Animal models of neglected parasitic diseases: In vivo multimodal imaging of experimental trypanosomatid infections.

African trypanosomiases and leishmaniases are significant neglected tropical diseases (NTDs) that affect millions globally, with severe health and socio-economic consequences, especially in endemic regions. Understanding the pathogenesis and dissemination of Trypanosoma brucei and Leishmania spp. parasites within their hosts is pivotal for the development of effective interventions. Whole-body bioluminescence and fluorescence imaging systems (BLI and FLI, respectively), are powerful tools to visualize and quantify the progression and distribution of these parasites in real-time within live animal models. By combining this technology with the engineering of stable T. brucei and Leishmania spp. strains expressing luciferase and/or fluorescent proteins, crucial aspects of the infection process including the parasites' homing, the infection dynamics, the tissue tropism, or the efficacy of experimental treatments and vaccines can be deeply investigated. This methodology allows for enhanced sensitivity and resolution, elucidating previously unrecognized infection niches and dynamics. Importantly, whole-body in vivo imaging is non-invasive, enabling for longitudinal studies during the course of an infection in the same animal, thereby aligning with the "3Rs" principle of animal research. Here, we detail a protocol for the generation of dual-reporter T. brucei and L. major, and their use to infect mice and follow the spatiotemporal dynamics of infection by in vivo imaging systems. Additionally, 3D micro-computed tomography (µCT) coupled to BLI in T. brucei-infected animals is applied to gain insights into the anatomical parasite distribution. This Chapter underscores the potential of these bioimaging modalities as indispensable tools in parasitology, paving the way for novel therapeutic strategies and deeper insights into host-parasite interactions.

Application of diffusion weighted multiple boli ASL to a murine model of human African trypanosomiasis.

Human African Trypanosomiasis (HAT) is a parasitic disease originating in sub-Saharan Africa. There is limited information about the changes in the blood brain barrier (BBB) during this infection. This study is the first to apply diffusion weighted ASL (DWASL) to examine changes in BBB impairment. No significant changes in water exchange across the BBB were found during the infection, even when a loss of barrier integrity was seen using Contrast Enhanced MRI (Gd-DTPA) during the late stage of the disease. Furthermore, using multiple boli ASL (mbASL), changes in cerebral blood flow (CBF) were found during the course of infection. Overall, this study highlights the need for further study of the BBB during HAT infection to understand the complex mechanisms behind impairment.

Correlation of PET/CT and Brain MRI Findings in Human African Trypanosomiasis Encephalitis.

ABSTRACT: A 49-year-old woman from Cameroon presented with history of several months of worsening headache, lethargy, left arm weakness, and generalized lymphadenopathy. Brain MRI demonstrated multifocal signal abnormality and enhancement involving the bilateral basal ganglia and cerebral white matter. FDG PET/CT performed as part of lymphadenopathy evaluation demonstrated patchy areas of increased metabolic activity of the brain parenchyma. Human African trypanosomiasis or African sleeping sickness is a protozoan infection caused by Trypanosoma brucei gambiense transmitted by the tsetse fly in sub-Saharan Africa. It is important to recognize the signs and symptoms in nonendemic countries to facilitate early diagnosis and treatment.

3 Tesla serial magnetic resonance imaging of human African trypanosomiasis (Trypanosoma brucei gambiense) and review of the literature.

We report serial magnetic resonance imaging (MRI) findings and follow-up in a case of human African trypanosomiasis (HAT) presenting with limited lesions followed by early and complete resolution. We searched the literature for documented cases and reviewed MRI findings before treatment. A 30-year-old Lebanese man, who had lived in Gabon for six years, presented with a two-year history of rash, anorexia, weight loss, arthralgia, paresthesia, and hypersomnia. Previously, the patient had received corticosteroid therapy for unconfirmed ANCA-associated vasculitis. Physical examination revealed a painless chancre on the left arm located at the site of an old insect bite, enlarged cervical, axillar and inguinal lymph nodes, hepatosplenomegaly and impaired concentration. Blood analysis showed an elevated protein level (90g/L) with hypoalbuminemia (24.2g/L) and elevated IgM (26.4g/L). Bone marrow aspirate and biopsy failed to detect any parasite. Polymerase chain reaction tests on blood and cerebrospinal fluid were positive for Trypanosoma. Serology tests confirmed the diagnosis of HAT due to Trypanosoma brucei gambiense infection. 3T MRI showed lesions in the hypothalamus and basal ganglia, the internal capsule, and the mesencephalon bilaterally. Follow-up MRI showed interval progression of the abnormalities. Treatment with melarsoprol was followed by clinical improvement with regression of the lesions on the three-month MRI, then total resolution at the 10-month follow-up. This case highlights a pattern of mild MRI lesions in T. brucei gambiense HAT with a total and rapid resolution under treatment. The literature review (16 HAT cases with sufficient radiological data, included ours) revealed an MRI pattern of brain lesion distribution that could be helpful for diagnosis and orienting biological tests.

FDG PET/CT in a Case of Human African Trypanosomiasis (Sleeping Sickness).

Human African trypanosomiasis imported to nonendemic countries is rare. It is very difficult to establish the correct diagnosis of human African trypanosomiasis in nonendemic areas. We present a case of human African trypanosomiasis with MRI and FDG PET/CT findings. Head MRI showed hyperintense areas in bilateral internal capsules. Abdominal and pelvic MRI showed hepatosplenomegaly and multiple enlarged lymph nodes. FDG PET/CT showed generalized hypermetabolic lymph nodes, diffuse FDG uptake of the spleen, and hepatosplenomegaly mimicking lymphoma. In addition, FDG PET/CT revealed decreased FDG uptake in the medial occipital cortex and cardiomegaly with pericardial effusion.

A new chimeric triple reporter fusion protein as a tool for in vitro and in vivo multimodal imaging to monitor the development of African trypanosomes and Leishmania parasites.

Trypanosomiases and leishmaniases, caused by a group of related protist parasites, are Neglected Tropical Diseases currently threatening >500 million people worldwide. Reporter proteins have revolutionised the research on infectious diseases and have opened up new advances in the understanding of trypanosomatid-borne diseases in terms of both biology, pathogenesis and drug development. Here, we describe the generation and some applications of a new chimeric triple reporter fusion protein combining the red-shifted firefly luciferase PpyREH9 and the tdTomato red fluorescent protein, fused by the TY1 tag. Expressed in both Trypanosoma brucei brucei and Leishmania major transgenic parasites, this construct was successfully assessed on different state-of-the-art imaging technologies, at different scales ranging from whole organism to cellular level, both in vitro and in vivo in murine models. For T. b. brucei, the usefulness of this triple marker to monitor the entire parasite cycle in both tsetse flies and mice was further demonstrated. This stable reporter allows to qualitatively and quantitatively scrutinize in real-time several crucial aspects of the parasite's development, including the development of African trypanosomes in the dermis of the mammalian host. We briefly discuss developments in bio-imaging technologies and highlight how we could improve our understanding of parasitism by combining the genetic engineering of parasites to the one of the hosting organisms in which they complete their developmental program.

Using microdialysis to analyse the passage of monovalent nanobodies through the blood-brain barrier.

BACKGROUND AND PURPOSE: Nanobodies are promising antigen-binding moieties for molecular imaging and therapeutic purposes because of their favourable pharmacological and pharmacokinetic properties. However, the capability of monovalent nanobodies to reach targets in the CNS remains to be demonstrated. EXPERIMENTAL APPROACH: We have assessed the blood-brain barrier permeability of Nb_An33, a nanobody against the Trypanosoma brucei brucei variant-specific surface glycoprotein (VSG). This analysis was performed in healthy rats and in rats that were in the encephalitic stage of African trypanosomiasis using intracerebral microdialysis, single photon emission computed tomography (SPECT) or a combination of both methodologies. This enabled the quantification of unlabelled and (99m) Tc-labelled nanobodies using, respectively, a sensitive VSG-based nanobody-detection elisa, radioactivity measurement in collected microdialysates and SPECT image analysis. KEY RESULTS: The combined read-out methodologies showed that Nb_An33 was detected in the brain of healthy rats following i.v. injection, inflammation-induced damage to the blood-brain barrier, as in the late encephalitic stage of trypanosomiasis, significantly increased the efficiency of passage of the nanobody through this barrier. Complementing SPECT analyses with intracerebral microdialysis improved analysis of brain disposition. There is clear value in assessing penetration of the blood-brain barrier by monovalent nanobodies in models of CNS inflammation. Our data also suggest that rapid clearance from blood might hamper efficient targeting of specific nanobodies to the CNS. CONCLUSIONS AND IMPLICATIONS: Nanobodies can enter the brain parenchyma from the systemic circulation, especially in pathological conditions where the blood-brain barrier integrity is compromised.

Computer-assisted tomographic findings in a patient with African trypanosomiasis.

A 5-year-old boy was diagnosed to have trypanosomiasis and responded to treatment with diminazine. However, 22 months later symptoms recurred, with evidence of cerebral trypanosomiasis. Computer assisted tomography (CAT) scans were subsequently performed on three occasions. He was treated with melarsoprol and to date has made a full recovery. This paper reports on the evolution of the cerebral lesions shown up by the CAT scans.

[Pseudo-tumoral human African trypanosomiasis due to Trypanosoma gambiense. Clinical and tomodensitometry study (author's transl)]. / Formes pseudo-tumorales de la trypanosomiase africaine à Trypanosoma gambiense. Etude clinique et tomodensitométrique.

The authors report an observation of african trypanosomiasis due to Trypanosoma Gambiense, clinical signs included massive and progressive hemiplegia, papillary edema and vascular shift from median line at arteriography. These pseudo tumoral clinical features are unusual in this disease. Asymetrical heterogenous hypodensities of the centrum semioval are dominant in the initial CT scanner aspect. The confrontation of CT scanner images to the clinical and evolutive data suggests the presence of associated cerebral edema and demyelination. With treatment, hypodensities were regressing while images of subcortical atrophy appeared. Lastly, in spite of severe general signs and the importance of neurological deficit, arsenical treatment associated with high doses of corticotherapy lead to a rapid improvement.