Doing more than testing and switching
May 2002
Cover Story
William Check, PhD
"New! Improved! HIV-1 Resistance Tests—Get One Today!"
Clinicians and laboratory scientists familiar with tests to detect resistance to drugs used to treat human immunodeficiency virus type 1 would unanimously reject this claim as extravagant and overwrought. Take away the "New!" and the exclamation marks, however, and they would probably give it their stamp of approval, including the implication that such testing should be routine for HIV-1-infected patients.
Greater acceptance and use of HIV-1 resistance tests rests not on any single dramatic breakthrough but on many incremental improvements:
But perhaps the strongest impetus to broader acceptance of HIV-1 resistance testing is an urgent need arising from growing rates of resistance in HIV-1 infections.
"There continues to be an accumulation of studies showing an increasing prevalence of drug resistance among newly infected individuals in North America," says Daniel Kuritzkes, MD, associate professor of medicine and microbiology in the division of infectious diseases at the University of Colorado Health Sciences Center and co-director of the Colorado AIDS Clinical Trials Unit. In one study, by Susan Little, MD, of the University of California at San Diego, resistance rates among newly HIV-1-infected persons were found to have quadrupled from 1995-1998 to 1999- 2000, with 12 to 14 percent of isolates in the latter period being resistant to at least one antiretroviral drug and several percent showing resistance to more than one drug. Dr. Kuritzkes calls these data "disturbing" because of the high numbers and, more important, because they show that "patients in treatment are not being effectively counseled about how to prevent transmission."
Sounding the same theme, David Hillyard, MD, director of molecular infectious disease testing at ARUP Laboratories in Salt Lake City, says, "HIV resistance testing is still in its infancy, as are clinical correlations to validate this testing. Yet," he says, "there is a growing realization that there is a real need [for these tests] due to emergence of resistant strains."
Dr. Hillyard cites reports that from 10 to 25 percent of treatment-naive persons have one or more mutations that may make them resistant to an antiretroviral drug. "So there is a tremendous need for tools to help guide physicians in this very difficult process of how you initially treat a patient," he says, "and in the even more difficult situation of a patient who has failed therapy and for whom you need to make a choice among a limited set of remaining drugs."
The data validate this approach, says Susan Eshleman, MD, PhD, associate professor of pathology and director of the HIV genotyping laboratory at Johns Hopkins Medical Institutions. "A number of retrospective studies in the literature support the use of resistance testing," she says. "And prospective studies are beginning to appear." Based on those clinical trials, three large networks—two in the U.S. and one in Europe—have recommended using resistance testing in various clinical settings. "Resistance testing is becoming part of routine management," Dr. Eshleman says.
Mark Wainberg, PhD, professor of medicine at McGill University and director of the McGill University AIDS Center, agrees: "There is a consensus that good clinical management of HIV-1-infected patients requires access to resistance tests."
Use of resistance testing may expand further now that one genotyping kit—Trugene from Visible Genetics—has received 510(k) market clearance from the FDA and a second genotyping kit—ViroSeq from Applied Biosystems—is under review. Dr. Kuritzkes calls approval of Trugene "a major advance" for the field. "It has helped to establish standards and to lend legitimacy to HIV resistance testing as part of patient management," he says.
Having an approved test should make reimbursement more widely available. "As tests become FDA cleared or approved, reimbursement becomes easier," notes James Versalovic, MD, PhD, director of microbiology at Texas Children’s Hospital and assistant professor of pathology at Baylor College of Medicine. "There is more widespread acceptance among insurers, including the federal government. Multiple CPT codes have been established."
In their current state, however, resistance tests provide limited information. "Existing resistance tests are better at telling you what drugs a patient won’t respond to than telling you what drug you could switch to that would be effective," Dr. Hillyard says. In the words of Graeme Moyle, MD, PhD, associate director of HIV research at Chelsea and Westminster Hospital, London, "Resistance test results today are probably more predictive of failure than of success."
Part of the problem is a limited number of therapeutic options. "Drug development in HIV is still a growth industry," Dr. Wainberg says. "We are treating people for life, and there is resistance out there to every available drug."
Another problem is the increased use of three- and four-drug treatment regimens. "Progress in resistance testing is slower because, with today’s complex regimens, it is quite difficult to sort out the contribution of each drug to resistance," says Charles Boucher, MD, PhD, clinical virologist at University Hospital in Utrecht, the Netherlands.
Interpreting resistance mutations is "extremely complex and somewhat unpredictable," Dr. Eshleman confirms. Mutations may work synergistically or additively. Some mutations cause cross-resistance to other drugs, while others may reverse resistance to other drugs or even cause hypersusceptibility. As a result, she says, "[Genotypic] resistance testing requires a commitment from the laboratory not only to the technical aspects of the work but also to interpretation of results."
Quality commercial systems are available to help with this work. "Whichever interpretive system you use should be current and needs to be reviewed on an ongoing basis by a panel of experts," Dr. Eshleman advises. "Every few months we learn more."
To improve technical quality, the CAP has instituted proficiency testing for genotypic resistance methods. The number of laboratories participating grew from 15 to 20 in 2000 to 56 in 2002. "We’ve really jumped," says Dr. Versalovic, who directs the CAP Survey. "Published studies have demonstrated an advantage to treating patients based on [genotypic] data, so physicians are demanding it and laboratories are implementing it."
While challenges to interpreting genotypic test results are obvious, "It is not that easy to interpret phenotypic resistance assays either," says Lisa Demeter, MD, associate professor of medicine at the University of Rochester School of Medicine. "There is a big controversy in the field now about how best to define phenotypic resistance thresholds."
Taken together, the rise of complex resistance testing and the increasing number of antiretroviral drugs make treatment by specialists more common, according to Robert Schooley, MD, head of the infectious diseases division at the University of Colorado Health Sciences Center. "There is less treatment now of HIV-infected persons by doctors who don’t do it most of the time," Dr. Schooley says.
Indications for resistance testing are well defined. Recommendations concur that resistance data be obtained after failure of first or subsequent drug regimens. "The patient who benefits most from resistance testing would be one who has failed a previous drug regimen or regimens but who still has treatment options," says Dr. Demeter. "For a patient who has been treated with all current antiretroviral drugs and has virus highly resistant to all those drugs, resistance testing is unlikely to help."
Resistance testing is also appropriate for treatment-naive persons who are diagnosed when acutely infected, because of the rise in the transmission of resistant virus and the clinicians’ tendency to treat individuals identified with early symptomatic infection. "I think most experts would recommend that you test those individuals but not delay treatment while you wait for results," she says. Treatment can be modified if resistance data make it necessary.
"The only group in whom resistance testing appears not to be indicated are chronically HIV-infected persons who have never been treated with an ARV [antiretroviral drug]," Dr. Demeter continues, "and there is even some controversy about this." Persons who are now chronically infected were initially infected when rates of transmitting resistant virus were much lower.
Dr. Moyle makes the opposite case, but cautiously. "A person may rebound on their first-line therapy because they started with a resistant virus strain," he says. "So there is a constructible argument to test before starting a patient on therapy. Although information is insufficient to say whether that is helpful."
Encouraged by official recommendations, use of resistance testing has risen, but not dramatically. "There has been a steady increase in the last few years in our clients ordering resistance testing," says ARUP’s Dr. Hillyard. "Still, a clear distinction should be made between resistance testing and viral load testing."
While all physicians who manage HIV-infected patients order viral load tests regularly, Dr. Hillyard observes considerable variation in the ordering of resistance tests. Some clinicians, including experts who are very astute in managing HIV disease, rely very little on resistance testing, he finds. "It should be remembered that people fail therapy for a variety of reasons," he says, "and only one part of the problem is access to a high-quality HIV resistance test."
Dr. Hillyard also notes the opposite—some clinicians with less expertise may rely too heavily on resistance testing. "They are tempted to see resistance testing as a stand-alone means to make decisions," he says, "while most clinicians would feel that HIV resistance testing is only one tool to use in making treatment decisions, and one that is in the early stages of proving itself."
In Dr. Boucher’s view, there has been "surprisingly slow pickup of genotypic testing throughout Europe and the U.S., which is mainly I think a function of reimbursement." He sees "a major disconnect" between published data and how widely testing is employed. "One other reason resistance testing is taking off slowly," he adds, "is the need to be an expert or full-time practitioner to use it well."
Results of several randomized prospective clinical trials that have been published or reported at meetings provide evidence that genotypic resistance testing yields clinical benefit. Data supporting a possible advantage to treatment guided by phenotypic analysis are still sparse.
In genotypic analysis, resistance is evaluated by determining the nucleic acid sequence of critical segments of viral genes for the two antiretroviral drug targets—reverse transcriptase and protease—and identifying mutations associated with resistance. In phenotypic analysis, segments of reverse transcriptase and protease genes are spliced into a standard HIV-1 genome and the recombinant clone is incubated in vitro with antiretroviral drugs, similar to assaying resistance to antibiotics. A medium-size to large molecular laboratory can set up genotypic analysis or samples can be sent to a reference laboratory. Specimens for phenotypic analysis must be sent to Virco’s laboratory in Mechelen, Belgium, to ViroLogic’s laboratory in South San Francisco, or to Specialty Laboratories in Santa Monica, Calif., which performs a version of phenotypic analysis developed by French firm Viralliance.
In the first two controlled trials of resistance testing, GART and VIRADAPT, after failure of a first antiretroviral combination regimen, viral suppression was significantly more frequent among patients whose next regimen was selected using results of genotypic resistance analysis. GART used a panel of three experts who interpreted genotypic information in conjunction with a patient’s history. Clinicians in VIRADAPT used a simple algorithm to interpret genotypic results themselves.
Similar benefit from use of genotyping was seen in the HAVANA trial, in which genotypic analysis was interpreted by a software package called RetroGram written by Dr. Boucher. "In HAVANA, there was also a strong suggestion that adding advice from experienced HIV physicians to genotyping provided further benefit," Dr Kuritzkes says.
In the VIRA 3001 study, guiding regimen changes by results of phenotypic analysis—Virco’s Antivirogram report—also improved clinical outcomes.
Specialists evaluate the degree of clinical advantage from resistance testing differently. "Knowledgeable physicians understand that it can be a valuable tool but it is just one small piece of the puzzle," says Dr. Hillyard. "So far the results are promising but somewhat modest in terms of clear-cut efficacy."
On the other hand, Dr. Demeter says, "The magnitude of effect you see in GART and VIRADAPT almost certainly underestimates the extent of benefit that you would get from genotypic resistance testing in clinical settings." Not all trials provided expert advice, she notes, and some provided advice that we now know was less than optimal. "So I think it was quite remarkable that we saw as big a difference as we did," she says.
Dr. Boucher calls beneficial results to date "relatively small," which he attributes to extensive cross-resistance within antiretroviral drug classes and to the interpretive challenge. "It is still somewhat of an art to properly interpret genotypes," he says. Better interpretation will be a function of better training and better software provided by vendors, he adds. Results of ongoing clinical trials relating genotypes to clinical outcomes will advance the interpretation software.
Optimizing these rich datasets in the laboratory will require in-house expertise, Dr. Hillyard says. "We have, as do most laboratories specializing in this area, an experienced HIV physician responsible for keeping up with the rapid changes in HIV sequence databases," he says. He calls such work "incredibly complex."
Even experienced HIV clinicians consult among themselves on how best to interpret genotypic results in terms of possible drug regimens. Dr. Moyle participates in such a group at his hospital, and Dr. Demeter’s practice group holds conferences every few months to discuss therapy in problem cases.
Beneficial interpretation of genotyping data assumes that laboratories have accurately determined the sequence. To advance this goal, the CAP’s HIV Surveys now include with each mailing one HIV genotyping challenge. Dr. Versalovic explains that each genotyping proficiency sample contains heat-inactivated HIV-1 that has been isolated from a patient.
"We have several HIV-1 strains with different sequences," he says, "and we use a single donor for each sample. The challenge for the laboratory is to identify the predominant mutations that may affect susceptibility of virus to antiretroviral drugs and to recognize quasi-species."
In the CAPHIV/HV2 Survey, laboratories are required only to report whether there are important mutations in either the reverse transcriptase or protease genes but do not have to specify the mutations. "Reporting requirements will continue to get more rigorous," Dr. Versalovic says.
With both the CAP and private companies providing standards and proficiency testing, there is now adequate proficiency testing available, Dr. Versalovic says.
In addition to trials in which resistance testing proved beneficial, there have been several in which it did not improve viral suppression. In NARVAL, conducted in France, no clinical benefit was seen with either genotypic or phenotypic analysis. Lack of benefit with genotypic analysis might have been due to the heavily pretreated status of many patients: In a post-hoc analysis, genotyping was beneficial in patients who had failed fewer previous regimens.
Even in this subgroup, however, phenotyping was not advantageous. Failure of phenotyping in NARVAL and in the CCTG 575 study, which employed ViroLogic’s Phenosense system, could have been due to inappropriate cutoffs for some commonly used nucleoside reverse transcriptase inhibitors, particularly dideoxyinosine (ddI), stavudine (d4T), and abacavir. In these studies, a 2.5-fold increase in IC50 was used as the definition of resistance. But in a retrospective analysis, treatment outcome was predicted only by a much lower cutoff, 1.6- to 1.7-fold. Such small shifts are difficult to measure, according to Dr. Kuritzkes.
"You are getting very close to the technical reproducibility of the assay," he notes. "These cutoffs may yield significant effects in the overall study population, but for an individual patient 1.7-fold could be 1.5-fold or 1.9-fold."
Estimates of the relative value of genotypic and phenotypic analysis vary. Dr. Schooley sees genotypic testing as generally more difficult to interpret. "You have to understand the predictive value of specific mutations," he says. While most laboratory reports state what genetic changes typically mean, he notes that these interpretations rely on algorithms, either based on rules selected by expert panels or generated by computerized decision trees. "Clinicians can work through these algorithms," Dr. Schooley says, "though it may take a few times to become conversant with them. And it can be helpful to have someone available who is experienced."
Phenotypic testing, on the other hand, "is analogous to what we have been doing for a long time with antibiotics," Dr. Schooley says. "It takes less time to assimilate."
To other specialists, however, the distinction is less clear-cut. Expressing a more common view, Dr. Demeter says genotyping is better at picking up early mutations that may signal evolving resistance. Also, she notes, "Phenotyping may be less sensitive at detecting resistance to ddI and d4T." In heavily pretreated patients who may have complex mutation patterns, phenotypic testing may offer an advantage.
"But in patients with limited experience, I really don’t see any advantage [to phenotypic analysis]," Dr. Demeter says. "Phenotyping is not the preferred initial test. If you look at prospective clinical trials, they provide stronger support for use of genotypic testing. So even if the cost of the two methods was similar, genotyping would have a slight edge." In fact, phenotyping is more expensive than genotyping.
Nor does Dr. Demeter think that interpreting results of phenotypic analysis is more intuitive. "I don’t think a phenotype is that much easier to interpret than a genotype," she says. "It is not completely straightforward. We still don’t fully understand how best to define IC50 thresholds."
Dr. Versalovic says the subset of patients in whom phenotypic analysis may be advantageous is not large. "When I was at the Massachusetts General Hospital," he says, "we reviewed the data and found that phenotyping would have helped in 10 percent or fewer cases. Resistance is a moving target," he acknowledges, "and with time there may be a greater need for phenotyping as patients are on treatment longer and more agents are used for treatment. But right now it is hard to justify the cost as a routine test."
Dr. Versalovic also questions whether it is really so easy to interpret phenotypic data. "Where do you set the cutoff?" he asks. "The IC50 for each drug, although based on correlative data, may be arbitrary." Cutoffs have been modified for some drugs based on accumulating data, Dr. Versalovic says. "Clinicians often prefer phenotypic analysis because the results appear easier to interpret," he continues. "But what does that mean in the patient?"
Dr. Moyle agrees. "Phenotypic analysis doesn’t always give us better numbers that we can more easily draw conclusions from." He notes that cutoffs can be of three kinds: a technical cutoff, based on assay reproducibility—is the tested virus significantly different from the reference virus?; a biological cutoff—is the patient’s isolate different from what we would find in a drug-naive population?; and a clinical cutoff—is the tested virus likely to respond to the drug we have tested against? Clinicians need to know the clinical cutoff. However, Dr. Moyle notes, "There are only a few drugs for which that is established." For other agents, the biological cutoff is reported.
But just as there are few drugs for which clinical cutoffs have been determined for phenotype, there are few data correlating the presence of specific mutations with loss of antiviral activity, Dr. Kuritzkes emphasizes. "In most cases," he says, "the significance of resistance mutations is judged by their correlation with phenotypes."
Whatever the advantages and disadvantages of each method, clinicians are voting with their order slips.
"When I survey treating physicians in large audiences at clinical meetings," Dr. Eshleman says, "I get a large show of hands for physicians who have ordered genotyping and a scant show of hands for those who have ordered phenotyping."
The ordering at ARUP would bear that out. "In our laboratory we have by far the greatest subscription to genotypic testing," Dr. Hillyard says. He attributes this to genotyping being more affordable and more rapid. Clinical data don’t overwhelmingly favor either method, in his view.
With approval of Visible Genetics’ genotyping kit and the possible future approval of Applied Biosystems’ kit, competition in this market should intensify. Is either product superior?
Dr. Demeter has experience in her research laboratory with both ViroSeq and Trugene. Both are robust and produce clean sequences from clinical specimens and both are labor-intensive. She sees a throughput advantage to the AB system. "But Trugene’s throughput is more than adequate for what most hospitals would require," she says.
"There is no clear winner in my opinion," says Dr. Versalovic, who is now setting up a new molecular diagnostics laboratory. "Both are quality systems for clinical use." One advantage of the Visible Genetics product is that it is FDA cleared. On the other hand, he says, "Technically I prefer AB’s capillary-based system."
Assuming that AB’s product is soon approved, Dr. Hillyard sees a problem that FDA’s way of setting product standards will pose for the interpretive software of both systems.
"Let’s say we have two FDA-approved tests," he hypothesizes. "Knowledge about resistance mutations is changing rapidly. How will their algorithms be updated?" He hopes the FDA will work out a process that won’t require full clinical studies every time an interpretive algorithm is updated.
HIV resistance testing will also be at the forefront of an FDA regulatory initiative regarding the large volume of molecular testing in clinical laboratories performed without FDA-approved kits. "How aggressive will FDA be in saying you must use approved tests?" Dr. Hillyard wonders.
Virco offers a combined genotypic-phenotypic resistance analysis called Virtual Phenotype. In this procedure, sequences from a patient isolate are matched against a database containing 28,000 patient isolates on which Virco has done genotypic and phenotypic analysis, yielding a probabilistic likelihood that the patient’s virus is susceptible to each drug.
"I think the Virtual Phenotype is a clever and potentially useful way to evaluate genotyping," says Dr. Kuritzkes. "Some studies, most recently at this year’s retrovirus meeting, found similar outcomes in patients managed by Virtual Phenotype as compared to patients managed by actual phenotyping." These studies were not large enough to establish true equivalence, he notes, but were probably accurate to a first approximation.
Virtual Phenotype’s advantage is that it is less expensive than actual phenotyping. A drawback is that it is not possible to match for every base pair in reverse transcriptase and protease, so an artificial intelligence program decides which codons to include. "Virtual Phenotype is most helpful when there are a large number of matches in the database and the distribution [of susceptibility or resistance] among those matches is clear-cut," Dr. Kuritzkes says. If, on the other hand, the database contains only 300 matches to a patient isolate and only 60 percent of the matching genotypes are sensitive (or resistant) to a particular drug, "in that case it’s more of a coin toss," Dr. Kuritzkes says. Virtual Phenotype provides all that information, so laboratorians and clinicians can look at it critically and decide whether to rely on it.
Virtual Phenotype is offered through Virco’s U.S. partners—LabCorp, Quest, and ARUP—which perform genotyping and send the sequence to Virco in Belgium for the VP analysis. An individual physician or medical center can also submit a sequence directly to Virco for VP analysis through a service called VircoNet.
Dr. Demeter’s clinic gets Virtual Phenotype as its standard resistance method through LabCorp. "It is a good test, and clinicians like it because it provides a genotype and an estimated IC50," she says. Whether they will continue to rely on VP now that Trugene has been approved has not been decided.
Tim Alcorn, PhD, director of the infectious disease laboratory at the LabCorp Center for Molecular Biology and Pathology, says ViroLogic also has a combined genotyping-phenotyping product called Phenosense GT, although it is not the same as Virtual Phenotype. "Using genotyping and phenotyping together probably gets you the best quantitative picture," Dr. Alcorn says, "although there are no data right now saying that having quantitative results allows better treatment. Qualitative information about reduced susceptibility seems to be as good."
While resistance testing can optimize regimen switches, it can’t create new possibilities when a patient’s isolate is resistant to all available drugs. Hence the continual search for better antiretroviral drugs.
"The challenge is to find new drugs with favorable pharmocokinetics or that are not susceptible to cross-resistance within drug classes," Dr. Eshleman says. She notes that resistance is not all-or-none but has to do with inhibiting viral replication at a particular plasma drug level. A new regimen may overcome resistance because of enhanced activity related to pharmacokinetics and attainable drug levels.
Says Dr. Schooley: "We now have a better idea about so-called cutoffs very much like what we see with antibiotics in bacteria. A result on an agent in vitro needs to be interpreted in terms of the amount of drug that will be delivered to the site of infection." One prominent example is that trough levels of protease inhibitors achieved in blood when these drugs are given as single agents may be only slightly above the susceptibility of wild-type viruses. However, trough levels are enhanced significantly by adding a second PI, most often ritonavir. "This allows you to use these agents in situations in which phenotypic susceptibility testing suggests they might not be useful," Dr. Schooley says.
Another advance has been the generation of correlations between resistance test results and clinical outcomes in the development programs of three recently approved antiretroviral drugs, the protease inhibitor ritonavir and the reverse transcriptase inhibitors tenofovir and abacavir. Clinical evaluation of these drugs included intensification studies in which the drug was added to a failing regimen, providing an estimate of the impact on viral suppression of phenotypic or genotypic resistance test results obtained at the time the drug was added. Based on such studies, Dr. Moyle notes, package inserts for these three antiretroviral drugs present suggested cutoffs for phenotypic test results and algorithms for establishing likely efficacy based on genotypic analysis. "This information can guide clinicians in making decisions about the likely utility of those drugs," he says.
This advance fits with the big push to define resistance based on clinical response. Says Dr. Kuritzkes, "It will be expected now for new drugs that come along, particularly for drugs being developed for use in salvage therapy, to have these correlations." He sees manufacturers of previously approved antiretroviral drugs now going back to find datasets to establish such correlations.
Dr. Kuritzkes points to a new class of drugs called entry inhibitors that are under investigation. Such agents "have great potential for improving salvage therapy," he says. For one of these, a fusion inhibitor called T20, phase III studies are nearly complete. Entry inhibitors interact with the HIV envelope protein, which is encoded by a large gene, with resistance mutations scattered across it. For this reason, Dr. Kuritzkes speculates, "It may be that phenotyping will have an edge over genotyping in determining susceptibility to entry inhibitors." At the retrovirus meeting, ViroLogic showed data using Phenosense to detect resistance to entry inhibitors. Virco is also developing a phenotyping assay for these agents. For some newer entry inhibitors, there may be differences in susceptibility even among patients who have never been exposed to them, so it may become important to use resistance testing before starting therapy with these agents, Dr. Kuritzkes says.
Finally, Dr. Eshleman has her eye on a laboratory enhancement. "One very interesting area in the future will be combining resistance testing with pharmacokinetic testing," she says. This will entail not only determining the patient’s viral phenotype or genotype but actually getting drug levels in a patient to determine what drug level suppresses replication in that person.
"In this way we will get a better idea of the in vivo relevance of resistance data," she says. And that, of course, is the purpose of the whole exercise.
William Check is a medical writer in Wilmette, Ill.