Seroepidemiology of human leptospirosis in subtropical Bhutan, 2024

Background Leptospirosis causes approximately 1.03 million cases and 58,900 deaths annually worldwide yet remains underrecognized because infections are often mild or self-limiting and require serological confirmation. In Bhutan, limited diagnostic capacity constrains surveillance, and data on human exposure in high-risk subtropical areas are limited. This study aimed to estimate seroprevalence, identify circulating serovars, map geographic distribution of seropositive cases, and assess potential risk factors for Leptospira exposure among adults in subtropical Bhutan. Methods/principal findings We conducted a cross-sectional study during September–October 2024 in six subtropical communities in southern Bhutan. A total of 503 adults were enrolled using probability-proportional-to-size (PPS) sampling; 492 were included in the final analysis after excluding 11 participants for inadequate blood sample or hemolysis. Serum samples were tested by IgM and IgG ELISA and by microscopic agglutination test (MAT), the reference standard used to define seropositivity for potential risk factor analysis and primary reacting serovar identification. MAT-based seroprevalence was 19.3% (95% CI: 16.0 – 23.1), compared with 22.4% (95% CI: 18.8 – 26.4) by ELISA IgM and 16.1% (95% CI: 13.0 – 19.7) by ELISA IgG. Among MAT-positive samples (n = 95), the predominant serovars were Australis (49%) and Lai-like (13%). Gelephu Thromde had the highest serovar diversity, while Australis and Patoc were detected in all six study areas. Multivariable logistic regression identified paddy farming (adjusted OR: 4.74; p < 0.001) and drinking unboiled water (adjusted OR: 2.34; p = 0.002) as independently associated with MAT seropositivity. In addition, residence in crowded urban (adjusted OR: 4.08; p = 0.001) or urban areas (adjusted OR: 2.94; p = 0.002) relative to rural areas and lower education (primary education: adjusted OR: 2.52; p = 0.013; unable to read and write: adjusted OR: 2.02; p = 0.034) were associated with MAT seropositivity. Conclusion/significance Approximately one in five adults in subtropical Bhutan had serological evidence of prior Leptospira spp. exposure, indicating that human exposure is more common than previously recognized. Given the cross-sectional design, these associations cannot establish temporality and self-reported exposure to potential risk factors may be subject to recall bias. These findings, however, provide baseline data on population exposure and point to actionable public health interventions: (1) educate communities on preventive behaviors, (2) promote use of protective gear during high-risk activities, (3) expand research and surveillance capacity to better understand the epidemiology of leptospirosis and (4) strengthen hospital-based diagnostics to enable early treatment and reduce complications from leptospirosis in Bhutan.