Testing for Drug Resistance

Background
Testing for HIV resistance to antiretroviral drugs is a rational adjunct to guide antiretroviral therapy. When combined with a detailed drug history and efforts aimed at maximizing drug adherence, these assays may help to maximize the benefits of antiretroviral therapy. Many studies in treatment experienced patients have shown strong associations between the presence of drug resistance (identified by either genotyping or phenotyping resistance assays) and failure of the antiretroviral treatment regimen to suppress HIV replication. Genotyping assays detect drug resistance mutations that are present in the relevant viral genes (i.e. RT and protease). Some genotyping assays involve sequencing of the entire RT and protease genes, while others utilize probes to detect selected mutations that are known to confer drug resistance. Genotyping assays can be performed relatively rapidly, such that results can be reported within 1-2 weeks of sample collection. Interpretation of test results requires an appreciation of the range of mutations that are selected for by various antiretroviral drugs, as well as the potential for cross-resistance to other drugs conferred by some of these mutations (see the http://hiv-web.lanl.gov web site). Consultation with an expert in HIV drug resistance is encouraged to facilitate interpretation of genotypic test results.

Phenotyping assays measure the ability of viruses to grow in various concentrations of antiretroviral drugs. Automated, recombinant phenotyping assays have recently become commercially available with turn-around times of 2-3 weeks; however, phenotyping assays are generally more costly to perform compared with genotypic assays. Recombinant phenotyping assays involve insertion of the RT and protease gene sequences derived from patient plasma HIV RNA into the backbone of a laboratory clone of HIV either by cloning or in vitro recombination. Replication of the recombinant virus at various drug concentrations is monitored by expression of a reporter gene and is compared with replication of a reference strain of HIV. The concentrations of drugs that inhibit 50% and 90% of viral replication (i.e. the IC₅₀ and IC₉₀) are calculated, and the ratio of the IC₅₀s of the test and reference viruses is reported as the fold increase in IC₅₀, or fold resistance. Interpretation of phenotyping assay results is complicated by the paucity of data on the specific level of resistance (fold increase in IC₅₀) that is associated with failure of different drugs; again, consultation with an expert may be helpful for interpretation of test results.

Further limitations of both genotyping and phenotyping assays include the lack of uniform quality assurance for all assays that are currently available, relatively high cost, and insensitivity for minor viral species; if drug-resistant viruses are present but constitute less than 10-20% of the circulating virus population, they will likely not be detected by current assays. This limitation is of particular importance when interpreting data about susceptibility to drugs that the patient has taken in the past but are not part of the current antiretroviral regimen. If drug resistance had developed to a drug that was subsequently discontinued, the drug-resistant virus can become a minor species because its growth advantage is lost (9). Consequently, resistance assays should be performed while the patient is taking his/her antiretroviral regimen, and data suggesting the absence of resistance should be interpreted carefully in relation to the prior treatment history.

Use of Resistance Assays in Clinical Practice
Resistance assays may be useful in the setting of virologic failure on antiretroviral therapy (see Table III), and in acute HIV infection. Recent prospective data supporting the use of resistance testing in clinical practice come from trials in which the utility of resistance tests were assessed in the setting of virologic failure. The VIRADAPT (10) and GART (11) studies compared virologic responses to antiretroviral treatment regimens when genotyping resistance tests were available to help guide therapy with those observed when changes in therapy were guided solely by clinical judgment. The results of both studies indicated that the short-term virologic response to therapy was significantly greater when results of resistance testing were available. Similarly, a recent prospective, randomized, multicenter trial has shown that therapy selected on the basis of phenotypic resistance testing significantly improves the virological response to antiretroviral therapy, compared with therapy selected without the aid of phenotypic testing (12). Thus, resistance testing appears to be a useful tool in selecting active drugs when changing antiretroviral regimens in the setting of virologic failure (BII). Similar rationale applies to the potential use of resistance testing in the setting of suboptimal viral load reduction, as detailed in "Criteria for Changing Therapy" (BIII). It should be noted that virologic failure in the setting of HAART (Highly Active Antiretroviral Therapy) is in some instances associated with resistance only to one component of the regimen (13); in this situation, it may be possible to substitute individual drugs in a failing regimen, although this concept requires clinical validation (see "Considerations for Changing a Failing Regimen" and Table 24). There are currently no prospective data to support the use of one type of resistance assay over the other (i.e. genotyping vs. phenotyping) in different clinical situations. Therefore, one type of assay is generally recommended per sample; however, in the setting of a complex prior treatment history, both assays may provide important and complementary information.

Transmission of drug-resistant strains of HIV has been documented, and may be associated with a suboptimal virologic response to initial antiretroviral therapy (14-17). Treatment of acute HIV infection is associated with improved immunological outcome (18, 19), and optimization of the initial antiretroviral regimen through the use of resistance testing is a reasonable albeit untested strategy (CIII). Because of its more rapid turnaround time, the use of a genotypic assay may be preferred in this setting; however, therapy should not be witheld while awaiting the results of resistance testing. The use of resistance testing prior to initiation of antiretroviral therapy in chronic HIV infection is not generally recommended (DIII) because of uncertainty about the prevalence of resistance in treatment-naive individuals and the fact that currently available resistance assays may fail to detect drug resistant species that were transmitted at the time of primary infection but became a minor species in the absence of selective drug pressure. The currently favored approach would be to reserve resistance testing for cases in which viral load suppression was suboptimal after initiation of therapy (see above), although this may change as more information becomes available on the prevalence of resistant virus in antiretroviral-näive individuals.

In general, recommendations for resistance testing in pregnancy should be the same as for non-pregnant patients: acute HIV infection, virologic failure on an antiretroviral regimen, or suboptimal viral load suppression after initiation of antiretroviral therapy are all appropriate indications for resistance testing. If an HIV+ pregnant woman is taking an antiretroviral regimen that does not include zidovudine, or if zidovudine was discontinued because of maternal drug resistance, intrapartum and neonatal zidovudine prophylaxis should still be administered to prevent mother-to-infant HIV transmission (see below, "Considerations for Antiretroviral Therapy in the HIV-Infected Pregnant Woman"). It is important to note that not all of zidovudine's activity in preventing mother-to-infant transmission of HIV can be accounted for by its effect on maternal viral load (20); furthermore, preliminary data indicate that the rate of perinatal transmission following zidovudine prophylaxis may not differ between those with and without zidovudine resistance mutations (21, 22). Further studies are needed to determine the best strategy to prevent mother-to-infant HIV transmission in the presence of zidovudine resistance.

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