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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.

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.

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 .

Background: Combination antiretroviral therapy (cART) has significantly reduced HIV morbidity and mortality in both developed and developing countries. However, the sustainability of cART may be compromised by the emergence of viral drug resistance mutations (DRM) and the cellular persistence of proviruses carrying these DRM. This is potentially a more serious problem in resource limited settings., Methods: DRM were evaluated in individuals with unsuppressed viral loads after first or multiple lines of cART at two sites in rural Limpopo, South Africa. Seventy-two patients with viral loads of >1000 copies/ml were recruited between March 2014 and December 2015. Complete protease (PR) and partial Reverse Transcriptase (RT) sequences were amplified from both plasma RNA and paired proviral DNA from 35 of these subjects. Amplicons were directly sequenced to determine subtype and DRM using the Stanford HIV Drug Resistance Interpretation algorithm., Results: Among the 72 samples, 69 could be PCR amplified from RNA and 35 from both RNA and DNA. Sixty-five (94.2%) viruses were subtype C, while one was subtype B (1.4%), one recombinant K/C, one recombinant C/B and one unclassified. Fifty-eight (84%) sequences carried at least one DRM, while 11 (15.9%) displayed no DRM. DRM prevalence according to drug class was: NRTI 60.8% NNRTI 65.2%, and PI 5.8%. The most common DRMs were; M184V (51.7%), K103N (50%), V106M (20.6%), D67N (13.3%), K65R (12%). The frequency of the DRM tracked well with the frequency of use of medications to which the mutations were predicted to confer resistance. Interestingly, a significant number of subjects showed predicted resistance to the newer NNRTIs, etravirine (33%) and rilpivirine (42%), both of which are not yet available in this setting. The proportion of DRM in RNA and DNA were mostly similar with the exception of the thymidine analogue mutations (TAMs) D67N, K70R, K219QE; and K103N which were slightly more prevalent in DNA than RNA. Subjects who had received cART for at least 5 years were more likely to harbour >2 DRM (p < 0.05) compared to those treated for a shorter period. DRM were more prevalent in this rural setting compared to a neighbouring urban setting., Conclusion: We found a very high prevalence of NRTI and NNRTI DRM in patients from rural Limpopo settings with different durations of treatment. The prevalence was significantly higher than those reported in urban settings in South Africa. The dominance of NNRTI based mutations late in treatment supports the use of PI based regimens for second line treatment in this setting. The slight dominance of TAMs in DNA from infected PBMCs compared to plasma virus requires further studies that should include cART subjects with suppressed virus. Such studies will improve our understanding of the pattern of drug resistance and dynamics of viral persistence in these rural settings.

Background: CCR5-using (r5) HIV-1 predominates during asymptomatic disease followed by occasional emergence of CXCR4-using (x4) or dual tropic (r5x4) virus. We examined the contribution of the x4 and r5 components to replicative fitness of HIV-1 isolates., Methods: Dual tropic r5x4 viruses were predicted from average HIV-1 env sequences of two primary subtype C HIV-1 isolates (C19 and C27) and from two patient plasma samples (B12 and B19). Chimeric Env viruses with an NL4-3 backbone were constructed from the B12 and B19 env sequences. To determine replicative fitness, these primary and chimeric dual tropic HIV-1 were then competed against HIV-1 reference isolates in U87.CD4 cells expressing CXCR4 or CCR5 or in PBMCs ± entry inhibitors. Contribution of the x4 and r5 clones within the quasispecies of these chimeric or primary HIV-1 isolates were then compared to the frequency of x4, r5, and dual tropic clones within the quasispecies as predicted by phenotypic assays, clonal sequencing, and 454 deep sequencing., Results: In the primary HIV-1 isolates (C19 and C27), subtype C dual tropic clones dominated over x4 clones while pure r5 clones were absent. In two subtype B chimeric viruses (B12 and B19), r5 clones were >100-fold more abundant than x4 or r5/x4 clones. The dual tropic C19 and C27 HIV-1 isolates outcompeted r5 primary HIV-1 isolates, B2 and C3 in PBMCs. When AMD3100 was added or when only U87.CD4.CCR5 cells were used, the B2 and C3 reference viruses now out-competed the r5 component of the dual tropic C19 and C27. In contrast, the same replicative fitness was observed with dualtropic B12 and B19 HIV-1 isolates relative to x4 HIV-1 A8 and E6 or the r5 B2 and C3 viruses, even when the r5 or x4 component was inhibited by maraviroc (or AMD3100) or in U87.CD4.CXCR4 (or CCR5) cells., Conclusions: In the dual tropic HIV-1 isolates, the x4 replicative fitness is higher than r5 clones but the x4 or x4/r5 clones are typically at low frequency in the intrapatient virus population. Ex vivo HIV propagation promotes outgrowth of the x4 clones and provides an over-estimate of x4 dominance in replicative fitness within dual tropic viruses.