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\begin{document}

\title{Clinical and laboratory profile of patients with anaphylaxis to fire ant
venom (Solenopsis sp) under specific subcutaneous immunotherapy}



\author[1]{Alexandra Watanabe}%
\author[1]{Marcelo Ferreira}%
\author[1]{Clóvis  Galvão}%
\author[2]{Jorge Kalil}%
\author[3]{Keity Santos}%
\author[4]{Fábio Castro}%
\affil[1]{University of Sao Paulo}%
\affil[2]{São Paulo University}%
\affil[3]{School of Medicine of Sao Paulo University}%
\affil[4]{School of Medicine of the University of São Paulo}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: Anaphylaxis to fire ant venoms (Solenopsis sp) is a
significant cause of systemic reaction caused by Hymenoptera stings in
children. There are only a few reports about the safety and efficacy of
specific immunotherapy. Objective: Evaluate clinical characteristics,
IgE and IgG4 specific responses of patients undergoing immunotherapy
with a whole-body extract of Solenopsis sp after one year of the
maintenance phase. Materials and methods: Thirty-three patients were
enrolled due to anaphylaxis by fire ant venom (Solenopsis sp) and
underwent specific immunotherapy. They were assessed at baseline and one
year after the beginning of the maintenance phase for skin test;
specific venom IgE and IgG4 antibodies; tryptase. Results: All patients
included presented a severe anaphylactic reaction. Although two patients
(6.25\%) presented a tryptase level higher than 11.4 ug/ml, systemic
mastocytosis was ruled out. There was no relationship between the
severity of the reaction with gender, tryptase level, atopy, previous
reactions, the allergen's concentration in the skin test or specific IgE
level. There was an increase in the specific IgG4/IgE ratio between the
two time points. Reactions were local, with only two mild systemic
reactions during the build-up phase. Twenty patients had accidental
stings during immunotherapy, with 3 presenting only urticaria.
Conclusions: This study is unprecedented in evaluating clinical and
laboratory data in the fire ant immunotherapy. Our results show that
after one year of the maintenance phase, patients did not develop any
severe reaction with only a few mild reactions and presented a
significant production of specific IgG4.%
\end{abstract}%



\sloppy


\textbf{Clinical and laboratory profile of patients with anaphylaxis to
fire ant venom (\emph{Solenopsis sp}) under specific subcutaneous
immunotherapy}

Alexandra Sayuri Watanabe MD\textsuperscript{1}(watanabe.ale@gmail.com),
Marcelo Alves Ferreira PhD\textsuperscript{2}(piktor@usp.br), Jorge
Kalil MD, PhD\textsuperscript{3,4,5}(jkalil@usp.br), Cl\selectlanguage{ngerman}óvis Eduardo
Santo Galvao MD, PhD\textsuperscript{3} (\emph{galvaoclovis@gmail.com}),
Keity Souza Santos PhD
\textsuperscript{3,4,5}(keitysouzasantos@gmail.com), Fabio Fernandes
Morato Castro MD,
PhD\textsuperscript{1.3}(fabio.castro@imabrasil.com.br)\textsuperscript{1}Serviço
de Imunologia Clinica e Alergia, Hospital das Clinicas HCFMUSP,
Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brazil
~\textsuperscript{2}Laboratorio de Biologia~Celular~ (LIM 59) Hospital
das Clinicas HCFMUSP,~ Faculdade de Medicina, Universidade de Sao Paulo,
Sao Paulo, SP, Brazil ~\textsuperscript{3}Disciplina de Imunologia
Clínica e Alergia, Faculdade de Medicina da Universidade de Sao Paulo,
São Paulo 01246-903, Brazil\textsuperscript{4}Laboratorio de Imunologia,
Instituto do Coracao, Hospital das Clinicas da Faculdade de Medicina da
Universidade de Sao Paulo, Sao Paulo 01246-903,
Brazil\textsuperscript{5}Institute for Investigation in Immunology
(iii), INCT, Sao Paulo 01246-903, Brazil\textbf{Corresponding Author:}
Alexandra Sayuri Watanabe email: watanabe.ale@gmail.com Address:
Hospital das Clínicas - Faculdade de Medicina da USP, Av. Dr. Eneas de
Carvalho Aguiar, 155, 8º floor, Block 3, Cerqueira César, Zip code:
05403-000 São Paulo, SP. Telephone: +55 (11) 2662-6225, +55(11)
2662-9571

\textbf{Conflict of interest's statement:} The authors declare that they
have no conflicts of interests to disclose.

\textbf{Abstract}

\textbf{Background:} Anaphylaxis to fire ant venoms (\emph{Solenopsis
sp} ) is a significant cause of systemic allergic reaction caused
by\emph{Hymenoptera} stings in children. There are only a few reports
about the safety and efficacy of specific immunotherapy.

\textbf{Objective:} Evaluate clinical characteristics, IgE and IgG4
specific responses of patients undergoing immunotherapy with a
whole-body extract of \emph{Solenopsis sp} after one year of the
maintenance phase.

\textbf{Materials and methods:} Thirty-three patients were enrolled due
to anaphylaxis by fire ant venom (\emph{Solenopsis sp} ) and underwent
specific venom immunotherapy. They were assessed at baseline and one
year after the beginning of the maintenance phase for skin test;
specific IgE and IgG4 antibodies to fire ant venom; tryptase.

\textbf{Results:} All patients included presented a severe anaphylactic
reaction. Although two patients (6.25\%) presented a tryptase level
higher than 11.4 ug/ml, systemic mastocytosis was ruled out. There was
no relationship between the severity of the reaction with gender,
tryptase level, atopy, previous reactions, concentration of the allergen
in the skin test or specific IgE level. There was an increase of the
specific IgG4/IgE ratio between the two time points. Reactions were
local, with only two mild systemic reactions during the build-up phase.
Twenty patients had accidental stings during immunotherapy, with 3
presenting only urticaria.

\textbf{Conclusions:} This study is unprecedented in evaluating clinical
and laboratory data in the fire ant immunotherapy. Our results show that
after one year of the maintenance phase, patients did not develop any
severe reaction with only a few mild reactions and presented a
significant production of specific IgG4.

\textbf{Abbreviations used:}

\textbf{FA: fire ant(s)}

\textbf{WBE: whole-body extract}

\textbf{VIT: venom immunotherapy}

\textbf{QoL: quality of life}

Keywords: fire ant, whole-body extract, Hymenoptera, immunotherapy

\textbf{Introduction}

Bees, wasps, and ants are the Hymenoptera involved in accidents causing
allergic manifestations. Bees and wasps' venoms are the most
well-characterized and purified commercial extracts are available for
testing and treatment in the United States and Europe, while ants are
much less studied. They are biodiverse, with 22 recognized subfamilies,
approximately 300 genera, and more than 14,000 species. However, only a
small number of them have been described to cause allergic reactions in
humans\textsuperscript{1}, including the so-called imported fire ants
(\emph{Solenopsis sp} .) present in Brazil\emph{.}

The clinical manifestations produced by \emph{Hymenoptera} venom can be
classified into local, large local, systemic anaphylactic and delayed
systemic reactions. The risk of a person being stung in an endemic area
is from 30\% to 60\%\textsuperscript{2,3}, with a 0.16\% to 16\% risk of
presenting severe systemic reactions\textsuperscript{4,5}.

The diagnosis of anaphylaxis to ant venom is based on clinical history,
testing for specific IgE antibodies to the venom, and skin tests after
at least 3 to 4 weeks after the acute event to reduce the likelihood of
a false-negative result. These tests not only confirm the diagnosis but
also identify the appropriate extract to be used in
immunotherapy\textsuperscript{6}.

Allergen-specific immunotherapy in children is indicated for the
treatment of allergic rhinitis, allergic asthma, some foods, and
Hymenoptera hypersensitivity\textsuperscript{7}. Besides being effective
in reducing subsequent systemic reactions in children and adults, this
treatment significantly increases the quality of life (QoL) of these
patients\textsuperscript{8}.

The treatment is considered to be safe and effective. The reported
efficacy for patients treated with bee venom is 77-84\%, and 91-96\% for
patients who received wasp venom\textsuperscript{9,10}.

Triplett\textsuperscript{11} was the first to report successful
immunotherapy with whole-body extracts for fire ant venom allergy in
1973. It is widely used in the USA by allergists\textsuperscript{12}.

There were no systematic studies on the efficacy of fire ant
immunotherapy with whole-body extract until Freeman et
al\textsuperscript{13} in 1992 used sting challenge to assess efficacy.
They demonstrated that immunotherapy with whole-body extracts provided a
high degree of protection to most patients.

Safe and effective of cluster, rush and ultra-rush schedules for fire
ant immunotherapy have been previously reported. Most of the studies
with a limited number of patients and the focus were mainly determined
whether prophylactic pretreatment with antihistamines and steroids
reduced the systemic reaction
rate\textsuperscript{14,15,16,17,18,19,20}.

Adams at al\textsuperscript{16} illustrated the safety and efficacy of
repeated fire ant rush immunotherapy in three patients. Beveridge et
al\textsuperscript{17} describe a cluster imported fire ant schedule in
a 66-year-old man.

Tankersley et al\textsuperscript{18} evaluated fifty-nine patients with
imported fire ant hypersensitivity randomized to placebo or twice-daily
terfenadine 60 mg, ranitidine 150 mg, and prednisone 30 mg before rush
protocol in a double-blinded study. Sting challenges were performed on
56 patients, and they concluded that rush immunotherapy is safe and
efficacious; the rate of mild systemic reactions was low.

Dietrich et al\textsuperscript{19} included 37 patients in a 1-day rush
protocol. Twenty-two were stung challenged, 21 did not result in any
signs or symptoms of a systemic reaction, giving an efficacy rate of
preventing systemic reactions of 95.5\% for the rush protocol.

Arseneau et al\textsuperscript{20} evaluate the safety and efficacy of
1-day rush immunotherapy with fire ant whole-body extract (0.5 mL 1:100
(wt/vol) maintenance injection), with a sting challenge on approximately
day 22 in 66 patients.

Brown et al\textsuperscript{21} reported that venom immunotherapy
with\emph{M pilosula} (jack jumper) was very effective in preventing
life-threatening sting anaphylaxis, and it could benefit the population
in areas of southeastern Australia where \emph{Myrmecia} ant stings
occur.

La Shell et al\textsuperscript{23} showed that three-year subcutaneous
immunotherapy with whole-body extract is safe; however, specific IgE and
IgG4 antibodies or tryptase before and after immunotherapy were not
evaluated.

Thus, the objective of this study was to evaluate side effects, efficacy
and specific IgE and IgG4 production of patients submitted to
subcutaneous fire ant immunotherapy with whole-body extracts
of\emph{Solenopsis sp.} during the first year of treatment\emph{.}

\textbf{Methods:}

\textbf{Types of participants enrolled in this study:}

Patients were attended at the ambulatory of anaphylaxis of the Clinical
Immunology and Allergy Service of the Hospital das Clínicas - Faculty of
Medicine -- Sao Paulo University. They presented a history of a severe
anaphylactic reaction (Muller's severity score: grade III or
IV\textsuperscript{24}) after fire ant sting of the
species\emph{Solenopsis sp} , and according to the following inclusion
criteria:

\selectlanguage{english}* Children or adults: age 5 to 65 year

\selectlanguage{english}* Diagnosis of type I hypersensitivity confirmed by positive
determination of specific IgE level (\emph{Solenopsis sp -} ImmunoCap(r)
i70) and a positive skin test (prick or intradermal test).

\selectlanguage{english}* Informed consent form signed by the patient or authorized
representatives approved by our institutional review board.

The following items were accepted as exclusion criteria: use of
immunomodulatory treatment in the last five years; previous treatment
with specific allergen immunotherapy; moderate or severe persistent
asthma and FEV1 \textless{}80\%; pregnancy; illiteracy; psychiatric
illness; continuous use of \selectlanguage{greek}β-\selectlanguage{english}blockers; cardiovascular diseases, organ
failure, and diseases that increase the risk of side effects of
adrenaline and other immunological, autoimmune diseases or malignancy.

\textbf{Research design:}

After fulfilling the inclusion criteria, patients underwent the skin
test. Prick test method were carried out with positive (histamine base)
and negative (saline) controls and the following concentrations of the
whole-body extract of the \emph{Solenopsis} mix (HollisterStier;
Spokane, USA) were utilized: 1:1,000,000 wt/vol; 1:100,000 wt/vol;
1:10,000 wt/vol; 1:1000 wt/vol, if needed. If the prick result was
negative, the prick test was followed by intradermal testing. The test
was considered positive when the wheal diameter was 5 mm or greater with
a flare of 11 mm or greater for the prick test and for the intradermal
test when the wheal enlargement was above 3 mm from the initial wheal
caused by the volume of the injected extract and flare greater than 11
mm\textsuperscript{18}.

After positivity to the skin test, the starting dose of the
immunotherapy was calculated as follows: if the positivity was in the
prick test, three concentrations were returned to start the treatment.
If this test was positive in the intradermal test, two concentrations
were returned. The protocol used for immunotherapy with whole-body
extract (\emph{Solenopsis} sp) was the cluster-modified schedule, whose
concentrations increased weekly until the maintenance phase, which in
this study was considered 1:100wt/vol, volume: 0.5mL. Maintenance doses
were subsequently spaced to once every 4 weeks. The approval of the
project by the Ethics Committee was signed on January 20, 2016 (Online
registration n\selectlanguage{ngerman}º: 14174). Figure 1 exemplifies the immunotherapy schedule
in the build-up phase used with an initial hypothetical dose:

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/372632/articles/490581-clinical-and-laboratory-profile-of-patients-with-anaphylaxis-to-fire-ant-venom-solenopsis-sp-under-specific-subcutaneous-immunotherapy}

\textbf{Figure 1.} Build-up phase of immunotherapy using a hypothetical
initial dose. Intervals between injections are of 30 minutes.

Skin testing and immunotherapy were performed with whole-body extract
(HollisterStier company, Spokane, USA) that consisted of
a\emph{Solenopsis invicta} and \emph{Solenopsis richteri} mix from a
50-mL 1:10 wt/vol stock bottle in 0.4\% phenol.

The following clinical characteristics: assessment of gender, atopy,
age, urban or rural area, and the following tests were performed at
baseline: measurement of total IgE, IgG, IgM, IgA antibodies;
measurement of serum IgE antibody for \emph{Solenopsis} mix (i70);
tryptase level, specific IgG4 antibody, and skin test.

After one year in the maintenance phase of the treatment, the following
parameters were evaluated: specific IgE antibody level (i70), specific
IgG4 antibody, adverse reactions to immunotherapy, accidental stings,
and which were reactions that occurred in those stings.

\textbf{Statistics}

Categorical data were summarized using percentages and analyzed using
Pearson's chi-square tests with Yates' continuity correction, Student's
t-test, and ANOVA with Tukey's adjustment or Wilcoxon test, whichever
most appropriate. Results were considered statistically significant for
P values less than 0.05.

\textbf{Results}

\textbf{Patients}

All 33 patients enrolled in the study had histories of systemic
reactions (Muller's severity score: grade III or IV) to fire ant stings
and positive skin test reactions to the whole-body extract
to\emph{Solenopsis} mix. Table 1

During the treatment, when they were in the maintenance phase, three
patients gave up the treatment: 2 of them reported that they had been
stung twice and had no reactions, added to the difficulty of
transportation. The third patient gave up for fear of losing a job.

The majority of systemic reactions (grade III or IV) to fire ants index
field stings resulted from 1 sting.

`Table 1: Demographics and clinical features of the patients:\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Age, years: Between 5 to 10 years Between 11 to 20 years Between 21 to
30 years Between 31 to 40 years \textgreater{} 41 years & 7 - 52 years:
6 patients (18,3\%) 19 patients (57,6\%) 4 patients (12,1\%) 2 patients
(6,0\%) 2 patients (6,0\%)\tabularnewline
\midrule
\endhead
Urban area & 31 patients (93,3\%)\tabularnewline
Male & 17 patients (51,5\%)\tabularnewline
Asthma & 7 patients (21,2\%)\tabularnewline
Tryptase level Reaction grade III (mean value) Reaction grade IV (mean
value) \textgreater{}11,4ng/mL & 3,63 ng/mL 4,76 ng/mL 2
patients*\tabularnewline
Grading system** Grade III Grade IV & 24 patients (72,7\%) 9 patients
(27,3\%)\tabularnewline
Index field reactions Grade III Grade IV & 1 sting: 23 patients
(69,70\%) 5 stings: 1 patient (3,03\%) 1 sting: 7 patients (21,21\%) 2
stings: 1 patient (3,03\%) 3 stings: 1 patient (3,03\%\tabularnewline
\bottomrule
\end{longtable}

* Patients were referred to the hematology sector, with systemic
mastocytosis ruled out.

**Systemic reactions measured using the Mueller grading system

\textbf{Possible risk factors associated with the severity of the
symptoms:}

\textbf{Gender:}

There was no correlation between gender and the severity of the reaction
(Pearson's Chi-squared test with Yates' continuity correction;
p=0.9151). Table 2

\textbf{Atopy}

Seventeen patients (51.51\%) reported a diagnosis of allergic rhinitis
and/or allergic asthma. The diagnosis of allergic rhinitis and/or asthma
did not prove to be a risk factor for the severity of the reaction, as
there was no relationship of this disease in patients who presented a
grade IV reaction. (p-value = 0.9233788). Table 2

\textbf{Tryptase level}

Patients with an anaphylactic reaction, grade III severity had a mean
basal tryptase level of 3.63 ng/mL, with a standard deviation of 3.06
ng/mL. Patients with grade IV anaphylactic reaction had an average of
4.76 ng / mL, with a standard deviation of 6.98 ng/mL. There was no
correlation between serum tryptase level and grade IV severity (Wilcoxon
rank with continuity correction; p = 0.4333). Table 2

\textbf{Previous reactions}

Considering only Grade-IV severe previous reactions, for natural history
assessment purposes, there was no correlation with any specific
reaction: 11.11\% had a previous grade-II severity reaction; 22.22\%,
Grade-IV; 55.56\%, local and 11.11\%, extensive local. In these
patients, there was no correlation between reaction severity in previous
stings and progression to a severe reaction in future stings (p =
1.207). Most of the patients reported a local reaction. Table 2

\selectlanguage{ngerman}\textbf{Ant fire IgE level (Immunocap i70®)}

Considering the correlation between the severity of the reaction and the
measurement of specific IgE levels against fire ant (ImmunoCap i70®),
there was no statistical difference between the groups (p = 0.776).
Patients with a grade III severity had a mean value of 26.3 kU/L, with a
standard deviation: 32.4, and patients with grade IV severity had a mean
value of 45.9 kU/L and standard deviation: 49.2. Table 2

\textbf{Skin test}

Patients presented the following results in the skin test (prick test
and intradermal test), with most of the results being positive in the
intradermal test (81.8\%), Figure 2.

There was no correlation between concentrations in positive skin test
with the severity of the reaction presented (chi-square test; p =
0.7354). Table 2

Figure 2:

(a) Percentage and number of patients (n) of positive skin tests (prick
test and intradermal test)

(b) examples of positive skin tests (outpatients Anaphylaxis of
HC-FMUSP)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Table 2: Baseline clinical and laboratory characteristics of the
patients\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\textbf{Clinical and laboratory characteristics of the patients}\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\textbf{Grade III}\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\textbf{Grade IV}\strut
\end{minipage} & \begin{minipage}[b]{0.22\columnwidth}\raggedright\strut
\textbf{p-value}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Male Female\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
12 patients (75\%) 11 patients (64,7\%)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
4 patients (25\%) 6 patients (35,3\%)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0.9151\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Asthma\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
5 patients (15,15\%)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
2 patients (6,06\%)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0.9233788\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Tryptase level (mean value)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
3.63 ng/mL\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
4,76 ng/mL\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0.4333\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Ant fire IgE level (mean value)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
26.3 kU/L\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
45.9 kU/L\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0.776\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Skin test Prick test Intradermal test\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
4 patients (12,12\%) 20 patients (60,06\%)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
2 patients (6,06\%) 7 patients (21,21\%)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0.7354\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Dynamic between specific IgE and IgG4 antibodies}

\textbf{Fire ant IgE antibody level: basal and in the maintenance phase}

When evaluating the ratio of the specific IgE level (Immunocap\selectlanguage{ngerman}® i70) for
each patient before and after one year in the maintenance phase, there
was a statistically significant difference (p= 0.0041). The specific IgE
antibody decreased in this interval of time in 73.3\% of patients (mean
baseline: 31.67 kU/L; after one year in the maintenance phase: 9.86
kU/L, mean).

\textbf{Serum determination of specific IgG4 antibody}

Serum specific IgG4 antibody was performed at baseline and 12 months
after the beginning of the maintenance phase (Figure 2a).

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/372632/articles/490581-clinical-and-laboratory-profile-of-patients-with-anaphylaxis-to-fire-ant-venom-solenopsis-sp-under-specific-subcutaneous-immunotherapy}

\textbf{Figure 2.} a) Specific IgG4 at baseline and after 12 months of
immunotherapy maintenance phase (n = 30). b) Comparison of the ratio
specific serum IgG4/IgE levels (baseline and after 12 months in the
maintenance phase), (Wilcoxon test, p = 0.0000318).

\textbf{Specific serum IgG4/IgE ratio}

When comparing the serum IgG4/IgE ratio to fire ant venom at baseline
and after one year from the beginning of the maintenance phase, there
was a statistically significant difference (Wilcoxon test, p =
0.0000318), Figure 2b.

\textbf{Adverse reactions to immunotherapy}

\textbf{During the build-up phase}

During the build-up phase, two patients (6.06\%) presented grade-I
reaction (only one organ involved limited to skin - mild urticaria), and
four patients (36.36\%) had local reactions\textgreater{} 50mm after
subcutaneous injections. In 57.58\%, there was a local reaction smaller
than 50mm or no reaction at all.

Considering the risk of reaction in each injection applied, in 1167
applications performed during the build-up phase, with 58.33\% of the
reactions occurring in dose 1:100wt/vol. The risk of systemic reaction
by injection was 0.017\% in this phase (Figure 3).

\textbf{During the maintenance phase:}

During the maintenance phase, five patients (15.15\%) presented local
reactions at the injection site \textgreater{}50mm, and 84.85\%
presented no reaction or had a reaction less than 50mm. No patient had a
systemic reaction in the maintenance phase of the treatment.

When considering the risk of reaction in each injection applied, we had
5 reactions (1,4\%) in 360 applications performed during the maintenance
phase, being only local reactions (Figure 3).

There was no need to use adrenaline at any time during treatment, and
the systemic reactions that occurred in the build-up phase were treated
only with antihistamines with improvement after 2 hours in the
observation period.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 3:} Number of adverse reactions (per injection) during
immunotherapy according to phase and reaction type.

\textbf{Field stings during the treatment}

During the treatment, 20 patients (60.6\%) presented accidental field
stings by fire ants.

During the build-up phase, five patients were stung: four local
reactions and one grade-I reaction (mild urticaria) that improved after
antihistamine, with no need to seek emergency care or use adrenaline
auto-injectable. Figure 4

In total, patients referred nine accidental field stings because they
have subsequent stings: 2 patients had experimented stings two times
during the build-up phase, one patient was stung three times, and two
patients had a single sting.

During one year of observation in the maintenance phase: nineteen
patients (57.6\%) were accidentally stung, with seventeen patients
(89.5\%) having experimented a local reaction and two patients (10.5\%)
having grade-I systemic reactions: urticaria, that improved after taking
an oral antihistamine, with no the need to seek emergency care or use
adrenaline auto-injectable. Figure 4

As some patients were stung more than once, 35 accidental stings were
observed during the first year of the maintenance phase. Figure 4\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 4:} Distribution of accidental sting per patient during
the build-up and maintenance phase according to the severity of the
reaction.

The patient 7 draws attention, he lives in a rural area, and he was
accidentally stung eight times, two times in the build-up phase, and six
times in the maintenance phase, with only one local reaction in all
events.

Considering only the patients who had a grade IV systemic reaction in
the index field sting (n=9), one patient had two field stings during the
build-up phase with local reaction. During the maintenance phase: three
patients had field stings (one, four and two stings) with local
reaction, and one patient had a single sting with a mild systemic
reaction, whose hives improved after taking 20mg Bilastine.

\textbf{Discussion}

Fire ant stings are frequent in pediatric age: 32\% to 54\% of the
surveyed population\textsuperscript{25}.

In this study, systemic reactions after fire ant stings were also more
prevalent in patients (n=17) under 15 years (51.51\%). If we expand the
age group up to 20 years old, the frequency of the systemic reactions
rises to 75.9\%. Among Grade-IV reactions, the patient's age has not
positively correlated with the grade of the sting reaction.

In literature, male sex is an independent risk factor for severe
systemic reactions to the field sting. The effect of male sex in insect
sting induced anaphylaxis presumably results from a selection effect.
Because of a different degree of exposure, adult men are stung more
frequently than women. They might, therefore, be at higher risk for
sensitization or severe allergic reactions to honeybee or wasp venom.
There is no data on fire ant venom\textsuperscript{26}. In this study,
there was no gender influence on the severity of the reaction; there was
only a slight predominance of males: 51.5\% over females: 48.5\%,
probably due to the greater exposure.

Concerning atopy, it is known that it is a risk factor for anaphylaxis
triggered by food, exercise, and latex\textsuperscript{27}. It has not
yet been established that atopic disease increases the risk of
anaphylaxis associated with \emph{Hymenoptera} venom allergic
reaction\textsuperscript{28}.

According to an international anaphylaxis consensus
(ICON)\textsuperscript{29}; cardiovascular disease and uncontrolled
asthma are well-recognized risk factors for severe anaphylaxis in
general. In the current study, there was no correlation between asthma
with a Grade-IV systemic reaction.

Considering the previous systemic reaction, in literature, 18\% of the
mild systemic reactions in previous stings evolved into severe systemic
reactions\textsuperscript{30}. In the current study, the percentage was
lower, 11.11\%. In the same study mentioned above, a third of patients
(24\%) developed the same severity as the previous
reactions\textsuperscript{30}; meanwhile, we obtained a percentage of
22.22\%, data similar to the literature.

Considering the previous large local reactions, Golden et
al\textsuperscript{31} observed that 7\% of children who had large local
reaction progressed to a systemic reaction in subsequent stings. Graft
and collaborators\textsuperscript{32} reported 2\% of systemic reaction
when they had large local reactions previously. Our findings showed that
11.11\% of the patients had experienced a large local reaction in the
previous sting. The vast majority (55.56\%) reported only local
reactions in previous reactions. Therefore, in our patients, there was
no correlation between having a certain severity of reaction in previous
stings and subsequently evolving to a more severe reaction in subsequent
stings (p = 1.207). The vast majority reported only a local reaction in
the sting prior to anaphylaxis.

In literature, a history of large local reactions carries a lower risk
of a systemic reaction than in all other sensitized patients. When a
large local reaction results after bee venom, the likelihood of
anaphylaxis from a future sting is approximately 5\%. For comparison,
when there is a history of anaphylaxis from a previous Hymenoptera sting
and the patient has positive skin test results to venom, at least 60\%
of adults and 20-32\% of children will develop anaphylaxis with a future
sting\textsuperscript{33}. A fact that draws attention in our study is
in 4 out of 18 patients, the first systemic reaction was already severe,
and that these patients were not referred to a specialist for
investigation. Only in the subsequent reaction, two patients were
referred to a specialist for investigation, and the other two patients
were not referred, looking for a specialist willingly.

Comparing the results of specific serum IgE measurements against fire
ant venom with skin tests results in patients with grade-IV systemic
reactions, our findings corroborate with the
literature\textsuperscript{34,35,36} that there is no correlation
between the degree of sensitization and severity of the reaction.

\textbf{Changes in specific IgE and IgG4 antibodies measurements}

The levels of fire ant IgE antibody decreased in the period observed in
73.3\% of the patients, while specific IgG4 increased in that period.
Thus, the specific IgE/IgG4 ratio at baseline and 12 months after the
maintenance phase showed significant differences.

After an initial increase in the first months of treatment, specific IgE
levels tend to decrease during immunotherapy\textsuperscript{37,38} and
generally remain low even after discontinuation of
treatment\textsuperscript{39}. On the other hand, specific serum IgG4
levels increase during immunotherapy with
venom\textsuperscript{40,41,42}. It is known that high levels of
specific IgG4 are positively correlated with the number of stings,
previously described in beekeepers\textsuperscript{43,44,45}. Thus, the
specific IgG4 antibody is considered a tolerance indicator in allergic
individuals because it has been suggested in the literature that these
antibodies can block the interaction between the allergen and the
specific IgE antibodies, thus preventing reactions with the
participation of IgE. These effects should be reflected in the specific
IgE/IgG4 ratio\textsuperscript{46,47}. The explanation for this is due
to the regulatory cell populations that form a suppressive environment:
a slight decrease in the production of allergen-specific IgE and early
switch to B cells to produce IgG4 and, consequently, an increase in IgG4
antibodies, which is a non-inflammatory agent in allergic
disorders\textsuperscript{7,48}.

\textbf{Serum tryptase levels}

In literature, about 80\% of patients with Muller's Grade-IV reaction
to\emph{Hymenoptera} venoms (bees and wasps) are diagnosed with
mastocytosis. In these severe reactions, 20\% of patients could not show
clinical signs of mastocytosis\textsuperscript{49}. Bonadonna et
al\textsuperscript{50} reported a correlation between a systemic
reaction to \emph{Hymenoptera} sting and mast cell tryptase. Three
hundred seventy-nine patients presented a history of systemic reactions
after being stung by these insects, 11.6\% had serum mast cell tryptase
that exceeded 11.4 ng/mL. In this group, the rate of anaphylaxis
(Muller's grade IV) was 70.5\%. Blum et al\textsuperscript{51} confirmed
these findings in a 5-year retrospective study, with 868 patients who
had severe reactions after insect stings (758 patients had both: total
IgE and baseline tryptase level above the reference values). Elevated
basal tryptase (\textgreater{} 11.4 ng/mL) was associated with severe
systemic reactions (p \textless{}0.03). Due to this strong association,
the guidelines on allergy to \emph{Hymenoptera} venoms always recommend
evaluating serum tryptase for these patients.

In this study, two patients (6.25\%) presented a tryptase level higher
than 11.4 ug/ml; however, due to the specific investigation of
hematology in our hospital, the diagnosis of Systemic Mastocytosis was
ruled out. As already mentioned, in literature, there is a high
correlation between severe anaphylaxis and \emph{Hymenoptera} stings but
being reported by bees and wasps. There is no data on fire ant venom so
far.

\textbf{Adverse reactions during immunotherapy}

Adverse events are frequently observed when unpurified extracts are
utilized, and the aqueous formulations tend to cause more local
reactions than depot diluents \textsuperscript{52,53,54}.

The main risk factors that provoke serious reactions in the
literature\textsuperscript{55} were very well-reviewed at each patient
visit and controlled in this study: dosage error, administration of the
injection without supervision by a trained professional, presence of
uncontrolled asthma, the high degree of sensitivity, concomitant use of
\selectlanguage{greek}β-\selectlanguage{english}blockers, systemic reactions prior to immunotherapy and use of lots
with new products.

When a local reaction was greater than 50mm, patients were instructed to
use fexofenadine 180mg (adults), and for children, the dose was adjusted
according to the weight presented. Only one adult patient took the oral
antihistamine because she had a bother local reaction; other patients
did not need the medication.

In a multicenter study\textsuperscript{56} evaluating data from 840
patients, side effects of immunotherapy (systemic reactions) with venom
(seventy-one percent were treated with Vespula-venom extract and 27\%
with honeybee-venom extract) were observed in a total of 20\% of
patients; 26,601 injections in 840 patients, systemic reactions were
observed in 1.9\% of the injections during the build-up phase and in
0.5\% the maintenance phase (p \textless{}0.05). Most of these reactions
were mild, and only a third of patients required medical treatment. A
similar frequency of systemic adverse effects was observed in a
published study analyzing data from 178 patients\textsuperscript{57}.
The build-up phase of venom immunotherapy has more adverse effects than
the maintenance phase.

In our study, the systemic reaction rate during immunotherapy was lower
than the literature, comparing extracts of honeybee or wasp venoms. Our
results showed a systemic reaction in 6.06\% during the build-up phase.
It was also lower when compared by injection (0.017\%). In the
maintenance phase, there were no systemic reactions. The similarity
remained in the slight severity of these reactions.

Thus, the severity of side effects due to immunotherapy does not
necessarily correlate with the severity of the treated allergic disease
symptoms. Systemic reactions induced by venom immunotherapy may occur,
but most patients tolerate this treatment without relevant side effects.

\textbf{Accidental Field stings}

Reaction evaluation after a sting during or after the treatment (either
by a challenge test with culprit insect or by accidental sting) is an
available method to determine the degree of response to venom
immunotherapy\textsuperscript{58}.

When the challenge test with culprit insect was performed in a series of
patients undergoing immunotherapy with bee and wasp venom, about 75 to
85\% \textsuperscript{8,59,60} or even more 95\%
\textsuperscript{61,62}of patients were protected.

Arseneau at el\textsuperscript{20} evaluated 66 patients submitted to
1-day rush immunotherapy protocol using fire ant whole-body extract, and
the conclusion was that the immunotherapy was efficacious (1 of 53
confirmed fire ant sting challenges (1.9\%) resulted in a reaction) and
had a low rate of systemic reactions.

In Brazil, for ethical reasons, we could not perform the provocation
test with the culprit insect; it is possible only to observe reactions
by accidental stings during treatment.

In this study, 20 patients were stung, 35 stings in total, as some of
them were stung more than once.

During the build-up phase, the patient still has an increased chance of
having systemic reactions if he is stung at that moment. The patient who
had a systemic reaction in the build-up phase in our study had a mild
reaction (only hives, with no need to seek immediate attention or use
adrenaline auto-injectable). Thus, we had one systemic reaction (in an
accidental sting), out of 5 patients stung during this phase: 20\%
treatment failure.

During the maintenance phase, two patients were accidentally stung. They
had systemic reactions (10.5\%) and described as mild reactions: only
hives, without the need to seek emergency care or the use of an
adrenaline auto-injectable.

The percentage of reactions in the maintenance phase (10.5\%) is lower
than the percentage found in the literature, which considers
approximately a 25\% risk of a new generalized systemic reaction in
subsequent stings in patients who received venom immunotherapy for 1 or
2 years\textsuperscript{63}.

The optimal length of treatment with fire ant immunotherapy is not
known. The treatment with fire ant immunotherapy commonly used often is
3--5 years based on extrapolation from flying
\emph{Hymenoptera}immunotherapy data. A 3- to 5-year course of flying
Hymenoptera (e.g., honey bee, hornet, wasp, and yellow jacket) venom
immunotherapy has been shown to provide protection against systemic
reactions\textsuperscript{64}.

Forester et al\textsuperscript{65} grouped patients into those who
received \textless{} 3 years (reduced course) and those who received
\textgreater{} 3 years (complete course) of imported fire ant
immunotherapy (IFA). All subjects on IFA immunotherapy received a
maintenance dose of 0.5 mL of a 1:100 w/v concentration of IFA
whole-body extract. No difference in the incidence or severity of
systemic reactions to field stings after immunotherapy discontinuation
for the two groups studied. The systemic reaction rate was low for both
the complete and the reduced course groups (7 and 6\%, respectively)
after discontinuation of IFA immunotherapy.

Our patients will be in immunotherapy for 3 years. After this period,
new data will be gathered.

\textbf{Conclusion}

Few studies have evaluated patients undergoing fire ant immunotherapy
with whole-body extracts. Our study is unprecedented in the evaluation
of clinical and laboratory data on these treatments.

The development of peripheral tolerance is the primary mechanism of
immunotherapy. In this study, we verified a significant difference at
the baseline and after one year of the maintenance phase in a specific
IgE/IgG4 ratio, reinforcing the hypothesis that the IgG4 antibody has a
mode of action ''blocking'' the specific IgE. It is important to note
that allergen-specific IgG can interfere not only in elucidating
allergic responses triggered by IgE in effectors but also in presenting
antigen IgE-mediated to T cells. It may be essential to decrease the
responses of allergen-specific TH2 cells. It is essential to consider
not only the individual values of each antibody but the ratio between
them at baseline and during immunotherapy. In this aspect, it would be
interesting to obtain further studies on specific T cells' behavior
during immunotherapy with fire ant venom.

The maintenance dose of 0.5mL in the concentration 1: 100 wt/vol
(Hollister Stier, USA) of a mixture of whole-body extract
of\emph{Solenopsis invicta} and \emph{richteri} was effective because of
the 20 patients accidentally stung during the treatment (1 year of
maintenance dose), two patients (10.5\%) had a systemic reaction
(urticaria), with improvement after taking an oral antihistamine.
Adverse reactions to immunotherapy were rare.

The lessons learned in this study should help to improve both the
effectiveness and the maintenance of tolerance to fire ant
immunotherapy. This treatment proved to be safe and effective.

Patients will complete treatment for three years from the maintenance
dose, and patients will be followed up after discontinuation of
treatment.

\textbf{References}

\begin{enumerate}
\tightlist
\item
  Golden DBK. Insect allergy. In: Adkinson NF Jr, Bochner BS, Busse WW,
  Holgate ST, Lemanske RF, Simons FER, editors. Adkinson: Middleton's
  Allergy: Principles and Practice. 8th ed. Philadelphia: Mosby
  Elsevier; 2014. p.1260-72 (p. 1261, Table 78-1).
\item
  deShazo RD, Butcher BT, Banks WA. Reactions to the stings of the
  imported fire ant. N Engl J Med. 1990;323:462-6.
\item
  deShazo RD, Williams DF, Moak ES. Fire ant attacks on residents in
  health care facilities: a report of two cases. Ann Intern Med.
  1999;131:424-9.
\item
  Stafford CT. Hypersensitivity to fire ant venom. Ann Allergy Asthma
  Immunol. 1996;77:87-95.
\item
  Triplett RF. The imported fire ant: health hazard or nuisance? South
  Med J. 1976;69:258-9.
\item
  Bilo BM, Rueff F, Mosbech H, Bonifazi F, Oude-Elberink JN. Diagnosis
  of Hymenoptera venom allergy. Allergy. 2005 Nov;60(11):1339-49.
\item
  Alvaro-Lozano M, Akdis CA, Akdis M, Alviani C, Angier E, Arasi S,
  Arzt-Gradwohl L, Barber D, Bazire R, Cavkaytar O, Comberiati P,
  Dramburg S, Durham SR, Eifan AO, Forchert L, Halken S, Kirtland M,
  Kucuksezer UC, Layhadi JA, Matricardi PM, Muraro A, Ozdemir C, Pajno
  GB, Pfaar O, Potapova E, Riggioni C, Roberts G, Rodr\selectlanguage{ngerman}íguez Del Río P,
  Shamji MH, Sturm GJ, Vazquez-Ortiz M. EAACI Allergen Immunotherapy
  User's Guide. Pediatr Allergy Immunol. 2020 May;31 Suppl 25:1-101.
  doi: 10.1111/pai.13189. PMID: 32436290.
\item
  Dhami S, Zaman H, Varga EM, Sturm GJ, Muraro A, Akdis CA, et al.
  Allergen immunotherapy for insect venom allergy: a systematic review
  and meta-analysis. Allergy 2017; 72:342-65.
\item
  Muller U, Helbling A, Berchtold E. Immunotherapy with honeybee venom
  and yellow jacket venom is different regarding efficacy and safety. J
  Allergy Clin Immunol 1992; 89:529-35.
\item
  Rueff F, Vos B, Elberink JO, Bender A, Chatelain R, Dugas-Breit S, et
  al. Predictors of clinical effectiveness of Hymenoptera venom
  immunotherapy. Clin Exp Allergy 2014.
\item
  Triplett RF. Sensitivity to the imported fire ant: successful
  treatment with immunotherapy. South Med J 1973- 66-477-80.
\item
  Stafford CT, Rhoades RB, Bunker-Soler AL,Thompson WO, Impson BS,
  Survey of whole-body extract immunotherapy for imported fire ant and
  other Hymenoptera sting allergy, J Allergy Clin Immunol 1989: 83:
  1107-11.
\item
  Freeman TM, Hylander R, Ortiz A, Martin ME. Imported Fire Ant
  Immunotherapy: Effectiveness of Whole Body Extracts. J Allergy Clin
  Immunol. 1992 Aug;90(2):210-5. doi: 10.1016/0091-6749(92)90073-b.
\item
  Duplantier JE, Freeman TM, Bahna SL, Good RA, Sher MR. Successful rush
  immunotherapy for anaphylaxis to imported fire ants. J Allergy Clin
  Immunol. 1998;101(6 Pt 1):855-856. doi:10.1016/s0091-6749(98)70318-5.
\item
  Judd CA, Parker AL, Meier EA, Tankersley MS. Successful administration
  of a 1-day imported fire ant rush immunotherapy protocol. Ann Allergy
  Asthma Immunol. 2008;101(3):311-315.
  doi:10.1016/S1081-1206(10)60497-8.
\item
  Adams KE, Johnson KS. Safety of Repeated Imported Fire Ant Ultra-Rush
  Protocols. Mil Med. 2019;184(5-6):e483-e485. doi:10.1093/milmed/usy275
\item
  Beveridge GB, Tankersley MS. Successful completion of an imported fire
  ant cluster immunotherapy protocol. Ann Allergy Asthma Immunol.
  2019;123(1):95-96. doi:10.1016/j.anai.2019.04.017
\item
  Tankersley MS, Walker RL, Butler WK, Hagan LL, Napoli DC, Freeman TM.
  Safety and efficacy of an imported fire ant rush immunotherapy
  protocol with and without prophylactic treatment. J Allergy Clin
  Immunol. 2002;109(3):556-562. DOI:10.1067/mai.2002.121956
\item
  Dietrich JJ, Moore LM, Nguyen S, Hagan LL, Tankersley MS. Imported
  fire ant hypersensitivity: a 1-day rush immunotherapy schedule without
  premedication. Ann Allergy Asthma Immunol. 2009;103(6):535e536.
  https://doi.org/10.1016/S1081-1206(10)60271-2.
\item
  Arseneau AM, Nesselroad TD, Dietrich JJ, et al. A 1-day imported fire
  ant rush immunotherapy schedule with and without premedication. J
  Allergy Clin Immunol. 2013;111(6):562e566.
  https://doi.org/10.1016/j.anai.2013.08.021.
\item
  Brown SG, Wiese MD, Blackman KE, Heddle RJ. Ant venom immunotherapy: a
  double-blind, placebo-controlled, crossover trial. Lancet.
  2003;361(9362):1001-1006. doi:10.1016/S0140-6736(03)12827-9
\item
  Wauters RH, Brooks DI, Schwartz DJ. Imported fire ant immunotherapy
  prescribing patterns in a large health care system during an 11-year
  period {[}published online ahead of print, 2020 Jun 13{]}. Ann Allergy
  Asthma Immunol. 2020;S1081-1206(20)30408-7.
  doi:10.1016/j.anai.2020.06.013
\item
  La Shell MS, Calabria CW, Quinn JM. Imported fire ant field reaction
  and immunotherapy safety characteristics: the IFACS study. J Allergy
  Clin Immunol. 2010;125(6):1294-1299. doi:10.1016/j.jaci.2010.02.041.
\item
  Mueller HL. Diagnosis and treatment of insect sensitivity. J Asthma
  Res 1996;3:331-3.
\item
  Stablein JJ, Lackey RF. Adverse reactions to ant stings. Clin Rev
  Allergy 1987;5:161.
\item
  Fehr D, Micaletto S, Moehr T, Schmid-Grendelmeier P. Risk factors for
  severe systemic sting reactions in wasp (Vespula spp.) and honeybee
  (Apis mellifera) venom allergic patients. Clin Transl Allergy. 2019
  October 11;9:54. doi: 10.1186/s13601-019-0292-5. eCollection 2019.
\item
  Simons FE, Ardusso LR, Bilo MB, El-Gamal YM, Ledford DK, Ring J, et
  al. World Allergy Organization anaphylaxis guidelines: summary. J
  Allergy Clin Immunol. 2011;127(3):587-93 e1-22.
\item
  Shaker MS, Wallace DV, Golden DBK, Oppenheimer J, Bernstein JA,
  Campbell RL, Dinakar C, Ellis A, Greenhawt M, Khan DA, Lang DM, Lang
  ES, Lieberman JA, Portnoy J et al. Anaphylaxis-a 2020 practice
  parameter update, systematic review, and Grading of Recommendations,
  Assessment, Development and Evaluation (GRADE) analysis. J Allergy
  Clin Immunol. 2020 January 28. pii: S0091-6749(20)30105-6. doi:
  10.1016/j.jaci.2020.01.017. {[}Epub ahead of print{]}
\item
  Simons FE, Ardusso LR, Bilo MB, Cardona V, Ebisawa M, El-Gamal YM, et
  al. International consensus on (ICON) anaphylaxis. The World Allergy
  Organization journal. 2014;7(1):9.
\item
  Fernandez J, Soriano V, Mayorga L, Mayor M. Natural history of
  Hymenoptera venom allergy in Eastern Spain. Clin Exp Allergy. 2005
  Feb;35(2):179-85.
\item
  Golden DBK, Kagey-Sobotka A, Norman PS, et al. Outcomes of allergy to
  insect stings in children with and without venom immunotherapy. N Engl
  J Med. 2004;351:668e674.
\item
  Graft DF, Schubert KC, Kagey-Sobotka A, et al. A prospective study of
  the natural history of large local reactions after Hymenoptera stings
  in children. J Pediatr. 1984;104:664e668.
\item
  Ochfeld EN, Greenberger PA. Stinging insect allergy and venom
  immunotherapy. Allergy Asthma Proc. 2019;40(6):372-375.
  doi:10.2500/aap.2019.40.4250
\item
  Sturm GJ, Kranzelbinder B, Schuster C, Sturm EM, Bokanovic D, Vollmann
  J, et al. Sensitization to Hymenoptera venoms is common, but systemic
  sting reactions are rare. J Allergy Clin Immunol.
  2014;133(6):1635--43.
\item
  Warrington R. Lack of Correlation between Severity of Clinical
  Symptoms, Skin Test Reactivity, and Radioallergosorbent Test Results
  in Venom Allergic Patients. Allergy Asthma Clin Immunol.
  2006;2(2):62--7.
\item
  Sturm GJ, Heinemann A, Schuster C, Wiednig M, Groselj-Strele A, Sturm
  EM, et al. Influence of total IgE levels on the severity of sting
  reactions in Hymenoptera venom allergy. Allergy. 2007;62(8):884--9.
\item
  Urbanek R, Kemeny DM, Richards D. Sub-class of IgG anti-bee venom
  antibody produced during bee venom immunotherapy and its relationship
  to long-term protection from bee stings and following termination of
  venom immunotherapy. Clin Allergy. 1986;16:317-322.
\item
  Rueff F, Wolf H, Schnitker J, Ring J, Przybilla B. Specific
  immunotherapy in honeybee venom allergy: a comparative study using
  aqueous and aluminium hydroxide adsorbed preparations. Allergy.
  2004;59:589-595.
\item
  van Halteren HK, van der Linden PW, Burgers JA, Bartelink AK.
  Discontinuation of yellow jacket venom immunotherapy: follow-up of 75
  patients by means of deliberate sting challenge. J Allergy Clin
  Immunol. 1997;100:767-770.
\item
  Ewan PW, Deighton J, Wilson AB, Lachmann PJ. Venom-specific IgG
  antibodies in bee and wasp allergy: lack of correlation with
  protection from stings. Clin Exp Allergy. 1993;23:647-660.
\item
  Golden DB, Lawrence ID, Hamilton RH, Kagey-Sobotka A, Valentine MD,
  Lichtenstein LM. Clinical correlation of the venom-specific IgG
  antibody level during maintenance venom immunotherapy. J Allergy Clin
  Immunol. 1992;90:386-393.
\item
  Wilson AB, Deighton J, Lachmann PJ, Ewan PW. A comparative study of
  IgG subclass antibodies in patients allergic to wasp or bee venom.
  Allergy. 1994;49:272-280.
\item
  Eich-Wanger C, Muller UR. Bee sting allergy in beekeepers. Clin Exp
  Allergy. 1998;28:1292-1298.
\item
  Chliva C, Aggelides X, Makris M, Katoulis A, Rigopoulos D, Tiligada E.
  Comparable profiles of serum histamine and IgG4 levels in allergic
  beekeepers. Allergy. 2015;70:457-460.
\item
  Hayashi Y, Hirata H, Watanabe M, et al. Usefulness of specific-IgG4 to
  Hymenoptera venom in the natural history of Hymenoptera stings. J
  Investig Allergol Clin Immunol. 2014;24:192-194.
\item
  W\textbackslash{}eurourtzen PA, Lund G, Lund K, Arvidsson M, Rak S,
  Ipsen H. A double-blind placebo-controlled birch allergy vaccination
  study II: correlation between inhibition of IgE binding, histamine
  release and facilitated allergen presentation. Clin Exp Allergy
  2008;38:1290-301.
\item
  Ejrnaes AM, Svenson M, Lund G, Larsen JN, Jacobi H. Inhibition of rBet
  v 1-induced basophil histamine release with specific
  immunotherapy-induced serum immunoglobulin G: no evidence that
  FcgammaRIIB signaling is important. Clin Exp Allergy 2006;36:273-82.
\item
  Kucuksezer UC, Ozdemir C, Akdis M, Akdis CA. Mechanisms of immune
  tolerance to allergens in children. Korean Journal of pediatrics.
  2013;56(12):505-513.
\item
  Przybilla B, Ruëff F. Hymenoptera venom allergy. J Dtsch Dermatol Ges.
  2010 Feb;8(2):114-27; quiz 128-30. doi:
  10.1111/j.1610-0387.2009.07125.x. Epub 2009 September 14.
\item
  Bonadonna P, Perbellini O, Passalacqua G, et al. Clonal mast cell
  disorders in patients with systemic reactions to Hymenoptera stings
  and increased serum tryptase levels. J Allergy Clin Immunol.
  2009;123:680-686.
\item
  Blum S, Gunzinger A, Muller UR, Helbling A. Influence of total and
  specific IgE, serum tryptase, and age on severity of allergic
  reactions to Hymenoptera stings. Allergy 2011;66:222-228
\item
  Bilò MB, Antonicelli L, Bonifazi F. Purified vs. non-purified venom
  immunotherapy. Curr Opin Allergy Clin Immunol 2010;(10):330-336.
  doi:10.1097/ACI.0b013e328339f2d1.
\item
  Bilò MB, Severino M, Cilia M, Pio A, Casino G, Ferrarini E, et al. The
  VISYT trial: Venom Immunotherapy Safety and Tolerability with purified
  vs non-purified extracts. Ann Allergy Asthma Immunol 2009;
  (103):57-61. doi:10.1016/S1081-1206(10)60144-5.
\item
  Bilò MB, Cinti B, Brianzoni MF, Braschi MC, Bonifazi M, Antonicelli L.
  Honeybee venom immunotherapy: a comparative study using purified and
  non-purified aqueous extracts in patients with normal Basal serum
  tryptase concentrations. J Allergy (Cairo) 2012; (2012):869243.
  doi:10.1155/2012/869243.
\item
  Cox L, Larenas-Linnemann D, Lockey RF, Passalacqua G. Speaking the
  same language: The World Allergy Organization Subcutaneous
  Immunotherapy Systemic Reaction Grading System. The Journal of allergy
  and clinical immunology. 2010;125(3):569-74, 74.e1-74.e7.
\item
  Mosbech H, Muller U. Side-effects of insect venom immunotherapy:
  results from an EAACI multicenter study. European Academy of
  Allergology and Clinical Immunology. Allergy, 2000; 55:1005--1010.
\item
  Wenzel J, Meissner-Kraemer M, Bauer R, et al. Safety of rush insect
  venom immunotherapy. The results of a retrospective study in 178
  patients. Allergy 2003; 58:1176--1179.
\item
  Rueff F, Przybilla B, Muller U, Mosbech H. The sting challenge test in
  Hymenoptera venom allergy. Allergy 1996; 51:216--225.
\item
  Haeberli G, Bronnimann M, Hunziker T, Muller U. Elevated basal serum
  tryptase and hymenoptera venom allergy: relation to severity of sting
  reactions and to safety and efficacy of venom immunotherapy. Clin Exp
  Allergy 2003;33:1216--1220.
\item
  Rueff F, Wolf H, Schnitker J, et al. Specific immunotherapy in
  honeybee venom allergy: A comparative study using aqueous and
  aluminium hydroxide adsorbed preparations. Allergy 2004; 59:589--595.
\item
  Golden DBK, Kagey-Sobotka A, Valentine MD, Lichtenstein LM. Dose
  dependence of Hymenoptera venom immunotherapy. J Allergy Clin Immunol
  1981; 67:370--374.
\item
  Urbanek R, Forster J, Kuhn W, Ziupa J. Discontinuation of bee venom
  immunotherapy in children and adolescents. J Pediatr 1985;
  107:367--371.
\item
  Golden DBK, Johnson K, Addison BI, Valentine MD, KageySobotka A,
  Lichtenstein LM. Clinical and immunologic observations in patients who
  stop venom immunotherapy. J Allergy Clin Immunol 1986;77:435-42.
\item
  Golden DB, Demain J, Freeman T, et al. Stinging insect
  hypersensitivity: A practice parameter update 2016. Ann Allergy Asthma
  Immunol. 2017;118(1):28-54. doi:10.1016/j.anai.2016.10.031.
\item
  Forester JP, Johnson TL, Arora R, Quinn JM. Systemic reaction rates to
  field stings among imported fire ant-sensitive patients receiving
  \textgreater{}3 years of immunotherapy versus \textless{}3 years of
  immunotherapy. Allergy Asthma Proc. 2007;28(4):485-488.
  doi:10.2500/aap.2007.28.3021.
\end{enumerate}

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