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r 2010 British HIV Association HIV Medicine (2010) SHORT COMMUNICATION Introduction of pharmacogenetic screening for thehuman leucocyte antigen (HLA) B*5701 variant inPolish HIV-infected patients M Parczewski, M Leszczyszyn-Pynka, A Wnuk, A Urban˜ska, K Fuksin˜ska, D Bander and A Boron˜-Kaczmarska Department of Infectious Diseases and Hepatology, Pomeranian Medical University, Szczecin, Poland ObjectiveProspective pharmacogenetic screening for the human leucocyte antigen (HLA) B*5701 allele cansignificantly reduce the number of cases of abacavir-related hypersensitivity among HIV-infectedpatients treated with this drug. The aim of this study was to establish the frequency of the HLAB*5701 variant in HIV-infected Poles.
MethodsThe sequence-specific primer (SSP) test was used to assess the feasibility of the introduction of suchtesting in clinical practice. For this purpose, 234 randomly selected HIV-positive patients werescreened using a low-resolution SSP assay, with HLA B*5701-positive results confirmed using ahigh-resolution test.
Results and ConclusionsThe HLA B*5701 variant was found in 11 of 234 subjects (4.7%). Testing with the selected methodproved quick and reliable.
Keywords: pharmacogenetics, abacavir hypersensitivity, HLA B*57, antiretroviral treatment Accepted 28 August 2009 drug in hypersensitive individuals can be fatal, with acuteanaphylaxis and hypotension [3].
Despite extensive research in the field of pharmaco- The hypothesis of a genetic factor-based mechanism for genetics, routine genetic marker testing for clinical abacavir hypersensitivity was proposed and supported in purposes is not common. One successful example of the 2002 by two independent research groups [4,5]. The implementation of such a test into practice is human presence of the HLA B*5701 variant was associated with leucocyte antigen (HLA) B*5701 testing among people increased risk of HSR development, which was confirmed living with HIV, prior to the introduction of the nucleoside in numerous studies [6–9]. Prospective screening was reverse transcriptase inhibitor abacavir to antiretroviral found to significantly reduce the number of HSRs noted, treatment. The drug was associated with hypersensitivity with HLA B*5701 testing having an overall positive reactions (HSRs), which were noted in up to 8% of prognostic value for clinically diagnosed HSRs of 61.2%, Caucasian individuals after challenge with the drug [1].
while the negative prognostic value was 95.5% [6].
Hypersensitivity can occur within 6 weeks of treatment Many countries introduced prospective HLA B*5701 initiation and most commonly manifests clinically as fever, testing as the standard of care for HIV-infected patients, rashes, respiratory and gastrointestinal symptoms or and this has been particularly successful in Australia and the malaise/lethargy [2]. The symptoms resolve quickly, within United Kingdom, allowing reductions in the number of 72 hours of drug discontinuation. Re-challenge with the adverse reactions observed, improvements in adherence totherapy and reductions in the number of abacavir dis-continuations [10,11]. Testing is cost effective, especially in Correspondence: Dr Mizosz Parczewski, Department of Infectious Diseases populations with higher frequencies of the HLA B*5701 and Hepatology, Pomeranian Medical University, Arkon˜ska 4, 71-455 allele (e.g. Caucasian populations), allowing reductions in Szczecin, Poland. Tel: 0048918139456; fax: 0048918139449; e-mail: costs related to HSR treatment [12]. For such populations, on 2 Parczewski et al.
average, only 14 tests would result in the prevention of one agarose gel (Sigma, St. Louis, MO, USA) stained with Gel- case of abacavir HSR [13]. HLA B*5701 testing is included in Star dye (Lonza, Rockland, Switzerland). Results were the European AIDS Clinical Society guidelines for clinical visualized under UV light (Transilluminator 4000; Strata- management and treatment of HIV-infected adults in gene, La Jolla, CA, USA) and recorded with a DS-34 Europe, with abacavir contraindicated if an individual tests Polaroid Direct Screen Camera.
positive for this variant (available online at Additionally, all B*57-positive samples were verified To avoid costly and time-consuming high-resolution using another CE marked assay performed using the Olerup sequencing, screening can be based on the sequence-specific SSP HLA-B* 57 high-resolution kit (Olerup SSP AB, amplification technique. This approach reduces both the cost Saltsjoebaden, Sweden), with subsequent electrophoresis of the test and the time needed to obtain results [14]. As and recording as described above.
validated tests become available, it might be expected thatthis field will develop rapidly in the near future.
In this study, we tested the HLA B*5701 allele frequency in a cohort of 200 HIV-positive individuals from the West In the studied group of 234 HIV-1-infected patients, 13 of Pomeranian region of Poland by means of sequence- 234 subjects (5.6%) tested positive for HLA B*5701 in the specific primer (SSP) polymerase chain reaction (PCR) low-resolution test (corresponding to serological type B57).
technology. The aim of the study was not only to provide The results were confirmed by the high-resolution test for allele frequency data for this group but also to determine 11 of these subjects (4.7%), while one individual was found the feasibility of widespread clinical implementation of to carry the HLA B*5703 variant and one patient B*5306.
genetic testing for this pharmacogenetic factor in Poland.
Six of the individuals (54.6%) carrying the HLA B*5701allele were male.
Example agarose gels demonstrating the presence of the Material and methods HLA B*5701 variant are shown in Figs 1 and 2.
The study group consisted of 234 randomly selectedpatients with confirmed HIV infection attending the Clinic The HLA B*5701 allele frequency found in the HIV-1- for Acquired Immunodeficiency Treatment, Department of positive group in this study is higher than the frequency Infectious Diseases and Hepatology, Szczecin, Poland. Mostof the individuals tested were male [male, 169 (72%);female, 65 (28%)]. The mean age (  standard error) of thestudied individuals was 40.9  9.5 years (median 39 years).
As the majority of patients attending the clinic are ofCaucasian origin (99.9%), for this study only Caucasianswere selected. All participants voluntarily consented toparticipate in the study.
DNA extraction and HLA typing Genomic DNA was extracted using the QIAamp DNA BloodMini Kit (Qiagen, Hilden, Germany) from whole bloodsamples previously collected in tubes containing ethylene-diaminetetraacetic acid (EDTA) anticoagulant. The extrac-tion was performed according to the manufacturer'sprotocol. DNA was resuspended in 200 mL of AE buffer(Qiagen) and stored at 20 1C for further analyses.
Fig. 1 Photograph of a gel showing polymerase chain reaction (PCR) For HLA B*5701 screening, the SSP HLA-Ready Gene products of the sequence-specific reaction allowing detection of the B5/57 Cross low-resolution kit (Inno-Train Diagnostik, human leucocyte antigen (HLA) B*57 allele (low-resolution test).
Kronberg, Germany) was used to perform an in vitro Lane 1, size marker Puc 8 (Fermentas, Vilnius, Lithuania). The upperband in lanes 2–8 represents the result of an internal positive control diagnostics validated, European Economic Area conformity reaction (product size 800 bp). The presence of lower bands in lanes mark (CE) marked test, according to the manufacturer's 2, 6 and 7 is specific for HLA B*57 (product sizes 100, 430 and 95 bp, protocol. PCR products were electrophoresed on a 3% r 2010 British HIV Association HIV Medicine (2010) HLA B*5701 in HIV-infected Poles 3 mechanism is involved. The prognostic value of suchscreening was also noted in a study by Waters et al., [11]with a reduction in HSR from 7.5% prior to the introductionof testing to 2% after the testing was introduced. However, itshould be noted that in one case an HLA B*5701-negativeindividual developed a strong HSR, which was confirmedimmunologically by skin-patch testing. Such an event maysuggest the involvement of additional immunologicalmechanisms in the development of symptoms; therefore,even if an individual is negative for HLA B*5701,counselling regarding HSR symptoms is necessary.
In a study by Saag et al., [19] based on retrospective patient record analysis with identification of patients withthe skin patch test confirmed abacavir HSR in subsequentHLA B*5701 testing 100% sensitivity in a white population Fig. 2 Photograph of a gel showing polymerase chain reaction (PCR) was observed. When HSRs were observed clinically but were products of the sequence-specific reaction allowing detection of the immunologically unconfirmed, the sensitivity decreased to human leucocyte antigen (HLA) B*5701 allele (high-resolution test).
44%, but the specificity remained high at 96%. This study Lanes 1 and 18, size marker Puc 8 (Fermentas, Vilnius, Lithuania). The confirms the need for and validity of HLA B*5701 testing in upper band in lanes 2–17 represents the result of an internal positivecontrol reaction (product size 800 bp for lanes 2, 5 and 11; for the clinical practice. Costs and the time required to provide a remaining lanes, the internal control reaction product size is valid result must also be considered. Results obtained using 1070 bp). The presence of lower bands in lanes 2, 3, 4 and 14 is SSP assays have been shown to be concordant with those specific for HLA B*5701 (product sizes 150, 100, 220 and 90 bp, obtained by sequencing [6,14]. The necessity for adequate quality assurance must be emphasized, as the test result is ofvital importance not only for HSR risk reduction but also previously reported by Nowak et al. [15] for the Polish from the perspective of therapeutic options available for the population (0.047 vs. 0.025, respectively; both studies patient [20]. For maximum accuracy, low-resolution HLA having the same sample size). Allelic frequencies of this B*5701 results should be confirmed with a high-resolution variant among European Caucasian populations vary from assay using a kit obtained from a different manufacturer, 0.007 in Romania to 0.071 among Andalusian Gypsies with only confirmed results provided to clinicians. In our (frequency data available online at opinion, such an approach provides good sensitivity and specificity of the results obtained. The cost of such testing is approximately eight-to-10-times lower than that of testing by HLA-B sequencing or PCR-SSP-based investigation of the entire B locus.
To summarize, we believe that HLA B*5701 testing based on the SSP test, with positive results confirmed by an The general aim of HLA B*5701 testing in Caucasian alternative, high-resolution test, is specific, accurate, fast populations is to reduce the risk of abacavir HSR, and and cost effective. As it could reduce the number of therefore the number of drug discontinuations and the abacavir HSRs, widespread use of this testing strategy in necessity for additional treatment. Such an approach HIV-positive patients should be encouraged.
increases patients' confidence in the safety of antiretroviraltreatment and significantly reduces not only the number ofobserved HSRs but also the number of treatment interrup- tions [18]. Results recently published for the PREDICT-1study showed that HLA B*5701 testing alone eliminated  Prospective (prior to the introduction of abacavir- immunologically confirmed reactions, with a reduction in containing therapy) genetic HLA screening for B*5701 the percentage of clinically observed cases in the in HIV-infected individuals in Poland is feasible and prospectively screened HLA B*5701-negative group to should be performed on a regular basis.
3.4% [6]. It is uncertain whether these clinically reported  Sequence-specific testing for HLA B*5701 has been HSRs are related to the concomitant with abacavir shown to be quick and reliable, especially where low- antiretroviral drugs, or whether another immunological resolution results are confirmed in a high-resolution test.
r 2010 British HIV Association HIV Medicine (2010) 4 Parczewski et al.
Acknowledgements and financial disclosures HIV-infected patients in Taiwan. J Antimicrob Chemother 2007; 60: 599–604.
The study was funded by the Department of Infectious 10 Lucas A, Nolan D, Mallal S. HLA-B*5701 screening for Diseases and Hepatology, Pomeranian Medical University, susceptibility to abacavir hypersensitivity. J Antimicrob Szczecin, Poland. Additional financial support was pro- Chemother 2007; 59: 591–593.
vided by the Association of Infectious Disease Prevention 11 Waters LJ, Mandalia S, Gazzard B, Nelson M. Prospective ‘Avicenna', Szczecin, Poland. No other external source of HLA-B*5701 screening and abacavir hypersensitivity: a single funding (e.g. funding from a pharmaceutical company) was centre experience. AIDS 2007; 21: 2533–2534.
involved in the study. The authors would like to express 12 Hughes DA, Vilar FJ, Ward CC, Alfirevic A, Park BK, their deep gratitude to Ms Dominika Tracz, Transfarm, Pirmohamed M. Cost-effectiveness analysis of HLA B*5701 Poland for her advice and support.
genotyping in preventing abacavir hypersensitivity.
Pharmacogenetics 2004; 14: 335–342.
13 Phillips EJ. Genetic screening to prevent abacavir hypersensitivity reaction: are we there yet? Clin Infect Dis 1 Hetherington S, McGuirk S, Powell G et al. Hypersensitivity 2006; 43: 103–105.
reactions during therapy with the nucleoside reverse 14 Martin AM, Nolan D, Mallal S. HLA-B*5701 typing by sequence- transcriptase inhibitor abacavir. Clin Ther 2001; 23: specific amplification: validation and comparison with sequence-based typing. Tissue Antigens 2005; 65: 571–574.
2 Cutrell AG, Hernandez JE, Fleming JW et al. Updated clinical 15 Nowak J, Mika-Witkowska R, Polak M et al. Allele and risk factor analysis of suspected hypersensitivity reactions to extended haplotype polymorphism of HLA-A, -C, -B, abacavir. Ann Pharmacother 2004; 38: 2171–2172.
-DRB1 and -DQB1 loci in Polish population and genetic 3 Escaut L, Liotier JY, Albengres E, Cheminot N, Vittecoq D.
affinities to other populations. Tissue Antigens 2008; 71: Abacavir rechallenge has to be avoided in case of hypersensitivity reaction. AIDS 1999; 30: 1419–1420.
16 Borghans JA, Mlgaard A, de Boer RJ, KeSmir C. HLA alleles 4 Hetherington S, Hughes AR, Mosteller M et al. Genetic associated with slow progression to AIDS truly prefer to variations in HLA-B region and hypersensitivity reactions to present HIV-1 p24. PLoS ONE 2007; 19: e920.
abacavir. Lancet 2002; 359: 1121–1122.
17 Migueles SA, Sabbaghian MS, Shupert WL et al. HLA B*5701 is 5 Mallal S, Nolan D, Witt C et al. Association between presence of highly associated with restriction of virus replication in a HLA-B*5701, HLA-DR7, and HLA-DQ3 and hypersensitivity to subgroup of HIV-infected long term nonprogressors. Proc Natl HIV-1 reverse-transcriptase inhibitor abacavir. Lancet 2002; Acad Sci USA 2000; 97: 2709–2714.
359: 727–732.
18 Zucman D, Truchis P, Majerholc C, Stegman S, Caillat-Zucman S.
6 Mallal S, Phillips E, Carosi G et al. for the PREDICT-1 Study Prospective screening for human leukocyte antigen-B*5701 Team. HLA-B*5701 screening for hypersensitivity to abacavir.
avoids abacavir hypersensitivity reaction in the ethnically mixed N Engl J Med 2008; 358: 568–579.
French HIV population. J Acquir Immune Defic Syndr 2007; 45: 7 Martin AM, Nolan D, Gaudieri S et al. Predisposition to abacavir hypersensitivity conferred by HLA-B*5701 and a 19 Saag M, Balu R, Phillips E et al. for the Study of haplotypic Hsp70-Hom variant. Proc Natl Acad Sci USA 2004; Hypersensitivity to Abacavir and Pharmacogenetic Evaluation 101: 4180–4185.
Study Team. High sensitivity of human leukocyte antigen- 8 Rauch A, Nolan D, Martin A, McKinnon E, Almeida C, Mallal S.
b*5701 as a marker for immunologically confirmed abacavir Prospective genetic screening decreases the incidence of hypersensitivity in white and black patients. Clin Infect Dis abacavir hypersensitivity reactions in the Western Australian 2008; 46: 1111–1118.
HIV cohort study. Clin Infect Dis 2006; 43: 99–102.
20 Hammond E, Almeida CA, Mamotte C et al. External quality 9 Sun HY, Hung CC, Lin PH et al. Incidence of abacavir assessment of HLA-B*5701 reporting: an international hypersensitivity and its relationship with HLA-B*5701 in multicentre survey. Antivir Ther 2007; 12: 1027–1032.
r 2010 British HIV Association HIV Medicine (2010)


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