Jered Singleton

A Simple, Inexpensive Device for Nucleic Acid Amplification without Electricity—Toward Instrument-Free Molecular Diagnostics in Low-Resource Settings

Background Molecular assays targeted to nucleic acid (NA) markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS) where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR) instrumentation (another is sample preparation). Methodology/Principal Findings In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heaters equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented. Conclusions/Significance We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA) heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes.

Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1

Background To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. Methodology/Significant Findings In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. Conclusion The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.

Non-Instrumented Incubation of a Recombinase Polymerase Amplification Assay for the Rapid and Sensitive Detection of Proviral HIV-1 DNA

Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25–43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS.

Electricity-Free Amplification and Detection for Molecular Point-of-Care Diagnosis of HIV-1

In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US

Non-instrumented nucleic acid amplification (NINA): Instrument-free molecular malaria diagnostics for low-resource settings

.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens.

Assessment of the availability of technology for trauma care in India

We have achieved the first complete, non-instrumented nucleic acid amplification test (NAAT) using a calcium oxide heat source thermally linked to an engineered phase change material. These two components alone maintain a thermal profile suitable for the loop-mediated isothermal amplification assay. Starting with computational fluid dynamics analysis, we identified nominal geometry for the exothermic reaction chamber, phase change material chamber, thermal insulation, and packaging. Using this model, we designed and fabricated an alpha prototype assay platform. We have verified the function of this multi-pathogen-capable platform with both fluorescent and visual turbidity indications using samples spiked with malaria DNA. Both the exothermically heated platform samples and samples heated on a Perkin-Elmer GeneAmp9600 thermocycler were first incubated at 62°C for 45 minutes, then heated to 95°C to terminate enzyme activity, then analyzed. Results from the exothermically heated, non-instrumented platform were comparable to those from the thermocycler. These developments will enable point-of-care diagnostics using accurate NAATs which until now have required a well-equipped laboratory. The aim of this research is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where assays such as immunochromatographic strip tests are successfully used but where there is no access to the infrastructure and logistics required to operate and maintain instrument-based diagnostics.

Precision chemical heating for diagnostic devices

BackgroundWe sought to assess the status of availability of technology for trauma care in a state in India and to identify factors contributing to both adequate levels of availability and to deficiencies. We also sought to identify potential solutions to deficiencies in terms of health system management and product development.MethodsThirty-two technology-related items were selected from the World Health Organization’s Guidelines for Essential Trauma Care. The status of these items was assessed at 43 small and large hospitals in Gujarat State. Site visits utilized direct inspection and interviews with administrative, clinical, and bioengineering staff.ResultsMany specific individual items could be better supplied, including many that were very low cost (e.g., chest tubes). Many deficiencies arose because of mismatch of resources, such as availability of equipment in the absence of personnel trained to use it. Several locally manufactured items were fairly well supplied: pulse oximetry, image intensification, and X-ray machines. Ventilators were often deficient because of inadequate numbers of units and frequent breakdowns.ConclusionsAvailability of a range of lower-cost items could be improved by better organization and planning, such as: better procurement and stock management; eliminating mismatch of resources, including optimizing training for use of existing resources; and by strengthening service contracts and in-house repair capabilities. From a product development viewpoint, there is a need for lower cost, more durable, and easier to repair ventilators. Promoting increased capacity for local manufacturing should also be considered as a potential method to decrease cost and increase availability of a range of equipment.

Non-Instrumented Nucleic Acid Amplification (NINA) for Rapid Detection of Ralstonia solanacearum Race 3 Biovar 2.

Decoupling nucleic acid amplification assays from infrastructure requirements such as grid electricity is critical for providing effective diagnosis and treatment at the point of care in low-resource settings. Here, we outline a complete strategy for the design of electricity-free precision heaters compatible with medical diagnostic applications requiring isothermal conditions, including nucleic acid amplification and lysis. Low-cost, highly energy dense components with better end-of-life disposal options than conventional batteries are proposed as an alternative to conventional heating methods to satisfy the unique needs of point of care use.

Performance of cryotherapy devices using nitrous oxide and carbon dioxide

We report on the use of a non-instrumented device for the implementation of a loop-mediated amplification (LAMP) based assay for the select-agent bacterial-wilt pathogen Ralstonia solanacearum race 3 biovar 2. Heat energy is generated within the device by the exothermic hydration of calcium oxide, and the reaction temperature is regulated by storing latent energy at the melting temperature of a renewable lipid-based engineered phase-change material. Endpoint detection of the LAMP reaction is achieved without opening the reaction tube by observing the fluorescence of an innovative FRET-based hybridization probe with a simple custom fluorometer. Non-instrumented devices could maintain reactions near the design temperature of 63°C for at least an hour. Using this approach DNA extracted from the pathogen could be detected at fewer than ten copies within a 25 μL reaction mix, illustrating the potential of these technologies for simple, powerful agricultural diagnostics in the field. Furthermore, the assay was just as reliable when implemented in a tropical environment at 31°C as it was when implemented in an air-conditioned lab maintained at 22°C, illustrating the potential value of the technology for field conditions in the tropics and subtropics.

Comparison of depth of necrosis using cryotherapy by gas and number of freeze cycles.

To compare temperatures reached by 4 different cryotherapy devices commonly used to treat precancerous cervical lesions in low‐resource countries using nitrous oxide (N2O) and carbon dioxide (CO2) as well as with and without a gas conditioner.