Clinical Antibacterial Efficacy and Treatment Outcome After Implementing Various Root Canal Irrigating Procedures
Phase: NA
Conditions: Apical Periodontitis, Root Canal Infection
Sponsor: National and Kapodistrian University of Athens

 
 
 
 
National and Kapodistrian University of Athens 
School of Dentistry 
 
 
 
 
RESEARCH PROTOCOL FOR APPROVAL 
TITLE OF PROPOSED PhD THESIS: 
 
Clinical efficacy of various irrigation parameters in the reduction of 
intracanal microbiota 
 
 
PhD STUDENT: 
Zervaki Agapi 
 
THREE-MEMBER ADVISORY COMMITTEE: 
 
1. 
Tzanetakis Giorgos (Supervisor) 
 
2. 
Georgopoulou Maria (Member) 
 
3. 
Kerezoudis Nikolaos (Member) 
 
DATE OF SUBMISSION: 

Athens __/_/2022 
 
Introduction  
Microbial infection is the main cause of pulp necrosis leading to subsequent establishment of 
apical periodontitis. The primary role of microorganisms on the development and 
maintenance of periapical disease has been long documented through classic and historic 
studies. A favorable endodontic treatment outcome is directly related to the eradication or 
the sufficient reduction of microbial load located inside the root canal system. 
Chemomechanical preparation provides a significant reduction of the intracanal microbial 
load. However, sterile root canals cannot be obtained, and sometimes the remaining bacteria 
may compromise the outcome (Barbosa-Ribeiro et al. 2021). Thus, the study and 
development of additional clinical approaches for further root canal disinfection is essential.  
Root canal irrigation is a fundamental part of chemomechanical procedure (Haapasalo 
et al. 2014). It complements mechanical preparation by the removal of residual bacteria, 
debris and necrotic pulp tissue, especially in areas that remain untouched by mechanical 
instrumentation procedure. Despite the great number of irrigants that have been proposed 
for use during clinical practice, sodium hypochlorite (NaOCl) remains the most commonly 
used irrigant due its tissue dissolution properties and its high antimicrobial capacity. 
Nevertheless, the optimal concentration of NaOCl (0,5-5%) for use in clinical practice is a 
matter of argument in endodontic research. Higher concentrations may exhibit better 
antimicrobial properties but the concomitant possible toxic effects on periradicular tissues 
should always be taken into consideration. So, an appropriate balance needs to be found 
between an adequate disinfection of the root canal system through the use of NaOCl and the 
minimal toxic effects of the irrigant on periapical tissues.   
Traditionally, culture-dependent approaches have been used to identify and quantify 
intracanal bacteria after different stages of root canal disinfection. However, these methods 
have major limitations such as low sensitivity, difficulty or inability of many species to grow 
and time consuming. The first two limitations have led to the underestimation of intracanal 
microbiota which may cause endodontic infection or persist after therapeutic procedures.  
Molecular methods have been applied to overcome the aforementioned disadvantages of 
culture-dependent approaches. They are characterized by high sensitivity and specificity, 

ability to detect both cultivable and uncultivable species without need to control anaerobic 
conditions. More specifically, Real-time PCR, also mentioned as Quantitative (qPCR) has been 
used to quantify single bacterial species or the total bacterial load of an infected root canal 
system. It is a very sensitive and highly efficient technique but it does not come without 
limitations; the main one is that the viability and pathogenicity of the detected 
microorganisms cannot be specified (Siqueira and Rôças 2005). 
Final irrigation with sodium hypolclorite is considered as one of the most important 
stages of root canal disinfection. The mechanical root canal preparation has been completed 
and final irrigation is anticipated to decrease further the microbial load under a level in which 
periapical disease can be eliminated and the healing process of the periapical tissues will 
begin.  Final irrigation can be categorized in three main parameters; the concentration and 
the volume of the main irrigant and the irrigation time. So far, few studies have investigated 
the effect of final irrigation time with NaOCl, whereas no study has quantified the volume and 
concentration of NaOCl that may lead to a sufficient decrease of the intracanal microbial load. 
Although some authors suggest that application time and volume of irrigant compensate the 
impact of concentration (Gazzaneo et al. 2019), the antimicrobial effect of various parameters 
of final irrigation has not been studied extensively yet. This highlights the need to investigate 
independently the parameters of final irrigation stage, namely the concentration, the 
application time and the volume of NaOCL and the contribution of each of them to the 
reduction of intracanal microbial load.  
The reduction of total microbial load during chemomichanical preparation will be 
estimated using Real-Time PCR based on 16S ribosomal RNA (rRNA). To expand the aims of 
the study, 2 bacterial species namely, Pseudoramibacter alactolyticus and Treponema 
Denticola were selected to be studied regarding their initial numbers and their quantitative 
changes throughout chemical disinfection. The rationale behind the selection of those specific 
species was to include both a Gram-positive and a Gram-negative species, the first 
(Pseudoramibacter alactolyticus) as most abundant in asymptomatic cases of apical 
periodontitis (Siqueira and Rôcas 2003) and the second (Treponema Denticola) as most 
abundant in symptomatic cases of apical periodontitis (Cavrini et al. 2008). In additions, both 
species has been shown that may survive during endodontic procedures and may be 
encountered in secondary infections. The latter as a result would be of outmost importance 
to be correlated with the clinical results of healing process after one year recall examination. 

Finally, the selected microorganisms have not been extensively studied in endodontic 
literature and yet in endodontic infections in Greek-living population. 
Aims of the Study 
The aim of the study is two-fold, first, to evaluate the clinical antibacterial efficacy of 
two different NaOCl concentrations (2,5% and 5%) under a predefined irrigant flow rate in 
teeth with pulp necrosis and apical periodontitis by using Real-time PCR. This will be examined 
through the calculation of the total bacterial load before any treatment procedure and the 
possible bacteria reduction after each treatment procedure (chemomechanical preparation, 
and final irrigation treatments). In addition, the antimicrobial efficacy of the above two 
different concentrations of NaOCl will be compared in terms of treatment outcome. All 
patients will be asked for one-year recall examination where the outcome of endodontic 
treatment will be evaluated through clinical and radiographic examination.  
Second, to evaluate the efficacy of final irrigation by assessing, if possible, a numerical 
definition for that “so called” as “copious irrigation”. For this purpose, the total amount of 
final irrigation will be divided in two equal parts of volume/ time (15ml for 5 minutes each). 
Total bacterial load that will remail after chemomechanical preparation will be compared to 
the number of bacteria calculated after the first and the second part of irrigation. It will be 
investigated if final irrigation provides to further microbial reduction and whether prolonged 
irrigation augments the antibacterial effect. The null hypothesis of this study is that increased 
volume/ time of irrigation will lead to similar bacteria reduction of shorter volume/ time but 
higher concentration, viz that higher NaOCl concentration compensates the antimicrobial 
effect of a more “copious” irrigation. 
Besides the total microbial load, the antibacterial efficacy of final irrigation procedure 
against two different bacterial species (namely Pseudoramibacter alactolyticus and 
Treponema denticola) will also be examined.  First, the prevalence of these specific species 
will be investigated in primary endodontic infections in Greek-living population. In addition, 
the effect of chemomechanical preparation and final irrigation procedure on the number of 
these species will be relatively examined though the reduction curves that will be obtained 
by qPCR.  
 
Materials and Methods: 

Study design: The present research will be designed as two-arm, parallel, randomized clinical 
study. The randomization of the teeth for sampling will be performed through the use of a 
special software (www.randomizer.org). All the treatment and sampling procedures will be 
carried out by the same investigator (PhD candidate Dr. Agapi Zervaki).   
 
Sample size calculation (Power analysis) 
      According to Rocas et al. (2016) and Rodriguez et al. (2017), use of NaOCl (concentration 
2.5%), may induce a reduction of total bacterial load of 55% (100% initially- 45% after 
chemomechanical preparation), in terms of percentage difference of Quantitative 
Polymerase Chain Reaction–positive samples. If we assume an expected reduction in bacterial 
load positive samples of 95%, for the novel intervention (NaOCl 5%), which accounts for a 
percentage difference in reduction between the interventions of 40%, with an alpha value of 
0.05 and an assumed power of 80%, the total sample required is 36 patients (18 per group). 
To account for any losses to follow- up, the final recruited number of patients will be raised 
to 22 per group, that is 44 patients in total. Sample size calculation is appeared below in detail.  
 
Estimated sample sizes for a two-sample proportions test 
Ho: p2 = p1 versus Ha: p2 != p1 
 
Study parameters: 
 
        alpha =    0.0500 
        power =    0.8000 
        delta =    0.4000  (difference) 
           p1 =    0.5500 
           p2 =    0.9500 
 
Estimated sample sizes: 
 
            N =        36 
  N per group =        18  
 
 
Study population: The study population will be comprised of patients who will referred for 
endodontic treatment at the Postgraduate program of the Department of Endodontics of 
School of Dentistry (National and Kapodistrian University of Athens).  Based on the estimated 
sample size, a total of 44 patients will participate in the study. Forty-four single rooted will be 
selected and randomly allocated to each experimental group.   

      A number of inclusion and exclusion criteria will be set for the participation of patients 
and eligibility of teeth in the study as follows:   
 
Inclusion criteria 
● Informed consent by the patients who wish to participate in the study. 
● Single-rooted teeth with pulp necrosis confirmed by pulp sensibility tests, negative 
response to both cold and electric pulp testing and radiographic evidence of apical 
periodontitis.  
● Teeth with relatively straight canals, complete root development and no pulp canal 
obliteration. 
Exclusion criteria  
● Patients who have received antibiotic treatment the last 3 months or need 
chemoprophylaxis for dental treatment.  
● Teeth with previous endodontic treatment. 
● Teeth with cracks or incomplete vertical root fracture which disturbs the integrity of 
the pulp chamber walls. 
● Teeth with periodontal pocket more than 4mm. (Paiva et al. 2012). 
 
Initial clinical and radiographic examination  
In the first appointment the whole process of treatment and sampling procedure will be 
explained to each patient, and they will sign an informed consent form. Firstly, the medical 
history of each patient will be recorded. Clinical examination will take place to a control tooth 
and the tooth of interest, and sensibility (cold and electric pulp test), palpation, percussion 
tests will be followed by periodontal examination. Finally, a periapical radiograph using 
parallel cone technique will complete the initial clinical and radiographic examination.  If a 
patient is likely to possess a recent CBCT taken for other reasons, it will be recorded. 
 
Treatment and sampling procedures: The compliance to aseptic techniques is essential 
throughout the entire experimental procedures (Rôças and Siqueira 2011a, b). Before the 
application of rubber dam, supragingival scaling will be carried out at the area of interest. 
Caries and defective restorations will be removed. Then, the specific tooth will be isolated. A 
double disinfecting procedure will follow at the operating field, the tooth and the clamp. 

Firstly, 3% hydrogen peroxide will be applied and then 2,5% NaOCl at all the above-mentioned 
surfaces. A sterile high-speed bur under constant sterile saline irrigation will be used for 
access cavity preparation. When the access is completed, the field including the access cavity 
will be disinfected again. Inactivation of NaOCl, will be carried out through the 5% sodium 
thiosulphate will be used and then a paper point will be scrubbed at the access cavity walls in 
order to take a sterility control sample. Only teeth of which their sterility samples are 
negative will be included in the study. 
       Immediately after access cavity preparation and the aforementioned decontamination 
procedure, the first microbiological sample will be taken (Sample S1). The root canal and the 
pulp chamber will be irrigated with sterile saline, a sterile K file #15 will be inserted in the 
canal to advance the irrigant and then the canal walls will be smoothly filed to the working 
length determined by using an apex locator. Three sterile paper points will be successively 
inserted in the same length for at least 1 minute, to absorb the fluid of the canal. The cutting 
part of the file and the paper points will aseptically be transferred to cyotubes containing Tris-
EDTA buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH = 7.6) and immediately be frozen at -
20oC. 
         Patients will be randomly divided into 2 experimental groups, each of 22 teeth, where 
different NaOCl concentrations 2,5% (Group A) and 5% (Group B) will be used. Working length 
(WL) will be established according to apex locator (Root ZX mini, Morita). Chemomechanical 
preparation will be completed in the same appointment. In the including teeth, Sx Protaper 
gold (Dentsply Maillefer, Ballaigues, Switzerland)) will be used for coronal flaring at 2/3 of the 
WL, 
Smarttrack 
(Nikinc, 
Eindhoven, 
Netherlands) 
17/.04-45/.04 
and 
Hyflex 
(Coltene/Whaledent, Altstätten, Switzerland.) 50/04 will be used for the instrumentation of 
the root canal, all at WL. Master apical file will range from 40/.04 to 50/.04, depending on the 
initial instrument of root canal. Six rotary files, apart from the orifice opener will be used in 
each case. Root canal mechanical instrumentation will be the same for both groups A and B. 
The volume of irrigant and the time of irrigation between files will be 2ml for 30 seconds for 
both groups leading therefore to a total volume of irrigant of 18ml for 270 seconds for the 
chemical preparation. A disposable luer lock syringe with a 27G needle (Endo Eze, Ultradent, 
South Jordan, UT) will deliver the irrigant throughout the procedure. The needle will be 
inserted up to 4 mm from working length. After the completion of preparation, root canals 
will be dried with paper points and flushed with 5ml of 5% sodium thiosulphate, for NaOCl 

deactivation. A small file will be inserted and instrumented the canal and a second sample 
(Sample S2) will be taken as described for Sample 1. After the chemomechanical preparation, 
an extra rinse will follow. The volume of irrigant used will be 15 ml and the time of irrigation 
5 minutes for both groups. Canals will be dried with paper points and Sample S3 will be taken 
with the exact procedure outlined above. Finally, another irrigation of 15ml in 5minutes will 
follow for each group and Sample S4 will be taken as described. 
All teeth will be obturated with warm vertical compaction with System/Obtura at the same 
appointment. Access cavities will be filled with temporary cement Cavit G and the patients 
will be referred for permanent restoration 
 
DNA extraction & Quantitative PCR analysis 
       The samples will be immediately transferred to laboratory. DNA from clinical samples will 
be extracted by using the QIAGEN DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA), 
according to the manufacturer’s instructions. Quantification of total bacteria before and after 
treatment procedures will be performed using 16S ribosomal RNA (rRNA) gene-targeted 
quantitative real-time PCR (qPCR) using the Mx3005P Real-Time PCR System (Agilent) and the 
SYBR Select Master Mix (Applied Biosystems, Foster City, CA) in a total reaction volume of 20 
mL, as described in literature (Rôças and Siqueira 2012, Antunes et al. 2015). Except for the 
universal primers, species-specific primers for P. alactolyticus and T. denticola will be used. 
 
Table 1. Primers used for qPCR assays, annealing temperatures and size (base pairs, bp) of the amplicons  
 
Target group  
Forward primer  
Reverse primer  
Annealing 
Temp   
Amplicon size 
total bacteria 1  
ACT CCT ACG GGA GGC AGC AG  
ATT ACC GCG GCT GCT GG  
53 °C  
200 bp  
Pseudoramibacter alactolyticus 2  CGAATAAGTCAG TGCCGG  
CTTCGCTTCCCTTTG TTCAG 
55°C  
421 pb  
Treponema denticola 3  
TAATACCGAATG TGCTCATTTACA T-3’  TCAAAGAAGCAT TCCCTCTTCTTCTTA 60  
316 bp  
 
        Each reaction will consist of 2x SYBR Green buffer MIX with Taq polymerase, 0.3-0.5 μM 
of each primer, 5μl of DNA template and water to 20μl. All reactions shall be prepared in 
duplicate for samples and standards. An initial denaturation at 95 °C for 3 minutes will be 
followed by 40 cycles of denaturation at 95 °C for 3-15 seconds, annealing at primer-specific 
temperatures indicated above (Table 1) for 30-60 seconds and amplicon extension at 72 °C 
for 60 seconds. Quantification of bacteria load will be performed based on the standard 
curves constructed using a DNA standard (ZymoBIOMICS Microbial Community DNA 
Standard). 

Recall examination 
      The outcome of endodontic treatment in all examined cases will be assessed. Clinical and 
radiographic evaluation will be performed as described in the initial examination. Using 
periapical index (PAI) (Orstavick et al. 1986), the score of each tooth at one year after 
treatment will be recorded. For scores ≤2 will be addressed as healed, whereas those showing 
a score ≥3 will be addressed as not healed. All the follow up examinations will be performed 
by the same clinician. However, the evaluation of follow-up radiographs will be performed by 
two independent and experienced endodontists who will be blinded to the treatment groups. 
The evaluators will be calibrated before the final radiographic evaluation and a Kappa 
coefficient will be provided for interexaminer agreement. 
 
Statistical Analysis 
Descriptive statistics will be performed for the pre-defined variables. Normality of distribution 
of residuals and homoskedacity will be checked first for the analyzed continuous data. A 
fisher’s exact test will be performed to assess any differences in the qPCR positive samples 
between the two interventions in terms of concentration and in terms of volume/ time. 
Univariable and multivariable (median) regression analysis will be performed to assess the 
effect of volume/ time and concentration of NaOCl parameters on the reduction of bacterial 
load in teeth with apical periodontitis. Interaction effects will be checked though the 
likelihood ratio test. Effect sizes and 95% Cis will also be presented.  The level of statistical 
significance will be set at 95%. All analyses will be conducted with Stata version 15.1 (Stata 
Corporation, College Station, Texas, USA). 
 
Clinical significance 
● To the best of our knowledge, this is the first randomized clinical study which 
evaluates the effect of different NaOCl concentrations on intracanal bacterial load.  
● In this study, there is the high expectation to define the ambiguous term “copious 
irrigation”. The separation of final irrigation in two time/ volume interval aims to find 
if there is a cutoff point where no further bacteria reduction will be performed.  
● The use of two different NaOCl concentrations in these time/ volume intervals intends 
to answer if higher concentrations compensate the effect of more “copious” 
irrigations. 

● The effect of NaOCl concentration on treatment outcome will be examined. 
● Bacteria load that will remain inside the root canal system after chemomechanical 
procedure will be correlated with treatment outcome, in order to set a threshold for 
bacteria reduction and success of endodontic treatment. 
● The results of this study will be of utmost clinical significance and may be directly 
applied to clinical practice of both endodontists and general practitioners.  
 
Expected outcomes 
According to the preexisting literature and to the study design a significant reduction in the 
total microbial load after chemomicanical procedure is anticipated. Irrigation with 5% sodium 
hypoclorite is expected to give better antimicrobial results. Also, final irrigation may lead to 
higher antimicrobial reduction until a cutoff point where no further antibacterial efficacy of 
irrigation could be detected. The antimicrobial effect of higher concentration of NaOCl may 
be compensated to higher volume/ time of irrigation, and this is also the null hypothesis of 
the study.   
In terms of treatment outcome, a tendency for higher success rate is expected in the higher 
NaOCl concentration group compared to lower NaOCl concentration group. 
Pseudoramibacter alactolyticus and Treponema Denticola are expected to be found in less 
than 50% of samples. Their counts are expected to reduce significantly but not eradication is 
expected in the after-treatment samples. 
 
Time schedule of the study  
1st YEAR 
1st semester: Literature Review 
2nd semester: Writing the Protocol  
3d semester: Protocol Presentation, Ethics Committee Approval 
4th semester: Treatment of patients, Writing general part of thesis 
 
 2nd YEAR 
1st semester: Treatment of patients, Writing general part of thesis 
2nd semester: Treatment of patients, DNA extraction 

3d semester: Treatment of patients, DNA extraction 
4th semester: Performing laboratory procedures (qPCR) 
3d YEAR 
1st semester: Performing laboratory procedures (qPCR) 
2nd semester: Statistical analysis, Thesis Writing, Paper writing 
 
3d semester: Thesis Writing  
4th semester: Thesis Presentation 
 
Place of Research 
The current PhD study will be performed at the Dental School of Athens UOA. Specifically, 
patients who proceed at the Department of Endodontics in the Undergraduate or 
Postgraduate Clinic for root canal treatment will be included. Endodontic treatments will be 
performed in the Postgraduate Endodontic Clinic. The storage of samples and the elaboration 
of laboratory procedures will take place at the Laboratory of Basic Science and Oral Biology. 
Real-time PCR will be performed at the Laboratory of Microbiology in the Dental School of 
Athens.  
 
Budget 
It is an entirely self-financed study. The estimated cost is 5000 euros. 
 
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