Fluorescence In Situ Hybridization (FISH) and Peptide Nucleic Acid Probe-Based FISH for Diagnosis of Q Fever Endocarditis and Vascular Infections

Abstract

Endocarditis and vascular infections are common manifestations of persistent localized infection due to Coxiella burnetii, and recently, fluorescence in situ hybridization (FISH) was proposed as an alternative tool for their diagnosis. In this study, we evaluated the efficiency of FISH in a series of valve and vascular samples infected by C. burnetii We tested 23 C. burnetii-positive valves and thrombus samples obtained from patients with Q fever endocarditis. Seven aneurysms and thrombus specimens were retrieved from patients with Q fever vascular infections. Samples were analyzed by culture, immunochemistry, and FISH with oligonucleotide and PNA probes targeting C. burnetii-specific 16S rRNA sequences. The immunohistochemical analysis was positive for five (17%) samples with significantly more copies of C. burnetii DNA than the negative ones (P = 0.02). FISH was positive for 13 (43%) samples and presented 43% and 40% sensitivity compared to that for quantitative PCR (qPCR) and culture, respectively. PNA FISH detected C. burnetii in 18 (60%) samples and presented 60% and 55% sensitivity compared to that for qPCR and culture, respectively. Immunohistochemistry had 38% and 28% sensitivity compared to that for FISH and PNA FISH, respectively. Samples found positive by both immunohistochemistry and PNA FISH contained significantly more copies of C. burnetii DNA than the negative ones (P = 0.03). Finally, PNA FISH was more sensitive than FISH (60% versus 43%, respectively) for the detection of C. burnetii We provide evidence that PNA FISH and FISH are important assays for the diagnosis of C. burnetii endocarditis and vascular infections.

Keywords: Coxiella burnetii; FISH; PNA probe; Q fever; infective endocarditis; oligonucleotide probe; vascular infections.

FIG 1

Histological analysis of a cardiac valve from a patient with Q fever endocarditis. (A) The image shows the abundant inflammatory infiltrate in valvular tissue, composed mainly of macrophages (hematoxylin-eosin-saffron; ×200 magnification). (B) Immunohistochemical detection of C. burnetii in a cardiac resected valve from a patient with Q fever endocarditis using a monoclonal antibody and hematoxylin counterstain. Note the intracellular location of the bacteria in the macrophage cytoplasm (×200 magnification).

FIG 2

Detection of C. burnetii in a mitral valve by FISH. The images correspond to the nuclei counterstained with DAPI in blue (A), the green channel (Alexa 488) showing hybridization of oligonucleotide probes CB-189 (B), the red channel (Alexa 550) showing hybridization of the universal probe EUB338 (C), and the merged signals (D). Bacteria are visualized directly in infected cells, mainly in the intracytoplasmic area, and appear in clusters as multiple rounded structures.

FIG 3

Detection of C. burnetii in aneurysm by FISH (A) and PNA FISH (B).

FIG 4

Comparison of IS1111 log10 DNA copies in cases that were FISH positive and FISH negative (A) and PNA FISH positive and PNA FISH negative (B).

FIG 5

Detection of C. burnetii in a mitral valve by PNA FISH. The images correspond to the nuclei counterstained with DAPI in blue (A), the green channel (Alexa 488) showing hybridization of specific C. burnetii probe CB189 (B), the red channel (Alexa 550) showing hybridization of the universal probe EUB338 (C), and the merged signals (D). Bacteria are visualized directly in infected cells, mainly in the intracytoplasmic area, and appear in clusters as multiple rounded structures.