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Antimicrobial Agents and Chemotherapy, June 2002, p. 1960-1965, Vol. 46, No. 6
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.6.1960-1965.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Method for Measuring Postantifungal Effect in Aspergillus Species

Roxana G. Vitale,1 Johan W. Mouton,2 Javier Afeltra,1 Jacques F. G. M. Meis,2 and Paul E. Verweij1*

Department of Medical Microbiology, University Medical Center Nijmegen,1 Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands2

Received 4 January 2002/ Returned for modification 2 February 2002/ Accepted 18 March 2002

An in vitro method for determination of postantifungal effect (PAFE) in molds was developed by using three isolates each of Aspergillus fumigatus, A. flavus, A. terreus, A. nidulans, and A. ustus. MICs of amphotericin B and itraconazole were determined by using National Committee for Clinical Laboratory Standards guidelines (M38-P). The inoculum was prepared in RPMI 1640 broth buffered with MOPS (morpholinepropanesulfonic acid) at pH 7.0, and conidia were exposed to amphotericin B and itraconazole at concentrations of 4, 1, and 0.25 times the MIC, each for 4, 2, and 1 h at 37°C. The same procedure was followed for controls with drug-free medium. Following exposure, the conidia were washed three times in saline and the numbers of CFU per milliliter were determined. Exposed and control conidia were then inoculated into microtitration plates and incubated at 37°C for 48 h in a spectrophotometer reader. The optical density (OD) was measured automatically at 10-min intervals, resulting in growth curves. PAFE was quantified by comparing three arbitrary points in the control growth curve, the first increase of OD and the points when 20 and 50% of the maximal growth were reached, with the growth curve of drug-exposed conidia. Amphotericin B induced PAFE in A. fumigatus at four times the MIC after 2 and 4 h of exposure ranging from 1.83 to 6.00 h and 9.33 to 10.80 h, respectively. Significantly shorter PAFEs or lack of PAFE was observed for A. terreus, A. ustus, and A. nidulans. Itraconazole did not induce measurable PAFE in the Aspergillus isolates at any concentration or exposure time tested. Further studies are warranted to investigate the implications of PAFE in relation to clinical efficacy and dosing frequency.

* Corresponding author. Mailing address: Department of Medical Microbiology, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: 31-24-3614356. Fax: 31-24-3540216. E-mail: p.verweij{at}mmb.azn.nl.

Antimicrobial Agents and Chemotherapy, June 2002, p. 1960-1965, Vol. 46, No. 6
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.6.1960-1965.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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