Phenotypic And Molecular Characterization Of Madurella TMMU3956, A Possible Novel Pathogenic Fungus Responsible For Eumycetoma

Mycetoma is a chronic granulomatous subcutaneous infection caused by true fungi (eumycetoma) or bacterial actinomycetes (actinomycetoma). The pathogens involved are found in the environment in certain types of soil and are directly inoculated into the subcutaneous tissues, commonly in the foot, through minor trauma or a thorn prick. Mycetoma has a prolonged, progressive, and indolent course and, if untreated, ultimately leads to destruction of deeper tissues and bone, resulting in deformity and disability which may necessitate amputation. The triad of a subcutaneous painless mass, sinuses, and grains discharged through the sinuses is the hallmark of mycetoma.Mycetoma has a worldwide but uneven distribution. The disease is endemic in tropical and subtropical regions and the African continent has the highest prevalence. Eumycetoma prevails in the mycetoma belt that stretches between the latitudes of 15? south and 30? north. The belt includes Sudan, Somalia, Senegal, India, Yemen, Mexico, Venezuela, Columbia, Argentina, and other countries. In Sudan, An admission of 1231 mycetoma cases to outpatient clinics throughout the country within a period of 2~5 years was observed. The disease has also been extensively reported in India. But it is seldom encountered in Western countries. Most of the imported cases involved immigrants who probably contracted the infection in their home countries. This complicates the clinical diagnosis since it is frequently unexpected. Reports on mycetoma have come from the USA, Ceylon, Germany, Egypt, Turkey, Philippines, Japan, Lebanon, Thailand, Iran, Netherlands, and Saudi Arabia. What is more, the disease didn't spare China. Between the decades of 1960~2009, 18 cases have been reported in China, of which 9 are eumycetoma and 9 are actinomycetoma.Mycetoma can affect any part of the body, and lesions are not restricted to the limbs only. Most cases are usually seen in the feet (70%), followed by hands (12%), then legs and knee joints. In highly endemic areas, other parts of the body might become affected as well. These include the arm, head and neck, thighs, and the perineum. Rarely, the chest and abdominal walls, facial bones, mandible, paranasal sinuses, eyelid, vulva, orbit, scrotum, and old surgical incisions might also be affected. The incidence of mycetoma infections of the head and neck is 3.75%. For unexplained reasons 80% craniofacial mycetoma was due to actinomycetes. Although the craniofacial mycetoma is rare, when affected it is the most difficult to treat. They can lead to serious complications arising from extension of swelling through the cranium that puts pressure on the brain. If the patient does not seek early medical care, the disease may run a rapidly fatal course.This study presents an extraordinary case of craniofacial eumycetoma extending from gum to lower jaw in a 27-year-old Chinese male. Our case is worth reporting not only by its rareness in China but also by its unusual affected site. Moreover, we have isolated a distinctive dematiaceous fungus from clinical specimens of this patient. By sequencing of the internal transcribed spacer (ITS) region, we found that it had maximum sequence identity with Madurella mycetomatis, one of the main microorganisms causing black-grain fungal mycetoma, but the similarity was less than 95%. It is proposed that the fungus is probably a novel species of genus Madurella and coded as TMMU3956 tentatively.Madurella species are well known agents responsible for eumycetoma. Two species are recognized, M. mycetomatis and M. grisea. The homogeneity of M. mycetomatis has been reported previously, at least in Sudan. By sequencing the ITS region with the ITS4 and ITS5 primers and using large-scale random amplification of polymorphic DNA(RAPD), those authors suggested that this fungus had a clonal origin. However, the same group that suggested a clonal origin of M. mycetomatis strains from Sudan has recently reported polymorphism within this species by using amplified fragment length polymorphism (AFLP). Those authors even suggested a possible relationship with clinical data such as lesion size. In the present study, the genus Madurella has been proven to be heterogeneous on the basis of rDNA small subunit (SSU) and ITS sequencing data. de Hoog et al. found that the ITS1+2 sequences of four M. mycetomatis clinical strains, CBS201.38, from Indonesia, CBS248.48, from New Mexico, USA, CBS216.29, from Italy, and CBS217.55, from Argentina, have significant difference (average 5.3%) and supposed that the sequence variability at ITS locus of'M. mycetomatis'might be related to the geographical area and the climatic environment. In addition, another investigation showed that 7 out of 15 M. mycetomatis strains diverged significantly from the type strain such as IP582.60, IP584.78, IP592.74 and IP1137.76 by comparison of the ITS2 regions. These strains were tentatively identified as Madurella sp. In fact, the CBS collection holds a number of strains from other climatic zones which showed more than 5% ITS difference and thus are likely to be a separate species. On the other hand, Madurella grisea were demonstrated to be more heterogeneous and closer to some Pyrenochaeta romeroi strains than to type M. grisea strains by comparison of the ITS sequences. Sequencing of the SSU rDNA gene of the authentic strain of M. mycetomatis (CBS247.48) and M. grisea (CBS331.55) revealed that M. mycetomatis was close to the genera Neurospora, Sordaria and Podospora, which all belong to the order Sordariales of ascomycetes, whereas M. grisea clustered with species of the genera Leptosphaeria, Cucurbidothis, Westerdykella and Sporormia, which are members of the order Pleosporales belong to ascomycetes. These data prove that'Madurella'has been currently applied as an umbrella term covering partly unrelated species. The taxonomic positions of M. grisea need improvement, and the identification of new species responsible for eumycetoma is warranted.In this study, we try to provide evidences for demonstrating that our strain is different from M. mycetomatis and M. grisea and to facilitate the timely administration of effective therapy by elucidating the morphological and physiological characteristics, antifungal susceptibility, experimental animal pathogenicity and rRNA gene sequence of M. TMMU3956. In concrete, we inoculated M. TMMU3956 onto plate and slide culture of different medium. Both gross morphological and microscopic observations were made in addition to scanning electron microscopy. The temperature test between 25~42℃and assimilation assay with API AUX 20C was carried out. Antifungal susceptibility test with itraconazole, voriconazole, amphotericin B, terbinafine, fluconazole and flucytosine was performed according to a modified CLSI 38-A method. Finally, the DNA extraction was conducted by the glass beads-salting out procedure and rDNA sequences were amplified with ITS, 18S and 28S primers. A Phylogenetic analysis tree was constructed based on the ITS1+2 sequences. In addition, an animal experiment with BALB/C mice and New Zealand white rabbits was attempted.The results showed that at least two ways of asexual multiplication, the phialospores and sporangiospores, were observed. The fungus could tolerate 42°C. Assimilation of glucose, arabinose, xylose, cellose, maltose and trehalose was positive. Antifungal susceptibility test indicated that it was highly susceptible to itraconazole, voriconazole and amphotericin B, moderately susceptible to terbinafine, and resistant to fluconazole and flucytosine. No characteristic black grains or detectable infection were observed in BALB/C mice and New Zealand white rabbits. The analyses of the ITS region and the 18S ribosomal gene sequences support a new species designation.In conclusion, the new strain is different from M. mycetomatis and M. grisea. It is suggested that this strain is a novel species of Madurella responsible for eumycetoma.