Monitoring resistance to human immunodeficiency virus type 1 protease inhibitors by pyrosequencing.
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.
O'Meara
D
D
Department of Biotechnology, Royal Institute of Technology (KTH), S-100 44 Stockholm, Sweden.
Wilbe
K
K
Leitner
T
T
Hejdeman
B
B
Albert
J
J
Lundeberg
J
J
D000163:Acquired Immunodeficiency Syndrome / Q000188:drug therapy*; D000328:Adult; D023241:Antiretroviral Therapy, Highly Active; D001483:Base Sequence; D003062:Codon; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004352:Drug Resistance, Microbial; D005260:Female; D005500:Follow-Up Studies; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D016333:HIV Protease / Q000235:genetics*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D016879:Salvage Therapy; D013997:Time Factors; D014779:Virus Replication / Q000187:drug effects*
D003062:Codon; D017931:DNA Primers; D004279:DNA, Viral; D017320:HIV Protease Inhibitors; D016333:HIV Protease