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Journal of Clinical Microbiology, August 2000, p. 2940-2942, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Detection of Paracoccidioides brasiliensis in Tissue Samples by a Nested PCR Assay

Ralf Bialek,1,* Aida Ibricevic,1 Christian Aepinus,2 Laura K. Najvar,3 Annette W. Fothergill,3 Jürgen Knobloch,1 and John R. Graybill3,4

Institute for Tropical Medicine1 and Department of Molecular Pathology,2 University Hospital Tübingen, D-72074 Tübingen, Germany, and Audie Murphy Hospital4 and University of Texas Health Science Center at San Antonio,3 San Antonio, Texas 78284

Received 11 January 2000/Returned for modification 6 March 2000/Accepted 31 May 2000

A nested PCR assay for the detection of Paracoccidioides brasiliensis DNA was evaluated, using a sequence of the immunogenic gp43 gene as a target. This gene encodes an outer membrane protein unique to this dimorphic fungus. DNA from six clinical isolates and the ATCC strain 60885 of P. brasiliensis, as well as DNA from closely related fungi, was examined to determine detection limits and cross-reactions. PCR was done on DNA extracts of lung homogenates from 23 experimentally P. brasiliensis-infected and two uninfected BALB/c mice and from 20 Histoplasma capsulatum-infected ICR mice. The results were compared to quantitative cultures. A detection limit of 0.5 fg of specific DNA was determined using cloned plasmid DNA. In all seven P. brasiliensis isolates, the expected 196-bp nested PCR product was found. Their sequences were 100% identical to the gp43 gene sequence in GenBank. DNA extracts of all other, related fungi were negative. The PCR assay was positive in 21 out of 23 culture-positive lung homogenates with concentrations of 1 × 103 to 1.3 × 107 CFU of P. brasiliensis per g of lung. Uninfected BALB/c mice and H. capsulatum-infected mice samples gave negative results. The high sensitivity and specificity of this new nested PCR assay for the detection of P. brasiliensis in tissue samples were demonstrated. The assay may be useful for diagnosis in human tissue samples.

* Corresponding author. Mailing address: Institut für Tropenmedizin, Universitätsklinikum Tübingen Keplerstrasse 15, D-72074 Tübingen, Germany. Phone: 49 7071 298 2367. Fax: 49 7071 29 5267. E-mail: ralf.bialek{at}med.uni-tuebingen.de.

Journal of Clinical Microbiology, August 2000, p. 2940-2942, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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