Effect of passive ultrasonic activation on microorganisms in primary root canal infection: a randomized clinical trial

Abstract Objective: This clinical study sought to evaluate the effectiveness of passive ultrasonic activation (PUA) in eliminating microorganisms in primary endodontic infection (PEI) after instrumentation of root canals using microbiological culture and checkerboard DNA-DNA hybridization. Methodology: Twenty root canals with PEI and apical periodontitis were selected. The root canals were instrumented and then randomly divided into 2 groups, according to the irrigation method: PUA and conventional needle irrigation (CNI). Microbiological samples were collected before instrumentation (S1), after instrumentation (S2) and after irrigation with 17% EDTA (S3). The samples were subjected to anaerobic culture technique and checkerboard DNA-DNA hybridization analysis. Results: A statistically significant difference was found between CNI (23.56%) and PUA (98.37%) regarding the median percentage values for culturable bacteria reduction (p<0.05). In the initial samples, the most frequently detected species was S. constellatus (50%), and after root canal treatment was E. faecalis (50%). Conclusion: Both treatments significantly decreased the number of bacterial species compared with the initial sample. However, no statistical difference in the total microbial load between PUA and CNI groups was detected. The number of cultivable anaerobic bacteria reduced significantly using PUA, and the bacterial composition and number of bacterial species after using either CNI or PUA was similar.


Introduction
The successful treatment of apical periodontitis depends on the maximum decrease in microorganisms and their by-products in root canals. Root canal preparation is associated with an irrigating solution to obtain maximal reduction in microbial load inside the root canal to prevent or eliminate apical periodontitis. [1][2][3] The irrigant used during instrumentation is supposed to act as a cleaning, disinfectant and lubricant agent. 4 Sodium hypochlorite (NaOCl) is the most widely used irrigant in endodontics, especially due to its antimicrobial activity 6 and organic tissue dissolution capacity. 4,5 In addition to NaOCl, the use of ethylenediaminetetraacetic acid (EDTA) is a common practice in endodontic treatment to remove the inorganic component or smear layer left in the canal during endodontic treatment. 6 However, the root canal system has some anatomical complexities such as apical ramifications, isthmus, and dentinal tubules, which may impede full disinfection. Studies have shown the presence of microorganisms in necrotic teeth not only in the main canal, but also throughout the root canal system, even after chemomechanical preparation. [7][8][9][10] The remaining bacteria may influence the treatment result and can be associated with persistent apical periodontitis. 11 Thus, all efforts have been made to obtain maximum bacterial elimination from the root canals before filling. 3 Conventional needle irrigation (CNI) is the most commonly performed irrigation system worldwide.
Despite its good control over the irrigant delivery, this technique seems to be unable to flush out organic and inorganic tissue remnants and to clean the apical third of the root canal. 12 Several adjunctive approaches have been developed to overcome the limitations of CNI. Passive ultrasonic activation (PUA) has been suggested to enhance root canal disinfection. 4 This technique improves the cleanliness of instrumented and uninstrumented areas using ultrasonic activation of the irrigant, which is expected to aid the delivery of irrigants into difficult-to-reach areas. 13 Despite the existence of several ex vivo studies assessing the antimicrobial effect of ultrasonic activation with NaOCl as an adjunctive step, they have been inconclusive regarding bacterial load reduction.
While some studies demonstrate better efficacy using the ultrasonic activation protocol, 14 others report absence of significant difference. 15,16

Sampling procedures
Instruments and all materials used in this study were treated with 60 Co gamma radiation (20 kGy for 6 hours) (EMBRARAD, Cotia, SP, Brazil). Samples were collected under aseptic conditions. The tooth was isolated with a rubber dam and had its crown and surrounding structures disinfected with 30% H 2 O 2 [volume/volume (V/V)] for 30 seconds, followed by 5.25% NaOCl for the same period and then inactivated with 5% sodium thiosulfate. 9 A two-stage access preparation was performed without using water spray but under manual irrigation with sterile/apyrogenic saline solution by using a sterile/apyrogenic high-speed diamond bur. The first stage was performed to promote a major removal of contaminants, including carious lesion and restoration.
In the second stage, the access cavity was again disinfected with 5.25% NaOCl and subsequently inactivated with 5% sodium thiosulfate before entering the pulp chamber. All procedures were performed aseptically.
Root canal samples were taken as follows: 3 sterile paper points were consecutively introduced into the full length of the canal, which was determined radiographically, and retained in position for 60 seconds, and then immediately placed into a sterile tube containing 1 mL VMGA III (Viability Medium Göteborg Agar) transport medium for microbiologic analysis. 23 After the first sample (S1 -baseline), the instrumentation was performed by one single operator using single-file reciprocation technique. According to the manufacturer's instructions, Reciproc R40 files (40.06) (VDW GmbH, Munich, Germany) were selected after confirming that for all teeth included the initial apical instrument was ISO size #20 hand file, which reached passively to working length. The file was adapted to an electric motor (VDW Silver, VDW GmbH, Munich, Germany) using preset adjustments.
The instrument was introduced into the root canal until resistance was felt and then activated. Next, the instrument was apically moved using in-and-

PUA Group
The root canals were irrigated with 4 mL of 2.5% NaOCl delivered by using a 31 gauge × 27 mm side port needle (NaviTip, Ultradent Products Inc., South Jordan, UT, USA) inserted up to 1 mm short of the WL, with PUA being performed for 30 seconds. The irrigating solution was renewed with 4 mL of 2.5% NaOCl and PUA was resumed for 30 additional seconds.
For inactivation of 2.5% NaOCl, the canal was irrigated with 5 mL of 5% sodium thiosulfate, followed by irrigation with 10 mL of saline solution. The second sample was collected (S2) in the same manner as the first sample (S1). The smear layer was removed by rinsing the canal with 17% EDTA, which remained inside the canal for 2 minutes and then was activated with PUA for 1 minute. After additional 2 minutes inside the root canal, 17% EDTA was removed by irrigation with 10 mL of saline solution. After the procedure, the third sample was collected (S3). The ultrasonic    The analysis results (CFUs/mL) are shown in Table 1.
Frequency and DNA concentration of each bacterial species investigated in S2 are shown in Figure 4.
The mean number of bacterial species in S2 was 10.7±6.7 and 8.6±6.9, respectively, when comparing PUA and CNI, as shown in Table 2 After endodontic treatment, S3, the number of bacterial species ranged from 1 to 23 (mean=8.7).
E. faecalis was the most frequently detected species (50%). No statistical difference in the number of detected species or the total bacterial load between S2 and S3 (p>0.05) was observed. The mean number of bacterial species in S3 was 7.6±5.5 and 9.8±6.3, respectively, when comparing PUA with CNI (Table   2), without statistical difference between the groups (p>0.05). Figure 5 shows the difference between the groups in the prevalence of microorganisms.
A significantly greater reduction in the number of bacterial species and in the total bacterial load was observed in the final sample (S3) with the use of PUA protocol, completely eliminating 14 bacterial species

PUA
The measurement of the outcome was defined by CBCT analysis with a 18-month follow-up. Four patients from the PUA group were absent from the recall visits. The reduction was detected for both treatments (p<0.043), considering the initial lesion volume. The mean final volumes were 39.0±45.3 and 39.3±27.9, for CNI and PUA, respectively. No differences between the groups were detected at this follow-up (p=0.614) ( Table 3).

Discussion
In this randomized clinical trial, PUA or CNI were assessed as final irrigation protocols. PUA has been suggested as an adjunctive procedure to increase the tissue dissolution 25 and, consequently, disinfection after instrumentation. Its benefits rely on the transmission of acoustic energy from a "noncutting" and oscillating tip to an irrigant inside the root canal.
The energy transmitted might lead to an acoustic streaming, cavitation, and/or warming of the irrigating substance, expanding its spectrum of action, especially on microorganisms in difficult-to-reach areas. 26,27 This study showed the presence of cultivable bacteria in all initial samples (S1). Data showed the use of both protocols reduced the number of cultivable bacteria after single-file reciprocation technique, using Before treatment (S1) After NaOCl irrigation (S2) After EDTA irrigation (S3) PUA* 9 ± 3.8 Aa 10.7 ± 6.7 Aa 7.6 ± 5.5 Aa CNI** 10.2 ± 5.9 Aa 8.6 ± 6.9 Aa 9.8 ± 6.3 Aa * PUA -Passive Ultrasonic Activation; **CNI -Conventional Needle Irrigation   Supporting this statement, our study showed a mean of 9.6 species per root canal in the baseline (S1) using the checkerboard DNA-DNA hybridization method. S.

S. constellatus, S. intermedius, and E. faecalis
remained in more than 45% of root canals in both groups, PUA e CNI, after endodontic treatment (S3).
Likewise, these findings demonstrate that Grampositive bacteria might be more resistant to endodontic treatment, as in Rôças and Siqueira 35 (2011). Besides, S. constellatus and S. intermedius are highly prevalent in primary endodontic infections, and, despite being commensal oral bacteria, they may be related to acute and invasive diseases when associated. 37 E. faecalis is also highly prevalent in primary endodontic infections due to its capacity to deeply penetrate into dentinal tubules 38 and its resistance to intracanal medication, thus being considered a microorganism highly resistant to endodontic treatment. Although enterococci are not considered highly virulent microorganisms, some authors suggest their pathogenicity can be more related to its resistance to several antimicrobial agents. 39,40 Moreover, synergistic interactions must be considered since their collective pathogenicity probably resulted from a combination of virulence factors. 36 The authors understand the similarity between groups, considering the checkerboard results (bacterial species identification), and therefore, a supposed similarity of our results to the CFU data may be questioned. This disparity between our outcomes (CFU × checkerboard) might be explained by two reasons: 1-the outcome is different due to the specificity of the analysis, or 2due to the sample size used for checkerboard analysis.
As the checkerboard was a complementary analysis in this study, one might assume it did not influence negatively the study. On the other hand, checkerboard, when used to detect microbiological profile between different types of endodontic infection, must be used as the main outcome and included in the sample size.
In this study, the primary outcome measurement was defined by CBCT analysis, which suggested both groups were effective in reducing periapical lesion. Moreover, both treatments resulted in clinical success considering the absence of pain, mobility, and fistula. As the power estimation to include patients considered the volume assessment, the above clinical considerations might be underpowered for granting such comparison, despite being an important outcome for the proposed treatments.

Conclusion
In conclusion, both treatments significantly decreased the number of bacterial species when compared with the initial sample. However, no statistical difference in the total microbial load between PUA and CNI groups was detected. The number of cultivable anaerobic bacteria significantly decreased using PUA; bacterial composition and number of bacterial species found after using CNI or PUA was similar.

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

Funding
The study was supported by the Brazilian agencies