High Sensitivity of the BioNote Anigen™ Rapid Rabies Antigen Test in Detection of Diverse Rabies Virus Variants Suggests Utility for Global Rabies Control
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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.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17633171.
This preprint evaluates the performance of the BioNote Anigen Rapid Rabies Antigen Test across a wide diversity of rabies virus variants, including both classical and atypical lineages. The authors report high sensitivity and specificity of the assay compared to gold-standard diagnostic methods, such as the direct fluorescent antibody (DFA) test and RT-qPCR. The study addresses an important global health issue: rabies remains a neglected zoonotic disease with nearly 60,000 human deaths annually, and accurate, field-deployable diagnostics are crucial for controlling transmission in low-resource regions. The paper is therefore both timely and globally relevant.
Some major issues that stood …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17633171.
This preprint evaluates the performance of the BioNote Anigen Rapid Rabies Antigen Test across a wide diversity of rabies virus variants, including both classical and atypical lineages. The authors report high sensitivity and specificity of the assay compared to gold-standard diagnostic methods, such as the direct fluorescent antibody (DFA) test and RT-qPCR. The study addresses an important global health issue: rabies remains a neglected zoonotic disease with nearly 60,000 human deaths annually, and accurate, field-deployable diagnostics are crucial for controlling transmission in low-resource regions. The paper is therefore both timely and globally relevant.
Some major issues that stood out to me relate to the scope and validation of the testing approach. The study involves a large number of geographically and genetically diverse RABV samples, but it would be helpful if the authors clarified how representative these samples are of the global viral diversity—especially from under-sampled regions in Asia and sub-Saharan Africa. This would strengthen the claim of "global utility." Additionally, while sensitivity and specificity values are impressive, the statistical treatment of confidence intervals could be expanded to show the robustness of performance across variant subgroups.
Another concern is the reliance on postmortem animal brain samples, which may not fully reflect the performance of the test in real-time surveillance or field-deployable conditions (e.g., testing in decomposed carcasses or non-invasive oral fluid samples). The authors briefly mention potential use in point-of-care (POC) surveillance, but there are no real-world deployment data presented to support this. Future studies might validate the test's performance under operational field conditions, including storage stability, temperature effects, and cross-reactivity with other lyssaviruses.
Overall, the manuscript is well-written, methodologically sound, and provides valuable data that could significantly impact rabies control efforts. Addressing the representativeness of the tested variants, including a broader discussion of field performance parameters, and clarifying statistical robustness would strengthen the conclusions and enhance its translational value for global rabies surveillance programs.
Competing interests
The author declares that they have no competing interests.
Use of Artificial Intelligence (AI)
The author declares that they used generative AI to come up with new ideas for their review.
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