Laboratory diagnosis of toxigenic Clostridium difficile by polymerase chain reaction: presence of toxin genes and their stable expression in toxigenic isolates from Japanese individuals

Abstract

Clostridium difficile causes pseudomembranous colitis and antibiotic-associated diarrhea. The definitive diagnosis of C. difficile infection is finally accomplished by the isolation of toxigenic C. difficile. However, only a small number of Japanese clinical laboratories are able to reach a definitive diagnosis of C. difficile infection, probably because simple reliable assays for toxins in the isolates are not available. In this study, we examined the compatibility of a polymerase chain reaction (PCR) assay and tissue culture assay to identify toxigenic C. difficile, in toxigenic and nontoxigenic C. difficile isolates from Japanese patients and healthy carriers. The specificity of PCR primers was demonstrated by restriction endonuclease digestion and seminested PCR in C. difficile VPI 10463 strain. No PCR product was amplified in the eight other clostridial species used to check the specificity of the PCR assay. The detection limit was 10(3) cells. Both toxin A and toxin B genes (the genes encoding the major virulence factors of C. difficile) were detected in 58 toxigenic C. difficile isolates, which showed a wide range of cytotoxic activity in tissue culture assays. Neither of the toxin genes was carried by 40 nontoxigenic strains of C. difficile. The results of this study strongly suggest that a definitive diagnosis of C. difficile infection can be accomplished by PCR detection of the toxin genes rather than by tissue culture assay of isolates.

The First Case of Antibiotic-associated Colitis by Clostridium difficile PCR Ribotype 027 in Korea

“…Because this strain is not universal, most laboratories are not equipped to perform PCR, which requires technical expertise, on a routine basis (21, 22). We suspected the presence of a highly virulent C. difficile strain, and thus the sample was referred to a research laboratory for detection the strain.…”

Prevalence of Clostridium spp. and Clostridium difficile in children with acute diarrhea in São Paulo city, Brazil

“…These findings indicate that both tcdA and tcdB or tcdB are stably expressed in C. difficile, suggesting that a definitive diagnosis of C. difficile infection can be accomplished by a multiplex-PCR detection of the toxin genes, rather than by tissue culture assay or enzyme immunoassay of isolates from stool (Karasawa et al 1999).…”

“…The major recognized cause of antibiotic-associated colitis is the cytotoxigenic C. difficile (Bartlett 1984). C. difficile produces 2 toxins: A (enterotoxin) and B (cytotoxin), which are the major virulence factors of this organism and which are encoded by 2 separated genes located in close proximity on the chromosome (Karasawa et al 1999). Toxin A causes fluid accumulation and mucosal damage in several animal models (rabbit ileal and colonic loops), as well as in hamster cecal segments and in mouse and rat intestines (Lyerly et al 1985).…”

National Clostridium difficile Standards Group: Report to the Department of Health

“…105 Alternative methods reported include reversed passive latex agglutination (applied to toxin A), 106 colony blot probe-hybridisation (applied to toxin B), 107 and PCR (toxins A and B). 108 These so-called 'second-look' cytotoxicity assays can be shown to detect toxigenic C. difficile in diarrhoeal specimens negative in the stool cytotoxicity assay, but the clinical significance of this finding requires confirmation.…”