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Mod Pathol. 2014 May;27(5):657-64. doi: 10.1038/modpathol.2013.184. Epub 2013 Nov 1.

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

Author information

1
Department of Pathology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
2
Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
3
Forensic Department, Queen Elizabeth Hospital, Sabah, Malaysia.
4
Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
5
School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia.

Abstract

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.

PMID:
24186135
DOI:
10.1038/modpathol.2013.184
[Indexed for MEDLINE]
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