Demonstration of toxin A and B by polymerase chain reaction and McCoy cell assay in clinical isolates of Clostridium difficile from Denmark

Abstract

Tvede, M. Demonstration of toxin A and B by polymerase chain reaction and McCoy cell assay in clinical isolates of Clostridium difficile from Denmark. APMIS 101: 18-22, 1993.A polymerase chain reaction (PCR) for demonstration of gene fragments of Clostridium dif;ficile was established. One hundred and sixty-eight clinical isolates of C. difjcile from three population groups were tested for production of cytotoxins by McCoy cell line assay (MCA) and for fragments of toxin A and B genes by PCR. The fragments for PCR amplification were at the 5' end of the toxin genes, which was found to be specific for C. difficife. Full agreement between the PCR and MCA results was found with respect to toxicity. Fifty-eight of the 168 strains were cytotoxin positive. Isolates from 41 normal healthy children did not differ regarding cytotoxicity compared to isolates from hospitalized children. In adult hospitalized patients a much higher frequency of toxin-producing isolates was found. In two strains from two children, not of the same family, only the toxin A gene fragment was demonstrated, indicating that some strains of C. difficile only harbour the gene for enterotoxin. When two isolates from different periods of time were tested from 36 of the healthy children, a variation in cytotoxicity was found: in seven children strains changed from non-toxic to toxic and in four vice versa. This may be explained by a fluctuating colonization of toxic and non-toxic C. dxficile strains, or it may indicate the need for examination of more than one strain from a positive faecal sample to demonstrate cytotoxicity.

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

“…Complete agreement between the presence of both toxin genes and cytotoxicity has also been found in C. difficile isolates from The Netherlands 12 and from Denmark. 13 These findings indicate that both the toxin A and B genes (or either one) are stably expressed in the C. difficile organism, suggesting that a definitive diagnosis of C. difficile infection can be accomplished by PCR detection of the toxin genes, rather than by tissue culture assay or enzyme immunoassay of isolates from stool.…”

“…12,13,[17][18][19] However, most of these studies involved DNA extraction steps that included boiling of cultures, which is laborious when a large number of strains are to be handled. In addition, polymorphisms in the toxin A and B genes have been reported.…”

Rapid Stool-Based Diagnosis of Clostridium difficile Infection by Real-Time PCR in a Children's Hospital

“…tcdA and tcdB encode exotoxins A (enterotoxin) and B (cytotoxin), respectively; tcdC and tcdD encode negative and positive regulators, respectively, that control the level of toxin production; and tcdE is purported to encode a holin-like protein thought to facilitate toxin release from the bacterial cell wall (44). Because toxins A and/or B are implicated in CDAD and genetic diversity of the PaLoc has been reported (37), we developed and clinically validated two separate hydrolysis probe real-time PCR assays targeting the tcdA and tcdB genes on the Roche LightCycler 1.0 platform (18,21,44). We subsequently multiplexed the tcdA and tcdB PCRs on the Roche LightCycler 2.0 platform.…”

Characterization of a Toxin A-Negative, Toxin B-Positive Strain of Clostridium difficile Responsible for a Nosocomial Outbreak of Clostridium difficile -Associated Diarrhea

“…The incidence of this disease ranges from 20 to 60 cases per 100,000 patient days (2,30), and the incidence appears to be increasing (14,30). C. difficile isolates associated with human disease are thought usually to produce both toxin A and toxin B (4,6,7,9,13,15,22,24), and genetic evaluations suggest coregulation of the two genes (10,35,40). Toxin B acts as a potent cytotoxin, but it does not cause plasma membranes of T84 cells to become leaky, nor does it disrupt tight junctions of these cells, whereas toxin A is an enterotoxin that has potent ability to disrupt the tight junctions of cultured human intestinal epithelial monolayers (11,12,16,17,28).…”