Environmental detection of Burkholderia pseudomallei and associated melioidosis risk: a molecular detection and case-control cohort study

Environmental exposure to Burkholderia pseudomallei, the causative agent of melioidosis, remains poorly characterised due to the limited sensitivity of conventional detection methods. This hinders accurate risk mapping and delays public health responses. Here, we developed CRISPR-BEEPs - a sensitive, equipment-light CRISPR-based assay - that demonstrated substantially improved sensitivity (93.5% vs 19.4%) and specificity (100% vs 98.0%) compared to conventional culture-based plate inspection techniques. We applied CRISPR-BEEPs to water samples collected from both natural and piped sources across 15,118 km{superscript 2} in northeast Thailand, including households of confirmed melioidosis patients and controls. B. pseudomallei was detected in 73.3% of groundwater samples, 32.4% of surface water, and 28.3% of piped water, with peak detection during the flood season. Importantly, the assays improved sensitivity enabled detection of a significant association between environmental B. pseudomallei detection within a 10 km radius of participants households and melioidosis risk (OR 2.74; 95% CI: 1.38- 5.48) - an association undetectable by conventional methods. These findings highlight major gaps in water treatment and distribution infrastructure and demonstrate the value of high-resolution environmental diagnostics. Strengthening water sanitation and surveillance systems is essential for mitigating melioidosis transmission and addressing the broader burden of waterborne diseases in vulnerable settings.