Utilização da Amplificação Isotérmica Mediada por Loop (LAMP) na detecção de genes de resistência em Enterobactérias produtoras de Carbapenemases

Abstract

Various methods have been proposed for identifying resistance genes in multiresistant bacteria. The most used for detecting acquired resistance is amplifying the target DNA through polymerase chain reaction (PCR). In small laboratories, acquiring this equipment is expensive, often making implementing the method unfeasible. Therefore, alternative methods for genotyping have been proposed, more notably the loop-mediated isothermal amplification (LAMP), which presents both a low operational cost and a good specificity and sensitivity for the alternative detection of bacterial resistance genes. The present study aimed to establish LAMP usage to detect the blaKPC gene by producing carbapenemase-type beta-lactamase. Seventy-six samples of Enterobacteriaceae isolated from clinical samples of patients of the Hospital Escola UFPel-Ebserh were isolated, phenotypically identified, and separated according to their resistance profile. The most frequent resistance marker detected was extended-spectrum beta-lactamases (ESBL) production, 51.32% (39/76), followed by the co-production of ESBL and carbapenemases, 35.53% (27/76), and carbapenemases alone, 11.84% (9/76). We found only one case of co-production of KPC+MBL, 1.31% (1/76). The 76 samples were analyzed by qPCR for the blaKPC and blaNDM-1 genes, obtaining 47.36% (36/76) positivity for the blaKPC gene and 18.42% (14/76) for the blaNDM-1 gene. Of the 36 samples that tested positive for the blaKPC gene, 25 agreed with the phenotypic test and were used to optimize the LAMP assays. In the LAMP assay, we verified the substantial concordance rate of LAMP with qPCR, with the Kappa index value equal to 0.761. Sensitivity was evaluated from the same dilutions used in qPCR. The sensitivity result showed that LAMP could detect 1.5 CFU mL-1 , ten times more sensitive than qPCR. Our findings indicate that the LAMP assay can be used in the bacteriology sector and confirm carbapenemases in routines and epidemiological studies.