Clinicopathological and ultrastructural characterization of periapical actinomycosis

Abstract

Background: The aim of the present study was to analyze the clinicopathological and the ultrastructural features of periapical actinomycosis (PA) cases.

Material and methods: Data from the files of an oral pathology laboratory were retrieved and the findings of histopathological analysis were evaluated. Hematoxylin-eosin (HE), a modified Brown & Brenn, and Grocott stains as well as ultrastructural analysis using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) were utilized.

Results: Six cases were obtained, 4 females and 2 males, with a mean age of 34 year-old. Two cases were symptomatic, lower teeth and the anterior region were more commonly affected, and all cases were characterized by periapical radiolucencies. All cases presented sulfur granules with a ray-fungus or club-shaped pattern of the Splendore-Hoeppli phenomenon in HE-stained sections, with filamentous gram-positive bacteria aggregates highlighted by the modified Brown & Brenn stain. SEM analysis revealed abundant packed rod-like and filamentous bacteria associated with an extracellular amorphous material. EDX analysis showed predominant picks of calcium and sulfur in actinomycotic colonies.

Conclusions: Our findings suggest that PA manifests either clinically and radiologically as a non-specific and heterogeneous condition and that the actinomycotic colonies consist in a calcium- and sulfur-rich matrix. Furthermore, the results highlight the importance of submitting periapical specimens after surgical removal to histopathological analysis.

Figure 1

Clinical and gross analysis. (A) Intraoperative view of case 4, showing an intraosseous cystic lesion emerging between the right lower central and lateral incisors roots. (B) Preoperative periapical radiograph showing a well-defined radiolucent lesion involving both periapical regions, which presented well-performed endodontic treatment. (C) Gross image of the surgically-removed specimen from the same case, showing a brownish and ovoid mass. (D) It can be observed a yellowish sulfur granule filling the whole cystic cavity of the lesion after the gross section.

Figure 2

Histopathological analysis. (A) Actinomycotic sulfur granule, on the left, surrounded by an intense infiltrate of inflammatory cells (HE; original magnification, ×50). (B) In detail, the periphery showing the ray-fungus appearance with the Splendore-Hoeppli phenomenon and mainly neutrophils as well as some plasma cells, macrophages and lymphocytes in close contact with the bacterial colony (HE; original magnification, ×100). (C) A periapical granuloma displaying a central actinomycotic colony with the Splendore-Hoeppli phenomenon (HE; original magnification, ×50). (D) In a high power view, it is possible to see the radiating club-pattern at the periphery of the sulfur granule with intermixed and surrounding immune cells (HE; original magnification, ×400).

Figure 3

Histochemical staining and ultrastructural analysis. (A) Brown & Brenn’s stain showing dense aggregates of filamentous actinomyces in the periphery (Taylor’s modified Brown & Brenn; original magnification, ×200). (B) Grocott’s stain showing argyrophilic filaments compatible with Actinomyces at the periphery of a sulfur granule (Grocott stain; original magnification, ×200). (C) The central area of a sulfur granule with diffuse bacterial aggregates (Taylor’s modified Brown & Brenn; original magnification, ×200). (D) A central area showing filamentous bacteria positive to Grocott stain (Grocott stain; original magnification, ×200). (E) Scanning electron micrograph showing packed rod-like and filamentous bacteria with interspersed coccoid and other bacterial types (original magnification, ×3000). (F) Energy dispersive X-ray spectroscopy graphic representing the analysis of the correspondent sulfur granule area (box in the upper right corner) showing picks of sulfur and calcium. The area shows characteristic filamentous Actinomyces associated with an amorphous extracellular matrix and nucleus of calcification.