Clinical Research

Influence of the Apical Preparation Size and the

Irrigant Type on Bacterial Reduction in Root
Canaltreated Teeth with Apical Periodontitis
Renata Costa Val Rodrigues, PhD,* Homan Zandi, DDS, Anne Karin Kristoffersen, PhD,
Morten Enersen, PhD, Ibrahimu Mdala, PhD,k Dag rstavik, PhD, Isabela N. R^
ocas, PhD,*
and Jose F. Siqueira, Jr, PhD*

Abstract
Introduction: This clinical study evaluated the influ-
ence of the apical preparation size using nickel-
titanium rotary instrumentation and the effect of a
disinfectant on bacterial reduction in root canaltreated
teeth with apical periodontitis. Methods: Forty-three
teeth with posttreatment apical periodontitis were
selected for retreatment. Teeth were randomly divided
into 2 groups according to the irrigant used (2.5% so-
dium hypochlorite [NaOCl], n = 22; saline, n = 21). Ca-
nals were prepared with the Twisted File Adaptive (TFA)
system (SybronEndo, Orange, CA). Bacteriological sam-
ples were taken before preparation (S1), after using the
first instrument (S2), and then after the third instrument
of the TFA system (S3). In the saline group, an additional
sample was taken after final irrigation with 1% NaOCl
(S4). DNA was extracted from the clinical samples and
subjected to quantitative real-time polymerase chain re-
action to evaluate the levels of total bacteria and strep-
tococci. Results: S1 from all teeth were positive for
bacteria. Preparation to the first and third instruments
from the TFA system showed a highly significant intraca-
nal bacterial reduction regardless of the irrigant
(P < .01). Apical enlargement to the third instrument
caused a significantly higher decrease in bacterial
counts than the first instrument (P < .01). Intergroup
comparison revealed no significant difference between
NaOCl and saline after the first instrument (P > .05).
NaOCl was significantly better than saline after using
the largest instrument in the series (P < .01). Conclu-
sions: Irrespective of the type of irrigant, an increase
in the apical preparation size significantly enhanced
root canal disinfection. The disinfecting benefit of NaOCl
over saline was significant at large apical preparation
sizes. (J Endod 2017;-:16)
Key Words
Chemomechanical preparation, endodontic retreatment, post-treatment apical peri-
odontitis, sodium hypochlorite, Twisted File Adaptive
T he major goal of treat-
ment/retreatment of in-
fected root canals of teeth
Signicance
The outcome of endodontic retreatment is depen-
dent on the effective control of root canal infection.
with apical periodontitis
is to eliminate bacterial This clinical study showed that the larger the apical
preparation size, the higher the bacterial reduction
populations as much as
in infected canals. The antibacterial effects of
possible (1). Chemome-
NaOCl were mostly observed after apical enlarge-
chanical preparation of
ment.
the root canal is a combi-
nation of the mechanical
effects of instrumentation and irrigation with chemical effects of irrigants to achieve
root canal cleaning, shaping, and disinfection. Mechanical preparation using irrigants
with no antimicrobial effects can significantly reduce the intracanal bacterial counts (2
4). However, the use of antimicrobial irrigants has been shown to significantly improve
disinfection during root canal preparation (57).
The apical width of preparation can be regarded as an important aspect of the
treatment of infected root canals. In vitro studies have revealed that the larger the apical
preparation size of infected canals, the greater the intracanal bacterial reduction (3, 8, 9).
However, clinical studies using culture (2, 4, 7, 10, 11) or microscopy (12) have shown
inconsistent results regarding the antibacterial benefits of apical enlargement. A recent
systematic review concluded that more evidence-based clinical research is needed on
the subject (13). Moreover, thus far, no clinical study has evaluated the antibacterial ef-
fects of apical enlargement during retreatment of teeth with apical periodontitis.
Intraradicular bacterial infection is the main cause of posttreatment apical periodon-
titis (1416). Although no specific species has been recognized as a risk factor for
posttreatment disease, several studies have shown that Streptococcus species are
among the most prevalent bacterial taxa identified in postpreparation samples (1618)
and retreatment cases (14, 15, 19, 20). Their high prevalence and dominance in the
canals of teeth with posttreatment apical periodontitis (1416) suggest that streptococci
can play an important role in persistent infections associated with this disease.

From the *Molecular Microbiology Laboratory, Department of Endodontics, Faculty of Dentistry, Estacio de Sa University; Department of Endodontics, Faculty of
Dentistry, Veiga de Almeida University, Rio de Janeiro, RJ, Brazil; and Department of Endodontics, Institute of Clinical Dentistry, Department of Oral Biology, Faculty of
Dentistry, and kDepartment of General Practice, University of Oslo, Oslo, Norway.
Address requests for reprints to Dr Renata Costa Val Rodrigues, Faculty of Dentistry, Estacio de Sa University, Av Alfredo Baltazar da Silveira, 580/cobertura, Recreio,
Rio de Janeiro, RJ, Brazil 22790-710. E-mail address: recostaval@gmail.com
0099-2399/$ - see front matter
Copyright 2017 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2017.02.004

JOE Volume -, Number -, - 2017 Bacterial Reduction during Retreatment 1

Clinical Research
This in vivo study was conducted to evaluate the effects of apical
preparation size using rotary nickel-titanium instruments and irrigation
with either sodium hypochlorite (NaOCl) or saline on intracanal bacte-
rial reduction during retreatment of teeth with posttreatment apical
periodontitis. A highly sensitive quantitative real-time polymerase chain
reaction (qPCR) assay was used to quantify total bacteria and Strepto-
coccus species levels before and after preparation steps.
Material and Methods
Case Selection
Approval for the study protocol was obtained from the Ethics Com-
mittee of the Estacio de Sa University, Rio de Janeiro, RJ, Brazil, and The
Regional Ethics Committee, University of Oslo, Oslo, Norway. Forty-
three patients (28 women and 15 men; mean age = 42 years; range,
2461 years) presenting to the endodontic clinic at 2 dental schools
(Estacio de Sa University and University of Oslo) were included in
this study for retreatment of teeth with posttreatment apical periodon-
titis. Only single-rooted teeth (39 patients) and roots with a single canal
from multirooted teeth (4 patients) were included in the study. All teeth
showed radiograph evidence of posttreatment apical periodontitis and
root canal fillings no more than 4 mm short of the apex. The initial treat-
ments were performed at least 4 years previously. No symptoms were
present. All teeth had adequate coronal restorations as determined clin-
ically and radiographically and no evidence of exposure of the root ca-
nal filling material to the oral cavity. Patients showed no significant
systemic disease. Exclusion criteria included teeth with periodontal
pockets deeper than 4 mm, teeth with severe crown destruction that
prevented proper rubber dam isolation, and teeth with intraradicular
posts.
Sample Taking and Treatment Procedures
A strictly aseptic technique for sample collection during endodon-
tic retreatment was performed. After an oral rinse with 0.12% chlorhex-
idine, supragingival plaque biofilms were removed by scaling and
cleansing with pumice. Caries and/or coronal restorations were
removed with sterile high-speed and low-speed burs. The tooth was iso-
lated with a rubber dam, and the operative field, which included the
tooth, clamp, and surrounding dam, was cleaned using 3% hydrogen
peroxide and disinfected with 2.5% NaOCl. After completing the access
preparation with sterile burs under sterile saline irrigation, the operative
field, this time also including the pulp chamber, was once again cleaned
and disinfected as described previously. The residual NaOCl was neutral-
ized with 10% sodium thiosulfate, and sterility control samples were
taken by scrubbing sterile paper points on the internal surface of the cav-
osurface angle of the access cavity as described previously (16, 21). This
included the area of the access cavity walls where the paper points used
later for taking root canal samples might accidentally touch. The paper
points used for sterility controls were transferred aseptically to a
cryotube containing Tris-EDTA buffer (10 mmol/L Tris-HCl, 1 mmol/L
EDTA, pH = 7.6) and immediately frozen at 20 C.
Gutta-percha fillings were removed using D-Race DR1 (size 30/.10
at 1000 rpm) and DR2 (size 25/.04 at 600 rpm) instruments (FKG Den-
taire, La Chaux-de-Fonds, Switzerland). At this point, irrigation was per-
formed with sterile saline solution, and no solvent was used. The working
length (WL) was established 1 mm short of the apical foramen with the
aid of an electronic apex locator (Novapex; Forum Technologies, Rishon
Le-Zion, Israel) and confirmed by radiographs. Next, the canal was left
filled with saline, and a small hand instrument was placed at the WL and
used to gently file the canal walls. An initial microbiologic sample (S1)
was taken from the root canal with sterile paper points consecutively
placed at the WL. Each paper point was left in the canal for about 1 minute
2 Rodrigues et al.
to absorb the canal content. Care was taken to avoid touching the access
cavity walls with the paper points used for sampling. Paper points were
transferred to cryotubes containing RNAlater (Ambion, Austin, TX),
stored at 4 C for 12 hours, and then frozen at 20 C.
For inclusion of a tooth in the study, sterility control samples had to
be negative, and S1 samples had to be positive for bacterial presence in
the qPCR assay described later. All 43 patients with 1 tooth each satisfied
these criteria and were included in the study. Teeth were randomly
distributed into 2 groups according to the irrigant used (2.5% NaOCl
or sterile saline solution). Chemomechanical procedures were
completed at the same appointment in all cases using the Twisted File
Adaptive system (TFA; SybronEndo, Orange, CA) for instrumentation.
The sequence of instruments was selected according to the root canal
anatomy and the manufacturers directions. Canals from single-rooted
teeth were prepared using the medium/large pack (2-color bands) of
TFA instruments (25/.08, 35/.06, and 50/.04), and molar canals were
treated using the small pack (1-color band) of TFA instruments (20/
.04, 25/.06, and 35/.04).
NaOCl Group
Twenty-two root canals were irrigated with 2.5% NaOCl during
preparation. The initial instrumentation with the DR2 instrument was
performed at the WL, and the canal was rinsed with 5 mL 2.5% NaOCl.
TFA instruments were operated in the Elements Motor (SybronEndo)
and used up to the WL. The first instrument in the kit was used, and
then the canal was irrigated with 6 mL 2.5% NaOCl, dried using sterile
paper points, and flushed with 1 mL 10% sodium thiosulfate for 1 min-
ute to inactivate NaOCl. Next, a sample (S2) was taken from the canals as
described for S1. The canal was irrigated with NaOCl, and the second
TFA instrument was used. After apical preparation with the third TFA in-
strument, the canal was irrigated with NaOCl, dried, and flushed with
sodium thiosulfate, and another sample (S3) was taken. The total vol-
ume of 2.5% NaOCl up to S3 was 23 mL. The irrigant was delivered using
disposable syringes and NaviTip needles (Ultradent, South Jordan, UT)
inserted up to 3 mm short of the WL. Twenty canals in this group were
enlarged to size 50/.04, and the other 2 canals were instrumented to size
35/.04.
Saline Group
Twenty-one teeth had their root canals prepared as described for
the NaOCl group but using saline (0.9% NaCl) as the irrigant and at the
same final volume as NaOCl. After S3 sample taking, irrigation with
10 mL 1% NaOCl was performed, the canal was dried and flushed
with 1 mL 10% sodium thiosulfate for 1 minute, and an additional sam-
ple (S4) was taken. Nineteen canals in this group were instrumented to
size 50/.04, and the other 2 were prepared to size 35/.04.
After preparation in both groups, EDTA was used for smear layer
removal, and the canal was medicated with a calcium hydroxide paste.
One week later, the canal was filled with gutta-percha and sealer, and
the tooth was coronally restored.
DNA Extraction and qPCR Analysis
Clinical samples were thawed to room temperature, and DNA was
extracted using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA)
following the protocol recommended by the manufacturer.
To quantify the total bacterial load and levels of Streptococcus
species before and after preparation steps, 16S ribosomal RNA gene
targeted qPCR was performed with Power SYBR Green PCR Master
Mix (Applied Biosystems, Foster City, CA) on an ABI 7500 Real-time
PCR instrument (Applied Biosystems) in a total reaction volume of
20 mL as described previously (14). The universal bacterial primers
JOE Volume -, Number -, - 2017
 Clinical Research
were 50 -GAT TAG ATA CCC TGG TAG TCC AC-30 and 50 -TAC CTT GTT

TABLE 1. The Total Bacterial Load in Root Canal Samples of Teeth with Post-treatment Apical Periodontitis Taken before (S1) and after Chemomechanical Preparation Using the First (S2) and Third (S3) Instruments in
ACG ACT T-30 (22). The primers for quantification of streptococci

Median (range)
0 (08.23 E+03)
were 50 -AGA TGG ACC TGC GTT GT-30 (group specific) and 50 -GCT
GCC TCC CGT AGG AGT CT-30 (universal) (23). Primers in a concen-

tration of 0.5 mmol/L each and DNA extract volume of 2 mL were added
to the PCR Master Mix in MicroAmp Optical 96-well reaction plates

S4
(Applied Biosystems).
Plates were sealed, centrifuged, and subjected to amplification.
Cycling conditions for the qPCR included 95 C/10 minutes and 40 re-

5.14 E+02
Mean
peats of the following steps: 95 C/1 minute, annealing for 1 minute

(52 C for the universal reaction and 60 C for the Streptococcus-spe-
cific reaction), and 72 C/1 minute. At each cycle, the accumulation of
polymerase chain reaction products was detected by monitoring the

8.95 E+01 (09.88 E+03)

0 (03.71 E+02)
increase in fluorescence of the reporter dye (double-stranded

Median (range)
DNA-binding SYBR Green, Applied Biosystems). All measurements
were taken in triplicate for samples and standards. In all experiments,
triplicates of negative controls containing no template DNA were sub-
jected to the same procedures.
Quantification was performed using standard curves made with

S3
known concentrations of genomic DNA extracted from Streptococcus
mutans ATCC 25175 (American Type Culture Collection, Manassas,
VA) (for both total bacteria and streptococci quantification). Extracted

6.48 E+02
7.71 E+01
DNA from 107 cells of S. mutans was 10-fold diluted up to 102 cells in

Mean
Tris-EDTA buffer and used for standard curve construction. After
amplification, melting curve analysis of PCR products was performed
to determine the specificity of the amplified products. The melting
curve was obtained from 60 C95 C, with continuous fluorescence

3.27 E+01 (02.80 E+04)

0 (03.10 E+04)
measurements taken at every 1% increase in temperature. Data acqui-

In the saline group, a sample was taken after the final rinse with NaOCl (S4). Data were obtained from quantitative polymerase chain reaction analysis.
Median (range)
sition and analysis were performed using ABI 7500 software v2.0.4
(Applied Biosystems).

Statistical Analysis S2
Sample size calculation revealed that a minimum of 21 teeth per
group would suffice to show a 5% difference in absolute bacterial
counts with a power of 90%. Data on bacterial counts were collected
at 4 different time points in the saline group and 3 different time points
2.10 E+03
1.65 E+03
Mean

in the NaOCl group for each patient. Comparisons were performed for
intragroup and intergroup analyses. The Poisson regression model is
the basic model for analyzing bacterial count data and was used as
described previously (24). Because data were highly correlated within
1.75 E+03 (1.47 E+012.42 E+05)
1.00 E+03 (1.06 E+027.80 E+05)

each patient, the Poisson regression model was extended by intro-

ducing patient random effects to account for data clustering. Inter-
group comparisons of bacterial counts were performed by adjusting
Median (range)

for bacterial counts in S1. Statistical analyses were performed using

StataSE 13 software (StataCorp LP, College Station, TX), and the signif-
icance level was established at P < .05.
S1

Results
Quantification data for total bacteria are depicted in Table 1. Bac-
teria were detected in all S1 samples from both groups. In the NaOCl
group, the number of bacterial cell equivalents in S1 samples was sub-
the Twisted File Adaptive System Kit

stantially decreased after using the first (S2 samples, 98% reduction)
1.98 E+04
8.24 E+04

and third instrument in the TFA kit (S3 samples, 99.9% reduction)
Mean

(P < .01). Twelve S2 and 15 S3 samples were rendered negative for

NaOCl, sodium hypochlorite.

total bacteria (Table 2). There was a 95% reduction from S2 to S3,
which was also highly statistically significant (P < .01).
In the saline group, the initial numbers of bacterial cell equiva-
NaOCl (22)
Groups (n)
Saline (21)

lents were significantly reduced in S2 (89% reduction) and S3

(97% reduction) (P < .01). Only 4 S2 and 7 S3 samples were rendered
negative to bacteria. There was a 69% reduction in bacterial counts
from S2 to S3 (P < .01). After a final rinse with 1% NaOCl, the number

JOE Volume -, Number -, - 2017 Bacterial Reduction during Retreatment 3

Clinical Research
TABLE 2. The Incidence of Quantitative Polymerase Chain Reactionpositive Results in Samples of Teeth with Posttreatment Apical Periodontitis Taken before (S1)
and after Chemomechanical Preparation Using the First (S2) and Third (S3) Instruments in the Twisted File Adaptive System Kit
Total bacteria
Groups S1 S2 S3
Saline 21/21 (100)* 17/21 (81)* 14/21 (67)*
NaOCl 22/22 (100)* 10/22 (45)* 07/22 (32)*
NaOCl, sodium hypochlorite.
In the saline group, a sample was taken after the final rinse with NaOCl (S4).
*The number of cases with a positive result/number of positive cases for total bacteria in S1 (%).
of negative cases increased to 13, and the total bacterial levels were
further significantly reduced (98% S1S4 reduction and 21% S3S4
reduction, P < .01) (Tables 1 and 2).
The reduction in bacterial counts by irrigation and instrumenta-
tion with the smaller size instrument (S1S2) was not significantly
different between NaOCl and saline (P = .07). However, irrigation
with NaOCl was significantly more effective than saline with the larger
instrument (S1S3, P < .01).
Streptococci occurred in 19 of 22 S1 samples from the NaOCl
group. After instrumentation with the first instrument, these bacteria
were still detected in 9 samples. The S1 to S2 99% count reduction
was highly significant (P < .01). After preparation to the third instru-
ment, streptococci were encountered in 6 samples (reduction of
99.9% from S1S3 and 91% from S2S3, P < .01). In the saline group,
streptococci were present in 14 of 21 S1 samples. After TFA instrumen-
tation using the first file of the kit, streptococci were still present in 11
samples, decreasing 87% in counts from S1 to S2 (P < .01). After com-
plete preparation, streptococci still remained in 10 cases (S1S3
reduction of 94% and S2S3 reduction of 54%, P < .01). After a final
rinse with 1% NaOCl, streptococci were found in 8 cases, with 96%
reduction from S1 and 38% from S3 (P < .01 for both) (Table 3). Sta-
tistically significant results for the reduction of streptococci were the
same as those for total bacteria, including intergroup comparisons.
Discussion
This clinical study evaluated the effects of apical enlargement and
the type of irrigant on bacterial reduction in infected root canals of teeth
with posttreatment apical periodontitis. All postpreparation samples
taken after the first or third instrument in the TFA series and irrigated
with either NaOCl or saline showed a significant reduction in the levels
of total bacteria and streptococci during retreatment. This is in agree-
ment with several previous studies that have shown the effectiveness of
preparation procedures in reducing bacterial populations from root ca-
naltreated teeth (16, 24, 25).
Intragroup comparisons showed that enlargement to the third in-
strument in the TFA kit (35/.04 or 50/.04) promoted significantly
higher bacterial elimination than the first instrument (20/.04 or 25/
.08, respectively), irrespective of the irrigant type. This agrees with other
studies that showed that larger preparations promote significantly
higher bacterial elimination (24, 710). This may be a result of
several factors. Large apical preparations increase the chances for
the instrument to touch more canal wall surfaces (26) and thus be
more effective in removing adhering biofilms and infected dentin. More-
over, the larger the preparation, the higher the probability of incorpo-
rating anatomic irregularities, fins, and recesses in the final canal shape.
The mechanical and chemical efficacy of the irrigation is also increased
because large preparations allow for a deeper penetration of the irriga-
tion needle, a larger volume of irrigants reaching the apical segment
(27), and better irrigant exchange in this region (28).
4 Rodrigues et al.
Streptococci
S4 S1 S2 S3 S4
8/21 (38)* 14/21 (67)* 11/21 (52)* 10/21 (48)* 08/21 (38)*
19/22 (86)* 9/22 (41)* 6/22 (27)*
The possibility also exists that better results with large prepara-
tions as observed in this study were related to the amount of irrigant
used. A larger volume of irrigant was used after the third instrument
when compared with the first one, with increased mechanical and
chemical (in the NaOCl group) effects on bacterial elimination. The
advantage of the experiment design used in this study is that each tooth
served as its own control and bacterial elimination could be evaluated
longitudinally. However, further studies on the subject should consider
using the same volume of irrigants for different apical sizes, obviously
using different teeth for comparisons.
Bacterial reduction can be used as a surrogate end point to
treatment outcome because there is a strong correlation between
bacterial elimination and healing of apical periodontitis (29). There
are not many studies available evaluating the effects of apical enlarge-
ment on treatment outcome (30). So far, the only randomized pro-
spective study on the subject reported on the effects of apical
preparation size in relation to the first apical binding file (31);
the healing rate of apical periodontitis was 48% (2 sizes larger),
71% (3 sizes), 80% (4 sizes), 85% (5 sizes), and 92% (6 sizes).
However, statistical analysis revealed that only 2 sizes larger showed
significantly less improvement than the others, and the authors
concluded that enlargement beyond 3 sizes larger may be of no
benefit. A systematic review concluded that there is only limited in-
formation on the subject and that best current available clinical ev-
idence suggests that large apical preparations improve the treatment
outcome of teeth with apical periodontitis (30).
It is important to point out that root canal preparation must be
large enough in the apical segment to increase cleaning and disinfection
and at the same time must be compatible with the root anatomy to avoid
accidents and not put the tooth at risk. For instance, overenlargement of
the coronal segment of the canal may weaken the root and predispose it
to fracture (32).
Saline was used in 1 group as the irrigant because this study was also
interested in the evaluation of the mechanical effects of preparation.
Although the mechanical effects were highly effective in reducing bacterial
populations, this study confirmed that chemical effects (using an antimi-
crobial agent) are fundamental to achieve better disinfection (57).
However, the significant improvement in disinfection was only
observed for larger preparations. There were no significant differences
in bacterial reduction between NaOCl and saline after the first
instrument. The possible explanations for the effects of the
antimicrobial irrigant to be evident only after large file sizes are as follows:
1. A larger volume of irrigant was used as the canal was enlarged to
greater sizes
2. The irrigant remained in the canal longer as the canal was enlarged
3. Larger preparations permit for a larger volume of irrigant in the ca-
nal, increasing the chances for improved chemical effects
Streptococcus species may be important members of persistent
endodontic infections (1420). Because several different species
JOE Volume -, Number -, - 2017
 Clinical Research
have been identified in postpreparation or retreatment samples,

6.91 E+01 (07.43 E+03)

primers that could evaluate the occurrence and counts of these

TABLE 3. Streptococcus Species Levels in Root Canal Samples of Teeth with Posttreatment Apical Periodontitis Taken before (S1) and after Chemomechanical Preparation Using the First (S2) and Third (S3)

Median (range)
bacteria at the genus level were selected. The present findings
confirmed the high prevalence of streptococci in root canaltreated
teeth (overall 33/43 samples, 77%). These bacteria remained

detectable in about one half of the cases irrigated with saline. Even
after enlargement to the third instrument in canals irrigated with
S4
NaOCl, streptococci were found in about one fourth of the samples.
These findings confirm that Streptococcus species are commonly
associated with posttreatment disease, and the implications of their
7.25 E+02 permanence in the canal at the time of filling remain to be clarified.
Mean

All sterility controls yielded negative results. These samples were

taken from the internal surface of the cavosurface angle of the access
cavities because these areas represent points of possible contact and
3.52 E+02 (08.78 E+03)
0 (03.66 E+02)

contamination of the paper point used for root canal sampling. The
negative results indicated that the double disinfecting approach with
Median (range)

hydrogen peroxide and NaOCl, which involved the whole tooth crown
including the access cavity walls and the pulp chamber, succeeded in
eliminating contaminating DNA from the tooth surfaces. Properly taken
sterility control samples are essential for this type of study. In the clin-
S3

ical setting using teeth with caries or restorations, control samples

should be taken immediately before root canal sampling and not
before the access cavity is prepared as recommended for intact teeth
1.17 E+03
6.83 E+01

(33). Otherwise, any contamination occurring during access cavity

Mean

preparation procedures would pass unnoticed and provide false re-

sults. Moreover, the second disinfection step must include the pulp
chamber, as opposed to what is proposed elsewhere (33), because
In the saline group, a sample was taken after the final rinse with NaOCl (S4). Data were obtained from quantitative polymerase chain reaction analysis.

bacteria present in the pulp chamber are not relevant for studies of
3.63 E+02 (02.63 E+04)
0 (01.03 E+04)

prevalence and antimicrobial effectiveness. Samples in this study

Median (range)

were taken by an experienced operator who was rigorously trained

to proceed with reduced risks of contamination. Care was taken to
avoid that the sample fluid overflowed to the pulp chamber and that
the paper points touched the access cavity walls during sampling.
S2

Attainment of negative sterility controls as determined by qPCR indi-

cates that this study reliably relies on the bacteriological conditions
of the root canals. The negative results in several postpreparation sam-
ples further confirmed that contamination from the tooth surfaces was
2.53 E+03
7.75 E+02
Mean

not a significant issue in this study.

The previous studies that evaluated the antibacterial
effects of apical enlargement were based on bacteriological culture
(2, 4, 7, 10, 11). The qPCR approach used in this study has been
1.85 E+03 (3.89 E+021.87 E+05)
6.38 E+02 (1.02 E+027.66 E+05)

recently used to evaluate the antibacterial effectiveness of treatment

and retreatment procedures (16, 21, 24). This method has a
recognized higher sensitivity than culture methods and can detect
Median (range)

not only cultivable bacterial species but also difficult-to-culture

and as-yet-uncultivated bacteria (34). Thus, a more accurate evalu-
ation of the antibacterial treatment effectiveness is expected. Howev-
Instruments in the Twisted File Adaptive System Kit

er, a reason of concern with DNA-based methods is their ability to

S1

detect DNA from dead cells (18). A study reported no significant

differences in bacterial levels after chemomechanical preparation
with NaOCl irrigation analyzed by culture or qPCR (35). It is highly
likely that DNA released by dead bacteria is rapidly degraded by
NaOCl in solution and rendered undetectable or it is washed away
1.81 E+04
9.08 E+04
Mean

by irrigation. The several postpreparation samples that were qPCR

negative suggest that DNA from dead cells were not a significant
NaOCl, sodium hypochlorite.

issue in this study either.

In conclusion, this clinical study showed that larger apical prep-
NaOCl (19)
Groups (n)
Saline (14)

aration sizes resulted in significantly improved root canal disinfection

regardless of the type of irrigant used. The antibacterial benefits of
NaOCl over saline were significantly evident at larger apical preparation
sizes.

JOE Volume -, Number -, - 2017 Bacterial Reduction during Retreatment 5

Clinical Research
Acknowledgments
The authors are grateful to SybronEndo for providing the TFA
instruments and accessory devices.
Supported by grants from the Fundac~ao Carlos Chagas Filho de
Amparo a Pesquisa do Estado do Rio de Janeiro and Conselho Na-
cional de Desenvolvimento Cientfico e Tecnologico, Brazilian
Governmental Institutions, and the University of Oslo.
The authors deny any conflicts of interest related to this study.
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3. Siqueira JF Jr, Lima KC, Magalhaes FA, et al. Mechanical reduction of the bacterial
population in the root canal by three instrumentation techniques. J Endod 1999;25:
3325.
4. rstavik D, Kerekes K, Molven O. Effects of extensive apical reaming and calcium
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5. Siqueira JF Jr, R^ocas IN, Favieri A, Lima KC. Chemomechanical reduction of the bac-
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