Eumycetoma

This presentation offers a concise yet comprehensive exploration of Eumycetoma, a rare and complex fungal infection. Delve into its origins, clinical manifestations, and contemporary treatment approaches. An invaluable resource for healthcare professionals and enthusiasts keen on understanding and addressing this intriguing medical condition.

1. WELCOME DR. SOURAV MD Resident, Phase-A, Year-1, Department of Microbiology & Immunology, BSMMU

2. EUMYCETOMA

3. WHAT IS MYCETOMA ? Mycetoma is a chronic granulomatous, progressive inflammatory disease that involves the subcutaneous tissue after a traumatic inoculation of the causative organism Clinically, it is manifested as a triad of swelling, discharging sinuses and presence of granules in the discharge Mycetoma is also known as Maduramycosis or Madura foot

4. HISTORY The first reference to mycetoma dates from the Byzantine period (300-600 A.D.), with evidence from an adult skeleton whose morphological bone structure was suggestive of a mycetoma infection First written account was found in an ancient Indian religious book,  Atharva Veda , where it was mentioned as “Anthill foot“ First described in Madras, South India, by a French missionary John Gill in 1842 naming it "Madura foot"

5. EPIDEMIOLOGY Mycetoma is found worldwide, but endemic in tropical and subtropical countries, mostly between the latitudes 15° south and 30° north, called “mycetoma belt” The endemic areas are characterized by short rainy seasons with little daily temperature fluctuations, followed by long dry seasons with broad daily temperature fluctuations (45-60°C to 15-18°C) Actinomycetoma is more prevalent in drier areas and eumycetoma in humid areas Male are prone to infection due to exposure to field work

6. In 1913, Pinoy observed that the disease was caused by two etiologic agents and divided the disease into two categories : Actinomycetoma (bacterial agents) and Eumycetoma (fungal agents) CLASSIFICATION

7. AGENTS OF MYCETOMA

8. CLINICAL MANIFESTATIONS Hallmark of mycetoma is presence of clinical triad consisting of: Tumor like swelling, i.e. tumefaction Discharging sinuses Discharge oozing from sinuses containing granules Feet are the most common site affected, although any site can be involved May be involvement of underlying fasciae and bones, producing osteolytic or osteosclerotic bony lesions

9. EUMYCETOMA ORGANISMS Most of the agents causing eumycetoma are saprotrophic environmental fungus Taxonomically these fungi are members of either Deuteromycetes or Ascomycetes About 25 fungal species causing eumycetoma Produce two types of grains: Black and White Black Grain Eumycetoma: Madurella mycetomatis Madurella grisea Exophiala jeanselmei Curvularia geniculata White Grain Eumycetoma: Pseudallescheria boydii Aspergillus nidulans Acremonium falciforme Fusarium species

10. PATHOGENESIS No known vector or animal reservoir The disease most likely initiates after a minor trauma that inoculates the causative microorganism in the subcutaneous tissue After inoculation of the causative agent, a subcutaneous infection develops The is usually painless and processes slowly, over months and years

11. PATHOGENESIS CONTD… Painless subcutaneous firm nodule observed Trauma of skin Massive swelling with skin rupture, and sinus tract formation Old sinuses close and new ones open, draining exudates with grains. Grains may sometimes be seen with naked eye

12. LABORATORY DIAGNOSIS Diagnosis of mycetoma is based on clinical presentation and identification of the etiologic agents Detailed and proper history about occupation, trauma , geographical area of the patient is valuable in diagnosis The most specific diagnostic tool is the examination of the grains discharged from sinuses

13. SPECIMEN COLLECTION Clinical sample is usually grains or granules Pus , exudates or biopsy may also be taken The lesions should be cleaned with antiseptics and the grains should be collected on sterile gauze by pressing the sinuses from periphery or by using a loop The samples can be obtained from any open sinus or by deep surgical biopsy

14. FINE-NEEDLE ASPIRATION CYTOLOGY Fine-needle aspiration cytology (FNAC) with cell blocks is also used for specimen collection In this technique, a needle attached to a syringe is inserted into the suspected mycetoma lesion and aspirated under negative pressure It should be performed in at least 3 different directions Figure: Fine-needle aspiration cytology collection technique

15. DIRECT EXAMINATION Granules are thoroughly washed in sterile saline; crushed between the slides and examined Macroscopic appearance of granules such as color, size, shape, texture may provide important clue to identify the etiological agent Grains contain fungi in KOH wet mount or on histopathological examination

16. MACROSCOPIC APPEARANCE Madurella mycetomatis  has black , big grains of 5mm or more. Initially, colonies are yellow or brown with a ridged surface and are frequently covered by a short aerial gray mycelium Madurella grisea  has black , oval or irregularly shaped grains that measure ≥ 1-2mm Scedosporium apiospermum  has hyaline white grains measuring 2-4 mm in diameter that vary in shape. Grains are soft and have septate hyphae and chlamydospores Madurella mycetomatis   Scedosporium apiospermum  

17. Figure: Surgical biopsy showing a well-encapsulated eumycetoma lesion with numerous black grains

18. MICROSCOPY KOH Preparation: Add few drops of KOH and grains on glass slide Put coverslip Crush gently by applying pressure over coverslip with glass rod or handle of loop Leave the wet mount for few hours in petridish with moist filter paper Examine under microscope to look for characteristic hyphae Grains are subjected to KOH mount , which reveals hyphae of 2–6 μm width along with chlamydospores at margin

19. Figure: KOH wet mount direct microscopic examination of M. mycetomatis grains showing its hyphal structure

20. HISTOPATHOLOGICAL STAINING Tissue sections stained with Hematoxylin & Eosin staining (H&E) and by special staining techniques, such as the Periodic-acid-Schiff (PAS) and Grocott-Gomori silver staining It reveals distinct cytological features, characterized by the presence of suppurative granulomas surrounding the characteristic grains of the causative organism

21. Figure: Eumycetoma (black grain and cement like substance) in hematoxylin-eosin staining

22. CULTURE Granules obtained from deep biopsies are the best specimen for culture as they contain live organisms The eumycetes culture must contain antibiotics Sabouraud’s dextrose agar media supplemented with antibiotics eg. penicillin G (20 U/ml), gentamicin sulphate (400 μg/ml), streptomycin (40 μg/ml), or chloramphenicol (50 μg/ml) used for culture

23. CULTURE CONTD… Washed and crushed granules are inoculated in several tube of SDA media The media incubated in 25°C and 37°C temperature

24. COLONY M. mycetomatis: Grows in Optimum temp. 37° valvatid or powdery. Usually cream color Diffuse brown pigment present M. grisea: Better grow at normal temperature Dark leathery, folded colony Acremnium falciforne : White colony with diffuse violet pigment Figure: M. mycetomatis growth in SDA media

25. SERODIAGNOSIS The tests are: Immunodiffusion Indirect hemagglutination assays ELISA etc. S erological assays have been developed only for M . mycetomatis and Pseudallescheria boydii For P . boydii , both an ID (Immunodiffusion ) assay with crude antigens and an IHA (indirect hemagglutination assays) were developed

26. MOLECULAR-BASED IDENTIFICATION METHODS PCR The technique based on the identification of the internal transcribed spacer (ITS) In order to identify all fungal mycetoma causative agents, the ITS regions are usually amplified with pan-fungal primers and sequenced Identification is based on comparing the resulting sequence with sequences already present in GenBank

27. FLOW CHART FOR THE DIAGNOSIS OF MYCETOMA

28. TREATMENT Management of mycetoma is highly challenging for the clinicians Many drugs have been tried with variable results Oral Ketoconazole 200mg twice daily and Itraconazole 100mg twice daily for 8-24 months Amphotericin B is recommended for Madurella and Fusarium species 

29. DIFFERENCE BETWEEN EUMYCETOMA AND ACTINOMYCETOMA Clinical manifestations Eumycetoma Actinomycetoma Tumor Single, well-defined margins Multiple tumor masses, ill-defined margins Sinuses Appear late, few in number Appear early, numerous with raised inflamed opening Discharge Serous Purulent Grains Black/White White/Red Bone Osteosclerotic lesions Osteolytic lesions Grains contain Fungal hyphae (> 2 um) Filamentous bacteria (< 2 um)

30. QUESTIONS? What is Eumycetoma? What are the etiological agents for eumycetomas? Difference between Eumycetoma and actinomycetoma? How to prepare grains sample for microscopic examination?

31. THANK YOU