Detection and quantification of the K103N mutation in HIV reverse transcriptase by pyrosequencing

Prolonged treatment of human immunodeficiency virus (HIV)-infected patients with nonnucleoside reverse transcriptase inhibitors (NNRTIs) might result in the selection of resistant mutants, the most frequent being the K103N mutation in reverse transcriptase. Resistance mutations are routinely detected by Sanger sequencing of the whole viral population, which does not detect sequence variants with frequencies below 20%. We have developed a pyrosequencing approach for the analysis of codon 103 of the HIV reverse transcriptase gene in the circulating viral population that detects variants below the limit of conventional sequencing. The method was tested with samples from 5 controls (not exposed to NNRTIs), 6 from patients exposed to NNRTIs and having a K103N mutant virus population detected by conventional sequencing, and 9 from patients previously exposed to NNRTIs that had a wild-type virus population by conventional sequencing. In 7 of 9, samples the mutation could not be detected by either the standard assay or pyrosequencing, while in 2 samples persistence of the mutation could be detected by pyrosequencing. The method might be of practical use in detecting minority variants of HIV in the clinical setting, in epidemiological studies with large numbers of samples, or as a complement to more complex approaches.