MULTIPLEX REAL TIME PCR FOR DETECTION OF COMMON CARBAPENEMASE GENES FROM DIFFERENT CLINICAL SPECIMENS
Soheir Abd El-Rahman, Yasser Ismail, Naglaa Fathy Alhusseini* and Maha Osman
Departments of Clinical and Chemical Pathology and *Medical Biochemistry, Faculty of Medicine, Benha University, Egypt
Background: Carbapenem resistant pathogens cause infections associated with significant morbidity and mortality.
Objective: This study evaluates the use of Multiplex PCR for rapid detection of most common carbapenemase genes among carbapenem resistant Gram negative bacilli. The assay was performed on clinical swabs which were processed for PCR analysis without prior culturing of bacterial species, allowing the output of results in hours from reception of swabs.
Material and Methods: A total of duplicate 250 clinical swabs were collected from intensive care units to isolate nosocomial gram negative bacilli. Of them, 55 duplicate swabs were selected according to reduced susceptibility to one or more carbapenems by disk diffusion or positive culture on Chromagar-KPC (Chromagar of Dr.A. Rambach).Of the 55 duplicate swabs, First swab was cultured and identified by phenotypic method, modified Hodge test (MHT), combined disc test (CDT) and were tested for the presence of carbapenemases (blaNDM-1, blaVIM, blaIMP ,blaGES, blaKPC and blaOXA genes) by Multiplex real-time PCR after isolation the other one was processed directly by Multiplex real-time PCR without culture step.
Results: Each one of the six carbapenemase genes tested presented a different melting curve after PCR amplification. The melting temperature (Tm) analysis of the amplicons identified was as follows: blaIMP type (Tm 80.1°C), blaOXA-48 (Tm 81.6°C), blaNDM-1 (Tm 84°C), blaGES type (Tm 88.6°C), blaVIM type (Tm 90.3°C) and blaKPC type (Tm 91.6°C). No amplification was detected among the negative samples. Out of the 55 gram negative isolates, 28 (50.9%) were Klebsiella pneumoniae, 15 (27.3%) were Enterobacter spp., 2 (3.6%) were Aceintobacter, 6 (10.9%) were Pseudomonas, 2 (3.6%) were Proteus mirabilis and 2(4%) were Serratia. The most common resistance genes detected were blaVIM (44/55) (80%) followed by blaKPC (41/55) (75%), the blaOXA gene (15/55) (27.25%), the blaGES (6/ 55) (2%),the blaIMP (3/55) (0.5%)and blaNDM (3/55) (0.5%). Direct PCR processing of clinical swabs without culture step was (98%) sensitivity, (100%) specificity, (100%) PPV, (83.3%) NPV in comparison with PCR processing after culture and isolation.
Conclusion: Accurate detection of the genes related with carbapenemase production by Molecular methods like Multiplex PCR overcome the limitations of the phenotypic methods.