Development of multiplex pyrosequencing for HLA-B*57:01 screening using single nucleotide polymorphism haplotype

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.

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.

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.

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.