Point-of-care isothermal nucleic acid amplification tests: progress and bottlenecks for extraction-free sample collection and preparation.

INTRODUCTION: Suitable sample collection and preparation methods are essential to enable nucleic acid amplification testing at the point of care (POC). Strategies that allow direct isothermal nucleic acid amplification testing (iNAAT) of crude sample lysate without the need for nucleic acid extraction minimize time to result as well as the need for operator expertise and costly infrastructure. AREAS COVERED: The authors review research to understand how sample matrix and preparation affect the design and performance of POC iNAATs. They focus on approaches where samples are directly combined with liquid reagents for preparation and amplification via iNAAT strategies. They review factors related to the type and method of sample collection, storage buffers, and lysis strategies. Finally, they discuss RNA targets and relevant regulatory considerations. EXPERT OPINION: Limitations in sample preparation methods are a significant technical barrier preventing implementation of nucleic acid testing at the POC. The authors propose a framework for co-designing sample preparation and amplification steps for optimal performance with an extraction-free paradigm by considering a sample matrix and lytic strategy prior to an amplification assay and readout. In the next 5 years, the authors anticipate increasing priority on the co-design of sample preparation and iNAATs.

A multiplexed, allele-specific recombinase polymerase amplification assay with lateral flow readout for sickle cell disease detection.

Isothermal nucleic acid amplification tests have the potential to improve disease diagnosis at the point of care, but it remains challenging to develop multiplexed tests that can detect ≥3 targets or to detect point mutations that may cause disease. These capabilities are critical to enabling informed clinical decision-making for many applications, such as sickle cell disease (SCD). To address this, we describe the development of a multiplexed allele-specific recombinase polymerase amplification (RPA) assay with lateral flow readout. We first characterize the specificity of RPA using primer design strategies employed in PCR to achieve point mutation detection, and demonstrate the utility of these strategies in achieving selective isothermal amplification and detection of genomic DNA encoding for the healthy ßA globin allele, or genomic DNA containing point mutations encoding for pathologic ßS and ßC globin alleles, which are responsible for most sickle cell disorders. We then optimize reaction conditions to achieve multiplexed amplification and identification of the three alleles in a single reaction. Finally, we perform a small pilot study with 20 extracted genomic DNA samples from SCD patients and healthy volunteers - of the 13 samples with valid results, the assay demonstrated 100% sensitivity and 100% specificity for detecting pathologic alleles, and an overall accuracy of 92.3% for genotype prediction. This multiplexed assay is rapid, minimally instrumented, and when combined with point-of-care sample preparation, could enable DNA-based diagnosis of SCD in low-resource settings. The strategies reported here could be applied to other challenges, such as detection of mutations that confer drug resistance.

An integrated isothermal nucleic acid amplification test to detect HPV16 and HPV18 DNA in resource-limited settings.

High-risk human papillomavirus (HPV) DNA testing is widely acknowledged as the most sensitive cervical cancer screening method but has limited availability in resource-limited settings, where the burden of cervical cancer is highest. Recently, HPV DNA tests have been developed for use in resource-limited settings, but they remain too costly for widespread use and require instruments that are often limited to centralized laboratories. To help meet the global need for low-cost cervical cancer screening, we developed a prototype, sample-to-answer, point-of-care test for HPV16 and HPV18 DNA. Our test relies on isothermal DNA amplification and lateral flow detection, two technologies that reduce the need for complex instrumentation. We integrated all test components into a low-cost, manufacturable platform, and performance of the integrated test was evaluated with synthetic samples, provider-collected clinical samples in a high-resource setting in the United States, and self-collected clinical samples in a low-resource setting in Mozambique. We demonstrated a clinically relevant limit of detection of 1000 HPV16 or HPV18 DNA copies per test. The test requires six user steps, yields results in 45 min, and can be performed using a benchtop instrument and minicentrifuge by minimally trained personnel. The projected per-test cost is <$5, and the projected instrumentation cost is <$1000. These results show the feasibility of a sample-to-answer, point-of-care HPV DNA test. With the inclusion of other HPV types, this test has the potential to fill a critical gap for decentralized and globally accessible cervical cancer screening.

Experimental Control of Macrophage Pro-Inflammatory Dynamics Using Predictive Models.

Macrophage activity is a major component of the healthy response to infection and injury that consists of tightly regulated early pro-inflammatory activation followed by anti-inflammatory and regenerative activity. In numerous diseases, however, macrophage polarization becomes dysregulated and can not only impair recovery, but can promote further injury and pathogenesis, e.g., after trauma or in diabetic ulcers. Dysregulated macrophages may either fail to polarize or become chronically polarized, resulting in increased production of cytotoxic factors, diminished capacity to clear pathogens, or failure to promote tissue regeneration. In these cases, a method of predicting and dynamically controlling macrophage polarization will enable a new strategy for treating diverse inflammatory diseases. In this work, we developed a model-predictive control framework to temporally regulate macrophage polarization. Using RAW 264.7 macrophages as a model system, we enabled temporal control by identifying transfer function models relating the polarization marker iNOS to exogenous pro- and anti-inflammatory stimuli. These stimuli-to-iNOS response models were identified using linear autoregressive with exogenous input terms (ARX) equations and were coupled with non-linear elements to account for experimentally identified supra-additive and hysteretic effects. Using this model architecture, we were able to reproduce experimentally observed temporal iNOS dynamics induced by lipopolysaccharides (LPS) and interferon gamma (IFN-γ). Moreover, the identified model enabled the design of time-varying input trajectories to experimentally sustain the duration and magnitude of iNOS expression. By designing transfer function models with the intent to predict cell behavior, we were able to predict and experimentally obtain temporal regulation of iNOS expression using LPS and IFN-γ from both naïve and non-naïve initial states. Moreover, our data driven models revealed decaying magnitude of iNOS response to LPS stimulation over time that could be recovered using combined treatment with both LPS and IFN-γ. Given the importance of dynamic tissue macrophage polarization and overall inflammatory regulation to a broad number of diseases, the temporal control methodology presented here will have numerous applications for regulating immune activity dynamics in chronic inflammatory diseases.