A novel ACE2-Based electrochemical biosensor for sensitive detection of SARS-CoV-2.

SARS-CoV-2 emerged in late 2019 and quickly spread globally, resulting in significant morbidity, mortality, and socio-economic disruptions. As of now, collaborative global efforts in vaccination and the advent of novel diagnostic tools have considerably curbed the spread and impact of the virus in many regions. Despite this progress, the demand remains for low-cost, accurate, rapid and scalable diagnostic tools to reduce the influence of SARS-CoV-2. Herein, the angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV-2, was immobilized on two types of electrodes, a screen-printed gold electrode (SPGE) and a screen-printed carbon electrode (SPCE), to develop electrochemical biosensors for detecting SARS-CoV-2 with high sensitivity and selectivity. This was achieved by using 1H, 1H, 2H, 2H-perfluorodecanethiol (PFDT) and aryl diazonium salt serving as linkers for SPGEs and SPCEs, respectively. Once SARS-CoV-2 was anchored onto the ACE2, the interaction of the virus with the redox probe was analyzed using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Aryl diazonium salt was observed as a superior linker compared to PFDT due to its consistent performance in the modification of the SPCEs and effective ACE2 enzyme immobilization. A distinct pair of redox peaks in the cyclic voltammogram of the biosensor modified with aryl diazonium salt highlighted the redox reaction between the functional groups of SARS-CoV-2 and the redox probe. The sensor presented a linear relationship between the redox response and the logarithm of SARS-CoV-2 concentration, with a detection limit of 1.02 × 106 TCID50/mL (50% tissue culture infectious dose). Furthermore, the biosensor showed remarkable selectivity towards SARS-CoV-2 over H1N1virus.

Prevalence of persistent SARS-CoV-2 in a large community surveillance study.

Persistent SARS-CoV-2 infections may act as viral reservoirs that could seed future outbreaks1-5, give rise to highly divergent lineages6-8 and contribute to cases with post-acute COVID-19 sequelae (long COVID)9,10. However, the population prevalence of persistent infections, their viral load kinetics and evolutionary dynamics over the course of infections remain largely unknown. Here, using viral sequence data collected as part of a national infection survey, we identified 381 individuals with SARS-CoV-2 RNA at high titre persisting for at least 30 days, of which 54 had viral RNA persisting at least 60 days. We refer to these as 'persistent infections' as available evidence suggests that they represent ongoing viral replication, although the persistence of non-replicating RNA cannot be ruled out in all. Individuals with persistent infection had more than 50% higher odds of self-reporting long COVID than individuals with non-persistent infection. We estimate that 0.1-0.5% of infections may become persistent with typically rebounding high viral loads and last for at least 60 days. In some individuals, we identified many viral amino acid substitutions, indicating periods of strong positive selection, whereas others had no consensus change in the sequences for prolonged periods, consistent with weak selection. Substitutions included mutations that are lineage defining for SARS-CoV-2 variants, at target sites for monoclonal antibodies and/or are commonly found in immunocompromised people11-14. This work has profound implications for understanding and characterizing SARS-CoV-2 infection, epidemiology and evolution.

Nucleic acid hybridization-based detection of pathogenic RNA using microscale thermophoresis.

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.

CRISPR dual enzyme cleavage triggers DNA and RNA substrate cleavage for SARS-CoV-2 dual gene detection.

The widespread dissemination of coronavirus 2019 imposes a significant burden on society. Therefore, rapid detection facilitates the reduction of transmission risk. In this study, we proposed a multiplex diagnostic platform for the rapid, ultrasensitive, visual, and simultaneous detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 1ab (ORF1ab) and N genes. A visual diagnostic method was developed using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a/Cas13a dual-enzyme digestion system integrated with multiplex reverse transcriptase-recombinase polymerase amplification (RT-RPA). Two CRISPR-Cas proteins (Cas12a and Cas13a) were introduced into the system to recognize and cleave the N gene and ORF1ab gene, respectively. We used fluorescent or CRISPR double digestion test strips to detect the digested products, with the N gene corresponding to the FAM channel in the PCR instrument or the T1 line on the test strip, and the ORF1ab gene corresponding to the ROX channel in the PCR instrument or the T2 line on the test strip. The analysis can be completed in less than 20 min. Meanwhile, we assessed the application of the platform and determined a sensitivity of up to 200 copies/mL. Additionally, dual gene validation in 105 clinical nasopharyngeal swab samples showed a 100% positive predictive value agreement and a 95.7% negative predictive value agreement between our method and quantitative reverse transcription-polymerase chain reaction. Overall, our method offered a novel insight into the rapid diagnosis of SARS-CoV-2.

Development of a Fluorescent Immunochromatographic Assay Based on Quantum Dot-Functionalized Two-Dimensional Monolayer Ti3C2 MXene Nanoprobes for the Simultaneous Detection of Influenza A Virus and SARS-CoV-2.

Accurate and rapid detection of the influenza A virus (FluA) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can effectively control their spread. We developed a colorimetric and fluorescent dual-functional two-channel immunochromatographic assay (ICA) biosensor to simultaneously detect the above-mentioned viruses. A unique two-dimensional Ti3C2-QD immunoprobe was established by adsorbing dense quantum dots (QDs) onto the light green monostromatic Ti3C2 MXene surface, resulting in light green colorimetric and superior fluorescence signals and guaranteeing high sensitivity, stability, and excellent liquidity for ICA detection. Rapid visual screening for FluA and SARS-CoV-2 infections was applicable via a green colorimetric signal. Sensitive and quantitative detection of viruses in their early stages of infection was performed by using the fluorescence signal. Our proposed Ti3C2-QD-ICA biosensor can simultaneously detect 1 ng/mL or 2.4 pg/mL FluA and 1 ng/mL or 6.2 pg/mL SARS-CoV-2 via its colorimetric or fluorescence signals, respectively, with a short testing time (20 min), good reproducibility, specificity, and accuracy. In addition, this method demonstrated sensitivity higher than that of the conventional AuNP-based ICA method in throat swab samples. Hence, our proposed Ti3C2-QD-ICA method can be potentially applied for the rapid, ultrasensitive, and multiplex detection of respiratory viruses.

Detection of SARS-CoV-2 RNA in wastewater from an enclosed college campus serves as an early warning surveillance system.

SARS-CoV-2, the causative agent of Covid-19, is shed from infected persons in respiratory droplets, feces, and urine. Using quantitative PCR (qPCR), our group hypothesized that we could detect SARS-CoV-2 in wastewater samples collected on a university campus prior to the detection of the virus in individuals on campus. Wastewater samples were collected 3 times a week from 5 locations on the main campus of the University of North Carolina Wilmington (UNCW) from July 24, 2020 to December 21, 2020. Post-collection, total RNA was extracted and SARS-CoV-2 RNA in the samples was detected by qPCR. SARS-CoV-2 signal was detected on campus beginning on August 19 as classes began and the signal increased in both intensity and breadth as the Fall semester progressed. A comparison of two RNA extraction methods from wastewater showed that SARS-CoV-2 was detected more frequently on filter samples versus the direct extracts. Aligning our wastewater data with the reported SARS-CoV-2 cases on the campus Covid-19 dashboard showed the virus signal was routinely detected in the wastewater prior to clusters of individual cases being reported. These data support the testing of wastewater for the presence of SARS-CoV-2 and may be used as part of a surveillance program for detecting the virus in a community prior to an outbreak occurring and could ultimately be incorporated with other SARS-CoV-2 metrics to better inform public health enabling a quick response to contain or mitigating spread of the virus.

An integrated microfluidic chip for nucleic acid extraction and continued cdPCR detection of pathogens.

This paper introduces an enclosed microfluidic chip that integrates sample preparation and the chamber-based digital polymerase chain reaction (cdPCR). The sample preparation of the chip includes nucleic acid extraction and purification based on magnetic beads, which adsorb nucleic acids by moving around the reaction chambers to complete the reactions including lysis, washing, and elution. The cdPCR area of the chip consists of tens of thousands of regularly arranged microchambers. After the sample preparation processes are completed, the purified nucleic acid can be directly introduced into the microchambers for amplification and detection on the chip. The nucleic acid extraction performance and digital quantification performance of the system were examined using synthetic SARS-CoV-2 plasmid templates at concentrations ranging from 101-105 copies per µL. Further on, a simulated clinical sample was used to test the system, and the integrated chip was able to accurately detect SARS-CoV-2 virus particle samples doped with interference (saliva) with a detection limit of 10 copies per µL. This integrated system could provide a promising tool for point-of-care testing of pathogenic infections.

Dispute simmers over who first shared SARS-CoV-2's genome.

Scientists suggest that GISAID, a prominent database of virus sequences, is rewriting pandemic history.

Share all SARS-CoV-2 data immediately.

When the first cases of human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were reported from Wuhan, China, in December 2019, there was quick agreement across scientific and health communities that understanding the facts about its emergence would help prevent future outbreaks. Never could I have imagined the degree of politicization that would cloud this quest. Over the past 39 months, while reported deaths from COVID-19 increased to nearly 7 million worldwide, science on the virus's origins has gotten smaller while the politics surrounding this question has grown ever bigger. Last month, the World Health Organization (WHO) learned that scientists in China possessed data on viral samples from Wuhan that had been gathered in January 2020, which should have been shared immediately-not 3 years later-with the global research community. The lack of data disclosure is simply inexcusable. The longer it takes to understand the origins of the pandemic, the harder it becomes to answer the question, and the more unsafe the world becomes.

If SARS-CoV-2 is blamed for causing meningoencephalitis, the virus must be detected in the CSF.

We have read with interest the article by Watroba and Bryda on a new-born male with SARS-CoV-2 associated meningo-encephalitis, post-inflammatory hydrocephalus and seizures [1]. Neuro-COVID in this patient was treated with a polypragmatic approach, including phenobarbital, acetazolamide, fluconazole, acyclovir, cefotaxime, and vancomycin [1]. The study is appealing but has limitations that raise concerns and should be discussed.

Codetections of Other Respiratory Viruses Among Children Hospitalized With COVID-19.

OBJECTIVES: To assess the clinical impact of respiratory virus codetections among children hospitalized with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: During March 2020 to February 2022, the US coronavirus disease 2019 (COVID-19)-Associated Hospitalization Surveillance Network (COVID-NET) identified 4372 children hospitalized with SARS-CoV-2 infection admitted primarily for fever, respiratory illness, or presumed COVID-19. We compared demographics, clinical features, and outcomes between those with and without codetections who had any non-SARS-CoV-2 virus testing. Among a subgroup of 1670 children with complete additional viral testing, we described the association between presence of codetections and severe respiratory illness using age-stratified multivariable logistic regression models. RESULTS: Among 4372 children hospitalized, 62% had non-SARS-CoV-2 respiratory virus testing, of which 21% had a codetection. Children with codetections were more likely to be <5 years old (yo), receive increased oxygen support, or be admitted to the ICU (P < .001). Among children <5 yo, having any viral codetection (<2 yo: adjusted odds ratio [aOR] 2.1 [95% confidence interval [CI] 1.5-3.0]; 2-4 yo: aOR 1.9 [95% CI 1.2-3.1]) or rhinovirus/enterovirus codetection (<2 yo: aOR 2.4 [95% CI 1.6-3.7]; 2-4: aOR 2.4 [95% CI 1.2-4.6]) was significantly associated with severe illness. Among children <2 yo, respiratory syncytial virus (RSV) codetections were also significantly associated with severe illness (aOR 1.9 [95% CI 1.3-2.9]). No significant associations were seen among children ≥5 yo. CONCLUSIONS: Respiratory virus codetections, including RSV and rhinovirus/enterovirus, may increase illness severity among children <5 yo hospitalized with SARS-CoV-2 infection.

New clues to pandemic's origin surface, causing uproar.

Genetic sequences from Wuhan market may point to animal that spread SARS-CoV-2, but data remain hidden.