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Assays for Antiretroviral Resistance
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Methods of Testing for HIV Drug Resistance
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Genotypic Resistance Testing
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transparent imageSample Processing and Sequencing
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transparent imageDideoxynucleotide Sequencing
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transparent imageHybridization Methods
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transparent imageDideoxynucleotide Sequencing Reproducibility
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transparent imageSequence Quality Control
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transparent imageIntersubtype Variability
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Phenotypic Resistance Testing
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Drug Resistance Interpretation
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transparent imageGeneral Principles
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transparent imageGenotypic Test Results
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transparent imageVirtualPhenotype
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transparent imageHIV RT and Protease Sequence Database
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transparent imagePhenotypic Interpretation
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Limitations of Drug Resistance Testing
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transparent imageComplex Relationship Between Drug Resistance and Disease Progression
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transparent imageQuasispecies Nature of HIV-1
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transparent imageCross-Resistance
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Acknowledgments
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References
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Tables
Table 1.Algorithms for Interpreting HIV-1 Protease and RT Sequences
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Table 2.Biological Cut-Offs Associated with Antivirogram and PhenoSense Drug Susceptibility Assays
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Table 3.Common Causes of Discordance Between Genotypic and Phenotypic Test Results
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Figures
Figure 1.Schematic Diagram Showing the Distribution of Drug-Resistance Mutations Within the HIV-1 Protease and RT Genes
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Figure 2.Chromatographic Tracings Showing the Automated DNA Sequence Analysis of the 10 Instances in which a Primary Resistance Mutation Was Detected by Just One of Two Laboratories in an Interlaboratory Comparison of 46 RT and Protease Sequences
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Figure 3.Schematic Representation of How the Protease Recognizes Nine Cleavage Sites to Create the Structural Proteins from the gag Gene and Enzymes from the pol Gene
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Methods of Testing for HIV Drug Resistance
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Drug resistance can be measured using either genotypic or phenotypic assays. Genotypic assays detect mutations that cause drug resistance. Phenotypic assays are drug susceptibility assays in which a fixed inoculum of HIV-1 is cultured in the presence of serial dilutions of an inhibitory drug. Current commercially available genotypic and phenotypic assays both test HIV-1 extracted from plasma. After extraction, the contiguous protease and reverse transcriptase (RT) genes are reverse transcribed to cDNA and then amplified using polymerase chain reaction (PCR) to generate sufficient DNA for genotypic or phenotypic testing. Because the segment of the HIV-1 genome used for genotypic testing (about 1500-2000 base pairs [bp]) is larger than that used for quantitative assays (about 100 bp), the sensitivity of most genotypic assays is lower (between 100 and 1000 RNA copies/mL depending on the assay) than most quantitative assays, which can typically detect <20 to 50 RNA copies/mL.

Genotypic testing is used more commonly than phenotypic testing because of its lower cost, wider availability, and shorter turnaround time. Genotypic testing provides early evidence of drug resistance within a virus population. Genotypic assays detect mutations present as mixtures even if the mutation is present at a level too low to affect susceptibility in a phenotypic assay, and detect transitional mutations that do not cause drug resistance by themselves but indicate the presence of selective drug pressure. Genotypic assays detect mutations even if the phenotypic effect of the mutation is suppressed by other mutations in the sequence. The clinical usefulness of genotypic testing has been demonstrated in four of five prospective randomized studies;(1,2,3,4,5) in contrast, phenotypic testing has been shown to be clinically useful in just one of four prospective randomized studies.(5,6,7,8)

Phenotypic testing in research settings is essential for establishing genotype-phenotype correlations, which provide the basis for interpreting genotypic tests and for designing new antiviral drugs that are effective against existing drug-resistant strains. Phenotypic testing in clinical settings is expected to be most useful for testing isolates with unusual combinations of drug resistance mutations. Phenotypic testing may also be useful in combination with therapeutic drug monitoring for designing salvage regimens in heavily treated patients whose viruses contain multiple drug resistance mutations.

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Genotypic Resistance Testing
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Sample Processing and Sequencing
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Because the half-life of HIV-1 in plasma is approximately 6 hours,(9) only actively replicating virus can be isolated from this source; thus the sequence of plasma virus represents the quasispecies most recently selected by antiretroviral drug therapy. The evolution of HIV-1 drug resistance mutations in proviral DNA in peripheral blood mononuclear cells (PMBCs) lags behind that in plasma HIV-1 RNA.(10,11,12-14) In patients with multiple virologic failures, proviral PBMC DNA may contain multiple archived mutations that are not present in plasma. However, the utility of PBMC sequencing has not been evaluated in either prospective or retrospective clinical trials.

Clonal sequencing of individual virus strains is performed in research settings to answer questions about the evolution of HIV-1 drug resistance. Direct PCR, or viral population-based sequencing is done in clinical settings because it is quicker and more affordable than testing multiple clones individually. For both population-based and clonal sequencing, the ability to detect minor variants is related to the proportion of those minor variants within the whole virus population. In direct PCR sequencing, a nucleotide mixture can be detected when the least common nucleotide is present in at least 20% of the sampled virus population.(15-19)

Figure 1 shows the distribution of known drug resistance mutations within HIV-1 protease and RT. Mutations that may individually confer resistance to one or more drugs are represented by tall lines. Accessory mutations that confer resistance only when present with other mutations are represented by short lines. Nearly all of the RT mutations are in the fingers and palm of the 5´ polymerase region of the enzyme. Sequencing for clinical purposes should encompass nearly all of the protease and positions 41- 236 of the RT. Mutations at position 318 have been shown to increase nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance when present with other mutations.(20) G333E is a naturally occurring polymorphism that facilitates zidovudine resistance in isolates from some patients receiving zidovudine and lamivudine that also have multiple zidovudine resistance mutations,(21) but dual resistance to these drugs usually emerges without this change.

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Dideoxynucleotide Sequencing
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Dideoxynucleotide sequencing is the standard approach to HIV genotyping; it involves the synthesis of a new DNA chain in the presence of a primer and a carefully created mix of deoxynucleotide triphosphates (dNTPs) and dideoxynucleotide triphosphate chain terminators (ddNTPs). New strand synthesis starts with the primer and continues until a ddNTP is incorporated in place of the appropriate dNTP. This reaction creates a set of DNA strands each differing from one another by the length of one nucleotide. The strands are separated using polyacrylamide gel electrophoresis, and the final nucleotide on each strand is read by an automated sequencer using fluorometric methods that depend upon the labeling of either the primer or the terminators.

Most diagnostic laboratories use their own "home-brew" methods: sequencing is done using reagents obtained from separate vendors, and polyacrylamide gel electrophoresis and sequencing reading is generally done using an automated sequencer. In 2001, a commercial HIV-1 RT and protease genotyping kit (TruGene, Visible Genetics, Toronto, Canada) was approved by the FDA for use in clinical settings.(22) A second kit is under consideration for FDA approval (ViroSeq, Applied Biosystems, Foster City, California).(23) Preliminary data suggest that results obtained with each assay are highly concordant.(24) The kits have stronger quality control and validation profiles than home-brew methods, making them preferable in clinical laboratories. However, they are more expensive and may not provide the versatility of home-brew methods.

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Hybridization Methods
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Sequencing by hybridization can be used to detect specific mutations, and in some situations, to determine the complete sequence of a gene. The Affymetrix GeneChip (Santa Clara, California) was designed to determine the complete sequence of HIV-1 protease and the first 1200 nucleotides of HIV-1 RT. Although this product is no longer available, DNA chip sequencing is likely to become increasingly common in the future and may be again applied to HIV genotyping. The INNO-LiPA HIV-1 line probe assays (Innogenetics, Ghent, Belgium) are point-mutation assays designed to detect specific HIV-1 protease and RT mutations.

The GeneChip was divided into several thousand segments each containing millions of similar probes designed to interrogate every nucleotide position in a test DNA or RNA molecule. Every nucleotide in the test molecule requires at least four sets of oligonucleotide probes to determine whether that nucleotide is an A, C, G, or T. DNA chip sequencing is also called "resequencing" because some prior knowledge of the sequence is required to design probes that can bind to nucleotides adjacent to the position being interrogated.

Because of genetic variability, sequencing HIV-1 by hybridization is challenging. Several studies compared the performance of the GeneChip with dideoxynucleotide sequencing and found that dideoxynucleotide sequencing was more reliable at detecting HIV-1 RT and protease mutations.(16,25-27) DNA chips were less capable of detecting insertions or deletions in viral sequences and of sequencing viral subtypes other than subtype B--the subtype on which the original chip tiling was based.(25,26)

Point mutation assays are inexpensive and have the potential to be highly sensitive for mutations present in only a small proportion of circulating viruses.(28,29) Because they require only simple laboratory equipment, they may be useful in areas that do not have ready access to expensive sequencers. The INNO-LiPA assays have probes for wild-type and mutant alleles of each codon, attached to a nitrocellulose strip.(30-32) Biotin-labeled RT-PCR product from the patient sample is hybridized to the strip. An avidin-enzyme complex and the enzyme substrate produce a color change on the paper strip where the PCR product has hybridized with a probe. This assay is limited because it can only detect a subset of drug resistance mutations and has had a 10% rate of uninterpretable results due to poor hybridization, particularly when uncommon mutations are present at key codons.(29,33)

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Dideoxynucleotide Sequencing Reproducibility
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Two large multicenter comparisons of sequence results obtained from samples containing mixtures of plasmid clones (ENVA-1) and spiked plasma samples (ENVA-2) have identified two important causes of interlaboratory variation: 1) variability in the quality of work done by different laboratories, and 2) the difficulty in consistently detecting minor variants even by laboratories performing high-quality work.(17,34) The variability in laboratory performance--which was independent of the particular sequencing methodology used--suggests a need for ongoing quality assurance testing. Although specific reasons for poor laboratory performance are not cited in the published literature, it is likely that cost-saving shortcuts (such as sequencing just one strand of DNA or using sequence primers that are too far apart to obtain consistent, high-quality electrophoretic peaks) may be responsible. The difficulty in consistently detecting minor variants is a function of the quasispecies nature of HIV-1 and is characteristic of all currently used susceptibility testing methods.

In experienced laboratories, dideoxynucleotide sequencing can be highly reproducible. In one study, 13 research laboratories were shipped cell pellets from cultured HIV-1 isolates.(35) The sequence concordance among laboratories was 99.7% at all nucleotide positions and 97% at positions associated with zidovudine resistance. Sequencing cultured cell pellets is simpler than sequencing plasma because RNA extraction and reverse transcription are not necessary and because cultured virus is more homogeneous than uncultured virus.(36,37) Nonetheless, the high interlaboratory concordance in this study attests to the intrinsic reliability of the dideoxy method for HIV-1 analysis.

In another study, two clinical laboratories also assessed the reproducibility of HIV-1 RT and protease sequencing using plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals.(38) Although both laboratories used sequencing reagents from the same commercial source, each used a different in-house protocol for plasma HIV-1 RNA extraction, reverse transcription, PCR, and sequencing. Overall sequence concordance between the two laboratories was 99.0%. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture when the second laboratory detected only one of the mixture's components. Discordance was significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels.

Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and, in every case in which one laboratory detected a mixture, the second laboratory detected either the same mixture or one of the mixture's components. The high concordance in detecting mixtures and the fact that most discordance between the two laboratories was partial suggest that most discordances were due to variation in sampling the HIV-1 quasispecies rather than to technical errors. Figure 2 shows that the few drug resistance mutations detected by one laboratory but missed by the other laboratory were present as minor variants.

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Sequence Quality Control
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Sequence quality control should aim to avoid PCR contamination and sample mix-ups. To prevent sample contamination with DNA from other sources, laboratories should use standard physical precautions,(39) run negative controls with each PCR step, and examine their final sequence results for the possibility of contamination with other samples sequenced at the same time.(40) Uracil N-glycosylase (UNG), which is used in Applied Biosystems' ViroSeq kit, has also been used to diminish the risk of PCR-based laboratory contamination. UNG works by destroying any PCR product from previous reactions at the start of each PCR assay.

Sequence analyses can be extremely useful for detecting both laboratory contamination and sample mix-up and should be incorporated into every laboratory's testing procedure. These analyses should compare each new sequence with other recently generated sequences to look for unexpectedly high levels of similarity. Phylogenetic trees can also be constructed to visually detect unexpectedly similar isolates. The HIV Sequence Database at Los Alamos National Laboratories has a tutorial to assist with sequence analysis for quality control purposes.(41) If previous sequences from a patient are available, then each new sequence should be compared with these earlier sequences. Although one would expect some change because of selective drug pressure, many aspects of the sequence, particularly polymorphic amino acids and silent nucleic acid positions, should undergo minimal change over time. Finally, if a sequence is incompatible with the patient treatment history, sequencing should be repeated on a new clinical sample.

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Intersubtype Variability
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During its spread among humans, group M HIV-1 has evolved into multiple subtypes that differ from one another by 10-30% along their genomes.(42,43) In North America and Europe, most HIV-1 isolates belong to subtype B, and the available antiretroviral drugs have been developed by drug screening and susceptibility testing using subtype B isolates. However, subtype B accounts for only a small proportion of HIV-1 isolates worldwide, and non-B isolates are being identified with increasing frequency, particularly in Europe.

It might be expected that the functional constraints on the protease and RT enzymes would prevent their genes from displaying much intersubtype variation. But several groups have shown that the protease and RT genes of global isolates are sufficiently different from one another to allow subtype grouping.(44,45,46,47) Although most intersubtype variation is caused by synonymous nucleotide substitutions, there are subtype-specific amino acid patterns.(44,45,46,47,48)

Protease intersubtype sequence variation is more likely to occur at positions associated with drug resistance than RT intersubtype variation. A protease substrate cleft mutation, V82I occurs commonly in subtype G isolates. Preliminary data suggest that V82I confers minimal or no resistance to the available protease inhibitors (PIs),(49-51) although it occasionally emerges during PI therapy.(52) Mutations at positions 20, 36, and 93 are accessory resistance mutations that occur more commonly in some non-B subtypes.(44)

A few studies have tested the in vitro susceptibility of non-subtype B HIV-1 isolates to antiretroviral drugs. Although group O isolates often demonstrate intrinsic resistance to the NNRTIs,(53,54) most studies have shown that non-B group M isolates are as susceptible as subtype B isolates to each of the three HIV drug classes.(55,56-58) Although limited data are available, there is no evidence yet for novel drug resistance mutations in non-B HIV-1 isolates.

Intersubtype genetic variability may complicate HIV-1 genotyping because primers used for reverse transcription, PCR, and sequencing may have a lower rate of annealing to non-B compared with subtype B sequences. The extent to which this occurs has not yet been reported. Preliminary studies confirm that many of the current sequencing methods are not optimized for amplifying and sequencing non-B isolates,(59) although one study using the ViroSeq kit on isolates showed a high sensitivity at detecting and sequencing subtype A, C, and D viruses from Uganda.(60)

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Phenotypic Resistance Testing
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Until the late 1990s, the most commonly used phenotypic assays were plaque reduction and PBMC assays. Plaque reduction assays were used primarily for testing laboratory isolates. Because they required the co-cultivation of virus with HeLa-CD4+ cells (which lack the CCR5 receptor), plaque reduction assays could only test the minority of clinical isolates that were syncytium inducing (CXCR4 tropic). In contrast, PBMC-based assays were suitable for testing most clinical isolates. PBMC assays, however, were laborious because they required a preliminary 10- to 14-day period to create a virus stock from a clinical isolate by cocultivating patient PBMCs with mitogen-stimulated PBMCs from HIV-seronegative individuals. Moreover, testing was expensive because virus growth in PBMCs cannot be monitored visually but requires the specific measurement of a virus product (eg, p24 antigen).

In the late 1990s, two companies developed standardized assays amenable to high-throughput performance: Tibotec-Virco (Antivirogram; Mechelen, Belgium) and ViroLogic (PhenoSense; South San Francisco, California).(61,62) Both assays use PCR to amplify the entire protease, much of RT and some of gag from HIV-1 RNA extracted from patient plasma. The amplified material is incorporated into a pol-deleted recombinant virus construct to create a recombinant HIV-1 isolate. A standardized virus inoculum is then used to infect a cell line, and virus replication is measured in the presence and absence of a range of concentrations of different antiretroviral drugs.

The first few steps of sample preparation (RNA extraction, reverse transcription, and PCR amplification) are the same for both of the phenotypic assays. After amplification, the ViroLogic PhenoSense assay, uses direct ligation, and the Tibotec-Virco Antivirogram assay uses homologous recombination in cell culture to combine the patient's pol gene with the pol-deleted HIV-1 vector. The ViroLogic assay tests the recombinant construct during a single cycle of replication using a luciferase reporter gene assay designed to be highly sensitive to HIV-1 replication. The Tibotec-Virco assay cultures the virus for several replication cycles using cell killing as the measure of virus replication.

Both companies report the concentration of drug (µM) required to inhibit HIV-1 replication by 50% (IC50), as well as how this concentration compares with the concentration of drug required to inhibit a wild-type HIV-1 clone. Both assays have undergone rigorous internal testing and have been shown to produce little variation (less than twofold) when the same sample is tested repeatedly.(63) However, there is only one comparative study of isolates with and without known drug- resistance mutations. A preliminary study in which both assays were run on 34 isolates from untreated persons and 16 isolates from treated persons demonstrated 92% concordance.(64) However, most of these isolates were wild type, so further comparisons of isolates with known genotypes would be useful.

Recombinant virus susceptibility assays have several advantages over older nonrecombinant assays. Recombinant virus assays can be done using plasma, whereas nonrecombinant assays require the isolation of PBMCs. Recombinant virus assays can be performed under highly uniform conditions because the backbone of the virus construct, which remains constant, can be tailored for replication in the cells used for susceptibility testing. Finally, recombinant virus assays use PCR to amplify the protease and RT genes, dramatically decreasing the need for virus culture.

The use of recombinant viruses for susceptibility testing, however, has a theoretical limitation for testing susceptibility to PIs that has not been adequately addressed in the published literature. PI resistance is modulated by mutations at gag-pol cleavage sites,(65-72) and although four of the nine cleavage sites in the recombinant virus come from the patient virus sample, five come from the laboratory virus construct (Figure 3). The cleavage sites with the most compensatory changes (p7/p1 and p1/p6) are included in both recombinant virus assays. However, if a patient's virus sample contains a critical compensatory mutation at one of the five cleavage sites absent from the recombinant virus, then the recombinant virus may fail to replicate or may produce inaccurate drug susceptibility results.

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Drug Resistance Interpretation
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General Principles
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HIV-1 drug resistance is rarely an all-or-none phenomenon. Clinicians treating infected patients usually need the answers to the following two questions: 1) Does the result suggest that the patient will respond to a drug in a manner comparable to a patient with a wild-type isolate?, and 2) Does the result suggest that the patient will obtain any antiviral benefit from the drug? The second question distinguishes antiviral susceptibility testing from antibacterial susceptibility testing. In the case of bacteria, it is usually possible to avoid using any drug with reduced susceptibility against a pathogen. This is usually not possible in the case of HIV, however, because of the extent of cross-resistance within each class of HIV drugs. To answer both these questions it is necessary to grade the extent of inferred resistance relative to wild type (eg, low-level, intermediate, high-level).

The clinical significance of both genotypic and phenotypic data have been difficult to establish for the following reasons: 1) all antiretroviral drugs are used in combinations, many of which are synergistic (reduced susceptibility to a drug may not interfere with the drug's beneficial effect on the antiretroviral activity of another drug used in the same regimen); 2) a drug may have some benefit even in the setting of resistance, because many drug-resistant variants are less fit than drug-susceptible variants; and 3) the serum levels of some drugs, particularly the PIs, can be highly variable. Low levels of PI resistance may be overcome in some cases if higher serum PI levels can be obtained. Therefore, most genotypic and phenotypic interpretations depend on a combination of in vitro as well as in vivo data.

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Genotypic Test Results
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Genotypic results bear little resemblance to those of a typical antimicrobial susceptibility assay. Rather than receiving a result such as "susceptible" or "resistant" for each of the available HIV drugs, the ordering clinician receives a list of mutations present in the virus isolate. The fact that genotypic interpretation is independent of the process of genotyping makes genotypic interpretation an ideal application for a computerized expert system. Laboratories doing HIV-1 genotyping can provide physicians with the option of receiving a file with the sequence data (string of nucleotides or list of amino acid differences from consensus). Such data can then be analyzed by interpretation systems other than those used by the sequencing laboratory.(73) Table 1 describes several of the most commonly used systems for HIV-1 genotypic interpretation. During the next 1-2 years, these algorithms will evolve and most likely converge through an ongoing process of interalgorithm comparison and validation using clinical data sets. This is because there is probably more concordance among clinical virologists than is currently reflected in published algorithms.

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VirtualPhenotype
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The VirtualPhenotype (Tibotec-Virco) is a pattern-matching algorithm that uses a large genotypic-phenotypic correlative database to infer phenotypic properties based on sequence data.(74) The analysis includes a tabulation of the number of matches in the database for each drug, and the distribution of phenotypes (fold increase in IC50) for the matching samples. The mean IC50 of the matching samples is interpreted using drug-specific cutoff values, providing a quantitative prediction of drug resistance. Although the VirtualPhenotype has been described in several abstracts, a complete description of the workings of this approach has not been published.

Specifically, it is not known which mutations are used to match a new sequence to those sequences that are already in the database. In addition, the database and pattern-matching algorithm are proprietary.

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HIV RT and Protease Sequence Database
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The HIV RT and Protease Sequence Database at Stanford University is an online database (http://hivdb.stanford.edu) that links sequence data to the HIV drug treatments of the patients from whom the sequenced isolates were obtained and to drug susceptibility results. The database also contains two sequence analysis programs. The first, HIV-SEQ, accepts user-submitted RT and protease sequences, compares them with a reference sequence, and uses the differences as query parameters for interrogating the sequence database.(75) This allows users to detect unusual sequence results immediately so that the person doing the sequencing can check the primary sequence output while it is still on the desktop. In addition, unexpected associations between sequences or isolates can be discovered by immediately retrieving data on isolates sharing one or more mutations with the sequence.

The second program, "Drug Resistance Interpretation," is an expert system that accepts user-submitted protease and RT sequences and returns inferred levels of resistance to the 16 FDA-approved anti-HIV drugs. Each drug resistance mutation is assigned a drug penalty score; the total score for a drug is derived by adding the penalty scores of each mutation associated with resistance to that drug. Using the total drug score, the program reports one of the following levels of inferred drug resistance: susceptible, potential low-level resistance, low-level resistance, intermediate resistance, and high-level resistance. Genotypic interpretations do not necessarily correlate with the inferred level of phenotypic resistance because the genotypic interpretation also uses correlations between genotype and clinical outcome in deciding how a drug's susceptibility should be graded. A listing of all mutation/drug score pairs can be found with the program's release notes.

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Phenotypic Interpretation
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To interpret a drug susceptibility result, it is important to know the reproducibility of the assay for a given drug ("technical cutoff" or "reproducibility cutoff"), the range of IC50's (or IC90's) required to inhibit wild-type viruses ("biological cutoff"), and the clinical significance associated with different levels of reduced drug susceptibility ("clinical cutoff"). There should ideally be at least two clinical cutoffs: the IC50 at which there is some reduction in drug activity and the IC50 at which a drug no longer has specific antiviral activity.

Both Tibotec-Virco and ViroLogic have published data on the reproducibility of their assay and the range in susceptibility results obtained in testing wild-type isolates from untreated persons.(76,77,78) However, the results are not readily accessible and may have been calculated differently for each assay (Table 2). Clinical cutoffs are still being developed, although important correlations between the level of susceptibility to a given drug and clinical outcome have recently been reported in the following settings: intensification with abacavir(79) or tenofovir,(80) switching from zidovudine to stavudine,(81) and salvage therapy with lopinavir and efavirenz in patients who developed virologic failure on an earlier PI-containing regimen.(82) Both assays have also recently lowered their clinical cut-points to detect low-level resistance to didanosine, zalcitibine, stavudine, and tenofovir.

The interpretation of phenotype results would also be aided by the publication of drug-susceptibility data obtained with both assays on isolates with common patterns of drug-resistance mutations. Although both the Antivirogram (Tibotec-Virco) and PhenoSense (ViroLogic) assays are recombinant virus assays, there are differences that might be expected to influence test performance. PhenoSense should theoretically be more reproducible because it measures drug activity over a single cycle of HIV replication using a very sensitive marker (Tat production measured by a luciferase reporter gene). In contrast, the Antivirogram compares the extent of cell killing in the presence and absence of a drug. Cell killing requires several rounds of replication (usually 4-7 days) and is likely to be a much less reproducible marker of HIV replication. However, it is difficult to determine for both assays the extent to which the technical and biological cutoffs overlap with the clinical cutoffs for these difficult-to-test drugs.

Three main factors explain apparently discordant results between genotypic and phenotypic susceptibility tests: 1) phenotypic tests are more likely to detect resistance caused by uncommon mutations that have not been included in genotypic interpretation algorithms; 2) genotypic tests are more likely to detect resistance caused by mutations present as part of a mixture, transitional mutations, and mutations that are suppressed by another mutation in the virus; and 3) genotypic tests are more likely to detect nucleoside reverse transcriptase inhibitor (NRTI) resistance caused by nucleotide excision mutations (NEMs). Examples of each of these scenarios are shown in Table 3.

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Limitations of Drug Resistance Testing
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Several factors limit the usefulness of both genotypic and phenotypic testing: 1) There is a complex relationship between drug resistance and clinical failure, often making it difficult to discern the contribution of drug resistance to virologic failure; 2) the HIV-1 population within an individual consists of innumerable variants and minor variants that often go undetected; 3) because of extensive cross-resistance within each drug class, the results of resistance testing often leave clinicians with few options for treatment.

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Complex Relationship Between Drug Resistance and Disease Progression
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Drug resistance is not the only cause of treatment failure. Nonadherence, the use of insufficiently potent treatment regimens, and pharmacokinetic factors that decrease the levels of one or more drugs in a treatment regimen also contribute to treatment failure. In addition, the natural history of HIV-1 infection is highly variable and dependent on a complex set of host-virus interactions.(83) In the absence of therapy some patients progress to advanced immune deficiency within 3 years after infection, whereas other patients remain healthy for more than 15 years. It is likely that the same host-virus interactions that so greatly influence disease progression in the absence of drug therapy also influence the risk of virologic failure in patients receiving antiretroviral therapy.

Two recent observations underscore the complexity of the relationship between drug resistance and disease progression. The first is that patients developing virologic failure on their first treatment regimen are usually found to have HIV-1 isolates with resistance to only one of the drugs in the regimen.(84,85,86,87,88,89) The drugs to which resistance most commonly develops in this situation are lamivudine and the NNRTIs; resistance to PIs and NRTIs other than lamivudine is less common in patients with initial virologic failure. The observation that virus becomes detectable and replication ensues despite the fact that the replicating virus remains sensitive to at least two drugs in the treatment regimen suggests that factors in addition to drug resistance are contributing to virologic failure. Possibly the remaining drugs in the regimen are not potent enough to fully suppress virus even though they remain active. Alternatively, one of the presumably active drugs in the regimen may be present at insufficient levels because of nonadherence or pharmacokinetic factors.

The second observation is that virologic failure in patients receiving HAART is not always followed by immunologic and clinical deterioration.(90-93) This may be because the immunologic benefits of virus suppression persist beyond the period of virus suppression or because drug-resistant viruses may be less virulent, particularly when they first emerge and are associated with fewer compensatory mutations.(94,95) However, the duration of immunologic benefit in the face of ongoing virus replication is not known. In patients experiencing their first virologic failure, treatment is usually changed because there is an ongoing risk of accumulating additional drug resistance mutations that would interfere with the success of salvage therapy.(96)

Several lines of evidence suggest that drug-resistant viruses are less fit than drug-susceptible viruses. First, in vitro experiments have consistently shown that isolates containing protease and/or RT drug resistance mutations replicate less well in cell culture, and that purified enzymes with these mutations usually have less activity than wild-type enzymes (reviewed in (97)). There are conflicting data, however, on whether multidrug-resistant variants are less cytopathic in specific types of cells (eg, thymus).(98-100) Second, drug resistance mutations are often replaced in vivo by wild-type variants within weeks to months after removal of selective drug pressure.(95,101-104) The rate at which this occurs depends on the extent to which archived wild-type viruses exist within an individual patient. If there are no archived wild-type viruses, a significant interlocking of primary and compensatory mutations may limit reversion to wild type.(105)

One clinical trial in patients with detectable viremia and multidrug-resistant virus showed that in those patients randomized to discontinue antiretroviral therapy, plasma HIV-1 RNA increased by 0.84 log10 and CD4 cell counts decreased by 128 cells/µL as the dominant virus population reverted to wild type.(91) This study suggests that the decreases in fitness of drug-resistant viruses seen in vitro are clinically significant, and that continuing drug therapy in the face of resistance may have utility in patients with few other therapeutic options. However, the possibility cannot be excluded that many of the isolates in this study retained some degree of susceptibility to one or more drugs in treatment regimens that were used.

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Quasispecies Nature of HIV-1
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A recognized limitation of HIV-1 drug susceptibility testing by either genotypic or phenotypic methods is the unreliability of these tests in detecting minority HIV-1 variants in the virus population of the patient tested. This limitation is particularly troublesome in patients with complicated treatment histories or those who have discontinued one or more antiretroviral drugs.(103,104,106) To maximize the likelihood of identifying drug-resistance mutations present within the virus population of a patient, it is important to obtain plasma samples for resistance testing before stopping or changing antiretroviral drugs and to consider a patient's treatment history when interpreting the results of resistance testing.

In some patients, the treatment history can be used to infer the presence of archived drug-resistance mutations. For example, if a patient previously received lamivudine as part of an incompletely suppressive treatment regimen, it is likely that M184V exists within the virus population of that patient, even if it is not detected at the time of genotyping. The same principle would apply to patients who received NNRTIs as part of an incompletely suppressive treatment regimen; however, in this situation, it would not be possible to know specifically which NNRTI mutations are likely to be archived. In contrast, patients receiving lamivudine and NNRTIs as part of completely suppressive treatment regimens are not expected to harbor variants resistant to these drugs.

If a patient once harbored drug-resistant variants, these variants may persist at low levels in latently infected cells even if a subsequent treatment regimen brings about complete virus suppression.(106,107-111) In patients in whom previous resistance tests have documented extensive drug resistance, the clinical usefulness of repeated resistance testing is likely to be minimal, because many resistant variants selected by previous treatment regimens will go undetected in future tests, yet are likely to emerge during attempts at salvage therapy.

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Cross-Resistance
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Most mutations arising during drug therapy contribute resistance to multiple drugs within the same drug class. Because there are only three drug classes available, and combinations of drugs from at least two classes are usually required to achieve durable HIV-1 suppression, cross-resistance is a significant problem. Genotypic assays frequently do not identify enough fully active non-cross-resistant drugs to completely block HIV-1 replication, and many patients changing regimens because of virologic failure will have to use a regimen containing drugs that are partially compromised at the start of therapy.

Although cross-resistance is not a direct limitation of genotypic or phenotypic testing, it limits the usefulness of resistance testing, particularly in heavily treated patients. Nonetheless, by providing prognostic data and helping to avoid unnecessary drugs, resistance testing can have a role even in these patients. When fully active drugs are not available, salvage therapy in heavily treated patients may include drug combinations that exploit antagonistic mutational interactions or generate high in vivo drug levels.

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Acknowledgments
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Robert W. Shafer is supported in part by NIH grant AI46148-01 and has received unrestricted educational grants from various pharmaceutical and diagnostic companies for maintenance of the HIV RT and Protease Sequence Database. A list of the companies who have supported the database can be found at http://hivdb.stanford.edu/pages/acknowledgements.html.

Matthew J. Gonzales assisted with manuscript preparation, including creation of many of the tables and figures, and is supported in part by a supplement to NIH grant AI46148-01.

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