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1.
Biosensors (Basel) ; 12(8)2022 Jul 25.
Article in English | MEDLINE | ID: covidwho-2023151

ABSTRACT

A silicon lab-on-chip, for the detection of nucleic acids through the integrated PCR and hybridization microarray, was developed. The silicon lab-on-chip manufactured through bio-MEMS technology is composed of two PCR microreactors (each volume 11.2 µL) and a microarray-hybridization microchamber (volume 30 µL), fluidically connected by buried bypass. It contains heaters and temperature sensors for the management and control of the temperature cycles during the PCR amplification and hybridization processes. A post-silicon process based on (i) plasmo-O2 cleaning/activation, (ii) vapor phase epoxy silanization, (iii) microarray fabrication and (iv) a protein-based passivation step was developed and fully characterized. The ssDNA microarray (4 rows × 10 columns) composed of 400 spots (spot size-70 ± 12 µm; spot-to-spot distance-130 ± 13 µm) was manufactured by piezo-dispense technology. A DNA microarray probe density in the range of 1310 to 2070 probe µm-2 was observed, together with a limit of detection of about 19 target µm-2. The performances of the silicon lab-on-chip were validated by the detection of the beta-globin gene directly from human blood. Remarkable sensitivity, multiplexing analysis and specificity were demonstrated for the detection of beta-globin and mycobacterium tuberculosis sequences.


Subject(s)
Lab-On-A-Chip Devices , Nucleic Acids , Oligonucleotide Array Sequence Analysis , Silicon , Humans , Nucleic Acids/analysis , Polymerase Chain Reaction , beta-Globins/analysis
2.
Diagnostics ; 12(8):1995, 2022.
Article in English | ProQuest Central | ID: covidwho-2023268

ABSTRACT

The complex and lengthy protocol of current viral nucleic acid extraction processes limits their use outside laboratory settings. Here, we describe a rapid and reliable method for extracting nucleic acids from viral samples using a rotating blade and magnetic beads. The viral membrane can be instantly lysed using a high-speed rotating blade, and nucleic acids can be immediately isolated using a silica magnetic surface. The process was completed within 60 s by this method. Routine washing and eluting processes were subsequently conducted within 5 min. The results achieved by this method were comparable to those of a commercially available method. When the blade-based lysis and magnetic bead adsorption processes were performed separately, the RNA recovery rate was very low, and the Ct value was delayed compared to simultaneous lysis and RNA adsorption. Overall, this method not only dramatically shortens the conventional extraction time but also allows for its convenient use outside the laboratory, such as at remote field sites and for point-of-care testing.

3.
Int Health ; 2022.
Article in English | Web of Science | ID: covidwho-2017965

ABSTRACT

BACKGROUND: During the coronavirus disease 2019 pandemic, a nucleic acid test is frequently conducted to identify positive cases. Compared with a hospital-based strategy, whole-community nucleic acid testing displays a unique advantage in rapid screening of a massive population. Yet a management plan to ensure ample and contamination-free sample collection is lacking.The objective of the current study was to establish an efficient operational mode of whole-community nucleic acid testing by management of a sample collection team and to provide a reference for joint prevention work to contain the spread of severe acute respiratory syndrome coronavirus 2. METHODS: The efficient operation of nucleic acid testing within the community was implemented by urgent setting up of sample collection teams, efficient allocation of medical supplies, optimization of management procedures and coordination among multiple working departments. RESULTS: A total of 21 585 nucleic acid samples were collected within 3 d, while no one was missed or experienced a cross infection. No falls, heatstroke, disputes or other adverse events occurred. CONCLUSIONS: Under the emergency setting of nucleic acid testing of a large population, a management system with orderly organization, clear division of responsibilities and standardized operational procedures should be formulated.

4.
Virulence ; 13(1):1471-1485, 2022.
Article in English | Web of Science | ID: covidwho-2017508

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen causing severe diarrhoea, dehydration, and death in nursing piglets and enormous economic losses for the global swine industry. Furthermore, it can infect multiple animal species including humans. Therefore, a rapid, definitive diagnostic assay is required for the effective control of this zoonotic pathogen. To identify PDCoV, we developed a nucleic acid detection assay combining reverse transcription recombinase-aided amplification (RT-RAA) with a lateral flow dipstick (LFD) targeting the highly conserved genomic region in the ORF1b gene. The RT-RAA-LFD assay exhibited good PDCoV detection reproducibility and repeatability and could be completed within 11 min. Ten minutes at 40 degrees C was required for nucleic acid amplification and 1 min at room temperature was needed for the visual LFD readout. The assay specifically detected PDCoV and did not cross-react with any other major swine pathogens. The 95% limit of detection (LOD) was 3.97 median tissue culture infectious dose PDCoV RNA per reaction. This performance was comparable to that of a reference TaqMan-based real-time RT-PCR (trRT-PCR) assay for PDCoV. Of 149 swine small intestine, rectal swab, and serum samples, 71 and 75 tested positive for PDCoV according to RT-RAA-LFD and trRT-PCR, respectively. The diagnostic coincidence rate for both assays was 97.32% (145/149) and the kappa value was 0.946 (p < 0.001). Overall, the RT-RAA-LFD assay is a user-friendly diagnostic tool that can rapidly and visually detect PDCoV.

5.
25th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2021 ; : 711-712, 2021.
Article in English | Scopus | ID: covidwho-2012173

ABSTRACT

The SARS-CoV-2 pandemic has elevated the development of novel diagnostic solutions, including rapid nucleic acid amplification tests (NAATs), to a global priority to meet the high demand for accurate, timely viral detection and diagnosis. However, ubiquitously implemented NAATs, such as polymerase chain reaction (PCR), consume hours of testing. We report a field-forward instrument capable of ultra-fast real-time PCR for amplification-based nucleic acid detection in a custom-designed microfluidic chip. Prudent selection and unconventional positioning of thermal cyclers relative to the microfluidic chip and a fluorescent detector permit ultra-fast simultaneous amplification and detection, with 40 cycles complete in under 10 minutes. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

6.
25th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2021 ; : 855-856, 2021.
Article in English | Scopus | ID: covidwho-2011960

ABSTRACT

Without global mass vaccination, COVID-19 will continue to infect and cause serious illness, disproportionately in low- and middle-income countries. Point-of-care and home-based nucleic acid amplification tests (NAATs) are valuable tools to control COVID-19 transmission. Here we present a rapid isothermal NAAT for duplexed detection of SARS-CoV-2 and an MS2 bacteriophage internal control. This assay amplifies RNA in less than 15 minutes, utilizes a low temperature of 39°C, and has fluorescence or visual lateral flow readout. This positions our assay for use in low-cost paper-based nucleic acid diagnostic devices for ultrasensitive and reliable COVID-19 detection in POC or home-based settings. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

7.
25th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2021 ; : 127-128, 2021.
Article in English | Scopus | ID: covidwho-2011604

ABSTRACT

We will present a microfluidic assay to detect SARS-CoV-2 RNA from nasopharyngeal swab samples. Our method leverages isotachophoresis (ITP) to integrate sample preparation, RT-LAMP, and CRISPR-based nucleic acid detection in an automatable chip. For the first time, we use ITP to purify, pre-concentrate and isothermally amplify target nucleic acids into a ~1 µL reaction volume on-chip. The device then transitions LAMP amplicons into an on-chip zone containing Cas12-gRNA complexes and reporter molecules to measure target-activated CRISPR activity. We will use our method to automatically detect COVID-19 from nasopharyngeal swab samples. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

8.
25th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2021 ; : 969-970, 2021.
Article in English | Scopus | ID: covidwho-2011590

ABSTRACT

Nucleic acid amplification detection is one of the most widely used molecular diagnostic techniques in recent years, which can rapidly and efficiently amplify the characteristic nucleotide sequences of pathogenic bacteria in the diagnosis of infectious diseases, it has been widely used in clinical diagnosis, disease screening and other fields. In this work, we report a micro-cavity digital PCR for rapid detection of pathogens on a silicon-based microfluidic chip. The device has the advantages of high flux, no pumping, rapid reaction, quantification and high sensitivity. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

9.
Journal of Xi'an Jiaotong University (Medical Sciences) ; 43(5):797-801, 2022.
Article in Chinese | EMBASE | ID: covidwho-2010485

ABSTRACT

Objective: To compare the clinical features of Omicron and Delta cases, so as to provide scientific basis for the prevention and treatment of COVID-19. Methods: The case-control study method was used to retrospectively analyze the clinical data of the Omicron cases admitted to the designated hospital for the treatment of COVID-19 in Xi'an from December 2021 to January 2022. and the Delta cases admitted during the same period were used as the control group. The demographic data, epidemiological history, vaccination status, clinical characteristics, laboratory tests, nucleic acid and antibody levels, and outcomes of patients in the two groups were collected and compared. Results: A total of 21 patients were included in the study, 5 were Omicron patients and 16 were Delta cases. The mean age of the patients in the two groups were (38.20±15.07) and (37.69±10.39) years, respectively.The time interval between the last vaccination and the diagnosis was (145.40±77.92) days and (159.00±99.74) days, respectively. For the initial symptoms, the patients with Omicron were mainly characterized by throat discomfort (3, 60%), cough and sputum (2, 40%), and the patients with Delta were mainly characterized by throat discomfort (5, 31.25%), fatigue (5, 31.25%), cough and sputum (4, 25%). On admission, laboratory tests showed that 60% of Omicron patients had low lymphocytes and elevated erythrocyte sedimentation rate, and 50% of patients in the delta group had elevated hemoglobin. The Ct values of ORFlab gene, N gene and E gene with Omicron were lower than those with Delta. And the difference of E gene between the two groups was statistically significant (t=-2.711, P=0.024). IgG antibody levels increased in both groups.The time for nucleic acid to turn negative with Omicron was (28.20±5.89) days, and it was (18.50±7.73) days with Delta, and the difference between the groups was statistically significant (t=2.565, P=0.019). The length of hospitalization with Omicron was (30.60±4.88) days, and that with Delta was (22.13±7.81) days, and the difference was statistically significant (t=2.270, P =0.035). Conclusions: The initial symptoms of Omicron patients are mainly throat discomfort, cough and sputum. The clinical manifestations are generally mild. The nucleic acid test Ct value is lower. The time for nucleic acid to turn negative and the time for hospitalization are longer, and the potential infectiousness is stronger. Those eligible for vaccination should complete the full course of vaccination and booster vaccination as soon as possible. At the same time, the management of "early detection, early reporting, early isolation, and early treatment" should be implemented.

10.
Frontiers in Pharmacology ; 13, 2022.
Article in English | EMBASE | ID: covidwho-2009899

ABSTRACT

Background: Our previous studies have shown that Yindan Jiedu granules (YDJDG) can effectively treat coronavirus disease 2019 (COVID-19);however, the high infectivity and the immune escape potential of the Omicron variant BA.2 make it more difficult to control, and patients with high-risk factors prone to progress rapidly. Purpose: To evaluate YDJDG’s efficacy in treating patients with the Omicron variant BA.2 with high-risk factors and compared it with that of Paxlovid. Methods: A total of 257 patients who fulfilled the inclusion criteria were allocated to the YDJDG (115 cases), Paxlovid (115 cases), and control (27 cases) groups. A Cox regression model was used to analyze the independent factors affecting the shedding time of nucleic acid in 14 days. Propensity score matching (PSM) was used to match the characteristics of individuals in the three groups, while the Kaplan-Meier method was used to compare the shedding proportion of nucleic acids. Results: Cox analysis showed that the vaccine booster (p = 0.006), YDJDG treatment (p = 0.020), and Paxlovid treatment (p < 0.0001) were independent predictors of nucleic acid shedding at 14 days. The median recovery time was 11.49 days in the YDJDG group, 10.21 days in the Paxlovid group, and 13.93 days in the control group. After PSM (3:1), the results showed that the nucleic acid shedding time of the YDJDG group (n = 53) was 2.47 days shorter than that of the control group (n = 21) (p = 0.0076), while the Paxlovid group (n = 44) had a 4.34 days shorter than that of the control group (n = 17) (p < 0.0001). After PSM (1:1), YDJDG and Paxlovid (76 pairs) were also analyzed. In the YDJDG group, nucleic acid shedding time was 1.43 days longer than that observed in the Paxlovid group (p = 0.020). At 10 and 14 days, the Paxlovid group showed a significant difference in the nucleic acid shedding proportion compared with the control group (p = 0.036, p = 0.0015). A significant difference was also observed between the YDJDG and control groups (p = 0.040) at 14 days. Conclusion: As a safe and convenient oral drug, YDJDG can be used as an alternative to antiviral therapy for such patients.

11.
Journal of Multidisciplinary Healthcare ; 15:1909-1919, 2022.
Article in English | EMBASE | ID: covidwho-2009781

ABSTRACT

Background: An epidemic of the Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began in March 2022, and over 600,000 cases were confirmed until early May 2022 in Shanghai, China. Data on Omicron infections are available in other countries, but the clinical features of patients in the Chinese population, especially in Shanghai, are still lacking. We collected data from a subset of asymptomatic and mildly ill patients to learn about the age and sex disparity of Omicron infection based on changes in cycle threshold values. Methods: The basic information of 325 patients who were consecutively admitted to the Shanghai Geriatrics Center was collected through medical records, and patients were tested for viral nucleic acid carriage using nasal swab samples during hospitalization. SAS 9.4 was used for data analysis, and a p value < 0.05% was considered statistically significant. Results: Among the 325 included patients, 58.8% were males, with a mean age of 47.2 years and 13.6 days of hospitalization on average. The average number of nucleic acid tests among female patients was 4.7, which was higher than that among male patients (4.1). The median value of the slope for cycle threshold (Ct) changes in the nucleic acid detection (NAD) test was 1.4. Logistic regression indicated that the proportion of slope for Ct changes >1.5 was slightly higher among male patients than among female patients (odds ratio (OR) = 1.06, 95% confidence interval (CI): 0.68–1.66), and patients aged <45 years and 45–59 years had a higher proportion of slope for Ct changes >1.5 than patients aged ≥60 years. Ct values were more variable in the early stages of infection and stabilized in the later stages of infection. Conclusion: Among patients with mild illness or asymptomatic infection, the Ct value is a good, timely, and cost-effective method to reflect the recovery progress of patients. The slope of Ct changes was steeper among younger patients and male patients, which indicates faster disease recovery.

12.
Annals of the Rheumatic Diseases ; 81:1689-1690, 2022.
Article in English | EMBASE | ID: covidwho-2009071

ABSTRACT

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load and its impact on disease outcome in patients with autoimmune rheumatic disease (ARD) are lacking. Also, whether patients with ARD receiving immunomodulators have different viral loads compared to the general population is unknown. Objectives: To compare the viral load of SARS-CoV-2 and its trending between patients without and with ARD. Methods: Retrospectively, patients with ARD infected with SARS-CoV-2 were matched by age and sex at a ratio of 1:2 to patients without ARD and not receiving immunosuppression or immunomodulator drugs. Viral load was determined by the cycle threshold (CT) value measured by a number of platforms: (a) Automated Platforms-the Roche Cobas 6800 system using the Cobas SARS-CoV-2 Test targeting the E and orf1a/b genes (Roche, Switzerland) and the Xpert Xpress SARS-CoV-2 targeting the E and N genes (Cepheid, USA);(b) Manual platforms-EZ1 (QIAGEN, USA), QIAsymphony (QIAGEN, USA), and Bioneer ExiPrepTM 96 Virus DNA/RNA kits Catalogue No K4614 (Bioneer, South Korea) extraction with thermal cycling using TaqPath™ PCR COVID-19 Combo Kit targeting the N, S and orf1a/b genes (Thermo Fisher Scientific, USA) on ABI 7500 thermal cyclers. Independent samples t-test was used to compare the mean CT values of the study groups at baseline and at 5 subsequent intervals (1-5.9, 6-11.9, 12-17.9, 18-23.9 and 24-30 days). Results: Mean age (SD) of 197 cases and 420 controls were 45.2 (11.8) and 44.1 (12.3) years, respectively. Females were predominant in both groups 60% vs. 52%, P=0.053. The most common ARD was rheumatoid arthritis in 82 cases (41.6%), followed by spondyloarthropathy in 33 (16.8%) and systemic lupus ery-thematosus in 31 (15.7%). Of the cases, 67% were on conventional synthetic disease modifying anti-rheumatic drugs (DMARDs), 15.2% on biological DMARDs and 4.6% patients were on rituximab. The mean CT values was signifcantly lower in the ARD group at baseline and persisted till day 24. Conclusion: Compared to patients without ARD, the viral load of SARS-CoV-2 in patients with ARD is signifcantly higher at baseline testing and persists till day 24. This fnding may indicate that patients with ARD are at higher risk of severe SARS-CoV-2 infection and prolonged potential transmission. Clinical outcome correlation is needed.

13.
Annals of the Rheumatic Diseases ; 81:961, 2022.
Article in English | EMBASE | ID: covidwho-2009057

ABSTRACT

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load and its impact on disease outcome in patients with autoimmune rheumatic disease (ARD) are lacking. Also, whether patients with ARD receiving immunomodulators have different viral loads compared to the general population is unknown. Objectives: To compare the viral load of SARS-CoV-2 and its trending between patients without and with ARD. Methods: Retrospectively, patients with ARD infected with SARS-CoV-2 were matched by age and sex at a ratio of 1:2 to patients without ARD and not receiving immunosuppression or immunomodulator drugs. Viral load was determined by the cycle threshold (CT) value measured by a number of platforms: (a) Automated Platforms-the Roche Cobas 6800 system using the Cobas SARS-CoV-2 Test targeting the E and orf1a/b genes (Roche, Switzerland) and the Xpert Xpress SARS-CoV-2 targeting the E and N genes (Cepheid, USA);(b) Manual platforms-EZ1 (QIAGEN, USA), QIAsymphony (QIAGEN, USA), and Bioneer ExiPrepTM 96 Virus DNA/RNA kits Catalogue No K4614 (Bioneer, South Korea) extraction with thermal cycling using TaqPath™ PCR COVID-19 Combo Kit targeting the N, S and orf1a/b genes (Thermo Fisher Scientific, USA) on ABI 7500 thermal cyclers. Independent samples t-test was used to compare the mean CT values of the study groups at baseline and at 5 subsequent intervals (1-5.9, 6-11.9, 12-17.9, 18-23.9 and 24-30 days). Results: Mean age (SD) of 197 cases and 420 controls were 45.2 (11.8) and 44.1 (12.3) years, respectively. Females were predominant in both groups 60% vs. 52%, P=0.053. The most common ARD was rheumatoid arthritis in 82 cases (41.6%), followed by spondyloarthropathy in 33 (16.8%) and systemic lupus ery-thematosus in 31 (15.7%). Of the cases, 67% were on conventional synthetic disease modifying anti-rheumatic drugs (DMARDs), 15.2% on biological DMARDs and 4.6% patients were on rituximab. The mean CT values was signifcantly lower in the ARD group at baseline and persisted till day 24. Conclusion: Compared to patients without ARD, the viral load of SARS-CoV-2 in patients with ARD is signifcantly higher at baseline testing and persists till day 24. This fnding may indicate that patients with ARD are at higher risk of severe SARS-CoV-2 infection and prolonged potential transmission. Clinical outcome correlation is needed.

14.
Annals of the Rheumatic Diseases ; 81:374, 2022.
Article in English | EMBASE | ID: covidwho-2008943

ABSTRACT

Background: The relevance of studying immune response after SARS-CoV-2 vaccination in patients with infammatory immune-mediated diseases (IMIDs) represents a deep concern regarding the risk estimation and management of patients with these diseases on immunomodulatory drugs. It is well known that certain treatments as anti CD20 therapies results in a diminished immunogenicity against common vaccines but it is a scarce data regarding the cellular protection obtained upon vaccination between patients with different IMID and between different treatments. Objectives: To compare a potential detriment on cellular and antibody-mediated protection upon SARS-CoV-2 vaccination in patients with IMIDs treated with immunosuppressive drugs. Methods: We recruited 73 patients with rheumatoid arthritis-RA-(n=49), spondy-larthritis-SpA-(n=19), infammatory bowel disease-IBD-(n=5), idiopathic juvenile arthritis-IJA-(n=2) and heterogenous group composed of sclerodermia, lupus, uveitis(n=6). They were treated mainly with rituximab (n=27), TNFi (n=37) or JAKi (n=3). We collected data of age,sex, csDMARDs, previous SARS-CoV-2 infection, last RTX infusion and prednisone use. After one month of vaccination, we assessed the humoral response performing the Thermo Scientific EliA SARS-CoV-2-Sp1 IgG Test (positivity cut-off >0.70 IU/ml) which was also compared with the data with of 35 healthy controls. In addition, in 40 patients who had serum antibody levels under 100UI/ml, we analysed the cellular response by the use of the QuantiFERON SARS-CoV-2 Starter Pack (Quiagen). A cut-off value of 0.15 IU/ml discriminate between positive or negative cell-mediated immune responses. We compared differences among the different IMIDs and between the different immu-nosuppressive treatments through non-parametric test (p<0.05) Results: Regarding demographic characteristics of patients, older patients (>56 years) and female sex were factors which were associated with low titles of serum antibodies. Anti-spike IgG antibodies were present in an 86% of the IMIDs patients and in 100% healthy controls with signifcant different IgG titre (median [IQR]): 51[11-184] vs 700[440-940];p<0.0001. The differences between (median [IQR]) serum antibody levels were statistically different between IMID type: 33[1-138] in RA vs 94[34-191] in SpA vs 204[187-204] in IBD vs 133[61-204] in IJA vs 13[1.5-31.8] in the rest;p=0.04. Remarkably, patients with IBD who had the highest antibodies titles were the youngest compared with the other patients. Target of the therapy played also an important role in serum antibody levels being these: 3.6 [0.7-51] in RTX patients vs 156 [45-204] in TNFi vs 40 [18-58] in JAKi patients;p<0.0001. In those patients who the last infusion of rituximab was, at least, one year before vaccination presented CD19+ B cells detected by fow cytometry and anti-spike IgG antibodies as well. Cell-mediated responses to SARS-CoV-2 were positive in 33% of IMIDs patients, indeterminated in 3% and negative in 65% of the patients. Strikingly, out of the 33% positive patients, 85% were treated with RTX. A 61% of the RTX patients had inducible cell-mediated responses vs 14% of the patients treated with TNFi;p<0.01. On the other hand, there were not differences in cell-mediated responses between positive and negative antibody patients. Conclusion: Titres of serum antibodies against spike protein of SARS-CoV-2 were lower in IMIDs patients than in controls. Patients with RTX had lower rates of positivity humoral response as well as lower serum titles than patients treated with other therapies regardless the patients 'age. Neverthless, in those patients in whom RTX infusion was delayed because of vaccination they conserved a humoral response. On the other hand, more patients treated with RTX had inducible cell-mediated responses compared with patients with TNFi.

15.
Annals of the Rheumatic Diseases ; 81:7-8, 2022.
Article in English | EMBASE | ID: covidwho-2008870

ABSTRACT

Background: An interferon gene signature (IGS) is present in approximately 50% of early, treatment naive rheumatoid arthritis (eRA) patients. We previously demonstrated it negatively impacts on initial disease outcomes. Objectives: To 1) reproduce previous fndings demonstrating the harmful effects of the IGS on early RA clinical outcomes, 2) identify which IFN class is responsible for the IGS and 3) seek evidence that IFN-a exposure contributes to harmful epigenetic footprint at disease onset. Methods: In a large multicentre inception cohort (n=190) of eRA patients (RA-MAP TACERA) whole blood transcriptome, IGS (MxA, IFI44L, OAS1, ISG15, IFI6) and circulating interferons (IFN)-a,-β,-y and-), was examined at baseline and 6 months in conjunction with disease activity and clinical characteristics. A separate eRA cohort of paired methylome and transcriptome from CD4 T and CD19 B cells (n=41 for each) was used to explore any epigenetic influence of the IGS. Results: The baseline IGS reproducibly and signifcantly negatively impacts on 6-month clinical outcomes. In the high IGS cohort there was increased DAS-28 (p=0.025) and reduced probability of achieving a good EULAR response (p=0.034) at 6-months. In addition, the IGS in eRA is shown for the frst time to predominantly refect raised circulating IFN-a protein, not other classes of IFN and examination of whole blood upstream nucleic acid sensors expression suggest a RNA trigger. Both the IGS and IFN-a signifcantly fell in parallel at 6 months (p<0.0001), whereas other classes of IFN remained statistically static. There was a signifcant association with IFN-a and RF titre but not ACPA. Comparison of CD4 T and CD19 B cells between IGS high and low eRA patients demonstrated differentially methylated CPG sites and altered transcript expression of disease relevant genes e.g. PARP9, STAT1, EPTSI1 which was similarly, and persistently altered 6 months in the separate TACERA cohort. Differentially methylated CPGs implicated altered transcription factor binding in B cells (GATA3, ETSI, NFATC2, EZH2) and T cells (p300, HIF1a) which cumulatively suggested IFN-a induced epigenetic changes promoting increased, and sustained, lymphocyte activation, proliferation and loss of anergy in the IGS high cohort. Conclusion: We validate that the IGS is a robust prognostic biomarker in eRA predicting poor therapeutic response. Its persistent harmful effects may be driven via epigenetic modifcations. These data have relevance for other IFN-a states, such as COVID-19, but also provide a rationale for the initial therapeutic targeting of IFN-a signalling, such as with JAKi, at disease onset in stratifed eRA subsets.

16.
Pathology International ; 2022.
Article in English | EMBASE | ID: covidwho-2008755

ABSTRACT

A 61-year-old woman without significant medical history developed fever 3 days after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination and went into shock the next day. She was negative for SARS-CoV-2 mRNA in real-time polymerase chain reaction (PCR). Finally, she died 10 days after vaccination. At autopsy, the heart showed moderate dilatation of both ventricles, and the myocardium showed an uneven color change and decreased elasticity. Histologically, severe myocarditis with extensive myocytolysis was observed. The myocarditis showed severe inflammatory cell infiltration with T-lymphocyte and macrophage predominance, and in addition to the inflammatory cells described above, vast nuclear dust accompanying neutrophilic infiltration was observed. In the bone marrow and lymph nodes, hemophagocytosis was observed. In postmortem examination, nucleic acids of any cardiotropic viruses including SARS-CoV-2 were not detected using multivirus real-time PCR system. We discussed the relationship between the possible immune reaction after vaccination and the myocarditis observed in this case from immunopathological viewpoints. This mRNA vaccine is the first applied nucleic acid vaccine for humans, and its mechanism of efficacy and immune acquisition remain unclear. We hope the accumulation of more detailed analyses of the similar cases to reveal the mechanism of this kind of adverse reaction.

17.
Journal of the American Chemical Society ; 2022.
Article in English | MEDLINE | ID: covidwho-2008246

ABSTRACT

We introduce a new method to generate an amplified signal in CRISPR-Cas-based detection. Target recognition activates a CRISPR-Cas complex, leading to catalytic cleavage of horseradish peroxidase (HRP)-labeled oligonucleotides from the surface of microbeads. We show that the HRP released into solution can be monitored through colorimetric, fluorometric, or luminescent approaches, yielding up to ∼75-fold turn-on signal and limits of detection (LODs) as low as ∼10 fM. Compared to Cas-based detection with a conventional fluorophore/quencher reporter, this strategy improves the LOD by ∼30-fold. As a proof-of-concept, we show the rapid (<1 h), PCR-free, and room temperature (25 °C) detection of a nucleic acid marker for the SARS-CoV-2 virus with the naked eye at clinically relevant concentrations. We further show that the probe set can be programmed to be recognized and activated in the presence of non-nucleic acid targets. Specifically, we show adenosine triphosphate (ATP) binding to an aptamer can activate CRISPR-Cas and trigger a colorimetric readout, enabling the analysis of ATP in human serum samples with sensitivity on par with that of several commercially available kits. Taken together, the strategy reported herein offers a simple and sensitive platform to detect analytes where target amplification is either inconvenient (e.g., PCR under point-of-care settings) or impossible.

18.
International Journal of Biological Macromolecules ; 219:1208-1215, 2022.
Article in English | EMBASE | ID: covidwho-2007740

ABSTRACT

The recent outbreak of one of the RNA viruses (2019-nCoV) has affected most of the population and the fatalities reported may label it as a modern-day scourge. Active research on RNA virus infections and vaccine development had more commercial impact which leads to an increase in patent filings. Patents are a goldmine of information whose mining yields crucial technological inputs for further research. In this research article, we have investigated both the patent applications and granted patents, to identify the technological trends and their impact on 2019-nCoV infection using biotechnology-related keywords such as genes, proteins, nucleic acid etc. related to the RNA virus infection. In our research, patent analysis was majorly focused on prospecting for patent data related to the RNA virus infections. Our patent analysis specifically identified spike protein (S protein) and nucleocapsid proteins (N proteins) as the most actively researched macromolecules for vaccine and/or drug development for diagnosis and treatment of RNA virus based infectious diseases. The outcomes of this patent intelligence study will be useful for the researchers who are actively working in the area of vaccine or drug development for RNA virus-based infections including 2019-nCoV and other emerging and re-emerging viral infections in the near future.

19.
Computational Biology and Chemistry ; : 107768, 2022.
Article in English | ScienceDirect | ID: covidwho-2007623

ABSTRACT

Nucleoside analogs/derivatives (NAs/NDs) with potent antiviral activities are now deemed very convenient choices for the treatment of coronavirus disease 2019 (COVID-19) arisen by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. At the same time, the appearance of a new strain of SARS-CoV-2, the Omicron variant, necessitates multiplied efforts in fighting COVID-19. Counteracting the crucial SARS-CoV-2 enzymes RNA-dependent RNA polymerase (RdRp) and 3′-to-5′ exoribonuclease (ExoN) jointly altogether using the same inhibitor is a quite successful new plan to demultiplicate SARS-CoV-2 particles and eliminate COVID-19 whatever the SARS-CoV-2 subtype is (due to the significant conservation nature of RdRps and ExoNs in the different SARS-CoV-2 strains). Successive in silico screening of know NAs finally disclosed six different promising NAs, which are riboprine/forodesine/tecadenoson/nelarabine/vidarabine/maribavir, respectively, that predictably can act through the planned dual-action mode. Further in vitro evaluations affirmed the anti-SARS-CoV-2/anti-COVID-19 potentials of these NAs, with riboprine and forodesine being at the top. The two NAs are able to effectively antagonize the replication of the new virulent SARS-CoV-2 strains with considerably minute in vitro anti-RdRp and anti-SARS-CoV-2 EC50 values of 189 and 408nM for riboprine and 207 and 657nM for forodesine, respectively, surpassing both remdesivir and the new anti-COVID-19 drug molnupiravir. Furthermore, the favorable structural characteristics of the two molecules qualify them for varied types of isosteric and analogistic chemical derivatization. In one word, the present important outcomes of this comprehensive dual study revealed the anticipating repurposing potentials of some known nucleosides, led by the two NAs riboprine and forodesine, to successfully discontinue the coronaviral-2 polymerase/exoribonuclease interactions with RNA nucleotides in the SARS-CoV-2 Omicron variant (BA.5 sublineage) and accordingly alleviate COVID-19 infections, motivating us to initiate the two drugs' diverse anti-COVID-19 pharmacological evaluations to add both of them betimes in the COVID-19 therapeutic protocols.

20.
Biosafety and Health ; 2022.
Article in English | ScienceDirect | ID: covidwho-2007566

ABSTRACT

The pandemic caused by SARS-CoV-2 has led to unprecedented social and economic disruption. Many Nucleic acid testing (NAT) laboratories in China have been established to control the epidemic better. This proficiency testing (PT) aims to evaluate the participants’ performance in qualitative and quantitative SARS-CoV-2 NAT and to explore the factors that contribute to differences in detection capabilities. Two different concentrations of RNA samples (A, B) were used for quantitative PT. Pseudovirus samples D, E (different concentration) and negative sample (F) were used for qualitative PT. 50 data sets were reported for qualitative PT, of which 74.00% were entirely correct for all samples. Forty-two laboratories participated in the quantitative PT. 37 submitted all gene results, of which only 56.76% were satisfactory. For qualitative detection, it is suggested that laboratories should strengthen personnel training, select qualified detection kits, and reduce cross-contamination to improve detection accuracy. For quantitative detection, the results of the reverse transcription digital PCR (RT-dPCR) method were more comparable and reliable than those of reverse transcription quantitative PCR (RT-qPCR). The copy number concentration of ORF1ab and N in samples A and B scattered in 85, 223, 50, and 106 folds, respectively. The differences in the quantitative result of RT-qPCR was mainly caused by the non-standard use of reference materials and the lack of personnel operating skills. Comparing the satisfaction of participants participating in both quantitative and qualitative proficiency testing, 95.65% of the laboratories with satisfactory quantitative results also judged the qualitative results correctly, while 85.71% of the laboratories with unsatisfactory quantitative results were also unsatisfied with their qualitative judgments. Therefore, the quantitative ability is the basis of qualitative judgment. Overall, participants from hospitals reported more satisfactory results than those from enterprises and universities. Therefore, surveillance, daily qualitiy control and standardized operating procedures should be strengthened to improve the capability of SARS-CoV-2 NAT.

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