Exhaled acetylcholine in breath condensate as a non-invasive biomarker of asthma control: Insights from patients treated with extrafine single-inhaler triple therapy.

A substantial proportion of patients with asthma remain uncontrolled despite high-dose ICS/LABA therapy, often due to persistent small-airway dysfunction and heightened cholinergic activation. Acetylcholine (ACh) produced by neuronal and non-neuronal airway cells amplifies inflammation, bronchoconstriction, and remodelling. Exhaled breath condensate (EBC) provides a non-invasive means to sample the distal airways, but ACh has never been quantified in EBC in asthma. Extrafine single-inhaler triple therapy (SITT) may modulate cholinergic activity through muscarinic blockade and improved small-airway deposition. To determine whether ACh concentration in EBC (ACh-EBC) distinguishes patients with not-well-controlled asthma on high-dose ICS/LABA from those achieving good control under extrafine SITT, and to evaluate its relationship with functional and inflammatory markers. In this cross-sectional observational study, 22 adults with asthma managed at the University Hospital of Bari were stratified by asthma control and treatment: uncontrolled asthma on high-dose ICS/LABA (n = 15) and controlled asthma on extrafine SITT (n = 7), alongside healthy controls (n = 7). ACh-EBC was quantified using a competitive high-sensitivity ELISA (A3574). Secondary assessments included FeNO50/FeNO350, spirometry, plethysmography, and impulse oscillometry (IOS). Group comparisons, ROC analysis, and discriminant analysis were performed. ACh-EBC exhibited a clear gradient: highest in uncontrolled ICS/LABA patients (3.23 pg), intermediate in SITT-controlled patients (2.97 pg), and lowest in healthy controls (2.75 pg; p = 0.001). Fres was significantly higher in uncontrolled asthma (18.80 vs. 13.50 Hz; p = 0.037), while FeNO50 was also elevated (p = 0.037). ROC analysis showed strong discriminatory accuracy for ACh-EBC (AUC 0.867; p = 0.007), with an optimal threshold of 3.000 pg. A multivariable discriminant model combining oscillometric and biochemical markers achieved 77.3% cross-validated accuracy. ACh-EBC is a promising non-invasive biomarker reflecting cholinergic activation and small-airway dysfunction in asthma. Elevated ACh-EBC identifies patients remaining uncontrolled on ICS/LABA, whereas lower levels in those receiving extrafine SITT mirror improved control and reduced cholinergic signalling. Integration of ACh-EBC with FeNO and IOS indices may support precision-medicine approaches aimed at identifying cholinergic-driven asthma and optimizing escalation to triple therapy.