A real-time PCR assay with improved specificity for detection and discrimination of all clinically relevant Bordetella species by the presence and distribution of three Insertion Sequence elements

<p>Abstract</p> <p>Background</p> <p>In Dutch laboratories molecular detection of <it>B. pertussis </it>and <it>B. parapertussis </it>is commonly based on insertion sequences <it>IS</it>481 and <it>IS</it>1001, respectively. Both IS elements are more widely spread among <it>Bordetella </it>species. Both <it>Bordetella </it><it>holmesii</it>, and <it>B. bronchiseptica </it>can harbour <it>IS</it>481. Also, <it>IS</it>1001 is found among <it>B. bronchiseptica</it>. <it>IS</it>481, and <it>IS</it>1001 based PCR thus lacks specificity when used for detection of specific <it>Bordetella spp</it>.</p> <p>Findings</p> <p>We designed a PCR based on <it>IS</it>1002, another IS element that is present among <it>Bordetella </it>species, and exploited it as a template in combination with PCR for <it>IS</it>481, and <it>IS</it>1001. In combining the PCRs for <it>IS</it>481, <it>IS</it>1001, and <it>IS</it>1002, and including an inhibition control, we were able to detect and discriminate all clinically relevant <it>Bordetella </it>species.</p> <p>Conclusions</p> <p>We developed an improved PCR method for specific detection of <it>B. pertussis</it>, <it>B. parapertussis, B. holmesii, and B. bronchiseptica.</it></p

Multi locus sequence typing of Chlamydiales: clonal groupings within the obligate intracellular bacteria Chlamydia trachomatis

<p>Abstract</p> <p>Background</p> <p>The obligate intracellular growing bacterium <it>Chlamydia trachomatis </it>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 <it>Chlamydophila pneumoniae</it>, 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.</p> <p>Results</p> <p>A collection of 26 strains of <it>C. trachomatis </it>of different serovars and clinical presentation and 18 strains of <it>C. pneumoniae </it>were included in the study. For comparison, sequences of <it>C. abortus, C. psittaci</it>, <it>C. caviae</it>, <it>C. felis</it>, <it>C. pecorum </it>(<it>Chlamydophila</it>), <it>C. muridarum </it>(<it>Chlamydia</it>) and of <it>Candidatus protochlamydia </it>and <it>Simkania negevensis </it>were also included. Sequences of fragments (400 – 500 base pairs) from seven housekeeping genes (<it>enoA</it>, <it>fumC</it>, <it>gatA</it>, <it>gidA</it>, <it>hemN</it>, <it>hlfX</it>, <it>oppA</it>) were analysed. Analysis of allelic profiles by eBurst revealed three non-overlapping clonal complexes among the <it>C. trachomatis </it>strains, while the <it>C. pneumoniae </it>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 <it>ompA </it>sequences. In one instance, exchange of <it>fumC </it>sequences between strains of different groups was observed. Cluster analyses of concatenated sequences of the Chlamydophila and Chlamydia species together with those of <it>Candidatus Protochlamydia amoebophila </it>and <it>Simkania negevensis </it>resulted in a tree identical to that obtained with 23S RNA gene sequences.</p> <p>Conclusion</p> <p>These data show that <it>C. trachomatis </it>and <it>C. pneumoniae </it>are highly uniform. The difference in genetic diversity between <it>C. trachomatis </it>and <it>C. pneumoniae </it>is in concordance with a later assimilation to the human host of the latter. Our data supports the taxonomy of the order of <it>Chlamydiales</it>.</p

Detection of novel chromosome-SCCmec variants in Methicillin Resistant Staphylococcus aureus and their inclusion in PCR based screening

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

平成25年度KALCSプログラム事業の概要

BACKGROUND:The objective of our study was the development of a semi-quantitative real-time PCR to detect uropathogens. Two multiplex PCR reactions were designed to detect Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Proteus mirabilis, Enterococcus faecalis, and Pseudomonas aeruginosa. 16S based PCR was performed in parallel to detect Gram-positive and Gram-negative bacteria. Firstly to identify non-targeted agents of infection in the same urine specimen, and secondly to quantify background flora. The method was evaluated in comparison with standard bacterial culture, and a commercial PCR kit for detection of uropathogens. FINDINGS:Analysis with a known panel of 116 clinical isolates yielded a PCR specificity of 100%. Analysis of urine specimens from 211 patients revealed a high correlation of PCR Cq values with both culture positivity and quantity. Concordance between PCR and culture was 98% when both methods yielded results. PCR was found to be more sensitive than culture. With a cut-off Cq value of 33, the negative predictive value of PCR was 94%. The 16S PCR confirmed most results. One specimen was positive by 16S PCR suggesting another cause of infection not detected by the specific PCR assays. CONCLUSION:We conclude that it is feasible to detect and identify uropathogens by multiplex real-time PCR assay

Molecular evidence for association of chlamydiales bacteria with epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer).

Epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer), previously associated with chlamydial bacterial infection using ultrastructural analysis, was further investigated by using molecular and immunocytochemical methods. Morphologically, all three species showed epitheliocystis cysts in the gills, and barramundi also showed lymphocystis cysts in the skin. From gill cysts of all three species and from skin cysts of barramundi 16S rRNA gene fragments were amplified by PCR and sequenced, which clustered by phylogenetic analysis together with other chlamydia-like organisms in the order Chlamydiales in a lineage separate from the family Chlamydiaceae. By using in situ RNA hybridization, 16S rRNA Chlamydiales-specific sequences were detected in gill cysts of silver perch and in gill and skin cysts of barramundi. By applying immunocytochemistry, chlamydial antigens (lipopolysaccharide and/or membrane protein) were detected in gill cysts of leafy seadragon and in gill and skin cysts of barramundi, but not in gill cysts of silver perch. In conclusion, this is the first time epitheliocystis agents of leafy seadragon, silver perch and barramundi have been undoubtedly identified as belonging to bacteria of the order Chlamydiales by molecular methods. In addition, the results suggested that lymphocystis cysts, known to be caused by iridovirus infection, could be coinfected with the epitheliocystis agent

Detection of novel chromosome-SCC<it>mec </it>variants in Methicillin Resistant <it>Staphylococcus aureus </it>and their inclusion in PCR based screening

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.</p

Analysis of results of PCR and the Seegene kit.

<p>Analysis of results of PCR and the Seegene kit.</p

Amplification of serial dilutions of bacterial cells.

<p>Standard curves (top) of PCRs for detection of <i>Citrobacter/Enterobacter spp</i>. (yellow), <i>Klebsiella spp</i>. (blue), <i>E</i>.<i>coli</i> (red), <i>E</i>.<i>faecalis</i> (brown), <i>P</i>.<i>mirabilis</i> (dark blue), and <i>P</i>.<i>aeruginosa</i> (purple). The Cq values per dilution are shown below the figure. Cq values that correspond with 10³ and 10⁵ cfu/ml were underlined in blue and red, respectively.</p

Graphic showing the correlation between positive cultures and PCR Cq values.

<p>Comparison of PCR and culture results for <i>E</i>. <i>coli</i>. Cq values of PCR are depicted in the blue bars ranging from Cq22-Cq40 on the left axis. The red bars depict positive cultures with growth of 10³, 10⁵, and >10⁵ cfu/ml on the right axis. Low Cq values correspond with high loads of micro-organisms, and thus with larger yield of positivity and quantities of cultures.</p