Deborah Money

Background: The vaginal microbiome is a dynamic environment colonized by a wide array of microorganisms. Although bacterial vaginosis (BV) is characterized by a disruption in the normal bacterial microbiome of the vagina, the factors contributing to recurrent BV remain unknown. In addition, very little is known about the role of viruses in the vaginal microbiome and associated dysbioses. Objectives: 1) characterize the vaginal bacteriome of women with recurrent BV using cpn60 sequencing, compare bacterial profiles to healthy-asymptomatic cohort, and correlate profiles to descriptive characteristics; and 2) characterize the vaginal virome of healthy-asymptomatic, HIV-positive women and women with recurrent BV, and correlate profiles to descriptive characteristics. Methods: Twenty-six women were recruited into the recurrent BV bacteriome study. Vaginal swabs were obtained for cpn60 sequencing and Gram stain Nugent scoring. Additionally, samples from 54 women were analyzed in the virome study: 21 healthy-asymptomatic, 25 HIV-positive and eight recurrent BV. The vaginal swabs were processed to enrich for viruses and then subjected to metagenomics shotgun sequencing. Demographic, behavioural and clinical information was collected for all participants, in both bacteriome and virome studies.Results: Bacteriome analyses detected 122 cpn60 operational taxonomic units (OTUs). Bacterial profiles clustered into six community state types (CSTs). Trends suggested a relationship between BV-associated CSTs and number of sexual partners (past year), oral sex, use of (hormonal) contraception, abnormal discharge (past 48 hours), lifetime history of trichomoniasis, and number of BV episodes (past two months and year). Virome analyses detected a total of 477 species. Viral profiles clustered into seven groups. Viral patterns were identified within bacteriome CSTs, Nugent scores, viral loads, between Lactobacillus-dominant, Lactobacillus iners-dominant, and heterogeneous profiles, and were associated with a number of descriptive characteristics.Conclusions: The vaginal microbiome is highly diverse and potentially associated with many clinical factors. Our ability to use the microbiome data to subdivide women into clusters, and detect trends between clusters and characteristics will expand our knowledge on the vaginal microbiome as a whole.

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Disruptions or imbalances of the vaginal microbiome can lead to negative reproductive health consequences for women, including an increased risk of sexually transmitted infections, pelvic inflammatory disease, and preterm birth. HIV-positive women may be particularly vulnerable to microbiome disruptions due to the immune dysfunction intrinsic to this disease. The objective of this study was to explore the vaginal microbiome in HIV-positive reproductive-aged women utilizing cpn60 metagenomic profiling and to correlate vaginal bacterial profiles with demographic/clinical variables. 54 HIV-positive women were recruited from the Oak Tree Clinic in Vancouver, BC. Demographic/clinical information was collected and vaginal gram-stains were assessed by Nugent’s scoring. Total DNA was extracted from vaginal swabs and PCR amplified using cpn60-specific universal primers. Cpn60-sequence libraries were generated with 454-GS-FLX Titanium pyrosequencing. 64 unique bacterial phylotypes were classified based on sequence similarity to known bacterial organisms and 10 common vaginal community clusters were generated. Fisher’s exact test and Wilcoxon signed-rank tests were utilized to conduct statistical analyses. The mean age of enrolled women was 36.6 years (range=22.3–48.8). The mean CD4 count for these women was 484 cells/mm³ (range=90-930 cells/mm³) while the mean viral load was 13,144 copies/mL (range=40-355,245 copies/mL). 63% of women had suppressed viral loads while 85% of women were on antiretroviral therapy. According to scored vaginal Gram stains, 30% (16/54) of women had bacterial vaginosis and 11% (6/54) had intermediate scores. 12/54 women were categorized as having abnormal vaginal discharge. Abnormal vaginal discharge significantly correlated with Dialister micraerophilus, Gardnerella vaginalis B, Gardnerella vaginalis D, Porphyromonas uenonis, Prevotella amnii, Prevotella buccalis, and Prevotella timonensis (p0.04 for all). Normal vaginal discharge significantly correlated with Lactobacillus crispatus (p0.0006). Viral loads 40 copies/mL significantly correlated with Lactobacillus crispatus and Lactobacillus gasseri while viral loads >40 copies/mL correlated with Atopobium vaginae, Gardnerella vaginalis D, Prevotella amnii, and 4 other potentially novel bacterial species (P0.03 for all). CD4 counts 350 cells/mm³ significantly correlated with Gardnerella vaginalis B and Lactobacillus iners (p0.05 for all). Cpn60-based sequence data demonstrated substantial variation in the vaginal microbiome among HIV-positive women, with species-specific differences dependent on vaginal discharge status, immune status and uncontrolled HIV replication.

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