Detection of genotype and toxin associated gene of hypervirulent Clostridium difficile clinical isolates / 国际检验医学杂志

Objective To investigate the genotype and variance of toxin associated genes of moxifloxacin‐resistant Clostridium difficile clinical isolates in Sydney .Methods Twenty‐two moxifloxacin‐resistant Clostridium difficile clinical isolates were collected from Sydney ,which were genotyped by using sequencer capillary gel electrophoresis based PCR‐ribotyping ,and toxin A and B cod‐ing gene tcdA and tcdB ,and binary toxin coding gene cdtA and cdtB were detected by using PCR method .Toxin regulator gene tc‐dC was analyzed by using PCR‐sequencing ,and was aligned with reference sequence of VPI 10463 (Genbank accession number :X92982) ,and the tcdC sequence types of all 22 isolates were identified by using blast tool in NCBI .Results Twenty‐one isolates were genotyped as hypervirulent PCR‐ribotypes 027 (RT027) ,and one isolate as RT078 ;all 22 isolates contained tcdA and tcdB for toxin A and B and cdtA and cdtB for binary toxin (tcdA+ tcdB+ cdtA+ cdtB+ ) .The tcdC sequence types of the 21 RT027 i‐solates belong to sc1 ,and that of the one RT078 isolate belongs to WA39 .Compared with tcdC reference sequence of VPI 10463 ,a consecutive 18 bp deletion (nt341 to 379) and one nucleotide deletion at position 117 were found in the 21 RT027 isolates ,and a consecutive 39 bp deletion (nt330 to 368) and one nucleotide mutation at position 184(C> T) were found in the one RT078 isolate . Conclusion Clostridium difficile hypervirulent RT027 was the common moxifloxacin resistant genotype ;Clostridium difficile hy‐pervirulent RT027 and RT078 clinical isolates contained genes for toxin A and B and binary toxin ,and contained gene sequence mu‐tation in toxin regulator gene tcdC .

Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

BACKGROUND: The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. METHODS: The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. RESULTS: A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. CONCLUSIONS: The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.

Clinical and Microbiologic Characteristics of Clostridium difficile Infection Caused by Binary Toxin Producing Strain in Korea / 감염과화학요법

BACKGROUND: Binary toxin-producing Clostridium difficile infections (CDI) are known to be more severe and to cause higher case fatality rates than those by binary toxin-negative isolates. There has been few data of binary toxin-producing CDI in Korea. Objective of the study is to characterize clinical and microbiological trait of CDI cause by binary-toxin producing isolates in Korea. MATERIALS AND METHODS: From September 2008 through January 2010, clinical characteristics, medication history and treatment outcome of all the CDI patients were collected prospectively. Toxin characterization, PCR ribotyping and antibiotic susceptibility were performed with the stool isolates of C. difficile. RESULTS: During the period, CDI caused by 11binary toxin-producing isolates and 105 toxin A & toxin B-positive binary toxin-negative isolates were identified. Comparing the disease severity and clinical findings between two groups, leukocytosis and mucoid stool were more frequently observed in patients with binary toxin-positive isolates (OR: 5.2, 95% CI: 1.1 to 25.4, P = 0.043; OR: 7.6, 95% CI: 1.6 to 35.6, P = 0.010, respectively), but clinical outcome of 2 groups did not show any difference. For the risk factors for acquisition of binary toxin-positive isolates, previous use of glycopeptides was the significant risk factor (OR: 6.2, 95% CI: 1.4 to 28.6, P = 0.019), but use of probiotics worked as an inhibitory factor (OR: 0.1, 95% CI: 0.0 to 0.8; P = 0.026). PCR ribotypes of binary toxinproducing C. difficile showed variable patterns: ribotype 130, 4 isolates; 027, 3 isolates; 267 and 122, 1 each isolate and unidentified C1, 2 isolates. All 11 binary toxin-positive isolates were highly susceptible to clindamycin, moxifloxacin, metronidazole, vancomycin and piperacillin-tazobactam, however, 1 of 11 of the isolates was resistant to rifaximin. CONCLUSIONS: Binary toxin-producing C. difficile infection was not common in Korea and those isolates showed diverse PCR ribotypes with high susceptibility to antimicrobial agents. Glycopeptide use was a risk factor for CDI by those isolates.