Original article / research
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Clinico-microbiological Study of b#bCandidab?b Infections Focusing on Risk Factors, Species Identification, Virulence Factors and Antifungal Susceptibility Patterns |
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MV Poornima, K Vidyalakshmi 1. Junior Consultant, Department of Microbiology, Baby Memorial Hospital, Calicut, Kerala, India. 2. Professor and Head, Department of Microbiology, Kasturba Medical College, Mangalore, Karnataka, India. |
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Correspondence
Address : Dr. MV Poornima, Sisiram, Kolaithazham, Karanthur, Kozhikode-673571a, Kerala, India. E-mail: poornimamv@yahoo.co.in |
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ABSTRACT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
: Infections caused by Candida species have been increasing along with the advancement in diagnostic and treatment interventions. Recently a shift from Candida albicans to non albicans species has also been observed. Infections by Candida species is aided by several virulence factors and prevalence of antifungal resistance. Aim: To speciate and study the virulence factors and determine susceptibility pattern of Candida isolates obtained from various clinical specimens. To determine the risk factors for infection with Candida species in patients admitted to a tertiary care hospital at Mangalore, Karnataka, India. Materials and Methods: This cross-sectional study was conducted at Department of Microbiology, Kasturba Medical College, Mangalore, Karnataka between October 2015 to September 2018. A total of 145 isolates from various clinical specimens were identified up to species level by germ tube test, chlamydospore formation, and growth characteristic on cornmeal agar, colour of colonies on chromogenic agar and carbohydrate fermentation tests. Virulence factors included were biofilm formation by microtitre plate method, phospholipase activity using egg yolk agar, proteinase activity using bovine serum albumin agar, haemolysin production, coagulase activity and production of true hyphae. Antifungal susceptibility test was performed as per CLSI 2009 M44-A2. The statistical analysis was done using statistical package version 16.0. The p-value less than 0.05 was considered as statistically significant. Results: Out of 145 Candida isolates C. tropicalis were 59 (40.6%) followed by C. albicans and other species. Presence of an intravenous catheter 124 (85.5%), treatment with broad spectrum antibiotics 121 (83.4%) and admission to ICU 113 (77.9%) were the most common risk factors identified in this study. Biofilm formation was detected in 80 (55.2%) of isolates, among which C. tropicalis (42 out of 59 71.1% p= 0.02) showed the highest propensity for biofilm formation. Phospholipase activity was highest among C. albicans 42 (79.2%, p= 0.001) whereas proteinase activity was highest in C. tropicalis 50 (84.7%, p=0.001). Coagulase production was detected in 14 isolates and 39 of our isolates had produced true hyphae. Production of true hyphae was observed more among C. tropicalis 25 (64.1%, p= 0.04). Non albicans species showed highest proteinase activity and true hyphae production of 69 (75.6% p= 0.002) and 25 (27.2% p=0.04) respectively. Fluconazole resistance was detected in 13.8% of the isolates. Conclusion: Study of risk factors and virulence factors will serve as a guideline for better understanding of the pathogenic mechanisms involved in fungal disease, which in turn will lead to better patient management and prognosis. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Keywords : Antifungal resistance, Biofilm, Candida albicans, Extracellular hydrolytic enzymes, Non albicans Candida species | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
INTRODUCTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
In the last few decades there has been a progressive upsurge in the incidence of fungal infections caused by Candida species (1),(2). This increase appears to run in parallel with the recent advances in medicine such as immunosuppressive therapy, increased use of surgical procedures, prostheses and broad-spectrum antibiotics (3). Several host factors such as diabetic mellitus, chronic kidney disease and malignancy also account for the development of Candida infection (3). Non-albicans Candida (NAC) species are now being recovered from the clinical samples with increasing frequency (3). The clinical presentation of patients infected with C. albicans and NAC species are indistinguishable. Accurate identification of the species causing infection is important because antifungal treatment is different for C. albicans and NAC infections (2). The transition of Candida albicans from commensal to a pathogen may be facilitated by a number of virulence factors like adhesion, biofilm production, extracellular hydrolytic enzyme activity and ability to form true hyphae as evidence by published literature (4). But there are only few literatures (3),(5) on virulence factors of NAC species, which also cause a significant number of infections. Study of virulence factors is important to understand pathogenesis and to explore new therapeutic options. The last decade has witnessed the development of new antifungal agents, as well as the emergence of antifungal resistance (1). Monitoring antifungal resistance among Candida species helps to detect the emergence of resistant strains; and also, to modify empirical treatment recommendations. We intend to study the common species of Candida causing infections in our patient population, their virulence factors and antifungal susceptibility patterns; as well as the risk factors which predispose patients to Candida infections. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MATERIAL AND METHODS | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
A cross-sectional study was conducted in the Central Laboratory, Department of Microbiology, Kasturba Medical College, Mangalore, Karnataka, India, during the period of October 2015 to September 2018. The sample size was calculated using the formulae 4PQ/d.2 .From the previous study done by Kaur R et al., taking P as proportion of Candida tropicalis as 40.8%, Q as 100-P with 95% confidence interval 80% power the calculated sample size was 145 (5). The study protocol was submitted to the Institutional Ethics Committee and approval was obtained before commencement of study (letter no: KMC MLR 09-15/185 dated 16/09/2015). A total of 145 consecutive isolates of Candida species from various clinical specimens were included in the study, isolates of Candida from exudate specimens were included based on the presence of inflammatory cells/ intracellular budding yeast cells/pseudohyphae in a Gram stained smear of the specimen. Isolates of Candida from urine specimens were included based on the presence of pus cells ≥5/HPF in a standard wet mount, along with oval budding yeast cells with pseudohyphae and a colony count of ≥105 CFU/ml in culture. All isolates of Candida from blood cultures were included. Isolates from respiratory and urine specimens that showed numerous epithelial cells suggesting contamination with saliva or vaginal secretions, respectively, were excluded from the study. The clinical data were collected from Medical Record Department using a proforma [Annexure 1]. Speciation was done by germ tube test (6), observing the morphology on cornmeal agar (7), CHRO Magar (Himedia laboratories Pvt., Ltd.,) (8) and carbohydrate fermentation test (2),(7). Assessment of Virulence Factors Biofilm formation Microtiter plate was used to evaluate biofilm formation by Candida isolates. Test isolates were grown in Saboraud’s Dextrose broth with 8% glucose. The broth was then diluted 1:100 in fresh medium and 150µl of it was inoculated into microtiter plate wells. The negative control wells had the sterile broth and Enterococcus faecalis ATCC29212 was employed as a positive control strain. The plates were incubated at 37°C for 48 hrs. After incubation content well was discarded and were washed with sterile distilled water twice, excess water was eliminated by tapping the plates on to blotting paper. Then the plates were stained with 200µl of 0.5% crystal violet for 15 minutes. Excess stain was washed off and allowed to dry. The dye bound to the adherent yeast cells was resolubilized by adding 200µl of ethanol: acetone mixture (80:20 w/v). Optical density (OD) of the wells were read using an ELISA plate reader (BioTek instruments, Made in USA, ELx800) at wavelength 570 nm. OD values were recorded for each well. Four wells were used for each test strain and its average were used for analysis (9),(10). The cut-off Optical density (ODc) for the microtiterplate is defined as three standard deviations above the mean OD of the negative control (11). Phospholipase Activity Phospholipase activity was determined by using egg yolk agar (65g Saboraud’s Dextrose Agar with 1 M NaCl, 0.005 M CaCl2 dissolved in 980ml distilled water and sterilized. A 20 mL of egg yolk was added to the cooled medium (45-50°C) mixed and dispensed in plates). Plates were inoculated with test isolates and incubated at 37°C for 5 days. The presence of phospholipase activity was determined by the formation of a precipitation zone around the yeast colonies (10). Reference strain C. albicans ATCC 60193 and C. krusei ATCC 6258 served as positive control. Proteinase Activity The protease production was tested in agar containing bovine serum albumin (Yeast carbon base 11.7g, yeast extract 0.1g, and bovine serum albumin 2g and 16g of agar-agar in 800mL of distilled water). Plates were inoculated with test isolates and incubated for 10 days at 37°C. Diameter of the lytic area surrounding the growth area on the medium was measured as enzymatic activity (12). Reference strain C. tropicalis ATCC 66029 and C. krusei ATCC 6258 served as positive control. Haemolysin Activity Haemolysin activity was assessed by using blood agar containing 7ml of human fresh blood in 100ml of SDA containing 3% glucose. Plates were inoculated with test isolates and incubated at 37°C. A transparent/semitransparent zone around the inoculation site after 48hrs of incubation was considered as positive haemolytic activity (10). Reference strain C. krusei ATCC 6258 served as positive control. Coagulase Activity Inoculate 0.1 mL of overnight SDA broth suspension to 0.5 mL of human plasma and incubated at 35°C-45°C. The clot formation was observed at 2 hours, 4 hours, 6 hours, and 24 hours by gently tilting and shaking the tubes. The S. aureus ATCC 25923 was used as a positive control (13). Hyphae Production An inoculating loop was loaded with the test organism and a well was formed at the top of a plate of cornmeal agar supplemented with 1% Tween 80. Dilution strokes were then made by cutting into the agar with the edge of the sterilized loop, holding it at an angle of 45 degrees to the surface of the agar. A sterile cover slip was placed such that it covered a part of the well some of the dilution strokes. The plates were incubated at 22°C for 48-72 hrs. Then the lid was removed and plates were placed on the microscopic stage, and the area under the coverslip as well as the edges of cover slip were observed for filamentous structures under high power magnification (6),(8). The reference strains, C. albicans ATCC 60193, C. tropicalis ATCC 66029, C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were included in each set of tests as quality control. Antifungal Susceptibility Testing Antifungal susceptibility was done on Muller Hinton Agar supplemented with 2% glucose and 0.5 µ/mL methylene blue to fluconazole (25 µg) and voriconazole (1 µg) (Hi-Media pvt., ltd.,) using disc diffusion method. Zone diameter was interpreted as per the M44/A2 protocol of CLSI guidelines 2009 (14). C. parapsilosis ATCC 22019 was included in each test as quality control isolates. STATISTICAL ANALYSIS The statistical analysis was done using statistical package 16.0. The p-value less than 0.05 was considered as statistically significant. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESULTS | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The various clinical specimens from which Candida species were isolated are shown in (Table/Fig 1). Overall, NAC species accounted for a higher number (92,63.4%) as compared to Candida albicans (53,36.5%). Among the individual species, Candida tropicalis accounted for the highest number 59 (40.7%) followed by Candida albicans 53 (36.5%) C. krusei 15 (10.3%), C. glabrata and C. parapsilosis 9 (6.2%). Presence of an intravenous catheter 124 (85.5%), treatment with broad spectrum antibiotics 121 (83.4%), admission to ICU 113 (77.9%), presence of urinary catheter 89 (61.4%), and diabetes mellitus 49 (33.8%) were the most common risk factors (Table/Fig 2). Ability to form biofilm in vitro was detected in 80 (55.2%) isolates. The highest propensity for biofilm formation was shown by Candida tropicalis 71.1% (42 out of 59). For the purpose of comparative analysis of test results, the adherence capabilities of the test strains were classified into the following under four categories: non-adherent- OD≤ ODc (0), weakly-ODc< OD≤ 2x ODc (+), moderately-2 x ODc< OD≤ 4 x ODc (++), or strongly adherent-4 x ODc< OD (+++), based upon the ODs of fungal films. Out of the 80 biofilm producing isolates, strong adherent biofilm production was detected in 41(28.3%) (Table/Fig 3). Phospholipase, proteinase and haemolysin activity were observed in 95 (65.5%), 95 (65.5%) and 116 (80%) respectively. Coagulase activity was observed in 14 (9.7 %) of isolates. True hyphae formation was observed in a total of 39 (26.9%) isolates. The comparison of virulence factors exhibited different Candida species, C. albicans and NAC species and their statistical significance are given in (Table/Fig 4),(Table/Fig 5). Out of 145 isolates, 20 (13.8%) were resistant to fluconazole and 5 (3.4%) showed dose-dependent susceptibility to fluconazole. The susceptibility pattern among the different Candida species was also compared and given in (Table/Fig 6). | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
DISCUSSION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The last few decades have witnessed a rise in the infections caused by Candida species, particularly those caused by the NAC species. This study focuses on the identification of various species of Candida obtained from clinical specimens of in-patients admitted to our hospital, risk factors contributing to infections, study of their virulence factors and antifungal susceptibility patterns. Among all isolates obtained in this study, NAC species accounted for the highest number and among them Candida tropicalis was the predominant species isolated (40.7%), followed by Candida albicans (36.5%). These findings were correlated with the studies done at different parts of India such as Kaur R et al., in which C. tropicalis (40.8%) and C. albicans (38.8 %) were the most common species isolated (5). Bhatt M et al., had reported 39% C. tropicalis, 20% C. parapsilosis, 14.7% each of C. krusei and C. albicans and 5.4% C. glabrata (15). C. albicans was found to be most common species isolated in studies conducted by Mnge P et al., in South Africa and Kiraz N and Oz Y in Turkey, but shift towards NAC species was reported [16,17]. The exact cause for the increase in NAC species is not known, however wide spread and inappropriate use of antifungal treatment could be contributing factor (15). Candida albicans is a normal flora in the various anatomical sites in a healthy individual. But condition worses when host immunity weakens, it behaves as an opportunistic pathogen. Candida infection can also occur in patients who had a prolonged hospital stay. The mechanism of transmission of infection may be through the hands of health care workers, or health care materials such as catheters (1). In a previous study done in our institution by Chowta MN et al., in 2007, 64% of patients with Candida infection had an intravenous catheter and 34.5% had prolonged exposure to antibiotics (18). In Bhatt M et al., work 100% of the patients were on broad spectrum antibiotics, 89% had indwelling venous catheters and 65% were mechanically ventilated (15). Thus, risk factors observed in our study is in accordance with another study. It is necessary to observe these risk factors extensively, and to start prophylaxis antifungal treatment according to Candida score in high-risk patients (19). Candida species infect the medical implants, by adhering to the surfaces and forming a colony. These microbial colonies are enclosed in a self-produced polymeric matrix called biofilm and represent a common mode of microbial growth. In the present study 55.2% of isolates were biofilm positive. Bhatt M et al., reported 64.7% and Bansal R et al., reported 66% of biofilm positive isolates from the blood culture (15),(20) and in the present study, it accounts for 54.8%. This shows blood culture isolates are more prone to develop biofilm and thus develops antifungal resistance which may lead to increase in mortality. In the current analysis Candida tropicalis was the highest biofilm producers (71.1%) when compared to other species. (p=0.02). The predominance of C. tropicalis to produce biofilm was reported by Bhatt M et al., (15). Phospholipases enables the invasion of the host cell by cleaving the phospholipid bonds (11). Thus, phospholipase enzyme can cause cell lysis and allow the hyphae to penetrate the cytoplasm (21). In our study, 65.5% were positive for phospholipase production. Highest phospholipase activity in this study was detected in C. albicans (p-value 0.001) (Table/Fig 2) which is in harmony with a study done by Das VM et al., (22). During our study the phospholipase activity of C. tropicalis was 69.4%. There is a wide variation in published reports of phospholipase production among C. tropicalis. Inci M et al., from Turkey reported that NAC species do not exhibit phospholipase activity, in either aerobic or anaerobic condition (9). However, Sachin CD et al., reported that phospholipase was the major virulence factor in C. tropicalis, which is in agreement with our study (21). Proteinases are capable of invading hosts epithelial and mucosal barrier proteins by hydrolyzing the peptide bond. Proteinase enzymes can disturb the significant proteins responsible host immune mechanism such as antibodies, complement, and cytokines (12). The proteinase activity of blood culture isolates in our study was 71%. These results are in accordance with Das VM et al., study which shows 74.56% of proteinase activity in blood culture isolates (22). Haemolysin production is also a virulence factor contributing to Candida pathogenesis. Secretion of haemolysin helps the organism to acquire iron and produce hyphae which facilitate invasion in disseminated candidiasis (23). In the recent study 80% of isolates were haemolysin positive which is in accordance with Inci M et al., (91.1%) (10) and Sachin CD et al., (51.8%) (21). Sachin CD et al., had reported maximum haemolytic activity from C. albicans in his work, and in the present study maximum haemolytic activity was observed in C. tropicalis (86.4%) and C. albicans (83%) (21). The ability of the Candida isolates to produce coagulase by using human plasma was analysed by Padmajakshi. G et al., in 2014 and they reported 4.3% of isolates producing coagulase activity in which three were C. albicans and one being C. tropicalis (13). None of the isolates produced coagulase by human plasma in another study done by Yigit N et al., (24). In our study, 14 isolates had produced coagulase using human plasma. Thompson D S et al., studied the virulence factors of Candida species and reported C. albicans, C. tropicalis and C. dubliniensis were major species of Candida that can produce true hyphae, which is in accordance with our study (25). Hyphae is capable of anchoring the cell layer and penetrate into the endothelial cells by which it can cause invasive infection. The Candida isolates showed 13.8% of resistance to fluconazole and 2.1% to voriconazole. But the percentage of resistance reported from Korea by Won EJ et al., was 2.6% to fluconazole and all the isolates were susceptible to voriconazole (26), Whereas the percentage resistance to fluconazole and voriconazole was more in two Indian studies done by Battacharjee P and Gupta S et al., (27),(28). In our study, 80% of the isolates of C. krusei were resistance to fluconazole (20% intermediate) which is consistent with other studies (18),(20),(22),(28). This shows antifungal resistance is becoming more prevalent. So, it is important to perform routine antifungal susceptibility testing for better therapeutic outcome. LIMITATION Since this an academic study: duration was only 18 months and confined to an institution so our sample size was only 145, this is a major limitation of this study. Accuracy of the study can be increased by incorporating more institution and more duration. Financial support from our institution was not sufficient to perform sugar assimilation test for speciation. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CONCLUSION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
A total of 145 consecutive isolates of Candida species were obtained from various clinical specimens. NAC species accounted for a higher number as compared to Candida albicans. Among the individual species, Candida tropicalis accounted for the highest number. Considering all virulence factors tested among all the isolates in this study, isolates of Candida tropicalis showed higher tendency to produce biofilm, proteinase, haemolysin, coagulase and true hyphae, compared to C albicans which showed higher tendency to produce phospholipase. Fluconazole resistance was detected in 13.8% of the isolates and 3.4% showed dose-dependent susceptibility. Three of the isolates were resistant to voriconazole and 7 isolates showed dose-dependent susceptibility. Presence of an intravenous catheter, treatment with broad spectrum antibiotics and admission to ICU were the most common risk factors. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ACKNOWLEDGEMENT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
We are grateful to Manipal University for the financial support in conducting this study. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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TABLES AND FIGURES | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||