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

<div><p>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 <i>Lactobacillus</i> 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 <i>G. vaginalis</i> 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. <i>L. iners</i> 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 <i>Lactobacillus</i> were detected in pre-conversion samples, possibly contributing to the decline in <i>Lactobacillus</i>. 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.</p></div

PB-qPCR generated microbial profiles of acute and post-treatment vaginal swabs of patients with histories of recurrent BV.

<p>Data is also provided as Table; these and the conversions to relative titers as described [71]. Patient 1 (P1) was sampled at 3 separate acute BV (aBV) episodes; 3a and 3b are samples of the 3^rd episode taken 5 days apart. P1 and P2 recurred during the study; P3 and P4 did not. P5 responded poorly and was ultimately diagnosed with BV at an intermediate Nugent score, 4 (P5-iBV). uc  =  uncultured.</p

Shifts in dominant species common to conversions in P1 and P2.

<p>Panels represent qPCR data using Mycoplasmatales primers (top) or <i>Enterococcus</i> primers (bottom). Amplicons were sequenced to identify species, or in some cases were identified by their distinguishing melt curves that matched those of the sequenced products.</p

Profiles and treatment regimens for recurrent BV patients.

<p>Note: Day 0 refers to day of enrollment as a longitudinal patient. Treatments were all for 7 days. A =  Amsel criteria, number positive; N =  Nugent score. n/a =  not available. *  =  Clue cells present but not above 20%; **  =  not clinically confirmed.</p

ΔCq values define conversion events before acute BV.

<p>Conversions (red bars) ΔCq values persistently <5 occurred 10–40 days before symptomatic BV in recurring patients P1 and P2, but not in non-recurring patients P3 and P4. Values remain <5 for most of the interval in P5. BV =  acute BV by Nugent and Amsel; M =  menses; C =  coitus.</p

ΔCq values in samples from RBV patients at visit 1 (acute BV, black bars) versus post-treatment visit 2 (grey bars).

<p>Patients who recurred rapidly recurring or responded poorly responding (P2, P5) are shown in dark grey; patients who did not recur or did so slowly are in light grey (P1, P2 after her 2^nd recurrence, P3, P4). Red to pink bars represent averages of a separate collection of patients (numbers in parentheses) who were only sampled at the initial acute BV and/or the post-treatment visits, with indicated standard deviations.</p

Microbial profiles of near-daily vaginal swabs from patients with histories of recurrent BV, characterized with 11 PB-qPCR targets (legend).

<p>Data are converted to % total titers and depicted on a log scale. Top panels of P1 and P2 show the expected rise to dominance of <i>Lactobacillus</i> after treatment, and conversions before acute BV. In both patients, sharp increases are seen in <i>G. vaginalis</i>, (2), <i>Prevotella</i> (4), <i>L. amnionii</i> (5), <i>BVAB2</i> (7), and <i>Mycoplasma</i> sp. (11). Patients P3 and P4, who did not recur, show sustained dominance of <i>Lactobacillus</i> after treatment, and their non-<i>Lactobacillus</i> populations remain generally at <1%, with frequent transient spikes. Red bar  =  menses;↓ =  coitus. Data is also presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082599#pone.0082599.s005" target="_blank">Table S4</a>.</p

Dense Genotyping of Immune-Related Loci Identifies Variants Associated with Clearance of HPV among HIV-Positive Women in the HIV Epidemiology Research Study (HERS)

<div><p>Persistent high-risk human papillomavirus (HR-HPV) is a necessary and causal factor of cervical cancer. Most women naturally clear HPV infections; however, the biological mechanisms related to HPV pathogenesis have not been clearly elucidated. Host genetic factors that specifically regulate immune response could play an important role. All HIV-positive women in the HIV Epidemiology Research Study (HERS) with a HR-HPV infection and at least one follow-up biannual visit were included in the study. Cervicovaginal lavage samples were tested for HPV using type-specific HPV hybridization assays. Type-specific HPV clearance was defined as two consecutive HPV-negative tests after a positive test. DNA from participants was genotyped for 196,524 variants within 186 known immune related loci using the custom ImmunoChip microarray. To assess the influence of each single-nucleotide polymorphism (SNP) with HR-HPV clearance, the Cox proportional hazards model with the Wei-Lin-Weissfeld approach was used, adjusting for CD4+ count, low risk HPV (LR-HPV) co-infection, and relevant confounders. Three analytical models were performed: race-specific (African Americans (n = 258), European Americans (n = 87), Hispanics (n = 55), race-adjusted combined analysis, and meta-analysis of pooled independent race-specific analyses. Women were followed for a median time of 1,617 days. Overall, three SNPs (rs1112085, rs11102637, and rs12030900) in the <i>MAGI-3</i> gene and one SNP (rs8031627) in the <i>SMAD3</i> gene were associated with HR-HPV clearance (p<10⁻⁶). A variant (rs1633038) in <i>HLA-G</i> were also significantly associated in African American. Results from this study support associations of immune-related genes, having potential biological mechanism, with differential cervical HR-HPV infection outcomes.</p> </div

Manhattan plot showing the association P-values of single nucleotide polymorphisms (SNPs) in the ImmunoChip with the time to clearance of HR-HPV.

<p>The X-axes display the chromosome on which the SNP is located, the Y-axes display −log₁₀ P-value. The dashed black line represents a significance level needed for multiple testing using the K effective method. Panel A.) Race-adjusted analysis B.) African Americans only C.) European Americans only, and D.) Hispanics only.</p

Demographic and clinical data 70 HIV/GBV-C co-infected women from the HERS cohort included in the current study.

<p>In parentheses are percentages unless otherwise noted. Numbers in brackets denote women with available data. P-values <0.10 are provided, although only p-values <0.05 are considered statistically significant. NS = not significant (p≥0.10); SD = standard deviation; IQR = interquartile range.</p><p>Demographic and clinical data 70 HIV/GBV-C co-infected women from the HERS cohort included in the current study.</p