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PMID: 19020057 Published · ppublish English Comparative Study Evaluation Study Journal Article Research Support, Non-U.S. Gov't

Development of an oligonucleotide array for direct detection of fungi in sputum samples from patients with cystic fibrosis.

Journal of clinical microbiology ·Vol. 47 ·No. 1 ·2009-01-00 ·Pages 142-52

Bouchara JP, Hsieh HY, Croquefer S, Barton R, Marchais V, Pihet M, Chang TC

Abstract

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.

MeSH Terms
Cystic Fibrosis/complications,microbiology DNA, Fungal/genetics DNA, Ribosomal Spacer/genetics Fungi/genetics,isolation & purification Humans Nucleic Acid Hybridization Oligonucleotide Array Sequence Analysis/methods Oligonucleotide Probes/genetics Polymerase Chain Reaction Sensitivity and Specificity Sputum/microbiology
Chemicals
DNA, Fungal DNA, Ribosomal Spacer Oligonucleotide Probes
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Bouchara Jean-Phillippe
Host-Pathogen Interaction Study Group, UPRES-EA 3142, Angers University, Angers, France.
Hsieh Hsin Yi
Croquefer Sabine
Barton Richard
Marchais Veronique
Pihet Marc
Chang Tsung Chain
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Article Info
Journal
Journal of clinical microbiology
Abbr.
J Clin Microbiol
ISSN
1098-660X
DOI
10.1128/JCM.01668-08
Published
2009-01-00
Epub
2008-00-19
Pages
142-52
Language
English
Region
United States
NLM ID
7505564
PMCID
PMC2620838
Subset
IM
External Links
PubMed source DOI: 10.1128/JCM.01668-08 PMC Full Text View journal details
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