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An Official Publication of the Indian Association of Oral and Maxillofacial Pathologists


 
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ORIGINAL ARTICLE  
Year : 2022  |  Volume : 26  |  Issue : 4  |  Page : 590
 

Comparative evaluation of prevalence and phenotypic variations of Candida species in patients of oral lichen planus and oral lichenoid lesions with healthy individuals - A prospective microbiological study


Department of Oral Pathology and Microbiology, MGM's Dental College and Hospital, Navi Mumbai, Maharashtra, India

Date of Submission14-Jul-2020
Date of Acceptance29-Oct-2021
Date of Web Publication22-Dec-2022

Correspondence Address:
Jigna Rakesh Pathak
Department of Oral and Maxillofacial Pathology, Oral Pathology, MGM Dental College & Hospital, MGM Institute of Health Science, Kamothe, Navi Mumbai, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jomfp.jomfp_229_21

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   Abstract 


Context: Candida isolates might exaggerate the development and advancement of oral lichen planus (OLP) alone or together with other factors. There is a differing pathogenic potential of various Candida species. Since Candida species isolated from OLP and oral lichenoid lesions (OLL) have shown to be resistant to commonly used antifungal agents, identification of Candida species may play a pivotal role in its successful clinical management.
Aim: The aim of the study was to evaluate and compare the prevalence and phenotypic variations of Candida species in patients of OLP and OLL with healthy controls.
Subjects and Methods: This prospective microbiological study was conducted in the institution using indirect microscopic examination. The sample comprised of 40 clinicohistopathologically diagnosed cases of the study group (OLP and OLL) and 40 cases of control group (healthy individuals).
Statistical Analysis Used: The data collected was statistically analyzed using the Chi-square test and Fisher's exact test with the SPSS 20.00 software.
Results: The prevalence of Candida albicans was higher in the control group (28.10%) as compared to the study group (24.60%) and this difference was statistically significant. An increased frequency of non C. albicans species was seen in the study group, in decreasing order of Candida glabrata (40.70%), Candida tropicalis (22.20%), Candida krusei (22.20%) and Candida guilliermondii (3.70%), as compared to the control group.
Conclusion: Non C. albicans species were the predominant pathogens associated with the study (OLP + OLL) group. It is important to identify the infecting strains of Candida because isolates of Candida species differ widely, both in their ability to cause infection and also in their susceptibility of resistance to antifungal agents. Thus, phenotypic speciation of Candida is emerging as a necessary trend to highlight the need of administering appropriate antifungal therapy.


Keywords: Antifungal agents, Candida albicans, non-Candida albicans, oral lichen planus, oral lichenoid lesion


How to cite this article:
Sarkate PR, Pathak JR, Patel S, Swain N, Sahu NK. Comparative evaluation of prevalence and phenotypic variations of Candida species in patients of oral lichen planus and oral lichenoid lesions with healthy individuals - A prospective microbiological study. J Oral Maxillofac Pathol 2022;26:590

How to cite this URL:
Sarkate PR, Pathak JR, Patel S, Swain N, Sahu NK. Comparative evaluation of prevalence and phenotypic variations of Candida species in patients of oral lichen planus and oral lichenoid lesions with healthy individuals - A prospective microbiological study. J Oral Maxillofac Pathol [serial online] 2022 [cited 2023 Jan 30];26:590. Available from: https://www.jomfp.in/text.asp?2022/26/4/590/364793





   Introduction Top


Oral lichen planus (OLP) is a T-cell mediated chronic inflammatory disorder of unknown etiology. The World Wide prevalence of OLP is approximately 2.2%[1] and in India, it is 2.6%.[2] OLP most commonly occurs at 30–80 years of age, with a female predilection. Two-thirds of OLP patients experience discomfort on having spicy and acidic foods.[3] There are varieties of lesions that resemble OLP, both clinically and histopathologically, which are referred to as oral lichenoid lesions (OLL).[4] If epithelial dysplasia is seen on initial diagnosis in cases of OLP and OLL, then, it is called as an oral lichenoid dysplasia (OLD). The overall rate of malignant transformation of OLP and OLL is reported to be 1.09% and 3.2%, respectively.[5] Candida isolates might exaggerate the development and advancement of OLP alone or together with other factors.[6] Besides Candida albicans, Non-C. albicans strains, such as Candida parapsilosis, Candida tropicalis, C. dubliniensis, Candida krusei and Candida glabrata, are frequently detected in OLP and OLL.[6],[7],[8],[9],[10],[11],[12],[13] Various studies are done which have shown that there is a differing capability of various strains to promote dysplasia by catalyzing the production of nitrosamine (carcinogenic) and also because of metabolism of ethanol to the carcinogenic acetaldehyde and induction of proinflammatory cytokines.[14] As there is a differing pathogenic potential of various Candida species, identification of species may play a pivotal role in successful clinical management. Furthermore, Non-C. albicans species isolated from OLP lesions have shown to be resistant to commonly used antifungal agents.[7] Thus, the following study was undertaken to comparatively evaluate the phenotypic variation of Candida species in patients of OLP and OLL with healthy individuals using indirect microscopic examination.


   Subjects and Methods Top


This prospective study was carried out from December 2018 to December 2019 in the Department of Oral and Maxillofacial Pathology and Microbiology.

All the individuals visiting the institution were clinically screened for OLP and OLL, and biopsy sample was obtained after an informed consent. The sample comprised 40 clinicohistopathologically diagnosed cases of OLP and OLL (study group), using modified WHO criteria 2003[1] and 40 clinically diagnosed cases of healthy individuals (control group).

All the individuals were assessed clinicopathologically and confirmed cases of OLP and OLL were chosen for the study. During the conduct of the study, all human ethical principles as per the World Medical Association-Declaration of Helsinki and the guidelines of Good Clinical Practice (ICMR) were observed. The Institutional Ethical Committee approval was taken (MGM/DCH/IERC/11/2017), and an informed consent was obtained from all individuals participating in the study.

Before tissue sampling under aseptic conditions, a swab of the respective area was taken using a disposable swab stick [Figure 1]. The swab sample collected was immediately inoculated on Sabouraud's dextrose agar (SDA) medium (HiMedia). After getting positive cultures [Figure 2], a small inoculum was incubated in serum at 37°C for about 3 h, and a few drops of the suspension were then examined under light microscope to observe germ tube (GT) formation [Figure 3] for the detection of C. albicans species (Raynaud's phenomenon).[15]
Figure 1: Swab culture from site of the lesion

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Figure 2: Swab culture inoculated on Sabouraud's dextrose agar media showing creamy white, convex colonies of Candida species after incubation

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Figure 3: Germ tube test showing production of filamentous extension form yeast cell indicating positive for Candida albicans

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For presence and speciation of non-C. albicans species, sugar fermentation test [Figure 4] and sugar assimilation test [Figure 5] were performed. Fermentation and assimilation test media were prepared with 2% sugar (Glucose, Sucrose, Lactose and Maltose) (HiMedia). Small inoculums were added into each tube and incubated for 48–72 h at 30°C. The ability to ferment sugar was shown by the presence of acid (indicator becomes pink) and gas trapped in the Durham tubes [Table 1]a. Sugar assimilation was depicted as a positive or a negative reaction for various sugars, as seen in [Table 1]b.[16]
Figure 4: Sugar fermentation test (positive for Candida tropicalis and C. kefyr)

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Figure 5: Sugar assimilation test (positive for Candida glabrata)

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Data obtained were presented using descriptive statistics in SPSS Inc. Version 20.00, Chicago, Illinois,USA. Values for P < 0.05 were considered statistically significant.


   Results Top


In the present study, the clinicohistopathological evaluation of the study group was done by using modified WHO criteria 2003.[1] The details of the evaluation of the cases of OLP, OLL and OLD are shown in [Table 2]. Microbiological evaluation for candida species of control group and study (OLP + OLL) group was done by indirect microscopic examination (SDA culture, GT test, sugar fermentation and sugar assimilation) as elaborated in [Table 3].
Table 2: Clinicohistopathological evaluation of study group (oral lichen planus and oral lichenoid lesions) by using modified WHO criteria 2003

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SDA swab culture showed 100% cases of study group (n = 40) and 42.5% cases of control group (n = 17) were positive for Candida species. On statistical comparison [Table 3]a, out of total cases in the present study (n = 80), the frequency of presence of Candida species was higher in study (OLP + OLL) group (50%) and absence of Candida species in control group (28.75%) and this difference was statistically significant (P < 0.0005) [Graph 1].



GT test showed that 40% cases (n = 16) of study (OLP + OLL) group were positive for C. albicans and the remaining 60% cases (n = 24) were non C. albicans. In the control group, 35% cases (n = 14) were C. albicans and only 7.5% cases (n = 3) were non C. albicans. On statistical comparison [Table 3]b, out of total positive cases (n = 57) in the present study, frequency of C. albicans (24.60%) was higher in control group and non C. albicans species (42.10%) in study (OLP + OLL) group and this difference was statistically significant (P < 0.0003) [Graph 2].



Sugar fermentation test and sugar assimilation test done for speciation of Candida showed 40% cases (n = 16) were C. albicans and 60% cases (n = 24) were non C. albicans. The non C. albicans included 45.8% cases (n = 11) of C. glabrata, 25% cases (n = 6) of C. tropicalis and C. krusei, 4.1% case (n = 1) of Candida guilliermondii. In control group 7.5% cases (n = 03) of non C. albicans species included 2.5% cases each one of C. glabrata (n = 01), C. tropicalis (n = 01) and C. krusei (n = 01). On statistical comparison [Table 3]c, species such as C. glabrata, C. tropicalis, C. krusei and C. guilliermondii were higher in study (OLP + OLL) group as compared to control group with statistically insignificant difference (P > 0.05).


   Discussion Top


Candida species are implicated as one of the risk factors in epithelial dysplasias in oral potentially malignant disorders such as leukoplakia, lichen planus and submucous fibrosis as well as in oral carcinogenesis.[17] Studies show an association of Candida species with OLP. Other than C. albicans, non-C. albicans strains, such as C. parapsilosis, C. tropicalis, C. dubliniensis, C. krusei and C. glabrata, are frequently detected in OLP. Furthermore, Non-C. albicans species isolated from OLP lesions have shown to be resistant to commonly used antifungal agents. A proper identification of the specific candidial strains should be performed due to the variable degree of invasiveness and antifungal sensitivity of the different species of Candida.[6] Emergence of drug-resistant strains of Candida has highlighted the need for reliable methods of candidial isolation and identification.[16] Thus, the above study was undertaken to comparatively evaluate the phenotypic variation of Candida species in patients of OLP and OLL with healthy individuals using indirect microscopic examination.

Among the various clinicohistopathological parameters evaluated as per modified WHO criteria 2003, study group included 35% cases (n = 14) of OLP and 65% cases (n = 26) of OLL in which 34.6% cases (n = 09) showed epithelial dysplasia. Shivhare et al. in 2016[17] believed that the modified classification of OLP 2003 is more practical and aids in the diagnosis of OLP better but it also has dysplasia as exclusion criteria for OLP. Patil et al.[18] in 2014 assumed that if OLP manifested bilaterally (clinically typical) and histologically only compatible (shows dysplasia), according to 2003 modified criteria, it should be labeled as an OLL. In fact, exclusion of all lesions that resemble OLPs but exhibit epithelial dysplasia may lead to an underestimation of the rate of malignant transformation of OLP. This fact was restated by Arora S et al. in 2006,[6] where they reported severe epithelial dysplasia and carcinoma in situ in their series of OLP. Rejecting a diagnosis of OLP solely due to the presence of dysplasia, therefore, requires consideration.[18]

In our study, on SDA swab culture, frequency of positive Candida species was higher in study (OLP + OLL) group and negative Candida species in control group. Several studies done by Arora S et al. 2016,[6] Lundström et al. 1984,[8] Zeng et al. 2009,[9] and Jainkittivong et al. 2017.,[10] show variable results in OLP and control group. The reason of such findings could be attributed to candidal growth on many preexisting pathological processes by inducible enzyme system that allows them to replicate using polycyclic aromatic hydrocarbon as their source of energy.[19] Negative culture in controls (28.75%) in our study indicates their absence in healthy host. Because microorganisms are continuously removed from the oral cavity by host clearance mechanism, Candida survival may not be always possible.[20]

On performing GT test for differentiation into C. albicans and non C. albicans species,[16] the frequency of C. albicans was higher in control group and non C. albicans species in study (OLP + OLL) group. To the best of our knowledge, no other studies show an increased number of non C. albicans in OLP and OLL as compared to healthy controls. Several studies were done by Mehdipour et al.[11] and Ebrahimi et al.[12] show variable results in OLP and control group. The formation of germ tubes is influenced by various environmental factors (temperature, concentration of inoculum, composition of the medium, pH and bacterial contamination) which may account for false readings.[16] One possible concern about this technique is that 95% of C. albicans isolates or some non C. albicans (C. dubliniensis, C. tropicalis and C. parapsilosis) can also generate germ tube or pseudohyphae in serum, leading to misidentification if only perfunctory tests are performed.[15]

In the present study, the frequency of non C. albicans species such as C. glabrata, C. tropicalis, C. krusei and C. guilliermondii was higher in study (OLP + OLL) group as compared to control group. Among non C. albicans species in study (OLP + OLL) group, the frequency of C. glabrata was higher and C. dubliniensis showing phenotypic similarity to C. albicans was not detected. The control group, however, showed the presence of only C. glabrata, C. tropicalis and C. krusei. Non C. albican species were also isolated in various other studies done by Lundström et al.[8] in 1984, Masaki et al.[13] in 2010, Mehdipour et al.[11] in 2010, Arora S et al.[6] in 2016 and Jainkittivong et al.[10] in 2017. All these studies isolated different types of non C. albican species in OLP cases such as C. glabrata, C. parapsilosis, Candida dubliniensis, C. krusei and C. fukuyamaensis, but unlike our study, all other studies showed higher frequency of C. albicans than non C. albican species. This variability could be attributed to different methodology used in various studies like PCR analysis and Chromagar culture isolation.[6],[7]

These non C. albicans species lack many of the virulence factors present in C. albicans, i.e., the ability to form hyphae and phenotypic switching. They have low adherence capacity to buccal epithelial and vascular endothelial surfaces. They secrete less proteinases.[21] However, C. glabrata is less susceptible to killing by human beta-defensins than is C. albicans and exhibits various degrees of resistance to the antifungal activity of salivary histatins and mucins.[19] In addition, C. glabrata possesses both innate and acquired resistance against antifungal drugs, due to its ability to modify ergosterol biosynthesis, mitochondrial function or antifungal efflux. This resistance allows for its relative overgrowth over other susceptible species. Furthermore, in a study, it was noted that biofilm-forming ability in C. tropicalis was greater as compared to C. albicans. Biofilm formation is implicated as an important virulence attribute of Candida species as it increases the ability to withstand host defenses and also confers significant resistance to antifungal therapy.[16],[21] Thus, although less virulent in nature, non C. albicans need to be differentiated and identified to emphasize the initiation of adequate and appropriate therapeutic modalities in treating OLP and OLL cases. Identification of different candida species may be the need of the hour as there are significant differences in their susceptibility to antimycotic drugs.[19],[21],[22]





A constant rise in immuno-suppressed patients, widening range of recognized pathogens and resistance to antifungal drugs are contributing factors which stresses the need for species identification of Candida, an opportunistic pathogen. Therefore, it is important to identify the infecting strains of Candida because isolates of Candida species differ widely, both in their ability to cause infection and also in their susceptibility to antifungal agents. Furthermore, it is important for successful clinical management and for determining appropriate control measures to prevent transmission of resistant candidal pathogens. Thus, phenotypic speciation of candida is emerging as a necessary trend to highlight the need of administering appropriate antifungal therapy. Studies with larger sample size are required to be done to substantiate this hypothesis.

Acknowledgments

We would like to thank all the staff members of Department of Oral and Maxillofacial Pathology and Microbiology and also Department of Medical Microbiology for their further support and guidance.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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