Exploiting the Clinical Potential of a Chemiluminescent Probe for <i>Mycoplasma pneumoniae</i> Diagnosis

<i>Mycoplasma pneumoniae</i> (MP) is a leading pathogen responsible for community-acquired pneumonia in the whole population, posing a life-threatening risk in severe disease. Current diagnostic methods, including culture, serology, and PCR, are limited by low sensitivity, delayed results, or operational complexity, highlighting the urgent need for rapid and reliable alternatives. Critically, MP metabolism produces hydrogen peroxide (H₂O₂), a key virulence factor, making it a promising biomarker for MP infection. Compared to other optical approaches, chemiluminescence-based one offers a powerful alternative for pathogen detection due to their high sensitivity, low cost, and rapid response. Herein, we report the first application of a chemiluminescent probe (MPCL) based on the established phenoxy-dioxetane scaffold with a 4-nitro-α-ketoamide trigger for direct MP detection. Our study demonstrated the practical value of this probe in distinguishing MP from other respiratory pathogens through quantitative H₂O₂ detection. MPCL enabled sensitive quantification and real-time imaging of H₂O₂ in MP-infected mice, and, when applied to clinical respiratory samples, achieved excellent diagnostic performance, with ROC analysis showing an area under the curve of 0.992 (95% CI: 0.974-1.000), 100% sensitivity, and 96.5% specificity. These results establish MPCL as a practical chemiluminescent tool with strong translational potential for rapid, low-cost, and reliable diagnosis of MP infections in both clinical and preclinical settings.