Imported fire ant allergy in young children: Skin reactivity and serum IgE antibodies to venom and whole body extract

Semi L. Bahna, MD,* John H. Strimas, MD,* Margaret A. Reed, MS,*+ and Brian T. Butcher, PhD** New Orleans, La.

We staled 19 young children (aged 15 to 39 rno~ths~ who had had a systemic allergic reaction cxfter importedJire ant (IFA) sting. Skin testing was performed with whole body extract (WBEi of both Solenopsis species; positive reactions to S. invicta was noted in 94% and to S. richteri in lOO%, Serum was tested for total 1gE level and for spec$c IgE antibodies by RAST with WBE and venom (V) of S. invicta. In vitro results were compared with those of 19 age and se-x-marched atopic control subjects. The range of total serwn fgE level was 31 to I720 IUimr (geometric mean 160 ~~~rn~~, compared with 7.5 to 6756 Ililml (geometric mean 85 ~~irn~~ in the control subjects. The d$ference between the two groups, however, was not statistically sign@zant (p > 0.05). In the IFA-allergic group, the IEA WBE RAST mean binding was 13.5% (range 2.0% to 30.2%), and the .IFA V RAST mean was 29.2% (range 7.8% to 46.7%). The control sera demonstrated low levels of activiry to both IFA WBE RAST (0.7% to 4.8Y~) and fFA V RAST (0.7% to 5.9%). IFA WBE RAST demonstrated that 26% of the IFA-allergic patients had cog& within the range of the atopic control subjects. In conmsr. the IFA V RAST clearly separated all IFA-allergic patients from their age and sex-matched atopic control slchjects. A scoring system for IFA V RAST is proposed. These results demonstrate superiority of IF*l V over IFA WBE RAST in the diagnosis of allergy to S. invicta. (J ALLERGY CLIN tMML’,NiJl 1988:82:419-24.)

Systemic allergic reactions to IFA are common in the southeastern United States. They occur mostly from the sting of two species of the genus ~~~en~~s~~, S. jnvjctu and S. richteri; the former is the more com- mon.‘* 2 A number of studies have emphasized the growing medical problem of hypersensitivity to these aggressive and pugnacious Hymenoptera.‘, 4 Surveys in the southeastern region revealed that anaphylactic shock occurred in about 4% of subjects who had a generalized allergic reaction to IFA.’

The stings are most frequent in the pediatric age group, 32% to 54% of the surveyed population.2 The estimated incidence of generalized reactions to IFA

From the *Section of Allergy and Immunology, Department of Pediatrics, Louisiana State University School of Medicine, and **Clinical Immunology Section, Department of Medicine, Tu- lane University Medical Center, New Orleans, La.

Supported by National Institutes of Iiealth Grant AI #2155 1. Received for vocation Oct. 14, 1987. Accepted for publication March 2, 1988. Reprint request% Sami L. Bahna, MD, Department of Allergy and

Immunology, Ckveland Clinic Foundation, A-72, 9500 Euclid Ave., Cleveland, OH 44195-5035.

Abbreviations used IFA: Imported fire ant

WBE: Whofe body extract V: Venom

G% Geometric mean

stings in children is two to three per l~,~.6 To the best of our knowledge, no studies have specifically to IFA allergy in childten. known about IFA allergy in the toddler ago group who are at an especially high risk for expose in the south- eastern region of the United States.

At the present time, the only test available in chn- ical practice to confirm IFA allergy is sk with the WE. A limited number of inve sug- gested superiority of the V extract over WX? in skin testing and in detecting serum-specific I@ antihod- ies7* * This observation, however, is not in agreement with that of another study.g

In the present study, which is limited to young children with IFA allergy, we comparnd the Ievels of serum-specific antibodies to IFA WBE and to IFA V

499

420 Bahna et al.

- isD

- Gi Gi

- 1sD 1SD

Alleyi; Pts Cod&ls

FIG. 1. Serum total IgE level in IFA-allergic children and in age and sex-matched atopic control children.

with each other in the study group and with those in atopic control subjects. The IFA used in these assays was S. invictu, the prevailing species in this region.

MATERIAL AND METHODS Subjects

The study group comprised 19 young children, with ages ranging from 15 to 39 months (mean 27.7 months). These patients had been referred from the Greater New Orleans area to the Allergy/Immunology Clinic of Louisiana State University Medical Center between 1981 and 1987. When they were first observed, each patient had a history of an IFA sting that was followed rapidly by one or more of the following manifestations: urticaria, angioedema, breathing difficulty, vomiting, colic, pallor, or fainting. The devel-

J. ALLERGY CLIN. IMMUNOL. SEPTEMBER 1989

TABLE I. Characteristics of IFA-allergic patients and atopic control subjects

Control Patients subjects p Value

No. 19 19 M:F 10:9 10:9 Age (mo)

Range 15-39 16-40 Mean 27.7 28.8 NS

Serum IgE (IU/ml) Range 31-1720 7.5-6756 GJZ 160 85 NS Median 157 74 NS

NS = not significant.

opment of more than one manifestation simultaneously was reported in 74% of subjects. In each instance, pustules char- acteristic of the IFA stings were either described by the parents or observed on physical examination.

Skin testing All subjects suspected of IFA allergy underwent skin

testing with commercial (Greer Laboratories, Lenoir, N.C.) WBE of both species of IFA (S. invictu and S. rich-i). Scratch testing was performed first with 1: 20 wtlvol glyc- erinated extract, and if the skin response was negative or equivocal, intradermal testing was performed with 1: 1000 wt/vol aqueous extract. Skin responses were scored 0 to 4 + , compared with the responses to a diluent and histamine (1 mglml for scratch and 0.1 mg/ml for intradermal tests). The wheal-and-flare response was scored 0, if it was similar to the response to diluent; 1 + , if it was 25% to 50% greater than the response to diluent; 2 + , if it was >50% above the response to diluent but smaller than the response to hista- mine; 3 + , if it was similar (75% to 125%) to the response to histamine; and 4 + , if it was > 125% above the response to histamine or if the wheal had pseudopods.

All 19 patients included in this study except one exhibited a skin response to S. invictu of at least 2 + (wheal diameter at least 5 mm above the negative control wheal diameter). The patient who had a negative skin test was included in this series because of the unequivocal association between the sting and rapid development of generalized urticaria and angioedema.

A blood sample was obtained from each patient by ve- nipuncture. The serum was stored at - 20” C after adding a preservative (sodium azide and e-aminocaproic acid).

Preparation of IFA antigens S. invictu WBE was prepared by a method developed in

our laboratory with live IFA from 12 separate colonies from the New Orleans area.“’ The species identification of the IFA collected from each colony was verified by an ento- mologist. The WBE was prepared by extracting 5 gm of

VOLUME 82

NUMBER 3, PART 1

l

. l .

l e em

f .

l

.

0

l

.

:

2SD

2SD e 5- -------s-e--- -,- 3SD

--f 0 IFA Atopic

Aller ic Pts Controls / 19) (19)

1SD

%

MD

FIG. 2. S. invicta V RAST in 19 IFA-allergic children and in 19 age and sex-matched control children.

IFA in 100 ml pyrogen-free phosphate-buffered saline (pH 7.4) in a Tissuemizer (Heat Systems Ultrasonic, Inc., Far- mingdale, N.Y.) with three l-minute bursts. This mixture was extracted overnight at 4” C, centrifuged at 10,000 tpm for 20 minutes to remove particulate material, and concen- trated on an Amicon YC-05 (Amicon Corp., Danvers, Mass.) ultrafllter (exclusion limit 500 daltons) to a final volume of approximately 5 ml. The final concentration was adjusted to 10 mg of dry weight per milliliter (after cor- recting for buffer content), and the material was sterile fil- tered, divided into aliquots, and stored at -70” C until it was used.

S. invicta V preparation was kindly provided by Vespa Laboratories, Spring Hills, Pa. The stated dry weight was 0.4 mg per vial. It was collected by an electric shock method and has been demonstrated by crossed immunoelectropho- resis to be antigenicalIy identical to the material collected directly from the V sacs.“’ The immunologic interrela- tionship between WBE and V has been reported pre- viously.‘, lo. ”

Total serum IgE antibody measurement Levels of the total IgE in the sera were determined by a

commercially available paper disk radioimmunoassay kit for

l

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l

0

.

l l e l

l ee

--w--e.

i

2SD

1SD

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Atopic Aller ic

d Pts Controls

19) 09) FIG. 3. S. invicta WBE RAST in 19 IFA-allergic children and in 19 age and sex-matched atopic control children.

IgE (Kallestad Laboratories, Austin, Texas), This compet- itive binding assay has a sensitivity of 5.0 IUlml.

RAST for rpe&fk JgE an Sera were tested by RAST to determine levels of IgE

antibodies specific to S. invicta V and WBE with the method previously reported.* The antigen extract was coupled at optimal concentration previousiy determind by titration studies*’ to CNBr-activated paper disks (Whetman No. 50 [Whatman, Inc., Clifton, N.J.]). The disks were incubated overnight with 100 p1 of serum and then washed three times with 2.5 ml of saline to remove unbound serum. The disk was incubated for another overnight with 100 ~1 (25,000 cpm) of ‘2’I-labeled anti-IgE (Kallestad Laboratories) and then washed three times to remove the unbound material. The bound radioactivity was measured in a gamma counter.

Control seca The control sera were obtained from 19 children with

atopic disease who had no history suggestive of lE4 allergy. The control children were also from the Greater New Or- leans area and were matched by age and sex with the IFA-

422 Bahna et al. J. ALLERGY CLIN. IMMUNOL. SEPTEMBER 1988

TABLE II. Comparison of Solenopsis invicta venom and WBE RAST in 19 IFA-allergic children and 19 atopic control children

% Bound

Group Range ii f SD p Value

V RAST

Patients 7.8-46.7 29.2 a 11.2 Control subjects 0.7-5.9 1.8 +-

I

<O.OOOl

WBE RAST NS <O.OOl

Control subjects 0.7-4.8 1.5 2 1.3 I 1.0 Patients 2.0-30.2 13.5

<0.0002 ” 9.7 I 1

NS = not significant.

TABLE III. Data on five children allergic to IFA (Solenopsis invicta) who had negative WBE RAST

Age ho)

Reaction to IFA sting

Skin test

Total IgE NJ/ml)

BAST (% bound)

V WBE VIWBE ratio

(%I

15 Anaphylaxis 2+ 92 32.0 2.0 1600 18 Anaphylaxis 3+ 70 13.7 3.7 370 21 Anaphylaxis 3+ 78 23.8 4.7 506 30 Urticaria ND 40 8.7 2.3 378 33 U&aria 3-t 31 7.8 2.8 279

allergic children (Table I), but they were not subjected to dent’s t test for the geometric means nor by the ranked- skin testing with IFA extracts. sum test.‘*

RESULTS Serum-specific IgE antibodies (RAST)

In this series of young children with hypersensitiv- ity to IFA sting, the reactions occurred predominantly between March and October, with a peak in July. In 73% of the patients, the parents were unaware of any previous reaction to IFA. A history of atopic disease was elicited in 47%, and a family history of atopy was present in 63%.

Data on RAST to S. invictu V and WBE are pre- sented in Table II and Figs. 2 and 3. RAST on control sera demonstrated low bound radioactivity to both V (0.7% to 5.9%; : 1.8%) and WBE (0.7% to 4.8%; ; 1.5%).

Skin test reactivity to IFA

All 19 patients but one demonstrated a positive response (2+ or greater) to skin testing with IFA WBE; four (22%), by scratch testing; and 14 (78%), by intradermal testing. A positive skin response to S. invictu was noted in 17 (94%) and in 18 (100%) to S. richteri.

Serum total IgE level

In the IFA-allergic group, the IgE range was 31 to 1720 IU/rnI (GZ 160 IU/ml; median 157 IU/ml), and in the atopic control subjects, it was 7.5 to 6756 IU/ml (GE 85 IU/ml; median 74 IU/ml) (Fig. 1). However, the difference between the two groups was not statistically significant (p > 0.05), neither by Stu-

WBE RAST demonstrated that 5 / 19 (26%) of IFA- allergic patients had counts within the range for the control subject. In contrast, V RAST demonstrated no overlap between IFA-allergic patients and control subjects. The IFA-allergic group demonstrated a mean bound activity to IFA V RAST 16.2 times that of the control subjects (29.2% versus 1.8%) respectively). The corresponding figures for the IFA WBE RAST were 13.5% for the patients and 1.5% for control subjects, that is, ninefold. Nevertheless, there was a significant correlation between the WBE RAST and V RAST counts in the patient group (r = 0.72; p C 0.001). Serum total IgE appeared to have a stron- ger correlation with WBE RAST (r = 0.8 1) than with V RAST (r = 0.67); the difference, however, was not statistically significant (p > 0.1).

The V RAST-bound activity in the control group demonstrated a mean + 3 SD of 5.9%, a cutoff level

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TABLE IV. Proposed scoring system for lFA* venom RAST

ifIt8tV8t Of bound t8diO8CtiV2ty

in the IFA-aliergic group

x -20rmoreSD 2 -1 SDto; -2SD xtox -1 SD iito: +I SD : cl SDtox i2SD

Actual

6.8 6.8-18.0

18.0-29.2 29.2-40.4 40.4-51.6

Q/m %ound venom RAST

&BpfOXh8te score

(6 i .t 6418 ; .,

1%<30 ,; + 30-<40 :* 40-<50 ,& 1.

*Solenapsis invicta.

that ~mpletely diffe~ntiat~ between the patients and the control subjects. In contrast, the WBE RAST- bound activity in the control group demonstrated a mean + 3 SD of 3.8%, a cutoff level that would in- clude five (26%) of the IFA-allergic patients. Data on those five patients are presented in Table IIII; all five patients had a definite systemic reaction, elevated total serum IgE level, and a V/WBE RAST count ratio of 279% to 1600%.

Based on our data on IFA-allergic patients and con- trol subjects (Figs. 2 and 3), a proposed scoring sys- tem for IFA V RAST is presented in Table IV

The single patient who did not demonstrate a pos- itive skin test (to either species of IFA) was a 24- month-old white male child who developed general- ized urticaria/ angioedema shortly after receiving mul- tiple 1FA stings. His condition improved when he was treated with ~p~~y~ne, and when he was ob- served several days later, he still had some pustules characteristics of IFA stings on the lower extremities. Skin testing with WBE was negative by scratch and intradermal testing to both IFA species. He had no previous allergic disease. His father, however, was highly allergic to IFA. This child’s serum demon- strated a total IgE level of 279 fU/ml and IEA V RAST of 46% bound activity, the highest level recorded in our laboratory to date. His WBE RAST bound activity was 26.3%. The child avoided exposure to IFA, and the u~c~a/angi~d~ never recurred during a 3- year follow-up. The parents refused to subject him to repeat skin testing or venipuncture.

DtSCUSSlON The results of this study indicate the superiority of

IFA V RAST over IFA WBE RAST for the diagnosis of allergy to S. invictu. V RAST in our study clearly differentiated between the IFA-allergic patients and the control subjects. In contrast, IFA WBE RAST was falsely negative in 26% of cases with definite severe clinical reactions.

Our endings differ from those of other workers9

who observed an overlap between ~A-cleric pa- tients and a group of frequently stung ~~~l~gi~ con- trol subjects in their levels of serum IgE antibodies to both WBE and V Our control group wss age and sex matched and had no history of significant reacticms to IFA sting, though conceivably more of them has been exposed to EFA. Another possible, yet more im- portant, difference between the two studies might be the degree of antigenic purity of the V extracts used, as suggested by the strong correlation between IFA WBE RAST and IFA V RAST noted in that study (r = 0.92), compared to the moderate ~o~lation noted in our study (r = 0.72).

Our data also indicate that allergy to ZFA in young children appears to be associated with higher specific IgE antibody levels than in adults When we compared the RAST data from this study with data from a similar study on adults and with the same r~geuts~a RAST binding by children’s sera was higher to both WBE (162%) and V (154%).

Although some of the previous studieP5 on allergy to IFA have included pediatric patients. our study is the first that addresses this high-risk ~~~l~~on. Al- though the parents did not recall a history of any previous IFA reaction in 73% of our patient group, previous studies27 4. “-” revealed that as many as 58% of the population in the southern United States ex- perienced IFA stings each year. It is very probable that most of our patient group has beeh repeatedly stung previously. It is not known, however, whether a high degree of exposure in this young age group had contributed to the high serum IgE antibody levels noted in them. We believe that most of our patients were selectively referred because of the parents’ con- cern and the severe reaction noted by the primary care physician. Information available suggests that this se- ries is mostly highly atopic, as indicated by the high incidence of family history of allergy, the early age of onset of the allergic reaction, and the remarkably elevated total serum IgE levels. Wi~out ap~~~~ate epidemiologic studies, however, the ~o~sibili~ that

424 Bahna et al. J. ALLERGY CLIN. IMMUNOL. SEPTEMBER 1989

atopic children are at a specially high risk of devel- oping hypersensitivity reactions to IFA sting cannot be excluded.

Our observation that the peak incidence of IFA re- actions was in July is probably a reflection of the concomitant remarkable seasonal increase in the pop- ulation of IFA in our region. It is possible also that there is a seasonal increased allergenic potency of the IFA VI8

tent of commerical IEA WBE extracts have been

The superiority of IFA V over IFA WBE has been demonstrated in skin testing as well.’ There appears to be therefore an urgent need for standardized, well- characterized IFA V extract to be available in clinical practice. Marked differences in antigen / allergen con-

4. deShazo RD, Grifting C, Kwan TH, Banks WA, Dvorak HF. Dermal hypersensitivity reactions to imported fire ants. J AL- LERGY CL~ I~UNOL 1984;74:841.

5. Triplett RF. The imported fire ant: health hazard or nuisance? South Med J 1976;69:258.

6. Ginsburg CM. Fire ant envenomation in children. Pediatrics 1984;73:689.

7. Strom GB, Boswell RN, Jacobs RL. In vivo and in vitro comparison of fire ant venom and fire ant whole body extract. J ALLERGY CLIN IMMUNOL 1983;72:46.

8. Butcher BT, deShazo RD, Grtiz AA, Reed MA. Superiority of Solenopsis invicta venom to whole body extract in RAST of diagnosis of imported fire ant allergy. Int Arch Allergy Appl Immunol 1988;85:458.

10. Butcher BT, Reed MA. Crossed immunoelectroohoretic studies

9. Paul1 BR, Coghlan TH, Vinson SB. Fire ant venom and whole body extract in the diagnosis of fire ant allergy. J ALLERGY CLIN IMMUNOL 1983;71:448.

noted.” Our observation of a patient with generalized urticaria/ angiodema, who had the highest RAST per- cent binding ever obtained in our laboratory, yet who failed to react to skin testing with the usual concen- trations of extract, may be at least partially due to the variability in composition of%ommerical IFA extracts. It is likely also that certain individuals have peculiar immunologic responses.

As in the case for allergy to other Hymenoptera,” it appears appropriate that efforts should be expended toward the development of well-characterized IFA V preparations for both skin testing and in vitro diag- nosis, and possibly for immunotherapy as well,

We thank Mr. M. Gnralnick, Vespa Laboratories, Spring Mills, Pa, for the generous donation of Solenopsis invictu venom, Mr. M. Andis, entomologist with the New Orleans Mosquito Control, New Orleans, La., for the identification of the IFA species, and Ms. Danelle Watkins, for technical assistance.

of whole body extracts and venom from the imported fire ant Solenopsis invicta. J ALLF,RGY CLIN IMMUNOL 1988;81:33-40.

11. Butcher BT, deShazo RD, Ortiz AA, Reed MA. Studies of the imported fire ant Solenopsis invicfa: RAST-inhibition studies of the imported fire ant SoZenopsis invicta with whole body extracts and venom preparation. J ALLERGY Ctm IMMUNOL 1988;81:1096-1100.

12. Elston RC, Johnson WD. Essential of biostatistics. Philadel- phia: FA Davis Co, 1987:141.

13. Lackey RF. Systemic reactions to stinging ants. J ALLERGY CLIN IMMUNOL 1974;54:132.

14. Rhoades RB, Schafer WL, Schmid WI-I, Wubbena PF, Dozier RM, Townes AW, Witting HJ. Hypersensitivity to the imported fire ant: a report of 49 cases. J ALLERGY CLIN IMMUNOL 1975;56:84.

15. Rhoades RB, Schafer WL, Newman M, Lackey R, Dozier RM, Wubbena PF, Townes AW, Schmid WH, Neder G, Brill T, Wittig HJ. Hypersensitivity to the imported fire ant in Flor- ida: report of 104 cases. J Fla Med Assoc 1977;64:247.

16. Yeager W. Frequency of 6re ant stinging in Lowndes County, Georgia. J Med Assoc Ga 1978;67:101.

17. Adams CT, Lofgren CS . Red imported fire ants (Hymenoptera: Formicidae): frequency of sting attacks on residents of Sumpter County, Georgia. 3 Med Entomol 1981;18:378.

18. Hannan CJ Jr, Stafford CT, Rhoades RB, Wray BB, Baer H,

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1. Rhoades RB. Medical aspects of the imported fire ant. Gaines- ville, Fla.: The University Presses of Florida, 1977.

2. Stablein JJ, Lackey RF. Adverse reactions to ant stings. Clin Rev Allergy 1987;5:161.

3. Triplett RF. Sensitivity to the imported fire ant: successful treatment with immunotherapy. South Med J 1973;66:477.

78:331. . 19. Butcher BT, Reed MA. Evaluation of commercial imported

fire ant extracts by crossed immunoelectrophoresis and ra- dioallergosorbent test [in press]. J ALLERGY CLIN IMMUNOL.

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