Phenotypic Assays

In the past, phenotypic assays required the isolation and preparation of high-titer stocks of virus from patient samples. This procedure was laborious and time-consuming, requiring 6 to 8 weeks to generate a result. Because primary clinical isolates grow best in peripheral blood mononuclear cells (PBMC), these assays made use of activated PBMC from seronegative donors. However, PBMC from different donors vary in their ability to support the growth of HIV-1, leading to significant interassay variation (5). For this reason, several groups have developed recombinant virus assays, in which the viral genes of interest (e.g., PR and RT) are introduced into a plasmid that carries all of the other viral genes needed for replication in cell culture (6; 7).

As with genotypic assays, the first step in a phenotypic assay involves extraction of HIV-1 RNA from plasma, followed by reverse transcription and PCR amplification of the PR and RT genes. These amplified genes are then inserted into vectors used to generate recombinant viruses that can be tested for susceptibility to protease and RT inhibitors. Because the viruses are each identical except for the protease and RT sequences, most of the interassay variation observed in older PBMC-based assays is eliminated, allowing for very precise determination of the fold-resistance of a particular viral isolate. As a result, differences in susceptibility of 2.5- to 4-fold compared to control usually are considered to be significant in the currently available assays.

Phenotypic assays are more complex and labor intensive than genotypic assays. Automation of these assays makes it possible to test many samples simultaneously, and allows for high through-put. However, the complexity of the automation limits the availability of these assays to only a few laboratories. at present phenotyping by recombinant virus assay is available from two sources: Virco (AntiVirogram; Mechelen, Belgium) and ViroLogic (PhenoSense; South San Francisco, CA). It is unlikely that these assays will ever be produced as "kits" that can be used to perform phenotypic tests at local hospital laboratories.

4/15/01

Copyright 2001 by HIV and Hepatitis.com. All Rights Reserved