Progress and Challenges towards Point-of-Care Diagnostic Development for Dengue

Pang, Junxiong; Chia, Po Ying; Lye, David C.; Leo, Yee Sin

doi:10.1128/jcm.00707-17

Cited by 44 publications

(50 citation statements)

References 57 publications

(41 reference statements)

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“…Dengue infection can be confirmed by isolating the virus and immunologically detecting viral protein Ns1 86 . Real-time PCR analysis is widely used for Ebola virus diagnostics, but expensive equipment and well-trained personnel are required to obtain accurate results 87 . Most importantly, the situation in the areas of outbreaks requires urgent action to identify infected persons and curb disease spread.…”

Section: Crispr-tools For Emerging and Re-emerging Infectious Diseasesmentioning

confidence: 99%

CRISPR-Cas Systems for Diagnosing Infectious Diseases

Kostyusheva¹,

Brezgin²,

Babin³

et al. 2020

Preprint

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Infectious diseases are a global health problem affecting billions of people. Developing rapid and sensitive diagnostic tools is key for successful patient management and curbing disease spread. Currently available diagnostics are very specific and sensitive but time-consuming and require expensive laboratory settings and well-trained personnel; thus, they are not available in resource-limited areas, for the purposes of large-scale screenings and in case of outbreaks and epidemics. Developing new, rapid, and affordable point-of-care diagnostic assays is urgently needed. This review focuses on CRISPR-based technologies and their perspectives to become platforms for point-of-care nucleic acid detection methods and as deployable diagnostic platforms that could help to identify and curb outbreaks and emerging epidemics. We describe the mechanisms and function of different classes and types of CRISPR-Cas systems, including pros and cons for developing molecular diagnostic tests and applications of each type to detect a wide range of infectious agents. Many Cas proteins (Cas9, Cas12, Cas13, Cas14) have been leveraged to create highly accurate and sensitive diagnostic tools combined with technologies of signal amplification and fluorescent, potentiometric, colorimetric, or lateral flow assay detection. In particular, the most advanced platforms -- SHERLOCK/v2, DETECTR, or CRISPR-Chip -- enable detection of attomolar amounts of pathogenic nucleic acids with specificity comparable to that of PCR but with minimal technical settings. Further developing CRISPR-based diagnostic tools promises to dramatically transform molecular diagnostics, making them easily affordable and accessible virtually anywhere in the world. The burden of socially significant diseases, frequent outbreaks, recent epidemics (MERS, SARS and the ongoing coronoviral nCov-2019 infection) urgently need the developing of express-diagnostic tools. Recently devised CRISPR-technologies represent the unprecedented opportunity to reshape epidemiological surveillance and molecular diagnostics.

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Section: Crispr-tools For Emerging and Re-emerging Infectious Diseasesmentioning

confidence: 99%

CRISPR-Cas Systems for Diagnosing Infectious Diseases

Kostyusheva¹,

Brezgin²,

Babin³

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…Recently, the portable, simple and compact POCKIT Nucleic Acid Analyzer (POCKIT, GeneReach, Taichung, Taiwan) which can automatically detect and interpret PCR results within one hour became available in mobile PCR laboratory formats [21][22][23]. A lightweight hand-held model, POCKIT Micro Plus Nucleic Acid Analyzer (POCKIT Micro Plus), that works with a built-in rechargeable battery is also available.…”

Section: Rt-pcr [Qrt-pcr])mentioning

confidence: 99%

An RT-PCR panel for rapid serotyping of dengue virus serotypes 1 to 4 in human serum and mosquito on a field-deployable PCR system

et al. 2019

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BackgroundDengue fever, a mosquito-borne disease, is caused by dengue virus (DENV) which includes four major serotypes (DENV-1, -2, -3, and -4). Some serotypes cause more severe diseases than the other; severe dengue is associated with secondary infections by a different serotype. Timely serotyping can provide early warning of dengue epidemics to improve management of patients and outbreaks. A mobile insulated isothermal PCR (iiPCR) system is available to allow molecular detection of pathogens near points of need.Methodology/Principle findingsIn this study, side-by-side comparison with the CDC DENV-1-4 Real Time RT-PCR (qRT-PCR) was performed to evaluate the performance of four singleplex DENV-1–4 serotyping reverse transcription-iiPCR (RT-iiPCR) reagents for DENV subtyping on the mobile PCR system. The four RT-iiPCRs did not react with Zika virus and chikungunya virus; tests with serial dilutions of the four DENV serotypes made in human serum showed they had detection endpoints comparable to those of the reference method, indicating great analytical sensitivity and specificity. Clinical performance of the RT-iiPCR reagents was evaluated by testing 40 serum samples each (around 20 target serotype-positive and 20 DENV-negative); all four reagents had high agreement (97.5–100%) with the reference qRT-PCR. Moreover, testing of mosquitoes separately infected experimentally with each serotype showed that the four reagents detected specifically their target DENV serotypes in mosquito.Conclusions/SignificanceWith analytical and clinical performance comparable to the reference qRT-PCR assay, the four index RT-iiPCR reagents on the field-deployable PCR system can serve as a useful tool for DENV detection near points of needs.

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“…Combined with automated RNA extraction protocols, RT-PCR can potentially facilitate fast and accurate diagnosis of dengue suspected cases with a single sample obtained during the patient's first visit to the clinician. However, the requirement of a pre-PCR nucleic acids extraction step, an expensive and not easy to use thermal cycler, and a highly trained technician make PCR technology not accessible in resource-limited regions where dengue is endemic [10,21]. Molecular diagnosis tools for dengue is still much needed at or near POC/PON, particularly for developing countries with limited public health resources.…”

Section: Introductionmentioning

confidence: 99%

A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos

Tsai¹,

Liu²,

Lin³

et al. 2019

PLoS ONE

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Background The insulated isothermal PCR (iiPCR) technology enables consistent PCR amplification and detection in a simple heating device. A pan-dengue virus (DENV) RT-iiPCR, targeting the 5’ untranslated region, was validated previously on the semi-automated POCKIT combo system (involving separate devices for nucleic acid extraction and PCR amplification/detection) to offer performance comparable to a laboratory real-time PCR. Working on the same technologies, a compact automated sample-in-answer-out system (POCKIT Central Nucleic Acid Analyser) has been available commercially for iiPCR, minimizing human error risks and allowing easy molecular bio-detection near points of need. Here, we evaluated the analytical and clinical performance of the pan-DENV RT-iiPCR on the fully automated system by comparison to those on the semi-automated system. Methodology/Principal findings Testing sera containing serial diluted DENV-1, -2, -3, or -4 cell culture stock, the pan-DENV RT-iiPCR system had similar 100% detection endpoints on the two systems; i.e. at 1, 10, 1 and 10 PFU/ml, respectively, on the fully automated system, and at 10, 1, 10 and 10 PFU/ml, respectively, on the semi-automated system. Furthermore, both fully automated and semi-automated PCR system can detect all four DENV serotypes in mosquitos. Clinical performance of the reagent on the two systems was evaluated by testing 60 human serum samples. Both systems detected the same 40 samples (ten DENV-1, -2, -3, and -4 positive each) and did not detect the other 20; 100% agreement (κ = 1) was found between the two systems. Conclusions/Significance With performance comparable to a previously validated system, the fully-automated PCR system allows applications of the pan-DENV reagent as a useful tool near points of need to facilitate easy, fast and effective detection of dengue virus and help mitigate versatile public health challenges in the control and management of dengue disease.

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Progress and Challenges towards Point-of-Care Diagnostic Development for Dengue

Cited by 44 publications

References 57 publications

CRISPR-Cas Systems for Diagnosing Infectious Diseases

CRISPR-Cas Systems for Diagnosing Infectious Diseases

An RT-PCR panel for rapid serotyping of dengue virus serotypes 1 to 4 in human serum and mosquito on a field-deployable PCR system

A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos

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