Microfluidic Immunoarray for Point-of-Care Detection of Cytokines in COVID-19 Patients.

The "cytokine storm" often induced in COVID-19 patients contributes to the onset of "acute respiratory distress syndrome" (ARDS) accompanied by lung infection and damage, multiorgan failure, and even death. This large increase in pro-inflammatory cytokines in blood may be related to severity. Rapid, on-demand cytokine analyses can thus be critical to inform treatment plans and improve survival rates. Here, we report a sensitive, low-cost, semiautomated 3D-printed microfluidic immunoarray to detect 2 cytokines and CRP simultaneously in a single 10 µL serum sample in 25 min. Accuracy was validated by analyzing 80 COVID-19 patient serum samples, with results well correlated to a commercial Meso Scale protein immunoassay. Capture antibodies immobilized in detection microwells in a flat well plate-type flow chamber facilitate the immunoassay, with a programmable syringe pump automatically delivering reagents. Chemiluminescence signals were captured in a dark box with a CCD camera integrated for 30 s. This system was optimized to detect inflammation biomarkers IL-6, IFN-γ, and CRP simultaneously in blood serum. Ultralow limits of detection (LODs) of 0.79 fg/mL for IL-6, 4.2 fg/mL for CRP, and 2.7 fg/mL for IFN-γ with dynamic ranges of up to 100 pg/mL were achieved. ROC statistical analyses showed a relatively good diagnostic value related to the samples assigned WHO COVID-19 scores for disease severity, with the best results for IL-6 and CRP. Monitoring these biomarkers for coronavirus severity may allow prediction of disease severity as a basis for critical treatment decisions and better survival rates.

Decreased progenitor TCF1 + T-cells correlate with COVID-19 disease severity.

COVID-19, caused by SARS-CoV-2, can lead to a severe inflammatory disease characterized by significant lymphopenia. However, the underlying cause for the depletion of T-cells in COVID-19 patients remains incompletely understood. In this study, we assessed the presence of different T-cell subsets in the progression of COVID-19 from mild to severe disease, with a focus on TCF1 expressing progenitor T-cells that are needed to replenish peripheral T-cells during infection. Our results showed a preferential decline in TCF1+ progenitor CD4 and CD8+ T-cells with disease severity. This decline was seen in various TCF1+ subsets including naive, memory and effector-memory cells, and surprisingly, was accompanied by a loss in cell division as seen by a marked decline in Ki67 expression. In addition, TCF1+ T-cells showed a reduction in pro-survival regulator, BcL2, and the appearance of a new population of TCF1 negative caspase-3 expressing cells in peripheral blood from patients with severe disease. The decline in TCF1+ T-cells was also seen in a subgroup of severe patients with vitamin D deficiency. Lastly, we found that sera from severe patients inhibited TCF1 transcription ex vivo which was attenuated by a blocking antibody against the cytokine, interleukin-12 (IL12). Collectively, our findings underscore the potential significance of TCF1+ progenitor T-cells in accounting for the loss of immunity in severe COVID-19 and outline an array of markers that could be used to identify disease progression.

Levels of Angiotensin and Kinin Metabolite Peptides Related to COVID-19 Severity.

In addition to crucial roles in normal human biology, peptide metabolites of the renin-angiotensin (RAS) and kallikrein-kinin systems (KKS) have been reported to be altered in COVID-19 patients. Here, we evaluate new data on RAS and KKS peptides in COVID-19 patient serum obtained from a recently developed, fully validated, and optimized stable isotope labeling LC-MS peptide assay. We found that the RAS peptides angiotensin (ANG) 1, 2, 1-5, and 1-7 were downregulated compared to COVID-free surrogate controls, while the KKS peptides Brad, Brad 1-8, and Brad 1-7 were upregulated. This paper focuses on uncovering the possible diagnostic value of these peptides using receiver operating characteristic (ROC) analyses of these data. ROC plots confirmed that all of the analyte peptides in 80 serum samples from COVID-19 patients were significantly altered from "normal" values of the control samples. The best diagnostic sensitivities and selectivities for COVID vs no COVID were found in ROC plots for Brad and Brad 1-7 (both 99% sensitivity, 100% selectivity). We then analyzed levels of all the peptides grouped according to preassigned values of the World Health Organization (WHO) COVID-19 Severity Index. ROC plots differentiated patients with a high WHO severity index from those with a low WHO severity index with moderate success, with BRAD (73% sensitivity, 79% selectivity) and Ang 1-7 (75% sensitivity, 65% selectivity) giving the best diagnostic performance. Results suggest the possible diagnostic value of these peptides as biomarkers to help identify moderate and serious COVID-19 cases at relatively early stages.

Safety and effectiveness concerns of lopinavir/ritonavir in COVID-19 affected patients: a retrospective series.

CONTEXT: Originally developed for treatment of human immunodeficiency virus (HIV), the antiviral combination lopinavir/ritonavir (LPV/r) is being repurposed for treating the novel coronavirus disease (COVID-19) despite minimal experience in this markedly different population and an in-vitro derived EC50 against SARS-CoV-2 several hundred-fold greater than for HIV. We present a case series including a case of severe hyponatremia and a 32-fold overdose raising safety and effectiveness concerns in COVID-19 patients. METHODS: We measured LPV trough concentrations in 12 patients and reviewed their clinical charts for side effects known to occur in HIV patients. FINDINGS: Compared to established LPV trough concentrations in HIV patients, concentrations in COVID-19 patients were 3-fold greater (19.37 ± 10.12 mcg/mL versus 6.25 mcg/mL). In addition, cholestasis and dyslipidemia toxicity thresholds were exceeded in 12/12 and 11/12 patients respectively. No patients achieved the presumed therapeutic concentration. Side effects included gastrointestinal symptoms (5/12), electrolyte imbalances (4/12), liver enzyme disturbances (5/12) and triglyceride elevations (2/12). CONCLUSION: No patients reached presumed therapeutic LPV concentrations despite experiencing side effects and exceeding cholestasis and dyslipidemia toxicity thresholds. This raises concerns for the safety and effectiveness of LPV/r. Clinicians should consider closely monitoring for side effects and not necessarily attribute them to COVID-19.