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Geretti AM, editor. Antiretroviral Resistance in Clinical Practice. London: Mediscript; 2006.

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Antiretroviral Resistance in Clinical Practice.

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Chapter 7Clinical cut-offs in the interpretation of phenotypic resistance

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Introduction

Resistance testing is a valuable tool in the management of antiretroviral therapy and its use in treatment failure is recommended by international guidelines [13]. A number of retrospective and prospective studies have shown that resistance testing has a beneficial influence on the choice of antiretroviral therapy in drug-experienced patients [4,5]. However, resistance assays are continually evolving and their role in the clinical management of HIV-1-infected individuals remains to be fully defined. At present, it is not clear under what circumstances phenotypic testing should be preferred to genotypic testing. The selection of one assay over another depends on specific factors such as access, cost, turnaround time and the availability of expert interpretation. It has been proposed that use of both phenotypic and genotypic tests combined may provide the best information for determining a salvage regimen in highly treatment-experienced patients with complex resistance patterns, but at present there are no objective data to support this belief.

With all resistance assays, the correct interpretation of results is paramount for the tests to be truly effective. Used correctly, resistance testing may help limit the development of further drug resistance and cross-resistance, and guide the optimal sequencing of antiretroviral regimens during the many years of therapy required to maintain the health of HIV-1-infected patients.

Definition of cut-off for the interpretation of resistance assays

Phenotypic tests

Commercially available phenotypic assays are based on the generation of recombinant viruses that express the reverse transcriptase (RT) and protease (PR) genes from HIV-1 isolates derived from the patient's plasma. The assays measure the ability of the recombinant viruses to replicate in cell culture (in vitro) in the presence of different drug concentrations, compared with a drug-susceptible reference recombinant virus (wild-type) [68]. The raw data output is the concentration of drug required to inhibit viral replication by 50% or 90% (IC50 or IC90, respectively) relative to the control. Results are usually expressed as the IC50 of the drug being tested for the patient-derived virus divided by the IC50 for the reference virus. The value of this ratio is commonly referred to as a fold change in susceptibility (see Figure 1). In principle, a fold change greater than 1 indicates that the patient's virus is less susceptible than the reference virus, whereas a fold change lower than 1 indicates that the patient's virus is more susceptible. The latter is known as phenotypic hypersusceptibility and is conventionally defined by the phenotype showing a fold change of <0.4 compared with the laboratory control virus. The mechanisms of this are only partially understood. It is likely that hypersusceptibility results from the antagonistic interaction between mutations, in which mutations that lead to resistance to one drug lead to increased susceptibility to another drug [9].

Figure 1. Phenotypic drug susceptibility curves.

Figure 1

Phenotypic drug susceptibility curves. The continous curve represents a wild-type drug-susceptible virus. The shift to the right of the dashed curve, representing the patient strain, indicates a reduction in drug susceptibility to a higher IC50 value. (more...)

Interpretation systems for phenotypic tests

The interpretation of phenotypic data is based on the measurement of the fold change for each antiretroviral drug tested against pre-defined cut-offs. The first important issue related to phenotype interpretation is determining the appropriate cut-offs for defining a clinical isolate as either drug susceptible or drug resistant. Until recently, `technical cut-offs' were in use, based on the reproducibility of the assay on repeat testing. One improvement over technical cut-offs was the introduction of `biological cut-offs', which are based on the distribution of the drug susceptibility of isolates from thousands of treatment-naive patients [10]. For interpretation of phenotypic data based on biological cut-offs, the clinical isolate is scored as susceptible to a certain drug if the fold change falls within the mean fold change observed with samples from treatment-naive patients, plus two standard deviations. A test result falling above the cut-off can be said to be above the normal susceptible range with 97.5% confidence. This provides a reference for comparison of the test virus with viruses circulating in the drug-naive population, although it does not provide information about the likelihood that the virus tested will respond to treatment with a particular drug. Thus, neither technical cut-offs nor biological cut-offs provide a link between drug susceptibility measured in vitro and the virological response observed in vivo.

Establishing clinical cut-offs

From a clinical perspective, the most pertinent method of interpretation of phenotypic data is based on the use of `clinical cut-offs'. These are derived using clinical response data from treatment-experienced patients by determining the relationship between fold changes measured at baseline and the reduction in viral load after a defined period of treatment. To reflect the gradual, rather than discrete, loss of drug activity observed with increasing resistance to the nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs), two phenotypic cut-off points are often derived for each drug. The first cut-off defines the fold change at which there is a reduction in antiviral activity (lower cut-off), whereas the second cut-off defines the fold change above which all drug activity is lost (upper cut-off) (see Figure 2 and Table 1).

Figure 2. Theoretical fold change–viral response curve based on drug susceptibility.

Figure 2

Theoretical fold change–viral response curve based on drug susceptibility. At the lower cut-off point (C1), a reduction in drug activity is noted and, at the upper cut-off point (C2), no activity would be expected. The zone between the two cut-off (more...)

Table 1. VircoTYPE clinical cut-offs [18].

Table 1

VircoTYPE clinical cut-offs [18].

The requirement for large samples of clinical data and the difficulty in extrapolating the activity of individual drugs within the context of combination therapies are important obstacles to the determination of clinical cut-offs. Despite the difficulties, a number of clinical cut-offs have been proposed and are currently in use in commercially available resistance assays. It should be noted that, in principle, cut-off values may differ depending on the phenotypic assay used [1113]. In practice, however, some assays appear to give comparable results, at least for some drugs [14]. Thus, it would seem that a cut-off determined by one phenotypic test can be used, under certain conditions, for the interpretation of another test. A potential limitation, however, applies to very low clinical cut-offs, where differences in the reproducibility of the test occur for some antiretroviral drugs.

To date, only a limited number of datasets has been defined to determine phenotypic cutoff points. Upper and a lower cut-offs for susceptibility to tenofovir (TDF) have been proposed based on data from the Gilead Sciences 902 and 907 studies [15]. In these trials, TDF was added to a failing combination regimen without other modifications to the regimen. Based on the observed decline in viral load after intensification, it appears that a fold change in IC50 of 1.4 differentiates full activity from partial activity, and a fold change of 3.8 marks the boundary line between partial activity and nearly complete loss of activity. A similar approach was used to define phenotypic cut-off points for didanosine (ddI) in the JAGUAR study. In a retrospective analysis of 168 patients who were randomly assigned to addition of ddI or placebo to a failing regimen, a lower cut-off point of 1.3-fold and an upper cut-off point of 2.2-fold identified patients with optimal, intermediate and poor responses to ddI intensification [16].

When defining phenotypic cut-offs for the PIs, it is important to differentiate between the use of ritonavir (RTV)-boosted or unboosted PIs, and to define clearly the PI dosing schedule. Pharmacokinetic boosting of PIs with RTV increases drug concentrations in the blood and by increasing the inhibitory quotient can overcome partial drug resistance. Therefore, cut-offs determined for an unboosted PI cannot be extrapolated for the same PI when used with RTV. In studies of PI-experienced, non-nucleoside reverse transcriptase inhibitor-naive patients who were treated with boosted lopinavir (LPV) plus NRTIs and efavirenz (EFV), the virological response remained relatively constant up to a fold change in IC50 of 10 for LPV. Viruses with higher fold changes had diminshed responses to this regimen, but patients continued to have some response up to fold changes of 40 [17]. Of note, the presence of EFV as a fully active drug in the regimen may have increased virological responses and led to an underestimation of the degree of resistance to LPV. Nonetheless, subsequent analyses have confirmed a lower clinical cut-off for LPV of about 10-fold and an upper cut-off of about 60-fold [18].

Genotypic assays

In clinical practice, genotypic resistance assays determine the presence of mutations that have previously been associated with drug resistance in vitro. The interpretation of results is based on the known phenotypic effects of resistance mutations, and therefore relies indirectly on the use of established clinical cut-offs. Information regarding the relationship between individual resistance mutations and susceptibility to approved antiretroviral agents is available on-line [1921]. The virtual phenotype is a commercial assay that provides genotype interpretation through the prediction of the viral phenotype by relating the patient's genotype to a large database of genotype–phenotype pairs. As with a real phenotype, clinical cut-offs have been determined from clinical response data. A potential limitation of this approach is that the level of confidence in the result depends on the number of matching genotypes in the database and on selecting the appropriate codons to incorporate into the search. Matches are based on positions preselected as relevant for each drug, not the entire sequence. Correlation between actual and virtual phenotype may be weaker for newer drugs or in cases in which there are fewer matches because of unusual genotypes.

Recommendations for clinical practice

  • Clinical cut-offs attempt to facilitate the interpretation of drug resistance by introducing artificial thresholds in drug activity. For most available antiretroviral drugs, development of resistance and loss of virological activity occur as a continuum; gradual loss of clinical activity is observed with increasing levels of resistance.
  • Phenotypic and genotypic test results should be interpreted by individuals with expert knowledge of antiretroviral therapy and drug resistance patterns. This is crucial to the development of successful sequencing of antiviral regimens.
  • Appropriate interpretation is the key to maximising the utility of available resistance assays, and their utility will continue to improve as clinical cut-off points and genotype algorithms are better defined.

References

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Bacheler L, Winters B, Harrigan R et al. Estimation of phenotypic clinical cutoffs for VircoTYPE through meta analyses of clinical trial and cohort data. 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2004, Abstr. H-1133.
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Stanford University HIV resistance database. http://hivdb​.stanford.edu (accessed on 30 January 2006).
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Los Alamos National Laboratory HIV databases. http://www​.hiv.lanl.gov (accessed on 30 January 2006).
Copyright © 2006, Mediscript.
Bookshelf ID: NBK2254PMID: 21249778

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