Comparison of LigAmp and an ASPCR assay for detection and quantification of K103N-containing HIV variants.
We compared the ability of the LigAmp assay and an ASPCR assay to detect and quantify K103N-containing HIV variants in samples from 63 women who received single-dose nevirapine in a clinical trial. Samples were first analyzed with the ViroSeq HIV Genotyping system, and ViroSeq PCR products were used as templates for the LigAmp and ASPCR assays. A cutoff of 0.5% K103N for detection of K103N was used for both assays. Results for the percentage K103N were similar for the two assays (R(2) = 0.92). Forty-six samples (73.0%) were positive for K103N by both assays and 13 samples (20.6%) were negative by both assays. Four samples (6.3%) were positive by ASPCR only. No samples were positive by LigAmp only. Eight discordant samples were analyzed in more detail. Sequence polymorphisms near oligonucleotide binding sites provided a possible explanation for the discordance in four of eight samples. The percentage K103N was also determined by analyzing 40 HIV clones from each of these eight samples, using a combined amplification/sequencing method (AmpliSeq). The percentage K103N determined by clonal analysis was consistent with the LigAmp result for five of eight samples, and was consistent with the ASPCR result for three of eight samples. Among 320 clones analyzed, we identified eight different codons at position 103 (mean = 3.8 codons/sample), which encoded six different amino acids, illustrating the extensive genetic diversity in HIV. Further studies are needed to compare performance of assays for detection and quantification of HIV drug resistance mutations in clinical samples.
Church
Jessica D
JD
Johns Hopkins University, Baltimore, Maryland 21205, USA.
Towler
William I
WI
Hoover
Donald R
DR
Hudelson
Sarah E
SE
Kumwenda
Newton
N
Taha
Taha E
TE
Eshleman
James R
JR
Eshleman
Susan H
SH
D019380:Anti-HIV Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001483:Base Sequence; D024882:Drug Resistance, Viral / Q000235:genetics; D005260:Female; D015751:Genes, pol; D014644:Genetic Variation; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D019829:Nevirapine / Q000494:pharmacology* / Q000627:therapeutic use; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D054302:pol Gene Products, Human Immunodeficiency Virus / Q000235:genetics
D019380:Anti-HIV Agents; D054302:pol Gene Products, Human Immunodeficiency Virus; D019829:Nevirapine