Section 1 -

Mycetoma - Pathology Michel Huerre U.R.E. Histotechnology and Pathology Unit Institut Pasteur Paris XV, France Eric Dannaoui CNR Mycologie et Antifongiques Unité de Mycologie Moléculaire. Institut Pasteur Paris Michel Develoux Tenon Hospital 4 Rue de la Chine Paris XX

Definition: Synonyms: madurafoot, maduramycosis. Mycetomas are chronic, localized, progressive infections of skin and subcutaneous tissues that may extend to muscle and bone. They are caused by exogenous aerobic actinomycetes (filamentous bacteria) and fungi (eumycotic). These microorganisms form grains of various sizes in tissues, these grains forming characteristic patterns, which can be used for diagnosis. This definition excludes some localized infections, such as actinomycoses resulting in granule formation in tissues, and infections caused by endogenous bacteria [1, 2, 3, 4, 5, 6] Epidemiology and geographic distribution: Mycetoma affects more men than women, with a ratio of approximately 5:1. There is no evidence of genetic predisposition and this difference between the sexes seems to be due to the greater exposure of men to the pathogen outdoors. No age group is exempt, but subjects aged between 20 and 40 years are most frequently affected. Most patients have been in direct contact with soil, but people with non-agricultural occupations may also be affected in endemic areas. Mycetoma occurs in the tropical and subtropical countries of Africa, Asia, South America and the Middle East, in areas from 15° south to 30° north of the equator, with a higher incidence in arid zones than in tropical forests. [7, 8, 9, 10] The species of eumycetes and actinomycetes that cause mycetoma are considered to be saprophytic, and persist in the soil from which they are isolated. Contamination mostly occurs through thorn pricks, generally on the legs, although other sites may be affected. The geographic distribution of the species involved has been studied for many years and the most frequently implicated species are listed in Table 1. It has been suggested that rainfall affects the distribution of etiologic agents. M. mycetomi, M. grisea, Leptosphaeria senegalensis, Actinomadura pelletieri, and Streptomyces somaliensis are observed in West Africa, which tends to have lower rainfall levels (50-800 mm per year), whereas Nocardia (Nocardia brasiliensis commonest in Mexico) is the predominant genus in South America, where precipitation levels are higher (2000 mm per year). Clinical patterns The clinical diagnosis of mycetoma is generally easy, for two reasons. First, the clinical presentation is usually monomorphic: chronic subcutaneous tumor with discharging sinuses, mostly in 20- to 40-year-old male patients. The presence of grains, visible to the naked eyes, in the discharged pus, provides additional support for the diagnosis. Second, most patients in endemic zones consult at a medical center an average of five years after the onset of symptoms. At this time, the lesions are typical. Clinical diagnosis may be difficult for extra-pedal localizations (20-30 % of cases in Africa) or cystic forms. When the diagnosis is clear, two questions must be addressed before microbiological and radiological examinations: is the mycetoma due to actinomycetes or fungi? Are there any complications and what is the extent of the disease? Various clinical presentations have been described, including black grain mycetoma (always eumycetoma), and actinomycetoma, and this classification is generally confirmed by histology (Table 1). However, the clinical presentation is not always sufficient to distinguish between etiological agents. Some complications, such as bacterial infections in patients with lymphatic involvement and discharging sinuses, are easy to diagnose. Mycetoma is usually painless. If the patient reports spontaneous pain, bone involvement should be suspected. Pathology: For all types of mycetoma, the tissue reaction is generally non specific, showing both pyogenic and granulomatous patterns. Abscess formation and necrosis with polymorphonuclear cells within the center of the mycotic granuloma may be seen, surrounded by lymphocytes and plasma cells. Fibrosis occurs in late lesions. A characteristic feature of mycetome is the presence of grains, which may or may not contain cement material, on sections. Indeed, the appearance of these grains on histological sections is so characteristic for several species that microscopic examination of hematoxylin and eosin-stained material can be used to identify the pathogen to species level in some cases. The presence of pigment is very useful for diagnosis. Black grains are always fungal. White grains may be caused by fungi or actinomycetes, but Eumycotic hyphae are generally visible within the grain at x250 and x400 magnifications, whereas actinomycete hyphae are not generally observed. Gomori's methenamine silver (GMS) staining is generally required to determine whether a grain is actinomycotic or eumycotic. Small grains are most frequently result from infection with Nocardia. The granules may be surrounded by club-like projections of eosinophilic Splendore-Hoeppli material and organic foreign material, such as thorns or splinters, may be seen. We indicate below the morphological patterns associated with the most frequent agents of mycetoma within tissue sections [2, 3, 4, 5, 6, 7, 8, 9, 10] (Table 1). Table 1: Classification, patterns and species I- Eumycetoma 1-1 Black grains: Frequent: Madurella mycetomi: 0.5-4 mm, brown pigment throughout its matrix, chlamydoconidia; two types of granules: a) compact, filamentous with cement throughout the granule, uniformly brown and b) vesicular type, irregular, cement and chlamydoconidia in the periphery. Madurella grisea: 0.3-0.6 mm grains, brown at the periphery, chlamydoconidia. Leptosphaeria senegalensis: 0.5-2 mm, larger than M. grisea, cement and black pigment at the periphery. Infrequent: Exophiala jeanselmei, Leptosphaeria tompkinsii, Pyrenochaeta (P) romeroi, P. mackinnonii, Chaetosphaeronema larense, Curvularia geniculata, C. lunata, Corynespora cassiicola, Plenodomus arramii 1-2 White grains: Frequent: Pseudallescheria boydii, Fusarium, Acremonium Rare: Neotestudina rosatii, Acremonium kiliense, A. recifei Aspergillus nidulans, Cylindrocarpon (C.) cyanescens, C. destructans Debatable: dermatophytic infections. Epidermophyton, Microsporon and Trichophyton [12] Actinomycetoma (no cement, except Streptomyces somaliensis) Frequent: Actinomadura madurae: grain 0.5-5 mm, dark violet or white-gray. Periphery of granules intensely hematoxylinophilic. A. pelletieri: 0.0-0.5 mm, the entire grain is red. Streptomyces somaliensis: 0.2-3 mm, regular, round or oval, with gray cement. Nocardia asteroides & brasiliensis. Small grains, white, 25-200 m Ziehl stain-positive. Rare: Nocardiopsis dassonvillei. N. transvalensis, N. otitiscaviarum Differential diagnosis On physical examination, the nodular and verrucous, often monstrously deformed lesions may be confused with chronic osteomyelitis, osseous tuberculosis, endemic Kaposi disease, other subcutaneous mycoses, soft-tissue tumors and various neoplasms, including skin metastasis. However, the diagnosis of mycetoma is generally obvious in endemic regions, unless only early lesions are present. On histological examination, the main differential diagnosis is bothriomycosis, a chronic, localized bacterial pseudomycosis of the skin and subcutaneous tissues caused by non filamentous gram-positive (cocci) and gram-negative bacteria (Enterobacteriaceae,) forming compact granules. These granules may be surrounded by Splendore-Hoeppli eosinophilic material. Histological differentiation can be achieved by tissue gram staining (Brown-Hopps and Brown- Brenn procedures). Although each species or group of species is characterized by a specific type of granule, definitive identification depends on isolation and characterization of the organism in culture. Very few specific antibodies are available (with the exception of antibodies against P. boydii, the most common agent of eumycotic mycetoma in the USA). Molecular techniques have recently been used. Molecular identification of fungal mycetoma agents [13, 14] The identification of black-grain mycetoma agents with standard mycological procedures is difficult because of poor or delayed sporulation. It is also time-consuming and requires expertise restricted to a small number of reference laboratories [13]. New diagnostic tools are therefore needed. Only a few studies have focused on the molecular identification of these fungi [14]. Recent studies have aimed to develop new molecular tools for identification of the various agents of mycetoma, particularly those responsible for black-grain mycetoma. PCR amplification and sequencing of the ITS1-5.8S-ITS2 region DNA has been shown to be a reliable method for identifying the causal agents to species level. A study of 56 strains made it possible to construct a sequence database and showed a low level of within-species variability for Madurella mycetomatis, Leptosphaeria senegalensis and L. thompkinsii, Curvularia lunuta and Exophiala jeanselmei. In contrast, strains of Madurella grisea and Pyrenochaeta romeroi seem to be more heterogeneous. These new molecular tools may facilitate the faster, more reliable identification of these poorly sporulating species and could be used to reassess the epidemiology of fungal mycetoma. Session Mycetoma. M.R. HUERRE, Institut Pasteur, Paris, France Case 1 Mauritania. 29-year-old man. Large tumor of the foot of five years' duration, producing large amounts of black grains. Pigment+++ Amputation of the whole foot required due to bone involvement. Case 2 Senegal. Man, 32 years old. Tumor of the knee, with fistulae and small grains. Biopsy. Case 3 Niger, 34-year-old man. Large, diffuse infiltration of the back of the neck. Biopsy. Case 4 Ethiopian woman, 35 years old, voluminous tumor of the foot. Case 5 Ethiopian man, farmer, 38 years old. Large tumor of the hand with numerous fistulae. Case 6 Brazil. Man, 31 years old, farmer. Tumor of the foot with fistulae. References Maghoub ES, Murray IG, Mycetoma, London, William Heinemann Medical Books, 1973 Winslow D.J. Mycetoma in : Baker RD et al eds. The pathologic anatomy of Mycoses. Human Infection with Fungi, Actinomycetes and algae. New york, Springer-Verlag, 1971, 589-613 Chandler FW, Kaplan W. Ajello L. A colour atlas and textbook of the Histopathology of Mycotic diseases, London, Wolfe medical publications, 1980, 76-82:222-239 Chandler F.W., Ajello L. Mycetoma ; Chapter 113:1035-1044. In Pathology of Infectious diseases. DH Connor, FW Chandler, H.J. Manz, JK Baird, D.A. 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