Diagnosis Of Rabies Research Articles

Analysis of rabies diagnosis in dogs and cats in the state of São Paulo, Brazil.

The genetic lineage of rabies virus (RABV) associated with dogs has not been found in the state of São Paulo since 1998, and all cases of rabies in domestic animals reported since then have involved the RABV lineage that circulates in bats. As there has been a change in the rabies transmission cycle in cats and dogs, we decided to analyze the tests used to diagnose rabies in these animals in the 15-year period from 2002 to 2016 in the state of São Paulo. During this period, 85,508 central nervous system (CNS) samples from dogs and cats were submitted to the Rabies Diagnosis Section at the Pasteur Institute of São Paulo for testing. All of the samples were tested by the fluorescent antibody test (FAT) and at least one of the following three tests: mouse inoculation test (MIT), rabies tissue culture infection test (RTCIT) and reverse transcription polymerase chain reaction (RT-PCR). Of all the samples tested, twenty were positive in at least one of these assays. Four other positive samples were identified at other institutions in the state of São Paulo. Of the twenty samples that tested positive at the Pasteur Institute of São Paulo, nine were tested by FAT, and the results were subsequently confirmed by other techniques; five gave inconclusive results, and therefore, other techniques had to be used as soon as possible in case the samples were positive; and six were negative by FAT and positive by one or more of the following tests: RTCIT, MIT and RT-PCR. Genetic typing of isolates from eighteen samples identified them as the lineage circulating in bats. In light of this finding, which indicates that genetic lineages associated with bats are circulating in domestic animals in the state of São Paulo, when the results of FAT carried out with samples from aggressive cats and dogs are inconclusive, complementary tests should be used. Decomposing samples and samples for which FAT was inconclusive should be tested using molecular techniques so that a definitive result can be obtained quickly and timely post-exposure prophylaxis can be administered to exposed individuals.

EXTENSIVE MUSCLE SPASMS AFTER UNPROVOKED DOG BITE: IS IT RABIES?

We report a 7-year-old incompletely immunized male child who presented with intermittent dyspnea, fever, and extensive muscle spasms following an alleged history of unprovoked Category III dog bite 20 days back with inadequate post-exposure prophylaxis. This case highlights the importance of detailed clinical evaluation including minor signs such as trismus, persistent tetanic muscle contraction in intervening period between muscle spasms and preservation of normal sensorium in the absence of aerophagia and hydrophobia in ruling out a possible diagnosis of rabies in this child, and establishment of diagnosis of less sinister treatable disease with better prognosis.

Rabies of canid biotype in wild dog (Lycaon pictus) and spotted hyaena (Crocuta crocuta) in Madikwe Game Reserve, South Africa in 2014-2015: Diagnosis, possible origins and implications for control.

Both domestic and wild carnivore species are commonly diagnosed with rabies virus (RABV) infection in South Africa. Although the majority of confirmed rabies cases in wild carnivore species are reported from the yellow mongoose (Cynictis penicillata), the rest are from other wild carnivores including the highly endangered wild dog (Lycaon pictus). Lyssavirus infection was confirmed in two wild dogs and a spotted hyaena (Crocuta crocuta) in the Madikwe Game Reserve, North West province in South Africa, in 2014 and 2015, using a direct fluorescent antibody test and immunohistochemistry. There had been no new wild dog introductions to the Madikwe Game Reserve for many years and the wild dogs were last vaccinated against rabies approximately 11 years prior to the incident. The first euthanised wild dog was the last surviving of a break-away pack of 6, and the second was the last of a larger pack of 18, the rest of which died with no carcasses being found or carcasses too decomposed for sampling. Subsequent antigenic typing of the lyssaviruses indicated that they were canid RABVs. The RABVs originating from 22 wild carnivore species, 7 dogs, and a caprine, mostly from the North West province, were genetically characterised by targeting a partial region of the nucleoprotein gene. The nucleotide sequence analyses of these viruses and two previously characterised RABVs confirmed that the outbreak viruses were also canid rabies, phylogenetically clustering with virus isolates originating from black-backed jackals recovered between 2012 and 2015 from the North West province, and domestic dogs from neighbouring communal areas. The source(s) of the mortalities and possible reservoir host(s) for the virus could only be speculated upon from data on specific predator numbers, movements and behaviour, kills, park management and the changing environmental ecology, which were monitored closely in Madikwe over several years. The most likely rabies sources were from boundary fence contacts between wild carnivores within the park, with domestic dogs or cats and/or naturally occurring wild carnivores outside the park. The associated risk of zoonotic infection and threat to important and endangered predators may be mitigated through regional rabies control primarily in domestic dogs and cats, as well as by preventative vaccination of at-risk park employees and their pets. The importance of ongoing prophylactic rabies protection by regular vaccination of highly endangered wildlife carnivores and the submission of carcasses for rabies diagnosis of any wild or domestic animals behaving uncharacteristically or found dead is emphasised.

Evaluation of Enzyme Linked Immuno-Sorbent Assay and Rapid Immuno-Diagnostic Test for Rabies Antigen Detection in Archived Dog Brain Tissues

Abstract Rabies urgently requires strengthening of new and existing diagnostic methodology in order to overcome the threat it poses. We evaluated the Enzyme Linked Immuno-Sorbent Assay (ELISA) and the Rapid Immunodiagnostic Test (RIDT) in detecting rabies viral antigens, comparing both tests with the Direct Fluorescent Antibody Test (DFAT) which is the gold standard in rabies diagnosis. Fifty dog brain tissues collected from the archives of the Central Diagnostic Laboratory, National Veterinary Research Institute, Vom, Nigeria, were utilized for this study. ELISA performed better than RIDT and recorded equivalent result with DFAT as compared with RIDT. There was a 96 % agreement between ELISA and DFAT for rabies antigen detection (concordance coefficient 78 % : 95 % C. I. 0.6366 to 0.8654) while there was a 54 % agreement between RIDT and DFAT (concordance coefficient 17 % : 95 % C. I. 0.05138—0.2752). Compared to DFAT, the sensitivities of ELISA and RIDT were 95.5 % and 47.6 %, respectively, and the specificities of ELISA and RIDT were 100 % and 87.5 % respectively. The simple Cohen’s kappa coefficient for ELISA related to the DFAT was found to be 0.834 (95 % C. I. 0.613—1.0). For RIDT, the Kappa value was 0.170 (95 % C. I. 0.003—0.337). The ELISA is as reliable a diagnostic method as the DFAT which is the gold standard for rabies diagnosis. It has an advantage of being able to analyse large number of samples at the same time, making it more suitable for epidemiological studies and for laboratories that cannot perform the DFAT. The unsatisfactory result of RIDT in this study reiterates the need to perform an adequate test validation before it can be used in the laboratory for rabies diagnosis.

Application and Comparative Evaluation of Fluorescent Antibody, Immunohistochemistry and Reverse Transcription Polymerase Chain Reaction Tests for the Detection of Rabies Virus Antigen or Nucleic Acid in Brain Samples of Animals Suspected of Rabies in India.

Accurate and early diagnosis of animal rabies is critical for undertaking public health measures. Whereas the direct fluorescent antibody (DFA) technique is the recommended test, the more convenient, direct rapid immunochemistry test (dRIT), as well as the more sensitive, reverse transcription polymerase chain reaction (RT-PCR), have recently been employed for the laboratory diagnosis of rabies. We compared the three methods on brain samples from domestic (dog, cat, cattle, buffalo, horse, pig and goat) and wild (leopard, wolf and jackal) animals from various parts of India. Of the 257 samples tested, 167 were positive by all the three tests; in addition, 35 of the 36 decomposed samples were positive by RT-PCR. This is the first study in which such large number of animal samples have been subjected to the three tests simultaneously. The results confirm 100% corroboration between DFA and dRIT, buttress the applicability of dRIT in the simple and rapid diagnosis of rabies in animals, and reaffirm the suitability of RT-PCR for samples unfit for testing either by DFA or dRIT.

Evaluation of monoclonal antibody–based direct, rapid immunohistochemical test for rabies diagnosis

Rabies is a major public health problem in developing countries in Asia and Africa. Although a number of laboratory diagnoses can be used for rabies control, the WHO and OIE recommended gold standard for rabies diagnosis is the direct fluorescent antibody test (FAT). However, FAT is not widely used in developing countries because of deficient financial sources to procure fluorescent microscope. Recently the direct rapid immunohistochemical test (dRIT) has been developed and has a worldwide promising application, particularly in developing countries, since its result can be read by inexpensive light microscopy, in addition to be consistent with that of FAT. However, no commercial conjugated antibody is available to meet the laboratory demand. We describe here the production of a monoclonal antibody (MAb) against rabies virus (RABV) N protein and its use as a biotinylated conjugate in a dRIT. Tested against a batch of 107 brain specimens representing a wide phylogenetic diversity of RABV collected from different animal species with multiple geographical origins in China, results showed that the dRIT had 100% specificity (95% CI 0.93–1.00) and 96.49% sensitivity (95% CI 0.88–1.00) as compared with the gold standard FAT. It therefore provides a simple, economical alternative to FAT, particularly for use in rabies diagnosis in developing countries.

Serological Survey of Rabies Virus Infection among Bats in Brazil

Rabies is a disease caused by a virus belonging to the family Rhabdoviridae, genus Lyssavirus (RABV), and is characterized as a progressive and acute encephalitis that presents high lethality. Between 2002 and 2012, 2149 wild animals were found positive for RABV in Brazil where 79% were associated with the aerial cycle of the disease. As part of the active surveillance of rabies in Brazil, the objective of this study was to investigate the presence of RABV infection in bats captured in two different regions of Brazil. Bat specimens were collected in (i) the metropolitan region of Rio de Janeiro city, (ii) Igrapiúna Municipality, Bahia State, and (iii) Serra do Tabuleiro State Park (PEST) in Santa Catarina State. Forty-four bats from the two first areas were tested for RABV at Institute Jorge Vaitsman (IJV), reference laboratory for rabies in Rio de Janeiro State. Of the individuals captured and sent for the diagnosis of rabies, a total of 15 were identified as being hematophagous, all of them belonging to the species Desmodus rotundus. Twenty-four individuals were considered as frugivorous, four as insectivores and one individual (Lonchophylla peracchii) identified as having a pollen/nectar feeding habit. All specimen sent to RABV diagnosis presented negative results. Considering the active surveillance for RABV in Brazil, studies of fauna survey with serological characterization are important in the strengthening of epidemiological surveillance and protection of human life.

Molecular approach for ante-mortem diagnosis of rabies in dogs.

Background & objectives:The ante-mortem diagnosis of rabies is of great significance in establishing the status of infection in dogs, especially since they are involved in exposure to human beings. The present study was, therefore, undertaken to elucidate the most appropriate secretion/tissue for reliable diagnosis of rabies in 26 living dogs suspected to be rabid.Methods:In the present study 26 dogs suspected to have rabies were included for ante-mortem diagnosis of rabies in clinical samples of skin and saliva by molecular approach viz. heminested reverse-transcriptase polymerase chain reaction (HnRT-PCR). Skin and saliva samples were collected from 13 dogs each.Results:Of the 13 clinically suspected dogs, fluorescent antibody technique (FAT) confirmed rabies in nine cases of dogs. Of these nine true-positive dogs, eight cases could be confirmed by HnRT-PCR from skin. Of the other 13 dogs clinically suspected for rabies, FAT confirmed rabies in 10 cases. Of these 10 true-positive dogs, rabies was detected ante-mortem by HnRT-PCR from the saliva in seven dogs. Thus, rabies was detected from skin with 90 per cent sensitivity, 100 per cent specificity and 92.85 per cent accuracy. With saliva, rabies was detected with a sensitivity of 76.92 per cent, specificity of 100 per cent and accuracy of 62.50 per cent. The positive predictive values were 100 per cent for both skin and saliva samples while negative predictive values were 80 and 50 per cent, respectively.Interpretation & conclusions:Skin biopsy may be more appropriate clinical sample as compared to saliva for ante-mortem diagnosis of rabies in dogs. HnRT-PCR can be employed for molecular diagnosis of rabies from skin in live dogs.

Comparison of Clinico-Pathological and Immunopathological Techniques for Diagnosis of Rabies in Dogs

Comparison of Clinico-Pathological and Immunopathological Techniques for Diagnosis of Rabies in Dogs

Results of laboratory diagnosis of rabies in herbivores (cattle and horses): a retrospective study

Rabies is a zoonosis of extreme importance to public health. Specifically in herbivores, it causes economic losses, considering its lethality. The diagnosis provides information to describe the epidemiology of the disease in the regional study. For this purpose, a survey of Central Nervous System (CNS) materials, received for diagnosis in the Center-West region of the State of Sao Paulo, by the Zoonosis Diagnostic Service (SDZ), Faculdade de Medicina Veterinaria e Zootecnia (FMVZ) - UNESP-Botucatu-SP. In the period from 2001 to 2015, a total of 643 samples were analyzed, being 489 (76.05%) of the bovine species, with 68 (13.9%) positive samples and 154 (23.95%) of the equine species with 17 (11.3%) positive. For the analyzed bovine samples, sensitivity and specificity values of the fluorescent antibody test (FAT) were 88.24 and 99.52% respectively and for equines, 47.06 and 100.0%. The frequency of samples received by species, month, and year and the results of the diagnostic tests recommended by the World Health Organization (WHO), fluorescent antibody test (FAT) and the mouse inoculation test (MIT), were analyzed.

Development and validation of an immunoperoxidase antigen detection test for improved diagnosis of rabies in Indonesia.

Rabies continues to pose a significant threat to human and animal health in regions of Indonesia. Indonesia has an extensive network of veterinary diagnostic laboratories and the 8 National laboratories are equipped to undertake diagnostic testing for rabies using the commercially-procured direct fluorescent antibody test (FAT), which is considered the reference (gold standard) test. However, many of the Indonesian Provincial diagnostic laboratories do not have a fluorescence microscope required to undertake the FAT. Instead, certain Provincial laboratories continue to screen samples using a chemical stain-based test (Seller’s stain test, SST). This test has low diagnostic sensitivity, with negative SST-tested samples being forwarded to the nearest National laboratory resulting in significant delays for completion of testing and considerable additional costs. This study sought to develop a cost-effective and diagnostically-accurate immunoperoxidase antigen detection (RIAD) test for rabies that can be readily and quickly performed by the resource-constrained Provincial laboratories. This would reduce the burden on the National laboratories and allow more rapid diagnoses and implementation of post-exposure prophylaxis. The RIAD test was evaluated using brain smears fixed with acetone or formalin and its performance was validated by comparison with established rabies diagnostic tests used in Indonesia, including the SST and FAT. A proficiency testing panel was distributed between Provincial laboratories to assess the reproducibility of the test. The performance of the RIAD test was improved by using acetone fixation of brain smears rather than formalin fixation such that it was of equivalent accuracy to that of the World Organisation for Animal Health (OIE)-recommended FAT, with both tests returning median diagnostic sensitivity and specificity values of 0.989 and 0.993, respectively. The RIAD test and FAT had higher diagnostic sensitivity than the SST (median = 0.562). Proficiency testing using a panel of 6 coded samples distributed to 16 laboratories showed that the RIAD test had good reproducibility with an overall agreement of 97%. This study describes the successful development, characterisation and use of a novel RIAD test and its fitness for purpose as a screening test for use in provincial Indonesian veterinary laboratories.

Evaluation of Direct Rapid Immunohistochemistry Test (DRIT) for Postmortem Diagnosis of Rabies

Direct fluorescent antibody test (DFAT) is considered as the gold standard for diagnosis of rabies in infected mammals as it has high sensitivity and specificity. However, high cost and technical demand limits its utilization, particularly in developing countries including India. Therefore, in this study we evaluated recently developed direct rapid immunohistochemistry test (DRIT) for diagnosis of rabies in comparison with the DFAT. A total of 109 brain samples received during the period of 6 years from different regions of India were tested following standard protocol. The results showed 100% correlation between the two tests.

Development of molecular confirmation tools for swift and easy rabies diagnostics

BackgroundAs rabies still represents a major public threat with tens of thousands of deaths per year, particularly in developing countries, adequate surveillance based on rapid and reliable rabies diagnosis for both humans and animals is essential. Rabies diagnosis relies on highly sensitive and specific laboratory tests for detection of viral antigens. Among those tests, at present the immunofluorescence antibody test is the “gold standard test” for rabies diagnosis, followed by virus isolation in either mice or cell culture. Because of the advantages of molecular assays in terms of sensitivity and applicability their approval as confirmatory diagnostic test by international organizations (OIE, WHO) is envisaged. Therefore, the objective was to develop and validate novel molecular assays and RNA extraction methods for rabies that reduce the turnaround time but remain highly sensitive and specific.MethodsHere, novel assays, i.e. HighSpeed RT-qPCR and isothermal recombinase polymerase amplification (RPA) were designed and tested. Furthermore, three magnetic bead-based rapid extraction methods for manual or automated extraction were validated and combined with the new downstream assays.ResultsWhile the conventional column based RNA extraction method showed the highest intra-run variations, all magnetic bead-based rapid extraction methods delivered nearly comparable sensitivity and efficiency of RNA recovery. All newly developed molecular tests were able to detect different rabies virus strains in a markedly reduced timeframe in comparison to the standard diagnostic assays. The observed detection limit for the HighSpeed RT-qPCR was 10 genome copies per reaction, and 1000 genome copies per reaction for the RPA assay.ConclusionMagnetic bead-based rapid RNA extraction methods are highly sensitive and show a high level of reproducibility and therefore, are particularly suitable for molecular diagnostic assays including rabies. In addition, with a detection limit of 10 genome copies per reaction, the HighSpeed RT-qPCR is suitable for rapid ante mortem rabies diagnosis in humans as well as confirmatory test in integrated bite management and subsequent post-exposure prophylaxis.

Development and evaluation of a RT-qPCR assay for fast and sensitive rabies diagnosis

Rabies virus is endemic to Russia, among other countries. It is therefore critical to develop a high-quality and high-precision diagnostic procedure for the control and prevention of infection.The main objective of the research presented here was to develop a reliable RT-qPCR assay for rabies diagnostics.For this purpose, a RABV strains from various biological and geographical origins were used. In addition, rabies-positive and rabies-negative samples, as well as nucleic acids from other viruses and DNA extracted from the brain tissues of mice, dogs, cats, bats and humans, were studied using the developed assay.The analytical sensitivity of the assay, as assessed using armored recombinant positive control dilutions, was 103 copies/ml, and the sensitivity measured using characterized strains was between 0.1 LD50/ml and 1.0 LD50/ml. A broad range of RNA from RABV strains circulating in different regions of Russia, as well as RNA from RABV-positive primary brain samples from 81 animals and two humans, was detected using the developed assay. No false-positive or false-negative results were obtained.Given that high analytical and diagnostic sensitivities and a high specificity were verified for this assay, it has high potential as a screening test that may be suitable for the epizootiological monitoring of animals and for the fast postmortem diagnosis of rabies.

Cost analysis: Rabies tissue culture infective test versus mouse inoculation test used in rabies diagnosis in Romania

Cost analysis: Rabies tissue culture infective test versus mouse inoculation test used in rabies diagnosis in Romania

Enhanced Rabies Surveillance to Support Effective Oral Rabies Vaccination of Raccoons in the Eastern United States.

Enhanced rabies surveillance (ERS) is essential for sound oral rabies vaccination (ORV) decisions to prevent the spread of specific rabies virus variants in meso-carnivores and to achieve disease elimination. Use of a direct rapid immunohistochemistry test (dRIT) in North America for timely, accurate rabies diagnosis in the field has facilitated greater ERS emphasis since 2005. ERS used in tandem with exposure-based public health surveillance provides a comprehensive understanding of the geographic distribution of rabies as an aid to formulate effective management strategies for raccoons and other meso-carnivores. In 2015, best management practices were implemented for improving, reinvigorating, and standardizing ERS. A point system for weighing ERS sample categories was evaluated, to determine whether sampling emphasis should be focused upon ill or strange-acting animals, the highest quality category. During 2016, 70.7% of rabid animals detected through ERS in raccoon rabies management states were obtained from strange-acting animals, followed by animals found dead (14.1%), road kills (9.1%), and nuisance-collected specimens (6.1%). Sample category weights may be adjusted based on additional evaluation to ensure continued emphasis on the highest value samples. High quality ERS, in conjunction with serologic evidence of population-based immunity, form the backbone for ORV decisions in the elimination of raccoon rabies.

Epidemiology of Rabies in Lesotho: The Importance of Routine Surveillance and Virus Characterization.

Rabies is widespread throughout Africa and Asia, despite the fact that the control and elimination of this disease has been proven to be feasible. Lesotho, a small landlocked country surrounded by South Africa, has been known to be endemic for rabies since the 1980s but the epidemiology of the disease remains poorly understood due to limited sample submission, constrained diagnostic capabilities, and a lack of molecular epidemiological data. Considering the existing challenges experienced in Lesotho, we aimed to evaluate the direct, rapid immunohistochemical test (DRIT) as an alternative to the direct fluorescent antibody (DFA) test for rabies diagnosis in Lesotho. Towards this aim, extensive training on the implementation and interpretation of the DRIT was hosted in Lesotho in April 2016 before both tests were applied to all samples subjected to routine rabies diagnosis at the Central Veterinary Laboratory (CVL). We found agreement between the DFA and DRIT assays in 90/96 samples (93.75%). The samples that produced inconsistent results (n = 6) were re-tested a further two times with both assays before being subjected to a real-time qPCR to confirm the diagnosis. Additionally, a statistically significant three-fold increase in the average number of samples submitted per month was observed after the DRIT implementation started, following continuous rabies awareness initiatives amongst the animal health professionals in the country over a 12-month period (p = 0.0279). Partial G-L intergenic regions of selected rabies-positive samples (n = 21) were amplified, sequenced, and subjected to phylogenetic analyses. Molecular epidemiological analyses, which included viruses from neighbouring provinces in South Africa, suggested that at least three independent rabies cycles within Lesotho were implicated in instances of cross-border transmission. This study has evaluated alternative methods for diagnosing and improving rabies surveillance in Lesotho, as well as providing new information that would be of importance in the planning of future disease intervention campaigns, not only in Lesotho, but also in neighbouring South Africa.

Development of an immunoenzyme test system for diagnosis if rabies

Rabies remains one of the most serious and pressing problems for veterinary science and medicine. According to the data of Rosselkhoznadzor Information and analytical center, the Russian Federation has been an area of natural focal endemicity for many years [13]. An important stage in the control and eradication of rabies in its laboratory diagnosis by detection of virus antigen in the pathological material. The aim of the study was to design an enzyme-linked immunosorbent assay (ELISA) test kit intended for the laboratory diagnosis of rabies in agricultural, domestic, and wild carnivores. Obtaining specific components for the kit, the GNKI “Sheep” master seed strain of rabies virus, the standard CVS strain of rabies virus, and white mice of the BALB line were used.In order to obtain high-activity of specific immunoglobulins (Igs ), the processes of antirabies serum production, immunoglobulin isolation, and purification have been improved as well as the conditions for immunoglobulin conjugation with peroxidase. It was found that the binding ratio should not to be lower than 0.45–0.6.A method of inactivation of rabies virus antigen has been developed, which eliminates the risk of being infected for laboratory workers and shows high specific activity in serological tests. The optimal concentrations of the reactants (immunoglobulin, antigen, conjugated) were determined; basic conditions for conducting the reaction were identified (pH of the carbonate/bicarbonate coating buffer, the optimum temperature, and duration of incubation) that ensure the required level of accuracy and sensitivity of the method.As a result of the conducted studies, a "Kit of preparations for the laboratory diagnosis of animal rabies in animals by ELISA method" was developed. The correlation between results obtained by the immunofluorescence (IF) method and the enzyme-linked immunoadsorbent assay (ELISA) methods was determined; the compliance coefficient constituted 98.8%, the sensitivity threshold of ELISA method being 3.3 Ig LD50 / ml. The sensitivity, specificity, and reproducibility of the developed test system were determined through laboratory and production tests.Â

Pathobiological investigation of naturally infected canine rabies cases from Sri Lanka

BackgroundThe recommended screening of rabies in ‘suspect’ animal cases involves testing fresh brain tissue. The preservation of fresh tissue however can be difficult under field conditions and formalin fixation provides a simple alternative that may allow a confirmatory diagnosis. The occurrence and location of histopathological changes and immunohistochemical (IHC) labelling for rabies in formalin fixed paraffin embedded (FFPE) canine brain is described in samples from 57 rabies suspect cases from Sri-Lanka. The presence of Negri bodies and immunohistochemical detection of rabies virus antigen were evaluated in the cortex, hippocampus, cerebellum and brainstem. The effect of autolysis and artefactual degeneration of the tissue was also assessed.ResultsRabies was confirmed in 53 of 57 (93%) cases by IHC. IHC labelling was statistically more abundant in the brainstem. Negri bodies were observed in 32 of 53 (60.4%) of the positive cases. Although tissue degradation had no effect on IHC diagnosis, it was associated with an inability to detect Negri bodies. In 13 cases, a confirmatory Polymerase chain reaction (PCR) testing for rabies virus RNA was undertaken by extracting RNA from fresh frozen tissue, and also attempted using FFPE samples. PCR detection using fresh frozen samples was in agreement with the IHC results. The PCR method from FFPE tissues was suitable for control material but unsuccessful in our field cases.ConclusionsHistopathological examination of the brain is essential to define the differential diagnoses of behaviour modifying conditions in rabies virus negative cases, but it is unreliable as the sole method for rabies diagnosis, particularly where artefactual change has occurred. Formalin fixation and paraffin embedding does not prevent detection of rabies virus via IHC labelling even where artefactual degeneration has occurred. This could represent a pragmatic secondary assay for rabies diagnosis in the field because formalin fixation can prevent sample degeneration. The brain stem was shown to be the site with most viral immunoreactivity; supporting recommended sampling protocols in favour of improved necropsy safety in the field. PCR testing of formalin fixed tissue may be successful in certain circumstances as an alternative test.

An inter- laboratory proficiency testing exercise for rabies diagnosis in Latin America and the Caribbean.

The direct fluorescent antibody test (DFA), is performed in all rabies reference laboratories across Latin America and the Caribbean (LAC). Despite DFA being a critical capacity in the control of rabies, there is not a standardized protocol in the region. We describe the results of the first inter-laboratory proficiency exercise of national rabies laboratories in LAC countries as part of the regional efforts towards dog-maintained rabies elimination in the American region. Twenty three laboratories affiliated to the Ministries of Health and Ministries of Agriculture participated in this exercise. In addition, the laboratories completed an online questionnaire to assess laboratory practices. Answers to the online questionnaire indicated large variability in the laboratories throughput, equipment used, protocols availability, quality control standards and biosafety requirements. Our results will inform actions to improve and harmonize laboratory rabies capacities across LAC in support for the regional efforts towards elimination of dog-maintained rabies.