Assessment of a standardized reverse-transcriptase PCR assay for quantifying HIV-1 RNA in plasma and serum

The analytical variability of the new commercially available Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay, Amplicor(TM) HIV-I Monitor(TM), has been assessed to establish criteria for assessing the significance of HIV-I RNA level measurements. Estimations of the standard deviations (SD) of log-copies in inter-assay (mean 0.09 log) and in inter-laboratory (mean 0.14 log) reproducibility experiments demonstrated that the assay can discriminate with 95% confidence between 3-fold (inter-assay) and 5-fold differences (inter-laboratory). The inter-lot reproducibility (mean 0.10 log) was similar to the inter-assay reproducibility. The HIV-I RNA concentrations measured in plasma collected in potassium EDTA anticoagulant were slightly higher than those measured in plasma collected in sodium citrate. The HIV-1 RNA concentrations measured in sera were about 50%, of the HIV-1 RNA concentrations measured in paired plasma samples. However, there was a strong correlation between these two measurements (P<0.0001). The assay was used to measure viral RNA in the plasma of 50 HIV-1 positive individuals at different stages of infection. All the individuals had detectable HIV-I RNA (300-957 000 copies/ml). There was no correlation between HIV-I RNA and Immune Complex Dissociated (ICD) p24 antigen, but HIV-I RNA was correlated with CD4+cell counts (P<0.0001) and the clinical stage (P=0.0042), with higher HIV-1 RNA concentrations in patients with a more advanced stage of the disease. The significant association of HIV-1 RNA with major markers of HIV infection and the reliability of this sensitive, easy-to-use RT-PCR assay indicate its suitability for use in clinical trials and suggest that this assay is appropriate for routine clinical applications.