Principles of HIV resistance testing and overview of assay performance characteristics

Transcription

1 Principles of HIV resistance testing and overview of assay performance characteristics Douglas D Richman Antiviral Therapy 5: Departments of Pathology and Medicine, San Diego VA Medical Center and University of California San Diego, 9500 Gilman Drive, La Jolla, CA , USA For correspondence: Tel: ; Fax: ; drichman@ucsd.edu HIV drug resistance testing is becoming an integral part of antiretroviral drug development and of patient management. The parameters that characterize the performance of both phenotypic and genotypic assays and the validation of this performance are essential to their proper use for these applications. Several principles and biological factors impacting drug resistance testing are summarized. Considerations regarding quality control and validation of the performance of genotypic and phenotypic assays are also addressed. HIV drug resistance testing is becoming an integral part of antiretroviral drug development and of patient management. The parameters that characterize the performance of both phenotypic and genotypic assays and the validation of this performance are essential to their proper use for these applications. Testing for drug resistance serves to guide the treatment of many infectious diseases. It predicts which drugs will not work and why they do not work, and it predicts which drugs might be useful. To help introduce the use of drug resistance testing and to help standardize communication about it, I will first provide a few definitions. Drug susceptibility is the phenotype of replication (or its inhibition) by various concentrations of drugs. The term phenotypic resistance is a tautology, since resistance is a phenotype. Genetic variations and mutations, the genotype, are what confer the phenotype. The terms sensitivity and resistance are value judgements as to whether a drug is likely to work based upon the results of the susceptibility assay and of data on drug exposure. Based on achievable drug concentrations, an isolate could be sensitive or resistant in vivo for the same IC 50 obtained in vitro. Wild-type is a strain of virus that has not been selected by drug treatment. A natural polymorphism is a genetic variant that can circulate in wild-type populations. HIV displays a remarkable amount of genetic variation. Usage of some amino acids that are natural polymorphisms in HIV isolates may be seen more frequently in drug-resistant isolates. A drug-resistance mutation is an amino-acid change conferring reduced susceptibility that is usually seen as a result of selection by drug treatment. The distinction between drug resistance mutations and natural polymorphisms can be important because many genetic variants may emerge with drug treatment that are also natural polymorphisms. The presence of variants that are natural polymorphisms cannot be used to make a claim that drug resistance is being transmitted. Drug-resistance assays for HIV, just as with other micro-organisms, cannot be expected to guarantee efficacy, because drug susceptibility is not the only predictor of treatment response. Other factors such as low drug potency, poor pharmacokinetics, high plasma-protein binding, poor patient adherence, amongst other factors, can result in treatment failure [1]. Resistance is thus a better predictor of failure than is sensitivity of success. There are two major applications of drug resistance testing: to evaluate drugs and to manage patients. There are multiple assays for drug resistance, and their utility requires standardization, quality assurance and validation for each of these applications. HIV raises several special challenges to the use of drug resistance testing: (i) no two strains of HIV are identical; (ii) within each individual, an HIV infection represents a mixture of genetic variants (a quasispecies) that is, each individual s virus is different from any other; (iii) this quasispecies is constantly evolving; (iv) drug treatment is selecting for additional variation and the drug treatment practices throughout the world are rapidly changing; (v) patient management often precedes what is in the published peer-reviewed literature and often involves investigational drugs. All of these factors affect the patient s quasispecies. The drug resistance assays that are being developed have to be useful and have to be valid in the context of this remarkable complexity. Several issues must be considered in selecting a drug 2000 International Medical Press /00/$

2 DD Richman resistance assay, including its intended use and its ability to evaluate virus from plasma, cells, semen or other tissues. For example, the Boom extraction methodology for HIV ribonucleic acid (RNA) made assays applicable to semen, while inhibitors in semen made other methods non-applicable. Also, no available assay routinely examines the clonal composition of a specimen. Both genotypic and phenotypic assays utilize bulk amplification of nucleic acid in the clinical specimen. Assays may be differentially applicable to different target pathogens. Analogous to quantitative assays for HIV RNA, the initial drug resistance assays have worked primarily for patients infected with clade B isolates. It is clear that these must work for other clades, as antiretroviral drug use is applied globally and also travel brings more non-clade B strains to Europe and North America. Different assays may provide different information. A genotypic assay, for example, could state that it is going to provide the sequence of 1500 nucleotides of pol or it might provide information regarding as few as 20 specific codons known to be relevant to drug resistance. A number of characteristics must be defined to validate the performance of drug resistance assays. There are different practical and regulatory issues with genotypic and phenotypic assays. The sensitivity of detection for different nucleotides or codons must be defined. Because of the sequence context around each codon, assays may differ in their ability to assess different codons [2]. Sensitivities may vary among assays with different plasma HIV RNA levels. Can the assay be applied to plasma from someone who has got a viral load of 5000, 500 or 50 copies/ml of HIV RNA plasma? What is its specificity of the assay? Does it generate the correct answer for each of several codons? Does the result get confounded by the presence of other infectious agents or by drugs or abnormal clinical chemistry values? For example, does the assay provide accurate results for a patient with circulating human T-cell leukaemia virus, HCMV, hepatitis B virus, hepatitis C virus, rheumatoid factor, renal failure, liver failure, hyperlipidemia and levels of various drugs in the plasma. Moreover, even given a well validated genotypic assay, a major variable is the performer of the assay, as discussed below. Despite all the issues regarding the challenge of HIV and the validation of the assays, it is important not to get discouraged. The assays are useful [3]. The desire for an ideal situation in which a test is validated for all situations and it works without need for change is not a realistic expectation if one is involved with a field like HIV. What is needed is to decide what criteria and what sort of validation provide sufficient comfort to implement the use of an assay. In generating standards for assay performance characteristics, a number of factors must be considered. With the complexity of HIV, certain arbitrary and finite decisions must be made about which codons to interrogate for validation. Validation must also be performed at selected, arbitrary concentrations of HIV RNA. A decision whether to test for mixtures of wildtype/mutant codon usage must be made, and if so at which proportions (50:50, 80:20). Should the standard be purified nucleic acid or purified virions? Should the test material be plasma spiked with these? These are very practical issues for developing standards and controls. There are two general approaches to genotyping. One is sequencing. Usually, but not always, reversetranscriptase (RT) PCR of products from patient plasma are assayed by either chain termination or some other variant of the Sanger sequencing method, or by a hybridization-based system. The Perkin Elmer and Visible Genetics systems exemplify the former approach, and hybridization based sequencing is exemplified by the microchip technology approach of Affymetrix. Point-mutation assays interrogate whether a wild-type or mutant amino acid is present at specific codons that are known to be important for drug resistance. Examples of this are the differential hybridization assay developed by the Chiron group and the Line Probe assay developed by Innogenetics. There are advantages and disadvantages to each genotyping approach. Sequencing has the advantage of interrogating the complete sequence of the amplified gene. Thus, it detects the unknown. For development of a new drug, this is clearly important. The limitations of sequencing include the magnitude of the data that are generated. Reviewing the sequence data for 1500 nucleotides from specimens obtained from multiple time points and patients may be difficult. Computer software to deal with this challenge is critical. Once this has been accomplished, the difficulty of interpreting the contributions of each genetic variation remains. This challenge is magnified by natural polymorphisms and mixtures. The codon-specific, differential hybridization assays have certain advantages. They are usually more sensitive in detecting minority species. The sequencing methodologies will often pick up mutant populations if they are present in the 20 50% range, at best [2]. The codon-specific assays will often detect as few as 2 5% in a mixture [4,5]. These assays are simpler to perform and usually simpler to interpret. Their limitations are that they only detect what they look for. Thus each application may have its benefits and limitations in different situations International Medical Press

3 Principles of HIV drug resistance testing With these issues in mind, the performance of assays for sequence determinations by using a panel of standards distributed to different laboratories in a blinded manner merits examination. In follow-up of the results of a first panel [6], a second panel was distributed to 35 laboratories in Europe and North America [7]. Plasma samples from HIV seronegative donors were spiked with infectious HIV that contained genotypically characterized compositions of clonal origin. These clones had five mutations each in the reverse transcriptase and the protease genes. Pure wild-type, pure mutant and various mixtures were examined at a viral load of copies. Each of the sites performed the assay according to whichever of the many sequencing methods that they happened to use. The results were reported as differences from a standard reference sequence by electronic entry into a database. The wide range of participants included academic and commercial laboratories that perform clinical service, some industry laboratories that perform sequencing for drug development and some small academic laboratories performing in-house research. The assay methods and kit types varied. Interlaboratory variation was examined for five resistance mutations in protease and five in reverse transcriptase. With 100% wild-type virus, the laboratories got almost all the answers right. With 100% mutant virus, all but two of the laboratories got all of the answers right, with those two getting nine of ten correct results. However, with various mixtures, success was varied: some laboratories got all of the answers right all of the time; some got most of the answers right all of the time; and some needed to work on their answers. Of note, the success or failure rate of various laboratories appeared to be independent of which of the kits or assays were used. The experience and expertise of the performer appears to be a critical component of the quality of results and cannot be addressed simply by excellent validation of genotyping reagents and methods. Another blinded study tried to determine if the sequence analysis of complex clinical isolates yielded comparable results from the two different experienced laboratories, one a commercial laboratory and one an academic laboratory [8]. Forty-four isolates from heavily pretreated individuals were aliquoted, distributed blinded and then sequenced. Examination of the sequences of over 4000 protease and RT amino-acid residues revealed close to 99% concordance of amino acid calls. Which of the many kits and methodologies for genotyping will be most used will be determined by accessibility, cost and emerging data regarding validation and clinical utility. Identifying expert and experienced assay performance is even more challenging. The situation with phenotypic assays is different. The initial assays for HIV susceptibility were expensive, cumbersome, labour-intensive, slow and relatively imprecise [9]. These methods which helped to identify and characterize the phenomenon of drug resistance are not really satisfactory for patient management or for high throughput generation of data. Two companies (Virco, Mechelen, Belgium and ViroLogic, South San Francisco, Calif., USA) have now developed assays that are relatively rapid, precise, reproducible and amenable to high throughput automated performance. Both of these assays amplify a segment of the gag/pol gene that incorporates protease, much of RT and some of gag from patient plasma HIV RNA. This amplified material is incorporated into a recombinant virus construct that is then used in a standardized highthroughput assay. Both of these assays use a reference laboratory strain of HIV as a control and then report the results of the test virus as fold-difference from control. One obvious potential limitation of these assays is that their utility is restricted to RT inhibitors and protease inhibitors. These targets represent all of the currently approved drugs; however, active programmes are in place to develop drugs against gp41-mediated fusion inhibitors, chemokine receptors and integrase. Another issue is that these are automated assays and are performed in-house; they are done at each of their sites with material shipped to them. Virco has analysed between 1700 and 2200 resistance determinations per approved drug. The variation was less than threefold for each of the assays over a period of a year compared to a wild-type reference strain (B Larder, personal communication). Regarding inter-assay reproducibility with 16 samples on 10 different occasions, the variation in IC 50 was between 1.2- and 2.5-fold. Regarding sensitivity, 95% of the plasma samples with a viral load over 1000 copies/ml were successfully amplified and assayed. Although the assay can reliably differentiate fourfold differences in IC 50, the clinical significance of changes of this magnitude have only been reported for abacavir and adefovir [10,11]. Amplification has been successful with all HIV-1 subtypes [12]. Validation of the ViroLogic assay has been performed by repeated testing of multiple patient plasma specimens (20 replicates per sample), with multiple operators, assay runs, reagent lots and repeated testing of drug-sensitive and drug-resistant viruses (N Hellmenn, personal communication). Twofold changes can be reliably differentiated by this assay for all drugs apart from zidovudine, for which a variability of two- to threefold is observed. A 2.5-fold change is thus reported as significantly different from the control. Once again, this is a laboratory definition Antiviral Therapy 5:1 29

4 DD Richman of reproducibility and not a definition of clinical significance. With the ViroLogic assay, none of the 80 seronegative samples produced a result, and testing of HIV samples containing multiple interfering substances had no impact. These conditions included high levels of triglycerides, haemoglobin, bilirubin, bacteria, fungi and other viruses. What is clinically important is the susceptibility that would predict success or failure with monotherapy with that drug. Generating such data will be challenging. From the transmission data summarized by Little [13] there are wild-type isolates that have eightfold reduced susceptibility to certain drugs. These isolates are not transmitted resistant virus; they represent wild-type variation. Additional assays have been performed on viruses from patients known to be treatment-naive, many of which were isolated in the 1980s to investigate the range of susceptibility of wild-type viruses circulating before antiretroviral drugs were available. For most drugs, isolates exhibit a variability in IC 50 that approaches unity and rarely exceeds a threefold change. For some drugs (especially nevirapine, delavirdine and nelfinavir) a minority of isolates show three- to 10-fold decreases in susceptibility [13]. They thus represent wild-type variation in susceptibility. The genotypic basis for this reduced susceptibility to non-nucleoside RT inhibitors was recently reported [14]. The implications for treatment response of these reduced susceptibility values is unknown. Regarding the ability of the assay to be performed in patients with low plasma viral loads, it appears to generate reliable results in 90% of patients with values above 540 copies/ml and 95% with 700 copies/ml or more. Artificial combinations of mixtures of wild-type and highly resistant virus for various drugs indicate that for most drugs, reduced susceptibility will be detected if at least 20% of the composition is resistant. With lamivudine-resistant virus however, the wild-type has much better growth characteristics and the majority of the mixture must be resistant before a phenotypic change is discerned. With more precise phenotypic assays, mutational interactions can be discerned, the significance of which needs to be defined. There are a number of mutations, especially in RT, but also in protease, that confer resistance to one drug and make the virus hypersensitive to others. The question arises whether this change will impact treatment response. Since the genotype accounts for the phenotype, they must correlate, but how well do they correlate with the available assays? In practice, correlations vary because of the limitations in our understanding and because of the complexities of some mutational interactions. Some correlations are black and white. A valine at 184 of RT confers absolute resistance to lamivudine and a methionine at 184 predicts sensitivity to lamivudine until new drug-resistance mutations are discovered. With a number of other drugs and mutations, especially the non-nucleoside RT inhibitors, certain mutations clearly predict high level resistance. Unfortunately, there are various shades of grey, because for many drugs, especially the protease inhibitors, multiple mutations are usually needed to generate high-level resistance, and there are very complex interactions which can be additive or suppressive in complex ways. The complexities of interpretation of genotypic data are discussed in more detail by D Aquila [14]. In conclusion, I would propose different approaches for different applications of drug resistance testing. For patient management, the determination of the most useful test that is, the test that gives the best predictive value for treatment efficacy considering cost, turnaround time, etc. requires the generation of more data. We do not yet have the information to say which test to use in which situation and, in fact, the answer may vary in different clinical situations like drug naive patients or highly drug experienced patients. In contrast, more straightforward recommendations can be made about the use of drug resistance assays for drug development. In terms of preclinical studies of a new drug candidate, one would want to assay for that drug s activity with a phenotypic assay against a series of laboratory strains, both wild-type and resistant viruses, that have been well characterized by both phenotype and genotype to other approved drugs. One would then also want to assay the drug susceptibility phenotype of various clinical isolates, both wild-type and those well-characterized for resistance to approved drugs. One would want to select for resistance in vitro, characterize the phenotype of that resistant virus against approved drugs to examine for cross-resistance, and then sequence this resistant virus to identify the mutations that have been generated. These mutations should then be characterized by site-directed mutagenesis to ascertain which ones contribute to resistance to that drug and to approved drugs. For clinical studies of new drugs, when patients fail to fully suppress HIV RNA with either monotherapy or combination therapy, the post-treatment isolates should be examined both for phenotypic changes to the candidate drug and to the other approved antiretroviral drugs, and for the acquisition of mutations. Previously uncharacterized mutations should then be evaluated by site-directed mutagenesis. Based upon the cross-resistance profile, the activity of this new drug should be tested against isolates from patients failing therapies. These isolates must be well characterized at International Medical Press

5 Principles of HIV drug resistance testing baseline for genotype and phenotype against this drug and against other drugs. This is a very important and practical problem. We know that a significant proportion of patients have detectable virus with resistance, and these patients need help with new drugs. We need to know how to use these new drugs most intelligently. Drug resistance testing when well validated and properly used should thus facilitate both patient management with available drugs and the optimal development and use of new drugs. Acknowledgements This work was supported by grants AI 27670, AI 38858, AI 43638, UCSD Center for AIDS Research (AI 36214), AI 29164, from the National Institutes of Health, and the Research Center for AIDS and HIV Infection of the San Diego Department of Veterans Affairs. I acknowledge Tom Gingeras (Affymetrix), Chris Petropoulous (ViroLogic), Brendan Larger (Virco), Nick Hellman (ViroLogic), and Rob Schuurman (Utrecht), who generously shared unpublished information. References 1. Hirsch MS, Conway B, D Aquila RT, Johnson VA, Brun- Vézinet F, Clotet B, Demeter LM, Hammer SM, Jacobsen DM, Kuritzkes DR, Loveday C, Mellors JW, Vella S & Richman DD. Antiretroviral drug resistance testing in adults with HIV infection. Journal of the American Medical Association 1998; 279: Günthard HF, Wong JK, Havlir DV & Richman DD. Comparative performance of high-density oligonucleotide sequencing and dideoxynucleotide sequencing of HIV type 1 pol from clinical samples. AIDS Research in Human Retrovirus 1998; 14: DeGruttola V, Dix L, D Aquila R, Holder D, Phillips A, Ait-Khaled M, Baxter J, Clevenbergh P, Hammer S, Harrigan R, Katzenstein D, Lanier R, Miller M, Para M, Yerly S, Zolopa A, Murray J, Patick A, Miller V, Castillo S, Pedneault L & Mellors J. 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