Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 24, Num. 4, 2006, pp. 273-279

Indian Journal of Medical Microbiology, Vol. 24, No. 4, October-December, 2006, pp. 273-279

Original Article

In-vitro susceptibility testing by agar dilution method to determine the minimum inhibitory concentrations of amphotericin B, fluconazole and ketoconazole against ocular fungal isolates

Therese KL, Bagyalakshmi R, Madhavan HN, Deepa P

Department of Microbiology, Vision Research Foundation, Sankara Nethralaya, Chennai - 600 006, Tamilnadu

Correspondence Address:Department of Microbiology, Vision Research Foundation, Sankara Nethralaya, Chennai - 600 006, Tamilnadu Email: lilyirudayam@gmail.com

Date of Submission: 27-Jun-2005
Date of Acceptance: 10-Jun-2006

Code Number: mb06079

Abstract

Keywords: Amphotericin B, fluconazole, ketoconazole, MIC, agar dilution method, antifungal susceptibility testing

Fungi are opportunistic in the eye, since they rarely infect healthy, intact ocular tissues. An overwhelming number of fungal genera and species have been implicated as cause of ophthalmic mycoses and this number is steadily increasing. Fungal keratitis and endophthalmitis are the major ocular infections in developing countries. Mycotic keratitis apparently occurs much more frequently in India than in developed countries.[1] Fungi were identified as the principal aetiological agent of corneal ulceration (44%) in India.[2]

Fungal keratitis contributes to 6 to 56%[3] of keratitis and fungal endophthalmitis contributes to 4 to 11% of all cases of endophthalmitis.[4]

The epidemiological pattern of corneal ulceration varies significantly from country to country and even from region to region. Fusarium species (47.1%) and Aspergillus species (16.1%) are the most common aetiological agents of corneal ulceration in India[5] and natamycin (5%) or amphotericin B (0.15%) remain the drug of choice for superficial keratitis.[1] The recent explosion in the rates of opportunistic fungal infections, combined with the increasing number of reports of resistance to the available antifungal agents has propelled interest in clinically relevant methods for antifungal sensitivity testing. The need for antifungal susceptibility testing increases beyond testing Candida species because resistance to antifungal drugs have been demonstrated against such diverse fungi as Cryptococcus neoformans , Aspergillus fumigatus, Aspergillus terreus, Histoplasma capsulatum and Trichosporon species. Hence it becomes evident that the need for meaningful susceptibility test result is very important for fungi as it is for bacteria.[6] Although antifungal susceptibility testing remains less well-established and utilized than antibacterial testing, the scientific support for its validity has benefited greatly by extrapolation from antibacterial testing.[7] The methods for antifungal sensitivity testing include National Committee for Clinical Laboratory Standards NCCLS (new name Clinical Laboratory Standards Institute (CLSI ) broth based methodology (M 27-A), CLSI methodology for moulds,[8] E-test agar based testing methods, flow cytometry and use of viability dyes. The above methods are time consuming and labour intensive, hence a more economical method such as agar dilution have been described.[9] There are only a limited number of antifungal susceptibility testing reports on ocular fungal isolates from India.[10],[11] The present study is focused on standardization of the in vitro agar dilution method for determination of minimum inhibitory concentration (MIC) of amphotericin B, ketoconazole and fluconazole on ocular fungal isolates.

Materials and Methods

The in-vitro antifungal susceptibility testing was carried out with the ocular fungal isolates obtained between December 1999 and May 2002 in Larsen and Toubro. Microbiology Research Centre, Sankara Nethralaya, Chennai, India.

Fungal strains

Standard strains of Candida species

Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, Candida albicans ATCC 24433, Candida albicans ATCC 90028, Candida tropicalis ATCC 750 and Candida parapsilosis ATCC 90018 were the ATCC strains included for standardization of in-vitro susceptibility testing, based on guidelines of CLSI[8] which states that ideal reference strains for quality control have MICs that fall near the mid-range of the concentration for all antifungal drugs.

Ocular isolates of fungi included in the study

Fifty ocular isolates of yeast and 130 filamentous fungi were included in the study. The distribution of the isolates of yeast and filamentous fungi from ocular specimens are shown in [Table - 1][Table - 2]. The fungi were isolated by the standard procedures[12] from corneal scrapings of keratitis and endopthalmitis patients. The yeast were identified by the standard germ tube test and fermentation and assimilation of carbohydrate tests. The filamentous fungi were identified based on colony morphology and by lactophenol cotton blue mount preparation of the slide cultures. The fungal isolates of donor corneal rim (DCR) and conjunctival swab were not associated with any clinical disease. However, these were also included to determine the MICs of amphotericin B, ketoconazole and fluconazole on ocular fungal isolates.

Preparation of stock solutions of antifungal agents

We have followed the guidelines of CLSI[8] for preparing the various concentrations of antifungal drugs. The active components in amphotericin B obtained was 78% and ketoconazole and fluconazole were 70%.

The stock I solution of amphotericin B (HiMedia, India) was prepared by using 10 mg of amphotericin B dissolved in 1000 ml of dimethylsulfoxide (DMSO).[13] The working solution was prepared by 1 in 1000 dilution. The range of concentrations of amphotericin B was 0.0625-8 mg/mL; DMSO was used to dissolve amphotericin B following CLSI Candida parapsilosis guidelines.[8]

Fluconazole ( Cipla Pharmaceuticals Limited, India) stock was 2 mg/mL. The aqueous stock solution was used directly without any further dilutions to obtain concentrations ranging from 0.2-102.4 μg/mL

Ketoconazole (Cadila Pharmaceuticals, India) stock solution was prepared by dissolving 2 mg of ketoconazole in 1 mL of methanol according to manufacturer′s instructions. Working solution was 1 in 100 dilution to obtain concentrations ranging from 0.025-12.8 μg/mL. When water was used to dissolve ketoconazole, it resulted in precipitation of the drug. Hence, methanol was used to dissolve ketoconazole and diluted 100 times from the stock solution. The intermediate solution was further diluted to the final strength in the test medium.

Medium

The medium yeast nitrogen base (Himedia, India) was dissolved in Phosphate buffer pH 7 and it was autoclaved at 110°C for 10 minutes.[14] The medium (10 mL) was dispensed in sterile screw capped (15 ml volume) glass bottles and the required concentrations of amphotericin B, fluconazole and ketoconazole (doubling dilutions) were incorporated in the medium. With each set a growth control without the antifungal agent and solvent control DMSO for amphotericin B and methanol control for ketoconazole were included.

Preparation of standard inoculum

The fungal strains were freshly subcultured on to Sabouraud dextrose agar and incubated at 25°C for three days. The yeast cells and spores were suspended in sterile distilled water and counted using Neubauer chamber to obtain 10 ⁵ cells/ mL. Ten microlitre of standardized suspension was inoculated onto the control plates and the media incorporated with the antifungal agents. The inoculated plates were incubated at 25°C for 48 hours. The readings were taken at the end of 48 hours and 72 hours.

Interpretation of results

The MIC was the lowest concentration of drug preventing growth of macroscopically visible colonies on drug-containing plates when there was visible growth on the drug-free control plates. The MIC readings were taken at the end of 48 hours of incubation, when growth appeared on the control plate. The time period was followed according to CLSI which recommends 48 hours for yeast and filamentous fungi and 72 hours for Cryptococcus species. Throughout the study, 48 hour, 72 hour and 96 hour readings (delayed growth) were taken and there was no change in the MIC values taken at 48 or 72 or 96 hours.

Criteria for susceptibility / resistance

The criteria for susceptibility / resistance for amphotericin B, fluconazole and ketoconazole[15] for the antifungal drugs is given in [Table - 3]. Percentile 90 and Percentile 50 of amphotericin B, fluconazole and ketoconazole refers to MIC 90 and MIC 50[16] respectively and was calculated for all ocular isolates.

Results

Standardization of in-vitro susceptibility testing by agar dilution method

The in-vitro susceptibility testing of amphotericin B, fluconazole and ketoconazole on fungi was standardized by agar dilution method with standard ATCC Candida species. The results of MICs of amphotericin B, fluconazole and ketoconazole on standard strains are given in [Table - 4].

The standardized in-vitro antifungal susceptibility testing technique was applied to determine the MICs of amphotericin B, fluconazole and ketoconazole on 180 ocular fungal isolates.

Results of MIC of amphotericin B on ocular fungal isolates

All the 50 Candida species tested were susceptible to amphotericin B. Among the 130 filamentous fungi tested, 124 (95.38%) were susceptible to amphotericin B while six (4.61%) were resistant to amphotericin B [Table - 5].

The six resistant strains were Aspergillus flavus (1) , Fusarium species (2), isolated from 3 corneal scrapings and Aspergillus terreus , Aspergillus niger , Penicillium species isolated from DCR (3). Among the yeasts, the MICs of amphotericin B were within the range of 0.0625-0.125 μg/mL in 42 (84%) while 91 (70%) filamentous fungi had a MIC range of 0.0625-0.25 μg /mL. The in-vitro susceptibility testing using amphotericin B on ocular isolates is shown in [Figure - 1]. Percentile 90 and Percentile 50 of amphotericin B are represented in chart 1.

Results of MICs of fluconazole on ocular fungal isolates

Of the 50 yeasts tested, 48 (96%) were sensitive to fluconazole with MIC < 51.2 μg/mL, while 2 (4%) were resistant (MIC of fluconazole 102.4-819.2 μg/mL).The two resistant yeasts (Candida krusei) were isolated from DCR and corneal scraping. Among the 130 filamentous fungi tested, 81 (62.3%) were sensitive to fluconazole while 49 (37.7%) were resistant to fluconazole [Table - 6].

The resistant strains were isolated from corneal scraping (25), corneal button (12) and DCR (12). Among the yeasts, 24 strains (48%) had MIC of fluconazole within the range of 0.2 - 6.4 μg /mL while 39 (30%) filamentous fungi had a MIC range of 25.6 or 51.2 μg /mL. The in-vitro susceptibility testing using fluconazole on ocular isolates is shown in [Figure - 2]. Percentile 90 and Percentile 50 of fluconazole have been calculated and represented in chart 2. Candida krusei had the highest percentile value of fluconazole among yeasts and A. flavus , A. niger, A. terreus and Fusarium species showed a higher percentile value for fluconazole.

Though the percentage of resistance to fluconazole reported is high,[17] in the present study 38% resistance to fluconazole was encountered.

Results of MICs of ketoconazole on ocular fungal isolates

Of the 50 yeasts tested 45 (90%) were sensitive to ketoconazole (MIC of ketoconazole < 0.8 µg/mL) while 5 (10%) were resistant to ketoconazole (MIC of ketoconazole > 0.8 µg/mL). The resistant strains were isolated from two corneal scrapings ( C. albicans 1, C. tropicalis 1) and three DCRs ( C. krusei- 2 and C. albicans- 1). Among the 130 filamentous fungi tested, 120 (92.3%) were sensitive to ketoconazole while 10 (7.6%) were resistant [Table - 7]. The resistant strains were isolated from three corneal scrapings ( A. flavus , A. terreus , Fusarium spp.), four corneal button ( A. flavus, A. terreus, Fusarium- 2) and three DCRs ( A. niger- 2, Penicillium spp. -1). Among the yeasts, 29 (58%) had MIC of ketoconazole within the range of 0.2-0.8 µg /ml while 71 (54.6%) filamentous fungi had a MIC range of 0.2-0.8 µg/mL. Percentile 90 and Percentile 50 of ketoconazole on ocular fungal isolates have been calculated and represented in Chart 3. Based on the percentile values of ketoconazole, A. niger and A. terreus were found to have higher values when compared to other filamentous fungi.

Among the yeasts tested C. krusei had a higher percentile value of ketoconazole. The distribution of 72 resistant strains of ocular fungal isolates to amphotericin B, ketoconazole and fluconazole are given in [Table - 8].

Discussion

The present study was designed to develop a simple, cost -effective procedure of agar dilution method to determine the MICs of antifungal drugs. Six ATCC strains were included in the study for correlation of the results with agar dilution method standardized in this study with the published reports of other standard methods of susceptibility testing. In a study conducted by Makimura et al[13] Candida albicans 90028 and Candida parapsilosis ATCC 90018 have been used to evaluate the standard dilution method against the frozen plate method. In yet another study[18] involving proficiency testing, the other Candida species, Candida parapsilosis ATCC 22019, Candida tropicalis ATCC 750 and Candida krusei ATCC 6258 were used to evaluate the standard dilution method against the frozen plate method.

Makimura et al[13] used DMSO as solvent for preparation of the stock solution of amphotericin B, which was adopted in the present study. Methanol was used instead of distilled water to prepare stock ketoconazole to prevent precipitation of the drug. The intermediate concentration of the ketoconazole (0.2 μg/mL) was further diluted to final concentration ranges in the test medium. This procedure avoided the dilution artifacts that resulted from precipitation of compounds with low solubility in aqueous media and the same dilution of methanol was used without the drug for the control plate to ensure that the particular concentration did not inhibit the growth of the fungal strains.

Interestingly, we found that majority of the ocular fungal isolates were susceptible to amphotericin B, followed by ketoconazole and fluconazole. The percentage of resistance to fluconazole and ketoconazole in the present study is comparatively lower than reported by Davey et al[19] and Yoshida et al.[9] The results of the MIC of the antifungal agents by agar dilution method on standard ATCC strains of Candida species standardized in this study and the published reports of MIC by CLSI broth dilution method were comparable, indicating the suitability of the agar dilution method. The CLSI broth micro dilution method (M-27a) is time consuming, expensive and technically difficult to perform. On the other hand the agar dilution method has two important advantages over the CLSI method. The first is the visual reading based on the intensity of growth showing the clear end point of inhibition.[20] The second is that susceptibility testing of large number of fungi is easier and economical as four strains can be tested with one set of agar plates in the agar dilution method whereas each strain needs one set of tubes in the CLSI method. The emergence of antifungal drug resistance has made susceptibility testing important [21] though the applicability of in-vitro antifungal sensitivity testing may not directly correlate well with the clinical outcome. The filamentous fungi resistant to amphotericin B in this study were A. flavus, A. terreus and Fusarium species. These findings correlated with the study conducted by Arikan et al [17] in which resistance to fluconazole was observed in 37.7% and to ketoconazole in 8.7% of the filamentous fungi. The percentage of resistance of fluconazole and ketoconazole correlated with the report published earlier.[20] In the present study, Fusarium species showed the highest resistance to fluconazole followed by A. flavus and Penicillium spp. An important finding in the present study is that all the resistant strains to the three antifungal drugs were from that of external ocular specimens and the percentage of resistance to amphotericin B, fluconazole and ketoconazole were comparable to that of the published reports.[1] Additionally, among filamentous fungi, A. niger followed by A. terreus exhibited higher percentage of resistance to amphotericin B, fluconazole and ketoconazole and among the yeasts, C. krusei exhibited higher resistance to fluconazole and ketoconazole. Majority of the ocular fungal isolates included in the study were susceptible to amphotericin B when compared to ketoconazole and fluconazole which were effective mainly against yeasts. However, MIC results obtained on ocular fungal isolates by in-vitro sensitivity testing are only meaningful when compared to the ocular tissue concentrations of the drugs obtained after oral, topical and parenteral administration. It should reliably predict the in-vivo response to therapy in human infections. However, drug pharmacokinetics and drug interactions, factors related to the host immune response and/or the status of the current underlying disease, proper patient management system and factors related to the virulence of the infecting organism and its interactions with both the host and therapeutic agents appear to have more value than the MIC as predictors of clinical outcome.

Conclusion

The present study indicates that the agar dilution method can be adopted for in-vitro antifungal sensitivity testing, as it is a simple, reproducible, cost effective and easy to perform technique in a routine clinical microbiology laboratory.

References

Copyright 2006 - Indian Journal of Medical Microbiology

The following images related to this document are available:

Photo images