Simple, easy-to-use, low-cost nucleic acid amplification tests (NAATs) are not available for diagnosis of infectious diseases in low-resource settings (LRS). Challenges to easy adoption or modification of existing NAAT technologies for LRS use include time-consuming sample preparation;cold chain requirements for reagent storage;and lack of instrumentation, electricity, and training at the point of care. Unfortunately, there are many infectious diseases rampant in LRS where the lack of appropriate NAATs is a critical barrier to timely diagnosis and treatment. PATH proposes a collaborative effort with the CDC that simultaneously merges scientific and technological capabilities with this clinical need to develop a calcium oxide (CaO)-heated DNA amplification kit that obviates the requirements for electricity and instrumentation at the point of care. PATH has demonstrated the use of CaO for heat and a proprietary engineered phase change material to stabilize an assay mixture within a narrow temperature range suitable for isothermal amplification. The CDC has developed a novel combination of lysis buffer, loop mediated isothermal amplification (LAMP) reaction and fluorescently-labeled primers to enable a simplified, extraction-free and lysis-free workflow. These recent innovations can be combined with a simple two-chamber, high-containment reaction tube prefilled with lyophilized LAMP mixture to create the first electricity-free, instrument-free, easy-to-use, low-cost NAAT kit. We envision multiple NAAT kits customized and validated for specific strains of infectious diseases such as tuberculosis, malaria, and HIV that will advance evidence-based medical practice at the point of care in LRS. We will develop, integrate, and validate components of this NAAT kit with the sensitivity of PCR, the simplicity of a strip test, and sufficient stability for storage out of the cold chain for long periods of time.
Aims 1 and 2 below describe development of the kit components.
Aim 3 describes the integration of these components into a stand-alone kit designed for low-infrastructure use. Finally, Aim 4 proposes the laboratory validation of this kit using HIV-1, a highly relevant pathogen afflicting many populations in LRS. " Aim 1: Specify and optimize the properties of the electricity-free incubator active materials using the existing incubator prototype. " Aim 2: Optimize CDC's innovations to LAMP HIV-1 assay, establish dry reagent formulations, and define sample. " Aim 3: Design and fabricate optimized kit hardware and consumables: the isothermal incubator and reaction containment tube. " Aim 4: Validate the electricity-free and instrument-free assay kit created in Aims 1-3.

Public Health Relevance

We propose to integrate two innovations to create and validate the first electricity-free, instrument-free, easy-to-use, low-cost, nucleic acid amplification test kit for diagnosis of infectious diseases in low-resource settings. PATH has demonstrated the use of calcium oxide and a proprietary engineered phase change material to stabilize the temperature of an assay mixture within a narrow range suitable for isothermal amplification. The Centers for Disease Control and Prevention have developed a novel combination of lysis buffer, loop mediated isothermal amplification (LAMP) reaction and fluorescently-labeled primers to enable a simplified, extraction-free and lysis-free workflow. These two technologies will combine with a simple, two-chamber, disposable, high containment reaction tube prefilled with lyophilized LAMP mixture.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB012641-03
Application #
8516365
Study Section
Instrumentation and Systems Development Study Section (ISD)
Program Officer
Korte, Brenda
Project Start
2011-09-19
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
3
Fiscal Year
2013
Total Cost
$517,834
Indirect Cost
$142,567
Name
Program/Appropriate/Technology/Health
Department
Type
DUNS #
103713624
City
Seattle
State
WA
Country
United States
Zip Code
98121
Mohon, Abu Naser; Lee, Lydia Da-Yeong; Bayih, Abebe Genetu et al. (2016) NINA-LAMP compared to microscopy, RDT, and nested PCR for the detection of imported malaria. Diagn Microbiol Infect Dis 85:149-53
Curtis, Kelly A; Rudolph, Donna L; Morrison, Daphne et al. (2016) Single-use, electricity-free amplification device for detection of HIV-1. J Virol Methods 237:132-137
Kemleu, Sylvie; Guelig, Dylan; Eboumbou Moukoko, Carole et al. (2016) A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections. PLoS One 11:e0165506
Buser, J R; Diesburg, S; Singleton, J et al. (2015) Precision chemical heating for diagnostic devices. Lab Chip 15:4423-32
Singleton, Jered; Osborn, Jennifer L; Lillis, Lorraine et al. (2014) Electricity-free amplification and detection for molecular point-of-care diagnosis of HIV-1. PLoS One 9:e113693
Kubota, Ryo; Labarre, Paul; Weigl, Bernhard H et al. (2013) Molecular diagnostics in a teacup: Non-Instrumented Nucleic Acid Amplification (NINA) for rapid, low cost detection of Salmonella enterica. Chin Sci Bull 58:1162-1168
Singleton, Jered; Zentner, Chris; Buser, Josh et al. (2013) Instrument-free exothermic heating with phase change temperature control for paper microfluidic devices. Proc SPIE Int Soc Opt Eng 8615:86150R
Kubota, Ryo; LaBarre, Paul; Singleton, Jered et al. (2011) Non-Instrumented Nucleic Acid Amplification (NINA) for Rapid Detection of Ralstonia solanacearum Race 3 Biovar 2. Biol Eng Trans 4:69-80
LaBarre, Paul; Boyle, David; Hawkins, Kenneth et al. (2011) Instrument-free nucleic acid amplification assays for global health settings. Proc SPIE Int Soc Opt Eng 8029: