HIV-1 Genotypic Drug Resistance Mutation Analysis, with Reflex to Phenotypic Drug Resistance Prediction, Plasma
NY State Approved Indicates the status of NY State approval and if the test is orderable for NY State clients.
Identification of key HIV genotypic mutations associated with resistance to nucleotide reverse-transcriptase inhibitors, nonnucleotide reverse-transcriptase inhibitors, and protease inhibitors
Reflex Tests Lists test(s) that may or may not be performed, at an additional charge, depending on the result and interpretation of the initial test(s)
|Test ID||Reporting Name||Available Separately||Always Performed|
|VPHIV||HIV-1 Phenotypic Resist Prediction||No||No|
Testing Algorithm Delineates situation(s) when tests are added to the initial order. This includes reflex and additional tests.
If HIV type 1 genotypic drug resistance mutation analysis detects drug resistance mutations, then VPHIV/88781 HIV-1 Phenotypic Drug Resistance Prediction (Add-On) will be performed at an additional charge.
See HIV Treatment Monitoring Algorithm in Special Instructions.
Special Instructions and Forms Describes specimen collection and preparation information, test algorithms, and other information pertinent to test. Also includes pertinent information and consent forms to be used when requesting a particular test
GHIVR/88782: Reverse Transcription-Polymerase Chain Reaction (RT-PCR)/DNA Sequencing
(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)
VPHIV/88781: Virtual Phenotype
Reporting Name A shorter/abbreviated version of the Published Name for a test; an abbreviated test name
HIV-1 Geno Reflex to Pheno Predict
Specimen Type Describes the specimen type needed for testing
Specimen Required Defines the optimal specimen. This field describes the type of specimen required to perform the test and the preferred volume to complete testing. The volume allows automated processing, fastest throughput and, when indicated, repeat or reflex testing.
Collection Container/Tube: Lavender top (EDTA)
Submission Container/Tube: Plastic vial
Specimen Volume: 2 mL
1. Spin down and separate plasma from cells within 6 hours of collection.
2. Freeze plasma immediately and ship specimen frozen on dry ice.
3. If shipment will be delayed for >24 hours, freeze plasma at -70 degrees C (up to 35 days) until shipment on dry ice.
1. This test is intended to be used to monitor known HIV-positive infections. It is not intended for primary detection of HIV infections.
2. Specimens submitted for HIV-1 genotyping should contain > or =1,000 copies/mL of HIV-1 RNA.
Specimen Minimum Volume Defines the amount of specimen required to perform an assay once, including instrument and container dead space. Submitting the minimum specimen volume makes it impossible to repeat the test or perform confirmatory or perform reflex testing. In some situations, a minimum specimen volume may result in a QNS (quantity not sufficient) result, requiring a second specimen to be collected.
Mild OK; Gross OK
Mild OK; Gross OK
Green-top (heparin) tube
Specimen Stability Information Provides a description of the temperatures required to transport a specimen to the laboratory. Alternate acceptable temperature(s) are also included.
|Plasma EDTA||Frozen||35 days|
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.
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.
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.
Clinical Reference 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
Method Description Describes how the test is performed and provides a method-specific reference
Genotyping drug resistance:
Trugene HIV-1 Genotyping assay, developed by Siemens Healthcare Diagnostics, Inc, determines the nucleotide bases by simultaneous bidirectional sequencing (1.9 kb total) of the viral reverse transcriptase (1,600 nucleotide) and protease (300 nucleotide) genes of HIV-1 in a blood sample and identifies any mutations. An automated DNA sequencing system (OpenGene computer software) compares the sample genotype to the known resistance mutations and generates a list of the mutations present and the antiviral drugs to which the mutations confer resistance.(Package insert: Trugene HIV-1 Genotyping Kit, Siemens Healthcare Diagnostics, Inc, Tarrytown, NY)
Phenotyping drug resistance:
HIV-1 target genomic sequence obtained from the genotyping test is analyzed by a proprietary software application with a large correlative database of approximately 30,000 matching clinical HIV-1 genotypes and phenotypes at Virco Lab Inc, USA (Bridgewater, NJ). The analysis identifies all the mutations that can affect resistance to each drug and then interrogates the database for phenotypic drug resistance patterns from previous clinical samples that match these patterns of genotypic mutations.
When all matches have been identified, the software further analyzes the data for all the matches using the Virtual Phenotype-LM linear regression modeling application, which takes into account the weighted contribution of individual mutations and mutation pairs to make fold change (FC) predictions for each drug. This predicted FC in IC50 for each drug is typically based on data from thousands of real phenotypes with the same patterns of genotypic mutations. Clinically relevant interpretation of predicted FC is based on comparison between the FC in IC50 and the clinical cutoff (CCO) values for the given drug. The CCO values are derived from a clinical outcomes database containing clinical data from more than 17,000 patients, including treatment history, current treatment, and changes in viral load and CD4 count after modifying treatment based on drug resistance testing results. When CCO values for a given drug are not available from the database, biological cutoff (BCO) value derived from in vitro susceptibility testing is used for comparison.(www.vircolab.com)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Varies; test will be performed in batches of 4
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
Performing Laboratory Location The location of the laboratory that performs the test
Test Classification Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer's instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR), Investigation Use Only (IUO) product, or a Research Use Only (RUO) product.
This test has been cleared or approved by the U.S. Food and Drug Administration and is used per manufacturer's instructions. Performance characteristics were verified by Mayo Clinic in a manner consistent with CLIA requirements.
CPT Code Information Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Medical Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.
87901-HIV-1 genotypic drug resistance
87900-HIV-1 phenotypic drug resistance prediction (add-on) (if appropriate)
LOINC® Code Information Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the result codes returned for this test or profile.
|Result ID||Reporting Name||LOINC Code|
|82340||HIV-1 Genotypic Drug Resistance, P||30554-0|
|21395||Reverse Transcriptase Mutations||30554-0|
|28076||Atazanavir with Ritonavir||49618-2|
|26784||Darunavir with Ritonavir||49630-7|
|26733||Fosamprenavir with Ritonavir||51409-1|
|26734||Indinavir with Ritonavir||49619-0|
|21532||Lopinavir with Ritonavir||42000-0|
|26735||Saquinavir with Ritonavir||49621-6|
|28201||Tipranavir with Ritonavir||49622-4|