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 the 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 widespread 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 according to the manufacturer’s 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 (RVVs). Test sensitivity and specificity were 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 types, and results suggest that the BioNote Anigen Test with the procedural changes would have broad benefits for rapid diagnosis in animals. IMPORTANCE Despite more than half a century of highly accurate rabies diagnostic methods, a global landscape analysis conducted in 2021 found that nearly all countries in Africa and Asia have inadequate rabies surveillance and testing programs. This dearth of surveillance and testing has been identified as a leading factor for rabies’ longstanding status as a neglected disease. Studies have consistently identified barriers with current diagnostic approaches, which render them unlikely to be utilized effectively in low- and middle-income countries, where an estimated 70,000 people die from the disease each year. In 2023, the World Organization for Animal Health cautioned against the use of unvalidated lateral flow assay tests outside of research or evaluation programs. Despite this statement of caution, numerous field deployments of these tests have been published. This evaluation found a high sensitivity and specificity for the BN-LFA, with values similar to those reported for other gold standard rabies tests.