Bloodstream infections remain one of the major challenges in intensive care units, leading to sepsis or even septic shock in many cases. Due to the lack of timely diagnostic approaches with sufficient sensitivity, mortality rates of sepsis are still unacceptably high. However a prompt diagnosis of the causative microorganism is critical to significantly improve outcome of bloodstream infections. Although various targeted molecular tests for blood samples are available, time-consuming blood culture-based approaches still represent the standard of care for the identification of bacteria. Here we describe the establishment of a complete diagnostic workflow for the identification of infectious microorganisms from seven septic patients based on unbiased sequence analyses of free circulating DNA from plasma by next-generation sequencing. We found significant levels of DNA fragments derived from pathogenic bacteria in samples from septic patients. Quantitative evaluation of normalized read counts and introduction of a sepsis indicating quantifier (SIQ) score allowed for an unambiguous identification of Gram-positive as well as Gram-negative bacteria that exactly matched with blood cultures from corresponding patient samples. In addition, we also identified species from samples where blood cultures were negative. Reads of non-human origin also comprised fragments derived from antimicrobial resistance genes, showing that, in principle, prediction of specific types of resistance might be possible. The complete workflow from sample preparation to species identification report could be accomplished in roughly 30 h, thus making this approach a promising diagnostic platform for critically ill patients suffering from bloodstream infections.

Long-lasting impairment of the immune system is believed to be the underlying reason for delayed deaths after surviving sepsis. We tested the hypothesis of persisting changes to the immune system in survivors of sepsis for the first time. In our prospective, cross-sectional pilot study, eight former patients who survived catecholamine-dependent sepsis and eight control individuals matched for age, sex, diabetes and renal insufficiency were enrolled. Each participant completed a questionnaire concerning morbidities, medications and infection history. Peripheral blood was collected for determination of i) immune cell subsets (CD4+, CD8+ T cells; CD25+ CD127- regulatory T cells; CD14+ monocytes), ii) cell surface receptor expression (PD-1, BTLA, TLR2, TLR4, TLR5, Dectin-1, PD-1 L), iii) HLA-DR expression, and iv) cytokine secretion (IL-6, IL10, TNF-α, IFN-γ) of whole blood stimulated with either α-CD3/28, LPS or zymosan. After surviving sepsis, former patients presented with increased numbers of clinical apparent infections, including those typically associated with an impaired immune system. Standard inflammatory markers indicated a low-level inflammatory situation in former sepsis patients. CD8+ cell surface receptor as well as monocytic HLA-DR density measurements showed no major differences between the groups, while CD4+ T cells tended towards two opposed mechanisms of negative immune cell regulation via PD-1 and BTLA. Moreover, the post-sepsis group showed alterations in monocyte surface expression of distinct pattern recognition receptors; most pronouncedly seen in a decrease of TLR5 expression. Cytokine secretion in response to important activators of both the innate (LPS, zymosan) and the adaptive immune system (α-CD3/28) seemed to be weakened in former septic patients. Cytokine secretion as a reaction to different activators of the immune system seemed to be comprehensively impaired in survivors of sepsis. Among others, this could be based on trends in the downregulation of distinct cell surface receptors. Based on our results, the conduct of larger validation studies seems feasible, aiming to characterize alterations and to find potential therapeutic targets to engage.

Sepsis still represents an important clinical and economic challenge for intensive care units. Severe complications like multi-organ failure with high mortality and the lack of specific diagnostic tools continue to hamper the development of improved therapies for sepsis. Fundamental questions regarding the cellular pathogenesis of experimental and clinical sepsis remain unresolved. According to experimental data, inhibiting macrophage migration inhibitory factor, high-mobility group box protein 1 (HMGB1), and complement factor C5a and inhibiting the TREM-1 (triggering receptor expressed on myeloid cells 1) signaling pathway and apoptosis represent promising new therapeutic options. In addition, we have demonstrated that blocking the signal transduction pathway of receptor of advanced glycation endproducts (RAGE), a new inflammation-perpetuating receptor and a member of the immunglobulin superfamily, increases survival in experimental sepsis. The activation of RAGE by advanced glycation end-products, S100, and HMGB1 initiates nuclear factor kappa B and mitogen-activated protein kinase pathways. Importantly, the survival rate of RAGE knockout mice was more than fourfold that of wild-type mice in a septic shock model of cecal ligation and puncture (CLP). Additionally, the application of soluble RAGE, an extracellular decoy for RAGE ligands, improves survival in mice after CLP, suggesting that RAGE is a central player in perpetuating the innate immune response. Understanding the basic signal transduction events triggered by this multi-ligand receptor may offer new diagnostic and therapeutic options in patients with sepsis.

Notch signaling, a highly conserved pathway in mammals, is crucial for differentiation and homeostasis of immune cells. Besides, this pathway is also directly involved in the transmission of immune signals. Notch signaling per se does not have a clear pro- or anti-inflammatory effect, but rather its impact is highly dependent on the immune cell type and the cellular environment, modulating several inflammatory conditions including sepsis, and therefore significantly impacts the course of disease. In this review, we will discuss the contribution of Notch signaling on the clinical picture of systemic inflammatory diseases, especially sepsis. Specifically, we will review its role during immune cell development and its contribution to the modulation of organ-specific immune responses. Finally, we will evaluate to what extent manipulation of the Notch signaling pathway could be a future therapeutic strategy.

Fungi are of increasing importance in sepsis. However, culture-based diagnostic procedures are associated with relevant weaknesses. Therefore, culture- and next-generation sequencing (NGS)-based fungal findings as well as corresponding plasma levels of β-d-glucan, interferon gamma (INF-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-2, -4, -6, -10, -17A, and mid-regional proadrenomedullin (MR-proADM) were evaluated in 50 septic patients at six consecutive time points within 28 days after sepsis onset. Furthermore, immune-response patterns during infections with Candida spp. were studied in a reconstituted human epithelium model. In total, 22% (n = 11) of patients suffered from a fungal infection. An NGS-based diagnostic approach appeared to be suitable for the identification of fungal pathogens in patients suffering from fungemia as well as in patients with negative blood cultures. Moreover, MR-proADM and IL-17A in plasma proved suitable for the identification of patients with a fungal infection. Using RNA-seq., adrenomedullin (ADM) was shown to be a target gene which is upregulated early after an epithelial infection with Candida spp. In summary, an NGS-based diagnostic approach was able to close the diagnostic gap of routinely used culture-based diagnostic procedures, which can be further facilitated by plasmatic measurements of MR-proADM and IL-17A. In addition, ADM was identified as an early target gene in response to epithelial infections with Candida spp.

Background: SARS-CoV-2 antigen rapid diagnostic tests (Ag-RDTs) are increasingly being integrated in testing strategies around the world. Studies of the Ag-RDTs have shown variable performance. In this systematic review and meta-analysis, we assessed the clinical accuracy (sensitivity and specificity) of commercially available Ag-RDTs. Methods and Results: We registered the review on PROSPERO (Registration number: CRD42020225140). We systematically searched multiple databases (PubMed, Web of Science Core Collection, medRvix and bioRvix, FIND) for publications evaluating the accuracy of Ag-RDTs for SARS-CoV-2 up until April 30 ^th , 2021. Descriptive analyses of all studies were performed and when more than four studies were available, a random-effects meta-analysis was used to estimate pooled sensitivity and specificity in comparison to reverse transcriptase polymerase chain reaction testing. We assessed heterogeneity by subgroup analyses, and rated study quality and risk of bias using the QUADAS 2 assessment tool. From a total of 14,254 articles, we included 133 analytical and clinical studies resulting in 214 clinical accuracy data sets with 112,323 samples. Across all meta-analyzed samples, the pooled Ag-RDT sensitivity was 71.2% (95% confidence interval [CI] 68.2 to 74.0) and increased to 76.3% (CI 73.1 to 79.2) if analysis was restricted to studies that followed the Ag-RDT manufacturers’ instructions. The LumiraDx showed the highest sensitivity with 88.2% (CI 59.0 to 97.5). Of instrument-free Ag-RDTs, Standard Q nasal performed best with 80.2% sensitivity (CI 70.3 to 87.4). Across all Ag-RDTs sensitivity was markedly better on samples with lower Ct-values, i.e., <20 (96.5%, CI 92.6 to 98.4) and <25 (95.8%, CI 92.3 to 97.8), in comparison to those with Ct ≥25 (50.7%, CI 35.6 to 65.8) and ≥30 (20.9%, CI 12.5 to 32.8). Testing in the first week from symptom onset resulted in substantially higher sensitivity (83.8%, CI 76.3 to 89.2) compared to testing after one week (61.5%, CI 52.2 to 70.0). The best Ag-RDT sensitivity was found with anterior nasal sampling (75.5%, CI 70.4 to 79.9) in comparison to other sample types (e.g., nasopharyngeal 71.6%, CI 68.1 to 74.9) although CIs were overlapping. Concerns of bias were raised across all data sets, and financial support from the manufacturer was reported in 24.1% of data sets. Our analysis was limited by the included studies’ heterogeneity in design and reporting, making it difficult to draw conclusions from. Conclusion: In this study we found that Ag-RDTs detect the vast majority of cases within the first week of symptom onset and those with high viral load. Thus, they can have high utility for diagnostic purposes in the early phase of disease, making them a valuable tool to fight the spread of SARS-CoV-2. Standardization in conduct and reporting of clinical accuracy studies would improve comparability and use of data. AUTHOR SUMMARY: Why was this study done? – Antigen rapid diagnostic tests (Ag-RDTs) are considered an important diagnostic tool to fight the spread of SARS-CoV-2 – An increasing number of Ag-RDTs is offered on the market, and a constantly growing body of literature evaluating their performance is available – To inform decision makers about the best test to choose, an up to date summary of their performance is needed What did the researchers do and find? – On a weekly basis, we search multiple data bases for evaluations of Ag-RDTs detecting SARS-CoV-2 and post the results on www.diagnosticsglobalhealth.org – Based on the search results up until April 30 ^th , 2021, we conducted a systematic review and meta-analysis, including a total of 133 clinical and analytical accuracy studies – Across all meta-analyzed studies, when Ag-RDTs were performed according to manufacturers’ recommendations, they showed a sensitivity of 76.3% (CI 73.1 to 79.2), with the LumiraDx (sensitivity 88.2%, CI 59.0 to 97.5) and of the instrument-free Ag-RDT Standard Q (74.9% sensitivity, CI 69.3 to 79.7) performing best. – Across all Ag-RDTs, sensitivity increased to 95.8% (CI 92.3 to 97.8) when restricting the analysis to samples with high viral loads (i.e., a Ct-value <25) and to 83.8% (CI 76.3 to 89.2) when tests were performed on patients within the first week after symptom onset What do these findings mean? – Ag-RDTs detect the vast majority of cases within the first week of symptom onset and those with high viral load. Thus, they can have high utility for diagnostic purposes in the early phase of disease – Out of all assessed tests, the Lumira Dx showed the highest accuracy. The Standard Q wasthe best performing test when only considering those that don’t require an instrument – A standardization of reporting methods for clinical accuracy studies would enhance future test-comparisons