Assessment of Lactobacillus species colonizing the vagina of apparently healthy Nigerian women, using PCR-DGGE and 16S rRNA gene sequencing

The identity of lactobacilli colonizing Africa-American women, have previously been based on culture-dependent methods. This led to some misleading speculations that black women lack lactobacilli in their vagina and are therefore highly susceptible to Bacterial vaginosis and STDs including HIV. In this study we used culture-independent procedures.About 241 vaginal swabs were obtained from ‘apparently’ healthy premenopausal women, between 18 and 48 years. Samples were Gram stained for the Nugent score evaluation. DNA was extracted from the bacteria on the vaginal swabs and amplified using polymerase chain reaction (PCR), with Lactobacillus primers. Samples with PCR products were separated with denaturing gradient gel electrophoresis (DGGE) and cut bands were re-amplified with Lactobacillus primers without the GC clamp. The re-amplified product was purified and sequenced with ABI PRISM Big-Dye Terminator. The sequence was identified with BLAST algorithm having the highest Gene Bank Accession number.Out of the 241 vaginal swab samples, 207 (85.8%) had PCR products, indicating the presence of lactobacilli, while 34 (14.2%) showed absence of lactobacilli and the Nugent scores were synonymous to either intermediate bacterial vaginosis or bacterial vaginosis (BV). Out of the 207 samples that had PCR product for Lactobacillus, 149 (72%) had sequence results as revealed by the BLAST algorithm. Most of the women (64%) were colonized by Lactobacillus iners as the predominant strain. Lactobacillus gasseri had 7.3%, followed by L. plantarum and L suntoryeus (6.0%) each. Others were colonized by Lactobacillus crispatus (3.0%), Lactobacillus rhamnosus (2.7%), Lactobacillus vaginalis (2.7%), Lactobacillus rennanqilfy (2.7%), followed by Lactobacillus fermentum (1.3%), Lactobacillus helveticus (1.3%), Lactobacillus johnsonii (1.3%) and Lactobacillus salivarus (1.3%). To our knowledge, this is the first report of Lactobacillus iners being the predominant species colonizing the vagina of the Nigerian women examined. This finding is in line with those from a recent study conducted among Canadian and Swedish women.     

Bacterial vaginosis among a group of married Jordanian women: occurrence and laboratory diagnosis

A total of 310 vaginal swabs collected from a group of married Jordanian women complaining of vaginal discharge were examined for bacterial vaginosis. The scoring system of Nugent for the interpretation of Gram staining was employed. This system revealed the presence of the condition in 29.7% of patients. Results obtained using the scoring system correlated significantly with the detection of clue cells and the scarcity of white blood cells in the vaginal discharge. An inverse relationship was found between bacterial vaginosis and Lactobacillus morphotypes determined by Gram staining. No definite relationship was detected between bacterial vaginosis and the recovery of Gardnerella vaginalis by culture as this organism was isolated from swabs which according to the Nugent criterion were negative for bacterial vaginosis. Bacterial vaginosis among the women investigated was more prevalent than vaginitis caused by Trichomonas vaginalis or yeasts.     

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.     

Impact of Bacterial Vaginosis,as Assessed by Nugent Criteria and Hormonal Status on Glycosidases and Lectin Binding in Cervicovaginal Lavage Samples

The objective of this study was to evaluate the impact of hormonal status and bacterial vaginosis (BV) on the glycosidases present and glycosylation changes as assessed by lectin binding to cervicovaginal lavage constituents. Frozen cervicovaginal lavage samples from a completed study examining the impact of reproductive hormones on the physicochemical properties of vaginal fluid were utilized for the present study. In the parent study, 165 women were characterized as having BV, intermediate or normal microflora using the Nugent criteria. The presence of glycosidases in the samples was determined using quantitative 4-methyl-umbelliferone based assays, and glycosylation was assessed using enzyme linked lectin assays (ELLA). Women with BV had elevated sialidase, α-galactosidase, β-galactosidase and α-glucosidase activities compared to intermediate or normal women (P<0.001, 0.003, 0.006 and 0.042 respectively). The amount of sialic acid (Sambucus nigra, P = 0.003) and high mannose (griffithsin, P<0.001) were reduced, as evaluated by lectin binding, in women with BV. When the data were stratified according to hormonal status, α-glucosidase and griffithsin binding were decreased among postmenopausal women (P<0.02) when compared to premenopausal groups. These data suggest that both hormonal status and BV impact the glycosidases and lectin binding sites present in vaginal fluid. The sialidases present at increased levels in women with BV likely reduce the number of sialic acid binding sites. Other enzymes likely reduce griffithsin binding. The alterations in the glycosidase content, high mannose and sialic acid binding sites in the cervicovaginal fluid associated with bacterial vaginosis may impact susceptibility to viruses, such as HIV, that utilize glycans as a portal of entry.     

Vaginal microbiome variances in sample groups categorized by clinical criteria of bacterial vaginosis

Background

One of the most common and recurrent vaginal infections is bacterial vaginosis (BV). The diagnosis is based on changes to the “normal” vaginal microbiome; however, the normal microbiome appears to differ according to reproductive status and ethnicity, and even among individuals within these groups. The Amsel criteria and Nugent score test are widely used for diagnosing BV; however, these tests are based on different criteria, and so may indicate distinct changes in the vaginal microbial community. Nevertheless, few studies have compared the results of these test against metagenomics analysis.

Methods

Vaginal flora samples from 77 participants were classified according to the Amsel criteria and Nugent score test. The microbiota composition was analyzed using 16S ribosome RNA gene amplicon sequencing. Bioinformatics analysis and multivariate statistical analysis were used to evaluate the microbial diversity and function.

Results

Only 3 % of the participants diagnosed BV negative using the Amsel criteria (A−) were BV-positive according to the Nugent score test (N+), while over half of the BV-positive patients using the Amsel criteria (A+) were BV-negative according to the Nugent score test (N−). Thirteen genera showed significant differences in distribution among BV status defined by BV tests (e.g., A − N−, A + N− and A + N+). Variations in the four most abundant taxa, Lactobacillus, Gardnerella, Prevotella, and Escherichia, were responsible for most of this dissimilarity. Furthermore, vaginal microbial diversity differed significantly among the three groups classified by the Nugent score test (N−, N+, and intermediate flora), but not between the Amsel criteria groups. Numerous predictive microbial functions, such as bacterial chemotaxis and bacterial invasion of epithelial cells, differed significantly among multiple BV test, but not between the A− and A+ groups.

Conclusions

Metagenomics analysis can greatly expand our current understanding of vaginal microbial diversity in health and disease. Metagenomics profiling may also provide more reliable diagnostic criteria for BV testing.

     

Impact of Bacterial Vaginosis on Perineal Tears during Delivery: A Prospective Cohort Study

Objective

Long term effects of perineal tears pose a major worldwide health issue for women during delivery. Since bacterial vaginosis is related to major obstacles in obstetrics the aim of this study was to determine the relationship between bacterial vaginosis and the occurrence of perineal tears during vaginal delivery.

Methods

Between June 2013 and December 2013 pregnant women delivering after 37 weeks were recruited at one University hospital / tertiary care referral center in the course of this single-center, prospective cohort study. Bacterial vaginosis was assessed according to Nugent score method. Logistic-regression model was used to estimate odds ratios, adjusted for other risk factors to test the relationship between bacterial vaginosis and the occurrence of 1st to 4th degree perineal tears in women undergoing vaginal delivery.

Results

A total of 728 woman were included, 662 analyzed with a complete Nugent Score of the vaginal swab. The prevalence of 1st to 4th degree perineal tears was 35.8% (95% Confidence Interval (95%CI) = [32.2; 39.6]). The presence of BV was not significantly associated to the incidence of perineal tears neither in the univariate analysis (crude Odds Ratio = 1.43; 95%CI = [0.79; 2.60]; p = 0.235) nor in the multivariate analysis (adjusted Odds Ratio = 1.65; 95%CI = [0.81; 3.36]; p = 0.167). Instrumental delivery was the most important risk factor for perineal lacerations.

Conclusions

There is no evidence that vaginosis is a risk factor for vaginal tears.

Trial Registration

ClinicalTrials.gov N° NCT01822782     

More Than Meets the Eye: Associations of Vaginal Bacteria with Gram Stain Morphotypes Using Molecular Phylogenetic Analysis

Bacterial vaginosis (BV) is a highly prevalent condition associated with adverse health outcomes. Gram stain analysis of vaginal fluid is the standard for confirming the diagnosis of BV, wherein abundances of key bacterial morphotypes are assessed. These Lactobacillus, Gardnerella, Bacteroides, and Mobiluncus morphotypes were originally linked to particular bacterial species through cultivation studies, but no studies have systematically investigated associations between uncultivated bacteria detected by molecular methods and Gram stain findings. In this study, 16S-rRNA PCR/pyrosequencing was used to examine associations between vaginal bacteria and bacterial morphotypes in 220 women with and without BV. Species-specific quantitative PCR (qPCR) and fluorescence in Situ hybridization (FISH) methods were used to document concentrations of two bacteria with curved rod morphologies: Mobiluncus and the fastidious BV-associated bacterium-1 (BVAB1). Rank abundance of vaginal bacteria in samples with evidence of curved gram-negative rods showed that BVAB1 was dominant (26.1%), while Mobiluncus was rare (0.2% of sequence reads). BVAB1 sequence reads were associated with Mobiluncus morphotypes (p<0.001). Among women with curved rods, mean concentration of BVAB1 DNA was 2 log units greater than Mobiluncus (p<0.001) using species-specific quantitative PCR. FISH analyses revealed that mean number of BVAB1 cells was 2 log units greater than Mobiluncus cells in women with highest Nugent score (p<0.001). Prevotella and Porphyromonas spp. were significantly associated with the “Bacteroides morphotype,” whereas Bacteroides species were rare. Gram-negative rods designated Mobiluncus morphotypes on Gram stain are more likely BVAB1. These findings provide a clearer picture of the bacteria associated with morphotypes on vaginal Gram stain.     

Longitudinal Analysis of Vaginal Microbiome Dynamics in Women with Recurrent Bacterial Vaginosis: Recognition of the Conversion Process

Bacterial vaginosis (BV) affects ∼30% of women of reproductive age, has a high rate of recurrence, and is associated with miscarriage, preterm birth, and increased risk of acquiring other sexually transmitted infections, including HIV-1. Little is known of the daily changes in the vaginal bacterial composition as it progresses from treatment to recurrence, or whether any of these might be useful in its prediction or an understanding of its causes. We used phylogenetic branch-inclusive quantitative PCR (PB-qPCR) and Lactobacillus blocked/unblocked qPCR (Lb-qPCR) to characterize longitudinal changes in the vaginal microbiota in sequential vaginal self-swabs from five women with recurrent BV, from diagnosis through remission to recurrence. Both patients with acute BV samples dominated by G. vaginalis recurred during the study with similar profiles, whereas the three patients with acute BV samples dominated by other anaerobes did not recur or recurred to an intermediate Nugent score. L. iners dominated remission phases, with intermittent days of abnormal microbial profiles typically associated with menses. The exception was a newly discovered phenomenon, a sustained period of abnormal profiles, termed conversion, which preceded symptomatic acute BV. Species known to have antagonistic activity towards Lactobacillus were detected in pre-conversion samples, possibly contributing to the decline in Lactobacillus. Lb-qPCR scores define two categories of response in the initial post-treatment visit samples; scores <5 may correspond with poor response to treatment or rapid recurrence, whereas scores >8 may predict delayed or no recurrence. Amsel criteria or Nugent scores did not have this potential predictive capability. Larger studies are warranted to evaluate the prognostic potential of detecting conversion and poor Lb-qPCR scores at the post-treatment visit of recurrent BV patients.     

Augmentation of antimicrobial metronidazole therapy of bacterial vaginosis with oral probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14: randomized, double-blind, placebo controlled trial

This study enrolled 125 premenopausal women diagnosed with bacterial vaginosis (BV) by presence of vaginal irritation, discharge and 'fishy' odor, and Nugent criteria and detection of sialidase enzyme. The subjects were treated with oral metronidazole (500 mg) twice daily from days 1 to 7, and randomized to receive oral Lactobacillus rhamnosus GR-1 (1 x 10(9)) and Lactobacillus reuteri RC-14 (1 x 10(9)) or placebo twice daily from days 1 to 30. Primary outcome was cure of BV as determined by normal Nugent score, negative sialidase test and no symptoms or signs of BV at day 30. A total of 106 subjects returned for 30-day follow-up, of which 88% were cured in the antibiotic/probiotic group compared to 40% in the antibiotic/placebo group (p<0.001). Of the remaining subjects, 30% subjects in the placebo group and none in the probiotic group had BV, while 30% in the placebo and 12% in the probiotic group fell into the intermediate category based upon Nugent score, sialidase result and clinical findings. High counts of Lactobacillus sp. (>10(5) CFU/ml) were recovered from the vagina of 96% probiotic-treated subjects compared to 53% controls at day 30. In summary, this study showed efficacious use of lactobacilli and antibiotic in the eradication of BV in black African women.     

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.     

治疗前后加德纳菌载量 对细菌性阴道病患者复发风险的预测价值

目的评价治疗前后加德纳菌载量预测细菌性阴道病(bacterial vaginosis,BV)患者复发风险的临床价值。方法将2017年1月至2018年1月于我院就诊的312例BV患者纳入研究,根据治疗后BV是否复发分为复发组(114例)及未复发组(198例),分别比较两组患者治疗前后阴道分泌物的pH值、Nugent评分、乳杆菌数量和加德纳菌载量,并应用受试者工作曲线(ROC)对上述指标预测BV复发的诊断价值及诊断效能进行评价。结果治疗前及治疗后复发组患者的pH值(t=2.187、3.822,均P0.05)、Nugent评分(t=1.976、5.611,均P0.05)、加德纳菌载量(t=5.089、16.204,均P0.05)均明显高于未复发组,乳杆菌数量(t=8.245、8.899;均P0.05)明显低于未复发组;多因素Logistic回归分析显示治疗后阴道分泌物Nugent评分及加德纳菌载量均是BV复发的独立危险因素,而乳杆菌数量为保护因素;对上述指标预测BV复发的相关性进行分析,ROC曲线显示,加德纳菌载量预测BV复发的AUC为0.892,明显高于pH值、Nugent评分、乳杆菌数量(AUC=0.580、0.648、0.772),诊断最佳截点为1.761×10~5 copy/mL,此时的敏感性和特异性分别为93.5%和75.4%。结论 BV患者治疗后的加德纳菌载量在预测其复发方面具有较高的敏感性及特异性,可用于预测BV复发。     

Lactic acid bacteria colonization and clinical outcome after probiotic supplementation in conventionally treated bacterial vaginosis and vulvovaginal candidiasis

This randomized double-blind placebo controlled study assessed the vaginal colonization of lactic acid bacteria and clinical outcome. Vaginal capsules containing L gasseri LN40, Lactobacillus fermentum LN99, L. casei subsp. rhamnosus LN113 and P. acidilactici LN23, or placebos were administered for five days to 95 women after conventional treatment of bacterial vaginosis and/or vulvovaginal candidiasis. Vulvovaginal examinations and vaginal samplings were performed before and after administration, after the first and second menstruation, and after six months. Presence of LN strains was assessed using RAPD analysis. LN strains were present 2–3 days after administration in 89% of the women receiving LN strains (placebo: 0%, p < 0.0001). After one menstruation 53% were colonized by at least one LN strain. Nine percent were still colonized six months after administration. Ninety-three percent of the women receiving LN strains were cured 2–3 days after administration (placebo: 83%), and 78% after one menstruation (placebo: 71%) (ns). The intervention group experienced less malodorous discharge 2–3 days after administration (p = 0.03) and after the second menstruation (p = 0.04), compared with placebo. In summary, five days of vaginal administration of LN strains after conventional treatment of bacterial vaginosis and/or vulvovaginal candidiasis lead to vaginal colonization, somewhat fewer recurrences and less malodorous discharge.     

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.     

The Vaginal Microbiota: What Have We Learned after a Decade of Molecular Characterization?

We conducted a systematic review of the Medline database (U.S. National Library of Medicine, National Institutes of Health, Bethesda, MD, U.S.A) to determine if consistent molecular vaginal microbiota (VMB) composition patterns can be discerned after a decade of molecular testing, and to evaluate demographic, behavioral and clinical determinants of VMB compositions. Studies were eligible when published between 1 January 2008 and 15 November 2013, and if at least one molecular technique (sequencing, PCR, DNA fingerprinting, or DNA hybridization) was used to characterize the VMB. Sixty three eligible studies were identified. These studies have now conclusively shown that lactobacilli-dominated VMB are associated with a healthy vaginal micro-environment and that bacterial vaginosis (BV) is best described as a polybacterial dysbiosis. The extent of dysbiosis correlates well with Nugent score and vaginal pH but not with the other Amsel criteria. Lactobacillus crispatus is more beneficial than L. iners. Longitudinal studies have shown that a L. crispatus-dominated VMB is more likely to shift to a L. iners-dominated or mixed lactobacilli VMB than to full dysbiosis. Data on VMB determinants are scarce and inconsistent, but dysbiosis is consistently associated with HIV, human papillomavirus (HPV), and Trichomonas vaginalis infection. In contrast, vaginal colonization with Candida spp. is more common in women with a lactobacilli-dominated VMB than in women with dysbiosis. Cervicovaginal mucosal immune responses to molecular VMB compositions have not yet been properly characterized. Molecular techniques have now become more affordable, and we make a case for incorporating them into larger epidemiological studies to address knowledge gaps in etiology and pathogenesis of dysbiosis, associations of different dysbiotic states with clinical outcomes, and to evaluate interventions aimed at restoring and maintaining a lactobacilli-dominated VMB.     

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.     

Resolution and Characterization of Distinct cpn60-Based Subgroups of Gardnerella vaginalis in the Vaginal Microbiota

Bacterial vaginosis (BV), characterized by a shift of the vaginal microbiota from a Lactobacillus-dominated community to a dense biofilm containing a complex mixture of organisms, is an important risk factor in poor reproductive health outcomes. The Nugent score, based on Gram stain, is used to diagnose BV and Gardnerella vaginalis abundance in the sample is one factor determining Nugent score. A high Nugent score is indicative of BV but does not always correspond to the presence of clinical symptoms. G. vaginalis is recognized as a heterogeneous group of organisms, which can also be part of the normal, healthy vaginal microbiome. In addition, asymptomatic BV and non-Gardnerella types of BV are being recognized. In an attempt to resolve the heterogeneous group of G. vaginalis, a phylogenetic tree of cpn60 universal target sequences from G. vaginalis isolates was constructed that indicates the existence of four subgroups of G. vaginalis. This subdivision, supported by whole genome similarity calculation of representative strains using JSpecies, demonstrates that these subgroups may represent different species. The cpn60 subgroupings did not correspond with the Piot biotyping scheme, but did show consistency with ARDRA genotyping and sialidase gene presence. Isolates from all four subgroups produced biofilm in vitro. We also investigated the distribution of G. vaginalis subgroups in vaginal samples from Kenyan women with Nugent scores consistent with BV, Intermediate and Normal microbiota (n = 44). All subgroups of G. vaginalis were detected in these women, with a significant difference (z = −3.372, n = 39, p = 0.001) in frequency of G. vaginalis subgroup B between BV and Normal groups. Establishment of a quantifiable relationship between G. vaginalis subgroup distribution and clinical status could have significant diagnostic implications.     

Endpiece: The noble profession

ObjectivesTo assess whether bacterial vaginosis or chlamydial infection before 10 weeks'' gestation is associated with miscarriage before 16 weeks.DesignProspective cohort study.Setting32 general practices and five family planning clinics in south London.Participants1216 pregnant women, mean age 31, presenting before 10 weeks'' gestation.Results121 of 1214 women (10.0%, 95% confidence interval 8.3% to 11.7%) miscarried before 16 weeks. 174 of 1201 women (14.5%, 12.5% to 16.5%) had bacterial vaginosis. Compared with women who were negative for bacterial vaginosis those who were positive had a relative risk of miscarriage before 16 weeks'' gestation of 1.2 (0.7 to 1.9). Bacterial vaginosis was, however, associated with miscarriage in the second trimester at 13-15 weeks (3.5, 1.2 to 10.3). Only 29 women (2.4%, 1.5% to 3.3%) had chlamydial infection, of whom one miscarried (0.32, 0.04 to 2.30).ConclusionBacterial vaginosis is not strongly predictive of early miscarriage but may be a predictor after 13 weeks'' gestation. The prevalence of Chlamydia was too low to assess the risk, but it is unlikely to be a major risk factor in pregnant women.

What is already known on this topic

Miscarriages are common and associated with considerable morbidity and costsBacterial vaginosis is associated with miscarriage after 16 weeks'' gestation and preterm birth but the role of chlamydial infection is uncertain

What this study adds

Bacterial vaginosis is not a strong predictor of miscarriage before 16 weeks'' gestation but may be associated with miscarriage at 13-15 weeks'' gestationThe prevalence of chlamydial infection was too low for it to be a major risk factor for miscarriage in this population of healthy pregnant womenNon-invasive screening for bacterial vaginosis and chlamydial infection by using self administered vaginal swabs is feasible in pregnant women in the community     

Consistent Condom Use Increases the Colonization of Lactobacillus crispatus in the Vagina

Background

Non-hormonal contraception methods have been widely used, but their effects on colonization by vaginal lactobacilli remain unclear.

Objective

To determine the association between non-hormonal contraception methods and vaginal lactobacilli on women’s reproductive health.

Methods

The cross-sectional study included 164 healthy women between 18–45 years of age. The subjects were divided into different groups on the basis of the different non-hormonal contraception methods used by them. At the postmenstrual visit (day 21 or 22 of the menstrual cycle), vaginal swabs were collected for determination of Nugent score, quantitative culture and real-time polymerase chain reaction (PCR) of vaginal lactobacilli. The prevalence, colony counts and 16S rRNA gene expression of the Lactobacillus strains were compared between the different groups by Chi-square and ANOVA statistical analysis methods.

Results

A Nugent score of 0–3 was more common in the condom group (93.1%) than in the group that used an interuterine device(IUD) (75.4%), (p = 0.005). The prevalence of H₂O₂-producing Lactobacillus was significantly higher in the condom group (82.3%) than in the IUD group (68.2%), (p = 0.016). There was a significant difference in colony count (mean ± standard error (SE), log₁₀colony forming unit (CFU)/ml) of H₂O₂-producing Lactobacillus between condom users (7.81±0.14) and IUD users (6.54±0.14), (p = 0.000). The 16S rRNA gene expression (mean ± SE, log₁₀copies/ml) of Lactobacillus crispatus was significantly higher in the condom group (8.09±0.16) than in the IUD group (6.03±0.18), (p = 0.000).

Conclusion

Consistent condom use increases the colonization of Lactobacillus crispatus in the vagina and may protect against both bacterial vaginosis (BV) and human immunodeficiency virus (HIV).     

Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes

Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.