Histopathological features of hind footpads.

A-B) Untreated control mouse at day 33 post-infection. A) Erosion, edema, and fibrinous exudates are observed (H&E x290). B) Aggregated acid-fast bacilli are observed primarily in the stroma, with some present in monocytes (arrowhead) (Fite-Faraco Staining x1750). C) Rifalazil (5mg/kg)-treated mouse at one-week of treatment. Some bacterial cells are fragmented (arrowheads) (Fite-Faraco staining x1750). D-E) Rifalazil (5mg/kg)-treated mouse at 15-weeks of treatment. D) Epithelioid cell granuloma with mild infiltration of lymphocytes is noted. Epidermal erosion, edema, fibrin, and neutrophil aggregation are not observed (H&E x120). E) Granularly degenerated acid-fast bacilli are observed in monocytes (arrowheads) (Fite-Faraco staining x1750). F-G) Rifalazil (5mg/kg)-treated mouse 15 weeks following termination of treatment. F) Epithelioid cell granuloma with mild infiltration of lymphocytes is noted. Epidermal erosion, edema, fibrin, and neutrophil aggregation are not observed (H&E x290). G) Completely degenerated acid-fast bacilli are observed in an epithelioid cell granuloma (Fite-Faraco staining x1750).</p

Log CFU numbers in the hind footpads.

† All of the mice in the untreated group reached to the endpoint at 4 weeks after the measurement was started. N.D Colony was not detected.</p

Reconstructed stereoscopic CT images of the maxilla.

<p>Two severely deformed cases, A (ID 9) and B (ID12), and two control cases, C (ID C1) and D (ID C3), are shown. The front, left side, and bottom view of the cranium are shown from top to bottom. The deformities of the anterior nasal spine and the alveolar process are easily observed in A and B. The result is that the retracted surface of the central maxilla and the regression of the alveolar process look nearly symmetric and crescentic. Arrows indicate the anterior nasal spine (ANS) or the corresponding point.</p

Workflow of Harmony software image analysis for intracellular <i>M</i>. <i>ulcerans</i>.

THP-1 cell cytoplasm, nuclei, and bacterial immunostaining input image were shown in Fig 4A. Cytoplasm with nucleus were detected by HCS Cell Mask Deep Red and Hoechst 33258 channel respectively (Fig 4B). Bacterial cells were identified as Alexa 488-staining objects within cytoplasm were specifically detected, and calculated the intensity of them (Fig 4C).</p

Gross skin erosion of hind footpads in untreated and rifalazil-treated <i>M</i>. <i>ulcerans</i> infected mice.

Gross skin erosion of hind footpads in untreated and rifalazil-treated M. ulcerans infected mice.</p

Comparative rifamycin efficacy against intracellular <i>M</i>. <i>ulcerans</i> with THP-1 human macrophage infection model.

The sum of fluorescence intensity (Alexa Flour 488) of intracellular bacterium after 72 hours incubation with RFP10μg/mL, RFP10μg/mL+SM10μg/mL, RLZ 10μg/mL and RLZ 10μg/mL+SM10μg/mL. Data are shown as mean ±SD. *p< 0.05 by unpaired t-test.</p

Gross skin reddening of hind footpads in untreated and rifalazil-treated <i>M</i>. <i>ulcerans</i> infected mice.

Gross skin reddening of hind footpads in untreated and rifalazil-treated M. ulcerans infected mice.</p

Thickness of hind footpads in <i>M</i>. <i>ulcerans</i> infected mice treated with or without RLZ.

Average of thickness of hind footpads (mean±SD, x10-2 mm). Parentheses indicate the number of mice tested. (XLSX)</p

Successive measurement of hind footpad thickness.

Measurements were started 33 days post-infection. † All of the mice in the untreated group reached to the endpoint at 4 weeks after the measurement was started.</p

Reference points and planes, and measurement lines on the CT images.

<p>A. Measurement lines on the median sagittal plane of the cranium. The volume of interest (VOI) was limited to the maxillofacial area and the anatomical landmarks are identified. The sum of the distance from ‘<i>n’</i> to the <i>‘alv</i> plane<i>’</i> (“<i>n-alv</i> distance”) and from the <i>‘alv</i> plane<i>’</i> to <i>‘ba’</i> (“<i>alv-ba</i> distance”) was defined as a reference of the cranial anterior-posterior length, termed “C_A-P”. The distance from <i>‘pr’</i> to the <i>‘alv</i> plane<i>’</i> was defined as the anterior-posterior length of the maxilla, termed “M_A-P”. <i>‘alv’</i>: the point where the midline of the palate is intersected by a straight tangent connecting the posterior borders of the alveolar crests. <i>‘n’</i>: the point of intersection of the nasofrontal suture and the mid-sagittal plane. <i>‘pr’</i>: the most anterior point on the alveolar border of the maxilla. <i>‘ba’</i>: the point where the anterior margin of the foramen magnum is intersected by the mid-sagittal plane. B. Definition of ‘<i>alv’</i> plane. The coronal plane which is orthogonal to the FH (Frankfurt horizontal plane) and parallel to the line connecting the left and right <i>‘po’</i>, and passes through <i>‘alv’</i>, was defined as the <i>‘alv</i> plane<i>’</i>.</p