We employed a multiplex polymerase chain reaction (PCR) coupled with capillary electrophoresis (mPCR-CE) targeting six Clostridium difficile genes, including tpi, tcdA, tcdB, cdtA, cdtB, and a deletion in tcdC for simultaneous detection and characterization of toxigenic C. difficile directly from fecal specimens. The mPCR-CE had a limit of detection of 10 colony-forming units per reaction with no cross-reactions with other related bacterial genes. Clinical validation was performed on 354 consecutively collected stool specimens from patients with suspected C. difficile infection and 45 isolates. The results were compared with a reference standard combined with BD MAX Cdiff, real-time cell analysis assay (RTCA), and mPCR-CE. The toxigenic C. difficile species were detected in 36 isolates and 45 stool specimens by the mPCR-CE, which provided a positive rate of 20.3% (81/399). The mPCR-CE had a specificity of 97.2% and a sensitivity of 96.0%, which was higher than RTCA (x = 5.67, P = 0.017) but lower than BD MAX Cdiff (P = 0.245). Among the 45 strains, 44 (97.8%) were determined as nonribotype 027 by the mPCR-CE, which was fully agreed with PCR ribotyping. Even though ribotypes 017 (n = 8, 17.8%), 001 (n = 6, 13.3%), and 012 (n = 7, 15.6%) were predominant in this region, ribotype 027 was an important genotype monitored routinely. The mPCR-CE provided an alternative diagnosis tool for the simultaneous detection of toxigenic C. difficile in stool and potentially differentiated between RT027 and non-RT027.
Clostridium Infections ; diagnosis ; Clostridium difficile ; genetics ; Electrophoresis, Capillary ; Feces ; microbiology ; Genes, Bacterial ; Humans ; Polymerase Chain Reaction ; Ribotyping ; Sensitivity and Specificity

BACKGROUND/AIMS: The incidence and severity of Clostridium difficile infection (CDI) have increased worldwide, resulting in a need for rapid and accurate diagnostic methods. METHODS: A retrospective study was conducted to compare CDI diagnosis methods between January 2014 and December 2014. The stool samples, which were obtained in presumptive CDI patients, were compared for their diagnostic accuracy and rapidity, including real-time polymerase chain reaction (PCR) of toxin genes, C. difficile toxin assay, and culture for C. difficile. RESULTS: A total of 207 cases from 116 patients were enrolled in this study and 117 cases (56.5%) were diagnosed as having CDI. Among the 117 cases, the sensitivities of real-time PCR, C. difficile toxin assay, and culture for C. difficile were 87.2% (102 cases; 95% CI, 80.7%–92.8%), 48.7% (57 cases; 95% CI, 41.0%–59.8%), and 65.0% (76 cases; 95% CI, 60.2%–78.5%), respectively (P < 0.005). Notably, 34 cases (29.0%) were diagnosed with CDI by real-time PCR only. The time required to obtain results was 2.27 hours (136.62±82.51 minutes) for real-time PCR, 83.67 hours (5,020.66±3,816.38 minutes) for toxin assay, and 105.79 hours (6,347.68±3,331.46 minutes) for culture (P < 0.005), respectively. CONCLUSIONS: We confirmed that real-time PCR of toxin genes is the most effective diagnostic method for accurate and early diagnosis of CDI. It also helps to diagnose hypervirulent CDI, such as ribotype 027 infection.
Clostridium difficile ; Clostridium ; Diagnosis ; Early Diagnosis ; Humans ; Incidence ; Methods ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Retrospective Studies ; Ribotyping

Since 2000, Clostridium difficile infection has increased substantially in both hospital-acquired and community-acquired diarrhea, not only in North America but also in Europe. There was a steady increase in the incidence and severity of C. difficile infection over the past decade, associated with significantly higher morbidity and mortality. The major risk factors for C. difficile infection appear to be the use of new antimicrobial therapy, long-term hospitalization in old age and emerging hypervirulent strains, such as various ribotypes. Rapid and accurate diagnosis of C. difficile infection is necessary for appropriate treatment as well as reliable epidemiological data. Currently available treatment options are withdrawal of the suspected offending antibiotics and then treating patients with highly effective antibiotics for C. difficile. Multiple recurrence or acute fulminant C. difficile infection could be treated with fecal microbiota transplantation. Promising therapies for treating C. difficile infection should always be equipped with high efficacy and safety in the future.
Anti-Bacterial Agents ; Clostridium ; Clostridium difficile ; Diarrhea ; Europe ; Hospitalization ; Humans ; Incidence ; Metagenome ; North America ; Recurrence ; Ribotyping ; Risk Factors ; Transplants

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.
Anti-Infective Agents ; Aza Compounds ; Clindamycin ; Clostridium ; Clostridium difficile ; Glycopeptides ; Humans ; Korea ; Leukocytosis ; Metronidazole ; Polymerase Chain Reaction ; Probiotics ; Prospective Studies ; Quinolines ; Ribotyping ; Risk Factors ; Sprains and Strains ; Treatment Outcome ; Vancomycin

OBJECTIVETo establish and compare the pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA) and automated ribotyping for subtyping of Citrobacter strains.

METHODSPFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system.

RESULTSWe optimized the plug preparation procedure, focused on the cell suspension concentration (turbidity of 2.5 to 3.5), SDS addition (no SDS needed) and lysis time (1 h), and selected the appropriate restriction enzyme (XbaI) and the electrophoretic parameters (1.0 s-20.0 s for 19 h) of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources (with the same species from the same place at the same time identified as the same source) and divided strains from different sources (from different years, places and hosts) into different subtypes.

CONCLUSIONPFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.

Automation ; Citrobacter ; classification ; genetics ; Electrophoresis, Gel, Pulsed-Field ; methods ; Minisatellite Repeats ; genetics ; Multilocus Sequence Typing ; methods ; Phylogeny ; Ribotyping ; methods

BACKGROUND: Clostridium difficile infection (CDI) is the predominant cause of hospital-acquired diarrhea. This study evaluated the sporicidal activities of several disinfectants against C. difficile spores. METHODS: We used toxigenic C. difficile strains with different ribotypes for our study. We compared the sporicidal activities of Cavicide (Metrex Research Corporation, USA), Cidex OPA (Advanced Sterilization Products, USA), 1% Rely+On Virkon (Dupont, UK), 0.25% Surfanios (Laboratoires Anios, France), sodium hypochlorite (Yuhan Clorox, Korea), and 70% ethyl alcohol (Duksan, Korea) by using dilution-neutralization method. The sporicidal activity of the disinfecting agents was considered to be the inactivation factor (IF). The IF was calculated as the log10 colony forming unit (CFU) reduction of the viable count from the initial inoculums. Disinfectants were considered to be sporicidal if they showed an IF> or =4. RESULTS: Cavicide, 70% ethyl alcohol, Rely+On Virkon, and Surfanios showed no reduction in spore counts at all exposure time. Solutions of sodium hypochlorite diluted 1:100 (> or =400 ppm available chlorine), 1:50, and 1:20 were sporicidal after 5 min, 2 min, and 30 s, respectively. Cidex OPA showed sporicidal activity after 30 min. CONCLUSION: To prevent the transmission of CDI, at least 1,000 ppm sodium hypochlorite solution should be used to disinfect the hospital environment. Contaminated endoscopes should be disinfected with Cidex OPA for more than 30 min.
Chlorophenols ; Clostridium ; Clostridium difficile ; Colony Count, Microbial ; Diarrhea ; Disinfectants ; Endoscopes ; Ethanol ; Glutaral ; Peroxides ; Ribotyping ; Sodium Hypochlorite ; Spores ; Stem Cells ; Sterilization ; Sulfuric Acids

BACKGROUND: Clostridium difficile infection (CDI) has markedly risen and is associated with hypervirulent ribotype 027 outbreaks in North America and Europe since 2003. The aims of this study were to determine the prevalence of ribotype 027 among C. difficile isolates in Korea, to characterize the ribotype 027 isolates, and to determine the clinical severity of CDI in patients infected with these isolates. METHODS: A total of 1,251 isolates of C. difficile recovered from stool specimens of suspected CDI patients at two tertiary-care hospitals and one commercial laboratory between 2002 and 2009. Genes for toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtA and cdtB) were detected by PCR. Mutation in the tcdC gene was detected by sequencing after PCR amplification. For molecular genotyping, we performed PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), and multilocus variable-number tandem-repeat analysis (MLVA). Minimum inhibitory concentrations of moxifloxacin were determined using Etest strips (AB bioMerieux, Sweden). RESULTS: We identified 7 isolates as ribotype 027. These isolates had the same tcdC mutation as the epidemic strain, and 6 of them were resistant to moxifloxacin. The isolates were categorized into 3 different PFGE types and 7 different MLVA types. All the 7 cases had occurred sporadically. CONCLUSIONS: C. difficile ribotype 027 is uncommon, but it has emerged in Korea. The spread of this ribotype should be closely monitored in order to avoid an outbreak of CDI in Korea.
Adult ; Aged ; Aged, 80 and over ; Bacterial Proteins/genetics/metabolism ; Bacterial Toxins/genetics/metabolism ; Clostridium difficile/genetics/*isolation & purification ; Drug Resistance, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Enterocolitis, Pseudomembranous/microbiology ; Enterotoxins/genetics/metabolism ; Feces/microbiology ; Female ; Humans ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Mutation ; Polymerase Chain Reaction ; Republic of Korea ; *Ribotyping

During the past decade, rates of Clostridium difficile infection (CDI) increased worldwide. Hypervirulent strains of C. difficile such as NAP1/BI/027 and PCR ribotype 078 have emerged that have changed the epidemiology of CDI. Especially, CDI rates also have increased in the community, in children previously thought to be at low risk. Recently, the use of gastric acid suppressant that facilitates intestinal transit of the bacteria and presence of inflammatory bowel disease has been reported as risk factors. Treatment for CDI usually relies on metronidazole or vancomycin, but recurrence rates remains high. New treatment options for multiple recurrence are challenging. In this article, we reviewed recent epidemiological changes, current knowledge of virulence factors, reasonable approach to the diagnosis, and optimal treatment of CDI. But, clinical guidelines for pediatric C. difficile disease have not been defined. It seems that the consensus and recommendations for managing pediatric CDI are urgently needed.
Bacteria ; Child ; Clostridium ; Clostridium difficile ; Consensus ; Gastric Acid ; Humans ; Inflammatory Bowel Diseases ; Metronidazole ; Polymerase Chain Reaction ; Recurrence ; Ribotyping ; Risk Factors ; Vancomycin ; Virulence Factors

Animals ; Clostridium difficile ; classification ; genetics ; Humans ; Molecular Typing ; Ribotyping

BACKGROUND: Clostridium difficile is a major cause of antibiotic-associated diarrhea. The objective of this study was to characterize clinical isolates of C. difficile obtained from various regions in Korea with regard to their toxin status, molecular type, and antimicrobial susceptibility. METHODS: We analyzed a total of 408 C. difficile isolates obtained between 2006 and 2008 from 408 patients with diarrhea in 12 South Korean teaching hospitals. C. difficile toxin genes tcdA, tcdB, cdtA, and cdtB were detected by PCR. Molecular genotyping was performed by PCR ribotyping. Antimicrobial susceptibilities of the 120 C. difficile isolates were assessed by agar dilution methods. RESULTS: Among 337 toxigenic isolates, 105 were toxin A-negative and toxin B-positive (A-B+) and 29 were binary toxin-producing strains. PCR ribotyping showed 50 different ribotype patterns. The 5 most frequently occurring ribotypes comprised 62.0% of all identified ribotypes. No isolate was susceptible to cefoxitin, and all except 1 were susceptible to piperacillin and piperacillin-tazobactam. The resistance rates of isolates to imipenem, cefotetan, moxifloxacin, ampicillin, and clindamycin were 25%, 34%, 42%, 51%, and 60%, respectively. The isolates showed no resistance to metronidazole or vancomycin. CONCLUSIONS: This is the first nationwide study on the toxin status, including PCR ribotyping and antimicrobial resistance, of C. difficile isolates in Korea. The prevalence of A-B+ strains was 25.7%, much higher than that reported from other countries. Binary toxin-producing strains accounted for 7.1% of all strains, which was not rare in Korea. The most prevalent ribotype was ribotype 017, and all A-B+ strains showed this pattern. We did not isolate strains with decreased susceptibility to metronidazole or vancomycin.
Clostridium Infections/microbiology ; Clostridium difficile/classification/*genetics/isolation & purification ; Diarrhea/microbiology ; *Drug Resistance, Bacterial ; Enterotoxins/*genetics ; Genetic Variation ; Genotype ; Hospitals, University ; Humans ; Microbial Sensitivity Tests ; Republic of Korea ; Ribotyping