Evaluation of in-house molecular assays for detection of HIV-1 proviral DNA

HIV-1 proviral DNA is a critical marker for the early diagnosis of vertical transmission, especially when viral load RNA is suppressed by antiretrovirals. Our objective was to evaluate the performance of a qualitative qPCR to detect HIV-1 proviral DNA. Using the TaqMan system, the assay targets the Integrase (IN), Long Terminal Repeat (LTR) regions of HIV-1, and the CCR5 gene as an internal control. To improve sensitivity, a Nested qPCR step was implemented. ACH-2 cells were used to construct a standard curve and standardize the assays. The assessment of the diagnostic sensitivity and specificity of qPCR was carried out on 125 clinical samples, with the performance of Nested qPCR evaluated on 47/125 clinical samples. qPCR had a detection limit of 40 copies/reaction of HIV-1 proviral DNA, a sensitivity of 85.3%, and a specificity of 100%. Nested qPCR increased sensitivity to 30 copies/reaction. The assays proved to be effective in detecting HIV-1 proviral DNA, identifying a variety of subtypes and CRFs of the virus circulating in Brazil, and applicable to a variety of cases, including those with suppressed plasma viremia.