Case 2 -

Nocardiosis consistent with infection due to due to cultured Nocardia caviae (now N. otitidiscaviarum) Randall T. Hayden St. Jude Children's Research Hospital Memphis, TN

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Clinical History The patient was a 74 year old man living in Louisiana with a history of diffuse actininc skin disease, severe arsenic related hyperkeratosis of palms and soles and tobacco related chronic pulmonary disease. He presented with new plaque-like excrescences on his left forearm. He had no lymphadenopathy. A punch biopsy and an excisional biopsy were performed on the forearm lesions. The punch biopsy was submitted for cultures which grew Staphylococcus on most plates and another species on one plate, both interpreted to be contaminants. On gross examination, the excisional biopsy consisted of a 3.7 x 2 cm ellipse of white skin. The surface displayed geographic thickening, pitting and grooving. There were areas of vascular congestion which extended into the cut surface. Representative sections were submitted for histology. Case 2 - Figure 1 - Skin excision specimen showing epidermal hyperplasia, hyperkeratosis, intraepidermal abscess and acute and chronic granulomatous dermatitis. (Hematoxylin and eosin, X4.) Case 2 - Figure 2 - Acid-fast branching filamentous microorganism in an area of necrosis in the dermis. (Fite-Faraco, X100.) Diagnosis: Nocardiosis consistent with infection due to due to cultured Nocardia caviae (now N. otitidiscaviarum) Description: Sections of skin show patchy parakeratotic hyperkeratosis, florid epidermal hyperplasia and epidermal and dermal microabcesses with multinucleated giant cells in the dermis. Sections stained with Brown Brenn tissue gram stain show slender branching filamentous gram-positive bacteria that are not visible on H&E-stained sections. The bacteria do not stain with GMS and are partially acid fast. No grains are seen. There are also gram-positive cocci in the surface keratin. Cultures were positive for Staphylococcus and a Nocardia species that was resistant to all beta-lactamases except imipenem and susceptible to all aminoglycosides, consistent with Nocardia caviae. Discussion: The major genera of actinomycetes that cause disease in humans include Nocardia, Actinomyces, Rhodococcus, Gordonia, Tsukamurella, Actinomadura ,Tropheryma, and Streptomyces. They are characterized by filamentous, partially branching bacteria that reproduce by either spore formation or fragmentation. They have a cell wall composition similar to other bacteria , are all gram-positive; and some, such as Nocardia spp. are partially acid fast. They are susceptible to inhibition by antibacterials. Actinomycetes are widespread environmental saprophytes. The first description of clinical disease caused by actinomyces appeared in 1878, and in 1888, Nocard described Nocardia as a cause of bovine granulomatous disease. Nocardia was first isolated from a human patient in 1890. This organism was designated as Nocardia asteroides in 1896. Waksman and Henrici classified and differentiated Nocardia and Actinomyces spp in 1943. The first description of Actinomadura ("Madura foot") appeared in 1894, and the causative agent was designated as "Streptothrix madurae". Streptomyces somaliensis was first described (as "Indiella somaliensis") in 1906. The genera Tsukamurella and Gordonia are more recent additions to actinomycetes (1988). Cervicofacial actinomycosis due Actinomyces israelii occurs in patients with poor dental hygiene or who have had recent oral surgery. It presents initially with perimandibular soft-tissue swelling that eventually spreads into adjacent tissues if untreated. Fistula formation may occur with the discharge of purulent material containing yellow "sulfur" granules. Other infections due to actinomycetes also result in the formation of grains, and are thus classified as mycetomas. Infections with grains are classified as "botryomycetoma" if the agent is nonfilamentous bacteria or as mycetoma if the agent is filamentous organisms. If the filamentous organisms are bacteria, it is called "Actinomycetoma". "Eumycotic mycetoma" is the term used for mycetoma due to pigmented or nonpigmented fungi. Certain species of the genera Nocardia, Streptomyces, Actinomyces, Actinomadura, and Madurella cause actinomycetoma. The most common anatomic location is the dorsal forefoot, the so-called Madura foot, but actinomycetoma can occur anywhere on the body. Foot and lower leg infections are most common. Hand is the next most common location. Trunk lesions are frequently caused by Nocardia species; whereas, head and neck lesions are usually caused by Streptomyces somaliensis. Bacteria from the environment enter through sites of local trauma. An initial neutrophilic response is followed by granulomatous inflammation. The process spreads through facial planes and sometimes involves the underlying bone. Hematogenous or lymphatic spread is uncommon. Actinomycetes can cause deep or disseminated infection, especially in immunocompromised hosts. Central nervous system actinomycosis occurs as a result of hematogenous spread from a distant site, such as lungs, abdomen, or pelvis, or from direct extension from cervicofacial actinomycosis. Thoracic actinomycosis occurs as a result of aspiration of oropharyngeal secretions, esophageal perforation, direct spread from an actinomycotic process of the neck or abdomen, or hematogenous spread from a distant lesion. Pulmonary infection, if untreated, can spread to the pleura, pericardium, and chest wall, ultimately leading to the formation of sinuses that discharge sulfur granules. Patients with abdominal actinomycetosis usually have a history of colonic perforation as a result of surgery, appendicitis, diverticulitis, trauma or ingestion of foreign bodies. A frequent presentation is a slowly growing tumor in the ileocecal region. Involvement of any abdominal organ, including the abdominal wall, can occur by direct spread, with eventual formation of draining sinuses. Actinomycosis of the pelvis most commonly occurs in association with a longstanding intrauterine contraceptive device.Whipple disease is characterized by the presence of the Tropheryma whippleii organisms within tissue macrophages. The classic presentation is intestine infection, often causing malabsorption. The infection may also infect joints, the central nervous system, or the cardiovascular system. The bacteria ingested by tissue macrophages are PAS-positive in tissue sections. Definitive diagnosis is made by electron microscopy or PCR. Actinomycetes have characteristic morphologic features in tissue sections. They are branching filaments, 1 mm in diameter and may be beaded or fragmented to form what appear to be chains or clusters of bacilli or coccobacilli. They are gram-positive on sections stained with either Brown-Brenn or Brown-Hopps. On sections stained with modified acid fast stains (Fite-Faraco or Coates-Fite) Nocardia species are positive and Actinomyces species are negative. In some infections, granules or grains form. Grains are aggregates of bacteria bordered by Splendore-Hoeppli material. In some cases, a presumptive identification may be made from the morphology of the grains. The grains of Actinomyces species stain intensely with H&E and have short radiating clubs around the periphery. Actinomadura madurae grains are large (1-5 mm) and have fringes that radiate out up to 50 micrometers. The grains of Actinomadura pelletieri stainred or pink on H&E sections. The grains of Streptomyces somaliensis stain poorly with H&E and have smooth surfaces with no radiations. Morphologic features of the bacterial filaments themselves are also useful in making an identification. Streptomyces somaliensis forms spores at the tips of the filaments, whereas the Actinomyces species do not form spores. Aggregates of the bacteria in the form of "sulfur granules" may be seen in direct examination of specimens submitted for culture. As in tissue sections, the actinomycetes are thin, gram-positive, filamentous, branched bacteria. Nocardia spp are partially acid fast with modified Kinyoun stain. Rhodococcus equi may be coccoid or coccobacillary; non-equi species may be more filamentous. For isolation of the organisms in culture, samples should be collected prior to treatment. Actinomycetes are usually facultatively anaerobic, but may be microaerophilic, aerobic, or anaerobic. Growth is supported by most non-selective bacterial, mycobacterial and fungal media. Because they are slow growing, media should be incubated for 2 to 3 weeks. Specimens collected from non-sterile sites may require selective enrichment to isolate actinomycetes, especially Nocardia. Nocardia isolated from culture are partially acid-fast and have a beaded appearance. The acid-fastness may be inconsistent, however, most notable with organisms grown on mycobacterial media. Aerial hyphae may be seen in Nocardia or Streptomyces. Cultures Streptomyces may produce coccoid forms that may be acid fast. Rhodococcus may produce coccobacilli with a "zigzag" appearance and is weakly acid-fast. Antimicrobial susceptibility testing is performed by broth microdilution, E-test, radiometric broth, or agar dilution methods. Optimal treatment depends on identification of the causative organism. The sensitivity of culture is limited, and many species are indistinguishable based on histologic and cytochemical features. Treatment is therefore often empiric. Limited studies have been performed demonstrating the development and use of immunohistochemistry to detect and identify actinomycetes. Antibodies are not commercially available. Most studies primarily used aspirates and smears; few studies used formalin-fixed, paraffin embedded tissue sections. The target antigens are cell wall and cytoplasmic polysaccharides. A limited spectrum of antibodies has been studied, most have been against A. israelii, with cross-reactivity shown among related organisms. Immunohistochemistry is not currently practical for routine diagnostic purposes. In situ hybridization is in use for differentiation of several bacterial, fungal and viral pathogens in tissue. The advantages are rapid turn-around-time, high specificity, improved sensitivity, amenability to automation, and long reagent shelf-life. In one study, 26 cases (13 Nocardia and 13 Actinomyces) were assayed with probes designed against the 16S rRNA genes of Nocardia and common Actinomyces spp. The results of in situ hybridization showed 100% correlation with histochemical stains, morphology, and culture. Although staining was weak in a few cases, there was no evidence of cross-reactivity or non-specific staining. In situ hybridization is a potential adjunctive value to aid in differentiation of Nocardia and Actinomyces spp. Most molecular amplification techniques are aimed at the 16S rRNA gene and variously employ PCR, Restriction endonuclease analysis, sequencing or PCR-RFLP. The HSP gene has also been targeted. The advantages of molecular techniques are a high degree of specificity and rapid turn-around-time compared to conventional methods. These techniques are difficult to adapt for use with tissue sections. References Brown JR. 1973. Human actinomycosis. A study of 181 subjects. Hum.Pathol. 4:319-330. Isotalo PA, Hayden RT, Qian X, Roberts GD, Lloyd RV. 2003. In Situ Hybridization for the Differentiation of Actinomyces and Nocardia in Tissue Sections. United States and Canadian Academy of Pathology Annual Meeting, Poster Presentation. McNeil MM, Brown JM. 1994. The medically important aerobic actinomycetes: epidemiology and microbiology. Clin.Microbiol.Rev. 7:357-417. Pollock PG, Valicenti JF Jr, Meyers DS, Frable WJ, Durham JB. 1978. The use of fluorescent and special staining techniques in the aspiration of nocardiosis and actinomycosis. Acta Cytol. 22:575-579. Steingrube VA, Wilson RW, Brown BA, Jost KC Jr, Blacklock Z, Gibson JL, Wallace RJ Jr. 1997. Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis. J.Clin.Microbiol. 35:817-822. Warren NG. 1996. Actinomycosis, nocardiosis, and actinomycetoma. Dermatol.Clin. 14:85-95.