Molecular Diagnosis of Urinary Tract Infections by Semi-Quantitative Detection of Uropathogens in a Routine Clinical Hospital Setting.

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.

Screening rectal swabs for carbapenemase genes.

In an outbreak setting, we screened 16,296 samples from 3,644 patients by PCR for the presence of blaOXA-48, blaVIM, blaIMP, blaNDM, and blaKPC. The blaOXA-48 gene was found in samples from 43 patients infected with 9 different species of Enterobacteriaceae. Five patients had Pseudomonas aeruginosa isolates containing blaVIM. The negative predictive value of screening was 100%, and the positive predictive value was 86%.

Multi-centre evaluation of real-time multiplex PCR for detection of carbapenemase genes OXA-48, VIM, IMP, NDM and KPC.

BACKGROUND: Resistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae. To prevent hospital transmission due to unnoticed carriage of carbapenemase producing micro-organisms in newly admitted patients, or follow-up of patients in an outbreak setting, a molecular screening method was developed for detection of the most prevalent carbapenemase genes; blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC. METHODS: A real-time multiplex PCR assay was evaluated using a collection of 86 Gram negative isolates, including 62 carbapenemase producers. Seven different laboratories carried out this method and used the assay for detection of the carbapenemase genes on a selection of 20 isolates. RESULTS: Both sensitivity and specificity of the multiplex PCR assay was 100%, as established by results on the strain collection and the inter-laboratory comparisons. CONCLUSIONS: In this study, we present a multiplex real-time PCR that is a robust, reliable and rapid method for the detection of the most prevalent carbapenemases blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC, and is suitable for screening of broth cultured rectal swabs and for identification of carbapenemase genes in cultures.

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.

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.

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.