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ORIGINAL ARTICLE
Year : 2015  |  Volume : 4  |  Issue : 3  |  Page : 170-175

A study on isolation, identification, and antifungal susceptibility of various oral candidal species in renal transplant patients


1 Department of Oral Pathology, Kamineni Institute Dental College and Hospital, Narketpally, Andhra Pradesh, India
2 SIBAR Institute of Dental Sciences, Guntur, Andhra Pradesh, India

Date of Web Publication15-Sep-2015

Correspondence Address:
Gontu Sridhar Reddy
Department of Oral Pathology, SIBAR Institute of Dental Sciences, Guntur, Andhra Pradesh
India
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Source of Support: Nil., Conflict of Interest: None declared.


DOI: 10.4103/2277-8632.165399

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  Abstract 

Background: The rationale of this study was to know the prevalence of candidal colonization in renal transplant individuals and to isolate and identify the various species and determine their antifungal susceptibility.
Materials and Methods: The study population was divided into Group I (renal transplant individuals, n =30) and Group II (healthy individuals, n =50) from whom oral rinse samples were collected in a container with sterile phosphate-buffered saline (PBS). All the collected samples were transported immediately and subjected to various mycological investigations. Statistical analysis was performed using Mann–Whitney U and Chi-square test.
Results: In Group I, 11 (36.67%) and in Group II, 9 (18%) showed positivity for Candida. Candida albicans formed the major species and it showed sensitivity to Fluconazole and Ketoconazole.
Conclusion: Prevalence of Candida species in the oral cavity of renal transplant recipients was higher than in immunocompetent control subjects. Administration of immunosuppressive drugs predisposes the development of an increased density of candidal colonies.

Keywords: Candida albicans, immunosuppressive drugs, oral candidiasis, renal transplants


How to cite this article:
Keerthi M, Reddy GS, Shekar PC, Chandra KL, Kumar KK, Reddy BV. A study on isolation, identification, and antifungal susceptibility of various oral candidal species in renal transplant patients. J NTR Univ Health Sci 2015;4:170-5

How to cite this URL:
Keerthi M, Reddy GS, Shekar PC, Chandra KL, Kumar KK, Reddy BV. A study on isolation, identification, and antifungal susceptibility of various oral candidal species in renal transplant patients. J NTR Univ Health Sci [serial online] 2015 [cited 2020 Apr 5];4:170-5. Available from: http://www.jdrntruhs.org/text.asp?2015/4/3/170/165399


  Introduction Top


Candida, a ubiquitous opportunistic fungal pathogen seen in immunocompromised patients, is a major cause of morbidity.[1] Although Candida albicans is the major species in candidiasis, other species such as Candida glabrata are recently emerging as possible etiological agents with greater risk of oral infection and other associated complications for the clinician.[2] Oral lesions may develop as a result of side-effects and drug interactions during immunosuppressive therapy.[3] Organ transplant patients who are treated with immunosuppressants have depressed cell-mediated immune response, due to which oral pathogens are more likely to cause local destruction and opportunistic infections. Oral candidiasis is seen in patients undergoing systemic steroid therapy and in patients with renal transplantation who received immunosuppressive therapy.[4],[5] Invasive fungal infections attributed to Candida species are particularly seen after the immediate post-organ transplant period.[6]Candida can trigger bloodstream, esophageal, gastrointestinal, respiratory, and urinary tract infections. In solid organ transplant recipients, the prevalence of Candida during the first 6 months ranges between 5 and 50% depending on the type of transplant (e.g. renal or liver). It was reported that up to 80% of candidal esophagitis episodes in renal transplant patients were preceded by oral thrush or colonization.[7]

The aims and objectives of the study were to know the prevalence of Candida colonizing in the oral cavity of the renal transplant patients, isolate and identify the Candida species and their antifungal susceptibility, and estimate the colony forming unit (CFU/ml) counts in both the control and study groups.


  Materials and Methods Top


A total of 80 cases were included in the study sample. Thirty (Group I) consecutive renal transplant patients were screened during routine, outpatient, follow-up visits at the transplant unit in Kidney Care Centre between February 2013 and March 2014. Fifty (Group II) controls attending outpatient department of the same hospital were included in the study. Subject selection criteria were as follows:

  1. Patients who had undergone renal transplant within a period of 6 months at the time of examination were included in Group I; and
  2. Patients who were on antibiotics for a period of 15 days before the sample collection or had a history of using corticosteroids or the subjects who were diabetic, anemic, or pregnant and had any known diseases or conditions that predispose to oral candidiasis were excluded from Group II.


Oral examination and microbial sampling

A complete oral examination was performed in all subjects. Unstimulated saliva was analyzed in this study and the saliva samples were collected in the morning between 9 a.m. and 2 p.m. Each individual was supplied with a universal container containing 10 ml of sterile phosphate-buffered saline (PBS 0.1 M, pH 7.2) solution and was asked to rinse the mouth for 60 s thoroughly, and the mouth rinse was expelled into a sterile container. The samples were transported immediately to the laboratory and were subjected to various mycological tests and processed according to the standard mycological procedures given by Milne in1996.[8]

Each sample was examined microscopically before culturing in the KOH wet mount and Gram's stained smear, which was done to identify the budding yeast cells and pseudohyphae [Figure 1]. The material was inoculated in Sabouraud dextrose agar (SDA) medium and incubated at 37°C for 1-3 days, and creamy white, smooth, pasty colonies were observed [Figure 2]. C. albicans isolates were confirmed by germ tube test [Figure 3] and chlamydospore production was confirmed on corn-meal agar by the Dalmau plate technique [Figure 4]. The isolates were then inoculated on CHROMagar Candida and incubated at 37°C in dark for 48 h. The colonies with color were considered for species identification [Figure 5]. Identification of various Candida species was done by carbohydrate fermentation test [Figure 6] and carbohydrate assimilation test.
Figure 1: Gram's staining of the smear showing budding yeast cells and pseudohyphae

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Figure 2: Candida colonies on Sabouraud dextrose agar

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Figure 3: Germ tube production by Candida albicans

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Figure 4: Growth of Candida albicans on corn-meal agar showing chlamydospores

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Figure 5: Colored Candida colonies on CHROMagar medium

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Figure 6: Fermentation of sugars by Candida species in sugar fermentation test

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Quantification of the colonies was done by the number of CFUs/ml, which was derived by the formula: CFU/ml = 1000 × number of colonies/4.

Antifungal susceptibility was assessed with azoles and polyenes drugs by disk diffusion method. For azoles, the zones must be measured up to colonies of normal size and for polyenes, the clear zone with no visible growth is measured [Figure 7].
Figure 7: Candida showing sensitivity to Fluconazole and Ketoconazole

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Statistical analysis

All the findings were compared between the two groups using Mann-Whitney U test and Chi-square test. P value <0.05 was considered significant.


  Results Top


In Group I, out of 30 cases, 11 (36.67%) were positive and in Group II, out of 50 cases, 9 (18%) were positive [Table 1]. In Group I, C. albicans (16.67%) was the major species followed by Candida krusei (3.33%), C. glabrata (3.33%), Candida parapsilosis (3.33%), mixed Candida species (3.33%), and C. albicans along with Aspergillus (3.33%). In Group II, C. albicans (16%) was the major species followed by C. parapsilosis (2%) [Table 2].
Table 1: Distribution of Study Subjects According to Presence and Absence of Culture

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Table 2: Distribution of Candidal Species Isolated from the Oral Cavities of Group I and Group II Subjects

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In Group I, the maximum (20% of patients) CFU/ml was 5000-10,000 followed by >10,000 CFU/ml (16.67% of patients). In Group II, the maximum (10% of patients) CFU/ml was 5000-10,000 followed by 1001-5000 CFU/ml (8% of patients) [Table 3].
Table 3: Distribution of Group I and Group II According to CFU/ML Counts

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The evaluation of antifungal drug susceptibility in Group I showed sensitivity to Fluconazole and Ketoconazole in nine (30%) cases and sensitivity to Fluconazole, Ketoconazole, and Itraconazole in two (6.67%) cases. In Group II, eight (16%) cases showed sensitivity to Fluconazole and Ketoconazole and one (2%) case showed sensitivity to Fluconazole, Ketoconazole, and Itraconazole. But none of the cases in groups I and II showed sensitivity to Amphotericin B [Table 4].
Table 4: Distribution of Group I and Group II Subjects According to Sensitivity

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  Discussion Top


The renal transplant patients undergoing long-term graft-preserving immunosuppressive therapy are predisposed to a variety of oral complications. Various investigators suggested that Candida can act as an opportunistic pathogen in the immunocompromised host. This concept has been reinforced by various studies in correlation with diabetes, human immunodeficiency virus (HIV) infections, leukemia, solid organ transplantation, and radiation therapy.[9]

Renal transplant patients have an increased susceptibility to infections of all types. Fungi such as Candida, Aspergillus, and Cryptococcus are opportunistic invaders that usually cause disease only in the host with altered defenses.[10]

In our study, the majority of Group I (53.33%) and Group II (38%) patients fell in the age group of 29-38 years, with the mean age of 37.67 and 34.96 years, respectively. Male predilection was seen in Group I (63.33%), whereas female predilection was seen in Group II (52%). This result was in accordance with that of Al-Mohaya et al.,[11] who reported male predilection with a mean age of 39.2 years. Lopez Pintor et al.[12] also reported male predilection in their study on oral lesions in renal transplant patients. Douglas et al.[13] stated that higher prevalence of fungal infection in middle-aged renal transplanted individuals is due to their immune-compromised state secondary to the drugs.

In our study, candidal colonies were present in the culture medium in 11 (36.67%) cases in Group I and 9 (18%) cases in Group II [Table 1]. Al-Mohaya et al.[11] reported that candidal colonization in renal transplants was 74.1% and in healthy individuals was 59.6%, and Güleç et al.[14] reported that candidal colonization in renal transplants was 25.5% and in healthy individuals was 12.5%.

Dignani et al.[15] stated that prevalence of candidal species as a human commensal may vary from place to place. The factors predisposing to candidal colonization rate in a normal individual may be poor oral hygiene, nutrition and immune status. However, sometimes it may reflect on sample selection. In the oral environment, the estimated prevalence of Candida species as human commensal varies considerably according to the nature, size and type of the sample and also according to the method of sampling adopted.

In our study, majority of the species in Group I consisted of C. albicans (16.67%) followed by C. krusei (3.33%), C. glabrata (3.33%), C. parapsilosis (3.33%), mixed Candida species (3.33%) and C. albicans along with Aspergillus (3.33%). In Group II, the majority of species consisted of C. albicans (16%) followed by C. parapsilosis (2%) [Table 2].

Güleç et al.[16] stated that mixed species of Candida were identified in a higher percentage of the transplant patients than in the control subjects. This finding may be indicative of the possibility that several species, which are normally more vulnerable to host immunity, are allowed to thrive under this immunosuppressed, more permissive host environment. The clinical significance of the presence of mixed Candida species in the oral cavity is not entirely understood. However, oral infections with mixed Candida species have been reported to be more persistent and more difficult to treat.

Al-Mohaya et al.[11] reported that non-albicans species were isolated more frequently from renal transplant patients than from healthy controls, which may be due to the immunosuppressed condition of the renal transplant patients.

Pozzilli et al.[17] suggested that immunosuppressive drugs act as predisposing factors to Candida infection by changing the oral flora, damaging the salivary gland function, and breaking the mucosal surface. Further, development of oral candidiasis presumably depends on the degree of immunosuppression in kidney transplantation. A determinant role in protection against infection of mucus membrane seems to be played by Th1 CD4+ cells, but the role of these Candida-specific antibodies is still controversial.

Our results were consistent with previous studies. Group I and II individuals showed higher prevalence of C. albicans. However, relatively higher percentage of C. albicans was seen in Group I than in Group II, and this may be due to immune suppression activity. Non-albicans species were uncommon in Group II individuals.

In the present study, Group I patients showed higher (>10,000 CFU/ml) CFUs than Group II (1001-5000 CFU/ml) individuals [Table 3]. Our results were consistent with Rebbecca et al.,[18] who suggested that higher CFUs are seen in renal transplantation individuals and are significantly associated. Similar results were found in the published reports of Al-Mohaya et al.[11] and Dongori-Bagtzoglou et al.[19]

In our study, analysis of drug susceptibility revealed that nine (30%) cases of Group I showed sensitivity to Fluconazole and Ketoconazole and two (6.67%) cases showed sensitivity to Fluconazole, Ketoconazole, and Itraconazole. Among Group II individuals, eight (16%) cases showed sensitivity to Fluconazole and Ketoconazole and one (2%) showed sensitivity to Fluconazole, Ketoconazole, and Itraconazole. None of the cases in groups I and II showed sensitivity to Amphotericin B [Table 4]. These results were consistent with Barry et al.,[20] who also reported the sensitivity of C. albicans to Fluconazole.


  Conclusion Top


The renal transplantation patients are at high risk of developing fungal infection. The most common oral lesion found in them is oral candidiasis. Immunosuppressive drugs are administered during transplantation and post-transplant period to avoid the graft rejection by suppressing the T cell activity of the host. However, this predisposes immune-compromised state in the individual; this later phenomenon changes the complete scenario of general health. C. albicans, which is a normal commensal of oral microflora, has beome an opportunistic pathogen due to deficient immune mechanism in the host. Oral lesions are gaining special attention due to simultaneous emergence of non-C. albicans species and disease severity. The crucial period of the first 6 months to 1 year of post-transplant period is at significantly high risk. Hence, periodic monitoring of oral health status and identification of species and appropriate antifungal drug administration may yield good prognosis.

 
  References Top

1.
Gupta KL, Ghosh AK, Kochhar R, Jha V, Chakrabarti A, Sakhuja V. Esophageal candidiasis after renal transplantation: Comparative study in patients on different immunosuppressive protocols. Am J Gastroenterol 1994;89:1062-5.  Back to cited text no. 1
    
2.
Li L, Redding S, Dongari-Bagtzoglou AI. Candida glabrata, an emerging oral opportunistic pathogen. J Dent Res 2007;86:204-15.  Back to cited text no. 2
    
3.
Parisi E, Glick M. Immune suppression and considerations for dental care. Dent Clin N Am 2003;47:709-31.  Back to cited text no. 3
    
4.
Lehner T. Oral thrush, or acute pseudo membranous candidiasis. Oral Surg Oral Med Oral Pathol 1964;18:27-37.  Back to cited text no. 4
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5.
Folb PI, Trounce JR. Immunological aspects of Candida infection complicating steroid and immunosuppressive drug therapy. Lancet 1970;2:1112-4.  Back to cited text no. 5
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6.
Patel R, Portela D, Badley AD, Harmsen WS, Larson-Keller JJ, Ilstrup DM, et al. Risk factor for invasive Candida and non-Candida fungal infections after liver transplantation. Transplantation 1996;62:926-34.  Back to cited text no. 6
    
7.
Badiee P, Kordbacheh P, Alborzi A, Zeini F, Mirhendy H, Mahmoody M. Fungal infections in solid organ recipients. Exp Clin Transplant 2005;3:385-9.  Back to cited text no. 7
    
8.
Milne LJ. Fungi, Mackie and McCartney Practical Medical Microbiology. In:Collee JG, editor. 14th ed, Vol. 2. Chapter 41. Edinburgh: Churchill Livingstone; 1996. p. 695-717.  Back to cited text no. 8
    
9.
Darwazeh AM, Lamey PJ, Fisher BM. Candidia in immunocompromised host. J Med Microbiol 1990;16:43-8.  Back to cited text no. 9
    
10.
Rifkind D, Marchioro TL, Schneck SA, Hill RB. Systemic Fungal Infections Complicating Renal Homotransplantation and Immunosuppressive Therapy: Clinical, Microbiological, Neurologic and Pathologic Features. Am J Med 1967;43:28-32.  Back to cited text no. 10
    
11.
Al-Mohaya MA, Darwazeh A, Al-Khudair W. Oral fungal colonization and oral candidiasis in renal transplant patients: The relationship to miswak use. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93:455-60.  Back to cited text no. 11
    
12.
Lopez Pintor R, Hernandez G, De arriba L, De Andres A. Comparision of oral lesion prevalence in renal transplant patients under immunosuppressive therapy and healthy controls. Oral Dis 2010;16:89-95.  Back to cited text no. 12
    
13.
Douglas L. Candida biofilms and their role in infection. Trends Microbiol 2003;11:30-7.  Back to cited text no. 13
    
14.
Güleç AT, Demirbilek M, Seçkin D, Can F, Saray Y, Sarifakioglu E, et al. Superficial fungal infections in 102 renal transplant recipients: A case-control study. J Am Acad Dermatol 2003;49:187-92.  Back to cited text no. 14
    
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Dignani MC, Solomkin J, Anaisse EJ. Candida. Textbook of Clinical Mycology. 1st ed. China: Churchill Livingstone;2003:195-239.  Back to cited text no. 15
    
16.
Güleç AT, Demirbilek M, Seçkin D, Can F, Saray Y, Sarifakioglu E, et al. Superficial fungal infections in 102 renal transplant recipients: A case-control study. J Am Acad Dermatol 2003;49:187-92.  Back to cited text no. 16
    
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Pozzilli C, Marinelli F, Romano S, Bagnato F. Cortico steroids treatment. J Neurol Sci 2004;223:47-51.  Back to cited text no. 17
    
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Gladdy RA, Richardson SE, Davies HD, Superina RA. Candida infection in pediatric liver transplant recipients. Liver Transpl Surg 1999;5:16-24.  Back to cited text no. 18
    
19.
Dongari-Bagtzoglou A, Dwivedi P, Ioannidou E, Shaqman M, Hull D, Burleson J. Oral Candida infection and colonization in solid organ transplant recipients. Oral Microbiol Immunol 2009;24:249-54.  Back to cited text no. 19
    
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Barry AL, Brown SD. Fluconazole disk diffusion procedure for determining susceptibility of Candida species. J Clin Microbiol 1996;34:2154-7.  Back to cited text no. 20
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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