Joaquín Sastre

A WAO - ARIA - GA²LEN consensus document on molecular-based allergy diagnostics

Molecular-based allergy (MA) diagnostics is an approach used to map the allergen sensitization of a patient at a molecular level, using purified natural or recombinant allergenic molecules (allergen components) instead of allergen extracts. Since its introduction, MA diagnostics has increasingly entered routine care, with currently more than 130 allergenic molecules commercially available for in vitro specific IgE (sIgE) testing.MA diagnostics allows for an increased accuracy in allergy diagnosis and prognosis and plays an important role in three key aspects of allergy diagnosis: (1) resolving genuine versus cross-reactive sensitization in poly-sensitized patients, thereby improving the understanding of triggering allergens; (2) assessing, in selected cases, the risk of severe, systemic versus mild, local reactions in food allergy, thereby reducing unnecessary anxiety for the patient and the need for food challenge testing; and (3) identifying patients and triggering allergens for specific immunotherapy (SIT).Singleplex and multiplex measurement platforms are available for MA diagnostics. The Immuno-Solid phase Allergen Chip (ISAC) is the most comprehensive platform currently available, which involves a biochip technology to measure sIgE antibodies against more than one hundred allergenic molecules in a single assay. As the field of MA diagnostics advances, future work needs to focus on large-scale, population-based studies involving practical applications, elucidation and expansion of additional allergenic molecules, and support for appropriate test interpretation. With the rapidly expanding evidence-base for MA diagnosis, there is a need for allergists to keep abreast of the latest information. The aim of this consensus document is to provide a practical guide for the indications, determination, and interpretation of MA diagnostics for clinicians trained in allergology.

EAACI Molecular Allergology User's Guide

The availability of allergen molecules (‘components’) from several protein families has advanced our understanding of immunoglobulin E (IgE)‐mediated responses and enabled ‘component‐resolved diagnosis’ (CRD). The European Academy of Allergy and Clinical Immunology (EAACI) Molecular Allergology Users Guide (MAUG) provides comprehensive information on important allergens and describes the diagnostic options using CRD. Part A of the EAACI MAUG introduces allergen molecules, families, composition of extracts, databases, and diagnostic IgE, skin, and basophil tests. Singleplex and multiplex IgE assays with components improve both sensitivity for low‐abundance allergens and analytical specificity; IgE to individual allergens can yield information on clinical risks and distinguish cross‐reactivity from true primary sensitization. Part B discusses the clinical and molecular aspects of IgE‐mediated allergies to foods (including nuts, seeds, legumes, fruits, vegetables, cereal grains, milk, egg, meat, fish, and shellfish), inhalants (pollen, mold spores, mites, and animal dander), and Hymenoptera venom. Diagnostic algorithms and short case histories provide useful information for the clinical workup of allergic individuals targeted for CRD. Part C covers protein families containing ubiquitous, highly cross‐reactive panallergens from plant (lipid transfer proteins, polcalcins, PR‐10, profilins) and animal sources (lipocalins, parvalbumins, serum albumins, tropomyosins) and explains their diagnostic and clinical utility. Part D lists 100 important allergen molecules. In conclusion, IgE‐mediated reactions and allergic diseases, including allergic rhinoconjunctivitis, asthma, food reactions, and insect sting reactions, are discussed from a novel molecular perspective. The EAACI MAUG documents the rapid progression of molecular allergology from basic research to its integration into clinical practice, a quantum leap in the management of allergic patients.

Chicken serum albumin (Gal d 5) is a partially heat-labile inhalant and food allergen implicated in the bird-egg syndrome*

Background: Chicken serum albumin (α‐livetin) has been implicated as the causative allergen of the bird‐egg syndrome. However, the clinical relevance of sensitization to this allergen has not been confirmed by specific challenge tests and environmental sampling. We investigated whether chicken albumin can be detected in air samples collected in a home with birds, and whether sensitization to this protein may cause respiratory and food allergy symptoms. The heat resistance of chicken albumin and the possible cross‐reactivity with conalbumin were also investigated.

Component-resolved in vitro diagnosis of hazelnut allergy in Europe

BACKGROUND Food allergy to hazelnut occurs both with and without concomitant pollen allergy. OBJECTIVE We sought to evaluate a panel of hazelnut allergens for diagnosis of hazelnut allergy in Spain, Switzerland, and Denmark. METHODS Fifty-two patients with a positive double-blind, placebo-controlled food challenge result with hazelnuts; 5 patients with a history of anaphylaxis; 62 patients with pollen allergy but hazelnut tolerance; and 63 nonatopic control subjects were included. Serum IgE levels to hazelnut extract, recombinant hazelnut allergens (rCor a 1.04, rCor a 2, rCor a 8, rCor a 11), and native allergens (nCor a 9, nCor a Bd8K, nCor a Bd11K) were analyzed by means of ImmunoCAP. RESULTS Among patients with hazelnut allergy, 91% (Switzerland/Spain, 100%; Denmark, 75%) had IgE to hazelnut extract, 75% to rCor a 1.04, 42% to rCor a 2, 28% to rCor a 8, and 2% to rCor a 11. The highest rate of sensitization to Cor a 1.04 was found in the northern regions (Switzerland/Denmark, 100%; Spain, 18%), whereas IgE to the lipid transfer protein rCor a 8 prevailed in Spain (Spain, 71%; Switzerland, 15%; Denmark, 5%). IgE to profilin rCor a 2 was equally distributed (40% to 45%). Among control subjects with pollen allergy, 61% had IgE to hazelnut extract, 69% to rCor a 1.04, 34% to rCor a 2, 10% to rCor a 8, and 6% to rCor a 11. CONCLUSION Component-resolved in vitro analyses revealed substantial differences in IgE profiles of hazelnut allergic and hazelnut tolerant patients across Europe.

Lack of allergic cross‐reactivity to cephalosporins among patients allergic to penicillins

There are some contradicting data about clinical allergic cross‐reactivity to cephalosporins among patients who have had a previous allergic reaction to penicillins.

Validation of the Spanish version of the Asthma Control Test (ACT).

Objective: Validation of the Spanish version of the Asthma Control Test (ACT). Methods: A total of 607 asthmatic patients were assessed. The psychometric properties of ACT were evaluated. The ACT capacity to predict the physicians assessment of asthma control was assessed using the area under the receiving operating characteristics (ROC) curve (AUC), sensitivity, specificity, and positive-negative predictive values. Results: ACTs Cronbach α was 0.84. The intraclass correlation coefficient was 0.85. The AUC was 0.86, with a sensitivity of 71% and a specificity of 85% for a score of ≤19. Conclusions: The Spanish version of ACT is shown to be a reliable and valid tool for evaluating and discriminating asthma control.

Greater epitope recognition of shrimp allergens by children than by adults suggests that shrimp sensitization decreases with age

BACKGROUND Shellfish allergy is a long-lasting disorder typically affecting adults. Despite its high prevalence, there is limited information about allergenic shrimp proteins and the epitopes implicated in such allergic reactions. OBJECTIVE We sought to identify the IgE-binding epitopes of the 4 shrimp allergens and to characterize epitope recognition profiles of children and adults with shrimp allergy. METHODS Fifty-three subjects, 34 children and 19 adults, were selected with immediate allergic reactions to shrimp, increased shrimp-specific serum IgE levels, and positive immunoblot binding to shrimp. Study subjects and 7 nonatopic control subjects were tested by means of peptide microarray for IgE binding with synthetic overlapping peptides spanning the sequences of Litopenaeus vannamei shrimp tropomyosin, arginine kinase (AK), myosin light chain (MLC), and sarcoplasmic calcium-binding protein (SCP). The Wilcoxon test was used to determine significant differences in z scores between patients and control subjects. RESULTS The median shrimp IgE level was 4-fold higher in children than in adults (47 vs 12.5 kU(A)/L). The frequency of allergen recognition was higher in children (tropomyosin, 81% [94% for children and 61% for adults]; MLC, 57% [70% for children and 31% for adults]; AK, 51% [67% for children and 21% for adults]; and SCP, 45% [59% for children and 21% for adults]), whereas control subjects showed negligible binding. Seven IgE-binding regions were identified in tropomyosin by means of peptide microarray, confirming previously identified shrimp epitopes. In addition, 3 new epitopes were identified in tropomyosin (epitopes 1, 3, and 5b-c), 5 epitopes were identified in MLC, 3 epitopes were identified in SCP, and 7 epitopes were identified in AK. Interestingly, frequency of individual epitope recognition, as well as intensity of IgE binding, was significantly greater in children than in adults for all 4 proteins. CONCLUSIONS Children with shrimp allergy have greater shrimp-specific IgE antibody levels and show more intense binding to shrimp peptides and greater epitope diversity than adults.

Current evidence and future research needs for FeNO measurement in respiratory diseases

Although not yet widely implemented, fraction of exhaled nitric oxide (FeNO) has emerged in recent years as a potentially useful biomarker for the assessment of airway inflammation both in undiagnosed patients with non-specific respiratory symptoms and in those with established airway disease. Research to date essentially suggests that FeNO measurement facilitates the identification of patients exhibiting T-helper cell type 2 (Th2)-mediated airway inflammation, and effectively those in whom anti-inflammatory therapy, particularly inhaled corticosteroids (ICS), is beneficial. In some studies, FeNO-guided management of patients with established airway disease is associated with lower exacerbation rates, improvements in adherence to anti-inflammatory therapy, and the ability to predict risk of future exacerbations or decline in lung function. Despite these data, concerns regarding the applicability and utility of FeNO in clinical practice still remain. This article reviews the current evidence, both supportive and critical of FeNO measurement, in the diagnosis and management of asthma and other inflammatory airway diseases. It additionally provides suggestions regarding the practical application of FeNO measurement: how it could be integrated into routine clinical practice, how its utility could be assessed and its true value to both clinicians and patients could be established. Although some unanswered questions remain, current evidence suggests that FeNO is potentially a valuable tool for improving the personalised management of inflammatory airway diseases.

Molecular Cloning of Paramyosin, a New Allergen of Anisakis simplex

Background: Anisakis simplex is a fish parasite that, when accidentally ingested by humans, may cause allergic reactions in sensitized individuals. The main objectives of our study were to: (1) construct a cDNA expression library of A. simplex; (2) identify clones producing specific IgE binding protein antigens, and (3) produce and purify the protein/s codified by the isolated clones produced in Escherichia coli. Methods: An expression cDNA library from the third stage larvae (L3) of A. simplex was constructed. This library was first screened with a rabbit anti A. simplex hyperimmune serum. The positive clones, identified using the rabbit serum, were rescreened with a pool of human sera containing high titers of IgE antibodies against A. simplex. Results: Two positive clones were isolated carrying the genes which codify for paramyosin. The paramyosin protein was produced in E. coli and purified. The partial sequence of a second paramyosin gene was also identified. The frequency of specific IgE binding to the recombinant and native forms of paramyosin using the sera of 26 A. simplex-sensitive individuals was 23 and 88%, respectively. Both paramyosins were able to inhibit 11% of the specific IgE binding to a total extract. Conclusions: We describe the primary structure of a paramyosin of A. simplex. It can be considered as an allergen based on its IgE binding capacity. We suggest that the recombinant protein does not maintain the complete allergenic properties of the native paramyosin, considering its lower IgE binding capacity of the recombinant protein. However, both proteins have the same specific IgE inhibition capacity. The recombinant protein can be produced in large quantities in E. coli. We propose the term Ani s 2 for this allergen.

Clinical cross‐reactivity between amoxicillin and cephadroxil in patients allergic to amoxicillin and with good tolerance of penicillin

In recent years, patients allergic to amoxicillin (AX) but with good tolerance of penicillin G (PG) have been described. It has been suggested that the epitope implicated in this type of sensitization might be located on the side‐chain of the AX molecule. Thus, cross‐reactivity between AX and cephadroxil (CEPH), a cephalosporin which shares an identical side‐chain with AX, is suspected. This study aimed to demonstrate clinical cross‐reactivity between AX and CEPH in patients allergic to AX and showing good tolerance of PG. In 76 of 576 subjects with suspected allergic reaction to PG and/or AX, the diagnosis of allergy was confirmed. All of these had specific IgE to PG, penicillin V, or AX, and/or positive skin tests to PPL (penicilloyl‐polylysine), or MDM (minor determinant mixture), or PG. or AX, and/or positive challenge tests with PG and/or AX. Sixteen subjects (21%) allergic to AX (11 with positive skin test and five with positive challenge test to AX) and good tolerance of PG (all with negative parenteral challenge test) were selected. These 16 patients were subsequently challenged with CEPH (up to 500 mg). Fourteen patients tolerated CEPH, and two (12%) had an immediate allergic reaction. Our study indicates that allergy to the side‐chain of aminopenicillins seems to have little clinical relevance in patients with allergic reactions to aminopenicillins but with good tolerance of PG, as 88% of patients with this clinical characteristic tolerate a cephalosporin which shares an identical side‐chain. It seems that IgE from most of these patients recognizes an epitope different from the side‐chain and the β‐Iactam ring.