Table 1.
List of compounds used to validate the assay.
Fig 1.
Representative images of the T. cruzi infected control, non-infected control, and 30 μM benznidazole treated infected cells (BNZ) for: (a) raw image acquisition (at 635 nm); (b) nuclei selection (selected in green and discarded in red; as described in Methods) and their cytoplasm boundaries identification (green lines); (c) discrimination of true amastigotes (green) from other non-parasitic cytoplasmic spots (red) amongst all cytoplasmic spots detected; (d) identification of infected cells (those with >1 amastigote inside, green) and non-infected cells (red); (e) scatter plots of all detected spots used to discriminate true (green dots) from false amastigote spots (red dots) based on their area and intensity (‘Spot area’ and ‘Corrected intensity’ thresholds lines are shown; ‘Corrected Intensity’ is expressed in arbitrary units). Bar is 30 μm.
Fig 2.
High resolution image of controls.
Representative T. cruzi infected and uninfected H9c2 pictures at 40X. Kinetoplastid kDNA (circle) and nDNA (bean-like) can be distinguished at this magnification. Bar is 30 μm.
Fig 3.
Discrimination of spots using ‘Corrected intensity’ as parameter.
A total of 1.18×106 spots were acquired from infected and non-infected control wells (five fields per well from 96 wells for each control) from 6 independent plates each. Spots were distributed in 9 groups and classified accordingly to their ‘Corrected intensity’ parameter based on the Hartigan-Wong algorithm [34]. The three parametric regions (low, intermediate and high) are indicated by red threshold lines in the box plot. Percentage of spots from infected and non-infected controls in each of the nine classes is shown below
Fig 4.
‘True amastigote’ discrimination thresholds based on their (a) ‘Corrected intensity’, and (b) ‘Spot area’ parameters were defined from the spots populations within both, infected and non-infected control wells (lognormal adjustments, blue lines; lower and upper ‘Corrected intensity’ thresholds (respectively at 48 and 274 a.u.), and the upper ‘Spot area’ cut off (51 px2), green lines). Inset in panel (a) zooms in at the spots distribution in the intermediate ‘Corrected intensity’ region (true amastigotes).
Fig 5.
Controls for T. cruzi infected (I) and non-infected (N) cells of six independent plates (P1–P6) are represented: (a) box plot of the number of amastigotes per cell for each control: I (infected untreated cells) and N (non-infected cells) (16 wells of each per plate), and BNZ treated infected cells (30 μM; 16 wells in one plate) is shown. The distribution of the populations is not normal, so the median value per plate (black line) is shown. The second (white box under median), third (white box over media) and forth (dashed line) quartiles and population outliers (dots) are represented. (b) Scatter plot correlating the number of host cells to the number of amastigotes per cell. Infected cells control wells of each plate (blue lines) and BNZ treated infected cells plate (red line).
Fig 6.
Representative curves of three independent experiments of BNZ, NFX, posaconazole and cyclohexymide normalized activity (%) against T. cruzi amastigotes per compound concentration (M) for each of the three assay outputs: ‘Am/Cell’ (blue), ‘%Infected’ (green) were normalized as described in [15]; ‘Cells’ (cytotoxic impact of the drugs; yellow) is expressed as percentage of live cells, considering non-treated T. cruzi infected wells as 100%.