Evaluation of the Etest Method Using Mueller-Hinton Agar with Glucose and Methylene Blue for Determining Amphotericin B MICs for 4,936 Clinical Isolates of Candida Species

The performance of the Etest using Mueller-Hinton agar supplementedwith glucose (2%) and methylene blue (0.5 µg/ml) (MH-GMB)for amphotericin B susceptibility testing of 4,936 isolatesof Candida spp. was assessed against that of Etest using RPMIagar with 2% glucose (RPG). MICs were determined by Etest inboth media for all 4,936 isolates and were read after incubationfor 48 h at 35°C. The Candida isolates included C. albicans(n = 2,728), C. glabrata (n = 722), C. parapsilosis (n = 666),C. tropicalis (n = 528), C. krusei (n = 143), C. lusitaniae(n = 54), C. guilliermondii (n = 39), C. pelliculosa (n = 17),C. kefyr (n = 15), C. rugosa (n = 11), C. dubliniensis (n =5), C. zeylanoides (n = 4), C. lipolytica (n = 3), and C. famata(n = 1). The Etest results with MH-GMB correlated well withthose with RPG. Overall agreement was 92.9%, and agreementsfor individual species were as follows: C. lusitaniae, 98.1%;C. albicans, 95.1%; C. glabrata, 94.3%; C. krusei, 91.6%; C.parapsilosis, 86.6%; and C. tropicalis, 86.4%. The Etest methodusing MH-GMB appears to be a useful method for determining amphotericinB susceptibilities of Candida species.

INTRODUCTION

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Introduction

The Etest stable agar gradient MIC method (AB BIODISK, Solna,Sweden) has been established as an excellent method for testingamphotericin B against Candida spp (4, 7, 12). Wanger et al.(12) and Law et al. (4) have demonstrated that the Etest provideda sensitive means for detection of amphotericin B-resistantstrains of Candida. Previously, we have confirmed this workand have shown that RPMI agar with 2% glucose (RPG) was superiorto other media, including antibiotic medium 3, Casitone, andunsupplemented Mueller-Hinton agar (MHA), for testing amphotericinB against Candida spp (7). Barry et al. (2) have shown thatMHA supplemented with 2% glucose and methylene blue (0.5 µg/ml)(MH-GMB) supported the growth of essentially all species ofCandida and was superior to RPG for fluconazole disk diffusiontesting. The National Committee for Clinical Laboratory Standards(NCCLS) has adopted MH-GMB as the medium of choice for diskdiffusion testing of Candida spp. (6). As both the disk diffusionand Etest methods are agar based, it is important to determinewhether both methods can be performed on the same agar medium.Although we have shown that MH-GMB works well for determiningMICs of fluconazole and voriconazole by the Etest (10), similardata for amphotericin B have not been published. In the presentstudy we compared MICs obtained with the Etest using MH-GMBwith those obtained with RPG by testing the susceptibilitiesof 4,936 clinical isolates of Candida spp. to amphotericin B.

MATERIALS AND METHODS

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Materials and Methods

Organisms. A total of 4,936 clinical isolates of Candida spp. were testedon both RPG and MH-GMB. The collection included 2,728 C. albicansisolates, 722 C. glabrata isolates, 666 C. parapsilosis isolates,528 C. tropicalis isolates, 143 C. krusei isolates, 54 C. lusitaniaeisolates, 39 C. guilliermondii isolates, 17 C. pelliculosa isolates,15 C. kefyr isolates, 11 C. rugosa isolates, 5 C. dubliniensisisolates, 4 C. zeylanoides isolates, 3 C. lipolytica isolates,and 1 C. famata isolate. The members of this collection wereall recent clinical isolates from geographically diverse medicalcenters throughout the world (11). The majority were isolatedfrom blood or normally sterile body fluids (11). The isolateswere identified by standard methods (3) and were stored as suspensionsin water at ambient temperature until used in the study. Priorto testing, each isolate was subcultured at least twice ontopotato dextrose agar (Remel, Lenexa, Kans.) to ensure purityand viability.

Susceptibility testing. Etest strips for amphotericin B were purchased from AB BIODISK.MICs were determined by Etest as described previously (7) withRPG (Remel) and an inoculum suspension adjusted to the turbidityof a 0.5 McFarland standard ( $~$ 10⁶ cells/ml). In addition, eachisolate was tested on MH-GMB. The MICs of amphotericin B onboth RPG and MH-GMB were read after 48 h of incubation at 35°Cand were determined to be at 100% inhibition of growth wherethe border of the elliptical inhibition zone intersected thescale of the strip edge (7, 9, 12). Quality control was ensuredby testing the NCCLS-recommended strains C. krusei ATCC 6258and C. parapsilosis ATCC 22019 (1, 5).

Analysis of results. The Etest MICs of amphotericin B determined on MH-GMB were comparedto those determined on RPG. Since the Etest scale has a continuousgradient of concentrations, the MICs between twofold dilutionswere raised to the next twofold level for comparison (8). Discrepanciesbetween MICs of no more than two dilutions were used to calculatethe percent agreement.

RESULTS AND DISCUSSION

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Results and Discussion

Table 1 summarizes the in vitro susceptibilities of 4,936 Candidaisolates to amphotericin B as determined by Etest using RPGand MH-GMB. The MICs obtained with RPG are consistent with thosereported previously for the individual Candida spp. (9). MICsobtained with MH-GMB were generally twofold lower than thosewith RPG.

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TABLE 1. Comparative amphotericin B susceptibility testing results for 4,936 isolates of Candida spp. determined by Etest using RPMI agar and Mueller-Hinton agar with glucose and methylene blue

Overall, 92.9% of the amphotericin B MICs obtained by the Etestin MH-GMB were within ±2 dilutions of those obtainedin RPG (Table 1). The agreement was >90% for all species,with the exception of C. parapsilosis (86.6%) and C. tropicalis(86.4%). When a discrepancy was observed between the resultsobtained with the two media, the MIC obtained with MH-GMB wasalways lower. The modal MICs obtained with both media rangedfrom 0.25 to 1 µg/ml for all species, with the exceptionof C. krusei, where the MICs tended to be elevated (2 to 4 µg/ml)with both RPG and MH-GMB.

The results of this study provide the first documentation ofthe applicability of MH-GMB for determining the in vitro susceptibilitiesof Candida spp. to amphotericin B by using the Etest stableagar gradient MIC method. As in previous studies (10), we foundthat MHA supplemented with glucose (2%) and methylene blue (0.5µg/ml) supported optimal growth of all species of Candidaand provided excellent agreement with MICs obtained on RPG.As was the case with fluconazole and voriconazole (11), theelliptical zones obtained on MH-GMB were sharp and easy to read.As with RPG, amphotericin B MICs obtained with the Etest inMH-GMB encompassed a much broader MIC range (0.002 to >32µg/ml) than those obtained with the reference broth microdilutionmethod (0.12 to 4 µg/ml) (data not shown).

In summary, we have provided the first evidence of the abilityof the Etest using MH-GMB to generate amphotericin B MIC datathat are comparable to those obtained with RPG. The importanceof this finding is that one may use the same agar medium toperform disk testing of fluconazole and voriconazole and toobtain amphotericin B MICs by using the Etest. The use of agar-basedtechnology for performing antifungal susceptibility testingof Candida provides a simple and flexible option for laboratoriesthat wish to test only a few agents selectively and do not wantto invest in broth microdilution testing.

ACKNOWLEDGMENTS

The excellent secretarial support of Linda Elliott is greatlyappreciated.

This study was supported in part by Pfizer Pharmaceuticals.

FOOTNOTES

* Corresponding author. Mailing address: Medical Microbiology Division, C606 GH, Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242. Phone: (319) 384-9566. Fax: (319) 356-4916. E-mail: michael-pfaller{at}uiowa.edu.

REFERENCES

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