International Association of Physicians in AIDS Care, February 2000 Journal
Clive Loveday, MB BS, PhD
Department of Retrovirology, Royal Free and University, College Medical School,
London, United Kingdom
| Introduction Viral Diversity and Drug Pressure Measurement of Resistance Evidence to Justify Resistance Testing Clinical Application of Resistance Testing References |
Drug resistance testing has supported therapy for infectious diseases for many years. The use of resistance testing in HIV/ AIDS has been limited until recently by a need for appropriate technology and a lack of evidence from randomized controlled trials. In addition, pressure from physicians and patient groups in this field of medicine can make it difficult to "perturb" the status quo even if it is based on additional scientific data.
A number of advances in HIV virology form the foundation for understanding the rationale for resistance testing to support clinical care of patients with HIV/AIDS. Primary HIV infection is associated with an initial high plasma viremia. Then, as immune responses develop in the host, viremia is modulated and reaches a "set point" that may well define the rate of disease progress in that patient from then onwards.1 However, this outline only takes account of viral numbers. It is important to realize that during primary infection a relatively homogeneous population of viruses initially infects the host. But as a consequence of high, error-prone replication (10 billion new virions per day), viral diversity increases over time. As a result, within a year or so of primary infection, the host is infected by a heterogeneous population of viruses that have evolved by natural selection.
These viruses represent the optimum organisms for survival in that given environment, that is, the patient. This process, obeying Darwinian laws of evolution, is the mechanism by which HIV may rapidly adapt to new environmental pressures. For example, if a patient is treated with antiretroviral therapies that fail to suppress viral replication completely, new viral populations ("quasispecies") evolve that carry mutant viruses resistant to therapies. However, the rate of HIV replication is so high that, even in the absence of drugs, single or double mutations can evolve de novo.2 This process--and sexual transmission of drug-resistant viruses--result in potential circulation of resistant viruses in drug-naive communities.
In this model of HIV diversification at disease onset, drug therapy will decrease viral replication (resulting in fewer virions) and also decrease viral diversity (allowing perhaps only a few quasispecies to replicate). If any therapy allows continued replication, a new population of viruses will evolve numerically and in diversity. These new populations are generally considered to be less fit than the original wild-type viruses, that is, they are thought to replicate less efficiently. If therapy is discontinued, wild-type viruses left as proviral DNA in cellular archives often redominate the viral population.
In summary, any replication allows evolution (of resistance). Resistance is a function of replication rate, the biology of infecting viruses, the duration of infection, and the nature and impact of the drug pressure. However, it must be understood that evolving resistance is only one cause of therapeutic failure. Equally important are "pharmacologic resistance" (low drug concentrations potentially caused by numerous factors) and poor adherence to therapy. Currently, these causes of therapeutic failure are not well defined or easy to evaluate.
For many years genome sequencing has been an integral part of virologic and molecular biological science. But research technologies have been time consuming, technically difficult, and cumbersome and have not lent themselves to easy use in a busy clinical service. However, recently companies have made enormous efforts to modify and adapt basic laboratory strategies to make resistance testing feasible in "real time" to support clinical care. These processes still require validation in the clinical setting. Two basic approaches exist for measuring resistance:
This type of assay has been superseded by recombinant virus technology in which selected RNA regions associated with drug resistance (reverse transcriptase [RT] and protease) from patient viruses are reverse transcribed and amplified by PCR before integration into an infectious laboratory strain of HIV-1. These model viruses are then evaluated for resistance in a drug susceptibility assay, and results are expressed as an IC50. These processes are difficult to carry out in local laboratories. Two companies now offer a clinical phenotyping service worldwide, Virco in Belgium and ViroLogic in the United States.
Modified sequencing technologies are available commercially from Visible Genetics, ABI/Perkin Elmer, and Affymetrix, and point mutation technologies from Innogenetics and Chiron. Sequence data generated by automated sequencing requires manual review; reporting is carried out by experienced virologists or persons with technical training, who give an opinion based on current scientific data.
Phenotyping and genotyping can be used to support care on a research footing, but additional information is required on all these technologies concerning clinical utility, quality controls, and performance data among diverse virus populations found in Europe and all points east.
Some data have emerged to justify use of resistance testing to support patient care. We have known for many years that viruses grown in multiple rounds of culture in the presence of an antiretroviral drug like zidovudine will rapidly evolve resistance-associated mutations (M41L, D67N, K70R, L210W, T215Y, and K219Q) in the RT gene. Further, these mutations singly or in combination in recombinant viruses confer changes in drug sensitivity (for example, T215Y: 60- to 70-fold decrease; K70R: 8-fold decrease; D67N + K70R + T215Y: 180-fold decrease).
Early in vivo data demonstrated that resistant viruses evolved in plasma as patients receiving monotherapy exhibited virologic failure.3,4 More recently, retrospective analyses of trials have shown associations between genotypic and phenotypic resistance at baseline and clinical and/or virologic failure.5,6 Conversely, the number of sensitive drugs in a combination is associated with the virologic success of that regimen. Meta-analyses of studies have shown that resistance measures are at least as good as other predictors of failure.7
Two randomized controlled trials recently confirmed the benefit of genotyping after virologic failure. The first, VIRADAPT, demonstrated a significantly better virologic response after 12 and 24 weeks of therapy with genotype-directed drug selection compared with standard of care (no genotyping).8 The second, GART, showed a significantly better virologic response after four to eight weeks of therapy with treatment based on genotyping and expert advice versus standard of care (no genotyping or expert advice).9 The patients in these studies were relatively drug experienced and had advanced disease. We still require long-term efficacy data with more than one year of follow-up in different patient groups (those with early disease and early in the course of therapy).
Within my own service we have been providing resistance genotypes for over one year to assist in patient care. Although we have run more than 1000 assays to date, this is not yet a full clinical service for technical, financial, and evidentiary reasons. We have established guidelines for patient sample storage during their therapy and mandate that plasma samples be stored at -70° C before commencing therapy and at every therapeutic crossroad thereafter. This allows retrospective testing of early samples when therapy becomes more complex or when it fails.
We recommend resistance testing to guide the next choice of drugs for patients in whom therapies are failing. Although in our service treatment of primary HIV infection is still limited to trials, a high community prevalence of resistance in drug-naive patients is an indication to screen patients for such viruses, but this should not delay starting treatment. In addition we consider sympathetically any requests for resistance testing in naive patients or those with a suboptimal virologic response. We provide resistance testing for pregnant women and for children, whose drug choices are currently more limited than those of adults. Resistance testing is also recommended following risk of exposure to HIV, but again therapy is not delayed but modified if necessary.
In conclusion resistance testing is coming into use as a direct request for support from physicians and patients. We are responding to those requests sympathetically, but there is a scientific need to continue to assess and evaluate the methods used, the patient groups treated, and the overall benefit in both clinical and financial terms.
1. Mellors JW, Rinaldo CR Jr, Gupta P, et al. Prognosis in HIV-1 infection predicted by the quantity of virus in plasma [see comments] [published erratum appears in Science 1997;275:14]. Science 1996;272:1167-1170.
2. Coffin JM. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science 1995;267:483-489.
3. Japour AJ, Welles S, D'Aquila RT, et al. Prevalence and clinical significance of zidovudine resistance mutations in human immunodeficiency virus isolated from patients after long-term zidovudine treatment. AIDS Clinical Trials Group 116B/117 Study Team and the Virology Committee Resistance Working Group. J Infect Dis 1995;171:1172-1179.
4. Loveday C, Kaye S, Tenant-Flowers M, et al. HIV-1 RNA serum-load and resistant viral genotypes during early zidovudine therapy. Lancet 1995;345:820-824.
5. Deeks SG, Hellmann HS, Grant RM, et al. Novel four-drug salvage treatment regimens after failure of a human immunodeficiency virus type 1 protease inhibitor-containing regimen: antiviral activity and correlation of baseline phenotypic drug susceptibility with virologic outcome. J Infect Dis 1999;179:1375-1381.
6. Zolopa AR, Shafer RW, Warford A, et al. HIV-1 genotypic resistance patterns predict response to saquinavir-ritonavir therapy in patients in whom previous protease inhibitor therapy had failed. Ann Intern Med 1999;131:813-821.
7. Hirsch MS, Brun-Vizenet F, D'Aquila R, et al. Antiretroviral drug resistance testing in adult HIV-1 infection: recommendations of the IAS-USA panel. In press.
8. Durant J, Clevenbergh P, Halfon P, et al. Drug-resistance genotyping in HIV-1 therapy: the VIRADAPT randomised controlled trial. Lancet 1999;353:2195-2199.
9. Baxter JL, Mayers DL, Wentworth DN, et al. A pilot study of the short-term effects of antiretroviral management based on plasma genotypic antiretroviral resistance testing (GART) in patients failing antiretroviral therapy. Presented at: 6th Conference on Retroviruses and Opportunistic Infections; January 31-February 4, 1999; Chicago. Abstract LB8.
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This information is designed to support, not replace, the relationship that exists between you and your doctor.