title: In situ hybridization to detect and identify Burkholderia pseudomallei in human melioidosis.
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.
authors.affiliation: Department of Pathology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Lin Chuan
authors.initials: LC
authors.lastname: Eu
authors.affiliation: Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Kien Chai
authors.initials: KC
authors.lastname: Ong
authors.affiliation: Forensic Department, Queen Elizabeth Hospital, Sabah, Malaysia.
authors.forename: Jessie
authors.initials: J
authors.lastname: Hiu
authors.affiliation: Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Jamunarani
authors.initials: J
authors.lastname: Vadivelu
authors.affiliation: School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia.
authors.forename: Sheila
authors.initials: S
authors.lastname: Nathan
authors.affiliation: Department of Pathology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Kum Thong
authors.initials: KT
authors.lastname: Wong
mesh_terms: D000818:Animals; D016957:Burkholderia pseudomallei / Q000302:isolation & purification*; D006801:Humans; D017403:In Situ Hybridization; D008554:Melioidosis / Q000175:diagnosis* / Q000473:pathology; D051379:Mice; D012680:Sensitivity and Specificity