Important note: Information in this article was accurate in June 2002. The state of the art may have changed since the publication date.
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Since the publication of a review of Antiretroviral Resistance Testing in the September 2000 HEPP News,1 resistance testing has become an important component of standard care for HIV -infected patients. This is largely due to the widespread availability of genotypic testing, as well as increased access to both the virtual and standard phenotype tests. Additionally, the increasing presence of resistance in both antiretroviral (ARV) experienced and ARV naïve individuals, combined with the availability of newer ARV agents capable of overcoming drug resistance, has fueled the need to assess for drug resistance in patients being treated for HIV infection. This article will provide an update on new developments in the area of HIV resistance and provide a guide to the implementation of resistance testing in correctional settings.
HIV resistance is increasing in the U.S. Richman, et al.2 reported in a study in late 2001 on the prevalence of resistance in the United States. In using the HIV Cost and Service Utilization Study database, 1,906 patients who were on treatment (receiving antiretroviral therapy) between 1996 and 1999 were identified. A phenotypic resistance assay was done on 1,209 patients whose viral load was >500 copies/mm3. Seventy eight percent (78%) had resistance to at least one ARV agent, 50% had resistance present in two classes of ARV agents, and 14% had three-class, or multi-drug resistant HIV (MDR-HIV). The majority of resistance was found in the nucleoside reverse transcriptase inhibitor (NRTI) class with lesser degrees in the protease inhibitor (PI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) classes, which may have been related to the date of the study (PIs and NRTIs were new when the study started). This degree of resistance likely reflects incomplete adherence to therapy (see Figure 1).
Equally disturbing is the increasing prevalence of ARV resistance in drug-naïve people who are recently HIV infected as first described by Little et al. and subsequently confirmed by others. Little et al. showed that 15% of acutely infected (naïve) patients had genotypic mutations at the time of their diagnosis. More importantly, these mutations in the reverse transcriptase and protease genomes persisted up to 303 days in the absence of ARV therapy3. Bennett et al. described the prevalence of genotypic resistance in a cohort of patients newly infected with HIV between 1998 and 20004. Ten percent of patients had NRTI resistance mutations, 4% had PI mutations, and 3% had NNRTI mutations; and 4% had resistance mutations in two classes of agents.
Use of genotypic or phenotypic resistance assays has been shown to improve response to rescue therapy in patients failing therapy.5,6 Combined with the above data, the current recommendation that resistance testing be performed in patients failing therapy or with incomplete viral suppression is warranted. And, although not yet generally recommended, the epidemiological data described above support the use of genotypic testing in recently infected patients for up to a year after their infection. However, clinical studies assessing this strategy have not been done (see HEPPigram, page 7 for guidance).
Resistance is best defined as reduced susceptibility of HIV to a specific ARV agent. As a result, resistance is not an all or none phenomenon. This relative reduction in susceptibility is best displayed through the use of phenotypic testing. The phenotypic assay reports the fold-increase in drug concentration needed to inhibit 50% of viral replication (IC50). The threshold defining reduced susceptibility for the nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs) is drug- specific. For example, a greater than 1.6-fold increase in the IC50 for stavudine confers resistance, whereas for tenofovir a 3.8-fold increase is necessary to confer resistance.
For non-ritonavir enhanced protease inhibitors (PIs), a greater than 2.5 - 4 fold increase in the IC50 (depending on the assay used) usually signifies an intermediate reduction in susceptibility, whereas a greater than 10-fold increase is required to confer complete resistance. With the introduction of co-formulated lopinavir/ritonavir (Kaletra), the standard PI thresholds no longer apply. The small amount of ritonavir in Kaletra leads to a large increase in lopinavir drug levels. Specifically, the lopinavir trough concentration (Cmin) greatly exceeds the IC50 in both wild-type and drug resistant HIV strains. Therefore the so-called inhibitory quotient (Cmin/ IC50) for lopinavir is quite large.7 Patients who failed three or more prior PI-containing regimens still responded to rescue therapy containing lopinavir/ritonavir as long as the fold-change in the IC50 was less than 40. Specific thresholds have not yet been defined for other ritonavir-enhanced regimens such as ritonavir plus indinavir, amprenavir, or saquinavir.
One last concept specific to phenotypic resistance testing is hypersusceptibility. Hypersusceptibility exists when there is a decrease in the amount of drug needed to inhibit viral replication. This is most commonly seen with the NNRTIs in the setting of significant prior NRTI use, but has also been seen with amprenavir. Clinically, Shulman et al has shown an enhanced anti-viral response in the presence of hypersusceptibility to efavirenz.8
In contrast to phenotypic testing, genotypic testing defines resistance based on the number of known resistance-conferring mutations present at the time of testing. The threshold differs for each drug because resistance is conferred by different mutational patterns. Each genotypic resistance-testing manufacturer sets up their own rules by which they interpret whether the mutations present are likely to confer reduced susceptibility. This can lead to significant differences in interpretations between testing kits, as well as confusion amongst clinicians inexperienced in interpreting genotypic resistance test results.9 Therefore, it is recommended that HIV experts assist with interpreting genotypic testing (see Table 1 for a comparison of genotypic and phenotypic testing).
Genotypic testing has led to the recently appreciated concept of NRTI class cross-resistance. Although the infrequently (1-3%) seen Q151M and T69S insertion mutations have been known to confer resistance across most of the NRTI class, work done by Whitcomb et al. at Virologic has shown that mutations previously associated with just zidovudine resistance actually confer resistance to all the NRTIs.10 These specific codon mutations-41, 67, 70, 210, 215, and 219-referred to as thymidine or nucleoside analogue mutations (TAMs or NAMs) cause varying degrees of resistance to all the nucleoside and nucleotide analogues (see HIV101, page 5). The more mutations present, the broader the resistance.
Despite all the data that may be derived about a patient's virus from the use of resistance assays, additional intricacies may limit their utility. First, discordance between genotypic and phenotypic testing exists. Parkin, et al. evaluated 200 patient samples with both testing modalities and found one-drug discordance in 75% and four-drug discordance in 22% of samples.11 Additionally, these in vitro assays may not translate into in vivo response. The reasons for this are multifactorial and include non-adherence, interpatient variability in absorption and metabolism of the agents (variability in therapeutic drug levels, see TDM below), and adverse drug-drug interactions. Finally, it should be understood that resistance test results reflect the pressure exerted by the current regimen on the virus. So if a patient who had previously developed the M184V (amino acid M replaced by amino acid V at position 184) mutation while on lamivudine is not taking it at the time resistance testing is repeated, this mutation may not be prevalent in adequate amounts to be detected (it may be present in an "archived" form in resting T cells). However if the patient is then started on a lamivudine- containing regimen that is not fully suppressive, the mutation will reappear and lead to virologic failure. Therefore, clinicians must utilize all available resistance test results and information about prior ARV therapy when planning rescue therapy.
Several years ago, Deeks et al. in San Francisco described their cohort of patients who had previously been undetectable on PI-based ARV therapy but subsequently had viral breakthrough. In this group, despite persistent viremia while on their failing regimen, most remained immunologically and clinically stable for many years.12 This study led many clinicians to maintain patients on their failing regimens. Coakley et al. recently reported on a cohort of patients who were receiving similar ARV therapy and who had rebounded with a viral load less than 1000 copies/mm3.13 These patients had detectable viremia for a mean of 22 months during which their CD4 count rose 97 cells/mm3 and viral load rose 61 copies/mm3. Forty patients had a genotype obtained which revealed that 90% had resistance to one or more of the ARVs they were taking. Six of seven patients whose viral load rose above 1000 copies/mm3 had resistance to all three agents compared with only nine of 33 whose viral load remained less than 1000 copies/mm3. Therefore, despite the lack of clinical and immunologic damage during low level viremia, viral evolution is ongoing and is likely to be clinically significant when rescue therapy is attempted. Thus, maintaining patients on failing regimens may be detrimental.
Newer testing modalities such as therapeutic drug monitoring (TDM) and viral fitness assays are not yet widely available, but are being studied in patients taking ARV therapy. When available, these will most likely be utilized in patients failing treatment and will therefore be combined with resistance assay results to further assist in the management of this expanding population.
TDM involves measuring plasma drug concentrations. The trough (or Cmin) is the concentration of drug just prior to administration of the next dose. Protease inhibitor trough levels have been linked to efficacy.14 This data, in conjunction with the unpredictable pharmacokinetic profile of the protease inhibitors, has led to clinical studies examining the role of TDM. Burger, et al. looked at the use of TDM with nelfinavir based regimens and found that use of TDM led to increasing the nelfinavir dose to achieve a better Cmin and virologic outcome.15 As mentioned earlier, ritonavir-boosted PIs achieve higher Cmin values and when TDM is readily available it will likely be combined with phenotypic data to adjust dosing in individuals to overcome resistance.
Viral fitness is the ability of the virus to replicate, infect, and kill T cells. Fitness varies between wild type and resistant strains. The acquisition of resistance mutations often leads to a period of decreased fitness, which in turn has been associated with prolonged immunologic stability in the setting of virologic failure.16 A fitness assay might be utilized to determine which patterns of resistance are less damaging. Clinicians can now begin receiving this type of information with a new replication assay, Replication Capacity (Virologic), that is now provided with Virologic's phenotype assays.
The use of resistance testing is illustrated by the following clinical case. A 38 year-old African-American inmate presented for HIV specialty care in March 2000. He had a history of HIV infection since 1996 but was asymptomatic and had a history of hepatitis C. In March 2000 he had a CD4 count of 375 cells/mm3 (15%) and a viral load of 736 copies. He was taking stavudine, lamivudine, and efavirenz since July 1999 but notably each drug had been started sequentially over a 2-year period. He had also received zidovudine, indinavir, and nelfinavir in the past. The decision at this time was to continue his current ARV given his overall stability.
He remained clinically stable on this regimen for the next 16 months and his CD4 count and viral load ranged from 324-414 cells/mm3 and <400-1571 copies/mm3 respectively. In August 2001 his viral load peaked at 3,080 copies/mm3 with a concurrent CD4 count of 399 (18%). A genotype was ordered and showed mutations at the following positions: 1) NRTI: 184, 2) NNRTI: 103, and 3) PI: 30, 63, 77, and 88 (see HIV101, page 5). These mutations were interpreted as conferring resistance to lamivudine, all the NNRTIs, and nelfinavir.
Due to the increase in his viral load and clear accumulation of resistance mutations, a change in ARV to lopinavir/ritonavir (Kaletra), stavudine (D4T or Zerit), didanosine (ddI, Videx), and abacavir (ABC, Ziagen) was made. Viral load two and six months later were 703 and <400 copies/mm3 respectively, and his CD4 remained stable at 336 (18%). He was subsequently paroled on stable ARV.
This case illustrates several important clinical issues regarding HIV resistance. First, sequential changes in ARV agents rather than changing the entire regimen when failing therapy will lead to incomplete suppression. This patient's current ARV regimen was not started simultaneously and the addition of agents with a low genetic barrier to resistance (due to the low number of mutations required to develop resistance, see HIV 101), ie. lamivudine and efavirenz, while there is incomplete suppression will lead to the development of resistance to these agents. Second, despite clinical and immunologic stability, viral evolution and accumulation of mutations will occur in the setting of incomplete viral suppression as was shown by Coakley et al. Any replication, even at low levels as in this patient, will allow the virus to evolve and develop mutations that sabotage the success of the regimen. Finally, resistance testing in the setting of virologic failure provides important information that leads to a greater likelihood of successful rescue therapy. As has been exhibited in several prospective studies, use of resistance assays allows clinicians to choose an ARV regimen with more active agents and therefore have a higher chance of suppressing viral replication.
Because of the significant prevalence of drug-resistant virus in both treatment-experienced and treatment-naïve patients, resistance testing has become an important component of the care of HIV-infected patients. It is important to keep in mind that patients with persistent low-level viremia continue to evolve drug-resistant viruses. Genotype and phenotype testing have been shown to increase patient response to rescue therapies in treatment-experienced individuals. The benefits of genotyping and phenotyping increase when the test results are used in conjunction with the consultation of an HIV expert to develop a treatment plan. Therapeutic drug-monitoring may play a greater role in the future, especially with drugs that have a low trough (Cmin) level.
Genotypic analysis involves: 1) amplication of the reverse transcriptase (RT) gene, protease (Pr) gene, or both by RT PCR; 2) DNA sequencing of amplicons generated for the dominant species (mutations are limited to those present in >20% of plasma virions); 3) reporting of mutations for each gene using a letter-number-letter standard, in which the first letter indicates the amino acid at the designated codon with wild type virus, the number is the codon, and the second letter indicates the amino acid substituted in the mutation (See HIV101, page 5 for a list of amino acid single letter abbreviations). Updated information on resistance testing can be obtained at http://hiv-web.lanl.gov. Genotypic assays include GeneSeq (Virologic), Truegene (Visible Genetics) and GenoSURE (LabCorp).
Phenotypic analysis involves insertion of the RT and protease genes from the patient's strain into a backbone laboratory clone by cloning or recombination. Replication is monitored at various drug concentrations and compared to a reference wild type virus. This assay is comparable to conventional in vitro tests of antimicrobial sensitivity, in which the microbe is grown in serial dilutions of antiviral agents. Results are reported as the IC50 for the test strain relative to that of a reference or wild type strain. The interpretation was previously based on a fixed ratio such as 4x to define resistance, meaning resistance is four-fold greater than that of the reference strain. The newer method individualizes by drug. For the Virco assay, the fold changes that define resistance are: zidovudine (AZT) - 4.0, lamivudine (3TC) - 4.5, didanosine (ddI) - 3.5, zalcitabine (ddC) - 3.5, stavudine (d4T) - 3.0, abacavir (ABC) - 3.0, nevirapine (NVP) - 8.0, efavirenz (EFV) - 6.0, indinavir (IDV) - 3.0, ritonavir (RTV) - 3.5, nelfinavir (NFV) - 4.0, saquinavir (SQV) - 2.5, amprenavir (APV) -2.5.
Virtual phenotype is a prediction of the phenotype of the test strain based on genotypic analysis. The mutational pattern of the test strain is compared with results of phenotypic assay results with strains showing similar mutations from a databank of >55,000 HIV isolates.
Phenotypic assays include Phenosense and Phenosense GT (genotype and phenotype in one) (Virologic), Antivirogram and Virtual Phenotype (Virco).
* Speaker's Bureau & Research Support: Bristol-Myers Squibb, Chiron, GlaxoSmithKline, Merck, Roche; Speaker's Bureau: Abbott, Agouron, Gilead
1. Paar D and Altice FL., "Antiretroviral Resistance Testing Here and Now", HEPP News 2000 Sep; 3(9): 1-4.
2. Richman DD, Bozzette S, Morton S, et al. 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago (USA) December 16-19, 2002. Abstract LB-17.
3. Little SJ, Daar ES, et al. "Persistence of Transmitted Drug Resistance among Subjects with Primary HIV Infection not Receiving Antiretroviral Therapy" 9th CROI, Seattle, WA. Feb. 24-28, 2002. Abstract 95.
4. Bennett D, Zaidi I, Heneine W, et al., "Prevalence of Mutations Associated with Antiretroviral Drug Resistance among Recently Diagnosed Persons with HIV, 1998-2000" 9th CROI. Seattle (USA). Feb. 23-28, 2002. Abstract 372-M.
5. Baxter JD, Mayers DL, Wentworth DN, Merigan TC. Antiviral Ther 1999; 4(Suppl): 43.
6. Cohen CJ, Hunt S, Sension S, et al. "A randomized trial assessing the impact of phenotypic resistance testing on antiretroviral therapy", AIDS 2002 Mar 8;16(4):579-88.
7. Piliero PJ. "The utility of inhibitory quotients in determining the relative potency of protease inhibitors", AIDS 2002 Mar 29;16(5):799-800.
8. Shulman N, Zolopa AR, Passaro D. "Phenotypic hypersusceptibility to non-nucleoside reverse transcriptase inhibitors in treatment-experienced HIV-infected patients: impact on virological response to efavirenz-based therapy", AIDS 2001 Jun 15;15(9):1125-32.
9. D'Aquila RT. "Limits of resistance testing", Antivir Ther 2000 Mar;5(1):71-6.
10. Whitcomb JE, Paxinos E, Huang W, et al., "The Presence of Nucleoside Analogue Mutations (NAMs)iIs Highly Correlated with Reduced Susceptibility to all NRTIs", 9th CROI. Seattle (USA). Feb. 23-28, 2002. Abstract 569-T.
11. Parkin NT, Chappey C, Maroldo L, et al., "Incidence and Nature of Phenotype-Genotype Discordance: Maximizing the Utility of Resistance Testing", 9th CROI. Seattle (USA). Feb. 23-28, 2002. Abstract 580-T.
12. Deeks SG. "Durable HIV treatment benefit despite low-level viremia: reassessing definitions of success or failure", JAMA 2001 Jul 11;286(2):224-6.
13. Coakley EP, Doweiko JP, Bellosillo E, et al. "HIV Drug Resistance Profiles and Clinical and Virologic Outcomes among HIV-Infected Subjects with Stable Detectable Plasma Viral Loads < 1000 Copies/mL for at least 12 Months", 9th CROI. Seattle (USA). Feb. 23-28, 2002. Abstract 556-T.
14. Drusano GL, Bilello JA, Preston SL, et al. "Hollow-fiber unit evaluation of a new human immunodeficiency virus type 1 protease inhibitor, BMS-232632, for determination of the linked pharmacodynamic variable", J Infect Dis 2001 Apr 1;183(7):1126-9.
15. Burger DM, Hugen PWH, et al. Program and abstracts of the 2nd Workshop on Clinical Pharmacology of HIV Therapy; April 2-4, 2001; Noordwijk, The Netherlands, Abstract 6.2b
16. Nijhuis M, Deeks S, Boucher C. "Implications of antiretroviral resistance on viral fitness", Curr Opin Infect Dis 2001 Feb;14(1):23-8.
17. From Bartlett JG, Gallant JE. 2001-2002 Medical Management of HIV Infection. Johns Hopkins University, Division of Infectious Diseases: 2001 (Chapter 2).
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©1997,1998,1999,2000,2001,2002. 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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