Antifungal susceptibility testing using the E test: comparison with the broth macrodilution technique

Journal of Antimicrobial Chemotherapy (996) 7, 65-7 Antifungal susceptibility testing using the E test: comparison with the broth macrodilution technique Sharon C. A. Chen, Maryann L. O'Donnell, Suzannah Gordon and Gwendolyn L. Gilbert Department of Clinical Microbiology, Centre for Infectious Diseases and Microbiology, Westmead Hospital, Hawkesbury Road, Westmead, New South Wales 5, Australia The National Committee for Clinical Laboratory Standards reference broth macrodilution method for antifungal susceptibility testing was compared with the E test by testing 86 clinical isolates of Candida spp. and Cryptococcus neoformans. The MIC agreement rates for the two methods for Candida spp. were 7-89% within ± doubling dilution and 87-00% within ± dilutions. For C. neoformans, agreement within ± dilution was > 70% for all the agents tested except for which agreement was 5%. Our data support the further evaluation of the E test as an alternative method for antifungal susceptibility testing. However, E test MICs of for C. neoformans, particularly C. neoformans var. gattii should be interpreted with caution, as falsely elevated MICs may occur. Introduction Fungal infections are important causes of morbidity and mortality in immunocompromised patients (Anaissie, Bodey & Rinaldi, 989; Beck-Sague et al., 99). The development of new antifungal agents, the rise in the incidence of infections caused by yeasts other than Candida albicans and reports of resistance to antifungal agents have increased the demand for in-vitro antifungal susceptibility testing (Smith et al., 986; Anaissie et al., 989; Wingard et al., 99; Cameron et al., 99; Rex et al., 99). However, such testing remains unstandardised. MICs of antifungal agents are influenced by many variables including medium composition and ph, incubation conditions, inoculum density and endpoint definition (Pfaller et al., 990; Fromtlingef a/., 99; Rex et al., 99). The National Committee for Clinical Laboratory Standards (NCCLS) has proposed a reference broth macrodilution method for susceptibility testing of yeasts (NCCLS, 99). However, the method is cumbersome and ill-defined endpoints with azole agents lead to difficulties in interpretation. A microdilution adaptation of the NCCLS M7-P proposed reference method (NCCLS, 99) has recently been shown to be comparable to the latter for susceptibility testing of yeasts (Pfaller & Barry, 99; Pfaller et al., 99). Simpler testing procedures are needed. The E test (AB Biodisk, Solna, Sweden) is an established method of susceptibility testing of bacteria. Recent studies suggest that it may be a convenient and reliable method of susceptibility testing of yeasts (Bolmstrom & Odds, 99; Espinel-Ingroff, Bolstrom & Jones, 99; Sewell, Pfaller & Barry, 99; Phone: +6()6655; Fax: +6()898659. 65 005-75/96/0065 + 09 $.00/0 '996 The British Society for Antimicrobial Chemotherapy

S. C. A. Chen et al. 99; Colombo et al., 995). However, in-vitro parameters have yet to be defined and experience with antifungal testing is limited. We compared the use of the E test with the reference NCCLS broth macrodilution method in determining the susceptibilities of 86 clinical yeast isolates to five antifungal agents including, and. Yeast isolates Materials and methods Eighty-six recent clinical yeast isolates from our laboratory's culture collection were tested. They comprised C. albicans (0 isolates), ten isolates each of Candida parapsilosis, Candida tropicalis, Torulopsis glabrata, Cryptococcus neoformans var. neoformans, C. neoformans var. gattii and six isolates of Candida krusei. Repeat isolates from the same patient were excluded. All isolates were recovered from clinical specimens, identified by standard methods (Warren & Shadomy, 99) and stored as a suspension in water at ambient temperature until used. Before to testing, each isolate was passaged at least twice on Sabouraud's dextrose agar (Difco Laboratories, Detroit, Michigan) at 0 C for -8 h to ensure optimal growth characteristics. Antifungal agents Five antifungal agents obtained as reagent grade powders were tested: (Bristol-Myers Squibb, Noble Park, Victoria), 5- (5FC; Roche Laboratories, Dee Why, NSW), (Pfizer Australia Pty Ltd, Ryde, New South Wales),' (Janssen-Cilag, Lane Cove, NSW) and intraconazole (Janssen-Cilag, Lane Cove, NSW). Stock solutions were prepared using deionised water as a solvent for 5FC and 00% dimethyl sulphoxide for the other compounds. The initial stock concentrations were 560 mg/l for all agents. Serial two-fold dilutions of each agent were prepared in RPMI-60 medium (Gibco laboratories, Grand Island, NY) buffered to ph 7.0 with 0.65 M MOPS buffer (Sigma, St. Louis, USA). Final drug concentrations in each case ranged from 0.06 to 6 mg/l. E test strips were obtained from Australian Laboratory Services, Rockdale, N.S.W. and stored at 0 C until used. MIC determinations Broth macrodilution. This was performed according to NCCLS (99) guidelines using the spectrophotometric method of inoculum preparation, a final inoculum concentration of -5 + 0 x 0 cells/ml, and RPMI-60 medium buffered to ph 70 with MOPS. Yeast inocula (0-9 ml) were added to x 75 mm polystyrene plastic tubes (Falcon 05, Becton Dickenson, NSW) containing 0. ml of doubling dilutions of each antifungal drug. Drug-free and yeast-free control tubes were included in each run. The tubes were incubated in air at 5 C; MIC endpoints were determined according to the NCCLS recommendations at 8 h for Candida spp. and 7 h for C. neoformans (NCCLS, 99). E test. This was performed according to the E test provisional technical guide available at the time of study (Anonymous, 99). Inoculum suspensions were prepared by homogenizing yeasts after culture for h on Sabouraud's dextrose agar in 0-85% NaCl to achieve a McFarland turbidity {Candida spp.). C. neoformans was cultured for 8-7 h and a 0 McFarland turbidity was obtained. In all cases, the inocula

Antifungal susceptibility testing 67 concentrations were adjusted to the appropriate turbidity with the aid of a spectrophotometer (50 nm wavelength). RPMI-60 agar with % glucose buffered with MOPS (ph 70) was used to test susceptibilities to and 5FC; potassium phosphate (ph 70) buffer was used in place of MOPS to prepare the E test plates for azole testing, as recommended by the manufacturers. Plates were then swabbed with the yeast suspension evenly in three directions. After allowing excess moisture to be absorbed by the agar, one E test strip was applied per plate. The plates were incubated at 5 C in air and read after h for Candida spp. and after 8-7 h for C. neoformans. E tests were additionally performed using Casitone agar (Difco, Detroit USA), in accordance with a more recent recommendation by the manufacturers (Anonymous, 99), to determine the susceptibility of C. neoformans to, after poor agreement with the reference method was noted using RPMI 60 medium. C. albicans ATCC 9008, C. albicans ATCC 9009, C. parapsilosis ATCC 9008, C. neoformans ATCC 90 and C. neoformans ATCC 90 were included as controls with each batch of organisms tested. For both test methods, MICs were recorded by two independent observers. E test MICs were rounded up to the next highest value to correspond with the standard two-fold dilution used in the reference broth method. To obtain reproducibility, the MIC determinations were repeated until at least two results were in agreement. E test MICs > mg/l and < 0.00 mg/l were also included in the analysis. Discrepancies among MIC endpoints of no more than two doubling dilutions were used to calculate the percentage agreement. Results The susceptibilities of the yeast isolates to the five antifungal agents using RPMI 60 medium are shown in Table I. In the E test, Candida spp. produced clearly detectable growth at h in RPMI-60 medium and MICs were determined at that time. Isolates of C. neoformans required 8-7 h incubation for detectable growth. A broad range of MIC values was obtained with all antifungal agents except for. The activities of each agent varied with the organism group tested. Broth microdilution MICs demonstrated, as expected, that all six isolates of C. krusei were resistant to with MICs ^6 mg/l. Most isolates of C. albicans and C. parapsilosis were susceptible, and C. glabrata and those of C. Iropicalis resistant to. The MICs of for two isolates of C. neoformans were 8 mg/l; these were obtained from patients infected with the human immunodeficiency virus (HIV) who had previously received for the treatment of oral candidiasis. For C. rropicalis isolates, broth macrodilution MIC90 values for the azole agents were ^ 6-fold higher than for C. albicans and C. parapsilosis, and two to eightfold higher than for C. glabrata. The MICs of all the azoles tested for one isolate was > mg/l; this was associated with azole treatment failure. Tables II and III summarise the percentage of MICs obtained by the E test that were within two dilutions of those obtained by the reference broth macrodilution method. The agreement between the two methods ranged from 7-89% within one dilution and 87-00% within two dilutions at 8 h for Candida spp., except C. tropicalis, for which the agreement was ^ 60% (± two dilutions; >0% ± one dilution). Agreement was best for C. glabrata (00% ± two dilutions and ^90% ± one dilution). For C. neoformans, agreement was >90% (±two dilutions) for all agents except, for which

Table I. MICs (mg/l) of five antifungal agents for 86 yeast isolates Species (no. tested) Antifungal agent MIC*, Macrodilution MIC* MIC (mg/l) Range MIC* E test MIG» Range s C. albicans (0) C. tropicalis (0) C. parapsilosis (0) C. glahrala(w) C. krusei (6) C. neuformans var. neoformuns (0) C. neoformans var. gatlii (0) 0-5 0 0-5 0 0 0-5 0 0 0 ^006 6 0-5 0 <;0O6 ^0-06 0 0-5 0-5 >6 6 0-5 0-5 0-5 6 6 0-5 8 0-5 0 0-- ^006->6 0->6 ^006-6 <;006-l 0-0-- l->6 ^0-06-6 <; 0-06- 0-5- ^006-0 5 0-5- 5S0-06-0-5 ^0-06- 0-5-6 <;0-06->6 ->6 0-5- - 0-5- ^0-06-6 ->6-0-5- <;0-06-0-5 0-8 0-5-8 :S 0-06- ; 006-0-5 5 0-06-0-0-5-8 0-8 0-- 0-05 0-06 0-5 ^0-06 006 0-5 0-5 006 05 0-5 006 ^6 05 006 > 0-5 > > 0 ^006 0 0-5 > ^0-06 0-5 0-5 0-5 > 05 0-06 > -5 > > 0-5 8 0 0-5 > 0- ^0-06- ^0O6-> 0-5-> ^006-> <; 0-06- 0-5- ^0-06- 05-> ^0-06-> 05-8 0-06- : 0-06-0-5 0-- ^006-006 ^006-0-5-> ^006-> l-> 09-> -> 0-5- ^006-> > - - : 0-06-0-5 0-> 006-> ^0-06- ^0-06- ^0-06-0-5 - 0-5-> 05- ^0-06-0-5 n > n e.

Table II. Comparison of E test MICs with broth dilution MICs for Candida spp. Antifungal agent Number of strains > - - Agreement broth - of E-test dilution same MICs with MICs" + + > + % within ± dilution % within ± dilutions Amphotericin B 5 Flucytosine Fluconazole Ketoconazole Itraconazole 9 " 7 Q*" V " 5 8 6 9 5 8 8 0 0 5 9 6 6 0 85 89 80 7 76 00 00 9 87 9 Numbers expressed as (log)i deviation from corresponding broth dilution MICs *C iropicalis strains. 'C. albicans (seven strains), C iropicalis (two strains). Table HI. Comparison of E test MICs with broth dilution MICs for C. neoformans a E Antifungal agent Number of strains > - - Agreement broth - of E-test dilution same MICs with MICs' + + > + % within ± dilution % within ± dilutions Amphotericin B 5 Flucytosine Fluconazole Ketoconazole Itraconazole 0 0 0 0 0 9 9 6 7 0 6 5 85 70 5 80 80 00 00 5 90 00 "Numbers expressed as (log): deviation from corresponding broth dilution MICs.

70 S. C. A. Chen et al. agreement was only 5%. These discrepancies were, generally, the result of lower MICs for C. tropicalis obtained by the E test; higher MICs for C. neoformans were noted for all the antifungal agents tested except, for which lower MICs were obtained using the E test. Overall agreement was best (00% ± two dilutions, >85%+one dilution) for and 5FC. Of the azoles, had the highest agreement followed by and for Candida spp. The converse was observed in testing C. neoformans; isolates of C. neoformans had E test MICs that were more than four-fold (two dilutions) higher than the broth dilution MICs. These comprised eight C. neoformans var. gatti and three C. neoformans var. neoformans. E test MICs > mg/l were noted; however the corresponding broth dilution MICs were < 0 mg/l. These isolates were obtained from patients who were successfully treated with indicating that the broth dilution method more accurately predicted clinical response. Repeat suscepibility testing of C. neoformans isolates to using Casitone agar resulted in an agreement of 65% (±two dilutions; 55% ± one dilution) between the two methods. However, E test MICs of > mg/l in six C. neoformans var. gattii isolates was consistently observed; the corresponding broth dilution MICs ranged from 0.5.0 mg/l. Of the Candida spp., the lowest agreement (60% + two dilutions, 0% ± one dilution) for was observed with C. tropicalis. Problems encountered with MIC determination of the azoles included the presence of trailing endpoints in both methods (Figure), especially for C. tropicalis. 'Light-bulb' Figure. Susceptibility of C. albicans todetermined by the E test, illustrating the phenomenon of trailing endpoint.

Antifungal susceptibility testing 7 phenomena and the presence of double zoning also occurred with the E test. In general, clearest end-points were seen with C. neoformans var. neoformans and the worst with C. tropical is. Discussion The present study supports the findings of previous comparisons between the E test and broth dilution techniques for antifungal susceptibility testing (Bolmstrom & Odds, 99; Sewell et al., 99; Colombo et al., 995). In general, there was a tendency to read E test MICs slightly lower; this may be due to the greater ease of visualising growth along a continuous gradient. The overall agreement (±two dilutions) between the E test and NCCLS reference method for azole susceptibility testing of Candida spp. was similar to that observed in two recent studies (Sewell et al., 99; Colombo et al., 995). Sewell et al. (99) noted that agreement, in susceptibility testing, for C. glabrata and C. tropicalis was poorest (7% and 56%, respectively). In contrast, we found little disparity in MICs obtained by the two methods for C. glabrata. MIC disagreements among Candida spp. tested indicated that C. tropicalis represented the major problem. Amphotericin B and 5FC gave the best agreement (± two dilutions) with the proposed NCCLS reference method in our study. In testing Candida spp., was superior to, which in turn outranked in percentage agreement achieved, as noted previously (Colombo et al., 995). However, gave the poorest agreement in testing C. neoformans, especially for C. neoformans var. gattii with 55% of E test MICs being more than four-fold higher than broth dilution MICs using RPMI medium. This contrasts with the findings of other investigators (Espinel-Ingroff et al., 99; Colombo et al., 995). Espinel-Ingroff et al. (99) demonstrated that, agreement was highest for (86-89%) and 5FC (90-9%) when tested against Candida spp. and C. neoformans. In their study, E test MICs were recorded using high resolution antifungal agar (HR Agar; Unipath, Basingstoke, England) as well as RPMI-60 agar. HR medium is reported to support better growth of yeasts and thus may affect the clarity of azole endpoint determinations (Rex et al., 99). The use of Casitone agar in our study improved but did not eliminate the marked disparity observed with E test and broth dilution MICs for in testing C. neoformans. The revised E test Technical Guide (Anonymous, 99) presently recommends Casitone agar as the medium for azole susceptibility testing for yeasts. We noted trailing endpoints frequently using both the reference broth macrodilution and E test in testing azole susceptibility; however, this was not evident in testing C. neoformans isolates. This was especially problematic with C. tropicalis, as observed in a recent report by Colombo et al. (995). One of the more important variables in the determination of MICs for azoles is the inoculum density and every effort was made to ensure that this was standardised. Further investigations are therefore warranted in determining the optimal medium for clearer azole end-point determinations by different laboratories. We read E test MICs for Candida spp. after h of incubation as others have noted that agreement is poorer if endpoints are recorded after ^8 h incubation for (Sewell et al., 99). For C. neoformans adequate growth for MIC determinations was evident at 8-7 h. This study adds important information comparing the E test with the reference broth macrodilution method for antifungal susceptibility testing. Overall, the agreement within two dilutions was ^87%, with the exception of in testing C. neoformans.

7 S. C. A. Chen et al. This occurred with the use of both RPMI 60 and Casitone medium in performing the E test. The E test appears to be a convenient and reliable alternative method for susceptibility testing of yeasts. However, at present, our data suggest caution in interpreting E test MICs of for C. neoformans, particularly C. neoformans var. gattii, as results may indicate false resistance. Acknowledgement We thank Mrs Rachel Motbey for typing the manuscript. References Anaissie, E. J., Bodey, G. P. & Rinaldi, M. G. (989). Emerging fungal pathogens. European Journal of Clinical Microbiology and Infectious Diseases 8, -0. Anonymous. (99). E Test technical guide no. : antifungal susceptibility testing of yeasts. AB B0DISK, Solna, Sweden. Anonymous. (99). E Test technical guide no. b: antifungal susceptibility testing of yeasts. AB BIODISK, Solna, Sweden. Beck-Sague, C. M. & Jarvis, W. R. and the National Nosocomial Infectious Surveillance System (99). Secular trends in the epidemiology of nosocomial fungal infections in the United States, 980-990. Journal of Infectious Diseases 67, 7-5. Bolmstrom, A. & Odds, F. (99). Comparison of antifungal susceptibility testing with E test and other methods for clinically documented isolates. In Program and Abstracts of the Twelth Congress of the International Society of Human and Animal Mycology, Adelaide, South Australia, 99. Abstract P., p.d.. Cameron, M. L., Schell, W. A., Bruch, S., Bartlett, J. A., Waskin, H. A. & Perfect, J. R. (99). Correlation of in-vitro resistance to Candida isolates in relation to therapy and symptoms in individuals seropositive for human immunodeficiency virus type I. Antimicrobial Agent and Chemotherapy 7, 9-5. Colombo, A. L., Barchiesi, F., McGough, D. A., Rinaldi, M. G. (995). Comparison of E test and National Committee for Clinical Laboratory Standards broth macrodilution method for azole susceptibility testing. Journal of Clinical Microbiology, 550. Espinel-Ingroff, A., Bolmstrom, A. & Jones, R. Collaborative evaluation of E test for antifungal susceptibility testing with 5 antifungal agents against pathogenic yeasts. In Abstracts of the Nmetv-Fourth General Meeting of the American Society for Microbiology, Las Vegas, 99. Abstract F-, p. 608. American Society for Microbiology, Washington, DC. Fromtling, R. A., Galgiani, J. N., Pfaller, M. A., Espinel-Ingnoff, A., Bartizal, K. F., Bartlett, M. S. et al. (99). Multicentre evaluation of a broth macrodilution antifungal susceptibility test for yeasts. Antimicrobial Agents and Chemotherapy 7, 95. National Committee for Clinical Laboratory Standards. (99). Reference Method for Broth Dilution Antifungal Susceptibility Testing for Yeasts. Proposed Standard Document M7-P. NCCLS, Villanova, PA. Pfaller, M. A. & Barry, A. L.(99). Evaluation of a novel colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates. Journal of Clinical Microbiology, 99-96. Pfaller, M. A., Grant, C, Morthland, G. V. & Rhine-Chalberg, J. (99). Comparative evaluation of alternative methods for broth dilution susceptibility testing of against Candida albicans. Journal of Clinical Microbiology, 506-9. Pfaller, M. A., Rinaldi, M. G., Galgiani, J. N., Bartlett, M. S, Body, B. A., Espinel-Ingnoff, A. et al. (990). Collaborative investigations of variables in susceptibility testing of yeasts. Antimicrobial Agents and Chemotherapy, 68-5. Rex, J. H, Pfaller, M. A., Rinaldi, M. C, Polak, A. & Galgiani, J N. (99). Antifungal susceptibility testing. Clinical Microbiological Reviews 6, 67-8.

Antifungal susceptibility testing 7 Sewell, D., Pfaller, M. A. & Barry, A. L. (99). Comparison of broth macrodilution, broth microdilution and E test antifungal susceptibility tests for. Journal of Clinical Microbiology, 099-0. Smith, K. J., Warnock, D. W., Kennedy, C. T., Johnson, E. M., Hopwood, V., Van Custem, J. et al. (986). Azole resistance in Candida albicans. Journal of Medical and Veterinary Mvcologv, -. Warren, N. G. & Shadomy, H. J. (99). Yeasts of medical importance. In Manual of Clinical Microbiology, 5th edn (Balows, A. Hausler, W. J., Hermann, K. L., Isenberg, H. D. & Shadomy, H. J., Eds), pp. 67-69. American Society for Microbiology, Washington, DC. Wingard, J. R., Merz, W. G., Rinaldi, M. G., Johnson, T. R., Karp, J. E. & Saral, R. (99). Increase in Candida krusei infection among patients with bone marrow transplantation and neutropenia treated prophylactically with. New England Journal of Medicine 5, 77. {Received 9 April 995; returned 5 June 995; revised June 995; accepted August 995)