Resistance testing: genotype

Lisa A. Spacek, M.D., Ph.D.

DEFINITION

  • HIV gene sequencing assays detect mutations that confer ARV drug resistance.
    • Reverse transcriptase and protease gene mutations confer NRTI, NNRTI, and PI resistance.
    • Integrase gene mutations confer integrase inhibitor (INSTI) resistance.
    • gp41 (envelope) gene mutations confer fusion inhibitor (enfuvirtide) resistance.
  • Assays identify mutations associated with clinical resistance and indicate which ARVs are unlikely to be effective.[2]
    • Absence of detectable resistance may be due to:
      • Presence of drug-resistant variants as minority populations. Sanger sequencing does not reliablly detect populations that are less than 10-20% of sample.[6]
      • Lack of drug-selective pressure if ART is discontinued prior to genotype testing.
      • Subtherapeutic drug levels, nonadherence to ART, or lab error.
      • Occurrence of relevant mutations outside the regions targeted by routine assays.
  • Co-receptor tropism assays predict co-receptor usage, both genotype and phenotype assays are available.[3]
    • Genotype assay: population sequencing with reflex to ultradeep sequencing if R5. Sequence of V3 loop of HIV gp120 is principle determinant of tropism.
    • Phenotype assay:
      • RNA assay for VL ≥1,000 c/ml
      • DNA assay for VL < 1,000 c/ml, determines co-receptor usage by proviral DNA from peripheral blood mononuclear cells
  • No commercial assay is available for resistance to post-attachment inhibitor, ibalizumab.

INDICATIONS

Acute or chronic infection, ART-naive patients.

  • Conventional assays require plasma viral load of at least 500 to 1,000 copies/ml. Genotype preferred over phenotype in ART-naive pts because of ability to detect mixtures that occur during reversion to wild-type.
  • Genotype testing is performed at time of diagnosis to detect transmitted resistant mutants, generally NNRTI- or NRTI-resistance.
    • GenoSure MG (Monogram BioSciences) includes drug resistance for NRTIs, NNRTIs, and PIs. GenoSure PRIme (MonogramBiosciences) includes drug resistance to NRTIs, NNRTIs, PIs, and INSTIs.
      • Baseline INSTI resistance testing is not routinely recommended, as transmitted resistance is rare.[8]
  • Recommended in acute and recent HIV infection[3]
  • Recommended for all pregnant women prior to ART initiation and for those entering pregnancy with detectable VL while on ART.[1]
  • Recommended to determine initial treatment especially if initiating NNRTI-based ART.[7][4]
  • Recommended in chronic HIV infection in ART-naive individuals, however, results may not influence the choice of regimen if starting an INSTI-based regimen.
    • If ART initiation is delayed, genotype testing may detect drug-resistant virus acquired since time of diagnosis (i.e., superinfection).

Established infection, ART-experienced patients.

  • Drug resistance testing results are used to select fully active drugs in:
    • Cases of virologic failure.
    • Suboptimal suppression of VL after ART initiation.
    • Pregnancy, if VL detectable on ART.
  • Drug resistance testing should be performed while patient is on ART, whenever possible, or within 4 wks after discontinuation. Longer delays after discontinuation may still allow detection of some thymidine analog mutations (TAMs) and NNRTI resistance, but ability to detect PI and 3TC/FTC resistance is reduced.
    • If virologic failure occurs on INSTI-containing therapy, use results of INSTI resistance testing to select next ART regimen.
    • Prior to use of CCR5 antagonist, test for co-receptor tropism. Genotype testing is as sensitive, and cheaper and faster than phenotype assay.
  • In the setting of virologic failure with VL >1000, standard assays recommended; if VL in 500-1000 range, standard assays may allow genotyping and should be considered.[3]
    • Research-based assays for next generation sequencing, such as allele-specific PCR and ultra-deep sequencing (Janssen Infectious Diseases BVBA, Beerse, Belgium), are used in studies to detect minority (< 20%) variants within the viral population.[14]
    • Although not commercially available, pre-salvage ultrasensitive sequencing (454) versus conventional (Sanger) sequencing has been shown to better predict viral failure in a subset of treatment-experienced pts with multidrug resistance.[12]
  • Low-level viremia, 50-1000 c/ml, may occur as blip or sustained low-level viremia.[13] With VL < 500 c/ml, conventional drug resistance testing is not usually recommended.
    • Evaluation of proviral DNA by next generation sequencing is commercially available (GenoSure Archive). In 48 paired plasma RNA and cell DNA samples, where discordance was defined as mutations seen in plasma RNA but not in DNA, 4/48 and 6/48 were discordant with historical RNA resistance tests in PR and RT regions, respectively. Major variant false omission rate ranged from 2-4%.[15]
    • Data based on RNA ’in-house’ assays suggest resistance testing conducted on samples with VL 250-500 c/ml were 90% successful and predicted future virologic outcomes in treatment-naive patients initiating first regimen, but commercial labs may not perform testing.[13]
    • Drug resistance mutations in proviral DNA, recovered from peripheral blood mononuclear cells, versus plasma viral RNA may show discordance in specific mutations, but this has not been sufficient to change ART decision-making.[5]
    • Data from Peninsula AIDS Research Cohort suggest that proviral DNA sequencing, specifically in setting of low level viremia or suppression, may have a role in monitoring of ART.[10]

Professional society guidelines

CLINICAL RECOMMENDATION

Important considerations

  • Review of past genotypes and past ART is of primary importance. Current drug resistance testing must be interpreted along with historical results. Cumulative rather than current genotype should be considered.
  • All prior and current drug-resistance testing results should be reviewed and considered when designing a new regimen for a patient experiencing virologic failure.[3]
  • Absence of resistance mutations may be due to limits of detection. Genotype assays may not detect minority variants or archived mutations that are transmitted or acquired on prior therapy.
  • Resistance detected reliably in species that make up >10-20% of viral pool. Mixtures of susceptible and resistant virus (present at >10-20%) can be detected with conventional sequencing (Sanger method) by genotype.
    • Minority variants not detected by standard assays.
      • Deep sequencing of V3 loop of HIV gp120 used to predict co-receptor tropism.
      • Next generation sequencing may improve diagnostic prediction of viral failure.[9] Available as GenoSure Archive assay from Monogram BioSciences.
      • Point mutation assays and clonal sequencing detect minority resistance, yet have unproven clinical utility.[14] Not commercially available.

Genotype vs. phenotype

  • Genotype
    • Less expensive than phenotype
    • Shorter turn-around time (1 to 2 wks)
    • May detect genetic mutations, including mixtures, before they result in phenotypic resistance
  • Phenotype (preferrably in combination with genotype)
    • Advantage in patients with more extensive resistance, where genotype interpretation can be complex, especially for PI resistance
    • May be useful for non-B subtypes
    • May be better for assessing susceptibility to newer agents, for which genotypic correlates of resistance are not completely determined
    • Phenotype alone may underestimate resistance due to mixtures. Genotype + phenotype preferred (Phenosense GT, Monogram BioSciences)

Tools for genotype interpretation

OTHER INFORMATION

  • Reflex testing can automatically run a resistance and/or tropism test if viral load meets or exceeds the viral load threshold for assay.
  • Web-based global surveillance map of drug resistance in ART-naive populations: https://hivdb.stanford.edu/page/surveillance-map/

Basis for recommendation

  1. Saag MS, Gandhi RT, Hoy JF, et al. Antiretroviral Drugs for Treatment and Prevention of HIV Infection in Adults: 2020 Recommendations of the International Antiviral Society-USA Panel. JAMA. 2020;324(16):1651-1669.  [PMID:33052386]

    Comment: Reverse transcriptase genotype testing recommended at diagnosis and viral failure. If failing INSTI-based regimen, then integrase genotype test recommended.

  2. Wensing AM, Calvez V, Ceccherini-Silberstein F, et al. 2019 update of the drug resistance mutations in HIV-1. Top Antivir Med. 2019;27(3):111-121.  [PMID:31634862]

    Comment: 2019 update adds bictegravir, cabotegravir, and doravirine. Authors note that magnitude of reduced susceptibility is modulated by the genetic context in which the mutation occurs.

  3. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents with HIV. Department of Health and Human Services. Available at https://clinicalinfo.hiv.gov/sites/default/files/inline-files/AdultandAdolescentGL.pdf.

    Comment: Drug resistance testing recommended at diagnosis, at ART initiation, and in pregnancy. Most genotypic assays involve conventional Sanger sequencing of the reverse transcriptase (RT), protease (PR), and integrase (IN) genes of circulating RNA in plasma to detect mutations that are known to confer drug resistance.
    Accessed 8/15/2021 at https://clinicalinfo.hiv.gov/en/guidelines/adult-and-adolescent-arv/drug-resistance-testing?view=full

References

  1. Bertagnolio S, Hermans L, Jordan MR, et al. Clinical Impact of Pretreatment Human Immunodeficiency Virus Drug Resistance in People Initiating Nonnucleoside Reverse Transcriptase Inhibitor-Containing Antiretroviral Therapy: A Systematic Review and Meta-analysis. J Infect Dis. 2021;224(3):377-388.  [PMID:33202025]

    Comment: Meta-analysis of 32 studies (31,441 individuals) assessed impact of baseline drug resistance testing in those initiating NNRTI-based ART. In resource-limited settings, despite rising prevalence of pretreatment drug resistance (PDR), baseline genotype testing is not standard practice. Evidence supports avoidance of NNRTI where PDR is >10%. In all included studies, HIV-DR was performed retrospectively on stored samples.
    Rating: Important

  2. Ellis KE, Nawas GT, Chan C, et al. Clinical Outcomes Following the Use of Archived Proviral HIV-1 DNA Genotype to Guide Antiretroviral Therapy Adjustment. Open Forum Infect Dis. 2020;7(1):ofz533.  [PMID:31915714]

    Comment: Retrospective review of pre- and post-switch data for 59 patients tested by next-generation sequencing-based assay of proviral DNA assessed clinical outcomes. Seventy-one percent were infected ≥ 10 years. Documented high level resistance to: DRV was seen in 2, RAL and/or EVG in 5, and DTG in 1. Given more potent ART, low-level resistance to first generation INSTIs, and increasing evidence for 2-drug or NRTI-sparing regimens, authors question utility of DNA GT.
    Rating: Important

  3. Parkin NT, Avila-Rios S, Bibby DF, et al. Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping. Viruses. 2020;12(7).  [PMID:32605062]

    Comment: Optimal threshold for low abdundance variants is debated. Authors compared pairwise results of next-generation sequencing to standard Sanger sequencing methods for low-abundance variants. Ten prepared specimens of HIV-1 subtypes B,C,D,or F were sequenced by 10 different labs. Quality assurance anomalies occurred most often at low-abdundance threshold of 5% and least often at 15 or 20%. The significance of LAV below 20% must first be shown to to increase predictive value of genotype testing for clinical outcomes.
    Rating: Important

  4. Soulie C, Santoro MM, Charpentier C, et al. Rare occurrence of doravirine resistance-associated mutations in HIV-1-infected treatment-naive patients. J Antimicrob Chemother. 2019;74(3):614-617.  [PMID:30476106]

    Comment: Drug resistance testing by bulk sequencing from 2010-2016 in Greece, Italy, and France included 9764 clinical sequences (53% B-subtypes) from treatment naive individuals found that presence of at least 1 DOR RAM or K103N/Y181C mutations was rare (N=5, 0.1%)

  5. Casadellà M, van Ham PM, Noguera-Julian M, et al. Primary resistance to integrase strand-transfer inhibitors in Europe. J Antimicrob Chemother. 2015;70(10):2885-8.  [PMID:26188038]

    Comment: Multi-center cross-sectional analysis conducted within European SPREAD HIV resistance surveillance study of 278 representative samples selected from 1950 ART-naive pts newly diagnosed in 2006-7 before INSTI drugs were commercially available. Population sequencing analysis and a subgroup of samples tested by 454 sequencing (N=56) found no signature mutations. Prevalence of transmitted drug resistance mutations for PIs, NRTIs, and NNRTIs were 2.5%, 9.1%, and 7.9%, respectively. Stanford HIVdb score ≥10 to at least one INSTI was seen in 11/278 (4%) and in 8/56 (14%) analyzed by 454 sequencing.

  6. Geretti AM, Paredes R, Kozal MJ. Transmission of HIV drug resistance: lessons from sensitive screening assays. Curr Opin Infect Dis. 2015;28(1):23-30.  [PMID:25501541]

    Comment: Review of assay technology for detecting HIV resistance includes pros and cons listing (Table 1) for conventional population (Sanger) sequencing, next generation deep sequencing, mutation-specific assays (e.g., allele-specific PCR assay). Authors emphasize importance of clinical validation.

  7. Derache A, Shin HS, Balamane M, et al. HIV drug resistance mutations in proviral DNA from a community treatment program. PLoS One. 2015;10(1):e0117430.  [PMID:25635815]

    Comment: Prospective study of 120 HIV+ pts in the Peninsula AIDS Research Cohort (Stanford) followed for mean of 39 wks. At entry, 38 were viremic (median VL 5,865 c/ml) and 82 were suppressed (VL< 50 c/ml). Of those viremic, vRNA versus proviral DNA genotypes showed concordance in 14/18 on ART and 18/20 off ART. Discordant results were seen in 6 pts with low mean VL of 697 c/ml, whereas concondant results were seen in 32 pts with mean VL of 31,252 c/ml. Of those suppressed and on ART (N=79), 21 had archived drug resistance mutations in proviral DNA. Three had NNRTI mutations detected by proviral DNA without documented exposure to NNRTIs.

  8. Drescher SM, von Wyl V, Yang WL, et al. Treatment-naive individuals are the major source of transmitted HIV-1 drug resistance in men who have sex with men in the Swiss HIV Cohort Study. Clin Infect Dis. 2014;58(2):285-94.  [PMID:24145874]

    Comment: Transmitted drug resistance (TDR) in the Swiss HIV Cohort Study was documented in 140 of 1674 (8.4%) MSM. Study illustrated transmission clusters with long TDR transmission chains. Authors emphasize importance of early test-and-treat strategies to reduce TDR.

  9. Pou C, Noguera-Julian M, Pérez-Álvarez S, et al. Improved prediction of salvage antiretroviral therapy outcomes using ultrasensitive HIV-1 drug resistance testing. Clin Infect Dis. 2014;59(4):578-88.  [PMID:24879788]

    Comment: Retrospective cohort analysis of 132 treatment-experienced pts with VL >5000, of whom 28/132 developed viral failure, tested with conventional drug resistance testing and ultrasensitive 454 sequencing, evaluated clinical relevance of ultrasensitive genotyping. Ultrasensitive genotyping by 454 sequencing reduced the genotypic susceptibility score (GSS) in 23-25% of pts. Cox regression showed GSS by 454 sequencing of < 3 (HR, 4.6; 95%CI, 1.5-14.0) and number of previous antiretrovirals per each additional drug (HR, 1.2; 95%CI, 1.1-1.3) were associated with viral failure.
    Rating: Important

  10. Gonzalez-Serna A, Min JE, Woods C, et al. Performance of HIV-1 drug resistance testing at low-level viremia and its ability to predict future virologic outcomes and viral evolution in treatment-naive individuals. Clin Infect Dis. 2014;58(8):1165-73.  [PMID:24429436]

    Comment: Historical prospective cohort study (British Columbia) of previously ART-naive patients who achieved undetectable VL before low-level viremia (HIV RNA 50-999 c/ml) while remaining on same ART, determined drug resistance mutations by ’in-house’ PCR method on 4915 plasma samples from 2492 pts (1996 to 2012). Those with GSS < 3 had increased risk of viral failure compared to those with GSS ≥3 (p=0.007). Samples with VL >250 c/ml had 90% success rate for genotyping, those with VL 50-249 c/ml had 74% success rate.
    Rating: Important

  11. Geretti AM, Conibear T, Hill A, et al. Sensitive testing of plasma HIV-1 RNA and Sanger sequencing of cellular HIV-1 DNA for the detection of drug resistance prior to starting first-line antiretroviral therapy with etravirine or efavirenz. J Antimicrob Chemother. 2014;69(4):1090-7.  [PMID:24284781]

    Comment: Retrospective analysis of baseline samples of pts entering the SENSE trial (Janssen-sponsored) to begin first-line ART with EFV or ETR-based ART. Plasma Sanger sequencing showed 8% of pts had ≥1 NRTI mutation (5%), NNRTI mutation (2%), or PI mutation (1%). No additional mutations were detected by allele-specific PCR and ultra-deep sequencing. Proviral DNA analyzed by Sanger sequencing increased detection of resistance by 2 additional samples.

  12. Toma J, et al: Drug resistance profiles derived from HIV-1 DNA in ARV suppressed patients correlate with historical resistance profiles obtained from HIV-1 plasma RNA. ICAAC, September 20, 2015, abstract presentation. Accessed November 16, 2015 at http://www.abstractsonline.com/Plan/ViewAbstract.aspx?mID=3798&sKey=f3c882f4-b11c-4b64-a64c-4b1276a4b52a&cKey=728a5a98-f969-44b6-8383-d095840d14a4&mKey=7a574a80-eab1-4b50-b343-4695df14907e.

    Comment: Abstract presentation at ICAAC describes GenoSure Archive (Monogram BioSciences, South San Francisco, CA), which amplifies cell-associated proviral DNA and uses next-generation sequencing to analyze the HIV-1 polymerase region including the full-length protease and integrase coding regions and amino acids 1-400 of reverse transcriptase. Assay targets those with low-level viremia (< 500 c/ml) or undetectable plasma RNA HIV on ART. In 48 paired samples, concordance was high, 92% for PR and 88% for RT regions.

Last updated: October 10, 2021