Monitoring resistance to human immunodeficiency virus type 1 protease inhibitors by pyrosequencing
2001 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 39, no 2, 464-473 p.Article in journal (Refereed) Published
The emergence of drug-resistant viral variants is the inevitable consequence of incomplete suppression of human immunodeficiency virus type 1 (HIV-1) replication during treatment with antiretroviral drugs. Sequencing to determine the resistance profiles of these variants has become increasingly important in the clinical management of HIV-1 patients, both in the initial design of a therapeutic plan and in selecting a salvage regimen. Here we have developed a pyrosequencing assay for the rapid characterization of resistance to HIV-1 protease inhibitors (PIs). Twelve pyrosequencing primers were designed and were evaluated on the MN strain and on viral DNA from peripheral blood mononuclear cells from eight untreated HN-l infected individuals. The method had a limit of detection of 20 to 25% for minor sequence variants. Pattern recognition (i.e., comparing actual sequence data with expected wild-type and mutant sequence patterns) simplified the identification of minor sequence variants. This real-time pyrosequencing method was applied in a longitudinal study monitoring the development of PI resistance in plasma samples obtained from four patients over a 2 1/2-year period. Pyrosequencing identified eight primary PI resistance mutations as well as several secondary mutations. This sequencing approach allows parallel analysis of 96 reactions in 1 h, facilitating the monitoring of drug resistance in eight patients simultaneously and, in combination with viral load analysis, should be a useful tool in the future to monitor HIV-1 during therapy.
Place, publisher, year, edition, pages
2001. Vol. 39, no 2, 464-473 p.
reverse-transcriptase, antiretroviral therapy, hiv-1 protease, drug design, plasma, infection, mutations, saquinavir, gene, dna
IdentifiersURN: urn:nbn:se:kth:diva-20333ISI: 000166776100007OAI: oai:DiVA.org:kth-20333DiVA: diva2:339028
QC 201005252010-08-102010-08-10Bibliographically approved