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Abstract Findings To facilitate automation, a novel DNA extraction method for MRSA was adopted. The MRSA specific chromosome-SCCmec PCR was adapted, additional primers were added, and the performance was validated. From various laboratories in The Netherlands we received a total of 86 MRSA clinical isolates, that were negative in commercially available tests. We identified 14 MRSA strains with new variant chromosome-SCCmec junctions by sequence analysis. These MRSA strains appeared to carry SCCmec sequences with a high degree of homology to SCC regions of S. epidermidis and S. haemolyticus. All were included for detection in chromosome-SCCmec based PCR. Background Efficient management of Methicillin Resistant Staphylococcus aureus (MRSA) in the hospital is needed to prevent dissemination. It is important that MRSA can be rapidly identified, and effective infection control measures can be initiated. Equally important is a rapid MRSA negative report, especially for patients in isolation. For negative screening we implemented fully automated high through-put molecular screening for MRSA. Conclusions Fourteen variant chromosome-SCCmec junctions in MRSA, that are not detected in commercially available MRSA detection kits were added to our PCR to detect all currently known variant SCC-mec types of MRSA.

Abstract Background In Dutch laboratories molecular detection of B. pertussis and B. parapertussis is commonly based on insertion sequences IS481 and IS1001, respectively. Both IS elements are more widely spread among Bordetella species. Both Bordetella holmesii, and B. bronchiseptica can harbour IS481. Also, IS1001 is found among B. bronchiseptica. IS481, and IS1001 based PCR thus lacks specificity when used for detection of specific Bordetella spp. Findings We designed a PCR based on IS1002, another IS element that is present among Bordetella species, and exploited it as a template in combination with PCR for IS481, and IS1001. In combining the PCRs for IS481, IS1001, and IS1002, and including an inhibition control, we were able to detect and discriminate all clinically relevant Bordetella species. Conclusions We developed an improved PCR method for specific detection of B. pertussis, B. parapertussis, B. holmesii, and B. bronchiseptica.

Abstract Background The obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome. The related Chlamydophila pneumoniae, of which no subtypes are recognized, causes respiratory infections worldwide. We developed a multi locus sequence typing (MLST) scheme to understand the population genetic structure and diversity of these species and to evaluate the association between genotype and disease. Results A collection of 26 strains of C. trachomatis of different serovars and clinical presentation and 18 strains of C. pneumoniae were included in the study. For comparison, sequences of C. abortus, C. psittaci, C. caviae, C. felis, C. pecorum (Chlamydophila), C. muridarum (Chlamydia) and of Candidatus protochlamydia and Simkania negevensis were also included. Sequences of fragments (400 – 500 base pairs) from seven housekeeping genes (enoA, fumC, gatA, gidA, hemN, hlfX, oppA) were analysed. Analysis of allelic profiles by eBurst revealed three non-overlapping clonal complexes among the C. trachomatis strains, while the C. pneumoniae strains formed a single group. An UPGMA tree produced from the allelic profiles resulted in three groups of sequence types. The LGV strains grouped in a single cluster, while the urogenital strains were distributed over two separated groups, one consisted solely of strains with frequent occurring serovars (E, D and F). The distribution of the different serovars over the three groups was not consistent, suggesting exchange of serovar encoding ompA sequences. In one instance, exchange of fumC sequences between strains of different groups was observed. Cluster analyses of concatenated sequences of the Chlamydophila and Chlamydia species together with those of Candidatus Protochlamydia amoebophila and Simkania negevensis resulted in a tree identical to that obtained with 23S RNA gene sequences. Conclusion These data show that C. trachomatis and C. pneumoniae are highly uniform. The difference in genetic diversity between C. trachomatis and C. pneumoniae is in concordance with a later assimilation to the human host of the latter. Our data supports the taxonomy of the order of Chlamydiales.

Abstract Background It has been reported that Chlamydophila (C.) pneumoniae is involved in the initiation and promotion of asthma and chronic obstructive pulmonary diseases (COPD). Surprisingly, the effect of C. pneumoniae on airway function has never been investigated. Methods In this study, mice were inoculated intranasally with C. pneumoniae (strain AR39) on day 0 and experiments were performed on day 2, 7, 14 and 21. Results We found that from day 7, C. pneumoniae infection causes both a sustained airway hyperresponsiveness and an inflammation. Interferon-γ (IFN-γ) and macrophage inflammatory chemokine-2 (MIP-2) levels in bronchoalveolar lavage (BAL)-fluid were increased on all experimental days with exception of day 7 where MIP-2 concentrations dropped to control levels. In contrast, tumor necrosis factor-α (TNF-α) levels were only increased on day 7. From day 7 to 21 epithelial damage and secretory cell hypertrophy was observed. It is suggested that, the inflammatory cells/mediators, the epithelial damage and secretory cell hypertrophy contribute to initiation of airway hyperresponsiveness. Conclusion Our study demonstrates for the first time that C. pneumoniae infection can modify bronchial responsiveness. This has clinical implications, since additional changes in airway responsiveness and inflammation-status induced by this bacterium may worsen and/or provoke breathlessness in asthma and COPD.

The genomic similarities of several Chlamydia isolates were investigated by examining genomic amplified fragment length polymorphism fingerprints. Cluster analysis of selected isolates from all species showed groups other than those based on sequence data from single genes but was in agreement with available DNA-DNA hybridization data. Cluster analysis of amplified fragment length polymorphism fingerprints of representatives of all species gave suggestions for a grouping of chlamydiae based on the analysis of the whole genome.

A study was conducted to determine the existence of additional members of the order Chlamydiales. A polymerase chain reaction was designed to determine all known Chalmydiaceae sequences in one reaction. Primers were chosen to amplify a fragment of the 16S rRNA gene. DNA was then isolated from peripheral blood mononuclear cells of health controls. Experimental results indicated a novel lineage separated from the genus Chlamydia with four known Chlamydial species.

Abstract: Mouse models have been frequently used in the study of Chlamydia pneumoniae (also known as Chlamydophila pneumoniae) infections. This gram-negative obligate intracellular bacterium causes respiratory infections, followed by dissemination of the bacterium to various organs throughout the body, including cardiovascular tissues, supporting the current hypothesis of a relationship between C. pneumoniae and atherosclerosis. Recently, clinical trials evaluated the effect of antichlamydial antibiotics on secondary cardiovascular events. Although small studies showed some effect, the large WIZARD study did not confirm these results, and the role of antichlamydial antibiotics in prevention of secondary events was questioned. To address these issues, data obtained from mouse models were systematically reviewed here. C. pneumoniae infections showed atherogenic properties in mice that were reproducible and confirmed by different research groups. However, antibiotic therapy was of limited value in these mouse models. Antibiotic therapy effectively cleared the acute infection, but did not influence the atherogenic properties of C. pneumoniae unless the therapy was started early during the acute infection. The results summarized here may help to better understand the results of the clinical antibiotic trials. [Copyright &y& Elsevier]

Background. Lymphogranuloma venereum (LGV) is a sexually transmitted disease (STD) and is rare in the Western world. Recently, 3 men who have sex with men presented with LGV proctitis at the Erasmus Medical Center, Rotterdam, The Netherlands. We investigated a possible outbreak in a sexual network of men who have sex with men (MSM). Methods. After active case finding, a total of 15 men presented and were investigated. Serum antibody titers to Chlamydia trachomatis were determined. Urine and rectum specimens were analyzed by polymerase chain reaction (PCR) for the presence of C. trachomatis. C. trachomatis-positive specimens were genotyped to detect the specific C. trachomatis serovars. All subjects underwent routine STD screening. Sociodemographic, clinical, and endoscopic characteristics were evaluated. Results. Thirteen subjects had high immunoglobulin (Ig) G and IgA titers to C. trachomatis, suggesting an invasive infection. Rectal specimens of 12 subjects were PCR-positive for C. trachomatis. All urine specimens were negative. Genotyping revealed serovars L2 (n = 8) and L1 (n = 1). An ulcerative proctitis was found in all subjects obtaining sigmoidoscopy (n = 9). Eleven of 13 subjects with an LGV diagnosis were seropositive for human immunodeficiency virus (HIV), 6 had another concomitant STD, and 1 had recently acquired a hepatitis C virus infection. Further sexual contacts were reported from The Netherlands, Germany, Belgium, the United Kingdom, and France. Conclusions. We revealed an outbreak of LGV proctitis among MSM in The Netherlands. The ulcerous character favors transmission of HIV, other STDs, and blood-borne diseases. From a public health perspective, it seems important to increase the awareness of possible LGV in MSM with symptomatic proctitis. [ABSTRACT FROM AUTHOR]

Presents letters written to the editor concerning medical topics. Outcomes of angioplasty versus thrombolysis by hospital angioplasty volume; Timing of antiretroviral treatment initiation; Chlamydia trachomatis and cervical squamous cell carcinoma; Effect of Y2K on the timing of human mortality.

Objective: Atherosclerotic lesions are characterized by an immune mediated chronic inflammation. Seroepidemiological studies support a relationship between atherosclerotic disease and infection with C. pneumoniae; an association further endorsed by immunocytochemical and DNA directed studies. However, the question arises whether C. pneumoniae acts as a causal antigen, or is merely a bystander. For this reason we have analyzed the T lymphocyte population of carotid atherosclerotic plaques of symptomatic patients for their response against C. pneumoniae. Methods: T cell lines were generated from carotid endarterectomy tissues obtained from eight patients with symptomatic disease. The response of these T cell lines against C. pneumoniae elementary bodies was analyzed by 3H-thymidine incorporation. T cell clones were generated by limiting dilution from the cell lines of three patients and tested for antigen specificity in the same manner. Furthermore, cytokine profiles (Th1/Th0/Th2) were established by measuring the production of IFN-γ and IL-4. Results: Of the eight T-cell lines five responded to C. pneumoniae. Eighteen of 69 CD4-positive clones, generated from three patients with a positive T cell lines response, responded to C. pneumoniae also. The majority (17/18, 96%) of these clones showed a Th1 cytokine profile. Conclusion: These results show that in a subpopulation of symptomatic patients C. pneumoniae can activate T cells within atherosclerotic plaques suggesting that a C. pneumoniae enhanced proinflammatory Th1 response contributes to plaque destabilization in these patients. [ABSTRACT FROM PUBLISHER]

Objective: To study whether replacement of nosocomial ampicillin-resistant Enterococcus faecium (ARE) clones by vancomycin-resistant E. faecium (VRE), belonging to the same genetic lineages, increases mortality in patients with E. faecium bacteremia, and to evaluate whether any such increase is mediated by a delay in appropriate antibiotic therapy., Design: Retrospective, matched-cohort study., Setting: The study included 20 Dutch and Danish hospitals from 2009 to 2014., Patients: Within the study period, 63 patients with VRE bacteremia (36 Dutch and 27 Danish) were identified and subsequently matched to 234 patients with ARE bacteremia (130 Dutch and 104 Danish) for hospital, ward, length of hospital stay prior to bacteremia, and age. For all patients, 30-day mortality after bacteremia onset was assessed., Methods: The risk ratio (RR) reflecting the impact of vancomycin resistance on 30-day mortality was estimated using Cox regression with further analytic control for confounding factors., Results: The 30-day mortality rates were 27% and 38% for ARE in the Netherlands and Denmark, respectively, and the 30-day mortality rates were 33% and 48% for VRE in these respective countries. The adjusted RR for 30-day mortality for VRE was 1.54 (95% confidence interval, 1.06-2.25). Although appropriate antibiotic therapy was initiated later for VRE than for ARE bacteremia, further analysis did not reveal mediation of the increased mortality risk., Conclusions: Compared to ARE bacteremia, VRE bacteremia was associated with higher 30-day mortality. One explanation for this association would be increased virulence of VRE, although both phenotypes belong to the same well-characterized core genomic lineage. Alternatively, it may be the result of unmeasured confounding.