Identification of the agent is important to guide treatment. Goodfellow and colleagues have described a stepwise procedure from the collection of specimens through to the isolation and identification of established causal agents of actinomycetoma.
In order to identify the causative agent, grains need to be isolated from the lesion. Grain material can be obtained by surgical excision or by extraction with a cotton swab from the sinuses or by fine needle aspiration (FNA). Deep-seated grains obtained by surgical excision are preferred over those extruded through sinuses because the latter are often inviable and contaminated.
A first indication of the causative agent is obtained by looking at the grain itself: the size, shape, color and consistency aid in identification of the organism; however, causal agents must be isolated for definitive identification. In direct examination, grains are mounted on a slide and crushed under a cover glass. The size of the filaments, septation, morphological characteristics and pigment formation can be used to differentiate between actinomycetoma and eumycetoma.
In actinomycetoma, fine filaments are seen and can be stained with Gram stain. In eumycetoma the filaments stain with Periodic Acid Schiff (PAS). Grains are plated onto appropriate culture media and incubated for at least 6 weeks. Identification based on colony morphology can be difficult, due to a large variety of colony types and to the fact that many species resemble each other. Lack of conidiation can further complicate matters.