Vaginal microbiota with bacterial vaginosis
Bacterial vaginosis is a polymicrobial clinical syndrome characterized by the replacement of the physiological lactobacillus microflora of the vagina with a spectrum of other microorganisms, mainly anaerobic. Numerous studies have shown that the key components of the vaginal microbiota with BV are Gardnerella vaginalis and Atopobium vaginae. A. vaginae are difficult to cultivate bacteria, and their association with BV has been described relatively recently using molecular methods. It was shown that these bacteria along with G. vaginalis are involved in the formation of biofilms on the vaginal epithelium. BV is characterized by a high recurrence rate, the main reasons for which are believed to be the resistance of BV-associated bacteria to antimicrobial agents, as well as the low susceptibility of biofilms to antibiotics.
Bacterial vaginosis (BV) is the most common disorder of the vaginal ecosystem in women of reproductive age. The main clinical manifestations of BV are specific secretions resulting from the decomposition of the normal vaginal mucin gel. BV is significantly associated with impaired reproductive health of women, such as cervicitis, an increased risk of infections caused by Neisseria gon- orrhoeae, Trichomonas vaginalis, Chlamydia trachomatis, Candida, HSV-2 and HIV, pelvic inflammatory diseases. BV is also associated with a number of pregnancy complications, including early and late miscarriages, premature labor, premature rupture of the membranes, low fetal weight, amnionitis, chorioamnionitis, and postpartum endometritis. BV is characterized by high rates of recurrence (about 30% within 3 months after treatment). Thus, the severity of BV complications dictates the need for timely and accurate diagnosis and effective treatment.
The difficulties of diagnosing and treating BV are largely determined by the fact that it is a polymicrobial state associated with a huge range of microorganisms, which differ in their biochemical, morphological, tinctorial characteristics, and in their sensitivity to antibacterial drugs. The use of molecular methods allowed us to identify a number of uncultivated or difficultly cultivated microorganisms involved in the development of BV. In addition, there is increasing evidence of the role of biofilms of bacterial communities tightly attached to the surface of the epithelium — in the pathogenesis of BV. This article presents the results of studies highlighting aspects of diagnosis and therapy, which are caused by the properties of key components of the vaginal microbiota in BV.
BV microbiology is very complex. BV is associated with significant changes in the microbiota of the vagina, namely a significant decrease in the number of lactobacilli and massive colonization by various bacteria, mainly obligate anaerobes. Using cultural methods, it has been shown that many microorganisms can be involved in the development of BV, such as Gardnerella vaginalis, Prevotella spp, Porphyromonas spp, Bacteroides spp, Peptostreptococcus spp, Mobiluncus spp, Mycoplasma hominis. However, the true diversity of microflora in BV was discovered only with the introduction of molecular-biological approaches, with the help of which many new (often uncultured) microorganisms were described, such as Atopobium vaginae, Megasphaera spp, Eggerthella, Leptotrichia spp, Dialister spp, bacteria, associated with bacterial vaginosis (bacterial vaginosis associated bacteria) BVAB1, BVAB2, BVAB3.
Although a whole spectrum of bacteria is associated with BV, the results of many studies have shown that the most important markers of BV are bacteria such as Gardnerella vaginalis and Atopobium vaginae. The role of G. vaginalis in the development of BV has been studied for many years, while the association of A. vaginae with BV has been described relatively recently. When gram-stained, A. vaginae appears as small cocci, round or oval, or sticks, localized as single cells, in pairs or short chains. A. vaginae are optional aerobic gram-positive bacteria that are difficult to isolate with traditional microbiological methods. Although the exact role of A. vaginae in the development of BV has not yet been established, the association of the microorganism with this disease is quite well defined, as well as its participation with G. vaginalis in the formation of biofilms on the vaginal epithelium, which are believed to play a key role role in pathogenesis bv.
A biofilm is a microbial community in which cells are attached to a surface and / or to each other and are enclosed in an interbacterial matrix of extracellular polymeric substances synthesized by them; Bacteria in biofilms have altered physiological properties. The microflora of a biofilm is more resistant to the effects of adverse factors of a physical, chemical, and biological nature as compared with free-floating (planktonic) bacteria. Under such conditions, bacteria are resistant to ultraviolet radiation, dehydration and viruses, antibiotics and immune protection factors. The stability factor of biofilms is the mucous-polymer layer, developed immediately after adhesion and including lipopolyaccharides, proteoglycans, glycoproteins, endopolysaccharides, similar to the substance of the cell wall, glycolicles and bacterial capsules.
Recent studies show that BV exists as a polymicrobial biofilm infection. G. vaginalis is believed to be the first to attach to the vaginal epithelium and then serve as a “framework” for the attachment of other bacteria. Alves P. et al. identified 30 species of bacteria associated with BV, and in model experiments characterized their virulence, defined as high adhesion, cytotoxicity, as well as a predisposition to form biofilms. It was shown that the majority of BV-associated bacteria tended to grow as biofilms, however G. vaginalis had the highest virulence.
Difficulties in the diagnosis and treatment of BV are largely determined by the fact that it is a polymicrobial state associated with a huge range of microorganisms, which differ in both biochemical, morphological, tinctorial characteristics, and sensitivity to antibacterial drugs.
Swidsinski A. et al. found biofilms in 90% of patients with BV and only in 10% of patients without BV. In the analysis of biofilms obtained in patients with BV, using the method of fluorescence in situ hybridization (fluorescence in situ hybridization, FISH) using probes for bacterial rRNA, it was shown that in most samples of G. vaginalis 60–90 % of the mass of the biofilm, A. vaginae - over 1–40% of the mass of the biofilm. Lactobacilli were found only in 20% of the samples, and their concentration was lower than 106 CFU / ml, which was 1–5% of the mass of the biofilm. Patients without BV either did not have biofilms, or had loose biofilms consisting mainly of species of the genus Lactobacillus.
Diagnosis of BV is performed using a number of clinical and laboratory methods. The main method of clinical diagnosis is the Amsel method, which involves the use of 4 criteria: 1) the presence of specific vaginal secretions (homogeneous, grayish-white, liquid, with an unpleasant odor); 2) the pH of the dischargeable vagina is above 4.5; 3) positive amine test; 4) identification of "key" cells (detached cells of the vaginal epithelium, the surface of which is dotted with bacteria, due to which they have a "granular" appearance) by microscopic examination of the native drug. If at least 3 criteria are met, the diagnosis of BV is established. The advantages of the Amsel method include the ability to diagnose BV at the time of a doctor's appointment and immediately appoint therapy. The disadvantages are the subjectivity and the inability to microscopically examine the native drug in most doctors. Clinical diagnostic methods for BV can be attributed to rapid tests (rapid tests), such as FemExam (trimethylamine test and pH measurement), gloves for measuring the patient’s pH, electronic nose (trimethylamine test), BVBlue (measuring sialidase activity), Pip Activity TestCard (measurement of lin-aminopeptidase activity). Rapid tests are not widely used in practice, mainly due to insufficiently high sensitivity and / or specificity.
The results of many studies suggest that G. vaginal and A. vaginae are almost universal markers of BV, while the use of quantitative thresholds significantly increases the specificity of tests for the identification of these microorganisms without a significant decrease in sensitivity.
The main method of laboratory diagnosis of BV is the Nugent method, which is based on the definition of three bacterial morphotypes: large gram-positive bacilli (lactobacillus morphotype), small gram-negative or gramvariable cocci and coccoacilli (Gardnerella morphotype), and gram-negative or gram-curved rods (morphological type). Mobiluncus). Depending on the sum of points, the samples are regarded as normocenosis (number of points from 0 to 3), intermediate version of microbiocenosis (number of points from 4 to 6) and BV (number of points from 7 to 10). The advantages of the Nugent method are relatively high sensitivity and specificity, a high degree of standardization, which ensures high reproducibility. The disadvantages include the complexity and the existence of an "intermediate" version of the vaginal microbiocenosis.
Microscopic examination of Gram-stained preparations also underlies the Ison-Hay methods and the WHO method, which are used much less frequently than the Nugent method. The cultural method for the diagnosis of BV is practically not used. This is due both to the difficulties of cultivating anaerobic microorganisms, and to the fact that bacteria associated with BV are usually components of the normal vaginal microflora.
The latter circumstance is also a major obstacle to the widespread use of tests based on the analysis of nucleic acids. However, since bacteria associated with BV are present in women with BV in much higher concentrations than in healthy women, this is a prerequisite for the development of molecular tests based on identifying clinically significant amounts of bacterial markers. BV. Although a wide range of bacteria is involved in BV development, only a limited number of microorganisms, namely G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2, Megasphaera type 1, Leptotrichia / Sneathia, have the potential to be used as targets for diagnosis BV. This is due to the fact that these species are present in the majority of patients with BV, which provides adequate diagnostic sensitivity. In this regard, it is interesting to note that species of the genus Mobiluncus (M. curtisii and M. mulieris), the definition of which is included in the Nugent test, although associated with BV, appear to be a poor diagnostic indicator due to low sensitivity. At the same time, the results of many studies suggest that G. vaginalis and A. vaginae are almost universal markers of BV, while the use of quantitative thresholds significantly increases the specificity of tests for the identification of these microorganisms without a significant decrease in sensitivity. So, in the work of Menard J. et al. It was shown that determining the quantitative threshold of A. vaginae (108 copies / ml) and G. vaginalis (109 copies / ml) is an accurate method for diagnosing BV with a sensitivity of 96% and a specificity of 99%. When determining the content of bacteria in the vagina of women with BV and without BV by the method of pyrosequencing, it was shown that a decrease in the content of lactobacilli with a simultaneous increase in the content of G. vaginalis and / or A. vaginae predicted BV with a sensitivity of 100% and specificity of 95%. High sensitivity and specificity (98.5% and 97%, respectively) were also shown for the method developed at the Central Research Institute of Epidemiology of the Federal Service for Supervision of Consumer Rights Protection and Human Services, which is based on determining the ratio of the concentrations of lactobacilli, G. vaginalis and A. vaginae Bacterial vaginosis).
Competent restoration normotion of Vaggines
The composition of normal vaginal contents includes various anaerobic or aerobic gram-positive and gram-negative microorganisms, the total titer of which is 108–1012 CFU / ml. Among them, facultative anaerobic bacteria are found at a concentration of 103–105 CFU / ml, while anaerobic bacteria are found at a concentration of 105–109 CFU / ml.
An important component of the vaginal indigenous flora are lactobacilli, bifidobacteria, and propionic acid bacteria. The evolution of the vaginal biotopes has led to the development of adaptive mechanisms that allow these microorganisms to actively develop in the vaginal environment and adhere to the epithelium, forming strong symbiotic bonds with it, and successfully compete with facultative and transient microflora. Vaginal Lactobacilli are capable to synthesize hydrogen peroxide, inhibit the growth of anaerobes obligat- GOVERNMENTAL genera Mobiluncus, Bacteroides, Peptococcus, Peptostreptococcus, Clostridium, and others. The toxicity of hydrogen peroxide is significantly increased in an acidic environment, whereby the spectrum of microorganisms inhibited significantly increases and extends to many optional - anaerobic and aerobic microorganisms (G. vaginalis, S. aureus, E. colli, Pseudomonas ssp., C. albicans, etc.). An important protective factor of the vaginal microflora is its ability to synthesize lysozyme and bacteriocins.
In the event of changes in the microecology of the vagina and an increase in pH, unfavorable conditions are created for the growth of lactobacilli and favorable conditions for the reproduction of bacteria that are associated with BV.
Bacterial vaginosis (BV) is one of the most common causes of abnormal white in women, mostly of reproductive age. BV is characterized by an excessively high concentration of obligate and facultative anaerobic conditional pathogenic microorganisms with a sharp decrease / absence of lactic acid bacteria (producing hydrogen peroxide) in the vagina. At the same time, the number of leukocytes does not increase significantly, which in general reflects the absence of a pronounced inflammatory reaction.
The prevalence of BV ranges from 16 to 65% and depends on the population studied. Statistically significant BV is more common in women under 25 years of age. In women of the Caucasian race, BV is diagnosed in 5–15% of cases, and in the Negroid race - 45–55%. BV, as a rule, is characteristic of women who have a large number of sexual partners, practicing unconventional sex and regular douching, who began sexually active at a young age. In outpatient gynecological practice, BV is detected in 15–25% of patients, among pregnant women, BV occurs in 20–46% of cases, in women with sexually transmitted infections - up to 40%, in women with inflammatory diseases. organs of the small pelvis - in 35%, with pathological white hair - up to 87%.
With BV, there is an increase in the concentration of aerobic and anaerobic microorganisms, against this background a decrease in the concentration of lactobacilli is noted, and this leads to a decrease in the amount of lactic acid, a decrease in the acidity of the vaginal contents and the growth of anaerobes that decarboxylate amino acids, increasing the concentration of amines in the vaginal secretion. Amines give a specific fishy smell to the secretions. Non-volatile salts of these compounds are converted to volatile amines with an alkaline pH value by adding to the drop of vaginal secretions KOH (amine test).
The main complaints at BV are:
the presence of liquid, homogeneous, sticky whitish-gray vaginal discharge, the number of which is usually insignificant. In the course of the long-term process of isolation, they acquire a yellowish-greenish color, become more dense, frothy;
the presence of an unpleasant "fishy" smell of discharge, which increases or appears after sexual contact, as well as during menstruation (alkalization of the environment);
rarely, discomfort, itching, burning sensation and dyspareunia (these symptoms are usually associated with inflammation, which is uncharacteristic for BV).
The duration of the symptoms may be from several months to several years. Asymptomatic BV occurs in about 50% of women. They either do not notice discharge or any other symptoms, or they are considered normal or associated with a violation of hygiene. The diagnosis is usually made in accordance with the criteria of Amsel or after microscopy of a smear with a Gram stain.