14th International HIV Drug Resistance Workshop 7-11 June 2005, Québec City, Canada

RESISTANCE MUTATIONS ARISE IN THE MAJORITY OF WOMEN PROVIDED SINGLE-DOSE NVP AND APPEAR TO DIFFER IN EMERGENCE AND PERSISTENCE

Antivir Ther. 10, Suppl 1:S13 (abstract no. 11)

JA Johnson¹, J-F Li¹, L Morris², N Martinson³, G Gray⁴, J McIntyre⁴ and W Heneine^1
1National Center for HIV, STD, and Tuberculosis Prevention, Centers for Disease Control and Prevention, Atlanta, GA, USA; ²National Institute for Communicable Diseases, Johannesburg, South Africa; ³Johns Hopkins University, Baltimore, MD, USA; ⁴Perinatal HIV Research Unit, University of Witwatersrand, Johannesburg, South Africa

BACKGROUND: Conventional sequence testing has shown that approximately 40% of women who receive peripartum single-dose nevirapine (SD-NVP) generate virus with resistance mutations to non-nucleoside reverse transcriptase inhibitors. However, the sensitivity limitation of sequencing prevents reliable detection of variants that comprise less than 20% of the sample virus population. Therefore, there is a need for sensitive assays that improve detection of drug-resistant virus emergence and persistence to better assess treatment implications.

METHODS: To identify low-frequency resistant variants, we developed HIV-1 subtype C real-time PCR assays for two common NVP mutations, K103N and Y181C. Assay evaluations of mutant plasmids in wildtype backgrounds established detection limits of 0.2% for K103N and 0.3% for Y181C, 40–100-times more sensitive than population-based sequencing. The realtime assays were used to reassess the emergence of drug resistance in women who received SD-NVP. We analysed genotyped matched pairs of pre-NVP and post-NVP plasma, collected 6–36 weeks postpartum, from 58 South African women. Of the post-NVP specimens, 40 had no detectable mutations, 16 had K103N, and 12 had Y181C by population-based sequencing. None of the pre-NVP specimens had evidence of resistance by sequencing.

RESULTS: Using real-time PCR analyses, neither K103N nor Y181C were detected in pre-treatment samples. The assays successfully identified the 16 post- NVP samples with sequence-detectable K103N and the 12 samples with Y181C. Of the post-NVP specimens negative for mutations by sequencing, real-time PCR testing found 16/40 (40%) were positive for K103N at 6–36 weeks post-exposure. Additionally, testing for Y181C found 3/40 (8%) samples were positive at 6–12 weeks postpartum. The K103N frequency appeared to peak ~3 months post-NVP. Clonal sequencing confirmed resistance mutations in all representative samples.

CONCLUSIONS: The finding of K103N and Y181C in an additional 43% of women with previously undetectable resistance suggests that resistant viruses emerge in the majority of women receiving SD-NVP. The disproportionately high numbers of K103N and its detection up to 36 weeks post-NVP suggests that in adults with HIV-1 subtype C this mutation has a selective advantage over Y181C and persists longer. These data emphasize the importance of sensitive assays to better assess the clinical implications of drug-resistant variants.

PRESENTING AUTHOR: JA Johnson

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2005-06-07
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