CONCLUSIONS: CD remains underdiagnosed in Germany despite the availability of national recommendations. Expanding targeted screening, increasing healthcare professional awareness, preventing non-vectorial transmission and improving health insurance coverage are essential to reduce the burden of CD.

Chagas disease is caused by infection with Trypanosoma cruzi and can result in chronic cardiomyopathy. However, the molecular programmes that define infected cardiac tissue, and how they change as the host-parasite relationship moves from acute pathogenic infection to long-term persistence, remain incompletely understood. Here, we used RNA sequencing to characterise host transcriptional responses in cardiac tissue during both acute and chronic infection in a mouse model. Cardiac parasite distribution was defined using bioluminescence imaging and histology, and RNA sequencing was performed on anatomically standardised cardiac tissue samples collected at both stages. Acute infection was associated with a broad interferon-driven immune activation, whereas during chronic infection this network contracted, with preservation of a tightly connected antigen presentation core centred on major histocompatibility complex genes and immunoproteasome components. Genes associated with extracellular matrix remodelling and fibrosis were not broadly activated. These findings indicate that reduced, but sustained immune activation, rather than widespread fibrotic transcriptional remodelling, represents a prominent feature of cardiac tissue in chronically infected mice. They identify a transition from an overtly inflammatory, pathogen-driven acute response to a more restricted chronic host-parasite equilibrium in which immune surveillance remains transcriptionally active despite very low and spatially intermittent cardiac infection. We identified a conserved 20-gene signature shared between acute and chronic stages. This overlap is consistent with continued or episodic parasite-derived stimulation and/or intrinsic host mechanisms that maintain cardiac immune surveillance. Thus, chronic T. cruzi infection can be viewed as a persistent host-parasite association in which biological coexistence does not imply benignity: immune containment and parasite persistence can occur alongside cumulative cardiac injury.

Evidence from bone marrow and intestinal lamina propria studies indicates that antibody-secreting cell (ASC) characteristics are shaped by the local microenvironment; yet whether secondary lymphoid organs harbor distinct ASC populations during infection remains unclear. Here, we investigated plasmablasts (PB) from the spleen and inguinal lymph nodes (LN) in acute Trypanosoma cruzi infection. Although PB from both tissues expressed canonical ASC markers, they displayed distinct transcriptional, phenotypic, and functional profiles. Splenic PB upregulated genes and proteins associated with inflammation, immunomodulation, and B-cell interactions. Activated and naive B cells also exhibited tissue-specific transcriptional programs, suggesting that local cues influence B-cell differentiation. Consistent with these differences, spleens from infected mice contained more T follicular helper cells, whereas LN were enriched in regulatory T cells. High-dimensional flow cytometry identified multiple PB subsets differentially represented between tissues. Functionally, splenic PB secreted higher levels of IgM, IgA, and IgG, including both parasite-specific and autoreactive antibodies. In contrast, LN PB preferentially produced IgG against peptide 72 of the shed acute-phase antigen (SAPA), despite lower overall T. cruzi-specific antibody production. These findings demonstrate that secondary lymphoid organs harbor distinct PB populations adapted to their local microenvironments during acute infection

Kinetoplastid parasites, including Trypanosoma cruzi, possess a highly organized subpellicular microtubule network essential for maintaining cell shape, organelle positioning, and dividing. Here we describe results obtained using immunofluorescence microscopy of cells processed using conventional ultrastructure expansion microscopy (U-ExM), that improved visualization of the cytoskeletal structures, with an ~4.5-fold expansion, as well as the recently described and iterative ultrastructure expansion microscopy (iU-ExM) with about 6.2-fold and 18-fold expansion of epimastigotes of T. cruzi. The optimized protocol involves the formation of an initial hydrogel crosslinked with N,N'(1,2-dihydroxyethylene) bisacrylamide (DHEBA), a temperature- and alkaline pH-sensitive cleavable crosslinker, followed by thermal denaturation at 85 degrees Celsius, intermediate immunostaining, neutral gel re-embedding, and the formation of a second hydrogel crosslinked with N,N' methylenebisacrylamide (BIS). Subsequent dissolution of the first hydrogel under alkaline conditions enables a second round of expansion. This approach achieved approximately 6.2-fold expansion during the first round and a final linear expansion of approximately 18-fold. Using conventional confocal microscopy, iU-ExM enabled high-resolution visualization of individual subpellicular microtubules and specialized internal microtubule arrangements, including those associated with the basal body, flagellar pocket, Golgi complex, and cytopharynx. Furthermore, the enhanced spatial resolution facilitated the examination of cytoskeletal reorganization at different stages of the cell cycle, revealing changes in microtubule organization associated with mitotic spindle formation and cytokinesis. Together, these findings establish iU-ExM as an accessible high-resolution imaging approach for investigating the complex subcellular architecture of T. cruzi, providing new opportunities to explore cytoskeletal organization and remodeling during parasite cell division using conventional confocal microscopy.

Trypanosoma brucei transmission through the tsetse fly does not require prior differentiation into the stumpy bloodstream form, revealing greater flexibility in the parasite life cycle.

Evidence from bone marrow and intestinal lamina propria studies indicates that antibody-secreting cell (ASC) characteristics are shaped by the local microenvironment; yet whether secondary lymphoid organs harbor distinct ASC populations during infection remains unclear. Here, we investigated plasmablasts (PB) from the spleen and inguinal lymph nodes (LN) in acute Trypanosoma cruzi infection. Although PB from both tissues expressed canonical ASC markers, they displayed distinct transcriptional, phenotypic, and functional profiles. Splenic PB upregulated genes and proteins associated with inflammation, immunomodulation, and B-cell interactions. Activated and naive B cells also exhibited tissue-specific transcriptional programs, suggesting that local cues influence B-cell differentiation. Consistent with these differences, spleens from infected mice contained more T follicular helper cells, whereas LN were enriched in regulatory T cells. High-dimensional flow cytometry identified multiple PB subsets differentially represented between tissues. Functionally, splenic PB secreted higher levels of IgM, IgA, and IgG, including both parasite-specific and autoreactive antibodies. In contrast, LN PB preferentially produced IgG against peptide 72 of the shed acute-phase antigen (SAPA), despite lower overall T. cruzi-specific antibody production. These findings demonstrate that secondary lymphoid organs harbor distinct PB populations adapted to their local microenvironments during acute infection