Rapid Immunohistochemical Test Research Articles

Use of a Direct, Rapid Immunohistochemical Test for Diagnosis of Rabies Virus in Bats

Rabies, a zoonotic encephalitis due to transmission of a lyssavirus, such as rabies virus (RABV), has the highest case fatality of any infectious disease. A global program for the elimination of human rabies caused by dogs is proposed for realization by 2030. Sensitive, specific, and inexpensive diagnostic tests are necessary for enhanced surveillance to detect infection, inform public health and veterinary professionals during risk assessments of exposure, and support overall programmatic goals. Multiple laboratory techniques are used to confirm a suspect case of rabies. One method for the detection of lyssavirus antigens within the brain is the direct rapid immunohistochemical test (dRIT), using light microscopy, and suitable for use under field conditions. Besides dogs, other major RABV reservoirs reside among mammalian mesocarnivores and bats. To date, use of the dRIT has been applied primarily for the diagnosis of RABV in suspect mesocarnivores. The purpose of this study was to assess the usefulness of the dRIT to the diagnosis of rabies in bats, compared to the gold-standard, the direct fluorescent antibody test (DFAT). Brains of 264 suspect bats, consisting of 21 species from Arizona and Texas, were used in the evaluation of the dRIT. The overall sensitivity of the dRIT was 100% (0.969–1.0, 95% CI) and the specificity was 94.6% (0.896–0.976, 95% CI), comparable to the DFAT. This preliminary study demonstrated the utility of the dRIT in the confirmation of RABV infection in bats. Future studies should include additional geographic, lyssavirus, and mammalian species representations for broader application during enhanced rabies surveillance, with incorporation of any potential adjustments to standard protocols, as needed.

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues.

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Local Surveillance and Control of Raccoon Rabies Virus in Striped Skunks (Mephitis mephitis) in Southwestern New Brunswick, Canada.

Targeted surveillance for raccoon rabies virus was conducted between February and May 2017, near Waweig, New Brunswick, Canada, in response to detection of a rabid striped skunk (Mephitis mephitis) on 8 February 2017. A total of six skunks, 11 raccoons (Procyon lotor), and two porcupines (Erethizon dorsatum) were live-trapped, euthanized, and tested for rabies virus antigens using the direct rapid immunohistochemical test. Of these, only two skunks tested positive for rabies. All three rabid skunks came from the same location, an abandoned barn used as a denning site. Four of five skunks removed from this barn were males. Feeding, aggression, extreme response to noise and light stimuli, and exposure to porcupine quills were observed in two rabid skunks. No additional cases of rabies in wildlife were detected in the area since 8 March 2017. A targeted surveillance approach that removed potentially infected wildlife followed by localized oral rabies vaccine distribution was implemented in this locality.

Development of biotinylated polyclonal anti-ribonucleoprotein IgG for detection of rabies virus antigen by direct rapid immunohistochemical test

The direct rapid immunohistochemical test (dRIT) has been recommended for laboratorial diagnosis of rabies, especially in developing countries. The absence of commercial primary antibodies, however, still represents a major limitation to its wider use in testing. We describe here the development of a biotinylated polyclonal antibody against Rabies lyssavirus (RABV) ribonucleoprotein (RNP) and its use as a primary reagent in dRIT. Anti-RNP polyclonal horse IgG was purified by ionic exchange chromatography followed by immunoaffinity column chromatography, and its affinity, diagnostic sensitivity, and specificity were evaluated. CNS samples (120) of suspected rabies cases in different animal species were tested by dRIT, with the positive (n = 14) and negative (n = 106) results confirmed by direct fluorescence antibody testing (dFAT). Comparing the results of dRIT and dFAT, we found that the biotinylated anti-RNP IgG delivered 100% diagnostic specificity and sensibility for rabies diagnosis. Our findings show that the biotinylated anti-RNP polyclonal IgG can be produced with the quality required for application in dRIT. This work represents an important step in efforts to diagnose rabies in developing countries.

A preliminary evaluation of a locally produced biotinylated polyclonal anti-rabies antibody for direct rapid immunohistochemical test (DRIT) in the Philippines

Rabies is a fatal zoonotic disease endemic in developing countries of Asia and Africa. Recently, the direct rapid immunohistochemical test (DRIT) was recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE) as a diagnostic test for rabies. Therefore, a biotinylated polyclonal antibody (pAb) against the rabies lyssavirus (RABV) nucleoprotein was developed using a plasmid cDNA vaccine derived from a challenge virus standard 11 strain. A preliminary evaluation on the efficacy of this reagent in recognizing the Philippine RABV strain was tested using banked canine hippocampal tissue samples with DRIT and the results were compared to dFAT. The effects of acetone and formalin fixation on DRIT were also assessed through immunoreactivity scores of the specimens. Of the 142 samples examined, 104 tested positive and 38 negative using both dFAT and DRIT, showing 100% agreement between the two diagnostic procedures. Moreover, no false positive or false negative results were observed using acetone and formalin fixation. Thus, locally prepared biotinylated pAb from plasmid cDNA can be used for DRIT, especially in resource-limited laboratories in the Philippines. However, these results should be confirmed with a more thorough evaluation of this technique, and the range of detection needs to be further evaluated in a larger panel of animal samples and on other lyssaviruses.

Detection of rabies virus antigen by the indirect rapid immunohistochemistry test in equines and comparisons with other diagnostic techniques.

Laboratory diagnosis of rabies in equines is essential for distinguishing the disease from other sources of encephalitis. Diagnosis by conventional techniques such as a direct fluorescent antibody test (dFAT) or viral isolation in mice or cell culture can be difficult, and the application of molecular biological methods may be necessary. We performed an indirect rapid immunohistochemistry test (iRIT) for the detection of the rabies virus (RABV) antigen in the central nervous system (CNS) of equines and compared the results with those of other diagnostic techniques. We reviewed result records from the Rabies Diagnosis Laboratory at Instituto Pasteur, São Paulo, Brazil, of 174 samples of equine CNS from July 2014 to June 2016, which were investigated by dFAT, rabies tissue culture infection test (RTCIT), mouse inoculation test (MIT) and reverse transcription-polymerase chain reaction (RT-PCR) followed by genetic sequencing. These samples, 29 presented divergent results among techniques and were selected for the performed in the iRIT. The detected positivity rate was 4/29 (14%) by dFAT, 5/28 (18%) by RTCIT, 10/29 (35%) by MIT and 26/27 (96%) by RT-PCR. We analysed 29 samples through imprints of the cortex, hippocampus, cerebellum and brainstem in slides fixed in 10% buffered formaldehyde. Eighteen samples were identified as positive (62%) by iRIT assay, representing a greater number of positive cases than that detected by dFAT, MIT and RTCIT but not by RT-PCR. Among the brain regions, the brainstem presented the highest positivity (78%), followed by the hippocampus (69%), cerebellum (67%) and cortex (67%). Our results provide evidence that iRIT can contribute to a rapid diagnosis of rabies in equines and that complementary tests should be used to improve diagnostic accuracy in this species.

Economic and feasibility comparison of the dRIT and DFA for decentralized rabies diagnosis in resource-limited settings: The use of Nigerian dog meat markets as a case study.

BackgroundRabies lyssavirus (RABV) is the aetiologic agent of rabies, a disease that is severely underreported in Nigeria as well as elsewhere in Africa and Asia. Despite the role that rabies diagnosis plays towards elucidating the true burden of the disease, Nigeria–a country of 180 million inhabitants–has a limited number of diagnostic facilities. In this study, we sought to investigate two of the World Organization for Animal Health (OIE)-recommended diagnostic assays for rabies–viz; the direct fluorescent antibody test (DFA) and the direct rapid immunohistochemical test (dRIT) in terms of their relative suitability in resource-limited settings. Our primary considerations were (1) the financial feasibility for implementation and (2) the diagnostic efficacy. As a case study, we used suspect rabies samples from dog meat markets in Nigeria.Methods/Principal findingsBy developing a simple simulation framework, we suggested that the assay with the lowest cost to implement and routinely use was the dRIT assay. The costs associated with the dRIT were lower in all simulated scenarios, irrespective of the number of samples tested per year. In addition to the cost analysis, the diagnostic efficacies of the two assays were evaluated. To do this, a cohort of DFA-positive and -negative samples collected from dog meat markets in Nigeria were initially diagnosed using the DFA in Nigeria and subsequently sent to South Africa for diagnostic confirmation. In South Africa, all the specimens were re-tested with the DFA, the dRIT and a quantitative real-time polymerase chain reaction (qRT-PCR). In our investigation, discrepancies were observed between the three diagnostic assays; with the incongruent results being resolved by means of confirmatory testing using the heminested reverse transcription polymerase reaction and sequencing to confirm that they were not contamination.Conclusions/SignificanceThe data obtained from this study suggested that the dRIT was not only an effective diagnostic assay that could be used to routinely diagnose rabies, but that the assay was also the most cost-effective option among all of the OIE recommended methods. In addition, the results of our investigation confirmed that some of the dogs slaughtered in dog markets were rabies-positive and that the markets posed a potential public health threat. Lastly, our data showed that the DFA, although regarded as the gold standard test for rabies, has some limitations—particularly at low antigen levels. Based on the results reported here and the current challenges faced in Nigeria, we believe that the dRIT assay would be the most suitable laboratory test for decentralized or confirmatory rabies diagnosis in Nigeria, given its relative speed, accuracy, cost and ease of use.

Evaluation of polyclonal anti-RNP IgG antibody for rabies diagnosis by indirect rapid immunohistochemistry test

Rabies still represents a major public health threat and estimated to cause 60,000 human deaths annually, particularly in developing countries. Thus, adequate surveillance based on rapid and reliable rabies diagnosis for both humans and animals is essential. The WHO and OIE recommended gold standard diagnostic technique for rabies is the direct immunofluorescence assay (dFAT). However, dFAT is expensive and requires a high level of expertise. As an alternative, the rapid immunohistochemistry technique is a promise to be a simple and cost effective diagnostic tool for rabies, and can be performed on field conditions prevalent in developing countries. However, no validated commercial conjugate antibody for rabies is available to meet the laboratory demand. Here, we evaluated the polyclonal anti-rabies virus ribonucleoprotein (RNP) IgG antibody for Rabies lyssavirus (RABV) detection by indirect rapid immunohistochemistry test (iRIT). We tested polyclonal anti-RNP IgG antibody against a batch of 100 brain specimens representing a wide phylogenetic origin in the State of São Paulo, Brazil. The purified IgG obtained 100% of diagnostic specificity and sensibility for RABV antigen detection in iRIT compared with the gold standard dFAT. In conclusion, our results demonstrate that the polyclonal anti-RNP IgG antibody may be used as a diagnostic reagent for rabies using iRIT, with the expectation of increase in availability and cost reduction of the epidemiological surveillance for developing countries.

Additional Progress in the Development and Application of a Direct, Rapid Immunohistochemical Test for Rabies Diagnosis.

Laboratory-based surveillance is fundamental to effective rabies prevention and control. The direct fluorescent antibody (AB) test (FAT) is the gold standard for rabies diagnosis. Recently, additional tests besides the FAT have been developed, such as the direct rapid immunohistochemical test (DRIT). In this study, our objective was to further refine technical aspects of the DRIT using a combination of two monoclonal ABs (MABs), 502 and 802, conduct additional testing among rabies reference laboratories using a diversity of animal species and rabies virus (RV) variants and compare the potential utility of the DRIT for end users via proficiency testing (PT) against the FAT. Considering the ideal molar ratios of biotin to AB in formulation of the DRIT conjugate, 3.9 was found to be superior to 7.4, for detection of RV antigens in the brain of a naturally infected raccoon. Optimization of the DRIT conjugate may also be dependent upon the apparent choice of specific viral antigens for testing, as a gray fox RV variant reacted less strongly than a raccoon RV variant in determining the working dilution of the MAB cocktail. Using the same MABs and protocol, the DRIT was compared to the FAT using more than 800 samples of mammalian brains, representative of more than 25 taxa, including in excess of 250 animal rabies cases from Europe and North America. Sensitivity was determined at 98% (96–100%, 95% CI) and specificity was calculated at 95% (92–96%, 95% CI). In a comparison among end users, PT of laboratory personnel resulted in values of 77–100% sensitivity and 86-100% specificity. Based upon these and previously reported results, the DRIT appears to be a suitable alternative to the FAT for use in lyssavirus diagnosis.

RABIES IN A SET OF EIGHT-WEEK OLD PUPPIES IN NIGERIA: THE NEED FOR REVIEW OF CURRENT DOG ANTIRABIES VACCINATION SCHEDULE.

Background:Rabies is endemic in Nigeria with clinical cases reported mainly in dogs and occasionally in livestock from all the geo-ecological zones of the country. Detection of rabies virus antigen in puppies at the age of five to ten weeks and in apparently healthy dogs shedding the virus in their saliva have been reported in some parts of Nigeria.Material and Method:This report describes a clinical rabies in a set of eight weeks old puppies confirmed by antigen detection using the direct fluorescent antibody test (DFAT), the direct rapid immunohistochemical test (DRIT), and RT-PCR.Results:it was positive for all test used including the RT-PCR which amplified at 750 bp from the gel electrophoresis.Conclusion:Occurrence of rabies in puppies of this age, within which they are acquired and owned by other unsuspecting members of the public, is of grave public health consequences. People that love puppies, especially children, who are fond of carrying and playing with them, are also faced with the risk of exposure to rabies. Consequently, review of the existing dog antirabies vaccination schedule at twelve weeks of age in Nigeria, is recommended to ensure effective immunization of this age group of dogs and for the overall safety of the vulnerable members of the public.

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.

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.

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.

Incorporating Direct Rapid Immunohistochemical Testing into Large-Scale Wildlife Rabies Surveillance.

Following an incursion of the mid-Atlantic raccoon variant of the rabies virus into southern Ontario, Canada, in late 2015, the direct rapid immunohistochemical test for rabies (dRIT) was employed on a large scale to establish the outbreak perimeter and to diagnose specific cases to inform rabies control management actions. In a 17-month period, 5800 wildlife carcasses were tested using the dRIT, of which 307 were identified as rabid. When compared with the gold standard fluorescent antibody test (FAT), the dRIT was found to have a sensitivity of 100% and a specificity of 98.2%. Positive and negative test agreement was shown to be 98.3% and 99.1%, respectively, with an overall test agreement of 98.8%. The average cost to test a sample was $3.13 CAD for materials, and hands-on technical time to complete the test is estimated at 0.55 h. The dRIT procedure was found to be accurate, fast, inexpensive, easy to learn and perform, and an excellent tool for monitoring the progression of a wildlife rabies incursion.

Enhanced diagnosis of rabies and molecular evidence for the transboundary spread of the disease in Mozambique.

Rabies is a neglected zoonotic disease with veterinary and public health significance, particularly in Africa and Asia. The current knowledge of the epidemiology of rabies in Mozambique is limited because of inadequate sample submission, constrained diagnostic capabilities and a lack of molecular epidemiological research. We wanted to consider the direct, rapid immunohistochemical test (DRIT) as an alternative to the direct fluorescent antibody (DFA) for rabies diagnosis at the diagnostic laboratory of the Central Veterinary Laboratory (CVL), Directorate of Animal Science, Maputo, Mozambique. Towards this aim, as a training exercise at the World Organisation for Animal Health (OIE) Rabies Reference Laboratory in South Africa, we performed the DRIT on 29 rabies samples from across Mozambique. With the use of the DRIT, we found 15 of the 29 samples (52%) to be negative. The DRIT-negative samples were retested by DFA at the OIE Rabies Reference Laboratory, as well as with an established real-time Polymerase chain reaction, confirming the DRIT-negative results. The DRIT-positive results (14/29) were retested with the DFA and subsequently amplified, sequenced and subjected to phylogenetic analyses, confirming the presence of rabies RNA. Molecular epidemiological analyses that included viruses from neighbouring countries suggested that rabies cycles within Mozambique might be implicated in multiple instances of cross-border transmission. In this regard, our study has provided new insights that should be helpful in informing the next steps required to better diagnose, control and hopefully eliminate rabies in Mozambique.

Evaluation of Direct Rapid Immunohistochemistry Test for Rabies Diagnosis in the Brain of Slaughtered Dogs in Northern Nigeria

This work was carried out in collaboration between all authors. Authors AG, JUU and AAD designed the study, ran the laboratory (FAT) work in Zaria, data analysis and drafted the initial manuscript and final review. Authors AG, MSA, PAO, AAO, AZ and FSU designed and involved in sample collection, shipment across the Niger State to Zaria, Nigeria and some literature search. Authors AG, LO, OVM, PY and MN, made necessary arrangements for the shipment of samples to CDC, Atlanta, USA, laboratory procedures (FAT and DRIT), literature review and reviewing of the final draft. All authors read and approved the final manuscript.

Comparison of direct, Rapid Immunohistochemical Test Performance with Direct Fluorescent Antibody Test for rabies diagnosis in Ethiopia

Background: Rabies continues to be a persistent and major public health problem for many developing countries. Rapid and accurate laboratory diagnosis is one of the basic elements in rabies control strategies. However, Laboratory diagnosis for animal and human rabies is severely constrained in much of the developing world including Ethiopia where rabies is endemic. Objective: To evaluate the performance of Direct, Rapid Immunohistochemical Test for rabies antigen detection from clinical samples in comparison with the Direct Fluorescent Antibody, gold-standard test. Method: The study was conducted from July, 2010- June, 2011 in Zoonoses Research Laboratory of Ethiopian Health and Nutrition Research Institute. During this study period a total of 200 brain tissue samples were collected from animals head submitted for routine rabies diagnosis. All the brain tissue samples were tested for rabies using Direct, Rapid Immunohistochemical Test, and Direct Fluorescence Antibody Test. Results: Above two-thirds (67.5%, 135 of 200) of the animal brain tissues examined using direct, Rapid Immunohistochemistry Test were found positive for rabies antigen that showed 100% sensitivity and specificity of direct, Rapid Immunohistochemistry Test compared to the results obtained by Direct Fluorescent Antibody test. Conclusion: The direct, Rapid Immunohistochemistry Test showed equivalent sensitivity and specificity as compared to Direct Fluorescent Antibody test under laboratory condition. Further study with large sample size and wide range of geographical representation of the country is needed to assess the ease of use and feasibility of this test at regional laboratories and under field conditions.

Antigen detection of rabies virus in brain smear using direct Rapid Immunohistochemistry Test

Rabies is zoonotic disease caused by a fatal, neurotropic virus. Rabies virus is classified into the Genus of Lyssavirus under the yang family of Rhabdoviridae . Rabies affecting hot- blooded animals, as well as human. Dogs, cats, monkeys are the vectors or reservoirs for rabies and the virus was transmitted through the saliva after infected animal’s bites. The aim of this study was to conduct rapid diagnosis to detect rabies viral antigen in brain smear using immunohistochemical (IHC) method namely direct Rapid Immunohistochemical Test (dRIT). A total number of 119 brain samples were achieved from Bukittinggi Veterinary Laboratory, West Sumatra. Standardisation and validation of the method were compared to Fluorescent Antibody Test (FAT) as a golden standard for rabies diagnosis. Results show that dRIT was a very good method, it can be performed within two hours without the need of fluorescent microscope. The samples were tested using FAT and from 119 samples tested, 80 (67.23%) samples were positive for rabies and 39 (32.77%) samples were negative for rabies whereas using dRIT showed that 78 (65.54%) samples were positive for rabies and 41 (34.45%) samples were negative for rabies. The dRIT results were validated by comparing them with FAT results as a golden standard for rabies. The relative sensitivity of dRIT to FAT was 97.5% and the relative specificity to FAT was 100% (with Kappa value of 0.976, stated as excellent). The achievement showed that dRIT is very potential diagnostic tool and is highly recommended to be used widely as a rapid diagnosis tool for rabies. Key Words: Rabies, Antigen Detection, Immunohistochemistry, Direct Rapid Immunohistochemical Test (dRIT)

Rural Rabies Prevention Project - A ‘One Health’ Experiment in India: An Overview

Aim: To assess the feasibility of implementing approach to prevent human rabies and control animal rabies in a rural community. Study Design: Health research in a rural setting. Place and Duration of Study: A medical college and a veterinary college along with an animal welfare organization delivered a wide array of integrated services in three villages' i.e. Kumbalagodu, Thagachikuppe and Gerupalya near Bangalore, India comprising a population of 10,220 persons for a period of two years from December, 2009 to November, 2011. The nearby three villages of Ramohally, Vinayakanagara and Bhimanakuppe with a population of 6,023 persons formed the control group, with no project inputs. Methodology: This consisted of household surveys at the beginning and end of two years ; rabies awareness campaigns; clinical and laboratory surveillance of rabies in dogs; rabies post-exposure prophylaxis (PEP) in humans; pre-exposure rabies prophylaxis (PrEP) by intradermal route in school children, pet dog owners and veterinarians ; mass dog vaccination and deworming; and sero-surveillance in both dogs and humans. Results: The ratio of veterinary and medical manpower in study villages was 1: 11 and dog to human ratio was 1: 23.The information, education and communication materials developed and used were domestic outdoor wall writings (11), domestic indoor annual wall calendar (2000), school book labels (1000); game charts (16); wall posters (65); flip chart (15); rabies educational DVD (1). There were 102 local cable television transmissions on rabies prevention. 69 persons received rabies PEP. PrEP was given to 368 school children, pet dog owners and veterinarians. Sixty one human serum samples were analyzed by rapid fluorescent focus inhibition test for rabies antibody detection. Sixteen veterinarians were trained to use direct rapid immunohistochemical test (dRIT) for rabies diagnosis. Six ruminants were confirmed rabid by dRIT. There were no cases of human rabies. The cost of entire project was US $ 85,958. Conclusion: A blend of medical, veterinary and animal welfare were successfully delivered through a health approach. Based on this success a conceptual model was evolved to propagate its replication in other rural communities across India.