Development and optimization of an easy to interpret loop-mediated isothermal amplification (LAMP) assay for the identification of bacterial pathogens causing childhood pneumonia.

INTRODUCTION: Pneumonia remains the leading infectious cause of death in children under five, especially in low-resource settings. Reducing mortality requires rapid, accessible, and reliable diagnostic tools. In this regard, the loop-mediated isothermal amplification (LAMP) technique has emerged as a fast and efficient alternative for simple pathogen detection. This study aimed to standardise and optimize a LAMP assay for detecting the main bacteria causing pneumonia in children, including Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Klebsiella pneumoniae, and Mycoplasmoides pneumoniae using a simple visual readout.

METHODS: Several fluorescent and colorimetric dyes were evaluated to identify those providing a clear readout visible to the naked eye. Once achieved, detection conditions for each pathogen in the panel were optimized, and the feasibility of the assay was assessed using respiratory clinical samples, including both confirmed positives and negatives for the bacteria targeted in the panel.

RESULTS AND DISCUSSION: SYBR Safe, Calcein-Mn2+, and SYTO 9 alone did not show a clear differentiation between positive and negative reactions. In contrast, the combination of hydroxynaphthol blue (HNB) and SYTO 9 proved suitable, providing a clear visual readout to the naked eye after optimization of concentrations and reaction conditions. The selected concentrations were 341.25 μM HNB and 0.75 μM SYTO 9, which enabled clear and stable fluorescence-based visualization of LAMP results, remaining visible for several months. The technique showed low detection limits: 3.9 ×103 CFU/mL for S. pneumoniae, 1.7 ×105 CFU/mL for S. aureus, 8.2 ×103 CFU/mL for H. influenzae, and 1.27 ×103 genome copies/reaction for M. pneumoniae. Primers designed to detect K. pneumoniae had high specificity and no cross-reactivity with a sensitivity of 1.5 × 104 CFU/mL. Detection times over 45-50 min may suggest colonization instead of active infection. The evaluation of the technique using clinical samples demonstrated its potential feasibility and applicability in real-world clinical settings. Although standardized under laboratory conditions, this LAMP technique shows promise for detecting major pneumonia-causing bacteria in children and could be particularly valuable in low-resource settings. Its rapid, sensitive, and affordable nature may help improve diagnostics and reduce pneumonia-related mortality. However, larger clinical validation studies are needed to confirm its performance and real-world applicability.