In situ identification of mycobacteria in Crohn's disease patient tissue using confocal scanning laser microscopy

Naser, Saleh A.; Shafran, Ira; Schwartz, D.; El-Zaatari, F. A. K.; Biggerstaff, John

doi:10.1006/mcpr.2001.0395

Cited by 42 publications

(30 citation statements)

References 36 publications

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“…Seiler and colleagues (18) used a polyclonal anti-M. bovis Bacille Calmette-Guerin serum for detection of cell wall-deficient M. tuberculosis in mouse tissue. Naser and colleagues (13) demonstrated M. avium subsp. paratuberculosis in tissue specimens from patients with Crohn's disease with a polyclonal antibody.…”

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confidence: 99%

Evaluation of Peptide Nucleic Acid-Fluorescence In Situ Hybridization for Identification of Clinically Relevant Mycobacteria in Clinical Specimens and Tissue Sections

et al. 2006

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With fluorescently labeled PNA (peptide nucleic acid) probes targeting 16S rRNA, we established a 3-h fluorescence in situ hybridization (FISH) procedure for specific visualization of members of the Mycobacterium tuberculosis complex, M. leprae, M. avium, and M. kansasii. Probe specificity was tested against a panel of 25 Mycobacterium spp. and 10 gram-positive organisms. After validation, probes were used to identify 52 mycobacterial culture isolates. Results were compared to conventional genotypic identification with amplificationbased methods. All isolates (M. tuberculosis complex, n ‫؍‬ 24; M. avium, n ‫؍‬ 7; M. kansasii, n ‫؍‬ 1) were correctly identified by FISH. In addition, the technique was used successfully for visualization of mycobacteria in biopsies from infected humans or animals. In conclusion, PNA-FISH is a fast and accurate tool for speciesspecific identification of culture-grown mycobacteria and for direct visualization of these organisms in tissue sections. It may be used successfully for both research and clinical microbiology.

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confidence: 99%

Evaluation of Peptide Nucleic Acid-Fluorescence In Situ Hybridization for Identification of Clinically Relevant Mycobacteria in Clinical Specimens and Tissue Sections

et al. 2006

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“…In this study IS6110 oligonucleotides DNA and PNA of 20 bp labeled with 6-carboxyfluorescein were built, following the same sequence as those used in the PCR test described by Lefmann et al [34] and Naser et al [35]. These oligonucleotides showed high specificity and sensitivity [44,45,48,49].…”

Section: Discussionmentioning

confidence: 99%

“…ISH is improved with the use of fluorescence (FISH) for both DNA or RNA probes [27,30,31,32,33,34,35,36,37]. However, conventional oligonucleotides (100 to 500 bp) used for DNA ISH or FISH have penetration problems because of the nature of the mycobacterial cellular surface, which shows low permeability for these molecules [11,27,28,38].…”

Section: Introductionmentioning

confidence: 99%

Mycobacterium tuberculosis complex detected by modified fluorescent in situ hybridization in lymph nodes of clinical samples

Rodriguez-Nuñez

Avelar

Márquez³

et al. 2011

J Infect Dev Ctries

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Introduction: Lymph node tuberculosis (TB) is the leading cause of extrapulmonary tuberculosis and is the most frequently identified type in Aguascalientes, Mexico. Conventional diagnosis has serious limitations for rapid detection of extrapulmonary tuberculosis in clinical samples. Here PCR and modified FISH have been tested as complementary diagnosis methods for extrapulmonary tuberculosis. Methodology: The specific insertion sequence IS6110 for Mycobacterium tuberculosis complex was used to perform PCR and build DNA and PNA FISH probes (20bp). PCR and modified DNA and PNA FISH assays were performed to evaluate 41 lymph node paraffin-embedded tissue samples, in comparison with the histopathology diagnosis, which was considered the gold standard (22 positive and 19 negative). Results: In comparison with histopathology diagnosis PCR showed 62.5 % sensitivity and 77.8 % specificity (χ 2 = 4.583 p< 0.05). Modified DNA FISH showed 71.4% sensitivity and 84.6% specificity (χ 2 = 11.21 p< 0.05). PNA FISH showed 66.7% sensitivity and 60.0% specificity (χ 2 = 2.93 p> 0.05). Ziehl Neelsen stain was positive in only four cases of 22 lymph node samples positive to histopathology. In contrast, PCR and modified DNA FISH were positive in 20 cases of the same group. The negative cases were coincident in all tests. Conclusions: PCR and DNA FISH showed a significant increase in the number of cases detected and also showed higher sensitivity and specificity compared with data reported by traditional methodology. In developing countries, these techniques could help to complement the early diagnosis and timely treatment of extrapulmonary tuberculosis.

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“…There has been a flurry of publications since then relating CD and MAP. [13][14][15][16][17][18][19][20][21][22][23][24][25] MAP infection in humans is difficult to detect. The organisms are intracellular and minimize their own immune recognition.…”

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In the search of a cause of Crohn’s disease

Makharia

Singh

2010

Indian J Gastroenterol

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While the exact cause of Crohn's disease (CD) is not known, it is thought that chronic inflammation in CD results from an inappropriate and chronic activation of the innate and adaptive mucosal immune systems in a genetically susceptible host, and that enteric microflorae play a pivotal role in the initiation and maintenance of the disease. 1 Organisms such as Pseudomonas maltophilia, Mycobacterium kansasii, Chlamydia trachomatis, Bacteroides fragilis, Listeria monocytogenes, Escherichia coli and Mycobacterium avium subspecies paratuberculosis (MAP) have been implicated and identified in the intestines of patients with CD; however, no other putative pathogenic organism except MAP causes a chronic granulomatous inflammation of the intestines in every other species in which it is present. 2 Moreover, because of remarkable clinical, morphological and epidemiological similarity between intestinal tuberculosis (caused by Mycobacterium tuberculosis) and CD in humans and Johne's disease (caused by Mycobacterium avium paratuberculosis) in cattle, a mycobacterial pathogen is thought to be a causative organism of CD. [3][4][5][6][7] An association and causality of Helicobacter pylori with peptic ulcer disease revolutionized the treatment of peptic ulcers; similar implication of MAP as a cause of CD has raised hopes amongst physicians, microbiologists and basic scientists for the cure of this disease. 8 In the first report almost 25 years back, Chiodini and colleagues isolated cell-wall deficient cells, called spheroplasts, from tissue samples after several months of incubation from patients with CD. These spheroplasts were sub-cultured and they later developed a cell wall that stained positively with Ziehl-Neelson staining; they were classified as mycobacterial like organisms. 9-11 These isolates were later confirmed as M. avium paratuberculosis by DNA hybridization. The unusual nature of the cell walldeficient MAP and the challenges in culture of MAP with its fastidious and slow growing requiring months to years to culture and multiple studies reporting failure to culture MAP from patients with CD dampened the initial zeal. 12 There was then a period of silence in the 1990s. With availability of better culture techniques and use of molecular techniques to identify MAP in 2000s, there was resurgence of interest of many laboratories in this field. There has been a flurry of publications since then relating CD and MAP. [13][14][15][16][17][18][19][20][21][22][23][24][25] MAP infection in humans is difficult to detect. The organisms are intracellular and minimize their own immune recognition. They are therefore difficult to isolate and propagate in culture and are relatively resistant to chemical and enzymatic lysis. Reliable access to their DNA is only achieved during sample processing by combining exposure to stringent lysis buffers with an additional optimised mechanical disruption step. Freezing samples and tissue extracts especially at -20°C substantially reduces the PCR detection rate of their GC-rich DNA. The identifi...

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In situ identification of mycobacteria in Crohn's disease patient tissue using confocal scanning laser microscopy

Cited by 42 publications

References 36 publications

Evaluation of Peptide Nucleic Acid-Fluorescence In Situ Hybridization for Identification of Clinically Relevant Mycobacteria in Clinical Specimens and Tissue Sections

Evaluation of Peptide Nucleic Acid-Fluorescence In Situ Hybridization for Identification of Clinically Relevant Mycobacteria in Clinical Specimens and Tissue Sections

Mycobacterium tuberculosis complex detected by modified fluorescent in situ hybridization in lymph nodes of clinical samples

In the search of a cause of Crohn’s disease

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