Comparative Study of Cytotoxin Assay, Enzyme Immunoassays: Techlab Toxins A/B,C.

Diff Quick for Glutamate Dehydrogenase (GDH) and PCRs for Detection of C. difficile in Adult Stool Specimens
W McCaffrey1, A Jissam1, P Jayaratne1,2, L Monkman1, C Kim1, G Broukhanski 4,5, DR Pillai4,5, CH Lee1,2
1Hamilton University of Toronto

Regional Laboratory Medicine Program-HamiltonHealth Sciences & St. Josephs Healthcare, 2Department of Pathology and Molecular Medicine, McMaster University, Hamilton, 3St. Josephs Healthcare, Hamilton 4Department of Laboratory Medicine & Pathobiology, University of Toronto, 5Ontario Agency for Health Protection and Promotion, Canada

INTRODUCTION
Clostridium difficile infection (CDI) is a leading cause of nosocomial diarrhea in adults. Symptoms can range from mild, self-limiting diarrhea to pseudomembraneous colitis to fulminant toxic megacolon, resulting in significant adverse outcomes. CDI requires rapid and reliable testing method for optimal patient care and infection control management. Current testing methods for the detection of CD toxin are limited. The cytotoxin assay (CTA) which was previously considered the gold standard may take up to 72 hours for the results to be obtained. Currently, the majority of clinical laboratories across North America employ enzyme immunoassay (EIA) for detection of the toxin A or both toxins A and B due to rapid turn-around-time and relative ease of operation. The sensitivity and specificity of EIAs, are variable.

METHOD CONTD
CTA: Vero cell tissue cultures were inoculated with fecal filtrates at neat 1:100, and 1:1000 dilutions, and observed for cytotoxic effect. Fecal filtrates positive for typical C. difficile cytotoxicity were confirmed with neutralization of cytotoxicity using a 1:10 dilution of fecal filtrate and C. sordellii antitoxin . Stool Culture and Subsequent PCR: the stool samples were alcohol shocked, cultured and plated on Cycloserine, Cefoxitin Fructose (CCFA) plates. The organisms were identified based on the typical horse barn odor, gram stain and gas liquid chromatography (GLC) pattern. The extraction of DNA from the culture isolates was done with a Qiagen DNA stool mini kit and Inhouse PCR test was performed for detection of Toxin B and triose phosphate isomerase (tpi )which is Clostridium species-specific). Each sample was repeated 2 times

CONCLUSION
As GDH is highly sensitive and less costly than PCR, it can be used as a screening tool to rule out C. difficile infection. For stool specimens which test positive for GDH, further confirmatory testing is required. For the confirmatory testing, PCR should be used rather than EIA.

ACKNOWLEDGEMENT
Astra for providing the PCR kits and Inverness for providing GDH EIA and PCR kits

STUDY OBJECTIVE
To compare cytotoxin assay with commercially available EIAs and PCR for the detection of CD toxins in clinical adult stool samples submitted to the laboratory for investigation of CDI.

RESULTS
Out of 470 samples, 20 tested positive by all methods ; 25 discrepant samples were cultured and tested for the presence of C. difficile toxin using in-house PCR. 21/25 samples were culture and toxin positive. Table 1: Test Performance characteristics of TOX A/B ; GDH; CTA and PCRs
Toxin A/B TECHLAB Sensitivity (%) Specificity (%) PPV (%) NPV (%) C. Diff QuickChek (GDH Antigen) PCR Toxin B proGastro Cd PCR Toxin A & B Astra

REFERENCES
1. McFarland LV, Brandmarker SA, Guandalini S. Pediatric Clostridium difficile: A phantom menace or clinical reality? Journal of Pediatric Gastroenterology and Nutrition 2000; 31:220-31. 2. Kader HA, Piccholi DA, Jawad AF, McGowan KL, Maller ES. Single toxin detection is inadequate to diagnose Clostridium difficile diarrhea in pediatric patients. Gastroenterology 1998; 115:1329-34. 3. Langley JM, LeBlanc JC, Hanakowski M, Goloubeva O. The role of Clostridium difficile and viruses as causes of nosocomial diarrhea in children. Infection Control and Hospital Epidemiology 2002; 23:66064. 4. Klein EJ, Boster DR, Stapp JR, Wells JG, Qin X, Clausen CR, Swerdlow DL, Braden CR, Tarr PI. Diarrhea etiology in a childrens hospital emergency department: a prospective cohort study. Clinical Infectious Diseases 2006; 43:807-13. 5. Bartlett J. Antibiotic-associated diarrhea, p.893-904. In MJ Blaser, PD Smith, JI Ravdin, HB Greenberg, and RL Guerrant (ed.), Infection of the gastrointestinal tract 1995. Raven Press, New York, NY. 6. Butterworth S, Koppert E, Clarke A, Wiggs B, MacFarlane J. Recent Trendsin Diagnosis and Treatment of Clostridium difficile in a Tertiary Care Facility. Amer J Surgery 1998. 175:403-407.

METHODS
Evaluation of 470 consecutive unformed stool specimens from adults with suspected CDI submitted to the microbiology laboratory at the Hamilton Health Sciences and St. Josephs Healthcare, Hamilton, Ontario, Canada was done using 2 EIAs: TOX A/B II (Techlab: Blacksburg, VA) C.diff Quick Check (Inverness, Ottawa, ON)for glutamate dehydrogenase detection. Cytotoxin assay (CTA) and PCR by proGastro Cd (Gen-Probe) Astra (Astra BIOTECH) for the detection of C. difficile toxin A and/or B were performed as per laboratory standard operating procedure and product insert procedure manuals. Stool culture and subsequent PCR for detection of toxin and house-keeping gene (tpi) from isolates were performed on the specimens with any discrepant results. Detection of C. difficile housekeeping gene (tpi) and toxins genes was done two ways: with real-time PCR - tpi and toxin B (tcdB) genes and standard PCR (tpi, toxin A, B and binary toxin genes tcdA, tcdB and cdtB). For this study, gold standard was defined as follows: 100% concordance between tests and for discrepant results, positive culture showing toxigenic strain

CTA

48.78 99.3 86.9 95.5

97.56 94.0 61.5 99.7

63.40 99.7 96.3 99.7

95.12 99.7 97.5 99.5

92.6 100 100 99.3