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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 3  |  Page : 513-517
Comparative study of adherence of oral Candida albicans isolates from HIV sero-positive individuals and HIV sero-negative individuals to human buccal epithelial cells


Department of Microbiology, SDM College of Medical Sciences and Hospital, Dharwad-580 009, Karnataka, India

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Date of Web Publication22-Oct-2010
 

   Abstract 

Aim: Candida albicans occurs as a commensal of the gastrointestinal tract. Under predisposing conditions, candida can produce a broad array of infections. HIV seropositive individuals show increased oral colonization compared to the HIV seronegative healthy individuals. C. albicans shows a variety of pathogenic factors. We have studied one such factor here; the adherence property of
C. albicans isolated from HIV seropositive individuals and HIV seronegative to Human Buccal Epithelial Cells (HBEC) of normal healthy individuals. Materials and Methods: Concentrated oral rinse specimen were collected from 50 healthy volunteers (control group) and 25 HIV positive individuals (test group) and used for isolation of C. albicans. Adherence assay was done using C. albicans isolates from both groups on HBEC collected from HIV sero-negative, normal individuals. The adherence assay method described by Kimura and Pearsall was used with minor modification. Statistical Analysis Used: The results of Adhesion assay were subjected to statistical analysis using student "t" test. Results: C. albicans isolated from both the groups were tested for their adherence property to normal HBEC. The isolates from test group showed more adherence to HBEC compared to those of the control group, with average rate of adherence being 56.6%. The control group showed average adherence rate of 29.1%. This was statistically significant with p value equal to 0.05. Conclusion: C. albicans from HIV infected individuals showed significant rise in degree of adhesion to the buccal epithelial cells than the isolates from healthy controls, suggesting the enhancement of virulence factors such as adherence in the presence of predisposing condition.

Keywords: Adherence, C. albicans, Human buccal epithelial cells, HIV, oral candida carriage, oral candidiasis

How to cite this article:
Jain PA, Veerabhadrudu K, Kulkarni R D, Ajantha G S, Shubhada C, Amruthkishan U. Comparative study of adherence of oral Candida albicans isolates from HIV sero-positive individuals and HIV sero-negative individuals to human buccal epithelial cells. Indian J Pathol Microbiol 2010;53:513-7

How to cite this URL:
Jain PA, Veerabhadrudu K, Kulkarni R D, Ajantha G S, Shubhada C, Amruthkishan U. Comparative study of adherence of oral Candida albicans isolates from HIV sero-positive individuals and HIV sero-negative individuals to human buccal epithelial cells. Indian J Pathol Microbiol [serial online] 2010 [cited 2019 Sep 17];53:513-7. Available from: http://www.ijpmonline.org/text.asp?2010/53/3/513/68300



   Introduction Top


Candida albicans occurs as a commensal in the gastrointestinal tract. Under predisposing conditions, candida can produce a broad array of infections, from superficial mild infections to deep seated fatal infections. Oral Candidiasis (OC) is one of the most common forms of candidiasis, which presents as mucocutaneous infections of the oral cavity involving mainly tongue, palate, gingival and fissures of the mouth. [1],[2] HIV infection is one of the common pre-disposing conditions for OC. C. albicans is the most commonly isolated species from OC and asymptomatic carriage in these patients. [3],[4],[5],[6]

OC in HIV-infected patients is studied extensively with its relation to CD4 + counts and considered to be an adjunct clinical marker of HIV disease progression. [7] Occurrence of OC and its progression in HIV is not clearly understood. [8] Several studies on oropharyngeal carriage of candida showed that, candida carriage rate and density are significantly higher in HIV-infected patients than in healthy HIV seronegative population. [4],[9] This higher colonization may be a pre-requisite for development of clinical candidiasis.

Phenotypical and molecular studies suggest that OC in HIV infection may be associated with the selection of strains with altered virulence determinants leading to colonization with candida, virulent in these hosts. [10],[11],[12]

The initial step in the establishment of colonization or disease is adherence of the fungus onto the host cells. The mechanisms by which a fungus attaches and maintains contact with host surface includes nonspecific mechanisms such as ionic and hydrophobic interactions as well as specific recognition between ligand and receptors. Components of the organisms which promote the host recognition and colonization are referred as adhesins. [13] Candidal adhesion on various host cells including human buccal epithelial cells (HBEC) is extensively studied so far. [14] Extensive number of studies have been done to isolate and characterize, putative adhesins of C. albicans, as well as ligands present on the HBEC. [15],[16],[17],[18],[19]

Several investigators have studied adhesion pattern of C. albicans isolates from HIV infected patients by in vitro methods. Conclusions of these studies vary in opinion. [20],[21],[22],[23] However, such studies may reveal important changes occurring in adherence process of candida to HBEC under the influence of immunocompromised condition of the host and may also help us to get an insight to the importance of adhesion molecules in the pathogenesis of OC in immunocompromised hosts. Very few studies of this kind are reported from Indian scenario. [11]

Aim

  • Detection and comparison of oral candida carriage rate in HIV seropositive and HIV seronegative individuals.
  • Know the difference in adherence of C. albicans from HIV infected individuals and HIV seronegative healthy subjects to HBEC obtained from normal healthy individuals.

   Materials and Methods Top


The study was performed over a period of three months from June to August 2008. Clearance from the ethical committee of the institute was obtained before the commencement of work.

Study Population and Data Collection

Test group: Twenty five HIV seropositive individuals, tested for HIV antibodies, according to the NACO guidelines were included as test subjects. The oral cavity was thoroughly examined for the presence of OC or any other oral lesions. These subjects were further divided into two groups,

  • Test Group 1: Subjects with OC: 6
  • Test Group 2: Subjects without OC: 19
Control Group: A total of 50 HIV seronegative healthy subjects were selected as controls. The examination of the oral cavity was done. The subjects treated with antibiotics and/or antifungal agents, under immunosuppressive therapy three months previous to the study and those having diabetes mellitus or any other kind of immunosuppression were excluded.

Collection of Samples

Samples were collected after obtaining informed consent from the subjects. Concentrated oral rinse method was used to collect the specimen according to the method explained by Samaranayake et al. [24] In brief, subject was instructed to keep 10 ml sterile Phosphate Buffer Saline (PBS) (pH: 7.2) in mouth for 1 minute and then spit it back in to a sterile container.

Culture

The oral rinse specimen was immediately transferred to the laboratory. The sample was centrifuged at 2000 rpm for 10 minutes. The supernatant was discarded and the pellets were further suspended in 1.0 ml of PBS and vortexed for 30 seconds. Of this suspension 100 μl was inoculated over the Sabouraud's Dextrose Agar (SDA) plate containing chloramphenicol. Plates were then incubated for 48 hrs at 37oC. The growth was studied for colony morphology and number of colony forming units (CFU) /ml. Gram stain was performed to confirm the growth of yeast. The candida colonies were further identified to species level by using the standard techniques like germ tube test, corn meal agar morphology and sugar assimilation tests. [2] The properly identified candida strains were stocked in SDA vials and preserved at 4oC for future use.

The Adherence Assay

The adherence assay method described by Kimura and Pearsall [25] was used with minor modifications.

Preparation of Cells for Adherence Assay

Collection and preparation of pooled HBECs from normal healthy individuals

HBECs were collected by scrapping the inner-side of the cheeks with sterile wooden spatulas from five healthy volunteers after obtaining consent. Fresh HBEC were used for the adhesion assay. The cell suspension from each volunteer was washed thrice in PBS (pH: 7.2), suspended in PBS and pooled [Figure 1]a. The HBECs were then adjusted to a concentration of 1x10 5 cells/ml using Neubaur's counting chamber.
Figure 1a: Gram's stain of buccal epithelial cells collected from normal healthy individuals (Gram's, ×450)
Figure 1b: Gram's stain preparati on showing the budding yeast cells adhered on HBEC (Gram's, ×1000)


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Preparation of Candida cells

The candida strains were sub cultured from the stock vials on SDA, incubated at 37oC for 48 hours to check purity and viability of the cells. The colonies were then inoculated in 5 ml of Yeast Nitrogen Broth (YNB) containing 0.05 M galactose and incubated at 37oC for 24 hours. The cells in the stationary phase were selected for the study. The yeast cells were washed thrice in PBS (pH: 7.2) and final suspension was prepared in PBS (pH: 7.2) to a concentration of approximately equal to 1 x 10 7 cells/ml using turbidometre.

Adherence Test

A 200 μl of the candida cells were mixed with 200 μl of HBECs, in a ratio of 5:1 ratio. The mixture was incubated at 37oC for one hour with gentle shaking. Epithelial cells were then washed with PBS to remove unattached yeasts. A smear was made from the mixture and stained by Gram's stain. Minimum of 100 epithelial cells were screened in each preparation. The number of epithelial cells showing yeast cells adhered on them was noted and percentage of adherence was calculated [Figure 1]b. The average number of yeast cells adherent per HBEC was also calculated. The experiment was repeated four times and average of the four readings was taken.


   Results Top


Oral Candida Carriage Rate

Oral candida carriage distribution is presented in [Table 1]. A total of 25 HIV reactive subjects (test group) were tested for oral candida carriage. Of these 25 subjects, 17 (68%) showed candida carriage, while the HIV non reactive healthy individuals showed a carriage rate of only 40% (20/50). Comparison of candida isolation in test subjects with OC (test Group:1) and test subjects without OC (test Group:2) showed that candida carriage rate was 100 % (6 out of 6) in test Group: 1, as against 57.89 % ( 11/19) in test Group: 2.
Table 1: Comparison of candida carriage in HIV reacti ve and HIV non-reacti ve individuals

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Candida Species Diversity

Prevalence of C. albicans vs Non Albicans Candida (NAC) among test and control groups, as well as in Test Group: 1 and 2 is shown in [Table 2]. A total number of 46 candida isolates were recovered from 75 subjects. Among this, 26 isolates were recovered from test individuals, while 20 isolations were made from control subjects. C. albicans was 35 out of 46 (76.08 %) were; 11 isolates were NAC. All six isolates recovered from test group: 1, were C. albicans. A total of 20 isolates were recovered from test group: 2. 35% of the isolates from this group were NAC. In control group of the 20 isolates recovered, 80% were C. albicans while 20% were NAC.
Table 2: Prevalence of C. albicans Vs. NAC in the study groups

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Species diversity among candida isolates from test and control groups, as well as in test group: 1 and 2 is shown in the [Figure 2]. Of the 11 NAC isolated in the study belonging to seven different species. C. guiellermondii which is known to show high MIC to fluconazole, as well as C. krusei which is intrinsically resistant to fluconazole were found among the isolates obtained from test group:1.
Figure 2: Species distributi on among study groups

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Adherence of C. albicans to Normal HBECs

Evaluation of adherence of C. albicans isolates from HIV infected subjects and healthy individuals to normal, pooled, buccal epithelial cells showed in [Table 3]. A total of 19 oral C. albicans isolates from test group and 16 from control group were tested for the adherence to pooled normal buccal epithelial cells. The isolates from test group showed more adherence to HBEC compared to that of control group, with average rate of adherence being 56.61 %. The control group showed average adherence rate of 29. 1 %. The average number of yeast cells adhered per buccal epithelial cells was 1.74 and 1.33 from test and control groups respectively. Adherence rate was less for isolates from test group: 1 (55.5%) compared to the isolates from test group: 2, (57.7%). However, the difference was not significant.
Table 3: Adherence of C. albicans to normal HBECs: Data shown is the average of 4 tests

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Standard strain of C. albicans MTCC 3017 showed 28.77% of adherence with 1.256 yeast cells adhered per HBEC.

The result from both the test and control groups was compared by using "t" test. C. albicans isolates from HIV infected individuals showed significant increase in the rate of adherence to the HBEC, compared to those isolates from control groups with p value equal to 0.05, which was statistically significant.


   Discussion Top


C. albicans is an endogenous pathogen which is the most virulent of all pathogenic Candida species and is the most common cause of human candidiasis in immunocompromised hosts. Previous data show higher rate of candida carriage and species variation in HIV positive patients than HIV negative subjects. This variation may be due to several factors such as diet, oral hygiene, lack of access for HAART, long term treatment with fluconazole etc. [3],[4],[5],[6],[9] Similar observations were made in the present study. The candida carriage rate in HIV reactive subjects was 68.0% and six different species of candida were recovered from them. In the HIV non-reactive subjects the carriage rate for candida was 40% and 4 different species of candida were recovered from them.

C. albicans was the most common isolate in both control and test groups of our study. Prevalence of NAC among HIV reactive individuals without OC was 35% vis-ΰ-vis 25% in HIV nonreactive subjects. Also there was greater variety of NAC isolated from asymptomatic HIV infected group, with five different species of candida; however, subjects with OC yielded only C. albicans. Increased association of C. albicans in the symptomatic stage may be resulting from the replacement of NAC by C. albicans, which may be attributed to its high adherence capacity to the HBEC in presence of altered host immune response. [8]

As recurrent oral infections with C. albicans are common in patients with HIV infection, we selected only C. albicans to perform adherence study. Previous studies showed, that C. albicans adheres to host cells more than any other species of candida. This implicates the relationship between the adherence capabilities and ability of C. albicans to colonize mucosal surfaces. [26]

In our study, C. albicans isolated from the HIV infected individuals had shown strong adherence to normal buccal epithelial cells compared to the isolates from healthy individuals. Macura and Bort (2001). [20] reported the strongest adherence between both fungi and epithelial cells isolated from a HIV positive subject in their study. [17] On the contrary, Pereiro et al. (1997) [21] reported that the adherence of C. albicans isolated from patients in the initial stages of AIDS to oral mucous cells was less compared to the isolates from subjects without HIV infection and adherence of C. albicans increased with the disease stage until it exceeded that of the normal subjects in proportion to the decrease in the CD4/CD8 ratio.

Interestingly, the percentage of adherence of C. albicans isolates from HIV positive subjects with OC (55.5 %) was marginally less than the isolates from HIV positive individuals without OC (57.7 %). Similar results from, Imbert-Bernard C et al. (1994) [22] show that the adherence of most of the C. albicans isolates from HIV positive patients with clinical OC to HBEC was weak. Whereas Sweet et al. [23] reported that, there is no significant difference in adherence detected between strains isolated from HIV-infected or AIDS subjects, or between strains isolated from C. albicans carriers (low salivary C. albicans counts) or subjects with OC.

Adhesion is the corollary of interaction between the receptors on host cells and adhesin molecules on the microbe. A variety of factors govern adhesion. Change in adherence may be associated with alteration of epithelial cell receptors in HIV infected subjects and change in the adhesins on candida strains. Thus studying the carriage rate of candida and their adherence throw light on the infection process. Findings from this study indicate that increased adherence plays an important role in the pathogenesis of oral candida infections in predisposing condition like HIV infection.

 
   References Top

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Correspondence Address:
Pavithra A Jain
Department of Microbiology, SDM College of Medical Sciences and Hospital, Dharwad -580 009
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.68300

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    Tables

  [Table 1], [Table 2], [Table 3]

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