A modified dentine infection model with fluorescent lipopolysaccharide and lipopolysaccharides sampling technique to compare XP-Endo finisher and passive ultrasonic irrigation.

AIM: The lipopolysaccharides-dentine-infection (LPS-dentine-infection) models and sampling techniques frequently used to evaluate LPS disinfection have limitations. In this study, a LPS-dentine-infection model was devised using fluorescent conjugate LPS. Secondly, a sampling technique using cryogenic grinding for intraradicular LPS analysis was evaluated. Thirdly, the effectiveness of the XP-endo Finisher (XP-EF) was compared with passive ultrasonic irrigation (PUI) in removing LPS from root canal system. METHODOLOGY: Sixty-nine mandibular premolars were submitted to dentine pretreatment and inoculated with fluorescent LPS conjugate (Alexa Fluor® 594). Twenty-three teeth were analysed under confocal laser scanning microscopy (CLSM) to validate this modified LPS-dentine-infection model. Forty-six teeth were randomly divided into two experimental groups: XP-EF (n = 23) and PUI (n = 23). All teeth were instrumented with XP-endo shaper (XPS; FKG Dentaire) and 2.5% NaOCl. The root canals were sampled with paper points before (s1) and after (s2) instrumentation and after supplemental treatment (s3) with XP-EF and PUI. After s3, all roots were cryogenically ground for intraradicular LPS analysis (s4). Limulus amebocyte lysate assay was used for LPS quantification. The Friedman test was used for differences in LPS among four time-points (s1, s2, s3, and s4). Dunn's test was used for pairwise testing of time-points. The significance level was set at 5% (p < .05). RESULTS: Fluorescent LPS conjugate was detected in 100% of the samples under CLSM with a penetration depth of approximately 400 µm into dentine. Chemo-mechanical preparation using XPS files significantly reduced LPS levels (p < .05). Both the XPS and PUI improved the LPS disinfection (p < .05), with no difference between them (p > .05). LPS was recovered from all samples after cryogenic grinding. The residual amount of LPS detected using the cryogenically sampling technique at s4 was approximately three times greater than with the paper-point sampling technique at s3. CONCLUSION: This study established a modified LPS-dentine-infection model using fluorescent conjugate LPS, and validated a LPS sampling technique for using cryopulverization intraradicular LPS analysis. Moreover, both the XP-EF and PUI further improved LPS disinfection from the root canals, and the innovative XP-EF was as effective as PUI.

Identification of Fusobacterium nucleatum in primary and secondary endodontic infections and its association with clinical features by using two different methods.

OBJECTIVE: Fusobacterium nucleatum is an important oral pathogen involved in endodontic infections. This study aimed to assess the frequency of Fusobacterium nucleatum in primary and secondary endodontic infections and its associations with the clinical features in a Brazilian population by using both culture and nested PCR methods. METHODS: A total of 100 microbial samples from patients with primary (n=50) and secondary endodontic infections (n=50) were analyzed by using culture and nested PCR methods. Strict anaerobic techniques were used for culture and identification of F. nucleatum. The DNA extracted from the samples was analyzed for the presence of target species by using species-specific primers. RESULTS: Culture and nested PCR methods detected F. nucleatum, respectively, in 11/100 and 82/100 root canals. F. nucleatum was isolated by culture from 10/50 (20%) root canals with primary infections and from 1/50 (2%) root canal with secondary/persistent infections. Nested PCR detected F. nucleatum in 42/50 (84%) root canals with primary infections and in 40/50 (80%) root canals with secondary/persistent endodontic infections. F. nucleatum was associated with spontaneous pain, tenderness to percussion, pain on palpation, swelling, tooth mobility, wet root canals, hemorrhagic exudate, tooth decay, inadequate restoration, and poor endodontic filling. CONCLUSION: F. nucleatum was found in more cases of primary endodontic infections than in cases of secondary/persistent ones. A higher prevalence of F. nucleatum was detected by using the nested PCR method than by using culture. The presence of F. nucleatum in the root canals was associated with several clinical features. CLINICAL RELEVANCE: The high prevalence of F. nucleatum in the root canals detected by molecular methods, and its association with several clinical features reveals the importance of these species in the development of apical pathologies and reinforces the need of an endodontic treatment directed to bacterial elimination.

Investigation of Filifactor alocis in primary and in secondary endodontic infections: A molecular study.

OBJECTIVE: Identification of specific bacteria in root canals (RCs) in distinct clinical conditions can support the comprehension of pathological processes. Thus, the objective of this clinical study was to investigate the presence of F. alocis in RCs of teeth with primary endodontic infection (PEI) and with persistent/secondary endodontic infection (SEI) by using molecular techniques. It was also aimed to associate its presence with the clinical features. In addition, the levels of F. alocis as well as the total bacterial cells in the samples were also quantitated. DESIGN: One hundred teeth (50 PEI and 50 SEI) were included. Microbial samples were performed using sterile paper points and assessed by using nested PCR and quantitative Real Time PCR (qPCR). The prevalence of F.alocis in RCs from PEI and SEI were compared by chi-square analysis. Fisher´s exact test or Pearson Chi-square, when appropriate, was used to test associations between clinical and radiographic features and the presence of F. alocis. Significance level was set at 5%. RESULTS: F. alocis was detected in 23 and 28 (PEI) and 12 and 11 (SEI) RCs using Nested PCR and qPCR, respectively. Statistically significant associations were found between the presence of F. alocis and PEI, pain, wet canals, swelling, abscess and purulent exudate (P < 0.05). Total bacterial count was similar in both conditions (P > 0.05). CONCLUSIONS: PEI harbour a significantly higher number of F. alocis than those with SEI. Filifactor alocis was significantly associated with clinical features in primary endodontic infections. Total bacterial count was similar in both clinical conditions.

In vivo biocompatibility and biomineralization of calcium silicate cements.

A new mineral trioxide aggregate (MTA) material has been developed with a modified composition that requires investigations to support its clinical use. This study evaluated the biocompatibility and biomineralization of this new MTA material and compared it with that of two other MTA cements over time. Tubes containing materials (or empty tubes as controls) were inserted into the subcutaneous tissues of 40 rats. On days 7, 15, 30, 60, and 90, the tubes were removed with the surrounding tissues, which were either stained with haematoxylin and eosin or von Kossa for further analyses or unstained for observation under polarized light. On days 7 and 15, moderate inflammation was observed in most specimens, and the fibrous capsule was thick. On day 30, there was mild inflammation in all groups, and the fibrous capsule was thin. On days 60 and 90, there was mild inflammation in the material groups, while the control group showed no inflammation, although no statistically significant difference between the groups was observed and the fibrous capsule was thin. All material groups showed structures that stained with von Kossa and could be observed under polarized light; this was not found for the control. In conclusion, the new MTA material had biocompatibility and biomineralization properties similar to those of the two existing MTA materials.

Correlation between crestal alveolar bone loss with intracanal bacteria and apical lesion area in necrotic teeth.

OBJECTIVE: This study aimed to analyze the correlation between crestal alveolar bone loss with the presence of some bacterial species in root canals and the apical lesion area of necrotic teeth. DESIGN: Data from 20 patients with diagnosis of pulp necrosis and acute apical abscesses, without active periodontal diseases, were evaluated. Patients with history of antibiotic usage three months prior to the study, with exposed pulp cavity, and with probing depth >3 mm were not included. The root size, the distance between the bone crest to the tooth apex in the mesial and distal surfaces, and the apical lesion area were measured from standard periapical radiographies by a calibrated examiner. Root canal samples were collected using sterilized paper points. In multirooted teeth, the largest root canal was sampled. Culture, microbial isolation and identification by phenotypic methods were performed. Spearman correlation and exact Fischer test were calculated between higher/lower existing bone crests, according to the median and the presence of specific bacteria. RESULTS: No statistically significant differences were found between occurrence of pathogenic bacteria, such as Porphyromonas gingivalis, Porphyromonas endodontalis, and Prevotella intermedia, and groups with higher/lower degree of bone loss (p > 0.05). A negative significant correlation was found between Parvimonas micra and periodontal bone loss (p = 0.02). Additionally, no statistically significant association was found between crestal bone loss and the apical lesion area. CONCLUSIONS: It was concluded that, in patients without active periodontitis, the presence of pathogenic bacteria in the root canal was not correlated with periodontal bone loss.

Resistance profiles to antimicrobial agents in bacteria isolated from acute endodontic infections: systematic review and meta-analysis.

Infected root canal or acute apical abscess exudates can harbour several species, including Fusobacterium, Porphyromonas, Prevotella, Parvimonas, Streptococcus, Treponema, Olsenella and not-yet cultivable species. A systematic review and meta-analysis was performed to assess resistance rates to antimicrobial agents in clinical studies that isolated bacteria from acute endodontic infections. Electronic databases and the grey literature were searched up to May 2015. Clinical studies in humans evaluating the antimicrobial resistance of primary acute endodontic infection isolates were included. PRISMA guidelines were followed. A random-effect meta-analysis was employed. The outcome was described as the pooled resistance rates for each antimicrobial agent. Heterogeneity and sensitivity analyses were performed. Subgroup analyses were conducted based upon report or not of the use of antibiotics prior to sampling as an exclusion factor (subgroups A and B, respectively). Data from seven studies were extracted. Resistance rates for 15 different antimicrobial agents were evaluated (range, 3.5-40.0%). Lower resistance rates were observed for amoxicillin/clavulanic acid and amoxicillin; higher resistance rates were detected for tetracycline. Resistance rates varied according to previous use of an antimicrobial agent as demonstrated by the subgroup analyses. Heterogeneity was observed for the resistance profiles of penicillin G in subgroup A and for amoxicillin, clindamycin, metronidazole and tetracycline in subgroup B. Sensitivity analyses demonstrated that resistance rates changed for metronidazole, clindamycin, tetracycline and amoxicillin. These findings suggest that clinical isolates had low resistance to ß-lactams. Further well-designed studies are needed to clarify whether the differences in susceptibility among the antimicrobial agents may influence clinical responses to treatment.

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.

Evaluation of cytotoxicity and physicochemical properties of calcium silicate-based endodontic sealer MTA Fillapex.

INTRODUCTION: The aim of the study was to evaluate the cytotoxicity, radiopacity, pH, and flow of a calcium silicate-based and an epoxy resin-based endodontic sealer, MTA Fillapex (Angelus, Londrina, PR, Brazil) and AH Plus (Dentsply, Konstanz, Germany), respectively. METHODS: Cytotoxicity, radiopacity, and flow evaluation were performed following ISO requirements. The pH level was measured at periods of 3, 24, 72, and 168 hours. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay to check the Balb/c 3T3 cells viability at 1- to 4-week periods. Data were statistically analyzed by analysis of variance and the Tukey test with a significance level of 5%. RESULTS: In all tested periods, MTA Fillapex was more cytotoxic than AH Plus (P < .05). Although AH Plus presented higher radiopacity than MTA Fillapex (P < .05), both sealers showed minimum required values. MTA Fillapex presented alkaline pH in all experimental times, whereas AH Plus cement showed a slightly neutral pH and a flow significantly lower than that of MTA Fillapex (P < .05). CONCLUSIONS: Although MTA Fillapex was more cytotoxic than AH Plus, it showed suitable physicochemical properties for an endodontic sealer.

Clustering behavior in microbial communities from acute endodontic infections.

INTRODUCTION: Acute endodontic infections harbor heterogeneous microbial communities in both the root canal (RC) system and apical tissues. Data comparing the microbial structure and diversity in endodontic infections in related ecosystems, such as RC with necrotic pulp and acute apical abscess (AAA), are scarce in the literature. The aim of this study was to examine the presence of selected endodontic pathogens in paired samples from necrotic RC and AAA using polymerase chain reaction (PCR) followed by the construction of cluster profiles. METHODS: Paired samples of RC and AAA exudates were collected from 20 subjects and analyzed by PCR for the presence of selected strict and facultative anaerobic strains. The frequency of species was compared between the RC and the AAA samples. A stringent neighboring clustering algorithm was applied to investigate the existence of similar high-order groups of samples. A dendrogram was constructed to show the arrangement of the sample groups produced by the hierarchical clustering. RESULTS: All samples harbored bacterial DNA. Porphyromonas endodontalis, Prevotella nigrescens, Filifactor alocis, and Tannerela forsythia were frequently detected in both RC and AAA samples. The selected anaerobic species were distributed in diverse small bacteria consortia. The samples of RC and AAA that presented at least one of the targeted microorganisms were grouped in small clusters. CONCLUSIONS: Anaerobic species were frequently detected in acute endodontic infections and heterogeneous microbial communities with low clustering behavior were observed in paired samples of RC and AAA.

Persistent extraradicular infection in root-filled asymptomatic human tooth: scanning electron microscopic analysis and microbial investigation after apical microsurgery.

INTRODUCTION: Procedural accidents have a negative effect on healing and might contribute to the persistence of infections in inaccessible apical areas, requiring surgical intervention. This report describes a case of persistent apical periodontitis of a lower left first molar associated with the sinus tract and a periapical lesion that required nonsurgical endodontic retreatment and apical surgery for resolution. METHODS: The tooth had received endodontic treatment 3 years ago and had to be retreated using the crown-down technique with chemical auxiliary substance (2% chlorhexidine gel), foramen patency, and enlargement and was filled in a single appointment. The occlusal access cavity was immediately restored with composite resin. After 1 month, it could be observed that the sinus tract persisted and, radiographically, the lesion remained unaltered. Therefore, endodontic microsurgery was indicated. Apical microsurgery was performed under magnification with the use of a dental operating microscope including apicectomy, root end with ultrasound, and sealing with mineral trioxide aggregate. A microbiological sample was collected from the apical lesion. The resected distal root apex was observed by scanning electron microscopy. RESULTS: The following species were detected: Actinomyces naeslundii and Actinomyces meyeri, Propionibacterium propionicum, Clostridium botullinum, Parvimonas micra, and Bacteroides ureolyticus; scanning electron microscopic analysis revealed bacterial biofilm surrounding the apical foramen and external radicular surface. Gutta-percha overfilling at the apex because of a zip caused during initial endodontic treatment could be observed. A 6-month follow-up showed apparent radiographic periapical healing, which progressed after 24 months. CONCLUSION: Gram-positive anaerobic bacteria and extraradicular biofilm seem to participate in the maintenance of persistent periapical pathology, and endodontic retreatment followed by periapical microsurgery proved to be a successful alternative in the resolution of persistent extraradicular infections.

Analysis of the antimicrobial susceptibility of anaerobic bacteria isolated from endodontic infections in Brazil during a period of nine years.

INTRODUCTION: The purpose of this study was to analyze the susceptibility of some anaerobic species isolated from a Brazilian population at different periods of time by determining a pattern of development of resistance to frequently prescribed antibiotics in endodontics. METHODS: Root canal samples were collected from infected teeth at different periods of time (2000-2002, 2003-2005, and 2007-2008) and microbiologically identified with conventional culture techniques. The susceptibility of Prevotella intermedia/nigrescens, P. oralis, Fusobacterium nucleatum, and P. micra isolated strains was determined by the minimum inhibitory concentration (MIC) of amoxicillin, amoxicillin + clavulanate, benzylpenicillin, clindamycin, erythromycin, and metronidazole by using the E-test method. RESULTS: Amoxicillin and amoxicillin + clavulanate were effective against the majority of species at the different periods of study. Overall, there were low statistical differences regarding the microbial susceptibility between the experimental periods. However, an increase in the anaerobic resistance to penicillin G and clindamycin was observed. Resistance to erythromycin was observed in all species, and there were statistically significant differences between 2000-2002 and 2003-2005 periods for F. nucleatum (P < .05) and between 2003-2005 and 2007-2008 periods for P. intermedia/nigrescens and P. oralis (P < .05). CONCLUSIONS: The antimicrobial resistance of anaerobes isolated from primary endodontic infections showed an increase throughout a period of time regarding a specific Brazilian population.

Treponema species detected in infected root canals and acute apical abscess exudates.

INTRODUCTION: Different microbial communities have been associated with acute endodontic infections. The majority of the microorganisms are as yet uncultivable or difficult to grow under current laboratory conditions. Treponema species are strict anaerobic bacteria that are involved in several oral diseases. The aim of this study was to detect the presence of Treponema species in infected root canals (RCs) and exudates related to acute apical abscesses (AAAs) as well as to determine positive association between targeted species and clinical signs/symptoms. METHODS: Paired samples of infected RCs and AAAs were collected from 20 subjects. Nested polymerase chain reaction assay with species-specific primers for 16S rDNA and downstream intergenic spacer region was used for microbial detection. The frequency of species and statistical associations between species and signs/symptoms of endodontic origin as well as their simultaneous detection in both milieus were investigated. RESULTS: The most frequently detected species were T. socranskii (RC, 17/20; AAA, 15/20), T. denticola (RC, 8/20; AAA, 11/20); T. medium (RC, 6/20; AAA, 9/20); and T. amylovorum (RC, 5/20; AAA, 9/20). Positive correlation was found for simultaneous presence of T. denticola in both RCs and AAAs (p = 0.01). Positive association was observed between T. medium and T. vincentii (p = .037). No positive statistical association was observed between the targeted species and signs/symptoms. CONCLUSIONS: The high incidence of Treponema species in RC and AAA samples from the same tooth indicated that they are important pathogens in acute endodontic infections.

Frequency, microbial interactions, and antimicrobial susceptibility of Fusobacterium nucleatum and Fusobacterium necrophorum isolated from primary endodontic infections.

This study assessed the prevalence and microbial interactions of Fusobacterium nucleatum and Fusobacterium necrophorum in primary endodontic infections from a Brazilian population and their antimicrobial susceptibility to some antibiotics by the E-test. One hundred ten samples from infected teeth with periapical pathologies were analyzed by culture methods. Five hundred eighty individual strains were isolated; 81.4% were strict anaerobes. F. nucleatum was found in 38 root canals and was associated with Porphyromonas gingivalis, Prevotella spp., and Eubacterium spp. F. necrophorum was found in 20 root canals and was associated with Peptostreptococcus prevotii. The simultaneous presence of F. nucleatum and F. necrophorum was not related to endodontic symptoms (p > 0.05). They were 100% susceptible to amoxicillin, amoxicillin/clavulanate, and cephaclor. Fusobacterium spp. is frequently isolated from primary-infected root canals of teeth with periapical pathologies. Amoxicillin is a useful antibiotic against F. nucleatum and F. necrophorum in endodontic infections and has been prescribed as the first choice in Brazil.

Microbial analysis of canals of root-filled teeth with periapical lesions using polymerase chain reaction.

The objective of the present study was to investigate the presence of nine bacterial species in root-filled teeth associated with periapical lesions using a polymerase chain reaction analysis and to correlate these species with clinical features of the cases. DNA was extracted from 45 canal samples of root-filled teeth with periapical lesions. A PCR assay using species-specific primers of 16S rDNA and the downstream intergenic spacer region was used for microbial detection. Enterococcus faecalis was the most prevalent species, detected in 77.8% of the study teeth, followed by Peptostreptococcus micros, detected in 51.1%. Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, and Prevotella nigrescens were detected in 35.6%, 22.2%, 11.1%, and 11.1% of the sampled teeth, respectively. Moreover, PCR detected Filifactor alocis in 26.7%, Treponema denticola in 24.4%, and Tannerella forsythia in 4.4% of the samples. T. denticola and P. micros were statistically associated with tenderness to percussion (p < 0.05). P. nigrescens was associated with the presence of spontaneous pain and abscess (p < 0.05). P. endodontalis and P. nigrescens were associated with purulent exudates (p < 0.05). Synergistic relationship was also observed between some species. The results of this study indicated that E. faecalis was the most frequently identified test species by PCR in teeth with failing endodontic treatment.

Molecular analysis of Filifactor alocis, Tannerella forsythia, and treponema denticola associated with primary endodontic infections and failed endodontic treatment.

The aim of this study was to investigate the presence of strict anaerobes such as Filifactor alocis, Tannerella forsythia, and Treponema denticola in primary and secondary root-infected canals with periapical lesions by molecular analysis and the association of these species with specific endodontic signs and symptoms. Microbial samples were taken from 100 root canals, 50 with necrotic pulp tissues (NPT, primary infection), and 50 with failed endodontic treatment (FET, secondary infection). DNA was extracted from the samples, which were analyzed for the presence of three endodontic pathogens using species-specific primers and PCR. F. alocis were isolated from 23 canals with NPT and 12 canals with FET; T. forsythia from 12 canals with NPT and three canals with FET; T. denticola from 19 canals with NPT and 12 canals with TEP. Suggested associations were found between primary infection and the presence of F. alocis and T. forsythia (both p < 0.05). In particular, associations were found between: pain and F. alocis; swelling and F. alocis; tenderness to percussion and T. forsythia; mobility and T. forsythia and T. denticola; wet canals and F. alocis, T. forsythia, and T. denticola; purulent exsudate and F. alocis, T. forsythia and T. denticola; abscess and F. alocis, T. forsythia, and T. denticola (all p < 0.05). The findings of this study indicated that F. alocis, T. forsythia, and T. denticola seem to be associated with endodontic signs and symptoms. Additionally, F. alocis and T. forsythia were detected more frequently in teeth with necrotic pulp than in teeth with failing endodontic treatment.

Enterococcus faecalis in dental root canals detected by culture and by polymerase chain reaction analysis.

OBJECTIVE: The objective of this study was to investigate the presence of Enterococcus faecalis in endodontic infections by culture and polymerase chain reaction analyses. STUDY DESIGN: Microbial samples were obtained from 50 teeth with untreated necrotic pulps (primary infection) and from 50 teeth with failing endodontic treatment (secondary infection). Culture techniques were used including serial dilution, plating, incubation, and biochemical identification. For PCR detection, samples were analyzed using a species-specific primer of the 16S rDNA and the downstream intergenic spacer region. RESULTS: Culture and PCR detected the test species in 23 of 100 and 79 of 100 of the teeth, respectively. E faecalis was cultured from 2 (4%) of 50 necrotic canals and from 21 (42%) of 50 root-treated canals. PCR detection identified the target species in 41 (82%) and 38 (76%) of 50 primary and secondary infections respectively. CONCLUSION: E faecalis was detected as frequently in teeth with necrotic pulp as in teeth with failing endodontic treatment when a PCR analysis was used.

Quantification of endotoxins in necrotic root canals from symptomatic and asymptomatic teeth.

The purpose of this investigation was to quantify the concentration of endotoxin in necrotic root canals and investigate the possible relationship between the concentration of endotoxin and endodontic signs and symptoms. Samples were collected from root canals of 50 patients requiring endodontic treatment due to necrosis of the pulpal tissue. Anaerobic techniques were used to determine the number of c.f.u. in each sample. A quantitative chromogenic Limulus amoebocyte lysate assay was used to measure the concentration of endotoxin in each sample. The presence of c.f.u. was detected by culture in all samples (range 10(2)-5x10(6)). In samples from cases of patients with spontaneous pain, the mean c.f.u. was 1.43x10(6) while in asymptomatic cases it was 9.1x10(4). Endotoxin was present in all the samples studied [range 2390.0-22100.0 endotoxin units (EU) ml-1]. The mean concentration of endotoxin in samples from patients with spontaneous pain was 18540.0 EU ml-1 while in asymptomatic cases it was 12030.0 EU ml-1. Asymptomatic cases generally had lower levels of endotoxin (i.e. a negative association). A positive association was found between endotoxin and symptomatic cases (e.g. spontaneous pain, tenderness to percussion, pain on palpation, swelling and purulent exudates). This study showed that endotoxin is present in high concentrations in root canals of symptomatic teeth. There was a positive correlation between the concentration of endotoxin in the root canal and the presence of endodontic signs and symptoms.