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

Alexandre Servat,Charles Rupprecht,Jordona Kirby,Richard Franka,Hildegund Ertl,Zhiquan Xiang

doi:10.3390/vetsci5020059

Abstract

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.

Highlights

IntroductionRabies is an acute progressive encephalitis caused by negative-stranded RNA viruses in the family
Rabies is an acute progressive encephalitis caused by negative-stranded RNA viruses in the familyRhabdoviridae, genus Lyssavirus and a major neglected zoonotic disease with substantial agricultural and public health burden [1]
The ratio of biotin conjugate to monoclonal ABs (MABs) affected the comparative detection of rabies virus (RV) inclusions within rabid raccoon brain impressions (Figures 1 and 2) greatly at 7.4 moles, so that RV inclusions were barely detectable at dilutions of or above 1:400 (Figure 1)

Summary

Introduction

Rabies is an acute progressive encephalitis caused by negative-stranded RNA viruses in the family. Rhabdoviridae, genus Lyssavirus and a major neglected zoonotic disease with substantial agricultural and public health burden [1]. The current gold standard for rabies diagnosis is the direct fluorescent antibody test (FAT), which detects viral antigens in the brain of affected mammals [2]. While the FAT is highly sensitive and specific, this test requires the use of a fluorescence microscope, which may limit its application in some resource-poor countries. In support of a global plan for the elimination of. 2018, 5, 59 canine rabies and for ongoing regional wildlife vaccination programs, additional diagnostic tests are needed [3] Sci. 2018, 5, 59 canine rabies and for ongoing regional wildlife vaccination programs, additional diagnostic tests are needed [3]

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Conclusion