Rabies is a fatal zoonotic disease that requires fast, accurate diagnosis to prevent disease in an exposed individual. The current gold standard for post-mortem diagnosis of human and animal rabies is the direct fluorescent antibody (DFA) test. While the DFA test has proven sensitive and reliable, it requires high quality antibody conjugates, a skilled technician, a fluorescence microscope and diagnostic specimen of sufficient quality. The LN34 pan-lyssavirus real-time RT-PCR assay represents a strong candidate for rabies post-mortem diagnostics due to its ability to detect RNA across the diverse Lyssavirus genus, its high sensitivity, its potential for use with deteriorated tissues, and its simple, easy to implement design. Here, we present data from a multi-site evaluation of the LN34 assay in 14 laboratories. A total of 2,978 samples (1,049 DFA positive) from Africa, the Americas, Asia, Europe, and the Middle East were tested. The LN34 assay exhibited low variability in repeatability and reproducibility studies and was capable of detecting viral RNA in fresh, frozen, archived, deteriorated and formalin-fixed brain tissue. The LN34 assay displayed high diagnostic specificity (99.68%) and sensitivity (99.90%) when compared to the DFA test, and no DFA positive samples were negative by the LN34 assay. The LN34 assay produced definitive findings for 80 samples that were inconclusive or untestable by DFA; 29 were positive. Five samples were inconclusive by the LN34 assay, and only one sample was inconclusive by both tests. Furthermore, use of the LN34 assay led to the identification of one false negative and 11 false positive DFA results. Together, these results demonstrate the reliability and robustness of the LN34 assay and support a role for the LN34 assay in improving rabies diagnostics and surveillance.

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...

Objectives: Each year, rabies virus infection results in the death of more than 50 000 persons worldwide. In the United States, the Centers for Disease Control and Prevention (CDC) reported 23 human rabies cases from May 1, 2008, through October 1, 2017. Although rabies testing in the United States is highly reliable, some specimens submitted to rabies laboratories do not have adequate tissues or may be substantially decomposed. In these instances, the specimen may be considered unsatisfactory for testing or produce indeterminate results using the gold standard direct fluorescent antibody test. The objective of this study was to evaluate the number of unsatisfactory samples or samples with indeterminate results that were positive for rabies virus after additional testing using real-time reverse transcriptase polymerase chain reaction (RT-PCR). Methods: In 2016, we retested all unsatisfactory specimens or specimens with indeterminate results using real-time RT-PCR. We further typed any sample that was real-time RT-PCR positive to identify the infecting rabies virus variant. Results: Of 210 retested unsatisfactory specimens or specimens with indeterminate results, 9 (4.3%) were positive for rabies. In each case, the animal was infected with a homologous rabies virus variant. Conclusion: These results confirm the recommendation by CDC and state public health laboratories that indeterminate results should be considered positive and justify the prompt treatment of exposed persons through an animal that is suspected to have rabies.