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Antimicrobial Agents and Chemotherapy, May 2002, p. 1583-1585, Vol. 46, No. 5
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.5.1583-1585.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

In Vitro Activities of Free and Lipid Formulations of Amphotericin B and Nystatin against Clinical Isolates of Coccidioides immitis at Various Saprobic Stages

Gloria M. González,^1,2* Rolando Tijerina,² Deanna A. Sutton,¹ John R. Graybill,^1,3 and Michael G. Rinaldi^1,3

Fungus Testing Laboratory, Department of Pathology, University of Texas Health Science Center at San Antonio,¹ Audie L. Murphy Division, South Texas Veterans Health Care System, San Antonio, Texas 78229-3900,³ Departamento de Microbiología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, México²

Received 31 January 2002/ Accepted 5 February 2002

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We investigated the susceptibilities of hyphal, mixed hyphal, ungerminated arthroconidial, and germinated arthroconidial populations of Coccidioides immitis to lipid formulations of amphotericin B and nystatin and their conventional preparations, utilizing the National Committee for Clinical Laboratory Standards M38-P broth macrodilution method. The differences in effects of the three different growth stages of the saprobic phase of C. immitis on the MIC/minimum lethal concentration (MLC) ratio were not statistically significant for any of the antifungal agents tested. These results suggest that either inocula could be used for in vitro susceptibility studies with C. immitis.

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Several strategies have been developed over the past few years in an effort to overcome the disadvantages associated with the clinical use of polyenes. The incorporation of amphotericin B and nystatin into phospholipid carriers has proven most useful for these agents, which have dose-limiting toxicities. The major considerations for developing drug carrier assemblage are related to their in vivo applicability. At present, four formulations have been developed. These four are amphotericin B lipid complex, amphotericin B colloidal dispersion, liposomal amphotericin B, and liposomal nystatin (4, 7). Limited data are available about the comparative in vitro activities of these lipid formulations against Coccidioides immitis.

In order to evaluate antifungal drugs in vitro, a standardized inoculum is required, as nonstandardized inocula cause variations in the MICs and minimum lethal concentrations (MLCs) (2, 9). In addition to inoculum size, one should specify the filamentous form of the fungus to be tested, as some authors cite differences depending upon the nature of the inoculum. Bezjak (1) compared conidia and hyphae of Aspergillus fumigatus and Aspergillus flavus by testing them with amphotericin B. He found no statistical difference in the MICs determined by using either conidial or hyphal suspensions as the starting inocula. Guarro et al. (3) evaluated the MICs and MLCs of amphotericin B, fluconazole, ketoconazole, flucytosine, miconazole, and itraconazole against filamentous opportunistic fungi such as Scopulariopsis species, Paecilomyces species, Cladosporium species, and Cladophialophora species by using a starting inoculum of either conidia or hyphal fragments. Unlike Bezjak, Guarro et al. demonstrated varying MICs and MLCs, depending upon the fungal form used, with hyphal inocula producing higher results (MIC/MLC ratio) than conidia inocula. Manavathu et al. showed that both germinated and ungerminated conidia of A. fumigatus were equally susceptible to the inhibitory and fungicidal activities of amphotericin B, itraconazole, voriconazole, and posaconazole (5). There is, therefore, substantial disagreement on the most appropriate form of fungi to be used for MIC/MLC ratio determinations.

Thus, the aims of this study were to (i) determine the in vitro activities of lipid-based preparations of polyenes against C. immitis isolates and to compare this activity against that for standard formulations of the compounds, and (ii) evaluate any potential influence that various forms of C. immitis may have on MIC/MLC ratio results.

Isolates. Twenty-five clinical isolates of C. immitis were used in this study. All were obtained from the Fungus Testing Laboratory, Department of Pathology, University of Texas Health Science Center at San Antonio. Isolates were identified as C. immitis by examination of macroscopic and microscopic morphologies and were confirmed by using a DNA probe (Gen-Probe Incorporated, San Diego, Calif.). Each isolate was maintained as a suspension in water at room temperature until testing was performed.

Growth of inocula. Isolates of C. immitis were grown in duplicate to check viability on potato flakes agar (PFA) slants prepared in-house at 35°C for 7 to 10 days. Isolates were evaluated after 4, 10, and 20 days of growth on PFA at 35°C to obtain three different growth stages, consisting of a predominately hyphal population, a mixed hyphal and ungerminated arthroconidial population, and a predominately arthroconidial population, respectively. The 20-day incubation was in Antibiotic Medium 3 (Difco, Detroit, Mich.) for 15 h to induce germination of arthroconidia.

Antifungal agents. Drugs were obtained from their respective manufacturers. They included conventional amphotericin B (Bristol-Myers Squibb, Princeton, N.J.), amphotericin lipid complex (The Liposome, Princeton, N.J.), amphotericin B colloidal dispersion (Sequus Pharmaceuticals, Menlo Park, Calif.), liposomal amphotericin B (Nexstar Pharmaceuticals, San Dimas, Calif.), liposomal nystatin (Aronex Pharmaceuticals, The Woodlands, Tex.), and nystatin (Sigma Chemical Co., St. Louis, Mo.). Antifungal drugs were reconstituted according to manufacturers' instructions. Serial twofold dilutions of each antifungal agent were prepared to obtain final drug concentration ranges of 0.06 to 16 µg/ml for all amphotericin B formulations and 0.06 to 32 µg/ml for free and liposomal nystatin. A 0.1-ml aliquot of each of the twofold serial dilutions was dispensed into a sterile plastic snap-cap tube (12 by 75 mm). Conventional amphotericin B and nystatin were then maintained at -70°C until needed; other drug dilutions were prepared the day of testing.

Antifungal susceptibility testing. Isolates of C. immitis were evaluated using the National Committee for Clinical Laboratory Standards broth macrodilution method M38-P, the reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi (proposed standard [8]). Briefly, the fungal growth was overlaid with sterile distilled water, and suspensions were made by gently scraping the colonies with wooden applicators. Heavy fragments were allowed to settle and the upper, homogeneous supernatant was transferred to sterile tubes. The hyphae-cell suspensions were vortexed and adjusted spectrophotometrically to 95% transmittance at 530 nm. Preliminary studies indicated that at 95% transmittance the starting inoculum was approximately 1 x 10⁴ to 5 x 10⁴ CFU/ml (G. M. González, R. Tijerina, D. A. Sutton, and M. G. Rinaldi, Abstr. 99th Gen. Meet. Am. Soc. Microbiol. 1999, abstr. C-172, p. 139-140, 1999). The different suspensions were further diluted 1:10 in antibiotic medium 3 for a final inoculum of 1 x 10³ to 5 x 10³. Tubes containing 0.1 ml of drug were inoculated with 0.9 ml of the suspensions. Tubes were incubated at 35°C and the MICs and MLCs were read at 24 and 48 h.

The MICs were defined as the first tube with complete absence of growth (score of 0). The MLC was determined by dispensing and streaking 100 µl of broth from the tubes with concentrations above the MIC onto Sabouraud dextrose plates and incubating at 35°C. The MLC was defined as the lowest concentration of antifungal compound causing growth of five colonies or less on the plates. All testing was performed in triplicate. A Paecilomyces variotii strain, UTHSC 90-459, was included with all testing. All studies were conducted following laboratory biosafety level 3 criteria for infectious agents (10).

Statistical analysis. The comparisons of the saprobic stages, the agents, and their interactions were tested by the generalized estimation equation (6), as responses were discrete and correlated within isolates. The P value was adjusted using Bonferroni's method (11); a P value of less than or equal to 0.05 was set as the significance level.

Table 1 displays the in vitro susceptibilities of 25 strains of C. immitis against free and lipid formulations of amphotericin B and nystatin. The 48-h P. variotii control strain MIC/MLC ratio ranges for conventional amphotericin B and nystatin were 0.5 to 1 and 16 to 32 µg/ml, respectively.

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TABLE 1. In vitro MICs and MLCs of free and lipid polyene formulations for strains of C. immitis in various stages of the saprobic phase

One of the concerns for in vitro susceptibility testing of filamentous fungi is the form of the filamentous fungi used in the inocula, but in our study the ranges and geometric means of the MICs and MLCs after 48 h of incubation showed that there was no statistically significant difference among the three different growth stages of C. immitis. Similarly, the in vitro data for germinated and ungerminated arthroconidia did not exhibit any significant differences between these two forms of inocula for the susceptibility testing of C. immitis.

Geometric means of the MICs and MLCs after 48 h of incubation of all lipid-based preparations of amphotericin B demonstrated statistically significant differences compared to the conventional formulation (P < 0.0001). Both the MIC and MLC values were lower for liposomal nystatin than for the standard formulation of nystatin; however, these variations were not statistically significant.

Although there were differences in study methodologies and the fungi evaluated, our in vitro data showed MIC results for hyphal and conidial inocula that were similar to those obtained by Bezjak (1) and Manavathu et al. (5) but different from those of Guarro et al. (3). One of the most important disadvantages when hyphae are used as inocula is that the procedure is time-consuming and it is difficult to dispense the inoculum homogeneously throughout the test samples. The use of ungerminated conidia for in vitro susceptibility testing is faster and easier than the use of geminated conidia and hyphae inocula.

Thus, our study has determined that the various saprobic stages of C. immitis utilized as inocula do not appear to exert any statistically significant effect on MIC/MLC ratios.

 
We thank Shukoo Lee, The University of Texas Health Science Center at San Antonio, for her assistance in the statistical analysis.

 
* Corresponding author. Mailing address: Facultad de Medicina, Universidad Autónoma de Nuevo León, Departamento de Microbiología, Madero y Dr. Eduardo A. Pequeño s/n, Colonia Mitras Centro, Monterrey, N.L. México. 64460. Phone: (52) 8329 4166. Fax: (52) 8348 5477. E-mail: gmglez{at}yahoo.com.mx.

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Antimicrobial Agents and Chemotherapy, May 2002, p. 1583-1585, Vol. 46, No. 5
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.5.1583-1585.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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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 HighWire Citing Articles via Google Scholar Google Scholar Articles by González, G. M. Articles by Rinaldi, M. G. Search for Related Content PubMed PubMed Citation Articles by González, G. M. Articles by Rinaldi, M. G.