ABSTRACT
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%.
Subject(s)
Bacterial Proteins/analysis , Enterobacteriaceae/enzymology , Gram-Negative Bacterial Infections/microbiology , Mass Screening/methods , Polymerase Chain Reaction/methods , Pseudomonas aeruginosa/enzymology , Rectum/microbiology , beta-Lactamases/analysis , Bacterial Proteins/genetics , Disease Outbreaks , Enterobacteriaceae/genetics , Gram-Negative Bacterial Infections/diagnosis , Gram-Negative Bacterial Infections/epidemiology , Humans , Molecular Diagnostic Techniques/methods , Predictive Value of Tests , Pseudomonas aeruginosa/genetics , beta-Lactamases/geneticsABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , Carbapenems , Drug Resistance, Bacterial/genetics , Enterobacteriaceae/genetics , Multiplex Polymerase Chain Reaction , beta-Lactamases/genetics , Humans , Real-Time Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
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.