Research Initiative Treatment Action (RITA!); Vol 5, No. 4 October 1999
Paul Simmons, RN, ACRN

In the mid-1990s, many in the HIV community thought, or at least hoped, that highly active antiretroviral therapy (HAART) could eradicate HIV. The clinical benefits of antiretrovirals were nothing short of staggering. Death and morbidity plummeted and it was not inconceivable that the drugs were powerful enough to cure.

Now, in the sober light of morning that follows every cocktail party, the limitations of antivirals are evident. The drugs do not cure; even in the presence of the most potent and durable combinations, residual virus and perhaps residual viral replication persist. Antiretrovirals are also limited in their mechanisms of action and patterns of resistance overlap between the available drugs.

In some clinical settings, the rate of virologic breakthrough exceeds fifty percent annually, presumably owing to the emergence of drug-resistant virus. In the absence of eradication, the community and healthcare providers need therapies effective over the long-term. Currently available antiretrovirals, however, too often demonstrate this short-term susceptibility to viral resistance.

How, then, can clinicians and patients make the best strategic use of their treatment options? For those who have already experienced viral rebound on therapy, what is the next, best regimen?

Researchers and clinicians have turned to resistance testing as a means to try to achieve the optimal use of antiretrovirals. The premise of such testing is simple. Select drug therapies to which the patient's virus is sensitive and avoid the agents for which there is evidence of viral resistance.

Drug resistance assays come in two forms: genotype and phenotype. Although both tests aim to support optimal decision-making, the tests differ in what they measure and in the type of information they provide.

Genotypic testing: how it works. The DNA of HIV is the repository of the virus's genetic material. Each gene carries information regarding the blueprint for making more viruses. This information is carried in four nucleotide bases called adenine (A), guanine (G), cytosine (C) and thymidine (T) that form the sequences of amino acids to form a particular protein. Three consecutive bases, termed a codon, code for a single amino acid. See Figure 1.

Viral enzymes like protease and reverse transcriptase are proteins. Proteins are made from long chains of amino acids. See Figure 2. The function of a protein is closely related to its shape and any change in the amino acid sequence will alter a protein's shape. The change may also prevent an anti-HIV drug from binding with the protein.

A genotypic assay examines the nucleotide base sequence in the genes that produce protease and reverse transcriptase. When the order of nucleotide bases in a patient's virus differs from the order in wild-type HIV, a mutation exists.

When a patient provides a blood sample for a genotype assay, the laboratory report will not show changes in the sequence of nucleotide bases. Instead, the report will show changes in the product of these bases. For example, M184V is the amino acid change most commonly associated with resistance to lamivudine (Epivir).¹ M184V indicates that the amino acid methionine, normally found at position 184 of reverse transcriptase in wild-type virus, has been replaced by valine. (The change in nucleotide base sequence is from the codon ATG to GTG.)

Researchers have identified more than one hundred amino acid substitutions associated with drug resistance. If unidentified mutations exist, a genotype test would fail to detect them. Moreover, if multiple, interacting amino acid substitutions are present in a single strain, the effect of the interaction on a particular drug may not be known.

A genotypic test generally costs approximately $500 and results are usually available within a few days. Generally, genotypic tests require a blood sample from an individual with a viral load of at least 1000 copies.²

Phenotypic testing: how it works. Phenotypic assays directly measure the ability of HIV to grow in the presence of drug. Specifically, a phenotypic test determines the concentration of drug—as measured in nanomoles (nM)³ —necessary to inhibit in vitro viral replication. The amount of drug necessary to inhibit either 50% or 95% of viral replication is known as the IC50 or IC95, respectively. (IC stands for inhibitory concentration.)

In phenotypic testing, the ability of a patient's HIV to grow in the presence of a drug is compared to the growth ability of a laboratory, or reference strain known to be drug sensitive. If it took 10nM to inhibit replication of the reference strain and 20nM to inhibit replication of the patient's specimen, the laboratory will report that the patient's specimen is 1 fold less sensitive to the drug than is the reference strain. Most experts believe that a 4 fold decrease in drug sensitivity is clinically significant.

A phenotypic tests generally costs approximately $900 and results may take anywhere from ten days to 2 weeks. The test requires a blood sample from an individual with a viral load of at least 1000 copies.

Limitations and clinical role of drug resistance testing. Understanding how these assays work is not the only challenge associated with them. It is also a challenge to determine the clinical value of these tests and the settings in which their use is most appropriate.

An HIV-infected patient carries a swarm of genetically distinct viral variants, and yet drug resistance tests—both genotypic and phenotypic—are presently unable to detect viral subpopulations that make up less than 20% of the quasispecies. See Figure3. If the assays detect evidence of resistance, this establishes that drug resistance is in fact present. But some of the minority variants not detected by these assays may also be drug resistant. While the tests are therefore undeniably useful in determining which drugs not to use, they may falsely suggest that a patient's virus is sensitive to a drug to which it is in fact resistant.

Moreover, some have noted the availability of a quicker, less expensive means of determining the drugs to which a patient's virus is resistant: read the drug history in the medical chart. Current treatment guidelines assume that viral rebound in the presence of drug either 1) indicates the emergence of resistant virus or 2) will result in resistant virus. Therefore, a patient who has experienced virologic breakthrough is generally counseled to change at least two, and preferably all three, of the drugs in her regimen. The national treatment guidelines state, "The safest approach remains to change all drugs in a failing regimen, regardless of the results of resistance testing."⁴

Suppose a patient has had an unquantifiable viral load on a regimen of zidovudine (Retrovir), lamivudine (Epivir) and indinavir (Crixivan). If a resistance assay shows the presence of mutations associated with decreased viral sensitivity to all three of these drugs, this is information the clinician could have surmised by taking note of the patient's drug regimen. If, on the other hand, the assay shows the presence of resistance to just lamivudine, few clinicians would change only that one drug.

So if resistance testing provides information that is either already known to the physician or information that the physician will not act on, what good is it?

Two important studies have sought to determine the clinical value of at least genotypic resistance testing.

Clinical trial data: GART. In February, the Community Programs for Clinical Research on AIDS (CPCRA) presented data from the GART study (Gentoypic Antiretroviral Resistance Testing). Investigators randomized 153 participants, all of who were failing a three-drug regimen, to one of two groups. In the first group, patients received genotypic resistance testing and their physicians received expert interpretation of the results. In the second group, the patients underwent resistance testing, but the results were withheld for 16 weeks.

After eight weeks on their new regimen, participants in group 1 had an average reduction in viral load of 1.2 logs. Participants in group 2 had an average viral load reduction of 0.6 log. Moreover, 50% of those in group 1 achieved unquantifiable viral loads⁵ compared to 23% in the second group.

The authors of the study concluded that the results of genotypic resistance testing, in combination with expert interpretation, improved short-term viral load responses. The long-term clinical significance of such responses requires elucidation and expert interpretation may not always be available to treating physicians.

Clinical trial data: VIRADAPT. In the twelve month VIRADAPT study funded by Visible Genetics Inc., makers of the TruGene HIV genotypic assay, investigators randomized one hundred eight participants, all of who had experienced viral rebound on treatment, to receive either a standard of care regimen or a regimen based on resistance testing.

At month six, the group basing treatment decisions on genotypic testing experienced a mean decrease of 1.15 log in viral load, whereas the control group saw a viral load reduction of only 0.67 log. Thirty-two percent of those in the testing group, versus 14% in the control arm, achieved unquantifiable viral loads.⁶ Moreover, 27% of patients in the genotype group did not follow published guidelines which call for changing at least two drugs in a failing regimen because the test showed that such a change was unnecessary.

The authors concluded "gentoypic resistance testing was beneficial for decisions about changes to treatment." (The Lancet, 535:9171, p. 2198, 1999.)

Summary. The long-term benefits of resistance testing for patients who have experienced viral breakthrough on their current regimen are not yet known. But the available data suggest that these assays improve clinical decision-making and may eventually become part of the routine management of HIV-infected patients.

In the meantime, resistance testing may find a niche in the care of patients who are recently infected and have never taken anti-HIV drugs. Data presented late last year at an international workshop on drug resistance monitoring indicate that between 5% and 20% of newly infected patients acquire strains of HIV resistant to one or more drugs. The assays may also prove useful in tailoring treatment regimens for healthcare workers exposed to HIV through needlesticks or other workplace accidents.

Whatever roles drug resistance assays end up playing in the management of HIV disease, a better understanding of resistance monitoring may lead to a greater respect for the mutability of HIV, a clearer sense of the limitations of antiretrovirals and a wider interest in studies that determine the optimal timing and sequencing of therapy.

¹ Although amino acid substitutions are clearly associated with resistance to antiretrovirals, the exact mechanism of resistance to nucleoside analogue reverse transcriptase inhibitors is not known.

² Recently, a new ultrasensitive genotypic assay was introduced by Visible Genetic Inc. that purports to be able to identify resistance mutations in the blood of individuals with a viral load as low as 60 copies. This ultrasensitive test is not yet commercially available but company officials expect to launch the product by the end of 1999 or beginning of 2000.

³ Nanomoles is simply a unit of measure, like pounds or ounces.

⁴ Guidelines for the use of antiretroviral agents in HIV-infected adults and adolescents, p. 10, May 5, 1999.

⁵ Limit of quantification was 500 copies.

⁶ Limit of quantification was 200 copies.

⁷ Soriano, et al., Second International Workshop on Salvage Therapy for HIV Infection, Abstract 014, 1999.

Assays: tests.

Wild-type: the typical form of an organism as it occurs in nature, as distinguished from mutant forms that may result from selective breeding.

In vitro: in the test tube.

Quasispecies: the population of genetically distinct viral variants that exist in an HIV-infected individual.

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Copyright © 1999 - Research Initiative Treatment Action (RITA!). Reproduced with permission. RITA! is published by The Center for AIDS. Contact Thomas Gegeny, MS, ELS, Editor, RITA! for permission to reproduce RITA!. tom@centerforaids.org. http://www.centerforaids.org

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