Vaginal Microbiota Molecular Profiling in Women with Bacterial Vaginosis: A Novel Diagnostic Tool.

Bacterial vaginosis (BV) is a most common microbiological syndrome. Multiplex next-generation sequencing (NGS) or molecular tests allow a complete and accurate vaginal microbiota profiling in order to determine the primary causative agent. Due to the high costs and limited availability of NGS, the multiplex real-time PCR draws more attention. The present study aimed to evaluate the microbial composition and dominant lactobacilli species in non-pregnant women with bacterial vaginosis using a multiplex RT-PCR test and determine its diagnostic significance. In total, 331 women complaining of vaginal discharge were included. BV was confirmed upon clinical examination and Nugent criteria. A real-time PCR test was carried out with a new Femoflor test, which identifies opportunistic bacteria, STD pathogens, and some viruses. According to the results, the rate of lactobacilli is significantly reduced in BV-affected patients when compared to healthy women. Moreover, the rate of L. crispatus significantly decreases, while the rate of L. iners remains high. Among obligate anaerobic bacteria, Gardnerella vaginalis was the most prevalent in women with BV. The Femoflor test demonstrated high sensitivity and specificity for diagnosing BV. Moreover, the test allows the identification of infection in women with intermediate vaginal microbiota, as well as STD pathogens, and viruses. Thus, the application of real-time PCR tests can be effectively used in vaginal microbiota evaluation in women with BV, intermediate vaginal microbiota, and healthy women. In addition, this test may be used as an alternative to the Amsel criteria and Nugent scoring method in diagnosing BV.

Local Immune Biomarker Expression Depending on the Uterine Microbiota in Patients with Idiopathic Infertility.

The endometrium has traditionally been considered sterile. Nowadays, active studies are performed on the female upper genital tract microbiota. Bacteria and/or viruses colonizing the endometrium are known to alter its functional properties, including receptivity and embryo implantation. Uterine cavity inflammation caused by microorganisms leads to disrupted cytokine expression, which, in turn, is mandatory for the successful implantation of the embryo. The present study assessed the vaginal and endometrial microbiota composition and its relation to the levels of cytokines produced by the endometrium in reproductive-aged women complaining of secondary infertility of unknown origin. The multiplex real-time PCR assay was applied for vaginal and endometrial microbiota analysis. The quantitative measurement of endometrial α-defensin (DEFa1), transforming growth factor (TGFß1), and basic fibroblast growth factor (bFGF2) was carried out using the ELISA (Cloud-Clone Corporation (Katy, TX, USA; manufactured in Wuhan, China). A reliable decline in endometrial TGFß1 and bFGF2 and an increase in DEFa1 were demonstrated in women with idiopathic infertility when compared to fertile patients. However, TGFß1, bFGF2, and DEFa1 expression correlated reliably only with the presence of Peptostreptococcus spp. and HPV in the uterine cavity. The obtained results highlight the importance of local immune biomarker determination in the assessment of certain bacteria and viruses' significance as causative agents of infertility.

Performance of the first commercial dual resistance assay, AmpliSens Mycoplasma genitalium-ML/FQ-Resist-FL, for detection of potential macrolide and quinolone resistance-associated mutations and prevalence of M. genitalium resistance mutations in St. Petersburg, Russia.

OBJECTIVES: Antimicrobial resistance in Mycoplasma genitalium (MG) is a poorly surveyed and controlled global health concern. We evaluated the first commercial dual resistance assay, AmpliSens M. genitalium-ML/FQ-Resist-FL assay, for detection of potential macrolide and quinolone resistance-associated mutations (MRAMs and QRAMs, respectively) and estimated the prevalence of these mutations in MG in St. Petersburg, Russia. METHODS: Urogenital samples positive (n=145 from 2007 to 2020) and negative (n=56 from 2021) for MG in routine diagnostics were retrospectively analysed using the AmpliSens M. genitalium-ML/FQ-Resist-FL assay (Central Research Institute of Epidemiology, Moscow, Russia) and Sanger sequencing for validation. RESULTS: The AmpliSens M. genitalium-ML/FQ-Resist-FL assay detected potential MRAMs and QRAMs with sensitivities of 100% (CI95% 83.9 to 100) and 92.3% (CI95% 66.7 to 99.6) and specificities of 99.2% (CI95% 95.6 to 100) and 100% (CI95% 97.2 to 100), respectively, in clinical specimens with ≥1000 MG geq/mL. In total, MRAMs were detected in 13.8% (CI95% 9.1 to 20.3) of samples, with 23S rRNA A2058G being the most prevalent mutation (45.0% (CI95% 25.8 to 65.8)). QRAMs were found in 9.0% (CI95% 5.3 to 14.7) of samples, with S83I the most frequent mutation (53.8% (CI95% 29.1 to 76.8)). Dual resistance was observed in 5.5% (CI95% 2.8 to 10.5) of samples. Potential MRAM and dual resistance rates significantly increased over time: from 0% in 2007-2008 to 25% (p trend =0.0009) and 10% (p trend =0.0447), respectively, in 2018-2020. QRAM rate appeared to increase (from 0% to 13%), but significance was not reached (p trend =0.0605). CONCLUSIONS: The rapid increase in MG antimicrobial resistance in St. Petersburg, especially prominent for MRAMs, necessitates implementation of macrolide resistance-guided therapy in Russia. The first commercial dual resistance assay, AmpliSens M. genitalium-ML/FQ-Resist-FL assay, was sensitive and specific for detection of potential MRAMs and QRAMs and could be valuable in macrolide resistance-guided therapies and possibly for surveillance of QRAMs. International surveillance of antimicrobial resistance-associated mutations in MG, further research into clinical relevance of several parC mutations and novel treatments are essential.

Significant shifts in the distribution of vaccine capsular polysaccharide types and rates of antimicrobial resistance of perinatal group B streptococci within the last decade in St. Petersburg, Russia.

Evaluation of circulating group B streptococcus (GBS) strains is important to assess the potential effects of GBS prevention strategies in a certain region. This study aimed at estimating the distributions of capsular polysaccharide (CPS) types and pilus profiles, and the rates of antimicrobial resistance among GBS strains isolated from colonized pregnant women and newborns in 2010-2011 and 2017-2018 in St. Petersburg, Russia. A total of 261 GBS isolates have been investigated. Antibiotic susceptibility testing was performed using the disc-diffusion method. CPS types and pilus profiles were determined by using PCR. Over the 9-year period, the resistance of GBS to both erythromycin and clindamycin has significantly increased, exceeding 30% in 2017-2018. The most prevalent CPS types were Ia, III, and V. Significant shifts were observed in the frequency of CPS types III (decreased) and V (increased), which resulted in a significant reduction (from 77 to 63%) in the potential coverage by a trivalent vaccine (including serotypes Ia, Ib, and III), whereas that of a pentavalent vaccine (including serotypes Ia, Ib, II, III, and V) remained largely unchanged (approximately 95%). The most common pilus profiles were PI-1/2a, PI-2a, and PI-1a/2b, and pilus genotype distribution has not changed with time. High and steadily growing resistance of perinatal GBS strains to clindamycin requires restricting its use to penicillin-allergic women at high risk for anaphylaxis and testing the GBS strains for their susceptibility to this antibiotic. A pentavalent CPS-based vaccine covers the vast majority of perinatal GBS strains in Russia.

Bacterial vaginosis-associated vaginal microbiota is an age-independent risk factor for Chlamydia trachomatis, Mycoplasma genitalium and Trichomonas vaginalis infections in low-risk women, St. Petersburg, Russia.

The large majority of studies investigating associations between bacterial vaginosis (BV) and sexually transmitted infections (STIs) have been conducted among predominantly young women with high risk for STIs. Since a risky sexual behavior is a significant risk factor for both STIs and BV, this creates a bias toward an increased association between BV and STIs. This study evaluated associations between BV-associated vaginal microbiota and STIs (Chlamydia trachomatis, Mycoplasma genitalium, Trichomonas vaginalis, and Neisseria gonorrhoeae) in a population of women with low risk for STIs and investigated STI outcomes depending on the dominating Lactobacillus species. Repository cervicovaginal samples collected from reproductive-age women from January 2014 to February 2019 were characterized for vaginal microbiota types and the STIs using multiplex real-time PCR assays. In total, 95 STI-positive and 91 STI-negative samples were included. A significant, age-independent association between BV-associated vaginal microbiota and the presence of C. trachomatis, M. genitalium, and T. vaginalis infections was identified (age-adjusted odds ratios 2.92 [95% confidence interval (CI) 1.24-7.03], 2.88 [95% CI 1.19-7.16], and 9.75 × 107 [95% CI 13.03-∞], respectively). Normal vaginal microbiota dominated by Lactobacillus crispatus, L. gasseri, or L. jensenii was a strong protective factor against C. trachomatis and/or M. genitalium infections, whereas L. iners-dominated microbiota was not significantly associated with C. trachomatis and/or M. genitalium positivity. The results of the present study confirm that STI prevention strategies should include interventions that also reduce the incidence of BV and promote a protective vaginal microbiota in both high- and low-risk women.

Quantitation of all Four Gardnerella vaginalis Clades Detects Abnormal Vaginal Microbiota Characteristic of Bacterial Vaginosis More Accurately than Putative G. vaginalis Sialidase A Gene Count.

BACKGROUND: Bacterial vaginosis (BV) is a vaginal disorder characterized by a depletion of the normal lactobacillus-dominant microbiota and overgrowth of mainly anaerobic bacteria. OBJECTIVES: The study aimed to evaluate the distribution and abundance of the Gardnerella vaginalis clades and sialidase A gene in vaginal samples from Russian women, and investigate if the G. vaginalis sialidase A gene count detects an abnormal vaginal microbiota characteristic of BV more accurately than G. vaginalis load. METHODS: Vaginal samples from 299 non-pregnant patients of gynecological clinics were examined using Nugent scores and G. vaginalis clade and sialidase A gene quantitative real-time polymerase chain reactions (PCRs). Discriminatory power for BV microbiota was evaluated with receiver operating characteristic (ROC) analysis. RESULTS: The vaginal microbiota was characterized by Nugent scores as normal, intermediate, and BV microbiota in 162, 58, and 79 women, respectively. G. vaginalis clades 1, 2, 3, 4, and the sialidase A gene were detected in 56% (51-62%), 40% (34-45%), 20% (16-25%), 94% (91-96%), and 70% (64-75%) of vaginal samples, respectively. The frequency and abundance of clades 1, 2, 4, and the sialidase A gene as well as clade multiplicity were significantly associated with abnormal microbiota. The sialidase A gene was present in all multi-clade samples, in all single-clade samples comprising clades 1, 2, and 3, and in four of 84 (5% [2-12%]) samples comprising clade 4 only. Total G. vaginalis load showed significantly higher discriminatory power for abnormal microbiota than sialidase A gene count (areas under ROC curves 0.933 vs. 0.881; p = 0.0306). CONCLUSIONS: Quantifying all four G. vaginalis clades discriminates between BV microbiota and normal microbiota more accurately than measuring G. vaginalis sialidase A gene. Clade 4 is strongly associated with BV microbiota, despite most clade 4 strains lacking the sialidase A gene.