Table 1.
Primers and probes used in the LN34 real-time RT-PCR assay.
Table 2.
List of the laboratories that participated in the LN34 assay evaluation and the country or region of sample origin.
Fig 1.
Analytical sensitivity of the LN34 assay.
A. Result of limit of detection analysis using serial dilutions of artificial positive control RNA. Estimated RNA copy number was plotted against the percent of runs in which amplification was observed (blue dots). A sigmoidal curve was fit to the data to estimate the limit of detection as the RNA copy number corresponding to 95% assay success (black line, gray shading indicates 95% confidence interval). B. LN34 assay Ct value upon 10 fold serial dilutions of RNA extracted from six lyssavirus isolates. Average Ct value and 95% confidence intervals are shown; several points are artificially offset along the x axis to avoid overlap. Linear regression analysis revealed the following slopes: DUVV -3.58, LBV -3.37, RABV1–3.64, RABV2–3.84, RABV3–3.66, RABV4–3.56. LN34 assay diagnostic cut-off for positive samples (Ct 35) is highlighted in yellow. Assay failure (Ct 45) is shown in red.
Fig 2.
LN34 assay repeatability and reproducibility.
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 25th and 75th quartiles. Whiskers extend to 1.5×(inter-quartile range); data outside whiskers are plotted individually. ** p < 0.01.
Table 3.
Variation between replicate, extraction, assay run, and laboratory observed during repeatability and reproducibility studies.
Table 4.
LN34 assay diagnostic algorithm for post-mortem brain stem samples.
Table 5.
LN34 and DFA test results for 1,020 U.S. samples tested in U.S. laboratories.
Fig 3.
A multi-laboratory international evaluation of the LN34 assay.
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.
Table 6.
LN34 and DFA test results for 2,978 samples tested in all laboratories during the LN34 assay evaluation.
Fig 4.
LN34 assay performance in different samples types.
Violin plots showing LN34 (A) and β-actin (B) Ct values observed for brain samples of varying reported sample conditions. Overlaid boxplots show median (thick line), 25th and 75th quartiles. Whiskers indicate 1.5×(inter-quartile range). Data points beyond the whiskers are plotted individually. Data are only shown for samples that exhibited amplification. The number of samples for each condition is indicated under the sample condition. ** p <0.01, *** p < 0.001.