Introduction

Only a few short years ago, viral load (HIV-1 RNA) testing was introduced as a new tool for HIV management. Many physicians, inside corrections as well as outside, delayed implementing the test. Though most of the arguments against its use included lack of standardization, inability to process specimens and shortage of specialists to interpret and utilize results in HIV management, the major unspoken obstacle was cost. In 2000, we now face a similar situation with antiretroviral resistance testing. Despite national guidelines for their use as the community standard of care in the US and favorable retrospective and prospective data, few correctional systems have embraced genotypic or phenotypic testing. This article will address specific issues in the use of resistance testing and provide an overview of clinical studies and potential application for their use.

Defining Resistance: The Causes of Viral Rebound

The presence of antiretroviral resistance to HIV medications may be signaled clinically by the observation of viral rebound. Viral rebound can be defined as any reproducible increase in the viral load determined to be threefold or greater that is not due to acute intercurrent infectious illness or vaccination. It is important to note that not all rebound phenomena are related to drug resistance. In fact, the most common cause of rebound is poor adherence. In studies of virologic rebound occurring in patients receiving a triple combination including a protease inhibitor, the largest percentage demonstrate no mutations at all, followed by mutations to the nucleoside reverse transcriptase inhibitor and then to the protease inhibitor.

Resistance is the result of two major characteristics of HIV:

1. its rapid turnover rate; and
   


2. its error prone RNA replication process.
   

HIV lacks a proofreading function that corrects the mistakes in viral replication that result in mutations. Within a given patient, HIV exists as a combination of multiple strains (quasispecies) that diverge from the original wild-type or unmutated virus. The quasispecies differ based on acquired mutations that are passed onto daughter viruses.

Most mutations that occur naturally in the course of viral replication result in no effect on viral susceptibility to ART, while others lead to death of the virus. In order to cause clinically important resistance, a mutation must:

1. decrease the viral sensitivity to the drug,
   


2. become the dominant quasispecies because of increased viral fitness in the setting of selective drug pressure, and
   


3. provide a competitive advantage over the wild-type of the virus and maintain viral replication by preserving enzyme function.

If one of several quasispecies has a mutation that results in resistance to a specific drug, then exposure to that drug acts as a selective pressure that allows the resistant quasispecies to replicate freely while the other quasispecies and wild-type virus that lack the resistance mutation are suppressed. The resistant quasispecies then becomes the predominant replicating strain. Clinically, the patient's viral load increases and treatment fails. The patterns and types of mutations associated with NRTIs, NNRTIs and PIs are described in the HIV 101, page 5.

The key to understanding the limitations of resistance testing is understanding that resistant quasispecies become the dominant strain when HAART is being used, while other forms of the virus are suppressed, including those that might be resistant to other drugs. That is, resistance to a given drug may not be detected if the patient is not taking that drug at the time that a resistance test is given. Since the selective pressure that favors replication of the resistant quasispecies over the susceptible strains has been removed, there may not be enough of the resistant quasispecies present to be detected by current resistance assays. Yet the resistant strain will rapidly re-emerge if the selective pressure (the drug) is re-instituted. Thus, knowledge of prior antiretroviral treatment may steer a clinician away from a drug that might appear effective when the results of resistance assays are interpreted without knowledge of prior treatment history.

Resistance to drugs may decrease the ability of the virus to replicate, as has been reported by a number of investigators. Drug resistance is associated with impaired protease and reverse transcriptase (RT) function and reduced replication capacity. In one report, Nelfinavir resistant viruses exhibited many protease cleavage defects and 70% of Nelfinavir-resistant viruses showed large reductions in viral replication1. In addition, some viruses exhibit hypersensitivity to selected drugs after developing mutations.2

Genotyping Versus Phenotyping

Resistance is measured by two methods: genotyping and phenotyping. Commercial assays using both of these methods are available. For example, TruGene (Visible Genetics) and ViroSequ (PE Applied Biosystems) provide genotyping information, and AntiVirogram (Virco) and PhenoSense (ViroLogic) provide phenotyping information. Genotypic assays provide information on mutations in the genes coding for reverse transcriptase and protease that confer drug. Phenotypic resistance is a direct measure of sensitivity and is similar to our current antibiotic sensitivity testing practices. Phenotypic assays rely on changes in the IC50, the minimum inhibitory concentration of the drug required to decrease viral replication by 50% in the particular cellular system used. The emergence of resistance is signaled by a significant increase in IC50 over baseline3, 4.

Both genotyping and phenotyping are complex technologies that utilize the polymerase chain reaction and other molecular techniques. They require specialized facilities staffed by well-trained laboratory personnel. Commercial assays using both methodologies are available, and the turn-around times for results are 1-2 weeks for genotyping and 2-4 weeks for phenotyping.

It is important to note that a plasma HIV RNA level above 1,000 copies/mL is necessary for either method to produce reliable results. Furthermore, neither method can routinely detect minority quasispecies; therefore, some resistant strains of virus may be missed. Although both types of assays are reproducible, both intra- and interlaboratory variability may be greater with genotypic assays. With regard to interpretation of results, complex mutational patterns detected by genotyping frequently require the interpretation of an expert whereas the results of phenotypic assays may be more easily interpreted by treating physicians. Phenotypic assays generally cost more than genotypic3.

Interpreting Genotypic and Phenotypic Resistance Assays

Interpretation of genotypic assays requires not only knowledge of the individual mutations, which confer resistance and cross-resistance to drugs within the same class, but also an understanding of the interactions of multiple resistance mutations. For example, a single mutation in the protease gene may confer high-level resistance for one PI, yet for another, it may require multiple mutations to confer resistance. However, the phenotypic expression of a combination of genotypically detected mutations cannot always be predicted3. To address this issue, Virco (Mechelen, Belgium), a manufacturer of one commercially available genotypic assay, has used a relational database of over 10,000 clinical isolates of HIV for which genotypic and phenotypic results are known, to assign a "virtual phenotype" to viral isolates based on mutational patterns. How this virtual phenotype correlates with response to antiretroviral therapy must be explored with appropriately designed clinical trials5.

Interpretation of phenotypic assay results suffers from a lack of clinical information regarding correlation of fold increase in resistance to in vivo activity of the various antiretroviral drugs. For example, a small fold increase in resistance to a protease inhibitor may be overcome by increasing serum levels of the protease inhibitor3.

Using Resistance Testing in Clinical Practice

The DHHS/Kaiser Guidelines4 for using antiretroviral agents recommend that resistance assays be used to modify antiretroviral therapy in the setting of virologic failure during ongoing HAART and in the case of suboptimal viral suppression after initiating a new regimen. Resistance testing should also be considered in antiretroviral naïve patients with acute HIV infection for whom treatment is planned. Suppression of viral replication during acute HIV infection may favorably alter the long-term course of HIV infection by allowing the immune system to develop antiviral responses that are otherwise impaired by unchecked viral replication during acute infection. This accounts for the recommendation of resistance testing for naïve patients with acute HIV infection, but not for naïve patients with established, chronic infection.

Studies Reveal Effectiveness of Resistance Testing

Several prospective studies provide information on the clinical utility of using HIV-1 resistance assays to direct therapy in patients who are failing an antiretroviral regimen. See Table 1 pg.2 for a summary. The GART6 and VIRADAPT7 studies used genotypic resistance assays. In the GART study, at eight weeks, the mean decline in HIV RNA was significantly greater in the group whose regimens were based on resistance testing than in the SOC group (-1.12 log vs. -0.52 log). Fifty-five percent of those in the resistance testing-based group had a viral load <500 copies/ml versus 25% in the SOC group. In the VIRADAPT study, at six months, the resistance testing group had a significantly greater decline in viral load than the SOC group (-1.15 log v. -0.67 log).

The VIRA 3001 Study8 and a study reported by Melnick, et al.8 used phenotypic resistance assays to direct a change in antiretroviral therapy. Using intent-to-treat analysis in which patients lost to follow up were counted as failures, the VIRA 3001 Study found no significant difference between the groups in the primary endpoint. In an alternative analysis using observed data, there was a significant difference in the groups (59% of those in the resistance-testing group had a viral load <400 copies/mL v. 42% in the SOC group, Melnick et al.) At four weeks, there was a statistically greater decline in viral RNA in the resistance-testing group than in the SOC group, but the difference was not sustained at 16 weeks. These two studies were conducted with participants who were more highly treatment-experienced than those in GART and VIRADAPT. Therefore, the number of available active agents was limited, particularly in the study reported by Melnick et al. in which even those on resistance-testing-based-regimens were on an average of less than three active drugs. This fact must be taken into account when interpreting these studies.

In the NARVAL study, 54110 highly treatment-experienced patients failing a 3 drug protease inhibitor containing regimen were randomized to therapy based either on genotyping, phenotyping, or SOC. At week 24, a greater percentage of participants in the genotyping-based group had HIV-1 RNA levels less than 200 copies/mL, but the difference was not statistically significant.

Although short in duration, GART and VIRADAPT clearly support the use of resistance assays to help direct antiretroviral therapy. VIRA 3001 and the study reported by Melnick, et al. are equivocal, while NARVAL does not support resistance testing. Because participants in these last three studies had greater prior treatment experience than in GART and VIRADAPT, one interpretation of these data is that resistance testing is less useful in highly treatment-experienced patients with few treatment options. Thus, one clear indication for use of resistance testing is after the first regimen fails.

Conclusion

In summary, the use of both genotypic and phenotypic resistance assays is expanding in clinical practice. Although specialized facilities and personnel are necessary to conduct these tests, commercially available kits have made the results reproducible, available in a timely fashion, and relatively affordable. Resistance testing to guide modifications in ongoing therapy is recommended in the setting of antiretroviral failure when a new regimen is anticipated and also in the setting of incomplete suppression of viral replication by a new regimen. It should also be considered when the decision is made to treat acute HIV infection. Several prospective clinical trials have demonstrated better suppression of viral replication in patients whose antiretroviral regimen has been guided by resistance testing, particularly in patients whose exposure to prior antiretroviral therapy has been limited, i.e. after the first regimen fails. Despite this benefit, resistance testing information must be combined with a complete medical history that details prior regimens, side effects to medications, and adherence with treatment. Such information is essential in selecting a regimen that is not only effective in suppressing viral replication but also acceptable to the individual patient.

References

*Speaker’s Bureau: Roche Pharmaceuticals

**Speaker’s Bureau: Agouron Pharmaceuticals, Bristol-Myers Squibb, DuPont, Glaxo Wellcome, Merck, Roche.

1. Wrin T. 4th International Workshop on HIV Drug Resistance and Treatment Strategies, Sitges, Spain, June 12, 2000.

2. Ziermann R, Limoli K, Das K, Arnold E, Petropoulos CJ, Parkin NT, A mutation in human immunodeficiency virus type 1 protease, N88S, that causes in vitro hypersensitivity to amprenavir, J Virol 2000 May;74(9):4414-9.

3. Deeks SG and Zolopa AR. Choosing and Using Resistance Assays. Available at: http://www.medscape.com/Medscape/HIV/AnnualUpdate/2000.

4. US Department of Health and Human Services and Henry J. Kaiser Family Foundation. Guidelines for the Use of Antiretroviral Agents in HIV-Infected Adults and Adolescents. Available at: http://www.hivatis.org/guidelines/adult/pdf/A&ajani.pdf.

5. Larder BA, Kemp SD, Hertogs K, et al. Quantitative prediction of HIV-1 phenotypic drug resistance from genotypes: The virtual phenotype (VirtualPhenotype). Antiviral Ther 2000; 5(Suppl 3):49.

6. Baxter JD, Mayers DL, Wentworth DN, Neaton JD, Hoover ML, Winters MA, Mannheimer SB, Thompson MA, Abrams DI, Brizz BJ, Ioannidis JP, Merigan TC. A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy. CPCRA 046 Study Team for the Terry Beirn Community Programs for Clinical Research on AIDS, AIDS 2000 Jun 16;14(9):F83-93.

7. Durant J, Clevenbergh P, Halfon P, et al. Drug-resistance genotyping in HIV-1 therapy: the VIRADAPT randomized controlled trial, Lancet 1999 Jun 26;353(9171):2195-9. Published erratum appears in Lancet 1999 Sep 25;354(9184):1128

8. Cohen C, Kessler H, Hunt S, et al. Phenotypic resistance testing significantly improves response to therapy: Final analysis of a randomized trial (VIRA3001). Antiviral Ther 2000; 5(Suppl 3):67.

9. Melnick D, Rosenthal J, Cameron M, et al.. Impact of Phenotypic Antiretroviral Drug Resistance Testing on the Response to Salvage Antiretroviral Therapy (ART) in Heavily Experienced Patients, 7th Conference on Retroviruses and Opportunistic Infections. Abstract 786.

10.. Meynard JL, Vray M, Morand-Joubert L, et al. Impact of treatment guided by phenotypic or genotypic resistance tests on the response to antiretroviral therapy: a randomized trial (NARVAL, ANRS 088). Antiviral Ther 2000; 5(S3):67-68.

2000-0910
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©1997,1998,1999,2000. The recently formed HIV Education Prison Project (HEPP) is a medical education program that targets a growing population, inmates in correctional facilities, that has been underserved in HIV care. It is part of the Brown University AIDS Program. Permission to use and reproduce portions of this newsletter is hereby granted provided that author and publication are fully credited and both copyright and permission notice appear with reprinted material. Inquiries may be directed to heppnews@brown.edu. Website: HIV Education Prison Project.

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