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Most point-of-care tests (POCT) use swabs for sampling and/or for applying a sample on the test. A variety of swabs differing in tip materials is commercially available. Different tip materials have different chemical and physical characteristics which might influence the specimen collection and release. We investigated properties of various types of swabs used in clinical diagnostics with focusing on two kinds of analytes, DNA and proteins, which are most often used targets in POCT. As the model samples we used diphtheria toxoid NIBSC 69/017 for investigating recovery of protein analytes such as antigens and bacterial strains of Escherichia coli ATCC 25922, diphtheria toxin-producing Corynebacterium diphtheriae NCTC 10648, and the clinical isolate nontoxigenic C. diphtheriae 5820/15 for investigating the recovery of nucleic acids. We investigated four types of swabs most commonly used in clinical diagnostics in terms of absorption capacity and efficiency of release of nucleic acids and proteins. Volume uptake was measured in milligrams. For DNA release various washing out buffers were used and the amount of released DNA was measured spectrophotometrically. The amount of protein released from the swabs were examined using the Lowry assay. We observed statistically significant differences (p < 0.05) in the mean weights of absorbed liquid, in the DNA recovery and protein recovery by the four variety of swab examined. However, the efficiency of DNA and protein release was not correlated to the absorbed volume of a sample, but rather to the properties of swabs. The swab composition and structure can have a significant impact on the collection and release efficiency of a sample. Therefore, validation of POCT in relation to the used swabs for sampling is really important. The use of inappropriate swabs could lead to false negative or misleading analysis results.

A Klebsiella pneumoniae epidemic strain that coproduced carbapenemase KPC-2 (K. pneumoniae carbapenemase 2) and 16S rRNA methylase ArmA has emerged in Poland. Four nonduplicate isolates from patients in a hospital in Warsaw, Poland, were found to carry the bla KPC-2 and armA genes on ca. 50-kb and 90-kb plasmids, respectively. Tn4401 with a 100-bp deletion in the variable region was detected in all the isolates. XbaI pulsed-field gel electrophoresis (PFGE) revealed 93.2% similarity of the isolates. All the isolates were resistant to carbapenems and 4,6-disubstituted 2-deoxystreptamines.

Rapid detection of highly pathogenic bacteria causing anthrax, plague and tularemia is crucial for the limitation of negative effects of a potential release (natural, accidental or deliberate). In the study, commercially available rapid tests for detection of Bacillus anthracis, Yersinia pestis and Francisella tularensis were investigated in terms of sensitivity, specificity and ease-to-perform. The study showed problems which could be faced during testing and results interpretation. Conclusions from this study should be helpful not only in selection of the most appropriate test for particular group of First Responders but also in undertaking decisions in situation of a contamination suspicion which have high social and economical impacts.

The ENGAGE project (http://www.engage-europe.eu/) was a collaboration between eight institutions across Europe. The aim was to boost the scientific cooperation to use whole genome sequencing (WGS) analysis in food safety and public health protection. ENGAGE focused on Escherichia coli (commensal E. coli) and different Salmonella spp. serotypes. A total of 3,360 genomes, 778 and 2,582 of E. coli and Salmonella, respectively, were produced. These genomes were stored and shared among partners in a temporary repository to be submitted to the European Nucleotide Archive by the end of the project. Generated genomes were used for benchmarking exercises to assess the possibility of replacing conventional typing with WGS for outbreak investigation. For the analysed strains, the benchmarking exercises showed that SPAdes assembly performed better than Velvet and that, by using different bioinformatics tools, WGS Salmonella serotyping and antimicrobial resistance genes detection, were largely in concordance with phenotypic data. Discrepancies were related to sequence quality and phenotype misclassification rather than to limitations of the bioinformatics tools.All partners were able to infer the expected phylogeny for the Salmonella and Campylobacter isolates in benchmarking exercises. Two WGS proficiency tests (assessing different genomic quality markers) were conducted among partners with satisfactory results. Guidelines including available bioinformatics tools and standard operating procedures (wet and dry lab) were prepared and posted online. Workshops, training courses and twinning programmes were conducted. The training focused on ENGAGE www.efsa.europa.eu/publications 2 EFSA Supporting publication 2018:EN-1431The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the author(s).online, Galaxy-based, and command line bioinformatics tools. To reach out beyond ENGAGE, an elearning course (17 videos) was developed and made available online. Several proof of concept projects were run and some outcomes published, e.g. the discovery of colistin resistance gene, mcr-5.Overall, the project showed that laboratories without previous WGS experience need a period of time to implement and perform WGS for foodborne pathogens routine analysis. All developed material will remain available on the ENGAGE website. The present document has been produced and adopted by the bodies identified above as author(s). In ...

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We characterized 17 clinical isolates of Klebsiella pneumoniae producing 16S rRNA methylase ArmA. The isolates originated in Poland from 2002 to May 2010 and encompassed four XbaI-PFGE clusters. All the isolates were resistant to amikacin, gentamicin and kanamycin (MIC range: 256-1024 mg l "1 ) and carried the armA gene on a large plasmid of approximately 90 or 130 kb in 15 and 2 isolates, respectively. The armA gene was found in a~10 kb ClaI restriction fragment of the large plasmid and was flanked by the same elements as in Tn1548. All the isolates carried the bla CTX-M gene for a CTX-M-type extended-spectrum b-lactamase. Our results show that ArmA has disseminated horizontally among K. pneumoniae isolates in Poland on the~90 kb plasmid of the pCTX-M3 family.

The presented study is focused on the development of repetitive, fast and versatile "on-off" signaling electrochemical genosensor for detection of pagA gene of Bacillus anthracis. Such a sensor will be further used in the portable devices dedicated to molecular diagnostic. The DNA molecular beacon probe was immobilized onto gold electrodes in its folded state through the alkanethiol linker at the 5'-end, while the 3'-end was labeled with methylene blue redox molecule. Initial optimization was conducted for HPLC purified cDNA. Nonetheless application of such prepared biosensor for unpurified PCR product required further investigations. As the sensor response was significantly dependent on the stem-loop architecture, the three different hairpin probes were analyzed. The detected sequence was interior to amplicon, identical as in case of cDNA. The developed "on-off" genosensor allows for detection, in 5 minute, the pagA gene sequence at the level of 5.7 nM with 22.9-86.0 nM linear detection range also in the great excess of other DNA presented in the sample. Both LOD and linear range were significantly improved in case of longer assay time. The biosensor proved its applicability also in detection of pagA gene of Bacillus anthracis in PCR samples amplified with reduced number of cycles.