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Objective Early stages with streptococcal necrotizing soft tissue infections (NSTIs) are often difficult to discern from cellulitis. Increased insight into inflammatory responses in streptococcal disease may guide correct interventions and discovery of novel diagnostic targets. Methods Plasma levels of 37 mediators, leucocytes and CRP from 102 patients with β-hemolytic streptococcal NSTI derived from a prospective Scandinavian multicentre study were compared to those of 23 cases of streptococcal cellulitis. Hierarchical cluster analyses were also performed. Results Differences in mediator levels between NSTI and cellulitis cases were revealed, in particular for IL-1β, TNFα and CXCL8 (AUC >0.90). Across streptococcal NSTI etiologies, eight biomarkers separated cases with septic shock from those without, and four mediators predicted a severe outcome. Conclusion Several inflammatory mediators and wider profiles were identified as potential biomarkers of NSTI. Associations of biomarker levels to type of infection and outcomes may be utilized to improve patient care and outcomes. publishedVersion

COVID-19 affects multiple organ-systems. Recent studies have indicated perturbations in the circulating metabolome linked to COVID-19 severity. However, several questions pertain with respect to the metabolome in COVID-19. We performed an in-depth assessment of 1129 unique metabolites in 27 hospitalized COVID-19 patients and integrated results with large-scale proteomic and immunology data to capture multi-organ system perturbations. More than half of the detected metabolic alterations in COVID-19 were driven by patient-specific confounding factors ranging from comorbidities to xenobiotic substances. Systematically adjusting for this, a COVID-19 specific metabolic imprint was defined which, over time, underwent a switch in response to SARS-CoV-2 seroconversion. Integration of the COVID-19 metabolome with clinical, cellular, molecular and immunological severity scales further revealed a network of metabolic trajectories aligned with multiple pathways for immune activation, and organ damage including neurological inflammation and damage. Altogether, this resource refines our understanding of the multi organ-system perturbations in severe COVID-19 patients. This article is protected by copyright. All rights reserved.

Introduction: Necrotizing Soft Tissue Infections (NSTI) are severe infections with high mortality affecting a heterogeneous patient population. There is a need for a clinical decision support system which predicts outcomes and provides treatment recommendations early in the disease course. Methods: To identify relevant clinical needs, interviews with eight medical professionals (surgeons, intensivists, general practitioner, emergency department physician) were conducted. This resulted in 24 unique questions. Mortality was selected as first endpoint to develop a machine learning (Random Forest) based prediction model. For this purpose, data from the prospective, international INFECT cohort (N = 409) was used. Results: Applying a feature selection procedure based on an unsupervised algorithm (Boruta) to the > 1000 variables available in INFECT, including baseline, and both NSTI specific and NSTI non-specific clinical data yielded sixteen predictive parameters available on or prior to the first day on the intensive care unit (ICU). Using these sixteen variables 30-day mortality could be accurately predicted (AUC = 0.91, 95% CI 0.88–0.96). Except for age, all variables were related to sepsis (e.g. lactate, urine production, systole). No NSTI-specific variables were identified. Predictions significantly outperformed the SOFA score(p < 0.001, AUC = 0.77, 95% CI 0.69–0.84) and exceeded but did not significantly differ from the SAPS II score (p = 0.07, AUC = 0.88, 95% CI 0.83–0.92). The developed model proved to be stable with AUC > 0.8 in case of high rates of missing data (50% missing) or when only using very early (

Background Necrotizing soft-tissue infections (NSTI) are life-threatening conditions often caused by β-hemolytic streptococci, group A Streptococcus (GAS) in particular. Optimal treatment is contentious. The INFECT cohort includes the largest set of prospectively enrolled streptococcal NSTI cases to date. Methods From the INFECT cohort of 409 adults admitted with NSTI to 5 clinical centers in Scandinavia, patients culture-positive for GAS or Streptococcus dysgalactiae (SD) were selected. Risk factors were identified by comparison with a cohort of nonnecrotizing streptococcal cellulitis. The impact of baseline factors and treatment on 90-day mortality was explored using Lasso regression. Whole-genome sequencing of bacterial isolates was used for emm typing and virulence gene profiling. Results The 126 GAS NSTI cases and 27 cases caused by SD constituted 31% and 7% of the whole NSTI cohort, respectively. When comparing to nonnecrotizing streptococcal cellulitis, streptococcal NSTI was associated to blunt trauma, absence of preexisting skin lesions, and a lower body mass index. Septic shock was significantly more frequent in GAS (65%) compared to SD (41%) and polymicrobial, nonstreptococcal NSTI (46%). Age, male sex, septic shock, and no administration of intravenous immunoglobulin (IVIG) were among factors associated with 90-day mortality. Predominant emm types were emm1, emm3, and emm28 in GAS and stG62647 in SD. Conclusions Streptococcal NSTI was associated with several risk factors, including blunt trauma. Septic shock was more frequent in NSTI caused by GAS than in cases due to SD. Factors associated with mortality in GAS NSTI included age, septic shock, and no administration of IVIG., This prospective study of streptococcal necrotizing soft-tissue infections comprised 126 Streptococcus pyogenes (GAS) cases and 27 Streptococcus dysgalactiae cases. Among the GAS cases, several factors were associated with mortality, including age, septic shock and no administration of intravenous immunoglobulin.

Background Necrotizing skin and soft-tissue infections (NSTI) are rare but potentially life-threatening and disabling infections that often require intensive care unit admission. Objectives To review all aspects of care for a critically ill individual with NSTI. Sources Literature search using Medline and Cochrane library, multidisciplinary panel of experts. Content The initial presentation of a patient with NSTI can be misleading, as features of severe systemic toxicity can obscure sometimes less impressive skin findings. The infection can spread rapidly, and delayed surgery worsens prognosis, hence there is a limited role for additional imaging in the critically ill patient. Also, the utility of clinical scores is contested. Prompt surgery with aggressive debridement of necrotic tissue is required for source control and allows for microbiological sampling. Also, prompt administration of broad-spectrum antimicrobial therapy is warranted, with the addition of clindamycin for its effect on toxin production, both in empirical therapy, and in targeted therapy for monomicrobial group A streptococcal and clostridial NSTI. The role of immunoglobulins and hyperbaric oxygen therapy remains controversial. Implications Close collaboration between intensive care, surgery, microbiology and infectious diseases, and centralization of care is fundamental in the approach to the severely ill patient with NSTI. As many aspects of management of these rare infections are supported by low-quality data only, multicentre trials are urgently needed.

Background Necrotizing soft tissue infections (NSTIs) are severe diseases with high morbidity and mortality. The diagnosis is challenging. Several guidelines recommend tissue biopsies as an adjunct diagnostic in routine management, but neither biopsy sampling nor classification is standardized or validated. We studied the quality of tissue biopsy examination as part of routine diagnostics in NSTIs. Methods This was a retrospective cohort study of adult patients undergoing surgery due to suspected NSTIs in which tissue biopsy was taken as part of routine management. Clinical data were reviewed. The biopsies were evaluated according to a proposed histopathologic classification system and independently assessed by 2 pathologists. Interrater reliability and diagnostic accuracy were determined. Results Tissue biopsies from 75 patients were examined, 55 NSTIs and 20 non-NSTIs cases. The cohorts were similar in clinical characteristics. Interrater reliability for histopathologic staging was moderate (0.53) and fair (0.37) for diagnosis. The sensitivity of histologic diagnosis was 75% and the specificity 80%. The positive predictive value was 91% and the negative predictive value 53%. Necrotizing Infection Clinical Composite Endpoint (NICCE) success was associated with a more severe histological stage, achieved by 42% and 71% of the cases in stage 1 and 2, respectively (P = .046). Conclusions Our findings suggest that tissue biopsies have low clinical accuracy. The interrater reliability among experienced pathologists is only fair to moderate. A histopathologically more severe stage was associated with favorable outcome. These findings discourage the use of histopathologic evaluation as part of contemporary management of patients with suspected NSTI.

Biofilm formation has been demonstrated in muscle and soft tissue samples from patients with necrotizing soft tissue infection (NSTI) caused byBacterial isolates and clinical data were obtained from NSTI patients enrolled in a multicenter prospective observational study. Biofilm forming capacity was determined using a microtiter plate assay.Among 57 cases, the three most frequently encounteredThe biofilm forming capacity of

Biofilm has recently been highlighted as a complicating feature of necrotizing soft tissue infections (NSTI) caused by Streptococcus pyogenes (i.e., group A Streptococcus [GAS]) contributing to a persistence of bacteria in tissue despite prolonged antibiotic therapy. Here, we assessed the standard treatment of benzylpenicillin and clindamycin with or without rifampin in a tissue-like setting. Antibiotic efficacy was evaluated by CFU determination in a human organotypic skin model infected for 24 or 48 h with GAS strains isolated from NSTI patients. Antibiotic effect was also evaluated by microcalorimetric metabolic assessment in in vitro infections of cellular monolayers providing continuous measurements over time. Adjunctive rifampin resulted in enhanced antibiotic efficacy of bacterial clearance in an organotypic skin tissue model, 97.5% versus 93.9% (P = 0.006). Through microcalorimetric measurements, adjunctive rifampin resulted in decreased metabolic activity and extended lag phase for all clinical GAS strains tested (P

Mucosa-associated invariant T (MAIT) cells recognize bacterial riboflavin metabolite Ags presented by MHC class Ib–related protein (MR1) and play important roles in immune control of microbes that synthesize riboflavin. This includes the pathobiont Staphylococcus aureus, which can also express a range of virulence factors, including the secreted toxin leukocidin ED (LukED). In this study, we found that human MAIT cells are hypersensitive to LukED-mediated lysis and lost on exposure to the toxin, leaving a T cell population devoid of MAIT cells. The cytolytic effect of LukED on MAIT cells was rapid and occurred at toxin concentrations lower than those required for toxicity against conventional T cells. Furthermore, this coincided with high MAIT cell expression of CCR5, and loss of these cells was efficiently inhibited by the CCR5 inhibitor maraviroc. Interestingly, exposure and preactivation of MAIT cells with IL-12 and IL-18, or activation via TCR triggering, partially protected from LukED toxicity. Furthermore, analysis of NK cells indicated that LukED targeted the mature cytotoxic CD57(+) NK cell subset in a CCR5-independent manner. Overall, these results indicate that LukED efficiently eliminates immune cells that can respond rapidly to S. aureus in an innate fashion without the need for clonal expansion, and that MAIT cells are exceptionally vulnerable to this toxin. Thus, the findings support a model where LukED secretion may allow S. aureus to avoid recognition by the rapid cell-mediated responses mediated by MAIT cells and NK cells.

Mucosa-associated invariant T (MAIT) cells recognize bacterial riboflavin metabolite antigens presented by MR1 and play an important role in antimicrobial immune defense. Staphylococcus aureus is a pathobiont expressing a range of virulence factors including the secreted toxin Leukocidin ED (LukED), which binds to certain chemokine receptors and causes cell death by osmolysis. Here, we investigated the effect of LukED on subsets of human T cells and NK cells that are involved in the early innate response to infection. MAIT cells were strikingly hypersensitive to LukED-mediated lysis and rapidly lost from the peripheral blood T cell pool upon exposure to the toxin, leaving a T cell population devoid of MAIT cells. The cytolytic effect of LukED on MAIT cells was rapid, occurred at lower LukED concentration compared to effects on the overall T cell pool, and coincided with extraordinarily high and uniform expression of CCR5. Furthermore, loss of MAIT cells was efficiently inhibited by the CCR5 inhibitor Maraviroc. Interestingly, pre-activation of MAIT cells with IL-12 and IL-18 also partially rescued these cells from LukED toxicity. Among NK cells, LukED targeted the more mature and cytotoxic CD57+ NK cell subset in a CXCR1-dependent manner. Overall, these results indicate that LukED efficiently eliminates cells of the human immune system that have the capacity to respond rapidly to S. aureus in an innate fashion, and that MAIT cells are exceptionally vulnerable to this toxin. Thus, the findings support a model where LukED functions as a S. aureus immune evasion mechanism to avoid recognition by the rapid cell-mediated responses mediated by MAIT cells and NK cells.

Necrotizing soft-tissue infections (NSTIs) have multiple causes, risk factors, anatomical locations, and pathogenic mechanisms. In patients with NSTI, circulating metabolites may serve as a substrate having impact on bacterial adaptation at the site of infection. Metabolic signatures associated with NSTI may reveal the potential to be useful as diagnostic and prognostic markers and novel targets for therapy. This study used untargeted metabolomics analyses of plasma from NSTI patients (n = 34) and healthy (noninfected) controls (n = 24) to identify the metabolic signatures and connectivity patterns among metabolites associated with NSTI. Metabolite-metabolite association networks were employed to compare the metabolic profiles of NSTI patients and noninfected surgical controls. Out of 97 metabolites detected, the abundance of 33 was significantly altered in NSTI patients. Analysis of metabolite-metabolite association networks showed a more densely connected network: specifically, 20 metabolites differentially connected between NSTI and controls. A selected set of significantly altered metabolites was tested in vitro to investigate potential influence on NSTI group A streptococcal strain growth and biofilm formation. Using chemically defined media supplemented with the selected metabolites, ornithine, ribose, urea, and glucuronic acid, revealed metabolite-specific effects on both bacterial growth and biofilm formation. This study identifies for the first time an NSTI-specific metabolic signature with implications for optimized diagnostics and therapies.

Since the outset of the COVID-19 pandemic, increasing evidence suggests that the innate immune responses play an important role in the disease development. A dysregulated inflammatory state has been proposed as key driver of clinical complications in COVID-19, with a potential detrimental role of granulocytes. However, a comprehensive phenotypic description of circulating granulocytes in SARS-CoV-2-infected patients is lacking. In this study, we used high-dimensional flow cytometry for granulocyte immunophenotyping in peripheral blood collected from COVID-19 patients during acute and convalescent phases. Severe COVID-19 was associated with increased levels of both mature and immature neutrophils, and decreased counts of eosinophils and basophils. Distinct immunotypes were evident in COVID-19 patients, with altered expression of several receptors involved in activation, adhesion and migration of granulocytes (e.g. CD62L, CD11a/b, CD69, CD63, CXCR4). Paired sampling revealed recovery and phenotypic restoration of the granulocytic signature in the convalescent phase. The identified granulocyte immunotypes correlated with distinct sets of soluble inflammatory markers supporting pathophysiologic relevance. Furthermore, clinical features, including multi-organ dysfunction and respiratory function, could be predicted using combined laboratory measurements and immunophenotyping. This study provides a comprehensive granulocyte characterization in COVID-19 and reveals specific immunotypes with potential predictive value for key clinical features associated with COVID-19.SignificanceAccumulating evidence shows that granulocytes are key modulators of the immune response to SARS-CoV-2 infection and their dysregulation could significantly impact COVID-19 severity and patient recovery after virus clearance. In the present study, we identify selected immune traits in neutrophil, eosinophil and basophil subsets associated to severity of COVID-19 and to peripheral protein profiles. Moreover, computational modeling indicates that the combined use of phenotypic data and laboratory measurements can effectively predict key clinical outcomes in COVID-19 patients. Finally, patient-matched longitudinal analysis shows phenotypic normalization of granulocyte subsets 4 months after hospitalization. Overall, in this work we extend the current understanding of the distinct contribution of granulocyte subsets to COVID-19 pathogenesis.

BACKGROUND Necrotizing soft-tissue infections (NSTIs) are rapidly progressing infections frequently complicated by septic shock and associated with high mortality. Early diagnosis is critical for patient outcome, but challenging due to vague initial symptoms. Here, we identified predictive biomarkers for NSTI clinical phenotypes and outcomes using a prospective multicenter NSTI patient cohort. METHODS Luminex multiplex assays were used to assess 36 soluble factors in plasma from NSTI patients with positive microbiological cultures (n = 251 and n = 60 in the discovery and validation cohorts, respectively). Control groups for comparative analyses included surgical controls (n = 20), non-NSTI controls (i.e., suspected NSTI with no necrosis detected upon exploratory surgery, n = 20), and sepsis patients (n = 24). RESULTS Thrombomodulin was identified as a unique biomarker for detection of NSTI (AUC, 0.95). A distinct profile discriminating mono- (type II) versus polymicrobial (type I) NSTI types was identified based on differential expression of IL-2, IL-10, IL-22, CXCL10, Fas-ligand, and MMP9 (AUC >0.7). While each NSTI type displayed a distinct array of biomarkers predicting septic shock, granulocyte CSF (G-CSF), S100A8, and IL-6 were shared by both types (AUC >0.78). Finally, differential connectivity analysis revealed distinctive networks associated with specific clinical phenotypes. CONCLUSIONS This study identifies predictive biomarkers for NSTI clinical phenotypes of potential value for diagnostic, prognostic, and therapeutic approaches in NSTIs. TRIAL REGISTRATION ClinicalTrials.gov NCT01790698. FUNDING Center for Innovative Medicine (CIMED); Region Stockholm; Swedish Research Council; European Union; Vinnova; Innovation Fund Denmark; Research Council of Norway; Netherlands Organisation for Health Research and Development; DLR Federal Ministry of Education and Research; and Swedish Children’s Cancer Foundation.

MAIT cell activation and decline in blood are associated with COVID-19 severity, features that dynamically recover in convalescence., Innate-like rapid responders Viral infections elicit host responses from conventional T cells, innate lymphoid cells, and innate-like lymphocyte subsets. Parrot et al. used blood from acute and convalescent COVID-19 patients to investigate how SARS-CoV-2 infection affects the innate-like mucosa-associated invariant T (MAIT) cells. Acute viral infection induced a profound decline in the number of blood MAIT cells and activation of the residual blood MAIT cells. The loss of circulating MAIT cells in acute COVID-19 patients coincided with enrichment of MAIT cells among T cells recovered from the respiratory tract. With convalescence, the number of blood MAIT cells and their activation status reverted toward normal. These findings indicate that circulating MAIT cells are mobilized early after SARS-CoV-2 infection and may contribute to both resolution and exacerbation of COVID-19–associated pneumonia., Severe coronavirus disease 2019 (COVID-19) is characterized by excessive inflammation of the lower airways. The balance of protective versus pathological immune responses in COVID-19 is incompletely understood. Mucosa-associated invariant T (MAIT) cells are antimicrobial T cells that recognize bacterial metabolites and can also function as innate-like sensors and mediators of antiviral responses. Here, we investigated the MAIT cell compartment in COVID-19 patients with moderate and severe disease, as well as in convalescence. We show profound and preferential decline in MAIT cells in the circulation of patients with active disease paired with strong activation. Furthermore, transcriptomic analyses indicated substantial MAIT cell enrichment and proinflammatory IL-17A bias in the airways. Unsupervised analysis identified MAIT cell CD69high and CXCR3low immunotypes associated with poor clinical outcome. MAIT cell levels normalized in the convalescent phase, consistent with dynamic recruitment to the tissues and later release back into the circulation when disease is resolved. These findings indicate that MAIT cells are engaged in the immune response against SARS-CoV-2 and suggest their possible involvement in COVID-19 immunopathogenesis.

Severe COVID-19 is characterized by excessive inflammation of the lower airways. The balance of protective versus pathological immune responses in COVID-19 is incompletely understood. Mucosa-associated invariant T (MAIT) cells are antimicrobial T cells that recognize bacterial metabolites, and can also function as innate-like sensors and mediators of antiviral responses. Here, we investigated the MAIT cell compartment in COVID-19 patients with moderate and severe disease, as well as in convalescence. We show profound and preferential decline in MAIT cells in circulation of patients with active disease paired with strong activation, as well as significant MAIT cell enrichment and pro-inflammatory IL-17A bias in the airways. Unsupervised analysis identified MAIT cell CD69high and CXCR3low immunotypes associated with poor clinical outcome. MAIT cell levels normalized in the convalescent phase, consistent with dynamic recruitment to the tissues and subsequent release with disease resolution. These findings indicate that MAIT cells are engaged in the immune response against SARS-CoV-2 and suggest their involvement in COVID-19 immunopathogenesis.One sentence summaryMAIT cells are strongly activated by SARS-CoV-2 infection in a manner associated with disease severity and outcome, they decline in blood, are enriched in the airways as a prominent IL-17A expressing subset, and dynamically recover in circulation during convalescence.

Monocytes and dendritic cells are crucial mediators of innate and adaptive immune responses during viral infection, but misdirected responses by these cells might contribute to immunopathology. A comprehensive map of the mononuclear phagocyte (MNP) landscape during SARS-CoV-2 infection and concomitant COVID-19 disease is lacking. We performed 25-color flow cytometry-analysis focusing on MNP lineages in SARS-CoV-2 infected patients with moderate and severe COVID-19. While redistribution of monocytes towards intermediate subset and decrease in circulating DCs occurred in response to infection, severe disease associated with appearance of Mo-MDSC-like cells and a higher frequency of pre-DC2. Furthermore, phenotypic alterations in MNPs, and their late precursors, were cell-lineage specific and in select cases associated with severe disease. Finally, unsupervised analysis revealed that the MNP profile, alone, could identify a cluster of COVID-19 non-survivors. This study provides a reference for the MNP response to clinical SARS-CoV-2 infection and unravel myeloid dysregulation associated with severe COVID-19.

Analyses of plasma collected pre- and postadministration of intravenous immunoglobulin (IVIG) from patients with group A Streptococcus necrotizing soft tissue infections demonstrated a negative correlation between IVIG dose and toxin-triggered T-cell proliferation (r = −.67, P Clinical Trials Registration. NCT 01790698 and NCT02111161.

Significance Bacterial toxins belonging to the family of superantigens are potent immunostimulatory antigens capable of activating T cells in a nonconventional manner. This results in an overzealous activation of immune cells and release of pathologic levels of pro-inflammatory cytokines, which underlies severe disease manifestations such as streptococcal toxic shock syndrome. Here, we provide evidence that mucosal-associated invariant T (MAIT) cells are major contributors to the overall cytokine response elicited by group A streptococci. Both streptococcal superantigens and surface-attached bacterial factors activate MAIT cells, but through different mechanisms. Furthermore, activated MAIT cells could be detected in patients during the acute phase of streptococcal toxic shock syndrome. Thus, this study identifies an actor and potential target for intervention in streptococcal toxic shock syndrome., Streptococcal toxic shock syndrome (STSS) is a rapidly progressing, life-threatening, systemic reaction to invasive infection caused by group A streptococci (GAS). GAS superantigens are key mediators of STSS through their potent activation of T cells leading to a cytokine storm and consequently vascular leakage, shock, and multiorgan failure. Mucosal-associated invariant T (MAIT) cells recognize MR1-presented antigens derived from microbial riboflavin biosynthesis and mount protective innate-like immune responses against the microbes producing such metabolites. GAS lack de novo riboflavin synthesis, and the role of MAIT cells in STSS has therefore so far been overlooked. Here we have conducted a comprehensive analysis of human MAIT cell responses to GAS, aiming to understand the contribution of MAIT cells to the pathogenesis of STSS. We show that MAIT cells are strongly activated and represent the major T cell source of IFNγ and TNF in the early stages of response to GAS. MAIT cell activation is biphasic with a rapid TCR Vβ2-specific, TNF-dominated response to superantigens and a later IL-12- and IL-18-dependent, IFNγ-dominated response to both bacterial cells and secreted factors. Depletion of MAIT cells from PBMC resulted in decreased total production of IFNγ, IL-1β, IL-2, and TNFβ. Peripheral blood MAIT cells in patients with STSS expressed elevated levels of the activation markers CD69, CD25, CD38, and HLA-DR during the acute compared with the convalescent phase. Our data demonstrate that MAIT cells are major contributors to the early cytokine response to GAS, and are therefore likely to contribute to the pathological cytokine storm underlying STSS.

A prominent feature of severe streptococcal infections is the profound inflammatory response that contributes to systemic toxicity. In sepsis the dysregulated host response involves both immunological and nonimmunological pathways. Here, we report a fatal case of an immunocompetent healthy female presenting with toxic shock and purpura fulminans caused by group B streptococcus (GBS; serotype III, CC19). The strain (LUMC16) was pigmented and hyperhemolytic. Stimulation of human primary cells with hyperhemolytic LUMC16 and STSS/NF-HH strains and pigment toxin resulted in a release of proinflammatory mediators, including tumor necrosis factor, interleukin (IL)-1β, and IL-6. In addition, LUMC16 induced blood clotting and showed factor XII activity on its surface, which was linked to the presence of the pigment. The expression of pigment was not linked to a mutation within the CovR/S region. In conclusion, our study shows that the hemolytic lipid toxin contributes to the ability of GBS to cause systemic hyperinflammation and interferes with the coagulation system.

Necrotizing soft tissue infections (NSTIs) are devastating infections caused by either a single pathogen, predominantly Streptococcus pyogenes, or by multiple bacterial species. A better understanding of the pathogenic mechanisms underlying these different NSTI types could facilitate faster diagnostic and more effective therapeutic strategies. Here, we integrate microbial community profiling with host and pathogen(s) transcriptional analysis in patient biopsies to dissect the pathophysiology of streptococcal and polymicrobial NSTIs. We observe that the pathogenicity of polymicrobial communities is mediated by synergistic interactions between community members, fueling a cycle of bacterial colonization and inflammatory tissue destruction. In S. pyogenes NSTIs, expression of specialized virulence factors underlies infection pathophysiology. Furthermore, we identify a strong interferon-related response specific to S. pyogenes NSTIs that could be exploited as a potential diagnostic biomarker. Our study provides insights into the pathophysiology of mono- and polymicrobial NSTIs and highlights the potential of host-derived signatures for microbial diagnosis of NSTIs., Necrotizing soft tissue infections (NSTIs) are caused by a single pathogen such as Streptococcus pyogenes or by multiple bacterial species. Here, the authors integrate microbial community profiling with host and pathogen transcriptional analysis in patient biopsies, and identify an interferon-related signature specific to S. pyogenes NSTIs.

Objectives Streptococcus pyogenes causes life-threatening invasive infections including necrotizing fasciitis (NF). Current treatment guidelines recommend the use of a cell-wall–active antibiotic combined with a protein synthesis inhibitor and surgical debridement in NF patients. Adjunctive therapy with intravenous immunoglobulin (IVIG) has been proposed for superantigen-associated streptococcal toxic shock syndrome. So far, benefits of IVIG treatment remain unclear and prospective clinical studies are scarce. Thus, we aimed to assess the effects of IVIG on virulence factor activity in vitro, ex vivo in patients and in vivo in a NF mouse model. Methods We investigated the effect of IVIG on the activity of the virulence factors streptolysin O (SLO), streptodornase 1 (Sda1), S. pyogenes cell envelope protease and streptococcal pyrogenic exotoxin B in vitro and ex vivo in patient sera. Additionally, we assessed the influence of IVIG on the clinical outcome in a murine NF model. Results In vitro, IVIG inhibited various streptococcal virulence factors. Further, IVIG treatment of group A Streptococcus–infected mice led to a reduced skin lesion size (median (interquartile range) day 3 intraperitoneal administration: 12 mm2 (9–14.5) vs. 4 mm2 (0.8–10.5), subcutaneous: 10.3 mm2 (6.9–18.6) vs. 0.5 mm2 (0.1–6.8)) and lower SLO activity. After treatment with IVIG, patient sera showed an elevated titre of specific SLO (7/9) and Sda1 (5/9) antibodies, reducing SLO and Sda1 activity. Conclusions The clear reduction in disease severity in IVIG-treated mice and inhibition of virulence factor activity in mouse and human sera suggest that IVIG may be beneficial in invasive group A Streptococcus infections such as NF in addition to streptococcal toxic shock syndrome.

Necrotizing Soft Tissue Infections (NSTIs), often reaching the deep fascia and muscle, are mainly caused by group A Streptococcus (GAS) and to a lesser extent by Staphylococcus aureus (SA). Conversely SA is a leading etiologic agent of pyomyositis suggesting that SA could have a specific tropism for the muscle. To assess the pathogenicity of these two bacterial species for muscles cells in comparison to keratinocytes, adhesion and invasion of NSTI-GAS and NSTI-SA were assessed on these cells. Bloodstream infections (BSI) SA isolates and non-invasive coagulase negative Staphylococci (CNS) isolates were used as controls.SA isolates from NSTI and from BSI exhibited stronger internalization into human keratinocytes and myoblasts than CNS or NSTI-GAS. While the median level of SA internalization culminated at 2% in human keratinocytes, it reached over 30% in human myoblasts due to a higher percentage of infected myoblasts (>11%) as compared to keratinocytes (psmα and RNAIII transcripts in NSTI group as compared to hematogenous group. However, the two groups were not discriminated at the genomic level. The cellular basis of high internalization rate in myoblasts was attributed to higher expression of α5β1 integrin in myoblasts as compared to keratinocytes. Major contribution of FnbpAB-integrin α5β1 pathway to internalization was confirmed by isogenic mutants.Our findings suggest the contribution of NSTI-SA severity by its unique propensity to invade and kill myoblasts, a property not shared by NSTI-GAS.ImportanceNecrotizing Soft Tissue Infection (NSTI) is a severe infection caused mainly by group A Streptococcus (GAS) and occasionally by S. aureus (SA); the latter being more often associated with pyomyositis. NSTIs frequently involve the deep fascia and may provoke muscle necrosis. The goal of this study was to determine the tropism and pathogenicity of these two bacterial species for muscle cells. The results revealed a high tropism of SA for myoblasts and myotubes followed by cytotoxicity as opposed to GAS that did not invade these cells. This study uncover a novel mechanism of SA contribution to NSTI with a direct muscle involvement, while in GAS NSTI this is likely indirect, for instance, secondary to vascular occlusion.

Summary SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. We here systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits robust, broad and highly functional memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19., Highlights 1. Acute phase SARS-CoV-2-specific T cells display an activated cytotoxic phenotype 2. Broad and polyfunctional SARS-CoV-2-specific T cell responses in convalescent phase 3. Detection of SARS-CoV-2-specific T cell responses also in seronegative individuals

During bacterial infections, damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) activate immune cells. Here, we investigated whether plasma and sputum levels of High Mobility Group Box 1 (HMGB1), a prototypic DAMP, are associated with disease severity and aetiology in community-acquired pneumonia (CAP). In addition, in patients with pneumococcal CAP, the impact of the level of sputum lytA DNA load, a PAMP, was investigated. We studied patients hospitalised for bacterial CAP (n = 111), and samples were collected at admission. HMGB1 was determined by enzyme-linked immunosorbent assays, and pneumococcal lytA DNA load was determined by quantitative polymerase chain reaction. Plasma and sputum HMGB1 levels did not correlate to disease severity (pneumonia severity index or presence of sepsis), but high sputum HMGB1 level was correlated to pneumococcal aetiology (p = 0.002). In pneumococcal pneumonia, high sputum lytA DNA load was associated with respiratory failure (low PaO2/FiO2 ratio; p = 0.019), and high sputum HMGB1 level was associated with bacteraemia (p = 0.006). To conclude, high sputum HMGB1 was not associated with severe disease, but with pneumococcal bacteraemia, indicating a potential role for HMGB1 in bacterial dissemination. High sputum lytA was associated with severe disease.

Group A Streptococcus causes severe invasive infections, including necrotizing fasciitis. The expression of an array of virulence factors targeting specific host immune functions impedes successful bacterial clearance. The virulence factor streptococcal DNase Sda1 was previously shown to interfere with the entrapment of bacteria through neutrophil extracellular traps and TLR9 signaling. In this study, we showed that plasmacytoid dendritic cells are recruited to the infected tissue during group A streptococcal necrotizing fasciitis. We found that the streptococcal DNase Sda1 impairs plasmacytoid dendritic cell recruitment by reducing IFN-1 levels at the site of infection. We found that streptococcal DNase Sda1 interferes with stabilization of the DNA by the host molecule HMGB1 protein, which may account for decreased IFN-1 levels at the site of infection.