BACKGROUND: Clostridium difficile is the major cause of antibiotic-associated diarrhea (AAD) and pseudomembranous colitis (PMC). The aim of this study was to develop the nested PCR assay for direct detection of toxigenic C. difficile in stool specimens and to evaluate the usefulness of the method. METHODS: Specificity of newly designed primers are tested with 36 reference strains of intestinal flora. Lower detection limit of nested PCR for B toxin gene in C. difficile was determined using 10-fold serial dilutions of C. difficile ATCC 9689. One hundred and two clinical stool samples were cultured for detection of C. difficile on cycloserine-cefoxitin- fructose agar and the PCR assay for detection of toxin B gene in C. difficile isolates was performed. Nested PCR assay for direct detection of toxin B gene in clinical samples was also performed. RESULTS: Nested PCR assay showed negative amplification results in intestinal floras except C. difficile ATCC 9689. Lower detection limit of nested PCR for toxin B gene was 10 4 CFU/mL. Sensitivity of nested PCR assay compared to culture method was 100% (29/29), and the specificity was 68% (50/73). CONCLUSION: Nested PCR assay showed high sensitivity in direct detection of toxin B gene in C. difficile isolates even after administration of metronidazole, so the assay could be used in initial diagnosis and follow-up tests of AAD and PMC.
Agar ; Clostridium difficile* ; Clostridium* ; Diagnosis ; Diarrhea ; Enterocolitis, Pseudomembranous ; Follow-Up Studies ; Fructose ; Humans* ; Limit of Detection ; Metronidazole ; Polymerase Chain Reaction* ; Sensitivity and Specificity

BACKGROUND: Clostridium difficile is the major cause of antibiotic-associated diarrhea (AAD) and pseudomembranous colitis (PMC). This study was designed to investigate predisposing factors of AAD or PMC and to evaluate the efficiency of nested PCR assay for direct detection of toxin B gene in the treatment and prognosis of these diseases. METHODS: From January to December, 2002, stool specimens from 142 patients in Kosin Medical Center, Busan, were tested for the detection of toxigenic C. difficile strains. Toxin B gene in C. difficile was detected by nested PCR. And chart review was performed to investigate the antibiotics or anticancer drug history, clinical symptoms, treatment regimens, and prognosis. RESULTS: Among 142 stool specimens, 56 specimens showed positive for the toxin B gene in C. difficile strains by PCR. Forty two percents (47/113) of stool specimens from patients with AAD and all of specimens from eight patiens with PMC were C. difficile toxin B gene positive. Administration of antibiotics or anticancer drugs was stopped in 92.7% of toxin B gene-positive cases, but those were stopped in only 48.5% of toxin B gene-negative cases. The cure rate was higher in positive cases (82%) than negative ones (71%). CONCLUSION: It is concluded that nested PCR assay for the direct detection of C. difficile toxin B gene was helpful in rapid diagnosis and treatment of AAD or PMC.
Anti-Bacterial Agents ; Busan ; Causality ; Clostridium difficile* ; Clostridium* ; Diagnosis* ; Diarrhea* ; Enterocolitis, Pseudomembranous* ; Humans ; Polymerase Chain Reaction ; Prognosis

BACKGROUND: Clostridium difficile is the major cause of antibiotic-associated diarrhea (AAD) and pseudomembranous colitis (PMC). This study was designed to investigate predisposing factors of AAD or PMC and to evaluate the efficiency of nested PCR assay for direct detection of toxin B gene in the treatment and prognosis of these diseases. METHODS: From January to December, 2002, stool specimens from 142 patients in Kosin Medical Center, Busan, were tested for the detection of toxigenic C. difficile strains. Toxin B gene in C. difficile was detected by nested PCR. And chart review was performed to investigate the antibiotics or anticancer drug history, clinical symptoms, treatment regimens, and prognosis. RESULTS: Among 142 stool specimens, 56 specimens showed positive for the toxin B gene in C. difficile strains by PCR. Forty two percents (47/113) of stool specimens from patients with AAD and all of specimens from eight patiens with PMC were C. difficile toxin B gene positive. Administration of antibiotics or anticancer drugs was stopped in 92.7% of toxin B gene-positive cases, but those were stopped in only 48.5% of toxin B gene-negative cases. The cure rate was higher in positive cases (82%) than negative ones (71%). CONCLUSION: It is concluded that nested PCR assay for the direct detection of C. difficile toxin B gene was helpful in rapid diagnosis and treatment of AAD or PMC.
Anti-Bacterial Agents ; Busan ; Causality ; Clostridium difficile* ; Clostridium* ; Diagnosis* ; Diarrhea* ; Enterocolitis, Pseudomembranous* ; Humans ; Polymerase Chain Reaction ; Prognosis