The two exciting developments in assay technology appear to be the measure of fitness or replicative capacity, and the ability to assess more sensitively minority viral populations through single genome sequencing.
Impact of minority viral populations and resistance using different replication assay techniques
Replication capacity can be measured by assessing viral growth kinetics over time or by a competition assay that combines mixtures of mutant and WT virus. The latter provides a comparative analysis of growth between the mutant and WT virus populations.
Researchers at Abbott Laboratories constructed four mutant clones with different genotypes from dual PI experienced patients receiving lopinavir/ritonavir therapy. When the samples were transfected alone in a single cycle assay, they were able to report
Detection of minority populations using a single genome sequencing approach
Single genome sequencing (SGS) provides a more sensitive analysis of viral diversity in patients. The NIH and NIAID together with John Mellors at Pittsburgh University have developed an SGS technique using RT-PCR sequence, cDNA from plasma RNA serially diluted to a single copy and a region from p6 gag, protease and RT to codon 300 amplified and sequenced. In this study plasma samples from 24 patients on a failing regimen or known to be infected with MDR virus were analysed with 15-46 single viral genomes from each sample. Whilst all the mutations detected by the standard composite genotype were detected by the SGS technique, not all the mutations found in SGS were confirmed by standard genotypic evaluation. In one example, a patient with 10 mutations conferring resistance to PIs, NRTIs and NNRTIs escaped detection by the conventional genotype method. These mutations were present in 5-20% of the 20 genomes sampled. Mutations detected by SGS but missed by standard genotyping included:
PI mutations: L10V, M46I, I84V, l90M, I93L
NRTI mutations: D67N, T215Y
NNRTI mutations: A98S, K101E, Y181C, G190A
They conclude that conventional genotyping strategies may prove ineffective in detecting viral quasi-species present at lower levels. Surprisingly, whilst it may be expected that standard genotyping would not detect mutations present in
Kearney M et al. Comparison of single-genome sequencing with standard genotype analysis for detection of HIV-1 drug resistance mutations. Antiviral Therapy 8: S96, 2003.
Mo H et al. The impact of minor populations of wild-type HIV on the replication capacity and phenotype of mutant variants in a single-cycle HIV resistance assay. Antiviral Therapy 8: S95, 2003.