ABSTRACT
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.
Subject(s)
Lyssavirus/genetics , RNA, Viral/genetics , Rabies , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , Diagnosis , Humans , Rabies/diagnosis , Rabies/geneticsABSTRACT
BACKGROUND: The Molecular Virology Proficiency Testing Program at the Wadsworth Center began the assembly and distribution of influenza virus panels to US public health labs (PHLs) in 2008. The program was created to assist PHLs in assessing their performance and in meeting CLIA regulations for mandated proficiency testing (PT). OBJECTIVES: To design and distribute proficiency testing panels containing influenza A virus subtypes H1N1 and H3N2, and influenza B; when H1N1pdm09 emerged it also was incorporated into the panels. A secondary objective was to determine the best matrix for long term storage of the molecular PT samples. STUDY DESIGN: Viruses were quantitated using TCID(50) and quantitative real-time RT-PCR. Reference laboratories were enlisted to verify viral identity in the panels and to help determine viral titers to be used in the PT panels sent to PHLs. RESULTS AND CONCLUSIONS: Of the 29 laboratories that participated the first year, 27 were able to correctly identify all of the virus types in the panel. Fifty-one PHLs participated in the program the second year when pandemic H1N1 was added, and 45 were able to correctly detect, type and subtype all of the viruses in the panel. In the program's third year, 60 laboratories participated; 58 correctly detected and subtyped all of the viruses in the panel. Annual surveys of assay techniques showed that the PHLs had shifted their extraction methods and PCR-thermocycler instrumentation to meet FDA-approved methods. The degradation study revealed that frozen viral stocks were stable for at least 30months, thus allowing ample time to prepare and pre-test panels.
