Basophil Histamine Release Induced by Amoxicilloyl-poly-L-lysine Compared With Amoxicillin in Patients With IgE-Mediated Allergic Reactions to Amoxicillin.

BACKGROUND: Amoxicillin (AX) is the ß-lactam most often involved in IgE-mediated reactions. Diagnosis is based mainly on skin testing, although sensitivity is not optimal. We produced a new AX derivative, amoxicilloyl-poly-L-lysine (APL), and analyzed its recognition of IgE using the passive histamine release test (pHRT). METHODS: The study population comprised patients (n=19) with confirmed AX allergy and specific IgE to AX and controls (n=10) with good tolerance to AX. pHRT was performed using "IgE-stripped" blood from a single donor that was sensitized in vitro by patient sera and incubated with AX or APL. Histamine release was determined and expressed as nanograms of histamine released per milliliter of blood. RESULTS: The clinical symptoms were anaphylaxis (n=9), urticaria (n=7), erythema (n=2), and nondefined immediate reactions (n=1). The median (IQR) time interval between reaction and study was 90 (60-240) days and between drug intake and development of symptoms 24 (10-60) minutes. The median sIgE level was 3.37 (0.95-5.89) kUA/L. The sensitivity of pHRT to APL was 79% and the specificity 100%, which were higher than data obtained with pHRT to AX (63% sensitivity and 90% specificity). There was a positive correlation between maximal histamine release levels obtained with AX and APL (r=0.63). CONCLUSIONS: In patients with immediate hypersensitivity reactions to AX, APL showed higher sensitivity and specificity than the culprit drug, AX, when tested in vitro by pHRT. This indicates that APL can improve the in vitro diagnostic accuracy of allergic reactions to AX. Further assessment of skin testing is necessary.

In vitro tests for drug hypersensitivity reactions: an ENDA/EAACI Drug Allergy Interest Group position paper.

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.

The role of IgE recognition in allergic reactions to amoxicillin and clavulanic acid.

Betalactam (BL) antibiotics are the drugs most frequently involved in IgE-mediated reactions. The culprit BL varies according to consumption patterns, with amoxicillin (AX) more prevalent in Southern Europe and penicillin V in Scandinavian countries. Nowadays, the combination of AX and clavulanic acid (CLV) is the most highly consumed BL containing medicine worldwide. Both BLs, AX and CLV, can independently be involved in reactions, which poses a diagnostic challenge. In patients with immediate allergic reactions to AX, two patterns of responses have been described, those responding to benzylpenicillin (cross-reactors) and those selective to AX. In addition, selective reactions to CLV account for around 30% of allergic reactions to the combination AX-CLV. These patterns of IgE recognition could be related to differences in the haptenation process, in the immunological response, or in the BL involved in the first sensitization. In this regard, patients with selective responses to CLV are generally younger than those allergic to AX or benzylpenicillin. So far, no evidence of cross-reactivity between CLV and other BLs has been reported. This shows the importance of an accurate diagnosis of CLV allergy, as patients with selective reactions to CLV could take other BLs including AX. Diagnosis can be performed in vivo and in vitro, although no immunoassay currently exists. Research regarding the CLV antigenic determinants and protein conjugates is essential to improve diagnosis. BLs need to covalently bind to a carrier protein to be immunogenic. The antigenic determinant of AX is the amoxicilloyl amide, but CLV leads to unstable structures, many of which are unknown. Moreover, the nature of the BL-protein conjugates plays an important role in IgE recognition. This review aims to summarize current knowledge on the immunochemistry, diagnostic approaches as well as chemical and proteomic studies for both AX and CLV.

Hypersensitivity reactions to ß-lactams: relevance of hapten-protein conjugates.

ß-Lactams (BL) are the drugs most frequently involved in allergic reactions. They are classified according to their chemical structure as penicillins, cephalosporins, monobactams, carbapenems, and clavams. All BL antibiotics have a BL ring that is fused to a 5-member or 6-member ring (except in monobactams) and has 1, 2 or 3 side chains (except in clavams). Differences in chemical structure mean that a wide range of BLs are recognized by the immune system, and patients may experience clinical reactions to one BL while tolerating others. Diagnosis is based on skin and in vitro testing, although both display low sensitivity, possibly because they are based on drugs or drug conjugates that are not optimally recognized by the immune system. BLs are haptens that need to bind to proteins covalently to elicit an immune response. These drugs have a high capacity to form covalent adducts with proteins through nucleophilic attack of amino groups in proteins on the BL ring. Allergenic determinants have been described for all BLs, although benzylpenicillin is the most widely studied. Moreover, formation of BL-protein adducts is selective, as we recently demonstrated for amoxicillin, which mainly modifies albumin, transferrin, and immunoglobulin heavy and light chains in human serum. Given the complexity of BL allergy, understanding the immunological mechanisms involved and optimization of diagnostic methods require multidisciplinary approaches that take into account the chemical structures of the drugs and the carrier molecules, as well as the patient immune response.

IgE to penicillins with different specificities can be identified by a multiepitope macromolecule: Bihaptenic penicillin structures and IgE specificities.

Quantitation of specific IgE by immunoassay is a recommended in vitro test for the diagnosis of immediate hypersensitivity reactions to betalactams (BLs), particularly when skin test results are negative. IgE antibodies that recognize the common nuclear structure of all BLs or the specific side chain structure can be mainly distinguished by immunoassays. The aim of this study was to develop an immunoassay system to detect IgE antibodies with different specificities. Cellulose discs conjugated with benzylpenicillin (BP), amoxicillin (AX) or both drugs, with poly-l-lysine (PLL) as carrier molecule, were used as solid phases in the radioallergosorbent test (RAST). Direct and inhibition radioimmunoassay studies were made to verify the structures recognized by serum IgE antibodies from penicillin-allergic patients. Our results indicated that the addition of both haptens did not decrease the capacity to capture IgE when serum specific to either BP or AX was used, at least in terms of sensitivity. In addition, the inclusion of two haptens improved significantly the levels of IgE detection in patients who recognized both BP and AX. Therefore, the use of a solid phase with a carrier molecule conjugated with two determinants (AX and BP) is helpful to recognize IgE antibodies against either of these determinants and is useful for screening sera with different specificities.

Hypersensitivity reactions to fluoroquinolones: analysis of the factors involved.

BACKGROUND: Hypersensitivity reactions to fluoroquinolones seem to be on the increase, especially immediate type reactions. OBJECTIVE: The aim of this study was to determine whether several conditions, including gender, age, type of reaction, time interval between the reaction and the study, type of symptoms, the specific fluoroquinolone involved in the reaction and previous confirmed hypersensitivity to betalactams or to other drugs were factors contributing to the development of hypersensitivity to fluoroquinolones. METHOD: We analysed retrospectively all patients attending our allergy department between January 2005 and December 2010 because of a reaction associated with fluoroquinolone administration. The diagnosis was confirmed by basophil activation test or drug provocation tests. In accordance with the results, patients were then classified as having hypersensitivity or non-hypersensitivity to fluoroquinolones. RESULTS: A group of 218 patients was evaluated; 69 were confirmed as having hypersensitivity, 146 as non-hypersensitivity and 3 were excluded. Comparisons between groups showed that the allergic patients more often had a previous confirmed hypersensitivity to betalactams (P = 0.029), immediate reactions (P = 0.001) and anaphylaxis (P = 0.000), and moxifloxacin was the fluoroquinolone most frequently involved (P = 0.027). The logistic regression analysis showed three factors associated with the diagnosis of hypersensitivity reactions to fluoroquinolones: previous hypersensitivity to betalactams (OR: 4.571; 95% CI: 0.987-21.171; adjusted OR: 23.654; 95% CI: 1.529-365.853), immediate reactions (OR: 17.333; 95% CI: 4.374-68.691; adjusted OR: 52.493; 95% CI: 6.621-416.200) and reactions induced by moxifloxacin (OR: 3.091; 95% CI: 1.160-8.239; adjusted OR: 13.610; 95% CI: 2.419-76.565). CONCLUSION: In patients who develop reactions to fluoroquinolones, hypersensitivity is more often confirmed in those with immediate reactions and when moxifloxacin is involved. Moreover, patients with hypersensitivity to betalactams are more prone to develop hypersensitivity reactions to fluoroquinolones.

Dendrimer-modified solid supports: nanostructured materials with potential drug allergy diagnostic applications.

Complex functional materials consisting of bioactive molecules immobilized on solid supports present potential applications in biosensoring. Advances in the fabrication of these surface materials are of growing interest for antibody-based diagnosis. This work exploits dendrimers as versatile nanostructures for templating sensor surfaces and the critical role of the immobilization protocol in the solid supports cellulose and zeolites, of organic and inorganic composition respectively. The fabrication and characterization, including the degree of functionalization and reproducibility, of different nanostructured materials are described. To validate the approach, the fabricated supports were further used as a solid phase for developing a radioimmunoassay to detect immunoglobulin E (IgE) specific to penicillin, the antibody involved in immediate allergy responses to this drug. The dendrimer-modified supports provide assays with significantly enhanced sensitivity, as well as increase the availability of biomolecules for specific interaction and minimize nonspecific adsorptions through appropriate functionalization protocols in each case. The manufacturing methodology involved the use of a long, flexible hydrophilic spacer in the cellulose materials, and a higher surface density of the immobilized dendrimers in the zeolite crystals. The ability of hybrid zeolite materials in such biosensing applications was evaluated for the first time. The assays were validated in human serum samples from patients allergic to penicillin and from non-allergic controls. The specificity and improved sensitivity of the dendrimer- modified supports make these strategies versatile for different bioactive molecules and could have significant implications for the quantification of a wide range of specific IgE antibodies and other biomolecules of diagnostic interest.

Immunoglobulin E-mediated hypersensitivity to amoxicillin: in vivo and in vitro comparative studies between an injectable therapeutic compound and a new commercial compound.

BACKGROUND: Skin testing with amoxicillin (AX) is necessary to diagnose immediate hypersensitivity reactions to this ß-lactam. A commercial AX (DIA-AX) has recently become available for skin testing. OBJECTIVE: The aim of this study was to compare DIA-AX with the injectable form (INJ-AX) in patients who have well-demonstrated IgE-mediated hypersensitivity to AX. METHODS: Chemical characterization using high-performance liquid chromatography of both DIA-AX and INJ-AX reagents was performed. Patients diagnosed with an immediate allergic reaction to AX and a positive skin test to INJ-AX (N=55) were re-evaluated within 6 months by performing skin testing with INJ-AX and DIA-AX. Basophil activation test (BAT) and Radioallergosorbent test (RAST) inhibition assay using both reagents were performed in a selected group of patients. RESULTS: The chemical analysis indicated that both DIA-AX and INJ-AX contained an AX compound with a purity above 95%. In the re-evaluation, 53 (96.4%) cases maintained skin test positivity to INJ-AX and were also positive to DIA-AX. Comparison of the papule area between the two reagents showed no significant differences between both reagents. BAT was performed in 30 samples and was positive to both compounds in 15 cases; no patient had a positive result to just one reagent. RAST inhibition studies using three individual cases and a pool of positive sera showed that the percentage inhibition detected with DIA-AX and INJ-AX was parallel and almost exactly the same. CONCLUSIONS: This study shows that DIA-AX is equivalent to INJ-AX in terms of skin test response, as well as with in vitro immunochemical and biological tests. DIA-AX can therefore be used in the diagnosis of immediate hypersensitivity reactions.

Role of minor determinants of amoxicillin in the diagnosis of immediate allergic reactions to amoxicillin.

BACKGROUND: Skin testing of subjects with immediate hypersensitivity to amoxicillin is performed using major and minor determinants of benzylpenicillin plus amoxicillin. However, sensitivity is not optimal, and other determinants need to be considered. We assessed the sensitivity of stable, well-characterized minor determinants of amoxicillin in subjects with immediate allergic reactions to amoxicillin to improve skin test sensitivity. METHODS: Amoxicillin, amoxicilloic acid, and diketopiperazine were prepared and characterized by reverse-phase HPLC, tested in vivo by skin testing and in vitro by basophil activation test and RAST inhibition assay. RESULTS: Patients with immediate hypersensitivity to amoxicillin were selected: Group A (n = 32), skin test positive just to amoxicillin; Group B (n = 19), skin test positive to benzylpenicillin determinants; Group C (n = 10), skin test negative and amoxicillin drug provocation test positive. In Group A, 27 subjects (81.8%) were skin test positive to amoxicillin, ten (30.3%) to amoxicilloic acid, two (6.1%) to diketopiperacine, and six (18.2%) negative. In Group B, nine (50%) were positive to amoxicillin, eight (42.1%) to amoxicilloic acid, none to diketopiperacine, and nine (50%) negative. In Group C, skin tests were negative. BAT was positive to amoxicillin in 26 patients (50.9%), to amoxicilloic acid in 15 (29.1%), and diketopiperazine in four (7.8%). RAST inhibition studies showed > 50% inhibition in all sera, with the highest concentration of amoxicillin and amoxicilloic acid. CONCLUSIONS: The combination of minor determinants of amoxicillin, amoxicilloic acid, and diketopiperazine seems to be of no greater value than the use of amoxicillin alone. Further efforts are needed to find new structures to improve sensitivity in the diagnosis of immediate hypersensitivity to betalactams.

Skin testing for immediate hypersensitivity to betalactams: comparison between two commercial kits.

INTRODUCTION: Skin testing with major and minor determinants of benzylpenicillin is the recommended standard practice to evaluate subjects with immediate hypersensitivity to betalactams. The withdrawal of these products from the market has set us back to the early days, before the introduction of reagents for in vivo testing. OBJECTIVES: To compare a recently released kit of benzylpenicillin conjugated to poly-l-lysine (PPL) and minor determinants mixture (MDM) with the previously existing kit in a positive control group of subjects sensitized to major and/or minor determinants of benzylpenicillin. METHODS: Skin tests with both kits were made in a group of positive subjects previously diagnosed with immediate hypersensitivity to penicillins and with positive results to PPL and/or MDM and in a negative control group. Radioallergosorbent test (RAST) inhibition assays with a pool of sera and individual samples were carried out to compare the inhibition capacity of PPL and MDM of both kits. RESULTS: Of 22 cases selected from our historical group, 14 were positive: eight to PPL, three to MDM and three to both. These results were equivalent for both kits. RAST inhibition studies showed similar potencies in the inhibition of PPL and MDM. CONCLUSIONS: Both tests show similar results in terms of RAST inhibition assays and skin tests sensitivity and specificity in the groups selected. The new assay can be used for the same purpose and indications as the previous test.