Urinary schistosomiasis is a waterborne parasitic infection caused by Schistosoma haematobium that affects approximately 30 million people annually in Nigeria. Treatment and eradication of this infection require effective diagnostics. However, current diagnostic tests have critical shortcomings and consequently are of limited value to stakeholders throughout the health care system who are involved in targeting the diagnosis and subsequent control of schistosomiasis. New diagnostic devices that fit the local health care infrastructure and support the different stakeholder diagnostic strategies remain a critical need. This study focuses on understanding, by means of Q-methodology, the context of use and application of a new diagnostic device that is needed to effectively diagnose urinary schistosomiasis in Oyo State, Nigeria. Q-methodology is a technique that investigates subjectivity by exploring how stakeholders rank-order opinion statements about a phenomenon. In this study, 40 statements were administered to evaluate stakeholder perspectives on the context of use and application of potential new diagnostic devices and how these perspectives or viewpoints are shared with other stakeholders. Potential new diagnostic devices will need to be deployable to remote or distant communities, be affordable, identify and confirm infection status before treatment in patients whose diagnosis of urinary schistosomiasis is based on self-reporting, and equip health care facilities with diagnostic devices optimized for the local setting while requiring local minimal infrastructural settings. Similarly, the context of use and application of a potential new diagnostic device for urinary schistosomiasis is primarily associated with the tasks stakeholders throughout the health care system perform or procedures employed. These findings will guide the development of new diagnostic devices for schistosomiasis that match the contextual landscape and diagnostic strategies in Oyo.

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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 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.

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Nucleic-acid detection is crucial for basic research as well as for applications in medicine such as diagnostics. In resource-limited settings, however, most DNA-detection diagnostic schemes are inapplicable since they rely on expensive machinery, electricity, and trained personnel. Here, we present an isothermal DNA detection scheme for the diagnosis of pathogenic DNA in resource-limited settings. DNA was extracted from urine and blood samples using two different instrument-free methods, and amplified using Recombinase Polymerase Amplification with a sensitivity of <10 copies of DNA within 15 minutes. Target DNA was bound by dCas9/sgRNA that was labelled with a DNA oligomer to subsequently induce Rolling Circle Amplification. This second amplification step produced many copies of a G-quadruplex DNA structure that facilitates a colorimetric readout that is visible to the naked eye. This isothermal DNA-detection scheme can be performed at temperatures between 20-45 °C. As an example of the applicability of the approach, we isothermally (23 °C) detected DNA from a parasite causing visceral leishmaniasis that was spiked into buffer and resulted in a sensitivity of at least 1 zeptomole. For proof of principle, DNA spiked into blood was coupled to the CRISPR-dCas9-based detection scheme yielding a colorimetric readout visible to the naked eye. Given the versatility of the guide-RNA programmability of targets, we envision that this DNA detection scheme can be adapted to detect any DNA with minimal means, which facilitates applications such as point-of-care diagnostics in resource-limited settings.

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Inadequate and nonintegrated diagnostics are the Achilles' heel of global efforts to monitor, control, and eradicate neglected tropical diseases (NTDs). While treatment is often available, NTDs are endemic among marginalized populations, due to the unavailability or inadequacy of diagnostic tests that cause empirical misdiagnoses. The need of the hour is early diagnosis at the point-of-care (PoC) of NTD patients. Here, we review the status quo of PoC diagnostic tests and practices for all of the 24 NTDs identified in the World Health Organization's (WHO) 2021-2030 roadmap, based on their different diagnostic requirements. We discuss the capabilities and shortcomings of current diagnostic tests, identify diagnostic needs, and formulate prerequisites of relevant PoC tests. Next to technical requirements, we stress the importance of availability and awareness programs for establishing PoC tests that fit endemic resource-limited settings. Better understanding of NTD diagnostics will pave the path for setting realistic goals for healthcare in areas with minimal resources, thereby alleviating the global healthcare burden.

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The rapid growth of point-of-care (POC) diagnostic tests necessitates a clear vision of when, where, and why a new POC diagnostic test needs to be developed and how it can be used in a way that matches a local health care context. Here, we present an innovative approach toward developing a concept target product profile (CTPP), which is a new mapping tool that helps researchers match a new diagnostic test to a specific local health care context early in the research and development process. As a case study, we focus on the diagnosis of visceral leishmaniasis (VL) in rural resource-limited regions of Kenya and Uganda. Our stepwise approach integrates elements of design thinking and uses a combination of literature reviews and field research for a context analysis of local health care systems and practices. We then use visual thinking in the form of Gigamaps and patient journeys to identify use case scenarios and to present our findings from the field research to key stakeholders. The use case scenarios describe the diagnostic scope of a new POC test based on the feasibility of the new test, the local need, and the contextual fit. For our case study of VL, we identify 2 valuable use case scenarios, namely test-of-cure and screening and confirmation, and we formulate a CTPP. We anticipate that a CTPP will enable researchers to match a new POC diagnostic test during the research and development process to the local health care context in which it will be used.

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The aim of this thesis was to develop a DNA-detection scheme for a point-of-care diagnostic test for Neglected Tropical Diseases (NTDs) for use within resource-limited settings. The scientific innovation is to develop an adaptable DNA-detection scheme, using CRISPR-dCas9 (catalytically inactive Cas9), that can detect the DNA of any pathogen in bodily fluids i.e. in a blood or urine sample. This detection of DNA of the pathogen will be much more reliable than antibody-based tests as it will work independently of the persons immune response. Unlike current antibody-based diagnostic tests, it will be able to distinguish between current and previous infections. Specifically for visceral leishmaniasis (VL), the current rk39 antigen-based rapid diagnostic test lacks specificity and sensitivity in sub-Saharan Africa, where VL remains prevalent. We aim for a DNA-detection scheme that does not require infrastructure, electricity, or skilled laboratory personnel to operate. Furthermore, the DNA-detection scheme will need to be functional at a broad temperature range, yet remain highly sensitive and specific. Such a DNA-detection scheme can be a promising tool for effective diagnoses of NTDs within resource-limited settings, though it needs to be further tested, incorporated into a packaged test format, and validated in the field. Integrating this DNA-detection scheme into a potentially low-cost diagnostic test is a very promising alternative to current diagnostic tests in both high-resource and resource-limited settings. @en

Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore experiments that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing. In our nanopore experiments, we observe the CRISPR-dCas9 proteins as local spikes that appear on top of the ionic current blockade signal of DNA molecules that translocate through the nanopore. The proteins exhibit a pronounced blockade signal that allows for facile identification of the targeted sequence. Even at the high salt conditions (1 M LiCl) required for nanopore experiments, dCas9 proteins are found to remain stably bound. The binding position of the target sequence can be read from the spike position along the DNA signal. We anticipate applications of this nanopore-based CRISPR-dCas9 biosensing approach in DNA-typing based diagnostics such as quick disease-strain identification, antibiotic-resistance detection, and genome typing.

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