Madurella mycetomatis is a fungus primarily reported in Central Africa as a causative agent of mycetoma in humans. This fungus has been misclassified for many years. Finally, following the improvement of molecular techniques, its current phylogenetic classification has been obtained and confirmed. Many methods exist to identify M. mycetomatis, both in lesions and in culture, including histological examination and molecular techniques. Histological examination is especially useful with respect to this fungus, as it has many unique morphological features. Strain-levels differences in response of M. mycetomatis antifungal agents is informative for treatment as well as laboratory isolation of cultures.
Madurella mycetomatis was not always the name of its described fungus, throughout the years it underwent many name changes ultimately leading to the currently accepted name. In 1901 Brumpt described the first recorded case of mycosis caused by M. mycetomatis, identifying black granules in association with mycetoma. In 1902 Laveran named the fungus Strepthothrix mycetomi, he identified the species from a mycetoma grain. Three years later, in 1905, Brumpt corrected its genus to Madurella, in turn changing its name to Madurella mycetomi. This fungus was then cultivated in-vitro by Brault in 1912, leading to the ability to study this fungus in culture. In 1977 the British Medical Research Council changed the name, to the currently accepted name, Madurella mycetomatis. Although the binomial name was finally determined, M. mycetomatis still remained incorrectly classified in the Pleosporales order. This error was eventually corrected, and M. mycetomatis was rightly placed in the Sorariales order.
The genus Madurella contains only two well defined species: M. mycetomatis and M. grisea. Roughly a dozen other species of uncertain validity have been described genus Madurella based on in vivo similarities and cultural sterility. Although similar, there remained some important physiological and morphological differences between the two well defined species, leading scientists to doubt their phylogeny. With the development of ribosomal sequencing and other molecular techniques, studies discovered that in fact, M. mycetomatis did not share a common ancestor with M. grisea. Thus lead to the understanding that M. mycetomatis belonged in the Sordariales order. This finding was further confirmed utilizing comparative genomics of a known species of the Sordariales order, Chaetomium thermophilum.
Genotypic variation can both help to explain geographical distribution of fungi, and differences in symptoms between two hosts affected by the same etiological agent. Restriction endonuclease assay (REA) and random amplification of polymorphic DNA (RAPD) were performed to characterize the different genotypes of M. mycetomatis. These methods identified 2 genotypic clusters in Africa, and 7 different genotypes from other continents. Further testing with amplified fragment length polymorphism (AFLP) found 3 clusters in Sudan, in contrast to the 2 clusters identified by RAPD, proving AFLP to be a more sensitive method. This understanding challenged the original believe that M. mycetomatis was genetically homogenous, and provides an explanation for the variability in host symptoms.
Physiology and ecology
Madurella mycetomatis has been identified in both soil and anthill samples, growing optimally at 37 ˚C, however can viably grow at up to 40 ˚C. This ability to grow at high temperatures is a feature that can be useful in identifying the fungus in culture. The fungus's ability, an inability, to break down various molecules can also be used to confirm its identity. Madurella mycetomatis is amylolytic yet is only weakly proteolytic, and has the ability to assimilate glucose, galactose, lactose and maltose, while unable to assimilate sucrose. Potassium nitrate, ammonium sulfate, asparagine and urea can also be used by the fungus. Madurella mycetomatis produces 1,8-dihydroxynapthalene a precursor to melanin – a protein extracellularly attached to proteins. Both molecules are responsible for the characteristic dark grain color. The melanin produced by the fungus has also been identified as a defense mechanism against processes such as hydrolytic enzymes, free radicals, redox buffering, antibodies and complement. The fungus also produces pyomelanin, a brown diffusible pigment.
Growth and morphology
The growth of M. mycetomatis is very slow and can be broken down into three stages. Initially the colony is dome shaped white-yellow or olivaceous brown in color. The mycelium is covered in grey down, giving it a woolly texture. Following the initial stage, brownish aerial mycelia (1 to 5 µm) form and the colony starts producing a diffusible pigment called pyomelanin, and becomes smooth in texture. Older colonies form masses of hyphae called sclerotia or grains. In nutritionally deficient or potato-carrot media, black grains (0.75 to 1 mm in diameter) with undifferentiated polygonal cells can be observed.
Grains of M. mycetomatis are hard and brittle, ranging between 0.5 and 1 mm (maximum being 2 mm), with masses from 2 to 4 mm. The grains are oval and often multi-lobed. They are reddish brown to black in color and texturally smooth or ridged.  The grains are made up of an internal mass of hyphae, 2 to 5 µm in diameter, with terminal cells swelling from 12 – 15 µm (maximum being 30 µm) in diameter. Overall two main types of grains are observed. The most common type is compact or filamentous, where a dark brown cement like amorphous, electron rich substance fills the voids surrounding the hyphal network. The hyphal network differs in growth between the cortical and medullar region, with radial versus multidirectional growth respectively. When stained with hematoxylin and eosin it appears rust-brown in color. In contrast, the second type, vesicular, has a light colored medulla and a brown cortical region filled with hyphae and vesicles 6 to 14 µm in diameter. Often it is difficult to determine the transition point from cortex to medulla. Lesions can have both the filamentous and vesicular type grains at the same time.
Although conidation, a form of asexual reproduction, in M. mycetomatis is rare, two main types can be described in-vitro. In the first type oval to pyriform conidia, 3 to 5 µm can be observed. The conidia have truncated bases and are on the tips of simple or branched conidiophores. In-vitro, this type of conidation can be observed in 50 % of cultures on soil extract, hay infusion or water agar. When grown on potato carrot agar or cornmeal agar the second type of conidation is observed. This type is characterized by small spherical conidia (3 µm in diameter) on tapered tips of flask shaped phialides and collarettes. On SDA media M. mycetomatis is sterile. No sexual stage has been identified for M. mycetomatis.
Madurella mycetomatis is the most common fungus with respect to causing mycetoma in humans, a chronic localized inflammatory disease. Madurella mycetomatis accounts for 70 % of mycetoma cases in central Africa, especially common in Sudan. Cases of mycetoma caused by this fungus have also been reported in West Africa, India, Venezuela, Curacao, Brazil, Peru and Argentina. The mode of entry for etiological agents of mycetoma, such as M. mycetomatis, is trauma, such as; snake bites, knives, splinters, thorns and insect bites. Thus having identified M. mycetomatis in soil and anthill samples substantiates its involvement in mycetoma. Infected hosts from samples in Sudan show variability in clinical symptoms, this corroborates the heterogenaity of M. mycetomatis genotypes.
There are various methods available for the purpose of differentiating fungal species. Histological examination allows for the exploitation of unique morphological features of M. mycetomatis, including but not limited to the unique cement like substance and multi-lobed morphology of the grains. Molecular analysis allows for a more sensitive technique to discriminate between morphologically similar species. An internal transcribed spacer (ITS) is amplified by polymerase chain reaction (PCR). Followed by restriction fragment length polymorphism (RFLP) digestion or gene sequencing to obtain the results. The highly variable nature of the ITS sequence between species not only allows for diagnosis, but also the identification of M. mycetomatis in soil samples. ITS has also been cited as the recommended method for isolation of M. mycetomatis. Additionally, molecular analysis lead to the determination that M. mycetomatis did not share a common ancestor with M. grisea, and belonged in the Sordariales class.
Understanding how a fungus might react to various anti-fungal agents in-vitro can be beneficial when wanting to study or isolate particular organisms in culture. The Clinical & Laboratory Standards Institute (CLSI) M38A methods indicate that conidial suspensions are to be used when testing anti-fungal susceptibility of filamentous fungi. When following these methods for M. mycetomatis, alternative suspensions of hyphal fragments are required, as conidial forms are exceedingly rare. Table 1 summarizes the minimal inhibitory concentrations at 90 % (MIC90) for various anti-fungal agents, with specific relation to M. mycetomatis.
|Anti-fungal drug||MIC90 (µg/mL)||MIC50 (µg/mL)||MIC range (µg/mL)|
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