Detection Of Rabies Virus Research Articles

<i>In vitro</i> methods for rabies virus detection, and evaluation of their use in the production of rabies immunoglobulin

Current highly sensitive methods for rabies virus and rabies antibodies detection in biological material can be used not only for diagnosis and experimental research, but also for the production of antirabies medicines used for postexposure prophylaxis. The aim of the study was to analyse existing methods for rabies virus and rabies antibodies detection and to assess the potential for using these methods at the control stages during production of heterologous antirabies immunoglobulin obtained from equine serum. The search for cutting-edge highly sensitive in vitro control methods that could compete with the biological method, which is the main method used in antirabies immunoglobulin control, is an important prerequisite for improvement of the production technology and the quality of antirabies medicines. The study demonstrated that the following test methods can be used in the production of antirabies immunoglobulin: fluorescent antibody technique, enzyme-linked immunosorbent assay, cell culture methods, atomic force microscopy, and flow cytometry. These methods could be used alone or as an alternative to the biological method in white mice. These methods were chosen because of their high sensitivity, specificity, rapid and easy implementation, cost-effectiveness, and automatic recording of test results.

A recombinase polymerase amplification assay for rapid detection of rabies virus

Rabies is a generally fatal encephalitis caused by a negative-sense single-stranded RNA lyssavirus transmitted to humans mainly from dog bite. Despite the recommendation by WHO and OIE to use the direct immunofluorescence test as standard method, molecular diagnostic assays like reverse transcription quantitative polymerase chain reaction (RT-qPCR) are increasing as a confirmatory method. However, both technologies are inaccessible in resource-limited settings. Moreover, the available point-of-need molecular assay is of poor detection limit for African strains. Herein, we developed a reverse transcription recombinase polymerase amplification (RT-RPA) assay as potential point-of-need diagnostic tool for rapid detection of various strains of rabies virus including locally isolated African strains. The sensitivity and specificity of the method was evaluated using a molecular RNA standard and different Rabies-related viruses belonging to the Rhabdoviridea family, respectively. The RABV-RPA performances were evaluated on isolates representative of the existing diversity and viral dilutions spiked in non-neural clinical specimen. The results were compared with RT-qPCR as a gold standard. The RABV-RPA detected down to 4 RNA molecules per reaction in 95% of the cases in less than 10 min. The RABV-RPA assay is highly specific as various RABV isolates were identified, but no amplification was observed for other member of the Rhabdoviridea family. The sample background did not affect the performance of the RABV-RPA as down to 11 RNA molecules were identified, which is similar to the RT-qPCR results. Our developed assay is suitable for use in low-resource settings as a promising alternative tool for ante-mortem rabies diagnosis in humans for facilitating timely control decisions.

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.

Active surveillance for antibodies confirms circulation of lyssaviruses in Palearctic bats

BackgroundPalearctic bats host a diversity of lyssaviruses, though not the classical rabies virus (RABV). As surveillance for bat rabies over the Palearctic area covering Central and Eastern Europe and Siberian regions of Russia has been irregular, we lack data on geographic and seasonal patterns of the infection.ResultsTo address this, we undertook serological testing, using non-lethally sampled blood, on 1027 bats of 25 species in Bulgaria, the Czech Republic, Poland, Russia and Slovenia between 2014 and 2018. The indirect enzyme-linked immunosorbent assay (ELISA) detected rabies virus anti-glycoprotein antibodies in 33 bats, giving an overall seroprevalence of 3.2%. Bat species exceeding the seroconversion threshold included Myotis blythii, Myotis gracilis, Myotis petax, Myotis myotis, Murina hilgendorfi, Rhinolophus ferrumequinum and Vespertilio murinus. While Myotis species (84.8%) and adult females (48.5%) dominated in seropositive bats, juveniles of both sexes showed no difference in seroprevalence. Higher numbers tested positive when sampled during the active season (10.5%), as compared with the hibernation period (0.9%). Bat rabies seroprevalence was significantly higher in natural habitats (4.0%) compared with synanthropic roosts (1.2%). Importantly, in 2018, we recorded 73.1% seroprevalence in a cave containing a M. blythii maternity colony in the Altai Krai of Russia.ConclusionsIdentification of such “hotspots” of non-RABV lyssavirus circulation not only provides important information for public health protection, it can also guide research activities aimed at more in-depth bat rabies studies.

Neuroglia infection by rabies virus after anterograde virus spread in peripheral neurons

The highly neurotropic rabies virus (RABV) enters peripheral neurons at axon termini and requires long distance axonal transport and trans-synaptic spread between neurons for the infection of the central nervous system (CNS). Recent 3D imaging of field RABV-infected brains revealed a remarkably high proportion of infected astroglia, indicating that highly virulent field viruses are able to suppress astrocyte-mediated innate immune responses and virus elimination pathways. While fundamental for CNS invasion, in vivo field RABV spread and tropism in peripheral tissues is understudied. Here, we used three-dimensional light sheet and confocal laser scanning microscopy to investigate the in vivo distribution patterns of a field RABV clone in cleared high-volume tissue samples after infection via a natural (intramuscular; hind leg) and an artificial (intracranial) inoculation route. Immunostaining of virus and host markers provided a comprehensive overview of RABV infection in the CNS and peripheral nerves after centripetal and centrifugal virus spread. Importantly, we identified non-neuronal, axon-ensheathing neuroglia (Schwann cells, SCs) in peripheral nerves of the hind leg and facial regions as a target cell population of field RABV. This suggests that virus release from axons and infected SCs is part of the RABV in vivo cycle and may affect RABV-related demyelination of peripheral neurons and local innate immune responses. Detection of RABV in axon-surrounding myelinating SCs after i.c. infection further provided evidence for anterograde spread of RABV, highlighting that RABV axonal transport and spread of infectious virus in peripheral nerves is not exclusively retrograde. Our data support a new model in which, comparable to CNS neuroglia, SC infection in peripheral nerves suppresses glia-mediated innate immunity and delays antiviral host responses required for successful transport from the peripheral infection sites to the brain.

Confirmation of Rabies in a Stray Dog in Umuahia, Nigeria – A Case Report.

Abstract Rabies is an important zoonotic disease and the increased numbers of stray dogs constitute a constraint on the prevention of human cases in Nigeria. This report reaffirms that rabies is endemic in Nigeria and stray dogs constitute a risk to humans. A stray Nigeria indigenous dog was presented to the Veterinary Teaching Hospital, Michael Okpara University of Agriculture, Umudike Nigeria with clinical manifestations of salivation, delirium, maniac and attack displays. It was quarantined for a period of 14 days, clinical manifestations persisted and a presumptive diagnosis of end stage furious form of rabies was made. The dog was then humanely euthanized, brain sample was collected and taken to the National Veterinary Research Institute (NVRI), Vom, Nigeria for rabies virus detection using direct fluorescent antibody test (DFAT). Result revealed that brain sample was positive for canine rabies depicted by apple green fuorescence. In conclusion, the stray dog was confirmed to be positive for rabies. To the authors’ knowledge, this is the first report of confirmed rabies case in a stray dog in Umuahia, Nigeria. Hence, there should be intense public awareness on the danger of rabies since the disease remains a public health concern in Nigeria.

Abortive vampire bat rabies infections in Peruvian peridomestic livestock.

Rabies virus infections normally cause universally lethal encephalitis across mammals. However, ‘abortive infections’ which are resolved prior to the onset of lethal disease have been described in bats and a variety of non-reservoir species. Here, we surveyed rabies virus neutralizing antibody titers in 332 unvaccinated livestock of 5 species from a vampire bat rabies endemic region of southern Peru where livestock are the main food source for bats. We detected rabies virus neutralizing antibody titers in 11, 5 and 3.6% of cows, goats and sheep respectively and seropositive animals did not die from rabies within two years after sampling. Seroprevalence was correlated with the number of local livestock rabies mortalities reported one year prior but also one year after sample collection. This suggests that serological status of livestock can indicate the past and future levels of rabies risk to non-reservoir hosts. To our knowledge, this is the first report of anti-rabies antibodies among goats and sheep, suggesting widespread abortive infections among livestock in vampire bat rabies endemic areas. Future research should resolve the within-host biology underlying clearance of rabies infections. Cost-effectiveness analyses are also needed to evaluate whether serological monitoring of livestock can be a viable complement to current monitoring of vampire bat rabies risk based on animal mortalities alone.

Comparison of five different laboratory techniques for the rabies diagnosis in clinically suspected cattle in Brazil

The direct-fluorescent antibody test (dFAT) is considered the “gold standard” assay to diagnose rabies. However, it is crucial to develop molecular techniques, such as RT-PCR and RT-qPCR, since many laboratories lack the needed supplies for performing complementary methods (viral isolation, for example). For this purpose, diagnostic techniques must be specific and sensitive to guarantee accuracy. This present investigation aimed to detect rabies virus (RABV) in 126 clinically suspected cattle in Brazil using different diagnostic tests [dFAT, mouse inoculation test (MIT), immunohistochemistry (IHC), RT-PCR and RT-qPCR] and to compare those results obtained under routine laboratory conditions. The results of the present investigation demonstrate that the molecular techniques are more sensitive and may detect low viral load, even though the non-homogeneous viral distribution caused a false-negative result in dFAT. We also observed a usual alteration in antigens distribution among regions of the central nervous system (CNS). By both dFAT and IHC assays, the most reliable CNS structures were thalamus and midbrain. Although this investigation demonstrated diagnostic sensitivity and specificity close to 100 % in all laboratory techniques employed, a dFAT auxiliary test is required for bovine specimens, such as molecular techniques, when there are poor sampling conditions (low viral load combined with unavailability of brainstem structures).

Detection of rabies virus antigen in brain tissue of dogs slaughtered for human consumption in Taraba State, Nigeria

Rabies as an ancient zoonosis constitutes a threat to public health by causing over 59,000 annual human mortalities worldwide. The aim of this study was to detect rabies virus in brain tissue of dogs slaughtered for human consumption in Taraba State, Nigeria. A total of 150 dogs comprising 136 adults and 14 puppies consisting of 82 males and 68 females was sampled from slaughter points in five Local Government Areas. Brain samples were collected from each dog in labeled sterile sample bottles and screened for rabies virus antigen using direct fluorescent antibody test (DFAT). Results showed that 3 out of the 150 (2%) brain samples screened were positive for rabies virus; out of which 2 were from Unguwan Kasa (14.3%) and 1 was from Quarter Five (7.1%). This therefore suggests the presence of rabies virus in dogs slaughtered for human consumption in Taraba State, Nigeria and their role as reservoirs of the virus. Therefore, there is need for awareness education on safe handling of dog meat to minimize the risk for butchers/meat handlers.
 Keywords: Brain samples, dogs, Prevalence, Rabies, Taraba State

Evaluation of veterinary laboratory proficiency based on results of interlaboratory comparisons organized by FGBI “ARRIAH” in 2018–2019

Laboratory diagnosis is a crucial component in implementation of the set of anti-epidemic measures aimed at contagious animal disease control. The need for unswerving trust in the quality of laboratory performance is a matter of importance not only for service providers and customers, but also for inspecting organiza­tions, accreditation bodies, etc. that establish performance requirements for diagnostic laboratories. Incorrect laboratory test results can lead to a misdiagnosis and, therefore, to grave consequences. One of the forms of experimental verification of a laboratory’s performance with a view to determine the laboratory’s competence and to verify its compliance with accreditation criteria as part of inspection control of the laboratory’s activities is interlaboratory comparison. The laboratory can prove its competence at a particular time, as well as clearly demonstrate how stable the quality of its test results is by summarizing and analyzing the results of its participation in interlaboratory comparisons. The analysis of the results of the interlaboratory comparisons (programmes for detection of causative agents or antibodies to the causative agents of avian influenza, Newcastle disease, rabies, classical swine fever, African swine fever, bluetongue, lumpy skin disease) organized by the FGBI “ARRIAH” for the veterinary laboratories of the Russian Federation in 2018–2019 is presented. The results showed that most of the laboratories had passed the tests successfully. The results submitted by participants were unsatisfactory in some interlaboratory comparison programmes (rabies virus detection using fluorescent antibody technique; detection of avian influenza, classical swine fever and lumpy skin disease viruses using polymerase chain reaction). That highlights the need for those participants who failed the tests to improve their laboratory testing quality.

Synthetic oligonucleotide primers to glycoprotein gene of rabies virus and based by nested polymerase chain reaction method for RNA Rabies virus detection

One of widely used methods to detect rabies virus RNA is a reverse transcription chain polymerase reaction (RT-PCR) allowing to determine a diagnosis in 5 hours. Thus, we have elaborated the method that allows to pursue effective elicitation of RNA strains and isolates of rabies virus in pathological and clinical material as well as to decrease research timespan to 6 hours, to lower by 9.8 times the diagnostics prime cost, labor expenditures – by 40 times. The way includes performance of nested RT-PCR with oligonucleotide primers having definite nucleotide sequences and synthesized to conservative glycoprotein gene. Wherein RT-PCR is pursued in two rounds. In the case of positive reaction, fragment corresponding to the size in the first round – 755 bp, in the second round – 259 bp, is synthesized.

Proficiency test for rabies serology: Adesign complying with international standards for a reliable assessment of participating laboratories.

BackgroundDomestic carnivores can introduce rabies into disease-free countries or areas if they are incubating the disease and transported during the pre-symptomatic period. For pets moved into the European Union, the European Commission decided to establish a system of community approval of laboratories willing to carry out the rabies serological controls to guarantee an effective control system. As the specific institute to coordinate the approval of the laboratories, designated by the European Commission in 2000, our laboratory organizes annual proficiency tests (PT) for laboratories already agreed or willing to be agreed to perform rabies serological controls (by detecting rabies virus neutralizing antibodies only) in the frame of international trade.Methodology/Principal findingsThe assessment criteria of this PT rely on the analysis of the specificity and the intra-laboratory consistency. The approach used to evaluate the degree of laboratory consistency is based on the use of compiled data obtained from previous PT campaigns, and is measured by the quality of a regression model. By using historical data for calculating assigned values and associated standard deviations, instead of values obtained from only one campaign, they became robust without any additional statistical treatment. In the present paper, more than 800 historical values were compiled for each of the regression parameters.Conclusions/SignificanceSince the beginning of these PT schemes in 1999, the overall percentage of failing laboratories remained stable over the years (4.1%) while the number of participants increased to 79 in 2018. This highlighted the robustness and the consistency of the statistical analyses used to assess the laboratory’s performance over the years. The improvements carried out and the consistency of our statistical analyses have resulted in the compliance of the rabies serology PT with the ISO/IEC 17043 and ISO 13528:2015 International Standards.

Increased detection of rabies virus in bats in Ceará State (Northeast Brazil) after implementation of a passive surveillance programme.

The intensification of dog, cat and livestock vaccination campaigns significantly reduced rabies cases in humans and domestic animals in Ceará State, Brazil. However, sylvatic animals-bats (order Chiroptera), wild canids, raccoons and non-human primates- remain as reservoirs for the virus. Our hypothesis is that surveillance and monitoring of rabies virus in bats, especially passive surveillance, is of fundamental importance, besides the implementation of health education and strengthening of surveillance actions in humans exposed to aggressions. Thus, we assessed the occurrence of rabies virus in animals focusing on bats, before and after launching of the Sylvatic Rabies Surveillance Program in 2010. Surveillance data from the 184 municipalities of Ceará State were analysed, collected during the periods 2003-2010 (active surveillance) and 2011-2016 (passive surveillance), respectively. A total of 13,543 mammalian samples were received for rabies diagnosis from 2003 to 2016. Of these, 10,960 were from dogs or cats (80.9%), 1,180 from bats (8.7%), 806 from other sylvatic animals (foxes, marmosets, raccoons; 6.0%) and 597 from herbivores (cattle, goats, sheep, equines, pigs; 4.4%). A total of 588 (4.3%) samples were positive for rabies. About 8.4% (99/1,180) of the bat samples were infected with rabies virus, 92 (92.9%) of these were from non-haematophagous bat species and 7 (7.1%) from haematophagous species. The number of bat samples received and infection rates increased considerably, after a shift from active surveillance (9/355 [2.5%] samples positive), to passive surveillance (90/825 [10.9%] samples positive). Surveillance of rabies virus in bats is fundamental for human and domestic animal health in Ceará State. Bats have to be considered as targets in surveillance and control programmes. Virus lineages should be characterized to increase knowledge on transmission dynamics of sylvatic rabies virus to domestic animals and the human population, and to provide additional evidence for planning and implementation of improved control measures.

Improving human rabies post-exposure prophylaxis

In The Lancet Infectious Diseases, Tineke Cantaert and colleagues 1 Cantaert T Borand L Kergoat L et al. A 1-week intradermal dose-sparing regimen for rabies post-exposure prophylaxis (RESIST-2): an observational cohort study. Lancet Infect Dis. 2019; (published online Sept 27)https://doi.org/10.1016/S1473-3099(19)30311-1 PubMed Scopus (12) Google Scholar illustrate a strong commitment in Cambodia to preventing human rabies by rapid intradermal delivery of post-exposure prophylaxis. The investigators included 116 people bitten by rabies virus-positive dogs and 20 people bitten by rabies virus-negative dogs who attended Institut Pasteur du Cambodge, Phnom Penh, Cambodia, between April 20, 2016, and Feb 9, 2018. Patients received intradermal vaccination on days 0, 3, 7, and 28, and serum samples were obtained for detection of rabies virus neutralising antibodies on days 0, 7, 28, and 42. This well designed cohort study showed that exposed people had rabies virus neutralising antibody titres after three rabies vaccine sessions (of two intradermal doses each) that were similar to titres observed immediately before a fourth vaccine session on day 28. The authors concluded that rabies post-exposure prophylaxis could be abridged to a 1-week (three sessions of two doses on days 0, 3, and 7) intradermal regimen. Recognising the possibility of long incubation periods, the investigators contacted all patients after 1 year and found that all were still alive. These compelling data help identify the best use of human rabies post-exposure prophylaxis in developing countries, which should be used in combination with mass vaccination of dogs. A 1-week intradermal dose-sparing regimen for rabies post-exposure prophylaxis (RESIST-2): an observational cohort studyThe fourth vaccine session on day 28 provides no additional benefit. Rabies post-exposure prophylaxis can be abridged to a two-dose, three-session, 1 week regimen to improve post-exposure prophylaxis coverage and equity at no risk to patients. Full-Text PDF

Evaluation of new antibodies for the detection of rabies virus in formalin fixed brain tissue samples

Fil: Jauregui, Gloria Rosa. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion en Ciencias Veterinarias y Agronomicas; Argentina

Reverse transcription recombinase polymerase amplification assay for rapid detection of canine associated rabies virus in Africa.

Rabies is a neglected disease mostly affecting the developing world. Accurate and reliable diagnostic and surveillance data forms the foundation for the formulation and monitoring of control strategies. Although various sensitive and specific tests are available for detection of rabies virus, implementation of these tests in low-resource settings are challenging and remains limited. In this study, we describe the developed of a reverse transcription recombinase polymerase amplification assay for the detection of rabies virus. The analytical sensitivity of this assay was determined to be 562 RNA copies and was performed in 20 minutes. The diagnostic sensitivity of the RT-RPA was 100% for detection of rabies virus in field samples. In conclusion, the RT-RPA assay allowed for very quick and sensitive detection of rabies virus and could be adapted for use in low-source settings.

Evaluation of Animal Rabies Surveillance System, Ekiti State, Nigeria, 2012-2017

Evaluation of Animal Rabies Surveillance System, Ekiti State, Nigeria, 2012-2017

Development and Validation of Heminested RT-PCR and qRT-PCR Assays for Comprehensive Detection of Rabies Virus in the Suspected Rabid Brain and Saliva Samples

Background: Rabies virus (RV) is one of the most dangerous zoonotic disease and major public health problems in most of the world, especially underdeveloped countries. Rabies is preventable by proper vaccination, even shortly after exposure. Today, it seems a fast, sensitive, and reliable rabies diagnostic method is required, which might reduce the financial burden of inappropriate diagnosis. Objectives: The aim of this study was to develop and validate two molecular techniques, including heminested RT-PCR and qRT-PCR assays, for comprehensive detection of rabies virus in the suspected rabid brain and saliva samples. Methods: In this study, we developed qRT-PCR as a fast, sensitive, and specific method for rapid detection of rabies virus in brain and saliva samples. Also, the sensitivity and specificity of the method were compared with heminested RT-PCR test and direct fluorescent antibody (dFA) as a serologic gold standard method of World Health Organization (WHO) and MIT (mouse inoculation test) as a confirmatory test. Results: A combination of compatible primers based on RNA-dependent RNA polymerase (L) gene of the Pasteur virus fixed strain (PV) (accession number. M13215) was used for developing the qRT-PCR assay. Primer and probes were designed according to other Iran circulating viruses genomes that were available in public databases (GenBank). The clinical sensitivities of qRT-PCR and heminested RT-PCR methods were determined 97.14% and 94.3%, respectively. In addition, the clinical specificities of qRT-PCR and heminested RT-PCR methods were determined 93.75% and 88.24%, respectively. Also, the analytical sensitivities of qRT-PCR and heminested RT-PCR methods were about 5 × 102 and 5 × 103 FFU/mL, respectively. Conclusions: In this study, qRT-PCR assay as a diagnostic molecular method with high sensitivity and specificity was developed for the detection of the rabies virus genome in both brain and saliva samples. Therefore, this rapid, accurate, and cost-effective detection and quantification method may be used as an investigative tool, which can be valid for detection of target viral genome in the research and diagnosis field.

Detection of rabies virus in organs unrelated to the central nervous system of experimentally-inoculated vampire bats

The aim of this research was to detect rabies virus in peripheral tissues in captive vampires. Vampire bats were inoculated with 106 MICLD50 of homologous rabies virus. Bats displayed clinical signs of rabies beginning on d 8 until the 19th d post-inoculation (pi). Rabies virus antigens were found in the brain of all rabid bats. Viral RNA was detected in brain, salivary gland and tongue tissue by RT-PCR and nested PCR (nPCR). Viral genome was also detected in organs unrelated to the central nervous system. Rabies virus was not detected in saliva nor documented from any tissues without occurrence of viral antigens in the brain. Host humoral response was most pronounced via the induction of viral neutralizing antibodies (VNA) from d 8 to 20 pi, having a peak at d 14 with 0.9 IU. Antibody levels were variable, but tended to remain high after inoculation, showing significant differences to the negative control group (P=0.001). This research is one of the few recent studies focused upon Desmodus rotundus and contributes to the basic knowledge of rabies virus pathogenesis, which is required for an understanding of perpetuation in a major viral reservoir in Latin America.

Heat induced epitope retrieval for rabies virus detection by direct fluorescent antibody test in formalin-fixed dog brain tissues.

There is a great need for a chemical method of tissue preservation that would allow sample storage for extended periods at room temperature. This study aimed at retrieving and detecting rabies virus antigen by direct fluorescent antibody test (DFAT) in formalin-fixed dog brain tissues. Forty fresh dog brain specimens were collected as paired samples from rabies suspected cases that were received for postmortem detection of rabies in the Central Diagnostic Laboratory, National Veterinary Research Institute, Vom. One portion of each paired sample was prepared for fresh fluorescent antibody testing and the other portion was prepared for epitope retrieval and florescent antibody testing following fixation in 10% neutral buffered formalin. DFAT on formalin-fixed tissue exhibited a sensitivity of 100% in comparison to DFAT on fresh-tissue. No false positive result was obtained in formalin-fixed DFAT procedure, demonstrating 100% specificity. There was no apparent difference in the intensity of fluorescence in DFAT on fresh sample and formalin-fixed DFAT following heat induced epitope retrieval (concordance = 98%; 95% C.I. 0.9660 to 0.9903). The strength of agreement between DFAT on formalin-fixed and DFAT on fresh tissue was very good (Cohen’s kappa coefficient value= 1.000; 95% C.I. 1.000-1.000). This study provides new information on the retrieval of rabies antigen by heat induced epitope retrieval for DFAT on formalinized tissues. Formalin could therefore, be used henceforth to fix tissues of rabies suspected cases for routine diagnosis, transportation or archival purposes. The heat induced epitope retrieval can be routinely used to retrieve rabies virus antigen for DFAT in cases where only formalin-fixed tissues are available or when preservation by freezing is difficult.