Examples of lesions presenting with both IS2404 qPCR positive and negative swabs.

<p>Lesions of six laboratory confirmed BU patients are shown, whereof four (# 1–4) presented with a ∆CT value ≤ 10, indicating a minimal to medium heterogeneity and two (# 5 and 6) presented with a ∆CT value > 10, indicative for a maximum heterogeneity. From each lesion four swabs have been taken in a clockwise manner and IS2404 qPCR has been performed.</p

Localisation of large clusters of AFB in the subcutis of BU lesions.

<p>Histological sections were stained with ZN (counterstain methylenblue). A: Cross sections through the excised tissue specimens from BU plaque lesions (A2-A4, A7-A8) or ulcerated BU lesions (A1, A5-A6) revealing large clusters of AFB located in the subcutis in different tissue depths (3 mm—10 mm). Boxed areas are shown in a higher magnification in (B). B: Large clusters of AFB. C: High magnification of AFB with typical rod shaped appearance.</p

Increase in the probability of obtaining one or more positive qPCR results with the number of swabs analyzed.

<p>Increase in the probability of obtaining one or more positive qPCR results with the number of swabs analyzed.</p

Correlation between direct smear microscopy and IS2404 qPCR results.

<p>Of the 123 IS2404 qPCR positive swab samples analyzed by direct smear microscopic analysis after ZN staining, 54 samples (43.9%) were only positive by qPCR, whereas 69 samples (56.1%) were positive for both methods applied. Up to a qPCR CT of 27.8 all swabs were positive for both methods. Between CT 27.9 and CT 33.8 results were variable and above CT 33.8 all samples were microscopy negative. A: AFB positive and negative swabs in correlation to the qPCR values. Each dot represents one swab sample. B: Percentage of AFB positive and negative swabs in correlation to qPCR value ranges.</p

Preferential location of <i>M</i>. <i>ulcerans</i> bacteria in the subcutis.

<p>A: In plaque lesions mainly large clusters of extracellular bacteria were found, whereas in ulcerated lesions single AFB and small clusters were more common. In both types of lesions AFB were primarily found in the subcutis. B: Examples for dispersed single bacteria (1) and small clusters of AFB (2) in histological sections stained with ZN (counterstain methylenblue).</p

Clinical features of the BU patients from whom punch biopsy samples were analysed in this work.

<p>The WHO category for each lesion was as follows: Category 1; a single small lesion or ulcer <5 cm in diameter, category 2; a single lesion 5–15 cm in diameter, category 3 a single lesion > 15 cm, multiple small lesions or lesions on the face. Data are n (%) or median (IQR).</p><p>Clinical features of the BU patients from whom punch biopsy samples were analysed in this work.</p

Histopathological features and biomarker expression in the skin of Buruli ulcer patients.

<p>The distribution of the data were assessed using the D'Agostino & Pearson normality test with a cut-off value of P<0.05. Semi-quantitative scoring was carried out as described in Materials and Methods. TM is normally expressed in the keratinocytes of the epidermis and the endothelial cells of the dermis and subcutis. Necrosis was scored according to the appearance of the tissue in H&E staining.</p><p>¹ TM staining was only scored in areas of lesions that had stained positively for SMA (endothelial cell marker), and a combined score derived from both the coverage and intensity was used.</p><p>²A major effect was defined as a score <1 for SMA and keratinocyte TM, <4 for endothelial TM combined score and >2 for fibrin staining and appearance of necrosis.</p><p>³A moderate effect was defined as a score of 1–2.5 inclusive for SMA and keratinocyte TM, combined endothelial TM score of 4–8 inclusive and 0.5–2 inclusive for fibrin staining and appearance of necrosis.</p><p>⁴No effect was defined as patients with maximum (SMA and TM) or zero (fibrin staining and appearance of necrosis) scores.</p><p>⁵In 11 (25%) of patients, TM was considered to be absent (combined TM score <1).</p><p>⁶Four of these patients did not show evidence of SMA staining in the subcutis and so TM was not quantified in these biopsies due to lack of intact endothelium.</p><p>⁷In 18 (50%) of patients, TM was considered to be absent (combined TM score <1)</p><p>Histopathological features and biomarker expression in the skin of Buruli ulcer patients.</p

Abundant fibrin deposition in the skin of patients with Buruli ulcer.

<p>A and B. Histological sections were stained with an α-fibrin antibody and counterstained with Haematoxylin. Slides were analyzed with a DM2500 Microscope (Leica). Pictures were taken with an Aperio scanner. Comparative staining of a healthy skin sample from an unaffected donor (A) or 4mm punch biopsies from 8 different laboratory confirmed BU patients (B) showing the variability in fibrin staining observed ranging from small isolated fibrin depositions (B1-B2), to large deposition seen only in the dermis or subcutis (B3-B6) and finally to extensive depositions covering the whole tissue sample (B7-B8). C and D. Scoring was carried out as described in Materials and Methods and are relative to a maximum of 3. The score for each individual biopsy analysed is shown, consisting of 31 patients with ulcers (red circles) and 9 patients with plaque lesions (green circles). The expected score for healthy tissue was 0 for both fibrin and necrosis (see A). In all cases, error bars show the median and 25–75% percentile scores. C. Fibrin staining in the dermis and subcutis. D. The degree of necrosis in each biopsy was scored according to appearance of the entire biopsy.</p

Mycolactone causes a profound depletion of thrombomodulin (TM) endothelial cell surfaces.

<p>Human dermal microvascular endothelial cells were exposed to various concentrations of mycolactone (MYC), 10ng/ml IL-1β or 0.025% DMSO (solvent control, equivalent to that in 125ng/ml MYC) for 24 hours (or other times as indicated). Cells were harvested and subjected to flow cytometry for TM and EPCR. A. Forward and side scatter plots. B. Histogram plots for TM and EPCR. Untreated unstained cells; filled grey, untreated cells with isotype control; dotted black line, untreated cells with TM or EPCR-PE; black line; MYC treated cells (7.8ng/ml) with TM or EPCR-PE, red line; IL-1β treated cells with TM or EPCR-PE, green line. C. Quantitation of TM and EPCR surface expression. MFIs are expressed as % of untreated cells with TM or EPCR-PE (mean±SEM, n = 3). Unstained and isotype bars are for untreated cells. D Quantitation of TM and EPCR surface expression over time in the presence of 7.8ng/ml mycolactone. MFIs are expressed as % of untreated cells with TM or EPCR-PE. DMSO control (0.08%) is after 48hrs exposure (mean±SEM, n = 3). *, P<0.05; **, P<0.01; ***, P<0.001.</p

Mycolactone causes detachment of endothelial cells, apoptosis in 3–4 days, and depletes CD31 (PECAM-1) and VE-cadherin the surface.

<p>Human dermal microvascular endothelial cells were exposed to various concentrations of mycolactone (MYC), 10ng/ml TNF, 1μM staurosporin or 0.025% DMSO (solvent control) as appropriate. A. Both attached and detached cells were subjected to Calcein/EtBr staining for live/dead cells. The proportion of cells that were either attached or detached and alive (Calcein +/EtBr-), and attached or detached and dead (Calcein-/EtBr +) are expressed as a % of the total population of cells. mean±SEM, n = 3. B. HDMVECs were exposed to 7.8ng/ml mycolactone over a timecourse. Cells were then analysed by confocal microscopy following staining of cells with no-wash reagents. CellEvent caspase-3/7 green detection reagent identified cells undergoing apoptosis, alongside PI and DRAQ5. The number of cells in late apoptosis (positive for both active caspase 3/7 and PI) were counted in 3 fields and expressed as a proportion of total cells (DRAQ5 stained) Mean±SEM (n = 3). C. Cells were harvested and subjected to flow cytometry for VE-Cadherin and CD31 (PECAM-1). MFIs are expressed as % of untreated cells (mean±SEM, n = 4). Unstained and isotype bars are for untreated cells. **, P<0.01; ***, P<0.001.</p