Point-of-care multiplexed molecular amplification and visual detection for HIV- 1 and beta-actin without electricity
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1 Point-of-care multiplexed molecular amplification and visual detection for HIV- 1 and beta-actin without electricity Dr. Jennifer Osborn, Technical Program Associate, Co-PI Paul LaBarre, Sr. Technical Officer, lead PI Photo: Lisa Stroux/PATH
2 Problem: Lack of Appropriate Diagnostic Tests Decentralized Centralized Low-Resource PCR Laboratory Laboratory Photo: PATH Page 2
3 Point-of-care Testing in Low-resource Settings Types of tests: Test settings Test users: RDT, molecular smears RDT, PCR, ELISA, microscopy RDT, handheld instruments RDT RDT Hospital Peripheral Lab Clinic or Health Post Community Home Hospital staff, lab tech Lab tech Clinical staff Minimally trained health worker Lay person Page 3
4 Molecular Diagnostics Technology Requirements Decentralized Low-Resource Centralized Laboratory PCR Laboratory Polymerase Chain Reaction WHO Diagnostic Criteria High cost Affordable Low cost Ideal POC Test Highly sensitive Sensitive Highly sensitive Highly specific Specific Highly specific Technically trained users required User friendly Minimal user steps required Days to months Rapid & robust Minutes to hours Power and equipment required Equipment free No power required, no complex equipment w_inside Photo: PATH Not portable, refrigeration required Deliverable to developing countries Highly portable, dried reagents Point-of-care molecular diagnostics could reduce the disease burden Page 4
5 Early Infant Diagnosis and Linkage to Care is Critical 370,000 new HIV infections from mother-to-child transmission (UNICEF, 2008) EID improves health outcomes CHER trial in South Africa: 76% reduction in morbidity 75% reduction in mortality Photo: March of Dimes
6 Challenges with Current Tests Available tests: rapid diagnostic tests (RDTs) PCR for infant testing with dried blood spots ~60% of infants are lost to follow-up Only 6-15% of HIV-exposed infants accessed testing (WHO, 2009) Maternal antibody interference Centralized laboratories Photo: PATH Photo: CDC
7 Defining Use Cases and Target Product Profiles (TPPs) Exploratory Stakeholder Interviews Use Case(s) TPP(s) Technical Specifications User Needs Assessments Alpha Prototype Design Iterations NIH RO1 Target product profile process helps to ensure technologies will meeting existing needs
8 PATH s Approach: A Non-Instrumented Nucleic Acid (NINA) Diagnostic Platform Exothermic Heat Isothermal Nucleic Acid Amplification Point-of-Care NINA Heater Device PCR Tubes Curtis et al. J. Med. Virol. 81: (2009) PCM MgFe/H2O Reaction Photo: PATH Isothermal amplification simplifies the power requirements for amplification Page 8
9 Benefits of NINA Compared to PCR Testing Centralized PCR Laboratory Gold Standard: PCR WHO Diagnostic Criteria NINA Heater + LFD High cost Affordable Low cost Highly sensitive Sensitive Sensitive Highly specific Specific Specific Point-of-Care NINA Heater Technically trained users required User friendly Minimal User Steps Days to months Rapid & robust ~60-90 minutes Power and complex equipment Equipment free No power or complex equipment Photo: oint/view_inside Not portable, refrigeration required Deliverable to developing countries Highly portable, dried reagents Photo: PATH The NINA heater is designed for nucleic acid detection in lowresource settings for improved acute and infant HIV diagnosis. Page 9
10 NINA Kit concept User-friendly Operation of the NINA Heater
11 NINA Heater Prototype Details
12 NINA Heater Performance 10 minute ramp up to 61.5⁰C ± 1.5⁰C Blue regions = outside specification Error bars show the range of the data Page 12
13 LAMP Assay Development: HIV-1 Real-Time Amplification Melt Curve Analysis Primers were designed by Hosaka et al. Loop F and BIP primers were modified for lateral flow detection Biotin & FITC tags Page 13
14 LAMP Assay Development: Human Beta-actin Real-Time Amplification Melt Curve Analysis Designed primers using PrimerExplorer V4 Software (Eiken Chemical Co. Ltd) Modified the FIP and BIP primers for lateral flow detection Biotin & Digoxigenin tags Page 14
15 LAMP Assay Development: Multiplex Melt Curve Analysis Lateral Flow Cassette Amplicons are generated for both HIV-1 (78⁰C) and beta-actin (89⁰C) Amplicons are detected on a lateral flow cassette (BioHelix Corp) Page 15
16 Next Steps Laboratory Continuing to evaluate isothermal assays for use with the NINA platform Collecting HIV samples for real specimen testing Evaluating detection strategies Engineering New device housing Integration of PCM and fuel cartridge Automation of heat activation Page 16
17 Potential Areas of Collaboration Isothermal HIV-1 assays with high performance Non-subtype B assays Novel nucleic acid extraction methods for the POC Feedback on the TPP for HIV diagnosis of acute and EID Josborn@path.org Plabarre@path.org Page 17 Photo: University of North Carolina Asheville
18 Acknowledgments NIH grant # R01EB A1 HIV experts and stakeholders CDC Michele Owen Kelly Curtis Donna Rudolph PATH team Paul LaBarre Will Price Jered Singleton Dylan Guelig Ken Hawkins Sarah McGray Rachel Johns Louise Downing Kendall Magnuson Bernhard Weigl David Boyle Gonzalo Domingo Page 18
19 Questions? Photo: PATH Page 19
20 Publications Curtis KA, Rudolph DL, Nejad I, et al. Isothermal amplification using a chemical heating device for point-of-care detection of HIV-1. Public Library of Science One. 2012;7(2):e Kubota R, LaBarre P, Singleton J, et al. Molecular diagnostics in a teacup: noninstrumented nucleic acid amplification (NINA) for rapid, low-cost detection of Salmonella enterica. Chinese Science Bulletin Kubota R, LaBarre P, Singleton J, Beddoe A, Weigl BH, Alvarez AM, Jenkins DM. Noninstrumented nucleic acid amplification (NINA) for rapid detection of Ralstonia solanacearum race 3 biovar 2. Biological Engineering Transactions. 2011;4(2): LaBarre P, Hawkins KR, Gerlach J, et al. A simple, inexpensive device for nucleic acid amplification without electricity toward instrument-free molecular diagnostics in lowresource settings. Public Library of Science One. 2011;6(5):e LaBarre P, Gerlach J, Wilmoth J, et al. Non-instrumented nucleic acid amplification (NINA): Instrument-free molecular malaria diagnostics for low-resource settings. Proceedings from the 32nd Annual International Conference of the IEEE Engineering in Medicine & Biology Society, August Page 20
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