Edula: Antifungal susceptibility of clinically significant candida species by disk diffusion method


Introduction

The Candida species are ubiquitous yeasts which are a part of normal flora of the alimentary tract of mammals and the mucocutaneous membranes of humans.1 It becomes an opportunistic pathogen as a result of one or more underlying pre-disposing factors.2 Until recently Candida albicans was the most common yeast isolated from infections. Epidemiologic data from the past decade reveal a paradigm shift in Candida infections with non -albicans Candida species such as C.glabrata, C.tropicalis, and C.krusei as emerging important pathogens.3, 4, 5, 6 This transition has had a significant clinical impact due to decreased susceptibility of these non -albicans yeasts to antifungal agents.5 Candida albicans and non-albicans species are closely related but differ from each other with respect to epidemiology, virulence characteristics, and fungal susceptibility. The Combined effects of increasing number of fungal infections, growing number of patients at risk, the increasing rate of fungal resistance, and the expanded antifungal armamentarium have led to an increased recognition of the need for standardized laboratory testing for antifungal drug susceptibility.7 Agar-based susceptibility testing methods have been focus of interest for many researchers, due to their simplicity, reproducibility and lack of requirements for specialized equipment.8 Various Indian workers9, 10, 11 used Mueller Hinton Agar supplemented with 2% Glucose and 0.5µg/ml Methylene Blue Dye for Antifungal Susceptibility testing by Disk Diffusion Method and reported an increased resistance of non –albicans Candida isolates, to Azole group of drugs. The present study was designed to perform antifungal susceptibility test on Candida isolates by Disk Diffusion Method and study its susceptibility pattern.

Materials and Methods

The present study was conducted in the department of Microbiology in a tertiary care hospital in Hyderabad from January 2013 to June 2014, with prior approval of the Institutional Ethics Committee. A total of 102 Candida isolates from different clinical samples were processed for species identification. Candida albicans was the major species isolated followed by C. tropicalis, C. parapsilosis, C. krusei, C. glabrata and C. dubliniensis. All the speciated Candida isolates were subjected to Antifungal susceptibility testing by disk diffusion method using Mueller-Hinton Agar + 2% Glucose and 0.5 μg/mL Methylene Blue Dye (GMB) Medium as per CLSI guidelines.12 Antifungal disc used: Amphotericin – B (100U), Clotrimazole (10µg), Fluconazole (25µg) and Voriconazole (1µg). 0.5 McFarland standards was used to standardize the inoculum density. C.albicans ATCC 90028 and C. parapsilosis ATCC 22019 were used as Controls. All the culture media, Antifungal disc, and control strains were obtained from Himedia Laboratories, Mumbai.

Results

The antifungal susceptibility pattern of candida species is shown in the following table.

Table 1

Antifungal susceptibility pattern of Candida species.

Candida spp.

Amphotericin –B

Clotrimazole

Fluconazole

Voriconazole

S

R

S

R

S

R

S

R

C.albicans (n =48)

48(100%)

-

45(93.75%)

3(6.25%)

46(95.83%)

2(4.16%)

47(97.91%)

1(2.08%)

C.tropicalis (n =34)

32(94.11%)

2(5.88%)

28(82.35%)

6(17.64%)

28(82.35%)

6(17.64%)

32(94.11%)

2(5.88%)

C.parapsilosis (n =8)

8(100%)

-

7(87.5%)

1(12.5%)

6(75%)

2(25%)

7(87.5%)

1(12.5%)

C.krusei (n =6)

5(83.33%)

1(16.66%)

4(66.66%)

2(33.33%)

0

6(100%)

5(83.33%)

1(16.66%)

C.glabrata (n =4)

4(100%)

-

2(50%)

2(50%)

1(25%)

3(75%)

4(100%)

-

C.dubliniensis (n =2)

2(100%)

0

1(50%)

1(50%)

2(100%)

0

2(100%)

0

Figure 1

Antifungal susceptibility pattern of Candida isolates on Mueller- Hinton Agar + GMB

https://s3-us-west-2.amazonaws.com/typeset-prod-media-server/c17c8189-4b17-488c-b400-425ff48ca5bcimage1.png
Figure 2

Antifungal sensitivity pattern of Candida isolates

https://s3-us-west-2.amazonaws.com/typeset-prod-media-server/c17c8189-4b17-488c-b400-425ff48ca5bcimage2.png
Figure 3

Antifungal Resistance pattern of Candida isolates

https://s3-us-west-2.amazonaws.com/typeset-prod-media-server/c17c8189-4b17-488c-b400-425ff48ca5bcimage3.png

Discussion

Candida species differ in their susceptibility to antifungal agents. On comparison of susceptibility pattern of C. albicans and non-albicans Candida species it was observed that C. albicans is more susceptible to all the antifungal agents tested. In the present study all the Candida albicans showed 100% susceptibility to Amphotericin-B. Similar findings were reported by other studies.13, 14 It was observed that C. albicans showed high fluconazole susceptibility rate (95.83%) in the present study, which is consistent with other reports by Akortha et al.,15Amar etal.,16 & Pahwa et al.,17 who also observed high Fluconazole susceptibility rates of 95.7%, 92.53% and 99% respectively. 92.53% of C. albicans were susceptible to Clotrimazole in a study by Amar et al.,16 which can be compared with the present study (93.7%). Pahwa et al.,17 reported 99% of C. albicans as susceptible to Voriconazole, which can be compared with 97.91% in present study.

Among non albicans Candida spp., susceptibility to Amphotericin–B and Voriconazole varied between 83.33% to 100 %, Clotrimazole between 50% to 87.5% and Fluconazole between 25% to 100%. Non-albicans Candida showed higher resistance to all the drugs than C. albicans. Maximum resistance was shown by C. krusei (100%) and C. glabrata (75%) in the present study. Findings from the present study indicate that all the isolates of C. krusei were resistant to Fluconazole. Similar findings were shown by Hamza et al.18 Non- albicans Candida has shown an increased resistance to azoles as in other studies.19, 20 Azole resistance candidiasis appears to be on the rise. The resistance to Fluconazole is of great concern because, it is most commonly used Azole for superficial as well as deep-seated candidiasis. It was observed that out of the total 19 isolates which showed resistance to Fluconazole,14 isolates were sensitive to Voriconazole. Voriconazole seemed to be superior to Fluconazole with a better susceptibility in the Fluconazole resistant strains also. This may be due to the more effective binding of Voriconazole to Cytochrome P450 isoenzyme of Candida species.

It was observed that infections with non-albicans Candida species (NAC) have shown decreased susceptibility to azole group of drugs.

Conclusion

Findings of the antifungal susceptibility test suggest that Candida spp., differ in their susceptibility to antifungal agents. On comparison of susceptibility pattern of C. albicans and non-albicans Candida species it was observed that non-albicans Candida showed higher resistance to all the drugs than C. albicans. Resistance to azole group of drugs was more pronounced in non-albicans candida spp. Antifungal susceptibility testing of Candida isolates will be helpful in guiding physicians to select the appropriate antifungal drug so that therapeutic failures can be avoided thus decreasing patient morbidity and mortality.

Acknowledgement

  1. I express my heartfelt gratitude to Dr. Maimoona Mustafa, Professor and HOD, Department of Microbiology, Deccan College of Medical Sciences, Hyderabad, who has always been a source of inspiration and guidance.

  2. I am thankful to the clinical and technical staff of the hospital for extending their cooperation.

Conflicts of Interest

All contributing authors declare no conflicts of interest.

Source of Funding

None.

References

1 

F C Odds Candida and Candidosis: A Review and Bibilography2nd Edn.London: Baillière Tindall1988

2 

E Segal D Elad WG Merz RJ Hay Candidiasis. In: Topley and Wilson’s Microbiology and Microbial Infections. Medical Mycology10th Edn.Hodder Arnold Publishers2005579623

3 

J Chander Candidiasis. In: A textbook of Medical Mycology3rd Edn.Mehta PublishersNew Delhi200926690

4 

D Greenspan Treatment of oral candidiasis in HIV infectionOral Surg Oral Med Oral Pathol 1994782211510.1016/0030-4220(94)90149-x

5 

VP Baradkar M Mathur S Kumar Hichrom candida agar for identification of candida speciesIndian J Pathol Microbiol 201053193510.4103/0377-4929.59192

6 

A Mane S Panchavalli S Bembalkar A Rishbud Species distribution and antifungal susceptibility of oral Candida colonising or infecting HIV infected individualsIndian J Med Res20101318368

7 

SG Revankar JR Graybill TF Patterson WG Merz RJ Hay Antifungal agents and antifungal susceptibility testingTopley and Wilson’s Microbiology and Microbial Infections. Medical Mycology. 10th Edn.Hodder Arnold PublishersLondon14763

8 

MJ Matar L Ostrosky-Zeichner VL Paetznick JR Rodriguez E Chen JH Rex Correlation between E-Test, Disk Diffusion, and Microdilution Methods for Antifungal Susceptibility Testing of Fluconazole and VoriconazoleAntimicrob Agents Chemother200347516475110.1128/aac.47.5.1647-1651.2003

9 

V Manjunath GS Vidya A Sharma Mridula Raj Prakash, Murugesh. Speciation of Candida by hicrome agar and sugar assimilation test in both hiv infected and non-infected patientsInt J Biol Med Res201232177882

10 

LR Patel JD Pethani P Bhatia SD Rathod PD shah Prevalence of Candida infection and its antifungal susceptibility pattern in tertiary care hospital, AhmedabadNatl J Med Res20122443941

11 

S Mondal A Mondal N Pal P Banerjee S Kumar D Bhargava Species distribution and in vitro antifungal susceptibility patterns of CandidaJ Inst Med Nepal201335145910.3126/jiom.v35i1.8897

12 

Clinical Laboratory Standards Institute (C.L.S.I.) Method for Antifungal Disk Diffusion Susceptibility of yeasts; Approved Guidelines- Second Edition. C.L.S.I. document M44-A2, Wayne, PA: Clinical and Laboratory standards Institute2009

13 

S Dharwad RM Saldanha Dominic Species Identification of Candida Isolates in Various Clinical Specimens with Their Antifungal Susceptibility PatternsJ Clin Diagn Res201156117781

14 

D Vijaya T R Harsha T Nagaratnamma Candida Speciation Using Chrom AgarJ Clin Diagn Res2011547557

15 

E E Akortha V O Nwaugo N O Chikwe Antifungal resistance among Candida species from patients with genitourinary tract infection isolated in Benin City, Edo state, NigeriaAfr J Microbiol Res20093116949

16 

AC Sajjan VV Mahalakshmi D Hajare Prevalence and Antifungal Susceptibility of Candida Species Isolated From Patients Attending Tertiary Care HospitalIOSR J Dent Med Sci201413544910.9790/0853-13524449

17 

N Pahwa R Kumar S Nirkhiwale A Bandi Species distribution and drug susceptibility of candida in clinical isolates from a tertiary care centre at IndoreIndian J Med Microbiol201432144810.4103/0255-0857.124300

18 

OJM Hamza MIN Matee MJ Moshi ENM Simon F Mugusi FHM Mikx Species distribution and in vitro antifungal susceptibility of oral yeast isolates from Tanzanian HIV-infected patients with primary and recurrent oropharyngeal candidiasisBMC Microbiol20088113510.1186/1471-2180-8-135

19 

MU Devi K H Sulochana Devi HL Singh A study on the prevalence, pattern of Antifungal susceptibility and co-relation of CD4 + T Lymphocytes amongst the Candida isolates from HIV patients in ManipurN J Basic Med Sci2432932

20 

SC Deorukhar S Saini Laboratory approach for diagnosis of Candida through agesInt J Curr Microbiol App Sci20143120618



jats-html.xsl

© This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


  • Article highlights
  • Article tables
  • Article images

Article History

Received : 28-04-2021

Accepted : 21-05-2021

Available online : 25-06-2021


View Article

PDF File   Full Text Article


Downlaod

PDF File   XML File   ePub File


Digital Object Identifier (DOI)

Article DOI

https://doi.org/10.18231/j.ijmmtd.2021.017


Article Metrics






Article Access statistics

Viewed: 165

PDF Downloaded: 70



Wiki in hindi