Resistance testing: phenotype
- In phenotypic resistance assays, RT, PR, integrase and envelope gene sequences derived from patient plasma HIV RNA are used to measure HIV replication at different concentrations of ARVs.
- Phenotypic co-receptor tropism assays detect CCR5, CXCR4, or dual/mixed (D/M)-tropic virus. Viral tropism is the ability of HIV to bind to different co-receptors on target cells to achieve viral entry, used to assess activity of CCR5 antagonist (maraviroc).
- Phenotypic resistance tests assess effect of all mutations, including mutational interactions. Most useful in pts with complex resistance patterns due to multiple mutations, especially to assess PI susceptibility, and for HIV-2, where genotyping is not easily interpretable.
- Minority populations < 10-20% are not reliably detected.
- Quantitatively measures drug resistance, assesses relative activity of ARVs with partial activity, which may be useful when treatment options are limited.
- Interpretation of phenotype based on two important clinical cutoffs: (1) lower cutoff: value above which clinical response diminished (partial susceptibility) and (2) upper cutoff: value above which no clinical response expected.
- Phenotype does not require understanding of genotypic correlates of resistance; therefore, may be especially useful for new drugs, non-B subtypes, and HIV-2.
- Measures hypersusceptibility.
- Consider phenotyping when treatment history complex and/or significant resistance expected, especially to PIs.
Professional Society Guidelines
- Genotype preferred over phenotype in ART-naive pts or patients who have interrupted ART. Genotype tests are cheaper and more likely to detect resistance due to wild-type/mutant mixtures, which can occur without selective drug pressure.
- Testing should be performed on ART or within 4 wks of stopping ART to detect drug-resistant populations that evolve under selective drug pressure.
- Phenotype may fail to detect resistance due to wild-type/mutant mixtures, especially in pts no longer on ART.
- Simultaneous genotype or combined phenotype/genotype (PhenoSense GT) should generally be ordered rather than phenotype alone, as genotype may sometimes detect resistance not detected by phenotype, especially in presence of wild type/mutant mixtures.
Phenotype Testing Methods
- Results reported as fold change in drug susceptibility of sequences derived from pt plasma vs. control. Susceptibility is measured by IC50, inhibitory concentration or amount of drug required to inhibit viral replication by 50%. Ratio of IC50 of pt sample to reference viruses is reported as the fold increase in IC50.
- Clinical cutoffs based on data from cohort studies and clinical trials to determine decline in susceptibility that results in reduced virologic response. When clinical cutoffs not available, biologic cutoffs provided, based on normal distribution of susceptibility to drug for wild-type strain from treatment-naive pts.
- Assays detect resistance only in species that make up >10-20% of viral population.
- Tropism testing: co-receptor tropism of patient-derived virus is confirmed by testing the susceptibility of the virus to specific CCR5 or CXCR4 inhibitors in vitro.
- Used to confirm CCR5 co-receptor tropism prior to use of a CCR5 co-receptor antagonist.
- Recommended for patients who experience virologic failure on CCR5 antagonist.
- Phenotype testing considered gold standard. Genotype assays are less expensive and require less time to perform, DHHS 2015 guidelines recommend tropism genotype test as alternative test.
- Proviral DNA recovered from peripheral mononuclear blood cells (PMBCs) can be used to determine tropism in patients with VL < 1,000 c/mL. Clinical uility remains to be determined.
Basis for recommendation
- Swenson LC, Chui CK, Brumme CJ, et al. Genotypic analysis of the V3 region of HIV from virologic nonresponders to maraviroc-containing regimens reveals distinct patterns of failure. Antimicrob Agents Chemother. 2013;57(12):6122-30. [PMID:24080655]
Comment: Analysis of MOTIVATE-1 and -2 virologic non-responders (n=181) to MVC by population-based and deep sequencing assessed changes in tropism and V3 sequences. All had R5 HIV by the original Trofile assay. From population sequencing, 90 pts had R5 and 91 had non-R5 HIV. For those with non-R5 HIV, the geno2pheno false positive rate fell from median of 20 at screening to 1.1 at failure. In 70% of cases, deep sequencing could detect a pretreatment CXCR4-using subpopulation at failure. Most pts with virologic failure and R5 virus were shown not to have phenotypic resistance and may have been nonadherent or had resistance to other components of the treatment regimen.
- Günthard HF, Aberg JA, Eron JJ, et al. Antiretroviral treatment of adult HIV infection: 2014 recommendations of the International Antiviral Society-USA Panel. JAMA. 2014;312(4):410-25. [PMID:25038359]
Comment: IAS-USA guidelines state baseline genotypic testing should be performed in all those ART-naive and in cases of confirmed viral failure.
- Vandamme AM, Camacho RJ, Ceccherini-Silberstein F, et al. European recommendations for the clinical use of HIV drug resistance testing: 2011 update. AIDS Rev. 2011;13(2):77-108. [PMID:21587341]
Comment: Genotype testing recommended in most clinical situations. Additional phenotype testing recommended for new drugs, in heavily pretreated patients, and for HIV-2 where genotyping is not easily interpretable. For phenotype interpretation, use clinical cutoff if available, if unavailable use biological cutoff.
- Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. Department of Health and Human Services. Available at http://www.aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf. Accessed 11/11/15.
Comment: Addition of phenotypic testing to genotypic testing recommended in persons with known or suspected complex drug resistance mutation patterns, particularly to PIs. May also provide useful clinical data in non-B subtypes.
- Heera J, Valluri S, Craig C, et al. First prospective comparison of genotypic vs phenotypic tropism assays in predicting virologic responses to Maraviroc (MVC) in a phase 3 study: MODERN. J Int AIDS Soc. 2014;17(4 Suppl 3):19519. [PMID:25394028]
Comment: Industry-sponsored prospective phase 3 RCT comparing use of genotype assays in ART-naive pts initiating ART with either MVC+DRV/r OR TDF/FTC+DRV/r by response of those R5 tropic pts with VL < 50 at week 48. Response rates were 81% versus 74% for genotype and phenotype, respectively (stratification adjusted difference, 7%; 95%CI, 1-15%).
- Pérez-Olmeda M, Alcami J. Determination of HIV tropism and its use in the clinical practice. Expert Rev Anti Infect Ther. 2013;11(12):1291-302. [PMID:24191978]
Comment: Comprehensive review of tropism assays developed worldwide describes phenotype assays as gold standard technology that directly measures viral entry on cell lines separately expressing CCR5 or CXCR4. However, due to cost and time-intensive methods, genotype testing is preferred as a less-expensive, easier, faster test.
- Vandekerckhove LP, Wensing AM, Kaiser R, et al. European guidelines on the clinical management of HIV-1 tropism testing. Lancet Infect Dis. 2011;11(5):394-407. [PMID:21429803]
Comment: Review of pros and cons of genotype versus phenotype tropism testing (Table 1). Comments include discussion of assay interpretation based on proviral DNA rather than viral RNA.
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