Additional file 1: of Prevalence of PTSD and other mental disorders in UK service personnel by time since end of deployment: a meta-analysis

Search strategy of UK studies that could be potentially relevant for inclusion in this study. (DOCX 195 kb

Polygala stipulacea

<p>Dot plots demonstrates ICAM-1 levels in inoculated mouse sera. (Mock inoculation; n = 2 per time point, <i>O. tsutsugamushi</i> inoculation; n = 6 per time point). Bars indicate median and error bars represent interquartile range. The asterisks indicate significant differences (<i>P</i><0.05).</p

Serum levels of circulating soluble VCAM-1.

<p>Dot plots demonstratesVCAM-1 levels in inoculated mouse sera. (Mock inoculation; n = 2 per time point, <i>O. tsutsugamushi</i> inoculation; n = 6 per time point). Bars indicate median and error bars represent interquartile range. The asterisks indicate significant differences (<i>P</i><0.05).</p

Detection of <i>O. tsutsugamushi</i> at the injection site (Panels A and B) and draining lymph node (Panel C) at 7 days post inoculation.

<p>In panel “A” <i>O. tsutsugamushi</i> in a mouse ear is demonstrated using peroxidase DAB reaction as brown colored dots (counterstain hematoxylin, original magnification x400). The immunofluorescence staining of <i>O. tsutsugamushi</i> is shown in panels “B” (mouse ear) and “C” (draining lymph node): <i>O. tsutsugamushi</i> with FITC (green) and DAPI nuclear counterstain in blue (original magnification x600).</p

Macroscopic evidence of regional lymphadenopathy in <i>O. tsutsugamushi</i> inoculated mice.

<p>Gross examination of draining lymph nodes (right superficial parotid lymph node) from <i>O. tsutsugamushi</i> inoculated mice at day 3 pi (left) and day 7 pi (right) shows enlargement of lymph nodes at day 7. Top panel: Karp-inoculated mice, Middle panel: Gilliam-inoculated mice, Bottom panel: Woods-inoculated mice.</p

The co-localisation pattern of <i>O. tsutsugamushi</i> with lymphocytes.

<p>Panel A: Perivascular cuff formation of predominantly mononuclear cells containing large numbers of CD3+ cells co-localising with <i>O. tsutsugamushi</i>. Panels B and C: Laser Scanning Micrographs. The association of T cells with <i>O. tsutsugamushi</i> was commonly by external attachment rather than definite intracellular location. Panel B depicts a 3-D stack projection showing external <i>O. tsutsugamushi</i> attachment to the T lymphocyte, in the lateral vertical sections. Panel B is a 0.6 µm thin section. Patient TM2193, magnification in panel A ×400, in panels B and C ×1000. Double-immunolabeling: CD3 in red, <i>O. tsutsugamushi</i> in green and DAPI nuclear counterstain in blue.</p

<i>O. tsutsugamushi</i> co-localize with CD68 positive macrophages in perivascular infiltrates.

<p>Panel A: Perivascular cuff formation and clusters of predominantly CD68+ macrophages, co-localizing closely with <i>O. tsutsugamushi</i>. Patient TM2193, magnification ×400. Magnification ×400, insert ×1000, double-immunolabeling: CD1a in red, <i>O. tsutsugamushi</i> in green and DAPI nuclear counterstain in blue. Panels B and C: Laser Scanning Micrographs with 3D stack projections of multiple 0.3 µm thin sections, depicting intact <i>O. tsutsugamushi</i> associated with CD68+ macrophages (panel B). Unambigous intracellular location of <i>O. tsutsugamushi</i> in a CD68+ cell (panel C). Patient TM2508, magnification ×1000, CD68 labeled in red, <i>O. tsutsugamushi</i> in green.</p

Ultrastructural examination of <i>O. tsutsugamushi</i> in U937 cells reveals microparticle formation.

<p>Panel A: Bizarre morphology of <i>O. tsutsugamushi</i> (B) in the U937 cell line, with corners and tent-like stretching within the cytoplasm with adjacent mitochondria (Mi). Bar length corresponds to 1 µm. Panel B: A number of <i>O. tsutsugamushi</i> (B) with associated microparticles (arrowheads) within the host cell cytoplasm. Bar represents 500 nm. Panel C: Enlargement of the enclosed bacterium from Panel B showing blebbing of the outer membrane (arrow). ‘mp’ - detached microparticle. Bar length corresponds to 100 nm. Panel D: Detail of two microparticles (mp) formed by the outer, but not inner leaflet of <i>O. tsutsugamushi</i>. These microparticles carry the surface antigens of the ‘mother’ <i>Orientia</i>, and their function remains unclear. Bar length corresponds to 100 nm. Insert - Enlargement of the enclosed area in D showing the structure of the limiting membrane of the microparticle. Bar length corresponds to 10 nm.</p

Multiple intracellular <i>O. tsutsugamushi</i> within antigen presenting cells (APCs) in the superficial dermis.

<p>APCs are characterised by MHC class II receptor (HLADR) positivity and were associated with intracellular <i>O. tsutsugamushi</i> in admission samples of an eschar from a patient with acute scrub typhus. Panel A: HLADR-positive cells in red, <i>O. tsutsugamushi</i> in green. Panels B and C: Laser Scanning Micrographs. Panel B depicts the same infected cell as in Panel A as a 0.3 µm thin section. Panel C shows a 3D stack projection (of 0.3 µm thin sections) of intracytosolic <i>O. tsutsugamushi</i> (in green), with small, high-density granules with high refractive index, staining positively for <i>O. tsutsugamushi</i> antigen. Patient TM2193, magnification ×400, LSM inserts ×1000, double-immunolabeling: HLADR in red, <i>O. tsutsugamushi</i> in green and DAPI nuclear counterstain in blue.</p

Histological features of eschars: Intra- and subepidermal changes.

<p>Panels A and B. Intraepidermal splitting and sub-epidermal blistering with basal vacuolar changes, inflammatory exocytosis and focal red blood cell extravasation in the superficial dermis. Patient TM2644 (Panel A) and TM2646 (Panel B), magnification ×400. Panel C. Acute inflammatory sloughing and artefactual separation of the epidermis. A demarcated area of ulceration and necrosis of the overlying epidermis with subepidermal vacuolation, necrosis and mononuclear infiltration, patient TM2663, magnification ×250. Panel D. Perivascular infiltrates and leucocytoclastic vasculitis. On the right hand side the cross-section of an arteriole, on the left, a venous vessel with intravascular obliteration and secondary thrombus. There is prominent perivascular cuffing with mononuclear cells and fibrinoid necrosis of the arteriolar wall. Patient TM2193, magnification ×250. All panels Haematoxylin & Eosin (H&E) staining.</p