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Predicting the severity of COVID-19 remains an unmet medical need. Our objective was to develop a blood-based host-gene-expression classifier for the severity of viral infections and validate it in independent data, including COVID-19. We developed a logistic regression-based classifier for the severity of viral infections and validated it in multiple viral infection settings including COVID-19. We used training data (N = 705) from 21 retrospective transcriptomic clinical studies of influenza and other viral illnesses looking at a preselected panel of host immune response messenger RNAs. We selected 6 host RNAs and trained logistic regression classifier with a cross-validation area under curve of 0.90 for predicting 30-day mortality in viral illnesses. Next, in 1417 samples across 21 independent retrospective cohorts the locked 6-RNA classifier had an area under curve of 0.94 for discriminating patients with severe vs. non-severe infection. Next, in independent cohorts of prospectively (N = 97) and retrospectively (N = 100) enrolled patients with confirmed COVID-19, the classifier had an area under curve of 0.89 and 0.87, respectively, for identifying patients with severe respiratory failure or 30-day mortality. Finally, we developed a loop-mediated isothermal gene expression assay for the 6-messenger-RNA panel to facilitate implementation as a rapid assay. With further study, the classifier could assist in the risk assessment of COVID-19 and other acute viral infections patients to determine severity and level of care, thereby improving patient management and reducing healthcare burden.

Background: Small intestinal bacterial overgrowth (SIBO), characterized by either increased numbers or presence of colonic type bacteria in the small bowel has been previously described in functional dyspepsia (FD), based on breath testing. In this study, we aim to examine the prevalence of SIBO among FD patients using small bowel aspirate culture. Methods: We prospectively enrolled outpatients fulfilling Rome IV criteria for FD. Severity of symptoms was graded using the patient assessment of upper gastrointestinal symptom severity index (PAGI-SYM) questionnaire. Patients underwent upper gastrointestinal endoscopy and duodenal fluid was aspirated in sterile traps. SIBO was defined as ≥103 colony forming units/mL of duodenal aspirate and/or presence of colonic type bacteria. Patients undergoing gastroscopy due to gastroesophageal reflux symptoms – control group (CG) – and patients with irritable bowel syndrome (IBS) fulfilling Rome IV criteria were also recruited. Results: We enrolled 227 FD subjects, 30 CG, and 90 IBS patients. Among FD patients, 144 (63.4%), 64 (28.2%), and 19 (8.4%) had postprandial distress syndrome (PDS), epigastric pain syndrome (EPS), and overlapping PDS-EPS syndrome, respectively. SIBO prevalence was 20.8%, 12.5%, and 31.6% among PDS, EPS, and overlapping PDS-EPS FD subtypes, respectively. Overall, SIBO prevalence was significantly higher in FD (44/227 [19.4%]) compared to CG (1/30 [3.3%]) (p = 0.037) and similar to IBS (44/227 [19.4%] vs. 15/90 [16.7%], p = 0.63) subjects. SIBO presence was associated neither with total nor with any subscale score of the PAGI-SYM questionnaire. Conclusion: In a cohort of Greek FD patients, SIBO prevalence was similar to that of IBS subjects and higher compared to that of controls.

BCG vaccination in children protects against heterologous infections and improves survival independently of tuberculosis prevention. The phase III ACTIVATE trial assessed whether BCG has similar effects in the elderly. In this double-blind, randomized trial, elderly patients (n = 198) received BCG or placebo vaccine at hospital discharge and were followed for 12 months for new infections. At interim analysis, BCG vaccination significantly increased the time to first infection (median 16 weeks compared to 11 weeks after placebo). The incidence of new infections was 42.3% (95% CIs 31.9%–53.4%) after placebo vaccination and 25.0% (95% CIs 16.4%–36.1%) after BCG vaccination; most of the protection was against respiratory tract infections of probable viral origin (hazard ratio 0.21, p = 0.013). No difference in the frequency of adverse effects was found. Data show that BCG vaccination is safe and can protect the elderly against infections. Larger studies are needed to assess protection against respiratory infections, including COVID-19 (ClinicalTrials.gov NCT03296423)., Graphical Abstract, Highlights • ACTIVATE is a prospective randomized trial of BCG vaccination in the elderly • BCG increased the time to first infection and decreased the incidence of new infection • Strongest protection was found against viral respiratory tract infections • Epigenetic reprogramming and increased cytokine production was found in monocytes, Interim analysis of the phase III ACTIVATE trial to evaluate protection against infection in elderly patients reveals that BCG vaccination is safe, increases the time to first infection, and shows protection against viral respiratory infections.

NLRP3 inflammasome is a multimolecular cytosol complex that, when activated, contributes to the cleavage of pro-interleukin (IL)-1β to IL-1β. To investigate NLRP3 inflammasome activation in inflammatory bowel disease. Peripheral blood mononuclear cells from Crohn’s disease (CD), ulcerative colitis (UC) patients and controls were stimulated with LPS in the absence or presence of MSU. After incubation, concentrations of IL-1β, IL-6, and TNFα were measured in cell supernatants and concentration of pro-IL-1β was measured in cell lysates. NLRP3 activation was defined as more than 30% increase in IL-1β production after MSU addition. In separate experiments, PBMCs were lysed for RNA isolation transcripts of IL-1β, TNFα, NLRP3, and CASP1 were measured by RT-PCR. DNA was isolated from CD patients for ATG16L1 gene genotyping. NLRP3 inflammasome was activated in 60% of CD patients compared to 28.6% of controls (p = 0.042); no significant difference was detected between UC and controls. Among UC patients, NLRP3 activation was associated (p = 0.008) with long-standing disease (>1.5 years). IL-1β levels were significantly higher in CD patents in comparison with controls (p = 0.032). No difference was detected in the levels of IL-6, TNFα, pro-IL-1β and in the numbers IL-1β, TNFα, NLRP3, and CASP1 transcripts among groups. IL-1β production was similar between carriers of wild-type and of SNP alleles of the rs2241880. NLRP3 inflammasome is activated in CD patients and in UC patients with long-standing disease.

This study was designed to identify changes in the monocytic membrane marker HLA-DR and heat shock proteins (HSPs) in relation to T-regulatory cells (T-regs) and other immunological marker changes in patients with systemic inflammatory response syndrome (SIRS) or sepsis/septic shock.Healthy volunteers, intensive care unit (ICU) patients with SIRS due to head injury and ICU patients with severe sepsis/septic shock were enrolled in the current study. Determination of CD14+/HLA-DR+ cells, intracellular heat-shock proteins and other immunological parameters were performed by flow cytometry and RT-PCR techniques as appropriate. Univariate and multivariate analysis examined associations of CD14/HLA-DR, HSPs, T-regs and suppressor of cytokine signalling (SOCS) proteins with SIRS, sepsis and outcome.Fifty patients (37 with severe sepsis and 13 with SIRS) were enrolled, together with 20 healthy volunteers used as a control group. Compared to healthy individuals, patients with SIRS and severe sepsis showed progressive decline of their CD14/HLA-DR expression (0% to 7.7% to 50% within each study subpopulation, p0.001). Mean fluorescent intensity (MFI) levels of HSP70 and HSP90 on monocytes and polymorphonuclear cells were significantly higher in SIRS patients compared to controls and fell significantly in severe sepsis/septic shock patients (p0.05 for all comparisons). There was no statistically significant difference between subgroups for levels of T-regulatory cells or relative copies of Suppressor of Cytokine Signalling 3 (SOCS3) proteins. In univariate models percent of CD14/HLA-DR was associated with mortality (OR: 1.8 95%CI 1.02-3.2, p=0.05), while in multivariate models after adjusting for CD14/HLA-DR only younger age and lower Acute Physiology and Chronic Health Evaluation II (APACHE II) scores were associated with increased chances of survival (beta -0.05, OR 0.9, 95% CI 0.9-0.99, p=0.038 for age and beta -0.11, OR 0.89, 95% CI 0.8-0.99, p=0.037 for APACHE II score).Significant associations with SIRS and sepsis were found for CD14/HLA-DR expression and monocyte and polymorphonuclear cell levels of HSP70 and 90. The role of these biomarkers in assessing the prognosis of sepsis needs to be further explored and validated in prospective studies.

To study the differences of monocyte activation by albicans and non-albicans species of Candida and its change in sepsis, peripheral blood mononuclear cells were isolated from 17 healthy volunteers and 26 patients with severe sepsis/shock, and incubated in the absence/presence of heat-killed (HK) isolates of four different Candida species and purified β-D-glucan from C.albicans. Experiments were repeated in the presence and absence of inhibitors of intracellular activation pathways. Expression of annexin V on cells membranes of monocytes and lymphocytes, cytoplasmic activity of caspase-3, and DNA fragmentation of monocytes were studied. Membrane expression of annexin V on viable monocytes of healthy volunteers decreased significantly after incubation with C.albicans but not with non-albicans species. The decrease was dose-dependent from the Candida inoculum and by the concentration of β-D-glucan. A relationship with inhibition of apoptosis was found as the activity of caspase-3 activity, and the level of DNA fragmentation were also decreased. Incubation in the absence/presence of inhibitors showed that the decrease by annexin V expression resulted by activation of the dectin-1 pathway and Raf-1 by β-D glucan. The decrease of annexin V(+)/PI(-) expression was not shown on monocytes of patients with severe sepsis/shock, where no effect of inhibitors was found. Decrease of annexin V binding on monocytes can be viewed as a selective response to C.albicans partly effected through activation of dectin-1. This response is down-regulated after a septic insult.

Proper management of COVID-19 mandates better understanding of disease pathogenesis. The sudden clinical deterioration 7-8 days after initial symptom onset suggests that severe respiratory failure (SRF) in COVID-19 is driven by a unique pattern of immune dysfunction. We studied immune responses of 54 COVID-19 patients, 28 of whom had SRF. All patients with SRF displayed either macrophage activation syndrome (MAS) or very low human leukocyte antigen D related (HLA-DR) expression accompanied by profound depletion of CD4 lymphocytes, CD19 lymphocytes, and natural killer (NK) cells. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production by circulating monocytes was sustained, a pattern distinct from bacterial sepsis or influenza. SARS-CoV-2 patient plasma inhibited HLA-DR expression, and this was partially restored by the IL-6 blocker Tocilizumab; off-label Tocilizumab treatment of patients was accompanied by increase in circulating lymphocytes. Thus, the unique pattern of immune dysregulation in severe COVID-19 is characterized by IL-6-mediated low HLA-DR expression and lymphopenia, associated with sustained cytokine production and hyper-inflammation.

To investigate the impact of carriage of single nucleotide polymorphisms (SNPs) of the Toll-like receptor-4 (TLR4) and of autophagy-related gene 16-like-1 (ATG16L1) in preterm delivery (PTD). A prospective cohort of 145 pregnant women was studied. Women were prospectively followed-up until delivery. Genotyping for rs4986790 (Asp299Gly transition) and rs4986791 (Thr399Ile transition) of TLR4 and for rs2241880 of ATG16L1 was done by PCR-restriction fragment length polymorphism. The primary study endpoint was the impact of carriage of minor alleles of TLR4 on early PTD before gestational week 32. Associations with human chorionic gonadotrophin (hCG) were also analyzed. Peripheral blood mononuclear cells were isolated from 15 healthy women and stimulated for cytokine production. No difference in clinical characteristics was observed between women delivering full term and preterm. The frequency of early PTD was 25% among women carrying minor alleles of TLR4 and 6.8% among women carrying major alleles (P: .032). Odds ratios for PTD were 3.85 among women carrying the GG genotype of rs2241880 and major alleles of TLR4 and 0.26 among carriers of GG genotype and minor alleles of TLR4 (P: .030). The co-presence of GG genotype of rs2241880 and hCG above 70 U/L was an independent variable for PTD. Stimulated production of interleukin-6 was greater among women with GG genotypes of rs2241880. Minor alleles of SNPs of TLR4 predispose to early PTD. The GG genotype of rs2241880 of ATG16L1 is associated with PTD when hCG is supra-elevated.

Heterogeneous results of published studies led to conduct a randomized clinical trial to assess the efficacy of a new formulation of four probiotics as prophylaxis for complications after colorectal surgery. A double-blind, placebo-controlled randomized study was conducted enrolling patients undergoing colorectal surgery for cancer. Capsules of placebo or of a formulation containing Lactobacillus acidophilus, L. p lantarum, Bifidobacterium lactis and Saccharomyces boulardii were administered starting one day before operation and continuing for another 15 days postoperatively. Patients were followed up for 30 days with the development of postoperative complications as the primary outcome. Gene expression and serum levels of cytokines were measured on postoperative day 4 ( www.clinicaltrials.gov NCT02313519). The study was prematurely stopped after enrolment due to efficacy in the primary outcome. Administration of probiotics significantly decreased the rate of all postoperative major complication (28.6 vs. 48.8 % of the placebo arm, p 0.010, odds ratio 0.42). Major benefit was found in the reduction of the rate of postoperative pneumonia (2.4 vs. 11.3 %, p 0.029), of surgical site infections (7.1 vs. 20.0 %, p 0.020) and of anastomotic leakage (1.2 vs. 8.8 %, p 0.031). The time until hospital discharge was shortened as well. Gene expression of SOCS3 was positively related with gene expression of TNF and of circulating IL-6 in the probiotic group but not in the placebo group. The studied probiotic formulation significantly decreased the risk of postoperative complications, namely mechanical ventilation, infections and anastomotic leakage. Modulation of the gene expression of SOCS3 is one suggested mechanism ( www.clinicaltrials.gov NCT02313519).

Based on previous findings for the role of single nucleotide polymorphisms (SNPs) of TNF for the predisposition for bloodstream infections, this study investigates the role of these SNPs at the promoter positions –376, –308, –238 in infective endocarditis (IE). In a case–control study, 83 patients with IE and 83 controls were enrolled. Blood genotyping for the presence of G or A alleles of the three SNPs was carried out using restriction fragment length polymorphisms. Haplotypes were calculated. Patients were mostly infected by Staphylococcus aureus (32.5%) and by species of enterococci (14.3%) and streptococci (14.3%). Carriage of the minor frequency A alleles at –238 of the promoter region of TNF was greater than in controls (8.4% versus 1.2%, p 0.003). The presence of any of the three GGA/GAA/AGA haplotypes was more frequent in patients with IE (OR 8.22, 95CI% 1.8–37.4, p 0.001). After multivariate logistic regression analysis, it was found that the only factor related to fatal outcome was carriage of the wild-type GGG haplotype (OR, 3.29, 95CI%, 1.05–10.29, p 0.04). GGA, AGA and GAA haplotypes were more frequent in patients with IE than in controls, suggesting a predisposition for IE and a potential protective role against fatal outcome, as the wild-type GGG haplotype was independently related with death.

Background TREM-1 (triggering receptor expressed on myeloid cells), a receptor expressed on neutrophils and monocytes, is upregulated in sepsis and seems to tune the inflammatory response. We explored the expression of TREM-1 at the gene level and on cell membranes of monocytes and association with clinical outcome. Methods Peripheral venous blood was sampled from 75 septic patients (39 patients with sepsis, 25 with severe sepsis and 11 with septic shock) on sepsis days 1, 3 and 7. TREM-1 on monocytes was measured by flow cytometry; gene expression of TREM-1 in circulating mononuclear cells was assessed by real-time PCR. sTREM-1 was measured in serum by an enzyme immunoassay. Results Although surface TREM-1, sTREM-1 and TREM-1 gene expression did not differ between sepsis, severe sepsis and septic shock on day 1, survivors had greater expression of surface TREM-1 on days 3 and 7 compared to non-survivors. sTREM-1 on non-survivors decreased on day 3 compared to baseline. Patients with increase of monocyte gene expression of TREM-1 from day 1 to day 3 had prolonged survival compared to patients with decrease of gene expression of TREM-1 from day 1 to day 3 (p: 0.031). Conclusions Early decrease of gene expression of TREM-1 in monocytes is associated with poor outcome. A reciprocal decrease of the pro-inflammatory surface receptor TREM-1 linked with sepsis-induced immunosuppression may be part of the explanation. Electronic supplementary material The online version of this article (doi:10.1186/s12865-014-0063-y) contains supplementary material, which is available to authorized users.