Development of a Highly Sensitive Protein Diagnostic Device using Isothermal Strand Displacement Amplification

Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP) tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA) device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus) or repetitive DNA (40 min, B. malayi and W. bancrofti) was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs.

We present a proof-of-concept design and preliminary data to demonstrate a novel syringe infusion pump that is low cost, nonelectric, reusable, and adjustable. This device addresses the need for infusion therapy in low- and middle-income countries (LMIC), where intermittent electrical power precludes the use of conventional electronic infusion pumps and limited financial resources make high costs of disposable infusion pumps impractical. Our design uses a pneumatically pressurized, hydraulic (air over oil) drive piston coupled to a closed-circuit flow restriction to drive a syringe plunger at a constant velocity, thus providing a constant volumetric flow rate to the patient. The device requires no proprietary or precision consumables, significantly reducing treatment costs compared with other methods. The highly adjustable device provides constant flow rates across the range of 0.5–8 mL/h when used with a 30-mL syringe. The user interface is simple and intuitive; the hardware is robust and portable. This novel technology platform has broad applications in addressing priority health needs in LMIC.

Environmental surveillance of poliovirus (PV) plays an important role in the global program for eradication of wild PV. The bag-mediated filtration system (BMFS) was first developed in 2014 and enhances PV surveillance when compared to the two-phase grab method currently recommended by the World Health Organization (WHO). In this study, the BMFS design was improved and tested for its usability in wastewater and wastewater-impacted surface waters in Nairobi, Kenya. Modifications made to the BMFS included the size, color, and shape of the collection bags, the filter housing used, and the device used to elute the samples from the filters. The modified BMFS concentrated 3-10 L down to 10 mL, which resulted in an effective volume assayed (900-3000 mL) that was 6-20 times greater than the effective volume assayed for samples processed by the WHO algorithm (150 mL). The system developed allows for sampling and in-field virus concentration, followed by transportation of the filter for further analysis with simpler logistics than the current methods. This may ultimately reduce the likelihood of false-negative samples by increasing the effective volume assayed compared to samples processed by the WHO algorithm, making the BMFS a valuable sampling system for wastewater and wastewater-impacted surface waters.

Early and accurate diagnosis of HIV is key for the reduction of transmission and initiation of patient care. The availability of a rapid nucleic acid test (NAT) for use at the point-of-care (POC) will fill a gap in HIV diagnostics, improving the diagnosis of acute infection and HIV in infants born to infected mothers. In this study, we evaluated the performance of non-instrumented nucleic acid amplification, single-use disposable (NINA-SUD) devices for the detection of HIV-1 in whole blood using reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) with lyophilized reagents. The NINA-SUD heating device harnesses the heat from an exothermic chemical reaction initiated by the addition of saline to magnesium iron powder. Reproducibility was demonstrated between NINA-SUD units and comparable, if not superior, performance for detecting clinical specimens was observed as compared to the thermal cycler. The stability of the lyophilized HIV-1 RT-LAMP reagents was also demonstrated following storage at -20, 4, 25, and 30°C for up to one month. The single-use, disposable NAT minimizes hands-on time and has the potential to facilitate HIV-1 testing in resource-limited settings or at the POC.

Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT), and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ μL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ μL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ μL). Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar's test: P = 0.0002), and the highest was with RT-PCR (87%, McNemar's test: P = 0.0523). Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay in malaria diagnosis in low to high parasite density settings.

Microscopy and field adaptable rapid diagnostic tests (RDTs) are not sensitive and specific in certain conditions such as poor training of microscopists, lack of electricity, or lower sensitivity in the detection of non-falciparum malaria. More sensitive point-of-care testing (POCT) would reduce delays in diagnosis and initiation of therapy. In the current study, we have evaluated the efficacy of noninstrumented nucleic acid amplification (NINA) coupled with loop-mediated isothermal amplification (LAMP) for detection of traveler's malaria (n=140) in comparison with microscopy, nested PCR, and the only Food and Drug Administration–approved rapid diagnostic test. NINA-LAMP was 100% sensitive and 98.6% specific when compared to nested PCR. For non-falciparum detection, NINA-LAMP sensitivity was 100% sensitive compared to nested PCR, whereas RDT sensitivity was 71%. LAMP is highly sensitive and specific for symptomatic malaria diagnosis regardless of species.

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.

Previous chemical heater designs for isothermal nucleic acid amplification have been based on solid-liquid phase transition, but using this approach, developers have identified design challenges en route to developing a low-cost, disposable device. Here, we demonstrate the feasibility of a new heater configuration suitable for isothermal amplification in which one reactant of an exothermic reaction is a liquid-gas phase-change material, thereby eliminating the need for a separate phase-change compartment. This design offers potentially enhanced performance and energy density compared to other chemical and electric heaters.

Malaria is a major public health problem in sub-Saharan African countries including Ethiopia. Early and accurate diagnosis followed by prompt and effective treatment is among the various tools available for prevention, control and elimination of malaria. This study aimed to evaluate the performance of non-instrumented nucleic acid amplification loop-mediated isothermal amplification (NINA-LAMP) compared to standard thick and thin film microscopy and nested PCR as gold standard for the sensitive diagnosis of malaria in Northwest Ethiopia.

Accurate diagnostic tools are essential for effective diagnosis, treatment, and tracking of individuals with infectious diseases. Diagnosis can be especially difficult in low-resource settings with limited infrastructure or unreliable access to electricity. Our previous work has demonstrated feasibility of a reusable configuration of an electricity-free, non-instrumented nucleic acid amplification platform (NINA) that complements isothermal assays for precise detection of a variety of pathogens. To maximize the potential impact of the NINA technology, we redesigned the technology in a disposable configuration to meet challenging cost, size, and ease of use requirements. This paper discusses the development of the single-use, disposable NINA platform. Key challenges included meeting repeatability, temperature stability, and precision targets. Through an iterative design process, we met these challenges using a novel design strategy including re-engineering of phase change materials (PCMs), addition of thermal enhancement additives to the PCM, and metered introduction of reactants using porous media. Manufacturing, performance, and reproducibility challenges were addressed, and the resultant design was rigorously tested in preparation for the production of a pilot batch of 150 disposable NINA devices that will be used for device and assay performance testing.

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$.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.

An increasing number of isothermal nucleic acid amplification test (NAAT) approaches are under development to liberate molecular diagnostics from the laboratory and enable efficient clinical treatment decision-making at the point-of-care (POC). Each of these approaches requires the optimization of individual reaction specifications for the most effective amplification of the target nucleic acids (NAs) including NA purity, primer/probe design and reagent composition, amplification reaction temperature window of performance, total amplification time to detect, and method of detection. As a result, significant variation exists in the critical specifications that need to be achieved. To provide better thermal management capability for NAAT POC use cases, we have improved the design of our previous NAAT enabling non-instrumented nucleic acid amplification (NINA) heater technology. Thermal modeling was used to define which specifications would have the greatest impact on overall system performance. As a result, we present an isothermal assay NINA heater platform with decreased warm-up time to assay temperature, increased thermal holdover time, and tighter adherence to the optimal assay target temperature for a malaria loop-mediated isothermal amplification assay.

Rapid sand filters are a familiar and mature technology, but the mechanical sophistication they incorporate in industrialized nations limits their sustainable application in developing countries. Conventional rapid sand filters require pumps, elevated tanks, or multiple filter units to generate high flow rates for backwashing. Stacked rapid sand filtration is introduced here as a more robust and sustainable alternative. A stacked rapid sand filter can backwash itself with no additional flow, which eliminates the need for pumps or other expensive equipment. This study presents laboratory and field proof-of-concept demonstrations of this novel technology. The multilayer configuration of stacked rapid sand filters allowed a laboratory unit to be loaded at 1.4-1.83 mm/s (120-160 m/day) per layer and backwashed at 10-11 mm/s (860-950 m/day) with the same or similar total flow rate. The filtered effluent met U. S. Environmental Protection Agency drinking water standards. The backwash cycle was also demonstrated, and flushing of contaminants from the sand bed was effective even with 5-10 NTU backwash water. A test stacked filter unit also demonstrated satisfactory filtration performance and effective backwashing at several water treatment plants in Honduras. DOI: 10.1061/(ASCE)EE.1943-7870.0000562. (C) 2012 American Society of Civil Engineers.