Development of an antigen detection test kit (Melioidosis-ATK) for point-of-care diagnosis of melioidosis

ABSTRACT Melioidosis is a fatal infectious disease caused by the environmental bacterium Burkholderia pseudomallei . Although it is highly endemic in tropical regions, melioidosis remains underdiagnosed. The disease presents with diverse clinical manifestations, but diagnosis relies on time-consuming bacterial culture. Many patients with poor outcomes are referred from community hospitals, where culture facilities are unavailable. Early and accurate diagnosis is critical for timely treatment and improved outcomes. We developed an antigen detection test kit (Melioidosis-ATK) that targets the 6-deoxy-heptan capsular polysaccharide (CPS) of B. pseudomallei . The diagnostic performance of the assay was evaluated using 88 clinical samples, including blood culture broth (obtained from incubated blood culture bottles) ( N = 17), sputum ( N = 33), pus ( N = 17), urine ( N = 14), and other body fluids ( N = 7) from 88 patients with melioidosis, 204 samples from 195 patients with other bacterial infections, and 38 samples from 38 patients with negative blood culture. The assay demonstrated a limit of detection of 4.33 × 10³ CFU/mL for B. pseudomallei and 0.2 ng/mL for purified CPS. The Melioidosis-ATK demonstrated high diagnostic performance, with a sensitivity of 100% for blood culture broth, 84.9% for sputum, 88.2% for pus, 100% for urine, and 85.7% for other body fluids. The assay also exhibited a specificity of 100% across all sample types evaluated. The Kappa coefficient of agreement between the antigen test and culture for all clinical specimens ranged from 0.77 to 1.00. These findings suggest that the Melioidosis-ATK is a promising point-of-care (POC) diagnostic tool for use in rural hospitals, offering rapid, simple, equipment-free testing with high performance and has the potential to improve patient outcomes. IMPORTANCE Melioidosis is a life-threatening but underrecognized infectious disease caused by Burkholderia pseudomallei , which predominantly occurs in regions with limited diagnostic capacity. A definitive diagnosis typically relies on bacterial culture, which is slow, technically demanding, and often unavailable in resource-constrained settings. To address this problem, we developed a rapid antigen detection assay (Melioidosis-ATK) that targets the 6-deoxy-heptan capsular polysaccharide (CPS) of B. pseudomallei and evaluated its performance using multiple clinical specimen types. The test demonstrated high sensitivity and specificity and can be performed as a point-of-care (POC) test. By enabling prompt diagnosis and timely initiation of effective therapy, this assay represents a practical tool to enhance clinical management and reduce disease-associated mortality in endemic regions.