In situ hybridisation for the identification of Helicobacter pylori in paraffin wax embedded tissue.

Bashir, Muiez; Lewis, F A; Quirke, Philip; Lee, A; Dixon, M. F.

doi:10.1136/jcp.47.9.862

Cited by 20 publications

(9 citation statements)

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“…In situ hybridization (ISH) using digoxigenin (DIG)-labeled oligonucleotide probes complementary to unique target sites on the 16S rRNA (rDNA) is a useful method for the detection and identification of microorganisms (Bashir et al 1994, Barrett et al 1997, Kwon & Chae 1999. Specific probes can be designed to locate organisms that cannot be cultured (Hayashi et al 1990, Komminoth & Werner 1997 or visualized by conventional methods, such as viruses (Lewis & Wells 1992), Chlamydiae (Campbell et al 1993) and Mycobacterium paratuberculosis (Hulten et al 2000).…”

Section: Introductionmentioning

confidence: 99%

PCR and in situ hybridization for the detection and localization of a new pathogen Francisella-like bacterium (FLB) in ornamental cichlids

Hsieh¹,

Wu²,

Tung³

et al. 2007

Dis. Aquat. Org.

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Archived formalin-fixed, paraffin-embedded tissues from 28 diseased ornamental cichlid fish associated with visceral granulomas were examined by polymerase chain reaction (PCR) and in situ hybridization (ISH) for detection of Francisella-like bacteria (FLB). The 16S rDNA FLB-specific primer pair 180f/465r was used on naturally infected ornamental cichlids, resulting in 11 positive cases (39%). Using DNA probes, all 28 cases (100%) showed a positive reaction, and most labeled cells were observed in the visceral granulomas of infected individuals. FLB was detected in cells morphologically resembling epithelioid and endothelioid macrophages. ISH was more sensitive than PCR or routine histopathological examination, based on the examination of archived formalin-fixed, paraffin-embedded tissues in this study. Furthermore, this technique located a new fish pathogen, FLB, in ornamental cichlids. The causative agent was similar to the pathogen inducing systemic granulomas in tilapia. KEY WORDS: Francisella-like bacterium · FLB · PCR · In situ hybridization · Cichlid · Ornamental fish Resale or republication not permitted without written consent of the publisherDis Aquat Org 75: [29][30][31][32][33][34][35][36] 2007 Disease Diagnostic Center [STAADDC] and collected between 1998 and 2002) of ornamental fish with visceral granulomas for investigation of the causative agent. A DIG-labeled DNA probe specific for a novel FLB partial 16S rRNA gene (base pairs 180 to 485) was developed for localization of the agent in the infected tissues of ornamental fish. This is the first report concerning granulomatous disease in ornamental fish due to infection with FLB, and comparing the sensitivities of ISH, PCR and histopathological assays. MATERIALS AND METHODSSampling. Twenty-eight diseased cichlid ornamental fish, consisting of 11 species: firebird Aulonocara rubescens, elegans Pseudotropheus elegans, zebra Pseudotropheus zebra, Rhodes's chilo Chilotilapia rhoadesii, Malawi eyebiter Dimidiochromis compressiceps, brown discus Symphysodon aequifasciatus, deep-water hap Haplochromis electra, electric blue hap Sciaenochromis fryeri, blue-white labido Labidochromis caeruleus, Placidochromis milomo and Frontosa cichlid Cyphotilapia frontosa were used as samples for study by PCR and ISH (Table 1). The disease had been tentatively diagnosed as FLB infection by cytological and histopathological examinations. Samples from 3 FLBfree firebird A. rubescens, as diagnosed by PCR and ISH, were selected as negative controls.Tissue processing. Brain, eye, gill arch, kidney, spleen, liver, heart and gastrointestinal (GI) tract were removed and cut into small pieces of ~5 mm 3 , and placed into neutral buffered formalin solution (20:1 [v/v] ratio of fixative to tissue) for 16 h. The fixed samples were dehydrated through an alcohol series and embedded in paraffin according to standard laboratory procedures. Serial sections 4 µm thick were cut, floated on the surface of a water-bath and mounted on positively charged slides (Muto Pure Chemic...

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Section: Introductionmentioning

confidence: 99%

PCR and in situ hybridization for the detection and localization of a new pathogen Francisella-like bacterium (FLB) in ornamental cichlids

Hsieh¹,

Wu²,

Tung³

et al. 2007

Dis. Aquat. Org.

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“…We believe the ability to visualize bacilli with this technique could reduce the likelihood of false positive results inherent in other molecular assays. In situ hybridization has been successfully applied to detect various bacteria, including Helicobacter pylori, 12 Haemophilus influenza, 13 and Klebsiella pneumoniae. 14 Recently, Hagen et al 15 reported a fluorescence in situ hybridization technique using a set of oligonucleotide probes for the identification of B. pseudomallei, B. mallei, and B. thailandensis in bacterial cell smears and experimentally infected mouse tissues.…”

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In situ hybridization to detect and identify Burkholderia pseudomallei in human melioidosis

Ong

Hiu

et al. 2014

Modern Pathology

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Burkholderia pseudomallei causes a potentially fatal infection called melioidosis. We have developed a nonfluorescent, colorimetric in situ hybridization assay using a specific probe to target 16s rRNA of B. pseudomallei in formalin-fixed, paraffin-embedded infected tissues for diagnostic purposes and to study infectious disease pathology. A 63-base pair DNA probe was synthesized and labeled with digoxigenin by PCR. Probe specificity was confirmed by BLAST analysis and by testing on appropriate microbial controls. The in situ hybridization assay was specifically and consistently positive for B. pseudomallei, showing strongly and crisply stained, single bacillus and bacilli clusters in mainly inflamed tissues in seven human acute melioidosis cases and experimentally infected mouse tissues. Intravascular and extravascular bacilli were detected in both intracellular and extracellular locations in various human organs, including lung, spleen, kidney, liver, bone marrow, and aortic mycotic aneurysm, particularly in the inflamed areas. Intravascular, intracellular bacteria in melioidosis have not been previously reported. Although the identity of infected intravascular leukocytes has to be confirmed, extravascular, intracellular bacilli appear to be found mainly within macrophages and neutrophils. Rarely, large intravascular, extracellular bacillary clusters/emboli could be detected in both human and mouse tissues. B. cepacia and non-Burkholderia pathogens (16 microbial species) all tested negative. Nonpathogenic B. thailandensis showed some cross-hybridization but signals were less intense. This in situ hybridization assay could be usefully adapted for B. pseudomallei identification in other clinical specimens such as pus and sputum.

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“…13,48 A sensitivity of 77% to 99% has been reported for the detection of H. pylori by histology. 15,23,28,30,37,45,49 Auxiliary methods, including immunohistochemistry, 2,18,30 in situ hybridization 5,22 and the polymerase chain reaction (PCR), 2,9,46,49 have been applied to improve the sensitivity to detect H. pylori in biopsy material. 47 Besides the direct visualization of the bacteria, a characteristic pattern of inflammation including neutrophils, mononuclear cells, as well as lymphoid follicles can be observed by histology; albeit only the detection of the bacteria unequivocally proves the diagnosis.…”

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Increased Rate of Helicobacter pylori Infection Detected by PCR in Biopsies With Chronic Gastritis

Zsikla¹,

Hailemariam²,

Baumann³

et al. 2006

American Journal of Surgical Pathology

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Histology is considered a sensitive method for detection of Helicobacter pylori, in gastric biopsies. We investigated the diagnostic potential of qualitative nested (nPCR) and quantitative PCR (qPCR) for detection of H. pylori using different primers on 126 archived gastric biopsies with inflammation and correlated the inflammatory changes with the presence and density of bacteria. H. pylori was detected in 42.8% biopsies by histology and PCR, an additional 15 samples were positive exclusively by PCR: nPCR was positive in all histologically positive samples, but qPCR failed to detect H. pylori in 10 biopsies. The inflammatory score was significantly higher in biopsies positive for H. pylori only by PCR showed a significant higher inflammatory score compared with negative biopsies (mean of neutrophils score, 1.60 vs. 0.90, P < 0.01; mean of mononuclear cells score, 2.27 vs. 1.67, P < 0.01), whereas the inflammatory score was similar compared with biopsies positive for H. pylori by histology (mean of neutrophils score, 1.60 vs. 1.56, not significant; mean of mononuclear cells score, 2.27 vs. 2.20, not significant). A weak correlation between inflammatory score and the density of H. pylori detected by histology was observed. The mean values of H. pylori DNA were significantly higher in histologic-positive than in histologic negative biopsies. We have shown that PCR can detect H. pylori in about 20% of histologic-negative gastric biopsies, indicating the clinical relevance of H. pylori detection by PCR in biopsies with characteristic inflammatory changes.

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In situ hybridisation for the identification of Helicobacter pylori in paraffin wax embedded tissue.

Cited by 20 publications

References 8 publications

PCR and in situ hybridization for the detection and localization of a new pathogen Francisella-like bacterium (FLB) in ornamental cichlids

PCR and in situ hybridization for the detection and localization of a new pathogen Francisella-like bacterium (FLB) in ornamental cichlids

In situ hybridization to detect and identify Burkholderia pseudomallei in human melioidosis

Increased Rate of Helicobacter pylori Infection Detected by PCR in Biopsies With Chronic Gastritis

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