Biomedical subjects

Isabela N Rôças

Publications and source records attributed to Isabela N Rôças.

At least 19 recordsLinked to original sources

PubMed2006

On the use of denaturing gradient gel electrophoresis approach for bacterial identification in endodontic infections.

Bacteria in infected root canals of teeth evincing chronic apical periodontitis lesions were identified by a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) approach. DNA was extracted from root canal samples, and part of the 16S rRNA gene of all bacteria was amplified by PCR and separated by DGGE, generating banding patterns representative of the community structure. Twenty visible bands were cut out of the gel, re-amplified, and sequenced to provide identification. Sequencing analysis revealed the presence of both cultivable and as-yet-uncultivated species in the samples analyzed, including representatives of the genera Fusobacterium, Bacteroides, Dialister, Synergistes, Prevotella, Eubacterium and Peptostreptococcus. Unambiguous identification was not always possible and the method's limitations are discussed. In general, the findings showed that PCR-DGGE can be useful for the identification of both cultivable and as-yet-uncultivated bacteria in endodontic infections.

Adult↗

PubMed2006

Characterization of Dialister species in infected root canals.

Members of the Dialister genus are asaccharolytic obligately anaerobic gram-negative coccobacilli that are culture-difficult or remain uncultivated. Their participation in endodontic infections has been only consistently demonstrated after advent of molecular biology approaches. This study was undertaken to characterize Dialister species in samples from primary endodontic infections using a devised 16S rRNA gene-based group-specific heminested PCR assay followed by sequencing of PCR products. Genomic DNA was isolated directly from clinical samples and used as template for PCR. Amplicons from positive specimens were sequenced and phylogenetically analyzed to determine species identity. Ten of 21 clinical samples yielded sequences with the highest percent similarities to oral Dialister species/phylotypes. Seven sequences were from Dialister invisus, and the other three sequences belonged to Dialister pneumosintes, Dialister oral clone BS095 and Dialister sp. clone IS013B24. Findings demonstrated that different Dialister species can take part in the microbiota associated with apical periodontitis lesions.

Adult↗

PubMed2006

Prevalence of selected bacterial named species and uncultivated phylotypes in endodontic abscesses from two geographic locations.

Evidence is mounting that the prevalence of some putative endodontic pathogens can significantly vary according to the geographic location in which samples were taken. This study aimed to provide additional knowledge on this subject by comparing the frequencies of 10 selected candidate endodontic pathogens in samples of acute apical abscesses obtained from two distinct geographic locations; Portland, OR, and Rio de Janeiro, Brazil. DNA was extracted from aspirates and used as template in nested PCR assays using 16S rRNA gene taxon-specific oligonucleotide primers. Of the target species/phylotypes, Treponema denticola (73% of the cases), Porphyromonas endodontalis (70%), and Tannerella forsythia (57%) were the most prevalent taxa found in Brazilian samples. Dialister invisus (70% of the cases), P. endodontalis (63%) and Dialister pneumosintes (55%) were the most frequent taxa in the Portland, OR samples. Data analysis revealed that T. denticola and T. forsythia were significantly more detected in Brazilian samples than in the Portland, OR samples. Although D. invisus, Filifactor alocis, and Synergistes oral clone W090 were detected in many more samples from the Portland, OR patients, differences were not found to be statistically significant. These findings confirmed that some bacterial taxa can markedly differ in the frequencies they occur in samples from different locations. It remains to be clarified whether this observation translates into relevant therapeutic implications.

Bacterial Infections↗

PubMed2006

Catonella morbi and Granulicatella adiacens: new species in endodontic infections.

OBJECTIVE: This study intended to examine samples from primary endodontic infections for the presence of Catonella morbi and Granulicatella adiacens, 2 species that have been recently suggested to be involved with infections in other oral sites. STUDY DESIGN: Genomic DNA was isolated directly from samples taken from teeth with different forms of apical periodontitis, and a devised culture-independent 16S rRNA gene-based heminested PCR assay was used to determine the prevalence of these 2 target species. Species-specific primers were developed by aligning closely related 16S rRNA gene sequences. Species-specificity for each primer pair was confirmed by running PCR against a panel of oral bacteria and by sequencing of DNA from representative positive samples. RESULTS: C morbi and G adiacens were detected in 33% and 19%, respectively, of the root canals associated with chronic apical periodontitis; 30% and 10%, respectively, of the cases diagnosed as acute apical periodontitis, and 16% and 11%, respectively, of the pus samples taken from acute apical abscesses. Overall, C morbi occurred in 26% and G adiacens in 14% of the samples taken from primary endodontic infections. CONCLUSIONS: Our findings demonstrate that C morbi and G adiacens can take part in the microbiota associated with primary endodontic infections, and their specific role in the disease process warrants further elucidation.

Adolescent↗

PubMed2005

Detection of novel oral species and phylotypes in symptomatic endodontic infections including abscesses.

This study was undertaken to investigate the occurrence of several uncultivated phylotypes and newly named bacterial species in symptomatic endodontic infections. Samples taken from cases clinically diagnosed as acute periradicular abscesses or acute periradicular periodontitis were surveyed for the presence of 12 taxa by means of a 16S rRNA-gene-based nested or hemi-nested PCR assay. The most prevalent of the target taxa were Dialister invisus, Olsenella uli, Granulicatella adiacens, and Synergistes clones BA121 and E3_33. Findings revealed that novel phylotypes and newly named species can take part in the microbiota associated with symptomatic endodontic infections and a pathogenetic role is suspected.

Actinobacteria↗

PubMed2005

Differences in prevalence of selected bacterial species in primary endodontic infections from two distinct geographic locations.

OBJECTIVE: This study intended to compare the prevalence of 7 putative endodontic pathogens in samples of primary endodontic infections taken from patients of 2 distant geographic locations. STUDY DESIGN: Samples from infected root canals associated with asymptomatic periradicular lesions or from pus aspirated from acute periradicular abscesses were collected from patients in Rio de Janeiro, Brazil, and Seoul, South Korea. South Korean samples were frozen and delivered to Brazil, where all steps in the molecular analysis were performed. DNA was extracted and a species-specific nested polymerase chain reaction assay was used to detect 7 target bacterial species. RESULTS: The most prevalent species detected in Brazilian samples were Porphyromonas endodontalis (79% of the cases) , Treponema denticola (79%), and Dialister pneumosintes (76%). The most prevalent species found in South Korean samples were Fusobacterium nucleatum (38% of the cases) , Tannerella forsythia (26%), and Treponema maltophilum (24%). Overall, P endodontalis, D pneumosintes, Filifactor alocis, T denticola, and T forsythia were significantly more detected in Brazilian samples than in South Korean samples ( P < .05). CONCLUSIONS: Findings indicated that the prevalence of some species in infections of endodontic origin may significantly differ from one geographic location to another.

Adolescent↗

PubMed2005

Periradicular status related to the quality of coronal restorations and root canal fillings in a Brazilian population.

OBJECTIVE: This cross-sectional study was undertaken to determine the prevalence of periradicular lesions in root-filled teeth from an urban adult Brazilian population, and to investigate the quality of root canal fillings and coronal restorations and their association with the periradicular status of these teeth. STUDY DESIGN: Root canal fillings from 2,051 teeth were categorized as adequate or inadequate on the basis of root canal filling length and homogeneity. Coronal restorations from the same teeth were categorized into adequate, inadequate, or absent. Results were analyzed statistically using the chi-squared test. RESULTS: The overall success rate of root-filled teeth was 49.7%. Cases with adequate endodontic treatment and adequate restorations had a success rate of 71%. When cases with adequate treatment and inadequate restoration were evaluated, the success rate was 65%. Cases with adequate treatment and absent restoration showed a success rate of 48%. Teeth with inadequate treatment combined with adequate restoration yielded a success rate of 38%, whereas the combination of inadequate treatment and inadequate restoration resulted in a success rate of 25%. Teeth with inadequate treatment and absent restoration showed the lowest success rate of this study (18%). Data analysis revealed that when the root canal filling appeared to be adequate, the quality of the restoration did not significantly influence the treatment outcome. However, when a coronal restoration was absent, the success rate of adequately treated canals was significantly reduced. The quality of the coronal restoration significantly affected the outcome of inadequately treated teeth. CONCLUSIONS: Our results revealed a high prevalence of periradicular lesions in root-filled teeth, which was comparable to that reported in other methodologically compatible studies from diverse geographical locations. In addition, even though the coronal restoration had a significant impact on the periradicular health, the quality of the root canal filling was found to be the most critical factor in this regard.

Adult↗

PubMed2005

Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of endodontic infections.

The recent expanding use of cultivation-independent techniques for bacterial identification is reliant on the lack of knowledge of the conditions under which most bacteria are growing in their natural habitat and the difficulty to develop culture media that accurately reproduce these conditions. A molecular method that has been recently used in several areas to examine the bacterial diversity living in diverse environments is the denaturing gradient gel electrophoresis (DGGE). In DGGE, polymerase chain reaction (PCR)-generated DNA fragments of the same length but with different base-pair sequences can be separated. Separation is based on electrophorectic mobility of a partially melted double-strand DNA molecule in polyacrylamide gels, which is decreased when compared with that of the completely helical form of the molecule. Molecules with different sequences may have a different melting behavior and will therefore stop migrating at different positions in the gel. Application of the PCR-DGGE method in endodontic research has revealed that there are significant differences in the predominant bacterial composition between asymptomatic and symptomatic cases. This suggests that the structure of the bacterial community can play a role in the development of symptoms. In addition, new bacterial phylotypes have been disclosed in primary endodontic infections. PCR-DGGE has also confirmed that intra-radicular infections are a common finding in root-filled teeth associated with persistent periradicular lesions. The microbiota in failed cases significantly vary from teeth to teeth, with a mean number of species far higher than previously shown by culturing approaches. Application of the PCR-DGGE technique in endodontic microbiology research has the potential to shed light on several aspects of the different types of endodontic infection as well as on the effects of treatment procedures with regard to infection control.

Bacteria↗

PubMed2004

Denaturing gradient gel electrophoresis analysis of bacterial communities associated with failed endodontic treatment.

OBJECTIVE: A great deal of evidence indicates that persistent infections of the root canal of human teeth play an important role in the failure of the root canal treatment. The present study was undertaken to apply the PCR-DGGE fingerprinting approach to examine the structure of the bacterial population infecting previously treated root canals of humans associated with persistent periradicular lesions. STUDY DESIGN: Samples were taken from 14 filled root canals, DNA was extracted, and part of the 16S rDNA of all bacteria was amplified by PCR and separated by DGGE, generating banding patterns representative of the community structure. Species-specific PCR for the detection of Enterococcus faecalis was also performed. RESULTS: The mean number of bands detected in the 16S rDNA community profiles was about 6, ranging from 1 to 26 bands. Each sample showed a unique structure of the microbial community. The species-specific PCR assay revealed the presence of E. faecalis in 10 of 14 samples, but DGGE analysis revealed it was not the dominant species. CONCLUSIONS: Results revealed that the intraradicular bacterial community associated with failed endodontic treatment significantly varied in composition from teeth to teeth. Persistent intraradicular infections were present in all root-filled teeth.

Adult↗

PubMed2004

Polymerase chain reaction-based analysis of microorganisms associated with failed endodontic treatment.

OBJECTIVE: In this study, we aimed to investigate the occurrence of several microbial species in cases of failed endodontic therapy by means of the polymerase chain reaction (PCR). Study design Root canal samples were taken from 22 root-filled teeth with persistent periradicular lesions selected for re-treatment. DNA was extracted from the samples and analyzed for the presence of 19 microbial taxa by using the polymerase chain reaction. RESULTS: All samples were positive for at least 1 of the target microbial species. Enterococcus faecalis was the most prevalent species-detected in 77% of the cases. The other most prevalent species were Pseudoramibacter alactolyticus (52%), Propionibacterium propionicum (52%), Dialister pneumosintes (48%), and Filifactor alocis (48%). Candida albicans was found in 9% of the samples. The mean number of species in samples filled up to 2 mm short of the radiographic apex was 3 (range, 1-5), whereas cases in which the filling was greater than 2 mm from the apex yielded a mean of 5 species (range, 2-11). This difference was statistically significant (P <.05). CONCLUSIONS: Microorganisms occurred in all cases of root-filled teeth associated with periradicular lesions, which lends strong support to the assertion that treatment failures are rather of infectious etiology, caused by persistent or secondary intraradicular infections. E faecalis was the most prevalent species, followed by 4 other anaerobic species: P. alactolyticus, P. propionicum, D. pneumosintes, and F. alocis. All examined samples harbored at least 1 of the following gram-positive bacterial species: E. faecalis, P. alactolyticus, or P. propionicum.

Adult↗

PubMed2004

Nested PCR detection of Centipeda periodontii in primary endodontic infections.

In recent years, molecular genetic methodologies have provided significant additional knowledge about components of the microbiota associated with infections of endodontic origin. Following this research line, the purpose of the present study was to investigate the prevalence of Centipeda periodontii in primary endodontic infections using a species-specific nested PCR assay. Samples were collected from fifty teeth having carious lesions, necrotic pulps, and different forms of periradicular diseases. DNA extracted from the samples was initially amplified using universal 16S rDNA primers, and a second round of amplification used the first PCR products to detect a specific fragment of C. periodontii 16S rDNA. This species was detected in 3 (13%) of 23 asymptomatic cases, in 1 (14%) of 7 cases diagnosed as acute apical periodontitis, and in 3 (15%) of 20 pus samples aspirated from acute periradicular abscesses. There was no significant association between C. periodontii and the presence of clinical symptoms. Overall, C. periodontii was detected in 14% of the cases of endodontic infections. This is probably the hitherto first study to detect C. periodontii in primary endodontic infections. The specific role played by this bacterial species in infections of endodontic origin awaits further clarification.

Adolescent↗

PubMed2004

Association of Enterococcus faecalis with different forms of periradicular diseases.

Data from culture studies have revealed that Enterococcus faecalis is occasionally isolated from primary endodontic infections but frequently recovered from treatment failures. This molecular study was undertaken to investigate the prevalence of E. faecalis in endodontic infections and to determine whether this species is associated with particular forms of periradicular diseases. Samples were taken from cases of untreated teeth with asymptomatic chronic periradicular lesions, acute apical periodontitis, or acute periradicular abscesses, and from root-filled teeth associated with asymptomatic chronic periradicular lesions. DNA was extracted from the samples, and a 16S rDNA-based nested polymerase chain reaction assay was used to identify E. faecalis. This species occurred in seven of 21 root canals associated with asymptomatic chronic periradicular lesions, in one of 10 root canals associated with acute apical periodontitis, and in one of 19 pus samples aspirated from acute periradicular abscesses. Statistical analysis showed that E. faecalis was significantly more associated with asymptomatic cases than with symptomatic ones. E. faecalis was detected in 20 of 30 cases of persistent endodontic infections associated with root-filled teeth. When comparing the frequencies of this species in 30 cases of persistent infections with 50 cases of primary infections, statistical analysis demonstrated that E. faecalis was strongly associated with persistent infections. The average odds of detecting E. faecalis in cases of persistent infections associated with treatment failure were 9.1. The results of this study indicated that E. faecalis is significantly more associated with asymptomatic cases of primary endodontic infections than with symptomatic ones. Furthermore, E. faecalis was much more likely to be found in cases of failed endodontic therapy than in primary infections.

Adolescent↗

PubMed2004

Selected endodontic pathogens in the apical third of infected root canals: a molecular investigation.

Bacteria located at the apical portion of the root canals are conceivably in a strategic position to induce damage to the periradicular tissues and resulting inflammatory diseases. This study sought to investigate the prevalence of 11 selected putative endodontic pathogens in the apical third of infected root canals associated with periradicular lesions. The apical root portion of 23 extracted teeth with carious pulpal exposures and attached periradicular lesions was sectioned, and the root canals were sampled for microbiological investigation. DNA was extracted from the samples and analyzed for the presence of 11 bacterial species using a nested polymerase chain reaction assay. The results showed that Pseuramibacter alactolyticus occurred in 10 cases (44%), Treponema denticola in 6 (26%), Fusobacterium nucleatum in 6 (26%), Porphyromonas endodontalis in 4 (17%), Filifactor alocis in 2 (9%), Dialister pneumosintes in 1 (4%), Porphyromonas gingivalis in 1 (4%), and Tannerella forsythensis in 1 (4%). No sample yielded Prevotella intermedia, Prevotella nigrescens, or Campylobacter rectus. Of the samples examined, 17 were positive for at least 1 of the target species. Occurrence of these bacterial species in the apical third of infected root canals suggests that they can be involved in causation of periradicular lesions.

Bacteria, Anaerobic↗

PubMed2004

Simultaneous detection of Dialister pneumosintes and Filifactor alocis in endodontic infections by 16S rDNA-directed multiplex PCR.

Dialister pneumosintes and Filifactor alocis have been recently considered as candidate endodontic pathogens. In this study, we devised a 16S rDNA-directed multiplex PCR protocol for simultaneous detection of these two bacterial species in endodontic infections. Samples were taken from infected root canals associated with asymptomatic periradicular lesions as well as from cases of acute periradicular abscesses. DNA extracted from the samples was used as template for simultaneous detection of D. pneumosintes and F. alocis through a multiplex PCR assay. Two fragments of the expected sizes, one specific for D. pneumosintes and the other for F. alocis, were simultaneously amplified from a mixture of reference genomic DNA containing DNA from both species. Clinical samples that were positive for the target species showed a single band of the predicted size for each species. D. pneumosintes was detected by multiplex PCR in 11 samples (7 asymptomatic and 4 abscesses) and F. alocis was identified in 9 cases (6 asymptomatic and 3 abscesses). Six samples (3 asymptomatic and 3 abscesses) shared the two species. Data from the present study confirmed that D. pneumosintes and F. alocis are common members of the microbiota present in primary endodontic infections and thereby may participate in the pathogenesis of periradicular lesions. The proposed multiplex PCR assay is a simple, rapid, and accurate method for the simultaneous detection of these two candidate endodontic pathogens.

Adult↗

PubMed2004

Polymerase chain reaction identification of microorganisms in previously root-filled teeth in a South Korean population.

A large body of evidence indicates that microorganisms are the primary causative agents of endodontic treatment failures. This study intended to assess the occurrence of nine putative endodontic pathogens in root-filled teeth associated with periradicular lesions in a South Korean population using a culture-independent molecular approach. Fourteen root-filled teeth with persistent periradicular diseases were selected for retreatment. After removal of the root canal filling, the canals were sampled, and a polymerase chain reaction assay using taxon-specific oligonucleotide primers was used for microbial detection. Bacteria were present in all cases, as revealed by amplification using ubiquitous 16S rDNA primers. The most frequently detected taxon was Enterococcus faecalis (64%), followed by Streptococcus spp. (21%) and Tannerella forsythensis (14%). The results of this study using a highly sensitive identification method are concurrent with those from other geographical locations using diverse identification methods in that E. faecalis is the main species found in cases of root-filled teeth associated with periradicular lesions.

Adolescent↗

PubMed2003

PCR-based identification of Treponema maltophilum, T amylovorum, T medium, and T lecithinolyticum in primary root canal infections.

OBJECTIVE: Molecular genetic methods have significantly contributed to the knowledge about the microbiota associated with infected root canals. Albeit spirochetes have been commonly observed in primary root canal infections, only recently they have been identified. The purpose of the present study was to investigate the occurrence of four treponemes-Treponema maltophilum, Treponema lecithinolyticum, Treponema amylovorum, and Treponema medium-in cases of primary endodontic infections associated with different forms of periradicular diseases through a 16S rDNA-based nested PCR assay. DESIGN: Samples were taken from thirty-one infected root canals associated with either asymptomatic or symptomatic apical periodontitis. DNA extracted from the samples was initially amplified using universal 16S rDNA primers, followed by a second round of amplification using the first PCR products to detect a specific fragment of the 16S rDNA of each target Treponema species. RESULTS: All cases were positive for the universal bacterial primers, indicating that samples contained bacterial DNA. Of the four target species, T. maltophilum was the most prevalent, being detected in 39% of the cases (33% of the asymptomatic cases and 50% of the symptomatic cases). T. lecithinolyticum was the next more prevalent among the species tested, being found in 26% of the samples (33% of asymptomatic cases and 10% of the symptomatic cases). T. amylovorum was found in 7% of the cases (5% of the asymptomatic cases and 10% of the symptomatic cases), while T. medium was in 13% of the cases (14% of the asymptomatic cases and 10% of the symptomatic cases). None of the species tested was significantly associated with clinical symptoms. CONCLUSIONS: This was possibly the hitherto first study to report the occurrence of T. lecithinolyticum, T. amylovorum, and T. medium in infections of endodontic origin. Overall, findings suggested that these oral treponemes, particularly T. maltophilum and T. lecithinolyticum, can be involved in the pathogenesis of periradicular diseases.

Adolescent↗

PubMed2003

PCR methodology as a valuable tool for identification of endodontic pathogens.

OBJECTIVES: This paper reviews the principles of polymerase chain reaction (PCR) methodology, its application in identification of endodontic pathogens and the perspectives regarding the knowledge to be reached with the use of this highly sensitive, specific and accurate methodology as a microbial identification test. DATA SOURCES: Studies published in the medical, dental and biological literature. STUDY SELECTION: Evaluation of published epidemiological studies examining the endodontic microbiota through PCR methodology. CONCLUSIONS: PCR technology has enabled the detection of bacterial species that are difficult or even impossible to culture as well as cultivable bacterial strains showing a phenotypically divergent or convergent behaviour. Moreover, PCR is more rapid, much more sensitive, and more accurate when compared with culture. Its use in endodontics to investigate the microbiota associated with infected root canals has expanded the knowledge on the bacteria involved in the pathogenesis of periradicular diseases. For instance, Tannerella forsythensis (formerly Bacteroides forsythus), Treponema denticola, other Treponema species, Dialister pneumosintes, and Prevotella tannerae were detected in infected root canals for the first time and in high prevalence when using PCR analysis. The diversity of endodontic microbiota has been demonstrated by studies using PCR amplification, cloning and sequencing of the PCR products. Moreover, other fastidious bacterial species, such as Porphyromonas endodontalis, Porphyromonas gingivalis and some Eubacterium spp., have been reported in endodontic infections at a higher prevalence than those reported by culture procedures.

Bacteria↗

PubMed2003

Polymerase chain reaction detection of Propionibacterium propionicus and Actinomyces radicidentis in primary and persistent endodontic infections.

OBJECTIVE: Propionibacterium propionicus and the recently described species Actinomyces radicidentis have been isolated from infections of endodontic origin; nevertheless, the possibility exists that their actual prevalence may have been underestimated by culture. The purpose of our study was to assess the occurrence of these 2 species in different types of endodontic infections by using the sensitive 16S rDNA-based nested polymerase chain reaction approach. STUDY DESIGN: To detect these 2 species, nested polymerase chain reaction was performed directly in samples taken from primary endodontic infections associated with asymptomatic periradicular lesions, acute apical periodontitis, or acute periradicular abscesses and in samples from patients in whom endodontic therapy had failed. DNA was extracted from the samples and initially amplified by using universal 16S rDNA primers. In the second round of amplification, the first polymerase chain reaction products were used to detect a specific 16S rDNA fragment of either P propionicus or A radicidentis. RESULTS: P propionicus was detected in 6/21 (29%) root canal samples from teeth with chronic periradicular lesions, in 5/10 (50%) cases diagnosed as acute apical periodontitis, and in 7/19 (37%) pus samples aspirated from acute periradicular abscesses. Overall, this species was found in 18/50 (36%) samples taken from primary endodontic infections. Of the root canal samples obtained from root-filled teeth with chronic periradicular lesions, P propionicus was detected in 7/12 (58%) cases. A radicidentis was detected in 1/21 (5%) root canal samples from teeth with chronic periradicular lesions and in 1/10 (10%) cases of acute apical periodontitis. No pus sample yielded this species. In general, A radicidentis was detected in 2/50 (4%) samples taken from primary endodontic infections and in 1/12 (8%) root canal samples taken from patients in whom endodontic treatment had failed. CONCLUSIONS: P propionicus was found in a relatively large number of patients with primary and persistent endodontic infections. This strengthens the assumption that this bacterial species is an endodontic pathogen associated with different forms of periradicular diseases. In contrast, A radicidentis was only occasionally detected in the patients examined. The role played by this species in endodontic infections remains to be clarified.

Actinomyces↗