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In Situ Hybridization for Coccidioides immitis 5.8S Ribosomal RNA Sequences in Formalin-fixed, Paraffin-embedded Pulmonary Specimens Using a Locked Nucleic Acid Probe: A Rapid Means for Identification in Tissue Sections

Montone, Kathleen T. MD*; Litzky, Leslie A. MD*; Feldman, Michael D. MD,PhD*; Peterman, Heather MD*; Mathis, Benjamin MD*; Baliff, Jeffrey MD*; Kaiser, Larry R. MD; Kucharczuk, John MD; Nachamkin, Irving Dr PH, MPH*

Diagnostic Molecular Pathology: June 2010 - Volume 19 - Issue 2 - pp 99-104
doi: 10.1097/PDM.0b013e3181b3aa55
Original Articles

Coccidioides immitis/Coccidioides posadasii are common causes of pulmonary infection in certain geographic areas, and are highly infectious when working with culture isolates in the laboratory. Rapid techniques to accurately identify this pathogen in tissues may be of benefit for diagnosis and in limiting the exposure of laboratory personnel to this agent. Locked nucleic acids (LNA) are modified nucleotides in which a ribonucleoside is linked between the 2′-oxygen and the 4′-carbon atoms with a methylene unit. LNA oligonucleotides exhibit increased thermal stability and make excellent probes for in situ hybridization (ISH). In this study, ISH utilizing a biotin-labeled LNA probe targeting Coccidioides sp. ribosomal RNA sequences in 6 formalin-fixed, paraffin-embedded pulmonary tissue specimens from 6 patients with culture positive or histologic findings suggestive of Coccidioides sp. infection is described. The cultures of the pulmonary specimens confirmed C. immitis in 3 of 6 patients. The ISH procedure with the LNA probe was positive in all 6 cases, although the number of organisms that were highlighted varied from rare to numerous. ISH with a biotin-labeled DNA probe of the same sequence was positive in 4 of the 6 cases and the signal intensity and number of organisms was much less than that observed with the LNA probe. Negative control tissues containing a variety of different fungal pathogens including Aspergillus sp., Fusarium sp., Blastomyces dermatitidis, Candida sp, Histoplasma capsulatum, and Zygomyces did not hybridize with the LNA and DNA probes. ISH with an LNA oligonucleotide probe targeting Coccidioides sp. ribosomal RNA is useful for rapid ISH. ISH could be rapidly performed when fungal pathogens are observed in tissue but cultures are negative or have not been performed.

*Department of Pathology and Laboratory Medicine

Department of Surgery, Division of Thoracic Surgery, University of Pennsylvania, Philadelphia, PA

Reprints: Kathleen T. Montone, MD, Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, 3400 Spruce Street, 6 Founders, Philadelphia, PA 19104 (e-mail:

© 2010 Lippincott Williams & Wilkins, Inc.