HIV resistance tests
enter everyday era
William Check, PhD
From the start of the AIDS epidemic, physicians who care
for HIV-infected persons have been early adopters. Soon after viral
load-the amount of HIV RNA in the blood-was demonstrated to be associated
with prognosis, assays to measure HIV RNA concentrations became
widely used to monitor disease stage and response to therapy. And
it was only a few years after multidrug combinations against the
virus became available that significant reductions in progression
to AIDS-defining illnesses and in mortality from HIV disease were
demonstrated on a population basis, reflecting broad deployment
of these highly active regimens.
Most recently, assays to detect resistance to antiviral drugs
have become de rigueur for optimal management of many HIV-infected
persons. An International AIDS Society-USA Panel on antiretroviral
drug resistance testing (JAMA. 2000;283:2417- 2426) and other
groups recommend them for use in several clinical situations. Emphasis
on the utility of such testing is much more emphatic in the panel’s
current statement than in its first report, published less than
two years ago. Says panel chair Martin Hirsch, MD, professor of
medicine at Harvard Medical School and director of clinical AIDS
research at Massachusetts General Hospital, "Over time new data
became available so that we could refine the suggestions with more
confidence than in the first go-around." Data have been published,
he says, "that validate the utility of genotypic analysis, and also,
but to a lesser extent, phenotypic analysis, to make decisions in
clinical situations that we face daily."
Adds panel member John Mellors, MD, chief of the Infectious Disease
Division and director, HIV/AIDS Programs, at the University of Pittsburgh
Medical Center, "There is no question that in the last year there
has been a steady accumulation of data supporting the use of resistance
testing." Retrospective analyses had consistently shown that, in
treatment-experienced patients, HIV drug resistance is predictive
of response to therapy. "That is unequivocal," Dr. Mellors says.
"Now," he adds, "there are several prospective studies showing
that if physicians are provided with resistance test results, their
patients respond better virologically to the prescribed antiretroviral
regimen compared to patients of physicians who don’t have such information."
Calling himself a "big fan of resistance testing," Mark Wainberg,
PhD, professor of medicine at McGill University, Montreal, director
of McGill’s AIDS Center, and immediate past president of the International
AIDS Society, agrees. "There is absolutely no doubt that resistance
testing is useful in the majority of cases. If we are judicious
in how we use this technique," he says, "it will improve patient
outcomes and save money."
Practice patterns already reflect study data to some degree, says
Tim Alcorn, PhD, director of infectious diseases at the Laboratory
Corporation of America Center for Molecular Biology and Pathology
in North Carolina. "There has been a significant increase in volume"
in resistance assays, Dr. Alcorn says. "We are seeing usage that
appears to be in compliance with the guidelines."
At Specialty Laboratories in Santa Monica, Calif., the volume
of HIV resistance tests also has grown in the past five years. "The
demand reflects clinical utility and a growing understanding of
the need for individualized treatment regimens," says Andreas Bakker,
PhD, Specialty’s director of genotyping and molecular technology.
But using the resistance assays optimally is not simple. "Certainly
adoption of resistance testing is going to increase the complexity
of HIV care," says panel member Daniel Kuritzkes, MD, associate professor
of medicine at the University of Colorado Health Sciences Center,
Denver, and co-director of the Colorado AIDS Clinical Trials Unit.
For example, the panel recommended "expert interpretation" of assay
And the degree of benefit from resistance assays is limited by
cross-resistance among classes of antiretroviral drugs. Says Graeme
Moyle, MD, PhD, director of HIV Research at Chelsea and Westminster
Hospital, London, "You can only get returns if you have drugs available
that are effective in that situation." Dr. Kuritzkes adds, "As new
drugs are developed and come on line, and assuming that full understanding
of resistance to these drugs is part of the package submitted to
FDA, that will drive the utility of these tests in more advanced
or experienced patients."
Data from three major prospective studies-GART, VIRADAPT,
and VIRA 3001-support the value of HIV resistance assays in patients
who fail their current regimens. In all three studies, virologic
response was superior among patients whose new therapy was selected
in accord with results from either genotypic or phenotypic resistance
Guiding therapy with the results of resistance assays reduced
HIV RNA by about a half log on average, which translates to about
a 30 percent reduction in the risk of disease progression. Is this
benefit clinically important? Yes, says Dr. Kuritzkes.
"Given that resistance testing will be applied in settings where
patients are already failing therapy," he says, "achieving complete
suppression of HIV replication is not likely. But a half-log reduction,
although not impressive compared to what we can achieve in treatment-naive
patients, confers a significant benefit."
Moreover, averages hide individual benefits. In GART and VIRADAPT,
about one-third of patients treated according to resistance data
achieved undetectable levels of HIV RNA (<200 or <500 copies/mL).
"And patients in VIRA 3001 did much better," Dr. Kuritzkes says.
About 60 percent of patients in VIRA 3001 who were being treated
based on resistance data had HIV RNA <400 copies/mL. "These were
first-time failures," Dr. Kuritzkes notes. "As assays are used more
often and earlier, they may enable greater benefit."
Dr. Mellors expresses a similar appreciation. "We are asking an
awful lot from a single test that only detects resistance in the
dominant viral species present in a patient to make a major difference
in treatment response over 24 weeks," he says. Other important variables
influence treatment response, such as disease stage, adherence to
medication, pharmacokinetics of individual drugs, and availability
of agents with activity against resistant strains. "Resistance tests
don’t improve adherence or enhance the drugs’ pharmacokinetics or
substitute for new drugs," Dr. Mellors says. "I think it is remarkable
that a single test result shows a difference in response. But we
also need to optimally manage patients, such as with medication
adherence support systems."
An additional trial from France, called NARVAL, found no advantage
of resistance testing at three months and a minimal advantage for
genotyping at six months. Dr. Wainberg calls NARVAL "an outlier."
"NARVAL is a very important study and seems to have been well
done," Dr. Wainberg acknowledges. But the majority of patients in
NARVAL had more advanced HIV disease than patients enrolled in GART,
VIRADAPT, or VIRA 3001. "So they may have had such complicated genomic
resistance patterns that expert interpretation and advice might
not have made as big a difference," Dr. Wainberg says. "In fact,"
he adds, "there is some feeling that phenotype has its best place
in populations where genotype is so complicated that you can’t tell
for sure what is going on."
Providing another perspective, Dr. Moyle points out that the group
in NARVAL that didn’t receive resistance testing took more "new" drugs-drugs
they had never taken before-than the group randomized to testing.
"Resistance testing enabled clinicians to recycle drugs into the patient’s
regimen and achieve a similar virological response without using as
many treatment options," he explains. So resistance testing may buy
time without using up effective drugs with which the physician might
be able to formulate a better regimen later when newer drugs emerge.
Genotyping assays have been thought to be less useful in persons
who have failed multiple regimens, as Dr. Wainberg noted. But, says
Charles Boucher, MD, PhD, clinical virologist at the University
Hospital, Utrecht, the Netherlands, "The HAVANNA trial [conducted
in Europe and involving genotype and expert advice by software]
showed that genotyping gave a better result throughout all the different
patient categories. We formerly thought if a patient had failed
their third regimen, phenotyping would be better. But in HAVANNA
we observed that genotyping still had an impact even in patients
who had cycled through one or two regimens already." Further work
will clarify these issues.
Based on these studies, HIV resistance assays are now recommended
for patients who experience treatment failure on their first or
subsequent regimen or who do not respond to a potent regimen. The
assays help to determine whether resistance is contributing to treatment
failure and what drugs to use in the next regimen to optimize response.
"In clinical trials, response to treatment is clearly and consistently
related to the number of new drugs prescribed to which the patient’s
virus is sensitive," Dr. Mellors says. And studies have shown that
physicians given results of a resistance test choose with greater
accuracy drugs that are active against a patient’s virus.
In addition, the panel’s statement says clinicians should "consider
testing" for HIV resistance in two groups of patients: those with
established infection that has not yet been treated and those with
primary or acute infection. In both types of patients, the value
of resistance assays remains to be proved but is a source of intense
Whether to test for resistance in untreated patients with established
infection of unknown duration is a difficult decision. If such patients
harbor a resistant virus strain, either acquired at primary infection
or generated by mutation during viral replication, it is likely
to be present as a minor quasi-species, probably below the sensitivity
of a resistance assay. Dr. Kuritzkes notes that many HIV-positive
persons not yet treated have been infected for a long time. Many
acquired their virus prior to the current therapeutic era, so they
are less likely to have a (primary) resistant virus. But some data
indicate that in cities where the epidemic has gone on longest and
treatment has been available longest, such as New York and Los Angeles,
there may be a much higher prevalence of primary resistance. "Eventually
we may need to establish guidelines based on local prevalence rates,"
"You could make an argument that everybody should get a genotype
before their first regimen is initiated," Dr. Wainberg says. He
cites the HIV care program in British Columbia, which he calls "the
longest running program in genotyping." In that program, genotyping
is done on almost everyone about to embark on their first course
of therapy, as well as those with treatment failure, Dr. Wainberg
Clinical researchers have become increasingly interested in
the second group-patients in the acute phase of HIV infection, within
30 to 60 days of acquisition and before seroconversion. Use of special
"detuned" assays makes detection of acute infection possible in patients
who come in with nonspecific symptoms of viral infection and a suspected
exposure. Work from Bruce Walker, MD, and colleagues at Massachusetts
General Hospital has showed that prompt reduction in viremia to undetectable
levels in these patients preserves immune function. "We don’t want
to make a mistake in prescribing therapy in that group," says Dr.
Mellors, "so we need to know the resistance profile of the strain
in those patients. There is enough primary resistance around to warrant
it. And the stakes are very high."
Data from Dr. Wainberg and
colleagues underscore this point. They genotyped viral isolates
from 81 untreated patients who were infected between 1997 and 1999.
Mutations associated with resistance to at least one class of antiretroviral
drug were found in 30 percent of patients; almost 10 percent had
genotypes suggestive of primary multidrug resistance. "Our results
are pretty consistent with other work," Dr. Wainberg says. "This
is an increasing problem. Drug-resistant viruses are being spread
sexually and through injecting drug use. And it is not just in Europe
and the U.S., but in developing countries." Transmission of drug-resistant
strains may become more problematic in developing countries because
of widespread use of less-suppressive one- and two-drug regimens,
which Dr. Wainberg calls "a prescription for resistance."
Testing for resistance in primary infection has two purposes,
Dr. Wainberg says: It shows how much resistance is present in a
population, which is important from a public health standpoint,
and it provides crucial treatment information. "You don’t want to
use drugs as first-line therapy in patients whose virus is resistant
to those compounds," he says.
Dr. Kuritzkes agrees that testing for resistance in patients with
acute infection is useful both for epidemiologic purposes and because
of "growing interest in providing immediate therapy for such patients."
Finding those patients is "challenging," he notes. Acute infection
looks like a general viral syndrome, so most patients will be seen
by a general internist or an emergency room physician. "More education
is needed for primary care practitioners," Dr. Kuritzkes says. "Physicians
do monospots at the drop of a hat, even though most come back negative.
There needs to be more thinking about HIV when there is a compatible
clinical presentation, even when the patient doesn’t seem to be
Dr. Hirsch is convinced that greater effort will go into detecting
persons with acute infection, largely because of Dr. Walker’s data.
"His work certainly suggests that treating very early has significant
advantages and might allow you to discontinue therapy down the road,"
Dr. Hirsch says. "These are very provocative observations and, if
confirmed by others-and I have no reason to think they won’t be,
will make identifying acute HIV infection extremely important."
Dr. Hirsch believes professional organizations for HIV care will
try to devise ways to detect HIV earlier. "We have been doing that
at this institution, as have a number of others, and it’s pretty
amazing how many such people you can find if you are looking very
hard for them," Dr. Hirsch says. "In that setting, susceptibility
patterns can be very helpful."
There may be some difference in the utility of genotypic
assays, which detect resistance based on sequencing of the viral
genome looking for known resistance mutations, and phenotypic assays,
which measure growth of virus in vitro in the presence of antiretroviral
drugs. In Dr. Alcorn’s laboratory, genotypic assays are ordered
two to three times as often as phenotypic assays, primarily because
genotypic assays are faster and less expensive and just as useful
for patients failing the first time with uncomplicated therapeutic
histories. "Phenotyping may be more useful for complicated therapeutic
histories," Dr. Alcorn says.
He believes genotyping may be favored even more with the introduction
of virtual phenotyping, a procedure made possible by the creation
of a large database containing genetic sequences of a great many clinical
HIV isolates along with their known phenotypes. A virtual phenotype
is obtained by matching the sequence of a patient’s virus with isolates
in the database that have a similar sequence, and extrapolating the
phenotype of the isolate in the database to the patient’s virus. Virtual
phenotypes strongly correlate to actual phenotypes, Dr. Alcorn says.
At Specialty Laboratories, too, genotyping is the more commonly
requested test. "Cost, time to result, and efficacy are the main
determinants of the test’s continued popularity," says Specialty’s
Dr. Bakker. Evidence is still lacking, he adds, that "phenotyping
guides therapy to superior patient outcome" when compared to treatment
based on appropriately interpreted genotypes.
Results from phenotypic testing may seem easier to read, especially
since they look like an antibacterial susceptibility test, Dr. Moyle
says. "Anything above 10-fold change in sensitivity is reported
from the laboratory as resistance," Dr. Moyle says. But, he points
out, the problem is that phenotypic results are not always clinically
relevant. For example, phenotypic resistance to stavudine (D4T)
is not always apparent, even when mutations are present and viral
load has rebounded. And with protease inhibitors (PIs) a 10-fold
increase in sensitivity may not impact efficacy when clinical exposure
to the drug is 70- to 80-fold above the effective concentration-which
may be true for booster PI regimens such as ritonavir 100 mg bid
with saquinavir, indinavir, or the recently approved PI lopinavir.
For the nonnucleoside reverse transcriptase inhibitor efavirenz,
too, clinical exposure may be much more than 10-fold higher than
the effective concentration, Dr. Moyle says.
Both types of tests are improving. Clinically relevant cutoffs
for drugs are being defined. "This is critical," Dr. Mellors says.
Dr. Kuritzkes and Richard D’Aquila, MD, of Harvard Medical School
are co-chairing a trial just getting underway in which both types
of tests will be compared in patients failing therapy.
Where the two types of tests will be done is another difference.
"Assays for phenotyping will continue to be done in specialized
laboratories," Dr. Kuritzkes says. Phenotyping relies on a technique
in which amplified protease and reverse transcriptase gene segments
from a patient’s isolate can be easily and quickly inserted into
a standard HIV vector to generate infectious viruses that are assayed
in an automated system. "Automation allows high throughput and makes
the assay economically feasible," Dr. Kuritzkes says. His laboratory
used to do "quite a number" of phenotypic assays, mostly for research
purposes. "We took six weeks from the time of getting the sample.
Now commercial assays can be done with an average two- to three-week
turnaround," he says.
Genotypic assays, on the other hand, can be done in a more decentralized
fashion, Dr. Kuritzkes says. Whether a laboratory does the genotypic
assay itself will depend to a great extent on economics, local demand,
and available expertise. "Applied Bioscience/Perkin-Elmer and Visible
Genetics are developing kits and systems that can be placed in a
hospital-based molecular biology laboratory to produce high-throughput
genotypes and generate reports useful for clinicians," he says.
Hospital laboratories and centers that see a large number of HIV-infected
individuals may find it more cost-effective to do assays in-house
rather than as sendouts, much like HIV RNA testing.
Before genotypic assays become routine in clinical laboratories, quality
control needs to be addressed, Dr. Boucher cautions. "QC is an issue
that people have largely ignored, because most genotyping has been
done in research laboratories, which normally are not as focused on
QA and QC as clinical laboratories," he says. A group of investigators
led by virologist Rob Schuurman, MD, of Utrecht, did a QC probe of
HIV resistance genotyping. They sent a panel of coded samples containing
various mixtures of clinically relevant resistance mutations to about
60 laboratories-"all the laboratories in the world doing this testing,"
Dr. Boucher says. "We observed that quite a number of laboratories
missed the correct interpretation if a mutation was present as a mixture
with wild type virus. We also observed that the more experienced the
laboratory and the better trained its people, the better were the
Missing a mutant virus present as a minor species would
be relevant to patients who have been on therapy for a short time
and those who have stopped therapy. "Generally, if a patient has
been on a stable regimen for a long time, you would expect most
mutations to become dominant in the viral population," Dr. Boucher
says. A minor mutant strain might also be present in a patient who
has been newly infected.
Dr. Boucher advises laboratories that are starting to do genotypic
HIV resistance testing to build in run controls and QC controls
and to adhere to an independent QC program. "I think it is good
that more clinically oriented laboratories are starting to do this
assay," Dr. Boucher says. "They are more used to thinking in terms
of QA and QC. But they must realize that it is a difficult assay
to do, probably the most error-prone assay in clinical virology."
Asked about the panel’s call for "expert interpretation"
of resistance assay results, Dr. Hirsch says, "In an ideal world
we could have not only the commercial laboratory’s interpretation
of the results it provides but also someone local with expertise
in interpreting HIV resistance test results to give practicing clinicians
useful guidance." Physicians in clinical practice who see hundreds
of patients don’t always have the time to keep up with the latest
advances in this complex area. "I have no doubt that [the amount
of expert advice] could be improved," Dr. Hirsch says. "In metropolitan
areas with major medical centers there are people with that kind
of expertise, but it is not available everywhere."
"We do need someone between the test result and the treating physician,"
Dr. Mellors says. "But I think what will happen is that the number
of people caring for HIV-infected patients will shrink and their
collective expertise will increase. Just as you don’t treat cancer
in general practice," he continues, "you shouldn’t be treating HIV
in general practice unless you do it enough so that you can interpret
the test results." Treating HIV disease "is not for the faint of
heart," Dr. Mellors says. In the meantime, he recommends consultation
with an expert. "If anybody calls me, I would speak with them,"
Obtaining such expert consultation depends on who is available
locally. "So-called experts don’t really have time to sit down and
deal with calls all day," Dr. Kuritzkes says. "They are busy doing
their own clinical or research work." He foresees systems that may
improve this situation, such as a move to set up a uniform rules-based
interpretation system. Experts would regularly review recent data
and make tables for suggested interpretation. Dr. Kuritzkes’ sense
from talking to companies making kits is that they would be happier
if an external body reviewed the data and formulated clinical significance
for various mutations. "We are trying to establish some standards
across industry so that automated reports would build in these interpretative
systems," Dr. Kuritzkes says, "but there are some regulatory issues."
He, too, acknowledges Virco’s (of Mechelen, Belgium) virtual phenotype,
a computer-based algorithm that relies on Virco’s large database
of linked phenotypic and genotypic data for individual isolates.
"It shows promise but clearly requires prospective validation,"
Dr. Kuritzkes says. "And anyway, our goal should be to determine
how a given genotype predicts the outcome with drugs A, B, and C,
not how well can I correlate genotype to phenotype."
Of the limited number of experts, Dr. Wainberg says, "I don’t
see a dilemma at all. I see an opportunity." It will be impossible
to instruct every practitioner on the intricacies of genotyping,
of course, and experts are busy people who cannot answer calls all
day about interpreting genotypes. "What we should be doing," he
says, "is training limited numbers of people to act as resource
individuals for physicians who can come to them with questions about
mutational profiles. And we can offer this as a reimbursable service."
Along this line, Dr. Hirsch notes that the Infectious Disease Society
of America has formed a subset of physicians who specialize in HIV
care. This group includes infectious disease specialists but also
those from other specialties who see a lot of HIV patients, such as
physicians in oncology and even general internal medicine and pediatrics.
"HIV care is a sub-branch of medicine now, and it will be increasingly
recognized as a specialty as things get more complex over time," he
With the large number of assays done by a commercial laboratory,
the demand for expert interpretation would be expected to be high.
Answering such questions can be complicated, Dr. Alcorn says. For
phenotypic assays, what is a relevant clinical cutoff? For genotypic
assays, there are so many combinations of mutations that need to
be interpreted. "Having said that," he points out, "evidence from
clinical trials, VIRADAPT in particular, where a rules-based algorithm
was used, demonstrate that even with complexity you can devise a
set of rules that will lead to clinically relevant results. Those
rules are being established," Dr. Alcorn adds. Physicians are becoming
aware of Web sites that help them interpret the results of resistance
assays, he notes: http://hivdb.stanford.edu and http:// idiotype.lanl.gov
(Los Alamos maintains the latter site). In any event, Dr. Alcorn
reports, "I don’t get a whole lot of calls asking about interpretation
An important milestone could be reached soon: The first FDA approval
of an HIV resistance assay. Visible Genetics has submitted its TruGene
genotyping kit for FDA approval. "It would be the first 510(k) HIV
resistance assay," Dr. Kuritzkes notes, "and it would make a difference."
A number of states are holding back on funding of these assays because
they are not approved. "When FDA approval arrives, it will drive
a number of states to fund these tests," Dr. Kuritzkes says. "I
don’t know how it will shift usage patterns," he adds. Many reference
laboratories are now using an in-house kit; how many will shift
to the approved kit is unclear.
Dr. Alcorn agrees that FDA approval of a genotyping kit would
speed up reimbursement policies. "We are now at the point where
it requires communication back and forth to payers about these tests,"
he says. Making them routinely reimbursable will go a long way toward
making them more widely used.
William Check is a freelance medical writer in Wilmette, IL