In September 2022, the Drug Discovery Unit at the University of Dundee, UK, organised an international meeting at the Wellcome Collection in London to explore the current clinical situation and... Show moreIn September 2022, the Drug Discovery Unit at the University of Dundee, UK, organised an international meeting at the Wellcome Collection in London to explore the current clinical situation and challenges associated with treating schistosomiasis. The aim of this meeting was to discuss the need for new treatments in view of the clinical situation and to ascertain what the key requirements would be for any potential new anti-schistosomals. This information will be essential to inform ongoing drug discovery efforts for schistosomiasis. We also discussed the potential drug discovery pathway and associated criteria for progressing compounds to the clinic. To date, praziquantel (PZQ) is the only drug available to treat all species causing schistosomiasis, but it is often unable to completely clear parasites from an infected patient, partially due to its inact i v i t y against juvenile worms. PZQ-mediated mass drug administration campaigns conducted in endemic areas (e.g., sub-Saharan Africa, where schistosomiasis is primarily prevalent) have contributed to reducing the burden of disease but wi l l not eliminate the disease as a public health problem. The potential for Schistosoma to develop resistance towards PZQ , as the sole treatment available, could become a concern. Consequently, new anthelmintic medications are urgently needed, and this Perspective aims to capture some of the learnings from our discussions on the key criteria for new treatments. Show less
During intestinal schistosomiasis, hundreds of parasite eggs transit across their mammalian host’s intestinal wall, triggering an exuberant immune response and considerable tissue damage. To limit... Show moreDuring intestinal schistosomiasis, hundreds of parasite eggs transit across their mammalian host’s intestinal wall, triggering an exuberant immune response and considerable tissue damage. To limit host pathology and thereby promote their own survival, schistosomes produce a variety of immunomodulatory molecules that modify host immunity. Schistosomes may also gain assistance from the intestinal microbiota. This thesis dissects the involvement of parasite, host, and microbial factors in the instruction of schistosome-associated immune responses. Using mouse models of egg producing and non-egg producing schistosomiasis, this thesis provides a concise narrative of schistosome-associated immune profiles over time and across multiple tissue sites, and offers insight into the contribution of key cell types (Bregs, CD11c+ cells) and signalling networks (Type I interferons) in immune response instruction. Focusing on the intestine, this thesis describes the impact of schistosome egg production on intestinal barrier integrity, microbiota structure and the colonic immune system. Finally, using germ-free mice and faecal transplants, this thesis provides evidence that the schistosome infection associated microbiota can influence the flavour of host Immunity. Together, these data elevate the mechanistic understanding of parasite-host-microbial relations and provides a strong platform for the future study of schistosome or microbial factors in the modulation of inflammatory disease. Show less
For many parasitic diseases, the microscopic examination of clinical samples such as urine and stool still serves as the diagnostic reference standard, primarily because microscopes are accessible... Show moreFor many parasitic diseases, the microscopic examination of clinical samples such as urine and stool still serves as the diagnostic reference standard, primarily because microscopes are accessible and cost-effective. However, conventional microscopy is laborious, requires highly skilled personnel, and is highly subjective. Requirements for skilled operators, coupled with the cost and maintenance needs of the microscopes, which is hardly done in endemic countries, presents grossly limited access to the diagnosis of parasitic diseases in resource-limited settings. The urgent requirement for the management of tropical diseases such as schistosomiasis, which is now focused on elimination, has underscored the critical need for the creation of access to easy-to-use diagnosis for case detection, community mapping, and surveillance. In this paper, we present a low-cost automated digital microscope-the Schistoscope-which is capable of automatic focusing and scanning regions of interest in prepared microscope slides, and automatic detection of Schistosoma haematobium eggs in captured images. The device was developed using widely accessible distributed manufacturing methods and off-the-shelf components to enable local manufacturability and ease of maintenance. For proof of principle, we created a Schistosoma haematobium egg dataset of over 5000 images captured from spiked and clinical urine samples from field settings and demonstrated the automatic detection of Schistosoma haematobium eggs using a trained deep neural network model. The experiments and results presented in this paper collectively illustrate the robustness, stability, and optical performance of the device, making it suitable for use in the monitoring and evaluation of schistosomiasis control programs in endemic settings. Show less
For many parasitic diseases, the microscopic examination of clinical samples such as urine and stool still serves as the diagnostic reference standard, primarily because microscopes are accessible... Show moreFor many parasitic diseases, the microscopic examination of clinical samples such as urine and stool still serves as the diagnostic reference standard, primarily because microscopes are accessible and cost-effective. However, conventional microscopy is laborious, requires highly skilled personnel, and is highly subjective. Requirements for skilled operators, coupled with the cost and maintenance needs of the microscopes, which is hardly done in endemic countries, presents grossly limited access to the diagnosis of parasitic diseases in resource-limited settings. The urgent requirement for the management of tropical diseases such as schistosomiasis, which is now focused on elimination, has underscored the critical need for the creation of access to easy-to-use diagnosis for case detection, community mapping, and surveillance. In this paper, we present a low-cost automated digital microscope-the Schistoscope-which is capable of automatic focusing and scanning regions of interest in prepared microscope slides, and automatic detection of Schistosoma haematobium eggs in captured images. The device was developed using widely accessible distributed manufacturing methods and off-the-shelf components to enable local manufacturability and ease of maintenance. For proof of principle, we created a Schistosoma haematobium egg dataset of over 5000 images captured from spiked and clinical urine samples from field settings and demonstrated the automatic detection of Schistosoma haematobium eggs using a trained deep neural network model. The experiments and results presented in this paper collectively illustrate the robustness, stability, and optical performance of the device, making it suitable for use in the monitoring and evaluation of schistosomiasis control programs in endemic settings. Show less
Pillay, P.; Downs, J.A.; Changalucha, J.M.; Brienen, E.A.T.; Ramarokoto, C.E.; Leutscher, P.D.C.; ... ; Lieshout, L. van 2020
Female Genital Schistosomiasis (FGS) is a neglected disease affecting millions, however challenging to diagnose. This explorative descriptive study compares Schistosoma real-time PCR analysis of... Show moreFemale Genital Schistosomiasis (FGS) is a neglected disease affecting millions, however challenging to diagnose. This explorative descriptive study compares Schistosoma real-time PCR analysis of cervico-vaginal lavages (CVL) with corresponding urine and stool samples of 933 women from five different previously described study populations. Sampling included 310 women from an S. mansoni endemic region in Mwanza, Tanzania and 112 women from a nearby S. haematobium endemic region. Findings were compared with samples collected from S. haematobium endemic regions in South Africa from 394 women and from 117 women from Madagascar of which 79 were urine pre-selected microscopy positive cases from highly-endemic communities and 38 were urine microscopy negatives from a low-endemic community. As anticipated, urine and stool microscopy and gynecological investigations varied substantially between study populations; however, the same Schistosoma real-time PCR was performed in one reference laboratory. Schistosoma DNA was detected in 13% (120/933) of the CVL, ranging from 3% in the S. mansoni Tanzanian endemic region to 61% in the pre-selected Malagasy urine microscopy positive cases. Detectable Schistosoma DNA in CVL was associated with Schistosoma DNA in urine but not with microscopic detection of eggs in urine or by cytological examination. This study confirmed real-time PCR for the detection of Schistosoma DNA in gynecological samples to be a valuable diagnostic tool to study the distribution of FGS within schistosomiasis endemic areas. Show less
Parasitic helminths modulate host immune responses. While the induction of type 2 immune responses is a widely recognized feature of helminth infections, a network of regulatory immune responses is... Show moreParasitic helminths modulate host immune responses. While the induction of type 2 immune responses is a widely recognized feature of helminth infections, a network of regulatory immune responses is often dominant during the chronic phase of infection. Suppression of the host immune system during helminth infections inhibits anti-parasite immunity, prevents tissue damage due to excessive inflammation and conveys spill-over suppression to inflammatory conditions such as allergy and asthma. The first part of this thesis focuses on the role of regulatory B cells, a prominent member of the immune regulatory network, in protection from allergic asthma by chronic Schistosoma (S.) mansoni infections. It furthermore identifies signals required for schistosome-induced regulatory B cell development. The second part of this thesis describes the protective effect of S. mansoni eggs, and a specific egg-derived glycoprotein, against allergic asthma in the absence of chronic infection. A better understanding how helminthes including S. mansoni modulate host immune responses, and the implications this has for inflammatory diseases such as allergic asthma, may provide valuable leads for the development of novel pharmaceutical agents for the treatment of allergic disorders. Show less
The detection of intestinal parasitic infections for routine diagnosis and for epidemiological research still depends mainly on microscopical examination of stool samples for the identification of... Show moreThe detection of intestinal parasitic infections for routine diagnosis and for epidemiological research still depends mainly on microscopical examination of stool samples for the identification of helminth eggs and protozoan trophozoites and cysts. Because microscopy has several limitations, additional diagnostic methods (e.g. culture, antigen detection) have been introduced to improve the detection of intestinal parasites. Although such additional methods increase sensitivity, the amount of hands-on time accumulates substantially. In chapter 1 various diagnostic methods are summarized and the diagnostic challenges are described for the most important intestinal parasitic infections. During the last years remarkable progress has been made in the development of molecular diagnostic methods based on the Polymerase Chain Reaction (PCR) technique. For the detection of intestinal parasites, DNA isolation from stool can be processed in a semi- or fully-automated system where after specific DNA of multiple targets can be amplified simultaneously, visualized on screen and semi-quantified in a closed tube system with a multiplex real-time PCR. In this thesis, a diagnostic approach using multiplex real-time PCR is assessed for the routine clinical diagnosis and epidemiology of intestinal parasites. In chapter 2 the diagnostic results that were obtained with multiplex-real-time PCR for the detection of Entamoeba histolytica, Giardia lamblia and Cryptosporidium parvum / C._hominis (HGC PCR) were compared with the results obtained by routine microscopy on faecal samples from patients visiting their general practitioner because of gastrointestinal symptoms. The study revealed that significant numbers of G._lamblia and Cryptosporidium infections remained undetected with microscopy, even with the use of a multiple sampling procedure of faecal specimens in combination with fixatives (also referred to as the triple faeces test (TFT) procedure). Parasites that had been detected with microscopy but not with real-time PCR consisted mainly of non-pathogenic parasites and Dientamoeba fragilis, of which its pathogenicity is still disputed upon. Hence, compared to the molecular approach, microscopy provided limited sensitivity in a routine diagnostic setting of general practice patients with gastro-intestinal complaints. For the same patient group as described in chapter 2, the results of Cryptosporidium detection with the HGC-PCR were studied in more detail in chapter 3. Microscopic examination for Cryptosporidium, which requires an additional staining procedure of the faecal smear, was specifically requested by general practitioners twenty-one times over a period of approximately seven months and was found positive in 13 cases. In contrast to the conventional diagnostic approach, with HGC PCR 80 cases were detected. The prevalence of Cryptosporidium in Dutch patients with gastro-intestinal complaints attending their general practitioner has so far exceeded the figures in previous studies. The highest infection rate has been detected among children aged under five years with a peak in the month of September: almost one-third of them had been infected with Cryptosporidium, mainly with the species Cryptosporidium hominis. The lack of request for an additional diagnostic procedure to detect Cryptosporidium is not the only reason for missing infections. Basic microscopic stool examination was often not requested, leaving a substantial number of gastro-intestinal parasitic infections undiagnosed. Besides the Dutch general practice patients group, the diagnostic approach with real-time PCR was also assessed for the routine diagnosis of intestinal parasites in returning travellers. Chapter 4 describes a prospective study where the results of 2591 microscopic examinations and antigen detections (i.e. care as usual) were compared with the results of those from the HGC-PCR and an additional Strongyloides stercoralis real-time PCR. The detection rates of all four targeted parasite species were increased with real-time PCR whereas the prevalence of ten additional pathogenic parasite species found with microscopy was 0.5% at most. A fully automated DNA isolation process and extension of the molecular diagnostic targets could further increase the detection rates of parasites while having considerable impact on cost-efficiency of the diagnostic procedures in the laboratory. Chapter 5 describes the molecular epidemiology of G. lamblia infections in the group of returned travellers. Symptoms of G._lamblia infections in this group, consisting mainly of adults, were highly variable and ranged from asymptomatic to the presence of severe gastro-intestinal complaints. Data in this study showed that although the G._lamblia Cycle-threshold (Ct)-values, which reflect the intensity of infection, correlated with the number of specified gastro-intestinal complaints, the parasite was also detected in 4.7 % of the travellers who did not have any intestinal symptoms. The reasons for the clinical heterogeneity of G._lamblia infections are still not fully understood. In previous studies the epidemiological role of G._lamblia assemblages (i.e. group of genotypes) have been suggested as an important factor associated with gastro-intestinal complaints, however, this could not be proven in this group of travellers. In chapter 6 a real-time PCR for the detection of Isospora belli was evaluated and in chapter 7 a multiplex real-time PCR was evaluated for the detection of Encephalitozoon intestinalis and Enterocytozoon bieneusi. These real-time PCRs have been developed in addition to the panel of molecular diagnostic assays to be used in a clinical setting and in epidemiological studies. As such, a phylogenetic study of E._bieneusi infections in persons with different clinical backgrounds is described in chapter 8. The results indicate a dynamic evolutionary process between genotypes of E._bieneusi. One specific genotype was restricted to transplantation patients receiving immuno-supressives and another genotype showed its preferential habitat in patients living with HIV/AIDS, which further emphasizes the predisposition for specific hosts by different E._bieneusi genotypes. Although Schistosoma mansoni and Schistosoma haematobium are living in the blood vessels and not in the intestinal lumen, this thesis also elaborates on real-time PCR for the detection of Schistosoma in humans. In chapter 9 a multiplex real-time PCR was evaluated for the detection of both S. mansoni and S._haematobium in stool samples collected in an area endemic for both Schistosoma species. The detected Ct-values correlated with the results from quantitative microscopy on stool and on urine samples. Furthermore, the use of ethanol-suspended stool samples for storage and transport at ambient temperatures, as was done in this study, makes collection of samples at remote areas more attractive. In chapter 10, the performance of the Schistosoma real-time PCR was evaluated for the diagnosis of female genital schistosomiasis on DNA isolated from vaginal lavages. The preliminary results in this study showed that the semi-quantitative outcome of the PCR can be used in the differential diagnosis of vaginal lesions and as a predictor of disease pathology. In conclusion, the comparative studies in this thesis revealed that the introduction of real-time PCR for routine detection of diarrhoea causing protozoa and helminths will improve the diagnostic efficiency of laboratories dealing with faecal samples. Standard diagnostic procedures can further be improved by the design of real-time PCR panels for specific patient groups containing parasitic, bacterial, fungal and / or viral targets. Furthermore, with the simple sample collection procedure and the high throughput potential, the multiplex real-time PCR showed to be a powerful tool for epidemiological studies, even in remote areas. The real-time PCR method has little value for clinical diagnostics in low-income countries but can certainly play a prominent role as a gold standard in quality control for the evaluation of new, inexpensive rapid tests for field diagnostics. Show less
