Jury :

Rapporteurs :

Isabelle DUFOUR- Professor - University of Bordeaux

Loïc FAVENNEC - University Professor - Hospital Practitioner - University of Rouen Normandy

Examiners :

Karim ADJOU - Professor - École Nationale Vétérinaire d'Alfort

Abdelkrim TALBI - Professor - École Centrale de Lille

Alexis VLANDAS - CNRS Research Fellow - IEMN, Lille

Thesis supervisor :

Jérôme FOLLET- Lecturer-Researcher ISA-JUNIA - Lille

Summary:

Parasites of the genus Cryptosporidium are the pathogens responsible for a gastroenteric disease marked by severe diarrhoea: cryptosporidiosis. This infection can be fatal in the most fragile individuals (young children, the elderly or immunocompromised people). Although cryptosporidiosis is present throughout the world, irrespective of a country's level of economic development, it has been described as the second most common cause of severe diarrhoea leading to death in children under the age of 2 in Africa and Asia. To date, no effective vaccine or treatment has been developed. The search for new therapeutic molecules has been hampered by the absence of standardised and automated tools, limiting the study of the parasite and the discovery of new treatments.

The aim of this thesis project is therefore to develop a microsystem based on impedance spectroscopy dedicated to the study of the parasite Cryptosporidium parvum and the screening of therapeutic molecules. In this context, the efficacy of paromomycin (the reference molecule used in the in vitro screening of molecules against C. parvum) was determined by standard methods (molecular and microscopic) in order to be compared with impedance spectroscopy.

To do this, HCT-8 cells from a human ileo-caecal adenocarcinoma were infected with C. parvum for 48 hours and treated with a range of increasing concentrations of paromomycin. The efficacy of paromomycin was determined first by microscopy and then by molecular method (qPCR). Parasite quantification on fluorescent microscopic images was performed automatically using the StarDist plugin (Fiji) and morphometric data on each parasite was obtained. Similarly, the median Inhibitory Concentrations of paromomycin (IC50) were obtained using these two methods, with values of 357µg/mL and 382µg/mL respectively. Impedance spectroscopy analysis enabled us to obtain a characteristic response from infected cells as early as 12 hours after infection, which could be used as an infectivity sensor and represent a suitable method for screening therapeutic molecules.

Abstract:

Cryptosporidium is a protozoan parasite which infects a wide range of hosts including Human Beings and has a global presence. Cryptosporidium is the etiologic agent of cryptosporidiosis, a disease characterised by profuse diarrhea. In vulnerable patients (infants, old people or immunodeficient persons) the prevalence and severity of infection increases and can result in death. In Africa and Asia, Cryptosporidium was identified as the second cause of diarrhea in children under 2 years old. To date no vaccine nor drug therapy is available. Research for new drugs against Cryptosporidium therapy has been impeded by the absence of standardized and automated tools enabling the study of the complete Cryptosporidium Life cycle and drug screening.

Therefore, this project aims to develop a microsystem based on impedance spectroscopy for the study of Cryptosporidium parvum and drugs screening. In this context, the efficacy of paromomycin (the gold standard used in drugs screening against C. parvum) was determined by standard methods (molecular and microscopic) to be compared with impedance spectroscopy.

Here, Human ileocecal colorectal adenocarcinoma cells (HCT-8) were grown to confluency and infected by C. parvum during 48 h with a range of paromomycin concentrations. Results were obtained by fluorescence microscopy and molecular method (real-time qPCR). The level of infection was assessed using fluorescent images combined with an automatic detection of parasites using a Fiji plugin (StarDist) and morphometric data for each parasite were recorded. The half maximal inhibitory concentrations (CI50) of paromomycin were obtained (357 µg/mL and 382 µg/mL respectively). Thus, the new approach using the electrical impedance-based device to quantify infectivity of HCT-8 infected by C. parvum showed a characteristic response of infected cells from 12 hours of infection, which could be used as an infectivity sensor, faster than current methods and could represent a suitable method for drug screening.