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Expansion of the use of lateral flow devices (LFD) for animal rabies diagnosis can help mitigate the widespread underreporting of rabies. However, this has been hindered by the limited number and small sample size of previous studies. To overcome this limitation, we conducted a multicenter study with a larger sample size to assess the diagnostic accuracy of the ADTEC LFD for postmortem rabies diagnosis in animals. Thirteen governmental animal diagnostic laboratories in the Philippines were involved in this study, and 791 animals suspected of having rabies were tested using both the direct fluorescence antibody test (DFAT) and ADTEC LFD between August 2021 and October 2022. The LFD demonstrated a sensitivity of 96.3% [95% confidence interval (CI): 94.1%-97.9%] and a specificity of 99.7% (95% CI: 98.4%-100%). Notably, false-negative results were more likely to occur in laboratories with lower annual processing volumes of rabies samples in the previous years (adjusted odds ratio 4.97, 95% CI: 1.49-16.53). In this multicenter study, the high sensitivity and specificity of the LFD for the diagnosis of animal rabies, compared to that of the DFAT, was demonstrated, yet concerns regarding false-negative results remain. In areas with limited experience in processing rabies samples, it is essential to provide comprehensive training and careful attention during implementation., Competing Interests: The authors declare no conflict of interest.

Molecular analysis of rabies virus can provide accurate diagnosis and information on its genetic diversity. The transportation of rabies brain samples from remote areas to a central laboratory is challenging owing to biohazard risks and decomposability. We investigated the utility of used lateral flow devices (LFDs) for subsequent molecular analysis and assessed the necessary storage temperatures. Using RNA extracted from used LFD strips, we performed conventional reverse transcription-PCR (RT-PCR) using an LN34 primer set to amplify short fragments (165 bp) for rabies virus detection and the P1-304 primer set to amplify long fragments of the entire N gene amplicon (1,506 bp) for phylogenetic analysis. Among 71 used LFDs stored in a refrigerator and 64 used LFDs stored at room temperature, the LN34 assay showed high sensitivities (96.2% and 100%, respectively) for the diagnosis of rabies, regardless of the storage temperature. A significant reduction in the sensitivity of rabies diagnosis was observed when using the P1-304 primer set for used LFDs stored at room temperature compared to those stored at refrigeration temperature (20.9% versus 100%; P  < 0.05). Subsequent sequencing and phylogenetic analysis were successfully performed using the amplicons generated by the P1-304 RT-PCR assays. Used LFDs are thus promising resources for rabies virus RNA detection and sequence analysis. Virus detection via RT-PCR, amplifying a short fragment, was possible regardless of the storage temperature of the used LFDs. However, refrigerated storage is recommended for RT-PCR amplification of long fragments for phylogenetic analysis.

Rabies is a fatal zoonotic disease caused by the rabies virus (RABV), with almost 100% mortality if proper post-exposure prophylaxis (PEP), consisting of rabies immunoglobulin (RIG) and rabies vaccine, is not applied in a timely manner. However, this is challenged by the limited availability of RIG, especially in resource-constrained countries. In this study, we assessed the scope of the antiviral drug favipiravir to treat rabies-infected mice as an alternative to RIG. Category III-like wounds were induced in RABV-challenged mice treated with favipiravir instead of RIG in the PEP regimen. The use of favipiravir followed by rabies vaccine provided complete protection against rabies-related death in 100% of mice, even after RABV propagated to the central nervous system during infection. Additionally, the virus-neutralizing antibody titer in the favipiravir and vaccine group was significantly higher than that of the RIG and vaccine recipients. The use of favipiravir with rabies vaccine seemingly prevents fatal outcomes and even rescues the cases that already express clinical symptoms. A clinical trial of this approach is warranted, especially in countries with low RIG availability., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: T. Komeno, N. Nakajima and Y. Furuta are employees of FUJIFILM Toyama Chemical Co., Ltd., which is the producer of favipiravir. A. Nishizono received the funding to perform this study from FUJIFILM Toyama Chemical Co., Ltd. K. Kimitsuki, S. Khan, R. Kaimori, T. Yahiro, N. Saito, K. Yamada, B. P. Quiambao, P. Virojanapirom, T. Hemachudha have no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Rabies is a type of acute fetal encephalitis caused by rabies virus (RABV). While it becomes incurable after symptom onset, it can be prevented by post-exposure prophylaxis (PEP) during the long incubation period. While preclinical diagnosis aids the appropriate PEP administration, it is mostly nonfeasible owing to the absence of viremia or a specific antibody response during the incubation period. Here, an attempt was made to identify a serum biomarker for the preclinical diagnosis of rabies. Using the serum from a mouse inoculated intramuscularly (i.m.) with 5 × 10 5 focus-forming units (FFU) of recombinant RABV expressing red firefly luciferase (1088/RFLuc) immediately before symptom onset, two-dimensional differential gel electrophoresis was conducted, followed by mass spectrometry, and it was confirmed that apolipoprotein A1 (ApoA1) was up-regulated. ELISA showed that the serum ApoA1 and specific antibody levels increased during the incubation period and on the day of symptom onset. Since a lower infectious dose can be used to induce the unstable and long incubation period generally observed in natural infection, the ApoA1 level in mice inoculated i.m. with 10 3  FFU of 1088/RFLuc was examined by monitoring viral dynamics using in vivo imaging. The serum ApoA1 and specific antibody levels were up-regulated in 50% and 58.3% of mice exhibiting robust RABV replication, respectively, but not in mice exhibiting weak RABV replication. In addition, it was reported that ApoA1 was found to be a biomarker for neuronal damage. Additional biomarker candidates will be needed for the effective preclinical diagnosis of rabies., (© 2021 The Authors. Microbiology and Immunology published by The Societies and John Wiley & Sons Australia, Ltd.)

Implementation of lateral flow devices (LFDs) for rabies antigen detection is expected to improve surveillance through the efficient detection of rabid animals in resource-limited settings; however, the use of LFDs for diagnosis remains controversial because some commercially available kits show low sensitivity. Therefore, we compared the diagnostic efficacy of three LFDs (ADTEC, Bionote, and Elabscience kits) paralleled with the direct fluorescent antibody test (dFAT) using fresh samples and investigated the diagnostic accuracies. To do so, we evaluated rabies-suspected samples submitted to the Regional Animal Disease Diagnostic Laboratory III, Philippines. Furthermore, we conducted real-time RT-PCR and sequencing to measure the accuracy of field laboratory diagnosis. The total number of animals submitted during this study period was 184 cases, including negative control samples. Of these, 53.9% (84 cases) were positive in the dFAT. Dogs were the most common rabies-suspected animal (n = 135). The sensitivities of the ADTEC and Bionote kits were 0.88 (74 cases) and 0.95 (80 cases), respectively. The specificity of both kits was 1.00 (100 cases). Furthermore, the sensitivity and specificity of the ADTEC kit after directly homogenizing the samples in assay buffer without dilution in phosphate-buffered saline (ADTEC kit DM) were 0.94 (79 cases) and 1.00 (100 cases), respectively. By contrast, there were no positive results using the Elabscience kit among all dFAT-positive samples. The sensitivity and specificity of LFDs make these tests highly feasible if properly used. Therefore, LFD tests can be used to strengthen the surveillance of rabies-infected animals in endemic and resource-limited settings., Competing Interests: The authors have declared that no competing interests exist.

Rabies virus (RABV) is a highly neurotropic virus and the causative agent of rabies, an encephalitis with an almost 100% case-fatality rate that remains incurable after the onset of symptoms. Favipiravir (T-705), a broad-spectrum antiviral drug against RNA viruses, has been shown to be effective against RABV in vitro but ineffective in vivo. We hypothesized that favipiravir is effective in infected mice when RABV replicates in the peripheral tissues/nerves but not after virus neuroinvasion. We attempted to clarify this point in this study using in vivo bioluminescence imaging. We generated a recombinant RABV from the field isolate 1088, which expressed red firefly luciferase (1088/RFLuc). This allowed semiquantitative detection and monitoring of primary replication at the inoculation site and viral spread in the central nervous system (CNS) in the same mice. Bioluminescence imaging revealed that favipiravir (300 mg/kg/day) treatment commencing 1 h after intramuscular inoculation of RABV efficiently suppressed viral replication at the inoculation site and the subsequent replication in the CNS. However, virus replication in the CNS was not inhibited when the treatment began 2 days after inoculation. We also found that higher doses (600 or 900 mg/kg/day) of favipiravir could suppress viral replication in the CNS even when administration started 2 days after inoculation. These results support our hypothesis and suggest that a highly effective drug-delivery system into the CNS and/or the enhancement of favipiravir conversion to its active form are required to improve favipiravir treatment of rabies. Furthermore, the bioluminescence imaging system established in this study will facilitate the development of treatment for symptomatic rabies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: The Philippines is ranked among the top countries with 200–300 annual deaths due to rabies. Most human rabies cases have been reported in remote areas, where dog surveillance is inadequate. Therefore, a strategy to effectively improve surveillance in remote areas will increase the number of detections. Detecting pathogens using portable real-time reverse transcription-polymerase chain reaction (RT-PCR) has the potential to be accepted in these areas. Thus, we aimed to develop an assay to detect the rabies virus (RABV) genome by combining the robust primer system LN34 with the PicoGene PCR1100 portable rapid instrument targeting RABV RNA (PCR1100 assay). Methods: Procedures were optimised using an LN34 primer/probe set, KAPA3G Plant PCR Kit (KAPA Biosystems), FastGene Scriptase II (NIPPON Genetics), and an artificial positive control RNA. Results: Positive control RNA showed an analytical limit of detection of 10 copies/µL without false positivity, generating results in approximately 32 min. Compared to dFAT or RT-qPCR using field samples, the sensitivity and specificity of the PCR1100 assay were 100%, and even lower copy numbers (approximately 10 copies/µL) were detected. Conclusions: This study demonstrated that the developed assay can detect rabies RNA in field samples. Because dog-mediated rabies is endemic in remote areas, the rapidity, mobility, and practicality of the PCR1100 assay as well as the high sensitivity of the LN34 system make it an ideal tool for the confirmation of rabies in these areas. [ABSTRACT FROM AUTHOR]