Molecular Microbiological Evaluation of Passive Ultrasonic Activation as a Supplementary Disinfecting Step: A Clinical Study

doi:10.1016/j.joen.2012.09.014

Journal of Endodontics

Volume 39, Issue 2, February 2013, Pages 190-194

https://doi.org/10.1016/j.joen.2012.09.014 Get rights and content

Abstract

Introduction

This in vivo study used molecular microbiology methods to evaluate the effects of passive ultrasonic irrigation (PUI) as a supplementary disinfecting step after root canal preparation.

Methods

Samples were taken from 10 necrotic root canals of teeth with apical periodontitis before (S1) and after rotary nickel-titanium instrumentation using 2.5% NaOCl as the irrigant (S2) and then after PUI for NaOCl activation (S3). The parameters examined included the incidence of positive broad-range polymerase chain reaction (PCR) results for bacterial presence, the impact on bacterial diversity evaluated by PCR-denaturing gradient gel electrophoresis (DGGE), the quantitative bacterial reduction determined by real-time PCR, and the identification of persistent species by clone library analysis.

Results

All S1 samples were positive for bacteria in all tests. Treatment procedures were significantly effective in reducing the incidence of positive results for bacteria, the number of bacterial cells (infectious bioburden), and the bacterial diversity (number of species and abundance). However, the supplementary PUI approach did not succeed in significantly enhancing disinfection beyond that achieved by chemomechanical preparation. Several bacterial species/phylotypes were identified in post-treatment samples that were positive for bacteria.

Conclusions

Findings from this clinical study including a small sample size suggest that PUI can be ineffective in significantly improving disinfection of the main root canal after chemomechanical procedures.

Section snippets

Clinical Material, Sample Taking, and Treatment Procedures

Samples from 10 patients used in a previous study were stored at −20°C and available for more comprehensive molecular analyses in the present study (19). For a more detailed description of the rigid inclusion/exclusion criteria, clinical material, and treatment procedures, the reader is referred to the previous publication (19). Briefly, under rubber dam isolation, the operative field was disinfected before and after completion of the access cavity, and a microbiologic sample was taken from the

qPCR

Data from qPCR analysis showed that all S1 samples were positive for bacterial presence. Six cases still had detectable bacteria in both S2 and S3. Of these, 5 cases maintained the positive status of S2 in S3, 1 case that was positive in S2 became negative in S3, and another case was negative in S2 but reverted to positive in S3. Quantitative data are depicted in Table 1. A mean bacterial load of 2.21 × 10⁶ cells was disclosed in S1 samples (range, 1.43 × 10⁴–1.54 × 10⁷). This number

Discussion

The antibacterial effects of PUI for the activation of NaOCl applied after chemomechanical debridement with rotary NiTi instrumentation and NaOCl irrigation were investigated in vivo by molecular microbiology techniques. Several microbiological parameters were taken into account by the distinct molecular analyses.

One of these parameters was the incidence of positive PCR results for bacteria after treatment procedures. This was a qualitative analysis based on qPCR findings, which showed that 60%

Acknowledgments

The authors deny any conflicts of interest related to this study.

References (45)

G.B. Shuping et al.
Reduction of intracanal bacteria using nickel-titanium rotary instrumentation and various medications
J Endod
(2000)
J.F. Siqueira et al.
Bacteriologic investigation of the effects of sodium hypochlorite and chlorhexidine during the endodontic treatment of teeth with apical periodontitis
Oral Surg Oral Med Oral Pathol Oral Radiol Endod
(2007)
I.N. Rôças et al.
In vivo antimicrobial effects of endodontic treatment procedures as assessed by molecular microbiologic techniques
J Endod
(2011)
I.N. Rôças et al.
Comparison of the in vivo antimicrobial effectiveness of sodium hypochlorite and chlorhexidine used as root canal irrigants: a molecular microbiology study
J Endod
(2011)
R.N. Weller et al.
Efficacy of ultrasonic cleaning
J Endod
(1980)
L.S. Gu et al.
Review of contemporary irrigant agitation techniques and devices
J Endod
(2009)
F.R. Alves et al.
Disinfecting oval-shaped root canals: effectiveness of different supplementary approaches
J Endod
(2011)
K. Carver et al.
In vivo antibacterial efficacy of ultrasound after hand and rotary instrumentation in human mandibular molars
J Endod
(2007)
J.F. Siqueira et al.
Exploiting molecular methods to explore endodontic infections: Part 2-redefining the endodontic microbiota
J Endod
(2005)
S.S. Paiva et al.
Supplementing the antimicrobial effects of chemomechanical debridement with either passive ultrasonic irrigation or a final rinse with chlorhexidine: a clinical study
J Endod
(2012)

F.R. Alves et al.

Bacterial community profiling of cryogenically ground samples from the apical and coronal root segments of teeth with apical periodontitis

J Endod

(2009)

S.F. Altschul et al.

Basic local alignment search tool

J Mol Biol

(1990)

P.N. Nair et al.

Microbial status of apical root canal system of human mandibular first molars with primary apical periodontitis after “one-visit” endodontic treatment

Oral Surg Oral Med Oral Pathol Oral Radiol Endod

(2005)

J. Vera et al.

One- versus two-visit endodontic treatment of teeth with apical periodontitis: a histobacteriologic study

J Endod

(2012)

H. Martin

Ultrasonic disinfection of the root canal

Oral Surg Oral Med Oral Pathol

(1976)

M. Ahmad et al.

Ultrasonic debridement of root canals: acoustic streaming and its possible role

J Endod

(1987)

M. Ahmad et al.

Ultrasonic debridement of root canals: an insight into the mechanisms involved

J Endod

(1987)

M. Ahmad et al.

Ultrasonic debridement of root canals: acoustic cavitation and its relevance

J Endod

(1988)

J.F. Siqueira et al.

Bacterial reduction in infected root canals treated with 2.5% NaOCl as an irrigant and calcium hydroxide/camphorated paramonochlorophenol paste as an intracanal dressing

J Endod

(2007)

I.N. Rôças et al.

Identification of bacteria enduring endodontic treatment procedures by a combined reverse transcriptase-polymerase chain reaction and reverse-capture checkerboard approach

J Endod

(2010)

M. Brundin et al.

Persistence of dead-cell bacterial DNA in ex vivo root canals and influence of nucleases on DNA decay in vitro

Oral Surg Oral Med Oral Pathol Oral Radiol Endod

(2010)

J.F. Siqueira et al.

Clinical implications and microbiology of bacterial persistence after treatment procedures

J Endod

(2008)

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Citation Excerpt :
There was a significant reduction in the bacterial load from pre-treatment (S1) to post-instrumentation (S2) and also from post-instrumentation to post-irrigation activation (S3). These results are in accordance with previous clinical studies that evaluated the effectiveness of chemo-mechanical procedures using 2.5 % NaOCl irrigation in reducing the bacterial load from root canal systems [32,33]. In the present study when activation of NaOCl using the various techniques were compared, ultrasonics and XP-endo Finisher were associated with the greatest reduction in CFUs with no significant difference between them.
To evaluate the effect of NaOCl irrigant activation using passive ultrasonic activation, F-files, XP-endo Finisher files and or needle irrigation alone on the reduction of intracanal bacteria from root canals in teeth undergoing root canal treatment.
Eighty patients with asymptomatic apical periodontitis were randomly divided into four groups. Access cavities were prepared and pre-treatment samples were taken (S1). Canals were then prepared and post-instrumentation samples (S2) taken. NaOCl was then activated with the various irrigant activation systems and post-irrigation samples (S3) taken. The samples were processed and colony forming units (CFUs) of bacteria were determined. The reduction in CFUs among the experimental groups were analysed using the Kruskal Wallis test and pairwise comparisons with the Mann-Whitney test. The Friedman test was used to compare the differences in the reduction of CFUs within the groups.
Overall, for all groups there was a significant reduction in the CFUs from S1 to S2 (P < 0.05) and from S2 to S3 (P < 0.05). Both XP-endo Finisher and passive ultrasonic activation techniques were associated with the greatest reduction in CFUs with no significant difference between them (P = 0.24). However, both were associated with significantly lower CFUs when compared to F-file activation and needle irrigation (P < 0.05). F-file activation reduced the CFUs significantly more compared to needle irrigation (P = 0.04).
XP-endo Finisher file activation and passive ultrasonic activation were equivalent in terms of reduction in CFUs. F-file activation did not reduce the CFUs to the same extent as the XP-endo Finisher file and ultrasonic activation but reduced the CFUs more than needle irrigation.
The present study highlights the effect of new XP-endo Finisher file and passive ultrasonic activation in reducing the bacterial load from the root canal system which is important for the success of root canal treatment.
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: Supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazilian Governmental Institutions.

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Clinical ResearchMolecular Microbiological Evaluation of Passive Ultrasonic Activation as a Supplementary Disinfecting Step: A Clinical Study

Abstract

Introduction

Methods

Results

Conclusions

Section snippets

Clinical Material, Sample Taking, and Treatment Procedures

qPCR

Discussion

Acknowledgments

J Endod

Oral Surg Oral Med Oral Pathol Oral Radiol Endod

J Endod

J Endod

J Endod

J Endod

J Endod

J Endod

J Endod

J Endod

J Endod

J Mol Biol

Oral Surg Oral Med Oral Pathol Oral Radiol Endod

J Endod

Oral Surg Oral Med Oral Pathol

J Endod

J Endod

J Endod

J Endod

J Endod

Oral Surg Oral Med Oral Pathol Oral Radiol Endod

Clinical implications and microbiology of bacterial persistence after treatment procedures

J Endod

Clinical Research
Molecular Microbiological Evaluation of Passive Ultrasonic Activation as a Supplementary Disinfecting Step: A Clinical Study