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Journal of Clinical Microbiology, February 2006, p. 553-560, Vol. 44, No. 2
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.2.553-560.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Rapid Susceptibility Testing of Medically Important Zygomycetes by XTT Assay

Charalampos Antachopoulos,1 Joseph Meletiadis,1 Emmanuel Roilides,1,2 Tin Sein,1 and Thomas J. Walsh1*

Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland,1 Third Department of Pediatrics, Aristotle University, Hippokration Hospital, Thessaloniki, Greece2

Received 9 September 2005/ Returned for modification 22 September 2005/ Accepted 10 November 2005

The XTT colorimetric assay quantifies fungal growth by measuring fungal metabolism and has been used successfully for susceptibility testing of Aspergillus species after 24 and 48 h of incubation. In the present study using 14 clinical isolates of Zygomycetes (Rhizopus oryzae [5 isolates], Cunninghamella spp. [3 isolates], Mucor spp. [3 isolates], and Absidia corymbifera [3 isolates]), significant metabolic activity was demonstrated before visual or spectrophotometric detection of fungal growth by performing the XTT assay as early as 6 h after inoculation. Testing of susceptibility to amphotericin B, posaconazole, and voriconazole was subsequently performed using the XTT method (100 µg/ml XTT, 25 µM menadione) at 6, 8, or 12 h after inoculation and the CLSI (formerly NCCLS) M38-A method with visual and spectrophotometric MIC determinations at 24 h after inoculation. Concentration-effect curves obtained with the use of the Emax model (a sigmoid curve with variable slope) were comparable between the early XTT and spectrophotometric readings at 24 h. Complete inhibition of early metabolic activity with the azoles was delayed in comparison to that with amphotericin B. Using appropriate cutoff levels, agreement was demonstrated between the early XTT and 24-h spectrophotometric or visual readings. In particular, for MIC-0 (the lowest drug concentration showing absence of visual growth) of amphotericin B, overall agreement levels were 90 to 93% for the 6-h XTT assay and 100% for the 8- and 12-h time points. For MIC-0 of posaconazole, agreement levels were 86% for the 6-h XTT and 93 to 100% for the 8- and 12-h time points. The overall agreement levels for MIC-0 and MIC-2 (the lowest drug concentration showing prominent reduction of growth compared with the control well) of voriconazole (compared with 24-h spectrophotometric readings) were 93 to 98% for the 8- and 12-h XTT assays. These results support the use of the XTT method for rapid MIC determination for Zygomycetes.

* Corresponding author. Mailing address: Pediatric Oncology Branch, National Cancer Institute, CRC, Rm. 1-5750, MSC 1100, 10 Center Drive, Bethesda, MD 20892. Phone: (301) 402-0023. Fax: (301) 480-2308. E-mail: walsht{at}mail.nih.gov.

Journal of Clinical Microbiology, February 2006, p. 553-560, Vol. 44, No. 2
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.2.553-560.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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