review and meta-analysis. The search strategies included search in electronic biomedical journal databases (MEDLINE, EMBASE,
CENTRAL) and handsearching records, using different matches of keywords for NaOCl, CHX and Enterococcus faecalis. From 41
in vivo studies, 5 studies met the inclusion criteria. In a sample containing 159 teeth, E. faecalis was detected initially in 16 (10%)
teeth by polymerase chain reaction (PCR) and 42 (26.4%) teeth by microbial culture techniques. After root canal disinfection, this
species was observed in 11 (6.9%) teeth by PCR and 12 (7.5%) teeth by culture. Risk differences of included studies were combined
as generic inverse variance data type (Review Manager Version 5.0 – Cochrane Collaboration, http://www.cc-ims.net, accessed 15
May 2008), taking into account the separate tracking of positive and negative cultures/PCR. The level of statistical significance was
set at p<0.05. In conclusion, NaOCl or CHX showed low ability to eliminate E. faecalis when evaluated by either PCR or culture
www.fob.usp.br/jaos or www.scielo.br/jaos
EFFICACY OF SODIUM HYPOCHLORITE AND
CHLORHEXIDINE AGAINST Enterococcus faecalis – A
Carlos ESTRELA1, Julio Almeida SILVA2, Ana Helena Gonçalves de ALENCAR3,
Claudio Rodrigues LELES4, Daniel Almeida DECURCIO2
1- DDS, MSc, PhD, Chairman and Professor of Endodontics, Federal University of Goiás, Goiânia, GO, Brazil.
2- DDS, MSc, Professor of Endodontics, Federal University of Goiás, Goiânia, GO, Brazil.
3- DDS, MSc, PhD, Professor of Endodontics, Federal University of Goiás, Goiânia, GO, Brazil.
4- DDS, MSc, PhD, Professor of Prevention and Oral Rehabilitation, Federal University of Goiás, Goiânia, GO, Brazil.
Correspondence address: Prof. Carlos Estrela - Centro de Ensino e Pesquisa Odontológica do Brazil (CEPOBRAS) - Rua C-245, Quadra 546, Lote 9,
Jardim América - 74.290-200, Goiânia, GO, Brazil - e-mail: firstname.lastname@example.org
Received: December 05, 2007 - Modification: June 16, 2008 - Accepted: June 17, 2008
he efficacy of the sodium hypochlorite (NaOCl) and chlorhexidine (CHX) on Enterococcus faecalis was evaluated by systematic
Key words: E. faecalis. Sodium hypochlorite. Chlorhexidine. Irrigating solutions. Systematic review.
The pathogenicity of endodontic microorganisms
responsible for stimulating apical periodontitis creates the
need for finding effective antimicrobial medicaments16.
Sodium hypochlorite (NaOCl) and chlorhexidine (CHX) are
the most frequently widely studied and employed
antimicrobial agents for treatment of root canal infection2,5.
These medicaments present chemical characteristics that are
particularly responsible for their distinct results when
compared1,6,20,23. These variations occur probably due to
differences in methodology, biological indicators,
concentrations, exposure time, the potential for different
anatomical and treatment differences between
The contemporary literature contains numerous reports
on the antimicrobial efficacy of NaOCl and CHX in several
experimental models - infected human teeth in vivo7,16-18,29,30,
infected human teeth ex vivo4, infected dog’s teeth in vivo5,21,
infected bovine teeth ex vivo10, biofilm model in membrane
filters22,25, direct contact and agar diffusion test6,9.
E. faecalis is an extensively evaluated biological
indicator12,15,19,26,27. Some factors can explain the concern with
this pathogen in endodontic infections. Its high prevalence
in cases with post-treatment disease associated with
virulence factors (aggregation substance, enterococcal
surface proteins (Esp), gelatinase, cytolysin toxin,
extracellular superoxide production, capsular
polysaccharides, antibiotic resistance determinant) can
facilitate the adherence of host cells and extracellular matrix,
tissue invasions, immunomodulation effect and cause toxin-
Actual thinking has recommended the implementation
of evidence-based dentistry, which valorizes studies
involving the systematic review or meta-analysis. Systematic
reviews use a strict methodological approach to search,
select, evaluate, and analyze original data from primary
sources. Good scientific evidence is mandatory to elaborate
clinical decisions, yet few systematic reviews or meta-
analysis have been developed in Endodontics14,21,28.
J Appl Oral Sci. 2008;16(6):364-8
Torabinejad and Babjri28 reported that the American Dental
Association has adopted the concept of evidence-based
healthcare, which requires the judicious integration of
systematic assessments of clinically relevant scientific
Previous studies using in vitro experimental models have
confirmed the antimicrobial efficacy of NaOCl and CHX
against E. faecali6,23, while others using different study
designs have not found same results4,5,7,16-18,29,30. Relevant
clinical questions based on evidence regarding the resistance
ofE. faecalis to NaOCl or CHX should be further discussed.
Therefore, the aim of this study was to review findings on
the antibacterial efficacy of NaOCl or CHX against E.
faecalis in endodontic infection using systematic review and
MATERIALS AND METHODS
This study was designed using an analysis of longitudinal
studies from a quantitative systematic review. Prospective
studies were selected towards the efficacy of NaOCl and
CHX against E. faecalis identified in endodontic infections
before and after root canal preparation. English-language
articles were retrieved from electronic biomedical journal
databases and handsearching records.
The following databases were searched on January 2nd,
2007: MEDLINE (without filter, from 1966 to January 2nd,
2007), EMBASE (without filter, from 1980 to January 2nd,
2007), Cochrane Oral Health Group Trials Register and
Cochrane Central Register of Controlled Trials
(CENTRAL). For the electronic search strategy, the
following terms were used as keywords in several
combinations: 1. faecalis and sodium hypochlorite OR, 2.
faecalis and chlorhexidine OR, 3. faecalis and root canal
infections OR, 4. faecalis and endodontics infections OR,
5. faecalis and root canal irrigants OR, 6. faecalis and
irrigating solution OR, 7. faecalis and endodontics irrigants
OR, 8. faecalis and intracanal irrigants.
A systematic review was conducted according to the
guidelines of the Cochrane Collaboration, which recommend
the search for the largest possible number of articles.
Handsearching was conducted by the review of the reference
lists of the eligible clinical trials and the review of author’s
personal databases of trial reports in an attempt to identify
any other relevant studies.
Inclusion and Exclusion Criteria
The selected articles were identified from titles and
abstracts by two independent reviewers, considering the
tabulated inclusion and exclusion criteria.. The inclusion
criteria were studies in humans, studies related to the efficacy
of NaOCl or CHX against E. faecalis, nonsurgical root canal
treatment performed during the study, subjects with a
noncontributory medical history, microbiological samples
collected before and after root canal preparation, English-
language articles. The exclusion criteria were in vitro and
animal studies, studies related to the efficacy of intracanal
irrigants and medications other than NaOCl or CHX, studies
without collection of microbiological samples before or
immediately after root canal preparation, non-English-
language articles, studies abstract only or no abstract,
literature reviews, studies involving primary teeth, case
reports, studies related only to microbial identification or
studies involving microorganisms other than E.faecalis.
Full-text reprints were obtained from all relevant and
potentially relevant studies, which seemed to meet the
inclusion criteria and from those that had insufficient data
in the title and abstract to make a clear decision.
The design of this meta-analysis was based on the
guidelines of the Cochrane Collaboration and on the
methodology of previous study21. Chi-square test was used
to analyze the differences between the studies. The essential
analysis of antibacterial efficacy was risk difference
(difference in the ratio of positive bacterial identification
TABLE 1- Studies included related to the efficacy of the NaOCl and CHX against E. faecalis
(n - number of samples, IET- initial endodontic treatment, RCP – root canal preparation)
EFFICACY OF SODIUM HYPOCHLORITE AND CHLORHEXIDINE AGAINST Enterococcus faecalis – A SYSTEMATIC REVIEW
obtained by PCR of culture techniques between pre- and
post-disinfection). Risk differences of included studies were
combined as generic inverse variance data type (Review
Manager Version 5.0 – Cochrane Collaboration, http://
www.cc-ims.net, accessed 15 May 2008), taking into account
the separate tracking of positive and negative cultures/PCR.
The level of statistical significance was set at p<0.05.
The search retrieved 229 related articles, being 6
literature reviews, 39 articles related to in vivo studies (27
in humans and 12 in animals), and 189 related to in vitro
studies. From the 39 in vivo studies, 5 met the inclusion
criteria. In these 5 papers, from a total of 159 teeth with
primary or secondary endodontic infections, E. faecalis was
initially detected in 16 (10%) teeth by PCR and 42 (26.4%)
teeth by culture. After disinfection (effect of root canal
enlargement associated with the action of chemical irrigants),
E. faecalis was identified in 11 (6.9%) teeth by PCR and 12
(7.5%) teeth by culture techniques. No longitudinal studies
in humans investigating the efficacy of NaOCl and CHX
against E. faecalis from endodontic infections were found
The outcomes of the 5 selected studies are shown in
Tables 2 and 3. The analysis was made between pre- and
post-disinfection of the same root canals. The 5 5 studies
were heterogeneous (Test of Homogeneity Chochran),
considering samples evaluated by culture technique (÷2
=45.85, df=4, p<0.00001) and PCR (÷2=1.65, df=1,
p=0.20). Thus, NaOCl or CHX showed low ability to
eliminate E. faecalis when evaluated by either culture or
The success of endodontic treatment is closely associated
to the control of endodontic microbiota. Several chemical
irrigants have been suggested for use in the treatment of
infected root canal1,2,4-7,9-11,16-18,20,22,23,25,28-30.
Studies with similar results have been reported when
NaOCl and CHX were compared9,30. However, other
investigations have shown that NaOCl presents better
antimicrobial activity than CHX1,20,23, or, conversely, that
CHX has better antimicrobial activity than NaOCl23.
Recently, it was demonstrated in a model of E. faecalis
biofilm in human root canals that ozonated water, 2.5%
NaOCl, 2% CHX and the application of gaseous ozone for
20 min were not sufficient to inactivate E. faecalis4. These
differences may have been caused by differences in the
experimental methodology, concentration, type of irrigating
solution, patient and anatomical differences in root canal
anatomy or the period of time used in the analysis.
It is important to emphasize the methodology used in
the present investigation. Studies based on scientific
evidence have stood out in dentistry14,21. Various advantages
of systematic review can be observed: explicit methods limit
bias in identifying and rejecting studies; conclusions are
more reliable and accurate; more information can be rapidly
assimilated by health care providers, researchers and
policymakers; delay among research discoveries,
implementation of effective diagnostic and therapeutic
strategies is potentially reduced; results of different studies
can be formally compared to establish generalization of
findings and consistency of results (lack of heterogeneity);
reasons for heterogeneity (inconsistency in results across
studies) can be identified and new hypotheses generated
about particular subgroups; quantitative systematic reviews
(meta-analyses) increase the accuracy of the overall result.
The investigation model adopted in the present essay
involved 5 studies, characterized by the heterogeneity of
the clinical protocols. The analysis was made between pre-
and post-disinfection of the same root canals considering
samples evaluated by culture or PCR techniques. Thus, the
NaOCl or CHX showed low ability to eliminate E. faecalis
when evaluate by both techniques. An identical problem
occurs when calcium hydroxide [Ca(OH)2] is tested against
E. faecalis14,21.Depending on the methodology - direct
contact, agar diffusion or contaminated dentin test - this
intracanal medicament may either present efficacy or not.
This fact was not confirmed in human studies7,17,18,29,30 (Table
1), but the limitations of the methodology employed in this
study should be considered. The application and validation
of the results of longitudinal studies, as far as evidence-
based view is concerned, are essential to determine the
scientific value of the selected studies. Knowledge of the
strategies to be applied for study selection is also important.
Therefore, planning and development of this study model
should be done with great caution8.
The first aspect to be considered is related to the bacteria
location. If bacteria are located only on the root canal surface,
where the intracanal medicaments can reach, NaOCl and
CHX may present efficacy against E. faecalis. However,
when bacteria are lodged within the dentinal tubules or in
deep layers, E. faecalis can be more resistant to the
antibacterial action of NaOCl and CHX.
Five studies met the inclusion criteria established for
the present investigation7,17,18,29,30. Peciuliene, et al.18 observed
E. faecalis in 25 asymptomatic teeth with secondary
infection. Avoiding contamination, microbiological samples
were collected from the canals before and after preparation
and irrigation with NaOCl and EDTA. E. faecalis was
isolated from 14 of those 20 culture positive teeth, usually
in pure culture or as a major component of the flora. Second
samples taken after preparation revealed growth in 7 of the
20 teeth. Five of the 7 cases were E. faecalis in pure culture.
Isolation of E. faecalis was not related to the use of any
specific root filling material in the original root filling.
Peciuliene, et al.29 determined the occurrence and role of
yeasts, enteric Gram-negative rods and Enterococcus species
in root-filled teeth with chronic apical periodontitis. After
collection of the first microbiological sample, the root canals
were prepared to a size 40 file using 2.5% NaOCl and 17%
EDTA as irrigating solutions. Microbes were isolated from
ESTRELA C, SILVA J A, ALENCAR A H G de, LELES C R, DECURCIO D A
33 of 40 teeth in the initial sampling. Yeasts were isolated
from 6 teeth, 3 of them together with E. faecalis.E. faecalis
was isolated from 21 of the 33 culture positive teeth, 11 in
pure culture. Growth was detected in 10 teeth of the second
samples. Six of the 10 cases were E. faecalis, with five being
a pure culture. Ferrari et al.7 detected enterococci, enteric
bacteria and yeast species from 25 root canals with primary
endodontic infections before and after canal preparation and
to test the antibiotic susceptibility of enterococcal strains
isolated. The canals were instrumented using a simple
stepback technique with Endo PTC cream associated 0.5%
NaOCl and EDTA. Microorganisms were isolated from 92%
of the samples following intracoronal access, 22% were
enterococci, enteric bacteria or yeast species. After
biomechanical preparation, these species were no longer
detected. After 7 days without intracanal dressing, 100% of
the canals contained microorganisms, 52% of which were
target species. E. faecalis and E. faecium were resistant to
removal by root canal preparation followed by intracanal
dressing. Zerella, et al.15 compared the effect of a slurry of
Ca(OH)2 mixed in aqueous 2% CHX versus aqueous
Ca(OH)2 slurry alone on the disinfection of the pulp space
of failed root-filled teeth during endodontic retreatment in
40 teeth. The root canal was then cleaned and shaped with
endodontic files using conventional endodontic technique.
A copious amount of 1.0% NaOCl solution was used for
irrigation. The results of this analysis were previously
reported. The teeth were nonsurgically retreated and
medicated over 3 treatment visits with 7-10-day intervals
with either Ca(OH)2 in water or Ca(OH)2 in 2% aqueous
CHX. Of the total sample population, 12 of 40 (30%) were
positive for bacteria before root filling. The control
medication disinfected 12 of 20 (60%) teeth including 2 of
4 teeth originally diagnosed with enterococci. The
experimental medication resulted in 16 disinfected teeth out
of 20 (80%) at the beginning of the third appointment. None
of the teeth originally containing enterococci showed
persistent bacterial growth. Canal dressing with a mixture
of 2% CHX and Ca(OH)2 slurry is as efficacious as aqueous
Ca(OH)2 on the disinfection of failed root-filled teeth.
Williams, et al.29 compared real-time quantitative PCR
(qPCR) assay to cultivation for E. faecalis detection and
quantification during endodontic treatment. Final shaping
and mechanical root canal debridement was achieved using
nickel-titanium files in a rotary crown-down technique. Teeth
were irrigated with 1.05% NaOCl between files and after
the final file. In primary infections, E. faecalis was present
in Sample 1 in 7% (1/15) of cases by cultivation and 13%
(2/15) by qPCR. No tooth was positive for the bacterium in
either Sample 2 or Sample 3 by cultivation, indicating the
removal of culturable E. faecalis by the instrumentation -
irrigation protocol. Using qPCR, 3 teeth (the two teeth
identified at Sample 1 and another tooth) harbored the
bacterium at both Sample 2 and Sample 3. As observed with
the primary infections, there was a nonsignificant trend for
the number of E. faecalis positive cases detected by qPCR
to increase to 57% (8/14) in Sample 2 and 50% (7/14) in
Sample 3. qPCR detected up to three times more E. faecalis
in Sample 1 than cultivation, but the difference was not
statistically significant. At collection times in Samples 2 and
3, qPCR identified more E. faecalis infections in refractory
lesions than cultivation (Table 1).
Based on the meta-analysis results, in the selected 5
studies9-13, from a total of 159 teeth with endodontic
infections, E. faecalis was detected initially in 16 (10%)
teeth by PCR and 42 (26.4%) teeth by microbial culture
techniques. Immediately after root canal preparation using
0.5% to 2.5% NaOCl, it was possibly to identify E. faecalis
in 11 teeth (6.9%) by PCR and in 12 teeth (7.5%) by culture.
No longitudinal studies in humans investigating the efficacy
of NaOCl and CHX against E. faecalis from endodontic
infections were found
The difficulty in comparing the studies retrieved in the
present search is due to differences on the methodological
design of each investigation: standardization of the limit of
preparation, choice of the preparation technique,
standardization of tooth type and sample size, time of the
initial endodontic treatment in cases of secondary infection,
quality control of the chemical irrigants and variation in their
concentration, criteria for the detection of the periapical
lesion etc, in addition to other important data that were not
mentioned in these studies (Table 1).
The selection of endodontic irrigants that aggregate the
largest possible number of ideal properties was a major point
in the present study. There was a concern in this systematic
review10 regarding to the process of making clinical decisions
in the control of microorganisms in endodontic infections.
In summary, the disinfection of the root canal system
produced by emptying, enlargement and action of NaOCl
reduces the remaining endodontic microbiota, which
optimizes the efficacy of the intracanal dressing and favors
the achievement of a higher level of success of the
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