Brief summaryNearly 50% patients of novel coronavirus (SARS-CoV-2)-infected pneumonia could not reach obvious clinical and radiological remission within 10 days after hospitalization. These refractory COVID-19 patients showed an obvious difference with the general patients in clinical characteristics. AbstractBackground: Since December 2019, novel coronavirus (SARS-CoV-2)-infected pneumonia (COVID-19) occurred in Wuhan, and rapidly spread throughout China.This study aimed to clarify the characteristics of patients with refractory COVID-19. Methods:In this retrospective single-center study, we included 155 consecutive patients with confirmed COVID-19 in Zhongnan Hospital of Wuhan University from January 1 st to February 5 th . The cases were divided into general and refractory COVID-19 groups according to the clinical efficacy after hospitalization, and the difference between groups were compared. Results:Compared with general COVID-19 patients (45.2%), refractory patients had an older age, male sex, more underlying comorbidities, lower incidence of fever, higher levels of maximum temperature among fever cases, higher incidence of breath shortness and anorexia, severer disease assessment on admission, high levels of neutrophil, aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and C-reactive protein, lower levels of platelets and albumin, and higher incidence of bilateral pneumonia and pleural effusion (P<0.05). Refractory COVID-19 patients were more likely to receive oxygen, mechanical ventilation, expectorant, and adjunctive treatment including corticosteroid, antiviral drugs and immune enhancer (P<0.05). After adjustment, those with refractory COVID-19 were also more likely to have a male sex and manifestations of anorexia and fever on admission, and receive oxygen, expectorant and adjunctive agents (P<0.05) when considering the factors of disease severity on admission, mechanical ventilation, and ICU transfer.Conclusion: Nearly 50% COVID-19 patients could not reach obvious clinical and radiological remission within 10 days after hospitalization. The patients with male sex, anorexia and no fever on admission predicted poor efficacy.

Quantitative real time PCR (RT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. However, due to the low viral load specimens and the limitations of RT-PCR, significant numbers of false negative reports are inevitable, which results in failure to timely diagnose, cut off transmission, and assess discharge criteria. To improve this situation, an optimized droplet digital PCR (ddPCR) was used for detection of SARS-CoV-2, which showed that the limit of detection of ddPCR is significantly lower than that of RT-PCR. We further explored the feasibility of ddPCR to detect SARS-CoV-2 RNA from 77 patients, and compared with RT-PCR in terms of the diagnostic accuracy based on the results of follow-up survey. 26 patients of COVID-19 with negative RT-PCR reports were reported as positive by ddPCR. The sensitivity, specificity, PPV, NPV, negative likelihood ratio (NLR) and accuracy were improved from 40% (95% CI: 27-55%), 100% (95% CI: 54-100%), 100%, 16% (95% CI: 13-19%), 0.6 (95% CI: 0.48-0.75) and 47% (95% CI: 33-60%) for RT-PCR to 94% (95% CI: 83-99%), 100% (95% CI: 48-100%), 100%, 63% (95% CI: 36-83%), 0.06 (95% CI: 0.02-0.18), and 95% (95% CI: 84-99%) for ddPCR, respectively. Moreover, 6/14 (42.9%) convalescents were detected as positive by ddPCR at 5-12 days post discharge. Overall, ddPCR shows superiority for clinical diagnosis of SARS-CoV-2 to reduce the false negative reports, which could be a powerful complement to the RT-PCR.

Background: Quercetin, a major flavonol, wildly exists in plantage, which has been reported to have an anti-apoptosis and anti-inflammation effects on vascular endothelial cells, but its underlying molecular mechanisms remain unclear. Objective: The aim of this study was to investigate the mechanisms of how quercetin inhibits tumor necrosis factor alpha (TNF-α) induced human umbilical vein endothelial cells (HUVECs) apoptosis and inflammation. Methods and Results: HUVECs were preconditioned with quercetin for 18 hours, and subsequently treated with TNF-α for 6 hours to induce apoptosis. The expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin, β-actin mRNA was then detected by RT-PCR. Flow cytometry was used to estimate the apoptosis rates, and the expression of activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB) was measured by Western blot. TNF-α induced elevated apoptosis rates and upregulation of VCAM-1, ICAM-1, and E-selectin were meaningfully reduced in HUVECs by pretreatment with quercetin. In addition, quercetin also inhibited the activation of AP-1and NF-κB. Conclusion: Results indicate that quercetin could suppress TNF-α induced apoptosis and inflammation by blocking NF-κB and AP-1 signaling pathway in HUVECs, which might be one of the underlying mechanisms in treatment of coronary heart disease.

32Background: Real-Time PCR (RT-PCR) is widely used as the gold standard for 33 clinical detection of SARS-CoV-2. However, due to the low viral load in patient 34 throat and the limitation of RT-PCR, significant numbers of false negative reports are 35 inevitable, which should not be ignored. 36 Methods: We explored the feasibility of droplet digital PCR (ddPCR) to detect 37 SARS-CoV-2 from 57 clinical pharyngeal swab samples and compared with RT-PCR 38 in terms of the sensitivity and accuracy. Among 57 samples, all of which were 39 reported as negative nucleic acid by officially approved clinical RT-PCR detection, 43 40 samples were collected from suspected patients with fever in clinic, and 14 were from 41 supposed convalescents who were about to discharge after treatment. The experiment 42 was double-blind. 43 Results: The lower limit of detection of the optimized ddPCR is at least 500 times 44 lower than that of RT-PCR. The overall accuracy of ddPCR for clinical detection is 45 94.3 %. 33 out of 35 negative pharyngeal swab samples checked by RT-PCR were 46 correctly judged by ddPCR based on the follow-up investigation. In addition, 9 out of 47 14 (64.2 %) supposed convalescents with negative nucleic acid test twice by RT-PCR 48 were positive by ddPCR detection. 49 Conclusions: ddPCR shows superiority for clinical detection of SARS-CoV-2 to 50 reduce the false negatives, which could be a powerful complement to the current 51 standard RT-PCR. Before the ddPCR to be approved for diagnosis, the current clinical 52 practice that the convalescent continues to be quarantined for 2 weeks is reasonable 53 and necessary. 54 55

Background: Viral clearance is one important indicator for the recovery of SARS-CoV-2 infected patients. Suboptimal T and B cell responses can delay viral clearance in MERS and SARS patients. The role of leukomonocytes in viral clearance of COVID-19 patients is not yet well defined.Methods: From January 26 to February 28, 2020, an observational study was launched at Zhongnan Hospital of Wuhan University, Wuhan, China. We enrolled 25 laboratory-confirmed COVID-19 patients, whose throat-swab specimens were tested positive for SARS-CoV-2 infection by qRT-PCR. We comprehensively analyzed clinical records, counts of lymphocyte subsets including CD3+, CD4+, CD8+ T cells, B cells and NK cells in the patients who successfully cleared SARS-CoV-2, and compared to those that failed to, after a standardized treatment of 8-14 days. Findings: In 25 enrolled COVID-19 patients, lymphopeniawas a common feature. After the treatment, 14 patients were tested negative for SARS-CoV-2. The patients that cleared the infection had restored the numbers of CD3+, CD4+, CD8+ T cellsand B cells as compared to the still viral RNA positive patients, while the recovered patients had a higher count of leukomonocytes. Conclusions: By comparison of leukomonocytes counts in COVID-19 patients at different stages of the disease, we found that CD3+, CD4+, CD8+ T cells and B cells appear to play important roles in viral clearance. The restoration of leukomonocytes counts from peripheral blood can be used as prognosis for the recovery of an COVID-19 infection. We propose that restoration of leukomonocytes counts can be added to the COVID-19 diagnostic guidanceas a criterion for releasing and discharging patients.