The following information offers an in-depth discussion of genotypic drug resistance testing and an overview of Virtual Phenotyping. You will learn how the genetic makeup of HIV can be used to detect drug resistance, how genotypic testing differs from other resistance testing methods, and how it can help physicians begin to overcome the challenges of drug resistance.

• How can genetics be used to determine drug resistance?
• How is genotypic resistance testing performed?
• What do the results of genotypic testing mean?
• What is Virtual Phenotype testing and how does it compare to genotypic testing?
• Why is genotypic resistance testing important?
• What are the advantages and disadvantages of genotypic testing?
• Is genotypic testing approved by the FDA?
• How can someone get a genotypic resistance test?
• How is genotypic testing impacting HIV therapy?

Understanding genotyping must begin with a basic explanation of genetics. The human body contains instructions for all of its parts and functions within molecules known as DNA. These instructions not only determine things like the color of our eyes, but also what medical challenges we may face as we age. The instructions held by DNA are translated into action by RNA in a process known as protein synthesis. Interpreting our genetic code means understanding how DNA and RNA cause our bodies to grow, develop, and perform. With this in mind, a knowledge of which genetic patterns are associated with drug resistance can be used to guide antiviral therapy.

The genetic makeup of HIV
The genetic makeup of an organism is called its genotype. In HIV, the genotype is determined by the sequence of nucleotides in the genes of the virus. Nucleotides are the molecular building blocks of RNA and DNA. Three-nucleotide subunits are called codons, which determine where an amino acid is placed on a protein chain. The placement of an amino acid defines the structure of the protein or enzyme that is produced during replication.

During replication, if a mutation occurs at a specific codon, the amino acid that is normally found at a particular point on the protein chain will be replaced by a different amino acid. Because HIV does not have the ability to correct such mistakes, this change will remain, leading to structural changes in the protein produced. This can result in a viral mutation that is less susceptible to antiviral medications.

Genetic mutations
Genetic mutations are labeled by their point on the protein chain, the amino acid that is normally produced there, and the amino acid that results due to mutation. This can be illustrated by examining the M184V mutation, which signals the potential for resistance to 3TC (lamivudine, Epivir). The number is the position of the mutation: codon 184. The first letter, "M" stands for methionine, which is the amino acid that is normally found at codon 184. The second letter, "V" for the amino acid valine, represents the amino acid that has been produced as a result of the mutation. In this example, the amino acid methionine at position 184 has been replaced by the amino acid valine.

This particular mutation appears on the reverse transcriptase enzyme. The reverse transcriptase enzyme plays an important role in HIV replication. It translates the viral RNA into DNA so that the human host cell can understand its instructions. If the reverse transcriptase enzyme is blocked by specific antiviral medications (nucleoside reverse transcriptase inhibitors [NRTIs] or non-nucleoside reverse transcriptase inhibitors [NNRTIs]), the virus cannot complete the replication process.

When amino acid replacements occur on the reverse transcriptase gene, as with mutation M184V, a structural change occurs in the enzyme produced. This change prevents the antiviral drug from binding with the enzyme. If the drug does not bind with the intended enzyme, it cannot successfully stop the virus from reproducing.

The use of genetics in drug resistance testing
Genetics can be used to determine the potential for drug resistance by looking at the reverse transcriptase and protease enzymes of a person's virus. If mutations are present at specific codons of these enzymes, certain protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs), or non-nucleoside reverse transcriptase inhibitors (NNRTIs) may be less effective for that individual. In the above example, if a person's virus has mutation M184V, the antiviral 3TC is less likely to work for him or her. It is also important to note that a single mutation may mean resistance in more than one drug or in multiple drug classes.

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There are two main types of genotypic resistance testing: sequencing assays and point mutation assays. Sequencing assays scan the complete sequence of a gene, whereas point mutation assays look for mutations at specific locations, or codons, in the gene sequence. Though these assays differ in how the genetic structure is analyzed, they both start by using polymerase chain reaction (PCR) technology to amplify (copy) the viral RNA or DNA present in a patient's blood sample. PCR technology allows researchers to amplify specific genetic sequences in a person's HIV to create a sufficient amount for genotypic testing.

How the test is performed
Step 1- Sample is taken

Step 2- Copies of the virus are made (Using PCR technology, the genetic material of a person's HIV is copied numerous times)

Step 3- Genes within the virus are evaluated for mutations (Once amplified, particular viral enzymes [most commonly the reverse transcriptase and protease enzymes], are evaluated to determine whether genetic mutations are present)

Step 4- Mutations found in the virus are compared to known resistance mutations (Genetic mutations present in the patient sample are compared to preestablished drug resistance mutation patterns)

Results: If the type and number of mutations present in a person's HIV match preestablished mutation patterns for a particular drug, that individual's virus has probably developed resistance to that drug.

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If genotypic testing reveals drug resistance mutations in a person's HIV, certain antiviral drugs may be less likely to work for him or her. The specific type and placement of the mutations dictates which drugs the virus may be resistant to. For example, we previously discussed the M184V mutation. If the M184V mutation is discovered in a person's HIV, the virus is probably resistant to 3TC. Once a physician understands which drugs are less likely to work for his or her patient, he or she can develop a treatment plan that avoids the use of those drugs. This results in a therapy regimen that is more likely to be effective for a longer period of time.

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Virtual Phenotyping is actually a genotype test that uses a database of phenotypes to enhance the genotype results. After determining the genotype, the results are compared to a database of resistance results. This database is made up of resistance test results where both genotyping and phenotyping were performed on each patient. If a person's virus has a genotype that matches a genotype in the database, it is assumed that the phenotype matched to the genotype in the database will be the same for the person being tested.

Although Virtual Phenotyping is a type of genotypic testing, it is generally considered to be an advancement over standard genotypic testing. However, Virtual Phenotyping should not be confused with phenotypic testing which is actually a direct measure of resistance that is performed by exposing a person's HIV directly to antiviral drugs. Virtual Phenotyping is not a direct measure, it is a prediction based on genotypic analysis and database matches.
*Virtual Phenotype is a registered trademark of Virco, Inc.

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Genotypic drug resistance testing is an important tool in HIV therapy that gives physicians a more complete picture of a person's health and therapy options. The results of genotypic testing can help physicians determine which drugs may not work in a patient's therapy, allowing them to create treatment plans that are more likely to suppress the virus for a longer period of time.

In addition to helping physicians select more effective therapies, there are long-term benefits that arise from using resistance testing to direct treatment decisions. These include avoiding the use of potentially ineffective drugs, and in turn, reducing the health risks associated with medication side effects. By optimizing therapy regimens, genotypic resistance testing can also reduce the amount of money wasted on purchasing unnecessary medications.

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The main advantage of genotypic analysis is the relative simplicity of the testing method itself. This translates into faster turnaround time as compared to phenotypic analysis-genotypic testing can take as little as a few days to deliver results, whereas certain phenotypic tests can take as long as eight weeks (recent advances in phenotypic testing have resulted in an assay that can take as little as 8 days). Because genotypic analysis is simpler to perform, it is also less expensive than phenotypic testing ($300-$600 vs. $700-$1000).

Though the reduced cost and turnaround time make genotypic analysis a favorable method of resistance testing, it does have some notable disadvantages. Above all, genotypic testing is an indirect measure of drug resistance. That means that the virus never comes in contact with the drug during testing. As such, it can be difficult to come to a definitive conclusion about how a person's HIV will respond to a particular drug. In some cases, a drug may still be viable treatment option, despite the presence of resistance mutations.

In addition, genotypic tests rely on expert interpretation of complex genetic mutations. The information is then compared to preestablished resistance mutations, which can pose an additional challenge: because new mutations are constantly developing, it is virtually impossible to maintain a complete list of resistance mutations.

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Drug resistance testing does not require FDA approval. In fact, the FDA has stated that it does not want to regulate this type of test at this time. However, laboratories that perform drug resistance tests are regulated by the Health Care Finance Administration (HCFA), according to the requirements of the Clinical Laboratory Improvement Act (CLIA).

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People who are interested in having genotypic resistance testing need to request the procedure from their doctor. Once the patient requests a test, a member of his or her medical team will draw a sample of blood and send it to a laboratory where the test is performed. Once the laboratory receives the sample, results will be delivered to the physician in as little as a few days.

Genotypic testing can cost anywhere from $300 to $600. Most third-party payors, including Medicare, currently cover resistance testing; however, individual insurance carriers make their own determinations regarding coverage. Physicians may need to check with a patient's insurance carrier before ordering a test.

There are several companies that perform HIV drug resistance testing. Some of these companies offer help lines, staffed with insurance specialists who can assist people with insurance preapproval or reimbursement. Because these companies understand the important role drug resistance testing can play in HIV therapy, many also offer special programs for individuals with low incomes and no health insurance.

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Because drug resistance has become a significant factor in antiviral therapy, the way HIV is treated is evolving to include a more widespread use of genotypic resistance testing. By looking at the genetic makeup of HIV, physicians can gain greater insight into a patient's therapy options, giving physicians the information they need to optimize treatment regimens.

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