Monitoring the emergence and patterns of antiretroviral drug resistance is crucial for the success and sustainability of treatment programmes. Moreover, as new drugs become available, there is a growing need to better characterize viruses from both drug naïve and virologically failing patients to better understand the suitability of these new drugs either as additional components of the current regimens or as salvage therapy. In this study we investigated the prevalence and pattern of drug resistance mutations in a cohort of HIV-1 subtype C-infected individuals failing therapy. In addition, since it has previously been suggested that suboptimal antiretroviral treatment or certain classes of drugs may select for the more virulent X4 virus variants 13
, we sought to identify correlates of viral tropism in HAART-naïve and therapy experienced virologically failing patients.
Our results show that in a South African setting where patients are receiving antiretroviral therapy according to national and WHO guidelines, 95% of patients failing therapy had at least one drug resistance mutation. The most common NRTI mutation was M184V/I in 87% of patients; with V106M/A (51%) and K103N (40%) the commonest NNRTI resistance mutations. These patterns are consistent with data from previous HIV-1C studies 10, 37–39
. However, we also found a high prevalence (13.3%) of the ETR-resistance associated M230L mutation, which occurs relatively rarely in NNRTI-failing patients. This finding may require follow-up studies. Data from this study also revealed that 55% of patients failing therapy had thymidine analog mutations (TAMs) compared to 32% of patients studied from the same city in 2005–2006 10
. Recent studies from Botswana, Malawi and South Africa have also reported similar high proportions of TAMs in persons failing therapy under public sector antiretroviral programmes with an apparent bias towards the TAM2 pathway in these subtype C studies 32, 40–41
. In contrast, a recent subtype B study showed that 65% of virologically failing subjects harboured TAM1 pathway mutations 8
suggesting that there may be subtype-specific differences in thymidine analog mutation pathways. Other reasons for TAM pathway mutation differences may include differences in antiretroviral regimens, nadir CD4+ T-cell count before HAART commencement, duration of treatment before failure and duration on a failing regimen. Overall, we found that more than 90% of patients failing therapy had at least one drug resistance mutation and 55% of patients harbored TAMs. To minimize the loss of future treatment options, improved adherence support mechanisms and better monitoring algorithms for patients on HAART in resource-poor settings will be required.
We also investigated here whether HAART-failing HIV-1C-infected patients had higher proportion of X4/dual/mixed viruses compared to HAART-naïve patients. Although we found this to be the case, the patients failing HAART had lower median (nadir) CD4+ counts compared to HAART-naïve patients, and individuals possessing X4/dual/mixed viruses had significantly lower CD4+ T cell counts compared to those with R5-only viruses, in both the HAART-naïve and HAART-failing arms of this study. These data therefore suggest that low CD4+ T-cell counts (and by extension the length of infection), rather than HAART is the possible main cause of X4/dual/mixed viruses, consistent with data from HIV-1 subtype B studies 12, 40
. However, only a longitudinal study can decisively determine whether there is higher proportion of emergence of X4/dual/mixed viruses in treated versus HAART-naïve patients with similar CD4+ T-cell counts. Nevertheless, our study underlines the importance of introducing CCR5 inhibitors relatively early in the course of HIV-1 subtype C infection for possible maximum benefit and to preserve other drugs for salvage use. Clinical trials are needed to determine the equivalence or superiority of CCR5 inhibitors as part of first line or early regimens, rather than as salvage therapy in HIV-1C settings.
Finally, the availability of virus phenotype and genotype data allowed us to assess the utility of V3 loop sequenced based methods for predicting viral tropism. Envelope sequence-based genotypic coreceptor prediction algorithms offer a simpler and less expensive means of analyzing viral tropism in patients and could facilitate easier and less expensive determination of whether a patient can be treated with CCR5 antagonists or not. Our data show that while genotypic methods are reliable for a majority of cases, they failed to correctly predict CXCR4-tropism in a significant minority of cases consistent with previous studies 42
. Although the Geno2Pheno and V3 charge methods achieved more than 80% sensitivity in predicting CXCR4/dual-tropism for this dataset, there remains an urgent need to further investigate and develop better predictive algorithms, perhaps taking into account sequences outside of the V3 and more detailed analysis of V3 loop sequences using newer technologies able to better characterize V3 loop quasispecies diversity.
In summary, our observations confirm and extend the body of knowledge in examining coreceptor tropism directly in patients failing currently recommended regimens, and comparing this with ARV-naïve patients in a HIV-1 subtype C setting. The presence of high proportions of patients with TAMs suggests that these mutations may be accumulating over time in this population as a result of inadequate viral suppression, perhaps as a consequence of poor immunological and/or clinically driven monitoring in the absence of viral load testing. These results may suggest that in situations where virologic monitoring is not possible, measures are needed to improve adherence and to develop better monitoring tools. Comparison of the prevalence of CXCR4-utilizing viruses between ARV-naive prior to initiating ART with the prevalence among treated patients revealed that there was a high prevalence of X4/dual/mixed viruses in patients failing treatment, possibly due to lower nadir CD4 counts in these patients, underlining the need to investigate the possible earlier use of CCR5 inhibitors before the development of X4/dual/mixed viruses. Our data also highlight the usefulness and limitations of genotypic coreceptor prediction methods in assessing whether HIV-1C infected patients can be put on regimens that include CCR5 inhibitors. Longitudinal studies on viral coreceptor evolution in HIV-1C infections are warranted.