Factors Affecting the Results of a Broth Microdilution Antifungal Susceptibility Testing In Vitro

Zbl. Bakt. 283,375-390 (1996) © Gustav Fischer Verlag, Stuttgart· Jena . New York

Factors Affecting the Results of a Broth Microdilution Antifungal Susceptibility Testing In Vitro* VLADIMiR BUCHTA 1 and MILOS OTCENAsEK2 1 Department

of Biological and Medical Sciences, Faculty of Pharmacy, Charles University, Hradec Knilove, Czech Republic 2 Institute of Experimental Biopharmaceutics, Joint Research Center of PRO.MED.CS and Academy of Sciences of the Czech Republic, Hradec Knilove, Czech Republic Received May 5, 1995 . Accepted August 3, 1995

Summary In experiments involving 10 antifungal drugs and 46 strains of potentially pathogenic fungi, the factors affecting the results of in vitro susceptibility testing were studied. The composition of the test medium, inoculum size, temperature and length of incubation were the most pronounced effects influencing the results of testing in vitro. Minimal inhibitory concentrations (MIC) of the antimycotics tested were lowest in complex media (Brain Heart Infusion, Antibiotic Medium 3, Sabouraud broth) except for 5-fluorocytosine which was most effective in Yeast Nitrogen Base medium. Inoculum sizes of 103 to 104 cfu· mL-I had no marked effect on MIC but starting from a final concentration of 105 cfu . mL-1, an abrupt increase in MIC in azole derivatives and 5-fluorocytosine was observed. There was a direct relationship between the duration of incubation and MIC of fungistatic antimycotics. The influence of the incubation temperature became generally manifest primarily in fungi with retarded growth at elevated temperature (>35°C). In these fungal species, a tendency towards a decrease in MIC with increasing temperature was apparent. The other factors studied (medium pH, buffer, solvent) had no substantial influence on the antifungal activity of the drugs tested.

Introduction The increasing incidence and a broadening spectrum of agents of mycotic infections in man in the last few decades was become a challenge for pharmaceutical research in seeking new and more effective antifungal drugs. At the same time, the requirements for the reliability, accuracy and rapidity of methods needed to evaluate the antimycotic activity of substances tested in vitro are increasing, whether within the screening of newly synthetized and isolated potential antimycotics or in determining the levels of

* This paper comprises data from the doctoral thesis of V. Buchta

376

V. Buchta and M. Otcenasek

antifungal drugs in body fluids and in establishing the susceptibility of clinical isolates of fungi. Antifungal susceptibility testing in vitro is generally characterized by a poor standardization and reproducibility of most of the methods used. The reasons for this lie primarily in a number of variables affecting the results of in vitro testing. The aim of the present study has been to evaluate the influence of test conditions on antifungal susceptibility results obtained in vitro with the micro dilution broth method.

Material and Methods

Fungi Forty-six fungal strains isolated mostly from samples of urine, blood, skin scales, swabs taken from rectum, vagina and buccal mucosa of patients with suspected or confirmed mycosis were included in the tests (numbers of strains given in parentheses): Candida albicans

(8), C. tropicalis (4), C. glabrata (3), C. krusei (2), C. parapsilosis (1), C. zeylanoides (1), Torulopsis candida (1), Cryptococcus neoformans (3), Geotrichum candidum (3), Trichosporon sp. (2), Saccharomyces cerevisiae (1), Rhodotorula rubra (1), Wangiella dermatitidis (1), Aspergillus fumigatus (3), A. flavus (2), A. niger (2), A. terreus (1), Absidia corymbifera (1), Mucor circinelloides (1), Rhizopus arrhizus (1), Trichophyton mentagrophytes

(4). Most of the strains had been obtained from culture collections of the Mycological Laboratory of the Institute of Experimental Biopharmaceutics at Hradec Kralove, a part of them from the microbiological departments of Charles University and five strains of C. albicans from the Department of Bacteriology and Mycology, Janssen Research Foundation at Beerse, Belgium. Fungal isolates were identified with the aid of standard methods, on the basis of morphological and biochemical characteristics. All strains were maintained on Sabouraud glucose agar (SGA) in the dark at 4°C and passaged into the same medium as filamentous fungi every three months and yeasts every six months. The yeast inoculum was prepared from 24-72 h colonies grown on SGA at 26°C or 37°C. Conidia from 5-10 day-old colonies were used to prepare suspensions of filamentous fungi. The cell density was adjusted, if not stated otherwise, by means of Burker's chamber to yield a final concentration of 1.0-5.0 X 104 cfu· mL-l.

Media Comparison of the minimal inhibitory concentration (MIC) of antimycotics determined with the micro dilution method was made in Sabouraud glucose broth - SBG (pH 5.6), Brain Heart Infusion (Difco) broth - BHI (pH 7.2), casitone broth - CAS (pH 6.7), Antibiotic medium 3 - ATB3 (pH 7.0), Yeast Nitrogen Base (Difco) with glucose and asparagine - YNB (pH 5.6) and tissue culture medium RPMI 1640 (USOL, Prague) - RPMI (pH 7.0). RPMI was buffered with 0.165 M morpholinepropanesulfonic acid (Sigma) - MOPS. The influence of the buffering system was studied in RPMI (pH 7.0) in the following buffers (all 0.165 M): Sorensen phosphate buffer, MOPS, TRIS (Serva) and HEPES (Serva).

Antifungal drugs The following substances and antimycotic preparations were used in the experiments: amphotericin B (Amphotericin B i. v., Squibb), nystatin (Serva), pimaricin (Pimafucin, GistBrocades), 5-fluorocytosin (Fluka), griseofulvin (Serva), clotrimazole (Bayer), ketoconazole (Janssen), itraconazole. (Janssen), miconazole (Daktarin, Janssen), fluconazole (Diflucan, Pfizer). All antimycotics were tested within a concentration range from 0.09 to 100 mg· L-t, except for 5-fluorocytosine (200 to 0.19 mg· L-1 ) and amphotericin B (0.045 to 50 mg· L-').

Antifungal Susceptibility Testing

377

Nystatin, griseofulvin, ketoconazole and itraconazole were dissolved in dimethylsulfoxide (DMSO) so that the final concentration of the solvent did not exceed 1 %. The other antimycotics were dissolved and diluted in distilled water. DMSO, DMSO + 6HCl and DMSO + 96% ethanol were used in the study of the influence of the solvent on the antimycotic activity of itraconazole.

Microdilution broth method The broth microdilution method was used for the determination of the MICs of the drugs tested. Each antimycotic was diluted in a two-fold dilution series (11 concentrations and medium control without drug) and dispensed in 0.2 mL volumes into wells of a polystyrene microtiter plate. Then, the wells were inoculated with 0.01 mL suspension of a given fungal strain to achieve a final concentration of 1.0 to 5.0 X 104 cfu . mL-1. MICs were read after 24, 48, 72, 120 and 168 h of incubation at 35°C. The effect of medium composition and pH (5.0 vs 6.5 vs 7.2), duration and temperature of incubation (26 vs 35 vs 39 0q, size of inoculum (10 3 vs 104 vs 10 5 vs 106 cfu . mL-1) on test results was studied. The influence of incubation temperature, pH of the medium and size of inoculum were tested in SGB.

Analysis of results The non-paired t-test was used for the evaluation of the effect of medium composition on the MIC of antimycotics after 24 hand 72 h (dermatophytes). The order of sensitivity of the individual fungal species to a given antimycotic was determined in each medium. The medium in which the lowest MIC value was achieved was given a score of 6 points, that with the highest MIC value was given a score of 1 point. If the MIC of a given antifungal drug was the same in two or more media, the points for the ratings were added and divided by the number of media with identical MIC. Finally, points for each medium and a given antimycotic were added for all fungal species and compared with one another using the non-paired t-test.

Results Amphotericin B, nystatin and clotrimazole were found to have the greatest in vitro activity against the majority of fungi tested, while 5-fluorocytosine, fluconazole and griseofulvin selectively inhibited some fungal species only (Table 1). Of individual mycotic agents, the lowest MIC values of the antimycotics tested were measured in the strains of C. glabrata, C. neoformans, C. candidum and Trichosporon sp. Contrastingly, the strains of C. albicans and zygomycetes were resistant to most antifungal drugs (Table 1). In complex media, the MICs of all antimycotics were generally lower than in welldefined media (YNB, RPMI) (Table 2). 5GB, ATB3 and BHI provided the lowest MICs in most yeast strains and filamentous fungi. In contrast to this, 5-fluorocytosine was most efficient in YNB and RPMI (Figure 5). There was a negligible difference between the sensitivity of T. mentagrophytes and zygomycetes depending on the composition of the medium used (Table 2). Testing of miconazole, ketoconazole, itraconazole and 5-fluorocytosine was pH affected a little only by the pH of the medium in the strains of C. tropicalis, C. glabrata, C. krusei, T. candida and Aspergillus spp. (Fig. 1). Differences in the MICs usually varied within a range of two to three dilutions. In azole derivatives, the MICs increased with increasing pH. 25

Zbl. Bakt. 283/3

0.09-0.19 0.13 0.19 0.19 0.04-0.19 0.18 0.19-0.39 0.20 0.04 0.04 0.04-0.39 0.14 0.19 0.19 0.09-1.56 0.32 0.04-0.09 0.06 0.17 0.38 0.14 0.21 0.12

Candida albicans (8) c. glabrata (3)

0.39-1.56 1.27 0.78 0.78 0.78 0.78 0.78-1.56 0.88 0.09-0.39 0.24 0.39-1.56 0.62 0.39-0.78 0.49 0.78-3.12 1.10 1.56 1.56 0.84 0.93 0.70 1.13 0.39

NYS 6.25-12.5 6.81 6.25 6.25 0.19-6.25 2.19 0.19-3.12 2.13 0.09 0.09 0.39-6.25 3.12 1.56-3.12 2.47 3.12-25 12.5 0.39-1.56 1.03 3.48 6.81 2.18 4.51 2.48

PIM 0.19-200 0.45 0.19 0.19 0.19-200 2.18 3.12-200 19.8 0.19-0.39 0.31 0.19 0.19 0.19-1.56 0.49 200 200 0.19 0.19 1.04 1.01 0.72 2.14 200

5FC 100 100 100 100 100 100 100 100 100 100 100 100 100 100 3.12 3.12 100 100 100 100 100 44.2 100

GRI

* 5FC tested in YNB (pH 5.6), r - range of MIC, G - geometric mean of MIC " Candida krusei (2), C. parapsilosis (1), C. zeylanoides (1) b Rhodotorula rubra (1), Saccharomyces cerevisiae (1), Torulopsis candida (1) C Geotrichum candidum (3), Trichosporon spp. (2) d Aspergillus fumigatus (3), A. flavus (2), A. niger (2), A. terreus (1) " Aspergillus spp. (8), T. mentagrophytes (4), G. candidum (3), Trichosporon spp. (2) 1 Absidia corymbifera (1), Mucor circinelloides (1), Rhizopus arrhizus (1)

r G r G C. tropicalis (4) r G Candida spp. r (4)" G Cryptococcus (3) r neoformans G Other yeasts (3)b r G Aspergillus r fumigatus (3) G Trichophyton men- r tagrophytes (4) G Yeast-like r fungi (5)C G Candida spp. (19) G Aspergillus spp. (8)d G Yeasts (25) G Hyphomycetes (17)" G Zygomycetes (3)1 G

AMB

Fungus (of strains)

AMB PIM CLZ KTZ FLZ

1.56-6.25 3.40 0.39-1.56 0.62 0.09-0.78 0.22 0.09 0.09 0.09 0.09 0.09-0.78 0.26 0.19-0.39 0.31 0.09-0.19 0.11 0.09 0.09 0.68 0.36 0.49 0.21 0.23

CLZ 25-50 45.8 0.39-0.78 0.62 0.19-50 2.19 0.19-12.5 0.54 0.09 0.09 0.09-6.25 0.47 3.12-6.25 3.93 0.78-6.25 1.31 0.09 0.09 4.81 1.86 2.26 0.58 39.7

KTZ

amphotericin B primaricin clotrimazole ketoconazole fluconazole

1.56-6.25 4.42 0.09 0.09 0.39-6.25 2.21 0.09-12.5 0.64 0.09 0.09 0.09-3.12 0.29 1.56 1.56 0.09-0.78 0.32 0.09 0.09 1.38 2.21 0.88 0.65 6.25

MCZ

Table 1. Susceptibility of potentially pathogenic fungi to 10 antimycotics in Sabouraud glucose broth (pH 6.0)*

NYS 5FC MCZ ITZ GRI

100 100 25 25 1.56-100 12.5 3.12-100 25.0 0.09 0.09 0.19-100 4.92 100 100 100 100 0.78-3.12 1.56 38.7 100 14.6 20.2 100

FLZ

nystatin 5-fluorocytosine miconazole itraconazole grisefulvin

100 100 0.39 0.39 0.09-100 0.90 0.09-100 0.52 0.09 0.09 0.09-100 1.52 0.09-0.19 0.12 0.09-0.19 0.13 0.09 0.09 5.11 0.14 2.72 0.12 100

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Antifungal Susceptibility Testing

379

The buffer had no marked influence on MIC changes in the individual antimycotics. In aspergilli, the MIC values of amphotericin B were higher by two dilutions in the phosphate buffer as compared to the other buffers. Similar results were obtained in testing 5-fluorocytosine in RPMI buffered with TRIS with Candida strains. No significant differences in the sensitivity of C. albicans (three strains), C. tropicalis (one strain), C. glabrata (one strain), C. neoformans (one strain) and A. fumigatus (two strains) were found on comparing the respective MICs of itraconazole dissolved in three different solvents. The effect of the incubation temperature was shown in part of the strains studied for all antimycotics and was most pronounced for amphotericin Band 5-fluorocytosine. Growth of the strains of C. zeylanoides, G. candidum, Trichosporon spp., R. rubra, W. dermatitidis and M. circinelloides at higher temperatures was however retarded (35°C) or completely inhibited (39 0C). In amphotericin B, the tendency towards a decrease in antimycotic efficacy with an increase in incubation temperature was apparent (Fig. 2). Contrastingly, a direct relationship between the antifungal effect and temperature was noted for 5-fluorocytosine and azole derivatives (Fig. 3). The MICs of the antimycotics tested increased with the length of the incubation period. The most pronounced differences, (up to a factor of 512) during the period studied (24-168 h) were found in C.glabrata, T. candida, A. terreus, A. niger and T. mentagrophytes. The magnitude of the MIC change varied with the composition of the medium, duration of incubation and inoculum size. The frequency of strains with considerable changes of MIC endpoints increased with increasing incubation periods (Table 3). At the same time, there were substantial differences in the number of the strains between the individual test media, especially when testing amphotericin Band 5-fluorocytosine. It should be noted that the results were affected by the fact that in respect of fluconazole, 5-fluorocytosine, miconazole and, partly, itraconazole, most of strains could not be evaluated because their MICs exceeded the maximal concentration tested (Table 3). The influence of the inoculum size was most considerable for imidazole and partially, triazole derivatives. To a lesser extent, i. e. within the range of one to two dilutions, it was found in 5-fluorocytosine, while the MIC of polyene antibiotics was affected in a negligible manner. Differences in the MIC of antimycotics between the individual concentrations of the inoculum tested increased during the incubation period. In general, the MICs of all antifungal drugs increased abruptly when an inoculum of 105 or 106 cfu· mL-l was tested. In the case of an inoculum lower than 105 cfu· mL-I, MIC changes usually did not exceed the range of two dilutions (Fig.4). Discussion The experiments have shown a critical influence of the individual test conditions on the results of evaluation of antimycotic activity in vitro. The factors can be divided into four categories: 1) methodological factors, 2) factors related to the testing system, 3) factors related to the fungus tested, 4) factors related to the drug tested (21). An assessment of the antifungal activity by means of broth techniques often involves difficulties in determining the MIC endpoints. Above all, this happens with azole derivatives and 5-fluorocytosine in whom partial inhibition of fungal growth can be observed regularly (19). In our experiments, this phenomenon has probably been responsible for the relative resistance of C. albicans to azole drugs because we

Candida spp. (19) Other yeasts (6) Aspergillus spp. (8) Other hyphomycetes (9) Zygomycetes (3)

Candida spp. (19) Other yeasts (6) Aspergillus spp. (8) Other hyphomycetes (9) Zygomycetes (3)

Candida spp. (19) Other yeasts (6) Aspergillus spp. (8) Other hyphomycetes (9) Zygomycetes (3)

Fungal species (no. isolates tested)

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100 100 100 1.56 100

200 200 200 200 200

0.04-6.25 0.04-6.25 0.04-6.25 0.04-6.25 0.04-3.12

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SIR Y/R Y,S/C S,NR SIN

Y,RIN Y,RIN YIN Y,RIN NIN

NY NY R,AIY S,NY A,RIY

RPMI (R)

3.37 1.90 4.82 4.96 1.97

0.81 0.68 0.10 0.62 0.05

AMPHOTERICIN B

YNB(Y)

0.83 1.55 84.1 12.4 > 200

~

11.4 0.09 1.43 0.30 39.7

69.1 1.89 14.9 1.55 > 100

MICONAZOLE

1.00 0.24 1.01 4.19 > 200

5-FLUOROCYTOSINE

MINIMAX

MIC (mglL)

~

1.38 0.09 1.56 0.12 6.25

46.3 15.5 77.1 35.4 > 200

0.17 0.07 0.38 0.10 0.12

SGB (S)

18.4 0.18 12.5 0.16 > 100

113.0 132.0 > 200 126.0 > 200

0.19 0.06 0.32 0.49 0.09

BHI (B)

G-MIC (mglL)

14.3 0.47 54.5 0.22 39.7

111.4 158.7 154.2 158.7 > 200

0.21 0.38 0.50 0.62 0.07

CAS (C)

Table 2. Effect of medium composition on in vitro susceptibility of potentially pathogenic fungi to 6 antimycotic drugs

0.08 0.04 0.13 0.17 0.04

11.9 0.21 19.3 0.10 31.5

93.0 126.0 > 200.0 147.0 > 200.0

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100 100 50 3.12 100

NIN SIN NIN SlY NIN

NIN N/R NIN N/R, Y S,BIY

SlY S, AIR B, AlY S, AIY c/Y

76.2 12.5 > 100 25.0 > 100

32.7 5.43 > 100 28.7 > 100

FLUCONAZOLE

26.0 0.88 0.32 0.88 31.5

22.3 1.16 0.17 0.64 9.65

8.18 0.63 4.62 1.24 19.8

ITRACONAZOLE

13.0 0.69 6.81 2.06 63.0

24.9 1.26 > 100 10.7 > 100

6.84 0.10 0.14 0.13 2.48

4.81 0.16 4.82 0.32 39.7

G-MIC - geometric mean after 24 h, except for C. neoformans (48 h) and T. mentagrophytes (72 h). 1 for all media tested, N - no significant difference among the media tested. MINIMAX - the medium in which the minimal/maximal MIC values were achieved. Other yeasts - C. glabrata (3), C. neoformans (3), R. rubra (1), S. cerevisiae (1). Other hyphomycetes - G. candidum (3), Trichosporon spp. (2), T. mentagrophytes (4). Zygomycetes - A. corymbifera (1), R. arrhizus (1), M. circinelloides (1).

Candida spp. (19) Other yeasts (6) Aspergillus spp. (8) Other hyphomycetes (9) Zygomycetes (3)

Candida spp. (19) Other yeasts (6) Aspergillus spp. (8) Other hyphomycetes (9) Zygomycetes (3)

Candida spp. (19) Other yeasts (6) Aspergillus spp. (8) Other hyphomycetes (9) Zygomycetes (3)

KETOCONAZOLE

50.0 4.41 > 100 5.35 > 100

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5.96 0.37 2.62 0.30 31.5

77.5 56.1 > 100 25.0 > 100

11.9 0.47 0.18 0.22 1.91

9.00 0.42 6.25 0.30 15.7

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pH

AF - Ajumigalus, AF1 - Ajlavus, AN - A.niger, AT - A.ferreus

Fig. 1. Effect of pH on MIC of miconazole against Aspergillus spp. after 48 hours.

chose as MIC endpoint criterion for all antimycotics the MICs optically clear well. In the case of fluconazole, another cause of its inadequate efficacy in vitro to be taken into account is the composition of the test medium and the buffer used (35). Although the National Committee for Clinical Laboratory Standards (NCCLS) recommends the tissue culture medium RPMI 1640 to be buffered with MOPS for testing of fluconazole, Werner et al. (35) found the MIC of the triazole in RPMI medium with MOPS to be higher as compared to other buffers (18). Our experiments have not confirmed these findings. Comparison of MIC of the antifungal compounds tested in four complex and two well-defined media has shown that the composition of the test medium plays an important role in the variability of antifungal susceptibility testing in vitro (Table 2). The interpretation of these data is not a simple one because, apart from a possible interference of some components (e. g. pyrimidines, purines) of undefined media with the antifungal effect, other factors such as pH and buffer capacity of the medium as well as nutrient requirements for growth of the individual fungi may be presumed (26). An increased physiological activity of the fungus may lead to its greater vulnerability towards the intervention of some antimycotics. This possibility is supported by the experiments demonstrating a higher sensitivity of C. albicans cells to azole derivatives in dependence on the growth phase (2). This could explain, at least in part, lower MIC values of azole derivatives and polyene antibiotics in SGB, BHI or ATB3. No substantial influence of the pH of the medium was found in our experiments as compared to previous studies (19). Nevertheless, we cannot exclude its share in the discrepancy between the results obtained in YNB (pH 5.6) and in the other media (pH

Antifungal Susceptibility Testing

383

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AFI58 AFlI08

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Fig. 2. Effect of temperature of incubation on susceptibility of Aspergillus strains to amphotericin B after 48 hours. 6.5 to 7.4), particularly in the case of testing the susceptibility of aspergilli to azole drugs (Table 2). In addition, in azole derivatives, the degree of protonization of their molecule which is higher at an acid pH and is indirectly proportional to their antifungal effect cannot be excluded (1). The influence of the buffer used on the MIC size of the antimycotic was found to be of less importance, though in the case of TRIS and the testing of 5-fluorocytosine, it need not necessarily playa negligible role (17). Recent papers studying the effect of the buffer of the testing system on the MIC of fluconazole point, however, to the contradictions and obscurities of its role in the testing system (35). The possibility of influencing the antimycotic activity by the solvent employed has so far been suggested for miconazole, itraconazole and nystatin (32, 34, 36). A comparison of the MICs itraconazole dissolved in three different solvents did not reveal any significant differences. This discrepancy to previous observations may be due to the use of a different testing system, particularly as far as the medium (composition, pH) and the method of testing are concerned. The incubation temperature plays a role in the testing of antimycotics, most frequently in connection with the growth optimum of the fungus studied, size of inoculum, stability of the antifungal drug and the choice of the method (4, 14, 16,20). The results of our experiments have shown that in the majority of yeast strains, a decrease in the MIC of amphotericin B can be seen with a drop of the incubation temperature. An opposite dependence has been found in C. candidum and Trichosporon spp. An indirect relationship between the temperature and the antimycotic activity of 5-flu-

384

V. Buchta and M. Otcenasek

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orocytosine and amphotericin B has been described in aspergilli (Fig. 2). Similar conclusions have been drawn from some other studies whose results confirmed a greater activity of nystatin against yeasts at a lower temperature and an increasing efficacy of 5-fluorocytosine with an increasing temperature (3, 13, 14,20). The higher temperature of incubation seems to have been involved in a more pronounced antimycotic effect of some drugs against our strains of C. candidum, Trichosporon spp., C. zeylanoides and most T. mentagrophytes strains. The strongly retarded or completely inhibited growth of these species at 35°C or 39 °C was in correspondence with this. A similar temperature effect was observed in T. mentagrophytes strains that were more sensitive to imidazole preparations at 37°C than at 32 °C (36). The incubation period has a substantial effect on the results of testing, especially in antimycotics of fungistatic nature (3, 4, 19). Our results were influenced by the fact that in polyenes, the changes of MIC endpoints could be read during the entire incubation period (168 h), while the MICs of azole derivatives and 5-fluorocytosine soon exceeded the maximal concentrations tested. This is the most probable explanation of a relatively high frequency of MIC changes in amphotericin B, particularly in complex media (Table 3). The effect of the incubation period on the stability of this polyene antibiotic in long-term experiments cannot be excluded either (6). The results of the testing of azole derivatives and partially, of 5-fluorocytosine have confirmed a direct relationship between the size of the inoculum and the MIC (3, 4, 12, 19). The MIC increased with the incubation period and it was apparent that MIC changes were more considerable in a dense inoculum (lOS and 106 cfu·mL-1 ) than in

Antifungal Susceptibility Testing

385

Table 3. Changes of MICs of systemic antimycotics in dependence on incubation period and composition of medium Medium

Drug YNB

AMB 5FC MZC KTZ ITZ FLZ ATM

BHI

SGB

CAS

ATB3

o o

MD

5 39 o 0

N

o

9

5

o

29

0

3

o

25

2

5

o

58

2

0

o

9

69

n

30 18

40 53

9 33

23 48

13 20

31 58

2 58

11 78

11 72

15 89

72

15 87

10 47

23 69

n

15 30

31 40

17 54

26 72

10

o

34 16

24 43

39 70

11 43

22 61

20 39

35 67

16 36

31 55

3 10 3 370

20 0

7 0

20 0

5 0

16 0

2 0

11 0

5 0

17 0

4 0

16 1

n

N N n

N

7

RPMI

0

o

47

0

n

14 35

31 50

0 39

9 36

5 29

22 39

10 31

17 41

13 28

17 46

4 33

11 41

8 32

18 42

n

5 61

9 77

12 52

33 71

56

5

18 64

56

7

22 63

4 61

15 78

13 57

26 67

8 57

21 70

12 24

22 39

8 30

23 38

7 17

25 30

32

9

27 42

8 34

25 46

7 34

25 44

N N n

N

AMB - amphotericin B, 5FC - 5-fluorocytosine, MZC - miconazole, KTZ - ketoconazole, ITZ - itraconazole, FLZ - fluconazole, ATM-all antimycotics tested, MD - all media tested n - cumulative % of strains with MIC increase by more than two dilutions after 48 h (tx ) and 168 h (ty) N - % of strains with MIC in excess of maximum concentration tested after 24 h (txl and 120 h (ty)

those of 104 and 105 cfu' mL-1. Improvement of intra- and interlaboratory reproducibility of the test results was demostrated in previous experiments using smaller inocula (10 3 vs. 104 cfu·mL-1) (9, 11). They led NCCLS to the recommendation to use a lower concentration of yeasts in the testing of antimycotics by a standard method (9, 30). However, problems with reading the results may arise when testing some yeasts with smaller inocula. If a concentration lower than 10 5 cfu' mL-1 was used in cryptococci, a very weak growth of this yeast was seen after 24 and 48 h of incubation, including that of the drug-free medium. This could explain a prominent susceptibility of our C. neoformans strains to most antimycotics tested. Similar results and recommendations to work on cryptococci with a denser inoculum and to prolong the period of incubation to 48-72 h were published by other authors (7, 11, 37). At present, systematic and coordinated efforts are being made to unify the methods of in-vitro testing of antifungal drugs. NceLS has suggested the macrodilution standard (M27-P) for the testing of yeasts and subsequent comparative studies have shown

386

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a high degree of agreement (>90%) for alternative microdilution methods (18, 22, 23, 24,25,28). Our experiments were performed with the aid of a microdilution broth assay based on the technique described by Espinel-Ingroff et al. (8), differing from the S-FLUOROCYTOSINE

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M27-P standard in the size of inoculum and its preparation. We used an inoculum of 1.0-5.0 X 104 cfu· mL-1 adjusted by means of Biirker's chamber instead of the photometric-method. In addition, no visible growth in wells was selected as MIC criterion which probably resulted in an underestimation of the effect of azole derivatives in our experiments. A more detailed knowledge of the influence of the individual factors on the variability of test results together with a determination of MIC endpoints is a basic prerequisite for the elaboration of standardized and reproducible methods of antifungal susceptibility testing. At the same time, the results of this methodology could provide clinically useful information. At present, unfortunately, no unequivocal conclusions can be made on the in-vivo efficacy of most antimycotics on the basis of their MICs. This is one of the reasons why generally accepted values of breakpoint concentrations for the individual antimycotics have not yet been determined. This reason, together with an unreliable correlation of results obtained in vitro and in vivo create an argument for a number of authors raising doubts about the justification of testing antimycotics in vitro, particularly in azole derivatives. In the case of a correlation between in-vitro and in-vivo results one should take into consideration that the outcome of treatment of a mycotic infection depends also on the factors which need not necessarily have a direct relationship with the antifungal effect of a given drug in vitro. This primarily refers to the pharmacokinetics of the compound (distribution, bioavailability at the site of infection, metabolic stability), therapeutic exertion of subinhibitory concentrations including possible interference with the mechanisms of virulence of the pathogen (adherence, morphological transformation), emergence of resistant strains, potential drug interactions and degree of alteration of the host immunity. These cirumstances may have their share in a hardly predictable outcome of therapy of mycotic infections based only on the laboratory data on the susceptibility of the etiologic agent. In spite of justified reservations as to the use of in-vitro tests to predict clinical results, the testing of antimycotics may be desirable in some cases. The usefulness of testing is supported by the present experience with the treatment of immuno-compromised patients, particularly those with AIDS, using amphotericin B and fluconazole. The argument in favour of the usefulness of testing is also corroborated by the growing number of therapeutic failures of antimycotic treatment in connection with the limited susceptibility of the given mycotic agent in vitro (5, 10, 15,22,27,29,31,33).

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24. Pfaller, M. A., Q. Vu, M. Lancaster, A. Espinel-Ingroff, A. Fothergill, C. Grant, M. R. McGinnis, L. Pasarell, M. G. Rinaldi, and L. Steelemoore: Multisite reproducibility of colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates. J. Clin. Microbio!. 32 (1994) 1625-1628 25. Pfaller, M. A., M. Bale, B. Buschelman, M. Lancaster, A. Espinel-Ingroff,]. H. Rex, and M. G. Rinaldi: Selection of candidate quality control isolates and tentative quality control ranges for in vitro susceptibility testing of yeast isolates by National Committee for Clinical Laboratory Standards proposed standard method. J. Clin. Microbio!. 32 (1994) 1650-1653 26. Polak, A. and M. Grenson: Interference between the uptake of pyrimidines and purines in yeast. Pathol.Microbio1.39 (1973) 37-38 27. Powderly, W. G., G. S. Kobayashi, G. P. Herzig, and G. Medoff: Amphotericin B-resistant yeast infection in severely immunocompromised patients. Am.J. Med. 84 (1988) 826-832 28. Rodriguez-Tudela, j. L. and ]. V. Martinez-Suarez: Improved medium for fluconazole susceptibility testing of Candida albicans. Antimicrob. Agents Chemother.38 (1994) 45-48 29. Sandven, P., A. Bjorneklett, A. Maeland, and The Norwegian Yeast Study Group: Susceptibilities of norwegian Candida albicans strains to fluconazole: emergence of resistance. Antimicrob. Agents Chemother. 37 (1993) 2443-2448 30. Sheehan, D.]., A. Espinel-Ingroff, L. Steele More, and C. D. Webb: Antifungal susceptibility testing of yeasts: A brief overview. Clin. Infect. Dis. 17 (Supp!. 2) (1993) S494-S500 31. Supparatpinyo, K., K. E. Nelson, W. G. Merz, B. j. Breslin, C. R. Copper, jr., C. Kamwan, and T. Sirisanthana: Response to antifungal therapy by human immunodeficiency virus-infected patients with disseminated Penicillium marneffei infections and in vitro susceptibilities of isolates from clinical specimens. Antimicrob. Agents Chemother.37 (1993) 2407-2411 32. Van Cutsem, j., F. Van Gerven, and P. A. j. Janssen: The in vitro evaluation of azoles. In: Iwata, K. and H. Vanden Bossche (eds.), In vitro and in vivo evaluation of antifungal agents, pp. 51-64. Elsevier Science Publishers (1986) 33. Velez,]. D., R. Allendoerfer, M. Luther, M. G. Rinaldi, and j. R. Graybill: Correlation of in vitro azole susceptibility with in vivo response in a murine model of cryptococcal meningitis. J. Infect. Dis. 168 (1993) 508-510 34. Wawrzkiewicz, K.: Aktywnosc fungistatyczna nystatyny w zeleznosci od srodowiska i rozpuszclnika. Polskie Arch. Welt 16 (1973) 85-94 35. Werner, E., M. Seibold, and E. Antweiler: Susceptibility testing of Candida species for fluconazole: the role of buffering in the agar dilution assay. Mycoses 36 (1993) 125130 36. Wright, L. R., E. M. Scott, and S. P. Gorman: The sensitivity of mycelium, arthrospores and microconidia of Trichophyton mentagrophytes to imidazoles determined by in-vitro tests. J. Antimicrob. Chemother.12 (1983) 317-327 37. Yamaguchi, H., T. Hiratani, and M. Plempel: In vitro studies of a new oral azole antimycotic BAY N-7133. J.Antimicrob. Chemother.11 (1983) 135-149

RNDr. Vladimir Buchta, CSc., Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 501 65 Hradec Kralove, Czech Republic