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Year : 2015  |  Volume : 1  |  Issue : 1  |  Page : 27-31

Detection of virulence factors and phenotypic characterization of Candida isolates from clinical specimens

1 Wellcome Trust Research Laboratory, CMC, Vellore, Tamil Nadu, India
2 ICMR-DHR-VDRL, Sri Venkateshwara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
3 Department of Microbiology, Pondicherry Institute of Medical Sciences, Pondicherry, India

Date of Submission23-Jun-2015
Date of Acceptance03-Sep-2015
Date of Web Publication9-Nov-2015

Correspondence Address:
Sheela Devi Chandrakesan
Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry
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Source of Support: None, Conflict of Interest: None

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Context: Infections associated with different Candida species have increased over the last decade especially with nonalbicans Candida. Species identification and antifungal susceptibility are vital for treatment and management of Candida infections. Virulence factors, including biofilm formation (BF) and secretion of hydrolytic enzymes have been demonstrated in Candida albicans and few other nonalbicans Candida.
Aim: The aim of this study was to speciate and detect virulence factors of different Candida species isolated from a clinical specimen. Methods: All clinically significant Candida species isolated during the study period from various clinical samples were speciated by standard phenotypic methods and using the chromogenic medium. BF, Phospholipase and protease activities were determined by tissue culture plate method, precipitation, and bovine serum albumin agar medium respectively.
Results: C. albicans was the most common species isolated followed by Candida tropicalis and Candida krusei. Chromogenic medium (CHROM agar Candida) was found to be 92% specific for identification of Candida species. BF was observed more in urine isolates of C. tropicalis followed by C. krusei. Phospholipase activity was seen only in C. albicans, whereas protease activity was observed in all four species of Candida but most commonly in C. tropicalis.
Conclusion: Rapid identification and speciation of Candida species is essential in guiding appropriate anti-fungal therapy. CHROM agar Candida is a simple and rapid test for speciation of Candida; however, when used along with other phenotypic methods they give more reliable results. Detection of virulence factors in Candida species might indicate invasiveness.

Keywords: Biofilm formation, Candida species, phospholipase activity, protease activity

How to cite this article:
Jose NV, Mudhigeti N, Asir J, Chandrakesan SD. Detection of virulence factors and phenotypic characterization of Candida isolates from clinical specimens. J Curr Res Sci Med 2015;1:27-31

How to cite this URL:
Jose NV, Mudhigeti N, Asir J, Chandrakesan SD. Detection of virulence factors and phenotypic characterization of Candida isolates from clinical specimens. J Curr Res Sci Med [serial online] 2015 [cited 2021 Jul 30];1:27-31. Available from: https://www.jcrsmed.org/text.asp?2015/1/1/27/168933

  Introduction Top

Over the last decade, fungal infections and range of yeasts associated with human infections have increased, especially with Candida.[1] Candidiasis has emerged as an alarming opportunistic infection with an increase in a number of patients who are immunocompromised, diabetics and the elderly. Extensive and prolonged use of antibacterial and cancer chemotherapeutic agents has further complicated the situation.[2]Candida albicans and related species in the recent times have developed resistance to anti-fungal agents, in particular to the azole compounds.[3] The emergence of drug-resistant Candida species is largely attributed to the use of prolonged and inappropriate empirical therapy which has further complicated the patient management and outcome. Hence, accurate species identification is important for the treatment of Candida infections, as the nonalbicans Candida continue to be increasingly documented.[4]

Pathogenicity of Candida is enhanced by several virulence determinants including adherence to the host surfaces, the formation of hyphae, and secretion of hydrolytic enzymes.[4] Extracellular phospholipase activity (PLA) has been recently demonstrated in nonalbicans Candida but in significantly lower amounts.[5],[6] The mortality rate associated with infections due to nonalbicans Candida is similar to C. albicans ranging from 15% to 35%.[7] However, the current understanding of virulence factors in Candia is limited due to various reasons, such as different species of Candida differ in their virulence and expression of these virulence factors has been studied mainly in C. albicans, than in other Candida species.[7] In this study, an attempt was made to speciate Candida isolated from clinical specimens and to detect its virulence factors such as phospholipase, protease, and biofilm formation (BF).

  Methods Top

A total of 100 Candida isolated from various clinical specimens (blood, clean catch midstream urine, pus, bronchoalveolar lavage, tracheal aspirates, and high vaginal swabs [HVSs]) submitted to the clinical microbiology laboratory from various Clinical Departments of Pondicherry Institute of Medical Sciences were included in the study. All suspected yeast colonies were confirmed by Gram staining and further speciated by conventional methods such as germ tube test (GTT), microscopic morphology on cornmeal agar (CMA), Sugar assimilation and fermentation test. Standard ATCC strains, C. albicans (ATCC90028), Candida parapsilosis (ATCC90018), and Candida krusei (ATCC6258) were used throughout the study as quality control strains.

All yeast isolates that grew on sheep blood agar were sub-cultured on chromogenic medium (HiCHROM Candida; HiMedia, Mumbai, India) and incubated at 37°C for 48 h. The isolates grew well and developed distinctive colored colonies after overnight incubation. Presumptive identification was made by noting the color of the colonies as per the manufacturer's Instructions (C. albicans-green, Candida tropicalis - blue, C. krusei - pink colonies with matt surface, C. parapsilosis - cream to pale pink).

The virulence factors such as the BF, phospholipase, and protease activity (PTA) were checked in vitro. Biofilm production was detected by standard tissue culture plate method as described by Chaudhury et al.[8] Based on optical density obtained, the strains were classified into one of the four categories; nonadherent (−), weakly adherent (+), moderately adherent (++), and strongly adherent (+++) [Figure 1].
Figure 1: Microtiter plates showing biofilm formation by spectrophotometric method

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The PLA was determined by growing the Candida on egg yolk agar and measuring the zone of precipitation. This activity was determined as the ratio of the diameter of the colony to the total diameter of the zone of precipitation.[9] The PTA was tested by growing the test organism on bovine serum albumin agar. The results were interpreted same as PLA [Figure 2].[10]
Figure 2: Precipitation zone around the colonies showing protease activity on bovine serum albumin agar

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As the study did not involve human subjects, waiver of informed consent was granted by the Institute Ethics Committee.

  Results Top

A total of 43 C. albicans and 57 nonalbicans Candida were identified based on GTT, chlamydospore formation, sugar assimilation, sugar fermentation tests, and by CHROM agar Candida. The majority of the isolates (92%) were identified accurately to the species level using CHROM agar Candida. Among the nonalbicans Candida species, C. tropicalis (37%) was the most common isolate, followed by C. krusei (18%) and C. parapsilosis (2%). PLA was demonstrated only in 33% (14/43) C. albicans whereas none of the nonalbicans Candida showed this activity. PTA was seen both in albicans and nonalbicans Candida. Among the nonalbicans Candida, PTA was noted in C. tropicalis (49%) followed by C. krusei (16%) and C. parapsilosis (2%) and in 33% of C. albicans. PTA (P = 0.05) was statistically significant in nonalbicans Candida when compared to C. albicans. Candida isolated from bloodstream infections showed highest percentage of phospholipase (50%) and protease (75%) enzyme production. Both C. albicans and nonalbicans Candida species exhibited BF. Among the 100 isolates of Candida, 69% were found to be biofilm producers. Among them, 42% were weakly adherent, 23% moderately adherent, and 4% were strongly adherent. Candida tropicalis was the most common biofilm producer (43%), followed by C. albicans (33%), C. krusei (22%) and C. parapsilosis (1%). BF (P = 0.04) was statistically significant in nonalbicans Candida when compared to C. albicans. Candida isolates from urine showed the highest percentage (80.4%) of BF followed by those wound swabs (72%) and blood (36%) as shown in [Table 1].
Table 1: Production of different virulence factors with respect to different Candida species and site of infection

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  Discussion Top

The infections caused by fungal agents have increased dramatically over the past decades. Candida species have emerged as an important pathogen and infections caused by them are severe, rapidly progressive and refractory to treatment especially in immunocompromised individuals. Of late nonalbicans Candida species have outnumbered the C. albicans as evident in various studies and some of them are inherently resistant to the commonly used antifungals.

In the present study, nonalbicans Candida isolates contributed to more than half (57%) of the candidial infections. Among the nonalbicans Candida isolates, C. tropicalis (37%) was found to be the predominant species followed by C. krusei (18%) and C. parapsilosis (2%). In our previous study, we also observed that the nonalbicans Candida was predominant (70%) as compared to C. albicans (30%), which indicate that the nonalbicans Candida infections are on the rise. Similar finding have been reported in the literature by different authors.[11],[12],[13],[14]

The chromogenic medium, CHROM agar Candida, is a simple and rapid method for identification of common Candida species with a sensitivity and specificity of 98 and 86–97%.[15],[16] In the present study, 92% of Candida isolates were identified accurately to the species level using CHROM agar Candida. Eight of the undifferentiated isolates were identified as C. tropicalis (5%) and C. albicans (2%) and C. parapsilosis (1%) by other phenotypic methods. CHROM agar appears to be quite accurate in identifying the most common Candida species, but it is not proposed as a substitute for the standard identification protocols. However, CHROM agar Candida can be used in conjunction with other phenotypic methods such as sugar assimilation and fermentation tests or microscopic findings on CMA for accurate speciation of Candida.

Candida species possess several virulence factors which help them to colonize, infect and evade the host defense mechanisms. Expression of virulence factors may vary depending upon the type of infection, the site, the stage of infection and the nature of the host response.[17] In the present study, the majority of the isolates were from urine samples (41%) followed by pus (25%) and HVS (13%). However, in a study conducted by Dharwad and Dominic, vaginal candidiasis was most common (38%) followed by candidemia (16%),[18] whereas Patel et al., reported that Candida species were most commonly associated with urinary tract infections (30.5%) followed by lower respiratory tract infections (28.9%) and candidemia (26%).[19]

Candida species have the ability to produce various hydrolytic enzymes that enhance their capacity to colonize mucosal or synthetic surfaces and to invade host tissue surfaces by disrupting host cell membranes. The most important hydrolytic enzymes produced by Candida species include phospholipases and proteases. Cerikcioglu et al. hypothesized that expression of these hydrolytic enzymes for long durations of colonization might contribute to the development of candidemia in preterm infants.[20] The absence or lowered expression of these enzymes may indicate the less virulent nature of Candida species.[21] Previous studies have shown increased PLA in samples from patients with invasive candidial infections than in those isolated from the flora of healthy individuals.[21] The present study also showed highest percentage of phospholipase (50%) and protease (75%) enzyme production in isolates from bloodstream infections.

Phospholipase production in nonalbicans Candida is a rare entity as reported in few studies.[22] In a recent study, PLA was found in all strains of C. albicans and 48% of them exhibited PTA, whereas none of the nonalbicans Candida showed PLA.[23] In concurrence with the report, none of the nonalbicans Candida isolates in our study showed PLA.

The Candida biofilms carries an important clinical risk of increased resistance to antifungal therapy and the ability of cells within the biofilm to evade host immune response. Reports have showed that majority of biofilms forming Candida isolates were from urine samples, followed by blood.[23],[24] Our clinical isolates of Candida showed highest percentage of biofilms forming Candida from urine and HVS samples (71%) followed by pus (64%) and blood stream (50%) infections. BF was demonstrated more frequently in C. tropicalis (82%) followed by C. krusei (65%) and C. albicans (53%), but a study done by Deorukhkar S has reported that BF most frequently occurred in isolates of C. krusei followed by C. tropicalis.[25] Most of the biofilms producing nonablicans Candida (82% of C. krusei and 81% of C. tropicalis) were also positive for PTA, whereas only 39% of C. albicans was positive for both. These results suggest that biofilms formation and PTA have much higher significance for nonalbicans Candida than that of C. albicans.

  Conclusion Top

Accurate species identification is important for the treatment of Candida infections, as most of the nonalbicans Candida are inherently resistant to antifungal agents especially to azoles. Hence rapid identification and speciation of Candida species is essential in guiding appropriate anti-fungal therapy. However, no single phenotypic test is highly effective in identifying Candida species. Therefore, a combination of tests is sometimes necessary for the identification. Virulence factors in Candida species may indicate the invasiveness of the infections. However, further studies need to be done with larger sample size in this field to establish their role in infections.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2]

  [Table 1]


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