Development and international multicenter evaluation of a second-generation immunochromatography test for the serological diagnosis of melioidosis

Abstract

Melioidosis is a life-threatening infectious disease caused by Burkholderia pseudomallei. Early and accurate diagnosis is critical for timely treatment and improved outcomes. Although highly endemic in tropical regions, it remains underdiagnosed due to limitations of current diagnostic methods. Bacterial culture, the diagnostic gold standard, is time-consuming, prone to misidentified species, requires specialized laboratory facilities, and has low sensitivity. The indirect hemagglutination assay is also unreliable due to poor sensitivity and specificity. We developed a second-generation immunochromatography test (Hcp1-ICT) to detect anti-Hcp1 IgG antibodies against hemolysin co-regulated protein 1 of B. pseudomallei and evaluated its diagnostic performance in an international multi-center study. A total of 1,838 stored serum samples from 601 culture-proven melioidosis patients, 598 healthy individuals and 639 patients with non-melioidosis infections in Thailand, Lao PDR, Vietnam, Malaysia, Sri Lanka, Cambodia, and Australia were analyzed for diagnosis of melioidosis. The sensitivities in Thailand, Lao PDR, Vietnam, Malaysia, Sri Lanka, Cambodia, and Australia were 92%, 77%, 76%, 80%, 74%, 90%, and 48%, respectively. The specificities with healthy donor samples were 96%, 79%, 98%, 100%, 100%, 87%, and 100%, while the specificities for cases with samples from other infections other than melioidosis were 97%, 87%, 98%, 98%, 100%, 90%, and 94%, respectively. These findings indicate that the Hcp1-ICT is a promising point-of-care tool for serodiagnosis of melioidosis. However, its variable performance across regions underscores the need for prospective validation locally in various regions to optimize its diagnostic utility and facilitate implementation in both referral centers and resource-limited settings. Author summary: Melioidosis, caused by B. pseudomallei, is a frequently fatal infection with an estimated 165,000 cases and 89,000 deaths annually. Diagnosis is delayed in many tropical regions because bacterial culture, the current gold standard, is slow, requires specialized facilities, and has limited sensitivity. The widely used indirect hemagglutination assay used for serodiagnosis of melioidosis performs poorly in endemic areas due to low sensitivity and specificity. Thus, rapid, accurate, point-of-care serological tests are urgently needed to guide early treatment and reduce mortality. We previously developed a first-generation Hcp1 Immunochromatographic test (Hcp1-ICT) to detect anti-Hcp1 IgG antibodies against hemolysin co-regulated protein-1 (Hcp1), a key virulence factor of B. pseudomallei. Although initial results in Thailand were promising, its performance required optimization and broader validation. In this study, we report the development of a second-generation Hcp1-ICT and its first international multicenter evaluation using 1,838 serum samples from patients in seven countries. Our findings demonstrate strong diagnostic potential, reveal regional variability in performance, and support the role of the second-generation Hcp1-ICT as a rapid, simple, suitable for both referral centers and resource-limited settings.

Original publication: PLOS Neglected Tropical Diseases (2026-07-06). Source. Source DOI: 10.1371/journal.pntd.0014484.