- HIV gene sequencing assays detect mutations that confer ARV drug resistance.
- Reverse transcriptase and protease genes: for evaluation of NRTI, NNRTI, and PI resistance
- Integrase genes: for evaluation of integrase inhibitor (INSTI) resistance
- gp41 (envelope) genes: for evaluation of fusion inhibitor (enfuvirtide) resistance
- Assays identify mutations associated with clinical resistance and indicate which ARVs are unlikely to be effective.
- Absence of detectable resistance may be due to: presence of drug-resistant variants as minority populations, lack of selection pressure if ART is discontinued prior to genotype testing, subtherapeutic drug levels, nonadherence to ART, lab error, occurrence of relevant mutations outside the regions targeted by routine assays. 
- Co-receptor tropism assays predict co-receptor usage; genotype and phenotype assays available. 
- 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
Acute or chronic infection, ART-naive patients.
- Genotype testing recommended: 
- At time of Dx, regardless of whether ART will be initiated. Reported rates of transmitted resistance range from 8% to 14%.   Transmission of resistant virus, persistence of transmitted resistant mutants, and demonstrated cost-effectiveness are reasons to perform resistance testing at time of Dx.
- When ART delayed, testing may detect drug-resistant virus acquired since Dx (i.e., superinfection).
- Recommended for all pregnant women prior to ART initiation and for those entering pregnancy with detectable VL while on ART. 
- Genotype preferred over phenotype in ART-naive pts because of ability to detect mixtures that occur during reversion to wild-type.
- INSTI resistance testing at baseline is not recommended, as transmitted resistance is rare.  
- Prior to use of CCR5 antagonist, test for co-receptor tropism. Genotype testing is as sensitive, but cheaper and faster than phenotype assay.
Established infection, ART-experienced patients.
- Drug resistance testing results used to select fully active drugs in:
- Cases of virologic failure.
- Suboptimal suppression of VL after ART initiation.
- Pregnancy, if VL detectable on ART.
- In setting of virologic failure on INSTI-based regimen, supplement standard resistance testing with INSTI genotypic resistance assay.  Assays that bundle NRTI, NNRTI, PI and INSTI mutations are available.
- 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.
- Treatment failure VL >1000, standard assays recommended; if VL in 500-1000 range, standard assays may allow genotyping and should be considered. 
- 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. 
- 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. 
- Low-level viremia, 50-1000 c/ml, may occur as blip or sustained low-level viremia.  With VL < 500 c/ml, drug resistance testing 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%. 
- 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. 
- 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.  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.
- Review of past genotypes and past ART is of primary importance. Test results must be interpreted along with historical results. Cumulative rather than current genotype should be considered. 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.  Available as GenoSure Archive assay from Monogram BioSciences.
- Point mutation assays and clonal sequencing detect minority resistance, yet have unproven clinical utility. Not commercially available.
- Minority variants not detected by standard assays.
Genotype vs. phenotype
- Less expensive.
- 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 (including combined test: Phenosense GT)
Tools for genotype interpretation
- Stanford HIV Drug Resistance Database, http://hivdb.stanford.edu/index.html
- International AIDS Society-USA (IAS-USA), http://www.iasusa.org/resistance_mutations
- Web-based global surveillance map of drug reistance in ART-naive populations: http://hivdb.stanford.edu…
Basis for recommendation
- Günthard HF et al: Antiretroviral treatment of adult HIV infection: 2014 recommendations of the International Antiviral Society-USA Panel. JAMA 312:410, 2014 Jul 23-30 [PMID:25038359]
Comment: IAS-USA Panel 2014 recommendations accessed at: https://www.iasusa.org/content/antiretroviral-treatment-adult-hiv-infection-2014-recommendations-international-antiviral-society-usa-panel.
- 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; Sections: Drug-Resistance Testing and Co-Receptor Tropism Assays; May 1, 2014.
Comment: Drug resistance testing recommended at diagnosis, at ART initiation, and in pregnancy. Resistance testing is recommened in the setting of suboptimal virologic response with HIV RNA >1,000 c/ml and should be considered, but may be unsuccessful with standard resistance testing, with HIV RNA >500 and < 1,000 c/ml. Due to inconsistencies in performance, drug resistance testing is not usually recommended with HIV RNA < 500 c/ml.
Accessed at: http://aidsinfo.nih.gov…. Accessed 11/4/15.
- Wensing AM et al: 2014 Update of the drug resistance mutations in HIV-1. Top Antivir Med 22:642, 2014 Jun-Jul [PMID:25101529]
Comment: Update emphasizes the importance of clinical context when interpreting genotype test results. Comments include assessing whether detected mutations are consistent with patient’s ART history, risk of false negative results is higher if sample collected in absence of ART, and failure of first-line ART most often results in drug resistance to 1 or 2 of the drugs in the regimen, e.g., 3TC, FTC or NNRTIs.
- Casadellà M et al: Primary resistance to integrase strand-transfer inhibitors in Europe. J Antimicrob Chemother 70:2885, 2015 [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.
- Derache A et al: HIV drug resistance mutations in proviral DNA from a community treatment program. PLoS One 10:, 2015 [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.
- Drescher SM 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 58:285, 2014 [PMID:24145874]
Comment: Transmitted drug resistance 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.
- Geretti AM 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 69:1090, 2014 [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.
- Geretti AM, Paredes R, Kozal MJ: Transmission of HIV drug resistance: lessons from sensitive screening assays. Curr Opin Infect Dis 28:23, 2015 [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.
- Gonzalez-Serna A 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 58:1165, 2014 [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.
- Panichsillapakit T et al: Prevalence of transmitted HIV drug resistance among recently infected persons in San Diego, California 1996-2013. J Acquir Immune Defic Syndr Sep 18 [PMID:26413846]
Comment: Reported prevalence of transmitted drug resistance measured in 496 treatment-naive recently HIV-infected members of San Diego Primary Infection Cohort was 13.5% (67/496), with an increasing trend from 1996-2013 for TDR to NNRTIs. TDR to NNRTIs was 8.5% (42/496), to protease inhibitors was 4.4% (22/496) and to NRTIs was 3.8% (19/496).
- Pou C et al: Improved prediction of salvage antiretroviral therapy outcomes using ultrasensitive HIV-1 drug resistance testing. Clin Infect Dis 59:578, 2014 [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.
- Swenson LC et al: HIV drug resistance detected during low-level viraemia is associated with subsequent virologic failure. AIDS Jan 21 [PMID:24451160]
Comment: First documented episode of low-level viremia (LLV) documented in 2176 patients was evaluated by resistance testing, 90% (N=1965) were successful, and of these, 263 were lost to follow-up or changed ART. Resistance was stratified based on genotype susceptibility scores (GSS). Higher GSS, being naive to treatment, LLV at later calendar year, and higher adherence significantly decreased risk for treatment failure.
- 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.
- Volpe JM et al: Five Antiretroviral Drug Class-Resistant HIV-1 in a Treatment-Naïve Patient Successfully Suppressed with Optimized Antiretroviral Drug Selection. J Int Assoc Provid AIDS Care 14:398, 2015 Sep-Oct [PMID:26188010]
Comment: Case report of transmitted resistance to 5 antiretroviral drug classes.
- Winand R et al: Assessing transmissibility of HIV-1 drug resistance mutations from treated and from drug-naive individuals. AIDS 29:2045, 2015 [PMID:26355575]
Comment: Authors illustrated the correlation of the prevalence of surveillance drug resistance mutations (SDRMs) in ART-naive pts with SDRMs in those on treatment with viral failure. Methods included identification of ’robust outliers’ with characterization of those outliers above the regression line as mutations found more often in ART-naive pts with greater transmissibility and lower reversion rate after transmission because of lower fitness cost. Whereas outliers below the regression line, occur less often in ART-naive pts than expected compared to those on ART with viral failure, and are transmitted less often and/or reverse faster because of higher fitness cost.
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