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\begin{document}

\title{Mollusk allergy in shrimp-allergic patients: still a complex diagnosis.
An Italian multicenter study}



\author[1]{Enrico Scala}%
\author[2]{Aruanno A}%
\author[3]{Boni Elisa}%
\author[4]{Ignazio Brusca}%
\author[5]{Cappiello Francesca}%
\author[1]{Caprini Elisabetta}%
\author[6]{Francesca Buzzulini}%
\author[3]{Gaia Deleonardi}%
\author[7]{Demonte A}%
\author[8]{Laura Farioli}%
\author[9]{Fabio Lodi-Rizzini}%
\author[8]{Laura Losappio}%
\author[10]{Donatella Macchia}%
\author[11]{Giuseppina Manzotti}%
\author[1]{Meneguzzi Giorgia}%
\author[7]{Montagni Marcello}%
\author[2]{Eleonora Nucera}%
\author[4]{Onida Rosa}%
\author[8]{Elide Anna Pastorello}%
\author[7]{silvia peveri}%
\author[10]{Anna Radice}%
\author[8]{Federica Rivolta}%
\author[2]{Angela Rizzi}%
\author[1]{Mauro Giani}%
\author[12]{Lorenzo Cecchi}%
\author[13]{Pinter E}%
\author[14]{Miglionico Marzia}%
\author[15]{Vantaggio L}%
\author[16]{Pravettoni V}%
\author[6]{Danilo Villalta}%
\author[17]{Riccardo Asero}%
\affil[1]{Istituto Dermopatico dell'Immacolata Istituto di Ricovero e Cura a Carattere Scientifico}%
\affil[2]{Fondazione Policlinico Universitario Agostino Gemelli IRCCS}%
\affil[3]{Azienda Unita Sanitaria Locale di Bologna}%
\affil[4]{Ospedale Buccheri La Ferla Fatebenefratelli}%
\affil[5]{Aziende Socio Sanitarie Territoriali di Lecco}%
\affil[6]{Ospedale Santa Maria degli Angeli di Pordenone}%
\affil[7]{Ospedale Guglielmo da Saliceto}%
\affil[8]{ASST Grande Ospedale Metropolitano Niguarda Dipartimento Funzionale Niguarda Cancer Center}%
\affil[9]{Azienda Ospedaliera Spedali Civili di Brescia Dipartimento di Salute Mentale}%
\affil[10]{Ospedale San Giovanni di Dio}%
\affil[11]{Casa Di Riposo Istituto Palazzolo}%
\affil[12]{Azienda USL Toscana centro Sedi di Prato}%
\affil[13]{Universita degli Studi di Roma La Sapienza Dipartimento di Medicina Molecolare}%
\affil[14]{Universita degli Studi di Roma La Sapienza}%
\affil[15]{Universita degli Studi di Roma La Sapienza Dipartimento di Scienze Medico-Chirurgiche e Medicina Translazionale}%
\affil[16]{Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico}%
\affil[17]{Clinica San Carlo}%


\vspace{-1em}



  
  \date{May 17, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
\textbf{Introduction \textbar{}}Shellfish allergy is an important cause
of food allergies worldwide. Both \emph{in vivo} and \emph{in vitro}
diagnostics failure nowadays is caused by the poor quality of the
extracts associated with the scarce availability of allergenic molecules
in the market. It is known that not all patients with shellfish
allergies experience adverse reactions to mollusks. It is still unclear
how to detect and diagnose correctly these patients. \textbf{Aim
\textbar{}}To investigate the features of shrimp-allergic patients
either reactive or tolerant to mollusks, with the currently available
diagnostic methods. \textbf{Methods\textbar{}} Nineteen centers,
scattered throughout Italy, participated in the study, enrolling
patients allergic to shrimp with or without associated reactions to
mollusks. Patients underwent skin tests using commercial extracts or
fresh raw and cooked foods, and IgE reactivity to currently available
allergenic extracts and molecules was measured \emph{in vitro}.
\textbf{Results\textbar{}} Two hundred and forty-seven individuals with
a history of adverse reaction to crustaceans participated in the study.
Only 47.8\% of them reacted after cephalopod or bivalve ingestion. None
of the tests used, either \emph{in vivo} or \emph{in vitro}, was able to
detect all selected patients. Accordingly, a great heterogeneity of
results was observed with an agreement between \emph{in vivo} and
\emph{in vitro} tests ranging between 52\% and 62\% of cases. Skin tests
were able to identify the cephalopod and bivalve reactors (p
\textless{}0.001), also using fresh cooked or raw food (p
\textless{}0.001). The reactivity profile of mollusk reactors was
dominated by Pen m 1, over Pen m 2 and Pen m 4 compared to the tolerant
subjects, but 33\% of patients allergic to shellfish were not detected
by any of the available molecules. A higher frequency of shrimp
hypersensitivity was recorded in northern Italy, while mollusk
reactivity was more frequent in the center-south. \textbf{Conclusion
\textbar{}}The current diagnostic methods are inadequate to predict the
cross-reactivity between crustaceans and mollusks. The detection of
mollusks hypersensitivity must still rely on skin tests with fresh
material. There is no need to exclude mollusks from shrimp allergic
patients' diet unless clinical history, the available diagnostic
instruments, and/or tolerance tests support such a decision. Primary
sensitization to mollusks seems possible.%
\end{abstract}%



\sloppy


\textbf{Mollusk allergy in shrimp-allergic patients: still a complex
diagnosis.}

\textbf{An Italian multicenter study}

Enrico, Scala MD (1); Arianna, Aruanno MD (2); Elisa, Boni MD (3);
Ignazio, Brusca MD (4); Francesca, Cappiello MD (5); Elisabetta,
Caprini, BD (1); Francesca, Buzzulini MD (6); Gaia, Deleonardi MD (7);
Antongiulio, Demonte MD (8); Laura, Farioli MD (9); Fabio, Lodi Rizzini
MD (10); Laura Michelina, Losappio MD (9); Donatella, Macchia MD (11);
Giuseppina, Manzotti MD (12); Giorgia, Meneguzzi MD (1); Marcello,
Montagni MD (8); Eleonora, Nucera MD (2); Rosa, Onida MD (4); Elide
Anna, Pastorello MD (9); Silvia, Peveri MD (8); Anna, Radice MD (11);
Federica, Rivolta MD (13); Angela, Rizzi MD (2); Mauro, Giani MD (1);
Lorenzo, Cecchi MD (14), Elena, Pinter MD (15), Marzia, Miglionico MD
(16), Lorenzo, Vantaggio MD (17), Valerio, Pravettoni MD (13); Danilo,
Villalta MD (6); Riccardo, Asero MD (18)

1 \textbar{} Clinical and Laboratory Molecular Allergy unit - IDI-IRCCS
\textbar{} FLMM - Roma, Italia

2 \textbar{} Fondazione Policlinico Universitario A. Gemelli IRCCS,
Universit\selectlanguage{ngerman}à Cattolica del Sacro Cuore -- ROMA, Italia

3 \textbar{} Allergologia e autoimmunità LUM AUSL Bologna

4 \textbar{} Clinical Pathology U.O.C., Buccheri La Ferla F.B.F.
Hospital, Palermo, Italy

5 \textbar{} Allergy unit ASST Lecco and Merate, Italy

6 \textbar{} SSD di Immunologia e Allergologia, PO S. Maria degli
Angeli, Pordenone, Italia

7 \textbar{} Allergologia e autoimmunità LUM AUSL Bologna

8 \textbar{} UOsD Allergologia, ospedale G. Da Saliceto, Piacenza,
Italia

9 \textbar{} Dipartimento di Allergologia, ASST Grande Ospedale
Metropolitano Niguarda, Milano, Italia

10 \textbar{} SSVD Allergologia Spedali Civili - Università Studi
Brescia, Brescia, Italia

11 \textbar{} SOS Allergologia e Immunologia Clinica, Ospedale San
Giovanni di Dio, Firenze, Italia

12 \textbar{} Servizio di Allergologia, Casa di Cura Beato Palazzolo,
Bergamo, Italia

13 \textbar{} Immunology and Allergy Department, IRCCS Foundation Ca'
Granda Ospedale Maggiore Policlinico, Milan, Italy

14 \textbar{} SOS Allergology and Clinical Immunology, USL Toscana
Centro, Prato, Italy

15 \textbar{} UOC Medicina interna e Immunologia Clinica, Department of
Molecular Medicine, Sapienza University of Rome, Rome, Italy

16 \textbar{} Department of Molecular Medicine, Sapienza University of
Rome, Rome, Italy

17 \textbar{} Department of Translational and Precision Medicine,
Sapienza University of Rome, Italy

18 \textbar{} Ambulatorio di allergologia, Clinica San Carlo, Paderno
Dugnano (MI), Italy

\textbf{Corresponding Author}

Enrico SCALA

Clinical and Laboratory MolecularAllergy Unit- IDI- IRCCS, Rome, Italy

via Monti di Creta 104, 00138 - Rome, Italy

Phone +39 348 7998657

email: \emph{e.scala@idi.it}

\textbf{1 \textbar{}} \textbf{Abstract}

\textbf{Introduction \textbar{}} Shellfish allergy is an important cause
of food allergies worldwide. Both \emph{in vivo} and \emph{in
vitro}diagnostics failure nowadays is caused by the poor quality of the
extracts associated with the scarce availability of allergenic molecules
in the market. It is known that not all patients with shellfish
allergies experience adverse reactions to mollusks. It is still unclear
how to detect and diagnose correctly these patients.

\textbf{Aim \textbar{}} To investigate the features of shrimp-allergic
patients either reactive or tolerant to mollusks, with the currently
available diagnostic methods.

\textbf{Methods\textbar{}} Nineteen centers, scattered throughout Italy,
participated in the study, enrolling patients allergic to shrimp with or
without associated reactions to mollusks. Patients underwent skin tests
using commercial extracts or fresh raw and cooked foods, and IgE
reactivity to currently available allergenic extracts and molecules was
measured \emph{in vitro} .

\textbf{Results\textbar{}} Two hundred and forty-seven individuals with
a history of adverse reaction to crustaceans participated in the study.
Only 47.8\% of them reacted after cephalopod or bivalve ingestion. None
of the tests used, either \emph{in vivo} or \emph{in vitro} , was able
to detect all selected patients. Accordingly, a great heterogeneity of
results was observed with an agreement between \emph{in vivo}
and\emph{in vitro} tests ranging between 52\% and 62\% of cases. Skin
tests were able to identify the cephalopod and bivalve reactors (p
\textless{}0.001), also using fresh cooked or raw food (p
\textless{}0.001). The reactivity profile of mollusk reactors was
dominated by Pen m 1, over Pen m 2 and Pen m 4 compared to the tolerant
subjects, but 33\% of patients allergic to shellfish were not detected
by any of the available molecules. A higher frequency of shrimp
hypersensitivity was recorded in northern Italy, while mollusk
reactivity was more frequent in the center-south.

\textbf{Conclusion \textbar{}} The current diagnostic methods are
inadequate to predict the cross-reactivity between crustaceans and
mollusks. The detection of mollusks hypersensitivity must still rely on
skin tests with fresh material. There is no need to exclude mollusks
from shrimp allergic patients' diet unless clinical history, the
available diagnostic instruments, and/or tolerance tests support such a
decision. Primary sensitization to mollusks seems possible.

\textbf{Key messages}

\begin{enumerate}
\tightlist
\item
  current diagnostic methods are inadequate to predict cross-reactivity
  between crustaceans and mollusks
\item
  the detection of mollusks hypersensitivity must still rely on skin
  tests with fresh material (and oral challenges where possible);
\item
  clinically, there is no need to exclude a priori mollusks from shrimp
  allergic patients' diet,
\item
  albeit rarely, primary sensitization to mollusks seems possible
\end{enumerate}

\textbf{Author contribution statement}

ES, EC, and GMe carried out the experiments and data collection. ES; AA;
EE; IB; FC; EC; FB; GD; AD; LF; FLR; LML; DM; GMa; GMe; MM; EN; RO; EAP;
SP; AR; FR; AR; MG; LC, VP; DV and RA recruited the patients. ES
performed the statistical analysis. ES and RA conceived the study and
assisted in data interpretation. All authors reviewed, edited and
approved the final manuscript.

\textbf{KeyWords}

Multiplex Analysis

IgE diagnosis

Tropomyosin

Crustaceans

Mollusks

Food Allergy

Urticaria/angioedema

Anaphylaxis

\textbf{2 \textbar{} INTRODUCTION}

Seafood is included among the ``Big Eight'' food groups responsible for
most cases of food allergy. It is estimated that depending on the
different geographic areas, about 3\% of the general population is
allergic to shellfish (1,2). This generic term includes more than 300
different invertebrate species which are divided into two large groups:
crustaceans (shrimps, crabs, lobsters etc.) and mollusks
(\emph{Bivalvia} , such as clam, mussels, scallops or oysters,
and\emph{Cephalopoda} , like squids, cuttlefish or octopuses
etc.)(3)(4). These taxonomic classifications are essential to predict
the structural, immunological and allergological similarities that
underlie a possible cross-reactivity. The availability and consumption
of seafood vary greatly in different parts of the world, representing
the second cause of primary food allergy in Italy, after Lipid transfer
proteins (5). Even eating habits, including the different methods of
food processing, may exert a strong impact on the incidence and severity
of allergy to crustaceans and seafood, since in some cases physical
treatments can increase or reduce IgE reactivity(6), depending on the
molecule involved in patient sensitization (7). So far, it has been
argued that due to the cross-reactivity among allergens in
invertebrates, primarily tropomyosin, the patient allergic to
crustaceans should also exclude cephalopods and Bivalvia from the diet.
However, recent studies seem to suggest that this is not the case; in
fact, a significant proportion of crustacean allergic patients report
that they usually tolerate other invertebrates (8). One of the main
diagnostic problems is that all the molecules commercially available for
\emph{in vitro} tests come from crustaceans and not from mollusks. As a
consequence, IgE diagnostic approach for mollusks adverse reaction is
always indirectly based on a presumptive but not fully demonstrated
cross-reactivity, unless tests with fresh, raw or cooked cephalopod or
Bivalvia are carried out, with all the drawbacks associated with such
approach.

The main aims of this study were (i) to investigate the prevalence of
patients allergic to crustaceans with or without cross-reactivity to
cephalopods and/or bivalve mollusks; (ii) to define the molecular
profile in both cross-reacting and non-cross-reacting individuals; (iii)
to highlight any differences in the severity of the allergic reactions,
and (iv) to investigate the geographical differences in seafood
sensitization profiles in Italy.

\textbf{3 \textbar{} PATIENTS AND METHODS}

Nineteen Italian allergy centers took part in a multicenter
cross-sectional observational clinical survey on crustacean allergy. In
every participating center, doctors consecutively enrolled the largest
possible number of patients with unequivocal crustacean (shrimp) and/or
mollusks (\emph{Bivalvia and/} or \emph{Cephalopoda} ) allergy, between
May 15th and November 15th, 2021. The patients with a history of food
allergy to shellfish confirmed by a positive skin prick test (SPT) with
commercial shrimp extract and/or with prick-prick test (PPT) with fresh
raw or cooked food were carefully interviewed about seafood, crustaceans
and/or mollusks tolerance. Diagnosis of tolerance was based on patient's
clinical history (i.e., consumption of the culprit food without
problems).

Shellfish-induced allergic reactions reported by patients included both
mild (such as oral allergy syndrome or isolated gastrointestinal
reactions) and moderate-severe reactions (i.e., urticaria/angioedema,
anaphylaxis). Given the observational nature of the study, no
randomization procedure was be implemented during enrollment. The
clinical histories were confirmed by a positive skin prick test (SPT)
with fresh food (either raw or cooked) and/or commercial extracts of
shrimp and mollusks currently available in Italy (Alk-Abello' s.p.a.,
Allergy Therapeutics Italia, Anallergo, Firma Srl, Roxall Italia,
Lofarma, and Stallergenes Italia Srl) and/or by the detection of IgE
specific for seafood extracts by ImmunoCAP (shrimp, mussel, clam,
octopus or squid), and Pen a 1 (tropomyosin) (Thermo Fisher, Uppsala,
Sweden). In a subset (98 individuals) a more in-depth evaluation was
carried out by the Allergy Explorer-ALEX2® (Macroarray Diagnostics,
Vienna) multiplex platform evaluating a broader profile of seafood
molecules (9), including Pen m 1 (tropomyosin), Pen m 2 (arginine
kinase), Pen m 3 (myosin light chain), and Pen m 4 (sarcoplasmic calcium
binding protein) all from Black-Tiger shrimp (\emph{Penaeus monodon} ),
Cra c 6 (the troponin-c from brown shrimp, \emph{Crangon crangon} ), Der
p 10 (tropomyosin), and Der p 11 (paramyosin) both
from\emph{Dermatophagoides pteronyssinus} , as well as of seafood
extracts, including crab, lobster, northern shrimp, white shrimp, squid,
mussel, oyster, clam, and scallop. IgE levels \textgreater{}0.3kUA/L
were considered positive as for manufacturer's instructions.

The diagnosis of seafood allergy was not confirmed by blinded or open
oral food challenges due both to the severity of several reactions and
the fact that many centers were not sufficiently equipped in terms of
facilities or personnel to manage possible severe allergic reactions.

\textbf{3.1 \textbar{} Statistics}

The sampled data were recorded and analyzed by SPSS (version 27.0.1.0
SPSS, Inc, Chicago, III).

In univariate analysis, the non-parametric Mann-Whitney U-test (two
groups) was first used to compare continuous IgE values in subjects with
or without a given clinical involvement; then, each variable of interest
was dichotomized (as negative or positive) to study the proportion of
subjects with symptoms in the two groups thus obtained.

Pearson's \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} test or Fisher's exact test (used for
two-by-two contingency tables with less than 50 cases) were used to
assess if paired observations on two variables expressed in a
contingency table, were independent of each other.

We performed multiple logistic regression for the clinical variables
with dichotomous scores (present, absent) to see whether the association
between clinical symptoms and different shellfish allergens reactivity
was present after simultaneously adjusting for the other variables of
interest.

The degree of relationship between the quantitative variables studied
was analyzed using Spearman Correlation (rho) test, and the most
commonly used bivariate correlation technique. A value of p \textless{}
0.05 was considered statistically significant.

\textbf{3.2 \textbar{} Ethical issues}

The study was approved by the Ethical Committee of the coordinating
centre (IDI-IRCCS CE \textbar{} 667/2021). Data collection takes place
anonymously, using only data obtained through routine specialist
surveys. Recruited patients gave informed consent to the use of their
clinical data in an anonymous form.

\textbf{4 \textbar{} RESULTS}

\textbf{4.1 \textbar{} General findings}

Two hundred forty-seven individuals (M 48.2\%, F 51.8\%; mean age 39 \selectlanguage{ngerman}±
17, range 2 - 79 years) enrolled in the 19 centers participating in the
study, represented the study group.

Forty-four per cent (n= 108) of the patients came from the
central-southern Italy , while the remaining 56\% (n= 139) of cases had
been recruited in northern Italy.

All patients selected for the study reported a clinical history
suggesting an adverse reaction to crustaceans confirmed by positive
in-vivo and/or in-vitro testing, but only 47.8\% of them (n=118)
reported adverse reactions after the ingestion of cephalopods or
bivalves; of these, 38.5\% (n=95) experienced urticaria/angioedema, and
9.3\% (n=23) anaphylaxis.

No age and sex differences were detected concerning symptom severity.

\textbf{4.2 \textbar{} \emph{In vivo} vs \emph{in vitro} diagnostic}

As previously shown(10), diagnostics with commercially available seafood
extracts did not always lead to the correct identification of sensitized
patients. None of the tests used, either \emph{in vivo} or \emph{in
vitro} , was able to detect all selected patients as positive. Indeed,
60,9\% per cent of the patients scored positive on SPT for shrimp;
78.3\% scored on skin testing with fresh food, 65,8\% with cooked food,
and 72,4\% were positive on the detection of specific IgE to shrimp by
ImmunoCAP. Thirty-nine (16.6\%) individuals were negative for skin tests
with both commercial extracts and raw or cooked shrimp but scored
positive on specific IgE assays.

As shown in Table 1, a concordance of commercial SPT and IgE assay for
crustaceans was found only in 56\% of patients; the concordance rose to
62\% between skin tests with raw or cooked fresh shrimp and the \emph{in
vitro} test. Comparing the in-vivo and in-vitro evaluations with
cephalopods (squid or octopus) or mollusks (mussels or clams), and even
higher heterogeneity of results was observed (Table 1).

No correlation was found comparing IgE levels to seafood extracts,
shrimp, cephalopods and mollusks, except for a moderate relationship
between cephalopods and mussels (rho 0.634, p \textless{}0.001)

To evaluate the prevalence of IgE reactivity to cephalopods or mollusks
in shrimp reactive subjects, our patients were extensively
studied\emph{in vitro} with both extracts and allergenic molecules,
using singleplex and multiplex methods. As shown in Table2, IgE
sensitization to shrimp was significantly associated with
hypersensitivity to cephalopods (p \textless{}0.005), but not to
mollusks on singleplex testing. Notably, specific IgE levels did not
differ in the two patient groups.

On the other hand, when shrimp immunoreactive patients were evaluated by
the multiplex system, they were also more frequently positive for crab,
lobster, northern and white shrimp, squid, mussel, and clam, but not for
oyster, and scallop (Table 2). Crustacean reactivity was accompanied by
significantly higher levels of specific IgE towards all extracts tested,
except for crab, oyster and scallop, where specific IgE levels did not
differ.

From the molecular point of view, IgE reactivity to crustaceans was
associated with a significantly higher frequency and higher levels of
specific IgE to tropomyosins (both Pen m 1 and Der p 10) and
sarcoplasmic calcium-binding protein (Pen m 4), in comparison with the
patient that scored negative for specific IgE to crustaceans (Table 2).

\textbf{4.3 \textbar{} Sensitization profiles and clinical correlations}

One hundred-eighteen (47,8\%) patients enrolled because allergic to
crustaceans, reported also moderate (80.5\%) to severe (19.5\%)
reactions after ingestion of mollusks.

There was no difference in the frequency of reactive episodes related to
sex or age.

Interestingly, a higher risk of developing a severe reaction after
mollusks intake was associated with skin test reactivity to commercial
extracts of cephalopod (OR: 4.81; CI 1.8-9.3; p \textless{}0.001) or
bivalve (OR: 14.970; CI 4.2-53.3; p \textless{}0.001). Similarly,
mollusk reactors showed a high frequency of reactivity to both raw (OR:
4.492; CI 1.9-10.4; p \textless{}0.001) and cooked squid (OR: 3.040; CI
1.3-7.3; p \textless{}0.001), and to both raw (OR: 20.907; CI 5.8-75.2;
p \textless{}0.001) and cooked clam (OR: 17.249; CI 3.1-41.1; p
\textless{}0.001).

The multiple logistic regression analysis, when simultaneously adjusted
for the presence of specific IgE to all the extracts studied, age, and
sex, showed a significant relationship between a history of adverse
reaction to mollusks and IgE reactivity to both cephalopods
(OR\textsubscript{adj} 4.0, 95\% CI 1.5-10.7, p \textless{}0,01) or
bivalve (OR\textsubscript{adj} 7.6, 95\% CI 3.1-18.6, p
\textless{}0.0001) extracts. Moreover, as shown in Table 3, allergy to
mollusks was associated with IgE reactivity to northern shrimp
(\emph{Litopaenaeus setiferus} ), squid (\emph{Loligo} spp.), mussel
(\emph{Mytilus edulis} ), oyster (\emph{Ostrea edulis} ), clam
(\emph{Ruditapes} spp.), and scallop (\emph{Pecten} spp.) extracts. Pen
m 1 (\emph{Penaeus monodon} tropomyosin) hypersensitivity was
significantly associated with an increased risk of severe reaction to
mollusks.

Three patients scoring SPT positive to crustaceans showed \emph{in
vitro} sensitization to bivalves (both oyster and mussel) in two cases
and squid in one case, with no sign of IgE reactivity against any the
extracts and molecules of crustaceans \emph{in vitro} , with only a low
reactivity in one case to paramyosin (0.24 kU/L) and tropomyosin (0.39
kU/L) from house dust mite (Der p 11 and Der p 10 respectively).

As summarized in Figure 1, the molecular sensitization profile was
dominated by Pen m 1, but more frequently in those who did not tolerate
mollusks. Moreover, mollusks reactors had also two times higher
frequency of sensitization to arginine kinase and sarcoplasmic
Ca\textsuperscript{++} binding protein (Figure 1). Interestingly, the
number of crustacean allergic patients who did not react to any of the
molecules currently available for \emph{in vitro} diagnostics was very
high, particularly among those who tolerated cephalopods and bivalves
(43\%).

\textbf{4.4 \textbar{}Geographical differences in seafood sensitization
profiles}

When we analyzed the data as a function of the geographical distribution
of patients, we found a significantly higher frequency of sensitization
to crustaceans in northern Italy, irrespective of the diagnostic tests
used (skin prick test, prick-prick test with fresh or cooked food, and
specific IgE measurement for allergen extracts), whilst in
centre-southern Italy, a more frequent sensitization to Bivalvia was
recorded (Figure 2 A-C). This observation was in keeping with a higher
frequency of reactions to bivalve in the south, whilst the sensitization
levels to cephalopod were comparable among south and north (Figure 2 D).

\textbf{5 \textbar{} Discussion}

In our multicenter study, involving 19 centers scattered throughout
Italy, we evaluated the prevalence and the sensitization profile of
allergic subjects to crustaceans reactive or not reactive also to
mollusks

We confirmed once more (11), that the diagnostics with both the extracts
and the molecules currently available in the market is inadequate to
detect satisfactorily patients hypersensitive to crustaceans and
mollusks. We observed an extreme heterogeneity of results from one
patient to another using the common diagnostic approaches, including
skin prick testing with commercial extracts, skin testing with fresh
material and specific IgE measurements. We previously showed the
unreliability of commercial extracts for SPT available on the market due
to differences in allergenic proteins concentrations (10), thus
potentially leading to confounding results.

The currently available molecular diagnostics for seafood shows two
major pitfalls. First, not all allergen molecules are present on the
diagnostic platforms and, second, practically all the molecules
available derive from crustaceans and none from mollusks. The diagnosis
of mollusks allergy is therefore always indirect, based on the
presumption of cross-reactivity with crustaceans. Of the 58 shellfish
molecules currently registered as allergens by the WHO/IUIS Allergen
Nomenclature Sub-Committee, only 8 belong to the mollusks (see Table in
the repository). If one considers, for instance, that the tropomyosins
from mollusks share no more than 60\% amino acid sequence identity with
the other allergenic tropomyosins isolated so far from crustaceans,
insects, mites, and fish (1), one can easily figure out that this might
lead to a failure in the detection of allergic patients (12). A 2018
study showed that in the pacific oyster extract, along with many
specific allergens of invertebrates, fish, and mites, can be isolated
allergens from other different biological sources such as pollen and
fungi, thus prompting interesting scenarios about possible unexpected
sources of sensitization(13).

Another interesting point of our study concerns the differences in
sensitization profiles observed in the different geographic areas of the
country. We detected that in the north sensitization to crustaceans was
much more frequent than in the center/south, where in turn sensitization
to cephalopods and bivalves was more prevalent. These differences might
be the result of different culinary habits between the North (where the
way of life is more similar to that in Central and Northern Europe) and
the South, where habits are similar to those in other Mediterranean
countries such as Greece or Spain(8). These geographical differences
could also underlie different sensitization mechanisms according to
different environmental exposures (11). This point needs to be addressed
in future studies.

The finding of three single patients who were mono-reactors to bivalves
(both oyster and mussel), suggests that primary sensitization to
mollusks is possible, albeit being an extremely rare event, sometimes
associated with sensitization also to house dust mites (14). A study
designed to verify whether reactivity is always secondary to
sensitization to mite allergens should be carried out, focusing
attention on a pediatric-only population.

Objective limitations of the study are the absence of oral food
challenge in patient selection, which was made only on the anamnestic
data, and the use of heterogeneous commercial preparations, both
for\emph{in vivo} and \emph{in vitro} diagnostics, where it is not
always declared. by the producers, the type of crustacean, prawn or
shrimp used to prepare the extract.

In conclusion, this study clearly shows the following: (i) current
diagnostic methods are inadequate to predict cross-reactivity between
crustaceans and mollusks due to the lack of specific mollusks allergens
and because these are only partially cross-reactive to crustacean
homologue proteins; (ii) the detection of mollusks hypersensitivity must
still rely on skin tests with fresh material (and oral challenges where
possible); (iii) clinically, there is no need to exclude a priori
mollusks from shrimp allergic patients' diet, unless the available
diagnostic instruments support such decision; (iv) albeit rarely,
primary sensitization to mollusks seems possible although the incomplete
spectrum of shrimp allergens does not allow us to exclude that shrimp
acts as primary sensitizer also in those few cases.

\textbf{Data Avaiability}

The data that support the findings of this study are available on
request from the corresponding author. The data are not publicly
available due to privacy or ethical restrictions.

\textbf{Conflict of interest}

ES has received consultant arrangements and speakers' bureau
participation from Stallergenes, Thermo Fisher Scientific, and
non-financial support from Microarray Diagnostics, Vienna, all outside
the submitted work. LC has received honoraria from Malesci, Menarini,
Mylan and Thermo Fisher Scientific. DV and RA received honoraria from
Thermo Fisher Scientific.

\textbf{Funding information}

This study was funded, in part, by the Italian Ministry of Health,
Current Research Program 2021-2023.

\textbf{Data Availability Statement information}

The data that support the findings of this study are available on
request from the corresponding author. The data are not publicly
available due to privacy or ethical restrictions.

\textbf{ETHICAL APPROVAL STATEMENT}

The research was conducted ethically following the World Medical
Association Declaration of Helsinki. All subjects have given their
written informed consent and that the study protocol was approved by the
institute's committee on human research. (CE: 667 \textbar{} 2021)

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/328414/articles/713685-mollusk-allergy-in-shrimp-allergic-patients-still-a-complex-diagnosis-an-italian-multicenter-study}

\textbf{Table 1.} Concordance among commercial SPT and IgE assay for
Crustaceans and Mollusks

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/328414/articles/713685-mollusk-allergy-in-shrimp-allergic-patients-still-a-complex-diagnosis-an-italian-multicenter-study}

\textbf{Table 2} . Mollusks extract and molecules IgE sensitization
frequency and levels as per multiplex and singleplex detection in
individuals with or without IgE reactivity to crustaceans. ns = not
significant

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/328414/articles/713685-mollusk-allergy-in-shrimp-allergic-patients-still-a-complex-diagnosis-an-italian-multicenter-study}

\textbf{Table 3} . Extract and molecules IgE sensitization frequency in
individuals tolerating or not tolerating mollusks. ns = not significant\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 1} Molecular sensitization profile in patients allergic
to crustaceans that were tolerant or reactors to cephalopods and/or
mollusks. Null refers to the percentage of patients not reactive to any
molecule tested.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 2.} Skin prick test (A), prick-prick test with fresh or
cooked food (C), and specific IgE measurement for allergen extracts(B)
in northern and central-southern Italy. Sensitization differences are
shown in D.

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