[Diagnostic algorithm for COVID-19 at the ER]. / Diagnostisch algoritme voor COVID-19 op de SEH.

OBJECTIVE: Evaluation of a diagnostic algorithm for estimating the risk of COVID-19 in patients who are referred to an emergency department for being suspected of having the disease. DESIGN: Retrospective study. METHOD: Patients with fever with no apparent cause and patients with recently developed respiratory symptoms, whether or not in combination with fever, were routinely given a PCR test, blood tests (lymphocyte count and LDH levels) and a chest CT scan. The CT scan was assessed according to the CO-RADS classification. Based on the findings, the patients were divided into 3 cohorts (proven COVID-19, strong suspicion of COVID-19, and low suspicion of COVID-19) and the appropriate isolation measures were taken. RESULTS: In the period from 8 to 31 March 2020, the algorithm was applied to 312 patients. COVID-19 was proven for 69 (22%) patients. COVID-19 was strongly suspected for 151 (48%) patients and suspicion was low for the remaining 92 (29%) patients. The percentage of patients with positive PCR results and the percentage of patients with abnormal laboratory test results increased as the CO-RADS score increased. Among patients with a CO-RADS score of 4 or 5, this percentage increased further when they also had lymphopenia or elevated LDH levels. We have adjusted the flowchart based on our findings. CONCLUSION: In case of patients who have been referred to an emergency department for suspected COVID-19, a good COVID-19 risk assessment can be made on the basis of clinical signs, laboratory abnormalities and low-dose CT scans. Even before the results of the PCR test are known and even if the results are negative, patients can be classified as 'proven COVID-19 patients' using the algorithm.

Serum concentrations of amoxicillin in neonates during continuous intravenous infusion.

Amoxicillin is commonly used for the treatment of neonatal bacterial infection with intermittent dosing (ID) regimens. However, increasing bacterial resistance, in addition to a lack of new antimicrobial agents, urges the optimization of current therapeutic options. Clinical studies in adults suggest continuous infusion (CI) regimens of beta-lactam antibiotics to be superior to ID. There are as yet no guidelines concerning the CI dosing of amoxicillin. The present study was developed to describe the CI pharmacokinetics and -dynamics of amoxicillin during the first 3 days of life in search of the optimal dosing regimen. Neonates with a gestational age above 34 weeks, at risk of neonatal infection and requiring amoxicillin therapy, were included. Serum concentrations of amoxicillin were measured during CI on days 1 and 3 in the steady state. Twenty-two serum samples of 11 patients were collected. All patients reached and retained serum concentrations of amoxicillin within the therapeutic range without exceeding the toxic concentration (serum concentrations on day 1 mean 55.4 mg/l, range 30.9-69.5, SD 10.5, and on day 3 48.8 mg/l, range 25.5-92.4, SD 18.4). There was no significant decrease in concentration from day 1 to day 3 (p = 0.38). This study showed therapeutic, nontoxic concentrations of amoxicillin in neonates on CI of amoxicillin in the first 3 days of life. Randomized controlled trials should reveal whether the clinical benefits of the CI of amoxicillin exceed those of ID regimens.

Serology in chronic Q fever is still surrounded by question marks.

Detection of antibodies using immunofluoresence tests (IFAT) is recommended for diagnosis of chronic Q fever, but other commercial antibody assays are also available. We compared an enzyme-linked immunosorbent assay (ELISA) (Virion/Serion) and a complement fixation test (CFT) (Virion/Serion) for the detection of Coxiella burnetii IgG phase I and IgA phase I in early- and follow-up serum samples from patients with chronic Q fever, diagnosed according to an algorithm that involves IFAT. For this, we tested sera of 49 patients, including 30 proven, 14 probable and five possible chronic Q fever cases. Sensitivity of CFT for diagnosis of chronic Q fever was suboptimal (85 %), as eight patients, including five with chronic Q fever, tested negative at time of diagnosis, whereas IgG phase I antibodies were detected in these five patients by ELISA. Sensitivity of ELISA was higher, although three probable patients were missed. No differences in ELISA IgA phase I detection between proven chronic Q fever and probable were observed; instead possible patients were in majority IgA negative (60 %). Serological examination using ELISA and CFT in follow-up sera from 26 patients on treatment was unsatisfactory. Like IFAT, all kinetic options were possible: decreasing, remaining stable or even increase during time. This study demonstrated that the sensitivity of CFT-based phase I antibody detection is low and therefore not recommended for diagnosis of chronic Q fever. Based on our results, serological follow-up to guide treatment decisions was of limited value.

A combination of interferon-gamma and interleukin-2 production by Coxiella burnetii-stimulated circulating cells discriminates between chronic Q fever and past Q fever.

Infection with Coxiella burnetii may lead to life-threatening chronic Q fever endocarditis or vascular infections, which are often difficult to diagnose. The present study aims to investigate whether measurement of in-vitro interferon-gamma (IFN-γ) production, a key cytokine in the immune response against C. burnetii, differentiates chronic from a past cleared infection, and whether measurement of other cytokines would improve the discriminative power. First, C. burnetii-specific IFN-γ production was measured in whole blood of 28 definite chronic Q fever patients and compared with 135 individuals with past Q fever (seropositive controls) and 908 seronegative controls. IFN-γ production was significantly higher in chronic Q fever patients than in controls, but with overlapping values between patients and seropositives. Secondly, the production of a series of other cytokines was measured in a subset of patients and controls, which showed that interleukin (IL)-2 production was significantly lower in patients than in seropositive controls. Subsequently, measuring IL-2 in all patients and all controls with substantial IFN-γ production showed that an IFN-γ/IL-2 ratio >11 had a sensitivity and specificity of 79% and 96%, respectively, to diagnose chronic Q fever. This indicates that a high IFN-γ/IL-2 ratio is highly suggestive for chronic Q fever. In an additional group of 25 individuals with persistent high anti-Coxiella phase I IgG titres without definite chronic infection, all but six showed an IFN-γ/IL-2 ratio <11. In conclusion, these findings hold promise for the often difficult diagnostic work-up of Q fever and the IFN-γ/IL-2 ratio may be used as an additional diagnostic marker.

Plasmodium falciparum malaria recrudescence occurring 2.5 years after leaving an endemic country.

Malaria tropica is almost exclusively diagnosed within two months after returning from an endemic country. We present here a male patient with severe P. falciparum malaria diagnosed 2.5 years after returning from Burkina-Faso. We speculate that our patient was chronically infected with PF malaria for more than 2 years, with an undetectable parasite index and without symptoms. Because of waning immunity clinically overt PF malaria was able to develop. This case illustrates the importance of malaria suspicion as a cause of illness in immigrants from malaria-endemic countries. Even when these immigrants did not travel for a long time, malaria should be considered in patients with typical symptoms.

Chronic Q fever: review of the literature and a proposal of new diagnostic criteria.

A review was performed to determine clinical aspects and diagnostic tools for chronic Q fever. We present a Dutch guideline based on literature and clinical experience with chronic Q fever patients in The Netherlands so far. In this guideline diagnosis is categorized as proven, possible or probable chronic infection based on serology, PCR, clinical symptoms, risk factors and diagnostic imaging.

von Willebrand factor activation, granzyme-B and thrombocytopenia in meningococcal disease.

SUMMARY BACKGROUND: During invasive meningococcal disease, severe thrombocytopenia is strongly associated with a poor outcome. OBJECTIVES: In order to elucidate the pathophysiological mechanism behind the development of thrombocytopenia, we studied the role of von Willebrand factor (VWF) in meningococcal disease. PATIENTS/METHODS: Thirty-two children with severe meningococcal disease admitted to our university hospital were included in this study. VWF and related parameters were measured and results were correlated with the development of shock and thrombocytopenia. RESULTS: At admission, all patients had increased levels of (active) VWF and VWF propeptide. The highest VWF propeptide levels were observed in patients with shock, indicating acute endothelial activation. Although VWF propeptide levels in patients with shock, with or without thrombocytopenia, were similar, increased active VWF was significantly lower in patients with thrombocytopenia as compared with patients without thrombocytopenia. ADAMTS13 was moderately decreased. However, the VWF multimeric pattern was minimally increased. We assume that these findings are explained by VWF consumption and perhaps by granzyme B (GrB). In vitro experiments showed that GrB is able to cleave VWF multimers in plasma, whereas GrB was high in patients with shock, who developed thrombocytopenia. CONCLUSIONS: Our results demonstrate that consumption of VWF, derived from endothelial cells, could be a key feature of meningococcal disease and primary to the development of thrombocytopenia during shock.

Serum bactericidal activity against Helicobacter pylori in patients with hypogammaglobulinaemia.

The two major primary antibody deficiency disorders are X-linked hypogammaglobulinaemia (XLA) and common variable immunodeficiency (CVID). CVID patients have an elevated risk for gastric cancer and extra-nodal marginal zone lymphoma. Both diseases are associated with Helicobacter pylori infection. We investigated whether antibody deficiency leads to defective serum bactericidal activity against H. pylori. We also investigated the correlation with immunoglobulin (Ig)M levels and observed the terminal complement complex (TCC) activity. Sera of 13 CVID patients (four H. pylori positive), one patient with hyper-IgM syndrome, one patient with Good syndrome (both H. pylori positive), five XLA patients, four H. pylori seropositive controls, four H. pylori seronegative controls and a sample of pooled human serum (PHS) were incubated in vitro with bacterial suspensions of H. pylori for 30 min. After 72 h of culture, colony-forming units were counted. TCC formation was measured by enzyme-linked immunosorbent assay. We found that normal human serum is bactericidal for H. pylori, whereas heat-inactivated serum shows hardly any killing of H. pylori. Serum (1%) of hypogammaglobulinaemia patients has a decreased bactericidal activity against H. pylori. Helicobacter pylori-positive (HP(+)) normal individuals show more than 90% killing of H. pylori, whereas CVID patients show 35% killing (P = 0.007) and XLA patients only 19% (P = 0.003). Serum (1%) of HP(+) volunteers showed significantly better killing compared with serum of H. pylori-negative (HP(-)) volunteers (P = 0.034). No correlation between (substituted) IgG levels and serum bactericidal activity was found, but a weak correlation between total serum IgM and serum bactericidal activity was found. In conclusion, serum bactericidal activity against H. pylori is decreased in patients with hypogammaglobulinaemia. Heat treatment of the serum abolished the bactericidal capacity, indicating that complement activity is essential for the bactericidal effect.

The expression of toll-like receptors 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of toll-like receptors 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells.

OBJECTIVE: To evaluate the expression of Toll-like receptors (TLRs) 3 and 7 in synovium and to study potential differences in the maturation and cytokine production mediated by TLR-2, TLR-3, TLR-4, and TLR-7/8 by dendritic cells (DCs) from rheumatoid arthritis (RA) patients and DCs from healthy controls. METHODS: Synovial expression of TLR-3 and TLR-7 in RA was studied using immunohistochemistry. Monocyte-derived DCs from RA patients and healthy controls were cultured for 6 days and subsequently stimulated for 48 hours via TLR-mediated pathways (lipoteichoic acid, Pam(3)Cys, and fibroblast-stimulating lipopeptide 1 for TLR-2, poly[I-C] for TLR-3, lipopolysaccharide and extra domain A for TLR-4, and R848 for TLR-7/8). Phenotypic DC maturation was measured using flow cytometry. The secretion of tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), IL-10, and IL-12 was measured using the Bio-Plex system. Cell lines expressing TLR-2 and TLR-4 were used for the detection of TLR-2 and TLR-4 ligands in serum and synovial fluid from RA patients. RESULTS: TLR-3 and TLR-7 were highly expressed in RA synovium. All TLR ligands elicited phenotypic DC maturation equally between DCs from RA patients and those from healthy controls. TLR-2- and TLR-4-mediated stimulation of DCs from RA patients resulted in markedly higher production of inflammatory mediators (TNFalpha and IL-6) compared with DCs from healthy controls. In contrast, upon stimulation of TLR-3 and TLR-7/8, the level of cytokine production was equal between DCs from RA patients and those from healthy controls. Remarkably, both TLR-3 and TLR-7/8 stimulation resulted in a skewed balance toward IL-12. Intriguingly, the combined stimulation of TLR-4 and TLR-3-7/8 resulted in a marked synergy with respect to the production of inflammatory mediators. As a proof of concept, TLR-4 ligands were increased in the serum and synovial fluid of RA patients. CONCLUSION: TLRs are involved in the regulation of DC activation and cytokine production. We hypothesize that various TLR ligands in the joint trigger multiple TLRs simultaneously, favoring the breakthrough of tolerance in RA.

Lethal Escherichia coli and Salmonella typhimurium endotoxemia is mediated through different pathways.

Despite the differences in the molecular structure between lipopolysaccharides (LPS) isolated from Escherichia coli, Klebsiella pneumoniae or Salmonella typhimurium, the potential differences in their biological effects in vivo have not been investigated. In the present study, TNF and LT double knock-out (TNF-/-LT-/-) mice were almost as susceptible as TNF+/+LT+/+ controls to S. typhimurium LPS, but they were significantly more resistant to lethal endotoxemia induced by E. coli or K. pneumoniae LPS. The effect was not due to endotoxin-associated proteins. In the knock-out mice, this difference in lethality was accompanied by decreased interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) production after challenge with E. coli LPS, whereas after S. typhimurium LPS more IL-1 and IFN-gamma were produced. In contrast, more IL-10 was produced after challenge of mice with E. coli LPS than with S. typhimurium LPS. The hypothesis that a combination of pro-inflammatory cytokines is responsible for the mortality after S. typhimurium LPS was suggested by experiments in mice deficient in IL-1beta-converting enzyme (ICE-/- mice). ICE-/-mice, lacking mature IL-1beta and IL-18, but also defective in IFN-gamma and TNF production, were completely protected against both E. coli and S. typhimurium LPS. Experiments in Toll-like receptor (TLR)-4 defective mice suggested that the difference is not due to differential activation of TLR4. In conclusion, TNF and LT play a central role in the lethality due to E. coli LPS, whereas the lethal effects of S. typhimurium LPS are mediated through mechanisms also involving other cytokines such as IFN-gamma, IL-1 and IL-18.

Contributions of Neisseria meningitidis LPS and non-LPS to proinflammatory cytokine response.

To determine the relative contribution of lipopolysaccharide (LPS) and non-LPS components of Neisseria meningitidis to the pathogenesis of meningococcal sepsis, this study quantitatively compared cytokine induction by isolated LPS, wild-type serogroup B meningococci (strain H44/76), and LPS-deficient mutant meningococci (strain H44/76[pLAK33]). Stimulation of human peripheral-blood mononuclear cells with wild-type and LPS-deficient meningococci showed that non-LPS components of meningococci are responsible for a substantial part of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta production and virtually all interferon (IFN)-gamma production. Based on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LPS in proteinase K-treated lysates of N. meningitidis H44/76, a quantitative comparison was made between the cytokine-inducing capacity of isolated and purified LPS and LPS-containing meningococci. At concentrations of >10(7) bacteria/mL, intact bacteria were more potent cytokine inductors than equivalent amounts of isolated LPS, and cytokine induction by non-LPS components was additive to that by LPS. Experiments with mice showed that non-LPS components of meningococci were able to induce cytokine production and mortality. The principal conclusion is that non-LPS parts of N. meningitidis may play a role in the pathogenesis of meningococcal sepsis by inducing substantial TNF-alpha, IL-1beta, and IFN-gamma production.