Emily C. Wagner

Characterization of the vaginal microbiota of healthy Canadian women through the menstrual cycle

BackgroundThe vaginal microbial community plays a vital role in maintaining women’s health. Understanding the precise bacterial composition is challenging because of the diverse and difficult-to-culture nature of many bacterial constituents, necessitating culture-independent methodology. During a natural menstrual cycle, physiological changes could have an impact on bacterial growth, colonization, and community structure. The objective of this study was to assess the stability of the vaginal microbiome of healthy Canadian women throughout a menstrual cycle by using cpn 60-based microbiota analysis. Vaginal swabs from 27 naturally cycling reproductive-age women were collected weekly through a single menstrual cycle. Polymerase chain reaction (PCR) was performed to amplify the universal target region of the cpn 60 gene and generate amplicons representative of the microbial community. Amplicons were pyrosequenced, assembled into operational taxonomic units, and analyzed. Samples were also assayed for total 16S rRNA gene content and Gardnerella vaginalis by quantitative PCR and screened for the presence of Mollicutes by using family and genus-specific PCR.ResultsOverall, the vaginal microbiome of most women remained relatively stable throughout the menstrual cycle, with little variation in diversity and only modest fluctuations in species richness. Microbiomes between women were more different than were those collected consecutively from individual women. Clustering of microbial profiles revealed the expected groupings dominated by Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii. Interestingly, two additional clusters were dominated by either Bifidobacterium breve or a heterogeneous mixture of nonlactobacilli. Direct G. vaginalis quantification correlated strongly with its pyrosequencing-read abundance, and Mollicutes, including Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum, were detected in most samples.ConclusionsOur cpn 60-based investigation of the vaginal microbiome demonstrated that in healthy women most vaginal microbiomes remained stable through their menstrual cycle. Of interest in these findings was the presence of Bifidobacteriales beyond just Gardnerella species. Bifidobacteriales are frequently underrepresented in 16S rRNA gene-based studies, and their detection by cpn 60-based investigation suggests that their significance in the vaginal community may be underappreciated.

A Study of the Vaginal Microbiome in Healthy Canadian Women Utilizing cpn60-Based Molecular Profiling Reveals Distinct Gardnerella Subgroup Community State Types.

The vaginal microbiota is important in women’s reproductive and overall health. However, the relationships between the structure, function and dynamics of this complex microbial community and health outcomes remain elusive. The objective of this study was to determine the phylogenetic range and abundance of prokaryotes in the vaginal microbiota of healthy, non-pregnant, ethnically diverse, reproductive-aged Canadian women. Socio-demographic, behavioural and clinical data were collected and vaginal swabs were analyzed from 310 women. Detailed profiles of their vaginal microbiomes were generated by pyrosequencing of the chaperonin-60 universal target. Six community state types (CST) were delineated by hierarchical clustering, including three Lactobacillus-dominated CST (L. crispatus, L. iners, L. jensenii), two Gardnerella-dominated (subgroups A and C) and an “intermediate” CST which included a small number of women with microbiomes dominated by seven other species or with no dominant species but minority populations of Streptococcus, Staphylococcus, Peptoniphilus, E. coli and various Proteobacteria in co-dominant communities. The striking correspondence between Nugent score and deep sequencing CST continues to reinforce the basic premise provided by the simpler Gram stain method, while additional analyses reveal detailed cpn60-based phylogeny and estimated abundance in microbial communities from vaginal samples. Ethnicity was the only demographic or clinical characteristic predicting CST, with differences in Asian and White women (p = 0.05). In conclusion, this study confirms previous work describing four cpn60-based subgroups of Gardnerella, revealing previously undescribed CST. The data describe the range of bacterial communities seen in Canadian women presenting with no specific vaginal health concerns, and provides an important baseline for future investigations of clinically important cohorts.

High Diversity and Variability in the Vaginal Microbiome in Women following Preterm Premature Rupture of Membranes (PPROM): A Prospective Cohort Study

Objective To characterize the vaginal microbiota of women following preterm premature rupture of membranes (PPROM), and determine if microbiome composition predicts latency duration and perinatal outcomes. Design A prospective cohort study Setting Canada Population Women with PPROM between 24+0 and 33+6 weeks gestational age (GA). Methods Microbiome profiles, based on pyrosequencing of the cpn60 universal target, were generated from vaginal samples at time of presentation with PPROM, weekly thereafter, and at delivery. Main Outcome Measures Vaginal microbiome composition, latency duration, gestational age at delivery, perinatal outcomes. Results Microbiome profiles were generated from 70 samples from 36 women. Mean GA at PPROM was 28.8 wk (mean latency 2.7 wk). Microbiome profiles were highly diverse but sequences representing Megasphaera type 1 and Prevotella spp. were detected in all vaginal samples. Only 13/70 samples were dominated by Lactobacillus spp. Microbiome profiles at the time of membrane rupture did not cluster by gestational age at PPROM, latency duration, presence of chorioamnionitis or by infant outcomes. Mycoplasma and/or Ureaplasma were detected by PCR in 81% (29/36) of women, and these women had significantly lower GA at delivery and correspondingly lower birth weight infants than Mycoplasma and/or Ureaplasma negative women. Conclusion Women with PPROM had mixed, abnormal vaginal microbiota but the microbiome profile at PPROM did not correlate with latency duration. Prevotella spp. and Megasphaera type I were ubiquitous. The presence of Mollicutes in the vaginal microbiome was associated with lower GA at delivery. The microbiome was remarkably unstable during the latency period.

Obstetrical and Neonatal Outcomes Among Women Infected With Hepatitis C and Their Infants

OBJECTIVES (1) To describe obstetrical and neonatal outcomes among a cohort of hepatitis C virus (HCV) infected women, comparing HCV RNA positive to HCV RNA negative women; (2) to characterize virologic and hepatic parameters associated with HCV infection during pregnancy; and (3) to describe the rate of HCV vertical transmission. METHODS We prospectively enrolled 145 HCV-positive pregnant women across British Columbia between 2000 and 2003. Participating women were monitored during pregnancy and their infants were followed to assess them for HCV infection. Maternal HCV RNA was assessed close to delivery. RESULTS Seventy percent of women reported injection drug use as their primary risk factor for HCV acquisition. Observed rates of intrauterine fetal death, preterm delivery, small for gestational age, and low birth weight infants were 3.4%, 17.9%, 11.3%, and 12.5%, respectively, without a significant association with maternal HCV RNA status. The rate of cholestasis was 5.6% in the HCV RNA-positive group (6/108) and 2.8% in the HCV RNA-negative group (1/37) (P = 0.496). Serum alanine aminotransferase levels decreased significantly through pregnancy, and were significantly higher in HCV RNA-positive women than in HCV RNA-negative women after controlling for cholestasis, co-infections, and alcohol consumption. Among the HCV RNA-positive women, the median FIB-4 score was 0.67 (IQR 0.56 to 0.76) in the first trimester, 0.74 (IQR 0.52 to 1.18) in the second trimester, and 0.89 (IQR 0.52 to 1.09) in the third trimester (P = 0.02). The median HCV viral load at delivery was 424 561 IU/mL. The vertical transmission rate was 4.7% in HCV RNA-positive women, with no cases in HCV RNA-negative women. CONCLUSION Because of the high rates of poor obstetrical outcomes found in this prospective cohort, population-level screening for HCV in pregnancy should be considered.

Understanding Men’s Perceptions of Human Papillomavirus and Cervical Cancer Screening in Kampala, Uganda

Purpose This preliminary study explores Ugandan men’s knowledge and attitudes about human papillomavirus (HPV), cervical cancer, and screening. Methods A local physician led an education session about cervical cancer for 62 men in Kisenyi, Kampala in Uganda. Trained nurse midwives administered surveys to assess knowledge and attitudes before and after the education session. Results From the pre-education survey, only 24.6% of men had heard of HPV previously, and 59% of men had heard of cervical cancer. Posteducation, 54.5% of men believed only women could be infected with HPV and 32.7% of men believed antibiotics could cure HPV. Despite their limited knowledge, 98.2% of men stated they would support their partners to receive screening for cervical cancer, and 100% of men surveyed stated they would encourage their daughter to get the HPV vaccine if available. Conclusions Knowledge of HPV and cervical cancer among Ugandan men is low. Even after targeted education, confusion remained about disease transmission and treatment. Ongoing education programs geared toward men and interventions to encourage spousal communication about reproductive health and shared decision making may improve awareness of cervical cancer prevention strategies.

Do protease inhibitor-containing combination antiretroviral therapy regimens increase risk of spontaneous preterm birth in pregnant HIV-Positive women?

administration with a meal or soft foods, and the impact of high doses of secnidazole on cardiac safety. SYM-1219 was administered in either applesauce, yogurt, or pudding, followed by 240 mL of water. Serial blood samples were collected to determine secnidazole plasma concentrations and PK parameters for each treatment group are reported. Safety assessments, ECGs, and vital signs were performed during each study. An in vitro metabolism program, including CYP metabolism, inhibition, and induction, substrate and inhibition potential for transporters, and the potential for secnidazole to inhibit ethanol metabolism was performed. RESULTS: A single 2-g oral dose of SYM-1219 achieves an average maximum plasma secnidazole concentration (Cmax) of 35.7 to 46.3 mg/mL approximately 2 to 3 hours after dosing (Tmax). Exposure, as assessed by area under the plasma-concentration time curve (AUC), increases linearly with dose. Secnidazole has a prolonged terminal elimination half-life (t1/2) of w17 hours. SYM-1219 can be administered in applesauce, pudding, or yogurt and the timing and content of a meal does not have an impact on drug bioavailability. Secnidazole is not a substrate or inhibitor of transporters nor does it induce or inhibit hepatic CYP450 enzymes, therefore the potential for clinically important interactions with CYP450 substrates, inhibitors, or inducers is minimal. In a clinical study, secnidazole had negligible impact on the PK of ethinyl estradiol or norethindrone, suggesting that a single dose of SYM-1219 will not impact the efficacy of oral contraceptives. Secnidazole does not inhibit aldehyde dehydrogenase in vitro and therefore does not alter ethanol metabolism. There were no clinically significant abnormalities in laboratory, vital signs, or ECGs following administration of SYM-1219 to study subjects. CONCLUSIONS: The favorable safety and efficacy profile of SYM-1219, coupled with its PK characteristics, are the foundation for this single-dose oral treatment regimen for BV.

Evidence of Subclinical mtDNA Alterations in HIV-Infected Pregnant Women Receiving Combination Antiretroviral Therapy Compared to HIV-Negative Pregnant Women

Introduction Combination antiretroviral therapy (cART) can effectively prevent vertical transmission of HIV but there is potential risk of adverse maternal, foetal or infant effects. Specifically, the effect of cART use during pregnancy on mitochondrial DNA (mtDNA) content in HIV-positive (HIV+) women is unclear. We sought to characterize subclinical alterations in peripheral blood mtDNA levels in cART-treated HIV+ women during pregnancy and the postpartum period. Methods This prospective longitudinal observational cohort study enrolled both HIV+ and HIV-negative (HIV-) pregnant women. Clinical data and blood samples were collected at three time points in pregnancy (13-<23 weeks, 23-<30 weeks, 30–40 weeks), and at delivery and six weeks post-partum in HIV+ women. Peripheral blood mtDNA to nuclear DNA (nDNA) ratio was measured by qPCR. Results Over a four year period, 63 HIV+ and 42 HIV- women were enrolled. HIV+ women showed significantly lower mtDNA/nDNA ratios compared to HIV- women during pregnancy (p = 0.003), after controlling for platelet count and repeated measurements using a multivariable mixed-effects model. Ethnicity, gestational age (GA) and substance use were also significantly associated with mtDNA/nDNA ratio (p≤0.02). Among HIV+ women, higher CD4 nadir was associated with higher mtDNA/nDNA ratios (p<0.0001), and these ratio were significantly lower during pregnancy compared to the postpartum period (p<0.0001). Conclusions In the context of this study, it was not possible to distinguish between mtDNA effects related to HIV infection versus cART therapy. Nevertheless, while mtDNA levels were relatively stable over time in both groups during pregnancy, they were significantly lower in HIV+ women compared to HIV- women. Although no immediate clinical impact was observed on maternal or infant health, lower maternal mtDNA levels may exert long-term effects on women and children and remain a concern. Improved knowledge of such subclinical alterations is another step toward optimizing the safety and efficacy of cART regimens during pregnancy.