Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections.

OBJECTIVE: The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. METHODOLOGY: Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). RESULTS: Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). CONCLUSIONS: The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Ex vivo evaluation of three instrumentation techniques on E. faecalis biofilm within oval shaped root canals.

The objective of the present study was to assess the effectiveness of reciprocating instrumentation in disinfecting oval-shaped root canals infected with Enterococcus faecalis. Forty-five human lower premolars were infected with a culture of E. faecalis (ATCC 29212) for 28 days. Five other teeth that were neither contaminated nor instrumented were used as controls. The 45 specimens were divided into three experimental groups (n = 15) based on the root canal preparation technique used: manual (K-type, Dentsply Maillefer, Ballaigues, Switzerland); rotary (MTwo, VDW GmbH, Munich, Germany); and reciprocating (Reciproc R50, VDW GmbH, Munich, Germany) instruments. During chemomechanical preparation, 21 mL of 2.5% NaOCl was used as the irrigating solution. Microbiological sampling was performed before (S1) and immediately after (S2) the chemomechanical preparation using sterilized paper points. Specimens were then cleaved, and 0.02 g of dentine chips was collected from the root thirds to verify the presence of microorganisms in dentinal tubules. All three preparation techniques reduced the number of microorganisms in the root canal lumen and dentine chips from the root thirds, but no significant differences were observed between the three groups (p > 0.05). Reciprocating instrumentation with Reciproc R50 was effective in reducing the number of microorganisms within the root canal system. Although this technique involves the use of only one file to perform the root canal therapy, it is as effective as conventional rotary instrumentation in reducing the E. faecalis biofilm from the root canal system. However, further clinical investigations are warranted in order to ratify these results.

Ex vivo evaluation of three instrumentation techniques on E. faecalis biofilm within oval shaped root canals

The objective of the present study was to assess the effectiveness of reciprocating instrumentation in disinfecting oval-shaped root canals infected with Enterococcus faecalis. Forty-five human lower premolars were infected with a culture of E. faecalis (ATCC 29212) for 28 days. Five other teeth that were neither contaminated nor instrumented were used as controls. The 45 specimens were divided into three experimental groups (n = 15) based on the root canal preparation technique used: manual (K-type, Dentsply Maillefer, Ballaigues, Switzerland); rotary (MTwo, VDW GmbH, Munich, Germany); and reciprocating (Reciproc R50, VDW GmbH, Munich, Germany) instruments. During chemomechanical preparation, 21 mL of 2.5% NaOCl was used as the irrigating solution. Microbiological sampling was performed before (S1) and immediately after (S2) the chemomechanical preparation using sterilized paper points. Specimens were then cleaved, and 0.02 g of dentine chips was collected from the root thirds to verify the presence of microorganisms in dentinal tubules. All three preparation techniques reduced the number of microorganisms in the root canal lumen and dentine chips from the root thirds, but no significant differences were observed between the three groups (p > 0.05). Reciprocating instrumentation with Reciproc R50 was effective in reducing the number of microorganisms within the root canal system. Although this technique involves the use of only one file to perform the root canal therapy, it is as effective as conventional rotary instrumentation in reducing theE. faecalis biofilm from the root canal system. However, further clinical investigations are warranted in order to ratify these results.

Influência da irrigação ultrassônica passiva na redução de bactérias e endotoxinas dos canais radiculares: um estudo clínico randomizado / Influence of passive ultrasonic irrigation in bacteria and endotoxins from root canals: a randomized clinical study

Este estudo clínico analisou os efeitos dos procedimentos endodônticos e da irrigação ultrassônica passiva (PUI) em bactérias e endotoxinas de canais radiculares. Cinquenta pacientes com dentes com periodontite apical primária foram divididos de forma randomizada em dois grupos: PUI (n=25) e irrigação convencional (IC) (n=25). O preparo químico-cirúrgico (PQC) foi realizado com instrumentos reciprocantes, utilizando-se NaOCl 2,5% durante o preparo; e EDTA 17%, para remoção do magma dentinário. Os canais radiculares foram preenchidos com pasta de hidróxido de cálcio por 14 dias e obturados. Foram realizadas coletas microbiológicas dos canais antes (S1) e após o PQC (S2), após os protocolos de irrigação (S3), após a medicação intracanal (S4) e após a reinstrumentação dos canais (S5). Durante o processamento das amostras, as coletas de 5 casos foram perdidas por fatores diversos. As amostras foram analisadas por PCR quantitativo para detecção e quantificação de bactérias e pelo teste turbidimétrico de LAL para detecção e determinação do nível de endotoxinas. Bactérias e endotoxinas foram observadas em 100% das amostras iniciais coletadas. Em ambos os grupos, houve diminuição significativa na concentração de endotoxinas entre uma etapa do tratamento e a etapa posterior (p<0,05). O mesmo foi observado quanto ao número de bactérias, exceto entre a remoção da medicação intracanal e a reinstrumentação antes da obturação (S5). A análise intergrupos demonstrou que, com relação às endotoxinas, não foram observadas diferenças significativas entre os grupos (p>0,05)...

The aim of this clinical study was to compare the effects of passive ultrasonic irrigation (PUI) and intracanal medication with calcium hydroxide in bacteria and endotoxins from root canal. Fifty teeth with apical periodontitis were randomly divided into two groups according to the irrigation protocol: PUI (n = 25) and conventional irrigation (CI) (n = 25). The root canal preparation of all the teeth was carried out with reciprocating files and 2.5% NaOCl during preparation; and 17% EDTA for smear layer removal. The root canals were medicated with calcium hydroxide for 14 days. Microbiological sampling were performed before (S1) and after the preparation (S2) after irrigation protocols (S3), and after intracanal medication (S4 and S5). During the processing of the samples, five cases were lost for several factors. The samples were analyzed by real time PCR, for the detection and quantification of bacteria, and the turbidimetric LAL assay, for the detection and analysis of the endotoxin levels. Bacteria and endotoxins were observed in 100% of the initial samples. In both groups, there was a significant decrease in the concentration of endotoxins between one step and the subsequent step of treatment (p < 0.05). The same was observed for the number of bacteria, with the exception of the reinstrumentation after the removal of the medication (S5). The intergroup analysis showed no significant differences between groups in endotoxin reduction ( p > 0.05 ). PUI was able to reduce the number of bacteria significantly better than CI (p < 0.05). No significant statistical difference was observed between groups regarding the occurrence of cases wielding positive results for bacteria or endotoxin. It was concluded that PUI was more effective than CI in reducing the number of bacteria but not the amount of endotoxin in the root canal. Furthermore, each step of the endodontic therapy was effective in reducing both the number of bacteria as the amount of endotoxin...

FE-SEM Evaluation of Dental Specimens Prepared by Different Methods for In Vitro Contamination.

Objective. To evaluate through FE-SEM the cleanliness and dentinal alterations promoted by different methods of dental sample preparation. Methods. Twenty-five human single-rooted teeth were used. The teeth were cleaned and autoclaved in wet medium and randomly divided into 5 groups (n = 5), according to the preparation methods employed-control group: no solutions applied; group 1: cement removal and irrigation with 5.25% NaOCl + 17 % EDTA for 4 minutes each; group 2: 17% EDTA + 2.5% NaOCl (4 minutes ultrasonic bath); group 3: cement removal and 17% EDTA + 5.25% NaOCl + phosphate buffer solution + distilled water (10 minutes ultrasonic); group 4: 17% EDTA + 5.25% NaOCl (3 minutes ultrasonic bath). Specimens were analyzed by field emission scanning electron microscope (FE-SEM), at 1500x magnification. Data were submitted to qualitative analysis according to a scoring system and submitted to Kruskal-Wallis test. Results. In ascending order, as to bind parameters, (i) cleanliness: control, group 2, group 3, group 5, and group 4, (ii) dentinal alterations: group 1, group 5, group 2, group 3, and group 4. Conclusion. The proposed protocol was suitable for subsequent microbiological contamination, because it showed less dentinal morphological alterations with increased removal of organic waste.

Desinfecção de canais radiculares preparados por diferentes técnicas de instrumentação e / Disinfection of root canals prepared by different techniques of instrumentation and irrigation regimes

O intuito do presente trabalho foi avaliar in vitro a desinfecção de canais radiculares de pré-molares inferiores humanos, variando a técnica de preparo e os regimes de irrigação utilizados. Para isso, 85 espécimes foram padronizados em comprimento e diâmetro apical e, após o devido preparo, receberam contaminação com Enterococcus faecalis e Candida albicans por 28 dias. Os canais foram divididos em 8 grupos: G1 - instrumentação manual associada à irrigação com solução fisiológica; G2 - instrumentação manual associada à irrigação com NaOCl à 1,0%; G3 - instrumentação manual associada à irrigação com NaOCl à 1,0% e AC à 15%; G4 - instrumentação manual associada à irrigação com NaOCl à 5,25%; G5 - instrumentação rotatória associada à irrigação com solução fisiológica; G6 - instrumentação rotatória associada à irrigação com NaOCl à 1,0%; G7 - instrumentação rotatória associada à irrigação com NaOCl à 1,0% e AC à 15%; G8 - instrumentação rotatória associada à irrigação com NaOCl à 5,25%. O grupo controle negativo foi composto por 5 dentes preenchidos com meio de cultura esterilizado. Todos os canais dos grupos experimentais foram preparados até instrumentos com diâmetro final de 0,50mm. Coletas microbianas foram realizada antes e após o PQC, com auxílio de pontas de papel esterilizadas. Raspas de dentina foram coletadas após o PQC com o intuito de verificar a ocorrência de MOs no interior dos túbulos dentinários. Não houve diferença estatística entre as técnicas de instrumentação, quanto à diminuição microbiana no interior dos canais. Quanto aos regimes de irrigação, o soro fisiológico produziu os piores resultados, seguido pelo NaOCl à 1,0%. Já quando acompanhado pelo AC, a desinfecção alcançada não demonstrou diferença estatística quanto aos grupos irrigados com NaOCl à 5,25%.

The purpose of this study was to evaluate the in vitro disinfection achieved in root canals of human mandibular premolars, varying the preparation technique and irrigation regimens. For this, 85 specimens were standardized in length and apical diameter, and after proper preparation were contaminated with Enterococcus faecalis and Candida albicans for 28 days. After this period, the canals were divided into eight experimental groups as follows: G1 - hand instrumentation with irrigation with sterile saline, G2 - hand instrumentation with irrigation with NaOCl to 1.0%, G3 - Manual instrumentation with irrigation with NaOCl to 1.0% and citric acid to 15%, G4 - instrumentation associated with irrigation with NaOCl at 5.25%; G5 - rotary instrumentation associated with irrigation with sterile saline and G6 - rotary instrumentation associated with irrigation with NaOCl to 1.0%; G7 - rotary instrumentation with irrigation with NaOCl to 1.0% and citric acid to 15%; G8 - rotary instrumentation with irrigation with NaOCl at 5.25%. Negative control group was composed of 5 teeth had their canals filled with sterile culture medium. All root canals of the specimens of experimental groups were prepared by instruments with a diameter equivalent to the tip end of 0.50 mm. Microbial samplings were performed before and after the PQC with the aid of sterilized paper points. Dentine chips were collected after the PQC in order to verify the occurrence of microorganisms inside the dentinal tubules. The results showed no statistical difference between the techniques of instrumentation, on the microbial reduction. As for systems of irrigation, saline produced the worst results followed by NaOCl 1.0%. When followed by citric acid, disinfection achieved no difference for the groups irrigated with NaOCl at 5.25%