Project Inform - September, 1998

Recently, HIV drug resistance tests have become more widely available with several laboratories offering a variety of these tests. Some of these laboratories appear to be more reliable in their ability to provide meaningful results whereas others have a poor reputation in their ability to perform these tests. None of these tests have been approved by the Food and Drug Administration and therefore are still considered experimental. Although laboratories are offering these tests, some insurance companies, Medicare and other sources for reimbursement may not cover them.

There are two ways of measuring resistance to HIV drugs. One, called genotypic resistance testing, seeks to determine any changes to a part of HIV's genetic structure, which change the way the virus makes key proteins (like protease or reverse transcriptase enzymes). Such changes are referred to as mutations. The other approach, called phenotypic resistance testing, is a more direct measure of resistance. It examines the amount of drug needed to inhibit the growth of HIV in a laboratory setting. In its natural state (wild type virus), when HIV is not resistant to a particular drug, known levels of the drug completely suppress HIV replication. Resistant HIV requires higher levels of the same drug to get an equal level of suppression. Both are likely to be useful, but neither test can tell with certainty which therapies people will get the most benefit from.

Genotypic Tests

Genotypic resistance testing examines samples of virus taken from a patient and looks for the presence of specific viral mutations which are known to be associated with resistance to certain drugs. Many laboratories are offering genotypic resistance testing. While most laboratories use essentially the same technology with some minor modifications, a few use completely different technologies. The most common method of measuring genotypic resistance involves taking a blood sample and using a machine that can read the specific sequence of different genes that are the targets of HIV drugs (i.e. the reverse transcriptase gene is the target for the nucleoside analogue and non-nucleoside analogue reverse transcriptase inhibitors and the protease gene for the protease inhibitors). The sequence results are then compared to those from a registry that holds the `true' genetic sequence of HIV. Certain changes from the 'true' sequence are known to correspond to resistance to certain drugs. These differences might be useful in helping to determine if someone has developed resistance to a specific drug, since almost all drugs follow a set pattern of mutations. For genotypic tests to be accurate, they generally require the use of a blood sample from a person with a viral load above 1,000 HIV RNA copies who is taking anti-HIV therapy. Otherwise, the pre-therapy strain of virus (wild type virus) will outgrow the mutant virus, and the results may show no mutations, although the mutant virus remains in the background and will quickly come back once the same therapy is re-started. Additionally, for these tests to show there are specific mutations, at least 20% of virus particles need to have that mutation to be able to reliably detect a change. In other words, these tests will not be able to pick up very low level resistant virus. The charts above show mutations associated with drug resistance (drugs being less effective).

Another technology used in genotypic resistance testing is the line probe assay (LiPA). This test utilizes different probes, or detectors of certain changes, which are designed to identify specific mutations. For instance, one probe would be designed specifically to detect any change at position 215 in the reverse transcriptase gene, which is known to be a mutation associated with resistance to AZT (zidovudine, Retrovir^®). However, given the number of mutations that are associated with HIV drug resistance, as shown by the chart below, this method of fishing for specific changes may be a less practical way to measure resistance.

Another method has merged computer chip technology with biotechnology. GeneChip technology uses a chip that has thousands of probes built into it which can detect changes in the reverse transcriptase and protease gene when a blood sample is placed on it. The chip is placed into a scanner, similar to those used in desktop publishing, which reads the results from the chip. The results of the scan are compiled by a computer program which shows whether there are specific mutations in the genes.

Phenotypic Tests

Phenotypic resistance testing grows samples of virus with genetic characteristics copied from blood samples submitted by individual patients. Cultures in which the virus is growing are then treated with various available antiviral therapies to determine how much drug is needed to inhibit the virus. The results are compared to the amount of drug needed to inhibit laboratory standard or wild type virus. If high drug levels are needed to keep the virus from replicating, this suggests the virus is no longer sensitive to the drug and is likely to have developed resistance. Because they directly test the patient's virus against the actual drugs used in treatment, phenotypic tests are considered the gold standard of resistance testing. This method is routinely used to test for antibiotics resistance before someone starts taking medications to determine if the bacteria are susceptible to the drug(s).

There are two phenotypic tests that are currently or soon to be commercially available. Both tests use a similar technology. Generally, with both, anything above a 2- to 4-fold change in resistance (2- to 4-fold higher or lower amount of drug needed to inhibit the virus by the same degree) is considered meaningful and outside the variability of the tests. With most drugs, anything above a 10-fold increase in resistance (sometimes referred to as a 10-fold decrease in sensitivity/susceptibility) is considered highly resistant and likely to mean that the drugs are no longer capable of blocking the virus from reproducing. A finding of 4- to 10-fold reduction in sensitivity to a drug is considered a moderate degree of resistance, one which might be overcome by higher drug levels. In order for phenotypic resistance test results to provide meaningful information, these tests generally require the use of a blood sample from people with a viral load of above 1,000 (but probably closer to the 5,000 range) copies HIV RNA who are currently taking anti-HIV therapies.

Availability

Different laboratories are currently offering tests identified in the following table.

RT = reverse transciptase 

Commentary

Overall, many questions remain about the use of these new tests. Perhaps most important, it is unclear when and how often to use them, and just how to make decisions based on the information they provide. Phenotypic testing is very expensive ($800-1,000 per test), while genotypic testing is merely expensive, ($300-500 per test). Certainly, for those with money to spare or a generous insurance company, the tests provide information you'd rather have than not have. But because of the high costs, careful decisions must be made about the true usefulness of the tests in guiding clinical practice. Unfortunately, it is still too early to know how much additional information these tests will provide patients and physicians. There are accumulating data that the phenotypic tests can show when a drug is no longer effective. However, there is very little data showing the phenotypic tests can predict if someone will benefit from a new regimen. In other words, phenotypic results may be useful in telling someone that a drug doesn't work, but at least for the time being, it is less well-proven that it can tell someone if a drug or combination will work. One potential downside for genotypic testing is that resistance to one drug sometimes results in increased sensitivity of another drug. Probably the most discussed has been between AZT and 3TC (lamivudine, Epivir^®), where resistance to 3TC reverses the resistance (increases sensitivity) of AZT against HIV. There are other examples of this phenomenon that may make it difficult for people to interpret genotypic results. Clearly, there are pros and cons to both types of resistance tests.

For more geno- and phenotypic information which includes resistance charts, please call Project Inform's National HIV/AIDS Treatment Hotline and request the Geno- and Phenotypic Resistance Tests Hotline Handout.

Genotypic Testing

Phenotypic Testing

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The original of this article can be found at http://www.projinf.org/pub/25/resistance.html