Lateral flow devices for samples collected by straw sampling method for postmortem canine rabies diagnosis

The direct fluorescent antibody test (dFAT) using brain sample after opening the skull is the standard rabies diagnostic test in animal rabies. However, it is not feasible in many resource-limited settings. Lateral flow devices (LFD) combined with a simple sampling methodology is quicker, simpler, and less hazardous than the standard test and can be a useful tool. We conducted a prospective on-site study to evaluate the diagnostic accuracy of the LFD with the straw sampling method compared with that of the dFAT with the skull opening procedure for post-mortem canine rabies diagnosis. We collected 97 rabies-suspected animals between December 1, 2020 and March 31, 2021. Among the 97 samples, 53 and 50 cases were positive tests for dFAT and LFD, respectively. The sensitivity and specificity of LFD with straw sampling method were 94.3% (95% confidence interval [CI], 84.3–98.8%) and 100% (95% CI, 92.0–100%), respectively. The performance of LFD by the straw sampling method showed relatively high sensitivity and 100% specificity compared with that of dFAT performed on samples collected after opening the skull. This methodology can be beneficial and is a strong tool to overcome limited animal surveillance in remote areas. However, because of our limited sample size, more data using fresh samples on-site and the optimizations are urgently needed for the further implementation in endemic areas.

Skin specimen as an alternative to brain tissue for post mortem rabies diagnosis in animals to scale up animal rabies surveillance

Background: Detection of the virus or some of its specific components using WHO and OIE recommended standard laboratory tests is the only way to get a reliable diagnosis of rabies. Brain tissue is the preferred specimen for post-mortem diagnosis of rabies in both humans and animals. Higher biosecurity requirements, skill and transportation facilities required for collection and transport of brain or whole carcass to the laboratory is one of the reasons for the poor rabies surveillance in animals. Point of care testing with simple, reliable and easy to operate devices would be an ideal approach for providing rapid results. Methods: The study evaluated diagnostic performance of two reference tests, DFAT and RTPCR on skin specimen, to assess its suitability as an alternative of brain tissue for post mortem rabies diagnosis in animals. Brain tissue and skin sample belonging to different species of animals (n=90) collected at necropsy were compared using Fluorescent Antibody Test and RT PCR, internationally approved methods for rabies diagnosis. Results: Validation of RT-PCR on skin and DFAT on skin in comparison with DFAT on brain as gold standard gave a sensitivity of 98% (95% CI:94.1-100) and 80% (95%CI:71.8-88.2) respectively. Specificity was 100% in both tests. Conclusion: The findings highlight the potential of skin specimen for improving rabies surveillance in animals especially in resource poor countries.

Background and descriptive features of rabies-suspected animals in Central Luzon, Philippines

Background The Philippines is one of the major endemic countries for canine rabies in Southeast Asia. However, detailed description and analysis of laboratory-confirmed animal rabies are limited. Highly accurate surveillance requires a thorough understanding of the target area-specific problems and obstacles. Therefore, we aim to describe and analyze the rabies suspect animals in Central Luzon, Philippines, to clarify the characteristics of management and clinical signs by conducting interviews with the owners. Methods We prospectively collected information on the rabies suspect animals submitted to the Regional animal laboratory in Central Luzon through passive laboratory-based rabies surveillance between 1st April 2019 and 30th September 2020. We performed active interviews directly or telephonically with the owner. The direct fluorescent antibody test was performed on the hippocampus, brain stem, and cerebellum for laboratory confirmation. Descriptive statistics were used to characterize the number of rabies cases according to management methods and characteristics of suspected animals during the observation period. Clinical symptoms of suspected rabid animals were analyzed by univariate logistic regression analysis. Results There were 292 sample submissions during the study period. Of these, 160 were positive for dFAT. Samples of pet animals (85.3%) provided by owners or their acquaintances (59.2%) accounted for the majority of laboratory confirmed cases. Case mapping showed that more rabies-suspected cases were sent from areas near the regional laboratory than from those far from the laboratory, despite the incidence of rabies being high in these areas. The management and clinical symptoms of 227 animal cases showed that most owners were managing their animals at home and were allowing them to roam outside (69.6%) and be unvaccinated (78.9%). Rabid animals were more likely to manifest aimless running, restlessness, and agitation. Conclusions Our study provided some features of animals with laboratory-confirmed rabies in Central Luzon. However, most of the samples were submitted from areas near the rabies diagnosis laboratory, and the number of samples submitted from remote areas was low. To improve the surveillance capacity, it is necessary to increase sample submissions from remote areas.

Laboratory Diagnosis of Rabies. Current State and Trends in Development

The review considers the relevant aspects of laboratory diagnosis of rabies. The methods of laboratory diagnostics of rabies infection, standardized by WHO in 2018, and their use in the Russian Federation are presented. The scheme of laboratory diagnostics of rabies, applied by specialists of the “48th CRI” of the Ministry of Defense of Russia, for the study of biological samples from deceased people is outlined. Between 2002 and 2018, the study of biomaterial was carried out using molecular-biological, virological methods of diagnosis and in some cases electron microscopy, which allowed to detect and identify the pathogen in 257 samples from 71 people, to certify and deposit new isolates of the rabies virus. Accumulation and analysis of the lessons learned in the application of molecular-biological method of rabies diagnosis allows us to recommend the use of RT-PCR, real-time RT-PCR sets (having a certificate of state registration) in healthcare and veterinary medicine practice to identify the causative agent of rabies infection. The use of molecular-biological methods is promising in terms of the development of rabies diagnosis to improve epidemiological surveillance and raise the efficiency of the biological protection of the population of the Russian Federation.

Fatal Case of Rabies in a Captive White-Tailed Deer: A Case Report from Chiapas, Mexico

Rabies is a fatal viral infection that causes enc ephalitis in warm-blooded animals, including humans. Dog-transmitted rabies is considered eradicated in Mexico; however, rabies is not being tested in livestock with neurological symptoms (one of the main manifestations of rabies disease). In this case report, we describe a rabies case in a white-tailed deer in the Santo Domingo ranch, in Catazajá, Chiapas, Mexico, where white-tailed deer are kept under captivity, and are meant for human consumption. This is the first report of a rabies case in white-tailed deer in Mexico. We also describe the challenges to obtain a rabies diagnosis and the lack of public health policies to ensure containment of the disease, as well as the lack of awareness among farmers in the area. One single confirmed case of rabies indicates that more animals are affected by the disease. The risk for human health and economical losses will remain unknown until rabies tests are routinely performed in animals that present neurological symptoms.

Multi-annual performance evaluation of laboratories in post-mortem diagnosis of animal rabies: Which techniques lead to the most reliable results in practice?

Rabies diagnosis proficiency tests on animal specimens using four techniques (FAT, RTCIT, conventional RT-PCR and real-time RT-PCR) were organised over 10 years (2009–2019). Seventy-three laboratories, of which 59% were from Europe, took part. As the panels were prepared with experimentally-infected samples, the error rate of laboratories on positive and negative samples was accurately estimated. Based on fitted values produced by mixed modelling including the variable “laboratory” as a random variable to take into account the longitudinal design of our dataset, the technique that provided the most concordant results was conventional RT-PCR (99.3%; 95% CI 99.0–99.6), closely followed by FAT (99.1%; 95% CI 98.7–99.4), real-time RT-PCR (98.7%; 95% CI 98.1–99.3) and then RTCIT (96.8%; 95% CI 95.8–97.7). We also found that conventional RT-PCR provided a better diagnostic sensitivity level (99.3% ±4.4%) than FAT (98.7% ±1.6%), real-time RT-PCR (97.9% ±0.8%) and RTCIT (95.3% ±5.1%). Regarding diagnostic specificity, RTCIT was the most specific technique (96.4% ±3.9%) followed closely by FAT (95.6% ±3.8%), real-time RT-PCR (95.0% ±1.8%) and conventional RT-PCR (92.9% ±0.5%). Due to multiple testing of the samples with different techniques, the overall diagnostic conclusion was also evaluated, and found to reach an inter-laboratory concordance level of 99.3%. The concordance for diagnostic sensitivity was 99.6% ±2.0% and for diagnostic specificity, 98.0% ±8.5%. Molecular biology techniques were, however, found to be less specific than expected. The potential reasons for such findings are discussed herein. The regular organisation of performance tests has contributed to an increase in the performance of participating laboratories over time, demonstrating the benefits of such testing. Maintaining a high-quality rabies diagnosis capability on a global scale is key to achieving the goal of eliminating dog-mediated human rabies deaths. The regular organisation of exercises on each continent using selected local strains to be tested according to the local epidemiological situation is one factor that could help increase reliable diagnosis worldwide. Rabies diagnosis capabilities could indeed be enhanced by providing adequate and sustainable proficiency testing on a large scale and in the long term

Evaluation of Two Real-Time, TaqMan Reverse Transcription-PCR Assays for Detection of Rabies Virus in Circulating Variants from Argentina: Influence of Sequence Variation

In rabies diagnosis, it is essential to count on a rapid test to give a quick response. The combined sensitivity and robustness of the TaqMan RT-PCR assays (qRT-PCR) have made these methods a valuable alternative for rabies virus (RABV) detection. We conducted a study to compare the applicability of two widely used qRT-PCR assays targeting the nucleoprotein gene (LysGT1 assay) and leader sequences (LN34 qRT-PCR assay) of RABV genomes, in all variants circulating in Argentina. A total of 44 samples obtained from bats, dogs, cattle, and horses, that were previously tested for rabies by FAT and conventional RT-PCR, were used in the study. All variants were successfully detected by the pan-lyssavirus LN34 qRT-PCR assay. The LysGT1 assay failed to detect three bat-related variants. We further sequenced the region targeted by LysGT1 and demonstrated that the presence of three or more mismatches with respect to the primers and probe sequences precludes viral detection. We conclude that the LysGT1 assay is prone to yield variant-dependent false-negative test results, and in consequence, the LN34 assay would ensure more effective detection of RABV in Argentina.

Effect of Postmortem Degradation on the Preservation of Viral Particles and Rabies Antigens in Mice Brains. Light and Electron Microscopic Study

Rabies diagnosis is mainly made on fresh brain tissue postmortem by means of the direct immunofluorescence test. However, in some cases, it is not possible to use this technique, given that the affected nervous tissue goes through a postmortem degradation process, due to problems in the handling and transport of the samples. For this reason, the preservation in time of the rabies virus inclusions was assessed, as well as the immunoreactivity and the ultrastructure of viral particles in tissue with postmortem degradation. Brains of mice inoculated with rabies virus and control mice were processed for conventional histology, immunohistochemistry, electron microscopy, and immunoelectron microscopy in different postmortem times. In the processed tissues for hematoxylin and eosin, the presence of eosinophilic inclusions was not observed beyond 12 h postmortem. Surprisingly, the immunoreactivity of the viral antigens increased with time, at least until 30 h postmortem. It was possible to easily recognize the viral particles by means of conventional electron microscopy until 12 h postmortem. Immunoelectron microscopy allowed us to identify the presence of viral antigens disseminated in the neuronal cytoplasm until 30 h postmortem, but immunoreactive viral particles were not observed. The rabies infection did not cause histological or ultrastructural alterations different from those in the control group as a result of the postmortem degradation. In conclusion, the immunohistochemistry is a reliable test for rabies diagnosis in samples with postmortem degradation and that have been fixed with aldehydes.