JOURNAL OF ENDODONTICS
Copyright © 2000 by The American Association of Endodontists
SCIENTIFIC ARTICLES
Occurrence of Candida albicans in Infections of
Endodontic Origin
J. Craig Baumgartner, DDS, PhD, Chad M. Watts, BS, and Tian Xia, DDS
Microorganisms are recognized as the etiological
agent for the majority of pulpal and periradicular
disease. Although bacteria have been the most
studied, fungi have also been associated with in-
fected root canals. The purpose of this study was
to evaluate the contents of infected root canals
and aspirates of cellulitis/abscesses of endodontic
origin for the presence of Candida albicans using
the polymerase chain reaction (PCR). PCR primers
specific for the 18S ribosomal RNA gene of C. al-
bicans were used to survey 24 samples taken from
infected root canals and 19 aspirates from perira-
dicular infections of endodontic origins. The pres-
ence of C. albicans was detected in 5 of 24 (21%)
samples taken from root canals, but none was de-
tected in the periradicular aspirates. The results
indicate that PCR is an extremely sensitive molec-
ular method that may be used to identify C. albi-
cans directly in samples from infections of end-
odontic origin.
Microbes are associated with virtually all diseases of the pulp and
periradicular tissues. The microbial ecosystem of infected root
canals may consist of bacteria, including spirochetes and fungi.
Several studies have observed or cultivated fungi from endodontic
infections (1– 4). In addition recent studies have associated the
presence of fungi with therapy-resistant endodontic infections (1,
3, 4). Fungi are considered normal inhabitants of the oral cavity,
but may produce disease when there are local or systemic factors
predisposing the individual to infection. Yeast is a general term
used to describe single-celled, usually rounded fungi that repro-
duce by budding. Some yeasts such as Candida albicans may
transform into a mycelia stage in certain environments. C. albicans
is the most encountered commensal and pathological yeast in the
oral cavity (5). Multiple unrelated commensal strains and/or dif-
ferent strains of C. albicans may be harbored in the same individ-
ual. The genus Candida is comprised of ⬃150 fungal species. C.
albicans has been the fungi most commonly isolated from the oral
695
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VOL. 26, NO. 12, DECEMBER 2000
cavity of both healthy and medically compromised individuals.
Previous studies using microscopy or culturing techniques have
inherent limitations in the identification of fungi in infected root
canals. The purpose of this study was to evaluate the contents of
infected root canals and aspirates of cellulitis/abscesses of end-
odontic origin for the presence of C. albicans using the polymerase
chain reaction (PCR).
MATERIALS AND METHODS
In accordance with guidelines of The Human Volunteers Re-
search Committee at the Oregon Health Sciences University, mi-
crobial samples were collected from 24 root canals and 19 aspirates
of cellulitis/abscesses of patients seen in the School of Dentistry,
Oregon Health Sciences University, Portland, OR. Selection was
based on clinical examination and the presence of a periapical
radiolucency that was determined to be of endodontic origin.
Microbial samples from the root canals were collected using strict
asepsis. A rubber dam was used to isolate each tooth. A cotton
applicator was used to clean the surface of the tooth and surround-
ing field with 30% hydrogen peroxide for 1 min. The tooth surface
was then disinfected by applying 5% iodine tincture for 1 min (6).
The iodine on the tooth surface was then inactivated with 5%
sodium thiosulfate. Access to the root canal was made using sterile
burs without water spray. The samples were collected under opti-
mal conditions and placed in sterilized pre-reduced transport fluid
(RTF) (7). If fluid was not present in the root canal after the access
opening, RTF was placed in the canal and a file used to disperse
the canal contents. The canal sample was collected with absorbent
paper points and sealed in a transport vial containing RTF.
Aseptic aspirates of cellulitis/abscesses of endodontic origin
were obtained after having the patient rinse with 15 ml of 0.12%
chlorhexidine gluconate (Zila Pharmaceuticals, Phoenix, AZ). The
mucosal surface was then disinfected with povidone-iodine swabs
(Purdue Frederick Co., Norwalk, CT). The contents of cellulitis/
abscesses were aspirated using a 16-gauge needle (Sherwood, St.
Louis, MO). For transportation to the laboratory, the aspirate was
immediately placed into a Port-A-Cul™ vial (Becton Dickinson,
Cockysville, MD).
All samples were frozen at ⫺70°C before analysis with PCR.
The DNA was isolated directly from each sample using a QiAamp
696 Baumgartner et al. Journal of Endodontics

Tissue Kit (Qiagen, Inc., Santa Clara, CA) after the method rec-
ommended by the manufacturer. This method included a preincu-
bation step with lysozyme to lyse the fungi cells before samples
were digested with proteinase K. After isolation, the DNA was
amplified using PCR.
The sequence for C. albicans was aligned using Geneworks
software (Version 2.5, Intelligenetics, Campbell, CA). The primer
pair used was designed to amplify a 276 base pair region from the
C. albicans 18S r-RNA gene (sense primer: CGATTCAGGG-
GAGGTAGTGAC, antisense primer: GGTTCGCCATAAATG-
GCTACCAG). The final step involved the extension of the primer.
PCR was prepared using methods outlined by Baumgartner et al.
(8). The 50-␮l reaction mixtures contained 2 units of Taq DNA
Polymerase (ProMega Corp., Madison, WI), 5 ␮l of 10⫻ PCR
buffer (ProMega Corp.), 1.5 mM MgCl, 0.4 ␮M sense and anti-
sense primers, 0.2 mM dNTPs, and a 15 ng sample of DNA.
Cycling conditions were as follows: 94°C for 1 min, 33 cycles at
94°C for 1 min, 55°C for 1 min, 72°C for 1.5 min, and a final 72°C
for 10 min. PCR products were then separated by 1.5% agarose gel
electrophoresis and visualized by ethidium bromide fluorescence.
Images were digitized using a Gel Doc 1000 system (Bio-Rad
Laboratories, Hercules, CA). A 100-bp DNA ladder (Life Tech-
nologies, Gaithersburg, MD) served as a size marker (8).

FIG 2. Sensitivity of PCR for the detection of C. albicans. Lane 1: 100

base pair DNA ladder. Lanes 2–7 were amplified using C. albicans
specific primers. Dilutions of C. albicans DNA from 10⫺1 ng to 10⫺6
was tested for sensitivity of detection.

RESULTS

The specificity of the primer pair designed to amplify the 18S

r-RNA gene of C. albicans was tested against several common
bacteria found within the root canal (Prevotella nigrescens 33563,
Prevotella intermedia 25611, Porphyromonas gingivalis 33277,
Porphyromonas endodontalis 35406, and Peptostreptococcus
anaerobius 27337). No DNA amplification other than the 276 base
pair amplicon for C. albicans was observed (Fig. 1). The sensitiv-
ity of PCR to detect C. albicans DNA was 10⫺4 ng (Fig. 2). These
tests confirmed that the PCR reactions were amplifying the tar-
geted regions of the 18S r-RNA gene of C. albicans (Fig. 3). Five
PCR products of the DNA extracted from the contents of 24 root
canals were found to be positive for the presence of C. albicans.
The 19 samples taken from cellulitis/abscesses were found to be
negative for the presence of C. albicans DNA.

FIG 1. Specificity of PCR for the detection of C. albicans. Lane 1: 100
base pair DNA ladder. Lanes 2– 8 were amplified using C. albicans
specific primers. Lanes 2 and 3 contained positive control DNA
(ATCC 44505) of C. albicans. Lane 4 contained Prevotella nigrescens
(ATCC 33563) DNA. Lane 5 contained Prevotella intermedia (ATCC
25611) DNA. Lane 6 contained Porphyromonas gingivalis (ATCC
33277) DNA. Lane 7 contained Porphyromonas endodontalis (ATCC
35406) DNA. Lane 8 contained Peptostreptococcus anerobius
(ATCC 27337) DNA.
DISCUSSION
This study confirms that C. albicans may be involved in root
canal infections more often then previously believed. In addition
the results of this study demonstrate that PCR is efficient in
detecting the presence of C. albicans. How pathogenic fungi are
associated with endodontic infections has yet to be determined.
However studies have identified the presence of fungi after ther-
Vol. 26, No. 12, December 2000
FIG 3. PCR survey of samples. Lane 1: 100 base pair DNA ladder.
Lanes 2–11 were amplified using C. albicans specific primers. Lane
2 contained positive control DNA (ATCC 44505) of C. albicans.
Lanes 3–11 contain DNA from clinical samples. Lanes 3 and 11
demonstrated the presence of C. albicans DNA.
apy-resistant endodontic treatment (1, 3, 4). Of 692 samples of
therapy-resistant chronic apical periodontitis, Waltimo et al. (4)
identified 48 fungi in 47 (7%) samples. Of the 20 yeast isolates
identified at the species level in that study, 16 (80%) were C.
albicans. Candida glabrata, Candida guilliermondii, and Geotri-
chum candidum were found in one root canal each, whereas one
strain of C. glabrata was isolated with C. albicans from a single
sample (4). C. albicans seems to be the most common species of
fungi infecting root canals.
Debelian et al. (9 –11) have recently reported that the frequency
of bacteremia detected by lysis filtration and cultivation in patients
undergoing endodontic therapy ranged from 31 to 54%. In one
patient a fungemia was detected when the yeast, Saccharomyces
cerevisiae, was recovered from both the root canal and the patients’
blood samples taken during and after endodontic therapy (10). S.
cerevisiae is not commonly isolated from the oral cavity, but is
used to make fermented beverages. Strains of Candida producing
a fungemia have not been detected. This would seem to be con-
sistent with our lack of detecting C. albicans in the samples taken
from aspirates of periradicular cellulitis/abscesses of endodontic
origin. Likewise, Nair (12) did not observe fungi in periapical
lesions associated with infected root canals.
Sen et al. (2) used scanning electron microscopy (SEM) to
observe bacteria and fungi in the root canals and dentinal tubules
of 10 extracted human molars with infected root canals. In four of
the specimens, the root canals were infected with fungi. The yeast
cells were identified as single spherical or oval cells ⬃4 to 6 ␮m
in diameter. Some branching hyphal networks were observed.
Candida may appear as both single yeast cells or with hyphae. In
one specimen they observed pseudohyphae of chains of budding
cells that apparently failed to separate (2).
C. albicans in Endodontic Infections 697
In a follow-up in vitro study, Sen et al. (13) used SEMs of
human root sections infected with C. albicans to observe its
growth patterns in relation to radicular dentin. The filamentous
hyphal form was dominant in their 5-day specimens. However
most of both the hyphae and the blastospores (yeast forms)
showed some penetration into dentinal tubules (13). The inves-
tigators concluded that C. albicans may be considered a denti-
nophilic microorganism.
Several studies have evaluated the effects of endodontic irri-
gants against C. albicans (14 –18). Smith and Wayman (15) evaluated
the antimicrobial effectiveness of citric acid and sodium hypochlorite
and found C. albicans to be more resistant than Streptococcus faecalis
or Bacillus sp. In addition citric acid was not as effective as NaOCl
(15). When Sen et al. (14) evaluated the antifungal properties of
chlorhexidine and NaOCl, they found C. albicans to be more resistant
in the presence of a smear layer than in the absence of a smear layer.
NaOCl alone started to show antifungal activity in the absence of a
smear layer after 30 min (14). They concluded that antimicrobial
effectiveness of irrigating solutions should be evaluated in patients
predisposed to oral candidiasis (14).
Candida often colonizes the oropharyngeal, gastrointestinal,
and vaginal microflora of healthy patients. In immunocompro-
mised patients fungi cause significant morbidity and mortality.
Unfortunately the number of immunocompromised patients has
increased during recent years. Until now diagnosis of fungal in-
fections has been based on cultivation, histology, or serology. It is
important to differentiate nondisease-producing colonization of
epithelial surfaces from disease producing invasion and infection
of normally sterile tissues (19, 20). Molecular methods such as
PCR now provide another means of detecting these organisms after
invasion and infection of normally sterile sites.
Dr. Baumgartner is chairman, Department of Endodontology, Oregon
Health Sciences University School of Dentistry, Portland, OR. Mr. Watts is an
undergraduate dental student at Oregon Health Sciences University School of
Dentistry, Portland, OR. Dr. Xia is a research assistant at Oregon Health
Sciences University School of Dentistry, Portland, OR. Address requests for
reprints to Dr. J. Craig Baumgartner, Department of Endodontology, Oregon
Health Sciences University School of Dentistry, 611 SW Campus Drive, Port-
land, OR 97201.
References
1. Nair PNR, Sjogren U, Krey G, Kahnberg KE, Sundqvist G. Intraradicular
bacteria and fungi in root-filled, asymptomatic human teeth with therapy-
resistant periapical lesions: a long-term light and electron microscopic fol-
low-up study. J Endodon 1990;16:580 –7.
2. Sen BH, Piskin B, Demirci T. Observation of bacteria and fungi in
infected root canals and dentinal tubules by SEM. Endod Dent Traumatol
1995;11:6 –9.
3. Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of
teeth with failed endodontic treatment and the outcome of conservative
re-treatment. Oral Surg 1998;85:86 –92.
4. Waltimo TMT, Sirén EK, Torkko HLK, Olsen I, Haapasalo MPP. Fungi in
therapy-resistant apical periodontitis. Int Endod J 1997;30:96 –101.
5. Fotos P, Hellstein J. Candida and candidosis: epidemiology diagnosis
and therapeutic management. Dent Clin North Am 1992;36:857–78.
6. Moller A. Microbiologic examination of root canals and periapical tis-
sues of human teeth. Odontol Tdskr 1966;74:63.
7. Syed SA, Loesche WJ. Survival of human dental plaque flora in various
transport media. Appl Microbiol 1972;24:638 – 44.
8. Baumgartner J, Bae K, Xia T, Whitt J, David L. Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and polymerase chain reaction for differ-
entiation of Prevotella intermedia and Prevetolla nigrescens. J Endodon 1999;
25:324 – 8.
9. Debelian G, Olsen I, Tronstad L. Anaerobic bacteremia and fungemia in
patients undergoing endodontic therapy: an overview. Ann Periodontol 1998;
3:281–7.
698 Baumgartner et al. Journal of Endodontics

10. Debelian GJ, Olsen I, Tronstad L. Observation of Saccharomyces
cerevisiae in blood of patient undergoing root canal treatment. Int Endod J
1997;30:313–7.
11. Debelian GF, Olsen I, Tronstad L. Bacteremia in conjunction with
endodontic therapy. Endod Dent Traumatol 1995;11:142–9.
12. Nair PNR. Light and electron microscopic studies of root canal flora
and periapical lesions. J Endodon 1987;13:29 –39.
13. Sen B, Safavi K, Spangberg L. Growth patterns of Candida albicans in
relation to radicular dentin. Oral Surg 1997;84:68 –73.
14. Sen H, Safavi K, Spangberg L. Antifungal effects of sodium hypochlo-
rite and chlorhexidine in root canals. J Endodon 1999;25:235– 8.
15. Smith JJ, Wayman BE. An evaluation of the antimicrobial effectiveness
of citric acid as a root canal irrigant. J Endodon 1986;12:54 – 8.
16. Harrison JW, Wagner GW, Henry CA. Comparison of the antimi-
crobial effectiveness of regular and fresh scent Clorox. J Endodon 1990;
7:328 –30.
17. Barbosa S, Spangberg L, Almeida D. Low surface tension calcium
hydroxide solution is an effective antiseptic. Int Endod J 1994;27:6 –10.
18. Pavelic B, Anic I, Najzar FD, Stilinovic B, Temmer K. The antimicrobial
efficiency of aqueous solutions of calcium hydroxide on S. mutans, S. faecalis,
and C. albicans. Acta Stomatol Croat 1991;25:207–12.
19. Heimdahl A, Nord C. Oral yeast infections in immunocompromised
and seriously diseased patients. Acta Odontol Scand 1990;48:77– 84.
20. Samaranayake L. Oral mycoses in HIV infection. Oral Surg 1992;73:
171– 80.

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