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Journal of Clinical Microbiology, October 2008, p. 3477-3478, Vol. 46, No. 10
0095-1137/08/$08.00+0     doi:10.1128/JCM.00610-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

In Vitro Natamycin Susceptibility of Ocular Isolates of Fusarium and Aspergillus Species: Comparison of Commercially Formulated Natamycin Eye Drops to Pharmaceutical-Grade Powder

P. Lalitha,¹ R. Vijaykumar,¹ N. V. Prajna,² and A. W. Fothergill^3*

Department of Ocular Microbiology, Aravind Eye Hospital, Madurai, India,¹ Department of Cornea, Aravind Eye Hospital, Madurai, India,² Department of Pathology, University of Texas Health Sciences Center, San Antonio, Texas³

Received 31 March 2008/ Returned for modification 12 May 2008/ Accepted 31 July 2008

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The Clinical and Laboratory Standards Institute susceptibility method prohibits the use of pharmacy preparations, but obtaining pure powders is difficult. The activity of natamycin against isolates of Aspergillus and Fusarium species isolated from keratitis was assessed by using both powder and pharmacy eye drop preparations. Eye drop preparations may be a viable option for testing natamycin activity.

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Keratitis is a leading cause of monocular blindness worldwide (11, 12, 14, 15), and recent reports suggest that there has been a steady increase in the percentage of infectious keratitis caused by fungus (1, 12, 17). Fusarium and Aspergillus species are the most common fungal isolates associated with this infection (1, 17). Classes of antifungals used for treatment of fungal keratitis include the polyenes, triazoles, and echinocandins (13). Natamycin (NAT), a tetraene polyene, has long been considered the mainstay of treatment for filamentous fungal keratitis and is the only available antifungal medication that has been approved for this indication by the U.S. Food and Drug Administration. Although previous reports regarding the efficacy of NAT in fungal keratitis are available (4-6), there are few studies that describe the in vitro activity of NAT against the common ocular isolates (2, 7, 8, 9).

The Clinical Laboratory and Standards Institute guidelines stress the use of pharmaceutical-grade powder solely for susceptibility testing. This is due to unknown purity and potency values and additives that may impact results (3). Pharmaceutical-grade powder is very difficult to obtain, especially for drugs that are not frequently used, such as NAT. We studied the in vitro activity of NAT against Fusarium and Aspergillus species isolated from cases of corneal ulcers seen at the Aravind Eye Hospital, Madurai, India, and compared the MICs obtained by using both pharmaceutical-grade NAT powder (NAT-P) and commercially available NAT eye drops (NAT-D).

A total of 100 fungal isolates recovered from clinical cases of corneal ulcer were evaluated in the present study. The fungal isolates included 41 Fusarium, 32 Aspergillus flavus, 18 Aspergillus fumigatus, 5 Aspergillus terreus, and 4 Aspergillus niger isolates. The reference strain of A. flavus ATCC 204304 was included and was tested with both formulations in each series of assays. Antifungal susceptibility testing was performed exactly as outlined in CLSI M38-A. Pharmaceutical-grade NAT was obtained from Alcon Laboratories (Ft. Worth, TX). A 5% NAT suspension of topical eye drops was purchased from Sun Pharmaceutical, Ltd. (Mumbai, India). Amphotericin B was used to assess quality control against A. flavus ATCC 204304 strains. Inocula were prepared without the use of Tween 20. NAT-P was weighed and dissolved in dimethyl sulfoxide. Stock solutions were stored frozen at –70°C until needed. Drug dilution tubes were prepared from a 3,200-µg/ml stock and diluted 1:2.5 with water to achieve a top concentration of 1,280 µg/ml. For NAT-D, 1 ml of 5% NAT suspension was mixed with 9 ml of 100% dimethyl sulfoxide (5,000 µg/ml). One part of this suspension was mixed with three parts of sterile water to give a concentration of 1,280 µg/ml. Final drug concentrations ranged from 128 to 0.25 µg/ml.

The MIC was defined as the lowest drug concentration that completely inhibited visual growth. NAT-P and NAT-D were tested simultaneously in the same plate for all of the isolates. The reference strain was tested in the same manner as the clinical isolates to verify the reproducibility for each run. The MICs of NAT-P and NAT-D were compared by using a Mann-Whitney test, and a P value of >0.05 was taken to mean that there was no significant difference found between groups. A P value of <0.05 meant a significant difference was found between groups.

The results of the MIC testing are given in Table 1. Comparison of the MICs between NAT-P and NAT-D showed perfect agreement, with 92.6% for Fusarium spp. (38 of 41 isolates) and 71.9% for A. flavus (23 of 32 isolates). Overall, for 79% of the isolates the NAT MICs were identical regardless of the drug formulation. Considering the allowable ±2 dilutions, there was only a one-dilution variation for 21% of the remaining isolates, bringing the overall agreement to 100% (Table 2).

View this table: [in this window] [in a new window]

TABLE 1. MICs of NAT-P and commercially available NAT-D for 100 corneal fungal isolates

View this table: [in this window] [in a new window]

TABLE 2. Comparison of MICs of NAT-P and commercially available NAT-D

Interestingly, susceptibility to NAT varied by species when the results for the aspergilli were examined. The NAT MICs for A. flavus were higher than for the other species (i.e., a NAT MIC₉₀ of 64 µg/ml compared to a NAT MIC₉₀ of 4 µg/ml for A. fumigatus). For other species, there were too few isolates to calculate the MIC₉₀.

The present study shows that the NAT had good activity against both Fusarium and Aspergillus spp., with slightly higher NAT MICs for Aspergillus spp. To date, susceptibility breakpoints have not been established for NAT, but the MICs obtained here are likely within the achievable levels obtained in the eye during standard therapy. For discussion purposes, the desirable target value of 16 µg/ml is used to represent susceptibility. Interestingly, the MICs obtained from both the pharmaceutical-grade powder and the eye drops were comparable.

In a recent study from China on the pattern of ocular fungal isolates, it was found that Fusarium was the predominant pathogen and that 93% of the isolates were sensitive to NAT, whereas 92% of the Aspergillus isolates were sensitive to itraconazole (16, 17).

Lalitha et al., examining the susceptibility of filamentous fungi isolated from keratitis to amphotericin B, NAT, caspofungin acetate, itraconazole, voriconazole, and posaconazole, found that triazoles and caspofungin had the lowest MICs against Aspergillus species; that voriconazole, amphotericin B, and posaconazole had the lowest MICs against Fusarium species; and that none of the Fusarium species were inhibited by itraconazole or caspofungin. The MICs of amphotericin B were significantly lower than those of NAT, but after correction for the typical prescription dose, NAT was found to be more effective (8). It is important that the in vitro studies be correlated with the clinical outcome. Such data are limited in cases of fungal keratitis, whereas in cases of bacterial keratitis the clinical outcomes have been correlated with the sensitivities of various antibiotics, and patients in whom the antibiotic MICs were higher demonstrated a poorer outcome.

One of the main deterrents in evaluating antifungal susceptibilities is the nonavailability of a pharmaceutical grade of the drug. Another of our other aims was to determine whether the MICs obtained with the pharmaceutical powder and the eye drops could produce comparable results. We found that the results were statistically comparable, leading us to believe that the option of using a commercial preparation of the antifungal agent might be an alternative to consider in evaluating antifungal susceptibility in situations where the pure form of the drug is not be available. Further studies with NAT-D from other manufacturers are required. In addition, similar studies with other antifungals should be performed.

 
* Corresponding author. Mailing address: University of Texas Health Science Center at San Antonio, 7780 Floyd Curl Dr., San Antonio, TX 78229-3900. Phone: (210) 567-6074. Fax: (210) 567-4076. E-mail: fothergill{at}uthscsa.edu

Published ahead of print on 13 August 2008.

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Journal of Clinical Microbiology, October 2008, p. 3477-3478, Vol. 46, No. 10
0095-1137/08/$08.00+0     doi:10.1128/JCM.00610-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lalitha, P. Articles by Fothergill, A. W. Search for Related Content PubMed PubMed Citation Articles by Lalitha, P. Articles by Fothergill, A. W.