Genotyping and antifungal susceptibility of human subgingival Candida albicans isolates

Archives of Oral Biology 47 (2002) 189–196

Genotyping and antifungal susceptibility of human subgingival Candida albicans isolates G. Pizzo a,∗ , F. Barchiesi b , L. Falconi Di Francesco b , G. Giuliana a , D. Arzeni b , M.E. Milici c , M. D’Angelo a , G. Scalise b a

Department of Oral Sciences, Section of Periodontology, University of Palermo, Via del Vespro 129, 90127 Palermo, Italy b Institute of Infectious Diseases and Public Health, University of Ancona, Via Conca-Torrette, 60020 Ancona, Italy c Department of Hygiene and Microbiology, University of Palermo, Via del Vespro 133, 90127 Palermo, Italy Accepted 25 September 2001

Abstract Subgingival colonization by Candida albicans has been described in human immunodeficiency virus (HIV)-infected individuals, but subgingival isolates have scarcely been characterized, particularly with respect to genotype and antifungal susceptibility. A series of 29 subgingival strains of C. albicans isolated from nine HIV-infected individuals was typed by electrophoretic karyotyping and tested for susceptibility to fluconazole, itraconazole, the new investigational triazole posaconazole and amphotericin B. DNA typing showed genetic heterogeneity within subgingival isolates, as almost every individual harbored his/her own specific isolate. Genetic identity was usually demonstrated within oral and subgingival isolates simultaneously collected from the same individual, but a number of DNA types were found to be unique to subgingival strains. These findings suggest that colonization is not just the result of Candida spreading from oral surfaces, and that subgingivally adapted strains could be involved. All isolates were susceptible to all the triazole drugs tested and amphotericin B. Additional studies on subgingival Candida colonization and further characterization of subgingival isolates are now required to clarify the role of Candida as opportunistic periodontal pathogen. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Candida albicans; Subgingival flora; Genotyping; Antifungal susceptibility

1. Introduction Candida albicans is usually encountered as a harmless commensal in the human mouth. Mucosal surfaces are the primary oral reservoir for this microorganism, but dental plaque can also harbor it (Cannon et al., 1995; Nikawa et al., 1998). Candidal colonization has been demonstrated in periodontal pockets (Slots et al., 1988; Dahlén and Wikström, 1995), refractory periodontitis (Slots et al., 1990; Listgarten et al., 1993; Olsvik et al., 1995), and failing dental implants (Leonhardt et al., 1999). The emergence of C. albicans in the subgingival flora may also occur as a result of the use of either antibiotics or radiotherapy (Rams Abbreviations: HIV, human immunodeficiency virus; NCCLS, National Committee for Clinical Laboratory Standards; MIC, minimum inhibitory concentration; PBS, phosphate-buffered saline ∗ Corresponding author. E-mail address: [email protected] (G. Pizzo).

et al., 1990; Helovuo et al., 1993; MacNeill et al., 1997; Leung et al., 1998). Further, the presence of C. albicans in the subgingival environment has been reported in human immunodeficiency virus (HIV)-infected individuals (Brady et al., 1996; Lamster et al., 1998; Chattin et al., 1999). The clinical significance of these observations is not yet fully understood, but there is increasing evidence that in immunocompromised patients Candida organisms may be implicated in opportunistic periodontal infections (Budtz-Jörgensen and Lombardi, 1996; Lamster et al., 1998; Velegraki et al., 1999; McKaig et al., 2000; Ryder, 2000). In recent years, several molecular typing methods have been used to characterize C. albicans isolates and delineate strain relatedness (Dahl et al., 1997; Barchiesi et al., 1995, 1998; Diaz-Guerra et al., 1998; Lischewski et al., 1999; Redding et al., 1999; Xu et al., 1999; Hannula et al., 2001; Waltimo et al., 2001). These methods have greatly enhanced knowledge about the epidemiology of oral candidosis, but thus far little information is available about subgingival

0003-9969/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 0 1 ) 0 0 1 1 4 - 5

190

G. Pizzo et al. / Archives of Oral Biology 47 (2002) 189–196

Candida colonization. The distribution of C. albicans biotypes in periodontal pockets was shown to be similar to that of oral surfaces (Rams and Slots, 1991). In HIV-positive patients, however, subgingival isolates appeared distinct from mucosal ones (Lamster et al., 1998). Subgingival Candida isolates are also poorly characterized with regard to their antifungal drug resistance. Recently, the National Committee for Clinical Laboratory Standards (NCCLS) has approved a reference method for antifungal susceptibility testing of yeasts (document M27-A; NCCLS, 1997). Despite the widespread use of this method, there are no studies, to the best of our knowledge, on the susceptibilities of subgingival isolates to antifungal agents. Our main aim now was to characterize a group of subgingival isolates of C. albicans recovered from HIV-positive patients. Molecular typing was performed by electrophoretic karyotyping, which has proved to be a reproducible and highly discriminatory technique for the genetic characterization of C. albicans (Pfaller, 1995; Barchiesi et al., 1998; Espinel-Ingroff et al., 1999). The susceptibilities of these isolates to a panel of antifungal agents were also determined by a broth microdilution method performed according to the NCCLS (1997) recommendations.

speed for 30 s. Oral rinse samples were obtained by asking the patients to rinse their mouths with 10 ml of PBS for 1 min and to spit the rinse into a sterile container. The oral rinse was centrifuged at 1700×g for 10 min, the supernatant discarded and the pellet resuspended in 1 ml of PBS. Samples were plated on to CHROMagarTM Candida plates (Becton Dickinson and Company, Franklin Lakes, NJ) in serial dilutions and incubated aerobically at 37 ◦ C for 48 h. Presumptive identification of C. albicans isolates were based on the light-green color of the colonies on these plates. The identity of isolates as C. albicans was confirmed by the following morphological and biochemical methods, germ-tube formation, chlamydospore production, and carbohydrate assimilation pattern by using the API 20C AUX identification system (BioMérieux, Marcy l’Etoile, France) (Williams and Lewis, 2000). Additional tests for growth at 45 ◦ C and reduction of 2,3,5-triphenyltetrazolium chloride were used to discriminate between C. albicans and C. dubliniensis (Giammanco et al., in press). Yeasts were stored in Sabouraud dextrose broth (Becton Dickinson and Company, Franklin Lakes, NJ) supplemented with sterile glycerol (20% final concentration) and frozen at −70 ◦ C until use. One to six C. albicans isolates from subgingival samples and one from the oral rinse sample were collected from each patient.

2. Materials and methods 2.1. Sources of isolates Isolates of C. albicans were collected from nine HIV-positive outpatients (six males and three females; age range, 27–44 years) consecutively referred to the Palermo University Department of Oral Sciences for routine diagnostic evaluation of their periodontal condition. Individuals included in the study had not received any periodontal therapy, antibiotics and antifungals during the previous 6 months, and none of them was a denture wearer. Female participants were neither pregnant nor taking oral contraceptives. An additional criterion for patient selection was the absence of HIV-associated periodontal lesions (Axéll et al., 1993). All participants gave informed consent for the procedure used to obtain subgingival and oral yeast isolates. 2.2. Sample collection Subgingival plaque samples were obtained with sterile fine paper points (Johnson & Johnson, Windsor, NJ). The sample sites were air-dried and isolated with sterile cotton rolls. Supragingival deposits were removed with sterile curettes and cotton pellets. Subgingival samples were taken from six teeth (one/sextant) per individual by inserting a single paper point to the depth of each of the sample sites (four/tooth). After placement for 10 s, all four paper points from a tooth were pooled in a vial containing 1 ml of phosphate-buffered saline (PBS) (0.1 M, pH 7.2) and sterile glass beads of 3 mm diameter. Organisms were mechanically dispersed from the paper points by vortexing at full

2.3. DNA typing Molecular typing of strains of C. albicans was accomplished by electrophoretic karyotyping. In brief, agarose plugs containing yeast DNA were prepared as described by Barchiesi et al. (1995). Chromosomes were resolved with a contour-clamped homogeneous electric-field system (CHEF DR-II; Bio-Rad, Hercules, CA) through 1% SeaKem Gold agarose (FMC BioProducts, BIOSPA, Milan, Italy) in 0.5 × TBE buffer (0.45 M Tris, 0.44 M boric acid, and 10 mM EDTA, pH 8.0) at 13 ◦ C. Electrophoresis was performed at 2 V/cm under the following conditions: 66 h during which the switch time was ramped from 60 to 300 s, and 60 h during which the switch time ramped from 420 to 900 s. Saccharomyces cerevisiae marker (Bio-Rad) was used as a DNA size standard and run in each gel. Ethidium bromide-stained gels were photographed under ultraviolet light. Each major and minor band was identified, and the distance from the origin of the gel relative to those of the molecular-weight standards was measured. Isolates were considered to be identical (same type) if all bands matched exactly. Isolates with electrophoretic karyotyping profiles differing by one band were considered to be similar and were identified as subtypes. Isolates were judged to have different DNA types if their profile differed by two or more bands. 2.4. Antifungal susceptibility testing In vitro susceptibility testing of C. albicans isolates to fluconazole (Pfizer Inc., New York, NY), itraconazole

G. Pizzo et al. / Archives of Oral Biology 47 (2002) 189–196

191

(Janssen Pharmaceutica, Beerse, Belgium), posaconazole (Schering-Plough Research Institute, Kenilworth, NJ), and amphotericin B (Bristol-Myers Squibb, Princeton, NJ) was performed by a broth microdilution, adhering to the NCCLS (1997) recommendations. Testing was performed in RPMI 1640 (Sigma–Aldrich, Milan, Italy) buffered to pH 7.0 with 0.165 mol/l morpholinepropanesulphonic acid buffer (Sigma–Aldrich). Stock solutions were prepared in dimethyl sulphoxide (amphotericin B), polyethylene glycol (posaconazole and itraconazole), and water (fluconazole). Serial dilutions of each antifungal agent and yeast inocula were prepared exactly as outlined in NCCLS document (1997). The final concentrations of the antifungal agents ranged from 0.125 to 64 ␮g/ml for fluconazole, from 0.0078 to 4.0 ␮g/ml for itraconazole and posaconazole, and from 0.03 to 8.0 ␮g/ml for amphotericin B. Minimum inhibitory concentrations (MICs) for fluconazole, itraconazole and posaconazole were defined as the first concentration of the agent at which turbidity in the well was ≥80% less than that in the control (drug-free) well. MIC for amphotericin B was defined as the first concentration of drug at which no growth was detected. Azole susceptibility patterns were defined according to the NCCLS criteria (NCCLS, 1997; Rex et al., 1997). Isolates were considered in vitro susceptible to amphotericin B if their MICs were ≤1.0 ␮g/ml (NCCLS, 1997). A significant variation in the susceptibility pattern was considered to have occurred if the MIC for a strain from a single participant was at least four times the MIC determined for the strains isolated from the same patient (Barchiesi et al., 1995). C. krusei ATCC 6258 was used as quality control strain in each run of the experiments.

Table 1 DNA types of 38 strains of Candida albicans isolated from nine HIV-infected individuals

3. Results A total of 38 C. albicans isolates were collected from the nine HIV-positive individuals. Twenty-nine isolates were recovered from the subgingival samples (one to six strains per participant), and nine strains were recovered from the oral rinse samples (one strain per participant). 3.1. DNA types The results for DNA types are summarized in Table 1. Under our electrophoretic conditions, the karyotyping revealed from six to nine bands (chromosomes) ranging in size from 700 to greater than 2200 kb (Fig. 1). Overall, the analysis of 38 clinical isolates of C. albicans revealed nine distinguishable DNA types (A–I). Among these, DNA types A, C, G–I had variant subtypes (Table 1). Four DNA types (A–C, and F) were unique to a single patient. DNA type G was isolated from participants 2 and 6, DNA type I from participants 3 and 4, and DNA type H from participants 4, 6, and 8. Six individuals (66.7%) harbored a single DNA type. The remaining three (33.3%)

Patient/isolatea

DNA typeb

1.S 1.OR

F F

2.S 2.OR

G G

3.S.a 3.S.b 3.S.c 3.S.d 3.S.e 3.OR

I2 I2 I2 I2 I2 I1

4.S.a 4.S.b 4.S.c 4.S.d 4.OR

I H I I I

5.S 5.OR

A A1

6.S.a 6.S.b 6.S.c 6.S.d 6.OR

G G1 E E H

7.S.a 7.S.b 7.S.c 7.S.d 7.S.e 7.S.f 7.OR

C1 C C1 C C C1 C

8.S.a 8.S.b 8.OR

H1 H H2

9.S.a 9.S.b 9.S.c 9.S.d 9.S.e 9.OR

B D B B B B

a Isolate designations beginning with the same number are from an individual participant. Upper-case letters indicate subgingival or oral strains, respectively; lower-case letters refer to multiple isolates from single participants. b Codes accompanied by a number strongly resemble the pattern type without number and denote DNA subtypes.

were colonized with two or three DNA types (participants 4 and 9, and participant 6, respectively). In participant 6, the strain isolated from the mouth proved to be genetically different from those recovered from subgingival samples.

192

G. Pizzo et al. / Archives of Oral Biology 47 (2002) 189–196

Fig. 1. Representative electrophoretic karyotyping profiles of 13 isolates of Candida albicans. Lanes 1–13: isolates 7.S.a, 7.S.b, 5.S, 3.S.a, 6.S.b, 2.OR, 5.OR, 8.OR, 9.S.a, 9.S.b, 8.S.a, 9.S.d, 9.S.e, respectively (as indicated in Table 1). Lower and upper arrows indicate 700 and 2200 kb, respectively.

3.2. Antifungal susceptibility patterns Antifungal susceptibility patterns of C. albicans isolates to the four antifungal agents are shown in Table 2. MICs for fluconazole ranged from 0.5 to 8.0 ␮g/ml, with an MIC50 and MIC90 (i.e. MICs at which 50 and 90% of the strains were inhibited, respectively) of 0.5 and 2.0 ␮g/ml, respectively. Itraconazole had a MIC range narrower than that observed for fluconazole, with an MIC50 and an MIC90 of 0.03 and 0.06 ␮g/ml, respectively. MICs for posaconazole ranged from 0.015 to 0.25 ␮g/ml, with an MIC50 of 0.06 ␮g/ml and an MIC90 of 0.125 ␮g/ml. MICs for amphotericin B ranged from 0.06 to 0.25 ␮g/ml, with the MIC50 and MIC90 of 0.125 ␮g/ml. Therefore, all isolates were in vitro susceptible to the antifungal agents under test (NCCLS, 1997; Rex et al., 1997). No significant variations in antifungal susceptibility patterns were noted among the strains isolated from individual patients.

4. Discussion Oral candidosis has increased markedly during the past two decades because of increasing numbers of immunocompromised patients. Along with this rising incidence, molecular typing techniques have become fundamental for studying the epidemiology of Candida isolates and for developing rational therapeutic strategies (Pfaller, 1995; Barchiesi et al., 1995, 1998; Dahl et al., 1997; Diaz-Guerra et al., 1998; Lischewski et al., 1999; Redding et al., 1999; Xu et al., 1999). Recently, subgingival Candida colonization has been reported to be substantial in HIV-positive individuals (Chattin et al., 1999; Ryder, 2000), but scant

research has been conducted on the epidemiological aspects of this event (Lamster et al., 1998). In the present study, we used electrophoretic karyotyping to characterize a group of subgingival C. albicans isolates recovered from HIV-positive patients. The DNA typing illustrates a number of important features of subgingival colonization by C. albicans. The majority of our participants (77.8%) harbored his/her own specific isolate (DNA type). This heterogeneity within subgingival isolates agrees with that reported for oral Candida colonization/infection (Miyasaki et al., 1992; Barchiesi et al., 1995, 1998; Dahl et al., 1997; Diaz-Guerra et al., 1998; Lischewski et al., 1999; Xu et al., 1999; Capoluongo et al., 2000; Waltimo et al., 2001). Two pairs of participants (4 and 8, and 3 and 4), indeed, shared similar DNA types (H and H1, and I and I2, respectively) among their subgingival isolates. These individuals were unrelated each to other and strains were isolated at different time intervals. Therefore, a mechanism of transmission from a common exogenous source should be excluded. It is conceivable that these particular DNA types represent common subpopulations within this species of Candida. The relatedness of isolates simultaneously collected from oral and subgingival sites was also evaluated. Genetic diversity among strains obtained simultaneously from different body sites of individual patients has already been documented (Dahl et al., 1997; Ruhnke et al., 1999; Xu et al., 1999). The relationships between oral and subgingival isolates, however, are as yet poorly understood and conflicting results have been reported. Rams and Slots (1991) used a biotype method for studying the variation of C. albicans isolates in adult periodontitis and showed that subgingival biotypes were strictly similar to those recovered from the

G. Pizzo et al. / Archives of Oral Biology 47 (2002) 189–196

193

Table 2 Antifungal susceptibilities of 38 strains of Candida albicans isolated from nine HIV-infected individuals Patient/isolatea

MIC (␮g/ml) Fluconazole

Itraconazole

Posaconazole

Amphotericin B

1.S 1.OR

0.5 0.5

0.06 0.06

0.125 0.03

0.25 0.125

2.S 2.OR

0.5 0.5

0.03 0.03

0.125 0.03

0.125 0.125

3.S.a 3.S.b 3.S.c 3.S.d 3.S.e 3.OR

0.5 0.5 0.5 0.5 0.5 0.5

0.03 0.03 0.03 0.03 0.03 0.06

0.125 0.125 0.125 0.125 0.125 0.015

0.125 0.125 0.125 0.125 0.125 0.06

4.S.a 4.S.b 4.S.c 4.S.d 4.OR

0.5 0.5 0.5 0.5 0.5

0.03 0.03 0.03 0.03 0.03

0.06 0.06 0.06 0.06 0.015

0.125 0.125 0.125 0.125 0.125

5.S 5.OR

0.5 0.5

0.03 0.03

0.125 0.03

0.125 0.125

6.S.a 6.S.b 6.S.c 6.S.d 6.OR

0.5 0.5 0.5 0.5 0.5

0.03 0.03 0.03 0.03 0.03

0.125 0.125 0.015 0.015 0.015

0.125 0.125 0.125 0.125 0.125

7.S.a 7.S.b 7.S.c 7.S.d 7.S.e 7.S.f 7.OR

4.0 8.0 2.0 2.0 2.0 2.0 2.0

0.06 0.125 0.06 0.06 0.06 0.06 0.125

0.125 0.25 0.125 0.125 0.125 0.125 0.25

0.25 0.06 0.06 0.06 0.06 0.06 0.06

8.S.a 8.S.b 8.OR

0.5 0.5 2.0

0.03 0.03 0.03

0.06 0.015 0.06

0.06 0.125 0.06

9.S.a 9.S.b 9.S.c 9.S.d 9.S.e 9.OR

0.5 0.5 0.5 0.5 0.5 0.5

0.03 0.03 0.03 0.03 0.03 0.125

0.06 0.06 0.06 0.03 0.03 0.125

0.125 0.125 0.125 0.125 0.125 0.06

a Isolate designations beginning with the same number are from an individual participant. Upper-case letters indicate subgingival or oral strains, respectively; lower-case letters refer to multiple isolates from single participants.

tongue and other mucosal surfaces. On the other hand, Lamster et al. (1998) used a DNA-fingerprinting technique and demonstrated that some fingerprints were unique to subgingival strains. This disagreement could be due to the different study populations evaluated (healthy subjects and HIV-positive individuals). Furthermore, it must be remembered that typing systems based on genetic techniques usually provide an overall higher sensitivity (Pfaller, 1995). In our series, the genotypes of the subgingival strains were

often related to those of the mucosal ones (participants 1–3, 5, 7, and 8), but in some instances differences between oral and subgingival isolates were noted. Participants 4 and 9, although harboring subgingival strains genetically related to those isolated from their mucosal surfaces, showed the contemporaneous presence of a different DNA type in subgingival sites. Interestingly, participant 6 was colonized with three DNA types, and the strain isolated from the mouth was genetically different from the subgingival strains. The

194

G. Pizzo et al. / Archives of Oral Biology 47 (2002) 189–196

genetic identity found between oral and subgingival isolates indicates that subgingival colonization could originate from the spread of yeasts from either saliva or dental plaque. On the other hand, the recovery of a unique DNA type in subgingival strains suggests that the presence of C. albicans could also be due to colonization with subgingivally adapted strains. As patterns of antifungal susceptibility among subgingival C. albicans isolates have, to the best of our knowledge, never been investigated, we measured the MICs of four antifungal agents, including three triazole drugs and the polyene derivative amphotericin B. Both fluconazole and itraconazole had potent antifungal activity against all the isolates tested. Our data suggest that these agents might be a useful adjunct to conventional therapy in the treatment of opportunistic periodontal infections by C. albicans, mainly in those patients at high risk of developing systemic candidosis (Budtz-Jörgensen and Lombardi, 1996; Velegraki et al., 1999; Ryder, 2000). Although susceptibilities to fluconazole and itraconazole varied among the isolates, all strains were susceptible in vitro to both antifungal agents (NCCLS, 1997; Rex et al., 1997). These findings correlated well with the clinical presentation of these patients, as none of them had received triazole therapy in the preceding 6 months. It is known that the emergence of resistant strains of C. albicans frequently occurs in patients previously exposed to azoles (Rex et al., 1995; Masiá Canuto et al., 2000). Posaconazole is a new, broad-spectrum, antifungal triazole currently under development. This agent has been shown to have potent in vitro and in vivo activities against several yeast species and filamentous fungi (Perfect et al., 1996; Oakley et al., 1997; Barchiesi et al., 2000). In this study, posaconazole had good inhibitory activity against C. albicans isolates (MIC range, 0.015–0.25 ␮g/ml). Interpretative criteria for MICs determined by NCCLS method, however, have not been defined for this antifungal. In accord with others (Pfaller et al., 1997; Barchiesi et al., 2000), we found that posaconazole was essentially as active as itraconazole and more active than fluconazole against all the isolates considered here, suggesting that this drug has significant potential for clinical development. Finally, we found that both subgingival and oral isolates were highly susceptible to amphotericin B, with identical MIC50 and MIC90 (0.125 ␮g/ml). In conclusion, we show genetic heterogeneity within subgingival C. albicans isolates recovered from HIV-positive individuals. Relatedness between oral and subgingival isolates recovered from the same individual was also demonstrated, but a number of DNA types were unique to subgingival strains. These findings suggest that colonization is not just the result of the spreading of Candida organisms from saliva and dental plaque, and that subgingivally adapted strains could be involved. All isolates were found susceptible to triazole derivatives (fluconazole, itraconazole and posaconazole) as well as to amphotericin B.

Additional studies on subgingival Candida colonization and further characterization of subgingival isolates are now required to clarify the role of Candida as an opportunistic periodontal pathogen.

Acknowledgements This investigation was supported in part by the Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST; ex-60% grants), Rome, Italy, and by a grant from Istituto Superiore di Sanità, Rome, Italy (III AIDS project, Grant no. 50C.29).

References Axéll, T., Azul, A.M., Challacombe, S.J., Ficarra, G., Flint, S., Greenspan, D., et al., 1993. EC-clearinghouse on oral problems related to HIV infection and WHO collaborating centre on oral manifestations of the human immunodeficiency virus: classification and diagnostic criteria for oral lesions in HIV infection. Journal of Oral Pathology and Medicine 22, 289–291. Barchiesi, F., Hollis, R.J., McGough, D.A., Scalise, G., Rinaldi, M.G., Pfaller, M.A., 1995. DNA subtypes and fluconazole susceptibilities of Candida albicans isolates from the oral cavities of patients with AIDS. Clinical Infectious Diseases 20, 634–640. Barchiesi, F., Arzeni, D., Del Prete, M.S., Sinicco, A., Falconi Di Francesco, L., Pasticci, M.B., et al., 1998. Fluconazole susceptibility and strain variation of Candida albicans isolates from HIV-infected patients with oropharyngeal candidosis. Journal of Antimicrobial Chemotherapy 41, 541–548. Barchiesi, F., Arzeni, D., Fothergill, A.W., Falconi Di Francesco, L., Caselli, F., Rinaldi, M.G., Scalise, G., et al., 2000. In vitro activities of the new antifungal triazole, SCH 56592, against common and emerging yeast pathogens. Antimicrobial Agents and Chemotherapy 44, 226–229. Brady, L.J., Walker, C., Oxford, G.E., Stewart, C., Magnusson, I., McArthur, W., 1996. Oral diseases, mycology and periodontal microbiology of HIV-1-infected women. Oral Microbiology and Immunology 11, 371–380. Budtz-Jörgensen, E., Lombardi, T., 1996. Antifungal therapy in the oral cavity. Periodontology 2000 (10), 89–106. Cannon, R.D., Holmes, A.R., Mason, A.B., Monk, B.C., 1995. Oral Candida: clearance, colonization, or candidiasis? Journal of Dental Research 74, 1152–1161. Capoluongo, E., Moretto, D., Giglio, A., Belardi, M., Prignano, G., Crescimbeni, E., et al., 2000. Heterogeneity of oral isolates of Candida albicans in HIV-positive patients: correlation between candidal carriage, karyotype and disease stage. Journal of Medical Microbiology 49, 985–991. Chattin, B.R., Ishihara, K., Okuda, K., Hirai, Y., Ishikawa, T., 1999. Specific microbial colonizations in the periodontal sites of HIV-infected subjects. Microbiology and Immunology 43, 847–852. Dahl, K.M., Keath, E.J., Fraser, V.J., Powderly, W.G., 1997. Molecular epidemiology of mucosal candidiasis in HIV-positive women. AIDS Research and Human Retroviruses 13, 485–491.

G. Pizzo et al. / Archives of Oral Biology 47 (2002) 189–196 Dahlén, G., Wikström, M., 1995. Occurrence of enteric rods, staphylococci and Candida in subgingival samples. Oral Microbiology and Immunology 10, 42–46. Diaz-Guerra, T.M., Martin-Suarez, J.V., Laguna, F., Valencia, E., Rodriguez-Tudela, J.L., 1998. Change in fluconazole susceptibility patterns and genetic relationship among oral Candida albicans isolates. AIDS 12, 1601–1610. Espinel-Ingroff, A., Vazquez, J.A., Boikov, D., Pfaller, M.A., 1999. Evaluation of DNA-based typing procedures for strain categorization of Candida spp. Diagnostic Microbiology and Infectious Diseases 33, 231–239. Giammanco, G.M., Pizzo, G., Pecorella, S., Distefano, S., Pecoraro, V., Milici, M.E. Identification of Candida dubliniensis among oral yeast isolates from an Italian population of human immunodeficiency virus-infected (HIV+) subjects. Oral Microbiology and Immunology, in press. Hannula, J., Dogan, B., Slots, J., Ökte, E., Asikainen, S., 2001. Subgingival strains of Candida albicans in relation to geographical origin and occurrence of periodontal pathogenic bacteria. Oral Microbiology and Immunology 16, 113–118. Helovuo, H., Hakkarainen, K., Paunio, K., 1993. Changes in the prevalence of subgingival enteric rods, staphylococci and yeasts after treatment with penicillin and erythromycin. Oral Microbiology and Immunology 8, 75–79. Lamster, I.B., Grbic, J.T., Mitchell-Lewis, D.A., Begg, M.D., Mitchell, A., 1998. New concepts regarding the pathogenesis of periodontal disease in HIV infection. Annals of Periodontology 3, 62–75. Leonhardt, A., Renvert, S., Dahlén, G., 1999. Microbial findings at failing implants. Clinical Oral Implants Research 10, 339–345. Leung, W.K., Jin, L.J., Samaranayake, L.P., Chiu, G.K.C., 1998. Subgingival microbiota of shallow periodontal pockets in individuals after head and neck irradiation. Oral Microbiology and Immunology 13, 1–10. Lischewski, A., Harmsen, D., Wilms, K., Baier, G., Gunzer, U., Klinker, H., et al., 1999. Molecular epidemiology of Candida albicans isolates from AIDS and cancer patients using a novel standardized CARE-2 DNA fingerprinting technique. Mycoses 42, 371–383. Listgarten, M.A., Lai, C.H., Young, V., 1993. Microbial composition and pattern of antibiotic resistance in subgingival microbial samples from patients with refractory periodontitis. Journal of Periodontology 64, 155–161. MacNeill, S., Rindler, E., Walker, A., Brown, A.R., Cobb, C.M., 1997. Effects of tetracycline hydrochloride and chlorhexidine gluconate on Candida albicans: an vitro study. Journal of Clinical Periodontology 24, 753–760. Masiá Canuto, M., Gutiérrez Rodero, F., Ortiz de la Tabla Ducasse, V., Hernández Aguado, I., Mart´ın González, C., Sánchez Sevillano, A., et al., 2000. Determinants for the development of oropharyngeal colonization or infection by fluconazole-resistant Candida strains in HIV-infected patients. European Journal of Clinical Microbiology and Infectious Diseases 19, 593–601. McKaig, R.G., Patton, L.L., Thomas, J.C., Strauss, R.P., Slade, G.D., Beck, J.D., 2000. Factors associated with periodontitis in an HIV-infected southeast USA study. Oral Diseases 6, 158– 165. Miyasaki, S., Hicks, J.B., Greenspan, D., Polacheck, I., MacPhail, L.A., White, T.C., et al., 1992. The identification and tracking of Candida albicans isolates from oral lesions in HIV-seropositive individuals. Journal of Acquired Immune Deficiency Syndromes 5, 1039–1046.

195

National Committee for Clinical Laboratory Standards, 1997. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard. NCCLS document M27-A. Villanova, PA. Nikawa, H., Hamada, T., Yamamoto, T., 1998. Denture plaque— past and recent concerns. Journal of Dentistry 26, 299–304. Oakley, K.L., Morrissey, G., Denning, D.W., 1997. Efficacy of SCH-56592 in a temporarily neutropenic murine model of invasive aspergillosis with an itraconazole-susceptible and an itraconazole-resistant isolate of Aspergillus fumigatus. Antimicrobial Agents and Chemotherapy 41, 1504–1507. Olsvik, B., Hansen, B.F., Tenover, F.C., Olsen, I., 1995. Tetracycline-resistant micro-organisms recovered from patients with refractory periodontal disease. Journal of Clinical Periodontology 22, 391–396. Perfect, J.R., Cox, G.M., Dodge, R.K., Schell, W.A., 1996. In vitro and in vivo efficacies of the azole SCH 56592 against Cryptococcus neoformans. Antimicrobial Agents and Chemotherapy 40, 1910–1913. Pfaller, M.A., 1995. Epidemiology of fungal infections: the promise of molecular typing. Clinical Infectious Diseases 20, 1535–1539. Pfaller, M.A., Messer, S., Jones, R.N., 1997. Activity of a new triazole, SCH 56592, compared with those of four other antifungal agents tested against clinical isolates of Candida spp. and Saccharomyces cerevisiae. Antimicrobial Agents and Chemotherapy 41, 233–235. Rams, T.E., Slots, J., 1991. Candida biotypes in human adult periodontitis. Oral Microbiology and Immunology 6, 191–192. Rams, T.E., Babalola, O.O., Slots, J., 1990. Subgingival occurrence of enteric rods, yeasts and staphylococci after systemic doxycycline therapy. Oral Microbiology and Immunology 5, 166–168. Redding, S.W., Zellars, R.C., Kirkpatrick, W.R., McAtee, R.K., Caceres, M.A., Fothergill, A.W., et al., 1999. Epidemiology of oropharyngeal Candida colonization and infection in patients receiving radiation for head and neck cancer. Journal of Clinical Microbiology 37, 3896–3900. Rex, J.H., Rinaldi, M.G., Pfaller, M.A., 1995. Resistance of Candida species to fluconazole. Antimicrobial Agents and Chemotherapy 39, 1–8. Rex, J.H., Pfaller, M.A., Galgiani, J.N., Bartlett, M.S., Espinel-Ingroff, A., Ghannoum, M.A., 1997. Development of interpretative breakpoints for antifungal susceptibility testing: conceptual framework and analysis on in vitro–in vivo correlation data for fluconazole, itraconazole, and Candida infections. Clinical Infectious Diseases 24, 235–247. Ruhnke, M., Grosch-Wörner, I., Lischewski, A., Neubauer, A., Steinmüller, A., Trautmann, M., et al., 1999. Genotypic relatedness of yeast isolates from women infected with human immunodeficiency virus and their children. Mycoses 42, 385– 394. Ryder, M.I., 2000. Periodontal management of HIV-infected patients. Periodontology 2000 (23), 85–93. Slots, J., Rams, T.E., Listgarten, M.A., 1988. Yeasts, enteric rods and pseudomonads in the subgingival flora of severe adult periodontitis. Oral Microbiology and Immunology 3, 47–52. Slots, J., Feik, D., Rams, T.E., 1990. Age and sex relationships of superinfecting microorganisms in periodontitis patients. Oral Microbiology and Immunology 5, 305–308. Velegraki, A., Nicolatou, O., Theodoridou, M., Monstrou, G., Legakis, N.J., 1999. Paediatric AIDS-related linear gingival

196

G. Pizzo et al. / Archives of Oral Biology 47 (2002) 189–196

erythema: a form of erythematous candidiasis. Journal of Oral Pathology and Medicine 28, 178–182. Waltimo, T.M.T., Dassanayake, R.S., Ørstavik, D., Haapasalo, M.P.P., Samaranayake, L.P., 2001. Phenotypes and randomly amplified polymorphic DNA profiles of Candida albicans isolates from root canal infections in a Finnish population. Oral Microbiology and Immunology 16, 106–112.

Williams, D.W., Lewis, M.A.O., 2000. Isolation and identification of Candida from the oral cavity. Oral Diseases 6, 4–11. Xu, J., Boyd, C.M., Livingston, E., Meyer, W., Madden, J.F., Mitchell, T.G., 1999. Species and genotypic diversities and similarities of pathogenic yeasts colonizing women. Journal of Clinical Microbiology 37, 3835–3843.