Understanding Drug Resistance & The Tests to Measure It

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What is drug resistance?

There are many types of germs, or pathogens, that can enter the human body. These include viruses, fungi, bacteria, and protozoa. Once inside the body, the primary goal of these germs is to survive and reproduce.

Pharmaceutical drugs are designed to target these germs and either kill them or prevent them from reproducing inside the body. If these germs continue to reproduce during treatment, they can alter themselves – or "mutate" – to avoid the drugs. This is called drug resistance.

When drug resistance occurs, the drug – or combination of drugs – loses its ability to block the germ from reproducing. Over time, the treatment can stop working completely.

How does HIV drug resistance occur?

HIV drug resistance means a reduction in the ability of a drug – or combination of drugs – to block HIV reproduction in the body.

Drug resistance occurs as a result of changes, or mutations, in HIV's genetic structure. HIV's genetic structure is in the form of RNA, a tight strand of proteins and enzymes needed by the virus to infect cells and produce new virus. Mutations are very common in HIV. HIV reproduces at an extremely rapid rate and does not contain the proteins needed to correct the mistakes it makes during copying.

Two of the most important HIV enzymes are reverse transcriptase and protease. Nucleoside analogues – also called Nucleoside Reverse Transcriptase Inhibitors (NRTIs) – and Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) target the reverse transcriptase enzyme. Protease Inhibitors (PIs) target the protease enzyme.

Another important protein is gp120, which is found out on the outer coat – the envelope – of HIV. This protein is the target of Fuzeon® (enfuvirtide; T-20), an anti-HIV drug that was approved in 2003. Fuzeon prevents HIV from binding to healthy T-cells in the body, which prevents these T-cells from becoming infected with the virus.

In order for these anti-HIV drugs to be effective, they must first 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 against the virus.

HIV drug-resistance mutations can occur both before and during anti-HIV drug therapy.

How do mutations occur before anti-HIV therapy is started?

Mutations that occur before anti-HIV drug therapy is started can happen in two ways: natural selection and transmission of drug-resistant virus.

Natural Selection

Soon after HIV enters the body, the virus begins reproducing at a rapid rate (approximately 10 billion 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, the body doesn't carry just one type of virus, but actually carries a large population of mixed viruses called quasi-species.

When HIV reproduces, it wants to be wild-type virus. This is the most natural and powerful form of the virus and, as a result, reproduces the best. Before anti-HIV therapy is started, wild-type virus is the most abundant in the body and dominates all other quasi-species.

When HIV makes mistakes during copying, mutated viruses – called variants – are produced. Some variants are too weak to survive and/or cannot reproduce. Other variants are strong enough to reproduce but are still not able to compete with the more fit wild-type virus. In turn, their numbers are less than wild-type virus in the body.

Some variants have mutations (sometimes called polymorphisms) that allow the virus to partly, or even fully, resist an anti-HIV drug. This is why HIV-positive people should never take just one anti-HIV drug (monotherapy). For example, HIV only requires one mutation in the reverse transcriptase enzyme – called "M184V" – to become completely resistant to Epivir® (3TC). The same problem holds for the non-nucleoside reverse transcriptase inhibitors Viramune® (nevirapine), Rescriptor® (delavirdine), and Sustiva® (efavirenz). The "K103N" mutation can cause the virus to become highly resistant to these drugs.

By the way, don't worry about needing to learn what "M184V" or "K103N" mean – these are just coded names that scientists have given to each genetic mutation they've identified while studying HIV drug resistance.

These HIV mutations occur randomly and there is no proven way to prevent them from occurring. Variants containing these mutations usually don't go on to develop additional mutations; doing so compromises their ability to stay alive in the body. Thus, while these variants may be completely resistant to one anti-HIV drug, they are almost always sensitive to other drugs used in a regimen. This is why three-drug regimens 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 same enzyme or different parts of the virus.

Transmission of Drug-Resistant Virus

Many HIV-positive people now take anti-HIV drugs. If someone has developed resistance to one or more of these anti-HIV drugs and has unprotected sex or shares needles with someone who is not infected with the virus, it is possible that they can infect their partner with a drug-resistant variant – a strain of HIV containing mutations that cause resistance to one or more anti-HIV drugs.

Here's an example:

Daniel is HIV-positive and has been taking a triple-drug anti-HIV regimen consisting of Crixivan, Retrovir, and Epivir. He does not know it, but he has a detectable viral load and his virus contains mutations associated with resistance to these three drugs. One night, he has unprotected sex with Tracy, an HIV-negative woman. Daniel's virus then enters Tracy 's body and begins reproducing. The end result is that Tracy has been infected with a multiple-drug-resistant (MDR) variant of HIV.

At first, the MDR variant in Tracy 's body would dominate all other viruses that are produced during copying. Over time, a wild-type virus will emerge and usually comes to dominate the MDR variant. But this does not mean that the MDR variant is gone; it has merely become a minority member of the entire population of HIV.

If Tracy were to start therapy a few years later with Crixivan, Retrovir, and Epivir, the wild-type HIV would diminish quickly, but would probably be replaced with the MDR variant already in her body. As a result, Tracy might have a difficult time reducing her viral load or keeping her viral load undetectable, because she was infected with a drug-resistant variant of HIV.

According to some studies, between 10% and 30% of all new HIV infections – defined as people infected with HIV over the past two years – involve strains resistant to at least one anti-HIV drug. To make matters worse, many researchers expect that this percentage will increase in the years to come, and that many more people will become infected with strains of HIV resistant to multiple drugs.

It might also be possible for someone who is already infected with HIV to be infected, again, with a drug-resistant strain of HIV. This is sometimes referred to as "reinfection" or "superinfection."

How do mutations occur during anti-HIV drug treatment?

Soon after anti-HIV drug treatment is started, the amount of virus in the body is reduced dramatically. Unfortunately, no anti-HIV drug – or combination of drugs – is able to completely stop HIV from reproducing. In other words, there is always a small population of virus in the body that continues reproducing, despite the presence of anti-HIV drugs.

In answering the previous question, "How do mutations occur before anti-HIV therapy is started?" we mentioned that there is a large mixture of virus in an HIV-infected person's body. Anti-HIV drug therapy reduces the amount of all HIV quasi-species in the body. The amount of wild-type virus is dramatically reduced and the number of variants is also decreased.

Even though wild-type virus is the most natural and powerful form of HIV, it is the most sensitive to anti-HIV drugs. Because of this, HIV variants in the body have a survival advantage over that of wild-type virus. In the presence of anti-HIV drug therapy, variants can become the dominant strain of HIV, even though there is a much smaller amount of HIV 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 stop it from reproducing. Once the virus has accumulated enough mutations, the anti-HIV 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, causing an undetectable viral load to become detectable again and increase over time. Should the drug-resistant virus continue to reproduce, it can acquire even more mutations to resist the anti-HIV drugs completely.

Mutations that emerge during therapy can be divided into two groups: primary mutations and secondary mutations. Each anti-HIV drug is associated with at least one primary mutation. This mutation is of greatest concern, as they are the ones that cause the greatest amount of drug resistance. Secondary mutations do not cause drug resistance unless a primary mutation is present. If both primary and secondary mutations are present, drug resistance can become more complicated.

While primary and secondary mutations can cause the virus to become resistant to anti-HIV drugs, they usually have a negative effect on the power of the virus. This is why some people who are experiencing an increase in their viral load might not see a decrease in their T-cell counts, at least not initially. In other words, the virus loses its ability to cause damage to the immune system if it contains drug-resistance mutations. However, some studies have shown that certain primary and secondary mutations can cause the virus to regain its power and, quite possibly, become even more powerful than wild-type virus. In turn, most experts recommend switching therapies before the virus accumulates any additional mutations.

Cross-resistance can also occur during therapy. When HIV becomes resistant to one drug, it can automatically become resistant to other drugs in the same class. For example, the primary and secondary HIV mutations that occur in someone who is taking the protease inhibitor Crixivan® (indinavir) are the same mutations that cause resistance to the protease inhibitor Norvir® (ritonavir). Even though the person hasn't yet taken Norvir, he or she will likely be cross-resistant to the drug and will not likely benefit from it.

What are some of the factors that contribute to the accumulation of drug-resistance mutations during therapy?

If there's one "golden rule" of anti-HIV drug treatment, it is: the less virus there is in the body, the less likely it is that the virus will continue reproducing and mutating. A powerful anti-HIV regimen is the most effective way to keep the level of virus low – preferably "undetectable" (<50 copies/mL as measured by a sensitive viral load test) – and to delay additional mutations from occurring.

Unfortunately, there are a number of factors that can prevent an anti-HIV drug regimen from being as powerful as it can be. These include:

My viral load has become detectable! Does this mean that drug resistance has occurred?

Figuring out if your anti-HIV drug regimen is no longer working properly can be determined in three ways:

1. Your viral load fails to go undetectable within the first few months after starting an anti-HIV drug regimen.
2. Your viral load goes from being undetectable to detectable (note: a one-time "blip" in viral load is not usually a sign that a drug regimen is no longer working).
3. Your detectable viral load continues increasing, even though you are still taking your prescribed anti-HIV drug regimen.

However, while viral load can help you determine whether or not your anti-HIV drug regimen is still working correctly, it cannot explain why your regimen is no longer working the way it should.

The fact is, a detectable or increasing viral load does not necessarily mean that drug-resistance mutations have occurred. A detectable viral load 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. Thus, it is important to act quickly and determine the reason why viral load is increasing soon after it becomes detectable.

If resistance mutations have developed, viral load tests cannot determine whether or not the virus is resistant to one specific drug or the entire regimen. Also, in a person with drug-resistant HIV, viral load testing cannot determine which drug or combination of drugs is likely to be the most effective in the future.

To look for drug resistance, there are two tests, or assays, available to people living with HIV and their healthcare providers. The first is called genotypic testing. Genotypic tests can help determine whether specific mutations are causing drug resistance and drug failure. The second method, called phenotypic testing, is a more direct measure of resistance and, more specifically, of the sensitivity of a person's HIV to particular anti-HIV drugs.

What is genotypic resistance testing?

Genotypic resistance testing examines the actual genetic structure – or genotype – of HIV taken from a patient (a standard blood sample is all that is required). The HIV is examined for the presence of specific genetic mutations that are known to cause resistance to certain drugs.

An example: As addressed under the previous question, "How do mutations occur before anti-HIV therapy is started?" researchers know that Epivir® (3TC) is not effective against forms of HIV that contain the mutation "M184V" in its reverse transcriptase enzyme. If a genotypic resistance test discovers a mutation at position M184V, chances are that the person's HIV is resistant to Epivir and is not likely to respond to the drug.

For many drugs, including the protease inhibitors and other nucleoside analogues, complex patterns of mutations are required for resistance to occur.

To conduct a genotypic test, laboratories use something called "PCR technology" to make many copies of, or "amplify," the HIV genetic material. Once amplification has been completed, the genetic sequences of particular viral enzymes – such as reverse transcriptase and protease – can be examined carefully for mutations. Depending on the type and number of mutations found, the laboratory 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: point-mutation assays and sequencing assays ("assay" is another word for "test"). 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 laboratories 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 the use of a blood sample from a person who is actively taking anti-HIV medication and has a viral load higher than 1,000.

If therapy is stopped before blood is drawn for a genotypic test, the wild-type virus in the body may outgrow the mutant virus. In turn, the results may not show any drug-resistant mutations, but the drug-resistant strain may still remain at very low numbers in the person's body and may quickly increase when therapy with the same drugs is restarted.

Genotypic resistance testing can take as little as a few days to complete. A single genotypic test can cost between $300 and $500, but they are usually covered by health insurance policies and other types or reimbursement programs.

What are some of the limitations of genotypic resistance testing?

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 causes the virus to become more resistant to anti-HIV drugs than other combinations of mutations. Researchers are still trying to determine which sequences of mutations are the most important.

It is also true that some genetic mutations have yet to be fully identified by researchers. Such is the case with drugs like Videx® (ddI), Viread® (tenofovir), and Kaletra® (lopinavir/ritonavir). In people who take these drugs, resistance certainly does occur. However, researchers are only beginning to determine the exact genetic mutations that cause HIV to become less sensitive to these drugs.

Mutations known to cause resistance to Retrovir® (AZT) and Epivir® (3TC) can also be misleading. For example, a genotypic resistance test may show that a person's HIV has several genetic mutations that cause resistance to Retrovir. However, if the person is also taking Epivir – which appears to increase HIV's sensitivity to Retrovir – such genetic mutations may not accurately reflect the amount of Retrovir resistance.

And here's another limitation to consider: genotypic tests do not evaluate the genetic structure of small HIV populations found in a blood sample. For example, there might be a population of HIV that contains a mutation at position "M184V" (the mutation that causes resistance to Epivir). Unless this particular strain accounts for more than 20% of the HIV population found in a blood sample, chances are that it will not be recognized by the test.

What is phenotypic resistance testing?

Unlike genotypic testing, which looks for particular genetic mutations that causes drug resistance, phenotypic testing directly measures the sensitivity – or phenotype – of a patient's HIV in response to particular antiviral drugs.

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

These results tell doctors how much of a particular drug is needed to stop the growth of HIV by 50% (compared to how much is needed to stop the wild-type virus by 50%). This is called IC50, where "IC" stands for "inhibitory concentration." In other words, a laboratory conducting a phenotypic test is trying to determine the amount of drug needed to stop HIV from reproducing. If it only takes a standard amount of the drug – a concentration equal to those commonly used by HIV-positive people – HIV is not resistant to the drug. If higher amounts of the drug are needed to stop HIV from reproducing, HIV is considered to be resistant to the drug being tested.

This allows doctors to "quantify" a patient's resistance level. If an patient shows resistance to a particular drug, that drug may still be effective, as long the patient’s level of resistance is not too high. The maximum level of resistance that someone can have before a drug is no longer considered to be effective is called a cutoff value. With this information, rather than immediately eliminating a drug because resistance is detected, doctors can evaluate a patient’s level of resistance to determine whether or not the drug is a still a viable treatment option.

Unlike genotypic tests, the phenotypic resistance test generally does not require a high viral load – the test can produce results even if your viral load is less than 1,000, but probably requires a detectable viral of over 500. Like genotypic testing, however, it is recommended that patients be taking anti-HIV therapies at the time blood is drawn for the test.

Because phenotypic testing directly measures the sensitivity of the virus to particular drugs, many researchers believe that these tests are more comprehensive and trustworthy than genotypic tests.

Sample Report.

Phenotypic Resistance Test: A Sample Report

What are some of the limitations of phenotypic resistance testing?

Phenotypic resistance testing procedures are relatively complex and can take longer than genotypic tests to produce accurate results – from 10 to 14 days. They are also more expensive than genotypic tests. A phenotypic test can cost between $750 and $900, but they are usually covered by health insurance policies and other types or reimbursement programs.

Like genotypic resistance tests, phenotypic tests cannot evaluate the sensitivity of small HIV populations found in a blood sample. For example, there might be a population of HIV that is not sensitive to Epivir. Unless this particular strain accounts for more than 10% to 20% of the HIV population found in a blood sample, chances are that it will not be recognized by the test.

Another challenge is that researchers still do not fully understand what level of resistance translates into a failure of treatment. As mentioned before, this is called the cutoff value – it's not always clear what this level is for each drug. 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 determines that a drug is no longer useful.

What about using genotypic and phenotypic tests together?

Using both tests together could certainly help deal with some of the weaknesses of each test administered individually.

One company, ViroLogic, will phenotype and genotype a blood sample and provide the results of both tests on the same lab report – called PhenoSense GT™. The results of both tests are usually available in the same amount of time it takes to run a phenotypic resistance test (approximately two weeks). Even though this combination of tests costs more and takes longer than a standard genotypic test, you and your doctor will have a more comprehensive resistance profile with which to make treatment decisions than if you'd only run a single test.

Another company, Tibotec-Virco, uses a test called VirtualPhenotype™. This test uses its own genotypic testing results to figure out what the virus's phenotype is, without actually performing a phenotypic test. To do this, it first analyzes HIV's genotype. Once the genotype has been determined, the laboratory searches a database containing the genotypes of several thousand HIV samples collected from other patients. It then retrieves the phenotypes – the IC50s – that correspond to these samples, averages the information together, and predicts the drugs that the current sample will be sensitive to or resistant to.

Can drug-resistance tests be used before someone starts anti-HIV therapy for the first time?

Maybe. Based on what is known about HIV's error-prone reproduction process, it is safe to assume that all HIV-infected people have at least a few forms of HIV 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 they stand a good chance of being killed off, once combination anti-HIV therapy is started. In other words, genotypic or phenotypic testing might not provide an accurate picture of drug resistance before therapy is started.

Drug-resistance tests might prove to be useful for people recently 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 of HIV emerges and dominates the viral population. Thus, in order for drug-resistance tests to be used, blood will probably need to be drawn soon after infection takes place (i.e., within several weeks, or possibly a couple of months after infection occurs). Unfortunately, only a small percentage of people know when they are infected or immediately go to see a healthcare provider. If a patient knows that they were infected recently and decides with their doctor to start treatment, they should start before resistance test results are available. Treatment regimens can then be adjusted within a few weeks if resistance to any drug is detected.

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. Drug-resistance tests, on the other hand, may play a valuable role in helping doctors and their patients understand why failure has occurred and what treatment options are still available.

If viral load fails to become undetectable after a new treatment regimen is started, or becomes detectable again after a period of being undetectable, drug-resistance testing may help determine the cause. If no mutations are present (using genotypic assays) or the HIV is still sensitive to the drugs being used (using phenotypic assays), the problem might be poor adherence/compliance or poor absorption. It is best to remedy these problems before resistance mutations develop.

If mutations are found or HIV is determined to be losing sensitivity to the drugs being used, drug-resistance tests can help determine which of the remaining anti-HIV drugs might be effective against the virus.

There have been a number of studies demonstrating that both genotypic tests and phenotypic tests can help patients and their healthcare providers choose a new regimen after an initial regimen has failed. Patients who use drug-resistance tests may be able to keep their viral load undetectable for a longer period of time than those who do not use the tests.

With drug resistance testing, it might also be possible to weed out the ineffective drug or drugs in a given combination. For example, in a study published in the Journal of the American Medical Association in January 2000 involving people taking an anti-HIV combination of Crixivan® (indinavir), Retrovir® (AZT) and Epivir® (3TC), 17 patients experienced viral load increases while receiving therapy. Although it would make sense to blame such viral load increases on multiple-drug resistance, resistance tests demonstrated that 14 patients had developed resistance to Epivir only – HIV in these patients could generally still be blocked by Crixivan.

Drug-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 simply add another drug [e.g., a low dose of Norvir® (ritonavir)] to increase the amount of Crixivan in the body. By increasing the amount of Crixivan, there is more drug available to combat the partially-resistant virus.

How can drug resistance be avoided?

There are a number of steps that HIV-positive people can take to prevent – or at least slow down – the development of resistance:

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