Th-1, Th-2 Cytokines Profile among Madurella mycetomatis Eumycetoma Patients.

Eumycetoma is a progressive and destructive chronic granulomatous subcutaneous inflammatory disease caused by certain fungi, the most common being Madurella mycetomatis. The host defence mechanisms against fungi usually range from an early non-specific immune response to activation and induction of specific adaptive immune responses by the production of Th-1 and Th-2 cytokines. The aim of this study is to determine the levels of Th-1 and Th-2 cytokines in patients infected with Madurella mycetomatis, and the association between their levels and disease prognosis. This is a descriptive cross-sectional study conducted at the Mycetoma Research Centre, University of Khartoum, Sudan, where 70 patients with confirmed M. mycetomatis eumycetoma were enrolled; 35 with, and 35 without surgical excision. 70 healthy individuals from mycetoma endemic areas were selected as controls. The levels of serum cytokines were determined by cytometric bead array technique. Significantly higher levels of the Th-1 cytokines (IFN-γ, TNF-α, IL-1ß and IL-2) were recorded in patients treated with surgical excision, compared to those treated without surgical excision. In contrast, the Th-2 cytokines (IL-4, IL-5, IL-6 and IL-10) were significantly lower in patients treated with surgical excision compared to those treated without surgical excision. In conclusion, the results of this study suggest that cell-mediated immunity can have a role to play in the pathogenesis of eumycetoma.

Use of immunoblotting in testing Madurella mycetomatis specific antigen.

BACKGROUND: Though serodiagnosis of actinomycetoma is established, that of eumycetoma due to Madurella mycetomatis is limited because of lack of pure antigen. Reliable rapid tests are needed to make an accurate timely diagnosis. The purpose of this study is to detect antigen parts of M. mycetomatis, which act specifically with M. mycetomatis antibodies. METHODS: Cytoplasmic antigen was prepared from molecularly identified cultures of M. mycetomatis by sonication, ultracentrifugation, dried, weighed and appropriately reconstituted. M. mycetomatis cytoplasmic antigen were separated using 12% sodium dodecyl sulfate-polyacrylamide gel, and immunoblotting to detect the reactive ones.Immunoblotting was carried out in nitrocellulose strips containing different molecular size. Sera from patients and co-patients as control were used. RESULTS: When stained with Coomassie brilliant blue R 250 seven molecular weights appeared but only three, 45, 60, 95 kDa reacted with M. mycetomatis patients few from control group, one from a malaria patient. No reactive band was observed with sera from actinomycetoma, Aspergillus flavus-associated aspergillosis, schistosomiasis, leishmaniasis, fungal sinusitis nor healthy controls. CONCLUSIONS: Specific fractions of M. mycetomatis antigen which were demonstrated by immunoblotting showed 75% sensitivity and 95% specificity. The true negative tests were 14 patients (32.5%). This also means that immunoblotting is reasonably reliable in diagnosis and follow-up of eumycetoma patients.

Fructose-bisphosphate aldolase and pyruvate kinase, two novel immunogens in Madurella mycetomatis.

Eumycetoma, a chronic granulomatous disease characterized by a subcutaneous mass, multiple sinuses and purulent discharge containing grains, remains difficult to diagnose and treat. Madurella mycetomatis is the most common causative agent of eumycetoma. Using a serum pool from patients with active mycetoma, we screened a M. mycetomatis-specific λgt11 cDNA library which was shown to contain 8% of cDNA inserts encoding proteins involved in glycolysis. Two of these enzymes, fructose-bisphosphate aldolase (FBA) and pyruvate kinase (PK), were produced in vitro and their antigenicity was studied with bead-based flow cytometry. It appeared that both FBA and PK IgG antibodies were present in eumycetoma patient sera. However, only FBA antibody levels were found to be significantly higher in eumycetoma patient sera when compared to healthy Sudanese controls. Furthermore, FBA and PK were also found to be expressed on the hyphae present in the mycetoma grain. In conclusion, this study presents two new antigenic proteins of M. mycetomatis next to the translationally controlled tumour protein (TCTP): the glycolytic enzymes FBA and PK. These antigens might be useful as vaccine-candidates in the prevention of mycetoma.