Intra- and Interlaboratory Study of a Method for Testing the Antifungal Susceptibilities of Dermatophytes

The National Committee for Clinical Laboratory Standards (NCCLS)M38-A standard for the susceptibility testing of conidium-formingfilamentous fungi does not explicitly address the testing ofdermatophytes. This multicenter study, involving six laboratories,investigated the MIC reproducibility of seven antifungal agentstested against 25 dermatophyte isolates (5 blinded pairs offive dermatophyte species per site for a total of 300 tests),using the method of dermatophyte testing developed at the Centerfor Medical Mycology, Cleveland, Ohio. The dermatophytes testedincluded Trichophyton rubrum, Trichophyton mentagrophytes, Trichophytontonsurans, Epidermophyton floccosum, and Microsporum canis.Seven antifungals with activity against dermatophytes were tested,including ciclopirox, fluconazole, griseofulvin, itraconazole,posaconazole, terbinafine, and voriconazole. InterlaboratoryMICs for all isolates were in 92 to 100% agreement at a visualendpoint reading of 50% inhibition as compared to the growthcontrol and 88 to 99% agreement at a visual endpoint readingof 80% inhibition as compared to the growth control. IntralaboratoryMICs between blinded pairs were in 97% agreement at a visualendpoint reading of 50% inhibition as compared to the growthcontrol and 96% agreement at a visual endpoint reading of 80%inhibition as compared to the growth control. Data from thisstudy support consideration of this method as an amendment tothe NCCLS M38-A standard for the testing of dermatophytes.

INTRODUCTION

Top

Introduction

There have been several multicenter studies involving filamentousfungi which have been used in the development of the NationalCommittee for Clinical Laboratory Standards (NCCLS) ReferenceMethod for Broth Dilution Antifungal Susceptibility Testingof Conidium-Forming Filamentous Fungi. Approved Standard M38-A(8). However, the published document does not address the antifungalsusceptibilities of dermatophytes such as Trichophyton, Microsporum,and Epidermophyton species. Earlier studies of dermatophytesusceptibility have compared various methods. Niewerth et al.(9) compared the agar macrodilution and broth microdilutionmethods, demonstrating consistently lower MICs with the microdilutionmethod. These lower MICs were more in line with skin tissuelevels achieved by usual treatment regimens. Granade and Artis(5) used standardized fragmented mycelia as an alternative toconidial suspensions to overcome the lack of conidiation insome dermatophyte strains. Fernandez-Torres et al. (4) comparedtwo different conidial suspension concentrations in the brothmicrodilution method and found no difference in the resultingMICs. Clearly, development and standardization of a method todetermine the susceptibility of dermatophytes are needed.

The purpose of this multicenter study was to determine the inter-and intralaboratory reproducibility of MIC testing of commondermatophyte species such as Trichophyton, Epidermophyton, andMicrosporum, using the microdilution method developed at theCenter for Medical Mycology. In order to develop such a method,Norris et al. (10) initially established the optimal growthconditions for the most common dermatophyte strains. These includedthe use of RPMI 1640 as the growth medium and 35°C for 4days as the optimal temperature and incubation time, respectively.However, one special problem posed by dermatophytes is thatconidium formation by Trichophyton rubrum is very poor on standardfungal isolation media. Subsequently, Jessup et al. (6) establishedthe use of oatmeal cereal agar as the optimal growth mediumfor inducing conidium formation in T. rubrum isolates.

The NCCLS M38-A standard for susceptibility testing of filamentousfungi describes both macro- and microdilution methodologies,and according to results achieved in the multicenter study offilamentous fungi by Espinel-Ingroff, (1), interlaboratory agreementwas demonstrated by both macro- and microdilution methods. Therefore,for this study the microdilution method was chosen because ofits greater ease of performance. The aim of this study was tovalidate this method as a prelude to inclusion as a referencemethod for determining the antifungal susceptibility of dermatophyteisolates.

MATERIALS AND METHODS

Top

Materials and Methods

Study participants. The following laboratories participated in this interlaboratorystudy: Department of Health, State of New York, Albany; MedicalCollege of Virginia/Virginia Commonwealth University, Richmond;Center for Medical Mycology, University Hospitals of Cleveland/CaseWestern Reserve University, Cleveland, Ohio; University of IowaCollege of Medicine, Iowa City; University of Texas Health ScienceCenter, Audie L. Murphy Memorial Veterans Hospital, San Antonio;and Centers for Disease Control and Prevention, Atlanta, Ga.

Study design. The six participating laboratories were supplied with frozenmicrotiter plates, prepared by Trek Diagnostics, Westlake, Ohio.These plates contained 10 serial dilutions of the antifungalsused, prepared in RPMI 1640, as well as two rows of RPMI 1640without antifungal agents, which acted as growth and sterilitycontrols. The individual antifungal ranges used were as follows:ciclopirox, 0.06 to 32 µg/ml; fluconazole, 0.125 to 64µg/ml; griseofulvin, 0.125 to 64 µg/ml; itraconazole,0.001 to 0.5 µg/ml; posaconazole, 0.015 to 8.0 µg/ml;terbinafine, 0.001 to 0.5 µg/ml; and voriconazole, 0.001to 0.5 µg/ml. Each well contained 100 µl of drugor RPMI 1640 alone. Ten-milliliter tubes of RPMI 1640 were alsosupplied for inoculum preparation.

Isolates. Isolates were chosen from the culture collection of clinicalisolates maintained at the Center for Medical Mycology, UniversityHospitals of Cleveland/Case Western Reserve University. Theseisolates had originally been subcultured onto potato dextroseagar (PDA) slants, incubated at 30°C until luxuriant, andfrozen at –80°C. Stored isolates were thawed and subculturedonto PDA plates, from which sets of new slants were prepared.Each laboratory was sent a set of five blinded pairs of fivedermatophyte strains, including T. rubrum, Trichophyton mentagrophytes,Trichophyton tonsurans, Epidermophyton floccosum, and Microsporumcanis. Three T. rubrum strains for which terbinafine MICs werelow were included, corresponding to the published MIC data byOsborne et al. (11) and Mukherjee et al. (7) that the majorityof terbinafine MICs for T. rubrum wild-type strains were 0.004and 0.002 µg/ml, respectively. Two T. rubrum strains forwhich MICs were elevated were also included.

Antifungal agents. Standard powders of seven antifungals were obtained from therespective manufacturers, including ciclopirox (Dermik/Aventis,Berwyn, Pa.), fluconazole (Pfizer, New York, N.Y.), griseofulvin(Sigma), itraconazole (Janssen, Titusville, N.J.), posaconazole(Schering-Plough Research Institute, Kenilworth, N.J.), terbinafine(Novartis, Basel, Switzerland), and voriconazole (Pfizer).

Susceptibility method. The method used to determine the antifungal susceptibilitiesof dermatophytes to these agents was based on the publicationsof Norris (10) and Jessup (6). Each dermatophyte isolate wassubcultured onto PDA and incubated at 30°C for 4 to 5 daysor until good conidiation was produced.

T. rubrum isolates were subcultured onto cereal (oatmeal) agarinstead of PDA in order to induce conidium production. A suspensionof conidia in sterile saline was made by gently swabbing thecolony surface with a sterile swab. The suspension was allowedto settle for 5 to 10 min, and conidia were counted with a hemacytometer.(For T. rubrum strains, turbidity produced by transference ofoatmeal agar precludes the use of McFarland standards.) Workingsuspensions of conidia were prepared in 10 ml of RPMI 1640 toa final concentration of 1 x 10³ to 3 x 10³ CFU/ml. Yeast controls,which included Candida parapsilosis ATCC 22019 and Candida kruseiATCC 6258, were subcultured onto PDA and incubated at 35°Cfor 24 h. Yeast inocula were prepared to a final concentrationof 0.5 x 10³ to 2.5 x 10³ CFU/ml. Microtiter plates were removedfrom the freezer and allow to thaw. Each drug concentrationwell and growth control well was inoculated with 100 µlof cell suspension by using a multichannel pipette. The finalvolume in each well was 200 µl. Dermatophyte plates wereincubated at 35°C for 4 days (yeast controls were incubatedfor 48 h.) Plates were examined visually for 50 and 80% growthinhibition as compared to the growth control. MIC results wererecorded in micrograms per milliliter.

Data analysis. All reported results were included in the data analysis. Themeasurement of interlaboratory agreement for each isolate (T.rubrum, T. mentagrophytes, etc.) was determined as a percentageof endpoints within 1, 2, and 3 dilutions (e.g., 0.5, 1.0, and2.0 µg/ml). Interlaboratory agreement for the entire groupof isolates, as well as intralaboratory agreement of blindedpair test results, was similarly determined. Results were consideredin agreement if they fell within 3 dilutions.

RESULTS

Top

Results

Table 1 shows the cumulative MIC data from all sites reportedby individual drug. All six participants detected two pairsof terbinafine-resistant T. rubrum isolates (one site had anadditional outlier). Two sites did not report MICs for fourisolates, either because of lack of conidiation, which occurredwith M. canis subcultures in particular, or contamination.

View this table:
[in this window]
[in a new window]
TABLE 1. Cumulative MIC parameter data from all sites

When read at the 50% endpoint, the MICs at which 50 and 90%of the isolates tested are inhibited (MIC₅₀ and MIC₉₀, respectively)for all antifungals were within 1 dilution of those read atthe 80% endpoint.

Table 2 summarizes the interlaboratory agreement of individualdrugs. Interlaboratory agreement within 3 dilutions ranged from92% (itraconazole) to 100% (voriconazole and posaconazole) atthe 50% inhibition endpoint and 87.8% (fluconazole) to 99.3%(posaconazole) at the 80% inhibition endpoint.

View this table:
[in this window]
[in a new window]
TABLE 2. Interlaboratory agreement summary

Table 3 summarizes the intralaboratory agreement or reproducibilityof MIC results of blinded pairs by individual drug. Intralaboratoryagreement within 3 dilutions ranged from 93.9% (fluconazole)to 100% (posaconazole) at the 50% inhibition endpoint and 89%(fluconazole) to 100% (ciclopirox and posaconazole) at the 80%inhibition endpoint.

View this table:
[in this window]
[in a new window]
TABLE 3. Intralaboratory agreement summary

DISCUSSION

Top

Discussion

Previously, griseofulvin had been the only antifungal availablefor the treatment of dermatophytoses. Recently, new antifungalshave been developed that have activity against dermatophytes,and a reliable susceptibility testing method is needed to helpphysicians manage patients with dermatophyte infections. Tothis end, we designed a susceptibility method for dermatophyteisolates by first establishing the optimal growth conditions,including medium, temperature, and incubation time (6, 10).The current multicenter study was conducted as a prelude tothe inclusion of this method as an NCCLS standard.

When establishing a susceptibility testing method, it is imperativeto ensure reproducibility of endpoints and detection of resistance.Several previous multicenter studies have been conducted toestablish guidelines for the susceptibility testing of yeastsand filamentous fungi (2, 3, 12, 13). The results from our studyare similar to those obtained in those previous studies, whichwere subsequently used to establish approved standards.

In the present study, reproducibility of MIC endpoints was veryhigh by both inter- and intralaboratory comparison. Lower percentagesof agreement within 3 dilutions were achieved with fluconazoleand itraconazole, although none were less than 87%. Since nosignificant differences were seen between 50 and 80% inhibitionendpoints, we recommend the 80% inhibition endpoint for easeof interpretation. Importantly, all sites were able to detectresistance in the two pairs of terbinafine-resistant T. rubrumisolates provided.

Based upon the results of this multicenter study, adoption ofthis method as an amendment to the NCCLS M38-A standard forthe testing of dermatophytes is warranted. Correlation of invitro dermatophyte MICs with clinical outcomes, as well as establishingquality control strains, remains to be determined.

ACKNOWLEDGMENTS

The Center for Medical Mycology thanks Dermik Laboratories,Pfizer, Inc., and Schering-Plough Research Institute for theirgenerous support of this study.

FOOTNOTES

* Corresponding author. Mailing address: Center for Medical Mycology, University Hospitals of Cleveland/Case Western Reserve University, 11100 Euclid Ave., Cleveland, OH 44106-5028. Phone: (216) 844-8580. Fax: (216) 844-1076. E-mail: mag3{at}cwru.edu.

REFERENCES

Top