Human rabies in India: an audit from a rabies diagnostic laboratory.

OBJECTIVES: Rabies, an acute progressive encephalomyelitis, continues to be a serious public health problem in India and many other countries in Asia and Africa. The low level of commitment to rabies control is partly attributable to challenges in laboratory diagnosis and lack of adequate surveillance to indicate the disease burden. A laboratory audit of human rabies cases was undertaken to disseminate information on the clinical, demographic, prophylactic and most importantly the laboratory diagnostic aspects of rabies. METHODS: A retrospective analysis of all clinically suspected human rabies cases, whose samples were received at a rabies diagnostic laboratory in South India in the last 3 years, was performed. Clinical and demographic details of patients were obtained. The clinical samples included cerebrospinal fluid (CSF), serum, saliva and nuchal skin biopsy collected antemortem, and brain tissue obtained post-mortem. Various laboratory tests were performed for diagnosis. RESULTS: Clinical samples from 128 patients with suspected rabies, from 11 states in India, were received for diagnostic confirmation. About 94% of the victims reported dog-bites, more than a third of them were children and most of the victims did not receive adequate post-exposure prophylaxis. Antemortem confirmation of rabies by a combination of laboratory diagnostic assays (detection of viral RNA in CSF, skin and saliva, and neutralising antibodies in CSF) could be achieved in 40.6% cases. CONCLUSIONS: Increasing awareness about adequate post-exposure prophylaxis, additional rabies diagnostic facilities, and enhanced human and animal rabies surveillance to indicate the true disease burden are essential to control this fatal disease.

Utility of real-time Taqman PCR for antemortem and postmortem diagnosis of human rabies.

Rabies, a fatal zoonotic viral encephalitis remains a neglected disease in India despite a high disease burden. Laboratory confirmation is essential, especially in patients with paralytic rabies who pose a diagnostic dilemma. However, conventional tests for diagnosis of rabies have several limitations. In the present study the utility of a real-time TaqMan PCR assay was evaluated for antemortem/postmortem diagnosis of rabies. Human clinical samples received for antemortem rabies diagnosis (CSF, saliva, nuchal skin biopsy, serum), and samples obtained postmortem from laboratory confirmed rabies in humans (brain tissue, CSF, serum) and animals (brain tissue) were included in the study. All CSF and sera were tested for rabies viral neutralizing antibodies (RVNA) by rapid fluorescent focus inhibition test (RFFIT) and all samples (except sera) were processed for detection of rabies viral RNA by real-time TaqMan PCR. All the 29 (100%) brain tissues from confirmed cases of human and animal rabies, and 11/14 (78.5%) CSF samples obtained postmortem from confirmed human rabies cases were positive by real-time TaqMan PCR. Rabies viral RNA was detected in 5/11 (45.4%) CSF samples, 6/10 (60%) nuchal skin biopsies, and 6/7 (85.7%) saliva samples received for antemortem diagnosis. Real-time TaqMan PCR alone could achieve antemortem rabies diagnosis in 11/13 (84.6%) cases; combined with RVNA detection in CSF antemortem rabies diagnosis could be achieved in all 13 (100%) cases. Real-time TaqMan PCR should be made available widely as an adjunctive test for diagnosis of human rabies in high disease burden countries like India.

Laboratory diagnosis of human rabies: recent advances.

Rabies, an acute progressive, fatal encephalomyelitis, transmitted most commonly through the bite of a rabid animal, is responsible for an estimated 61,000 human deaths worldwide. The true disease burden and public health impact due to rabies remain underestimated due to lack of sensitive laboratory diagnostic methods. Rapid diagnosis of rabies can help initiate prompt infection control and public health measures, obviate the need for unnecessary treatment/medical tests, and assist in timely administration of pre- or postexposure prophylactic vaccination to family members and medical staff. Antemortem diagnosis of human rabies provides an impetus for clinicians to attempt experimental therapeutic approaches in some patients, especially after the reported survival of a few cases of human rabies. Traditional methods for antemortem and postmortem rabies diagnosis have several limitations. Recent advances in technology have led to the improvement or development of several diagnostic assays which include methods for rabies viral antigen and antibody detection and assays for viral nucleic acid detection and identification of specific biomarkers. These assays which complement traditional methods have the potential to revolutionize rabies diagnosis in future.

Dendritic poly(ether imine) based gene delivery vector.

The nonviral vector based gene delivery approach is attractive due to advantages associated with molecular-level modifications suitable for optimization of vector properties. In a new class of nonviral gene delivery systems, we herein report the potential of poly(ether imine) (PETIM) dendrimers to mediate an effective gene delivery function. PETIM dendrimer, constituted with tertiary amine branch points, n-propyl ether linkers and primary amines at their peripheries, exhibits significantly reduced toxicities, over a broad concentration range. The dendrimer complexes pDNA effectively, protects DNA from endosomal damages, and delivers to the cell nucleus. Gene transfection studies, utilizing a reporter plasmid pEGFP-C1 and upon complexation with dendrimer, showed a robust expression of the encoded protein. The study shows that PETIM dendrimers are hitherto unknown novel gene delivery vectors, combining features of poly(ethylene imine)-based polymers and dendrimers, yet are relatively nontoxic and structurally precise.

Mycophenolic acid inhibits replication of Japanese encephalitis virus.

BACKGROUND: Japanese encephalitis is a major public health problem in several parts of Asia, particularly India, Nepal, Sri Lanka and Myanmar (Burma). Despite its public health implications, there are no effective antiviral drugs available. METHODS: The present study evaluated the effect of mycophenolic acid on Japanese encephalitis virus (JEV) using an in vitro cytopathic effect inhibition assay, plaque reduction assay and virus yield reduction assay, and its therapeutic potential was also assessed in vivo in a mouse model. RESULTS: Analysis of the results obtained in the in vitro and in vivo experiments suggests that mycophenolic acid has significant antiviral activity against JEV, with an IC(50) of 3.1 µg/ml, a therapeutic index of 16 and a 75% protection against lethal challenge of JEV. CONCLUSION: The study concludes that this compound significantly inhibited the replication of JEV in vitro and protected mice in vivo.

Pentoxifylline inhibits replication of Japanese encephalitis virus: a comparative study with ribavirin.

Several investigations have shown that pentoxifylline possesses broad-spectrum antiviral activity against a range of RNA and DNA viruses. However, its ability to inhibit Japanese encephalitis virus (JEV) replication has not yet been studied. The present study was designed to investigate the antiviral activity of pentoxifylline against JEV in vitro and in vivo. The activity of pentoxifylline against JEV was evaluated in vitro using cytopathic effect inhibition and plaque reduction assays. Pentoxifylline was able to inhibit JEV replication in a dose-dependent manner at a 50% inhibitory concentration (IC(50)) of 50.3microg/mL (0.00018microM) and a therapeutic index (TI) of 10. Experiments to study the mechanism of antiviral action of pentoxifylline using in vitro translation of viral mRNA suggested that the drug did not interfere either with early or late protein synthesis but most likely exerted its action on virus assembly and/or release. Furthermore, the in vivo study showed that pentoxifylline at a concentration of 100mg/kg and 200mg/kg body weight was able to protect completely mice challenged with 50 x 50% lethal dose (LD(50)) of JEV.

Development and evaluation of a competitive ELISA for estimation of rabies neutralizing antibodies after post-exposure rabies vaccination in humans.

OBJECTIVES: Currently three tests are approved for the estimation of neutralizing antibodies after rabies vaccination: the mouse neutralization test (MNT), the rapid fluorescent focus inhibition test (RFFIT), and the fluorescent antibody virus neutralization (FAVN) test. Performance of these tests requires a lot of expertise and is generally carried out in reference laboratories and, hence, they are not available to many people. The aim of the present study was to develop and evaluate a competitive ELISA (C-ELISA) for estimation of neutralizing antibodies in order to make this testing more widely available. METHODS: The C-ELISA was designed based on competition between a murine neutralizing monoclonal antibody (Mab) and the antibodies in serum of vaccinated people. The test was initially standardized using known negative and known positive serum samples for determining the optimal dilution of the Mab as well as the cut-off value (%) for ascertaining the level of inhibition. Nine hundred and ninety serum samples were tested from 250 people who had been administered purified chick embryo cell vaccine (PCECV). Serum samples were collected on days 0, 14, 30 and 90 post-vaccination, and were tested by C-ELISA. RESULTS: All the serum samples that were positive by RFFIT were also positive by C-ELISA. The titers obtained with C-ELISA were marginally higher than the RFFIT titers, but a significant correlation was noted between the two tests (r=0.897). None of the negative controls were detected to be positive for rabies antibodies by either of these tests. Therefore the C-ELISA was found to be 100% specific and sensitive in comparison to RFFIT. Further, the initial rise and fall of antibody titers on different days post-vaccination was comparable for both tests. CONCLUSIONS: The C-ELISA described herein can be used to quantify rabies neutralizing antibody levels after vaccination. This test is simple and can be conveniently used under field conditions for monitoring seroconversion after post-exposure rabies vaccination. Moreover it does not require handling of infectious virus by the end user.

Injecting rabies immunoglobulin (RIG) into wounds only: A significant saving of lives and costly RIG.

An increasing number of dog bite victims were being presented to public hospitals in Himachal Pradesh in 2014 amidst virtual non availability of any rabies immunoglobulin (RIG). Only a small quantity of equine rabies immunoglobulin (eRIG) was available from the government owned Central Research Institute (CRI) Kasauli. This available eRIG was used in 269 patients as an emergency response and only for local infiltration of severe bite wounds by suspected rabid dogs. This was followed by rabies vaccination, using the WHO approved intra-dermal Thai Red Cross Society vaccination schedule. A subgroup of 26 patients were later identified who had been severely bitten by laboratory confirmed rabid dogs. They were followed for more than one year and all were found to be alive.

Local infiltration of rabies immunoglobulins without systemic intramuscular administration: An alternative cost effective approach for passive immunization against rabies.

Presently the dose of rabies immunoglobulin (RIG) which is an integral part of rabies post exposure prophylaxis (PEP) is calculated based on body weight though the recommendation is to infiltrate the wound(s). This practice demands large quantities of RIG which may be unaffordable to many patients. In this background, we conducted this study to know if the quantity and cost of RIG can be reduced by restricting passive immunization to local infiltration alone and avoiding systemic intramuscular administration based on the available scientific evidence. Two hundred and sixty nine category III patients bitten by suspect or confirmed rabid dogs/animals were infiltrated with equine rabies immunoglobulin (ERIGs) in and around the wound. The quantity of ERIG used was proportionate to the size and number of wounds irrespective of their body weight. They were followed with a regular course of rabies vaccination by intra-dermal route. As against 363 vials of RIGs required for all these cases as per current recommendation based on body weight, they required only 42 vials of 5ml RIG. Minimum dose of RIGs given was 0.25 ml and maximum dose given was 8 ml. On an average 1.26 ml of RIGs was required per patient that costs Rs. 150 ($3). All the patients were followed for 9 months and they were healthy and normal at the end of observation period. With local infiltration, that required small quantities of RIG, the RIGs could be made available to all patients in times of short supply in the market. A total of 30 (11%) serum samples of patients were tested for rabies virus neutralizing antibodies by the rapid fluorescent focus inhibition test (RFFIT) and all showed antibody titers >0.5 IU/mL by day 14. In no case the dose was higher than that required based on body weight and no immunosuppression resulted. To conclude, this pilot study shows that local infiltration of RIG need to be considered in times of non-availability in the market or unaffordability by poor patients. This preliminary study needs to be done on larger scale in other centers with long term follow up to substantiate the results of our study.

Natural Rabies Infection in a Domestic Fowl (Gallus domesticus): A Report from India.

BACKGROUND: Rabies is a fatal encephalitis caused by viruses belonging to the genus Lyssavirus of the family Rhabdoviridae. It is a viral disease primarily affecting mammals, though all warm blooded animals are susceptible. Experimental rabies virus infection in birds has been reported, but naturally occurring infection of birds has been documented very rarely. PRINCIPAL FINDINGS: The carcass of a domestic fowl (Gallus domesticus), which had been bitten by a stray dog one month back, was brought to the rabies diagnostic laboratory. A necropsy was performed and the brain tissue obtained was subjected to laboratory tests for rabies. The brain tissue was positive for rabies viral antigens by fluorescent antibody test (FAT) confirming a diagnosis of rabies. Phylogenetic analysis based on nucleoprotein gene sequencing revealed that the rabies virus strain from the domestic fowl belonged to a distinct and relatively rare Indian subcontinent lineage. SIGNIFICANCE: This case of naturally acquired rabies infection in a bird species, Gallus domesticus, being reported for the first time in India, was identified from an area which has a significant stray dog population and is highly endemic for canine rabies. It indicates that spill over of infection even to an unusual host is possible in highly endemic areas. Lack of any clinical signs, and fewer opportunities for diagnostic laboratory testing of suspected rabies in birds, may be the reason for disease in these species being undiagnosed and probably under-reported. Butchering and handling of rabies virus- infected poultry may pose a potential exposure risk.

Atypical rabies encephalitis in a six-year-old boy: clinical, radiological, and laboratory findings.

A 6-year-old boy from India developed an atypical form of rabies following a stray dog bite and as a consequence of not receiving the standard World Health Organization recommended post-exposure prophylaxis for category III wounds. Serial rising rabies virus neutralizing antibody titres in serum and cerebrospinal fluid by rapid fluorescent focus inhibition test helped confirm the diagnosis of rabies. The child has survived for 4 months since the onset of illness, albeit with neurological sequelae.

Cellular immune response following pre-exposure and postexposure rabies vaccination by intradermal and intramuscular routes.

PURPOSE: Immunization against rabies in humans induces protective neutralizing antibodies; however, the induction of type 1 or type 2 cytokine mediated cellular immune responses following rabies vaccination is not understood. Hence, the present study investigated cellular cytokine responses in vaccinated individuals. MATERIALS AND METHODS: The study groups included healthy rabies antigen naive controls (n=10), individuals who received intradermal primary (n=10) or booster pre-exposure vaccination (n=20) and subjects who received postexposure rabies vaccination either by intradermal (n=18) or intramuscular (n=20) routes. The antigen specific cellular responses were analyzed by stimulating peripheral blood mononuclear cells with a rabies vaccine antigen in the interferon-γ (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunospot (ELISpot) assay. These responses were compared to the rabies virus neutralizing antibody (RVNA) titers that were measured by rapid fluorescent focus inhibition test. RESULTS: We observed that cellular and humoral immune responses to primary intradermal rabies vaccination could be greatly enhanced by a booster vaccine; and both type 1 and type 2 cytokine responses were significantly elevated. The magnitude of type 1 and type 2 cytokine responses did not differ significantly among the intramuscular and intradermal routes of postexposure vaccination. The number of cells producing IFN-γ and IL-4 correlated significantly with the levels of RVNA. CONCLUSION: Both type 1 and type 2 cellular cytokine responses are strongly induced after rabies vaccination and directly correlate with levels of RVNA titers. The neutralizing antibody as well as the type 1 and type 2 cytokine responses may be important for vaccine induced protective responses against rabies.

In vitro inactivation of the rabies virus by ascorbic acid.

OBJECTIVE: The current recommended inactivating agent for the rabies virus, beta propiolactone (BPL) is very expensive and potentially carcinogenic. There is a need to evaluate alternative chemicals, which will inactivate the virus without affecting its antigenicity. In this study the effect of ascorbic acid on the infectivity of the rabies virus has been investigated. METHOD: Vero cell grown fixed rabies virus CVS strain was treated with 0.1 mg/ml, 0.5 mg/ml and 1mg/ml final concentrations of ascorbic acid and 5 microg/ml of copper sulfate and kept at 4 degrees C along with untreated virus material. Each aliquot was titrated after various intervals for viral infectivity using both mice inoculation and titration in vero cells. The antigenicity of the virus material was determined by antibody induction in mice and modified NIH tests in parallel with virus material inactivated with a 1:4000 concentration of BPL. RESULTS: An optimal concentration of 0.5 mg/ml of ascorbic acid and 5 microg/ml of copper sulfate completely inactivated the virus after 72 hours. The inactivated virus retained good antigenicity and potency value, which was comparable with using BPL. CONCLUSION: These findings suggest that ascorbic acid can be used as an inactivating agent for fixed rabies virus grown in cell culture particularly for the preparation of diagnostic reagents. Further studies are required to evaluate its effect on the cell associated virus, probable therapeutic potential and feasibility of replacing BPL in production of inactivated rabies vaccine.

Rapid diagnosis of rabies in humans and animals by a dot blot enzyme immunoassay.

OBJECTIVES: The presently advocated tests for rapid diagnosis of rabies, such as the fluorescent antibody test (FAT) are expensive and require expertise to carry out and interpret the results. In this study, a simple direct dot blot enzyme immunoassay (DIA) has been developed and evaluated to detect the rabies antigen in brain specimens of animals and humans. The utility of this test in the ante-mortem diagnosis of human rabies has also been evaluated. METHODS: Brain homogenates of suspected rabid animals (n = 250), humans (n = 16) and clinical samples like saliva (n = 12) and cerebrospinal fluid (CSF) (n = 12) were directly spotted on polyvinylidene difluoride membrane (PVDF) and the absorbed rabies nucleoprotein antigen was detected using biotinylated antinucleoprotein antibody followed by treatment with streptavidin peroxidase conjugate and color development with diamino benzedine (DAB). Rabies-infected and normal mouse brain homogenates were used as positive and negative controls, respectively. The results of this test were evaluated with fluorescent antibody technique (for brain samples) and mouse inoculation test (for saliva and CSF samples). RESULTS: A distinct dark brown color was seen in the positive control and all positive samples, while there was no color development with either the negative control or the negative samples. The concordance between the fluorescent antibody test (FAT) and dot immunoassay was 98.4% for brain samples, 83.3% for saliva and 91.6% for CSF samples. The specificity of the test was found to be 100%. CONCLUSIONS: The dot blot enzyme immunoassay (DIA) test described here is a sensitive, specific and rapid test for the post-mortem diagnosis of rabies in animals and humans. The utility of this test for the ante-mortem diagnosis of rabies needs to be further evaluated.

Survival from rabies encephalitis.

Rabies is a major public health problem in Asia and Africa, with nearly 60,000 deaths every year, and represents a substantial economic burden. Neurologists frequently encounter atypical cases, and need to make informed decisions regarding diagnosis and management. No therapy has been shown to unequivocally improve survival in rabies till date. Despite the overwhelmingly fatal nature of this disease, a small number of patients have been reported to survive acute rabies encephalitis with varying degrees of neurological sequelae. This paper presents the eleventh documented case of survival from rabies, which developed after being bitten by a stray dog, albeit with severe neurological residua. Similar to patients in previous reports, this man demonstrated a robust immune response as indicated by peripheral viral clearance and very high serum and cerebrospinal fluid antibody titres. Immunologically-mediated virus clearance therefore appears to be a prerequisite for survival. A detailed review of previously reported survivors, as well as descriptions of the host response and viral clearance in human rabies, current therapy for this disease and future directions in improving the currently dismal prognosis are provided.

Intradermal vaccination for rabies prophylaxis: conceptualization, evolution, present status and future.

Rabies is a fatal viral encephalitis which can be effectively prevented by prophylactic measures. The currently available cell culture vaccines used for rabies prophylaxis are expensive for use by the standard intramuscular route of administration. In the last 3 decades, intradermal (ID) routes of vaccination using lesser amounts of vaccine as compared to that used for standard intramuscular vaccination have been used extensively in some Asian countries which has reduced the economic burden of rabies prophylaxis and also contributed in achieving a decline in the incidence of human rabies. ID vaccination is based on sound immunological principles and has been found to be safe and immunogenic. New short duration regimens to further economize the cost and enhance patient compliance, and novel non-invasive devices for ID vaccine delivery are being evaluated. Considering the success of ID rabies vaccination in Asian countries, its implementation in rabies endemic African countries should be encouraged.

Immunogenicity and efficacy of a plasmid DNA rabies vaccine incorporating Myd88 as a genetic adjuvant.

PURPOSE: Myeloid differentiation factor 88 (Myd88), a ubiquitous Toll-like receptor adaptor molecule, has been reported to play important roles in B cell responses to infections and vaccination. The present study evaluated the effects of genetic adjuvanting with Myd88 on the immune responses to a plasmid DNA rabies vaccine. MATERIALS AND METHODS: Plasmids encoding rabies glycoprotein alone (pIRES-Rgp) or a fragment of Myd88 gene in addition (pIRES-Rgp-Myd) were constructed and administered intramuscularly or intrademally in Swiss albino mice (on days 0, 7, and 21). Rabies virus neutralizing antibody (RVNA) titres were estimated in the mice sera on days 14 and 28 by rapid fluorescent focus inhibition test. The protective efficacy of the constructs was evaluated by an intracerebral challenge with challenge virus standard virus on day 35. RESULTS: Co-expression of Myd88 increased RVNA responses to pIRES-Rgp by 3- and 2-folds, following intramuscular and intradermal immunization, respectively. pIRES-Rgp protected 80% of the mice following intramuscular and intradermal immunizations, while pIRES-Rgp-Myd afforded 100% protection following similar administrations. CONCLUSION: Genetic adjuvanting with Myd88 enhanced the RVNA responses and protective efficacy of a plasmid DNA rabies vaccine. This strategy might be useful for rabies vaccination of canines in the field, and needs further evaluation.

Enhancement of immunogenicity and efficacy of a plasmid DNA rabies vaccine by nanoformulation with a fourth-generation amine-terminated poly(ether imine) dendrimer.

PURPOSE: Delayed onset of, and low magnitude of, protective immune responses are major drawbacks limiting the practical utility of plasmid vaccination against rabies. In this study we evaluated whether nanoformulation with the novel poly(ether imine) (PETIM) dendrimer can enhance the immunogenicity and efficacy of a plasmid-based rabies vaccine. MATERIALS AND METHODS: A plasmid vaccine construct (pIRES-Rgp) was prepared by cloning the full-length rabies virus glycoprotein gene into pIRES vector. Drawing upon the results of our previous study, a dendriplex (dendrimer-DNA complex) of pIRES-Rgp was made with PETIM dendrimer (10:1 w/w, PETIM:pIRES-Rgp). In vitro transfection was done on baby hamster kidney (BHK)-21 cells to evaluate expression of glycoprotein gene from pIRES-Rgp and PETIM-pIRES-Rgp. Subsequently, groups of Swiss albino mice were immunized intramuscularly with pIRES-Rgp or PETIM-pIRES-Rgp. A commercially available cell culture rabies vaccine was included for comparison. Rabies virus neutralizing antibody (RVNA) titers in the immune sera were evaluated on days 14, 28, and 90 by rapid fluorescent focus inhibition test. Finally, an intracerebral challenge study using a challenge virus standard strain of rabies virus was done to evaluate the protective efficacy of the formulations. RESULTS: Protective levels of RVNA titer (≥0.5 IU/mL) were observed by day 14 in animals immunized with pIRES-Rgp and its dendriplex. Notably, PETIM-pIRES-Rgp produced 4.5-fold higher RVNA titers compared to pIRES-Rgp at this time point. All mice immunized with the PETIM-pIRES-Rgp survived the intracerebral rabies virus challenge, compared with 60% in the group which received pIRES-Rgp. CONCLUSION: Our results suggest that nanoformulation with PETIM dendrimer can produce an earlier onset of a high-titered protective antibody response to a plasmid-based rabies vaccine. PETIM dendriplexing appears to be an efficacious nonviral delivery strategy to enhance genetic vaccination.

Feasibility of reducing rabies immunoglobulin dosage for passive immunization against rabies: results of In vitro and In vivo studies.

Passive immunization is a crucial parameter for prevention of human rabies. Presently as World Health Organization (WHO) strongly advocates local infiltration of rabies immunoglobulin in and around the bite wound, we feel that there is no basis for calculating the dose of immunoglobulin based on body weight. Keeping this in view we conducted both in vitro and in vivo studies to know whether the dose of immunoglobulin can be reduced and still obtain complete neutralization of the virus. In vitro neutralization studies were conducted using CVS strain of virus and BHK 21 cells. In vivo experiments were conducted in 4 weeks old Swiss albino mice by initial challenge with CVS followed by infiltration with increasing dilutions of either human rabies immunoglobulin (HRIG) and equine rabies immunoglobulin (ERIG). In vitro studies showed that a dose of 100 FFD 50 of CVS was neutralized by increasing dilution of both HRIG and ERIG and 100% neutralization was observed with HRIG and ERIG in as low quantities as 0.025 IU. In mice studies there was 100% survival of mice infiltrated with 0.025 IU of both HRIG and ERIG compared with 100% mortality in mice infiltrated with normal saline. These results suggest that it is possible to reduce the dose of rabies immunoglobulins by at least 16 times the presently advocated dose. These findings needs to be further evaluated using larger animal models and street viruses prevalent in nature but cannot serve as recommendations for use of RIG for passive immunization in humans.

Evaluation of a direct rapid immunohistochemical test (dRIT) for rapid diagnosis of rabies in animals and humans.

Presently the gold standard diagnostic technique for rabies is the direct immunofluorescence assay (dFA) which is very expensive and requires a high level of expertise. There is a need for more economical and user friendly tests, particularly for use in developing countries. We have established one such test called the direct rapid immunohistochemical test (dRIT) for diagnosis of rabies using brain tissue. The test is based on capture of rabies nucleoprotein (N) antigen in brain smears using a cocktail of biotinylated monoclonal antibodies specific for the N protein and color development by streptavidin peroxidase-amino ethyl carbazole and counter staining with haematoxollin. The test was done in parallel with standard FAT dFA using 400 brain samples from different animals and humans. The rabies virus N protein appears under light microscope as reddish brown particles against a light blue background. There was 100 % correlation between the results obtained by the two tests. Also, interpretation of results by dRIT was easier and only required a light microscope. To conclude, this newly developed dRIT technique promises to be a simple, cost effective diagnostic tool for rabies and will have applicability in field conditions prevalent in developing countries.