Apical periodontitis: preliminary assessment of microbiota by 16S rRNA high throughput amplicon target sequencing.

BACKGROUND: Apical periodontitis includes periapical granulomas and radicular cysts, which are histologically distinguished by the absence and the presence of an epithelial lining, respectively. The main cause of apical periodontitis is the bacterial colonization of the root canal space. This research aimed at assessing whether and how periapical granulomas and radicular cysts differ in terms of microbiota using high throughput amplicon target sequencing (HTS) techniques. METHODS: This study included 5 cases of Periapical Granulomas (PGs) and 5 cases of Radicular Cysts (RCs) selected on the base of histology out of 37 patients from January 2015 to February 2016. Complete medical history, panoramic radiograms (OPTs) and histologic records of each patient were assessed. Only lesions greater than 1 cm in diameter and developed in proximity to teeth with bad prognosis were included. The microbiota present in periapical granulomas and radicular cysts thus retrieved was finely characterized by pyrosequencing of the 16S rRNA genes. RESULTS: The core of OTUs shared between periapical granulomas and radicular cysts was dominated by the presence of facultative anaerobes taxa such as: Lactococcus lactis, Propionibacterium acnes, Staphylococcus warneri, Acinetobacter johnsonii and Gemellales. L. lactis, the main OTUs of the entire datasets, was associated with periapical granuloma samples. Consistently with literature, the anaerobic taxa detected were most abundant in radicular cyst samples. Indeed, a higher abundance of presumptive predicted metabolic pathways related to Lipopolysaccharide biosynthesis was found in radicular cyst samples. CONCLUSIONS: The present pilot study confirmed the different microbial characterization of the two main apical periodontitis types and shade light on the possible role of L. lactis in periapical granulomas.

Actinomyces israelii in radicular cysts: a molecular study.

OBJECTIVE: To investigate whether the microscopic filamentous aggregates observed in radicular cysts are associated with the molecular identification of Actinomyces israelii. Moreover, to verify whether this bacterium can be detected in radicular cyst specimens not presenting aggregates. STUDY DESIGN: Microscopic colonies suggestive of Actinomyces were found in 8 out of 279 radicular cyst samples (case group). The case and control groups (n = 12; samples without filamentous colonies) were submitted to the semi-nested polymerase chain reaction to test the presence of A israelii. DNA sequencing was performed to validate polymerase chain reaction results. RESULTS: Two and 3 samples in the case and control groups, respectively, did not present a functional genomic DNA template and were excluded from the study. A israelii was identified in all samples of the case group and in 3 out of 9 samples of the control group. CONCLUSIONS: Although A israelii is more commonly identified in radicular cysts presenting filamentous aggregates, it also appears to be detected in radicular cysts without this microscopic finding.

Bilateral Radicular Cyst with Severe Destruction of the Buccal Cortical Plate Secondary to Endodontic Failure.

Large apical radiolucencies after endodontic treatment are usually advised orthograde retreatment, apical surgery and that too with a guarded prognosis. Microsurgical techniques, the use of ultrasonic and magnification aides, have been in use in the developed countries for the past few decades and have shown significant improvement in long-term success of such cases. MTA is proposed as one of the best materials for sealing root end surfaces. In this report, the healing response of cases of 2 large apical radiolucencies, found separately in the anterior maxilla, which were treated by orthograde as well as retrograde endodontic treatments and guided tissue regeneration techniques.

Extraradicular infection as the cause of persistent symptoms: a case series.

INTRODUCTION: This article describes 3 cases that presented persistent symptoms after appropriate endodontic treatment. Histopathologic and histobacteriologic investigation were conducted for determination of the cause. METHODS: Three cases are reported that presented with persistent symptoms after endodontic retreatment (cases 1 and 2) or treatment (case 3). Periapical surgery was indicated and performed in these cases. The biopsy specimens, consisting of root apices and the apical periodontitis lesions, were subjected to histopathologic and histobacteriologic analyses. RESULTS: Case 1 was an apical cyst with necrotic debris, heavily colonized by ramifying bacteria, in the lumen. No bacteria were found in the apical root canal system. Case 2 was a granuloma displaying numerous bacterial aggregations through the inflammatory tissue. Infection was also present in the dentinal tubules at the apical root canal. Case 3 was a cyst with bacterial colonies floating in its lumen; bacterial biofilms were also seen on the external apical root surface, filling a large lateral canal and other apical ramifications, and between layers of cementum detached from the root surface. No bacteria were detected in the main root canal. CONCLUSIONS: Different forms of extraradicular infection were associated with symptoms in these cases, leading to short-term endodontic failure only solved by periapical surgery.

Bacteriological findings in radicular cyst and keratocystic odontogenic tumour fluids from asymptomatic patients.

OBJECTIVE: In this study the potential presence of bacteria in radicular cyst (RC) and keratocystic odontogenic tumour(KCOT) fluids from clinically asymptomatic patients was investigated. MATERIALS AND METHODS: Cyst fluids were collected by needle aspiration from 16 patients with asymptomatic osteolytic lesions (10 RCs and 6 KCOTs) undergoing surgery. All samples were transferred into tubes containing pre-reduced transport medium, delivered to the microbiology laboratory and processed within 1h. The cysts, surgically enucleated, were sent for standard histopathological examination. Cyst fluid samples were cultured on selective and differential media in anaerobic (for about 2 weeks) and aerobic (for 24-48 h) conditions to detect viable microorganisms. After incubation, the colonies were counted, Gram-stained and identified by biochemical tests. RESULTS: Cultures were positive for the presence of bacteria in 15 (9 RCs, 6 KCOTs) out of 16 cases. RCs and KCOTs generally yielded low bacterial counts (10(2)-10(4) CFU/ml) and were predominantly colonized by obligate anaerobes (64%), whereas less commonly by facultative anaerobes (36%). No significant differences in the detection frequencies of obligate and facultative anaerobes were evidenced between RCs and KCOTs. Propionibacterium acnes was the most common obligate anaerobe recovered both in RC and KCOT fluids. Among facultative anaerobes, Gemella morbillorum was more frequently isolated in KCOTs, whereas Staphylococcus spp. in RCs. CONCLUSIONS: Bacteria may be present and persist within fluids of clinically asymptomatic jaw cystic lesions. The influence of bacteria and latent bacterial infection within cystic jaw lesions should be reconsidered in odontogenic cyst progression.

The predominant bacteria isolated from radicular cysts.

PURPOSE: To detect predominant bacteria associated with radicular cysts and discuss in light of the literature. MATERIAL AND METHODS: Clinical materials were obtained from 35 radicular cysts by aspiration. Cultures were made from clinical materials by modern laboratory techniques, they underwent microbiologic analysis. RESULTS: The following are microorganisms isolated from cultures: Streptococcus milleri Group (SMG) (23.8%) [Streptococcus constellatus (19.1%) and Streptococcus anginosus (4.7%)], Streptococcus sanguis (14.3%), Streptococcus mitis (4.7%), Streptococcus cremoris (4.7%), Peptostreptococcus pevotii (4.7%), Prevotella buccae (4.7%), Prevotella intermedia (4.7%), Actinomyces meyeri (4.7%), Actinomyces viscosus (4.7%), Propionibacterium propionicum (4.7%), Bacteroides capillosus (4.7%), Staphylococcus hominis (4.7%), Rothia denticariosa (4.7%), Gemella haemolysans (4.7%), and Fusobacterium nucleatum (4.7%). CONCLUSIONS: Results of this study demonstrated that radicular cysts show a great variety of anaerobic and facultative anaerobic bacterial flora. It was observed that all isolated microorganisms were the types commonly found in oral flora. Although no specific microorganism was found, Streptococcus spp. bacteria (47.5%) - especially SMG (23.8%) - were predominantly found in the microorganisms isolated. Furthermore, radicular cysts might be polymicrobial originated. Although radicular cyst is an inflammatory cyst, some radicular cyst fluids might be sterile.

Investigation of cultivable bacteria isolated from longstanding retreatment-resistant lesions of teeth with apical periodontitis.

INTRODUCTION: The objective of this research was to investigate the presence of viable bacteria in tissue samples from persistent apical lesions and to correlate the microbiological findings with the histopathological diagnosis of the lesion. METHODS: Twenty persistent apical lesions associated with well-performed endodontic retreatment were collected. Tissue samples were processed through culture techniques including serial dilution, plating, aerobic and anaerobic incubation, and biochemical tests for microbial identification followed by histopathological diagnosis. RESULTS: Cysts were more frequently diagnosed (13/20). Strict anaerobic species predominated in both cysts (80.4% of the species detected) and granulomas (65% of the species detected). Viable gram-positive bacteria were frequently recovered from apical lesions (cysts = 70.6%, granulomas = 84.4%). Gemella morbillorum and Propionibacterium acnes were the most frequently recovered species from cysts and granulomas, respectively. At least 1 gram-positive bacterial species was present in almost every sample (cysts = 12/13, granulomas = 7/7). No significant correlation was found between histologic findings and bacterial species. CONCLUSIONS: In conclusion, although cysts were more frequent than granulomas in cases of failure of endodontic retreatment, bacteria were isolated from both types of lesions, with a predominance of gram-positive species, suggesting that these species can survive outside the root canal and might be related to the persistence of the pathological process even after accurate endodontic retreatment.

Specific in situ visualization of plasma cells producing antibodies against Porphyromonas gingivalis in gingival radicular cyst: application of the enzyme-labeled antigen method.

The enzyme-labeled antigen method was applied to visualize plasma cells producing antibodies to Porphyromonas gingivalis, flora of the human oral cavity. Antibodies to P. gingivalis have reportedly been detected in sera of patients with periodontitis. Biotinylated bacterial antigens, Ag53, and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro, and Lys-hgp) were prepared by the cell-free protein synthesis system using the wheat germ extract. In paraformaldehyde-fixed frozen sections of rat lymph nodes experimentally immunized with Ag53-positive and Ag53-negative P. gingivalis, plasma cells were labeled with biotinylated Arg-hgp and Lys-hgp. Antibodies to Ag53 were detected only in the nodes immunized with Ag53-positive bacteria. In two of eight lesions of gingival radicular cyst with inflammatory infiltration, CD138-positive plasma cells in frozen sections were signalized for Arg-hgp and Lys-hgp. An absorption study using unlabeled antigens confirmed the specificity of staining. The AlphaScreen method identified the same-type antibodies in tissue extracts but not in sera. Antibodies to Ag53, Arg-pro, and Lys-pro were undetectable. In two cases, serum antibodies to Arg-hgp and Lys-hgp were AlphaScreen positive, whereas plasma cells were scarcely observed within the lesions. These findings indicate the validity of the enzyme-labeled antigen method. This is the very first application of this novel histochemical technique to human clinical samples.

Biofilms and apical periodontitis: study of prevalence and association with clinical and histopathologic findings.

INTRODUCTION: This study evaluated the prevalence of bacterial biofilms in untreated and treated root canals of teeth evincing apical periodontitis. The associations of biofilms with clinical conditions, radiographic size, and the histopathologic type of apical periodontitis were also investigated. METHODS: The material comprised biopsy specimens from 106 (64 untreated and 42 treated) roots of teeth with apical periodontitis. Specimens were obtained by apical surgery or extraction and were processed for histopathologic and histobacteriologic techniques. RESULTS: Bacteria were found in all but one specimen. Overall, intraradicular biofilm arrangements were observed in the apical segment of 77% of the root canals (untreated canals: 80%; treated canals: 74%). Biofilms were also seen covering the walls of ramifications and isthmuses. Bacterial biofilms were visualized in 62% and 82% of the root canals of teeth with small and large radiographic lesions, respectively. All canals with very large lesions harbored intraradicular biofilms. Biofilms were significantly associated with epithelialized lesions (cysts and epithelialized granulomas or abscesses) (p < 0.001). The overall prevalence of biofilms in cysts, abscesses, and granulomas was 95%, 83%, and 69.5%, respectively. No correlation was found between biofilms and clinical symptoms or sinus tract presence (p > 0.05). Extraradicular biofilms were observed in only 6% of the cases. CONCLUSIONS: The overall findings are consistent with acceptable criteria to include apical periodontitis in the set of biofilm-induced diseases. Biofilm morphologic structure varied from case to case and no unique pattern for endodontic infections was identified. Biofilms are more likely to be present in association with longstanding pathologic processes, including large lesions and cysts.

The clinical spectrum of Actinomyces-associated lesions of the oral mucosa and jawbones: correlations with histomorphometric analysis.

OBJECTIVES: To characterize the clinical manifestations of Actinomyces-associated lesions of the oral mucosa and jawbones, and to correlate the clinical course and treatment requirements with the findings of histomorphometric analysis. STUDY DESIGN: The study was a 10-year retrospective analysis of archived cases with microscopic identification of Actinomyces infection. Actinomyces colonies were identified, using hematoxylin-eosin, Gram, and periodic acid-Schiff stains, exhibiting filamentous morphology with color variation between center and periphery. Only colonies with adjacent tissue reaction (inflammation, fibrosis) were analyzed. Actinomyces density (AD) was calculated by dividing total number of colonies by tissue surface, Actinomyces relative surface (ARS) was calculated by dividing total bacterial surface by tissue surface. RESULTS: The study included 106 cases (48 male, 58 female; aged 13-84 years, mean 50.5 years). Cases presented a wide clinical spectrum, involving jawbone and/or oral soft tissues. Cases included osteomyelitis associated with bisphosphonates, osteoradionecrosis, osteomyelitis unrelated to radiation or bisphosphonates, periapical lesions, odontogenic cysts, periimplantitis, and lesion mimicking periodontal disease. The AD correlated with median length of antibiotic treatment (R = 0.284; P = .028). CONCLUSIONS: Because we were able to identify 106 such cases, the results indicate that Actinomyces-associated lesions may not be as rare as would be expected from the relatively low number of cases in the literature. Actinomyces-associated lesions presented in a wide spectrum of clinical settings and a variety of contributing factors. Quantitative analysis of the number of bacterial colonies (representing bacterial load) could help in evaluating the aggressive potential of the lesion and help in treatment planning.

Nonsurgical root canal therapy of large cyst-like inflammatory periapical lesions and inflammatory apical cysts.

It is a general belief that large cyst-like periapical lesions and apical true cysts caused by root canal infection are less likely to heal after nonsurgical root canal therapy. Nevertheless, there is no direct evidence to support this assumption. A large cyst-like periapical lesion or an apical true cyst is formed within an area of apical periodontitis and cannot form by itself. Therefore, both large cyst-like periapical lesions and apical true cysts are of inflammatory and not of neoplastic origin. Apical periodontitis lesions, regardless of whether they are granulomas, abscesses, or cysts, fail to heal after nonsurgical root canal therapy for the same reason, intraradicular and/or extraradicular infection. If the microbial etiology of large cyst-like periapical lesions and inflammatory apical true cysts in the root canal is removed by nonsurgical root canal therapy, the lesions might regress by the mechanism of apoptosis in a manner similar to the resolution of inflammatory apical pocket cysts. To achieve satisfactory periapical wound healing, surgical removal of an apical true cyst must include elimination of root canal infection.

T lymphocyte activation and cytokine expression in periapical granulomas and radicular cysts.

INTRODUCTION: Radicular cysts (RCs) are periapical lesions resulting in jaw bone destruction. The inflammatory dental periapical granuloma (PG) is considered to be the origin of RC formation; however the mechanism of RC development remains unclear. METHODS: Cell suspension from the surgically extirpated tissue of 27 RCs and 25 PGs was obtained. Bacteriological analysis of the PG tissue samples was performed in order to define two major groups of PG according to the prevailing causative bacterial infection: the streptococcal PG (PG-S, n=10) and the anaerobe PG (PG-A, n=9) group. The inflammatory response of tissue infiltrating lymphocytes was assessed by following T lymphocyte activation (HLA-DR expression) as well as interferon gamma (IFN-gamma) and interleukin 4 (IL-4) production which were evaluated by the flow cytometry. RESULTS: In comparison to RC both types of PG contained a higher proportion of activated T cells (HLA-DR) and lower proportion of IL-4 producing cells. PG-A tissue contained increased percentage of CD3 cells and increased percentage of T helper 1 (Th1) cells in comparison with PG-S. In RC the IFN-gamma production is higher than in streptococcal PG-S but similar as in PG-A. DISCUSSION: Tissue infiltration by Th2 cells and IL-4 production is likely to play an etiopathogenic role in RC formation.

Anatomic and microbiologic challenges to achieving success with endodontic treatment: a case report.

This article describes a case of persistent apical periodontitis that required several nonsurgical and surgical approaches for resolution. A 28-year-old woman presented with a large symptomatic apical periodontitis lesion associated with the maxillary left lateral incisor that persisted in spite of previous nonsurgical and surgical endodontic treatment. Root canal retreatment was accomplished, but the lesion only showed a slight decrease in size after 18 months. Forty-six months after retreatment, the patient returned because of reemergence of symptoms. Radiographically, the lesion remained relatively unaltered. Periradicular resurgery was performed, and a biopsy specimen consisting of the lesion and the apical part of the root was collected for analysis. Radiographic and clinical reevaluation after 4 years revealed complete healing. Histopathologic and histobacteriologic observations demonstrated that the lesion was a cyst, and that the probable reason for failure was the occurrence of bacteria within dentinal tubules and in a lateral canal slightly coronal to the amalgam root-end filling. This case report clearly illustrates the difficulties imposed by anatomic complexities in attaining a disinfection level that is compatible with periradicular tissue healing.

Epithelium and bacteria in periapical lesions.

OBJECTIVE: The purpose of this study was to evaluate 50 human periapical lesions for bacteria and epithelium in a case study in dental practice. STUDY DESIGN: Specimens were obtained from the extraction of 50 untreated teeth that had lesions attached to their apices. The specimens were histologically evaluated using serial sections. RESULTS: Bacteria were found in all teeth, colonizing necrotic tissue in the main canal, dentinal tubules, or apical ramifications, and in the body of the periapical lesion in 18 abscesses or cysts. Twenty-one lesions were epithelialized; 14 abscesses, 20 granulomas, and 16 cysts were distinguished. In 18 root canals inflamed tissue was found in the apical part of the canal. A single foramen was present in 13 cases while apical ramifications were found in 37 cases. CONCLUSIONS: Granulomas were most common, and most epithelialized lesions were cysts. Bacteria were only detected periapically in abscesses or cysts. Inflamed tissue was present in the apical root canal in one third of cases.

Periapical actinomycosis: a clinicopathologic study.

OBJECTIVE: We sought to evaluate the incidence and clinical outcome of an accidental finding of actinomycotic colonies in periapical lesions submitted for histologic examination. STUDY DESIGN: The study included all periapical biopsy specimens submitted for histologic examination between 1997 and 2000. Sections of paraffin-embedded tissues, 5 microm, were cut and stained by using hematoxylin and eosin, periodic acid-Schiff, and the Gram stain. The presence of typical branching colonies of filamentous bacteria staining positive for periodic acid-Schiff and Gram stain was indicative of Actinomyces. RESULTS: Typical actinomycotic colonies were identified in 17 of 963 (1.8%) periapical biopsy specimens. The mean patient age was 42, and males were predominant (65%). The maxilla was the most frequently involved site (65%), with equal distribution in the anterior and posterior areas. Radiographically, most cases presented as well-demarcated radiolucent lesions. Malignancy was suspected in 3 cases. Of the periapical lesions, 15 were epithelialized, and in 4 cases, a true epithelial-lined lumen was found, which was diagnosed as a radicular cyst. A residual cyst was diagnosed in 1 case, and in 1 case, an epithelial lining was not identified. Treatment included surgical curettage and a short course of antibiotic therapy. Healing was uneventful in all cases. CONCLUSIONS: Periapical actinomycosis is not common. Its outcome is favorable after surgical curettage supplemented by short-term antibiotic treatment. The relationship of periapical actinomycosis with the more serious cervicofacial actinomycosis should be evaluated.

Ciliated epithelium-lined radicular cysts.

OBJECTIVE: This report describes 3 cases of ciliated epithelium-lined radicular cysts among 256 apical periodontitis lesions and also illustrates the occurrence of an Actinomyces-infected periapical cyst. STUDY DESIGN: Serial and step serial sections of 256 plastic-embedded root apices with attached apical periodontitis lesions that were prepared for a previous investigation were reviewed for the presence of ciliated epithelium-lined radicular cysts. The lesions that were found to have such epithelial lining were examined in a transmission electron microscope to elaborate the fine structure of the ciliated cells. RESULTS: A total of 3 ciliated columnar epithelium-lined cysts was found among the 256 apical periodontitis lesions examined. Two of the lesions also contained stratified squamous epithelium. All 3 lesions affected maxillary premolars. One of the lesions was a true cyst, and the other 2 were periapical pocket cysts. The lumen of 1 of the latter revealed the presence of typical "ray-fungus" actinomycotic colonies. CONCLUSION: Although the stratified squamous component of the epithelia that lined the radicular cysts reported here may be derived from the cell rests of Malassez, the ciliated epithelial cells may be of sinus origin. Microbial agents from diseased root canals can advance into radicular cysts, particularly in pocket cysts, with the possible threat of such infection in upper posterior teeth spreading into the maxillary sinus.

[Bacteriologic study of the contents of radicular cysts]. / Bakteriolosko ispitivanje sadrzaja radikularnih cista.

INTRODUCTION: Radicular cysts are, most often, oral tissue cysts. The basis for their development are the remains of Hertwig's epithelial sheath, which is stimulated to proliferate by infectious agents, mostly periapical granuloma or pulp necrosis. The cyst contents can be liquid, either clear or milky-white, or thick and yellowish-caseous pus. Bacteriological and histopathological researches have shown that development of radicular cysts from periapical lesions is a consequence of penetration of normal bacterial flora of the oral cavity. MATERIAL AND METHODS: The study has bacteriologically examined contents of 50 radicular cysts. They were operated between 1993 and 1995 at the Department of Oral Surgery of the Outpatient Health Care Centre Banja Luka. Pathological substrate of the cyst was punctured by a sterile needle and bacteriologically examined at the Microbiological Laboratory of the Clinical Centre in Banja Luka. DISCUSSION AND CONCLUSION: Radicular cysts contents most often consisted of: alpha-hemolytic streptococcus, Streptococcus pneumoniae, Staphyilococcus epidermidis, Streptococcus group B and alpha-hemolytic Streptococcus. It is concluded that normal oral cavity flora is present in the cysts contents, the infection penetrated through the root canal to periapex, where, due to the course of time, radicular cyst develops from periapical granuloma.

Calculus-like deposit at apex of tooth with refractory apical periodontitis.

It is generally accepted that bacteria in or outside the root canal are the reason for apical periodontitis and endodontic failures. This case report presents a 60-year-old woman with a periapical lesion and a fistulous tract which did not respond to conventional root canal treatment. During periapical surgery, granulomatous tissue was removed and a calculus-like deposit was observed on the root surface. A radicular cyst was diagnosed. Nine months after this calculus-like deposit had been removed and the cyst enucleated, complete recovery of the bone had occurred. It is suggested that the presence of the calculus-like deposit at the apex of the tooth or its effects may in part have delayed the healing of the periapical inflammation in spite of apparently adequate endodontic treatment.