Assessment of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Pathogenic Bacteria from Respiratory Samples in Patients with Hospital-Acquired Pneumonia

Abstract

Rapid identification of the causative agent of hospital-acquired pneumonia (HAP) will allow an earlier administration of a more appropriate antibiotic and could improve the outcome of these patients. The aim of this study was to develop a rapid protocol to identify the main microorganisms involved in HAP by loop-mediated isothermal amplification (LAMP) directly from respiratory samples. First of all, a rapid procedure (<30 min) to extract the DNA from bronchoalveolar lavage (BAL), endotracheal aspirate (EA) or bronchoaspirate (BAS) was set up. A specific LAMP for Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii was performed with the extracted solution at 65 °C for 30–40 min. Overall, 58 positive BAL and 83 EA/BAS samples were tested. The limits of detection varied according to the microorganism detected. Validation of the LAMP assay with BAL samples showed that the assay was 100% specific and 86.3% sensitive (positive predictive value of 100% and a negative predictive value of 50%) compared with culture. Meanwhile for BAS/EA samples, the assay rendered the following statistical parameters: 100% specificity, 94.6% sensitivity, 100% positive predictive value and 69.2% negative predictive value. The turnaround time including sample preparation and LAMP was circa 1 h. LAMP method may be used to detect the most frequent bacteria causing HAP. It is a simple, cheap, sensitive, specific and rapid assay.

Evaluation of a Loop-Mediated Isothermal Amplification Assay to Detect Carbapenemases Directly From Bronchoalveolar Lavage Fluid Spiked With Acinetobacter spp.

“…We visualize the following workflow for diagnosis of hospital-acquired pneumonia (HAP): when the sample arrives to the clinical microbiology laboratory, rapid identification of the bacteria causing HAP is performed also using an in-house LAMP reaction approach ( Vergara et al, 2020a ); if A. baumannii is identified as the pathogen causing the infection, the method to detect specific carbapenemases in Acinetobacter described in this study is performed. However, if Enterobacterales is detected, the same approach can be applied ( Vergara et al, 2020b ).…”

Principles and Recent Advances in Biosensors for Pathogens Detection

“…Under isothermal conditions, the amplification is carried out between 332 K and 338 K. The amplicons employed in loopmediated isothermal amplification are combinations of various sizes of stem loop DNAs, which are used to amplify the target nucleic acid of interest. [142][143][144] This reaction can be enhanced by the use of one or two loop primers. The amplification of nucleic acid sequences typically produces an approximately 100-fold higher amplification product than the traditional PCR method.…”

“…With the naked eye, the reaction product obtained as a result of loop-mediated isothermal amplification can be seen using SYBR Green I dye instead of the traditional gel electrophoresis. [142][143][144] In the presence of amplicons, the colour of the solution turns green, while for blends without any amplification, it appears orange. This approach is extensively used to detect ultra-low concentrations of various microbial pathogens, including SARS-CoV-2 virus, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, Salmonella spp., Vibrio parahaemolyticus, Yersinia enterocolitica, Phalaenopsis orchid virus and Campylobacter jejuni.…”

Rapid Detection to Differentiate Hypervirulent Klebsiella pneumoniae (hvKp) From Classical K. pneumoniae by Identifying peg-344 With Loop-Mediated Isothermal Amplication (LAMP)

“…The reaction mixture was composed of 3 primers, viz., a pair of inner primers (FIP and BIP, 80pmol of each), a pair of outer primers (F3 and B3, 5pmol of each), a pair of loop primers (LB and LF, 20 pmol of each), which would speed up the LAMP reaction, 1 µL of Bst DNA polymerase (8 units), 2 µL of DNA template, and 12.5 µL of the reaction mix available in the kit. The LAMP reaction was carried out under isothermal conditions, at 65 • C, for 60 min and then stopped at 85 • C for 5 min (Vergara et al, 2020).…”