Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates.
BACKGROUND
Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE).


METHODS
116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes.


RESULTS
Among the carbapenem-resistant isolates the genes bla KPC, bla VIM, bla NDM or bla OXA were detected. Both RT-PCR assays detected all bla KPC, bla VIM and bla NDM in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials.


CONCLUSION
The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.
['Smiljanic|M|M|', 'Kaase|M|M|', 'Ahmad-Nejad|P|P|', 'Ghebremedhin|B|B|']
D000900:Anti-Bacterial Agents D001426:Bacterial Proteins D004755:Enterobacteriaceae D004756:Enterobacteriaceae Infections D006090:Gram-Negative Bacteria D016905:Gram-Negative Bacterial Infections D006801:Humans D008826:Microbial Sensitivity Tests D060888:Real-Time Polymerase Chain Reaction D001618:beta-Lactamases
Carbapenemases RT-PCR Acinetobacter baumannii MDR gram-negative bacteria