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.

Eficácia de limpeza dos sistemas rotatórios de NiTi, comparados aos instrumentos manuais, em canais radiculares estreitos e achatados / Cleanness efficacy of NiTi rotary systems compared to manual instruments in narrow and flattened root canals

OBJETIVO: comparar, ex vivo, a eficácia de limpeza de três sistemas rotatórios (Mtwo, K3 e ProTaper) e um manual (K-Flexofile), em canais radiculares estreitos e achatados, com base na remoção de corante das paredes de dentina. MÉTODOS: os canais radiculares de quarenta dentes humanos foram preenchidos com tinta preta e, após a secagem do corante, os dentes foram divididos aleatoriamente em quatro grupos (n = 10), de acordo com o sistema de instrumentação utilizado: G1 = Mtwo; G2 = K3; G3 = ProTaper e G4 = K-Flexofile. Após a instrumentação, os dentes foram clivados longitudinalmente. A análise qualitativa baseou-se na quantidade de corante remanescente aderido às paredes de dentina nos terços apical, médio e coronal do canal radicular, e em sua totalidade, de acordo com quatro escores. Para a análise quantitativa, cada hemissecção foi digitalizada e analisada por meio do softwareImage Tool. A eficácia de limpeza foi determinada por meio da quantificação da diferença entre a área total de cada canal radicular e a área não instrumentada, em mm2. Os dados foram submetidos aos testes Kruskal-Wallis ou One-way ANOVA e Bonferronipost hoc (p < 0,05). RESULTADOS: não foram evidenciados canais radiculares completamente limpos. Não foi observada diferença significativa entre os sistemas de instrumentação, à análise qualitativa (p > 0,05). CONCLUSÕES: Em relação à análise quantitativa, os instrumentos Mtwo apresentaram uma eficácia de limpeza significativamente superior, quando comparados aos outros sistemas (p < 0,05). em geral, a eficácia de limpeza do sistema Mtwo foi ligeiramente superior, comparada à dos instrumentos K3, ProTaper e K-Flexofile, dentro dos parâmetros do presente estudo e independentemente das limitações.

Macrophage cell activation with acute apical abscess contents determined by interleukin-1 Beta and tumor necrosis factor alpha production.

INTRODUCTION: This clinical study has investigated the antigenic activity of bacterial contents from exudates of acute apical abscesses (AAAs) and their paired root canal contents regarding the stimulation capacity by levels of interleukin (IL)-1 beta and tumor necrosis factor alpha (TNF-α) throughout the root canal treatment against macrophage cells. METHODS: Paired samples of infected root canals and exudates of AAAs were collected from 10 subjects. Endodontic contents were sampled before (root canal sample [RCS] 1) and after chemomechanical preparation (RCS2) and after 30 days of intracanal medication with calcium hydroxide + chlorhexidine gel (Ca[OH]2 + CHX gel) (RCS3). Polymerase chain reaction (16S rDNA) was used for detection of the target bacteria, whereas limulus amebocyte lysate was used to measure endotoxin levels. Raw 264.7 macrophages were stimulated with AAA exudates from endodontic contents sampled in different moments of root canal treatment. Enzyme-linked immunosorbent assays were used to measure the levels of TNF-α and IL-1 beta. RESULTS: Parvimonas micra, Porphyromonas endodontalis, Dialister pneumosintes, and Prevotella nigrescens were the most frequently detected species. Higher levels of endotoxins were found in samples from periapical exudates at RCS1 (P < .005). In fact, samples collected from periapical exudates showed a higher stimulation capacity at RCS1 (P < .05). A positive correlation was found between endotoxins from exudates with IL-1 beta (r = 0.97) and TNF-α (r = 0.88) production (P < .01). The significant reduction of endotoxins and bacterial species achieved by chemomechanical procedures (RCS2) resulted in a lower capacity of root canal contents to stimulate the cells compared with that at RCS1 (P < .05). The use of Ca(OH)2 + CHX gel as an intracanal medication (RCS3) improved the removal of endotoxins and bacteria from infected root canals (P < .05) whose contents induced a lower stimulation capacity against macrophages cells at RCS1, RCS2, and RCS3 (P < .05). CONCLUSIONS: AAA exudates showed higher levels of endotoxins and showed a greater capacity of macrophage stimulation than the paired root canal samples. Moreover, the use of intracanal medication improved the removal of bacteria and endotoxins from infected root canals, which may have resulted in the reduction of the inflammatory potential of the root canal content.

Quantification of endotoxins in infected root canals and acute apical abscess exudates: monitoring the effectiveness of root canal procedures in the reduction of endotoxins.

INTRODUCTION: This clinical study was conducted to measure the endotoxin levels in infected root canals (RCs) and exudates related to acute apical abscesses (AAAs). In addition, the effectiveness of RC procedures in reducing the endotoxin levels in RCs was monitored. METHODS: Paired samples of infected RCs and exudates from AAAs were collected from 10 subjects by using paper points. RCs samples were collected before (RCS1) and after chemomechanical preparation (CMP) (RCS2), after 17% EDTA (RCS3), and after 30 days of intracanal medication (Ca[OH]2 + chlorhexidine) (RCS4). A turbidimetric kinetic limulus amebocyte lysate assay was used for the measurement of endotoxins. RESULTS: Endotoxins were detected in 100% of the baseline samples of AAAs and RCs (RCS1) with median values of 175 EU/mL and 41.5 EU/mL, respectively (P < .05). After CMP (RCS2), endotoxins were reduced to a median value of 0.54 EU/mL (P < .05). Subsequent irrigation with EDTA (RCS3) failed to present a significant effectiveness in reducing the endotoxin levels (median= 0.37 EU/mL) (P = .07). However, intracanal medication for 30 days (RCS4) reduced endotoxins to median values of 0.03 EU/mL (P < .01). CONCLUSIONS: The present study revealed a strong association between the high levels of endotoxins found in AAAs and RCs collected from the same tooth. Moreover, the effectiveness of CMP in reducing the endotoxin levels from RCs in acute endodontic infection was improved by the use of RC medication.

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.

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.