Phenotypic and Genotypic Assays for HIV-1 Drug Resistance
Oct., 1999
Genotypic and Phenotypic Assays for HIV-1 Drug Resistance
I.
Introduction:
The virus population in an HIV-1 infected individual has quasispecies nature just
like any other RNA viruses. So-called quasispecies refers to the existence of genetically
distinct viral variants that evolve from the initial virus inoculum. For retroviruses, such
as HIV-1, this is mainly due to its reverse transcriptase does not have the DNA
proofreading mechanisms and the rapid viral turnover yyy Eigen, 1993 Sci. Am:269, 4249; Drake, PNAS, 1993 90: 4171-4175; Mansky, 1995 JV, 69:5087-5094 kkk. The size
of HIV-1 genome is about 104 nucleotides, the error rate of reverse transcriptase is about
1/105 nucleotides and it is estimated that there are 109 viral particles produced per day
and the rounds of replication is about 107, therefore, about 3300 newly produced viruses
will carry a particular mutation each day yyy Coffin, 1995, Science Vol. 267: 483-489;
Kuritzkes, 1999 HIV Clinical Management, Vol.13 kkk.
Although the usage of combination therapy of potent antiretroviral drugs in HIV1 infected individuals has produced significantly reductions of HIV-1 RNA in plasma to
below detectable levels and resulted in dramatic decrease in disease progression and
mortality in the developed world yyy Palella, 1998 NEJM 338:853-860 kkk there is
increasing evidence demonstrated the failure of such potent combination therapy.
Numerous factors could contribute to the failure of antiretroviral therapy including
limited potency of the regimen, poor adherence, pharmacological factors, limited
penetration of drug into sanctuary sites, and progressive immunologic decline, but drugresistance variants may be the results of all of above and most likely to be responsible for
the failure of combination therapy yyy Hirsch, 1998, JAMA vol. 279: 1984-1991;
Kuritzkes, 1999 HIV Clinical Management, Vol.13 kkk. When antiviral drug selective
pressure is applied to viral quasispecies in an infected person, preexisting minor viral
species resistant to that drug rapidly become predominant and are selected as the most fit
species in the presence of drug. In addition, persistent viral replication permits further
viral evolution leading to high-level drug resistance by cumulative mutation acquisition
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Phenotypic and Genotypic Assays for HIV-1 Drug Resistance
Oct., 1999
yyy Coffin, 1995, Science Vol. 267: 483-489; Hirsch, 1998, JAMA vol. 279: 1984-1991
kkk.
Resistance to antiretroviral drugs is determined by mutations in the genetic
regions that encode the protease (PR) and reverse transcriptase (RT). There are two types
of mutations: primary and secondary mutations. Primary mutations alter the binding sites
of drug to its target and result in an increase in the amount of drug necessary to inhibit the
function of enzymes. Secondary mutations increase the level of resistance by improving
the fitness of viruses carrying primary mutations, such mutations have little or no effect
to resistance in the absence of the primary mutations yyy Kuritzkes, 1999 HIV Clinical
Management, Vol.13 kkk. There are also two types of resistance:
1. Genotypic resistance: a prediction of resistance based on the virus gene sequences
and a database containing the phenotypic resistance information for virus gene
sequences.
2. Phenotypic resistance: an increase in the concentration of drug required inhibiting
a virus relative to a drug sensitive reference virus.
yyy Dutko (you wrote this in the outline) kkk
II.
Why is it important to measure drug resistance?
Currently, there are very few laboratory tools that clinician could use to guide the
selection of antiretroviral therapy in treatment-experienced patients with virologic
failure. Although treatment history and presumed patterns of cross-resistance provide
some guideposts for making rational treatment decisions, history alone may be a poor
indicator of the extent of resistance in the virus population in an infected individual.
Drug resistance testing of HIV-1 could be a valuable tool in patient management in
such circumstances. As mentioned above, the quasispecies nature of HIV-1 plays a
very important role in the emerging of drug resistance virions. It is likely that the
success of combination drug therapy (such as HAART) has been determined even
before it starts. Performing drug resistance tests up-front may provide the most
valuable information to maximize the effect of any combination therapy yyy Perrin,
1998 Science Vol. 280:1871-1873 kkk. Therefore, the significance of drug resistance
tests including the choice of initial treatment regimen, explaining and managing
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Phenotypic and Genotypic Assays for HIV-1 Drug Resistance
Oct., 1999
treatment failure, tracking the prevalence of drug resistance in HIV-1 primary
infection, and guiding the choice of salvage therapy in the treatment failure patients
yyy Kuritzkes, 1999 HIV Clinical Management, Vol.13 kkk. Recent advances in
drug susceptibility testing and preliminary data from pilot studies provide
encouraging results that may speed the implementation of these assays yyy Durant,
1999 The Lancet Vol. 353: 2195-2199; Saag, 1999 39th ICAAC kkk.
III.
How is resistance measured?
1. Genotypic assays for drug resistance: to sequence the HIV-1 protease and reverse
transcriptase genes and to predict the potential drug resistance based on the deduced
amino acid sequences of these proteins. Such assays are depend on the amplification
of the interested genes from HIV-1 RNA in plasma and previously demonstrated data
to formulate any prediction.
2. Phenotypic assays for drug resistance: to test the susceptibility of HIV-1 to inhibition
by a particular drug. Drug susceptibility is defined by determining the amount of
drug required to inhibit
virus production in vitro
by
50%
(IC50),
90%
(IC90), or 95% (IC95),
most commonly used is
IC50. Figure 1 shows a
drug susceptibility profile
difference between drug
sensitive and resistance
patients (adapted from
brochure of PhenoSense, ViroLogic Inc.).
3. Concordance
and
discordance
between
Figure 1
genotypic
and
phenotypic
assays.
Concordance results are the ones that genotypic and phenotypic testings are in
agreement. This is heavily depending on the depth of our knowledge on how the
protease and reverse transcriptase works and all the potential interactions in the
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Phenotypic and Genotypic Assays for HIV-1 Drug Resistance
Oct., 1999
enzyme coding regions. Discordance is the phenotypic testing results are different
from the predicated results obtained from genotypic testing. Such discordance is
mainly caused by the lack of understanding of all possible mutational interactions
(primary and secondary mutations, known and unknown mutations). For example,
some naturally occurring codons within the PR or RT genes may modulate the
enzyme functions (phenotype) when known drug resistance mutations present on
these genes and produce drug sensitive virions and vise versa.
4. Advantages and disadvantages of genotypic and phenotypic assays. As shown in
figure 2 (modified from Kuritzkes, 1999 HIV Clinical Management, Vol.13), both
tests have advantages and disadvantages when come to clinical utilities.
Figure 2
IV.
Companies involved in phenotying and genotyping.
1. ViroLogic Inc.
PhenoSenseTM HIV. This assay basically performing a phenotypic assay based on the
genetic information that obtained from patient plasma samples.
Almost a
combination between genotypic and phenotypic tests, PhenoSenseTM HIV amplifies
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Phenotypic and Genotypic Assays for HIV-1 Drug Resistance
Oct., 1999
the nucleic acid from patient plasma first and then incorporates the patient-derived
segment into a resistance test vector that has an indicator gene (Luciferase). Such
vectors are then introduced into host cells through transfection, and virus particles are
collected from the transfected cells and used for infecting target cells.
Drug
susceptibility is measured by comparing luciferase activity produced in the presence
and absence of antiviral drugs. PhenoSenseTM HIV needs about 14 days to get results
which is longer than genotypic test (usually a few days) and shorter than phenotypic
test (usually 4to 6 weeks). The assay can readily perform on patients with viral load
as low as 500 copies per ml. It was estimated that over 90% of successful rate if the
patient’s viral load is 500 to 1000 copies per ml. Minority resistance species can also
be detected if it is over 10% to 40% of total viral population yyy ViroLogic Brochure
on PhenoSenseTM HIV kkk.
The latest results have shown that this assay can provide consistent and
reproducible results when assessed twice over a median of 30 days in patients on
stable antiretroviral therapy yyy Haubrich, Abstract 417, 39th ICAAC, 1999 kkk.
Another study headed by Dr. Saag of University of Alabama at Birmingham using
this assay showed that the test more accurately predicated resistance to antiretroviral
drugs than the patient’s previous track record of treatment yyy Saag, 1999 39th
ICAAC kkk.
2. Virco/Laboratory Corporation of America
Genotypic assay: VircoGENTM. Nucleic acid sequence based assay and its software is
connected to the VIRCO’s database to quickly identify the potential phenotypes of
drug resistance. The preliminary data showed that patients had sensitive viruses
determined by this assay were four times likely to respond to drug. The turn-around
time is about five to ten working days therapy yyy LabCorp/VIRCO News Releases
June, 1998 kkk.
Phenotypic assay: AntivirogramTM. In vitro cell culture based assay the turn-around
time is about three to four weeks yyy LabCorp/VIRCO News Releases June, 1998
kkk.
3.
Visible Genetics
5
Phenotypic and Genotypic Assays for HIV-1 Drug Resistance
Genotypic assay: TruGeneTM.
Oct., 1999
Nucleic acid amplification and sequence analysis
based assay in combination with Roche Amplicor extraction methods. The only
genotypic test that can analyze viral load as low as 200 copies per ml or less and still
provide useful information. The latest results showed that TruGeneTM testing has a
significant benefit on the virological response when choosing a therapeutic alternative
yyy Visible Genetics Inc., News releases, June, 1999; Durant the Lancet, 1999 Vol.
353:2195-2199 kkk.
4. Specialty Laboratories
Genotypic assay: HIV-1 GenotypRTM Plus. Nucleic acid amplification (PCR) and
sequencing analysis based assay. The turn-around time is about 7-9 days.
Phenotypic assay: a probe based technology to detect drug resistance phenotypically.
The turn-around time is about four weeks and cost ($600 per sample) less than other
phenotypic tests (average $1000 per sample) yyy Specialty Laboratories Brochure,
1999 kkk.
V.
Comparison among different assays:
1. Genotypic assays: VircoGENTM,. TruGeneTM, and GenotypRTM Plus:
All three assays cost about the same, around $500 per sample and the turn-around
time is about five to ten days. TruGeneTM is the most sensitive assay (can go below
200 copies per ml) and VircoGENTM has direct access to an extensive database for
data interpretation. GenotypRTM Plus takes about 7 to 9 days that may save a day or
two compare to the other two assays.
2. Phenotypic assays: PhenoSenseTM HIV,. AntivirogramTM, and Phenotypic assay by
Specialty Labs.:
PhenoSenseTM HIV is the fastest one (turn-around time 14 days or less compares
to four to six weeks) and has been demonstrated to be able to provide very predicative
results for selection of drug therapy.
AntivirogramTM is produced by same
manufacturer as genotypic assay VircoGENTM and both tests results could be done by
one shipment of samples and obtain complementary results from one source. The
phenotypic assay offered by Specialty Labs. is the most cost-effective one among
these three assays, $600 per sample compares to $1000 per sample.
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Phenotypic and Genotypic Assays for HIV-1 Drug Resistance
Oct., 1999
3. Genotypic vs. phenotypic assays:
Genotypic assays are less expensive, take less time and easier to perform, and
offer results faster. But such assays need expert interpretation of the results and it
still is difficult to predict the consequences of mutational interactions on the
phenotype. Although it takes longer and more difficult and expensive to run a
phenotypic test, phenotypic assays can provide susceptibility data even the genetic
basis of a particular resistance has not been identified or understood. Importantly,
phenotypic assay measures the net effect of the sum of different mutations and crossresistance even some of the interactions are poorly understood.
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Phenotypic and Genotypic Assays for HIV-1 Drug Resistance
VI.
Oct., 1999
Drug resistance categories
There are three types of drug resistance: to PIs (protease inhibitors), to NNRTIs
(non-nucleoside reverse transcriptase inhibitors), and to NRTIs (nucleoside reverse
transcriptase inhibitors). Figure 3 shows the key codons for all three types of drug
resistance.
Figure 3
Figure 3 Shows the most common HIV-1 mutations selected by PIs (A), NRTIs (B), and
NNRTIs (C). For each amino acid residue listed, the letter above the listing indicates the wild
type (drug sensitive) amino acid in that position and the italicized letter below the residue
indicates the substitution that confers drug resistance. The black bars represent drug selected
primary mutations, the white bars represent drug selected secondary mutations, and the blackand-white bar indicates a mutation selected in vitro, but rarely seen in specimens from patients in
whom therapy fails.
1. Key codons for resistance to protease inhibitors (PIs)

Indinavir (Crixivan): M46I, L; V82A,F,T (sensitive position resistance)

Ritonavir: V82A,F,T

Saquinavir: G48V

Nelfinavir: D30N

Amprenavir: I50V
Besides the drug resistance to individual ptotease inhibitor, there are many studies
showed that cross-resistance among PIs present another big challenge for antiretroviral
therapy drug selection and patient management yyy Condra, 1995 Nature Vol374:569-571;
Condra, 1998 Drug Resistance Updates; Palmer, 1999 AIDS, Vol 13:661-667 kkk. As high
as 77-95% of patient plasma-derived recombinant HIV-1 isolates showed broad crossresistance among different protease inhibitors has been reported yyy Hertogs, Abstract
42195, 12th World AIDS Conference, Geneva, 1998 kkk
2. Key codons for resistance to NRTIs

Zidovudine: K70R; T215Y

Didanosine: L74V

Zalcitabine: L74V; T69D
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Phenotypic and Genotypic Assays for HIV-1 Drug Resistance
Oct., 1999

Lamivudine: M184V

Stavudine: V75T

Abacavir: M184V

Multinucleoside resistance: Q151M; A62V; V75I; F77L; F116Y
3. Key codons for resistance to NNRTIs (key resistance codons)

Navirapine: K103N; V106A; V108I; V181C,I; Y188C; G190A

Delavirdine: K103N; Y181C,I

Efavirenz: K103N
VI.
Clinical results
There are several studies have been published using drug resistance testing. Some of the
studies were retrospective and some of them were prospective. The following are two
examples:
1. VIRADAPT: a prospective randomized study comparing genotype-guided therapeutic
decision-making vs. standard of care in 108 patients on a stable failing antiretroviral
regimen. In this study, TruGeneTM by Visible Genetics Inc. was used as the drug
resistance test.
As shown in Figure 4 the use of drug resistance test could be
beneficial as measured by viral loads in plasma from both control and genotypeguided patients yyy Durant, et al., Lancet, vol.353, No. 9171: 2195, 1999; Garraffo, et
al., Abstract 1166, 39th ICAAC, 1999; Chaix, et al., Abstract 466, 39th ICAAC, 1999
kkk.
The VIRADAPT Trial: HIV-1 RNA
Mean Change From BL at 3 and 6
Months
The VIRADAPT Trial: Patients With
Undetectable HIV-1 RNA at 3 and 6 Months
Genotypic Group
Control Group
35
Control Group
Genotypic Group
0
30
–0.20
25
–0.40
Plasma
HIV-1 RNA
(log10 )
% Patients With
HIV-1 RNA
<200 Copies/mL
–0.60
–0.80
20
15
10
–1.00
5
–1.20
0
0
–1.40
0
3
Time, months
6
3
6
Time, months
Durant et al. Lancet. 1999;353:2198.
Durant et al. Lancet. 1999;353:2197.
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Phenotypic and Genotypic Assays for HIV-1 Drug Resistance
Oct., 1999
Figure 4
2. GART
study (Genotypic Antitretroviral Resistance testing): a multicenter,
randomized pilot study to determine the short-term effects of genotypic analysis in
management of patients failing antiretroviral therapy. ABI sequencing instrument
was used to perform the genotypic analysis. Total 153 patients were enrolled and
divided to two groups, one GART, the other one no-GART.
With expert
interpretation, GART group was superior to no-GART as measured by short-term
viral load response (-1.14 log vs. –0.65 log) yyy Baxter, 1999 Antiviral Therapy
Vol.4: 43 kkk.
VII.
Conclusions
The latest promising results by several groups (Saag, Durant, and Baxter) showed
that either genotypic or phenotypic drug resistance tests are feasible and could be very
beneficial in guiding the choice of treatment regimen in patients failing current therapy if
they were done correctly. However, there are a number of unanswered questions will
present big challenge for such tests including to establish an uniform criteria for data
interpretation, quality control for all different assays, development of more sensitive
ways to detect minor mutations, time-consuming issue, and costly issue. Despite these
known limitations, drug resistance tests in combination with development of new classes
of antiviral drugs may be the only ways that can fight treatment failure, at least for now.
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