Test Id : HIVDR

For information see HIV Treatment Monitoring Algorithm

This test is intended for detection and identification of drug resistance-associated HIV-1 genotypic mutations in plasma specimens of individuals prior to or while receiving combination antiretroviral therapy.

Prior to requesting this test, patients must have a confirmed plasma HIV-1 RNA level (ie, viral load) of 1000 copies/mL or higher within the preceding 30 days. HIVQN / HIV-1 RNA Detection and Quantification, Plasma is available to provide this prerequisite test result. Alternately, if the patient's viral load is unknown, order HIQDR / HIV-1 RNA Quantification with Reflex to Genotypic Drug Resistance to Reverse Transcriptase, Protease, and Integrase Inhibitors, Plasma, which will perform viral load followed by genotype, if appropriate.

For initial diagnosis of HIV, order HIVDX / HIV-1 and HIV-2 Antigen and Antibody Diagnostic Evaluation, Plasma.

If shipment will be delayed for more than 24 hours, freeze plasma specimen at -70 degrees C (up to 60 days) until shipment on dry ice.

The following ask-at-order entry question must be answered at the time of test ordering (mark answer on the test request form if not ordering electronically):

HIV-1 RNA level copies/mL in last 30 days = (select answer option)

<1000

1000 to 1,000,000

1,000,001 to 10,000,000

>10,000,000

Note: Test requests for submitted specimens with less than 1000 copies/mL (not sufficient amount for testing), “No,” or no response entered will be canceled.

Supplies: Sarstedt Aliquot Tube, 5 mL (T914)

Collection Container/Tube: Lavender top (EDTA)

Submission Container/Tube: Plastic vial

Specimen Volume: 2.2 mL

Collection Instructions:

1. Centrifuge blood collection tube and aliquot plasma into plastic vial per collection tube manufacturer's instructions (eg, centrifuge and aliquot within 2 hours of collection for BD Vacutainer tubes).

2. Freeze aliquoted plasma for shipment.

Additional Information: Specimens submitted for HIV-1 genotyping must contain 1000 copies/mL or more of HIV-1 RNA.

• HIV Treatment Monitoring Algorithm

If not ordering electronically, complete, print, and send a Microbiology Test Request (T244) with the specimen.

0.8 mL

Identifying HIV-1 genotypic mutations associated with resistance to nucleotide and non-nucleoside reverse-transcriptase inhibitors, protease inhibitors, and integrase strain transfer inhibitors

Guiding initiation or change of combination antiretroviral therapy in individuals, including children, with HIV-1 infection

For information see HIV Treatment Monitoring Algorithm

Antiviral resistance may compromise the efficacy of antiretroviral therapy (ART) in patients receiving such therapy for HIV1 infection. When combination therapy fails, detection and analysis of antiviral drug resistance-associated viral genotypic mutations can guide necessary changes to ART to suppress viral replication (ie, reduce 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 ART (combination of nucleoside and nonnucleoside reverse-transcriptase inhibitors, protease inhibitors, and/or integrase strain transfer inhibitors) may be effective in reducing the viral load, genotypic mutations arising in the drug-targeted HIV-1 genome due to selective pressure from antiviral therapy will 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 variants 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-1 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-1 genotypic drug resistance mutations.

An interpretive report will be provided.

Detectable HIV-1 genotypic mutations conferring resistance to an antiviral drug are reported as amino acid codon changes (eg, M184V) resulting from the nucleic acid base alterations, according to the interpretative algorithm of the Stanford HIV Database program. The codon mutations are categorized and interpreted in relation to previously published data of phenotypic antiviral susceptibility tests on virus that harbor such mutations. Each codon mutation is assigned a drug penalty score. The total score generated from all mutations relevant to the specific antiviral drug is used to estimate the level of resistance to that drug. These interpretive rules may be updated periodically by the Stanford HIV Database Team after reviewing newly published data on HIV-1 genotypic drug resistance-associated codon mutations.

Susceptible (SUSC) indicates that the codon mutations present in patient's HIV-1 strain have not been associated with resistance to the specific drug (Stanford HIVdb total score 0 to 9).

Potential Low-Level Resistance (PLR) indicates that codon mutations detected have been associated with possible reduction in susceptibility to the specific drug (Stanford HIVdb score 10 to 14).

Low-Level Resistance (LR) indicates that codon mutations detected have been associated with reduction in susceptibility to the specific drug (Stanford HIVdb score 15 to 29).

Intermediate Resistance (IR) indicates that codon mutations detected have been associated with reduction in susceptibility to the specific drug (Stanford HIVdb score 30 to 59).

High-level Resistant (HR) indicates that codon mutations detected have been associated with maximum reduction in susceptibility to the specific drug (Stanford HIVdb > or = 60).

Unable to genotype indicates that the sequence data obtained are of poor quality to determine the presence or absence of resistance-associated codon mutations in the patient's HIV-1 strain. Probable causes of such poor sequence data include polymorphism in the region of the sequencing primers interfering with primer binding and subsequent sequencing reaction, or low viral load (ie, <1000 copies/mL).

Inconclusive indicates inability of the assay to reliably determine antiviral resistance because of the presence of polymerase chain reaction inhibitors or ambiguous or incomplete viral target sequences generated from the assay.

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 individual.

The HIV-1 genotypic test is not a direct measure of drug resistance. Although genotypic testing can detect variants in the relevant HIV-1 genome, the significance of these variants requires careful interpretation to predict drug susceptibility. This assay's ability to amplify the target and detect genotypic mutation is poor and unreliable when the plasma HIV-1 viral load (VL) is less than 1000 copies/mL. Specimens submitted for this test should contain greater than or equal to 1000 copies/mL of HIV-1 RNA. Per assay manufacturer claims, the assay's ability to detect minor drug-resistant HIV-1 variants among 90% or more of HIV-1 group M strains varies depending on the VL in the tested plasma specimen; 20% or higher at VL of 1000 copies/mL, 10% or higher at VL of 5000 copies/mL, and 5% or higher at VL of 15,000 copies/mL.

The list of drug resistance-associated codon mutations and interpretive rules used by the Stanford HIV database are updated periodically by the Stanford HIV Database team. Therefore, the test results do not necessarily include all resistance-associated codon 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).

1. Weber J, Volkova I, Sahoo MK, Tzou PL, Shafer RW, Pinsky BA. Prospective evaluation of the Vela Diagnostics next-generation sequencing platform for HIV-1 genotypic resistance testing. J Mol Diagn. 2019;21(6): 961-970. doi: 10.1016/j.jmoldx.2019.06.003

2. Avila-Rios S, Parkin N, Swanstrom R, et al. Next-generation sequencing for HIV drug resistance testing: laboratory, clinical, and implementation considerations. Viruses. 2020;12(6):617.doi: 10.3390/v12060617

3. Raymond S, Nicot F, Abravanel F, et al: Performance evaluation of the Vela Dx Sentosa next-generation sequencing system for HIV-1 DNA genotypic resistance. J Clin Virol. 2020;122:104229. doi: 10.1016/j.jcv.2019.104229

4. Panel on Antiretroviral Guidelines for Adults and Adolescents: Guidelines for the use of antiretroviral agents in adults and adolescents with HIV. US Department of Health and Human Services. Updated May 26, 2023. Accessed September 1, 2023. Available at https://clinicalinfo.hiv.gov/sites/default/files/guidelines/documents/adult-adolescent-arv/guidelines-adult-adolescent-arv.pdf

This test utilizes the US Food and Drug Administration-approved, commercially available Sentosa SQ HIV-1 Genotyping Assay, which is a next-generation sequencing assay based on a "sequencing by synthesis" method. The assay is designed to generate 2 amplicons (approximately 1500 base pairs [bp] and approximately 1000 bp in length) spanning the PR / RT- and INT-coding regions, respectively, of the HIV-1 genome for sequencing. Codon positions 1 to 99, 1 to 387, and 1 to 288 in the PR-, RT-, and INT-coding regions, respectively, are subsequently analyzed by the assay software for clinically relevant codon substitutions.

Clinical plasma specimens are subjected to automated HIV-1 RNA extraction and purification, followed by reverse-transcription polymerase chain reaction of HIV-1 target sequences, with both a system control and a positive control included in each assay run for quality control purposes. Automated DNA library preparation is performed using the amplified products, including enzymatic shearing, adapter ligation, purification, and normalization, prior to DNA template preparation and sequencing. Sequencing reactions are conducted with the Sentosa SQ301 sequencer, and the assembled sequence data are analyzed using proprietary analysis and interpretive software applications. HIV-1 antiviral drug-resistance interpretations are based on algorithms implemented in the most current version of the Stanford University HIV Drug Resistance Database (HIVdb; Stanford University) using a 5% variant detection cutoff threshold set by the assay manufacturer.(Instruction manual: Sentosa SQ HIV-1 Genotyping Assay User Manual. Vela Diagnostics; Version 1.3, 05/2023)

Monday through Friday

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• Prospective clients should contact their account representative. For assistance, contact Customer Service.

This test has been cleared, approved, or is exempt by the US 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.

0219U