Principle
of the PHENOSCRIPT™ assay
The PHENOSCRIPT™ report
Important parameters
Comparison with other commercially available tests
PHENOSCRIPT™ is a novel and rapid HIV phenotypic test used to measure
resistance to antiretroviral drugs used in HIV therapy.
More precisely, PHENOSCRIPT™ is an in vitro single cycle recombinant
virus assay which provides a direct measure of the ability of virus derived
from the patient to replicate in the presence of drug. This assay is
able to test the susceptibility of the virus to all antiretroviral molecules
approved to date – protease inhibitors, reverse transcriptase inhibitors
and entry inhibitors – and for several molecules under development.
The technique is particularly rapid as the test needs only five days
in the laboratory after the PCR amplification of the viral sequences
(which requires two days). The test is based on a single cycle of viral
replication making it rapid and ensuring an accurate reflection of the
patient’s plasma virus population. The separate amplification of
the protease and the reverse transcriptase (rather than amplification
of one long fragment) enhances the sensitivity of the test (500 copies/
ml).
PHENOSCRIPT™ has been compared to other HIV phenotypic resistance
tests available by the EuroGuidelines group (5th International Workshop
on Drug Resistance and Treatment Strategies). Excellent correlation between
the tests was observed.
PHENOSCRIPT™ can be used for:
 Aiding the selection of optimal therapy for HIV infected individuals.
Pre-clinical and clinical monitoring of resistance and cross-resistance.
Screening of new compounds against wild-type and multi-drug resistant
viruses.
Study of the replication capacity of the virus.
Evaluating the neutralising antibody responses to potential vaccine
products.
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Principle of the PHENOSCRIPT™ assay
Three steps are involved in the assay:
Isolation and amplification of viral sequences involved in drug resistance
(Figure 1).
Single cycle recombinant viral assay (scRVA) (Figure 2).
Data interpretation (Figure 3).
Plasma is obtained from the patient's blood sample (HIV RNA >500 copies/ml),
viral RNA is extracted and three regions – gag-protease (GP), reverse-transcriptase
(RT) and envelope (ENV) – are separately amplified to test protease
inhibitors (PIs), reverse transcriptase inhibitors (RTIs) and entry inhibitors
(EI) respectively. The amplification is performed separately for each
region in order to increase the sensitivity of the PCR (Figure 1). Different
primer combinations are possible to optimise the amplification of highly
mutated specimens and different subtypes.
Figure 1: Viral RNA extraction and amplification of regions involved
in drug resistance
Each PCR product is then separately co-transfected into
producer cells along with the corresponding PHENOSCRIPT™ plasmid.
For the PI and RTI assays, the single cycle of infection is ensured
by the deletion of the envelope encoding region of the HIV plasmid.
The envelope of the recombinant virus is provided by the G protein
of the Vesicular Stomitis virus (VSV-G protein), for which the genetic
information is carried on a separate plasmid.
For the measurement of susceptibility to PIs, the transfected producer
cells are treated with serial dilutions of drugs and the resulting
recombinant viruses are then used to infect indicator cells (Figure
2).
For the measurement of susceptibility to RTIs, indicator cells are
pre-treated with serial dilutions of drugs four hours before infection
with the recombinant viruses harvested from the producer cells (Figure
2).
For the measurement of susceptibility to EIs, indicator cells expressing
CCR5 or CXCR4 co-receptors are treated with serial dilutions of drugs
and infected with recombinant viruses harvested from the producer cells
(Figure 2).
The indicator cells contain the LacZ gene under HIV-1 LTR control.
When the cells are infected the production of ß-galactosidase
is detected using a CPRG based colorimetric assay and measured by Optical
density (Figure 2).
Figure 2: Principle of the single cycle recombinant assay (5 days)
Optical density readings are used to calculate the percent
reduction in infectious virus relative to the drug free control at
each drug concentration tested. Inhibitory concentrations (ICs, 90%
for PIs and 50% for RTIs and EIs) are interpolated from a plot of log
drug concentration versus percent reduction in infectious virus using
software developed in house (ODALIS®).
The results are reported as a resistance index (RI). This RI represents
the fold difference between the drug susceptibility of the studied
virus and the drug susceptibility of the wild type drug susceptible
control virus (NL4-3), which is run in each PHENOSCRIPT™ assay
to control the inter-assay variation. In each assay, controls derived
from cloned samples, chosen to present different resistance patterns
for each class of antiretroviral, are also systematically added to
control the inter-assay variation.
PHENOSCRIPT™ provides information about the potential
contribution of the tested drug to the overall response to a new treatment
combination. This is reported as “Likely”, “Possible”,
or “Unlikely”. The method of interpretation is based on
threshold parameters or cut-off values (Figure 3).
Figure 3: Determination of viral susceptibility
(IC, RI) to conclude
about the contribution of the tested drug to the
virologic response
:
“Likely”, “Possible” or “Unlikely”.
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The PHENOSCRIPT™ report
Figure 4: The PHENOSCRIPT™ report
The interpretation of results is based on the comparison
of the resistance index with different threshold parameters or cut-offs
as represented in Figure 3:
- Technical cut-offs are based on the reproducibility of the assay.
They represent the 95% confidence intervals of the assay for repeat
assays carried out on drug susceptible and drug resistant samples.
A RI greater than the technical cut off indicates significantly lower
drug susceptibility than the control drug-susceptible virus (NL4-3).
- Clinical cut-offs: For some drugs it has been possible to define
clinically relevant cut-offs based on an analysis of the relationship
between baseline phenotype and reduction in viral load at month 3 in
the ANRS 088 clinical trial.
The use of these cut-offs provides a more precise estimation of the
potential contribution of any one drug to the response to a multiple
component therapy. Studies are in progress to verify these cut-offs
and to define cut-offs for other drugs.
NB Kaletra and Viread’s cut offs are those published
by the manufacturers.
- Treatment Naive Range (TNR): For those drugs where a clinical cut-off
has not yet been determined, biological cut-offs have been established:
The susceptibility of the virus is compared with the natural range
of susceptibilities seen for viruses from treatment naive individuals.
Interpretation of the Resistance Index value is performed according
to the following rules:
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Important parameters
Quality controls and reproducibility
Both negative and positive controls are included for all RNA extraction,
reverse transcriptase polymerase chain reaction (RT-PCR) and nested
PCR.
A wild-type drug sensitive control PCR product (NL4-3) is included
in every recombinant virus assay (RVA) and is used to evaluate the
accuracy of the assay. The geometric mean IC50 or IC90 for NL4-3 in
twenty repeat assays, multiplied by twice the standard deviation is
used to define validation limits for the test. Thus if the IC50 or
IC90 obtained for NL43 falls outside these limits for any one drug,
the assay is repeated.
The reproducibility of the RVA step of the PHENOSCRIPT™ in terms
of RI has been followed since September 2000 by the inclusion of control
sample PCR products. These controls are derived from mutated viruses
that were chosen to present different resistance patterns for each
class of drug. These results provide the assay validation limits.
The reproducibility of the PHENOSCRIPT™ assay in terms of RI
has also been tested using an in house panel of samples and one from
a national genotyping quality control study in France (The AC11 1999
CQ panel). Similar levels of reproducibility were seen with both panels.
Data available on demand.
Sensitivity
The sensitivity of the test depends on a number of factors:
The efficiency of the PCR amplification step, which is affected by
numerous factors including viral load and sequence homogeneity with
primers.
The efficiency of homologous recombination, which is affected by sequence
homogeneity of the overlapping recombination regions.
The ability to detect the recombinant virus produced, which is affected
by the infectivity of the virus, which can be significantly reduced
in multi-resistant viruses.
The results obtained with the PHENOSCRIPT™ assay so far show
a success rate of 90% of cases, regardless of viral load (a further
7.5% of cases a result was obtained for either PIs or RTIs).
Excellent results are obtained for viral loads higher than 500
copies per ml. The rate of success is reduced below this level.
Data available
on demand.
Non B sub-types
As several choices of amplification primers are available, the study
of highly mutated samples and non-B subtypes is possible. The PHENOSCRIPT™ assay
has been tested with a panel of 23 viruses of sub-types A, B, C,
D, E, F, G, H, J and some mixtures (5th International Workshop on
HIV Drug Resistance and Treatment Strategies) with a success rate
of 75% for the PI assay and 95% for the RTI assay.
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Comparison with other commercially available tests
PHENOSCRIPT™ has been compared with other commercially available
phenotypic HIV susceptibility assays in two independent studies: Prof3020
conducted by Glaxo Wellcome (Dam et al, 2001, Poster 158, 5th International
Workshop on HIV Drug Resistance and Treatment Strategies) and a comparative
study conducted by the EuroGuidelines group (Miller et al, 2001, Poster
169, 5th International Workshop on HIV Drug Resistance and Treatment
Strategies).
In the Glaxo Wellcome study PROF3020, VIRalliance and Virco
performed RVA testing on identical aliquots of plasma (360 tests).
Both laboratories
reported a success rate of 90%. For most drugs tested individually,
the correlation between log transformed RI values from the 2 assays
was good. Significant R2 values (p <0.01) were obtained for most
of the drugs.
In the EuroGuidelines study VIRalliance reported results
for all 30 samples whereas Virco and ViroLogics were unable to amplify
one
sample
each. The overall correlation between the three laboratories was
good, particularly between VIRalliance and ViroLogic.
The majority of discordant results seen were for the nucleoside RTIs,
most often with Virco giving a low RI for samples that were classified
as resistant by genotype and by the other two phenotypic tests. The
exception was 3TC where 6 samples classified as sensitive by genotype
gave a resistant phenotype in all cases with the ViroLogic assay
and in four cases with the VIRalliance assay (Figure 5). Data available
on demand.
Figure 5 : Presentation of the correlation
between the three commercially
available HIV phenotyping assays. [Miller
et al: “Comparison of HIV-1
drug susceptibility (phenotype) results
reported by three major laboratories“.
Abstract 169, Antiviral
Ther, Scottsdale, 2001; 6 (suppl 1): 129].
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