المؤلفون: Astrid Versluis, Thuy‐My Le, Francine C. van Erp, Mark A. Blankestijn, Geert F. Houben, André C. Knulst, Harmieke van Os‐ Medendorp

المصدر: Clinical and Translational Allergy, Vol 12, Iss 2, Pp n/a-n/a (2022)

مصطلحات موضوعية: erwachsene, diät einhaltung, ernährungsberatung, nahrungsmittelallergien, provokationstestung, Immunologic diseases. Allergy, RC581-607

الوصف: Abstract Background After a positive food challenge (FC), patients receive dietary advice regarding avoidance of the culprit food. We examined the frequency and variables associated with dietary adherence after a positive FC in adults. Methods In this prospective daily practice study, adults with a positive FC were included. After every FC, dietary advice was given consisting of three options: (1) strict avoidance, (2) avoidance but products with precautionary allergen labelling (PAL) allowed and (3) (small) amounts allowed. Questionnaires about dietary adherence and associated variables were completed prior to and 6 months after the FC(s). Results 41 patients (with 58 positive FCs) were included. Overall, patients adhered to the advised diet after 31% of the FCs. After 33 FCs, the advice was strict avoidance, whereof 82% followed a less strict diet. After 16 FCs, the advice was avoidance but products with PAL allowed, whereof 19% followed a less strict and 25% a stricter diet. In 9 FCs with the least strict advice, “(small) amounts allowed’’, 67% followed a stricter diet. Three variables were associated with adherence: misremembering dietary advice, impaired health‐related quality of life (HRQL) on domain “Emotional impact’’ and the need for dietary change after the FC. Conclusion After one third of the positive FCs, patients adhered to the dietary advice. Variables associated with adherence were misremembering dietary advice, impaired HRQL on domain “Emotional impact’’ and the need for dietary change after the FC. It seems important that healthcare professionals should more frequently apply adherence‐enhancing strategies to improve dietary adherence.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2045-7022Test

الوصول الحر: https://doaj.org/article/24379545fddb4794a3aefd655fe1eae2Test

المؤلفون: Anna M. Ehlers, Madlen Rossnagel, Bettina Brix, Mark A. Blankestijn, Thuy-My Le, Waltraud Suer, Henny G. Otten, André C. Knulst

المصدر: Clinical and Translational Allergy, Vol 9, Iss 1, Pp 1-5 (2019)

مصطلحات موضوعية: Diagnostics, Food allergy, IgE hypersensitivity3 oleosins, Sesame, Immunologic diseases. Allergy, RC581-607

الوصف: Abstract Background In daily practice, one-third of sesame allergic patients, confirmed by clinical history or food challenge, do not show any detectable specific IgE using current diagnostics. Currently used sesame extracts are water-based and therefore lacking hydrophobic proteins like oleosins. Oleosins, the stabilizer of lipid droplets in plants, are described as allergens in sesame, peanut and hazelnut. In this study, we examine the role of oleosins in sesame allergy and their potential cross-reactivity between sesame and (pea)nuts. Methods Specific IgE and IgG sensitisation to native and heterologously expressed sesame components and oleosins from other nuts, free of seed storage proteins, was assessed by line blot and sera from 17 sesame allergic patients without detectable specific IgE sensitisation to sesame, and compared to 18 sesame allergic and 13 tolerant patients with specific IgE sensitisation to sesame. Results Sesame allergic patients without sensitisation showed no specific IgE to the tested sesame oleosins or components. Low levels of specific IgE to sesame oleosins were detected in 17% of sesame allergic and 15% of tolerant patients with sIgE sensitisation. Oleosins were recognised by serum IgG from multiple patients confirming immune reactivity and excluding technical issues leading to lack of specific IgE-binding to oleosins. Conclusion Sesame oleosins are minor allergens and appear to have no additonal value in diagnosing sesame allergy in adults based on sIgE and sIgG detection. There is a high need for additional diagnostic tools in those patients to minimize the number of required food challenges.

وصف الملف: electronic resource

العلاقة: http://link.springer.com/article/10.1186/s13601-019-0271-xTest; https://doaj.org/toc/2045-7022Test

الوصول الحر: https://doaj.org/article/7ca31c986b4c420f9328a0665a7ff0a3Test

المؤلفون: Mark A. Blankestijn, André C. Knulst, Edward F. Knol, Thuy-My Le, Heike Rockmann, Henny G. Otten, Rob J. B. Klemans

المصدر: Clinical and Translational Allergy, Vol 7, Iss 1, Pp 1-8 (2017)

مصطلحات موضوعية: ImmunoCAP ISAC, Food allergy, PR-10, Specific IgE, Storage proteins, Immunologic diseases. Allergy, RC581-607

الوصف: Abstract Background The extent of co-sensitization within and between food protein families in an adult population is largely unknown. This study aimed to identify the most frequently recognized components in the PR-10 and storage protein family, as well as patterns in (co-)sensitization, in a birch-endemic area. Methods Results of ImmunoCAP ISAC, performed during routine care in Dutch adult outpatients suspected of food allergy, were collected. Results A total of 305 patients were selected, aged 16–79 years (median 32 years). Sensitization to one or more PR-10 proteins was most frequent (74% of all subjects), followed by 35% to storage protein and 15% to nsLTPs. Within the PR-10 family, subjects were most often sensitized to Bet v 1 (73% of 305), Cor a 1.04 (72%) and Mal d 1 (68%). Sensitization to PR-10s from soy, celery and kiwi occurred distinctively less often ( 70%). Subjects sensitized to these ‘less common PR-10 proteins’ were sensitized to more food and inhalant components on the ISAC, compared to subjects sensitized to ‘common PR-10 proteins’ (median 22 vs 13 out of 112, p

وصف الملف: electronic resource

العلاقة: http://link.springer.com/article/10.1186/s13601-017-0177-4Test; https://doaj.org/toc/2045-7022Test

الوصول الحر: https://doaj.org/article/074b38feaf9243228f089713f41c6cbfTest

المؤلفون: André C. Knulst, Harmieke van Os-Medendorp, Astrid Versluis, Yolanda Meijer, Francine C. van Erp, Mark A. Blankestijn, Thuy-My Le

المصدر: Clinical and Experimental Allergy

مصطلحات موضوعية: 0301 basic medicine, Adult, Male, medicine.medical_specialty, Adolescent, Immunology, Aftercare, Anxiety, Culprit, food challenge, 03 medical and health sciences, 0302 clinical medicine, Quality of life, Food allergy, Internal medicine, Surveys and Questionnaires, medicine, Immunology and Allergy, Humans, Clinical significance, Prospective Studies, Prospective cohort study, Clinical Allergy, reintroduction, Aged, Univariate analysis, food allergy, business.industry, Feeding Behavior, Middle Aged, medicine.disease, 030104 developmental biology, 030228 respiratory system, Telephone interview, Food, Quality of Life, Original Article, Female, medicine.symptom, ORIGINAL ARTICLES, business, Food Hypersensitivity

الوصف: BACKGROUND: Reintroduction of a food after negative food challenge (FC) faces many obstacles. There are no studies available about this subject in adults. OBJECTIVE: To investigate the frequency, reasons and risk factors of reintroduction failure in adults. METHODS: In this prospective study, adult patients received standardized follow-up care after negative FCs including a reintroduction scheme and supportive telephone consultations. Data were collected by telephone interview (2 weeks after FC) and questionnaires (at baseline and 6 months after FC(s)): food habits questionnaire, State-Trait Anxiety Inventory, Food Allergy Quality of Life Questionnaire-Adult Form and Food Allergy Independent Measure. Frequency and reasons of reintroduction failure were analysed using descriptive statistics and risk factors with univariate analyses. RESULTS: Eighty patients were included with, in total, 113 negative FCs. Reintroduction failed on short-term (2 weeks after FC) in 20% (95% CI: 13%-28%). Common reasons were symptoms upon ingestion during the reintroduction scheme (50%) and no need to eat the food (23%). On the long-term (5-12 months after FC(s)), reintroduction failure increased to 40% (95% CI: 28%-53%). Common reasons were atypical symptoms after eating the food (59%) and fear for an allergic reaction (24%). Five risk factors for long-term reintroduction failure were found: if culprit food was not one of the 13 EU regulated allergens, reintroduction failure at short-term, atypical symptoms during FC, a lower quality of life and a higher state anxiety. CONCLUSIONS AND CLINICAL RELEVANCE: Reintroduction failure after negative FCs in adults is common, increases over time, and is primarily due to atypical symptoms. This stresses the need for more patient-tailored care before and after negative food challenges.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::cacc1a980571149e2982eb65894f5233Test
http://europepmc.org/articles/PMC7187472Test

المؤلفون: Mark A. Blankestijn, Anna M. Ehlers, André C. Knulst, Marco Klinge, Henny G. Otten

المصدر: Clinical and Experimental Allergy, 49(1), 17. Wiley-Blackwell
Clinical and Experimental Allergy

مصطلحات موضوعية: 0301 basic medicine, B‐cell epitopes, Allergy, medicine.drug_class, Immunology, Reviews, Review, Immunoglobulin E, Monoclonal antibody, Epitope, 03 medical and health sciences, 0302 clinical medicine, Food allergy, medicine, Animals, Humans, Immunology and Allergy, B-Lymphocytes, food allergy, biology, business.industry, Allergens, medicine.disease, epitope mapping, 030104 developmental biology, Epitope mapping, 030228 respiratory system, Polyclonal antibodies, B-cell epitopes, biology.protein, Epitopes, B-Lymphocyte, monoclonal antibodies, Antibody, business, Food Hypersensitivity

الوصف: Summary In vitro allergy diagnostics are currently based on the detection of specific IgE binding on intact allergens or a mixture thereof. This approach has drawbacks as it may yield false‐negative and/or false‐positive results. Thus, we reviewed the impact of known B‐cell epitopes of food allergens to predict transience or persistence, tolerance or allergy and the severity of an allergic reaction and to examine new epitope mapping strategies meant to improve serum‐based allergy diagnostics. Recent epitope mapping approaches have been worthwhile in epitope identification and may increase the specificity of allergy diagnostics by using epitopes predominately recognized by allergic patients in some cases. However, these approaches did not lead to discrimination between clinically relevant and irrelevant epitopes so far, since the polyclonal serum IgE‐binding epitope spectrum seems to be too individual, independent of the disease status of the patients. New epitope mapping strategies are necessary to overcome these obstacles. The use of patient‐derived monoclonal antibodies instead of patient sera for functional characterization of clinically relevant and irrelevant epitope combinations, distinguished by their ability to induce degranulation, might be a promising approach to gain more insight into the allergic reaction and to improve serum‐based allergy diagnostics.

وصف الملف: image/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::4487bbea1f39e52351c842efba09c761Test
https://doi.org/10.1111/cea.13291Test

المؤلفون: Edward F. Knol, Alf Weimann, Henny G. Otten, Waltraud Suer, André C. Knulst, Mark A. Blankestijn

المصدر: Clinical and Experimental Allergy, 48(1), 60. Wiley-Blackwell

مصطلحات موضوعية: Male, 0301 basic medicine, Peanut allergy, Immunoglobulin E, Epitope, law.invention, Epitopes, 0302 clinical medicine, law, Immunology and Allergy, heterocyclic compounds, Sensitization, chemistry.chemical_classification, biology, Chemistry, peanut allergy, food and beverages, Middle Aged, Blot, medicine.anatomical_structure, storage proteins, Recombinant DNA, Female, lipids (amino acids, peptides, and proteins), 2S Albumins, Plant, Adult, Adolescent, Immunology, Cross Reactions, Ara h 7, 03 medical and health sciences, 2S albumin, Ara h 6, Food allergy, Ara h 2, Journal Article, medicine, Humans, Storage protein, Peanut Hypersensitivity, Aged, food allergy, Antigens, Plant, biochemical phenomena, metabolism, and nutrition, medicine.disease, Molecular biology, carbohydrates (lipids), 030104 developmental biology, 030228 respiratory system, biology.protein

الوصف: Background Little is known on the clinical relevance of peanut 2S albumin Ara h 7. Objective To investigate the discriminative ability of Ara h 7 in peanut allergy and assess the role of cross-reactivity between Ara h 2, 6 and Ara h 7 isoforms. Methods Sensitization to recombinant peanut storage proteins Ara h 1, 2, 3, 6 and 7 was assessed using a line blot in sera from 40 peanut tolerant and 40 peanut allergic patients, based on food challenge outcome. A dose-dependent ELISA inhibition experiment was performed with recombinant Ara h 2, 6 and Ara h 7 isoforms. Results For Ara h 7.0201 an area under the ROC curve was found of 0.83, comparable to Ara h 2 (AUC 0.81) and Ara h 6 (AUC 0.85). Ara h 7 intensity values strongly correlated with those from Ara h 2 and 6 (rs=0.81). Of all patients sensitized to 2S albumins Ara h 2, 6 or 7, the majority was co-sensitized to all three (n=24, 68%), although mono-sensitization to either 2S albumin was also observed in selected patients (Ara h 2: n=6, 17%; Ara h 6: n=2, 6%; Ara h 7: n=2, 6%). Binding to Ara h 7.0101 could be strongly inhibited by Ara h 7.0201, but not the other way around. Conclusions & Clinical Relevance Specific IgE against Ara h 7.0201 has a predictive ability for peanut allergy similar to Ara h 2 and 6, and possesses unique IgE epitopes as well as epitopes shared between the other Ara h 7 isoform and Ara h 2 and 6. While co-sensitization to all three 2S albumins is most common, mono-sensitization to either Ara h 2, 6 or 7 occurs in selected patients, leading to a risk of misdiagnosis when testing for a single 2S albumin. This article is protected by copyright. All rights reserved.

وصف الملف: image/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::cc8b18c8fdaba9d6a9a4ba436e28c8e7Test
https://doi.org/10.1111/cea.13030Test

المؤلفون: W. Marty Blom, Rob J.B. Klemans, Steve L. Taylor, André C. Knulst, Joseph L. Baumert, Mark A. Blankestijn, Geert F. Houben, Benjamin C. Remington

المصدر: Journal of Allergy and Clinical Immunology: In Practice, 5(2), 376–380. Elsevier

مصطلحات موضوعية: Adult, Male, Risk, Allergy, medicine.medical_specialty, Walnut, No-observed-adverse-effect level, Food industry, Population, Administration, Oral, Juglans, Toxicology, 03 medical and health sciences, 0404 agricultural biotechnology, 0302 clinical medicine, Food Labeling, Food allergy, Journal Article, medicine, Humans, Immunology and Allergy, Prospective Studies, Adverse effect, education, education.field_of_study, business.industry, 04 agricultural and veterinary sciences, Allergens, Eliciting doses, medicine.disease, 040401 food science, Surgery, Allergen thresholds, Lowest-observed-adverse-effect level, Threshold dose, 030228 respiratory system, Threshold dose distributions, Female, Immunization, Nut Hypersensitivity, business

الوصف: Background In food allergy, eliciting doses (EDs) of foods on a population level can improve risk management and labeling strategies for the food industry and regulatory authorities. Previously, data available for walnut were unsuitable to determine EDs. Objective The objective of this study was to determine EDs for walnut allergic adults and to compare with previously established threshold data for peanut and tree nuts. Methods Prospectively, adult subjects with a suspected walnut allergy underwent a low-dose double-blind, placebo-controlled food challenge. Individual no observed and lowest observed adverse effect levels were determined and log-normal, log-logistic, and Weibull models were fit to the data. Estimated ED values were calculated for the ED5, ED10, and ED50, the dose respectively predicted to provoke an allergic reaction in 5%, 10%, and 50% of the walnut allergic population. Results Fifty-seven subjects were challenged and 33 subjects were confirmed to be walnut allergic. Objective symptoms occurred in 20 of the positive challenges (61%). The cumulative EDs in the distribution models ranged from 3.1 to 4.1 mg for the ED05, from 10.6 to 14.6 mg walnut protein for the ED10, and from 590 to 625 mg of walnut protein for the ED50. Conclusions Our data indicate that population EDs for walnut are slightly higher compared with those for peanut and hazelnut allergy. Currently available data indicate that the ED values for hazelnut could be used as a conservative temporary placeholder when implementing risk management strategies for other tree nuts where little or no food challenge data are available.

وصف الملف: image/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::f4c401216b94ccb530371acdbea86dc1Test
https://doi.org/10.1016/j.jaip.2016.12.005Test

المؤلفون: Henderikus G. Otten, Mignon T. van den Elzen, André C. Knulst, M. F. C. L. Go, H. van Os-Medendorp, Mark A. Blankestijn

المصدر: Clinical Reviews in Allergy & Immunology, 54(3), 412. Humana Press
Clinical Reviews in Allergy & Immunology

مصطلحات موضوعية: medicine.medical_specialty, Angiotensin-Converting Enzyme Inhibitors, Omalizumab, Pharmacology, Bradykinin, Article, 03 medical and health sciences, Ecallantide, chemistry.chemical_compound, 0302 clinical medicine, Refractory, Icatibant, medicine, Humans, Immunology and Allergy, 030212 general & internal medicine, Angioedema, business.industry, Wheals, General Medicine, Idiopathic, medicine.disease, Dermatology, Treatment, Treatment Outcome, Tranexamic Acid, 030228 respiratory system, chemistry, Angiotensin-converting enzyme inhibitor, Hereditary angioedema, Fresh frozen plasma, Progestins, medicine.symptom, business, Tranexamic acid, medicine.drug

الوصف: Non-hereditary angioedema (AE) with normal C1 esterase inhibitor (C1INH) can be presumably bradykinin- or mast cell-mediated, or of unknown cause. In this systematic review, we searched PubMed, EMBASE, and Scopus to provide an overview of the efficacy of different treatment options for the abovementioned subtypes of refractory non-hereditary AE with or without wheals and with normal C1INH. After study selection and risk of bias assessment, 61 articles were included for data extraction and analysis. Therapies were described for angiotensin-converting enzyme inhibitor-induced AE (ACEi-AE), for idiopathic AE, and for AE with wheals. Described treatments consisted of ecallantide, icatibant, C1INH, fresh frozen plasma (FFP), tranexamic acid (TA), and omalizumab. Additionally, individual studies for anti-vitamin K, progestin, and methotrexate were found. Safety information was available in 26 articles. Most therapies were used off-label and in few patients. There is a need for additional studies with a high level of evidence. In conclusion, in acute attacks of ACEi-AE and idiopathic AE, treatment with icatibant, C1INH, TA, and FFP often leads to symptom relief within 2 h, with limited side effects. For prophylactic treatment of idiopathic AE and AE with wheals, omalizumab, TA, and C1INH were effective and safe in the majority of patients.

وصف الملف: image/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::76684d06cb7b812e508c342324064445Test
https://dspace.library.uu.nl/handle/1874/376369Test

المؤلفون: Kitty C.M. Verhoeckx, André C. Knulst, Marco Gaspari, W.M. Blom, Henderikus G. Otten, C. F. den Hartog Jager, Mark A. Blankestijn, G. A.H. de Jong, Geert F. Houben

المصدر: Clinical and Experimental Allergy, 48(9), 1206. Wiley-Blackwell

مصطلحات موضوعية: 0301 basic medicine, Male, Allergy, medicine.disease_cause, Immunoglobulin E, Mass Spectrometry, 11s globulin, 0302 clinical medicine, Allergen, immunoblot, Immunology and Allergy, Walnut allergy, Sensitization, Plant Proteins, Immunoassay, education.field_of_study, biology, Predictive value, medicine.anatomical_structure, Female, Nut Hypersensitivity, walnut allergy, Juglans, Adult, Population, Immunology, double blind placebo controlled food challenge, Cross Reactions, Immunoblot, Sensitivity and Specificity, 03 medical and health sciences, Young Adult, medicine, Double blind placebo controlled food challenge, Humans, Jug r 4, education, Skin Tests, Nutrition, business.industry, Plant Extracts, Antigens, Plant, medicine.disease, biology.organism_classification, 030104 developmental biology, 030228 respiratory system, biology.protein, business, Chromatography, Liquid

الوصف: Background: The role of sensitization to commercially available allergens of English walnut (Juglans regia) Jug r 1, 2 and 3 in walnut allergy has been previously investigated in walnut allergic adults and was unable to explain all cases of walnut allergy. Objectives: Identify recognized walnut allergens, other than the ones previously investigated (Jug r 1-3), in walnut allergic adults and determine the sensitization frequency and diagnostic value. Methods: Three different in-house walnut extracts were prepared and analysed on SDS-PAGE blots to identify allergenic walnut proteins. Immunoblots and immunoprecipitation, followed by LC-MS analysis, were performed to screen for, and confirm, IgE binding to walnut allergens in selected walnut allergic adults. In a cohort of 55 walnut challenged adults, including 33 allergic and 22 tolerant, sensitization to native and recombinant walnut allergen Jug r 4 was assessed using immunoblotting and immuno-line blot (EUROLINE), respectively. Results: Screening of sera of 8 walnut allergic adults identified Jug r 4 as an allergen in our population. In the total cohort of 55 subjects, 5 were positive for Jug r 4 on immunoblot and 10 on EUROLINE. All but one EUROLINE positive subject had a positive food challenge (sensitivity 27%, specificity 95%, PPV 90%, NPV 47%). All 5 subjects positive on immunoblot were also positive on EUROLINE. LC-MS analysis showed a lack of Jug r 4 in the ImmunoCAP extract. Co-sensitization to other 11S albumins (eg hazelnut Cor a 9) was common in Jug r 4 sensitized subjects, potentially due to cross-reactivity. Conclusions: Walnut 11S globulin Jug r 4 is a relevant minor allergen, recognized by 27% of walnut allergic adults. It has a high positive predictive value of 90% for walnut allergy. Specific IgE against Jug r 4 occurred mostly with concomitant sensitization to other walnut components, mainly Jug r 1. © 2018 The Authors. Clinical & Experimental Allergy Published by John Wiley & Sons Ltd

وصف الملف: application/pdf; image/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ae8eb5189e831f2ee845fce17107139eTest
http://resolver.tudelft.nl/uuid:3f723994-0f0e-4f1c-8482-3ba8113cd27eTest

المؤلفون: André C. Knulst, Mark A. Blankestijn, Henny G. Otten, Thuy My Le, Rob J.B. Klemans, Edward F. Knol, Heike Röckmann

المصدر: Clinical and Translational Allergy, 7(1). BioMed Central
Clinical and Translational Allergy, Vol 7, Iss 1, Pp 1-8 (2017)
Clinical and Translational Allergy

مصطلحات موضوعية: 0301 basic medicine, Pulmonary and Respiratory Medicine, Intoxicative inhalant, Allergy, Immunology, Adult population, Storage proteins, 03 medical and health sciences, 0302 clinical medicine, Food allergy, medicine, Storage protein, Immunology and Allergy, PR-10, Sensitization, chemistry.chemical_classification, Food protein, business.industry, Research, RC581-607, medicine.disease, 030104 developmental biology, medicine.anatomical_structure, Specific IgE, 030228 respiratory system, chemistry, Concomitant, Immunologic diseases. Allergy, ImmunoCAP ISAC, business

الوصف: Background The extent of co-sensitization within and between food protein families in an adult population is largely unknown. This study aimed to identify the most frequently recognized components in the PR-10 and storage protein family, as well as patterns in (co-)sensitization, in a birch-endemic area. Methods Results of ImmunoCAP ISAC, performed during routine care in Dutch adult outpatients suspected of food allergy, were collected. Results A total of 305 patients were selected, aged 16–79 years (median 32 years). Sensitization to one or more PR-10 proteins was most frequent (74% of all subjects), followed by 35% to storage protein and 15% to nsLTPs. Within the PR-10 family, subjects were most often sensitized to Bet v 1 (73% of 305), Cor a 1.04 (72%) and Mal d 1 (68%). Sensitization to PR-10s from soy, celery and kiwi occurred distinctively less often ( 70%). Subjects sensitized to these ‘less common PR-10 proteins’ were sensitized to more food and inhalant components on the ISAC, compared to subjects sensitized to ‘common PR-10 proteins’ (median 22 vs 13 out of 112, p

وصف الملف: image/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::38626657f2e7528f4e2539f07fb750fcTest
https://dspace.library.uu.nl/handle/1874/357650Test