A New PNA-FISH Probe Targeting Fannyhessea vaginae

Bacterial vaginosis (BV) is the most common vaginal infection in women of reproductive age and has been associated with serious health complications, mainly in pregnant women. It is characterized by a decrease in the number of Lactobacillus species in the healthy vaginal microbiota and an overgrowth of strict and facultative anaerobic bacteria that develop a polymicrobial biofilm. Despite over 60 years of research investigating BV, its etiology is not fully understood. Gardnerella spp. is a crucial microorganism that contributes to the formation of the biofilm and the development of BV, but the role of other BV-associated bacteria is not clear. Nevertheless, Fannyhessea vaginae (previously known as Atopobium vaginae) is a highly specific species for BV, and co-colonization with Gardnerella is thought to be a very specific diagnostic marker. The diagnosis of BV still presents some limitations, since currently used methods often fail to accurately detect BV. This work aims to develop a novel peptide nucleic acid (PNA) probe targeting F. vaginae. This probe was further validated in a multiplex assay, which included a Gardnerella-specific PNA probe, as a possible method for diagnosis of BV, and was compared with quantification by qPCR. The new PNA probe showed excellent sensitivity and specificity and could discriminate F. vaginae-Gardnerella biofilms, confirming the potential to be used for the detection of BV-associated pathogens.

Quantification of Gardnerella vaginalis, Atopobium vaginae and Lactobacillus spp. in bacterial vaginosis

Introduction: The aim of the study was to investigate prevalence of bacteria most frequently associated with bacterial vaginosis using Amsel’s criteria as well as to quantify these bacteria by real-time PCR and to explore the difference in their quantity between healthy and bacterial vaginosis samples. Methodology: For classification of vaginal discharge samples Amsel’s criteria have been used. To detect and quantify Gardnerella vaginalis Atopobium vaginae, Lactobacillus spp. and total vaginal microbiome, real-time PCR has been applied. Results: According to results of our study Amsel’s criteria matched well with real-time PCR diversification of healthy women and women with BV. Nevertheless, real-time PCR has been more sensitive in diagnosis of bacterial vaginosis. DNA quantification of bacteria demonstrated that mutual abundance of G.vaginalis and A. vaginae was good bacterial vaginosis marker . On the contrary, Lactobacillus spp. was present in high amount in both healthy and bacterial vaginosis samples, but ratio of investigated bacteria was different between them. In fact, G. vaginalis and A. vaginae comprised only 0.1% of total microbiome in healthy, whereas Lactobacillus spp. took 99.3% of it. Nonetheless, in bacterial vaginosis, G. vaginalis and A. vaginae made up 34.4% of total microbiome, while Lactobacillus spp. was 21.6%. Conclusions: According to the results of our study real-time PCR analysis was more sensitive in diagnosis of bacterial vaginosis than Amsel’s method, as well as it represented fine tool in making a difference between microbial entities in healthy and bacterial vaginosis samples.

Association of Female Genital Schistosomiasis with the Cervicovaginal Microbiota and Sexually Transmitted Infections in Zambian Women

Background The cervicovaginal microbiota, including sexually transmitted infections (STI), have not been well-described in female genital schistosomiasis (FGS). Methods Women (aged 18-31, sexually active, non-pregnant) were invited to participate at the final follow-up of HPTN 071 (PopART) Population Cohort in January-August 2018. We measured key species of the cervicovaginal microbiota (Lactobacillus crispatus, L. iners, Gardnerella vaginalis, Atopobium vaginae and Candida) and STI (Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium) using quantitative PCR (qPCR). We evaluated associations of microbiota and STI presence and concentration with FGS (qPCR-detected Schistosoma DNA in any of three genital specimens). Results The presence and concentration of key cervicovaginal species did not differ between participants with (n=30) or without FGS (n=158). A higher proportion of participants with FGS had T. vaginalis compared to FGS negative women (p=0.08), with further analysis showing that T. vaginalis was more prevalent among women with ≥2 Schistosoma qPCR positive genital specimens (50.0%, 8/16) than among FGS negative women (21.5% 34/158, p=0.01). Conclusions We found weak evidence of an association between T. vaginalis presence and FGS, with a stronger association in women with a higher burden FGS infection. Additional research is needed on potential between-parasite interactions, especially regarding HIV-1 vulnerability.

LONG-TERM OUTCOMES OF DENTAL TREATMENT OF PATIENTS WITH BACTERIAL VAGINOSIS AND THEIR SEXUAL PARTNERS

Patients with bacterial vaginosis are often diagnosed as having chronic generalized inflammatory and inflammatory-dystrophic periodontal diseases including chronic catarrhal gingivitis, mild and moderate periodontitis. The aim of this study was to compare the effectiveness of the treatment of inflammatory and inflammatory-dystrophic periodontal diseases in women with bacterial vaginosis and their sexual partners. The study included 28 heterosexual couples aged 18-45. Clinical dental check-up was performed during the treatment and in 3 months after the treatment completion. Green-Vermilion indices, РMA (in Parma modification), complex periodontal index (CPI) according to Leus, Svrakov's iodine number were determined. Both groups of patients were examined for the presence of Gardnerella vaginalis and Atopobium vaginae in the oral cavity by PCR testing. The therapeutic course was the same for all groups of patients. General therapy included "Fluconazole" in a dose of 50 mg once a day for 7 days, "Clindamycin" in a dose of 150 mg every 6 hours for 5 days, probiotic "Symbiter acidophilus concentrated" was prescribed to be taken starting from the 3rd day of antibiotic therapy for 21 days, and Calcium-D3-Nycomed (1 tab. during dinner for 30 days). Local treatment included oral baths of the drug "Stomatophyte" after morning and evening hygienic procedures for 7 days, "Metrogil-dent" ointment for gum application for 7 days, the chewing drug "Lizak" (1 tab. every 6 hours for 5 days), "Symbiter omega" in silicone caps for gum application overnight following the for 21 days. Chronic generalized catarrhal gingivitis was detected in 10.71% of women, mild chronic generalized periodontitis – in 64.29% and moderate chronic generalized periodontitis – in 25% of the women. In men who were carriers of pathogens of bacterial vaginosis, chronic generalized catarrhal gingivitis was found in 7.14% of individuals, mild chronic generalized periodontitis – in 60.71% and moderate chronic generalized periodontitis – in 32 male subjects. General and local dental treatment of women with bacterial vaginosis and their sexual partners showed a positive evidence-based dynamics of dental status. 3 months after clinical and laboratory observations, no complaints were presented and the oral hygiene status improved significantly. Before the treatment, the Green-Vermilion index mean values in women with bacterial vaginosis were 1.323 ± 0.035; by the end of 3 month therapy they equalled to 1.032 ± 0.021. A similar dynamics was seen in the men: from the initial index values of 1.336 ± 0.041, the Green-Vermilion index lowered to 1.048 ± 0.036. By the end of 3 month therapy, the PMA decreased by 49.03% in women and by 51.95% in men; no significant difference between the indicators of groups I and II was registered, while the difference between the results in the middle of groups I and II is significant. The analysis of the СPI index values demonstrated the women had a significant difference between baseline and post-treatment outcomes of 1.98 scores, while the men had 2.01 scores. No significant difference was found between the findings of groups I and II both before and after the therapy. A similar positive dynamics was confirmed by the Svrakov's iodine number indicator and according to this indicator obtained before and after the therapy, difference between groups was not detected. The significant difference between the indicators in the group of women and men was 2,789 and 2,831, respectively. The detection Gardnerella vaginalis and Atopobium vaginae in the oral cavity before and after the therapy did not demonstrate a significant difference between the group of men and women, but there was a clear difference between the parameters obtained at the initial examination and 3 months after the therapy. Gardnerella vaginalis was detected by 67.87% and 61.59% less, Atopobium vaginae – by 65.79% and 58.65% less in the women and men, respectively. This patented treatment regimen for patients with bacterial vaginosis contributes to the regression of inflammatory manifestations, improves oral hygiene, and is effective for the treatment of women with this comorbidity and for the therapy of their sexual partners. The dynamics of reducing the percentage of detection of bacterial vaginosis pathogens reflects the dynamics of the clinical picture of periodontal disease in both women with bacterial vaginosis and men who are their sexual partners that confirms the effectiveness of the therapy for both gender groups.

Atopobium vaginae and Prevotella bivia Are Able to Incorporate and Influence Gene Expression in a Pre-Formed Gardnerella vaginalis Biofilm

Bacterial vaginosis (BV) is associated with a highly structured polymicrobial biofilm on the vaginal epithelium where Gardnerella species presumably play a pivotal role. Gardnerella vaginalis, Atopobium vaginae, and Prevotella bivia are vaginal pathogens detected during the early stages of incident BV. Herein, we aimed to analyze the impact of A. vaginae and P. bivia on a pre-established G. vaginalis biofilm using a novel in vitro triple-species biofilm model. Total biofilm biomass was determined by the crystal violet method. We also discriminated the bacterial populations in the biofilm and in its planktonic fraction by using PNA FISH. We further analyzed the influence of A. vaginae and P. bivia on the expression of key virulence genes of G. vaginalis by quantitative PCR. In our tested conditions, A. vaginae and P. bivia were able to incorporate into pre-established G. vaginalis biofilms but did not induce an increase in total biofilm biomass, when compared with 48-h G. vaginalis biofilms. However, they were able to significantly influence the expression of HMPREF0424_0821, a gene suggested to be associated with biofilm maintenance in G. vaginalis. This study suggests that microbial relationships between co-infecting bacteria can deeply affect the G. vaginalis biofilm, a crucial marker of BV.

Association Between Leptotrichia Amnionii and Atopobium Vaginae as A Risk Factor For Miscarriage States in A Specimens of Iraqi Women

Miscarriage is one of the fundamental complications occurring in pregnant women. Many fastidious and uncultivated bacterial species are related to miscarriage and have a significant role in the infection. The association between inverse pregnancy outcomes and infections of abnormal bacteria has been rarely investigated. Therefore, this study aimed to determine Leptotrichia amnionii and Atopobium vaginae as a risk factor for miscarriage states. A total of 80 cervical swabs and blood samples were obtained from females (34 non-pregnant with recurrent spontaneous miscarriage, 11 pregnant who had previous miscarriage, and 35 without miscarriage as control) who were referred to a hospital in Baghdad city. The relationship between demographic characteristics and miscarriage groups indicates a positive association between the age of 30 years and >45 years and miscarriage states, while no significant association (p>0.05) was found for the other parameters compared with the control. Also, no association was observed between the levels of TORCH IgM , antiphospholipid (APL) IgM, and anticardiolipidin (ACL) IgM antibodies and spontaneous miscarriage. The findings of RT-PCR detection revealed that, out of 45 samples of recurrent spontaneous miscarriage, 17 were detected with high-risk of L. aminonia compared with two samples from the control group. Also, 7 samples of recurrent spontaneous miscarriage were shown to be infected with A. vaginae compared with five samples from the control group. The adjusted odd ratios (ORs) of L. anmionii for spontaneous miscarriage and pregnancy states were 7.89 (CI 95%, 1.60 - 39.00) and 9.43 (CI 95%, 1.43 - 61.9), respectively, with highly significant difference between the study groups compared with the control. While no significant result was recorded between the adjusted ORs of A. vaginae for spontaneous miscarriage and pregnancy states, which were 1.23 (CI 95%, 0.416 - 3.67) and 0.636 (CI 95%, 0.083 - 4.88), respectively, compared with the control.

Predominance of Atopobium vaginae at Midtrimester: a Potential Indicator of Preterm Birth Risk in a Nigerian Cohort

ABSTRACT Preterm birth (PTB) is the largest contributor to infant death in sub-Saharan Africa and globally. With a global estimate of 773,600, Nigeria has the third highest rate of PTB worldwide. There have been a number of microbiome profiling studies to identify vaginal microbiomes suggestive of preterm and healthy birth outcome. However, studies on the pregnancy vaginal microbiome in Africa are sparse with none performed in Nigeria. Moreover, few studies have considered the concurrent impact of steroid hormones and the vaginal microbiome on pregnancy outcome. We assessed two key determinants of pregnancy progression to gain a deeper understanding of the interactions between vaginal microbiome composition, steroid hormone concentrations, and pregnancy outcome. Vaginal swabs and blood samples were prospectively collected from healthy midtrimester pregnant women. Vaginal microbiome compositions were assessed by analysis of the V3-V5 region of 16S rRNA genes, and potential functional metabolic traits of identified vaginal microbiomes were imputed by PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states) analysis, while plasma estradiol (E2) and progesterone (P1) levels were quantified by the competitive enzyme-linked immunosorbent assay (ELISA). PTB vaginal samples were characterized by increased microbial richness, high diversity, and depletion of lactobacilli compared to term delivery samples. Women who delivered preterm were characterized by an Atopobium vaginae-dominated vagitype. High relative abundance of Atopobium vaginae at the midtrimester was highly predictive of PTB (area under the receiving operator characteristics [AUROC] of 0.983). There was a marked overlap in the range of plasma E2 and P1 values between term and PTB groups. IMPORTANCE Giving birth too soon accounts for half of all newborn deaths worldwide. Clinical symptoms alone are not sufficient to identify women at risk of giving birth too early, as such a pragmatic approach to reducing the incidence of preterm birth entails developing early strategies for intervention before it materializes. In view of the role played by the vaginal microbiome and maternal steroid hormones in determining obstetric outcome, we assessed the vaginal microbiome composition and steroid hormone during pregnancy and examined their relationship in predicting preterm birth risk in Nigerian women. This study highlights a potential early-driver microbial marker for prediction of preterm birth risk and supports the notion that vaginal microbiome composition varies across populations. A knowledge of relevant preterm birth microbial markers specific to populations would enhance the development of personalized therapeutic interventions toward restoring a microbiome that optimizes reproductive health fitness, therefore reducing the incidence of preterm birth.