Discussion
This multinational study indicates that, based on fish PV- and
extract-sIgE detection for 263 fish-allergic patients, up to 90% may
tolerate cartilaginous fish. In addition, up to 21% of fish-allergic
patients may tolerate some bony fish species. Multiplex in vitroIgE quantification, using purified allergens and extracts from various
fish species, may be utilized to identify patient candidates and fish
species for FCs, with the ultimate goal to confirm the clinical
tolerance of some species.
Fish allergens from different species share limited similarities. While
some IgE epitopes of parvalbumins are highly conserved, species-specific
epitopes have also been identified.18,19 Additionally,
not all species have equal allergenic potential.20 We
previously showed the low reactivity to cartilaginous fish among
bony-fish allergic patients due to the evolutionary distance between
beta- and alpha-PVs from bony and cartilaginous fish,
respectively.4 In addition, a significant proportion
of fish-allergic patients may tolerate certain bony
fish.5 Besides PVs, other fish allergens have been
identified (e.g. tropomyosin, lactate dehydrogenase, glucose-6-phosphate
isomerase, creatine kinase) but their cross-reactivity potential is
unknown.21,22
In addition to species and allergen diversity, eating habits differ
across the world. Fish is subject to different cooking methods, which
may impact allergen stability and IgE reactivity.23,24Beta-PVs demonstrated higher heat stability than enolase and
aldolase.3 The variety of species, allergens and
processing methods present a challenge for diagnosis. Certain
region-specific important species may be absent from common diagnostic
tests, and routine approaches often focus on limited number of
species.25 High-risk patients with fish allergy are
recommended strict avoidance of all fish, which is often
unnecessary.25 Next-generation diagnostic approaches
are hence necessary and multiplex molecular allergy diagnosis emerges as
a promising tool.26,27
A research version of the ALEX2 multiplex assay (Macro
Array Diagnostics), with values of sIgE strongly correlated to those
obtained by ImmunoCAP-ISAC (Thermo Fischer)28 was used
here to quantify total, fish extract- and PV-sIgE in fish-allergic
patients’ sera. The ALEX2 platform was previously
successfully utilized in quantifying serum IgE of patients with atopic
dermatitis, dust mite allergy and nut allergy.29-31 We
first investigated the IgE levels to PVs from 10 fish species. The
species were selected based on published phylogenetic analyses, covering
species relevant for consumption, and those responsible for
monosensitizations.3,32 The median sIgE values were
the highest for tuna and mackerel PVs (>10 kU/L), followed
by herring, carp, salmon and swordfish (> 8kU/L), ocean
perch (6 kU/L), cod (5 kU/L), sole (2.5 kU/L) and ray (<0.3
kU/L) (Figure 1A ). High IgE levels to tuna and swordfish PVs
were unexpected, considering previous reports on their lower
allergenicity due to low PV abundance in their dark
muscles.33,34 Our data indicate the presence of shared
IgE epitopes with other beta-PVs. However, the clinical relevance of
these findings, and their clinical cross-reactivity require future
investigations. Significantly lower IgE for cod PV than for several
other beta PVs demonstrated that cod PV, a commonly used diagnostic
marker, may not be sufficient for accurate fish allergy diagnosis. In
this study, 22% of the patients were sensitized to ray PV
(Figure 1B ), showing that tolerance of cartilaginous fish may
be a possibility for many but not all bony-fish sensitized individuals.
Fish consumption differs across geographic regions in terms of quantity
and processing.8 According to the Food and Agriculture
Organization, China was the top fish consumer in 2017 worldwide based on
total supply. Among the countries from our study, the highest per capita
fish consumption was estimated for Norway (51.4 kg) followed by Spain
(42.5 kg) and China (38.8 kg).35 IgE to PVs from
different species may reflect exposure and indicate the species most
relevant for diagnosis. Interestingly, we observed the highest IgE to
all PVs for cohorts from Norway, China and Spain, and each tested
country displayed weaker IgE to ray and sole PVs than to others
(Figure 1D ). However, IgE levels to most commonly used PVs in
diagnostics differed depending on the country. Cod PV-sIgE was overall
low and the highest for patients from Spain and Norway, while tuna
PV-IgE was high (in comparison to other PVs) in all cohorts. Currently
we cannot reach conclusions about the clinical relevance of IgE binding
to tuna PV.34 Nevertheless, tuna PV appears as highly
cross-reactive protein to other beta PVs and its potential use in
diagnostic assays should be investigated in the future. Although the
country-specific distribution of IgE to different PVs may in part be a
result not only from IgE specific to single PVs but also cross-reactive
IgE, our data indicate important PVs for fish-allergy diagnosis for each
country.
The diversity of PV-sIgE levels was analyzed next (Figure 2 ).
Shannon’s diversity did not depend on patient’s symptom severity, age or
gender, suggesting that IgE profiles to different PVs are largely
independent of these factors, and each patient should be subjected to a
wide-range diagnosis with multiple fish species.
In our study, up to 41% of patients were negative to some of the bony
fish PVs and up to 97% to ray PV (Figure 3A ). Previous studies
indicated that up to 30% of patients may tolerate some fish
species.3 This variable reactivity to different PVs
emphasized the importance of including allergens from diverse fish
families into diagnostic approaches. Furthermore, based on probability
calculations (Figure 3B ), we demonstrated that tuna PV may be
utilized to predict whether patients may be negative to other PVs when
negative to this protein.
Additional allergens such as enolase, aldolase or collagen may be
important for some patients. Our previous study on 101 fish-allergic
patients showed sensitization to fish collagen by
21%.36 Another study reported the varying prevalence
of sensitization to enolase and aldolase depending on fish species,
being as high as 70% for cod enolase.5 Some allergens
lost their IgE-binding capacity upon heat treatment.37In contrast, HEs may contain gelatin, still able to bind
IgE.38,39 In this study, lower IgE levels to extracts
were observed compared to PVs (Figure 4 ), indicating that for
patients with predominant sensitization to PV, the dilution factor of
the parvalbumin in whole extracts coated on allergen-detection systems
may yield a negative test result. In contrast, to determine the
sensitization to other known and yet unknown allergens, whole extracts
may be required. This was emphasized by negative result for PVs but
positive results for extracts for some patients. Additionally, for most
of the species, correlation between IgE levels to PVs and extracts was
weak, indicating that all three components (PV, RE and HE) are required
for diagnosis.
Finally, we investigated patients with no IgE to PV, RE and HE from the
same fish, as potential candidates for the tolerance-confirming FCs. As
many as 90% of the patients were detected as candidates for future FCs
with ray and up to 21% with specific bony fish species (Figure
5A ). Negativity to ray was confirmed for 6 of 8 tested Chinese patients
by PPT. For the PPT, the Dipturus innominatus ray, available in
China, was used. Although Dipturus innominatus and Raja
clavata (used in the ALEX2 assay) belong to the same
fish family (Rajidae)40 and high IgE cross-reactivity
is expected, the possibility of unshared epitopes on some allergens
cannot be excluded, possibly explaining the 2 positive PPT outcomes.
Larger studies are required to confirm negative in vitro IgE data
using functional assays or FC, and to calculate the predictive values of
the multiplex in vitro diagnosis. When IgE quantification for
many species cannot be performed, our calculations of probability to be
negative to some fish if known to be negative to another (Figure
5B ) may be used as a guideline for selecting the panel for in
vitro testing. A recently published prospective clinical study showed
development of fish tolerance with age.41 Our data may
therefore also be used in the context of determining the species for
testing tolerance development over time. A limitation of our study is
the absence of FCs for tolerance confirmations. Schulkes et al.
indicated a frequent serological, but limited clinical cross-reactivity
between fish species.42 Although a further
investigation of the clinical significance of our data is required, we
provide a clear direction for future studies which should carefully
choose the species for specific patients to explore tolerance.
Together, the presented data demonstrate the need for fish extracts in
both raw and heated form, and the PVs from several evolutionary distant
species for next-generation fish allergy diagnosis, which will enable
identification of potentially safe-to-consume species for individual
patients. Ultimately, combining the knowledge about the important
species and allergens with novel bioinformatic approaches will permit
the design of region-specific diagnostic arrays, which will
significantly improve safety and wellbeing of fish-allergic individuals.