clotet.qxp 4/2/09 11:38 am Page 57
HIV Drug Resistance Testing
Methods to determine viral tropism include recombinant phenotypic allow tropism determinations in subjects with undetectable viraemia
tests, such as the Monogram Trofile™ assay, as well as genotype- who might require switching to a CCR-5 antagonist for toxicity or
based predictors, heteroduplex tracking assays and flow-cytometry- tolerability reasons.
based methods. Currently, the best evidence supports the use of
phenotypic methods, although other methods of screening for HIV co- An alternative in-house phenotypic method to determine co-receptor
receptor usage prior to the administration of CCR-5 antagonists may tropism is co-cultivation of patient-derived peripheral blood
reduce costs and increase turn-around time over phenotypic methods. mononuclear cells (PBMCs) with MT2 cells. The MT2 cells are human
HTLV-1-transformed lymphoblasts isolated from cord blood from
Phenotypic Tropism Assays subjects with human T-cell leukaemia and co-cultured with cells from
Phenotypic tropism assays require the amplification of env sequences patients with adult T-cell leukaemia. The MT2 cells are CD4+ and have
from plasma HIV-1 RNA and the construction of viral pseudotypes or the CXCR-4 co-receptor but lack CCR-5, being susceptible to infection
infectious recombinant viruses that express the patient-derived env by X4 HIV-1. Infection with HIV is cytopathogenic on MT2 cells, being
sequences along with a reporter gene.
11
These pseudotyped viruses or detected by the induction of syncytia. Direct co-cultivation of IL-2 and
viral recombinants are then inoculated onto cells that express CD4 phytohaemagglutinin (PHA)-stimulated patient-derived PBMCs with
along with CCR-5 or CXCR-4. Co-receptor tropism is determined by MT2 cells is considered the most sensitive non-commercial phenotypic
measuring the abilities of these pseudovirus populations to efficiently method to detect X4 viruses. Overall, direct comparisons have shown
infect CD4+/U87 cells expressing either the CXCR-4 or CCR-5 co- equivalent results between this method and the earlier version of the
receptor. Viruses exclusively and efficiently infecting Trofile assay; comparisons with the enhanced sensitivity Trofile are
CXCR4+/CD4+/U87 cells are designated X4-tropic. Conversely, viruses under way. The turn-around time of MT2 assays is about three to four
exclusively and efficiently infecting CCR5+/CD4+/U87 cells are weeks. The main limitation of the MT2 assays is that infectious viruses
designated R5-tropic. Viruses capable of infecting CXCR4+/CD4+/U87
and CCR5+/CD4+/U87 cells are designated dual/mixed-tropic. HIV-1
isolates that use CC-R5 exclusively are termed R5 viruses, those that
An alternative in-house phenotypic
use only CXCR-4 are termed X4 viruses, and those that use both are
termed R5/X4 or dual-tropic viruses. Because these assays do not
method to determine co-receptor
distinguish between the presence of truly dual-tropic viruses and a
tropism is co-cultivation of patient-
mixture of R5 and X4 viruses, samples that can infect both CCR-5- and
CXCR-4-expressing cells are often termed dual–mixed viruses.
1
derived peripheral blood mononuclear
cells with MT2 cells.
Tropism testing generally requires a plasma sample with an HIV-1 level
of ≥1,000 copies/ml. The assay used in most clinical trials of CCR-5
antagonists is the Trofile™ assay (Monogram Biosciences). This is the may be difficult to obtain from stored frozen PBMCs and, possibly,
only clinically validated assay to identify tropism and is considered the may be more difficult in subjects with prolonged undetectable
current gold standard. All large CCR-5 antagonist clinical trials were viraemia. Conversely, this technology is cheaper than commercial
performed using the older version of this assay, which was able to phenotypic assays and can be performed in most HIV laboratories with
detect CXCR-4-using viruses when they constituted at least 5–10% of a relatively high throughput. Because the source of virus is PBMCs
the virus population.
11
Recent technical improvements allow the new instead of plasma, MT2 assays can be performed in subjects with
version of Trofile (also known as the Enhanced Sensitivity [ES]-Trofile undetectable viraemia, but there is little information on the clinical
assay) to detect down to 0.3% CXCR-4-using or dual-mixed virus.
12
value of MT2 assays in these subjects. Also, it might be difficult to
The enhanced sensitivity of the Trofile assay has important implications recover infectious viruses in more than 60–70% of aviremic subjects.
for the clinical management of HIV-infected subjects. A re-analysis of
baseline plasma specimens of the vicriviroc ACTG A5211 study
13
and Genotypic Tropism Assays
the maraviroc MOTIVATE 1 and 2
14,15
and MERIT
16
trials showed that, The main genetic determinant of co-receptor tropism in the HIV
respectively, roughly 10, 8 and 15% of subjects who had been envelope is the V3-loop region in gp120. However, changes in this
classified as harbouring R5-using HIV-1 actually had X4 or dual–mixed region alone are not always necessary or sufficient to confer a particular
viruses.
17
The higher sensitivity of the ES-Trofile assay narrows the phenotype in viruses expressing engineered gp120 proteins, since
number of patients to whom CCR5 antagonists can be prescribed, but changes in other regions of gp120, particularly in V1/V2
18-22
and C4,
23
improves the outcome of CCR-5 antagonist therapy. Using the first have been shown to influence phenotype either alone or in conjunction
version of the Trofile assay to evaluate viral tropism, the MERIT trial with V3. In addition, isolates with identical V3 sequences can have
found maraviroc to be inferior to efavirenz, both with an AZT/3TC dissimilar patterns of co-receptor usage, cell tropism or replication
backbone, as initial therapy for drug-naïve individuals.
16
A subsequent capacity.
20,21,24,25
Typically, specific changes in these other regions do
re-analysis of the MERIT trial showed that if participants had been not have consistent effects in a wide range of sequence contexts.
screened for X4-using viruses with the enhanced assay, more subjects
harbouring X4 viruses would have been excluded, but both arms Genotypic approaches to determining co-receptor use are thus based on
would likely have reached comparable virological outcomes.
17
The amplifying the envelope gene and sequencing the V3 loop and,
main limitation of the Trofile assay is its cost and the need to ship sometimes, additional regions such as V2. Different algorithms and
patient samples to a central laboratory in San Francisco, US, which interpretation rules that attempt to infer a co-receptor tropism
slows the turn-around time. Also, the current Trofile assay does not phenotype from the genotypic information provided are available online.
EUROPEAN INFECTIOUS DISEASE 57
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