A low‐cost, sensitive and specific PCR‐based tool for rapid clinical detection of HLA‐B*35 alleles associated with delayed drug hypersensitivity reactions

HLA (HLA) alleles are risk factors for CD8+ T‐cell‐mediated drug hypersensitivity reactions. However, as most HLA associations are incompletely predictive and/or involve risk alleles at low frequency, costly sequence‐based typing can elude an economically productive cost: benefit ratio for clinical validation studies and diagnostic and/or preventative screening. Hence rapid and low‐cost detection assays are now required, both for single alleles but also across risk loci associated with broader multi‐disease risk; exemplified by associations with diverse alleles in HLA‐B*35, including HLA‐B*35:01 and green tea‐ or co‐trimoxazole‐induced liver injury. Here, we developed a cost‐effective (<$10USD) qPCR assay for rapid (<2.5 h) clinical detection of HLA‐B*35 alleles. The assay was validated using 430 DNA samples with previous American society for histocompatibility and immunogenetics‐accredited sequence‐based high‐resolution HLA typing, positively detecting all HLA‐B*35 allelic variants in our cohort, and as expected by primer design, the six samples that expressed low‐frequency B*78:01. The assay did not result in positive detection for any negative control allele. With expected detection of B*35 and B*78, our assay sensitivity (95% CI, 95.07%–100.00%) and specificity (95% CI, 98.97%–100.00%) of 100% using as low as 10 ng of DNA provides a reliable HLA‐B*35 screening tool for clinical validation and HLA–risk‐based prevention and diagnostics.