Emergence of HIV drug resistance
The rate of any nucleoside reverse transcriptase inhibitor (NRTI)-related resistance mutation increased over time from the end of year 1 (9.6%) to the end of year 6 (30.0%, P < 0.001 for trend; Fig. s1a, http://links.lww.com/QAD/A337). Among the NRTI-based drug-resistant mutations, T215FSY was the most frequently seen at all time points. M41L, M184V and L210W were also commonly seen, with the rates for these four mutations increasing over time (P < 0.001 for trend). Thymidine analogue-associated mutation type 1 (TAM1) mutations (M41L/L210W/T215Y) were more common than TAM2 mutations (D67N/K70R/T215F/K219QE) after 1 year post-cART initiation, and became twice as frequent as the latter from year 4 on. Non-nucleoside reverse transcriptase inhibitor (NNRTI)-related drug-resistant mutations appeared early and peaked in year 4 at 42.2%, finishing at 35.7% in year 6 (Fig. s1b, http://links.lww.com/QAD/A337). The most common NNRTI-resistance mutations detected were K103NS, Y181CI, G190AS and K101EP.
We further analysed mutation patterns when drug resistance was firstly detected in a given year of treatment. More than 95% of patients had NNRTI-related mutations at the first detection. However, the proportion of NRTI-related mutations was quite different between years (P < 0.001; Fig. 2a); persons with detectable drug-resistant mutations within the first year of cART had the lower rate with 23.5% of NRTI-related mutations, whereas those who had drug resistance detected beyond the first year had 50.4% of NRTI-related mutations. The differences were mainly found in the occurrence of TAMs (Fig. s2, http://links.lww.com/QAD/A337), such as M41L (P = 0.001), D67N (P = 0.001), K70R (P = 0.009) and T215Y (P = 0.005). When drug resistance was detected for the first time, the median viral load of patients differed significantly depending upon whether HIVDR appeared within the first year of cART (4.9 log copies/ml) or after the first year (4.1 log copies/ml, P < 0.001; Fig. 2b).
Our cohort study was conducted among patients who were predominantly farmers and FPDs in two counties in central China. They acquired HIV-1 through unhygienic plasmapheresis practices (reinfusion of pooled red blood cells) in the mid-1990s. In 2004, they were the first patients in China to receive free cART through the National Free ART Program. After 6 years of follow-up on first-line cART in our longitudinal study, adverse events had become commonplace: a cumulative three-quarters had failed virologically, nearly two-thirds developed drug resistance mutations, over half had failed immunologically and one-fifth had died. For patients with adverse events, median intervals of about 18 months each separated virologic failure (at 17.5 months) from HIVDR (at 36.6 months) from immunologic failure (at 55.2 months). Hence, if a patient is suboptimally virally suppressed, our real-world experience in a poor, rural region of China suggests about a year and half between each of these three adverse events: virologic failure, HIVDR and immunologic failure. Our disappointing drug resistance finding (a cumulative 64.4% of the cohort) highlights the challenges found with an unfamiliar chronic disease receiving lifelong treatment in a subsistence farming culture.
Many studies have examined the relationship between virologic failure and drug resistance [14–16], virologic failure and immunologic failure [17–19], virologic failure and death [17,19,20], drug resistance and death , immunologic failure and resistance , and immunologic failure and death [19,20]. Our study has the advantage of having examined thoroughly these relationships in one single cohort analysis; our median clinical follow-up of over 6 years in the absence of widely available second-line treatment offers data unique in the literature. It is notable that individuals experienced virologic failure before the emergence of drug resistance in the cohort study. Reasons for such a sequence may include a relatively low adherence or insufficient dosing with which drug concentration could not reach the threshold for drug resistance viruses outgrowing wild viruses . The limitation of consensus genotypic testing inhibits the detection of minor viral quasispecies with an abundance of less than 10–20% . Nevertheless, our results underscore the importance of viral load measurement as the primary marker of treatment efficacy and suggest the importance of switching cART regimens when there is evidence of virologic failure, to prevent the accumulation of drug resistance.
The cumulative rate of drug resistance was 24.2% at 12 months of treatment and 37.9% at 24 months, higher than the rates reported in other low and middle-income countries scaling-up cART. Malawi, for example, had 12% drug resistance in patients on treatment for more than 6 months . Cameroon had resistance levels of 4.4% after 12 months of treatment and 16.9% at 24 months . The reasons for these differences are likely multifactorial and may be related to provider experience, structural factors such as convenience of medication refills, patient adherence, stigma and levels of patient disclosure to others, and initial treatment regimen used, including drug side effects and tolerance [27,28]. In a multicentre study among antiretroviral-naive patients in China, persons on a 3TC-based treatment regimen had much higher rates of virologic suppression (less than 50 copies/ml) than those on a ddI-based regimen (68.2 vs. 39.7%, P < 0.001) .
In our analysis of risk factors associated with mortality, advanced immunosuppression at treatment initiation was the strongest risk factor, consistent with global experiences [8,30]. Our study also demonstrated an association between HIVDR and mortality, consistent with previous studies [21,31]. In addition, patients who developed HIVDR during treatment were nearly two-fold more likely to die than those who developed HIVDR later. The need for early adherence education and monitoring when starting ART must be stressed, as it was the earliest treatment failures who suffered the most adverse clinical outcomes. It also underscores the WHO HIVDR monitoring strategy, putting an emphasis on the first year of cART.
When HIVDR viruses were first detected, we found that drug resistance mutation patterns were quite different between patients acquiring detectable HIVDR in the first year and those who developed HIVDR later. Three-quarters of people developing HIVDR in their first year of cART had solitary NNRTI-resistant mutations, whereas half of the patients with later onset of HIVDR had both NRTI and NNRTI-resistant mutations. Some NRTI-resistant mutations, especially TAMs including M41L, K70R, T215FY and L210W, can reduce viral replicative fitness . Even though we do not know whether replicative fitness differences are an important contributor to the higher mortality seen, there was significantly higher viral load in the group with detectable HIVDR in the first year of treatment than the later onset HIVDR group. In individuals receiving NNRTI-based cART, persons with lower (<75%) medication adherence had a higher risk of HIVDR than those with moderate-to-high (75–100%) level of adherence . Early onset of drug resistance suggests poor adherence and hence incomplete viral suppression, likely from near the beginning of cART use, and persons with early HIVDR consequently have a higher risk of other adverse events and death.
Careful selection of the proper initial first-line regimens can improve both effectiveness and sustainability of cART. This is especially crucial for the public health scale-up of cART, wherein large numbers of patients are on the same regimens and there are fewer monitoring resources for follow-up of individuals and for community-wide assessments. Different levels of antiretroviral drug toxicity, drug–drug interactions, varying pharmacokinetics and pharmacogenomics, and socioeconomic limitations all affect cART adherence dramatically . At the early stage of China's National Free Antiretroviral Treatment Program, suboptimal regimens were chosen due to limited resources and accessibility of the best antiretroviral drugs. The initially used regimens, NVP and ddI along with ZDV or d4T, may have less potency and more side effect than other later regimens (TDF and an NNRTI along with 3TC, for example). A lack of optimized regimens likely contributes to lower medication adherence. In addition, NVP has a much longer half-life than other drug components; low adherence may result in NVP monotherapy and predispose to virologic failure and drug resistance.
Countries with limited resources may still want to use more potent and less toxic first-line cART regimens, as the long-term costs may be offset by maximizing the effects and sustainability of the first-line treatment, minimizing or delaying emergence of HIVDR, and easing the burden on intensive treatment monitoring. When to change virologically failing treatment regimens in resource-limited settings with limited second-line treatment options is a challenging question, given realities of drug availability and cost . Our study explored the time to virologic failure, drug resistance, immunologic failure and death in a remote rural area before the availability of second-line cART regimens. Because drug-resistant mutations impair viral fitness and replication, thereby partially suppressing plasma viral load [32,35,36], there may be benefit of continuing failing treatment regimens in patients when they have no access to second-line treatment options, especially in those with advanced HIV. Our study suggested as much, as patients who had detectable drug resistance beyond the first year of cART (later onset) seemed to benefit from partially active first-line regimens, experiencing reduced mortality compared with persons with earlier onset of HIVDR (within the first year of cART). However, our study also showed that the continuation of a virologically failing regimen was associated with an increased risk of resistance mutation accumulation. The decision of when to change regimens needs to balance these risks and benefits, as well as consider economic factors and long-term effects of first-line and second-line treatment .
The principal strength of our study is the long 6-year follow-up of our patients for the continuum of adverse virologic, immunologic and clinical outcomes in the context of HIVDR; other cohorts have been followed for only 12–24 months to study emergence of HIVDR. We also had a follow-up rate of more than 95% over 6 years and were able to assess laboratory specimens quickly, despite the remote rural setting. There are two notable limitations to our study. The first is that our study has uncertain generalizability, as it was conducted in two locations among FPDs. It is likely that this cohort is representative of FPDs because they generally had similar demographics and were infected with the same strain of HIV-1 at a short window period of blood contamination between 1993 and 1995 [9,38]. Furthermore, a recent analysis of HIV mortality in China found similar mortality risks among a variety of at-risk cohorts . Although adherence rates may vary among different populations and therefore the specific timing of HIVDR emergence may vary, the overall sequence of events from virologic failure to death is consistent with the current literature. A second limitation is that about half of the patients were missing baseline CD4+ cell count and viral load data due to the sudden start of cART to save lives, even before laboratory services were established. However, an analysis between those with and without baseline data showed no significant differences in either demographics or mortality rate; so, biases should be minimal (Table 2).
In conclusion, our clinical cohort study examined the frequency, sequence and timing of the emergence of virologic failure, genotypic resistance, immunologic failure and death in rural Chinese farmers on cART over 6 years. Failing treatment regimens were not changed due to limited resources and availability of second-line drugs. The cohort was stopped when second-line drugs became available and the seriousness of their unavailability was made clear from the data that emerged from our study. The median timing of these events was about one and half years between adverse milestones, but the interval times between these benchmarks would differ, we believe, on the basis of specific population characteristics. Still, this timetable of adverse outcomes may be helpful for healthcare practitioners and planners. Prevention of drug resistance in the very first year of treatment is important to lower long-term mortality, especially in resource-limited settings in which second-line therapies are limited. It remains a high research priority to optimize the timing of a switch to second or even third-line treatment regimens in the setting of limited resources.
We appreciate the contribution of all persons participating in this study. We thank the staff from provincial and county CDCs for sample collection and assistance. We thank Drs Xihong Lin, Peter Gilbert and Haiqun Lin for statistical suggestions. This work was supported by grants from the Ministry of Science and Technology of China (2012ZX10001-002), National natural Science Foundation in China (81101281), and Chinese State Key Laboratory for Infectious Disease Develop Grant (2011SKLID102), and the Canadian International Development Research Centre (104519-010).
S.Y., X.H., R.Y., L.L. conceived and designed the study; S.B., W.Z., R.Y., W.X., L.Z., L.Y., Y.S. and Z.Q. did the field investigation, data collection and experiment performance; L.L., X.H., S.B., W.Z., R.Y., C.R., V.S. and S.Y. did the data analysis and interpretation; and L.L., C.R., V.S. and S.Y. did the manuscript composing; all authors reviewed and approved the final version.
Conflicts of interest
All authors have no conflicts of interest to declare.
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antiretroviral therapy; China; cohort; drug resistance; immunologic failure; mortality; virologic failure
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