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Antimicrobial Agents and Chemotherapy, July 2009, p. 2965-2973, Vol. 53, No. 7
0066-4804/09/$08.00+0 doi:10.1128/AAC.01672-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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
Andrea Hauser,1,2
Kizito Mugenyi,3
Rose Kabasinguzi,3
Kerstin Bluethgen,2
Claudia Kuecherer,2
Gundel Harms,1 and
Andrea Kunz1*
Institute of Tropical Medicine and International Health, Charité-University Medicine Berlin, Berlin, Germany,1 Robert Koch Institute, Berlin, Germany,2 MoH/GTZ PMTCT Project Western Uganda, Fort Portal, Uganda3
Received 19 December 2008/ Returned for modification 12 February 2009/ Accepted 30 April 2009
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.
* Corresponding author. Mailing address: Institute of Tropical Medicine and International Health, Charité-University Medicine Berlin, Spandauer Damm 130, 14050 Berlin, Germany. Phone: 49-30-30116 700. Fax: 49-30-30116 710. E-mail: andrea-ursula.kunz{at}charite.de
Published ahead of print on 11 May 2009.
Antimicrobial Agents and Chemotherapy, July 2009, p. 2965-2973, Vol. 53, No. 7
0066-4804/09/$08.00+0 doi:10.1128/AAC.01672-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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