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Journal of Clinical Microbiology, February 2003, p. 712-716, Vol. 41, No. 2
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.2.712-716.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Simple Method for Screening Candida Species Isolates for the Presence of Secreted Proteinases: a Tool for the Prediction of Successful Inhibitory Treatment

Jií Dostál,¹ Petr Hamal,² Libue Pavlíková,¹ Milan Souek,¹ Tomá Ruml,³ Iva Pichová,¹ and Olga Hruková-Heidingsfeldová^1*

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,¹ Institute of Biochemistry and Microbiology and Center for Integrated Genomics, Institute of Chemical Technology, Prague,³ Institute of Microbiology, Faculty of Medicine, Palack University, Olomouc, Czech Republic²

Received 8 July 2002/ Returned for modification 18 August 2002/ Accepted 11 November 2002

The yeasts of the genus Candida are opportunistic pathogens associated with the rising incidence of life-threatening infections in immunocompromised individuals. Secretion of aspartic proteinases has been determined to be one of the virulence factors of the pathogenic Candida species. To analyze the extracellular proteolytic activities of a large number of Candida clinical isolates, we developed a screening system based on a solid medium containing hemoglobin as the sole nitrogen source. The cleavage of hemoglobin by the secreted proteinases results in formation of clearance zones. The visibility of such zones was enhanced by addition of an acid-base indicator. Using this system, we assessed 245 clinical isolates of Candida from patients in the hospital of the Faculty of Medicine, Palacky University, Olomouc, Czech Republic, for the presence of secreted aspartic proteases (Saps). We also used the test plates for rapid semiquantitative testing of Sap inhibitors. Most of the pepstatin analogs affected the formation of the zones of clearance as well as the growth of Candida albicans, C. tropicalis, and C. parapsilosis colonies. By contrast, the human immunodeficiency virus proteinase inhibitors saquinavir, ritonavir, nelfinavir, and indinavir had no effect on the Candida strains tested. These results are in agreement with the inhibition constants obtained for the individual inhibitors with purified Saps. Thus, the plates containing hemoglobin proved to be an appropriate tool for the rapid and reliable assessment of Sap production and inhibition.

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* Corresponding author. Mailing address: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo namesti 2, 166 10 Prague 6, Czech Republic. Phone: (00420) 220183249. Fax: (00420) 224310090. E-mail: olga-hh{at}uochb.cas.cz.

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Journal of Clinical Microbiology, February 2003, p. 712-716, Vol. 41, No. 2
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.2.712-716.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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