Further Standardization of Broth Microdilution Methodology for In Vitro Susceptibility Testing of Caspofungin against Candida Species by Use of an International Collection of More than 3,000 Clinical Isolates

The influence of test variables on in vitro susceptibility testingof caspofungin was examined with 694 isolates of Candida albicansincluding seven laboratory-derived glucan synthesis mutants.The conditions providing the greatest separation between themutant strains and the clinical isolates were RPMI medium, MICend point criterion of partial inhibition, and incubation for24 h. These testing conditions were then applied to 3,322 isolatesof Candida spp. (3,314 clinical isolates and eight glucan synthesismutants). Among the 11 isolates for which caspofungin MICs were $≥$ 2 µg/ml, eight were accounted for by the glucan synthesismutants. The MICs for >99% of isolates were $≤$ 1 µg/ml,and thus these isolates were differentiated from strains withreduced in vitro and in vivo susceptibilities to caspofungin.

INTRODUCTION

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Introduction

Caspofungin is an echinocandin-class antifungal agent with potentin vitro and in vivo activities against Candida spp (1, 4, 7,10, 11). Caspofungin has recently been approved for primarytreatment of candidemia and other forms of invasive candidiasis(4, 11).

In vitro susceptibility studies using National Committee forClinical Laboratory Standards (NCCLS) broth microdilution (BMD)methods have documented the excellent spectrum and potency ofcaspofungin versus a wide range of Candida spp. (3, 13, 16,17). For the most part these studies have employed 48-h incubationin RPMI 1640 medium and a conservative MIC end point criterionof complete inhibition of growth (MIC-0) relative to controltubes or wells (2). By the use of these criteria, C. albicans,C. glabrata, and C. tropicalis have been shown to be the mostsusceptible species (MIC at which 90% of the isolates testedare inhibited [MIC₉₀], $≤$ 0.5 µg/ml; 99% of MICs were $≤$ 1 µg/ml)and C. parapsilosis (MIC₉₀, 4 µg/ml) and C. guilliermondii(MIC₉₀, >8 µg/ml) have been shown to be the speciesleast susceptible to caspofungin (16).

It is well known that lower MICs of caspofungin may be obtainedwith Candida spp. when testing is performed in antibiotic medium3 (AM3) rather than RPMI medium (2, 13). To some extent thismedium effect may be due to falsely elevated MICs caused bytrailing growth patterns in RPMI that are not observed in AM3(2, 8, 13). Indeed, earlier studies using scanning electronmicroscopy to study the interaction between Candida spp. andthe echinocandin LY303366 demonstrated that apparent trailingin RPMI broth beyond the concentration at which a prominentdecrease in growth was observed was due to nonviable cellulardebris rather than intact yeast cells (8). Thus, the MIC endpoint criterion for echinocandins should probably be less stringentthan MIC₀ to avoid falsely high MICs due to dead organisms andcellular debris (2, 8).

A recent multicenter (17 laboratories) study by Odds and colleaguesindicated that highly reproducible MIC results (>80% of MICswithin ±1 dilution of the modal MIC) were obtained whenCandida spp. were tested against caspofungin by the NCCLS BMDmethod with RPMI 1640 broth, incubation for no longer than 24h, and a MIC end point criterion that specified the concentrationat which the first prominent reduction in growth (MIC-2 or $≥$ 50%inhibition relative to control growth) was observed (14). Furthermore,these test conditions were sufficient to differentiate isolateswith "normal" susceptibilities from glucan synthesis mutantstrains with "low" susceptibilities to caspofungin. Althoughsimilar results were obtained when AM3 was used in place ofRPMI medium, concerns regarding batch-to-batch variability withAM3 make this a less acceptable choice of medium (2, 14).

The present study was designed to address several of the findingsand concerns raised by the studies noted above. First (phase1), the issue of medium choice, duration of incubation, andMIC end point criterion was examined using a large collectionof C. albicans clinical isolates supplemented with seven laboratory-derivedglucan synthesis mutants of C. albicans as markers of reducedin vitro and in vivo susceptibilities to caspofungin (5, 9).Second (phase 2), the optimal testing conditions, as describedby Odds et al. (14) and confirmed by our initial (phase 1) studies,were used to test an international collection of more than 3,000clinical isolates of Candida spp. encompassing nine differentspecies. Again, the seven glucan synthesis mutants of C. albicans,plus an additional strain of C. krusei with reduced in vitroand in vivo susceptibilities to caspofungin (14), were usedto identify a potentially resistant category.

MATERIALS AND METHODS

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Materials and Methods

Organisms. (i) Phase 1. A total of 687 clinical isolates of C. albicans representinga broad geographical distribution were used to assess the effectof test medium, MIC end point criterion, and duration of incubation.These isolates were obtained from various surveillance studiesconducted by the University of Iowa, and each isolate representedan individual infectious episode (15). In addition, seven laboratory-derivedglucan synthesis mutants (mutation in the FKS1 gene) of C. albicanswere included to represent strains with reduced in vitro andin vivo susceptibilities to caspofungin (2, 5, 9). The isolateswere identified by standard methods (6) and stored as watersuspensions until they were used in the study. Prior to testing,each isolate was passaged at least twice on potato dextroseagar (Remel, Lenexa, Kans.) and CHROMagar Candida (Hardy Laboratories,Santa Monica, Calif.) to ensure purity and viability.

(ii) Phase 2. A total of 3,314 clinical isolates obtained from more than 100different medical centers internationally were tested (17).The collection included 1,993 strains of C. albicans, 481 ofC. glabrata, 310 of C. tropicalis, 361 of C. parapsilosis, 100of C. krusei, 27 of C. guilliermondii, 24 of C. lusitaniae,9 of C. kefyr, and 9 of C. pelliculosa. The isolates were allrecent clinical isolates, and each represented an individualinfectious episode (17). In addition, the seven glucan synthesismutants of C. albicans described above (2) and one strain ofC. krusei with reduced susceptibility (14) were included asmarkers of decreased susceptibility to caspofungin. The isolateswere identified by standard methods (6) and stored as watersuspensions until used in the study. Prior to testing, eachisolate was passaged at least twice on potato dextrose agar(Remel) and CHROMagar Candida to ensure purity and viability.

Antifungal agents. Caspofungin reference powder was obtained from the manufacturer(Merck Co., Whitehouse Station, Pa.). A stock solution was preparedin water, and serial twofold dilutions were prepared exactlyas outlined in NCCLS document M27-A2 (12). Final dilutions weremade in RPMI 1640 medium (Sigma, St. Louis, Mo.) buffered topH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer(Sigma) (phases 1 and 2) and in AM3 (Becton Dickinson, Sparks,Md.) (phase 1).

Antifungal susceptibility studies. BMD testing was performed in accordance with the guidelinesin NCCLS document M27-A2 (12). In phase 1, both RPMI and AM3broth were used, whereas in phase 2 only RPMI broth was used.The trays were incubated at 35°C, and MIC end points wereread visually. Drug-free and yeast-free controls were included.

In phase 1, the 694 isolates of C. albicans (687 clinical isolatesand seven glucan synthesis mutants) were tested in both RPMIand AM3 broth and MICs were determined after 24 and 48 h ofincubation. MICs were determined at each time point with twovisual criteria: complete inhibition, or MIC-0, the lowest caspofunginconcentration that supported no visible growth (a clear well),and partial inhibition, or MIC-2, the lowest caspofungin concentrationthat caused a significant diminution ( $≥$ 50%) of growth below controlgrowth levels (12).

In phase 2, the 3,322 isolates of Candida spp. (3,314 clinicalisolates plus eight glucan synthesis mutants [seven of C. albicansand one of C. krusei]) were tested in RPMI broth and the partialinhibition (MIC-2) end point was determined after 24 h of incubation.

Quality control. Quality control was performed by testing the NCCLS-recommendedstrains C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 (12).

RESULTS AND DISCUSSION

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Results and Discussion

Phase 1. Table 1 summarizes the caspofungin MIC distribution for 694isolates of C. albicans (clinical isolates plus glucan synthesismutants) tested in both RPMI and AM3 broth. Little differencewas observed in the MIC distributions between 24- and 48-h readingsirrespective of the testing conditions, although the 48-h completeinhibition MICs in RPMI tended to be higher than those readat 24 h. As expected, the partial inhibition end point criterionresulted in lower MICs with both media. The MICs determinedin AM3 were also lower than those determined in RPMI. Notably,all of the testing conditions differentiated the mutant strainswith decreased susceptibilities to caspofungin from the clinicalisolates tested. The conditions producing the greatest separationbetween the mutant strains and the clinical isolates were RPMImedium, the partial inhibition end point criterion, and 24-hincubation. These findings are consistent with those reportedby Odds et al. (14) and suggest that this approach may be usefulin testing other species of Candida versus caspofungin.

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TABLE 1. Caspofungin MIC distribution for 694 isolates of C. albicans: influence of test medium, reading time, and end point criterion

Phase 2. Table 2 summarizes the in vitro susceptibilities of 3,322 isolatesof Candida spp. (3,314 clinical isolates and eight glucan synthesismutants) to caspofungin when tested in RPMI 1640 medium with24-h incubation and the partial inhibition end point criterion.Among the 11 isolates for which caspofungin MICs were $≥$ 2 µg/ml,eight were accounted for by the glucan synthesis mutants.

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TABLE 2. Caspofungin MIC distribution for 3,322 isolates of Candida spp. tested in RPMI 1640 broth with 24-h incubation and a partial inhibition end point criterion

These data indicate that the use of RPMI medium, 24-h incubation,and the partial inhibition end point criterion provides an invitro test method that reliably differentiates strains of Candidaspp. with known FKS1 mutations and reduced in vitro and in vivosusceptibilities to caspofungin from the vast majority of clinicalisolates of Candida spp.

The MIC distributions generated for 3,314 clinical isolatesof Candida spp. (Table 2) reveal two important findings. First,isolates for which caspofungin MICs exceed 1 µg/ml rarelyoccur in clinical infections. Only three (two of C. parapsilosisand one of C. guilliermondii) out of 3,314 (0.09%) clinicalisolates exhibited decreased susceptibilities to caspofunginwith MICs ( $≥$ 2 µg/ml) comparable to those observed withthe laboratory-derived glucan synthesis mutants (Table 2). Second,among the nine species tested in phase 2 of the study thereappear to be two groups that can be differentiated by the degreeof susceptibility to caspofungin. Group I includes three commonspecies, C. albicans, C. glabrata, and C. tropicalis (as wellas the less common C. kefyr and C. pelliculosa), and exhibitsexquisite susceptibility to caspofungin (MIC₉₀, 0.06 µg/ml),whereas group II includes C. parapsilosis and less common speciessuch as C. krusei, C. lusitaniae, and C. guilliermondii andis approximately 10-fold less susceptible (MIC₉₀, 0.5 µg/ml)to caspofungin. All of these species appear to respond equallywell clinically to caspofungin treatment (11), and MICs for>99% of isolates in both groups are $≤$ 1 µg/ml; thus,these isolates are differentiated from strains with documentedFKS1 gene mutations (Table 2). However, the differences in theMIC distributions of the two groups suggest possible biologicaldifferences in the way that they respond to caspofungin thatwarrant further investigation.

In summary, we have provided further validation of in vitromethods for determining the susceptibilities of Candida spp.to caspofungin. Using a large international collection of clinicalisolates of Candida spp., we have shown that the use of RPMImedium, 24-h incubation, and the partial inhibition end pointcriterion provides an in vitro test method that reliably differentiatesstrains of Candida spp. with known glucan synthesis mutationsand reduced in vivo susceptibilities to caspofungin from thevast majority (99.9%) of clinical isolates of Candida spp. Wehave also defined potentially important differences in the degreeof susceptibility of two broad groups of Candida spp. headedby C. albicans and C. parapsilosis, respectively. The clinicalusefulness of this testing method must ultimately be validatedby clinical outcomes; however, for now the use of this standardizedmeans of testing caspofungin will be important in monitoringsusceptibility trends of this new agent over time.

ACKNOWLEDGMENTS

We thank Linda Elliott and Shanna Duffy for excellent secretarialassistance in the preparation of the manuscript.

This study was supported in part by Merck & Company.

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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