Genetic relatedness of commensal strains of Candida ... - Springer Link

Mycopathologia (2007) 164:255–263 DOI 10.1007/s11046-007-9052-5

Genetic relatedness of commensal strains of Candida albicans carried in the oral cavity of patients’ dental prosthesis users in Brazil Regina Helena Pires-Gonçalves Æ Elaine Toscano Miranda Æ Lilian Cristiane Baeza Æ Marcelo Teruyuki Matsumoto Æ Jose´ Eduardo Zaia Æ Maria Jose´ Soares Mendes-Giannini

Received: 1 April 2007 / Accepted: 29 August 2007 / Published online: 29 September 2007 Springer Science+Business Media B.V. 2007

Abstract The aim of this study is to describe the degree of yeast-colonization in diabetic and hemodialysed-users of dental prostheses. Individuals (306) were examined using an oral rinse technique in order to evaluate the incidence of yeast-carriage, and genotype of C. albicans. Yeasts were isolated from 68.4% (91/133) individual’s dental prostheses users. Dental prostheses were found to be a significant factor for the yeast colonization (P \ 0.05). Overall, the intensity of carriage was higher in diabetic patients as compared with health and hemodialysed individuals (P \ 0.05). The isolation rates were: C. albicans (51.7%), C. parapsilosis (20.9%), C. tropicalis (14.3%), C. glabrata (6.6%), C. krusei (3.3%), C. rugosa (1.1%), and Pichia (Pichia ohmeri, 2.2%). Ready-To-Go RAPD Analysis Beads were used and primer OPJ 6 distinguished the C. albicans isolates found in prostheses users. All the isolates were grouped into 11 RAPD profiles in four main clusters and, the average SAB for the entire collection R. H. Pires-Gonçalves E. T. Miranda L. C. Baeza M. T. Matsumoto M. J. S. Mendes-Giannini Departamento de Ana´lises Clıńicas, Faculdade de Cieˆncias Farmaceûticas, UNESP, Araraquara, SP, Brazil J. E. Zaia Universidade de Franca, Franca, SP, Brazil M. J. S. Mendes-Giannini (&) R. Expediciona´rios do Brasil, CEP 14802–901, Araraquara, SP 1621, Brazil e-mail: [email protected]

of 47 C. albicans isolates were 0.779 ± 0.178. Over 85% of isolates had a similarity level higher than or equal to 0.8 reinforcing the idea that the use of dental prostheses, independently of the host’s clinical condition, probably provides the necessary conditions for these strains to gain a growth-specific advantage over others. Keywords Oral Candida species Randomly amplified polymorphic DNA (RAPD) C. albicans genotype Dental prostheses Oral carriage

Introduction Candida albicans is a part of the normal microbial flora that colonizes mucocutaneous surfaces of the oral cavity, gastrointestinal tract, and vagina of the healthy human host. Although Candida does not normally cause disease, when immune defenses are compromised or the normal microflora balance is disrupted, C. albicans transforms itself into an opportunistic pathogenic, leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients and of oropharyngeal disease in AIDS patients. On the other hand, dental prostheses provide a solid base favorable to yeast adherence and colonization [1], resulting in an inflammatory process in the oral soft tissue, especially in the subjacent area covered by complete or partial

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dentures [2]. Current studies indicate that other factors within the oral environment such as saliva, pH, bacteria, hyphal formation, systemic diseases, and defects in the immune system have been showed to influence adhesion of Candida species to surface in the mouth [3, 4]. In addition, Diabetes mellitus and others high-risk patients could be considered as an additional variable that might influence not only oral Candida carriage, but also the ability of isolates to enhance the expression of virulence attributes [5]. Despite the high frequency of commensal carriage of C. albicans and its prominence as a major fungal pathogen, little is known about its genetic homogeneity, evolution, and persistence during commensalism or parasitism. The development of genotype techniques have helped to delineate subtypes of colonizing C. albicans strains and facilitated epidemiological analysis [6, 7]. For C. albicans, it has been demonstrated that RAPD analysis is highly effective in discriminating among completely unrelated strains, identifying the same strain, and discriminating microevolution in a single colonizing strain. RAPD is relatively cost-effective (for large numbers of isolates) and matches the resolving power of electrophoretic karyotyping. This, together with availability of computer-assisted software systems that generate dendrograms of genetic relatedness among C. albicans isolates and it has significantly advanced lineage studies over progressive infective episodes or during asymptomatic carriage [8, 9]. In addition, it has been found, by a variety of molecular methods that strains of C. albicans tend to be genetically similar if they are isolated from the same specific groups, such as HIV positive and negative patients [10], from geographically-related locations [10] or from several sites on the same or related patients [11–13]. Unfortunately, there have been very few studies of the genetic composition of commensal populations of infectious fungi. On the other hand, there are very little data in Brazil, on the incidence of yeast carriage and the identity of yeast species in the oral cavities of diabetic and hemodialysed patients and/or denture wearers. The main aim of this investigation was to assess the quantity, species and characterize the subtypes of oral C. albicans isolates from welldefined cohorts of diabetic and hemodialysed patients and healthy individual’s dental prostheses users.

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Mycopathologia (2007) 164:255–263

Methods Patients Ethical approval for the project was granted by the Research Ethics Committee of the Post-Graduate Department and Research Department of the University of Franca, Saõ Paulo State, Brazil (protocol 001/ 99) and it included: 175 insulin-using diabetes mellitus patients attending the diabetic clinics at the ´ nico Endocrinology Department of SUS (Sistema U de Sau´de, the Public Health System) in the state of Saõ Paulo, Brazil, and 77 patients with chronic renal failure who were receiving regular hemodialysis (the mean duration of hemodialysis therapy was three years, the range being two month to eight years) and 54 blood donors from the same region, who formed the healthy individuals group. All patients received a detailed oral examination to verify the integrity of the buccal mucosa at the time of sampling. Patients with clinical signs and symptoms of candidiasis were excluded from the study. The biological material was collected from November 13, 2000 to November 25, 2001. Patients were questioned about the use of partial or complete dentures, and these were noted.

Collection of yeasts isolates and growth conditions A total of 91 yeast isolates was obtained from the cohort of 91 dental-prosthesis users. The microorganisms were recovered using the oral-rinse technique of Samaranayake et al. [14]. In brief, the patients were requested to rinse the mouth with 10 ml phosphate-buffered saline PBS for 60s, and the liquid was collected in plastic containers and centrifuged; pelleted material was re-suspended in 1 ml PBS and 50 ll was spread on Sabouraud Dextrose Agar (SDA, Sigma-Aldrich, Dorset, UK) plus chloramphenicol and incubated at 35–37C for 48–72 h. The number of colony-forming units (CFU) for each sample was quantified (CP 600 Plus colony counter; Phoenix Luferco, Brazil), and one colony per sample was randomly selected and subcultured to obtain a pure growth. Morphologically distinct colonies from each culture was subcultured and stored on Sabouraud’s dextrose slant for species differentiation, DNA fingerprinting and storage. All isolates


were identified by morphology, physiological, and biochemical characteristics [15]. The yeasts identified as C. albicans were screened for their ability to grow on SDA at 45C for 48 h and xylose assimilation, to distinguish C. dubliniensis [16]. They were then evaluated with the API 20C Aux kit (BioMe´rieux SA, France). Isolates from a single sample yielding identical API 20C AUX profiles were considered the same yeast strain.

Genotyping of C. albicans isolates Preparation of DNA for RAPD Only C. albicans isolates from patients using dental prostheses (removable and total superior and/or inferior) were subcultured on Sabouraud agar with chloramphenicol and incubated at 37C for 48 h. One to three loops of cells from each colony were subcultured in 15 ml of YPG broth (1% of yeast extract, 2% of peptone, and 1% of glucose), constantly stirred for 18 h at 37C, and the cultures used for the genomic DNA extraction. This was performed with the Puregene DNA Isolation Kit (Gentra Systems INC., Minneapolis, USA), following the maker’s recommendations. DNA yield and integrity were verified by electrophoresis in 1% agarose gel.

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give a final reaction volume of 25 ll. Thermocycling was performed in a GeneAmp 9700 machine (Perkin Elmer Corporation, Foster City, USA), as follows: an initial denaturation step at 95C for 5 min, followed by 45 cycles consisting of denaturation at 95C for 1 min, annealing at 36C for 1 minute and with a final extension step at 72C for 5 min. Control tubes without template DNA were included in each run and reproducibility was checked for each reaction [7, 18]. The PCR products were electrophoresed in agarose gels (2%) in TBE buffer (Tris base 54 g, boric acid 27.5 g, EDTA 0.5 M 20 ml; distilled water to 1,000 ml) · 1 at 150 volts for 2 h at room temperature. Amplicons in the gel were stained with ethidium bromide (0.5 lg/ml) and visualized under ultraviolet transillumination. Gels were visually analyzed and, in order to standardize the quality of the images, recorded under UV light using the Image Master VDS System (Amersham Pharmacia Biotech, UK). All visible and well-defined bands obtained by visual analysis and confirmed by the software were included in the analysis. Dendrograms were used to determine the relationship among isolates on the basis of RAPD bands. Gels were normalized using Saccharomyces cerevisiae chromosomal DNA standards as a reference. All experiments were carried out in duplicate to assess reproducibility.

Randomly amplified polymorphic DNA analysis

Computer-assisted analysis of data and dendrogram generation

RAPD analysis was performed using Ready-To-Go RAPD Analysis Beads (Amersham Biosciences Corp., Piscataway, NJ, USA), as described by the manufacturer, carefully observing factors affecting reproducibility. Previous studies [17] were developed with the all ready to-go kit primers and the most discriminatory primer OPJ 6 (50 -d[CCCGTCAGCA]30 ) was selected for use with all strains. The reproducibility of the method was verified by repeating all amplifications from two isolates of the same strain a minimum of three times with selected strains. One ll of the DNA template (containing approximately 25 ng) was mixed with 19 ll of distilled water, 5.0 ll (25 pmol) of OPJ 6, Ready-To-Go RAPD Analysis Bead, dNTPs (0.4 mM), 1 unit of AmpliTaq polymerase DNA, PCR-solution, 3 mM MgCl2, 30 mM KCl, and 10 mM Tris (pH = 8.3) to

RAPD profiles were analyzed by GelCompar software Version 2.0 (Applied Maths). The similarity coefficient (SAB) between patterns for every pair of isolates A and B was computed with the formula SAB = 2E/(2E + a + b), where E is the number of common bands in the patterns of A and B, a is the number of bands in pattern A with no correlates in pattern B, and b is the number of bands in pattern B with no correlates in pattern A. Dendrograms based on SAB values were generated by UPGMA, implemented in the GelCompar software. An SAB value of 1.00 indicates that the banding patterns for strain A are identical with that of strain B; SAB values of 0.80–0.99 represent highly similar, but nonidentical, strains, and may suggest the occurrence of microevolution in a single strain; and SAB values below 0.80 represent unrelated strains [8, 19].

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Statistical analysis

counts, which was confirmed by the Dunn’s multiple comparisons method (P \ 0.05). The frequency of yeasts among dental prosthesis users carriers related to age are displayed in Table 3, where it is seen that C. albicans predominated, and most isolates of C. parapsilosis are concentrated in group A2 (‡51-years-old). C. albicans was the most commonly recovered species, isolated from 61.7% (87/141) of the overall individuals. Among the 91 dental prosthesis users who carried yeasts, the isolation frequency was: C. albicans (51.7%), C. parapsilosis (20.9%), C. tropicalis (14.3%), C. glabrata (6.6%), C. krusei (3.3%), C. rugosa (1.1%), and Pichia (Pichia ohmeri, 2.2%). The group of diabetic patients was most frequently colonized (68.1%) and showed diversity of strains. A total of 47 C. albicans isolates recovered from the oral cavities of dental prosthesis users were submitted to RAPD analysis with OPJ 6. All the isolates were grouped into 11 RAPD profiles in four main clusters showing 50% of similarity (Fig. 1). The SAB ranged from 0.5 to 1 with an average 0.779 ± 0.178. Eleven genotypes and four major

The significance of differences among multiple groups was assessed using the chi-squared test and an analysis of variance. When the data sets failed the normality test, the Kruskal–Wallis was used. Posthoc pair wise comparison between groups was tested for significance using Dunn’s method with the level of significance set at P \ 0.05. Data are reported as means ± SEM [18].

Results Out of the 306 patients screened for oral carriage of yeasts, 46% (141/306) were colonized. There were 68.4% carriers (91/133) among 133 dental prosthesis users (Table 1), and the rate of colonization varied significantly between these two subgroups (P \ 0.05). Only eight patients with prosthesis were not colonized. The total viable counts (CFU/ml of oral rinse) of yeast isolated are summarized in Table 2. By comparing the results it is noticeable that diabetic had higher viable

Table 1 Frequency of yeasts isolated from oral rinse samples of dental prosthesis users of three groups: diabetic; hemodialysed, and healthy group Individuals

Diabetic

a

Hemodialised Healthy Total a

Oral carriers (Dental prosthesis users)

b

c

d

Rate of colonization (%)

Oral carriers (without a prosthesis)

Rate of colonization (%)

62/83

74.7

30/92

32.6

24/39

61.5

6/38

15.8

05/11

45.5

14/43

32.6

91/133

68.4*

50/173

28.9

Diabetic group, n = 175

b

Hemodialised group, n = 77

c

Healthy individuals, n = 54

d

Total, n = 306

*This was significantly (x2 = 47.26, P \ 0.05) different when compared with patients without a prosthesis

Table 2 Number of yeast colony-forming units per ml in oral rinse samples of three groups: diabetic; hemodialysed, and healthy group n Healthy individuals

19

Range

Mean (Std error of mean)

2

10 –3.0 · 10 2

3

1,078.9 ( 208.5) 3

Median 800

Hemodialysed patients

30

3 · 10 –68.0 · 10

8,853.3 (2871.2)

1350

Diabetic patients

92

2 · 102–57.6 · 103

14,321.8 (1587.4)

8500*

*The Kruskal–Wallis with a post-hoc pair wise comparison (Dunn’s method) showed that the yeast CFU/ml in diabetic patients group was significantly (P \ 0.05) higher than other groups

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Table 3 Proportions of yeast species as a function of age assessed by sugar assimilation patterns Age group

Organism

% of isolates

A1

C. albicans

59.2

C. tropicalis

18.5

C. glabrata

7.4

C. krusei

7.4

C. parapsilosis

3.7

C. rugosa A2

3.7

C. albicans

48.4

C. parapsilosis

28.1

C. tropicalis

12.5

C. glabrata

6.2

P. ohmeri

3.2

C. krusei

1.6

*Group A1 £ 50-years-old (n = 27 individuals) Group A2 ‡ 51-years-old (n = 64 individuals)

genotypic clusters (I, II, III and IV) were derived at a threshold SAB of 80%. The cluster I isolates had approximately 80% similarity and were distributed into two groups IA and IB included 40 (85.0%) of the isolates. One of them (IB) had a coefficient of similarity of 90% containing 26 isolates, which comprised two subgroups with a high degree ([90%) of similarity between them. Both subgroups had a total of 26 isolates showed SAB values of 1.00, representing identical strains. The averages SAB of the hemodialysed and diabetic isolates were 0.828 ± 0.126 and 0.741 ± 0.191, respectively. Clusters II, III and IV included only seven strains of diabetic patients, with SAB £ 0.70, suggesting unrelated isolates (Fig. 1).

Discussion Yeast cells, especially Candida species, are common in oral cavities and in immunocompromised and immunocompetent individuals [20], with a predominance of C. albicans [1–3, 10, 21]. On the other hand, little is known about the microbiota of diabetic [22]. and hemodialysed individuals in Brazil. Published reports of the frequency of isolation of oral yeasts show considerable variation between similar sites and populations. A number of relevant factors have been associated with oral carriage of yeast in the

population and certain groups of individuals, with varying degrees of immunodeficiency, have an increased risk of being infected by Candida. The methodology also influences in the obtained data. The concentrated rinse culture was simple to perform, equally sensitive and superior in quantifying yeast carriage than the imprint culture technique and, it is suggested that this technique could be preferentially employed in investigations to obtain comparable data from different centers [14]. Therefore, some controversy still remains regarding the role of diabetes in the colonization of the oral cavity by yeasts [23]. On the other hand, hemodialysed patients are especially group of individuals who had ‘special needs’ and few studies have been developed to evaluate their oral microbiota. Specific factors (physiological, tegument continuity injury, nutritional factors, chemical and radiotherapy, surgical procedures and others, like poor oral hygiene) are associated with the increasing frequency of Candida species in the oral cavity [20, 24]. Previous studies [21, 22] have shown great variability in the number of patients whose oral cavities are colonized by yeasts. This may be due, at least in part, to the sampling method [20] and to the fact that the number of colony forming units per ml of patient saliva does not correlate with clinical evidence of oral candidiasis [20, 25]. This could be concluded from the present research, since the apparent amount of yeast (CFU/ml) among the diabetic (mean = 14,321.8 ± 1,587.4), hemodialysed (mean = 8,853.3 ± 2,871.2) and healthy patients (mean = 1,078.9 ± 208.5) has no relationship with oral mucosal disease. The high number of Candida colonies seen in diabetic and hemodialysed patients could reflect host-Candida species interaction(s) favorable to colonization. It is important to note that diabetics, particularly those who are prosthesis wearers have an increased burden of asymptomatic colonization of the oral cavity in comparison to their controls. We have also demonstrated that the presence of C albicans and the cohabitation of different Candida species was more frequent in denture wearers. Barbeau et al. [2], have pointed out the presence of yeast on dentures was significantly associated with the extent of the inflammation. On the other hand, our findings suggest that the presence of dentures influenced the oral yeast carriage of diabetic and hemodialysed patients.

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Dice (Opt:1.50%) (Tol 3.0%-3.0%) (H>0.0%S>0.0%) [0.0%-100.0%]

RAPD 100

90

80

70

60

RAPD 50

Fig. 1 UPGMA dendrogram generated from the similarity coefficient (SAB) which shows interindividual similarity. SABs were determined by DICE coefficients calculated for RAPD patterns of 47 isolates of C. albicans taken from 47 dental prosthesis users in three groups: diabetic (CD); hemodialysed (HD), and healthy group (HC). Vertical dashes line mark the position of SAB = 0.8, and subdivides the dendrogram into 2 large clusters. The cluster I isolates had approximately 80% similarity and were distributed into two subclusters IA (12 isolates) and IB (28 isolates). One of them (IB), containing 26 isolates, which comprised two subgroups with a high degree ([90%) of similarity between them. A high discriminatory power was found with primer 6, which produced up to 10 bands, as seen in RAPD patterns of isolates CD 131 and CD 130


CD104 CD116 HD49 HD40 HD31

IA

CD56 CD103 HD38 CD158 CD61 CD79 HD68 CD33 CD43 CD23 CD85 CD163 CD166 HD39

I

HD63 HD69 HD13 CD22 CD40 CD119 CD142 CD50 HD03 HD30 HD23 HD32 HD75 HD73 CD154 CD156 HC10 HC39

IB

HC50 CD86 HD59 CD131 CD130

II

CD45 CD94

III

CD108 CD24 CD30

IV

In agreement with previous studies [2, 24, 25] but not with that of Al-Karaawi et al. [25], the presence of C. albicans on the oral mucosa in 66.9% of the studied patients must be considered as a representative high prevalence of this microorganism in persons

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wearing dental prosthesis. Patients with either partial or complete dentures were more often colonized, in some studies, as well as, in our work, twice or three times as often, than patients without dentures [24]. This may be explained by the fact that in denture


wearers, the fitting surface of the denture limits masticator movements, and is covered by a layer of microbial plaque that candidal species can readily colonize; also, when dentures are not properly adapted, they induce to tissue maceration, causing low-intensity and long-lasting local traumatisms, which can reduce tissue resistance against infections, increasing the permeability of the epithelium to soluble candidal antigens and toxins [26–29]. The oral lesions among elderly people are frequent and commonly related to the use of dentures [30] and, the principal risk factors for colonization were a dental prosthesis, poor oral hygiene and the use of antibiotics [31, 32]. There is clear evidence that C. albicans adheres to oral surfaces including acrylic dentures and mucosa [3] and, frequently colonize the oral mucous of patients wearing dental prosthesis, as well as the prosthesis [2, 24, 32]. C. albicans is the most adapted species as a commensal within the oral niche [2, 22, 33]. The present results suggest that for this test population, the C. albicans carriage (61.7%; 87/141) is more frequent than that of other species. Even so, the percentage of carriage non-albicans species was high (36.4%; 50/137), which could be explained by the larger percentage (68.1%) of diabetic patients than other groups, since previous studies associated diabetic patients with oral carriage of a range of Candida species [34, 35]. Another factor that has influenced this result was age, since 70.3% (64/91) were ‡51years-old. The frequency of C. albicans in patients with increasing age decreases while non-C. albicans yeasts increases [36, 37]. Previous research [3, 12, 24, 36] suggests that intensity of carriers were greater among older individuals than in a lower age range (15–18 years-old). Besides that, 94.3% (133/141) were dental prosthesis users and in previous research, more diverse yeast species were isolated from the oral cavities of patients with dentures (either full or partial) than from dentate patients [27, 36–39] and, in the current study, the fact that selective media (that produce differential staining of candidal species and highlight morphological variations of colonies) were not used may have reduced the sensitivity of detection of that diversity. Understanding of species identification, commensalism and pathogenicity, person-to-person spread and the development of antifungal resistance within specific strains has been greatly enhanced by the

261

utilization of molecular epidemiological methodology [6–8, 21]. Thus, the aim of this study was to analyze the genetic similarity of C. albicans isolates associated with dental prosthesis in diabetic, hemodialysed patients, and healthy individuals and the prevalence of C. albicans colonization in these individuals. RAPD is already recognized as a valuable tool for studying genetic epidemiology Candida infections [9, 20, 40–43]. Reproducibility in the patterns obtained by RAPD-PCR was assessed by carrying out the experiments in duplicate; no significant differences in profiles were observed. The Ready-To-Go RAPD analysis beads used, resulted in reproducible and stable banding patterns. As the RAPD technique is simple, rapid and rather cheap, it is already recognized as a valuable tool for studying genetic epidemiology. Interestingly, most of strains could be placed into major cluster (I) with a similarity index SAB ‡ 0.80 (mean SAB = 0.779 ± 0.178), including diabetic, hemodialysed and control group. On the other hand, 88.9% of the isolates from the subgroup IB yielding RAPD profiles with high similarity ‡90%, demonstrating that these isolates are highly related. Among them, two types were found with strains of clonal origin. It has been shown that a single cluster of genetically related infection causing C. albicans isolates usually predominates in a given patient population in a given geographical locale [44, 45]. Alternatively it is possible that the predominant groups described in these studies are local representatives of one group, geographically widespread and causing infection in a variety of patient types. These strains, from different individuals, may perhaps adapt to the similar oral conditions in diverse individuals. These results are consistent with genetic relation studies of strains of C. albicans, in which different populations can have the same or similar genotypes [44, 45]. On the other hand, there is evidence that C. albicans is reproduced in a clonal way [46–48]. Organisms which reproduce clonally adapt to their environments if such niches are stable. Alternatively, when these microorganisms are exposed to frequent environmental changes they respond through microevolutionary selection based on their capacity to adapt to varied niches [48]. Thus, in our study, we noticed that 55.3% (26/47) of the individuals had highly similar but not identical strains in their oral

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cavities, which suggests that these strains could have adapted to the oral environmental conditions and are suffering microevolution. According to Chong et al. [18], an SAB value of 0.80–0.99 represents highly similar (but not identical) strains, suggesting that these infections were caused by a single strain that underwent microevolution. Some particular species present a selective advantage to specific locations in the human body [44, 49]. When analyzing the dendrogram, we noticed that over 85% of isolates had a similarity level higher than or equal to 0.8 reinforcing the idea that the use of dental prostheses probably provides the necessary conditions for these strains to gain a growth-specific advantage over others; this was also reported by Hossain et al. [21] and Song et al. [50]. These findings may be attributed to oral conditions specific to these groups of patients. Upto our knowledge, this is the first molecular epidemiological study from Brazil that characterize the genotypic distribution of C. albicans isolates, obtained from oral cavities of hemodialysed, diabetic, and healthy carriers who use dental prostheses.


7.

8. 9.

10.

11.

12.

13.

14. Acknowledgments This study was supported by CNPq (process 476595/2003). We are most grateful to Dr Anı´bal Moyse´s Simaõ Juńior, Dr Jose´ Vaner Pedigone and Dr Marco Antoˆnio Benedetti Filho and their teams for their contributions to this study.

References 1. Na¨rhi TO. Salivary yeasts, saliva and oral mucosa in the elderly. J Dent Res 1993;72:1009–14. 2. Barbeau J, Se´guin J, Goulet JP, Koninck L, Avon SL, Lalonde B, Rompre´ P, Deslauriers N. Reassessing the presence of Candida albicans in denture-related stomatitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:51–9. 3. Webb BC, Thomas CJ, Willcox MD, Harty DW, Knox KW. Candida-associated denture stomatitis. Aetiology and management: a review. Part1. Factors influencing distribution of Candida species in the oral cavity. Aust Dent J 1998;43:45–50. 4. Webb BC, Thomas CJ, Willcox MD, Harty DW, Knox KW. Candida-associated denture stomatitis. Aetiology and management: a review. Part 2. Oral diseases caused by Candida species. Aust Dent 1998;43:160–6. 5. Manfredi M, McCullough MJ, Al-Karaawi ZM, Vescovi P, Porter SR. In vitro evaluation of virulence attributes of Candida spp. Isolated from patients affected by diabetes mellitus. Oral Microbiol Immunol 2006;21:183–9. 6. Bostock A, Khattak MN, Mathews R, Burnie J. Comparison of PCR fingerprinting, by random amplification of

123

15. 16.

17.

18. 19.

20. 21.

22.

polymorphic DNA, with other molecular typing methods for Candida albicans. J Gen Microbiol 1993;139:2179–84. Lehmann PF, Lin D, Lasker BA. Genotypic identification and characterization of species and strains within the genus Candida by using random amplified polymorphic DNA. J Clin Microbiol 1992;30:3249–54. Soll DR. The ins and outs of DNA fingerprinting the infectious fungi. Clin Microbiol Rev 2000;13:332–70. Samaranayake YH, Samaranayake LP, Dassanayake RS, Yau JY, Tsang WK, Che BP, Yeung KW. ‘‘Genotypic shuffing’’ of sequential clones of Candida albicans in HIV infected individuals with and without symptomatic oral candidiasis. J Med Microbiol 2003;52:349–59. Xu J, Vilgays R, Mitchell TG. Lack of genetic differentiation between two geographic samples of Candida albicans isolated from patients infected with human immunodeficiency virus. J Bacteriol 1999;181:1369–73. Xu J, Boyd CM, Livingston E, Meyer W, Madden JF, Mitchell TG. Species and genotypic diversities and similarities of pathogenic yeasts colonizing women. J Clin Microbiol 1999;37:3835–43. Kleinegger CL, Lockhart SR, Vargas K, Soll DR. Frequency, intensity, species, and strains of oral Candida vary as a function of host age. J Clin Microbiol 1996;34: 2246–54. Schmid J, Rotman M, Reed B, Pierson CL, Soll DR. Genetic similarity of Candida albicans strains from vaginitis patients and their sexual partner. J Clin Microbiol 1993;31:39–46. Samaranayake LP, MacFarlane TW, Lamey PJ, Ferguson MM. A comparison of oral rinse and imprint sampling techniques for the detection of yeast, coliform and Staphylococcus aureus carriage in the oral cavity. J Oral Pathol 1986;15:386–8. Kurtzman CP, Fell JW. The yeasts: a taxonomic study. Amsterdam: Elsevier; 1998. Gales AC, Pfaller MA, Houston AK, Joly S, Sullivan DJ, Coleman DC, Soll DR. Identification of Candida dubliniensis based on temperature and utilization of xylose and amethyl-D-glucoside as determined with API 20C AUX and Vitek YBC Systems. J Clin Microbiol 1999;37:3804–8. Matsumoto MT, Fusco-Almeida AM, Baeza LC, Melhem Mde S, Mendes-Giannini MJ. Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from Sao Paulo State, Brazil. Rev Inst Med Trop Sao Paulo 2007;49:41–7. Zar JH. Biostatistical analysis, 4th ed. New Jersey: Prentice Hall; 1999. Chong PP, Lee YL, Tan BC, Ng KP. Genetic relatedness of Candida strains isolated from women with vaginal candididasis in Malaysia. J Med Microbiol 2003;52:657–66. Odds FC. Candida and Candidosis: a review and bibliography, 2nd ed. London: Ballie`re Tindall; 1988. Hossain H, Ansari F, Schulz-Weidner N, Wetzel WE, Chakraborty T, Domann E. Clonal identity of Candida albicans in the oral cavity and gastrointestinal tract of preschool children. Oral Microbiol Immunol 2003;18:302–8. Gonçalves RH, Miranda ET, Zaia JE, Giannini MJ. Species diversity of yeast in oral colonization of insulin-treated diabetes mellitus patients. Mycopathologia 2006;162: 83–89.

Mycopathologia (2007) 164:255–263 23. Soysa NS, Samaranayake LP, Ellepola AN. Diabetes mellitus as a contributory factor in oral candidosis. Diabet Med 2006;23:455–9. 24. Abu-Elteen KH, Abu-Alteen RM. The prevalence of Candida albicans populations in the mouth of complete denture wearers. New Microbiol 1998;21:41–8. 25. Al-Karaawi ZM, Manfredi M, Waugh ACW, McCullough MJ, Jorge J, Scully C, Porter SR. Molecular characterization of Candida spp isolated from the oral cavities of patients from diverse clinical settings. Oral Microbiol Immunol 2002;17:44–9. 26. Ghannoum MA, Abu-Elteen KH. Pathogenicity determinants of Candida. Mycoses 1990;6:265–282. 27. Budtz-Jo¨rgensen E, Stenderup A, Grabowski M. An epidemiologic study of yeasts in elderly denture wearers. Community Dent Oral Epidemiol 1975;3:115–8. 28. Coulter WA, Strawbridge JL, Clifford T. Denture induced changes in palatal plaque microflora. Microbiol Ecol Health Dis 1990;3:77–85. 29. Cannon RD. Colonization is a crucial factor in oral candidiasis. J Dent Educ 2001;65:785–7. 30. Triantos D. Intra-oral findings and general health conditions among institutionalized and non-institutionalized elderly in Greece. J Oral Pathol Med 2005;34:577–82. 31. Fanello S, Bouchara JP, Sauteron M, Delbos V, Parot E, Marot-Leblond A, Moalic E, Le Flohicc AM, Brangerd B. Predictive value of oral colonization by Candida yeasts for the onset of a nosocomial infection in elderly hospitalized patients. J Med Microbiol 2006;55:223–8. 32. Baena-Monroy T, Moreno-Maldonado V, Franco-Martinez F, Aldape-Barrios B, Quindos G, Sanchez-Vargas LO. Candida albicans, Staphylococcus aureus and Streptococcus mutans colonization in patients wearing dental prosthesis. Med Oral Pathol Oral Cir Bucal 2005;10:27–39. 33. Calderone RA. Candida and Candidiasis. Washington DC: ASM Press; 2002. 34. Aly FZ, Blackwell C, Mackenzie DA, Weir DM. Identification of oral yeast species isolated from individuals with diabetes mellitus. Mycoses 1995;38:107–10. 35. Manfredi M, Al-Karaawi Z, McCullough MJ, Hurel S, Porter SR. The isolation, identification and molecular analysis of Candida spp. isolated from the oral cavities of patients with diabetes mellitus. Oral Microbiol Immunol 2002;17:181–5. 36. Qi QG, Hu T, Zhou XD. Frequency, species and molecular characterization of oral Candida in hosts of different age in China. J Oral Pathol Med 2005;34:352–6. 37. Vandenbussche M, Swinne D. Yeasts oral carriage in denture wearers. Mykosen 1984;27:431–5. 38. Willis AM, Coulter WA, Sullivan DJ, Coleman DC, Hayes JR, Bell PM, Lamey PJ. Isolation of C. dubliniensis from insulin-using diabetes mellitus patients. J Oral Pathol Med 2000;29:86–90.

263 39. Torres SR, Peixoto CB, Caldas DM, Silva EB, Magalha˜es FA, Uzeda M, Nucci M. Clinical aspects of Candida species carriage in saliva of xerostomics subjects. Med Mycol 2003;41:411–5. 40. Clemons KV, Feroze F, Holmberg K, Stevens DA. Comparative analysis of genetic variability among Candida albicans isolates from different geographic locales by three genotypic methods. J Clin Microbiol 1997;35:1332–6. 41. Pujol C, Joly S, Lockhart S, Noel S, Tibayrenc M, Soll DR. Parity of MLEE, RAPD and Ca3 hybridization as fingerprinting methods for Candida albicans. J Clin Microbiol 1997;35:2348–58. 42. Boerlin P, Boerlin-Petzold F, Goudet J, Durussel C, Pagani JL, Chave JP, Bille J. Typing Candida albicans oral isolates from human immunodeficiency virus-infected patients by multilocus enzyme electrophoresis and DNA fingerprinting. J Clin Microbiol 1996;34:1235–48. 43. Metzgar D, Van Belkum A, Field D, Haubrich R, Willis C. Random amplification of polymorphic DNA and microsatellite genotyping of pre-and posttreatment isolates of Candida spp. From human immunodeficiency virus-infected patients on different fluconazole regimens. J Clin Microbiol 1998;36:2308–13. 44. Schmid J, Herd S, Hunter PR, Cannon RD, Yasin M, Salleh M, Samad S, Carr M, Parr D, McKinney W, Schousboe M, Harris B, Ikram R, Harris M, Restrepo A, Hoyos G, Singh KP. Evidence for a general-purpose genotype in Candida albicans, highly prevalent in multiple geographical regions, patient types and types of infection. Microbiology 1999;45:2405–13. 45. Xu J, Mitchell TG, Vilgalys R. PCR-RFLP analyses reveal both extensive clonality and local genetic differentiation in Candida albicans. Mol Ecol 1999;8:59–73. 46. Caugant DA, Sandven P. Epidemiological analysis of Candida albicans strains by multilocus enzyme electrophoresis. J Clin Microbiol 1993;31:215–20. 47. Pujol C, Reynes J, Renaud F, Raymond M, Tibayrenc M, Ayala FJ, Janbon F, Mallie´ M, Bastide JM. The yeast Candida albicans has a clonal mode of reproduction in a population of infected human immunodeficiency viruspositive patients. Proc Nat Acad Sci USA 1993;90:9456–9. 48. Tibayrenc M. Are Candida albicans natural populations subdivided? Trends Microbiol 1997;5:253–7. 49. Soll DR, Galask R, Schimd J, Hanna C, Mac K, Morrow B. Genetic dissimilarity of commensal strains of Candida spp carried in different anatomical locations of the same healty women. J Clin Microbiol 1991;29:1702–10. 50. Song X, Sun J, Store G, Eribe ERK, Hansen BF, Olsen I. Genotypic relatedness of yeasts in thrush and denture stomatitis. Oral Microbiol Immunol 2006;21:301–8.

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