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J Clin Microbiol ; 39(2): 464-73, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11158091

ABSTRACT

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 HIV-1-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.


Subject(s)
Acquired Immunodeficiency Syndrome/drug therapy , Drug Resistance, Microbial , HIV Infections/virology , HIV Protease Inhibitors/pharmacology , HIV Protease/genetics , HIV-1/drug effects , HIV-1/genetics , Polymorphism, Genetic , Virus Replication/drug effects , Adult , Antiretroviral Therapy, Highly Active , Base Sequence , Codon , DNA Primers , DNA, Viral/genetics , DNA, Viral/isolation & purification , Female , Follow-Up Studies , HIV Infections/drug therapy , HIV-1/physiology , Humans , Male , Middle Aged , Polymerase Chain Reaction/methods , Salvage Therapy , Time Factors
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