Important note: Information in this article was accurate in September 2001. The state of the art may have changed since the publication date.
HIV Drug Resistance and Drug-Resistance Testing: Just the FAQs
ACRIA Update, Vol. 10, No. 4, Fall 2001 Tim Horn
Just when everyone starts getting used to viral load tests, with all their confusing "logs" and "copies" and "undetectable levels," another family of lab tests with its own bewildering lingo arrives on the scene. Drug-resistance tests are, in fact, the most sophisticated forms of technology to be incorporated into the routine care of people living with HIV. In order to make sense of what these tests are, how they work, and the information they provide, it's necessary to understand why they are actually needed - to help demystify why HIV treatment sometimes fails and what can be done about it.
This article is based on a question-and-answer lesson plan used by CRIA to help people living with HIV and service providers better understand HIV drug resistance, the most common and frustrating reason for treatment failure. As with all of the articles in ACRIA Update, we encourage readers to use this information to communicate better with their healthcare providers. No question about drug resistance - including what you can do to help prevent it and the options you have if it occurs - is unimportant. We hope this review of frequently asked questions (FAQs) will help you and your healthcare provider decide when and how to use these tests and to make the most of the results they yield.
What is drug resistance?
Many germs can enter the human body and cause harm, including viruses, fungi, bacteria, and protozoa. Once inside your body, the primary goal of a germ is to survive and reproduce.
Most pharmaceutical drugs are designed to kill these germs or prevent them from reproducing. If a germ continues to reproduce during treatment, it can alter itself - or mutate - to avoid the drug. This is called drug resistance.
When drug resistance occurs, the drug - or combination of drugs - can't keep the germ from reproducing. Over time, the treatment can stop working completely.
How does HIV drug resistance occur?
HIV drug resistance means a loss in the ability of a drug - or combination of drugs - to block HIV reproduction in the body.
Drug resistance occurs due to mutations in HIV's genetic structure, which is in the form of RNA, a tight strand of proteins needed by the virus to infect cells and produce new virus.
HIV reproduces very rapidly and can't correct mistakes made during the copying of genetic material, so mutations are very common.
Two of the most important HIV enzymes are reverse transcriptase and protease.
Nucleoside analogues and non-nucleoside reverse transcriptase inhibitors target the reverse transcriptase enzyme, while protease inhibitors target the protease enzyme.
In order for these antiretroviral drugs to be effective, they must attach themselves to the necessary enzyme. Certain mutations can prevent a drug from binding with the enzyme and, as a result, make the drug less effective.
How do mutations occur before starting therapy?
Natural selection: Soon after HIV enters the body, the virus begins reproducing at a rapid rate - billions of new viruses are produced every day. In the process, HIV produces both perfect copies of itself (wild-type virus) and copies containing errors (mutated virus). In other words, there is no single virus in the body but, instead, a large population of mixed viruses called quasispecies.
Wild-type virus is the most natural and usually most powerful form of HIV and, as a result, reproduces the best. Before therapy is started, wild-type virus is the most abundant in the body and dominates all other quasispecies.
When HIV makes mistakes during copying, mutated viruses - called variants - are produced. Some variants are too weak to survive and/or can't reproduce. Others are strong enough to reproduce but still can't compete with the more fit wild-type virus. As a result, their numbers are less than wild-type virus in the body.
Some variants have mutations that allow the virus to partly, or even fully, resist an antiretroviral drug. This is why people living with HIV should never take just one antiretroviral (monotherapy). For example, HIV only requires one mutation to become completely resistant to Epivir (3TC). Similarly, a single mutation can cause resistance to all of the non-nucleosides.
These mutations occur randomly and there's no known way to prevent them. Variants usually don't go on to develop additional mutations; doing so compromises their ability to stay alive in the body. So, while these variants may be completely resistant to one drug, they're almost always sensitive to other drugs in a regimen. This is why three-drug combinations work better: a variant may be resistant to one of the drugs but doesn't stand much of a chance when facing two other drugs that bind to different parts of the virus.
Transmission of drug-resistant virus: According to some studies, between 10% and 30% of all new HIV infections (people infected within the past two years) involve strains resistant to at least one drug.
Many HIV-positive people now take or have taken antiretroviral therapy. If someone who has developed resistance to one or more of the antiretrovirals transmits the virus to someone else, their partner could now have a multiple-drug-resistant (MDR) variant of HIV.
If this person were to start therapy later on with any of the antiretrovirals that the first person had developed resistance to, it might be difficult to reduce viral load or keep viral load undetectable.
How do mutations occur during therapy?
Soon after antiretroviral therapy is started, the amount of virus in the body is reduced dramatically. Unfortunately, no combination completely stops HIV from reproducing. There's always a small population of virus that continues to reproduce.
Therapy reduces the amount of all HIV quasispecies in the body. The amount of wild-type virus is dramatically reduced, as is the number of variants.
Because wild-type virus is usually the most sensitive to antiretrovirals, HIV variants in the body may have a survival advantage. In the presence of therapy, variants can become the dominant strain of HIV, even though there is much less virus in the body.
Over time, variants accumulate additional mutations. Some of these mutations will harm the virus, while others will further limit a drug's ability to block reproduction. Once the virus has accumulated enough mutations, the drugs lose their ability to bind to it and prevent it from reproducing. As the drugs become weaker, the amount of drug-resistant virus in the body increases. An undetectable viral load can become detectable again and increase over time. If the drug-resistant virus continues to reproduce, it can acquire even more mutations to resist the antiretrovirals completely.
Mutations that emerge during therapy can be divided into primary mutations and secondary mutations. Each antiretroviral is associated with at least one primary mutation. Primary mutations cause the greatest amount of drug resistance. Secondary mutations don't cause drug resistance unless a primary mutation is present. If both primary and secondary mutations are present, drug resistance becomes more complicated.
Primary and secondary mutations usually have a negative effect on the power of the virus. This is why some people who experience an increase in their viral load might not see a decrease in their CD4+ cell counts, at least not at first. In other words, the virus may lose its ability to cause damage to the immune system if it contains drug-resistance mutations. However, studies show that some mutations can cause the virus to regain its power and possibly become even more powerful than wild-type virus.
Cross-resistance can also occur during therapy. When HIV becomes resistant to one drug, it can automatically be resistant to other drugs in the same class. For example, the primary and secondary mutations that occur in someone who is taking the protease inhibitor Crixivan (indinavir) are the same mutations that cause resistance to Norvir (ritonavir). Even though the person hasn't taken Norvir, he or she will likely be cross-resistant to the drug and probably wouldn't benefit from it.
The key to avoiding the accumulation of mutations that cause resistance and cross-resistance is to keep the amount of virus in the body as low as possible, for as long as possible.
What factors contribute to the accumulation of drug-resistance mutations during therapy?
Don't forget the golden rule: the less virus there is in the body, the less likely it is that mutations will develop. A powerful regimen is the most effective way to keep the level of virus low - preferably undetectable - and to delay additional mutations from occurring.
There are a number of factors that can prevent your regimen from being as powerful as it can be:
Poor adherence: For drugs to work correctly, they must be taken exactly as prescribed. This means taking the correct number of pills each day, being careful to take them a certain number of hours apart, while also following dietary requirements.
Skipping doses or taking medication incorrectly can cause the trough level of a drug to decrease in the body. The trough level is the amount of drug left in your body just before another dose is taken. If the trough level becomes too low, HIV can reproduce more freely and accumulate additional mutations.
According to a few research reports, an HIV-positive person must be more than 95% adherent with his or her regimen in order for it to continue working properly. This means missing less than one dose a month.
Poor absorption: Not only must antiretrovirals be taken on schedule, they also need to be absorbed effectively into the bloodstream. A drug - or combination of drugs - that isn't absorbed properly can result in trough levels that are too low and, ultimately, allow HIV reproduction and the accumulation of drug-resistance mutations.
Some drugs have specific dietary requirements. For example, people taking standard doses of Crixivan must take the drug every eight hours on an empty stomach. This means not eating within two hours before or one hour after taking the drug. (Note: If Crixivan is taken in combination with Norvir, food restrictions don't apply.) Conversely, Fortovase (saquinavir) should be taken with food, preferably food containing a moderate amount of fat. If dietary requirements aren't followed, drug levels in the body will decrease.
Diarrhea and vomiting can cause medications to be expelled from the gut too quickly, reducing the amount of drug absorbed into the bloodstream.
Varying pharmacokinetics: Pharmacokinetics is a term used to mean how a drug is absorbed, distributed, metabolized, and removed from the body.
Even though two people might receive the exact same dose of a drug, the amount of drug may be higher in one person's bloodstream than in the other's bloodstream. Factors such as body weight, height, age and gender can contribute to this difference. Some people also process, or metabolize, drugs faster or slower than others do. This can speed up - or slow down - the rate at which a drug is cleared from the body.
A drug's correct dose - the dose dispensed by pharmacists - is the average dose found to be safe and effective in clinical trials. In other words, some people may be able to keep their viral load undetectable using lower doses of the drug, while others might require higher doses.
In the future, healthcare providers may perform blood tests to measure the amount of drug in their patients' bodies. This is called therapeutic drug monitoring and it may help determine whether or not you have the correct trough level of each medication.
Does a rebound in viral load mean that drug resistance has occurred?
Figuring out if a regimen isn't working properly can be determined in three ways:
A viral load that fails to go undetectable within the first few months of therapy.
A viral load that goes from being undetectable to detectable (note: a one-time "blip" in viral load isn't usually a sign that a drug regimen is no longer working).
A detectable viral load continues to increase, even though you're still on therapy.
While a viral load test can help determine whether or not a regimen is still working correctly, it can't explain why a regimen is no longer working the way it should.
A detectable or increasing viral load doesn't necessarily mean that drug-resistance mutations have developed. It may be due to poor adherence or poor absorption. While these can eventually lead to the emergence of drug-resistance mutations, viral load can become detectable before they develop. It's important to determine the reason why viral load is increasing soon after it becomes detectable.
If resistance mutations have developed, viral load tests can't determine whether or not the virus is resistant to one specific drug or the entire regimen. Moreover, if you have drug-resistant HIV, viral load testing can't determine which drug or combination of drugs is likely to be the most effective in the future.
There are two tests, or assays, that look for drug resistance. Genotypic testing can help determine whether specific mutations are causing drug resistance and drug failure. Phenotypic testing is a more direct measure of resistance and, more specifically, the actual sensitivity of your HIV to individual antiretrovirals.
What is genotypic testing?
Genotypic resistance testing examines the actual structure - or genotype - of your HIV (a standard blood sample is all that's required). The HIV is examined for the presence of specific mutations that are known to cause resistance to certain drugs.
For example, we know that Epivir isn't effective against HIV that contains the M184V mutation in its reverse transcriptase enzyme. If a genotypic test discovers this mutation, chances are that your HIV is resistant to Epivir and won't respond to the drug.
Many drugs, including the protease inhibitors, require complex patterns of mutations for resistance to occur.
With a genotypic test, the genetic sequences of particular viral enzymes - such as reverse transcriptase and protease - are examined carefully for mutations. Depending on the type and number of mutations found, the lab can determine whether someone has developed resistance to a specific drug, since almost all drugs follow a set pattern of mutations.
There are actually two types of genotypic tests: sequencing assays and point-mutation assays. Sequencing assays look for any mutation in either the reverse transcriptase or protease enzymes. Point-mutation assays look for key mutations in these enzymes that are known to cause drug resistance. Most labs use point-mutation assays, as they are easier (and cheaper) to perform and their results are easier to interpret.
For genotypic tests to be accurate, they generally require a blood sample from someone who is currently on therapy and has a viral load higher than 1,000 copies/mL.
If you stop therapy before blood is drawn, the wild-type virus in your body may outgrow the mutant virus. This would result in the genotypic test "reading" the wild-type strain, which wouldn't show any signs of resistance. In other words, it's important that you're still taking your drugs at the time of genotypic testing.
Genotypic testing can take as little as a few days to complete, and a single genotypic test costs between $300 and $500. Many public and private health insurance programs cover the cost.
How are genotypic test results reported?
When genotypic testing results come back from the lab, they list the mutations that were found in the virus' reverse transcriptase and protease enzymes. It's important to understand how these mutations are reported.
An example: The M184V mutation is responsible for causing resistance to Epivir. The 184 refers to the amino acid position, or codon, in the reverse transcriptase enzyme. The M (methionine) is the amino acid at position 184 of a wild-type virus' reverse transcriptase enzyme. The V (valine) refers to the mutation that results in drug resistance. In other words, the amino acid methionine at position 184 has been replaced by a valine, which prevents Epivir from binding with the enzyme to keep the virus from reproducing.
While researchers have identified a number of mutations that can cause drug resistance, they don't know everything there is to know about these mutations. We know that some combinations of mutations cause HIV to become more resistant than other combinations of mutations. Researchers are still trying to determine which sequences of mutations are the most important.
Some genetic mutations have yet to be fully identified. This is the case with Videx (ddI) and Zerit (d4T). Resistance certainly occurs with these drugs, but researchers are only beginning to determine which mutations cause HIV to become less sensitive to them.
Mutations known to cause resistance to Retrovir (AZT) and Epivir can also be misleading. A genotypic test may show that your HIV has several mutations that cause resistance to AZT. However, if you're also taking Epivir - which seems to increase HIV's sensitivity to AZT - these mutations may not accurately reflect the degree of AZT resistance.
Another limitation: genotypic tests don't evaluate the genetic structure of small HIV populations found in a blood sample. Unless a particular strain accounts for more than 20% of the HIV in a sample, chances are that it won't be recognized by the test.
What is phenotypic testing?
Unlike genotypic testing, which looks for particular genetic mutations that cause drug resistance, phenotypic testing directly measures the sensitivity - or phenotype - of your HIV in response to particular antiviral drugs.
Phenotypic resistance tests measure the concentration of a drug required to inhibit viral replication in the test tube by a defined amount such as 50% or 95%. This is called IC50 or IC95 (IC stands for inhibitory concentration). If it only takes a standard amount of the drug to stop HIV from reproducing - a concentration equal to the usual dose - HIV isn't resistant to the drug. If higher amounts of the drug are needed, HIV is considered to be resistant to that drug.
The concentration of drug necessary to inhibit virus replication is expressed in nanomoles (nM). For example, if 100nM of a particular drug is needed to suppress wild-type HIV, but it takes 400nM to suppress virus found in your blood sample, your HIV is said to be fourfold resistant to the drug being tested. In other words, your HIV is four times less sensitive to the drug.
Unlike genotypic tests, phenotypic tests generally don't require a high viral load. Like genotypic tests, however, it is recommended that you be taking therapy when blood is drawn for phenotyping.
Because phenotypic testing directly measures the sensitivity of HIV to particular drugs, many researchers believe that these tests are more comprehensive and trustworthy than genotypic tests.
Phenotypic testing procedures are relatively complex and can take longer than genotypic tests to produce accurate results - from ten days to several weeks. They're also more expensive. A single phenotypic test can cost between $700 and $900. Not all public and private health insurance programs cover phenotypic testing - be sure to check with your healthcare provider before having the test done.
Phenotypic tests can't evaluate the sensitivity of small HIV populations found in a blood sample. Unless a particular strain accounts for more than 10% to 20% of the HIV population in the sample, chances are that it won't be recognized.
Another challenge is that we still don't fully understand what level of resistance translates into treatment failure. For example, a five-, six-, or sevenfold reduction in the sensitivity of HIV to a protease inhibitor is considered "moderate." But is there a significant difference between a fivefold reduction and a sevenfold reduction? Researchers are still trying to figure out what level of resistance means that a drug is no longer useful.
Can drug-resistance tests be used before you first start therapy?
Maybe. Based on what is known about HIV's error-prone reproduction process, it's safe to assume that everyone has at least a few strains of virus that are resistant to individual drugs before therapy is started. However, these strains are often too limited in number and strength to compete with wild-type virus and stand a good chance of being killed off when you start therapy. In other words, genotypic or phenotypic testing might not provide an accurate picture of drug resistance before therapy is started.
Drug-resistance tests might be useful for people infected with multiple-drug-resistant (MDR) strains of HIV. Soon after an MDR strain enters the body, it begins reproducing. Over time, a wild-type strain dominates the viral population. In order for resistance tests to be useful, blood will probably need to be drawn soon after infection takes place (within a few weeks). Only a small percentage of people know when they're infected or immediately see a healthcare provider.
Can drug-resistance tests be used to choose a new drug regimen after an initial one fails?
Yes. Viral load tests can help determine whether or not drug failure is occurring, but resistance tests may play an invaluable role in helping you and your doctor understand why failure has occurred and what treatment options are still available.
If viral load fails to become undetectable or becomes detectable again after being undetectable, resistance testing may help determine the cause. If no mutations are present (using genotypic assays) or the HIV is still sensitive to the drugs you're taking (using phenotypic assays), the problem might be poor adherence or poor absorption. It's best to resolve these problems before resistance mutations develop.
If mutations are found or HIV is determined to be losing sensitivity to the drugs being used, resistance tests can help determine which of the remaining antiretrovirals might be effective.
Without resistance tests, it's recommended that anyone who appears to be failing a combination should switch to an entirely new batch of drugs. This can be frustrating, as many people don't have three or more untried drugs to choose from. It may also be a wasteful decision if your virus is still sensitive to some of the drugs you're currently taking. A number of studies conducted over the past few years have shown that, when viral load rebounds while on treatment, the virus is usually not resistant to all three (or more) drugs being used. So it's always good to know which drugs the virus has become resistant to and which drugs the virus remains sensitive to.
Resistance tests may be able to help you and your doctor choose a new regimen after an initial regimen has failed, possibly helping to weed out the ineffective drug or drugs in a given combination.
Resistance testing can also help determine what can be done about partial resistance. For example, a phenotypic test might determine that HIV is partially - as opposed to completely - resistant to a certain protease inhibitor (e.g., Crixivan). In this case, it might be possible to add a low dose of Norvir to increase the amount of Crixivan in the body. By increasing Crixivan levels, there's more drug available to combat the partially resistant virus.
How can drug resistance be avoided?
Learn as much as possible about anti-HIV drugs. The more you know, the easier it will be to make treatment choices that can help avoid drug resistance.
Start treatment with a powerful anti-HIV regimen. The first drug regimen you take may be your best chance to fully suppress the virus and prevent the development of drug resistance.
Follow instructions. It's very important to take your medications exactly as prescribed. Missing doses, not taking the right number of pills, or eating when pills need to be taken on an empty stomach, can all cause viral load to increase and drug-resistance mutations to develop.
Good communication with a healthcare provider. Asking questions and reporting any problems to your healthcare provider are important for avoiding drug resistance.
Regular viral load testing matters. An increasing viral load is often the first sign that drug resistance is developing. Monitoring viral load regularly is a good way to guard against resistance.
Tim Horn is executive editor of the PRN Notebook, published by Physicians' Research Network in New York.
AEGiS is made possible through unrestricted grants from Boehringer Ingelheim, iMetrikus, Inc., the National Library of Medicine, and donations from users like you. Always watch for outdated information. This article first appeared in 2001. This material is designed to support, not replace, the relationship that exists between you and your doctor.
AEGiS presents published material, reprinted with permission and neither endorses nor opposes any material. All information contained on this website, including information relating to health conditions, products, and treatments, is for informational purposes only. It is often presented in summary or aggregate form. It is not meant to be a substitute for the advice provided by your own physician or other medical professionals. Always discuss treatment options with a doctor who specializes in treating HIV.
Important note: Information in this article was accurate in September 2001. The state of the art may have changed since the publication date.
