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Comparative Study on DNA Amplification Methods for Detection of Carbapenem-Resistant Enterobacteriaceae (CRE)

Background: Carbapenem-resistant Enterobacteriaceae (CRE) is a resistant group of gram-negative bacteria that produces carbapenemase destroying carbapenem molecules causing drug resistances. Among them, the incidence of New Delhi metallo-beta-lactamases (blaNDM-1), a metallocarbapenemases or class B carbapenemases or metallo-β-lactamases (MBL), are increasing worldwide.

Objective: To focused on comparative study on DNA amplification methods for detection of CRE in terms of analytical sensitivity and specificity.

Materials and Methods: CRE strains (Escherichia coli, Escherichia cloacae, Citrobacter freudii, and Klebsiella pneumonia) were collected from HRH Maha Chakri Sirindhorn Medical Center, Srinakharinwirot University, Nakhon Nayok, Thailand. All specimens were initially screened for CRE isolates by using carbapenem disks inhibition test. The polymerase chain reaction (PCR), loop mediated isothermal amplification (LAMP) and recombinant polymerase amplification (RPA) assays were further employed for identification of drug resistance blaNDM-1 gene among CRE isolates. The analytical sensitivity and specificity of the three amplification assays were compared and analyzed.

Results: The analytical sensitivity test of PCR, LAMP and RPA assays revealed that the limit of detection were 0.74 ng/μL, 7.4 pg/μL, and 0.74 ng/μL, respectively. Specificity test demonstrated that all three assays showed no cross hybridization to the other 15 related bacterial isolates.

Conclusion: The data pointed out that LAMP assay was 100 times more sensitive than PCR and RPA, which could be suitable for application as early detection test for carbapenem resistant bacteria. Hence, the physician can select the proper antibiotics to treat the CRE infectious bacteria, which can reduce morbidity and mortality rates in patients. In the future, LAMP will be combined with lateral flow dipstick (LFD) to improve the efficacy of the assay as not only the early detection test with high sensitivity and specificity for carbapenem resistant bacteria, but also as the convenient and rapid screening test.

Keywords: Enterobacteriaceae; Carbapenem; Carbapenem-resistant Enterobacteriaceae (CRE); New Delhi metallo-beta-lactamase (blaNDM-1)