A Pan-<i>Lyssavirus</i> Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable <i>Rabies virus</i> and Other Lyssaviruses

<div><p>Rabies, resulting from infection by <i>Rabies virus</i> (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the <i>Lyssavirus</i> genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the <i>Lyssavirus</i> genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional <i>Lyssavirus</i> species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay.</p></div

<i>Rabies</i> virus positive samples used to validate the sensitivity of the LN34 real-time RT-PCR assay.

<p><i>Rabies</i> virus positive samples used to validate the sensitivity of the LN34 real-time RT-PCR assay.</p

List of primers and probes used in this study.

<p>List of primers and probes used in this study.</p

Positive clinical samples from human (ante mortem and postmortem) and animal (postmortem) specimen.

<p>Positive clinical samples from human (ante mortem and postmortem) and animal (postmortem) specimen.</p

Sequence alignment of the LN34 assay target region from isolates of 14 <i>Lyssavirus</i> species.

<p>Sequence alignment of the LN34 assay target region from isolates of 14 <i>Lyssavirus</i> species.</p

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

<div><p>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 <i>Lyssavirus</i> 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.</p></div

LN34 assay diagnostic algorithm for post-mortem brain stem samples.

<p>LN34 assay diagnostic algorithm for post-mortem brain stem samples.</p

LN34 assay repeatability and reproducibility.

<p>A–B. Comparison of replicate Ct values for the same RNA sample tested in the same assay run for LN34 (A) and β-actin (B) assays. Ct value for replicate 1 is plotted against replicate 2. Gray line indicates identity (y = x). Results of linear regression analysis is shown in the upper left corner. Vertical red lines indicate the diagnostic cut-off values for positive samples for each assay. Points are transparent; darker color indicates overlapping points. Samples that failed to amplify are plotted at Ct 0. C. LN34 Ct values reported for positive control RNA tested in 12 laboratories shown as a beeswarm plot. Each dot represents the average value for one assay run; points are plotted according to Ct value (y-axis), then offset along the x-axis to show the distribution of points at each Ct value. Orange dots indicate Ct values observed in one laboratory using a PCR machine with decreased sensitivity, and green dots indicate Ct values reported from the same laboratory using a different PCR machine. D. Comparison of LN34 Ct value for a panel of 14 samples tested in three real-time PCR machines. Machine 2 was determined to produce significantly higher Ct values than either Machine 1 or 3, for the same sample. Boxplots show median (thick line) and 25^th and 75^th quartiles. Whiskers extend to 1.5×(inter-quartile range); data outside whiskers are plotted individually. ** p < 0.01.</p

A multi-laboratory international evaluation of the LN34 assay.

<p>A. Proportion of samples originating and tested in the United States (US), originating and tested outside the U.S. (INT), or originating outside the U.S. and tested in the U.S. (US-INT). B. Frequency of LN34 Ct values for DFA positive samples. C. Frequency of β-actin Ct values for all 2,978 samples tested. Diagnostic cut-off values for positive samples (Ct 35 for LN34 and Ct 33 for β-actin) are highlighted by red vertical lines.</p

Variation between replicate, extraction, assay run, and laboratory observed during repeatability and reproducibility studies.

<p>Variation between replicate, extraction, assay run, and laboratory observed during repeatability and reproducibility studies.</p