Mihaela Zidarn

Recombinant allergen-based IgE testing to distinguish bee and wasp allergy

BACKGROUND The identification of the disease-causing insect in venom allergy is often difficult. OBJECTIVE To establish recombinant allergen-based IgE tests to diagnose bee and yellow jacket wasp allergy. METHODS Sera from patients with bee and/or wasp allergy (n = 43) and patients with pollen allergy with false-positive IgE serology to venom extracts were tested for IgE reactivity in allergen extract-based tests or with purified allergens, including nonglycosylated Escherichia coli-expressed recombinant (r) Api m 1, rApi m 2, rVes v 5, and insect cell-expressed, glycosylated rApi m 2 as well as 2 natural plant glycoproteins (Phl p 4, bromelain). RESULTS The patients with venom allergy could be diagnosed with a combination of E coli-expressed rApi m 1, rApi m 2, and rVes v 5 whereas patients with pollen allergy remained negative. For a group of 29 patients for whom the sensitizing venom could not be identified with natural allergen extracts, testing with nonglycosylated allergens allowed identification of the sensitizing venom. Recombinant nonglycosylated allergens also allowed definition of the sensitizing venom for those 14 patients who had reacted either with bee or wasp venom extracts. By IgE inhibition studies, it is shown that glycosylated Api m 2 contains carbohydrate epitopes that cross-react with natural Api m 1, Ves v 2, natural Phl p 4, and bromelain, thus identifying cross-reactive structures responsible for serologic false-positive test results or double-positivity to bee and wasp extracts. CONCLUSION Nonglycosylated recombinant bee and wasp venom allergens allow the identification of patients with bee and wasp allergy and should facilitate accurate prescription of venom immunotherapy.

High sensitivity of CAP-FEIA rVes v 5 and rVes v 1 for diagnosis of Vespula venom allergy

and proline, IgE produced against HWPs probably cross-reacts with natural wheat proteins. In fact, preincubation of sera with HWP-A clearly revealed a decreased binding of IgE to natural wheat proteins. In conclusion, measurement of basophil CD203c expression induced by various preparations of wheat proteins is highly useful in predicting causative allergens in patients with WDEIA. Furthermore, the basophil activation test based on the expression of CD203c might help determine causative allergens for a wide variety of food allergies.

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.

Hereditary Angioedema Nationwide Study in Slovenia Reveals Four Novel Mutations in SERPING1 Gene

Hereditary angioedema (HAE) is a rare autosomal dominant disease characterized by swelling of the face, lips, tongue, larynx, genitalia, or extremities, with abdominal pain caused by intra-abdominal edema. HAE is caused by mutations affecting the C1 inhibitor gene, SERPING1, resulting in low levels of C1 inhibitor (Type I HAE) or normal levels of ineffective C1 inhibitor (Type II HAE). A nationwide survey identified nine unrelated families with HAE in Slovenia, among whom 17 individuals from eight families were recruited for genetic analyses. A diagnosis of HAE was established in the presence of clinical and laboratory criteria (low C1 inhibitor antigenic levels and/or function), followed up by a positive family history. Genetic studies were carried out using PCR and sequencing to detect SERPING1 mutations in promoter, noncoding exon 1, the 7 coding exons, and exon-intron boundaries. Multiplex ligation-dependent probe amplification was performed in order to search for large deletions/duplications in SERPING1 gene. A mutation responsible for HAE was identified in patients from seven families with the disease. In HAE type I families, one previously reported substitution (Gln67Stop, c.265C>T) and four novel mutations were identified. The new mutations included two missense substitutions, Ser128Phe (c.449C>T), and Glu429Lys (c.1351G>A), together with two frameshift mutations, indel (c.49delGinsTT) and deletion (c.593_594delCT). Both families with HAE type II harbored the two well-known substitutions affecting the arginyl residue at the reactive center in exon 8, Arg444Cys (c.1396C>T) and Arg444His (c.1397G>A), respectively. In one patient only the homozygous variant g.566T>C (c.-21T>C) was identified. Our study identified four novel mutations in the Slovenian HAE population, highlighting the heterogeneity of mutations in the SERPING1 gene causing C1 inhibitor deficiency and HAE. In a single patient with HAE a homozygous variant g.566T>C (c.-21T>C) might be responsible for the disease.

Safety and efficacy of immunotherapy with the recombinant B-cell epitope–based grass pollen vaccine BM32

Background: BM32 is a grass pollen allergy vaccine based on recombinant fusion proteins consisting of nonallergenic peptides from the IgE‐binding sites of the 4 major grass pollen allergens and the hepatitis B preS protein. Objective: We sought to study the safety and clinical efficacy of immunotherapy (allergen immunotherapy) with BM32 in patients with grass pollen–induced rhinitis and controlled asthma. Methods: A double‐blind, placebo‐controlled, multicenter allergen immunotherapy field study was conducted for 2 grass pollen seasons. After a baseline season, subjects (n = 181) were randomized and received 3 preseasonal injections of either placebo (n = 58) or a low dose (80 &mgr;g, n = 60) or high dose (160 &mgr;g, n = 63) of BM32 in year 1, respectively, followed by a booster injection in autumn. In the second year, all actively treated subjects received 3 preseasonal injections of the BM32 low dose, and placebo‐treated subjects continued with placebo. Clinical efficacy was assessed by using combined symptom medication scores, visual analog scales, Rhinoconjunctivitis Quality of Life Questionnaires, and asthma symptom scores. Adverse events were graded according to the European Academy of Allergy and Clinical Immunology. Allergen‐specific antibodies were determined by using ELISA, ImmunoCAP, and ImmunoCAP ISAC. Results: Although statistical significance regarding the primary end point was not reached, BM32‐treated subjects, when compared with placebo‐treated subjects, showed an improvement regarding symptom medication, visual analog scale, Rhinoconjunctivitis Quality of Life Questionnaire, and asthma symptom scores in both treatment years. This was accompanied by an induction of allergen‐specific IgG without induction of allergen‐specific IgE and a reduction in the seasonally induced increase in allergen‐specific IgE levels in year 2. In the first year, more grade 2 reactions were observed in the active (n = 6) versus placebo (n = 1) groups, whereas there was almost no difference in the second year. Conclusions: Injections of BM32 induced allergen‐specific IgG, improved clinical symptoms of seasonal grass pollen allergy, and were well tolerated.

The culprit insect but not severity of allergic reactions to bee and wasp venom can be determined by molecular diagnosis

Background Allergy to bee and wasp venom can lead to life-threatening systemic reactions. The identification of the culprit species is important for allergen-specific immunotherapy. Objectives To determine a panel of recombinant bee and wasp allergens which is suitable for the identification of bee or wasp as culprit allergen sources and to search for molecular surrogates of clinical severity of sting reactions. Methods Sera from eighty-seven patients with a detailed documentation of their severity of sting reaction (Mueller grade) and who had been subjected to titrated skin testing with bee and wasp venom were analyzed for bee and wasp-specific IgE levels by ImmunoCAPTM. IgE-reactivity testing was performed using a comprehensive panel of recombinant bee and wasp venom allergens (rApi m 1, 2, 3, 4, 5 and 10; rVes v 1 and 5) by ISAC chip technology, ImmunoCAP and ELISA. IgG4 antibodies to rApi m 1 and rVes v 5 were determined by ELISA and IgE/IgG4 ratios were calculated. Results from skin testing, IgE serology and IgE/IgG4 ratios were compared with severity of sting reactions. Results The panel of rApi m 1, rApi m 10, rVes v 1 and rVes v 5 allowed identification of the culprit venom in all but two of the 87 patients with good agreement to skin testing. Severities of sting reactions were not associated with results obtained by skin testing, venom-specific IgE levels or molecular diagnosis. Severe sting reactions were observed in patients showing < 1 ISU and < 2kUA/L of IgE to Api m 1 and/or Ves v 5. Conclusion We identified a minimal panel of recombinant bee and wasp allergens for molecular diagnosis which may permit identification of bee and/or wasp as culprit insect in venom-sensitized subjects. The severity of sting reactions was not associated with parameters obtained by molecular diagnosis.

Differences in Reporting the Ragweed Pollen Season Using Google Trends across 15 Countries

Background: Google Trends (GT) searches trends of specific queries in Google, which potentially reflect the real-life epidemiology of allergic rhinitis. We compared GT terms related to ragweed pollen allergy in American and European Union countries with a known ragweed pollen season. Our aim was to assess seasonality and the terms needed to perform the GT searches and to compare these during the spring and summer pollen seasons. Methods: We examined GT queries from January 1, 2011, to January 4, 2017. We included 15 countries with a known ragweed pollen season and used the standard 5-year GT graphs. We used the GT translation for all countries and the untranslated native terms for each country. Results: The results of “pollen,” “ragweed,” and “allergy” searches differed between countries, but “ragweed” was clearly identified in 12 of the 15 countries. There was considerable heterogeneity of findings when the GT translation was used. For Croatia, Hungary, Romania, Serbia, and Slovenia, the GT translation was inappropriate. The country patterns of “pollen,” “hay fever,” and “allergy” differed in 8 of the 11 countries with identified “ragweed” queries during the spring and the summer, indicating that the perception of tree and grass pollen allergy differs from that of ragweed pollen. Conclusions: To investigate ragweed pollen allergy using GT, the term “ragweed” as a plant is required and the translation of “ragweed” in the native language needed.

Recommendations for the management of asthma patients at primary and specialist pulmonary levels in Slovenia

The purpose of this paper is to implement the guidelines proposed by GINA in the Slovenian healthcare system, and to describe the cornerstones of the management of this disease. The document is meant to serve as an agreed approach to the management of asthma patients.

Identification and treatment of patients with suspected penicillin allergy.

Background: Penicillin antibiotics are the most common medicines that are suspected to cause allergy. True penicillin allergy is rarely diagnosed in clinical practice. Many patients with a personal history of penicillin allergy are unnecessarily denied the benefits of penicillin and are given broader-spectrum antibiotics. Such unnecessary prescription of extended-spectrum antibiotics could contribute to the development and spread of multiple drug-resistant bacteria. The aim of our study was to check the identification and treatment of patients with a reported history of penicillin allergy. Methods: In the first part of the study, 21 pharmacies were included. Pharmacists asked adults who came to the pharmacy with a prescription for a non-penicillin antibiotic whether they are penicillin allergic and whether the allergy was confirmed with tests. In the second part of the study the antibiotic prescription pattern was surveyed in patients with ruled-out penicillin allergy. Results: 355/435 (81.6%) subjects, who came with the prescriptions for non-penicillin antibiotic, were patients – direct users of the prescribed antibiotic. Out of 124 patients with non-penicillin prescriptions, 26 (21%) claimed penicillin hypersensitivity, however, only in 7 (26.9%) it had been confirmed by allergy tests. Out of 272 patients with presumed penicillin hypersensitivity, diagnosis was confirmed in only 22 (8%). 61.4% patients with ruled-out penicillin allergy got a prescription for antibiotic in the previous year. However, in only 43.9% of cases these were penicillin antibiotics. Conclusion: We confirmed that many patients report penicillin allergy, however, the allergy is rarely confirmed by tests. Also, after tests that rule out penicillin allergy have been performed, penicillin antibiotics are still prescribed in lesser percent than other antibiotics. We found out that the vast majority of adults who come in the pharmacy with an antibiotic prescription are direct consumers of the prescribed antibiotic. Therefore, there is an opportunity to develop and implement a program of pharmaceutical care also for antibiotic treatment.

Sensitization to inhalant alergens in patients with allegic airway disease in Slovenia

Background: Skin prick testing is the basic diagnostic method for IgE-mediated allergies. To define a standard battery of allergens, data about local sensitization pattern are needed. The aims of the study were to define the prevalence of sensitization to allergens recommended as Pan European standard prick test panel in Slovenija, to define a minimum battery of allergens for epidemiological studies and to asses sensitization to those allergens across the country. Another aim was to define the usefulness of testing with extensively cross-reactive allergens and the use of allergen mixtures. Methods: The prevalence of sensitization for Pan-European standard prick test panel was assessed at single referral allergy centre, where the same patients were also tested with cross reactive allergens and allergen mixtures. A minimum battery for epidemiological study was defined from this data. The prevalence of sensitization to those allergens was than assessed in 13 centers in Slovenija. Results: All allergens suggested as Pan-European standard prick test panel shoved a prevalence of more than 2 %, so all of them should be used in clinical setting. Additional three allergens are needed for Slovene standard skin prick test panel. Eight allergens are needed for a minimum battery of allergens needed in epidemiological studies. Conclusion: Slovene standard panel for skin prick tests should include: Alternaria, Ambrosia, Artemisia, Aspergillus, Betulacea, Blatella, Cat, Cladosporium, Cypres, Dermatophagoides farinae, Dermatophagoides pteronyssinus, Dog, Grass, Olive, Parietaria, Penicillium, Plane, Plantago, Rumex, Urtica.. Further studies are needed to confirm the importance of added allergens and to define other allergens that might be locally important.