Detection of Streptococcus pneumoniae in Urine by Loop-Mediated Isothermal Amplification

Abstract

Objective To assess the loop-mediated isothermal amplification (LAMP) to detect cell-free DNA from Streptococcus pneumoniae in urine samples from children with pneumococcal pneumonia.

Methods LAMP reactions using four primers (backward inner primer, forward inner primer, B3, and F3) targeting conserved regions of the S. pneumoniae ply gene and DNA from the recombinant plasmid pTrc99A-ply were optimized for temperature (65 degrees C) and MgSO4 concentration (8mM) conditions. Urine samples from 71 patients with symptoms of pneumonia and from 17 healthy children were tested side by side using the isothermal methodology LAMP and the commercial urinary antigen test, BinaxNOW S. pneumoniae assay. Percentages of sensitivity, specificity, positive predictive value (PPV), negative predictive value, and positive (LR) were calculated to compare both tests.

Results The specificity of the LAMP reaction was confirmed against several species of bacteria and yeast that can cause pneumonia or urine infections. The suitability of the LAMP assay was evaluated in urine samples from 71 patients and 17 healthy children. All patients (100%) with confirmed pneumococcal pneumonia were positive for the LAMP assay. Among patients with possible/probable pneumonia, 74.1% were identified as positive using the LAMP test. Notably, a higher specificity (95.4%), PPV ( 94.1%) and positive LR (21.7) were found compared with the urinary antigen test.

Conclusion The presence of S. pneumoniae cell-free DNA in urine samples of pediatric patients can be used as a specific diagnostic biomarker for community-acquired pneumonia by using the LAMP methodology.