Development of multiplex pyrosequencing for HLA-B57:01 screening using single nucleotide polymorphism haplotype

Summary What is known and objective Abacavir (ABC) is a commonly used nucleoside reverse‐transcriptase inhibitor with potent antiviral activity against HIV‐1. The US Food and Drug Administration and international HIV treatment guidelines recommend HLA‐B*57:01 screening before initiating treatment with ABC. The current standard method for HLA‐B*57:01 screening is limited by its high‐cost, time‐consuming and labour‐intensive procedure with the requirement of a specialized laboratory. Our study aims to develop a more reliable screening test by selecting rs3093726 as an additional single nucleotide polymorphism (SNP) to combine with rs2395029 for multiplex pyrosequencing development. It offers high‐accuracy, cost‐effective and rapid detection. Methods Multiplex pyrosequencing was developed for HLA‐B*57:01 screening using rs2395029 and rs3093726 as a surrogate marker and tested in 130 Thai subjects in parallel with singleplex pyrosequencing of each SNP and the standard sequence‐based method. Results and discussion Multiplex pyrosequencing showed 100% concordance when compared with both singleplex pyrosequencing and standard sequence‐based method. This method showed 100% of negative predictive value (NPV), positive predictive value (PPV), specificity and sensitivity. What is new and conclusion Multiplex pyrosequencing is a powerful tool for HLA‐B*57:01 screening using the rs2395029 and rs3093726 haplotype genotyping as surrogate marker for this HLA‐B. The assay provides accurate, cost‐effective and rapid detection of this haplotype. It can be applied for ABC hypersensitivity screening of the Thai population before initiating treatment with ABC. Multiplex pyrosequencing is a powerful tool for HLA‐B*57:01 screening by using the rs2395029 and rs3093726 haplotype genotyping as surrogate marker for this HLA‐B. The assay provides accurate, cost effective and rapid detection of this haplotype. It can be applied for ABC hypersensitivity screening of the Thai population before initiating treatment with ABC.