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

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 <i>S. pneumoniae</i>, 1.7 ×10⁵ CFU/mL for <i>S. aureus</i>, 8.2 ×10³ CFU/mL for <i>H. influenzae,</i> and 1.27 ×10³ genome copies/reaction for <i>M. pneumoniae</i>. Primers designed to detect <i>K. pneumoniae</i> had high specificity and no cross-reactivity with a sensitivity of 1.5 × 10⁴ CFU/mL. Detection times over 45<b>-</b>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.