case_images: PMC11251414_01
case_images: map of Brazil with the State of Maranhao highlighted. Geographical location of the patients is indicated in the map. The patients (1 and 2) reside in cities located within an ecotone region of state. Map created in QGIS v.3.24.3 (http://www.qgis.org).
case_images: 1678-8060-mioc-119-e240026-gf1.jpg
case_images: PMC11251414_01_1678-8060-mioc-119-e240026-gf1.jpg
case_images: f1
case_images: In October 2020, she presented a single ulcer lesion in the lower limb [ (Fig. 1)], and the TL diagnosis was confirmed by Giemsa-stained slides from skin scraping.
case_images: PMC11251414_02
case_images: light microscopy of Giemsa-stained preparations of BG03 (A) and BG05 (B) parasites showing different promastigote morphotypes: leptomonad (l) metacylic (m) nectomonad (n), procyclic (p) and dividing-procyclics. n: nucleus; k: kinetoplast; f: flagellum. Scale-bars: 10 microm.
case_images: 1678-8060-mioc-119-e240026-gf2.jpg
case_images: PMC11251414_02_1678-8060-mioc-119-e240026-gf2.jpg
case_images: f2
case_images: Promastigote cultures were established from each clinical isolate (Fig. 2), and the gDNA was isolated for molecular typing. We first performed a multiplex PCR targeting the kDNA minicircle as previously described and the PCR analysis indicated that both isolates belong to the L. (L.) donovani complex [ (Fig. 2)].
case_images: sequence alignment of the ITS of BG03 and BG05 isolates. Nucleotide sequences of Leishmania (Leishmania) infantum (MHOM/BR/1972/LD), L. (L.) amazonensis (MHOM/BR/1973/M2269) and L. (V.) braziliensis (MHOM/BR/1975/M2309) are included in the aligment and were obtained from GenBank (acession numbers MW538642, AJ000316.1 and AJ300483.1, respectively). The similarity between the nucleotides is highlighted in greyscale. The multiple sequence alignment was performed using GeneDoc version 2.7.
case_images: 1678-8060-mioc-119-e240026-gf3.jpg
case_images: PMC11251414_02_1678-8060-mioc-119-e240026-gf3.jpg
case_images: f3
case_images: The full nucleotide sequences of BG03 and BG05 showed at least 99.8% identity with L. (L.) infantum LD strain sequence, as well as 99.8% identity to each other (Fig. 3). Size analysis of parasites revealed two different populations: non-metacyclic forms (FSChigh) and metacyclic forms (FSClow) (see gating strategy on  (Fig. 3).
case_images: in vitro development of Leishmania (Leishmania) infantum LD strain and BG03, BG05 clinical isolates. Growth curve (A) and metacyclogenesis profile (B) of the strain and isolates. Results from three and two independent experiments with technical triplicates, respectively; data represent means +- standard error of the mean (SEM). Statistical analysis was performed by two-way analysis of variance (ANOVA).
case_images: 1678-8060-mioc-119-e240026-gf4.jpg
case_images: PMC11251414_02_1678-8060-mioc-119-e240026-gf4.jpg
case_images: f4
case_images: All growth curves showed similar profiles, in which promastigotes grew logarithmically on days 2 and 3 and reached the stationary phase on day 4 (Fig. 4A). No statistically significant differences were observed in cell densities between LD strain and BG03 and BG05 isolates (Fig. 4A). We used parasites on the 2nd day of culture as negative control for metacyclogenesis, given the low frequency of metacyclic forms observed during the log. phase of growth.   No differences were found on day 2 between the LD strain and the two clinical isolates (Fig. 4B). The frequency of metacyclic promastigotes for LD strain and BG03 increased slightly from fourth to sixth day of culture (Fig. 4B). Interestingly, the BG05 isolate showed higher frequency of metacyclic forms on the fourth day compared to LD strain and BG03 isolate, but this frequency drastically decreased on the sixth day (Fig. 4B).
case_images: in vitro infectivitiy of Leishmania (Leishmania) infantum BG03 and BG05 isolates and MHOM/BR/1972/LD strain in distict macrophages. In vitro infection of bone marrow-derived macrophages (BMDM) with early-stationary (A, B) and late-stationary (C, D) promastigotes. Results from three independent experiments for BMDM in triplicate and data represent means +- standard error of the mean (SEM). In vitro infection of human monocyte-derived macrophage with early-stationary promastigotes (E, F). Results from two independent experiments for macrophage-like THP-1 and data represent means +- SEM. Photomicrograph of BMDM and macrophage-like THP-1 cells infected with L. infantum laboratory strain and isolates on day- 4 and 6 of culture. Yellow arrow indicates intracellular amastigotes. Scale-bars: 10 microm. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post-hoc test.
case_images: 1678-8060-mioc-119-e240026-gf5.jpg
case_images: PMC11251414_02_1678-8060-mioc-119-e240026-gf5.jpg
case_images: f5
case_images: The percentage of infected macrophages with early stat-phase promastigotes was 43.7 +- 1.0% and 45.8 +- 2.4% for BG03 and BG05 isolates, respectively. This data showed that promastigotes of the clinical isolates are more infective on day 4 compared to L. (L.) infantum LD strain (31.9 +- 1.9%) (Fig. 5A). No statistically significant differences were observed in the average of number of amastigotes per macrophage between the LD strain and the clinical isolates using early stat-phase promastigotes (Fig. 5B). Different results were observed for infections with late stat-phase promastigotes (Fig. 5C). LD strain was the most infective (60.7 +- 1.7%), while the percentage of infected macrophages for BG03 and BG05 isolates was 50.1 +- 2.9% and 40.5 +- 2.9%, respectively (Fig. 5C). An increase in the average number of amastigotes per cell was observed when using promastigotes on day 6 compared to day 4 (Fig. 5B-D). However, no statistically significant differences were observed in the number of amastigotes per macrophage between LD strain and clinical isolates using late stat-phase promastigotes (Fig. 5D). A similar infection profile in THP-1-derived macrophages was observed using early-stat promastigotes, where BG03 (44.8 +- 3.1%) and BG05 (45.5 +- 3.4%) were more infective compared to LD strain (29.7 +- 2.8%) (Fig. 5E). Although there was no significant difference in the number of amastigotes per macrophage observed in this cell model, the average parasite count was slightly higher compared to BMDM (Fig. 5B, F).