Supportive Data

Accuracy:

A total of 100 clinical stool specimens submitted to Mayo between 11/2015 and 03/2016 for testing by a commercial multiplex gastrointestinal panel (that includes norovirus) were aliquoted, blinded, and tested within 24 to 48 hours of receipt using the norovirus G1/G2 real-time polymerase chain reaction (PCR) assays. Specimens yielding discordant results between the multiplex panel and the real-time PCR were submitted to an outside reference lab and the Minnesota Department of Health (MDH) for norovirus molecular testing.

Table 1. Comparison of results following discordant analysis at an outside reference laboratory and MDH

Adjusted Sensitivity (95% confidence interval [CI])=96.9% (88.8, 99.8)

Adjusted Specificity (95% CI)=100% (88.2, 100)

Adjusted Agreement (95% CI)=98% (92.6, 99.9)

a. Samples showing discordant results between the norovirus real-time PCR and FilmArray were tested by a molecular method at Focus Diagnostics and MDH.

b. Samples that showed agreement between the norovirus real-time PCR and FilmArray were not tested further.

c. Four of these 63 samples were identified as G1 by the LDT; the remaining 59 were identified as norovirus G2.

d. One of these 2 samples was positive for norovirus at Focus Diagnostics and MDH, and upon repeat testing in triplicate by the LDT, was also positive. The second sample was positive for norovirus at MDH, but negative by Focus and the LDT upon repeat testing.

Testing of clinical stool samples yielded 59 specimens that were positive for norovirus G2 by the LDT; however, only 4 samples were determined to be positive for norovirus G1. In order to supplement the clinical data and increase the number of samples positive for norovirus G1, spiking studies were performed. Analyte-negative stool samples (n=26) were spiked with norovirus G1 RNA (ATCC) at 1 dilution above the defined limit of detection (LOD) (Table 2).

Table 2. Results of spiking studies for norovirus GI in stool specimens.

Analytical Sensitivity:

The limit of detection of both the G1 and G2 assays was determined to be 5 copies/mcL (5000 copies/mL) of stool.

Analytical Specificity:

A comprehensive specificity panel, consisting of bacteria (n=16), parasites (n=5), or viruses (n=5) known to cause gastrointestinal disease was tested by the norovirus G1/G2 assays. In addition, human DNA was tested. All members of the specificity panel were negative by both the norovirus G1 and G2 assays. In addition, a BLAST (basic local alignment search tool) analysis was performed on the primer and probe sequences and did not reveal significant cross-reactivity with organisms that may be present in stool samples.