This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bialek, R.
Right arrow Articles by Hohle, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bialek, R.
Right arrow Articles by Hohle, R.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, May 2002, p. 1644-1647, Vol. 40, No. 5
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.5.1644-1647.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Evaluation of Two Nested PCR Assays for Detection of Histoplasma capsulatum DNA in Human Tissue

Ralf Bialek,1* Antje Feucht,1 Christian Aepinus,2,{dagger} Gudrun Just-Nübling,3 Valerie J. Robertson,4 Jürgen Knobloch,1 and Rolf Hohle5

Institute for Tropical Medicine,1 Department of Molecular Pathology, University Hospital Tübingen, Tübingen,2 Department of Infectious Diseases, University of Frankfurt, Frankfurt am Main, Germany,3 Department of Medical Microbiology,4 Department of Histopathology, University of Zimbabwe, Harare, Zimbabwe5

Received 5 November 2001/ Returned for modification 4 February 2002/ Accepted 13 February 2002

In order to evaluate the diagnostic relevance of two nested PCR assays for diagnosis of histoplasmosis in clinical specimens, 100 paraffin-embedded biopsy specimens were examined. Upon microscopy of tissue, 50 biopsy specimens were histoplasma positive and 50 were negative. Due to destruction by formalin fixation, successful extraction of amplifiable human DNA was limited to 29 and 33 samples, respectively. A product of the Histoplasma capsulatum nested PCR assay targeting the gene encoding the unique fungal 100-kDa-like protein was detected in 20 histopathologically positive biopsy specimens but in none of the microscopically negative samples. Sequencing revealed that all 20 products of 210 bp were identical to the sequence of H. capsulatum in the GenBank database. In contrast, the nested PCR assay targeting the fungal 18S rRNA genes amplified products in 26 histopathologically positive but also in 18 microscopically negative biopsy specimens. However, sequencing revealed that only 20 of these 44 PCR products (231 bp) were identical to the sequence of H. capsulatum. The remaining 24 sequences were homologous to those of several Euascomycetes. These PCR products were detected only in tissues possibly colonized by nonpathogenic fungi, possibly causing these nonspecific amplifications. The detection limit of both H. capsulatum nested PCR assays was 1 to 5 fungal cells per sample. The two assays were similarly sensitive in identifying H. capsulatum. In this preliminary study, the novel 100-kDa-like-protein gene nested PCR revealed a specificity of 100% without requiring sequencing, which was necessary for identification of the 18S ribosomal DNA nested PCR products in order to avoid a high rate of false-positive results.

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

{dagger} Present address: Hygieneinstitut der Universität Würzburg, 97070 Würzburg, Germany.

Journal of Clinical Microbiology, May 2002, p. 1644-1647, Vol. 40, No. 5
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.5.1644-1647.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Martin-Davila, P., Fortun, J., Lopez-Velez, R., Norman, F., Montes de Oca, M., Zamarron, P., Gonzalez, M. I., Moreno, A., Pumarola, T., Garrido, G., Candela, A., Moreno, S. (2008). Transmission of Tropical and Geographically Restricted Infections during Solid-Organ Transplantation. Clin. Microbiol. Rev. 21: 60-96 [Abstract] [Full Text]  
  • Cloud, J. L., Bauman, S. K., Pelfrey, J. M., Ashwood, E. R., Wheat, L. J., LeMonte, A. (2007). Biased Report on the IMMY ALPHA Histoplasma Antigen Enzyme Immunoassay for Diagnosis of Histoplasmosis. CVI 14: 1389-1391 [Full Text]  
  • Bialek, R, Konrad, F, Kern, J, Aepinus, C, Cecenas, L, Gonzalez, G M, Just-Nubling, G, Willinger, B, Presterl, E, Lass-Florl, C, Rickerts, V (2005). PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue. J. Clin. Pathol. 58: 1180-1184 [Abstract] [Full Text]  
  • de Aguirre, L., Hurst, S. F., Choi, J. S., Shin, J. H., Hinrikson, H. P., Morrison, C. J. (2004). Rapid Differentiation of Aspergillus Species from Other Medically Important Opportunistic Molds and Yeasts by PCR-Enzyme Immunoassay. J. Clin. Microbiol. 42: 3495-3504 [Abstract] [Full Text]  
  • Ren, P., Sridhar, S., Chaturvedi, V. (2004). Use of Paraffin-Embedded Tissue for Identification of Saccharomyces cerevisiae in a Baker's Lung Nodule by Fungal PCR and Nucleotide Sequencing. J. Clin. Microbiol. 42: 2840-2842 [Abstract] [Full Text]  
  • Bialek, R., Kern, J., Herrmann, T., Tijerina, R., Cecenas, L., Reischl, U., Gonzalez, G. M. (2004). PCR Assays for Identification of Coccidioides posadasii Based on the Nucleotide Sequence of the Antigen 2/Proline-Rich Antigen. J. Clin. Microbiol. 42: 778-783 [Abstract] [Full Text]  
  • Martagon-Villamil, J., Shrestha, N., Sholtis, M., Isada, C. M., Hall, G. S., Bryne, T., Lodge, B. A., Reller, L. B., Procop, G. W. (2003). Identification of Histoplasmacapsulatum from Culture Extracts by Real-Time PCR. J. Clin. Microbiol. 41: 1295-1298 [Abstract] [Full Text]  
  • Guedes, H. L. d. M., Guimaraes, A. J., Muniz, M. d. M., Pizzini, C. V., Hamilton, A. J., Peralta, J. M., Deepe, G. S. Jr., Zancope-Oliveira, R. M. (2003). PCR Assay for Identification of Histoplasma capsulatum Based on the Nucleotide Sequence of the M Antigen. J. Clin. Microbiol. 41: 535-539 [Abstract] [Full Text]  
  • Schofield, D. A., Westwater, C., Paulling, E. E., Nicholas, P. J., Balish, E. (2003). Detection of Candida albicans mRNA from Formalin-Fixed, Paraffin-Embedded Mouse Tissues by Nested Reverse Transcription-PCR. J. Clin. Microbiol. 41: 831-834 [Abstract] [Full Text]  
  • Bialek, R., Cascante Cirera, A., Herrmann, T., Aepinus, C., Shearn-Bochsler, V. I., Legendre, A. M. (2003). Nested PCR Assays for Detection of Blastomyces dermatitidis DNA in Paraffin-Embedded Canine Tissue. J. Clin. Microbiol. 41: 205-208 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»