Do contexto epidemiológico em Manaus-AM à novas estratégias diagnósticas: avanço na detecção imunológica e molecular da esporotricose

Abstract

Sporotrichosis, a neglected mycosis caused by the fungus Sporothrix, represents a growing public health challenge in Brazil, driven primarily by the endemic species S. brasiliensis and its zoonotic transmission through felines. This study presents a set of methodological innovations aimed at more effective diagnosis. The first chapter of this thesis addresses an epidemiological assessment of the spread of sporotrichosis in Manaus between 2020 and 2023, which revealed an exponential increase in cases. A total of 4,301 cases were confirmed, 3,403 in animals (99.6% in cats) and 898 in humans. The strong correlation between animal and human cases highlights the role of felines as important reservoirs and disseminators of the disease, emphasizing the need for population and environmental control. In response to diagnostic deficiencies, Chapters II, III, and IV of this study focused on the development of immunoassays and a molecular test for the detection of Sporothrix sp.. The recombinant ssCBF protein (Sporothrix schenckii cell wall and cell-bound fractions) was produced and purified and used in an indirect ELISA immunoassay to detect IgG antibodies against the native ssCBF protein in cat serum. The ELISA test was able to detect patient negative results throughout treatment, positioning itself as a test for treatment monitoring. Additionally, a prototype immunochromatographic test was developed, demonstrating its potential as a rapid and accessible diagnostic tool. The simple agreement percentage between ELISA and RDT was 90.2%, with a Cohen's kappa coefficient of 0.79, indicating near-perfect agreement. The same recombinant protein was used to produce IgY polyclonal antibodies, and their recognition of S. brasiliensis was confirmed by immunofluorescence, emphasizing the potential of the developed tool for detecting the native antigen. Another approach used was the development of a MIRA-LFD molecular assay for detecting the presence of Sporothrix sp. DNA, with a limit of detection ("LOD") of 2.1 pg/μL, without cross- reactivity with fungi from other species evaluated. This methodology represents an advance in result delivery time and laboratory infrastructure when compared to traditional amplification techniques. Thus, the results presented here provide a solid foundation for the development of diagnostic and monitoring tools that can directly impact sporotrichosis control in endemic areas, paving the way for more efficient health strategies.