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

Lin Chuan Eu, Kien Chai Ong, Jessie Hiu, Jamunarani Vadivelu, Sheila Nathan, Kum Thong Wong

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)657-664
Number of pages8
JournalModern Pathology
Volume27
Issue number5
DOIs
Publication statusPublished - 2014

Fingerprint

Melioidosis
Burkholderia pseudomallei
Bacillus
In Situ Hybridization
Infected Aneurysm
Burkholderia cepacia
Digoxigenin
Suppuration
Aortic Aneurysm
DNA Probes
Embolism
Sputum
Base Pairing
Paraffin
Formaldehyde
Communicable Diseases
Neutrophils
Leukocytes
Spleen
Bone Marrow

Keywords

  • 16s rRNA
  • Burkholderia pseudomallei
  • DNA probe
  • in situ hybridization
  • melioidosis

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Medicine(all)

Cite this

In situ hybridization to detect and identify Burkholderia pseudomallei in human melioidosis. / Eu, Lin Chuan; Ong, Kien Chai; Hiu, Jessie; Vadivelu, Jamunarani; Nathan, Sheila; Wong, Kum Thong.

In: Modern Pathology, Vol. 27, No. 5, 2014, p. 657-664.

Research output: Contribution to journalArticle

Eu, Lin Chuan ; Ong, Kien Chai ; Hiu, Jessie ; Vadivelu, Jamunarani ; Nathan, Sheila ; Wong, Kum Thong. / In situ hybridization to detect and identify Burkholderia pseudomallei in human melioidosis. In: Modern Pathology. 2014 ; Vol. 27, No. 5. pp. 657-664.
@article{c7e374bdaffe449f89383cbff969ed93,
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.",
keywords = "16s rRNA, Burkholderia pseudomallei, DNA probe, in situ hybridization, melioidosis",
author = "Eu, {Lin Chuan} and Ong, {Kien Chai} and Jessie Hiu and Jamunarani Vadivelu and Sheila Nathan and Wong, {Kum Thong}",
year = "2014",
doi = "10.1038/modpathol.2013.184",
language = "English",
volume = "27",
pages = "657--664",
journal = "Modern Pathology",
issn = "0893-3952",
publisher = "Nature Publishing Group",
number = "5",

}

TY - JOUR

T1 - In situ hybridization to detect and identify Burkholderia pseudomallei in human melioidosis

AU - Eu, Lin Chuan

AU - Ong, Kien Chai

AU - Hiu, Jessie

AU - Vadivelu, Jamunarani

AU - Nathan, Sheila

AU - Wong, Kum Thong

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

KW - 16s rRNA

KW - Burkholderia pseudomallei

KW - DNA probe

KW - in situ hybridization

KW - melioidosis

UR - http://www.scopus.com/inward/record.url?scp=84899988813&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84899988813&partnerID=8YFLogxK

U2 - 10.1038/modpathol.2013.184

DO - 10.1038/modpathol.2013.184

M3 - Article

C2 - 24186135

AN - SCOPUS:84899988813

VL - 27

SP - 657

EP - 664

JO - Modern Pathology

JF - Modern Pathology

SN - 0893-3952

IS - 5

ER -