As presented at 112th General Meeting of the American Society for Microbiology, Session C-173 June 16 19, 2012, San Francisco, California Evaluation of the Analytical Reactivity (Inclusivity) and Analytical Specificity (Cross-Reactivity) of the BD MAX Cdiff Assay A New Automated Molecular Assay. N. Paquette, I. Paradis, C. Lehouillier, R. Therrien, C. Roger-Dalbert GeneOhm Sciences Canada Inc. (BD Diagnostics), Quebec City, Quebec, CANADA ABSTRACT Objective: The BD MAX Cdiff Assay performed on the BD MAX System is an automated in vitro diagnostic test for the direct, qualitative detection of the Clostridium difficile toxin B gene (tcdb) in human liquid or soft stool specimens from patients suspected of having a C. difficile infection (CDI). The BD MAX Cdiff Assay is intended to aid in the diagnosis of CDI. The first objective of this study was to evaluate the analytical specificity (cross reactivity) of the BD MAX Cdiff Assay towards organisms found in stools. The second objective was to challenge the BD MAX Cdiff Assay with a large variety of toxigenic C. difficile strains in the analytical reactivity study (inclusivity). Methods: The analytical specificity testing has been performed with four non toxigenic C. difficile strains, two C. difficile strains of Toxinotype XI lacking tcdb gene and 30 other-clostridium strains (including four strains of Clostridium sordellii), along with 99 closely related organisms and other pathogenic and commensal flora found in the gut and stools (representing globally 90 species). All strains were tested at a concentration of 1X10 8 CFU/mL. Moreover, cross reactivity testing has been performed with seven viruses potentially present in stools. All viruses were tested at a concentration of 1X10 5 PFU/mL. The analytical reactivity testing has been performed on 64 toxigenic C. difficile strains (including 23 Toxinotypes), present in over 21 countries, from well-characterized clinical isolates or public collections. Some hypervirulent strains (NAP1) were also tested. C. difficile strains were tested at a concentration corresponding to 3 times the LoD95% of the assay. Results: None of the bacterial species or viruses used in the analytical specificity study tested positive with the BD MAX Cdiff Assay. The assay correctly identified all 64 C. difficile strains carrying the tcdb gene in the analytical sensitivity study. Conclusion: No cross reactivity has been observed with the BD MAX Cdiff Assay even with C. sordellii, which Lethal Toxin is genetically similar to C. difficile Toxin B. All toxigenic C. difficile strains tested from different geographical areas were successfully detected with the BD MAX Cdiff Assay. This new molecular assay for the detection of C. difficile tcdb gene demonstrated a high level of sensitivity and specificity. introduction C. difficile is the etiologic agent of a spectrum of diseases. Epidemiology studies demonstrate that an increased number of C. difficileassociated outbreaks have been reported worldwide 1, some with increased mortality and morbidity 2,3. Disease symptoms range from mild diarrhea to severe colitis, and even bowel perforation and death. This pathogen is the major cause of antibiotic-associated diarrhea (AAD) and pseudomembranous colitis. 4 The bacterium C. difficile, like the other bacteria comprising the intestinal flora, are killed by antibiotic therapy but not the C. difficile spores, which are insensitive to the majority of antibiotics. The BD MAX Cdiff Assay performed on the BD MAX System is an automated in vitro diagnostic test for the direct, qualitative detection of the Clostridium difficile toxin B gene (tcdb) in human liquid or soft stool specimens from patients suspected of having a C. difficile infection (CDI). The test, performed directly on the specimen, utilizes real-time polymerase chain reaction (PCR) for the amplification of C. difficile toxin B gene DNA and fluorogenic target-specific hybridization probes for the detection of the amplified DNA. The BD MAX Cdiff Assay is intended to aid in the diagnosis of CDI.
methods The objective of the analytical specificity (cross reactivity) study was to assess the analytical specificity of the BD MAX Cdiff Assay with isolates of phylogenetically related species (Clostridium other that C. difficile) and other organisms (bacteria and viruses) likely to be found in stool specimens. A total of 36 non toxigenic Clostridium species, as well as 98 other bacterial strains and seven viruses phylogenetically related to C. difficile or commonly found in human stools were tested. Human DNA was also tested. FIGURE 1. BD MAX System Bacterial strains were tested with the BD MAX Cdiff Assay at a concentration of 1X10 8 CFU/mL or 1X10 8 elementary bodies/ ml for intracellular bacteria. Viral suspension were tested at a concentration of 1X10 5 PFU/mL. Purified genomic DNA were tested at 1X10 5 cp/ Sample buffer tube. The objective of the analytical reactivity (inclusivity) study was to determine whether the BD MAX Cdiff Assay is able to detect different strains from various geographic origins, various toxinotypes 5, 6, 7, NAP1 outbreaks and various temporal diversities. A total of 64 toxigenic strains of Clostridium difficile were tested with the BD MAX Cdiff Assay, at a concentration equivalent to 3x LoD 95%. FIGURE 2. BD MAX reagents and consumable. A. Unitized Reagent Strip B. PCR Cartridge Tests were performed on BD MAX System (Figure 1) using Unitized reagent strip and cartridge (Figure 2) according to BD MAX Cdiff Assay workflow (Figure 3). FIGURE 1. BD MAX Cdiff Assay Workflow Steps 1 and 2. Recover and discharge stool Step 3. Load samples Step 4. Snap in reagents Step 5. Load PCR cartridge Step 6. Start run
results None of the bacterial species or viruses used in the analytical specificity study tested positive with the BD MAX Cdiff Assay (Table 1). TABLE 1. Analytical specificity (Cross-reactivity) Gender and Species Reference ID Assay Result Clostridium beijerinckii ATCC 8260 NEG Clostridium bifermentans NCTC 6929 NEG Clostridium bolteae BAA-613 NEG Clostridium butyricum CCRI-11128 NEG Clostridium chauvoei ATCC 11957 NEG Clostridium difficile 1 ATCC 700057 NEG Clostridium difficile 1 ATCC 43593 NEG Clostridium difficile 1 ATCC 43601 NEG Clostridium difficile 2 IS58 NEG Clostridium difficile 2 R11402 NEG Clostridium difficile 1 NCTC11206 NEG Clostridium fallax ATCC 19400 NEG Clostridium haemolyticum ATCC 9650 NEG Clostridium histolyticum ATCC 19401 NEG Clostridium innocuum CCRI-9927 NEG Clostridium nexile ATCC 27757 NEG Clostridium novyi ATCC 19402 NEG Clostridium orbiscindens ATCC 49531 NEG Clostridium paraputrificum ATCC 25780 NEG Clostridium perfringens ATCC 13124 NEG Clostridium ramosum ATCC 25582 NEG Clostridium scindens ATCC 35704 NEG Clostridium septicum ATCC 12464 NEG Clostridium sordellii ATCC 9714 NEG Clostridium sordellii NCCB 87107 NEG Clostridium sordellii CIP 103710 NEG Clostridium sordellii ATCC 9715 NEG Clostridium lavalense CCRI-9842 NEG Clostridium lavalense CCRI-9929 NEG Clostridium sphenoides ATCC 19403 NEG Clostridium spiroforme ATCC 29899 NEG Clostridium sporogenes ATCC 15579 NEG Clostridium symbiosum ATCC 14940 NEG Clostridium symbiosum CCRI-9928 NEG Clostridium tertium ATCC 14573 NEG Clostridium tetani ATCC 19406 NEG Abiotrophia defectiva ATCC 49176 NEG Acinetobacter baumannii ATCC 19606 NEG Acinetobacter lwoffii CDCF 3697 NEG Aeromonas hydrophila ATCC 7966 NEG Alcaligenes faecalis subsp. faecalis ATCC 15554 NEG Anaerococcus tetradius ATCC 35098 NEG Bacillus cereus ATCC 14579 NEG Gender and Species Reference ID Assay Result Bacillus cereus ATCC 13472 NEG Bacteroides caccae ATCC 43185 NEG Bacteroides stercoris ATCC 43183 NEG Bifidobacterium adolescentis ATCC 15703 NEG Bifidobacterium longum ATCC 15707 NEG Campylobacter coli ATCC 43479 NEG Campylobacter jejuni subsp. jejuni ATCC 33292 NEG Candida albicans ATCC 10261 NEG Candida catenulata ATCC 10565 NEG Cedecea davisae ATCC 33431 NEG Citrobacter amalonaticus ATCC 25405 NEG Citrobacter freundii ATCC 8090 NEG Citrobacter koseri ATCC 27028 NEG Citrobacter sedlakii ATCC 51115 NEG Collinsella aerofaciens ATCC 25986 NEG Corynebacterium genitalium LSPQ 3583 NEG Desulfovibrio piger ATCC 29098 NEG Edwardsiella tarda ATCC 15947 NEG Eggerthella lenta CCRI-9926 NEG Enterobacter aerogenes ATCC 13048 NEG Enterobacter cloacae subsp. cloacae ATCC 13047 NEG Enterococcus casseliflavus CCRI-1566 NEG Enterococcus cecorum ATCC 43198 NEG Enterococcus dispar ATCC 51266 NEG Enterococcus faecalis ATCC 19433 NEG Enterococcus faecium VanA ATCC 700221 NEG Enterococcus gallinarum CCRI-1561 NEG Enterococcus hirae ATCC 8043 NEG Enterococcus raffinosus ATCC 49427 NEG Escherichia coli Top10 NEG Escherichia coli ATCC 23511 NEG Escherichia coli ATCC 25922 NEG Escherichia fergusonii ATCC 35469 NEG Escherichia hermannii ATCC 33650 NEG Fusobacterium varium ATCC 8501 NEG Gardnerella vaginalis ATCC 14019 NEG Gemella morbillorum ATCC 27824 NEG Hafnia alvei ATCC 13337 NEG Helicobacter cinaedi NH 454 NEG Helicobacter pylori ATCC 43504 NEG Klebsiella oxytoca ATCC 33496 NEG Klebsiella oxytoca ATCC 13182 NEG Klebsiella pneumoniae subsp. pneumoniae ATCC 13883 NEG Lactobacillus acidophilus ATCC 4356 NEG
Gender and Species Reference ID Assay Result Lactobacillus reuteri ATCC 23272 NEG Lactococcus lactis subsp. lactis ATCC 11454 NEG Leminorella grimontii ATCC 33999 NEG Listeria grayi ATCC 19120 NEG Listeria innocua ATCC 33090 NEG Listeria monocytogenes L374 NEG Mitsuokella multacida ATCC 27723 NEG Mobiluncus curtisii subsp. holmesii ATCC 35242 NEG Moellerella wisconsensis ATCC 35017 NEG Morganella morganii subsp. morganii ATCC 25830 NEG Neisseria gonorrhoeae ATCC 35201 NEG Parabacteroides merdae ATCC 43184 NEG Peptoniphilus asaccharolyticus ATCC 14963 NEG Peptostreptococcus anaerobius ATCC 27337 NEG Plesiomonas shigelloides ATCC 14029 NEG Porphyromonas asaccharolytica ATCC 25260 NEG Prevotella melaninogenica ATCC 25845 NEG Proteus mirabilis ATCC 25933 NEG Proteus penneri ATCC 35198 NEG Providencia alcalifaciens ATCC 9886 NEG Providencia rettgeri ATCC 9250 NEG Providencia stuartii ATCC 33672 NEG Pseudomonas aeruginosa ATCC 35554 NEG Pseudomonas putida LCDC D7172 NEG Ruminococcus bromii ATCC 27255 NEG Salmonella enterica subsp. enterica ATCC 14028 NEG Salmonella enterica subsp. arizonae ATCC 13314 NEG Salmonella enterica subsp. enterica ATCC 7001 NEG Serratia liquefaciens ATCC 27592 NEG Gender and Species Reference ID Assay Result Serratia marcescens ATCC 13880 NEG Shigella boydii ATCC 9207 NEG Shigella dysenteriae 2933 NEG Shigella sonnei ATCC 29930 NEG Staphylococcus aureus subsp. aureus ATCC 43300 NEG Staphylococcus epidermidis ATCC 14990 NEG Stenotrophomonas maltophilia ATCC 13637 NEG Streptococcus agalactiae ATCC 13813 NEG Streptococcus dysgalactiae subsp. dysgalactiae ATCC 43078 NEG Streptococcus intermedius ATCC 27335 NEG Streptococcus uberis ATCC 19436 NEG Trabulsiella guamensis ATCC 49490 NEG Veillonella parvula ATCC 10790 NEG Vibrio parahaemolyticus ATCC 17802 NEG Yersinia bercovieri ATCC 43970 NEG Yersinia rohdei ATCC 43380 NEG Homo sapiens Hsap-25 NEG Vibrio cholerae CCRI-10454 NEG Adenovirus Type 14 CCRI-15991 NEG Rotavirus, strain WA 308140 NEG Norovirus group 1 308128 NEG Enterovirus CCRI-17000 NEG Echovirus 19 (Strain Burke) CCRI-15850 NEG Coxsackie virus B1 (Com-5) CCRI-15869 NEG Cytomegalovirus Strain AD-169 307018 NEG Chlamydia trachomatis Serovar D NEG 1 Non-toxigenic Clostridium difficile 2 Clostridium difficile XIa and XIb (lacking tcdb gene) The BD MAX Cdiff Assay detected (Table 2): -all the tested C. difficile toxinotypes (0, I, II, IIIa, IIIb, IV, V, VI, VII, VIII, IX, X, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XXI, XXIII, XXIV); -strains representing a NAP1 outbreak; -strains representing various temporal diversity -strains representing various geographic origins (21 countries).
TABLE 2: Analytical reactivity (Inclusivity) Country of origin Strain reference ID Toxin Expression Tox Assay result USA ATCC 700792 A+B+ 0 POS ND 3 ATCC 17858 A+B+ 0 POS USA ATCC 51695 A+B+ 0 POS Belgium ATCC 43600 A+B+ 0 POS Belgium ATCC 43599 A+B+ 0 POS Belgium ATCC 43596 A+B+ 0 POS Belgium ATCC 43594 A+B+ 0 POS ND 3 ATCC 17857 A+B+ 0 POS Switzerland ATCC BAA-1382 A+B+ 0 POS USA 141 NAv 1 ND 3 POS USA CD11 NAv 1 ND 3 POS France 301 NAv 1 ND 3 POS Canada CD 246 NAv 1 ND 3 POS New Zealand NCTC 11382 A+B+ ND 3 POS UK NCTC 11204 A+B+ ND 3 POS Turkey NI1 NAv 1 ND 3 POS Germany CI3 NAv 1 ND 3 POS Italy FI9 NAv 1 ND 3 POS Switzerland LI6 NAv 1 ND 3 POS Spain KI3 NAv 1 ND 3 POS The Netherlands GII1 NAv 1 ND 3 POS Poland HI1 NAv 1 ND 3 POS Ireland DII1 NAv 1 ND 3 POS UK QI1 NAv 1 ND 3 POS Sweden MI23 NAv 1 ND 3 POS Hungary EII1 NAv 1 ND 3 POS Greece PI2 NAv 1 ND 3 POS France AC008 A+B+ II POS France SE844 A+B+ IIIa POS Belgium 55767 A+B+ IV POS Belgium 51377 A+B+ VI POS Belgium 57267 A+B+ VII POS Belgium 51680 A+B+ IX POS UK 8864 A-B+ X POS USA CH6223 A+B+ XXI POS Canada M14614 A+B+ 0 POS Canada M39177 A+B+ 0 POS Canada M16256 A+B+ I POS Canada M25097 A+B+ IV POS Canada M30761 A+B+ VI POS Canada M46722 A+B+ IX POS Canada M40767 A+B+ IX POS Canada M26479 A+B+ XII POS Canada M13883 A+B+ XXI POS France SE881 A+B+ V POS Canada M14473 A+B+ III 2 POS
Country of origin Strain reference ID Toxin Expression Tox Assay result Canada M28681 A+B+ III 2 POS Canada M37992 A+B+ III 2 POS Canada M41124 A+B+ III 2 POS Canada M1137 A+B+ III 2 POS ND 3 IS25 A+B+ XII POS USA R9367 A+B+ XIII POS UK R10870 A+B+ XIV POS USA R9385 A+B+ XV POS Indonesia SUC36 A-B+ XVI POS Japan J9965 A-B+ XVII POS Korean K095 A+B+ XVIII POS Japan TR13 A+B+ XIX POS USA 8785 A+B+ XXIII POS Kuwait 597B A+B+ XXIV POS ND 3 ATCC 43255 A+B+ 0 POS ND 3 ATCC 9689 A+B+ 0 POS ND 3 ATCC BAA-1805 A+B+ IIIb 2 POS ND 3 ATCC 43598 A-B+ VIII POS 1 NAv: Non available 2 Strains identified as NAP1 and associated with CDI outbreaks 3 ND: Not Determined Conclusion The BD MAX Cdiff assay performed well in the presence of phylogenetically related species. No cross reactivity has been observed for the BD MAX Cdiff Assay with other phylogenetically related species or other organisms likely to be found in stool specimens A variety of clinically relevant toxigenic Clostridium difficile strains were included in this study taking into account geographic origin, toxinotypes, NAP1 outbreaks and temporal diversity. Sixty-four (64) strains including 23 toxinotypes and representing 21 countries were tested, including strains from public collections and from well-characterized clinical isolates. The assay correctly identified all tested toxigenic C. difficile strains. References 1 Cloud J and Kelly CP. Update on Clostridium difficile associated disease. Curr Opin Gastroenterol. 2007 Jan; 23 (1):4-9. 2 Warny M et al. Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. The Lancet. 2005 Sep 24-30; 366 (9491):1079-84. 3 Gould C V and McDonald L C. Bench-to-bedside review: Clostridium difficile colitis. Critical Care. 2008; 12 (1):203. 4 Bartlett, John G. MD, Clostridium difficile: Old and New Observations. J of Clin Gastroent. May/June 2007; 41 Supplement 1:S24-S29. ACKNOWLEDGEMENTS C. difficile strains were kindly provided by: M. Rupnik, Institute of Public Health Maribor, SI-2000 Maribor, Slovenia F. Barbut, National Reference Laboratory for C. difficile, Paris, France M. Delmée, Université Catholique de Louvain, Brussels, Belgium J. Pepin, University of Sherbrooke, Sherbrooke, Quebec, Canada E. Frost, University of Sherbrooke, Sherbrooke, Quebec, Canada 223601