Molecular Identification of Cervical Microbes in HIV-Negative and HIV-Positive Women in an African Setting Using a Customized Bacterial Vaginosis Microbial DNA Quantitative PCR (qPCR) Array.

Bacterial vaginosis (BV) is a common polymicrobial vaginal disorder that is associated with sexually transmitted infections (STIs), including HIV. Several studies have utilized broad-range 16S rRNA gene PCR assays with sequence analysis to characterize cervicovaginal bacterial communities of women with healthy and diseased conditions. With the high burden of BV and STIs among African women, there is a need for targeted PCR assays that can rapidly determine the true epidemiological profile of key cervical microbes, including BV-associated bacteria, and a need to explore the utility of such assays for microbiological diagnosis of BV. Here, we used a taxon-directed 16S rRNA gene quantitative PCR (qPCR) assay to examine the prevalences and determinants of specific cervical microbes among African women with and without HIV infection. Cervical samples were collected using a cytobrush from 162 women (aged ≥30 years) attending a community-based clinic in Eastern Cape, South Africa. The samples were screened for specific microbes (i.e., STIs, emerging sexually transmitted pathogens [pathobionts], and BV-associated bacteria) using a customized bacterial vaginosis microbial DNA qPCR array. Statistical analyses were performed using GraphPad Prism v6.01. Chi-square/Fisher's exact tests were used to evaluate the determinants associated with specific cervical microbes. Only 145 women had any detectable microbes and were included in the analysis. Lactobacillus iners (62.8%) and specific BV-associated bacteria, namely, Gardnerella vaginalis (58.6%), Atopobium vaginae (40.7%), and the pathobiont Ureaplasma parvum (37.9%), were the most prevalent microbes. Hierarchical clustering analysis revealed that 42.8% of the women (62/145) had a diverse array of heterogeneously distributed bacteria typically linked to BV. Women with detectable Lactobacillus species, specifically Lactobacillus crispatus and Lactobacillus jensenii, and to a lesser extent L. iners, had very low prevalence of BV-associated bacteria. Although the cumulative burden of STIs/pathobionts was 62.8%, Chlamydia trachomatis (3.4%), Neisseria gonorrhoeae (4.8%), and Trichomonas vaginalis (4.8%) were detected at low rates. HIV infection was associated with the presence of STIs/pathobionts (P = 0.022) and L. iners (P = 0.003). Prevalent STIs/pathobionts were associated with having multiple partners in the past 12 months (n ≥ 2, P = 0.015), high number of lifetime sexual partners (n ≥ 3, P = 0.007), vaginal sex in the past month (P = 0.010), and decreasing age of women (P = 0.005). C. trachomatis was associated with increasing age among HIV-positive women (P = 0.016). The pathobiont Ureaplasma urealyticum was inversely associated with age of women in the whole cohort (P = 0.018). The overall prevalence of STIs/pathobionts was high and was associated with HIV infection and sexual behavior. Our study helps us to understand the epidemiological trend of STIs and pathobionts and highlights the need to understand the impact of sexual networks on STI and pathobiont transmission and prevention among women in an African setting. IMPORTANCE Bacterial vaginosis (BV), whose etiology remains a matter of controversy, is a common vaginal disorder among reproductive-age women and can increase the risk for sexually transmitted infections (STIs). African women bear a disproportionately high burden of STIs and BV. Using a targeted quantitative PCR (qPCR) assay, a customized bacterial vaginosis microbial DNA qPCR array, we examined the prevalences and determinants of key cervical microbes, including BV-associated bacteria and emerging sexually transmitted pathogens (pathobionts) among women of African descent aged between 30 and 75 years. High-risk behaviors were associated with a higher prevalence of STIs/pathobionts, suggesting the need to better understand the influence of sexual networks on STI and pathobiont transmission and prevention among women. Our molecular assay is important in the surveillance of BV-associated bacteria, pathobionts, and STIs as well as diagnostic microbiology of BV. Furthermore, our research contributes to a better understanding of the epidemiology of STIs and pathobionts in Africa.

Detection of sexually transmitted pathogens and co-infection with human papillomavirus in women residing in rural Eastern Cape, South Africa.

BACKGROUND: South African women of reproductive age have a high burden of sexually transmitted infections (STIs), including human papillomavirus (HPV) infection. However, there is limited information on the prevalence of sexually transmitted pathogens in women from rural Eastern Cape Province, South Africa. The study aims at determining the prevalence of sexually transmitted pathogens and co-infection with high-risk (HR) HPV among women from rural Eastern Cape Province, South Africa. METHODS: A total of 205 cervical specimens were collected from women aged ≥ 30 years from a rural community-based clinic. The samples were tested for a panel of pathogenic STIs [Chlamydia trachomatis (serovars A-K & L1-L3), Haemophilus ducreyi, Herpes Simplex Virus (Types 1 & 2), Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis (TV), and pathobionts [Mycoplasma genitalium (MG), Mycoplasma hominis (MH) and Ureaplasma spp. (UP)] using a multiplex PCR STD direct flow chip assay through a manual Hybrispot platform (Master Diagnostica, Granada, Spain). HR-HPV detection was performed by Hybrid Capture-2 assay. RESULTS: High-risk HPV prevalence was 32.2% (66/205) and HIV-1 prevalence was 38.5% (79/205). The overall prevalence of six pathogenic STIs was 22.9% (47/205), with TV having the highest prevalence (15.6%; 32/205). UP (70.2%, 144/205) and MH (36.6%, 75/205) were the most frequently detected pathobionts. Co-infection with ≥ 2 pathogens pathobionts was observed among 52.7% (108/205) participants. Of the six pathogenic STIs, three participants had more than one STI (1.46%) with the presence of MH and UP. HSV-2 (OR: 4.17, CI [1.184-14.690]) and HIV infection (OR: 2.11, CI [1.145-3.873]) were independent STIs associated with HR-HPV infection. CONCLUSIONS: The high prevalence of pathogenic STIs underscores the need to improve syndromic management policy by implementing effective strategies of prevention, screening tests, and management. HSV-2 and HIV positive remain strongly associated with HR-HPV infection.

Acceptability of self- collection for human papillomavirus detection in the Eastern Cape, South Africa.

Human papillomavirus (HPV) testing on vaginal self-collected and cervical clinician-collected specimens shows comparable performance. Self-sampling on FTA cards is suitable for women residing in rural settings or not attending regular screening and increases participation rate in the cervical cancer screening programme. We aimed to investigate and compare high-risk (HR)-HPV prevalence in clinician-collected and self-collected genital specimens as well as two different HPV tests on the clinician collected samples. A total of 737 women were recruited from two sites, a community health clinic (n = 413) and a referral clinic (n = 324) in the Eastern Cape Province. Cervical clinician-collected (FTA cards and Digene transport medium) and vaginal self-collected specimens were tested for HR-HPV using the hpVIR assay (FTA cards) and Hybrid Capture-2 (Digene transport medium). There was no significant difference in HR-HPV positivity between clinician-collected and self-collected specimens among women from the community-based clinic (26.4% vs 27.9%, p = 0.601) or the referral clinic (83.6% vs 79.9%, p = 0.222). HPV16, HPV35, and HPV33/52/58 group were the most frequently detected genotypes at both study sites. Self-sampling for HPV testing received a high positive response of acceptance (77.2% in the community-based clinic and 83.0% in referral clinic). The overall agreement between hpVIR assay and HC-2 was 87.7% (k = 0.754). The study found good agreement between clinician-collected and self-collected genital specimens. Self-collection can have a positive impact on a cervical screening program in South Africa by increasing coverage of women in rural areas, in particular those unable to visit the clinics and women attending clinics where cytology-based programs are not functioning effectively.

The Penile Microbiota in Uncircumcised and Circumcised Men: Relationships With HIV and Human Papillomavirus Infections and Cervicovaginal Microbiota.

While the human microbiota especially that of the gut, cervix, and vagina continue to receive great attention, very little is currently known about the penile (glans, coronal sulcus, foreskin, and shaft) microbiota. The best evidences to date for the potential role of the penile microbiota in human immunodeficiency virus (HIV) and other sexually transmitted infections (STIs) acquisition have come from studies examining medical male circumcision. We are still at the foothills of identifying specific penile bacteria that could be associated with increased risk of STI/HIV acquisition. In this review, we summarize the available literature on the human penile microbiota and how it is impacted by circumcision. We also discuss the potential role of penile microbiota in STIs and its impact on cervicovaginal microbiota. Taken together, the findings from the penile microbiota studies coupled with observational studies on the effect of male circumcision for reduction of STI/HIV infection risk suggest that specific penile anaerobic bacteria such as Prevotella spp. potentially have a mechanistic role that increases the risk of genital infections and syndromes, including bacterial vaginosis in sexual partners. Although penile Corynebacterium and Staphylococcus have been associated with healthy cervicovaginal microbiota and have been found to increase following male circumcision, further investigations are warranted to ascertain the exact roles of these bacteria in the reproductive health of men and women. This review aims to address existing gaps and challenges and future prospects in the penile microbiota research. The information described here may have translational significance, thereby improving reproductive health and management of STI/HIV.

The penile microbiota of Black South African men: relationship with human papillomavirus and HIV infection.

BACKGROUND: To date, the microbiota of the human penis has been studied mostly in connection with circumcision, HIV risk and female partner bacterial vaginosis (BV). These studies have shown that male circumcision reduces penile anaerobic bacteria, that greater abundance of penile anaerobic bacteria is correlated with increased cytokine levels and greater risk of HIV infection, and that the penile microbiota is an important harbour for BV-associated bacteria. While circumcision has been shown to significantly reduce the risk of acquiring human papillomavirus (HPV) infection, the relationship of the penile microbiota with HPV is still unknown. In this study, we examined the penile microbiota of HPV-infected men as well as the impact of HIV status. RESULTS: The penile skin microbiota of 238 men from Cape Town (South Africa) were profiled using Illumina sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene. Corynebacterium and Prevotella were found to be the most abundant genera. Six distinct community state types (CSTs) were identified. CST-1, dominated by Corynebacterium, corresponded to less infections with high-risk HPV (HR-HPV) relative to CSTs 2-6. Men in CST-5 had greater relative abundances of Prevotella, Clostridiales, and Porphyromonas and a lower relative abundance of Corynebacterium. Moreover, they were significantly more likely to have HPV or HR-HPV infections than men in CST-1. Using a machine learning approach, we identified greater relative abundances of the anaerobic BV-associated bacteria (Prevotella, Peptinophilus, and Dialister) and lower relative abundance of Corynebacterium in HR-HPV-infected men compared to HR-HPV-uninfected men. No association was observed between HIV and CST, although the penile microbiota of HIV-infected men had greater relative abundances of Staphylococcus compared to HIV-uninfected men. CONCLUSIONS: We found significant differences in the penile microbiota composition of men with and without HPV and HIV infections. HIV and HR-HPV infections were strongly associated with greater relative abundances of Staphylococcus and BV-associated bacterial taxa (notably Prevotella, Peptinophilus and Dialister), respectively. It is possible that these taxa could increase susceptibility to HIV and HR-HPV acquisition, in addition to creating conditions in which infections persist. Further longitudinal studies are required to establish causal relationships and to determine the extent of the effect.

Human papillomavirus prevalence and risk factors among HIV-negative and HIV-positive women residing in rural Eastern Cape, South Africa.

OBJECTIVES: To investigate the prevalence of high-risk (HR) human papillomavirus (HPV) and factors associated with HR-HPV infection among women from rural Eastern Cape, South Africa. METHODS: HPV prevalence was determined by Hybrid Capture 2 assay in cervical specimens from 417 women aged ≥30 years (median 46 years) recruited from the community health clinic in the Eastern Cape. RESULTS: HR-HPV prevalence was 28.5% (119/417), and HIV-positive women had significantly higher HR-HPV prevalence than HIV-negative women (40.6%, 63/155 vs 21.4%, 56/262, respectively; p = 0.001). HIV-positive status (odds ratio (OR) 2.52, 95% confidence interval (CI) 1.63-3.90), having ≥3 lifetime sexual partners (OR 2.12, 95% CI 1.16-3.89), having ≥1 sexual partner in the last month (OR 1.89, 95% CI 1.21-2.92), ≥4 times frequency of vaginal sex in the past 1 month (OR 2.40, 95% CI 1.32-4.35), and having a vaginal discharge currently/in the previous week (OR 2.13, 95% CI 1.18-3.85) increased the risk of HR-HPV infection. In the multivariate analysis, HIV positivity remained strongly associated with HR-HPV infection (OR 1.94, 95% CI 1.17-3.22). CONCLUSIONS: Risk factors related to sexual behaviors play a significant role in HR-HPV infection in this population. This report will inform health policymakers on HPV prevalence and contribute to discussions on the use of HPV testing as the primary cervical cancer screening test in South Africa.

Factors associated with the composition and diversity of the cervical microbiota of reproductive-age Black South African women: a retrospective cross-sectional study.

BACKGROUND: Lactobacillus spp. are common bacteria in the cervical and vaginal microbiota (CVM) and are thought to represent a "healthy" cervicovaginal state. Several studies have found an independent association between ethnicity/race and cervical and vaginal microbiota (CVM) composition. Women of sub-Saharan African descent appear to be significantly more likely to have non-Lactobacillus-dominated CVM compared to women of European descent. The factors contributing to these differences remain to be fully elucidated. The CVM of Black South African women and factors influencing their CVM remain understudied. In this study, we characterized the cervical microbiota of reproductive-age South African women and assessed the associations of these microbiota with participants' metadata. METHODS: The cervical microbiota from cervical DNA of 62 reproductive-age women were profiled by Ion Torrent sequencing the V4 hypervariable region of the bacterial 16S ribosomal RNA (rRNA) gene and analyzed with the Quantitative Insights Into Microbial Ecology (QIIME), UPARSE, and metagenomeSeq tools. Associations between cervical microbiota and participants' metadata were assessed using GraphPad Prism, R packages and an in-house script. RESULTS: The cervical microbiota clustered into three distinct community state types (CSTs): Lactobacillus iners-dominated cervical microbiota (CST I (38.7%, 24/62)), unclassified Lactobacillus-dominated cervical microbiota (CST II (4.8%, 3/62)), and diverse cervical microbiota (CST III (56.5%, 35/62)) with an array of heterogeneous bacteria, predominantly the bacterial vaginosis (BV)-associated Gardnerella, Prevotella, Sneathia, and Shuttleworthia. CST III was associated with BV (p = 0.001). Women in CST I were more likely to be on hormonal contraception, especially progestin-based, compared to women in CST III (odds ratio: 5.2 (95% CI [1.6-17.2]); p = 0.005). Women on hormonal contraception had a significantly lower alpha (Shannon indices: 0.9 (0.2-1.9) versus 2.3 (0.6-2.3); p = 0.025) and beta (permutational multivariate analysis of variance (PERMANOVA) pseudo-F statistic =4.31, p = 0.019) diversity compared to non-users. There was no significant difference in the alpha (Shannon indices: 1.0 (0.3-2.2) versus 1.9 (0.3-2.2); p = 0.483) and beta (PERMANOVA pseudo-F statistic = 0.89, p = 0.373) diversity in women with versus without human papillomavirus infection. CONCLUSIONS: The majority of Black women in our study had non-Lactobacillus-dominated cervical microbiota. Additional studies are needed to examine whether such microbiota represent abnormal, intermediate or variant states of health. Lastly, the association of hormonal contraception with L. iners dominance requires further in-depth research to confirm this association, determine its biological mechanism and whether it has a beneficial effect on the cervicovaginal health.

Evolutionary dynamics of ten novel Gamma-PVs: insights from phylogenetic incongruence, recombination and phylodynamic analyses.

BACKGROUND: Human papillomaviruses (HPVs) are genetically diverse, belonging to five distinct genera: Alpha, Beta, Gamma, Mu and Nu. All papillomaviruses have double stranded DNA genomes that are thought to evolve slowly because they co-opt high-fidelity host cellular DNA polymerases for their replication. Despite extensive efforts to catalogue all the HPV species that infect humans, it is likely that many still remain undiscovered. Here we use the sequences of ten novel Gammapapillomaviruses (Gamma-PVs) characterized in previous studies and related HPVs to analyse the evolutionary dynamics of these viruses at the whole genome and individual gene scales. RESULTS: We found statistically significant incongruences between the phylogenetic trees of different genes which imply gene-to-gene variation in the evolutionary processes underlying the diversification of Gamma-PVs. We were, however, only able to detect convincing evidence of a single recombination event which, on its own, cannot explain the observed incongruences between gene phylogenies. The divergence times of the last common ancestor (LCA) of the Alpha, Beta, Mu, Nu and Gamma genera was predicted to have existed between 49.7-58.5 million years ago, before splitting into the five main lineages. The LCA of the Gamma-PVs at this time was predicted to have existed between 45.3 and 67.5 million years ago: approximately at the time when the simian and tarsier lineages of the primates diverged. CONCLUSION: Consequently, we report here phylogenetic tree incongruence without strong evidence of recombination.

The cervical microbiota in reproductive-age South African women with and without human papillomavirus infection.

In this study we examined potential associations of HPV infection with the cervical microbiota. Cervical samples were collected from 87 HIV-seronegative reproductive-age Black South African women. Microbiota were characterized by Illumina sequencing of the V3-V4 hypervariable regions of the bacterial 16S rRNA gene. Thirty seven (42.5%) and 30 (34.5%) of the women had prevalent HPV and high-risk (HR)-HPV, respectively. Only 23 women (26.4%) had cervical microbiota dominated by a single Lactobacillus species (L. crispatus (2/87 (2.3%)), L. jensenii (2/87 (2.3%)), and L. iners (19/87 (21.8%)). The majority of the women (56/87 (64.4%)) had diverse cervical microbiota consisting of mainly bacterial vaginosis-associated bacteria. The remaining women (8/87 (9.2%)) had microbiota dominated by Aerococcus, Streptococcus, Chlamydia or Corynebacterium. Women with HR-HPV had significantly higher relative abundances of Aerococcaceae, Pseudomonadaceae and Bifidobacteriaceae compared to those with low-risk (LR)-HPV or no HPV-infection (LDA score >2.0, p < 0.05, q < 0.2). Gardnerella, Sneathia, and Atopobium were also found at greater relative abundances in HR-HPV-infected women compared to those with low-risk (LR)-HPV or no HPV-infection (LDA score >2.0, p < 0.05), although the difference was not significant after FDR-adjustment (q > 0.2). Further investigations of the bacterial taxa significantly enriched in HR-HPV-infected women are warranted.

Discovery, characterisation and genomic variation of six novel Gammapapillomavirus types from penile swabs in South Africa.

Six novel human papillomaviruses from penile swabs were characterised. Multiple full genome clones for each novel type were generated, and complete genome sizes were: HPV211 (7253bp), HPV212 (7208bp), HPV213 (7096bp), HPV214 (7357), HPV215 (7186bp) and HPV216 (7233bp). Phylogenetically the novel papillomaviruses all clustered with Gammapapillomaviruses: HPV211 is most closely related to HPV168 (72% identity in the L1 nucleotide sequence) of the Gamma-8 species, HPV212 is most closely related to HPV144 (82.9%) of the Gamma-17 species, HPV213 is most closely related to HPV153 (71.8%) of the Gamma-13 species, HPV214 is most closely related to HPV103 (75.3%) of the Gamma-6 species, HPV215 and HPV216 are most closely related to HPV129 (76.8% and 79.2% respectively) of the Gamma-9 species. The novel HPV types demonstrated the classical genomic organisation of Gammapapillomavirusess, with seven open reading frames (ORFs) encoding five early (E1, E2, E4, E6 and E7) and two late (L1 and L2) proteins. Typical of Gammapapillomavirusess the novel types all lacked the E5 ORF and HPV214 also lacked the E6 ORF. HPV212 had nine unique variants, HPV213 had five and HPV215 had four variants. Conserved domains observed among the novel types are the Zinc finger Binding Domain and PDZ domains. A retinoblastoma binding domain (pRB) binding domain in E7 protein was additionally identified in HPV214. This study expands the knowledge of the rapidly growing Gammapapillomavirus genus.