Basophil Activation Test in the Diagnosis of Anisakis Allergy: An Observational Study from an Area of High Seafood Consumption in Italy.

The rising popularity of undercooked or raw seafood containing larvae of the Anisakis parasite has led to issues of public health concern due to allergic manifestations. We conducted an observational study on the use of an innovative Anisakis allergy diagnostic algorithm in a convenience sample of 53 allergic outpatients recruited in Western Sicily, between April 2021 and March 2022. We included individuals with an anamnesis suggestive of IgE sensitization to Anisakis reporting clinical manifestation in the last month due to allergic reactions after eating fresh fish, or in subjects at high exposure risk with sea products while abstaining from fish ingestion, excluding those with documented fish sensitization. Outpatients were tested via Skin Prick Test, IgE-specific dosage and Basophil Activation Test (BAT). Twenty-six outpatients were diagnosed with Anisakis, while 27 with Chronic Urticaria (CU). We found a seven-fold excess risk for Anisakis (p4) positivity in the Anisakis allergic outpatients, as compared to the CU ones. BAT showed the best diagnostic accuracy (92.45%) and specificity (100%), while specific IgE to Ascaris (p1) documented the best sensitivity (92.31%) but a very low specificity (37.04%). In conclusion, our findings may represent a potentially useful contribution to the future development of updated clinical guidelines.

Comparison of current methods for anti-dsDNA antibody detection and reshaping diagnostic strategies.

Anti-double-stranded DNA antibodies (anti-dsDNA) are considered a specific marker for systemic lupus erythematosus (SLE). Though the Farr technique was once the reference method for their detection, it has been almost entirely replaced by more recently developed assays. However, there is still no solid evidence of the commutability of these methods in terms of diagnostic accuracy and their correlation with the Crithidia luciliae immunofluorescence test (CLIFT). Anti-dsDNA antibody levels were measured in 80 subjects: 24 patients with SLE, 36 disease controls drawn from different autoimmune rheumatic diseases (14 systemic sclerosis, 10 Sjögren's syndrome, nine autoimmune myositis, three mixed connective tissue disease), 10 inflammatory arthritis and 10 apparently healthy blood donors by eight different methods: fluorescence enzyme immunoassay, microdot array, chemiluminescent immunoassay (two assays), multiplex flow immunoassay, particle multi-analyte technology immunoassay and two CLIFT. At the recommended manufacturer cut-off, the sensitivity varied from 67% to 92%, while the specificity ranged from 84% to 98%. Positive agreement among CLIFT and the other assays was higher than negative agreement. Mean agreement among methods assessed by the Cohen's kappa was 0.715, ranging from moderate (0.588) to almost perfect (0.888). Evaluation of the concordance among quantitative values by regression analysis showed a poor correlation index (mean r2, 0.66). The present study shows that current technologies for anti-dsDNA antibody detection are not fully comparable. In particular, their different correlation with CLIFT influences their positioning in the diagnostic algorithm for SLE (either in association or sequentially). Considering the high intermethod variability, harmonization and commutability of anti-dsDNA antibody testing remains an unachieved goal.

Mollusk allergy in shrimp-allergic patients: Still a complex diagnosis. An Italian real-life cross-sectional multicenter study.

Introduction: Shellfish allergy is an important cause of food allergies worldwide. Both in vivo and in vitro diagnostics failure nowadays is caused by the poor quality of the extracts associated with the scarce availability of allergenic molecules in the market. It is known that not all patients with shellfish allergies experience adverse reactions to mollusks. It is still unclear how to detect and diagnose these patients correctly. Aim: To investigate the features of shrimp-allergic patients either reactive or tolerant to mollusks, with the currently available diagnostic methods. Methods: Nineteen centers, scattered throughout Italy, participated in the real-life study, enrolling patients allergic to shrimp with or without associated reactions to mollusks. Patients underwent skin tests using commercial extracts or fresh raw and cooked shrimp and mollusks, and IgE reactivity to currently available allergenic extracts and molecules was measured in vitro. Results: Two hundred and forty-seven individuals with a self reported adverse reactions to shrimp participated in the study; of these 47.8% reported an adverse reaction to mollusks ingestion (cephalopod and/or bivalve). Neither of the tests used, in vivo nor in vitro, was able to detect all selected patients. Accordingly, a great heterogeneity of results was observed: in vivo and in vitro tests agreed in 52% and 62% of cases. Skin tests were able to identify the mollusk reactors (p < 0.001), also using fresh cooked or raw food (p < 0.001). The reactivity profile of mollusk reactors was dominated by Pen m 1, over Pen m 2 and Pen m 4 compared to tolerant subjects, but 33% of patients were not detected by any of the available molecules. Overall, a higher frequency of IgE rectivity to shrimp was recorded in northern Italy, while mollusk reactivity was more frequent in the center-south. Conclusion: The current diagnostic methods are inadequate to predict the cross-reactivity between crustaceans and mollusks. The detection of mollusks hypersensitivity should still rely on skin tests with fresh material. The exclusion of mollusks from shrimp allergic patients' diets should occur when clinical history, available diagnostic instruments, and/or tolerance tests support such a decision.

Current technologies for anti-ENA antibody detection: State-of-the-art of diagnostic immunoassays.

BACKGROUND: Autoantibodies against extractable nuclear antigens (ENA) play a pivotal role in the diagnosis and classification of systemic autoimmune rheumatic diseases (SARD). In recent years, newly developed methods have enabled the simultaneous and quantitative detection of multiple anti-ENA reactivities. However, data regarding the comparability of results obtained using different technologies across different platforms are scarce. In this study we compared eight different immunoassays, commonly used in current laboratory practice for detection of anti-ENA antibodies. METHODS: Sixty patients suffering from different SARD, 10 inflammatory arthritis patients (disease controls) and 10 healthy blood donors were included in this comparative study. Sera were collected in 15 centers belonging to the Study Group on Autoimmune Diseases of the Italian Society of Clinical Pathology and Laboratory Medicine. We evaluated the analytical sensitivity, specificity and diagnostic accuracy of each method for antibodies to Sm, RNP, Ro60, Ro52, Scl70, CENP-B and Jo1. Cohen's kappa was used to analyze the agreement among methods. RESULTS: Average agreement among methods was 0.82, ranging from substantial (k = 0.72) to almost perfect (k = 0.92). However, while the specificity was very good for all methods, some differences emerged regarding the analytical sensitivity. CONCLUSIONS: Diagnostic performance of current technologies for anti-ENA antibody detection showed good comparability. However, as some differences exist among methods, laboratory scientists and clinicians must be aware of the diagnostic accuracy of the testing method in use.

Recognizing the Emergent and Submerged Iceberg of the Celiac Disease: ITAMA Project-Global Strategy Protocol.

Coeliac disease (CD) is frequently underdiagnosed with a consequent heavy burden in terms of morbidity and health care costs. Diagnosis of CD is based on the evaluation of symptoms and anti-transglutaminase antibodies IgA (TGA-IgA) levels, with values above a tenfold increase being the basis of the biopsy-free diagnostic approach suggested by present guidelines. This study showcased the largest screening project for CD carried out to date in school children (n=20,000) aimed at assessing the diagnostic accuracy of minimally invasive finger prick point-of-care tests (POCT) which, combined with conventional celiac serology and the aid of an artificial intelligence-based system, may eliminate the need for intestinal biopsy. Moreover, this study delves deeper into the "coeliac iceberg" in an attempt to identify people with disorders who may benefit from a gluten-free diet, even in the absence of gastrointestinal symptoms, abnormal serology and histology. This was achieved by looking for TGA-IgA mucosal deposits in duodenal biopsy. This large European multidisciplinary health project paves the way to an improved quality of life for patients by reducing the costs for diagnosis due to delayed findings of CD and to offer business opportunities in terms of diagnostic tools and support.

Diagnosing and Monitoring Celiac Patients with Selective IgA Deficiency: Still an Open Issue.

Although, the association between celiac disease (CD) and selective immunoglobulin A deficiency (SIgAD) has been known for more than fifty years, the procedures for diagnosing and monitoring patients with both conditions are still far from definitive. When serological markers were introduced as pre-bioptic investigations, it was immediately clear that searching for specific IgA antibodies without checking total serum IgA could lead to a failure in diagnosing IgA-deficient CD patients, while specific IgG antibodies could be useful as additional tests, because they are frequently found in the serum of affected patients. Nonetheless, until recently the diagnosis of CD in IgA-deficient patients was based on the few, fragmentary and often contradictory data available in literature. The introduction of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) guidelines in 2012 provided the current criteria for diagnosing CD in IgA-deficient patients, although some issues remained open, such as the selection of patients who should undergo specific IgG antibody testing and the choice of the most reliable IgG-based test for both diagnosis and follow-up. A real-life study recently assessed the impact of the 2012 ESPGHAN guidelines in diagnosing and monitoring CD in SIgAD patients, highlighting several pitfalls that can lead to operational uncertainties and difficulties in patient management. In the present report, the evolution of diagnostic tools and criteria for CD in SIgAD patients has been critically assessed, both strengths and open issues have been highlighted, and future perspectives for improving the current diagnostic protocols have been suggested.

Allergy to Polyethilenglicole of Anti-SARS CoV2 Vaccine Recipient: A Case Report of Young Adult Recipient and the Management of Future Exposure to SARS-CoV2.

The main contraindication to the anti-SARS CoV2 vaccine is an anaphylactic reaction to a vaccine component. The need to vaccinate allergic people who are at higher risk can be of public health interest and this report shows a case of an allergic reaction to PEG of a HCW who had received the first dose of anti-SARS CoV2 vaccine. For 5 h after the administration of the vaccine, she had the appearance of erythematous spots on the face and neck, and a feeling of a slurred mouth and hoarseness. In order to treat the event, she was administered 8 mg intravenous dexamethasone, 1 vial intravenous chlorphenamine maleate, 250 mL intravenous 0.9% NaCl, and conventional oxygen therapy (2 L/min) with complete resolution of the suspected adverse drug reaction. According to the contraindication to the cutaneous test for this patient, BAT was used for further investigations. The patient who suffered the adverse reaction to the COVID-19 vaccine and other five allergic patients who did not report any adverse reaction after the vaccination were tested. There was a significant activation of the vaccine-reactive patient's basophils with 14.79 CD203chigh% at the concentration of 0.2 mg/mL, while other patients were negative. People who have a confirmed reaction to a vaccine component should undergo further investigation to discover other possible cross-reactions and select the right vaccine to immunize them.

A new diagnostic algorithm for pattern-oriented autoantibody testing according to the ICAP nomenclature: A pilot study.

The commercial tests currently available as second-level tests to detect ANA sub-specificities are generally used independently from the ANA immunofluorescence (IIF) pattern. The aim of this study was to evaluate the efficacy of the use of a customizable pattern-oriented antigenic panel by immunoblot (IB) using the International Consensus on ANA Patterns (ICAP) classification scheme, in order to introduce a novel and updated autoimmune diagnostic flowchart. 710 sera referred for routine ANA testing were selected on the basis of the ANA pattern according to the ICAP nomenclature (nuclear speckled AC-2,4,5; nucleolar AC-8,9,10,29; cytoplasmic speckled AC-18,19,20) and on an IIF titer ≥1:320. They were then assayed by three experimental IB assays using a panel of selected antigens. ICAP-oriented IB detected 515 antibody reactivities vs. 457 of traditional anti-ENA in the nuclear speckled pattern group, 108 vs. 28 in the nucleolar pattern group, and 43 vs. 34 in the cytoplasmic speckled pattern. This pilot study may lead the way for a new approach introducing an ICAP pattern-oriented follow up testing as a valid alternative to the existing standard panels, thus enabling more patients with autoimmune rheumatic disease to be accurately diagnosed.

Emerging Allergens in Goji Berry Superfruit: The Identification of New IgE Binding Proteins towards Allergic Patients' Sera.

The goji berry (Lycium barbarum L.) (GB) is gaining increasing attention with high consumption worldwide due to its exceptional nutritional value and medicinal benefits displayed in humans. Beyond their beneficial properties, GBs contain renowned allergenic proteins, and therefore deserve inclusion among the allergenic foods capable of inducing allergic reactions in sensitive consumers. GB allergy has been frequently linked to the panallergen lipid transfer protein (LTP), especially across the population of the Mediterranean area. Methods: In this study, we investigated the protein profile of GBs focusing on the most reactive proteins against immunoglobulins E (IgE) of allergic patients' sera, as ascertained by immunoblot experiments. The protein spots displaying a clear reaction were excised, in-gel digested, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by data searching against a restricted database for a reliable protein identification. Results: According to our data, three main spots were identified in GB extract as IgE binding proteins after immunoblot analysis. Some major proteins were identified and the three proteins that provided the highest reactivity were putatively attributed to vicilin and legumin proteins followed by a protein matching with 11S globulin belonging to the cupin superfamily. Finally, the whole GB protein extract was also submitted to bottom-up proteomics followed by a software-based database (DB) screening and a more exhaustive list of GB proteins was compiled.

Anti-DFS70 antibodies detected by specific methods in patients with thrombosis or recurrent pregnancy loss: no evidence of an association.

A dense fine speckled pattern (DFS) caused by antibodies to the DFS70 kDa nuclear protein is a relatively common finding while testing for anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) on HEp-2 cells. However, despite many efforts and numerous studies, the clinical significance of anti-DFS70 antibodies is still unknown as they can be found in patients with various disorders and even in healthy subjects. In this study we aimed at verifying whether these antibodies are associated with thrombotic events or with unexplained recurrent pregnancy loss (RPL). We studied 443 patients with venous or arterial thrombosis or RPL and 244 controls by IIF on HEp-2 cells and by a DFS70-specific chemiluminescent immunoassay (CIA). The DFS pattern was observed in IIF in 31/443 (7.0%) patients and in 6/244 (2.5%) controls (p = 0.01) while anti-DFS70 specific antibodies were detected by CIA in 11 (2.5%) patients and in one (0.4%) control (p = 0.06). Positive samples, either by IIF or by CIA, were then assayed by a second DFS70-specific line-immunoassay (LIA) method: 83.3% of the CIA positive samples were confirmed DFS70 positive versus only 29.7% of the IIF positive samples. These findings show that IIF overestimates anti-DFS70 antibody frequency and that results obtained by specific CIA and LIA assays do not indicate that venous or arterial thrombosis or RPL are linked to a higher prevalence of anti-DFS70 antibodies.

Evaluation of a new multiplex assay for allergy diagnosis.

BACKGROUND: Molecular allergy has significantly improved the quality of allergy diagnosis; however, the positioning of singleplex and multiplex assays in the diagnostic algorithm is still a matter of debate. METHODS: Aim of the study was to test the analytical performance of the recently commercialized Allergy Explorer-ALEX® in a selected population (105 allergic patients and 15 negative controls), comparing it with the reference ImmunoCAP® method and with skin prick test (SPT). RESULTS: Inter-assay qualitative comparison showed a substantial agreement between ALEX® and SPT (k = 0.64). A substantial agreement between ALEX® and ImmunoCAP® was shown on the detection of IgE to extracts (k = 0.64 for inhalants and k = 0.51 for food allergens), whereas a higher agreement was shown on detection of molecular components (k = 0.92 for inhalants and k = 0.72 for food allergens). Quantitative comparison showed a poor correlation between ALEX® and ImmunoCAP®. CONCLUSION: The simultaneous detection of both extracts and molecular components with ALEX® assay can potentially overcome some of the major limitations of the multiplex assay currently in use. However, before using ALEX® as routine method, the analytical performance (in particular for extracts) needs to be further investigated on a larger scale.

The extract, the molecular allergen or both for the in vitro diagnosis of peach and peanut sensitization?

INTRODUCTION: Identifying the target molecule in food allergies, helps to assess the risk of anaphylaxis in a patient. Lipid Transfer Protein is the most frequent cause of food allergies in the Mediterranean area. The diagnosis based on allergenic extracts, suffers from a high variability in the results because some important allergenic molecules are lacking. This study was disegned to assess whether Pru p 3 and Ara h 9 molecules are quantitative and qualitative enough present in their whole allergenic extracts. METHODS: 943 patients with a clinical history of suspected peach and/or peanut food allergies were recruited and underwent measurement of a specific serum IgE (ImmunoCAP system (Thermofisher/Phadia Diagnostics, Uppsala, Sweden) to the following allergens and molecules: peach (f95) and/or peanut (f13), Pru p 3 (f420), Pru p 1 (f419), Pru p 4 (f421), Ara h 1 (f422), Ara h 2 (f423) Ara h 3 (f424) and Ara h 9 (f427). RESULTS: Out of the 943 patients included in this study, 122 were positive to sIgE to peanut extract. At a cut-off point of 0.35 kIU/L, 62 patients were positive to sIgE to Ara h 9 but negative to peanut extract. Increasing the cut-off point of Ara h 9 at 10 kIU/L, 15 patients were only positive to sIgE to Ara h 9. 244 out of the 943 patients were positive to sIgE to peach extract. At a cut-off point of 0.35 kIU/L, 27 patients were negative to sIgE to Pru p 3 and positive to sIgE to peach extract, whilst 11 patients were peach extract sIgE positive and sIgE negative to Pru p 1, Pru p 3 and Pru p 4. Only 12 patients resulted positive to Pru p1 and/or Pru p 4. CONCLUSION: Our data strongly suggests to include the measurement of sIgE to Ara h 9 into the diagnostic algorithm of peanut sensitization. 4.5% of the sicilian population suspected of peach sensitization were positive to peach extract and negative to all the available molecules.

IL-33/s-ST2 ratio, systemic symptoms, and basophil activation in Pru p 3-sensitized allergic patients.

BACKGROUND: Although IL-33/ST2 axis is involved in the development of allergic diseases, its contribution in food allergy is still unknown. METHODS: In this study, we assessed the serum levels of IL-33 and its s-ST2 receptor in 53 control patients (without allergic diseases), 47 peach (Pru p 3)-sensitized allergic patients (SAP), and in 68 non-Pru p 3-SAP. Basophil activation test (BAT) was used to assess the basophil activation due to allergen exposure before and after the addition of s-ST2 to the blood samples from 5 Pru p 3-SAP. RESULTS: IL-33 levels in Pru p 3-SAP were higher than in non-Pru p 3-SAP and in normal controls. Lower s-ST2 levels were found in Pru p 3-SAP than in non-Pru p 3-SAP. IL-33/s-ST2 ratio was higher in Pru p 3-SAP than in both non-Pru p 3-SAP and controls. Higher IL-33/s-ST2 ratio was observed in Pru p 3-SAP with severe than in those with mild systemic symptoms. BAT analysis in Pru p 3-SAP showed a decrease in basophil activation due to Pru p 3 exposure after the addition of s-ST2 to the blood samples. CONCLUSIONS: An imbalance in the baseline levels of IL-33/ST2 pathway is present in Pru p 3-SAP. The measurement of this pathway might be helpful to detect patients at a higher risk of developing severe systemic symptoms.

Safety of Oats in Children with Celiac Disease: A Double-Blind, Randomized, Placebo-Controlled Trial.

OBJECTIVE: To evaluate the long-term validity and safety of pure oats in the treatment of children with celiac disease. STUDY DESIGN: This noninferiority clinical trial used a double-blind, placebo-controlled, crossover design extended over 15 months. Three hundred six children with a biopsy-proven diagnosis of celiac disease on a gluten-free diet for ≥2 years were randomly assigned to eat specifically prepared gluten-free food containing an age-dependent amount (15-40 g) of either placebo or purified nonreactive varieties of oats for 2 consecutive 6-month periods separated by washout standard gluten-free diet for 3 months. Clinical (body mass index, Gastrointestinal Symptoms Rating Scale score), serologic (IgA antitransglutaminase antibodies, and IgA anti-avenin antibodies), and intestinal permeability data were measured at baseline, and after 6, 9, and 15 months. Direct treatment effect was evaluated by a nonparametric approach using medians (95% CI) as summary statistic. RESULTS: After the exclusion of 129 patients who dropped out, the cohort included 177 children (79 in the oats-placebo and 98 in the placebo-oats group; median, 0.004; 95% CI, -0.0002 to 0.0089). Direct treatment effect was not statistically significant for clinical, serologic, and intestinal permeability variables (body mass index: median, -0.5; 95% CI, -0.12 to 0.00; Gastrointestinal Symptoms Rating Scale score: median, 0; 95% CI, -2.5 to 0.00; IgA antitransglutaminase antibodies: median, -0.02; 95% CI, -0.25 to 0.23; IgA anti-avenin antibodies: median, -0.0002; 95% CI, -0.0007 to 0.0003; intestinal permeability test: median, 0.004; 95% CI, -0.0002 to 0.0089). CONCLUSIONS: Pure nonreactive oat products are a safe dietary choice in the treatment of children with celiac disease. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00808301.

The ANA-reflex test as a model for improving clinical appropriateness in autoimmune diagnostics.

Reflex tests are widely used in clinical laboratories, for example, to diagnose thyroid disorders or in the follow-up of prostate cancer. Reflex tests for antinuclear antibodies (ANA) have recently gained attention as a way to improve appropriateness in the immunological diagnosis of autoimmune rheumatic diseases and avoid waste of resources. However, the ANA-reflex test is not as simple as other consolidated reflex tests (the TSH-reflex tests or the PSA-reflex tests) because of the intrinsic complexity of the ANA test performed by the indirect immunofluorescence method on cellular substrates. The wide heterogeneity of the ANA patterns, which need correct interpretation, and the subsequent choice of the most appropriate confirmatory test (ANA subserology), which depend on the pattern feature and on clinical information, hinder any informatics automation, and require the pathologist's intervention. In this review, the Study Group on Autoimmune Diseases of the Italian Society of Clinical Pathology and Laboratory Medicine provides some indications on the configuration of the ANA-reflex test, using two different approaches depending on whether clinical information is available or not. We further give some suggestions on how to report results of the ANA-reflex test.

Computer-Assisted Classification Patterns in Autoimmune Diagnostics: The AIDA Project.

Antinuclear antibodies (ANAs) are significant biomarkers in the diagnosis of autoimmune diseases in humans, done by mean of Indirect ImmunoFluorescence (IIF) method, and performed by analyzing patterns and fluorescence intensity. This paper introduces the AIDA Project (autoimmunity: diagnosis assisted by computer) developed in the framework of an Italy-Tunisia cross-border cooperation and its preliminary results. A database of interpreted IIF images is being collected through the exchange of images and double reporting and a Gold Standard database, containing around 1000 double reported images, has been settled. The Gold Standard database is used for optimization of a CAD (Computer Aided Detection) solution and for the assessment of its added value, in order to be applied along with an Immunologist as a second Reader in detection of autoantibodies. This CAD system is able to identify on IIF images the fluorescence intensity and the fluorescence pattern. Preliminary results show that CAD, used as second Reader, appeared to perform better than Junior Immunologists and hence may significantly improve their efficacy; compared with two Junior Immunologists, the CAD system showed higher Intensity Accuracy (85,5% versus 66,0% and 66,0%), higher Patterns Accuracy (79,3% versus 48,0% and 66,2%), and higher Mean Class Accuracy (79,4% versus 56,7% and 64.2%).