Abstract
Rabies is a lethal and notifiable zoonotic disease for which diagnostics have to meet the highest standards. In recent years, an evolution was especially seen in molecular diagnostics with a wide variety of different detection methods published. Therefore, a first international ring trial specifically designed on the use of reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus genomic RNA was organized. The trial focussed on assessment and comparison of the performance of conventional and real-time assays. In total, 16 European laboratories participated. All participants were asked to investigate a panel of defined lyssavirus RNAs, consisting of Rabies virus (RABV) and European bat lyssavirus 1 and 2 (EBLV-1 and -2) RNA samples, with systems available in their laboratory.The ring trial allowed the important conclusion that conventional RT-PCR assays were really robust assays tested with a high concordance between different laboratories and assays. The real-time RT-PCR system by Wakeley et al. (2005) in combination with an intercalating dye, and the combined version by Hoffmann and co-workers (2010) showed good sensitivity for the detection of all RABV samples included in this test panel. Furthermore, all used EBLV-specific assays, real-time RT-PCRs as well as conventional RT-PCR systems, were shown to be suitable for a reliable detection of EBLVs. It has to be mentioned that differences were seen in the performance between both the individual RT-PCR systems and the laboratories. Laboratories which used more than one molecular assay for testing the sample panel always concluded a correct sample result.Due to the markedly high genetic diversity of lyssaviruses, the application of different assays in diagnostics is needed to achieve a maximum of diagnostic accuracy. To improve the knowledge about the diagnostic performance proficiency testing at an international level is recommended before using lyssavirus molecular diagnostics e.g. for confirmatory testing.
Highlights
Rabies as a notifiable zoonotic disease is an acute, progressive and incurable viral encephalitis which is clinically characterized by central nervous disorders that lead to death
Nine different published and five unpublished assays mostly targeting the nucleoprotein gene were used in the frame of this ring trial, including real-time reverse transcription polymerase chain reaction (RT-PCR) (14 labs) as well as conventional RT-PCR (5 labs) techniques (Tables 2, 3, and 4)
During the recent validation of a fluorogenic probe-based realtime RT-PCR for rabies virus (RABV), it had become evident, that a single assay was not sufficient to detect all tested RABV strains due to a high degree of genetic diversity [17]
Summary
Rabies as a notifiable zoonotic disease is an acute, progressive and incurable viral encephalitis which is clinically characterized by central nervous disorders that lead to death. The disease is caused by different Lyssavirus species of the Rhabdoviridae family [1], with classical rabies virus (RABV) being responsible for tens of thousands of deaths per year [2]. In Europe, alongside sylvatic rabies in foxes, bat rabies is prevalent in a number of different bat species, mainly caused by the European bat lyssaviruses type 1 and. From single rabid bats e.g. West Caucasian for RT-PCR for the detection of lyssavirus genomic RNA, e.g. RABV and EBLV, focussing on an assessment and comparison of the performance of conventional and real-time RT-PCR assays established in different European laboratories. In an animal case laboratory confirmation of rabies via RT-PCR is on the one hand important for the identification of new lyssavirus species. On the other hand if human contacts occurred with this rabid animal adequate post exposure prophylaxis (PEP) must be initiated
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