Collaborative study to evaluate a candidate World Health Organization international standard for chikungunya virus for nucleic acid amplification technique (NAT)-based assays

Collaborative Study Group - Portugal:Maria João Alves, Líbia Zé-Zé (National Institute of Health Dr. Ricardo Jorge, Center for Vectors and Infectious Diseases Research)This report describes the World Health Organization (WHO) project to develop an international standard (IS) for Chikungunya virus (CHIKV) RNA for use with nucleic acid amplification technique (NAT)-based assays. An international collaborative study was conducted to determine the potency of the candidate standard using a range of NAT-based assays for CHIKV, and to evaluate the suitability of the candidate for the calibration of secondary reference materials and the standardization of CHIKV viral load measurements. The candidate standard consisted of a heat inactivated CHIKV strain of the East/South/Central African genotype (ESCA), also known as the Indian Ocean Lineage, isolated from a patient returning from India to the United States in 20061 , diluted in human negative plasma. The lyophilized candidate preparation (Sample 1), the corresponding liquid-frozen bulk material (Sample 2) and three different clinical samples (Sample 3, Sample 4 and Sample 5) were included in the collaborative study. Twenty-five laboratories representing 14 countries participated in the study to evaluate the material using their routine CHIKV NAT assays. Twenty-four laboratories returned 31 data sets from 17 commercial assays and 14 in-house methods. Of these 31 methods, 11 were quantitative and 20 were qualitative. The results of the study indicate the suitability of the candidate material of the CHIKV strain of ESCA genotype (Sample 1) as the proposed 1st WHO IS for CHIKV. It is therefore proposed that the candidate material (PEI code 11785/16) is established as the 1st WHO IS for CHIKV RNA for NAT-based assays with an assigned potency of 2,500,000 International Units (IU)/mL when reconstituted in 0.5 mL of nuclease-free water. On-going studies for real-time and accelerated stability of the proposed IS indicate that the preparation is stable and suitable for long-term use under the proposed storage conditions.info:eu-repo/semantics/publishedVersio

Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions

Drug-induced anaphylaxis—elicitors, mechanisms and diagnosis

Drugs are one of the major causes of anaphylaxis. For example 2346 cases of drug-induced anaphylaxis were reported to the anaphylaxis register as of March 2019. The most common triggers of drug-induced anaphylaxis were nonsteroidal anti-inflammatory drugs (NSAIDs; n = 902) and antibiotics (n = 721). Drug-induced anaphylaxis can be caused by IgE-dependent (e.g., penicillins) and IgE-independent mechanisms. Recently MRG-PX2 has been identified as a receptor for non-IgE-dependent mechanisms. Drug-induced anaphylaxis results more frequently in lethal reactions and is more commonly associated with cardiovascular symptoms. Also therapy refractory anaphylaxis is more frequently triggered by drugs. For the diagnosis of drug-induced anaphylaxis current national and international guidelines should be followed including provocation tests to avoid future reactions

First WHO International Reference Panel containing hepatitis B virus genotypes A-G for assays of the viral DNA

Background: WHO International Standards (IS) are provided for the calibration and validation of diagnostic and screening assays, e.g. for hepatitis B virus (HBV). HBV forms numerous subgenotypes and the current IS for HBV DNA reflects subgenotype A2. Objective: A reference panel with the most prevalent subgenotypes should facilitate evaluation of genotype-specific detection efficiencies. Study design: 215 HBV positive plasma samples collected worldwide were characterized for HBV markers and sequenced. Fifteen subgenotype A1, A2, B2, B4, C2, D1, D3, E, F2 and G samples were selected for the panel. The lyophilized samples were tested in parallel with the IS in an international collaborative study with 16 laboratories using 13 different nucleic acid amplification techniques (NATs). Results: Eight of 13 NAT had a HBV DNA detection efficiency which was independent of the genotype and consistent with the IS, while with five assays, certain deviations were noted, particularly with genotype F which was under quantitated or even missed by three assays. The panel was accepted by the WHO as the " 1st WHO International Reference Panel for HBV Genotypes for HBV NAT-Based Assays". Conclusions: The evaluation of HBV DNA assays should include many different genotypes. The WHO Reference Panel is universally available for manufacturers of HBV DNA assays, diagnostic laboratories and control authorities to facilitate standardized validation of HBV genotype specific detection efficiency of both diagnostic (quantitative and qualitative) and screening NAT assays.Fil: Chudy, M.. No especifíca;Fil: Hanschmann, K. M.. No especifíca;Fil: Kress, J.. No especifíca;Fil: Nick, S.. No especifíca;Fil: Campos, Rodolfo Hector. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wend, U.. No especifíca;Fil: Gerlich, W.. No especifíca;Fil: Nübling, C. M.. No especifíca

Assessment of component-resolved in vitro diagnosis of celeriac allergy

BACKGROUND: Previous studies have demonstrated insufficient sensitivity of commercially available celeriac extract reagents in the diagnosis of celeriac allergy. OBJECTIVE: We sought to assess the diagnostic performance of specific IgE determination based on recombinant and purified natural celeriac allergens in comparison with an extract-based assay and to investigate interference by IgE to cross-reactive carbohydrate determinants and its biologic activity. METHODS: Twenty-four subjects with a positive double-blind, placebo-controlled food challenge result to celeriac; 20 atopic control subjects with birch pollen allergy who tolerated celeriac; and 20 nonatopic subjects were enrolled. IgE binding was investigated for celeriac allergens (rApi g 1.01, rApi g 4, and nApi g 5), extract reagents (celeriac, birch, mugwort, and timothy grass pollen), birch pollen allergens (rBet v 1 and rBet v 2), and cross-reactive carbohydrate determinants by means of ImmunoCAP analysis. Biologic activity of allergens was determined based on basophil mediator release. RESULTS: Component-resolved ImmunoCAP analysis considerably increased the sensitivity to detect celeriac-specific IgE by 20%. Sensitization to carbohydrate structures was detected in 38% of patients with celeriac allergy, and there was an excellent correlation between sensitization to the glycoprotein Api g 5 and isolated glycan. Positive results among atopic control subjects were mainly caused by protein allergens, whereas the effect of carbohydrate epitopes was marginal. The ability of allergens to induce mediator release decreased in the order Bet v 1 > Api g 1 > Api g 5, confirming the low biologic activity of IgE to carbohydrate epitopes. CONCLUSION: Component-resolved diagnosis allowed an increase in diagnostic sensitivity from 67% to 88% compared with extract-based diagnosis. Sensitization to Api g 5 was attributable to its glycan moieties but did not interfere with diagnostic specificity