Evaluation of LAMP assay using phenotypic tests and conventional PCR for detection of bla NDM-1 and bla KPC genes among carbapenem-resistant clinical Gram-negative isolates

Abstract

Carbapenem-resistant pathogens cause infections associated with significant morbidity and mortality. This study evaluates the use of the loop-mediated isothermal amplification (LAMP) assay for rapid and cost-effective detection of bla NDM-1 and bla KPC genes among carbapenem-resistant Gram-negative bacteria in comparison with conventional PCR and existing phenotypic methods. A total of 60 carbapenem-resistant clinical isolates [Escherichia coli (15), Klebsiella pneumoniae (22), Acinetobacter baumannii (23)] were screened for the presence of carbapenemases (bla KPC and bla NDM-1) using phenotypic methods such as the modified Hodge test (MHT) and combined disc test (CDT) and molecular methods such as conventional PCR and LAMP assay. In all, 47/60 isolates (78.3 %) were MHT positive while 48 isolates were positive by CDT [46.6 % positive with EDTA, 30 % with 3′ aminophenylboronic acid (APB) plus EDTA and 1.6 % with APB alone]. Isolates showing CDT positivity with EDTA or APB contained bla NDM-1 and bla KPC genes, respectively. bla NDM-1 was present as a lone gene in 28 isolates (46.7 %) and present together with the bla KPC gene in 19 isolates (31.7 %). Only one E. coli isolate had a lone bla KPC gene. The LAMP assay detected either or both bla NDM-1 and bla KPC genes in four isolates that were missed by conventional PCR. Neither gene could be detected in 12 (20 %) isolates. The LAMP assay has greater sensitivity, specificity and rapidity compared to the phenotypic methods and PCR for the detection of bla NDM-1 and bla KPC. With a turnaround time of only 2–3 h, the LAMP assay can be considered a point-of-care assay.