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Journal of Clinical Microbiology, November 2008, p. 3690-3702, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.00917-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Detection of a Molecular Biomarker for Zygomycetes by Quantitative PCR Assays of Plasma, Bronchoalveolar Lavage, and Lung Tissue in a Rabbit Model of Experimental Pulmonary Zygomycosis{triangledown}

Miki Kasai,1,{dagger} Susan M. Harrington,2,{dagger},{ddagger} Andrea Francesconi,1 Vidmantas Petraitis,1,3 Ruta Petraitiene,1,3 Mara G. Beveridge,1 Tena Knudsen,1 Jeffery Milanovich,1 Margaret P. Cotton,1 Johanna Hughes,1 Robert L. Schaufele,1 Tin Sein,1 John Bacher,4 Patrick R. Murray,2 Dimitrios P. Kontoyiannis,5 and Thomas J. Walsh1*

NCI, NIH, Bethesda, Maryland,1 Department of Laboratory Medicine, Warren Grant Magnuson Clinical Center, Bethesda, Maryland,2 LASP, SAIC-Frederick, Inc., Frederick, Maryland,3 Division of Veterinary Resources, Office of Research Services, NIH, Bethesda, Maryland,4 University of Texas M. D. Anderson Cancer Center, Houston, Texas5

Received 13 May 2008/ Returned for modification 29 June 2008/ Accepted 2 September 2008

We developed two real-time quantitative PCR (qPCR) assays, targeting the 28S rRNA gene, for the diagnosis of zygomycosis caused by the most common, clinically significant Zygomycetes. The amplicons of the first qPCR assay (qPCR-1) from Rhizopus, Mucor, and Rhizomucor species were distinguished through melt curve analysis. The second qPCR assay (qPCR-2) detected Cunninghamella species using a different primer/probe set. For both assays, the analytic sensitivity for the detection of hyphal elements from germinating sporangiospores in bronchoalveolar lavage (BAL) fluid and lung tissue homogenates from rabbits was 1 to 10 sporangiospores/ml. Four unique and clinically applicable models of invasive pulmonary zygomycosis served as surrogates of human infections, facilitating the validation of these assays for potential diagnostic utility. For qPCR-1, 5 of 98 infarcted lung specimens were positive by qPCR and negative by quantitative culture (qCx). None were qCx positive only. Among 23 BAL fluid samples, all were positive by qPCR, while 22 were positive by qCx. qPCR-1 detected Rhizopus and Mucor DNA in 20 (39%) of 51 serial plasma samples as early as day 1 postinoculation. Similar properties were observed for qPCR-2, which showed greater sensitivity than qCx for BAL fluid (100% versus 67%; P = 0.04; n = 15). The assay detected Cunninghamella DNA in 18 (58%) of 31 serial plasma samples as early as day 1 postinoculation. These qPCR assays are sensitive and specific for the detection of Rhizopus, Mucor, Rhizomucor, and Cunninghamella species and can be used for the study and detection of infections caused by these life-threatening pathogens.

* Corresponding author. Mailing address: Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute, Building 10-CRC, Room 1-5740, Bethesda, MD 20892. Phone: (301) 402-0023. Fax: (301) 480-2308. E-mail: walsht{at}mail.nih.gov

{triangledown} Published ahead of print on 24 September 2008.

{dagger} M.K. and S.M.H. contributed equally to this work.

{ddagger} Present address: Albany Medical Center, 43 New Scotland Ave., Albany, NY 12208.

Journal of Clinical Microbiology, November 2008, p. 3690-3702, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.00917-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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