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

Journal Article · · PLoS ONE
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  1. Centers for Disease Control and Prevention (CDC), Atlanta, GA (United States); OSTI
  2. Pennsylvania Department of Health, Exton, PA (United States)
  3. Wisconsin State Laboratory of Hygiene, Madison, WI (United States)
  4. Centers for Disease Control and Prevention (CDC), Atlanta, GA (United States)
  5. Maryland Department of Health, Baltimore, MD (United States)
  6. New Mexico Department of Health, Santa Fe, NM (United States)
  7. Department of Health, Research Institute for Tropical Medicine, Alabang Muntinlupa City, Manila (Philippines)
  8. New York State Department of Health, Albany, NY (United States)
  9. University of Georgia, Athens, GA (United States)
  10. California Department of Public Health, Sacramento, CA (United States)
  11. Public Health Institute of Chile, Santiago (Chile)
  12. Public Health Command Europe, Landstuhl (Germany)
  13. Ministère de l’Agriculture, Port-au-Prince (Haiti)
  14. Karnataka Veterinary, Animal and Fisheries Sciences University, Bangalore (India)
  15. Animal Health Centre, Ministry of Agriculture, Abbotsford (Canada)
+ Show Author Affiliations

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.

Research Organization:
Oak Ridge Insitute. for Science and Education (ORISE), Oak Ridge, TN (United States)
Sponsoring Organization:
Centers for Disease Control and Prevention; USDOE Office of Science (SC)
Grant/Contract Number:
SC0014664
OSTI ID:
1904894
Journal Information:
PLoS ONE, Journal Name: PLoS ONE Journal Issue: 5 Vol. 13; ISSN 1932-6203
Publisher:
Public Library of ScienceCopyright Statement
Country of Publication:
United States
Language:
English

References (71)

Development of real-time reverse transcriptase polymerase chain reaction methods for human rabies diagnosis journal August 2009
A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories journal March 2014
Ante mortem diagnosis of human rabies using saliva samples: Comparison of real time and conventional RT-PCR techniques journal May 2006
A simple method for detection of rabies viral sequences in 16-year old archival brain specimens with one-week fixation in formalin journal June 2006
Use of filter paper (FTA®) technology for sampling, recovery and molecular characterisation of rabies viruses journal March 2007
Comparative detection of rabies RNA by NASBA, real-time PCR and conventional PCR journal August 2011
A universal real-time assay for the detection of Lyssaviruses journal October 2011
Detection of rabies viral RNA by TaqMan real-time RT-PCR using non-neural specimens from dogs infected with rabies virus journal September 2012
Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus journal May 2017
Rescue of infectious classical swine fever and foot-and-mouth disease virus by RNA transfection and virus detection by RT-PCR after extended storage of samples in Trizol® journal June 2000
Rabies virus detection by RT-PCR in decomposed naturally infected brains journal June 2002
Detection of Australian bat lyssavirus using a fluorogenic probe journal December 2002
A rapid, low-cost, and microfluidic chip-based system for parallel identification of multiple pathogens related to clinical pneumonia journal July 2017
Rapid, Affordable and Portable Medium-Throughput Molecular Device for Zika Virus journal December 2016
A Reliable Diagnosis of Human Rabies Based on Analysis of Skin Biopsy Specimens journal December 2008
The use of Confidence or Fiducial Limits Illustrated in the case of the Binomial journal January 1934
Receiver-operating characteristic curves and likelihood ratios: improvements over traditional methods for the evaluation and application of veterinary clinical pathology tests journal March 2006
Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy journal November 2010
Comparative Analysis of Rabies Virus Reverse Transcription-PCR and Virus Isolation Using Samples from a Patient Infected with Rabies Virus journal August 2010
Improved PCR Methods for Detection of African Rabies and Rabies-Related Lyssaviruses journal November 2010
Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses journal November 1997
Development of a Real-Time, TaqMan Reverse Transcription-PCR Assay for Detection and Differentiation of Lyssavirus Genotypes 1, 5, and 6 journal June 2005
Lyssaviruses and rabies: current conundrums, concerns, contradictions and controversies journal January 2017
Emerging Technologies for the Detection of Rabies Virus: Challenges and Hopes in the 21st Century journal September 2009
Comparison of Biotinylated Monoclonal and Polyclonal Antibodies in an Evaluation of a Direct Rapid Immunohistochemical Test for the Routine Diagnosis of Rabies in Southern Africa journal September 2014
Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection journal July 2016
Dose-Response Analysis Using R journal December 2015
Influence of canine brain decomposition on laboratory diagnosis of rabies journal February 1999
Estimating the global burden of endemic canine rabies text January 2015
Rabies Virus in the Decomposed Brain of an Ethiopian Wolf Detected by Nested Reverse Transcription-Polymerase Chain Reaction journal October 1997
Requirements for Minimum Sample Size for Sensitivity and Specificity Analysis journal January 2016
Development of real-time reverse transcriptase polymerase chain reaction methods for human rabies diagnosis journal August 2009
ggplot2 book January 2009
Rescue of infectious classical swine fever and foot-and-mouth disease virus by RNA transfection and virus detection by RT-PCR after extended storage of samples in Trizol® journal June 2000
Rabies virus detection by RT-PCR in decomposed naturally infected brains journal June 2002
Detection of Australian bat lyssavirus using a fluorogenic probe journal December 2002
A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories journal March 2014
Ante mortem diagnosis of human rabies using saliva samples: Comparison of real time and conventional RT-PCR techniques journal May 2006
A simple method for detection of rabies viral sequences in 16-year old archival brain specimens with one-week fixation in formalin journal June 2006
Use of filter paper (FTA®) technology for sampling, recovery and molecular characterisation of rabies viruses journal March 2007
Comparative detection of rabies RNA by NASBA, real-time PCR and conventional PCR journal August 2011
A universal real-time assay for the detection of Lyssaviruses journal October 2011
Detection of rabies viral RNA by TaqMan real-time RT-PCR using non-neural specimens from dogs infected with rabies virus journal September 2012
Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus journal May 2017
Development of a hemi-nested RT-PCR method for the specific determination of European Bat Lyssavirus 1 journal May 2004
Rabies journal November 2017
A rapid, low-cost, and microfluidic chip-based system for parallel identification of multiple pathogens related to clinical pneumonia journal July 2017
Rapid, Affordable and Portable Medium-Throughput Molecular Device for Zika Virus journal December 2016
A Reliable Diagnosis of Human Rabies Based on Analysis of Skin Biopsy Specimens journal December 2008
The use of Confidence or Fiducial Limits Illustrated in the case of the Binomial journal January 1934
Diagnosis of Rabies by Polymerase Chain Reaction with Nested Primers journal January 1993
Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples journal July 2006
Receiver-operating characteristic curves and likelihood ratios: improvements over traditional methods for the evaluation and application of veterinary clinical pathology tests journal March 2006
Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy journal November 2010
Comparative Analysis of Rabies Virus Reverse Transcription-PCR and Virus Isolation Using Samples from a Patient Infected with Rabies Virus journal August 2010
Improved PCR Methods for Detection of African Rabies and Rabies-Related Lyssaviruses journal November 2010
Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses journal November 1997
Development of a Real-Time, TaqMan Reverse Transcription-PCR Assay for Detection and Differentiation of Lyssavirus Genotypes 1, 5, and 6 journal June 2005
pROC: an open-source package for R and S+ to analyze and compare ROC curves journal March 2011
Lyssaviruses and rabies: current conundrums, concerns, contradictions and controversies journal January 2017
Emerging Technologies for the Detection of Rabies Virus: Challenges and Hopes in the 21st Century journal September 2009
Comparison of Biotinylated Monoclonal and Polyclonal Antibodies in an Evaluation of a Direct Rapid Immunohistochemical Test for the Routine Diagnosis of Rabies in Southern Africa journal September 2014
Estimating the Global Burden of Endemic Canine Rabies journal April 2015
Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection journal July 2016
A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses journal January 2017
Development and validation of an immunoperoxidase antigen detection test for improved diagnosis of rabies in Indonesia journal November 2017
Dose-Response Analysis Using R journal December 2015
Ante- and post-mortem diagnosis of rabies using nucleic acid-amplification tests journal March 2010
Influence of canine brain decomposition on laboratory diagnosis of rabies journal February 1999
Evaluation of a Direct, Rapid Immunohistochemical Test for Rabies Diagnosis journal February 2012
Rabies Virus in the Decomposed Brain of an Ethiopian Wolf Detected by Nested Reverse Transcription-Polymerase Chain Reaction journal October 1997

Cited By (2)

Bat rabies in Washington State: Temporal-spatial trends and risk factors for zoonotic transmission (2000–2017) journal October 2018
Using the LN34 Pan-Lyssavirus Real-Time RT-PCR Assay for Rabies Diagnosis and Rapid Genetic Typing from Formalin-Fixed Human Brain Tissue journal January 2020

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