CLINICAL ASPECTS OF CANDIDA SPECIES CARRIAGE IN SALIVA OF XEROTOMIC

Medical Mycology October

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Taylor&Francis healthsciences

a'

2003,4 1 , 41 1 4 15

Clinical aspects of Candida species carriage in saliva of xerotomic subjects S. R. TORRES*, C . B. PEIXOTO*, D. M . CALDAS*, E. B. SILVAT, F. A. C . MAGALHAES-J-, M. UZEDA-J-& M. NUCCI$ *Department of Oral Pathology and Oral Diagnostic, School of Dentistry Universidade Federal do Rio de janeiro; "fral Microbiology Laboratory Microbiology Institute, Universidade Federal do Rio de janeiro; $Department of Internal Medicine, University Hospital, Universidade Federal do Rio de janeiro, Brazil

In order to investigate the clinical factors that might influence the diversity and the degree of Candida species carriage in saliva, we conducted a cross-sectional study with 133 patients with complaints of xerostomia. Anamnesis, oral examination and collection of chewing-stimulated whole saliva were performed. The samples of saliva were kept refrigerated until they were plated onto CHROMagar candidag7'; cfu were counted and Candida species were identified by standard methods. There was a high prevalence of niixed Candida colonization. N o relationship was found between total Candida cfu counts and variables like gender, age, place of origin, underlying diseases, exposure to medications (except antibiotics), daily habits and salivary flow rates. Oral candidiasis, antibiotic exposure and dental prosthesis wearing were associated with relatively high Candida counts in saliva. Low salivary flow rates predisposed to intense colonization by C. albicans and C. parapsilosis. Keywords

Candida , colonization, hyposalivation, saliva, salivary flow rate

Introduction

Candidiasis is the most frequent fungal infection in the mouth. Factors that predispose to oral candidiasis are well documented [I], but little attention has been given to factors that influence Candida oral colonization. Xerostomia is a symptom characterized by a feeling of dry mouth [2,3]. It is a complaint associated with many systemic diseases [4-71, as well as a frequent drug side-effect [7-91. It may be caused by many factors, including hyposalivation [3,10], sialochemistry alterations [3] and changes in oral moisture perception [7,8]. When xerostomia is associated with hyposalivation, it may predispose to oral candidiasis [7,11]. Furthermore, other factors such as poor oral hygiene, denture wearing, oral epithelial dysplasia, smoking habits, orthodontic appliances, mouth breathing and chronic

irritants have been found to be local factors that predispose to candidiasis [I,121. Likewise, systemic conditions such as malignancy, endocrine diseases and immunodeficiencies, as well as exposure to antibiotics, corticosteroids and radiation therapy are risk factors for Candida infection [I]. Some authors [13,14] have suggested that high oral colonization predisposes to candidiasis, but most of these studies evaluated only colonization by Candida albicans. It has also been reported that local predisposing factors are associated with a greater diversity of Candida species colonizing the mouth [12]. The aim of this study was to investigate the clinical factors that might influence the diversity and the degree of Candida spp. carriage in saliva. Materials and methods

Received September 200 1; Accepted 23 September 2002 Correspondence: S. R. Torres, Rua Alinirante Gomes Pereira 130, apto. 202, Urca, Rio de Janeiro, Brazil CEP 22.291 -170. Tel.: + 55 21 2295 3687; Fax: f 5 5 21 2537 3432; E-mail: sandrator@ odonto.ufnj.br @ 2003 ISHAM

Downloaded from https://academic.oup.com/mmy/article-abstract/41/5/411/1005178 by guest on 05 June 2018

Six hundred and ninety-nine randomly selected patients from the University Hospital and the School of Dentistry of the Universidade Federal do Rio de Janeiro (SDIUFRJ) were invited to answer a questionnaire containing 10 questions regarding complaints of DOI: I 0.1 0801??????????????

Torres et a/.

xerostomia, and were further evaluated if they had answered 'yes' to at least one of the questions. The patients were evaluated by means of oral examination, anamnesis and saliva collection at the Oral Diagnosis Clinics (ODC) SDIUFRJ. Chewing-stimulated whole saliva [15,16] was collected in sterile test tubes for 5 min. The samples of saliva were kept refrigerated and taken to the Oral Microbiology Laboratory, UFRJ, within 2 h. The samples were heated at 55 "C for 2 min to release microorganisms from salivary clumps, and were homogenized in a vortex (Supermixer&, Lab-Line Instruments, Melrose Park, IL, USA). A 0.1-ml sample of pure and diluted saliva was plated onto CHROMagar ~andida' (Chromagar Company, Paris, France) in 90-mm diameter plates, and incubated at 37 "C for 72 h. Colonies were counted and yeasts were identified. Total and colony-color-specific cfu were counted after 72 h. One representative cfu of Candida of each color was isolated and identified on the basis of germ tube formation, chlamydospore formation in cornmeal agar, and growth at 37 "C and 45 "C on Sabouraud agar [17]. The identification of other Candida species was performed at the Mycology Laboratory, University Hospital, UFRJ, and was based on characteristic patterns of fermentation and assimilation of carbohydrates [18,19]. In order to analyze possible factors associated with colonization by Candida spp., we compared patients with and without colonization in regard to demographics, habits and clinical characteristics. Furthermore, we evaluated the association between these variables and the degree of colonization (median cfu counts). Clinical diagnosis of intraoral candidiasis was based on criteria published previously [20]. Patients with a diagnosis of candidiasis or other oral diseases were treated appropriately and were followed up at ODCI SDIUFRJ. For the comparison of dichotomous variables, Fischer test or chi-square test were used as appropriate. For continuous variables, we used the Student t-test or the Wilcoxon test. Results

Four hundred and ninety-eight patients answered 'yes' to at least one of the survey questions, but after screening, only 133 patients were inducted into the study and examined at ODCISDIUFRJ. The median age of these subjects was 51 years (range 14-81 years). There were 84 (63%) females and 49 (37%) males. Almost all patients (98%) had some source of underlying disease (Table 1) and 118 (89%) were taking

Table 1

Underlying diseases in 133 patients

Underlying diseases

Num ber

"/o

None Cancer Diabetes Collagen diseases Cardiovascular diseases Endocrille and metabolic diseases Respiratory diseases Neurological diseases HIV infectioii Gastrointestinal diseases Other The sum exceeds 133 because there were patients who had more than one underlying disease.

medications. Daily habits were noted in 57 subjects (43%). The most frequent habits were drinking coffee (38%) and smoking cigarettes (13%). The oral mucosa was altered in 39 patients (29%); the most frequent alteration was coated tongue (7%). Oral lesions consistent with the diagnosis of candidiasis were present in four cases (3%). Dental prosthesis wearing was registered in 60 patients (45%). The median chewingstimulated salivary flow of all 133 patients was 0.78 ml m i n ' (range 0.01-3.0 ml inin-'). There were 93 (70%) subjects colonized by Candida. The median cfu of Candida in saliva was 500 cfu ml(range 0-85 200 cfu ml-'). No correlation was observed between Candida counts in saliva and any specific underlying disease, or in regard to age, gender, place of birth, daily habits, oral mucous alterations, salivary flow rates or exposure to medication, except for the use of antibiotics. Antibiotic use showed a weak, marginally insignificant possible trend for cfu counts higher than those found in patients who were not exposed to antibiotics (median cfu counts 1780 cfu ml- vs. 370 cfu ml- l , P = 0.059). Patients with a clinical diagnosis of oral candidiasis had median Candida cfu counts in saliva significantly higher than those of patients without this finding (10 100 cfu ml-' vs. 400 cfu ml-', P=0.008). The yeast isolates obtained fi-om the four cases of oral candidiasis were C. albicans alone in two cases, and C. albicans plus C. krusei in two cases. Table 2 shows the clinical aspects, species identification and colony counts for these four patients. The median cfu count of Candida in the saliva of patients wearing dental prostheses was significantly higher than that of patients without prostheses (1065 cfu ml- vs. 30 cfu ml P = 0.02). There was no significant correlation between dental prosthesis wearing and colonization by multiple Candida species (P = 0.25).

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@ 2003 ISHAM, Medical Mycology 4 1 , 4 1 1 -415


Table 2 Clinical aspects, species identification and cfu counts for the four patients with candidiasis Patient Total cfu Species and counts cfu counts

Species and cfu counts

1

10 060

-

2

6480

3

10 140

4

30 000

C. albicans 10 060

Clinical aspects

Treatment

Erythematous candidiasis posterior dorsal tongue and hard palate C. albicans 6000 C. krusei 480 Erythematous candidiasis associated to dental prosthesis wearing C. albicans 10 130 C. krusei 10 Erythematous candidiasis posterior dorsal tongue C. albicans 30000 Pseudomembranous and erythematous candidiasis disseminated through oral mucosa -

-

-

Species identification was performed for samples from 64/93 colonized patients (Table 3). Thirty-four (53%) patients were colonized by a single Candida species, whereas 30 patients (47%) had mixed colonization (24 patients with colonization by two species and six patients with colonization by three species). The most commonly identified species were C. albicans (57 patients, 89%), C. parapsilosis (21 patients, 33%) and C. tropicalis (nine patients, 13%). Comparing the median Candida cfu counts of the three most common species we observed that cfu counts were higher for C. tropicalis (3200 cfu ml - l), than for C. albicans (2000 cfu ml - l ) and C. parapsilosis (420 cfu ml - I ) ( P = 0.001). Table 4 shows the median cfu counts of the Candida species. There was an inverse relationship between salivary flow rates and intensity of colonization by C. albicans and C. parapsilosis (P = 0.0002 and P = 0.01, respectively) (Table 5). Furthermore, there was a positive

Table 3 Species distribution of the 64 subjects who had Candidu species identified Carzdida species

Number

-

Topical iniconazole (14 days) Topical iniconazole (7 days) Topical miconazole (14 days) Topical miconazole (14 days)

relationship between colonization by C. albicans and female gender ( P = 0.02). Discussion

This study showed that 70% of the patients were colonized by Candida . The prevalence of yeast carriage seen in saliva in previous studies has ranged between 25% and 75% of the subjects, depending on the population of patients studied [21]. It seems unclear why some subjects are carriers of Candida and others are not, but some factors like nutrition, blood group, immune response and species phenotype have been cited in the literature as having some influence in Candida carriage [20,21]. The high prevalence of Candida colonization in this study may reflect the clinical status of the patients and the method for collecting the samples, both associated with high carriage rates [2 1,221. In the present study, we did not observe any relationship between total Candida cfu counts and variables like gender, age, place of origin, underlying disease, exposure to medications (except for antibiotics), daily

%I

albicans parapsilosis tropicalis albicalzs + C. parapsilosis albicalzs + C. tropicalis albicalzs + C. krusei albicans + C. glabrata albicans + C. guilliernio~zdii parapsilosis + C. tropicalis parapsilosis + C. krusei albicans + C. parapsilosis + C. tropicalis albicans + C. parapsilosis + C. krusei albicans + C. tropicalis + C. glabmta albicans + C. tropicalis C. krusei albicans + C. tiropicalis+ C. guilliemzoizdii albicans + C. tropicalis C. norcegensis

+ +

@ 2003 ISHAM, Medical Mycology 4 1 , 4 1 1 -4 15


Table 4 Candida cfu counts in saliva of the 64 patients with Cmdida species identified Candida sp.

Number*

57 C. albicans 21 C. parapsilosist 9 C. tropicalis:: 6 C. krusei 3 C. glabrata C. guillier~zolzdii 3 1 C. norvegelzsis

Median, cfu ml 2000 420 3200 235 400 100 2240

-

'

Range, cfu ml -

'

10-85 200 10-7200 20-17 800 10-500 100-4200 40- 110 2240

*The sum exceeds 64 because there were 30 patients (47%))with mixed colonization. ?P = 0.001 comparing C. parapsilosis with C. albicans and C. tropicalis. I P = 0.6 comparing C. albicans with C. tropicalis.

Torres et a/.

Table 5

Salivary flow rates (ml inin-

I)

and Ca~zdiducfu counts (cfu inl-

Species

Median salivary flow rates

C. alhicans C. parapsilosis C. tropicalis

0.6 0.47 0.7

I)

of the three most frequent species, and P value for this relationship

Salivary flow rates range

habits and salivary flow rates. As expected, patients with oral candidiasis had significantly higher median cfu counts in saliva than did patients without candidiasis. It has been suggested that smoking significantly increases carriage of Candida in the mouth [22]. However, some studies [20] do not show such correlation. In the present study, we did not observe any correlation between smoking and Candida carriage or Candida cfu counts. Similar to other studies [23], our study showed a correlation between wearing a dental prosthesis and high intensity of Candida colonization. Regarding the species colonizing patients with dental prosthesis, Beighton et al. [24] found a high frequency of mixed colonization in denture wearers, as well as in patients with denture stomatitis. We did not find a significant correlation between dental prosthesis wearing and single vs. mixed colonization. In another study [25], dental prosthesis wearing was shown to be a predisposing factor for colonization by C. glabrata. As our study included only three patients colonized by C. glabrata, we were not able to demonstrate such a relationship. None of the most common species found in the present study was statistically associated with the wearing of a dental prosthesis. Jorge et al. [12] found a great diversity of Candida species in individuals with local predisposing factors, as compared to controls. We observed a high frequency of mixed colonization by Candida species (47%) in our subjects, possibly because of the characteristics of the population studied. Stenderup [21] found a greater diversity of species in hospital patients than in healthy people. Other studies [24-261 using the chromogenic medium CHROMagar showed rates of mixed colonization varying between 26.3% and 60%. Therefore, not only the population studied, but also the method for processing the samples may influence the frequency of detection of mixed colonization. As expected, C. albicans was the most frequent species identified. It was found either as single or mixed colonization in 89% of the 64 patients who had Candida species identified. We found a correlation between female gender and high C. albicans cfu counts, but other species did not

Median Cundida cfu

Candida cfu range

P value

show this trend. The data of Arendorf & Walker [22] also appeared to show higher C. albicam carriage in females than males, but on statistical analysis, the difference was not significant. We do not have a clear explanation for this discrepancy with our study. Saliva has antimicrobial capacity and low salivary flow rates have been related to greater C. albicans cfu counts [14]. In the present study, we found a relationship between low salivary flow rates and high cfu counts of C. albicans and C. parapsilosis. Thirty-three per cent of the patients were colonized by C. parapsilosis (single or mixed), a prevalence much higher than that observed in other studies (0.4-8.3%) [27]. It is possible that the actual prevalence of C. parapsilosis colonization may have been underestimated in previous studies, because the chromogenic medium CHROMagar Candida' was not used for cfu counting in those studies, as it was in ours. However, in studies that used the same methodology as we did, the frequency of C. parapsilosis colonization ranged from zero to 55% [24-26,281. As we observed a correlation between low salivary flow rates and high C. parapsilosis carriage, it is possible that salivary flow rate is a significant variable determining the degree or frequency of C. parapsilosis colonization. This study has some limitations. First, as we did not investigate healthy individuals, our data cannot be extrapolated to the whole population. Furthermore, because not all isolates were available for species identification, it was not possible to study species characteristics in a larger sample size. Clinicians must be aware of clinical factors that influence the frequency and the diversity of Candida spp. in the saliva. Further studies must be performed in order to compare Candida species carriage in healthy individuals with that of patients with specific underlying diseases. Moreover, the significance of elevated cfu levels and increased species diversity in oral Candida carriage must be investigated in relation to the development of candidiasis.

We thank Tiyomi Akiti, Maria da Gloria Carvalho Barreiros and Viviane Souza Pinto for laboratory @ 2003 ISHAM, Medical Mycology 4 1 , 41 1 -41 5


Candida carriage In saliva

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CLINICAL ASPECTS OF CANDIDA SPECIES CARRIAGE IN SALIVA OF XEROTOMIC

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