Development of loop-mediated isothermal amplification (LAMP)-based assay for rapid detection of fosfomycin resistance
Nazareno Scaccia1,2,3, Inneke Marie van der Heijden Natário4,5, Letícia Silva Figueiredo4
1Centre for Antimicrobial Optimisation Network (CAMO-Net) Brazil, Departamento de Infectologia e Medicina Tropical, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Av. Dr. Enéas de Carvalho Aguiar, 470, SP, BR, 05403- 000, Cerqueira César, Sao Paulo, Brazil. nazareno.scaccia@usp.br.
Abstract:
Fosfomycin is an old treatment agent that has been reintroduced as a first-line antibiotic for the treatment of acute uncomplicated urinary tract infections caused by multidrug-resistant bacteria. However, resistance to fosfomycin is rising, and limited options for its detection are available. In this study, we developed a molecular technique based on loop-mediated isothermal amplification (LAMP) as a rapid and easy-to-perform method for the detection of the fosA resistance gene. LAMP primers were designed using the Primer Explorer V5 software. A total of 63 bacterial strains (34 Klebsiella pneumoniae and 29 Escherichia coli) previously characterized by whole genome sequencing, with different MLST and resistant to fosfomycin were used to evaluate the performance of the new LAMP assay. The fosA LAMP-based assay was standardized for optimum primer concentration and time of reaction. Its sensitivity and specificity were also characterized. LAMP products were detected by visual detection (positive reactions turned yellow while negative controls remained pink) and by electrophoresis. Positive LAMP reaction occurred with incubation at 65 °C for 40 min. Nevertheless, amplification products were already observed at 20 min. The overall sensitivity and specificity of the LAMP assay were 90.5% and 90.9%, respectively. For Klebsiella pneumoniae, sensitivity reached 100%, while specificity could not be determined due to the absence of negative samples. In Escherichia coli, sensitivity was 43% and specificity was 90.9%. The limit of detection of LAMP reaction was 8 pg/µL. To the best of our knowledge, this is the first study to develop a LAMP methodology to detect the fosA gene. This LAMP-based assay is a promising rapid molecular method for fosfomycin detection, which could be implemented for on-site analysis in resource-limited settings, with the potential to improve antibiotic therapy interventions for urinary tract infections.
