Antigens and antibodies in sporotrichosis: characterization and application on diagnosis.
Sporotrichosis is an important subcutaneous mycosis, with increasing numbers of cases all over the world. However, only few data is available regarding its immunological aspects, particularly the humoral response. Consequently, there is a lack of standardized immunoassays. In this work an enzyme-lynked immunosorbent assay was developed for specific antibody detection in serum samples obtained from 90 patients with different clinical forms of sporotrichosis. Potential cross-reactions were analyzed with 72 heterologous sera from patients with histoplasmosis, paracoccidioidomycosis, cryptococcosis, aspergillosis, tuberculosis and American tegumentary leishmaniasis. The assay was developed with mycelial-phase Sporothrix schenckii exoantigens, an antigenic complex presenting two proteins with homology to STB2 protein from Saccharomyces cerevisiae, as analyzed by mass spectrometry. We found a sensitivity of 97% and a specificity of 89% in this assay. Some cross reactions were seen, as observed in other immunoassays for the diagnosis of sporotrichosis. The ELISA appears to be especially useful for cutaneous forms of disease, since they are not promptly diagnosed with available immunoprecipitation or agglutination techiniques. Using this standardized protocol we also measured immunoglobulin G, IgM and IgA in sera from 41 patients with sporotrichosis before antifungal treatment and from another 35 patients with sporotrichosis during itraconazole treatment course. IgA antibodies against S. schenckii exoantigens were present in the majority of patients before treatment (95.1%), as well as IgM and IgG (85.4%). IgG presented higher frequency in patients under treatment (91.4%). Percentages of positive samples for IgM (71.4%) and IgA (88.6%) were lower in patients under treatment and differences in mean optical densities were significant. Seventy-eight percent of sporotrichosis patients without treatment had detectable levels of all isotypes tested and this percentage dropped to 62.9% in patients receiving itraconazole. Percentage of patients with at least two detectable isotypes was 92.7% before treatment and 88.6% after, showing that detection of these three isotypes together improves sensitivity in the serodiagnosis of sporotrichosis. Reactivity of 94 sera from patients with other diseases and healthy individuals was also tested and some cross reactivity was seen, especially with IgM in leishmaniasis patients. In general, 8.5% of these heterologous sera were positive for at least two isotypes and only one (1.1%) was positive for the three isotypes. These results suggest that the ELISA using mycelial-phase S. schenckii exoantigens is a very sensitive tool for the serodiagnosis of sporotrichosis and can be used in conjunction with conventional methods of diagnosis for this infection.
Advisor:Rosely Maria Zancopé Oliveira
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Enzyme-Linked Immunosorbent Assay
Date of Publication:08/03/2007