Detection of HLA-B*57:01 By Real-Time PCR: Implementation into Routine Clinical Practice and Additional Validation Data

Abstract

Aim:HLA-B*57:01 status needs to be determined before initiating abacavir therapy. We developed a pharmacogenetic real-time (Q)-PCR screening test using two sets of sequence specific primers. This test has been implemented into routine clinical practice. Materials & methods: HIV-infected patients admitted at our University Hospital were thus genotyped using the above mentioned test. A panel of 80 DNA samples with a known genotype were used to characterize Q-PCR conditions using different master mixes. Results: A total of 353 patients were genotyped, detecting 15 (4.25%) HLA-B*57:01 positive carriers. Among the negative patients, 17.2% were treated with abacavir without any hypersensitivity reaction. Using different Q-PCR master mixes, significantly lower cutoff Ct values were found, thus new analytical settings are provided. Conclusion: The pharmacogenetic test developed in our laboratory for the fast screening of HLA-B*57:01 can be successfully implemented into routine clinical practice. All 16 sequences (including an additional six) currently known for the HLA-B*57:01 allele are detected by sequence specific primers used in this test. The Brilliant II SYBR® Green QPCR MM (Stratagene) can safely replace the master mix originally used to develop the test. Original submitted 2 August 2013; Revision submitted 2 December 2013

High Frequency of Human Leukocyte Antigen-B*57:01 Allele Carriers among HIV-Infected Patients in Serbia

“…One could speculate that the rather high prevalence of HLA-B * 57: 01 in HIV-infected patients in Serbia found in our study might be attributed to false-positive detection, as described in some previous studies [11] . However, existing studies comparing commercially available real-time PCR tests for HLA-B * 57: 01 detection demonstrated their high concordance and reliability, and there is only a single report of possibly false-negative results published to date [30] . On the other hand, the use of buccal swabs as primary samples facilitated easy access of a noninfective source of DNA in our study.…”

Diffusion of complementary evolving pharmaceutical innovations: The case of Abacavir and its pharmacogenetic companion diagnostic in Italy

“…The EQA program showed the proficiency of all the virology units in providing correct HLA-B*57:01 genotyping by inexpensive assays, mostly based on Q-PCR techniques (Meini et al, 2016). During the EQA one of the research teams reported the poor performance of a commercial test (Dello Russo et al, 2013). This occurrence was confirmed by another virology research centre on a larger sample using a similar technological platform (Falasca et al, 2016).…”

HLA and Delayed Drug-Induced Hypersensitivity

“…Genomic DNA sequencing-based typing, which is based on the Sanger method, is one of the most common methods, and is the gold standard for HLA-B*57:01 identification [100,101,102]. However, this method is expensive, not always available, and can yield ambiguous results [100,101,103]. Other methods include sequence-specific primer PCR and real-time PCR coupled with melting curve analysis.…”

“…Other methods include sequence-specific primer PCR and real-time PCR coupled with melting curve analysis. Although once popular, sequence-specific primer PCR can also generate ambiguous results and requires constant sequence updates [101,103]. Real-time PCR, by contrast, is less prone to ambiguity and is highly specific, sensitive, and efficient [100].…”