Feasibility and acceptability of frequent vaginal self-sampling at home by Rwandan women at high risk of urogenital tract infections.

OBJECTIVES: To establish temporal links between vaginal microbiota (VMB) data and incident clinical events, frequent longitudinal vaginal sampling is required. Self-collection of swabs at the participant's home may be useful to avoid overburdening research clinics and participants. One-off vaginal self-sampling for STI or cervical cancer screening programmes has been shown to be feasible and acceptable to women in multiple studies, including in sub-Saharan Africa, but the feasibility and acceptability of frequent longitudinal vaginal sampling in the context of VMB sequencing studies is unknown. METHODS: Twelve participants of a randomised clinical trial in Kigali, Rwanda, self-collected vaginal swabs three times a week for a month. We studied feasibility by comparing DNA concentrations, proportions of samples with >1000 16S rRNA amplicon sequencing reads and VMB composition outcomes of self-collected swabs with clinician-collected swabs. We evaluated the acceptability of self-collection using structured face-to-face interviews and a focus group discussion. RESULTS: The participants collected vaginal swabs at 131 different time points. One woman stopped self-sampling after one try due to a social harm. All self-sampled swabs generated >1000 rRNA amplicon sequencing reads, and the DNA concentration of self-sampled swabs and clinician-sampled swabs did not differ significantly (Kruskal-Wallis p=0.484). Self-sampled and clinician-sampled swabs generated similar VMB composition data. Participants reported feeling very comfortable during self-sampling (11/12; 91.7%) and that self-sampling had become easier over time (12/12; 100%). They mentioned reduced travel time and travel costs as advantages of self-sampling at home. CONCLUSIONS: Frequent longitudinal vaginal sampling at home is feasible and acceptable to participants, even in the context of a low-resource setting, as long as adequate counselling is provided. TRIAL REGISTRATION NUMBER: NCT02459665.

Pathobionts in the Vaginal Microbiota: Individual Participant Data Meta-Analysis of Three Sequencing Studies.

Sequencing studies have shown that optimal vaginal microbiota (VMB) are lactobacilli-dominated and that anaerobes associated with bacterial vaginosis (BV-anaerobes) are commonly present. However, they overlooked a less prevalent but more pathogenic group of vaginal bacteria: the pathobionts that cause maternal and neonatal infections and pelvic inflammatory disease. We conducted an individual participant data meta-analysis of three VMB sequencing studies that included diverse groups of women in Rwanda, South Africa, and the Netherlands (2,044 samples from 1,163 women in total). We identified 40 pathobiont taxa but only six were non-minority taxa (at least 1% relative abundance in at least one sample) in all studies: Streptococcus (54% of pathobionts reads), Staphylococcus, Enterococcus, Escherichia/Shigella, Haemophilus, and Campylobacter. When all pathobionts were combined into one bacterial group, the VMB of 17% of women contained a relative abundance of at least 1%. We found a significant negative correlation between relative abundances (ρ = -0.9234), but not estimated concentrations (r = 0.0031), of lactobacilli and BV-anaerobes; and a significant positive correlation between estimated concentrations of pathobionts and BV-anaerobes (r = 0.1938) but not between pathobionts and lactobacilli (r = 0.0436; although lactobacilli declined non-significantly with increasing pathobionts proportions). VMB sequencing data were also classified into mutually exclusive VMB types. The overall mean bacterial load of the ≥20% pathobionts VMB type (5.85 log10 cells/µl) was similar to those of the three lactobacilli-dominated VMB types (means 5.13-5.83 log10 cells/µl) but lower than those of the four anaerobic dysbiosis VMB types (means 6.11-6.87 log10 cells/µl). These results suggest that pathobionts co-occur with both lactobacilli and BV-anaerobes and do not expand as much as BV-anaerobes do in a dysbiotic situation. Pathobionts detection/levels were increased in samples with a Nugent score of 4-6 in both studies that conducted Nugent-scoring. Having pathobionts was positively associated with young age, non-Dutch origin, hormonal contraceptive use, smoking, antibiotic use in the 14 days prior to sampling, HIV status, and the presence of sexually transmitted pathogens, in at least one but not all studies; inconsistently associated with sexual risk-taking and unusual vaginal discharge reporting; and not associated with vaginal yeasts detection by microscopy. We recommend that future VMB studies quantify common vaginal pathobiont genera.

Vaginal probiotic adherence and acceptability in Rwandan women with high sexual risk participating in a pilot randomised controlled trial: a mixed-methods approach.

OBJECTIVES: To evaluate adherence and acceptability of intermittent vaginal probiotic or antibiotic use to prevent bacterial vaginosis (BV) recurrence. DESIGN: Repeated adherence and acceptability assessments using mixed methods within a pilot randomised controlled trial. SETTING: Research clinic in Kigali, Rwanda. PARTICIPANTS: Rwandan women with high sexual risk. INTERVENTIONS: Women diagnosed with BV and/or trichomoniasis were randomised to four groups (n=17 each) after completing metronidazole treatment: behavioural counselling only, or behavioural counselling plus 2-month intermittent use of oral metronidazole, Ecologic Femi+ (EF+) vaginal capsule or Gynophilus LP (GynLP) vaginal tablet. OUTCOME MEASURES: Adherence and acceptability were assessed by structured face-to-face interviews, semi-structured focus group discussions and in-depth interviews, daily diaries and counting of used/unused study products in randomised women (n=68). Vaginal infection knowledge was assessed by structured face-to-face interviews in randomised women and women attending recruitment sessions (n=131). RESULTS: Most women (93%) were sex workers, 99.2% were unfamiliar with BV and none had ever used probiotics. All probiotic users (n=32) reported that insertion became easier over time. Triangulated adherence data showed that 17/17 EF+ users and 13/16 GynLP users used ≥80% of required doses (Fisher's exact p=0.103). Younger age (p=0.076), asking many questions at enrolment (p=0.116), having menses (p=0.104) and reporting urogenital symptoms (p=0.103) were non-significantly associated with lower perfect adherence. Women believed that the probiotics reduced BV recurrence, but reported that partners were sometimes unsupportive of study participation. Self-reported vaginal washing practices decreased during follow-up, but sexual risk behaviours did not. Most women (12/15) with an uncircumcised steady partner discussed penile hygiene with him, but many women found this difficult, especially with male clients. CONCLUSIONS: High-risk women require education about vaginal infections. Vaginal probiotic acceptability and adherence were high in this cohort. Our results can be used to inform future product development and to fine-tune counselling messages in prevention programmes. TRIAL REGISTRATION NUMBER: NCT02459665.

Intermittent Lactobacilli-containing Vaginal Probiotic or Metronidazole Use to Prevent Bacterial Vaginosis Recurrence: A Pilot Study Incorporating Microscopy and Sequencing.

Bacterial vaginosis (BV) is associated with HIV acquisition and adverse pregnancy outcomes. Recurrence after metronidazole treatment is high. HIV-negative, non-pregnant Rwandan BV patients were randomized to four groups (n = 17/group) after seven-day oral metronidazole treatment: behavioral counseling only (control), or counseling plus intermittent use of oral metronidazole, Ecologic Femi+ vaginal capsule (containing multiple Lactobacillus and one Bifidobacterium species), or Gynophilus LP vaginal tablet (L. rhamnosus 35) for two months. Vaginal microbiota assessments at all visits included Gram stain Nugent scoring and 16S rRNA gene qPCR and HiSeq sequencing. All interventions were safe. BV (Nugent 7-10) incidence was 10.18 per person-year at risk in the control group, and lower in the metronidazole (1.41/person-year; p = 0.004), Ecologic Femi+ (3.58/person-year; p = 0.043), and Gynophilus LP groups (5.36/person-year; p = 0.220). In mixed effects models adjusted for hormonal contraception/pregnancy, sexual risk-taking, and age, metronidazole and Ecologic Femi+ users, each compared to controls, had higher Lactobacillus and lower BV-anaerobes estimated concentrations and/or relative abundances, and were less likely to have a dysbiotic vaginal microbiota type by sequencing. Inter-individual variability was high and effects disappeared soon after intervention cessation. Lactobacilli-based vaginal probiotics warrant further evaluation because, in contrast to antibiotics, they are not expected to negatively affect gut microbiota or cause antimicrobial resistance.

Impact of oral metronidazole treatment on the vaginal microbiota and correlates of treatment failure.

BACKGROUND: Metronidazole is the first-line treatment for bacterial vaginosis, but cure rates are suboptimal and recurrence rates high. OBJECTIVES: To evaluate the impact of a standard course of oral metronidazole treatment (500 mg twice per day for 7 days) on the vaginal microbiota of Rwandan bacterial vaginosis patients using microscopy and 16S rRNA gene sequencing, and to evaluate correlates of treatment failure. STUDY DESIGN: HIV-negative, nonpregnant women aged 18-45 years with bacterial vaginosis and/or Trichomonas vaginalis (N=68) were interviewed and sampled before and after metronidazole treatment. They were also screened, and treated if applicable, for other urogenital infections. The vaginal microbiota was assessed by Gram stain Nugent scoring, Illumina 16S rRNA HiSeq sequencing (relative abundances), and BactQuant 16S gene quantitative polymerase chain reaction (estimated concentrations). Only women with a pretreatment Nugent score of 7-10 and a valid posttreatment Nugent score (N=55) were included in metronidazole treatment failure analyses, with treatment failure defined as a posttreatment Nugent score of 4-10. RESULTS: The bacterial vaginosis cure rate by Nugent scoring was 54.5%. The mean total vaginal bacterial concentration declined from 6.59 to 5.85 log10/µL (P<.001), which was mostly due to a reduction in mean bacterial vaginosis-associated anaerobes concentration (all bacterial vaginosis-associated anaerobe taxa combined) from 6.23 to 4.55 log10/µL (P<.001). However, only 16.4% of women had a bacterial vaginosis anaerobes concentration reduction of more than 50%, and only 3 women had complete eradication. The mean concentration of lactobacilli (all species combined) increased from 4.98 to 5.56 log10/µL (P=.017), with L. iners being the most common species pre- and posttreatment. The mean concentration of pathobionts (defined as Proteobacteria, streptococci, staphylococci, enterococci, and a few others) did not change significantly: from 1.92 log10/µL pretreatment to 2.01 log10/µL posttreatment (P=.939). Pretreatment pathobionts concentration, and having a pretreatment vaginal microbiota type containing more than 50% Gardnerella vaginalis (compared with less than 50%), were associated with increased likelihood of treatment failure, but the latter did not reach statistical significance (P=.044 and P=.084, respectively). CONCLUSIONS: Metronidazole alone may not cure women with high G. vaginalis relative abundance, potentially due to biofilm presence, and women with high pathobionts concentration. These women may benefit from additional biofilm-disrupting and/or pathobiont-targeting treatments.

Human papillomavirus infection and cervical dysplasia in HIV-positive women: potential role of the vaginal microbiota.

OBJECTIVES: To assess the associations between microbiological markers of vaginal dysbiosis and incident/cleared/type-swap/persistent high-risk human papillomavirus (hrHPV) infection; and incident/cured/cleared/persistent high-grade cervical intraepithelial neoplasia (CIN2+) while controlling for persistent hrHPV infection. DESIGN: Two nested case-control studies (N = 304 and 236) within a prospective cohort of HIV-positive women in Johannesburg, South Africa. METHODS: Participants were examined for hrHPV type (INNO-LiPA), cervical dysplasia (histology), and vaginal microbiota (VMB) composition (V3-V4 Illumina HiSeq 2x300 bp) at baseline and endline, a median of 16 months later. RESULTS: Women with incident hrHPV compared to those who remained hrHPV-negative were less likely to have an optimal Lactobacillus crispatus or jensenii-dominated VMB type at end-line [relative risk ratio (RRR) 0.125, P = 0.019], but not at baseline. Having different hrHPV types at both visits was associated with multiple anaerobic dysbiosis markers at baseline (e.g. increased bacterial vaginosis-associated anaerobes relative abundance: RRR 3.246, P = 0.026). Compared to women without CIN2+, but with hrHPV at both visits, women with incident CIN2+ had increased Simpson diversity (RRR 7.352, P = 0.028) and nonsignificant trends in other anaerobic dysbiosis markers at end-line but not baseline. These associations persisted after controlling for age, hormonal contraception, and CD4 cell count. Current hormonal contraceptive use (predominantly progestin-only injectables) was associated with increased CIN2+ risk over-and-above persistent hrHPV infection and independent of VMB composition. CONCLUSIONS: hrHPV infection (and/or increased sexual risk-taking) may cause anaerobic vaginal dysbiosis, but a bidirectional relationship is also possible. In this population, dysbiosis did not increase CIN2+ risk, but CIN2+ increased dysbiosis risk. The CIN2+ risk associated with progestin-only injectable use requires further evaluation.

Targeted point-of-care testing compared with syndromic management of urogenital infections in women (WISH): a cross-sectional screening and diagnostic accuracy study.

BACKGROUND: Sexually transmitted and urogenital infections are typically managed by WHO-recommended syndromic algorithms in resource-poor countries, and presumptively in Europe. However, algorithms for vaginal discharge and lower abdominal pain perform poorly in women. The women's improvement of sexual and reproductive health (WISH) study in Kigali, Rwanda, sought to improve case-finding and infection management in women by introducing point-of-care tests. The main aim was to compare the performance of the WISH algorithms and the WHO vaginal discharge and lower abdominal pain algorithms with gold standard testing. METHODS: This cross-sectional screening and diagnostic accuracy study recruited women aged 18 years or older with or without urogenital symptoms at risk of acquiring sexually transmitted infections in Kigali, Rwanda. Recruitment activities were implemented by study staff with the help of community mobilisers at health centres, pharmacies, markets, women's organisations, and at "umuganda" community meetings. At the study visit, participants had a face-to-face interview that included questions about current urogenital symptoms. Participants were first asked without prompting (spontaneous reporting), followed by questions about 14 specific symptoms (structural reporting). Next, the WISH algorithms were implemented. All participants had point-of-care tests for bacterial vaginosis (vaginal pH of 5·0 or above) and Trichomonas vaginalis (immunoassay) regardless of symptom reporting. Women with a positive risk score had point-of-care tests for Chlamydia trachomatis and Neisseria gonorrhoea (nucleic acid amplification tests). Vulvovaginal candidiasis was treated presumptively. Nucleic acid amplification tests for C trachomatis, N gonorrhoeae, T vaginalis, bacterial vaginosis, and vulvovaginal candidiasis were the gold standard, and all patients provided swabs for these. FINDINGS: Participants were recruited between July 5, 2016, and March 14, 2017. 705 participants were enrolled in the study and completed a study visit, and 51 attended 53 additional visits. Prevalence by gold standard testing was 8·5% for C trachomatis, 7·1% for N gonorrhoeae, 16·1% for T vaginalis, 18·1% for bacterial vaginosis, and 8·6% for vulvovaginal candidiasis. The WISH algorithms identified similar numbers of C trachomatis, N gonorrhoeae, and T vaginalis infections, but much higher numbers of bacterial vaginosis and vulvovaginal candidiasis infections. Compared with gold standard testing, the WISH algorithms had a good sensitivity and high specificity for C trachomatis (sensitivity 71·7%, specificity 100%), N gonorrhoeae (sensitivity 76·0%, specificity 100%), and T vaginalis (sensitivity 68·5%, specificity 97·4%), high sensitivity but low specificity for bacterial vaginosis (sensitivity 95·2%, specificity 41·2%), and moderate sensitivity and specificity for vulvovaginal candidiasis (sensitivity 64·4%, specificity 69·4%). The performance of vaginal pH testing for bacterial vaginosis improved by increasing the cutoff to 5·5, followed by confirmatory testing (sensitivity 73·6%, specificity 100%). The WHO algorithms had moderate sensitivity and poor specificity for all infections compared with gold standard testing: C trachomatis sensitivity 58·3%, specificity 44·7%; N gonorrhoeae sensitivity 66·0%, specificity 45·2%; T vaginalis sensitivity 60·4%, specificity 45·6%; bacterial vaginosis sensitivity 61·6%, specificity 46·0%; and vulvovaginal candidiasis sensitivity 74·6%, specificity 50·6%. Two participants attended additional visits because they had a mild allergic reaction to metronidazole. Staff and participants considered point-of-care testing feasible and acceptable. INTERPRETATION: Point-of-care testing for urogenital infections might improve case-finding and infection management and is feasible in resource-poor settings. Point-of-care tests should be further developed, including those targeting multiple conditions. Additional studies in other populations, including populations with low prevalence of sexually transmitted and urogenital infections, are warranted. FUNDING: European and Developing Countries Clinical Trials Partnership.

The impact of hormonal contraception and pregnancy on sexually transmitted infections and on cervicovaginal microbiota in african sex workers.

BACKGROUND: The observed association between Depo-Provera injectable use and increased HIV acquisition may be caused by hormone-induced increased susceptibility to other sexually transmitted infections (STIs) or changes in the cervicovaginal microbiota (VMB), accompanied by genital immune activation and/or mucosal remodeling. METHODS: Rwandan female sex workers (n = 800) were interviewed about contraceptive use and sexual behavior and were tested for STIs, bacterial vaginosis by Nugent score and pregnancy, at baseline. A subset of 397 HIV-negative, nonpregnant women were interviewed and tested again at regular intervals for 2 years. The VMB of a subset of 174 women was characterized by phylogenetic microarray. Outcomes of STI and VMB were compared between women with hormonal exposures (reporting oral contraceptive or injectable use, or testing positive for pregnancy) and controls (not reporting hormonal contraception and not pregnant). RESULTS: Oral contraceptive use was associated with increased human papillomavirus prevalence (adjusted odds ratio [aOR], 3.10; 1.21-7.94) and Chlamydia trachomatis incidence (aOR, 6.13; 1.58-23.80), injectable use with increased herpes simplex virus-2 prevalence (aOR, 2.13; 1.26-3.59) and pregnancy with lower HIV prevalence (aOR, 0.45; 0.22-0.92) but higher candidiasis incidence (aOR, 2.14; 1.12-4.09). Hormonal status was not associated with Nugent score category or phylogenetic VMB clustering, but oral contraceptive users had lower semiquantitative vaginal abundance of Prevotella, Sneathia/Leptotrichia amnionii, and Mycoplasma species. CONCLUSIONS: Oral contraceptive and injectable use were associated with several STIs but not with VMB composition. The increased herpes simplex virus-2 prevalence among injectable users might explain the potentially higher HIV risk in these women, but more research is needed to confirm these results and elucidate biological mechanisms.

Hormonal contraception decreases bacterial vaginosis but oral contraception may increase candidiasis: implications for HIV transmission.

OBJECTIVE: A 2012 WHO consultation concluded that combined oral contraception (COC) does not increase HIV acquisition in women, but the evidence for depot medroxyprogesterone acetate (DMPA) is conflicting. We evaluated the effect of COC and DMPA use on the vaginal microbiome because current evidence suggests that any deviation from a 'healthy' vaginal microbiome increases women's susceptibility to HIV. METHODS: We conducted a systematic review and reanalysed the Hormonal Contraception and HIV Acquisition (HC-HIV) study. Vaginal microbiome outcomes included bacterial vaginosis by Nugent scoring, vaginal candidiasis by culture or KOH wet mount and microbiome compositions as characterized by molecular techniques. RESULTS: Our review of 36 eligible studies found that COC and DMPA use reduce bacterial vaginosis by 10-20 and 18-30%, respectively. The HC-HIV data showed that COC and DMPA use also reduce intermediate microbiota (Nugent score of 4-6) by 11% each. In contrast, COC use (but not DMPA use) may increase vaginal candidiasis. Molecular vaginal microbiome studies (n=4) confirm that high oestrogen levels favour a vaginal microbiome composition dominated by 'healthy' Lactobacillus species; the effects of progesterone are less clear and not well studied. CONCLUSION: DMPA use does not increase HIV risk by increasing bacterial vaginosis or vaginal candidiasis. COC use may predispose for vaginal candidiasis, but is not believed to be associated with increased HIV acquisition. However, the potential role of Candida species, and vaginal microbiome imbalances other than bacterial vaginosis or Candida species, in HIV transmission cannot yet be ruled out. Further in-depth molecular studies are needed.