X-ray :

xray

Molecular Identification of Mycetoma Causative Organisms:

Most of the fungi that cause mycetoma grow slowly and culture plates should be incubated for at least four weeks before being discarded as negative. Identification of the isolated fungus is based on thegross morphology of fruit bodies and conidia, if present. Conventional mycological identification is difficult in some species such as T. grisea, Madurella mycetomatis, Neotestudina rosatii, Pyrenochaeta mackinnonii, and Pyrenochaeta romeroi. Most of these species donot sporulate readily, and identification is based on molecular techniques, particularly sequencing of the rDNA Internal Transcribed Spacer (ITS) region.

The molecular diagnosis of a number of eumycetoma causing agents has been developed. The black yeast Exophiala jeanselmei and Phaeoacremonium are recognisable by sequence data of the rDNA Internal Transcribed Spacer (ITS) region.  SEQ CHAPTER \h \r 1rDNA Restriction Fragment Length Polymorphism and specific ITS-PCR were used for species identification of Madurella mycetomatis. Although Scedosporium apiospermum cultures can be identified by culture morphology, PCR-based assays for rapid diagnosis of infections from infected tissue was found useful.

Ahmed SA, van de Sande WW, Desnos-Ollivier M, Fahal AH, Mhmoud NA, de Hoog GS. Application of Isothermal Amplification Techniques for the Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma. J Clin Microbiol. 2015 Aug 5. pii: JCM.01544-15.

Ahmed AO, Mukhtar MM, Kools-Sijmons M, Fahal AH, de Hoog S, van den Ende BG, Zijstara, ED, Verburgh H, Abugroun ESA, EL Hassan AM, van Beklkum A. Development of a species-specific PCR RFLP procedure for the identification of Madurella mycetomatis. J Clin Microbiol. 1999; 37(10):3175-8.

 

Histopathology of Mycetoma:

The identification of Mycetoma causative agents using histopathological reporting has been performed since 1950s. Interestingly, in many cases, the surgical biopsy may be the only tool to establish the diagnosis of mycetoma especially when the lesion is small. Various stains can be used for microscopical diagnosis of the mycetoma for the identification of the causative organism and for demonstration of the host reaction against to causative organism. Hematoxylin and eosin stains is the commonest stain in use, also Periodic acid Schiff (PAS) can be used especially when studying the pale grains of mycetoma; gram stain is valuable in the identification of Nocardia and Streptomyces spp.

Processing of the sample toward Histopathological diagnosis of Mycetoma:

Excisional biopsy

This is one of the techniques that used for obtaining a biopsy from the lesions and it provided that the entire lesion is completely and deeply removed till the subcutis plane, after that the wound is then closed by sutures.

Minimal handling of tissue

Transfer of tissue from the biopsy wound to the formalin container should be carried out skillfully, with minimal handling of tissue. When the tissue is crushed between the blades of forceps, the tissue is compressed laterally giving rise to compression artifact after processing.

Washing the specimen off excess blood

Blood collected on and around the biopsy specimen should be washed with normal saline.

Fixation of tissue biopsy:

After obtaining the tissue it should be immediately immersed in 10% neutral buffer formalin after that transported to the histopathology lab for further processing for the preparation of tissue blocks and histopathological examinations.

Grains appearance:

Microscopical appearance:

Histopathological Appearance of Grains Using H and E staining technique:

Eumycetoma:

Madurella mycetomi grains tend to be large, light to dark brown in color, irregular outlines and tend to be fracture. There are three structural forms of the grains; filamentous, vesicular, and mixed. The size of the grain ranges between 200 and 900µm in diameter. They are rounded to oval in shape; some of them may be bilobated or trilobated. The grains look uniform each consists from outer cortex and inner medulla. The hyphae of the filamentous grains are large, segmented and contains chlamydospores, both terminal and intercalary.

The whole grain is embedded in hard brown cement like material that is produced and secreted by the organism. Electron microscope examination of the cement material show it consist of an amorphous electron dense material with area containing small to large membrane bound vesicular inclusion.

The hyphae have a thick well defined wall which measured about 2µm, spectated and branched. Some of the hyphae appear empty and in some eosinophilic granular materials are found. The chlamydospores are large and measured about 15µm and their cell wall is thick and eosinophilic.

In the less common vesicular form of the grains, the whole grains is composed of a large cell about 7µm in diameter, the brown pigment is retained but is more prominent in the cortex part compared to medullary part. Vesicular grains are rare and the cause of the grain vesiculation in unknown.

hysto 1

Photo shows a vesicular type of grain. The hyphae are mainly in the periphery and are swollen. The center of the grain consists of cement substance (H&E×40).

Actinomycetoma:

Nocardia brasiliensis: the grains of Nocardia brasiliensis is failed to be stained with routine H and E staining technique, they are none pigmented. Its shape is varying from small clump like a ball to large irregular clumps with irregular outlines. The grains can easily be differentiated from that of other actinomycete in that it is Ziehl neelson (ZN) positive.

hysto 2

Photomicrograph showing Nocardia in tissue section surrounded by inflammatory cells (Zeilh Neelson X40).

Streptomyces Somaliensis: the grains are rounded to oval in shape, with homogenous appearance in tissue section, they appear as faint yellow in unstained section, and the grains are not well stained with H and E. Moreover, as a result of sectioning they may show longitudinal cracks, the hyphae are fine (measured between 0.5 - 2µm in diameter), closely package and embedded in cement matrix.

hysto 3

Photomicrograph showing Streptomyces Somaliensis in tissue section surrounded by inflammatory cells (H&E X10).

Actinomadura Pelletieri: the grains are smalls round to oval and semicircular and sickle like where been observed, the filamentous structure are pretty difficult to be detected however a careful and meticulous examination to the periphery of the grains may show some of them, this type of the grains have a characteristic of deep violet stain using H and E which allow the definitive diagnosis without a need for culturing techniques.

hysto 4

Photomicrograph showing Actinomadura Pelletieri in tissue section (H&E X10).

 
Siddig EE, Mhmoud NA, Bakhiet SM, Abdallah OB, Mekki SO, El Dawi NI, et al. (2019) The Accuracy of Histopathological and Cytopathological Techniques in the Identification of the Mycetoma Causative Agents. PLoS Negl Trop Dis 13(8): e0007056.

 

 

Magnetic Resonance Imaging of Mycetoma:  

MRI

MRI is a non-invasive imaging, it can characterise the soft tissue masses of mycetoma and aid in early diagnosis. In mycetoma, the MRI appearance is in the form of the dot-in-circle sign which is aunique pathological feature of mycetomaand it is highly specific sign for mycetoma. In advanced cases, MRI is essential to plan the treatment options.

 EL Shamy, ME, Fahal AH, Shakir MY, Homedia MMA. New MRI Grading System for the Diagnosis and Management of Mycetoma. Trans R Soc Trop Med Hyg. 2012; 106(12):738-42.