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Background: Leptospirosis, a zoonosis caused by species in the spirochete genus Leptospira, is endemic to the Yaeyama region in Okinawa, subtropical Japan. Species of the P1 subclade "virulent" group, within the genus Leptospira, are the main etiological agents of leptospirosis in Okinawa. However, their environmental persistence is poorly understood. This study used a combination of bacterial isolation and environmental DNA (eDNA) metabarcoding methods to understand the eco-epidemiology of leptospirosis in this endemic region. Findings: Polymerase chain reaction (PCR) characterized twelve human clinical L. interrogans isolates belonging to the P1 subclade "virulent" subgroup and 11 environmental soil isolates of the P1subclade "low virulent" subgroup (genetically related to L. kmetyi, n = 1; L. alstonii, n = 4; L. barantonii, n = 6) from the Yaeyama region targeting four virulence-related genes (lipL32, ligA, ligB and lpxD1). Clinical isolates were PCR positive for at least three targeted genes, while all environmental isolates were positive only for lipL32. Analysis of infected renal epithelial cells with selected clinical and environmental strains, revealed the disassembly of cell-cell junctions for the Hebdomadis clinical strain serogroup. Comparison of leptospiral eDNA during winter and summer identified operational taxonomic units corresponding to the species isolated from soil samples (L. kmetyi and L. barantonii) and additional P2 subclade species (L. licerasiae, L. wolffii-related, among others) that were not detected by soil cultivation. Total Leptospira read counts were higher in summer than in winter and the analysis of leptospiral/animal eDNA relationship suggested Rattus spp. as a potential reservoir animal. Conclusion: Our study demonstrated high environmental Leptospira diversity in the Yaeyama region, particularly during summer, when most of the leptospirosis cases are reported. In addition, several Leptospira species with pathogenic potential were identified that have not yet been reported in Yaeyama; however, the environmental persistence of P1 subclade species previously isolated from human clinical cases in this region was absent, suggesting the need of further methodology development and surveillance. Author summary: Leptospirosis is a widespread bacterial zoonosis and one of the most important acute febrile infectious disease in tropical and subtropical regions, which is difficult to differentiate from other infectious diseases common in these regions. Leptospirosis is endemic to Okinawa prefecture, the southernmost prefecture of Japan, where the infection occurs mainly after recreational activities in rivers in the northern part of Okinawa Main Island and the Yaeyama region. This study combined several methods such as bacterial isolation from soil and environmental DNA metabarcoding from river water samples to understand the persistence of Leptospira outside the human host, leptospiral diversity in the environment, and their potential reservoir animals in the wild environment of the Yaeyama region. Although this study didn't confirm the environmental persistence of Leptospira species previously isolated from clinical cases, several newly reported Leptospira species with pathogenic potential from the Yaeyama region suggested the need for continual surveillance to improve leptospirosis control and prevention in this region. [ABSTRACT FROM AUTHOR]

Background: Leptospirosis is considered an endemic disease among agricultural workers in Okinawa Prefecture, which is the southernmost part of Japan and has a subtropical climate, but data on the current status and trend of this disease are scarce. Methodology/principal findings: We conducted a retrospective study of clinically suspected leptospirosis patients whose sample and information were sent to the Okinawa Prefectural Institute of Health and Environment from November 2003 to December 2020. Laboratory diagnosis was established using culture, nested polymerase chain reaction (PCR), and/or microscopic agglutination test (MAT) with blood, cerebrospinal fluid, and/or urine samples. Statistical analyses were performed to compare the epidemiological information, clinical features, and sensitivities of diagnostic methods among laboratory-confirmed cases. Serogroups and the species of Leptospira isolates were determined by MAT using 13 antisera and flaB sequencing. A total of 531 clinically suspected patients were recruited, among whom 246 (46.3%) were laboratory confirmed to have leptospirosis. Among the confirmed cases, patients aged 20–29 years (22.4%) and male patients (85.7%) were the most common. The most common estimated sources of infection were recreation (44.5%) and labor (27.8%) in rivers. Approximately half of the isolates were of the L. interrogans serogroup Hebdomadis. The main clinical symptoms were fever (97.1%), myalgia (56.3%), and conjunctival hyperemia (52.2%). Headache occurred significantly more often in patients with Hebdomadis serogroup infections than those with other serogroup infections. The sensitivities of culture and PCR exceeded 65% during the first 6 days, while the sensitivity of MAT surpassed that of culture and PCR in the second week after onset. PCR using blood samples was a preferable method for the early diagnosis of leptospirosis. Conclusions/significance: The results of this study will support clinicians in the diagnosis and treatment of undifferentiated febrile patients in Okinawa Prefecture as well as patients returning from Okinawa Prefecture. Author summary: Leptospirosis is an important but largely under-recognized public health problem in the tropics. Recreational leptospirosis is a major cause of infection; people contract leptospirosis via recreational activities in rivers or lakes. Okinawa Prefecture is located in the southernmost part of Japan, has a subtropical climate, and is a major tourist destination, with an estimated 10 million visitors annually. In this study, we revealed that the trend of leptospirosis in this prefecture has changed. Leptospirosis was considered an endemic disease among agricultural workers; however, during the last 17 years, most of the confirmed cases in Okinawa Prefecture were attributable to recreation or labor in rivers in the summer in the northern part of the main island of Okinawa and the Yaeyama region, which are designated as national parks with abundant nature. Since recreation in rivers is the most popular activity among tourists in Okinawa Prefecture, leptospirosis is a health concern not only for residents of Okinawa Prefecture but also for tourists. [ABSTRACT FROM AUTHOR]