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Keywords:

  • cross-reactivity;
  • Hymenoptera venom;
  • hypersensitivity;
  • IgE antibodies;
  • Polistes

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The American Polistes species venom mixture – that of P. annularis, P. fuscatus, P. metricus and P. exclamans– was the only commercially available mixture for diagnosis and therapy until 1996. However, these species of Polistes are not present in Europe, where P. dominulus and P. gallicus and to a lesser extent P. nimphus are widespread. The aim of this study was to assess the allergenic differences among the commercial American mix, P. dominulus and P. gallicus venom in European patients and therefore to verify if this mixture is suitable for diagnosis in these patients. We carried out skin tests, radioallergosorbent tests (RAST) and RAST inhibition in Italian patients with adverse reactions to Polistes stings. RAST inhibition results demonstrated that cross-reactivity between the American and European species is only partial and that P. dominulus and P. gallicus venoms have exclusive allergens. Skin tests and direct RAST confirmed these results and also showed that European Polistes venom is more suitable than the American mix in Italian patients. Moreover, we found a high rate of cross-reactivity between P. dominulus and P. gallicus. To conclude, P. dominulus and/or P. gallicus venoms are necessary for diagnosis and therefore in the therapy of European patients.

Polistes is the most common genus among social waSPS. Polistes dominulus (Pd), Polistes gallicus (Pg) and, to a lesser extent Polistes ninphus, are the most widely spread in Europe (1). Only a mixture of the American Polistes species (A Pm) –P. annularis, P. fuscatus, P. metricus and P. exclamans– was commercially available until 1996, when Anallergo (Florence, Italy) started the production of Pd venom. The American species are not present in Europe.

The aim of this study was to assess the allergenic differences among the A Pm, Pd and Pg venoms in European patients. We carried out skin tests, radioallergosorbent test (RAST) and RAST inhibition in Italian patients with adverse reactions to Polistes stings.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients

We studied 21 patients with a history of adverse reactions to Hymenoptera stings (Table 1). Patients 1–10 had positive tests only to Polistes genus. Patients 11–21 also had positive tests to other genera. However, in the latter group of patients, Polistes venom was recognized as the ‘primary venom’ (2): in three patients (no. 17, 18 and 19), RAST inhibition (not presented) showed that the other positivities were attributable to cross-reactivity with Polistes venom. For the remaining eight patients, the data – identification of the culprit insect and/or the results of the skin tests and serum specific IgE – indicated primary sensitization to Polistes. Beekeepers monopositive to Apis mellifera formed the control group.

Table 1.   clinical data, skin tests and serologic results
Patient no.Pat.Age (years)SexAtopyReaction (Mueller grade)Intradermal tests (threshold in μg/ml)*Serum specific IgE (% retained radioactivity)†Total IgE (kU/l)
Apis m.Vespula spp. V. crabro APm Pd PgPmCSPdCSPgCS
  1. *Lowest venom concentration able to induce at least a 5-mm diameter's wheal with erythema.

  2. †We consider positive (bold type) the values superior to double of control serum.

  3. ‡CAP-System Pharmacia.

  4. LLR = large local reaction; APm = American Polistes mix; Pd = Polistes dominulus; Pg = Polistes gallicus; CS = control serum (pool of sera of patients with negative skin tests); N.D. = not done.

1RS73MNOIVNEGNEGNEG10.010.013.21.95.11.34.81.612
2CR37FNOLLRNEGNEGNEGNEG0.10.13.01.94.01.34.61.650
3BL62FNOIVNEGNEGNEG0.10.10.19.31.617.01.112.71.021
4MS16MNOLLRNEGNEGNEG0.10.10.129.41.640.61.137.81.0194
5MA49FNOIINEGNEGNEG0.0010.0010.00132.61.642.81.134.51.0180
6MP69FNOIVNEGNEGNEGNEG113.31.73.72.43.52.675
7PS33MYESLLRNEGNEGNEG0.10.10.018.91.710.52.411.22.631
8SI53MYESINEGNEGNEG10.10.115.51.638.91.137.41.081
9SG20MYESIIINEGNEGNEG0.01N.D.N.D.36.01.651.81.148.31.0437
10CM38MYESIVNEGNEGNEGNEG0.1N.D.NEG‡NEG‡N.D.N.D.102
11FL33MNOIIINEGNEG110.10.17.71.613.11.111.71.071
12TS35MNOIIINEGNEG110.10.110.41.616.61.112.01.0105
13LF47MNOIVNEG1NEGNEG113.71.95.61.35.91.690
14SDR17MNOIINEG0.010.010.10.010.019.81.615.81.112.71.0402
15ML25MNOIVNEGNEG10.1113.51.92.01.32.51.6104
16PF43MNOIVNEG10.10.10.10.17.01.68.11.14.91.026
17GG42MNOIV10.00010.010.10.010.0139.82.250.31.448.91.2366
18BG35MNOIV10.110.10.010.0115.72.223.91.419.31.2315
19DL49MNOIV10.010.010.010.0010.0124.52.224.31.424.81.2134
20NS17MYESIIINEG10.10.010.010.00111.91.635.71.118.41.0554
21CD50MNOIIINEG10.10.10.010.015.71.69.41.17.71.0152

Venoms

Apis mellifera, Vespula spp. and A Pm venoms were purchased from Stallergènes (Milan, Italy) and Alk-Abellò (Milan, Italy). The Vespa crabro venom was prepared by Anallergo. Pd and Pg venoms were obtained from nests collected in Tuscany and stored at −70°C. The insects were individually identified by an entomologist. After defreezing, the venom was collected by dissection and gently squeezing the sac (3). The protein content was assessed using the Lowry method and the yield was 60 μg per sac. The venoms were dialysed for in vivo use by removing components with molecular weight under 1000 Da using Centripor protein concentrator tubes from Anallergo. They were then lyophilized and refrigerated at −20°C for further use.

Skin tests

Skin tests were performed as previously described (4).

Serum-specific IgE

To compare values among the A Pm, Pd and Pg venoms, RAST was performed using solid-phase nitrocellulose according to Ceska's method (5), and the same concentration for all the three allergens was used (Anallergo).

The test was considered positive when values were more than double those obtained with a pool of sera from patients with negative skin tests to Hymenoptera venom, using the same allergen in the solid phase: ‘control serum’ (Table 1). The values are expressed as a percentage: bound radioactivity divided by total radioactivity. All the tests were performed twice in the same session and the mean value was reported.

RAST inhibition

We used sera from 10 patients with sufficiently high serum-specific IgE (no. 3, 4, 5, 8, 9, 11, 12, 14, 19 and 21). At low specific IgE levels, RAST inhibition is hardly reliable (6). RAST inhibition was carried out according to Hoffman's method (7). Homologous RAST inhibition was performed for Pd and Pg: the same venom was used in the solid phase and as an adsorbent. In the same session, specific IgE binding to solid-phase Pd and Pg was inhibited through serum adsorption with the A Pm. Reciprocal RAST inhibition was also carried out between Pd and Pg.

Statistical analysis

Significance was assessed by the Mann–Whitney U-test. Differences below 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Skin tests

Fifteen of the 20 patients (75%) showed greater skin reactivity to Pd and/or Pg than to the A Pm (Table 1). In four of the 20 patients, reactivity was of the same order. Patient no. 15 had greater skin reactivity to A Pm; he had been stung when on holiday in the USA. In patient no. 9, intradermal tests with the European Polistes venoms were not performed.

It should be noted that in four patients, skin tests were negative to the A Pm and positive to the European Polistes (no. 2, 6, 10 and 13). Patient no. 10 had urticaria pigmentosa and a history of very severe anaphylactic shock; the patient was diagnosed in another Allergy Unit with ‘non-IgE mediated insect sting anaphylaxis in subjects with urticaria pigmentosa’ because a skin test with European Polistes sp. had not been performed. Comparison between Pd and Pg showed the same skin reactivity in 16 of the 19 patients. In the controls, skin tests were negative.

Serum-specific IgE

In all RAST-positive patients (18/21), IgE titres to the European species were higher than those to the A Pm (P < 0.001). IgE mean values were: Pd: 18.4%, Pg: 13.5%, A Pm: 10.7%. In three patients, RAST was negative to the A Pm and positive to the European species (no. 1, 2 and 13). Comparison between Pd and Pg serum-specific IgE titres showed a high correlation (r = 0.97).

RAST inhibition

Nine of 10 patients clearly had specific IgE to allergens of the European species which are exclusive because adsorption of serum with the A Pm only partially inhibited the binding of specific IgE to European Polistes (Figs 1 and 2). In these nine patients, RAST for Pd was inhibited by homologous venom by 80.9% on average, whereas it was inhibited by the A Pm by 46.1% on average (Fig. 3). RAST to Pg was inhibited by homologous venom by 86.6% on average, whereas it was inhibited by the A Pm by 47.5% on average (Fig. 3). In one of the 10 patients, cross-reactivity was complete (Figs 1 and 2: DL): that is, specific IgE tended only towards allergens common to both American and European species. Moreover, serum IgE titres in this patient were virtually the same towards the American and European species (DL; no. 19).

image

Figure 1.  RAST inhibition. IgE binding to Polistes dominulus venom inhibited by adsorption with American Polistes mix in 10 patients.

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image

Figure 2.  RAST inhibition. IgE binding to Polistes gallicus venom inhibited by adsorption with American Polistes mix in 10 patients.

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image

Figure 3.  RAST inhibition: mean values of 10 patients. IgE binding to Polistes dominulus venom inhibited by American Polistes mix (DA) and by P. dominulus (DD). IgE binding to P. gallicus inhibited by American mix (GA) and by P. gallicus (GG).

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Reciprocal RAST inhibition between Pd and Pg, carried out in six patients, showed, in all of them, high cross-reactivity between the two species. Specific IgE to Pd were adsorbed by Pg venom and the values varied from 75% to 98%– average 84% (not presented). Specific IgE to Pg were adsorbed by Pd venom and the values varied from 62% to 96%– average 81% (not presented). This confirms the results of skin tests and serum-specific IgE.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Among the genus Polistes, P. dominulus, P. gallicus and, to a lesser extent, P. nimphus are the most widely spread in Europe, while P. annularis, P. fuscatus, P. metricus and P. exclamans are present in the USA (1). The latter are not present in Europe.

Recent systematic studies (8, 9) have confirmed that North American and European species of Polistes belong to different subgenera and are phylogenetically distant. The most common European species –P. dominulus, P. gallicus, P. nimphus and P. biglumis– belong to the subgenus Polistes sensu stricto while the most common North American native species –P. exclamans, P. annularis and P. metricus and P. fuscatus– are included in the subgenera Aphanilopterus and Fuscopolistes (g). We already know that cross-reactivity among European species of Polistes is very strong whereas that between European and American species is weaker (4). Our RAST inhibition studies demonstrate that adsorption with the A Pm only slightly inhibits IgE binding to the European venoms. This, therefore, suggests the presence of exclusive allergens in the latter.

No in vivo studies in European patients have been carried out to compare the venoms from the American and European Polistes. RAST inhibition studies, carried out in only four patients, compared Pd with single American species but not with the commercial A Pm (10). Our study is the first to describe in vivo tests with Pd and Pg venom, carried out in European patients.

Our in vivo results confirm the data previously obtained in vitro on the allergenic differences between American and European Polistes (10–13). It is particularly important to note that our four patients, whose skin tests were negative to the A Pm, showed positive skin tests to European Polistes venoms; two of them had severe reactions – grade IV of Mueller classification – and skin tests positive to 1 μg/ml. This further stresses that there is no correlation between skin test reactivity and severity of the reaction and also the need for performing skin tests up to this concentration (4).

The man suffering from urticaria pigmentosa, with a history of a very severe anaphylactic shock after a Polistes sting (positively identified by the patient) is an emblematic case. It clearly demonstrates that omitting to perform skin tests for Pd venom would have repeated the wrong diagnosis of non IgE-mediated anaphylaxis and deprived the patient of life-saving immunotherapy (14).

So far we have treated 130 patients with the same Pd venom used for in vivo and in vitro tests (Anallergo). As regards safety, we have never observed systemic reactions or other side effects (15). Our data also demonstrate an excellent degree of protection from field stings, numbering 59 of 60 field stings without adverse reactions in 26 patients (M. G. Severino, pers. obs.).

In conclusion, our data raise the problem of the need to introduce the now commercially available European Pd venom extract into clinical practice.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We thank Maurizio Valentini and Barbara Pantera for their excellent technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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