Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria
© 2020 National Academy of Sciences. All rights reserved. Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 parasites per microliter blood) that work in...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Proceedings of the National Academy of Sciences
2021
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| Online Access: | https://hdl.handle.net/1721.1/136151 |
| _version_ | 1811072078765359104 |
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| author | Lee, Rose A Puig, Helena De Nguyen, Peter Q Angenent-Mari, Nicolaas M Donghia, Nina M McGee, James P Dvorin, Jeffrey D Klapperich, Catherine M Pollock, Nira R Collins, James J |
| author_facet | Lee, Rose A Puig, Helena De Nguyen, Peter Q Angenent-Mari, Nicolaas M Donghia, Nina M McGee, James P Dvorin, Jeffrey D Klapperich, Catherine M Pollock, Nira R Collins, James J |
| author_sort | Lee, Rose A |
| collection | MIT |
| description | © 2020 National Academy of Sciences. All rights reserved. Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care diagnostics are also lacking for nonfalciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods, such as PCR, have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS. Here we describe a CRISPR-based diagnostic for ultrasensitive detection and differentiation of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, using the nucleic acid detection platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking). We present a streamlined, fieldapplicable, diagnostic comprised of a 10-min SHERLOCK parasite rapid extraction protocol, followed by SHERLOCK for 60 min for Plasmodium species-specific detection via fluorescent or lateral flow strip readout. We optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction, and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization. Our P. falciparum and P. vivax assays exhibited 100% sensitivity and specificity on clinical samples (5 P. falciparum and 10 P. vivax samples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid point-of-care clinical diagnostic for nonfalciparum malaria species and low parasite density P. falciparum infections. |
| first_indexed | 2024-09-23T09:00:34Z |
| format | Article |
| id | mit-1721.1/136151 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T09:00:34Z |
| publishDate | 2021 |
| publisher | Proceedings of the National Academy of Sciences |
| record_format | dspace |
| spelling | mit-1721.1/1361512021-10-28T04:49:47Z Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria Lee, Rose A Puig, Helena De Nguyen, Peter Q Angenent-Mari, Nicolaas M Donghia, Nina M McGee, James P Dvorin, Jeffrey D Klapperich, Catherine M Pollock, Nira R Collins, James J © 2020 National Academy of Sciences. All rights reserved. Asymptomatic carriers of Plasmodium parasites hamper malaria control and eradication. Achieving malaria eradication requires ultrasensitive diagnostics for low parasite density infections (<100 parasites per microliter blood) that work in resource-limited settings (RLS). Sensitive point-of-care diagnostics are also lacking for nonfalciparum malaria, which is characterized by lower density infections and may require additional therapy for radical cure. Molecular methods, such as PCR, have high sensitivity and specificity, but remain high-complexity technologies impractical for RLS. Here we describe a CRISPR-based diagnostic for ultrasensitive detection and differentiation of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, using the nucleic acid detection platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking). We present a streamlined, fieldapplicable, diagnostic comprised of a 10-min SHERLOCK parasite rapid extraction protocol, followed by SHERLOCK for 60 min for Plasmodium species-specific detection via fluorescent or lateral flow strip readout. We optimized one-pot, lyophilized, isothermal assays with a simplified sample preparation method independent of nucleic acid extraction, and showed that these assays are capable of detection below two parasites per microliter blood, a limit of detection suggested by the World Health Organization. Our P. falciparum and P. vivax assays exhibited 100% sensitivity and specificity on clinical samples (5 P. falciparum and 10 P. vivax samples). This work establishes a field-applicable diagnostic for ultrasensitive detection of asymptomatic carriers as well as a rapid point-of-care clinical diagnostic for nonfalciparum malaria species and low parasite density P. falciparum infections. 2021-10-27T20:31:06Z 2021-10-27T20:31:06Z 2020 2021-08-25T18:01:29Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/136151 en 10.1073/PNAS.2010196117 Proceedings of the National Academy of Sciences of the United States of America Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf Proceedings of the National Academy of Sciences PNAS |
| spellingShingle | Lee, Rose A Puig, Helena De Nguyen, Peter Q Angenent-Mari, Nicolaas M Donghia, Nina M McGee, James P Dvorin, Jeffrey D Klapperich, Catherine M Pollock, Nira R Collins, James J Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria |
| title | Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria |
| title_full | Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria |
| title_fullStr | Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria |
| title_full_unstemmed | Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria |
| title_short | Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria |
| title_sort | ultrasensitive crispr based diagnostic for field applicable detection of plasmodium species in symptomatic and asymptomatic malaria |
| url | https://hdl.handle.net/1721.1/136151 |
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