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Among patients developing viremia on first-line HAART in a South African cohort, 41% resuppressed without regimen switch. At first detection of viremia, 66% had any resistance mutations and 4% had thymidine analogue mutations (TAMs). After 12 months of viremia, 14% had TAMs.
Episodes of viremia are common in African ART programs. We sought to describe viremia, resuppression, and accumulation of resistance during first-line combination antiretroviral therapy (cART) in South Africa.
Retrospective analysis of a cohort receiving zidovudine, lamivudine, and either efavirenz or nevirapine with 6 monthly HIV RNA monitoring. We assessed viremia (HIV RNA >1000 c/mL after initial HIV RNA response) and resuppression (HIV RNA <400 c/mL after viremia). Genotypic resistance testing was performed using stored plasma on a subset at first detection of viremia and subsequently among patients with persistent viremia.
3,727 patients initiated cART (median CD4 147 cells/mm3) between 2002 and 2007. Of 1007 patients who developed viremia, 41% (331/815), with subsequent HIV RNA assays, resuppressed without regimen switch. At identification of viremia, 45/68 (66%) had HIV-1 drug resistance; 62% had NNRTI resistance, 37% had M184V/I, and 4% had multi-nucleoside analogue drug mutations. By 12 months of persistent viremia among a subset with resistance testing to 12 months, this increased to 78%, 57%, and 14% respectively. Resistance was associated with a reduced probability of resuppression; however 50% of patients with NNRTI resistance resuppressed while on an NNRTI.
The majority of patients had NNRTI resistance mutations at detection of viremia. However, 41% resuppressed without regimen switch. Our study supports maximizing first-line use while minimizing risk of significant cross resistance by implementing intensive adherence support and repeat HIV RNA testing 3–6 months after detecting viremia with regimen switch only if viremia persists.
The recent availability of antiretroviral therapy in Africa has had a dramatic impact on suppressing HIV and reducing mortality [1;2]. Within 12 months of initiating combination antiretroviral therapy (cART), 65 to 90% of individuals achieve HIV RNA levels below the limit of detection [3–7]. However not all individuals maintain virologic control ; those who do not are at risk for generation of drug resistant HIV and eventual CD4 decline. In order to identify patients who are not maintaining virologic control, treatment guidelines used in the United States recommend HIV RNA monitoring 2 – 4 weeks after treatment initiation and every 4 – 8 weeks until the HIV RNA level is <50 copies/mL . Guidelines used in resource-limited regions recommend monitoring of HIV RNA and/or CD4 every six months and make provision for situations in which no laboratory monitoring is available [9;10].
As a consequence of the monitoring strategies available in resource-limited countries, identification of HIV viremia may be delayed. Furthermore, because of limited cART options, relative immune preservation despite viremia, and limited data to guide decision making, many providers are hesitant to switch therapy when viremia is identified. When HIV viremia is detected or decisions to switch therapies are made, multiple HIV resistance mutations may be present [11;12]. However, minimal data are available on patterns of HIV viremia, re-suppression, and the accumulation of resistance during persistent viremia; therefore, we evaluated these issues in an HIV treatment cohort in South Africa.
Patients included in this study were enrolled in a workplace HIV programme in South Africa and met the following criteria: 1) initiated cART between November 2002 and May 2006, 2) had at least one HIV RNA assay while on therapy, 3) were ≥18 years old, 4) were ART naive at cART initiation, and 5) received a cART regimen of zidovudine, lamivudine, and a non-nucleoside reverse transcriptase inhibitor (NNRTI), either efavirenz or nevirapine. The observation period for each patient was from cART initiation until the earlier of June 2008 or last follow-up HIV RNA assay.
This ART program has been described elsewhere . In brief, cART eligibility was based on modified WHO criteria: CD4 count <250 cells/mm3; WHO stage 3 and CD4 count <350 cells/mm3; or WHO stage 4. CD4 count and HIV RNA levels were determined before cART initiation, after six weeks on cART, and then every six months. Programme guidelines recommended switch to second-line therapy if two sequential HIV RNA assays were >1000 copies/mL (c/mL) and the provider assessed that good adherence was achievable. Regimen changes were recorded on a standardized clinic visit form. In addition, central pharmacy distribution records were used to verify medications. The second-line regimen was abacavir, didanosine, and lopinavir/ritonavir.
The adherence strategy was based on individual structured counseling with 1 to 2 visits at each of the following time points: cART preparation, initiation, maintenance, and treatment failure, and suspected poor adherence. Routine adherence assessment included number of pills missed in the preceding 3 days, time of day pills were missed in the preceding 7 days, and the reason pills were missed.
All patients included in this study signed informed consent and ethical approval for this study was obtained from the Research Ethics Committees of the Anglo-Gold Health Service and the London School for Hygiene and Tropical Medicine, the University of KwaZulu-Natal, and the Institutional Review Board at Johns Hopkins University School of Medicine.
HIV viremia: HIV RNA >1000 c/mL while on first-line therapy after an initial drop >1 log10 c/mL from the pre-cART level.
Persistent viremia: HIV viremia >1000 c/mL detected on at least two consecutive HIV RNA assays while on first-line therapy.
Resuppression: HIV RNA to <400 c/mL while remaining on first-line cART after at least one HIV RNA assay demonstrating viremia.
WHO CD4 failure criteria: fall of CD4 count to pre-cART baseline, 50% fall from on-cART peak value, or persistent CD4 levels <100 cells/mm3 for >12 months . We modified these criteria and did not use persistent CD4 levels <100 cells/mm3 as a criterion for treatment failure because of the number of individuals in this study with virologic failure before 12 months.
Resistance mutation: Resistance mutations were defined based on the IAS-USA 2008 resistance guide . Thymidine associated mutations (TAMs) were M41L, D67N, K70R, L210W, T215Y/F, K219Q/E and multi-NRTI mutations included TAMS, K65R, and L74V.
In 2007 we selected every third individual with viremia based on study number. We selected specimens for genotyping from first detection of viremia and, for those patients with persistent viremia, subsequent time points for which samples were available. Subsequently, additional follow-up data were obtained. To assess if patients whose samples were genotyped were representative of the larger population we compared, using the chi-square or t test, patients with genotype results with patients who also developed viremia but did not have genotyping.
HIV RNA was assayed with Amplicor HIV-1 Monitor Test, Roche Diagnostics. Sequencing was performed by extracting viral RNA from stored plasma and amplifying a 1.7Kb fragment spanning the pol gene by nested PCR using the Thermoscript TM RT-PCR System. PCR products were sequenced by using BigDyeTerminators and an ABI 310 DNA Sequencer (Applied Biosystems, Foster City, CA) . Consensus sequences from all genotyped individuals were aligned and manually edited using the Sequencher version 4.5 program (GeneCodes, Ann Arbor, MI). Multiple alignments were performed using Clustal X version 2.0 (Conway Institute, University College, Dublin). Phylogenetic analysis of nucleic acid sequences was carried out with Mega version 4.0 . Reference sequences were downloaded from the Los Alamos database (www.hiv.lanl.gov).
Logistic regression was used to identify variables associated with the presence of i) NNRTI resistance and ii) the M184V/I mutation at first detection of viremia and to evaluate associations with resuppression within 6 months of detecting viremia among subjects with an HIV RNA result within this time frame. 95% confidence intervals for proportions with resistant virus were calculated with exact binomial methods. Bar graphs were used to display the accumulation of mutations (any NNRTI, M184V/I, or any TAM) at detection of viremia and at 6, 12, and 18 months of persistent viremia on first-line cART for patients with multiple resistance tests at least one of which was ≥12 months after first detection of viremia. All HIV RNA values were log10 transformed for analysis.
A total of 3,727 patients met our inclusion criteria, of whom 3,479 (93%) were male with a median age of 42 years and median CD4 count at cART initiation of 147 cells/mm3 (interquartile range [IQR]: 80–216)(Table 1). The median duration between HIV RNA assays was 4 months (interquartile range, IQR, 2.2–5.6). The median follow-up was 17.4 months (IQR: 6.0–31) with 6118 person-years of follow-up.
A total of 3,432 subjects (92%) had >1 log10 decline in HIV RNA between the time of cART initiation and the end of the observation period of whom 3,159 suppressed to <400c/mL. Of the 3,432 with >1 log10 HIV RNA decline, 1,007 (29%) developed HIV viremia detected a median of 11 months (IQR 6.2–18) after cART initiation. A total of 815 (81%) of those with HIV viremia had follow-up HIV RNA laboratory results on first-line therapy (Figure 1). Reasons for absence of repeat HIV RNA assays on first-line therapy amongst the other 192 patients were cART was stopped or switched, they left the cART program, died, or had no additional HIV RNA assays during the observation period. Of the 815 patients with subsequent HIV RNA results, 331 (41%) resuppressed (HIV RNA <400 copies/mL) without a regimen switch, of these 224 achieved an HIV RNA <50 c/mL. Only 46 of the 1,007 patients with viremia were switched to second-line therapy during the observation period.
At the time of first detection of HIV viremia, 211 patients (21% of patients with HIV viremia) met modified WHO CD4 criteria for treatment failure. Another 110 patients with viremia fulfilled the modified criteria at a later time point. Of these 321 (211+110) patients with modified WHO CD4 failure and viremia, 111 (34%) resuppressed without a change in regimen.
We selected 138 patients for genotyping; samples from 18 could not be amplified and insufficient sample was available from 30 patients. As a result, 90 patients underwent genotyping. Individuals with genotype results were similar in terms of gender, age, weight at cART initiation, median CD4 at cART initiation, median HIV RNA at cART initiation, WHO stage, and NNRTI received to those viremic individuals who were not genotyped (all p>0.1). We used a neighbor joining tree to assess subtype clustering. One patient clustered with subtype A HIV-1, the rest clustered with subtype C.
68 subjects had genotypic resistance tests done at first detection of HIV viremia (the remaining 22 had insufficient sample at this time point). Of these 68, 45 (66%) had at least one HIV resistance mutation in reverse transcriptase. NNRTI mutations were most common, 42/68 (62%), followed by the M184V/I mutation, 25/68 (37%)(Table 2). Only 4/68 (6%) had one or more multi-NRTI mutations at detection of viremia.
In unadjusted analysis, the presence of an NNRTI mutation and the M184V/I mutation at first detection of viremia were both associated with higher HIV RNA at cART initiation (Table 3). However, neither was associated with HIV RNA level at the time viremia was identified. Sex, age, CD4 count, NNRTI agent, modified WHO CD4 failure criteria, and WHO stage at cART initiation were not associated with the presence of either NNRTI mutations or the M184V/I mutation (all, p>0.1). Higher weight at cART initiation (p=0.02), modified WHO CD4 failure criteria (p=0.06), and HIV RNA at cART initiation (p=0.03) were associated with a reduced odds of the M184V/I mutation. Results from multivariate modeling for the M184V/I including weight and baseline HIV RNA were as follows: HIV RNA at baseline, compared to < 4.6 log10 c/mL; 4.6–4.9 c/mL, odds ratio (OR) 2.7 (0.64–11); and >4.9 c/mL, OR 5.9 (1.4–26), p trend=0.04; weight per 10kg increase, OR 0.44 (0.23–0.83), p=0.01.
We restricted analysis of the accumulation of mutations during HIV viremia to the 30 patients with multiple genotypes with the latest at least 12 months from first detection of viremia while remaining on first-line cART. At detection of viremia 71% (5/7) of tested patients had NNRTI resistance mutations; this increased to 89% (8/9) by 6 months, 78% (11/14) by 12 months, and 94% (15/16) by 18 months (Figure 2). For lamivudine, at detection of viremia 43% (3/7) had the M184V/I mutation, by 6 months 44% (4/9), by 12 months 57% (8/14), and by 18 months 80% (12/15). At detection of viremia none had a TAM, by 6 months 11% (1/9), by 12 months 14% (2/14), and by 18 months 31% (4/13) had developed at least 1 TAM. Of subjects with a TAM by 18 months, 2 had 1 or 2 TAMs and 2 had 4 or 5 TAMs. Among all subjects with resistance testing, median time to 1, 2, and ≥3 TAMs was 8.4 (range 5.6–11.3), 2.7 (range: 0.9–18.3) and 10.3 months (range: 5.5–18.4), respectively.
Assessing the 655 patients (299 of whom resuppressed) with HIV RNA testing within 6 months of first detection of viremia (among the total 815 subjects with viremia and any subsequent test results), we found a trend toward resuppression and lower log10 HIV RNA at cART initiation (compared to log10 HIV RNA ≤4.5 copies/mL; log10 HIV RNA 4.6–4.9 c/mL, OR: 0.98, 95% CI 0.65–1.47; and log10 HIV RNA ≥5 copies/mL, OR: 0.67, 95% CI: 0.44–1.0; p=0.08) and associations with higher baseline CD4 count (per 50 cell increase, OR: 1.1, 95% CI 1.0–1.2; p=0.01) and WHO stage at cART initiation (compared to stage 1 or 2; stage 3, OR: 0.62, 95% CI 0.42–0.90; and stage 4, OR: 0.63, 95% CI 0.39–1.0, p =0.03). In multivariate analysis, WHO stage was not included due to colinearity with CD4 count. In this analysis, baseline CD4 remained associated with resuppression (adjusted OR 1.1 per 50 cell/mm3 increase, 95% CI: 1.0–1.2, p=0.01) but baseline log10 HIV RNA lost significance (p=0.5). In addition, among patients with resistance testing and follow-up HIV RNA testing (n=74) the absence of resistance mutations at first detection of viremia was associated with a greater chance of resuppression (OR 4.7, 95% CI: 1.5–15, p=0.009). There was no evidence of an association between resuppression and sex, age, weight at cART initiation, HIV RNA at detection of viremia, or modified WHO CD4 failure criteria (all, p>0.1).
We also evaluated the impact of resistance on resuppression by comparing the level of resistance among patients who did and did not resuppress. We identified 11 of 22 (50%; 95% CI: 28–72%) patients who resuppressed on the first-line and had genotyping that had NNRTI resistance. Among 52 patients with genotyping who did not resuppress, 42 (79%, 95% CI: 65–89%) had detectable resistance at first detection of viremia (Figure 1). Of the patients achieving resuppression despite detectable resistance, all had NNRTI resistance mutations: K103N (8), V106M (5), P225H (1), or G190A (1).
This study provides important information on the accumulation of drug resistance during HIV viremia while remaining on first-line cART. Unlike previous studies of cART in Africa, we have performed serial resistance testing during HIV viremia. In addition, we describe resuppression after viremia while remaining on first-line cART.
At first detection of viremia, the majority of patients had NNRTI resistance; lamivudine resistance was the next most common. TAMs and multi-NRTI mutations accumulated after longer duration viremia. The burden of resistance at detection of viremia is consistent with previous studies in which prevalence of NNRTI mutations ranged from 45 to 100%, the M184V/I mutation from 24 to 95%, and any TAM from 0 to 75% [5;11;12;18–22]. We hypothesize that the greater proportion of TAMs reported by some cohorts reflects longer delays in detecting viremia. Our rate of TAM accumulation is consistent with clinical trials of dual NRTI therapy from industrialized countries [23–25]. Of note, we used a cut-off of 400 copies/mL as a definition of resuppression for consistency with other studies and to avoid including blips as failures. Future studies evaluating lower level viremia (50–400 c/mL) are important for resource-limited settings.
An unusual contribution of our study is the repeated resistance testing on patients with persistent viremia while receiving first-line cART. This enabled us to assess the accumulation of resistance. This information is useful for informing clinical decisions, such as how rapidly to switch to second-line after detecting viremia. We found that, at the time of detection of viremia, a patient has a high probability of NNRTI resistance. When viremic for an additional 6 months on first-line therapy most patients had an NNRTI resistance mutation and approximately two-thirds had the M184V/I mutation. Importantly, only approximately 10% had any TAM at six months. As multiple TAMs are required for significant resistance to thymidine analogues (zidovudine and stavudine) and cross-resistance to other NRTI agents, we found that cross resistance was minimal six months after viremia was detected.
Importantly, among patients who developed viremia, 41% resuppressed while remaining on first-line therapy. However, had they been rapidly switched to a second-line regimen, benefit from the first-line regimen would have been prematurely abbreviated and the option of having an additional therapy available for the future would have been eliminated. High rates of resuppression after developing viremia have been reported, in abstract form, from other African cohorts [26;27].
We further found that even when NNRTI resistance mutations were present, successful resuppression occurred among some patients continuing the first-line regimen. This finding may seem surprising. However, resuppression on an NNRTI-based regimen despite NNRTI resistance has been noted in a treatment interruption study  and among individuals with transmitted resistance in a clinical trial using NNRTI based regimens .
Several limitations of our study are worth noting. First, we did not sequence HIV samples at the time of cART initiation. As a result, patients may have had resistance mutations prior to cART. We believe this is unlikely because 1) no patients reported prior ART, 2) 95% of the cohort was men and thus potential for exposure through prevention of mother to child transmission was minimal, 3) our cohort started cART in 2002, two years before the South African national cART rollout, and 4) the levels of transmitted resistance in South Africa at this time were low . A second weakness is that we lacked resources to sequence specimens from all patients who developed viremia.
Additional limitations reflect the nature of our cohort. Our cohort was mostly men who worked in mines and received HIV care through workplace HIV programmes. As a result, aspects of the cohort may differ from other cohorts. However, we believe that overall accumulation of resistance during viremia is likely to be similar across cohorts with subtype C HIV. In addition, this was an observational cohort and decisions regarding when to switch therapy were likely affected by perceptions of patient adherence. Thus, it is possible that individuals who developed viremia and were thought to have good adherence were switched more rapidly than those with perceived poor adherence. However, the impact of such as bias is likely to be minimal because only 46 of the 1007 patients who developed viremia were switched to second-line therapy. Furthermore, as an observational cohort using local standard of care, we lacked the frequency of HIV RNA monitoring to accurately describe the time to resuppression after developing viremia.
In ART programs with access to intermittent HIV RNA assays and limited treatment options, our results support added emphasis on adherence when viremia is identified followed by regimen switch only if viremia persists for 3–6 months. This strategy leads to limited risk of cross-resistance while maximizing the opportunity for resuppression on first-line therapy. This contrasts starkly with management in high-income countries where testing resources and the availability of many ART options allow rapid regimen switch and resistance testing when viremia is first identified. However, our proposed strategy fits resource-limited settings because it helps to avoid potentially premature switches to costlier and less tolerable regimens without risking the emergence of high-level cross resistance.
Support: This work was supported by the Aurum Institute and the National Institute for Communicable Diseases, South Africa. CJH was supported by NIH DK074348, REC by NIH AI5535901 and AI016137, and ADG by a UK Department of Health Public Health Career Scientist Award.
We thank Drs. Rami Kantor and David Katzenstein for constructive critiques.
Conflicts of interest: CJH: none, SC: none, JS: none, TP: none, CI: none, KLF: none, GJC: none, REC: none, LM: none, ADG: none
Note: This work was presented in part at the 16th Conference on Retroviruses and Opportunistic Infections, Montreal, Canada, February 7–11, 2009.