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BackgroundIn 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.FindingsWe 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.ConclusionsWe developed an improved PCR method for specific detection of B. pertussis, B. parapertussis, B. holmesii, and B. bronchiseptica.

Herpes simplex virus type 1 (HSV-1)-related disease ranges from a localized, self-limiting illness to fatal disease in immunocompromised individuals. The corneal disease herpetic keratitis may develop after reactivation of a latent virus or reinfection with an exogenous herpesvirus. Molecular analysis of the virus involved may allow distinction between these two options. The HSV-1 genome contains several hypervariable regions that vary in numbers of reiterating regions (reiterations I to VIII [ReI to ReVIII]) between individual strains. Twenty-four HSV-1 clones, derived by subcloning of HSV-1 (strain F) twice in limiting dilutions, were tested in a PCR-based assay to analyze the stabilities of ReI, ReIII, ReIV, and ReVII. ReI and ReIII proved to vary in size upon subcloning, whereas ReIV and ReVII were stable. Subsequently, 37 unrelated isolates and 10 sequential isolates from five patients, all with HSV-1-induced keratitis, were genotyped for ReIV and ReVII. Of the 37 unrelated samples, 34 (92%) could be discriminated, while the genotypes of the viruses in sequential samples were identical for each individual. Conclusively, the data show that the approach presented allows the rapid and accurate discrimination of HSV-1 strains in studies that address the transmission and pathogenesis of HSV-1 infections.

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.

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