International interlaboratory trials on rabies diagnosis: An overview of results and variation in reference diagnosis techniques (fluorescent antibody test, rabies tissue culture infection test, mouse inoculation test) and molecular biology techniques
French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Nancy Laboratory for Rabies and Wildlife, WHO Collaborating Centre for Research and Management in Zoonoses Control, OIE Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies, European Union Reference Institute for Rabies Serology, Technopôle Agricole et Vétérinaire, BP 40009, 54220 Malzéville cedex, France.
Interlaboratory trials on rabies diagnosis were organised in 2009 and in 2010 by the European Union Reference Laboratory (EURL) for rabies. In 2009, two panels of virus samples were sent to participating laboratories to compare results on reference diagnosis techniques and on RT-PCR. A single panel was sent in 2010 to test FAT (fluorescent antibody test), RTCIT (rabies tissue culture infection test) and RT-PCR techniques. The virus panels included the RABV, EBLV-1, EBLV-2 and ABLV strains. Results revealed that laboratories produced the highest proportion of concordant results using RT-PCR (90.5%) and FAT (87.1%), followed by RTCIT (70.0%) and MIT (35.0%) in 2009 and in FAT (85.0%) and RT-PCR (80.6%) followed by RTCIT (77.3%) in 2010. Errors were only observed in bat strains (i.e. none in the RABV strain) for the RT-PCR or FAT techniques, highlighting the need to improve diagnosis most specifically in such strains. RT-PCR was the technique showing the lowest rate of false negative results in either trial year, while RTCIT and MIT (performed in 2009 only) were the techniques with the lowest proportion of false positive results. Nevertheless, the FAT technique represented a good compromise with both satisfactory sensitivity and specificity, as only a few false positive (1.6% in 2009, 5.8% in 2010) and false negative results (1.6% in both 2009 and 2010) were detected. The analysis of technical questionnaires describing the protocols used by participating laboratories revealed variation in the methods used that may induce inconsistencies in the results. In this study, the number of readers for FAT slide examination was identified as a factor affecting significantly the results of laboratories, suggesting that two independent readers are necessary for routine rabies diagnosis. Our findings highlight the need for all rabies diagnostic laboratories to improve harmonisation of procedures.
"Samples obtained through participation to consecutive an interlaboratory proficiency tests organised by the European Union reference laboratory of rabies (ANSES, Nancy, France) between 2009 and 2013. Samples were reconstituted in 1 mL sterile, nuclease-free, and distilled water (Robardet et al., 2011 ). 2 Van Gucht et al., 2013 . 3 Bourhy et al., 1992 . "
[Show abstract][Hide abstract] ABSTRACT: A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.
"The nature of the FITC labelled anti-rabies antibody conjugate, whether mono or polyclonal, is also one of these critical variables (Rudd et al., 2005). While in-house products are used occasionally by laboratories (Trimarchi and Debbie, 1974; Tzianabos et al., 1976; Ribas Antunez et al., 2005; Caporale et al., 2009) most of the conjugates used are commercial reagents (Robardet et al., 2011). An assessment on a broader international level has never been conducted . "
[Show abstract][Hide abstract] ABSTRACT: Twelve national reference laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies conjugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive brain tissues infected with various lyssavirus species as well as negative samples were analyzed blindly using a standardized FAT procedure. Conjugates B, C, D, and E were found to be significantly more effective than conjugate A for GS7 (French RABV) diluted samples (1/8 and 1/100) while the frequency of concordant results of conjugates C and D differ significantly from conjugates A, B and E for CVS 27. For detection of EBLV-1 strains, conjugates C and D also presented a significantly lower frequency of discordant results compared to conjugates A, B and E. Conjugates B, C and D were found to be significantly more effective than conjugates E and A for EBLV-2 and ABLV samples. In view of these results, conjugates C and D set themselves apart from the others and appeared as the most effective of this 5-panel conjugates. This study clearly demonstrates that the variability of conjugates used by National Reference Laboratories can potentially lead to discordant results and influence assay sensitivity. In case of false negative results this could have a dramatic impact if the animal under investigation is responsible for human exposure. To avoid such situations, confirmatory tests should be implemented.
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Lyssaviruses have been known for centuries to cause lethal encephalitis in animals and humans, representing a serious public health problem especially in developing countries. Little is known about the way that lyssaviruses in general, and Duvenhage virus in particular cause disease. Studies of pathogenesis have been hampered by the fact that the virus has not yet been propagated and characterized extensively. In this paper, we describe the characterization of Duvenhage virus in vitro. Further, we characterized the virus in BALB/c mice. We compared Duvenhage virus with a wild type rabies virus (silver-haired bat rabies virus) and we found that while in vitro the differences of these two viruses were not significant, the in vivo characteristics of these two viruses differed significantly. Histological analyses of infected mouse brains suggest that differences in virulence may be associated with difference in tropism. Elucidating the differences in pathogenesis between different lyssaviruses might help us in the design of novel treatment protocols.
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