Isabela N. Rôças

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.

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.

Actinomyces Species, Streptococci, and Enterococcus faecalis in Primary Root Canal Infections

The purpose of this study was to evaluate the prevalence of Actinomyces species, streptococci, and Enterococcus faecalis in primary root canal infections by using a molecular genetic method. Samples were obtained from 53 infected teeth, of which 27 cases were diagnosed as acute periradicular abscesses. DNA was extracted to evaluate the occurrence of 13 bacterial species by using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Polymerase chain reaction using an ubiquitous bacterial primer was undertaken to check the presence of bacterial DNA in clinical samples. All root canal samples contained bacteria as demonstrated by polymerase chain reaction. The checkerboard DNA-DNA hybridization assay allowed the detection of streptococci in 22.6% of the samples, Actinomyces species in 9.4%, and E. faecalis in 7.5%. The most prevalent species were members of the Streptococcus anginosus group. With regard to the asymptomatic lesions, the most prevalent species were S. intermedius (11.5% of the cases), E. faecalis (11.5%), and S. anginosus (7.7%). S. constellatus was the most prevalent species in pus samples (25.9% of the cases). The other most prevalent species in abscessed teeth were A. gerencseriae (14.8%), S. gordonii (11.1%), S. intermedius (11.1%), A. israelii (7.4%), S. anginosus (7.4%), and S. sanguis (7.4%). S. constellatus was the only species positively associated with acute periradicular abscess (p < 0.01).

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.

Detection of Putative Oral Pathogens in Acute Periradicular Abscesses by 16S rDNA-Directed Polymerase Chain Reaction

A 16S rDNA-directed polymerase chain reaction method was used to assess the occurrence of four black-pigmented anaerobic rods, Treponema denticola, and Actinobacillus actinomycetemcomitans in acute periradicular abscesses. Pus was collected by aspiration from 10 cases diagnosed as acute abscesses of endodontic origin. DNA was extracted from the samples and analyzed using a polymerase chain reaction-based identification assay. The method allowed detecting black-pigmented anaerobes in 80% of the examined abscesses. Porphyromonas endodontalis was found in 70%, T. denticola in 50%, Porphyromonas gingivalis in 40%, and Prevotella intermedia in 10% of the cases. P. gingivalis was always found associated with P. endodontalis. Prevotella nigrescens and A. actinomycetemcomitans were not found in any pus sample. The high prevalence of P. endodontalis, T. denticola, and P. gingivalis suggests that they can play an important role in the etiology of acute periradicular abscesses.

Elimination of Candida albicans Infection of the Radicular Dentin by Intracanal Medications

Fungi have been associated with cases of secondary or persistent root canal infections. The purpose of this study was to evaluate the effectiveness of four intracanal medications in disinfecting the root dentin of bovine teeth experimentally infected with Candida albicans. Infected dentin cylinders were exposed to four different medications: calcium hydroxide/glycerin; calcium hydroxide/0.12% chlorhexidine digluconate; calcium hydroxide/camphorated paramonochlorophenol/glycerin; and 0.12% chlorhexidine digluconate/zinc oxide. Specimens were in contact with the medications for 1 h, 2 days, and 7 days. The viability of C. albicans after exposure was evaluated by specimen incubation in culture medium to compare the effectiveness of the medications in disinfecting dentin. Results showed that the specimens treated with calcium hydroxide/camphorated paramonochlorophenol/ glycerin paste or with chlorhexidine/zinc oxide paste were completely disinfected after 1 h of exposure. Calcium hydroxide/glycerin paste only consistently eliminated C. albicans infection after 7 days of exposure. Calcium hydroxide mixed with chlorhexidine was ineffective in disinfecting dentin even after 1 week of medication exposure. Among the medications tested, the calcium hydroxide/camphorated paramonochlorophenol/glycerin paste and chlorhexidine digluconate mixed with zinc oxide were the most effective in eliminating C. albicans cells from dentinal specimens.

Comparison of 16S rDNA-based PCR and checkerboard DNA-DNA hybridisation for detection of selected endodontic pathogens.

Molecular methods have been used recently to investigate the bacteria encountered in human endodontic infections. The aim of the present study was to compare the ability of a 16S rDNA-based PCR assay and checkerboard DNA-DNA hybridisation in detecting Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Peptostreptococcus micros, Porphyromonas endodontalis, Por. gingivalis and Treponema denticola directly from clinical samples. Specimens were obtained from 50 cases of endodontic infections and the presence of the target species was investigated by whole genomic DNA probes and checkerboard DNA-DNA hybridisation or taxon-specific oligonucleotides with PCR assay. Prevalence of the target species was based on data obtained by each method. The sensitivity and specificity of each molecular method was compared with the data generated by the other method as the reference--a value of 1.0 representing total agreement with the chosen standard. The methods were also compared with regard to the prevalence values for each target species. Regardless of the detection method used, T. denticola, Por. gingivalis, Por. endodontalis and B. forsythus were the most prevalent species. If the checkerboard data for these four species were used as the reference, PCR detection sensitivities ranged from 0.53 to 1.0, and specificities from 0.5 to 0.88, depending on the target bacterial species. When PCR data for the same species were used as the reference, the detection sensitivities for the checkerboard method ranged from 0.17 to 0.73, and specificities from 0.75 to 1.0. Accuracy values ranged from 0.6 to 0.74. On the whole, matching results between the two molecular methods ranged from 60% to 97.5%, depending on the target species. The major discrepancies between the methods comprised a number of PCR-positive but checkerboard-negative results. Significantly higher prevalence figures for Por. endodontalis and T. denticola were observed after PCR assessment. There was no further significant difference between the methods with regard to detection of the other target species.

Fungal Infection of the Radicular Dentin

Although fungi have been detected in infected root canals, their precise role as endodontic pathogens has not yet been elucidated. The purpose of this study was to investigate the pattern of radicular dentin colonization by five fungal species. Bovine root sections were infected with each of the following fungal species: Candida albicans, Candida glabrata, Candida guilliermondii, Candida parapsilosis, and Saccharomyces cerevisiae. After 14 days, the sections were fixed in glutaraldehyde, split into two halves, critical point-dried in CO2, sputter-coated with gold, and examined under scanning electron microscopy. Regardless of the species, single or budding yeast cells were the only fungal forms observed. C. albicans colonized most of the specimens. On the other hand, the other four fungal species presented discrete or no colonization of the radicular dentin. C. albicans showed different patterns of dentin infection. In some specimens, colonization of the dentinal surface was slight and no penetration within dentinal tubules was observed. In the other specimens, some areas of the root canal walls were covered with large colonies of yeast cells and some dentinal tubules were heavily infected. The results suggested that whereas C. albicans showed the ability to colonize dentin, the other four fungal species did not. This can help to explain why C. albicans is the fungal species most often found in endodontic infections.

Bacteroides forsythus in Primary Endodontic Infections as Detected by Nested PCR

The purpose of this study was to investigate the prevalence of Bacteroides forsythus in primary endodontic infections using a species-specific nested polymerase chain reaction assay. Samples were collected from 50 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. A second round of amplification used the first polymerase chain reaction products to detect a specific fragment of B. forsythus 16S rDNA. B. forsythus was detected in 13 of 22 asymptomatic cases (59.1%), 4 of 10 root canals associated with acute apical periodontitis (40%), and 9 of 18 cases diagnosed as acute periradicular abscesses (50%). There was no relationship between the presence of B. forsythus and the occurrence of symptoms. In general, this bacterial species was detected in 26 of 50 samples of endodontic infections (52%). The findings of this study support the assertion that this bacterial species is associated with infections of endodontic origin and suggest that B. forsythus may be involved in the pathogenesis of different forms of periradicular lesions.

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.