Insights from the WHO Tricycle-aligned Fleming Fund EQASIA External Quality Assessment (EQA) programme detecting ESBL-, AmpC- and carbapenemase-producing Escherichia coli in South and South-East Asia

Objectives: The increasing prevalence of antimicrobial resistance (AMR) in Escherichia coli, notably those producing AmpC, ESBL and carbapenemases among food-producing animals, poses a significant public health threat, especially in low- and middle-income countries. With support from the Fleming Fund and in alignment with the WHO, FAO and WOAH tripartite Tricycle project, the EQASIA Matrix External Quality Assessment (EQA) scheme aimed at evaluating laboratories’ proficiency from different One Health (OH) sectors in detecting resistant E. coli from food-like matrices in South and South-East Asia. Methods: Between 2021 and 2024, four Matrix EQA rounds were implemented across OH sectors. Each round involved four lyophilized simulated meat samples spiked with well-characterized E. coli strains expressing β-lactam resistance phenotypes. Participating laboratories performed selective isolation, species identification, antimicrobial susceptibility testing (AST) and phenotypic classification using routine methods. Results were submitted via a secure online platform and evaluated against expected outcomes, using CLSI-based interpretive criteria and a standardized error categorization system. Results: Twenty-one laboratories from 10 countries participated across the four rounds. Accuracy in E. coli identification declined from 75% in 2021 to 23.1% in 2024. Only one laboratory achieved full identification accuracy in any round. AST performance showed inter-laboratory variation, with deviation rates ranging from 0.5% to 41%. Misclassification of resistance phenotypes, particularly AmpC and combined ESBL + AmpC, was common. Quality control testing improved modestly over time but remained incomplete. Conclusions: The Matrix EQA revealed critical insights into regional laboratory capacity and highlighted persistent technical gaps in detecting complex resistance mechanisms from food matrices. With the conclusion of the Fleming Fund, maintaining these gains will require sustained national investment, EQA integration into AMR strategies and continued regional coordination to support Tricycle-aligned surveillance.