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Purpose: Leptospirosis is an endemic disease in India and uveitis is its late complication. Several Indian reports showed diversity of serovars, changing patterns and existence of new serovars. Failure to add new serovars in testing panel result in increased false-negativity in serology. Aim: To analyse seroprevalence, changing patterns and to discuss the resulting challenges in diagnosis. Methods: In this retrospective study covering the period from 1994 to 2020, we analysed data from laboratory records of patients diagnosed with leptospiral uveitis in South India. Microscopic agglutination Test (MAT) and/or Enzyme-Linked Immunosorbent Assay (ELISA) were performed on clinically diagnosed leptospiral uveitis cases from our hospital, as well as on systemic leptospirosis patients from government and private hospitals. Results: Out of a total of 87 216 new uveitis cases with varying causes over 27 years, 3,658 (4.1%) were clinically diagnosed as leptospiral uveitis. Among them, 1,268 (34.7%) patients were seropositive. In 1994, 92% of clinically diagnosed leptospirosis patients were seropositive in the MAT performed at the Centers for Disease Control and Prevention in Atlanta. However, the positivity rate gradually declined to 35% over the years. The predominant serovars identified were L. autumnalis, L. icterohaemorrhagiae, and L. australis. There were notable variations in the distribution of serovars over the years. Conclusions: The data suggest a declining sensitivity of MAT and ELISA, possibly due to the emergence of new serovars. Customizing the panel based on local isolates could enhance the performance of MAT. Critical need is the addition of advanced molecular techniques to improve the diagnosis. [ABSTRACT FROM AUTHOR]