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/17993130.
Summary
This preprint evaluates the performance of the Xpert MTB/XDR assay for rapid detection of drug resistance in Mycobacterium tuberculosis (Mtb) beyond rifampicin, specifically targeting fluoroquinolone and aminoglycoside resistance. The authors compiled a multi-site panel of clinical specimens across multiple geographic regions and compared Xpert MTB/XDR results against conventional phenotypic drug-susceptibility testing (DST). The study reports high sensitivity and specificity for several resistance-associated markers, suggesting that this assay could substantially reduce time to diagnosis of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis and enable more …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17993130.
Summary
This preprint evaluates the performance of the Xpert MTB/XDR assay for rapid detection of drug resistance in Mycobacterium tuberculosis (Mtb) beyond rifampicin, specifically targeting fluoroquinolone and aminoglycoside resistance. The authors compiled a multi-site panel of clinical specimens across multiple geographic regions and compared Xpert MTB/XDR results against conventional phenotypic drug-susceptibility testing (DST). The study reports high sensitivity and specificity for several resistance-associated markers, suggesting that this assay could substantially reduce time to diagnosis of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis and enable more rapid treatment decisions. The preprint highlights the importance of expanding rapid molecular diagnostics beyond rifampicin resistance alone, particularly given rising global rates of second-line drug resistance. The authors argue that Xpert MTB/XDR could serve as a practical, near–near-point-of-care diagnostic tool, especially in decentralized and resource-limited settings.
Major Concerns
Unclear specimen types and clinical relevance
The manuscript does not clearly state whether the tested samples were cultured isolates, direct patient specimens (e.g., sputum), or a mixture of both.
Actionable suggestion: The authors should explicitly specify, for each sample (or sample group), whether it was a cultured isolate or a direct clinical specimen. If direct specimens were used, details should be provided on smear status (smear-positive vs. smear-negative), specimen type (sputum, extrapulmonary), and any preprocessing steps.
If the panel is enriched for resistant isolates, this should be clearly acknowledged.
Actionable suggestion: The authors should report the proportion of resistant vs. susceptible samples and discuss how enrichment may affect positive and negative predictive values in routine clinical populations.
Limited description of geographic and epidemiological diversity
While the study includes samples from multiple sites, there is insufficient detail about the diversity of Mtb lineages, resistance-conferring mutations, and patient backgrounds.
Actionable suggestion: The authors should include a table or supplementary material describing, for each specimen or site: Geographic origin, Mtb lineage, Specific resistance-associated mutations detected, and Prior treatment status (new case vs. previously treated)
This information is necessary for readers to assess how generalizable the results are to different global TB settings.
Performance in low bacterial load and heteroresistance not addressed
The manuscript does not evaluate or discuss assay performance in cases of low bacterial burden or heteroresistance (mixed susceptible and resistant populations), which are common in smear-negative, extrapulmonary, or early TB.
Actionable suggestion: The authors should either:
Present stratified performance data by smear status or bacterial load (if available), or
Explicitly state that such data were not collected and discuss this as a limitation.
Additionally, discussion of heteroresistance detection (or lack thereof) should be included, along with implications for missed resistance.
Lack of information on limits of detection and sample processing
No data are provided on the assay's limit of detection (LOD) or the impact of sample quality and processing on assay performance.
Actionable suggestion: The authors should report any available LOD data or reference existing validation studies. They should also describe sample processing protocols (e.g., decontamination methods) and discuss how these may influence sensitivity in real-world settings.
Minor Concerns
Terminology and definitions
Terms such as "non-rifampicin drug resistance," "MDR," and "XDR" are used without a clear definition.
Actionable suggestion: The authors should define all resistance categories according to current WHO guidelines and clarify whether resistance refers to mono-resistance, multidrug resistance, or resistance across multiple drug classes.
Clinical interpretation and limitations
The discussion does not sufficiently address clinical contexts in which molecular assays may underperform or mislead.
Actionable suggestion: The authors should expand the discussion to explicitly address: Paucibacillary and extrapulmonary disease, Heteroresistance, Discordance between molecular resistance markers and phenotypic DST
They should also comment on the potential risk of overdiagnosis or overtreatment when molecular resistance detection does not correlate with phenotypic resistance, and suggest confirmatory testing strategies or follow-up studies.
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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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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