r 2010 British HIV Association
HIV Medicine (2010)
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 .
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 .
while the negative prognostic value was 95.5% .
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 . 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 . 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 . 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 . 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., 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.,  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.  for the Polish
from the perspective of therapeutic options available for the
population (0.047 vs. 0.025, respectively; both studies
patient . 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 . 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% . 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.
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r 2010 British HIV Association HIV Medicine (2010)
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