J. Med. Microbiol. - Voi. 38 (1993), 384-387 0 1993 The Pathological Society of Great Britain and Ireland Detection of C/mtridium difficile enterotoxin gene in clinical specimens by the polymerase chain reaction HELEN S. BOONDEEKHUN, V. GURTLER", MARYANN L. ODD, VICKI A. WILSON and B. C. MAYALL Department of Microbiology, Heidelberg Repatriation Hospital, Heidelberg West 308 I, Victoria, Australia Summary. A rapid assay was developed for detection of the Clostridium dzficile enterotoxin gene in stool specimens by means of the polymerase chain reaction (PCR). The PCR primers amplified a 63-bp repetitive sequence of the enterotoxin gene, thereby generating a distinctive ladder pattern of DNA bands following electrophoresis. Crude DNA extracts from stools containing C. dzficile produced one (63-bp) or more bands of the characteristicladder. Of 172 stool specimens from 58 patients, 37 gave positive results by culture (15 specimens) or cytotoxin assay (36 specimens). When 36 available " positive" specimens were tested by the PCR assay, 34 (94%) gave positive results-24 by direct testing, and 10 after extraction of DNA by the Qiagen procedure. Insufficient material of the remaining two specimens was available for DNA extraction. Of 2 1 stools " negative " for C. dzficile by culture or cytotoxin assay, one gave a positive result by PCR and seven produced atypical bands. The rapid PCR detection technique for C. dzficile was more sensitive than standard culture, and of a sensitivity similar to cytotoxin testing. The method has the potential for adoption in routine laboratory practice. Introduction Pseudomembranous colitis and antibiotic-asso- ciated diarrhoea in man are associated closely with Clustridium dzfici1e.l- Two major toxins are produced by C. dzflcile, an enterobxin and a cytoto~in,~,~ of which the enterotoxin is thought to be the main cause of the disease symptom^.^ Current culture-baseddetec- tion methods for C. dzficile in human stools are slower and less sensitive than detection of cytotoxin; however, the latter requires cell-culture facilities, and is not entirely specific for C. dzficile. Various ELISA techniques have been developed, but these lack sen- sitivity, specificity or both.6 The C. dificife enterotoxin gene has been sequenced, and repeat sequences have been characterised.'~~ One of the repeat sequences has been used in conjunction with the polymerase chain reaction (PCR) to dem- onstrate the enterotoxin gene in isolates of C. dificile,' thereby providing the basis for an alternative, highly specific, rapid assay. This paper describes the ap- plication of such an assay to the direct detection of the enterotoxin gene of C. dzficile in clinical specimens. Materials and methods Clinical specimens and bacteria Specimens used in this study were loose or liquid stools from patients at Heidelberg Repatriation Hos- pital; most of these patients had received antibiotic therapy and were suspected of having C. dz@cile- related diarrhoea. All stool samples were cultured for enteric pathogens, including C. dzficile, and were tested for C, dzficile cytotoxin. Stool samples were stored at -20°C before assaying by PCR. The type strains of bacteria used were C. dzficile strain ATCC 9689, and C. sordellii strains ATCC 9714 and CDC- 14337. C. dzficile strain 630 was obtained from Dr H. Hachler, University of Zurich, Switzerland. All other C. dzficile strains, and one strain of C. sordellii, were clinical isolates from Heidelberg Repatriation Hos- pi tal, Heidelberg, Australia. Culture method C. dzficile was isolated from human stools by inoculation on to C. dificile Agar (Oxoid), supple- mented with cycloserine 125 mg/L and cefoxitin 4mg/L (Oxoid), and incubation at 36°C in an anaerobic atmosphere for 48 h (T. Riley, personal communication). Identification was confirmed by gas- liquid chromatography and biochemical tests.' Received 2 July 1992; revised version accepted 25 Nov. 1992. * Correspondence should be sent to Mr V. Gurtler. 384