Heparin-mediated PCR interference in SARS-CoV-2 assays and subsequent reversal with heparinase I

K Edwards; T Corocher; Y Hersusianto; D Campbell; K Subbarao; J A Neil; P Monagle; P Ho

doi:10.1016/j.jviromet.2024.114944

Heparin-mediated PCR interference in SARS-CoV-2 assays and subsequent reversal with heparinase I

J Virol Methods. 2024 Apr 20:327:114944. doi: 10.1016/j.jviromet.2024.114944. Online ahead of print.

Authors

K Edwards¹, T Corocher², Y Hersusianto³, D Campbell⁴, K Subbarao⁵, J A Neil⁶, P Monagle⁷, P Ho⁸

Affiliations

¹ Northern Pathology Victoria, Northern Health, Epping, VIC, Australia; NorthErn Clinical diagnostics and ThrombovAscular Research (NECTAR) Centre, Northern Health, Epping, VIC, Australia. Electronic address: kira.edwards@nh.org.au.
² Northern Pathology Victoria, Northern Health, Epping, VIC, Australia; NorthErn Clinical diagnostics and ThrombovAscular Research (NECTAR) Centre, Northern Health, Epping, VIC, Australia; Infectious Diseases, Northern Health, Epping, VIC, Australia.
³ Northern Pathology Victoria, Northern Health, Epping, VIC, Australia; Infectious Diseases, Northern Health, Epping, VIC, Australia.
⁴ Hospital without Walls, Northern Health, Epping, VIC, Australia; Department of Medicine - Southern Clinical School, Monash University, Clayton, VIC, Australia.
⁵ Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia; WHO Collaborating Centre for Reference and Research on Influenza, Peter Doherty Institute for Infection and Immunity, Melbourne, Vic, Australia.
⁶ Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.
⁷ Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia; Murdoch Children's Research Institute, Melbourne, VIC, Australia; Department of Haematology, Royal Children's Hospital, Parkville, VIC, Australia; Kids Cancer Centre, Sydney Children's Hospital, Randwick, NSW, Australia.
⁸ Northern Pathology Victoria, Northern Health, Epping, VIC, Australia; NorthErn Clinical diagnostics and ThrombovAscular Research (NECTAR) Centre, Northern Health, Epping, VIC, Australia; Department of Medicine - Northern Health, University of Melbourne, Epping, VIC, Australia.

PMID: 38649069
DOI: 10.1016/j.jviromet.2024.114944

Abstract

Heparin is postulated to block the interaction of SARS-CoV-2 with highly glycosylated proteins which are critical for binding the angiotensin-converting enzyme 2 (ACE2), an essential mechanism for host-cell entry and viral replication. Intranasal heparin is under investigation for use as a SARS-CoV-2 preventative in the IntraNasal Heparin Trial (INHERIT, NCT05204550). Heparin directly interferes with real-time quantitative polymerase chain reaction (RT-qPCR), the gold standard for SARS-CoV-2 detection. This study aimed to investigate the magnitude of heparin interference across various clinical laboratory testing platforms, and the reversal of any interference by degradation of heparin using the heparinase I enzyme in nasopharyngeal swab (NP) samples for SARS-CoV-2 analysis by RT-qPCR. Heparin-mediated PCR interference was evident at heparin concentrations as low as 10 IU/mL across all platforms tested, with the exclusion of the Hologic Panther Aptima SARS-CoV-2 assay. Rates of false negative or invalid results increased with increasing heparin concentrations on all platforms, except the Hologic Panther Aptima and Roche Cobas LIAT. Heparinase I reversed heparin-mediated PCR inhibition across in all samples tested, except those with initial Ct values >35. Our study shows that the use of heparin-containing nasal sprays interferes with the detection of SARS-CoV-2 in NP swab samples by RT-qPCR, a phenomenon that is not well recognised in the literature. Furthermore, this study has also demonstrated that heparin-mediated PCR inhibition can be prevented through heparinase I treatment, demonstrating restoration of clinically significant results with Ct values <35.

Keywords: Heparin; Heparinase I; INHERIT; Intranasal heparin trial; PCR interference; SARS-CoV-2.