Abdalla O. A. Ahmed

Mycetoma caused by Madurella mycetomatis: a neglected infectious burden

Tropical eumycetoma is frequently caused by the fungus Madurella mycetomatis. The disease is characterised by extensive subcutaneous masses, usually with sinuses draining pus, blood, and fungal grains. The disease affects individuals of all ages, although disability is most severe in adults who work outdoors. Compared with major diseases such as tuberculosis, malaria, and HIV, disease from M mycetomatis is underestimated but socioeconomically important. Many scientific case reports on mycetoma exist, but fundamental research was lacking until recently. We present a review on developments in the clinical, epidemiological, and diagnostic management of M mycetomatis eumycetoma. We describe newly developed molecular diagnostic and gene typing procedures, and their application for management of patients and environmental research. Fungal susceptibility tests have been developed as well as a mouse model of infection. These advances should greatly further our understanding of the molecular basis of eumycetoma.

Environmental Occurrence of Madurella mycetomatis, the Major Agent of Human Eumycetoma in Sudan

ABSTRACT Madurella mycetomatis is the main causative agent of human eumycetoma, a severe debilitating disease endemic in Sudan. It has been suggested that eumycetoma has a soil-borne or thorn prick-mediated origin. For this reason, efforts were undertaken to culture M. mycetomatis from soil samples (n = 43) and thorn collections (n = 35) derived from areas in which it is endemic. However, ribosomal sequencing data revealed that the black fungi obtained all belonged to other fungal species. In addition, we performed PCR-mediated detection followed by restriction fragment length polymorphism (RFLP) analysis for the identification of M. mycetomatis DNA from the environmental samples as well as biopsies from patients with mycetoma. In the case of the Sudanese soil samples, 17 out of 74 (23%) samples were positive for M. mycetomatis DNA. Among the thorn collections, 1 out of 22 (5%) was positive in the PCR. All PCR RFLP patterns clearly indicated the presence of M. mycetomatis. In contrast, 15 Dutch and English control soil samples were all negative. Clinically and environmentally obtained fungal PCR products share the same PCR RFLP patterns, suggesting identity, at least at the species level. These observations support the hypothesis that eumycetoma is primarily environmentally acquired and suggest that M. mycetomatis needs special conditions for growth, as direct isolation from the environment seems to be impossible.

Testing of the In Vitro Susceptibilities of Madurella mycetomatis to Six Antifungal Agents by Using the Sensititre System in Comparison with a Viability-Based 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-5- [(Phenylamino)Carbonyl]-2H-Tetrazolium Hydroxide (XTT) Assay and a Modified NCCLS Method

ABSTRACT The in vitro susceptibilities of 36 clinical isolates of Madurella mycetomatis, the prime agent of eumycetoma in Africa, to ketoconazole, itraconazole, fluconazole, voriconazole, amphotericin B, and flucytosine were determined by the Sensititre YeastOne system. This system appeared to be a rapid and easy test, and by use of hyphal suspensions it generated results comparable to those of a modified NCCLS method. After 10 days of incubation, the antifungal activities of ketoconazole (MIC at which 90% of isolates were inhibited [MIC90], 0.125 μg/ml), itraconazole (MIC90, 0.064 μg/ml), and voriconazole (MIC90, 0.125 μg/ml) appeared superior to those of fluconazole (MIC90, 128 μg/ml) and amphotericin B (MIC90, 1 μg/ml), with MICs in the clinically relevant range. All isolates were resistant to flucytosine (all MICs above 64 μg/ml). Based on the relatively broad range of MICs obtained for the antifungal agents, routine testing of M. mycetomatis isolates for susceptibility to antifungal agents seems to be relevant to adequate therapeutic management.

In Vitro Susceptibilities of Madurella mycetomatis to Itraconazole and Amphotericin B Assessed by a Modified NCCLS Method and a Viability-Based 2,3-Bis(2-Methoxy-4-Nitro-5- Sulfophenyl)-5-[(Phenylamino)Carbonyl]-2H- Tetrazolium Hydroxide (XTT) Assay

ABSTRACT Susceptibilities of Madurella mycetomatis against amphotericin B and itraconazole in vitro were determined by protocols based on NCCLS guidelines (visual reading) and a 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay for fungal viability. The XTT assay was reproducible and sensitive for both antifungals. Itraconazole (MIC at which 50% of the isolates tested are inhibited [MIC50]) of 0.06 to 0.13 mg/liter) was superior to amphotericin B (MIC50 of 0.5 to 1.0 mg/liter).

Phylogeny and typification of Madurella mycetomatis, with a comparison of other agents of eumycetoma

The genus Madurella, described for non‐sporulating agents of human mycetoma, is proven to be heterogeneous on the basis of rDNA small subunit (SSU) and Internal Transcribed Spacer (ITS) sequencing data. Madurella mycetomatis, the main agent of mycetoma in arid zones of Central and East Africa, probably belongs to the ascomycete order Sordariales. Madurella mycetomatis, the generic type species, is neotypified. Madurella grisea, with worldwide occurrence, is likely to be a member of the order Pleosporales, just as the mycetoma agents of Leptosphaeria, Pseudochaetosphaeronema, and Pyrenochaeta. Neotestudina rosatii belongs to the order Dothideales. Judging from ITS data, M. mycetomatis and N. rosatii are species complexes. The ex‐type strain of N. rosatii, from a human mycetome, has an ITS sequence that deviates from that of environmental strains of the species.

Molecular Detection and Identification of Agents of Eumycetoma: Detailed Report of Two Cases

ABSTRACT We describe two cases of eumycetoma in the legs. The infections could not be adequately diagnosed by classical mycology, but the causative agents were successfully identified as Madurella mycetomatis by species-specific PCR and DNA sequencing.

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.

A murine model of Madurella mycetomatis eumycetoma

Eumycetoma due to Madurella mycetomatis is a major mycological health problem in endemic areas. We infected BALB/c mice (male or female) with various amounts of M. mycetomatis mycelium, containing sterilized soil as a natural adjuvant or Freunds incomplete adjuvant. Mice differed with respect to age and immune status. Intraperitoneal, subcutaneous and intravenous inoculation was explored and survival was monitored. Mice were killed at various intervals after inoculation, checked for the presence of the characteristic black grains, and organs were cultured for M. mycetomatis. Infected organs were subjected to histopathological examination. Immunocompetent male mice were as susceptible as immunocompromised female mice, but showed higher mortality rates. In conclusion, a reproducible mouse model of intraperitoneal M. mycetomatis infection with characteristic black grains in immunocompetent adult or young female mice was developed. Although this experimental model does not simulate macroscopic features of the subcutaneous M. mycetomatis infection in humans, the histopathological characteristics of the lesions and the development of black grains are clearly representative for the human infection. This model will enable further studies on the pathogenesis as well as prevention and treatment of the fungal infection.

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

ABSTRACT 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.

Madurella mycetomatis Strains from Mycetoma Lesions in Sudanese Patients Are Clonal

ABSTRACT Molecular diversity among clinical isolates of Madurella mycetomatis, the prime fungal agent of human mycetoma in Sudan, could possibly explain the diverse clinical presentations of this severely debilitating infectious disease. In addition, culture-independent DNA-mediated typing tests need to be developed for this organism, since M. mycetomatis DNA, but not the organism itself, can be identified in soil, the material from which infections are thought to originate. A collection of 38 different clinical M. mycetomatis isolates was characterized by large-scale random amplification of polymorphic DNA using 20 different primer species. These analyses, involving at least 2,600 annealing sites, showed a complete lack of DNA fingerprint variation among the various isolates. From the resulting homogeneous DNA fingerprints, seven fragments were cloned and sequenced, and novel, species-specific PCR restriction fragment length polymorphism (RFLP) tests were designed. The seven PCR RFLP tests were successfully performed on the 38 different M. mycetomatis strains. However, again all M. mycetomatis DNA patterns obtained appeared to be identical, whereas patterns produced using DNAs from other fungal species were clearly discriminatory. These results suggest that there is little genetic variation among clinically relevant M. mycetomatis strains from Sudan. The data tentatively imply that different manifestations of mycetoma are due to differences in host susceptibility rather than differential virulence of the causative agent.