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Several studies have reported persistent infection of monocytes in virally suppressed individuals; however, others failed to detect HIV in this subset. These discrepancies are likely explained by the diversity of the methods used to isolate monocytes and to detect HIV infection. In this study, we show that only flow cytometry cell sorting yields a highly pure population of monocytes largely devoid of CD4 contaminants. Using this approach in a longitudinal cohort of HIV-infected individuals before and during ART, we demonstrate that HIV is rarely found in monocytes from untreated and treated HIV-infected individuals. 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