M. W. Egan, D. A. Spratt, Y.-L. Ng, J. M. Lam, D. R. Moles & K. Gulabivala
Departments of Conservative Dentistry and Oral Pathology, Eastman Dental Institute for Oral Health Care Sciences, University College London, London, UK.

Introduction.
Yeasts are ubiquitous in the environment, being found in humans, animals, fruit, vegetables and other plant material. Some yeasts live as normal inhabitants in humans without any clinical effects. Symptom-free oral carriage of Candida organisms has been recognized for many years (Scully et al. 1994). The transformation of yeasts from innocuous commensals to harmful pathogens may depend on factors other than the attributes of the organism (Samaranayake & Yaacob 1990). Local or systemic predisposing factors in the host may be of equal or greater importance in the pathogenesis of the disease (Shepherd 1992). Immuno-compromised hosts suffering from diseases such as diabetes (Lamey et al. 1988), malignancy (Jobbins et al. 1992) or HIV infection are obvious systemic host factors. Unfortunately, some of the life-saving medical advances, including the use of broad-spectrum antibiotics, immuno-suppressive drugs and intensive cancer chemotherapy, also predispose the patients to a variety of fungal infections (Dixon et al. 1996).

Table 1. Literature relating to isolation of yeasts from root canals.

The study of fungal infection in the oral cavity has focused mainly on various presentations of candidiasis of the oral mucosa such as pseudomembranous candidiasis (oral thrush), chronic atrophic candidiasis (denture stomatitis), angular cheilitis, acute atrophic candidiasis and chronic hyperplastic candidiasis ( Candida leukoplakia ) (Samaranayake & Yaacob 1990). The prevalence and diversity of yeasts associated with periapical diseases have not however, been studied in any depth. Their presence has been demonstrated by cultivation or microscopy in untreated root caries ( Jackson & Halder 1963, Wilson & Hall 1968, Q en et al. 1995), dentinal tubules (Kinirons 1983, Damm et al. 1988), treated root canals associated with persistent apical periodontitis (Nair et al. 1990, Molander et al. 1998, Sundqvist et al. 1998), apical root surfaces of teeth with asymptomatic apical periodontitis (Lomçsali et al. 1996) and in periapical tissues (Tronstad et al. 1987).
The presence of yeasts in root canals has usually been reported during the course of microbial investigations of root canal systems with a prevalence ranging from 0.6% to 10% in untreated cases (Slack 1953, 1975, MacDonald et al. 1957, Leavitt et al. 1958, Hobson 1959, Goldman & Pearson 1969, Kessler 1972) and 3.7–10% in treatment-resistant cases (Tronstad et al. 1987, Molander et al. 1998, Sundqvist et al. 1998). Only a few studies have specifically sought to investigate the prevalence of yeasts in root canal infections using cultivation techniques (Table 1). Using a variety of culture media, they have demonstrated a higher prevalence ranging from 7% in treated teeth (Waltimo et al. 1997) to 55% in untreated teeth (Najzar-Fleger et al. 1992). The majority of the recovered yeasts were Candida with C. albicans being the most prevalent (Waltimo et al. 1997). In agreement with these findings, the presence of C. albicans has been detected in 21% of infected root canals using 18S rRNA directed species-specific primers (Baumgartner 2000). Other species such as C. glabrata , C. guillermondii , C. incospicia were also isolated by Waltimo et al. (1997). Factors affecting the colonization of the root canal by yeasts derived from the oral environment have not been specifically investigated. A number of factors do however, appear to predispose to this process; immunocompromising diseases such as cancer (Damm et al. 1988), the use of intracanal medicaments (Jackson & Halder 1963), local (Wilson & Hall 1968) and systemic antibiotics (Matusow 1981) and previous root canal treatment (Sirén et al. 1997, Sundqvist et al. 1998). Although the collective picture appears to suggest a higher prevalence of yeasts in untreated canals, it has been hypothesized that the reduction of specific groups of bacteria in the canal during treatment may allow yeasts to overgrow and predominate in the low nutrient environment (Sirén et al. 1997, Sundqvist et al. 1998). Another possibility is that the yeasts may gain access during treatment as a result of poor asepsis.
The aims of this study were:

• to determine the relative prevalence and diversity of yeasts in saliva and root canals from the same patients; and
• to correlate these findings with medical and antibiotic histories of the patient, as well as the endodontic and restorative status of the involved teeth.

Materials and methods.

Patient selection.
Sixty teeth, from 55 consecutive patients attending the Department of Conservative Dentistry, Eastman Dental Hospital (London) for nonsurgical root canal treatment were included in this study. Previously root-treated ( n = 25) and untreated ( n = 35) teeth associated with radiographic evidence of periapical disease were selected for investigation.

Clinical data.
The patients’ medical history was obtained and none had a history of prolonged antibiotic or steroid therapy, anaemia, diabetes or any condition or treatment known to promote the candidal carrier state. The antibiotic history was recorded and corroborated by correspondence with their general dental and medical practitioners. The condition of the restoration margins was assessed to establish the presence or absence of restoration leakage. The presence of caries, fractured restorations, probe-able restoration margins or marginal staining were used as positive indicators. The nature of the canal contents and the periodontal condition of the tooth were noted.

Saliva and root canal sampling.
A 2-mL sample of whole unstimulated saliva was collected from each patient into a sterile container before sampling from the root canal(s). The target teeth were scaled, polished and isolated with rubber dam. The sampling field was decontaminated by scrubbing with 30% hydrogen peroxide (v/v) (Sigma Chemical Ltd, Poole, UK), followed by soaking with 10% iodine (w/v) (Betadine ®, Seton Health Care Group PLC, Oldham, UK) for 1 min. The iodine was inactivated by 5% sodium thiosulphate (w/v) (Sigma Chemical Ltd). The decontamination procedures were repeated following access cavity preparation. Any previous poorly condensed root canal filling was removed prior to sampling using sterile Hedstrom files (Kerr UK Limited, Peterborough, UK) alone; solvent (chloroform) was necessary for the removal of guttapercha prior to sampling in only three cases. In most cases the canals were negotiable to their full length after removal of gutta-percha as judged by the apex locator and radiographic confirmation. The previously untreated canals were negotiated with small files (sizes 6, 8, 10) to their full length, again judged by apex locator and radiographic confirmation. If the canals were dry, sterile phosphate buffered saline was introduced and the canals filed (using a size appropriate to the canal) to release debris (including bacteria) into the fluid. The debris-laden fluid was soaked up using three sterile paper points, each being left in the canal for at least 1 min. They were immediately transferred aseptically into vials containing reduced transport fluid (RFT) (Syed & Loesche 1972) and taken to the microbiology laboratory for processing within 3 h.

Laboratory processing of samples.
The saliva and root canal samples were vortexed (Vortex, Scientific Industries Inc., Springfield, NY, USA) for 1 min and 10-fold serially diluted to 10 –2 in RTF. From the undiluted sample and the dilutions, 50 L aliquots were spread on sabouraud dextrose agar (SAB) plates (Oxoid Ltd, Basingstoke, UK) using a sterile glass spreader. The plates were incubated at 30 C for 3 days. Yeast colonies were counted and colony forming units per millilitre were calculated. Pure yeast cultures were obtained by further subculturing on SAB media.

Identification of yeasts.
Preliminary identification of yeasts from bacterial cell colonies was based on the growth characteristics and colony morphology (Warren & Hazen 1995).
The yeast isolates were further characterized and speciated based on the following:

• Germ tube formation test.
  Candida albicans and C. dubliniensis were differentiated from other Candida species by their ability to form germ tubes. A loopful of inoculum from the yeast colony was suspended in 0.5 mL of sterile horse serum, incubated for 2–3 h at 37 C and the culture was examined under 40 magnification (Carl Zeiss, Jena, Germany) for germ tube growth from the yeast cells.
• Hyphal morphology.
  Pure yeast isolates were cultured on starch-containing cornmeal agar plates which stimulated the production of true hyphae, pseudohyphae, arthrospores and chlamydospores (Campbell et al. 1996).
• Biochemical tests.
  The Rapid ID32C® (Biomérieux UK Limited, Basingstoke, UK) system for yeast identification was used. The strips provided in the kits were inoculated and developed, and results were recorded according to the manufacturer’s instructions. Identification was obtained using a computer software package (APILAB Plus, Biomérieux) to interpret the data generated by the kit. The results were correlated with the germ tube formation test and hyphal morphology.

Statistical analysis.
Fifty-five cases were selected for statistical analyses. For those patients who gave two root canal samples, only the first set of data was included for analysis. All statistical analyses were made with a computer program, STATA 5 (STATA version 5. STATA Corporation, College Station, TX, USA 1995). Logistic regression models were used to investigate the factors associated with the presence of yeasts in root canals.

Yeast species recovered from saliva and root canal samples.
Fifty-nine saliva and 60 root canal samples were obtained from the 55 patients. Five patients had two teeth sampled and four patients gave a second saliva sample (Table 2). Yeasts were more frequently recovered from saliva (19/59 or 32.2%) than root canal (6/60 or 10%) samples. Of the six yeast-positive root canals, two had no previous root treatment (out of a total of 35) and four had previous root treatment (out of a total of 25).
Twenty-three isolates were recovered from saliva and eight from the root canal samples. They were identified to the species level (Table 3) with the majority belonging to the genus Candida (29% of patients or 74% of isolates). Of the yeast species isolated from saliva, C. albicans was the most prevalent (17/23 or 73.9%) followed by R. mucilaginosa (2/23), C. dubliniensis (1/23), C. tropicalis (1/23) and Cryptococcus humicolus (1/23) (Table 3). The yeast species recovered from root canals were R. mucilaginosa (4/8), C. albicans (3/8), and C. sake (1/8) (Table 3). The clinical details of the patients with yeast-positive root canal samples are presented in Table 4. The medical history indicated that none of these patients was immuno-compromised but all had received at least one course of antibiotics within the previous 12 months.

Table 2. Summary of number of patients involved in the study and results from sampling.



Table 3. Yeast species recovered from saliva and root canal samples and their concentration in colony forming unit (CFU) counts.
a. The Rapid ID result did not correspond with hyphal morphology.
b. The Rapid ID result did not correspond with germ tube formation test.



Table 4. Summary of clinical details from patients with yeast-positive root canal samples.

Statistical analysis.
The associations between the presence of yeasts in root canals (dependent variable) and the presence of yeasts in saliva, leakage of restorations, previous root canal treatment and antibiotic history (explanatory variables) are presented in Tables 5–9.
The relationship between the presence of yeasts in root canals and saliva is presented in Table 5. Their association was highly significant ( P = 0.021), with canals being 13.8 times (95% CI = 1.5–129.9) more likely to have yeasts isolated when they were also present in saliva (Table 6). Further logistic regression analysis also revealed that the significant effect of yeasts in saliva on the presence of yeasts in root canals was maintained when the effect of potential confounders was controlled (Table 7). The effects of restoration leakage ( P = 0.08) and previous root canal treatment ( P = 0.123) were equivocal (Table 6). The effect of systemic antibiotic history could not be analysed, due to the absence of samples with positive yeasts in root canal and negative antibiotic history (Table 8). The history of antibiotic therapy (Table 9) was not associated with the presence of yeasts in saliva (OR = 1.1).

Table 5. Summary of the relationship between prevalence of yeasts in root canals and saliva.



Table 6. Univariate logistic regression models for individual explanatory variables (yeasts in saliva, restoration leakage, previous root canal treatment) given separately.


*Confidence interval.

Table 7. Logistic regression models for the effect of yeasts in saliva adjusted for restoration leakage or/and previous root treatment.



Table 8. Summary of relation between the presence of yeasts in root canals and antibiotic history (n = 55 patients)



Table 9. Summary of relation between the presence of yeasts in saliva and antibiotic history (n = 55 patients).


Crude odds ratio = 1.1.

The sample collection (imprint culture, mucosal swab, mouth rinse, saliva) (Arendorf & Walker 1980, Samaranayake et al. 1986) and cultivation techniques (Najzar-Fleger et al. 1992) appear to influence the recovery of yeasts. The saliva culture technique used in this study was one of the most sensitive methods for assessing Candidal carriage in healthy individuals (Oliver & Shillitoe 1984), except that it does not allow study of the intraoral distribution of yeasts as would be demonstrated by imprint culture (Arendorf & Walker 1980). Most of the studies using non-selective blood agar (Tronstad et al. 1987, Najzar-Fleger et al. 1992, Sundqvist et al. 1998, Molander et al. 1998) have reported a lower prevalence of yeasts in root canals compared to the present (10%) and other (7.6–55%) studies (Jackson & Halder 1963, Wilson & Hall 1968, Najzar-Fleger et al. 1992) using selective sabouraud dextrose agar for cultivation.
The prevalence of yeasts in untreated cases in this study was 5.7%, in contrast to the 1.9% (Wilson & Hall 1968) and 26% (Jackson & Halder 1963) reported in previous studies. The prevalence increased to 6.8% and 33.6%, respectively, after placement of an intracanal dressing in the above studies; this would appear to substantiate the hypothesis that inadequate treatment may allow yeasts to overgrow (Sirén et al. 1997, Sundqvist et al. 1998). It is possible the differences were due to coronal status of the tooth, details of which were not given. Investigation of treatment-resistant cases by Waltimo et al. (1997) found a prevalence of 4.9% in 967 retreatment cases compared to the 16% recovered in this study. In their study, the sampling was carried out by a number of general dental practitioners following written instructions on the sampling procedure, the samples being processed 1–3 days following acquisition. The low recovery of yeasts may also be attributed to the use of tryptic-soyagar medium for isolation and the method used for preliminary identification of yeasts. A range of identification protocols were used in the present study, ranging from the germ tube formation test to microscopic examination on corn meal agar to rapid identification kits (Rapid ID32C ® ). The correlation of the identifications was good for 30 out of 32 isolates, the tests validating each other, implying a robustness and consistency.
Of the Candida species isolated from saliva samples in this study, C. albicans was the most prevalent (17/23), corroborating the findings of others (Odds 1979). However, Rodotorula mucilaginosa, Candida dubliniensis, Cryptococcus humicolus and Trichosporan mucoides, which are usually associated with immuno-compromised patients (Campbell et al. 1996) and possess increased virulence in susceptible hosts (McCullough & Hume 1995), were recovered from healthy individuals in this study. C. dubliniensis was recently identified by Sullivan et al. (1995) and is believed to be strongly associated with human immunodeficiency virus infection (Coleman et al. 1997). More recently, it has also been recovered from HIVnegative individuals (Odds et al. 1998, Pinjon et al. 1998). In the present study, C. albicans was recovered from one patient (patient 1) at his initial visit, whilst C. dubliniensis was recovered from the saliva a month later. This may be explained by the fact that C. albicans and C. dubliniensis are closely related (Sullivan et al. 1995), with the colonies appearing identical on SAB plates; both strains may have been present on each occasion and not detected.
All five patients (six teeth) with yeasts present in the root canal sample were healthy and had no history of immunodeficiency, but had received a course of antibiotics within the previous 12 months. The root canal systems of five teeth had some communication with the oral cavity, via coronal restoration leakage, sinus tract or oro-antral communication (Table 4). The sixth case had a history of post crown decementation and incision/drainage of a swelling associated with the tooth in question some months earlier, although the corresponding saliva sample was negative for yeasts. The periodontal probing profile associated with these teeth was less than 4 mm for all the cases, except patient 2, who had a deep vertical bony defect that communicated with the periapical lesion.
Of the yeast species isolated from the root canals, R. mucilaginosa (4/8) and C. albicans (3/8) were almost equally prevalent, whilst C. sake (1/8) was recovered as a single strain from only one tooth (patient 3). The isolation of Rhodotorula species from three root canals and the saliva of two patients (patients 1 and 5) was unusual, because its presence in root canals has not been reported previously.
Yeasts were shown to be more prevalent in previously treated (16%) than untreated (5.7%) canals; in either case, they were strongly associated with their presence in saliva (odds ratio = 13.8, P = 0.021, 95% CI = 1.5–129.9). Although the number of yeast-positive samples is small, the small P-value indicates that the detected difference is likely to be real, whilst the wide confidence interval suggests that the true magnitude of this difference is difficult to estimate with precision. The yeasts may have invaded root canals during initial treatment via leaking restorations or by colonization of dentinal tubules. Once Candida species enter the root canal, they may further penetrate into root canal dentinal tubules by adoption of a range of growth patterns (blastospores and hyphae) and interact with other microorganisms to form a complex biofilm (Bagg & Silverwood 1986, Jenkinson et al. 1990, Qen et al. 1997, 1999). Fungi, unlike most bacteria, are highly resistant to routine root canal irrigants such as sodium hypochlorite (Qen et al. 1999, Waltimo et al. 1999) and medication (calcium hydroxide) (Waltimo et al. 1999). The antifungal effect of sodium hypochlorite may be further hindered by the presence of a smear layer (Qen et al. 1999). Mechanical instrumentation of canals may help to disrupt and expose biofilm organisms to irrigating solutions but at the same time may produce a smear layer that may enhance the growth of surviving yeast cells. On the basis of this, a case could be made for ethylenediaminetetraacetic acid (EDTA) irrigation to remove the smear layer and perhaps an antifungal medicament for recalcitrant cases. Furthermore, EDTA has been shown to have a potent antifungal effect (Qen et al. 2000), possibly due to its ability to chelate calcium ions which have a critical role in morphogenesis and pathogenicity of C. albicans.
The role of yeasts in root canal infection and periapical disease is still poorly understood and warrants further investigation.

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