Despite more than 50 years of vaccination, pertussis is still an endemic disease, with regular epidemic outbreaks. With the exception of Poland, European countries have replaced whole-cell vaccines (WCVs) by acellular vaccines (ACVs) in the 1990s. Worldwide, antigenic divergence in vaccine antigens has been found between vaccine strains and circulating strains. In this work, 466 Bordetella pertussis isolates collected in the period 1998–2012 from 13 European countries were characterised by multi-locus antigen sequence typing (MAST) of the pertussis toxin promoter (ptxP) and of the genes coding for proteins used in the ACVs: pertussis toxin (Ptx), pertactin (Prn), type 2 fimbriae (Fim2) and type 3 fimbriae (Fim3). Isolates were further characterised by fimbrial serotyping, multi-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). The results showed a very similar B. pertussis population for 12 countries using ACVs, while Poland, which uses a WCV, was quite distinct, suggesting that ACVs and WCVs select for different B. pertussis populations. This study forms a baseline for future studies on the effect of vaccination programmes on B. pertussis populations.Electronic supplementary materialThe online version of this article (doi:10.1007/s10096-014-2297-2) contains supplementary material, which is available to authorized users.

Significance and Impact of the Study: The number of opportunistic infections caused by Corynebacteria is increasing due to increase in number of immunocompromised patients. New Corynebacterium species and new human infections, caused by this group of bacteria, has been described recently. However, identification of Corynebacteria is still a challenge despite application of sophisticated laboratory methods. In the study we present possibilities and limitations of various commercial systems for identification of Corynebacteria. AbstractThe Corynebacterium is a genus of bacteria of growing clinical importance. Progress in medicine results in growing population of immunocompromised patients and growing number of infections caused by opportunistic pathogens. A new infections caused by new Corynebacterium species and species previously regarded as commensal micro-organisms have been described. Parallel with changes in Corynebacteria infections, the microbiological laboratory diagnostic possibilities are changing. But identification of this group of bacteria to the species level remains difficult. In the paper, we present various manual, semiautomated and automated assays used in clinical laboratories for Corynebacterium identification, such as API Coryne, RapID CB Plus, BBL Crystal Gram Positive ID System, MICRONAUT-RPO, VITEK 2, BD Phoenix System, Sherlock Microbial ID System, MicroSeq Microbial Identification System, Biolog Microbial Identification Systems, MALDI-TOF MS systems, polymerase chain reaction (PCR)-based and sequencing-based assays. The presented assays are based on various properties, like biochemical tests, specific DNA sequences, composition of cellular fatty acids, protein profiles and have specific limitations.

In the present study, clinical isolates of Bordetella pertussis collected in Poland from 1960 to 2005 were analyzed by pulsed-field gel electrophoresis (PFGE) according to protocols recommended in previous studies. Among the 110 isolates from 1995 to 2005, 59 PFGE patterns were found, most of which were different from those currently circulating in other European Union (EU) countries for which data are available. The PFGE patterns of currently disseminating B. pertussis clones were found within PFGE groups III and IV, as elsewhere in the EU, and in newly identified clusters A and C. Up to 70, 26, and 4%, respectively, of the currently isolated strains in Poland harbored ptxA1-prn1, ptxA1-prn2, and ptxA1-prn3 allele combinations, and most (82%) were found to be of the Fim2 phenotype. Differences in the extent of heterogeneity estimated by PFGE typing in B. pertussis populations circulating in Poland in comparison to other EU countries may be due to the different vaccine composition strategy, since routine pertussis vaccination was initiated in Poland in 1960.

Only three Corynebacterium species are known to produce a lethal exotoxin called diphtheria toxin. These are C. diphtheriae, C. ulcerans and C. pseudotuberculosis. The diphtheria toxin gene (tox) is carried in a family of closely related corynebacteriophages and therefore the toxin can be produced only through lysogenisation, in which the corynephage encoding tox is stably inserted into the chromosome. However, ‘nontoxigenic tox gene-bearing’ (NTTB) strains, which are genotypically tox-positive but do not express the protein, have been described. The emergence of NTTB strains was first observed during the 1990s diphtheria epidemic in Eastern Europe and nowadays such isolates have been detected in many countries in the world. Recently, novel species of Corynebacterium genus have been described which might have the potential of producing the diphtheria toxin due to the possession of the diphtheria toxin gene but it has not produced toxin in laboratory tests. The circulation of NTTB strains could be related to the increased risk for diphtheria disease arising from the risk of re-emerging toxin expression. The article presents the mechanism of diphtheria toxin expression and action, recently described novel species of NTTB corynebacteria as well as the taxonomic changes within the C. diphtheriae group.

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Background: Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts. The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains. Methods: Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria. Results: The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/μl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 min of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks. Conclusion: The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections as well as for other infections caused by diphtheria-toxin producing Corynebacterium species. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.

BackgroundCorynebacterium diphtheriae is a re-emerging pathogen in Europe causing invasive infections in vaccinated persons and classical diphtheria in unvaccinated persons. In the presented study we analysed genetic changes in C. diphtheriae isolates collected in Poland from the period before the introduction of the mass anti-diphtheria vaccination to the present time when over 98% of the population is vaccinated.MethodsA total of 62 C. diphtheriae isolates collected in the 1950s–1960s, 1990s and 2000–2016 in Poland were investigated. Examined properties of the isolates included toxigenic status, presence of tox gene, biotype, MLST type (ST) and type of infection.ResultsA total of 12 sequence types (STs) were identified among the analysed C. diphtheriae isolates. The highest variability of STs was observed among isolates from diphtheria and asymptomatic carriers collected in the XX century. Over 95% of isolates collected from invasive and wound infections in 2004–2016 belonged to ST8. Isolates from the XX century represented all four biotypes: mitis, gravis, intermedius and belfanti, but the belfanti biotype appeared only after the epidemic in the 1990s. All except three isolates from the XXI century represented the biotype gravis.ConclusionsDuring a diphtheria epidemic period, non-epidemic clones of C. diphtheriae might also disseminate and persist in a particular area after the epidemic. An increase of the anti-diphtheria antibody level in the population causes not only the elimination of toxigenic strains from the population but may also influence the reduction of diversity of C. diphtheriae isolates. MLST types do not reflect the virulence of isolates. Each ST can be represented by various virulent variants representing various pathogenic capacities, for example toxigenic non-invasive, nontoxigenic invasive and nontoxigenic non-invasive.

Rapid and accurate detection and identification of pathogens in clinical samples is essential for all infection diseases. However, in the case of epidemics, it plays a key role not only in the implementation of effective therapy but also in limiting the spread of the epidemic. In this study, we present the application of two nucleic acid isothermal amplification methods—reverse transcription helicase dependent amplification (RT-HDA) and reverse transcription loop-mediated amplification (RT-LAMP)—combined with lateral flow assay as the tools for the rapid detection of SARS-CoV-2, the etiological agent of COVID-19, which caused the ongoing global pandemic. In order to optimize the RT-had, the LOD was 3 genome copies per reaction for amplification conducted for 10–20 min, whereas for RT-LAMP, the LOD was 30–300 genome copies per reaction for a reaction conducted for 40 min. No false-positive results were detected for RT-HDA conducted for 10 to 90 min, but false-positive results occurred when RT-LAMP was conducted for longer than 40 min. We concluded that RT-HDA combined with LFA is more sensitive than RT-LAMP, and it is a good alternative for the development of point-of-care tests for SARS-CoV-2 detection as this method is simple, inexpensive, practical, and does not require qualified personnel to perform the test and interpret its results.