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Rabies is an acute viral infection and causes encephalitis which affects most warm blooded animals including man. It is caused by a single stranded RNA virus of the genus Lyssavirus and family Rhabdoviridae. It is still a major public health problem in most developing countries including India where an estimated 20 000 human deaths and 17 million animal bites are reported every year[9]. The main vector of rabies in India is dog in over 95 % of human cases but other animals like cats, monkeys, mongooses and wild animals also transmit the disease. Though rabies is almost 100 % fatal, it is also successfully preventable if the currently recommended state of the art prophylactic measures are instituted soon after the exposure[11]. Prior confirmation of the rabid status of the biting animal can guide the physician to start or withhold the post exposure treatment as the presently available vaccines and immunoglobulin's are quite expensive and often in short supply. The presently recommended gold standard technique for post mortem diagnosis of rabies is the direct immunofluorescence assay (dFA)[1]. The performance of this technique is presently restricted to 2-3 laboratories in India as it requires an expensive fluorescence microscope, a specific rabies conjugate as well as technical expertise. Thus there is a need for a rapid diagnostic technique that can be adapted to field conditions, is economical for the resource constraint countries but is also as sensitive and specific as dFA. Recently the Centers for Disease Control (CDC) Atlanta, has developed a simple and raid technique for rabies diagnosis which is based on the principle of immunohistochemistry where the rabies virus nucleoprotein (N) antigen in the suspect brain smear is captured by a cocktail of biotinylated anti-N monoclonal antibody and subsequent color development. This technique, which is known as the direct rapid immunohistochemical test (dRIT), has been found to be as specific and sensitive as the gold standard dFA and has undergone limited field trials in Africa, China, Afghanistan and Iraq[2, 3, 8, 10]. Based on the results of these trials, the South-East Asia Regional Office (SEARO) of the WHO wanted to assess the utility of this test in other Asian countries, particularly the Indian subcontinent where rabies is highly endemic. Thus, the department of Neurovirology at the National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, India, which is also a WHO collaborating centre for reference and research on rabies, was supplied with reagents and technical input from CDC, Atlanta, USA. Subsequently, the department successfully established the test procedure and has evaluated this test in comparison to dFA using a number of animal brains from different species as well as some human brains. In this report we communicate our results which clearly establish that this new test dRIT is as sensitive and specific as dFA and has the potential to replace the dFA in resource constrained developing countries.
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Before studying the test slides, positive and negative controls stained by the two techniques were examined using appropriate microscopes. In the dFA test, normal mouse brain smears did not show any fluorescence and in the dRIT, only a background light blue colour was observed (Fig. 1). In the dFA test, the positive smears showed bright green fluorescence particles of varying size either scattered or within the neurons. In the dRIT, positive brains showed dark red particles scattered all over the field or within the neurons (Fig. 1). The comparative results of the two tests in a positive brain smear are shown in Fig. 2. The brains of the animals which were positive by dFA were also positive by dRIT and the negative brains were negative by both the tests. Thus there was 100 % concordance between the two tests. Interestingly 3 dog brains which were faintly positive by dFA were very strongly positive by dRIT. Generally it was easier to interpret a smear stained by dRIT than by dFA. In case of fresh brains, the ease of interpretation was similar with both the tests but with brains preserved in 50 % glycerol saline, interpretation was much easier with the dRIT technique. The results of different brain samples tested by the two techniques is depicted in Table 1.
Table 1. Correlation between direct rapid immunohistochemistry test (dRIT) and direct Fluorescent antibody assay (dFA)