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Journal of Clinical Microbiology, August 2002, p. 3112-3113, Vol. 40, No. 8
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.8.3112-3113.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Positive IS900 In Situ Hybridization Signals as Evidence for Role of Mycobacterium avium subsp. paratuberculosis in Etiology of Crohn's Disease


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LETTER
 
A long-term scientific debate questions the possible role of Mycobacterium avium subsp. paratuberculosis in the etiology of Crohn's disease (CD). The article of Sechi et al. (5) describes the detection of these mycobacteria by an in situ hybridization (ISH) technique using IS900 as a probe. Most intestinal biopsy specimens taken from CD patients were found positive by paraffin section (82% of the patients), whereas the positive hybridization signal was not observed in any of the biopsy specimens from non-CD patients tested. However, the IS900-targeted PCR and Ziehl-Neelsen staining of material from CD- and non-CD patients invariably yielded a negative result. These discrepant results led the authors to the interpretation that cellular forms of Mycobacterium avium subsp. paratuberculosis are highly prevalent in intestinal tissues of CD patients, but not in those of non-CD patients.

We have some serious doubts regarding these conclusions.

First, it is hard to imagine that the IS900 ISH results shown in the figures of the article in question can be interpreted as truly positive. Staining patterns for infectious diseases should be interpreted in the histopathological context of infected cells. For Mycobacterium avium subsp. paratuberculosis, an intracellular bacterium, this means an intracytoplasmic granular staining pattern of infected immune cells in which the cell nucleus is still visible (1). In contrast to this staining pattern, the phosphatase reaction deposit in the representative figure on IS900 hybridization in the Sechi study is a large rounded dot that exceeded the size of a single cell. Such deposits are typical in sections with (little) cracks as can be observed in these illustrations. Hybridization of other types of intracellular bacteria, such as Chlamydia pneumoniae, has been described as granula restricted to the cytoplasm (2, 3).

Second, if the authors' interpretation regarding the high rate of positive IS900 hybridization signals is correct, a large amount of genomic DNA should be present given the enormous strong signal of this staining, and hence, at least some of the IS900-targeted PCRs should have been positive. However, the authors did not find any positive IS900 PCR results. It is not likely that their assumption of a low sensitivity of the IS900-targeted PCR due to the occurrence of nicks in the DNA caused by formalin fixation is correct. At least a control PCR assay on the integrity of the DNA should have been included to confirm this possible artifact, which was attributed to the pretreatment of the material. Such a control, for instance, one of the human housekeeping genes (4), would also provide evidence for the presence or absence of PCR inhibitors. The conclusions drawn on the basis of the described IS900 PCR protocol are therefore not justified.

With this single unconfirmed and questionable observation, we believe that the IS900 ISH results in this study should be interpreted with great care and, consequently, do not contribute to the answer of the important scientific questions related to the possible involvement of (cell wall-deficient) Mycobacterium avium subsp. paratuberculosis in the etiology of CD. Multidisciplinary studies are needed with congruous and conclusive results to examine the true nature of the etiology of CD.


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REFERENCES
 
    1
  1. Dowell, S. F., R. W. Peeling, J. Boman, G. M. Carlone, B. S. Fields, J. Guarner, M. R. Hammerschlag, L. A. Jackson, C. Kuo, M. Maass, T. O. Messmer, D. F. Talkington, M. L. Tondella, S. R. Zaki, and the C. pneumoniae Workshop Participants. 2001. Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada). Clin. Infect. Dis. 33:492-502.[CrossRef][Medline]
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  2. Meijer, A., P. J. M. Roholl, S. K. Gielis-Proper, Y. F. Meulenberg, and J. M. Ossewaarde. 2000. Chlamydia pneumoniae in vitro and in vivo: a critical evaluation of in situ detection methods. J. Clin. Pathol. 53:904-910.[Abstract/Free Full Text]
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  3. Meijer, A., P. J. M. Roholl, S. K. Gielis-Proper, and J. M. Ossewaarde. 2000. Chlamydia pneumoniae antigen and fragmented DNA in macrophages in atherosclerotic lesions. J. Clin. Pathol. 53:911-916.[Abstract/Free Full Text]
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  4. Meijer, A., A. Van der Vliet, P. J. M. Roholl, S. K. Gielis-Proper, A. De Vries, and J. M. Ossewaarde. 1999. Chlamydia pneumoniae in abdominal aneurysms. Abundance of membrane components in the absence of heat shock protein 60 and DNA. Arterioscler. Thromb. Vasc. Biol. 19:2680-2686.[Abstract/Free Full Text]
    5. 5
  5. Sechi, L. A., M. Mura, F. Tanda, A. Lissia, A. Solinas, G. Fadda, and S. Zanetti. 2001. Identification of Mycobacterium avium subsp. paratuberculosis in biopsy specimens from patients with Crohn's disease identified by in situ hybridization. J. Clin. Microbiol. 39:4514-4517.[Abstract/Free Full Text]
Paul J. M. Roholl*
Laboratory of Pathology and Immunobiology

Arnold Herrewegh
Dick van Soolingen
Laboratory of Infectious Diseases and Perinatal Screening, National Institute of Public Health and the Environment, P.O. Box 1, 3720BA Bilthoven, The Netherlands

* E-mail: p.roholl{at}rivm.nl.


Authors' Reply


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LETTER 
 
We are grateful for the interest shown by Drs. Roholl, Herrewegh, and van Soolingen in our recent article in this journal. In our study (4), we used the in situ hybridization technique with IS900 as a probe for the detection of Mycobacterium avium subsp. paratuberculosis DNA in intestinal tissue samples from patients who had Crohn's disease and patients who did not have Crohn's disease. We found that 72% of the tissue samples from Crohn's disease patients gave positive results, whereas none of the samples from patients who did not have Crohn's disease hybridized with the probe used. Ziehl-Neelsen staining and IS900 PCR on the same samples gave negative results.

Regarding the first point of Dr. Roholl et al., on the interpretation of the staining patterns, what he states is true (intracellular bacteria have an intracytoplasmic granular staining pattern), but we would like to observe that, in other works, a pattern similar to what we observed was reported (3); Hulten et al. stated that "spheroplasts are present both as single organisms or in large fluorescent aggregates" and that "These sections showed dark purple dots or larger purple aggregates corresponding to single spheroplast or groups of several organisms, respectively." That is, the intracellular bacterium Chlamydia pneumoniae, to which Dr. Roholl refers, has a different pattern inside cells, whereas mycobacteria grow as aggregates due to the complexity of their cell wall. Moreover, the presence of spheroplast forms of mycobacteria could be an explanation of the negative results with acid-fast mycobacterial staining.

Indeed, we obtained several examples of positive hybridization in samples from Crohn's patients, and although in one of the published pictures (4), a little crack can be observed, there are other examples of positive hybridization with no cracks at all and hybridization "as granula restricted to the cytoplasm," as shown in Fig. 1.



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  		FIG. 1. Example of a IS900-positive hybridization within an ileum tissue sample of a Crohn's disease patient.

Dr. Roholl did not mention that, for a control, we used another probe, IS6110, which is specific for the Mycobacterium tuberculosis complex, against the same tissue samples. We did not observe hybridization when this probe was used, and again we did not obtain hybridization with other tissue samples from patients who did not have Crohn's disease.

Regarding the second point of discussion, PCR negativity, we increased the number of patients studied up to 100 patients (analyzing 108 samples [unpublished data]). We found 7% of the tissue samples gave positive results by IS900 PCR analysis. The explanation that we used in interpreting the results (degradation of DNA in paraffin-embedded tissue samples) is accepted in several works (1). The control proposed by Dr. Roholl would provide evidence for the presence of PCR inhibitors, but it would not be definitive. The major problem with our PCR was the length of the fragment amplified. To amplify an internal region of IS900, we used the same primers used to prepare the probe. The amplified fragment is larger than 100 bp, which is the maximum size recommended in this type of experiments. Discrepancies, experimental difficulties, and laboratory-to-laboratory variation have plagued PCR detection of M. paratuberculosis (2). We wanted to add new techniques to search for the presence of this microorganism in the intestinal tissues of Crohn's disease patients.


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REFERENCES 
 
    1
  1. El-Zaatari, F. A. K., M. S. Osato, and D. Y. Graham. 2001. Etiology of Crohn's disease: the role of Mycobacterium avium paratuberculosis. Trends Mol. Med. 7:247-252.[CrossRef][Medline]
    2. 2
  2. Hermon-Taylor, J., T. J. Bull, J. M. Sheridan, J. Cheng, M. L. Stellakis, and N. Sumar. 2000. Causation of Crohn's disease by Mycobacterium avium subspecies paratuberculosis. Can. J. Gastroenterol. 14:521-539.[Medline]
    3. 3
  3. Hulten, K., T. J. Karttunen, H. M. T. El-Zimaity, S. A. Naser, A. Almashhrawi, D. Y. Graham, and F. A. K. El-Zaatari. 2000. In situ hybridization method for studies of cell wall deficient M. paratuberculosis in tissue samples. Vet. Microbiol. 77:513-518.[CrossRef][Medline]
    4. 4
  4. Sechi, L. A., M. Mura, F. Tanda, A. Lissia, A. Solinas, G. Fadda, and S. Zanetti. 2001. Identification of Mycobacterium avium subsp. paratuberculosis in biopsy specimens from patients with Crohn's disease identified by in situ hybridization. J. Clin. Microbiol. 39:4514-4517.
L. A. Sechi
M. Mura
S. Zanetti
Dipartimento di Scienze Biomediche, Sezione di Microbiologia, Università di Sassari, 07100 Sassari, Italy

F. Tanda
A. Lissia
Istituto di Anatomia e Istologia Patologica, Università degli Studi di Sassari, Viale S. Pietro, 07100 Sassari, Italy

G. Fadda
Istituto di Microbiologia, Facoltà di Medicina e Chirurgia "Agostino Gemelli", Università Cattolica del Sacro Cuore, 00168 Rome, Italy

Journal of Clinical Microbiology, August 2002, p. 3112-3113, Vol. 40, No. 8
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.8.3112-3113.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Amand, A. L. St., Frank, D. N., De Groote, M. A., Pace, N. R. (2005). Use of Specific rRNA Oligonucleotide Probes for Microscopic Detection of Mycobacterium avium Complex Organisms in Tissue. J. Clin. Microbiol. 43: 1505-1514 [Abstract] [Full Text]  

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