Detection and quantification of minor human immunodeficiency virus type 1 variants harboring K103N and Y181C resistance mutations in subtype A and D isolates by allele-specific real-time PCR.
Nevirapine (single dose), commonly used to prevent the mother-to-child transmission of human immunodeficiency virus (HIV) in developing countries, frequently induces viral resistance. Even mutations which occur only in a minor population of the HIV quasispecies (<20%) are associated with subsequent treatment failure but cannot be detected by population-based sequencing. We developed sensitive allele-specific real-time PCR (ASPCR) assays for two key resistance mutations of nevirapine. The assays were specifically designed to analyze HIV-1 subtype A and D isolates accounting for the majority of HIV infections in Uganda. Assays were evaluated using DNA standards and clinical samples of Ugandan women having preventively taken single-dose nevirapine. Lower detection limits of drug-resistant HIV type 1 (HIV-1) variants carrying reverse transcriptase mutations were 0.019% (K103N [AAC]), 0.013% (K103N [AAT]), and 0.29% (Y181C [TGT]), respectively. Accuracy and precision were high, with coefficients of variation (the standard ratio divided by the mean) of 0.02 to 0.15 for intra-assay variability and those of 0.07 to 0.15 (K103N) and 0.28 to 0.52 (Y181C) for inter-assay variability. ASPCR assays enabled the additional identification of 12 (20%) minor drug-resistant HIV variants in the 20 clinical Ugandan samples (3 mutation analyses per patient; 60 analyses in total) which were not detectable by population-based sequencing. The individual patient cutoff derived from the clinical baseline sample was more appropriate than the standard-based cutoff from cloned DNA. The latter is a suitable alternative since the presence/absence of drug-resistant HIV-1 strains was concordantly identified in 92% (55/60) of the analyses. These assays are useful to monitor the emergence and persistence of drug-resistant HIV-1 variants in subjects infected with HIV-1 subtypes A and D.
Institute of Tropical Medicine and International Health, Charité-University Medicine Berlin, Spandauer Damm 130, Berlin, Germany.
Andrea
A
Hauser
Kizito
K
Mugenyi
Rose
R
Kabasinguzi
Kerstin
K
Bluethgen
Claudia
C
Kuecherer
Gundel
G
Harms
Andrea
A
Kunz
D019380:Anti-HIV Agents / Q000494:pharmacology* / Q000627:therapeutic use; D024882:Drug Resistance, Viral / Q000235:genetics*; D005260:Female; D015658:HIV Infections / Q000188:drug therapy; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D009154:Mutation; D019829:Nevirapine / Q000494:pharmacology* / Q000627:therapeutic use; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
D019380:Anti-HIV Agents; D019829:Nevirapine