Genotyping vs Phenotypking: Advantages and Disadvantages


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Genotyping vs Phenotyping: Advantages and Disadvantages

Genotypic and phenotypic assays each have specific advantages and disadvantages. Because many laboratories are capable of performing automated DNA sequencing, genotypic testing is available from many more laboratories than is phenotyping. As a rule, genotyping is less complex, faster, and less expensive than phenotyping. In addition, in some circumstances a key resistance mutation may be detected by genotyping even though no change in the phenotype is produced (e.g., emergence of an L90M mutation in protease). Such changes might be the first step along the path to high-level resistance, and detection of these mutations might prompt a change in therapy in a patient with detectable plasma viremia.

Despite these advantages, there are important disadvantages to genotyping. Perhaps the greatest disadvantage is the complexity of the data generated by these assays. Keeping track of which mutations correspond to resistance to which drug poses an enormous challenge. Moreover, mutations that cause resistance to one drug might improve viral sensitivity to different drug (for example, the 184V mutation causes resistance to 3TC but sensitizes HIV-1 to AZT (8)). Another potential disadvantage of genotyping is inter-laboratory variation. An international study compared the performance of laboratories on a blinded panel of samples containing wild-type or mutant viruses in different proportions (9). Nearly all the labs provided the correct sequence for specimens that were completely wild-type. However, mutations in RT were detected only 66% of the time, and mutations in protease were detected only 71% of the time in samples that contained only mutant virus. The expected mutations were identified correctly in fewer than half the laboratories when mutant and wild-type virus were present as a 50:50 mixture. Results of this study showed that the experience of the technician actually doing the assay was critical to laboratory performance.

Phenotyping has the advantage of providing susceptibility data in a format familiar to most clinicians (i.e., the IC50 or IC90). In addition, because susceptibility is measured directly, the effects of mutational interactions are more easily sorted out. As a result, there is less need for expert interpretation of phenotypic resistance tests. Because these assays are performed in a few central reference laboratories, interlaboratory variation is less problematic. A study conducted by the CDC compared results on paired plasma samples tested by the Antivirogram and PhenoSense assays (10). The two assays gave similar results in over 90% of samples tested. Most of the samples on which the labs disagreed had phenotypes that were close to the cut-offs (e.g., 2.5- or 4-fold resistance). Further comparisons of a larger number of resistant isolates are planned.

Phenotypic assays also have potential disadvantages. Because these tests are less widely available, there is often a longer time until results are available. In addition, phenotypic assays are significantly more expensive than genotypic assays. Another limitation of phenotypic assays is that clinically significant "cut-offs" or "break points" for distinguishing sensitive and resistant isolates have not been defined for most drugs (see below). Moreover, phenotypic tests may fail to detect very small shifts in susceptibility that accompany the presence of only one or two key resistance mutations that nevertheless result in reduced drug activity (e.g., the 90M mutation for saquinavir or the 74V mutation for ddI).

Shared limitations

Genotyping and phenotyping share some common technical limitations as well. Samples with <500-1000 HIV-1 RNA copies/mL usually fail to yield a result. In addition, both types of tests are relatively insensitive to the presence of minor variants. As a rule, a mutant needs to make up at least 20-30% of the viral quasispecies before it is detected by genotyping or can exert a noticeable effect on the phenotype. Because genotyping and phenotyping both rely on a PCR step to amplify PR and RT gene sequences, cross-contamination is a significant concern. Even though diagnostic laboratories take elaborate precautions to prevent contamination, problems can occur even in the best laboratories. Therefore, if a resistance test result does not seem to make sense in the context of a patient's current or former treatment regimen the test should be repeated.

4/15/01