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 Marchetti, G.
Right arrow Articles by Franzetti, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Marchetti, G.
Right arrow Articles by Franzetti, F.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, June 1998, p. 1512-1517, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Evaluation of PCR in Detection of Mycobacterium tuberculosis from Formalin-Fixed, Paraffin-Embedded Tissues: Comparison of Four Amplification Assays

Giulia Marchetti,1,* Andrea Gori,1 Lidia Catozzi,1 Luca Vago,2 Manuela Nebuloni,2 M. Cristina Rossi,1 Anna Degli Esposti,1 Alessandra Bandera,1 and Fabio Franzetti1

Clinic of Infectious Diseases1 and Pathology Unit,2 "Luigi Sacco" Hospital, University of Milan, Milan, Italy

Received 24 September 1997/Returned for modification 18 December 1997/Accepted 4 March 1998

We compared the sensitivities and specificities of four nested PCR assays for the detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues. Thirty-seven autopsy samples from human immunodeficiency virus-positive patients were analyzed: 15 were M. tuberculosis positive, 11 served as negative controls, and 11 were Ziehl-Neelsen positive without cultural confirmation of M. tuberculosis. Three genomic sequences (mtp40, 65-kDa antigen gene, and IS6110) with different molecular masses and numbers of repetitions within the M. tuberculosis genome were targeted. On the IS6110 sequence, two fragments of different sizes (106 and 123 bp, respectively) were amplified with two separate pairs of primers. The highest sensitivity rates were obtained by amplifying the highly repetitive IS6110 insertion sequence, and the different primers tested showed a sensitivity ranging from 80 to 87%. Amplification of the large 223-bp fragment of the mtp40 sequence present in a single copy in the M. tuberculosis genome yielded a high rate of false-negative results, ranging from 66 to 80%. A poor sensitivity (from 47 to 60%) was also shown by PCR amplification of the 142-bp 65-kDa antigen gene. All the PCRs except that for the 65-kDa antigen gene showed a specificity of 100%. Moreover, different results were obtained with different dilutions of DNA, and DNA concentrations of 1 and 3 µg yielded the highest sensitivities depending upon which protocol was used. Application of the PCRs to the Ziehl-Neelsen-positive, culture-negative samples confirmed the sensitivities of the PCRs obtained with the control samples. In conclusion, PCR can successfully be used to detect M. tuberculosis from paraffin-embedded tissues and can be particularly useful in the validation of a diagnosis of tuberculosis in clinical settings in which the diagnosis is uncertain. However, the efficacy of PCR strictly depends on several amplification parameters such as DNA concentration, target DNA size, and the repetitiveness of the amplified sequence.

* Corresponding author. Mailing address: Clinic of Infectious Diseases, "Luigi Sacco" Hospital, University of Milan, Via G.B. Grassi, 74, 20157 Milan, Italy. Phone: 39 2 35799677. Fax: 39 2 3560805. E-mail: a.gori{at}imiucca.csi.unimi.it.

Journal of Clinical Microbiology, June 1998, p. 1512-1517, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Nopvichai, C, Sanpavat, A, Sawatdee, R, Assanasen, T, Wacharapluesadee, S, Thorner, P S, Shuangshoti, S (2009). PCR detection of Mycobacterium tuberculosis in necrotising non-granulomatous lymphadenitis using formalin-fixed paraffin-embedded tissue: a study in Thai patients. J. Clin. Pathol. 62: 812-815 [Abstract] [Full Text]  
  • Ortega, N., Navarro, J. A., Nicolas, L., Buendia, A. J., Caro, M. R., Del Rio, L., Martinez, C. M., Cuello, F., Salinas, J., Gallego, M. C. (2007). Evaluation of Chlamydophila abortus DNA extraction protocols for polymerase chain reaction diagnosis in paraffin-embedded tissues. jvdi 19: 421-425 [Abstract] [Full Text]  
  • Zink, A R, Nerlich, A G (2004). Molecular strain identification of the Mycobacterium tuberculosis complex in archival tissue samples. J. Clin. Pathol. 57: 1185-1192 [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]  
  • Brown-Elliott, B. A., Wallace, R. J. Jr. (2002). Clinical and Taxonomic Status of Pathogenic Nonpigmented or Late-Pigmenting Rapidly Growing Mycobacteria. Clin. Microbiol. Rev. 15: 716-746 [Abstract] [Full Text]  
  • Fukunaga, H., Murakami, T., Gondo, T., Sugi, K., Ishihara, T. (2002). Sensitivity of Acid-Fast Staining for Mycobacterium tuberculosis in Formalin-fixed Tissue. Am. J. Respir. Crit. Care Med. 166: 994-997 [Abstract] [Full Text]  
  • ZINK, A., HAAS, C. J., REISCHL, U., SZEIMIES, U., NERLICH, A. G. (2001). Molecular analysis of skeletal tuberculosis in an ancient Egyptian population. J Med Microbiol 50: 355-366 [Abstract] [Full Text]  
  • Ruiz-Manzano, J., Manterola, J.-M., Gamboa, F., Calatrava, A., Monso, E., Martinez, C., Ausina, V. (2000). Detection of Mycobacterium tuberculosis in Paraffin-Embedded Pleural Biopsy Specimens by Commercial Ribosomal RNA and DNA Amplification Kits. Chest 118: 648-655 [Abstract] [Full Text]  
  • Rossi, M. C., Gori, A., Zehender, G., Marchetti, G., Ferrario, G., De Maddalena, C., Catozzi, L., Bandera, A., Esposti, A. D., Franzetti, F. (2000). A PCR-Colorimetric Microwell Plate Hybridization Assay for Detection of Mycobacterium tuberculosis and M. avium from Culture Samples and Ziehl-Neelsen-Positive Smears. J. Clin. Microbiol. 38: 1772-1776 [Abstract] [Full Text]  
  • Lew, M. A., Graeme-Cook, F. M. (1999). Case 27-1999- An 82-Year-Old Woman with Numerous Chronic Disorders and a Recent Febrile Illness. NEJM 341: 827-834 [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»