Detection of drug‐resistant HIV minorities in clinical specimens and therapy failure

• Published in: HIV medicine Vol. 9; no. 3; pp. 133 - 141
• Main Authors: Louvel, S, Battegay, M, Vernazza, P, Bregenzer, T, Klimkait, T, Hamy, F
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.03.2008
• Subjects: Alleles; allele‐specific PCR; Anti-HIV Agents - therapeutic use; Drug Resistance, Viral - genetics; Genotype; HIV Infections - drug therapy; HIV Infections - genetics; Humans; minor viral population; Mutation - genetics; Phenotype; real‐time PCR; replicative phenotyping; Reverse Transcriptase Polymerase Chain Reaction - methods; Treatment Failure; viral fitness
• ISSN: 1464-2662; 1468-1293
• DOI: 10.1111/j.1468-1293.2007.00529.x

Objective Particularly for therapy‐experienced patients, resistance assessment by genotypic or phenotypic methods produces discordances. This study seeks proof that differences may arise from the fact that genotyping produces a single summary sequence whereas replicative phenotyping (rPhenotyping) functionally detects and assigns resistances in mixed HIV populations. Methods For validation, defined mixes of wild‐type and M184V mutant were analysed by rPhenotyping or standard genotyping. Allele‐specific and quantitative polymerase chain reaction (PCR) set detection and quantification limits for minor virus populations in vitro and in authentic clinical samples showing geno‐/pheno‐discrepant lamivudine resistance. Results Allele‐specific and real‐time PCR methods detected down to 0.3% of mutant M184V. The functional assessment was sensitive enough to reveal <1% of mutant M184V in mixed samples. Also in discordant samples from the diagnostic routine, in which rPhenotyping had identified drug resistance, real‐time PCR confirmed minute amounts of mutant M184V. Conclusion By utilizing the replication dynamics of HIV under drug pressure, a rPhenotyping format potently reveals relevant therapy‐resistant minority species, even of HIV known to possess reduced replicative fitness. With its rapid turnaround of 8 days and its high sensitivity, our rPhenotyping system may be a valuable diagnostic tool for detecting the early emergence of therapy‐threatening HIV minorities or the persistence of residual resistant virus.