Coamplification of HIV-1 proviral DNA and viral RNA in assays used for quantification of HIV-1 RNA

Elevated HIV-1 viral load (VL) observed in specimens frozen in situ in plasma preparation tubes (PPTs) compared to EDTA plasma specimens may affect therapeutic monitoring of HIV-infected patients. The increase in viral load is cell associated and minimized when plasma from the PPT is aspirated or recentrifuged prior to freezing. This study investigates the contribution of integrated HIV-1 proviral DNA to elevated VL in the quantification of HIV-1 RNA in plasma. Fifty paired specimens collected in EDTA tubes and PPTs frozen in situ were used for analysis. HIV-1 VL was measured using the COBAS Amplicor Monitor ultrasensitive test version 1.5. Contaminating proviral DNA was detected using a nested PCR targeting the Alu repeat in human genomic DNA and HIV pol gene simultaneously. Treatment of the specimen with DNase resulted in significantly lower quantifiable HIV-1 RNA in specimens from PPTs compared to the corresponding EDTA tubes (P = 0.004). After the RNA was destroyed by heat treatment, the mean HIV-1 RNA VL decreased by 79% in the EDTA tube compared to 65% in the PPT. The nested PCR amplified integrated proviral DNA in nucleic acid extracted from plasma in PPT and EDTA specimens with high viral load values. Likewise, a semiquantitative densitometric analysis revealed that the total amount of genomic DNA in the PPT was higher than that in the EDTA tube. Our investigation clearly shows that both proviral DNA and intracellular RNA are amplified simultaneously in the COBAS Amplicor HIV-1 Monitor assay and that proviral DNA contributes to the elevated VL in plasma frozen in PPTs.