A combination of appropriate microbiological and pathological studies is essential for proper diagnosis. A high index of suspicion should be communicated to the microbiology diagnostic laboratory, along with material from draining sinuses, from deep-needle aspiration, or from biopsy specimens. It is important to avoid antimicrobial therapy prior to obtaining a specimen. Anaerobic culture is required, and no selective media are available to restrict overgrowth of the slow-growing Actinomyces by associated microflora. The presence, in pus or tissue specimens, of non-acid-fast, gram-positive organisms with filamentous branching is very suggestive of the diagnosis. The characteristic morphology of sulfur granules and the presence of gram-positive organisms within are helpful. In tissue sections stained with hematoxylin and eosin, sulfur granules are round or oval basophilic masses with a radiating arrangement of eosinophilic terminal "clubs." However, Actinomyces species are infrequently visible in sections stained with hematoxylin and eosin; visualization is facilitated by special stains such as Grocott-Gomori methenamine silver, p-aminosalicylic acid, McCallen-Goodpasture, and Brown-Brenn. Multiple biopsy sections from different tissue levels are recommended to improve histopatho-logic diagnosis. The granules must be distinguished from similar structures that are sometimes produced in infections and that are caused by Nocardia, Monosporium, Cephalosporium, Staphylococcus (botryomycosis), and others. Actinomyces and Arachnia generally can be differentiated from other gram-positive anaerobes by means of growth rate (slow), by catalase production (negative, except A. viscosus), and by gas-liquid chromatographic detection of acetic, lactic, and succinic acids produced in peptone-yeast-glucose broth. Direct fluorescent antibody conjugates and immunofluorescence testing can be used but are not readily available to clinical microbiology laboratories.

Imaging methods such as conventional radiography, CT, and magnetic resonance imaging do not provide a specific diagnosis but allow more accurate definition of the dimensions and extension of the infection.

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