Contemporary microbiology and identification of Corynebacteria spp. causing infections in human.

The 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, semi-automated 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. 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.

Comparison of eleven commercially available rapid tests for detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis.

UNLABELLED: Yersinia pestis, Bacillus anthracis and Francisella tularensis cause serious zoonotic diseases and have the potential to cause high morbidity and mortality in humans. In case of natural outbreaks and deliberate or accidental release of these pathogens rapid detection of the bacteria is crucial for limitation of negative effects of the release. In the present study, we evaluated 11 commercially available rapid test kits for the detection of Y. pestis, B. anthracis and F. tularensis in terms of sensitivity, specificity and simplicity of the procedure. The results revealed that rapid and easy-to-perform lateral flow assays for detection of highly pathogenic bacteria have very limited sensitivity. In contrast, the immunofiltration assays showed high sensitivity but limited specificity and required a too complicated procedure to be applied in the field by nonlaboratory workers (e.g. First Responders like fire, police and emergency medical personnel). Each sample - whether tested negative or positive by the rapid tests - should be retested in a reference laboratory using validated methods. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Incidence and factors predicting whooping cough due to parapertussis diagnosis among patients referred to general practitioners, Poland, 2009-2011.

Parapertussis leads to similar symptoms as pertussis, both being caused by bacteria from the genus Bordetella. Poland does not routinely diagnose nor conduct surveillance for parapertussis. We estimated parapertussis incidence and determined predictors of parapertussis diagnosis in the Polish population. Between July 2009 and April 2011, we conducted a prospective cohort study among patients attending 78 general practices. We included patients aged ≥ 3 years, with cough lasting >2 weeks, interviewed patients and collected a nasopharyngeal swab. We confirmed cases by real-time PCR. We estimated parapertussis incidence rates by dividing the number of cases by the summed person-time of observation in respective practices. We assessed predictors of PCR-confirmed parapertussis by comparing cases with patients testing negative. Using logistic regression, we calculated odds ratios (ORs) and 95% confidence intervals (95%CI). We identified 78 cases among 1,231 patients meeting inclusion criteria. The incidence rate was 39/100,000 person-years (95%CI 31-49). The highest rates (140/100,000; 95%CI 74-239), were among children 3-5 years of age and the lowest (24/100,000; 95%CI 13-40) among persons aged 20-39 years of age. Boys aged 3-5 years (7.1; 2.1-25.3) and women aged >40 years (4.1; 1.4-11.7) or living in crowded households (4.3; 1.4-12.9) or contacting persons with prolonged cough (2.3; 1.1-4.5) were more likely to be diagnosed. Our results suggest that laboratory diagnosis could be prioritized for children in the preschool age and women aged over 40 who were referred to their GP with prolonged cough. In the absence of vaccine, post-exposure prophylaxis for close contacts of parapertussis cases could an adequate preventative measure.

The utility of the PCR melting profile technique for typing Corynebacterium diphtheriae isolates.

UNLABELLED: Selection of appropriate typing method depends on a number of factors, including the scale of the investigation, the rapidity required of the results and the financial and technical resources available. Several typing methods have been applied to Corynebacterium diphtheriae genotyping, but most are laborious and time-consuming or require expensive equipment. We report an evaluation of the utility of the PCR melting profile technique for simple and easy-to-perform genotyping of C. diphtheriae. We compared the method with ribotyping-the 'gold standard' for C. diphtheriae typing-and PFGE, MLST, AFLP, RAPD and spoligotyping. SIGNIFICANCE AND IMPACT OF THE STUDY: Occurrence of Corynebacterium diphtheriae infections-in the form of diphtheria in endemic countries and in the form of invasive infections in countries with high antidiphtheria vaccination coverage-indicates the need for maintenance of ability to genotype this pathogen by laboratories. Application of an appropriate typing method is essential not only in outbreak investigations for understanding and predicting epidemics but also in monitoring of the evolution and spread of epidemic clones of C. diphtheriae. The PCR melting profile method presented in the study is a good alternative for C. diphtheriae typing.

Incidence of pertussis in patients of general practitioners in Poland.

We estimated the incidence of pertussis in patients consulting general practitioners (GPs). Between July 2009 and April 2011, we conducted a prospective cohort study of patients attending 78 general practices (158 863 persons overall). We included patients aged ≥ 3 years, with cough lasting 2-15 weeks, who gave informed consent. GPs interviewed eligible patients, collected a blood specimen, and a nasopharyngeal swab. At follow-up 30-60 days after the initial visit, physicians collected a second blood specimen and conducted patient interview. Cases were confirmed by specific IgA and/or IgG antibody titre exceeding significantly the general population background level or detection of bacterial DNA by real-time PCR. During the study period, 3864 patients with prolonged cough consulted the participating GPs, of those 1852 met the inclusion criteria, 1232 were recruited, and 288 were confirmed as pertussis cases (4% by PCR, 96% by serology). The adjusted incidence rate was 201.1/100 000 person-years [95% confidence interval (CI) 133.9-302.0], ranging from 456.5 (95% CI 239.3-870.8) in the 15-19 years group to 94.0 (95% CI 33.4-264.5) in the 25-29 years group. The reporting ratio was 61, ranging from 4 in those aged 3-5 years, to 167 in those aged 65-69 years. The study confirmed high incidence of pertussis in all age groups in the general population, in particular in adults, not appropriately documented by the existing surveillance system.

Comparison of seven commercial enzyme-linked immunosorbent assays for the detection of anti-diphtheria toxin antibodies.

Determination of immune status of patients to diphtheria toxin is based mainly on the results of commercially available ELISA kits. The aim of the present study was to compare the results obtained by ELISAs from seven different manufacturers: Mikrogen, Immunolab, Sekisui Virotech, NovaTec, Virion\Serion, IBL International and Euroimmun. All assays were performed according to the manufacturers' instructions. The concentrations of the anti-diphtheria toxin antibodies in 72 serum samples were calculated on the basis of curves constructed from standards supplied by manufacturers and the new reference material-International Standard for Diphtheria Antitoxin (10/262). The repeatability and reproducibility of all the ELISA kits tested were good. Number of sera with concentrations of the anti-diphtheria toxin antibodies below the WHO-recommended level of protection (0.1 IU/ml) were dependent on the ELISA used: Mikrogen, 20/72 samples (27.7%); Immunolab, 11/72 samples (15.3%); Sekisui Virotech, 0/72 samples (0%); NovaTec 18/72 samples (25.0%); Serion 12/72 samples (16.7%); IBL International, 7/72 samples (9.7 %); and Euroimmun, 17/72 samples (23.6%). However, the results obtained in particular ELISAs, with the exception of Sekisui Virotech, were much more consistent when the concentrations of the anti-diphtheria toxin antibodies in 72 sera measured by using curves constructed from the International Standard 10/262. The data obtained clearly demonstrated that manufacturer-dependent differences between anti-diphtheria IgG ELISA kits exist. The differences in recommendations accepted by the individual manufacturers together with differences shown in our studies in sensitivity greatly affect the clinical interpretation of results.

Characterization and comparison of invasive Corynebacterium diphtheriae isolates from France and Poland.

Corynebacterium diphtheriae, the agent of diphtheria, is rarely responsible for bacteremia. However, high numbers of bacteremia have been reported in countries with extensive immunization coverage. Here, we used molecular and phenotypic tools to characterize and compare 42 invasive isolates collected in France (including New Caledonia) and Poland over a 23-year period.

Phosphatidylserine liposomes can be tethered by caldesmon to actin filaments.

Rotary shadowing electron microscopy revealed that attachment of caldesmon to phosphatidylserine (PS) liposomes was mainly through its C-terminal end. To determine the PS-binding sites of caldesmon, we have made use of synthetic peptides covering the two C-terminal calmodulin binding sites and a recombinant fragment corresponding to the N-terminal end of the C-terminal domain that contains an amphipathic helix. Interactions of these peptides with the PS liposomes were studied by nondenaturing gel electrophoresis and fluorescence spectroscopy. The results showed that both calmodulin-binding sites of caldesmon were able to interact with PS. The affinity (Kd) of PS for these sites was in the range of 1.8-14.3 x 10(-5) M, compared to 0.69 x 10(-5) M for the whole caldesmon molecule. Fragments located outside of calmodulin-binding sites bound PS weakly (3.85 x 10(-4) M) and thus may contain a second class of lipid-binding sites. Binding of PS induced conformational changes in regions other than the C-terminal PS-binding sites, as evidenced by the changes in the susceptibility to proteolytic cleavages. Most significantly, the presence of caldesmon greatly increased binding of PS to F-actin, suggesting that caldesmon may tether PS liposomes to actin filaments. These results raise the possibility that caldesmon-lipid interactions could play a functionally important role in the assembly of contractile filaments near the membranes.

Characterization of chicken gizzard calcyclin and examination of its interaction with caldesmon.

Using a procedure developed to purify calcyclin from mouse Ehrlich ascites tumor cells calcyclin was purified from smooth muscle of chicken gizzard. Chicken gizzard calcyclin bound to phenyl-Sepharose in a calcium dependent manner as did mouse EAT cells and rabbit lung calcyclin but appeared to be more acidic than its mammalian counterparts as revealed by ion exchange chromatography on Mono Q. Chicken gizzard calcyclin bound 45Ca2+ on nitrocellulose filters and exhibited a shift in electrophoretic mobility on urea-PAGE depending on Ca2+ concentration. Crosslinking experiments with BS3 showed that chicken gizzard calcyclin was able to form noncovalent dimers. As indicated by a decrease in maximum tryptophan fluorescence emission of caldesmon (about 14% at 1:1 molar ratio) and displacement of calmodulin from its complex with caldesmon, chicken gizzard calcyclin binds caldesmon. This binding was, however, much weaker than that of calmodulin and could not influence the interaction of caldesmon with actin. In consequence, calcyclin was unable to reverse the inhibitory effect of caldesmon on actin-activated Mg(2+)-ATPase activity of myosin in the presence of Ca2+.