In Vitro Activities of New Antifungal Agents against Chaetomium spp. and Inoculum Standardization

Chaetomium is an unusual etiological agent of human infections,but the mortality rate among immunocompromised patients is considerablygreater than that among nonimmunocompromised individuals. Weinvestigated the in vitro antifungal susceptibilities to novelantifungal agents of 19 strains belonging to three species ofChaetomium which have been involved in human infections, i.e.,Chaetomium globosum, C. atrobrunneum, and C. nigricolor, andone strain of the closely related species Achaetomium strumarium.A modification of the NCCLS reference microdilution method (M38-A)was used to evaluate the in vitro activities of ravuconazole,voriconazole, albaconazole, and micafungin. Micafungin was notactive at all, while the geometric mean MICs and minimum effectiveconcentrations of the three triazoles were less than 0.5 and0.4 µg/ml, respectively.

INTRODUCTION

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Introduction

Several species of the ascomycete genus Chaetomium are ableto cause human infections, and these mainly affect the nailsor skin (5). However, disseminated infections have also beenreported, especially in neutropenic patients (11). Despite antifungaltreatments with amphotericin B (AMB) alone or in combinationwith itraconazole, the mortality rate among patients with severeinfections caused by Chaetomium is high (M. A. Barron, D. A.Sutton, R. Veve, J. Guarro, M. Rinaldi, E. Thompson, P. J. Cagnoni,and N. E. Madinger, Abstr. 42nd Intersci. Conf. Antimicrob.Agents Chemother., abstr. 870, 2002). The most prevalent clinicalspecies is Chaetomium globosum, although other species havealso been reported (11). Clinical and in vitro data on the activitiesof antifungal drugs against the different species of Chaetomiumare scarce. In previous studies, using broth macro- and microdilutionmethods, we demonstrated the inhibitory activities of miconazole,ketoconazole, and itraconazole in vitro (11, 17). However, thesusceptibility of Chaetomium to the novel antifungal drugs ispractically unknown.

In this work we have evaluated the in vitro activities of thenew triazoles albaconazole (ABC), voriconazole (VRC), and ravuconazole(RVC) and the echinocandin micafungin (MFG) against a representativenumber of strains of Chaetomium species of clinical interestand one strain of Achaetomium strumarium, which has also beeninvolved in human infections. We followed the guidelines ofNCCLS reference method M38-A for antifungal susceptibility testingof molds (14). However, since this reference method does notapply to fungi that reproduce by ascospores instead of conidia,we have made a few modifications to the type and the size ofthe inoculum and the incubation conditions.

MATERIALS AND METHODS

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Materials and Methods

Isolates. Nineteen isolates of Chaetomium spp. (11 isolates of C. globosum,7 isolates of C. atrobrunneum, 1 isolate of C. nigricolor) and1 isolate of A. strumarium were tested; all of them were fromclinical sources. The isolates were stored on oatmeal agar (OA;oatmeal, 30 g; agar, 20 g; tap water, 1 liter) slants coveredwith paraffin oil, subcultured on OA plates, and incubated at30°C for 15 to 20 days. Paecilomyces variotti ATCC 36257was included in each batch of tests as a reference strain.

Antifungal agents. Antifungal agents were obtained as pure powders. AMB (USP, Rockville,Md.), ABC (J. Uriach & Cía, Barcelona, Spain), VRC(Pfizer Inc., Madrid, Spain), and RVC (Bristol-Myers SquibbCompany, New Brunswick, N.J.) were diluted in dimethyl sulfoxide(Panreac Química S.A., Barcelona, Spain). MFG (FujisawaPharmaceutical Co. Ltd., Osaka, Japan) was diluted in steriledistilled water. Microplates were prepared as described in documentM38-A (14). Final drug concentrations ranged from 32 to 0.06µg/ml for MFG and from 16 to 0.03 µg/ml for theother drugs.

Inoculum preparations. Inocula were prepared by scraping the fungal colonies growingon the agar plates with a culture loop and suspending them insterile saline. The fungal suspensions were vigorously vortexedfor 15 s and filtered through sterile gauze to remove the hyphaeand obtain suspensions composed almost exclusively of ascospores.These suspensions were adjusted with a hemacytometer to 10⁷ascospores/ml. The viability of the ascospores was verifiedby plating 100 µl of serial dilutions of each inoculumonto Sabouraud dextrose agar (Pronadisa, Madrid, Spain) plates.The adjusted suspensions were centrifuged, the supernatant wasdiscarded, and the ascospore pellets were resuspended in thesame volume of saline. The optical densities (ODs) of the ascosporesuspensions were determined spectrophotometrically at 530 nm.All the isolates were tested three times.

MIC determination. The MIC was the lowest drug concentration that produced a completeabsence of growth. MIC readings were taken visually with theaid of an inverted mirror.

MEC determination. The minimum effective concentration (MEC) was the lowest drugconcentration that produced morphological changes in fungalhyphae. Macroscopically it was recognized as the observationof very compact and round colonies in the microtiter wells.MEC readings were made with the aid of a stereoscopic microscope.

MLC determination. The minimum lethal concentration (MLC) was the lowest drug concentrationthat resulted in 99.9% killing of spores. Briefly, 10-µlaliquots were subcultured from each well that showed completeinhibition of growth (an optically clear well) onto Sabourauddextrose agar plates. The plates were incubated at 30°Cfor 96 h. The MLC was the lowest antifungal concentration thatresulted in either no growth or fewer than three colonies perplate (6).

MICs, MECs, and MLCs were determined after 72 h of incubationat 30°C. The MIC and MEC ranges, the corresponding geometricmeans, and the MICs and MECs at which 90% of the isolates testedwere inhibited (MIC₉₀s and MEC₉₀s, respectively) were determinedfor each combination of fungal species and drug and for allthe isolates and drugs. For MLCs, only geometric means weredetermined.

RESULTS

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Results

Inocula standardization. The ODs of the ascospore suspensions adjusted to 10⁷ ascospores/mlwith a hemacytometer ranged from 1.05 to 1.50 (Table 1). Theascospore germination rate was very low for all species, withC. globosum being the species that showed the highest mean germinationrate (15.3%). The range of growth for all isolates tested was5 x 10⁵ to 4.4 x 10⁶ CFU/ml.

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TABLE 1. Correlation between ODs of ascospore suspensions^a and the number of CFU per milliliter

Antifungal data. All isolates tested produced detectable growth at 72 h of incubation.Therefore, MICs were read after this time. Table 2 shows theMICs, MECs, and MLCs of the five antifungal drugs for the 20isolates tested. AMB showed the broadest MIC range (0.5 to 8µg/ml). In general, the MICs, MECs, and MLCs of this drugwere high. The geometric mean MICs were identical to the geometricmean MECs, and the MIC₉₀s and MEC₉₀s were also identical. MFGshowed a very high mean MIC (64 µg/ml), although the meanMEC was considerably lower (9.2 µg/ml). The MLCs of MFGwere not evaluated because the MICs were always greater thanthe highest concentration tested. The three azoles were generallyactive. The mean MICs of these drugs, with a few exceptions,were similar to each other and were less than 0.5 µg/ml.The mean MECs of the three azoles were also similar to eachother and were only slightly lower than the mean MICs. The MLCsof all antifungals tested were high and generally exceeded theconcentrations normally achievable in the host.

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TABLE 2. MICs, MECs, and MLCs of five antifungal agents for 20 strains of Chaetomium and Achaetomium isolates

In general, for the two species for which more than one isolatewas assayed, all the drugs tested, with the exception of MFG,displayed slightly lower (1 or 2 dilutions) MICs and MECs forC. atrobrunneum than for C. globosum.

DISCUSSION

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Discussion

Inoculum standardization by counting with a hemacytometer forantifungal susceptibility testing of filamentous fungi has beenconsidered an accurate and reproducible method. After the suspensionswere cultured on agar plates, the spore counts and the correspondingCFU obtained by this method differed, in general, by less than10% (1, 10, 16), but this was true only for inocula made upof conidia. When the molds tested were reproduced as ascospores,such as Chaetomium, the situation was different. Conidia arepropagules that spread and that are produced in massive numbers,and they germinate quickly when the environmental conditionsare favorable. By contrast, ascospores are resistant cells whichcan remain dormant for a long time, and a germination activatormay be required. In coprophilous fungi, for example, the activationof the ascospores comes after passage through the gut of ananimal. In the laboratory, some alcoholic, acid, or phenolicsolutions or simply heating can induce ascospore germination(8). With adequate activation, most of the spores will germinate;if not, they will never abandon their dormant state. As no pretreatmentwas applied to inocula in our study, their size had to be increasedto 10⁷ spores/ml in order for the number of germinated ascosporesto fit into the range recommended by the NCCLS (14). A preliminarytest, performed with some of the strains used in the presentstudy, allowed us to determine the rates of germination of thedifferent species, which proved to be wide and variable, butin general very low (approximately 10%), with the exceptionof that of C. nigricolor, whose germination rate was considerablylower.

We also developed a spectrophotometric procedure in order tofacilitate inoculum preparation for the testing of these typesof fungi in future studies. Most strains of Chaetomium and Achaetomiumproduce soluble dark pigments, however, which can influencethe OD readings. To rectify this interference, inoculum suspensionswere centrifuged and the supernatant was changed and replacedby the same volume of sterile saline. By this procedure, theOD required to obtain the final inoculum concentration suggestedby the NCCLS was 1.3, which was considerably higher than thatrequired for fungi that produce conidia (0.09 to 0.17). Theseimportant differences can be explained by the fact that theinocula of ascospores are 10 times more concentrated than thosemade up of conidia and by the fact that the ascospores of Chaetomiumare bigger than most conidia of any of the genera included inthe M38-A document. By this procedure, the inoculum used totest isolates of Chaetomium and related genera can be easilystandardized.

The antifungal activity of AMB obtained here agrees with thosefound by other investigators (4, 11, 13). In a previous studywe reported AMB MICs similar to those obtained here (MIC₉₀,9.23 µg/ml at 72 h of incubation), even though the resultswere generated by the macrodilution method. The activities ofthe azoles ABC and VRC against C. globosum were previously testedby Capilla et al. (4) and McGinnis and Pasarell (13), respectively,and their results were also comparable to ours.

In this study, in addition to the more traditional MICs andMLCs, we have also used MECs to evaluate the antifungal activitiesof the drugs tested. The MEC was defined by Kurtz et al. (12)for the testing of lipopeptides. Morphological changes can bescored macroscopically in the wells, by recording the presenceof very compact colonies forming clumps (2, 7, 12), or by detectingmore subtle changes such as variations in the breadth of thehyphae when they are observed microscopically (9). MECs canbe 1 to 2 dilutions lower when the last criterion is used (7).The method that is the more predictive of the clinical outcome,however, has not been determined. We used the macroscopic readingbecause it is easier to perform in a clinical laboratory. Itis remarkable that, in our study, while the MICs of MFG werealways out of range, the MECs of this drug were as low as 0.5µg/ml for some of the isolates. Although MEC endpointswere initially limited to the testing of lipopepticidal antifungalagents, they have recently been used for azoles and AMB (3,18). In our study, the MECs of AMB were identical to the MICsand the MECs of the azoles were similar to the MICs, which generallyconfirmed the results obtained by other investigators (3, 15,18).

Neither AMB deoxycholate, alone or in combination with itraconazole,nor its liposomal formulation has been effective treatment forany of the six patients with Chaetomium infections treated withantifungal drugs (Barron et al., 42th ICAAC). The good in vitroactivities of the three triazoles obtained in our study encourageus to perform further studies with animal models to confirmtheir potential for the treatment of Chaetomium infections,for which there is no effective therapy.

FOOTNOTES

* Corresponding author. Mailing address: Unitat de Microbiologia, Facultat de Medicina, Universitat Rovira i Virgili. Carrer Sant Llorenç, 21.43201 Reus, Spain. Phone: 977-759359. Fax: 977-759322. E-mail: umb{at}fmcs.urv.es.

REFERENCES

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