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Comparison of the Virulence of Different Candida albicans Samples Isolated from HIV-Positive Patients and Denture Stomatitis Lesions: In Vitro and In Vivo Tests

Fernanda Freire*, Felipe de Camargo Ribeiro, Damara da Silva Ávila, Cristiane Aparecida Pereira, Juliana Campos Junqueira, Antonio Olavo Cardoso Jorge

Department of Biosciences and Oral Diagnosis, Institute of Science and Technology, UNESP–University, Estadual Paulista, São José dos Campos, Brazil

*Corresponding Author:
Fernanda Freire
Department of Biosciences and Oral Diagnosis; Institute of Science and Technology
UNESP–Univ Estadual Paulista, São José dos Campos, Brazil
Tel: +55 12 39479033
Fax: +55 12 39479010
E-mail: [email protected]

Received date: 12/12/2016; Accepted date: 30/01/2017; Published date: 07/02/2017

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The objective of this study was to evaluate the in vitro and in vivo virulence of candidiasis induced by Candida albicans. Four clinical samples, two from HIV-positive patients (14/60) and two from denture stomatitis (DS) lesions (32/62), and one reference strain were used in this study. Initially was evaluated the in vitro secretion of hemolysin, lipase, phospholipase and proteinase enzymes; and cell surface hydrophobicity. Following, mice were immunosuppressed and inoculated with C. albicans suspensions. After six days, the animals were euthanized and the tongues were removed for macroscopic and histological analysis. All samples produced the virulence factors; however the samples from HIV-positive patients were more virulent. In the macroscopic analysis, all groups showed candidiasis lesions, without significant difference between them. The group inoculated with isolates of C. albicans from HIV-positive patients had higher amount of yeasts and hyphae (p=0.0036), and more tissue damage (p=0.0016). The inflammatory infiltrate showed statistical difference between the strains 14 and 62, and also between samples 62 and 32 (p<0.0001). Based on these results it can be concluded that the clinical samples from HIVpositive patients were more virulent. The experimental model of this work was essential to increase our understanding of the pathogenicity of C. albicans.


Candida albicans, Oral candidiasis, Immunosuppression, Experimental infection, Mice model


Fungal infections in the oral cavity are mainly caused by Candida albicans species [1,2], which constitutes about 70% of Candida spp. in mouth isolated [2]. Colonization and infection by C. albicans yeast are mediated by the formation of a biofilm, which is composed of a heterogeneous mixture of blastoconidia, pseudohyphae and hyphae embedded in extracellular polymeric substances that form channels and pores and exhibit different phenotypic characteristics than planktonic Candida. Biofilm can maintain the integrity of the cells, protecting from phagocytosis and limiting the diffusion of antifungal [3]. In addition, other virulence characteristics of C. albicans, such as adhesion to mucosal cells, ability to convert from a single-celled yeast to a filamentous form and the secretion of hydrolytic extracellular enzymes, which are utilized in the process of host tissue invasion and liberation of nutrients, make C. albicans a pathogen that causes a broad spectrum of infections in different host sites [4].

C. albicans biofilms are frequently associated with the occurrence of denture stomatitis [5,6]. Denture stomatitis is a common inflammatory reaction, multifactorial etiology, which is usually associated with Candida species, particularly C. albicans, due to its high virulence, ability to adhere and form biofilms on oral cavity tissues and denture surfaces [7].

In HIV patient, oral manifestations are the most important and earliest indicators. There are seven cardinal signs of HIV infection: oral candidiasis, hairy leukoplakia, Kaposi sarcoma, linear gingival erythema, necrotizing ulcerative gingivitis, necrotizing ulcerative periodontitis, and non-Hodgkin lymphoma. The features mentioned above are present in 50% of patients with HIV infection and 80% in patients with AIDS [8]. Candidiasis is caused due to C. albicans [9]. There are four forms of Candida infection which includes pseudomembranous candidiasis, erythematous candidiasis, hyperplastic candidiasis, and angular cheilitis. It has been noted that one or more combination of the above said may be present in patients [8]. Low CD4 count is present in all the above four forms of candidiasis [9].

The animal model in mice has been useful in the development of experimental candidiasis, because it has no Candida spp. as a constituent of the microbiota and secondary immune response against this microorganism. In addition, this animal is easily obtained in large numbers, presents the immune system more similar to humans and their maintenance is cheaper [10,11].

The importance of analyzing clinical isolates is because they may differ in virulence factors. Therefore, a detailed characterization of C. albicans virulence factors is necessary not only to understanding the process of infection in detail but also to generating new and more effective anti-fungal compounds [12].

Thus, the objective of this study was to evaluate the production of different virulence factors in vitro, and compare candidiasis induced by clinical isolates of C. albicans in an experimental mice model.

Materials and Methods

Ethics Committee

The Animal Research Ethics Committee from the Institute of Science and Technology at UNESP, approved this study under protocol number 11/2014- CEUA/ICT-CJSC-UNESP.


The Four clinical samples of C. albicans were used, two from HIV patients (14 and 60) and two from other individuals with denture stomatitis lesions (32 and 62). One reference strain C. albicans (ATCC 18804) (American Type Culture Collection-ATCC) was also included in this study. The clinical samples were previously isolated and identified [13,14], and together with the reference strain, were maintained in our laboratory stock collection at –80°C.

In Vitro Virulence Tests

For the assessment of enzymatic and hemolytic activity, each sample was seeded in Sabouraud dextrose agar (SDA; Difco, Le Pont de Claix, France) and incubated at 37ºC for 24 h. Next, the samples were equidistantly spot-inoculated (~6 mm) on the specific medium for each enzyme studied. Each sample was tested 8 times, and the plates were incubated at 37°C for 2-7 days.

Proteinase secretion was assessed using a mix of two culture media [15]. The medium A, sterilized by autoclaving, was composed of C6H12O6, KH2PO4, MgSO4 (Labsynth, Diadema, SP, Brazil) agar (Difco, Le Pont de Claix, France) and distilled water. The medium B, sterilized by filtration, was composed of bovine albumin fraction V, riboflavin, nicotinic acid, thiamine hydrochloride (Sigma-Aldrich, Milwaukee, WI, USA) and distilled water.

Phospholipase production [16] by the Candida isolates was assessed using SDA containing NaCl, CaCl2 (Labsynth, Diadema, SP, Brazil), and sterile egg yolk emulsion without the addition of potassium tellurite (Himedia, Mumbai, India).

Lipase activity [17] was determined by growing the sample on a medium containing peptone (HiMedia), CaCl2, NaCl (Labsynth), agar (Difco), Tween 80 (Sigma-Aldrich, Milwaukee, WI, USA), and distilled water.

Hemolytic activity [18] was assessed using SDA (Difco) supplemented with C6H12O6 (Labsynth) and fresh sheep blood (Cecon, São Paulo, SP, Brazil).

The enzymatic and hemolytic activities were determined after the incubation period. The colony diameter and the total diameter of the colony and precipitation zone (Pz) were measured, and the enzymatic activity was scored using the method described by Price et al. [16]. The Pz value representing the ratio of the colony alone to the diameter of the colony plus the precipitation zone. The results were classified as follows: no activity (Pz=1), moderate activity (0.64 ≥ Pz<1) and strong activity (Pz<0.64).

The CSH (Cell Surface Hydrophobicity) [19] of each sample was determined through addition of 1 ml of xylene (Sigma-Aldrich, Milwaukee, WI, USA) in each suspension of Candida spp. Suspension without xylene was utilized as control. After incubation in a water bath at 37°C for 40 min, the lower aqueous phase was carefully collected and measured directly by spectrophotometric readings at 520 nm. CSH was expressed as a percentage of yeast adherence to xylene and was determined by the formula [(C0) CH)/C0] × 100, where C0 is the result of the control tube and CH is the result of the test tube. Each sample was tested in triplicate. The highly hydrophobic samples were those with values greater than 50%, whereas those that were moderately hydrophobic exhibited values between 20 and 50%, and hydrophilic samples were those with values below 20%.

Induction of Oral Candidiasis

The fungal inoculum preparation: Before the oral candidosis induction, we did the collection in the mouth of the mice with swab, sowed on Sabouraud dextrose agar and it was incubated at 37°C for 48 h to verify the absence of Candida. The five samples were seeded onto SDA (Difco) and incubated at 37°C for 24 h. Next, each sample was grown in Yeast Nitrogen Base liquid medium (Himedia, Mumbai, India) with 100 μM of glucose added (Vetec, Rio de Janeiro, Brazil), and then incubated at 37°C for 18 h. Cells were collected by centrifugation and washed three times with Phosphate Buffered Saline (PBS). The pellet was resuspended in 10 ml of PBS and adjusted to 108 viable cells/ml after counting in a Neubauer chamber (Laboroptik GMBH, Bad Homburg, Germany).

Experimental animals: Fifty adult male mice (Mus musculus, Albinus, Swiss), weighing 30 to 60 g and with no Candida in their buccal cavities, were included in the study. The food and water source were ad libitum, and the animals were kept in ventilated racks with a capacity of 5 animals. Ten animals were used for the study of experimental candidiasis induced by the C. albicans standard sample, and forty animals in the study of experimental candidiasis induced by the clinical isolates, divided among the groups: 14, 60, 32 and 62 (n=10 for each group).

The methodology described by Takakura et al. [10] was used to induce experimental candidiasis with some modifications. Briefly, the animals were immunosuppressed with 2 subcutaneous injections of prednisolone (Depo-Medrol, Laboratórios Pfizer Ltda., Guarulhos, SP, Brazil) at a dose of 100 mg/kg of body weight 1 day before and 3 days after infection with Candida. Tetracycline chloride (Terramicina, Laboratórios Pfizer Ltda., Guarulhos, SP, Brazil) was administered in the drinking water at a concentration of 0.83 mg/ml beginning 1 day before infection and maintained throughout the experiment. Intramuscular injection of chlorpromazine chloride (10 mg/kg of body weight; Amplictil, Sanofi Aventis, Suzano, SP, Brazil) was used to sedate the animals.

A sterile swab (Absorve, Cral, São Paulo, SP, Brazil) soaked in the C. albicans suspension was used to inoculate the sedated mice by rubbing the swab for 1 minute on the tongue dorsum in order to induce oral candidiasis.

The euthanasia of mice was performed within 7 days after the first immunosuppression. This procedure was performed by administration of an overdose of anesthetic. Tongues were then removed for macroscopic and microscopic analysis.

Macroscopic analysis of candidiasis on the tongue dorsum of mice: Characteristic lesions of candidiasis on the tongue dorsum were observed using a stereomicroscope (Zeiss, Göttingen, Germany). In order to quantify the number of lesions on each tongue dorsum, scores were assigned from 0 to 4: 0, normal; 1, white patches on less than 20% of the surface; 2, white patches covering between 21% and 90% of the surface; 3, white patches on more than 91% of the surface; and 4, thick white patchy pseudo membranes covering more than 91% of the surface [10].

Optical microscopy of the tongue dorsum of mice: For the purpose of microscopic analysis of the lesions, the tongues were fixed in 10% formalin for 24 hours. After embedding in paraffin, 5 μm tissue slices were cut and stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS). The presence of candidiasis was analyzed using optical microscopy (Olympus, CX41, and Tokyo, Japan) at X400 magnification.

Candidiasis lesions were quantified by counting the number of hyphae and epithelial lesions in histological sections stained with PAS and HE, respectively. For each stain, two histological sections were randomly selected for each animal. In each histological section, 25 histologic fields were analyzed in an anteroposterior direction, resulting in a total of 50 histologic fields analyzed.

The presence of yeasts and hyphae was quantified according to the methodology of Junqueira et al. [20], attributing the following scores to histologic fields: 1, 1 to 5 yeasts/hyphae; 2, 6 to 15 yeasts/hyphae; 3, 16 to 50 yeasts/hyphae; and 4, more than 50 yeasts/hyphae. For statistical analysis, a median of the scores obtained from the 50 histologic fields was determined per animal. The intensity of the tissue lesions was evaluated by counting the number of histologic fields with the presence of epithelial lesions, such as flaking, loss of filiform papillae, loss of stratification, epithelial hyperplasia, exocytosis, spongiosis, acantholysis, hyperkeratosis, disorganization of the basal layer and intraepithelial micro abscesses development. The mean of the number of histologic sections with epithelial lesions was determined per animal for statistical analysis.

Both analyses, macroscopic and microscopic, were performed by a blinded researcher.

Statistical analysis

The data obtained were analyzed statistically with the aid GraphPad Prism 5 software (GraphPad Software, Inc, La Jolla, CA, USA). Kruskal-Wallis and Dunn's multiple comparison (p ≤ 0.05) tests were applied to results.


In Vitro Virulence Tests

All samples produced the virulence factors evaluated in vitro (Table 1). The samples that had the lowest pz values, indicating greater enzymatic activity were C. albicans 60 and C. albicans 14, both isolated from HIV-positive patients.

Virulence Factor C. albicans
C. albicans (ATCC) C. albicans 60 C. albicans 14 C. albicans 32 C. albicans 62
Hemolysin1 0.47±0.04AB 0.46±0.05AB 0.41±0.03AB 0.52±0.08B 0.50±0.07AB
Lipase1 0.27±0.04AB 0.24±0.04B 0.27±0.05AB 0.31±0.07AB 0.32±0.08B
Phospholipase1 0.71±0.08A 0.67±0.02AB 0.61±0.05B 0.68±0.02A 0.70±0.03A
Proteinase1 0.45±0.06A 0.33±0.03B 0.34±0.03B 0.44±0.04AC 0.39±0.06C
CSH2 29.79±4.08A 47.13±6.13B 48.84±3.40B 39.15±3.23C 40.37±3.70C
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