Utility of immunohistochemical staining technique as a routine tool in Rabies Diagnosis

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Kalima-Munalula, Mukatimui N
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Rabies is one of few diseases that cause anxiety to both the exposed individual and the health authorities responsible for its prevention and control. Diagnostic procedures in rabies have involved histopathological detection of the Negri body, immunofluorescence and mouse inoculation.Brain specimens from suspected and confirmed rabies cases were obtained from the University of Zambia, School of Veterinary Medicine, Lusaka. These specimens were taken from dogs, cats, cows and horses. The specimens were tested for rabies using the Direct Immunofluorescent Antibody Technique (DFAT) and the results compared to those obtained using the indirect avidin-biotin technique using Streptavidin alkaline phosphatase technique and routine haematoxylin eosin (HE) staining for Negri bodies. Fixation in formalin was done for varying lengths of time from 24 to 72 hours. Sections from the paraffin blocks were cut using a sliding microtome at a thickness of 3 micrometers and fished onto clean slides coated with Poly-L-Lysine to avoid detachment during the incubation and rinsing steps. Enzyme digestion using trypsin was applied to sections to unmask antigenic sites concealed during formalin fixation. Fast red TR salt was the chromogen used of the 39 cases tested the DFAT gave positive results in 29 cases and negative results in 10 cases. The immunohistochemical test gave positive results in 25 cases and negative results in 14 cases. The HE staining gave positive results in 16 cases and negative results in 23 cases. Only in one case did the DFAT give a negative result and the immunohistochemical technique give a positive result. Using DFAT as the gold standard, the sensitivity and specificity of the immunohistochemical test and histopathology were calculated. However, these two means of comparison were not sufficient so the level of agreement between the methods was also calculated and a kappa value for each obtained. The sensitivity of the immunohistochemical technique was found to be 82.7%, specificity was 90%. The level of agreement was 85% with a positive predictive value (PPV) of 0.96, and negative predictive value (NPV) of 0.64. The calculated kappa value was 0.64. The histopathological test gave sensitivity of 55.1% and specificity 100%. The level of agreement was 67% with PPV = 1.00, NPV = 0.43. The calculated kappa value was 0.39. It was possible to show that the indirect immunohistochemical technique using Streptavidin alkaline phosphatase can successfully be used to diagnose rabies. This technique is an adjunct to DFAT, and is an alternative in places where it is not possible to keep specimens frozen or to get them to a diagnostic laboratory in a fresh state. Other instances where the immunohistochemical technique can be applied are those where the dark field microscope with an ultraviolet source is not available, or in retrospective studies. It was also shown that trypsin digestion greatly improves the staining quality of the sections. However, digestion beyond 90 minutes was found to result in unacceptable tissue destruction and loss of antigenicity. Thus it was important to establish a time limit for the optimal digestion time. In this work, this was found to be between 30 and 60 minutes using a final concentration of 1 mg/ml in Tris buffer and at an incubation temperature of 37°C. Another aspect that this work attempted to cover was the effect of fixative and fixation time on the results obtained by the immunohistochemical technique. This was done by experimentally infecting mice with rabies virus and the brain collected and fixed in four different fixatives for varying lengths of time. It was possible to obtain positive reaction from tissues fixed in all four fixatives. However, the number of samples obtained was limited because of high number of deaths caused by trauma. Also the mice included in this part of the study were collected at a late time of infection. The virus titres are thus expected to be high. There seemed to be no difference following fixation for different time periods.
Rabies---Diagnosis , Rabies Vaccination