Recent advances in RNA sample preparation techniques for the detection of SARS-CoV-2 in saliva and gargle.

Molecular detection of SARS-CoV-2 in gargle and saliva complements the standard analysis of nasopharyngeal swabs (NPS) specimens. Although gargle and saliva specimens can be readily obtained non-invasively, appropriate collection and processing of gargle and saliva specimens are critical to the accuracy and sensitivity of the overall analytical method. This review highlights challenges and recent advances in the treatment of gargle and saliva samples for subsequent analysis using reverse transcription polymerase chain reaction (RT-PCR) and isothermal amplification techniques. Important considerations include appropriate collection of gargle and saliva samples, on-site inactivation of viruses in the sample, preservation of viral RNA, extraction and concentration of viral RNA, removal of substances that inhibit nucleic acid amplification reactions, and the compatibility of sample treatment protocols with the subsequent nucleic acid amplification and detection techniques. The principles and approaches discussed in this review are applicable to molecular detection of other microbial pathogens.

CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern.

The continuing evolution of the SARS-CoV-2 virus has led to the emergence of many variants, including variants of concern (VOCs). CRISPR-Cas systems have been used to develop techniques for the detection of variants. These techniques have focused on the detection of variant-specific mutations in the spike protein gene of SARS-CoV-2. These sequences mostly carry single-nucleotide mutations and are difficult to differentiate using a single CRISPR-based assay. Here we discuss the specificity of the Cas9, Cas12, and Cas13 systems, important considerations of mutation sites, design of guide RNA, and recent progress in CRISPR-based assays for SARS-CoV-2 variants. Strategies for discriminating single-nucleotide mutations include optimizing the position of mismatches, modifying nucleotides in the guide RNA, and using two guide RNAs to recognize the specific mutation sequence and a conservative sequence. Further research is needed to confront challenges in the detection and differentiation of variants and sublineages of SARS-CoV-2 in clinical diagnostic and point-of-care applications.

On-Site Viral Inactivation and RNA Preservation of Gargle and Saliva Samples Combined with Direct Analysis of SARS-CoV-2 RNA on Magnetic Beads.

Samples of nasopharyngeal swabs (NPS) are commonly used for the detection of SARS-CoV-2 and diagnosis of COVID-19. As an alternative, self-collection of saliva and gargle samples minimizes transmission to healthcare workers and relieves the pressure of resources and healthcare personnel during the pandemic. This study aimed to develop an enhanced method enabling simultaneous viral inactivation and RNA preservation during on-site self-collection of saliva and gargle samples. Our method involves the addition of saliva or gargle samples to a newly formulated viral inactivation and RNA preservation (VIP) buffer, concentration of the viral RNA on magnetic beads, and detection of SARS-CoV-2 using reverse transcription quantitative polymerase chain reaction directly from the magnetic beads. This method has a limit of detection of 25 RNA copies per 200 µL of gargle or saliva sample and 9-111 times higher sensitivity than the viral RNA preparation kit recommended by the United States Centers for Disease Control and Prevention. The integrated method was successfully used to analyze more than 200 gargle and saliva samples, including the detection of SARS-CoV-2 in 123 gargle and saliva samples collected daily from two NPS-confirmed positive SARS-CoV-2 patients throughout the course of their infection and recovery. The VIP buffer is stable at room temperature for at least 6 months. SARS-CoV-2 RNA (65 copies/200 µL sample) is stable in the VIP buffer at room temperature for at least 3 weeks. The on-site inactivation of SARS-CoV-2 and preservation of the viral RNA enables self-collection of samples, reduces risks associated with SARS-CoV-2 transmission, and maintains the stability of the target analyte.

Immunogenicity, durability, and safety of an mRNA and three platform-based COVID-19 vaccines as a third dose following two doses of CoronaVac in China: A randomised, double-blinded, placebo-controlled, phase 2 trial.

Background: More effective vaccine candidates against variants of concern as a booster dose are needed in people primed with two-dose inactivated COVID-19 vaccines. Methods: This randomised, double-blinded, investigator-initiated phase 2 trial aims to evaluate immunogenicity, durability, and safety of an mRNA vaccine candidate (RQ3013) and three other platform vaccines (an adenovirus-vectored vaccine candidate [ChAdTS-S], a recombinant protein vaccine candidate [ZR202-CoV], and an inactivated vaccine [CoronaVac]) as a booster. 250 eligible volunteers, who had received a prime two-dose CoronaVac (3 to 5 weeks apart) vaccination 100-270 days before, were randomly assigned in a 1:1:1:1:1 ratio to receive a third dose of RQ3013 (30 µg mRNA per 0.15 mL), ChAdTS-S (5×1010 viral particles per 0.5 mL), ZR202-CoV (25 µg prefusion-stabilized Spike ectodomain trimer per 0.5 mL), CoronaVac (3 µg inactivated CN02 strain of SARS-CoV-2 per 0.5 mL) or placebo (0.5 mL of 0.9% sodium chloride solution) via intramuscular injection into the upper arm at a single clinical site in Kunming, China. Participants, investigators, and immunogenicity laboratory were masked to group assignment. The primary immunogenicity outcomes were geometric mean titres (GMTs) of neutralising antibodies against live SARS-CoV-2 (wild-type, delta and omicron) virus at day 0 (before vaccination), day 7, day 14 and day 28 after vaccination, as analysed in a modified intention-to-treat (mITT) population (all participants who completed their booster doses and had at least one post-dose immunogenicity data). Secondary outcomes include T cell responses against the wild-type and omicron SARS-CoV-2 Spike protein. The primary safety outcome was incidence of adverse events within 14 days after the booster vaccination. This trial is registered with ChiCTR.org.cn, ChiCTR2200057758. Findings: Between January 1, 2022, and February 28, 2022, 235 eligible participants were enrolled and vaccinated, and the primary analysis included 234 participants. At baseline, neutralising antibodies against wild-type virus, the delta, or omicron variants were low or undetectable in all groups. After the booster vaccination, GMTs of neutralising antibodies ranged from 75.4 (95% confidence interval [CI] 61.4-92.5) in CoronaVac to 950.1 (95% CI 785.4-1149.3) in RQ3013 against live wild-type SARS-CoV-2, and from 8.1 (95% CI: 6.1-10.7) in CoronaVac to 247.0 (95% CI 194.1-314.3) in RQ3013 against the omicron variant at day 14. Immunogenicities of all heterologous regimens were superior to that of homologous regimen in neutralisation against all tested SARS-CoV-2 strains, with RQ3013 showing the highest geometric mean ratios (GMRs) of 12.6, 14.7, and 31.3 against the wild-type, the delta variant and the omicron variant compared to CoronaVac at day 14 post-vaccination, respectively. Durability analysis at day 90 showed that >90% of participants in RQ3013 and ZR202-CoV were seropositive for the omicron variant while ZR202-CoV with adjuvants containing CpG showed a slightly better durability than RQ3013. T cell responses specific to the omicron variant were similar to that of the wild-type, with RQ3013 showing the highest boosting effect. Any solicited injection site or systemic adverse events reported within 14 days after vaccination were most commonly observed in RQ3013 (47/47, 100%), followed by ZR202-CoV (46/47, 97.9%) and ChAdTS-S (43/48, 89.6%), and then CoronaVac (37/46, 80.4%) and placebo (21/47, 44.7%). More than 90% of the adverse events were grade 1 (mild) or 2 (moderate) with a typical resolution time of 3 days. No grade 4 adverse events or serious adverse events were reported by study vaccines. Interpretation: Although all study vaccines boosted neutralising antibodies with no safety concerns, RQ3013 showed much stronger cross-neutralisation and cellular responses, adding more effective vaccine candidates against the omicron variant. Funding: Yunnan Provincial Science and Technology Department China (202102AA100051 and 202003AC100010), the Double First-class University funding to Yunnan University, National Natural Science Foundation of China (81960116, 82060368 and 82170711), Yunnan Natural Science Foundation (202001AT070085), High-level Health Technical Personnel Project of Yunnan Province (H-2018102) and Spring City Plan: The High-level Talent Promotion and Training Project of Kunming.

The impact of medical infrastructure on regional innovation: An empirical analysis of China's prefecture-level cities.

Because of public health emergencies, such as the COVID-19 pandemic, having an optimal medical infrastructure is an important way to maintain the normal operation of society and stimulate vitality in regional innovation. Based on the data on 260 cities at the prefecture level and above in China from 2001 to 2018, this paper investigates the characteristics and mechanisms of medical infrastructure on regional innovation. After a series of regressions, we robustly find that medical infrastructure has a significantly positive impact on regional innovation. In addition, based on the mediating effect model, the mechanism test shows that medical infrastructure can promote regional innovation through the channels of the natural population growth rate, educational level, and the environmental greening level. Finally, considering the urban heterogeneity, we find that the positive impact of medical infrastructure on regional innovation is reflected mainly in eastern and central cities, non-sub-provincial cities, and non-resource-based cities. These conclusions not only enrich the theoretical research on regional innovation from the perspective of medical infrastructure but also shed light on how to better promote regional innovation for China or even other countries.

Optimization of Logistics System with Fuzzy FMEA-AHP Methodology

The COVID-19 pandemic broke out and the global logistics industry suffered severe losses;therefore, the Fuzzy FMEA-AHP (Fuzzy Failure Mode and Effects Analysis-Analytic Hierarchy Process) method is proposed to analyze the failure reasons of the logistics system in the COVID-19 pandemic. In this article, we have made an optimization on the basis of the FMEA method: the fuzzy is integrated into the FMEA algorithm, referred to as F-RPWN (fuzzy risk priority-weighted number). Meanwhile, the AHP is used to determine the weights of risk indicators. In this article, we consider new logistics failures, such as the failure modes and failure reasons of the logistics system under the COVID-19 pandemic. There are 12 failures that have been determined, and relevant preventive and corrective measures have been recommended to cut off the path of failure propagation and reduce the impact of failures. In addition, the proposed method can help logistics firms, their supply chain partners, and customers with risk management issues during the COVID-19 pandemic.

Evaluating the influence of constant source profile presumption on PMF analysis of PM2.5 by comparing long- and short-term hourly observation-based modeling.

Hourly PM2.5 speciation data have been widely used as an input of positive matrix factorization (PMF) model to apportion PM2.5 components to specific source-related factors. However, the influence of constant source profile presumption during the observation period is less investigated. In the current work, hourly concentrations of PM2.5 water-soluble inorganic ions, bulk organic and elemental carbon, and elements were obtained at an urban site in Nanjing, China from 2017 to 2020. PMF analysis based on observation data during specific pollution (firework combustion, sandstorm, and winter haze) and emission-reduction (COVID-19 pandemic) periods was compared with that using the whole 4-year data set (PMFwhole). Due to the lack of data variability, event-based PMF solutions did not separate secondary sulfate and nitrate. But they showed better performance in simulating average concentrations and temporal variations of input species, particularly for primary source markers, than the PMFwhole solution. After removing event data, PMF modeling was conducted for individual months (PMFmonth) and the 4-year period (PMF4-year), respectively. PMFmonth solutions reflected varied source profiles and contributions and reproduced monthly variations of input species better than the PMF4-year solution, but failed to capture seasonal patterns of secondary salts. Additionally, four winter pollution days were selected for hour-by-hour PMF simulations, and three sample sizes (500, 1000, and 2000) were tested using a moving window method. The results showed that using short-term observation data performed better in reflecting immediate changes in primary sources, which will benefit future air quality control when primary PM emissions begin to increase.

Lockdown-induced Urban Aerosol Change over Changchun, China During COVID-19 Outbreak with Polarization LiDAR.

Depending on various government policies, COVID-19 (Corona Virus Disease-19) lockdowns have had diverse impacts on global aerosol concentrations. In 2022, Changchun, a provincial capital city in Northeast China, suffered a severe COVID-19 outbreak and implemented a very strict lockdown that lasted for nearly two months. Using ground-based polarization Light Detection and Ranging (LiDAR), we detected real-time aerosol profile parameters (EC, extinction coefficient; DR, depolarization ratio; AOD, aerosol optical depth), as well as air-quality and meteorological indexes from 1 March to 30 April in 2021 and 2022 to quantify the effects of lockdown on aerosol concentrations. The period in 2022 was divided into three stages: pre-lockdown (1-10 March), strict lockdown (11 March to 10 April), and partial lockdown (11-30 April). The results showed that, during the strict lockdown period, compared with the pre-lockdown period, there were substantial reductions in aerosol parameters (EC and AOD), and this was consistent with the concentrations of the atmospheric pollutants PM2.5 (particulate matter with an aerodynamic diameter ≤ 2.5 µm) and PM10 (particulate matter with an aerodynamic diameter ≤ 10 µm), and the O3 concentration increased by 8.3%. During the strict lockdown, the values of EC within 0-1 km and AOD decreased by 16.0% and 11.2%, respectively, as compared to the corresponding period in 2021. Lockdown reduced the conventional and organized emissions of air pollutants, and it clearly delayed the time of seasonal emissions from agricultural burning; however, it did not decrease the number of farmland fire points. Considering meteorological factors and eliminating the influence of wind-blown dust events, the results showed that reductions from conventional organized emission sources during the strict lockdown contributed to a 30% air-quality improvement and a 22% reduction in near-surface extinction (0-2 km). Aerosols produced by urban epidemic prevention and disinfection can also be identified using the EC. Regarding seasonal sources of agricultural straw burning, the concentrated burning induced by the epidemic led to the occurrence of heavy pollution from increased amounts of atmospheric aerosols, with a contribution rate of 62%. These results indicate that there is great potential to further improve air quality in the local area, and suggest that the comprehensive use of straw accompanied by reasonable planned burning is the best way to achieve this.

Family Matters: Examine the Role of Family Financial Socialization to Improve Consumer Financial Well-Being

This paper compares family financial socialization with school financial education in their roles to improve consumer financial well-being. Using a national sample of US adult respondents (at the age of 18 and above) from the 2016 National Financial Well-Being Survey, this study proposes a structural equation model. It shows that family socialization and school education play equally important and distinctively separate roles in elevating financial well-being. Family socialization functions through a mediating factor in helping people cultivate good financial habits in saving, planning, and budgeting. This study contributes to the research of the direct and indirect relationship between family financial socialization, financial literacy, financial habits, and financial well-being.

On-Site Viral Inactivation and RNA Preservation of Gargle and Saliva Samples Combined with Direct Analysis of SARS-CoV-2 RNA on Magnetic Beads

Samples of nasopharyngeal swabs (NPS) are commonly used for the detection of SARS-CoV-2 and diagnosis of COVID-19. As an alternative, self-collection of saliva and gargle samples minimizes transmission to healthcare workers and relieves the pressure of resources and healthcare personnel during the pandemic. This study aimed to develop an enhanced method enabling simultaneous viral inactivation and RNA preservation during on-site self-collection of saliva and gargle samples. Our method involves the addition of saliva or gargle samples to a newly formulated viral inactivation and RNA preservation (VIP) buffer, concentration of the viral RNA on magnetic beads, and detection of SARS-CoV-2 using reverse transcription quantitative polymerase chain reaction directly from the magnetic beads. This method has a limit of detection of 25 RNA copies per 200 μL of gargle or saliva sample and 9–111 times higher sensitivity than the viral RNA preparation kit recommended by the United States Centers for Disease Control and Prevention. The integrated method was successfully used to analyze more than 200 gargle and saliva samples, including the detection of SARS-CoV-2 in 123 gargle and saliva samples collected daily from two NPS-confirmed positive SARS-CoV-2 patients throughout the course of their infection and recovery. The VIP buffer is stable at room temperature for at least 6 months. SARS-CoV-2 RNA (65 copies/200 μL sample) is stable in the VIP buffer at room temperature for at least 3 weeks. The on-site inactivation of SARS-CoV-2 and preservation of the viral RNA enables self-collection of samples, reduces risks associated with SARS-CoV-2 transmission, and maintains the stability of the target analyte.

The CRISPR-Cas toolbox for analytical and diagnostic assay development.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems have revolutionized biological and biomedical sciences in many ways. The last few years have also seen tremendous interest in deploying the CRISPR-Cas toolbox for analytical and diagnostic assay development because CRISPR-Cas is one of the most powerful classes of molecular machineries for the recognition and manipulation of nucleic acids. In the short period of development, many CRISPR-enabled assays have already established critical roles in clinical diagnostics, biosensing, and bioimaging. We describe in this review the recent advances and design principles of CRISPR mediated analytical tools with an emphasis on the functional roles of CRISPR-Cas machineries as highly efficient binders and molecular scissors. We highlight the diverse engineering approaches for molecularly modifying CRISPR-Cas machineries and for devising better readout platforms. We discuss the potential roles of these new approaches and platforms in enhancing assay sensitivity, specificity, multiplexity, and clinical outcomes. By illustrating the biochemical and analytical processes, we hope this review will help guide the best use of the CRISPR-Cas toolbox in detecting, quantifying and imaging biologically and clinically important molecules and inspire new ideas, technological advances and engineering strategies for addressing real-world challenges such as the on-going COVID-19 pandemic.

Integrating Reverse Transcription Recombinase Polymerase Amplification with CRISPR Technology for the One-Tube Assay of RNA.

CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.

Polyketone metabolites isolated from Rhodiola tibetica endohytic fungus Alternaria sp. HJT-Y7 and their SARS-CoV-2 virus inhibitory activitives.

Six new polyketone metabolites, compounds (1-6) and seven known polyketone compounds (7-13) were isolated from Rhodiola tibetica endophytic fungus Alternaria sp. The structural elucidation of five new polyketone metabolites were elucidated on the basis of spectroscopic including 2D NMR and HRMS and spectrometric analysis. Inhibition rate evaluation revealed that compounds 1(EC50 = 0.02 mM), 3(EC50 = 0.3 mM), 6(EC50 = 0.07 mM), 8(EC50 = 0.1 mM) and 9(EC50 = 0.04 mM) had inhibitory effect on the SARS-CoV-2 virus.

CRISPR technology incorporating amplification strategies: molecular assays for nucleic acids, proteins, and small molecules.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein systems have transformed the field of genome editing and transcriptional modulation. Progress in CRISPR-Cas technology has also advanced molecular detection of diverse targets, ranging from nucleic acids to proteins. Incorporating CRISPR-Cas systems with various nucleic acid amplification strategies enables the generation of amplified detection signals, enrichment of low-abundance molecular targets, improvements in analytical specificity and sensitivity, and development of point-of-care (POC) diagnostic techniques. These systems take advantage of various Cas proteins for their particular features, including RNA-guided endonuclease activity, sequence-specific recognition, multiple turnover trans-cleavage activity of Cas12 and Cas13, and unwinding and nicking ability of Cas9. Integrating a CRISPR-Cas system after nucleic acid amplification improves detection specificity due to RNA-guided recognition of specific sequences of amplicons. Incorporating CRISPR-Cas before nucleic acid amplification enables enrichment of rare and low-abundance nucleic acid targets and depletion of unwanted abundant nucleic acids. Unwinding of dsDNA to ssDNA using CRISPR-Cas9 at a moderate temperature facilitates techniques for achieving isothermal exponential amplification of nucleic acids. A combination of CRISPR-Cas systems with functional nucleic acids (FNAs) and molecular translators enables the detection of non-nucleic acid targets, such as proteins, metal ions, and small molecules. Successful integrations of CRISPR technology with nucleic acid amplification techniques result in highly sensitive and rapid detection of SARS-CoV-2, the virus that causes the COVID-19 pandemic.

Texture feature-based machine learning classifier could assist in the diagnosis of COVID-19.

PURPOSE: Differentiating COVID-19 from other acute infectious pneumonias rapidly is challenging at present. This study aims to improve the diagnosis of COVID-19 using computed tomography (CT). METHOD: COVID-19 was confirmed mainly by virus nucleic acid testing and epidemiological history according to WHO interim guidance, while other infectious pneumonias were diagnosed by antigen testing. The texture features were extracted from CT images by two radiologists with 5 years of work experience using modified wavelet transform and matrix computation analyses. The random forest (RF) classifier was applied to identify COVID-19 patients and images. RESULTS: We retrospectively analysed the data of 95 individuals (291 images) with COVID-19 and 96 individuals (279 images) with other acute infectious pneumonias, including 50 individuals (160 images) with influenza A/B. In total, 6 texture features showed a positive association with COVID-19, while 4 features were negatively associated. The mean AUROC, accuracy, sensitivity, and specificity values of the 5-fold test sets were 0.800, 0.722, 0.770, and 0.680 for image classification and 0.858, 0.826, 0.809, and 0.842 for individual classification, respectively. The feature 'Correlation' contributed most both at the image level and individual level, even compared with the clinical factors. In addition, the texture features could discriminate COVID-19 from influenza A/B, with an AUROC of 0.883 for images and 0.957 for individuals. CONCLUSIONS: The developed texture feature-based RF classifier could assist in the diagnosis of COVID-19, which could be a rapid screening tool in the era of pandemic.

Differentiation between COVID‐19 and bacterial pneumonia using radiomics of chest computed tomography and clinical features

To develop and validate an effective model for distinguishing COVID‐19 from bacterial pneumonia. In the training group and internal validation group, all patients were randomly divided into a training group (n = 245) and a validation group (n = 105). The whole lung lesion on chest computed tomography (CT) was drawn as the region of interest (ROI) for each patient. Both feature selection and model construction were first performed in the training set and then further tested in the validation set with the same thresholds. Additional tests were conducted on an external multicentre cohort with 105 subjects. The diagnostic model of LightGBM showed the best performance, achieving a sensitivity of 0.941, specificity of 0.981, accuracy of 0.962 on the validation dataset. In this study, we established a differential model to distinguish between COVID‐19 and bacterial pneumonia based on chest CT radiomics and clinical indexes.

Clinical characteristics of hospitalized mild/moderate COVID-19 patients with a prolonged negative conversion time of SARS-CoV-2 nucleic acid detection.

BACKGROUND: The impact of COVID-19 has been devastating on a global scale. The negative conversion time (NCT) of SARS-CoV-2 RNA is closely related to clinical manifestation and disease progression in COVID-19 patients. Our study aimed to predict factors associated with prolonged NCT of SARS-CoV-2 RNA in mild/moderate COVID-19 patients. METHODS: The clinical features, laboratory data and treatment outcomes of COVID-19 patients were retrospectively analyzed. Then univariate and multivariate analysis were used to screen out risk factors of influencing prolonged NCT of SARS-CoV-2 RNA. RESULTS: Thirty-two hospitalized mild/moderate COVID-19 patients were enrolled. The general clinical symptoms were cough (78.1%), fever (75%), diarrhea (68.8%), expectoration (56.3%), and nausea (37.5%). More than 40% of the patients had decreased erythrocyte, hemoglobin and leucocyte and 93.8% patients were detected in abnormalities of chest CT. The median NCT of SARS-CoV-2 RNA was 19.5 days (IQR: 14.25-25). Univariate analysis found fever, nausea, diarrhea and abnormalities in chest CTs were positively associated with prolonged NCT of viral RNA (P< 0.05). The multivariate Cox proportional hazard model revealed that fever [Exp (B), 0.284; 95% CI, 0.114-0.707; P<0.05] and nausea [Exp (B), 0.257; 95%CI, 0.096-0.689; P<0.05] were two significant independent factors. CONCLUSIONS: Fever and nausea were two significant independent factors in prolonged NCT of viral RNA in mild/moderate COVID-19 patients, which provided a useful references for disease progression and treatment of COVID-19.