Composition of the Vaginal Microbiota in Women of Reproductive Age – Sensitive and Specific Molecular Diagnosis of Bacterial Vaginosis Is Possible?

Background and Objective

Bacterial vaginosis (BV) is the most common vaginal disorder, characterized by depletion of the normal lactobacillus-dominant microbiota and overgrowth of commensal anaerobic bacteria. This study aimed to investigate the composition of the vaginal microbiota in women of reproductive age (healthy women and women with BV), with the view of developing molecular criteria for BV diagnosis.

Materials and Methods

Vaginal samples from 163 women (79 control, 73 BV and 11 intermediate (Lactobacillary grade II flora) cases) were analyzed using 454 pyrosequencing of the hypervariable regions V3–V4 of the 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays. Sensitivities and specificities of potential BV markers were computed using the Amsel criteria as reference standard for BV. The use of quantitative thresholds for prediction of BV, determined for both relative abundance measured with 454 pyrosequencing and bacterial load measured with qPCR, was evaluated.

Results

Relative to the healthy women, the BV patients had in their vaginal microbiota significantly higher prevalence, loads and relative abundances of the majority of BV associated bacteria. However, only Gardnerella vaginalis, Atopobium vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 detected at or above optimal thresholds were highly predictable for BV, with the best diagnostic accuracy shown for A. vaginae. The depletion of Lactobacillus species combined with the presence of either G. vaginalis or A. vaginae at diagnostic levels was a highly accurate BV predictor.

Conclusions

Quantitative determination of the presence of G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Measurements of abundance of normal and BV microbiota relative to total bacteria in vaginal fluid may provide more accurate BV diagnosis, and be used for test-of-cure, rather than qualitative detection or absolute counts of BV related microorganisms.     

Deep Sequencing of the Vaginal Microbiota of Women with HIV

Background

Women living with HIV and co-infected with bacterial vaginosis (BV) are at higher risk for transmitting HIV to a partner or newborn. It is poorly understood which bacterial communities constitute BV or the normal vaginal microbiota among this population and how the microbiota associated with BV responds to antibiotic treatment.

Methods and Findings

The vaginal microbiota of 132 HIV positive Tanzanian women, including 39 who received metronidazole treatment for BV, were profiled using Illumina to sequence the V6 region of the 16S rRNA gene. Of note, Gardnerella vaginalis and Lactobacillus iners were detected in each sample constituting core members of the vaginal microbiota. Eight major clusters were detected with relatively uniform microbiota compositions. Two clusters dominated by L. iners or L. crispatus were strongly associated with a normal microbiota. The L. crispatus dominated microbiota were associated with low pH, but when L. crispatus was not present, a large fraction of L. iners was required to predict a low pH. Four clusters were strongly associated with BV, and were dominated by Prevotella bivia, Lachnospiraceae, or a mixture of different species. Metronidazole treatment reduced the microbial diversity and perturbed the BV-associated microbiota, but rarely resulted in the establishment of a lactobacilli-dominated microbiota.

Conclusions

Illumina based microbial profiling enabled high though-put analyses of microbial samples at a high phylogenetic resolution. The vaginal microbiota among women living with HIV in Sub-Saharan Africa constitutes several profiles associated with a normal microbiota or BV. Recurrence of BV frequently constitutes a different BV-associated profile than before antibiotic treatment.     

The complex vaginal flora of West African women with bacterial vaginosis

Background

The spectrum of bacteria associated with bacterial vaginosis (BV) has recently expanded through taxonomic changes and the use of molecular methods. These methods have yet to be used in large-scale epidemiological studies in Africa where BV is highly prevalent.

Methods

An analysis of samples obtained during a clinical trial of the management of vaginal discharge in four West African countries. Samples were available from 1555 participants; 843 (54%) had BV. Nucleic acids of 13 bacterial genera or species potentially associated with BV were detected through the polymerase chain reaction.

Results

The associations between various components of the vaginal flora were complex. Excluding Lactobacillus, the other 12 micro-organisms were all associated with each other at the p≤0.001 level. The prevalence of various bacterial genera or species varied according to age, sexual activity and HIV status. In multivariate analysis, the presence of Gardnerella vaginalis, Bifidobacterium, Megasphaera elsdenii, Dialister, Mycoplasma hominis, Leptotrichia, and Prevotella were independently associated with BV as was the absence of Lactobacillus and Peptoniphilus. However, Mobiluncus, Atopobium vaginae, Anaerococcus, and Eggerthella were not independently associated with BV. Unexpectedly, after treatment with a regimen that included either metronidazole or tinidazole, the proportion of patients with a complete resolution of symptoms by day 14 increased with the number of bacterial genera or species present at enrolment.

Conclusions

Numerous bacterial genera or species were strongly associated with each other in a pattern that suggested a symbiotic relationship. BV cases with a simpler flora were less likely to respond to treatment. Overall, the vaginal flora of West African women with BV was reminiscent of that of their counterparts in industrialized countries.     

Bacterial vaginosis (BV) candidate bacteria: associations with BV and behavioural practices in sexually-experienced and inexperienced women

Background

In recent years several new fastidious bacteria have been identified that display a high specificity for BV; however no previous studies have comprehensively assessed the behavioural risk associations of these bacterial vaginosis-candidate organisms (BV-COs).

Methods

We examined the associations between 8 key previously described BV-COs and BV status established by Nugent''s score (NS). We also examined the sexual practices associated with each BV-CO. We incorporated 2 study populations: 193 from a sexually-inexperienced university population and 146 from a highly sexually-active clinic population. Detailed behavioural data was collected by questionnaire and vaginal smears were scored by the Nugent method. Stored samples were tested by quantitative PCR assays for the 8 BV-COs: Atopobium vaginae, Gardnerella vaginalis, Leptotrichia spp., Megasphaera type I, Sneathia spp., and the Clostridia-like bacteria BVAB1, BVAB2 and BVAB3. Associations between BV-COs and BV and behaviours were examined by univariate and multivariable analyses.

Results

On univariate analysis, all BV-COs were more common in BV compared to normal flora. However, only Megasphaera type I, BVAB2, A. vaginae and G. vaginalis were significantly independently associated with BV by multivariable analysis. Six of the eight BV-COs (Megasphaera type I, BVAB2, BVAB3, Sneathia, Leptotrichia and G. vaginalis) were rare or absent in sexually-unexposed women, and demonstrated increasing odds of detection with increasing levels of sexual activity and/or numbers of lifetime sexual partners. Only G. vaginalis and A. vaginae were commonly detected in sexually-unexposed women. Megasphaera type I was independently associated with women-who-have-sex-with women (WSW) and lifetime sexual partner numbers, while unprotected penile-vaginal-sex was associated with BVAB2 detection by multivariate analysis.

Conclusions

Four of eight key BV-COs were significantly associated with BV after adjusting for the presence of other BV-COs. The majority of BV-COs were absent or rare in sexually-unexposed women, and associated with increasing sexual exposure, suggesting potential sexual transmission of BV-COs.     

Exploring the diversity of Gardnerella vaginalis in the genitourinary tract microbiota of monogamous couples through subtle nucleotide variation

Background

Bacterial vaginosis (BV) is an enigmatic disease of unknown origin that affects a large percentage of women. The vaginal microbiota of women with BV is associated with serious sequelae, including abnormal pregnancies. The etiology of BV is not fully understood, however, it has been suggested that it is transmissible, and that G. vaginalis may be an etiological agent. Studies using enzymatic assays to define G. vaginalis biotypes, as well as more recent genomic comparisons of G. vaginalis isolates from symptomatic and asymptomatic women, suggest that particular G. vaginalis strains may play a key role in the pathogenesis of BV.

Methodology/Principal Findings

To explore G. vaginalis diversity, distribution and sexual transmission, we developed a Shannon entropy-based method to analyze low-level sequence variation in 65,710 G. vaginalis 16S rRNA gene segments that were PCR-amplified from vaginal samples of 53 monogamous women and from urethral and penile skin samples of their male partners. We observed a high degree of low-level diversity among G. vaginalis sequences with a total of 46 unique sequence variants (oligotypes), and also found strong correlations of these oligotypes between sexual partners. Even though Gram stain-defined normal and some Gram stain-defined intermediate oligotype profiles clustered together in UniFrac analysis, no single G. vaginalis oligotype was found to be specific to BV or normal vaginal samples.

Conclusions

This study describes a novel method for investigating G. vaginalis diversity at a low level of taxonomic discrimination. The findings support cultivation-based studies that indicate sexual partners harbor the same strains of G. vaginalis. This study also highlights the fact that a few, reproducible nucleotide variations within the 16S rRNA gene can reveal clinical or epidemiological associations that would be missed by genus-level or species-level categorization of 16S rRNA data.     

Risks for Acquisition of Bacterial Vaginosis Among Women Who Report Sex with Women: A Cohort Study

Background

Bacterial vaginosis (BV) is common in women who have sex with women. While cross-sectional data support a role for sexual transmission, risks for incident BV have not been prospectively studied in this group.

Methodology/Principal Findings

We studied risks for BV acquisition in a prospective cohort study of women (age 16–35 years) who reported sex with other women (≥1 partner, prior year). Women were followed for one year with examinations at quarterly visits and for genital symptoms at any time. Species-specific 16S rRNA gene PCRs for BV-associated bacteria (BVAB) were applied to vaginal fluid obtained at enrollment. Sexual behaviors were ascertained by computer-assisted interview. Of 335 participants, 239 had no BV at baseline; 199 were seen in follow-up (median follow-up 355 days, 4.0 visits/subject). Forty women experienced ≥1 BV episode. Risks for incident BV were presentation ≤14 days since onset of menses (hazard ratio (HR) 2.3 (95% CI, 1.2–4.7), report of new sex partner with BV history (HR 3.63 (1.1–11.9)), change in vaginal discharge (HR 2.6 (1.3–5.2)) and detection of any of several BVAB in vaginal fluid at enrollment, including BVAB1 (HR 6.3 (1.4–28.1)), BVAB2 (HR 18.2 (6.4–51.8)), BVAB3 (HR 12.6 (2.7–58.4)), G. vaginalis (HR 3.9 (1.5–10.4)), Atopobium vaginae (HR 4.2 (1.9–9.3)), Leptotrichia spp (9.3 (3.0–24.4)), and Megasphaera-1 (HR 11.5 (5.0–26.6)). Detection of Lactobacillus crispatus at enrollment conferred reduced risk for subsequent BV (HR 0.18 (0.08–0.4)). Detailed analysis of behavioral data suggested a direct dose-response relationship with increasing number of episodes of receptive oral-vulvovaginal sex (HR 1.02 (95% CI, 1.00–1.04).

Conclusions/Significance

Vaginal detection of several BVAB in BV-negative women predicted subsequent BV, suggesting that changes in vaginal microbiota precede BV by weeks or months. BV acquisition was associated with report of new partner with BV; associations with sexual practices – specifically, receptive oral sex – require further investigation.     

Comparative Genomics of Gardnerella vaginalis Strains Reveals Substantial Differences in Metabolic and Virulence Potential

Background

Gardnerella vaginalis is described as a common vaginal bacterial species whose presence correlates strongly with bacterial vaginosis (BV). Here we report the genome sequencing and comparative analyses of three strains of G. vaginalis. Strains 317 (ATCC 14019) and 594 (ATCC 14018) were isolated from the vaginal tracts of women with symptomatic BV, while Strain 409-05 was isolated from a healthy, asymptomatic individual with a Nugent score of 9.

Principal Findings

Substantial genomic rearrangement and heterogeneity were observed that appeared to have resulted from both mobile elements and substantial lateral gene transfer. These genomic differences translated to differences in metabolic potential. All strains are equipped with significant virulence potential, including genes encoding the previously described vaginolysin, pili for cytoadhesion, EPS biosynthetic genes for biofilm formation, and antimicrobial resistance systems, We also observed systems promoting multi-drug and lantibiotic extrusion. All G. vaginalis strains possess a large number of genes that may enhance their ability to compete with and exclude other vaginal colonists. These include up to six toxin-antitoxin systems and up to nine additional antitoxins lacking cognate toxins, several of which are clustered within each genome. All strains encode bacteriocidal toxins, including two lysozyme-like toxins produced uniquely by strain 409-05. Interestingly, the BV isolates encode numerous proteins not found in strain 409-05 that likely increase their pathogenic potential. These include enzymes enabling mucin degradation, a trait previously described to strongly correlate with BV, although commonly attributed to non-G. vaginalis species.

Conclusions

Collectively, our results indicate that all three strains are able to thrive in vaginal environments, and therein the BV isolates are capable of occupying a niche that is unique from 409-05. Each strain has significant virulence potential, although genomic and metabolic differences, such as the ability to degrade mucin, indicate that the detection of G. vaginalis in the vaginal tract provides only partial information on the physiological potential of the organism.     

Associations between Vaginal Pathogenic Community and Bacterial Vaginosis in Chinese Reproductive-Age Women

Background

Bacterial vaginosis (BV) is one of the most common urogenital infections among women of reproductive age that represents shifts in microbiota from Lactobacillus spp. to diverse anaerobes. The aim of our study was to evalute the diagnostic values of Gardnerella, Atopobium, Eggerthella, Megasphaera typeI, Leptotrichia/Sneathia and Prevotella, defined as a vaginal pathogenic community for BV and their associations with vaginal pH and Nugent scores.

Methods and Findings

We investigated the vaginal pathogenic bacteria and Lactobacillus spp. with species-specific real-time quantitative PCR (qPCR) in 50 BV-positive and 50 BV-negative Chinese women of reproductive age. Relative to BV-negative subjects, a siginificant decline in Lactobacillus and an obvious increase in bacteria in the vaginal pathogenic community were observed in BV-postive subjects (P<0.05). With the exception of Megasphaera typeI, other vaginal pathogenic bacteria were highly predictable for BV with a better sensitivity and specificity. The vaginal pathogenic community was positively associated with vaginal pH and Nugent scores, while Lactobacillus spp., such as L. iners and L. crispatus was negatively associated with them (P<0.05).

Conclusions

Our data implied that the prevalance of vaginal pathogenic bacteria as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Vaginal microbiota shifts, especially the overgrowth of the vaginal pathogenic community, showed well diagnostic values in predicting BV. Postive correlations between those vaginal pathogenic bacteria and vaginal pH, Nugent score indicated the vaginal pathogenic community rather than a single vaginal microorganism, was participated in the onset of BV directly.     

Presence of a Polymicrobial Endometrial Biofilm in Patients with Bacterial Vaginosis

Objective

To assess whether the bacterial vaginosis biofilm extends into the upper female genital tract.

Study Design

Endometrial samples obtained during curettage and fallopian tube samples obtained during salpingectomy were collected. Endometrial and fallopian tube samples were analyzed for the presence of bacteria with fluorescence-in-situ-hybridisation (FISH) analysis with probes targeting bacterial vaginosis-associated and other bacteria.

Results

A structured polymicrobial Gardnerella vaginalis biofilm could be detected in part of the endometrial and fallopian tube specimens. Women with bacterial vaginosis had a 50.0% (95% CI 24.0–76.0) risk of presenting with an endometrial Gardnerella vaginalis biofilm. Pregnancy (AOR  = 41.5, 95% CI 5.0–341.9, p<0.001) and the presence of bacterial vaginosis (AOR  = 23.2, 95% CI 2.6–205.9, p<0.001) were highly predictive of the presence of uterine or fallopian bacterial colonisation when compared to non-pregnant women without bacterial vaginosis.

Conclusion

Bacterial vaginosis is frequently associated with the presence of a structured polymicrobial Gardnerella vaginalis biofilm attached to the endometrium. This may have major implications for our understanding of the pathogenesis of adverse pregnancy outcome in association with bacterial vaginosis.     

Longitudinal qPCR Study of the Dynamics of L. crispatus,L. iners,A. vaginae, (Sialidase Positive) G. vaginalis,and P. bivia in the Vagina

Background

To obtain more detailed understanding of the causes of disturbance of the vaginal microflora (VMF), a longitudinal study was carried out for 17 women during two menstrual cycles.

Methods

Vaginal swabs were obtained daily from 17 non-pregnant, menarchal volunteers. For each woman, Gram stains were scored, the quantitative changes of 5 key vaginal species, i.e. Atopobium vaginae, Lactobacillus crispatus, L. iners, (sialidase positive) Gardnerella vaginalis and Prevotella bivia were quantified with qPCR and hydrogen-peroxide production was assessed on TMB+ agar.

Results

Women could be divided in 9 subjects with predominantly normal VMF (grades Ia, Ib and Iab, group N) and 8 with predominantly disturbed VMF (grades I-like, II, III and IV, group D).VMF was variable between women, but overall stable for most of the women. Menses were the strongest disturbing factor of the VMF. L. crispatus was present at log7–9 cells/ml in grade Ia, Iab and II VMF, but concentrations declined 100-fold during menses. L. crispatus below log7 cells/ml corresponded with poor H₂O₂-production. L. iners was present at log 10 cells/ml in grade Ib, II and III VMF. Sialidase negative G. vaginalis strains (average log5 cells/ml) were detected in grade I, I-like and IV VMF. In grade II VMF, predominantly a mixture of both sialidase negative and positive G. vaginalis strains (average log9 cells/ml) were present, and predominantly sialidase positive strains in grade III VMF. The presence of A. vaginae (average log9 cells/ml) coincided with grade II and III VMF. P. bivia (log4–8 cells/ml) was mostly present in grade III vaginal microflora. L. iners, G. vaginalis, A. vaginae and P. bivia all increased around menses for group N women, and as such L. iners was considered a member of disturbed VMF.

Conclusions

This qPCR-based study confirms largely the results of previous culture-based, microscopy-based and pyrosequencing-based studies.     

Screening of Compounds against Gardnerella vaginalis Biofilms

Bacterial vaginosis (BV) is a common infection in reproductive age woman and is characterized by dysbiosis of the healthy vaginal flora which is dominated by Lactobacilli, followed by growth of bacteria like Gardnerella vaginalis. The ability of G. vaginalis to form biofilms contributes to the high rates of recurrence that are typical for BV and which unfortunately make repeated antibiotic therapy inevitable. Here we developed a biofilm model for G. vaginalis and screened a large spectrum of compounds for their ability to prevent biofilm formation and to resolve an existing G. vaginalis biofilm. The antibiotics metronidazole and tobramycin were highly effective in preventing biofilm formation, but had no effect on an established biofilm. The application of the amphoteric tenside sodium cocoamphoacetate (SCAA) led to disintegration of existing biofilms, reducing biomass by 51% and viability by 61% and it was able to increase the effect of metronidazole by 40% (biomass) and 61% (viability). Our data show that attacking the biofilm and the bacterial cells by the combination of an amphoteric tenside with the antibiotic metronidazole might be a useful strategy against BV.     

Prevalence and Correlates of Bacterial Vaginosis in Different Sub-Populations of Women in Sub-Saharan Africa: A Cross-Sectional Study

Background

Clinical development of vaginally applied products aimed at reducing the transmission of HIV and other sexually transmitted infections, has highlighted the need for a better characterisation of the vaginal environment. We set out to characterise the vaginal environment in women in different settings in sub-Saharan Africa.

Methods

A longitudinal study was conducted in Kenya, Rwanda and South-Africa. Women were recruited into pre-defined study groups including adult, non-pregnant, HIV-negative women; pregnant women; adolescent girls; HIV-negative women engaging in vaginal practices; female sex workers; and HIV-positive women. Consenting women were interviewed and underwent a pelvic exam. Samples of vaginal fluid and a blood sample were taken and tested for bacterial vaginosis (BV), HIV and other reproductive tract infections (RTIs). This paper presents the cross-sectional analyses of BV Nugent scores and RTI prevalence and correlates at the screening and the enrolment visit.

Results

At the screening visit 38% of women had BV defined as a Nugent score of 7–10, and 64% had more than one RTI (N. gonorrhoea, C. trachomatis, T. vaginalis, syphilis) and/or Candida. At screening the likelihood of BV was lower in women using progestin-only contraception and higher in women with more than one RTI. At enrolment, BV scores were significantly associated with the presence of prostate specific antigen (PSA) in the vaginal fluid and with being a self-acknowledged sex worker. Further, sex workers were more likely to have incident BV by Nugent score at enrolment.

Conclusions

Our study confirmed some of the correlates of BV that have been previously reported but the most salient finding was the association between BV and the presence of PSA in the vaginal fluid which is suggestive of recent unprotected sexual intercourse.     

Correlates of HIV-1 genital shedding in Tanzanian women

Background

Understanding the correlates of HIV shedding is important to inform strategies to reduce HIV infectiousness. We examined correlates of genital HIV-1 RNA in women who were seropositive for both herpes simplex virus (HSV)-2 and HIV-1 and who were enrolled in a randomised controlled trial of HSV suppressive therapy (aciclovir 400 mg b.i.d vs. placebo) in Tanzania.

Methodology

Samples, including a cervico-vaginal lavage, were collected and tested for genital HIV-1 and HSV and reproductive tract infections (RTIs) at randomisation and 6, 12 and 24 months follow-up. Data from all women at randomisation and women in the placebo arm during follow-up were analysed using generalised estimating equations to determine the correlates of cervico-vaginal HIV-1 RNA detection and load.

Principal Findings

Cervico-vaginal HIV-1 RNA was detected at 52.0% of 971 visits among 482 women, and was independently associated with plasma viral load, presence of genital ulcers, pregnancy, bloody cervical or vaginal discharge, abnormal vaginal discharge, cervical ectopy, Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, an intermediate bacterial vaginosis score and HSV DNA detection. Similar factors were associated with genital HIV-1 RNA load.

Conclusions

RTIs were associated with increased presence and quantity of genital HIV-1 RNA in this population. These results highlight the importance of integrating effective RTI treatment into HIV care services.     

Characteristic male urine microbiomes associate with asymptomatic sexually transmitted infection

Background

The microbiome of the male urogenital tract is poorly described but it has been suggested that bacterial colonization of the male urethra might impact risk of sexually transmitted infection (STI). Previous cultivation-dependent studies showed that a variety of non-pathogenic bacteria colonize the urethra but did not thoroughly characterize these microbiomes or establish links between the compositions of urethral microbiomes and STI.

Methodology/Findings

Here, we used 16S rRNA PCR and sequencing to identify bacteria in urine specimens collected from men who lacked symptoms of urethral inflammation but who differed in status for STI. All of the urine samples contained multiple bacterial genera and many contained taxa that colonize the human vagina. Uncultivated bacteria associated with female genital tract pathology were abundant in specimens from men who had STI.

Conclusions

Urine microbiomes from men with STI were dominated by fastidious, anaerobic and uncultivated bacteria. The same taxa were rare in STI negative individuals. Our findings suggest that the composition of male urine microbiomes is related to STI.     

Longitudinal study of the dynamics of vaginal microflora during two consecutive menstrual cycles

Background

Although the vaginal microflora (VMF) has been well studied, information on the fluctuation of the different bacterial species throughout the menstrual cycle and the information on events preceding the presence of disturbed VMF is still very limited. Documenting the dynamics of the VMF during the menstrual cycle might provide better insights. In this study, we assessed the presence of different Lactobacillus species in relation to the BV associated species during the menstrual cycle, assessed the influence of the menstrual cycle on the different categories of vaginal microflora and assessed possible causes, such as menstruation and sexual intercourse, of VMF disturbance. To our knowledge, this is the first longitudinal study in which swabs and Gram stains were available for each day of two consecutive menstrual cycles, whereby 8 grades of VMF were distinguished by Gram stain analysis, and whereby the swabs were cultured every 7^th day and identification of the bacterial isolates was carried out with a molecular technique.

Methods

Self-collected vaginal swabs were obtained daily from 17 non pregnant, menarchal volunteers, and used for daily Gram staining and weekly culture. Bacterial isolates were identified with tDNA-PCR and 16 S rRNA gene sequencing.

Results

Nine women presented with predominantly normal VMF and the 8 others had predominantly disturbed VMF. The overall VMF of each volunteer was characteristic and rather stable. Menses and antimicrobials were the major disturbing factors of the VMF. Disturbances were always accompanied by a rise in Gram positive cocci, which also appeared to be a significant group within the VMF in general.

Conclusions

We observed a huge interindividual variability of predominantly stable VMF types. The importance of Gram positive cocci in VMF is underestimated. L. crispatus was the species that was most negatively affected by the menses, whereas the presence of the other lactobacilli was less variable.     

A Multi-Omic Systems-Based Approach Reveals Metabolic Markers of Bacterial Vaginosis and Insight into the Disease

Background

Bacterial vaginosis (BV) is the most common vaginal disorder of reproductive-age women. Yet the cause of BV has not been established. To uncover key determinants of BV, we employed a multi-omic, systems-biology approach, including both deep 16S rRNA gene-based sequencing and metabolomics of lavage samples from 36 women. These women varied demographically, behaviorally, and in terms of health status and symptoms.

Principal Findings

16S rRNA gene-based community composition profiles reflected Nugent scores, but not Amsel criteria. In contrast, metabolomic profiles were markedly more concordant with Amsel criteria. Metabolomic profiles revealed two distinct symptomatic BV types (SBVI and SBVII) with similar characteristics that indicated disruption of epithelial integrity, but each type was correlated to the presence of different microbial taxa and metabolites, as well as to different host behaviors. The characteristic odor associated with BV was linked to increases in putrescine and cadaverine, which were both linked to Dialister spp. Additional correlations were seen with the presence of discharge, 2-methyl-2-hydroxybutanoic acid, and Mobiluncus spp., and with pain, diethylene glycol and Gardnerella spp.

Conclusions

The results not only provide useful diagnostic biomarkers, but also may ultimately provide much needed insight into the determinants of BV.     

Efficacy of oral metronidazole with vaginal clindamycin or vaginal probiotic for bacterial vaginosis: randomised placebo-controlled double-blind trial

Background

To determine if oral metronidazole (MTZ-400mg bid) with 2% vaginal clindamycin-cream (Clind) or a Lactobacillus acidophilus vaginal-probiotic containing oestriol (Prob) reduces 6-month bacterial vaginosis (BV) recurrence.

Methods

Double-blind placebo-controlled parallel-group single-site study with balanced randomization (1∶1∶1) conducted at Melbourne Sexual Health Centre, Australia. Participants with symptomatic BV [Nugent Score (NS) = 7–10 or ≥3 Amsel''s criteria and NS = 4–10], were randomly allocated to MTZ-Clind, MTZ-Prob or MTZ-Placebo and assessed at 1,2,3 and 6 months. MTZ and Clind were administered for 7 days and Prob and Placebo for 12 days. Primary outcome was BV recurrence (NS of 7–10) on self-collected vaginal-swabs over 6-months. Cumulative BV recurrence rates were compared between groups by Chi-squared statistics. Kaplan-Meier, log rank and Cox regression analyses were used to compare time until and risk of BV recurrence between groups.

Results

450 18–50 year old females were randomized and 408 (91%), equally distributed between groups, provided ≥1 NS post-randomization and were included in analyses; 42 (9%) participants with no post-randomization data were excluded. Six-month retention rates were 78% (n = 351). One-month BV recurrence (NS 7–10) rates were 3.6% (5/140), 6.8% (9/133) and 9.6% (13/135) in the MTZ-Clind, MTZ-Prob and MTZ-Placebo groups respectively, p = 0.13. Hazard ratios (HR) for BV recurrence at one-month, adjusted for adherence to vaginal therapy, were 0.43 (95%CI 0.15–1.22) and 0.75 (95% CI 0.32–1.76) in the MTZ-Clind and MTZ-Prob groups compared to MTZ-Plac respectively. Cumulative 6-month BV recurrence was 28.2%; (95%CI 24.0–32.7%) with no difference between groups, p = 0.82; HRs for 6-month BV recurrence for MTZ-Clind and MTZ-Prob compared to MTZ-Plac, adjusted for adherence to vaginal therapy were 1.09(95% CI = 0.70–1.70) and 1.03(95% CI = 0.65–1.63), respectively. No serious adverse events occurred.

Conclusion

Combining the recommended first line therapies of oral metronidazole and vaginal clindamycin, or oral metronidazole with an extended-course of a commercially available vaginal-L.acidophilus probiotic, does not reduce BV recurrence.

Trial Registration

ANZCTR.org.au ACTRN12607000350426     

Factors Associated with Participation and Attrition in a Longitudinal Study of Bacterial Vaginosis in Australian Women Who Have Sex with Women

Objective

A number of social and sexual risk factors for bacterial vaginosis (BV) have been described. It is important to understand whether these factors are associated with non-participation or attrition of participants from longitudinal studies in order to examine potential for recruitment or attrition bias. We describe factors associated with participation and attrition in a 24-month prospective cohort study, investigating incident BV among Australian women who have sex with women.

Study Design and Setting

Participants negative for prevalent BV were offered enrolment in a longitudinal cohort study. Participants self-collected vaginal samples and completed questionnaires 3-monthly to endpoint (BV-positive/BV-negative by 24 months). Factors associated with participation in the cohort study were examined by logistic regression and factors associated with attrition from the cohort were examined by Cox regression.

Results

The cross-sectional study recruited 457 women. 334 BV-negative women were eligible for the cohort and 298 (89%, 95%CI 85, 92) enrolled. Lower educational levels (aOR 2.72, 95%CI 1.09, 6.83), smoking (aOR 2.44, 95%CI 1.13, 5.27), past BV symptoms (aOR 3.42, 95%CI 1.16, 10.10) and prior genital warts (aOR 2.71, 95%CI 1.14, 6.46) were associated with non-participation; a partner co-enrolling increased participation (aOR 3.73, 95%CI 1.43, 9.70). 248 participants (83%, 95%CI 78, 87) were retained to study endpoint (BV-negative at 24 months or BV-positive at any stage). Attrition was associated being <30 yrs (aHR 2.15, 95%CI 1.13, 4.10) and a male partner at enrolment (aHR 6.12, 95%CI 1.99, 18.82).

Conclusion

We achieved high participation and retention levels in a prospective cohort study and report factors influencing participation and retention of participants over a 24-month study period, which will assist in the design and implementation of future cohort studies in sexual health and disease.     

Pattern of infection and antibiotic activity among Streptococcus agalactiae isolates from adults in Mashhad,Iran

Background:

One of the main causes of sexually transmitted diseases is group B β- hemolytic streptococci (GBS) multiplying in the genital tracts. Penicillin is the most common drug for the treatment of infections caused by these bacteria, but in patients suffering from Penicillin allergy, Erythromycin and Clindamycin are used as alternative therapeutic drugs against GBS. Recently, resistance to these drugs has been reported more often. In this study, efforts have been made to determine the prevalence and antibiotic resistance of GBS.

Methods:

Modified Christie Atkins Munch-Petersen (CAMP) test was conducted on over 2400 samples of urine and discharge taken from vagina, urethra and prostate. The drug sensitivity was performed by double disk sensitivity tests to Bacitracin, Trimethoprim, and Sulfamethoxazole and then the resistant samples were investigated by E-test to determine the minimal inhibitory concentrations (MICs) value.

Results:

Twenty-three vaginal and 10 urethral discharge, 27urine and 6 prostatic secretion samples were GBS positive. The most symbiotic microorganisms with GBS were strains of Enterococci (90%), Staphylococcus saprophyticus (25%) and Candida albicans (6%). The disk diffusion method showed 18 cases with Penicillin resistance (MIC: 1.5 mg/ml).

Conclusion:

Taken together, GBS carriers’ rate in this study was found 20.65% (8.24% men and 12.4% women). Furthermore, findings showed high-level resistance to Erythromycin and Clindamycin.Key Words: Antibiotic resistance, Genitourinary system, Streptococcus agalactiae, minimal inhibitory concentration (MIC)     

Performance of swabs, lavage, and diluents to quantify biomarkers of female genital tract soluble mucosal mediators

Background

Measurement of immune mediators and antimicrobial activity in female genital tract secretions may provide biomarkers predictive of risk for HIV-1 acquisition and surrogate markers of microbicide safety. However, optimal methods for sample collection do not exist. This study compared collection methods.

Methods

Secretions were collected from 48 women (24 with bacterial vaginosis [BV]) using vaginal and endocervical Dacron and flocked swabs. Cervicovaginal lavage (CVL) was collected with 10 mL of Normosol-R (n = 20), saline (n = 14), or water (n = 14). The concentration of gluconate in Normosol-R CVL was determined to estimate the dilution factor. Cytokine and antimicrobial mediators were measured by Luminex or ELISA and corrected for protein content. Endogenous anti-HIV-1 and anti-E. coli activity were measured by TZM-bl assay or E. coli growth.

Results

Higher concentrations of protein were recovered by CVL, despite a 10-fold dilution of secretions, as compared to swab eluents. After protein correction, endocervical swabs recovered the highest mediator levels regardless of BV status. Endocervical and vaginal flocked swabs recovered significantly higher levels of anti-HIV-1 and anti-E. coli activity than Dacron swabs (P<0.001). BV had a significant effect on CVL mediator recovery. Normosol-R tended to recover higher levels of most mediators among women with BV, whereas saline or water tended to recover higher levels among women without BV. Saline recovered the highest levels of anti-HIV-1 activity regardless of BV status.

Conclusions

Endocervical swabs and CVL collected with saline provide the best recovery of most mediators and would be the optimal sampling method(s) for clinical trials.