Recent progress in CRISPR-based microfluidic assays and applications

In the past few years, CRISPR-based (Clustered regularly interspaced short palindromic repeats-based) detection techniques have arisen in the field of bioanalysis, molecular biology and clinical diagnosis. However, there are still some limitations, such as high cost, high technical requirements, complexity, cross contamination, lack of quantification strategies, etc. Microfluidic platforms, as emerging technol-ogies, have the potential to take CRISPR-based technology to the new level in the future. In this review, we illustrate the recent progress as well as our work and collaborative work in CRISPR-based microfluidic detection assays based on the categories of microfluidic chips, including paper-based microfluidics, centrifugal microfluidics, digital microfluidics, electrochemical microfluidics and wearable microfluidics, mainly focusing on how microfluidic platforms improve CRISPR-based detection method in the aspect of visualization, detection throughput, anti-contamination, simple operation, sensitivity, specificity and quantification, etc. And we outlook the future trends of CRISPR-based microfluidic detection methods in both innovation of CRISPR-based and design of microfluidic platforms. (c) 2022 Elsevier B.V. All rights reserved.

Impact of high/low-volume hospitals of ECMO before the COVID-19 pandemic era on ECMO outcomes in patients with fatal COVID-19

Introduction: The impact of treatment experience with ECMO prior to the COVID-19 pandemic era on the outcomes of ECMO during the COVID-19 pandemic has not been well investigated. The aim of this study was to investigate the differences in the outcomes of ECMO between highvolume hospitals in Japan before the COVID-19 pandemic and low-volume hospitals. Method(s): This is a prospective observational cohort study of patients with fatal COVID-19 pneumonia in Japan receiving ECMO. The association between patient background, high/low-volume hospitals, and survival from February 2020 to November 2021 were evaluated. Prognostic factors were assessed by Kaplan-Meier and Cox proportional hazards analyses. High-volume hospitals were defined as those with a total of at least 20 cases prior to the COVID-19 pandemic era. Result(s): Ten hospitals (1.1% of a total of 900 ICUs in Japan) were classified as high-volume hospitals, with a median number of ECMO cases of 35.5 (IQR, 25.5-40.5). There were a total of 1,214 ECMO cases during the COVID-19 pandemic, of which 1030 (85%) were treated in low-volume hospitals and 184 (15%) were treated in high-volume hospitals. Kaplan-Meier analysis demonstrated slightly better survival in high-volume hospitals in 30-day (p=0.062) and 60-day (p=0.11) survivals, whereas, in 90-day survival, the two groups were almost identical (p=0.87). Cox proportional hazards analysis also failed to demonstrate that high-volume hospital before the COVID-19 pandemic era was independent prognostic factor (HR, 0.97;95%CI, 0.74-1.28). Conclusion(s): In Japan, high-volume hospital prior to the COVID-19 pandemic era was not independent prognostic factor.

Low-volume ultrasound-guided supraclavicular block in a multicomorbid patient for emergency vascular surgery - In COVID-19 era.

Supraclavicular block is the most commonly used block in upper limb surgeries, right from the day it was introduced into clinical practice in Germany by Kulenkampff in 1911. The block underwent many changes in its application due to the advent of peripheral nerve stimulator and ultrasonographic application in regional anesthesia. This case report focuses on supraclavicular block's application in a multicomorbid patient, the drug dose required, and how the scope of regional anesthesia can be extended in times of pandemic, like coronavirus disease 2019 (COVID-19), in coming future.

SARS-CoV-2 surveillance in indoor and outdoor size-segregated aerosol samples.

We aimed to determine the presence of SARS-CoV-2 RNA in indoor and outdoor size-segregated aerosol samples (PM10-2.5, PM2.5). Five outdoor daily samples were collected between November and December 2020 in an urban/industrial area with relatively high PM10 levels (Maliaño, Santander, Spain) by using a PM impactor (air flowrate of 30 L/min). In a non-hospital indoor sampling surveillance context, 8 samples in classrooms and 6 samples in the central library-Paraninfo of the University of Cantabria (UC) were collected between April and June 2021 by using personal PM samplers (air flowrate of 3 L/min). Lastly, 8 samples in the pediatric nasopharyngeal testing room at Liencres Hospital, 6 samples from different single occupancy rooms of positive patients, and 2 samples in clinical areas of the COVID plant of the University Hospital Marqués de Valdecilla (HUMV) were collected between January and May 2021. N1, N2 genes were used to test the presence of SARS-CoV-2 RNA by RT-qPCR. SARS-CoV-2 positive detection was only obtained from one fine fraction (PM2.5) sample, corresponding to one occupancy room, where a patient with positive PCR and cough was present. Negative results found in other sampling areas such as the pediatric nasopharyngeal testing rooms should be interpreted in terms of air sampling volume limitation and good ventilation.

INCREASING THE LOW-RISK THRESHOLD FOR PATIENTS WITH UPPER GASTROINTESTINAL BLEEDING DURING THE COVID-19 PANDEMIC: A PROSPECTIVE, MULTICENTRE, FEASIBILITY STUDY

BACKGROUND: The differences in clinical outcomes following emergency medical services between high and low-volume centers with respect to acute lower gastrointestinal bleeding (ALGIB) remain unknown. In this study, we aimed to compare clinical outcomes and management strategies between high and low-volume centers in emergency medical services. METHODS: In this retrospective study, propensity score matching was used to compare high and low-volume hospitals with respect to emergency medical services. We identified 10,550 cases of ALGIB from 43 hospitals including one prefectural group between May 2002 and August 2020. After excluding duplicated cases, 8,286 cases were included and divided into two groups (high and low-volume centers) according to the number of emergency medical services performed in 2019. Hospitals with more than 5,000 cases of emergency medical services in 2019 were categorized as high-volume centers. The remaining centers were considered to be low-volume centers. Age;sex;history of colectomy and colonic diverticular bleeding (CDB);and comorbidities, including Charlson Comorbidity Index, vital signs at admission, laboratory data, and use of antithrombotic agents were used to calculate propensity scores that were matched one-to-one using the nearest neighbor method and applied to the high and the low-volume centers. We compared the two groups in terms of the diagnostic and treatment strategies used, which included computed tomography (CT) and colonoscopy, as well as the clinical outcomes thereof. RESULTS: A total of 2,652 patients were matched in each group. Although CDB was the most common cause of ALGIB in both groups, the proportions of definitive and presumptive CDB were both significantly higher in the low-volume centers (22% vs. 16%, P < 0.0001;45% vs. 41%, P=0.001). Both CT and enhanced CT were performed with greater frequency in the high-volume centers (80% vs. 66%, P < 0.0001;77% vs. 67%, P < 0.0001), but colonoscopy was not (84% vs. 94%, P < 0.0001). The proportion of patients who underwent either early colonoscopy (performed within 24 hours after admission) or endoscopic therapy was significantly lower in the high-volume centers (56% vs. 72%, P <0.0001;24% vs. 30%, P < 0.0001). The incidence of early rebleeding was not significantly different between the groups (16% vs. 18%, P=0.097). The median values (interquartile range) of PRBCs transfused were highest in the high-volume centers (0 [4] vs. 0 [2], P < 0.0001). However, a significant difference was not observed in LOS (7 [7] vs. 7 [6], P=0.069). CONCLUSION: Although clinical strategies for the management of ALGIB varied between the hospitals with high or low volumes of emergency medical services, early rebleeding did not differ between the two groups.

Rapid Detection of SARS-CoV-2 Using Duplex Reverse Transcription-Multienzyme Isothermal Rapid Amplification in a Point-of-Care Testing.

In December 2019, a severe acute respiratory syndrome caused by SARS-CoV-2 spread rapidly worldwide. Portable nucleic acid tests of SARS-CoV-2 are critically important for diagnostics. In this study, we used an isothermal amplification method-Multienzyme Isothermal Rapid Amplification (MIRA)-for rapid detection of SARS-CoV-2. We designed the primers and probes in ORF1ab and N gene of SARS-CoV-2. The amplicons could be monitored by lateral flow dipsticks (LFDs). The reaction temperature, time, concentrations of primers and probes, and working volume were optimized. Four commercial swab collection buffers were used to test the amplification efficacy of our assay without RNA extraction. Our assay was able to amplify duplex targets of SARS-CoV-2 in one single reaction using one-step RT-MIRA. The assay worked well in a low volume of 10 µl at 38°C for 20 min. Using three collection buffers without guanidinium, our assay was able to amplify efficaciously without RNA extraction. The 95% limit of detection (LoD) of the RT-MIRA assay was 49.5 (95% CI, 46.8-52.7) copies/ml for ORF1ab gene and 48.8 (95% CI, 46.5-52.6) copies/ml for N gene. There is no cross-reaction with other human respiratory pathogens, such as SARS-CoV, MERS-CoV, influenza A virus, influenza B virus, human adenovirus, respiratory syncytial virus, human parainfluenza virus, and coronavirus 229E in our assay. The precision evaluation revealed that the C50-20% to C50+20% range bounds the C5-C95 interval. This assay also showed high anti-interference ability. The extraction-free RT-MIRA and qPCR detection results of 243 nucleic acid specimens from suspected patients or national references showed a 100.0% (95% confidence interval, 94.2%-100.0%) positive predictive value and a 100.0% (95% confidence interval, 92.7%-100.0%) negative predictive value. Compared with qPCR, the kappa value of the two assays was 1.00 (P < 0.0001). In conclusion, we provide a portable and visualized method for detection of SARS-CoV-2 without RNA extraction, allowing its application in SARS-CoV-2 on-site detection.

A robust intelligent zero-day cyber-attack detection technique.

With the introduction of the Internet to the mainstream like e-commerce, online banking, health system and other day-to-day essentials, risk of being exposed to various are increasing exponentially. Zero-day attack(s) targeting unknown vulnerabilities of a software or system opens up further research direction in the field of cyber-attacks. Existing approaches either uses ML/DNN or anomaly-based approach to protect against these attacks. Detecting zero-day attacks through these techniques miss several parameters like frequency of particular byte streams in network traffic and their correlation. Covering attacks that produce lower traffic is difficult through neural network models because it requires higher traffic for correct prediction. This paper proposes a novel robust and intelligent cyber-attack detection model to cover the issues mentioned above using the concept of heavy-hitter and graph technique to detect zero-day attacks. The proposed work consists of two phases (a) Signature generation and (b) Evaluation phase. This model evaluates the performance using generated signatures at the training phase. The result analysis of the proposed zero-day attack detection shows higher performance for accuracy of 91.33% for the binary classification and accuracy of 90.35% for multi-class classification on real-time attack data. The performance against benchmark data set CICIDS18 shows a promising result of 91.62% for binary-class classification on this model. Thus, the proposed approach shows an encouraging result to detect zero-day attacks.

In Vivo Study of Aerosol, Droplets and Splatter Reduction in Dentistry.

Oral health care workers (OHCW) are exposed to pathogenic microorganisms during dental aerosol-generating procedures. Technologies aimed at the reduction of aerosol, droplets and splatter are essential. This in vivo study assessed aerosol, droplet and splatter contamination in a simulated clinical scenario. The coolant of the high-speed air turbine was colored with red concentrate. The red aerosol, droplets and splatter contamination on the wrists of the OHCW and chests of the OHCW/volunteer protective gowns, were assessed and quantified in cm2. The efficacy of various evacuation strategies was assessed: low-volume saliva ejector (LV) alone, high-volume evacuator (HV) plus LV and an extra-oral dental aerosol suction device (DASD) plus LV. The Kruskal-Wallis rank-sum test for multiple independent samples with a post-hoc test was used. No significant difference between the LV alone compared to the HV plus LV was demonstrated (p = 0.372059). The DASD combined with LV resulted in a 62% reduction of contamination of the OHCW. The HV plus LV reduced contamination by 53% compared to LV alone (p = 0.019945). The DASD demonstrated a 50% reduction in the contamination of the OHCWs wrists and a 30% reduction in chest contamination compared to HV plus LV. The DASD in conjunction with LV was more effective in reducing aerosol, droplets and splatter than HV plus LV.

Low Volume in Vitro Diagnostic Proton NMR Spectroscopy of Human Blood Plasma for Lipoprotein and Metabolite Analysis: Application to SARS-CoV-2 Biomarkers.

The utility of low sample volume in vitro diagnostic (IVDr) proton nuclear magnetic resonance (1H NMR) spectroscopic experiments on blood plasma for information recovery from limited availability or high value samples was exemplified using plasma from patients with SARS-CoV-2 infection and normal controls. 1H NMR spectra were obtained using solvent-suppressed 1D, spin-echo (CPMG), and 2-dimensional J-resolved (JRES) spectroscopy using both 3 mm outer diameter SampleJet NMR tubes (100 µL plasma) and 5 mm SampleJet NMR tubes (300 µL plasma) under in vitro diagnostic conditions. We noted near identical diagnostic models in both standard and low volume IVDr lipoprotein analysis (measuring 112 lipoprotein parameters) with a comparison of the two tubes yielding R2 values ranging between 0.82 and 0.99 for the 40 paired lipoprotein parameters samples. Lipoprotein measurements for the 3 mm tubes were achieved without time penalty over the 5 mm tubes as defined by biomarker recovery for SARS-CoV-2. Overall, biomarker pattern recovery for the lipoproteins was extremely similar, but there were some small positive offsets in the linear equations for several variables due to small shimming artifacts, but there was minimal degradation of the biological information. For the standard untargeted 1D, CPMG, and JRES NMR experiments on the same samples, the reduced signal-to-noise was more constraining and required greater scanning times to achieve similar differential diagnostic performance (15 min per sample per experiment for 3 mm 1D and CPMG, compared to 4 min for the 5 mm tubes). We conclude that the 3 mm IVDr method is fit-for-purpose for quantitative lipoprotein measurements, allowing the preparation of smaller volumes for high value or limited volume samples that is common in clinical studies. If there are no analytical time constraints, the lower volume experiments are equally informative for untargeted profiling.