Rapid, Near Point-of-Care Assay for HLA-B*57:01 Genotype Associated with Severe Hypersensitivity Reaction to Abacavir

Objective The nucleoside reverse transcriptase inhibitor abacavir is commonly used to treat young children with HIV infection. Abacavir can trigger a severe hypersensitivity reaction in people who are homozygous or heterozygous for HLA-B*57:01 . Testing for HLA-B*57:01 prior to abacavir initiation is standard-of-care in high-resource settings, but current tests are too costly for resource-limited settings. To address this gap, we developed an inexpensive, simple-to-use rapid assay to detect HLA-B*57:01 . Methods We designed and optimized a multiplexed PCR to amplify HLA-B*57 subtypes and the human beta-globin gene. Subsequently, probes annealed to the amplicon and were ligated when specific for the HLA-B*57:01 allele. Ligated products were detected by immunocapture in a lateral flow strip. Cell lines with known HLA genotypes were used to optimize the assay. The assay was then evaluated by comparing the genotype of clinical specimens (n=60) enriched for individuals with HLA-B*57:01 by the new assay to that from sequencing. Results The optimized multiplex PCR for B*57 and beta-globin resulted in a 40-minute, 35-cycle amplification, followed by a 20-minute ligation reaction and 15-minute detection step. Evaluation of the HLA-B*57:01 oligonucleotide ligation assay using clinical specimens had a sensitivity of 100% (n=27/27 typed as B*57:01 ) and specificity of 100% (n=33/33 typed as non- B*57:01 ) by visual interpretation of lateral flow strips. Conclusions This rapid and economical assay can accurately detect the presence of HLA-B*57:01 in clinical specimens. Use of this assay could expand access to HLA-B*57:01 genotyping and facilitate safe same-day initiation of abacavir-based treatment. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Support for this project came from the National Institutes of Health (NIH) including R01 AI110375 (LMF); R01 AI145486 (BRL); the Clinical and Retrovirology Research Core and the Molecular Profiling and Computational Biology Core of the University of Washington Fred Hutch Center for AIDS Research [P30 AI027757]; and the International Maternal Pediatric Adolescent AIDS Clinical Trials Network (IMPAACT). IMPAACT is funded by the National Institute of Allergy and Infectious Diseases (NIAID) with co-funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) and the National Institute of Mental Health (NIMH), all components of NIH under Award Numbers UM1AI068632 (IMPAACT LOC), UM1AI068616 (IMPAACT SDMC) and UM1AI106716 (IMPAACT LC), and by NICHD contract number HHSN275201800001I. The work was supported in part by funds from the Canadian Institutes of Health Research (grants PJT-148621 and PJT-159625), received by SAR, as well as funds from the Mexican Government (Comision de Equidad y Genero de las Legislaturas LX-LXI y Comision de Igualdad de Genero de la Legislatura LXII de la H. Camara de Diputados de la Republica Mexicana), received by SAR. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funders. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Recruitment of the Mexican samples was evaluated and approved by the Ethics Committee of INER (protocol code E02-17). All participants were adults (>18 years) and gave written informed consent before blood sample donation. Seattle Primary Infection Cohort was reviewed and approved by the UW Institutional Review Board, and all subjects provided written consent All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as [ClinicalTrials.gov][1]. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Genbank submission pending [1]: http://ClinicalTrials.gov