Application and Comparative Evaluation of Fluorescent Antibody, Immunohistochemistry and Reverse Transcription Polymerase Chain Reaction Tests for the Detection of Rabies Virus Antigen or Nucleic Acid in Brain Samples of Animals Suspected of Rabies in India

Abstract

Accurate and early diagnosis of animal rabies is critical for undertaking public health measures. Whereas the direct fluorescent antibody (DFA) technique is the recommended test, the more convenient, direct rapid immunochemistry test (dRIT), as well as the more sensitive, reverse transcription polymerase chain reaction (RT-PCR), have recently been employed for the laboratory diagnosis of rabies. We compared the three methods on brain samples from domestic (dog, cat, cattle, buffalo, horse, pig and goat) and wild (leopard, wolf and jackal) animals from various parts of India. Of the 257 samples tested, 167 were positive by all the three tests; in addition, 35 of the 36 decomposed samples were positive by RT-PCR. This is the first study in which such large number of animal samples have been subjected to the three tests simultaneously. The results confirm 100% corroboration between DFA and dRIT, buttress the applicability of dRIT in the simple and rapid diagnosis of rabies in animals, and reaffirm the suitability of RT-PCR for samples unfit for testing either by DFA or dRIT.

An inter-laboratory trial as a tool to increase rabies diagnostic capabilities of Sub-Saharan African Veterinary laboratories

“…Another advantage of the molecular approach is a better sensitivity in case of putrefied samples, which are often unfit for the detection of viral antigens [31]. This is an important factor in the African setting, where the arrival of field samples to diagnostic facilities can be delayed due to transportation issues, samples can be incorrectly stored [10] or suspected animals can be exhumed several weeks after their burial following suspicion of human rabies cases [32].…”

“…This is an important factor in the African setting, where the arrival of field samples to diagnostic facilities can be delayed due to transportation issues, samples can be incorrectly stored [10] or suspected animals can be exhumed several weeks after their burial following suspicion of human rabies cases [32]. Although real-time RT-PCR protocols that detect smaller fragments is a better option for detection of the viral genomes in case of advanced decomposition [32], a recent study from India [31] showed that out of thirty six [36] field putrefied samples unfit for detection of viral antigens by either DFA test or dRIT, thirty five [35] of them still tested positive by mean of a well-known hemi-nested RT-PCR also targeting rabies virus N gene [33]. In any case, although opting for molecular testing increases the possibility to identify the virus in putrefied material, it is not exempt from misdiagnosis.…”

Detection of rabies virus antigen by the indirect rapid immunohistochemistry test in equines and comparisons with other diagnostic techniques

“…Prabhu et al. (2018) compared the dRIT with the dFAT and RT‐PCR techniques performed in brain from wild and domestic animals from various parts of India, among them, only five horses. These authors demonstrated that 167/257 (65%) samples were found positive by all three tests; however, the viral variant was not mentioned.…”

“…The dRIT was developed in the late 1990s at the Centers for Disease Control and Prevention (CDC) in Atlanta (USA) (Rupprecht et al., 2014). Subsequent investigations have demonstrated that the dRIT presents equal or higher diagnostic sensitivity and specificity than the dFAT (Coetzer, Sabeta, Markotter, Rupprecht, & Nel, 2014; Dürr et al., 2008; Madhusudana, Subha, Thankappan, & Ashwin, 2012; Mani & Madhusudana, 2013; Middel, Fehlner‐Gardiner, Pulham, & Buchanan, 2017; Patrick et al., 2019; Prabhu et al., 2018).…”

“…The dRIT is based on rabies virus (RABV) antigen detection using biotin‐conjugated primary antibody in central nervous system (CNS) samples, which may be fresh, frozen or conserved in glycerol (Lembo et al., 2006). The technique has some advantages, such as low cost and time requirements and easy interpretation, and it is widely used an alternative diagnostic tool for rabies diagnosis, especially in countries with limited financial resources (Dürr et al., 2008; Madhusudana et al., 2012; Middel et al., 2017; Prabhu et al., 2018). Almost all reagents needed for the dRIT can be purchased commercially, with the exception of the biotinylated monoclonal or polyclonal antibodies (which may be self‐produced or obtained from OIE/WHO rabies reference laboratories).…”