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.