Molecular methods to distinguish between classical rabies and the rabies-related European bat lyssaviruses

Black, Elizabeth M; McElhinney, Lorraine M.; Lowings, J. P.; Smith, Jean S.; Johnstone, P.; Heaton, Paul R.

doi:10.1016/s0166-0934(00)00159-2

Cited by 30 publications

(18 citation statements)

References 16 publications

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“…Only five of all previous PCR-based methods for rabies diagnosis have a similarly wide range of specificity (2,13,14,26,31), and only two of these provide virus identification by product sequencing (13,14). As the only bat lyssavirus known so far in Spain is EBV1, the primers described here were optimized for the detection of this particular virus, and sensitivity to the other lyssaviruses may be suboptimal.…”

Section: Discussionmentioning

confidence: 99%

Screening of Active Lyssavirus Infection in Wild Bat Populations by Viral RNA Detection on Oropharyngeal Swabs

et al. 2001

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Brain analysis cannot be used for the investigation of active lyssavirus infection in healthy bats because most bat species are protected by conservation directives. Consequently, serology remains the only tool for performing virological studies on natural bat populations; however, the presence of antibodies merely reflects past exposure to the virus and is not a valid marker of active infection. This work describes a new nested reverse transcription (RT)-PCR technique specifically designed for the detection of the European bat virus 1 on oropharyngeal swabs obtained from bats but also able to amplify RNA from the remaining rabies-related lyssaviruses in brain samples. The technique was successfully used for surveillance of a serotine bat (Eptesicus serotinus) colony involved in a case of human exposure, in which 15 out of 71 oropharyngeal swabs were positive. Lyssavirus infection was detected on 13 oropharyngeal swabs but in only 5 brains out of the 34 animals from which simultaneous brain and oropharyngeal samples had been taken. The lyssavirus involved could be rapidly identified by automatic sequencing of the RT-PCR products obtained from 14 brains and three bat oropharyngeal swabs. In conclusion, RT-PCR using oropharyngeal swabs will permit screening of wild bat populations for active lyssavirus infection, for research or epidemiological purposes, in line not only with conservation policies but also in a more efficient manner than classical detection techniques used on the brain.

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Section: Discussionmentioning

confidence: 99%

Screening of Active Lyssavirus Infection in Wild Bat Populations by Viral RNA Detection on Oropharyngeal Swabs

et al. 2001

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“…In order to develop a large-scale routine animal testing method that could become an adjunct to, or potentially replace, the DFA test, we sought a robotic system that would handle larger numbers of specimens per batch. Although the sensitivity of the molecular detection methods has been shown to be greater than the sensitivity of DFA (15,16), there has yet to be a molecular strategy assembled for use in the United States that is cost-effective, concise in its implementation, and ensures the detection of all prevalent rabies variants by selected primer/probe combinations.…”

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confidence: 99%

Comparison of Automated Quantitative Reverse Transcription-PCR and Direct Fluorescent-Antibody Detection for Routine Rabies Diagnosis in the United States

et al. 2015

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Rabies virus found worldwide and prevalent throughout the United States continues to be a public health concern. Direct-fluorescent antibody (DFA) detection remains the gold standard for rabies virus diagnostics. Assessing the utility of a high-throughput molecular platform such as the QIAsymphony SP/AS, in conjunction with quantitative reverse transcription-PCR (qRT-PCR), to augment or potentially replace the DFA test, was the focus of this project. Here we describe a triplex qRT-PCR assay, including assembly and evaluation for sensitivity, specificity, and ability to detect variants. Additionally, we compared the qRT-PCR assay to the gold standard direct fluorescent-antibody test. More than 1,000 specimens submitted for routine rabies diagnosis were tested to directly compare the two methods. All results were in agreement between the two methods, with one additional specimen detected by qRT-PCR below the limits of the DFA sensitivity. With the proper continued validation for variant detection, molecular methods have a place in routine rabies diagnostics within the United States. Rabies virus, in the Lyssavirus genus of the Rhabdoviridae family, is found worldwide and has been described since antiquity (1). The fatality rate of clinical rabies infections remains greater than 99%. There are at least 14 recognized species of lyssaviruses circulating in the world (2), yet in the Western Hemisphere, only classical rabies virus has been identified in terrestrial mammals and bats.The cost of rabies control in the United States exceeds 300 million dollars annually (http://www.cdc.gov/rabies/location/usa /cost.html). A great portion of this expense results from the cost of administering postexposure prophylaxis (PEP) to individuals meeting risk assessment guidelines for contracting rabies virus after a contact with a rabid or suspect rabid animal. The need for accurate and timely diagnostic tools to identify positive rabies cases will continue, as this disease is prevalent in the continental United States, Canada, and Mexico (3). The direct-fluorescent antibody test (DFA) (4) is a rapid and sensitive method for diagnosing rabies infection, and as a World Organisation for Animal Health (OIE)/World Health Organization (WHO)-prescribed rabies test, it is considered the worldwide gold standard for rabies diagnosis. The accuracy of the DFA relies upon the examination of fresh brain tissue, the experience of a trained microscopist with access to a quality fluorescence microscope, and the availability of high-quality antirabies diagnostic conjugates.The Rabies Laboratory of the New York State Department of Health (NYSDOH) performs diagnostic testing on approximately 7,000 animal specimens a year. During the summer months, the specimen submission rate can approach 150 to 200 samples per day, making a high-throughput method of detection a necessity. In most laboratories, the DFA can be completed within 3 h from receipt of specimen, and it has proven to be highly specific, sensitive, and reliable. Because a rabies laboratory can...

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“…Furthermore, there is no test for specificity included in the assay (2). To overcome this, several methods have been developed for rabies diagnosis, including hybridization (28), restriction fragment length polymorphism (RFLP), PCR-enzyme-linked immunosorbent assay (ELISA) (5), in situ hybrid-ization (10), and sequencing. The latter has become widely used, as the sequences obtained can be used for further genetic characterization (21).…”

mentioning

confidence: 99%

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy

Hoffmann

Freuling

Wakeley³

et al. 2010

J Clin Microbiol

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To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs, an improved diagnostic sensitivity and reliability can be ascertained for postmortem and intra vitam real-time RT-PCR analyses in rabies reference laboratories.

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Molecular methods to distinguish between classical rabies and the rabies-related European bat lyssaviruses

Cited by 30 publications

References 16 publications

Screening of Active Lyssavirus Infection in Wild Bat Populations by Viral RNA Detection on Oropharyngeal Swabs

Screening of Active Lyssavirus Infection in Wild Bat Populations by Viral RNA Detection on Oropharyngeal Swabs

Comparison of Automated Quantitative Reverse Transcription-PCR and Direct Fluorescent-Antibody Detection for Routine Rabies Diagnosis in the United States

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy

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