Antiretroviral Resistance Testing for Clinical Management

Transcription

1 AIDS Rev 2002;4:3-12 Antiretroviral Resistance Testing for Clinical Management Bryan E. Youree and Richard T. D Aquila Vanderbilt University Medical Center, Nashville, Tennesse, USA Abstract The advent of highly active antiretroviral therapy (HAART) has dramatically changed the management of HIV disease. These powerful regimens of drugs have reduced morbidity and mortality as well as number of hospitalizations among individuals with HIV. Nevertheless, many patients fail to achieve viral suppression. Various factors underlie this problem, but resistance to current medications has been increasingly recognized as an important element. Several methods of assessing antiretroviral resistance in HIV-1 are currently available. Phenotype assays directly assess the level of susceptibility of a virus to specific drugs. Genotype assays determine the presence of viral mutations that may affect viral susceptibility to a particular drug or class of drugs. An increasing amount of data indicates that antiretroviral resistance testing may improve response to therapy and increase the likelihood of achieving viral suppression. In this paper we review the available data regarding the role of antiretroviral resistance testing and compare the two different assays. We also discuss limitations of the current assays, interpretation of the data, and current consensus guidelines. Although many questions remain, antiretroviral resistance testing along with expert opinion can aid in the management of HIV disease. Key words Genotype. Phenotype. Antiretroviral Resistance Testing. Introduction medications, failure to achieve complete viral suppression is common in clinical practice, occurring The introduction of highly active antiretroviral therapy (HAART) has had a profound impact on the at a rate of 40 to 70 percent 1-3. management of HIV infection No part in developed of this countries. For the first time, suppression of HIV RNA to publication The reasons may for drug be failure are multi-factorial. Poor penetration of drugs into certain body compartments, insufficient adherence, and variable undetectable levels in plasma is possible. This, in turn, leads to decreased morbidity reproduced and mortality or photocopying pharmokinetics within and between individuals contribute to subtherapeutic drug levels in vivo. This through reduction in the incidence of opportunistic infections and prolonged survival. Currently, there allows for selection of either preexisting or newly without the prior written are nineteen drugs approved by the FDA for the generated drug-resistant permission mutants. HIV drug resistance is a product of the high replication and muta- treatment of HIV, including 16 unique HIV-1 inhibitors (Table 1). Despite the availability of these tion rates of HIV. The half-life of plasma virions can of publisher be as little as 6 h, and more than 10 billion copies can be generated in a single day in untreated individuals 4. Because the HIV reverse Correspondence to: Permanyer Publications 2010 transcriptase Richard T. D Aquila Vanderbilt University Medical Center lacks proofreading capability, an average of 1 muta- Nashville, Tennesse occurs during each replication cycle 5. Drug USA resistance results from the substitution of amino 3A I D S R E V I E W S

2 AIDS Rev 2002;4 Table 1. FDA-approved antiretroviral drugs Class Generic Name (abbreviation) Brand Name Nucleoside Analogue Zidovudine (AZT, ZDV) Retrovir Reverse Transcriptase Didanosine (ddi) Videx Inhibitors Zalcitabine (ddc) Hivid (NRTI) Stavudine (d4t) Zerit Lamivudine (3TC) Epivir Zidovudine/Lamivudine (AZT/3TC) Combivir Abacavir (ABC) Ziagen Zidovudine/Lamivudine/Abacavir Trizivir (AZT/3TC/ABC) Nonnucleoside Reverse Nevirapine (NVP) Viramune Transcriptase Inhibitors Delavirdine (DLV) Rescriptor (NNRTI) Efavirenz (EFV) Sustiva Nucleotide Analogue Tenofovir Viread Reverse Transcriptase Inhibitor Protease Inhibitors (PI) Saquinavir (SQV, hgc) Invirase Saquinavir (SQV, sgc) Fortovase Ritonavir (RTV) Norvir Indinavir (IDV) Crixivan Nelfinavir (NFV) Viracept Amprenavir (APV) Agenerase Lopinavir/Ritonavir (LPV/RTV) Kaletra 4A I D S R E V I E W S acids that encode the reverse transcriptase and drug. Primary mutations often render viral isolates protease enzymes. Although many of these resistance mutations are harmful to viral replication in the mutations generally develop in viruses with previous less fit than the original wild-type virus. Secondary absence of drug, they can be selected because primary mutations. However, they also can occur as they allow HIV to replicate better in the presence of natural polymorphisms in viruses from drug-naïve the antiretroviral therapy. Once selected, the capacity of HIV for genomic integration and dormancy have little discernable effect on viral susceptibility to patients. Individually, secondary mutations may means that the mutants may persist, perhaps for later re-selection. satory, resulting in increased fitness of the particular a drug in vitro. They are predominantly compen- The reading frames for both the reverse transcriptase and protease enzymes are found in the HIV-1 mutations can be shared by several agents within a viral isolate. Unlike primary mutations, secondary pol gene. Some specific single mutations can drug class. For patients who have undergone antiretroviral therapy, resistance selected by previously directly diminish drug binding to the enzyme s active site and thereby impact on a drug s inhibitory used drugs can lead to cross-resistance to other effect. This is exemplified by the M184V and RT drugs and have a significant impact on the efficacy Y181C mutations that confer resistance to lamivudine and nevirapine respectively. Other mutations patients, infection with drug-resistant virus may lim- of salvage regimens. Even in antiretroviral-naïve have more complex mechanisms that cause resistance, distinct from a direct effect on drug binding. initial combination regimen. Several methods of it the virological and immunological benefits of the Resistance to some antiretrovirals requires the assessing antiretroviral resistance in HIV-1 are currently available to aid clinicians in the selection of accumulation of many mutations. Zidovudine, for example, selects multiple, sequential mutations combination regimens. The two different types of No part of this publication may be before high-level resistance occurs 6. Even when drug resistance tests available are assays for genotype and phenotype 4,8. Genotype assays provide several mutations are present, there is minimal decrease in binding of the active reproduced form of zidovudine or photocopying information about viral mutations that may result in to the mutant RT. Likewise, some protease inhibitors changes in viral susceptibility to a particular drug or need 3 or more specific mutations to lose efficacy class of drugs. Phenotype assays directly quantitate the level permission of susceptibility of a patient s virus to and many of those mutations without do not the directly prior impair written drug binding 7. The combination of continued viral specific drugs in vitro. replication and selective drug pressure for several months can select for multiple mutations. of the publisher Resistance mutations have been classified as primary or Phenotypic assays secondary. Permanyer Primary mutations develop Publications during the course of virologic failure. They are usually laboratory of a recombinant virus through insertion Phenotypic assays begin by 2010 construction in the drug specific and have a measureable impact of the protease and reverse transcriptase genes of the in vitro susceptibility of HIV-1 to a particular a patient s virus pool into a reference strain of virus.

3 Bryan E. Youree and Richard T. D Aquila: Antiretroviral Resistance Testing for Clinical Management This pool of recombinant virus is tested in vitro for the amount of a particular drug needed to inhibit virus replication by 50% (IC 50 ). Results are presented as the fold-change in IC 50 for each isolate as compared with a drug-susceptible control strain 9. subpopulations could nevertheless contribute to drug failure. Genotypic assays The measurement can also be compared to a prior Genotypic resistance assays employ DNA specimen from the same patient. This assay is a direct measurement of the actual sensitivity of a patient s HIV to specific drugs. Currently, there are two recombinant virus phenotypic assays available in the US from commercial laboratories operated by ViroLogic, Inc. (South San Francisco) and Virco (Belgium). (A third laboratory, VirAlliance in France, has begun offering such an assay in Europe more recently.) These assays use reporter genes to measure the level of recombinant virus replication at a given drug concentration 9. An increase in IC 50 beyond a certain level above that of the reference strain is categorized as less susceptible sequencing techniques to investigate the reverse transcriptase and protease regions of the HIV-1 genome for all previously identified resistanceassociated mutations. Similar to phenotypic assays, genotypic assays require amplification of HIV-1 sequences from plasma containing at least 500 to 1000 HIV RNA copies/ml and may detect a mutant present in plasma at a level above 10 to 50% of the total population of viruses 13. Several forms of genotyping assays are available. Direct sequencing is most commonly used 4. This is usually based on a cycle sequencing methodology, but can also be based on chip-based sequencing by hybridization. 10 or reduced susceptibility 11. The cut-off Recent studies comparing cycle sequencing and points for a clinical criterion of resistance, rather than technical criterion of reduced susceptibility are discussed below (under Interpretation of Assays ). These assays require greater than > HIV-1 RNA copies/ml in plasma. The ViroLogic assay incorporates several protease cleavage sites in gag, as well as all of the protease and the RT coding region to codon The Virco assay includes all the protease and RT coding region with coverage to RT codon The phenotypic assays cost approximately $700 to $900 per sample, and the average processing time is 2-8 weeks 4,8. The time until a report is available is usually longer than that of genotypic assays, which are described below. Due to the complexity and biologic hazard of the technology, as well as the level of infrastructure and operator expertise required, most clinical reference laboratories or local hospital laboratories are likely to never be capable of performing phenotypic testing. The phenotypic drug susceptibility assay has several limitations. The major limitation is how to clinically interpret these results (discussed more under Interpretation of Assays ). More data are needed about delineating the clinical cut-off for an sequencing by hybridization demonstrated good nucleotide concordance rates overall. However, higher rates of discordance were observed among resistance-associated codons 14,15. These studies suggest that the presence of genetic mixtures may influence hybridization-based sequencing. Another hybridization-based method, the line-probe assay, is more rapid and cheaper than direct sequencing, although it analyzes only a limited subset of the positions in which resistance mutations may occur. Viral genome sequencing can be performed using cycle-sequencing based kits, such as the TRUGENE HIV-1 Genotyping Kit, by Visible Genetics, Inc. (VGI), or the ViroSeq Kit, by Applied Biosystems, Inc. (ABI). Several reference laboratories also have developed their own assays, often referred to as home-brew assays. Although reference laboratory based assays are now more readily available, they are not required to undergo a process of FDA approval. This process of rigorous external review is likely to improve standardization and consistency of results. The TRUGENE HIV-1 Genotyping Kit received FDA approval on September 26, 2001; the ABI kit is not yet approved at this writing. The cost of a genotype assay is approximately $400 per sample. Most reference laboratories have a turnaround time of 2 or more weeks; this might be short- IC 50 for each drug; e.g., how much resistance is enough to overcome the inhibitory effects of the ened if testing was available in local hospital-based drug in vivo. There are a few small studies that laboratories. Availability for local hospital-based laboratories as well as result standardization and consis- address the IC 50 associated with loss a plasma virus load response. Preliminary No part data of about this such publication a tency may also be may expected be to improve with recent clinical cut-off for an IC 50 are now available for introduction of an FDA-approved kit. The major limitation of the genotypic assays is interpretation of the only 2 drugs; no data are reproduced available about other or photocopying drugs. Questions also remain whether in vivo drug results. The clinical utility of the resistance-associated mutations identified by genotyping is dependent levels can be achieved in at least some individuals that may overcome the without level of in vitro the phenotypic prior written on a database permission that correlates sequence variation in resistance associated, on average, with loss of HIV protease and reverse transcriptase with phenotypic resistance to antiretroviral drugs. Difficulties response. Moreover, in vitro data concerning single drugs may not account for possible of synergy the publisher arise in estimating the consequences of the interaction of some drug combinations. Another limitation of of multiple mutations on phenotype and the both phenotype Permanyer and genotype assays is Publications that extent of cross-resistance among 2010 drugs in a class 4. minority subpopulations of drug-resistant virus As an example, some mutations (RT L74V, associated with didanosine resistance; RT M184V, associat- that are less than 10-50% of the total population may not be detected 8,9. These undetected minor ed with lamivudine resistance; and RT Y181C, asso- 5A I D S R E V I E W S

4 6A I D S R E V I E W S AIDS Rev 2002;4 ciated with nevirapine resistance) have been shown starting from the wild type RT 215T. Since these to restore susceptibility of HIV-1 to zidovudine, even special mutants may also signal that there is an in the presence of several resistance mutations previously selected by zidovudine 16. The interpretation possibility of rapid re-selection of RT T215 Y or F. archived RT T215Y or F, they may also suggest the of information from genotypic assays benefits from Thus, these non-resistance mutations may serve expert opinion, although there are other more practical approaches (see Interpretation of Assays ). tion of resistance. as sentinels suggesting relatively easier acquisi- Several issues are still topics of research but will Another issue requiring further research involves be mentioned here because of their future clinical a preliminary report that certain rare NNRTI-resistant viruses can grow better in vitro in the presence relevance. While essential to understanding biology and epidemiology of resistant virus, the concepts of of an NNRTI than in the absence of the drug 23. Such primary and secondary mutations are more mutants might exhibit increased replication on drug applicable to clinical management of some drugs in vivo; e.g. drug-stimulated replication. This trait than others. Recently, the International AIDS Society seems analogous to vancomycin-dependent enterococci and just as alarming, but it has not yet been USA Resistance Testing Consensus Panel has stopped differentiating between primary and conclusively demonstrated. secondary mutations for reverse transcriptase inhibitors (see This was intended to emphasize the generalization that a larger number of mutations (including secondary mutations) The current genotypic and phenotypic assays Limitations would be associated with higher levels of resistance require plasma with copies of HIV-1 and potential cross-resistance to both nucleoside RNA per milliliter. Thus, viral load testing remains RT inhibitors (NRTI) and nonnucleoside reverse the method of choice to monitor for drug failure. The transcriptase inhibitor (NNRTI). In other words, secondary mutations cannot be ignored, or considered common clade B viruses of Europe and North resistance assays were developed using the more unimportant, by clinicians interpreting genotypic America. The genetically diverse HIV strains from resistance tests. It now seems inadequate to rely different geographic regions may be less well only on primary mutations for identifying resistance. amplified for genotyping or phenotyping. This is an This concept has not yet been applied to protease important concern in caring for patients who inhibitors (PI) by the IAS-USA group. Management acquire HIV in non-western countries, but the limited available data suggest at least a majority of non- of some of the PIs, such as nelfinavir and saquinavir, can probably still be based on whether or not primary mutations are present, with less attention paid the current methods optimized for clade B virus. clade B specimens can be genotyped with some of to secondary mutations. However, other protease Furthermore, detection of clinically significant inhibitors, such as indinavir or lopinavir, are better minority subpopulations of virus remains a problem. managed by attention to the total number of mutations present, as now suggested for reverse transcrip- subpopulations that are > 20% of the total viral pop- Both genotypic and phenotypic assays detect only tase inhibitors. Moreover, specific primary mutations ulation. This value may vary depending upon the are difficult to categorize for indinavir and lopinavir individual mutation 4,8. Detection of small subpopulations is particularly important in patients who have and need not be present for resistance to occur; an accumulation of secondary mutations may be all been heavily treated and have often changed therapy. The predominant HIV-1 population may shift that is required for resistance. A second current research topic involves a special category of mutation that has been described in type virus lacking these mutations within a few from a virus with drug-specific mutations to the wild RT codon 215. Mutations that encode neither the weeks of discontinuing a drug; the resistant mutants wild type amino acid, threonine (T), nor the resistant may then be an undetectable subpopulation. Testing at this time will give the appearance of suscep- mutant amino acids, tyrosine (Y) or phenylalanine (F) can be seen at RT codon 215. Several studies tible, or more susceptible, virus. However, resistance will reemerge quickly upon reintroduction of show that RT 215 D, C, or S can be present in specimens from drug-naïve persons (in up to 10% the medication by rapid re-selection of a minor of drug-naïve subjects No in some part studies). of this These publication subpopulation. may Therefore, be performance of either a mutants have evolved by a single nucleotide genotypic or phenotypic assay while the patient is change from the zidovudine reproduced resistant mutant allele or photocopying on a failing regimen will minimize falsely negative (RT 215 Y or F). These mutations in codon 215 do results. not cause detectable decreased susceptibility to Another significant limitation to the current assays zidovudine in vitro, without and confer better the replicative prior written is adequate permission standardization and clinical validation. capacity on the virus in the absence of drug than Errors in specimen processing, contamination during PCR, and laboratory technical performance RT T215Y or F mutations In vitro data suggests more rapid selection of zidovudine resistant of the RTpublisher may compromise the quality and validity of the data. 215 Y or F from one of these special mutants than A study evaluating the detecting capabilities of 23 from the wild Permanyer type 22. This is presumably because Publications laboratories demonstrated large 2010 interlaboratory differences in the quality of the results 13. This stresses the single nucleotide change to RT 215Y or F is more easily acquired from these special mutants the importance of quality controls. More recent surveys have shown improvement in these areas, than are the two nucleotide changes needed if per-

5 haps, in part related to better standardization of genotype methods as sequencing kits have been developed 24. Interpretation of assays The most challenging aspect of the resistance assays lies in the interpretation of their data. This is especially true with genotypic assays. The literature on this topic is expansive and continuously changing. Several academic groups, as well as companies, have developed software that uses a rulesbased interpretation of the identified mutations to interpret their expected effects on antiretroviral susceptibility. This method of interpretation may use both in vivo virologic and in vitro phenotypic response data for predicting effects of both reverse transcriptase and protease sequences on antiretroviral drug resistance. The home-brew assays of individual reference laboratories may use such an approach to reporting results. However, they may differ in regards to specific rules used, how up to date with current literature those rules are, whether the rules are periodically revised, and whether acknowledged experts are involved in such updates. One academic web site has compared some of these rules-based algorithms 25. One of the rules often used in such comparisons was developed some years ago by the Resistance Collaborative Group (RCG), has never been updated, and is inadequate for clinical use 26. The FDA has approved the Visible Genetics algorithm with the condition that it be updated every 6 months by an independent panel of experts. One reference laboratory, Virco, has created another approach to interpreting genotype assays. Termed virtual phenotype, it consists of a proprietary database of results from patient specimens that have been both genotyped and phenotyped for each drug. This data base is used to calculate the probability of a resistant phenotype for individual drugs according to the genotype considered to be relevant for each drug 27,28. The match is based on certain selected mutations and not the entire sequence. The interpretation quality is likely to be better when there are a larger number of prior specimens in the database that match the mutations classed as relevant for each drug in the specimen being queried. It appears to be similar to rulesbased algorithms in retrospective analyses and does provide somewhat more quantitative information about probable magnitude of resistance than the current rules-based approaches. There have not been any prospective studies reported to date comparing the different methods of genotype interpretation. Currently, a Resistance Mutations Project Panel exists as a subgroup of the IAS-USA Resistance Testing Guidelines Panel. This panel provides regular updates on the mutations that affect the clinical utility of currently approved antiretroviral drugs. They are also beginning to create a database of the scientific evidence of the potential effects of resistance mutations in clinical settings. The eventual Bryan E. Youree and Richard T. D Aquila: Antiretroviral Resistance Testing for Clinical Management goal is to provide a complete catalog of mutations and their interactions, with supporting literature citations. Consensus updates can be found at Despite differences in their techniques to generate recombinant virus and to test drug susceptibility, phenotypic assays report their results as an IC 50 for each drug. This represents a fold change relative to the IC 50 of a reference virus. However, it is still under study if different values would be obtained in different laboratories using different methods from the same specimen. One study of predominantly drug-susceptible isolates found fairly good concordance between the two US assays 29. The most challenging aspect of phenotypic assays is determining the relevant cutoff for fold change in IC 50. A technical cutoff is based on variability in repeated testing of the same specimen (precision) while a biologic cutoff is based on the range of IC 50 of wild type isolates never exposed to drugs. A clinical cutoff is defined as a fold change that is needed to overcome the effects of drug levels present in vivo. Currently, data for clinical cutoffs are available only for abacavir and lopinovir/retonivir 30,31. The degree of variability in the assay (technical cutoff) should be lower than either the biological or clinical cutoffs in order for the testing of a single specimen to reliably determine whether the IC 50 is above or below the biological or clinical cutoff. Clinical trial data Data concerning the usefulness of drug resistance testing are available from both prospective and retrospective studies. The first prospective trial suggesting a virologic benefit to genotype testing in the clinical setting was VIRADAPT 33. This was an open, controlled study of 108 HIV-1-infected patients randomized in a 2:3 ratio to either a control arm (n = 43) or a genotype arm (n = 65). Entry requirements included HIV-1 RNA > 10,000 copies/ml despite at least 3 months of protease inhibitor use and > 6 months of nucleoside reverse transcriptase inhibitor use. Patients in the control arm were prescribed new regimens based on the International AIDS Society (IAS) published guidelines on antiretroviral treatment 33. Patients in the genotype arm were given a new regimen after physician review of the genotype results 32. An algorithm demonstrating associations between specific mutations and their effects on specific drugs directed interpretation of the detected mutations in this group. No expert advice about which specific drugs to use was routinely given in VIRADAPT, although patients previously failing 2 or 3 protease inhibitors had their cases discussed by 2 or 3 study physicians before a decision was made on the next treatment. Viral load, CD4-cell count, and genotypic resistance assay were performed at randomization and every 3 months. Treatment was adjusted after 3 months of a new regimen if HIV-1 RNA remained > 10,000 copies/ml or decreased < 0.5 log 10 from baseline; genotype testing was available at each No part of this publication may be reproduced or photocopying without the prior written permission of the publisher Permanyer Publications A I D S R E V I E W S

6 8A I D S R E V I E W S AIDS Rev 2002;4 adjustment for the genotype group but not for the control group. New medications were incorporated in the therapeutic regimen as they became available during the study 32. By month 3, the mean change in plasma HIV-1RNA level was significantly greater in the genotypic assay arm of the study There have also been prospective studies analyzing the adequacy of phenotypic HIV-1 resistance testing. The VIRA3001 study was an unblinded, randomized trial that compared the Antivirogram assay (Virco) with standard of care 36. Enrolled patients were NNRTI naïve and exhibited virologic failure on ( 1.04 log 10 ) than in the standard care arm ( 0.46 their first protease inhibitor-containing regimen. log 10 ). Furthermore, HIV-1 RNA was undetectable Patients were required to have 2000 copies/ml (<200 copies/ml) in significantly more patients in HIV-1 RNA at study entry. Analysis at week 16 the genotypic assay arm compared to the standard care arm (29 vs 14% of patients, respectively). demonstrated that patients randomized to the phenotypic-assay arm had a significantly greater Results at 6 months showed similar differences decrease in viral load ( 1.23 log 10 vs 0.87 log 10 ). (change in virus load, 1.15 vs log 10 ; proportion of patients with undetectable virus load, 32 vs achieved undetectable viral loads (<400 copies/ml) This arm also had significantly more patients that 14%, respectively). At 6 months, resistance test information was made available for all patients with detectable viral loads. Analysis at 1 year demonstrated that the benefits of resistance testing were maintained and were still possible even if strategy implementation was delayed for 6 months. The GART (Genotypic Antiviral Resistance Testing) study assessed the short-term effects of genotypic resistance testing in the management of patients for whom treatment with a protease inhibitor and two nucleoside reverse transcriptase inhibitors was failing. Study entry criteria required that participants had been on their current therapy for 16 weeks before experiencing virologic failure, which was defined as a 3-fold rise in plasma HIV-1 RNA from the nadir while on therapy. A total of 153 individuals at week 16 (59 vs 42%). It should be noted that of the > 270 patients randomized in this study, only 140 were available for the interim analysis at week 16. The Kaiser Permanente study used the Antivirogram assay to compare phenotype with standard of care in heavily treated patients 37. Entry requirements included antiretroviral therapy for > 3 months with HIV-1 RNA > 2000 copies/ml. Roughly 75% of the patients enrolled in this study had failed 2 protease inhibitors. The study did demonstrate a significantly higher number of sensitive drugs prescribed in the phenotype group compared to the standard of care arm. However, no significant difference in decline of HIV-1 RNA between the phenotype and standard of care arms was observed at week 16. The original study design called for a larger sample, were randomized to GART (N = 78) or standard of but slow accrual prevented full enrollment. 38 This care (N = 75). Specific changes in drug choices were recommended by an expert panel for the genotype-assigned group based on discussion of that individual s genotype result and other factors. The change in plasma HIV-1 RNA level averaged at 4 and 8 weeks was significantly better for the patients in the genotypic-assay study arm than for and the limited drug options available for these treatment-experienced patients, prevents drawing any significant conclusions from this study. CCTG 575 randomized 256 patients to phenotype testing using the Phenosense (ViroLogic) assay versus standard of care. Study patients had experienced > 6 months of antiretroviral therapy with as least 1 protease inhibitor and had entry HIV-1 RNA >400 controls ( 1.19 log 10 vs 0.61 log 10 ); control group subjects also did not get expert advice on drug copies/ml 39. The median time of prior antiretroviral choices. The proportion of patients with undetectable virus loads (<500 copies/ml) was significantly higher in the GART arm than in the standard of care arm at weeks 4 (45 vs 23%, respectively) and week 8 (55 vs 25%, respectively). However, the significant difference was lost by week 12. It is worthwhile to note that half of the patients in the genotype-assigned group did not receive the therapy therapy was 36 months, and 76% were NNRTI naïve. The reduction in HIV-1 RNA and the proportion achieving < 400 copies/ml were not significantly different between the two arms at 6 and 12 months. However, it was noted that the number of susceptible NNRTI and protease inhibitors at baseline were independent predictors of the percentage of patients reaching <400 copies/ml at 6 and 12 recommended by the expert panel. Further months. Results have led to revisions in IC 50 cut-offs analysis demonstrated no significant difference in the Phenosense assay for some of the most commonly prescribed between the two study No arms in part clinics of where this expert publication may drugs; be the cut-offs used in the panel recommendations were followed less than study may have not predicted drug effectiveness 60% of the time. This suggests reproduced that both the genotype test and expert advice were helpful. an advantage. or photocopying well enough for the phenotype test to have provided The Havanna study sought to further elucidate Various retrospective clinical studies have indicated that antiretroviral permission resistance testing may have the role of expert advice without in interpretation of prior a genotype written test result. Patients were randomized to 1 of clinical utility in estimating the probability of suc- four arms: no genotype / no advice (control), genotype without advice, advice without genotype, of the andpublisher studies using a standard primary endpoint of virocess of salvage regimens. A reanalysis of these both genotype and advice. Data from 24 weeks logic failure and time frame was performed 26. Baseline genotypic and phenotypic revealed that the Permanyer best response (defined as HIV-1 Publications 2010 drug resistance predicted virologic failure whether analyzed separately RNA < 400 copies/ml) occurred in the arm receiving both genotype testing and expert advice, and that or adjusted for other confounders such as viral load genotype without advice was better than control 35. and the type of new drugs in the regimen. In the

7 majority of studies, there was a 2-fold reduction in the likelihood of virologic failure for each additional drug in the regimen to which the virus was susceptible by genotyping methods and a 2- to 3-fold reduction for each additional drug to which the virus was susceptible by phenotyping. Bryan E. Youree and Richard T. D Aquila: Antiretroviral Resistance Testing for Clinical Management benefit over only on of the assays for the most drug-experienced subjects. Cost-effectiveness The cost of any new study or treatment plays a role in its clinical utility. The cost to treat HIV-1- infected adults in the United States has been estimated to be approximately $7 billion yearly 42. This Comparing genotypic and phenotypic resistance assays analysis assessed the cost to be roughly $20,000 Much debate has arisen regarding which type of per patient per year. The expense of the antiretroviral medications accounts for most of these costs. assay is more clinically useful. Genotypic assays provide mutations that are predominant in a Cohort studies have demonstrated that roughly patient s circulating virus population. However, the 50% of patients fail initial suppression or develop reports are difficult to interpret by non-experts; rebound in HIV RNA 43,44. Each failure and regimen the interpretation algorithms vary and may not be change accrues further costs with additional clinic up to date; and knowledge about the interactions of visits, laboratory tests and new prescriptions. It has multiple mutations is incomplete. This is improving been estimated that each change in regimen costs as assays become used in a wider array of laboratories and by more clinicians, and methods / inter- Several studies have evaluated the cost-effec- between $500 and $1000. pretation schemes become more standardized, tiveness of HIV treatment regimens 45,46. One study Phenotypic assay results appear easier to interpret projected the total discounted cost over a 5 year as they provide direct in vitro sensitivity data similar period to be $5100 lower in patients receiving triple to MICs for bacteria. However, these assays do not antiretroviral therapy compared with double therapy 45. This savings was attributed to delayed disease account for drug-drug interactions, and questions remain regarding relevant clinical cutoff values progression. However, the cost of triple-therapy for fold change in IC 50. The latter is improving as became more expensive over time due to longer more data become available. There are also differences in price and turn around times that favor increase in monthly cost of caring for HIV patients survival. A more recent study demonstrated an genotyping. Theoretically, mutations that do not treated with highly active antiretroviral therapy themselves confer any change in IC 50 but that can (HAART) 46. Again, the majority of cost came from be sentinels for more rapid resistance emergence antiretroviral medications. The study noted that (eg, non-resistance associated mutations in RT most of these costs were offset by a marked codon 215) might provide an advantage for genotype. In addition, some mutations may give similar The costs accumulated during the VIRADAPT trial decrease in inpatient-related costs. increases in IC 50 in vitro, but be associated with differences in viral load responses in vivo, as has assay was assessed as $500 in this analysis. At 1 have been evaluated 47,48. The cost of a genotypic been suggested in regard to PR D30N versus year, there proved to be no significant difference in L90M for nelfinavir 40. the mean cost of care between the genotyping arm The NARVAL trial (ANRS 088) investigated the and the standard of care arm ($18,484 vs $20,412). clinical utility of using genotype or phenotype The costs of resistance testing were outweighed by assays compared with the standard of care (no reduced use of protease inhibitors in the VIRADAPT testing) for choosing salvage regimens in heavily trial 47,48. treated patients 41. Patients were stratified at baseline according to viral load and prior antiretroviral assess the cost-effectiveness of genotypic resis- A recent study used an HIV simulation model to therapy. Phenotype and genotype testing was performed again on patients in their respective arm infected patients using data from randomized trials, tance testing 49. This model simulated 1 million HIV- who failed to achieve at least a 1 log decrease in the Multicenter AIDS Cohort Study, the national their viral load at week 12. The proportion of AIDS Cost and Services Utilization Survey, the Red patients with HIV-1 RNA < 200 copies/ml was not Book, and an institutional cost-accounting system. No part of this publication significantly different at week 12 between no testing, genotype, and phenotype arms. A benefit was CPCRA 046 and VIRADAPT. The simulation com- The trials used may to generate be the model included observed in the genotype arm reproduced regarding the percentage of patients with HIV-1 RNA < 200 genotypic testing strategies, incorporating CD4 cell or photocopying pared standard of care treatment strategies with copies/ml at weeks 12 and 24, but this was not count and HIV RNA levels as predictors of disease maintained at week 36. without This study the has been prior criticized for using heavily pre-treated subjects with expectancy and incremental cost per quality- written progression. permission Measured outcomes included life few drug options and for having inadequate criteria for interpreting both genotype and of phenotype. the publisher ondary resistance testing (e.g., at time of virologic adjusted life-years. The analysis found that sec- Other trials to compare the 2 test modalities have failure of a drug regimen) resulted in increased life been limited to Permanyer date by poor accrual, as testing Publications expectancy of 3 months. The 2010 incremental cost per has become available in routine practice outside quality-adjusted life-year gained was $17,900 when of trials. Another question is also now supplanting the GART strategy was utilized and $16,300 using this comparison: will both assays provide added data from VIRADAPT. These values were compara- 9A I D S R E V I E W S

8 A I D S R E V I E W S 10 AIDS Rev 2002;4 ble to the widely accepted intervention of Pneumocystis carinii (PCP) prophylaxis ($16,000 per QALY gained) and less than that for prophylaxis against Mycobacterium avium ($35,000 per QALY gained) 50. The longer quality-adjusted life expectancy associated with testing leads to increased cost of care over a longer term (largely due to antiretroviral drugs). However, this is within the range accepted for other expensive medical or surgical interventions. Indeed, the difference in cost between genotyping versus phenotyping is relatively small compared to the total increased costs. The authors suggest that there may not be a difference in costeffectiveness between the two modalities unless one is proven in the future to be more effective than the other. One can also speculate that if using both modalities improves effectiveness of treatment, this might balance the increased cost. Recommendations for clinical use Several agencies have provided guidelines and recommendations for the use of antiretroviral resistance testing in clinical practice, which are outlined in table 2 8,51,52. The goal of each group was to summarize the current data and provide a framework for the use of antiretroviral resistance testing. Testing is recommended if sufficient data exists or considered if fewer data is available. In general, all of the treatment guidelines are similar in their recommendations. All reach a consensus in regards to acutely infected patients, pregnant women, and patients switching therapy due to treatment failures. The EuroGuidelines also include recommendations for pediatric patients and post-exposure prophylaxis. The US Department of Health and Human Services guidelines recommend that resistance testing in pregnancy should be the same as for non-pregnant patients. None of the guidelines currently advocate the use of one type of resistance assay over another. Several studies have attempted to quantify the increasing concern of primary infection by HIV strains with drug resistance mutations The rates of resistance transmission have varied from 3 to 35% by these reports. Some of the higher rates represent reduced susceptibility by phenotypic assay and may not represent complete resistance. The majority of mutations discovered tend to be directed against reverse transcriptase inhibitors, and the rate of resistance to all three classes of medications in a single specimen has been universally low to date. The rates demonstrated in many studies vary by geographic locale. At least one study found that mode of transmission, gender of the patient, and race had no effect on the prevalence of resistance 54. One recent study demonstrated that in drug-naïve patients, the majority of baseline mutations had no negative effect on treatment outcomes up to 18 months 53. Most of the panel guidelines recommend consideration of testing in acute infection, especially if there is suspicion that the virus was transmitted from an individual that has been treated. However, there is less consensus regarding chronic infections, e.g., subjects who present for initial care with an indeterminate and antiretroviral-free period since HIV was acquired. A major concern has been that resistant mutants acquired at initial infection may be out-competed by wild type viruses over a long time off-drug and therefore, be only rarely detectable with current methods that have limited ability to detect minor virus subpopulations. However, a recent study found that resistance mutations were identifiable in more than 15% of drug-naïve subjects presenting after an indeterminate time of acquiring infection 58. This suggests the need for further study on this topic. These guidelines are likely to continually change as more data becomes available. As with any consensus guidelines, the decision to use antiretroviral resistance testing should be based on the clinical context in which the patient presents. In the case of post-exposure prophylaxis, treatment should not be delayed to await the results of testing. Evidence also exists that treatment should not be delayed in the setting of acute HIV infection as it may have an impact on long-term immune control 57,59. Final thoughts While a reasonable amount of data for antiretroviral resistance testing exists, many questions remain. Prospective trials of genotypic assays (GART, Viradapt, Havanna) have found a significant small advantage for genotyping with advice 32,34,35. The data for phenotypic assays (VIRA3001, Kaiser, CCTG575, NARVAL) has been less consistent, with only the VIRA3001 study showing a significant No part of this publication may be Table 2. Treatment guidelines and recommendations for the use of antiretroviral drug resistance testing reproduced or Recommendation photocopying without the prior written permission Clinical Situation IAS-USA Panel EuroGuidelines Department of Health and Human Services Primary HIV-1 infection Consider Consider Consider Chronic infection Consider Consider Not generally recommended Post-exposure prophylaxis No recommendations of the publisher Recommend No recommendation Treatment failure Recommend Recommend Recommend Permanyer Publications 2010 Pregnancy Recommend Recommend No distinction between pregnant and non-pregnant patients Pediatrics No recommendations Recommend No recommendation

9 advantage 36,37,39,41. Current data is limited to only short-term virologic responses. Most studies provide data for 6 months follow up, with VIRADAPT following patients for 1 year. The existing data also emphasizes the importance of expert opinion. How this translates to physicians who are not experts in HIV remains to be seen. Interpretation of the assays can be difficult, but should improve as more data becomes available correlating genotypic and phenotypic resistance. The use of phenotype versus genotype assays remains a debate. We recommend using genotype for 1 st or 2 nd regimen failure. Phenotype may help after genotype for 3 rd or more regimen failures. Regardless, the maintenance of longitudinal records including prior assay results is vital. This will prevent missing mutants selected by a drug that failed previously. New areas of investigation include therapeutic drug monitoring and inhibitory quotient. Inhibitory quotient (C trough /IC 50 ) describes the association between drug exposure and drug susceptibility of a pathogen. Whether the inhibitory quotient is a better predictor of susceptibility than IC 50 will be another important area for future research. Acknowledgements We are grateful to Cindy Gay for careful review of the manuscript. References 1. Ledergerber B, Egger M, Opravil M, et al. Clinical progression and virological failure on highly active antiretroviral therapy in HIV-1 patients: a prospective cohort study. Lancet 1999; 353: Deeks S, Barbour J, Martin J, et al. Sustained CD4+ T cell response after virologic failure of protease inhibitor-based regimens in patients with human immunodeficiency virus infection. J Infect Dis 2000;181: Lucas G, Chaisson R, Moore R. Highly active antiretroviral therapy in a large urban clinic: risk factors for virologic failure and adverse drug reactions. Ann Intern Med 1999;131: Wilson J, Bean P. 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Predicting HIV-1 phenotypic resistance from genotype using a large phenotype-genotype relational database. Antivir Ther 1999;4: Qari S, Respess R, Weinstock H, et al. Comparative analysis of two commercial phenotypic assays for drug susceptibility testing of human immunodeficiency virus type 1. J Clin Microbiol 2002;40: Lanier E, Hellmann N, Scott J, et al. Determination of clinically relevant phenotypic resistance cutoff for abacavir using the Phenosense assay. 8 th CROI. Chicago, IL. February 4-8, 2001 [abstract 54]. 31. Kempf D, Isaacson J, King M, et al. Genotypic correlates of reduced in vitro susceptibility to ABT-378 in HIV isolates from No part of this publication may be reproduced or photocopying without the prior written permission of the publisher Permanyer Publications 2010 A I D S R E V I E W S 11