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Fluoroquinolone prophylaxis’s (FQ-P) usefulness in patients with neutropenia is controversial. In recent decades, Italian epidemiological data has shown worrisome rates of FQ resistance. A single-center cohort study on 136 autologous stem cell transplantations (ASCTs) and 223 allogeneic hematopoietic stem cell transplantations (allo-HSCTs) was performed from January 2018 to December 2020. Piperacillin/tazobactam was the first-line therapy for febrile neutropenia (FN). Since February 2019, FQ-P has been omitted. We evaluated the day +30 posttransplant cumulative incidence function (CIF) of gram-negative bacteria pre-engraftment bloodstream infections (PE-BSIs) and any changes in antimicrobial resistance, FN, and infection-related mortality (IRM). In ASCTs, ≥1 FN episode occurred in 74.3% of transplants, without differences among groups (P = .66). CIF of gram-negative bacteria PE-BSI was 10.1%, with a significant difference according to FQ-P (0% [LEVO-group] vs 14.1% [NO-LEVO-group], P = .016). CIF of IRM was 0% in both groups. In allo-HSCTs, ≥1 FN episode occurred in 96.4% of transplants, without differences among groups (P = .72). CIF of gram-negative bacteria PE-BSI was 28%, significantly higher without FQ-P (14.7% [LEVO-group] vs 34.4% [NO-LEVO-group], P = .003). CIF of IRM was 5%, superimposable in both groups (P = .62). Comparing antimicrobial resistance among gram-negative bacteria of allo-HSCT setting, in the group without FQ-P, a significantly higher proportion of pathogens was susceptible to piperacillin/tazobactam (71% vs 30%, P = .026), FQ (49% vs 10%, P = .03), and carbapenems (95% vs 50%, P = .001). FQ-P discontinuation increased gram-negative bacteria PE-BSI but did not impact IRM, both in the ASCT and allo-HSCT settings; importantly, it concurred to significantly decrease antimicrobial resistance in gram-negative bacteria.

The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2 locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.

IntroductionThe integrity of the gut barrier (GB) is fundamental to regulate the crosstalk between the microbiota and the immune system and to prevent inflammation and autoimmunity at the intestinal level but also in organs distal from the gut such as the pancreatic islets. In support to this idea, we recently demonstrated that breakage of GB integrity leads to activation of islet-reactive T cells and triggers autoimmune Type 1 Diabetes (T1D). In T1D patients as in the NOD mice, the spontaneous model of autoimmune diabetes, there are alterations of the GB that specifically affect structure and composition of the mucus layer; however, it is yet to be determined whether a causal link between breakage of the GB integrity and occurrence of autoimmune T1D exists.MethodsHere we restored GB integrity in the NOD mice through administration of an anti-inflammatory diet (AID- enriched in soluble fiber inulin and omega 3-PUFA) and tested the effect on T1D pathogenesis.ResultsWe found that the AID prevented T1D in NOD mice by restoring GB integrity with increased mucus layer thickness and higher mRNA transcripts of structural (Muc2) and immunoregulatory mucins (Muc1 and Muc3) as well as of tight junction proteins (claudin1). Restoration of GB integrity was linked to reduction of intestinal inflammation (i.e., reduced expression of IL-1β, IL-23 and IL-17 transcripts) and expansion of regulatory T cells (FoxP3+ Treg cells and IL-10+ Tr1 cells) at the expenses of effector Th1/Th17 cells in the intestine, pancreatic lymph nodes (PLN) and intra-islet lymphocytes (IIL) of AID-fed NOD mice. Importantly, the restoration of GB integrity and immune homeostasis were associated with enhanced concentrations of anti-inflammatory metabolites of the ω3/ω6 polyunsaturated fatty acids (PUFA) and arachidonic pathways and modifications of the microbiome profile with increased relative abundance of mucus-modulating bacterial species such as Akkermansia muciniphila and Akkermansia glycaniphila.DiscussionOur data provide evidence that the restoration of GB integrity and intestinal immune homeostasis through administration of a tolerogenic AID that changed the gut microbial and metabolic profiles prevents autoimmune T1D in preclinical models.

Background and aims Vaccines, to limit SARS-CoV-2 infection, were produced and reliable assays are needed for their evaluation. The WHO produced an International-Standard (WHO-IS) to facilitate the standardization/comparison of serological methods. The WHO-IS, produced in limited amount, was never tested for reproducibility. This study aims at developing a reproducible and accessible working standard (WS) to complement the WHO-IS. Materials and methods Sera from vaccinated individuals were used to produce the WSs. The WHO-IS, the WSs and single serum samples (n = 48) were tested on 6 quantitative serological devices. Neutralization assays were performed for the 48 samples and compared with their antibody titers. Results The WS carry an antibody titer 20-fold higher than the WHO-IS. It was reproducible, showed both good linearity and insignificant intra- and inter-laboratory variability. However, the WSs behave differently from the WHO-IS. Analysis of the 48 samples showed that single correlation factors are not sufficient to harmonize results from different assays. Yet, all the devices predict neutralization activity based on the antibody titer. Conclusions A reproducible and highly concentrated WS, specific for IgG against SARS-CoV-2 Spike-glycoprotein was produced. Such characteristics make it particularly suited for the harmonization of commercially available assays and the consequent evaluation of post-vaccinated individuals.

Fluoroquinolone prophylaxis (FQ-P) usefulness in neutropenic patients is controversial. In recent decades, Italian epidemiological data has shown worrisome rates of FQ resistance. A single-center cohort study on 136 autologous stem cell transplantations (ASCTs) and 223 allogeneic hematopoietic stem cell transplantations (allo-HSCTs) was performed from Jan-2018 to Dec-2020. Piperacillin/tazobactam was the first-line therapy for febrile neutropenia (FN). Since Feb-2019, FQ-P was omitted. We evaluated the day +30 post-transplant cumulative incidence function (CIF) of Gram-negative bacteria pre-engraftment blood-stream infections (PE-BSIs) and any changes in antimicrobial resistance, FN and infection-related mortality (IRM). In ASCTs, ≥1 FN episode occurred in 74.3% of transplants, without differences among groups [p=0.66]. CIF of Gram-negative bacteria PE-BSI was 10.1% with a significant difference according to FQ-P [0% (LEVO-group) versus 14.1% (NO-LEVO-group), p=0.016]. CIF of IRM was 0% in both groups. In allo-HSCTs, ≥1 FN episode occurred in 96.4% of transplants, without differences among groups [p=0.72]. CIF of Gram-negative bacteria PE-BSI was 28% and it was significantly higher without FQ-P [14.7% (LEVO-group) versus 34.4% (NO-LEVO-group), p=0.003]. CIF of IRM was 5%, superimposable in both groups [p=0.62]. Comparing antimicrobial resistance among Gram-negative bacteria in the setting of allo-HSCT, in the group without FQ-P a significantly higher proportion of pathogens was susceptible to piperacillin/tazobactam (71% versus 30%, p=0.026), FQ (49% versus 10%, p=0.03) and carbapenems (95% versus 50%, p=0.001). FQ-P discontinuation increased Gram-negative bacteria PE-BSI, but it did not impact IRM, both in ASCT and allo-HSCT setting; importantly, it concurred to decrease significantly antimicrobial resistance in Gram-negative bacteria.

SARS-CoV-2 is a positive single-stranded RNA virus that interacts with proteins of infected cells at different stages of its life cycle. These interactions are necessary for the host to recognize and block the replication of the virus. Yet, if cells fail to block SARS-CoV-2, host proteins are recruited to translate, transcribe and replicate the genetic material of the virus. To identify the host proteins that bind to SARS-CoV-2 RNA, we adopted the RNA-Protein Interaction Detection coupled to Mass Spectrometry (RaPID-MS) technology, which allows the purification and identification by MS-based proteomics of the proteins associated with a specific RNA of interest expressed in mammalian cells. We specifically investigated proteins associated with the 5′ and 3′ end regions of SARS-CoV-2 RNA. As associations might involve non-physical protein-RNA interactions, we defined a set of reliable protein-RNA interactions by exploiting the predictive power of the catRAPID algorithm that assesses the direct binding potential of proteins to a given RNA region. Among these specific SARS-CoV-2 RNA end interactors, we identified the pseudouridine synthase PUS7 that binds to both 5′ and 3′ ends of viral RNA, which harbor the canonical consensus sequence modified by PUS7. We corroborated our results through SARS-CoV-2 RNA analysis by nanopore direct RNA sequencing. Indeed, these PUS7 consensus regions were found highly modified on viral RNAs, as demonstrated by ionic current features that are significantly different compared to the unmodified in vitro transcribed RNA. Overall, our data map the specific host protein interactions of SARS-CoV-2 RNA and point to a role for cellular pseudouridine synthases and the post-transcriptional pseudouridine modifications in the viral life cycle.

In patients with severe or critical Coronavirus disease 2019 (COVID-19) manifestations, a thromboinflammatory syndrome, with diffuse microvascular thrombosis, is increasingly evident as the final step of pro-inflammatory cytokines storm. Actually, no proven effective therapies for novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection exist. Preliminary observations on anticoagulant therapy appear to be associated with better outcomes in moderate and severe COVID-19 patients with signs of coagulopathy and in those requiring mechanical ventilation. The pathophysiology underlying the prothrombotic state elicited by SARS-CoV-2 outlines possible protective mechanisms of antithrombotic therapy (in primis anticoagulants) for this viral illness. The indications for antiplatelet/anticoagulant use (prevention, prophylaxis, therapy) are guided by the clinical context and the COVID-19 severity. We provide a practical approach on antithrombotic therapy management for COVID-19 patients from a multidisciplinary point of view., Highlights • An endothelial thromboinflammatory syndrome is involved in severe COVID-19 manifestations. • A pathophysiological rationale (anti-inflammatory and direct antiviral effects) motivates the use of antithrombotic therapy • Anticoagulant therapy is associated with better outcomes in case of coagulopathy and mechanical ventilation. • A potential role for antithrombotic therapy in COVID-19 prevention and treatment needs further investigation.

The extremely rapid spread of the SARS-CoV-2 has already resulted in more than 1 million reported deaths of coronavirus disease 2019 (COVID-19). The ability of infectious particles to persist on environmental surfaces is potentially considered a factor for viral spreading. Therefore, limiting viral diffusion in public environments should be achieved with correct disinfection of objects, tissues, and clothes. This study proves how two widespread disinfection systems, short-wavelength ultraviolet light (UV-C) and ozone (O3), are active in vitro on different commonly used materials. The development of devices equipped with UV-C, or ozone generators, may prevent the virus from spreading in public places.

The dependence of development and homeostasis in animals on the interaction of hundreds of extracellular regulatory proteins with the peri- and extracellular glycosaminoglycan heparan sulfate (HS) is exploited by many microbial pathogens as a means of adherence and invasion. Heparin, a widely used anticoagulant drug, is structurally similar to HS and is a common experimental proxy. Exogenous heparin prevents infection by a range of viruses, including S-associated coronavirus isolate HSR1. Here, we show that heparin inhibits severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) invasion of Vero cells by up to 80% at doses achievable through prophylaxis and, particularly relevant, within the range deliverable by nebulisation. Surface plasmon resonance and circular dichroism spectroscopy demonstrate that heparin and enoxaparin, a low-molecular-weight heparin which is a clinical anticoagulant, bind and induce a conformational change in the spike (S1) protein receptor-binding domain (S1 RBD) of SARS-CoV-2. A library of heparin derivatives and size-defined fragments were used to probe the structural basis of this interaction. Binding to the RBD is more strongly dependent on the presence of 2-O or 6-O sulfate groups than on N-sulfation and a hexasaccharide is the minimum size required for secondary structural changes to be induced in the RBD. It is likely that inhibition of viral infection arises from an overlap between the binding sites of heparin/HS on S1 RBD and that of the angiotensin-converting enzyme 2. The results suggest a route for the rapid development of a first-line therapeutic by repurposing heparin and its derivatives as antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.

M. P. Doyle, J. R. Genualdi, A. L. Bailey, N. Kose, et al. (mBio 13:e00512-22, 2022, https://doi.org/10.1128/mBio.00512-22), report on the cloning of a panel of fully human monoclonal antibodies (mAbs) directed against yellow fever virus (YFV). In particular, mAb YFV-136 is endowed with interesting cross-YFV substrain-neutralizing features. The importance of YFV-136 and other mAbs with similar characteristics is related not necessarily only to their possible future use in the clinic but also to their role in a better understanding of the biology of YFV (as well as of other flaviviruses) for the development of effective therapeutic and prophylactic strategies. The emergence and reemergence of different flaviviruses worldwide in the last decades certainly make this a compelling clinical priority.

(1) Background: Our aim is the evaluation of the neutralizing activity of BNT162b2 mRNA vaccine-induced antibodies in different in vitro cellular models, as this still represents one of the surrogates of protection against SARS-CoV-2 viral variants. (2) Methods: The entry mechanisms of SARS-CoV-2 in three cell lines (Vero E6, Vero E6/TMPRSS2 and Calu-3) were evaluated with both pseudoviruses and whole virus particles. The neutralizing capability of sera collected from vaccinated subjects was characterized through cytopathic effects and Real-Time RT PCR. (3) Results: In contrast to Vero E6 and Vero E6/TMPRSS2, Calu-3 allowed the evaluation of both viral entry mechanisms, resembling what occurs during natural infection. The choice of an appropriate cellular model can decisively influence the determination of the neutralizing activity of antibodies against SARS-CoV-2 variants. Indeed, the lack of correlation between neutralizing data in Calu-3 and Vero E6 demonstrated that testing the antibody inhibitory activity by using a single cell model possibly results in an inaccurate characterization. (4) Conclusions: Cellular systems allowing only one of the two viral entry pathways may not fully reflect the neutralizing activity of vaccine-induced antibodies moving increasingly further away from possible correlates of protection from SARS-CoV-2 infection.

The purpose of this study was to evaluate the efficacy and safety of the Moderna-1273 mRNA vaccine for SARS-CoV-2 in patients with immune-mediated diseases under different treatments. Anti-trimeric spike protein antibodies were tested in 287 patients with rheumatic or autoimmune diseases (10% receiving mycophenolate mofetil, 15% low-dose glucocorticoids, 21% methotrexate, and 58% biologic/targeted synthetic drugs) at baseline and in 219 (76%) 4 weeks after the second Moderna-1273 mRNA vaccine dose. Family members or caretakers were enrolled as the controls. The neutralizing serum activity against SARS-CoV-2-G614, alpha, and beta variants in vitro and the cytotoxic T cell response to SARS-CoV-2 peptides were determined in a subgroup of patients and controls. Anti-SARS-CoV-2 antibody development, i.e., seroconversion, was observed in 69% of the mycophenolate-treated patients compared to 100% of both the patients taking other treatments and the controls (p < 0.0001). A dose-dependent impairment of the humoral response was observed in the mycophenolate-treated patients. A daily dose of >1 g at vaccination was a significant risk factor for non-seroconversion (ROC AUC 0.89, 95% CI 0.80–98, p < 0.0001). Moreover, in the seroconverted patients, a daily dose of >1 g of mycophenolate was associated with significantly lower anti-SARS-CoV-2 antibody titers, showing slightly reduced neutralizing serum activity but a comparable cytotoxic response compared to other immunosuppressants. In non-seroconverted patients treated with mycophenolate at a daily dose of >1 g, the cytotoxic activity elicited by viral peptides was also impaired. Mycophenolate treatment affects the Moderna-1273 mRNA vaccine immunogenicity in a dose-dependent manner, independent of rheumatological disease.

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5′ cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5′ cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

Background: Cefiderocol is a siderophore cephalosporin that exhibits antimicrobial activity against most multi-drug resistant Gram-negative bacteria, including Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia. Methods: A total of 20 multidrug-resistant A. baumannii strains were isolated from 2020 to 2021, molecularly characterized and tested to assess the in vitro antibacterial activity of cefiderocol. Thirteen strains were carbapenem-hydrolysing oxacillinase OXA-23-like producers, while seven were non-OXA-23-like producers. Minimum inhibitory concentrations (MICs) were determined by broth microdilution, considered as the gold standard method. Disk diffusion test was also carried out using iron-depleted CAMHB plates for cefiderocol. Results: Cefiderocol MICs ranged from 0.5 to 1 mg/L for OXA-23-like non-producing A. baumannii strains and from 0.25 to >32 mg/L for OXA-23-like producers, using the broth microdilution method. Cefiderocol MIC90 was 8 mg/L. Diameter of inhibition zone of cefiderocol ranged from 18 to 25 mm for OXA-23-like non-producers and from 15 to 36 mm for OXA-23-like producers, using the diffusion disk method. A large variability and a low reproducibility were observed during the determination of diameter inhibition zone. Molecular characterization showed that all isolates presented the ISAba1 genetic element upstream the blaOXA-51. Among OXA-23-like non-producers, four were blaOXA-58 positive and two were negative for all the resistance determinants analyzed. Conclusions: Cefiderocol showed in vitro antimicrobial activity against both carbapenem-susceptible and non-susceptible A. baumannii strains, although some OXA-23-like producers were resistant. Further clinical studies are needed to consolidate the role of cefiderocol as an antibiotic against MDR A. baumannii.

SARS-CoV-2 proximal origin is still unclear, limiting the possibility of foreseeing other spillover events with pandemic potential. Here we propose an evolutionary model based on the thorough dissection of SARS-CoV-2 and RaTG13 – the closest bat relative – spike dynamics, kinetics and binding to ACE2. Our results indicate that both spikes share nearly identical, high affinities for Rhinolophus affinis bat and human ACE2, pointing out to negligible species barriers directly related to receptor binding. Also, SARS-CoV-2 spike shows a higher degree of dynamics and kinetics optimization that favors ACE2 engagement. Therefore, we devise an affinity-independent evolutionary process that likely took place in R. affinis bats and limits the eventual involvement of other animal species in initiating the pandemic to the role of vector.

The COVID-19 pandemic caused by the β-coronavirus SARS-CoV-2 has made the development of safe and effective vaccines a critical global priority. To date, four vaccines have already been approved by European and American authorities for preventing COVID-19 but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle, a technology previously utilized for cancer vaccines. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 Spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax – a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein RBD – induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function and significantly lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started in Italy.

SummaryThe humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in COVID-19. The present study was designed to conduct a systematic investigation of the interaction of humoral fluid phase pattern recognition molecules (PRM) with SARS-CoV-2. Out of 10 PRM tested, the long pentraxin PTX3 and Mannose Binding Lectin (MBL) bound the viral Nucleoprotein and Spike, respectively. MBL bound trimeric Spike, including that of variants of concern, in a glycan- dependent way and inhibited SARS-CoV-2 in threein vitromodels. Moreover, upon binding to Spike, MBL activated the lectin pathway of complement activation. Genetic polymorphisms at the MBL locus were associated with disease severity. These results suggest that selected humoral fluid phase PRM can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.

SummaryThe COVID-19 outbreak driven by SARS-CoV-2 has caused more than 2.5 million deaths globally, with the most severe cases characterized by over-exuberant production of immune-mediators, the nature of which is not fully understood. Interferons of the type I (IFN-I) or type III (IFN-III) families are potent antivirals, but their role in COVID-19 remains debated. Our analysis of gene and protein expression along the respiratory tract shows that IFNs, especially IFN-III, are over-represented in the lower airways of patients with severe COVID-19, while high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity; also, IFN expression varies with abundance of the cell types that produce them. Our data point to a dynamic process of inter- and intra-family production of IFNs in COVID-19, and suggest that IFNs play opposing roles at distinct anatomical sites.

Growing evidence suggests that conventional dendritic cells (cDCs) undergo aberrant maturation in COVID-19 and this negatively affects T cell activation. The presence of functional effector T cells in mild patients and dysfunctional T cells in severely ill patients suggests that adequate T cell responses are needed to limit disease severity. Therefore, understanding how cDCs cope with SARS-CoV-2 infections can help elucidate the mechanism of generation of protective immune responses. Here, we report that cDC2 subtypes exhibit similar infection-induced gene signatures with the up-regulation of interferon-stimulated genes and IL-6 signaling pathways. The main difference observed between DC2s and DC3s is the up-regulation of anti-apoptotic genes in DC3s, which explains their accumulation during infection. Furthermore, comparing cDCs between severe and mild patients, we find in the former a profound down-regulation of genes encoding molecules involved in antigen presentation, such as major histocompatibility complex class II (MHCII) molecules, β2 microglobulin, TAP and costimulatory proteins, while an opposite trend is observed for proinflammatory molecules, such as complement and coagulation factors. Therefore, as the severity of the disease increases, cDC2s enhance their inflammatory properties and lose their main function, which is the antigen presentation capacity. In vitro, direct exposure of cDC2s to the virus recapitulates the type of activation observed in vivo. Our findings provide evidence that SARS-CoV-2 can interact directly with cDC2s and, by inducing the down-regulation of crucial molecules required for T cell activation, implements an efficient immune escape mechanism that correlates with disease severity.

Severe COVID-19 is characterized by overproduction of immune mediators, but the role of interferons (IFNs) of the type I (IFN-I) or type III (IFN-III) families remains debated. We scrutinized the production of IFNs along the respiratory tract of COVID-19 patients and found that high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity. Production of specific IFN-III, but not IFN-I, members, denotes patients with a mild pathology and efficiently drives the transcription of genes that protect against SARS-CoV-2. In contrast, compared to subjects with other infectious or non-infectious lung pathologies, IFNs are over-represented in the lower airways of patients with severe COVID-19 that exhibit gene pathways associated with increased apoptosis and decreased proliferation. Our data demonstrate a dynamic production of IFNs in SARS-CoV-2-infected patients and show IFNs play opposing roles at distinct anatomical sites., An in-depth analysis of interferons in COVID-19 reveals differences in their roles based on anatomical location, viral load, age and disease severity. In the upper respiratory tract, high levels of IFN-III are protective and result in mild disease in spite of higher SARS-CoV-2 viral burden while the lower airways of patients with severe COVID-19 demonstrate elevated IFN-I and III, cell death and a reduction in interferon stimulated genes.

Growing evidence suggests that conventional dendritic cells (cDCs) undergo aberrant maturation in COVID‐19, which negatively affects T‐cell activation. The presence of effector T cells in patients with mild disease and dysfunctional T cells in severely ill patients suggests that adequate T‐cell responses limit disease severity. Understanding how cDCs cope with SARS‐CoV‐2 can help elucidate how protective immune responses are generated. Here, we report that cDC2 subtypes exhibit similar infection‐induced gene signatures, with the upregulation of interferon‐stimulated genes and interleukin (IL)‐6 signaling pathways. Furthermore, comparison of cDCs between patients with severe and mild disease showed severely ill patients to exhibit profound downregulation of genes encoding molecules involved in antigen presentation, such as MHCII, TAP, and costimulatory proteins, whereas we observed the opposite for proinflammatory molecules, such as complement and coagulation factors. Thus, as disease severity increases, cDC2s exhibit enhanced inflammatory properties and lose antigen presentation capacity. Moreover, DC3s showed upregulation of anti‐apoptotic genes and accumulated during infection. Direct exposure of cDC2s to the virus in vitro recapitulated the activation profile observed in vivo. Our findings suggest that SARS‐CoV‐2 interacts directly with cDC2s and implements an efficient immune escape mechanism that correlates with disease severity by downregulating crucial molecules required for T‐cell activation. This article is protected by copyright. All rights reserved

Routine biochemical and hematological tests have been reported to be useful in the stratification and prognostication of pediatric and adult patients with diagnosed coronavirus disease (COVID-19), correlating with poor outcomes such as the need for mechanical ventilation or intensive care, progression to multisystem organ failure, and/or death. While these tests are already well established in most clinical laboratories, there is still debate regarding their clinical value in the management of COVID-19, particularly in pediatrics, as well as the value of composite clinical risk scores in COVID-19 prognostication. This document by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Task Force on COVID-19 provides interim guidance on: (A) clinical indications for testing, (B) recommendations for test selection and interpretation, (C) considerations in test interpretation, and (D) current limitations of biochemical/hematological monitoring of COVID-19 patients. These evidence-based recommendations will provide practical guidance to clinical laboratories worldwide, underscoring the contribution of biochemical and hematological testing to our collective pandemic response.

Background: The role of microbiota in Lynch syndrome (LS) is still under debate. We compared oral and fecal microbiota of LS saliva and stool samples with normal healthy controls (NHC). Methods: Total DNA was purified from feces and saliva to amplify the V3–V4 region of the 16s rRNA gene. Sequences with a high-quality score and length >250 bp were used for taxonomic analysis with QIIME software. Results: Compared to NHC, LS fecal samples demonstrated a statistically significant increase of Bacteroidetes and Proteobacteria and a significant decrease of Firmicutes at the phylum level and of Ruminococcaceae at the family level. Moreover, LS oral samples exhibited a statistically significant increase of Veillonellaceae and Leptotrichiaceae and a statistically significant decrease of Pasteurellaceae. A beta-diversity index allowed differentiation of the two groups. Conclusions: A peculiar microbial signature is associated with LS, similar to that of sporadic colorectal cancer and Crohn’s disease. These data suggest a possible role of proinflammatory bacteria in tumor development in a condition of genetic predisposition, such as LS.

Plenty of serologic tests for SARS-CoV-2 have been developed so far, thus documenting the importance of evaluating the relevant features of the immune response to this viral agent. The performance of these assays is currently under investigation. Amongst them, LIAISON® SARS-CoV-2 S1/S2 IgG by DiaSorin and Elecsys Anti-SARS-CoV-2 cobas® by Roche are currently used by laboratory medicine hospital departments in Italy and many other countries. In the present study, we have firstly compared two serologic tests on serum samples collected at two different time points from forty-six laboratory-confirmed COVID-19 subjects. Secondly, eighty-five negative serum samples collected before the SARS-CoV-2 pandemic were analyzed. Thirdly, possible correlations between antibody levels and the resulting neutralizing activity against a clinical isolate of SARS-CoV-2 were evaluated. Results revealed that both tests are endowed with low sensitivity on the day of hospital admission, which increased to 97.8 and 100% for samples collected after 15 days for DiaSorin and Roche tests, respectively. The specificity of the two tests ranges from 96.5 to 100%, respectively. Importantly, a poor direct correlation between antibody titers and neutralizing activity levels was evidenced in the present study.

The global coronavirus disease 2019 (COVID-19) has presented major challenges for clinical laboratories, from initial diagnosis to patient monitoring and treatment. Initial response to this pandemic involved the development, production, and distribution of diagnostic molecular assays at an unprecedented rate, leading to minimal validation requirements and concerns regarding their diagnostic accuracy in clinical settings. In addition to molecular testing, serological assays to detect antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are now becoming available from numerous diagnostic manufacturers. In both cases, the lack of peer-reviewed data and regulatory oversight, combined with general misconceptions regarding their appropriate use, have highlighted the importance of laboratory professionals in robustly validating and evaluating these assays for appropriate clinical use. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Task Force on COVID-19 has been established to synthesize up-to-date information on the epidemiology, pathogenesis, and laboratory diagnosis and monitoring of COVID-19, as well as to develop practical recommendations on the use of molecular, serological, and biochemical tests in disease diagnosis and management. This review summarizes the latest evidence and status of molecular, serological, and biochemical testing in COVID-19 and highlights some key considerations for clinical laboratories operating to support the global fight against this ongoing pandemic. Confidently this consolidated information provides a useful resource to laboratories and a reminder of the laboratory’s critical role as the world battles this unprecedented crisis.

Objectives The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Task Force on COVID-19 conducted a global survey to understand how biochemistry laboratories manage the operational challenges during the coronavirus disease 2019 (COVID-19) pandemic. Materials and methods An electronic survey was distributed globally to record the operational considerations to mitigate biosafety risks in the laboratory. Additionally, the laboratories were asked to indicate the operational challenges they faced. Results A total of 1210 valid submissions were included in this analysis. Most of the survey participants worked in hospital laboratories. Around 15% of laboratories restricted certain tests on patients with clinically suspected or confirmed COVID-19 over biosafety concerns. Just over 10% of the laboratories had to restrict their test menu or services due to resource constraints. Approximately a third of laboratories performed temperature monitoring, while two thirds of laboratories increased the frequency of disinfection. Just less than 50% of the laboratories split their teams. The greatest reported challenge faced by laboratories during the COVID-19 pandemic is securing sufficient supplies of personal protective equipment (PPE), analytical equipment, including those used at the point of care, as well as reagents, consumables and other laboratory materials. This was followed by having inadequate staff, managing their morale, anxiety and deployment. Conclusions The restriction of tests and services may have undesirable clinical consequences as clinicians are deprived of important information to deliver appropriate care to their patients. Staff rostering and biosafety concerns require longer-term solutions as they are crucial for the continued operation of the laboratory during what may well be a prolonged pandemic.

Objectives A global survey was conducted by the IFCC Task Force on COVID-19 to better understand how general biochemistry laboratories manage the pre-analytical, analytical and post-analytical processes to mitigate biohazard risks during the coronavirus disease 2019 (COVID-19) pandemic. Methods An electronic survey was developed to record the general characteristics of the laboratory, as well as the pre-analytical, analytical, post-analytical and operational practices of biochemistry laboratories that are managing clinical samples of patients with COVID-19. Results A total of 1210 submissions were included in the analysis. The majority of responses came from hospital central/core laboratories that serve hospital patient groups and handle moderate daily sample volumes. There has been a decrease in the use of pneumatic tube transport, increase in hand delivery and increase in number of layers of plastic bags for samples of patients with clinically suspected or confirmed COVID-19. Surgical face masks and gloves are the most commonly used personal protective equipment (PPE). Just >50% of the laboratories did not perform an additional decontamination step on the instrument after analysis of samples from patients with clinically suspected or confirmed COVID-19. A fifth of laboratories disallowed add-on testing on these samples. Less than a quarter of laboratories autoclaved their samples prior to disposal. Conclusions The survey responses showed wide variation in pre-analytical, analytical and post-analytical practices in terms of PPE adoption and biosafety processes. It is likely that many of the suboptimal biosafety practices are related to practical local factors, such as limited PPE availability and lack of automated instrumentation.

Interferons interfere with lung repair Interferons (IFNs) are central to antiviral immunity. Viral recognition elicits IFN production, which in turn triggers the transcription of IFN-stimulated genes (ISGs), which engage in various antiviral functions. Type I IFNs (IFN-α and IFN-β) are widely expressed and can result in immunopathology during viral infections. By contrast, type III IFN (IFN-λ) responses are primarily restricted to mucosal surfaces and are thought to confer antiviral protection without driving damaging proinflammatory responses. Accordingly, IFN-λ has been proposed as a therapeutic in coronavirus disease 2019 (COVID-19) and other such viral respiratory diseases (see the Perspective by Grajales-Reyes and Colonna). Broggi et al. report that COVID-19 patient morbidity correlates with the high expression of type I and III IFNs in the lung. Furthermore, IFN-λ secreted by dendritic cells in the lungs of mice exposed to synthetic viral RNA causes damage to the lung epithelium, which increases susceptibility to lethal bacterial superinfections. Similarly, using a mouse model of influenza infection, Major et al. found that IFN signaling (especially IFN-λ) hampers lung repair by inducing p53 and inhibiting epithelial proliferation and differentiation. Complicating this picture, Hadjadj et al. observed that peripheral blood immune cells from severe and critical COVID-19 patients have diminished type I IFN and enhanced proinflammatory interleukin-6– and tumor necrosis factor-α–fueled responses. This suggests that in contrast to local production, systemic production of IFNs may be beneficial. The results of this trio of studies suggest that the location, timing, and duration of IFN exposure are critical parameters underlying the success or failure of therapeutics for viral respiratory infections. Science , this issue p. 706 , p. 712 , p. 718 ; see also p. 626

Comprehensive characterization of the urinary and urothelium-bound microbiomes in bladder cancer (BCa) and healthy state is essential to understand how these local microbiomes may play a role in BCa tumorigenesis and response to therapy, as well as to explain sex-based differences in BCa pathobiology. Performing 16 s rDNA microbiome analysis on 166 samples (urine and paired bladder tissues) from therapy-naive BCa patients undergoing radical cystectomy and healthy controls, we defined (1) sex-specific microbiome differences in the urine and bladder tissue, and (2) representativeness of the tissue microenvironment by the voided urinary microbiome. The genus Klebsiella was more common in the urine of female BCa patients versus healthy controls, while no clinically relevant bacteria were found differently enriched in men. In tissues, the genus Burkholderia was more abundant in the neoplastic versus the non-neoplastic tissue in both sexes, suggesting a potential role in BCa pathobiology. Lastly, we found that the urinary microbiome shares >80% of the bacterial families present in the paired bladder tissue, making the urinary microbiome a fair proxy of the tissue bacterial environment. Patient summary We identified specific bacteria present in the urine and tissues of male and female bladder cancer patients. These novel data represent a first step toward understanding the influence of the bladder microbiome on the development of bladder cancer and on the response to intravesical and systemic therapies.

Next-generation sequencing techniques have enabled identification of the microorganisms colonizing mucosal tissues. The International Congress "MicrobiotaMi 2020" (Milan, February 2020) will focus on the mechanisms of microbiota-related functions in health and disease and their clinical application.

Infections are a major cause of morbidity and mortality in hematological patients. We prospectively tested a new molecular assay (Verigene®) in 79 consecutive hematological patients, with sepsis by gram-negative bacteria. A total of 82 gram-negative microorganisms were isolated by blood cultures, of which 76 cases were mono-microbial. Considering the bacteria detectable by the system, the concordance with standard blood cultures was 100%. Resistance genes were detected in 20 of the isolates and 100% were concordant with the phenotypic antibiotic resistance. Overall, this new assay correctly identified 66/82 of all the gram-negative pathogens, yielding a general sensitivity of 80.5%, and providing information on genetic antibiotic resistance in a few hours. This new molecular assay could ameliorate patient management, resulting in a more rational use of antibiotics.

A 74-year old woman presented to the emergency room with difficulties in speaking and standing. The patient was not able to answer the clinicians’ questions, but her husband explained that she had been ill with a fever and a worsening cough for the last 2 weeks and had taken a fall 2 days before

Answer: Streptococcus pneumoniae. The figure in the photo quiz shows an uncommon Gram stain of a Streptococcus pneumoniae isolate. The subcultured isolate did not grow either on blood agar (BBL Columbia agar with 5% sheep blood; Becton Dickinson) or on chocolate agar (chocolate agar-PolyViteX; bioM

Herpes simplex virus 1 (HSV-1) and HSV-2 can evade serum antibody-mediated neutralization through cell-to-cell transmission mechanisms, which represent one of the central steps in disease reactivation. To address the role of humoral immunity in controlling HSV-1 and HSV-2 replication, we analyzed serum samples from 44 HSV-1 and HSV-2 seropositive subjects by evaluating (i) their efficiency in binding both the purified viral particles and recombinant gD and gB viral glycoproteins, (ii) their neutralizing activity, and (iii) their capacity to inhibit the cell-to-cell virus passage in vitro. All of the sera were capable of binding gD, gB, and whole virions, and all sera significantly neutralized cell-free virus. However, neither whole sera nor purified serum IgG fraction was able to inhibit significantly cell-to-cell virus spreading in in vitro post-virus-entry infectious assays. Conversely, when spiked with an already described anti-gD human monoclonal neutralizing antibody capable of inhibiting HSV-1 and -2 cell-to-cell transmission, each serum boosted both its neutralizing and post-virus-entry inhibitory activity, with no interference exerted by serum antibody subpopulations. IMPORTANCE Despite its importance in the physiopathology of HSV-1 and -2 infections, the cell-to-cell spreading mechanism is still poorly understood. The data shown here suggest that infection-elicited neutralizing antibodies capable of inhibiting cell-to-cell virus spread can be underrepresented in most infected subjects. These observations can be of great help in better understanding the role of humoral immunity in controlling virus reactivation and in the perspective of developing novel therapeutic strategies, studying novel correlates of protection, and designing effective vaccines.

Background: Despite being actively investigated for several other malignancies, the impact of the microbiome on tumorigenesis, response to therapy, and patient outcomes has not been thoroughly assessed in urothelial bladder cancer (BCa). Of note, the relationship between urinary and bladder tissue microbiomes has not yet been investigated. Herein, we aimed to assess: i) the gender-specific microbiome differences in the urine and bladder tissue; ii) the extent to which paired urine samples mirror the bladder tissue-associated microbiome. Methods: A total of 166 biologic samples were analyzed: morning, mid-stream voided urines from 49 therapy-naïve patients (36 males, 13 females) undergoing radical cystectomy for MIBC and from 59 age-matched healthy controls (34 males, 25 females), plus bladder tissue specimens (paired BCa/non-BCa tissues) of 29 patients (21 males, 8 females). Exclusion criteria included history of recurrent urinary tract infections, positive urine dipstick test at the time of sample collection, recent antibiotic therapy, history of intravesical or neoadjuvant treatment for BCa. Microbiome was analyzed by amplicon-based approach. Sequences with a high-quality score >Q30 and length >250bp were used for the taxonomic analysis and were processed using the QIIME (Quantitative Insights Into Microbial Ecology) software package (v1.9.1). Intra- and interdiversity between samples and identification of taxonomic biomarkers by using the linear discriminant analysis (LDA) effect size (LEfSe) were considered significant at p-value ≤0.05. Results: Comparing neoplastic vs. non-neoplastic paired tissues, beta-diversity did not show distinct clustering of the samples. At the taxonomic level, the genus Burkholderia was enriched in the neoplastic specimens in both genders. When we compared urines of patients vs. controls, the male and female urinary microbiome was dominated by members of the three major bacterial phyla Proteobacteria, Firmicutes, and Bacteroidetes. At lower taxonomic levels, the bacterial taxa differently enriched in BCa vs. controls were gender-specific, with 18 and 36 bacterial taxa differentially represented in BCa and controls in men and women, respectively. Comparing paired urinary and tissue-associated microbiome, we defined the “common BCa microbiome,” representing 34 and 16 bacterial families (>80% of total relative abundance) shared in the urines and tissues of male and female patients, respectively. Conclusions: We provide novel characterization of the gender-specific microbiome in the urines and paired bladder tissues of BCa patients. A gender-specific “common BCa microbiome” was detailed, highlighting potentially actionable bacterial taxa. Citation Format: Filippo Pederzoli, Roberto Ferrarese, Virginia Amato, Irene Locatelli, Elisa Alchera, Roberta Lucianò, Manuela Nebuloni, Alberto Briganti, Andrea Gallina, Valentina Murdica, Renzo Colombo, Andrea Necchi, Massimo Clementi, Francesco Montorsi, Nicasio Mancini, Andrea Salonia, Massimo Alfano. Intratissutal and urinary microbiome in therapy-naive bladder cancer patients: Definition of a gender-specific common microbiome [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A33.

PN-SIA28 is a human monoclonal antibody (Hu-MAb) targeting highly conserved epitopes within the stem portion of the influenza virus hemagglutinin (HA) (N. Clementi, et al, PLoS One 6:e28001, 2011, http://dx.doi.org/10.1371/journal.pone.0028001). Previous in vitro studies demonstrated PN-SIA28 neutralizing activities against phylogenetically divergent influenza A subtypes. In this study, the protective activity of PN-SIA28 was evaluated in mice inoculated with lethal influenza A/WSN/33 (H1N1), A/Quebec/144147/09 (H1N1)pdm09, and A/Victoria/3/75 (H3N2) viruses. At 24 h postinoculation (p.i.), animals received PN-SIA28 intraperitoneally (1 or 10 mg/kg of body weight) or 10 mg/kg of unrelated Hu-MAb (mock). Body weight loss and mortality rate (MR) were recorded for 14 days postinfection (p.i.). Lung viral titers (LVT) were determined at day 5 p.i. In A/WSN/33 (H1N1)-infected groups, all untreated and mock-receiving mice died, whereas MRs of 87.5% and 25% were observed in mice that received PN-SIA28 1 and 10 mg/kg, respectively. In influenza A(H1N1) pdm09-infected groups, an MR of 75% was recorded for untreated and mock-treated groups, whereas the PN-SIA28 1-mg/kg and 10-mg/kg groups had rates of 62.5% and 0%, respectively. In A/Victoria/3/75 (H3N2)-infected animals, untreated and mock-treated animals had MRs of 37.5% and 25%, respectively, and no mortalities were recorded after PN-SIA28 treatments. Accordingly, PN-SIA28 treatments significantly reduced weight losses and resulted in a ≥1-log reduction in LVT compared to the control in all infection groups. This study confirms that antibodies targeting highly conserved epitopes in the influenza HA stem region, like PN-SIA28, not only neutralize influenza A viruses of clinically relevant subtypes in vitro but also, more importantly, protect from a lethal influenza virus challenge in vivo. Copyright © 2015, American Society for Microbiology. All Rights Reserved. PN-SIA28 is a human monoclonal antibody (Hu-MAb) targeting highly conserved epitopes within the stem portion of the influenza virus hemagglutinin (HA) (N. Clementi, et al, PLoS One 6:e28001, 2011, http://dx.doi.org/10.1371/journal.pone.0028001). Previous in vitro studies demonstrated PN-SIA28 neutralizing activities against phylogenetically divergent influenza A subtypes. In this study, the protective activity of PN-SIA28 was evaluated in mice inoculated with lethal influenza A/WSN/33 (H1N1), A/Quebec/144147/09 (H1N1)pdm09, and A/Victoria/3/75 (H3N2) viruses. At 24 h postinoculation (p.i.), animals received PN-SIA28 intraperitoneally (1 or 10 mg/kg of body weight) or 10 mg/kg of unrelated Hu-MAb (mock). Body weight loss and mortality rate (MR) were recorded for 14 days postinfection (p.i.). Lung viral titers (LVT) were determined at day 5 p.i. In A/WSN/33 (H1N1)-infected groups, all untreated and mock-receiving mice died, whereas MRs of 87.5% and 25% were observed in mice that received PN-SIA28 1 and 10 mg/kg, respectively. In influenza A(H1N1) pdm09-infected groups, an MR of 75% was recorded for untreated and mock-treated groups, whereas the PN-SIA28 1-mg/kg and 10-mg/kg groups had rates of 62.5% and 0%, respectively. In A/Victoria/3/75 (H3N2)-infected animals, untreated and mock-treated animals had MRs of 37.5% and 25%, respectively, and no mortalities were recorded after PN-SIA28 treatments. Accordingly, PN-SIA28 treatments significantly reduced weight losses and resulted in a ≥1-log reduction in LVT compared to the control in all infection groups. This study confirms that antibodies targeting highly conserved epitopes in the influenza HA stem region, like PN-SIA28, not only neutralize influenza A viruses of clinically relevant subtypes in vitro but also, more importantly, protect from a lethal influenza virus challenge in vivo.

Despite the clinical need of novel and safe anti-herpetic compounds effective for treating both primary infections and reactivations of Herpes Simplex Virus type 1 (HSV-1) and type 2 (HSV-2), the development of novel antivirals approved for clinical administration has been limited in the last decades to improvements of nucleoside analogues compounds. In this context, targeting different steps of the herpesvirus life cycle, including entry and cell-to-cell infection, can represent an important starting point for obtaining more efficient infection inhibition, and for overcoming both drug resistance and toxicity. Under these perspectives, testing possible synergy between drugs currently in clinical use and novel immunotherapeutics, such as neutralizing human monoclonal antibodies, represents a fascinating option. In the study here described we tested for the first-time possible combinations of inhibitors of Herpesvirus DNA synthesis and a human neutralizing IgG able to block also cell-to-cell infection, by analysing experimental results with different mathematical models. The present study clearly highlights the synergism between all anti-herpetic drugs tested in combination with the mAb; this strongly suggests possible reduction of anti-herpetic drugs combined with the IgG for overcoming drug-related side effects, as indicated by Drug Reduction Index.