AIDSmeds.com - Understanding Drug Resistance : What Is Phenotypic Resistance Testing?

Unlike genotypic testing, which looks for particular genetic mutations that cause drug resistance, phenotypic testing directly measures the behavior—or phenotype—of a patient's HIV in response to particular antiretrovirals. Because of the way phenotypic tests work and the results they provide, many experts believe that these tests are more comprehensive and trustworthy than genotypic tests, especially when testing samples from patients who have tried and failed a number of HIV drugs in the past.

Using the simplest terms, phenotypic testing is performed by placing samples of a patient's HIV in test tubes with each HIV drug to observe how the virus reacts. The ability of the virus to grow (or not grow) in the presence of each drug is evaluated. The virus is exposed to varying strengths, or concentrations, of each drug. The ability of the patient's virus to grow in the presence of the drugs is compared with some wild-type virus that is known to be 100 percent susceptible to all HIV drugs. The comparison between the patient's virus and the wild-type virus provides the phenotyping results.

These results tell doctors how much of a particular drug is needed to reduce HIV replication. In other words, the laboratory conducting a phenotypic test is trying to determine the amount, or concentration, of drug needed to stop HIV from reproducing.

For example, if four times as much of the NRTI Ziagen (abacavir) is needed to control HIV replication, the virus is said to have "fourfold resistance" to the drug. If seven times as much Ziagen is needed, the virus is sevenfold resistant to the drug.

When phenotypic tests first became available, interpreting these fold changes was difficult. It wasn't clear what a fold change meant in terms of the virus being fully sensitive, less sensitive, or not sensitive to a specific HIV drug. As a result, companies conducting phenotypic tests began working closely with researchers to better understand fold changes and what they really mean in terms of resistance to available medications. After several years of extensive research, these companies have developed "clinical cutoffs"—an important component of phenotypic testing that allows for much easier interpretation of fold changes as they relate to the sensitivity of HIV to many of the available medications.

The PhenoSense lab report also includes information on an HIV sample's replication capacity (RC). RC measures how well a patient's virus is able to replicate compared to a wild-type reference virus. Generally speaking, wild-type virus replicates the best inside the human body. When the virus accumulates drug-resistance mutations, it allows the virus to continue replicating in the presence of HIV treatment. However, these mutations can affect HIV's fitness—its ability to cause damage to the immune system at the same rate as wild-type HIV.

Knowing HIV's RC may help to determine when to delay, start, switch or interrupt therapy. For example, in highly treatment-experienced patients with multi-drug resistance, viruses with low RC values may be associated with stable or increasing CD4 counts, which reduces the need for changing a regimen. However, more information from clinical trials is needed to better understand how best to use RC in making such treatment decisions.

Returning to the example of Ziagen, using Monogram Bioscience's PhenoSense HIV assay, the lower clinical cutoff is 4.5-fold resistance and the upper clinical cutoff is 6.5-fold resistance. In other words, HIV that is fourfold resistant to Ziagen is still technically sensitive to the drug, whereas HIV that is sevenfold resistant to Ziagen means that the virus is much less sensitive to the drug and, as a result, not a good treatment choice.

As for fold changes that fall between the lower and upper cutoffs, this means partial resistance (the higher the fold change, the less sensitive HIV is to the drug being used). While it is always best to use antiretrovirals that your virus is fully sensitive to, it is sometimes necessary to use (or reuse) medications your HIV is partially resistant to.

Each HIV drug has different clinical cutoffs, which can be confusing. To help make sense of these cutoffs and to make it easier for health care providers to interpret the results, laboratories conducting these tests provide detailed reports for every test conducted. Check out what a phenotypic resistance test report looks like by clicking on the following link:

There are two "conventional" phenotypic tests available: Monogram Bioscience's PhenoSense assay and Virco Lab's Antivirogram. Both tests evaluate the fold changes for all of the available NRTIs, Protease Inhibitors, and NNRTIs. Monogram Biosciences has a separate phenotypic assay, called PhenoSense Entry, which tests HIV's sensitivity to the entry inhibitor Fuzeon.

Another test is Virco Lab's vircoTYPE HIV-1 assay. This is actually a "predictive" phenotypic test, using genotypic testing results to figure out what the virus's phenotype is, without actually performing a phenotypic test. To do this, labs use genotyping testing to determine if an HIV sample has mutations known to cause drug resistance. Once the genotype has been determined, the laboratory searches a database maintained by Virco containing the genotypes of several thousand HIV samples collected from other patients. It then retrieves the phenotypes—the fold changes—that correspond to these samples, averages the information together and predicts the drugs that the current sample will be more or less sensitive to.

It is important to note that Monogram Biosciences and Virco calculate their cutoffs differently. As a result, the cutoffs determined for one company's test (e.g., PhenoSense) do not apply to the cutoffs determined for the other company's test (e.g., vircoTYPE).