This thesis presents the development of quantitative imaging tools to study parasite migration. Since migration is crucial for malaria parasites to continue their life cycle, factors influencing... Show moreThis thesis presents the development of quantitative imaging tools to study parasite migration. Since migration is crucial for malaria parasites to continue their life cycle, factors influencing their migration capability may also impact the efficacy of malaria vaccine candidates. Here, imaging of parasite migration was used to gain insights that can support the development of antiparasitic vaccines. SMOOT (Sporozoite Motility Orienting and Organizing Tool) was developed and established as a quantitative software analysis tool for tracking the migration of malaria sporozoites in vitro and in human skin explant. This tool provides a readout with high kinematic detail, enabling the quantitative characterization of novel factors influencing the migration capability of malaria sporozoites. Subsequently, the study of sporozoite migration was expanded beyond in vitro and ex vivo models. A hybrid tracer labeling approach for malaria sporozoites was developed and used to reveal the in vivo dissemination of malaria sporozoites in a murine model. This multimodal imaging approach was also applied to investigate human skin invasion by helminth larvae. This thesis concludes with a review of the broader potential for imaging technology to advance the development of new diagnostic methods, therapeutic interventions and vaccines for combating parasitic infections. Show less
Background Microscopic examination of thick and thin blood films is the gold standard in current guidelines for the diagnosis of malaria, but guidelines do not uniformly agree on which combination... Show moreBackground Microscopic examination of thick and thin blood films is the gold standard in current guidelines for the diagnosis of malaria, but guidelines do not uniformly agree on which combination of other methods should be used and when. Methods Three questionnaires were sent between March 2018 and September 2019 to laboratories subscribing to the external quality assessment scheme for the diagnosis of blood and intestinal parasites of the Dutch Foundation for Quality Assessment in Medical Laboratories in order to investigate how much variation in the laboratory diagnosis of malaria between different clinical laboratories is present in the Netherlands. Results The questionnaires were partially or fully completed by 67 of 77 (87%) laboratories. Only 9 laboratories reported 10 or more malaria positive patients per year. Most laboratories use a different diagnostic strategy, within office versus outside office hours depending on the screening assay result. Within office hours, 62.5% (35/56) of the responding laboratories perform an immunochromatographic test (ICT) in combination with microscopic examination of thick and thin blood films without additional examinations, such as Quantitative Buffy Coat and/or rtPCR analysis. Outside office hours 85.7% (48/56) of laboratories use an ICT as single screening assay and positive results are immediately confirmed by thick and thin blood films without additional examinations (89.6%, 43/48). In case of a negative ICT result outside office hours, 70.8% (34/48) of the laboratories perform microscopic examination of the thick film the next morning and 22.9% (11/48) confirm the negative ICT result immediately. Furthermore, substantial differences were found in the microscopic examinations of thick and thin blood films; the staining, theoretical sensitivity of the thick film and determination of parasitaemia. Conclusions This study demonstrated a remarkably high variation between laboratories in both their diagnostic strategy as well as their methods for microscopic examination for the diagnosis of malaria in a clinical setting, despite existing national and international guidelines. While the impact of these variations on the accuracy of the diagnosis of malaria is yet unknown, these findings should stimulate clinical laboratories to critically review their own diagnostic strategy. Show less
Skin-penetrating parasites have something in common; they all need to evade the initial immune response in the skin in order to avoid being evicted by their hostile host and establish an infection.... Show moreSkin-penetrating parasites have something in common; they all need to evade the initial immune response in the skin in order to avoid being evicted by their hostile host and establish an infection. To do so, they are equipped with the necessary cunning stratagems. For example, they can act directly on immune cells to alter their function and they can optimize their migration patterns to their hostile environment. This thesis is aimed at unravelling those mechanisms. We study two devastating parasitic diseases: Malaria and Schistosomiasis. Both deadly and debilitating parasitic diseases, with over 200 million (malaria) and over 240 million (schistosomiasis) cases annually; the need for potent vaccines is evident. Whole weakened parasites can be used to vaccinate individuals against parasitic diseases like malaria. However, delivery of these parasites in the skin, as is commonly done in vaccinations, reduces their protectivity. We hypothesize that this reduction is caused by parasite-mediated immune-regulatory mechanisms that are initiated upon their first encounter with immune cells in the skin. We investigated whether skin penetrating parasites exploit these existing mechanisms in human skin in order to enhance their survival. Show less
Controlled human infection (CHI) models are an important research tool. Healthy volunteers are experimentally infected with a pathogen. In malaria research the model has been used for decades. Here... Show moreControlled human infection (CHI) models are an important research tool. Healthy volunteers are experimentally infected with a pathogen. In malaria research the model has been used for decades. Here, the model was used to test new Plasmodium falciparum strains, NF135.C10 an NF166.C8, and compare these with the commonly used strain NF54. In addition, a genetically modified malaria vaccine, PfSPZ-GA1, was tested. Unfortunately only few volunteers were protected against malaria.For schistosomiasis, a controlled human schistosomiasis infection (CoHSI) model was developed and hereafter a dose finding study with single-sex male only cercariae was performed. This study showed that in 80% of volunteers 20 cercariae were effective to induce an infection. Although the use of 30 cercariae resulted in 100% infection rate two out of three volunteers developed Katayama syndrome. These side effects were less after infection with 20 cercariae.At last suggestions were made to further improve the CHI model by the use of historical controls. Although this study design can only be used in CHI’s with well-know outcomes these study will be safer by reducing the cumulative risks as less volunteers can be used for these trials. Show less
To maintain momentum towards improved malaria control and elimination, a vaccine would be a key addition to the intervention toolkit. Two approaches are recommended: (1) promote the development and... Show moreTo maintain momentum towards improved malaria control and elimination, a vaccine would be a key addition to the intervention toolkit. Two approaches are recommended: (1) promote the development and short to medium term deployment of first generation vaccine candidates and (2) support innovation and discovery to identify and develop highly effective, long-lasting and affordable next generation malaria vaccines. Show less
Background: Medical schools offer students the opportunity to perform international electives. This study aimed to assess health risks among medical students, to tailor institutional guidelines... Show moreBackground: Medical schools offer students the opportunity to perform international electives. This study aimed to assess health risks among medical students, to tailor institutional guidelines.Methods: Multicenter study at Dutch and Belgian universities, among medical students who visited low- or middle-income countries. Students completed four questionnaires: once before the elective and two weeks, three- and six months after return.Results: Data was complete for 479 students (follow-up rate 84%). Most traveled to Surinam (29%) and South-Africa (14%). Half of the students encountered difficulties in adapting to local culture. Almost 40% visited malaria endemic countries. Nearly all (87%) used chemoprophylaxis as prescribed. Definite needle-stick or splash injuries were reported by 7%. All were dealt with adequately in accordance with national guidelines. However, less than half of 24 possible incidents were handled adequately. Two-and-a-half percent had unprotected sex with a new partner. The incidence of travelers' diarrhea (TD) was 46%. In those with TD, the incidence of post-travel new-onset abdominal complaints was 3%. Three percent were involved in a minor traffic accident, 18% were injured during leisure activities, 5% were threatened or experienced physical violence. Only half of the students visiting a highly endemic country were screened for tuberculosis post-travel. For schistosomiasis this was 6%.Conclusions: Students abroad are exposed to medical and non-medical challenges, which should be addressed during pre-travel counseling. Contact details of a professional back home should be provided, so students can confer in case of problems while abroad. Lastly, we recommend a centrally organized post-travel health check. Show less
Using a variety of anti-malaria tools has resulted in a steady decline of malaria in several endemic countries worldwide. An effective vaccine will be critical to halt malaria or even succeed to... Show moreUsing a variety of anti-malaria tools has resulted in a steady decline of malaria in several endemic countries worldwide. An effective vaccine will be critical to halt malaria or even succeed to final eradication. In that perspective, we studied the potential of whole sporozoite immunization by bites of P. falciparum infected mosquitoes under chemoprophylaxis (CPS). In this thesis we further explored this CPS model and assessed different immunizing doses, type of chemoprophylaxis and immunological determinants of disease and protection. We found a clear dose dependent efficacy, independent of type of chemoprophylaxis, found CD107a and CD8 T cells producing granzyme B related to protective immunity. In the field many genetically different strains circulate and a future vaccine should be able to cover multiple strains. We re-challenged volunteers with a different strain and found modest heterologously protection.We retrospectively assessed the parasitological dynamics and adverse events using a positive qPCR rather than thick smear and found reduced the clinical symptoms of malaria for volunteers after challenge.Successful malaria eradication will be more likely to be achieved with a multi-disciplinary approach. Additionally, sufficient and continuous funds will proof to be of tremendous necessity. Show less
Background The complex life cycle of malaria parasites requires well-orchestrated stage specific gene expression. In the vertebrate host the parasites grow and multiply by schizogony in two... Show moreBackground The complex life cycle of malaria parasites requires well-orchestrated stage specific gene expression. In the vertebrate host the parasites grow and multiply by schizogony in two different environments: within erythrocytes and within hepatocytes. Whereas erythrocytic parasites are well-studied in this respect, relatively little is known about the exo-erythrocytic stages. Methods In an attempt to fill this gap, genome wide RNA-seq analyses of various exo-erythrocytic stages of Plasmodium berghei including sporozoites, samples from a time-course of liver stage development and detached cells were performed. These latter contain infectious merozoites and represent the final step in exo-erythrocytic development. Results The analysis represents the complete transcriptome of the entire life cycle of P. berghei parasites with temporal detailed analysis of the liver stage allowing comparison of gene expression across the progression of the life cycle. These RNA-seq data from different developmental stages were used to cluster genes with similar expression profiles, in order to infer their functions. A comparison with published data from other parasite stages confirmed stage-specific gene expression and revealed numerous genes that are expressed differentially in blood and exo-erythrocytic stages. One of the most exo-erythrocytic stage-specific genes was PBANKA_1003900, which has previously been annotated as a "gametocyte specific protein". The promoter of this gene drove high GFP expression in exo-erythrocytic stages, confirming its expression profile seen by RNA-seq. Conclusions The comparative analysis of the genome wide mRNA expression profiles of erythrocytic and different exo-erythrocytic stages could be used to improve the understanding of gene regulation in Plasmodium parasites and can be used to model exo-erythrocytic stage metabolic networks toward the identification of differences in metabolic processes during schizogony in erythrocytes and hepatocytes. Show less
Nijhuis, R.H.T.; Lieshout, L. van; Verweij, J.J.; Claas, E.C.J.; Wessels, E. 2018
In this thesis we describe a set of studies, performed using rodent models of malaria, aimed to identify methods to improve vaccines consisting of live attenuated sporozoites, in particular... Show moreIn this thesis we describe a set of studies, performed using rodent models of malaria, aimed to identify methods to improve vaccines consisting of live attenuated sporozoites, in particular genetically attenuated parasites (GAP) vaccines. Studies in rodents and humans have shown that immunization with live-attenuated sporozoites can generate protective immunity, however induction of sterile protection in humans has required immunization with multiple vaccine doses and each dose consisting of relatively high numbers of sporozoites. Increasing the immunogenicity of whole sporozoite (wsp) vaccines can both reduce the number of sporozoites per dose and the number of vaccine doses. In the studies described in this thesis we attempted to increase GAP immunogenicity by: (i) adding adjuvants during GAP immunization; (ii) introducing genes encoding putative immunomodulatory proteins in the GAP genome to create ‘self-adjuvanting’ parasites; (iii) generating GAPs that arrest late into liver-stage development (LA-GAP) to increase antigen load and diversity during immunization; and (iv) exploring possibilities to genetically modify parasite to express vaccine antigens from different life cycle stages, in order to test the ability of parasites to induce immune responses against multiple life cycle stages and to inform the creation of a ‘multi-stage’ GAP vaccine. Show less
Geographical differences in the prevalence of allergic and chronic inflammatory diseases as well as in the efficacy of vaccines have been described. To improve our understanding of... Show moreGeographical differences in the prevalence of allergic and chronic inflammatory diseases as well as in the efficacy of vaccines have been described. To improve our understanding of immunological differences between various human populations, the innate and adaptive immune system and their responses to stimulation have been compared between Europeans and Africans. Furthermore, IgG glycosylation profiles have been compared between several populations and in response to vaccination. In addition, the immune response to controlled human malaria infection has been determined in Europeans and Africans by mass cytometry as well as the role of γδ T cells and CD4+ T cells in immune responses during natural malaria infection in Indonesian children. The results indicated striking differences that need to be taken into account. It might be necessary to develop population-specific vaccines and treatments against infectious, allergic, and chronic inflammatory diseases. Show less
Prasetyani, M.A.; Mast, Q. de; Afeworki, R.; Kaisar, M.M.M.; Stefanie, D.; Sartono, E.; ... ; Ven, A.J. van der 2017
The work presented in this thesis aimed at increasing our understanding of the effect of helminths on Plasmodium spp. immune response in co-infected individuals living in endemic countries. It... Show moreThe work presented in this thesis aimed at increasing our understanding of the effect of helminths on Plasmodium spp. immune response in co-infected individuals living in endemic countries. It presents data from studies conducted in rural and semi urban areas of Lambaréné (Gabon) where the burden of malaria and helminths is particularly important. Although scarce previous studies have indicated an effect of helminths on malaria outcomes and immune response to Plasmodium spp. parasite in co-infected subjects. However it is still debated how consistent is this effect across study sites and teams and what its immunological basis is. Show less
Low birth weight including preterm birth and intrauterine growth retardation, remains important in sub-Saharan Africa and particularly highly prevalent in Gabon. Among the risk factors of... Show more Low birth weight including preterm birth and intrauterine growth retardation, remains important in sub-Saharan Africa and particularly highly prevalent in Gabon. Among the risk factors of low birth weight in sub-Saharan Africa are very young maternal age, first pregnancy, poor gestational nutrition and small stature of the mother. In Gabon, besides malaria, the other two major parasitic infections namely urogenital schistosomiasis and the filarial infection Loa loa, are common in pregnant women. Maternal schistosomiasis like malaria showed to be associated with higher proportions of low birth weight babies. Mefloquine as an alternative preventive treatment, despite showing no difference with sulphadoxine – pyrimethamine in preventing low birth weight, was however more effective in preventing malaria infection and anaemia. Mefloquine administered for the prevention of malaria was effective against concomitant urogenital schistosomiasis, suggesting that mefloquine could seriously be considered as a combined intervention for both malaria and schistosomiasis during pregnancy, and an alternative to praziquantel. Maternal infection with L. loa was associated with expansion in the neonatal cord blood of functionally activate Tregs that kept Th1 and Th17 immune responses in check, providing some insights on the impact of in utero exposure on the offspring’s development and health. Show less
