In the last issue, we brought you the first half of this update about HIV resistance testing. In that article, the authors discussed genotyping.

Although combination therapy has greatly reduced AIDS-associated mortality, the problem of HIV resistance leading to treatment failures has led to a need for antiviral-resistance testing to optimize treatment options.

Phenotyping

HIV Phenotyping is an in vitro assessment of the ability of the virus to replicate in the presence of varying concentrations of antiviral drugs. Early phenotypic assays involved the isolation of peripheral blood mononuclear cells from a patient. HIV virus from the patient cells was then grown in co-culture with uninfected PHA-stimulated donor lymphocytes. This was followed by culture of virus stock in the presence of antiviral compounds where, growth was measured by P24 production.1 These earlier assays were time consuming (they took 8-12 weeks), labor intensive, and not very reproducible, all of which made them impractical for routine clinical use.

The Virtual Phenotypeª from Virco (Mechelen, Belgium), a biotechnology company focused on HIV resistance testing, uses a genotype/phenotype relational database to analyze clusters of mutations. Clusters of mutations are compared to a database of samples that has been both phenotyped and genotyped. The numbers of samples with matching mutation clusters are then interrogated for their phenotypic response to a drug. The result of this type of analysis is a "virtual phenotype."2

The phenotypic assays more commonly used today are the recombinant viral assays (RVA) first developed by Kellam & Larder.3 These assays use PCR to amplify sections of the HIV genome that are then inserted into a laboratory vector to make a replicating recombinant virus. This recombinant virus is then grown in the presence of antiviral compounds to determine its phenotype.

As with the genotyping assays discusses in the last issue, the genetic region of interest in phenotypic assays includes the protease and the first amino acids of the reverse transcriptase encoding region. Reverse transcription and polymerase chain reaction are used to create sufficient amplicon for subsequent steps. The amplicon is combined with a laboratory viral construct, an HIV viral DNA sequence lacking the relevant protease and reverse transcriptase segment, through either homologous recombination or ligation to make a replicating recombinant virus. A reporter gene construct is used to allow for rapid detection of viral replication. In addition, the constructs have well-defined infection and growth characteristics in cell culture, allowing these assays to be reproducible and automated despite their complex nature. For each antiviral drug, a range of concentrations is tested to generate a dose-response curve. The inhibitory concentration required to inhibit 50% of virus replication (IC50) is then calculated. Typically, these assays take 2-4 weeks to complete. The IC50 of a given patient-derived recombinant is compared with the IC50 of a wild-type reference strain and a fold change in IC50 is calculated. Reduced susceptibility is defined as a ³2.5 or ³4 fold change in IC50, depending on the laboratory providing the service.

Recombinant viral phenotyping assays are still quite expensive and require significant expertise. Typically, they cost between $750 and $1,000 and are currently available commercially from only two companies, ViroLogic (South San Francisco, CA)4 and Virco (Mechelen, Belgium).5 The assays are currently provided as a service, and because of their complexity will not be available in kit format in the foreseeable future. There are some minor technical differences between the two assays. For example, the ViroLogic assay uses ligation to insert the protease and reverse transcriptase amplicon into the vector, whereas Virco's assay uses recombination. ViroLogic uses detection of light from a reporter luciferase gene, which has been cloned into the vector, to measure viral replication.4 Virco, on the other hand, uses an alternate reporter gene and high-resolution optics to measure viral replication.5 And ViroLogic's assay requires only a single round of replication as an endpoint, whereas several rounds are required for Virco's assay.

Although phenotypic results are easy to read, many questions remain about the interpretation of changes in susceptibility to a particular antiviral drug in the context of patient care. Resistance or decreased susceptibility is defined strictly in laboratory terms as folds increase in IC50 as used by Virco Laboratories (see Table 1).

As yet, there are no clinically defined breakpoints that relate the measured degree of drug susceptibility to the concentration of drug needed to treat the patient. There is some emerging data for protease inhibitors,6,7 but none for reverse transcriptase inhibitors. Depending on achievable active drug concentration in the patient, a four or five fold increase in IC50 may or may not cause in vivo clinically significant resistance. For example, moderate nevirapine resistance does not preclude the use of nevirapine in multidrug combination treatments.8 In addition, phenotypic assays do not take into consideration drug-drug interactions that increase the active concentration of a drug. The addition of ritonavir to a saquinavir-containing combination can increase the active concentration of saquinavir,9 which may or may not overcome moderate levels of saquinavir resistance.

Resistance Assay Sensitivity

Because all resistance assays, whether genotypic or phenotypic, start with a PCR amplification of HIV viral RNA from patient plasma, the result is a "populational" view of resistance as opposed to a clonal analysis. Only those viral variants that represent a significant percentage of the population (>10%-20%) will be detected. Since many of the resistance-associated mutations decrease the fitness of the virus to replicate,10,11 removal of the selective drug pressure may result in the resistant variant quickly becoming a minor species in the viral population. These resistant variants may not be detected by current resistance assays.12 Therefore testing is recommended while a patient is still on therapy and while selective pressure is being maintained.

Although current viral load assays are sensitive to 50 copies/mL,13 resistance assays are generally only sensitive to 500-1,000 copies. At lower viral concentrations, the resistance analysis could become more "clonal" and not representative of the population of viral variants in the sample.

This would result in decreased reproducibility and increased assay-to-assay variation.14 Although it may be possible to get amplified material from samples with viral loads below 500-1,000 copies/mL, the results may not be a representation of the patient's total viral population.

Genotype, Phenotype, or Both?

Both a genotype and a phenotype may provide helpful information in assessing a patient's viral resistance. For many situations, a single test may be sufficient. In some cases, the use of the two tests in combination may be needed. A well-performed genotype may be able to recognize mixtures sooner than a phenotype, thus giving an early indication of developing resistance. In addition, a genotype may give more information about the accumulation of mutations that precedes the development of a particular phenotype. For example, genotypic assays can detect mutations at codon 82, which precede the development of significant clinical resistance to ritonavir and indinavir.15 For newer antiviral drugs, all the mutations associated with resistance have not been identified and published, and so a phenotype may be far more informative. Among patients with complex treatment histories and numerous resistance-associated mutations, genotype interpretation may become too complicated and a phenotype might be more helpful. 

 References

     1.   Japour AJ, Mayers DL, Johnson VA, et al. Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. The RV-43 Study Group; the AIDS Clinical Trials Group Virology Committee Resistance Working Group. Antimicrob Agents Chemother. 1993;37:.

     2.   Nersesian RC, Fong DM, Kolmodin RM, et al. Development of a microarray-based HIV-1 drug resistance/genotyping assay. 7th Conference on Retroviruses and Opportunistic Infections; January 30-February 2, 2000; San Francisco, CA. [Abstract 794]

     3.   Kellam P and Larder BA. Recombinant virus assay: a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates. Antimicrob Agents Chemother. 1994;38:23-30.

     4.   Petropoulos CJ, Parkin NT, Limoli KL, et al. A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1. Antimicrob Agents Chemother. 2000;44:.

     5.   Hertogs K, de Bethune MP, Miller V, et al. A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs. Antimicrob Agents Chemother. 1998;42:.

     6.   Garraffo R, Durant J, Clevenbergh,P et al. Relevance of protease inhibitor plasma levels in patients treated with genotypic adapted therapy: pharmacological data from the Viradapt study. Antiviral Therapy. 1999;4(suppl 1):75. [Abstract 109]

     7.   Merry C, Back D, Barry M, et al. Therapeutic drug monitoring (TDM) of protease inhibitors (Pis): what to measure and when. 7th Conference on Retroviruses and Opportunistic Infections; January 30-February 2, 2000; San Francisco, CA. [Abstract 104]

     8.   Conway B. Development of drug resistance in patients receiving combinations of zidovudine, didanosine, and nevirapine. 39th Interscience Conference on Antimicrobial Agents and Chemotherapy; September 26-29, 1999. San Francisco, CA. [Abstract 436]

     9.   Hsu A, Granneman GR, Cao G, et al. Pharmacokinetic interactions between two human immunodeficiency virus protease inhibitors, ritonavir and saquinavir. Clin Pharmacol Ther. 1998;63:.

     10.  Zhang YM, Imamichi H, ImamichiT, et al. Drug resistance during indinavir therapy is caused by mutations in the protease gene and in its Gag substrate cleavage sites. J Virol. 1997;71: .

     11.  Maeda Y, Venzon DJ, Mitsuya H, et al. Altered

drug sensitivity, fitness and evolution of human immunodeficiency virus type 1 with pol gene

mutations conferring multi-dideoxynucleoside

resistance. J Infect Dis. 1998;177:.

     12.  Devereux HL, Youle M, Johnson MA, Loveday C. Rapid decline in detectability of HIV-1 drug resistance mutations after stopping therapy. AIDS. 1999;13:F123-F127.

     13.  Mulder J, Resnick R, Saget B, et al. A rapid and simple method for extracting human immunodeficiency virus type 1 RNA from plasma: enhanced sensitivity. J Clin Microbiol. 1997;35:.

     14.  Shafer RW, Warford A, Winters MA, et al. Reproducibility of human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase sequencing of plasma samples from heavily treated HIV-1 infected individuals. J Virol Methods. 2000;86:.

     15.  Molla A, Korneyeva M, Gao Q, et al. Ordered accumulation of mutations in HIV protease confers resistance to ritonavir. Nat Med. 1996;2:.