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Virulence Factors of Candida albicans Isolates from the Oral Cavities of HIV-1-Positive Patients Tatiany O.A. Menezes1, Luciana C.S. Gillet1, Sílvio A.F. Menezes1, Rosimar N.M. Feitosa3, Marluísa O.G. Ishak3, Ricardo Ishak3, Sílvia H. Marques-da-Silva2 and Antonio C.R. Vallinoto*,3 1
Federal University of Pará (Universidade Federal do Pará – UFPA), Health Sciences Institute, School of Dentistry; Evandro Chagas Institute, Bacteriology and Mycology Section, Mycology Laboratory, Ananindeua, Pará, Brazil; 3 Federal University of Pará, Institute of Biological Sciences, Virology Laboratory, Belém, Pará, Brazil 2
Abstract: The present study assessed the phenotypic aspects of oral-cavity Candida albicans isolates from 300 HIV-1positive patients, relating the most commonly investigated virulence factors (enzyme typing and germ-tube formation) to the most common morphotypes. The samples were seeded into specific media for isolation and subsequent identification using the automated Vitek 2 system. The following assays were performed for phenotypic characterization: morphotyping, germ-tube formation and enzyme typing. Out of 300 collected samples, 144 tested positive for yeasts of the Candida genus, 98 (32.7 %) of which were identified as C. albicans. The latter samples were attributed to seven different morphotypes; the three most common morphotypes were 7208 (49 %), 7308 (14.3 %) and 3208 (13.3 %). All of the C. albicans isolate samples formed germ tubes and produced the enzymes proteinase and phospholipase, with an activity classified as intermediate to high. Due to the identification of virulence factors among the analyzed samples, monitoring of HIV-1-positive patients colonized by different morphotypes must be established because these morphotypes are extremely pathogenic and can trigger severe fungal infections.
Keywords: HIV-1, Co-infection, Candida albicans, oral cavity, virulence factors. INTRODUCTION The Candida genus comprises approximately 200 species, of which Candida albicans is most commonly related to colonization processes and pathogenicity in the oral cavity, whereas the other species (C. tropicalis, C. krusei, C. guillermondii, C. glabata and C. parapsilosis) are less commonly observed [1-3]. Candidiasis is an opportunistic disease of fungal etiology caused by the proliferation of Candida yeasts and is asymptomatic in approximately 30 to 50 % of individuals [4, 5]. Moreover, approximately 90 % of HIV-1-seropositive patients suffer from oral mucosal candidiasis at least once during the course of infection by the virus [6-9]. The virulence of C. albicans arises from the synergistic action of several aggression mechanisms [10, 11], such as the high production capacity of host tissue-degrading exoenzymes, morphological dimorphism (mycelium formation), and phenotypic switching. These mechanisms can lead to an altered adherence to epithelial cells, susceptibility to antifungals, fungicidal activity of neutrophils and production of toxins, which, combined with a weak host immune response, may lead to candidiasis [1215]. Phenotypic-morphological alteration and the secretion of proteinase and phospholipase are of paramount importance *Address correspondence to this author at the Universidade Federal do Pará, Instituto de Ciências Biológicas, Laboratório de Virologia, Rua Augusto Correa no. 1, 66075-910, Belem, Para, Brasil; Tel: +55 91 8115 85 78; Fax: +55 91 3201 75 87; E-mail:
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in the process of host invasion [16, 17], and several typing methods, such as morphotyping, germ-tube testing and enzyme typing, have been developed to better identify and characterize Candida isolates, as well as to determine the virulence of the yeast [13, 18-20]. In several studies, the severity and chronicity of oral candidiasis in HIV-1-positive patients has been attributed exclusively to the deficient immune response of the host rather than to the virulence of the yeast itself [17, 21]. In contrast, a recent study on phenotyping and genotyping suggested that oral infection is mainly associated to C. albicans isolates with a greater ability to modify virulence factors in HIV-1-positive individuals, thus rendering these morphotypes more virulent [22]. In this context, the aim of the present study was to assess the phenotypic profile of oral-cavity C. albicans isolates from HIV-1-positive patients, identifying the main virulence factors of the yeasts and the most common morphotypes for use as risk indicators for oral candidiasis. MATERIALS AND METHODS Population and Samples Collection The present work was a cross-sectional study with the sample size calculated according to the percentage of prevalence of HIV-1-positive patients colonized by C. albicans in the oral mucosa, which was estimated at 60 % according to the scientific literature [22-29]. Samples were collected after having obtained approval from the Research Ethics Committee of the Health Sciences Institute at the Federal University of Pará (process No. © 2013 Bentham Science Publishers
2 Current HIV Research, 2013, Vol. 11, No. 4
120/09) and the signature of an informed consent form from the patients who chose to participate in the study. A total of 300 HIV-1-positive patients of both genders who were over 18 years old and being treated at the Unit for Special Infectious and Parasitic Diseases (URE-DIPE) in Belém, State of Pará, Brazil, were included in the study, provided that they presented no clinical manifestation of oral candidiasis and were not receiving antimicrobial treatment. Isolation and Identification of C. albicans To isolate Candida yeasts, saliva was collected from the jugal mucosa with sterile swabs. Duplicate samples were then seeded on Sabouraud dextrose agar with chloramphenicol (MERCK, Darmstadt, Germany) and transported to the Mycology and Bacteriology Laboratory at the Evandro Chagas Institute (IEC/SVS/MS). The developing moist white-yellowish yeast-like colonies were kept in the mycological collection at room temperature for 15 days. The identification of Candida yeasts was performed using the automated Vitek 2 system (BioMérieux, Grenoble, France).
Menezes et al.
and Price et al. [34], respectively. Suspensions of approximately 5 L of each isolate in sterilized water equaling 1 McFarland standard were incubated at equidistant points in proteinase agar (DIFCO, Baltimore, USA) or phospholipase agar (DIFCO, Baltimore, USA) for proteinase and phospholipase assays, respectively. The plates were incubated for 7 days (proteinase) and 4 days (phospholipase) at 37 °C; the tests were performed in duplicate. The enzymatic activity of each sample was indicated by the formation of an opaque halo of precipitation around the point of the yeast-sample inoculation and quantified by calculating the Pz value, i.e., dividing the colony diameter (cd) by the diameter of the zone of precipitation (dp), measured in centimeters: Pz = cd/dp. The results were divided into the following levels to better distinguish and visualize the enzymatic activity of Candida yeasts: Pz = 1.00 (no production), 0.99 Pz 0.75 (weak production), 0.74 Pz 0.50 (intermediate production) and 0.49 Pz 0.25 (high production). RESULTS Isolation and Identification of C. albicans
Morphotyping Morphotyping was performed according to Phongpaichit et al. [30] and Hunter and Fraser [31]. C. albicans yeasts were cultivated in Sabouraud dextrose agar (MERCK, Darmstadt, Germany) for 48 h at 25 oC. Subsequently, suspensions were made in sterile saline solution (the inoculum was adjusted to an equivalent of 3 McFarland standards). Each suspension was spread over a malt-extract agar (MERCK, Darmstadt, Germany) plate with a sterilized swab to generate a standardized 8 mm wide by 60 mm long streak. The plates were then incubated for 10 days at 25 oC (each assay was performed in duplicate). The readout was made based on the macromorphological properties of the fringe and the topography of the colony. The samples were then classified with the four-digit sequence proposed by Hunter and Fraser [31]. Germ-Tube Test A suspension containing 0.5 mL fetal bovine serum (Cultilab, São Paulo, Brazil) was obtained from the isolated colony and incubated in a test tube for a maximum of 3 h at 37 ºC. During this period, a drop of suspension from each sample was collected at hourly intervals, placed between a slide and cover slip and examined under a microscope (40x objective). The presence of germ tubes, i.e., small filaments projecting from blastoconidia without any constriction at the parent cell, allows the presumptive identification of C. albicans [32]. Enzyme Typing The production of the enzymes proteinase and phospholipase was assessed according to Ruchel et al. [33]
Yeast-like Candida colonies were isolated from 144 of the 300 collected samples, of which 32.7 % (98) were biochemically identified as C. albicans. Among these samples, 53.1 % of the patients were female, and 46.9 % were male. The average age was 38 years, and the age range was between 18 and 66 years, with a pronounced peak in the number of patients between 31 and 59 years (Table 1). Phenotypic Characteristics of C. albicans Morphotyping Morphotyping of the 98 oral-cavity C. albicans isolates from HIV-1-positive individuals allowed the characterization of 7 different morphotypes (Table 2). The most common morphotypes were 7208 (49 %), 7308 (14.3 %) and 3208 (13.3 %). Germ-Tube Test The formation of germ tubes by C. albicans in the presence of fetal bovine serum was observed after the 2 h interval in all of the oral-cavity isolates from HIV-1-positive individuals. Enzyme Typing All of the C. albicans samples isolated from the oral cavities of HIV-1-positive individuals produced proteinase and phospholipase with intermediate to high activity (Table 3). To assess the virulence of the most common morphotypes (7208, 7308 and 3208), the relationship between the enzymatic activity of proteinase and phospholipase and the morphotypes was studied. For this purpose, the mean and standard deviation of each morphotype was calculated, and the samples were subjected to an analysis of variance. There was no statistically significant difference among the morphotypes (Table 4).
Candida albicans Isolates from HIV-1-Positive Patients
Table 1.
Current HIV Research, 2013, Vol. 11, No. 4
Demographic Data and Laboratory Distributions of the Oral Cavity Candida albicans Isolates from HIV1-Positive Individuals Variables
N
%
Candida albicans
98
32.7
Candida non-albicans
46
15.3
Negative
156
52
Total
300
100
18 - 30
18
18.4
31-59
78
79.6
> 60
2
2.0
Total
98
100
Identification of Candida
Table 3.
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Activity Profile of Proteinase and Phospholipase Produced by the Oral Cavity Candida albicans Isolates from HIV-1-Positive Individuals Biochemical Variable
N
%
HP
45
45.9
LP
6
6.1
IP
47
48.0
Total
98
100
HP
52
53.1 %
LP
2
2.0 %
IP
44
44.9 %
Total
98
100.0 %
Proteinase
Age Range (Years)*
Phospholipase
Gender Female
52
53.1
Male
46
46.9
Total
98
100
Yes
80
81,63
No
18
18,36
Total
98
100
Candida albicans
392.73 ±264.49
-
Candida non-albicans
463.98 ±292.01
-
Negative
452.94 ±274.00
-
HP = high production 0.49 Pz 0.25; LP = low production 0.99 Pz 0.75; IP = intermediate production 0.74 Pz 0.50; N = sample size.
Table 4.
Relation of the Most Common Morphotypes with the Production of Proteinase and Phospholipase
Use of Antiretrovirals
CD4+ T Cells (Mean/±SD)
Candida albicans
4,044.46 ±16,840.15
-
Candida non-albicans
11,657.89 ±43,371.81
-
397.25 ±958.13
-
N = sample size; *individuals with Candida albicans
Table 2.
Morphotype Distribution of the Oral Cavity Candida albicans Isolates from HIV-1-Positive Individuals
Morphotypes
Fringe Characteristics
N
%
0008
Hairy only
3
3.1
1208
Discontinuous fringe, 3 to 5 mm, untextured, hairy
7
7.1
2208
Discontinuous fringe, 2 mm or less, untextured, hairy
2
2.0
3208
Discontinuous fringe, 2 mm or less, untextured, hairy
13
13.3
5338
Continuous fringe, 3 to 5 mm, intermediate texture, hairy
11
11.2
7208
Continuous fringe, 2 mm or less, untextured, hairy
48
49.0
7308
Continuous fringe, 3 to 5 mm, untextured, hairy
14
14.3
98
100
Total
Proteinase Mean ± SD
Phospholipase Mean ± SD
7208
0.4925 ± 0.1609
0.4731 ± 0.1165
7308
0.4264 ± 0.1713
0.5286 ± 0.1276
3208
0.4169 ± 0.1744
0.5385 ± 0.1271
p = 0.2088 (ANOVA); p = 0.1179 (ANOVA); SD = standard deviation.
DISCUSSION
HIV-1 Viral Load (Mean/±SD)
Negative
Morphotypes
Oral candidiasis caused by C. albicans is considered one of the most common opportunistic infections in HIV-1positive individuals. C. albicans is the most commonly isolated species, and it can trigger systemic infections in immunocompromised individuals. Several studies have suggested an increased colonization of the oral cavity of HIV-1-positive individuals by C. albicans, rather than by non-albicans species, ranging between 51.56 % and 93 % [9, 23, 24, 27, 35-38]. These findings are in agreement with the findings of the present study, in which 68 % of the 144 isolated Candida yeasts were identified as C. albicans. No studies relating age range to gender with respect to the colonization of HIV-1-positive individuals by C. albicans could be found. In the present study, 79.6 % of the colonized individuals were between 31 and 59 years of age, and approximately half of the patients were females (53.1 %). This pattern may occur because 20 to 30 % of all women bear a colonization of the vaginal mucosa that can potentially develop in other sites of the body, especially during adulthood [14]. The expression of the parasite/host relationship depends on the balance between the virulence of the microorganism and the host defense. For the host, colonization by yeasts
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prior to the decrease in the capacity of the immune response of HIV-1-positive individuals seems to promote oral candidiasis [19, 22]. However, in addition to the factors inherent to the host, it has been proposed that there are differences in the pathogenicity of Candida sp. isolates. Specifically, the fungus is not merely a passive participant in the infectious process, but rather, several virulence factors are proposed regarding the oral cavity, which are poorly investigated to date. During the course of the investigation, a combination of several methods was necessary to assess the virulence of the yeast. This combination is in agreement with Menezes et al. [35], Vasconcellos et al. [39] and De Luca et al. [17], who have noted that the phenotypic methods commonly used in laboratories to characterize yeasts are based primarily on the observation of microscopic structures (morphotyping), enzyme-activity tests, germ-tube formation and carbonsource assimilation. In the present study, 7 different C. albicans morphotypes were characterized following the typing method developed by Hunter and Fraser [31]. The morphotype with continuous fringe, parallel filaments of 2 mm length or shorter, no texture and a surface topography with hairs was the most commonly detected morphotype in HIV-1-positive individuals, followed by the morphotype with continuous fringe and filaments of 3 to 5 mm length. Among the reviewed studies [19, 39-41], there was a consensus that the C. albicans morphotypes with continuous fringe formation were the most common; thus, this formation is considered as an attribute of virulence that favors the fungal adherence capacity, rendering the fungus more virulent. However, it must be noted that each study identified a different morphotype. This diversity of the results may be because a variety of populations was studied, including children with Down syndrome and diabetic adults, and may also be due to the use of different typing methods. Despite the discrepancies, the results are acceptable given the scarcity of studies regarding morphotypes in HIV-1-positive patients. The ability of C. albicans to form hyphae has been suggested to be an important pathogenicity factor because it increases the capacity to adhere and penetrate human cells, thus promoting fungal dissemination [32, 42]. In the present study, all of the C. albicans isolates presented germ-tube formation during the established time period, corroborating previous studies [10, 19, 43, 44]. Exoenzyme (proteinase and phospholipase) production by C. albicans yeasts constitutes a critical virulence factor of this fungus [12, 15, 19, 20, 22, 45]. To better evaluate the exoenzymatic behavior of the C. albicans isolates, the precipitation zones were classified into four categories with regard to the production levels of proteinase and phospholipase: absent, weak, intermediate and high production. Among HIV-1-positive individuals, all of the C. albicans isolates were shown to produce proteinase and phospholipase at intermediate to high levels. This finding is in agreement with the findings of several previous studies [12, 14, 15, 19, 22, 42], which detected isolates with high production levels of proteinase and phospholipase in patients with different pathologies. The authors of the cited articles
Menezes et al.
have noted the extreme importance of these enzymes for host invasion because these enzymes promote the degradation of physical barriers and the evasion of the fungus from lymphocytes and phagocytic cells, ultimately favoring the colonization process. To assess the virulence factors of oral-cavity C. albicans isolates from HIV-1-positive individuals and to apply these data to the monitoring of colonized patients, the relationships between the main morphotypes and the production of the enzymes proteinase and phospholipase were chosen as riskfactor indicators for oral candidiasis. However, no direct relationship between the analyzed morphotypes (7208, 7308 and 3208) and the production of the exoenzymes could be found because all of the isolates were considered to be highly pathogenic due to high enzymatic activity. The identification of the virulence factors of oral cavity C. albicans isolates from HIV-1-positive individuals in this study suggests the importance of systematically studying a combination of factors related to patients. This research could help integrate the results of studies such as those of Owotade et al. [38], who highlighted a correlation between the increased fungal colonization of immunocompromised individuals and both the use of ill-fitting oral prostheses and the absence of antiretroviral therapy. Accordingly, clinical and dental monitoring must be established for HIV-1-positive individuals colonized by different C. albicans morphotypes, given that these morphotypes are extremely pathogenic and can trigger oral candidiasis and be a source for fungal dissemination in the host. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGMENTS We thank the patients who participated in this study. The present study was supported by funds from the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPQ, process no. 471629/2011-1). PATIENT CONSENT Declared none. HUMAN/ANIMAL RIGHTS Declared none. REFERENCES [1]
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Accepted: June 17, 2013
