Virulence Factor Genes in Invasive Escherichia coli Are Associated with Clinical Outcomes and Disease Severity in Patients with Sepsis: A Prospective Observational Cohort Study.

BACKGROUND: Escherichia coli harbours virulence factors that facilitate the development of bloodstream infections. Studies determining virulence factors in clinical isolates often have limited access to clinical data and lack associations with patient outcome. The goal of this study was to correlate sepsis outcome and virulence factors of clinical E. coli isolates in a large cohort. METHODS: Patients presenting at the emergency department whose blood cultures were positive for E. coli were prospectively included. Clinical and laboratory parameters were collected at admission. SOFA-score was calculated to determine disease severity. Patient outcomes were in-hospital mortality and ICU admission. Whole genome sequencing was performed for E. coli isolates and virulence genes were detected using the VirulenceFinder database. RESULTS: In total, 103 E. coli blood isolates were sequenced. Isolates had six to 41 virulence genes present. One virulence gene, kpsMII_K23, a K1 capsule group 2 of E. coli type K23, was significantly more present in isolates of patients who died. kpsMII_K23 and cvaC (Microcin C) were significantly more frequent in isolates of patients who were admitted to the ICU. Fourteen virulence genes (mchB, mchC, papA_fsiA_F16, sat, senB, iucC, iutA, iha, sfaD, cnf1, focG, vat, cldB, and mcmA) significantly differed between patients with and without sepsis. CONCLUSIONS: Microcins, toxins, and fimbriae were associated with disease severity. Adhesins and iron uptake proteins seemed to be protective. Two genes were associated with worse clinical outcome. These findings contribute to a better understanding of host-pathogen interactions and could help identifying patients most at risk for a worse outcome.

Feasibility and safety of synchrotron-based X-ray phase contrast imaging as a technique complementary to histopathology analysis.

X-ray phase contrast imaging (X-PCI) is a powerful technique for high-resolution, three-dimensional imaging of soft tissue samples in a non-destructive manner. In this technical report, we assess the quality of standard histopathological techniques performed on formalin-fixed, paraffin-embedded (FFPE) human tissue samples that have been irradiated with different doses of X-rays in the context of an X-PCI experiment. The data from this study demonstrate that routine histochemical and immunohistochemical staining quality as well as DNA and RNA analyses are not affected by previous X-PCI on human FFPE samples. From these data we conclude it is feasible and acceptable to perform X-PCI on FFPE human biopsies.

An observational study on lifestyle and environmental risk factors in patients with acute appendicitis.

Purpose: Acute appendicitis is a common abdominal emergency worldwide. This study aimed at characterizing environmental risk factors influencing the development and severity of acute appendicitis. Methods: Patients from a Belgian acute appendicitis cohort (n = 374) and healthy controls from the 500 functional genomics (500FG) cohort (n = 513) were compared. Individuals with a history of appendectomy (n = 1067) and without a history of appendectomy (n = 8656) were available from the Nijmegen Biomedical Study (NBS). Questionnaires on demographics, lifestyle and environment were available. Binary logistic regression was used for prediction models. Results: Fifteen risk factors for developing acute appendicitis were identified. Binary logistic regression showed that 7 were independent risk factors: family history of acute appendicitis, having grown up in a rural environment, having a lower education, probiotic use as well as antibiotic use increased the risk of developing appendicitis. Fruit and fiber-rich vegetable consumption decreased the risk. Findings on vegetable consumption, smoking and level of education were replicated in the NBS population. Independent risk factors for complicated appendicitis were being male, higher age, and a delay to diagnosis of more than 48 h. Conclusions: Environmental exposures influence the risk of developing appendicitis. Further research into these factors is needed.

Causative agents of bloodstream infections in two Croatian hospitals and their resistance mechanisms.

Blood samples were collected alongside with routine blood cultures (BC) from patients with suspected sepsis, to evaluate the prevalence of different causative agents in patients with bacteraemia. Among 667 blood samples, there were 122 positive BC (18%). Haemoglobin content, platelet number, and systolic blood pressure values were significantly lower in patients with positive BC, whereas serum lactate levels, CRP, creatinine and urea content were significantly higher in patients with positive BC. The rate of multidrug (MDR) or extensively drug resistant (XDR) bacteria was 24% (n = 29): Klebsiella pneumoniae (9), Pseudomonas aeruginosa (9), Acinetobacter baumannii (4), Escherichia coli (1), vancomycin resistant Enterococcus spp (VRE) (3), and methicillin-resistant Staphylococcus aureus MRSA (3). The dominant resistance mechanisms were the production of extended-spectrum ß-lactamases, OXA-48 carbapenemase, and colistin resistance in K. pneumoniae, VIM metallo-ß-lactamases in P. aeruginosa and OXA-23-like oxacillinases in A. baumannii. The study revealed high rate of MDR strains among positive BCs in Zagreb, Croatia.

A prospective observational cohort study to identify inflammatory biomarkers for the diagnosis and prognosis of patients with sepsis.

BACKGROUND: Sepsis is a life-threatening organ dysfunction. A fast diagnosis is crucial for patient management. Proteins that are synthesized during the inflammatory response can be used as biomarkers, helping in a rapid clinical assessment or an early diagnosis of infection. The aim of this study was to identify biomarkers of inflammation for the diagnosis and prognosis of infection in patients with suspected sepsis. METHODS: In total 406 episodes were included in a prospective cohort study. Plasma was collected from all patients with suspected sepsis, for whom blood cultures were drawn, in the emergency department (ED), the department of infectious diseases, or the haemodialysis unit on the first day of a new episode. Samples were analysed using a 92-plex proteomic panel based on a proximity extension assay with oligonucleotide-labelled antibody probe pairs (OLink, Uppsala, Sweden). Supervised and unsupervised differential expression analyses and pathway enrichment analyses were performed to search for inflammatory proteins that were different between patients with viral or bacterial sepsis and between patients with worse or less severe outcome. RESULTS: Supervised differential expression analysis revealed 21 proteins that were significantly lower in circulation of patients with viral infections compared to patients with bacterial infections. More strongly, higher expression levels were observed for 38 proteins in patients with high SOFA scores (> 4), and for 21 proteins in patients with worse outcome. These proteins are mostly involved in pathways known to be activated early in the inflammatory response. Unsupervised, hierarchical clustering confirmed that inflammatory response was more strongly related to disease severity than to aetiology. CONCLUSION: Several differentially expressed inflammatory proteins were identified that could be used as biomarkers for sepsis. These proteins are mostly related to disease severity. Within the setting of an emergency department, they could be used for outcome prediction, patient monitoring, and directing diagnostics. TRAIL REGISTRATION NUMBER: clinicaltrial.gov identifier NCT03841162.

Studying the clinical, radiological, histological, microbiological, and immunological evolution during the different COVID-19 disease stages using minimal invasive autopsy.

The WHO defines different COVID-19 disease stages in which the pathophysiological mechanisms differ. We evaluated the characteristics of these COVID-19 disease stages. Forty-four PCR-confirmed COVID-19 patients were included in a prospective minimal invasive autopsy cohort. Patients were classified into mild-moderate (n = 4), severe-critical (n = 32) and post-acute disease (n = 8) and clinical, radiological, histological, microbiological and immunological data were compared. Classified according to Thoracic Society of America, patients with mild-moderate disease had no typical COVID-19 images on CT-Thorax versus 71.9% with typical images in severe-critical disease and 87.5% in post-acute disease (P < 0.001). Diffuse alveolar damage was absent in mild-moderate disease but present in 93.8% and 87.5% of patients with severe-critical and post-acute COVID-19 respectively (P = 0.002). Other organs with COVID-19 related histopathological changes were liver and heart. Interferon-γ levels were significantly higher in patients with severe-critical COVID-19 (P = 0.046). Anti-SARS CoV-2 IgG was positive in 66%, 40.6% and 87.5% of patients with mild-moderate, severe-critical and post-acute COVID-19 respectively (n.s.). Significant differences in histopathological and immunological characteristics between patients with mild-moderate disease compared to patients with severe-critical disease were found, whereas differences between patients with severe-critical disease and post-acute disease were limited. This emphasizes the need for tailored treatment of COVID-19 patients.

Organ-specific genome diversity of replication-competent SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is not always confined to the respiratory system, as it impacts people on a broad clinical spectrum from asymptomatic to severe systemic manifestations resulting in death. Further, accumulation of intra-host single nucleotide variants during prolonged SARS-CoV-2 infection may lead to emergence of variants of concern (VOCs). Still, information on virus infectivity and intra-host evolution across organs is sparse. We report a detailed virological analysis of thirteen postmortem coronavirus disease 2019 (COVID-19) cases that provides proof of viremia and presence of replication-competent SARS-CoV-2 in extrapulmonary organs of immunocompromised patients, including heart, kidney, liver, and spleen (NCT04366882). In parallel, we identify organ-specific SARS-CoV-2 genome diversity and mutations of concern N501Y, T1027I, and Y453F, while the patient had died long before reported emergence of VOCs. These mutations appear in multiple organs and replicate in Vero E6 cells, highlighting their infectivity. Finally, we show two stages of fatal disease evolution based on disease duration and viral loads in lungs and plasma. Our results provide insights about the pathogenesis and intra-host evolution of SARS-CoV-2 and show that COVID-19 treatment and hygiene measures need to be tailored to specific needs of immunocompromised patients, even when respiratory symptoms cease.

Audit of empirical antibiotic therapy for sepsis and the impact of early multidisciplinary consultation on patient outcomes.

OBJECTIVES: To perform an audit of empirical antibiotic therapy (EAT) of sepsis at the emergency department and to analyse the impact of an antimicrobial stewardship (AMS) programme on process and patient outcomes. PATIENTS AND METHODS: A prospective, single-centre cohort study including patients with sequential organ failure assessment (SOFA) score ≥2 from whom blood cultures were taken was conducted between February 2019 and April 2020. EAT was assessed using eight applicable inpatient quality indicators (IQIs) for responsible antibiotic use. Patient outcomes were hospital length-of-stay (LOS), ICU admission, ICU LOS, and in-hospital mortality. RESULTS: The audit included 900 sepsis episodes in 803 patients. Full guideline adherence regarding choice and dosing was 45.9%; adherence regarding choice alone was 68.1%. EAT was active against all likely pathogens in 665/787 (84.5%) episodes. In the guideline non-adherent group, choice of EAT was inappropriate in 122/251 (48.6%) episodes. Changes within 3 days occurred in 335/900 (37.2%) episodes. Treating physicians changed administration route more often, whereas microbiological/infectious disease (ID)/AMS consultant advice resulted in de-escalation and discontinuation (P = 0.000). Guideline-adherent choice was associated with significantly shorter LOS (6 (4-11) vs. 8 (5-15) days). Full adherence was associated with significantly lower mortality (23 (6.4%) vs. 48 (11.3%)) and shorter LOS (6 (4-10) vs. 8 (5-14) days). CONCLUSION: Five global quality indicators of EAT were measurable in routine clinical practice. Full adherence to guidelines was only moderate. Adherence to guidelines was associated with better patient outcomes.

Risk Factors for Mortality, Intensive Care Unit Admission, and Bacteremia in Patients Suspected of Sepsis at the Emergency Department: A Prospective Cohort Study.

BACKGROUND: There is a clear need for a better assessment of independent risk factors for in-hospital mortality, intensive care unit admission, and bacteremia in patients presenting with suspected sepsis at the emergency department. METHODS: A prospective observational cohort study including 1690 patients was performed. Two multivariable logistic regression models were used to identify independent risk factors. RESULTS: Sequential organ failure assessment (SOFA) score of ≥2 and serum lactate of ≥2mmol/L were associated with all outcomes. Other independent risk factors were individual SOFA variables and systemic inflammatory response syndrome variables but varied per outcome. Mean arterial pressure <70 mmHg negatively impacted all outcomes. CONCLUSIONS: These readily available measurements can help with early risk stratification and prediction of prognosis.

Differentiating influenza from COVID-19 in patients presenting with suspected sepsis.

There is a need for a quick assessment of severely ill patients presenting to the hospital. The objectives of this study were to identify clinical, laboratory and imaging parameters that could differentiate between influenza and COVID-19 and to assess the frequency and impact of early bacterial co-infection. A prospective observational cohort study was performed between February 2019 and April 2020. A retrospective cohort was studied early in the COVID-19 pandemic. Patients suspected of sepsis with PCR-confirmed influenza or SARS-CoV-2 were included. A multivariable logistic regression model was built to differentiate COVID-19 from influenza. In total, 103 patients tested positive for influenza and 110 patients for SARS-CoV-2, respectively. Hypertension (OR 6.550), both unilateral (OR 4.764) and bilateral (OR 7.916), chest X-ray abnormalities, lower temperature (OR 0.535), lower absolute leukocyte count (OR 0.857), lower AST levels (OR 0.946), higher LDH (OR 1.008), higher ALT (OR 1.044) and higher ferritin (OR 1.001) were predictive of COVID-19. Early bacterial co-infection was more frequent in patients with influenza (10.7% vs. 2.7%). Empiric antibiotic usage was high (76.7% vs. 84.5%). Several factors determined at presentation to the hospital can differentiate between influenza and COVID-19. In the future, this could help in triage, diagnosis and early management. Clinicaltrial.gov Identifier: NCT03841162.

The clinical value of minimal invasive autopsy in COVID-19 patients.

BACKGROUND: Minimally invasive autopsy (MIA) is a validated and safe method to establish the cause of death (COD), mainly in low-resource settings. However, the additional clinical value of MIA in Coronavirus disease (COVID-19) patients in a high-resource setting is unknown. The objective was to assess if and how MIA changed clinical COD and contributing diagnoses in deceased COVID-19 patients. METHODS AND FINDINGS: A prospective observational cohort from April to May 2020 in a 981-bed teaching hospital in the epicenter of the COVID-19 pandemic in Belgium was established. Patients who died with either PCR-confirmed or radiologically confirmed COVID-19 infection were consecutively included. MIA consisted of whole-body CT and CT-guided Tru-Cut® biopsies. Diagnostic modalities were clinical chart review, radiology, microbiology, and histopathology which were assessed by two independent experts per modality. MIA COD and contributing diagnoses were established during a multi-disciplinary meeting. Clinical COD (CCOD) and contributing diagnosis were abstracted from the discharge letter. The main outcomes were alterations in CCOD and contributing diagnoses after MIA, and the contribution of each diagnostic modality. We included 18 patients, of which 7 after intensive care unit hospitalization. MIA led to an alteration in 15/18 (83%) patients. The CCOD was altered in 5/18 (28%) patients. MIA found a new COD (1/5), a more specific COD (1/5), a less certain COD (1/5), or a contributing diagnosis to be the COD (2/5). Contributing diagnoses were altered in 14/18 (78%) patients: 9 new diagnoses, 5 diagnoses dismissed, 3 made more specific, and 2 made less certain. Overall, histopathology contributed in 14/15 (93%) patients with alterations, radiology and microbiology each in 6/15 (40%), and clinical review in 3/15 (20%). Histopathology was deemed the most important modality in 10 patients, radiology in two patients, and microbiology in one patient. CONCLUSION: MIA, especially histological examination, can add valuable new clinical information regarding the cause of death in COVID-19 patients, even in a high-resource setting with wide access to premortem diagnostic modalities. MIA may provide important clinical insights and should be applied in the current ongoing pandemic. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT04366882.

The Clinical Impact of Rapid Molecular Microbiological Diagnostics for Pathogen and Resistance Gene Identification in Patients With Sepsis: A Systematic Review.

Fast microbiological diagnostics (MDx) are needed to ensure early targeted antimicrobial treatment in sepsis. This systematic review focuses on the impact on antimicrobial management and patient outcomes of MDx for pathogen and resistance gene identification compared with blood cultures. PubMed was searched for clinical studies using either whole blood directly or after short-term incubation. Twenty-five articles were retrieved describing the outcomes of 8 different MDx. Three interventional studies showed a significant increase in appropriateness of antimicrobial therapy and a nonsignificant change in time to appropriate therapy. Impact on mortality was conflicting. Length of stay was significantly lower in 2 studies. A significant decrease in antimicrobial cost was demonstrated in 6 studies. The limitations of this systematic review include the low number and observed heterogeneity of clinical studies. In conclusion, potential benefits of MDx regarding antimicrobial management and some patient outcomes were reported. More rigorous intervention studies are needed focusing on the direct benefits for patients.

An observational study of innate immune responses in patients with acute appendicitis.

Acute appendicitis is a common surgical emergency worldwide. Exaggerated immune responses could be associated with appendicitis. This study aimed at characterizing immune responses towards a large variety of gut commensals and pathogens, and pattern recognition receptor (PRR) ligands, and investigating the course of systemic inflammation in a prospective cohort of acute appendicitis patients. PBMC responses of 23 patients of the cohort and 23 healthy controls were characterized more than 8 months post-surgery. Serum cytokine levels were measured in 23 patients at the time of appendicitis and after one month. CRP, WBC and percentage of neutrophils were analyzed in the total cohort of 325 patients. No differences in PBMC responses were found between patients and controls. Stronger IL-10 responses were found following complicated appendicitis. A trend towards lower IL-8 responses was shown following gangrenous appendicitis. Serum IL-10 and IL-6 were significantly elevated at presentation, and IL-6, IL-8 and TNF-α levels were higher in complicated appendicitis. Routine biomarkers could predict severity of appendicitis with high specificities, but low sensitivities. Cytokine responses in patients following acute appendicitis did not differ from healthy controls. Higher serum cytokine levels were found in acute complicated and gangrenous cases. Further research into discriminative biomarkers is warranted.

Epidemiology of colistin-resistant, carbapenemase-producing Enterobacteriaceae and Acinetobacter baumannii in Croatia.

Colistin is a last-resort antibiotic for the treatment of infections caused by multidrug and carbapenem-resistant Gram-negative bacteria. Colistin resistance has been emerging and multiple outbreaks have been reported in Europe and elsewhere. It has been most frequently reported in carbapenem-resistant K. pneumoniae. In this study, 24 multidrug and colistin-resistant clinical isolates (14 K. pneumoniae, one E. aerogenes, one E. cloacae, and eight A. baumannii) were collected from four hospitals in Croatia from 2013 to 2018, in order to analyse the molecular epidemiology and mechanisms of antibiotic resistance. ß-lactamase and carbapenemase genes were detected by PCR. Genotyping was done on selected isolates by rep-PCR. Whole genome sequencing (WGS) was performed to discover possible molecular mechanisms for the observed colistin resistance. All isolates, except two K. pneumoniae isolates, were extensively drug resistant. Ten out of 16 (63%) K. pneumoniae isolates possessed blaOXA-48, which is the most common carbapenem resistance gene in Croatia and in other parts of Europe. All A. baumannii isolates possessed the OXA-23-like carbapenem hydrolysing oxacillinase and five turned out to be pandrug-resistant. Colistin resistance was most likely chromosomally mediated. After sequence analysis, none of the isolates were found to possess any of the mcr gene variants. Several previously reported mutations were found in PmrB, PhoP, PhoQ, and MgrB, which are associated with colistin resistance. In the global phylogenetic analysis, DNA mutations causing mutations in the MgrB protein were present mostly in lineages comprising colistin resistant isolates, and the second most prevalent mutation (K3X) was also encountered in our isolates. In addition, based on genotyping by rep-PCR, the spread of colistin resistance is most likely to be clonal. Most importantly, the presence of colistin resistance together with carbapenemase genes in extensively drug resistant isolates poses real threats in the use of carbapenems and colistin to fight infections.

Lesion size is associated with genetic polymorphisms in TLR1, TLR6, and TIRAP genes in patients with major abscesses and diabetic foot infections.

Genetic variation in Toll-like receptors (TLRs) has previously been associated with susceptibility to complicated skin and skin structure infections (cSSSIs). The aim of this study was to investigate associations between the severity of cSSSIs, i.e., major abscesses and diabetic foot infections (DFIs), and a set of genetic polymorphisms in the Toll-like receptor pathway. A total of 121 patients with major abscesses and 132 with DFIs participating in a randomized clinical trial were genotyped for 13 nonsynonymous single-nucleotide polymorphisms (SNPs) in genes coding for TLRs and the signaling adaptor molecule TIRAP. Infection severity was defined by lesion size at clinical presentation for both types of infections. The PEDIS infection score was also used to define severity of DFIs. Linear regression models were used to study factors independently associated with severity. In patients with large abscesses, hetero- or homozygosity for the allelic variant TLR6 (P249S) was associated with significantly smaller lesions while homozygosity for the allelic variant TLR1 (R80T) was associated with significantly larger lesions. PRRs genes were not significantly associated with PEDIS. However, patients with DFI hetero- or homozygous for the allelic variant TLR1 (S248N) had significantly larger lesions. Polymorphisms in TLR1 and TLR6 influence the severity of cSSSIs as assessed by the lesion size of major abscesses and DFIs. ClinicalTrial.gov Identifier: NCT00402727.