The ribotyping of Clostridioides difficile is one of the basic methods of molecular epidemiology for monitoring the spread of C. difficile infections. In the Czech Republic, this procedure is mainly available in university hospitals. The introduction of ribotyping in a tertiary health care facility such as Liberec Regional Hospital not only increases safety in the facility but also supports regional professional development. In our study, 556 stool samples collected between June 2017 and June 2018 were used for C. difficile infection screening, followed by cultivation, toxinotyping, and ribotyping of positive samples. The toxinotyping of 96 samples revealed that 44.8% of typed strains could produce toxins A and B encoded by tcdA and tcdB, respectively. The ribotyping of the same samples revealed two epidemic peaks, caused by the regionally most prevalent ribotype 176 (n = 30, 31.3). C. difficile infection incidence ranged between 5.5 and 4.2 cases per 10,000 patient-bed days. Molecular diagnostics and molecular epidemiology are the two most developing parts of clinical laboratories. The correct applications of molecular methods help ensure greater safety in hospitals.

• Keywords
• Clonal spreading, Clostridioides difficile, Health care facility, Ribotyping, Toxinotyping,
• MeSH
• Bacterial Toxins * MeSH
• Clostridioides difficile * genetics   MeSH
• Clostridioides MeSH
• Clostridium Infections * diagnosis   epidemiology   MeSH
• Humans MeSH
• Hospitals, University MeSH
• Delivery of Health Care MeSH
• Ribotyping MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Toxins * MeSH

We aimed to characterize Clostridioides difficile isolates cultured during a six-month single-center study from stool samples of patients with C. difficile infection (CDI) genotyped by the Xpert®C. difficile/Epi assay by polymerase chain reaction (PCR) ribotyping, toxin genes' detection and multi-locus variable number tandem repeats analysis (MLVA). The susceptibility to metronidazole, vancomycin and moxifloxacin was determined by agar dilution. In addition, the presence of Thr82Ile in the GyrA and a single nucleotide deletion at position (Δ117) in the tcdC gene were investigated. Between January 1 and June 30, 2016, of 114 CDIs, 75 cases were genotyped as presumptive PCR ribotype (RT) 027 infections using a commercial assay. C. difficile isolates cultured from presumptive RT027 stool samples belonged to RT176. These isolates carried genes for toxin A (tcdA), B (tcdB), binary (cdtA/B) and had Δ117 in the tcdC gene. Using MLVA, the 71/75 isolates clustered into two clonal complexes (CCs). Of these, 39 isolates (54.9%) were from patients hospitalized in acute care and 32 isolates (45.1%) were isolated from patients hospitalized in the long-term care department. All isolates were susceptible to metronidazole and vancomycin, and 105 isolates were resistant to moxifloxacin (92%) carrying Thr83Ile in the GyrA. An outbreak of RT176 CDIs, suspected as RT027, was recognized in a Slovakian hospital. In order to monitor the emergence and spread of RT027-variants, the identification of a presumptive RT027 CDI should be confirmed at a strain level by PCR ribotyping.

• Keywords
• MLVA, Slovakia, Thr82Ile, binary toxin, ribotyping, tcdC, Δ117,
• Publication type
• Journal Article MeSH

OBJECTIVES: To investigate the relationship between Clostridium (Clostridioides) difficile strain characteristics and C. difficile infection (CDI) outcome. METHODS: Between October and December 2017, 16 hospitals collected epidemiological data according to the European Centre for Disease Prevention and Control (ECDC) surveillance protocol for CDI. C. difficile isolates were characterized by ribotyping, toxin genes detection and antibiotic susceptibility testing to metronidazole, vancomycin and moxifloxacin. RESULTS: The overall mean CDI incidence density was 4.5 [95% CI 3.6-5.3] cases per 10,000 patient-days. From the 433 CDI cases, 330 (76.2%) were healthcare-associated, 52 (12.0%) cases were community-associated or of unknown origin and 51 (11.8%) CDI cases recurrent; a complicated course of CDI was reported in 65 cases (15.0%). Eighty-eight (20.3%) of patients died and 59 of them within 30 days after the CDI diagnosis. From the 379 C. difficile isolates, the most prevalent PCR ribotypes were 001 (n = 127, 33.5%) and 176 (n = 44, 11.6%). A total of 186 (49.1%) isolates showed a reduced susceptibility to moxifloxacin (> 4 mg/L) and 96.4% of them had Thr82Ile in the GyrA. Nineteen isolates revealed reduced susceptibility to metronidazole and two isolates to vancomycin (> 2 mg/L). A fatal outcome was associated with a reduced susceptibility to moxifloxacin, the advanced age of the patients and a complicated course of CDI (p<0.05). No association between ribotype, binary toxin and a reduced susceptibility to moxifloxacin and complicated course or recurrent CDI was found. CONCLUSIONS: A reduced susceptibility to moxifloxacin, in causative C. difficile strains was associated with fatal outcome of the patients, therefore it is an important marker in surveillance of CDI.

• Keywords
• Clostridioides difficile infection, Clostridium difficile infection, Czech Republic, Mortality, Moxifloxacin, PCR ribotype 001, PCR ribotype 176,
• MeSH
• Anti-Bacterial Agents therapeutic use   MeSH
• Clostridioides difficile drug effects   MeSH
• Feces microbiology   MeSH
• Cross Infection MeSH
• Clostridium Infections drug therapy   microbiology   mortality   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Moxifloxacin therapeutic use   MeSH
• Ribotyping MeSH
• Aged MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Moxifloxacin MeSH

This study aimed to implement a toxigenic culture as an optional third diagnostic step for glutamate dehydrogenase (GDH)-positive and toxin A/B-negative diarrheal stool samples into a diagnostic algorithm for Clostridioides (Clostridium) difficile infection (CDI), and to characterise C. difficile isolates for epidemiological purposes. During the 5-month study, 481 diarrhoeal stool samples from three Slovak hospitals were investigated and 66 non-duplicated GDH-positive stool samples were found. Of them, 36 were also toxin A/B-positive. Twenty-three GDH-positive and toxin A/B-negative stool samples were shown subsequently to be positive following toxigenic culture (TC). Molecular characterisation of C. difficile isolates showed the predominance of PCR ribotype (RT) 001 (n = 37, 56.1%) and the occurrence of RT 176 (n = 3, 4.5%). C. difficile RT 001 isolates clustered to eight clonal complexes (CCs) using multiple-locus variable-number tandem repeats analysis (MLVA). Interestingly, one third of RT 001 isolates clustering in these CCs were cultured from toxin A/B-negative stool samples. Our observations highlight the need of use multiple step diagnostic algorithm in CDI diagnosis in order to detect all CDI cases and to avoid the spread of C. difficile in healthcare settings.

• MeSH
• Algorithms MeSH
• Bacterial Proteins analysis   MeSH
• Clostridioides difficile classification   isolation & purification   MeSH
• Molecular Diagnostic Techniques MeSH
• Enterotoxins analysis   deficiency   MeSH
• Feces microbiology   MeSH
• Glutamate Dehydrogenase analysis   MeSH
• Clostridium Infections diagnosis   epidemiology   microbiology   MeSH
• Humans MeSH
• Minisatellite Repeats genetics   MeSH
• Molecular Typing * MeSH
• Multilocus Sequence Typing MeSH
• Hospitals MeSH
• Polymerase Chain Reaction * MeSH
• Diarrhea diagnosis   epidemiology   microbiology   MeSH
• Ribotyping methods   MeSH
• Cluster Analysis MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Geographicals
• Slovakia epidemiology   MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Enterotoxins MeSH
• Glutamate Dehydrogenase MeSH

The aim of the study was to provide an update on the epidemiology of C. difficile infection (CDI) in a representative number of hospitals within the Czech Republic in 2015. In 2015, twenty-eight Czech hospitals were invited to participate in a CDI study. Laboratories sent the first 20 consecutive C. difficile isolates for characterization by capillary-electrophoresis (CE) ribotyping and the presence of toxin genes and collected patient data on previous hospitalization, antibiotic treatment, the number of CDI episodes and the course of CDI. The mean incidence of CDI was 5.2 [95% CI 4.2-6.2] cases per 10,000 patient-bed days in 27 hospitals in 2015. Of 490 C. difficile isolates, the prevalent PCR ribotypes (RTs) were 001 (n = 164, 33.5%) and 176 (n = 125, 25.5%) followed by 014 (n = 37, 7.6%), 012 (n = 17, 3.5%), 020 (n = 16, 3.3%), 017 (n = 14, 2.9%) and 002 (n = 11, 2.2%). A severe course of CDI was reported in 104 cases (21.2%) with significant association with RT001 infection (p = 0.03). CDI recurrence was 10.8% (n = 53). The previous use of fluoroquinolones was associated with RTs 001 and 176 CDIs (p = 0.046 and p = 0.041). We observed a persistence of RTs 001 and 176 CDIs in the Czech Republic that was associated with the previous use of fluoroquinolones. This highlights the need for a reduction in fluoroquinolone use in Czech hospital settings.

• Keywords
• CDI surveillance, Capillary electrophoresis ribotyping, Clostridium difficile, Clostridium difficile infection, Fluoroquinolones, Toxin genes,
• MeSH
• Anti-Bacterial Agents therapeutic use   MeSH
• Drug Resistance, Bacterial physiology   MeSH
• Clostridioides difficile classification   genetics   isolation & purification   MeSH
• Fluoroquinolones therapeutic use   MeSH
• Cross Infection epidemiology   microbiology   MeSH
• Humans MeSH
• Hospitals MeSH
• Enterocolitis, Pseudomembranous drug therapy   epidemiology   microbiology   MeSH
• Ribotyping MeSH
• Aged MeSH
• Check Tag
• Humans MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Fluoroquinolones MeSH

In 2011-2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacterial Toxins analysis   genetics   MeSH
• Clostridioides difficile classification   genetics   isolation & purification   MeSH
• Disk Diffusion Antimicrobial Tests MeSH
• Incidence MeSH
• Clostridium Infections epidemiology   microbiology   MeSH
• Humans MeSH
• Minisatellite Repeats MeSH
• Molecular Typing * MeSH
• Hospitals MeSH
• Ribotyping MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Bacterial Toxins MeSH

BACKGROUND: Clostridium difficile infection (CDI) is a major cause of antibiotic-associated diarrhoea. Given an increasing CDI incidence and global spread of epidemic ribotypes, a 1-year study was performed to analyse the molecular characteristics of C. difficile isolates and associated clinical outcomes from patients diagnosed with CDI in the Internal Medicine department at University Hospital Motol, Prague from February 2013 to February 2014. RESULTS: A total of 85 unformed stool samples were analysed and CDI was laboratory confirmed in 30 patients (6.8 CDI cases per 10,000 patient bed days and 50.6 CDI cases per 10,000 admissions). The CDI recurrence rate within 3 months of treatment discontinuation was 13.3% (4/30). Mortality within 3 months after first CDI episode was 26.7% (8/30), with CDI the cause of death in two cases. 51.9% of C. difficile isolates belonged to PCR-ribotype 176. MLVA of ribotype 176 isolates revealed two clonal complexes formed by 10/14 isolates. ATLAS scores and Horn's index were higher in patients with ribotype 176 infections than with non-ribotype 176 infections. CONCLUSION: This study highlights the clinical relevance of C. difficile PCR-ribotype 176 and its capacity to spread within a healthcare facility.

• MeSH
• Survival Analysis MeSH
• Anti-Bacterial Agents administration & dosage   adverse effects   MeSH
• Clostridioides difficile classification   genetics   isolation & purification   MeSH
• Feces microbiology   MeSH
• Clostridium Infections epidemiology   microbiology   mortality   pathology   MeSH
• Humans MeSH
• Hospitals, University MeSH
• Polymerase Chain Reaction MeSH
• Diarrhea chemically induced   epidemiology   microbiology   pathology   MeSH
• Recurrence MeSH
• Ribotyping * MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH