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

Corneal herpes simplex virus type 1 superinfection in patients with recrudescent herpetic keratitis

PURPOSE: Herpetic keratitis is a common sequel of a corneal infection with herpes simplex virus (HSV)-1. Recrudescent herpetic keratitis (RHK) may result in irreversible damage to the cornea. Recurrences may be caused by reactivation of endogenous HSV-1 or reinfection with exogenous HSV-1. The objective of this study was to determine the incidence and risk factors involved of HSV-1 superinfection in patients with RHK. METHODS: From 30 patients with RHK, sequential corneal HSV-1 isolates were genotyped by PCR amplification of the hypervariable regions located within the HSV-1 genes US1, US10/11, and US12. The clinical data from the patients obtained retrospectively were: ophthalmologic history, clinical picture during recurrences, number and time points of penetrating keratoplasty (PKP), and steroid or acyclovir treatment. RESULTS: Whereas the sequential corneal HSV-1 isolates of 19 (63%) of 30 patients had the same genotype (designated as group 1), the sequential isolates of 11 patients (37%) were genetically different (designated as group 2). Among the clinical data analyzed, only the time point of PKP was significantly different between the patient groups. A

Review of a major epidemic of methicillin-resistant Staphylococcus aureus: The costs of screening and consequences of outbreak management

Background: A major outbreak of methicillin-resistant Staphylococcus aureus (MRSA) occurred in locations C and Z of our hospital and lasted for several years. It affected 1,230 patients and 153 personnel. Methods: Outbreak management was installed according to the Dutch "search and destroy" policy. A rapid, high-throughput method for molecular screening of potential MRSA carriers was implemented. Outbreak isolates were retrospectively genotyped by pulsed field gel electrophoresis. Costs of molecular screening were compared with screening by culture. Results: Genotyping results revealed 4 distinct epidemic MRSA clones. Three were present in hospital C. Because of a merger of hospitals, these clones spread to hospital Z. Another clone of MRSA affected other health care-related institutions in the region. Because of the implementation of strict containment measures of the "search and destroy" policy, the annual number of tests decreased from 100,000 to 18,000. The disposables and reagents used in polymerase chain reaction technology are more expensive than those of conventional methods. However, the clinical and economic benefits of fast results in regard to expenses of the hospital clearly outweigh the higher costs of screening. Conclusion: The implementation of a rapid, high-throughput molecular screening system greatly contributed to the effectiveness of strict containment measures of the "search and destroy" policy. The major epidemic clones of MRSA in the outbreak were eradicated by this strategy

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

Recombination in transformation of bacillus subtilis

This thesis describes experiments aimed at advancing our knowledge concerning the process of transformation in Bacillus subtilis. To this purpose mutants, impaired in transformation, were isolated. Both in these mutants and in the highly - transformable wild - type synchronously transforming at low temperature the fate of transforming DNA was studied. ... Zie: Summary

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