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Journal of Clinical Microbiology, January 2009, p. 142-152, Vol. 47, No. 1
0095-1137/09/$08.00+0     doi:10.1128/JCM.01668-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Development of an Oligonucleotide Array for Direct Detection of Fungi in Sputum Samples from Patients with Cystic Fibrosis{triangledown} ,{dagger}

Jean-Phillippe Bouchara,1,2,{ddagger} Hsin Yi Hsieh,3,{ddagger} Sabine Croquefer,1 Richard Barton,4 Veronique Marchais,1 Marc Pihet,1 and Tsung Chain Chang3*

Host-Pathogen Interaction Study Group, UPRES-EA 3142, Angers University,1 Laboratory of Parasitology and Mycology, Angers University Hospital, Angers, France,2 Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China,3 Mycology Reference Centre, Department of Microbiology, Leeds General Infirmary, Leeds, LS1 3EX, United Kingdom4

Received 27 August 2008/ Returned for modification 19 October 2008/ Accepted 12 November 2008

Cystic fibrosis (CF) is the most common inherited genetic disease in Caucasian populations. Besides bacteria, many species of fungi may colonize the respiratory tract of these patients, sometimes leading to true respiratory infections. In this study, an oligonucleotide array capable of identifying 20 fungal species was developed to directly detect fungi in the sputum samples of CF patients. Species-specific oligonucleotide probes were designed from the internal transcribed spacer (ITS) regions of the rRNA operon and immobilized on a nylon membrane. The fungal ITS regions were amplified by PCR and hybridized to the array for species identification. The array was validated by testing 182 target strains (strains which we aimed to identify) and 141 nontarget strains (135 species), and a sensitivity of 100% and a specificity of 99.2% were obtained. The validated array was then used for direct detection of fungi in 57 sputum samples from 39 CF patients, and the results were compared to those obtained by culture. For 16 sputum samples, the results obtained by the array corresponded with those obtained by culture. For 33 samples, the array detected more fungal species than culture did, while the reverse was found for eight samples. The accuracy of the array for fungal detection in sputum samples was confirmed (or partially confirmed) in some samples by cloning and resequencing the amplified ITS fragments. The present array is a useful tool for both the simultaneous detection of multiple fungal species present in the sputa of CF patients and the identification of fungi isolated from these patients.

* Corresponding author. Mailing address: Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, 1 University Rd., Tainan 701, Taiwan. Phone: 886-6-2353535, ext. 5790. Fax: 886-6-2363956. E-mail: tsungcha{at}mail.ncku.edu.tw

{triangledown} Published ahead of print on 19 November 2008.

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/.

{ddagger} Jean-Phillippe Bouchara and Hsin Yi Hsieh contributed equally to this study.

Journal of Clinical Microbiology, January 2009, p. 142-152, Vol. 47, No. 1
0095-1137/09/$08.00+0     doi:10.1128/JCM.01668-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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