HIV Resistance Workshop 2003: advances in diagnostic techniques

Yasmin Halima
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This article is more than 21 years old.

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.

Glossary

assay

A test used to measure something.

capacity

In discussions of consent for medical treatment, the ability of a person to make a decision for themselves and understand its implications. Young children, people who are unconscious and some people with mental health problems may lack capacity. In the context of health services, the staff and resources that are available for patient care.

replication

The process of viral multiplication or reproduction. Viruses cannot replicate without the machinery and metabolism of cells (human cells, in the case of HIV), which is why viruses infect cells.

protease inhibitor (PI)

Family of antiretrovirals which target the protease enzyme. Includes amprenavir, indinavir, lopinavir, ritonavir, saquinavir, nelfinavir, and atazanavir.

genome

The complete set of genes or genetic material (information) present in a cell or organism.

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

References

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.

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