up to February 10, 2020. Epidemiological, demographic, and clinical data were collected. The clinical course of survivors and non-survivors were compared. Risk factors for death were analyzed. Results: A total of 107 discharged patients with COVID-19 were enrolled. The clinical course of COVID-19 presented as a tri-phasic pattern. Week 1 after illness onset was characterized by fever, cough, dyspnea, lymphopenia, and radiological multi-lobar pulmonary infiltrates. In severe cases, thrombocytopenia, acute kidney injury, acute myocardial injury, and adult respiratory distress syndrome were observed. During week 2, in mild cases, fever, cough, and systemic symptoms began to resolve and platelet count rose to normal range, but lymphopenia persisted. In severe cases, leukocytosis, neutrophilia, and deteriorating multi-organ dysfunction were dominant. By week 3, mild cases had clinically resolved except for lymphopenia. However, severe cases showed persistent lymphopenia, severe acute respiratory dyspnea syndrome, refractory shock, anuric acute kidney injury, coagulopathy, thrombocytopenia, and death. Older age and male sex were independent risk factors for poor outcome of the illness. Conclusions: A period of 7-13 days after illness onset is the critical stage in the COVID-19 course. Age and male gender were independent risk factors for death of COVID-19.

A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The VHH gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double mutant gene. The Nb28-AP construct was transformed into E. coli BL21(DE3)plysS and soluble expression in bacteria was confirmed by SDS-PAGE and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 ng/mL and 0.04 ng/mL, respectively, with a linear range of 0.06–0.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.

A sensitive indirect competitive nanobody-based enzyme linked immunosorbent assay (Nb-ELISA) for ochratoxin A (OTA) with high resistance to cereal matrix interference was developed. Nanobodies against OTA (Nb15, Nb28, Nb32, Nb36) were expressed in E. coli cells and their thermal stabilities were compared with that of an OTA-specific monoclonal antibody 6H8. All nanobodies could still retain their antigen-binding activity after exposure to temperature 95 °C for 5 min or to 90 °C for 75 min. Nb28 that exhibited the highest sensitivity in ELISA was selected for further research. An indirect competitive ELISA based on Nb28 was developed for OTA, with an IC50 of 0.64 ng/mL and a linear range (IC20-IC80) of 0.27–1.47 ng/mL. Cereal samples were analyzed following a 2.5 fold dilution of sample extracts, showing the good resistance to matrix interference of the Nb-ELSIA. The recovery of spiked cereal samples (rice, oats, barley) ranged from 80% to 105% and the Nb-ELISA results of OTA content in naturally contamined samples were in good agreement with those determined by a commercial ELISA kit. The results indicated the reliablity of nanobody as a promising immunoassay reagent for detection of mycotoxins in food matrix and its potential in biosensor development.

Phage display-mediated immuno-polymerase chain reaction (PD-IPCR) is an ultrasensitive detection technology that combines the advantages of immuno-PCR and phage display. The phage particle, which displayed antibody fragments including single-chain fragment variable (scFv), variable domain of heavy-chain antibodies (VHH), and antigen-binding fragment (Fab) on the surface can be directly used in IPCR, supplying both the detection antibody and deoxyribonucleic acid (DNA) template. In this work, we used ochratoxin A (OTA) as a model system to study the capacity of PD-IPCR in the detection of toxic small molecular weight compounds, especially mycotoxins. An alpaca-derived VHH library was constructed and subjected to four cycles of panning. In total, 16 clones with four unique sequences were selected by competitive binding with OTA. The clone VHH-28 resulted in the lowest 50% inhibitory concentration of 0.31 ng/mL in the phage enzyme-linked immunosorbent assay (ELISA) and was selected to develop the VHH phage-based real-time immuno-PCR (RT-IPCR). The detection limit of the VHH phage-based RT-IPCR was 3.7 pg/L, with a linear range of 0.01–1000 pg/mL. This method was compared with conventional ELISA, and validation results indicated the reliability of VHH phage-based RT-IPCR in the detection of OTA in cereal samples. This study provides a new idea for the ultrasensitive detection of mycotoxins and other toxic small molecular weight compounds.