Actinomycosis is not Frequent in the Periapex But is a Persistent Lesion

a typical extra-radicular infection. This study determined the frequency and correlated the content of bacteria colonies with the of periapical actinomycosis size. The study comprised a total of 218 periapical lesions (PL) (cysts, granulomas or abscess). The specimens embedded in paraffin were sliced into 4-μm sections and stained with hematoxylin-eosin, Gram, Periodic Acid–Schiff (PAS) and Grocott’s stain. The presence of bacterial colonies composed of filamentous structures labeled with the histochemical stains were described as Actinomyces, and for each case, the bacterial colonies were counted and measured. The correlation between the number and size of bacterial colonies and the size of PL was tested using Pearson’s adjusted correlation coefficient. From 218 PL, bacterial colonies were identified in 64 biopsies. Seven cases (0.3%) fulfill the criteria for diagnosis of periapical actinomycosis. All of cases were therapy-resistant and did not showed periapical repair after 12 months of follow-up. Periapical surgery or dental extraction was performed. The correlation test indicated no correlation between the number of bacterial colonies and the lesion size (p=0.752, r=-0.148). However, a larger bacterial colony size generally resulted in a larger periapical lesion (P=0.000, r=0.657). The frequency of periapical actinomycosis was low, and this lesion should be included in the differential diagnosis of PL. The size of the Actinomyces colonies seemed to contribute to increase the size of the periapical lesion. Actinomycosis is not Frequent in the Periapex But is a Persistent Lesion


Introduction
Tooth carious exposure following bacterial invasion on the pulp constitutes the most common cause of pulp necrosis (1).A consequence of infection of the root canal system involves progressive stages of inflammation and changes of periapical bone structure, characterized by apical periodontitis (2).Apart from endodontic origin (3,4), periapical lesions may be of non-endodontic origin (5).
The maintenance of root canal infection is the main cause of apical periodontitis in endodontically treated teeth.The aetiology of post-treatment disease have been associated with microbial aetiologic factors (intraradicular and extraradicular infection -bacteria, fungi) and nonmicrobial aetiologic factors (endogenous -true cysts and exogenous -foreign-body reaction) (2)(3)(4).
Actinomycosis is an infectious disease caused by gram-positive anaerobic pathogen, being that Actinomyces israelii (6)(7)(8)10) is the most common isolated species in human actinomycosis.In the head and neck region, the most common form of actinomycosis is the cervicofacial form, which is often difficult to distinguish from other inflammatory lesions in this region (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15).The occurrence of such infections in the oral cavity begins after the loss of mucosal integrity caused by local anesthesia, bone or tooth fractures, tooth extraction or eruption, periodontal disease, pulp exposure, or even endodontic treatment.The rupture of the mucosa facilitates the invasion of pathogens into the soft tissues and their proliferation due to the anaerobic conditions established (9,16,17).
In new imaging exams have been emphasized as the high prevalence of apical periodontitis after root canal treatment (22), even against the better understanding of the factors involved in the persistence of periapical lesions and the contemporary root canal therapeutic techniques (21).During the establishment of a diagnostic hypothesis associated with persistent periapical lesions, usually the periapical actinomycosis has not been mentioned, especially due to the lack of data (2)(3)(4).Therefore, the objective of the present study is to determine the frequency of periapical actinomycosis in 218 periapical lesions and to correlate the content of bacteria colonies with the periapical lesion size.

Material and Methods
The study comprised a total of 218 periapical lesions (cysts, granulomas or abscesses) microscopically identified in the laboratory of surgical pathology at the Dental School of Universidade Federal da Bahia, BA, Brazil and the Universidade Federal de Goiás, Brazil, from 2002-2014.The experimental design was based on previous study made by Hirshberg et al. (9).Clinicopathological data as gender, age, location, teeth, previous root canal treatment, symptom, clinical diagnosis, microscopic diagnosis, size of lesion and treatment were retrieved from the charts and/ or pathological reports.The study design was approved by institutional Ethics Committee.
The material was fixed in 10% formalin, embedded in paraffin and sliced into 4-µm sections for morphological analysis.After staining with hematoxylin-eosin, gram, Periodic acid-Schiff (PAS), and Grocott's stain, all slides were reviewed by an experienced pathologist who recorded the morphological aspects of the lesions to confirm the diagnosis.The presence of bacterial colonies composed of filamentous structures aggregated to form a mass with color variations by hematoxylin-eosin stain ("sun ray effect") surrounded by inflammation and/or fibrosis and labeled with the histochemical stains were described as Actinomyces (9).For each case, the bacterial colonies were counted and measured in the larger diameters (mm) and their mean number in each case was expressed.An analysis of the histological sections was performed using AxioVisionTM Rel 4.8.6 2009 software (Zeiss, Göttingen, Germany) coupled a Zeiss Axio Scope microscope (Carl Zeiss MicroImaging, Inc., Göttingen, Germany).
The correlation between the number and size of bacterial colonies and the size of periapical lesions was tested using Pearson's adjusted correlation coefficient.A level of significance of 5% was adopted (p<0.05).The statistical analysis was performed using the Minitab program for Windows, version 14 (Minitab, Inc., USA).

Results
From a total of 218 periapical lesions, bacterial colonies were identified in 64 periapical biopsies.However, seven fulfill the criteria for periapical actinomycosis.The lesions affected women more frequently and the mean age of patients was 54.4 years.The mandible was more often affected in the anterior region.Radiographically, the lesions presented with well-defined radiolucency and were located in the periapical region; most patients had undergone prior root canal treatment (Table 1).All of cases were therapyresistant periapical lesions and did not show periapical repair after 12 months of follow-up.Thus, periapical surgery (surgical enucleation of lesion followed by apicoectomy) or dental extraction was performed.
The main clinical suspicion of these persistent secondary infections was apical periodontitis (Figs.1A, 1B and 2A, 2B).Periapical actinomycosis was not mentioned as a differential diagnosis suspicion in any of the cases.Pain symptoms in the region involved were described for three cases.All cases were histopathologically diagnosed as radicular cysts associated with inflammation and actinomycosis infection due to the presence of bacterial colonies characteristic of actinomycosis.In all cases observed Actinomyces colonies were represented by small or large isolated masses forming rosettes, often surrounded by an acute, chronic, or mixed inflammatory infiltrate inside the cystic lumen (Figs.1C, 1D, 1E, 1G, 1H, 2C and 2D) varying from 1-9 colonies per case.The size ranged from 1-4 cm.Histochemical techniques for PAS and Grocott highlighted bacterial colonies (Figs.1F, 2E and 2F).After the histopathological diagnosis of periapical actinomycosis, 1.5 g amoxicillin was administered daily for 10 days.
The correlation test indicated no correlation between the number of bacterial colonies and the lesion size (p=0.752,r=-0148).However, a larger bacterial colony size generally resulted in a larger periapical lesion (p=0.000,r=0.657).
Studies have shown periapical actinomycosis in 1.8% -4.4% of the cases of periapical lesions (8,9).Hirshberg et al. (9) evaluated the incidence of an accidental finding of actinomycotic colonies in periapical lesions and actinomycotic colonies were identified in 17 of 963 (1.8%) periapical biopsy specimens.The cases of periapical actinomycosis had a favorable outcome after conservative surgical curettage with short-term antibiotic treatment.
Interestingly, none of the periapical biopsies in our study included any mention of actinomycosis infection in clinical suspicion.Therefore, the presence of this type of bacteria was a surprising finding.The presences of extra-radicular bacterial plaque or actinomycotic colonies are conditions that often maintain a periapical lesion after root canal treatment (18)(19)(20)(21).Sunde et al. (19) analyzed the periapical microbiota of 36 teeth with refractory apical periodontitis.A variety of microorganisms, comprising facultative and anaerobic bacteria as well as yeasts, remained in refractory periapical endodontic lesions after long-term-root-canal treatment with calcium hydroxide (and systemic antibiotic treatment).Sulfur granules, containing Actinomyces species and other bacteria, were detected in 9 lesions (25%), and many of the granules were calcified.Ricucci & Siqueira (21) reported a case of extraradicular actinomycosis that formed a continuum with the intraradicular infection and based on a critical review of the literature they stressed there is no evidence that apical actinomycosis is indeed an independent entity leading to persistent apical periodontitis lesions.
In this case series, women were more frequently affected, and most lesions were located in the mandible, especially in the anterior region.These findings are different from those reported in other studies in which men and the maxilla were more frequently affected (9).In addition, painful symptoms have been reported, 58% of the cases described here were asymptomatic.It is important to note that all biopsies were performed due to the presence of persistent alterations on radiographic images after root canal treatment and it has been reported that the Actinomyces leads to the development of asymptomatic and persistent periapical infection.
Radiographically, the lesions presented with welldefined periapical radiolucency and all lesions involved teeth that had undergone endodontic treatment.This finding is consistent with a previous report (9); however, we had difficulty analyzing the radiographic images because not all biopsies were accompanied by imaging exams.According Kukiane et al. (23), regarding periapical radiographic examinations, the average size of lesions in which Actinomyces is present is greater than that of lesions in which bacterial colonization is not present.
Histopathologically, all cases fulfilled the criteria for a radicular cyst (24), but all cases were associated with an Actinomyces infection.These colonies were present in the lumen or adjacent to the cyst, had different sizes, were isolated or sometimes overlapping, and were characterized by filamentous structures radiating from the center that were positive on PAS and Grocott staining and surrounded by an inflammatory infiltrate.These findings are similar to those of previous authors (25).It is important to state there are other means of identifying an Actinomyces infection.However, according to Hirshberg et al (9), in any case in which a non-healing and endodontically nonresolving lesion is associated with histopathological identification of actinomycosis colonies in the periapical specimen, the diagnosis of periapical actinomycosis should be applied.
This study showed that the average size of radicular   cysts with Actinomyces colonization was 1.78 cm, which is similar to the value reported by a previous study (24) in which the average size of these cysts was nearly 2.0 cm.When this result was compared with those previously described by Kukiane et al. (23), although that study used a method different from the one applied in the present study, the presence of Actinomyces did not appear to increase the size of the radicular cyst lesions.In addition, the number of bacterial colonies per lesion was also similar to that observed by Hirshberg et al. (9).It could be hypothesized that a larger colony size resulted in a larger radicular cyst as correlation test was significant; however, the bacterial colonies were inside the cystic lumen but a greater number of cases could clarify this matter.In summary, root canal infections are frequently responsible for apical periodontitis, but the frequency of periapical actinomycosis was low in this study.However, periapical actinomycosis should be included in the differential diagnosis of persistent periapical lesions.In addition, other studies are needed to better understand whether the size of the Actinomyces colonies contributes to increase the size of the periapical lesion

Figure 1 .
Figure 1.Case of periapical actinomycosis.A: Periapical radiograph of tooth #32 showing apical periodontitis associated with root canal treatment failure; B: tooth extraction and implant placement; C: large bacterial colony within the cystic lumen; D: high magnification showing the peripheral edge of thick radiating clubs; E: high magnification showing the accumulation of leukocytes around the bacterial colonies; F: high magnification of Figure D showing PAS-positive colonies; G: bacterial colonies of different sizes surrounded by numerous neutrophils; H) high magnification showing a more basophilic core and peripheral radiating clubs.

Figure 2 .
Figure 2. Case of periapical actinomycosis.A: Large well-circumscribed radiolucent image involving the periapical region of tooth #22; B: periapical image after apicoectomy; C: histopathologic analysis showing periapical actinomycosis (note the large bacterial colony with peripheral buddings); D: periphery with radiating actinomycetes (note the neutrophils surrounding the bacterial colony); E: Grocott silver-positive actinomycetes; F: high magnification showing thin and superimposed clubs.