181. Loop-mediated Isothermal Amplification Assay for ß-lactamase Identification on Clinical Isolates of Pseudomonas aeruginosa
Session: Poster Abstract Session: Diagnostics: Bacteriology, Sequencing, and Resistance
Thursday, October 27, 2016
Room: Poster Hall
Background: Rapid evaluation of antimicrobial susceptibility is of great clinical importance in the treatment of nosocomial Pseudomonas aeruginosa infections, which increasingly carry carbapenemases and metallo-beta-lactamases. We developed a loop-mediated isothermal amplification (LAMP)-based assay to detect six ß-lactamase genes (KPC, GES, NDM, IMP, VIM and OXA-48). We evaluated this new assay using previously genotypically and phenotypically characterized clinical P. aeruginosa isolates that collected between 2004 and 2012 from diverse geographic locations including Argentina, Brazil, China, Colombia, Croatia, France, Germany, Greece, India, Philippine, Mexico, Romania, and Spain (Antimicrob. Agents Chemother 2015).

Methods: The LAMP-based assay was evaluated using reference strains having each of six ß-lactamase genes; including Klebsiella pneumoniae, Escherichia coli, P. aeruginosa, and Acinetobacter bereziniae. A collection of 47 clinical P. aeruginosa isolates, previously fully characterized by next-generation sequencing (NGS) were used for evaluation of our new LAMP method. The results were compared with those obtained using the conventional PCR method (J Antimicrob Chemother 2012).

Results: The LAMP assay was able to detect as few as 10 copies of the gene in a sample, which was comparable to the conventional PCR method. The LAMP assay correctly detected ß-lactamase genes in all collected clinical isolates (47/47). In contrast, PCR did not detect four IMP ß-lactamase genes (IMP-4, 13, 15 and 18) in the clinical isolates (43/47). Among those PCR failed to detect, one isolate was susceptible to carbapenem and three were carbapenem-resistant isolates. Compared to the NGS results; the sensitivity and specificity of the IMP ß-lactamase genes LAMP assay were 100% (4/4) and 100% (43/43), whereas those of the conventional PCR were 0% (0/4) and 100% (43/43).

Conclusion: The established LAMP assay proved to be an appropriate tool for the detection of six different ß-lactamase genes. The test demonstrated higher sensitivity and similar specificity compared with conventional PCR. The LAMP needs the minimum technology for the assay. The test would be useful for routine simple confirmation of ß-lactamase producing P. aeruginosa.

Mitsuko Seki, DDS, PhD1, Daisuke Omagari, DDS, PhD1, Paul Kilgore, MD, MPH2, Dong Wook Kim, PhD3, Tatsushi Adachi, MSc4, Robert Mclaughlin, PhD5, Humphrey Gardner, MD FCAP5, Abdulbaset Salim, MBChB, MPH2 and Satoshi Hayakawa, MD, PhD1, (1)Nihon University, Tokyo, Japan, (2)Wayne State University, Detroit, MI, (3)Hanyang University, Ansan, South Korea, (4)Kaneka Co.ltd, Takasago, Japan, (5)AstraZeneca, Waltham, MA

Disclosures:

M. Seki, Kaneka Co. Ltd: Collaborator , Research grant

D. Omagari, None

P. Kilgore, None

D. W. Kim, None

T. Adachi, Kaneka Co. Ltd: Employee , Salary

R. Mclaughlin, Astrazeneca: Employee , Salary

H. Gardner, Astrazeneca: Employee , Salary

A. Salim, None

S. Hayakawa, Kaneka Co. Ltd: Collaborator , Research grant

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