[13] INCREASED SENSITIVITY OF DETECTION OF K103N RESISTANCE VARIANTS BY REAL-TIME PCR IN RNA AND DNA AFTER SINGLE-DOSE NEVIRAPINE

14th International HIV Drug Resistance Workshop

7-11 June 2005, Québec City, Canada

INCREASED SENSITIVITY OF DETECTION OF K103N RESISTANCE VARIANTS BY REAL-TIME PCR IN RNA AND DNA AFTER SINGLE-DOSE NEVIRAPINE

Antivir Ther. 10, Suppl 1:S15 (abstract no. 13)

S Loubser¹, P Balfe², G Sherman³, S Hammer², L Kuhn² and L Morris¹
¹AIDS Virus Research Unit, National Institute for Communicable Diseases, Johannesburg, South Africa; ²Division of Infectious Diseases, Columbia University, NY, USA; ³Department of Molecular Medicine and Haematology, National Health Laboratory Service and the University of the Witwatersrand, Johannesburg, South Africa

BACKGROUND: Nevirapine resistance mutations (NVPR) are selected following single-dose treatment (sdNVP) in women (22–75% 1–2 weeks; 15–48% 6–8 weeks) and babies (39–53% 4–8 weeks). Standard genotyping fails to detect mixtures below ~20% suggesting that NVPR is substantially underestimated. Real-time PCR methods improve sensitivity of detection and quantification of minority populations. The most common high-level NVPR selected in women following sdNVP is K103N in HIV-1 reverse transcriptase (RT) which confers cross-resistance to other NNRTI’s and can persist for many months. We have developed a real-time PCR assay for the detection and semi-quantification of K103N in RNA and DNA collected 6–48 weeks post-sdNVP from women enrolled in a pMTCT program.

METHOD: Viral RNA and DNA was used to amplify RT sequences by nested (RT)-PCR. Diluted amplicons were used for real-time PCR detection of wild-type (K, lysine, AAA, AAG) and mutant codons (N, asparagine, AAC, AAT) at position 103. Threshold cycles (Ct) were used to derive relative K103N frequencies. Increased sensitivity of real-time PCR detection was assessed by comparison with samples previously genotyped (“in-house”).

RESULTS: K103N was identified in 52% (n=31) of RNA samples previously genotyped and in 87% by real-time PCR (sensitivity=0.2%). Genotyping failed to detect mutants below ~10%. Cross-sectional analysis of RNA collected 6–48 weeks post-sdNVP showed 87% (n=32) had K103N at 6 weeks, which declined to 67% (n=27), 35% (n=37) and 13% (n=54) at 12, 21 and 48 weeks respectively. In DNA collected at 6 and 48 weeks post-sdNVP, 53% (n=43) had K103N at 6 weeks and 4.2% (n=48) at 48 weeks. Longitudinal analysis of RNA 6–48 weeks post-sdNVP for 16 women showed similar decay rates but higher levels of K103N persisted longer.

CONCLUSION: Real-time PCR detection of K103N was more sensitive than genotyping. An additional 73% (n=15) of RNA samples previously identified as wild-type by genotyping had detectable K103N. Cross-sectional analysis showed that K103N was detectable in RNA in the majority of women 6 weeks post-sdNVP which declined over time and was present in a minority at 48 weeks. In DNA, K103N was detectable in ~50% of samples at 6 weeks post-sdNVP and was essentially absent at 48 weeks.

PRESENTING AUTHOR: S Loubser

Download PDF of this abstract.

2005-06-07
13

Copyright © 2005 - International Medical Press Ltd. Reproduction of this abstract (other than one copy for personal reference) must be cleared through the International Medical Press Ltd. 2-4 Idol Lane, London EC3R 5DD UK.