Clinical evaluation of an in-house reverse transcription competitive PCR for quantitation of human immunodeficiency virus type 1 RNA in plasma

An in-house reverse transcription (RT)-competitive PCR (RT-cPCR) for the qu antitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma sam ples was developed and validated. The procedure involves (i) extraction of RNA with spin columns, (ii) ready-to-use bead-mediated RT, (iii) competitiv e PCR in a microtiter plate, (iv) agarose gel electrophoresis of the reacti on products, and (v) densitometric analysis of the digitized image of the g el. Quadruplicate tests and dilution studies showed that the sensitivity an d interest coefficient of variability of the RT-cPCR are comparable to thos e of the reference AMPLICOR HIV-1 MONITOR test. The results obtained by the two assays with a panel of 45 clinical samples were in good agreement (mea n difference, 0.36 +/- 0.25 log units). Analysis of 1,982 clinical samples by the in-house RT-cPCR yielded the typical range of plasma HIV-1 RNA level s with the expected inverse correlation between CD4 counts and HIV-1 RNA ti ters, In addition, testing of plasma from 36 subjects at weeks 0 and 4 with respect to the time of initiation of protease inhibitor therapy detected a significant decrease in HIV-1 viremia, The mean reduction in the HIV-1 RNA level was 0.914 log unit for those receiving saquinavir (P = 0.0210), 1.58 4 log units for those receiving indinavir (P = 0.0047), and 1.904 log units for those receiving ritonavir (P < 0.0001), The in-house RT-cPCR assay is simple to develop and perform and allows quantitation of HIV-1 RNA in 100 t o 200 samples per operator per week. Since the cost is 1/8 to 1/10 of those of reference commercial assays, this procedure could be conveniently used in medium-scale laboratories.