With increasing access to ART in LMIC, increased numbers of patients are failing first-line therapy and thus being switched to second-line therapy.[15
] In higher income countries, both choice of initial therapy and switch to second-line are done with the use of individual genotypes.[7
] In LMIC, a public health approach, with more limited first and second-line treatment options, has been used. In this setting it is critical to understand the evolution of resistance patterns, and whether the current treatment regimens are adequate.
The majority of virus in this South African sample, in both naive and non-naïve patients, was clade C.[24
] In both the naïve and non-naïve group there were a number of mutations in the protease enzyme, possibly indicating divergence from clade B virus. The impact of these on viral drug susceptibility is uncertain, but many, including L10I/V, K20R, M36I, L63P, A71V/T and V77I, are not expected to cause major drug resistance. [28
] These mutations are similar to those noted in other African clade C virus and clade C consensus sequences.[16
] Other mutations that are more likely to impact on the future use of a protease inhibitor, including D30N, M46I, I47V, I50V and V82A/F, were present, but in a minority of individuals, with only a single mutation noted per individual. The effect of such single mutations on the use of lopinavir in second line is not clear in our population.
There were very few mutations noted in the reverse transcriptase enzyme in the naive group. The two mutations that are likely to reduce susceptibility to NRTIs if transmitted, T215C and M41L, were not seen in either the naïve or non-naïve groups. [28
] The multiplicity of NNRTI mutations seen in the non-naïve group make NNRTI resistance the most likely to be transmitted in our population. Although no K103N mutation, expected to have the most impact on the use of NNRTIs, was seen in the naïve samples, there were 2 individuals (1.6%) each with a single mutation (Y181C and G190E) that would have some impact on NNRTI susceptibility. Although the currently recommended treatment regimens for first-line remain appropriate, ongoing surveillance of NNRTI-resistant virus remains important.[3
The samples in the non-naïve group were taken from 110 individuals failing initial NNRTI therapy in the South African public sector. Previous data have shown that the rate of confirmed virological failure in this cohort was 5.6% at 32 months.[15
] Adherence is monitored by tablet count in all public-sector clinics in South Africa and any viral load increase should initiate a “stepped-up” adherence package including counsellor-driven re-education sessions, more regular clinic visits with emphasis on the use of a pill-box as a reminder system, as well as a home visit to assess living circumstances where the resources are available for this service.[15
] Seventy-five percent of those with an initial viral load breakthrough of >1000 copies/ml again achieved suppression after structured adherence interventions.[15
] For those in whom failure was confirmed with a second specimen >1000 copies/ml, the median time from treatment commencement to noting initial virological breakthrough was 9 months.
The focus on adherence may explain the relatively small number of individuals with virological failure who did not have a significant drug-resistant mutation. Only seven individuals (6.4%) had wild type virus at genotype, a smaller proportion than seen in the DART study (10%).[20
] Most of these individuals with confirmed failure had resistance mutations which would exclude use of two of the antiretrovirals used in first-line regimens, i.e. 3TC (83%), a similar proportion to that seen in the DART study (70%), and the NNRTIs (86%).[20
] Resistance to both NNRTIs and 3TC (M184V) develops rapidly after initial virological breakthrough.
Resistance to the third drug in the regimen, the thymidine analogue, occurred more slowly. While 23% of the group had at least one TAM, relatively few had two or three TAMs and the majority of individual remained susceptible to both AZT and d4T. A trend towards TAM accumulation with prolonged time on failing therapy was noted, but was not-significant. People with lower CD4 counts at the time of genotype were also more likely to have acquired a TAM, perhaps indicative of a longer time on failing therapy than noted here, due to the length of time between viral loads in this cohort. Thymidine analogues are currently recycled in second-line therapy in South Africa (AZT, ddI and lopinavir/ritonavir), so TAM accumulation may reduce the efficacy of this therapy. However, if failure is identified before acquisition of TAMs, second-line therapy may remain more efficacious.
The increased presence (9%) of the K65R mutation in the non-naive samples was unexpected, given the absence of abacavir or tenofovir in the South African treatment regimens. There is emerging evidence that non-subtype B virus may have a propensity to develop the K65R more readily compared to subtype B.[10
] Doualla-Bell noted that d4T also selected for K65R in subtype C virus in Botswana, and that the mutation developed within 3 months of tenofovir therapy, unlike in subtype B where the K65R tends to emerge slowly in a small proportion of individuals on tenofovir.[10
] It is also possible that 3TC may select for the K65R mutation as recently described.[22
]With the registration of tenofovir in South Africa in 2007, there is a push for the widespread use of this agent to replace d4T, initially in those experiencing adverse effects, but with the view to broad-spectrum first-line use. The likely rapid emergence of resistance to tenofovir in clade C virus should be of concern for treatment programmes, as the presence of this mutation reduces susceptibility to all NRTIs except AZT, and thus would limit the choice of NRTIs for second-line therapy.[30
A limitation of this study is the 4 to 6 month window between viral load samples in the South African antiretroviral programme. Some individuals may have failed within weeks of their last suppressed viral load and others within days of their first raised viral load. Time from first virological breakthrough to time of genotype may therefore be an underestimate.
Resistance to the reverse transcriptase enzyme after exposure to NNRTI-containing first-line therapy follows a pattern that is predictable and similar to that of clade B: initial resistance to antiretrovirals that require a single point mutation, followed by slower development of resistance to drugs with a higher genetic barrier to resistance, such as the thymidine analogues.
Had second-line treatment been commenced within 6 months of initial virological breakthrough in the non-naïve group in this study, the likelihood of accumulating TAMs may have been reduced, with a potential increase in the efficacy of the recycled thymidine analogue in second-line therapy. Identification of and rapid response to virological failure is thus important to maintain the full benefit of second-line therapy. This would suggest clinical value to regular viral load testing to identify virologic failure soon after it occurs, in contrast to a recently published model.[31
] Due to the unexpected emergence of the K65R mutation in a substantial proportion of the cohort, tenofovir should be introduced cautiously with careful assessment of its impact on the emergence of resistance.
This study suggests that, at present, it is not critical in the context of the South African National ART programme to have routine access to genotypes at baseline, as the vast majority of naïve samples continue to be wild type. In contrast, the development of extensive resistance in those failing first-line therapy suggests that viral load monitoring is critical and there may well be a role for individual genotypes in those failing first-line therapy, particularly if second-line therapy is likely to be compromised by resistance to first-line therapy. Increased availability of low cost assays for identifying resistance in patients in South Africa would be clinically valuable.