Objectives:We attempted to define optimal conditions for amplification of low copy number HIV-1 RNA sequences in plasma samples, applying improved conditions for nucleic acid extraction and amplification.Methods:Several methodologic parameters were evaluated, including methods of RNA extraction, volumes of plasma samples, proportion of extracted RNA used as a template for amplification, and reverse transcriptase-DNA polymerase enzyme combination employed in cDNA synthesis and polymerase chain reaction amplification.Results:With this improved assay, we were able to obtain sufficient amounts of amplified material for direct sequencing in 97% of all plasma samples in our study, including 88% of samples with viral loads <80 copies/mL, 78% of samples with viral loads <50 copies/mL, and even 2 (67%) of 3 samples with <20 copies/mL.Conclusions:This procedure could be useful for testing resistance mutations in patients undergoing highly active antiretroviral therapy, in which the viral load is commonly <400 copies/mL, and even if it is <20 RNA copies/mL.Journal of Human Virology 2000;3:27-34 © Lippincott Williams & Wilkins, Inc.