Ribotyping of Clostridioides difficile in the Liberec Regional Hospital: a tertiary health care facility.

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.

Multi-branch cooperation on SARS-CoV-2 omicron capture: a case report.

Shortly after the WHOs first notice a suspected case of omicron SARS-CoV-2 was reported in Liberec, Czech Republic. The primary goal of the following actions was to test the presence of the variant and stop the spread of the virus variant. On November 25 a sixty-year-old lady, who had recently returned from Namibia, visited a GP with flu-like symptoms and a rash on her chest. The antigen test was positive for SARS-CoV-2, a PCR test was planned. At that time, it was not known that a new variant of concern was spreading from Africa. On November 26 in the morning the GP announced a suspected omicron case to the Regional public health authority, who organized the following steps. A mobile sampling team was sent to the patient's home immediately, sample transported into the regional hospital and analyzed with the help of the national reference laboratory. The captured virus SARS-CoV-2 fitted the description of the omicron variant, was shared in the GISAID database and named hCoV-19/Czech Republic/KNL_2021-110119140/2021. Contact tracing was started immediately, eleven persons were tested and quarantined. One of them positive with no further spread. It is the first documented omicron case in the Czech Republic and one of the first cases in Europe, with an excellent systemic response to the alert. The laboratory was able to detect the omicron variant instantly after the request. This case also demonstrates how easily the virus spreads on long distances and how important it might be to increase the uptake of the booster vaccine.

Effect of Restriction of Fluoroquinolone Antibiotics on Clostridioides difficile Infections in the University Hospital Hradec Králové.

Clostridioides difficile is the most common pathogen responsible for hospital-acquired diarrhea. This complication of antibiotic treatment mainly endangers the health of elder patients. Preventing the development of C. difficile infections (CDI) is still a challenge that needs to be addressed. In our study, the results of 872 C. difficile positive stool samples were used to describe the epidemiological situation affected by a change in the prescription of fluoroquinolone antibiotics. In a total, 93 of strains were typed by polymerase chain reaction (PCR) and capillary gel electrophoresis. Between years 2014 and 2018 the decline in the fluoroquinolones consumption was 69.3 defined daily dose (DDD) per 1000 patient-days (from 103.3 to 34.0), in same period CDI incidence declined by 1.3 cases per 10,000 patient-bed days (from 5.6 to 4.3). Results of epidemiologic and statistical analysis shows that decline in fluoroquinolones consumption has significant influence on CDI incidence and prevalence of hypervirulent strains. In the University Hospital Hradec Králové properly managed antibiotic stewardship policy has reduced CDI incidence by 23.2% and lowered rate of hypervirulent ribotypes 001 and 176.

[First experiences with detection of the tcdB gene of Clostridioides difficile using Simplexa C. difficile Direct Kit].

OBJECTIVE: Clostridioides difficile (formerly Clostridium difficile) is one of the main pathogens causing nosocomial infections today. It colonizes the intestines of patients receiving antibiotic therapy, causing unpleasant or even life-threatening conditions (diarrhea, toxic megacolon). Rapid and correct detection of strain toxigenicity is essential for treatment and isolation of patients. Simplexa C. difficile Direct Kit is a real-time PCR kit detecting the tcdB gene of C. difficile. The kit does not require DNA isolation; stool eluates are directly used for the reaction. The study aimed to verify the analytical properties of the kit by its comparison with culture and in-house multiplex PCR methods. MATERIAL AND METHODS: A total of 164 stool samples were prospectively tested using two immunoenzymatic kits (C. diff Quik Chek Complete and LIAISON C. difficile GDH, Toxins AandB). In 39 samples, the results were discrepant or unclear (GDH+TOX-). These samples were tested using in-house multiplex PCR and the Simplexa kit. RESULTS: The Simplexa kit had 94.7% sensitivity, 100% specificity, 100% positive predictive value and 95.2% negative predictive value. These parameters were calculated from the numbers of true-/false-positive and true-/false-negative results. True results were determined based on the consensus of culture and in-house multiplex PCR results. Another outcome of the study was comparison of the Quik Chek and LIAISON kits. CONCLUSION: The analytical properties of the Simplexa kit were tested on 39 samples. These samples were selected for their unclear (GDH+TOX-) or discrepant results yielded by immunoenzymatic methods. Compared with culture and subsequent in-house PCR detection of the tcdB gene, the Simplexa kit showed properties declared by the manufacturer. An important advantage of the kit was the absence inhibitions when stool eluates were directly used for PCR reactions.

Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes.

Bacterial amidases and nitrile hydratases can be used for the synthesis of various intermediates and products in the chemical and pharmaceutical industries and for the bioremediation of toxic pollutants. The aim of this study was to analyze the expression of the amidase and nitrile hydratase genes of Rhodococcus erythropolis and test the stereospecific nitrile hydratase and amidase activities on chiral cyanohydrins. The nucleotide sequences of the gene clusters containing the oxd (aldoxime dehydratase), ami (amidase), nha1, nha2 (subunits of the nitrile hydratase), nhr1, nhr2, nhr3 and nhr4 (putative regulatory proteins) genes of two R. erythropolis strains, A4 and CCM2595, were determined. All genes of both of the clusters are transcribed in the same direction. RT-PCR analysis, primer extension and promoter fusions with the gfp reporter gene showed that the ami, nha1 and nha2 genes of R. erythropolis A4 form an operon transcribed from the Pami promoter and an internal Pnha promoter. The activity of Pami was found to be weakly induced when the cells grew in the presence of acetonitrile, whereas the Pnha promoter was moderately induced by both the acetonitrile or acetamide used instead of the inorganic nitrogen source. However, R. erythropolis A4 cells showed no increase in amidase and nitrile hydratase activities in the presence of acetamide or acetonitrile in the medium. R. erythropolis A4 nitrile hydratase and amidase were found to be effective at hydrolysing cyanohydrins and 2-hydroxyamides, respectively.