A Multicenter Study of the Revogene C. difficile System for Detection of the Toxin B Gene from Unformed Stool Specimens

Clostridioides difficile is the leading cause of diarrhea in hospitalized U.S. patients and results in over 400,000 cases of C. difficile infection per year. C. difficile infections have mortality rates of 6 to 30% and significantly increase health care costs, because of increased length of stay and increased frequency of readmissions due to recurrences. Efforts to reduce the spread of C. difficile in hospitals have led to the development of rapid sensitive diagnostic methods.

care costs by more than $1.5 billion per year (5). Active infections can be treated with the use of vancomycin, fidaxomicin, or even fecal transplantation, in a small number of cases; therefore, accurate diagnostics for active infections are necessary to improve patient care. However, because 2 to 17% of healthy patients can be colonized (2,6), the use of clinical assessment criteria to guide proper testing or testing algorithms to differentiate between colonization and active infection is advised.
To optimize patient management, the European Society of Clinical Microbiology and Infectious Diseases recommends first performing a sensitive diagnostic procedure such as a glutamate dehydrogenase (GDH) assay or a tcdB nucleic acid amplification test, followed by toxin detection with either a toxin A/B enzyme immunoassay or a toxigenic culture (TC) (7). Molecular assays have high sensitivity and specificity, but the detection of a toxigenic C. difficile strain does not automatically indicate that patient treatment is required, because this can lead to unnecessary treatment of colonized patients (8,9). Consequently, it remains crucial to clinically assess patients and to submit specimens only from patients at high risk of having CDI, to help avoid unnecessary testing. If proper clinical assessment is performed, based on preestablished institutional criteria, then the use of standalone molecular methods is sufficient (10). In addition, Infectious Diseases Society of America (IDSA) guidelines suggest that C. difficile testing should be performed only for patients presenting with recent unformed stools occurring Ն3 times in a Ͻ24-h period.
Here, we present a multisite investigational evaluation of the Revogene C. difficile test for use in detection of the toxin B gene from toxigenic strains of C. difficile. The Meridian Bioscience Revogene instrument is a real-time PCR (rtPCR)-based assay system that allows easy molecular detection of C. difficile in up to 8 specimens after a single heating step. In this study, a total of 2,461 residual stool specimens from 7 sites within the United States and Canada were enrolled for molecular detection of both C. difficile and tcdB. . All specimens were deidentified before enrollment and after completion of standard-of-care (SOC) testing. In total, 797 specimens were prospectively enrolled; 1,664 specimens were retrospective specimens that had been stored at -80°C. Microbiology Specialists Inc. (Houston, TX, USA) performed the reference testing. All sites obtained appropriate institutional review board approval or waivers, as specified by their local human subjects research committees.

Collection
Toxigenic culture. TC was performed at a single reference site (Microbiology Specialists Inc.). Specimens were shipped daily in anaerobic transport medium (Anaerobe Systems, Morgan Hill, CA, USA) and both tested directly and enriched within 4 days after collection. Direct TC testing involved inoculating the sample onto a standard cycloserine-cefoxitin-fructose agar (CCFA) plate (Anaerobe Systems), which was incubated for 48 h at 35°C to 37°C under anaerobic conditions. The CCFA plate was then examined for any colonies resembling C. difficile. Enrichment was performed like direct TC but included an additional incubation for 48 h at 35°C to 37°C in cycloserine-cefoxitin-mannitol broth with taurocholate and lysozyme, under anaerobic conditions. If direct culture identified C. difficile colonies, then enrichment testing was discontinued. Possible C. difficile isolates were grown in chopped meat carbohydrate (CMC) broth (Anaerobe Systems) and then identified using gas-liquid chromatography (GLC).
Cell cytotoxicity neutralization assay. All GLC-positive C. difficile isolates were tested for toxigenicity, from the same CMC broth, using a cell cytotoxicity neutralization assay, according to a Microbiology Specialists Inc. protocol (protocol no. ST040). In summary, cell-free culture filtrate was generated by centrifugation of CMC broth at 3,000 rpm for 1 h, filtered, and diluted 1:5 with sterile diluent. The diluted culture filtrate was further diluted with sterile diluent or C. difficile antitoxin (1:10 dilution) and incubated at room temperature for 30 min. Diluted specimen filtrate (with or without antitoxin) was then used to inoculate HFF-1 culture tubes (final dilution, 1:40), which were incubated for 48 h at 35°C in a rolling tube incubator. If cells in specimen filtrate with antitoxin had normal morphology but cells in specimen filtrate appeared rounded, then C. difficile toxin B was reported to be present.
Revogene C. difficile testing. Testing with the Revogene C. difficile assay was performed by following the package insert. Briefly, the stool specimens were eluted with a 5-l loop into a buffer, which was then used to inoculate a microfluidic molecular device (PIE; GenePOC) with 150 l of buffered sample. The PIEs were then added to the Revogene instrument, which can hold up to 8 PIEs and performs nucleic acid extraction followed by amplification and detection of the target sequence using rtPCR. The Revogene C. difficile test employs C. difficile primers and probe to detect a 263-bp target region of the toxin B (tcdB) gene of C. difficile. Results from the rtPCR were interpreted by the Revogene software and determined to be positive or negative for tcdB.
SOC testing. SOC test results were also collected for each specimen. The SOC test methods used in this study included the Xpert C. difficile assay (sites 2, 3, and 7), the BD MAX Cdiff assay (sites 1, 4, 6, and 7), the Illumigene C. difficile assay (site 4), the BD GeneOhm Cdiff assay (site 5), and the Luminex xTAG gastrointestinal pathogen panel (site 4). Site 1 performed BD MAX Cdiff testing followed by enzyme immunoassay testing (TechLab CDiff Quik Chek) if BD MAX Cdiff test results were positive. All testing was performed by following the laboratories' policies, based on the manufacturers' package inserts.
Statistical analysis. Results from the Revogene C. difficile assay were compared to those of direct and enriched cell culture. Performance characteristics, including positive percent agreement (PPA) and negative percent agreement (NPA), were calculated using standard methods. Ninety-five percent confidence intervals (CIs) were established by using a binomial expansion.

RESULTS
Specimen characteristics. A total of 2,461 specimens were enrolled and tested with combined C. difficile culture methods, to characterize the performance of the Revogene C. difficile assay. Specimens were obtained from a range of age groups, including Ͻ2 years of age (n ϭ 9), 3 to 18 years of age (n ϭ 105), 19 to 60 years of age (n ϭ 1,199), and Ͼ60 years of age (n ϭ 1,148) ( Table 1). The overall positivity rate for toxigenic C. difficile (tcdB) averaged 13.5%. The rates of toxigenic CDI varied among the age groups, from 11.6% to 31.1%, with the highest rate being observed for the Ͻ2-year-old population (n ϭ 9). Carriage of C. difficile is common among infants, and samples generally should not be submitted for C. difficile testing (11).
Comparison of Revogene C. difficile testing and the reference method for identification of toxigenic C. difficile. The overall PPA and NPA for the Revogene C. difficile assay, compared to the combined reference method, were 85.0% and 97.2%, respectively ( Table 2). All sites displayed Revogene tcdB NPA of Ͼ91% and PPA of Ͼ83% except for site 6, which had a lower PPA of 74.0%. Of the 2,461 specimens enrolled, 59 were positive by the molecular assay and negative by the reference method (false-positive [FP] samples), and 50 were negative by the molecular assay but positive by the reference method (false-negative [FN] samples).
Performance of Revogene C. difficile testing according to patient location. Data were stratified based on patient location, i.e., inpatient, outpatient, long-term care facility, or emergency department (Table 3). Three true-negative (TN) specimens were removed from this analysis because the location was unknown. The rates of tcdB identification varied somewhat among patients from the various locations, with specimens from emergency department patients having the lowest rate and specimens from long-term care facility patients having the highest rate (8.0% and 30.3%, respectively) ( Table 3). Most specimens originated from hospitalized patients (inpatients) (n ϭ 1,743 [70.8% of total specimens]), while specimens from patients in long-term care   were negative by SOC testing. Additionally, results for 51.0% of the FP specimens (26/51 specimens) agreed with the Revogene C. difficile assay results. Collectively, use of the Revogene C. difficile molecular assay, in comparison to all of the SOC testing at the enrollment sites, resulted in increased PPA (88.2%) ( Table 4).

DISCUSSION
The literature demonstrates variability in the performance characteristics of currently available molecular assays for C. difficile detection, with sensitivities and PPA, compared to TC, ranging from 81.6% to 100% (9,(12)(13)(14)(15)(16)(17). This wide range is likely due to a variety of factors, including bacterial load, targeted patient populations, and the sensitivity of the comparative culture method, since there is no agreed-upon or approved standard method. Utilization of molecular assays has suggested that many "gold standard" methods miss samples due to low tcdB copy numbers that are below the limit of detection for the culture and antigen methods (14,15,18). The PPA observed in our study (85%) was comparable to those in other studies addressing the performance of molecular detection of C. difficile, including studies with the Xpert C. difficile assay (82.8% or 90%) (14,15), the BD MAX Cdiff assay (81.6% to 89.3%) (12,14,15,17), the Simplexa C. difficile assay (87.4%) (17), and the Illumigene C. difficile assay (82.4% or 86.7%) (15,18). However, other studies regularly observed sensitivities and PPA over 90% for C. difficile molecular assays, including the Xpert C. difficile assay (93.5% to 100%) (9,12,16,17), the Illumigene C. difficile assay (91.8%) (19), the BD GeneOhm Cdiff assay (95.5%) (18), and the Verigene CDF assay (5.2%) (17). Importantly, it must be mentioned that none of the aforementioned studies determined the performance of the molecular assays in comparison to a reference method that combined direct culture and enriched culture, as employed by this study. The reference method used to evaluate a new assay can have large effects on overall performance, which is likely the cause of variability between studies.
A weakness of this study was the lack of semiquantitative data for C. difficile culture, to enable a comparison with FN samples and specimens with low bacterial loads. The lower PPA found in this study is likely due to the rigorous reference testing used for the evaluation. The reference method included direct TC testing and enrichment broth culture, which promoted cellular growth and thus increased low levels of toxin. Among FN specimens, 41/50 specimens (82%) were positive only when enrichment broth culture was performed; compared to direct TC only, the PPA increased from 85.0% to 95.3% (data not shown). The latter PPA correlates better with those of other molecular assays, because the same methodology of direct culture was used for the comparison  (12,15,16,18,19). There are at least two explanations for the difference between enriched culture and direct culture. Because enrichment broth culture was used to detect low levels of organisms from specimens, some FN results might be due to organisms being present at concentrations below the limit of detection for both the Revogene C. difficile assay and direct plating. Alternatively, because enrichment broth culture requires only a few CFU or spores to grow to high bacterial counts, contamination could have occurred between inoculation of specimens, resulting in a FN molecular test report. Low levels of starting organisms likely played a role, as enrichment broth culture detected an additional 59 true-positive (TP) specimens that direct culture alone would have missed. A strength of this study was the multicenter approach, with specimens collected throughout North America. While no ribotyping was performed, the C. difficile strains found in this study are most likely a diverse representation of C. difficile strains, providing a more realistic comparison to the highly sensitive TC. The potentially diverse strains represented in this study might have been a factor in the level of sensitivity observed (8,16). A multicenter study utilizing the Xpert C. difficile molecular assay found that the C. difficile subtype played a role in the detection of tcdB, as sensitivities ranged from 75% to 100% among 8 ribotypes (16).
The Revogene molecular assay for tcdB detection in C. difficile requires Ͻ5 min of hands-on time and produces results with a turnaround time of 70 min, with a high negative predictive value. Additionally, the Revogene assay can accommodate up to 8 microfluidic cartridges (PIEs), to facilitate batch testing of 1 to 8 specimens per run. This instrumentation would be suitable for any laboratory that requires rapid results from rtPCR technology and the flexibility for small-batch testing. The incorporation of a molecular assay into a multistep algorithm in testing for C. difficile can be justified by earlier reporting of CDI cases, thus preventing further spread of nosocomial CDI, with shortened length of stay for CDI patients (8,16). Additionally, the high negative predictive value and reduced turnaround time with molecular testing can reduce the practice of multiple test ordering, as well as the total numbers of C. difficile toxin assays ordered (12). The impact of the Revogene C. difficile assay on patient management will likely be similar to those of other molecular assays; when combined with proper clinical assessment of patients at risk of CDI or used as a frontline test in a multistep testing approach, it could improve patient management, infection control, and the reliability of surveillance data (10,12,13). The IDSA guidelines enforce this view, as they specifically recommend a stool toxin test, as well as a molecular assay or GDH assay, as part of a multistep testing algorithm if no preestablished institutional criteria for patient stool submission are in place. Because this is the first study to directly evaluate the performance of the Revogene C. difficile assay, compared with TC, additional studies should be performed to fully assess the clinical performance of this assay, relative to other commercially available molecular tests.