Rapid Detection of Predominant SARS-CoV-2 Variants Using Multiplex High-Resolution Melting Analysis.

Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a considerable threat to global public health. This study developed and evaluated a rapid, low-cost, expandable, and sequencing-free high-resolution melting (HRM) assay for the direct detection of SARS-CoV-2 variants. A panel of 64 common bacterial and viral pathogens that can cause respiratory tract infections was employed to evaluate our method's specificity. Serial dilutions of viral isolates determined the sensitivity of the method. Finally, the assay's clinical performance was assessed using 324 clinical samples with potential SARS-CoV-2 infection. Multiplex HRM analysis accurately identified SARS-CoV-2 (as confirmed with parallel reverse transcription-quantitative PCR [qRT-PCR] tests), differentiating between mutations at each marker site within approximately 2 h. For each target, the limit of detection (LOD) was lower than 10 copies/reaction (the LOD of N, G142D, R158G, Y505H, V213G, G446S, S413R, F486V, and S704L was 7.38, 9.72, 9.96, 9.96, 9.50, 7.80, 9.33, 8.25, and 8.25 copies/reaction, respectively). No cross-reactivity occurred with organisms of the specificity testing panel. In terms of variant detection, our results had a 97.9% (47/48) rate of agreement with standard Sanger sequencing. The multiplex HRM assay therefore offers a rapid and simple procedure for detecting SARS-CoV-2 variants. IMPORTANCE In the face of the current severe situation of increasing SARS-CoV-2 variants, we developed an upgraded multiplex HRM method for the predominant SARS-CoV-2 variants based on our original research. This method not only could identify the variants but also could be utilized in subsequent detection of novel variants since the assay has great performance in terms of flexibility. In summary, the upgraded multiplex HRM assay is a rapid, reliable, and economical detection method, which could better screen prevalent virus strains, monitor the epidemic situation, and help to develop measures for the prevention and control of SARS-CoV-2.

A multiplex method for detection of SARS-CoV-2 variants based on MALDI-TOF mass spectrometry.

The recent outbreak of the coronavirus disease 2019 (COVID-19) pandemic and the continuous evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have highlighted the significance of new detection methods for global monitoring and prevention. Although quantitative reverse transcription PCR (RT-qPCR), the current gold standard for diagnosis, performs excellently in genetic testing, its multiplexing capability is limited because of the signal crosstalk of various fluorophores. Herein, we present a highly efficient platform which combines 17-plex assays with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), enabling the targeting of 14 different mutation sites of the spike gene. Diagnosis using a set of 324 nasopharyngeal swabs or sputum clinical samples with SARS-CoV-2 MS method was identical to that with the RT-qPCR. The detection consistency of mutation sites was 97.9% (47/48) compared to Sanger sequencing without cross-reaction with other respiratory-related pathogens. Therefore, the MS method is highly potent to track and assess SARS-CoV-2 changes in a timely manner, thereby aiding the continuous response to viral variation and prevention of further transmission.

A Multiplex Method for Detection of SARS-CoV-2 Variants Based on MALDI-TOF Mass Spectrometry

The recent outbreak of the coronavirus disease 2019 (COVID-19) pandemic and the continuous evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have highlighted the significance of new detection methods for global monitoring and prevention. Although quantitative reverse transcription PCR (RT-qPCR), the current gold standard for diagnosis, performs excellently in genetic testing, its multiplexing capability is limited because of the signal crosstalk of various fluorophores. Herein, we present a highly efficient platform which combines 17-plex assays with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), enabling the targeting of 14 different mutation sites of the spike gene. Diagnosis using a set of 324 nasopharyngeal swab or sputum clinical samples with SARS-CoV-2 MS method was identical to that with the RT-qPCR. The detection consistency of mutation sites was 97.9% (47/48) compared to Sanger sequencing without cross-reaction with other respiratory-related pathogens. Therefore, the MS method is highly potent to track and assess SARS-CoV-2 changes in a timely manner, thereby aiding continuous response to viral variation and prevention of further transmission.

Post-Acute COVID-19 Respiratory Symptoms in Patients With Asthma: An Electronic Health Records-Based Study.

BACKGROUND: Post-viral respiratory symptoms are common among patients with asthma. Respiratory symptoms after acute COVID-19 are widely reported in the general population, but large-scale studies identifying symptom risk for patients with asthma are lacking. OBJECTIVE: To identify and compare risk for post-acute COVID-19 respiratory symptoms in patients with and without asthma. METHODS: This retrospective, observational cohort study included COVID-19-positive patients between March 4, 2020, and January 20, 2021, with up to 180 days of health care follow-up in a health care system in the Northeastern United States. Respiratory symptoms recorded in clinical notes from days 28 to 180 after COVID-19 diagnosis were extracted using natural language processing. Cohorts were stratified by hospitalization status during the acute COVID-19 period. Univariable and multivariable analyses were used to compare symptoms among patients with and without asthma adjusting for demographic and clinical confounders. RESULTS: Among 31,084 eligible patients with COVID-19, 2863 (9.2%) had hospitalization during the acute COVID-19 period; 4049 (13.0%) had a history of asthma, accounting for 13.8% of hospitalized and 12.9% of nonhospitalized patients. In the post-acute COVID-19 period, patients with asthma had significantly higher risk of shortness of breath, cough, bronchospasm, and wheezing than patients without an asthma history. Incident respiratory symptoms of bronchospasm and wheezing were also higher in patients with asthma. Patients with asthma who had not been hospitalized during acute COVID-19 had additionally higher risk of cough, abnormal breathing, sputum changes, and a wider range of incident respiratory symptoms. CONCLUSION: Patients with asthma may have an under-recognized burden of respiratory symptoms after COVID-19 warranting increased awareness and monitoring in this population.

Repurposing of clinically approved drugs for treatment of coronavirus disease 2019 in a 2019-novel coronavirus-related coronavirus model.

BACKGROUND: Medicines for the treatment of 2019-novel coronavirus (2019-nCoV) infections are urgently needed. However, drug screening using live 2019-nCoV requires high-level biosafety facilities, which imposes an obstacle for those institutions without such facilities or 2019-nCoV. This study aims to repurpose the clinically approved drugs for the treatment of coronavirus disease 2019 (COVID-19) in a 2019-nCoV-related coronavirus model. METHODS: A 2019-nCoV-related pangolin coronavirus GX_P2V/pangolin/2017/Guangxi was described. Whether GX_P2V uses angiotensin-converting enzyme 2 (ACE2) as the cell receptor was investigated by using small interfering RNA (siRNA)-mediated silencing of ACE2. The pangolin coronavirus model was used to identify drug candidates for treating 2019-nCoV infection. Two libraries of 2406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on Vero E6 cells by GX_P2V infection. The anti-viral activities and anti-viral mechanisms of potential drugs were further investigated. Viral yields of RNAs and infectious particles were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and plaque assay, respectively. RESULTS: The spike protein of coronavirus GX_P2V shares 92.2% amino acid identity with that of 2019-nCoV isolate Wuhan-hu-1, and uses ACE2 as the receptor for infection just like 2019-nCoV. Three drugs, including cepharanthine (CEP), selamectin, and mefloquine hydrochloride, exhibited complete inhibition of cytopathic effects in cell culture at 10 µmol/L. CEP demonstrated the most potent inhibition of GX_P2V infection, with a concentration for 50% of maximal effect [EC50] of 0.98 µmol/L. The viral RNA yield in cells treated with 10 µmol/L CEP was 15,393-fold lower than in cells without CEP treatment ([6.48 ±â€Š0.02] × 10vs. 1.00 ±â€Š0.12, t = 150.38, P < 0.001) at 72 h post-infection (p.i.). Plaque assays found no production of live viruses in media containing 10 µmol/L CEP at 48 h p.i. Furthermore, we found CEP had potent anti-viral activities against both viral entry (0.46 ±â€Š0.12, vs.1.00 ±â€Š0.37, t = 2.42, P < 0.05) and viral replication ([6.18 ±â€Š0.95] × 10vs. 1.00 ±â€Š0.43, t = 3.98, P < 0.05). CONCLUSIONS: Our pangolin coronavirus GX_P2V is a workable model for 2019-nCoV research. CEP, selamectin, and mefloquine hydrochloride are potential drugs for treating 2019-nCoV infection. Our results strongly suggest that CEP is a wide-spectrum inhibitor of pan-betacoronavirus, and further study of CEP for treatment of 2019-nCoV infection is warranted.

All-in-one in situ colorimetric RT-LAMP assay for point-of-care testing of SARS-CoV-2.

The ongoing outbreaks of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have resulted in unprecedented challenges to global health. To effectively contain the COVID-19 transmission, rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. To address the huge need for ever-increasing tests, we developed a facile all-in-one nucleic acid testing assay by combining Si-OH activated glass bead (aGB)-based viral RNA fast extraction and in situ colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection in a single tube. aGBs demonstrate a strong ability to capture viral RNA in a guanidinium-based lysis buffer, and the purified aGBs/RNA composite, without RNA elution step, could be directly used to perform RT-LAMP assay. The assay was well characterized by using a novel SARS-CoV-2-like coronavirus GX/P2V, and showed a limit of detection (LOD) of 15 copies per µL in simulated clinical samples within 50 min. We further demonstrated our assay by testing simulated SARS-CoV-2 pseudovirus samples, showing an LOD of 32 copies per µL and high specificity without cross-reactivity with the most closely related GX/P2V or host DNA/RNA. The all-in-one approach developed in this study has the potential as a simple, scalable, and time-saving alternative for point-of-care testing of SARS-CoV-2 in low-income regions, as well as a promising tool for at-home testing.

PASCLex: A comprehensive post-acute sequelae of COVID-19 (PASC) symptom lexicon derived from electronic health record clinical notes.

OBJECTIVE: To develop a comprehensive post-acute sequelae of COVID-19 (PASC) symptom lexicon (PASCLex) from clinical notes to support PASC symptom identification and research. METHODS: We identified 26,117 COVID-19 positive patients from the Mass General Brigham's electronic health records (EHR) and extracted 328,879 clinical notes from their post-acute infection period (day 51-110 from first positive COVID-19 test). PASCLex incorporated Unified Medical Language System® (UMLS) Metathesaurus concepts and synonyms based on selected semantic types. The MTERMS natural language processing (NLP) tool was used to automatically extract symptoms from a development dataset. The lexicon was iteratively revised with manual chart review, keyword search, concept consolidation, and evaluation of NLP output. We assessed the comprehensiveness of PASCLex and the NLP performance using a validation dataset and reported the symptom prevalence across the entire corpus. RESULTS: PASCLex included 355 symptoms consolidated from 1520 UMLS concepts of 16,466 synonyms. NLP achieved an averaged precision of 0.94 and an estimated recall of 0.84. Symptoms with the highest frequency included pain (43.1%), anxiety (25.8%), depression (24.0%), fatigue (23.4%), joint pain (21.0%), shortness of breath (20.8%), headache (20.0%), nausea and/or vomiting (19.9%), myalgia (19.0%), and gastroesophageal reflux (18.6%). DISCUSSION AND CONCLUSION: PASC symptoms are diverse. A comprehensive lexicon of PASC symptoms can be derived using an ontology-driven, EHR-guided and NLP-assisted approach. By using unstructured data, this approach may improve identification and analysis of patient symptoms in the EHR, and inform prospective study design, preventative care strategies, and therapeutic interventions for patient care.

Transcriptome analysis of cepharanthine against a SARS-CoV-2-related coronavirus.

Antiviral therapies targeting the pandemic coronavirus disease 2019 (COVID-19) are urgently required. We studied an already-approved botanical drug cepharanthine (CEP) in a cell culture model of GX_P2V, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related virus. RNA-sequencing results showed the virus perturbed the expression of multiple genes including those associated with cellular stress responses such as endoplasmic reticulum (ER) stress and heat shock factor 1 (HSF1)-mediated heat shock response, of which heat shock response-related genes and pathways were at the core. CEP was potent to reverse most dysregulated genes and pathways in infected cells including ER stress/unfolded protein response and HSF1-mediated heat shock response. Additionally, single-cell transcriptomes also confirmed that genes of cellular stress responses and autophagy pathways were enriched in several peripheral blood mononuclear cells populations from COVID-19 patients. In summary, this study uncovered the transcriptome of a SARS-CoV-2-related coronavirus infection model and anti-viral activities of CEP, providing evidence for CEP as a promising therapeutic option for SARS-CoV-2 infection.

COVID-19 severity in asthma patients: a multi-center matched cohort study.

OBJECTIVE: The evidence pertaining to the effects of asthma on Coronavirus disease 2019 outcomes has been unclear. To improve our understanding of the clinically important association of asthma and Coronavirus disease 2019. METHODS: A matched cohort study was performed using data from the Mass General Brigham Health Care System (Boston, MA). Adult (age ≥18 years) patients with confirmed Coronavirus disease 2019 and without chronic obstructive pulmonary disease, cystic fibrosis, or interstitial lung disease between March 4, 2020 and July 2, 2020 were analyzed. Up to five non-asthma comparators were matched to each asthma patient based on age (within 5 years), sex, and date of positive test (within 7 days). The primary outcomes were hospitalization, mechanical ventilation, and death, using multivariable Cox-proportional hazards models accounting for competing risk of death, when appropriate. Patients were followed for these outcomes from diagnosis of Coronavirus disease 2019 until July 2, 2020. RESULTS: Among 562 asthma patients, 199 (21%) were hospitalized, 15 (3%) received mechanical ventilation, and 7 (1%) died. Among the 2686 matched comparators, 487 (18%) were hospitalized, 107 (4%) received mechanical ventilation, and 69 (3%) died. The adjusted Hazard Ratios among asthma patients were 0.99 (95% Confidence Internal 0.80, 1.22) for hospitalization, 0.69 (95% Confidence Internal 0.36, 1.29) for mechanical ventilation, and 0.30 (95% Confidence Internal 0.11, 0.80) for death. CONCLUSIONS: In this matched cohort study from a large Boston-based healthcare system, asthma was associated with comparable risk of hospitalization and mechanical ventilation but a lower risk of mortality.

COVID-19 prevention and control scheme in an ENT endoscopy unit in China

Due to the increasing spread of the novel coronavirus disease (COVID-19), prevention and control measures have become increasingly important. As a key location for diagnosing and treating upper airway diseases, strict precautions are required in ear nose and throat (ENT) endoscopy units. Endoscopy workers have a high risk of occupational exposure. Therefore, procedures must be strictly performed according to the prevention and control plan. The prevention and control requirements for COVID-19, as directed by the National Health Commission of China, have been universally deployed in our hospital. We have carefully analyzed the risk factors of infection during the epidemic period and subsequently formulated a prevention and control scheme for COVID-19 based on the infection control measures in the ENT endoscopy unit. These have helped to avoid cross-infection in the hospital and ensure the safety of patients and medical staff during the COVID-19 epidemic.