Performance of immunodiffusion and dot blot assays using autochthonous antigens: A decade of paracoccidioidomycosis diagnosis in microbiologically proven cases

Although serological testing may be influenced by several factors, it remains essential for diagnosing paracoccidioidomycosis (PCM) and monitoring patients' response to treatments. This retrospective, multicenter study evaluated the diagnostic accuracy of the double immunodiffusion (ID) and dot blot (DB) assays using antigenic extracts from the reference strain (RAgPb) and 10 autochthonous Paracoccidioides spp. isolates. We analyzed sera from 370 microbiologically confirmed PCM cases registered in Argentina over a 10-year period (2013-2022). While 318 (86%) showed an identity reaction using ID with the RAgPb, 52 (14%) sera were initially identified as false negatives by referral centers. Upon retesting, 34/52 turned positive, highlighting the operator- and procedure-dependent variability that impacts ID sensitivity. The remaining 18 (5%) sera were tested using autochthonous antigens and showed reactivity by ID and DB, whereas all remained negative when RAgPb was used. These findings underscore the impact of antigenic variability and support the use of native antigens to enhance sensitivity. The DB assay demonstrated higher sensitivity, faster results, and simplicity, making it also suitable for routine diagnosis. No statistically significant differences (P > .05; odds ratio = 2.69) were observed between PCM clinical form and ID results. This study emphasizes the importance of including region-specific antigens and standardized protocols to improve PCM diagnosis and minimize false negatives in endemic areas.