Multi-site evaluation of the LN34 pan-lyssavirus real-time RT-PCR assay for post-mortem rabies diagnostics

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.

Clinical Presentation and Serologic Response during a Rabies Epizootic in Captive Common Vampire Bats (Desmodus rotundus)

“…After collection, saliva samples were placed in vials containing 0.5 mL of viral transport media and stored at −80 • C until further testing. These swabs were tested at the NWHC following the LN34 pan-lyssavirus real-time RT-PCR [17,18], with some modifications to the protocol, such as using 8.5 µL of RNA per reaction and using an iCycler instrument (BioRad, Hercules, CA, USA). Cycle threshold (Ct) values ≤ 35 were considered positive (i.e., RABV RNA present), whereas values 35-40 were considered inconclusive and may indicate a low virus load, insufficient sample, or possible cross contamination.…”

Using the LN34 Pan-Lyssavirus Real-Time RT-PCR Assay for Rabies Diagnosis and Rapid Genetic Typing from Formalin-Fixed Human Brain Tissue

“…The improved sensitivity of the LN34 assay has been demonstrated using multiple types of tissues including fresh, frozen, and fixed tissues and tissues in poor condition [19,20]. In this reported case, the LN34 assay was able to detect viral nucleic acid in a formalin-fixed clinical sample, and the LN34 amplicon sequencing provided genetic typing information for the cases using fragmented RNA.…”

“…The formalin-fixed tissue outlined in this study tested positive by IHC and the results were promptly reported to implement appropriate measures and prevent additional human cases; however, the RABV strain characterization was not performed due to limits of IHC to distinguish RABV strains. A new molecular approach, the LN34 real-time RT-PCR assay, has recently demonstrated the ability to amplify and detect a 165 bp fragment of the RABV genome in formalin-fixed tissues [20]. We successfully generated RABV amplicons using the LN34 assay in seven out of eight samples, with Ct values ranging from 26.1 to 35.…”

“…To confirm RABV infection, nucleic acid was extracted from three areas of the brain tissue wherein the IHC detected high concentrations of RABV antigen, three areas with low concentration, and two from gray and white matter areas that were untested by IHC. Approximately 0.01 g of tissue was dissociated mechanically using ceramic beads in 360 µL of lysis buffer with 40 µL proteinase K (DNeasy Blood and Tissue Kit-Qiagen, Hilden, Germany) and incubated overnight at 55 • C. Then 100 µL of homogenized tissue was mixed with 400 µL of Trizol; the RNA extraction and the LN34 assay were performed as described in detail previously [19,20].…”

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“…Por outro lado, o sistema de RT-qPCR utilizando a sonda de hidrólise fluorogênica 5' exonuclease (TaqMan), assegura uma alta especificidade analítica em decorrência da reação de hibridização intrínseca da sonda (HUGHES et al, 2004;WAKELEY et al, 2005;WACHARAPLUESADEE et al, 2008;NADIN-DAVIS et al, 2009). Entretanto a significante diversidade de sequências entre as diferentes variantes virais torna difícil o desenvolvimento de um único, robusto e simples ensaio para diagnóstico da raiva (GIGANTE et al, 2018 Fonte: Souza, T.C.P.(2019). Legenda: A) Encéfalo antes do início do processamento com aspecto sanguinolento, indicando a presença de hemorragia.…”