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| Year : 2012 | Volume
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| Issue : 4 | Page : 490-497 |
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| Comparative study of Candida by conventional and CHROMagar method in non-denture and denture wearers by oral rinse technique |
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Shruti Nayak1, B Kavitha2, G Sriram3, TR Saraswathi4, B Sivapathasundharam2, AL Dorothy5
1 Department of Oral Pathology and Microbiology, Yenepoya Dental College, Yenepoya University, Deralakatte, Mangalore, India
2 Department of Oral Pathology and Microbiology, Meenakshi Ammal Dental College, Maher University, Alapakkam Main Road, Madhuravoyal, Chennai, India
3 Faculty of Dental Sciences, National University of Singapore, Singapore
4 Department of Oral Pathology and Microbiology, Vishnu Dental College, Bhimavaram, Andhra Pradesh, India
5 Department of Microbiology Meenakshi Ammal Dental College, Maher University, Alapakkam Main Road, Madhuravoyal, Chennai, India
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| Date of Web Publication | 20-Dec-2012 |
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 Abstract | | |
Introduction: Candidal species colonizes the oral cavities of healthy individuals without dentures and also of denture wearers. Soft liners and tissue conditioning materials have been found to support the growth of Candida albicans which may predispose to lesions. The most important and common candidal species are C. albicans, C. tropicalis, and C. glabrata. C albicans is usually isolated from both the fitting surface of the denture and the denture-bearing mucosa of the affected patients. The aim of this study was to isolate, quantify, and speciate candidal species in non-denture wearers (controls) and denture wearers (study group) by the oral rinse technique. Isolation was done using Sabouraud dextrose agar (SDA). Speciation was done using conventional methods like the germ tube test, carbohydrate fermentation test, urease test, as well as the CHROMagar method.
Aims and Objective: 1) To assess the prevalence of Candida in non-denture wearers and in denture wearers by oral rinse technique, with isolation on SDA; 2) to speciate and quantify Candida in non-denture wearers and denture wearers by using conventional methods (germ tube test, carbohydrate fermentation test, urease test) and the CHROMagar method; 3) to assess the influence of smoking and diabetes on candidal species among the denture wearers; and 4) to assess the sensitivity and specificity of SDA and CHRO Magar
Materials and Methods: Salivary samples for Candida evaluation were collected from the subjects in sterile sample containers, using the oral rinse technique.
Results: C glabrata was the most commonly found species among denture wearers and non-denture wearers both by conventional and CHROMagar methods. In males, C. albicans was the predominant species, whereas C. glabrata was the predominant species in females. Candidal colonization was higher in denture wearers compared to non-denture wearers, especially among females. The CHROMagar method was more rapid compared to conventional methods. In the present study, CHROMagar Candida showed 100% specificity and 100% sensitivity when compared to SDA and conventional methods. Keywords: Candida , CHROMagar, conventional, denture, species
How to cite this article:
Nayak S, Kavitha B, Sriram G, Saraswathi T R, Sivapathasundharam B, Dorothy A L. Comparative study of Candida by conventional and CHROMagar method in non-denture and denture wearers by oral rinse technique. Indian J Dent Res 2012;23:490-7 |
How to cite this URL:
Nayak S, Kavitha B, Sriram G, Saraswathi T R, Sivapathasundharam B, Dorothy A L. Comparative study of Candida by conventional and CHROMagar method in non-denture and denture wearers by oral rinse technique. Indian J Dent Res [serial online] 2012 [cited 2023 Mar 21];23:490-7. Available from: https://www.ijdr.in/text.asp?2012/23/4/490/104956 |
The fungi are a diverse group of saprophytic and parasitic eukaryotic organisms. [1] The biological kingdom of the fungi is composed of approximately 70000 known species of fungi, characterized by pronounced differences in structure, physiology, and modes of reproduction. About 300 species of fungi have been implicated directly as agents of human or animal disease, with less than a dozen of these species causing about 90% of all fungal infections. [2]
The most important oral yeasts belong to the genus Candida. Candida albicans is the predominant yeast species, followed by Candida glabrata, Candida krusei, Candida tropicalis, Candida guilliermondii, Candida kefyr, and Candida parapsilosis. [3],[4] Candida species are normal commensals present in a large percentage of healthy individuals. [5] Candida albicans is a common organism in denture wearers. The germ tube test is used for identification of C. albicans and differentiation from other Candida species. Its limitations have promoted the search for alternatives. C albicans can be rapidly and accurately identified by using fluorgenic and colorimetric enzyme substrates after primary isolation or directly on differential isolation plates (CHROMAgar Candida or Chromalbicans agar). [6],[7]
The medium most widely used for the isolation of Candida and other yeast species from clinical specimens is Sabouraud dextrose/glucose agar, [8] a general-purpose medium that supports the growth of most pathogenic fungi. A new commercial product CHROMagar Candida is now available that can be used for the isolation and presumptive identification of C. albicans, C. krusei, and C. tropicalis. This medium greatly facilitates the detection of specimens containing mixtures of yeast species. [9] Acrylic dentures are important predisposing factors for oral candidiasis. When they are ill fitting and there is suboptimal hygiene they act as reservoirs of infection. High salivary yeast counts are more common in denture wearers than in dentate individuals [10]
| Aims and Objectives | |  |
- To assess the prevalence of Candida by oral rinse technique, with isolation on Sabouraud dextrose agar (SDA) and CHROMagar, in non-denture wearers and in denture wearers.
- To speciate and quantify Candida in non-denture wearers and denture wearers by using conventional methods (germ tube test, carbohydrate fermentation test, urease test) and the CHROMagar method.
- To assess the influence of smoking and diabetes on candidal species among denture wearers.
- To assess the sensitivity and specificity of SDA and CHROMagar
| Materials and Methods | | |
The present study was performed in the microbiology laboratory of the Meenakshi Ammal Dental College and Hospital, Chennai. Subjects were enrolled from among the patients attending the departments of Prosthodontics and Oral Medicine, Meenakshi Ammal Dental College and Hospital, Chennai. In all, 100 subjects were enrolled for the study. Among these, 60 had dentures (males: 30, females: 30) and 40 (males: 20, females: 20) were without dentures and had normal, clinically healthy, mouths and no other systemic diseases or habits; the latter formed the control group. The ages ranged from 30 to 70 years. Patients with ill-fitting or broken dentures, who had come for replacement of the dentures, were also included in the study group.
Procedure
Subjects were given 10 ml of phosphate-buffered saline (PBS; 0.1M, pH 7.2) and were requested to rinse the mouth thoroughly for 60 s. The rinses were collected in a sterile sample container. From this, 100 μl of sample was transferred to 1 ml of Sabouraud broth and incubated overnight at 37°C. 100 μl of sample from 1: 10 dilution was plated and evenly spread using an 'L' rod on both SDA (supplemented by chloramphenicol capsule) and CHROMagar plates simultaneously. The plates were then incubated overnight at 37°C. After incubation, the colonies were counted and quantified by counting the colony-forming units (cfu) [Figure 1]. Speciation of Candida was done by the germ tube test, urease test, and carbohydrate fermentation test, as well as by the CHROMagar method.
| Results | | |
In this study, a total of 100 subjects were included, of whom 40 were non-denture wearers and 60 were denture wearers. The subjects included both males and females between the ages of 30 and 70 years. Among the 60 denture wearers, 33 (20 males and 13 females) were complete denture wearers. Among the 27 RPD (removable partial dentures) wearers, 10 were males and 17 were females [Table 1].
The prevalence of Candida among denture wearers and non-denture wearers was assessed using the presence/absence of growth on SDA and CHROMagar method [Table 2]. Out of the 60 denture wearers, 95% (n=57) showed candidal growth both in SDA and CHROMagar. Similarly, out of 40 non-denture wearers, 75% (n=30) showed candidal growth both in SDA and CHROMagar. This greater prevalence of Candida in the denture wearers was statistically significant (P=0.004). | Table 2: Prevalence of Candida among all denture wearers and non-denture wearers
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Speciation of Candida by conventional method using the germ tube test, urease test, and carbohydrate fermentation test was done. Among the denture wearers and non-denture wearers, the various species seen in order of decreasing frequency were C. glabrata, C. tropicalis, C. albicans, and C. krusei. Even though the order of frequency of various Candida species was same among denture wearers and non-denture wearers, the denture wearers showed a statistically significant increased frequency of all Candida species compared to the non-denture wearers (P=0.98). C glabrata was the most common species isolated and was present in 68.3% and 37.5% of denture wearers and non-denture wearers, respectively, C. krusei was the least common species found and was present in 36.7% and 20% of denture wearers and non-denture wearers, respectively [Table 3]. | Table 3: Speciation among denture and non-denture wearers by SDA and biochemical tests
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The prevalence of Candida among male and female denture wearers and non-denture wearers was assessed using the presence/absence of growth on SDA and CHROMagar method. Among females, 96.7% of denture wearers showed candidal growth both in SDA and CHROMagar. This increased prevalence of Candida among denture wearers was statistically significant (P=0.008). Both SDA and CHROMagar were equally efficient in the detection of Candida in denture wearers and non-denture wearers. In the present study, most of the female denture wearers used RPD and, moreover, wore it continuously throughout the day and night. In contrast, most males had complete dentures, and they wore it only during the daytime.
Speciation of Candida using CHROMagar was based on the color exhibited by the colonies. Green-colored colonies indicated C. albicans, and blue, pink, and white/violet indicated C. tropicalis, C. krusei, and C. glabrata, respectively. Speciation of Candida was done among the denture wearers and non-denture wearers. C. glabrata was the most common species isolated and C. krusei was the least common species found in denture wearers and in non-denture wearers [Table 4]. The various species seen in order of decreasing frequency were C. glabrata, C. tropicalis, C. albicans, and C. krusei. Even though the order of frequency of various Candida species was the same among denture wearers and non-denture wearers, the denture wearers showed a statistically significant increased frequency of all Candida species compared to the non-denture wearers (P=0.9). C glabrata was the most common species isolated and was present in 68.3% (n=41) and 37.5% (n=15) of denture wearers and non-denture wearers, respectively, C. krusei was the least common species found and was present in 36.7% (n=22) and 20% (n=8) of denture wearers and non-denture wearers, respectively, by the CHROMagar method [Table 4]. | Table 4: Speciation among denture and non-denture wearers by CHROMagar method
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In the CHROMagar method, C. albicans was the most common species isolated in males and was present in 66.7% (n=20) and 40% (n=8) in denture wearers and non-denture wearers, respectively. C krusei was the least common species found and was present in 20% (n=8) and 20% (n=4) of denture wearers and non-denture wearers, respectively. Similarly, in females, C. glabrata was the most predominant species and was present in 76.7% (n=23) and 35% (n=7) in denture and non-denture wearers, respectively. C albicans was the least common species found in females and was present in 30% (n=9) and 15% (n=3) of denture wearers and non-denture wearers, respectively. Similar findings were obtained by the conventional methods also.
Distribution of colony counts (cfu/ml) among denture wearers and non-denture wearers was examined. The range of colony-forming units among denture wearers and non-denture wearers in SDA and CHROMagar is given below:
Denture wearers (cfu/ml)
- SDA : 60×10 3 to 2048×10 3
- CHROMagar : 50×10 3 to 2264×10 3
Non-denture wearers (cfu/ml)
- SDA : 4×10 3 to 2680×10 3
- CHROMagar : 7.8×10 3 to 1724×10 3
Among the 60 denture wearers, 46.7% (n=28; males-13 and females-15) had no disease. Of the remaining 53.3% (n=32; males-17 and females-15) of denture wearers some had a single disease and others had multiple diseases. Diabetes was the most common disease, followed by cardiovascular disease, hypertension, asthma, and gastric problems. Among the total 30 male denture wearers, the commonest disease was diabetes and was seen in 50% (n=15) of these males. Similarly, among the total 30 females, 50% (n=15) had no systemic disease; among the remaining 15 females, diabetes was the most common disease, being present in 30% (n=9). Among the 40 (males-20, females-20) non-denture wearers, both males and females were free from any of the above mentioned systemic diseases, including diabetes. [Table 5]
Analysis of candidal species among non-denture wearers and denture wearers with and without diabetes using conventional methods showed that out of 60 denture wearers, 36 (males-15, females-21) subjects without diabetes showed growth and 24 (males-15, females-9) subjects with diabetes showed candidal growth. Among nondiabetic denture wearers, C. tropicalis and C. glabrata were the commonest, being seen in 22.8% (n=8) cases each; C. krusei was the least common, being seen in only 14.3% (n=5). Similarly, among diabetic denture wearers, C. albicans (52%; n=13) was the most commonly isolated species, whereas C. krusei (8%; n=2) was the least common. Among the 40 non-denture wearers, 30 showed candidal growth and 10 did not. Speciation showed C. glabrata (37.5%; n=15) to be the most common species among non-denture wearers.
Analysis of the prevalence of different candidal species among male denture wearers and male non-denture wearers according to smoking status, done using conventional method, showed that C. albicans was the most common species in all groups of subjects. Among smoking male denture wearers, C. albicans showed predominance of 60% (n=6) over the other three candidal species. The least common species were C. krusei and C. glabrata, being seen in only 30% (n=3) each. Similarly, among male non-denture wearers, both C. albicans and C. glabrata showed equal predominance, being seen in 40% (n=8) each; C. krusei, seen in 20% (n=4), was the least common species. [Table 6]
| Discussion | | |
Candida l colonization was seen in 95% of denture wearers, which is in accordance with the study done by Sylvie et al.[11] In non-denture wearers, 75% showed candidal growth, which is consistent with the study done by Daniluk et al.[12] In this study, male non-denture wearers showed more candidal growth than females. However, in denture wearers, Candida was more common in females than males, which is concordant with the study done by Pires et al.[13] In the present study, most of the female denture wearers used RPD and, moreover, wore it continuously throughout day and night. In contrast, males mostly had complete dentures, and they wore it only during the daytime. This is the main reason for the increased candidal colonization in females. Other reasons for increased candidal growth are the higher incidence of edentulism among females and the greater tendency to seek dental treatment in females than males. Also, the poor oral hygiene conditions in females causes increased candidal growth. Similar findings have been reported by Ikebe et al. [14]
Candidal colonization was more common in the older denture users, especially over the age of 60 years; this is probably attributable to the reduced salivary flow. A study done by Ikebe et al.[14] showed similar findings. In a study done by Zaremba et al.[15] it was found that the prevalence of candidal colonization was higher in adults without dentures compared to those with dentures, and in denture wearers it is seen more in the elderly age-group. Dentures predispose to infection, with Candida seen in 65% of elderly people wearing upper complete dentures. Wearing of dentures produces a microenvironment conducive to the growth of Candida, which flourishes in a low-oxygen, low-pH, and anaerobic environment. The increased colonization by Candida in denture wearers may be due to enhanced adherence of Candida species to acrylic, reduced saliva flow under the surfaces of the denture fittings, improperly fitted dentures, and poor oral hygiene. [16]
In the present study, oral hygiene in non-denture wearers were categorized as good, fair, or poor as per their hygiene estimated by the simplified oral hygiene index (S-OHI). Oral hygiene status was assessed in only RPD wearers. For complete denture wearers, the oral hygiene index was not assessed because they were edentulous, but the status of their complete dentures were assessed based on the presence of deposits on the tissue fitting surface and the stains on acrylic teeth. Absence of deposits and stains indicated good oral hygiene, whereas their presence indicated poor oral hygiene. In this study, poor oral hygiene and poor maintenance of the prosthesis was seen to increase the chances of colonization by Candida. These findings correlated with the study done by Daniluk et al.[12] and Compagnoni et al.17 Maintenance of good denture hygiene and removal of dentures at night reduces candidal growth according to Akpan et al.[18]
The conventional methods used for isolation, quantification, and speciation of Candida include the germ tube test, urease test, carbohydrate fermentation test, and culturing in SDA [Figure 2]. The germ tube test is closely associated with dimorphism in yeasts. It is the common phenomenon, occurring over the course of 1-3 h for C. albicans and, therefore, can be used as rapid presumptive identification test [Figure 3]. [19] The urease test is done to differentiate species like C. krusei, C. lipolytica, and C. humicola from C. albicans. This test detects the ability of yeast to produce the enzyme urease. In the presence of a suitable substrate, urease splits urea, producing ammonia, which raises the pH and causes a color shift to pinkish-red from colorless at 25-30°C for up to 4 days. [20] | Figure 2: Petri dish showing confluent growth of cream-colored candidal colonies in SDA
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The carbohydrate fermentation test shows that fermentative yeasts recovered from clinical specimens produce carbon dioxide and alcohol. Production of gas, and not a pH shift, is indicative of fermentation. Positive fermentation is evidenced by gas bubbles being trapped in the tubes. There is a color change noted from original purple color to a yellow color [Figure 4]. The test assesses the ability of various species of yeast to form gas from different carbohydrates. [21] | Figure 4: Test tubes 1 to 5 (purple) indicates negative control and test tubes 6, 9, and 10 (yellow) indicate the positive reaction
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The species isolated in denture wearers did not significantly differ from those in dentate individuals or in edentulous patients not wearing dentures, with C. albicans being the most prevalent species. [22] Speciation was done by conventional and CHROMagar methods simultaneously. Conventional tests based on germ tube formation and fermentation of sugars (dextrose, maltose, sucrose, lactose) and the urease test were used to identify C. albicans, C. tropicalis, C. krusei, and C. glabrata. Samples positive for germ tube and which show positive fermentation of dextrose and maltose indicate presence of C. albicans. Presence/absence of germ tube formation and fermentation of all these sugars (dextrose, maltose, and sucrose) indicate C. tropicalis. Absence of germ tube formation, fermentation of dextrose alone, and positive urease test indicate the presence of C. krusei. Absence of germ tube formation, fermentation of dextrose alone, and negative urease test indicate the presence of C. glabrata [Table 7].
In the study done by White et al., [22] the fungi isolated from the human mouths predominantly belonged to the genus Candida. There are more than 350 candidal species, and approximately ten of these can colonize the oral cavity. In the present study, C. glabrata was the most prevalent species, followed by C. tropicalis, C. albicans, and C. krusei, in both denture wearers and non-denture wearers. In the study done by White et al.[22] in non-denture wearers and by Jean Barbeau et al.[23] in denture wearers, it was found that C. albicans was the most prevalent species, followed by C. glabrata and C. tropicalis. According to Letica et al.[24] and Giovanni. [25] the most common species in denture wearers was C. albicans, followed by C. tropicalis, C. krusei, C. guilliermondii, C. parapsilosis, C. glabrata, and C. lusitaniae.
CHROMagar Candida is a special media that yields microbial colonies of varying pigmentation, secondary to chromogenic substrates that react with enzymes secreted by the microorganisms. We used this technique also, with speciation of Candida done by the color exhibited by each species on CHROMagar: green colonies indicated C. albicans, blue indicated C. tropicalis, pink indicated C. krusei, and white/violet indicated C. glabrata [Figure 5] and [Figure 6]. The conventional methods and the CHROMagar method were compared and were found to give similar results. There was no difference in results between conventional and CHROMagar methods, in total number of all the subjects and gender wise distribution of species. In the present study, CHROMagar Candida showed 100% specificity and 100% sensitivity when compared to SDA and conventional methods. This was similar to the findings of Larone et al.[26] who reported 81% sensitivity and 100% specificity for CHROMagar. In the study by Pires et al.[13] C. albicans was the most common species, responsible for almost 70% of the isolates, and it was also the predominant species found in denture users. [13] Studies done by Daniluk et al., [12] Golecka et al., [27] and Zaremba et al.[15] also found that C. albicans was the predominant species in both non-denture wearers and denture wearers. | Figure 5: Petri dish showing colonies of different colors: C albicans as green, C. tropicalis as blue, C. krusei as pink, and C. glabrata as white/violet
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 | Figure 6: Petri dish showing different colonies of different species: green indicates C. albicans, blue indicates C. tropicalis, pink indicates C. krusei, and white/violet indicates C. glabrata
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In this study we found greater candidal growth in denture wearers, especially in females. Speciation of Candida by conventional methods and CHROMagar among denture wearers and non-denture wearers showed C. glabrata to be the most common species, followed by C. tropicalis, C. albicans, and C. krusei. Among males, both denture wearers and non-denture wearers showed C. albicans as the predominant species and C. krusei as the least predominant species. Similarly, among females, in both denture wearers and non-denture wearers, C. glabrata was the most commonly isolated species and C. albicans the least common.
Both SDA and CHROMagar were equally effective in detecting the presence of Candida among denture and non-denture wearers. In the present study, CHROMagar Candida showed 100% specific and 100% sensitive results when compared to SDA and conventional. This is concordant with the results of Yucecoy et al., [28] which showed 99.4% sensitivity and 100% specificity for CHROMagar, and Larone et al., [26] who reported 81% sensitivity and 100% specificity.
Quantification of the colonies was done by analyzing the colony forming units per milliliter (cfu/ml) in SDA and CHROMagar plates simultaneously in both denture wearers and non-denture wearers. The number of colonies formed was multiplied by 1000 (which was represented as cfu/ml). [29] Colony count was calculated by the calibrated loop method of Myers and Koshi. In the present study, distribution of colony counts among denture wearers and non-denture wearers ranged from 60000-2048000 in SDA and 50000-2264000 in CHROMagar. Similarly, in non-denture wearers it ranged from 4000-2680000 in SDA and 78000-1724000 in CHROMagar. Isaac et al.[30] found that the relationship of candidal counts to anatomical sites was more than 10 3 cfu/ml. Also, in a study done by Astrid, [31] the yeast carriage in healthy denture wearers by swabbing the colony counts ranged from 50 to 10 4 cfu/ml.
The influence of smoking, which was the most common habit in denture werarers nd non-denture wearers, was analyzed. We found that candidal colonization and growth was increased in smoking denture wearers compared to nonsmoking denture wearers. The finding of the study done by SeopEun et al.[32] had led them to suggest that smoking dictates oral Candida carriage in healthy individuals. A study done by Shulman [33] found that smoking increased candidal colonization in maxillary complete/partial and mandibular complete denture wearers but not in mandibular partial denture wearers. The relationship between candidal growth and smoking was put forward by Sitheeque et al., [34] who found that tobacco smoking favors Candida colonization due to induction of increased epithelial keratin, reduced IgA and, possible, depression of PMNL (polymorphonuclear leukocytes) function. [34] Scully et al.[35] reviewed a study done by Tagaki et al., who hypothesized that cigarette smoking provides nutrition for C. albicans, the fungus replicating by using polycyclic aromatic hydrocarbons as a source of carbon and energy. [35]
The influence of smoking on candidal species in male denture wearers and non-denture wearers showed that C. albicans is the most predominant among the four candidal species and C. krusei was the least predominant species. C tropicalis and C. glabrata showed the highest (and equal) predominance among nondiabetic denture wearers. Eun et al. [32] and Shulman [33] found that smoking increased candidal colonization. Diabetic denture wearers showed C. albicans as the predominant species. Among non-denture wearers C. glabrata was the predominant species. In all the three groups, C. krusei showed least predominance. The findings of the present study were consistent with that of the studies done by Pires [13] and Akpan et al.[18]
A number of systemic diseases increase the susceptibility to oral candidiasis and the harmful effects of mechanical irritation. Nutritional deficiencies such as deficiency of amino acids, iron, and certain vitamins of the B complex group are reported to lower the resistance of the oral mucosa. In this study it was found that diabetic denture wearers showed a higher rate of Candida carriage. This correlated with the studies done by Pires [13] and Akpan et al.[18] The reasons for high oral candidal carriage in patients with diabetes are the high salivary glucose levels and adhesion of Candida to buccal epithelial cells in diabetics. [15] Daniluk et al. reported that the frequency of oral candidal isolates was greater in diabetic subjects with dentures compared to diabetic subjects without dentures. [12]
| Conclusion | | |
To conclude, both conventional and CHROMagar methods can be used for isolation and speciation of Candida, but the CHROMagar method is more rapid compared to the conventional methods. Candidal colonization was higher in denture wearers compared to non-denture wearers, especially in females. The influence of smoking and diabetes on candidal growth was found to be greater in denture wearers compared to non-denture wearers, with C. albicans being the most predominant species in male denture-wearing smokers and diabetic denture wearers. This study presents a preliminary comparison of the conventional methods with the CHROMagar method for isolation and speciation of Candida.
| Acknowledgment | | |
We authors would like to express our thanks and gratitude to Sabarinath B, Senior lecturer and Subhasini D, Microbiologist for their invaluable help, guidance and support.
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Correspondence Address:
Shruti Nayak
Department of Oral Pathology and Microbiology, Yenepoya Dental College, Yenepoya University, Deralakatte, Mangalore
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0970-9290.104956
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7] |
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