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 »  Home  »  Endodontic Articles 4  »  Prevalence of yeasts in saliva and root canals of teeth associated with apical periodontitis
Prevalence of yeasts in saliva and root canals of teeth associated with apical periodontitis
Introduction - Materials and methods.



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.

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.