Paper‐Origami‐Based Multiplexed Malaria Diagnostics from Whole Blood

Abstract We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper‐folding technique of origami to enable the sequential steps of DNA extraction, loop‐mediated isothermal amplification (LAMP), and array‐based fluorescence detection. A low‐cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a “sample‐to‐answer” diagnosis from a finger‐prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold‐standard polymerase chain reaction (PCR) assay in an operator‐blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples.

[1]  David G Lalloo,et al.  UK malaria treatment guidelines. , 2007, The Journal of infection.

[2]  T. Notomi,et al.  Loop-mediated isothermal amplification of DNA. , 2000, Nucleic acids research.

[3]  Jaclyn A. Adkins,et al.  Recent developments in paper-based microfluidic devices. , 2015, Analytical chemistry.

[4]  M. Dou,et al.  A Versatile PDMS/Paper Hybrid Microfluidic Platform for Sensitive Infectious Disease Diagnosis , 2014, Analytical chemistry.

[5]  George M Whitesides,et al.  "Paper Machine" for Molecular Diagnostics. , 2015, Analytical chemistry.

[6]  K. Jaton,et al.  Detection of Four Plasmodium Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays , 2004, Journal of Clinical Microbiology.

[7]  M. Ndao,et al.  Multiplexed Real-Time PCR Assay for Discrimination of Plasmodium Species with Improved Sensitivity for Mixed Infections , 2009, Journal of Clinical Microbiology.

[8]  A. Heath,et al.  Establishment of the 1st World Health Organization International Standard for Plasmodium falciparum DNA for nucleic acid amplification technique (NAT)-based assays , 2008, Malaria Journal.

[9]  Cristina Stoyanov,et al.  Measuring malaria by passive case detection: a new perspective based on Zambian experience , 2013, Malaria Journal.

[10]  Shenguang Ge,et al.  Photoelectrochemical lab-on-paper device equipped with a porous Au-paper electrode and fluidic delay-switch for sensitive detection of DNA hybridization. , 2013, Lab on a chip.

[11]  M. McMorrow,et al.  Diagnosis of Malaria , 2008, Molecular Diagnosis & Therapy.

[12]  Yasuyoshi Mori,et al.  Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products , 2008, Nature Protocols.

[13]  J. Reboud,et al.  Rapid ultrasonic isothermal amplification of DNA with multiplexed melting analysis – applications in the clinical diagnosis of sexually transmitted diseases. , 2015, Chemical communications.

[14]  C. Rogier,et al.  Changing Malaria Epidemiology and Diagnostic Criteria for Plasmodium falciparum Clinical Malaria , 2012, PloS one.

[15]  G. Whitesides,et al.  Diagnostics for the developing world: microfluidic paper-based analytical devices. , 2010, Analytical chemistry.

[16]  George M Whitesides,et al.  Folding analytical devices for electrochemical ELISA in hydrophobic R(H) paper. , 2014, Analytical chemistry.

[17]  S. Ramachandran,et al.  A low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami. , 2012, Lab on a chip.

[18]  Eun-Taek Han,et al.  Detection of Four Plasmodium Species by Genus- and Species-Specific Loop-Mediated Isothermal Amplification for Clinical Diagnosis , 2007, Journal of Clinical Microbiology.

[19]  R. Gilman,et al.  Polymerase chain reaction detection of Plasmodium vivax and Plasmodium falciparum DNA from stored serum samples: implications for retrospective diagnosis of malaria. , 2007, The American journal of tropical medicine and hygiene.

[20]  W. Jarra,et al.  High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. , 1993, Molecular and biochemical parasitology.

[21]  G. Whitesides,et al.  Three-dimensional microfluidic devices fabricated in layered paper and tape , 2008, Proceedings of the National Academy of Sciences.

[22]  Chris Drakeley,et al.  Overdiagnosis of malaria in patients with severe febrile illness in Tanzania: a prospective study , 2004, BMJ : British Medical Journal.

[23]  S. P. Kachur,et al.  The silent threat: asymptomatic parasitemia and malaria transmission , 2013, Expert review of anti-infective therapy.

[24]  P. Chiodini,et al.  External quality assessment schemes raise standards: evidence from the UKNEQAS parasitology subschemes , 2003, Journal of clinical pathology.

[25]  Thomas C Evans,et al.  Simultaneous multiple target detection in real-time loop-mediated isothermal amplification. , 2012, BioTechniques.

[26]  R. Crooks,et al.  Three-dimensional paper microfluidic devices assembled using the principles of origami. , 2011, Journal of the American Chemical Society.

[27]  A. Moody.  Rapid Diagnostic Tests for Malaria Parasites , 2002, Clinical Microbiology Reviews.

[28]  N. Engel,et al.  Point-of-Care Testing for Infectious Diseases: Diversity, Complexity, and Barriers in Low- And Middle-Income Countries , 2012, PLoS medicine.