Discussion
Fish allergy is common, often life-long, and has a major impact on the
pediatric population and their families. Fish allergy has long been an
umbrella term, while clinical management of fish allergy is particularly
complex due to the huge diversity of edible fish but extensive
cross-reactivity, and the presence of multiple fish allergens with
sensitization dependent on culture, dietary habits and cooking methods.
Precision diagnosis for specific fish allergy and tolerance has major
implications for proper patient labeling, reducing unnecessary food
avoidance and reintroducing fish into patients’ diets. Our study is the
first to present a fish allergenicity ladder based on both clinical and
molecular data, and validated by oral food challenges. We also, for the
first time, identified IgE-binding epitopes of parvalbumins from salmon,
cod, grouper, and grass carp with patients diagnosed by DBPCFC for
predicting clinical cross-reactivity and fish-specific allergy.
The fish allergenicity ladder presented in this study is robustly
constructed based on both the sIgE levels of a large cohort of 166
physician-diagnosed fish-allergic and sensitized subjects against nine
fishes, as well as the patients’ clinical reactivity against a vast
panel of marine and freshwater fishes. Grass carp and salmon were the
two most common fishes first introduced as solid foods (in congee) in
our traditional Chinese diet, and the difference in the percentage of
reported tolerance was obvious (40.2% for salmon and 7.5% for grass
carp). Likely subsequent to the allergic episode to these fishes
followed by a positive SPT and/or sIgE test during clinical check-ups, a
majority of our fish-sensitized subjects avoided (i.e. naïve) other
fishes like tuna, halibut, cod, grouper, catfish and tilapia that were
usually introduced later in our pediatric cohort. Interestingly, the
pattern of allergy and tolerance to these fishes was still clear and
strongly correlated with the sIgE levels to the respective fishes.
Similar tolerance pattern was also reported in Singaporean fish allergic
subjects, by which 75.9% fish allergic children tolerated some fish
species with salmon (37.0%), tuna (24.1%) and cod (22.2%) being the
leading tolerated fishes 26 . Our oral food
challenge results from 58 fish-sensitized subjects also conformed well
with our analysis on patients’ self-reported history. Apart from a much
higher incidence of grass carp than salmon allergy and the high
proportion of partial tolerance to salmon in grass carp allergic
patients (60.4%), ED10 to grass carp was also almost 35-fold lower
comparing to that of salmon. Similar DBPCFC results were also reported,
by which 6% of subjects reacted to salmon only comparing to 20% who
reacted to cod only, and 11% reacted to both cod and mackerel comparing
to none reacted to salmon and mackerel 27 . In
subjects with fish allergy, differences in the specificity of
allergenicity is a critical consideration in the process of fish
reintroduction.
Our findings on the high parvalbumin to extract ratio and strong
correlation between sIgE to fish extract and parvalbumins suggested that
parvalbumin is the major allergen in our cohort as in most studied
populations 7,28 . We showed that the relative
level of parvalbumin increases along the allergenicity ladder
constructed based on sIgE levels and clinical history. For instance,
parvalbumin content increases from tuna, halibut, salmon, cod, grouper,
catfish, grass carp and tilapia as validated by both proteomics and
transcriptomic analysis. Such findings agreed with previous reports that
large migratory fishes with more dark muscle have lesser parvalbumins
for continuous swimming, while sedentary fishes have more white muscle
and parvalbumin for short burst swimming 29 .
Parvalbumin content was as low as <1mg/g in salmon and tuna
while the difference can be 48 times comparing between splendid
alfonsino and bigeye tuna. Our study here describes beyond such
difference and further showed that fish parvalbumins present the same
molecular allergenicity by testing eight recombinant parvalbumins from
tuna, salmon, cod, grouper, and the freshwater fishes with 37 allergic
samples. Our findings also demonstrated the strong and positive
correlation of parvalbumin content with sIgE reactivity and also
incident of self-reported history in allergic patients, which emphasize
the clinical attribution of parvalbumin content to differential
sensitivity to fishes. A case study of fish oral immunotherapy (OIT)
with stepwise increase of parvalbumin from 1mg to 66mg over two years
led to reduced parvalbumin-specific IgE and elevated level of IgG430 . While “conventional” fish OIT ingesting
the same fish over years can put patient at risk of heavy metal toxicity
and develop food anxiety, gradually introducing a wider spectrum of
fishes along the allergenicity ladder can be an alternative form of fish
OIT. Regular administration of food allergen along the ladder is likely
to attain similar immune changes in OIT that assist to expand patients’
diet and establish tolerance. For instance, milk and egg ladders that
were originally outlined for managing non-IgE-mediated food allergy have
been extended to the management of IgE-mediated allergies31,32 . A handful of studies have now been
published illustrating the safety and efficacy of milk and egg ladders
that participants could tolerant more allergenic foods just after a year33-39 . Even consuming baked goods regularly in
step 1 of the food ladder also promoted tolerance40,41 . There is increasing recognition that
children with fish allergy can tolerate some fish species while lessons
from egg and milk allergy inform the possible resolution of food allergy
through a step-wise progression from extensively heated (low
allergenicity) to less heated (high allergenicity) foods.
Fish ladder can thus embark on fish allergy management through
facilitating dietary expansion and encouraging the resolution of fish
allergy.
Based on such fish allergenicity ladder, we further extended our
analysis for IgE-binding epitopes with the specific emphasis to predict
clinical cross-reactivity and fish-specific allergy/tolerance. To our
knowledge, this is the first study to identify IgE-binding epitopes of
parvalbumins from four different fish species of differential
allergenicity at each step (salmon, cod, grouper, and grass carp) with
sizable samples from fish allergic (n=11), partial fish tolerant (n=12)
and complete fish tolerant (n=5) subjects diagnosed by DBPCFC, the gold
standard of food allergy diagnosis. We identified Epi_c_64-78 from
grouper parvalbumin as major cross-reactive IgE-binding epitopes to
identify “general” fish allergy. This epitope strongly and
specifically reacted with grass carp and grouper allergic subjects but
none of the tolerant patients, and overlaps with the previously
described regions in salmon 42 . This epitope
sequence shared an average sequence homology of 61.2% with other tested
parvalbumins, and 69.7% similarity with the homologous sequence in
grass carp parvalbumin. We also, for the first time, identified an
IgE-binding region at Sal_s_β1_19-33 that stand as a novel biomarker
to differentiate salmon tolerance among subjects allergic to grass carp
and/or grouper. We also importantly showed that such prediction could
not be achieved by fish- or parvalbumin-specific IgE levels of these
individuals but only their sIgE reactivity to this epitope region.
Predicting such tolerance in fish allergic patients is important when
considering the beneficial effects of fish in young children. For
instance, oily fish such as salmon is rich in omega-3 fatty acids that
can protect against heart disease and support neuronal growth and brain
development 43 . These two epitopes will be of
great value for precision diagnosis of fish allergy, then better advise
on fish reintroduction in allergic patients. Further testing and
validation of the epitopes identified in this study in larger and
multinational cohorts of fish allergic patients with known fish-specific
allergy and tolerance are warranted.
We are aware that the present study comprises only Chinese patients, and
analyzed parvalbumin content and fishes available on the ImmunoCAP
system only. The applicability of the allergenicity ladder in other
populations with different dietary habits and sensitization to other
important fish allergens have yet to be considered. Epitope validation
and extending this fish allergenicity ladder to a wider spectrum of
fishes, and consider also different cooking methods and time for a more
comprehensive ladder will the next steps to move fish allergy care into
the next millennium and improve the quality of life of fish allergic
individuals and their families. It is worth mentioning that this is the
first attempt to compare the relative expression of fish allergens with
a transcriptomic approach. Interestingly, the expression of aldolase has
a “reverse” pattern contrary to parvalbumin, by which the relative
expression of aldolase peaks in salmon, followed by yellowfin tuna,
halibut and grouper, and appears low in freshwater fishes and cod.
Aldolase, enolase and collagen are important fish allergens and
mono-sensitization to these allergens has been well-documented6,44,45 . Unlike sera from patients with
parvalbumin-specific allergy that reacted strongly with fishes of high
parvalbumin content and weakly with fishes of low parvalbumin content,
those with collagen-specific allergy reacted similarly to all 22 species
of fishes despite the varying levels of parvalbumin46 . It is therefore important to test for the
major sensitizing allergen in patients implicated with fish allergy,
followed by screening epitope-specific IgE for possible fish tolerance
under the framework of precision diagnosis. While parvalbumin accounts
for >80% sensitization in fish allergic individuals, most
patients can rely on this propose fish ladder based on parvalbumin
levels. Our two identified epitopes and fish allergenicity ladder are
clinically useful in these patients with parvalbumin-specific allergy
for selecting fish in each step of the ladder for further IgE testing,
to inform which step of the fish ladder to start with during
reintroduction, and to guide fish reintroduction by gradually stepping
up the ladder from tolerant fishes or low allergenicity fishes to
increase the threshold dose to fish parvalbumin and achieve remission of
fish allergy.
In summary, the parvalbumin epitopes and fish allergenicity ladder
presented in this study can serve as a new compass to guide IgE testing
and fish reintroduction. These can benefit a majority of fish allergic
patients considering the role of parvalbumin as a major fish allergen
across different geographical populations. Particularly in the era of
telehealth, fish ladder guiding allergenic food reintroduction at home
is convenient, and can greatly reduce burden on clinical care and ease
financial strain.