Erratum: Prevalence and distribution of selected cervical human papillomavirus types in HIV infected and HIV uninfected women in South Africa, 1989-2021: A narrative review.

[This corrects the article DOI: 10.4102/sajid.v37i1.363.].

Molecular epidemiology of SARS-CoV-2 in Northern South Africa: wastewater surveillance from January 2021 to May 2022.

Introduction: Wastewater-based genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) provides a comprehensive approach to characterize evolutionary patterns and distribution of viral types in a population. This study documents the molecular epidemiology of SARS-CoV-2, in Northern South Africa, from January 2021 to May 2022. Methodology: A total of 487 wastewater samples were collected from the influent of eight wastewater treatment facilities and tested for SARS-CoV-2 RNA using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). SARS-CoV-2 positive samples with genome copies/mL ≥1,500 were subjected to allele-specific genotyping (ASG) targeting the Spike protein; 75 SARS-CoV-2 positive samples were subjected to whole genome sequencing (WGS) on the ATOPlex platform. Variants of concern (VoC) and lineages were assigned using the Nextclade and PangoLIN Software. Concordance for VoC between ASG and WGS analyses was determined. Sequence relationship was determined by phylogenetic analysis. Results: Seventy-five percent (365/487) of the influent samples were positive for SARS-CoV-2 RNA. Delta and Omicron VoC were more predominant at a prevalence of 45 and 32%, respectively, and they were detected as early as January and February 2021, while Beta VoC was least detected at a prevalence of 5%. A total of 11/60 (18%) sequences were assigned lineages and clades only, but not a specific VoC name. Phylogenetic analysis was used to investigate the relationship of these sequences to other study sequences, and further characterize them. Concordance in variant assignment between ASG and WGS was seen in 51.2% of the study sequences. There was more intra-variant diversity among Beta VoC sequences; mutation E484K was absent. Three previously undescribed mutations (A361S, V327I, D427Y) were seen in Delta VoC. Discussion and Conclusion: The detection of Delta and Omicron VoCs in study sites earlier in the outbreak than has been reported in other regions of South Africa highlights the importance of population-based approaches over individual sample-based approaches in genomic surveillance. Inclusion of non-Spike protein targets could improve the specificity of ASG, since all VoCs share similar Spike protein mutations. Finally, continuous molecular epidemiology with the application of sensitive technologies such as next generation sequencing (NGS) is necessary for the documentation of mutations whose implications when further investigated could enhance diagnostics, and vaccine development efforts.

Prevalence and distribution of selected cervical human papillomavirus types in HIV infected and HIV uninfected women in South Africa, 1989-2021: A narrative review.

Background: Human papillomavirus infection, a causative agent of cervical cancer, is of great concern, more so in populations with high HIV prevalence, such as South Africa. Aim: This review aimed to examine the prevalence and distribution of selected cervical human papillomavirus (HPV) types in HIV infected and HIV uninfected women in South Africa. Methods: PubMed and Web of Science databases were searched using key words. For data integrity, data was assessed by two authors independently. The study inclusion criteria comprised records on cervical HPV, HPV genotyping and HPV type distribution among South African women. Statistical analysis was performed using Social Science Statistics. Results: Sixty-nine articles met the inclusion criteria for analysis. Data on cervical HPV prevalence and type distribution was available only for five of the nine provinces of South Africa. Only 4/69 studies used sequencing as an approach to identify HPV types. In a general population, HPV type 16 was the most frequent (8.80%), followed by types 35 (4.86%), 18 (4.14%), 58 and 52 with the frequency of 3.65% and 3.62%, respectively. Furthermore, the least frequent type was HPV 70 (0.74%). Both HIV infected and HIV uninfected populations had a higher prevalence of high-risk human papillomavirus (hrHPV) types 16, 18 and 35 than other HPV types; while HPV types 6, 11 and 70 were the least frequent types from these populations. Lastly, HPV 16 was the most predominant type among women with normal (2.03%) and abnormal cervical cytology (6.60%). Conclusion: Expanding on HPV genotyping will improve the knowledge in patterns of HPV type distribution in South Africa that will further help in decision making to improve current diagnostics, and future vaccine development and assessment.

Drug Resistance Mutations in a Population Before Antiretroviral Therapy Initiation in Northern South Africa.

South Africa introduced the "diagnose and treat" universal HIV treatment program in September 2016. This program enables all identified HIV-positive patients to immediately start first-line antiretroviral therapy (ART). However, the presence of drug-resistant (DR) viruses in the drug-naive population complicates the choice of ART. We used next-generation sequencing (NGS) to determine the prevalence and diversity of HIV DR mutations in patients entering HIV treatment programs in northern South Africa. RNA was isolated from plasma of drug-naive HIV-1-infected patients. Using reverse transcriptase polymerase chain reaction, the HIV-1-pol gene comprising the complete protease (PR) and the first 900 bp of reverse transcriptase (RT) was amplified and sequenced on an Illumina MiniSeq platform. Consensus sequences were derived at >20% threshold and at >5% threshold using Geneious PRIME® software version 2020.1.2. HIV-1 surveillance drug resistance mutations (SDRM) were inferred using Calibrated Population Resistance tool in HIV Drug Resistance Database. Viral subtypes were determined using REGA and RIP genotyping tools. The HIV PR/RT region was successfully sequenced from 241 patients. From these, 23 (9.5%) had at least one SDRM detected at >20% threshold, with a prevalence of 9.5% (n = 18), 3% (n = 7), and 0.4% (n = 1) for non-nucleoside reverse transcriptase inhibitors (NNRTI), nucleoside reverse transcriptase inhibitors (NRTI), and protease inhibitors (PI), respectively. The number of patients with SDRM increased to 31 (12.9%) when minority variants were accounted for at >5% threshold. The most frequent SDRMs based on drug class were; K103N (7.9%-NNRTI), K65R (2.5%-NRTI), and D30N (0.8%-PI). Four cases of dual NRTI/NNRTI mutations were identified. All consensus sequences were subtype C, except three, which were C/A1, C/F1, and C/G recombinants. NGS analysis confirms that individuals entering HIV treatment programs in northern South Africa, habor moderate levels of SDRM, including cases of dual-class drug resistance. Further SDRM studies may be required to better understand resistance in the drug-naive population in the era of "diagnose and treat" in Limpopo Province, South Africa.

Undisclosed exposure to antiretrovirals prior to treatment initiation: An exploratory analysis.

BACKGROUND: The proportion of individuals with a history of exposure ('pre-exposure') to antiretrovirals (ARVs) prior to formal initiation into antiretroviral treatment (ART) is unknown. OBJECTIVES: This study describes the detection of ARVs in plasma and/or hair, of persons who self-reported no pre-exposure to ART at their first-time initiation onto ART in three clinics in the province of Limpopo, South Africa (SA). METHOD: Concentrations of tenofovir (TDF), emtricitabine (FTC) and efavirenz (EFV) in the plasma and hair of individuals initiating ART were analysed using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Next generation sequences of HIV polymerase gene were analysed with Geneious software 11.15, and drug resistance (DR) mutations were determined according to the Stanford HIV Drug-Resistance database. Participants' demographic data were collected on a structured questionnaire. Data that describe prior exposure to ARV were also collected by this self-reporting method. RESULTS: Paired blood and hair samples were collected from 77 individuals newly initiated onto ART from 2017 to 2019. We detected at least one of the drugs in the plasma or hair of 41/77 (53.2%) patients who responded with a 'no' to the question 'have you received ARVs before initiation onto ART?' Thirty-one participants (n = 31/77, 40.3%) had TDF in either plasma or hair. Emtricitabine and EFV were found in the plasma or hair of 12/77 (15.6%) and 25/77 (32.4%) of participants respectively. Six (n = 6/77, 7.792%) had all three ARVs in plasma or hair. Prevalence of DR mutations at the > 5% significance threshold level in those known to have had ARV-exposure determined by LC-MS/MS prior to ART-initiation was not significant (χ2 = 0.798; p = 0.372), when compared to those who had no prior exposure but still showed DR. CONCLUSION: Antiretroviral levels in the hair of individuals initiating treatment imply prolonged prior-exposure to that ARV. The presence of ARV in plasma and hair of persons living with HIV (PLWH) who deny ARV-use, requires an explanation. A larger study at multiple sites and regular DR surveillance of ART-naïve PLWH will be necessary to confirm the generalisability of these findings to the wider South African population.

Potential challenges to sustained viral load suppression in the HIV treatment programme in South Africa: a narrative overview.

BACKGROUND: South Africa, with one of the highest HIV prevalences in the world, introduced the universal test and treat (UTT) programme in September 2016. Barriers to sustained viral suppression may include drug resistance in the pre-treated population, non-adherence, acquired resistance; pharmacokinetics and pharmacodynamics, and concurrent use of alternative treatments. OBJECTIVE: The purpose of this review is to highlight potential challenges to achieving sustained viral load suppression in South Africa (SA), a major expectation of the UTT initiative. METHODOLOGY: Through the PRISMA approach, published articles from South Africa on transmitted drug resistance; adherence to ARV; host genetic factors in drug pharmacokinetics and pharmacodynamics, and interactions between ARV and herbal medicine were searched and reviewed. RESULTS: The level of drug resistance in the pre-treated population in South Africa has increased over the years, although it is heterogeneous across and within Provinces. At least one study has documented a pre-treated population with moderate (> 5%) or high (> 15%) levels of drug resistance in eight of the nine Provinces. The concurrent use of ARV and medicinal herbal preparation is fairly common in SA, and may be impacting negatively on adherence to ARV. Only few studies have investigated the association between the genetically diverse South African population and pharmacokinetics and pharmacodynamics of ARVs. CONCLUSION: The increasing levels of drug resistant viruses in the pre-treated population poses a threat to viral load suppression and the sustainability of first line regimens. Drug resistance surveillance systems to track the emergence of resistant viruses, study the burden of prior exposure to ARV and the parallel use of alternative medicines, with the goal of minimizing resistance development and virologic failure are proposed for all the Provinces of South Africa. Optimal management of the different drivers of drug resistance in the pre-treated population, non-adherence, and acquired drug resistance will be beneficial in ensuring sustained viral suppression in at least 90% of those on treatment, a key component of the 90-90-90 strategy.

HIV-1 subtype C predicted co-receptor tropism in Africa: an individual sequence level meta-analysis.

BACKGROUND: Entry inhibitors, such as Maraviroc, hold promise as components of HIV treatment and/or pre-exposure prophylaxis in Africa. Maraviroc inhibits the interaction between HIV Envelope gp120 V3-loop and CCR5 coreceptor. HIV-1 subtype C (HIV-1-C) is predominant in Southern Africa and preferably uses CCR5 co-receptor. Therefore, a significant proportion of HIV-1-C CXCR4 utilizing viruses (X4) may compromise the effectiveness of Maraviroc. This analysis examined coreceptor preferences in early and chronic HIV-1-C infections across Africa. METHODS: African HIV-1-C Envelope gp120 V3-loop sequences sampled from 1988 to 2014 were retrieved from Los Alamos HIV Sequence Database. Sequences from early infections (< 186 days post infection) and chronic infections (> 186 days post infection) were analysed for predicted co-receptor preferences using Geno2Pheno [Coreceptor] 10% FPR, Phenoseq-C, and PSSMsinsi web tools. V3-loop diversity was determined, and viral subtype was confirmed by phylogenetic analysis. National treatment guidelines across Africa were reviewed for Maraviroc recommendation. RESULTS: Sequences from early (n = 6316) and chronic (n = 7338) HIV-1-C infected individuals from 10 and 15 African countries respectively were available for analyses. Overall, 518/6316 (8.2%; 95% CI 0.7-9.3) of early sequences were X4, with Ethiopia and Malawi having more than 10% each. For chronic infections, 8.3% (95% CI 2.4-16.2) sequences were X4 viruses, with Ethiopia, Tanzania, and Zimbabwe having more than 10% each. For sequences from early chronic infections (< 1 year post infection), the prevalence of X4 viruses was 8.5% (95% CI 2.6-11.2). In late chronic infections (≥ 5 years post infection), X4 viruses were observed in 36% (95% CI - 16.3 to 49.9), with two countries having relatively high X4 viruses: South Africa (43%) and Malawi (24%). The V3-loop amino acid sequence were more variable in X4 viruses in chronic infections compared to acute infections, with South Africa, Ethiopia and Zimbabwe showing the highest levels of V3-loop diversity. All sequences were phylogenetically confirmed as HIV-1-C and clustered according to their co-receptor tropism. In Africa, Maraviroc is registered only in South Africa and Uganda. CONCLUSIONS: Our analyses illustrate that X4 viruses are present in significantly similar proportions in early and early chronic HIV-1 subtype C infected individuals across Africa. In contrast, in late chronic infections, X4 viruses increase 3-5 folds. We can draw two inferences from our observations: (1) to enhance the utility of Maraviroc in chronic HIV subtype C infections in Africa, prior virus co-receptor determination is needed; (2) on the flip side, research on the efficacy of CXCR4 antagonists for HIV-1-C infections is encouraged. Currently, the use of Maraviroc is very limited in Africa.

Characterization of APOBEC3 variation in a population of HIV-1 infected individuals in northern South Africa.

BACKGROUND: The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) genes A3D, A3F, A3G and A3H have all been implicated in the restriction of human immunodeficiency virus type 1 (HIV-1) replication. Polymorphisms in these genes are likely to impact viral replication and fitness, contributing to viral diversity. Currently, only a few studies indicate that polymorphisms in the A3 genes may be correlated with infection risk and disease progression. METHODS: To characterize polymorphisms in the coding regions of these APOBEC3 genes in an HIV-1 infected population from the Limpopo Province of South Africa, APOBEC3 gene fragments were amplified from genomic DNA of 192 HIV-1 infected subjects and sequenced on an Illumina MiSeq platform. SNPs were confirmed and compared to SNPs in other populations reported in the 1000 Genome Phase III and HapMap databases, as well as in the ExAC exome database. Hardy-Weinberg Equilibrium was calculated and haplotypes were inferred using the LDlink 3.0 web tool. Linkage Disequilibrium (LD) for these SNPS were calculated in the total 1000 genome and AFR populations using the same tool. RESULTS: Known variants compared to the GRCh37 consensus genome sequence were detected at relatively high frequencies (> 5%) in all of the APOBEC3 genes. A3H showed the most variation, with several of the variants present in both alleles in almost all of the patients. Several minor allele variants (< 5%) were also detected in A3D, A3F and A3G. In addition, novel R6K, L221R and T238I variants in A3D and I117I in A3F were observed. Four, five, four, and three haplotypes were identified for A3D, A3F, A3G, and A3H respectively. CONCLUSIONS: The study showed significant polymorphisms in the APOBEC3D, 3F, 3G and 3H genes in our South African HIV1-infected cohort. In the case of all of these genes, the polymorphisms were generally present at higher frequencies than reported in other 1000 genome populations and in the ExAC exome consortium database .

Next generation sequencing reveals a high frequency of CXCR4 utilizing viruses in HIV-1 chronically infected drug experienced individuals in South Africa.

BACKGROUND: Entry inhibitors, such as Maraviroc, bind to CCR5 inhibiting entry of CCR5 utilizing viruses (R5 viruses). In the course of HIV infection, CXCR4 utilizing viruses (X4 viruses) may emerge and outgrow R5 viruses, and potentially limit the effectiveness of Maraviroc. The use of Maraviroc is reserved for salvage therapy in South Africa. OBJECTIVE: In this study, we examined the frequency of R5 and X4 viruses, using next generation sequencing, in patients under treatment to draw inferences on the utility of Maraviroc in a South African population. STUDY DESIGN: Proviral DNA was isolated from peripheral blood mononuclear cells (PBMC) of 72 chronically HIV infected patients on antiretroviral treatment. HIV V3 loop gene was amplified and sequenced on an Illumina MiniSeq platform. Viral subtypes were determined by the jumping profile Hidden Markov Model (jpHMM) and REGA genotyping tools. De Novo consensus sequences were derived for the majority and minority populations for each patient using Geneious® software version 8.1.5. HIV-1 tropism was inferred using PSSMsinsi, Geno2pheno and Phenoseq-C web-based tools. RESULTS: Quality V3 loop sequences were obtained from 72 patients, with 5 years (range: 0-16) median duration on treatment. Subtypes A1, B and C viruses were identified at frequencies of 4% (3/72), 4% (3/72) and 92% (66/72) respectively. Fifty four percent (39/72) of patients exclusively harboured R5 viral quasispecies; and 21% (15/72) exclusively harbored X4 viral quasispecies. Twenty five percent of patients (18/72) harbored dual/mixture of R5X4 quasispecies. Of these 18 patients, about 28% (5/18) harbored the R5+X4, a mixture with a majority R5 and minority X4 viruses, while about 72% (13/18) harbored the R5X4+ mixture with a majority X4 and minority R5 viruses. The proportion of all patients who harbored X4 viruses either exclusively or dual/mixture was 46% (33/72). Thirty-five percent (23/66) of the patients who were of HIV-1 subtype C harboured X4 viruses (χ2 = 3.58; p = .058), and 57% of these (13/23) harbored X4 viruses exclusively. CD4+ cell count less than 350 cell/µl was associated with the presence of X4 viruses (χ2 = 4.99; p = .008). CONCLUSION: The effectiveness of Maraviroc as a component in salvage therapy may be compromised for a significant number of chronically infected patients harboring CXCR4 utilizing viruses.