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A recent survey of the European Academy of Allergy and Clinical Immunology (EAACI) Drug Allergy Interest Group (DAIG) on how European allergy specialists deal with beta-lactam (BL) hypersensitivity demonstrated a significant heterogeneity in current practice, suggesting the need to review and update existing EAACI guidelines in order to make the diagnostic procedures as safe and accurate, but also as cost-effective, as possible. For this purpose, a bibliographic search on large studies regarding BL hypersensitivity diagnosis was performed by an EAACI task force, which reviewed and evaluated the literature data using the GRADE system for quality of evidence and strength of recommendation. The updated guidelines provide a risk stratification in BL hypersensitivity according to index reaction(s), as well as an algorithmic approach, based on cross-reactivity studies, in patients with a suspicion of BL hypersensitivity and an immediate need for antibiotic therapy, when referral to an allergist is not feasible. Furthermore, the update addresses availability and concentrations of skin test (ST) reagents, ST and drug provocation test (DPT) protocols, and diagnostic algorithms and administration of alternative BL in allergic subjects. Specifically, distinct diagnostic algorithms are suggested depending on risk stratification of the patient into high and low risk based on the morphology and chronology of the reaction, immediate (ie, occurring within 1-6 hours after the last administered dose) or nonimmediate (ie, occurring more than 1 hour after the initial drug administration), and the reaction severity. Regarding the allergy workup, the main novelty of this document is the fact that in some low-risk nonimmediate reactions ST are not mandatory, especially in children. For DPT, further studies are necessary to provide data supporting the standardization of protocols, especially of those regarding nonimmediate reactions, for which there is currently no consensus., (© 2019 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

An accurate diagnosis of β-lactam (BL) allergy can reduce patient morbidity and mortality. Our aim was to investigate the availability of BL reagents, their use and test procedures in different parts of Europe, as well as any differences in the diagnostic workups for evaluating subjects with BL hypersensitivity. A survey was emailed to all members of the EAACI Drug Allergy Interest Group (DAIG) between February and April 2016, and the questionnaire was meant to study the management of suspected BL hypersensitivity. The questionnaire was emailed to 82 DAIG centres and answered by 57. Amoxicillin alone or combined to clavulanic acid were the most commonly involved BL except in the Danish centre, where penicillin V was the most frequently suspected BL. All centres performed an allergy workup in subjects with histories of hypersensitivity to BL: 53 centres (93%) followed DAIG guidelines, two national guidelines and two local guidelines. However, there were deviations from DAIG recommendations concerning allergy tests, especially drug provocation tests. A significant heterogeneity exists in current practice not only among countries, but also among centres within the same country. This suggests the need to re-evaluate, update and standardize protocols on the management of patients with suspected BL allergy., (© 2019 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Background: β-Lactam hypersensitivity has been classified according to the phenotype and function of drug-specific T cells. However, new T-cell subsets have not been considered., Objective: The objective of this study was to use piperacillin as a model of β-lactam hypersensitivity to study the nature of the drug-specific T-cell response induced in the blood and skin of hypersensitive patients and healthy volunteers., Methods: Drug-specific T cells were cloned from blood and inflamed skin, and cellular phenotype and function were explored. Naive T cells from healthy volunteers were primed to piperacillin, cloned, and subjected to the similar analyses., Results: PBMC and T-cell clones (n = 570, 84% CD4 + ) from blood of piperacillin-hypersensitive patients proliferated and secreted T H 1/T H 2 cytokines alongside IL-22 after drug stimulation. IL-17A secretion was not detected. Drug-specific clones from inflamed skin (n = 96, 83% CD4 + ) secreted a similar profile of cytokines but displayed greater cytolytic activity, secreting perforin, granzyme B, and Fas ligand when activated. Blood- and skin-derived clones expressed high levels of skin-homing chemokine receptors and migrated in the presence of the ligands CCL17 and CCL27. Piperacillin-primed naive T cells from healthy volunteers also secreted IFN-γ, IL-13, IL-22, and cytolytic molecules. Aryl hydrocarbon receptor blockade prevented differentiation of the naive T cells into antigen-specific IL-22-secreting cells., Conclusion: Together, our results reveal that circulating and skin-resident, antigen-specific, IL-22-secreting T cells are detectable in patients with β-lactam hypersensitivity. Furthermore, differentiation of naive T cells into antigen-specific T H 22 cells is dependent on aryl hydrocarbon receptor signaling., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The risk of developing hypersensitivity to alternative antibiotics is a concern for penicillin hypersensitive patients and healthcare providers. Herein we use piperacillin hypersensitivity as a model to explore the reactivity of drug-specific IgG against alternative β-lactam protein adducts. Mass spectrometry was used to show the drugs (amoxicillin, flucloxacillin, benzyl penicillin, aztreonam, and piperacillin) bind to similar lysine residues on the protein carrier bovine serum albumin. However, the hapten-specific IgG antibodies found in piperacillin hypersensitive patient plasma did not bind to other β-lactam protein conjugates. These data outline the fine specificity of piperacillin-specific IgG antibodies that circulate in patients with hypersensitivity.

Background: Delayed-type β-lactam hypersensitivity develops in subset of patients. The cellular immunological processes that underlie the drug-specific response have been described; however, little is known about involvement of the humoral immune system. Thus, the aim of this study was to utilize piperacillin hypersensitivity as an exemplar to (i) develop cell culture methods for the detection of drug-specific B-cell responses, (ii) characterize drug-specific IgG subtypes and (iii) assess reactivity of IgG antibodies against proteins modified to different levels with piperacillin haptens., Methods: IgG secretion and CD19 + CD27 + expression on B cells were measured using ELISPOT and flow cytometry, respectively. A piperacillin-BSA adduct was used as an antigen in ELISA antibody binding studies. Adducts generated using different ratios of drug to protein were used to determine the degree of conjugation required to detect IgG binding., Results: B cells from hypersensitive patients, but not controls, were stimulated to secrete IgG and increase CD27 expression when cultured with soluble piperacillin. A piperacillin-BSA adduct with cyclized and hydrolysed forms of the hapten bound to eight lysine residues was used to detect hapten-specific IgG 1-4 subclasses in patient plasma. Hapten inhibition and the use of structurally unrelated hapten-BSA adducts confirmed antigen specificity. Antibody binding was detected with antigens generated at piperacillin/BSA ratios of 10:1 and above, which corresponded to a minimum epitope density of 1 for antibody binding., Conclusion: These data show that antigen-specific B lymphocytes and T lymphocytes are activated in piperacillin-hypersensitive patients. Further work is needed to define the role different IgG subtypes play in regulating the iatrogenic disease., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Covalent modification of protein by drugs may disrupt self-tolerance, leading to lymphocyte activation. Until now, determination of the threshold required for this process has not been possible. Therefore, we performed quantitative mass spectrometric analyses to define the epitopes formed in tolerant and hypersensitive patients taking the β-lactam antibiotic piperacillin and the threshold required for T cell activation. A hydrolyzed piperacillin hapten was detected on four lysine residues of human serum albumin (HSA) isolated from tolerant patients. The level of modified Lys 541 ranged from 2.6 to 4.8%. Analysis of plasma from hypersensitive patients revealed the same pattern and levels of modification 1-10 d after the commencement of therapy. Piperacillin-responsive skin-homing CD4 + clones expressing an array of Vβ receptors were activated in a dose-, time-, and processing-dependent manner; analysis of incubation medium revealed that 2.6% of Lys 541 in HSA was modified when T cells were activated. Piperacillin-HSA conjugates that had levels and epitopes identical to those detected in patients were shown to selectively stimulate additional CD4 + clones, which expressed a more restricted Vβ repertoire. To conclude, the levels of piperacillin-HSA modification that activated T cells are equivalent to the ones formed in hypersensitive and tolerant patients, which indicates that threshold levels of drug Ag are formed in all patients. Thus, the propensity to develop hypersensitivity is dependent on other factors, such as the presence of T cells within an individual's repertoire that can be activated with the β-lactam hapten and/or an imbalance in immune regulation., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Drug hypersensitivity reactions (DHRs) are a matter of great concern, both for outpatient and in hospital care. The evaluation of these patients is complex, because in vivo tests have a suboptimal sensitivity and can be time-consuming, expensive and potentially risky, especially drug provocation tests. There are several currently available in vitro methods that can be classified into two main groups: those that help to characterize the active phase of the reaction and those that help to identify the culprit drug. The utility of these in vitro methods depends on the mechanisms involved, meaning that they cannot be used for the evaluation of all types of DHRs. Moreover, their effectiveness has not been defined by a consensus agreement between experts in the field. Thus, the European Network on Drug Allergy and Drug Allergy Interest Group of the European Academy of Allergy and Clinical Immunology has organized a task force to provide data and recommendations regarding the available in vitro methods for DHR diagnosis. We have found that although there are many in vitro tests, few of them can be given a recommendation of grade B or above mainly because there is a lack of well-controlled studies, most information comes from small studies with few subjects and results are not always confirmed in later studies. Therefore, it is necessary to validate the currently available in vitro tests in a large series of well-characterized patients with DHR and to develop new tests for diagnosis., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: For certain HLA allele-associated drug hypersensitivity reactions, the parent drug has been shown to associate directly with the risk allele. In other forms of hypersensitivity, HLA risk alleles have not been identified and T cells are activated in an allele unrestricted manner. Chemically reactive drug metabolites bind to multiple proteins; thus, it is assumed that the derived peptide antigens interact with a number of HLA molecules to activate T cells; however, HLA restriction of the drug metabolite-specific T-cell response has not been studied., Objective: To utilize T cells from sulfamethoxazole (SMX) hypersensitive patients with cystic fibrosis to examine the HLA molecules that interact with nitroso SMX (SMX-NO)-derived antigens., Methods: T-cell clones were generated from 4 hypersensitive patients. Drug-specific proliferative responses and cytokine secretion were measured. Anti-human class I and class II antibodies were used to analyse HLA restriction. Antigen-presenting cells expressing different HLA molecules were used to determine the alleles involved in the presentation of SMX-NO-derived antigens to T cells., Results: A total of 976 clones were tested for SMX-NO reactivity. Thirty-nine CD4+ clones were activated with SMX-NO and found to proliferate and secrete cytokines. The SMX-NO-specific response was blocked with an antibody against HLA-DQ. SMX-NO-specific responses were detected with antigen-presenting cells expressing HLA-DQB1*05:01 (patient 1) and HLA-DQB1*02:01 (patient 2), but not other HLA-DQB1 alleles., Conclusion and Clinical Relevance: HLA-DQ plays an important role in the activation of SMX-NO-specific CD4+ T cells. Detection of HLA-DQ allele-restricted responses suggests that T cells are activated by a limited repertoire of SMX-NO-modified peptides., (© 2015 John Wiley & Sons Ltd.)

Background: β-Lactam hypersensitivity has been classified according to the phenotype and function of drug-specific T cells. However, new T-cell subsets have not been considered. Objective: The objective of this study was to use piperacillin as a model of β-lactam hypersensitivity to study the nature of the drug-specific T-cell response induced in the blood and skin of hypersensitive patients and healthy volunteers. Methods: Drug-specific T cells were cloned from blood and inflamed skin, and cellular phenotype and function were explored. Naive T cells from healthy volunteers were primed to piperacillin, cloned, and subjected to the similar analyses. Results: PBMC and T-cell clones (n5570, 84% CD4+) fromblood of piperacillin-hypersensitive patients proliferated and secreted TH1/TH2 cytokines alongside IL-22 after drug stimulation. IL-17A secretion was not detected. Drug-specific clones from inflamed skin (n 5 96, 83% CD4+) secreted a similar profile of cytokines but displayed greater cytolytic activity, secreting perforin, granzyme B, and Fas ligand when activated. Blood- and skin-derived clones expressed high levels of skin-homing chemokine receptors and migrated in the presence of the ligands CCL17 and CCL27. Piperacillin-primed naive T cells from healthy volunteers also secreted IFN-γ, IL-13, IL-22, and cytolytic molecules. Aryl hydrocarbon receptor blockade prevented differentiation of the naive T cells into antigen-specific IL-22-secreting cells. Conclusion: Together, our results reveal that circulating and skin-resident, antigen-specific, IL-22-secreting T cells are detectable in patients with β-lactam hypersensitivity. Furthermore, differentiation of naive T cells into antigenspecific TH22 cells is dependent on aryl hydrocarbon receptor signaling. [ABSTRACT FROM AUTHOR]

Drug hypersensitivity reactions ( DHRs) are a matter of great concern, both for outpatient and in hospital care. The evaluation of these patients is complex, because in vivo tests have a suboptimal sensitivity and can be time-consuming, expensive and potentially risky, especially drug provocation tests. There are several currently available in vitro methods that can be classified into two main groups: those that help to characterize the active phase of the reaction and those that help to identify the culprit drug. The utility of these in vitro methods depends on the mechanisms involved, meaning that they cannot be used for the evaluation of all types of DHRs. Moreover, their effectiveness has not been defined by a consensus agreement between experts in the field. Thus, the European Network on Drug Allergy and Drug Allergy Interest Group of the European Academy of Allergy and Clinical Immunology has organized a task force to provide data and recommendations regarding the available in vitro methods for DHR diagnosis. We have found that although there are many in vitro tests, few of them can be given a recommendation of grade B or above mainly because there is a lack of well-controlled studies, most information comes from small studies with few subjects and results are not always confirmed in later studies. Therefore, it is necessary to validate the currently available in vitro tests in a large series of well-characterized patients with DHR and to develop new tests for diagnosis. [ABSTRACT FROM AUTHOR]