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 allowan earlier administration of a more appropriate antibiotic and could improve the outcome of thesepatients. The aim of this study was to develop a rapid protocol to identify the main microorganismsinvolved 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 forStaphylococcusaureus,Escherichia coli,Klebsiella pneumoniae, Pseudomonas aeruginosa,Stenotrophomonas maltophiliaandAcinetobacter baumanniiwas 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 themicroorganism 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 valueof 50%) compared with culture. Meanwhile for BAS/EA samples, the assay rendered the followingstatistical 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 circa1 h. LAMP method may be used to detect the most frequent bacteria causing HAP. It is a simple,cheap, sensitive, specific and rapid assay.