A Single Center’s Experience with a Novel Nucleic Acid Amplification Test for the Detection of the Clostridium difficile Toxin B Gene from Clinical Samples C. Dang 1 , S. Lewis 2 , M. Olson 1 , A. Romesburg 1 , J. Liffers 1 , B. C. Ellis 1 , K. C. Carroll 2 1 Johns Hopkins Hospital, Baltimore, MD , 2 Johns Hopkins University School of Medicine, Baltimore, MD Background: Clostridium (now Clostridioides) difficile remains a formidable pathogen. While controversial, many laboratories use a molecular test for the diagnosis of C. difficile infection (CDI). We report a single center’s experience from a multi -center study for FDA 510K approval of a new qualitative real-time PCR assay, i.e. the GenePOC™ CDiff assay (GenePOC Inc., Québec, Canada). Methods: The Johns Hopkins Hospital (JHH) Microbiology Laboratory tested 200 unformed stool specimens. To run the GenePOC CDiff test, approximately 5 μl of the stool sample was tested according to the manufacturer’s package insert. 500 µl of the stool sample was transferred to an Anaerobe Transport Medium and shipped to an off-site reference lab for direct culture on CCFA agar and enriched culture using CCMB-TAL broth in case direct culture was negative. Recovered isolates were determined to be cytotoxin positive by a cell culture cytotoxin neutralization assay. In addition, since the standard-of-care in our clinical laboratory is the BD MAX™ Cdiff test (BD Diagnostics, Inc., USA), the GenePOC CDiff assay was also compared with the BD MAX results. Results: Two hundred samples were tested at JHH. Eleven samples were eliminated due to an exceeded transit time to the reference lab for culture. Thirteen samples were excluded because SBT and PIEs were mixed with different lot numbers. Therefore, a total of 176 stool specimens had valid test results. Twelve samples were positive for toxigenic C. difficile by direct culture. All direct culture positive samples were positive by GenePOC CDiff (sensitivity, 100%). An additional nine specimens were direct culture negative, but positive by the GenePOC CDiff assay (specificity, 94.5%). Twenty-three samples were positive by combined culture, sixteen of which were positive by the GenePOC assay (69.6% sensitivity). There were five GenePOC positive, culture negative samples for a specificity of 96.7%. Compared to the BD MAX (n = 189), the sensitivity and specificity of the GenePOC test were 95.5% and 100%, respectively. Conclusion: The GenePOC CDiff test compares favorably to direct culture on CCFA and to another FDA-cleared NAAT that detects the toxin B gene in unformed fecal samples. Clostridium (now Clostridioides) difficile continues to cause significant morbidity and mortality. Accurate and rapid diagnosis is essential in the appropriate treatment of patients and in preventing transmission of this infection in the hospital environment. While the optimum diagnostic method is still a matter of debate, the updated guidelines from the Infectious Diseases Society of America and the Society for Healthcare Epidemiology (1) recommend either a two-step algorithm based upon testing for glutamate dehydrogenase or nucleic acid test (NAT) followed by a toxin test or, in institutions where when there are pre- agreed institutional criteria for patient stool submission, a NAT alone may be acceptable. NATs are currently the most sensitive, but least specific tests available for the diagnosis of C. difficile. Studies have shown a reduction in transmission over time when NATs are used appropriately in hospitalized patients who meet clinical criteria for C. difficile disease combined with optimum infection control measures (2). The GenePOC™ CDiff assay is a qualitative in vitro diagnostic test that utilizes real-time polymerase chain reaction (PCR) to detect tcdB of toxigenic C. difficile. The goal of this project was to compare the GenePOC CDiff assay to a reference method (toxigenic culture) as well as to the standard of care molecular test (BD MAX Cdiff assay) currently in use in the Johns Hopkins Hospital Microbiology Laboratory. Test Method: GenePOC CDiff Assay GenePOC Instrument Comparative Methods ABSTRACT (AMENDED) MATERIALS AND METHODS • The GenePOC™ CDiff assay is comparable to direct toxigenic culture for C. difficile detection, but less sensitive than enriched culture (CCMB-TAL). • The GenePOC™ CDiff assay is comparable to the FDA-cleared BD MAX™ Cdiff test, a NAAT that detects the toxin B gene found in C. difficile. • The GenePOC™ CDiff assay requires little sample preparation for testing and will provide accurate results within 70 minutes, allowing for rapid diagnosis of C. difficile infection. Controls: • Each PIE contains reagents for the detection of tcdB and process controls to monitor processing, amplification, and the absence of reaction inhibitors. • Positive external controls, provided by GenePOC, were tested each day. • 150 μL of SBT was used as a negative external control. Testing Algorithm: • The stool samples were placed in the SBTs for testing. Afterwards, the SBTs were placed at 2-8°C in the event repeat testing is required. If the sample did not require repeat testing, the SBT was frozen at -70±5°C or colder. • 500 μl of stool sample was transferred into an Anaerobe Transport Medium kept at room temperature for shipment to a reference lab (Microbiology Specialists, Inc.) for toxigenic culture (reference method). • The remaining (minimum of 250 μL) of the homogenized stool specimen was frozen at - 70±5°C (or colder) for further testing (if needed). GenePOC Specimen Processing: • 5μl of unformed stool was transferred into the appropriately labeled Sample Buffer Tube (SBT) using a disposable inoculating loop. • The SBT was closed and vortexed as described in the GenePOC CDiff test package insert. • Mixed SBT was transferred into the PIE using the disposable transfer pipette. • The PIE was loaded onto the GenePOC instrument and run. • The specimen identifier, SBT barcode, and PIE barcode were scanned prior to insertion of the PIE. • The PIE was inserted into the instrument carousel. • A maximum of eight samples can be processed in a single run including external controls. • For runs with less than eight patient samples, the empty places were filled with PIEs containing external controls. • Results were available in 70 min. Toxigenic Culture (Reference Method) • Specimens were inoculated onto a CCFA plate for direct culture and a CCMB-TAL broth for enriched culture. • After incubation, the broth was sub- cultured to another CCFA plate. • The plates were examined for colonies characteristic of C. difficile. • Suspicious isolates were confirmed as C. difficile by gas liquid chromatography after incubation in chopped meat carbohydrate broth for 48 h. • Toxigenicity of isolates was determined by cell culture cytotoxicity neutralization assay. BD MAX (Standard of care) • Specimens were inoculated into SBT tube, vortexed, and run on BD MAX instrument. • Real-time PCR for the amplification of C. difficile toxin B gene. • Detection of the amplified DNA uses fluorogenic target specific hybridization probes. • Results are available in 100 minutes. Table 3: GenePOC™ r esults compared to BD MAX™ results Specimen Inclusion Criteria: • At least 1.25ml of unformed stool from patients suspected of having CDI • Fresh specimens were tested with the GenePOC and the reference method within 48 h • Specimens were residual, de-identified, and kept at 2-8 C prior to testing Table 1: GenePOC™ CDiff results compared to direct culture results. References 1. McDonald LC, Gerding DN, Johnson S, et al. 2018. Clinical practice guidelines for Clostridium difficile infection in adults and children: 2017 update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA). Clin Infect Dis 66:987-94. 2. Napierala M, Munson E, Skonieczny P, et al. 2013. Impact of toxigenic Clostridium difficile polymerase chain reaction testing on the clinical microbiology laboratory and inpatient epidemiology. Diagn Microbiol Infect Dis. 76:534-38. DIRECT CULTURE POS NEG GENEPOC™ CDIFF ASSAY POS 12 9 NEG 0 155 Table 4: Analysis of GenePOC™ results versus comparative methods. GenePOC™ Cdiff vs Direct Culture GenePOC™ Cdiff vs Combined Culture GenePOC™ CDiff vs BD MAX™ Cdiff Sensitivity 100% Sensitivity 69.6% Sensitivity 95.5% Specificity 94.5% Specificity 96.7% Specificity 100% Saturday - 288 Johns Hopkins University 410-955-5077 [email protected] Table 2: GenePOC™ r esults compared to BD MAX™ results COMBINED CULTURE POS NEG GENEPOC™ CDIFF ASSAY POS 16 5 NEG 7 148 BD MAX™ CDIFF ASSAY POS NEG GENEPOC™ CDIFF ASSAY POS 21 0 NEG 1 152