A study of the genital microbiotas of black South African women and men: associations with human papillomavirus and HIV infections

Persistent genital infection with oncogenic or high-risk human papillomavirus (HPV) is causally associated with cervical cancer in women and some penile cancers in men. The role of the complex genital microbiota in HPV infection has not been extensively addressed. This study characterised the genital microbiotas of heterosexually-active Black South African women and men, predominantly of the Xhosa ethnicity, recruited from a community in Cape Town, South Africa. The association of the genital microbiotas with prevalent HPV, HIV, demographic, behavioural, and clinical characteristics of the participants was examined. In Chapter 2 the bacterial communities in cervicovaginal samples from 62 HIVseronegative South African women were profiled by Ion Torrent PGM sequencing of the V4 hypervariable region of the bacterial 16S rRNA gene (IT-V4 method). The cervicovaginal microbiotas (CVMs) were found to cluster into three distinct community state types (CSTs): Lactobacillus iners-dominated CVMs (CST I (38.7%, 24/62)), unclassified Lactobacillusdominated CVMs (CST II (4.8%, 3/62)), and diverse CVMs (CST III (56.5%, 35/62)) with an array of heterogeneous bacteria, predominantly the bacterial vaginosis (BV)-associated Gardnerella, Prevotella, Sneathia, and Shuttleworthia. The majority of the women had nonLactobacillus-dominated CVMs. Lactobacilli are recognised as protective against sexually transmitted infections. Among the Lactobacillus species detected in the women, L. iners was the most prevalent and abundant. This species is recognised as the least protective amongst the vaginal lactobacilli. Women in CST I were more likely to be on hormonal contraception compared to women in CST III (relative risk (RR): 2.6 [95% CI 1.3-5.3]; p=0.005). Further research is required to confirm this association and to determine the biological mechanism. Microbiome research methodologies are constantly improving and in Chapter 3 the performance of two bacterial 16S rRNA gene amplicon-based methodologies were compared. The CVMs of 19 women were characterised using the IT-V4 method (Chapter 2) and using the Illumina 16S rRNA metagenomics method (IM-V3/V4 method). The latter method involves sequencing the V3 and V4 hypervariable regions of the 16S rRNA gene on the Illumina MiSeq platform. The two methods showed a high degree of correlation (r=0.89, p3, p3, p3, p 2, p<0.05) Men with BV-negative female sexual partners (66.5% (157/236)) had higher relative abundances of Lactobacillus in their penile microbiotas than men with BV-positive female partners (p=0.007). Atopobium, Sneathia, and Saccharofermentans were significantly more prevalent in men with BV-positive female partners than men with BV-negative partners (p<0.020). The main limitations of our study include relatively small sample size of women, insufficient participant information such as host genetics, other STIs (e.g., herpes simplex virus) and abnormal vaginal flora (e.g., aerobic vaginitis), using a less sensitive method to diagnose BV in women, and inherent biases evident in any retrospective study. Moreover, we did not adjust for confounding factors in our analysis due to the small sample size. Despite the underscored limitations, our findings provide insight into the baseline genital microbiotas of the Black South African women and men. The associations identified in this cross-sectional study between specific microbiota members and HPV infection, particularly the association between Sneathia and HPV/high-risk HPV infection, identified in both women and men, are hypothesis-generating and warrant further investigation. The study forms a critical starting point for future longitudinal confirmatory association studies and studies examining these bacteria as potential biomarkers or risk factors for HPV infection

Influence of non-synonymous sequence mutations on the architecture of HIV-1 clade C protease receptor site : docking and molecular dynamics studies

Despite the current interventions to avert contagions and AIDS-related deaths, sub-Saharan Africa is still the region most severely affected by the HIV/AIDS pandemic, where clade C is the dominant circulating HIV-1 strain. The pol-encoded HIV-1 protease enzyme has been extensively exploited as a drug target. Protease inhibitors have been engineered within the framework of clade B, the commonest in America, Europe and Australia. Recent studies have attested the existence of sequence and catalytic disparities between clades B and C proteases that could upset drug susceptibilities. Emergence of drug-resistant associated mutations and combinatorial explosions due to recombination thwarts the attempt to stabilize the current highly active antiretroviral therapy (HAART) baseline. The project aimed at identifying the structural and molecular mechanisms hired by mutants to affect the efficacies of both FDA approved and Rhodes University (RU)-synthesized inhibitors, in order to define how current and or future drugs ought to be modified or synthesized with the intent of combating drug resistance. The rationale involved the generation of homology models of the HIV-1 sequences from the South African infants failing treatment with two protease inhibitors: lopinavir and ritonavir (as monitored by alterations in surrogate markers: CD4 cell count decline and viral load upsurge). Consistent with previous studies, we established nine polymorphisms: 12S, 15V, 19I, 36I, 41K, 63P, 69K, 89M, and 93L, linked to subtype C wild-type; some of which are associated with protease treatment in clade B. Even though we predicted two occurrence patterns of M46I, I54V and V82A mutations as V82A→I54V→M46I and I54V→V82A→M46V, other possibilities might exist. Mutations either caused a protracted or contracted active site cleft, which enforced differential drug responses. The in silico docking indicated susceptibility discordances between clades B and C in certain polymorphisms and non-polymorphisms. The RU-synthesized ligands displayed varied efficacies that were below those of the FDA approved protease inhibitors. The flaps underwent a wide range of structural motions to accommodate and stabilize the ligands. Computational analyses unravelled the need for these potential drugs to be restructured by (de novo) drug engineers to improve their binding fits, affinities, energies and interactions with multiple key protease residues in order to target resilient HIV-1 assemblages. Accumulating evidences on contrasting drug-choice interpretations from the Stanford HIVdb should act as an impetus for the customization of a HIVdb for the sub-Saharan subcontinent

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance.

Investment in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing in Africa over the past year has led to a major increase in the number of sequences that have been generated and used to track the pandemic on the continent, a number that now exceeds 100,000 genomes. Our results show an increase in the number of African countries that are able to sequence domestically and highlight that local sequencing enables faster turnaround times and more-regular routine surveillance. Despite limitations of low testing proportions, findings from this genomic surveillance study underscore the heterogeneous nature of the pandemic and illuminate the distinct dispersal dynamics of variants of concern-particularly Alpha, Beta, Delta, and Omicron-on the continent. Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve while the continent faces many emerging and reemerging infectious disease threats. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

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

Surveillance of HIV-1 <i>pol</i> transmitted drug resistance in acutely and recently infected antiretroviral drug-naïve persons in rural western Kenya

<div><p>HIV-1 transmitted drug resistance (TDR) is of increasing public health concern in sub-Saharan Africa with the rollout of antiretroviral (ARV) therapy. Such data are, however, limited in Kenya, where HIV-1 drug resistance testing is not routinely performed. From a population-based household survey conducted between September and November 2012 in rural western Kenya, we retrospectively assessed HIV-1 TDR baseline rates, its determinants, and genetic diversity among drug-naïve persons aged 15–59 years with acute HIV-1 infections (AHI) and recent HIV-1 infections (RHI) as determined by nucleic acid amplification test and both Limiting Antigen and BioRad avidity immunoassays, respectively. HIV-1 <i>pol</i> sequences were scored for drug resistance mutations using Stanford HIVdb and WHO 2009 mutation guidelines. HIV-1 subtyping was computed in MEGA6. Eighty seven (93.5%) of the eligible samples were successfully sequenced. Of these, 8 had at least one TDR mutation, resulting in a TDR prevalence of 9.2% (95% CI 4.7–17.1). No TDR was observed among persons with AHI (n = 7). TDR prevalence was 4.6% (95% CI 1.8–11.2) for nucleoside reverse transcriptase inhibitors (NRTIs), 6.9% (95% CI 3.2–14.2) for non- nucleoside reverse transcriptase inhibitors (NNRTIs), and 1.2% (95% CI 0.2–6.2) for protease inhibitors. Three (3.4% 95% CI 0.8–10.1) persons had dual-class NRTI/NNRTI resistance. Predominant TDR mutations in the reverse transcriptase included K103N/S (4.6%) and M184V (2.3%); only M46I/L (1.1%) occurred in the protease. All the eight persons were predicted to have different grades of resistance to the ARV regimens, ranging from potential low-level to high-level resistance. HIV-1 subtype distribution was heterogeneous: A (57.5%), C (6.9%), D (21.8%), G (2.3%), and circulating recombinant forms (11.5%). Only low CD4 count was associated with TDR (p = 0.0145). Our findings warrant the need for enhanced HIV-1 TDR monitoring in order to inform on population-based therapeutic guidelines and public health interventions.</p></div

Prevalence of TDR in selected regions of East Africa.

<p>Prevalence of TDR in selected regions of East Africa.</p

Predicted antiretroviral (ARV) drug responses of eight persons with TDR mutations.

<p>Drug responses were based on the Stanford HIVdb v7.0 report. The y-axis indicates the percentage number of sequences with TDR while the x-axis indicates the different ARV drug classes that were affected by the TDR mutations. Drugs that were unaffected by any particular TDR were excluded from the analyses. TDR—Transmitted Drug Resistance; PIs—Protease Inhibitors; NRTIs—Nucleoside Reverse Transcriptase Inhibitors; NNRTIs—Non-Nucleoside Reverse Transcriptase Inhibitors; ATV/r—Boosted Atazanavir; FPV/r—Boosted Fosamprenavir; IDV/r—Boosted Indinavir; LPV/r—Boosted Lopinavir; NFV—Nelfinavir; TPV/r—Boosted Tipranavir; r—Ritonavir; 3TC—Lamivudine; ABC—Abacavir; AZT—Zidovudine; D4T—Stavudine; DDI—Didanosine; FTC—Emtricitabine; TDF—Tenofovir; EFV—Efavirenz; ETR—Etravirine; NVP—Nevirapine; RPV—Rilpivirine.</p

Demographic and clinical characteristics of acutely and recently HIV-1 infected antiretroviral-naïve persons in the Ndhiwa cohort, Kenya, 2012.

<p>Demographic and clinical characteristics of acutely and recently HIV-1 infected antiretroviral-naïve persons in the Ndhiwa cohort, Kenya, 2012.</p