings (+); Pseudohyphae (+), Nugent score: 3, AV score: 2. Figure 5-2B: Flora density 1+, flora diversity 1+, dominant bacteria: Gram-positive bacilli, pathogen: Trichomonas infection (-), fungal infec-tion: Spores (+); yeast buddings (+); Pseudohyphae (-), Nugent score: 1, AV score: 0.Figure 5-3 Candida pseudohyphae (Gram Staining, 1000×)Notes: The white arrow points to Candida spores, and the red arrow points to Candida pseudo-hyphae. Figure 5-3A: Flora density 2+, flora diversity 2+, dominant bacteria: Gram-positive bacilli, pathogen: Trichomonas infection (-), fungal infection: Spores (-); yeast buddings (-); Pseudohyphae (+), Nugent score: 3, AV score: 0. Figure 5-3B: Flora density 1+, flora diversity 1+, dominant bacteria: Gram-positive bacilli, pathogen: Trichomonas infection (-), fungal in-fection: Spores (+); yeast buddings (+); Pseudohyphae (+), Nugent score: 2, AV score: 0.
Vulvovaginal Candidiasis 73easy to perform and provide accurate identification, but they are time-consuming and cumbersome. Another method is chromogenic media, which is simpler and offers better sensitivity and specificity compared to traditional culture methods. These two methods are commonly used in primary care hospitals. Another widely used method is the yeast identification system, which can typically identify most Candida species within 48 hours. This system can identify 16 types of Candida species, but it takes longer to yield results.5.5 Diagnosis and Antidiastole5.5.1 DiagnosisThe typical case is not difficult to diagnose. Diagnosis can be made based on medical history, triggers, symptoms, signs, and laboratory examinations. If C.albicans is found in vaginal discharge, the diagnosis can be confirmed.5.5.2 AntidiastoleCytolytic Vaginosis (CV) CV is caused by the overgrowth of normally functioning lactobacilli, leading to a lowered vaginal pH, excessive acidity, itching, pain in the vulva, discomfort during intercourse, and vulvar discomfort during urination. It presents with thick or thin, white, cheese-like vaginal discharge. The clinical symptoms are similar to VVC. However, in CV patients, microscopic examination of vaginal discharge shows a large number of lactobacilli, broken epithelial cells (indicating cytolysis), bare nuclei, and a lack of Candida spores, budding spores, or pseudohyphae. Culture of vaginal discharge may reveal a normal or abundant growth of lactobacilli, with no Candida growth. In contrast, in VVC patients, Candida is typically detectable through both microscopic examination and culture of vaginal discharge.Bacterial Vaginosis (BV) BV is a clinical syndrome primarily caused by anaerobic bacteria. It can lead to a watery, thin vaginal discharge, often with a fishy odor. Vaginal congestion is not prominent, and pathological features do not show inflammatory changes. BV can coexist with VVC or trichomoniasis.Trichomoniasis Trichomoniasis is a vaginal mucosal infection caused by Trichomonas vaginalis. It shares many symptoms with VVC, sometimes differing only in severity. In
74 Chapter 5terms of itching, pain, and burning sensations, VVC is typically more severe than trichomoniasis, while urinary symptoms are more commonly associated with trichomoniasis. The main distinction between the two conditions lies in the characteristics of the vaginal discharge. VVC discharge is often described as cottage cheese-like or curd-like, while trichomoniasis discharge is typically thin, purulent, gray-green, and foamy.5.6 Treatment and Follow-UpThe key to treatment is actively eliminating the underlying causes. Antifungal medications should be selected and applied either locally or systemically based on the patient’s condition, following standardized protocols. Regular follow-up is essential.5.6.1 Eliminating the Underlying CausesIf a VVC patient has diabetes, active treatment is recommended, along with promptly discontinuing broad-spectrum antibiotics, estrogen, and corticosteroids. Underwear should be changed frequently, and used clothing, underwear, basins, and towels should be boiled to disinfect them. Vaginal Candida infections often involve cross-infection with other areas, such as the mouth and intestines. If necessary, samples should be taken from these areas for fungal microscopy or culture. Treatment should be initiated promptly if there are typical clinical symptoms or positive fungal cultures. For patients with persistent or frequently recurrent VVC, sexual transmission is also a potential cause of recurrence. Approximately 10% of male partners of symptomatic female patients have Candida-associated urethritis and should be treated to prevent cross-infection. After defecation, wiping should be done from front to back to avoid transferring fungi from the anus to the vagina.5.6.2 Drug TreatmentThe main treatment is antifungal therapy, which includes both local and systemic medications. Systemic medication is generally used for those who cannot tolerate local treatments, unmarried women, or those who prefer not to use local therapy. The treatment regimen varies depending on the classification of VVC.Uncomplicated VVC a. Vaginal medications • Miconazole soft gel capsules 1200mg, single dose; • Miconazole soft gel capsules 400mg, 1 dose per night for 3 days;
Vulvovaginal Candidiasis 75• Miconazole suppositories 200mg, 1 dose per night for 7 days; • Clotrimazole suppositories or tablets 500mg, single dose; • Clotrimazole suppositories 100mg, 1 dose per night for 7 days; • Nystatin 100,000 U, 1 dose per night for 14 days.b. Oral medications • Fluconazole 150mg, single dose.The above treatment options can be selected based on the patient's specific condition and availability of medications.Severe VVC For severe VVC, the treatment course should be extended based on the regimen for uncomplicated VVC. For example, Fluconazole 150mg, single dose, with a repeat dose 3 days later. In cases of more severe symptoms, low-concentration corticosteroid ointment or imidazole-based creams can be applied locally. Itraconazole can also be used, with a recommended course of 5-7 days. VVC in Pregnancy In early pregnancy, medications should be used cautiously. Select azole-based vaginal medications that are considered safe for the fetus, and oral antifungal medications are generally avoided. The primary treatment should be local therapy, following the regimen for uncomplicated VVC, but longer treatment courses tend to be more effective than short ones.RVVC When a VVC patient experiences fungal culture-confirmed symptoms after clinical symptoms and signs have disappeared and the fungal test was negative, this is termed recurrence. If the patient experiences 4 or more episodes within a year, it is classified as RVVC. VVC is more likely to recur before menstruation, so vaginal discharge examination should be done before the next menstrual period following treatment. The recurrence rate is generally around 5%, and while some cases are triggered by identifiable factors, the mechanisms for most recurrences are unclear. The treatment principles include intensive therapy and maintenance therapy, with medication chosen based on culture results and drug sensitivity tests. After achieving a negative fungal culture following intensive therapy, maintenance therapy should be given for six months.a. Intensive therapy: Either oral or local treatment can be selected, but therapy should only proceed if fungal tests show effectiveness. The specific regimens are as follows:
76 Chapter 5• Oral therapy: Fluconazole 150mg, single dose, on days 1st, 4th, and 7th. • Vaginal therapy: 1. Miconazole suppositories or soft gel capsules 400mg, one dose per night for 6 days. 2. Miconazole suppository 1200mg, on days 1st, 4th, and 7th. 3. Clotrimazole suppositories or tablets 500mg, on days 1st, 4th, and 7th. 4. Clotrimazole suppository 100mg, 1 dose per night for 7-14 days. b. Maintenance therapy: Currently, there are no well-established protocols for maintenance therapy, but since VVC typically recurs before menstruation, the treatment regimen can be selected based on the patient’s recurrence pattern. • For those with regular monthly recurrences, preventive treatment is recommended before each episode, for 6 months. This is known as monthly therapy. • For irregular recurrences, a weekly regimen can be used for 6 months to prevent further episodes, known as weekly therapy. Short-course medications are preferred for this, such as Clotrimazole vaginal tablets/suppositories 500mg, one dose per week.Sexual Partner TreatmentGenerally, sexual partners do not require routine treatment. However, sexual partners of patients with RVVC should be examined, and treatment should be provided if necessary. Approximately 15% of men who have contact with women with VVC may develop balanitis, characterized by redness of the glans penis, along with itching and irritation. Therefore, symptomatic male partners should undergo timely examination and treatment to prevent reinfection in women.Treatment Precautionsa. Use antifungal medications in a standardized manner. The initial treatment is a critical period for proper management. b. Routine vaginal douching is not recommended. c. During the acute phase of VVC, avoid sexual intercourse or use condoms if intercourse occurs. d. Emphasize individualized treatment. e. Be mindful of mixed infections and treat them promptly. f. For long-term oral antifungal therapy, monitor liver and kidney function and other potential toxicities
Vulvovaginal Candidiasis 775.6.3 Follow-UpPost-treatment follow-up is crucial. Follow-up should be conducted 7–14th days after treatment and after the next menstrual period. If two consecutive fungal cultures are negative, the treatment is considered effective. For RVVC patients, follow-up should occur 7–14th days, 1st month, 3th months, and 6th months after the end of treatment, with a follow-up after each subsequent menstrual period.References[1] Infectious Diseases Collaboration Group, Chinese Society of Obstetrics and Gynecology. Revised Guidelines for Diagnosis and Treatment of Vulvovaginal Candidiasis (VVC). Chinese Journal of Practical Gynecology and Obstetrics, 2012, 28(6): 401-402. [2] Dai Zhang, Zhaohui Liu, Qinping Liao, et al. Study on Etiology and Factors Associated with Disease Severity of Vulvovaginal Candidiasis. Journal of Practical Obstetrics and Gynecology, 2010, 26(12): 906-909.[3] A.M.Powell, P.Nyirjesy. Recurrent vulvovaginitis. Best Pract Res Clin Obstet Gynecol, 2014, 28(7): 967-976
C H A P T E R - 6Trichomoniasis VaginalisWenjie ZhouTranslator: Xin-ai Yue6. 6Trichomoniasis Vaginalis (TV) is a vaginal infection caused by the protozoan Trichomonas vaginalis. This organism is a flagellated protozoan that primarily parasitizes the mucosal surfaces of the female genital and urinary tract, and is also found in the male urethra. It is one of the most common non-viral sexually transmitted infections (STIs). Over 200 million new and recurrent cases of trichomoniasis are reported worldwide annually, although the incidence in China is relatively low. After infection with Trichomonas vaginalis, the body triggers a series of cellular and humoral immune responses. The infection causes the accumulation of a large number of neutrophils at the infection site, which leads to inflammation. Therefore, vaginitis, cervicitis, urethritis, and prostatitis in men are common in patients with Trichomonas vaginalis infection. Additionally, Trichomonas vaginalis infections in women significantly increase the risk of pelvic inflammatory disease (PID), and in pregnant women, the infection increases the risk of adverse perinatal outcomes such as preterm birth, premature rupture of membranes, and low birth weight. Neonates born via vaginal delivery are highly susceptible to Trichomonas vaginalis infection during passage through the birth canal, which can lead to neonatal respiratory and urinary tract infections. Trichomoniasis may also increase the risk of mental health disorders, particularly in patients with treatment-resistant infection.Trichomoniasis is both a sexually transmitted disease (STD) and a parasitic infection, and it is one of the most common causes of vaginitis. Persistent Trichomonas vaginalis infection is a major clinical challenge in treatment. The global incidence of Trichomoniasis vaginalis in women is about 8%, with approximately 90% of cases occurring in socioeconomically underdeveloped areas. The prevalence of the disease varies widely across different racial and regional populations, ranging from 0.3% to 20%, and in some areas, it can be as high as 52%. Due to the lack of an effective global monitoring system for this disease, the actual epidemiological incidence of Trichomonas vaginalis-
Trichomoniasis Vaginalis 79associated vaginitis is not well-known. The relatively slow progress of research on Trichomoniasis vaginalis in earlier years may be one reason for the lack of public awareness. However, with the development and application of bioinformatics and genomic technologies, recent genomic research has indicateed that the immune-inflammatory response in urinary and genital tract epithelial cells caused by Trichomonas vaginalis infection is associated with the occurrence, invasion, and metastasis of cancers, such as cervical and prostate cancer. Moreover, some studies have indicated that women infected with Trichomonas vaginalis may have a twofold increased risk of acquiring HIV compared to those without the infection. Therefore, Trichomonas vaginalis infection deserves greater attention, and there is a need for increased research efforts, as well as improved prevention and management strategies.6.1 Etiology of Trichomoniasis6.1.1 Introduction to Trichomoniasis VaginalisTrichomoniasis vaginalis was first discovered in 1836 by the French physician Alfred François Donné, and it was not until 1916 that the pathogen responsible for trichomoniasis was definitively identified as Trichomonas vaginalis. This protozoan is one of the oldest eukaryotic organisms, classified in the kingdom Protista, phylum Sarcomastigophora, class Mastigophorea, and order Trichomonadida. Trichomonas vaginalis is a single-celled, flagellated protozoan with various morphological forms. It primarily parasitizes the epithelial cells of the female urogenital tract but is also commonly found in the male urogenital tract. The parasite thrives in environments that are close to anaerobic conditions, although it is a microaerophilic organism, requiring low levels of oxygen.Origin of Trichomonas Vaginalis The origin of Trichomonas vaginalis has been a subject of debate and ontroversy. For a long time, people mistakenly believed that Trichomonas vaginalis and Trichomonas tenax, the protozoan inhabiting the human oral cavity, were closely related. However, molecular evolutionary and phylogenetic studies have shown that Trichomonas vaginalis and Trichomonas tenax are each more closely related to species of Trichomonas that parasitize birds, rather than to each other. Interestingly, their relationship with each other is relatively distant (see Figure 6-1). This indicates that these two human parasites have different origins and are
80 Chapter 6distinct zoonotic parasites.Therefore, comparative studies of the genomes and host-parasite interactions of the Trichomonas species found in humans and birds would be highly valuable in determining the molecular basis of their respective pathological biology.Figure 6-1 Molecular phylogenetic identification of avian parasites as sister groups of Trichomonas vaginalisNotes: The molecular phylogeny based on the Rpb1 protein (the largest subunit of RNA polymerase II) focuses on trichomonads, and the phylogenetic relationships are consist-ent with those derived from other genes. Species isolated from humans are highlighted with arrows, while all other species are isolated from birds (indicated with bird spe-cies and country names). Notably, a sequence from a bird isolate is highly similar to the Trichomonas vaginalis sequence, possibly representing a transfer between humans and birds. Figure source: Hirt and Sherrard. Trichomonas vaginalis origins, molecular pathobiology and clinical considerations. Sexually transmitted diseases 2015; 28(1): 72-79.
Trichomoniasis Vaginalis 81Genomics of Trichomonas Vaginalis Trichomonas vaginalis possesses a large genome, consisting of six chromosomes, which can encode up to 6000 proteins. Based on differences in their genomes, common strains of Trichomonas vaginalis include G3, T1, C1:NIH, C16, B7RC2, among others. For example, the G3 strain has a genome length of 176,441,227 base pairs. The genome of T. vaginalis contains a large number of histone genes, and introns have been identified in 65 highly conserved gene sequences. Within the nucleus of T. vaginalis, there are five small nuclear RNAs (snRNA-U1, U2, U4, U5, and U6) that, together with proteins, form the spliceosome.RNA polymerase II is responsible for the transcription of protein-coding genes and also synthesizes snRNA-U1, U2, U4, and U5, while snRNA-U6 is transcribed by RNA polymerase III. Unlike many eukaryotic cells, T. vaginalis lacks TATA-box-like promoters. In most cases, its promoter elements are located in the non-coding region of the 5’ end of protein-coding genes. In eukaryotic cells, such promoter elements are the only ones capable of accurately transcribing like the TATA box. These promoter elements are located within approximately 20 base pairs before the start codon. Studies have shown that they share a common motif with the sequence TCA+1Py(T/A) surrounding the transcription initiation site.The protein that binds to T. vaginalis promoter elements is a 39 kDa peptide (Inr-binding protein 39, or IBP39). This binding protein has two domains connected by a hydrolytic protein. The N-terminal region of the binding protein interacts with the promoter element, while the C-terminal region binds to a subunit of RNA polymerase II. The IBD domain is a DNA-binding domain unique to the T. vaginalis family, and it is considered a specific transcription factor for T. vaginalis.In addition, the genes encoding transport proteins and pathogenic proteins that enable T. vaginalis to engulf host proteins, bacteria, and even host cells during its lifecycle are crucial for the parasite’s endocytosis and phagocytosis processes.Within the gene family of T. vaginalis, there is noticeable amplification of genes encoding proteins, particularly those for unique eukaryotic protein kinases, atypical protein kinases, cytosolic-like tyrosine kinase families (excluding receptor serine/threonine kinases), heterodimeric guanine nucleotide-binding proteins, and proteins involved in the mitogen-activated protein kinase (MAPK) pathway and yeast-like signaling pathways. The eukaryotic protein kinase domains play an important role in the signaling pathways of T. vaginalis.
82 Chapter 6Compared to other higher eukaryotic cells, the regulation of protein kinases and cellular signaling in T. vaginalis is relatively simpler.Surface Proteomics Proteomic studies of Trichomonas vaginalis reveal that it has over 300 surface proteins across ten different families, with at least one family containing transmembrane domains that share one or more characteristics with surface proteins from other pathogens. Three primary surface protein families have been identified: BspA-like proteins, GP63-like proteins, and adhesins, with the BspA-like protein family being the largest.The BspA-like protein family has the largest gene set encoding surface proteins, with each gene containing a unique leucine-rich repeat sequence. The GP63-like protein family, the second largest, encodes 77 isoforms, 55 of which have transmembrane potential. GP63-like proteins are metalloproteases belonging to the metzincin class of enzymes, characterized by the HExxHxxGxxH motif (where "x" represents any amino acid), which binds zinc and forms a catalytic site. Other proteins in this family include 28 subtilisin-like serine proteases, 9 different serine proteases, and 5 calpain-like cysteine proteases. Calpain-like cysteine proteases have 22-23 identifiable transmembrane domains and play a role in transporting important surface protein fragments or producing small peptides during amino acid oxidation-reduction for energy generation.The third family of surface proteins has structural domains similar to those found in other mucosal pathogens. Members of this protein family appear at different stages of mucosal infection, aiding T. vaginalis in evading the host’s adaptive immune response.Morphology The life cycle of Trichomonas vaginalis consists only of the trophozoite stage, with no cyst stage. The trophozoite is colorless, transparent, and has a refractive quality, displaying a highly variable shape and strong motility. When fixed and stained, the organism appears pear-shaped, with individual trophozoites ranging from 10 to 20 micrometers in length and 2 to 14 micrometers in width.Using Giemsa stain, T. vaginalis is observed to have five flagellar bases arranged in a circular pattern at the anterior end. From these flagellar bases, five flagella extend; four of them are located at the front, freely moving and providing propulsion for the organism. The fifth flagellum extends towards the middle of
Trichomoniasis Vaginalis 83Figure 6-2 Morphology of Trichomonas vaginalis under Gram staining (1000×, arrow indicates T. vaginalis)Notes: Flora density: 2+, flora diversity: 3+, Dominant bacteria: Gram-positive cocci, Patho-gen: Trichomoniasis (+), Fungal infection: Spores (-); Germinating spores (-); Hyphae (-), Nugent score: 6, AV score: 6 (Mixed infections of TV and AV).Figure 6-3 Morphology of Trichomonas vaginalis under Gram staining (1000×, arrow indicates T. vaginalis)Notes: Flora density: 2+, flora diversity: 2+, Dominant bacteria: Gram-negative short rods, Pathogen: Trichomonas (+), Fungal infection: Spores (-); Germinating spores (-); Hyphae (-), Nugent score: 5, AV score: 6 (Mixed infections of TV and AV).
84 Chapter 6the body and connects to the undulating membrane along the lateral side of the body. The undulating membrane is thin, transparent, and runs along the length of the body, assisting in the organism’s movement with a wave-like motion that is non-directional.Under scanning electron microscopy, T. vaginalis exhibits surface folds, with some showing pseudopodia or distinct invaginations. The posterior flagellum extends in a wavelike manner along the left side of the undulating membrane towards the rear of the body but does not form the outer edge of the undulating membrane. At the posterior end of the organism, a rod-like structure, the axostyle, extends out of the body, forming a tail-like projection. The axostyle is composed of microtubules, is thin and transparent, and runs longitudinally from the anterior to the posterior end of the body. Its primary function is to assist in attachment to epithelial cells and to participate in cell division.The anterior third of the body contains an oval-shaped, vacuolated nucleus, which contains a large nucleolus. Surrounding the nucleus, there are fibrous structures called paracentriolar fibers. The cytoplasm of T. vaginalis is granular, containing numerous hydrogenase bodies that are distributed along the axostyle (Fig. 6-2).During the infection process, when T. vaginalis adheres to the epithelial cells of the host, its appearance changes. At this point, the morphology of T. vaginalis becomes varied, with its flagella retracting. The organism takes on an amoeboid form and adheres to the surface of the host's epithelial cells.Biological Characteristics The growth of T. vaginalis is influenced by several factors, including temperature, pH, and anaerobic conditions. The optimal temperature for its growth and reproduction in vitro is 35-37°C. At 22°C, T. vaginalis significantly reduces its metabolic activity and becomes enlarged, surviving 120–145 hours at 22-25°C. It can tolerate low temperatures, surviving for 7 hours at -10°C, but it is not resistant to high temperatures, surviving only 2 hours at 40±1°C and dying within 4 minutes at 50°C or higher. T. vaginalis is very sensitive to changes in environmental pH, thriving between pH 4.9 and 7.5. In culture media, it reproduces most readily between pH 5.0 and 7.5, with an optimal range of 5.5–6.0. A slightly acidic environment supports its growth, but a pH below 5.0 inhibits its growth and can kill the organism. In vitro experiments show that T. vaginalis pH requirements vary with the culture medium; for instance, in liver extract broth, the optimal pH is 5.6–5.8, while in Diamond’s TYM medium, it is 5.8–6.0.
Trichomoniasis Vaginalis 85The life cycle of T. vaginalis is relatively simple, involving only the trophozoite stage without cyst formation. The trophozoite stage is both the infective and pathogenic phase. T. vaginalis reproduces by longitudinal or multiple fission, with one nucleus capable of dividing up to five times, resulting in up to 16 parasites. T. vaginalis mainly resides in the female vagina, often in the posterior fornix, but it can also inhabit the urethra, bladder, paraurethral glands, uterus, and Bartholin’s glands. In males, it primarily parasitizes the prostate but can also be found in the epididymis, sub-preputial area, and urethra. Transmission occurs through both direct and indirect routes: direct transmission is primarily through sexual contact, while indirect transmission can occur through shared public bath facilities and similar means (Figure 6-4).Figure 6-4 Host and Trichomonas vaginalis Interaction and Its Interaction with Other MicroorganismsNotes: a. After the trophozoite of Trichomonas vaginalis (pear-shaped cells) contacts hu-man tissues [epithelial cells and extracellular matrix proteins], it quickly differentiates into a ameboid form (pancake-shaped cells), greatly increasing the surface contact. This is considered one of the key cellular processes critical to long-term infection. The binding of T. vaginalis lipopolysaccharide (LPS) and surface proteins mediates attachment to host tissues. Some T. vaginalis proteins are transferred to the surface of host cells through T. vaginalis exosomes, which are thought to affect the attachment of the parasite (these details
86 Chapter 6are discussed in the text). b. The complex interactions of bacteria, viruses, and eukaryot-ic organisms in the human-microbe interactions. This figure shows the global context of human-microbe interactions in a pregnant woman infected with T. vaginalis and her part-ner, in their respective urogenital tracts. T. vaginalis infection can affect the health of both the adult and embryo (e.g., preterm birth and HIV transmission in utero). Other common eukaryotic microorganisms encountered in the urogenital tract include Candida species. T. vaginalis, TVV, mycoplasma, and other microorganisms can regulate the inflammato-ry state of the urogenital tract, which in turn may alter an individual’s susceptibility to other infections, such as HIV—see text for more details. TVV refers to Trichomonas vag-inalis virus. Figure source: Hirt and Sherrard. Trichomonas vaginalis origins, molecular pathobiology and clinical considerations. Sexually transmitted diseases 2015; 28(1): 72-79.Energy Metabolism Trichomonas vaginalis is a highly dependent and host-predatory parasite, primarily ingesting bacteria, vaginal epithelial cells, and red blood cells through mechanisms such as phagocytosis and surface penetration to obtain nutrients. Several trophozoites can adhere simultaneously to the same epithelial cell, extending polymorphic pseudopodia or engulfing host cell microvilli or other components on the surface through surface invagination, damaging the epithelium. They also uptake the abundant mucopolysaccharides on the surface of epithelial cells via pinocytosis, storing them in the form of glycogen. Due to its inability to synthesize macromolecules, especially purines, pyrimidines, and lipids including cholesterol, Trichomonas vaginalis relies on carbohydrates as its main energy source. Whether in an aerobic or anaerobic vaginal environment, Trichomonas vaginalis can utilize carbohydrates via glycolysis in the cytoplasm. Through the direct pathway involving pyruvate kinase, or through bypass pathways involvwing oxaloacetate and malate pathways, phosphoenolpyruvate is converted to pyruvate.Trichomonas vaginalis is a microaerophilic parasitic protozoan, but it lacks mitochondria. This means it lacks the cytochromes, mitochondrial respiratory chain, and DNA associated with higher eukaryotic aerobic metabolism. However, it possesses a unique energy-generating organelle with a bilayer membrane structure, called the hydrogenosome. The hydrogenosome has a diameter of 0.5–1.0 micrometers and contains four conserved functional domains with different H-cluster complexes, along with mitochondrial-like FeS-cluster complexes and cytoplasmic auxiliary proteins. Trichomonas vaginalis uses the hydrogenosome in the cytoplasm to catalyze glycolysis and utilize carbohydrates. In the direct pathway, pyruvate kinase
Trichomoniasis Vaginalis 87plays a role, while in bypass pathways involving oxaloacetate and malate, phosphoenolpyruvate is converted to pyruvate. The hydrogenosome's primary function is to decarboxylate pyruvate to generate acetyl-CoA while producing ATP, coupled with electron transfer mediated by iron-sulfur proteins. The end products of hydrogenosome metabolism are H2O, acetate, H2, and CO2.Trichomonas vaginalis has strong transport capabilities, facilitated by the cation-chloride symporter family, which aids in the transmembrane transport of complex carbohydrates and amino acids. This allows it to respond to changes in mucosal permeability and maintain its survival. Additionally, Trichomonas vaginalis has an unusual system for synthesizing inositol phosphate ceramides. In the low-oxygen environment in which it resides, Trichomonas vaginalis uses its own redox system and antioxidant mechanisms to resist damage caused by oxygen fluctuations in the environment. It also transcribes and translates a variety of protective substances, including superoxide dismutase, thioredoxin reductase, peroxidase, and erythropoietin.6.1.2 PathogenesisThe virulence of Trichomonas vaginalis is associated with factors such as toxins secreted by the parasite, its adhesive properties, mechanical actions, and phagocytic activity. Adhesion to host cells is a key factor in the pathogen's ability to cause disease. It is a prerequisite for the parasite's survival, initiation, and maintenance of infection. The trophozoite of Trichomonas vaginalis attaches to the mucosal surface of the vagina, primarily through longitudinal binary fission, with some also undergoing transverse binary fission and a small number undergoing multiple fission to proliferate. In vitro experiments have confirmed that the latent period of Trichomonas vaginalis trophozoites is between 4 and 28 days. Once successfully invading the host, Trichomonas vaginalis can survive in the acidic vaginal environment, which is generally unfavorable for most organisms.Trichomonas vaginalis adheres to host vaginal epithelial cell surface proteins, extracellular matrix proteins, and other microorganisms on the mucosal surface through a different mechanism than other pathogens. This adhesion is mediated by surface adhesion proteins, including phospholipid glycosides, cytokines, and cytoskeletal protein α-actin. During the interaction between the trophozoite and the host, enzymes and molecules secreted by the parasite play an important role in mediating the adhesion of the parasite to the host epithelial cells and inducing cytotoxicity, while also triggering local immune responses in the host.
88 Chapter 6Adhesion During the menstrual cycle, changes in sex hormones lead to periodic shedding of vaginal epithelial cells, resulting in continuous changes in the vaginal environment. To adapt to these environmental changes, Trichomonas vaginalis employs various strategies for colonization in the vagina. After adhering to host cells, Trichomonas vaginalis transforms into a flattened, tightly attached, amoeba-like form on the target cell surface. This transformation increases the surface area of contact with the cell, and through the action of its own mucins, the trophozoite’s cytoplasmic extensions intertwine with the target cells. Trichomonas vaginalis releases cysteine proteases into the vaginal environment, causing the desquamation of vaginal and cervical epithelium. This cell-to-cell interaction enables Trichomonas vaginalis to effectively release cytotoxic substances onto host cells. Studies on the molecular mechanisms of adhesion between Trichomonas vaginalis and human cells indicate that phospholipid glycosides are the primary adhesion factors synthesized by the parasite. In addition to phospholipid glycosides, several proteins are expressed during the adhesion process, including adhesion proteins, fibronectin-binding proteins, laminin-binding proteins, α-actin, enolase, phosphoglucomutase, and conserved GTP-binding proteins. During adhesion, the expression levels of four major adhesion proteins (AP65, AP51, AP33, and AP23), GAPDH, and several hypothesized proteins are upregulated in a receptor-ligand manner. The extent of upregulation depends on factors such as time, temperature, and pH, which causes Trichomonas vaginalis to become more flattened and adhesive to host cells.Laminin and fibronectin are important extracellular matrix glycoproteins. Laminin is a dimer involved in the formation of the basement membrane, while fibronectin is a trimer that includes collagen, fibrinogen, and heparan sulfate proteoglycans, which are key adhesion glycoproteins. Both play significant roles in maintaining the integrity of the urogenital tract walls. Trichomonas vaginalis mediates adhesion via adhesion proteins and recognizes the host epithelial cell’s laminin. During adhesion, the expression of carbohydrates capable of recognizing laminin and proteins that degrade fibronectin-binding proteins is significantly upregulated.Iron, calcium, and phosphatases are essential nutrients for the growth and colonization of Trichomonas vaginalis. During the adhesion process, differential gene expression allows the parasite to acquire these nutrients from the parasitic environment, thereby enabling its colonization of the vaginal environment.During adhesion to host cells, the Trichomonas vaginalis ap65-1 gene
Trichomoniasis Vaginalis 89encodes a 65 kDa enzyme, enolase, which is closely related to adhesion. When polyclonal antibodies against ap65 are used to treat the parasite and incubated with epithelial cells, the adhesion ability of the parasite to the epithelial cells is significantly reduced. This may occur because the antibodies compete with the adhesion molecules on the epithelial cell surface, indicateing that ap65 is a key adhesion protein for Trichomonas vaginalis. The transcription of the ap65-1 gene is regulated by two similar but oppositely acting DNA regulatory elements, MRE-1/MRE-2r and MRE-2f. This gene regulation is related to iron concentration. The promoter element of the ap65-1 gene is a target site for multiple Myb transcription factors, including Myb2 and Myb3 (transcription activators) and Myb1 (transcription repressor), which together control the iron-induced transcription of ap65-1. When iron concentrations increase, Myb1 binds to the proximal site of the ap65-1 gene promoter. Conversely, when iron is scarce, Myb2 binds to the proximal site. Another DNA regulatory protein, Myb3, only interacts with the MRF-1 element. The activation of transcription by Myb2 and Myb3, and repression by Myb1, indicates that Trichomonas vaginalis regulates its adhesion protein expression in response to iron levels.Since Trichomonas vaginalis cannot synthesize lipids, red blood cells may be a primary source of fatty acids and iron for the parasite. Iron is also a crucial nutrient for the trophozoite's survival. 6.1.3 Cysteine Proteases and Cytotoxic Molecules The cysteine proteases of Trichomonas vaginalis form a large family, and studies by foreign researchers have demonstrated that these cysteine proteases are important virulence factors of the parasite. These cysteine proteases exhibit trypsin-like activity by degrading target cell proteins (such as laminin, fibronectin, and other components), which helps to separate host cells from tissues and mucosal surfaces. A small fraction of the cysteine proteases in Trichomonas vaginalis (with molecular masses of 25, 27, and 34 kDa) can specifically hydrolyze proteins containing arginine-arginine residues, while the remaining cysteine proteases have a broader substrate hydrolysis activity. The four different cysteine protease genes of Trichomonas vaginalis enable the parasite to cross the protective mucin barrier of the host epithelium under the action of these proteases. Therefore, cysteine proteases play a significant role in the destruction of target cell proteins and in the dissemination of infection within the host during the pathogenic process.In addition to cysteine proteases, Trichomonas vaginalis also produces several
90 Chapter 6other cytotoxic molecules and releases these molecules by damaging the target cell membrane. These cytotoxic molecules have pore-forming activities and can create pores in the red blood cell membrane. Trichomonas vaginalis also secretes lytic factors with varying phospholipase A2 activities, which damage nucleated cells and red blood cells, and specifically degrade phosphatidylcholine. This mechanism is part of its unique pathogenicity.6.1.4 Host Response and Innate Immune Mechanisms Trichomonas vaginalis escapes the host's immune system through several strategies, including mediating the destruction of complement proteins, molecular mimicry, and coating itself with host plasma proteins. The immune response generated by humans against Trichomonas vaginalis infection appears to be minimal. Trichomonas vaginalis has a unique and abundant surface lycosylphosphatidylinositol (GPI)-anchored protein, which is similar to prokaryotic ribosomal complexes. During the parasite's adhesion to the extracellular matrix, these GPI-anchored molecules are key factors in both cytotoxicity and the host immune response. They trigger the host’s white blood cells to secrete interleukin-8 (IL-8), parasite-specific immunoglobulin G (IgG), immunoglobulin A (IgA), Th1 cytokines, leukotrienes, reactive nitrogen intermediates, and macrophage inflammatory protein 3α (MIP-3α). Additionally, Trichomonas vaginalis induces the expression of nitric oxide synthase (iNOS), activates helper T cells, and promotes the migration of neutrophils across endothelial cells.Trichomonas vaginalis also produces immunosuppressive cytokines, including IL-10 and TGFβ, which lead to apoptosis of T cells, macrophages, and dendritic cells mediated by aspartate-specific cysteine proteases.Recent comprehensive analyses of the composition and structure of Trichomonas vaginalis have revealed that GPI-anchored molecules contain specific pro-inflammatory structural domains. These domains, with external branched sugars and ceramide-phosphoinositide glycans (CPI-GC), activate NFκB, ERK1/2, and MEK1/2 signaling pathways. Additionally, Trichomonas vaginalis induces the expression of cyclooxygenase-2 (COX-2) via the p38 mitogen-activated protein kinase (MAPK) pathway, and upregulates and activates toll-like receptors (TLR2, TLR4, and TLR9). The ceramide-phosphoinositide glycans contain terminal poly-N-acetylglucosamine, which means they have ligands for animal lectins, known as galactose-binding lectins. Cervical and vaginal epithelial cells release galectin-1 and galectin-3, which
Trichomoniasis Vaginalis 91have opposing roles in Trichomonas vaginalis infection and the associated inflammatory response. Galectin-1 inhibits leukocyte responses to inflammation, while galectin-3 enhances leukocyte responses to inflammatory signals. Furthermore, galectin-1 promotes viral adhesion and favors intracellular viral replication in HIV-1-infected cells, while galectin-3 and certain cytokines inhibit its expression. As a result, Trichomonas vaginalis infection initiates the activation of acquired immune responses.6.2 Epidemiology of TrichomoniasisTrichomoniasis may be the most common non-viral sexually transmitted infection worldwide. Although it is not a reportable disease, the World Health Organization estimated that in 2008, approximately 276.4 million people were infected, with nearly 90% of the cases occurring in low-income populations with limited healthcare resources. The infection rate of Trichomonas vaginalis is higher than that of Chlamydia trachomatis, Neisseria gonorrhoeae, and Treponema pallidum, and it is more easily transmitted in epidemics. It is estimated that the global prevalence of trichomoniasis in women is 8.1%, while in men, it is approximately 1.0%. However, these prevalence rates are likely underestimated, as the data mainly comes from literature reports, and most detection methods rely on microscopy, which is less sensitive compared to nucleic acid amplification techniques. Since trichomoniasis is not a reportable disease, there is a lack of an effective surveillance system, and the true epidemiology of the disease is still not fully understood.It is well known that due to differences in population and geography, the prevalence rates in different regions of the world can only be estimated based on literature reports and related epidemiological surveys. In the United States, studies using PCR detection on two large populations found that the prevalence of Trichomoniasis was 2.3% among adolescents and 3.1% among women aged 14–49 years. In Africa, there are significant regional variations in the prevalence of Trichomoniasis, with some areas showing high rates of infection. For example, in Zimbabwe, the prevalence based on antibody testing was approximately 9.5% in both men and women. In Tanzania, using more sensitive nucleic acid amplification techniques, the positivity rate for trichomoniasis among men was 11%. In Papua New Guinea, the prevalence of trichomoniasis in women seems to be very high, ranging from 21% in pregnant women to 42.6% in the general non-pregnant female population. According to the available literature and research, the prevalence of Trichomoniasis in other parts of the
92 Chapter 6world is relatively low among women of reproductive age when detected using more sensitive nucleic acid amplification techniques (e.g., 1% in rural areas of Vietnam, 0.37% in Flanders, Belgium, and 2.9% in Shandong Province, China). Typically, researchers use the positive rate of trichomoniasis vaginalis detected during prenatal screenings or family planning visits to estimate the prevalence in a given region. Studies have also found that the incidence of Trichomoniasis is regionally uneven, with prevalence ranging from 3.2% to 52% in underdeveloped areas, while in developed regions such as the United States, the prevalence is between 7.6% and 12.6%. Therefore, the prevalence of Trichomoniasis varies widely, depending on the region and the level of economic and medical development.Regarding racial susceptibility, existing studies indicate that African or African-descendant populations tend to have a higher prevalence of Trichomoniasis. For instance, sub-Saharan Africa, as well as African-descendant populations in Central America (e.g., Californians of African descent) and African Americans in the United States, show higher infection rates. In the U.S., the prevalence of Trichomoniasis among African American women is the highest, with infection rates ranging from 13% to 51%. The incidence among African American women is ten times higher than among white women, highlighting a significant racial health disparity. Other risk factors for Trichomoniasis include increasing age, incarceration, intravenous drug use, commercial sex work, and bacterial vaginosis.6.3 Impact of Trichomoniasis on Reproductive HealthIn women, Trichomoniasis attaches to and damages vaginal epithelial cells, leading to vaginitis. Among all women infected with Trichomonas vaginalis, the majority are asymptomatic. Women with trichomoniasis may experience a variety of complications, including adverse pregnancy outcomes, preterm or premature birth, low birth weight, premature rupture of membranes, an increased risk of tubal infertility, atypical pelvic inflammatory disease, an increased risk of HIV infection, and even cervical cancer.6.3.1 Pregnancy OutcomesTrichomoniasis is associated with adverse birth outcomes, such as low birth weight, preterm birth, pelvic inflammatory disease, and premature rupture of membranes. Studies have shown that maternal infection with Trichomonas vaginalis can lead to vaginal and respiratory infections in newborns. The
Trichomoniasis Vaginalis 93infection or the host’s inflammatory response to Trichomonas may reduce the strength of the chorioamniotic membrane, making pregnant women more susceptible to premature rupture of membranes and preterm birth. 6.3.2 HIV Infection RiskWomen with Trichomoniasis are at a higher risk of acquiring HIV compared to healthy women. Trichomonas vaginalis has become an auxiliary factor in the transmission of HIV. The mechanisms that increase susceptibility include: • The immune response to Trichomonas infection induces the recruitment of HIV target cells, increasing the number of target cells (i.e., CD4+ T cells) in the genital tract. • HIV protective factors, such as secretory leukocyte protease inhibitors, are disrupted. • Direct and indirect cytotoxic effects of the parasite itself.• The cytokines secreted by Trichomonas cause mucosal punctate hemorrhaging, damaging the structural integrity and defense barriers of the epithelial cells, thereby weakening their ability to resist HIV invasion.• Trichomoniasis can alter the normal vaginal microbiota, making it more susceptible to bacterial vaginosis, which increases the risk of HIV infection.Research also shows an association between Trichomoniasis and conditions like cervicitis, urethritis, bacterial vaginosis, vulvovaginal candidiasis, Herpes Simplex Virus Types I and II, Chlamydia, Gonorrhea, and Syphilis. Trichomoniasis appears to have a bidirectional association with Herpes Simplex Virus Type II (HSV-II), similar to its relationship with HIV-1. The co-infection of Trichomoniasis with HSV-2 is associated with worse outcomes, and women with Trichomoniasis have a higher incidence of HSV-2. Trichomoniasis is also linked to Human Papillomavirus (HPV) infection, indicateing an indirect connection between Trichomoniasis and cervical cancer.6.4 Clinical Features of TrichomoniasisHumans are the only natural host for Trichomonas vaginalis. There are two other types of trichomonads—Trichomonas tenax and Trichomonas hominis—that can infect humans but do not cause disease. The evidence for sexual transmission of Trichomonas is clear, with the highest infection rates observed among women who have multiple sexual partners, including both heterosexual and homosexual relationships. Additionally, Trichomonas vaginalis can also be transmitted vertically through vaginal delivery.
94 Chapter 6Most women (85%) and men (77%) infected with Trichomonas vaginalis are asymptomatic, with a latent period of 4 to 28 days after sexual contact. About one-third of asymptomatic women will develop symptoms within 6 months. In men, symptoms may include non-gonococcal urethritis (e.g., urethral discharge, irritative symptoms, or dysuria), such as urethral discharge and pain during urination.In women, the most common sites of infection are the vagina, urethra, and cervical endometrium. Symptoms of Trichomonas vaginalis infection in women include an increase in vaginal discharge with changes in its characteristics (typically becoming thin, purulent, frothy, foul-smelling, yellow-green), as well as discomfort during intercourse, dysuria, frequent urination, vulvar pruritus, vulvar irritative symptoms (vulvar vaginal erythema, edema), and lower abdominal pain. The most typical symptom is the alteration in the characteristics of vaginal discharge. The normal vaginal pH is 4.5, and when the vaginal pH exceeds 5, the likelihood of Trichomonas vaginitis infection significantly increases. During physical examination, erythema and edema of the vulva and vagina may be observed, along with frothy yellow-gray or green vaginal discharge, and an elevated pH (pH > 6). About 5% of women with trichomonas vaginitis may present with yellowish cervicitis or "strawberry cervix." If colposcopy were more widely used, this percentage could potentially rise to nearly 50%.Trichomonas vaginalis has been confirmed to be isolated from the vagina, cervix, urethra, paraurethral glands, Bartholin's glands, bladder, fallopian tubes, uterorectal pouch, prostate, and kidneys. Therefore, other complications of infection may include adnexitis, endometritis, paraurethral gland inflammation, and Bartholin's gland abscess. In men, it may cause epididymitis, prostatitis, and reduced sperm motility.6.5 Diagnosis and Treatment of Trichomoniasis6.5.1 DiagnosisTrichomoniasis vaginitis is diagnosed based on clinical features and laboratory tests. Although a variety of typical symptoms may appear following infection with Trichomonas vaginalis, these symptoms are highly similar to those of other sexually transmitted infections and cannot be used as specific clinical signs to diagnose trichomoniasis. Therefore, accurate, reliable, convenient, and inexpensive laboratory diagnostic tests play a key role in diagnosing trichomoniasis. These diagnostic tests include older techniques (Papanicolaou smear, Gram staining of vaginal discharge, and saline wet mount), updated technologies (culture and RNA methods), and the latest technologies (rapid
Trichomoniasis Vaginalis 95antigen testing and nucleic acid amplification). For women, cervical and vaginal swab samples are more reliable, while the most common sample for male diagnostic tests is urine. However, Trichomonas vaginalis is difficult to survive in acidic environments. In vitro, when the pH is less than 5.0, Trichomonas vaginalis rapidly dies and lyses. The pH of vaginal samples in women with trichomoniasis is typically greater than 4.5, and when 10% potassium hydroxide solution is added to the vaginal discharge of patients, amines are released, resulting in a distinct foul odor (commonly known as the amine test).Optical Microscopy Examination For symptomatic patients, immediate optical microscopy of vaginal discharge in the laboratory is a very simple, rapid, and cost-effective method. Under a warm saline wet mount examination, trichomonads appear oval or round with flagella, alongside a large number of polymorphonuclear leukocytes. Since trichomonads resemble leukocytes in size and shape, a diagnosis can be made when trichomonads are observed shaking or rotating under the microscope. Notably, for symptomatic patients, wet mount microscopy and culture methods both show excellent efficacy in detecting trichomonads.Despite these advantages, optical microscopy has a sensitivity of approximately 60% compared to PCR methods. Factors leading to false-negative results include low levels of trichomonad infection, sample collection site, sample storage time, duration of microscopy, and the skill level of the clinician. Optical microscopy is less likely to detect low-level infections, especially when the trichomonad load in the sample is below 10^4 cells/mL and if the entire slide is not examined. Delays from sample collection to testing significantly decrease the sensitivity of microscopy; when microscopy lasts over 10 minutes, sensitivity drops to 20%. Therefore, laboratories that cannot perform testing within one hour after sample collection should consider alternative methods. This is particularly important for laboratories that rely on centralized testing facilities and transport samples remotely, as delayed examination may lower the sensitivity due to decreased motility of the parasites. When trichomonads cannot move, their shape and size resemble lymphocytes, making them harder to distinguish.Staining methods include Gram, Wright’s, or Giemsa staining and fluorescent staining. Traditional staining methods often do not yield satisfactory results. These methods can be improved to facilitate the detection and identification of Trichomonas vaginalis. Vaginal discharge can be heat-fixed, followed by Gram staining (see Chapter 14, Section 1, "Pre-analytical procedures"). By adding cedar oil and observing the Gram-stained smear under a 10×100 high-power
96 Chapter 6lens, good results can be obtained. The Gram-staining method for discharge is not temperature-sensitive; even with reduced or lost trichomonad motility, their structures remain visible post-staining. This method is easy to perform in the laboratory, with clear and identifiable morphology, requires a short detection time, and is suitable for trichomoniasis screening. Additionally, the sample is long-lasting without fading.Culturing Method Culturing is considered the gold standard for diagnosing trichomoniasis. However, this method requires incubating vaginal discharge samples for 3 to 5 days and performing daily microscopic examinations to identify Trichomonas vaginalis. Due to time constraints, this method is not routinely used in clinical laboratories and is mainly applied in research settings. Compared to direct optical microscopy, culturing in liquid media provides a higher sensitivity due to the effective reproduction of Trichomonas vaginalis. Commonly used culture media include Diamond's modified medium and InPouch TV medium.The procedure for Diamond’s modified culture medium involves immediately inoculating the vaginal discharge sample (from a vaginal swab) into the medium within 1 hour of collection. The culture is incubated at 37°C, and daily microscopic observations are made for up to 5 days until motile trichomonads are detected. A positive result is typically seen within the first 3 days of inoculation. Several points should be noted during the culturing process:a. A slightly acidic pH of 5.2–5.4 in the medium is optimal for Trichomonas vaginalis reproduction.b. The ideal incubation temperature for Trichomonas vaginalis growth is 36°C–37°C. During subculture, handling, and transportation, temperature fluctuations must be minimized to avoid excessive parasite death.c. Penicillin and streptomycin must be added to the culture medium before use to prevent contamination from E. coli and other bacterial strains, which can lead to culture failure.d. Adding fetal calf serum to the culture medium promotes parasite growth. Fetal calf serum contains high levels of pantothenic acid, which, along with vitamins, supports the growth of Trichomonas vaginalis, ensuring optimal culture results.e. When taking samples from the culture, it is recommended to collect from the bottom of the medium, as most live trichomonads are concentrated there.InPouch TV Medium is a transparent, rectangular plastic aerobic culture pouch with a small passageway between the upper and lower chambers. After repeatedly rinsing the sample in the upper chamber, the medium is squeezed into the lower chamber, and the pouch is sealed and incubated at 37°C. The result can
Trichomoniasis Vaginalis 97be observed under a microscope without opening the pouch. InPouch TV can be stored at room temperature before incubation at 37°C, and the inoculated pouch can be kept at room temperature for up to 48 hours .Compared to traditional culture media, InPouch TV is a more ideal and convenient culture method developed in recent years, with several advantages: a. High Sensitivity: It can detect as few as 4 microorganisms per milliliter of medium. b. Antibacterial and Antifungal Properties: It has resistance to a range of bacteria and fungi. c. Long Shelf Life: It can be stored at room temperature for up to one year. d. Suitable for Transport: It is easy to transport, making it useful for remote testing. e. Ease of Use: The procedure is straightforward, and it is easy to observe under a microscope.However, the InPouch TV medium is still relatively expensive and requires experienced physicians to perform daily microscopic observations, which increases costs. Additionally, the culture period is about 7 days, which is relatively long compared to other diagnostic methods.Antigen Detection Compared to traditional wet mount microscopy and culturing methods, which require careful handling of samples (e.g., timely processing, transportation, and testing to maintain parasite viability), recent years have seen the emergence of diagnostic techniques that can quickly detect Trichomonas vaginalis antigens or nucleic acids without the need for culturing. These methods are less demanding in terms of sample storage, transport, and environmental temperature. Two of the most representative techniques are the OSOM Trichomonas Rapid Antigen Test and the Kalon Trichomonas Vaginalis Latex Agglutination Test.The OSOM Trichomonas Rapid Antigen Test is a common point-of-care test (POCT) that uses an immunochromatographic test strip with specific antibodies to detect Trichomonas protein antigens. When the vaginal trichomonas antigen binds with the specific antibody, a blue line appears on the test strip. This method requires no sample preparation or related instruments and provides results within 30 minutes. The Kalon Trichomonas Vaginalis Latex Agglutination Test is also a POCT method that involves the use of latex beads coated with specific antibodies, which bind to the Trichomonas protein antigen. After antigen-antibody binding, latex agglutination forms on the slide. This method also requires no sample preparation or specialized equipment and gives results within 10 minutes. These rapid tests are particularly suitable for areas with high population mobility or emergency departments, where quick results are crucial.The sensitivity of these POCT tests is comparable to or slightly higher than
98 Chapter 6wet mount microscopy and is close to that of culturing methods. According to the literature, the sensitivity of these tests ranges from 40% to 95%. These rapid diagnostic tests are highly specific for patients with clinical symptoms of trichomoniasis, with clinical specificity ranging from 92% to 100%. Vaginal swab samples meet the requirements for both the OSOM Trichomonas Rapid Antigen Test and the Kalon Trichomonas Vaginalis Latex Agglutination Test. Therefore, these tests are not only suitable for symptomatic women or those suspected of having trichomoniasis, but can also be used to screen and assess asymptomatic women or men for Trichomonas vaginalis infection.Molecular Diagnostic Techniques Similar to other sexually transmitted infections, nucleic acid amplification techniques (NAAT) have provided an important new tool for the diagnosis of trichomoniasis due to their high sensitivity and specificity. Nucleic acid amplification techniques include PCR, transcription-mediated amplification (TMA), and other biochemical methods that replicate and amplify specific DNA or RNA target sequences of Trichomonas vaginalis to millions of copies for analysis. This amplification process inherently gives nucleic acid amplification techniques higher analytical sensitivity compared to optical microscopy, culturing methods, and rapid antigen detection tests.The high specificity of nucleic acid amplification tests is based on the use of nucleotide primers and probe sequences that are specifically designed for the target organism, ensuring that results are not interfered with by the presence of other pathogens during the testing process.Due to the significant increase in sensitivity compared to non-amplified nucleic acid tests, as well as the lower requirements for sample handling (only nucleic acids need to be preserved, not the viability of the parasite), nucleic acid amplification tests offer significant advantages in diagnosing trichomoniasis in both men and women. The sensitivity of nucleic acid amplification tests for detecting Trichomonas vaginalis ranges from 76% to 100%, making it highly suitable for screening and detecting infection, particularly in cases where trichomoniasis is at a low level with a small number of parasites present. NAAT can be used for various types of urogenital samples, including vaginal, urethral, and cervical swabs collected by clinicians, as well as non-invasive urine samples, minimally invasive self-collected vaginal swabs, and cervical cell samples used for liquid-based cytology.Since nucleic acid amplification tests do not require the preservation of parasite viability, they have fewer stringent requirements regarding sample
Trichomoniasis Vaginalis 99collection, storage, handling, transportation, and environmental temperature. As a result, NAAT can be applied in both clinical and research settings, as well as in epidemiological studies of populations. samples used for NAAT testing for trichomoniasis are typically also suitable for the detection of other sexually transmitted infections, including Neisseria gonorrhoeae and Chlamydia trachomatis.However, while nucleic acid amplification tests offer high sensitivity and are useful for diagnosis, they have some limitations in monitoring treatment efficacy. Although NAAT may remain positive for several days after treatment, other tests may turn negative within two weeks following treatment for trichomoniasis, while nucleic acid tests may still show a positive result for several days after the infection has been cured. Summary In recent years, the diagnosis of trichomoniasis has become increasingly convenient and precise. The wet mount method using optical microscopy has been used for decades to diagnose trichomoniasis. This method is cost-effective, easy to operate, and requires relatively simple equipment, making it suitable for point-of-care testing. However, its sensitivity is relatively low, typically around 50%-70%, and even experienced laboratory personnel typically achieve no more than 80% sensitivity.Culturing methods have higher sensitivity compared to wet mount microscopy, but due to their inherent characteristics, they are expensive and require longer testing periods. Antigen detection methods, which were approved by the FDA and became available in 2011, offer higher sensitivity, but their application still requires further validation. Nucleic acid amplification technologies offer high sensitivity, rapid testing, and moderate to high costs, but they require specialized testing equipment, limiting their use in point-of-care settings.6.5.2 TreatmentSince the 1960s, oral nitroimidazole drugs, including metronidazole and tinidazole, have been the mainstay for the treatment of trichomoniasis, with metronidazole being the first-line treatment. Metronidazole belongs to the 5-nitroimidazole family, and studies have reported a success rate of approximately 95% in treating trichomoniasis and similar infections with related drugs such as tinidazole and secnidazole. The drug enters Trichomonas vaginalis through simple diffusion, where it preferentially receives electrons from the
100 Chapter 6hydrogenase system, inhibiting hydrogen molecule production. Iron-sulfur proteins transfer electrons directly to the drug, which is reduced and activated to generate cytotoxic nitro intermediates that bind briefly with DNA, causing DNA damage and cell death.The treatment guidelines for trichomoniasis provided by the World Health Organization (WHO), the Centers for Disease Control and Prevention (CDC) in the United States, and China recommend metronidazole or tinidazole as the first-line therapy. The recommended dose for both is 2 grams as a single oral dose, or metronidazole 400-500 mg taken orally twice a day for 7 days as an alternative regimen. During the 48 hours after taking metronidazole or 72 hours after taking tinidazole, alcohol consumption should be strictly avoided. Common side effects of nitroimidazole drugs include nausea, headache, dizziness, skin itching, discomfort, fatigue, thirst, frequent urination, watery vaginal discharge, vaginal bleeding, and vaginal itching. Given that the resistance rate of Trichomonas vaginalis to metronidazole ranges from 4.3% to 13.3%, with the resistance rate steadily increasing, tinidazole can be used for patients who fail metronidazole treatment. Tinidazole offers several advantages over metronidazole: a. Stronger Antitrichomonas Activity: The minimum lethal concentration (MLC) for metronidazole over 24 hours is 1.6-3.2 mg/L, and for 72 hours, it is 1.60-3.83 mg/L. In vitro data show that 60% of Trichomonas vaginalis isolates have a lower MLC for tinidazole compared to metronidazole. b. Longer Effective Duration: Tinidazole has a plasma half-life of 12–14 hours, compared to metronidazole. c. Better Tolerance: Patients generally tolerate tinidazole better than metronidazole.The cure rate for trichomoniasis with metronidazole treatment is between 84% and 98%, while tinidazole's cure rate ranges from 92% to 100%. Tinidazole offers higher cure rates, a longer half-life, higher tissue concentrations, and a lower minimum lethal concentration. If treatment fails and there is no history of sexual contact, a drug resistance test should be conducted. Because urinary tract infections are often concurrently present, systemic therapy is more effective than local treatments.It is recommended to treat sexual partners concurrently. The treatment options for sexual partners include either tinidazole or metronidazole two grams as a single dose. Unprotected sexual contact should be avoided until cure is confirmed.Treatment of Trichomoniasis in Pregnant and Lactating WomenThe use of metronidazole during pregnancy has been a subject of controversy. Since metronidazole easily crosses the placenta, some researchers have raised
Trichomoniasis Vaginalis 101concerns about its potential teratogenicity. There have been a few case reports of facial defects in infants born to mothers who took metronidazole during weeks 6 to 7 of pregnancy, although most retrospective cohort studies have not found any such correlation. Metronidazole is classified as a Category B drug by the FDA, and some meta-analyses have indicateed that it is safe to use during all stages of pregnancy. Tinidazole, however, has not been evaluated in pregnant women and is still classified as a Category C drug. The Centers for Disease Control and Prevention (CDC) in the United States recommends the use of 2 grams of metronidazole at any point during pregnancy, while the World Health Organization (WHO) and Chinese guidelines do not recommend treating trichomoniasis in the first trimester unless the benefits outweigh the potential risks and can be shown to prevent adverse pregnancy outcomes.Regarding treatment during breastfeeding, it is generally recommended to temporarily stop breastfeeding for 12 to 24 hours during and after the last dose of metronidazole to minimize the newborn’s exposure. For lactating women taking tinidazole, it is advised to temporarily halt breastfeeding for 3 days during and after the last dose.Treatment of Trichomoniasis in Women with HIV For women who are at risk of HIV infection and have trichomoniasis, studies have shown that repeated metronidazole treatment is more effective than a single dose. However, recent studies have found that for women with AIDS who are undergoing antiretroviral therapy, the effectiveness of metronidazole treatment is often suboptimal.Treatment of Recurrent/Persistent InfectionsRecurrent infections are quite common, with an incidence rate ranging from 5% to 31%. Potential causes of positive test results after treatment include reinfection from an untreated partner, new exposure to an infected partner, or treatment failure. Studies on the origins of recurrent infections have shown that treatment failure is the most common cause. Possible reasons for early recurrent trichomoniasis include drug resistance, non-adherence to treatment, clinical treatment failure, or reinfection from an untreated partner. Therefore, the sexual partners of patients with trichomoniasis should also receive treatment.If reinfection is suspected, a single dose of metronidazole two grams or tinidazole 2 grams can be administered. If the recommended treatment fails, and reinfection is ruled out, an alternative option is tinidazole 2 grams orally once daily for 7 days. If the above treatments fail and reinfection or poor adherence has been excluded, it may be advisable to perform metronidazole and tinidazole
102 Chapter 6susceptibility testing, if possible, and consider a high-dose or ultra-high-dose tinidazole regimen:• High-dose tinidazole regimen: one grams orally every 8 hours for 14 days (total dose 42 grams).• Ultra-high-dose tinidazole regimen: 2 grams orally every 12 hours for 14 days (total dose 56 grams).• For both high-dose and ultra-high-dose tinidazole regimens, additional vaginal administration of metronidazole or other supportive medications may be used.• High-dose or ultra-high-dose regimens should be handled by specialists. Since these doses exceed standard recommendations, patients should provide informed consent, and off-label usage should be documented, if possible.• The sexual partner should receive regular treatment, and treatment efficacy should be assessed. Clinicians often advise patients to undergo testing and treatment with their sexual partners. However, current testing methods for detecting and monitoring male infections lack high sensitivity, and further improvement in these techniques is needed.Follow-up and Evaluation of Treatment Effectiveness The effectiveness of treatment is assessed based on whether the trichomoniasis test is positive or negative at follow-up. It is recommended to retest 2-4 weeks post-treatment to evaluate efficacy. It is important to distinguish between reinfection and treatment failure. Using NAAT to detect trichomonas DNA post-treatment, the median time to a negative test result is 7 days (ranging from 0 to 84 days), with 85% of cases turning negative by day 21 post-treatment. There is no evidence to support repeated testing of sexual partners.References[1] Thomas Edwards. Trichomonas vaginalis: Clinical relevance, pathogenicity and diagnosis. Crit Rev Microbiol. 2016, 42(3): 406-17[2] Küng E, Fürnkranz U, Walochnik J. Chemotherapeutic options for the treatment of human trichomoniasis[J]. Int J Antimicrob Agents. 2019, 53(2): 116-127[3] Alessio C, Nyirjesy P. Management of resistant trichomoniasis[J]. Curr Infect Dis Rep. 2019, 21(9): 31-38[4] Bouchemal K, Bories C, Loiseau PM. Strategies for prevention and
Trichomoniasis Vaginalis 103treatment of trichomonas vaginalis infections[J]. Clin Microbiol Rev, 2017, 30(3): 811-825[5] Infectious Diseases Collaborative Group, Obstetrics and Gynecology Branch, Chinese Medical Association. Guidelines for the Diagnosis and Treatment of Trichomoniasis Vaginitis (Revised Edition 2021). Chinese Journal of Obstetrics and Gynecology, 2021, 56(1): 7-10.
C H A P T E R - 7Aerobic VaginitisWenjie ZhouTranslator:Tingting Zhu7. 7There are two types of bacteria that cause vaginal bacterial infections. One type is bacterial vaginosis (BV) caused by anaerobic bacteria and facultative anaerobic bacteria, and the other type is caused by aerobic bacteria, which is clinically called aerobic vaginitis (AV). AV was officially reported by Donders et al. in 2002, and the etiology and pathogenesis are still unclear at present. It is generally believed that it is caused by the reduction or absence of the number of lactobacilli in the vagina and the significant increase of aerobic bacteria, resulting in vaginal inflammation. The vaginal pathogens in AV patients are complex, including Gram-positive and Gram-negative aerobic bacteria. The clinical manifestations include increased yellow vaginal discharge, abnormal discharge odor, congestion and edema of the vaginal wall mucosa, accompanied by dyspareunia, vulvovaginal itching, etc., and it is often combined with other vaginal inflammations. AV not only causes vulvovaginal discomfort in patients but is also currently considered to be closely related to the following conditions: sexually transmitted diseases (such as human papillomavirus, human immunodeficiency virus, Trichomonas vaginalis and Chlamydia trachomatis), pelvic inflammatory disease, infertility, as well as adverse pregnancy outcomes such as miscarriage, preterm birth, premature rupture of membranes, chorioamnionitis, neonatal infection, and puerperal infection. These are all several diseases that were previously considered to be closely related to BV. More importantly, due to the significant differences in the pathogenesis and diagnostic methods of these two infections, the treatment methods are also different. Domestic clinicians have insufficient understanding of AV, and the diagnosis and treatment levels are uneven. Therefore, the differential diagnosis of AV and BV is crucial. The treatment of AV is classified and managed based on the clinical characteristics and microscopic examination results of patients, including antibacterial drug treatment, treatment for vaginal mucosal inflammatory reactions, and restoration of vaginal microecology. For aerobic
Aerobic Vaginitis 105infection, the corresponding antimicrobial agents were selected individually according to the background microflora of gram-negative bacteria or gram-positive bacteria.7.1 OverviewIn order to accurately describe the microecological imbalance of the vagina, Donders et al. first proposed the academic term AV in 2002. Its proposal complements the gap for another vaginal microecological imbalance in addition to the existing BV. But going back in history, in the process of understanding the disease, it was named "exudative vaginitis" in 1956 and "desquamative inflammatory vaginitis (DIV)" in 1965. AV is one of the common vaginal infectious diseases, the incidence of different countries and regions due to different groups of patients, races, and diagnostic methods vary. Foreign studies have reported that the incidence of AV is 4.9%~11.8%. In China, the proportion of AV in vaginal inflammation fluctuated from 9.4% to 23.7%.The diagnosis of AV is currently made using the Donders score system, which is based on phase contrast microscopy analysis of vaginal secretions in wet sheets, including the dominant level of Lactobacillus (designated as "lactobacillus grade"), the characteristics and number of inflammatory cells, the background flora, and the presence or absence of immature epithelial cells from the underlying vaginal mucosa. This can be combined to calculate the score, used to assess AV severity and then grading. The most serious AV includes DIV.By introducing the concept of AV and related diagnostic criteria, we can effectively explain several questions that have not been clearly answered before: What is the "intermediate microflora"? Why do women with BV show a range of inflammatory symptoms of varying degrees based on the Nugent score? What is so-called "inflammatory BV"? What can cause DIV (severe AV)?The Nugent score was first proposed by Robert Nugent in the 1990s and is now accepted by most researchers and clinicians. The Nugent score is the most accepted of the existing criteria for the diagnosis and evaluation of efficacy of bacterial vaginosis, but it does not cover the actual clinical situation perfectly. At present, the definitions of BV (Nugent score 7 and above) and normal flora (Nugent score 3 and below) are more clearly defined by Nugent scoring criteria, but the microecological characteristics of "intermediate BV" determined by this scoring criteria (Nugent score 4 to 6 points) are more clearly defined by Nugent scoring criteria. The concept of "between normal flora and BV flora" is vague. The microecological characteristics of
106 Chapter 7this intermediate type could not represent the excessive state of normal and abnormal microecology. This is because in clinical practice, what doctors observe through the microscope is that there is a subtle difference between intermediate BV and simple BV, that is, there is partial anaerobic bacterial overgrowth. An analysis of this subset of cases found that a subset of women lacking Lactobacillus also had inflammatory responses and varying degrees of atrophy of the vaginal epithelium and did not have a vaginal microecology characterized by typical BV cue cells. This has led to the emergence of a new definition for this particular type of vaginitis: aerobic vaginitis.In short, by introducing the concept of AV, we can explain the previously known abnormal vaginal flora and many mysteries that BV cannot explain.7.2 EtiologyIn a healthy vaginal microenvironment, balance and interaction between microbes are critical. This balance can shift into a state of disorder called abnormal vaginal flora (AVF) or dysbiosis, which can lead to diseases such as BV, AV, or VVC. The microflora of the vaginal microenvironment of BV is mainly composed of anaerobic bacteria, while the microflora of AV is composed of aerobic microorganisms commonly found in the gut. Common bacteria that cause aerobic vaginitis are: Escherichia coli(E. coli), Staphylococcus aureus(S. aureus), coagulase negative staphylococcus (e.g. Staphylococcus epidermidis), Group B streptococcus (Streptococcus agalactis, GBS) and Enterococcus faecalis. There are also some scholars' research results that indicate that Streptococcus virescens is often found in AV, and this point is still controversial. At present, it is not clear whether the presence of aerobic flora in the vaginal microenvironment is necessarily related to pathogenicity, as some scholars have proposed that AV is an immune disorder of the vaginal flora, an imbalance of the vaginal microenvironment, rather than a bacterial infection in a strict sense. The current consensus is that AV does have varying levels of inflammation (measured not only by the number of white blood cells, but also by the proportion of so-called "toxic white blood cells", i.e. the number of white blood cells with toxic particles to the total number of white blood cells), as well as varying levels of parbasal layer cells or immature epithelial cells.7.3 PathophysiologyAs with BV, the pathogenesis of AV is not fully understood. There are many theories that try to explain this complex problem.
Aerobic Vaginitis 1077.3.1 The Microflora, Mainly Lactobacillus, DecreasedAt present, it is known that there are up to 200 kinds of microorganisms in the female vagina, which are mainly bacteria, and at least 6 to 8 kinds of microorganisms can be isolated in each woman's vagina. Various microorganisms as well as microorganisms and the host between the mutual restriction, mutual coordination, to maintain the balance of the vaginal microecological environment. In the vaginal flora of healthy women, Lactobacillus is the dominant bacterium, and its isolation rate reaches 80% to 100%, accounting for more than 95% of the vaginal microorganisms, which is the core of maintaining the ecological balance of the microbiome.Because the vagina's healthy microbiome is mainly composed of Lactobacillus, which can inhibit the adhesion of various pathogens to the mucous membranes of the urogenital tract, including Group B streptococcus and S.aureus. In addition, colonization of Lactobacillus curvatus (L. curvatus) and the surface proteins of Lactobacillus lactis and Lactobacillus jensenii (L. jensenii) were able to strongly inhibit the growth of E. coli. Direct physical contact is an important way that Lactobacillus can effectively inhibit the secretion of toxic shock syndrome toxins by S.aureus and the secretion of inflammatory cytokines induced by staphylococcus in epithelial cells. It has been found that Lactobacillus can competitively bind to receptors on host epithelial cells, while different species and strains differ in their ability to adhere to host cells. In fact, in addition to the theory that Lactobacillus competitively binds to epithelial cells and thus inhibits the adhesion of other pathogens to vaginal epithelial cells, two theories have been proposed.This theory can at least answer how Lactobacillus in the vaginal wall of premenopausal women inhibits the growth of other microorganisms, the mechanism of action mainly includes: competition for nutrition; Production of antimicrobial substances, such as bacteriocin, hydrogen peroxide (H2O2) and lactic acid. The bacteriocin secreted by some strains of Lactobacillus was found to inhibit the growth of E. coli, while the bacteriocin produced by other strains of Lactobacillus prevented the growth of S. aureus. In in vitro studies, proteins secreted by L. curvatus and/or L. jensenii inhibit the growth of E. coli in cervicovaginal fluid. Vaginal pH value is one of the main indicators of vaginal microecological environment assessment, and it has a high sensitivity in the diagnosis of female lower reproductive tract infections.Vaginal interleukin (IL)-1b concentration was associated with Lactobacillus rating. Even in the presence of abnormal flora and BV, H2O2 secreted
108 Chapter 7by Lactobacillus reduces IL-1b levels, suggesting that H2O2 secreted by Lactobacillus can act as an immunomodulator even when many other bacteria are present in the vagina. In particular, H2O2 secreted by L. curvatus and L. jensenii can effectively inhibit the expression of IL-1b, while ordinary Lactobacillus does not have this ability. Under normal circumstances, the vaginal pH of healthy women is 3.8 to 4.5, and this acidic environment is mainly maintained by the lactic acid and H2O2 produced by Lactobacillus in the vagina. When the vaginal infection, microecological environment imbalance, vaginal pH also changes with the change of vaginal flora. Other studies have found that Lactobacillus also reduces IL-6 expression. In vitro studies demonstrated that probiotic Lactobacillus reuteri RC-14, alone or in combination with Lactobacillus rhamnosusGR-1, positively regulated the secretion of IL-8 and interferon-R-induced protein 10 (IP-10) secreted by VK2 epithelial cells. And once the protection of these protective lactobacilli is lost, there will be a high inflammatory state of AV.7.3.2 Local Immunomodulatory ImbalanceThe diagnosis of AV in women has been described in terms of local immune dysregulation. IL-1b concentration was much higher in AV than in BV and significantly higher than in normal controls. In the vaginal environment, a decrease in the number of Lactobacillus in the vagina is directly related to an increase in inflammatory cytokines. Elevated IL-8 levels and enhanced sialidase activity are often seen in AV. While IL-6 is an important trigger that triggers the inflammatory cascade, IL-8 is a chemokine that attracts and activates neutrophils to migrate to the site of inflammation. In addition to inducing cell migration, IL-8 can also release granzyme to induce phagocytosis of leukocytes. More chemotactic granulocytic pro-inflammatory factors were increased in AV, while the anti-inflammatory levels were not high, suggesting that AV is an infectious disease that is different from BV and mainly caused by vaginosis.Most of the cytokines are the products of the body's immune response and play a biphasic role: When AV occurs, the vaginal mucosa on the one hand secretes a large number of pro-inflammatory cytokines and chemotaxis neutrophils to clear pathogens, on the other hand, the excessive imbalance of inflammatory mediators will cause congestion and edema of the vaginal mucosa, causing vulvovaginal discomfort in patients. The concentration of proinflammatory cytokines in the vagina increased significantly in severe AV, indicating that the imbalance of vaginal cytokines is related to the severity of the disease. Similar
Aerobic Vaginitis 109symptoms can also be seen after severe AV (e.g. DIV), erosive lichen planus, and corticosteroid hormone therapy, so AV patients have significantly higher levels of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12p70, IL-17, and TNF-α in vaginal secretions than healthy women. It is suggested that the pathogenesis of AV patients is related to the imbalance of vaginal local immunity. The levels of pro-inflammatory cytokines were significantly increased in severe AV, suggesting that the up-regulated expression of multiple pro-inflammatory cytokines in the vaginal inflammatory microenvironment was associated with the increase in the severity of AV disease. The host immune status is an important factor in the pathogenesis of AV, and it is good to consider the change of immune regulatory balance as the starting point for the pathogenesis of AV or DIV, but it still needs to be further confirmed. Colonization of aerobic bacteria was an important discovery of AV. Group B streptococcus, S.aureus and coagulase negative staphylococcus were the most common gram-positive bacteria in the vaginal secretions of symptomatic AV women, and Enterococcus faecalis was also frequently detected. And E. coli is the most common gram-negative bacteria.The aerobic pathogenicity of AV is not only related to the composition of bacteria, but also the concentration of bacteria. In the study, the total amount of colonized aerobic bacteria detected in the vaginal secretions of women with AV-positive patients was more than ten times that of women with AV-negative patients, and the total amount of Lactobacillus in the vagina of normal women was more than 1000 times that of patients with AV. However, there are still some shortcomings in the existing theories, so it is impossible to fully and reasonably explain the immunomodulatory mechanism.7.3.3 SialidaseSome strains or strains produce sialidase enzymes that degrade host defense molecules (such as IgA). The main function of the defense molecules degraded by sialidase is to remove sialic acid that sticks to mucosal and epithelial cells. Sialidase can effectively remove sialic acid by avoiding secretory IgA proteolysis, which is the mechanism of reducing local immune response. Elevated sialidase activity usually indicates an increased risk factor for preterm birth. Although sialidase positivity is a marker of BV, sialidase is also produced in some bacteria associated with AV, such as Streptococcus agalactis.7.3.4 The Role of Local Estrogen LevelsSmears of normal vaginal discharge should contain only mature epithelial cells.
110 Chapter 7In AV cases, the absence of estrogen results in the emergence of intermediate epithelial cells in the exfoliated epithelium or even a lower layer of parbasal and basal cells.A wet smear examination of vaginal secretions in DIV women also reveals a large increase in the number of parbasal cells, and studies have shown that the vaginal mucosa is still deficient in estrogen even when high levels of estrogen are detected in the peripheral blood.Most of the current research on vaginal acidification has focused on the role of Lactobacillus, and host epithelial cells may also be involved in vaginal acidification. The idea that host epithelial cells may also participate in vaginal acidification is proposed based on the following two points: 1) Cervical vaginal epithelial cells secrete a large amount of H+ ions under the action of estrogen; 2) Host epithelial cells convert glycogen into lactic acid and fatty acids. As a result, host epithelial cells work with lactic acid-producing bacteria to acidize the vagina and create an environment conducive to lactic acid bacteria (Lactobacillus, Streptococcus) and fungi.Kallikrenase-related (KLK) peptidases are another important factor in the vaginal defense system. The KLK family consists of 15 genes located on chromosome 19q13.4. These genes code for the production of serine peptidase, which plays a key role in epithelial cell shedding. Vaginal epithelial cells regulate KLK expression, especially after exposure to estrogen and glucocorticoids.Many exogenous or endogenous factors can lead to the imbalance of the vaginal microecological environment, such as age, pregnancy, menstrual cycle, frequency of sexual life and the number of sexual partners, vaginal irrigation, long-term use of antibacterial drugs, etc. These influencing factors can easily lead to the occurrence of vaginal infectious diseases, the main clinical manifestations of various types of vaginitis. Therefore, the pathogenesis of AV still needs to be further studied.7.4 EpidemiologyAV accounts for 5% to 23.74% in patients with vulvovaginal symptoms, and the incidence rate in pregnant women is 8.3% to 10.8%, which is a common vaginal inflammation in clinical practice. At present, there are few epidemiological studies on the prevalence of AV, and due to the lack of understanding of the disease and the limitation of diagnostic methods, the data given in this part cannot fully reflect the prevalence of AV. A 2013 meta-analysis suggested that the prevalence of AV in non-pregnant women was between 5% and 10%, but this
Aerobic Vaginitis 111meta-analysis was based on data from only two papers and did not mention the grade of AV.7.4.1 Prevalence of AV in Non-Pregnant WomenA routine gynecological exam in Kampala, Uganda, graded according to microscopic observations, found that 11% of women had moderate or severe AV. According to a retrospective study, the prevalence of AV in the Bulgarian population was found to be 11.8%. The incidence of mild, moderate, and severe AV in asymptomatic non-pregnant women in Moscow, Russia was 13.4%, 7.2%, and 3.1%, respectively, with an average prevalence of 13.1%. The prevalence of moderate to severe AV in Portuguese women was 7.4%. The prevalence of AV among symptomatic patients in Chinese women was 23.7%, accompanied by co-infection with bacterial vaginosis, candida disease, or trichomoniasis vaginalis. Therefore, the prevalence of severe AV among non-pregnant women in Europe, Asia, and Africa is estimated to be between 7% and 13%, which is similar to the prevalence of BV.7.4.2 Prevalence of AV in Pregnant WomenThe incidence of AV during pregnancy appears to be much lower than in non-pregnant women. According to the literature, the overall prevalence of low-risk pregnant women in Belgium was 8.3%, while in Russia, the prevalence was only 4.3%. Although the prevalence of AV in pregnant women has so far been poorly reported in the literature, the incidence of AV during pregnancy appears to be low, estimated in the range of 4-8%.There are currently few risk factors for AV. A survey of Chinese women found that independent risk factors for AV include: being unmarried, long-term use of Iuds, use of antibiotics, and frequent vaginal douching. These risk factors were found to be almost consistent with the risk factors for BV. Condom use and a good educational background (higher education) are protective factors for AV.7.5 Effects of Aerobic Vaginitis on Reproductive Health7.5.1 Miscarriage and Premature BirthThe results of animal studies have confirmed that the increase of lipopolysaccharide (LPS) derived from E. coli and its associated anti-inflammatory cytokines leads to the failure of embryo implantation in the endometrium. Although there is no large data to confirm a possible association
112 Chapter 7between spontaneous abortion and the presence of symptomatic AV or AV flora, sporadic cases have been reported that some pathogenic serotypes of E. coli are associated with recurrent abortion. The local immune response induced in the host is unique in each case. Concentrations of the pro-inflammatory cytokines IL-1β, IL-6 and IL-8 were significantly correlated with LBG in pregnant women and were inversely proportional to the number of Lactobacillus. In general, moderately elevated levels of the pro-inflammatory cytokine IL-1β do not induce subsequent increases in IL-6 and IL-8. The concentration of IL-1β detected in the vagina in AV is much higher than in BV, but the concentration of IL-6 should be a cause for alarm. Because IL-6 and IL-8 are associated with adverse pregnancy outcomes, patients with AV have a higher risk index for preterm birth.7.5.2 Amniotic Rupture and ChorioamniitisThe occurrence of amniotic rupture and chorioamniitis may be related to the increased concentration of IL-1β, IL-6 and IL-8 induced by AV. The most common pathogens that typically cause symptomatic AV include: Group B streptococcus, E.coli and S. aureus. E. coli is one of the most common aerobic bacteria in vaginal secretions and the most common cause of AV. It is also easily detected in the placental tissue of patients with chorioamniotic infection and is the only pathogen that has been detected. In one study, E. coli and S.aureus were found in vaginal swabs obtained early in labor, predicting preterm birth. Other studies have linked vaginal colonization of ureaplasma Urealyticum and Mycoplasma hominis to preterm birth, intrauterine infections, and preterm birth. Therefore, the abnormality of aerobic bacteria in early pregnancy means a significant risk for premature birth and chorioamniotic infection of newborns.At present, there are some shortcomings in the diagnosis and treatment of vaginitis caused by bacteria. Classifying women's vaginal microbiota as "normal," "intermediate," or "bacterial vaginosis" can result in women with major infectious diseases not being detected, leading to inappropriate and incomplete treatment. Especially during pregnancy, the lack of proper diagnostic norms has led to many misconceptions and treatment failure studies, which are themselves aimed at reducing infection-related pregnancy complications such as preterm birth, chorioamniotic meningitis, and premature rupture of membranes in the third trimester. Through the understanding and diagnosis of AV, these problems can be explained more comprehensively. The reduction of lactic acid bacteria in the vagina, increased inflammatory cell response, the presence of opportunistic or pathogenic bacteria, sialidase production, and estrogen
Aerobic Vaginitis 113deficiency may explain the pathogenesis and pathology of AV. During pregnancy, women with AV in early pregnancy are at increased risk of preterm delivery and have a higher risk of premature rupture of membranes, chorioamnionitis, and necrotizing umbilicitis in the third trimester.Studies of AV treatment options suggest that antimicrobial therapy (antibacterial drugs), topical estrogen therapy, corticosteroids and probiotics, as well as some physical therapies, are effective in relieving symptoms and reducing recurrence, and once it is clear that AV is not BV or other vaginal inflammatory diseases, the choice of intravaginal and systemic medication is more targeted. It can significantly reduce the treatment time and improve the curative effect, especially for a series of pregnancy complications caused by vaginitis. The specific treatment plan must be adjusted according to the results of the microscope and the needs of the patient in order to achieve the best treatment effect. For AV, more precise diagnosis, pathogenesis, treatment and prevention are expected to be further studied.7.6 Clinical Manifestations10% to 20% of AV patients are asymptomatic. The main clinical symptoms of AV are changes in the characteristics of vaginal secretions, increased vaginal secretions, pain with intercourse, vulvovaginal pruritus and so on. The vaginal manifestations of wall mucosa hyperemia and edema, vaginal secretions typical characteristics of yellow thin, non-fishy odor odor, the increase of purulent secretions, a large increase in the number of white cells, amine test negative. Gynecological examination can see vulva red swelling, vulva vaginal congestion, severe can see scattered hemorrhagic points or ulcers. These features are different from BV, BV has no vaginal redness, swelling, inflammation and other manifestations, occasionally gray secretions, clue cells can be seen under the microscope, and the amine test of BV is positive. Symptoms may last for a long time and fluctuate in the severity of symptoms that will occur. Gynecological examination found that the vaginal mucosa and vestibule have different degrees of redness and swelling, and the vaginal mucosa in patients with severe AV can appear blood stasis and erosion. DIV is a severe form of AV characterized by severe vaginal discomfort and purulent discharge. Many patients have large amounts of vaginal purulent discharge, vestibulovaginal irritation, sexual discomfort, and vaginal inflammation or erythema for 12 months or longer. If the normal vaginal flora is replaced, then the process of converting glycogen into lactic acid in the vaginal epithelium is inhibited, which may eventually lead to
114 Chapter 7an increase in the pH of the vaginal environment. When pH > 4.5, trichomonas vaginitis (TV), AV, or co-infection with AV and BV should be considered at the same time.In the clinical gynecological specialty examination, the vaginal speculum examination may observe a yellowish discharge and a small area of ulcers on the surface of the vagina with a reddish tinge. The scope of individual patients is wider, most commonly in the anterior vaginal wall. Due to the thinning of the vaginal walls and the appearance of ulcers, AV may make women more susceptible to HIV or other sexually transmitted pathogens, as earlier data suggest that the missing of Lactobacillus is closely associated with gonorrhea, chlamydia, syphilis, and trichomoniasis infections.In addition to causing vulvovaginal discomfort in patients, AV can also cause gynecological complications such as upper reproductive infection leading to pelvic inflammatory disease, infertility, ectopic pregnancy, chorioamnitis, abortion, premature rupture of membranes, umbilical cord inflammation and neonatal sepsis.7.7 DiagnosisDonders' microscopic wet film examination of vaginal discharge is the diagnostic method of choice for AV. In 2004, Tempera et al. proposed the diagnostic criteria of combining clinical features and wet smear characteristics with clinical and microbiological diagnosis, i.e., clinical symptoms and/or signs and an AV score of ≥3. In 2021, Fengxia Xue, et al. proposed the combined diagnostic criteria of AV by combining clinical features with a Gram-stained smear, which solves the limitations of the microscopic wet smear method, and is more practical and easy to be promoted. It is more practical and easy to popularize.7.7.1 Saline Wet Slides Diagnostic Method At present, the more widely used at home and abroad is the saline wet film diagnostic standard of vaginal secretion proposed by Donders (see Table 7-1), evaluating the lactobacillary grade, the number of leukocytes, the proportion of leukocytes containing poisonous particles, the background colonies and the proportion of parabasal epithelial cells by phase contrast microscope, and scoring the five items respectively. The advantages of this diagnostic criterion are that it can reflect the characteristics of AV flora, inflammatory reaction and vaginal mucosal atrophy; the disadvantages are that it requires high requirements for testing personnel and equipment, saline wet film is not easy to be stored, it
Aerobic Vaginitis 115is not possible to repeat the reading of the film, and it is not combined with the patient's clinical symptoms and physical signs. When making the diagnosis of AV, in addition to an AV score of ≥3, the diagnosis should be made by combining the clinical symptoms and signs of the patient.Table 7-1 Donders’ scoring criteria for aerobic vaginitis microscopic wet film method (phase contrast microscope, 400×)AV scoresLBGLeukocyte countingPercentage of leukocytes containing toxic granulesBackground floraPercentage of PBCs0Grade I or Grade IIa≤10/HPFabsent or scatteredinconspicuous or lysogenousabsent or <1%1 Grade IIb>10/HPF and ≤10 around 1 epithelial cell≤50% leukocytesEnterobacte-riaceae-like bacilli1% ~ 10%2 Grade III>10 per epi-thelial cell>50% leuko-cytesCoccidioidomy-cetes or strepto-coccus>10%Notes: LBG: lactobacillary grade, Grade I: abundant polymorphic lactobacilli, no other bac-teria; Grade IIa: mixed flora, but dominated by lactobacilli; Grade IIb: mixed flora, but the proportion of lactobacilli is significantly reduced, less than other bacteria; Grade III: lactoba-cilli are severely reduced or absent, and other bacteria are overpopulated. HPF: high power field; PBC: parabasal epitheliocytes.Tempera et al. in 2004 proposed the combination of clinical features and wet film characteristics, the AV diagnostic criteria in this method are as follows: a. abnormal vaginal yellow discharge; b. elevated vaginal pH, most of the pH > 5.0; c. discharge has a bad smell (but negative KOH test); d. vaginal secretions high magnification microscopy of a large number of leukocytes (400 ×); c. the use of the Donders classification score to determine. The Donders’ scoring was used to determine. One of them, lactobacillary grade (LBG), was divided into I, IIa, IIb and III. The specific methodology was based on an independent scoring system
116 Chapter 7of five microscopic items, two of which were related to the characterization of the vaginal flora, one of which was the LBG (LBG-I-III), which was assessed microscopically and allowed the identification of the LBG (Ⅰ, Ⅱa, Ⅱb, or Ⅲ) by using the phase contrast at a magnification of 400× (see Table 7-1, notes). Depending on the relative dominance of Lactobacillus or other bacteria, they can be classified into mildly disturbed, relatively normal (LBG-IIa) and moderately disturbed, relatively abnormal (LBG-IIb) colonies. LBG-III colonies consist of many other bacteria with no Lactobacillus present (Figure 7-1). Figure 7-1 Phase contrast microscope image of lactobacillary grade (LBG) (400×)Notes: 7-1A: LBG I; 7-1B: LBG IIa; 7-1C: LBG IIb; 7-1D: LBG III. Image credit: Donders, Gilbert G G; Bellen, Gert; Grinceviciene, Svitrigaile; Aerobic vaginitis: no longer a stranger.[J].Res Microbiol.2017,168(9-10):845-858.LBG is the base of the 5 scores, reflecting the level of probiotics in the organism, and its score is inversely proportional to the level of probiotics. The second is the type of background flora, and an increase in the background flora score reflects the proliferation of vaginal aerobic bacteria. The sum of these two scores can be used as an indicator of the degree of microecological dysbiosis in the AV (see Table 7-1 for scoring methods). The other three of the five items are as follows: a. White blood cell count. b. The proportion of leukocytes containing toxic granules to the total number of leukocytes (toxic granular leukocytes are swollen leukocytes with cytoplasmic granules displaying lysosomal activity; see
Aerobic Vaginitis 117Figure 7-2). c. Proportion of parabasal cells to epithelial cells (see Figure 7-3). The scoring method of the above three items is still shown in Table 7-1. The higher the score, the more severe the inflammatory response and the degree of inflammation in the AV, and the sum of the above three scores can be used as an indicator of the degree of inflammation in the AV. The increase in these three scores corresponds to the yellow vaginal discharge and vaginal congestion seen in the AV clinic. These three scores essentially reflect the fact that the causative organisms of AV have triggered an immune response in the host, and therefore AV is truly inflammatory. This is where AV differs markedly from BV. The local immune response of the classical BV host is suppressed. There is a lack of white blood cells under the microscope and no clinical inflammatory response, so BV is called a disease rather than inflammation. The classic Nugent scoring system of BV is purely microecological. The five scores are added together to get a total score, which is then used to diagnose AV: 0~2 scores indicate no AV, 3~4 scores indicate mild AV, 5-6 scores indicates moderate AV, and 7 to 10 scores indicates severe AV (see Chapter 2, Vaginal Microecological Evaluation System: Figures 2-3 and 2-4). In some studies, moderate/severe AV (MSAV, AV score of 5 to 10) has been considered pathological. The proportion of AV diagnosed in patients with vaginitis disease is increasing through advancing understanding of this disorder.Figure 7-2 Phase contrast microscopy image of vaginal discharge from a patient with aerobic vaginitis (400×)Notes: 7-2A: Microbiota of Lactobacillus-deficiency morphotypes (LBG III) and spherical bacteria. 7-2B: Streptococcal strands, a typical feature of AV caused by Streptococcus or Staphylococcus. 7-2C: Toxic granular leukocytes filled with lysosomal granules. Image credit: Donders, Gilbert G G; Bellen, Gert; Grinceviciene, Svitrigaile; Aerobic vaginitis: no longer a stranger.[J].Res Microbiol.2017,168(9-10). 845-858.
118 Chapter 7Figure 7-3 Phase contrast microscopy images of epithelial cells (400×)Notes: 7-3A: superficial epithelial cells; 7-3B: middle epithelial cells; 7-3C: parabasal cells. Image credit: Donders, Gilbert G G; Bellen, Gert; Grinceviciene, Svitrigaile; Aerobic vagini-tis: no longer a stranger.[J].Res Microbiol.2017,168(9-10). 845-858.7.7.2 AV Combined Diagnostic Criteria Based on Gram Staining Smear and Clinical Features In 2021, Fengxia Xue, et al. found that saline wet films were difficult to provide a homogenized and standardized preparation and reading system due to the fluidity of the medium and the dynamic change of the microscopic image, which made it more difficult to achieve intelligent diagnosis of AV. In addition, the AV saline wet film scoring method is a purely laboratory diagnosis, the diagnosis of asymptomatic AV patients can be up to 10% to 20%, the part of the experimental diagnosis of “patients” whether clinical intervention and intervention is over-treatment, it is worth questioning. Another situation is that in clinical practice, some patients are found to have obvious clinical manifestations of vaginitis and abnormal vaginal cleanliness, but they do not meet the diagnostic criteria for AV diagnosis by saline wet-slice scoring method. Therefore, there are some limitations in the application of the saline smear method in screening patients for potential clinical interventions, guiding treatment, and determining the regression of inflammation. Therefore, the combined diagnostic criteria of AV with Gram stained smears and clinical features were proposed. The combined diagnostic criteria consisted of five indicators, including: leukocyte count (400×micrograph), PBC percentage (400×micrograph), LBG (1000×micrograph), background colonization (1000×micrograph), and clinical features (vaginal pH, congestion of the vaginal mucosa, and yellow secretion). Each indicator is scored 0-2 points, with a total score of 10, and the diagnosis of AV is made when the score is ≥4.
Aerobic Vaginitis 1194-5 points is mild AV, 6-7 points is moderate AV, and 8-10 points is severe AV, as shown in Table 7-2.Table 7-2 AV combined diagnostic criteria based on Gram staining smear and clinical features (Gram-stained, 1000×)AV score LBGLeukocyte countingClinical featuresBackground floraPercentage of PBCs0Grade I and IIa≤10/HPFpH ≤4.5 and no abnor-mal signsNo obvious Miscellane-ous bacteriaNone or <1%1 Grade IIb>10/HPF and ≤10/HPF around 1 epithelial cellpH >4.5 or any 1 or 2 abnormal signsE. coli-like bacteria1% ~ 10%2 Grade III>10/HPF around 1 epithelial cellpH>4.5 or any 1 or 2 abnormal signsCoccus or streptococ-cus>10%Notes: 1. AV score = LBG score + leukocyte count score + background colony score + PBC percentage score + clinical characteristic score. 2. Abnormal signs: 1) vaginal mucosal con-gestion; 2) yellow discharge. 3. AV score range 0-10 points, ≥4 points to diagnose AV (still need to combine with abnormal signs), normal reference value range 0-3 points; 4. LBG, background colonization and PBC percentage were assessed under 1000× microscope (oil microscope), and the number of leukocytes was assessed under high power microscope (HPF).The AV combined diagnostic criteria based on Gram staining smear and clinical features, using ordinary optical microscope, is easy to operate, has low equipment and personnel requirements, is easy to popularize and promote, is cost-effective, and can realize the simultaneous diagnosis of four common vaginal inflammatory diseases on the same smear, which improves the diagnostic efficiency, and has good practicality and promotable value. It takes into account both laboratory indicators and clinical features with high accuracy and reliability, and by incorporating clinical scoring items that are closely related to AV, it makes it more likely that patients who really need clinical intervention will
120 Chapter 7be diagnosed and treated in a timely manner. In addition to optimizing the AV diagnostic system, the proposal and application of this diagnostic standard also lays the foundation for the development of a model for artificial intelligence to diagnose AV.7.7.3 Functional TestingElevated pH, reduced H2O2, positive leukocyte esterase, positive β-glucuronidase or positive coagulase have a certain auxiliary role in the diagnosis of AV. Mardh et al. developed a bedside test for diagnosing AV based on five enzyme indexes: • hydrogen peroxide activity, reflecting the growth status of peroxide-producing Lactobacillus peroxidans; • leukocyte esterase activity, indicating the presence of inflammation; • sialidase activity, produced by a large number of bacteria associated with BV and AV, such as Atopobium vaginalis, Gardnerella vaginalis, Prevotella, and Streptococcus anisopliae; • β-glucuronidase activity, which is specific for E. coli infections; and • coagulase activity, which indicates the presence of S.aureus and E. coli. Their test reported a sensitivity of 90%, but the study lacked specificity analysis. The above functional tests cannot yet be used as separate criteria for the diagnosis of AV. Functional tests must be combined with morphological tests. When functional and morphologic results are inconsistent, the morphologic test results take precedence.7.7.4 Molecular DiagnosisSome scholars have evaluated the application of quantitative polymerase chain reaction (qPCR) in the diagnosis of AV. The concentrations of Lactobacillus, aerobic and anaerobic microorganisms are used as variables in a mathematical formula to accurately detect AV cases. However, the limitation of qPCR in the diagnosis of AV is that the researchers did not take into account the inflammation and immaturity of the epithelial cells that may affect the results. The lack of sensitivity and specificity analysis is also a shortcoming of this type of study.A sequencing method for vaginal microbiome analysis has also been recommended. This technique targets the highly variable V1V3 region of the vaginal bacterial 16S rDNA gene for microbiome analysis using a deep sequencing approach (i.e., approximately 30,000 reads per sample), thereby detecting and quantifying smaller numbers of microorganisms, which are
Aerobic Vaginitis 121often important vaginal microbiota components. However, despite its ability to investigate the vaginal microbial fraction, this method is not currently applicable to AV clinical diagnosis due to its excessive complexity and laboratory-based nature. Due to the high sensitivity of 90%, pH testing may be proposed as a surrogate test for AV. However, this technique lacks specificity because BV, trichomonas infections, Pseudomonas aeruginosa infections, menstrual blood, and spermatozoa are also commonly associated with higher pH levels. In general, pH testing can only be used as an alternative screening tool for abnormal vaginal flora in countries where other diagnostic tools are not available. Similarly, microscopic characterization of spherical flora can be used as a proxy for AV, however, because these methods pick out only a defined subpopulation of AV and cannot include information on inflammation and vaginal epithelial atrophy components, they are limited to a rapid screening technique.7.7.5 Culture MethodsPatients with AV have dysfunctional vaginal microecology and relatively complex pathogenic bacteria, and may have pathogenic species that are difficult to culture by traditional culture methods. Therefore, bacterial culture is not recommended for the diagnosis of AV, but drug sensitivity tests can be used to guide treatment and follow-up.AV patients are easy to combine with other vaginal inflammation, when diagnosing AV, attention should be paid to exclude the mixed infection of other common vaginal inflammation, AV needs to be differential diagnosis with BV, and attention should also be paid to whether there is mixed infection of AV and BV. In addition, the vaginal secretions of AV patients are mostly yellow purulent, similar to the signs of patients with cervicitis and pelvic inflammatory diseases, and attention should also be paid to the exclusion of abnormal secretions caused by cervical and upper genital tract infections when diagnosing AV.7.8 Treatment of AVThere is no effective standard treatment program. The etiology of any disease should be clearly identified, and for infectious vaginal diseases, topical medications maintained at appropriate doses may be superior to systemic medications for safe and rapid onset of action while reducing systemic absorption.The treatment of AV is currently antimicrobial, hormonal, nonsteroidal anti-inflammatory, and/or probiotic therapy, either topically or systemically. It is recommended to categorize the management of patients
122 Chapter 7according to their clinical characteristics and microscopic findings. Treatment for aerobic infections: empirical antimicrobials are selected and can be administered according to the microscopic characteristics, corresponding to the background flora of gram-negative bacilli, gram-positive cocci, or an increase in both. For those with poor efficacy or recurrent cases, the drugs can also be adjusted according to the results of vaginal bacterial culture and drug sensitivity.7.8.1 Antimicrobial Therapy The optimal treatment of AV in pregnant and non-pregnant women is controversial and there is no uniform gold standard. Antimicrobial susceptibility of vaginal aerobic strains in women of childbearing age was investigated in a hospital-based cross-sectional study. Group B streptococcus had a 20% resistance rate to ciprofloxacin, norfloxacin, erythromycin and clindamycin. Gram-negative bacilli (E. coli, Pseudomonas, and Enterobacteriaceae) had a high rate of resistance to cotrimoxazole (57.1%-80%), tetracycline (57.1%-73.3%), and amoxicillin (80%-85.7%). Similarly, the resistance rate of S. aureus to tetracycline, amoxicillin and cotrimoxazole was 67% to 83%. These data tell us that care must be taken in the use of antimicrobials for the treatment of vaginal infections, and that unregulated use or even abuse of antimicrobials should be avoided to avoid the occurrence of very high rates of resistance. It has been demonstrated that the use of metronidazole is not effective in improving the status of AV because the causative organisms of AV are not anaerobes in the BV, but aerobic (intestinal) bacterial microorganisms. Therefore, when metronidazole is found to be ineffective in the treatment of symptomatic vulvovaginitis in clinical practice, consideration should be given to the presence of AV or a combination of AV.Clindamycin Clindamycin is also effective against several aerobic Gram-positive cocci due to its broad-spectrum properties, and its inherent anti-inflammatory effects appear to have a more profound effect in women with severe AV. A 2% clindamycin ointment 5 g was used, administered vaginally once/day for 7-21 days. For severe AV (DIV), treatment with 2% clindamycin 5 g, vaginally, 1 time/day may be used, and after symptoms resolve, maintenance therapy may be provided with the drug 1 to 2 times per week for 2 to 6 months, which may reduce recurrent disease episodes. It should be noted that the oily matrix in clindamycin cream (within 5 days of use) or clindamycin vaginal suppositories (within 72 hours
Aerobic Vaginitis 123of use) may diminish the protective effect of latex condoms, and patients are advised to avoid sexual intercourse during treatment.If clindamycin is used as the drug of choice one should be aware and vigilant of its disadvantages: although control of the infection is possible for a short period of time, it may be transient, and its antimicrobial spectrum does not encompass all AV-causing organisms. Moreover, a large number of clinical studies have shown that in the course of repeated treatment of recurrent disease, the use of clindamycin as an antimicrobial agent in the treatment of AV is highly susceptible to the development of drug resistance, the most common methicillin-resistant S.aureus and Group B streptococcus.Quinolones The antibacterial spectrum of the 3rd generation quinolones covers some Gram-positive and negative bacteria, and levofloxacin 200 mg can be used orally, 2 times/day for 7 days. The 4th generation quinolones not only have anti-Gram-negative activity, but also have stronger anti-Gram-positive activity. Moxifloxacin can be used orally to treat the efficacy of AV. Moxifloxacin 400 mg orally once/day for 6 days may be used. Most patients with AV are cured with a course of moxifloxacin. The vaginal microecology was effectively improved in most patients after one course of treatment. A second course of treatment was given to those women whose clinical symptoms did not improve, and after two courses of treatment, more than 10% of the women still did not show significant clinical improvement. This result was most likely due to a decrease in efficacy as the duration of treatment was increased and/or the dose of antimicrobials was increased, possibly due to the loss of protective live Lactobacilli as a result of intensive treatment.Other Antimicrobial TherapyS.aureus is the most common vaginal pathogen in patients aged 31-40 years, followed by enteric gram-negative bacilli and other gram-positive cocci. Of the routinely available aminoglycosides, third-generation cephalosporins, penicillins, quinolones, sulfonamides, and tetracyclines antimicrobials, there is sufficient effective activity (more than 80% of strains are killed) against these common aerobic vaginal pathogens. Cefuroxime belongs to the 2nd generation of cephalosporins and is similar to the 1st generation against gram-positive cocci and more active against gram-negative bacilli than the 1st generation. Cefuroxime 250 mg orally 2 times/day for 7 days. Kanamycin: kanamycin has strong activity against gram-negative aerobic bacilli, and also has some antibacterial effect on staphylococcus spp. (methicillin-sensitive strains), and
124 Chapter 7has no significant effect on lactobacilli. Kanamycin vaginal suppository 100 mg can be administered vaginally once/day for 6 days. Correlative data showed that kanamycin-treated women maintained clinical symptomatic relief with normal vaginal pH and LBG-Ⅰ. The kanamycin-treated group had a decrease in vaginal leukocytes, a significant decrease in Enterobacteriaceae, and a decrease in vaginal mucosal burning and itching. Homeostasis of the intravaginal environment (normalization of pH and presence of Lactobacillus) was more pronounced in the kanamycin-treated group.Combinations of carbapenems and clavulanic acid-lactams (clavulanice β-lactam combinations) have also been proposed.Overseas Treatment Program Unlike the domestic is currently foreign bactericidal treatment of two studies showed that the vaginal treatment with dequalinium chloride (dequalinium chloride) can significantly reduce the clinical symptoms of AV, reduce the presence of relevant pathogenic microorganisms in vaginal cultures, and well tolerated. Studies have demonstrated the in vitro antimicrobial activity of dequalinium chloride against different pathogens associated with vaginal infections, including aerobic bacteria such as staphylococci, streptococci and E.coli. The total symptom scores of these AV patients treated with dequalinium chloride decreased from high scores on admission to normal levels at follow-up, respectively. A randomized, open cohort study confirmed the favorable clinical efficacy of diazinonium chloride in the treatment of aerobic bacterial vaginal infections.Nifuratel 50 intravaginally for 10 days showed good clinical efficacy in AV, especially in cases of mixed infections. 500 mg was superior to the 250 mg dose in terms of clinical efficacy. Vaginal douching with povidone iodine (povidone iodine) may have some clinical therapeutic effect because the product inhibits the growth of most of the major Gram-positive and Gram-negative intestinal flora that are associated with AV. Studies have demonstrated the effectiveness of this technique in preventing the incidence of infections in women after miscarriage or cesarean section. Although the regeneration of Lactobacillus in women with BV treated with povidone-iodine is quite rapid, there is now definitive evidence to confirm the role of povidone-iodine in the treatment of women with AV.7.8.2 Combination Therapy with Non-Antimicrobial Drugs It is unlikely that any of the above antimicrobial drugs will have a long-term positive effect on the vaginal environment. Therefore, it should only
Aerobic Vaginitis 125be considered for initial use with a short course of therapy to control acute symptoms in complicated and severe cases such as staphylococcal or streptococcal xanthococcal vaginitis, which are rare and specific subclasses of severe AV. Clindamycin was first used to treat DIV, and after 1 or 2 courses of therapy, two-thirds of the women showed efficacy. In the remaining one-third of women with AV who exhibit atrophy of the vaginal mucosa, topical estrogen, such as estradiol valerate 0.1% twice weekly, may be used to further improve symptoms. The efficacy of clindamycin is also comparable to that of clindamycin with the use of chloroquinaldo-prostaglandin vaginal tablets. Chloroquinaldo is a broad-spectrum antimicrobial agent, and prostaglandin acts on the mucous membranes of the lower genital tract to produce a topical estrogenic effect, with the specific regimen of 1 tablet/day, vaginally at bedtime, for a total of 12 days. Contraindications to hormone use, such as breast cancer and a history of previous thromboembolism, should be noted when using estrogenic drugs.Although continuous treatment usually relieves symptoms, it is not curative. And clinical practice and summary and experience tell us that most treatments are chosen based on microscopic findings: if inflammation is predominant corticosteroid hormone therapy may be chosen, with the following regimen: hydrocortisone 300-500 mg, vaginally at bedtime, once/day for 7-21 days. For those with symptomatic improvement, a maintenance regimen can be chosen, i.e., hydrocortisone 300-500 mg vaginally at bedtime, once or twice a week for 2-6 months; or clobetasol propionate vaginally at bedtime, once/day for 1 week. In maintenance therapy, the addition of fluconazole 150 mg orally 1 time/week may be considered to prevent vaginal fungal sensation in those at risk for fungal infection.Estrogen may be used if the vaginal epithelial cell maturation index is low. Antimicrobials may be indicated for large numbers of cocci (for more infectious types). In most women with significant vaginal atrophy, increased parabasal cellularity (more than 10% of epithelial cells) is found by microscopic examination of vaginal secretions, and in most cases local intravaginal application of estrogen (e.g., 0.1% estradiol valerate) is very helpful. This is usually effective in young women during the postpartum period, on low-dose combined contraceptive or progestin therapy, or in older women during perimenopause or untreated menopause. However, in women with contraindications to steroid hormone use, such as patients with breast cancer or a history of thromboembolism, this method is not likely to be used. There have been recent clinical trials confirming the use of vaginal products combined with probiotics (107 live Lactobacillus acidophilus cells) and an ultra-low dose of
126 Chapter 730 mg estriol (E3) for the treatment of severe symptomatic vaginal atrophy in menopausal women, as well as in women suffering from hormone deficiencies, and in women with sex hormone receptor-positive breast cancer who are taking anti-hormonal aromatase inhibitors.7.8.3 Probiotic Treatment In addition to using antimicrobial drugs to treat infectious agents in the vagina, you should also actively restore the vaginal micro-ecosystem and immunity. Research has confirmed that probiotics can maintain vaginal microecological stability and promote the balance of immune regulation. Lactobacillus is important for maintaining the vaginal environment because some Lactobacillus strains are able to counteract pathogen colonization by secreting antimicrobials, lactic acid, and hydrogen peroxide through competitive adhesion to the vaginal epithelium. In chronic vaginitis such as AV, it is necessary to maintain a defensive flora by administering chemicals such as metronidazole or clindamycin in addition to exogenous Lactobacillus on a regular basis (e.g., monthly). Frequent and prolonged use of antimicrobials can produce unfavorable side effects. However, exogenous Lactobacillus application may be a safer alternative to prevent recurrence In a randomized, placebo-controlled, double-blind clinical trial, 360 patients with vaginal infections including BV, pseudomycosis, trichomonas, and Chlamydia trachomatis were studied for enrollment. All patients received standardized anti-infective therapy 2 to 3 days prior to study enrollment and were randomized into two groups: 240 received the probiotic Gynoflor (containing 107 live Lactobacillus acidophilus and 0.03 mg of estriol) and 120 received placebo. Patients were treated with a vaginal ring every night for 6 consecutive days, and postmenopausal patients were treated for 12 days. Two assessments were performed 3-7 days and 4-6 weeks after the end of treatment. Assessments included vaginal pH, total symptom scores, and counts of Lactobacillus, pathogenic microorganisms, and leukocytes. The results showed significant improvement in the vaginal microcosm after administration of the probiotic Gynoflor compared to the placebo group. Therefore, AV actively restores the damaged flora using probiotics after treatment with antimicrobial drugs.7.8.4 Traditional Chinese Medicine (TCM)Traditional Chinese medicine is different from western antimicrobial drugs, its efficacy is relatively mild, drug resistance is relatively rare, and it has certain efficacy for AV, providing a new direction for AV treatment.
Aerobic Vaginitis 1277.8.5 Treatment of AV During PregnancyAV is an important cause of pregnancy complications. The incidence of AV in pregnancy varies depending on the study area, the population, the week of pregnancy, and the presence or absence of symptoms, and fluctuates from 4% to 9%, which is generally lower than that of the non-pregnant population. There is an interaction between pregnancy status and AV. On the one hand, elevated estrogen and progesterone levels, imbalance of local vaginal immunoregulation, and increased cervical mucus and vaginal secretions during pregnancy increase the risk of reproductive tract infections in pregnant women; on the other hand, the common causative organisms of AV are mostly vaginal colonizers, and when the vaginal flora is disturbed, these colonizers can be transformed into AV-causing organisms that can be upstreamed to cause adverse pregnancy outcomes.Principles of screening and treatment of AV in pregnancy: There is a lack of evidence-based medical support for screening and treatment of AV in pregnancy. Referring to the "Expert Consensus on the Diagnosis and Treatment of Aerobic Vaginitis (2021 Edition)", it is recommended that symptomatic individuals should be screened for AV, and asymptomatic women with a history of infection-associated miscarriage or preterm labor should be screened for AV in high-risk pregnancies. Given the available research suggesting that AV in pregnancy can lead to adverse pregnancy outcomes, AV in pregnancy should be treated in a manner that weighs the benefits of treatment against the potential risks."The Expert Consensus on the Diagnosis and Management of Aerobic Vaginitis (2021 Edition)" recommends the application of medications that target aerobic bacteria and are safe in pregnancy, with the following regimen: cefuroxime 250 mg orally 2 times/day for 7 days.Available studies have shown a significant reduction in the incidence of preterm labor in women treated with clindamycin for AV. And in patients with intra-amniotic infections caused by AV, erythromycin and tetracycline treatment can be helpful in improving the pregnancy outcome if chosen. However, there is no large sample size data on the use of clindamycin, erythromycin and tetracycline during pregnancy as mentioned in the above study.AV is a common vaginal infection in pregnancy and is associated with a high incidence of preterm rupture of membranes.Screening for vaginal microecology may be helpful in improving pregnancy outcomes and may reduce preterm labor, preterm rupture of membranes and other adverse pregnancy outcomes.
128 Chapter 77.8.6 Management of Sexual PartnersMale sexual partners of AV patients do not require routine screening and treatment.7.8.7 Follow-UpPatients with AV who have persistent or recurrent symptoms need to be followed up. If possible, vaginal microecology testing can be repeated after AV treatment to assess the recovery of vaginal flora and the efficacy of treatment.References[1] Donders GG. Aerobic vaginitis: no longer a stranger. Res Microbiol, 2017,168:845-858[2] Cha Han.Aerobic vaginitis in late pregnancy and outcomes of pregnancy.Eur J Clin Microbiol Infect Dis, 2019, 38:233-239[3] Cheng V. Advances in the etiology, diagnosis and treatment of aerobic vaginitis. Hainan Med, 2020, 31(4):523-528[4] Infectious Diseases Collaborative Group of the Obstetrics and Gynecology Section of the Chinese Medical Association. Expert consensus on the diagnosis and treatment of aerobic vaginitis (2021 edition). Chinese Journal of Obstetrics and Gynecology, 2021, 56(1): 11-14. [5] Paavonen J, Brunham RC. Bacterial vaginosis and desquamative inflammatory vaginitis. N Engl J Med, 2018,379(23):2246-2254
C H A P T E R - 8Vulvovaginitis in Young GirlsJing ZhangTranslator:Tingting Zhu8. 8Reproductive tract infections in young girls is a common and frequent disease in infants and young children, seriously jeopardizing the physical and mental health of infants and young children, which includes vulvovaginitis and internal genital infections, vulvovaginitis is predominantly occurring in children between the ages of 1 and 9 years. Vulvovaginitis in Young Girls is most commonly caused by bacterial infection, and common pathogens include Escherichia coli, Staphylococcus, and Streptococcus. It has also been suggested that Vulva vaginitis in young girls is mostly a non-specific infection. Due to the complexity of the causes, and the age of the children can not accurately express the disease, parents do not pay enough attention to this disease, diagnosis and treatment is not standardized in time, repeated use of antibacterial drugs, etc., may lead to local bacterial imbalance, bacteria resistant to antibacterial drugs, resulting in delayed condition of the children, and some of them have recurrent episodes.For vulvovaginitis in young girls, the integrity of the hymen should be taken into account in the treatment of medication, and care should be taken to avoid the stimulation and damage caused by medication because of the poor development of the vulva and vagina. Topical medication only works on the vulva and cannot reach the vagina well, and it is very difficult to put medication inside the vagina, and systemic medication is feared to have greater side effects, so clinical treatment is more difficult. Generally, after treatment, the inflammation can be controlled, if pay attention to local hygiene, maintain good hygiene habits, the probability of recurrence will be greatly reduced. If you do not pay attention to local hygiene and develop bad habits, it may cause recurrent vulvovaginitis and develop into recurrent vulvovaginitis.8.1 EtiologyIn recent years, vulvovaginitis in young girls has become a common and easily recurring childhood disease in clinic. Normal pediatric vaginal flora include:
130 Chapter 8Staphylococcus, rod-shaped bacillus, Lactobacillus, E.coli, Streptococcus and so on. Because of the difference between the development of vulva and the level of estrogen in the body and adults, the anatomical characteristics and physiological environment of the vagina of young girls, coupled with poor hygiene habits, when the body's resistance drops or infections occur, the normal bacterial flora imbalance, pathogenic bacteria or conditionally pathogenic bacteria reproduce in large quantities, which can easily lead to vulvovaginitis.8.1.1 Physiological and Anatomical Characteristics of VaginaYoung girls are prone to vulvovaginitis because of their age and physiological state. The epithelial tissue of the vulva and vagina of young girls is thin and easy to be damaged. The vulvar fat pad is immature and lacks fat pads and pubic hair. The labia are small and thin, which cannot completely cover the vaginal vestibule and have a weak protective effect on the vestibule. The hymen opening is small and easily blocked by sticky secretions, which is not conducive to drainage.The gonadal function of young girls is in a quiescent state, due to the lack of estrogen, the epithelium of the vaginal mucosa is thin, Lactobacillus is not a dominant bacteria, the pH value of the vagina is neutral or alkaline, about 6.5-7.5, the antimicrobial capacity is weak, which in turn leads to pathogenic microorganisms to grow, and easy to occur pathogenic bacterial infections.8.1.2 Poor Local HygieneYoung girls may not be able to keep their vulva clean due to lack of knowledge of proper hygiene and cleanliness by parents or caregivers, or poor care. Common bad habits include:• Wiping in the wrong direction after stools, which should be done from front to back to avoid contamination of the vaginal opening.• Young girls may wear crotchless pants and sit or lie down in dirty places, thus contaminating the vulva or allowing foreign objects to enter the vagina.• Parents use strong alkaline soap or other cleansing products to wash the vulva of young girls, which disrupts the micro-ecological balance of the vulva and vagina. In recent years, due to the increasing incidence of sexually transmitted diseases (STDs) in adults, many pathogens can be transmitted through mother-to-child transmission and sexual contact, which has led to some parents overdoing it and paying too much attention to the
Vulvovaginitis in Young Girls 131vulvar cleansing of their young daughters. Mothers of children who may have had genital tract infections themselves may over-use vulvovaginal cleansers on their children, who are generally negative for pathogens in vaginal secretions. The use of prophylactic sitz baths for these children is also inadvisable, and some topical cleansers can damage the vulvar skin and vaginal mucosa of young girls, resulting in injurious vulvovaginitis or vaginitis.• Out of curiosity, young girls may insert foreign objects into the natural orifices of their bodies, resulting in the retention of foreign objects, which may repeatedly cause inflammation.• Young girls may wear poor-quality diaper pads or tight, non-breathable clothes or underwear, fail to clean the vulva every day and change the underwear in a timely manner, and share bathtubs with adults, etc., all of which can induce infections.8.1.3 Neighboring Organ InfectionYoung girls anus adjacent to the vagina, the distance between the two small, pediatric local hygiene awareness is poor, urine and feces are easy to contaminate the local, play vulva and the ground or dirty hand contact, resulting in the vagina is susceptible to fecal contamination. Such as pinworm infection, pinworms can crawl from the host anus to the vulva-vagina, bring the bacteria of the digestive tract here.8.1.4 Systemic DiseasesYoung girls due to obesity, systemic infectious diseases, immune diseases, other vulvar skin diseases, long-term use of antibacterial drugs can lead to secondary infections.8.2 Clinical ManifestationsAll the affected children have vulvar or vaginal itching, burning sensation, swelling, increased vaginal discharge, some of them are accompanied by frequent urination, urinary urgency and other symptoms, burning sensation during urination, gynecological examination reveals congestion of the inner labia minora, yellowish-white or yellowish-green viscous secretion around the hymen, which can also be seen in the underpants.
132 Chapter 88.2.1 Vulvovaginal SymptomsWhen acute vulvovaginitis occurs in children, due to their young age and lack of expression, they usually cannot accurately state their special symptoms, and must rely on the attentive observation of parents or caregivers. Children may cry for unknown reasons and become easily agitated when they feel unwell. This may be manifested by the child crying when sitting down and touching the pubic area. The child may also scratch the pubic area, resulting in redness, swelling and scratches. When vaginal discharge increases, the child's underwear may be contaminated with the discharge and sometimes blood may be seen. Examination of the vulva and vagina may reveal redness, discharge and odor.8.2.2 Urinary Tract SymptomsDiscomfort in urination due to localized irritation by urine, burning sensation or severe pain, crying during urination, reluctance to urinate, and in severe cases, difficulty in urination. children under 2 years of age mostly cry during urination and defecation, are easily frightened, and have disturbed sleep, while older children may show repeated pulling of the diaper or rubbing of the vulva on hard surfaces. children above 3 years of age may show difficulty in urination, and may complain of pain in urination, vulvar pain, and a bad odor. Children over 3 years old may have difficulty urinating and may complain of painful urination, vulvar pain, vulvar itching and other discomforts.8.2.3 Recurrent Vaginal DischargeWhen the child has a foreign body in the vagina, it is manifested as recurrent vaginal discharge. Children with recurrent vulvovaginitis have a history of recurrent episodes of vulvovaginitis, and after repeated episodes of vulvovaginitis, labial adhesions may be formed, and those with obvious adhesions may cause a change in the direction of the urinary line, which may aggravate the inflammation of the vulva.8.3 DiagnosisThe diagnosis of vulvovaginitis in young girls is different from that of adolescent girls and adults, often the correct history cannot be obtained directly from the child, and the child often does not cooperate with the physical examination, the following diagnostic steps can be followed: the child has vulvar itching, increased vaginal secretions with or without odor; clinical examination shows
Vulvovaginitis in Young Girls 133redness and swelling of the vulvovaginal orifice or a large amount of abnormal secretions from the vaginal orifice; laboratory examination, smear of vaginal secretions taken from the vagina by cotton swabs Laboratory examination, smear of vaginal secretion taken with a cotton swab is positive for pathogens, and culture of vaginal discharge + drug sensitivity test is performed to confirm the diagnosis and administration of medication in persistent cases.8.3.1 History TakingWhen receiving the child, we should actively communicate with the child to gain the child's trust and eliminate her panic. Pay attention to the language skills when talking to the child, and communicate closely with the parents to ask questions to obtain the medical history. When taking a medical history, one should look for the cause of the symptoms and must note the duration, color, amount, consistency, and odor of the discharge. Fishy or unpleasant-smelling discharge often suggests an anaerobic infection.For recurrent vulvovaginitis in young girls, previous diagnosis and treatment are of great importance, and the child should be asked for a detailed medical history. The child's past examination results should also be carefully asked, if the child has had a bacteriological examination of vaginal secretions, the results of the examination, whether or not to obtain pathogenic bacteria, whether or not to carry out antimicrobial treatment and the efficacy of the treatment should be carefully asked and recorded. At the same time, parents should be carefully asked about the child's poor hygiene habits, such as whether the cleaning method after urination and defecation is correct, whether tight or breathable clothes are often worn, whether the vulva is often touched with dirty hands or whether the vulva is often in contact with the unclean ground, whether there is a slight sassafras injury on the vulva due to outdoor activities, and whether the diaper is often changed and the type of diapers are used in the children who use diapers.Other important things to look for include a history of systemic infections such as upper respiratory tract infections, gastrointestinal infections, skin diseases, and reproductive tract infections in the child and family, a history of recent medications, a moist or dirty living environment, and cleanliness of the home's bathtubs. The possibility of child sexual abuse should also be considered.8.3.2 Physical ExaminationVulvovaginitis in young girls should be examined more comprehensively.
134 Chapter 8During the examination, in order to reduce the anxiety of the child and to obtain the child's cooperation, the child can be held by the mother or other parents, and adopt the frog-legged position, and the examiner can gently separate the labia majora downward for better observation. During the examination, attention is paid to the labia majora and minora, vaginal opening, hymen, clitoris, urethral opening, anus and lower part of the vagina. Discharge should be taken for microscopic examination and bacteriological examination should be performed at the same time. Attention should be paid to the presence of bleeding or vaginal foreign body, if vaginal foreign body is suspected, parents may be advised to arrange for speculum examination under anesthesia for the child. Parental consent and cooperation should be obtained during the examination.Vulvovaginitis in young girls presents in a variety of ways. There may be no discharge and no obvious signs of infection. When discharge is present, it may be a small, dry remnant of vulvovaginal secretion or a large amount from the vaginal opening. The color of the discharge varies and may be clear, white, yellow, green, or grayish brown. In some children, signs of poor hygiene may be seen, such as debris in the labial folds or fecal contamination of the vulva. Sometimes congestion, edema and erythema of the vulva are also seen, with vascularization of the vaginal opening and vestibular surface.It should also be noted that in cases of child sexual abuse, the shape of the hymen needs to be noted, and signs of injury may be seen, such as scarring, lacerations, or an incomplete hymen. Examination should be done by gently parting the labia majora and pressing the perineum downward, which helps to visualize the lower part of the vagina and locate secretions or foreign bodies behind the hymen.The anus and its surrounding skin are also examined for signs of inflammation or skin lesions or other injuries. If pinworm infection is suspected, the perianal skin and anus may be pressed early in the morning using a clean instrument, such as a film, to extract perianal secretions, and stool screening may be performed to check for characteristic pinworm eggs. If necessary, gentle anal and abdominal examination as well as ultrasonography of the pelvic region can be performed to help determine whether there are vaginal foreign bodies, pelvic masses.8.3.3 Etiologic ExaminationSample Collection In order to clarify the etiology of vulvovaginitis in young girls, examination
Vulvovaginitis in Young Girls 135of vulvovaginal discharge is necessary. Vaginal discharge should be removed by non-invasive methods, and sterilized cotton swabs can be used to collect samples directly. Smears and, if necessary, bacterial cultures should be obtained after collection of the sample, with attention to anaerobic cultures depending on the nature of the discharge. Appropriate bacteriologic examination may help to obtain a correct diagnosis. Another method of sample collection is to use a syringe to inject saline into the vagina and collect the fluid that flows into the lower vaginal area and vestibule. Collection can also be performed using the cannula technique, in which a 14- or 16-gauge plastic intravenous catheter is placed inside another short 12-gauge plastic or rubber catheter, and the other end is attached to a syringe of sterile saline containing 1 to 2 mL of saline. The examiner gently places the red rubber catheter into the opening of the hymen and pushes and sucks on the syringe repeatedly to collect the vaginal secretions.Microscopic Examination of Vulvovaginal Discharge The examiner microscopically observes the content of leukocytes, squamous epithelial cells, and lactobacilli, and is careful to look for Neisseria gonorrhoeae, Trichomonas, yeast-like fungi, and clue cells, etc. The morphology of vaginal secretions from the laboratory microscope for common vulvovaginitis in young girls is illustrated in Fig. 8-1. Strains of bacteria obtained by bacterial cultures are also often found in the normal vaginal flora. Unless evidence of infection by Neisseria gonorrhoeae, Trichomonas, yeast-like fungi, or clue cells is found, most vulvovaginitis in young girls is due to dysbiosis caused by poor local hygiene. The causative organisms are most commonly β-hemolytic Group A streptococcus (streptococcus pyogenes), followed by Haemophilus influenzae, S.aureus, Moraxella catarrhalis, streptococcus pneumoniae, Neisseria meningitidis, and, less commonly, Shigella pneumoniae.Cultures The cultures of vaginal discharge of young girls include general bacterial cultures and cultures of gonococcus, ureaplasma urealyticum (UU), mycoplasm (MH) and chlamydia (CT). Biopsies of vulvar adnexa are taken and sent for pathologic examination. Bacterial cultures are obtained for strains often seen in the normal vaginal flora. The causative organisms are most commonly β-hemolytic group A streptococcus (Streptococcus pyogenes), followed by β-hemolytic group B streptococcus (Streptococcus agalactiae), coagulase-positive staphylococcus (S.aureus), Haemophilus influenzae, E.coli, Enterococcus faecalis, and Shigella pneumoniae.
136 Chapter 8Figure 8-1 Gram-stained microscopic morphology of vulvovaginitis in young girls (1000×)Notes: Flora density: 3+, flora diversity: 3+, Dominant organism: gram-negative short bacilli, Pathogen: trichomonas infection (-), Fungal infection: spores (-); budding spores (-); hyphae (-), Nugent score: 6, AV score: 2.8.4 Classification of Vulvovaginitis in Young Girls8.4.1 Bacterial VulvovaginitisBacterial vulvovaginitis in young girls with atypical presentation is usually not clearly categorized clinically. Usually E.coli, Haemophilus spp., Streptococcus grass-green, Enterococcus spp., Aspergillus spp. and Streptococcus hemolyticus infections are the main ones. However, there is not a big difference in the rate of isolation of the vaginal flora of the children with vulvovaginitis comparing with that of the normal young girls, which means that the bacterial-induced vulvovaginitis is mainly related to the pre-pubertal hormone level and the dysfunction of the intra-vaginal flora.Clinical symptoms and signs are usually characterized by redness and swelling of the vulva, profuse vaginal discharge with pain and itching, and ulceration of the skin or frequent urination, urinary urgency or even difficulty in urination in some of the affected children.Laboratory examination of vaginal discharge of young girls, microscopic leukocytosis, check the clue cells, etc. In addition, vaginal discharge culture can be carried out to clarify the causative agent.8.4.2 Pseudomonas VulvovaginitisPseudomonas vulvovaginitis in young girls is mainly caused by Candida A B
Vulvovaginitis in Young Girls 137albicans. The small amount of pseudohyphae that normally exists in the vulvovagina of young girls usually does not cause any symptoms, but when the stability of the host's internal environment is out of order, especially when antimicrobial drugs interfere with it or immune dysfunction occurs, the pseudohyphae that exists in the vagina of young girls can become the causative organisms. In addition, the vulva of young children is poorly developed, and ovarian function is not yet sound, the natural defense function of young children's vagina has not yet been established, poor resistance, more susceptible to infection, resulting in young girls suffering from Pseudomonas vulvovaginitis. According to the literature, as the incidence of Pseudomonas vulvovaginitis in women increases, the incidence of mother-to-child transmission of Pseudomonas vulvovaginitis also tends to increase.Young children or their parents do not pay attention to hygiene, letting young children share baths, tubs, towels and underwear, mixing young children's clothes with those of adults, and letting young children wear wet clothes, and so on, the above bad hygiene habits can easily infect young children with Candida species. In addition, some bad hygiene habits of young children can also cause this disease: not wearing underwear, wearing tight underwear for a long time, nylon pants, long-term pubic use of pads, diapers. The use of non-strictly sterilized toilet paper or toilet paper sprayed with perfume, etc., the above bad habits make the female perineum temperature, humidity, acidity increase, so that hosted in the human skin, mucous membranes, Candida albicans reproduces in large quantities, and when it reaches a certain amount, it can lead to the human body to develop the disease. There is also improper education of young children, there is curiosity to touch the vulva, put foreign objects into the vagina, as well as with the parents' knowledge of hygiene care methods are not appropriate have a certain relationship.Clinical symptoms are mainly characterized by vulvar itching, burning pain, and vaginal secretions in the form of white soya bean dregs. Laboratory examination of vaginal secretions, microscopic detection of pseudofilamentous yeast.8.4.3 Trichomoniasis VaginalisThe incidence of trichomoniasis vaginalis in young girls is low before the age of 12, and it increases significantly after menarche. The clinical manifestations are redness and swelling of the vulva and clitoris, congestion of the vestibular mucous membrane, and purulent secretion from the vaginal opening; in severe
138 Chapter 8cases, the vulvar skin is ulcerated, and the mucous membrane of the urethral opening is congested and edematous.8.4.4 Gonococcal VaginitisGonorrhea is an infectious disease of the genitourinary system caused by gonococcal infection, which is currently the most common type of sexually transmitted disease in China before puberty, and spreads almost exclusively by sexual contact in adults. In recent years, with China's opening up to the outside world, frequent international exchanges have led to the rapid spread of sexually transmitted diseases (STDs), which were basically eliminated in China in the 1960s, plus the fact that the vaginal mucosa of girls is not composed of squamous epithelium but of columnar epithelium, and the chemical composition of its mucosal secretion is different from that of adults, making it easy to be infected.Neisseria gonorrhoeae infection in young girls: a. to public places, especially public baths, touching everywhere, sitting and other direct contact by infection. b. and Neisseria gonorrhoeae family members share contaminated bedding, pots and pans. c. Direct infection due to sexual assault, not many domestic reports. The fetus is infected by contact with the contaminated secretions or amniotic fluid of the sick mother when passing through the birth canal. d. Medical infection.Symptoms usually appear within 1 week after exposure to the infection, young girls in the clinical mainly sub-clinical state, manifested as redness, swelling, burning, burning itching vulva, vagina and urethra congestion, edema, labia major and minora flushing, swelling, vaginal secretions increased, purulent, due to the urethra is short, urinary drainage, urinary tract irritation symptoms are atypical, and sometimes redness, swelling, and burning pain in anus can occur. It should be noted that gonorrhea is often combined with mycoplasma urealytium infection. Should be more parts of the material, in addition to the vulva, vagina, at the same time on the rectum, oropharyngeal secretions for smear examination or culture to help confirm the diagnosis.8.4.5 Mycoplasma InfectionsMycoplasma urealytium infection in young girls is often transmitted indirectly from mother to child, through the placenta and the birth canal to the newborn, resulting in the newborn carrying the microorganism at delivery, which may
Vulvovaginitis in Young Girls 139also be the initial source of carriage in the normal healthy population. The neonatal carriage rate has been reported to be 28.57% in healthy females and 5% in prepubertal females, and the colonization rate of mycoplasma urealytiumin children is less than 10%. There may be differences in mycoplasma urealytium between the transplacental and cesarean section populations. Mothers infected with mycoplasma urealytium and transvaginal deliveries can result in a significantly higher detection rate of mycoplasma urealytium, which may be related to the passage of the newborn through the birth canal during labor and delivery, where exposure through the birth canal allows more opportunities for transmission of mycoplasma urealytium to the mucous membranes of the genitourinary tract of the newborn. However, there is not a single factor that affects the detection rate of mycoplasma urealytium in newborns, and it is also affected by many external factors during growth.Mycoplasma urealytium is a prokaryotic microorganism, spherical rod-shaped, size 125-250mμm, molecular weight 4.5x108, highly polymorphic, no cell wall, can produce ureolytic enzymes to break down urea. It is resistant to penicillin, specific antibody can inhibit its growth, it has affinity for cell membrane, and it needs steroid substance when growing and reproducing. The cause of cellular damage by mycoplasma urealytium may be due to the fact that the mycoplasma, which adheres to the surface of the host cell, obtains lipids and cholesterol from the cell membrane and thus obtains nutrients. Mycoplasma urealytium contains the enzyme urease, which hydrolyzes urea to produce large amounts of ammonia, and through its metabolism produces toxic substances that cause cellular damage.Possible routes of transmission of mycoplasma urealytium include: • Intrauterine infection. Intrauterine infection is the main route of transmission of perinatal mycoplasma urealytium infection. Mycoplasma urealytium may infect the amniotic membrane, amniotic fluid, and fetus from the cervix or vagina upstream. In addition to this, mycoplasma urealytium may be transmitted from the placenta to the fetus via the maternal bloodstream. • Infection during labor. Since mycoplasma urealytium mainly colonizes the female lower genital tract, it may cause infection when a newborn is delivered vaginally, but it is often difficult to distinguish it from intrauterine infection. • Horizontal or medical transmission. There are a few newborns who are negative for mycoplasma urealytium at birth, but turn positive one week after birth, suggesting that there may be horizontal or medical transmission.
140 Chapter 8Laboratory tests: • Enzyme-linked immunosorbent assay (ELISA) can be used for the detection of mycoplasma urealytium. ELISA is a highly specific and sensitive method for the detection of antigens and antibodies, and can detect immunoglobulin isoforms with a high degree of reproducibility. ELISA is based on the cellular proteins of the solubilized mycoplasma urealytium as the antigen, and because of the presence of a specific common antigen among the various types of mycoplasma urealytium, ELISA is not a reliable test, so it is a good choice. Because there is a specific common antigen between all types of Mycoplasma urealyticum, ELISA cannot detect type-specific antibodies in human serum, but rabbit anti-mycoplasma urealytium type antibodies can be used to serotype mycoplasma urealytium. This may be due to the fact that mycoplasma urealytium has different antigenic effects on humans and rabbits and produces different antibodies. • Direct or indirect immunofluorescence test on solid medium can also be used for the detection of mycoplasma urealytium, which can be used for serotyping of clinical samples of mycoplasma urealytium, and for identification of mixed serotypes of strains grown on the surface of the same solid medium. This method is simple and easy to implement, but sometimes the results are not easy to determine, and subjective factors have a greater impact. PCR combined with liquid-phase hybridization can identify the biota of Mycoplasma urealyticum. Clinical isolation of wild Mycoplasma urealyticum strains for serotyping often need to be cultured, often need a few days or longer, which is not conducive to large-scale clinical strain grouping or typing research. Overseas, there is a combination of liquid hybridization method with the help of urease gene to identify the biota directly with the amplified samples within 1 day. This method is rapid, relatively objective, and can detect low levels of mycoplasma urealytium, which can be identified directly from amplified samples within 1 day.Mycoplasma urealytium infection in young girls is characterized by a chronic, prolonged increase in plasma yellowish-white vaginal discharge and varying degrees of self-consciousness.8.4.6 Chlamydia Trachomatis InfectionChlamydia trachomatis is the main pathogen of non-gonococcal inflammation of the genitourinary tract, Chlamydia grows and reproduces in the host cell, there are two different kinds of particles: small and dense protozoa and sparse
Vulvovaginitis in Young Girls 141protozoa, the protozoa are more stable outside the host cell, without the ability to reproduce, but with a strong infective potential, invade into the host cell and gradually develop into a protozoa to reproduce in a divisive way, but without infective potential, the development of a mature offspring protozoa is released from the infected cell that is damaged, and then infected with new infected cells. The mature progenitor is released from the destroyed infected cells and reinfects new susceptible cells, leading to a series of pathological changes.Young girls lack estrogen, vaginal epithelium is thin, easy to cause Chlamydia trachomatis infection, the incidence rate of 1.04/100,000, Chlamydia trachomatis mainly invades the columnar epithelial cells of the genitourinary tract, does not infect the squamous epithelium, the incubation period of 10-20 days, slow onset of disease. Pregnant women with genital Chlamydia trachomatis can infect the fetus and newborn through the intrauterine or birth canal; infection can also occur through the hands, clothing, bath tub and other ways.Clinical manifestations are increased vaginal discharge, young girls complain of painful urination, vulvar itching, yellowish-white sticky discharge around the hymen of the vaginal opening, redness and swelling of the vaginal opening, vestibule and labia major and minor, leading to vestibular adenitis, cervicitis, and in severe cases, subacute pelvic inflammatory disease, and children with mild abdominal pain and low-grade fever, and most of the patients are delayed in the diagnosis because of no obvious symptoms. Most of the patients are delayed due to lack of obvious symptoms. Most of them can be diagnosed clearly according to clinical symptoms, high-risk factors of contacting infected people, combined with chlamydia culture and enzyme-linked immunosorbent assay.8.4.7 Pinworm VulvovaginitisPinworm vulvovaginitis occurs in young girls under normal circumstances is not common, if the clinical diagnosis of other types of vulvovaginitis, but according to its etiology of treatment, clinical treatment is ineffective, and children with unexplained friction, scratching the vulva, to the itching at night, it is important to consider pinworm vulvovaginitis. Laboratory tests: with ordinary cotton swabs or tongue depressor wrapped in cellophane in the morning before the stool swabbing perianal skin folds for 1 week, laboratory microscopy to see pinworm eggs can be confirmed.
142 Chapter 88.4.8 Diaper DermatitisDiaper dermatitis is a common skin disease in newborns and infants, mainly manifested as erythema, or even blisters, vesicles or ulcers.8.4.9 Contact InflammationInflammation of vulvar skin due to contact with certain irritating substances or allergic substances, or due to obesity, vulvar skin, skin between the thighs rubbing against each other with fluid exudation, burning sensation in the contact area, rash, blisters, and in severe cases, necrosis and ulceration.8.4.10 Vaginal Foreign Body InfectionIf the vulvovaginitis of young girls is not cured for a long time, recurrence, especially when the vaginal discharge is bloody or purulent, sometimes accompanied by foul smell should consider the possibility of vaginal foreign body, often because of the curiosity of young girls or to relieve the itching of the vulva, put the foreign body into the vagina, resulting in secondary infections, and even the formation of ulcers and granulation tissue. It is difficult for the child to admit to a history of vaginal foreign body insertion and for the parents to be aware of it.8.5 TreatmentThe vaginal microbial ecosystem in children with clinical signs of vulvovaginitis is complex and variable, and the presence of a microorganism does not necessarily mean that it is the cause of the infection. Therefore, the diagnosis of vulvovaginitis in girls requires a complex and comprehensive approach. When infection analysis suggests vaginitis, verification by microbial culture should also be considered, along with a judgment call in relation to symptoms. Especially in young children, if medication is needed, the choice of antibiotic should be based on culture sensitization as much as possible for safety and efficacy reasons. Pre-pubertal girls with poor reproductive tract infections should be given sufficient attention for early detection and treatment, and hygiene habits should be emphasized and symptomatic treatment should be provided.8.5.1 Remove the Cause of the DiseaseWhen the causes of vulvovaginitis in young girls are clearly excluded, such as vaginal foreign bodies and sexual abuse, the vast majority of children can be
Vulvovaginitis in Young Girls 143cured by improving vulvar hygiene. For children with recurrent vulvovaginitis, special attention should be paid to proper hygiene practices. Parents or caregivers should be told very clearly that proper hygiene practices are necessary to improve local vulvar hygiene and the overall hygiene of the child. The child should be bathed every day, and should have a special bath tub; the water used for washing hair should no longer be used for bathing, and the application of strong alkaline soap or shower gel should be avoided; after bathing, the water around the vulva should be absorbed with clean towels, and the underwear should be changed; excessively tight pants should be avoided, especially nylon pants, and cotton underpants should be worn, with ventilation and air circulation to keep the vulva clean, dry and to minimize friction; hygiene habits should be adopted for hand-washing after urination and defecation; the child should be told very clearly that correct hygiene habits are necessary to improve the local hygiene of the vulva and the overall personal hygiene. Wash hands after urination and defecation; the direction of wiping after urination and defecation should be from the front to the back, and should be wiped clean with paper, and cleaned with water after defecation if possible.8.5.2 Antibacterial Drugs and Symptomatic Treatment• Bacterial vulvovaginitis in young girls, first empirical treatment, negative positive drugs, antibacterial drugs or other anti-infective treatment should be carried out when the bacterial culture obtains the above causative organisms or when there is clear evidence of specific infection. Antimicrobial drugs are best selected based on culture and drug sensitivity test results: Group B α-hemolytic streptococcus and Haemophilus influenzae infections can be selected amoxicillin; S.aureus infections can be selected cephalosporin or amoxicillin, sulbactam. • Children with trichomonas vulvovaginitis can be treated with sitz baths with 1:20 iodophor and oral metronidazole.• Pseudomonas vulvovaginitis can be treated with sitz bath with 1:20 iodophor and topical application of clotrimazole cream at a dose of 1 time/day for 10 days.• Children with gonococcal vulvovaginitis can be treated with sitz bath 1:20 iodophor and intramuscular injection of cefotrizine, and oral azithromycin.• Children with Mycoplasma and Chlamydia vulvovaginitis can be treated with sitz baths of 3% boric acid solution and oral azithromycin.• Children with pinworm vulvovaginitis should have their intestinal pinworms
144 Chapter 8completely eradicated to prevent recurrence. Give the child oral bis (hydroxynaphthyl) thiopyrimidine. Or use anti-pinworm spirit anal ointment (containing 3% of bis (hydroxynaphthyl) thiopyrimidine), wash the vulva and perianal area before going to bed. Squeeze out a little ointment coated around the anus. Then insert the plastic injection tube into the anus 1 cm and squeeze out 1 g of ointment. 7 consecutive days.• Children with diaper dermatitis should keep the vulva clean, breathable and dry. Use cotton diapers to avoid irritation, wipe dry after local warm water cleaning, and apply vegetable oil or zinc oxide ointment directly.• Children with contact inflammation should remove allergens, avoid irritants, and use topical zinc oxide ointment; severe cases can be cured with oral anti-allergy drugs and topical glucocorticoids.• For children without pathogenic microorganisms, 3% boric acid solution can be used in sitz bath, the dose is 2 times / day, continuous treatment for 7 to 10 days.8.5.3 Treatment of Special InfectionsFor children suspected of sexual abuse or recurrent episodes of vulvovaginitis, especially those with obvious vaginal discharge, special attention should be paid to the presence of Neisseria gonorrhea, Chlamydia trachomatis and other infections. Neisseria gonorrhoeae infection should be treated with sensitive antimicrobial drugs such as third-generation cephalosporins. Chlamydia trachomatis infection requires the use of macrolides antibacterial drugs, for children over 8 years old can also consider doxycycline.8.5.4 Traditional Chinese Medicine(TCM)Due to the physiological structure of young girls, most of the topical drugs are not suitable for infants and young children, traditional Chinese medicine prescription due to its small toxicity, few side effects and other characteristics, in the clinic also has a relatively wide range of applications.If use Chinese medicine sitz bath treatment, Chinese medicine prescription for: snake bed seed, bitter ginseng, cypress, cloud ling, raw Baibu, white ringworm skin, Xia Gu Cao plus subtraction. 1000 ml water micro-flame decoction for 15 minutes, remove the dregs, fumigation sitz bath for 20~30 minutes, 2 times / day, for 7 days as a course of treatment. Generally, the Chinese medicinal liquid is diluted with povidone-iodine. Some studies have also shown that the
Vulvovaginitis in Young Girls 145method of low-pressure vaginal douching is more effective in the treatment of vulvovaginitis in young girls, choosing the appropriate caliber catheter during douching can avoid damaging the vaginal mucosa and hymen, and at the same time, it can make the drugs directly contact the lesion, which can play a better therapeutic effect, in addition, through low-pressure douching, fully clearing vaginal secretions, which can reduce the recurrence of the chance of recurrence.8.5.5 PsychotherapyIn addition to anti-infective treatment and symptomatic treatment of vulvovaginal infections in young girls, children who are confirmed to have been sexually abused also need professional psychotherapy and judicial intervention.8.5.6 Speculum Diagnosis and TreatmentYoung girls with recurrent episodes of vulvovaginitis and unsatisfactory results of antibacterial treatment, especially the recurrence of fishy odor or unpleasant odor of the discharge suggestive of anaerobic bacterial infections, we need to pay attention to the possibility of foreign bodies in the vagina. Endoscopy should be actively considered in children in whom palpation or speculum examination does not reveal a foreign body. Consent should be obtained from the child's legal guardian and the procedure should be performed by an experienced physician. It is performed under intravenous anesthesia and can be performed using a small-bore hysteroscope to examine the vagina and avoid damage to the hymen. The speculum can clearly diagnose or remove the vaginal foreign body, both diagnostic and therapeutic significance, with good clinical results.8.5.7 Determination of Therapeutic EffectVulvovaginitis in young girls is cured, which is manifested as the disappearance of uncomfortable symptoms, no abnormal secretion from the vaginal opening and urethral opening, and normal color of mucous membrane. Significantly reduced symptoms, reduced or disappeared vaginal and urethral secretion, reduced or subsided urethral and vaginal mucous membrane redness and swelling. Effective manifestation is symptomatic relief, vaginal and urethral discharge reduction, urethral and vaginal mucosal erythema and discharge reduction is not obvious. Ineffective in that there is no reduction or aggravation of symptoms. Recurrence of the disease within 2 weeks after the patient stops treatment is considered as relapse.
146 Chapter 88.6 PreventionBecause the vagina and anus are very close, lack of protective pubic hair, labia fat pads, and lack of estrogen, so keep the vulva clean, dry is the prerequisite for the cure of vulvovaginitis, and develop good hygiene habits is the key to preventing this disease. For girls in the pre-pubertal period, due to the ovarian function began to activate, in the body under the influence of estrogen physiological leukorrhea increase, the vaginal discharge is manifested as a plasma, yellowish-white, non-stimulating, no odor, panties can have yellowish-brown stains. Therefore, it is important to observe the characteristics of vaginal dischage of young girls with vulvovaginitis and avoid unnecessary medical interventions before vaginal dischage examinations.Explain the anatomy and physiological characteristics of young girls to the parents of the affected children, and explain to the parents the causes of the disease, symptoms, treatment measures, methods of care, and the time of follow-up, so that the parents of the affected children can understand the disease and actively cooperate with the treatment. Teach parents how to take care of young girls to avoid vulvovaginitis:• Instruct parents to cultivate their children's good living habits from childhood, wash the vulva every day, keep the vulva of young girls clean and hygienic and change the panties frequently, and give the young girls to wear cotton panties with good breathability, do not wear nylon or chemical fiber panties,.• After defecation, you should wipe from front to back and wash with warm water, do not use wet paper towel instead.• Young girls should also use diapers or diapers during daytime play, and wear crotch-sealing pants for young girls as early as possible to protect the perineal skin and help prevent vulvovaginitis and vaginitis in young girls.• Wash and bathe tools exclusively for each person, use separate towels, tubs and towels for washing the perineum, and wash clothes separately from family members, do not mix and wash.• Try to take a shower or a basin bath and then a shower, avoid sharing the bath with the mother, and do not go swimming in public pools without health checks.• Children's underwear should be washed every day and placed in the sun or boiled in boiling water to sterilize.• Avoid eating spicy and stimulating foods.• Educate young children to be aware of self-protection and not to put foreign
Vulvovaginitis in Young Girls 147objects into the vagina.• If the young girl has pinworms, take active treatment measures, and should take anthelmintic medicine in time to avoid itching and redness of the vulva caused by pinworms.• Observe the vulva of young girls, and treat them promptly if any abnormality is found.References[1] Chengzhe Gao, Jianchun Zhao, Yuanyuan Cao. Analysis of factors associated with vulvovaginitis in young girls. Clinical Medicine Practice, 2018,02: 105-107.[2] Xing Xie, Beihua Kong, Tao Duan. Obstetrics and gynecology. Beijing:People's Health Publishing House, 2018.7.[3] Xueling Huang. Observation on the effect of health education intervention in young girls with vulvovaginitis. Journal of Aerospace Medicine, 2017, 28(7):872-873.[4] Kapila S, Bradford J, Fischer G. Share Vulvar psoriasis in adults and children: a clinical audit of 194 cases and review of the literature. j Low Genit J Low Genit Tract Dis, 2012, 16(4):364-371.
C H A P T E R - 9Atrophic VaginitisJing ZhangTranslator: Tingting Zhu9. 9Atrophic vaginitis, also known as senile vaginitis, is an inflammatory disease caused by atrophy of the vaginal epithelium secondary to decreased peripheral estrogen levels in women. It is common in postmenopausal older women, and about 25%-50% of postmenopausal women suffer from atrophic vaginitis. Due to the decline of ovarian function, estrogen level decreases, the vaginal wall atrophies, the mucous membrane thins, the glycogen content in the epithelial cells decreases, the pH value in the vagina rises, the local resistance decreases, and the pathogenic bacteria are easy to invade and reproduce causing inflammation, which makes the disease difficult to heal and recurring. The main symptoms are increased vaginal discharge and vulvar itching, burning sensation. Examination shows that the vagina is aged, the folds disappear, and the vaginal wall becomes smooth and thin. The vaginal mucosa is congested with small bleeding spots and sometimes superficial ulcers are seen. If the ulcerated surface is adherent to the opposite side, the adhesion can be separated during vaginal examination and cause bleeding, and when the adhesion is severe, it can cause vaginal stenosis or even atresia, and poor drainage of inflammatory secretions can form vaginal pus or even uterine pus.9.1 History and Epidemiology of Atrophic Vaginitis9.1.1 HistoryAs early as 1888, Kellogg reported that in "postmenopausal women there is often a syndrome of increased vaginal discharge accompanied by intense itching and pain". Later in 1963, Robert Wilson reported a more accurate description of the disease, "A significant proportion of older women experience a negative condition ......, in which the vaginal walls atrophy and eventually close to disappearing; the vagina loses its dilatation and elasticity, and becomes shorter and easier to break. The epithelium of the mucous membranes of the vulva
Atrophic Vaginitis 149and vagina is thin and reddened with congestion, and the mucous membranes sometimes bleed and ulcerate. Pathologic changes in the mucous membranes (of the vulva and vagina) make them more susceptible to infection, and geriatric vaginitis tends to cause vaginal adhesions or irritating discharges, and even fungal or trichomonas infections". This was the first time the term geriatric vaginitis appeared. Later, in order to emphasize the pathological changes of vulvar and vaginal atrophy, the disease was finally named “atrophic vaginitis”.9.1.2 EpidemiologyAtrophic vaginitis is a geriatric gynecological disease, which is more common in clinical practice, and symptoms usually appear 4-5 years after menopause. Worldwide, about 25%~50% of postmenopausal women suffer from atrophic vaginitis. The prevalence of this disease ranges from 30% to 58.6% domestically and reaches up to 98.5% abroad, and with the increasing trend of population aging in China, the incidence of this disease is on the rise. Some studies have also shown that 3% of women of childbearing age experience vaginal dryness, with the proportion of symptoms increasing to 4% in early perimenopause, 21% in late perimenopause, and 47% within three years of menopause. In addition to vaginal symptoms, some women also experience genitourinary symptoms, which can vary. Among older women with urogenital symptoms, 27% present with vaginal or vulvar dryness; 18.6% with vaginal or vulvar tingling; 11.1% with increased vaginal or vulvar discharge; and 5.2% with dysuria. Despite the high prevalence, atrophic vaginitis continues to be underdiagnosed. Because diagnosis involves inquiries about reproductive tract symptoms, some women may be too shy to talk about their symptoms, and only 20% to 25% of patients are willing to seek help from a doctor, while even fewer women in relatively undeveloped areas are aware of the relevant scientific knowledge. In recent years, with the development and wide application of Internet information, the epidemiologic survey of atrophic vaginitis is more immediate and real. Some surveys show that 45% of postmenopausal women have vaginal symptoms, but only 4% of participants can realize that such symptoms are related to menopause.9.2 Etiology and Risk FactorsAtrophic vaginitis is common in women after natural menopause or ovarian decline, but also in women with postpartum amenorrhea or pharmacologic pseudo-menopausal treatment. Due to the decline of ovarian function, the level of estrogen decreases, the vaginal wall atrophies, the mucosa thins, the glycogen
150 Chapter 9content in the epithelial cells decreases, and the pH value in the vagina rises, mostly to 5.0-7.0. At this time, Lactobacillus is no longer the dominant bacteria in the vagina, and due to the decrease of the local resistance, other pathogenic bacteria reproduce too much or invade easily to cause vaginal inflammation. Atrophic vaginitis can also occur secondary to a hypoestrogenic state, such as after ovariectomy, after treatment with gonadotropin-releasing hormone agonists, hypoestrogenic amenorrhea, or during the course of radiation, chemotherapy, and endocrine therapy for various cancers. Since the above conditions can make patients enter menopause rapidly, and the trend of recent years is more and more youthful, the timely management of menopause-related symptoms in the above patients has become the focus of social concern.9.2.1 Effects of Decreasing Estrogen Levels on the Vagina The upper 3/4 of the vagina is differentiated from the embryonic mesoderm, and the terminal 1/4 is differentiated from the endoderm, which also forms the urogenital ridge. The inner layer of the vagina is covered with a complex squamous epithelium, the middle layer is muscular and the outer layer is fibrous. The vulva also differentiates from the urogenital ridge, but the labia majora epithelium differentiates from ectoderm. The estrogen receptors (ERs, α and β) are prevalent in the vagina, vulva, pelvic floor musculature, pelvic fascia, urethra, and bladder triangle, and their levels decline with menopause, with a gradual down-regulation of receptor function. Estrogen receptor density is highest in the vagina and decreases in the vulva. The distribution of androgen receptors in women is the opposite, with the highest density of androgen receptors in the vulvar skin and a gradual decrease in the density of androgen receptors in the vagina. In women of childbearing age, the vaginal mucosa is well supplied with endogenous estrogen and exhibits a thickened and moist fold-like appearance. Estrogen receptors (α and β) are expressed in the vaginal tissues of women of childbearing age, whereas estrogen receptor-β is not expressed in the vaginal tissues of postmenopausal women.Estrogen is the main physiological regulator of the vagina, and the complex squamous epithelium of the vagina is thickened under the influence of estrogen to form abundant mucosal folds. At the same time, the epithelial cells contain a large amount of glycogen, which, through its own glycolysis and that of the symbiotic Lactobacillus vaginalis, produces lactic acid and other components with bacteriostatic effects, such as hydrogen peroxide, preventing the invasion of exogenous pathogens.
Atrophic Vaginitis 151The synthesis and secretion of estrogen in the body decreases significantly after menopause, and the level of estradiol decreases from 40~400 pg/ml before menopause to about 20 pg/ml after menopause, with an activity of only 1/3 of that before menopause, which is relatively poor in its action in the body and shows a decrease in the level of estrogen. Androstenedione becomes an important source of androgens in the postmenopausal period, and most of the former are directly converted through the ovary to form estradiol. As a result of the decline in endogenous estrogen levels, the vulvovaginal skin and mucosa show atrophic changes from year to year. The vaginal epithelium atrophies and shortens, the wrinkled walls tend to spread, and the mucosa is thin, pale, or covered with small hemorrhages and erythema. With ageing, the blood supply of the vaginal mucosa declines progressively, collagen fibers become swollen and dissolved and glassy, elastic fibers break down and decompose, the elasticity around the vagina disappears, and connective tissue increases. Vaginal shortening and narrowing, ductility significantly reduced, so that the vaginal elasticity nearly disappeared, and even ultimately cause vaginal tightness and stiffness, causing sexual dysfunction and a series of symptoms.9.2.2 Vaginal Flora DysbiosisNormal vagina is usually moist, lubrication, with obvious suitable for the survival of vaginal microorganisms in the environment, the existence of a variety of microbial communities, the largest number of which is Lactobacillus, up to 95% of the total number of about. Lactobacilli are mainly distributed on the epithelial surface of the vaginal mucosa, they adhere closely and form a dense bacterial film, preventing the adhesion of other pathogenic bacteria, thus effectively protecting the vaginal wall. The ratio of anaerobic bacteria to aerobic bacteria in normal flora is 5:1, and the parthenogenetic anaerobic Lactobacillus, which can produce lactic acid and hydrogen peroxide, predominates, which maintains the vaginal pH below 4.5. In this environment, the pathogenic bacteria lose part of their activity and have poor adherence, and the local immunity in the vagina is stronger, and the microecological environment in the vagina is maintained to impede the growth of other microorganisms, which is crucial for the prevention of vaginal infections. Role. After menopause, the vaginal lactobacillus decreases, Lactobacillus is replaced by other anaerobic bacteria, the survival environment deteriorates, the number is decreasing, the vaginal mucosa lacks the protection of lactobacillus, which provides an opportunity for the further reproduction and invasion of pathogenic bacteria, leading to easy infection and morbidity.
152 Chapter 99.2.3 The Vaginal pH Value Rises Vaginal pH value reveals the acid-base situation inside the vagina, illustrates the advantages and disadvantages of the survival environment of the vaginal micro-ecology, and can be an important indicator for detecting vaginal infections. Under normal circumstances, under the joint action of sex hormones and bacteria in the body, the vaginal epithelial cells shed and release glycogen, the latter being converted into lactic acid by the probiotics in the vagina, forming and maintaining an acidic vaginal environment with a pH of 3.8~4.5, which protects the organism from inflammation and infectious diseases.Due to the lack of normal microbial communities in the vagina, the vaginal microenvironment is out of balance, resulting in abnormal vaginal secretions and an inability to effectively maintain the balance of the vaginal environment. Because the vaginal pH value changes, it will definitely destroy the vaginal defense barrier, resulting in the normal flora in the vagina to lose the ability to self-organize, so that the overall population imbalance, and the elderly menopausal women, due to the weakening of the effect of estrogen receptors in the vagina, estrogen level is low, the vaginal epithelium is thinning, the cellular detachment is reduced, and the content and activity of lactose dehydrogenase are also decreasing, resulting in the increase of the pH, the vaginal environment is gradually transformed into neutral or even alkaline. The vaginal environment gradually changes to neutral or even alkaline. The accumulation of toxic substances such as indole, pulse and amine in the vagina stimulates the overgrowth of pathogenic bacteria and the reduction of normal flora, leading to inflammation and infection, and even cancer may occur. Therefore, lowering vaginal pH can maintain the vaginal microecological environment. Combined with the factors affecting vaginal pH changes, the treatment improves the patient's vaginal environment, effectively inhibits the growth of pathogenic bacteria, and enhances the resistance of the patient's vaginal mucosa.9.2.4 Expression of Vaginal Exfoliated CellsNormal adult women's vaginal mucosa is covered by layers of squamous epithelial cells, including the base layer, the middle layer and the surface layer, with different cell morphologies, gradually maturing from the base layer to the surface layer. In a hypoestrogenic environment, there is a lack of superficial cells in the vaginal epithelium and a significant increase in the proportion of cells in the intermediate and basal layers. The lack of glycogen-rich vaginal epithelial cells leads to a decrease or disappearance of Lactobacillus, a decrease in lactic
Atrophic Vaginitis 153acid synthesis, and an increase in vaginal pH. The loss of the above protective mechanisms makes the vagina more susceptible to contamination from skin and intestinal bacteria, causing an increase in vaginal discharge and associated symptoms of infection.9.2.5 Localized Immune Deficiency The immune system of female vaginal mucosa is regulated by estrogen and progesterone at the same time, which constitutes the first line of defense of female reproductive tract. As the level of estrogen and progesterone decreases to different degrees in elderly women, the IgA antibody synthesized by the plasma cells of the vagina and cervix decreases, which leads to easy invasion and reproduction of pathogenic bacteria, causing inflammatory changes in the vagina.9.2.6 Other Influencing FactorsSmoking has a direct impact on the vaginal complex squamous epithelial cells, smoking may inhibit the biological effects of estrogen, reduce the vaginal mucosal blood perfusion, and promote vaginal atrophic changes. The level of androgens in a woman's body will also have a certain effect on the vagina, androgens in the postmenopausal body are mainly testosterone and androstenedione. Postmenopausal women with higher levels of androgens are less likely to experience vaginal atrophy and are able to maintain relatively better sexual function. In addition, differences in the mode of delivery may also affect vaginal status and correlate with the severity of genital symptoms of atrophic vaginitis, which are more severe in women who have not had a vaginal delivery than in those who have had a vaginal delivery. In recent years, with advances in molecular microecological techniques, vaginal commensal microorganisms have been found to be associated with the development and symptoms of atrophic vaginitis.9.3 Clinical ManifestationsThe most common symptom is vaginal dryness, other symptoms include vaginal burning and itching sensation, increased vaginal discharge, but thin texture, yellowish, pus and bloody leukorrhea can be seen in severe infection. Some patients are accompanied by pain during intercourse and vaginal bleeding after intercourse. Neurologically, the decrease in the number of sympathetic
154 Chapter 9nerves impairs vasodilation, leading to vaginal dryness and reduced lubrication. Hyperplasia of the nerve fibers innervating the terminal pain receptors further aggravates the pain of intercourse as the patient becomes more sensitive to pain. Decreased activity of the sebaceous glands in the vagina and decreased mucus production further reduce vaginal lubrication, causing the mucosa to become dry and bleed easily. Examination shows age-related changes in the vagina, with epithelial atrophy, loss of folds, and smooth, thin vaginal walls. The vaginal mucosa is congested with scattered small hemorrhagic spots or punctate hemorrhagic spots, and sometimes superficial ulcers are seen. If the ulcerated surface is adherent to the opposite side, if the adherent part is touched during examination it may cause separation and cause bleeding, and in severe cases it may cause vaginal stenosis or even atresia, and poor drainage of inflammatory secretions may form vaginal pus or even. In the urinary system, atrophy of urethral tissues and reduction of smooth muscle function of urethra and bladder lead to the emergence of symptoms such as urinary frequency and urgency (see Table 9-1).All of these symptoms manifest themselves in various forms and to varying degrees in different patients. Sometimes patients have atypical symptoms and may present with complaints of lower abdominal pain and discomfort. Symptoms often precede physical signs, with some patients experiencing decreased vaginal lubrication and impaired sexual arousal before gynecologic examination reveals any abnormalities. As with other menopausal syndromes, the symptoms worsen and persist with age, seriously affecting women's quality of life and sexual health. It is therefore important to recognize the early symptoms of atrophic vaginitis on an individual basis.9.4 DiagnosisThe diagnosis of atrophic vaginitis is mainly based on symptoms and signs. The diagnosis of atrophic vaginitis is not difficult and is based on whether or not the patient is menopausal, a history of ovarian surgery, pelvic radiation therapy, or pharmacologic amenorrhea, in combination with clinical manifestations and the exclusion of other diseases.Traditionally, there are two main indicators for diagnosing atrophic vaginitis and evaluating the effectiveness of treatment: vaginal pH and vaginal maturation index (VMI). In recent years, another test, patient self-assessment and complaints, has also been included as an important diagnostic and efficacy element. Patient self-assessment mainly includes six symptoms that are most
Atrophic Vaginitis 155Table 9-1 Genitourinary Tract Symptoms, Signs, and Anatomical Changes Associated with Estrogen DeficiencySite Symptoms, signs and anatomical changesVulva Loss of vulvar fat padVulvar contraction, loss of labia majora and minora boundariesShortening of the clitoral prepuce and exposure of the clitorisReduction or disappearance of pubic hairSusceptibility to chemical irritation, mechanical injury and infectionUrinary systemVaginal dryness, tingling, decreased blood supplyItching, burning sensationThinning of vaginal tissue, abnormal keratinizationMucosal lesions, including petechiae, cracked ulcers and inflammationShortening and narrowing of the vagina, loss of the fornixLoss of mucosal folds, loss of elasticityElevated vaginal pHSusceptibility to mechanical injury and slow healing, sexual dysfunctionUrinary systemReduced bladder capacity, increased residual urineDecreased maximum intravesical pressure during urinationDecreased urethral closing pressure, decreased urethral urine flowUrinary urgency, urinary incontinenceRecurrent urinary tract infections
156 Chapter 9disturbing and affect patients' quality of life, including vaginal dryness, vaginal pain, vaginal irritation, postcoital vaginal bleeding, sexual dysfunction, and dyspareunia, which are categorized into three to four grades according to their severity.Atrophic vaginitis is associated with estrogen deficiency, and its main changes are reflected in the changes in the reproductive system due to estrogen deficiency. Performing a meticulous physical examination and taking vaginal secretions for morphologic testing (see Figs. 9-1, 9-2) is very important in the diagnosis of this disease. At the same time, it is necessary to exclude the associated inflammation caused by specific infections and similar signs and symptoms of vulvar skin-related lesions from the differential diagnosis, such as infectious diseases: bacterial vaginosis, vulvovaginal candidiasis, trichomoniasis vaginalis, gonorrhea, chlamydia and mycoplasma infections, syphilis infections, etc.; skin irritation: medical irritation, urinary irritation, contact dermatitis, etc.; and vulvovaginal lesions: Vesicular lichen planus, atrophic sclerosing lichen planus, mucosal aspergillosis, and so on. There are also desquamative vaginitis, vulvodynia, and pubic neuralgia. Figure 9-1 Gram-stained microscopic morphology of atrophic vaginitis-A (1000×)Notes: Flora density: no bacteria were seen, flora diversity: no bacteria were seen, dominant bacteria: no bacteria were seen, pathogens: trichomonas infection (-), fungal infection: spores (-); budding spores (-); hyphae (-), Nugent score: 4, AV score: 1.A B
Atrophic Vaginitis 157Figure 9-2 Gram-stained microscopic morphology of atrophic vaginitis-B (1000×)Notes: It’s flora density: 2+, flora diversity: 1+, dominant organisms: gram-positive cocci, pathogens: trichomonas infection (-), fungal infection: spores (-); budding spores (-); hyphae (-), Nugent score: 4, AV score: 7, it also can be diagnosed as aerobic vaginitis(AV).9.5 TreatmentThe principles of treatment are inhibition of bacterial growth, estrogen supplementation, and enhancement of vaginal resistance. Estrogen supplementation therapy is the current measure of choice for atrophic vaginitis. The primary goal of treatment is to reduce the patient's symptoms, but the patient's needs must also be taken into account. Antimicrobials are usually given to inhibit the growth of pathogenic microorganisms. In non-sexually active postmenopausal women, symptomatic relief is the mainstay, and the first line of treatment includes the use of long-acting vaginal lubricants and low-dose vaginal estrogen to improve the weakness of the vaginal mucosa and enhance vaginal epithelial chemotaxis. Improving the microecological environment can be achieved by giving microbial agents. For patients with atrophic vaginitis who are sexually active, in addition to symptomatic improvement, patients are helped to improve their quality of sexual life through dynamic follow-up of their condition and adjustment of their treatment regimen. Currently available treatment options are non-hormonal vaginal lubricants used during intercourse, long-acting vaginal moisturizers, and estrogen therapy (systemic or topical). Clinicians should be adaptable in the actual treatment process, and can flexibly choose or combine drugs according to the patient's condition.A B
158 Chapter 99.5.1 Antibacterial Drug TreatmentInhibit bacterial growth, apply antibacterial drugs locally in the vagina, and choose appropriate drugs according to the symptoms and signs and vaginal secretion examination. Atrophic vaginitis often coexist with BV, AV, TV, according to the specific circumstances of the selection of the appropriate antibacterial drugs, the choice of suppositories, creams, gels and other specialized dosage forms are more appropriate. 9.5.2 Estrogen TreatmentFor the cause, supplemental estrogen is the main treatment method for atrophic vaginitis. After menopause, the estrogen level in women's body decreases, and perimenopausal symptoms appear one after another, and supplemental treatment through estrogen is the current preferred measure for atrophic vaginitis. Estrogen alone or estrogen-progestin combination can effectively improve menopause-related symptoms, including atrophic vaginitis. When systemic hormone supplementation programs are chosen to treat the remaining menopause-associated symptoms, atrophic vaginitis symptoms are often effectively improved as well. However, there are still approximately 10 to 15 percent of women who do not experience relief of vaginal symptoms when treated with a systemic hormone supplementation regimen and require additional low-dose estrogen to be given locally in the vagina. Topical estrogen preparations can improve the signs and symptoms of atrophic vaginitis that do not respond to nonhormonal therapy. Studies have shown that topical estrogen therapy in the vagina is effective in improving the symptoms of atrophic vaginitis, as well as lowering vaginal pH, increasing the number of Lactobacillus vaginalis, improving the vaginal epithelial maturation index, and improving stress urinary incontinence and urinary tract infections.Topical estrogen therapy, such as estrogen tablets, suppositories, softgels, and creams, is preferred when only atrophic vaginitis symptoms or urinary tract symptoms are present. Options include estriol cream, estriol vaginal suppositories, conjugated estrogen ointment, protien vaginal tablets, protien vaginal soft capsules, and protien cream. The methods of use are similar, all are once daily vaginal medication. Gradually reduce the number of times of use after a week, the maintenance amount is generally once every 1~2 weeks. Such as estriol cream, vaginal administration, 1mg / day, a total of 1 week, 1 week after the estriol cream to 1mg each time, 2 times a week, and then can continue to reduce the amount according to the specific circumstances, once a week or
Atrophic Vaginitis 159once every two weeks. Estriol promotes the growth of vaginal mucosa without stimulating the endometrium, so it is indicated for the treatment of symptoms of genitourinary atrophy caused by estrogen deficiency. Or combined with estrogen ointment topical application, 1~2 times a day for 14 days, and then reduce the amount, once a week or once every two weeks can be. However, it should be noted that the combination of estrogen ointment topical application will also be absorbed into the bloodstream, the advantage is that it also has the effect of alleviating the systemic symptoms of menopause, but long-term use of the endometrium needs to be monitored, and the need to combine with progesterone, if necessary. Prostaglandins are strictly topical estrogens with minimal absorption into the bloodstream.Some women still have concerns about the safety of vaginal estrogen therapy. In general, low-dose topical vaginal estrogen is considered to have fewer side effects and risks than systemic estrogen because the body's blood levels of estrogen are very low in the case of topical vaginal administration. It has also been shown that the use of low-dose vaginal estrogen does not increase the risk of venous thromboembolism. The chances of developing endometrial hyperplasia or endometrial cancer are also low when serum estradiol levels are kept at normal postmenopausal levels. Although vaginal estrogen therapy is usually safe for most postmenopausal women with localized symptoms, the lack of prospective, large-sample randomized controlled trials suggests that estrogen preparations should be used with caution in some high-risk groups such as patients with undiagnosed vaginal or uterine bleeding and those with estrogen-dependent tumors, with breast cancer, or with endometrial cancer.For patients who also need systemic hormone supplementation therapy, systemic estrogen-progestin therapy is given after excluding contraindications. According to the specific situation, individualized selection of drug plan.9.5.3 Antibacterial Drugs Combined with Estrogen TreatmentAt present, the more commonly used clinical method is the local application of antibacterial drugs plus a small dose of estrogen, such as chloroquinaldolprostene (chloroquinaldol is a broad-spectrum bactericidal agent), long-term use of small doses of local estrogen to maintain the improvement of symptoms. The purpose is not only to inhibit the growth of pathogenic bacteria, but also to enhance the resistance of the vaginal mucosa and reduce the possibility of reoccurrence.
160 Chapter 99.5.4 Biological Agent Treatment In order to improve the vaginal environment and make it reach the healthy standard, it can be realized by supplementing the normal microbial flora, and this method is also a direction of gradual deepening in the clinic. Lactobacillus vaginal preparation with active Lactobacillus as the main ingredient, by supplementing and propagating the normal flora in the patient's body, ensuring the restoration of the integrity and functionality of the vaginal flora barrier, reducing the adhesion of pathogenic bacteria to the epithelium of the vaginal mucous membrane, and maintaining a normal cleanliness and a good micro-ecological balance within the vagina, is currently considered by domestic researchers and scholars to be used in the treatment of atrophic vaginitis and, compared with the traditional single-use antimicrobial drug treatment, has a lower recurrence rate. It has a lower recurrence rate.9.5.5 Chinese Medicine TreatmentInternal Treatment Chinese medicine soup is an important means used in clinical Chinese medicine for the treatment of senile vaginitis. Commonly used Chinese medicines include turtle shells, cypress, hyssop, geoderma lucidum, cornelian cherry, yam, shengdi, mulberry parasites and licorice, etc. Commonly used formulas include Zhi Bai Di Huang Pills and Zi Yin Gu Chong Soup, among which Zi Yin Gu Chong Soup is one of the most commonly used traditional Chinese medicines soups. Internal dosage forms mainly include soup, pill and powder. In addition, Zhi Bai Di Huang Pills is also one of the internal treatments for the disease, which is a derivative of Liu Wei Di Huang Pills and has the effects of reducing fire, nourishing yin and clearing heat.External TreatmentExternal therapy is an important means of treatment for atrophic vaginitis with significant efficacy and high safety, including douching, fumigation and vaginal medication. Among them, Chinese medicine fumigation is the first treatment method, which includes Poria cocos, fresh eucalyptus leaves, whole herb of Cnidium, fresh peach leaves, whole herb of Cocklebur, Cranesbill buds, ice tablets, and Sichuan peppercorns, which can nourish yin and nourish blood as well as clear away heat and dampness. In addition, vaginal suppositories also belong to the Chinese medicine clinical treatment of atrophic vaginitis is one of the important means, through the mechanism of supporting the positive and
Atrophic Vaginitis 161expel the evil, regulate the immune system can play a good swelling and pain relief, anti-infection, maintenance of the vaginal micro-ecology, repair of the damaged mucous membrane and other effects.9.5.6 Other TherapiesLaser can activate the repair function of the organism, and laser treatment can promote the healing and repair of damaged tissues. Some studies show that symptoms of atrophic vaginitis, such as burning sensation, dryness, itching sensation and difficulty in sexual intercourse in postmenopausal patients have been significantly improved after receiving laser treatment. However, as this treatment has only just begun to be used in clinical practice, its clinical efficacy and long-term complications and effects need to be further evaluated, and more studies and data are needed to further evaluate this new treatment option. In addition, microwave can promote the absorption of drugs, microwave therapy is an effective adjunctive method for the treatment of this disease. Nanosilver is a new type of antimicrobial material, which can kill the pathogens inducing vaginitis. Nanosilver, the active ingredient of nanosilver ointment, has extremely strong broad-spectrum anti-microbial properties, which plays a better role in killing pathogenic microorganisms by blocking the microbial respiratory enzyme system and eliminating its activity. The treatment of atrophic vaginitis with nano-silver antimicrobial gel has been reported to be more effective and safer than metronidazole plus combined with estrogen treatment.9.6 Integrated Care InterventionsComprehensive nursing care includes psychological care, dietary care and vaginal care. Nursing intervention is a necessary supplement to the disease treatment of patients with atrophic vaginitis, and is of great significance to patients' disease recovery and physical and mental health.9.6.1 Psychological CareIf the physical and mental health of certain patients is seriously affected because of the disease, it may affect the patients' active cooperation with the doctor for treatment, so the treatment effect is not obvious. If the patient is depressed for a long time, it will not only affect the therapeutic effect, but also bring other problems in psychology, greatly affecting the physical and mental health of the patient, so it is necessary to take effective nursing methods. Nursing staff
162 Chapter 9should timely and actively communicate with the patient, observe the patient's mood, psychological state of change, find the reasons for the patient's bad mood, take targeted methods, channeling the patient's negative emotions, at the same time, actively allow the patient's family to participate in the cooperation, good communication, so that the patient better cooperate with the treatment and care.9.6.2 Dietary CareDuring the treatment period, focus on giving patients a variety of nutritional supplements to enhance the body's resistance. Supplement foods with high protein content, such as milk, lean meat, etc.; prohibit stimulating foods, such as too greasy, too cold and spicy foods; eat more fresh fruits and vegetables, and consume more light and easy-to-digest foods, to better replenish the nutrients in the patient's body; at the same time, the patient should take the habit of eating less and more often, and should not overeat.9.6.3 Vaginal CarePatients with atrophic vaginitis should be Informed of the importance of vaginal care and suggest sitting in the basin.9.7 PreventionWith the global increase in human life expectancy, especially for women, which is significantly higher than that of men, one-third of most women's lives will be spent after menopause. Menopause marks the physiological decline of ovarian function, depletion of primordial follicles, decreased secretion of steroid hormones, functional deterioration of target tissues, and finally a series of clinical symptoms, including severe psychological and physical changes, the current situation of which has attracted the attention of the World Health Organization. Reproductive health care for postmenopausal women should be actively carried out, good health behavior strategies should be formulated, individualized principles of medical interventions should be clarified, safer, more effective and controllable treatments should be introduced, and publicity and education on popular science should be strengthened to ensure that the goal of reproductive health for older women is successfully achieved. However, atrophic vaginitis, as a common and frequent disease among postmenopausal women, is often characterized by recurrent episodes, so there are still more difficulties and questions about its effective prevention and control.
Atrophic Vaginitis 163First of all, patients should pay attention to the washing and cleaning of the vulva, and should not use scalding water to wash to relieve the itching because of vulvar itching, which will cause the dryness of the vulvar skin and aggravate the degree of itching. If the patient occurs vulvar itching, should use weak acid formula of professional vulvar care solution to wash the vulvar area; change underwear diligently, wear loose, cotton comfortable underwear and pants, avoid wearing tight-fitting underwear and pants; discomfort symptoms should be diagnosed and treated in a timely manner, and can not be used indiscriminately on their own medication. The causative agent of atrophic vaginitis is not exactly the same as that of women of childbearing age, mainly Staphylococcus, E. coli, etc., and the wrong medication will not only be ineffective, but also counterproductive.Postmenopausal women should pay more attention to personal hygiene, usually do not use soap, shower gel, liquid soap, etc. to wash the vulva, to avoid causing dryness and atrophy of the vulvar skin, damage to the vulvar skin, resulting in the invasion of bacteria; selection of qualified manufacturers of toilet paper; underpants cleaned and often light sun or ironing; do not share with others washing basin, towels, etc.; underpants and socks and other clothing should be separated from the washing to ensure that all hygiene and cleanliness, to reduce the chance of bacteria invasion. Reduce the chance of bacterial invasion. Postmenopausal women's vaginal mucosa is thin, vaginal tissues lack of elasticity, sexual life may cause vaginal damage and bacterial infection, so appropriate to reduce sexual life or use lubricants during sex to reduce the damage to the vagina. Supplement estrogen when necessary, either locally or systemically. In terms of diet, pay attention to the balanced and sufficient intake of various nutrients, and use vitamins and other supplements when necessary. It is advisable to consume light and nutritious food, such as milk, beans, fish, vegetables, fruits, etc.; it is advisable to consume grains and miscellaneous cereals, such as round-grained rice, glutinous rice, black sesame seeds, black soybeans, etc.; it is advisable to consume food that replenishes the spleen and kidneys, such as mussels, walnuts kernels, animal livers, eggs, etc.; and it is advisable to consume less or avoid eating spicy, sweet, and stimulating food, such as chocolates, chili peppers, and seafood, etc.9.8 Re-Recognition of Vaginal Microecology in Menopausal WomenIn recent years, with the continuous advancement and development of the human metagenome project and molecular microecology technology, the study
164 Chapter 9of vaginal microbial communities has also gradually received attention. The potential role and value of commensal microorganisms in maintaining women's vaginal health has also begun to be emphasized. The symbiotic microbial community of the female reproductive tract colonizes the mucosal epithelium in an orderly manner, forming a biological barrier that protects women against the adhesion and colonization of foreign pathogenic bacteria, restricts the overgrowth of various conditionally pathogenic bacteria, and plays a very important role in maintaining the balance of the microecological environment in the vagina. The vaginal microenvironment controls the flora, and the flora also has an important influence on the vaginal microenvironment. Once this equilibrium is disrupted, the health of the organism will be threatened, and the risk of bacterial vaginosis, gonorrhea, chlamydia, vulvovaginal pseudohyphae, and HIV invasion will then increase. Traditional theory suggests that the normal symbiotic flora of healthy women of childbearing age is mainly Lactobacillus, which can decompose glycogen to produce lactic acid, making the vagina an acidic environment of pH 3.8~4.5 and inhibiting the invasion of foreign pathogens. In older women, vaginal atrophy and reduced glycogen content in the epithelium lead to a lack of Lactobacillus, the vaginal pH rises and the ability to kill pathogenic bacteria decreases, making it easier to develop infectious diseases of the reproductive tract, which are not easy to cure.The female vagina is not a sterile environment, but there are a large number of symbiotic microorganisms. In recent years, with the launch of the Human Metagenome Project, researchers working on reproductive health have turned their attention to the study of symbiotic microorganisms in the human body. Currently, the new view of the human body is that the human body is a “super organism” composed of symbiotic microorganisms, and the sum of the genetic information of the symbiotic microorganisms is called the metagenome, which is also known as the “second genome” of the human body. The number of symbiotic microorganisms in the human body is about 10 times the number of human cells, the number of genes for the human body is about 100 times, the microbiota in the body to assist in digestion, maintain normal metabolism, maintain the body's immune balance, as well as to prevent the invasion of pathogens, to maintain the normal physiological function of the human body plays an important role. Female vaginal symbiotic microorganisms colonize the mucosal epithelium in an orderly manner, constituting a biofilm-like community structure, forming an effective biological barrier that prevents the adhesion and colonization of foreign pathogenic bacteria, restricts the overgrowth of various
Atrophic Vaginitis 165conditionally pathogenic bacteria, and plays a very important role in maintaining a balanced micro-ecological environment in the lower genital tract. Once the microecological status of the vagina is imbalanced or damaged, the risk of bacterial vaginosis, vulvovaginal pseudomycosis, gonorrhea and other sexually transmitted diseases, as well as HIV invasion, will be significantly increased.References[1] Xing Xie, Beihua Kong, Tao Duan. Obstetrics and Gynecology Ninth Edition . Beijing: People's Health Publishing House, 2018.7.[2] Shirong Li. Research progress of drug therapy for senile vaginitis. Journal of Practical Gynecological Endocrinology. 2019, 6(4): 16.[3] Wang X. Discussion on the application effect of metronidazole combined with Lactobacillus and estriol in the treatment of geriatric vaginitis. China Practical Medicine,2018,13(27):94-95.[4] Schiavi M C, Sciuga V, Giannini A, et al. Overactive bladder syndrome treatment with ospemifene in menopausal patients with vulvovaginal atrophy. Improvement of sexuality? Gynecological Endocrinology, 2018, 34(8):1-4. [5] Salvatore S, Katlein França, Lotti T, et al. Early Regenerative Modifications of Human Postmenopausal Atrophic Vaginal Mucosa Following Fractional CO2 Laser Treatment. Macedonian Journal of Medical Sciences, 2018, 6(1 Global Dermatology 2):6-14.
C H A P T E R - 10Cytolytic VaginitisZhengqiang HuTranslator: Yunxia Li10. 1Lactobacillus species is the most important member of the normal vaginal flora. In women of childbearing age, it is the dominant bacteria of normal vaginal microecosystem. By producing lactic acid, decreasing vaginal pH value, and competing for adhesion, Lactobacillus species produce H2O2 and other inhibitory substances such as lactocin, stimulating the immune system to inhibit overgrowth of Escherichia coli, Candida albicans, Gardnerella vaginitis and Mobiluncus, maintaining vaginal microecological balance, so as to inhibit the happening of vaginal inflammation. Because of the overgrowth of lactobacilli, some women of childbearing age have the phenomenon of dissolution and rupture of vaginal epithelial cells, which lead to clinical symptoms similar to vulva-vaginal candidiasis (VVC), such as vulva itching, burning sensation, a large number of white vaginal discharge. A large number of lactobacilli, naked nucleus and epithelial cell debris can be found under microscopic observation of vaginal discharge, which is known as cytolytic vaginosis (CV) . CV is a common ‘disease’ of the female genital tract. It is characterized by overgrowth of lactobacilli and lysis of squamous epithelial cells, including the presence of cytoplasmic debris and an intact cellular naked nucleus. However, as people do not have sufficient understanding of this disease, and its symptoms are very similar to VVC, CV is often ignored or misdiagnosed in clinical diagnosis and treatment, so that many patients can not be released from the pain of the disease as soon as possible.10.1 The Origin of the Name of CVCV used to be named Doderlein's cytolysis. Due to the special anatomical and etiological characteristics of the vagina, it has always attracted the attention of many microbiological experts. In May 1891, Doderlein read a paper named "About vaginal discharge and vaginal bacteria" at the fourth German Obstetrics Congress in Bonn, and later known as Doderlein Bacillus, the current name
Cytolytic Vaginitis 167Lactobacillus species. In his paper, Doderlein correctly described his long, large gram-positive bacilli. He believes that these bacilli are bacteria present in normal vaginal discharge and are often detected from the vagina at the same time with yeast (39%). He found that the bacteria was antagonistic to Staphylococcus growth, and noted that the bactericidal effect of vaginal discharge was due to lactic acid produced by the bacterium.Jotten (1922) once named Lactobacillus as Lactobacillus vaginalis, which was confirmed by Thomas (1928). According to the description of bacterial culture and biological characteristics by Doderlein and the research of later scholars, it believed that this bacterium is the present Lactobacillus acidophilus (L. acidophilus). This bacterium will decompose the glycogen contained in the vaginal epithelial cells and produce lactic acid products. Due to the accumulation of these lactic acid, the vagina will be acidic (pH3.5~4.0). This acidic environment makes most pathogenic bacteria unable to reproduce and grow, so women can avoid vaginal infections. While the bacilli proposed by Doderlein is actually a mixture of 7-8 types of vaginal Lactobacillus species, including L. acidophilus. Therefore, in order to avoid the misunderstanding of the disease caused by its naming, in 1991, Cibley L, et al. named the phenomenon of rupture of vaginal epithelial cells and naked nucleus as cytolytic vaginosis (CV).10.2 Pathology and Clinical Manifestations10.2.1 The Causes of CVCV is a common disease. Previously, it is known as "Lactobacillus hyperplasia syndrome" or "Doderlein cytolysis" because of the typical epithelial cell rupture and lactobacilli digestion activities. It is currently widely recognized because of lactobacilli overgrowth. A large number of lactobacilli growth and reproduce and decompose glycogen contained by vaginal epithelial cells, producing a lot of lactic acid, and causing epithelial cells dissolved rupture, resulting vulva itching, pain, sexual intercourse, urination vulva discomfort and other clinical symptoms.10.2.2 The Pathogenesis of CVFemale vagina is a complex microecosystem, which is a dynamic equilibrium system composed of multiple microorganisms. The composition of vaginal flora will change during pre-puberty, puberty, fertility, pregnancy and menopause. In addition, different menstrual cycles also affect the vaginal microecological environment. Lactobacilli is the dominant bacteria of normal vaginal flora of reproductive age women. By producing lactic acid, hydrogen peroxide
168 Chapter 10and nutrient competition, it inhibit the growth of other bacteria. At the same time, other bacteria can also limit the growth of lactobacilli through nutrient competition, which makes the vaginal normal flora in a symbiotic antagonistic relationship, maintaining vaginal micro ecological balance. When the conditions limiting the growth of lactobacilli are reduced, such as long-term anti-fungal drugs, acidic liquid vaginal flushing, lactobacilli will overgrowth and reproduce, resulting in a large number of lactobacilli decompose vaginal epithelial cells, resulting in cytolysis.Women of childbearing age have strong estrogen secretion, and vaginal mucosa glycogen in epithelial cells are rich in storage. After shedding or disintegration, low pH value is formed by enzymes and flora, which is also conducive to the growth of lactobacilli. Although at present we could not define the mechanism by which the overgrowth of lactobacilli may lead to changes in the vaginal microenvironment, but the overgrowth of lactobacilli lead to fermentation glucose produce lactic acid, carbon dioxide, ethanol, formic acid, acetic acid and hydrogen peroxide, and too much acid stimulation will cause chronic periodic vaginal burning and itching symptoms. Further research on the pathogenesis of CV is needed.10.2.3 The Clinical Manifestations of CVVulval pain is one of the most common complaints in patients with CV. Moreover, the vulval pain in patients with CV has obvious characteristics, that is, the pain is significantly aggravated after sexual intercourse, which is generally the heaviest on the second day of sexual intercourse. Traditional drugs can only temporarily relieve the pain of patients, and then relapse immediately after drug withdrawal. In addition, patients often report vulvar pruritus pain, dyspareunia, vulvar discomfort during urination, thick or thin white cheese vaginal discharge.The clinical manifestations of patients with CV have obvious periodicity, that is, significantly aggravated in the luteal phase. In clinical examination, it will be found that: a. the patients have normal vulva or slight erythema and edema in the vulva; c. the pH value of vaginal discharge is 3.5~4.0; b. wet smear microscopic examination found the unique manifestations: the presence of a large number of lactobacilli, ruptured epithelial cells (i. e., cytolysis), cell naked nucleus and lacking of Candida species, clue cells or white blood cells. Sometimes, lactobacilli severely overgrows, covering epithelial cells and appearing "false clue cells". The culture of vaginal discharge can show normal vaginal flora or large growth of lactobacilli without Candida growing.
Cytolytic Vaginitis 16910.3 Diagnosis and Treatment of CV10.3.1 The Diagnosis of CV The diagnosis of CV is generally relatively simple and does not require complicated laboratory tests, but the key is to exclude infections from other pathogens. General diagnostic criteria include: a. typical periodic clinical manifestations; b. high clinical suspicion: vaginal discharge may be white, cheese-like, foam; pH value is 3.5-4.0; An increased number of lactobacilli was visible on the smear, often adhering to the epithelial cells, forming "false clue cells"; A small amount of white blood cells; evidence of cytolysis: bare nuclei and epithelial cell debris; lack of Trichomonas, Gardnerella, Candida (spores, yeasts with budding, and pseudohyphae). Patients with the above symptoms can generally be clearly diagnosed. The microscopic morphology of the vaginal discharge smear of CV is shown in Figure Figure 10-1.Figure 10-1 Micromorphology of cytolytic vaginosis (Gram stain, 1000×)Notes: Flora density: 3 +; flora diversity: 1 +; dominant flora: lactobacilli; trichomonas infection (-); fungal infection: spore (-); yest-like budding (-); hyphae (-); Nugent score: 0; AV score:0; visible on smear: epithelial cell debris and “naked nucleus”.10.3.2 The Antidiastole of CV The clinical symptoms of CV are similar to VVC, and attention should be paid to differentiate CV from VVC. Microscopic smear of vaginal discharge from patients with VVC showed Candida spores, yeasts with budding or
170 Chapter 10pseudohyhae; positive fungal culture with the growth of C. Albicans or non-albicans Candida species.10.3.3 The Treatment of CV CV is a simple and complex disease, and the key to treatment is to clarify the diagnosis and symptomatic treatment. For most people, it can be cured with low-cost treatment, and there is no danger to patients.The treatment of CV is local irrigation with sodium bicarbonate or sitz bath to improve the pH value of the vagina. Irrigation solution can be made by adding 30~60g sodium bicarbonate to 1 liter of heating water, 2~3 times a week. The patients with CV can also be treated according to the need of 1~2 times a week, 2 weeks for a course of treatment. These patients can be washed 24 to 48 hours before symptoms under the guidance of a doctor. The clinical symptoms of patients with CV are very similar to those of vulvovaginal candidiasis. There are pruritus vulvae, sexual intercourse discomfort, white cheese vaginal discharge and other symptoms in patients with CV and VVC. If without careful microscopic examination, it is easy to misdiagnose only with clinical symptoms, so doctors should pay enough attention to CV.10.4 SummaryCV is currently a disease with unclear pathogenesis. The clinical symptoms of CV are obvious, and there is a certain morbidity. Although it does little harm to women's physical health, it may cause unnecessary mental burden and injury due to clinical neglect and misdiagnosis. Lactobacilli is the dominant bacteria of the normal vaginal flora of women of childbearing age, which inhibits the growth and reproduction of other bacteria through the production of lactic acid, hydrogen peroxide and nutrient competition. Other bacteria can also limit the growth and reproduction of lactobacilli through nutrient competition, so that the normal vaginal flora is in a symbiotic and antagonistic relationship, and maintain the balance of vaginal microecosystem. When the conditions that limit the growth of lactobacilli are reduced, such as long-term use of antifungal drugs, acidic liquid vaginal flushing, it will overgrow and reproduce, resulting in a large number of lactobacilli decomposition of vaginal epithelial cells and cytolysis. Women of childbearing age have strong estrogen secretion, and vaginal mucosa glycogen in epithelial cells are rich in storage. After shedding or disintegration, low pH value is formed by enzymes and flora, which also favors the growth of lactobacilli. However, the pathogenesis of CV needs to be
Cytolytic Vaginitis 171further studied, including the investigation of the incidence of clinical patients, the investigation of disease correlation, the culture and identification of bacteria, the quantification of vaginal colonies, and the symbiotic relationship between bacterial communities.References[1] Chong Wang, Zhongming zeng . Progress in cytolytic vaginosis. Journal of China Maternal and Child Health Care. 2009,24(28):4048-4049.(Chinese)[2] Demirezen S. Cytolytic vaginosis: examination of 2947 vaginal smears. 2003,11(1):23-24.[3] Cibley LJ, Cibley LJ. Cytolytic vaginosis. American Journal of Obstetrics & Gynecology, 1991,165:1245-1249.[4] Shuhua Y, Ying L, Jianhong W, et al.Variation of the Vaginal Lactobacillus Microbiome in Cytolytic Vaginosis. Journal of Lower Genital Tract Disease. 2020, 24(4): 417-420.[5] Zhengqiang Hu, Wei Zhou, Liyuan Mu, et al. Identification of Cytolytic Vaginosis Versus Vulvovaginal Candidiasis. Journal of Lower Genital Tract Disease. 2015, 19 (2): 152-155.
C H A P T E R - 11Mixed VaginitisMeng chen, Hongwei LiuTranslator: Yiduo Zhang11. 1The mixed vaginitis refers to an inflammatory condition of the vagina caused by the invasion of multiple pathogens, such as fungi, bacteria, and Trichomonas vaginalis. Due to the presence of multiple pathogenic organisms, the clinical manifestations and laboratory characteristics of mixed vaginitis are more complex and diverse, which poses certain challenges for clinical diagnosis and treatment. Additionally, mixed vaginitis may also be associated with infections by other pathogens in the vulva or cervix, including herpes simplex virus, Chlamydia trachomatis, Neisseria gonorrhoeae, and human papillomavirus. The presence of these pathogens undoubtedly adds further complications to the diagnosis and management of mixed vaginitis. In recent years, mixed vaginitis has received increasing attention; however, research on the ecological characteristics of mixed vaginitis remains relatively limited.11.1 EpidemiologyThe reported incidence of mixed vaginitis varies in the literature, fluctuating approximately between 7.33% and 41.87%. The discrepancies in incidence rates across different studies are attributed not only to the biological exposure rates of the studied populations but also to researchers' understanding of vaginitis and the experimental techniques and conditions employed. For example, aerobic vaginitis (AV) was introduced as a concept in 2002, and it has been less than 20 years since its recognition. Many studies' insufficient understanding of AV may lead to missed diagnoses. Some laboratories may not conduct comprehensive testing, leading to the misclassification of mixed infections as simple vaginitis. Additionally, the knowledge level of laboratory personnel regarding mixed vaginitis, as well as the experimental techniques and equipment conditions, may result in the detection of only one pathogenic organism, thus overlooking the possibility of two or more mixed infections. Overall, in clinical practice, infections caused by multiple pathogens are more common than those caused by
Mixed Vaginitis 173a single pathogen. Therefore, clinicians and laboratory technicians should fully consider the potential for mixed infections when assessing patients, striving for accurate and comprehensive diagnoses.Mixed vaginitis often begins with an early manifestation of vaginal microecological imbalance, followed by the infection of a primary pathogen, which then allows for subsequent infections by secondary or tertiary pathogens. Cases of simultaneous infection by two or more pathogens are relatively rare; if such instances occur, factors such as diabetes and immunocompromised states should also be considered. The pathogens associated with mixed vaginitis are diverse, potentially comprising combinations of bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), aerobic vaginitis (AV), and trichomoniasis vaginalis (TV). Generally, the incidence of pathogenic combinations in mixed vaginitis correlates with the incidence of simple vaginitis. Based on the incidence of simple vaginal infections, common combinations in mixed vaginitis include bacterial vaginosis with vulvovaginal candidiasis, aerobic vaginitis with vulvovaginal candidiasis, vulvovaginal candidiasis with trichomoniasis, and bacterial vaginosis with trichomoniasis.11.2 Key Points in Clinical Diagnosis and TreatmentDue to the characteristics of having two or more types of vaginitis, mixed vaginitis presents a diverse range of clinical manifestations. Patients may exhibit symptoms primarily related to one type of vaginitis or may display symptoms from multiple types, or even atypical clinical presentations. In summary, the clinical manifestations predominantly include abnormal vaginal discharge and/or vulvovaginal itching. Because of the variety of clinical presentations associated with mixed vaginitis, the rates of missed and misdiagnoses based solely on clinical experience are high. Clinical practitioners must improve their understanding of mixed vaginitis and should not rely solely on experience to determine testing parameters or diagnose a specific type of vaginitis for treatment. The diagnosis of mixed vaginitis relies heavily on laboratory evidence as well as clinical signs. To accurately diagnose the various forms of vaginitis and reduce the likelihood of missed diagnoses of mixed vaginitis, examinations of vaginal microecology are essential. Accurate and timely assessments of microecology not only aid in diagnosis but also provide guidance for subsequent treatment and prognosis. Diagnosis of mixed vaginitis requires (1) the presence of at least two pathogens or the fulfillment of diagnostic criteria for two or more types of vaginitis, and (2) the concurrent presence of symptoms and signs
174 Chapter 11corresponding to two or more types of vaginitis that necessitate combined pharmacological treatment. Additionally, examinations of mixed vaginitis may reveal poorer cleanliness, higher vaginal pH, and reduced lactobacilli.Currently, there is no standardized treatment or follow-up plan for mixed vaginitis, both domestically and internationally. Treatment needs to be highly individualized, placing high demands on clinicians. In principle, treatment should reference protocols for each type of simple vaginitis. Different combinations of pathogens warrant the selection of various combined pharmacological therapies. Efforts should be made to minimize unnecessary use of antimicrobial agents to reduce drug toxicity and side effects, as well as to prevent increased rates of drug resistance. While targeting specific pathogens with appropriate treatments, attention should also be given to the results of vaginal microecological assessments, prompting timely use of vaginal microecological modulators to correct imbalances. If mixed infections are not treated promptly, there is a risk of treatment failure and recurrent infections. Due to the inherent complexity of pathogen infections in mixed vaginitis compared to simple vaginitis, along with a more severe imbalance of vaginal microecology, relapse rates are higher. As such, treatment durations for mixed vaginitis are generally longer, and cure standards should not solely rely on the absence of pathogens; instead, the establishment and restoration of vaginal microecological homeostasis should also be emphasized to minimize recurrence. Moreover, alongside pharmacological treatment for mixed vaginitis, management of sexual partners, emphasizing follow-up during treatment, and establishing a comprehensive treatment plan are essential.The following section will focus on one of the most common forms of mixed vaginitis: bacterial vaginosis combined with vulvovaginal candidiasis.11.3 Bacterial Vaginosis Combined with Vulvovaginal Candidiasis11.3.1 Clinical ManifestationsEarly-stage bacterial vaginosis combined with vulvovaginal candidiasis may be asymptomatic. As the vaginal microecological imbalance progresses, symptoms may gradually appear, primarily manifesting as abnormal vaginal discharge and/or vulvovaginal itching. Increased vaginal discharge is a common characteristic shared by both BV and VVC; however, the nature of the discharge may exhibit features of both conditions, such as cottage cheese-like discharge accompanied by a foul or fishy odor, or watery discharge. Vulvovaginal itching is a common result of irritation from the discharge of both BV and VVC. Although congestion
Mixed Vaginitis 175of the vaginal or vulvar mucosa is rare in isolated BV cases, VVC can lead to congestion, redness, and even ulceration of the mucosa. Therefore, under the combined influence of both conditions, clinical presentations of congestion and mucosal inflammation may be observed. Analyzing the clinical manifestations based solely on itching, congestion, and characteristics of discharge may often overlook BV, leading to missed diagnoses of mixed vaginitis.11.3.2 Laboratory TestingLaboratory testing focuses on vaginal microecology assessment, including microscopic examination of vaginal discharge, pH measurement, and various biochemical indicators. This also includes the amine test and, when necessary, culture of the discharge.Vaginal Microecological Assessmenta. Microscopic examination of vaginal discharge: This method allows for direct observation and diagnosis of the pathogenic bacteria involved in vaginitis. After smearing, wet mount or Gram stain microscopy can be performed. The presence of clue cells and hyphae or budding yeast can aid in the diagnosis of BV and VVC. Wet mount examination for VVC pathogens requires 10% KOH, with positive rates slightly lower than that of Gram staining. Microscopic examination also helps determine the cleanliness of vaginal discharge and the white blood cell count. Typically, in isolated BV cases, the white blood cell count is low; however, if BV is diagnosed but the microscopy shows an increased white blood cell count, mixed infection, including cervical or uterine infections, should be considered.b. Vaginal pH value testing: Normal vaginal pH ranges from 3.8 to 4.5, where an acidic environment is more favorable for the proliferation of lactobacilli. When BV and VVC occur concurrently, the balance of vaginal microecology is disrupted, leading to an inability to maintain normal pH levels, which generally results in increased vaginal pH. Elevated pH levels favor the growth of anaerobic bacteria such as Gardnerella, further exacerbating the disruption of vaginal microecology, leading to a vicious cycle that complicates and makes mixed vaginitis more challenging to treat.c. Functional testing: Relevant functional indicators associated with BV and VVC include: hydrogen peroxide levels, leukocyte esterase, sialidase, and N-acetylglucosaminidase. Hydrogen peroxide concentration indicates whether lactobacilli function is normal; generally, BV is hydrogen peroxide positive and sialidase positive. In VVC cases, certain aspartic proteases and
176 Chapter 11N-acetylglucosaminidase may be positive. In isolated BV, leukocyte esterase is negative, but in mixed vaginitis, a positive leukocyte esterase indicates a high presence of white blood cells performing anti-infection functions; thus, leukocyte esterase levels can reflect the severity of inflammation.Amine Test When 10% KOH is added to vaginal discharge, a characteristic foul "fishy" or ammonia odor is released, indicating a positive amine test. This test holds significant diagnostic value for BV; however, its sensitivity is low, and a negative result does not exclude BV. This test is considered the most specific yet least sensitive indicator for diagnosing BV.Culture of Vaginal DischargeCulture takes longer than microscopic examination, which can delay early diagnosis and treatment of vaginitis; therefore, its routine clinical use is rare. However, for recurrent or difficult-to-treat cases of vaginitis, culture may be recommended to clarify the nature of the pathogens, and sensitivity testing should be performed alongside culture to facilitate targeted treatment. Generally, culture is not conducted for BV, while it is reserved for difficult cases of recurrent VVC.11.3.3 DiagnosisIn general, diagnosing bacterial vaginosis combined with vulvovaginal candidiasis is straightforward when clinical symptoms and/or signs of vaginitis are present alongside laboratory evidence. The diagnosis primarily relies on laboratory indicators; mixed vaginitis must fulfill the diagnostic criteria for at least two forms of vaginitis.VVC Positive identification of pseudohyphae or yeast buddings during microscopic examination of vaginal discharge is sufficient for diagnosis. Any of the following methods may be used: (1) 10% KOH wet mount microscopy; (2) Smear method: Gram staining microscopy; or (3) Culture method, accompanied by drug-sensitivity testing.BVDiagnosis of BV requires meeting at least one of the following criteria: • Amsel criteria: meeting three out of four criteria is sufficient for diagnosis (see Chapter 4, 4.5);
Mixed Vaginitis 177• Nugent score of ≥7; • Hay/Ison criteria: microscopy results are grade III or, if grade II, vaginal pH > 4.5.11.3.4 TreatmentThe treatment for BV combined with VVC necessitates individualized and combined pharmacotherapy due to the presence of multiple pathogens. Treatment should focus on covering the relevant pathogens and maintaining vaginal microecological balance. However, even with these considerations, the treatment duration and cure rates for mixed vaginitis remain lower than for simple vaginitis, and the likelihood of recurrence is higher.Pathogen Treatment For VVC + BV, both antifungal agents and nitroimidazole medications should be administered concurrently. Common treatment regimens include topical combined use of antifungal agents and nitroimidazole medications, oral combined use of both antifungal agents and nitroimidazole medications, and combinations such as oral nitroimidazole plus topical antifungal or oral antifungal plus topical nitroimidazole. Local formulations containing both classes of medications, such as those containing both metronidazole and clotrimazole, may also be utilized (for specific regimens, refer to Chapter 4, 4.6, and Chapter 5, 5.5).Vaginal Microecological RegulationIn addition to targeting pathogens, correcting disruptions in vaginal microecology is crucial for shortening treatment duration, improving cure rates, and reducing recurrence rates. Furthermore, while antimicrobial agents are used to eradicate pathogens, they may also disrupt the vaginal microenvironment and worsen microecological imbalances; thus, the use of lactobacilli preparations can help restore vaginal microecological balance. Specific medications may include lactobacilli-based microecological preparations and traditional Chinese medicine(TCM).Interpretation of CureClinical cure is defined as the absence of symptoms, positive signs, and pathogens in patients. However, both BV and VVC are prone to recurrence, and the recurrence rate is even higher when both conditions are present together. While current clinical cure standards do not include vaginal microecological
178 Chapter 11indicators as evaluation criteria, the normalcy of vaginal microecological indicators during laboratory assessments is often closely related to the recurrence of vaginitis. Therefore, when evaluating cures, attention should be paid to the vaginal microecological indicators, and timely supplementation with microecological preparations should be administered to maintain stability and reduce recurrence.Identifying and Managing Potential Triggers The balance of vaginal microecology is influenced by various factors, including female hormone levels, menstruation, sexual activity, contraceptive methods, hygiene habits, and various medical conditions such as diabetes, post-kidney transplantation, and cancer treatments. Consequently, while actively treating mixed vaginitis and adjusting vaginal microecological balance, attention should also be given to potential triggers that may disrupt vaginal microecology. Maintaining a healthy lifestyle, enhancing the body's immunity, avoiding habits that may negatively impact vaginal microecology, and reducing the likelihood of mixed vaginitis recurrence are essential.11.3.5 Follow-Up Currently, there are no specific follow-up standards for BV+VVC. It is recommended to refer to the follow-up requirements for VVC. Follow-up should occur 7 to 14 days after treatment and after the next menstrual cycle. For patients with recurrent VVC + BV, follow-up times should be increased, typically at 7 to 14 days after treatment, and then once after one month, three months, and six months post-menstruation.References[1] Paladine HL, Desai UA. Vaginitis: Diagnosis and Treatment. Am Fam Physician, 2018, 97(5): 321-329.[2] Neal CM, Kus LH, Eckert LO, Peipert JF. Vulvovaginitis: Screening for and Management of Trichomoniasis, Vulvovaginal Candidiasis, and Bacterial Vaginosis. Am J Obstet Gynecol. 2020 Feb;222(2):114-122.[3] Zhan Zhang and Zhaohui Liu.. Mixed vaginitis and vaginal microecology. Chinese Journal of Practical Gynecology and Obstetrics, 2020,36(2):185-189.[4] Collaborative Group on Infectious Diseases of the Chinese Society of Obstetrics and Gynecology. Revised guidelines for diagnosis and treatment
Mixed Vaginitis 179of vulvovaginal candidiasis (VVC). Chinese Journal of Practical Gynecology and Obstetrics, 2012,28(6):401-402. [5] Collaborative Group on Infectious Diseases of the Chinese Society of Obstetrics and Gynecology. Guidelines for diagnosis and treatment of bacterial vaginosis (2021 revised edition). Chinese Journal of Obstetrics and Gynecology, 2021,56(1):3-6. [6] Chen Wang, Huihui Wang, Huanrong Li, et al. Interpretation of the 2018 European Association for the Control of Sexually Transmitted Diseases/World Health Organization Guidelines on the Management of Vaginal Discharge (Vaginitis). Chinese Journal of Practical Gynecology and Obstetrics, 2018,12:1360-1365.[7] Collaborative Group on Infectious Diseases of the Chinese Society of Obstetrics and Gynecology. Expert consensus on clinical application of vaginal microbiota evaluation. Chinese Journal of Obstetrics and Gynecology, 2016,51(10):721-723. [8] Collaborative Group on Infectious Diseases of the Chinese Society of Obstetrics and Gynecology. Expert Consensus on Diagnosis and Treatment of Mixed Vaginitis (2021 edition). Chinese Journal of Obstetrics and Gynecology,2021, 56 (1):15-18.
C H A P T E R - 12Vaginal Microbiota InhibitionYuanting TangTranslator: Yiduo Zhang12. 1According to the "Expert Consensus on the Clinical Application of Vaginal Microecological Evaluation (2016)", vaginal microbiota inhibition refers to a significant reduction of bacteria in vaginal discharge. This condition is characterized by the absence of dominant bacteria or the presence of only a small number of bacteria, with a flora density of ≤ I grade, meaning that there are an average of 1 to 9 bacteria per oil immersion field or no bacteria observed at all. The flora diversity is also ≤ I grade, indicating that only 1 to 3 types of bacteria can be identified under the microscope, or no bacteria are observed at all. Women experiencing microbiota inhibition have a lower quantity of beneficial bacteria in the vaginal microbiota, making them more susceptible to pathogenic microorganisms and increasing the risk of developing other types of vaginitis.12.1 Causes of Vaginal Microbiota InhibitionThe urinary and reproductive tracts in women are distinct pathways, whereas in men, they are a common channel. Therefore, there are significant differences in the urinary and reproductive tract microbiota between men and women. With the advancements in science and technology, recent analyses using 16S rDNA gene amplification sequencing, real-time quantitative PCR, and traditional microbial culture methods have been conducted on samples from different sites of the reproductive tract in healthy women of childbearing age, including the lower third of the vagina, the posterior fornix, the cervical canal, the uterine cavity, the fallopian tubes, and pelvic fluid. These studies investigated the distribution of microbiota in the pelvic and upper reproductive tracts in women and their association with reproductive system diseases. The findings challenge the traditional notion that "the pelvis and upper reproductive tract are sterile environments", revealing that the normal pelvic and upper reproductive tracts in women also harbor microorganisms. This indicates a certain continuity in the microbial structure from the vagina through the cervical canal, uterine cavity,
Vaginal Microbiota Inhibition 181fallopian tubes, and into the pelvis, highlighting the significant association between the pelvic and female reproductive tract microecological environment and the health of the reproductive system as well as related diseases.The vagina is not a closed system, nor is it strictly an anaerobic environment; rather, it contains a large number of both strict and facultative anaerobes. This is due to the coexistence of various bacteria within the vagina, where aerobic bacteria consume a significant amount of oxygen during metabolism, creating favorable conditions for the growth and reproduction of strict and facultative anaerobic bacteria. Although there are numerous microorganisms in the vagina, the dominant bacteria in the vagina of most healthy women of childbearing age are lactobacilli. Under the influence of estrogen, vaginal epithelial cells rapidly produce glycogen, which is then broken down into lactic acid by lactic acid-producing bacteria (mostly Lactobacillus species) in the vagina, resulting in an acidic environment. This acidic condition is conducive to the growth of lactobacilli while inhibiting the growth of non-acidophilic microorganisms, thereby forming a positive feedback loop that promotes the dominant growth of lactobacilli. Consequently, the vaginal microbiota predominantly consists of lactobacilli.Lactobacillus are Gram-positive, non-spore-forming bacteria that can appear as slender, curved, or rod-shaped. They are facultative anaerobes, containing oxidase and peroxidase, and they ferment glucose, producing lactic acid, acetic acid, and carbon dioxide as final products. Their metabolic byproducts include bacteriocins, and some Lactobacillus can produce hydrogen peroxide (H2O2). Several types of Lactobacillus are commonly found in the vagina, including Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners. In addition to bacteria, the normal vaginal microbiota also includes fungi and mycoplasma. Under normal circumstances, fungi and mycoplasma do not proliferate in large quantities, thus not causing disease. However, if certain factors lead to a decrease in lactobacilli, resulting in changes in vaginal pH and a loss of inhibitory effects on fungi and mycoplasma, it may lead to corresponding diseases.Under normal conditions, the microbial communities in the vagina are in a balanced state, with the presence of Lactobacillus playing a crucial role in maintaining the stability of the vaginal environment for the host. However, when certain factors disrupt the vaginal environment, leading to changes in the microbial population that exceed physiological limits, the quantity, types, ratios, and distribution of the vaginal microbiota may alter, resulting in dysbiosis of the vaginal microbiome.
182 Chapter 12Figure 12-1 Morphology of Lactobacillus under a wet mount using an automated analyzer microscope (400× magnification; Lactobacillus was indicated by arrows).When there is a significant reduction in bacteria in the vaginal discharge, with no dominant species or only a small number of bacteria present, and the density and diversity of bacteria are classified as level I or below, this is termed vaginal microbiota inhibition. It is important to note that the vaginal microbiota is not constant throughout a woman’s life and can vary at different stages of life. Newborn infants have no bacteria in the vagina, but microbes can be detected shortly after birth. As women reach sexual maturity, the bacterial population in most women’s vaginas gradually develops into a Lactobacillus-dominated community. After menopause, the decline in estrogen levels inhibits the growth of Lactobacillus in the vagina, which often leads to vaginal microbiota inhibition in older women.These changes related to age, physiological status, and other intrinsic factors are termed endogenous microbiota inhibition. Meanwhile, there are also cases of exogenous microbiota inhibition caused by the extensive use of broad-spectrum antibiotics, the application of certain spermicides, prolonged vaginal douching, or following chemotherapy and radiotherapy.
Vaginal Microbiota Inhibition 18312.1.1 Endogenous Vaginal Microbiota InhibitionSuppression of Vaginal Microbiota in Women of Different Agesa. Vaginal microbiota in prepubertal girls: The composition of vaginal microbiota changes significantly throughout a woman's life. The human lifespan can be divided into neonatal, childhood, puberty, reproductive, perimenopausal, and elderly stages. Immediately after birth, the newborn is influenced by estrogen produced by the mother’s placenta and ovaries, resulting in a vaginal epithelium rich in glycogen. Within 24 hours after birth, the primary colonizers of the vagina are Lactobacillus species.There are two hypotheses regarding the colonization of Lactobacillus: one suggests that the newborn acquires Lactobacillus through the birth canal during vaginal delivery; the other proposes that the newborn obtains Lactobacillus from the external environment through the oral route, which then colonizes the reproductive tract after passing through the gastrointestinal tract. However, neither hypothesis has been rigorously proven.A few weeks after birth, as the estrogen levels influenced by maternal factors decline and the gonads and adrenal glands have yet to develop, the glycogen levels in the vaginal epithelium begin to decrease, resulting in thinning and atrophy. Consequently, the number of Lactobacillus diminishes, and the overall bacterial count in the vagina becomes low, with only a few Gram-positive cocci occasionally detectable. This condition typically persists until puberty.Research indicates that prior to menarche, the most common bacterial species in females are Corynebacterium spp., coagulase-negative staphylococci, and Escherichia coli. Some studies have also found that vaginal cultures from adolescents can yield Lactobacillus, particularly strains that produce hydrogen peroxide. Overall, prepubertal healthy females generally have fewer vaginal microbiota, which may be in a state of suppression.However, there is limited research on the vaginal microbiota status of healthy, asymptomatic females, especially in infancy, indicating a need for further studies to understand the vaginal microbiota status in prepubertal healthy females.
184 Chapter 12Figure 12-2: Vaginal discharge smear of a 4-year-old girl (Gram staining, 400×).Notes: Flora density: 2+, flora diversity: 3+, dominant bacteria: Gram-negative rods, No sig-nificant Lactobacillus identified. Pathogens: Trichomoniasis (-), fungal infection: spores (-); budding spores (-); hyphae (-). Nugent score: 6. AV score: 3.b. Vaginal microbiota in perimenopausal and postmenopausal women: Due to the decline in estrogen levels in perimenopausal women, the expression levels of estrogen receptors in the vagina decrease. This leads to a reduction in glycogen production by vaginal epithelial cells influenced by estrogen. Consequently, the production of lactic acid by lactobacilli, which utilize glycogen, also decreases. The rise in vaginal pH results in the loss of the acidic environment that is conducive to lactobacilli growth. The average species diversity and quantity of vaginal microbiota during the perimenopausal period are lower compared to women of reproductive age; however, some perimenopausal women may show compensatory growth of cocci that can produce lactic acid.As age increases, the density of vaginal lactobacilli declines, especially after menopause, where there is a sharp reduction in the number of lactobacilli in the vagina, leading to a suppressed microbiota. By the time of menopause, not only is there a decrease in lactobacilli but also in other common facultative anaerobic bacteria, anaerobes, and Candida species compared to women of reproductive age, indicating a state of microbial suppression. However, in some women, the number of Escherichia coli in the vagina may dominate, possibly related to the migration of pathogenic microbes from the gut to the vagina.
Vaginal Microbiota Inhibition 185Postmenopausal women are found to have fewer lactobacilli compared to premenopausal women, while women undergoing hormone replacement therapy show a significant increase in vaginal lactobacilli. We believe that the suppression of vaginal microbiota in perimenopausal and postmenopausal women is related to the decline in estrogen levels.Figure 12-3: Vaginal discharge smear of a 65-year-old woman (Gram staining, 1000×). Notes: Flora density 1+, flora diversity 1+, dominant bacteria are Gram-positive cocci, lac-tobacilli not observed. Pathogens: Trichomonas infection (-), fungal infection: spores (-), budding spores (-), hyphae (-), Nugent score: 4, AV score:2.Suppression of Vaginal Microbiota in Physiological Activitiesa. Impact of Sexual Activity on Female Vaginal Microbiota: Numerous factors influence the vaginal microbiota in daily life, with sexual activity being the primary one. Male semen is an alkaline fluid, and after sexual intercourse, there can be changes in vaginal pH. The vaginal pH can rise to 7.2 post-intercourse and remain elevated for 6 to 8 hours. This disrupts the vagina's self-cleansing mechanism and ecological balance, making it easier for infections to occur. Generally, healthy women can self-regulate and quickly restore normal conditions; however, frequent sexual activity may further worsen the internal environment, leading to the suppression of lactobacilli. Additionally, some women may excessively rinse the vagina after sexual activity, which could reduce the quantity and diversity of the vaginal microbiota, further contributing
186 Chapter 12to microbial suppression.b. Impact of Pregnancy and Lactation on Female Vaginal Microbiota: The vaginal microbiota can change with factors such as pregnancy and ovarian function. During pregnancy, glycogen levels in the vagina increase, promoting robust bacterial growth, making microbial suppression less likely. However, in the postpartum lactation period, due to decreased estrogen levels, the number of lactobacilli may decline, potentially leading to vaginal microbiota inhibition. Studies show that 6–8 weeks postpartum, the proportion of breastfeeding women experiencing vaginal microbiota inhibition can be as high as 40 times that of non-pregnant women undergoing health check-ups during the same period (22.9% vs. 0.2%). Interestingly, research indicates that the rate of microbiota inhibition among breastfeeding mothers is significantly higher than that of artificially-fed mothers. This phenomenon may be related to the higher levels of prolactin in breastfeeding women and lower estrogen levels compared to non-breastfeeding women. However, it is noteworthy that generally, women with suppressed vaginal microbiota during this period do not experience symptoms of vaginal discomfort. The phenomenon of vaginal microbiota inhibition in women 6–8 weeks postpartum relates to the physiological decline in estrogen and is temporary. As breastfeeding ends and the menstrual cycle resumes, the microbiota inhibition gradually normalizes. Therefore, unless breastfeeding women experience vaginal discomfort, routine treatment is not recommended.Impact of Sex Hormones on Female Vaginal MicrobiotaEstrogen binds to the estrogen receptors in vaginal epithelial cells, initiating a series of physiological activities, such as thickening of the vaginal epithelium and an increase in glycogen content, which provides ample nutrients for microbial growth. Simultaneously, estrogen enhances the adhesion of bacteria to the vaginal epithelium by affecting the bioelectric potential between the bacteria and the vaginal epithelium. Vaginal lactobacilli break down glycogen into lactic acid, creating a normal acidic environment in the vagina; this acidic environment further promotes the growth of lactobacilli. Therefore, under the influence of estrogen, lactobacilli become the dominant bacteria in the normal vaginal microbiota of healthy women, maintaining the dynamic balance of vaginal microecology.The effect of progesterone on the vaginal microbiota is opposite to that of estrogen. Increased progesterone can lead to the shedding of surface vaginal epithelial cells, which carries away the attached vaginal bacteria, resulting in a
Vaginal Microbiota Inhibition 187reduction of the vaginal microbiota. The premenstrual phase is characterized by peak progesterone secretion, which is a common physiological stage for vaginal microbiota inhibition. At the beginning of the menstrual cycle, the viable counts of aerobic and facultative anaerobic bacteria decrease continuously, potentially dropping by approximately 100 times compared to the bacterial counts before the next menstruation. During menstruation, non-lactobacilli bacteria proliferate significantly, while the quantity of lactobacilli decreases or remains roughly the same; throughout the menstrual cycle, lactobacilli are continuously in the process of recovering growth.12.1.2 Pathological Suppression of Vaginal MicrobiotaImpact of Long-Term Use of Antibacterial Drugs on Vaginal MicrobiotaThe long-term irrational use of antibacterial drugs is an important factor in the suppression of vaginal microbiota. Broad-spectrum antibacterial agents indiscriminately kill all bacteria in the vagina, ultimately leading to microbiota inhibition. Research shows that different antibacterial drugs have varying effects on vaginal lactobacilli. It has been reported that lactobacilli exhibit different sensitivities to various cephalosporins, being very sensitive to penicillin, while vancomycin, doxycycline, metronidazole, and most antifungal agents targeting vulvovaginal candidiasis show no significant inhibitory effect on lactobacilli. Clindamycin ointment does have an inhibitory effect on vaginal lactobacilli. Therefore, when using probiotics in conjunction with antibacterial treatment for vaginal infections, the effects of antibacterial drugs on lactobacilli need to be considered. More research is underway to explore additional antibacterial drugs that can effectively kill other pathogenic microorganisms in the vagina while having minimal impact on lactobacilli, thereby restoring the vaginal microecology to a normal state.Impact of Contraceptive Methods on Vaginal MicrobiotaCurrently, reports on the effects of contraceptive pills on vaginal microbiota are contradictory. Some studies indicate that the use of oral contraceptives, spermicides, lubricants, or intrauterine devices does not significantly change the quantity of aerobic and anaerobic bacteria in the vagina. However, other reports present opposing viewpoints, which may be due to the varying effects of different contraceptive methods on vaginal microbiota, with only certain spermicides capable of causing microbiota inhibition. Some studies
188 Chapter 12suggest that intrauterine devices may disrupt the homeostasis of the vaginal environment, particularly during the perioperative period, making the vaginal microecology more prone to imbalance. Conversely, condoms can block alkaline semen from entering the acidic vaginal environment, which is beneficial for the growth of lactobacilli. Therefore, it is evident that different contraceptive methods and medications can produce varying effects on vaginal microbiota, with some causing microbiota inhibition.Impact of Personal Hygiene Habits on Vaginal MicrobiotaDifferences in living environments, economic conditions, and awareness of reproductive health can lead to varied personal hygiene habits. Scholars abroad have compared women who choose different menstrual hygiene products and concluded that there are no significant differences in the impact of various products on the normal vaginal microbiota during menstruation. As early as the 15th century, literature discussed that some women would douche to maintain personal hygiene and prevent infections or even blindly to avoid pregnancy or the transmission of sexually transmitted diseases. However, current studies indicate that frequent vaginal douching can lower vaginal acidity, decrease normal vaginal flora, and cause significant disruption of the vaginal microbiota, leading to microbiota inhibition. Furthermore, the flow and pressure of the flushing may enable pathogenic bacteria from the lower reproductive tract to ascend and cause inflammation in the upper reproductive tract. Early literature suggested that although vaginal douching reduced vaginal acidity, there was no significant difference in the detection rate of lactobacilli, possibly due to insufficient sample size or selection bias.Some studies have shown that various antiseptic-containing douching products exhibit strong inhibitory effects on all tested vaginal microorganisms within a short time (less than one minute). Among them, three vinegar-containing douching solutions selectively inhibit vaginitis associated with bacterial vaginosis, Group B Streptococcus vaginitis, and candidiasis, with the inhibited pathogens including Gardnerella, Mobiluncus, Mycoplasma, Ureaplasma, Bacteroides, and Group B Streptococcus, but not Lactobacillus. It can be observed that the antibacterial effects of commercial douching products vary among different brands and tested microbial species. However, these studies typically have insufficient sample sizes, lack rigor, and present unclear logical relationships, necessitating further quality research to clarify the relationship between douching products and vaginal microbiota.
Vaginal Microbiota Inhibition 189Suppression of Vaginal Microbiota in Gynecological DiseasesVaginal microbiota varies among different gynecological diseases. In conditions such as vulvovaginal candidiasis, the proportion of patients with vaginal microbiota inhibition can be 10 to 30 times higher than that of healthy women, and in some regions, it can reach 80 to 100 times. However, similar to healthy women, in women presenting with vaginal inflammation, the quantity of vaginal lactobacilli gradually decreases with age, and the proportion of women with vaginal microbiota inhibition increases, peaking during the perimenopausal period.Studies indicate that, compared to healthy women, the preoperative proportion of vaginal microbiota inhibition is higher in infertile patients, particularly in those with endometriosis, where the suppression rate is greater than in cases of hydrosalpinx, pelvic adhesions, tubal occlusion, tubal torsion, and pelvic inflammatory disease.In patients undergoing postoperative radiotherapy or chemotherapy for gynecological malignancies, evaluations of vaginal microecology show that over 65% exhibit vaginal microbiota inhibition, with 40% in the ≤55 age group and 80% in the >55 age group. In women with gynecological malignancies who have undergone major surgeries or radiotherapy/chemotherapy, the surgical removal of ovaries and/or the impact of radiotherapy and chemotherapy can alter hormonal levels, disrupt the vaginal microecological environment, and lead to microbiota inhibition. However, the level of microbiota inhibition in these women is not uniform. Although all underwent radiotherapy or chemotherapy, the proportion of women with normal vaginal microecology is significantly higher in the ≤55 age group compared to the >55 age group. This may be related to differences in estrogen levels, duration of chemotherapy, or frequency of radiotherapy; for instance, some early-stage younger patients may have retained part of their ovaries during surgery, or the ≤55 age group may have a higher body mass index, with fat converting to estrogen under the influence of aromatase.Research suggests that vaginal microecological suppression may be related to the occurrence and progression of gynecological malignancies; however, the causal relationship remains unclear, as factors like age and estrogen level have not been adequately controlled. Further research is needed to confirm this. However, it is clear that conditions such as AIDS, tumors, or the use of immunosuppressants, along with radiotherapy, can impair the immune system, reduce lactobacilli, and lead to vaginal microbiota inhibition.
190 Chapter 1212.2 Symptoms and Signs of Vaginal Microbiota InhibitionThe symptoms and signs of women with vaginal microbiota inhibition can vary significantly. Healthy asymptomatic women can also experience vaginal microbiota inhibition, and women with this condition may not exhibit any symptoms. It is also noteworthy that vaginal microbiota inhibition often occurs concurrently with pathogenic microbial infections, leading to a diverse array of clinical symptoms.Common symptoms of isolated vaginal microbiota inhibition (without coexisting pathogenic microbial infections) include increased vaginal discharge, which is typically thin and may appear light yellow; in severe cases, it can manifest as purulent or purulent-bloody discharge. Patients with purulent discharge often present with an inflammatory response. Many patients may also experience vulvar itching, burning sensations, and lower abdominal pain or discomfort. Due to the generally lower estrogen levels in women with vaginal microbiota inhibition, these patients may also exhibit symptoms associated with low estrogen levels, displaying signs of atrophic vaginitis (see Chapter 9, 9.4).In summary, all these symptoms in patients with microbiota inhibition vary in presentation and severity among different individuals. Some patients may be completely asymptomatic, while those with mild symptoms might present atypically, primarily complaining of vaginal dryness, discomfort, or reduced discharge. In more severe cases, reproductive and urinary system symptoms may arise, even affecting systemic health and the overall quality of life for women.12.3 Diagnosis of Vaginal Microbiota InhibitionThe diagnosis of vaginal microbiota inhibition is generally not difficult based on the medical history, clinical presentation, and laboratory tests. However, it is important to rule out other vaginal diseases before making a diagnosis. When no pathogens such as Trichomonas, clue cells, or yeast are detected, and routine examination of vaginal discharge indicates a significant reduction or absence of lactobacilli, a preliminary diagnosis of vaginal microbiota inhibition can be made. This may be accompanied by a large number of leukocytes and basal epithelial cells, and the vaginal microbiota can also be assessed through a microbiota evaluation system.The clinical evaluation system of vaginal microecology comprehensively assesses the vaginal microecological environment through five aspects: the density and diversity of the vaginal microbiota, dominant bacteria, inflammatory response of the body, and the morphology of pathogenic bacteria. This is
Vaginal Microbiota Inhibition 191combined with functional indicators such as vaginal pH, hydrogen peroxide, and leukocyte esterase (See Chapter 2, "Evaluation System of Vaginal Microecology," 2.1, "Morphological Examination").When the density of the vaginal microbiota is classified as levels II-III, diversity is also II-III, the dominant bacteria are lactobacilli, vaginal cleanliness is rated as grade I, lactobacilli function normally (i.e., H2O2 positive), and vaginal pH is between 3.8 and 4.5, the vaginal microecology is considered normal. In cases of microbiota inhibition, the microbiota status shows a density of I or none, diversity of I or none, with no dominant bacteria or only a small number of bacteria, including a few Gram-positive bacilli, Gram-positive cocci, and Gram-positive streptococci. No positive findings for pathogens are identified, i.e., Trichomonas negative, Candida negative, and clue cells negative. Nugent scores may range from 2 to 4, and functional assessments can show H2O2 positive, with sialidase negative or positive, and leukocyte esterase negative or positive. pH measurements generally range between 4.5 and 5.0 (see Figure 13-4).Some patients with vaginal microbiota inhibition have a decrease in estrogen levels. The maturity of vaginal squamous epithelial cells is positively correlated with estrogen levels in the body. Estrogen promotes the proliferation of basal and suprabasal cells in the vaginal epithelium, which gradually differentiate into intermediate and superficial cells. The ratio of different layers of squamous epithelial cells in the vagina reflects the estrogen levels in the body. Estrogen plays a leading role in maintaining the dynamic balance of vaginal microecology. The vaginal cell maturation index (the percentage of superficial cells among squamous epithelial cells) can indirectly reflect estrogen levels in women and is a simple method that has some reference value in the diagnosis of vaginal microbiota inhibition.
192 Chapter 12Figure 13-4: Vaginal microbiota inhibition (Gram Staining, 1000×)Notes: Figure 13-4 a: No flora density, no flora diversity, no dominant bacteria, white blood cells visible;Figure 13-4 b: Flora density grade I, flora diversity grade I, dominant bacteria are Gram-pos-itive short rods, mucus threads visible in the background;Figure 13-4 c: Flora density grade I, flora diversity grade I, dominant bacteria are Gram-pos-itive cocci, cell debris visible in the background;Figure 13-4 d: Flora density grade I, flora diversity grade I, dominant bacteria are Gram-pos-itive cocci, basal squamous epithelial cells and white blood cells visible in the background;Figure 13-4 e: No flora density, no flora diversity, no dominant bacteria, basal squamous epi-thelial cells visible;Figure 13-4 f: No flora density, no flora diversity, no dominant bacteria, superficial squamous epithelial cells and white blood cells visible.12.4 Treatment for Inhibition of Vaginal Microbiota12.4.1 Restoring the Vaginal Acidic EnvironmentThe normal vagina maintains a slightly acidic environment (pH 3.8-4.5), which serves as a natural barrier. When the vaginal microbiota is suppressed, the pH rises to slightly alkaline levels, typically between 4.5 and 5.0. To restore the vaginal pH, acidic washes or solutions containing acidic substances can be used. Physical methods such as rinsing the vagina with a solution of 1% lactic acid or 0.5% acetic acid can be performed daily. Additionally, chemical agents
Vaginal Microbiota Inhibition 193that regulate the acid-base balance can be applied to increase the number of lactic acid bacteria in the vagina. Highly acidic substances, such as polyphenol aldehyde, can also protect the growth of Lactobacillus, maintain the acidic environment of the vagina, promote tissue regeneration, and facilitate epithelial re-covering. Furthermore, vaginal lactic acid gels (e.g., Bayer Canesbalance, used at 5 mL/day for 7 consecutive days), oligolactic vaginal suppositories (e.g., Vagisan, 1 capsule each night), and lactate-releasing vaginal rings can effectively restore the vaginal acidic environment.12.4.2 Restoring the Dominant Vaginal MicrobiotaCurrently, there are many probiotic formulations available for clinical use; however, most are designed for gut health, with few dedicated to the female reproductive tract. Probiotics for vaginal application must meet five criteria: a. They should contain an adequate number of probiotics to ensure sufficient quantity upon reaching the vagina. Oral Lactobacillus formulations should not contain fewer than 10^8 live bacteria per day, while vaginal Lactobacillus formulations should contain at least 0.25 × 106 CFU daily; b. They must adhere well to vaginal epithelial cells, allowing them to settle and proliferate in the vagina to form a normal, balanced microbiota; c. They should prevent the adhesion of pathogenic microorganisms in the vagina; d. They must produce acidic substances, H2O2, bioactive substances, and/or cytokines to inhibit the growth of pathogenic microorganisms; e. They must be safe, without side effects, non-invasive, and non-carcinogenic. Due to these stringent requirements, there are currently few Lactobacillus products that meet these criteria.Studies have shown that Lactobacillus rhamnosus (L. rhamnosus, GR-1) and Lactobacillus fermentum (RC-14) meet the aforementioned criteria. GR-1 can survive in nonoxynol-9, a spermicide, while RC-14 secretes surfactants that prevent pathogenic microbial adhesion in the vagina. These characteristics make these two strains superior candidates for treating vaginal microbial disorders. Both strains are commercially available and were isolated from healthy women, having been used as dietary supplements clinically without observed adverse reactions, and have received safety qualification certification (QPS) from the European Food Safety Authority.In China, vaginal probiotic formulations are commonly used. The commonly used vaginal Lactobacillus strain, DM8909, developed by the Department of Microecology at Dalian Medical University, is derived from the Lactobacillus delbrueckii subsp. bulgaricus DM8909 strain, which has been commercially
194 Chapter 12produced. The DM8909 strain is isolated from vaginal discharge of healthy women and does not carry plasmids. Additionally, there are lactobacillus vaginal capsules that utilize live enterococci; both strains have been shown to produce lactic acid, bacteriocin, and H2O2. The difference is that live enterococci cannot colonize but can promote the growth of existing Lactobacillus, while the vaginal Lactobacillus preparation can colonize temporarily. Despite the absence of long-term colonizing Lactobacillus formulations, clinical microecological regulators primarily use live bacteria.Due to challenges such as transport, storage difficulties, poor stability, and incompatibility with sensitive antibacterial agents faced by live bacterial formulations, inactivated probiotics have recently garnered attention. In studies evaluating the adhesion of Lactobacillus to vaginal epithelial and cervical epithelial cell lines (HeLa), results indicated no significant difference in the adhesion of live and heat-inactivated Lactobacillus to scraped vaginal epithelial cells, whereas the adhesion index for heat-inactivated Lactobacillus to HeLa cells was significantly higher than that of live bacteria. This demonstrates that heat-inactivated Lactobacillus has sufficient adhesive properties for vaginal and cervical epithelium, making it a potential safe and effective option for easier administration as a micro-ecological preparation.12.4.3 Restoring Vaginal Estrogen LevelsAs most patients with inhibited vaginal microbiota also exhibit low estrogen levels, estrogen supplements can be specifically administered. Estrogen formulations can be given orally or locally. Currently, several vaginal estrogen preparations are available, including suppositories, tablets, creams, and silicone ring release systems (see Chapter 9, 9.6 "Atrophic Vaginitis Treatment"). The application of estrogen can enhance vaginal resistance, promote vaginal epithelial proliferation, and restore vaginal microbiota.12.4.4 Improving unhealthy lifestyle habitsFrequent sexual intercourse can lead to vaginal microecological imbalance. Vaginal douching or indiscriminate use of cleansing solutions can alter the vaginal pH, washing away indigenous flora and causing microbiota inhibition. Prolonged irrational use of antibacterial agents can also lead to vaginal microbiota inhibition, and a suppressed vaginal environment increases the risk of various vaginitis.
Vaginal Microbiota Inhibition 195Therefore, it is important to maintain moderation in sexual activity and avoid unclean practices. Vaginal douching or indiscriminate use of cleansing solutions should be avoided; it is recommended to clean the vulva 1-2 times daily with water. Attention should be given to the hygiene of used utensils, and it is advisable to shower daily if possible rather than taking baths. After experiencing vaginitis, timely medical consultation and reasonable treatment are necessary, and self-administered douching should be avoided. It's essential to maintain hygiene during menstruation, the postpartum period, and after abortions. Antibacterial agents should be used judiciously to prevent misuse. Additionally, attention should be given to restoring local defense mechanisms and establishing and maintaining a normal microecological environment in the vagina.References[1] Collaborative Group on Infectious Diseases of the Chinese Society of Obstetrics and Gynecology. Expert consensus on clinical application of vaginal microecological evaluation. Chinese Journal of Obstetrics and Gynecology, 2016,51(10):721-723.[2] Xing Xie, Beihua Kong, Tao Duan. Obstetrics and Gynecology. 9th edition. Beijing: People’s Healthy Publishing House, 2018.[3] Collaborative Group on Infectious Diseases of the Chinese Society of Obstetrics and Gynecology. Guidelines for diagnosis and treatment of bacterial vaginosis (2021 revised edition). Chinese Journal of Obstetrics and Gynecology, 2021,56(1):3-6.
C H A P T E R - 13Vaginal Microecological TreatmentYuanting TangTranslator: Yiduo Zhang, Zhengqiang Hu13. 1Microecological treatment refers to the adjustment of microecological imbalances, maintenance of microecological equilibrium, and enhancement of host health or improvement of health status through the use of formulations made from beneficial normal microorganisms and/or their metabolic products and/or growth-promoting substances, based on the principles of microecology. As awareness of vaginal microecology becomes more comprehensive and in-depth, the use of vaginal microecological formulations has become increasingly widespread.Once the ecological balance of the vagina is disrupted or exogenous pathogens invade, inflammation can occur. Additionally, prolonged use of antibacterial agents that inhibit the growth of Lactobacillus or a weakened immune system can allow other pathogenic bacteria to dominate, leading to inflammation. The "Guidelines for the Diagnosis and Treatment of Bacterial Vaginosis in China" and the "Diagnosis and Treatment Strategy for Vaginal Infectious Diseases in China" indicate that microecological preparations for restoring vaginal microecological balance can be selected. Thus, the treatment trend for vaginal infections includes two aspects: first, the effective elimination of various pathogens, and second, the restoration of vaginal defense functions and reconstruction of the vaginal microecology. Only through these measures can recurrent vaginal infections be prevented.The vaginal microecological system is primarily composed of four parts: the normal anatomical structure of the vagina, the endocrine regulation system, the local vaginal immune system, and various vaginal microbial communities. Clinically, there is a general lack of comprehensive and in-depth understanding of microecological preparations, leading to instances of unreasonable treatments. Currently, the treatment concept for vaginitis emphasizes not only the elimination of pathogenic microorganisms but also the importance of vaginal microecological treatment, shifting the treatment philosophy from pure
Vaginal Microecological Treatment 197antibacterial approaches to a dual focus on both antibacterial actions and the promotion of microecological balance.Currently, vaginal microecological treatment typically includes several aspects: a. Establishment of Dominant Vaginal Flora: This primarily refers to the application of probiotic preparations. b. Nutritional Support for Dominant Vaginal Flora: This mainly involves the use of prebiotic formulations. c. Repair of Vaginal Mucosa: This includes the use of nutrients for epithelial health. d. Regulation of Vaginal discharge: This mainly refers to the application of estrogen. e. Correction of Vaginal pH: This includes the use of acidic washes. Additionally, traditional Chinese medicine (TCM) can also be chosen to assist in the restoration of the vaginal microecological environment.13.1 Probiotic PreparationsOne of the goals of vaginal microecological treatment is to establish and restore the dominant vaginal flora, with lactic acid bacteria preparations, primarily Lactobacillus formulations, being the most commonly used in clinical practice. The following sections will summarize the development history of lactic acid bacteria preparations, the characteristics of Lactobacillus, the transfer and colonization of Lactobacillus, the mechanisms of action of Lactobacillus, guidelines on the application of Lactobacillus preparations, precautions for combined medications, and the application of Lactobacillus in different types of vaginitis. This will enable clinicians to use Lactobacillus preparations more rationally in treatment, improve the cure rates of vaginitis and related diseases, reduce the recurrence of vaginitis, and enhance the quality of life for women. At the same time, it is important to avoid inappropriate overuse of lactic acid bacteria preparations to reduce cost-effectiveness ratios and minimize patients' time and economic burdens.13.1.1 Development History of Lactobacillus PreparationsLactobacillus species are not a classification term in microbiology; instead, they refer to a group of bacteria that can ferment sugars to produce lactic acid. The history of lactic acid bacteria dates back to 1857 when Louis Pasteur first described the presence of tiny organisms in sour milk. In 1873, Joseph Lister isolated and identified microorganisms from sour milk, naming them Lactobacillus species . In 1891, at the Fourth Congress of German Women Scientists, Albert Doederlein presented a lecture titled "On Vaginal discharge and Vaginal
198 Chapter 13Bacteria." Doederlein pointed out that vaginal discharge from healthy pregnant women could cultivate a Gram-positive rod that had antagonistic effects on the growth of Staphylococcus, attributing this bactericidal effect to lactic acid produced by the bacteria (in 1928, Stanley Thoms identified it as Lactobacillus acidophilus, although this was subject to some debate and varied reports). By 1973, it was generally accepted that facultative anaerobic lactobacilli were the dominant bacteria in the vaginas of healthy women of childbearing age, with a separation rate in vaginal discharge of 80% to 100% and the highest concentrations (107 to 108 CFU/mL of vaginal discharge).Research has identified over 100 types of Lactobacillus that can be isolated from the vagina, including: Lactobacillus iners (L. iners), Lactobacillus crispatus (L. crispatus), Lactobacillus gasseri (L. gasseri), Lactobacillus jensenii (L. jensenii), Lactobacillus acidophilus (L. acidophilus), Lactobacillus delbrueckii(L. delbrueckii), Lactobacillus rhamnosus (L. rhamnosus),and so on.Which specific Lactobacillus strain is dominant? Initially, it was believed to be L. acidophilus, but later researchers employed DNA probe technology to identify Lactobacillus strains in the vagina of healthy women. This research concluded that the dominant vaginal Lactobacillus species are L. crispatus and L. jensenii, which are capable of producing hydrogen peroxide (H2O2). Subsequently, many scholars confirmed, through various molecular biology methods, that L. crispatus, L. gasseri, L. iners, and L. jensenii are the most common types of Lactobacillus species in the vaginas of women of childbearing age. Notably, the main hydrogen peroxide-producing strains are primarily L. crispatus, L. gasseri, L. jensenii, and L. acidophilus (as illustrated in Figure 13-1).Research indicates that there are differences in the types of Lactobacillus found in the vaginas of women from different regions. However, early studies, such as the 2002 comparison analysis by Pavlova on vaginal discharge from women in seven countries, including Argentina, Belgium, and China, suggested that there were no significant differences in the dominant Lactobacillus species in the vagina. With advancements in research techniques, particularly the application of various molecular technologies, more scholars have used multiple methods to study the vaginal microbiota of healthy women in countries such as China, Belgium, Brazil, the United States, Canada, Germany, the Netherlands, Japan, Italy, and Nigeria. These studies have shown that there are differences in the dominant vaginal bacteria among women from different regions. However, Lactobacillus crispatus is one of the most common dominant vaginal bacteria, and in Caucasian women, Lactobacillus crispatus and Lactobacillus jensenii are
Vaginal Microecological Treatment 199more prevalent than other Lactobacillus species. Additionally, culture, behavior, dietary habits, racial genetics, and differences in innate or acquired immune systems can all influence the vaginal microbiota of women.Figure 13-1 Morphology of Lactobacillus under a microscope (Gram staining, 1000×).Notes: Flora density 3+, Flora diversity 1+, Dominant bacteria: Lactobacillus, Pathogens: Trichomonas infection (-), Fungal infection: spores (-), budding spores (-), hyphae (-), Nugent score: 0, AV score: 0.With the continuous development of molecular biology techniques and the application of high-throughput sequencing technology, the current popular approach is to use 16S rDNA microbial diversity sequencing to type the vaginal microbiota structure of healthy women of childbearing age. The most famous classification is the five types of vaginal microbiota communities by Petrova MI, which include Types I to V. Type I is dominated by L. crispatus, with a vaginal pH of 4.0, capable of producing lactic acid and hydrogen peroxide. Type II is dominated by L. gasseri, with a vaginal pH of 4.3, producing lactic acid and hydrogen peroxide. Type III is dominated by L. iners, with a vaginal pH of 4.4. Type IV-A has multiple dominant bacteria, with Lactobacillus species being more abundant than other bacteria, and a pH range of 4-5. Type IV-B has multiple dominant bacteria, with Lactobacillus species being less abundant than other bacteria, and a vaginal pH often at 5.3. Type V is dominated by L. jensenii,
200 Chapter 13with a pH of 4.7 and the ability to produce hydrogen peroxide. There are certain differences in the bacterial species colonized in the vaginas of women from different races and geographical environments. Additionally, lactate-producing cocci can be isolated from the vaginal discharge of healthy women. These cocci can synergize with Lactobacillus species to enhance the ability to inhibit and kill pathogenic bacteria, expanding the range of antibacterial activity. They are also a type of probiotic involved in maintaining vaginal microbiota balance.Since Alexander completed the whole-genome sequencing of the first lactic acid bacterium, Lactococcus lactis subsp. IL1403, more genotypes of lactic acid bacterium have been identified one by one, including Lactobacillus rhamnosus (L. rhamnosus) LGG strain, also known as L. rhamnosus GG (ATCC 53103). LGG is currently one of the most famous and extensively studied strains in the world, and it is also the most representative strain among L. rhamnosus species. The research on lactic acid bacterium can generally be divided into four stages: The first stage is isolation and cultivation, using culture and biochemical methods to isolate and screen for excellent strains of Lactobacillus and lactate-producing cocci, typically from the vaginal discharge of healthy women. The second stage is in vitro experiments, using in vitro tests that simulate the vaginal environment and/or animal models to verify the efficacy of lactic acid bacteria. Unfortunately, no in vitro test or animal vaginal environment model that completely simulates the human vaginal environment has been found so far. The third stage is clinical trials, applying strains with excellent probiotic properties verified in animal models to clinical controlled trials. Although there have been numerous trials of different scales in recent years to test the effects of lactic acid bacteria, the results have not been consistent, and even completely opposite conclusions have been drawn. After many effective and ineffective clinical trials, it has gradually been recognized that the efficacy of probiotics may not be uniform. Therefore, some researchers have begun to focus on the fourth stage—research on mechanisms of action. With the development of high-throughput sequencing technology, it has been found that the human vagina is colonized by a diverse range of microorganisms, influenced by many factors, and is in a state of dynamic equilibrium. Can the mechanisms of action be revealed to discover how lactic acid bacteria function in the host vagina, thereby applying different functional probiotics to different beneficiary populations for precise treatment? In summary, current research has gradually shifted from basic screening and efficacy verification to in-depth exploration of mechanisms of action. The vagina, as a complex and open microecological environment, is influenced by too many
Vaginal Microecological Treatment 201factors. More in-depth and effective results are still needed, and the research on probiotic preparations remains a long and arduous task.Currently, there are many types of probiotic preparations used in clinical practice, but most are for the gut, with fewer specifically designed for the female reproductive tract. For detailed information on the application of probiotic preparations, please refer to Chapter 12, 12.4.13.1.2 Characteristics of Lactobacillus StrainsLactobacillus strains is a Gram-positive rod-shaped bacterium, without spores, flagella, or capsule, and appears slender and curved or in the form of spherical rods or rods. It is a facultative anaerobe, capable of surviving in aerobic conditions but grows better under anaerobic conditions. The optimal growth temperature is 35°C-38°C, and it can grow in a pH range of 3.0-8.0, with the optimal growth pH in vitro being 6.0. As the culture time extends, the pH of the culture medium gradually decreases. Among them, Lactobacillus delbrueckii DM8909 is sensitive to temperature and can be inactivated in liquid at 60°C within 10 minutes, while it can survive for 30 minutes at 60°C in dried powder form. It has strong acid tolerance and can survive in a pH range of 2.5-8.0. Different antimicrobial agents have varying inhibitory effects on vaginal Lactobacillus. Most Lactobacillus strains show different sensitivities to cephalosporins, are highly sensitive to penicillin, and are not significantly inhibited by vancomycin, doxycycline, and metronidazole. Clindamycin ointment has inhibitory effects on vaginal Lactobacillus. Specifically, DM8909 is resistant to metronidazole but highly sensitive to ampicillin, penicillin, erythromycin, and gentamicin, and moderately sensitive to polymyxin and amikacin.13.1.3 Transfer and Colonization of Lactobacillus StrainsResearch has shown that orally ingested Lactobacillus strains can enter the gastrointestinal tract as live bacteria, travel through the rectum to the vagina, and colonize there to exert their biological functions. Once in the vagina, probiotics need to adhere to the mucosal surface to colonize and function. However, due to the continuous shedding and renewal of mucosal cells, the adhered bacteria will eventually detach from the mucosal surface. Therefore, it is generally believed that the colonization of exogenous probiotics in the body is transient. DNA fingerprinting analysis of fecal samples from subjects who ingested GR-1 and RC-14 strains revealed that these strains could be detected in fecal samples
202 Chapter 13during the intake period and up to 7 days after cessation. However, in a longer-term study where subjects were given GR-1 and RC-14 strains orally for 60 days, although higher numbers of Lactobacillus were detected during the intervention period, no significant differences in vaginal Lactobacillus counts were observed between the intervention and control groups 30 days after the intervention ended. This indicates that while orally ingested GR-1 and RC-14 strains can colonize the vagina, this colonization is temporary and cannot persist long-term without continued supplementation.Can Lactobacillus strains directly administered to the vagina achieve long-term colonization? Unfortunately, the answer is no. Direct vaginal administration of Lactobacillus does not extend the survival and colonization time of live bacteria in the vagina. In a study where subjects were administered a capsule containing 10^9 CFU of GR-1 and RC-14 strains vaginally for 3 consecutive days, both strains were detectable in the vagina during the administration period. However, after cessation, the number of vaginal Lactobacillus decreased over time and could no longer be detected after 30 days. In contrast, using Lactobacillus rhamnosus GG (LGG) as a control strain, it was found that GR-1 and RC-14 strains had better colonization and survival than LGG, indicating that the adhesion of Lactobacillus to vaginal epithelial mucosa is strain-specific.Another study showed that after vaginal administration of a single dose of DM8909 strain (with a live bacterial count of at least 2.5×10^5 CFU), the medication completely dissolved in the vagina within 2 hours, with no change in live bacterial count. After 8 hours, the live bacterial count decreased by 10-fold, and after 24 hours, it dropped to 0.8×10^6 CFU/mL. When administered daily for 10 days according to the product instructions, the strain could still be isolated 4 days after discontinuation, but the exogenous Lactobacillus gradually disappeared over time. Therefore, more research is needed to identify exogenous Lactobacillus preparations that can grow and reproduce long-term in the vagina.13.1.4 Mechanisms of Action of LactobacillusProducing of Lactic Acid by LactobacillusVaginal epithelial cells deposit glycogen under the influence of estrogen, and the glycogen stored in the vaginal mucosal epithelium serves as the primary nutrient source for Lactobacillus. Glycogen is metabolized by Lactobacillus to produce lactic acid, acetic acid, and other acidic substances (confirmed by proton nuclear magnetic resonance), which play a crucial role in maintaining the vaginal
Vaginal Microecological Treatment 203pH. Lactic acid helps maintain the normal acidic environment of the vagina, inhibiting the growth and reproduction of non-acidophilic microorganisms such as trichomonads, certain aerobic and anaerobic bacteria, and others.In healthy women, the concentration of lactic acid in the vagina (mass/volume) is (1.0±0.2)% (≤110 mmol/L), with a pH range of 3.8 to 4.5. Different Lactobacillus species produce varying amounts of lactic acid. For example, Lactobacillus crispatus produces the most lactic acid, while Lactobacillus jensenii produces the least. Both can produce L- and D-lactic acid; Lactobacillus iners produces only L-lactic acid, and Lactobacillus gasseri produces only D-lactic acid. In addition to Lactobacillus, vaginal epithelial cells can also produce lactic acid under the influence of estrogen, accounting for only 15% of the total vaginal lactic acid and producing only L-lactic acid. In addition to lactic acid, the vagina also contains small amounts of other short-chain fatty acids such as propionic acid, butyric acid, and succinic acid. However, the acid dissociation constant (pKa) of lactic acid is significantly higher than that of other organic acids, with a pKa of 3.89. When dysbiosis occurs, such as in bacterial vaginosis, the concentration of lactic acid in the vagina decreases significantly, while other organic acids increase, leading to a rise in pH and a weakened protective effect on the vaginal environment.Producing of Antimicrobial Factors by LactobacillusAntimicrobial factors produced by Lactobacillus, such as hydrogen peroxide (H₂O₂), bacteriocins, bacteriocin-like substances, and biosurfactants, exert a biological antagonistic effect in the vagina, maintaining the dominance of Lactobacillus and resisting pathogen invasion.a. Mechanism of H₂O₂ antimicrobial action: Vaginal Lactobacillus can be classified into H₂O₂-producing and non-producing strains. H₂O₂-producing strains are commonly found in Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus crispatus. H₂O₂ can directly kill microorganisms or, in the presence of peroxidase, oxidize halogen compounds to form hypohalous acids with strong antimicrobial properties. The H₂O₂ produced by Lactobacillus significantly inhibits the growth of non-acidophilic microorganisms in the vagina, showing antagonistic effects against Staphylococcus and Enterobacteriaceae, thereby maintaining the dominance of Lactobacillus and preventing vaginal dysbiosis.b. Mechanism of Bacteriocins and Bacteriocin-Like Substances: Lactobacillus can produce antimicrobial substances similar to antibiotics, known as bacteriocins and bacteriocin-like substances. Bacteriocins are peptide or peptide-sugar/lipid complex proteins, such as lactococcin, enterocin, and
204 Chapter 13pediocin, which typically act on strains closely related to the producing strain. Bacteriocin-like substances describe antagonistic compounds that do not fully meet the definition of bacteriocins and have a broader antimicrobial spectrum. Numerous studies have shown that bacteriocin-like substances produced by Lactobacillus exhibit strong antimicrobial activity, inhibiting the growth of pathogens such as Candida albicans, Escherichia coli, and Gardnerella vaginalis, and synergistically enhancing the antimicrobial effect of H₂O₂.For example, studies on the inhibition of Listeria monocytogenes by Lactobacillus found that cell-free supernatants of Lactobacillus UAL11, Leuconostoc UAL14, and Pediococcus acidilactici PAC1.0 retained inhibitory activity after neutralization, ultrafiltration, or heat treatment, suggesting that bacteriocins were responsible for this antimicrobial effect. The strain GR-1 produces a bacteriocin-like substance distinct from lactic acid and H₂O₂, which inhibits the growth of uropathogenic E. coli (UPEC). Agglutination of GR-1 with UPEC is also an important defense mechanism against pathogen infection. The supernatants of GR-1 and RC-14 strains can inhibit the activity of two UPEC adhesin promoters (type 1 and P fimbriae) and upregulate the activity of two outer membrane porin promoters involved in UPEC membrane stress. These antimicrobial factors provide an effective, non-specific protective mechanism that is crucial for maintaining normal vaginal ecological balance and preventing infections.c. Mechanism of Biosurfactants: Biosurfactants are surface-active substances obtained through microbial biocatalysis and biosynthesis, such as glycolipids, amino acids, and proteins. Biosurfactants produced by Lactobacillus not only directly inhibit the growth of Gardnerella vaginalis, trichomonads, Candida, Staphylococcus, and other pathogens but also reduce the adhesion capacity of vaginal pathogens. The mechanism involves the formation of biofilms by pathogens, which can hinder the penetration of antimicrobial drugs and reduce treatment efficacy. Biosurfactants secreted by Lactobacillus can disrupt pathogen adhesion and biofilm formation, thereby enhancing the antimicrobial effect of antibiotics.At least 15 Lactobacillus species have been identified as producers of biosurfactants. For example, GR-1 and RC-14 strains produce biosurfactants that can bind to collagen types III and IV on epithelial cells to form a more stable biofilm, thereby clearing and replacing biofilms formed by pathogens. Additionally, treating Candida-infected cells with supernatants of GR-1 and RC-14 strains upregulates the production of interleukin-8 (IL-8) and interferon-
Vaginal Microecological Treatment 205γ-inducible protein 10 (IP-10) by infected cells. These cytokines attract more immune cells to the site of infection, facilitating rapid clearance of the infection.High Cellular Adhesion of Lactobacillus in the Vaginal MucosaAfter adhering to and colonizing the surface of vaginal mucosal epithelial cells, Lactobacillus adjusts its gene expression and secretes large amounts of extracellular polysaccharides to form biofilms during growth and reproduction. Lactobacillus competes for binding sites on vaginal mucosal epithelial cell receptors, maintaining colonization resistance of the vaginal epithelium. This spatial occupation can even displace other bacteria originally adhering to the vaginal epithelium, replacing them and preventing pathogen adhesion and invasion. Additionally, Lactobacillus can interfere with pathogen colonization on the vaginal epithelium by altering the adhesiveness of pathogens. This adhesion is regulated by estrogen levels, which stimulate the movement of surface charges on vaginal epithelial cells, altering the surface potential of vaginal cells. This potential change affects bacterial adhesion and thus influences the composition of the vaginal microbiota.Adhesion capacity is one of the most important criteria for screening probiotic strains, determining their ability to colonize mucosal surfaces, antagonize pathogenic microorganisms, regulate immune functions, and promote the repair of vaginal epithelial mucosal cells. The adhesion of Lactobacillus is also related to glycoproteins and carbohydrates on the bacterial cell wall surface. In an acidic environment with a low pH, Lactobacillus can better exclude the adhesion of other bacteria. In vitro experiments have shown that GR-1 and RC-14 strains can adhere well to cell surfaces, especially to the epithelial cells of the urogenital tract.Immune Modulation by LactobacillusThe female reproductive tract is part of the systemic mucosal immune system. When the vaginal epithelium is stimulated by various internal and external factors, it continuously produces inflammatory and chemotactic factors. For example, when stimulated by pathogenic bacteria, the expression of IL-1 and IL-8 is upregulated, which attracts neutrophils to the vaginal mucosa to combat the invading pathogens. Researchers used gene chip technology to study the correlation between the Lactobacillus rhamnosus GR-1 strain and host immune function. They found that instilling Lactobacillus into the vagina upregulated the expression of 24 genes related to vaginal immune responses, including antimicrobial peptides, Toll-like receptors (TLRs), and cytokine receptors, by
206 Chapter 13more than fivefold. This suggests that the GR-1 strain can modulate the innate immune defense barrier of the vaginal mucosa.The immune modulatory effects of Lactobacillus are manifested in two aspects:a. Non-specific Immunity: Lactobacillus plays an early role in non-specific immune defense against infections. This includes: • Enhancing mucosal barrier function; • Strengthening the phagocytic capacity of macrophages. For example, Lactobacillus johnsonii Lj1 and Bifidobacterium lactis Bb12 can enhance the phagocytic activity of phagocytes against Escherichia coli in vitro. • Stimulating the production of various cytokines. For instance, Lactobacillus can stimulate macrophages to produce anti-inflammatory cytokine IL-10, thereby reducing systemic inflammation. • In vitro studies using placental trophoblast cells as a model have shown that GR-1 can inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNF-α) production by increasing IL-10 through the phosphorylation of signal transducer and activator of transcription 3 (STAT-3) and mitogen-activated protein kinase p38. • Lactobacillus species can increase the expression of surface receptors CR1, CR3, and FcR on neutrophils in healthy individuals, enhancing their phagocytic capacity. However, in allergic individuals, the expression of these receptors is reduced, leading to decreased phagocytic activity and a weakened allergic response.b. Specific Immune Response: Lactobacillus can enhance humoral immunity by increasing the levels of IgA, IgM, and IgG on mucosal surfaces and in serum. It also promotes the proliferation and maturation of T and B lymphocytes, thereby strengthening cell-mediated immunity. Additionally, Lactobacillus can modulate the Th1/Th2 balance. This effect is achieved through bacterial genomic DNA stimulation and induction by peripheral blood mononuclear cells (PBMCs). Lactobacillus can also enhance the proliferative capacity of B cells in response to mitogens.Nutritional Competitive Advantage of LactobacillusLactobacillus, which colonizes the vagina in large numbers, has a competitive nutritional advantage. Lactobacillus utilizes glycogen from vaginal epithelial cells for growth, thereby reducing the nutrient supply available to other microbial communities and interfering with their growth. As glycogen is consumed, the production of lactic acid and other substances further inhibits the growth of non-
Vaginal Microecological Treatment 207acidophilic pathogens, including non-acidophilic bacteria and trichomonads. This consolidates the dominant position of Lactobacillus, establishing a virtuous cycle that maintains a healthy vaginal microbial environment.13.1.5 Instructions for the Use of Lactobacillus Preparations in Diagnosis and Treatment The current guidelines for the diagnosis and treatment of vaginitis in our country, such as the Guidelines for the Diagnosis and Treatment of Trichomoniasis Vaginitis and the Revised Guidelines for the Diagnosis and Treatment of Vulvovaginal Candidiasis (VVC), do not recommend the use of microecological agents in their treatment plans; the Guidelines for the Diagnosis and Treatment of Bacterial Vaginosis mentioned that preparations to restore normal vaginal flora can be used, but it does not specify the duration or dosage. In 2015, the U.S. Centers for Disease Control and Prevention (CDC) guidelines on the diagnosis and treatment of vaginal inflammation noted that some studies have evaluated the clinical and microbiological efficacy of vaginal Lactobacillus preparations in treating bacterial vaginosis (BV) and restoring normal flora. However, there is no research supporting any Lactobacillus preparation or probiotics as a supplementary or alternative treatment for BV. The role of these preparations in the treatment and prevention of BV still requires further study. In the clinical treatment of various types of vaginitis, whether oral or vaginal administration of antibiotics, especially broad-spectrum antibiotics, may lead to the disappearance of symptoms. However, the vaginal micro-ecosystem has not been restored, making it more susceptible to pathogenic bacteria and prone to recurrence. For example, BV has a clinical cure rate of 90%, but it is likely to recur within three months. Repeated use of antibiotics after multiple episodes can easily lead to antibiotic resistance, which in turn reduces the cure rate and increases the recurrence rate, ultimately forming a vicious cycle. Despite the severity of the clinical situation, the clinical promotion of microecological agents has not been ideal, and the main reasons may include: Lacking of Reliable Animal Models Currently, there is no direct evidence on the effectiveness of probiotics. It is well known that the most fundamental direct evidence for assessing microbial efficacy, and the first step in probiotic research, is animal experimentation.
208 Chapter 13However, unlike intestinal microbiota, which can be studied using pig gut microbiota similar to human gut microbiota, a reliable vaginal microbiota animal model has not yet been established. The reason for this is that the human vaginal microbiome is diverse and forms a microbial community. To date, no animal vaginal microenvironment similar to the human vaginal microbiome has been discovered. Most current animal experiments involve disrupting the natural vaginal microbiota of animals and then artificially inoculating one or several bacteria to simulate the human vaginal microbiota, which still differs from the true human microenvironment. Therefore, the reliability of probiotic research remains insufficient. Currently, animal models most similar to the human vaginal microbiota often use Beagle dogs and rhesus monkeys, but with the development of organoids and high-throughput sequencing, a reliable model will eventually emerge. Lacking of Reliable Large-Sample Multicenter Randomized Controlled Triple-Blind Trials Currently, there is no rigorous research on the application of Lactobacillus preparations using large-sample multicenter randomized controlled triple-blind trials with high-quality studies in terms of strict randomization methods, concealed assignment schemes, and intention-to-treat analysis. Many studies also lack standardized and comprehensive evaluation criteria for drug efficacy, which affects the vaginal microenvironment due to various confounding factors such as age, frequency of sexual activity, educational level, and their impact on drug efficacy and recurrence. Most studies have not controlled for these confounding factors. Therefore, the evidence for the use of Lactobacillus appears insufficient, requiring more large-sample, high-quality randomized controlled trials to support it; Lacking of Excellent Commercialized Lactobacillus Preparations in Clinical Treatment. Currently, there are few Lactobacillus preparations available, and no research has found a Lactobacillus preparation that can colonize the vagina long-term after use. The currently used live Lactobacillus preparations can only colonize the vagina for a short period. Once the use stops, exogenous Lactobacillus preparations will gradually disappear and cannot become the dominant vaginal flora. Lactobacillus preparations cannot colonize the vagina; they can only promote the growth of existing Lactobacillus in the vagina. Additionally, in 2013, Petrova MI used second-generation sequencing technology to analyze
Vaginal Microecological Treatment 209the vaginal microbiota diversity of healthy women and identified five types of vaginal microecological structures. The dominant bacteria in these five types are Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, Multiple Dominant Bacteria, and Lactobacillus jensenii. The dominant vaginal flora varies among different races. For example, the dominant vaginal flora in healthy black women is primarily Type IV, which is dominated by multiple dominant bacteria. Does this mean that vaginal Lactobacillus preparations are needed for Type IV vaginal microecology? Moreover, the vaginal Lactobacillus preparations currently used in clinical practice are not common dominant bacteria in the vagina of healthy women. How significant is the application of non-dominant vaginal flora within the body?Insufficient Evidence for High Cost-Benefit RatiosFrom the perspective of cost-benefit ratios, most vaginal infections with identified pathogens currently use lactobacillus preparations as part of combination therapy, which increases both the economic burden and time costs for patients. Is this worth it when considering the cost-benefit ratio? These are pressing issues that need to be addressed. 13.1.6 Influence of Combination Drugs on Lactobacillus Combination Therapy with Antibiotics and Probiotics The combination therapy of antibiotics and probiotics includes two methods: First, combined therapy, where antibiotics and probiotics are used simultaneously. This method has better patient compliance, making it the most commonly used clinical approach. However, it is important to note that since multiple antibiotics can kill or inhibit lactobacilli, when using both antibiotics and probiotics, they should generally be administered at intervals of 12 hours in the morning and evening; otherwise, the live bacterial count of lactobacilli will be affected, reducing their efficacy. Second, sequential therapy, which involves first using antibiotics to control infection and then using probiotics to restore micro-ecological balance. In a systematic review evaluating the efficacy and safety of Lactobacillus preparations for treating bacterial vaginosis, 11 randomized controlled trials (RCTs) involving 1,316 patients reached the following conclusions: compared to monotherapy with metronidazole, the combination of metronidazole and lactobacilli reduced BV recurrence by 95%, while sequential treatment reduced BV recurrence by 75%. Another systematic
210 Chapter 13review comparing the effects of monotherapy with miconazole and sequential treatment with lactobacillus preparations for simple vulvovaginal candidiasis included 8 RCTs involving 5,156 patients and concluded that the recurrence rate of monotherapy was 3.7 times higher than that of sequential treatment, and the recurrence rate of monotherapy was 12.9 times higher than that of simultaneous combined treatment. However, in the analysis of effects, the subgroup analysis of simultaneous treatment within the combined treatment group used a random effects model due to the inability to exclude heterogeneity, which weakened the impact of large-sample trials and amplified the influence of small-sample trials. In summary, studies have shown that the combination of antibiotics and probiotics is more effective, while monotherapy with antibiotics has a higher recurrence rate. Alkaline Wash Solution The alkaline wash will increase the pH value of the vagina, which is unfavorable to the growth of Lactobacillus and leads to a decrease in colonization in the vagina. It is recommended that alkaline wash should not be used in the treatment of lactobacillus. Apart from the above two drugs, no reports have been seen of other drugs affecting lactobacillus preparations. Additionally, if using live Lactobacillus preparations, to ensure their growth and colonization in the vagina, it is necessary to avoid factors such as temperature and pH that could affect the live bacteria. Transportation and storage should be carried out according to the manufacturer's instructions for cold chain.13.1.7 The Application of Lactobacillus Preparations in the Treatment of VaginitisStudies have shown that vaginal inflammation (disease) is better treated with routine treatment and supplemented with Lactobacillus preparations, resulting in lower recurrence rates. The increase of vaginal lactobacillus is more conducive to the recovery of vaginal microecological balance. Application of Lactobacillus in BV Treatment BV refers to the reduction or absence of dominant lactobacilli in the vagina, leading to the overgrowth and adhesion of anaerobic bacteria such as Gardnerella on the edges of epithelial cells, causing the cell margins to become indistinct, forming characteristic "clue cells." BV is a common reproductive tract infection in women of childbearing age, with an infection
Vaginal Microecological Treatment 211rate of about 15%-50% in China. The treatment usually involves the use of antibiotics, but the recurrence rate after discontinuation is high, reaching up to 70% at four weeks follow-up. Prolonging the duration of antibiotic use also fails to prevent recurrence. Numerous studies have shown that combining antibiotics with live lactobacilli can significantly reduce the recurrence rate after discontinuation. Therefore, one of the key aspects of microecological therapy in BV is restoring the dominance of vaginal Lactobacillus to achieve a normal vaginal microecological balance.As early as 1996, Shalev et al. used Lactobacillus probiotic preparations to treat BV and found that drinking yogurt containing active Lactobacillus could significantly reduce the occurrence of BV. However, subsequent studies showed that not all Lactobacillus strains have the same biological activity, leading to conflicting results. It wasn't until 2001 that Reid first reported that after orally ingesting defatted cow's milk containing>109 CFU of Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14, two specific Lactobacillus strains, daily in the morning and evening, Lactobacillus reached the urogenital tract from the digestive tract, settled and proliferated there, restoring normal vaginal flora while altering the gut microbiota environment. This intervention inhibited and replaced pathogenic microorganisms in the gastrointestinal tract, reducing the likelihood of these microorganisms transferring from the gastrointestinal tract, especially the rectum, to the reproductive tract, preventing the adhesion and growth of pathogenic microorganisms in the vagina. Subsequent studies targeting BV patients with a Nugent score of 7-10 showed that oral administration of Lactobacillus combined with antibiotics helped normalize the Nugent scores of the subjects over both short-term and long-term periods, and also improved patient tolerance. Currently, the main approach in China is to use vaginal Lactobacillus preparations. Studies have found that vaginal Lactobacillus can significantly reduce the number of Enterobacteriaceae and yeast in the vagina, demonstrating that exogenous lactobacilli enhance the acidic environment of the vagina, maintaining its acidic barrier and biological defense, effectively inhibiting pathogenic microorganisms. Clinical trials on BV patients have shown that after using lactobacillus preparations, the number of vaginal Lactobacillus has increased significantly, correcting the imbalance of vaginal flora, inhibiting the growth and reproduction of pathogenic bacteria, and effectively improving the vaginal microenvironment, with good therapeutic effects on BV and no significant side effects. In a comparative analysis of BV treatment efficacy, the
212 Chapter 13combination of lactobacillus and antibiotics showed higher effectiveness rates compared to antibiotic therapy alone, lower incidence of adverse reactions, and significantly lower recurrence rates at 1 and 3 months post-treatment. Microscopic examination revealed an increase in lactobacilli numbers, with rising levels of vaginal lactobacilli, gradually restoring the balance of the microenvironment and eliminating microbial dysbiosis. The combined use of Lactobacillus preparations and antibiotics can effectively replenish the number of vaginal lactobacilli, restore the balance of the vaginal microenvironment, and achieve higher cure rates and overall effectiveness compared to antibiotic therapy alone. Therefore, both oral and vaginal Lactobacillus preparations have certain effects on BV, whether they are used alone or in combination. The Application of Lactobacillus in the Treatment of Vulvovaginal Candidiasis (VVC) Candida species is a conditional pathogen; 10-20% of healthy non-pregnant women and 30% of healthy pregnant women can have Candida isolated from their vaginas. Colonized Candida does not cause clinical symptoms, but when the body's resistance decreases or local mucosa is damaged, the overgrowth of yeast can lead to vulvovaginal candidiasis. Currently, the treatment for vulvovaginal candidiasis generally involves topical vaginal or (and) oral antifungal medications such as clotrimazole. The use of antifungal drugs may have certain adverse reactions. Can Lctobacillus preparations be used to treat vaginal candidiasis? There is currently significant debate on this topic. Early epidemiological studies showed that the vaginal bacterial culture of VVC women was predominantly Lactobacillus, and some studies suggested no significant correlation between Lactobacillus and Candida species. Lactobacillus can coexist with Candida without inhibiting or destroying each other. Another study found no significant difference in the incidence of vulvovaginal candidiasis between daily consumption of Yogurt containing Lactobacillus acidophilus(L. acidophilus) and that of pasteurized Yogurt. However, there are scholars who hold opposite views. They studied patients with recurrent vulvovaginal candidiasis (RVVC) who consumed 8 ounces of Yogurt containing L. acidophilus for six months, which significantly reduced vaginal yeast infections and colonization. Unfortunately, this trial lacked a control group, and the high dropout rate led to a small final sample size. Opinions vary on whether Lactobacillus is related to Candida. Recently, domestic researchers used second-generation sequencing 16S rDNA methods to find that the abundance of Lactobacillus in the vagina of women with VVC was significantly lower
Vaginal Microecological Treatment 213than in healthy women, suggesting that Candida albicans (C.albicans) inhibits Lactobacillus growth through the production of mycotoxin-like substances (gliotoxin). Subsequently, using multi-species mixed culture techniques and colony-forming unit cultivation techniques, it was discovered that Lactobacillus inhibit cellular synthesis of C.albicans, possibly by regulating the pH of the vagina and culture medium; some researchers explored the mechanism by which Lactobacillus inhibits C.albicans from becoming pathogenic, suggesting that Lactobacillus may directly inhibit the budding of C.albicans, competitively inhibit its adhesion to vaginal epithelial cells, and compete for nutrients with C.albicans; moreover, Lactobacillus produces H2O2, lactic acid, lactoferrin B, antimicrobial peptides, and other substances that inhibit C. albicans growth and reduce vaginal pH. However, only a few Lactobacillus strains isolated from healthy women's vaginas, including L. acidophilus, Lactobacillus casei, Lactobacillus salivarius, and Lactobacillus jensenii, can inhibit C.albicans growth, with L. acidophilus showing the strongest inhibitory effect against C.albicans, which may also be related to the production of hydrogen peroxide by L. acidophilus. Unfortunately, commonly used Lactobacillus preparations such as GR-1, RC-14, and DM8909 do not inhibit Candida species and are almost ineffective in treating or alleviating VVC. However, literature reports that Lactobacillus can help restore vaginal flora imbalance after the use of antifungal drugs. Studies on the combination of antifungal drugs and Lactobacillus have shown that patients receiving Lactobacillus have higher overall treatment efficacy, faster symptom resolution; lower recurrence rates, especially after 12 weeks; and fewer adverse reactions. It can be concluded that supplementing with Lactobacillus effectively prevents vaginal flora imbalance during antifungal therapy, does not increase adverse reactions, and reduces recurrence rates. However, there is still considerable controversy regarding the effectiveness of Lactobacillus preparations in treating VVC, and more basic and clinical research is needed to confirm these findings. Application of Lactobacillus in the Treatment of Trichomoniasis Vaginitis (TV)The number of lactobacilli in vagina with TV is generally significantly reduced. Some studies suggest that Trichomonas vaginalis may swallow lactobacilli as nutrition or directly act on lactobacilli to kill it; or act on the protein secreted by lactobacilli to make it inactive. The role of Lactobacillus in treating TV is currently controversial. Some studies suggest that Lactobacillus
214 Chapter 13has a significant inhibitory effect on the growth of Trichomonas vaginalis in the vagina. Researchers found that both metronidazole and Lactobacillus preparations showed similar short-term efficacy in treating patients with TV, but the cure rates at 3 and 6 months were higher for the Lactobacillus group compared to the metronidazole group. The overall effectiveness of Lactobacillus preparation was high, with a noticeable increase in vaginal lactobacilli counts and a significant decrease in vaginal pH levels after treatment. It is believed that exogenous Lactobacillus enhances the acidic environment of the vagina and inhibits Trichomonas growth. This effect is related to the concentration of lactobacilli; when the concentration is 10^7 CFU/ml, Trichomonas can consume Lactobacillus for nutrition, leading to a reduction in lactobacilli numbers and a higher infection rate, similar to the condition in human vaginas; however, at 10^9 CFU/ml, Lactobacillus does not provide an ideal environment for Trichomonas growth. Instead, Lactobacillus competes with Trichomonas and stimulates the immune system, producing H2O2, thus reducing the survival rate of Trichomonas. High concentrations of Lactobacillus can inhibit Trichomonas growth, but this is not always positively correlated; at a concentration of 0.5×10^8/ml, the mortality rate of Trichomonas is highest. Therefore, within a certain concentration range, Lactobacillus has the strongest inhibitory effect on Trichomonas. In summary, more evidence is needed to prove the applicability of Lactobacillus preparation in treating TV. The application of Lactobacillus in the treatment of aerobic vaginitis (AV) AV was first described by Donders in 2002. Unlike traditional BV, which is caused by anaerobic bacteria such as Gardnerella, AV is typically due to vaginal flora imbalance caused by facultative anaerobes and concurrent vaginitis. In severe cases, vaginal discharge may contain parabasal cells. There is no consensus on the description of AV, but it is generally considered a conditionally pathogenic state. Common opportunistic pathogens for AV include Escherichia coli, Group B Streptococcus (GBS), Enterococcus faecalis, Staphylococcus aureus, etc. Some studies suggest that Lactobacillus has inhibitory effects on AV pathogens such as GBS and Escherichia coli. In vitro experiments show that Lactobacillus primarily inhibits GBS growth through protonated lactic acid. Oral administration of Lactobacillus microecological preparations can turn GBS-positive women from 35 to 37 weeks of pregnancy negative, reducing the colonization of GBS in the gut and vagina. Currently, more research is needed on the therapeutic effects of Lactobacillus in treating AV.
Vaginal Microecological Treatment 215The Application of Lactobacillus in Vaginal Microbiota Inhibition According to "the Expert Consensus on the Clinical Application of Evaluation of Vaginal Microecology of China (2016)", vaginal microbiota inhibition refers to a significant reduction in bacteria in vaginal discharge, characterized by the absence or presence of dominant bacteria and a low flora density, with the density being less than 1-9 per oil immersion microscopy. The flora diversity is also below grade I, meaning that only 1-3 types can be identified. Women with suppressed vaginal microbiota have fewer bacteria in their vagina, making them more susceptible to pathogenic microorganisms. Moreover, women with suppressed vaginal microbiota often have low levels of estrogen. Treatment options include estrogen-based drugs like norethisterone, antibiotics primarily composed of metronidazole, and microecological agents such as Lactobacillus vaginal capsules. During clinical treatment, the supplementation of estrogen and local application of antibiotics are limited by their adverse reactions. Lactobacillus can directly replenish the dominant vaginal bacteria. Both Lactobacillus and lactic acid-producing bacterial preparations generate lactic acid, maintaining an acidic environment in the vagina, which further promotes the recovery of the vaginal microecology. Studies have shown that using Lactobacillus vaginal capsules can improve clinical symptoms in patients with suppressed microbiota, reduce vaginal pH levels, and enhance vaginal cleanliness. The Application of Lactobacillus in the Treatment of Vaginitis During Pregnancy and Perinatal Period Studies on the treatment of vaginitis during mid-to-late pregnancy and its perinatal outcomes show that using lactobacilli for vaginal inflammation has a high overall effectiveness rate. It also reduces the incidence of miscarriage, preterm birth, premature rupture of membranes, low birth weight infants, neonatal infections, and puerperal infections. Other studies have reported that changes in vaginal microecology and the occurrence of genital tract infections are the primary causative factors leading to premature rupture of membranes. Therefore, using lactobacilli to adjust the vaginal microecology is of great significance for the optimal health of patients with vaginitis during pregnancy. Other Applications of Lactobacillus Studies show that the use of vaginal Lactobacillus may reduce the persistent infection rate of high-risk human papillomavirus (HR-HPV) in the cervix, primarily observed within the first 1 to 2 years of treatment, which is consistent
216 Chapter 13with the reversal rate of cervical intraepithelial neoplasia stage I (CIN I). The mechanism may be due to lactobacilli adjusting the vaginal environment, thereby increasing the clearance rate and reducing high-risk factors for HR-HPV infection and CIN formation. Moreover, the combination of Lactobacillus preparations, interferon α-2b suppositories, and LEEP therapy for CIN with HR-HPV infection may promote postoperative wound healing, reduce vaginal bleeding, shorten the duration of vaginal discharge, and decrease postoperative complications. In summary, using Lactobacillus preparations to increase the number of lactobacilli in the vagina can restore the weakly acidic environment of the vagina, promote the balance of vaginal microecology and immune regulation. Moreover, it is non-irritating to the vaginal mucosa, with no significant adverse reactions reported. It can be used for regulating the vaginal microecology in patients with various types of vaginitis, improving the cure rate of vaginitis, and reducing its recurrence rate. This is an inevitable trend in the treatment of reproductive tract infections and has significant importance for enhancing women’s quality of life. 13.2 PrebioticsIn 2017, the International Scientific Association for Probiotics and Prebiotics (ISAPP) defined prebiotics as substances selected by the host’s microflora that are beneficial to the host’s health. In simpler terms, prebiotics refer to food for probiotics, which can be utilized by the normal vaginal flora but cannot be digested by the human body. They selectively stimulate the growth and activity of beneficial bacteria in the vagina while inhibiting the growth of harmful bacteria. Therefore, prebiotics play a crucial role in restoring the ecological balance of the vaginal microbiota. 13.2.1 The Application Conditions that Meet the Prebiotic Preparations The application conditions for prebiotic formulations must include at least three aspects: First, the prebiotics must be able to reach the vagina; oral prebiotics cannot be digested and absorbed by the body and have the ability to resist host digestive enzymes; Second, the prebiotics can be utilized by vaginal bacteria and fermented by vaginal microorganisms; Third, the prebiotics should selectively stimulate the growth of health-related vaginal bacteria and not be utilized by pathogenic vaginal microorganisms. For intestinal prebiotics, three types of prebiotics are generally recognized as meeting the requirements: inulin,
Vaginal Microecological Treatment 217fructooligosaccharides, and galactooligosaccharides. Additionally, some soluble dietary fibers and oligosaccharides also meet these requirements. Extensive research has confirmed that using vaginal microecological preparations, such as vaginal lactobacillus gel (containing 225 mg of lactic acid and 5 g of glycogen-type prebiotics), can effectively treat BV and prevent recurrence. 13.2.2 The Research Stage of Prebiotic Preparation Research on prebiotics can roughly be divided into three stages. The first stage involves the isolation and screening of probiotics, which is the most fundamental work. This stage uses pure culture and biochemical methods to isolate and screen out high-quality strains of Lactobacillus and other probiotic bacteria. The second stage involves in vitro experiments simulating the vaginal environment and animal model studies to verify the probiotic benefits of prebiotics on probiotics. The third stage conducts clinical trials, applying prebiotics with excellent probiotic properties from animal models to clinical control trials. In recent years, various population-scale trials have been conducted to test the effects of prebiotics. However, the results of clinical studies are not consistent. The next direction for research should focus on understanding the mechanisms by which prebiotics work, identifying how they function in the host's vagina, and selecting more effective components for specific individuals or scenarios to enhance the performance of prebiotics. 13.2.3 Clinical Types of Prebiotic Preparations Prebiotic formulations are typically oligosaccharides, such as lactulose, fructooligosaccharides, glucose, galactose, and soy oligosaccharides. This includes oligosaccharide gels, etc. In addition to the carbohydrate prebiotics mentioned earlier, there are also plant protein prebiotics. Gynecological fluid dressings (plant proteins, soy peptides, carrot extracts, tea tree oil, etc.) are a new type of microecological regulator containing prebiotic-plant proteins, which serve as nutrients for Lactobacillus. They can stimulate the growth of Lactobacillus in the vagina, restoring its dominant position among beneficial bacteria. Their pH ranges from 3.8 to 4.5, which is the physiological pH value for the vagina. Using gynecological fluid dressings for irrigation therapy can quickly restore the normal pH environment of the vagina, promote the growth of autologous Lactobacillus, and inhibit the proliferation of pathogenic bacteria. The trace amounts of carrot extract in gynecological fluid dressings
218 Chapter 13can exert estrogen-like effects, nourish and repair damaged vaginal mucosa, and enhance the immunity and resistance of the mucosa. Therefore, gynecological fluid dressings can simultaneously regulate the three core elements of vaginal microecology, working together with antibiotics to effectively reduce the recurrence of gynecological inflammation. 13.2.4 Clinical Application of Prebiotics Preparations Studies have shown that the combined use of antibiotics and prebiotics can improve the recovery rate in patients with BV, RVVC, etc., and reduce recurrence. Although the overall effectiveness is not significantly higher than using antibiotics alone, after prebiotic treatment, patients exhibit low levels of vaginal pH, high numbers of lactobacilli, and low levels of leukocyte esterase. After BV patients were cured with oligosaccharide gel, L.salivarius strain, L.gasseri strain, and L.crispatus strain were isolated from vaginal discharge, indicating that oligosaccharide gel is an effective new microecological regulator for treating BV, capable of restoring a normal vaginal flora dominated by Lactobacillus species. In summary, there are currently fewer prebiotics that can be applied to the vagina, and their evidence is not yet sufficient or comprehensive. In the future, the use of prebiotics should be based on the characteristics of the host's vaginal microbiota, with targeted interventions conducted after a thorough understanding of the composition and structure of the vaginal microbiota. Focus on the interaction between prebiotics and the host's indigenous microbiota, identify the microbial types responsible for prebiotic effects, and explore the impact of prebiotics on the host's physiological metabolism, thereby achieving personalized prebiotic interventions.13.3 Repair of Vaginal MucosaVaginal microecological imbalance is often accompanied by vaginal mucosal damage, promoting mucosal proliferation, helping damaged mucosa to heal, and helping local epithelial immune function recovery are also the direction of vaginal microecological treatment. 13.3.1 Estrogen Estrogen is a crucial factor in the vaginal defense system. It stimulates the proliferation and division of basal cells, promotes keratinocyte formation, repairs
Vaginal Microecological Treatment 219damaged vaginal mucosa, enhances the resistance of the vaginal mucosa, and maintains vaginal elasticity. At the same time, estrogen helps maintain the micro-ecological environment of the vagina, stimulating the thickening and glycogen-rich growth of vaginal epithelium. The generated glycogen is broken down by lactobacilli in the vagina, lowering the pH value and inhibiting the proliferation of pathogenic bacteria. 13.3.2 Hyaluronic acid Hyaluronic acid is the primary polysaccharide in the extracellular matrix, responsible for nourishing and hydrating epithelial cells, involving tissue repair processes, regulating inflammatory responses, and promoting the formation of epithelial cells. Studies on the effects of hyaluronic acid on the growth of vaginal epithelial cell lines, VK2 cells (ATCC-CRL2616), have found that a hyaluronic acid derivative vaginal gel at a concentration of 24mg/ml can promote Th1-mediated cellular immune responses and repair vaginal mucosal epithelial cells; while the role of hyaluronic acid vaginal gel in castrated rat vaginal epithelium indicates that it can effectively alleviate vaginal atrophy, improve the local microenvironment of the vagina, and enhance the repair capacity of vaginal epithelium. 13.3.3 Collagen Collagen is a biomolecule and the most abundant functional protein in the human body. Collagen is the primary component of skin and mucous membranes, providing elastic fibers and building blocks for collagen formation, acting as ligands or binding to fibroblast receptors to stimulate hyaluronic acid synthesis.There are several types of collagen, with common forms including Type I, II, III, V, and XI. In normal skin tissue, collagen primarily exists in the form of Type I and III collagen fibers. Type I and III collagen are closely related to the process and quality of skin injury repair. In fetal skin, Type III collagen accounts for 60%, and the lack of a scar healing mechanism in fetuses is due to their strong ability to synthesize Type III collagen. As growth and development occur, Type III collagen decreases while Type I collagen increases. In adult skin, the collagen composition is 80% Type I and 20% Type III. Recombinant human collagen is based on the original gene sequence of type III human skin collagen. It involves optimizing and splicing parts with strong water solubility and high biological activity, resulting in a new recombinant human
220 Chapter 13type III collagen sequence. This sequence can be produced on a large scale using biotechnology fermentation techniques. Further optimization enhances the hydrophilicity and activity of the collagen. Recombinant human collagen contains a large number of hydrophilic genes, which gives it excellent film-forming properties and helps retain moisture in the stratum corneum of the skin. Additionally, its directional guidance ability allows epithelial cells to quickly enter damaged areas, effectively accelerating skin regeneration and shortening wound healing time, thereby restoring the skin barrier function. When applied to the vagina, it can restore vaginal elasticity, alleviate vaginal atrophy, repair damaged elastic fibers, and speed up tissue and wound healing; its design mimics physiological extracellular matrix, improving vaginal atrophy, enhancing vaginal elasticity, and restoring the youthful state of the reproductive tract. The main component of recombinant human collagen vaginal gel is recombinant human collagen, supplemented with poloxamer and sodium hyaluronate. Collagen and sodium hyaluronate have the function of relieving vaginal dryness. Poloxamer is a new type of high molecular biological material that naturally forms a uniform gel upon contact with body temperature, diffusing evenly over the tissue surface to form a protective layer. This allows the recombinant human collagen to maintain prolonged contact with the tissue surface, enhancing its bioavailability and achieving repair effects. Using recombinant human collagen vaginal gel to treat atrophic vaginitis has shown significant efficacy, but its effectiveness alone is not as good as when combined with other drugs. The mechanism involves the formation of a mesh-like structure on the mucosal surface by the recombinant human collagen vaginal gel. After vaginal atrophy, the gaps between keratinocytes increase. Similar to the keratinocyte structure, collagen can penetrate into the keratin layer to form a film, replenishing the collagen deficiency in the dermis and restoring elasticity. It also promotes fibroblast proliferation. However, there is limited research on recombinant human collagen vaginal gel, and more studies are needed to demonstrate its efficacy in vaginal microecological therapy.13.4 Regulation of Vaginal EndocrineThe vaginal micro-ecosystem is a dynamic equilibrium system influenced by various factors, including ovarian function, pregnancy, and the menstrual cycle. Estrogen thickens the vaginal epithelium and increases intracellular glycogen, providing ample nutrients for microbial growth. Estrogen not only binds to estrogen receptors on the vaginal epithelium but also affects bacterial
Vaginal Microecological Treatment 221adhesion by influencing the bioelectric potential between bacteria and the vaginal epithelium; progesterone can cause the shedding of surface vaginal cells, leading to the detachment of vaginal bacteria attached to these cells, resulting in instability of the vaginal flora. When estrogen levels are low, lactobacilli decrease. Therefore, when treating vaginitis with reduced estrogen levels, if there are no contraindications for estrogen use (such as endometrial disease where estrogen use increases the risk of endometrial cancer), estrogen can be used for treatment (see Chapter 9 "Atrophic Vaginitis" 9.6 "Treatment").13.5 Correction of Vaginal pH ValueThe vagina is a slightly acidic environment (pH 3.8-4.5), serving as a natural barrier. Once this barrier is compromised, it becomes susceptible to infections caused by pathogenic microorganisms, leading to various vaginal inflammations. To restore the pH of the vagina, methods include using mild acidic washes and other physical means, employing chemical agents that regulate pH levels; increasing the number of lactobacilli in the vagina, such as using medications containing acidic substances like 1% lactic acid or 0.5% acetic acid for vaginal irrigation; using highly acidic substances like polychlorosulfonated paraffin, which can destroy bacteria through high acidity and protein coagulation, selectively causing necrosis or deformation of diseased tissues and columnar epithelial cells, facilitating the selective coagulation and removal of necrotic or diseased tissue while leaving normal squamous epithelium unaffected, promoting tissue regeneration and re-epithelialization; these methods also eliminate various pathogenic microorganisms within the vagina while protecting the growth of physiological flora, maintaining the acidic environment of the vagina. Studies show that lactic acid and ionized lactate in the vagina are in a dynamic equilibrium. In a healthy vaginal environment with a pH <4.5, ionized lactate exerts strong bactericidal effects, with a pH threshold of 3.9-4.0. When the vaginal microecosystem is disrupted and the pH> 4.5, lactate anions lose their bactericidal activity. To maintain lactate as ionized form, foreign countries commonly use vaginal lactate gels such as Bayer canesbalance, low-polymer lactate vaginal suppositories like vagisan, and releasable lactate vaginal rings. Vaginal lactate gels have better tolerance compared to antibiotics, and when combined with antibiotic therapy, they show significant efficacy and can significantly reduce recurrence. Lactic acid remains protonated below pKa 3.9 and lactate ion form above pKa 3.9. Protonated lactic acid has membrane permeability; once inside
222 Chapter 13cells, it disrupts the cell membranes of Gram-negative bacteria, altering their permeability and causing cellular dysfunction, ultimately leading to bacterial death. Additionally, studies have found that in BV women, lactate levels and neutrophil gelatinase-associated lipocalin (neutrophil gelatinase-associated lipocalin, NGAL) are significantly lower compared to normal women. Lactate is positively correlated with NGAL levels, which can inhibit the uptake of iron by Gram-negative bacteria as a nutrient, suggesting that lactate may prevent BV by increasing NGAL levels. The inhibitory effect of lactic acid on Candida albicans is not yet clear. In women with VVC, the concentrations of L-and D-type lactic acid in the vagina are similar to those in normal women. In vitro experiments show that the supernatant of Lactobacillus from healthy women's vaginas has no inhibitory effect on Candida albicans, but Candida albicans can promote the production of extracellular matrix metalloproteinase inducer (extracellular matrix metalloproteinase inducer, EMMPRIN) by vaginal epithelial cells. EMMPRIN can work together with MCT on the surface of vaginal epithelial cells to transport lactic acid into or out of the cell, maintaining normal levels of lactic acid within the cell and inhibiting lactic acid-mediated cell death. Besides vaginal epithelial cells, MCT is also expressed on the surface of fungal cells, which can work together with EMMPRIN produced by vaginal epithelial cells to regulate the level of lactic acid inside fungal cells, protecting fungi from the influence of lactic acid. On the other hand, both sugars and lactic acid can serve as energy sources for Candida albicans. Compared to sugars, Candida albicans grown using lactic acid (Concentration: 178 mmol/L, pH 3.0 ~ 7.5) shows reduced growth rate and inhibited hypha formation; physiological levels of lactic acid in the vagina (Concentration: 22 mmol/L, pH 4.2) can enhance the inhibitory effect of clotrimazole on fungi; lactic acid (Concentration: 65 mmol/L, pH 4.5) affects gene transcription in Candida albicans, upregulates iron transporters, reduces intracellular iron concentration, and downregulates RNA and ribosome synthesis. The effects of lactic acid on Candida albicans and VVC require further research. In summary, restoring the normal pH environment of the vagina can promote the growth of autologous Lactobacillus and inhibit the proliferation of pathogenic microorganisms.13.6 The Role of Chinese Medicine in Vaginal Microecology TreatmentVaginal microecological imbalance is categorized as "leukorrheal diseases" or "vulvar itching" in traditional Chinese medicine (TCM). The term "leukorrheal
Vaginal Microecological Treatment 223diseases" first appeared in the‘Suwen: Discussion on Bones and Organs’, where it states, "When the Conception Vessel is diseased... women experience leukorrhea with masses." Physiological leukorrhea is a clear, transparent fluid of moderate volume, odorless, and free from fishy or foul smells, secreted by healthy females. In TCM, leucorrhea is associated with dampness; treatment primarily focuses on drying dampness; or due to spleen and stomach deficiency leading to yang qi sinking, which is treated by strengthening the spleen, uplifting yang, and stopping leukorrhea; or caused by dampness. Internal causes include spleen and kidney yang deficiency, where yang deficiency leads to water retention and dampness, affecting the lower burner; external causes involve invasion by cold-dampness, causing damage to the belt vessel. Vulvar itching refers to external genital itching in women. In TCM, internal causes of vulvar itching are yin deficiency and liver-kidney dysfunction, while external causes are damp-heat generating parasites and their toxins. TCM supports vaginal microecological therapy through both oral and topical applications. 13.6.1 Oral Medication According to reports, TCM internal medications for vaginitis include softshell turtle shell, achyranthes root, tree peony bark, and yam, with commonly used formulas such as Zhibai Dihuang Pills and Nourishing Yin and Consolidating Chong Decoction. Internal dosage forms mainly include decoctions, pills, and powders. Taking Zhibai Dihuang Pills as an example, this medicine originates from‘Medical Mirror’, which is the Six-Ingredient Dihuang Pills plus Anemarrhena and Phellodendron. In the formula, Anemarrhena clears heat and nourishes yin, Phellodendron purges fire, dries dampness, detoxifies, and reduces fever, Rehmannia tonifies the kidneys, fills essence, nourishes yin, and replenishes blood, Cornus officinalis astringes yin and consolidates, Poria strengthens the spleen, promotes diuresis, and calms the mind, Moutan bark clears heat and cools blood, and Alisma promotes diuresis and clears heat. The combination of these herbs treats both symptoms and root causes, tonifies liver and kidney functions, nourishes yin, clears heat, and removes dampness. However, overall, there is less research on the use of TCM internal medications for treating vaginitis, and self-medication is not recommended. 13.6.2 External Medicine According to the different methods of using topical Chinese medicine for vaginitis, they can be divided into irrigation, fumigation and washing, sitz baths,
224 Chapter 13and vaginal administration. Among these, common herbs used in irrigation and fumigation and washing sitz baths include Cnidium monnieri, Geranium, Smilax glabra, Phellodendron bark, and Borneol, with the main therapeutic principles being to nourish yin, clear heat, kill parasites, and dry dampness. Depending on clinical needs, decoctions such as Baijie Wash or TCM preparations like Bao Fu Kang Suppositories can be used. Currently, the application rate of topical medications is higher than that of oral Chinese medicine in clinical practice, which is related to the convenience of use and direct action on the affected area. More importantly, topical medications do not need to be metabolized by the liver, kidneys, and other metabolic organs, significantly enhancing their safety compared to oral medications. The Combination of Vaginal Medication and External Washing with Chinese MedicineBao Fu Kang suppositories can be used in combination with external herbal washes to treat vaginal microecological imbalance. Composition of the fumigation and washing medicine: Sophora flavescens 30g, alum 15g, Dictamnus dasycarpus 30g, Cnidium monnieri 50g, Euphorbia peplus 30g, Kochia scoparia 30g, Phellodendron amurense 20g. Starting from the third day after menstruation ends, first rinse the external genitalia with warm water, then take about 1000mL of the medicinal solution to fumigate and wash the external genitalia and vagina, followed by a sitz bath for 20 minutes, and place one Bao Fu Kang suppository in the vagina once daily for a course of 7 days.Vaginal Douche Ligustrum Wash can treat vaginal microecological imbalance. The formula of Ligustrum Wash consists of: 600g Ligustrum, 500g Sophora, 500g Phellodendron, 500g Cassia Seed, and 50g Menthol Oil. Dilute with physiological saline, then wipe the external genitalia and rinse the vagina. Use once daily for 7 consecutive days. Fumigation and Sitting Bath Method The fumigation method accelerates local blood circulation, facilitating the absorption of medication and aiding its direct delivery to the affected area. Yinyao Le can be used for local fumigation. The formula composition of Yinyao Le is: Phellodendron bark 100g, Sophora flavescens 120g, Cnidium monnieri 100g, Thunderball 30g, Areca nut 30g. Boil the above herbs to obtain 250mL of decoction, then add 1000mL of boiling water. When the temperature is high, steam the external genitalia; when it cools down to a suitable temperature,
Vaginal Microecological Treatment 225take a sitting bath. Another formula composition and method can also be used: Cnidium monnieri, Sophora flavescens, Phellodendron bark each 30g, Sichuan pepper 15g. Boil these herbs in 2000mL of water until boiling, filter, and steam the external genitalia at high temperatures. When the water temperature cools down to a suitable level, take a sitting bath. Place one Dakenning suppository in the vagina once daily for seven consecutive days. Although TCM and prepared Chinese medicines are currently used in clinical settings, the pharmacological effects of TCM cannot yet be fully explained. Can our next research efforts clearly identify the specific components that play a role in TCM and further isolate and purify them? For example, studies using propolis tincture to observe its efficacy against Escherichia coli vaginitis in mice have found that the number of bacteria in the vagina of mice treated with propolis tincture was significantly reduced compared to those in the control group, and the degree of mucosal inflammation recovery in the treatment group was significantly better than in the control group. Traditional Chinese medical literature records that propolis has the functions of moisturizing the skin, promoting tissue regeneration, detoxifying, antibacterial, and relieving pain. Western medicine has discovered that propolis contains flavonoids, phenols, lactones, aldehydes, ketones, steroids, and other compounds, which are non-toxic, non-irritating, and non-carcinogenic, and possess antibacterial, antiviral, immune-stimulating, analgesic, and tissue-regenerative properties. The integration of traditional Chinese and Western medicine is beneficial for the promotion and development of TCM. Guided by TCM diagnostic methods and the concept of "treating before disease," we can start from syndrome research and intervene with TCM to leverage its role in public health and well-being.13.7 SummaryCurrently, the treatment of vaginal microecological imbalance primarily involves using sensitive antibiotics to inhibit or eliminate pathogenic microorganisms. The targets for elimination include overgrowing aerobic or anaerobic bacteria, budding spores, or pseudohyphae such as Candida and Trichomonas vaginalis. Vaginal microecological therapy is conducted simultaneously with or after the elimination of these pathogens, aiming to restore the vaginal microenvironment, which is the ultimate goal in treating vaginitis. If the vaginal microecological environment has not returned to normal, the suppressed pathogens or introduced pathogens may reproduce again, leading to recurrence or new cases of vaginitis. Under the regulation of estrogen and the promotion of nutrients such as
226 Chapter 13prebiotics, probiotics are supplemented to thrive as dominant microorganisms in the vaginal microbiota. They bind with receptors on the vaginal epithelium, utilizing glycogen to produce lactic acid and other acidic substances or supplementing exogenous acidic substances to correct the vaginal pH level. Probiotics can also generate antimicrobial factors that protect the vaginal mucosa, working synergistically with hyaluronic acid, collagen, and other protective agents to repair the vaginal mucosa and enhance resistance against pathogens. Alternatively, relevant TCM preparations can be used to promote the restoration of the vaginal microenvironment to a normal state. By addressing these five aspects simultaneously, the ultimate goal is to ensure the health of women's reproductive tracts. References[1] Xie Xing, Kong Beihua, Duan Tao. Obstetrics and Gynecology. 9th edition. Beijing: People's Health Publishing House, 2018.7 [2] Collaborative Group on Infectious Diseases of the Chinese Society of Obstetrics and Gynecology. Guidelines for the diagnosis and treatment of bacterial vaginosis (2021 revised edition). Chinese Journal of Obstetrics and Gynecology, 2021,56(1):3-6 [3] Collaborative Group on Infectious Diseases of the Chinese Society of Obstetrics and Gynecology. Expert consensus on the clinical application of vaginal microecological evaluation. Chinese Journal of Obstetrics and Gynecology, 2016,51(10):721-723.
C H A P T E R - 14Operation Specification and Quality Control of Vaginal Microecological ExaminationsYuanting Tang, Zhengqiang Hu, Xiaojuan Liu Translator: Zhengqiang Hu14. 1 Vaginal microecological examination means that, by microscopic examination and functional tests of vaginal discharge, we can observe the morphological indicators such as flora density, flora diversity, dominant bacteria, vaginal cleanliness, Trichomonas Vaginalis, yeast-like fungus (spores, yeasts with budding, and pseudohyphae), Gram-negative (G-) diplococcus and clue cells, and functional indicators such as vaginal pH value, hydrogen peroxide, leukocyte esterase and sialidase. Vaginal microecological examinations are to aid in the clinical understanding of the vaginal microecosystem changes, diagnosing of vulvovaginal candidatosis (VVC), trichomonas vaginitis (TV), bacterial vaginosis (BV), aerobic vaginitis(AV), gonorrhea vaginitis, cytolytic vaginosis (CV), and microecological changes such as flora inhibition and flora overgrowth, putting forward clinically feasible reference opinions. Vaginal microecological examination must be conducted in accordance with the correct operation specifications, and the process includes issuing the application form from the clinician to the laboratory to complete the tests. Quality control is a series of methods and measures to ensure the quality of operation specification of the whole process, including pre-analysis, analysis and post-analysis process.14.1 Pre-analysis ProcessThe pre-analysis process starts from the doctor's prescription and ends with the start of the analytical procedure, including patient preparation and sample processing (Collection, transportation, processing and storage of vaginal discharge samples). The quality of pre-analysis process is a prerequisite for ensuring the correctness and effectiveness of the test results.
228 Chapter 1414.1.1 Sample Collection and ReceptionCollection of Vaginal Discharge Samples a. Collecting equipment • Clean, dry, covered sampling bottles (tubes) and (or) clean slides within the validity period;• One-time sampling swabs or sterile sampling swabs and sampling tubes equipped with instruments. Collection tools include scraper, cotton swab, nylon flocking swab, silicone swab, etc.."Operating Procedures of Clinical Laboratory of China" (Fourth Edition) recommend using cotton swabs soaked with normal saline. Other international and foreign guidelines including International Union against sexually transmitted infections (IUSTI), World Health Organization (WHO), and Centers for Disease Control and Prevention (CDC) of USA do not specify sampling tools. However, it should be noted that if the cotton swab is not soaked in physiological saline before sampling but used directly for sampling, some patients' vaginal discharge are attached to the cotton fibers and difficult to wash off, which may cause insufficient sample amount.• Equipment requirements: the sampling bottle (tube) must be clean, dry and covered. b. Sample Collectors: Vaginal discharge collectors are usually healthcare professionals including clinical doctors from gynecology obstetrics and reproductive medicine. The British association for sexual health and HIV(BASHH) and Australia sexual health alliance (ASHA) consider self-sampling of vaginal discharge to be equivalent to sampling by healthcare professionals. Therefore patients can also be recommended to sample themselves under the guidance of healthcare professionals.c. Sampling method• The doctor will put a disposable sterile endoscope into the vagina, expose the cervix, and use a sampling swab to scrape enough vaginal discharge from the upper 1/3 of the vaginal wall. • For self-sampling, the flow chart is as follows• The appropriate amount of vaginal discharge is directly and evenly coated on sterile slides, the thickness of which must be uniform and appropriate to have no overlapping cells after staining. The smear is put into the sampling bottle, cover to prevent contamination and immediately send it for inspection; or the sampling swab of appropriate vaginal discharge is put into the matching sampling tube directly for inspection.
Operation Specification and Quality Control of Vaginal Microecological Examinations 229Figure 14-1 Flow chart of self-sampling of vaginal discharged. Sample collecting site: The "Operating Procedures of Clinical Laboratory of China" (Fourth Edition) recommends that the collecting site for tests of vaginal discharge should be the deep part of vagina, the posterior fornix, or the upper 1/3 lateral wall of the vagina. Studies have suggested that the vaginal flora on the vaginal upper 1/3 lateral wall best reflects the true vaginal microecological status in women, while the flora from other sites are influenced by various factors. To avoid vaginal injury during sampling in young girls, it is advisable to use sterile swabs to wipe the discharge from both inner and outer labia for sampling. Additionally, the collection sites recommended by the IUSTI and WHO management guidelines of vaginal discharge are the vaginal wall. The BASHH British National Guidelines for the Management of Vulvovaginal Candidiasis recommend collecting vaginal discharge from the anterior fornix to check for Candida species; the BASHH British National Guidelines for the Management of trichomonas vaginitis recommend collecting vaginal discharge from the posterior fornix to check for Trichomonas; the ASHA Guidelines for the Management of Sexually Transmitted Diseases specifically mention that high vaginal swabs should be collected for vulvovaginal candidiasis and trichomoniasis.Therefore, it can be seen that collecting different sites according to different vaginal inflammation may improve the detection rate of the disease, and different
230 Chapter 14collecting sites can be selected according to the symptoms of clinical suspected vaginitis. e. Precautions of sample collection: Good communication between the laboratory and the clinic is an important guarantee to obtain accurate test results. The laboratory must ensure that every clinician knows the patient's precautions before ordering a vaginal microbiome test: • Sample quantity must be sufficient. that is to say, be visible to the naked eye; • Sample collection should avoid menstrual period. It is recommended to take samples from patients 3-7 days after clean menstruation; • Sexual intercourse should not be performed 48 hours before sample collection. • Before sample collection, the topical drugs should be stopped 3 days, and the antibacterial drugs should be stopped 7 days; • Tub baths and vaginal douching should be avoided before sample collection. It should be submitted for tests immediately after collection. The placement time in room temperature should not exceed 2 hours. Vaginal discharge samples that cannot be detected in time should be kept in a 2~8℃ environment, and the tests should be completed as soon as possible. Trichomonas is active at 25~42℃, so simple heat preservation measures should be taken and samples should be sent for examination as soon as possible after sample collection to improve the detection rate. Preservatives, disinfectants or detergents should not be added to the discharge samples. Vaginal discharge that need to be tested functionally should be kept away from direct sunlight.The receipt of the vaginal discharge samples a. Verification of information. When receiving samples, the inspector should check at least two kinds of patient information, such as name, age or registration number, to avoid confusing the samples. If a secondary separation of the sample is required, at least two kinds of patient information should be noted on the separated sample. b. The rejection of samples: The rejection criteria for vaginal discharge include: • Vaginal discharge samples are minimal or absent; • Vaginal discharge smears are extremely thick with severe overlap of cells and bacteria, which affects the microscopic observation; • The samples are contaminated by feces, urine, or the samples are added with disinfectant, etc. • The samples are placed for more than 2 hours without 2-8℃.
Operation Specification and Quality Control of Vaginal Microecological Examinations 231• Sample smears are covered with external drugs, affecting staining and observation. Vaginal discharge samples that meet the above rejection criteria should be rejected. If the clinician requests the test or the patient insists on it, the laboratory should note the rejection of the samples in the remarks column after completing the test, and record the rejection information, notification record and treatment measures in "the Record of Sample Rejection". 14.1.2 Sample Handling Smears Need Gram StainingAfter the smears that need Gram staining is sent to the laboratory, it is dried naturally and fixed before Gram staining. Fixation method: Before Gram staining, the slide can be fixed with ethanol or other methods, but the commonly used method is heating: fix the vaginal discharge smears on the slide heater for about 1 minute at a temperature of 70-80°℃ to prevent deformation of microorganisms or cells due to water evaporation. The purpose of fixation is not only to kill microorganisms or parasites but also to maintain the original morphology and structure of cells, bacteria, and parasites, facilitating staining and observation; it also ensures that bacteria, cells, and parasites are stably attached to the microscope slide to avoid being washed off during staining.Vaginal Swab Samples for Functional Tests or Automatic AnalysisFor vaginal swab samples for vaginal microecological function testing or automated leukorrhea analysis, please add the supporting diluent of corresponding instrument for instrument inspection.14.1.3 Gram Staining Principle of Gram StainingThe Gram staining method, established by Danish bacteriologists Christain Gram, et al in 1884, is widely used in microbial teaching, scientific research and clinical practice, as well as in the diagnosis of various inflammation and diseases in the intestine and vagina. Therefore, it is of great theoretical and practical significance to deeply understand the principle of Gram staining. At present, the principle of Gram staining is not very clear. The dyeing mechanism is more complex. Only one theory can not accurately clarify the mechanism of gram staining. It requires the integration of various theories: a. Physics theory: The bacteria do not form new compounds in combination
232 Chapter 14with dyes, but rather on their own functions. First, adsorption effect: different bacteria can choose different dye liquid ions and show different staining results. Second, the dye solution enters the cells by absorption. b. Chemical theory: Gram staining solution is crystal violet-ammonium oxalate solution. Crystal violet is a tribenzane dye, formula C25H30ClN3. It is a dark green powder, with metallic luster, soluble in water, ethanol, chloroform, insoluble in ether. Due to the σ-π super pooling in the presence of submethyl group in the triphenylmolecules, the transfer of electron cloud to the benzene ring makes the secondary methyl group very active for a certain degree of ionization. After ionization, the negative C ion formed is unstable and electron rearrangement occurs, resulting in increased electron cloud density on N atoms and the loss of electrons, making N atoms with a positive charge, and SP3 hybrid occurs at the same time. The change of structure before and after ionization brings about the change of color, ranging from pH 0.1 to 1.5 to 3.2. Because after ionization of H+, a conjugated base is presented, and because it is purple in the higher range of pH or alkaline environment, it is also called alkaline purple. Crystal violet-ammonium oxalate is ionized in aqueous solution, with a positive charge, electrostatic binding with the negatively charged group in bacterial cells, thus appearing purple. At the same time, because the submethyl group in the crystal violet is affected by the three phenyl groups, it is in a common alkali state under physiological conditions, so it can compete against hydrogen ions with the microbial enzyme system, so that the enzyme becomes an inactive oxidation state and kills bacteria. Ammonium oxalate in solution mainly acts to stabilize and balance the crystal violet structure and the ionization state. c. Isoelectric point theory: The isoelectric point of Gram-positive bacteria is known to be pH value 2~3, and the isoelectric point of Gram-negative bacteria is pH value 4~5. It indicates that the Gram-positive bacterial band has more negative charge than the Gram-negative bacteria and has a strong binding force with crystal violet-ammonium oxalate. Moreover, after dyeing with potassium iodide, the ionic dissociation of groups on bacterial cell walls and inside cells that can be ionized in acid was promoted by the alkaline action of potassium iodide, which decreased its isoelectric point. However, due to the difference in the number of dissociation groups, the isoelectric point of Gram-positive bacteria decreases more than that of Gram-negative bacteria, so it binds more firmly with the dye and has stronger resistance to ethanol decolorization, resulting in the complex formed by the bacterial body and the dye being difficult to be extracted by ethanol and appearing purple. At the same time, Gram-negative bacteria
Operation Specification and Quality Control of Vaginal Microecological Examinations 233have weak binding force with the dye, and their bodies are easily extracted as colorless complexes with ammonium oxalate-violet and iodine-potassium iodide.d. Permeation theory: Ethanol can dehydrate the cell wall peptidoglycan of Gram-positive bacteria, narrowing the cell wall gap and permeability, so that part of the crystalline violet-iodine complex into the bacteria can be retained, and the bacteria body appears purple. Gram-negative bacteria contain less peptidoglycan, little change in the cell wall, and the permeability is not affected. The crystal violet-iodine complex entering the bacteria is easy to be exposed and be removed, and the bacteria are redyed red by compound red.Gram Staining Solution The first solution: crystal violet-ammonium oxalate solution (primary dye). The second liquid: iodine-potassium iodide solution (media colorant). The third liquid: acetone alcohol (Decolorizer, it is recommended to mix anhydrous ethanol with acetone in a 7:3 ratio). The fourth liquid: alkaline compound or sand yellow solution (redye solution). Staining Methods The following staining methods are suitable for both manual staining and automatic instrument staining: a. Primary staining: Add crystal violet-ammonium oxalate solution to the fixed smear, cover the sample film, stain for about 30 seconds, and wash with water; b. Medium staining: After the smear is dried of excess water, add iodine-potassium iodide solution, stain for about 30~60 seconds, and wash with water; c. Decolorization: After the smear is dried of excess water, add the decolorization solution, until no obvious purple continues to fall off, wash with water; d. Re-staining: After the smear is dried of excess water, add alkaline fuchsin, stain for about 10~30 seconds, wash with water; e. Drying: The smear is dried naturally or baked at 70-80℃, and examined under a microscope after drying.Quality Control of the Gram Staininga. Reagent requirements: The commercial reagents should be stored according to the conditions required by the manufacturer's instructions (including temperature, humidity, etc.). The validity period of the reagent is generally changed after bottle opening. For example, the validity period of Gram stain solution is generally 1 month, with the possibility of growth of bacteria exceeds
234 Chapter 141 month. Therefore, the reagent after opening should be affixed with the reagent label. The label content is: reagent name, replacement date, expiry date after opening, replacement personnel, etc.. The self-dispensing reagents should be standardized according to the process of the self-dispensing reagent. b. Preparation of quality control tablets: Take one strain of standard Staphylococcus aureus and one strain of standard Escherichia coli, transfer them to blood agar plates for incubation at 35℃18-24 hours, then use cotton swabs to pick several colonies from each strain, mix them in sterile saline solution, and sterilize them by autoclaving at 121℃15 minutes before storage. Use a inoculation loop to transfer the suspension onto clean microscope slides, dry it with a 70-80℃ slides heating. Perform Gram staining test, ensure that the smear is appropriately thick, clearly distinguish between Gram-negative and Gram-positive bacteria, indicating that the quality control slide is qualified, and store it for later use.c. Quality control method: Take the above quality control tablet, fixing and gram staining it, as a control for Gram-positive bacteria and Gram-negative bacteria. Standard Staphylococcus aureus is stained Gram-positive and standard Escherichia coli is stained Gram-negative are as controlled. Standard Staphylococcus aureus is stained Gram-negative and standard Escherichia coli is stained Gram-positive are as out of control. After the out of control of Gram staining, vaginal discharge smear is shown in Figure 14-2 and Figure 14-3.The Precautions of Gram Staininga. The staining results are often affected by the operator's technology or the performance of the dyeing machine, so the quality control must be done daily to ensure the correctness of the staining results. b. Smear or bacterial liquid should not be too thick, otherwise the film will fall off or not easy to decolinate in the dyeing process, thus lead to false positive. On the other hand, smears should not be too thin, or too few samples can lead to false negatives. c. Strictly control the staining and discoloration time. The first dying time is too long, prone to appear false Gram-positive. The discoloration time is not enough, prone to the bacteria appear false Gram-positive. The decolorization time is too long, the positive bacteria will be dyed negative. If there is water on the smear, the decolorization force is become weaker, easy to form a false positive, so the smear must be drained of excess water and then discoloration. d. Automatic Gram staining instruments are recommended to standardize Gram staining. The Gram staining instruments shall be maintained, with
Operation Specification and Quality Control of Vaginal Microecological Examinations 235Figure 14-2 False positives resulting from insufficient Gram stain discoloration (1000×)Figure 14-3 False negative resulting from insufficient staining of crystal vio-let-ammonium oxalate or excessive discoloration (1000×)
236 Chapter 14adequate reagents and lotion, to ensure that the instrument is in a normal and optimal staining state. e. If the staining is out of control or the effect is not good, the staining slide should be reproduced. f. When flushing, the water should not be perpendicular to the smear, so as not to flush away the film. Staining ResultsGram-positive bacteria are purple, and Gram-negative bacteria are red.14.2 Analyzing ProcessAnalyzing process includes procedures and operating specifications of vaginal microbiological examination, and internal quality control (IQC) of morphological examination and functional tests. It is recommended to conducting IQC at least once a day and form a laboratory-specific IQC chart. For each IQC, the positive IQC product, negative IQC product and routine samples should be operated with the same method and same operator, and the routine examination can only be carried out after the IQC is under control. If the IQC is out of control, the quality control product should be replaced immediately. If it is still out of control, the new batch of reagents should be replaced immediately until the routine experiment can be carried out after under control. If necessary, the samples between the last under control and the latest out of control should be retested, and if the retest results are not consistent, the report issued should be recalled.14.2.1 Vaginal Microbiological Examination ProcessThe vaginal microecological examination process are shown in Figure 14-4.Fresh vaginal discharge samples should be used necessarily. If there is no test within 2 hours after collection, the sample swabs shall be sealed and refrigerated in 2℃ ~8℃ environment. Before testing, the sample shall be restored to room temperature and mix the vaginal discharge samples well.
Operation Specification and Quality Control of Vaginal Microecological Examinations 237Figure 14-4 Analysis process of vaginal microecological examination14.2.2 Vaginal Microecological Evaluation System According to "the Expert Consensus on clinical Application of vaginal microecological evaluation", the vaginal microecological evaluation system includes two parts: morphological indicators and functional indicators. Morphological indicators include vaginal flora density, vaginal flora diversity, dominant bacteria, flora inhibition and overgrowth, pathogenic microorganisms (Candida species, Trichomonas Vaginalis, clue cells, Gram-negative diplococcus, etc.), Nugent scoring, aerobic vaginitis Donders scoring (AV scoring), white blood cells, etc.. Functional tests include vaginal pH value, hydrogen peroxide (H2O2), leukocyte esterases (LE), sialidase (SNa), β-glucuronosidase (GUS), coagulase, β-N-acetaminoglucosidase (NAG), proline aminopeptidase (PAP), et al. (Detailed introduction was in Chapter 2: Vaginal Microecological Evaluation System ).14.2.3 Operation Specifications of Vaginal Microecological Instruments Vaginal Discharge Analyzer Based on Microscope Imaging Before testing, the vaginal discharge analyzer based on microscope imaging should be checked whether the suction needle is stained with water droplets and dirty, whether it is bent and blocked, whether the cleaning pipeline is dirty or blocked, and whether the reagents and consumables need to be added. When testing, put the test card and other consumables into the designated location of the vaginal discharge analyzer, then, input sample information, select the test items, input the sample rack, place the sample rack in the sample dish, start
238 Chapter 14the testing operation. The system will automatically transfer the sample on the sample rack, automatic mixing, automatic drop sample, automatic smear, automatic transmit to microscope for photographing, microscope automatic conversing to high power field, microscope automatic focus, automatically in the video cell identification, automatic drop samples into detection card, automatic photograph the used detection cards, automatic waste detection card, automatic cleaning sampling and loading devices and pipeline. The staffs then automatically identified cells, bacteria and pathogens through software, and the sample test results were obtained through observation and analysis of the images. Vaginal Discharge Analyzer Based on Flow Cytometric Imaging Vaginal discharge analyzer based on flow cytometric imaging is used for automated analysis of routine tests of vaginal discharge samples. Before testing, check whether the sample suction needle is stained with water droplets and dirty, whether it is bent and blocked, whether the cleaning pool and staining pool are dirty or blocked. Check the amount of reagents and consumables. Focus before daily detection according to the operation manual (in order to make the morphological parts of the vaginal discharge be clearly photographed and imaged when passing through the focusing plane of the camera hole), quality control (including negative and positive quality control). Calibrate the analysis system per month with standard liquid. During the test, put the disposable sampler equipped with the patient's vaginal discharge nylon flocking swab on the test rack and put the test rack on the right side of the sample transmission unit of the analysis system. After clicking start, the test tube rack automatically removes the lid, mixes and transfers to the sampling position of the analytical system for testing. 14.2.4 Quality Control of the Morphological Examinations Manual Method The "Operating Procedures of Clinical Laboratory of China" (Fourth Edition) recommended physiological saline smear method to observe vaginal samples or use physiological saline hanging drop method to observe Trichomonas. The wet smear method should pay special attention to the quality of the samples. In addition to the wet smear method, Gram staining is also a very common method. Manual Gram staining and automatic mechanical Gram staining can all be used. When using the automatic staining machine, we should pay attention to the cleaning of the pipeline after daily work to avoid blocking the pipeline. The
Operation Specification and Quality Control of Vaginal Microecological Examinations 239addition of reagents and the filling of pipeline before staining to color the quality control smear and patient smears satisfactorily. The purpose of IQC is to control the precision and consistency of the instrument and personnel testing. Morphological quality control adopts the personnel comparison method (All morphological items are double sampling compared, and the detection results of negative and positive judgment is consistent, the difference is not more than one degree level for the results in control). It is recommended to randomly select 3-5 samples every day (trying to include each negative and positive result in the reports) and compare them by the reporting reviewers on the same day, filling in the "Vaginal Microecology Internal Quality Control Record". The consistency of the results of the personnel comparison should be > 80%. The inconsistent results should be discussed by increasing the number of testing personnel to ensure consistency of results. All the standard morphology of common bacteria, fungi, parasites and cells in vaginal discharge are consulted in "Female Vaginal Microecological Map" (Reference [1] of Chapter 1) .Strengthening personnel training, regularly and promptly conducting morphological comparisons and assessments among laboratory staffs are also important quality control methods. Morphological comparisons and assessments should be conducted at least once every 6 months (at least once every 3 months for new employees), with at least 5 samples evaluated each time, including normal and abnormal smears, and records of comparisons or assessments must be kept. All morphological examination staffs must undergo comparisons and assessments. Comparison and assessment qualified judgment criteria: there should be ≥ 80% of the results in agreement.The Instrument Method Ideally positive control should contain all the components in the vaginal discharge. But at present, the formation of multiple quality control products mostly use standardized red blood cells for quality control, and the formation of multiple quality control products may contain standardized red blood cells, white blood cells and epithelial cells. The quality control should be conducted at least once a day. Positive quality control results and negative quality control results should be within the scope of quality control products specified by the manufacturer. Before using the quality control products, the results of 20 times (usually 2 times a day, continuing 10 days or 4 times a day, continuing 5 days) should be accumulated to form the target value and standard deviation of the laboratory, and draw the quality control map according to the situation of the laboratory.
240 Chapter 1414.2.5 Quality Control of Functional (Dry Chemistry) Tests Manual Method a. Quality control of pH value: Prepare the dry chemistry negative (or positive) quality control products and reagents according to the reagent instructions (the dry powder products need to be fully dissolved and restored to room temperature before use), and gently press the prepared positive and negative quality control products onto two pH test strips to allow the quality control products to moisten the test strips. After adding the products for 30 seconds, read the pH value within 1 minute by referring to the standard colorimetric card. b. Quality control of other functional (dry chemistry) tests: Add one drop of the positive and negative quality control products to each of the dry chemistry reagent reacting holes, such as β-N-acetylglucosaminidase (NAG); sialidase (SNa); leukocyte esterase (LE); β-glucuronidase (GUS); hydrogen peroxide (H2O2), and gently mix it. Place the reaction plate into the incubator that comes with the kit, and incubate at the specified temperature of 35-37°℃ for 15 minutes. Immediately read the results within 2 minutes after incubation.The judgment criteria: • NAG: no color indicates negative; yellow color indicates positive. • SNa: no color or yellow color indicates negative; blue or green color indicates positive. • LE: no color indicates negative, blue or green color indicates positive. • GUS: no color indicates negative; blue or green color indicates positive. • H2O2: purplish red color indicates negative; no color indicates positive. Instrument Method Prepare the dry chemistry negative (or positive) quality control products and reagents according to the reagent instructions (the dry powder products need to be fully dissolved and restored to room temperature before use). Place the configured negative and positive quality control products in the designated position of the instrument, put the dry chemical test cards according to the actual use requirements, then carry out the quality control tests according to the instrument operation instructions, and automatically interpret the results. The positive results of the instrument method can be graded, and the quality control map can be drawn according to the results. The judgment criteria: negative control results can not be positive; positive control results can not be negative; positive results can be permitted to have a grade difference; weak positive results are regarded as positive results.
Operation Specification and Quality Control of Vaginal Microecological Examinations 241The instrument should be used with two quality control products, negative and positive, for daily IQC. The test results from any reagent module can be permitted to have a qualitative grade difference from the expected target value of the quality control solution. The results exceeding this range or jumping between normal and abnormal (negative and positive) should be considered out of control.Quality control frequency: including morphological comparison and functional tests. According to the number of samples, usually once a day. Treatment of out of control: Fill in the out of control report, analyze the causes of out of control, and timely correct and track. Evaluate the test report before the out of control of IQC. If the causes of out of control affects the test results, the report before the out of control should be recalled, and the results should be retested after the out of control is corrected. If the control is caused by accidental error and has no impact on the test results, recalling the report before the out of control is not needed. Treatment process of out of control was expressed in Figure 14-5.Figure 14-5 Treatment process of out of control for automated analysis of vaginal discharge
242 Chapter 1414.2.6 External Quality Assessment(EQA) The objective of EQA was to control the accuracy of the test results. Method of EQARelated testing items (such as epithelial cells, clue cells, fungi, Trichhomonas Vaginalis, Nugent scoring, AV scoring, etc.) were to participate in the EQA activities organized by a third party organization, or make a comparison with other laboratories (alternative experiment). The Process for Out of Control Treatment Fill in the out of control report, analyze the cause of out of control, timely correct it(complete it within the specified time) to avoid the recurrence of out of control. The Replacement Scheme of the EQA (Inter-laboratory Comparison) The selection principle of the comparison laboratory was stipulated: the same level or higher level laboratory which obtained laboratory accreditation. If the comparison laboratory does not obtain the laboratory accreditation, only the same level or higher level laboratory must be selected. The quantity of comparison samples are at least 5, including normal and abnormal microecological samples. The inter-laboratory comparison frequency is at least twice a year. The judgment criteria: over 80% of the comparison results should be consistent. 14.2.7 Vaginal Microecological Report Contents and Methods of the Vaginal Microecological Report There are various forms of vaginal microecological reports. According to the specific situation of each hospital, communicating with clinicians, vaginal microecological reports that meet the quality requirements of clinical and laboratory departments were established. The contents and methods of the following vaginal microecological examination report meet the quality requirements of the microecological examinations of the hospital of the Chief Editors (Table 14-1), including the status of vaginal discharge samples, morphological examination, flora status and flora function tests. Comments according to the results of the microecological examinations to indicate the clinician can also be added.
Operation Specification and Quality Control of Vaginal Microecological Examinations 243Table 14-1 Contents and methods of vaginal microecological examination report[The sample status]Smearing thick thin normalSampling good Bad[1]Staining methodGram staining[Morphological examinations]Vaginal cleanliness[2]Grade I Grade II Grade III Grade IVEpithelial cells full field 1/2 field <1/2 field absentWhite blood cells0~4/HP 5~15/HP 16~30/HP >30/HPLactobacilli >30/oil field 6~30/oil field 1~5/oil field <1/oil field Yeast spores Absent Absent Present PresentYeasts with buddingAbsent Absent Present PresentYeast pseudohyphaeAbsent Absent Present PresentG- diplococcus Absent AbsentPresent inside and outside of leukocytesTrichomonas vaginalisAbsent Absent present presentRatio of clue cellsAbsent 1-20% >20% >20%[Flora situation]Flora density[3] Absent 1+ 2+ 3+ 4+Flora diversity[4]Absent 1+ 2+ 3+ 4+
244 Chapter 14Dominant bacteria[5]Lactobacilli, et alNugent scoring [6]0-3 (Normal)4~6 (Intermediate BV)≥7(BV)AV scoring [7]0-3 (Normal)4~5 (Mild AV)6~7 (Moderate AV)8~10 (Severe AV)Lactobacillary grade[8]Grade Ⅰ Grade Ⅱa Grade Ⅱb Grade Ⅲ[Flora functional tests]pH Value[9] ≤4.5 >4.5H2O2 Negative Weak Positive PositiveSNa Negative Weak Positive PositiveLE Negative Weak Positive 1+ 2+ 3+NAG Negative Weak Positive PositiveGUS Negative Weak Positive PositiveCoagulase Negative Weak Positive PositiveMicroecological reference opinions[10]Microecological color map 1 Microecological color map 2Notes: [1]: Bad materials include few samples or non-vaginal discharge (such as drug residue, crystallization, etc.); [2] Vaginal cleanliness report should refer to Table 2-2 and Table 2-3 of Chapter 2 in this book. [3] [4]: Their reporting methods are described in Table 2-1, Chapter 2 in this book; [5]: The dominant bacteria reports include: Lactobacillus, G+ b (gram-positive bacteria), G- b(s) (Gram-negative short bacteria), G- b(v) (gram-negative vibrio), G+ co (Gram-positive coccus), G+ Streptococcus, G- b (L) (Gram-negative long bacilli), G- diplococcus. [6]: The Nugent scoring methods are shown in Chapter 2, Table 2-4. [7]: The AV scoring methods are shown in Chapter 7, Table7-2. [8]: Lactobacillary grade criteria are shown in Table 14-2. [9]: The pH value reports the specific value. [10]: Microecological reference opinions are divided into morphological results (Table 14-3) and dry chemistry results (Table 14-4).
Operation Specification and Quality Control of Vaginal Microecological Examinations 245Introduction of Parts of the Vaginal Microecological Report Contentsa. Nugent scoring by Gram staining: The Nugent scoring was first proposed by Robea Nugent in the 1990s and later regarded as the gold standard for laboratory diagnosis of bacterial vaginosis (BV), to see Chapter 2 ‘Vaginal Microecological Evaluation System’, Table 2-4. b. Lactobacillary grade: The Lactobacillary grade is based on the elimination relationship and their dominance situation between vaginal lactobacilli and other flora. Its grade is inversely proportional to the proportion of Lactobacillus species, including GradeⅠ, Ⅱa, Ⅱb and Ⅲ (See Table 14-2). Table 14-2 The Lactobacillary gradeLactobacillary grade Situation under microscopeGrade ⅠMany multiform Lactobacillus species , and no other bacteriaGrade ⅡaMixed flora, but mainly Lactobacillus speciesGrade ⅡbMixed flora, but the proportion of Lactobacillus species was significantly reduced, and other flora were predomi-nantGrade ⅢLactobacillus species was severely reduced or absent, and other flora were overgrewc. The indicative significance of vaginal microecological morphological results (See Table 14-3). Table 14-3 The indicative significance of morphological results Items Indications of the resultsWhite blood cells(WBC)WBC >15/HP indicates of inflamma-tion
246 Chapter 14Lactobacillus speciesIt’s predominance indicates that the vaginal flora is normal (Except for young girls and postmenopausal wom-en)Yeast spores are present It indicates fungal colonizationYeasts with budding are presentIt indicates VVC, potential of a non-al-bicans Candida infectionYeast pseudohyphaes are presentIt indicates VVC, potential of a C. al-bicans infectionG- diplococcus are present inside and outside of leukocytesIt is recommended to send a culture of cervical discharge for diagnosis of gonococcal infectionTrichomonas vaginalis are present It indicates trichomonas vaginitis (TV)Clue cells are presentThe ratio >20% indicates bacteria vag-inosis (BV)d. The indicative significance of the dry chemistry results are shown in Table 14-4. Table 14-4 The indicative significance of the dry chemistry resultsItems Indications of the resultspH value (3.8-4.5 is normal)The more the pH value deviates from the normal value, the greater the de-gree of vaginal microecological dam-ageH2O2It’s negative indicates of normal flo-ra, normal function of lactobacilli, or presence of lactococcus; It’s positive indicates of decline of Lactobacillus function and vaginal microdysbiosis.Sialidase (SNa) It’s positive indicates of BV
Operation Specification and Quality Control of Vaginal Microecological Examinations 247Leukocyte esterase (LE)It’s positive indicates of vaginal in-flammatory or previous inflammatory statusβ-N-acetylglucosaminosidase (NAG)It’s positive and pH≥4.8, indicates po-tential infection of Trichomonas vagi-nalisIt’s positive and pH≤4.6, indicates po-tential infection of VVCβ-glucuronidase (GUS)It’s positive indicates infection of aer-obic vaginitis (AV)CoagulaseIt’s positive also indicates infection of AV14.2.8 Performance Evaluation and Maintenance of Automatic Analyzer of Vaginal Discharge Performance EvaluationBefore the instrument is put into use, it should be evaluated through the evaluation of the analysis system to verify whether its performance meets the performance requirements specified in the equipment manual (the performance requirements specified by the manufacturer should meet the clinical needs). The evaluation contents includes accuracy, precision, carryover rate and instrument comparison. Maintenance of Instruments a. Daily maintenance: A complete standard operating procedure (SOP) should be established and be strictly implemented. Timely register the daily condition and occurrence of the instrument problems, and make a comprehensive inspection of the instrument (waste reagent plate, waste liquid, waste glass slides, etc.). After the daily measurement, clean the instrument and microscope body with chlorine-containing disinfectant, remove the waste reagent plate, waste liquid and waste glass slide in time, and shut down the instruments. b. Weekly maintenance or monthly maintenance: Weekly maintenance or monthly maintenance should be done for all kinds of automatic analyzer and microscope according to the specific situation of the instruments.
248 Chapter 14c. Regular calibration: Regular calibration should be done for automatic analyzers of vaginal discharge (half-year cycle). d. Temporary maintenance: Instruments should be calibrated and maintained in time after installation, movement and fault repair. e. Record: All maintenance and calibration should be recorded and kept in the instrument file or the instrument record file.14.3 Post-Analysis Process14.3.1 Preservation of the Vaginal Discharge Samples Generally, vaginal discharge samples are kept in the sample cryo-depository (2-8℃) for one week after the results are reported. They are treated uniformly as medical waste, and are recorded and signed. The pictures on the software of the vaginal discharge automatic analyzer are generally automatically deleted after a month. The smears with morphological significance can be used to wipe off the lens oil, and seal the film for a long time. 14.3.2 Report ReviewChecking Patient InformationPlease checking patient information and test items carefully, and promptly verify and correct the non-compliance with the clinical information. This information includes the patient's name, gender, age, hospitalization ID number, application form ID number, etc.. Historical Results Reviewing The previous results and clinical diagnosis of the patients should be compared. If there is any discrepancy, we should communicate with the clinical department through telephone, laboratory information system(LIS), visit to wards and other forms to discuss the possible factors affecting the abnormal results until the problem is solved before reviewing and issuing the report.Quality Control ReviewingThe results of IQC should be reviewed on the same day. Only when the microscopic examination or instrument automation analysis of IQC results is under control can reports be reviewed and issued. If IQC results is out of control, a report of out-of-control should be promptly filled out, the reasons for the out-of-control analysis should be analyzed, and quality control corrections should be made in a timely manner. Reports issued before the QC correction should be evaluated. If the reasons for the out-of-control do not affect the patient's report results, the reports
Operation Specification and Quality Control of Vaginal Microecological Examinations 249can continue to be issued; if the reasons for the out-of-control do affect the patient's report results, the results from the last IQC results under control until the current IQC results out of control should be promptly retrieved, and the report should be reissued after the quality control is back to under control.14.3.3 Clinical Contacts Visit the Diagnosis Room Routine vaginal discharge tests and vaginal microecological examinations are usually ordered by the obstetrics and gynecology diagnostic rooms. Therefore, laboratory personnel should frequently visit the diagnostic rooms to contact the ordering physician to know whether the sampling equipment meets clinical requirements? Whether it causes discomfort to the patient? Whether the physician follows the sample collection precautions? Has the Examination Manual been distributed to the physicians? What is the physician's opinion on the consistency between testing equipment, testing methods, and clinical results? What are the physician's suggestions for improving and perfecting vaginal microecological examinations? etc.Clinical Consultations For some difficult cases (such as mixed infections of various vaginitis or vaginosis), when clinically necessary, consultations and discussions can be held between the laboratory department and clinical department doctors. Clinical diagnosis can be given according to the patient's medical history and clinical symptoms, combined with the laboratory test results. False positives or false negatives caused by sample collection factors, test methods or test instrument factors can be corrected through clinical consultation. The laboratory can also find the problems in the vaginal microecological operation process through communication with clinicians and correct them in time. Telephone ContactingThe laboratory can also find the problems in the tests (such as missed diagnosis or misdiagnosis) through the telephone contact with clinicians, correct them in time through review, and improve the test method or operation process. The laboratory can also directly contact the patient by telephone, ask the patient's history and clinical symptoms, then make the results of the test reports more accurate and reliable. References
250 Chapter 14[1] Collaborative Group on Infectious Diseases of the Chinese Society of Obstetrics and Gynecology. Expert consensus on the clinical application of vaginal microecological evaluation. Chinese Journal of Obstetrics and Gynecology, 2016,10 (50): 721-723.[2] Department of Medical Administration, Ministry of Health, PRC. National Operating Procedures of Clinical Laboratory. Beijing: People's Publishing House. 2015: 196-198. [3] Hongli Wang. Experimental diagnostics. Beijing: People's Health Publishing House, 2001.
C H A P T E R - 15Interpretation of Vaginal Microecology ReportHongwei Liu, Ying WangTranslator: Yunxia Li15. 1The test report is an important weapon for clinicians to diagnose diseases, and the correct interpretation of the report is the basic skill of doctors. Vaginal infection is a common clinical disease, and the main test method is vaginal discharge examination. From the early simple routine examination of leucorrhea, to the current gradually mature and recognized vaginal microecological examination, every test report is of great significance for diagnosis. This chapter explains the interpretation of the microecological reports.15.1 Overview Vaginal microecological examination includes two morphological and functional parts. To get the test reports, we need to understand the basic situation of the reports, especially the reports of other hospitals. At present, the methods, items and forms of vaginal microecological examination in different hospitals may be inconsistent. Doctors should first be familiar with the content and form of their reports of own hospital, and can skillfully interpret them. And then, they should gradually get familiar with and interpret the reports of other hospitals. General procedure for interpreting microecological reports is as follow: 15.1.1 Report on the Profile Whether the report is a report of their own hospital or not? The interpretation of the report of other hospital should be more careful, and a familiar process should be added to understand the content and expression method of the report. 15.1.2 Basic Information of the Patients The age, chief complaint (diagnosis), different physiological periods, whether be treated or not, especially vaginal medication of the patients should be learned about.
252 Chapter 1515.1.3 Determine the Arrangement of the Reports The contents of the report generally include the basic information of the sample, the sampling situation, the staining method, the morphological and functional indicators, the microphotography and other large contents.15.2 Morphological ExaminationMorphological examination was developed based on the classical routine examination of leucorrhea, including cleanliness, flora density, flora diversity, dominant bacteria (gram-positive cocci, gram-negative bacilli, gram-negative vibrio, etc.), the body inflammatory reactivity (leukocytes, pyocytes, etc.), epithelial cells, pathogenic microorganisms (Trichomonas vaginalis, yeast spores, yeasts with budding, pseudohyphe, gram positive or negative cocci, bacilli, vibrio, etc.), clue cells, Lactobacillary grade, Nugent score, AV score, etc.. It is the major part of the microecological examination, and is the classic examination methods of vaginal discharge. It is also important for the identification of vaginal infections.15.3 Functional TestsFunctional tests are test methods of vaginal discharge gradually developed in recent years, and its specificity and sensitivity still need to be accumulated through experience. Therefore, when morphological and functional test results are contradictory, it is indicated to focus on morphology. Functionality mainly includes the following three aspects: 15.3.1 Function of the Lactobacillus Species• The pH value reflects the function of lactic acid produced by Lactobacillus species and Lactococcus to maintain the acidic vaginal environment. • Hydrogen peroxide (H2O2) is a metabolite of Lactobacillus species and Lactococcus species, reflecting the number and function of lactic acid bacteria. 15.3.2 Function of Pathogenic Bacteria There are several enzymes or metabolites commonly follows. With technological advances, there may be more tests going into clinical applications. • Sialidase (SNa): It is a more specific functional indicator of BV. • β-glucuronidase (GUS): it is a functional enzyme for aerobic bacteria. • Coagulase: It is mainly produced by Staphylococcus aureus, indicating the
Interpretation of Vaginal Microecology Report 253proliferation of aerobic bacteria in the vagina. • β-N-acetylglucosaminidase (NAG): It should be determined by combining it with vaginal pH value. When NAG is positive, and pH≥4.8, it often indicates Trichomonas vaginalis or anaerobic bacteria infection; when NAG is positive,and pH≤4.6, it indicates Candida species infection or its previous infection. • Proline aminopeptidase (PIP): It is a non-specific indicator, with PIP positive in some Gardnerella vaginalis, mobiluncus and Candida albicans infections.15.3.3 Reactivity of Organism The positive of Leukocyte esterase (LE) indicates that a large number of multi-nucleated white blood cells in vaginal discharges are destroyed, thus releasing this enzyme. There is an inflammatory response, indicating that a variety of reproductive tract inflammation is possible, such as cervicitis, pelvic inflammation, vaginitis, etc., so comprehensive analysis should be combined with clinical indicators.15.4 Case Analysis15.4.1 Normal Vaginal Microecology【Case 1】The patient was a 31-year-old female, which was reviewed after treatment of vulvovaginal candidosis. Microecological report was shown in Table 15-1. Table 15-1 Case 1- normal vaginal microecology reportTest items Results Reference value1. Smear Normal Normal2. Sampling Good Good3. Staining Gram staining Gram staining【Morphological examination】4. Vaginal Cleanness Grade II Grade I~II5. Epithelium 1/2 field ≥1/2 field
254 Chapter 15Test items Results Reference value6. White Blood Cells 5-15 /HP <15/HP7. Lactobacillus species >30 /oil field ≥6/oil field8. Yeast spores Absent Absent or present9. Yeasts with budding Absent Absent10. Yeast pseudohyphae Absent Absent11. G- diplodocus Absent Absent12. Trichomonas vaginalis Absent Absent13. Ratio of clue cells Absent Absent【Flora status】14. Flora density 3+ 2+ ~ 3+15. Flora diverity 1+ 2+ ~ 3+16. Dominant bacteria Lactobacillus species Lactobacillus species17. Nugent score 0 0 ~ 618. AV score 0 0 ~ 319. Lactobacillary grade Grade I Grade I~IIa【Microbiota functional tests】20. pH value 4.4 <4.521. H2O2 Negative Negative22. Sialidase Negative Negative23. Leukocyte esterase Negative Negative24. β-glucuronidase Negative Negative25. β-N-acetylglucosaminidase Negative NegativeInterpretation of the report: a. The patient was child-bearing age, after vaginitis treatment; b. Good sampling materials, indicating the reliability of the results; c. The results included morphological and functional contents, which were vaginal microecological examination; d. The results of morphological
Interpretation of Vaginal Microecology Report 255examination were not abnormal, the flora was basically normal, and e. the functional test results were normal. Therefore, on the whole, this was a basically normal vaginal microecological report. It was recommended not to do special treatment, but it should be comprehensively considered according to the patient's clinical data, previous treatment and other conditions.15.4.2 Mixed Vaginitis 【Case 2】The patient was a 31-year-old female, G4, P0+4, required pre-pregnancy examination. Yellow leukorrhea was for a month, and vaginal mucosa’s congestion was found in physical examination. There was light yellow foam-like vaginal discharge, and cervical discharge is slightly yellow. Microecological reports are shown in Table 15-2. Table 15-2 Case 2-Microecological report of mixed vaginitisTest items Results Reference value1. Smear Normal Normal2. Sampling Good Good3. Staining Gram staining Gram staining【Morphological examination】4. Vaginal Cleanness Grade IV Grade I~II5. Epithelium Full field ≥1/2field6. White Blood Cells 15-30/HP <15/HP7. Lactobacillus species <1 /oil field ≥6/oil field8. Yeast spores Present Absent or present9. Yeasts with budding Present Absent10. Yeast pseudohyphae Present Absent11. G- diplodocus Absent Absent12. Trichomonas vaginalis Absent Absent13. Ratio of clue cells >20% Absent
256 Chapter 15Test items Results Reference value【Flora status】14. Flora density 3+ 2+ ~ 3+15. Flora diverity 3+ 2+ ~ 3+16. Dominant bacteria G- short bacillus Lactobacillus species17. Nugent score 9 0 ~ 618. AV score 3 0 ~ 319. Lactobacillary grade Grade III Grade I~IIa【Microbiota functional tests】20. pH value 5.1 <4.521. H2O2 Positive Negative22. Sialidase Weak positive Negative23. Leukocyte esterase Positive Negative24. β-glucuronidase Negative Negative25. β-N-acetylglucosaminidase Weak positive NegativeInterpretation of the report: a. The patient was child-bearing age, and to do a pre-pregnancy examination; b. Abnormal vaginal discharge recently. c. History of previous multiple pregnancies, but atoke; d. Good sampling materials, which showed that the reliability of the results is good; e. The results includes two large contents of morphological and functional examination. Morphological examination was by Gram staining, which indicated that it was vaginal microecological examination; f. Morphological examination: (1) Poor cleanliness, more white blood cells, loss of lactobacilli and increasing number of pathogenic bacteria; (2) Flora status: the flora density and flora diversity were at the upper limit of normal, (3) The dominant bacteria was G-bacilli, (4) Lactobacillary grade: grade III, reflecting the lack of lactobacilli; (5) Pathogenic bacteria: yeast-like spores, yeast-like pseudohyphae and yeasts with budding all were present, being consistent with the laboratory diagnosis of vulvovaginal Candidiasis; (6) Nugent score was 9, meeting the laboratory diagnostic criteria of bacterial vaginosis (BV); (7) The ratio of clue cells was >20%, which was
Interpretation of Vaginal Microecology Report 257one of the Amsel diagnostic criteria for BV, and was a necessary standard; g. Functional tests: (1) Function of lactobacilli: pH value was 5.1, reflecting the under acidity of the vagina, lactobacilli or lactococcus were poorly functional; Hydrogen peroxide: positive, also reflected the poor function of lactobacilli or lactococcus; (2) Pathogen function tests: sialidase was weak positive, reflecting more anaerobic bacteria in vaginal discharge; NAG: weak positive, but the pH value was 5.1, which indicated that it may be related to trichomonas or anaerobic bacteria; (3) Body reactivity item: leukocyte esterase was positive, indicating that leukocytes performed the phagocytic function, being an indicator of the inflammatory response. In summary, this report was an abnormal vaginal microecological result, abnormal flora, absence of lactobacillus, the presence of inflammatory response, pathogens were Candida species through morphological and functional tests, anaerobic bacteria was positive, so the diagnosis of this patient was mixed vaginitis: VVC+BV. Combined with the clinical presentation of the patient, this diagnosis was supported. In addition, paying attention to the description of cervical discharge, whether there were cervical infection problems was alerted. Relevant examinations for comprehensive analysis should be combined with. The VVC+BV patient reviewed after treatment, and poor function of lactobacilli was found. Microecological reports are shown in Table 15-3.Table 15-3 Case 3-Microecological report after mixed vaginitis treatmentTest items Results Reference value1. Smear Normal Normal2. Sampling Good Good3. Staining Gram staining Gram staining【Morphological examination】4. Vaginal Cleanness Grade II Grade I~II5. Epithelium 1/2 field ≥1/2field6. White Blood Cells 5-15/HP <15/HP7. Lactobacillus species 5-30 /oil field ≥6/oil field8. Yeast spores Absent Absent or present
258 Chapter 15Test items Results Reference value9. Yeasts with budding Absent Absent10. Yeast pseudohyphae Absent Absent11. G- diplodocus Absent Absent12. Trichomonas vaginalis Absent Absent13. Ratio of clue cells Absent Absent【Flora status】14. Flora density 2+ 2+ ~ 3+15. Flora diverity 1+ 2+ ~ 3+16. Dominant bacteria Lactobacillus species Lactobacillus species17. Nugent score 1 0 ~ 618. AV score 0 0 ~ 319. Lactobacillary grade Grade IIa Grade I~IIa【Microbiota functional tests】20. pH value 5.1 <4.521. H2O2 Positive Negative22. Sialidase Negative Negative23. Leukocyte esterase Negative Negative24. β-glucuronidase Negative Negative25. β-N-acetylglucosaminidase Negative NegativeInterpretation of the report: After treatment, the morphological indicators were basically normal. Functional tests: pathogen and body reactivity indicators were all recovered. The hydrogen peroxide was positive and pH value was 4.7, which indicated that the function of lactobacilli was not recovered. 15.4.3 RVVC【Case 3】The patient was a female of 31-year-old with recurrent
Interpretation of Vaginal Microecology Report 259vulvovaginal Candidosis (RVVC). She reviewed on February 11,2019. On the same day, after fungal culture of vaginal discharge, there were Candida glabrata isolates grew. On February 11th, 2019, the vaginal microecological report of pretreatment for patients with RVVC was shown in Table 15-4. Table 15-4 Case 3-Microecological report of patients with RVVCTest items Results Reference value1. Smear Normal Normal2. Sampling Good Good3. Staining Gram staining Gram staining【Morphological examination】4. Vaginal Cleanness Grade III Grade I~II5. Epithelium 1/2 field ≥1/2field6. White Blood Cells 5-15/HP <15/HP7. Lactobacillus species 6-30 /oil field ≥6/oil field8. Yeast spores Present Absent or present9. Yeasts with budding Present Absent10. Yeast pseudohyphae Absent Absent11. G- diplodocus Absent Absent12. Trichomonas vaginalis Absent Absent13. Ratio of clue cells Absent Absent【Flora status】14. Flora density 2+ 2+ ~ 3+15. Flora diverity 2+ 2+ ~ 3+16. Dominant bacteria Lactobacillus species Lactobacillus species17. Nugent score 2 0 ~ 6
260 Chapter 15Test items Results Reference value18. AV score 0 0 ~ 319. Lactobacillary grade Grade IIa Grade I~IIa【Microbiota functional tests】20. pH value 4.8 <4.521. H2O2 Positive Negative22. Sialidase Negative Negative23. Leukocyte esterase Negative Negative24. β-glucuronidase Negative Negative25. β-N-acetylglucosaminidase Negative NegativeInterpretation of the report: a. The patient was child-bearing age; b. Review of RVVC; c. Good sampling materials, which indicated that the reliability of the results was good; d. The results included two large contents of morphological and functional, and morphological examination was by Gram staining, which indicated that it was a vaginal microecological examination; e. Morphological examination: (1) Vaginal cleanliness: grade III; (2) Microflora: the flora density and flora diversity were normal, (3) The dominant bacteria were lactobacilli; (4) The lactobacillary grade was grade IIa, in normal range; (5) Pathogenic bacteria: yeast-like spores and yeasts with budding were present, being consistent with the diagnosis of vulvovaginal candidiasis. Because of the presence of yeasts with budding and the absent of pseudohyphae, non-Candida albicans infection was suspected; (6) Nugent score was 4, which indicated the intermediate type of bacterial vaginosis. f. Functional tests: (1) The function of lactobacilli: pH value was 4.8, reflecting the lack of vaginal acid. Lactobacilli of acid producing were less or poor function. The hydrogen peroxide was positive which also reflected the poor function of lactobacilli; (2) pathogen functional tests all were negative; (3) Body reactivity indicator also was negative. In summary, this report was an abnormal vaginal microecological result: morphological examination of yeast-like spores and yeasts with budding supported the diagnosis of vulvovaginal Candidosis, and tended to non-Candida albicans infection. Combined with the patient's fungal culture of vaginal discharge, medical history and clinical
Interpretation of Vaginal Microecology Report 261manifestations, this diagnosis was supported. At the same time, the Nugent score of this sample was 4, and the function of lactobacilli was abnormal. Combined with the medical history of RVVC, we should be alert to mixed vaginal infection. November 3rd, 2019, The review report of vaginal microecology after multiple courses of treatment. It was shown in Table 15-5. Table 15-5 Case 3-Microecological report after multiple courses of treatment for patients with RVVCTest items Results Reference value1. Smear Normal Normal2. Sampling Good Good3. Staining Gram staining Gram staining【Morphological examination】4. Vaginal Cleanness Grade II Grade I~II5. Epithelium full field ≥1/2field6. White Blood Cells 0-5/HP <15/HP7. Lactobacillus species 1-5 /oil field ≥6/oil field8. Yeast spores Absent Absent or present9. Yeasts with budding Absent Absent10. Yeast pseudohyphae Absent Absent11. G- diplodocus Absent Absent12. Trichomonas vaginalis Absent Absent13. Ratio of clue cells Absent Absent【Flora status】14. Flora density 2+ 2+ ~ 3+15. Flora diverity 2+ 2+ ~ 3+16. Dominant bacteria G+ short bacillus Lactobacillus species17. Nugent score 6 0 ~ 6
262 Chapter 15Test items Results Reference value18. AV score 1 0 ~ 319. Lactobacillary grade Grade IIb Grade I~IIa【Microbiota functional tests】20. pH value 4.6 <4.521. H2O2 Positive Negative22. Sialidase Negative Negative23. Leukocyte esterase Negative Negative24. β-glucuronidase Negative Negative25. β-N-acetylglucosaminidase Negative NegativeInterpretation of the report: After treatment, Candida species had turned negative, but the dominant bacteria were abnormal, pH value was 4.6, and hydrogen peroxide was positive, reflecting that the number and function of acid-producing bacteria were not recovered. The Nugent score was 6, so the treatment of the patient could not be terminated, and the problem of RVVC and mixed infection should be considered and treated individually. 15.4.4 Bacterial Vaginosis【Case 4】The patient was a 54-year-old female, with loose menstruation, itchy vulva, and foam-like vaginal discharge for a week. Microecological reports are shown in Table 15-6. Table 15-6 Case 4-Vaginal microecological report of bacterial vaginosisTest items Results Reference value1. Smear Normal Normal2. Sampling Good Good3. Staining Gram staining Gram staining
Interpretation of Vaginal Microecology Report 263Test items Results Reference value【Morphological examination】4. Vaginal Cleanness Grade III Grade I~II5. Epithelium 1/2 field ≥1/2field6. White Blood Cells >30/HP <15/HP7. Lactobacillus species <1 /oil field ≥6/oil field8. Yeast spores Absent Absent or present9. Yeasts with budding Absent Absent10. Yeast pseudohyphae Absent Absent11. G- diplodocus Absent Absent12. Trichomonas vaginalis Absent Absent13. Ratio of clue cells >20% Absent【Flora status】14. Flora density 3+ 2+ ~ 3+15. Flora diverity 2+ 2+ ~ 3+16. Dominant bacteria G- short bacillus Lactobacillus species17. Nugent score 8 0 ~ 618. AV score 8 0 ~ 319. Lactobacillary grade Grade III Grade I~IIa【Microbiota functional tests】20. pH value 4.8 <4.521. H2O2 Positive Negative22. Sialidase Weak positive Negative23. Leukocyte esterase Positive Negative24. β-glucuronidase Negative Negative25. β-N-acetylglucosaminidase Weak positive Negative
264 Chapter 15Interpretation of the report: a. The patient was perimenopausal women; b. Good sampling materials, which indicated that the reliability of the results was good; c. The results includes two large contents of morphology and function, and the morphological content was acquired by Gram staining, which indicated a vaginal microecological examination; d. Morphological examination: (1) Vaginal cleanliness was Grade III, lactobacillus was deleted, and there were more white blood cells; (2) Flora status: increased flora density, reducing flora diversity; (3) The dominant bacteria was G- short bacillus; (4) Lactobacillary grade was grade III, reflecting the lack of lactobacillus; (5) Pathogenic bacteria: the ratio of clue cells was >20%, and the Nugent score was 8, which met the laboratory diagnostic criteria of bacterial vaginosis (BV). (6) The AV score was 7, which indicated the diagnosis of aerobic vaginitis (AV). Please combined it with her clinical symptoms. e. Functional tests: (1) Lactobacillus function: pH value was 4.8, reflecting the lack of vaginal acid. Lactobacilli of acid producing were less or poor function; hydrogen peroxide was positive, which also reflected the poor function of lactobacilli; (2) Pathogen functional tests: sialidase was weak positive, which was related to BV; (3) leukocyte esterase was positive, which indicated inflammatory reaction; (4) β -N-acetylglucosaminosidase was weak positive and pH value was 4.8, which indicated the presence of trichomonas or anaerobic bacteria. In summary of this report: this was an abnormal vaginal microecological result, and the morphology and function of anaerobic bacteria indicators were positive, so the diagnosis of BV was considered. But the morphological and functional indicators show inflammatory reaction. combined with the characteristics of the vaginal discharge and AV score, the diagnosis of this patient was mixed vaginitis: BV+AV. 【Case 5】The patient was a 38-year-old female. Her menstruation volume was less for 7 years, and no contraception for 1 year. Her vaginal discharge was thin and foam-like. Microecological reports are shown in Table 15-7. Table 15-7 Case 5-Vaginal microecological report of bacterial vaginosisTest items Results Reference value1. Smear Normal Normal2. Sampling Good Good
Interpretation of Vaginal Microecology Report 265Test items Results Reference value3. Staining Gram staining Gram staining【Morphological examination】4. Vaginal Cleanness Grade III Grade I~II5. Epithelium 1/2 field ≥1/2field6. White Blood Cells 0-5/HP <15/HP7. Lactobacillus species <1 /oil field ≥6/oil field8. Yeast spores Absent Absent or present9. Yeasts with budding Absent Absent10. Yeast pseudohyphae Absent Absent11. G- diplodocus Absent Absent12. Trichomonas vaginalis Absent Absent13. Ratio of clue cells >20% Absent【Flora status】14. Flora density 4+ 2+ ~ 3+15. Flora diverity 2+ 2+ ~ 3+16. Dominant bacteria G- vibrion Lactobacillus species17. Nugent score 8 0 ~ 618. AV score 2 0 ~ 319. Lactobacillary grade Grade III Grade I~IIa【Microbiota functional tests】20. pH value 4.6 <4.521. H2O2 Positive Negative22. Sialidase Weak positive Negative23. Leukocyte esterase Negative Negative24. β-glucuronidase Negative Negative
266 Chapter 15Test items Results Reference value25. β-N-acetylglucosaminidase Negative NegativeInterpretation of the report: a. The patient was child bearing age; b. She had birth requirements; c. Good sampling materials, which indicated that the reliability of the results was good; d. The results includes two large contents of morphology and function, and the morphological content was acquired by Gram staining, which indicated a vaginal microecological examination; e. Morphological examination: (1) Vaginal cleanliness was Grade III, lactobacillus was deleted, and there were few white blood cells; (2) Flora status: increased flora density and reducing flora diversity; (3) The dominant bacterium is G- vibrion, (4) Lactobacillary grade was grade III, reflecting the lack of lactobacillus; (5) Pathogenic bacteria: the ratio of clue cells was >20%, and the Nugent score was 8, which met the laboratory diagnostic criteria of BV; f. Functional tests: (1) Lactobacillus function: pH value was 4.6, reflecting the lack of vaginal acid. Lactobacilli of acid producing were less or poor function; hydrogen peroxide was positive, which also reflected the poor function of lactobacilli; (2) pathogen functional tests: sialidase was weak positive, which was related to BV; (3) leukocyte esterase was negative, which indicated low inflammatory reaction; In summary, this was an abnormal vaginal microecological report. The pathogen is positive for anaerobic bacteria through morphology examination and functional teats, and the inflammatory reaction is not obvious, therefore the diagnosis of BV was considered. Combined with the characteristics of vaginal discharge, BV was the clinical diagnosis for this patient.References[1] 1.Infectious Diseases Cooperative Group of The Obstetrics and Gynecology Branch of the Chinese Medical Association. Expert consensus on the clinical application of vaginal microecological evaluation. Chinese Journal of Obstetrics and Gynecology, 2016,51 (10): 721- -723. [2] 2.Infectious Diseases Cooperative Group of China Branch of Obstetrics and Gynecology. Revised specification of diagnosis and treatment of Candida vulvovagina (VVC). Chinese Journal of Practical Gynecology and Obstetrics, 2012,28 (6): 401-402.
Interpretation of Vaginal Microecology Report 267[3] Infectious Diseases Cooperative Group of The Obstetrics and Gynecology Branch of the Chinese Medical Association. Guidelines for the Diagnosis and Treatment of Bacterial Vaginosis 2021 Revision. Chinese Journal of Obstetrics and Gynecology, 2021,56 (1): 3-6. [4] Infectious Diseases Cooperative Group of Obstetrics and Gynecology Branch of Chinese Medical Association, expert consensus on diagnosis and Treatment of mixed vaginitis (2021 edition), 56 (1): 15-18.
C H A P T E R - 16Progress in Diagnosis of Vaginal Microecology --Automated Detection and Artificial IntelligenceWenjie ZhouTranslator: Zhengqiang Hu16. 1The President of the International Committee for Clinical Chemistry (IFCC), Mr.Maurizio Ferrari, pointed out in a report titled "How to imagine the future of laboratory medicine" that future developments in laboratory medicine will rely on electronic computers and information technology. From the creation and innovation of the internet to the interpretation of artificial intelligence test results, especially the application of new technologies such as automatic cell recognition, these can play a significant role in the future development and construction of laboratory medicine. The future opportunities for laboratory medicine include standardization, automation, and intelligence, as well as the close integration of big data with the internet. Intelligence and artificial intelligence, as platforms, can achieve automatic judgment and review in the field of laboratory medicine in the near future. 16.1 The History and Status Quo of Automatic Detection of Vaginal MicroecologyAs part of vaginal microbiota examination, routine vaginal discharge tests are the most common and widely used laboratory tests in gynecological disease diagnosis, now widely available in hospitals at all levels across the country. However, most hospitals, especially primary care facilities, still use manual wet mount microscopy for vaginal discharge testing. The current standard procedure for collecting vaginal samples is also one of the most basic clinical operations: a doctor uses a cotton swab to scrape an appropriate amount of vaginal discharge from the patient's vagina, and the patient then sends the sample to the laboratory department on their own. Laboratory personnel process the samples submitted by patients (in some primary care facilities, this task is performed by obstetricians in the consultation room), placing the cotton swabs containing vaginal discharge into test tubes and adding 0.9% sodium chloride solution. Once the cotton swabs are fully moistened, a clean slide is taken and the saline vaginal discharge
Progress in Diagnosis of Vaginal Microecology --Automated Detection and Artificial Intelligence 269solution is evenly spread over it. Laboratory physicians observe multiple fields under high magnification with an optical microscope and provide corresponding microscopic examination conclusions. Due to the numerous factors affecting samples transportation (such as patients not completing samples submission within the shortest time), the manual processing of samples is complex and cumbersome, making it unsuitable for large-scale population screening. Microscopic examination is time-consuming and labor-intensive, requiring a high number of personnel with professional qualifications and relevant work experience. As the number of samples submitted for testing increases, prolonged and intense samples processing work can lead to operational errors, significantly increasing the risk of misjudgment. Additionally, differences in the experience of laboratory personnel may result in inconsistent test conclusions, not only delaying diagnosis and treatment but also necessitating substantial investment in training and comparison items for relevant personnel in hospital laboratories. Nowadays, with the increasingly close integration of digital image processing and medical testing, there has been rapid progress in automatic recognition and classification technology for biomedical images. Using machine vision technology to share part of the testing tasks with inspectors, or even completely replacing their work, has become a development trend in modern medical testing. The interdisciplinary integration of digital image processing and medicine can greatly facilitate health diagnosis and treatment. The automatic recognition and classification of formed elements in medical microscopic images have always been the main focus of research. Researchers both domestically and internationally are making efforts to develop intelligent analysis systems for medical microscopic images. In the early stages of research, medical imaging devices generally had limited functions and could not perform intelligent recognition on the collected images; they could only display real-time images captured by cameras on computers. For example, in the early 1980s, the Yollow IRIS urine smart analyzer developed in the United States was led by International Remote Imaging Systems Co., Ltd. It could display microscopic images of urine sediment from high-speed cameras in real-time on computer screens, allowing professional medical personnel to identify formed elements in the images. With the continuous development of the in vitro diagnostic equipment industry and the rapid advancement of digital image processing technology, medical image processing techniques have significantly improved. Since the 1990s,
270 Chapter 16more and more intelligent medical image analysis devices have been applied in real life both domestically and internationally. For instance, the VetLabUA urine analyzer and ProCyte Dx fully automatic blood cell analyzer launched by American Idemitsu Biotech Co., Ltd., and the SEDTRON automatic sediment analyzer introduced by German company Bauschmann (Boehringer Mannheim). The promotion and application of these devices in daily life have reduced the workload of medical personnel and greatly improved people's medical care experiences. At the same time, from the early 1980s to the present, scholars both domestically and internationally have made increasingly sophisticated advancements in medical image processing algorithms. These developments have evolved from traditional cell morphology-based segmentation to the use of artificial neural networks for identifying and classifying formed elements, as well as employing CNN convolutional neural networks through deep learning to achieve efficient recognition. In 1980, M. D. Levine et al. proposed an algorithm based on a Markov chain model, which enabled the tracking of red blood cells in the blood and analyzed their movement. In 1996, J. P. Thiran et al. provided reference data for determining the benign or malignant nature of tumors by combining geodesic theory and morphological characteristics of cells with microscopic images of cancerous tissue. In 2013, M. Farhan et al. proposed an algorithm to segment overlapping cells using their concave points. In 2013, Wenhui Li from Nanchang University of Aeronautics and Astronautics used pattern recognition technology based on image processing fundamentals to achieve automatic segmentation and recognition of formed elements in saline vaginal discharge.The in vitro diagnostic equipment industry began to sprout in the early 1980s. Initially, it could only display microscopic images on a computer in real time. With the rapid development of digital image processing technology, today's devices can not only display but also intelligently analyze and identify formed elements in microscopic images, greatly facilitating medical diagnosis and patient care. However, to date, there have been no reports of fully automatic vaginal discharge analyzers abroad, nor has there been any discovery of such devices with integrated functions for full automatic microscopy, intelligent identification of formed elements, and detection of vaginal biochemical markers in the domestic market.Most of the vaginal discharge analyzers currently available on the domestic market only have functions for detecting vaginal biochemical substances and
Progress in Diagnosis of Vaginal Microecology --Automated Detection and Artificial Intelligence 271displaying real-time microscopic images of vaginal discharge. Some manufacturers have even achieved the recognition and analysis of vaginal discharge images. Some Vaginal Discharge Analyzers uses vaginal biochemical markers to assist doctors in assessing the cleanliness of vaginal discharge (Figure 16-1) .Figure 16-1 Automated vaginal discharge analyzerThe automated instrument processes samples and injects them into specific analytical plates, achieving precise automatic control of the microscope and CCD lens for full-field scanning and real-time image acquisition. It then uses a digital imaging system to digitally capture and intelligently identify formed elements in the samples. The instrument’s automatic focusing algorithm integrates recognition and clarity operators to ensure the sharpness of the captured images. Advanced deep convolutional neural network technology is employed, combining deep learning with intelligent detection of formed elements in samples. This allows for automatic identification of epithelial cells, red blood cells, white blood cells, poisoned white blood cells, clue cells, Candida, fungal spores, yeast buddings, and trichomonads, thus screening common components such as cells, fungi, and trichomonads. Samples can be automatically mixed and stained before further testing. Pathological components are effectively labeled, achieving coloring marks for cocci (single, double, chain), bacilli (long, short, coccobacilli, lactobacilli, Gardnerella), etc., enhancing detection rates and classification accuracy. It can also automatically evaluate flora density, lactobacillary ratio,
272 Chapter 16flora diversity, AV scoring, and Nugent scoring. It fully meets the requirements for microscopic examination of vaginal discharge, with recognition rates continuously improving as learning progresses. In functional testing, it enables automatic card distribution (such as β-N-acetylglucosaminidase, sialidase, leukocyte esterase, β-glucuronidase, hydrogen peroxide, pH value test cards) for measurement, precise droplet placement, automatic layered incubation, and automatic interpretation of dry chemistry test results. Morphological content of suspicious samples must be highlighted to the operator for manual review before issuing a report. 16.2 Introduction to Automated Technology for the Formation of Vaginal Discharge16.2.1 Application of Artificial Intelligence in Sample Automatic Transmission Logistics System Automatic sample transfer has become a reality in many hospitals. Laboratory departments can quickly and promptly obtain samples from various wards, outpatient clinics, emergency rooms, or blood collection windows through track or pneumatic pipeline systems and intelligent logistics management systems. Currently, the fastest pneumatic pipeline transfer system can reach speeds of up to 600 meters per minute. Clinical physicians can also use HIS to timely access information such as sampling time, submission time, receipt time by the laboratory department, testing time, and issuance time. Some hospital laboratories are equipped with automated sample delivery robots that transport samples received at the window to different work areas or operator stations along predetermined routes. In the future, drones will be used to deliver samples across buildings and floors. An intelligent automated sample transfer logistics system is also an essential configuration for modern hospitals. 16.2.2 Microscopic Imaging Optical System for Saline Vaginal Discharge The optical system for microscopic imaging of saline vaginal discharge consists of a microscope and a camera. This system requires that the microscopic images of saline vaginal discharge be clear with high contrast, where the edges of various formed elements are distinct and their shapes are clearly visible, facilitating subsequent segmentation and extraction of these elements. The smallest contaminating bacteria in the formed elements have a diameter range of 0.5 to 1 μm, which can only be clearly seen under a 40x objective lens on an
Progress in Diagnosis of Vaginal Microecology --Automated Detection and Artificial Intelligence 273optical microscope. Currently, automated vaginal discharge detection platforms on the market, considering cost factors, typically opt for ordinary optical systems and choose the Graph EXCCD series CCD cameras based on image quality and cost-effectiveness. The current system uses automatic focusing by the microscope, where the process involves image processing to control the motor operation of the focus axis movement to the focal point position through the results of the image processing. During the automatic focusing process, the microscope projects the sample's image onto the CCD, and the pixel information captured by the CCD is converted into digital signals via an image acquisition card and displayed on the computer screen. After image processing, the clarity of the image is analyzed to determine whether it is out-of-focus or focused. Then, operation commands are sent via serial port to control the motor-driven focus axis to adjust in the corresponding direction until the focus point is found. This system achieves precise automatic control between the microscope and the CCD lens, enabling fully automatic, full-field scanning, and real-time image acquisition (Figures 16-2 to 16-7).Figure 16-2 White blood cells in vaginal discharge under an optical microscope of an automatic analyzer (400x)
274 Chapter 16Figure 16-3 White blood cells and Trichomonas vaginalis in vaginal discharge under an optical microscope of an automatic analyzer (400x)Figure 16-4 Red blood cells in vaginal discharge under an optical microscope of an automatic analyzer (400x)
Progress in Diagnosis of Vaginal Microecology --Automated Detection and Artificial Intelligence 275Figure 16-5 Fungal yeast buddings and spores in vaginal discharge under an optical microscope of an automatic analyzer (400x)Figure 16-6 Fungal hyphae (shown in the blue rectangles) in vaginal discharge under an optical microscope of an automatic analyzer (400x)
276 Chapter 16Figure 16-7 Clue cells (shown in the blue squares) in vaginal discharge under an optical microscope of an automatic analyzer (400x)16.2.3 Determination of Vaginal Cleanliness by Artificial Neural Network Model Artificial neurons are a simulation of biological neurons, aiming to achieve most of the functions of biological neurons.An artificial neural network is an abstract and simplified model that mimics the human brain, consisting of many interconnected artificial neurons with a connection structure similar to that of biological neurons in the brain. When different values are input into the artificial neural network, it performs weighted sum operations on all inputs based on the network weights, then outputs the result value. The concept of artificial neural networks can be traced back to the early 1940s, when American psychologist Warren McCulloch (Warren McCulloch) and mathematician Walter Pitts (Walter Pitts) jointly invented a simplified neural network model based on mathematical threshold logic, known as the MP neural model. A single neuron has a relatively simple structure and cannot perform complex functions. However, if each neuron is used as a basic unit and a large number of neurons are connected according to certain special arrangement models to form an artificial neural network, it can handle various types of information and solve more challenging problems. In practical applications, multiple neurons are typically arranged in layers to form an artificial neural network.
Progress in Diagnosis of Vaginal Microecology --Automated Detection and Artificial Intelligence 277Based on this theory and applying it to vaginal discharge analysis, since the cleanliness of vaginal discharge is determined by the number and proportion of formed elements such as epithelial cells, white blood cells, pus cells, bacilli, and miscellaneous bacteria in the discharge, vaginal discharge cleanliness is classified into four grades: Grade I, II, III, and IV. Therefore, neural network training is conducted by extracting the characteristics of these formed elements-epithelial cells, white blood cells, pus cells, bacilli, and miscellaneous bacteria-at different levels of vaginal discharge cleanliness.16.2.4 Cell State Recognition Based on Deep Convolutional Neural Network The formed elements in vaginal discharge include epithelial cells, white blood cells, pus cells, bacilli, and miscellaneous bacteria. The types and quantities of formed elements in human vaginal discharge are numerous and complex. The types of epithelial cells vary with the organs from which they shed; the epithelial cells in vaginal discharge mainly come from the shedding of vaginal epithelial cells, specifically squamous epithelial cells. There are many types of cells, and the white blood cells in vaginal discharge are primarily neutrophils, although lymphocytes and monocytes can also be present. Neutrophils have a diameter range of 10-14 μm, are round or oval in shape, have indistinct cell nucleus boundaries that appear blurry, and contain prominent granular material within the cytoplasm. The standard range for white blood cells in vaginal discharge is 0-5 per HP. If the number of white blood cells exceeds this range, it may indicate a decrease in vaginal cleanliness or signs of vaginitis. The first step is to extract candidate regions on cell samples through the proposed regional method. The second step is to extract convolutional neural network features through the training model of convolutional neural network. Finally, the support vector machine detector scores each candidate region and obtains the final score of each candidate region. Region convolutional neural network (region-convolutional neural network, R-CNN), as a popular target detection method at present, has achieved good results on natural images, and is much better than other traditional methods in various public data sets. It can effectively detect a single target, multiple targets or multiple overlapping targets. Deep convolutional neural networks have been applied in the field of image classification for some time. Compared to other methods, their ability to learn joint features and classify is what allows them to achieve higher classification accuracy on large-scale datasets. This breakthrough in large-scale image
278 Chapter 16classification mainly comes from AlexKrizhevsky's paper published in 2012, where their team proposed AlexNet, which achieved the best results on ILSVRC 2012, improving by over ten percentage points. After the success of AlexNet, many similar teams began to make significant improvements based on it to enhance classification accuracy, such as reducing the size of convolution filters or increasing the depth of the network. For databases with a large number of categories in image classification, building a hierarchical classifier is a common strategy. The most popular approach for hierarchical classifiers is to build tree structures, allowing targets within the same category to share information. Another method involves constructing hierarchical deep convolutional neural networks (HD-CNNs) by embedding deep neural networks, dividing the classification task into two parts: first, performing coarse classification to identify simple categories, and then gradually classifying more challenging categories. Partial region-based convolutional neural networks can not only recognize the entire target but also its local information, such as the categories of different parts, making them a branch application of regional convolutional neural networks.At present, domestic instruments use deep convolutional neural network technology to explore artificial intelligence technology for deep learning. The entire deep learning model of their instrument consists of three parts: the backbone network, RPN network, and Fast-RCNN + mask. The backbone network is composed of a ResNet + FPN structure, which generates feature maps; the RPN network generates proposal regions; Fast-RCNN performs classification and regression; and the mask branch classifies and regresses targets at the pixel level (i.e., segmentation). The backbone network is implemented through the cascading of convolutional networks and multi-scale detection algorithm FPN. FPN upsamples high-level feature maps by a factor of two, then applies weighting. Through this connection method, each layer's feature map integrates different resolutions and intensities of semantic information from higher layers. These integrated feature maps are used for target detection at different resolutions, ensuring that each layer has strong semantic information and an appropriate resolution. This connection method significantly increases the probability of detecting small targets. The RPN network primarily functions by generating k anchor boxes of different sizes at each position using a sliding window on the feature layer output by the CNN network. These anchor boxes are then fed into the convolutional neural network to extract features for target classification and bounding box regression. In RPN, target classification
Progress in Diagnosis of Vaginal Microecology --Automated Detection and Artificial Intelligence 279only determines whether the anchor boxes on the feature map belong to the background or foreground, while bounding box regression corrects the anchor boxes that belong to the foreground, removing overlapping and inaccurate anchor boxes. Fast-RCNN uses SOFTMAX for target classification and mask generation to generate target frames. The division of cell detection and state recognition into two parts is also derived from hierarchical classification, which first identifies whether it is a cell and then identifies the category of the cell. This can avoid unnecessary computation and achieve lower error rate.16.3 Application and Prospect of Artificial Intelligence Technology-- Continue to Improve the Morphological Artificial Intelligence Equipment Recognition AbilityThe progress of artificial intelligence in morphological examination has been mentioned earlier, but most devices can only perform preliminary screening at present. In clinical practice, there are still many difficult issues and challenging cells that intelligent devices cannot identify or recognize; they require manual identification, sometimes even the expertise of highly experienced specialists. To continuously improve the morphological recognition and accurate identification capabilities of intelligent devices for various samples, it is necessary to expand digital image databases, refine algorithms, add feature parameters, conduct deep learning training, introduce special staining techniques, and integrate other auxiliary technical methods. Only then can improvements and enhancements be achieved. The modernization level of a hospital should not only be reflected in strong medical technology, advanced equipment, and effective management, but also in the degree of informatization and intelligence.References[1] Shimin Zhang. Application and prospect of artificial intelligence technology in medical laboratory science. International Journal of Laboratory Medicine, 2018,39(5):513-520. [2] Fan Deng. Cell detection and state recognition based on CNN for microscopic image sequences. Zhejiang: Zhejiang University of Technology, 2016. [3] Quan Zhou, Suwen Qi, Bin Xiao, et al. Artificial intelligence to assist the development of laboratory medicine. Journal of Southern Medical University. 2020,40(2):287-297.
AFTERWORDFemale’s reproductive tract infection is a social and public health problem in China and even globally. Almost all the reproductive tract infections are accompanied by the change of the female genital tract microecological environment. In recent years, new progress has been made in the diagnosis and treatment of reproductive tract infections in China. The development and application of vaginal microecological detection system provides the possibility for the accurate treatment of female reproductive tract infection, so the standardized examination of female reproductive tract infection and vaginal microecology has also been paid more and more attention from experts and scholars in related fields. However, the diagnostic technology represented by the vaginal micro-ecological evaluation system has not been widely used. The promotion of the diagnosis and treatment norms and key technologies of reproductive tract infection, training the technical level and service ability of grassroots doctors and testing personnel are the main work of the prevention and treatment of reproductive tract infections in China. Therefore, there is still a long way to go to popularize the clinical application of vaginal microecological examination in the whole process. Xiaojuan Liu Translator: Zhengqiang Hu, January, 2025
