HIV-1 Genotypic Drug Resistance Mutation Analysis, with Reflex to Phenotypic Drug Resistance Prediction, Plasma
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Antiviral resistance may compromise highly active antiretroviral therapy (HAART) in HIV-infected patients receiving HAART. When combination therapy fails, detection and analysis of HIV genotypic mutations can guide necessary changes to antiretroviral therapy and decrease HIV viral load, thereby improving patient outcome.
HIV-1 is an RNA virus that infects cells and is then converted to complementary DNA by the action of the viral reverse transcriptase (RT) gene product. RT has little proofreading capacity and therefore incorporates errors in the proviral DNA. These errors are transcribed into infectious viral particles when the proviral DNA is transcribed into RNA. Similarly, the enzyme protease catalyzes a polyprotein to produce peptides necessary for active viral replication. Although HAART (combination of nucleoside analog, nonnucleoside agent and/or protease inhibitor) may be effective in reducing the viral load, genotypic mutations arising in the drug-targeted HIV gene loci due to selective pressure from antiviral therapy result in antiviral resistance that may compromise such therapy.
Amplification and analysis of drug-targeted HIV gene sequence allows identification of changes in nucleotide bases and associated amino acid codons that may cause antiviral drug resistance. Such genotypic changes are deemed as mutations by comparing the sequence data of the patient's HIV strain to those of a wild-type HIV strain. The significance of these genotypic mutations in relation to antiviral resistance is then determined by a set of interpretive rules developed by a consensus panel of leading experts in the field of HIV resistance. Relevant data presented at a recognized scientific conference or published in peer-reviewed journals are considered by the consensus panel in developing these rules. When necessary, reliable unpublished drug resistance data known to consensus panel members may be considered in the process. The interpretive rules are updated by the consensus panel annually after reviewing newly published data on HIV genotypic drug resistance mutations.
Phenotypic assays for antiviral drug susceptibility determine the amount of drug needed to inhibit viral growth in cell culture. The amount of drug needed to inhibit virus growth by 50% is called the 50% inhibitory concentration or IC50. Similarly, the concentration of drug that inhibits virus growth by 95% is known as the IC95. Testing a particular drug against a large number of isolates from patients who never received antiretroviral therapy can determine the average IC50 for wild-type HIV-1 isolates. Viruses that are inhibited by the same or lower concentrations of that drug are considered susceptible or sensitive, while those that are inhibited only at higher drug concentrations are considered resistant. Results of phenotypic assays are typically expressed as a fold change in IC50 of each drug against the patient's virus when compared to the IC50 for the reference wild-type HIV-1 strain. For example, if the IC50 of zidovudine (AZT) for the reference wild-type stain is 2 nM and the drug shows an IC50 of 20 nM for the patient's isolate, then the patient's virus would be 10-fold more resistant than the wild-type strain to this drug. However, fold changes in IC50 must be interpreted in relation to the concentration of drug that can be achieved in the plasma and the clinical response to the drug in question.
Identification of key HIV genotypic mutations associated with resistance to nucleotide reverse-transcriptase inhibitors, nonnucleotide reverse-transcriptase inhibitors, and protease inhibitors
Detectable HIV-1 genotypic mutations conferring resistance to an antiviral drug are reported as amino acid codon changes (eg, M184V) resulting from the mutations.
Genotypic drug resistance:
-Susceptible indicates that the genotypic mutations present in patient's HIV-1 strain have not been associated with resistance to the specific drug in question.
-Resistant indicates that genotypic mutations (see specific list in corresponding result comment) detected have been associated with maximum reduction in susceptibility to the specific drug.
-Possibly resistant indicates that genotypic mutations detected have been associated with 1 or both of the following outcomes:
- Diminished virologic response in some, but not all, patients having virus with these mutations
- Intermediate decrease in susceptibility of the virus to the specific drug
-Insufficient evidence indicates that there is inadequate direct or indirect evidence to determine susceptibility of the virus to the specific drug on the basis of the genotypic mutations present, according to the opinion of the consensus panel of leading experts in the field of HIV resistance.
-Unable to genotype result indicates that the sequence data obtained are of poor quality to determine the presence or absence of genotypic resistant mutations in the patient's HIV strain. Possible causes of such poor sequence data include low HIV viral load (i.e. <1,000 copies/mL) and polymorphism in the region of the sequencing primers interfering with primer binding and subsequent sequencing reaction.
Phenotypic drug resistance prediction:
-For each drug, fold change (FC) is predicted by statistical comparison of the IC50 from all of the clinical virus specimens matching the patient's genotypic mutation data to that of the wild-type HIV-1 strain (susceptible reference virus). For example, an FC value of 3.0 for a specific drug indicates that the IC50 of that drug for the patient's virus is predicted to be 3 times higher than that of the susceptible reference virus.
-Predicted FC values should be interpreted with reference to clinical cutoff (CCO) or biological cutoff (BCO) values. CCO values are based on clinical observations of virologic response (change in viral load) in treated patients, indicating how that response is affected by viral resistance. BCO values are derived from laboratory observations indicating the normal range of in vitro susceptibility of wild-type viruses. CCO1 is the baseline FC associated with a 20% loss of the wild-type virologic response due to drug resistance, whereas CCO2 is the baseline FC associated with an 80% loss of the wild-type virologic response due to drug resistance.
Resistance analysis for a given drug is based on the magnitude of the predicted FC in IC50 relative to the CCO or BCO values:
-Susceptible indicates that the predicted FC in IC50 is < or =BCO value for the given drug.
-Resistant indicates that the predicted FC in IC50 is >BCO value for the given drug.
-Maximal response indicates that the predicted FC in IC50 is < or =CCO1 value for the specific drug.
-Reduced response indicates that the predicted FC in IC50 is >CCO1 but < or =CCO2 values for the specific drug.
-Minimal response indicates that the predicted FC in IC50 is >CCO2 value for the specific drug.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
Phenotyping (Virco Type HIV-1) cannot be performed when no clinically relevant drug resistance mutations are detected by HIV-1 genotypic drug resistance testing.
Due to the complexity of the results generated, the International AIDS Society-USA Panel recommends expert interpretation of genotyping and phenotype test results for patient care management. A patient's response to antiviral therapy depends on multiple factors, including the percentage of patient's viral populations that is drug resistant, patient compliance with the prescribed drug therapy, patient access to adequate care, drug pharmacokinetics, and drug interactions. Drug resistance test results should be interpreted only in conjunction with clinical presentation and other laboratory markers when making therapeutic decisions.
Absence of resistance to a drug does not rule out the presence of reservoirs of drug-resistant virus in the infected patient.
The HIV-1 genotypic test is not a direct measure of drug resistance. Although genotypic testing can detect mutations in the relevant HIV-1 genome, the significance of these mutations requires careful interpretation to predict drug susceptibility. This assay's ability to amplify the target and detect genotypic mutations is poor and unreliable when the plasma HIV-1 viral load is <1,000 copies/mL. Specimens submitted for this test should contain > or =1,000 copies/mL of HIV-1 RNA.
This assay has been optimized for genotypic analysis and interpretation of HIV-1 group M subtype B, which is the majority of HIV-1 isolates infecting patients in the United States and Europe. The protease and reverse transcriptase gene regions examined in this assay are not well correlated with the envelope gene, which is the defining gene sequence used for subtyping. Other subtypes of group M HIV-1 have been tested and validated to a limited extent by this assay. Therefore, genotypic mutations in groups N and O, and some group M non-B subtype HIV-1 isolates may or may not be detected using this assay, and it is not known whether drug resistance mutation interpretation for group M subtype B isolates apply to these other groups and subtypes of HIV-1.
The genotypic mutation database and interpretive rules used by this commercial assay are updated periodically by the assay manufacturer. Therefore, the test results do not necessarily include all of the drug-related mutations described in the current medical literature.
Possible causes of treatment failure other than the development of drug resistance are poor adherence to medication regimen, drug potency, and individual variation in pharmacokinetics (eg, inadequate phosphorylation of nucleosides).
The Virco Type HIV-1 result is a phenotypic resistance prediction based on statistical analysis of the genotypic mutations using a proprietary genotype-phenotype correlative database. This analysis is not a direct measure of phenotypic drug resistance.
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
Clinical References Provides recommendations for further in-depth reading of a clinical nature
1. Hanna GJ: HIV-1 genotypic and phenotypic resistance. Clin Lab Med 2002;22:637-649
2. Cavert W, Balfour HH: Detection of antiretroviral resistance in HIV-1. Clin Lab Med 2003;23:915-928
3. Cohen CJ, Hunt S, Sension M, et al: A randomized trial assessing the impact of phenotypic resistance testing on antiretroviral therapy. AIDS 2002;16:579-588
4. Mazotta F, Caputo SL, Torti C, et al: Real versus virtual phenotype to guide treatment in heavily pretreated patients: 48-week follow-up of the Genotipo-Fenotipo di Resistenza (GenPheRex) trial. J Acquir Immune Defic Syndr 2003;32:268-280
5. Perez-Elias MJ, Garcia-Arata I, MuAoz V, et al: Phenotype or virtual phenotype for choosing antiretroviral therapy after failure: a prospective, randomized study. Antivir Ther 2003;8:577-584