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      Diagnosing point-of-care diagnostics for neglected tropical diseases

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          Abstract

          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.

          Author summary

          Diagnostic practices are crucial for neglected tropical diseases (NTDs). In this review, we critically discuss each of 24 NTDs as defined in the 2021–2030 roadmap of the World Health Organization (WHO), to identify their specific point-of-care (PoC) diagnostic needs. In doing so, we sketch possible solutions to meet the diagnostic needs by providing realistic technical solutions that will enable early diagnosis of NTDs. We group NTDs in accordance to the common solutions for their PoC diagnostic needs so that joint interventions can be applied for multiple NTDs, in order to lessen the global burden of NTDs.

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          Most cited references97

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          Field-deployable viral diagnostics using CRISPR-Cas13

          Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in <2 hours. We further demonstrate that SHERLOCK can distinguish the four DENV serotypes, as well as region-specific strains of ZIKV from the 2015-2016 pandemic. Finally, we report the rapid (<1 week) design and testing of instrument-free assays to detect clinically relevant viral single-nucleotide polymorphisms.
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            SHERLOCK: nucleic acid detection with CRISPR nucleases

            Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.
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              CRISPR/Cas Systems towards Next-Generation Biosensing

              Beyond its remarkable genome editing ability, the CRISPR/Cas9 effector has also been utilized in biosensing applications. The recent discovery of the collateral RNA cleavage activity of the Cas13a effector has sparked even greater interest in developing novel biosensing technologies for nucleic acid detection and promised significant advances in CRISPR diagnostics. Now, along with the discovery of Cas12 collateral cleavage activities on single-stranded DNA (ssDNA), several CRISPR/Cas systems have been established for detecting various targets, including bacteria, viruses, cancer mutations, and others. Based on key Cas effectors, we provide a detailed classification of CRISPR/Cas biosensing systems and propose their future utility. As the field continues to mature, CRISPR/Cas systems have the potential to become promising candidates for next-generation diagnostic biosensing platforms.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                17 June 2021
                June 2021
                : 15
                : 6
                : e0009405
                Affiliations
                [001]Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, The Netherlands
                Foundation for Innovative New Diagnostics (FIND), SWITZERLAND
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0003-0825-4411
                https://orcid.org/0000-0002-6160-6896
                https://orcid.org/0000-0001-6273-071X
                Article
                PNTD-D-20-01984
                10.1371/journal.pntd.0009405
                8211285
                34138846
                6fdf5488-d8e3-438f-a1b6-ddd15c878c20
                © 2021 Bharadwaj et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Page count
                Figures: 5, Tables: 1, Pages: 21
                Product
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001831, Technische Universiteit Delft;
                Award Recipient :
                This work was supported by Delft Global Initiative ( https://www.tudelft.nl/global) CD received the grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Review
                Medicine and Health Sciences
                Medical Conditions
                Tropical Diseases
                Neglected Tropical Diseases
                Medicine and Health Sciences
                Diagnostic Medicine
                Medicine and Health Sciences
                Diagnostic Medicine
                Virus Testing
                Biology and Life Sciences
                Biochemistry
                Biomarkers
                Biology and Life Sciences
                Veterinary Science
                Veterinary Diseases
                Medicine and Health Sciences
                Medical Conditions
                Tropical Diseases
                Neglected Tropical Diseases
                Leishmaniasis
                Medicine and Health Sciences
                Medical Conditions
                Parasitic Diseases
                Protozoan Infections
                Leishmaniasis
                Medicine and Health Sciences
                Medical Conditions
                Infectious Diseases
                Zoonoses
                Leishmaniasis
                Medicine and Health Sciences
                Medical Conditions
                Tropical Diseases
                Neglected Tropical Diseases
                Dengue Fever
                Medicine and Health Sciences
                Medical Conditions
                Infectious Diseases
                Viral Diseases
                Dengue Fever
                Medicine and Health Sciences
                Oncology
                Cancer Treatment
                Antibody Therapy
                Medicine and Health Sciences
                Clinical Medicine
                Clinical Immunology
                Antibody Therapy
                Biology and Life Sciences
                Immunology
                Clinical Immunology
                Antibody Therapy
                Medicine and Health Sciences
                Immunology
                Clinical Immunology
                Antibody Therapy

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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