Low anti-SARS-CoV-2 S antibody levels predict increased mortality and dissemination of viral components in the blood of critical COVID-19 patients.

BACKGROUND: Anti-SARS-CoV-2 S antibodies prevent viral replication. Critically ill COVID-19 patients show viral material in plasma, associated with a dysregulated host response. If these antibodies influence survival and viral dissemination in ICU-COVID patients is unknown. PATIENTS/METHODS: We studied the impact of anti-SARS-CoV-2 S antibodies levels on survival, viral RNA-load in plasma, and N-antigenaemia in 92 COVID-19 patients over ICU admission. RESULTS: Frequency of N-antigenaemia was >2.5-fold higher in absence of antibodies. Antibodies correlated inversely with viral RNA-load in plasma, representing a protective factor against mortality (adjusted HR [CI 95%], p): (S IgM [AUC ≥ 60]: 0.44 [0.22; 0.88], 0.020); (S IgG [AUC ≥ 237]: 0.31 [0.16; 0.61], <0.001). Viral RNA-load in plasma and N-antigenaemia predicted increased mortality: (N1-viral load [≥2.156 copies/ml]: 2.25 [1.16; 4.36], 0.016); (N-antigenaemia: 2.45 [1.27; 4.69], 0.007). CONCLUSIONS: Low anti-SARS-CoV-2 S antibody levels predict mortality in critical COVID-19. Our findings support that these antibodies contribute to prevent systemic dissemination of SARS-CoV-2.

Comparison of real-time and droplet digital PCR to detect and quantify SARS-CoV-2 RNA in plasma.

BACKGROUND: The presence of SARS-CoV-2 RNA in plasma has been linked to disease severity and mortality. We compared RT-qPCR to droplet digital PCR (ddPCR) to detect SARS-CoV-2 RNA in plasma from COVID-19 patients (mild, moderate, and critical disease). METHODS: The presence/concentration of SARS-CoV-2 RNA in plasma was compared in three groups of COVID-19 patients (30 outpatients, 30 ward patients and 30 ICU patients) using both RT-qPCR and ddPCR. Plasma was obtained in the first 24h following admission, and RNA was extracted using eMAG. ddPCR was performed using Bio-Rad SARS-CoV-2 detection kit, and RT-qPCR was performed using GeneFinder™ COVID-19 Plus RealAmp Kit. Statistical analysis was performed using Statistical Package for the Social Science. RESULTS: SARS-CoV-2 RNA was detected, using ddPCR and RT-qPCR, in 91% and 87% of ICU patients, 27% and 23% of ward patients and 3% and 3% of outpatients. The concordance of the results obtained by both methods was excellent (Cohen's kappa index = 0.953). RT-qPCR was able to detect 34/36 (94.4%) patients positive for viral RNA in plasma by ddPCR. Viral RNA load was higher in ICU patients compared with the other groups (P < .001), by both ddPCR and RT-qPCR. AUC analysis revealed Ct values (RT-qPCR) and viral RNA load values (ddPCR) can similarly differentiate between patients admitted to wards and to the ICU (AUC of 0.90 and 0.89, respectively). CONCLUSION: Both methods yielded similar prevalence of RNAemia between groups, with ICU patients showing the highest (>85%). RT-qPCR was as useful as ddPCR to detect and quantify SARS-CoV-2 RNAemia in plasma.

Viral RNA load in plasma is associated with critical illness and a dysregulated host response in COVID-19.

BACKGROUND: COVID-19 can course with respiratory and extrapulmonary disease. SARS-CoV-2 RNA is detected in respiratory samples but also in blood, stool and urine. Severe COVID-19 is characterized by a dysregulated host response to this virus. We studied whether viral RNAemia or viral RNA load in plasma is associated with severe COVID-19 and also to this dysregulated response. METHODS: A total of 250 patients with COVID-19 were recruited (50 outpatients, 100 hospitalized ward patients and 100 critically ill). Viral RNA detection and quantification in plasma was performed using droplet digital PCR, targeting the N1 and N2 regions of the SARS-CoV-2 nucleoprotein gene. The association between SARS-CoV-2 RNAemia and viral RNA load in plasma with severity was evaluated by multivariate logistic regression. Correlations between viral RNA load and biomarkers evidencing dysregulation of host response were evaluated by calculating the Spearman correlation coefficients. RESULTS: The frequency of viral RNAemia was higher in the critically ill patients (78%) compared to ward patients (27%) and outpatients (2%) (p < 0.001). Critical patients had higher viral RNA loads in plasma than non-critically ill patients, with non-survivors showing the highest values. When outpatients and ward patients were compared, viral RNAemia did not show significant associations in the multivariate analysis. In contrast, when ward patients were compared with ICU patients, both viral RNAemia and viral RNA load in plasma were associated with critical illness (OR [CI 95%], p): RNAemia (3.92 [1.183-12.968], 0.025), viral RNA load (N1) (1.962 [1.244-3.096], 0.004); viral RNA load (N2) (2.229 [1.382-3.595], 0.001). Viral RNA load in plasma correlated with higher levels of chemokines (CXCL10, CCL2), biomarkers indicative of a systemic inflammatory response (IL-6, CRP, ferritin), activation of NK cells (IL-15), endothelial dysfunction (VCAM-1, angiopoietin-2, ICAM-1), coagulation activation (D-Dimer and INR), tissue damage (LDH, GPT), neutrophil response (neutrophils counts, myeloperoxidase, GM-CSF) and immunodepression (PD-L1, IL-10, lymphopenia and monocytopenia). CONCLUSIONS: SARS-CoV-2 RNAemia and viral RNA load in plasma are associated with critical illness in COVID-19. Viral RNA load in plasma correlates with key signatures of dysregulated host responses, suggesting a major role of uncontrolled viral replication in the pathogenesis of this disease.

Author Correction: Combined quantification of procalcitonin and HLA-DR improves sepsis detection in surgical patients.

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Combined quantification of procalcitonin and HLA-DR improves sepsis detection in surgical patients.

Early recognition of sepsis is a key factor to improve survival to this disease in surgical patients, since it allows prompt control of the infectious source. Combining pro-inflammatory and immunosupression biomarkers could represent a good strategy to improve sepsis detection. Here we evaluated the combination of procalcitonin (PCT) with gene expression levels of HLA-DRA to detect sepsis in a cohort of 154 surgical patients (101 with sepsis and 53 with no infection). HLA-DRA expression was quantified using droplet digital PCR, a next-generation PCR technology. Area under the receiver operating curve analysis (AUROC) showed that the PCT/HLA-DRA ratio outperformed PCT to detect sepsis (AUROC [CI95%], p): PCT: 0.80 [0.73-0.88], <0.001; PCT/HLA-DRA: 0.85 [0.78-0.91], <0.001. In the multivariate analysis, the ratio showed a superior ability to predict sepsis compared to that of PCT (OR [CI 95%], p): PCT/HLA-DRA: 7.66 [1.82-32.29], 0.006; PCT: 4.21 [1.15-15.43] 0.030. Multivariate analysis was confirmed using a new surgical cohort with 74 sepsis patients and 21 controls: PCT/HLA-DRA: 34.86 [1.22-995.08], 0.038; PCT: 5.52 [0.40-75.78], 0.201. In conclusion, the combination of PCT with HLA-DRA is a promising strategy for improving sepsis detection in surgical patients.

Traffic Through the Trans-Golgi Network and the Endosomal System Requires Collaboration Between Exomer and Clathrin Adaptors in Fission Yeast.

Despite its biological and medical relevance, traffic from the Golgi to the plasma membrane (PM) is one of the least understood steps of secretion. Exomer is a protein complex that mediates the trafficking of certain cargoes from the trans-Golgi network/early endosomes to the PM in budding yeast. Here, we show that in Schizosaccharomyces pombe the Cfr1 and Bch1 proteins constitute the simplest form of an exomer. Cfr1 co-immunoprecipitates with Assembly Polypeptide adaptor 1 (AP-1), AP-2, and Golgi-localized, gamma-adaptin ear domain homology, ARF-binding (GGA) subunits, and cfr1+ interacts genetically with AP-1 and GGA genes. Exomer-defective cells exhibit multiple mild defects, including alterations in the morphology of Golgi stacks and the distribution of the synaptobrevin-like Syb1 protein, carboxypeptidase missorting, and stress sensitivity. S. pombe apm1Δ cells exhibit a defect in trafficking through the early endosomes that is severely aggravated in the absence of exomer. apm1Δ cfr1Δ cells exhibit a dramatic disorganization of intracellular compartments, including massive accumulation of electron-dense tubulovesicular structures. While the trans-Golgi network/early endosomes are severely disorganized in the apm1Δ cfr1Δ strain, gga21Δ gga22Δ cfr1Δ cells exhibit a significant disturbance of the prevacuolar/vacuolar compartments. Our findings show that exomer collaborates with clathrin adaptors in trafficking through diverse cellular compartments, and that this collaboration is important to maintain their integrity. These results indicate that the effect of eliminating exomer is more pervasive than that described to date, and suggest that exomer complexes might participate in diverse steps of vesicle transport in other organisms.

The AP-2 complex is required for proper temporal and spatial dynamics of endocytic patches in fission yeast.

In metazoans the AP-2 complex has a well-defined role in clathrin-mediated endocytosis. By contrast, its direct role in endocytosis in unicellular eukaryotes has been questioned. Here, we report co- immunoprecipitation between the fission yeast AP-2 component Apl3p and clathrin, as well as the genetic interactions between apl3Δ and clc1 and sla2Δ/end4Δ mutants. Furthermore, a double clc1 apl3Δ mutant was found to be defective in FM4-64 uptake. In an otherwise wild-type strain, apl3Δ cells exhibit altered dynamics of the endocytic sites, with a heterogeneous and extended lifetime of early and late markers at the patches. Additionally, around 50% of the endocytic patches exhibit abnormal spatial dynamics, with immobile patches and patches that bounce backwards to the cell surface, showing a pervasive effect of the absence of AP-2. These alterations in the endocytic machinery result in abnormal cell wall synthesis and morphogenesis. Our results complement those found in budding yeast and confirm that a direct role of AP-2 in endocytosis has been conserved throughout evolution.

Membrane organization and cell fusion during mating in fission yeast requires multipass membrane protein Prm1.

The involvement of Schizosaccharomyces pombe prm1(+) in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell-cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1(+) and the dni(+) genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell-cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell-cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion.

Regulation of cell wall synthesis by the clathrin light chain is essential for viability in Schizosaccharomyces pombe.

The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization; when grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, ß(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodelling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis, and suggest that this is the reason for the lethality of clc1Δ cells in the absence of osmotic stabilization.

The integrity of the cytokinesis machinery under stress conditions requires the glucan synthase Bgs1p and its regulator Cfh3p.

In yeast, cytokinesis requires coordination between nuclear division, acto-myosin ring contraction, and septum synthesis. We studied the role of the Schizosaccharomyces pombe Bgs1p and Cfh3p proteins during cytokinesis under stress conditions. Cfh3p formed a ring in the septal area that contracted during mitosis; Cfh3p colocalized and co-immunoprecipitated with Cdc15p, showing that Cfh3p interacted with the contractile acto-myosin ring. In a wild-type strain, a significant number of contractile rings collapsed under stress conditions and this number increased dramatically in the cfh3Δ, bgs1cps1-191, and cfh3Δ bgs1/cps1-191. Our results show that after osmotic shock Cfh3p is essential for the stability of the (1,3) glucan synthase Bgs1p in the septal area, but not at the cell poles. Finally, cells adapted to stress; they repaired their contractile rings and re-localized Bgs1p to the cell surface some time after osmotic shock. A detailed analysis of the cytokinesis machinery in the presence of KCl revealed that the actomyosin ring collapsed before Bgs1p was internalized, and that it was repaired before Bgs1p re-localized to the cell surface. In the cfh3Δ, bgs1/cps1-191, and cfh3Δ bgs1/cps1-191 mutants, which have reduced glucan synthesis, the damage produced to the ring had stronger consequences, suggesting that an intact primary septum contributes to ring stability. The results show that the contractile actomyosin ring is very sensitive to stress, and that cells have efficient mechanisms to remedy the damage produced in this structure.