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


CONCLUSIONS
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.
Louvel
S
S
Institute for Medical Microbiology, University of Basel, Basel, Switzerland. severine.louvel@unibas.ch
Battegay
M
M
Vernazza
P
P
Bregenzer
T
T
Klimkait
T
T
Hamy
F
F
D000483:Alleles; D019380:Anti-HIV Agents / Q000627:therapeutic use*; D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D015658:HIV Infections / Q000188:drug therapy* / Q000235:genetics; D006801:Humans; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017211:Treatment Failure
D019380:Anti-HIV Agents