doi:10.1111/iej.
12730
REVIEW
Prevalence and nature of fungi in root canal
infections: a systematic review and meta-analysis
€
I. F. Persoon1, W. Crielaard1 & A. R. Ozok 2
1
Department of Preventive Dentistry; and 2Department of Endodontology, Academic Centre for Dentistry Amsterdam (ACTA),
University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
Abstract
€ performed using MetaXL. The screening of 1041 titles
Persoon IF, Crielaard W, Ozok AR. Prevalence and
nature of fungi in root canal infections: a systematic review
and meta-analysis. International Endodontic Journal, 50, 1055–
1066, 2017.
The role of bacteria in causing apical periodontitis has
been widely established, whilst the role of other
microorganisms is studied less thoroughly. This sys-
tematic review and meta-analysis reviewed the litera-
ture for the prevalence and diversity of fungi in root
canal infections. An extensive literature search was
carried out in the Cochrane databases, EMBASE, MED-
LINE, LILACS, SciELO and Web of Science. Additional
studies were identified from six endodontic journals,
four main endodontic textbooks and references of rele-
vant papers. Selected clinical studies included sampling
of necrotic pulps in permanent teeth and microbial
analysis of these samples. Studies were critically
appraised using the Joanna Briggs Institute Prevalence
Introduction
Apical periodontitis is a persistent inflammatory reac-
tion in the periapical tissues caused by microorgan-
isms infecting the root canal system (Kakehashi et al.
1965, Dahlen et al. 1982). The microorganisms colo-
nize the root canal system from the oral cavity after
ecological selection (Hsiao et al. 2012). The contribu-
tion of bacteria to these infections has been widely
Correspondence: Ilona F. Persoon, Academic Centre for Den-
tistry Amsterdam (ACTA), Gustav Mahlerlaan 3004, 1081
LA Amsterdam, The Netherlands (e-mail: i.persoon@acta.nl)
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
Critical Appraisal Checklist. Meta-analysis was
and abstracts and full-text reading yielded 54 studies.
The overall prevalence of fungi in root canal infections
was 7.5% (CI 95%: 3.6–11.8%) in the inverse variance
fixed effect heterogeneity model. Candida albicans was
the most frequently isolated species. Significant hetero-
geneity was observed (P < 0.001, I2 = 85.04%).
Subgroup analyses based on geographical location,
period of publication, type of infection, state of general
health, communication with the oral cavity, type of
sample and identification method revealed no factor
influencing the prevalence. Better standardized tech-
niques and a comprehensive analysis will reveal a
more detailed and accurate representation of the preva-
lence and nature of fungi in root canal infections.
Keywords: apical periodontitis, Candida albicans,
fungi, microbiology, mycology, root canal infection.
Received 18 April 2016; accepted 28 November 2016
€
established (Sundqvist 1976, Ozok et al. 2012). The
contribution of microorganisms from other kingdoms,
such as viruses (Sabeti et al. 2003), bacteriophages
(Stevens et al. 2009), archaea (Vianna et al. 2006)
and fungi (Morse & Yates 1941), has been examined
less thoroughly.
Fungi are often associated with oral disease (de
Carvalho et al. 2006, Dagistan et al. 2009, Canabarro
et al. 2013), although newer research points to a
function in health as well (Ghannoum et al. 2010).
Fungi are highly prevalent and diverse within the
healthy oral cavity (Ghannoum et al. 2010). They
can be found within root canal infections as well
(Egan et al. 2002), and their contribution to
International Endodontic Journal, 50, 1055–1066, 2017 1055
 Fungi in root canal infections Persoon et al.
periapical disease might be significant. Fungi are con-
sidered to be more prevalent in persistent root canal
infections and reinfections that have communication
with the oral cavity and in immunocompromised indi-
viduals (Egan et al. 2002).
As the current available evidence is contradictory,
more accurate data on fungi are necessary to pro-
vide data on their relevance and the potential neces-
sity for targeted treatment. This study systematically
reviewed the literature on the prevalence and diver-
sity of the fungi found in root canal infections and
factors possibly contributing to their differential
presence.
Materials and methods
Information sources and search strategy
The review protocol was drafted in advance and was
based on the protocols from the Cochrane Collabora-
tion (Higgins & Green 2008) and the Joanna Briggs
Institute (JBI; Joanna Briggs Institute 2014). The
search strategy was set up with the help of a librarian
and tailored to each separate database. Electronic
database searches of the Cochrane library, EMBASE,
LILACS via BIREME, MEDLINE via PubMed, SciELO
and Web of Science via Web of Knowledge were per-
formed up to and including February 2016. The
search strategy identified the terms ‘apical periodonti-
tis’ and ‘fungi’ and was tailored to each database
(Table S1). Additional studies were identified from cit-
ing papers and bibliographies of articles included after
full-text reading and relevant reviews, a database
search of six main endodontic journals (Australian
Endodontic Journal, Dental Traumatology, Endodontic
Topics, International Endodontic Journal, Journal of
Endodontics, Oral Surgery, Oral Medicine, Oral
Pathology, Oral Radiology and Endodontics) and from
relevant chapters of four endodontic textbooks (Ingle
& Bakland, Endodontics 6, 2008; Hargreaves &
Cohen: Pathways of the Pulp, 2011; Bergenholtz &
Horsted-Bindslev & Reit: Textbook of Endodontology
2, 2010; Ørstavik & Pitt Ford: Essential Endodontol-
ogy: Prevention and Treatment of Apical Periodonti-
tis, 2007).
Study selection
Two reviewers (ARO, IFP) independently screened the
articles on title and abstract according to three eligi-
bility criteria. Disagreements between the reviewers
1056 International Endodontic Journal, 50, 1055–1066, 2017
were resolved by discussion. The article had to study
infections within the root canal (i), because the
samples taken from infections spreading outside the
root canal were deemed impossible to sample and
control for contamination. Only articles specifically
studying fungi or studying the whole field of microbi-
ology were included (ii), because many articles clas-
sify fungi with bacteria. Only studies collecting
clinical specimens from human teeth were considered
(iii). No language restrictions were set. When no
abstract was available or it was unclear if the article
met the inclusion criteria, it was considered for full-
text reading.
In the consecutive round, the same two reviewers
screened the full texts; articles were included based
on seven eligibility criteria. The study had to focus
on fungi or on fungi alongside other microorganisms
(i). Exclusively root canals associated with an
infected necrotic pulp and an apical lesion were stud-
ied, or it had to be possible to extract these data sep-
arately; the apical periodontitis had to be confirmed
radiographically and/or clinically (ii). The infection
had to be within the confinement of the root canal
(iii). Clinical samples from human teeth had to be
taken within the described study, thereby excluding
reviews and in vitro, in vivo or animal studies (iv).
The samples had to be taken from permanent teeth
(v). The teeth had to be sampled immediately after
endodontic access to prevent iatrogenic contamina-
tion (vi). Adequate aseptic procedures, such as clean-
ing of the tooth, application of rubberdam and a
disinfectant, had to be reported (vii). Finally, data
had to be reported complete enough to distil data of
the number of fungi infected teeth of all sampled
teeth (viii).
Data collection
A data extraction sheet was developed, pilot-tested
on 10 key articles and optimized accordingly. The
first reviewer (IFP) extracted all the data from the
included studies, and the second reviewer (ARO)
checked these data. Disagreements between the
reviewers were resolved by discussion. Two authors
were approached for further information, of which
one responded. Data extracted from the studies
included the following: (i) participants’ characteristics
(age, gender, comorbidity, type of infection, clinical
setting), (ii) methods (type of sample, controls, identi-
fication technique) and (iii) results (prevalence and
species of fungi).
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd

Figure 1 Flow diagram of study selection.
Risk of bias assessment
Risk of bias of individual studies was assessed using
the JBI Prevalence Critical Appraisal Checklist (Joanna
Briggs Institute 2014).The two criteria on appropriate
statistical analysis and objective criteria for identifica-
tion of subgroups were excluded, as they were
deemed irrelevant for the included studies. The same
two reviewers assessed all studies. Disagreements
between the reviewers were resolved by discussion.
The assessment was used to retrieve more insight into
the variance of risk of bias of individual studies, but
these were not used for subgroup analyses.
Statistical analysis
The estimated prevalence of teeth infected with fungi
was calculated using the inverse variance (fixed effect)
heterogeneity (IVhet) model with double arcsine trans-
formations (Barendregt et al. 2013, Doi et al. 2015).
This model corrects for studies with low prevalence
rates, increased sample size and increased heterogene-
ity. This conservative model will give a reserved out-
come, giving only a slight chance of overestimating
the true prevalence rate. The diversity of the reported
fungal species was not used within the statistical anal-
ysis. Heterogeneity was assessed using I2. Subgroup
analyses were performed to identify possible causes of
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
Persoon et al. Fungi in root canal infections
heterogeneity. In case of statistical significant differ-
ences between subgroups, meta-regression was per-
formed. Risk of bias across studies was visually
assessed using a Doi plot, which is similar to a funnel
plot. This can be quantitatively assessed using the Luis
Furuya-Kanamori (LFK) index, where values of <1
indicate symmetry, 1–2 indicate minor asymmetry
and >2 indicate major asymmetry. All analyses were
performed, and graphs were drafted using MetaXL 3.1
(http://www.epigear.com) within Microsoft Excel ver-
sion 14 (Microsoft Corporation 2010).
Results
The total search resulted in 1041 unique titles and
abstracts. After screening, 167 remained for full-text
reading, after which 54 studies were selected for
inclusion in the review (Fig. 1 and Table S2). No
unpublished studies were obtained.
In the combined 54 studies, a total of 3603 partici-
pants were included. Thirty-four of the included stud-
ies examined primary root canal infections, whereas
16 examined persistent or reinfections and four exam-
ined both types of infection. Further study character-
istics, prevalence data with confidence intervals and
identified fungal species are shown in Table 1.
Risk of bias within studies was assessed using the
JBI Critical Appraisal Checklist (Table S3). Overall,
International Endodontic Journal, 50, 1055–1066, 2017 1057
 Fungi in root canal infections Persoon et al.
Table 1 Main characteristics and findings of included studies
1058 International Endodontic Journal, 50, 1055–1066, 2017 © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Persoon et al. Fungi in root canal infections
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 50, 1055–1066, 2017 1059
 Fungi in root canal infections Persoon et al.
most studies had low risk of bias because subjects had
been recruited from representative settings, such as
regular and referral dental clinics, and inclusion and
exclusion criteria were described. Also, most studies
had data analysis with sufficient coverage and objec-
tive measurement criteria. Sample size in 50 of 54
included studies gave high risk of bias, because it was
well below the calculated sample size of N = 190 (as-
suming a prevalence of 7.5% and a precision half of
that). Risk of bias was rated as unclear for the repre-
sentation of the target population and confounders,
because of missing data. Measurement reliability was
often scored as unclear because measurements were
not controlled for contamination of the operative field
using sterility checks, many samples were excluded
after contaminated sterility checks or results of steril-
ity checks were not reported. If many samples were
discarded, this negatively influenced the coverage of
the data analysis.
The combined prevalence of fungal species in root
canal infections is 7.5% (95% confidence interval:
4.5–10.8%) in the conservative IVhet model. When
studies applied different identification techniques, only
the results with the highest fungal presence were
included in the analysis. Subgroup analysis regarding
the period of publication, geographical location, type
of infection, state of general health, communication
with the oral cavity, type of sample or identification
method revealed no factor influencing the prevalence
(Fig. 2). Because of the large heterogeneity and no
significant differences between subgroups, no further
meta-regression was performed.
Significant heterogeneity was observed (P < 0.001,
I2 = 85.04%). Risk of bias across studies was visual-
ized in a Doi plot, indicating slight asymmetry
(Figure S1). The LFK index was 1.81, also indicating
minor asymmetry. Studies with a higher prevalence
showed more diversity in reported prevalence rates,
although this does not point to a specific source of
bias.
Discussion
For the first time, the literature has been systemati-
cally reviewed on the prevalence and nature of fungi
in root canal infections. Within the limitations of the
included studies concerning heterogeneous study pro-
tocols and increased risk of bias, 7.5% of all root
canal infections were positive for fungi. The subgroup
analysis identified no factor significantly influencing
the prevalence. It appears that fungi play a role in a
1060 International Endodontic Journal, 50, 1055–1066, 2017
proportion of root canal infections, although the body
of evidence is not strong.
The inclusion criteria were strict to identify samples
that could represent the root canal infection most
accurately. If samples were taken outside the confine-
ment of the root canal, the chance of interference
with the microorganisms is substantial. For instance,
aspergillosis of the maxillary sinus is associated with
infected root canal fillings, although it remains
unclear whether the microorganisms originate from
the root canal or the nasal cavity (Mensi et al. 2007).
Not all studies sampled the root canal immediately
after the endodontic access, but only in the next ses-
sion of the root canal treatment. This allows for iatro-
genic contamination, leakage of the temporary seal or
outgrowth of a species that profits from microbial
inactivation of other species (Barthel et al. 2001, Fer-
rari et al. 2005, Niazi et al. 2010). Several relevant
studies had to be excluded because of missing infor-
mation about the study design and execution. This is
a frequent limitation of systematic reviews, but neces-
sary to decrease heterogeneity. Still, 54 studies with a
total of 3603 participants could be included to
increase precision as compared to a minor single
study.
Despite the rather strict inclusion criteria, large
heterogeneity was observed. Firstly, large heterogene-
ity can be caused by a nonrepresentative sample.
Sample size influences the determined prevalence,
especially with a sample size below 15 (Jovani & Tella
2006). Systematic reviews are particularly relevant
when the separate studies are limited by small sample
sizes as a larger representation of the population is
generated through combining data. In this meta-ana-
lysis the IVhet model was applied, which corrects for
the influence of sample size (Doi et al. 2015).
Apart from sample size, the state of included sam-
ples might influence the determined prevalence. This
might vary considerably, similar to the variety
between root canal infections. The availability of
nutrients, the microbial inoculation from the oral cav-
ity and the duration of the apical periodontitis can
substantially affect the microbial community in the
root canal (Fabricius et al. 1982, Ch avez de Paz
2007). Besides, fungi are larger than bacteria and not
motile, and therefore less capable of invading the root
canal via dentinal tubules (Waltimo et al. 2000).
Fungi should then invade the root canal via minor
communication with the oral cavity, such as cracks
or minimal dentine layers underneath deep carious
lesions, during treatment or afterwards via defective
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Persoon et al. Fungi in root canal infections

Figure 2 Overall and subgroup prevalence rates of fungi in root canal infections. Prevalence rates grouped according to period of
publication (a), geographical location (b), type of infection (c), state of general health (d), communication with the oral cavity (e),
type of sample (f) and identification method (g). Prevalence values (95% confidence intervals) are shown on the right side.

coronal sealing. Communication of the infection with
the oral cavity might provide access for fungi and
drive an ecology suitable for their colonization,
although none of the included studies that were com-
paring this factor (Hobson 1959, Winkler & Van
Amerongen 1959, Pinheiro et al. 2003a, Chu et al.
2005, Zbidi et al. 2005) or the subgroup analysis of
this review were able confirm this. Endodontic treat-
ment might also provide access to fungi, meaning
they would be more prevalent in persistent infections
or reinfections (Waltimo et al. 1997). However, this
could not be confirmed by the included studies (Win-
kler & Van Amerongen 1959, M€ oller 1966, Egan
et al. 2002, Dumani et al. 2012) or the subgroup
analysis for this review. The state of general health
has also been speculated to influence fungal preva-
lence within root canal infections, as a compromised
host response can lead to more and more severe
infections involving fungi (Dagistan et al. 2009,
Brown et al. 2012). Results from this review cannot
confirm this, although some studies report fungi
abundantly when the immune response is compro-
mised. Some of these health conditions were alco-
holism (Matusow 1979), diabetes (Zbidi et al. 2005,
Gomes et al. 2010), blood anaemia (Zbidi et al.
2005), autoimmune diseases (Gomes et al. 2010) and
human immunodeficiency virus infection (Ferrari
2004, Brito et al. 2012). Furthermore, no geographi-
cal location or period of the study was associated with
a higher fungal prevalence.
Secondly, large heterogeneity can be caused by
errors unrelated to the sample population. The wide
range of sample types and techniques each has their
limitations and may have contributed to the large
heterogeneity, although no specific tendency was
observed. Each technique has a threshold of

© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 50, 1055–1066, 2017 1061
 Fungi in root canal infections Persoon et al.
detection, where DNA techniques are more sensitive
than cultivation (Zambon & Haraszthy 1995). Espe-
cially microscopy is more prone to overlook part of
the sample as it is not entirely analysed. However,
fungi can be well identified using microscopy with-
out making a distinction on identity. Cultivation and
DNA techniques may be intentionally or uninten-
tionally targeted to a specific species, which limits
the possibility of identifying all fungi. No medium
contains nutrients to cultivate all fungi, just as some
microorganisms are uncultivable in a laboratory
environment either because of nutritional require-
ments or metabolic state (Zambon & Haraszthy
1995). Many DNA techniques are not open-ended,
so that only the DNA of pre-specified species is anal-
ysed. Analysis of universally evolutionary conserved
genes of the RNA, such as 18S or internal tran-
scribed spacer, circumvents this problem (Ghannoum
et al. 2010). Additionally, paper point sampling is
associated with significantly lower microbial yield
than when the whole tooth is analysed, both in
abundance and in prevalence (Akpata 1974). When
samples are taken in vivo using paper points, the risk
of contamination from the operating field is substan-
tial. Therefore, sterilization of the operating field and
an aseptic sampling technique are essential. Because
sterility is not always accomplished, it is important
to check for this (Ng et al. 2003) and otherwise dis-
card the sample. Many studies were unclear on
whether a sterility check was performed or how the
results thereof were handled and thus scored higher
concerning their risk of bias.
Risk of bias was assessed using the JBI Prevalence
Critical Appraisal Checklist. Proper risk of bias assess-
ment tools for prevalence studies is rare, and many of
the items used to assess other study designs are not
applicable (Munn et al. 2014). A limitation of the cur-
rent checklist is its limited discriminative power,
because many items had low or high endorsement
rates. However, this checklist has been developed by
experts and is accompanied by an extensive descrip-
tion of how to rate each item. Therefore, it was
deemed as the most suitable available checklist for
this study.
Candida and, to a lesser extent, Saccharomyces,
Aspergillus and Rhodotorula were the most frequently
isolated fungal genera within root canal infections.
This is similar to studies into general fungal disease
(Brown et al. 2012) and local presence within the
oral cavity (Ghannoum et al. 2010, Munguia-Perez
et al. 2012, Dupuy et al. 2014, Monteiro-da-Silva
1062 International Endodontic Journal, 50, 1055–1066, 2017
et al. 2014, Mukherjee et al. 2014). Candida albicans
was the most frequently isolated species. It is a dimor-
phic fungus, as it exists both as single cells (yeasts)
and in filamentous forms (hyphae). Hyphae enable
the fungus to colonize and penetrate host tissues
(Waltimo et al. 2000), but also allow potent
immunostimulation and tissue damage (Moyes et al.
2016). However, thus far, most studies only reflect a
superficial composition of the fungal component of
root canal infections. Deep sequencing will allow a
higher fungal diversity to be identified, similar to
studies in the bacterial component of root canal infec-
€
tions (Ozok et al. 2012).
Despite a low prevalence, fungi might still be
highly relevant. The prevalence and abundance of
rare microorganisms can change over time. The
wide range of fungal prevalence (0–100%) deter-
mined by the studies included in this review might
allude to this. Rare microorganisms can significantly
contribute to changes over time, influence ecological
behaviour and modulate the host response (Shade
et al. 2014, Rolig et al. 2015). For marginal peri-
odontitis, a complex evolution in the ecological
development of the microbiome has been hypothe-
sized, where keystone organisms play a major role
in initiating the disease process, which later is domi-
nated and controlled by others (Lamont & Hajishen-
gallis 2015). To orchestrate such evolutionary
processes, communication and interaction between
the microorganisms are essential. Cross-kingdom
interaction between bacteria and fungi might be
essential to raise virulence to such limits where it is
harmful or even lethal to the host (Schlecht et al.
2015).To reveal the possibilities and necessity to
develop treatment strategies targeted at fungi, fur-
ther research which clarifies the contribution of
fungi to the microbial ecology in infected root canals
and the challenge to the host in the periapical tis-
sues is essential.
Conclusion
Within the limitations of the studies included accord-
ing to strict criteria but still presenting heterogeneous
study protocols and increased risk of bias, fungi con-
tributed to 7.5% of root canal infections. No factor
was identified to significantly influence this preva-
lence. Better standardized techniques and a compre-
hensive analysis will reveal a more detailed and
accurate representation of the prevalence and nature
of fungi in root canal infections.
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd

Acknowledgements
The authors would like to thank C. Boutsioukis for
his assistance with drafting the review protocol,
R.H.J. Otten for his assistance with the database
search, J.G.P. Bouwman for his assistance with the
retrieval of the articles and A.J. van Wijk for his assis-
tance with the statistical analysis.
Conflict of interest
The authors have stated explicitly that there are no
conflict of interests in connection with this article.
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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Figure S1. Doi plot.
Table S1. Customized database search strategy for
MEDLINE via PubMed and EMBASE.
Table S2. Specification of excluded full-text arti-
cles.
Table S3. Critical appraisal of included studies.
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd