Antiretroviral therapy (ART) consisting of the nonnucleoside reverse transcriptase inhibitor (NNRTI), efavirenz, and tenofovir disoproxil fumarate combined with lamivudine or emtricitabine is a WHO recommended choice for first-line ART  and have become the mainstay of the large-scale rollout of ART in the Southern African region. The WHO has formulated criteria for determining levels of transmitted drug resistance (TDR) recommending the inclusion of asymptomatic and recently diagnosed individuals under 25 years of age  in an attempt to detect resistant viral strains before reversion to levels undetectable by population sequencing. However, this approach is logistically challenging. Testing at the time of commencing first-line ART is more commonly performed , referred to as pre-ART drug resistance (PDR). As not all patients presenting for first-line ART initiation are truly ART naive, PDR is a combination of detectable TDR and other effects, including undisclosed ART exposure, both programmatically important. PDR should be distinguished from WHO-defined TDR surveillance [3–5]. Nevertheless, in a universal test and treat setting a large proportion of patients presenting for first-time ART may be recently infected, likely providing a good estimate of TDR.
Models predict that TDR would increase with higher therapy coverage and a longer time since therapy scale-up . Routine viral load monitoring to identify patients experiencing ART failure, could limit TDR by early identification of patients who require interventions to achieve viral load suppression before they could infect others . Conversely, delayed therapy switches of patients failing ART may contribute to an increase in TDR . Uniform surveillance for drug resistance is based on a list of surveillance drug resistance mutations (SDRM), which excludes subtype-associated polymorphisms . Recently, an increase in PDR has been described in South Africa [10,11]. An increase of PDR against NNRTI has also recently been reported from the larger Sub-Saharan African region, the Caribbean/Latin America, United States, and upper-income Asian countries, whereas levels remained stable at moderate levels in Europe [12–15].
The Cape Winelands Health District has an antenatal clinic HIV prevalence of 15.0% . The public sector rollout of ART started in 2004, with the study clinic currently providing approximately 800 individuals with ART. Current national guidelines recommend a first-line regimen of a fixed-dose combination of tenofovir disoproxil fumarate, emtricitabine, and efavirenz. In the area surrounding the clinic, ART coverage has been enhanced further through a community randomized trial implementing universal test and treat since January 2014 which aims to reduce transmission by limiting infectivity of infected individuals [17,18]. The extent to which high local treatment coverage would limit transmission may, however, be dependent on the role of acutely infected individuals, not yet on ART, and whether sexual networks include a large proportion of individuals from low ART coverage settings outside of the intervention community [19–21].
We, therefore, investigated PDR at a primary healthcare clinic in the Drakenstein subdistrict, Cape Winelands District, in an area with high-ART coverage, and assessed whether transmission was locally clustered or part of a large regional epidemic, and whether there was evidence of clusters of TDR.
Sequential patients referred for ART initiation after testing HIV-positive in the surrounding community or at the clinic and who provided informed consent were included. Patients with a self-reported history of previous ART were excluded. The study was approved by the Stellenbosch University Health Research Ethics Committee (N15/09/079).
HIV viral load testing was performed in the regional state laboratory using the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 (Roche Molecular Diagnostics, Pleasanton, California, USA).
Genotypic HIV drug-resistance testing was performed by an in-house population sequencing assay covering amino acids in protease 1–99 and reverse transcriptase 1–262 . Drug resistance was identified with the Stanford database calibrated population resistance tool  using the 2009 SDRM list  and HIV-1 subtype with the REGA HIV-1 Subtyping Tool Version 3.0 .
Samples were coded for anonymized phylogenetic analysis to detect clusters as follows: subtype; sex; age category (16–24, 25–30, 31–40, and 41–60); SDRM; number (if more than one sequence with the same coding characteristics); coding fields were separated by underscores.
A phylogenetic tree was constructed in Geneious 9.1.5 using a Tamara Neighbor Joining Tree algorithm (Fig. 1).
In total, 64 patients who presented for ART initiation at the study clinic between 6 June 2016 and 10 January 2017 were recruited. Median CD4+ cell count was 337 (interquartile range: 181–474) cells/μl. Genotyping was unsuccessful in four of 64 patients, three of which had HIV viral loads less than 100 copies/ml and one that had a viral load of 130 copies/ml, leaving 60 for analysis. These four individuals were diagnosed as infected by two positive rapid HIV antibody tests, the individual with a viral load of 130 copies/ml in addition confirmed with a fourth generation serological assay. In the latter, history excluded prior ART suggesting a likely natural controller, whereas clinical history suggested possible undisclosed prior ART in two of the other individuals. Of the 60 patients included, 31 (52%) were women; overall mean age was 34 years, 38 for female and 29 for male patients. In total, 58 samples (97%) were classified as HIV-1 subtype C and two (3%) as CD recombinants circulating recombinant form (CRF)10.
For 59 patients, loci sequenced included all SDRM (including protease positons 1–99 and at least reverse transcriptase 1–239); for one patient only a partial sequence was obtained (protease 1–99 and reverse transcriptase 47–262), which included all NNRTI, protease inhibitor (PI), and nucleos(t)ide reverse transcriptase inhibitor SDRM except M41L. Details of SDRM detected are provided in Table 1. In summary, six of 60 patients (10%) had an SDRM. None had any PI-associated SDRM, five of 60 (8.3%) NNRTI SDRM, and one of 60 (1.7%) NRTI SDRM; GenBank accession numbers MF285802–MF285860).
From an anonymized neighbor joining tree (Fig. 1) there are neither large transmission clusters, nor linkage among cases with PDR. When analysed using the Los Alamos database, sequences mapped to other sequences across the Southern African region (49% closest match was in South Africa; 25% Zambia; 12% Botswana; and 14% other, mostly in Sub-Sahara Africa). Two participants, ‘C_F_25–30_none’ and ‘C_M_41–60_none’, shared almost identical sequences and could represent sexual partners linked by a recent transmission event; other sequences belonging to clusters of 2–3 participant sequences could represent less recent or indirect transmission events.
Similar to a recent national surveillance study of HIV drug resistance , we found a prevalence of 10% PDR, with mutations affecting NNRTI sensitivity the most prevalent. Similarly, an increase in NNRTI–PDR has been observed in Latin America and the United States . K103N, found in five of six cases with any PDR, has been observed in 55% of patients from lower middle income countries with any drug resistance mutation . Although the PDR prevalence is not above the 15% WHO threshold warranting an alternative first-line ART regimen, any PDR, even when present as minor variants, may increase the chance of ART failure .
Our study was limited by performing population sequencing in patients who may have been infected for years, therefore, the levels of PDR were likely underestimated. Also, some patients may not have disclosed previous exposure to ART. A strength of the study was that participants were enrolled at a primary healthcare site, limiting referral bias.
There was neither evidence of clusters of TDR nor large transmission clusters. Sequences were overall very diverse, mapping to many different other HIV-1 subtype C sequences from the region. Considering this, it is unclear to what extent the high regional ART coverage has effectively limited local transmission clusters, while clearly not impacting on transmissions from outside the region.
Moderate levels of PDR may impact on ART success and, therefore, influence choice of first-line ART. However, other considerations may be just as important for choosing the best first-line regimen. Ideally high durability regimens should be used as first line: good tolerability and low toxicity could improve adherence and quality of life of treated individuals, and high genetic barriers to resistance limit TDR. The availability of fixed-dose generic formulations and their cost are pivotal in resource-limited settings. High regimen effectiveness and durability could result in programmatic cost savings as these regimens may require less frequent viral load monitoring and less frequent switches to less tolerable second-line regimens.
Based on these considerations there are efforts to obtain integrase inhibitors, especially high genetic barrier dolutegravir, for first-line ART in Southern Africa, with Botswana first to implement dolutegravir-based regimens as preferred first line for patients newly initiated on ART . When applied in a large-scale rollout, this may not only increase individual therapy success but also reduce onward transmission and TDR. More durable, effective, and better tolerable first-line regimens could curb the HIV epidemic, especially if the overall cost of treatment (which includes program management, individual care, and treatment monitoring) could be reduced to allow sustainability of treatment programs in regions with a high prevalence and generalized HIV epidemics.
Research assistance support: Kirsten Veldsman, Mary Grace Katusiime.
The article was funded by South African MRC Collaborating Centre (TygHIVLab) and NHLS Research Trust.
Conflicts of interest
There are no conflicts of interest.
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