Rapid Detection of Toxigenic Clostridium difficile in Fecal Samples by Magnetic Immuno PCR Assay

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1994, p Vol. 32, No /94/$ Copyright ) 1994, American Society for Microbiology Rapid Detection of Toxigenic Clostridium difficile in Fecal Samples by Magnetic Immuno PCR Assay MAURICE J. H. M. WOLFHAGEN,l 2* AD C. FLUIT,1'3 RUURD TORENSMA,' 3 MIRIAM J. J. G. POPPELIER,"3 AND JAN VERHOEF' Eijkman-Winkler Laboratory for Medical Microbiology, University Hospital Utrecht, 3508 GA Utrecht, 1 Hanzelaboratory for Medical Microbiology and Infectious Diseases, 8021 AM Zwolle,2 and U-Gene Research by, 3584 CJ Utrecht,3 The Netherlands Received 17 November 1993/Returned for modification 7 February 1994/Accepted 5 April 1994 Rapid detection of toxigenic Clostridium dificile in fecal samples was accomplished with the magnetic immuno PCR assay (MIPA). Elaborate DNA extraction techniques were unnecessary. First, we generated a mouse monoclonal antibody (MAb) reactive with only C. difficile, Clostridium sordellii, and Clostridium bifermentans. Then, magnetic beads were coated with the MAb, incubated with fecal samples to allow binding with C. difficile, extracted from the stool with a magnet, and processed in the PCR with primers specific for the toxin B gene. After optimizing MIPA by raising the number of PCR cycles from 35 to 40 and adding Chelex 100 to the PCR mixture, we found a sensitivity of 96.7%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 94.1% when compared with the culture of cytotoxic C. difficile from fecal samples. MIPA is a rapid, easy, and sensitive PCR method for demonstrating the presence of toxigenic C. difficile in stool samples and avoids the disadvantage of elaborate extraction of DNA from fecal samples. Toxigenic Clostridium difficile can be the cause of various clinical syndromes, ranging from asymptomatic colonization via mild antibiotic-associated diarrhea to pseudomembranous colitis, which can progress to toxic megacolon and subsequent death (3). Although diseases caused by this organism have been known since 1974 (25), the diagnosis of the cause of antibiotic-associated diarrhea still poses many problems. The toxin B assay of stool samples by the tissue culture cytotoxicity assay is still considered to be the "gold standard" (3), eveni though Peterson et al. (23) found a sensitivity of only 67% and a specificity of 99% for the demonstration of toxin B in stool samples, while they found a sensitivity of culture of stool samples for C. difficile of 97% and a specificity of 93% (23). Furthermore, pseudomembranous colitis can be diagnosed by proctosigmoidoscopy in patients without a positive stool cytotoxicity assay result (13). In contrast to the tissue culture cytotoxicity assay, demonstration of the presence of C. difficile in stool cultures can easily be performed by using commercially available culture media based on the formula described by George et al. (11). However, both nontoxigenic and toxigenic C. difficile strains can be cultured in this medium. This means that for a definite diagnosis, the capacities of these isolates to produce toxin must still be determined. This can be done not only by the tissue culture cytotoxicity assay but also by demonstrating the presence of the genes encoding either toxin A or toxin B or both. We and several others have found that the presence of the genes encoding for toxin A and toxin B correlates with the presence of toxins, with very few exceptions (10, 17, 22, 31-34). Two methods for the direct identification of the genes encoding for toxins have been described: colony blot hybridization, which is performed on replica primary plates inoculated with stool samples (29), and the detection of toxigenic C. difficile in stool samples by the PCR (15, 18). Both methods * Corresponding author. Mailing address: Eijkman-Winkler Laboratory for Medical Microbiology, University Hospital Utrecht, P.O. Box 85500, room G , Heidelberglaan 100, 3508 GA Utrecht, The Netherlands. Phone: Fax: have drawbacks. Colony blot hybridization requires that the agar plate be incubated for 24 h, while the PCR itself is simple, but the sample preparation is laborious (4, 14, 15, 18). Recently, a magnetic immuno PCR assay (MIPA) was described. The MIPA allowed for the detection of Salmonella spp. in fecal samples (28) and avoided the need to extract DNA from the stool sample. This method uses magnetic beads coated with antibodies to extract the target organism from the sample. In this way, the advantages of PCR can be exploited without the disadvantage of elaborate sample preparation. To test the applicability of this methodology for the detection of C. difficile, we first produced a mouse monoclonal antibody (MAb) reactive with all known C. difficile serotypes (7, 8) and then used MIPA to demonstrate toxigenic C. difficile directly in fecal samples. MATERIALS AND METHODS Bacterial strains. The C. difficile reference strains, including the 10 serotypes that are now known, were purchased from the American Type Culture Collection (ATCC 9689, ATCC 43593, ATCC 43594, and ATCC to ATYC 43603) (7, 8). The other strains and clinical isolates were kindly donated by various Dutch hospitals and the Dutch National Institute for Public Health and Environmental Protection (Rijksinstituut voor Volkgezondheid en Milieuhygiene), Bilthoven, The Netherlands. Clinical specimens were frozen immediately upon receipt. To isolate strains from clinical specimens, fecal samples were plated onto C. difficile agar (Oxoid, Basingstoke, United Kingdom) containing 500 mg of cycloserine per liter and 16 mg of cefoxitin (Oxoid) per liter, and the plates were incubated anaerobically for 48 h at 37 C. Cultures suspect for the presence of C. difficile were identified as described by Holdeman et al. (16). Tissue culture cytotoxicity assay. The tissue culture cytotoxicity assay was performed on all fecal specimens as described by Allen (1). Briefly, cytotoxicity was determined on Vero cells grown in 96-well plates. The assay was considered positive

2 1630 WOLFHAGEN ET AL. when cells showed at least a 50% cytopathic effect and could be neutralized by Clostridium sordellii antitoxin (Wellcome, Temple Hill, United Kingdom). Generation of MAbs. Female BALB/c mice (ages, 6 to 8 weeks) were used for the generation of MAbs. These mice were immunized weekly, first subcutaneously with the different live C. difficile strains from ATCC and later intraperitoneally with only toxin-negative strains. A total of 10 CFU per immunization was used. Spleen cells obtained from immunized mice were collected and fused with the Sp2/0 myeloma cell line. Supernatants from wells containing growing hybridomas were screened for agglutinating antibodies by slide agglutination, with live ATCC strains used as the antigen. Immunoblots. The reactivities of the strains in immunoblots were tested as described by McKay et al. (21). Briefly, a loopful of C. difficile ATCC 9689, cultured overnight on Reinforced Clostridial Agar (Oxoid), was boiled in sample buffer (4% sodium dodecyl sulfate [SDS], 5% dithiothreitol, and 40% glycerol in 0.1 M Tris-HCl [ph 7.0]) for 5 min. SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (19). Proteins were analyzed after blotting onto nitrocellulose filters (26). Filters were blocked with 3% gelatin and 0.05% Tween 20 in phosphate-buffered saline (ph 7.4) and were probed with the supernatant of hybridoma-containing wells. After washing, a goat anti-mouse immunoglobulin G (IgG) labeled with horseradish peroxidase (Southern Biotechnology Associates, Birmingham, Ala.) was added and the filters were incubated for 1 h at 37 C. Final results were obtained by incubating the filters in 3,3-diaminobenzidine tetrahydrochloride (0.5 mg/ml)-0.075% H202. Magnetic immuno separation. Magnisort M magnetic chromium dioxide particles coated with goat immunoglobulins specific for murine immunoglobulin (IgG and IgM; Dupont, Wilmington, Del.) were incubated with 75,ul of hybridoma culture supernatant at room temperature for 15 min with continuous shaking. The magnetic particles were recovered by magnetic force, and the supernatant was discarded. Stool specimens were diluted 1:20 with saline and vigorously shaken to obtain a homogenized sample. After the sample was allowed to settle, 100,ll of supernatant was incubated with the magnetic particles for 15 min at room temperature with continuous shaking. The particles were extracted by magnetic force, washed three times with saline, and then resuspended in 100,ul of distilled water with or without 5% (wt/vol) Chelex 100 (Bio-Rad, Richmond, Calif.) (28). This was then processed for PCR. PCR. The PCR was performed as described by Fluit et al. (9, 10). In short, the resuspended beads were heated for 5 min at 95 C to lyse the bound bacteria. The sample was briefly centrifuged, and the supernatant was used in the PCR. PCRs were performed in 50 mm KCl-1.5 mm MgCl2-10 mm Tris-HCl (ph 8.3)-0.01% (wt/vol) gelatin-100 pum (each) deoxynucleoside triphosphate-50 pmol of each appropriate primer-1,ug of T4 gene 32 protein (Boehringer, Mannheim, Germany)-1.0 U of Taq polymerase (Cetus, Emeryville, Calif.). The sequences of the primers for toxin B were 5'- TAATAGAAAACAGTTAGAAA-3' (nucleotides 410 to 429) and 5'-TCCAATCCAAACAAAATGTA-3' (complementary to nucleotides 691 to 710), on the basis of the sequence of the toxin B gene (2), and resulted in a DNA fragment of 301 bp. Samples were subjected to either 35 or 40 cycles of amplification. Amplification was carried out in a DNA thermal cycler (Perkin-Elmer, Norwalk, Conn.), with each cycle consisting of 1 min at 94 C, 1 min at 50 C, and 1 min at 72 C. Amplified DNA was detected by agarose gel electrophoresis (20) in the presence of ethidium bromide. Confirmation of the amplification products was obtained by using a digoxigenin-labeled (Boehringer) probe as described by Widjojoatmodjo et al. (27). The probe was synthesized by PCR, with DNA of C. difficile ATTC 9689 used as a template and digoxigenin-dutp added as a label. The primers used were 5'-GTCAGAGAATACTGT AGTCG-3' (nucleotides 508 to 527) and 5'-TCCAATCCAAA CAAAATGTA-3' (complementary to nucleotides 691 to 710), resulting in a digoxigenin-labeled probe with a length of 203 bp Ḋetection of toxigenic C. difficile in spiked samples. Serial 10-fold dilutions from overnight cultures of C. difficile ATCC 9689 were mixed with a negative stool sample, which was characterized by a negative cytotoxicity assay result and from which no C. difficile was cultured. These spiked samples were processed by the MIPA as described above. A direct PCR on these samples was also performed. RESULTS J. CLIN. MICROBIOL. Generation of MAbs. One agglutinating MAb (MAb 95.25; IgG3) that reacted with all tested C. difficile strains was obtained. These strains consisted of both toxigenic and nontoxigenic reference strains (ATCC 9689, ATCC 43593, ATCC 43594, and ATCC to ATCC 43603) and clinical isolates (Table 1). Furthermore, the reactivity of MAb was tested on other microorganisms which are either normally present in the gut or pathogenic and which are the possible cause of diarrhea (Table 1). Apart from agglutination with C. difficile, agglutination was also found with C. sordellii and Clostridium bifermentans. The MAb was not reactive in the immunoblot. Detection of toxigenic C. dificile in spiked samples. To establish the detection limit of the MIPA, we compared MIPA with PCR performed directly on fecal samples spiked with C. difficile (Fig. 1). Both amplification steps consisted of 35 cycles. Using MIPA, we could detect 3 x 10 toxigenic C. difficile per g of feces, while direct PCR was negative with up to 1 x 10 C. difficile per g of feces. This means that at least a 104-fold increase in the detection level of toxigenic C. difficile was obtained. Detection of toxigenic C. dijicile in clinical samples. We processed 46 fecal samples. Of these 46 samples, 23 were positive in the cytotoxicity assay and yielded toxigenic C. difficile in culture, 8 yielded toxigenic C. difficile in culture but were negative in the cytotoxicity assay, 2 were positive for culture of nontoxigenic C. difficile and negative in the cytotoxicity assay, and 13 were negative in both assays. The first results obtained with the 35-cycle MIPA without Chelex 100 were disappointing (see the low sensitivities listed in Table 2). Four samples tested by MIPA were false negative in comparison with the results of the cytotoxicity assay, while nine samples were false negative in comparison with the results of culture for toxigenic C. difficile. To improve the sensitivity and specificity of the MIPA, we increased the number of cycles from 35 to 40 (data not shown). Although this improved the outcome of the assay, several amplification products were vaguely visible in the agarose gel, which made the result difficult to interpret. After adding Chelex 100 and using the 40-cycle MIPA, we obtained bright, clear-cut bands in the agarose gel (Fig. 2). The results were confirmed by Southern blot hybridization. The correlation of MIPA plus Chelex 100 with culture for toxigenic C. difficile was excellent. Seven samples were positive by using MIPA plus Chelex 100, while they were negative by the cytotoxicity assay. Samples with noncytotoxic C. difficile remained negative in all MIPAs.

3 VOL. 32, 1994 TABLE 1. Agglutination patterns of several Clostridium strains with MAb (mouse anti-c. difficile IgG3)a Species No. of isolates Typeb Toxin C. difficile 1 A + C. difficile 1 B C. difficile 1 A + C. difficile 1 C + C. difficile 1 D C. dificile 1 F + C. difficile 1 G + C. difficile 1 H + C. difficile 1 I - C. difficile 1 K C. difficile 1 X C. difficile 24 + C. difficile 24 C. difficile 8 NDC C. bifermentans 3 C. sordellii 2 All strains described in the table were positive for agglutination. The following strains were negative for agglutination: Clostridium perfringens (n = 11), Clostridium butyricum (n = 1), Clostridium cadaveris (n = 1), Clostridium histolyticum (n = 1), Clostridium innocuum (n = 1), Clostridium paraputnfucium (n = 1), Clostridium putnrucium (n = 1), Clostridium septicum (n = 1), Clostridium sporogenes (n = 1), Clostridium sphenoides (n = 1), Bacteroides fragilis (n = 5), Bacteroides vulgatus (n = 10), Bacteroides thetaiotaomicron (n = 2), Bacteroides levii (n = 5), Bacteroides capillosus (n = 3), Bacteroides ovatus (n = 2), Bacteroides loescheii (n = 2), Bacteroides caccae (n = 1), Bacteroides bivius (n = 1), Bacteroides merdae (n = 1), Shigella flexneri (n = 3), Shigella sonnei (n = 2), Shigella boydii (n = 3), Shigella dysenteriae (n = 3), Salmonella serotype Durazzo (n = 1), Salmonella paratyphi (n = 1), Salmonella typhimurium (n = 1), Salmonella serotype Heidelberg (n = 1), Salmonella abortusequi (n = 1), Salmonella serotype Derby (n = 1), Salmonella serotype Reading (n = 1), Salmonella serotype Schwarzengrund (n = 1), Salmonella abortusbovis (n = 1), Salmonella serotype Nigeria (n = 1), Salmonella serotype Virchow (n = 1), Salmonella serotype Amersfoort (n = 1), Salmonella serotype Glostrup (n = 1), Salmonella serotype Takoradi (n = 1), Salmonella serotype Newport (n = 1), Salmonella serotype Virginia (n = 1), Salmonella serotype Amherstiana (n = 1), Salmonella serotype Kentucky (n = 1), Salmonella serotype Emek (n = 1), Salmonella serotype Eimsbuettel (n = 1), Salmonella serotype Dublin (n = 1), Salmonella serotype Panama (n = 1), Salmonella typhi (n = 2), Salmonella enteritidis (n = 1), Salmonella meleagridis (n = 1), Salmonella anatum (n = 1), Salmonella Cerro (n = 1), Escherichia coli serotypes 078 (n = 1), 0127 (n = 1), 0166K? (n = 1), 0111K- (n = 1), 06K? (n = 1), 021K2 (n = 1), 033K- (n = 1), 023K18 (n = 1), 025 (n = 1), 0128K- (n = 1), 0111K- (n = 1), and 048K- (n = 1), Campylobacterjejuni (n = 10), Yersinia enterolitica (n = 3), Enterococcus faecalis (n = 4), and Vibrio cholerae (n = 1). b All strains with known serotype are ATCC strains. c ND, not done. DISCUSSION Direct PCR of feces is known to be less sensitive than PCR of purified DNA because of the presence of inhibitory compounds in feces like bilirubin, urobilinogens, and bile salts (28). To avoid these inhibitory factors, several investigators (4, 15, 18) perform PCR with stool samples after DNA extraction, a method that is rather laborious. This can be circumvented by magnetic immuno separation of the microorganisms from the stool sample. To achieve magnetic immuno separation, we made an agglutinating MAb reactive with all known C. difficile serotypes (7, 8) and various clinical isolates collected from all over The Netherlands, and various strains obtained from one hospital have been shown to be different by restriction restriction fragment length polymorphism analysis (30). Our MAb (MAb 92.25), however, was not specific for C. difficile alone; it also agglutinated C. sordellii and C. bifermentans. For the past several years, a commercially available agglutination test, the MicroScreen C. difficile latex slide agglutination test (Mercia Diagnostics, Guildford, United Kingdom), for the demonstration of C. difficile has been marketed. Like our MAb, this assay RAPID DETECTION OF TOXIGENIC C. DIFFICILE 1631 B M _ -a - b c -d e- -9 -a v= ~~~~~b -c -d -e _g~~~~~~- -f M FIG. 1. Detection limit of MIPA (35 cycles) (A) compared with that of PCR performed directly on spiked fecal samples (B). (A) (MIPA). Lane 1, positive control; lane 2, negative control; lanes 3 to 8, 10-fold dilutions of spiked fecal samples starting with 3 x 107 in lane 3 through 3 x 102 in lane 8. (B) Same samples as described for panel A. Markers (lanes M) were as follows: a, 11,497 bp; b, 2,838 bp; c, 1,700 bp; d, 1,159 and 1,093 bp; e, 805 bp; f, 514 bp; g, 339 bp. also cross-reacts with C. sordellii and C. bifernentans (5). In 1981, Poxton and Byrne (24) described the antigenic relationship of EDTA-extracted antigens from these microorganisms; cross-reactions were found with antisera to various Clostridium spp. However, antibodies reacting with C. difficile, C. irregularis, C. sordellii, and C. bifermentans did not react with other Clostridium spp. Therefore, this group of Clostridium spp. TABLE 2. Comparison of tissue culture cytotoxicity assay and culture of cytotoxic C. difficile strains with MIPA Percent Assay Positive Negative Sensitivity Specificity predictive predictive value value TCCAV versus MIPA for 35 cycles TCCA versus MIPA with Chelex 100 for 40 cycles Culture of toxigenic C. difficile versus MIPA for 35 cycles Culture of toxigenic C. difficile versus MIPA with Chelex 100 for 40 cycles a TCCA, tissue culture cytotoxicity assay.

4 1632 WOLFHAGEN ET AL. A b- 9- a B C FIG. 2. MIPA (40 cycles) plus Chelex 100 performed on clinical fecal samples. (A) Lane 1, positive control; lane 2, markers; lanes 3 to 14, positive fecal samples. (B) Lane 1, negative control; lane 2, markers; lanes 3 to 14, negative fecal samples. Markers: a, 11,497 bp; b, 2,838 bp; c, 1,700 bp; d, 1,159 and 1,093 bp, e, 805 bp, f, 514 bp; g, 339 bp. probably shares common antigens (24). Even though the MAb described here does not discriminate between toxigenic and nontoxigenic isolates, its cross-reactivity does not influence the outcome of MIPA because the shortcomings of the MAb are compensated for by the selective amplification of the toxin B gene specific for toxigenic C difficile. The sensitivity, specificity, positive predictive value, and negative predictive value of MIPA compared with the tissue culture cytotoxicity assay and culture of cytotoxic C. difficile strains are given in Table 2. From the data in Table 2, it can be concluded that the 40-cycle MIPA with Chelex 100 is the most sensitive assay for the detection of cytotoxic C. difficile. These findings agree with previous results (28) that showed that the addition of Chelex 100 contributes to the sensitivity of PCR on fecal samples. The addition of Chelex 100 probably leads to the more efficient lysis of C. difficile, the protection of DNA at high temperatures, and the inactivation of polymerase inhibitors (6). Seven samples were positive by MIPA but were negative for cytotoxin in feces, resulting in a specificity of 69.6% for MIPA versus the tissue culture cytotoxicity assay (Table 2). This may be explained by the fact that, in some instances, no free toxin is present in feces, even though toxigenic C. difficle are present. Recently, Gerding and Brazier (12) stated that antibiotic-associated diarrhea caused by C difficile can be diagnosed in a patient with diarrhea after antibiotic treatment only when toxins or toxigenic C. difficile strains in stool samples, or both, are detected. This supports the use of MIPA for diagnosing antibiotic-associated diarrhea and questions the use of the tissue culture cytotoxicity assay as the gold standard. In an earlier study (29), toxigenic C. we described the detection of difficile directly after culture on the agar plate as a J. CLIN. MICROBIOL. sensitive and simple method. Even though colony blot hybridization can be done reliably in any laboratory because it does not need expensive equipment like a thermocycler and enzymes, we think that PCR will become a generally accepted technique in the very near future. Kato et al. (18) detected toxigenic C. difficile in fecal samples by PCR by using an elaborate DNA purification method. However, the sensitivity of the assay described by that group was equal to that of the cytotoxicity assay (18). Gumerlock et al. (15) detected 103 toxigenic C. difficile in fecal samples using an elaborate DNA extraction method and PCR. Our method displayed a lower detection limit than that described by Kato et al. (18). It was also as sensitive as the method described by Gumerlock et al. (15) but was much easier to perform and could be performed more rapidly. The time needed to perform the MIPA as described above, including sample preparation and analyses, is less than 7 h, which can be shortened considerably by using an improved system for detecting the amplicons. Although the present study showed promising results, more elaborate clinical studies need to be performed to establish the role of MIPA among routine diagnostic methods. REFERENCES 1. Allen, S. D Clostridium, p In E. H. Lenette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 2. Barroso, L. A., S. Z. Wang, C. J. Phelps, J. L. Johnson, and T. D. Wilkins Nucleotide sequence of Clostridium difficile toxin B gene. Nucleic Acids Res. 18: Bartlett, J. G Antibiotic-associated diarrhea. Clin. Infect. Dis. 15: Boondeekhun, H. S., V. Gurtler, M. L. Odd, V. A. Wilson, and B. C. Mayall Detection of Clostridium difficile enterotoxin gene in clinical specimens by the polymerase chain reaction. J. Med. Microbiol. 38: Bowman, R. A., S. A. Arrow, and T. V. Riley Latex particle agglutination for detecting and identifying Clostridium difficile. J. Clin. Pathol. 39: de Lamballerie, X., C. Zandotti, C. Vignoli, C. Bollet, and P. de Micco A one step microbial DNA extraction method using "Chelex 100" suitable for gene amplification. Res. Microbiol. 143: Delmee, M., and V. Avesani Virulence of ten serogroups of Clostridium difficile in hamsters. J. Med. Microbiol. 33: Delmee, M., Y. Laroche, V. Avesani, and G. Cornelis Comparison of serogrouping and polyacrylamide gel electrophoresis for typing Clostridium difficile. J. Clin. Microbiol. 24: Fluit, A. C., M. N. Widjojoatmodjo, A. T. A. Box, R. Torensma, and J. Verhoet Rapid detection of salmonellae in poultry with the magnetic immuno-polymerase chain reaction assay. Appl. Environ. Microbiol. 59: Fluit, A. C., M. J. H. M. Wolfiagen, G. P. H. T. Verdonk, M. Jansze, R. Torensma, and J. Verhoef Nontoxigenic strains of Clostridium difficile lack the genes for both toxin A and toxin B. J. Clin. Microbiol. 29: George, W. L., V. L. Sutter, D. Citron, and S. M. Finegold Selective and differential medium for isolation of Clostridium difficile. J. Clin. Microbiol. 9: Gerding, D. N., and J. S. Brazier Optimal methods for identifying Clostridium difficile infections. Clin. Infect. Dis. 16(Suppl. 4):S439-S Gerding, D. N., M. M. Olson, L. R. Peterson, D. G. Teasley, R. L. Gebhard, M. L. Schwartz, and J. T. Lee, Jr Clostridium difficile-associated diarrhea and colitis in adults. A prospectic case-controlled epidemiologic study. Arch. Intern. Med. 146: Gumerlock, P. H., Y. J. Tang, F. J. Meyers, and J. Silva, Jr Use of the polymerase chain reaction for the specific and direct detection of Clostridium difficile in human feces. Rev. Infect. Dis. 13:

5 VOL. 32, 1994 RAPID DETECTION OF TOXIGENIC C. DIFFICILE Gumerlock, P. H., Y. J. Tang, J. B. Weiss, and J. Silva, Jr Specific detection of toxigenic strains of Clostridium difcile in stool specimens. J. Clin. Microbiol. 31: Holdeman, L. V., E. P. Cato, and W. E. C. Moore Anaerobe laboratory manual. Virginia Polytechnic Institute and State University, Blacksburg. 17. Kato, N., C.-Y. Ou, H. Kato, S. L. Bartley, V. K. Brown, V. R. Dowell, Jr., and K. Ueno Identification of toxigenic Clostridium difficile by the polymerase chain reaction. J. Clin. Microbiol. 29: Kato, N., C.-Y. Ou, H. Kato, S. L. Bartley, C.-C. Luo, G. E. Killgore, and K. Ueno Detection of toxigenic Clostridium difficile in stool specimens by the polymerase chain reaction. J. Infect. Dis. 167: Laemmli, U Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: Maniatis, T., E. F. Fritsch, and J. Sambroolk Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 21. McKay, I., J. E. Coia, and I. R. Poxton Typing of Clostridium difficile causing diarrhoea in an orthopaedic ward. J. Clin. Pathol. 42: McMillin, D. E., L. L. Muldrow, and S. J. Laggette Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction. Can. J. Microbiol. 38: Peterson, L. R., M. M. Olson, C. J. Shanholtzer, and D. N. Gerding Results of a prospective, 18-month clinical evaluation of culture, cytotoxin testing, and culturette brand (CDT) latex testing in the diagnosis of Clostridium difficile-associated diarrhea. Diagn. Microbiol. Infect. Dis. 10: Poxton, I. R., and M. D. Byrne Immunological analysis of the EDTA-soluble antigens of Clostridium difficile and related species. J. Gen. Microbiol. 122: Tedesco, F. J., R. W. Barton, and D. H. Alpers Clindamycinassociated colitis. Ann. Intern. Med. 81: Towbin, H., T. Staehelin, and J. Gordon Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: Widjojoatmodjo, M. N., A. C. Fluit, R. Torensma, B. H. I. Keller, and J. Verhoef Evaluation of the magnetic immuno PCR assay (MIPA) for rapid detection of Salmonella. Eur. J. Clin. Microbiol. Infect. Dis. 10: Widjojoatmodjo, M. N., A. C. Fluit, R. Torensma, G. P. T. H. Verdonk, and J. Verhoef The magnetic immuno polymerase chain reaction assay for direct detection of salmonellae in fecal samples. J. Clin. Microbiol. 30: Wolfhagen, M. J. H. M., A. C. Fluit, M. Jansze, C. M. A. Rademaker, and J. Verhoef Detection of toxigenic Clostridium difficile in fecal samples by colony blot hybridization. Eur. J. Clin. Microbiol. Infect. Dis. 12: Wolfhagen, M. J. H. M., A. C. Fluit, R. Torensma, M. Jansze, A. F. A. Kuypers, E. A. E. Verhage, and J. Verhoef Comparison of typing methods for Clostridium difficile isolates. J. Clin. Microbiol. 31: Wren, B. W., C. L. Clayton, N. B. Castledine, and S. Tabaqchali Identification of toxigenic Clostridium difficile strains by using a toxin A gene-specific probe. J. Clin. Microbiol. 28: Wren, B. W., C. L. Clayton, and S. Tabaqchali Rapid identification of toxigenic Clostridium difficile by polymerase chain reaction. Lancet 335: Wren, B. W., C. L. Clayton, and S. Tabaqchali Nucleotide sequence of Clostridium difficile toxin A gene fragment and detection of toxigenic strains by polymerase chain reaction. FEMS Microbiol. Lett. 58: Wren, B. W., S. R. Heard, A. I. Al-Saleh, and S. Tabaqchali Characterisation of Clostridium difficile strains by polymerase chain reaction with toxin A- and B-specific primers. J. Med. Microbiol. 38: Downloaded from on October 3, 2018 by guest