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

Thesis (Master's)--University of Washington, 2018The lateral flow test (LFT) is the preferred tool for detection of protein biomarkers in low-resource settings. While low-cost and rapid, these immunoassay devices often suffer from limited sensitivity in the single chemical step format. Our group has addressed this deficiency by coupling a lateral flow test to isothermal strand displacement amplification (iSDA), a nucleic-acid amplification test (NAAT). This novel method is called LFT-iSDA and uses an oligonucleotide-labelled detection antibody in the immunoassay as the target template for iSDA. In its current embodiment, LFT-iSDA is ~10,000x more sensitive than gold nanoparticle-based LFTs, but is highly manual and relies on laboratory infrastructure. Here we present preliminary development of a diagnostic device integrating the highly sensitive LFT-iSDA into a cartridge supporting rapid time-to-results and minimal user-steps— a format amenable to decentralized test settings. We characterized performance limitations of this assay, developed a real-time fluorescence detection system, and used a two-dimensional paper network (2DPN) to semi-automate this multi-step chemical process. These results show important progress toward enabling highly sensitive protein detection in a user-friendly, low-cost device, at the point of care

Evaluation of non-instrumented nucleic acid amplification by loop-mediated isothermal amplification (NINA-LAMP) for the diagnosis of malaria in Northwest Ethiopia

BACKGROUND: 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. METHODS: A cross-sectional study was conducted in North Gondar, Ethiopia from March to July 2014. Eighty-two blood samples were collected from malaria suspected patients visiting Kola Diba Health Centre and analysed for Plasmodium parasites by microscopy, NINA-LAMP and nested PCR. The NINA-LAMP method was performed using the Loopamp™ Malaria Pan/Pf detection kits for detecting DNA of the genus Plasmodium and more specifically Plasmodium falciparum using an electricity-free heater. Diagnostic accuracy outcome measures (analytical sensitivity, specificity, predictive values, and Kappa scores) of NINA-LAMP and microscopy were compared to nested PCR. RESULTS: A total of 82 samples were tested in the primary analysis. Using nested PCR as reference, the sensitivity and specificity of the primary NINA-LAMP assay were 96.8% (95% confidence interval (CI), 83.2% - 99.5%) and 84.3% (95% CI, 71.4% - 92.9%), respectively for detection of Plasmodium genus, and 100% (95% CI, 75.1% - 100%) and 81.2% (95% CI, 69.9% - 89.6%), respectively for detection of P. falciparum parasite. Microscopy demonstrated sensitivity and specificity of 93.6% (95% CI, 78.5% - 99.0%) and 98.0% (95% CI, 89.5% - 99.7%), respectively for the detection of Plasmodium parasites. Post-hoc repeat NINA-LAMP analysis showed improvement in diagnostic accuracy, which was comparable to nested PCR performance and superior to microscopy for detection at both the Plasmodium genus level and P. falciparum parasites. CONCLUSION: NINA-LAMP is highly sensitive for the diagnosis of malaria and detection of Plasmodium parasite infection at both the genus and species level when compared to nested PCR. NINA-LAMP is more sensitive than microscopy for the detection of P. falciparum and differentiation from non-falciparum species and may be a critical diagnostic modality in efforts to eradicate malaria from areas of low endemicity

Design of a Novel, Adjustable Flow Rate, Reusable, Electricity-Free, Low-Cost Syringe Infusion Pump

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.</jats:p

Design of a New Type of Compact Chemical Heater for Isothermal Nucleic Acid Amplification

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

Design of a New Type of Compact Chemical Heater for Isothermal Nucleic Acid Amplification.

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

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$.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

Performance at elevated temperatures.

<p><b>(a)</b> The temperature-time curve (<math><mrow>X<mo stretchy="true">¯</mo></mrow></math> ± s) shows smoother ramp-up and extended holdover between 57°C and 62°C (dashed lines) at a 45°C ambient temperature compared to 22°C. (n = 3) <b>(b)</b> Ramp-up time decreases by 1 minute (**p = 0.006) and holdover time increases by 60% compared to room temperature (*p = 0.04). (n = 3).</p

Ramp-up and holdover times.

<p><b>(a)</b> Ramp-up times (<math><mrow>X<mo stretchy="true">¯</mo></mrow></math> ± s) are reduced (*p = 0.018) and holdover times (<math><mrow>X<mo stretchy="true">¯</mo></mrow></math> ± s) show no change with the addition of sodium chloride to the magnesium-iron fuel pack. (n = 3) <b>(b)</b> In contrast, the addition of copper (II) chloride does not significantly affect ramp-up, but significantly reduces holdover (**p = 0.005). (n = 3) <b>†</b> indicates identical data points.</p

A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections.

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