CONSORT RANDOMIZED CLINICAL TRIAL

Homan Zandi, DDS,*†

Outcome of Endodontic Nikola Petronijevic, DDS,*
Ibrahimu Mdala, MSc, PhD,‡
Retreatment Using 2 Root Anne K. Kristoffersen, MSc,
PhD,† Morten Enersen, DDS,
Canal Irrigants and Influence of PhD,† Isabela N. Ro ^ças, DDS,
MSc, PhD,§ Jose
F. Siqueira, Jr.,
Infection on Healing as DDS, MSc, PhD,§ and
Dag Ørstavik, DDS, PhD*
Determined by a Molecular
Method: A Randomized Clinical
Trial

ABSTRACT
SIGNIFICANCE
Introduction: This study compared the clinical and radiographic outcome of endodontic
retreatment of teeth with apical periodontitis using either 1% sodium hypochlorite (NaOCl) or Retreatment of infected teeth
2% chlorhexidine digluconate (CHX) as the irrigant. The influence of residual infection detected using either sodium
by a molecular method on the outcome was also examined. Methods: Fifty-two root-filled hypochlorite or chlorhexidine
teeth with apical periodontitis were randomly assigned into 2 groups according to the irrigant as irrigants showed similar
used during retreatment. Root canal microbiological samples taken before (S1) and after (S2) clinical and radiographic
preparation using either NaOCl or CHX irrigation and after calcium hydroxide medication (S3) outcomes. Molecular findings
were subjected to 16S ribosomal RNA gene-based real-time quantitative polymerase chain revealed a better prognosis
reaction (qPCR) to quantify total bacteria. The periapical status was scored using the when bacteria were absent at
periapical index and dichotomized as healed (,3) or not healed (
3) at the 1- and 4-year the time of filling or the bacterial
follow-up. Results: Forty-five (NaOCl, n 5 20; CHX, n 5 25) and 33 teeth (NaOCl, n 5 16; load was reduced below
CHX, n 5 17) were available at the 1- and 4-year follow-up, respectively. After 1 year, 65% approximately 103 cell counts.
in the NaOCl group and 64% in the CHX group healed, with no differences between them
(P . .05). At the later follow-up, the corresponding figures were 81% and 82%, respectively
(P . .05). Canals that yielded qPCR-negative results in S3 had a higher healing rate (79%)
than qPCR-positive canals (45%, P , .05). The mean bacterial load increased from S2 to S3 in
half of the unhealed cases (P , .05). All S3-positive canals containing ,3.12 ! 103 bacterial
cell counts healed. Increasing the apical level of the root canal filling influenced the outcome
(P , .05). Conclusions: No significant differences in the clinical outcome between 1%
NaOCl and 2% CHX were found. Bacterial persistence at the time of filling as detected by
qPCR significantly affected the outcome. (J Endod 2019;-:1–10.)
From the *Department of Endodontics,
Institute of Clinical Dentistry, †Department
KEY WORDS of Oral Biology, Faculty of Dentistry, and
‡
Department of General Practice,
Apical limit of obturation; chlorhexidine; endodontic retreatment; retreatment outcome; University of Oslo, Oslo, Norway; and
x
root-filled teeth with infection; sodium hypochlorite Department of Endodontics, Esta
cio de
S
a University, Rio de Janeiro, Brazil
Address requests for reprints to Dr
Homan Zandi, Department of
Endodontics, Institute of Clinical Dentistry,
Antibacterial irrigation in association with mechanical debridement of the root canal is essential to treat University of Oslo, PO BX 1109 Blindern,
endodontic infections1. Sodium hypochlorite (NaOCl, 0.5%–5.25%) has been the most widely used N-0317 Oslo, Norway.
E-mail address: homanz@odont.uio.no
irrigation solution in root canal treatment/retreatment, and clinical findings have shown similar 0099-2399/$ - see front matter
antibacterial effects when different concentrations of this substance are used2,3. A recent randomized
Copyright © 2019 American Association
clinical study also reported no significant differences in the clinical outcome between high (5%) and low of Endodontists.
(1%) NaOCl concentrations4. High concentrations of NaOCl have been shown to have deleterious effects https://doi.org/10.1016/
on vital tissue5. Chlorhexidine digluconate (CHX) is another efficient antibacterial irrigant6. Substantivity to j.joen.2019.05.021

JOE  Volume -, Number -, - 2019 Endodontic Retreatment Using 2 Root Canal Irrigants 1
TABLE 1 - Data Sorted by Categories, Age, Sex, Diagnosis, and Tooth Type in 45 Patients with Teeth with
Posttreatment Apical Periodontitis
Group
Age*

50
,50
Sex
Male
Female
Diagnosis
CAP with symptoms
CAP without symptoms
Tooth type
Anterior and premolar
Molar
CAP, chronic apical periodontitis.
*Mean age 5 51 years.
† prepared to the working length with hand
The 2 treatment groups showed a similar distribution in the different categories (P values from .42–.94).
dentin7 and low tissue irritation8 are some of
the benefits of using CHX. However, CHX
lacks tissue-dissolving ability when compared
with NaOCl9. Clinical studies comparing the
antibacterial efficacy of NaOCl (2.5%–5.25%)
with CHX (0.12%–2%) in the treatment of
single-rooted teeth with primary infection,
either by using culture-dependent10–13 or
culture-independent6,14,15 techniques, and in
the retreatment of teeth with persistent
infection16 have shown conflicting results. In
addition, none of these studies has correlated
the microbiological results with the clinical
outcome.
Culture-dependent methods have
been traditionally used to evaluate the
antibacterial effectiveness of treatment
procedures, and results from several studies
have shown that bacterial persistence at the
time of filling can project a poor
outcome17,18. Because culture-independent
molecular methods are more sensitive than
culture and can detect bacteria that are
difficult to grow or even uncultivable, they
can provide more reliable results regarding
the antimicrobial effects of endodontic
treatment procedures19,20. To date, no study
has assessed the correlation between
molecular microbiology results and the
endodontic treatment/retreatment
outcome.
Therefore, the main aim of the present
randomized clinical study was to compare the
clinical and radiographic outcome of root
canal retreatment using 1% NaOCl or 2%
CHX as the irrigant in teeth with posttreatment
apical periodontitis at a 1- and 4-year
follow-up. This study also evaluated if
bacterial detection at the time of filling by a
molecular method can project the retreatment
outcome.
2 Zandi et al.
(quantitative polymerase chain reaction
[qPCR]) were as described previously16.
Briefly, 3 root canal samples were taken: 1
NaOCl† (n 5 20) CHX† (n 5 25) immediately after root canal filling removal for
retreatment (S1), 1 after instrumentation using
12 12 either 1% NaOCl or 2% CHX as the irrigant
8 13 (S2), and 1 after removal of the
interappointment calcium hydroxide dressing
11 16 (S3). Sterility controls were taken at each
9 9 treatment session. Retreatment and obturation
procedures were as follows: gutta-percha
7 7
filling was initially removed with Gates Glidden
13 18
burs (Dentsply Maillefer, Ballaigues,
9 12 Switzerland), PreRaCe burs (FKG Dentaire,
8 13 La Chaux/de/Fonds, Switzerland), and/or
hand files. No solvent was used during the
retreatment procedures. Root canals were
nickel-titanium files (NitiFlex, Dentsply Maillefer)
to sizes 40 to 60. NaOCl or CHX was used for
irrigation, and a paste of calcium hydroxide
MATERIALS AND METHODS mixed with sterile saline was applied to the
canals using Lentulo spiral fillers (Dentsply
Patient Selection and Treatment
Maillefer). The coronal portions of the canal
Fifty-two consecutive patients (32 men and 20
orifices were sealed off with a 2-mm plug of
women; mean age 5 52 years; range, 25–91
Cavit G (3M ESPE, St Paul, MN), and a thick
years), each presenting a root-filled tooth with
layer of IRM (Dentsply, York, PA) was used as a
persistent apical periodontitis with a score of 3
temporary filling on top. Two to 3 weeks later,
or more on the periapical index (PAI) scale,
the temporary restoration was removed, and
were recruited (Table 1). The patients are a
the calcium hydroxide paste was removed
subset of the material described previously16.
using files and a small amount of sterile saline
Briefly, the randomization process was as
under magnification of an operative
follows. After explaining the procedure and
microscope. Canals were irrigated with either
obtaining informed consent, patients were
irrigation solutions in each group and
randomly assigned to the 1% NaOCl or 2%
obturated with gutta-percha and AH Plus
CHX irrigation groups by flipping a coin.
sealer (Dentsply Maillefer) using the cold lateral
Blinding the treatment provider and the dental
compaction technique.
assistant was not feasible because of the odor
of the NaOCl solution compared with CHX.
The teeth had histories of root canal treatment
Bacteriologic Data
from at least 3 years back in time showing no
The bacteriologic data specific for the teeth in
signs of healing. Radiographically, the
patients presenting for the 1-year follow-up
diameter of the initial size of the
were extracted from the original data set
periapical lesions ranged from 2.4–10.2 mm
published previously16. The antibacterial
(mean 5 5.7 mm, median 5 5.7 mm). Previous
effectiveness as shown previously16 and the
root canal fillings ended from 0–5.7 mm short
clinical outcomes of the 2 irrigants were not
of the radiographic apex (mean 5 1.9 mm,
significantly different (shown in this study).
median 5 1.8 mm) with no overfillings. The
Therefore, both irrigation groups were pooled
teeth had intact coronal restorations with no
together to investigate the effect of the residual
obvious exposure of the root canal filling
total bacterial counts evaluated by a molecular
material to the oral cavity. The distribution of
method on the outcome of the retreatment of
age, sex, endodontic diagnosis, and the tooth
teeth with apical periodontitis. The bacterial
types was not significantly different in the 2
load was categorized as 102–106 bacterial cell
irrigation groups (Table 1). Complications
equivalents. In 4 teeth, S1 samples yielded
during retreatment, such as the use of
negative results for bacterial DNA; hence, they
antibiotics, a contaminated working field,
were removed from the analysis. In addition,
extraction, or apical surgery, led to the loss of 7
1 tooth with much higher bacterial counts
cases, leaving 20 and 25 cases in the NaOCl
above the average of 5.7 ! 105 bacterial cell
and CHX groups, respectively. A flowchart of
equivalents in S1 was excluded as an outlier.
the study is shown in Figure 1. Details on the
Forty patients remained for analyses involving
treatment procedures, bacteriologic sampling,
bacteriologic data. The parameters used for
and molecular microbiology analysis
analyses of associations with treatment
JOE  Volume -, Number -, - 2019
FIGURE 1 – A flowchart of the study.
outcome and relationships are provided
in Table 2.
Follow-up and Outcome
Assessments
Patients were recalled after 1 year
(average 5 14 months) for clinical and
radiographic examination. Clinical signs and
symptoms were recorded (sensitivity to
percussion or palpation, pain, deep
periodontal pocket, sinus tract, or swelling).
All teeth had received either a permanent
restoration or a crown. Digital radiographs
were taken with the same x-ray beam holder,
long-cone technique, and standardized
exposure in order to obtain optimal diagnostic
quality. All patients were also invited for a later
recall, with an average follow-up of
approximately 4 years postoperatively.
JOE  Volume -, Number -, - 2019
Restorations were checked clinically, and all
appeared to be of good quality during the
whole follow-up period.
To avoid examiner bias, a strategy was
developed for the evaluation of the periapical
status of the treated teeth. Root canal fillings,
restorations, and crowns were digitally
masked, leaving only the root and apical bone
structure visible for analysis. Next, the
radiographs were coded and randomly mixed.
Two endodontists (D.Ø. and N.P.) analyzed
the periapical status using the PAI scoring
system21. The observers were calibrated by
scoring a standard set of 100 radiographs
twice, obtaining a Cohen kappa score of 0.8,
which indicated very good reproducibility. All
radiographs were scored independently. In
case of disagreement, the evaluators
discussed divergent scores to reach a
consensus. If no consensus was reached, a
Endodontic Retreatment Using 2 Root Canal Irrigants
score given by a third endodontist was chosen
as the final result. For each tooth, the
preoperative (PAI 0), 1-year follow-up (PAI 1),
and 4-year follow-up (PAI 4) radiographs were
scored. PAI scores were dichotomized as
“healed” (PAI 1 and 2) or “not healed” (PAI 3–5)
at the follow-ups. Cases scored as healed
radiographically did not show any clinical signs
or symptoms.
For analysis of the size of the apical
periodontitis lesions, 1 observer (N.P.)
measured the largest diameter of the periapical
radiolucencies twice for each case. The
measurements were performed using the
software used for acquisition of the
radiographs and recorded in millimeters to an
accuracy of 0.1 mm. The average size of the
lesions was then recorded at the following time
points: preoperatively, at the 1-year follow-up,
and at the 4-year follow-up. For some
3
TABLE 2 - A List of the Target and the Independent Variables with Codes and the Data Types Investigated in 45 To investigate the association between lesion
Patients with Root-filled Teeth with Posttreatment Apical Periodontitis size and counts of bacteria on the treatment
outcome, a binary logistic regression model
Variables Data type was fitted to the data. The binary logistic model
Target variables was also used to investigate the association
Reduction in lesion size from start to 1-y follow-up L0–L1 Numeric between lesion size at the start and the
Reduction in lesion size from start to 4-y follow-up L0–L4 Numeric reduction in lesion size of 2 mm or .2 mm at
Reduction in lesion size from 1-y to 4-y follow-up L1–L4 Numeric the 1-year follow-up. A Spearman rank order
PAI ,3/PAI .2 Healed/not healed Binary correlation was used to determine the
Independent variables relationship between the decrease of the root
Bacteriologic data
canal filling distance to the apex and the
Bacterial sample at start S1 Numeric
decrease of the lesion size. All statistical
Bacterial sample after chemomechanical S2 Numeric
preparation analyses were performed using StataSE13
Bacterial sample after intracanal medication S3 Presence/ (StataCorp LP, College Station, TX), and the
absence significance level was set at P , .05.
Change in bacterial counts from start to S1–S3 Numeric
after medication
Change in bacterial counts from instrumentation S2–S3 Numeric
to after medication
RESULTS
Lesion size General
Lesion size at start L0 Numeric After 1 year, all 45 patients returned for the
Lesion size at start . or 5 mm LL/LS Binary clinical and radiographic examination. At the
Lesion size at 1-y follow-up L1 Numeric
later follow-up, 33 (73%) patients were
Lesion size at 4-y follow-up L4 Numeric
available; 7 individuals were lost to follow-up, in
Apical level of the root filling
Apical level of the root filling at start GP1 Numeric 4 the unhealed roots had been resected or
Apical level at start dichotomized as . or 1 mm GP1L/GP1S Binary extracted because of vertical root fracture, and
Apical level of the root filling postoperatively GP2 Numeric in 1 apical surgery had been performed on the
Increasing the apical level by at least 0.5 mm GP1–GP2
0.5 mm Numeric unhealed root. Of the 45 teeth, 29 (64%) were
Periapical score index assessment classified as healed. Details regarding
PAI at start PAI 0 Ordinal 1–5 exclusions and losses are listed in the
PAI at 1-y follow-up PAI 1 Ordinal 1–5 flowchart shown in Figure 1. PAI score
PAI at 4-y follow-up PAI 4 Ordinal 1–5 changes from the preoperative period to the
GP1, measurement taken preoperatively; GP1L, apical level 2 mm short of the apex; GP1S, apical level .2 mm short of
1- and 4-year follow-ups are provided in
the apex; GP2, measurement taken after treatment; L0, preoperative lesion size; L1, 1-year follow-up lesion size; L4, Supplemental Figure S1 and Table S1
4-year follow-up lesion size; LL, large apical periodontitis lesion (,5 mm in diameter); LS, small apical periodontitis lesion (available online at www.jendodon.com).
(5 mm in diameter); PAI, periapical index; S1, root canal sample taken after root canal filling removal for retreatment;
S2, root canal sample taken after instrumentation using either 1% sodium hypochlorite or 2% chlorhexidine digluconate;
S3, root canal sample taken after removal of the interappointment calcium hydroxide dressing.
Influence of Irrigant Type
After 1 year, 13 of 20 (65%) teeth in the
analyses, the apical periodontitis lesions were were classified as “not increased.” The NaOCl-treated group and 16 of 25 (64%) teeth
categorized as “small” if they were 5 mm or technical quality of the root canal fillings was in the CHX-treated group healed, with no
“large” if they were .5 mm in diameter. The evaluated according to Jordal et al22 by statistical differences between groups
change in the size of the lesion was calculated assigning a score from 1 to 4 (score 1 5 the (P 5 .94). At the later follow-up, healing was
and used as a continuous variable in other most adequate and score 4 5 the least observed in 13 of 16 (81%) teeth treated with
analyses. adequate). NaOCl and 14 of 17 (82%) teeth treated with
The same observer (N.P.) also CHX, with no significant differences between
measured the distance from the end point of the 2 irrigants (P . .05). The increase in the
the root canal filling material to the radiographic Statistical Analysis success rate in both irrigation groups from 1
apex, twice for each case. The average of the 2 Associations between categoric variables were year to the later follow-ups was not statistically
measurements was used for analyses. The established using the chi-square and Fisher significant (P . .05).
measurements preoperatively and after exact tests. The independent t test was used In the NaOCl group, 4 teeth were not
retreatment were called GP1 and GP2, to compare the means of the initial sizes of available for the later follow-up. Of the 11 teeth
respectively. GP1 and GP2 were dichotomized apical periodontitis between the healed and that scored as healed after 1 year, 2 teeth
as long (ie, apical level 2 mm short of the not healed groups, whereas the Mann-Whitney (18%) reversed to not healed after 4 years. Of
apex) or short (ie, apical level .2 mm short of test was used to assess the median the 5 teeth classified as not healed after 1 year,
the apex). The influence of the apical level of differences of GP1 and GP2 and to evaluate 4 (80%) healed at the later recall time. In the
filling after (GP2) retreatment on the outcome the differences of bacterial counts from S2 to CHX group, 8 teeth were lost at the later
was evaluated. The length of the root canal S3 between the healed and not healed groups. follow-up. Of the 15 teeth scored as healed
filling in retreated teeth was classified as The Poisson regression model was used to after 1 year, 2 (13%) reversed to not healed
“increased” if the apical level of filling in GP1 assess the differences in bacterial counts after 4 years. One of the 2 teeth classified as
increased by at least 0.5 mm in GP2. All values between large and small lesions and also not healed after 1 year healed after 4 years
,0.5 mm and lengths that were decreased between long and short root canal fillings in S1. (Table 3).

4 Zandi et al. JOE  Volume -, Number -, - 2019

TABLE 3 - Periapical Changes Followed from the Start through 4 Years in 45 Teeth with Posttreatment Apical Periodontitis

1-y follow-up 4-y follow-up Positive reversals Negative reversals

success, n (%) success, n (%) after 4 y,* n (%) after 4 y,† n (%)
1% NaOCl 13 (65) 13 (81) 4 (80) 2 (18)
n 20 16 5‡ 11x
2% CHX 16 (64) 14 (82) 1 (50) 2 (13)
n 25 17 2k 15{
P value ..05 ..05

CHX, chlorhexidine digluconate; NaOCl, sodium hypochlorite.

*Positive reversals: teeth scored as not healed (periapical index .2) at the 1-year follow-up scored as healed (periapical index ,3) at the 4-year follow-up.
†
Negative reversals: teeth scored as healed (periapical index ,3) at the 1-year follow-up scored as not healed (periapical index .2) at the 4-year follow-up.
‡
Two patients missing.
x
Two patients missing.
k
Seven patients missing.
{
One patient missing.

Influence of Residual Bacteria
Twenty-three of 29 teeth (79%) with negative
bacterial detection in S3 samples healed. By
contrast, healing was observed in only 5 of
11 (45%) cases showing bacterial presence
in S3. This difference was significant
(P 5 .037) (Table 4). In the 11 teeth positive
for bacteria in S3, the bacterial load was
significantly higher in S1 than in the 29 teeth
negative for bacteria in S3 (P , .01) (Fig. 2).
In the 29 root canals negative for bacteria in
S3, the mean bacterial load decreased by an
average of 104 bacterial cell equivalents from
S1, whereas in the 11 root canals positive for
bacteria, bacterial counts remained with an
TABLE 4 - The Effect of Preoperative and Intraoperative Factors on Outcome in the Retreatment of 40 Root-filled Teeth
with Posttreatment Apical Periodontitis
Prognostic factor
Preoperative
Root filling length before retreatment
Long (2 mm)
Short (.2 mm)
Initial size of lesions
Small (5 mm)
Large (.5 mm)
Intraoperative
Root filling after retreatment
Long (2 mm)
Short (.2 mm)
Apical level of root filling: change
from GP1 to GP2
Increased
Not increased
Bacterial presence at the time of
root filling
S3 positive
S3 negative
Bacterial load from S2 to S3
Remains negative or decreasing
Increasing
GP1, measurement taken preoperatively; GP2, measurement taken after treatment; S2, root canal sample taken after
instrumentation using either 1% sodium hypochlorite or 2% chlorhexidine digluconate; S3, root canal sample taken after
removal of the interappointment calcium hydroxide dressing.
Bold values indicate statistical significance.
average of 104 cells in S3 (Fig. 2). Of the 11 (P 5 .023). The bacterial load in S3 was
qPCR-positive cases in S3, 6 unhealed cases significantly higher in the not healed than in the
after 1 year harbored an average of 104 healed cases (P , .05) (Fig. 3).
bacterial counts in S3, whereas the 5 healed S2 became negative in 23 of 40
cases contained an average of 103 bacterial patients. In 20 of these cases, S3 remained
counts in S3. negative; 15 of these cases healed. In the 3
In both the healed and not healed cases in which S3 reversed to positive, none
groups, the mean total bacterial counts healed. S2 remained positive in 17 of 40
reduced significantly from S1 to S2 and from patients. Nine of these cases reversed to
S1 to S3 (P , .01). There were no significant negative in S3; 8 of these teeth healed. In the 8
differences between the healed and not healed cases in which S3 remained positive, 5
groups (P . .05). However, although the healed (Fig. 4).
bacterial load decreased from S2 to S3 in the
healed group (P 5 .007), it increased Influence of Apical Periodontitis
significantly in the group that did not heal Lesion Size
Large lesions harbored significantly higher
bacterial counts than small lesions (P , .01)
(Supplemental Table S2 is available online at
www.jendodon.com). However, the lesion
size did not influence the retreatment outcome
Healed, n (%) Not healed, n (%) P value
(P . .05). The mean size of the apical
periodontitis lesions before retreatment and
after follow-ups and the relative changes are
13 (62) 8 (38) .31 shown in Supplemental Tables S3–S7
15 (79) 4 (21)
(available online at www.jendodon.com).
10 (71) 4 (29) 1.00
18 (69) 8 (31) Influence of the Apical Level of
Filling
The apical level of the root canal filling after
26 (70) 11 (30) 1.00 retreatment (GP2) did not influence the outcome
2 (66) 1 (34) (P . .05). However, increasing the filling length
by at least 0.5 mm during retreatment
significantly improved the healing rate (81% vs
22 (81) 5 (19) .02
46%, P 5 .02; Table 4). The root canal filling
6 (46) 7 (54)
quality score increased from an average of 2
before retreatment to an average of 1.2 after
5 (45) 6 (55) .03 retreatment; this did not influence the outcome.
23 (79) 6 (21)
24 (77) 7 (23) .09
DISCUSSION
4 (44) 5 (56) The present randomized study compared the
clinical and radiographic outcome of the
retreatment of teeth with posttreatment apical
periodontitis using 1% NaOCl or 2% CHX as
the irrigant. At the 1-year follow-up, the

JOE  Volume -, Number -, - 2019 Endodontic Retreatment Using 2 Root Canal Irrigants 5
FIGURE 2 – The number of bacteria present/absent with their mean counts of bacteria (*) during retreatment (S1), after chemomechanical preparation (S2), and after intracanal
medication (S3) in 40 root-filled teeth with apical periodontitis. In the 11 teeth positive for bacteria in S3, the bacterial load was significantly higher in S1 than in the 29 teeth negative for
bacteria in S3 (P , .01). In the 29 root canals negative for bacteria in S3, the mean bacterial load decreased by an average of 104 bacterial cell equivalents from S1, whereas in the 11
root canals positive for bacteria, bacterial counts remained with an average of 104 cells in S3. Of the 11 qPCR-positive cases in S3, 6 not healed cases after 1 year still harbored an
average of 104 bacterial counts in S3, whereas the 5 healed cases contained an average of 103 bacterial counts in S3.

success rate was 65% and 64% in the NaOCl-
and CHX-treated teeth, respectively. The
success rate increased to 81% and 82%,
respectively, after 4 years. The differences
between groups in both evaluation periods
were not significant. The number of cases in
this study is limited, which means that only
relatively large differences are likely to reach
statistical significance.
Previous studies on the retreatment of
teeth with persistent infections that used 0.5%
NaOCl as the irrigant have reported similar
outcome results (58%–84%)18,23–26, which
has been shown to be poorer than the
treatment of teeth with primary infections25. To
date, no study has compared the clinical
outcome of treatments using NaOCl or CHX as
the irrigation solutions.
All complications between 1 and 4
years, such as vertical fractures, extractions,
and surgeries, occurred in the group of teeth
that had not healed after 1 year. Roots that
fractured later may well have been fractured
earlier and would have been diagnosed as not
healed after 1 year. Teeth that were lost to
follow-up may also have not healed earlier and
could be the reason for no compliance at the
later recall. Regardless, the majority of the
healed cases after 1 year maintained this
status after 4 years, and a small
proportion of teeth needed a longer time to
heal. This is in accordance with previous
studies that have suggested a follow-up period

6 Zandi et al. JOE  Volume -, Number -, - 2019

 implication of this is important because some
persistent bacteria, especially gram-positive
facultative bacteria (Streptococcus and
Enterococcus species), are highly prevalent in
root-filled teeth with infection16,34–36. For these
reasons, 2% CHX was selected as the other
irrigant solution for comparison. Two recent
clinical randomized studies using the same
molecular methodology, 1 in the treatment of
single-rooted teeth with primary infections
(2.5% NaOCl vs 2% CHX)6 and the other in the
retreatment of single-/multirooted teeth with
persistent infections (1% NaOCl vs 2% CHX)16,
FIGURE 3 – Log10 of the mean total bacterial counts reduced significantly after chemomechanical preparation (S2) in found no significant differences between the
both the healed (n 5 28) and not healed (n 5 12) cases (P , .05), with no differences between the healed and not irrigants in terms of the number of root canals
healed groups (P . .05). Although the bacterial load continued to reduce after intracanal medication (S3) in the healed negative for bacteria and the reduction in the
group (P , .05), these counts increased significantly from S2 to S3 in the not healed groups (P 5 .023). The bacterial total bacterial counts.
load in S3 was also significantly higher in the not healed than in the healed cases (P , .05, n 5 40). So far, no studies have reported on a
correlation between molecular findings and
of at least 4 to 5 years for full periradicular not vary much and high concentrations may be treatment outcomes. This was another
healing to occur1,25. harmful to the periradicular tissues29,30, 1% purpose of this study, and our findings showed
Several in vivo studies have compared NaOCl solution was used in our study. Two that bacteria present at the time of root canal
the effects of different NaOCl concentrations on percent CHX has excellent antibacterial filling (S3) as detected by qPCR significantly
disinfection2,3,27, postoperative pain28, and the properties and has been used in clinical impaired the prognosis of retreatment. The
clinical and radiographic outcome4. All these culture11,12 and molecular6,14,16 studies. difference of the healing rate was 34%
studies revealed no significant differences Results from some in vitro studies have between qPCR-negative and qPCR-positive
between the different NaOCl concentrations. indicated that the effectiveness of NaOCl and canals. These results are in agreement with
Because the antibacterial effects and the clinical CHX against several candidate endodontic several previous qualitative culture-dependent
outcome of different NaOCl concentrations do pathogens may differ31–33. The clinical studies on primary infections that have shown

FIGURE 4 – After chemomechanical preparation (S2), 23 root canals of 40 became negative. In 20 of these cases, the root canals remained negative after calcium hydroxide dressing
(S3); 15 of these teeth healed. In the 3 root canals in which S3 reversed to positive, none healed. S2 remained positive in 17 of 40 root canals. Nine of these cases reversed to negative
in S3; 8 of these teeth healed. In the 8 cases in which S3 remained positive, 5 teeth healed. * depicts mean counts of bacteria.

JOE  Volume -, Number -, - 2019 Endodontic Retreatment Using 2 Root Canal Irrigants 7
an association between positive cultures and a
poor outcome17,18,23,37–39. Molecular studies
are more sensitive, accurate, and specific than
culture studies40, presumably being a more
accurate outcome predictor for persistent
infections.
Findings revealed that the outcome was
also influenced by the initial (S1) and final (S3)
bacterial load. A previous study16 showed that
83% of the root canals became bacteria free at
S2 when the initial bacterial load was below
2.7 ! 104 bacterial cell equivalents. The initial
bacterial load was significantly higher in
S3-positive root canals than in S3-negative
ones. Of the root canals that yielded negative
qPCR results in S2, 13% reversed to positive
after calcium hydroxide medication; this may
have occurred because of recontamination
between appointments or regrowth of the
remaining bacteria. All these reversals resulted
in not healing after 1 year. In the root canals
positive for bacteria in S2, half of the cases
showed a significant increase in the bacterial
load from an average level of 102 in S2 to 104
cell counts in S3. These cases also resulted in
not healing after 1 year. All cases in which the
bacterial load was reduced and remained at
levels below 3.12 ! 103 cell counts healed.
The findings are compatible with the concept
of pathogenic threshold for residual bacteria to
influence the development or maintenance of
apical periodontitis41. Peters and Wesselink42
speculated that failures were related to higher
numbers of cultivable bacteria in the canals
€gren et al17
(.103 cells at the time of filling). Sjo
reported that the majority of successful cases
had cultivable bacterial counts below 103 cells.
It is noteworthy that 21% of the S3-negative
cases did not heal. The reasons for the failure
of bacteria-negative teeth may be the inability
of paper point sampling techniques to access
bacteria located in ramifications, isthmuses,
and dentinal tubules, in addition to
extraradicular infections43.
The present study confirmed findings
from other studies showing a higher bacterial
load and more taxa in the canals of teeth with
larger lesions1,44. However, no correlation was
observed between the initial size of apical
periodontitis and the retreatment outcome.
Previous studies have reported conflicting
results in relation to the effect of the
preoperative lesion size on the outcome23–25.
The apical level of filling did not influence
the outcome. It is important to point out that no
overfillings or gross underfillings occurred, and
all cases were retreated following
contemporary and highly accepted standards.
However, an increase in the apical level of filling
during retreatment resulted in a significantly
better outcome (81%) when compared with
cases in which such an increase did not
happen. This suggests that by advancing the
antimicrobial treatment procedures in the
apical direction, better infection control in the
very apical part of the canal was achieved.
In conclusion, this study showed similar
clinical and radiographic outcomes for the
retreatment of teeth with apical periodontitis
using 1% NaOCl or 2% CHX as the main
irrigant. However, despite the very small
difference (1%) in outcomes, one should be
aware of the relatively small sample size
evaluated. Furthermore, cases that showed
qPCR-negative results for total bacteria at the
time of filling resulted in a significantly better
healing rate. The results also indicated that a
reduction of the intracanal bacterial counts to
levels below 3.12 ! 103 cells increased the
chances for a better outcome. Finally, a better
prognosis was achieved by increasing the
apical level of the root canal filling during
retreatment.
ACKNOWLEDGMENTS
The authors thank Mrs Ronak Karimi, the
dental assistant in the private practice, for her
valuable support in sample taking and also the
logistics to carry on this study.
Supported by the University of Oslo,
Fundaça ~o Carlos Chagas Filho de Amparo a 
Pesquisa do Estado do Rio de Janeiro, and
Conselho Nacional de Desenvolvimento
Científico e Tecnolo
gico.
The authors deny any conflicts of
interest related to this study.
SUPPLEMENTARY MATERIAL
Supplementary material associated with this
article can be found in the online version at
www.jendodon.com (https://doi.org/10.1016/
j.joen.2019.05.021).

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33. Siqueira Jr JF, Batista MM, Fraga RC, et al. Antibacterial effects of endodontic irrigants on black-
pigmented gram-negative anaerobes and facultative bacteria. J Endod 1998;24:414–6.

34. Rodrigues RC, Antunes HS, Neves MA, et al. Infection control in retreatment cases: in vivo
antibacterial effects of 2 instrumentation systems. J Endod 2015;41:1600–5.
35. Zandi H, Kristoffersen AK, Ørstavik D, et al. Microbial analysis of endodontic infections in root-
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37. Molander A, Warfvinge J, Reit C, et al. Clinical and radiographic evaluation of one- and two-visit
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16S rRNA gene sequencing. Oral Microbiol Immunol 2007;22:266–71.

10 Zandi et al. JOE  Volume -, Number -, - 2019

SUPPLEMENTAL MATERIAL

SUPPLEMENTAL FIGURE S1 – PAI changes after (A ) 1 year in 45 patients and (B) 4 year in 33 patients with root-filled teeth with apical periodontitis. Patients lost to follow-up at
the 4-year recall: PAI preoperative 5 (n 5 2), PAI preoperative 4 (n 5 4), and PAI preoperative 3 (n 5 6).

JOE  Volume -, Number -, - 2019 Endodontic Retreatment Using 2 Root Canal Irrigants 10.e1
SUPPLEMENTAL TABLE S1 - Periapical Index (PAI) Scores at 1- and 4-year Follow-ups in 45 Patients with Root-
filled Teeth with Posttreatment Apical Periodontitis

PAI scores at 1- and 4-year follow-ups

PAI 5 PAI 4 PAI 3 PAI 2 PAI 1
PAI scores
(preoperative) 1y 4 y* 1y 4 y* 1y 4 y* 1y 4 y* 1y 4 y*
PAI 5 (n 5 2) 1 0 1 0 0 0 0 0 0 0
PAI 4 (n 5 17) 0 0 2 0 4 2 6 4 5 6
PAI 3 (n 5 26) 0 0 1 0 7 3 7 4 11 13

*Lost to follow-up at the 4-year recall: PAI preoperative 5 (n 5 2), PAI preoperative 4 (n 5 4), and PAI preoperative 3 (n 5 6).

SUPPLEMENTAL TABLE S2 - The Initial Mean Bacterial Load in Short- and Long Root Fillings and in Small and Large
Periapical Lesions in the Retreatment of 40 Root-filled Teeth With Posttreatment Apical Periodontitis

Mean bacterial load in S1 (log10)

Initial apical level of the root filling
Long (2 mm) 4.13 ! 105 (5.81)
n 5 21 (median, 1.42 ! 104; range 1.78 ! 102–7.43 ! 106)
Short (.2 mm) 8.79 ! 104 (4.94).
n 5 19 (median, 1.29 ! 104; range 1.02 ! 102–8.33 ! 105)
Initial sizes of apical
periodontitis lesion
Small (5 mm) 2.33 ! 104 (4.37)
n 5 14 (median, 1.13 ! 104; range 1.11 ! 103–8.33 ! 104)
Large (.5 mm) 3.85 ! 105 (5.59)
n 5 26 (median, 1.97 ! 104; range 1.02 ! 102–7.4 ! 106)

S1, root canal sample taken after root canal filling removal for retreatment.
P , .01.

10.e2 Zandi et al. JOE  Volume -, Number -, - 2019

SUPPLEMENTAL TABLE S3 - Periapical Lesions in Root-filled Teeth with Posttreatment Apical Periodontitis .5 mm
in Diameter Were Associated with a Greater Reduction in Size by 2 mm Compared with Lesions ,5 mm in Diameter (P
5 .03, n 5 36*)

Reduction of size of lesions

at 1-y follow-up (mm)
Initial sizes of apical periodontitis lesion 2 .2
5 mm 9 3
.5 mm 8 16

*Cases in which lesion sizes reduced after the 1-year follow-up.

SUPPLEMENTAL TABLE S4 - Changes in the Periapical Lesion Sizes before Retreatment and after the 1-year Follow-up and the Bacterial Load and Presence in S1 and S3 Samples
in Both Small (5 mm) and Large (.5 mm) Lesions in 40 Patients with Root-filled Teeth with Posttreatment Apical Periodontitis

Lesion sizes £5 mm preoperatively (n 5 14) Lesion sizes .5 mm preoperatively (n 5 26)

Lesion size (mm) Before retreatment After 1 year Before retreatment After 1 year
Mean (range) Mean (range) Mean (range) Mean (range)
3.93 (2.42–4.88) 2.60 (0.0–4.13) 6.95 (5.02–10.20) 4.12 (0.0–11.91)
Median 5 4.06 Median 5 2.99 Median 5 6.45 Median 5 4.51
Bacterial load in S1
Mean 2.33 ! 104 3.85 ! 105
Range (1.11 ! 103–8.33 ! 104) (1.02 ! 102–7.43 ! 106)
Bacterial load in S2
Mean 8.33 ! 101 9.44 ! 102
Range (0.0–4.95 ! 102) (0.0–6.64 ! 103)
Bacterial load in S3
Mean 7.65 ! 102 3.91 ! 103
Range (0.0–4.71 ! 103) (0.0–9.11 ! 104)
Bacterial presence in S3, n 6 5
Mean GP1 1.88 mm 2.19 mm
Mean GP2 1.17 mm 0.78 mm
Healed cases (%) 10/14 (71%) 18/26 (69%)

GP1, measurement taken preoperatively; GP2, measurement taken after treatment; S1, root canal sample taken after root canal filling removal for retreatment; S2, root canal sample taken
after instrumentation using either 1% sodium hypochlorite or 2% chlorhexidine digluconate; S3, root canal sample taken after removal of the interappointment calcium hydroxide dressing.

JOE  Volume -, Number -, - 2019 Endodontic Retreatment Using 2 Root Canal Irrigants 10.e3
 SUPPLEMENTAL TABLE S5 - The Amount of Bacterial Species in S1 and S2 Samples; the Mean Total Bacterial Counts in S1, S2, and S3 Samples; Periapical Lesion Size Preoperatively and at the 1- and 4-year Follow-up; Root Filling Distance to
10.e4

the Apex Preoperatively and Postoperatively; Apical Size at 1 and 4 mm from the Apex Preoperatively and Postoperatively; and Periapical Index (PAI) Scores at the Start and at the 1- and 4-year Follow-up in 8 Patients with Root-filled Teeth with Apical
Periodontitis
Zandi et al.

Root filling Root filling Root filling

Lesion Lesion distance distance Apical size at Apical size Root filling size at 4
Species Species Mean total Mean total Mean total Lesion size size (1-y size (4-y to the apex to the apex 1 mm from at 1 mm from size at 4 mm mm from
S1 S2 bacterial bacterial bacterial (preoperatively) follow-up) follow-up) (preoperatively) (postoperatively) the apex the apex from the apex the apex PAI PAI PAI
ID Group (n 5 159) (n 5 66) counts (S1) counts (S2) counts (S3) (L0)* (L1)* (L4)* (GP1)* (GP2)* (preoperatively)* (postoperatively)* (preoperatively)* (postoperatively)* (start) (1 y) (4 y)
8 NaOCl 20 15 5.60 ! 104 0 0 9.69 6.97 0.00 1.33 1.34 0.82 0.81 0.90 0.96 4 3 1
9 CHX 4 10 3.05 ! 105 7.47 ! 102 5.93 ! 102 5.20 2.39 1.79 0.00 0.32 0.37 0.51 0.44 0.96 3 3 3
11 CHX 15 5 7.43 ! 106 6.64 ! 103 8.34 ! 103 6.10 1.75 0.00 0.11 0.00 0.27 0.68 0.54 1.04 3 1 1
18 NaOCl 33 4 5.69 ! 105 2.56 ! 102 0 5.25 3.93 2.27 2.06 0.44 0.60 0.56 0.72 3 1
35 NaOCl 27 6 7.84 ! 103 2.61 ! 102 1.41 ! 103 4.88 4.13 1.79 2.12 1.97 0.16 0.50 0.30 0.68 3 1 1
45 CHX 37 8 6.66 ! 103 1.33 ! 102 0 5.05 0.00 0.00 0.34 0.92 0.41 0.47 0.47 0.82 3 1 1
55 NaOCl 8 10 5.86 ! 104 2.80 ! 103 7.21 ! 102 6.04 4.90 0.00 2.69 0.00 0.64 0.85 0.72 1.08 3 1 2
56 CHX 15 8 5.13 ! 105 4.56 ! 103 0 8.89 2.27 0.00 0.00 0.00 0.31 1.10 1.18 1.82 3 1 1
Average 19.875 8.25 1.12 ! 106 1.93 ! 103 1.38 ! 103 6.39 3.29 0.51 1.11 0.82 0.43 0.69 0.64 1.01 3.13 1.50 1.43

CHX, chlorhexidine digluconate; GP1, measurement taken preoperatively; GP2, measurement taken after treatment; L0, preoperative lesion size; L1, 1-year follow-up lesion size; L4, 4-year follow-up lesion size; NaOCl, sodium hypochlorite; S1, root
canal sample taken after root canal filling removal for retreatment; S2, root canal sample taken after instrumentation using either 1% sodium hypochlorite or 2% chlorhexidine digluconate; S3, root canal sample taken after removal of the interappointment
calcium hydroxide dressing.
*Values given in (mm)
JOE  Volume -, Number -, - 2019
SUPPLEMENTAL TABLE S6 - The Total Bacterial Counts in S1, S2, and S3 Samples of 40 Teeth with Posttreatment Apical Periodontitis; Periapical Lesion Sizes at the Start and at
the 1- and 4-year Follow-ups; Root Filling Distance to the Apex Preoperatively and Postoperatively; and Success Rates at 1 and 4 Years Postoperatively (N 5 40)

S3 positive,* Healed at Healed at

S1 S2 S3 n (%) L0 L1 L4 GP1 GP2 1 y, n (%) 4 y, n (%)
Total
Mean 2.58 ! 105 6.42 ! 102 2.81 ! 103 11 (27) 5.79 3.62 0.91 2.08 0.92 28 (70) 26 (83)†
n 40 40 40 40 40 40 30 40 40 40 31
NaOCl
Mean 9.20 ! 104 3.59 ! 102 4.13 ! 102 4 (23) 6.02 3.20 1.27 2.80 1.30 12 (71) 12 (86)‡
n 17 17 17 17 17 17 14 17 17 17 14
CHX
Mean 3.81 ! 105 8.53 ! 102 4.58 ! 103 7 (30) 5.61 3.36 0.60 1.55 0.63 16 (67) 14 (82)x
n 23 23 23 23 23 23 16 23 23 23 17

GP1, the distance from the end point of the root filling to the apex before retreatment give in millimeters; GP2, the distance from the end point of the root filling to the apex after retreatment
given in millimeters; L0, the mean lesion size measured in millimeters before retreatment; L1, the mean lesion size measured in millimeters at the 1-year follow-up; L4, the mean lesion size
measured in millimeters at the 4-year follow-up; S1, the mean bacterial counts of samples taken before chemomechanical preparation; S2, the mean bacterial counts of samples taken
after chemomechanical preparation; S3, the mean bacterial counts of samples taken before intracanal medication
*S3-positive samples for quantitative polymerase chain reaction.
†
Nine patients were lost to follow-up.
‡
Three patients were lost to follow-up.
x
Nine patients were lost to follow-up.

SUPPLEMENTAL TABLE S7 - Changes of the Apical Level of the Root Fillings before and after Retreatment, Changes of the Periapical Lesion Sizes before Retreatment and after the
1- and 4-tear Follow-Up, and Success Rates at the 1- and 4-tear Follow-Up in the Retreatment of 40 Teeth with Posttreatment Apical Periodontitis Using 1% Sodium Hypochlorite
(NaOCl) and 2% Chlorhexidine Digluconate (CHX) as Irrigants

Healed at Healed at
GP1–GP2* L0–LP1† L0–L4‡ L1–L4x 1 y, n (%) 4 y, n (%)
Total
Mean 1.17 2.49 5.03 2.32 28 (70) 26 (83)k
Range (20.76 to 5.34) (21.19 to 6.62) (20.82 to 9.69) (20.48 to 8.60)
Median 0.73 1.71 4.76 2.24
n 40 40 30 30 40 31
NaOCl
Mean 1.50 2.82 5.07 2.10 12 (71) 12 (86){
Range (20.76 to 5.34) (20.58 to 6.57) (1.03–9.69) (2010 to 6.97)
Median 0.86 1.76 5.13 2.13
n 17 17 14 14 17 14
CHX
Mean 0.92 2.25 4.99 2.51 16 (70) 14 (82)#
Range (20.58 to 3.87) (21.19 to 6.62) (20.82 to 8.89) (20.48 to 8.60)
Median 0.62 1.53 4.72 2.29
n 23 23 16 16 23 17

*The difference between the apical level of the root filling to the apex before and after retreatment.
†
The difference between the sizes of the periapical lesions before retreatment and after the 1-year follow-up.
‡
The difference between the sizes of the periapical lesions before retreatment and after the 4-year follow-up.
x
The difference between the sizes of the periapical lesions from the 1-year to the 4-year follow-up.
k
Nine patients were lost to follow-up after 4 years.
{
Three patients were lost to follow-up after 4 years.
#
Six patients were lost to follow-up after 4 years.

JOE  Volume -, Number -, - 2019 Endodontic Retreatment Using 2 Root Canal Irrigants 10.e5