Development of Integrated Systems for On-Site Infection Detection.

The modern healthcare system faces an unrelenting threat from microorganisms, as evidenced by global outbreaks of new viral diseases, emerging antimicrobial resistance, and the rising incidence of healthcare-associated infections (HAIs). An effective response to these threats requires rapid and accurate diagnostic tests that can identify causative pathogens at the point of care (POC). Such tests could eliminate diagnostic uncertainties, facilitating patient triaging, minimizing the empiric use of antimicrobial drugs, and enabling targeted treatments. Current standard methods, however, often fail to meet the needs of rapid diagnosis in POC settings. Culture-based assays entail long processing times and require specialized laboratory infrastructure; nucleic acid (NA) tests are often limited to centralized hospitals due to assay complexity and high costs. Here we discuss two new POC tests developed in our groups to enable the rapid diagnosis of infection. The first is nanoPCR that takes advantages of core-shell magnetoplasmonic nanoparticles (MPNs): (i) Au shell significantly accelerates thermocycling via volumetric, plasmonic light-to-heat conversion and (ii) a magnetic core enables sensitive in situ fluorescent detection via magnetic clearing. By adopting a Ferris wheel module, the system expedites multisamples in parallel with a minimal setup. When applied to COVID-19 diagnosis, nanoPCR detected SARS-CoV-2 RNA down to 3.2 copy/µL within 17 min. In particular, nanoPCR diagnostics accurately identified COVID-19 cases in clinical samples (n = 150), validating its clinical applicability. The second is a polarization anisotropy diagnostic (PAD) system that exploits the principle of fluorescence polarization (FP) as a detection modality. Fluorescent probes were designed to alter their molecular weight upon recognizing target NAs. This event modulates the probes' tumbling rate (Brownian motion), which leads to changes in FP. The approach is robust against environmental noise and benefits from the ratiometric nature of the signal readout. We applied PAD to detect clinically relevant HAI bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus). The PAD assay demonstrated detection sensitivity down to the single bacterium level and determined both drug resistance and virulence status. In summary, these new tests have the potential to become powerful tools for rapid diagnosis in the infectious disease space. They do not require highly skilled personnel or labor-intensive analyses, and the assays are quick and cost-effective. These attributes will make nanoPCR and PAD well-aligned with a POC workflow to aid physicians to initiate prompt and informed patient treatment.

Effect of asthma and asthma medication on the prognosis of patients with COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has spread worldwide rapidly. However, the effects of asthma, asthma medication and asthma severity on the clinical outcomes of COVID-19 have not yet been established. METHODS: The study included 7590 de-identified patients, who were confirmed to have COVID-19 using the severe acute respiratory syndrome coronavirus 2 RNA-PCR tests conducted up to May 15, 2020; we used the linked-medical claims data provided by the Health Insurance Review and Assessment Service. Asthma and asthma severity (steps suggested by the Global Initiative for Asthma) were defined using the diagnostic code and history of asthma medication usage. RESULTS: Among 7590 COVID-19 patients, 218 (2.9%) had underlying asthma. The total medical cost associated with COVID-19 patients with underlying asthma was significantly higher than that of other patients. Mortality rate for COVID-19 patients with underlying asthma (7.8%) was significantly higher than that of other patients (2.8%; p<0.001). However, asthma was not an independent risk factor for the clinical outcomes of COVID-19 after adjustment, nor did asthma medication use and asthma severity affect the clinical outcomes of COVID-19. However, use of oral short-acting ß2-agonists was an independent factor to increase the total medical cost burden. Patients with step 5 asthma showed significant prolonged duration of admission compared to those with step 1 asthma in both univariate and multivariate analysis. CONCLUSIONS: Asthma led to poor outcomes of COVID-19; however, underlying asthma, use of asthma medication and asthma severity were not independent factors for poor clinical outcomes of COVID-19, generally.

Fluorescence polarization system for rapid COVID-19 diagnosis.

Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis. Termed CODA (CRISPR Optical Detection of Anisotropy), the method combined isothermal nucleic acid amplification, activation of CRISPR/Cas12a, and signal generation in a single assay, eliminating extra manual steps. Importantly, signal detection was based on the ratiometric measurement of fluorescent anisotropy, which allowed CODA to achieve a high signal-to-noise ratio. For point-of-care operation, we built a compact, standalone CODA device integrating optoelectronics, an embedded heater, and a microcontroller for data processing. The developed system completed SARS-CoV-2 RNA detection within 20 min of sample loading; the limit of detection reached 3 copy/µL. When applied to clinical samples (10 confirmed COVID-19 patients; 10 controls), the rapid CODA test accurately classified COVID-19 status, in concordance with gold-standard clinical diagnostics.

Fast detection of SARS-CoV-2 RNA via the integration of plasmonic thermocycling and fluorescence detection in a portable device.

The diagnosis of severe acute respiratory syndrome 2 (SARS-CoV-2) infection by quantitative PCR with reverse transcription (RT-qPCR) typically involves bulky instrumentation in centralized laboratories and an assay time of 1-2 h. Here, we show that SARS-CoV-2 RNA can be detected in 17 min via a portable device integrating reverse transcription, fast thermocycling (via plasmonic heating through magneto-plasmonic nanoparticles) and in situ fluorescence detection following magnetic clearance of the nanoparticles. The device correctly classified all nasopharyngeal, oropharyngeal and sputum samples from 75 patients with COVID-19 and 75 healthy controls, with good concordance in fluorescence intensity with standard RT-qPCR (Pearson coefficients > 0.7 for the N1, N2 and RPP30 genes). Fast, portable and automated nucleic acid detection should facilitate testing at the point of care.

Management of Allergic Patients During the COVID-19 Pandemic in Asia.

Although a viral infection is a major triggering factor of asthma and allergic diseases, asthma is suggested to be not a predisposing condition for coronavirus disease 2019 (COVID-19) infection. However, patients with severe asthma/allergic disease requiring systemic corticosteroids or immunosuppressive agents may be at higher risk of more severe clinical course of this infectious disease. For allergic patients who have been followed up at an allergy clinic in our region, it is recommended that they (patients with asthma, rhinitis, atopic dermatitis or chronic urticaria) continue to receive maintenance therapy and be in a well-controlled status. Patients who have used biologics (currently available for targeting type 2 inflammation) and allergen immunotherapy should continue the treatment while minimizing hospital and face-to-face visits. It is essential to wear protective equipment for the protection of health care workers as well as patients. We report this consensus to support allergists and clinical immunologists to make optimal decisions under the urgent situation in Asia.