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1 The Clinical Utility of Pharmacogenetic Tests in HIV Therapy David Boettiger Erasmus MC Rotterdam, Department of Virology The introduction of HAART has had a remarkably positive impact on HIV patient survival. However individual genetic variability can significantly affect patient response to drugs. A number of associations between genotype and antiretroviral response have been identified, none more important than that of HLA-B*5701 and abacavir hypersensitivity. The abacavir example highlights the quality of evidence required for a pharmacogenetic test to be accepted into clinical practice. This review summarizes the current, confirmed pharmacogenetic associations in HIV therapy and provides an assessment of their clinical utility based on the evidence to date. True individualization of antiretroviral treatment remains a distant hope. Nevertheless, with careful consideration of the requirements for a pharmacogenetic test to gain acceptance into clinical practice and advanced scientific techniques we may soon see rapid development in the utilization of pharmacogenetics in HIV therapy. Keywords HIV, HAART, antiretroviral, pharmacogenetics, pharmacogenetic testing, abacavir Introduction Since the introduction of combined drug treatment for HIV in the form of Highly Active Antiretroviral Therapy (HAART) patient survival has improved enormously. When HIV/AIDS was first recognized in the early 1980s it typically killed patients within 1-2 years. Now, newly infected patients in the developed world can generally expect to live at least another 30 years.1 The first drug recognized to be efficacious against HIV was the nucleotide reverse transcriptase inhibitor, zidovudine.2, 3 Unfortunately, monotherapy with zidovudine and lack of experience with HIV led to the rapid emergence of resistance.4 Since then numerous compounds have been added to the nucleotide/side reverse transcriptase (NRTI) drug class and several other antiretroviral classes developed. With a current first-line HAART regimen, typically consisting of two NRTIs and a nonnucleotide reverse transcriptase inhibitor (NNRTI) or protease inhibitor, patients can achieve an undetectable viral load and almost normal CD4 cell level, as well as dramatically slow the emergence of HIV drug resistance. The recent introduction of CCR5 inhibitors and integrase inhibitors has also provided additional second- and third-line treatment options. Current treatment guidelines for HIV are based largely on the efficacy, cost, availability and toxicity of antiretrovirals, as well as the clinical experience with their use.5-7 While these are logical parameters on which to base such guidelines, their ‘generizability’ to individuals is limited. For example, a new patient may reasonably be prescribed an affordable and available first-line antiretroviral, which is known to be effective in 90% of patients and cause serious nephrotoxicity in only 0.5% of users. Whilst an appropriate choice for most, there remains a 10% chance the drug will not be efficacious, and it is impossible to distinguish whether the patient is likely to be one of the unlucky few to suffer drug-induced nephrotoxicity. 2 A range of factors may contribute to variability in antiretroviral response. Age, gender, ethnicity, drug interactions, drug resistance, concomitant disease states, pregnancy and individual genetic variation are all known to influence pharmacodynamics. Pharmacogenetics examines the effect of specific genetic variants on drug response. To date, most pharmacogenetic studies have evaluated associations as a function of variation in the genes encoding proteins important in pharmacokinetics and/or drug toxicity (i.e. transport proteins, metabolizing enzymes, and immune response proteins). Pharmacogenetic research ultimately aims to individualize patient treatment. Pre-treatment screening for genetic variants known to influence the efficacy or toxicity of a drug may lead to more elaborate treatment guidelines that prevent unnecessary side effects or regimen failure. The best example of this in HIV medicine is the pharmacogenetic test for Human Leukocyte Antigen (HLA)B*5701 to avoid abacavir toxicity. Abacavir causes a severe, potentially life-threatening hypersensitivity reaction in a small percentage of users. Based on evidence that certain HLA genotypes were more prevalent in these patients,8-10 various authors hypothesized (and later confirmed) that genotypic testing could prospectively identify those most likely to succumb to abacavir hypersensitivity.11-14 Despite the large number of approved antiretrovirals available, abacavir provides the only current example of a clinically useful pharmacogenetic test in HIV treatment. Other clear genotypephenotype links exist, however research must prove a genetic test is clinically relevant, affordable, convenient and acceptable to patients and healthcare workers before it will be implemented into clinical practice. This review summarizes the confirmed, clinically important and potentially important pharmacogenetic associations identified by HIV research to date, and assesses their clinical utility based on the current evidence. ‘Confirmed’ associations are defined here as those involving a particular gene, single nucleotide polymorphism, allele or haplotype with at least two, independent supporting studies and minimal or no conflicting evidence. ‘Clinically important and potentially important’ associations are defined here as those known or suspected to be related to significant improvement or inhibition of drug efficacy, or significant increase or decrease in the likelihood of an adverse event, in a large number of patients. For a thorough list of recognized, suspected and controversial associations the reader is directed to the HIV Pharmacogenomics website (www.hivpharmacogenomics.org).15 Search Strategy PubMed and Embase were searched for relevant literature. Appendix 1 is a list of the keywords used in this search. Associations were only considered in detail if current evidence indicated they were of clinical significance or likely to be of clinical significance. If a reported association was not supported by other independent pharmacogenetic or general population data, or there existed any more than minor contrary evidence of the association, it was not considered in detail. Included studies needed to evaluate a pharmacogenetic association in HIV positive patients using at least one antiretroviral drug. Studies were required to evaluate at least one pharmacogenetic association involving a specific gene but not necessarily a specific mutation in that gene. All study designs were considered for inclusion. Review articles, articles not written in English, studies in non-human subjects and in vitro experiments were excluded. 3 The reference lists of articles selected from the abovementioned procedure were individually screened for additional studies of relevance. The HIV pharmacogenetics website’s list of pharmacogenetic associations was also searched for studies not already captured.15 Introducing a Pharmacogenetic Test into Clinical Practice Box 1 details the requirements of a pharmacogenetic test before it will be incorporated into practice. Importantly, evidence and opinion must clearly favor the clinical relevance of such a test before cost effectiveness is evaluated, a robust assay and reporting system developed, patient and health care worker education initiated, and finally, recommendation given by clinical guidelines. CLINICALLY RELEVANT High quality data must show a clear pharmacogenetic association and that patients, preferably from a wide range of ethnic backgrounds, will gain significant benefit from a test based on this association. COST EFFECTIVE The cost of testing must be outweighed by the benefit it will provide. ROBUST ASSAY The test assay must exhibit acceptable sensitivity and specificity. QUICK SIMPLE REPORT The test must generate an easily interpretable report in a timely manner. SUFFICIENT EDUCATION Patients and health care professionals need to be aware of testing implications and limitations. GUIDELINE RECOMMENDATION Guideline recommendation will ensure the test is incorporated into local protocol. Box 1. Requirements for introduction of a pharmacogenetic test into clinical practice Nucleoside/Nucleotide Reverse Transcriptase Inhibitors NRTIs are structurally similar to natural nucleoside/nucleotides used in nucleic acid synthesis only they lack a 3’ hydroxyl group. After phosphorylation NRTIs are incorporated by HIV reverse transcriptase into viral DNA and effectively terminate DNA elongation as 5’ to 3’ phosphodiester linkage can no longer occur. Hepatic glucuronidation is thought the main metabolic pathway of zidovudine, while lamivudine and tenofovir are renally excreted virtually unchanged. Abacavir is predominantly metabolized in the liver by alcohol dehydrogenase and glucoronidation. Two members of the ATP-binding cassette super family, multidrug resistance protein (MRP) 4 and breast cancer related protein, are important NRTI transport proteins that regulate intracellular drug concentration. The immune response protein, HLA, plays a strong role in the hypersensitivity reaction occasionally seen with abacavir use. 4 Abacavir hypersensitivity (HLA-B*5701 & HCP5 T335G) Abacavir is a well-tolerated antiretroviral that appears to exhibit similar or slightly inferior efficacy compared to more frequently used NRTIs such as tenofovir and emtricitabine.16-19 The main treatment limiting toxicity associated with abacavir is a hypersensitivity reaction that typically occurs during the first 6 weeks of treatment in approximately 5% of patients.20 It is characterized by nonspecific symptoms, such as fever, rash, and gastrointestinal and respiratory manifestations, which make clinical diagnosis difficult.20 Symptoms usually subside within 24 hours of ceasing the drug however re-challenge is contra-indicated as it can result in severe morbidity or death.21 The recognition that abacavir hypersensitivity reaction (AHR) occurs in few individuals shortly after initiating treatment and that those who do not develop the reaction remain at low risk despite continued therapy led researchers to believe genetic susceptibility may be involved. Early work supported this theory by demonstrating a familial connection to AHR,22 as well as an association with Caucasians, but not blacks.10 This was followed by two further studies independently establishing an association between AHR and the major histocompatibility complex class I allele, HLA-B*5701.8, 9 Although the precise mechanism remains unknown, biological evidence that AHR is mediated by HLAB*5701 restricted CD8+ lymphocytes came from cellular and ex vivo studies showing strong tumour necrosis factor-α and interferon-γ responses, and CD8+ proliferation after ex vivo exposure to abacavir.23, 24 The increasing body of evidence supporting the relationship between HLA-B*5701 and AHR encouraged observational research assessing the utility of HLA-B*5701 screening in clinical practice.11, 14 The positive results gleamed from this work necessitated a randomized trial. The PREDICT-1 study was a randomized, double-blind, controlled trial that enrolled 1956 HIV infected patients of various (mainly Caucasian) ethnic backgrounds.13 Both HLA-B*5701 negative and unscreened (control) patients were treated with an abacavir regimen. To confirm a diagnosis of AHR, patients were exposed to an epicutaneous patch test. Screening prevented all immunologically confirmed AHR (0% screened group vs. 2.7% control group), and significantly reduced clinically diagnosed cases (3.4% screened group vs. 7.8% control group). The authors therefore concluded that a pharmacogenetic test for HLA-B*5701 prevents AHR. Further research confirmed the feasibility of introducing HLA-B*5701 screening into clinical practice. The results from Saag et al (2008)12 showed that HLA-B*5701 screening is a sensitive and specific marker for immunologically confirmed AHR in black and white populations, despite observing only a very small number of reactions in black patients (n=5). Hammond et al (2007)25 demonstrated that sequence specific PCR amplification could provide similarly accurate results to the more expensive and laborious full high resolution HLA typing used to prove the clinical relevance of HLA-B*5701 screening. Two studies have indicated the cost-effectiveness of HLA-B*5701 testing.26, 27 The latest of these applied data from PREDICT-1 to simulate a cohort of patients receiving HIV therapy.26 This allowed the authors to perform a cost-effectiveness assessment of HLA-B*5701 testing for guiding selection of first-line HIV regimens in the United States. First-line abacavir, lamivudine, and efavirenz without prior HLAB*5701 testing was compared to the same regimen with prior HLA-B*5701 testing and a 5 first-line regimen of tenofovir, emtricitabine and efavirenz. Abacavir-based treatment with HLAB*5701 testing was estimated to cost $36,700 per quality adjusted life year (QALY) gained when compared to no testing, below the commonly accepted thresholds in the United States of $50,000$100,000/QALY.26 Initiating treatment with a tenofovir-based regimen increased costs without improving QALYs. Interestingly, especially given recent data indicating abacavir-based HAART exhibits inferior efficacy to tenofovir-based therapy16, 17, HLA-B*5701 testing remained the preferred strategy only if abacavir-based treatment had equal efficacy and cost less per month than tenofovir-based treatment. Results were also sensitive to the cost of HLA-B*5701 testing and the prevalence of HLA*B5701. Since 2008, US and UK guidelines have indicated that abacavir should not be used without prior HLAB*5701 screening and should be avoided in patients that test positive.6, 28 Importantly, both guidelines warn that HLA-B*5701 negative patients may rarely experience AHR and hence it remains appropriate to monitor for signs of abacavir hypersensitivity after genetic testing. The most recent World Health Organization guidelines on the other hand refrain from commenting on HLA-B*5701 testing, instead stating that abacavir adds additional cost and complexity that adds serious constraint to its use in first-line regimens and prevents its recommendation as a preferred second-line agent.5 This highlights an important weakness of pharmacogenetic testing. Despite the clear benefit of HLAB*5701 testing, it adds an additional requirement to prescribing. Combined with data from randomized controlled trials indicating the inferior efficacy16, 17 and cardiovascular safety19 of abacavir compared to other NRTIs, it makes for an unattractive option to physicians. Interestingly, new research into AHR has discovered that the HLA complex P5 (HCP5) single nucleotide polymorphism, T335G, is an accurate surrogate marker of HLA-B*5701.29, 30 When compared with HLA-B*5701 testing in a study of 245 HIV infected patients, HCP5 testing showed a 93% positive predictive value and 100% negative predictive value.30 As genotyping HCP5 T335G is cheaper and easier than HLA-B*5701 testing, further confirmation of the sensitivity and specificity of this assay may prove it to be a good alternative for predicting AHR. Tenofovir-associated renal tubulopathy (MRP2) Tenofovir exhibits strong antiretroviral efficacy, a good safety profile and a long half-life that permits once-daily dosing. Unsurprisingly therefore, it is a very popular drug in first-line HIV treatment. Although randomized controlled trials in HIV infected patients indicated tenofovir exhibits good renal safety,31-34 rare cases of renal tubular dysfunction have been reported in tenofovir users which raises concern about long-term use of the drug.35-41 Tenofovir undergoes extensive renal clearance by a combination of glomerular filtration and active tubular secretion. It is taken up at the proximal tubule basolateral membrane by human organic anion transporter 1 and transported into the urine by MRP4, and possibly MRP2, mediated cellular efflux at the apical membrane.42 The mechanism underlying tenofovir-associated renal tubulopathy remains undetermined however it could be that dysfunctional transport protein function leads to tenofovir accumulation in renal proximal tubular cells and subsequent toxicity. Retrospective analysis has shown little association between genetic variants of human organic anion transporter 1 and tenofovir pharmacokinetics suggesting they do not contribute to tenofovir- 6 associated renal toxicity.43 On the other hand, a small case-control study by Izzedine et al (2006)44 found that the G1249A single nucleotide polymorphism and four polymorphism haplotype, CATC, of the ABCC2 gene (encoding MRP2) are both associated with tenofovir-induced renal tubulopathy. In support of this finding, a recent study by Rodriguez-Novoa et al (2009)45 found that in a population of tenofovir treated HIV patients (n=115), the percentage of patients with tenofovir-associated renal tubulopathy (n=19) was higher among those with genotype CC at position 24 of ABCC2 than among those with genotypes CT or TT (24% vs. 6%). This preliminary work suggests a pharmacogenetic test involving the ABCC2 gene may be useful for preventing tenofovir use in those susceptible to developing renal tubulopathy. However more convincing research is required to confirm this association, define a specific variant of the ABCC2 gene involved in this association, and to elucidate the likely mechanism/s involved. NRTI-associated pancreatitis (CFTR & SPINK-1) Pancreatitis is a known adverse event of NRTIs, particularly didanosine.46 Cystic fibrosis transmembrane regulator (CFTR) and serine protease inhibitor kazal-1 (SPINK-1) variants have been associated with chronic and idiopathic pancreatitis in the general population.47-49 The mechanism/s by which CFTR mutation may lead to pancreatitis has not been established. SPINK-1 is a potent protease inhibitor which might protect against pancreatitis by preventing activation of the pancreatic digestive enzyme cascade.50 Polymorphisms in SPINK-1 could therefore inhibit this function. Felley et al (2004)51 conducted a study in HIV infected patients to compare the frequency of various CFTR mutations and the SPINK-1 polymorphism, N34S, in patients with hyperamylasemia and matched controls. Ten patients were diagnosed with acute pancreatitis, four of whom had CFTR mutations or the SPINK-1 polymorphism (40%) compared with 9 of 90 controls or asymptomatic hyperamylasemia patients (10%). This study indicated that CFTR mutations and the N34S polymorphism in SPINK-1 may further increase susceptibility to pancreatitis in HIV patients treated with NRTIs. Currently this association is only supported by data from the general population studies mentioned above. Furthermore, much larger, higher quality studies are required involving analysis of CFTR mutations alone, SPINK-1 polymorphism alone, or co-presence of these genetic variants. NRTI-associated peripheral neuropathy (Mitochondrial haplotypes) Stavudine and didanosine exhibit numerous serious safety concerns that limit their clinical use.5 Unfortunately, in many resource poor settings stavudine remains a first-line option due to its availability and affordability. One of the serious toxicities of stavudine and didanosine (and to a lesser extent other NRTIs) is the development of peripheral neuropathy.52 The mechanism by which NRTIs cause peripheral neuropathy and other long-term toxicity (such as lipoatrophy, hepatic steatosis and lactic acidosis) is probably associated with NRTI-induced mitochondrial injury. Although NRTIs predominantly inhibit viral nucleic acid synthesis, they have also been shown to inhibit mitochondrial DNA polymerase.53, 54 Mitochondrial DNA depletion and subsequent mitochondrial dysfunction leads to impaired oxidative phosphorylation and tissue injury.55, 56 Similarities between the clinical manifestations of inherited mitochondrial DNA diseases and the long-term adverse effects of NRTIs prompted researchers to look for mitochondrial DNA variants that may explain susceptibility to peripheral neuropathy among HIV infected patients. 7 Combinations of polymorphisms define mitochondrial haplogroups which are found with varying frequency in different continental populations. Approximately 15% of European descendants express mitochondrial haplogroup T, which is distinguished by a signature pattern of three polymorphisms, C7028T, G10398A, and G13368A.57 A 2005 case-control study by Hulgan et al found that among 137 white subjects that received didanosine plus stavudine, 20.8% of those who developed peripheral neuropathy belonged to mitochondrial haplogroup T compared to 4.5% of control subjects.58 The authors followed this up with a similar study in the same population indicating the single nucleotide polymorphism, A4917G, found almost exclusively in haplogroup T, was also associated with NRTIinduced peripheral neuropathy.59 Although only confirming the likely importance of mitochondrial DNA variants rather than any specific haplogroup, a study published this year by Canter et al found that an African subhaplogroup is also implicated in susceptibility to NRTI-associated peripheral neuropathy.60 These preliminary findings suggest a genetic test of mitochondrial DNA prior to NRTI initiation may be able to identify those at risk of peripheral neuropathy. Non-Nucleoside Reverse Transcriptase Inhibitors NNRTIs have a strong affinity for a hydrophobic pocket located close to the catalytic domain of HIV reverse transcriptase. When bound they cause a conformational change in the catalytic site that blocks the enzymes ability to synthesize DNA. Efavirenz is predominantly metabolized by CYP2B6 and to a lesser extent by CYP3A4. Nevirapine is predominantly metabolized by CYP2B6 and CYP3A4 with a minor contribution from CYP3A5. Although some pharmacogenetic studies have implicated the transmembrane efflux drug transporter, MRP1, as an important protein in the pharmacokinetics of NNRTIs, this remains controversial.61, 62 Similar to the case for abacavir, HLA proteins appear to be involved in the hypersensitivity reaction occasionally seen in response to nevirapine. Nevirapine-associated hypersensitivity (HLA-DRB1*0101 & HLA-Cω8) Nevirapine is frequently prescribed as part of first-line HAART and for preventing mother-to-child transmission owing to its good efficacy and affordability. Unfortunately however the NNRTI induces a potentially fatal hypersensitivity reaction characterized by fever, rash and/or hepatotoxicity in approximately 5% of users.63 As for abacavir, hypersensitivity to nevirapine usually presents within the first 6 weeks of treatment and re-challenge after a confirmed reaction is contra-indicated.64 However, nevirapine hypersensitivity seems quite complex as both HLA class I and II alleles have been implicated in different populations65-68 and the reaction may be dependent on CD4+ cell count.64 Nevirapine hypersensitivity has been shown to occur more frequently in treatment naïve women and men with a CD4+ cell count greater than 250 cells/mm3 and 400 cells/mm3, respectively, whilst low CD4+ cell count appears to be protective.64 Further, Martin et al (2005)66 identified an association between nevirapine hepatic or systemic reactions and the HLA-DRB1*0101 allele which was limited to patients with a CD4+ cell count greater than 25% (of total white cell count). In contrast, this study also found the occurrence of nevirapine-associated isolated rash was not associated with CD4+ cell count or HLA-DRB1*0101. A more recent, although poor quality, study based on a French cohort reported that 83% (5 of 6) of participants presenting with isolated rash were HLA-DRB1*0101 positive versus only 7% (1 of 15) in the nevirapine-tolerant group and that this association was not 8 dependent on CD4+ cell count.68 Hence, it appears different mechanisms could be underlying the risk of nevirapine-associated isolated rash and nevirapine related hepatic reactions. While the role of HLA-DRB1*0101 in isolated skin reactions to nevirapine is controversial, HLA-DRB1*0101 testing in conjunction with a CD4+ cell count may be useful for averting nevirapine sensitivity-associated hepatotoxicity. In addition to the abovementioned, the class I HLA allele, HLA-Cω8, has also been implicated in nevirapine hypersensitivity.65, 67 Following a small study showing the association between HLACω8/HLA-B14 haplotype and nevirapine hypersensitivity reaction in a Sardinian population,67 a similar study was undertaken in a Japanese population in whom the HLA-B14 allele is not present. Gatanaga et al (2007)65 reported that the frequency of HLA-Cω8-positive patients in the nevirapinehypersensitive group (n=12) was 42%, which was significantly higher than those of the nevirapinetolerant group (10%, n=29) and the general Japanese population (9–14%). Further supporting data is required to fully determine the clinical significance of this association. Nevirapine-associated hepatitis (MRP1 C3435T) As well as causing an immune-mediated hepatic sensitivity reaction in a small percentage of patients, nevirapine can cause hepatotoxicity in the form of hepatitis and asymptomatic serum transaminase elevations.63 This reaction mostly occurs within the first 12 weeks of therapy.69 Whilst the paucity of knowledge on nevirapine transport in hepatocytes makes it difficult to hypothesize the mechanism by which the drug may cause hepatitis it has been suggested that altered efflux activity in the gut associated with MRP1 variants may influence disposition of nevirapine and/or its metabolites in a way that affects intracellular drug concentrations and toxicity.70 It is uncertain whether nevirapine or efavirenz are substrates for MRP1, however the C3435T polymorphism in MRP1 has been associated with improved virological outcome in patients using efavirenz-containing regimens. While it does not confer an amino acid change, C3435T may reduce MRP1 mRNA stability71 or may be linked to other functional polymorphisms.72 In 2006, two independent studies reported that C3435T in MRP1 reduces the risk of nevirapineassociated hepatotoxicity.70, 73 The larger of these studies (n=161) used data from a randomized, double-blind trial conducted in South Africa and found that genotypes at MRP1 position 3435 in case patients and controls were CC in 83% and 67%, CT in 13% and 28%, and TT in 4% and 6%, respectively.70 Encouragingly, a recently published case-control study in a Mozambique population concurs with these findings.74 Additional supporting literature is required to determine the clinical utility of MRP1 C3435T testing for predicting patients (un)likely to develop nevirapine-associated hepatitis. Efavirenz and increased plasma levels (CYP2B6 G516T) Efavirenz exhibits comparable efficacy to nevirapine and is frequently used in first-line HAART despite its higher cost. It is associated with central nervous system side effects in as many as 50% of users75. While the milder, more common adverse effects such as dizziness, insomnia, somnolence and impaired concentration often resolve without intervention within the first 12 weeks of therapy, 76 more severe central toxicities such as psychosis and depression have been reported.77-80 Various authors have indicated that adverse effects of efavirenz on the central nervous system frequently 9 correspond with high drug plasma concentrations.81-84 Efavirenz also exhibits a long half-life and low genetic barrier to resistance,85, 86 hence high initial plasma levels could effectively lead to extended monotherapy and rapid development of HIV drug resistance after ceasing a typical 2xNRTI + efavirenz regimen.82 Dysfunctional CYP2B6, the main metabolizing enzyme of efavirenz, leads to reduced hepatic clearance of the NNRTI.87 Studies in populations of various ages and ethnicities have repeatedly shown CYP2B6 G516T to be associated with increased plasma efavirenz levels.85, 86, 88-98 Moreover, elevated plasma efavirenz concentrations in patients expressing the CYP2B6 G516T genotype have been associated with central nervous system side effects.80, 93, 99-101 Studies showing a relationship between CYP2B6 G516T and the development of efavirenz resistance are currently lacking, however using clinical trial data Ribaudo et al (2006)86 predicted that plasma efavirenz levels would remain elevated for >21 days in 29% of subjects with the TT genotype. Taken together, the abovementioned studies imply pharmacogenetic testing of CYP2B6 may help guide optimal efavirenz dosing. In fact, a recent study out of Japan by Gatanaga et al (2007)102 found that dose reduction in CYP2B6 C516T carriers resulted in improvement of central nervous system side effects for 10 of 14 patients, and that a lower starting dose of 400mg could be successfully initiated in efavirenz-naïve carriers. Despite this, a recent Cochrane review concluded that current data does not support routine use of therapeutic drug monitoring in patients receiving HAART citing generally poor uptake of recommendations as one of the likely reasons for this outcome.103 A key limitation to CYP2B6 G516T pharmacogenetic testing is that the mutation does not lead to increased efavirenz plasma concentrations in all carriers.90, 93, 100 It has also been suggested that providing G516T heterozygous patients with a lower dose of efavirenz may increase rates of virological failure possibly due to the slow-metabolizer phenotype protecting against poor adherence.101 Current research has identified genetic variants of proteins such as MRP1, CYP2A6, CYP3A4 and uridine diphosphate glucuronosyl-transferase (UGT) 2B7 that can account for some of the variability in efavirenz plasma concentrations not explained by CYP2B6 mutation.89, 104-107 Formulation of a predictive algorithm that combines the effects of some or all known factors influencing efavirenz pharmacokinetics could be used to individualize dosing, however more work is required in this area. Protease Inhibitors The cleavage of precursor proteins by protease is essential for assembly of infectious HIV particles. Inhibition of protease results in the release of structurally disorganized and non-infectious virus. Protease inhibitors mimic the structure of precursor protein cleavage sites and hence bind and inhibit the catalytic domain of protease. They act as both substrates and inhibitors of the CYP3A enzymes. Commonly, protease inhibitors are co-administered with low-dose ritonavir, a protease inhibitor that potently antagonizes the CYP3A sub-family, to enhance the bioavailability of the primary protease inhibitor. They are substrates for MRP1 and bind heavily to the plasma proteins, albumin and α1-acid glycoprotein. Several proteins, especially various apolipoprotein subtypes, are believed to play a role in the dyslipidemia and insulin resistance known to be associated with protease inhibitor use. 10 Atazanavir- & indinavir-associated hyperbilirubinemia (UGT1A1*28) Atazanavir is one of the preferred ritinovir-boosted protease inhibitors (along with boosted lopinavir) recommended by the WHO due to its superior safety, affordability and ease of administration compared to other protease inhibitors.5 Compared with atazanavir, indinavir exhibits similar efficacy, is not as well tolerated and is more expensive.5 Patients receiving atazanavir or indinavir have an increased risk of developing unconjugated hyperbilirubinemia. Although reversible and benign, some patients may develop overt jaundice which can lead to them discontinuing antiretroviral treatment. The mechanism behind this reaction is probably due to indinavir and/or atazanavir competitively inhibiting the conjugation of bilirubin by UGT1A1.108, 109 Reduced UGT1A1 activity caused by mutation in the promoter region of the enzyme (UGT1A1*28) causes Gilberts syndrome, a common condition characterized by elevated bilirubin. Numerous studies in patients of Caucasian, African and Asian decent have shown a clear association between the UGT1A1*28 allele and atazanavir and/or indinavir induced hyperbilirubinemia.110-114 In the one study that looked at the effect of both protease inhibitors in HIV patients, atazanavir increased average bilirubin levels by 15mmol/L and indinavir increased average bilirubin levels by 8mmol/L.114 UGT1A1*28 homozygosity increased bilirubin levels by an average of 5.2mmol/L. When combined, 67% of individuals homozygous for UGT1A1*28 and receiving atazanavir or indinavir had ≥2 episodes of hyperbilirubinemia in the jaundice range versus 7% of those with the wild-type allele and not receiving either drug. In addition to the UGT1A1*28 allele, MRP1 variants appear to increase atazanavir plasma levels (probably due to reduced clearance) and the subsequent risk of developing hyperbilirubinemia.112, 115 Combined genotyping of UGT1A1 and MRP1 prior to atazanavir or indinavir administration may identify patients most at risk of developing hyperbilirubinemia. Although it is one of the stronger pharmacogenetic associations in HIV medicine, the benign nature of hyperbilirubinemia may limit the clinical acceptability of a UGT1A1 ± MRP1 test prior to atazanavir or indinavir treatment. Protease inhibitor-associated hypertriglyceridemia (ABCA1, APOA5, APOC3, APOE) Protease inhibitors and other antiretroviral classes are associated with an increased risk of adverse cardiovascular outcome, mediated at least in part by dyslipidemia.116-118 Antiretroviral-mediated dyslipidemia may include hypertriglyceridemia, decreased HDL cholesterol, and/or increased LDL cholesterol.119 Hypertriglyceridemia associated with HIV drug therapy most frequently occurs with regimens containing ritonavir.117, 120 The mechanism by which protease inhibitors may lead to hypertriglyceridemia is not known. However, the fact that not all patients develop hypertriglyceridemia, despite similar protease inhibitor exposure and comparable demographic, immunologic, and virologic characteristics suggests involvement of a genetic component. Polymorphisms in genes known to be associated with dyslipidemia in the general population have been studied to elucidate their effect on lipid regulation in HIV infected patients. Numerous authors have established that there is an association between various apolipoprotein C3 (APOC3) and apolipoprotein E (APOE) genotypes and hypertriglyceridemia in patients using a ritonavir-containing regimen.119, 121-125 In a study of 329 antiretroviral treated, HIV infected patients, Tarr et al (2005)121 found that patients using ritonavir who expressed an unfavorable genotype for both APOC3 and 11 APOE had a median plasma triglyceride level of 7.33mmol/L, compared with 3.08mmol/L in the absence of antiretroviral therapy. Studies have also confirmed an association between genetic variants of ATP-binding cassette subfamily A (ABCA1) and apolipoprotein A5 (APOA5), and protease inhibitor-associated hypertriglyceridemia.119, 125 Arnedo et al (2007)119 incorporated these genotypes into a scoring algorithm for evaluating likely susceptibility to antiretroviral induced dyslipidemia. Patients received a predictive score based on their composite ABCA1/APOA5/APOC3/APOE/cholesterol ester transferase protein genotype and the type of antiretroviral therapy they were using (cholesterol ester transferase protein variants have been implicated in antiretroviral dyslipidemia though this association has not been confirmed)119. Although this algorithm is yet to be validated in other study populations, it illustrates how clinicians may someday identify individuals at increased risk of antiretroviral-related hypertriglyceridemia or dyslipidemia and thereby reduce the risk of atherosclerotic disease in HIV infected patients. Assessing the Clinical Utility of Pharmacogenetic Associations in HIV Therapy A growing interest in pharmacogenetics and the potential for individualized therapy has led to the discovery of a vast number of genotype-drug response associations in HIV therapy. While their unveiling is of great intrigue to researchers, clinician and patient interest lies primarily with the potential for pharmacogenetic testing to improve patient outcome. Table 2 assesses the clinical utility of the confirmed associations discussed above based on the criteria outlined in Box 1. The assessment of clinical relevance is split into parts, similar to previous evaluations of pharmacogenetic testing (Table 3).126 The first part assumes a routine pharmacogenetic test based on the association is available and provides an evaluation of the strength of evidence to support using this test in practice. The second classifies the clinical significance of the association based on the severity of the condition involved, as it most frequently presents. The evaluation of evidence is based on the WHO 2006 assessment of evidence criteria127 in that each pharmacogenetic test is given a level A (recommended), B (consider), or C (optional) recommendation followed by a score of I,II, III or IV based on the evidence to support this recommendation. A score of I indicates high level evidence including at least one randomized controlled trial with clinical, laboratory or programmatic end-points; II at least one high-quality study or several adequate studies with clinical, laboratory or programmatic end-points; III observational cohort data, one or more case-controlled or analytical studies adequately conducted; and the lowest level of evidence, IV, indicates expert opinion based on evaluation of other evidence. The clinical significance of each association is given a score of 1 if the condition involved is immediately lifethreatening; 2 if it causes irreversible morbidity; 3 if it causes reversible morbidity requiring immediate medical attention; and 4 if it causes reversible morbidity requiring medical attention. Where there exists uncertainty in allocating a score, consideration is also given to the frequency at which the condition occurs. For example, if a condition may reasonably be allocated a score of 2 or 3 but occurs commonly (>10%) it is scored as 2. If the condition occurrs infrequently (<10%) it would receive a score of 3. The remaining columns of Table 2 are the author’s evaluation based on current literature. For a summary of the strengths and limitations of each individual study included in this evaluation, refer to Appendix 2. 12 Drug Class Drug(s) NRTIs Abacavir Tenofovir All NNRTIs Protease Inhibitors Nevirapine Genotype Causal Association Mechanism Clinical Significance Current Status References HLA-B*5701 Increased incidence of abacavir hypersensitivity reaction Unknown but probably an enhanced HLA-mediated immune response to abacavir Genetic testing to prevent use of abacavir in those most susceptible to abacavir hypersensitivity Association supported by a strong body of observational data in racially diverse populations, and one well-conducted randomized trial. Testing recommended by US and UK guidelines since 2008 6, 8, 9, 11-14, 2328 HCP5 T335G Alternative marker for HLAB*5701 screening Linkage with HLA-B*5701 allele Cheaper and easier testing procedure than HLA-B*5701 testing Association is as for HLA-B*5701. Assay requires further confirmation of sensitivity and specificity 29, 30 MRP2 Increased incidence of tenofovir-induced renal tubulopathy Undetermined but possibly modulation of MRP2 function leading to impaired tenofovir elimination and subsequent toxicity Genetic test to indicate those not suitable for tenofovir therapy Requires further verification of clinical relevance. Association supported by limited observational data. Specific variant of the ABCC2 gene involved in this association needs to be confirmed 44, 45 CFTR & SPINK-1 Increased incidence of pancreatitis in the general population Pancreatitis is an adverse effect of NRTIs thus CFTR and SPINK-1 mutations may further increase risk Genetic test to isolate those more susceptible to pancreatitis Requires further verification of clinical relevance. Association only supported by one case-control study and data from the general population. Evidence in HIV patients includes a combined SPINK-1/CFTR endpoint 47-49, 51 Mitochondrial haplogroups Increased susceptibility to NRTI-associated peripheral neuropathy Certain haplotypes may be more susceptible to NRTI induced mitochondrial depletion/dysfunction Genetic test of mitochondrial haplogroup to identify those at risk of peripheral neuropathy with NRTI use Requires further verification of clinical relevance. Association supported by data generated from two case-control studies. No confirmation of association with a particular haplogroup in different populations 58-60 HLACw8 HLA-DRB1*0101 Increased incidence of hypersensitivity reaction Unknown but probably an enhanced HLA-mediated immune response to nevirapine Genetic testing to prevent use of nevirapine in those susceptible to nevirapine hypersensitivity Requires further verification of clinical relevance. Each allele association supported by limited observational data. HLA-DRB1*0101 association complicated by involvement of CD4+ cell count HLACw8 - 65, 67 HLA-DRB1*0101 - 66, 68 MRP1 C3435T Decreased risk of hepatotoxicity Unknown but possibly altered MRP1 efflux of nevirapine in the gut Genetic test may be able to predict patients most (un)likely to develop nevirapine associated hepatotoxicity Requires further verification of clinical relevance. Association supported by data from three case-control studies undertaken in various ethnicities 70, 73, 74 Efavirenz CYP2B6 G516T Increased plasma levels and central nervous system toxicity Impaired hepatic metabolism of efavirenz Genetic test to guide appropriate dosing Strong association supported by a large volume of observational data generated from populations of various ages and ethnicities. Other less significant genetic markers require verification of clinical relevance to aid creation of a pharmacogenetic algorithm 80, 85, 86, 88102, 104-107 Atazanavir & Indinavir UGT1A1*28 MRP1 variants Increased risk of hyperbilirubinemia Impaired UGT1A1 bilirubin conjugation exaggerates the dosedependent increase in bilirubin caused by atazanavir and indinavir. MRP1 mutation probably impairs atazanavir elimination leading to increased bilirubin Unknown UGT1A1*28 genetic test may identify those most at risk of developing hyperbilirubinemia, especially if combined with MRP1 testing Strong association (for UGT1A1*28) supported by numerous observational studies in a range of ethnically diverse populations. Clinical acceptability of a genetic test may be low because of the benign nature of hyperbilirubinemia UGT1A1*28 +/MRP1 variants 110-114 All ABCA1, APOA5, APOC3, APOE Increased likelihood of protease inhibitor associated hypertriglyceridemia MRP1 C3435T 115 Genetic test incorporating relevant risk alleles to form a genetic score may predict those at risk of hypertriglyceridemia Strong association supported by a number of well-conducted observational studies. Further verification of clinical relevance is made difficult by the complexity of factors that affect lipid levels Table 1. Summary of clinically important or potentially important, confirmed pharmacogenetic associations in HIV therapy 119, 121-125 13 Despite the success of introducing genetic testing for abacavir, some doubt remains regarding the cost-effectiveness of routine HLA-B*5701 testing due to the inferior efficacy of abacavir-containing regimens. Nevirapine hypersensitivity and NRTI-associated peripheral neuropathy are clinically significant conditions however evidence supporting predictive pharmacogenetic testing for these and all other candidate tests discussed is currently lacking. The associations between efavirenz plasma levels and CYP2B6 G516T, and protease inhibitor-associated hypertriglyceridemia and unfavorable ABCA1, APOA5, APOC3 and APOE genotypes are supported by large volumes of literature but exhibit complexity that may limit their utility for pharmacogenetic testing. The connection between atazanavir-/indinavir- associated hyperbilirubinemia and the UGT1A1*28 genotype is also well supported by the literature however the clinical significance of hyperbilirubinemia is relatively low. LEVELS OF EVIDENCE CLINICAL SIGNIFICANCE Recommendation Supporting Evidence A (recommended) I - high level evidence including at least one randomized controlled trial with clinical, laboratory or programmatic end-points 1 – Immediately life threatening B (consider) II- at least one high-quality study or several adequate studies with clinical, laboratory or programmatic end-points 2 – Causes irreversible morbidity C (optional) III - observational cohort data, one or more case-controlled or analytical studies adequately conducted 3 – Causes reversible morbidity requiring urgent medical attention IV - indicates expert opinion based on evaluation of other evidence 4 - Causes reversible morbidity requiring medical attention Table 3. Assessment of clinical relevance. Levels of evidence are based on the WHO 2006 assessment of evidence criteria.127 Clinical significance evaluates the seriousness of the condition involved in the pharmacogenetic association. Future of HIV Pharmacogenetics Confirmed pharmacogenetic associations in HIV medicine have only been established for NRTIs, NNRTIs and protease inhibitors. Numerous unconfirmed and insignificant associations for these antiretroviral classes have been reported,15 and some early work has led to insignificant pharmacogenetic findings for the new compounds, raltegravir128 and maraviroc.129 Future research is likely to reveal numerous, as yet undiscovered pharmacogenetic associations for both old and new antiretrovirals. This review has summarized the literature on HLA-B*5701 genetic testing to prevent abacavir hypersensitivity and other promising pharmacogenetic associations in HIV therapy. An assessment of the clinical utility of these associations revealed that, except in the case of abacavir, their clinical relevance remains to be established. Notably, this evaluation also casts doubt over the cost-effectiveness of HLA-B*5701 testing for abacavir. Some of the associations discussed appear too complex for genetic testing in clinical practice. However evidence-based algorithms can accommodate the multifactorial nature of drug responses. Such an approach has already been attempted for antiretroviral-associated dyslipidemia119 and evidence suggests it may also be useful for predicting efavirenz toxicity.89, 104-107 HIV pharmacogenetics must overcome various hurdles before genetic individualization of HAART therapy becomes realistic. Many published studies in this field are underpowered and of low quality (see appendix 2). Future work should evaluate associations most likely to be clinically relevant (see box 2) in large, ethnically diverse populations.130, 131 Results should then be validated in independent patient populations. Attention also needs to be given to ensuring studies are directed towards 14 Clinically Relevant Cost Effective A Robust Assay Quick Simple Report Sufficient Education Guideline recom. Comment Level of evidence Clinical Significance Abacavir hypersensitivity (HLA-B*5701) AI 1 ? √ √ √ √ Successful introduction to clinical practice. Recent data indicating the inferior efficacy of abacavir compared to other NRTIs places doubt over the cost-effectiveness of HLA-B*5701 testing. Abacavir hypersensitivity (HCP5 T335G as a marker for HLAB*5701) AI 1 ? ? X X X May serve as a cheaper, easier alternative to HLA-B*5701 testing pending further insight into assay specificity and sensitivity. Tenofovir-induced renal tubulopathy (MRP2) CIII 3 ? X X X X Insufficient evidence to confirm clinical relevance. NRTI-associated pancreatitis (CFTR & SPINK-1) CIII 3 ? X X X X Insufficient evidence to confirm clinical relevance. NRTI-associated peripheral neuropathy (Mitochondrial haplogroups) CIII 2 ? X X X X Insufficient evidence to confirm clinical relevance. Nevirapine hypersensitivity (HLA-DRB1*0101 & HLA-Cω8) CIII 1 ? X X X X Insufficient evidence to confirm clinical relevance. Hepatic reaction association to HLA-DRB1*0101 allele complicated by association of CD4+ cell count. Nevirapine-associated hepatitis (MRP1 C3435T) CIII 3 ? X X X X Insufficient evidence to confirm clinical relevance. Efavirenz and increase plasma levels (CYP2B6 G516T) CII 3 ? X X X X Significant evidence to support association. An algorithm to account for major sources of inter-individual variability may be useful. Atazanavir- and indinavirassociated hyperbilirubinemia (UGT1A1*28 & MRP1 variants) CII 4 ? X X X X Strong association in the case of UGT1A1*28 however benign nature of hyperbilirubinemia may limit clinical acceptability. Combined UGT1A1*28/MRP1 genetic testing may be more predictive. Protease inhibitor-associated hypertriglyceridemia (ABCA1, APOA5, APOC3, APOE) CII 3 ? X X X X Significant supportive evidence of association. An algorithm may be useful to account for complexity of genetic and non-genetic factors involved in lipid regulation. Table 2. Assessment of the clinical utility of pharmacogenetic associations in HIV therapy. Clinical relevance is assessed by the WHO 2006 levels of evidence criteria and clinical significance as outlined in Table 2. The remaining columns are allocations made by the author based on current literature. √ = Yes/Available, ? = Uncertain/Unknown, X = No/Not available. 15 acquiring guideline recommendation by generating clinically relevant information such as diagnostic test criteria (eg. sensitivity and specificity) and cost-effectiveness figures. With high quality, clinically applicable data, influential groups such as regulatory agencies and the pharmaceutical industry are far more likely to become involved in educating, encouraging and enforcing the use of a pharmacogenetic test.131, 132 It must also be demonstrated that the pharmacogenetic test does actually improve clinical outcome outside the controlled settings of a randomized trial. Positive follow-up data on the incidence of abacavir hypersensitivity reactions since the introduction of guideline recommendations for HLA-B*5701 testing will almost certainly increase physician compliance with this recommendation and is also likely to improve the acceptance of pharmacogenetic testing in HIV medicine. DRUG CHARACTERISTICS Narrow therapeutic index Difficulty predicting response or adverse event Large interindividual variability in response Box 2. Characteristics of drugs most likely to benefit from a pharmacogenetic test. Adapted from Swen et al (2007)131 Consistent pharmacokinetic-pharmacodynamic relationship Long-term treatment The future of HIV pharmacogenetics extends beyond the prospective identification of those susceptible to a specific antiretroviral drug response. Kaslow et al (2001)133 reported that the HLAB*27 and B*57 alleles were associated with a favorable immune response to an experimental HIV vaccine. Cressy & Lallemant (2010)134 attempted to identify genetic factors that lead to prolonged nevirapine exposure and hence rapid selection of resistant virus in women given the antiretroviral for prevention of mother-to-child HIV transmission. Genetic testing could also become useful for identifying patients most likely to succeed on HAART or more importantly, a particular HAART regimen. 85, 110, 135-148 The current shift from isolated analyses of specific genetic variants to genome wide association studies should enhance our understanding of the complex genetic mechanisms underlying variability in drug response. Furthermore, numerous new potential targets for pharmacogenetic research may be revealed. More affordable methods of individual patient testing will also be important, particularly for HIV pharmacogenetics. While cost-effectiveness of a pharmacogenetic test is important for its implementation into healthcare systems of the developed world, it is absolutely essential if such methods are ever to become accessible to the developing world where the demand for antiretroviral therapy is greatest. 16 Appendix 1 – Keyword Search Databases searched: PubMed and Embase Last search: 8 August 2010 Search variables: #1 'HIV' OR 'human immunodeficiency virus' OR 'AIDS' OR 'acquired immunodeficiency syndrome' #2 'ARV' OR 'antiretroviral' OR 'HAART' OR 'highly active antiretroviral therapy' OR 'ART' OR 'antiretroviral therapy' OR 'NRTI' OR 'nucleoside reverse transcriptase inhibitor' OR 'nucleotide reverse transcriptase inhibitor' 'NNRTI' OR 'non-nucleoside reverse transcriptase inhibitor' OR 'PI' OR 'protease inhibitor' OR 'fusion inhibitor' OR 'integrase inhibitor' OR 'CCR5' OR 'cellular coreceptor 5' OR 'lamivudine' OR 'abacavir' OR 'azidothymidine' OR 'AZT' OR 'stavudine' OR 'didanosine' OR 'tenofovir' OR 'emtricitabine' OR 'efavirenz' OR 'nevirapine' OR 'lopinavir' OR 'atazanavir' OR 'indinavir' OR 'nelfinavir' OR 'ritinovir' OR 'saquinavir' #3 'pharmacogenetic' OR 'pharmacogenomic' OR 'genetic' OR 'genetic testing' OR 'HLA-B*5701' #1 and #2 and #3 Appendix 2 – Individual study evaluation Studies describing the associations discussed above were assessed for methodological quality. Studies were evaluated based on the US Preventive Services Task Force Quality Rating Criteria for randomized controlled trials (RCTs), cohort studies, and case control studies.149 Cross sectional studies were evaluated with the criteria for cohort studies without consideration of follow-up. The single non-randomized trial was evaluated with the criteria for RCTs without consideration of randomization. Case reports were not evaluated but are mentioned in the table. The criteria are briefly described: RCTs and cohort studies Adequate randomization (RCT), or consideration of confounders (cohort) Maintenance of comparable groups No important differential loss to follow-up or high rate of loss to follow-up Equal, reliable and valid measurements Clearly defined intervention Important outcome/s considered Intention to treat analysis (RCT), or adequate adjustment for potential confounders (cohort) Good – Meets all criteria. Comparable groups assembled initially and maintained throughout the study (follow-up ≥80%); reliable and valid measurement instruments used and applied equally to both groups; interventions are clearly indicated; important outcomes considered; and appropriate attention to confounders in analysis. Fair – Meets most of the criteria without any of the important limitations mentioned in the “poor” category. Generally comparable groups initially assembled but some question remains regarding 17 differences that occurred in follow-up; measurement instruments acceptable (although not ideal) and generally applied equally; some but not all important outcomes considered; and some but not all potential confounders are accounted for. Poor – If any of the following limitations are present: Groups assembled initially are not comparable or maintained throughout the study; unreliable or invalid measurement instruments used or not applied equally among groups (including not masking outcome assessment); or key confounders are given little attention. Case control studies Accurate ascertainment of cases Non-biased selection of cases and controls Adequate response rate Diagnostic testing procedure applied equally to each group Accurate measure of exposure applied equally to each group Appropriate attention given to potential confounders Good – Appropriate ascertainment of cases and non-biased selection of cases and controls; exclusion criteria applied equally to cases and controls; response rate equal to or greater than 80%; diagnostic procedures and measurements are accurate and applied equally to cases and controls; and appropriate attention to confounders. Fair – Recent, relevant, without major apparent selection or diagnostic work-up bias but with response rate less than 80% or attention to some but not all important confounders. Poor – Major selection or diagnostic work-up biases; response rate <50%; or inattention to confounders. 18 Study Characteristics Drug Class NRTIs Drug(s) Abacavir Genotype Comments on Methodology Grading Ref Fair 8 Well defined outcome. Potential for unblinding of HLA status. Single physician deciding outcome. Good 9 Well defined outcome. Potential for unblinding of HLA status. Good 11 Immunologically confirmed HSR Separate analysis of Blacks and Whites. Immunologically confirmed outcome. Very low number of confirmed outcomes in Black patients (n=5) Good 12 1956 abacavir naive patients from 19 countries Immunologically confirmed HSR Large study population. Immunologically confirmed outcome and clinically diagnosed outcome. No multi-ethnic analyses owing to large white dominance in study group. Good 13 Case control & Cohort 49 French patients previously exposed to abacavir (case control) & 137 French patients due to begin abacavir (cohort) Criteria defined HSR Unspecified diagnostic criteria that appears to exaggerate HSR incidence. Limited consideration of confounders. Time separated and small control group. Poor 14 Martin et al (2004) Case control 200 patients from Mallal et al (2002) and an extra 48 patients from the same clinic Immunologically confirmed HSR Consideration of Hsp70-Hom variant. White patients only. Good 23 Phillips et al (2005) Case control 7 patch test positive patients and 11 abacavir tolerant controls Patch test response Repeat testing to confirm outcome. HLA-B*5701 status not the primary determinant. Small sample size. Fair 24 Colombo et al (2008) Cross sectional 1103 participants in the Swiss HIV Cohort Study HCP5 T335G status Well defined HSR status. Evaluation of discordant patients. Large study population. Potential for unblinding of HLA status. Good 29 Rodriguez-Novoa et al (2010) Cross sectional 245 abacavir naive patients HCP5 T335G status Evaluation of discordant patient. Loosely defined HSR status. Fair 30 Author (year) Design Population Primary Outcome Hetherington et al (2002) Case control 85 patients, 115 controls from the GSK database of clinical trials for abacavir Clinically diagnosed HSR Mallal et al (2002) Nested cohort First 200 patients exposed to abacavir in the Western Australia HIV Cohort Study Criteria defined HSR Rauch et al (2006) Case control 131 cases, 140 controls from the Swiss HIV Cohort study Criteria defined HSR Saag et al (2008) Case control 130 white cases, 202 white controls. 69 black cases, 206 black controls Mallal et al (2008) RCT Zucman et al (2007) HLA-B*5701 HCP5 T335G Confounders well adjusted for. Imperfect matching between cases and controls. Limited analysis of confirmed HSR. 19 Tenofovir All MRP2 CFTR & SPINK1 Izzedine et al (2006) Case control 13 case patients, 17 tenofovir treated controls Tenofovir-induced renal tubulopathy Rodriguez-Novoa et al (2009) Case control 115 consecutive patients presenting at a single clinic Kidney tubular dysfunction Felley et al (2004) Case control 51 patients with hyperamylasemia, 51 matched controls Hulgan et al (2005) Case control Canter et al (2008) Fair 44 Well defined outcome. Large number of confounders considered. Small number of cases (n=19). Good 45 Hyperamylasemia Pancreatitis not the primary outcome. Consideration of a large number of confounders. Low number of pancreatitis cases (n=10). Poor 51 147 patients with any grade of peripheral neuropathy, 362 control patients without peripheral neuropathy Peripheral neuropathy of any grade Long follow-up of cases and controls (3 yrs). No consideration of potentially important confounders such as smoking or diabetes. Exclusion of patients with prior peripheral neuropathy. Fair 58 Case control 70 patients with any grade of peripheral neuropathy, 180 control patients without peripheral neuropathy Peripheral neuropathy of any grade No consideration of potentially important confounders such as smoking or diabetes. Exclusion of patients with prior peripheral neuropathy. White study participants only. Fair 59 Canter et al (2010) Case control 51 non-Hispanic black patients from the AIDS Clinical Trials Group study 384 who had developed peripheral neuropathy, 105 controls from the same trial Peripheral neuropathy of any grade No consideration of potentially important confounders such as smoking or diabetes. Black study participants only. Fair 60 Gatanaga et al (2007) Case control 11 case patients, 29 nevirapine using controls Nevirapine HSR Small study population. Imprecise definition of primary outcome. Patients only of Japanese ethnicity. Fair 65 Littera et al (2006) Case control 13 nevirapine hypersensitive case patients, 36 tolerant controls Nevirapine HSR Small study population. Patients only of Sardinian ethnicity. Exclusion of patients with skin rash or late side effects (>6wks). Confounding variables well accounted for. Good 67 Martin et al (2005) Case control 26 cases of nevirapine HSR from the Western Australia HIV cohort, 209 controls from same study Nevirapine HSR Determinant was interaction between HLA-DRB1*0101 and CD4+ count. Well defined outcome but not verified by another physician and potential for unblinding of HLA status. Fair 66 Vitezica et al (2008) Cohort 21 patients receiving efavirenz or nevirapine from a French cohort Cutaneous drug reaction Combined exposure (efavirenz or nevirapine). Poorly defined outcome. Small number of cases (n=6). Poor 68 Haas et al (2006) Case Control 53 cases and 108 matched controls from the Gilead study protocol FTC-302 Grade 3 or 4 hepatotoxicity Well defined exposure and outcome. Small number of nonblack races included in analysis. Good 70 Mitochondrial haplogroups NNRTIs Nevirapine HLACw8 HLADRB1*0101 Small study population. Prior renal function not considered in analyses. MRP1 C3435T 20 Efavirenz Ritchie et al (2006) Case control 20 case patients using their first NNRTI and 49 matched controls from the Comprehensive Care Center in Nashville, Tennessee. Ciccacci et al (2010) abstract only Case control 78 cases and 78 controls from Mozambique Nevirapine-induced hepatotoxicity Hasse et al (2005) Case report 33yo Thai, female patient using efavirenz Severe psychosis Haas et al (2005) Cohort 504 patients participating in the Adult AIDS Clinical Trials Group study 384 genetic analysis Long-term response to treatment Ribaudo et al (2006) Cross sectional 152 evaluable subjects from the AIDS Clinical Trials Group (ACTG) study Efavirenz pharmacokinetics Saitoh et al (2007) Cohort 71 child patients in Pediatric AIDS Clinical Trials Group 382 (PACTG 382), Efavirenz pharmacokinetics and resistance Selection criteria removes children that may have switched from efavirenz prior to 6mths of therapy. Ethnicities combined in analysis. Motsinger et al (2006) Cohort 304 ACTG 384 participants randomized to receive efavirenz Efavirenz pharmacokinetics and response to treatment Separation of data from different races. Efavirenz plasma levels analyzed as a dichotomous outcome. Good 89 Rodriguez-Novoa et al (2005) Cohort 100 consecutive patients initiating efavirenz at a clinic in Madrid Efavirenz plasma concentration White only participants. Exclusion criteria not appropriate (eg. removed those with poor compliance who may be more likely to exhibit high plasma levels). Most confounders removed by exclusion criteria. Fair 90 Tsuchiya et al (2004) Cross sectional 35 patients on efavirenz being treated at the AIDS clinical entre, IMCJ Efavirenz plasma concentration Japanese only patients. Exclusion criteria not appropriate (eg. removed those with poor compliance who may be more likely to exhibit high plasma levels). Most confounders removed by exclusion criteria. Fair 91 Hepatotoxicity Uncertainty regarding the sourcing of controls. Exposure included nevirapine and efavirenz in analysis. Lose definition of hepatotoxicity. Poor 73 Many uncertainties in methodology owing to limited information available in abstract. In particular with regard to participant selection and outcome measure. Not possible 74 N/A N/A 80 Increased plasma level or CNS symptoms not the primary outcome. Long follow up. Thorough analysis of different races present in study population. Could have included more confounders in analysis (eg. Concomitant medications, smoking status) Fair 85 Good 86 Fair 88 CYP2B6 G516T Model applied to predict likely plasma concentrations rather than actual clinical measurements 21 Cross sectional 163 patients recruited prospectively in a previous study and 6 additional individuals characterized by very high plasma efavirenz levels identified through routine TDM Efavirenz plasma concentration CYP2B6 G516T grouped with other loss of function alleles. Limited consideration of confounders. Poor 92 Cohort 202 patients randomized to receive efavirenz in study A5097s from the Adult AIDS Clinical Trials Group Questionnaire identified CNS side effects Well defined exposure and outcome. Questionable clinical relevance of using a questionnaire. Good 93 Chen et al (2010) Cross sectional 159 patients receiving antiretroviral therapy (70 receiving efavirenz) at Shanghai Public Health Clinical Center Efavirenz plasma concentration No consideration for confounding variables. Poor 94 Gupta et al (2008) Cross sectional 13 hemodialysis patients receiving efavirenz Efavirenz pharmacokinetics CYP2B6 516G>T status not the focus of this study. No consideration of confounders. Very small study population (n=11 with genotypic data available using efavirenz) Poor 95 Leger et al (2009) Cohort 45 Haitian patients receiving efavirenz Efavirenz pharmacokinetics Confounding adjusted for. Results validated in a separate group of African American patients. Good 96 Nyakutira et al (2008) Cross sectional 74 outpatients from clinics in Harare receiving efavirenz Efavirenz pharmacokinetics Sex the only confounder considered. Fair 97 Ramachandran et al (2009) Cross sectional 15 patients receiving efavirenz from an outpatient clinic in India Efavirenz pharmacokinetics No confounder accounted for. No validation of plasma concentrations. Very small study size. Patients may have been on therapy for a min of 1 week. Poor 98 Lowenhaupt et al (2007) Case report 12yo white female Psychosis N/A N/A 99 Rotger et al (2005) Cross sectional (pharmacokinetic study)/Cohort (CNS toxicity study) 167 patients receiving efavirenz in the Swiss HIV Cohort Study Efavirenz pharmacokinetics and CNS toxicity Questionable clinical significance of reporting CNS symptoms by questionnaire. No evaluation of confounders in PK side of study. Not stated when assessment of CNS toxicity performed. Poor 100 Rotger et al (2007) Haas et al (2004) 22 Protease Inhibitors Atazanavir & Indinavir Ribaudo et al (2010) Cross sectional (pharmacokinetic)/Cohort (treatment response) Patients from the AIDS Clinical Trials Group. 317 pharmacokinetics study and 643 for treatment response Efavirenz pharmacokinetics and treatment response Thorough evaluation of confounders. Assessment of CNS toxicity truncated at 24 weeks. Good 101 Gatanaga et al (2007) Non-randomized trial 456 patients receiving or due to receive efavirenz at hospitals in Japan Efavirenz plasma concentration after dose adjustment (if required) Follow up appeared sufficient. Clear definition of criteria for dose change. Good 102 di Iulio et al (2009) Cross sectional 169 patients previously characterized for their CYP2B6 genotype Efavirenz plasma concentration and its primary metabolites Primary determinant was CYP2A6 status. Confounding well accounted for. Fair 104 Fair 105 Kwara et al (2009) Cohort 94 Ghanaian patients Efavirenz plasma concentration Significant percentage (60%) of participants had tuberculosis. Primary determinant included CYP2A6 and UGT2B7 status. Confounding well accounted for. Blood sampling not performed at consistent time points (4 and 8wks for 66 pts, 2wks for 28 pts). Kwara et al (2009) Cohort 74 patients enrolled in a pilot trial of a new antiretroviral regimen undertaken in Ghana Efavirenz plasma concentration Significant percentage (46%) of participants had tuberculosis. Primary determinant included CYP2A6 status. Confounding well accounted for. Fair 106 Arab-Alameddine et al (2009) Cohort 169 patients from the Swiss HIV Cohort Study Efavirenz pharmacokinetics CYP2B6 status evaluated along with CYP2A6 and CYP3A4/5. Confounders well accounted for. Good 107 Anderson et al (2006) Cohort 33 antiretroviral-naive patients who participated in a randomized pharmacological study of indinavir, lamivudine, and zidovudine. Antiretroviral pharmacokinetics Limited assessment of confounders (sex, weight, race). Number of patients from non-white races too low for accurate stratified analysis. Multiple genes analyzed (CYP3A5, MRP1, MRP2, MRP4, BCRP, UGT1A1). Fair 110 Boyd et al (2006) Cohort 96 Thai patients receiving indinavir Hyperbilirubinemia Clinically relevant outcome but not compared with plasma levels in this study. Confounding not considered. Outcome poorly defined (total bilirubin >1 mg/dl). Poor 111 Cohort 129 Korean patients initiating atazanavir at Seoul National University Hospital hyperbilirubinemia MRP1 influence also considered. Exclusion of patients with poor virological response may have excluded those with adverse effects to treatment. Atazanavir concentration not measured. 6 patients that developed jaundice not included in analyses. Poor 112 UGT1A1*28 +/- MRP1 Park et al (2010) 23 Patients with poor adherence excluded introducing a possible selection bias. Thorough analysis of possible confounders. Atazanavir plasma levels compared against bilirubin levels. Poor 113 Hyperbilirubinemia Long follow-up. Thorough analysis of possible confounders. Clinically relevant outcome (jaundice). Atazanavir and indinavir users combined in analysis. Outcome compared between PI users with UGT1A1 *28and non-PI users without UGT1A1 *28. More relevant to compare PI users with UGT1A1 *28 to PI users without UGT1A1 *28. Good 114 Cohort 74 antiretroviral-experienced patients initiating atazanavir with 2 NRTIs at a clinic in Madrid Plasma atazanavir and bilirubin concentrations Patients with poor adherence excluded introducing a possible selection bias. Lack of explanation as to why plasma levels only measurable in 57 patients. Confounders well accounted for. Atazanavir and bilirubin levels measured. Poor 115 Arnedo et al (2007) Cohort 419 antiretroviral treated participants of the Swiss HIV Cohort study Dyslipidemia Thorough consideration of confounders. Well defined outcome. Scoring system needs validation in an independent population. Good 119 Tarr et al (2005) Cohort 329 antiretroviral treated patients enrolled in the Swiss HIV Cohort study Triglyceride, HDL and Non-HDL concentrations, and lipoatrophy Confounding well accounted for. Low drop-out rate. LDL is a more commonly used lipid marker than non-HDL. Good 121 Fauvel et al (2001) Cross sectional 60 consecutive male patients using at least one PI attending an HIV follow-up consultation Lipids, apolipoproteins, lipoparticle levels were determined, together with glucose and markers of insulin secretion. Exclusion criteria selected patients less likely to exhibit dyslipidemia which may not be evenly distributed across genotypes tested. Limited consideration of confounders. Only men included in study. Cross sectional design not well suited to outcome. Poor 122 Foulkes et al (2006) Cross sectional 440 patients enrolled in selected AIDS Clinical Trial Group studies on stable PI therapy Plasma lipids Cross sectional design not well suited to outcome. Analyses stratified by race. Consideration of numerous possible confounders. Fair 123 Bonnet et al (2008) Cross sectional 40 white, male patients attending a follow-up consultation at University Hospital in Southern France Plasma lipids and lipoatrophy Cross sectional design not well suited to outcome. Limited consideration of confounders in final analysis. Poor 124 Rotger et al (2009) Cohort 745 patients from the Swiss HIV Cohort study Total cholesterol, HDL cholesterol, and triglycerides Confounders thoroughly accounted for. Long duration. 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