ORIGINAL RESEARCH Detection of drug-resistant HIV minorities in clinical

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 o1 % 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