Evaluation of RT-PCR Assay for Routine Laboratory Diagnosis of Rabies in Post Mortem Brain Samples from Different Species of Animals

Abstract

Rabies in domestic and wild animals continues to be a major public health threat in India. Rapid and accurate diagnosis of rabies in animals is therefore of utmost importance as the individuals who were in contact with the rabid animals are at a greater risk. A significant amount of diagnostic tissue samples submitted to our laboratory are often autolysed and the WHO recommended direct fluorescent antibody test (FAT) for rabies diagnosis cannot be used in such samples. In this pilot study we have evaluated three different diagnostic primer sets for rapid sensitive and specific detection of rabies genome from the brain samples of different species of animals. We have validated a sensitive RT-PCR assay using brain tissue samples from different species of animals such as cat, cattle, dog, mouse and human, for routine diagnosis of rabies. Our results show the potential of this assay as a confirmatory test when the FAT results are unreliable and also as an alternative diagnostic test in circumstances when the diagnostic samples are unsuitable for use in FAT. Furthermore the nucleotide sequence of nucleoprotein gene amplified using this assay can also be used for the molecular epidemiological study of the rabies viruses in India.Keywords Rabies Á India Á Diagnosis Á FAT Á RT-PCR Animal and human rabies are endemic throughout the Indian subcontinent with the exception of Andaman and Nicobar Islands. The causative agent, rabies virus (family: Rhabdoviridae; genus: Lyssavirus) is largely maintained in two ecologically inter-related disease cycles; urban and sylvatic (wildlife). Although, dog is primarily responsible for rabies transmission to humans in India [3], wildlife plays a much lesser but not insignificant role in transmission [15]. It has been reported that more than 96 % of rabies incidence in India are the result of contact with infected dogs. In addition, rabies was reported as a result of contact with infected jackals in 1.7 %, cats in 0.8 %, monkeys in 0.4 %, mongooses in 0.4 %, and foxes in 3 % of the cases [3]. Therefore ante and post mortem diagnosis of rabies in animals is of utmost importance as the animals and human beings who come in contact with these rabid animals are at a greater risk of contracting the disease. The most widely used diagnostic test for rabies is the fluorescent antibody test (FAT), which is recommended by both WHO and OIE [17]. This 'gold standard' test may be used directly on a brain tissue impression smear and can also be used to confirm the presence of rabies antigen in cell culture or in brain tissue of mice that have been inoculated for diagnostic purposes. Although, the FAT gives reliable results on fresh specimens within a few hours in more than 95-99 % of the cases [17], autolysed tissue samples can reduce the sensitivity of this test and often are unsuitable for confirming the presence of rabies antigen. Classical reverse transcription-polymerase chain reaction (RT-PCR)

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

“…A simple conventional RT-PCR has been found to be highly congruent to DFA in declaring positivity for rabies [ 35 , 41 , 42 ]. An example of the RT-PCR profile is depicted in Figure 2 .…”

Continued circulation, recombination and evolution of the ancient subcontinent lineage despite predominance of the recent arctic-like lineage rabies viruses (RABV) in India

“…The samples were tested at the rabies unit of Madras Veterinary College for rabies confirmation by fluorescent antibody test (FAT) [17]. In addition to FAT, N-gene specific RT-PCR [18] of 20% homogenate of the respective brain samples in phosphate buffered saline was performed to confirm the presence of rabies viral genomes. Sample collection from animals was performed in full compliance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) regulations and the study was approved by the Institutional Animal Ethics Committee (IAEC) of Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai, India.…”

Pathobiological investigation of naturally infected canine rabies cases from Sri Lanka

“…These methods, have been reported to give 100% specificity and sensitivity [2] and Taqman RT-PCR is considered capable of detecting divergent, novel Lyssaviruses [16, 24]. PCR techniques are important confirmatory tests, often used in tandem with FAT [21], which may also be used when brain tissue is severely autolysed [8, 25].…”

“…Heminested reverse transcription polymerase chain reaction (HnRT-PCR) and real time, Taqman reverse transcription polymerase chain reaction (Taqman RT-PCR) assays have been described [ 16 , 17 , 34 ]. These methods, have been reported to give 100% specificity and sensitivity [ 2 ] and Taqman RT-PCR is considered capable of detecting divergent, novel Lyssaviruses [ 16 , 24 ]. PCR techniques are important confirmatory tests, often used in tandem with FAT [ 21 ], which may also be used when brain tissue is severely autolysed [ 8 , 25 ].…”