High Sensitivity of the BioNote Anigen™ Rapid Rabies Antigen Test in Detection of Diverse Rabies Virus Variants Suggests Utility for Global Rabies Control

Abstract Point-of-care, immunochromatographic tests have not reliably detected rabies virus and have not utilized a consistent diagnostic protocol. International diagnostic standards established by the World Organization for Animal Health require strategic and large-scale validation studies prior to widescale use. The United States (US) Centers for Disease Control and Prevention, National Rabies Reference Laboratory undertook a large-scale validation of the BioNote Anigen™ Rapid Rabies Antigen Test in collaboration with eight US public health laboratories and three international laboratories. Modifications were made to manufacturer instructions to maximize antigen concentration in the test diluent. A total of 1,399 samples underwent paired testing with one of three US gold standard tests, consisting of 31 types of animals and 9 rabies virus variants. Test sensitivity and specificity was 97.11% (CI: 95.21% - 98.27%) and 99.89% (CI: 99.36% - 99.98%). Fourteen samples resulted in false-negative results, primarily impacting dogs that were euthanized early or shown to have a low viral load. Limit of detection studies found that false-negative results often occurred when the sample had a PCR Ct value >23. The BioNote Anigen™ Test performed well across diverse RVVs found in North America. Sensitivity of the test was slightly lower than, but not statistically inferior to, the minimum 98% value established for the current gold-standard tests. This is the largest systematic evaluation of a rabies point-of-care test that includes diverse RVV and animal type and results suggest that the BioNote Anigen™ Test with the procedural changes would have broad benefits for rapid diagnosis in animals. Competing Interest Statement The authors have declared no competing interest. Funding Statement This study did not receive any funding Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. 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. 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, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data produced in the present study are available upon reasonable request to the authors