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Background: Short term efficacy of combination antiretroviral therapy (cART) in resource-constrained settings is comparable to that found in western studies. However, long term data are limited. India has the third largest HIV infected population in the world but the long-term outcome of first line therapy according to the national guidelines has not been evaluated yet. Therefore, we conducted a long-term longitudinal analysis of the efficacy of the national first-line therapy in India from an observational cohort of Indian patients in two different clinical settings. Methodology/Principal Findings: A total 323 patients who had been on ART for a median of 23 months and achieved virological suppression <100 copies/ml by their study baseline visit, were included and followed for two years. Blood samples were collected every six months for viral load and CD4 count. Drug resistance genotyping was performed when the viral load was >2000 copies/mL. Adherence and treatment interruptions (>48 h) were assessed via self-report. In the studied patients, the median duration of viral suppression was 44 months; 15.8% of patients showed viral rebound, and 2.8% viral failure. Viral rebound or failure was significantly negatively related to perfect adherence (100% adherence and no treatment interruption >48 hrs). Virological re-suppression in the subsequent visit was observed in three patients without any change in therapy despite the presence of key mutations. Conclusion/Significance: Our study reports for the first time, a good long-term response to the first line therapy for a median of nearly four years although a less than perfect adherence increases the risk for treatment failure and subsequent drug resistance development. The empirical findings in this study also indicate the overall success of the Indian ART program in two different settings which likely are representative of other clinics that operate under the national guidelines. [ABSTRACT FROM AUTHOR]