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Case Reports
. 2018 Nov 26;8(1):17373.
doi: 10.1038/s41598-018-35775-9.

Ultrastructural Visualization of Orientia Tsutsugamushi in Biopsied Eschars and Monocytes From Scrub Typhus Patients in South Korea

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Case Reports

Ultrastructural Visualization of Orientia Tsutsugamushi in Biopsied Eschars and Monocytes From Scrub Typhus Patients in South Korea

Hyun-Joo Ro et al. Sci Rep. .
Free PMC article

Abstract

Scrub typhus, which is caused by Orientia tsutsugamushi, is a public health problem in the Asian-Pacific region and is the third most frequently reported infectious disease in South Korea. While ultrastructural studies have been performed on O. tsutsugamushi in murine fibroblasts, its variable locations in patients have hampered similar studies in humans. Two patients with scrub typhus agreed to provide an eschar biopsy and peripheral blood, respectively. Transmission electron microscopy was performed separately on the necrotic crust and perifocal skin of the eschar, the peripheral blood, and the infected murine L cells. O. tsutsugamushi was located within or adjacent to the outermost layer of the perifocal inflamed skin of the eschar but not in the necrotic centre. O. tsutsugamushi in peripheral blood monocytes exhibited the characteristic features of O. tsutsugamushi in L cells, namely, nearly round shaped bacteria with a size of 1-2 µm and a double membrane bearing a clear halo-like outer layer. The findings confirmed that the bacterium was predominantly located in the inflamed skin around the eschar and that the bacterium had the same ultrastructural features in human monocytes as in L cells. These findings suggest that the perifocal area, not the necrotic centre, should be sampled for diagnosis.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Relative quantification of the O. tsutsugamushi 56 kDa gene in PBMCs and eschars. The expression level of the 56 kDa gene was evaluated by nested qRT-PCR. The data were normalized to the Gapdh dCT value of the non-infected sample of normal subject #1. The error bars represent the SD of three technical replicates of each sample.
Figure 2
Figure 2
(a) Eschar biopsy of a scrub typhus patient (case 1) that was used for transmission electron microscopy (TEM). (b) Schematic depiction showing how the eschar biopsy was divided into the central necrotic crust (Enec) and the perifocal inflamed skin (Eskin). TEM micrographs showing that O. tsutsugamushi (O.tsutsu) is absent in (c) Enec and frequent in (d) Eskin. Scale bars, 1 µm.
Figure 3
Figure 3
TEM image of scrub typhus patient eschar: (b,d,e) O.tsutsugamushi in perifocal skin (Eskin) marked with black asterisks; (a,c) enlarged image of (b,f) high magnification TEM image of O.tsutsugamushi in Eskin. Black arrows, black arrowhead and white arrowhead point to the electron lucent halo, outer membrane (OM) and plasma membrane (PM), respectively. (b,d) Scale bar, 1 µm. (a,c,e) Scale bar, 500 nm. (f) Scale bar, 200 nm.
Figure 4
Figure 4
Transmission electron microscope images of O. tsutsugamushi (black asterisks) in (a) cultured murine L cells and (b,c) peripheral blood mononuclear cells isolated from a scrub typhus patient (case 2). (d,e) enlarged images of (b,c), respectively. N: nucleus; M: mitochondria. Scale bars: 1 µm for (ac) and 500 nm for (d,e).

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