Patch testing with the European baseline series and 10 added allergens: Single-centre study of 748 patients.

BACKGROUND: The European baseline series (EBS) of contact allergens is subject to change. An allergen is considered for inclusion when routine patch testing of patients with suspected contact dermatitis results in ≥0.5% prevalence rate. OBJECTIVES: We aimed to determine the frequency of sensitizations to 30 EBS allergens and 10 locally added allergens. Additionally, we assessed the strength and evolution of reactions to all tested allergens and co-reactivity of additional allergens. METHODS: Patch testing with our baseline series of 40 allergens was done in 748 consecutive adults. Tests were applied to the upper back and removed by patients after 48 h. Readings were done on Day 3 (D3) and D6 or D7 (D6/7). Positive reactions fulfilled the criteria of at least one plus (+) reaction. A retrospective analysis was done. RESULTS: Eight allergens not listed in the EBS had ≥0.5% prevalence rate (i.e., cocamidopropyl betaine, thiomersal, disperse blue mix 106/124, 2-bromo-2-nitropropane-1,3-diol, diazolidinyl urea, propylene glycol, Compositae mix II and dexamethasone-21-phosphate), and 16.6% of positive reactions would have been missed without D6/7 readings. CONCLUSION: We propose further studies to evaluate whether cocamidopropyl betaine, disperse blue mix 106/124, 2-bromo-2-nitropropane-1,3-diol, diazolidinyl urea and Compositae mix II need to be added to the EBS.

Biomarkers of the Severity of Honeybee Sting Reactions and the Severity and Threshold of Systemic Adverse Events During Immunotherapy.

BACKGROUND: A biomarker that could identify individuals at high risk for severe honeybee sting allergic reaction and/or systemic adverse events (SAEs) during venom immunotherapy (VIT) would improve the management of patients with honeybee (HB) venom allergy. OBJECTIVE: To identify biomarkers for risk of severe sting reactions or SAEs during VIT. METHODS: We recruited 332 patients undergoing HB VIT. We ascertained predictors of the severity of the field-sting reaction and the severity and threshold of SAEs during VIT. We assessed the use of cardiovascular medications; baseline serum tryptase (BST) levels; specific IgEs to HB venom, rApi m 1, and rApi m 10; and basophil activation test (BAT) response. RESULTS: Significant and independent predictors of a severe HB field-sting reaction were age (P = .008), an absence of skin symptoms (P = .001), BST (P = .014), and BAT response at an HB venom concentration of 0.1 µg/mL (P = .001). Predictors of severe SAEs during HB VIT were age (P = .025), BST (P = .006), and BAT response (P = .001). BAT response was also an individual and significant predictor of any SAEs and SAEs at a low cumulative allergen dose (median, 55 µg) during VIT build-up (P < .001). The use of ß-blockers and angiotensin-converting-enzyme inhibitors and specific IgE levels were not associated with the severity of HB field-sting reactions or VIT SAEs. CONCLUSIONS: BST and basophil activation are independent risk factors for severe HB sting anaphylaxis and SAEs during HB VIT. BAT response was the best biomarker for any SAEs and a lower threshold of SAEs during HB VIT. These risk factors can help guide recommendations for VIT and overcome systemic reactions to HB VIT.

Routine KIT p.D816V screening identifies clonal mast cell disease in patients with Hymenoptera allergy regularly missed using baseline tryptase levels alone.

BACKGROUND: Clonal mast cell disorders and elevated basal serum tryptase (BST) levels with unknown cause(s) are associated with severe Hymenoptera venom-triggered anaphylaxis (HVA). However, some individuals with clonal disease have a normal BST level (<11.4 ng/mL). OBJECTIVE: Our aim was to evaluate whether screening for KIT p.D816V in the blood is a useful clinical tool to risk-stratify patients with venom allergy. METHODS: We prospectively recruited 374 patients with Hymenoptera allergy and no overt signs of mastocytosis who were referred to our center during the years 2018 and 2019. KIT p.D816V was determined in their peripheral blood by quantitative PCR, and tryptase genotyping was performed by droplet digital PCR. RESULTS: In all, 351 patients (93.9%) had normal levels of BST, and KIT p.D816V was detected in 8% of patients (28 of 351), predominantly in patients with the most severe Mueller grade IV anaphylaxis (18.2% [24 of 132] vs 1.8% in patients with lower grades [4 of 88 with grade III and 0 of 131 with other grades]; P < .001). In grade IV patients with a normal BST level, KIT p.D816V was associated with more severe symptoms, including a significantly higher frequency of loss of consciousness (58.3% [14 of 24] vs 34.3% [37 of 108]; P = .03) and absence of skin symptoms (41.7% [10 of 24] vs 15.7% [17 of 108]; P = .004). Among patients with a normal BST level, KIT p.D816V (OR = 10.25 [95% CI = 3.75-36.14]; P < .0001) was the major risk factor associated with severe HVA. Hereditary α-tryptasemia (HαT) due to increased germline copies of TPSAB1 encoding α-tryptase was the most common cause (65.2% [15 of 23]) of elevated BST level in patients with HVA, and together with KIT p.D816V, it accounted for 90% of BST level elevations (20 of 23) in patients with HVA. CONCLUSION: These results indicate that routine KIT p.D816V screening identifies clonal disease in high-risk patients with HVA who are regularly missed when BST level is used alone.

Chemokines during anaphylaxis: the importance of CCL2 and CCL2-dependent chemotactic activity for basophils.

BACKGROUND: The role of chemokines in anaphylaxis is unclear. METHODS: We prospectively recruited 49 patients presenting to the emergency department with an acute episode of anaphylaxis and 28 healthy subjects. We measured serum levels of the chemokines CCL2, CCL5, CCL7, CCL8, CCL11, CCL13, CCL17, CCL21, CCL22, CCL24, and CCL26, tryptase, the absolute number of circulating basophils, monocytes, lymphocytes, and PMNs, and whole blood FCER1A, CPA3 and HDC gene expression at two time points: during the anaphylactic episode and in convalescent samples collected approximately 3 months later. We then investigated the in vitro chemotactic activity of chemokines induced during anaphylaxis for the in vitro migration of the corresponding cells. RESULTS: Only CCL2 chemokine levels were significantly increased in anaphylaxis samples (median 514 pg/ml) compared to convalescent samples (284 pg/ml, P < 0.0001) and healthy subjects (279 pg/ml, P < 0.0001); there was no significant difference in any of the other chemokines. There was a significant positive correlation between the rates of increase of serum CCL2 (median [range]: 106.0% [- 44.7% to 557.4%]) and tryptase (133.8% [- 6.6% to 893.4%]; r = 0.68, P < 0.0001) and between the acute concentration of serum CCL2 and the acute concentration of serum tryptase (r = 0.77, P < 0.0001). The number of circulating basophils, but not other blood cells, significantly decreased during anaphylaxis (median 5.0 vs. 19.1 cells/µl in convalescent samples; P < 0.0001); a decrease in whole-blood gene expression of basophil markers (P ≤ 0.0018) confirmed these changes. Anaphylactic serum enhances the in vitro migration of basophils via CCL2-dependent chemotactic activity; in contrast, no CCL2-dependent chemotactic activity was observed for convalescent samples. CONCLUSIONS: Our findings imply an important and specific role for CCL2-mediated chemotactic activity in the pathophysiology of human anaphylaxis.

Distinct Contributory Factors Determine Basophil-Allergen Sensitivity in Grass Pollen Rhinitis and in Anaphylactic Wasp Venom Allergy.

BACKGROUND: We sought to determine whether basophil-allergen sensitivity could be transferred to donor basophils by passive IgE sensitisation in allergic rhinitis and anaphylactic Hymenoptera venom hypersensitivity. METHODS: We studied 15 wasp venom-, 19 grass pollen- and 2 house dust mite-allergic patients, 2 healthy donors, and 8 wasp venom-allergic donors. In all subjects, we first evaluated the initial basophil response to wasp venom, grass pollen, or house dust mite allergen. Donor basophils were then stripped, sensitised with the different patients' serum IgE, and challenged with the corresponding allergen. The CD63 response of donor basophils was then compared with initial basophil responses. RESULTS: In wasp venom-allergic subjects, the IgE transfer did not reflect the initial basophil-allergen sensitivity, because the venom IgE of subjects with high or low basophil sensitivity induced comparable responsiveness in healthy donor basophils. Furthermore, vice versa, when we sensitised the donor basophils of wasp venom-allergic individuals with different wasp venom or house dust mite IgE, we demonstrated that their response was predictable by their initial basophil allergen sensitivity. In the rhinitis allergy model, the IgE transfer correlated with the patients' initial basophil responsiveness because the grass pollen IgE of the subjects with high basophil allergen sensitivity induced significantly higher responsiveness of donor basophils than the IgE of subjects with initially low basophil allergen sensitivity. CONCLUSIONS: Our results suggest that basophil allergen sensitivity evaluated by flow-cytometric CD63 analysis depends on two distinct contribution factors. In anaphylactic Hymenoptera allergy, the major factor was intrinsic cellular sensitivity, whereas in pollen allergy, the major factor was allergen-specific IgE on the cell surface.

Omalizumab in chronic urticaria: our experience and literature review.

INTRODUCTION: Chronic urticaria (CU) severely affects quality of life. If symptoms are not controlled by antihistamines, patients need immunomodulatory drugs. Recent studies show a tremendous effect of omalizumab, a monoclonal antibody against human IgE in refractory CU. METHODS: We report on the use of omalizumab in four patients with CU. By reviewing medical files, we estimated the proportion of CU patients that are candidates for such treatment. We reviewed the literature to compare the dosing schedules and outcome measures used in different studies. RESULTS: Up to 14% of CU patients referred to a tertiary center are candidates for omalizumab. Four of our CU were patients treated with doses of 150 mg/month or less, and all responded with nearly complete remission of symptoms. In the literature, 90% of patients respond to treatment, the response being obvious in days. Half of patients were able to stop all other medications, including antihistamines. More than half of patients responded well to doses of 150 mg of omalizumab every 4 to 8 weeks. In the majority of patients, the disease relapsed after discontinuation of omalizumab. CONCLUSIONS: Omalizumab should be offered to patients with refractory CU. The duration of treatment is not known.

Down-regulation of FcεRI-mediated CD63 basophil response during short-term VIT determined venom-nonspecific desensitization.

BACKGROUND: We recently showed a desensitization of FcεRI-mediated basophil response after short-term VIT. Our aim was to evaluate the allergen specificity of this desensitization. METHODS: In 11 Hymenoptera-venom double positive subjects, basophil threshold sensitivity (CD-sens) to anti-FcεRI, honeybee, and Vespula venom was assessed at the beginning and just before the first maintenance dose (MD) of single ultra-rush VIT. In some patients we also monitored CD-sens to rApi m 1 and/or rVes v 5 or other co-sensitizations (i.e., grass pollen). In additional 7 patients, basophils were stripped and sensitized with house dust mite (HDM) IgEs at the same time points. RESULTS: We demonstrated a marked reduction of CD-sens to anti-FcεRI and VIT-specific venom before the first MD in all 18 subjects included. Furthermore, in 10 out of 11 double positive subjects, a significant and comparable decrease before the first MD was also evident for non-VIT venom; this nonspecific decrease was further supported by the opposite recombinant species-specific major allergen. In one subject with additional grass pollen allergy, a decrease of CD-sens to grass allergen was also demonstrated. Similarly, in 7 cases of patients with passively HDM-sensitized basophils, a significant reduction of CD-sens was also evident to de novo sensitized HDM allergen. CONCLUSIONS: Short-term VIT induced basophil desensitization to VIT-specific as well as to VIT-nonspecific venom. As opposed to long-term VIT, which induces venom-specific changes, the effect of short-term VIT seems to be venom-nonspecific.

Clinical routine utility of basophil activation testing for diagnosis of hymenoptera-allergic patients with emphasis on individuals with negative venom-specific IgE antibodies.

BACKGROUND: Previous reports suggest the usefulness of basophil activation testing (BAT) in Hymenoptera-allergic patients with negative venom-specific IgE antibodies. We sought to evaluate the diagnostic utility of this testing in a routine clinical laboratory setting. MATERIALS AND METHODS: Twenty-one patients with anaphylactic reactions to Hymenoptera sting (median grade III) and negative venom-specific IgE were routinely and prospectively tested with BAT. RESULTS: We were able to diagnose 81% (17 of 21) of patients with BAT and 57% (12 of 21) with intradermal skin testing. Three wasp venom-allergic patients showed IgE positivity to rVes v 5. Four patients (19%) were negative for all tests. In the case of double-positive BAT, the culprit insect correlated with the venom that induced a significantly higher basophil response. CONCLUSIONS: BAT allows the identification of severe Hymenoptera-allergic patients with negative specific IgE and skin tests. The routine use of this cellular test should facilitate prescription of venom immunotherapy in complex cases with inconclusive diagnostic results.

Carbohydrate epitopes as a cause of cross-reactivity in patients allergic to Hymenoptera venom.

BACKGROUND: Among patients with allergy to insect stings, double positivity in tests for IgE antibodies specific to honey bee and wasp venoms is a frequent diagnostic problem. True double sensitization and possible cross-reactivity of venom hyaluronidases and with carbohydrate determinants must be considered in such patients. We studied the frequency of sensitization to carbohydrate determinants and the role of these in double positivity in tests for specific IgE antibodies. MATERIALS AND METHODS: A group of 66 patients (41 men, 25 women; 16-66 years) with double positivity for wasp and bee venoms were tested in the FEIA inhibition test in order to distinguish true double sensitization from cross-reactivity. Patients were tested for the presence of IgE antibodies specific to oilseed rape (OSR) pollen and MUXF3 allergens, both of which are rich in cross-reacting carbohydrate epitopes. RESULTS: Inhibition tests revealed true double sensitization in 37 patients (56.1%) and cross-reactivity in 29. Among those showing cross-reactivity, five were sensitized to honey bee venom and 24 to wasp venom. The median value of IgE specific for OSR pollen in patients sensitized to honey bee venom was 4.350 IU/ml, in patients sensitized to wasp venom 0.61 IU/ml, and in patients with double sensitization 0.25 IU/ml (P = 0.028, Kruskal-Wallis test). Findings for IgE specific for MUXF3 were similar. Discordance between OSR pollen positivity and MUXF3 positivity was found in 11.1% of the patients. CONCLUSION: The values of IgE specific for OSR pollen and MUXF3 in patients with primary sensitization to either honey bee venom or wasp venom were significantly higher than in patients with double sensitization. These results confirm that IgE antibodies against carbohydrates epitopes are a frequent cause of double positivity in tests for anti-venom IgE antibodies.

The local and systemic side-effects of venom and inhaled-allergen subcutaneous immunotherapy.

BACKGROUND: Although immunotherapy is effective in allergic rhinitis, conjunctivitis, asthma and stinging insect hypersensitivity, it carries a risk of anaphylactic reactions. METHODS: In a 4-year retrospective survey, we investigated 1257 adult patients who had received venom or inhaled-allergen subcutaneous immunotherapy. The dose-increase phase was performed as the 2-day rush protocol for venom immunotherapy and the 6-week protocol for inhaled-allergen immunotherapy. RESULTS: A total of 904 patients received venom immunotherapy and 353 patients inhaled-allergen immunotherapy. The prevalence of systemic reactions was 13.6%. The frequency of systemic reactions was higher during the maintenance phase than in the dose-increase phase (9.6% vs. 5.9%) and was highest in both phases of treatment with honeybee venom (P < 0.001). The majority of systemic reactions were mild. Five (0.4%) patients had reaction with a fall of blood pressure and were treated with adrenaline. There was no fatal outcome. The systemic side-effects during the dose-increase phase of venom immunotherapy occurred at a median dose of 46 microg (range 2-100 microg). Large local reactions occurred in 13.9% of patients without any significant difference between the allergens. CONCLUSIONS: We have shown that systemic reactions are not rare even during maintenance phase in patients with a well tolerated dose-increase phase of treatment. The most prominent risk factor for systemic reactions was immunotherapy with honeybee extract.

Basophil sensitivity in patients not responding to venom immunotherapy.

BACKGROUND: Treatment failure of venom immunotherapy (VIT) is not rare and the risk and pathogenic factors for those failures are so far poorly understood. For that reason we evaluated allergen-specific basophil sensitivity in patients who did not tolerate field re-stings after completed VIT treatment. METHODS: Basophil responsiveness was evaluated by flow cytometry analyses of basophil CD63 surface expression induced by different concentrations of bee or wasp venom (1, 0.1 and 0.01 microg/ml) in 14 treated patients who had experienced systemic allergic reactions (Muller grades II-III) and 17 treated patients who had no reactions after the field re-stings. We also included a group of 28 Hymenoptera venom-allergic patients who had not received VIT. RESULTS: In 14 patients who still reacted to bee or wasp sting, basophil response at a venom concentration of 0.1 microg/ml was significantly higher than in patients who tolerated field re-stings (p = 0.03; t test). Basophil response was also slightly higher at a concentration of 1 microg/ml, but not to statistical significance (p = 0.12; t test). There was no difference in the response to direct cross-linking of the IgE and in venom-specific IgE and IgG4 serum concentrations between those 2 groups (p > 0.8; Fisher's exact test, t test). Patients who tolerated field re-stings have also significantly lower basophil response in comparison to patients who had not received VIT, both at 0.1 and 1 microg/ml of venom concentrations (p < 0.001; t test). CONCLUSIONS: The results suggest that basophil venom-specific sensitivity is associated with the efficiency of VIT.

The influence of antimicrobial therapy on the sensitivity of Legionella PCR.

The aim of our study was to establish the sensitivity of Legionella DNA detection in lower respiratory tract samples in 3 cases of Legionnaires' disease after initiation of specific antibiotic therapy. The results showed that Legionella amplicon intensity was highest in the sputum or bronchial aspirates collected at or before the start of appropriate therapy and decreased markedly within 3 days of therapy. PCR testing was negative within 4 to 6 days of therapy. These data suggest that within a few days specific antimicrobial therapy induces a significant drop of bacterial concentration in respiratory secretions reaching the detection limit of PCR assay. Respiratory samples for Legionella PCR should be obtained before or early after initiating antimicrobial therapy.

Wasp venom is appropriate for immunotherapy of patients with allergic reaction to the European hornet sting.

AIM: To identify whether it is the yellow jacket (Vespula germanica) or European hornet (Vespa crabro) venom that induces sensitization in patients with IgE-mediated allergic reaction to the venom from the sting of a European hornet. Since these patients usually have positive skin tests and specific IgE to all vespid venoms, it would be useful to distinguish cross-reactors from non-cross-reactors to perform immunotherapy with the venom that induced the sensitization. METHODS: We performed inhibition tests in 24 patients who had experienced anaphylactic reaction after being stung by a European hornet. RESULTS: Of 24 patients with allergic reaction after Vespa crabro sting, 17 were sensitized only to epitopes of Vespula germanica venom. Only 4 out of 24 patients were sensitized to epitopes completely cross-reactive with Dolichovespula arenaria venom. CONCLUSION: In Slovenia, the vast majority of patients with anaphylactic reaction to Vespa crabro sting seem to be sensitized to Vespula germanica venom. We consider wasp venom an appropriate immunotherapeutic agent for such patients, except for those with proven primary sensitization to specific epitopes of Vespa crabro venom. Fluorescence enzyme immunoassay inhibition should be considered a convenient tool for the identification of primary sensitization in patients allergic to vespid venoms.