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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
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by HIV and Hepatitis.com. All Rights Reserved
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