Multicenter evaluation of Verigene Enteric Pathogens Nucleic Acid Test for detection of gastrointestinal pathogens

We investigated the efficiency of the Verigene Enteric Pathogens Nucleic Acid Test (Verigene EP test), which is an automated microarray-based assay system that enables rapid and simultaneous genetic detection of gastrointestinal pathogens and toxins, including those in the Campylobacter Group, Salmonella species, Shigella species, the Vibrio Group, Yersinia enterocolitica, Shiga toxin 1 and 2, norovirus GI/GII, and rotavirus A. Three clinical laboratories evaluated the Verigene EP test, using 268 stool samples for bacterial and toxin genes and 167 samples for viral genes. Culture-based reference methods were used for the detection of bacteria and toxins, while a different molecular assay was used for viral detection. The overall concordance rate between the Verigene EP test and the reference methods for the 1940 assays was 99.0%. The overall sensitivity and specificity of the Verigene EP test were 97.0% and 99.3%, respectively. Of the 19 samples with discordant results, 13 samples were false positives and six were false negatives. The Verigene EP test simultaneously detected two targets in 11 samples; overall, the test demonstrated high efficiency in detecting crucial diarrheagenic pathogens, indicating its suitability for clinical practice.


Scientific Reports
| (2021) 11:3033 | https://doi.org/10.1038/s41598-021-82490-z www.nature.com/scientificreports/ Verigene EP test. The Verigene EP test, which has been approved for use in clinical settings by the Food and Drug Administration (FDA) in the United States and by the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan, was used according to the manufacturer's instructions. The test requires a processor, a reader, and single-use consumables. To prepare the fecal suspension for the assay, a swab dipped into stool was mixed with Stool Prep Buffer for 15-20 s using a vortex mixer. Extraction Tray, Tip Holder Assembly, Amplification Tray, and Test Cartridge were loaded into the processor. After centrifugation, 200 µL of the suspension was loaded into the Sample Loading Well of the Extraction Tray. Nucleic acid extraction, amplification, and nanoparticle-based microarray hybridization were automatically performed on the processor, and the data were analyzed by the reader. The Verigene EP test is aimed at the genetic detection of Campylobacter Group (C. jejuni, coli, and lari), Salmonella species, Shigella species (S. boydii, sonnei, flexneri, and dysenteriae), Vibrio Group (V. cholerae and parahaemolyticus), Yersinia enterocolitica, Shiga toxin (Stx) 1 and 2, norovirus GI/GII, and rotavirus A 3,4,6 .
The test does not target protozoa.
Culture methods. Culture-based methods for the detection of bacteria and toxins were performed based on the guidelines for the diagnostic testing of enteric infections, textbooks, and the manufacturer's instructions [7][8][9] . The media used and the identification methods are listed in Supplemental as follows 14,15 . Briefly, a 10% (wt/vol) stool suspension was prepared with distilled sterile water and centrifuged at 3000×g for 20 min. Nucleic acid was extracted from 140 μL of the stool suspension using NucleoMag (Takara Bio Inc.) or QIAamp Virus RNA Mini Kit (Qiagen) according to the manufacturer's instructions. The extracted nucleic acid was eluted with 60 μL of diethyl pyrocarbonate-treated water and stored at -80 °C until use. After reverse transcription using PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio Inc.) according to the manufacturer's instructions, multiplex PCR was performed using the QuantiTect Multiplex PCR kit (Qiagen) on a LightCycler 480 System (Roche Diagnostics). Amplification was carried out in 50 μL reaction mixture containing 5 μL nucleic acid sample. The reaction steps were as follows: 15 min at 95 °C and 40 cycles consisting of 1 min at 94 °C and 90 s at 60 °C. Primers and probes for the detection of norovirus GI/GII and rotavirus A genes have been described elsewhere 14,15 .

Reference methods and discrepant analysis.
Culture-based methods were used as reference methods for the detection of bacteria and toxins. The xTAG GPP was used as a reference method for viral detection. For the detection of bacteria and toxins, a discrepant analysis was performed by re-examination using the Verigene EP test for the samples with false-negative results or by the assay using the xTAG GPP for the samples with falsepositive results. Discrepant analysis for each virus was performed using multiplex real-time PCR.
Efficiency of the Verigene EP test. We calculated the sensitivity and specificity of the Verigene EP test in detecting targets, based on results from the reference methods, and determined the 95% confidence intervals using the software R version 3.5.1 16 .
Assessment using characterized stool samples. To 13 and six were false positives and false negatives, respectively. Seven of the 13 false-positive samples were positive, and one out of the six false-negative samples was negative in the discrepant analysis ( Table 1).
The Verigene EP test simultaneously detected two targets in 11 samples (Table 2). Of these 11 samples, five samples showed fully concordant results between the Verigene EP test and the reference methods, whereas six samples exhibited partial concordance between the two (Table 2). Table 1. Efficiency of the Verigene Enteric Pathogens Nucleic Acid Test to detect pathogens and toxin genes for clinical stool samples. Data were compared to reference methods and expressed as numbers or percentages (95% confidence interval). TP true positive, TN true negative, FP false positive, FN false negative. a Campylobacter jejuni, coli, and lari. b Shigella boydii, sonnei, flexneri, and dysenteriae. c Vibrio cholerae and parahaemolyticus. d All samples were positive in the discrepant analysis. e The sample was negative in the discrepant analysis. f All samples were positive in re-examination using the Verigene EP test. g One sample was positive and two samples were negative in the discrepant analysis. h One sample was positive and the other was negative in the discrepant analysis.

Discussion
Our study results demonstrated that the Verigene EP test accurately detected the genes of enteric pathogens and toxins in clinical samples with high sensitivity and specificity, which is consistent with previous reports 3,4 . Due to the ability of the test to detect multiple targets in a single assay, the Verigene EP test can avoid overlooking of any crucial gastrointestinal pathogens. Furthermore, the Verigene EP test enables rapid diagnosis with less work or training for laboratory technicians 12 ; on the other hand, conventional bacterial cultures are labor-and time-intensive. Additionally, unnecessary use of antimicrobial drugs and the duration of patient isolation may be reduced because of rapid diagnosis. A previous study reported that the xTAG GPP for gastrointestinal pathogens increased laboratory testing costs but decreased total costs for hospitalization by reducing patient isolation days 3,17 . The Verigene EP test might have similar effects; however, further studies are required.
While the Verigene EP test showed 19 discrepant results compared to the reference methods, the discrepant analysis proved that the Verigene EP test might have provided true results, at least in some samples with discrepant results. If bacteria were detected by molecular method but not cultured, we should recognize both possibilities; that the results of molecular methods may be false positives and that the sensitivity of cultures is lower than that of molecular methods [18][19][20] . A previous study described that the culture method failed to detect Campylobacter in 30% of positive clinical samples compared to non-cultural methods, including molecular assays 21 . Additionally, if the xTAG GPP was not the best test in some cases, as described previously 11 , the false-positive results of the Verigene EP test could result from the false-negative results of the xTAG GPP in the discrepant analysis.
Although each institution needs to consider how best to use the Verigene EP test effectively in daily practice, the test may be suitably used for community-acquired diarrhea requiring hospitalization to rapidly screen unmissable pathogens. Conversely, for hospital-acquired diarrhea, we might have to initially perform the Verigene Clostridium difficile nucleic acid test, which is a separate test for a single pathogen and performed using the same Verigene System 12,22 .
This study has some limitations. First, we could not adequately assess the ability of this test to detect Shigella species in clinical samples because the number of positive samples was limited. Instead, the Verigene EP test could successfully detect S. sonnei in characterized stool samples, and previous studies reported that the sensitivity and specificity of the Verigene EP test for the detection of Shigella species was 87.5-95.4% and 99.1-99.8%, respectively 3,4 . Furthermore, because we did not collect patient information and did not test all samples submitted to our laboratories during the study period, we could not validate the pathogenicity of the detected pathogens and the incidence of infections. Reportedly, some Stx 1 and 2 subtypes are linked to mild diseases or are rarely involved in human diseases 23 . Finally, for detecting bacteria and toxins, we could not assess the efficiency of the Verigene EP test compared to other molecular assays because another molecular method was used only for the discrepant analysis.
In conclusion, the Verigene EP test is useful for detecting crucial diarrheagenic pathogens, and may have applications in daily clinical practice. Further studies are needed to establish practical diagnostic strategies for the complementary use of molecular assays and traditional methods.