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1.
Front Public Health ; 10: 864197, 2022.
Article in English | MEDLINE | ID: covidwho-1877515

ABSTRACT

Objective: To explore the current knowledge and application of vital sign zero and the identify-isolate-inform (3I) system among healthcare workers in China in order to provide a reference for future improvement of healthcare workers' awareness of personal protection and prevention and control measures of infectious diseases. Methods: The questionnaire was used to investigate the basic information of health care workers, their knowledge and application of Vital sign zero and the 3I system. A total of 602 forms of health care workers from tertiary hospitals were randomly collected and included for analysis. Results: The survey showed that 45.30% and 57.30% of the healthcare workers from Chinese tertiary hospitals know about vital sign zero and 3I system while 51.80% and 57.30% of them applied these measures in their clinical practices. Logistics regression analysis results showed that healthcare workers aged 35 years old and below were less aware of vital sign zero than those above 50 years old (OR = 0.405, 95% CI: 0.174-0.942, P = 0.036). Compared with those in Northwest China, healthcare workers who worked in East China (OR = 0.147, 95% CI: 0.031-0.702, P = 0.016), Central China (OR = 0.085, 95% CI: 0.018-0.403, P = 0.002), Southwest China (OR = 0.083, 95% CI: 0.014-0.48, P = 0.006) and North China (OR = 0.201, 95% CI: 0.042-0.966, P = 0.045) were less aware of vital sign zero while the healthcare workers in Northeast China (OR=9.714, 95% CI: 1.091-86.521, P = 0.042), East China (OR = 18.049, 95% CI: 2.258-144.259, P = 0.006), Central China (OR = 25.560, 95% CI: 3.210-203.502, P = 0.002), South China (OR = 11.141, 95% CI: 1.395-88.947, P = 0.023), Southwest China (OR = 23.200, 95% CI: 2.524-213.286, P = 0.005) and North China (OR = 14.078, 95% CI: 1.756-112.895, P = 0.013) had a better understanding of the 3I system than those in Northwest China. Healthcare workers with more than 20 years of working experience showed less knowledge of the 3I system than those with less than 5 years of working experience (OR = 0.409, 95% CI: 0.215-0.77, P = 0.006). Conclusion: The current levels of knowledge and application of vital sign zero and the 3I system in the healthcare workers of Chinese tertiary hospitals need to be improved. The concept of vital sign zero should be incorporated into the prevention triage system of infectious diseases.


Subject(s)
Communicable Diseases , Health Personnel , Adult , Health Knowledge, Attitudes, Practice , Humans , Middle Aged , Tertiary Care Centers , Vital Signs
2.
ACS Synth Biol ; 11(5): 1811-1823, 2022 May 20.
Article in English | MEDLINE | ID: covidwho-1815478

ABSTRACT

Objectives: Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a great threat and burden to global public health. Here, we evaluated a clustered regularly interspaced short palindromic repeat-associated enzyme 12a (CRISPR-Cas12a)-based method for detecting major SARS-CoV-2 variants of concern (VOCs) in SARS-CoV-2 positive clinical samples. Methods: Allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of SARS-CoV-2 are designed. A total of 59 SARS-CoV-2 positive oropharyngeal swab specimens were used to evaluate the performance of the CRISPR-Cas12a-mediated assay to identify major SARS-CoV-2 VOCs. Results: Compared with Sanger sequencing, the eight allele-specific crRNAs analyzed can specifically identify the corresponding mutations with a positive predictive value of 83.3-100% and a negative predictive value of 85.7-100%. Our CRISPR-Cas12a-mediated assay distinguished wild-type and four major VOCs (Alpha, Beta, Delta, and Omicron) of SARS-CoV-2 with a sensitivity of 93.8-100.0% and a specificity of 100.0%. The two methods showed a concordance of 98.3% (58/59) with a κ value of 0.956-1.000, while seven (11.9%) samples were found to be positive for extra mutations by the CRISPR-based assay. Furthermore, neither virus titers nor the sequences adjacent to the signature mutations were associated with the variation of fluorescence intensity detected or the false-positive reaction observed when testing clinical samples. In addition, there was no cross-reaction observed when detecting 33 SARS-CoV-2 negative clinical samples infected with common respiratory pathogens. Conclusions: The CRISPR-Cas12a-based genotyping assay is highly sensitive and specific when detecting both the SARS-CoV-2 wild-type strain and major VOCs. It is a simple and rapid assay that can monitor and track the circulating SARS-CoV-2 variants and the dynamics of the coronavirus disease 2019 (COVID-19) pandemic and can be easily implemented in resource-limited settings.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Humans , Mutation , SARS-CoV-2/genetics
3.
Biosens Bioelectron ; 205: 114098, 2022 Jun 01.
Article in English | MEDLINE | ID: covidwho-1693895

ABSTRACT

BACKGROUND: The newly emerged SARS-CoV-2 variant of concern (VOC) Omicron is spreading quickly worldwide, which manifests an urgent need of simple and rapid assay to detect and diagnose Omicron infection and track its spread. METHODS: To design allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of Omicron variant, and to develop a CRISPR-Cas12a-based assay to specifically detect Omicron variant. RESULTS: Our system showed a low limit of detection of 2 copies per reaction for the plasmid DNA of Omicron variant, and could readily detect Omicron variant in 5 laboratory-confirmed clinical samples and distinguish them from 57 SARS-CoV-2 positive clinical samples (4 virus isolates and 53 oropharyngeal swab specimens) infected with wild-type (N = 8) and the variants of Alpha (N = 17), Beta (N = 17) and Delta (N = 15). The testing results could be measured by fluorescent detector or judged by naked eyes. In addition, no cross-reaction was observed when detecting 16 clinical samples infected with 9 common respiratory pathogens. CONCLUSIONS: The rapid assay could be easily set up in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests and implemented routinely in resource-limited settings to monitor and track the spread of Omicron variant.


Subject(s)
Biosensing Techniques , COVID-19 , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Humans , SARS-CoV-2/genetics
4.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-313731

ABSTRACT

Background: Real-world evidence (RWE) of a vaccine supplements clinical trial data by providing information in populations differing from clinical trial populations, under different epidemiological situations, on alternative outcomes, or against different pathogen lineages. To date, RWE on inactivated COVID-19 vaccines against the highly transmissible SARS-CoV-2 B.1.617.2 (Delta) variant is limited, leaving an important gap in the evidence base of inactivated COVID-19 vaccines for use by immunization programs. Methods: Between May and June 2021, an outbreak of the B.1.617.2 variant was discovered and traced in Guangdong, China. Before this outbreak, Guangdong province had started mass vaccination using inactivated vaccines approved by China’s regulator for use in adults. Using surveillance and vaccination data from the outbreak, we assessed the real-world effectiveness of inactivated vaccines against pneumonia and severe illness caused by the B.1.617.2 variant. We enrolled 10813 subjects who were close contacts of laboratory-confirmed cases, categorizing them as an unvaccinated group, a partially vaccinated (1-dose) group, and a fully vaccinated (2-dose) group. We estimated relative risk (RR) and vaccine effectiveness (VE) of the vaccinated groups in relation to the unvaccinated group. Findings: Unadjusted and adjusted VE of full vaccination against pneumonia were 77·7% (95% CI 45·1–90·9) and 69·5% (95% CI 42·8–96·3), respectively. Full vaccination was 100% effective against severe illness. Unadjusted and adjusted VE of partial vaccination against pneumonia were 1·4% (95% CI -79·7–45·9) and 8·4% (95% CI -47·6–64·4). Interpretation Full vaccination with inactivated vaccines is effective against pneumonia, severe, and critical illness caused by the B.1.617.2 variant. Effort should be placed to ensure full vaccination of target populations. Funding: National Natural Science Foundation of China and Key-Area Research and Development Program of Guangdong.Declaration of Interest: None to declare. Ethical Approval: This study was approved by the institutional ethics committee of the Guangdong Provincial Center for Disease Control and Prevention (GDCDC). Data in the study were collected per administrative requirements of disease control and surveillance, and were anonymized for analysis. Participants were informed about the requirements of disease surveillance and provided oral consents.

5.
Ann Intern Med ; 175(4): 533-540, 2022 04.
Article in English | MEDLINE | ID: covidwho-1677759

ABSTRACT

BACKGROUND: Real-world evidence on inactivated COVID-19 vaccines against the highly transmissible B.1.617.2 (Delta) variant of SARS-CoV-2 is limited, leaving an important gap in the evidence base about inactivated COVID-19 vaccines for use by immunization programs. OBJECTIVE: To estimate inactivated vaccine effectiveness (VE) against the B.1.617.2 variant. DESIGN: Retrospective cohort study. SETTING: The study was based on the first outbreak of the B.1.617.2 variant in mainland China that was discovered and traced in Guangdong in May and June 2021. PARTICIPANTS: 10 805 adult case patients with laboratory-confirmed infection and close contacts. MEASUREMENTS: Participants were categorized as unvaccinated, partially vaccinated (1 dose), and fully vaccinated (2 doses). We estimated VE against the primary outcome of pneumonia and the secondary outcomes of infections, symptomatic infections, and severe or critical illness associated with the B.1.617.2 variant. RESULTS: Results are reported in the order of outcome severity. Of 10 805 participants, 1.3% contracted infections, 1.2% developed symptomatic infections, 1.1% had pneumonia, and 0.2% had severe or critical illness. The adjusted VEs of full vaccination were 51.8% (95% CI, 20.3% to 83.2%) against infection, 60.4% (CI, 31.8% to 88.9%) against symptomatic infection, and 78.4% (CI, 56.9% to 99.9%) against pneumonia. Also, full vaccination was 100% (CI, 98.4% to 100.0%) effective against severe or critical illness. By contrast, the adjusted VEs of partial vaccination against infection, symptomatic infection, and pneumonia were 10.7% (CI, -41.2% to 62.6%), 6.8% (CI, -47.4% to 61.0%), and 11.6% (CI, -42.6% to 65.8%), respectively. LIMITATION: Observational study with possible unmeasured confounders; insufficient data to do reliable subgroup analyses by age and vaccine brand. CONCLUSION: Full vaccination with inactivated vaccines is effective against the B.1.617.2 variant. Effort should be made to ensure full vaccination of target populations. PRIMARY FUNDING SOURCE: National Natural Science Foundation of China and Key-Area Research and Development Program of Guangdong Province.


Subject(s)
COVID-19 Vaccines , COVID-19 , Adult , COVID-19/epidemiology , COVID-19/prevention & control , Cohort Studies , Critical Illness , Humans , Retrospective Studies , SARS-CoV-2/genetics , Vaccines, Inactivated
6.
Front Microbiol ; 12: 789062, 2021.
Article in English | MEDLINE | ID: covidwho-1581272

ABSTRACT

Mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have made this virus more infectious. Previous studies have confirmed that non-structural protein 13 (NSP13) plays an important role in immune evasion by physically interacting with TANK binding kinase 1 (TBK1) to inhibit IFNß production. Mutations have been reported in NSP13; hence, in the current study, biophysical and structural modeling methodologies were adapted to dissect the influence of major mutations in NSP13, i.e., P77L, Q88H, D260Y, E341D, and M429I, on its binding to the TBK1 and to escape the human immune system. The results revealed that these mutations significantly affected the binding of NSP13 and TBK1 by altering the hydrogen bonding network and dynamic structural features. The stability, flexibility, and compactness of these mutants displayed different dynamic features, which are the basis for immune evasion. Moreover, the binding was further validated using the MM/GBSA approach, revealing that these mutations have higher binding energies than the wild-type (WT) NSP13 protein. These findings thus justify the basis of stronger interactions and evasion for these NSP13 mutants. In conclusion, the current findings explored the key features of the NSP13 WT and its mutant complexes, which can be used to design structure-based inhibitors against the SARS-CoV-2 new variants to rescue the host immune system.

7.
Microbiol Spectr ; 9(3): e0101721, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1522923

ABSTRACT

A big challenge for the control of COVID-19 pandemic is the emergence of variants of concern (VOCs) or variants of interest (VOIs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be more transmissible and/or more virulent and could escape immunity obtained through infection or vaccination. A simple and rapid test for SARS-CoV-2 variants is an unmet need and is of great public health importance. In this study, we designed and analytically validated a CRISPR-Cas12a system for direct detection of SARS-CoV-2 VOCs. We further evaluated the combination of ordinary reverse transcription-PCR (RT-PCR) and CRISPR-Cas12a to improve the detection sensitivity and developed a universal system by introducing a protospacer adjacent motif (PAM) near the target mutation sites through PCR primer design to detect mutations without PAM. Our results indicated that the CRISPR-Cas12a assay could readily detect the signature spike protein mutations (K417N/T, L452R/Q, T478K, E484K/Q, N501Y, and D614G) to distinguish alpha, beta, gamma, delta, kappa, lambda, and epsilon variants of SARS-CoV-2. In addition, the open reading frame 8 (ORF8) mutations (T/C substitution at nt28144 and the corresponding change of amino acid L/S) could differentiate L and S lineages of SARS-CoV-2. The low limit of detection could reach 10 copies/reaction. Our assay successfully distinguished 4 SARS-CoV-2 strains of wild type and alpha (B.1.1.7), beta (B.1.351), and delta (B.1.617.2) variants. By testing 32 SARS-CoV-2-positive clinical samples infected with the wild type (n = 5) and alpha (n = 11), beta (n = 8), and delta variants (n = 8), the concordance between our assay and sequencing was 100%. The CRISPR-based approach is rapid and robust and can be adapted for screening the emerging mutations and immediately implemented in laboratories already performing nucleic acid amplification tests or in resource-limited settings. IMPORTANCE We described CRISPR-Cas12-based multiplex allele-specific assay for rapid SARS-CoV-2 variant genotyping. The new system has the potential to be quickly developed, continuously updated, and easily implemented for screening of SARS-CoV-2 variants in resource-limited settings. This approach can be adapted for emerging mutations and implemented in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests using existing resources and extracted nucleic acid.


Subject(s)
COVID-19 Testing/methods , COVID-19/virology , CRISPR-Cas Systems , SARS-CoV-2/classification , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Alleles , COVID-19/diagnosis , Databases, Nucleic Acid , Humans , Mass Screening , Mutation , Polymerase Chain Reaction , Public Health , Spike Glycoprotein, Coronavirus/genetics
8.
Front Microbiol ; 12: 703145, 2021.
Article in English | MEDLINE | ID: covidwho-1337656

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been continuously mutating since its first emergence in early 2020. These alterations have led this virus to gain significant difference in infectivity, pathogenicity, and host immune evasion. We previously found that the open-reading frame 8 (ORF8) of SARS-CoV-2 can inhibit interferon production by decreasing the nuclear translocation of interferon regulatory factor 3 (IRF3). Since several mutations in ORF8 have been observed, therefore, in the present study, we adapted structural and biophysical analysis approaches to explore the impact of various mutations of ORF8, such as S24L, L84S, V62L, and W45L, the recently circulating mutant in Pakistan, on its ability to bind IRF3 and to evade the host immune system. We found that mutations in ORF8 could affect the binding efficiency with IRF3 based on molecular docking analysis, which was further supported by molecular dynamics simulations. Among all the reported mutations, W45L was found to bind most stringently to IRF3. Our analysis revealed that mutations in ORF8 may help the virus evade the immune system by changing its binding affinity with IRF3.

9.
J Virol Methods ; 292: 114141, 2021 06.
Article in English | MEDLINE | ID: covidwho-1142107

ABSTRACT

In this study, we developed and evaluated a luciferase immunosorbent assay (LISA) for quantitative detection of IgG antibody against SARS-CoV-2 nucleoprotein (NP). Anti-SARS-CoV-2 NP antibody in serum or plasma samples was captured by protein G-coated microtiter plate and detected using the crude cell lysates expressing Nanoluc luciferase (Nluc) enzyme fused with SARS-CoV-2 NP. After the addition of furimazine substrate, the levels of anti-SARS-CoV-2 NP IgG antibody were quantitatively measured as luciferase light units. As expected, SARS-CoV-2 NP showed cross-reactivity with the monoclonal antibodies against SARS-CoV NP, but not MERS-CoV NP-specific monoclonal antibodies or the monoclonal antibodies against SARS-CoV Spike protein. LISA for detecting murine monoclonal antibody against SARS-CoV NP showed a low limit of detection of 0.4 pg/µl and linear detection range from 0.4 pg/µl to 75 pg/µl. Furthermore, LISA had a sensitivity of 71 % when testing COVID-19 patients at the second week post onset and a specificity of 100 % when testing healthy blood donors.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin G/blood , Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Antibodies, Monoclonal/immunology , Cross Reactions , Humans , Luciferases
10.
Virus Res ; 296: 198350, 2021 04 15.
Article in English | MEDLINE | ID: covidwho-1091608

ABSTRACT

The open reading frame 8 (orf8) is an accessory protein of SARS-CoV-2. It has 121 amino acids with two genotypes, orf8L and orf8S. In this study, we overexpressed the orf8L and orf8S of SARS-CoV-2 as well as the orf8b of SARS-CoV to investigate their roles in the regulation of endoplasmic reticulum (ER) stress and the inhibition of interferon beta (IFNß) production. We found that the two genotypes of SARS-CoV-2 orf8 are capable of inducing ER stress without significant difference by triggering the activating transcription factor 6 (ATF6) and inositol-requiring enzymes 1 (IRE1) branches of the ER stress pathway. However, the third branch of ER stress pathway, i.e. the protein kinase-like ER kinase (PERK), was unaffected by the overexpression of SARS-CoV-2 orf8L or orf8S. Moreover, both orf8L and orf8S of SARS-CoV-2 are capable of down regulating the production of IFNß and interferon-stimulated genes (ISG), ISG15 and ISG56 induced by polyinosinic-polycytidylic acid (poly (I:C)). Moreover, we also found decreased nuclear translocation of Interferon regulatory factor 3 (IRF3), after overexpressing orf8L and orf8S induced by poly (I:C). Our data demonstrated that SARS-CoV-2 orf8 protein could induce ER stress by activating the ATF6 and IRE1 pathways, but not the PERK pathway, and functions as an interferon antagonist to inhibit the production of IFNß. However, these functions appeared not to be affected by the genotypes of SARS-CoV-2 orf8L and orf8S.


Subject(s)
Endoplasmic Reticulum Stress/physiology , Immune Evasion , Interferon-beta/antagonists & inhibitors , Viral Proteins/physiology , Activating Transcription Factor 6/physiology , Endoribonucleases/physiology , HEK293 Cells , Humans , Interferon-beta/biosynthesis , Sequence Alignment , Signal Transduction/physiology , Unfolded Protein Response , Viral Proteins/chemistry , X-Box Binding Protein 1/physiology , eIF-2 Kinase/physiology
11.
Virology ; 551: 26-35, 2020 12.
Article in English | MEDLINE | ID: covidwho-799506

ABSTRACT

BACKGROUND: SARS-CoV-2 is a novel coronavirus and the cause of COVID-19. More than 80% of COVID-19 patients exhibit mild or moderate symptoms. In this study, we investigated the dynamics of viral load and antibodies against SARS-CoV-2 in a longitudinal cohort of COVID-19 patients with severe and mild/moderate diseases. METHODS: Demographic and clinical information were obtained. Serial samples of blood, nasal and pharyngeal and anal swabs were collected at different time points post-onset. SARS-CoV-2 RNA and anti-SARS-CoV-2 antibodies were measured by qRT-PCR and immunoassays, respectively. RESULTS: Respiratory SARS-CoV-2 RNA was detectable in 58.0% (58/100) COVID-19 patients upon admission and lasted for a median of 13 days post-onset. In addition, 5.9% (1/17) and 20.2% (19/94) of the blood and anal swab specimens were positive for SARS-CoV-2 RNA, respectively. Anal viral RNA was more frequently detected in the patients who were positive for viral RNA in the respiratory samples upon admission. Specific anti-SARS-CoV-2 antibody developed within two weeks after onset, reached peak approximately 17 days post-onset and then maintained at relatively high level up to 50 days we analyzed in most patients. However, the levels of antibodies were variable among the patients. High titers of antibodies appeared to be associated with the severity of the disease. Furthermore, viral proteins from different sources showed significant difference of serological sensitivity especially during the first week post-onset. CONCLUSIONS: Our results indicate rapid clearance or self-elimination of viral RNA in about half of the COVID-19 patients upon admission. Viral RNA shedding of SARS-CoV-2 occurred in multiple tissues including the respiratory system, blood, and intestine. Variable levels of specific anti-SARS-CoV-2 antibody may be associated with disease severity. These findings have shed light on viral kinetics and antibody response in COVID-19 patients and provide scientific evidence for infection control and patient management.


Subject(s)
COVID-19/virology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Adult , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/blood , COVID-19/diagnosis , Female , Humans , Kinetics , Longitudinal Studies , Male , Middle Aged , Nasopharynx/virology , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Viral Load , Virus Shedding
12.
Sci Total Environ ; 728: 138890, 2020 Aug 01.
Article in English | MEDLINE | ID: covidwho-139423

ABSTRACT

A COVID-19 outbreak emerged in Wuhan, China at the end of 2019 and developed into a global pandemic during March 2020. The effects of temperature on the dynamics of the COVID-19 epidemic in China are unknown. Data on COVID-19 daily confirmed cases and daily mean temperatures were collected from 31 provincial-level regions in mainland China between Jan. 20 and Feb. 29, 2020. Locally weighted regression and smoothing scatterplot (LOESS), distributed lag nonlinear models (DLNMs), and random-effects meta-analysis were used to examine the relationship between daily confirmed cases rate of COVID-19 and temperature conditions. The daily number of new cases peaked on Feb. 12, and then decreased. The daily confirmed cases rate of COVID-19 had a biphasic relationship with temperature (with a peak at 10 °C), and the daily incidence of COVID-19 decreased at values below and above these values. The overall epidemic intensity of COVID-19 reduced slightly following days with higher temperatures with a relative risk (RR) was 0.96 (95% CI: 0.93, 0.99). A random-effect meta-analysis including 28 provinces in mainland China, we confirmed the statistically significant association between temperature and RR during the study period (Coefficient = -0.0100, 95% CI: -0.0125, -0.0074). The DLNMs in Hubei Province (outside of Wuhan) and Wuhan showed similar patterns of temperature. Additionally, a modified susceptible-exposed-infectious-recovered (M-SEIR) model, with adjustment for climatic factors, was used to provide a complete characterization of the impact of climate on the dynamics of the COVID-19 epidemic.


Subject(s)
Coronavirus Infections/epidemiology , Pneumonia, Viral/epidemiology , Temperature , Betacoronavirus , COVID-19 , China/epidemiology , Humans , Pandemics , SARS-CoV-2
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