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  Table of Contents    
BRIEF COMMUNICATION  
Year : 2014  |  Volume : 57  |  Issue : 4  |  Page : 595-597
Evaluation of antifungal susceptibility testing in Candida isolates by Candifast and disk-diffusion method


1 Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Microbiology, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India

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Date of Web Publication11-Oct-2014
 

   Abstract 

With the increase in invasive fungal infections due to Candida species and resistance to antifungal therapy, in vitro antifungal susceptibility testing is becoming an important part of clinical microbiology laboratories. Along with broth microdilution and disk diffusion method, various commercial methods are being increasingly used for antifungal susceptibility testing, especially in the developed world. In our study, we compared the antifungal susceptibility patterns of 39 isolates of Candida to three antifungal drugs (fluconazole, amphotericin B, ketoconazole) by Candifast and disk diffusion method. The following resistance pattern was found by Candifast: Fluconazole (30.8%), ketoconazole (12.8%), amphotericin B (0%). The results obtained by disk diffusion method were in complete agreement with Candifast results.

Keywords: Antifungal, Candida, Candifast, disk diffusion

How to cite this article:
Giri S, Kindo AJ. Evaluation of antifungal susceptibility testing in Candida isolates by Candifast and disk-diffusion method . Indian J Pathol Microbiol 2014;57:595-7

How to cite this URL:
Giri S, Kindo AJ. Evaluation of antifungal susceptibility testing in Candida isolates by Candifast and disk-diffusion method . Indian J Pathol Microbiol [serial online] 2014 [cited 2019 Dec 11];57:595-7. Available from: http://www.ijpmonline.org/text.asp?2014/57/4/595/142680



   Introduction Top


0Candida species are one of the leading causes of invasive fungal infections world-wide. [1],[2] Despite the introduction of newer antifungal drugs for the treatment of infections by Candida species, the occurrence of invasive fungal infections and resistance to antifungal therapy is on the rise. [3] Hence, in vitro antifungal drug susceptibility testing for Candida species has become important in the detection of resistance as well as in effective patient management. [3],[4] Standardized methods for in vitro antifungal susceptibility testing of yeasts by broth microdilution and disk diffusion methods are now available from the Clinical and Laboratory Standards Institute (CLSI). [5],[6] Disk diffusion method is a commonly used method for antifungal susceptibility testing in busy clinical microbiology laboratories. Commercial systems like Candifast, Fungitest, Sensititre-Yeast One, etc., are now being used by laboratories for antifungal susceptibility testing, especially in the developed world.

The present study was undertaken to compare Candifast with disk diffusion method for the detection of resistance pattern in clinical isolates of Candida. The Candifast kit can be used for the identification as well as susceptibility testing of Candida isolates.


   Materials and methods Top


Antifungal susceptibility testing was performed for 39 clinical isolates of Candida (29 Candida tropicalis, 4 Candida albicans, 3 Candida parapsilosis, 2 Candida krusei, and 1 Candida glabrata). All the isolates were collected from blood samples of patients at a University teaching hospital in Chennai. The Candida isolates were identified to species level using various phenotypic tests such as fermentation, assimilation, tetrazolium reduction medium, Candifast, and CHROMagar Candida (France) medium. The methods used for susceptibility testing were Candifast and disk diffusion.

Candifast

The Candifast kit (International Microbio, France) allows the identification of Candida species and testing of their susceptibility to various antifungal agents. It is a 20well tray with two rows, one for identification and the other for susceptibility testing. The determination of the resistance of yeasts to antifungal agents is based on growth or absence of growth of the yeasts in the presence of various antifungal agents. An isolated colony of Candida was inoculated into reagent-1 (R1) bottle and mixed well. The turbidity of the suspension was compared with the turbidity control. 100 μl of inoculated R1 was added to R2. 100 μl of R2 was then added to each of the wells, 2 drops of paraffin oil was added to each well and the test tray was sealed and incubated at 37 °C for 24 hours. Reading was taken once the yeast grew in the control well. The indicator used is phenol red. A yellow or orange-yellow color in the susceptibility test row, due to glucose fermentation, indicated that the yeast was able to grow in the presence of the antifungal agent and hence was resistant to that drug. If the color in the well was red or pink, the isolate was inhibited by the drug in that well [Figure 1] and so was sensitive to that drug. The susceptibility testing by Candifast kit can be done for the following anti-fungal drugs: Amphotericin B (4 μg/ml), fluconazole (16 μg/ml), ketoconazole (16 μg/ml), nystatin (200 units/ml), flucytosine (35 μg/ml), econazole (16 μg/ml), and miconazole (16 μg/ml). Quality control testing was also performed using C. albicans ATCC (American Type Culture Collection) 90028 to check the efficacy of the kit.
Figure 1: Antifungal susceptibility testing by Candifast

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Disk-diffusion method

Antifungal susceptibility testing by disk diffusion method was performed according to CLSI guidelines (CLSI document M44-A2) and manufacturer's instructions. [6] The media and antifungal disks used in the testing were from HiMedia Laboratories, Mumbai. The standard medium used for disk diffusion test was Mueller-Hinton agar supplemented with 2% dextrose and 0.5 μg/ml methylene blue [Figure 2]. Incorporation of methylene blue in the medium has been found to improve the yeast growth and provide sharp zones of inhibition for the azole group of drugs. [6] The colonies were suspended in 5 ml of sterile 0.85% saline, and the turbidity was adjusted to yield 1 × 10 5 -1 × 10 6 cells/ml (0.5 McFarland standard). The antifungal disks tested were amphotericin B (100 units), fluconazole (25 mcg) and ketoconazole (10 mcg). Quality control tests using reference strain C. albicans (ATCC 90028) were set up on each day that susceptibility tests were performed to check for the precision and accuracy of the results of disk diffusion testing.
Figure 2: Antifungal susceptibility testing by disk diffusion method

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   Results Top


The antifungal susceptibility testing of the 39 Candida isolates by Candifast method showed the following resistance pattern: 12 Candida isolates (30.8%) were resistant to fluconazole, while 5 (12.8%) and 0 (0%) isolates were resistant to ketoconazole and amphotericin B, respectively [Table 1]. The resistance pattern found with the disk diffusion method was in complete concordance with the Candifast results. Out of the 12 (30.8%) Candida isolates resistant to fluconazole, there were 7 isolates of C. tropicalis, 2 each of C. parapsilosis, C. krusei, and 1 C. glabrata. Ketoconazole resistance was found in 2 isolates each of C. tropicalis and C. parapsilosis and 1 isolate of C. glabrata. All the isolates of C. albicans were susceptible to the three antifungal drugs. Both the methods showed 100% agreement in antifungal susceptibility patterns for C. albicans and non-C. albicans isolates.
Table 1: Antifungal susceptibility testing results of Candida isolates by Candifast method

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   Discussion Top


Antifungal susceptibility testing in vitro is playing an important role in antifungal drug selection, as an aid in drug development and as a means of tracking the development of resistance. Disk diffusion is the most commonly used method for susceptibility testing of Candida isolates and is preferred in clinical microbiology laboratories. The M44-A2 document of the CLSI gives guidelines for susceptibility testing of yeasts using this method. [6] Many clinical laboratories, especially those in the developed world, prefer to use commercial systems for determining susceptibility to various antifungal agents. Many of these methods are more rapid and easier to perform compared to the microdilution method of susceptibility testing which is the gold standard. Among the commonly used commercial methods for antifungal susceptibility testing are Candifast, Fungitest, Sensititre Yeast One method etc. [7] These methods have been evaluated for their agreement with the microdilution method and intralaboratory reproducibility in various studies. [8]

In our study, we evaluated the antifungal susceptibility pattern of 39 Candida isolates from blood samples of patients using the Candifast and disk diffusion method. There was complete agreement in the susceptibility pattern of Candida isolates by both the methods. All Candida isolates were found to be susceptible to amphotericin B. Fluconazole and ketoconazole resistance was seen in 30.8% and 12.8% of Candida isolates, respectively. There was 100% agreement between disk diffusion method and Candifast when the susceptibility pattern of C. albicans and non-C. albicans were compared. Candifast has been used for antifungal susceptibility testing of Candida isolates in some studies on candidemia. [9] There are very few studies which have compared the susceptibility pattern of isolates of various Candida species by different methods. In a recent study from Egypt, it was found that for amphotericin B, the agreement of Candifast with standard broth microdilution method was 100% in all species except C. glabrata (85.7%). [4] The same study also showed a 100% agreement for fluconazole between the two methods in all species except C. albicans, C. glabrata which was 92.8% and 85.7%, respectively. [4] In another study by Schmalreck et al., it was found that the correlation between Candifast and microdilution method of testing susceptibility of clinical yeast isolates to fluconazole was 83%. [10] However, another multicenter study by Morace et al., comparing six commercial systems (Candifast, disk diffusion, Etest, Fungitest, Integral System Yeasts, and Sensititre-Yeast One) with the broth microdilution method for fluconazole susceptibility testing of Candida species did not find a good agreement between Candifast and the broth microdilution method. [8] Hence, further studies involving the evaluation of various commercial methods for anti-fungal susceptibility testing are necessary. An added advantage of using Candifast for susceptibility testing is that it can also simultaneously be used for speciation of Candida isolates. [11]


   Conclusion Top


Our study found Candifast to be an easy and rapid method for the identification and susceptibility testing of Candida isolates and can be used in busy clinical microbiology laboratories.

 
   References Top

1.
Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: A persistent public health problem. Clin Microbiol Rev 2007;20:133-63.  Back to cited text no. 1
    
2.
Cannon RD, Lamping E, Holmes AR, Niimi K, Baret PV, Keniya MV, et al. Efflux-mediated antifungal drug resistance. Clin Microbiol Rev 2009;22:291-321.  Back to cited text no. 2
    
3.
Pfaller MA. Antifungal drug resistance: Mechanisms, epidemiology, and consequences for treatment. Am J Med 2012;125:S3-13.  Back to cited text no. 3
[PUBMED]    
4.
El-Mashad N, Mahmoud MT. Commercial tests for in vitro antifungal susceptibility testing of Candida species compared to standard (NCCLS) broth microdilution. Int J Med Med Sci 2011;3:100-4.  Back to cited text no. 4
    
5.
Rex JH, Alexander BD, Andes D, Skaggs BA, Brown SD, Chaturvedi V, et al. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard. 3 rd ed. Pennsylvania: Clinical and Laboratory Standards Institute (CLSI); 2008. p. 1-25.  Back to cited text no. 5
    
6.
Rex JH, Ghannoum MA, Alexander BD, Andes D, Brown SD, Diekema DJ, et al. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts: Approved Guideline. 2 nd ed. Pennsylvania: Clinical and Laboratory Standards Institute (CLSI); 2009. p. 1-23.  Back to cited text no. 6
    
7.
Osoba AO, Al-Mowallad AW, McAlear DE, Hussein BA. Candidemia and the susceptibility pattern of Candida isolates in blood. Saudi Med J 2003;24:1060-3.  Back to cited text no. 7
    
8.
Morace G, Amato G, Bistoni F, Fadda G, Marone P, Montagna MT, et al. Multicenter comparative evaluation of six commercial systems and the national committee for clinical laboratory standards M27-A broth microdilution method for fluconazole susceptibility testing of Candida species. J Clin Microbiol 2002;40:2953-8.  Back to cited text no. 8
    
9.
Akbar DH, Tahawi AT. Candidemia at a University Hospital: Epidemiology, risk factors and predictors of mortality. Ann Saudi Med 2001;21:178-82.  Back to cited text no. 9
    
10.
Schmalreck AF, Kottmann I, Reiser A, Ruffer U, Scharr E, Vanca E. An evaluation of seven methods of testing in vitro susceptibility of clinical yeast isolates to fluconazole. Mycoses 1995;38:359-68.  Back to cited text no. 10
    
11.
Gündes SG, Gulenc S, Bingol R. Comparative performance of Fungichrom I, Candifast and API 20C Aux systems in the identification of clinically significant yeasts. J Med Microbiol 2001;50:1105-10.  Back to cited text no. 11
    

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Correspondence Address:
Sidhartha Giri
Wellcome Trust Research Laboratory, Christian Medical College, Vellore, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.142680

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