Since the introduction in 2002 of government-sponsored antiretroviral therapy (ART) in South Africa in 2002, nearly 1 million patients have received ART, and the death rate from HIV/AIDS has declined. Although early accounts of treatment success reported high rates of adherence and viral suppression, rates of virologic failure (VF) have begun to approximate those observed in resource-rich countries. Because virologic monitoring is less frequent than in the US, there is more opportunity for resistance mutations to accumulate before VF is detected and ART switched to a 2nd-line regimen. As a result, the efficacy of 2nd-line therapy may be compromised. To date, there has not been a comprehensive study of the prevalence and risk factors associated with drug resistance in persons failing 1st-line ART in South Africa. Likewise, the effect of minority drug-resistant variants on the efficacy of non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens has not been studied except in women who received single-dose nevirapine (NVP) for prevention of mother-to-child transmission of HIV-1. Because transmitted resistance remains rare in South Africa, drug- resistant minority variants detected in this population may be assumed to reflect the spontaneous generation of such mutants by the error-prone HIV-1 RT. We propose to study the patterns and predictors of HIV-1 drug resistance after VF of 1st-line ART and the clinical significance of minority drug-resistant minority variants in peri-urban and rural settings in KwaZulu-Natal (KZN), South Africa. Specific aims of this proposal are as follows: 1) To determine the red blood cell ARV levels associated with VF and HIV-1 drug resistance at the time of 1st-line ART failure in KZN; 2) To determine the risk factors for VF and HIV-1 drug resistance in rural and peri-urban settings in the KZN province, South Africa; 3) To determine the effect of spontaneously arising drug- resistant minority variants on the risk of VF in patients receiving 1st-line ART. Subjects starting ART will be enrolled from clinics in two settings in KZN-peri-urban and rural. The number and patterns of HIV drug resistance mutations for patients who experience virologic failure of a 1st-line regimen of TDF plus lamivudine and EFV or NVP will be compared to resistance patterns reported for subtype B virus. A case-control design will be used to identify risk factors for suboptimal adherence and virologic failure with or without drug resistance in different settings in KZN province. The prevalence of minority drug resistant variants in the virus population will be determined in pre-treatment samples using allele-specific PCR and next-generation sequencing using the Illumina Solexa platform; the effect of these resistance mutations on the risk of virologic failure will be determined. Exploratory analyses will compare the frequency distribution of minority drug resistance mutations with the frequency predicted from population genetic theory to estimate the fitness cost of these mutations compared to the wildtype in the absence of drugs. The probability that a minority variant becomes fixed in the population and leads to resistance and VF will also be determined.