Detection and sampling methods for isolation of Candida spp . from oral cavities in diabetics and non-diabetics

The purpose of this study was to detect Candida spp. on the tongue and in the subgingival sites in healthy and type 2 diabetes (T2D) patients with chronic periodontitis (CP), and to compare the accuracy of sampling methods. This study included 131 patients divided into four groups: healthy control (group A), nondiabetics + CP (Group B), diabetics with good metabolic control + CP (group C) and diabetics with poor glycoregulation + CP (Group D). Cotton swab samples from tongue and subgingival samples were obtained from each patient with help of sterile paper points and a sterile curette. Swab cultures were made on Sabouraud dextrose agar. The number of CFUs was counted. The sampling methods for subgingival plaque were compared by Receiving Operator Curve (ROC). The presence of Candida spp. on the tongue was statistically significant among groups (group D vs. others three groups: χ2: p < 0.005 for each group). Positive findings of subgingival Candida spp. did not differ among the groups. There were no significant differences in the quantification of Candida spp., neither on the tongue, nor in the subgingival samples. 17.2% of diabetic patients revealed the presence of Candida spp. in the subgingival samples, with negative finding on tongue. There was a significant difference in the sampling methods for subgingival plaque (p = 0.000). Candida spp. is more prevalent on the tongue of diabetics. The sampling of subgingival plaque by a sterile curette is more accurate than with paper points. Subgingival plaque may represent a reservoir of commensals. It is necessary to standardize the sampling of subgingival plaque.


Introduction
Type 2 diabetes mellitus (T2D) is a chronic metabolic disorder that leads to progressive defects in insulin secretion based on insulin resistance. 1 For thirty years periodontitis has been acknowledged as the sixth chronic complication of diabetes. 2,3Candida infections are chronic opportunistic infections related to diabetic patients.The presence of Candida spp. in oral cavities of diabetics varies between 50-80%. 4,5,6,7Yeasts commonly inhabit tongue, palate and buccal mucosa, and it has recently been found in the subgingival sites. 8The periodontium may represent a reservoir of opportunistic microorganism, especially in immunocompromised patients. 9The presence of yeasts in subgingival sites was examined in relationship to general health and periodontal status.It varies between 30-50% in the case of diabetics. 4,7,10Yeasts 11 and viruses 12 could have a significant role in the pathogenesis of periodontal diseases.The immunological response around hyphae of Candida spp. is similar to the response to periopathogens of bacterial origin, and consists of chronic mononuclear inflammatory cells with sporadic neutrophil leucocytes. 11The potential role of yeasts in the pathogenesis of periodontitis is especially important for diabetic patients, because antibiotics are commonly used in the treatment of periodontitis.
The purpose of this study was to detect Candida spp. on the tongue and in subgingival sites in healthy and T2D patients with chronic periodontitis (CP), and to compare the accuracy of sampling methods.

Inclusion and exclusion criteria
The exclusion criteria were: presence of any disease except T2D, aggressive periodontitis, usage of medication that might affect the periodontium, e.g.corticosteroids, antibiotics, antiseptics, history of oral candidiosis treatment, pregnancy, lactation and periodontal treatment in the last 1.5 year.

Anamnesis data and biochemical/hematological analysis
Self-reported information about blood type, everyday intake of sweets and smoking habits were recorded.According to the blood type, patients were divided into O vs. A+B+AB blood type. 13Patients were classified according to their smoking status as "non-smokers" and "smokers".Fasting plasma glucose levels (FPG), glycated hemoglobin (HbA 1 c), hematological parameters (RBC, Hgb, HCT, MCV, MCH, MCHC, RDW) and sedimentation rate were measured.

Diagnosis of CP and T2D
T2D was diagnosed according to the criteria of the American Diabetes Association 1 by measuring glycaemia during 2 h 75 g oral glucose tolerance test (OGTT), as well as HbA 1 c values.Nondiabetics exhibited normal parameters on OGTT and HbA 1 c < 6.5%.Glycoregulation was classified, according to HbA 1 c, as satisfactory (HbA 1 c ≤ 7.5%) and as with poor metabolic control (HbA 1 c > 7.5%).
Full mouth clinical examinations were performed at six sites per tooth and evaluated on each tooth in order to access periodontal parameters: plaque index-Silness Loe (PI), dichotomous bleeding on probing (BOP), probing pocket depth (PPD) and clinical attachment loss (CAL).Two calibrated doctors performed examinations using periodontal probe (XP 23/UNC-15, Hu-Friedy, Chicago, USA).Periodontitis was diagnosed if subject exhibited CAL > 1 mm and PPD > 3 mm at least at three sites in two quadrants. 14,15Patients without clinical signs of periodontitis exhibited PPD < 3 mm and CAL = 0 mm.

Sample collection and cultivation
Samples were collected a day after periodontal examination.Oral swabs were collected by swabbing ten times from the dorsum of the tongue with the help of a dry sterile cotton stick.Swab cultures were immediately inoculated on Sabouraud dextrose agar (SDA) (Oxoid, Basingstoke, UK).The tooth with deepest PPD was isolated by means of sterile cotton rolls and the supragingival plaque was removed by using sterile gauze and a curette.Two sterile paper points with a size of 30 were placed into the pocket for 30s until a mild resistance appeaed.Paper points contaminated by blood were not included in the analysis.Subgingival samples were obtained from the same pocket by means of a sterile curette (S4L/4R SS G.Hartzell&Son, Concord, California).Both subgingival samples were inoculated in sterile plastic tubes containing 1 mL of Sabouraud dextrose broth (Oxoid, Basingstoke, UK).The plastic tubes were vortexed for 1 min and 20ml of suspended broth was streaked on SDA.Samples were inoculated at 37°C for 48 h.The cultural and microscopic qualities of the yeasts were examined, and the germ-tube production test, as well as the carbohydrate and potassium nitrate assimilation tests, were performed when needed.After incubation, one calibrated microbiologist counted the growth density and number of Colony Forming Units (CFUs).The yeast growth density from tongue samples was defined as rare, medium or dense.The number of CFUs was measured for samples taken by means of paper points.Depending on the CFU/ml there were defined three groups: 1: < 500, 2: 500 -2500 and 3: > 2500.

Statistical analysis
Statistical analyses were carried out by using SPSS 18.0 software package for Windows (SPSS inc., Chicago, USA) and MedCalc for Windows, version 13.3.30(MedCalc Software, Mariakerke, Belgium) for the Receiving Operators Curve (ROC) analysis.
Descriptive data were presented as Mean ± SD or the percentage for discrete measures.t-test and One Way ANOVA were used for normally distributed data.Non-parametric data were analyzed using by using the Kruskall-Wallis and Mann-Whitney test.Categorical variables were compared using the Chi Square Test (χ 2 ).The relationship between CFU and clinical parameters was determined by Spearman's correlation coefficient.The linear regression model was used to determine predictors of the presence of Candida spp.ROC analysis was carried out in order to compare sampling methods for subgingival plaque collection for isolation of Candida spp.Differences were considered significant when p-value was < 0.05.

Results
Demographic, clinical and biochemical data are presented in Table 1.Groups were matched by age, gender and smoking status.
Differences between groups C and D were observed for HbA1c (Mann-Whitney, p = 0.000) and FPG (Mann-Whitney, p = 0.000).Hematological parameters did not differ between groups.Groups were matched according to their blood type (χ 2 , p = 0.541).
PI differed between groups (Table 2).BOP was different between group A and other groups (Bonferroni: B vs. C = B vs. D = C vs. D: p = 1.000).PPD did not differ significantly between groups B, C and D (Bonferroni: B vs. C = B vs. D = C vs. D, p = 1.000).
The quantification of Candida spp. on the tongue did not differ between groups.The univariate logistic regression model was applied, in order to identify parameters that could predict positive finding of Candida spp. on tongue.Age, gender, blood type, everyday intake of sugars, smoking habits, number of teeth, diabetes duration, treatment mode, FPG level, HbA1c, RBC, Hgb, MCV, HCT, MCH, MCHC and RDW were analyzed as potential predictors.There was found no predictor for the positive finding of yeasts on tongue.
There was no relation between presence of subgingival Candida spp.and clinical periodontal parameters.The quantification of subgingival Candida spp. was not different between groups.
In the case of diabetic patients, there was a positive correlation between the presence of subgingival Candida spp.and HbA1c (Spearman correlation coefficient, r = 0.276, p = 0.025).
Logistic regression analysis did not identify any parameter that could predict the presence of Candida spp. in subgingival samples.
18/131 (12.9%) patients presented negative Candida spp.findings on the tongue and positive findings in subgingival samples.
There was a statistical difference regarding sampling methods for subgingival plaque collection and yeast detection (Table 3).The ROC curve was used to compare the diagnostic techniques of both collecting methods.The referent sampling method was by means of a sterile curette.There was a difference between methods (p = 0.000).Sensitivity was 0.576 and specificity was 0.919.The area under the curve was 0.747.Asymptomatic 95% Confidence Interval was 0.638-0.857(Figure 2).

Discussion
The proposed microbiological etiologies of the periodontal disease have been changing for decades.There is increasing evidence about the involvement of microorganisms other than bacteria (e.g.viruses 11 and yeasts 8,12 ) in the pathogenesis of periodontal disease.
Candida spp. is a common oral saprophyte.Yeast may form biofilm, which is an essential strategy for their survival in oral milieu. 16,15Beside biofilm formation, this genus is able to produce exoenzimes, proteinases and metabolites in order to adhere to epithelial cells and inhibit the function of polymorphonuclears. 17,18,19t can be isolated in about 50% of healthy population without clinical signs of infection. 13In the case of diabetics, this prevalence is higher.
The prevalence and quantification of Candida spp. on tongue and subgingival samples were examined in diabetic and non-diabetic patients.To the best of our knowledge, this is the first study that examined patients with a clinically healthy periodontium, subjects diagnosed with periodontitis and diabetics.In our study, the overall prevalence of Candida on the tongue was 27.3%.The most frequent finding was in a group of poorly controlled diabetics.Differences in the prevalence of Candida spp. on tongue in the case of diabetic patients is in accordance with other studies, 6,20,21 but the percentage (37.9%)was lower compared to other studies, where findings of Candida on tongue varied between 59-77%. 4,5,7The quantification of yeast growth on tongue did not differ between groups, which is contrary to other studies 6,22 probably because denture wearers were included and sampling methods were different.
Candida spp. was a common ly occurring microorganism in samples from subgingival sites.Statistical differences of yast presence were not observed between groups or between diabetics vs. non-diabetics.Sardi et al. 20 found differences in subgingival findings of yeasts between well-controlled insulin dependent T2D patients and control group of healthy CP patients.When comparing the prevalence of subgingival positive findings in good vs. poorly controlled diabetics, Melton et al. 4 found no significant differences.Studies examining the prevalence of subgingival yeasts in healthy patients according to their periodontal status, demonstrated the impact of the periodontal probing depth on the presence of subgingival yeasts 8 which was not the case in our study.Candida spp. is an opportunistic pathogen, and it is considered as marker of immunocompromised patients.Periodontitis itself has been recognized as a state of disturbed cellular and humoral immune local response 23 and patients with diagnosed diabetes are also considered immunocompromised.Our results, which show a similar prevalence of subgingival yeasts in healthy patients and healthy patients with diagnosed CP, are in contrast with these facts.We examined only the presence of yeasts which does not always lead to clinical infection.Some authors indicate that the presence of yeasts in subgingival sites is transient. 24The reaction of host immunity around yeasts was not a subject of investigation or the exact species of Candida genus.Candida spp. is capable of adhering to epithelial cells and inducing inflammation. 25here is an increasing number of studies investigating subgingival prevalence of Candida spp.Some studies used a sterile curette as sampling method, 7,24 while others used sterile paper points. 8,9To the best of our knowledge, there is no explanation in any of these studies about the choice of the sampling method.In an attempt to answer this question, we used both methods in the same pocket and ROC analysis was carried out.A sterile   curette method was defined as the "golden standard".Specificity, which represents true negative results, was excellent (0.919) but sensitivity, which represents the probability that test results will be positive when the disease is present, was 0.576.The area under the curve shows a fair accuracy of the test.According to this ROC analysis, it may be concluded that subgingival plaque sampling by means of a sterile curette is more accurate than sampling by means of sterile paper points.This is in agreement with the fact that Candida spp.forms its colonies on the surface of the epithelial cells 26 , i.e. it is necessary to "scratch" with the help of a curette in order to ensure the accuracy of the results.On the other hand, sampling by sterile paper points is more appropriate if it is necessary to quantify Candida spp.Sampling with paper point can be standardized in terms of paper point size, duration of the presence of the paper point in the pocket and paper point pressure in the pocket.In different studies, there are differences in paper point size and insertion duration when sampling the subgingival plaque.Considering that numerous studies use paper points as sampling method, methodology should be standardized in order to compare results.It has already been proven that subgingival sites may be a reservoir of Candida spp. 9 In our study 12.9% of all patients harbored yeast in subgingival sites with no presence on tongue.The subgingival area is beneficial for Candida growth. 16e detection of yeast prevalence and their quantification, as well as the recognition of different species, virulence factors and drug resistance in subgingival biofilm are important because of the emerging usage of antibiotics as adjuvant periodontitis therapy.The usage of a broad spectrum antibiotics may lead to Candida opportunistic infections and periodontal destruction.Some authors indicate that the presence of yeasts in the subgingival area is transient, 24 i.e. the study to be carried out should be rather longitudinal than cross-sectional.

Conclusions
Candida spp. is more prevalent on tongue in the case of diabetics than in the healthy control group, regardless of periodontal status.In addition to that, diabetics with poor glycoregulation exhibited more yeast than patients with good metabolic control.The subgingival area may represent reservoir of commensals.However, longitudinal studies are needed to confirm these results.Correspondingly, it is necessary to standardize sampling methods for the collection of subgingival plaque.

Figure 2 .
Figure 2. ROC curve for sampling methods of subgingival plaque by sterile curette and by sterile paper points.

Table 1 .
Demographic and biochemical data of patients.
Positive finding of candida spp. in subgingival samplesPositive finding of candida spp. on tongue Figure 1.Presence of Candida spp. on tongue and in subgingival samples.

Table 3 .
Comparison of sampling methods for subgingival plaque collection.