Monitoring resistance to human immunodeficiency virus type 1 protease inhibitors by pyrosequencing

The emergence of drug-resistant viral variants is the inevitable consequenc e of incomplete suppression of human immunodeficiency virus type 1 (HIV-1) replication during treatment with antiretroviral drugs. Sequencing to deter mine the resistance profiles of these variants has become increasingly impo rtant in the clinical management of HIV-1 patients, both in the initial des ign of a therapeutic plan and in selecting a salvage regimen. Here we have developed a pyrosequencing assay for the rapid characterization of resistan ce to HIV-1 protease inhibitors (PIs). Twelve pyrosequencing primers were d esigned and were evaluated on the MN strain and on viral DNA from periphera l blood mononuclear cells from eight untreated HN-l infected individuals. T he method had a limit of detection of 20 to 25% for minor sequence variants . Pattern recognition (i.e., comparing actual sequence data with expected w ild-type and mutant sequence patterns) simplified the identification of min or sequence variants. This real-time pyrosequencing method was applied in a longitudinal study monitoring the development of PI resistance in plasma s amples obtained from four patients over a 2 1/2-year period. Pyrosequencing identified eight primary PI resistance mutations as well as several second ary mutations. This sequencing approach allows parallel analysis of 96 reac tions in 1 h, facilitating the monitoring of drug resistance in eight patie nts simultaneously and, in combination with viral load analysis, should be a useful tool in the future to monitor HIV-1 during therapy.