CASE REPORT

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A 57-year-old male patient from Belgaum, south India, had sustained a trauma by a thorn 10 to 12 years earlier. The thorn was removed but a pustule developed at the site some time after the injury. The pustule discharged serosanguineous fluid and eventually healed. Later, new lesions developed, and this process was repeated over a period of 11 years. In 1990, when first seen by the senior authors, the patient was admitted to the hospital with slight swelling of the left foot, pain, and draining sinuses and was diagnosed as having pale white grain eumycetoma based on histopathologic examination of the biopsy tissue specimen and isolation of a hyaline, hyphomycetous fungus that could not be identified at that time.

During his most recent hospital admission in June 1997, the left foot was swollen and the skin over the foot was darkly pigmented. There were three discharging sinuses, two on the dorsum and the third on the plantar aspect of the foot, and several scars were visible. The sinus openings were slightly elevated from the surrounding tissue, showing a small amount of discharge (Fig. 1). The foot was hard and woody to the touch. There were no signs of acute inflammation. A general physical examination was normal. The X-ray examination of the foot showed no bone involvement but showed soft tissue swelling. Routine clinical chemistry and hematological investigations and serology for human immunodeficiency virus were within normal limits.

View larger version (105K): [in this window] [in a new window]   FIG. 1.   Swollen left foot of the patient, showing discharging sinuses.

After a thorough cleaning of the lesions, a Pasteur pipette was introduced into one of the sinuses and the fluid was aspirated. The aspirated material was rinsed in sterile saline and allowed to settle. A few creamy white, soft, white granules measuring 0.5 to 1.2 mm in diameter were observed. The granules were separated, washed in sterile saline, and when examined microscopically in KOH mounts, showed hyaline, septate hyphae, and chlamydospores. Several washed granules were cultured on plates of Sabouraud dextrose agar with chloramphenicol (Sab+C) incubated at room temperature (25 to 30°C). Fungal growth was apparent after 12 days of incubation. Colonies were slow growing, white, and downy, producing a light wine-reddish pigment which diffused into the medium. Microscopic examination of the slide culture preparations on potato dextrose agar after 3 weeks of incubation at 25°C showed sterile, septate, hyaline hyphae. Attempts to induce sporulation were unsuccessful. Two unstained histologic slides and a subculture were sent to one of us (A.A.P.) for further examination.

Histopathologic examination. The slides were stained by hematoxylin and eosin and Gomori's methenamine silver stain procedures. The overlying epidermis was intact and of normal thickness. In the deep dermis and subcutaneous tissue, there were multifocal abscesses and burrowing sinus tracts, surrounded by varying numbers of epithelioid histiocytes and multinucleated giant cells. The granulomatous component blended imperceptibly with peripheral granulation tissue that was infiltrated with mixed inflammatory cells. The suppurative centers of abscesses and sinus tracts contained discrete, spherical, oval and lobular mycotic granules, 0.5 to 1.0 mm in diameter, composed of compact, interwoven, hyaline hyphae and fewer vesicular, thick-walled chlamydospores up to 15 µm in diameter. Hyphae that formed the granules were branched, septate, 4.0 to 6.5 µm wide, and often radially oriented, especially at the periphery of the granules where fungal elements, including chlamydospores, were more numerous and compact. The central portions of some of the granules consisted of a loose network of disintegrated, poorly stained, randomly oriented hyphae and intact or degenerated inflammatory cells. Cement-like substance was not observed within the matrix of the granules (Fig. 2). Each granule was entirely or partially bordered by brightly eosinophilic, dentate to smoothly contoured, Splendore-Hoeppli material. Mycelial elements sometimes extended into this material, which was intimately surrounded by intact neutrophils. There was no histologic evidence of a coexisting infection, and foreign materials such as thorns were not detected either within or near the granules.

View larger version (138K): [in this window] [in a new window]   FIG. 2.   Section of granule showing a loose network of branched, septate hyphae and vesicular chlamydospores in peripheral area of the granule, Gomori methenamine silver stain. Magnification, ×250.

Mycology. The isolate was given accession number CDC 534-96. Colonies on Sabouraud dextrose agar were slow growing, raised in the center, downy, and white at first, becoming grayish buff after 2 weeks at 25°C. They measured 6 to 7.5 mm in diameter after 2 weeks at 25°C and produced reddish brown pigment on the reverse of the colonies that diffused into the medium. Colonies at 37°C measured 3.5 to 4.5 mm in diameter. The isolate did not grow at 40°C. Microscopic examination of slide culture preparations on potato dextrose agar after 3 weeks at 25°C showed hyaline, septate, branched hyphae 2.5 to 5.0 µm wide, producing chlamydospores but devoid of conidia.

View larger version (89K): [in this window] [in a new window]   FIG. 3.   Colony of Cylindrocarpon sp. (UAMH 8935) on potato dextrose agar incubated at room temperature. (Top) After 11 days, showing presence of some diffusible pigment. (Bottom) Same plate after 3 months, showing strong reddish brown diffusing pigment.

View larger version (78K): [in this window] [in a new window]   FIG. 4.   Microscopic morphology of Cylindrocarpon sp. (UAMH 8935). (A) Septate phialide and cylindrical conidia that formed on oatmeal salts slide culture after 14 weeks. (B) Chlamydospores formed on Takashio agar after 22 weeks. Magnification, ×580.

Discussion. Eumycetoma is a long-term, slowly progressing infection that develops in individuals usually following traumatic implantation of fungus-contaminated plant material. Thirteen species of fungi (3 belonging to the Ascomycota and 10 belonging to the Deuteromycota) are known to cause white grain eumycetoma (12). Some of these fungi can be difficult to identify in vitro because of poor sporulation, and we know little about their plant hosts, on which they may potentially produce conidia of their life cycle. Host response to the presence of the fungus and adaptation to growth in human tissue (including bone) may induce phenotypic changes, resulting in isolates which appear atypical in culture. Similar aberrant morphology characterized by absence of conidia and restricted colonies has been observed particularly among isolates of Aspergillus fumigatus and other species of Aspergillus recovered from patients with chronic infections (13; L. Sigler, M. A. Viviani, U. Magrini, R. Epis, V. Fregoni, A. Grancini, and A. Pastorini, International Society for Human and Animal Mycology, abstr. P396, 1997).