Dear Henry, resistance is OLD NEWS. Here is a good summary, so that you may leave the subject.
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Volume 7
October 1997
Number 5
A continuing medical education service for physicians who treat persons living with HIV/AIDS sponsored by The University of Alabama School of Medicine
Resistance in HIV
The HIV Management Council
This publication reflects the latest data on management of HIV. It is published by World Health CME, a division of World Health Communications Inc., 41 Madison Avenue, New York, New York 10010-2202; E-mail: whcme@whci.41mad.com. The opinions or views expressed in this educational publication are those of the participants and do not necessarily reflect the opinions or recommendations of the University of Alabama School of Medicine or World Health Communications Inc.
Contents may be reproduced freely for educational (noncommercial) purposes.
Printed in USA October 1997
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Definition and overview
Resistance to antiretroviral drugs is the greatest obstacle to effective therapy of HIV disease. HIV has repeatedly demonstrated its ability to escape every antiretroviral agent available to date by developing resistance mutations.
Mathematical models estimate that 10 billion virions are produced daily, providing ample opportunity for every possible mutation to develop. The reason for this high mutation rate is that single-stranded RNA viruses such as HIV-1 copy themselves in the absence of DNA proofreading mechanisms, leading to errors in replication.
Through a Darwinian selection process, mutant viral strains that can survive and replicate through their resistance to current agents can flourish and even completely replace wild-type strains in the presence of antiretroviral drugs, sometimes in weeks.
Broadly stated, antiretroviral resistance is the outcome of any mutation that enhances viral replication in the presence of an inhibitor, regardless of mechanism. Three conditions drive the emergence of resistance to HIV therapeutics:
High replication rate, or large number (1010) of daily viral replication cycles
Tendency toward mutation, or the notable lack of genomic fidelity, leading to a large number of "errors" in transcribing the viral genome during replication and resulting in a large number of daily mutations
Selective pressure, in which a drug blocks the growth of wild-type virus but allows the proliferation of mutant variants; this promotes selection of mutant variants at the expense of wild-type, or susceptible, strains
When drugs are used in situations in which near-full suppression is not achieved, selective pressure for the emergence of resistant isolates is applied. These situations may include (1) inappropriate selection of drugs, (2) nonadherence, or (3) pharmacokinetic interactions. Each factor may result in incomplete suppression that may lead to resistance. It may also be possible for an individual to acquire a resistant isolate via sexual, percutaneous, or vertical routes of transmission.
Reverse transcriptase, protease, and other potential drug targets within HIV have shown extreme mutability, characterized by metamorphosis into drug-resistant forms without loss of function. The end result is treatment failure.
In the era of highly active antiretroviral therapy (HAART), concerns about the emergence of multidrug resistance underscore the importance of individually designing therapeutic regimens that provide maximal viral suppression, thereby delaying or preventing the selection of resistant mutants.
Resistance assays
Rapid phenotypic and genotypic assay systems are currently in development but are not widely available for clinical use.
By sequencing viral DNA, genotypic assays identify mutations such as the 184 mutation associated with 3TC resistance or the L90M mutation associated with protease inhibitor resistance.
Genotypic resistance assays enable the clinician to identify the specific resistance mutations associated with treatment failure and perform susceptibility testing to identify specific resistant variants in treatment-naive patients.
Phenotypic resistance assays determine the ability of HIV to replicate in the presence of specific drug concentrations. In the clinic, phenotypic resistance assays may guide the selection of treatment options to account for known resistance patterns. The performance of phenotypic assays has involved a time-consuming and labor-intensive process that required virus isolation, expansion, titration, and susceptibility testing in primary lymphocytes. In attempts to streamline this process, rapid phenotypic assays have been developed, but they are not yet commercially available.
Importantly, the correlation between genotypic resistance, phenotypic resistance, and clinical response remains unclear. These issues need to be resolved to determine the appropriate roles for genotypic and phenotypic resistance assays in clinical practice.
An inherent limitation of resistance assay systems is that, although they may indicate that a viral variant will be unresponsive to specific drugs, they cannot predict to which drugs the virus will be sensitive. Moreover, both phenotypic and genotypic assays assess only the predominant viral quasi-species circulating in plasma. Because of issues related to viral fitness, resistant variants selected by drug pressure may quickly contract to a level below the threshold of detection in plasma when selective pressure is removed. The resistant variants remain as archival quasi-species in a proviral form and will return to detectable levels quickly if selective pressure is reapplied. Currently available genotypic and phenotypic assays cannot detect minority populations, presenting a serious shortcoming and a major barrier to their clinical utility.
At present, these technologies for the measurement of drug resistance are confined to the research setting.
Until such assays become widely available clinically, the most practical method for evaluating drug resistance in the context of treatment failure will remain the measurement of quantitative plasma HIV-1 RNA levels.
Complete viral suppression: the best strategy for preventing resistance
Suboptimal, or partly suppressive, drug regimens facilitate the selection of resistant variants, while completely suppressive regimens prevent mutants from ever arising. Even combination therapy delays or prevents resistance only when viral replication is completely suppressed. In clinical practice, a rebound in viral load may herald the emergence of resistance. Inadequate therapy ultimately results in resistance, viral rebound, and therapeutic failure.
In a HAART regimen (AZT + 3TC + either ritonavir, indinavir, or nelfinavir), incomplete suppression led to detectable resistance mutations. By contrast, resistance mutations have not been observed in patients who achieve complete viral suppression on HAART regimens. After 68 weeks of treatment with AZT + 3TC + indinavir, resistance mutations to 3TC or indinavir were detected only rarely in patients whose viral load was suppressed below the limit of detection at <500 copies/mL.
Results of the Multicenter AIDS Cohort Study (MACS) demonstrated that viral load reliably predicts patient prognosis irrespective of CD4 cell count. Similarly, clinical trials have shown that antiretroviral therapy promotes reductions in viral load that correlate with improved prognosis. For example, a 1-log (10-fold) reduction in viral load in response to antiretroviral therapy is associated with a 65% reduction in the risk of disease progression. In the absence of clinically available resistance assays, persistent viral replication, as indicated by plasma viral load above 200 copies/mL, is predictive of drug resistance and treatment failure.
Results of studies comparing dual- and triple-therapy regimens indicate that the more complete suppression obtained with triple combination therapy forestalls resistance, drug failure, and disease progression. At present, the best strategy to prevent resistance is the prescription of a HAART regimen that achieves maximal viral suppression. In tailoring such regimens to individual patients, the clinician should consider the following factors:
Nonoverlapping toxicities
Pharmacokinetic interactions
Absence of cross-resistance
Divergent or beneficial mutation profiles
Importantly, drug concentrations should be suppressive in all potentially infected compartments, including lymphoid tissue (macrophages) and plasma (lymphocytes). An agent that crosses the blood-brain barrier should be included, because brain macrophages are an important reservoir of virus.
Drug failure unrelated to resistance
Clinical failure of protease inhibitors, as well as of reverse transcriptase inhibitors, may occur independently of resistance mutations. In a large military cohort study, 21% of isolates from patients not responding to combination therapy showed no detectable resistance mutations in either reverse transcriptase or protease genes; 33% had evidence of resistance to protease inhibitors; and 46% showed evidence of resistance to both nucleosides and protease inhibitors. Thus, more than 50% of the patients did not respond to treatment with protease inhibitors without evidence of resistance to these drugs. Apparently sensitive virus may be unresponsive to therapy for several reasons apart from resistance, including
The patient's intolerance of the drug(s)
Altered intracellular metabolism or individual variation in pharmacokinetics
Malabsorption
Drug interactions
Change in viral phenotype (ie, non-syncytium-inducing to syncytium-inducing)
Progressive immune decline
Nonadherence to the regimen
Failure to adhere to the therapeutic regimen may be the most significant of these factors. Confronted with apparent treatment failure, practitioners should question patients about their adherence. It may be possible to restore the activity of a "failing" agent by improving adherence. On the other hand, resistant viral strains may already have emerged because of incomplete suppression of replication.
Response despite resistance
The presence of resistance does not always coincide with treatment failure, raising questions about the interpretation of currently available resistance assays in clinical practice.
Virus present in some body compartments may remain sensitive to a particular drug although demonstrating resistance in other compartments. For example, resistant virus may be present in CD4 lymphocytes in plasma, while sensitive virus may be harbored in sanctuary sites such as brain-tissue macrophages (astrocytes). Therefore, the drug may still demonstrate a degree of clinical efficacy.
Enzyme mutability
Currently, 143 distinct mutations have been identified in HIV-1 that are associated with the development of resistance to one or more antiretroviral agents. To date, 23 amino acid substitutions have been linked to resistance to nucleoside analog reverse transcriptase inhibitors (5 of them to multidrug resistance), 35 to resistance to nonnucleosides, and 42 of 99 possible substitutions to resistance to protease inhibitors. Extensively pretreated patients may be particularly vulnerable to multiple concurrent reverse transcriptase and protease resistance mutations, often within a single viral clone.
Protease inhibitors
Initially, it was believed that four characteristics of the viral protease would make it less susceptible to mutations than reverse transcriptase. These characteristics include multiple substrate cleavage sites, the small size of the enzyme (99 amino acids), the presence of five highly conserved regions, and its homodimer structure. These assumptions, however, proved premature; the protease enzyme is highly mutable.
Protease inhibitors are not a "magic bullet" capable of overcoming nucleoside resistance. The recommended standard of care, triple combination HAART regimens containing two nucleosides and a protease inhibitor, is capable of holding viral replication in check and preventing or delaying the emergence of resistance in most patients. In this connection, the importance of strict adherence to the dose schedule to maintain continuous viral suppression cannot be overemphasized. Long-term experience is accumulating in evaluating whether triple combination therapy can durably suppress virus for prolonged periods without the occurrence of breakthrough resistance or additive toxicity.
Cross-resistance
Protease inhibitor resistance is further complicated by cross-resistance. As documented by genotypic analyses of resistant virus, substitutions at pivotal amino acid sites in protease may result in cross-resistance so that patients treated with one protease inhibitor develop virus resistant to others.
In general, the greater the number of mutations in the protease gene associated with a specific protease inhibitor, the greater the potential for that drug to be associated with cross-resistance.
In clinical practice, cross-resistance limits the efficacy of protease inhibitors as a class in that it restricts future options for patients for whom initial nucleoside/protease inhibitor combination regimens have failed.
Resistant isolates can be selected by all of the available protease inhibitors. The initial mutations may vary among protease inhibitors, but cross-resistance increases as additional mutations emerge. Although in some protease inhibitors the initial mutation appears to be less important in terms of cross-resistance with other protease inhibitors, it is not clear how frequently patients are detected with a single mutation in a clinical setting. The ultimate proof of this concept rests on demonstrating the frequency and durability of responses to subsequent agents after treatment failure. In the meantime, the initial regimen has the greatest chance of fully suppressing virus. Therefore, drugs should be chosen with a view toward sustaining efficacy and preventing the emergence of resistance as opposed to anticipating failure and choosing the primary drug based on the anticipation of secondary regimens.
Preexisting resistance: implications for transmission
If unchecked by antiretroviral therapy, high rates of viral replication persist throughout all stages of HIV infection, and a period of true virologic latency does not exist.
In view of the high mutation rate of HIV, it is hardly surprising that resistance mutations have been documented in treatment-naive individuals in relation to every class of approved antiretroviral drug, as confirmed by genotypic sequencing analyses. For example, the Y181 protease-resistant variant is estimated to preexist in 1 in 1000 previously untreated patients. The addition of an antiretroviral drug in the context of preexisting resistance promotes the selection of mutant strains. In this situation, drug-resistant strains may become dominant within a matter of weeks (ie, 3TC and nonnucleosides) or months to years (ie, AZT and protease inhibitors), causing high-level resistance and rising viral load and leading to treatment failure.
Although HAART regimens have been shown to hold viral replication in check, the increasing utilization of these combination regimens has raised concerns about the emergence and transmission of multidrug-resistant strains. For example, individuals who fail to adhere to rigorous long-term HAART regimens may develop multidrug-resistant strains that are fully replication-competent. If these nonadherent individuals subsequently fail to observe transmission-prevention strategies (ie, condom use, avoidance of needle sharing, prenatal AZT prophylaxis), then increasing numbers of individuals are likely to become infected with multidrug-resistant variants unresponsive to current treatments.
Conclusion
Although the use of potent therapy in early-stage HIV disease may delay the emergence of resistance, it has not yet been eliminated as a clinical problem. Consequently, therapeutic failures continue to occur.
Nonadherence, often resulting in insufficient suppression of viral replication, has emerged as a key factor in the development of resistance. Therefore, clinicians should continually reinforce the importance of following the drug schedule. Even patients with the best intentions may occasionally lapse, and nonadherence is bound to remain a significant concern, given the long-term nature and increasing complexity of combination regimens.
In the future, resistance issues will undoubtedly become more complex for several reasons:
Extensive use of combination regimens that may involve even greater numbers of drugs
Emergence of increasingly complex patterns of cross-resistance
Transmission of resistant virus via sexual, percutaneous, and vertical routes
As a major and continuing obstacle to effective HIV therapy, resistance challenges the research and treatment communities to develop new technologies and therapeutic approaches capable of overcoming it.
SELECTED WEB SITES
AIDS Clinical Trials Information Service
actis.org
AIDS Research Information Center
critpath.org
AIDS Treatment Data Network
health.nyam.org
AIDS Treatment News Online
immunet.org
CDC AIDS Clearinghouse
cdcnac.org
HIV/AIDS Treatment Information Service
hivatis.org
HIV Information Network Online
hivline.com
IAPAC (International Association of Physicians in AIDS Care)
iapac.org
NIAID AIDS Gopher
gopher://odie.niaid.nih.gov:70/11/aids
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GENOTYPIC vs PHENOTYPIC
The term genotypic resistance is a misnomer. So-called genotypic resistance is actually the presence in the viral genome of mutations associated with resistance. These mutations may or may not be accompanied by measurable resistance to antiretroviral agents. On the other hand, phenotypic resistance is defined as replication of HIV in the presence of drug. This may be defined in vitro by an increase in IC50 of the drug (the concentration that inhibits viral replication by 50%) or in vivo by a return of viral load toward pretreatment (baseline) levels in the presence of therapeutic concentrations of the drug.
Genotypic evidence of resistance is not always accompanied by phenotypic resistance. Some resistance mutations may merely raise the threshold of sensitivity of the virus, so that higher drug levels may still be effective. In some cases, increasing the dose of the drug will counter resistance.
In some instances, a mutation may result in clinical benefit by restoring viral susceptibility to a second drug (eg, the 3TC-induced mutation at codon 184 that restores AZT sensitivity).
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RESISTANCE REBOUND: A CASE REPORT
An anecdotal case report suggests that after resistance develops, it persists. The resistant variants simply remain latent following discontinuation of therapy, only to rapidly reappear subsequent to drug reintroduction after a long "washout" period.
In that case report, a patient who initially responded to indinavir monotherapy developed 30-fold resistance, characterized by six resistance mutations, after long-term treatment. Six months after the discontinuation of indinavir, all mutations had returned to wild-type, and the predominant viral isolate again appeared sensitive to indinavir. However, within 3 weeks after rechallenge with indinavir, high-level resistance reemerged, characterized by the same six mutations previously identified. It appears that the viral genotype with this resistance mutation pattern had "gone underground" but had not disappeared during the period of drug discontinuation. It was able to resurface under selective pressure from indinavir.
Whether the rapid reemergence of resistance of this patient is an isolated phenomenon or typical of protease resistance remains to be determined.
Clinical Insight gratefully acknowledges the help of Murex Diagnostics in providing meeting transcripts on which part of this issue was based.
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Suggested reading
Abstracts of the Sixth International Workshop on HIV Drug Resistance, Treatment Strategies and Eradication; June 25-28, 1997; St. Petersburg, FL.
de Jong JJ, Foudraine N, Huismans R, et al. First-line treatment with zidovudine/lamivudine or stavudine/lamivudine rapidly leads to lamivudine resistance even in patients with a serum HIV RNA load below 5,000 copies/mL. Abstract 68.
Emini EA, Holder DJ, Schleif WA, et al. Evidence for the prevention of new HIV-1 infection cycles in patients treated with indinavir plus zidovudine plus lamivudine. Abstract 128.
Hammer SM, Demeter LM, Fischl MA, et al. Clinical, immunological and virological outcomes in ACTG 320, a randomized, placebo-controlled trial of indinavir in combination with two nucleosides in HIV-1-infected persons with CD4+ cell counts 200/mm3. Abstract 67.
Holder DJ, Shivaprakash M, Danovich RM, et al. Duration of HIV-1 load suppression in patients treated with indinavir who experience virus load declines to <500 vRNA copies/mL. Abstract 129.
Markowitz M, Cao Y, Vesanen M, et al. Intensive virological assessment of aggressively treated subjects with recent and chronic HIV infection. Abstract 126.
Mayers DL, Gallahan DL, Martin GJ, et al. Drug resistance genotypes from plasma virus of HIV-infected patients failing combination drug therapy. Abstract 80.
Opravil M, DeMasi R, Hill A. Prediction of long-term HIV RNA suppression during zidovudine/lamivudine treatment. Abstract 60.
Shafer RW, Winters MA, Merigan TC. Multiple concurrent RT and protease mutations and multidrug resistance in heavily treated HIV-1-infected patients. Abstract 39.
Young B, Johnson S, Bakhtiari M, et al. Genotypic analysis of HIV-1 protease from patients failing highly active anti-retroviral therapy: preliminary analysis. Abstract 65.
Carpenter CCJ, Fischl MA, Hammer SM, et al. Antiretroviral therapy for HIV infection in 1997: updated recommendations of the International AIDS Society-USA Panel. JAMA. 1997;277:1962-1969.
Condra JH, Emini EA. Preventing HIV-1 drug resistance. Sci Med. 1997;4:14-23.
Condra J, Schleif WA, Blahy OM, et al. Evidence for the existence of long-lived genetic reservoirs of HIV-1 in infected patients. Presented at the Fourth International Workshop on HIV Drug Resistance; July 6-9, 1995; Sardinia, Italy. Abstract 82.
D'Aquila RT. HIV-1 chemotherapy and drug resistance. Clin Diagn Virol. 1995;3:299-316.
Feinberg M. Hidden dangers of incompletely suppressive antiretroviral therapy. Lancet. 1997;349:1408-1409.
Havlir DV, Richman DD. Viral dynamics of HIV: implications for drug development and therapeutic strategies. Ann Intern Med. 1996;124:984-994.
Mayers DL. Resistance and cross-resistance to nucleoside reverse transcriptase inhibitors. HIV. 1997;6:15-21.
McMahon D, Mellors JW. Resistance and cross-resistance to protease inhibitors. HIV. 1997;6:9-14.
Perno CF, Aquaro S, Cenci A. Development of resistance to anti-HIV drugs in primary macrophages. Presented at the Tenth International Conference on Antiretroviral Research; April 6-11, 1997; Atlanta, GA. Abstract 58.
Schinazi RF, Larder BA, Mellors JW. Mutations in retroviral genes associated with drug resistance. International Antiretroviral News. 1996;4:95-107.
Spruance W, Pavia AT, Mellors JW, et al. Clinical efficacy of monotherapy with stavudine compared with zidovudine in HIV-infected, zidovudine-experienced patients. A randomized, double-blind, controlled trial. Bristol-Myers Squibb Stavudine/019 Study Group. Ann Intern Med. 1997;126:355-363.
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