Effective high-throughput RT-qPCR screening for SARS-CoV-2 infections in children.

Systematic SARS-CoV-2 testing is a valuable tool for infection control and surveillance. However, broad application of high sensitive RT-qPCR testing in children is often hampered due to unpleasant sample collection, limited RT-qPCR capacities and high costs. Here, we developed a high-throughput approach ('Lolli-Method') for SARS-CoV-2 detection in children, combining non-invasive sample collection with an RT-qPCR-pool testing strategy. SARS-CoV-2 infections were diagnosed with sensitivities of 100% and 93.9% when viral loads were >106 copies/ml and >103 copies/ml in corresponding Naso-/Oropharyngeal-swabs, respectively. For effective application of the Lolli-Method in schools and daycare facilities, SEIR-modeling indicated a preferred frequency of two tests per week. The developed test strategy was implemented in 3,700 schools and 698 daycare facilities in Germany, screening over 800,000 individuals twice per week. In a period of 3 months, 6,364 pool-RT-qPCRs tested positive (0.64%), ranging from 0.05% to 2.61% per week. Notably, infections correlated with local SARS-CoV-2 incidences and with a school social deprivation index. Moreover, in comparison with the alpha variant, statistical modeling revealed a 36.8% increase for multiple (≥2 children) infections per class following infections with the delta variant. We conclude that the Lolli-Method is a powerful tool for SARS-CoV-2 surveillance and can support infection control in schools and daycare facilities.

Diagnostic performance of rapid influenza antigen assays in patients infected with the new influenza A (H1N1) virus.

BACKGROUND: The new swine influenza (H1N1) requires fast and accurate diagnosis. Currently, there are few reports about the diagnostic performance of influenza antigen tests with regard to the H1N1 virus infection. We evaluated the reactivity of eight commercially available rapid antigen tests in samples from confirmed infected patients. METHODS: From 23 patients tested positive by polymerase chain reaction (PCR), parallel testing with the antigen tests QuickVue Influenza A+B (Quidel Corporation), Binax NOW Flu A and B (Binax), Clearview Exact Influenza A and B (Inverness medical), TRUFLU (Meridian Bioscience), Directigen EZ Flu A+B (Becton Dickinson), Influenza A and B (Diagnostik Nord), Xpect Flu A and B (remel) and Influenza Antigen Quick (Generic Assays) was performed. RESULTS: Only few antigen tests showed positive reactivity in six of the 23 samples. The virus load of the reactive samples as indicated by the PCR cycle threshold was high. Negative results of rapid influenza tests do not rule out infection with the new influenza A (H1N1) virus. Their analytical sensitivity with reference to the virus load is low and not reproducible. CONCLUSIONS: As reliable laboratory results during a pandemic are essential, the diagnostic use of rapid influenza tests seems to be questionable.

Cord blood allergen-specific IgE is associated with reduced IFN-gamma production by cord blood cells: the Protection against Allergy-Study in Rural Environments (PASTURE) Study.

BACKGROUND: It is currently discussed whether allergic sensitization may start in utero under the influence of the maternal immune system and environmental determinants. OBJECTIVE: To investigate the relationship between allergen-specific cord blood (CB) IgE levels, parental sensitization, CB cytokine production, and environmental influences. METHODS: As part of an ongoing multicenter birth cohort study, allergen-specific IgE antibodies against 20 common seasonal, perennial, and food allergens were measured in blood samples from 922 neonates, 922 mothers, and 835 fathers. Supernatants from stimulated CB cells were assessed for the production of IL-5, IFN-gamma, IL-10, and TNF-alpha. RESULTS: Allergen-specific IgE antibodies were detectable in 23.9% of newborns. Contamination with maternal serum was excluded by several means of analyses, including the absence of IgA antibodies. Clear correlation between maternal and fetal IgE was found only for hen's egg, cow's milk, and soybean allergen. Fetal IgE correlated negatively with the level of IFN-gamma production, but not with IL-5 and IL-10. CONCLUSION: Allergen-specific IgE antibodies most probably of fetal origin are detectable in CB and correlate with a lowered CB IFN-gamma production.

Prenatal exposure to a farm environment modifies atopic sensitization at birth.

BACKGROUND: Previous cross-sectional surveys have suggested that maternal exposure to animal sheds during pregnancy exerted a protective effect on atopic sensitization in children lasting until school age. OBJECTIVE: We sought to evaluate the effects of maternal exposure to animal sheds and other farm-related exposures during pregnancy on cord blood IgE levels in a prospective birth cohort. METHODS: Pregnant women living in rural areas in Austria, Finland, France, Germany, and Switzerland were recruited in the third trimester of pregnancy. Information on maternal farm-related exposures, nutrition, and health during pregnancy was obtained by means of interviews. Specific IgE levels for food and common inhalant allergens were assessed in cord blood of 922 children and peripheral blood samples of their mothers. RESULTS: Different sensitization patterns in cord blood of farm and nonfarm children were observed. In multivariable analysis consumption of boiled, but not unboiled, farm milk during pregnancy was positively associated with specific IgE to cow's milk independently from maternal IgE. In contrast, there was an inverse relationship between maternal exposure to animal sheds and cord blood IgE levels against seasonal allergens (adjusted odds ratio, 0.38; 95% CI, 0.21-0.70). This association was not confounded by maternal IgE levels. Maternal contact with hay enhanced the protective effect of exposure to animal sheds on IgE levels to grass pollen in cord blood. CONCLUSIONS: Maternal exposure during pregnancy influences atopic sensitization patterns in cord blood. The (microbial) context of allergen contact possibly modifies the risk of atopic sensitization.

Interference in a homogeneous assay for low-density lipoprotein cholesterol by lipoprotein X.

BACKGROUND: Homogeneous assays for cholesterol in low-density lipoprotein (LDL) are currently in wide use for guideline-based diagnosis and monitoring of dyslipaemic or coronary conditions. In some sera from patients with impaired liver function, we measured implausibly low LDL concentrations using a sugar compound-based assay [LDL-cholesterol (LDL-C), Roche Diagnostics]. We investigated whether an interfering factor, possibly associated with cholestasis, is consistently responsible for this disturbance. METHODS: We compared results of the LDL-C assay in samples with implausible (n=158) and plausible (n=65) LDL concentrations with those of another assay based on two selective detergents (LDLD, Beckman Coulter) and with sequential density ultracentrifugation. We measured total bilirubin, triglycerides, bile acids and lipoprotein X (Lp X) concentrations in samples with the described disturbance and examined the effect of bile salt addition to normal samples. RESULTS: The LDL-C assay was negatively biased compared to the LDLD assay (bias -0.63 mmol/L) and sequential density ultracentrifugation (bias -0.85 mmol/L) in samples with an implausible lipoprotein profile, but showed good method agreement in all other samples. The bile acid concentration did not correlate with the LDL bias, and addition of bile acids showed no interference with the LDL-C assay. The Lp X concentration correlated with the bias between the LDL-C and LDLD assays (R=0.66, p<0.0001); there was no interference with the LDLD assay, even at high Lp X concentrations. CONCLUSIONS: We conclude that the LDL-C assay is subject to interference by Lp X and can provide grossly negatively biased results in cholestatic conditions. In such patients, LDL measurement with an assay based on a different method should be performed.

Analysis of hemostasis alterations in sepsis.

This laboratory study tested new methods to analyze hemostasis alterations in septic patients. Samples of ethylenediamine tetraacetic acid (EDTA) plasma and citrated plasma were collected from 62 patients with clinical diagnosis of sepsis. Additionally, a subset of EDTA-plasma samples from each patient was stabilized 1 + 1 with 2.5 mol/l arginine, pH 8.6, to conserve the real hemostasis activation state. EDTA-arginine plasma, EDTA plasma and citrated plasma samples were tested in duplicate. The patients at admission to the intensive care unit had 36 +/- 26 (normal, 0.8 +/- 0.2) ng/ml global endotoxin reactivity, 188 +/- 66% (normal, 100 +/- 20%) fibrinogen function, 179 +/- 66% (normal, 100 +/- 20%) fibrinogen antigen, 4.0 +/- 3.6 (normal, 0.049 +/- 0.025) microg/ml D-dimer, 313 +/- 307% (normal, 100 +/- 30%) plasmin-antiplasmin complex, 8.7 +/- 11.4 (normal, 1.1 +/- 0.7) U/ml plasminogen activator inhibitor-1, 12.1 +/- 10.5 (normal, 1.3 +/- 0.4) ng/ml thrombin-antithrombin III complex, 173 +/- 62% (normal, 100 +/- 20%) thrombin, 568 +/- 225 (normal, 140 +/- 42) pg/ml tissue factor, and 2.56 +/- 2.48 (normal, 0.19 +/- 0.04) microg/ml soluble intercellular adhesion molecule-1. Endotoxin (lipopolysaccharide and/or beta-glucan) reactivity (EDTA plasma), fibrinogen function + antigen + ratio and plasminogen activator inhibitor-1 (citrated plasma), and D-dimer, soluble intercellular adhesion molecule-1, thrombin activity (EDTA-arginine-stabilized plasma) presented large aberrations in septic patients when compared with normal values and may therefore be particularly interesting as markers of hemostasis alteration. Whether the observed alterations are of clinical significance has to be determined in well defined patient groups.

Brain natriuretic peptide for prediction of Cheyne-Stokes respiration in heart failure patients.

Cheyne-Stokes respiration (CSR) is indicative of adverse outcome in patients with chronic heart failure (CHF). We evaluated the use of brain natriuretic peptide (BNP) plasma levels to predict CSR in CHF patients. In this cross-sectional study, overnight polygraphy and cardiac work-up were performed and neurohumoral activation was determined in 102 consecutive CHF patients (25-82 years). Demographic characteristics did not significantly differ among patients with (n=38) or without CSR (n=64); BNP (median: 377 vs. 142 pg/ml, p<0.001) and norepinephrine levels (459+/-283 vs. 346+/-204 pg/ml, p=0.02) were significantly increased in patients with CSR. BNP concentrations were significantly associated with the central apnoea/hypopnoea index (y=253+/-5.3x; r=0.26, p=0.01). The area under the ROC curve that used BNP to predict CSR was 0.780 (95% CI: 0.688 to 0.873). Using established cut-off limits of BNP plasma levels, heart failure patients with BNP levels >500 pg/ml displayed a 13 fold increased risk of CSR (95% CI: 2.34-73.50; p=0.03) compared to patients with BNP levels <100 pg/ml. In multiple logistic regression analysis p(a)CO2 (point estimate 0.84, 95% CI: 0.72 to 0.98; p=0.02) and higher BNP class (point estimate 3.14, 95% CI: 1.38-7.144; p=0.006) emerged as parameters independently predicting the presence of CSR in our cohort of CHF patients. In conclusion, CSR is associated with neurohumoral activation in CHF patients. Specifically, BNP levels are associated with the severity of cardiac and sleep-related disease, and may be helpful in the diagnosis of CSR and more appropriate use of polysomnography in CHF patients.

B-type natriuretic peptide levels predict event-free survival in patients with implantable cardioverter defibrillators.

INTRODUCTION: We examined whether B-type natriuretic peptide (BNP) levels predict outcome in heart failure patients with implantable cardioverter defibrillators (ICD) using a combined endpoint of malignant tachyarrhythmias, death or heart transplantation. METHODS AND RESULTS: BNP levels were measured in 123 ICD patients with chronic heart failure (age: 63+/-12 years, ejection fraction: 29+/-10%). After a median follow-up of 25 months, the combined endpoint was reached in 28 patients (first tachyarrhythmic event, n=16; death, n=11; heart transplantation, n=1). BNP levels were significantly lower in patients with event-free survival compared to patients reaching the combined endpoint of this study (median: 140 vs. 373 pg/ml; p<0.001). Multivariable Cox regression analysis revealed that BNP levels predict adverse outcome (RR 1.002 per pg/ml increment; 95% CI: 1.001-1.003; p<0.001) and use of beta-blockers was associated with favourable outcome (RR 0.319; 95% CI 0.151-0.670; p=0.004). LV ejection fraction (p=0.66) did not significantly predict event-free survival in multivariable analysis. CONCLUSIONS: BNP plasma levels are useful markers to predict event-free survival in ICD patients with heart failure. Of note, malignant tachyarrhythmias appear responsible for about 50% of fatal outcomes. Our findings suggest that determination of BNP plasma levels is more valuable than determining LV ejection fraction to anticipate event-free survival in this population.

Diagnostic and analytical performance of a screening panel for allergy.

Worldwide, allergic diseases are increasing in prevalence and incidence. Early assessment of the immunoglobulin E (IgE) sensitisation status has a major impact on clinical outcome and selection of therapeutic options. Recently, a number of new IgE-detecting test systems have entered the market, including screening tests allowing identification of a wide spectrum of sensitising allergens. We evaluated the analytical and diagnostic performance of the newly developed Allergy Screen test panel for atopy (Mediwiss Analytic, Moers, Germany). The evaluation was performed for four major respiratory and four major nutritional allergens in 142 patients with clinical suspicion of respiratory and/or food allergies. For all allergens, the test showed acceptable concordance to the skin-prick test and the in vitro IgE CAP system (Pharmacia, Freiburg, Germany). The analytical performance was acceptable, with CVs between 2 and 8% in the positive range and good dilution linearity (R=0.9735). Imprecision in the low IgE concentration range dramatically improved by lowering the cut-off value to 0.2 IU/mL IgE. In conclusion, the Allergy Screen panel yields reliable results in the detection of allergic sensitisation to common allergens.

Value of heart rate variability to predict ventricular arrhythmias in recipients of prophylactic defibrillators with idiopathic dilated cardiomyopathy.

This study investigated the relation between heart rate variability (HRV) measured as standard deviation of normal to normal RR intervals (SDNN) on baseline 24-hour ambulatory electrocardiogram (ECG) and subsequent appropriate implantable cardioverter defibrillator (ICD) interventions in 70 patients with idiopathic dilated cardiomyopathy (IDC) in whom ICDs were implanted prophylactically in the presence of a low left ventricular ejection fraction (LVEF). During 43 +/- 26 months of follow-up, 26 of 70 (37%) study patients with IDC received one or more appropriate ICD interventions for sustained ventricular tachycardia (VT) or ventricular fibrillation (VF) documented by electrograms stored in the ICD. Mean SDNN at ICD implant was 94 +/- 33 ms. No difference was found between patients with (90 +/- 25 ms) versus without (96 +/- 37 ms) appropriate ICD interventions for VT or VF during follow-up. Multivariate Cox regression analysis of baseline clinical characteristics including age, gender, LVEF, NYHA functional class, nonsustained VT on Holter, history of syncope, left bundle branch block, baseline medication and HRV revealed LVEF as the only significant predictor of arrhythmia. These findings do not support the use of HRV measured as SDNN on 24-hour ambulatory ECG to select patients with IDC for prophylactic ICD therapy.