Diagnostic Timelines in Acute Leptospirosis: A Four-Patient Case Series

Acute leptospirosis is difficult to diagnose because its manifestations are nonspecific and serological tests may be negative early. We retrospectively analyzed four eligible patients with confirmed leptospirosis identified through an electronic medical record search at two hospitals and reconstructed their diagnostic timelines. Paired microscopic agglutination testing (MAT) independently confirmed leptospirosis in all four patients. Acute-phase plasma metagenomic next-generation sequencing (mNGS), subsequent reverse-transcription quantitative polymerase chain reaction (RT-qPCR) of stored acute-phase serum, and paired enzyme-linked immunosorbent assay (ELISA) and MAT results were analyzed. All patients developed fever and systemic inflammation, and three developed septic shock. The original mNGS reports contained 4–42,125 reads assigned to the reported Leptospira species and provided the first etiological clues to leptospirosis during clinical care. RT-qPCR was positive in all acute-phase sera, with cycle threshold values of 30.80, 33.52, 26.92, and 34.70. Acute-phase MAT was negative in all patients. ELISA IgM and IgG were both negative in three, whereas Case 4 was IgM-positive but IgG-negative on day 8. During convalescence, ELISA IgM and IgG were positive in all patients, and MAT titers rose to 1:3200, 1:1600, 1:400, and 1:800. These findings emphasize that molecular and serological results should be interpreted according to sampling time.