EVALUATION OF THREE DIAGNOSTIC METHODS FOR RABIES VIRUS DETECTION IN FRESH, FROZEN ARCHIVED AND CITRATE BUFFER TREATED FORMALIN-FIXED DOG BRAIN TISSUES

EVALUATION OF THREE DIAGNOSTIC METHODS FOR RABIES VIRUS DETECTION IN FRESH, FROZEN ARCHIVED AND CITRATE BUFFER TREATED FORMALIN-FIXED DOG BRAIN TISSUES

Abstract:

Rabies is recognized as a global zoonosis yet it is remarkably neglected, despite thehigh fatality of the disease following onset of clinical signs. It remains a threat under-appreciated by healthcare practitioners in many endemic areas, often owing to lack of post-mortem evaluation and public health reporting. This study aimsto evaluate rabies virus detection by Direct Fluorescent Antibody Test (DFAT), Enzyme Linked Immuno-Sorbent Assay (ELISA) and Rapid Immuno-Diagnostic Test (RIDT) in fresh, frozen archived and citrate buffer treated formalin-fixed dog brain tissues.A total of fifty confirmed DFAT positive and negative dog brain tissues preserved at -20°C were collected from the archives of the Central Diagnostic Laboratory, National Veterinary Research Institute, Vom, Nigeria for analysis using DFAT, ELISA and RIDT. This study also evaluated the rabies status of fresh brain specimens from 40 rabies-suspected cases. A comparable brain specimen from each case was fixed in neutral buffered formalin and examined by the DFAT following treatment with sodium citrate buffer. We found 96% agreement of ELISA and DFAT for rabies antigen detection [Cohen‘s kappa coefficient (κ) = 0.834; 95% C.I.,Concordance coefficient =78%: 95% C.I.] and 54% agreement of RIDT and DFAT (κ = 0.170; 95% C.I., concordance coefficient= 17%: 95% C.I.). The sensitivities of ELISA and RIDT were 95.5% and 47.6% compared to DFAT respectively, while the specificities of ELISA and RIDT were 100% and 87.5% respectively. ELISA performed better than RIDT and recorded equivalent result with DFAT. Following treatment with sodium citrate buffer, DFAT on formalin-fixed brain tissues exhibited a sensitivity of 100% in comparison to DFAT on fresh-tissues. No false positive resultwas obtained in formalin-fixed DFAT procedure, demonstrating 100% specificity. There was no apparent difference in the intensity of fluorescence between DFAT on fresh samples and formalin-fixed DFAT (κ = 1.000; 95% C.I.,concordance = 98%; 95% C.I.). 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 analyze large number of samples at the same time, making it more suitable for epidemiological studies and for laboratories that cannot perform 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.Rabies antigen retrieval from formalin-fixed brain tissues with heated sodium buffer and its subsequent detection by DFAT is as valid and reliable a procedure for rabies diagnosis as the DFAT on fresh samples.Formalin could therefore, be used henceforth to fix tissues of rabies suspected cases for routine diagnosis, transportationor archival purposes

EVALUATION OF THREE DIAGNOSTIC METHODS FOR RABIES VIRUS DETECTION IN FRESH, FROZEN ARCHIVED AND CITRATE BUFFER TREATED FORMALIN-FIXED DOG BRAIN TISSUES

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