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Kinome analysis of Madurella mycetomatis identified kinases in the cell wall integrity pathway as novel potential therapeutic drug targets in eumycetoma caused by Madurella mycetomatis.

Eumycetoma is a neglected tropical subcutaneous disease most commonly caused by the fungus Madurella mycetomatis. Currently, eumycetoma is treated by a combination of antifungal therapy and surgery, with limited success rates. To identify novel drug targets we used an in silico approach to determine the kinases present in M. mycetomatis genome and rank them as potential drug targets. In total 132 predicted kinases were identified in M. mycetomatis, of which 21 were predicted to be essential for fungal viability and 4 of these had no human orthologues. Two were linked to the Cell Wall Integrity (CWI) signalling pathway and were expressed in a Galleria mellonella infection model. Several kinase inhibitors were identified after in silico modelling, however only 8 were able to inhibit growth. Five had predicted binding affinity with components of the CWI. Altogether, the CWI shows potential as a drug target for further evaluation.

Madurella↗

[Black-grain eumycetoma due to Madurella grisea. A report of 2 cases].

Two cases of black grains eumycotic mycetoma, occurring on a foot, are reported. Both proceeded from the state of Bahia (Brazil), and in both the etiologic agent was Madurella grisea Mackinnon et al., 1949. The grains structure as well as the micromorphologic characteristics of the fungus in saprophytic life were studied. It is the author's belief that these observations correspond to the 7th and 8th cases reported in the Brazilian medical literature. The authors do consider the following Madurella species as nomen dubium or nomina confusa: M. ramiroi, M. oswaldoi, M. bovoi, M. tozeuri, M. mansonii, M. brumpti, M. reynieri, M. americana, M. lackawanna e M. ikedae and the same for Rubromadurella mycetomi. The only valid species must be Madurella mycetomatis McGinnis, 1980 (= Madurella mycetomi Brumpt, 1905) and Madurella grisea Mackinnon et al., 1949. Treatment with itraconazole in both reported cases, for a 3 month duration, did not produce any regression of the lesions, the clinical improvement being meager.

Adult↗

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.

DNA Primers↗

Potential utility of random amplified polymorphic DNA (RAPD) and restriction endonuclease assay (REA) as typing systems for Madurella mycetomatis.

Two molecular methods were compared, random amplified polymorphic DNA (RAPD) and restriction endonuclease analysis (REA), in order to evaluate their ability to discriminate, and to characterize Madurella mycetomatis strains isolated from human mycetomas in different parts of the world. Both methods were able to cluster the Madurella mycetomatis isolates into the same number of distinct typing groups. However, RAPD, presenting several advantages over REA such as its rapidity, simplicity, and the accessibility for implementation in the laboratory, is a more sensitive and reproducible tool for the study of Madurella mycetomatis epidemiology than REA.

DNA Restriction Enzymes↗

Madura's foot: report of a case caused by Madurella mycetomatis.

We present a case of mycetoma by Madurella mycetomatis on the foot of a Chinese young man, living in Italy for more than ten years. Clinically the lesion closely resembled and was initially misinterpreted as a vascular neoformation. We analyze the histological and morphological features of the Madurella mycetomatis infection through which we managed to type the etiological agent. Our case is worth reporting because of the rarity of this disease in Europe and the unusual clinical presentation. It also offers the opportunity to stress the need for the clinical suspicion of this dermatosis, considering the increase of immigration towards our regions.

Adult↗

[Madurella grisea eumycetoma. Presentation of a case].

One case of mycetoma produced by Madurella grisea localised on the foot of a house wife is reported. The identification of the strain was made through the following features: aspect of the grains, micro e macromorphology in Sabouraud agar and capacity of assimilation of the sugars. This is the fifth case of mycetoma by Madurella grisea reported from Brasil.

Adult↗

Maduromycosis (Madurella mycetomatis) in a horse.

A case of maduromycosis mycetoma caused by Madurella mycetomatis is reported. The horse presented with multiple subcutaneous swelling over the right scapula. There were no discharging fistulae present. Macroscopically the lesion contained a black granular material embedded in a granulomatous mass. Microscopically large numbers of microcolonies were present containing segmented hyphae. No typical chlamydospores were observed. Cultures yielded a fungus identified as Madurella mycetomatis. Treatment consisted of surgical excision and local treatment with thiabendazole powder.

Animals↗

A case of eumycetoma due to Madurella grisea in northern Brazil.

A case of mycetoma caused by the black fungus Madurella grisea in northern Brazil is reported. The lesion was located on the patient's right foot without bone involvement. Clinical samples were collected by opening the fistules with a scalp and the grains removed for microbiological and histopathological analyses. Although mycetoma caused by M. grisea has been previously reported in Brazil, this is the first time that of this fungus has been identify in this particular region of the country.

Adult↗

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 Freund's 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.

Animals↗

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.

Animals↗

Atypical presentation of Madurella mycetomatis mycetoma in a renal transplant patient.

A renal transplant patient presented with a swelling on his foot. Fungal hyphae were found in the abscess, and culture demonstrated Madurella mycetomatis, an agent of mycetoma. There were no black grains in the pus and no overlying sinuses. This was an atypical presentation of maduromycosis in a renal transplant patient.

Drug Therapy, Combination↗

Eumycetoma due to Madurella mycetomatis acquired in Jamaica.

We report a case of eumycetoma due to Madurella mycetomatis affecting the left dorsal forefoot in a 35-year-old woman. She had spent her childhood in Jamaica, and had been resident in the U.K. for 20 years prior to her presentation. She gave a highly suggestive history for a mycetoma, having observed the intermittent discharge of black grains from the lesion. The diagnosis was confirmed by histological and mycological analysis of the grains, and a magnetic resonance imaging scan excluded osteomyelitis. She has responded very favourably to treatment with itraconazole. Mycetomas due to M. mycetomatis have not previously been reported from Jamaica.

Adult↗

Isolation and identification of Madurella mycetomatis from two cases of black grain mycetoma in Libya.

Two cases of mycetoma of the foot caused by Madurella mycetomatis are reported in which identification of the etiologic agents was confirmed by culture. A Libyan male aged 40 years and a Chadian female aged 30 years, exhibited eumycetoma of the foot 1 and 2 years, respectively, after a local injury. The diagnoses were based on the clinical presentation and confirmed by mycologic analysis of the grains and culture isolation of the etiologic agent. Prior to this report M. mycetomatis has not been recorded in Libya as a cause of eumycetoma.

Adult↗

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.

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 [MIC(50)]) of 0.06 to 0.13 mg/liter) was superior to amphotericin B (MIC(50) of 0.5 to 1.0 mg/liter).

Amphotericin B↗

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.

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 microg/ml), itraconazole (MIC90, 0.064 microg/ml), and voriconazole (MIC90, 0.125 microg/ml) appeared superior to those of fluconazole (MIC90, 128 microg/ml) and amphotericin B (MIC90, 1 microg/ml), with MICs in the clinically relevant range. All isolates were resistant to flucytosine (all MICs above 64 microg/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.

Antifungal Agents↗

Development of a species-specific PCR-restriction fragment length polymorphism analysis procedure for identification of Madurella mycetomatis.

Madurella mycetomatis is the commonest cause of eumycetoma in Sudan and other countries in tropical Africa. Currently, the early diagnosis of mycetoma is difficult. In attempting to improve the identification of M. mycetomatis and, consequently, the diagnosis of mycetoma, we have developed specific oligonucleotide primers based on the sequence of the internal transcribed spacer (ITS) regions spacing the genes encoding the fungal ribosomal RNAs. The ITS regions were amplified with universal primers and sequenced, and then two sets of species-specific primers were designed which specifically amplify parts of the ITS and the 5.8S ribosomal DNA gene. The new primers were tested for specificity with DNA isolated from human mycetoma lesions and DNA extracted from cultures of M. mycetomatis reference strains and related fungi as well as human DNA. To study the genetic variability of the ITS regions of M. mycetomatis, ITS amplicons were obtained from 25 different clinical isolates and subjected to restriction fragment length polymorphism (RFLP) analysis with CfoI, HaeIII, MspI, Sau3AI, RsaI, and SpeI restriction enzymes. RFLP analysis of the ITS region did not reveal even a single difference, indicating the homogeneity of the isolates analyzed during the current study.

Humans↗

Environmental occurrence of Madurella mycetomatis, the major agent of human eumycetoma in Sudan.

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.

DNA, Fungal↗