Diagnosis of Apis dorsata venom allergy: use of recombinant allergens of Apis mellifera and a passive basophil activation test.

BACKGROUND: Allergy to Apis dorsata (Giant Asian Honeybee) venom is the commonest insect allergy in Sri Lanka and South East Asia. However, laboratory diagnosis is difficult as the pure venom and diagnostic reagents are not commercially available. OBJECTIVE: This study assessed the use of four recombinant allergens of A. mellifera venom and the passive basophil activation test in the diagnosis of A. dorsata venom anaphylaxis. METHODS: Serum IgE levels to four recombinant allergens of A. mellifera, rApi m 1, 2, 5 and 10 were assessed and compared with serum IgE to the crude venom of A. mellifera or V. vulgaris by Phadia ImmunoCAP, in patients who developed anaphylaxis to A. dorsata stings. Basophil activation in response to venom of A. dorsata or V. affinis was assessed using a passive basophil activation test. Association of the severity of the reaction with basophil activation was compared. RESULTS: rApi m 1 and 10 combinedly had significant correlation (r = 0.722; p < 0.001) with the crude venom of A. mellifera (Western honeybee) and a higher positivity rate of 90% (27/30). Whereas, IgE reactivity to rApi m 2 or 5 had significant correlation (p = 0.02 and p = 0.005 respectively) with V. vulgaris crude venom. All 30 (100%) were positive to A. dorsata venom in passive BAT; 70% (21/30) had over 80% activation, 96.7% (29/30) had over 60% activation and 100% had over 50% activation. Percentage activation of basophils in patients who had mild or moderate reactions (n = 20) was significantly low (p = 0.02) from that of patients who had severe reactions (n = 10). CONCLUSIONS: rApi m 1 and 10 when combined was sensitive for the diagnosis of A. dorsata allergy. This combination had the lowest cross-reactivity rate with Vespula vulgaris. The passive BAT is highly sensitive in A. dorsata allergy. The basophil reactivity was significantly higher in severe anaphylaxis compared to mild/moderate anaphylaxis. This finding should be further explored in further studies.

Diagnosis of Vespa affinis venom allergy: use of immunochemical methods and a passive basophil activation test.

BACKGROUND: Allergy to Vespa affinis venom is common in the Asia Pacific region. Venom preparations for diagnosis are not commercially available for this species. METHODS: The prominent allergens in V. affinis venom were identifiedusing immunochemical methods. Use of ImmunoCAP of Vespula vulgaris crude venom/its components and a passive basophil activation test (BAT) in the diagnosis of patients who had anaphylaxis to V. affinis venom (n = 30) were also accessed. The IgE double-positivity rates (positive to both hornet and honeybee) in ImmunoCAP and the passive BAT were determined. RESULTS: High IgE reactivity was seen with the five allergens in V. affinis venom; 96% (29/30) for 34 and 24 kDa, 93% (28/30) for 45 kDa and 90% (27/30) reactivity for the 100 and 80 kDa respectively. IgE cross-reactivity was low with ImmunoCAP using V. vulgaris venom (43%; 13/30) and Ves v1 (3%; 1/30), but relatively high with Ves v5 (73%; 22/30). All patients (100%) were positive to V. affinis venom in passive BAT. In ImmunoCAP, a high double-positivity rate (76%; 23/30) was detected while no double-positivity was detected in passive BAT. CONCLUSIONS: High IgE reactivity for five allergens of V. affinis points to the potential of using these allergens in component resolved diagnosis (CRD). The passive BAT has shown its importance as a promising diagnostic tool with high accuracy. It would be particularly useful in cases with doubtful double-positive results of other diagnostic tests.

Aetiology of anaphylaxis in patients referred to an immunology clinic in Colombo, Sri Lanka.

BACKGROUND: The aetiology of anaphylaxis differs according to types of foods consumed, fauna and foliage and cultural practices. Although the aetiology of anaphylaxis in Western countries are well known, the causes in South Asian countries have not been reported. We sought to determine the causes of anaphylaxis in patients referred to an immunology clinic in Colombo, Sri Lanka. METHODS: 238 episodes of anaphylaxis were reviewed in 188 patients who were referred and skin prick tests and in vitro tests (ImmunoCap) were carried out to assess the presence of allergen specific IgE. Clinical features and severity of anaphylaxis was also recorded along with treatment received. RESULTS: Anaphylaxis to food either following direct exposure 90/238 (37.5%) or after exercise in the form of food dependent exercise induced anaphylaxis 29/238 (12.2%) was the predominant cause of anaphylaxis. Allergy to cow's milk and red meat, after immediate exposure, accounted for 66/238 (27.7%) of instances of all episodes of anaphylaxis and 66/90 (73.33%) of anaphylaxis due to food. Vaccines accounted for 28/238 (11.8%) of instances of anaphylaxis, especially among children. Of those who developed anaphylaxis to the MMR (n = 14), 71.4% of them had specific IgE to cow's milk and 35.7% of them had specific IgE to beef. Of those who developed anaphylaxis to insect stings, 27/42 of these episodes occurred following stings of ants (family Formicidae). The predominant cause of anaphylaxis changed with the age, with food allergy being the most frequent trigger of anaphylaxis in childhood, while drug allergy and idiopathic anaphylaxis being more frequent after 30 years of age. CONCLUSIONS: In this cohort, anaphylaxis to red meat appears to be the predominant cause of food induced anaphylaxis and presence of beef specific IgE and cow's milk, appears to be a predisposing factor for vaccine induced anaphylaxis.

Sensitization to bovine serum albumin as a possible cause of allergic reactions to vaccines.

BACKGROUND: Immediate type hypersensitivity to vaccines containing bovine/porcine excipients, such as the measles, mumps and rubella (MMR) vaccine is probably due to sensitization to bovine/porcine gelatin. Most patients with such reactions in Sri Lanka have cow's milk (CM) or beef allergy. OBJECTIVES: We investigated whether those who had beef and CM allergy had a higher incidence of hypersensitivity reactions to vaccines and the possible trigger of such reactions. MATERIAL AND METHODS: Twenty patients with immediate type hypersensitivity reactions to vaccines containing bovine/porcine excipients, controls with allergy to beef/pork (n=11) or CM (n=11), and 8 non atopic controls were recruited. Total serum IgE, specific IgE to beef, CM, casein, beta lactoglobulin, gelatin and bovine serum albumin (BSA) by Phadia ImmunoCap and IgE to porcine gelatin by Western blot were evaluated. RESULTS: 11/20, 5/20, 2/20, 2/20, 1/20 and 1/20 patients reported allergic reactions to measles containing, JE, rabies primary chick embryo, pentavalent, diphtheria and tetanus, and adult diphtheria and tetanus vaccines, respectively. Only one patient with allergy to vaccines had gelatin specific IgE, whereas IgE to BSA was seen in 73.3%, 90%, 66.6% and 0 of vaccine, beef or CM allergic and non-atopic controls, respectively. The mean IgE to BSA was higher in patients with allergy to vaccines, although not significant. Specific IgE to BSA was present in 54.7% of children with allergy to CM, of whom 11.8% had high levels (>17.5kUA/L). In contrast, 66.6% of these children did not have specific IgE to ß-lactoglobulin, which is one of the major components of whey protein. CONCLUSION AND CLINICAL RELEVANCE: Gelatin does not appear to play a major role in Sri Lankan children with allergy to vaccines. In contrast, due to the higher levels of BSA specific IgE, sensitization to BSA is possibly playing a role.

IgE cross-reactivity of phospholipase A2 and hyaluronidase of Apis dorsata (Giant Asian Honeybee) and Apis mellifera (Western Honeybee) venom: Possible use of A. mellifera venom for diagnosis of patients allergic to A. dorsata venom.

Diagnostic and therapeutic reagents are unavailable for anaphylaxis arising from stings by Apis dorsata. Venom profiles and cross-reactivity of A. dorsata and Apis mellifera were compared, to ascertain whether venom of A. mellifera can be used for diagnosis in A. dorsata allergy. Both venom profiles were similar by High Performance Liquid Chromatography and SDS-PAGE. Sera of 29 of 30 (96.7%) patients with anaphylaxis to A. dorsata stings had IgE to the phospholipase-2 (PLA2) doublet (15 and 16 kDa) of A. dorsata venom by immunoblot, compared to 26 of 30 (86.7%) with the PLA2 of A. mellifera and a purified preparation of PLA2. Twelve patients (40%) with severe anaphylaxis had IgE reactivity to a 39 kDa protein band of venom of both species, a third band, identified in immunoblot as hyaluronidase. The cross-reactivity of PLA2 and hyaluronidase of A. dorsata and A. mellifera were further confirmed by immunoblot inhibition results. Twenty five of 30 (83.3%) of our patients had positive venom specific IgE (>0.35 KUA/L) reactivity to Phadia ImmunoCAPs of A. mellifera venom. The observed IgE cross reactivity suggests the possibility of using A. mellifera venom as a diagnostic test for A. dorsata venom allergy.