Mycetoma A thorn in flesh!!Mycetoma is a slowly progressive, chronic granulomatous infection of skin and subcutaneous tissues

Mycetoma is a slowly progressive, chronic granulomatous infection of skin and subcutaneous tissues with involvement of underlying fasciae and bones, usually affecting extremes
INTRODUCTION
HISTORY
EPIDEMOLOGY
CLASSIFICATION
CLINICAL FEATURES
RADIOLOGICAL FEATURES
LABORATORY DIAGNOSIS

1. Mycetoma A thorn in flesh PRESENT BY DR. RASIKA GUIDE: DR CHAYA

2. CONTEXT INTRODUCTION HISTORY EPIDEMOLOGY CLASSIFICATION CLINICAL FEATURES RADIOLOGICAL FEATURES LABORATORY DIAGNOSIS

3. INTRODUCTION Mycetoma is a slowly progressive, chronic granulomatous infection of skin and subcutaneous tissues with involvement of underlying fasciae and bones, usually affecting extremes

4. HISTORY It is mentioned in Atharvaveda as Padavalmika i.e. anthill foot In 1842, John Gill described this disease for the first time in India in a dispensary at Madurai, T amil Nadu In 1846, Colebrook confirmed Gill’s observation and term ‘ Madura Foot ’

5. HISTORY Carter – 1874 – “ T he Fungus disease of India ”. Henry Vandyke Carter   english anatomist, surgeon, and anatomical artist

6. HISTORY Pinoy in 1913 divided into “ Actinomycetoma ” &“ Eumycetoma ” Chalmers & Archibald - 1918/19 the term Maduromycosis and Actinomycosis Mackinnon -1954/1956 - enumerated and classified agents of these two types .

7. EPIDEMIOLOGY The causative organisms of  mycetoma  are distributed worldwide but are endemic in tropical and subtropical areas in the ‘ Mycetoma  belt ’, (latitudes 15° S and 30° N) which includes the Bolivarian Republic of Venezuela, Chad, Ethiopia, India, Mauritania, Mexico, Senegal, Somalia, Sudan and Yemen.

8. EPIDEMIOLOGY

9. EPIDEMIOLOGY 33.3% 16.8% 13.2%

10. EPIDEMIOLOGY In India, 65% of cases are due to aerobic Actinomycetales mainly A.madurae , N.brasiliensis and few A.pelletieri 35% are mainly due to Madurella mycetomatis . Regional variation Eumycetoma in north India Actinomycotic mycetoma in south I ndia

11. CLASSIFICATION Mycetoma Eumycetoma Actinomycetoma

12. CAUSATIVE AGENTS: B acterial agents Actinomadura madurae Actinomadura pelletieri Nocardia brasiliensis Nocardia caviae Nocardia asteroids Nocardiopsis dassonvillei Streptomyces somaliensis

13. CAUSATIVE AGENTS: Fungal agents Madurella mycetomatis Madurella grisea Curvularia geniculate Madurella pseudomycetomatis Madurella Tropicana Madurella fahalii

14. MODES & SITES OF INFECTION: Any site subjected to traumatic implantation of soil saprophytes into subcutaneous tissues.

15. Cardinal features: T R I A D

16. Draining sinuses G ranules Swelling

17. DISTRIBUTION 17 Global Burden of Human Mycetoma : A Systematic Review and Meta-analysis. Wendy W. J. van de Sande.Published : November 7, 2013

18. AGE WISE DISTRIBUTION Global Burden of Human Mycetoma : A Systematic Review and Meta-analysis. Wendy W. J. van de Sande.Published : November 7, 2013

19. Clinical manifestation Single, small, painless subcutaneous nodule. Nodule slowly increases in size, becomes fixed to the underlying tissue. After several months, the middle of the lesion caves in, ulcerates and develop the sinus tracts which open to the skin surface discharging sanguineous, seropurulent , or purulent exudates containing grains. Deformity develops as the disease spreads through fascial planes to involve and destroy muscles, fascia, and bones in the contiguous area. The affected extremity is grossly swollen with club shaped masses of cystic areas. Muscle and bone destruction can be extensive in advanced disease, causing incapacitation. Secondary bacterial infection is common.

20. CLINICAL FEATURES Actinomycetoma Eumycetoma Causative organism Aerobic actinomycetes Hyaline and phaeoid hyphomycetes Tumor mass Multiple, diffuse with ill defined margins Usually single, with well-defined margins Sinuses Appear early and more in number Appear late and less in number Opening of sinuses Rasied ,inflamed and flared up Flat opening & not flared up

21. CLINICAL FEATURES Actinomycetoma Eumycetoma Flap of opening Easily removed Not easily removed Discharge Usually purulent Serous or sero - sanguineous Grains white Black or white Extent of involvement Most extensive & obliterates with hypertrophic, punched out osteolytic lesions of bone Less extensive ,only osteosclerotic lesions

22. CLINICAL LESION ACTINOMYCETOMA EUMYCETOMA

23. Radiological examination ACTINOMYCETOMA R adiograph of the foot showing multiple lytic lesions involving the mid foot bones and joints

24. Radiological examination The recently described "dot-in-circle" sign on magnetic resonance imaging (MRI) is easy to recognize and highly specific

25. Radiological examination USG showing medial aspect of distal foot showing hypoechoic lesions with central hyperechoic fungal grains (arrow)

26. Differential diagnosis Cutaneous TB Syphilis Yaws Sporotrichosis B otryomycosis, coccidiomycosis , Bacterial osteomyelitis, C hromoblastomycosis , and Bone or soft tissue tumors.

27. • In tissues, M. mycetomatis forms numerous black sclerotia (grains) • Grains are vegetative aggregates of the fungal mycelia embedded in a hard brown matrix • This matrix consists of extra-cellular cement (1,8-dihydroxynaphthalene melanin) in combination with host tissue debris • Rigid matrix might act as a barrier protecting the fungus from the natural immunity of the host and antifungal agents • Melanins are thought to be protective in circumstances of host-induced oxidative stress Pathology of Mycetoma

28. LABORATORY DIAGNOSIS History Occupation Trauma G eographical location

29. LABORATORY DIAGNOSIS Sample: Grains or granules Pus and exudates Biopsy FNAC

30. LABORATORY DIAGNOSIS Methods of collection of grains: Sterile gauze Wire Sterile petri dish photograph showing sequestrum ( circle) being held by the forceps and black coloured granules – which represent colonies in the case of madura foot (arrow ).

31. LABORATORY DIAGNOSIS Gross examination Gross - Size, shape, texture and color of grains provide clue to etiological agents

32. Color of grains Eumycetoma Black grains - Madurella spp., Leptosphaeria spp., Curvularia spp., Exophiala spp., Phaeoacremonium spp., Phialophora verrucosa , Pyrenochaeta mackinnonii , P. romeroi Pale grains (white to yellow) - Pseudallescheria boydii ( Scedosporium apiospermum ), Acremonium spp., Aspergillus spp., Fusarium spp., Neotestudina rosatii Actinomycetoma Pale grains (white to yellow) - Actinomadurae madurae , Nocardia spp. Yellow to brown grains - Streptomyces spp. Red to pink grains - Actinomadurae pelletieri

33. LABORATORY DIAGNOSIS Direct examination Examination of crushed grains/pus/exudate – KOH , Gram staining , Modified AFB Staining

34. LABORATORY DIAGNOSIS Direct examination Gram’s stain: Actinomycetoma Eumycetoma Gram positive Gram negative 0.5-1 filaments 2-5 hyphae Septate fine branching filaments Septate hyphae Stained better with Gram stain Stained better with Gomorimethamine silver or PAS stains

35. Crushed grain Pus or exudate Gram’s stain

36. KOH mount

37. Crush grain Pus or exudate Modified AFB stain

38. Histopathological examination H& E , PAS,GMS Histologically, mycetoma lesions are similar . Granules , often coated with a homogenous material and surrounded by polymorphonuclear cells . Surrounding these abscesses are areas of dense fibrosis and granulomatous inflammation Actinomycetoma -neutrophilic infiltrate surrounding the actinomycotic colony with occasional giant cell ( H and E, ×250)

39. Eumycetoma -thick club shaped structures (<) ( H and E, ×1250)

40. Histopathlogical examination Splendore – Hoeppli phenomenon:- Granules with surrounding radially arranged amorphous eosinophilic hyaline material amorphous , eosinophilic, hyaline  material  ( H&E×100). Fungal element

41. LABORATORY DIAGNOSIS

42. LABORATORY DIAGNOSIS Culture

43. LABORATORY DIAGNOSIS Culture

44. LABORATORY DIAGNOSIS Temperature and conditions for culture For Actinomycotic mycetoma – Blood agar or BHI agar, incubated at 37  C both aerobically and anaerobically For Eumycotic mycetoma – SDA without cycloheximide , incubated at 25 C

45. LABORATORY DIAGNOSIS Actinomadura madurae Granule size :1- 5mm colour : white- pink shape : spherical and angular consistency : soft/ easily crushed

46. LABORATORY DIAGNOSIS Actinomadura madurae Culture: waxy, cerebriform at center with flat peripheral zone, membranous and tough Colour :white to yellow

47. LABORATORY DIAGNOSIS Actinomadura pelletieri Granule size :300-500µm colour : Ruby red shape : spherical/oval, irregular consistency : firmly resistant to pressure

48. LABORATORY DIAGNOSIS Actinomadura pelletieri Culture LJ medium: small with surface variously folded, wrinkled or tuberculated SDA: Covered with short white aerial hyphae Colour : red on LJ medium

49. Haematoxylin and eosin (H&E) stained tissue section showing red grained actinomycotic mycetoma caused by actinomadura pelletierii . (Original sections courtesy Prof. R. Vanbreuseghem , Antwerp, Belgium). Red grains

50. LABORATORY DIAGNOSIS Streptomyces somalinesis Granule size :0.5-1mm colour : white- pink shape : spherical/ lobulated consistency : smooth and hard crushed breaks into angular fragments

51. LABORATORY DIAGNOSIS Streptomyces somaliansis Culture: grows better LJ medium LJ medium: folded/ cerebriform Colour : ochre yellow SDA: grey- brown with diffusible brown pigment

52. . Haematoxylin and eosin (H&E) stained tissue section showing white grained actinomycotic mycetoma caused by Streptomyces somaliensis . (Original sections courtesy Prof. R. Vanbreuseghem , Antwerp, Belgium White grain

53. LABORATORY DIAGNOSIS Nocardia brasiliensis Granule size :<1 mm colour : white- yellow shape : spherical, irregular often lobulated consistency :soft

54. Noca r dia : Modified AFB stain 100X oil immersion

55. LABORATORY DIAGNOSIS Nocardia brasiliensis Culture LJ medium: small heaped up, wrinkled or folded Colour :orange

56. LABORATORY DIAGNOSIS Nocardia asteroides Culture SDA agar without antibiotics: buff or waxy cerebriform chalky white Glucose- neopeptone agar: chalky white colonies at 50  C Colour :chalky white or orange

57. N asteroides : Gram stain 100X oil immersion

58. LABORATORY DIAGNOSIS Nocardia caviae Culture SDA agar without antibiotics: similar to N. asteroids sparse with short aerial hypae Colour :yellow to orange Decomposes xanthine Survive when expose to 50 C for 8 hours

59. LABORATORY DIAGNOSIS BACTERIAL AGENT CASEIN TYROSINE XANTHINE UREASE AFB GROWTH IN GELATIN A madurae + + - - - - A pelletier + + - - - - N brasilensis + + - + + + UTLIZATION OF PHYSIOLOGICAL CHARATERSTICS OF ACTINOMYCETOMA

60. LABORATORY DIAGNOSIS UTLIZATION OF PHYSIOLOGICAL CHARATERSTICS OF ACTINOMYCETOMA BACTERIAL AGENT CASEIN TYROSINE XANTHINE UREASE AFB GROWTH IN GELATIN N caviae - - + + + - N asteroides - - - + + - N dassonvillei + + + + + - S somaliensis + + - - - -

61. LABORATORY DIAGNOSIS Madurae mycetomi Granule size :5 mm colour : brown-black shape : spherical/oval consistency :firm

62. Grains of Madurella mycetomatis (tissue micro colonies ) are brown or black, 0.5 to 1.0 mm in size, round or lobed, hard and brittle, composed of hyphae which are 2 to 5 um in diameter, with terminal cells expanded to 12 to 15 (30) um in diameter Fungal hyphae neutrophils

63. Haematoxylin and eosin (H&E) stained tissue section showing black grained eumycotic mycetoma caused by Madurella mycetomatis . (Courtesy Dr O'Keefe, School of Public Health and Tropical Medicine, N.S.W.). Black grain/ hyphae

64. LABORATORY DIAGNOSIS Madurae mycetomi Culture Developed within few days as a white mycelium which turn to yellow-brown Brown diffusible pigment appears Colour : yellow- brown

65. LABORATORY DIAGNOSIS Madurae pseudomycetomatis Culture Developed within few days as a is slow-growing, granular, and cauliflower-like Brown diffusible pigment appears Colour : yellow- brown

66. Fungal hyphae growing toward the periphery of the grain. PAS staining. Magnification, ×200 .. Madurella pseudomycetomatis

67. Fungal grains seen microscopically in KOH mounts. The grains were composed of many dematiaceous hyphae with some brown, swollen cells . Madurella pseudomycetomatis

68. LABORATORY DIAGNOSIS Madurae grisea Granule size :>1 mm colour :pale center with brown interstitial material shape : spherical/oval consistency :soft when young firm when larger

69. LABORATORY DIAGNOSIS Madurae grisea Culture : 3-5 mm diameter, 8-12 mm high Cerebriform at the center and radially folded toward periphery Mycelial matrix is dark grey covered with grey aerial hyphae Colour : red- brown(old culture)

70. LABORATORY DIAGNOSIS Phialophora jeanselmei Granule size : 1mm colour :brown shape : irregular

71. Phialophora jeanselmei Typical accumulation of the oval to ellipsoidal annelloconidia around the top, and along the sides of the annellophore . (1000X, LPCB)

72. LABORATORY DIAGNOSIS Culture : Initially moist soon becomes velvety Colour : Olive green Phialophora jeanselmei

73. LABORATORY DIAGNOSIS Curvularia geniculate Culture : Rapid growth Woolly texture Whitish colonies becoming olive brown on the surface and reverse Colour : Olive brown

74. Curvularia geniculate Septate hyphae, generally brown in appearance Conidiophores brown, erect, simple or branched. Geniculate producing conidia in sympodial order C onidia have transverse septa,

75. LABORATORY DIAGNOSIS FUNGAL AGENT GLUCOSE LACTOSE MALTOSE SUCROSE STARCH HYDROLYSIS PROTEASE M mycetomatis + + + - + + M grisea + - + + + + P jeanselmei + + + + + - UTLIZATION OF PHYSIOLOGICAL CHARATERSTICS OF EUMYCETOMA

76. . N o reliable serologic test available for diagnosis of mycetoma . Lack of standardized preparation of the antigen(s) has hampered performance of such a Test M any etiological agents of mycetoma require independent testing with several antigens or the use of a polyvalent antigen preparation. S erodiagnosis

77. . Immunodiffusion (ID) and counter immunoelectrophoresis (CIE ) have been the most widely used tests for detection of antibodies in mycetoma patients; both have provided inconsistent results due to poor specificity & sensitivity due to cross reactivity

78. Photograph showing a counter immunoelectrophoresis test with positive bands.

79. Enzyme linked immunosorbent assay (ELISA) is more sensitive and reproducible than ID and CIE but has the limitation that patients from endemic areas may also show elevated antibody titers by ELISA

80. Fructose-bisphosphate aldolase and pyruvate kinase, two novel immunogens in  Madurella mycetomatis   Nele de Klerk  Corné de Vogel Ahmed Fahal  Alex van BelkumWendy W. J. van de Sande Medical Mycology , Volume 50, Issue 2, 1 February 2012, Pages 141-153 Serological assays performed with pure antigens of  M .  mycetomatis  were an ELISA based on the recombinant-produced translationally controlled tumour protein ( TCTP) The Luminex assays based on TCTP, fructose-bisphosphate aldolase (FBA), and pyruvate kinase (PK) . Although patients had higher levels of antibodies, the same levels were also detected in healthy controls, making the techniques unsuitable as diagnostic tools 

81. Due to the use of non-standardized and poorly prepared crude antigens, cross reactivity and false positive and negative results hamper the diagnostic value of these tests  

82. PCR • Targeting ribosomal gene complex with universal primers • Have been detected from biopsy as well as soil samples ITS (internal trasnscriber spacer) region and 5.8S ribosomal DNA

83. RFLP RFLP analysis with Cfol,Haell,Mspl,Sau3Al,Rsal and Spel restriction enzymes .

84. TREATMENT Eumycetoma : oral ketoconazole 200mg twice daily for 3 months Itraconazole 100mg twice daily for 3 months conventional and liposomal formulation of Amphotericin B

85. TREATMENT Actinomycetoma : Sulphonamides 100-200mg/ day single dose for 3-6 months Streptomycin (14mg/kg/day)for first month in addition to long course of TMP-SMX (150mg TMP+ 800mg SMX) Amoxycillin + clavulanate : 500mg for 6 months Amikacin 15mg/kg/day for 3 weeks

86. TREATMENT Welsh regimen Inj . Amikacin 15 mg/kg/iv divided into 2 doses for 21 days. Constitutes 1 cycle 1–3 such cycle at the interval of 15 days were given Tab . Trimethoprim-sulfamethoxazole (7 and 35  mg/kg/day resp ) for a period of 6 months .

87. TREATMENT Modified Welsh regimen Oral rifampicin (10 mg/kg/day) is added as a third drug along with trimethoprime and sulfomethoxazole (T-S), in the welsh regime .

88. Prognosis Response to therapy in 70% to 90% of actinomycetomas 40% rate of cure in Eumycetomas 80% recurrence rate in absence of surgical intervention

89. K e y learning points: An ideal diagnostic tool for mycetoma is lacking. A combination of techniques is always required to reach a diagnosis. The direct microscopy technique is rapid but lacks specificity. Cytological examination of a cytological smear is a rapid and simple tool .

90. Ke y learning points The histopathological technique can give accurate results provided that the grains are available in the tissue section. Grain culture in sterile conditions and in expert hands has high yield. Molecular techniques in well-equipped centres provide an accurate identification of the causative agents of mycetoma . There is an urgent need for simple, accurate, reliable, cost-effective and field-friendly diagnostic tests.

91. …….could dramatically reduce the incidence!!!

92. Causative agent Texture Colour Size Shape Culture Actinomadura madurae soft/ easily crushed white- pink 1- 5mm spherical and angular waxy, cerebriform Actinomadura pelletieri firmly resistant to pressure Ruby red 300-500µm spherical/oval, irregular small with surface folded, wrinkled or tuberculated Nocardia Spp. soft white- yellow <1 mm spherical, irregular often lobulated small heaped up, wrinkled or folded Streptomyces somaliensis smooth and hard white- pink 0.5-1mm spherical/ lobulated folded/ cerebriform

93. Causative agent Texture Size colour Shape Culture Madurella mycetomatis firm 5 mm brown-black spherical/oval white mycelium which turn to yellow-brown Madurella grisea soft >1 mm brown spherical/oval Cerebriform at the center and radially folded toward periphery Curvularia geniculate hard 0.5-1mm brown oval Whitish colonies becoming olive brown on the surface Madurella pseudomycetomatis firm 5mm brown spherical granular, and cauliflower-like Phialophora jeanselmei soft 0.3-0.5mm brown irregular Moist velvety

94. References Rippon JE. Medical Mycology. 3rd edn . London: WB Saunders Company; 1988. pp. 80-118.    Robert Branscomb. Mycetoma : An Overview :CE update microbiology and virology and histology The Fifth International Conference on Mycetoma : 2011  Chander , Jagdish , Textbook of Medical Mycology(Third Edition), © 2011 Dr. Jagdish Chander , Mehta Publishers, New Delhi Duaner . R. Hospenthal . Agents of Mycetoma Ahmed Hassan Fahal .  Review Mycetoma , Khartoum Medical Journal (2011) Vol. 04, No. 01, pp. 514 - 523

95. Thank you!!!