Moderate Levels of Pre-Treatment HIV-1 Antiretroviral Drug Resistance Detected in the First South African National Survey

<div><p>Background</p><p>In order to assess the level of transmitted and/or pre-treatment antiretroviral drug resistance to HIV-1, the World Health Organization (WHO) recommends that regular surveys are conducted. This study’s objective was to assess the frequency of HIV-1 antiretroviral drug resistance in patients initiating antiretroviral treatment (ART) in the public sector throughout South Africa.</p><p>Methods</p><p>A prospective cross-sectional survey was conducted using probability proportional to size sampling. This method ensured that samples from each province were proportionally collected, based on the number of patients receiving ART in each region. Samples were collected between March 2013 and October 2014. <i>Pol</i> sequences were obtained using RT-PCR and Sanger sequencing and submitted to the Stanford Calibrated Population Resistance tool v6.0.</p><p>Results</p><p>A total of 277 sequences were available for analysis. Most participants were female (58.8%) and the median age was 34 years (IQR: 29–42). The median baseline CD4-count was 149 cells/mm³ (IQR: 62–249) and, based on self-reporting, participants had been diagnosed as HIV-positive approximately 44 days prior to sample collection (IQR: 23–179). Subtyping revealed that 98.2% were infected with HIV-1 subtype C. Overall, 25 out of 277 patients presented with ≥1 surveillance drug resistance mutation (SDRM, 9.0%, 95% CI: 6.1–13.0%). Non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations were the most numerous mutations detected (n = 23). Only two patients presented with a protease inhibitor (PI) mutation. In four patients ≥4 SDRMs were detected, which might indicate that these patients were not truly ART-naïve or were infected with a multi-resistant virus.</p><p>Conclusions</p><p>These results show that the level of antiretroviral drug resistance in ART-naïve South Africans has reached moderate levels, as per the WHO classification. Therefore, regular surveys of pre-treatment drug resistance levels in all regions of South Africa is highly recommended to monitor the changing levels of pre-treatment antiretroviral drug resistance.</p></div

Detailed antiretroviral drug resistance mutation profiles detected in 25 of the 277 pre-treatment patient samples.

<p>Detailed antiretroviral drug resistance mutation profiles detected in 25 of the 277 pre-treatment patient samples.</p

Prevalence of surveillance drug resistance mutations (SDRM) amongst HIV-1 infected individuals throughout South Africa.

<p>Prevalence of surveillance drug resistance mutations (SDRM) amongst HIV-1 infected individuals throughout South Africa.</p

Long-term persistence of RAM.

<p>Of the 16 patients who presented with RAM at baseline, we analyzed plasma specimens collected at later time points (“follow-up 1”, “follow-up 2”) for the presence of RAM. We detected three different scenarios, including persistent RAM, disappearing RAM, and newly emerging RAM (some samples appear in more than one scenario). Time to follow-up sample is indicated in months. In some cases, no PCR product from plasma samples could be generated. The investigation for long-term stability of RAM is insofar incomplete as plasma specimens collected at later time points were not available (“n.a.”) for all patients, referred to as “n.d.” (not determined).</p

Phylogenetic analysis of RT and PR sequences from the Tanzanian cohort.

<p>A neighbour-joining phylogenetic tree <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023091#pone.0023091-Saitou1" target="_blank">[30]</a> was constructed from the 88 patient derived HIV-1 sequences from the Tanzania cohort and the two partner-derived sequences. Reference sequences were obtained from the Los Alamos HIV sequence database. The analyzed 1302 bp region includes the complete Protease and Reverse Transcriptase coding region. The tree was constructed using Mega software version 4, and the evolutionary distances were calculated using the Kimura 2-parameter method. The bootstrap consensus tree was inferred from 50000 replicates and values greater than 70% are indicated on the branch lengths. The scale at the bottom left indicates the calculated genetic distances between the branches of the phylogenetic tree. Circles represent the 88 samples from our cohort. Black-dotted circles are without RAM, open-circled sequences are with RAM, open triangles are sequences with RAM from HIV-infected partners of two study subjects, which were not included in the determination of HIVDR as these patients received ART. Sequences without symbols are subtype reference sequences derived from Los Alamos database. The subtype is indicated at the end of each sequence name. Relative subtype frequency: A1: 34%, A1D: 7%, C: 26%, CRF10_CD: 4%, D: 28%, B: 1%. Sequences isolated from two couples (couple I, couple II) with NVP resistances.</p

HIVDR in the Mwanza cohort.

<p>RAM according to the Stanford HIV Drug Resistance Database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023091#pone.0023091-Stanford1" target="_blank">[17]</a> in 16/88 baseline samples. Mutations associated with a score of 60 were attributed as high-level resistance-associated mutations (RAM) and mutations with a score of 10–35 were attributed as low-level RAM. Mutations listed for WHO HIVDR surveillance <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023091#pone.0023091-Bennett3" target="_blank">[18]</a> are indicated in bold. Superscripted numbers identify the reasons for excluding the respected mutations from the HIVDR list: 1) nonpolymorphic, but at highly polymorphic position; 2) polymorphic position in subtypes B, F, CRF01_AE; 3) polymorphic in multiple subtypes. ABC: Abacavir; ATV: Atazanavir; AZT: Zidovudine; ddI: Didanosine; DLV: Delaviridine; D4T: Stavudine; EFV: Efavirenz; FTC: Emtricitabine; LPV: Lopinavir; NFV: Nelfinavir; NVP: Nevirapine; SQV: Saquinavir; 3TC:Lamivudine <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023091#pone.0023091-Joint1" target="_blank">[29]</a>. <u>Underlined</u> drugs are part of the local first line regimens, double line underlined drugs are part of local second line regimens. Data derived from 88 sequenced samples. Therapeutic drug monitoring (TDM) was performed from plasma samples collected at baseline for NNRTIs (Efavirenz and Nevirapine) and PIs (Nelfinavir, Saquinavir, Atazanavir and Lopinavir).</p

NVP resistance and PMTCT.

<p>Patients with NVP resistance mutations were analyzed regarding PMTCT (with NVP monotherapy) as a possible cause for the emergence of the mutation. PMTCT as a cause for the mutation's appearance is discussed as “possible” if the mother received PMTCT; it is discussed as “unclear” if the PMTCT status and the dates of birth of the children are unknown; for plausibility reasons PMTCT was excluded (“no”) as a trigger of the NVP mutation if the patient is either male or if a female patient presented with unknown PMTCT history combined with the HIV infection being diagnosed only since the date of birth of the youngest child.</p

HIVDR affects efficacy of local first- and second-line ART regimens.

<p>Effects of HIVDR (high- and low-level resistances) on drugs included in the local antiretroviral regimens. A: Effects on local first-line ART regimen. HIVDR that affects at least one of the drugs included in first-line therapy (AZT/D4T plus 3TC plus NVP/EFV) scored positive. B: Effects on local second-line ART regimen. HIVDR that affects at least one of the drugs included in second-line therapy (ddI/ABC plus LPV/SQV plus RTV) scored positive. C: Proportion of patients with HIVDR that affects first-line ART regimen who also carry HIVDR that affects second-line ART regimen.</p

The frequency of HIVDR is age-dependent.

<p>HIVDR was determined by bulk sequencing from ART-naïve patients. A.: Frequency of WHO-defined HIVDR in patients aged under 25 years (n = 20, left bar), patients aged over 25 years (n = 68, middle bar), and in the total study population (n = 88, right white bar). B: Frequency of WHO-defined HIVDR peaks in different age groups. Numbers in brackets indicate the number of individuals tested in each age group. Data as means ± S.E.M.. For statistical analysis, Fisher's exact test was performed. Differences with a P<0.05 were regarded as statistically significant. C: Number of WHO-defined RAM per individual (“RAM burden”). D: Number of affected antiretroviral drug classes (NRTI, NNRTI, PI) per individual in relation to the number of WHO-defined RAM per individual.</p

Demographic patient characteristics of the study population.

<p>The study population consisted of 120 ART-naive HIV-1 infected adults. PBMC- or plasma samples from 88 patients yielded amplicons for the bulk sequencing reaction. Data are expressed as means ± S.D. and range in parentheses. Patients with CD4 counts <200/ml at sample date initiated ART if CD4 counts remained below 350/ml four weeks later.</p