Interleukin-17 and matrix metalloprotease-9 expression in the mycetoma granuloma.

Mycetoma is a persistent, progressive granulomatous inflammatory disease caused either by fungi or by bacteria. Characteristic of this disease is that the causative agents organise themselves in macroscopic structures called grains. These grains are surrounded by a massive inflammatory reaction. The processes leading to this host tissue reaction and the immunophenotypic characteristics of the mycetoma granuloma are not known. Due to the massive immune reaction and the tissue remodeling involved, we hypothesised that the expression levels of interleukin-17 (IL-17) and matrix metalloprotease-9 (MMP-9) in the mycetoma granuloma formation were correlated to the severity of the disease and that this correlation was independent of the causative agent responsible for the granuloma reaction. To determine the expression of IL-17 and MMP-9 in mycetoma lesions, the present study was conducted at the Mycetoma Research Centre, Sudan. Surgical biopsies from 100 patients with confirmed mycetoma were obtained, and IL-17 and MMP-9 expression in the mycetoma granuloma were evaluated immunohistochemically. IL-17 was mainly expressed in Zones I and II, and far less in Zone III. MMP-9 was detected mainly in Zones II and III, and the least expression was in Zone I. MMP-9 was more highly expressed in Actinomadura pelletierii and Streptomyces somaliensis biopsies compared to Madurella mycetomatis biopsies. MMP-9 levels were directly proportional to the levels of IL-17 (p = 0.001). The only significant association between MMP9 and the patients' characteristics was the disease duration (p<0.001). There was an insignificant correlation between the IL-17 levels and the patients' demographic characteristics.

Role of a NSAID in the apparent cure of a fungal mycetoma.

UNLABELLED: We report the case of a fungal mycetoma due to Madurella mycetomatis that failed to respond to surgery and antifungal treatment but responded strongly to the addition of a non-steroidal anti-inflammatory drug (NSAID). This African patient was born in Mauritania in 1972. He was a herdsman, living close to the Senegal River. The first nodules appeared on the left foot at the age of 13years (1985). The patient suffered frequent flare-ups with the appearance of black grains and underwent surgery in 1988 and 1992 in Senegal. After remission for several months after surgery, new fistulae occurred. The patient emigrated to France in 1995 and underwent a third surgical intervention in 1996. M. mycetomatis was cultured from the black grains. The patient was otherwise in good health, with no diabetes, and HIV tests were negative. We saw the patient for the first time in 2005, at which time he had flare-ups every two to three months. Imaging disclosed an absence of bone involvement. The patient underwent a fourth operation in October, 2005, and voriconazole treatment was initiated. A new flare-up occurred in February, 2006. CT, MRI, and PET scans revealed calcaneus and tarsal involvement, and posaconazole then replaced voriconazole. Flucytosine was added four months later, due to an absence of improvement. New flares-ups occurred and a fifth surgical intervention was performed in September, 2006. The pain, which had been present for three years, worsened; the patient had to stop working and was no longer able to walk without crutches. Amputation of the foot was considered. Empiric treatment with a NSAID, diclofenac (Voltaren(®); 100mg/day), was added to the antifungal treatment in November 2006, to treat the patient's pain and inflammation. A major improvement was observed within one week. The patient was able to walk without crutches one month later. After two months, clinical examination was normal: no pain, inflammation, nodules or fistulae. Flucytosine was stopped after six months of treatment, in January 2007, diclofenac after 10months, in October 2007, and posaconazole after 18.5months, also in October 2007. No relapse has occurred during the eight years of follow-up since treatment ended. The patient seems to have been cured and has normal CT, MRI, and PET scans. IN SUMMARY: This eumycetoma, which had progressed over 20years despite surgery and antifungal treatments, seems to have been cured by the addition of a NSAID. This observation suggests that inflammation plays a major role in the pathogenesis of fungal mycetoma. Clinical studies of treatments including an NSAID should be conducted to confirm this finding.

Mycetoma caused by Madurella mycetomatis: a completely neglected medico-social dilemma.

Mycetoma is a debilitating disease with a highly particular geographical distribution. The mycetoma belt circles the entire world just above the equator and defines the region with the highest prevalence and incidence. Although the disease is seen in Central America, India and all across Africa, Sudan seems to be the homeland of mycetoma. Mycetoma is an infectious disease caused either by bacteria (actinomycetoma) or true fungi (eumycetoma). In Sudan most cases are caused by the fungal species Madurella mycetomatis. The precise natural habitat of this fungus is still an enigma, but its DNA can easily be found in soil and plant samples in endemic areas. Although the entire human population in these areas are in regular contact with the fungus, most individuals are unaffected. Thus mycetoma is an ideal clinical and experimental model system for the study of host-pathogen interactions. Also, given its relative importance locally, improvements in clinical and laboratory diagnostics and knowledge of the epidemiology of the disease are badly needed. This chapter describes the current state of affairs in the field of eumycetoma caused by M. mycetomatis. The value of laboratory research on this disease and future perspective for control and prevention of the infection are discussed.

Melanin biosynthesis in Madurella mycetomatis and its effect on susceptibility to itraconazole and ketoconazole.

One of the hallmarks of eumycetoma is the formation of fungal grains, which are secreted by multiple sinuses in infected tissues. Madurella mycetomatis grains are black. This black colour was shown to be due to the presence of melanin. Melanin can be produced through various biochemical pathways. It appeared that M. mycetomatis melanisation could be blocked by inhibitors of the pyo- and dihydroxynaphthalene (DHN)-melanin pathways but not by inhibitors of the dihydroxyphenylalanine (L-DOPA)-melanin pathway. Melanin isolated from M. mycetomatis cells provides in vitro protection against the killing effects of the oxidant permanganate and several antifungals. When melanin was added to the culture medium, MICs were found to be 16-fold elevated in the case of itraconazole and 32-fold for ketoconazole. MICs for amphotericin B, fluconazole and voriconazole were not affected. Since itraconazole and ketoconazole are the main antifungal agents used to treat mycetoma, the clinical relevance of the in vitro rise in MIC should be studied further.

Translationally controlled tumor protein from Madurella mycetomatis, a marker for tumorous mycetoma progression.

About 40 years ago Abs against the fungus Madurella mycetomatis were first demonstrated to be present in eumycetoma patients, a disease characterized by tumorous swellings. To date nothing is known about the individual immunoreactive Ags present in this fungus. In the present study, we identify its first immunogenic Ag, a protein homologous to the translationally controlled tumor protein (TCTP), a well-conserved histamine release factor in a range of eukaryotes. The gene for this Ag was demonstrated to be present in two variants in M. mycetomatis, with 13% aa difference between the two proteins encoded. In vitro, TCTP was secreted into the culture medium. In vivo, it was found to be expressed on hyphae present in developing stages of the eumycetoma-characteristic black grain. Significant IgG and IgM immune responses, against the whole protein and selected M. mycetomatis-specific peptides, were determined. The Ab levels correlated with lesion size and disease duration. Overall, the patients with the largest lesions had the highest Ab level, which lowered with decreasing size of the lesion. After 6-15 years of disease duration the Ab levels were the highest. TCTP is the first well-characterized immunogenic Ag, simultaneously the first monomolecular vaccine candidate, identified for the fungus M. mycetomatis.

Genotyping of Madurella mycetomatis by selective amplification of restriction fragments (amplified fragment length polymorphism) and subtype correlation with geographical origin and lesion size.

One of the causative organisms of mycetoma is the fungus Madurella mycetomatis. Previously, extensive molecular typing studies identified Sudanese isolates of this fungus as clonal, but polymorphic genetic markers have not yet been identified. Here, we report on the selective amplification of restriction fragment (AFLP) analysis of 37 Sudanese clinical isolates of M. mycetomatis. Of 93 AFLP fragments generated, 25 were polymorphic, and 12 of these 25 polymorphic fragments were found in a large fraction of the strains. Comparative analysis resulted into a tree, composed of two main (clusters I and II) and one minor cluster (cluster III). Seventy-five percent of the strains found in cluster I originated from central Sudan, while the origin of the strains in cluster II was more heterogeneous. Furthermore, the strains found in cluster I were generally obtained from lesions larger than those from which the strains found in cluster II were obtained (chi-square test for trend, P = 0.03). Among the 12 more commonly found polymorphisms, 4 showed sequence homology with known genes. Marker A7 was homologous to an endo-1,4-beta-glucanase from Aspergillus oryzae, 97% identical markers A12 and B3 matched a hypothetical protein from Gibberella zeae, and marker B4 was homologous to casein kinase I from Danio rerio. The last marker seemed to be associated with strains originating from central Sudan (P = 0.001). This is the first report on a genotypic study where genetic markers which may be used to study pathogenicity in M. mycetomatis were obtained.