- Visibility 227 Views
- Downloads 18 Downloads
- Permissions
- DOI 10.18231/j.ijmmtd.2021.054
-
CrossMark
Speciation of candida species isolated in clinical samples in a tertiary health care centre in Northern India
Abstract
Aim and Objective: The purpose of this study is to isolate, identify and specification of various the Candida species from various clinical samples in a tertiary care hospital, and to characterize various the isolated Candida species.
Materials and Methods: A study was conducted on people of different age groups from January 2019 to December 2019. Candida species isolated from different patients by using Potassium Hydroxide mount and processed by BacTalert 3D (Biomerieux) automated blood culture system. Further culture identification of Candida species were done on Sabouraud dextrose agar (SDA). Speciation of Candida was done using Germ tube test, CHROM agar Candida Medium, Cornmeal agar, Sugar Fermentation test and Sugar Assimilation test.
Results: In our study Candida albicans was the most common species isolated, among non albicans Candida i.e. 21 (38.9%); 19(35.2%) of C.tropicalis was the most common followed by 9(16.7%) of C.glabrat and 5(9.3%) of C.krusei. Maximum number of Candida isolates were obtained from NICU i.e. 27(50.0%) followed by 11 from Med (20.3%), 7 from E/W (13.0%), 2 from BICU (3.7%), 2 from Skin (3.7%), 1 from PICU (1.9%), and 1 from R/R (1.9%).
Conclusion: Our study showed that Candida albicans is the most common isolates species. Among non albicans Candida, C.tropicalis was found to be the most common isolate followed by C.glabrata, C.krusei. Children less than 1 year are most affected with maximum number of Candida species were obtained from NICU department. HiChrom Candida is proven to be more useful as differential agar.
Introduction
Candida is yeast like fungus. It is a ubiquitous human commensal. It becomes pathogenic and causes infections when the host's resistance to infection is lowered either locally or systemically. [1]
The genus Candida comprises of about 150 yeast species, which is composed of a heterogeneous group of organisms & consists of more than 17 different Candida species that are responsible for different human infections. [2] The commonest pathogenic species of this genus is Candida albicans, it has namely A and B, on basis of their differences between mannan components of cell wall. [3]
Candida is the most opportunistic fungal pathogen, which results in various manifestations ranging from mucocutaneous lesions to life threatening invasive diseases. Although Candida albicans is the most common cause of candidiasis accounting for about 60-80% of infections, a shift towards non albicans Candida species is evident in recent years. [4] It include various species such as C.glabrata, C.tropicalis, C.parapsillosis. Among these, C.tropicalis is one of the most common non albicans Candida species isolated from various clinical types of candidiasis. [5]
The morbidity and mortality associated with candidal infections are significant and have emerged as important public health problems. Increasing use of broad-spectrum antibiotics, intravascular catheters, cytotoxic chemotherapies, invasive surgical procedures and long duration of hospital stay are few of the pre-disposing risk factors. [6]
The emergence of non albicans Candida species may represent selection of less susceptible species like C. glabrata and C.krusei. C. glabrata is less susceptible and C. krusei is intrinsically resistant to Fluconazole. C. tropicalis has the highest adherence rate to inanimate materials such as urinary and vascular catheters, and is often involved in biofilm formation, that is more resistant to antifungal agents. Resistance to azoles in C. tropicalis and C. albicans has also been increasingly reported. [7]
The azole drugs have been commonly used to treat many forms of Candida infections for a long time. Resistance to the azole is seen more in non albicans Candida species as compared to C. albicans. Although many new antifungal drugs have been licensed in recent years, their resistance is becoming a major concern during treatment of such patients. [8]
Among the most studied Candida species, Candida albicans has several known virulence factors contributing to its pathogenicity that include adherence to epithelial and endothelial cells, proteinase production, pseudohyphae formation, phenotypic switching, phospholipase production and antigenic modulation as a result of pseudo hyphae formation. After yeast cells of Candida encounter a particular host tissue, colonization takes place at the local sites or it invades deeper into the host tissue. [3]
Candidiasis is an opportunistic fungal disease found in humans who affect the skin, nails, mucosa and various internal organs. There are several types of candidiasis including mucosal candidiasis, cutaneous candidiasis and systemic candidiasis and the incidence of all these infections has been increased due to many factors such as more people living with HIV, the excessive use of antibiotics, organ transplantation and the use of invasive devises. [9]
The purpose of this study is to isolate and identify the Candida species from various clinical samples in a tertiary care hospital, to identify the spectrum of the Candida infections in clinical samples and to characterize the isolated Candida species.
Materials and Methods
A study was conducted in the department of Microbiology in our institute. Various samples obtained from people of different age groups with suspected candidiasis from IPD & OPD from January 2019 to December 2019 were processed in mycology section.
Candida species isolated from different patients were included in the study. The samples were collected using aseptic conditions and the patient’s information such as name, age, sex, occupation, duration of hospitalization, ward, underlying medical conditions, associated risk factors such as presence of urinary catheter, mechanical ventilation, central line insertion, duration of antibiotic therapy, antifungal prophylaxis, exposure to invasive procedures, and use of corticosteroids were obtained from clinical records and analyzed.
Various tests performed for the identification of Candida species are given below-:
Direct examination
Direct microscopic examination was done by Potassium Hydroxide mount (KOH) to see the presence of fungal elements. It was prepared from Potassium hydroxide (10gm), Glycerol (10ml) and Distilled Water (80ml).
Procedure
The concentration of KOH was increased depending upon nature of clinical materials
The wet mount of KOH was prepared by the following two methods:
1) Slide KOH 2) Tube KOH
Slide KOH Procedure
Placed a small amount of sample on a clean glass slide.
Poured a drop of 10%KOH on the specimen and placed a coverslip over it.
Passed the slide gently over flame.
Left the slide for few minutes.
Examined first under low power lens (10x) of light microscope then under high power lens (40x).
Tube KOH procedure
This procedure was used for nail clippings and skin biopsies which dissolves with difficultly.
The homogenized tissue material was dissolved in 10% KOH in test tube and examined after overnight incubation at 37°C. [3]
Blood culture
Blood samples (5 -10ml) were collected in blood culture bottles using aseptic and antiseptic precautions and incubated in BacTalert 3D (Biomerieux) automated blood culture system. Samples were processed using standard mycological procedure as shown in [Figure 1]. [4]
Culture Identification of candida
For the isolation of Candida species the samples were inoculated on Sabouraud dextrose agar (SDA) with antibiotics and incubated aerobically one at 22°C in Biological Oxygen Demand (BOD) and other at 37°C in incubator for 48hrs.[10]
The growth was identified as yeast like organisms by smooth, cream colored, white and pasty colonies on SDA after 3 to 4 days as shown in [Figure 2]. The further confirmation was done by Gram Staining to observe the presence of yeast cells and pseudohyphae as shown in [Figure 3], Germ tube test, Chlamydospore formation on Corn meal agar, growth on CHROM agar and the biological test were sugar fermentation, sugar assimilation for final confirmation of species. [4]
Speciation of candida
Speciation of Candida was done by following methods-
Germ tube test: This was used for the preliminary identification of Candida albicans. It is also known as Reynold Braude phenomenon as shown in [Figure 4]. [10]
CHROM agar Candida Medium: CHROM agar is new chromogenic differential isolation medium that facilitates presumptive differentiation of some of clinically important yeast like organisms and can be used for simultaneous isolation and presumptive identification of various Candida species like Candida albicans, C. krusei, C. tropicalis, C. glabrata, C. parapsilopsis and C. dubliniensis.The CHROM agar Candida shows different colors of colonies after incubation of 48 to 72 hours at 30°C as shown in table 1 and [Figure 5].
Cornmeal agar: It is a nutritionally deficient medium being low in nutrients and hence suppresses vegetative growth and stimulates sporulation in fungi. It is also used for preservation of fungi as stock cultures as shown in [Figure 6], [Figure 7]. [10]
Sugar Fermentation test: This test was done to observe the ability of Candida species to ferment a number of carbohydrates and thus produces acid, gas and hence pink color in presence of Andrade’s indicator. [11]
Sugar Assimilation test: Yeasts and yeast like fungi utilize specific carbohydrates substrate alone. Organisms were inoculated into a carbohydrate free medium and carbohydrate containing filter paper discs were placed. Utilization was determined by presence of growth around the discs. Characteristic carbohydrate utilization profiles were used to identify species of yeast. [11]
Results
In the present study, a total of 54 Candida species were isolated from various clinical samples which included 31 from blood (57.4%), 17 from sputum (31.5%), 4 from urine (7.4%), 1 from catheter tip (1.9%) and 1 from skin (1.9%).
Candida albicans was the most common species isolated among non albicans Candida i.e. 21 (38.9%); 19(35.2%) of C.tropicalis was the most commonest followed by 9(16.7%) of C.glabrat and 5(9.3%) of C.krusei.
Candida isolates were present in 35 males (64.8%) and 19 females (35.2%). The rate of isolation of the Candida species was more in male patients than female patients.
The youngest patient was less than one year old and the oldest one was 60 years old. Maximum number of patients i.e. 29 (53.7%) belonged to the age group of <1 year and minimum number of patients i.e. 4 (7.4%) belonged to the age group of >60 years.
Maximum number of Candida isolates were obtained from NICU i.e. 27(50.0%) followed by 11 from Med (20.3%), 7 from E/W (13.0%), 2 from BICU (3.7%), 2 from Skin (3.7%), 1 from PICU (1.9%), and 1 from R/R (1.9%).
The results of the Conventional method & HI Chrome Candida differential agar for various species are as shown in the [Table 2].
|
Candida species |
Color change |
|
C .albicans |
Light green |
|
C.glabrata |
Pink to Purple |
|
C.krusei |
Pink |
|
C.tropicalis |
Blue with pink hole |
|
Candida species |
No. of Candida species identified by Conventional method |
No. of Candida species identified using CHROM agar |
|
Candida albicans |
22 |
21 |
|
Candida tropicalis |
19 |
19 |
|
Candida glabrata |
9 |
9 |
|
Candida krusei |
4 |
5 |
|
Total |
54 |
54 |
Discussion
Candida species are one of the major fungal pathogens of humans causing a diverse range of diseases. Most notable among invasive candidiasis is candidemia. Nosocomial bloodstream infections due to Candida species are associated with a mortality rate of 5% to 71%. According to the findings of the Centers for Disease Control and Prevention, Candida infections are held responsible for 11% of all nosocomial infections. Candida albicans remains the leading cause of Candida bloodstream infections; however, the prevalence of non albicans Candida infections has increased worldwide, particularly C. tropicalis, C. glabrata, C. parapsilosis and C. krusei as seen by Bac N.D et al.[12]
In this study, most of the Candida isolates were found to be higher in male patients (55.10%) as compared to female patients (44.8%).This correlates well with the study of RA Kashid et al in 2011 who reported that the isolation of Candida species were higher in males (55.10%) as compared to females (44.8%) with male to female ratio of 1:0.81 and Renuka Devi et al [13] in which 54.6% were in males and 45.3% in females. Unlike this study from Amar C.S et al [14] in 2013, isolated Candida species were more from female(60.2%) than male (39.8%) patients in ratio of 0.6:1 (M>F).
In this study, Candida was isolated most commonly in the age group of 0<1year (53.7%), followed by 41-60years (31.5%), 21-40years(7.5%) and >60years (7.4%) which is similar to the study conducted by L. Sumitra Devi et al [15] in which Candida was mostly isolated in the age group of 0-<1year old (60.9%), followed by 21-40 years (12.5%), >60 years (10.9%),1-20 years (7.8%) and 41-60 years (7.8%). This study does not correlate with the study don by Soumya Kaup et al [16] in which majority of Candida isolates from patients aged between 61-70 years followed by 41-50 & 51-60 years.
In this study, 54 Candida species were isolated from various clinical sample which include blood (57.4%) followed by sputum (31.5%), urine (7.4%) ,Catheter tip(1.9%), Skin(1.9%). Maximum number of Candida species were isolated from blood sample. This study correlates with the observation of other authors who showed that majority of Candida were isolated from blood 33.6% by Tavleen Jaggi et al, [6] 50% by Manmeet Gill et al [17] and 57.7% by Raminder Sandhu et al. [18] This study is not similar with Sankarankutty Jaya et al [19] who proved that majority of Candida isolates were from urine (48%) followed by respiratory sample (17%).
Data from surveillance and control of pathogens of epidemiological importance (SCOPE) surveillance system confirms that Candida species have become the fourth leading cause of blood stream infections. A recent study done by MN Chowta et al [20] shows that candidemia is associated with increased cost and attributable mortality of 38%. Although Candida albicans is the most frequently encountered organism, a number of reports have documented non albicans Candida species such as C. tropicalis, C. glabrata, C. parapsilosis and C. krusei and other filamentous fungi as emerging pathogens in recent years. Intravascular catheters, broad-spectrum antibiotics therapy, mucosal colonization, neutropenia, previous surgical procedures (particularly complicated abdominal surgery), total parenteral nutrition and concomitant bacteremia have been identified as significant risk factors for invasive candidal infection in various epidemiologic studies.
In this study, out of 54 Candida isolates obtained from various clinical samples, majority were Candida albican (38.9%). Among non albicans Candida, C. tropicalis (35.2%) followed by C. glabrata (16.7%), C. krusei (9.3%) which is similar with another study conducted by Tavleen Jaggi et al [6] in which Candida albicans was the commonest species isolated causing (44%) of the infection followed by Candida tropicalis, Candida parapsilosis causing (26.4%) and (12.8%) of the infection respectively and also by other authors such as B.S.G Sailaja et al[21] that showed higher incidence of Candida albicans (64%) over non albicans Candida(36%) but it is discordant with the study carried out by Mokaddas et al in 2007 showed that non albicans Candida incidence (60.5%) were higher than that of C.albicans (39.5%) and also by Rachana Mehta et al in 2016 showed that the isolation rate of non albicans Candida (59.1%) were higher as comparatively C.albicans (40.9%).
In this study, all the 54 isolates of Candida species namely C. albicans, C. tropicalis, C. glabrata, and C. krusei were identified by conventional method. Candida isolates were inoculated on HI Chrome Candida differential agar. We observed that performance of HI Chrome Candida differential agar for identification of the above four species were exactly parallel to that of conventional method. Similar findings were observed in various studies like Amar C.Sajjan et al[22] in 2014 and Mr Joginder et al[23] in 2020
In this study, maximum number of Candida isolates were obtained from various departments including NICU (50.0%) followed by Medicine (20.3%), E/W (13.0%), BICU (3.7%), Skin (3.7%), PICU (1.9%), R/R (1.9%). Similar studies were conducted by other authors such as Asifa Nazir et al[24] in which the isolation rate of Candida from neonatal ICU was 18.86% and by Raminder Sandhu et al [18] in 2015 who also observed that the majority of Candida isolates were obtained from NICU(24%) followed by respiratory medicine ward(11%), MICU(7%).
Conclusions
This study emphasizes the requirement of precise isolation and identification of Candida species from various clinical samples. Prevalence of candidiasis was found to be higher in patients associated with predisposing factors like indwelling vascular catheters, prolonged antibiotic therapy and diabetes mellitus. Our study showed that Candida albicans is the most common isolates species. Among non albicans Candida, C.tropicalis was found to be the most common isolate followed by C.glabrata, C.krusei. Children less than 1 year are most affected with maximum number of Candida species were obtained from NICU department.
HiChrom Candida is proven to be more useful as differential agar, as it provides for the rapid isolation and identification of medically important candida species in a resource-limited setting and it is cost effective.
Source of Funding
The authors declare that we have received no financial support for the research, authorship, and/or publication of this article.
Conflicts of Interest
The authors declare no potential conflict of interest with respect to research, authorship, and/or publication of this article.
References
- Kanna BV, Kumar GA, Swapna M, Easow JM. Isolation and identification of Candida species from various clinical samples in a tertiary care hospital. Int J Res med Sci. 2017;5(8):3520-2. [Google Scholar]
- Azad M, Das S, Kumar A. Isolation,Identification &Antifungal susceptibility testing of Candida species from various clinical specimens of ICU patients in a tertiary care hospital of Bhagalpur, Bihar. Int J Res Rev. 2019;6(9):115-22. [Google Scholar]
- Chander J. Textbook of Medical Mycology. 3rd Edn.. 2018. [Google Scholar]
- Kaur J, Sharma P, Sharma S. Emergence of non albicans Candida species in critical care patients of tertiary care hospital. India J Microbical Research. 2016;3(3):398-400. [Google Scholar]
- Mokaddas EM, Al-Sweih NA, Khan Z. Species distribution and antifungal susceptibility of Candida bloodstream isolates in Kuwait: a 10-years study. J Med Microbiol. 2007;56(Pt 2):255-9. [Google Scholar] [Crossref]
- Jaggi T, Urhekar AD, Pai C, Hodiwal BA, Gore S, Kar H. Study of Candida species in various clinical samples in a tertiarty care hospital. DHR Int J Med Sci. 2014;5(2):83-8. [Google Scholar]
- Samyukthaa A, Saikumar C. Isolation, Identification &speciation from various clinical specimen in a tertiary care hospital in Chennai. Scholar J Appl Med Sci. 2017;5(8F):3460-8. [Google Scholar]
- Toure AO, Ama IB, AE, NBH, SK, Berenger AA. Species identification of Candida isolates in various clinical specimen& their antifungal susceptibility pattern in Cote d”Ivoire. Afr J Microbiol Res. 2016;10(2):66-72. [Google Scholar]
- Jasim ST, Hassan AA, Flayyih TM. Isolation and Identification of Candida species from different clinical specimens and study the virulence factors. World J Pharm Pharm Sci. 2016;5(7):121-37. [Google Scholar]
- Colle GJ, Fraser GA, Marmion PB, Simmons A. Mackie&McCartney Practical Medical Microbiology 14th edn.. . 2014. [Google Scholar]
- Moore G, Jaciow D. Mycology for the clinical laboratory. . 1979. [Google Scholar]
- Bac ND, Anh LT, Quang LB, Luc NK, Nga TTT, Nagi M. Prevalence of Candida blood stream isolates from patients in two hospitals in Vietnam. Iran J Microbiol. 2019;11(2):108-13. [Google Scholar]
- Devi RA, Hymavathi R, Mounika G. Candida species isolation ,identification &biofilm detection at a tertiary care hospital. Int J Contemp Med Res. 2019;6(4):6-9. [Google Scholar]
- Amar CS, Ashish J, Hajare V, Belagali. Study of prevalence &antifungal susceptibility of Candida. Int J Pharm Bio-Sci. 2013;4(2):361-81. [Google Scholar]
- Devi SL, Maheshwari M. Speciation of Candid species isolated from clinical specimens by using Chrome agar and Conventional methods. Int J Scientific Res Publication. 2014;4(3):1-5. [Google Scholar]
- Kaup S, Sankarankutty J, Balasubrahmanya HV, Kulkarni S, Nirmala M. Speciation of Candida using Hichrome Candida Differential Agar. Int J Curr Microbiol Appl Sci. 2016;5(7):267-74. [Google Scholar]
- Gill M, Sharma S, Khanna A. Emergence of non albicans Candida in a tertiary care hospital of north India. Indian J Microbiol Res. 2018;5(2):244-8. [Google Scholar]
- Sandhu R, Dahiya S, Sharma KR. Isolation & Identification of Candida & non albicans Candida species using chromogenic medium. Int J Biomed Res. 2015;6(12):958-62. [Google Scholar]
- Jaya S, Harita V. Candida species isolation from various clinical samples & their susceptibility patterns to antifungal. J Med Microbial Infect Dis. 2013;1(1):22-6. [Google Scholar]
- Chowta MN, Adhikari P, Rajeev A, Shenoya K. Study of risk factors and prevalence of invasive candidiasis in a tertiary care hospital. India J Crit Care Med. 2007;11(2):67-73. [Google Scholar]
- Sailaja B, Prasad PD. A study on isolation of Candida species in various clinical samples in a tertiary health care unit. Indian J Microbial Res. 2019;6(3):258-60. [Google Scholar]
- Sajjan AC, Mahalakshmi VV, Hajare D. Prevalence &Antifungal susceptibility of Candida species isolated from patient attending tertiary care hospital. J Dent Med Sci. 2014;13(5):44-9. [Google Scholar]
- Jogender, Kulshrestha A. A study on identification &Speciation of medically important Candida species isolated from various clinical samples by using HiChrome Candida. Int J Sci Res. 2020;9(2):44-6. [Google Scholar]
- Nazir A, Masoodi T. Spectrum of Candidal species isolated from neonatal admitted in an Intensive Care Unit of teaching hospital of Kashmir, North India. J Physicians. 2018;10(3):255-9. [Google Scholar]