|Year : 2014 | Volume
| Issue : 1 | Page : 45-51
To assess candidal colonization and species diversity in the oral cavity of diabetic and nondiabetic denture wearers and correlation with the presence of candida on finger tips of the patients: An in vivo study
Navjot Kaur Boparai1, Ulhas N Amasi1, Raghunath Patil1, Sheetal U Harakuni2
1 Departments of Prosthodontics and Crown and Bridge, Viswanath Katti Institute of Dental Sciences, Belgaum, Karnataka, India
2 Department of Microbiology, J.N. Medical College, KLE University's, Belgaum, Karnataka, India
|Date of Web Publication||2-Jul-2014|
Navjot Kaur Boparai
Departments of Prosthodontics and Crown and Bridge, KLE University's Viswanath Katti Institute of Dental Sciences, JNMC Campus, Nehru Nagar, Belgaum - 590 010, Karnataka
Source of Support: None, Conflict of Interest: None
Human oral cavity in health is normally colonized by different microbiological flora predominantly with Candida species. Wearing a removable denture and an immunologically deranged health is known to increase oral candidal colonization. The purpose of this study was to assess candidal colonization and species diversity in the oral cavity of diabetic and nondiabetic denture wearers and correlation with the presence of Candida on finger tips of the patients. 120 subjects in the age group of 40-70 years were included in the study. Three groups were formed, Group I: Diabetic, denture wearer, Group II: Nondiabetic denture wearer, Group III: Nondiabetic, nondenture wearer subjects. The oral samples from all the subjects were collected with oral rinse technique and were then inoculated on to the CHROMagar. For fingertip sampling, subjects placed their hands on sabouraud dextrose plate for 1 min and candidal growth was assessed and species were isolated using auxanometric plate method of sugar assimilation test. The mean number of colony forming unit (CFU) of Candida in Group I, Group II and Group III was 892.50, 590.00 and 152.50, respectively. Most commonly isolated species in diabetic denture wearer group were Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei, Candida kefyr, Candida gulliermondi and Candida parapsilosis. The mean number of colonies for fingertip contamination was more for diabetic denture wearers (1.50) as compared to nondiabetic denture wearers (1.00). In conclusion, candidal carriage (based on the CFU) was more in diabetic denture wearer patients than the nondiabetic denture wearers. C. krusei and C. kefyr were the two species of Candida found only in diabetic subjects. Furthermore, there was a correlation in the fingertip contamination and oral candidal colonization.
Keywords: Candida , CHROMagar, denture wearer, diabetic and nondiabetic, sabouraud agar
|How to cite this article:|
Boparai NK, Amasi UN, Patil R, Harakuni SU. To assess candidal colonization and species diversity in the oral cavity of diabetic and nondiabetic denture wearers and correlation with the presence of candida on finger tips of the patients: An in vivo study. Indian J Health Sci Biomed Res 2014;7:45-51
|How to cite this URL:|
Boparai NK, Amasi UN, Patil R, Harakuni SU. To assess candidal colonization and species diversity in the oral cavity of diabetic and nondiabetic denture wearers and correlation with the presence of candida on finger tips of the patients: An in vivo study. Indian J Health Sci Biomed Res [serial online] 2014 [cited 2019 Aug 24];7:45-51. Available from: http://www.ijournalhs.org/text.asp?2014/7/1/45/135041
| Introduction|| |
In healthy individuals, oral cavity harbors many different spectra of micro-organisms. Among them, Candida species is predominantly observed to be present (in 30-70%). If the human host defense system gets disturbed by diseases or conditions, which alter the immune status, Candida species become virulent and cause candidiasis, which manifests through various clinical forms, involving one or more oral sites and can even disseminate into invasive forms.
Candida species have the capacity to adhere to the oral mucosa because of different factors related to fungi (such as the transition from blastoconia to hyphae, the production of several extra cellular enzymes such as proteinases and phospholipases) and the oral microenvironment conditions (temperature, pH, carbohydrates concentration, etc.). Several oral bacteria such as Streptococcus sanguis, Streptococcus salivarius, Streptococcus mutans, Fusobacterium nucleatum and Actinomyces viscosus too promote the adhesion of yeasts to the oral epithelium. 
Candida species also have the affinity to adhere to, and subsequently colonize, the denture acrylic resin material.  Candida adheres directly or via a layer of denture plaque to denture base (polymethylmethacrylate). 
Diabetics also have an increased predisposition to the occurrence of candidiasis, which is associated with poor glycemic control and therapeutic dentures. This predisposition is also caused by xerostomia, which may be due to the increased glucose levels in oral fluids or immune dysregulation. 
Candida species have been shown to contaminate the toothbrush of oral Candida carriers through direct contact with saliva and to contaminate the hands of hospital personnel. Removable denture wearers frequently use their fingers to take their prostheses out of their mouths, either to cleanse or simply check them. Oral Candida, if present, may even contaminate the wearers' fingers. 
Furthermore, there is a change in the candidal species from being oral commensals to a pathogen.
Identification of Candida species is very important to understand the candidal pathogenicity and the related complications.
Taking the above mentioned literature into consideration, the aim of the present study was to assess the candidal colonization and species diversity in the oral cavity of diabetic and nondiabetic denture wearers and its correlation with the presence of Candida on the fingertips of the patients.
| Materials and Methods|| |
The study was carried out in the Department of Prosthodontics and Crown and Bridge, Vishwanath Katti Institute of Dental Sciences, Belgaum and Department of Microbilology, Jawaharlal Nehru Medical College, Karantaka Lingayat Education Society University, Belgaum. One hundred and twenty subjects in the age group of 40-70 years, attending to the Department of Prosthodontics and Crown and Bridge, KLE Vishwanath Katti Institute of Dental Sciences, Belgaum, were included in the study.
Three groups were formed, with 40 subjects (20 males and 20 females) in each group.
- Edentulous, denture wearer, noninsulin dependent diabetic subjects (fasting blood sugar [FBS] >130 mg/dL)
- Edentulous, denture wearer, nondiabetic subjects (FBS <110 mg/dL)
- Edentulous, nondenture wearer, nondiabetic subjects (FBS < 110 mg/dL).
The diabetic status of the patients was determined by the history of previous diagnosis of diabetes and their blood glucose levels were also determined before the sample collection using calorimetric method. The denture wearer patients selected were using the dentures since at least 1-year. All dentures were fabricated of heat cure acrylic resin.
The patients in the control group (Group III) were completely edentulous since 1-month at least and have not worn a denture earlier in their life. All the subjects in three groups were selected from the patients attending the Department of Prosthodontics, Vishwanath Katti Institute of Dental Sciences, KLE University Belgaum. The purpose and objectives of the study were explained to all subjects and the informed consent was also obtained from them in the vernacular language.
The study was approved by the ethical committee KLE University, Belgaum.
Information on demographics, medical variables, denture handling and denture wearing habits; and hand washing habits after denture handling was obtained using a questionnaire. Patients with history of smoking, any systemic disease, use of broad spectrum antibiotics, Corticosteroid therapy and anti-fungal therapy, antiseptic mouthwashes were excluded from the study.
Collection of sample
For microbiologic sampling from the finger tips, each subject in Group I and Group II was asked to press the fingertips of both of his/her hands on a plate of sabouraud dextrose agar (SDA) (HiMedia) for 1 min. This fingertip sampling was performed after seating the subject on the dental chair and before he/she touched his/her dentures [Figure 1]. 
Oral yeast colonization was assessed with the concentrated oral rinse technique as described by Samaranayake et al.  Subject was instructed not to eat and drink 2 h prior to the sample collection. Each individual was given 10 mL of sterile phosphate buffer saline solution (0.1 M solution with7.2 pH) and was asked to rinse mouth for 60 s with this solution. Then that mouth rinse was collected in sterile containers and was sent to the microbiological laboratory [Figure 2]. To prevent the circadian variation, samples were collected between 10 am to 11 am.
Oral rinse samples were then inoculated onto CHROMagar plates [Figure 3] and [Figure 4]. The chromogenic agar plates were prepared according to the manufacturer's instructions. The oral rinse sample was vertex mixed prior to plating. A drop of oral rinse sample was taken with 4 mm internal diameter inoculating loop. From this, the inoculum was spread in a line across the entire plate crossing the first inoculum's streak numerous times to produce isolated colonies. The plates were then incubated aerobically for 48 h at 37°C. The subject was designated as candidal carrier if there was any growth of candidal colonies. The number of colonies on each plate were counted after 48 h of incubation and the number of Colony forming unit/milliliter (CFU/mL) of oral rinse was derived from the formula: 
CFU/mL = 100 × number of colonies.
The different species of Candida were identified based on the color displayed by them on CHROMagar [Figure 5]. 
- Candida albicans = light green
- Candida glabrata = white pink, purple
- Candida tropicalis = dark blue to blue gray (with dark halo in agar)
- Candida gulliermondi = pale pink
- Candida kefyr = pink
- Candida krusei = pale pink, purple (rough with spongy pale edges)
- Candida parapsilosis = white, pale pink.
Saboraud dextrose agar plate in which finger tips were pressed, was incubated aerobically for 48 h at 37°C and candidal growth was assessed [Figure 6].
The suspected growth of Candida species was confirmed by Gram staining. Speciation of each isolate was performed by germ tube test, and morphology of growth on Corn meal agar. Confirmation of species was done by auxanometric plate method [Table 1] of sugar assimilation test [Figure 7]. 
| Results|| |
- The number of CFUs was higher in the diabetic denture wearer group when compared to the nondiabetic group, but it was not statistically significant (P = 0.0613)
- Significant difference was found in CFU count in diabetic denture wearer group and the control (P = 0.00001), and also in nondiabetic denture wearer group and the control (P = 0.0004) [Table 2]
- C. albicans was the most commonly isolated species in all the three groups. It was followed by common isolation of C. tropicalis (30%), C. glabarta (25%), C. krusei (15%), C. kefyr (10%), C. gulliermondi (7.50%) and C. parapsilosis (5%) in diabetic denture wearer subjects. The higher isolation and colonization of nonalbicans Candida species was seen in diabetic group when compared to nondiabetic group [Table 3]
- Comparison was made for the isolation of Candida colonies on the 4. SDA plates, using Mann-Whitney U-test. Number of colonies was found to be more for diabetic denture wearer group (1.50) when compared to nondiabetic denture wearer group (1.00) [Table 4]
- Only three species of Candida that is, C. albicans, C. glabrata and C. tropicalis, were isolated from the fingertip samples of the subjects. Though, significant difference was found in the occurrence of C. albicans (P = 0.0309) [Table 5]
- The Candida colony count on SDA varied according to the denture handling habits of the subjects. Of 80 subjects, 63 patients have the habit of handling their denture twice or thrice and 17 subjects handle their denture >3 times. Mean colony count in Group I for patients who handled their denture for 2-3 times and >3 times was 1.20 and 2.40, respectively. For Group II, it was 0.75 and 3.14, respectively.
|Table 2: Comparison of Group I, Group II and Group III with respect to Candida CFU counts by Kruskal-Wallis ANOVA-test|
Click here to view
|Table 3: Comparison of different Candida species on hi-chrome in Group I, Group II and Group III|
Click here to view
|Table 4: Comparison of Group I and Group II with respect to number of Candida colonies on SDA by Mann-Whitney U-test|
Click here to view
|Table 5: Comparison of Group I, Group II, and Group III with respect to different Candida species on SDA|
Click here to view
| Discussion|| |
Candida is present in the oral cavity of almost half of the population.  On CHROMagar the differentiation is done on the basis of strongly contrasted colony colors produced by reactions of species specific enzymes with a proprietary chromogenic substrate thus giving the presumptive identification of the yeast species. 
In this study, the number of CFU of Candida is found to be more in diabetic denture wearer subjects when compared to the nondiabetic denture wearer or control group. These results are similar to the few previous studies done evaluating the same factor. According to Daniluk et al.  also, the occurrence rate of oral C. albicans in patients with dentures was higher than in patients without dentures (P < 0.05). A study by Lotfi-Kamran et al.  also revealed that diabetes mellitus increased the colonization of Candida in denture and mouth.
The results of this study can be explained on the basis of two factors:
- Local: Presence of acrylic prosthesis
- Systemic: Diabetes.
Acrylic dentures act as a predisposing factor in the occurrence of oral candidal infection. The dentures can act as a reservoir of infection, because of the surface irregularities, micro porosities, improper fit and suboptimal hygiene.  Acrylic resins are pervious and exhibit more water sorption. This may help the candidial cells to adhere or to even penetrate the surface of acrylic resin. Surface irregularities of acrylic resin acts as a factor in the entrapment of microorganisms. Candida species possess surface free energy closer to that of polymethyl methacrylate. This makes possible for the yeasts to nest and make difficult to eliminate bacteria by mechanical and chemical hygiene maneuver. ,
In this study, the number of CFU of Candida was found to be more in diabetic subjects than the nondiabetic groups. These results are similar to the report by Tapper-Jones et al.  that stated the higher candidal density in diabetics. With multiple imprint culture technique, they have found that 60% of the diabetic group harboured C. albicans, which was similar to the carrier rate determined by the swabs and was considerably more than the value of 41% found by a mouthwash technique. The mean salivary flow rate in nondiabetic subjects was found to be 1.14 mL/min and 0.95 in diabetic subjects. Furthermore, the salivary buffering capacity was found to be 5.80 and 5.26 in nondiabetic and diabetic subjects respectively. 
Even the higher colonization of nonalbicans Candida species in diabetic group found in this study is supported by other studies. Martinez et al.  isolated Candida species in Type II Diabetics, and found that C. albicans, C. glabrata ad C. kruzei were most commonly present. These species are less susceptible to common anti-fungal drugs than C. albicans.
As found in this study, a significant proportion of denture wearer subjects positive for oral Candida also had the Candida colonization on their hands. This suggests that their hands may have been contaminated with yeast during denture handling. However it was found that one subject (nondiabetic, denture wearer, female) had Candida colonization on her finger tips though her oral cavity is found to be free of yeast. This suggests the presence of other sources of contamination. Furthermore, some of the subjects were positive for certain Candida species on the fingertips of their hands, that did not match the oral candidal species. In certain previous studies also, C. cryptohumicolus and C. rugose were isolated from the fingertips, but these species are not the common mycological commensals of the oral cavity.  Further studies are required to identify the Candida species to the strain level and to find out the other possible sources of fingertip contamination with a clinical significance.
This study results also showed that the patients who handled their denture >3 times a day had higher rate of candidal colonization when compared to the patients who handled their denture 2-3 times/day. This demonstrates an association between the poor oral hygiene and oral and fingertip colonization.
Most frequently used drugs against the Candida infection are ketoconazole, fluconazole, miconazole, polyenes (nystatin), imidazole derivatives (clotrimazole) and chlorhexidine mouth washes. The identification of multiple species of Candida has made the delineation of drug susceptibilities to Candida species or strains to anti-fungal drugs very important. Also, due to the identification of multi-azole-resistant strains of Candida. 
In this study, major attention was given to the maintenance of denture hygiene in denture wearer patients. To improve oral mucosal health, the participants were informed to control their blood glucose level and to regularly clean their dentures and keep them dry overnight. This is a simple and an efficient way to control yeast colonization in denture wearers. 
The importance of denture cleansing by the mechanical and chemical methods was demonstrated. The most commonly used mechanical methods are brushing with dentifrice or neutral soap and use of ultrasonic devices. The chemical methods involve denture immersion in chemical products, such as sodium perborate and sodium hypochlorite, hydrochloride, phosphoric and benzoic acids, chlorhexidine digluconate, and enzymes, such as proteases and mutanases. 
| Conclusion|| |
The following conclusions are drawn from the study:
- Candidal carriage and colonization (based on the CFU) was more in diabetic denture wearer patients than the nondiabetic denture wearers
- The common species of Candida isolated in diabetic denture wearer patients are: C. albicans (87.50%), C. tropicalis (30.0%), C. glabrata (25.0%), C. krusei (15%), C. kefyr (10%), C. gulliermondi (7.50%), and C. parapsilosis (5%)
- The common species of Candida isolated in nondiabetic denture wearer patients are: C. albicans (80.0%), C. tropicalis (17.0%), C. glabrata (22.0%), C. gulliermondi (5.0%), and C. parapsilosis (1%)
- C. krusei and C. kefyr are the two species of Candida found only in diabetic subjects
- The candidal colonization on the fingertips of the subjects was found to be more in diabetic denture wearer group as compared to nondiabetics. The commonly isolated Candida species from the finger tips were: C. albicans, C. glabrata and C. tropicalis
- The candidal colonization was found to be less in the subjects who handled their dentures for 2-3 times for insertion/removal than the subjects who handled their dentures >3 times
- The possible role of other means of fingertip contamination rather than the denture handling as in few subjects the fingertip candidal contamination was present even in the absence of oral infection. In few subjects, the species of Candida found on finger tips did not match their oral Candida species.
| References|| |
|1.||Darwazeh AM, Al-Refai S, Al-Mojaiwel S. Isolation of Candida species from the oral cavity and fingertips of complete denture wearers. J Prosthet Dent 2001;86:420-3. |
|2.||Pereira-Cenci T, Del Bel Cury AA, Crielaard W, Ten Cate JM. Development of Candida-associated denture stomatitis: New insights. J Appl Oral Sci 2008;16:86-94. |
|3.||Lotfi-Kamran MH, Jafari AA, Falah-Tafti A, Tavakoli E, Falahzadeh MH. Candida colonization on the denture of diabetic and non-diabetic patients. Dent Res J (Isfahan) 2009;6:23-7. |
|4.||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. |
|5.||Mackie and McCartney Practical Medical Microbiology. Vol. 2. |
|6.||Odds FC, Bernaerts R. CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species. J Clin Microbiol 1994;32:1923-9. |
|7.||Larone DH. Medically Important Fungi: A Guide to Identification. 5 th ed.; 2011. |
|8.||Radford DR, Sweet SP, Challacombe SJ, Walter JD. Adherence of Candida albicans to denture-base materials with different surface finishes. J Dent 1998;26:577-83. |
|9.||Beighton D, Ludford R, Clark DT, Brailsford SR, Pankhurst CL, Tinsley GF, et al. Use of CHROMagar Candida medium for isolation of yeasts from dental samples. J Clin Microbiol 1995;33:3025-7. |
|10.||Daniluk T, Tokajuk G, Stokowska W, Fiedoruk K, Sciepuk M, Zaremba ML, et al. Occurrence rate of oral Candida albicans in denture wearer patients. Adv Med Sci 2006;51 Suppl 1:77-80. |
|11.||Tapper-Jones LM, Aldred MJ, Walker DM, Hayes TM. Candidal infections and populations of Candida albicans in mouths of diabetics. J Clin Pathol 1981;34:706-11. |
|12.||Cristina de Lima D, Nakata GC, Balducci I, Almeida JD. Oral manifestations of diabetes mellitus in complete denture wearers. J Prosthet Dent 2008;99:60-5. |
|13.||Martinez RF, Jaimes-Aveldañez A, Hernández-Pérez F, Arenas R, Miguel GF. Oral Candida spp carriers: Its prevalence in patients with type 2 diabetes mellitus. An Bras Dermatol 2013;88:222-5. |
|14.||McElhaney-Feser GE, Raulli RE, Cihlar RL. Synergy of nitric oxide and azoles against Candida species in vitro. Antimicrob Agents Chemother 1998;42:2342-6. |
|15.||Pellizzaro D, Polyzois G, Machado AL, Giampaolo ET, Sanitá PV, Vergani CE. Effectiveness of mechanical brushing with different denture cleansing agents in reducing in vitro Candida albicans biofilm viability. Braz Dent J 2012;23:547-54. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]