BIG DATA TAKES AIM AT A HUMAN PROBLEM Dr Ashley Waardenberg - A James Cook University scientist is part of an international team that’s used new ‘big data’ analysis to achieve a major advance in understanding neurological disorders such as Epilepsy, Alzheimer’s and Parkinson’s disease. Dr Ashley Waardenberg a Theme Leader from JCU’s Centre for Tropical Bioinformatics and Molecular Biology, said scientists from JCU, The Children’s Medical Research Institute, Sydney, University of Southern Denmark and Bonn University (Germany) looked at how neurons in the brain communicated with each other. Read full Article. http://getstem.com.au/big-data-takes-aim-at-a-big-human- problem/ GENETIC TESTING AT CAIRNS BASE HOSPITAL AIMS TO CATCH DEADLY DISEASES Prof John McBride and Dr Matt Field- James Cook University researchers will be trialling the use of handheld genetic testing devices at Cairns and Townsville public hospitals to catch deadly diseases before they get out of control. DR ASHLEY WAARDENBERG PROF JOHN MCBRIDE & DR MATT FIELD http://online.isentialink.com/townsvillebulletin.com.au/201 9/03/07/f1f9c4f3-c20f-4ee6-af73-b268186f7b4b.html CENTRE FOR MOLECULAR THERAPEUTICS MARCH NEWS UPDATE
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BIG DATA TAKES AIM AT A HUMAN PROBLEM
Dr Ashley Waardenberg - A James Cook University
scientist is part of an international team that’s
used new ‘big data’ analysis to achieve a major
advance in understanding neurological disorders such
as Epilepsy, Alzheimer’s and Parkinson’s disease.
Dr Ashley Waardenberg a Theme Leader from JCU’s
Centre for Tropical Bioinformatics and Molecular Biology,
said scientists from JCU, The Children’s Medical Research
Institute, Sydney, University of Southern Denmark and
Bonn University (Germany) looked at how neurons in the
brain communicated with each other.
Read full Article.
http://getstem.com.au/big-data-takes-aim-at-a-big-human-
problem/
GENETIC TESTING AT
CAIRNS BASE HOSPITAL
AIMS TO CATCH DEADLY
DISEASES
Prof John McBride and Dr Matt Field- James Cook
University researchers will be trialling the use of
handheld genetic testing devices at Cairns and
Townsville public hospitals to catch deadly diseases
before they get out of control.
DR ASHLEY WAARDENBERG
PROF JOHN MCBRIDE & DR MATT FIELD
http://online.isentialink.com/townsvillebulletin.com.au/201
9/03/07/f1f9c4f3-c20f-4ee6-af73-b268186f7b4b.html
CENTRE FOR MOLECULAR THERAPEUTICS
MARCH NEWS UPDATE
PROF ANDREAS LOPATA
EATING FISH MAY HELP
PREVENT ASTHMA
Prof Andreas Lopata - A James Cook University scientist
says an innovative study has revealed new evidence
that eating fish can help prevent asthma.
Professor Andreas Lopata from JCU’s AITHM took part in
the study which tested 642 people who worked in a fish
processing factory in a small village in South Africa.
Read the full Article
http://getstem.com.au/eating-fish-may-help-prevent-
asthma/
MARCH SUBMISSIONS
Mohadeseh Dastpeyman
Structure-Function Relationships of Disulphide-Rich Peptides
RIGHT: Professor Norelle Daly & Mohadeseh at the morning tea celebration.
Bemnet Amare
Molecular and Immunological characterization of
Schistosoma acetylcholinesterases and their evaluation as
a potential immunogenic vaccine candidates.
Stephanie Ryan
Investigating the Immunomodulatory Properties of Hookworm Excretory / Secretory Products Produced in a Cell-Free Protein Expression System
RIGHT: Prof Alex Loukas, Dr Mark Pearson, Stephanie Ryan and Bemnet Amare
CMT PUBLICATIONS THIS QUARTER
Berger, Maximus, Taylor, Sean, Harriss, Linton, Campbell, Sandra, Thompson, Fintan, Jones, Samuel, Sushames, Ashleigh, Amminger, G.
Paul, Sarnyai, Zoltan, and McDermott, Robyn (2019) Hair cortisol, allostatic load, and depressive symptoms in Australian Aboriginal
and Torres Strait Islander people. Stress.
Chasland, Lauren C., Knuiman, Matthew W., Divitini, Mark L., Murray, Kevin, Handelsman, David J., Flicker, Leon, Hankey, Graeme
J., Almeida, Osvaldo P., Golledge, Jonathan, Ridgers, Nicola D., Naylor, Louise H., Green, Daniel J., and Yeap, Bu B. (2019) Higher circulating
androgens and higher physical activity levels are associated with less central adiposity and lower risk of cardiovascular death in older
men. Clinical Endocrinology, 90 (2). pp. 375-383
Cooper, Martha M., Loiseau, Claire, McCarthy, James S., and Doolan, Denise L. (2019) Human challenge models: tools to accelerate
the development of malaria vaccines. Expert Review of Vaccines.
Currell, Scott Derek, Liaw, Andrew, Blackmore Grant, Peter Donald, Esterman, Adrian, and Nimmo, Alan (2019) Orthodontic
mechanotherapies and their influence on external root resorption: a systematic review. American Journal of Orthodontics and Dentofacial
Orthopedics, 155 (3). pp. 313-329.
Holt, Michael R., Miles, John J., Inder, Warrick J., and Thomson, Rachel (2019) Exploring immunomodulation by endocrine changes in Lady
Windermere Syndrome. Clinical and Experimental Immunology. (In Press)
Harriss, Linton R., Thompson, Fintan, Lawson, Kenny, O'Loughlin, Mary, and McDermott, Robyn (2019) Preventable hospitalisations in
regional Queensland: potential for primary health? Australian Health Review. AH18033. (In Press)
Kreuter, Roxane, Wankell, Miriam, Ahlenstiel, Golo, and Hebbard, Lionel (2019) The role of obesity in inflammatory bowel
disease. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1865 (1). pp. 63-72.
Liaw, A., Miller, C., and Nimmo, A. (2019) Comparing the periodontal tissue response to non‐surgical scaling and root planing alone,
adjunctive azithromycin, or adjunctive amoxicillin plus metronidazole in generalized chronic moderate‐to‐severe periodontitis: a preliminary
randomized controlled trial. Australian Dental Journal. (In Press)
Law, Soi Cheng, Haigh, Oscar L., Walpole, Carina M., Colm, Keane, Miles, John J., Ghandi, Maher K., Radford, Kristen J., and Steptoe,
Raymond J.(2019) Simple, rapid and inexpensive typing of common HLA class I alleles for immunological studies. Journal of Immunological
Methods, 465. pp. 72-76.
Nugraha, Ari Satia, Pratoko, Dwi Koko, Damayanti, Yuvita Dian, Lestari, Nadya Dini, Laksono, Tinton Agung, Addy, Hardian Susilo, Untari,
Ludmilla Fitri, Kusumawardani, Banun, and Wangchuk, Phurpa (2019) Antibacterial and anticancer activities of nine lichens of Indonesian
Java Island. Journal of Biologically Active Products from Nature, 9 (1). pp. 39-46.
O'Loughlin, Mary, Harriss, Linton, Thompson, Fintan, McDermott, Robyn, and Mills, Jane (2019) Exploring factors that influence adult
presentation to an emergency department in regional Queensland: a linked, cross‐sectional, patient perspective study. Emergency Medicine
Australasia, 31 (3). pp. 67-75.
Rowland, Joshua, Akbarov, Artur, Eales, James, Xu, Xiaoguang, Dormer, John P., Guo, Hui, Denniff, Matthew, Jiang, Xiao, Ranjzad,
Parisa, Nazgiewicz, Alicja, Ribeiro Prestes, Prescilla, Antczak, Andrzej, Szulinska, Monika, Wise, Ingrid A., Zukowska-Szczechowska,
Ewa, Bogdanski, Pawel, Woolf, Adrian S., Samani, Nilesh J., Charchar, Fadi J., and Tomaszewski, Maciej (2019) Uncovering genetic
mechanisms of kidney aging through transcriptomics, genomics, and epigenomics. Kidney International, 95 (3). pp. 624-635.
Vuckovic, Slavica, Minnie, Simone A., Smith, David, Gartlan, Kate H., Watkins, Thomas S., Markey, Kate A., Mukhopadhyay,
Pamela, Guillerey, Camille, Kuns, Rachel D., Locke, Kelly R., Pritchard, Antonia L., Johansson, Peter A., Varelias, Antiopi, Zhang,
Ping, Huntington, Nicholas D., Waddell, Nicola, Chesi, Marta, Miles, John J., Smyth, Mark J., and Hill, Geoffrey R. (2019) Bone marrow
transplantation generates T cell–dependent control of myeloma in mice. Journal of Clinical Investigation, 129 (1). pp. 106-121.
Van den Berghe, Femke, Paris, Monique C.J., Sarnyai, Zoltan, Briggs, Michael B., Millar, Robert P., Ganswindt, Andre, and Paris, Damien
B.B.P.(2019) Social rank does not affect sperm quality in male African wild dogs (Lycaon pictus). Reproduction, Fertility and Development.
(In Press)
Walker, Sarah, Wankell, Miriam, White, Rose, Ho, Vikki, Deo, Nikita, Devine, Carol, Dewdney, Brittany, Bhathal, Prithi, Govaere,
Olivier, Roskams, Tania, Qiao, Liang, George, Jacob, and Hebbard, Lionel (2019) Targeting mTOR and Src restricts hepatocellular carcinoma
growth in a novel murine liver cancer model. PLoS One, 14 (2). e0212860.
Zijlema, Wilma L., Stasinska, Ania, Blake, David, Dirgawati, Mila, Flicker, Leon, Yeap, Bu B., Golledge, Jonathan, Hankey, Graeme
J., Nieuwenhuijsen, Mark, and Heyworth, Jane (2019) The longitudinal association between natural outdoor environments and mortality
in 9218 older men from Perth, Western Australia. Environment International, 125. pp. 430-436.
Ruethers T, Taki AC, Nugraha R, Cao TT1, Koeberl M, Kamath SD, Williamson NA, O'Callaghan S, Nie S, Mehr SS, Campbell DE, Lopata AL, Variability of allergens in commercial fish extracts for skin prick testing. Please see attached PDF
Le TTK, Nguyen DH, Vu ATL, Ruethers T, Taki AC, Lopata AL A cross-sectional, population-based study on the prevalence of food allergies among children in two different socio-economic regions of Vietnam. Please see attached PDF
“Probably Science” live podcast Cairns event: Show Report
If you missed the show the podcast is now available as episode 327 and available on the link below.
https://probablyscience.libsyn.com/episode-327-live-from-cairns-with-drs-michael-smout-and-claudia-cobos
Under the Microscope - Kim Miles
1. Tell me about your area of research?I am working as a research assistant for the ParagenBio Project which is looking at finding a treatment for inflammatory
bowel disease using proteins derived from hookworm secretions.
2. What interests you about working in this area?This is an exciting project as it translational, I can imagine that the basic science we are doing could help create a therapy
which could be used in the clinic.
3. How do you see your research developing in the future?Oh, if only I knew! My role is very flexible but grant dependent. I have been working as a research assistant for 10 years
and in that time, I have worked in six different labs, with everything from a few months of work after getting my degree
to working in a lab in Cardiff, UK. I have gained so many different technical skills but there are still so many more I could
learn.
4. What are the 5 most important techniques you use in your research?Being a team member. Documenting everything, clearly. Aseptic technique. Developing the ability to set your timer,
leave it behind and still come back with 30sec to spare. Multitasking.
5. What advice do you have for science students who are considering medical research as a career?Try and get some work experience in a few different labs. If you can sit down and talk to the researchers, see what day
to day life is like in the lab and be prepared to learn a lot of acronyms.
6. What do you see as the benefits of being part of the Centre for Molecular Therapeutics (CMT)?Being surrounded by people who are enthusiastic to be at work and love what they do.
7. What would you like to do in the future?I would like to keep working in Cairns at JCU, I haven’t worked here long but it’s great. The campus is in such a wild
setting, the people are lovely and I’d like to bring up my family here.
8. Tell me 5 things you dislike?Liquorice, Liars, Relentless wind, Cane Toads, Gympie road in Brisbane.
9. Tell me 5 things that make you happy?Cooking, Gardening, Going to the Beach, Playing with my kids and Gin
10. Tell me about the highlights of your professional career so far?
I got to work in the Wellcome Institute at Heath Hospital in Cardiff for 3 years, it was fantastic to work outside of
Australia for a while. Also, a few years ago I took an image of a gut section for my supervisor which went on to win a
prize.
https://cosmosmagazine.com/biology/microscopic-secrets-your-immune-system-unveiled
Pediatr Allergy Immunol. 2019;1–8. wileyonlinelibrary.com/journal/pai | 1© 2019 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.
Received:28August2018 | Revised:17January2019 | Accepted:18January2019DOI: 10.1111/pai.13022
O R I G I N A L A R T I C L E
A cross‐sectional, population‐based study on the prevalence of food allergies among children in two different socio‐economic regions of Vietnam
Thu T. K. Le1,2 | Duy H. Nguyen3 | An T. L. Vu4 | Thimo Ruethers1,2,5 | Aya C. Taki1,2,5 | Andreas L. Lopata1,2,5
Abbreviations: CI, confidence interval; EAACI, European Academy of Allergy and Clinical Immunology; FA, food allergy; IgE, immunoglobulin E; OFC, oral food challenge.
1Molecular Allergy Research Laboratory, College of Public Health, Medical and VeterinarySciences,JamesCookUniversity,Townsville, Qld, Australia2Australian Institute of Tropical Health andMedicine,JamesCookUniversity,Townsville, Qld, Australia3Kindergarten 2 thang 9, Tien Giang, Vietnam4Faculty of Food Science and Technology,NongLamUniversityofHoChiMinh City, Ho Chi Minh City, Vietnam5Centre for Food and Allergy Research, Murdoch Children’s Research Institute,Parkville,Vic,Australia
CorrespondenceAndreas L. Lopata, Molecular Allergy Research Laboratory, College of Public Health, Medical and Veterinary Sciences, JamesCookUniversity,Townsville,Qld,Australia.Email: [email protected]
Funding informationThisworkwasfundedbygrantsfromthe Australian Research Council, and the National Health and Medical Research Council (ID: 1086656) to AL. TL is the recipient of the Endeavour Postgraduate Scholarship. TR is a PhD full‐time scholar of the Centre for Food and Allergy Research, Australia.
Editor:JenniferKoplin
AbstractBackground: There is a paucity of data on the prevalence of food allergy (FA) in Vietnam. A cross‐sectional, population‐based study was conducted to evaluate the current prevalence of FA among 2‐ to 6‐year‐old children in two different regions in Vietnam.Method: A structured, anonymous questionnaire, modified from published FA epide‐miologic studies and based on EAACI guidelines, was distributed to parents/guardi‐ans of participating children in Hue City (urban area) and Tien Giang Province (rural area). Data collected from the survey were statistically analyzed to generate the prevalence of self‐reported and doctor‐diagnosed FA and overarching pattern of food allergens.Results: A total of 8620 responses were collected (response rate 81.5%). Children in Tien Giang reported more than twice the food‐induced adverse reactions seen in childreninHue(47.8%vs. 20.5%). In contrast, children in Hue showed higher self‐re‐ported FA (9.8%) and doctor‐diagnosed FA rates (8.4%) than children in Tien Giang (7.9%and5.0%,respectively).Crustaceanwasthepredominantallergy‐inducingfoodinbothareas(330of580cases,56.9%),followedbyfish,mollusk,beef,milk,andegg.However, substantial variations of FA patterns were seen between the study sites. Geographiclocationandco‐morbiditiesofotherallergicdiseaseswerekeyriskfac‐tors for FA (P < 0.001).Conclusions: The prevalence of FA in Vietnamese children seems to be higher than previously reported from other Asian countries. Crustacean is the predominant al‐lergy‐inducing food among participating preschool children in Vietnam. The variation of reported food allergen sources across different socio‐economic locations could imply different eating habits or the participation of indoor and outdoor allergen exposure.
K E Y W O R D S
children, crustacean allergy, fish allergy, food allergy, prevalence, Vietnam
2 | LE Et aL.
1 | INTRODUC TION
Food allergy (FA), an adverse immune reaction to food proteins, has a wide spectrum of clinical presentations, ranging frommild skinproblems to severe systematic reactions. In the most severe case, FA can lead to anaphylaxis and might result in death within minutes. FA is estimated to affect about 8% of children and 5% of adults in the general population worldwide.1
Childrenaremorelikelytodevelopfoodallergiesthanadultsdueto remaining controversial causes, including the immature immune system in childhood and/or the inappropriate food introductory prac‐tices.2,3 Eight food groups, often referred as the “Big 8,” account for over90%offoodallergicreactionsandincludecow’smilk,egg,pea‐nut,treenuts,soy,wheat,fish,andshellfish.Exceptforcow’smilkandegg allergy which are often outgrown, most other FAs often persist for life.1 So far, no cure is available and childhood FA imposes a sub‐stantial health and economic burden for children and their caregivers.
The common food commodities accounting for FA in children are cow’smilk,egg,peanut,treenuts,andfish,4 while the first three foods are the leading causes for pediatric anaphylaxis in Western countries.5 In Asia, the prevalence of pediatric FA seems to vary between 1.11% and 7.65%,6andthepatternsofFAshowedmarkeddifferencefromotherparts of the world.1Recentstudiesamong2‐to7‐year‐oldchildrenfromSingapore, Thailand, the Philippines, and Hong Kong demonstrated that shellfish allergy was dominant, but not milk, egg, or peanut.7,8 Furthermore, fish was reported to be the predominant allergen in ad‐olescents in the Philippines, Singapore, and Thailand.9 Within Asia, studies fromJapanandKorea showeddifferentFApatterns tomostavailable FA data, with wheat allergy particularly common in East Asian countries.6 These data were supported by a recent study from Australia, where large differences in allergy/anaphylaxis risk and trigger weredemonstrated between children born in Australia and children born in Asia.10 The variation of FA patterns throughout Asia indicates that re‐gion‐specific and accurate data on FA prevalence and clinical patterns are crucial for an effective FA management program in any community.
In Vietnam, about 4.4 million children aged 2‐6 years attend kindergartens,accountingover90%ofallchildrenatthisagegroupin 2016.11 The first population‐based study on FA in adults was re‐cently performed by our group, revealing a high rate of FA in this population, self‐reported FA (18.0%) and doctor‐diagnosed FA (5.8%),12 while no data on children are available so far. We sought to evaluate the epidemiologic and clinical features of FA in Vietnamese preschool children. The possible variations of childhood FA preva‐lenceanditsassociatedriskfactorsinsocio‐economicallydifferentregions in Vietnam were also investigated.
2 | METHODS
2.1 | Study design and subjects
A cross‐sectional, population‐based study was conducted in pre‐school children aged from 2 to 6 years in 2016. Survey participants
were randomly selected using the cluster sampling method from alistof25kindergartensinHueCityand14kindergartensinCaiBe District, Tien Giang Province, representing a total of 104 602 preschool children in two regions.11 The paper‐based question‐naires were distributed to parents/guardians of children at their kindergartens.Mostof theanswer sheetswerecollectedon thesame day. The response rate was calculated based on the num‐ber of returned answer sheets divided by the total distributed questionnaires.
2.2 | Sample size calculation
To obtain a statistical estimation of the prevalence of FA, the mini‐mum sample size was calculated based on the current estimated prevalence of FA in children (8%) in the general population13; the chosen precision of the estimation d = 1/5p was calculated with a statistical confidence of two standard errors of the mean z = 1.96 (95% confidence interval (CI), P < 0.05). The minimum necessary sample size calculated for children was 1825 participants.
2.3 | Study locations
The study was conducted in two different regions of Vietnam: Hue City and Cai Be District of Tien Giang Province. Hue City is in the Central region of Vietnam with a population density of 5011 per squarekilometer.ThemaineconomicactivitiesinHuearetourism,industry,andaquaculture.Urbanizationhasquicklytakenplacedinthis city due to the rapid development of tourism. Hue has an aver‐agetemperatureof25.4°C,averagehumidityof87%,andatotalof1754.2hoursofsunshineperyear.
CaiBeDistrict isaruralarea intheMekongDeltaofsouthernVietnam.Thisriver‐landmixedtownhasapopulationdensityof657per square kilometer. Themajor economic activities inCaiBe areaquaculture, rice, and fruit farming. Cai Be‐Tien Giang has an aver‐age temperature of 28.2°C, average humidity of 80.4%, and a total of 2104.6 hours of sunshine per year.14
Inthisstudy,takingintoconsiderationtheeffectsofpopulationdensity, living lifestyle, and environmental conditions, we defined participants in Hue City as living in urban area and participants from Cai Be District as living in rural area.
2.4 | Questionnaire design
The questionnaire,modified frompublished studies in theUnitedStates and Asia,7,9 had twoparts: Part I asked theparticipant de‐mographic information, and part II contained ten questions on FA (Appendix S1). The questionnaire was translated into Vietnamese. The content of the questionnaire and its translation were reviewed andapprovedbytheHumanResearchEthicsCommitteeatJamesCookUniversity (ID:H6437).Byanswering thequestionnaire, theparents/guardians gave the informed consent to the study and the permission to use obtained child health information for research publications and reports.
| 3LE Et aL.
2.5 | Definitions
We established a set of criteria to define self‐reported and doc‐tor‐diagnosed FA in this survey based on the most recent European Academy of Allergy and Clinical Immunology (EAACI) guidelines on FA and anaphylaxis.15 In specific, the suggestive symptoms of FA were considered including persistent symptoms toward food inges‐tion and the co‐occurrence of two or more different clinical presen‐tations.16 The typical symptoms for IgE‐mediated FA included hives/urticaria or angioedema or vomiting or gastrointestinal symptoms or anaphylactic reactions (ie, reduced blood pressure, loss of con‐sciousness, chest pain, and weak pulse) after food intake. In thisstudy, children with only one symptom of hives/angioedema were also defined as food allergic and included.
Self‐reported FA was the group of participants who fulfilled the above criteria and reported having FA.
Doctor‐diagnosed FA was the group of participants with self‐re‐ported FA, which was clinically confirmed by a medical practitioner.
Food‐induced adverse symptoms: any abnormal clinical response that occurs following ingestion of a food or food component.
Family history of FA was defined when the participant had in their immediate family a member with FA.
Coexisting other allergic diseases was defined when the partici‐pant had any other allergic diseases including pollen allergy, antibi‐otic allergy, asthma, eczema.
2.6 | Statistical analysis
The survey data were analyzed and plotted using the IBM SPSS StatisticsforWindows(IBMCorp,Armonk,NY,USA),version24.0,andGraphPadPrism,version7.03.Continuousvariableswerepre‐sented as median and inter‐quartile range (IQR). Categorical data were compared by using either Fisher’s exact test or chi‐square test with a 2‐tailed P‐value. The Wilson/Brown method was performed to provide a 95% CI of proportions. Multivariable logistic regression modelwasusedtostudytheassociationbetweenmultipleriskfac‐tors and the incidence of having doctor‐diagnosed FA. A P‐value of <0.05 was considered as statistically significant for all tests.
3 | RESULTS
3.1 | Participants
A total of 8620 questionnaires were completed and returned from the two survey sites (response rate 81.5%). The survey in Hue gained a higher response rate (93.5%) than in Tien Giang (69.5%). Minimal difference in gender distribution was observed across the two survey sites. The age median (IQR) of the participants was 4 (2‐6) years in Hue and 6 (2‐6.5) years in Tien Giang. The de‐mographic characteristics of participating children are presented in Table 1.
Variable, n (%) Hue Tien Giang Difference, PTotal study population
Total 4443 4177 8620
Female 2206 (49.6) 2120 (50.8) 0.2860 4326 (50.2)
Male 2239 (50.4) 2055 (49.2) 0.2860 4294 (49.8)
Age group (years)
2 to <3 1140(25.7) 52 (1.3) <0.0001 1192 (13.8)
3 to <4 1365(30.7) 655(15.7) <0.0001 2020 (23.4)
4 to 6 1940 (43.6) 3467(83.0) <0.0001 5407(62.7)
Age, median (IQR) 4 (2‐6) 6 (2‐6.5)
Reported adverse reactions to food
911 (20.5) 1994(47.8) <0.0001 2905(33.7)
Self‐reported FA 433 (9.8) 330(7.9) 0.0026 763(8.9)
Seekingmedicaladvice for FAa
394 (91.6) 250(76.7) <0.0001 644 (84.4)
Doctor‐diagnosed FA 373(8.4) 207(5.0) <0.0001 580(6.7)
FA to 1 food group 328(87.9) 125 (60.4) <0.0001 453(78.1)
FA to 2 different food groups
40(10.7) 36(17.4) 0.9084 76(13.1)
FA to more than 2 different food groups
4 (1.1) 37(17.9) <0.0001 41(7.1)
The Fisher's exact test was performed using GraphPad Prism for Windows (GraphPad Software, La Jolla,CA,USA)toobtainP‐values.aAmong subjects with self‐reported FA.
TA B L E 1 Demographics of participating children in Hue and Tien Giang
4 | LE Et aL.
3.2 | Comparison of reported food‐induced adverse symptoms between children in Hue and Tien Giang
Children in Tien Giang were reported to have twice the food‐inducedadversesymptoms thanchildren inHue (47.8%vs20.5%)(Table 1). However, self‐reported FA in Hue (9.8%) was higher than in TienGiang(7.9%)(Table2).IntheperceivedFAgroup,morechildrenin Hue presented to doctors for medical advice, 91.6% compared to 76.7%inTienGiang.Overall,theprevalenceoflifetimedoctor‐diag‐nosed childhood FA in Hue was 8.4%, nearly double the rate of 5.0% in Tien Giang (P < 0.0001).
Suspected FA children in Hue reported less concurrent episodes than those in Tien Giang (an average of 1.4 episodes compared to 2.0 episodes, respectively). Hives, diarrhea, and nausea or vomiting were the most predominant clinical presentations reported. Ten partici‐pants (0.2%) in Tien Giang experienced severe symptoms (ie, loss of consciousness, drop inbloodpressure, chestpain, andweakpulse)due to FA, while in Hue, only one case was reported (0.02%) (Figure 1).
3.3 | Distribution of the major food allergens in FA children in Hue and Tien Giang
Mostoftheaffectedsubjects(78.1%)reportedfoodadversesymp‐toms to only one food item; 13.1% reported adverse reactions to twodifferentfooditems,and7.1%hadreactionstomorethantwodifferent food groups. Crustacean was the most predominant al‐lergy‐causing food type in both Hue (50.1%) and Tien Giang (30.6%), while the distribution of the remaining “Big 8” food groups was very different (Figure 2). Statistically significant differences were seen in theprevalenceof crustacean,mollusk,beef,milk,wheat, and treenut allergies between children in Hue and Tien Giang (P < 0.05) (Table 2).
3.4 | Contribution of environmental factors to FA incidence
Genetic and environmental factors are reported to play a role in the development of FA.1,17 In this study, we analyzed the contribution of geographic location, gender, and family history of FA as well as coexisting other allergic diseases to the FA incidence by using multi‐variable logistic regression model. A strong influence of participant locationandatopicconditionstoFAriskwasobservedinthisstudy.Children living inHue (urbanarea)haveahigher riskofhavingFAthan children living in Tien Giang (OR: 3.902, P < 0.001). The FA rate was found to be 3.428 times higher in participants with other ex‐isting allergic diseases (P < 0.006). Gender and family history of FA showednoimpactonFAriskinthisstudypopulation(Table3).
4 | DISCUSSION
This population‐based survey is the first to establish the preva‐lenceofself‐reportedFA(8.9%)anddoctor‐diagnosedFA(6.7%)in TA
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0.28
451.
24 (1
.03‐
1.50
)
Mollusk
0.90
(0.6
6‐1.
22)
2.13(1.73‐2.61)
<0.0
001
1.50(1.26‐1.78)
0.72(0.51‐1.01)
1.36(1.05‐1.76)
0.00
381.03(0.84‐1.27)
Beef
0.34
(0.2
0‐0.
56)
2.32
(1.9
1‐2.
82)
<0.0
001
1.30
(1.0
8‐1.
56)
0.27(0.15‐0.47)
1.46(1.14‐1.87)
<0.0
001
0.85(0.67‐1.06)
Milk
0.81
(0.5
9‐1.
12)
0.26(0.15‐0.47)
0.00
060.55(0.41‐0.72)
0.70(0.49‐0.99)
0.22
(0.1
1‐0.
41)
0.00
120.
46 (0
.34‐
0.63
)
Egg
1.15(0.87‐1.51)
1.10(0.83‐1.47)
0.9187
1.13(0.92‐1.37)
0.95(0.70‐1.28)
0.74(0.52‐1.05)
0.3471
0.85(0.67‐1.06)
Whe
at0.07(0.02‐0.20)
0.50(0.33‐0.77)
0.00
010.
28 (0
.19‐
0.41
)0.07(0.02‐0.20)
0.38
(0.2
4‐0.
62)
0.00
20.
22 (0
.14‐
0.34
)
Pean
ut0.47(0.31‐0.72)
0.36
(0.2
2‐0.
59)
0.50
460.
42 (0
.30‐
0.58
)0.27(0.15‐0.47)
0.31
(0.1
8‐0.
53)
0.7226
0.29
(0.2
0‐0.
43)
Soy
0.18
(0.0
9‐0.
35)
0.26(0.15‐0.47)
0.49
340.
22 (0
.14‐
0.34
)0.
16 (0
.08‐
0.32
)0.17(0.08‐0.35)
>0.9
999
0.16(0.10‐0.27)
Tree
nut
s0.07(0.02‐0.20)
0.43(0.27‐0.68)
0.00
060.24(0.16‐0.37)
0.02
(0.0
0‐0.
13)
0.31
(0.1
8‐0.
53)
0.00
070.16(0.10‐0.27)
Oth
er fo
ods
0.41
(0.2
6‐0.
64)
0.53
(0.3
5‐0.
80)
0.43
140.
46 (0
.34‐
0.63
)0.
25 (0
.14‐
0.44
)0.
29 (0
.16‐
0.50
)0.
8354
0.27(0.18‐0.40)
All
data
wer
e an
alyz
ed u
sing
Gra
phPa
d Pr
ism
for W
indo
ws.
The
Wils
on/B
row
n m
etho
d w
as u
sed
to c
alcu
late
the
95%
CIs
. Fis
her's
exa
ct te
st a
nd c
hi‐s
quar
e te
st w
ere
used
(whe
re a
ppro
pria
te) t
o co
mpa
re
the
prev
alen
ce in
two
stud
y gr
oups
. A P
‐val
ue o
f <0.
05 w
as d
enot
ed a
s st
atis
tical
sig
nific
ance
(hig
hlig
hted
in b
old)
.
| 5LE Et aL.
F I G U R E 1 Proportion of reported clinical symptoms in participating children in Hue and Tien Giang. A, Reported food‐induced adverse symptoms (n = 2905). B, Reported adverse symptoms in self‐reported FA participants (n = 763).C,Reportedadversesymptomsindoctor‐diagnosed FA participants (n = 580)
Hue Tien Giang0
5
10
15
20
25
30
40
60
80
100
Perc
enta
ge o
f cas
esReported food-induced
adverse symptoms
Hue Tien Giang0
5
10
15
20
25
30
40
60
80
100
Perc
enta
ge o
f cas
es
Self-reported FA
Hue Tien Giang0
5
10
15
20
25
30
40
60
80
100
Perc
enta
ge o
f cas
es
Doctor-diagnosed FA(A) (B) (C)
F I G U R E 2 Comparison of the distribution of reported food groups eliciting clinical reactions in participating children in Hue and Tien Giang: A, Reported food‐induced adverse symptoms in Hue (number of participants n = 911); B, self‐reported FA in Hue; C, doctor‐diagnosed FA in Hue; D, reported food‐induced adverse symptoms in Tien Giang; E, self‐reported FA in Tien Giang; F, doctor‐diagnosed FA in Tien Giang. The total number of reported food groups is presented for each study area and symptom group
Hue
Tien Giang
Reported food-induced adverse symptoms
Self-reported FA Doctor-diagnosed FA
(A)
(D)
(B)
(E)
(C)
(F)
n = 911 n = 433 n = 373
n = 1,994 n = 330 n = 207
Total = 1,073 Total = 496 Total = 425
Total = 2,192 Total = 580 Total = 382
6 | LE Et aL.
Vietnamese children. Our findings indicate large variations of FA prevalence between two survey sites with different socio‐economic backgrounds. The population living in the urban area presented ahigher prevalence of FA but also had a higher rate of doctor consul‐tationtodiagnoseFA.Mostparticipants (78.1%)reportedadversesymptoms to only one food group, with crustacean the dominating food allergen. Hives and gastrointestinal tract problems were the most commonly reported clinical symptoms for both regions.
We observed a higher rate of self‐reported FA (8.9%) than doctor‐diagnosedFA(6.7%),consistentwithpreviousassessmentsofques‐tionnaire‐based FA rates in Asian populations (1.11%‐7.65%).6 This variation appears to be determined by the complex pathophysiology of adverse reactions to food and the perception of respondents of this disease. Common etiology in pediatrics with food‐related adverse symptoms is immune‐mediated FAs and non‐immune–mediated food intolerance.18There isa lackofstrongevidencetodifferentiateFAfrom food intolerance exclusively based on reported clinical history, especiallyinAsiancommunities.Amongdoctor‐diagnosedmilkaller‐gic participants, two‐thirds of participants presented gastrointestinal symptoms, which might imply the contribution of other food‐induced disordersratherthantrueFA.Afoodoutbreak,suspectedtobeanacute allergic reaction to a new formula product, was recorded in 19 out of 229 hospitalized children in 2009.19Unfortunately, noaller‐gens were identified due to the constraint of diagnostic capacity in Vietnam. Further investigations will exclude other non‐immunoglob‐ulin E (IgE)–mediated FAs, such as food protein‐induced enterocolitis syndrome and eosinophilic esophagitis in the pediatric population to give an accurate estimation of true FA prevalence.20
Patient’s clinical history of FA is the initial motive for further diagnostic analysis; however, only 4 to 5% of the self‐reporting FA population is generally confirmed as true FA.4 Parent‐reported FA in Thai children was found to be 9.3%, but reduced to 1.1% when con‐firmed by oral food challenge (OFC).21 A survey of Singapore‐born children aged 4‐6 years showed the variation of self‐reported FA to shellfishwith7.22%ascomparedtoarateof1.19%withconvincinghistory FAs7 As it was consistently concluded in previous studies, an accurate diagnostic procedure of IgE‐mediated FA must comprise multiple tests including skin prick testing,measurement of serumspecific IgE, and OFC.22 However, only limited services are available
in Vietnam for diagnosing FA, particularly in rural areas. Most commercial diagnostic tests that are readily available in Western countries, including IgEquantificationandskinpricktests,arenotregistered or partially available to private patients and in specialized clinics. In the presented study, data could not be collected for the onset of adverse symptoms that might have better supported dif‐ferentiating between IgE‐mediated and non‐IgE–mediated FA. This is one of the biggest challenges in studying the prevalence of FA in a country where only a few people have access to correct FA diag‐nosis. This paper‐based survey on health conditions was thought to be a rather new practice for most Vietnamese, so we aimed and suc‐ceededatkeepingthequestionnaireassimpleaspossibletoachievea high response rate (81.5%).
This study revealed a distinct distribution of the “Big 8” food allergens inVietnamesechildren.Unlike thepatternsofchildhoodFA from Western populations, previous studies in Asian populations showed the predominance of shellfish and fish allergy rather than egg,cow’smilk,andpeanut,6,7,9 and this tendency was also deter‐mined in this survey. Children from rural and urban Vietnam re‐portedhigheradversereactionratestoseafood,thenbeef,milk,andegg. The predominance of seafood allergy in Asia might be claimed for the availability and high consumption of this food commodity.23 InVietnam, theaverage fishconsumption iswith33kgpercapitaper annum much higher than the world’s average consumption of 21kg.23 The impact of ethnic characteristics to seafood allergy in Asian communities was validated in a study among expatriate and local Singaporean children, revealing the predominance of shellfish allergy in local children compared with expatriate children.7
Considering ethnic characteristics and cultural dietary practices, we found considerable variations of FA prevalence among urban and rural population in Vietnam. Crustacean and milk allergy arepredominant in children in Hue (urban area). However, there were insufficient data on the consumption of these commodities between thetwoareastopostulateFArisk.Thehighincidenceofshellfishal‐lergy in urban children might be related to higher exposure to indoor allergens as discussed in the current literature.24 For instance, in‐door mites were documented to cross‐react with the major shellfish allergen tropomyosin,25 and storage mites were identified in indoor environments in the north of Vietnam.26 In contrast, children in the TienGiangProvinceshowedamuchhigherprevalenceofmollusk,wheat,treenuts,andbeef.RecentstudiesintheUnitedStatesandSwedendocumented theassociationof redmeatallergywith tickbites,27,28 which was explained by the cross‐reactivity of a carbohy‐drate oligosaccharide galactose‐alpha‐1,3‐galactose in mammalian meatandasimilarcomponentfound inthesalivaoftick.Childrenfromruralareasaremorelikelytohavetickbitesthanthoseinthecity,29 and therefore, environmental factors might contribute to the high rate of beef allergy in children in Tien Giang. Similarly, the high incidence of wheat and tree nut allergy in this subpopulation might be explained by the possible cross‐reactivity of these food allergens with other aeroallergens abundant in the rural area. It should be noted that wheat is not a staple food in Vietnam and no data on gluten intolerance or celiac disease have been reported so far in this
TA B L E 3 Multivariable logistic regression analysis of demographic factors for FA
Risk factor OR P‐value
Gender (Female/Male) 1.567 0.172
FamilyhistoryofFA(Yes/No) 1.018 0.961
Coexisting other allergic diseases (Yes/No)
3.428 0.006
Participant location (Hue/Tien Giang)
3.902 <0.001
Binary logistic regression was performed in SPSS Statistics for Windows to generate ORs. A P‐value of <0.05 was considered as statistically sig‐nificant (highlighted in bold).
| 7LE Et aL.
population. This will be of interest for further investigation into the influence of environmental factors to FA.
The data from the multivariable logistic regression analysis of demographic risk factors (gender, family history of FA, coexistingother allergic diseases, and geographic location) demonstrated a strong contribution of coexisting other allergic diseases (OR = 3.428, P < 0.006) to FA incidence, but not a family history of FA (OR = 1.018, P = 0.961). FA is thought to run in a family.17 However, the contribu‐tionofafamilyhistoryofFAtotheriskofFAdevelopmentremainsinconsistent among studies.30,31 In the present study, we did not apply any additional logistic regression models to further assess in‐dividualriskfactorsforFA.
The strengths of this study are the large population‐based data‐set (n = 8620) collected at two different socio‐economic survey sites and the high response rate (81.5%). The limitations of this study are the self‐administered data on FA, and therefore, the response might contain recall bias. Our target population was children aged from 2 to 6 years, and the information on children outside this age group with potentially different FA rates has not been included. There are several factors such as the disparity of the medical facilities among rural and urban areas in Vietnam and the economic circumstances of participants that might contribute to the variation on reported FA rates among the two study sites.
In conclusion, this study contributes to the current paucity of FA data in the broader Asian population and is the first to profile this emerging epidemic in Vietnam. Our study clearly showed that FA is prominent in Vietnam, but unexpected patterns of food allergies are perceived. A large variation of FA incidence was observed in subpop‐ulations from rural and urban regions, implying possible impacts of living conditions. Further investigations are necessary to confirm the true prevalence of FA and possible cross‐reactivities between differ‐ent allergen sources for a precise diagnosis and better management of this serious childhood illness.
COMPETING INTERESTS
The authors declare that they have no competing interests.
AUTHORS’ CONTRIBUTIONS
TL and AL developed the concept and study design. DN and TL con‐ducted the on‐site survey. AV and TL processed survey data. TL per‐formed the statistical analysis. TL wrote the manuscript. AT, TR, and AL edited the final manuscript. All authors contributed to the devel‐opment of the manuscript and approved the final version.
ETHIC S APPROVAL
This study was approved by the Human Research Ethics Committee (HREC)atJamesCookUniversity(ID:H6437).
CONSENT FOR PUBLIC ATION
All authors have approved the manuscript for submission.
AVAIL ABILIT Y OF DATA AND MATERIAL S
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
ORCID
Thu T. K. Le https://orcid.org/0000‐0001‐9811‐0328
Thimo Ruethers https://orcid.org/0000‐0002‐0856‐3452
Aya C. Taki https://orcid.org/0000‐0003‐3489‐4367
Andreas L. Lopata https://orcid.org/0000‐0002‐2940‐9235
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2. Tran MM, Lefebvre DL, Dai D, et al. Timing of food introduction and development of food sensitization in a prospective birth cohort. Pediatr Allergy Immunol.2017;28(5):471‐477.
3. NeelandMR,KoplinJJ,DangTD,etal.Earlylifeinnateimmunesig‐natures of persistent food allergy. J Allergy Clin Immunol.2017;14:14.
4. NwaruBI,HicksteinL,PanesarSS,etal.Theepidemiologyoffoodallergy in Europe: a systematic review and meta‐analysis. Allergy. 2014;69(1):62‐75.
5. SamadyW,TrainorJ,SmithB,GuptaR.Food‐inducedAnaphylaxisin Infants and Children. Ann Allergy. 2018;31:31.
6. LeeAJ,ThalayasingamM,LeeBW.FoodallergyinAsia:howdoesitcompare? Asia Pac Allergy. 2013;3(1):3‐14.
7. Shek LP, Cabrera‐Morales EA, Soh SE, et al. A population‐basedquestionnaire survey on the prevalence of peanut, tree nut, and shellfish allergy in 2 Asian populations. J Allergy Clin Immunol. 2010;126(2):324‐331,31e1–7.
8. LeungTF,YungE,WongYS,LamCW,WongGW.Parent‐reportedadverse food reactions in Hong Kong Chinese pre‐schoolers: epide‐miology,clinicalspectrumandriskfactors.Pediatr Allergy Immunol. 2009;20(4):339‐346.
9. ConnettGJ,GerezI,Cabrera‐MoralesEA,etal.Apopulation‐basedstudy of fish allergy in the Philippines, Singapore and Thailand. Int Arch Allergy Immunol. 2012;159(4):384‐390.
10. WangY,AllenKJ,SuainiN,PetersRL,PonsonbyAL,KoplinJJ.Asianchildren living in Australia have a different profile of allergy and ana‐phylaxis than Australian‐born children: a State‐wide survey. Clin Exp Allergy. 2018;19:19.
11. Statistical Publishing House. Statistical Yearbook of Vietnam 2015 ‐ Education [Internet]. 2016. file:///C:/Users/jc303254/Downloads/10.%20Giao%20duc.pdf.AccessedApril,272017.
12. Le T, Tran T, Ho H, Vu A, Lopata AL. Prevalence of food allergy in Vietnam: comparison of web‐based with traditional paper‐based survey. World Allergy Organ J. 2018;11(1):16.
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14. Vietnam GSOo. Administrative Unit, Land and Climate. Ha Noi, Vietnam: General Statistics Office of. Vietnam; 2016.
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15. Muraro A, Werfel T, Hoffmann‐Sommergruber K, et al. EAACI food allergy and anaphylaxis guidelines: diagnosis and management of food allergy. Allergy. 2014;69(8):1008‐1025.
16. Halken S, Muraro A. Food Allergy. Global Atlas of Allergy. Zurich, Switzerland: EAACI; 2014:199‐201.
17. Lack G. Update on risk factors for food allergy. J Allergy Clin Immunol.2012;129(5):1187‐1197.
18. Turnbull JL,AdamsHN,GorardDA.Reviewarticle: thediagnosisand management of food allergy and food intolerances. Aliment Pharmacol Ther. 2015;41(1):3‐25.
19. Vo TH, Le NH, Patel MS, Phan LT, Tran Minh NN. Acute allergic reactionsinVietnamesechildrenafterdrinkinganewmilkproduct.Foodborne Pathog Dis. 2012;9(2):156‐159.
20. Mehr S, Frith K, Barnes EH, Campbell DE, Group FS. Food protein‐induced enterocolitis syndrome in Australia: A population‐based study, 2012–2014. J Allergy Clin Immunol.2017;140(5):1323‐1330.
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23. FAOSTAT. Per Capita consumption of Fish and Shellfish for Human Food, by region and country [Internet]. 2013. http://www.fao.org/faostat/en/#data/BL.AccessedApril,102017.
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SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article.
How to cite this article: Le TTK, Nguyen DH, Vu ATL, RuethersT,TakiAC,LopataAL.Across‐sectional,population‐based study on the prevalence of food allergies among children in two different socio‐economic regions of Vietnam. Pediatr Allergy Immunol. 2019;00:1–8. https://doi.org/10.1111/pai.13022
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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/all.13748 This article is protected by copyright. All rights reserved.
MR. THIMO RUETHERS (Orcid ID : 0000-0002-0856-3452)
Article type : Original Article: Experimental Allergy and Immunology
Variability of allergens in commercial fish extracts for skin prick testing
Short title: Analysis of fish extracts for allergy diagnostics
Thimo Ruethers1,2,3,4, Aya C. Taki1,2,3,4, Roni Nugraha1,3,4,5, Trúc T. Cao1, Martina Koeberl6,
Sandip D. Kamath1,2,3,4, Nicholas A. Williamson7, Sean O’Callaghan7, Shuai Nie7,
Sam S. Mehr2,8,9, Dianne E. Campbell2,9,10, Andreas L. Lopata1,2,3,4
1Molecular Allergy Research Laboratory, College of Public Health, Medical and Veterinary
Sciences, James Cook University, Townsville, Queensland, Australia; 2Centre for Food and Allergy Research, Murdoch Children’s Research Institute, Melbourne,
Victoria, Australia; 3Australian Institute of Tropical Health and Medicine, James Cook University, Townsville,
Queensland, Australia; 4Centre for Sustainable Tropical Fisheries and Aquaculture, Faculty of Science and
Engineering, James Cook University, Townsville, Queensland, Australia; 5Department of Aquatic Product Technology, Bogor Agricultural University, Bogor, Jawa
Barat, Indonesia; 6Technical Development and Innovation Group, National Measurement Institute, Melbourne,
Australia;
7Bio21 Mass Spectrometry and Proteomics Facility, The Bio21 Molecular Science and
Biotechnology Institute, The University of Melbourne; 8Children’s Hospital at Westmead, Allergy and Immunology, Sydney, New South Wales,
Australia; 9Department of Allergy and Immunology, Royal Children’s Hospital Melbourne, Melbourne,
Victoria, Australia;
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This article is protected by copyright. All rights reserved.
10Discipline of Paediatrics and Child Health, University of Sydney, Sydney, New South
Wales, Australia
Corresponding author: Andreas L. Lopata; Pharmacy and Medical Research, Bldg. 47,
1 James Cook Drive, James Cook University, Townsville, QLD 4811, Australia; phone:
+61(07)47814563; fax: +61(07)47816078; e-mail: [email protected]
Acknowledgements
TR is holder of a full-time PhD scholarship from the Centre for Food and Allergy Research
and James Cook University, Australia. SK is a National Health and Medical Research
Council (NHMRC) Peter Doherty Early Career Research Fellow (GNT1124143). The study
was financially supported by an Australian Research Council fellowship to AL and an
NHMRC grant (APP1086656) to AL and DC.
Conflicts of interest
The authors declare that they have no relevant conflicts of interest.
Author contributions
TR performed the study and wrote the manuscript. AL led the study. TR, AT, SK, SM, DC,
and AL designed the study. SM and DC recruited the patients and acquired the commercial
fish extracts. TR, RN, TC, MK, SN, SO, and NW contributed significantly to the generation
of the data. All co-authors were substantially involved in the analyses and interpretation of all
data and critically reviewed the manuscript.
Abstract
Background: Commercial allergen extracts for allergy skin prick testing (SPT) are widely
used for diagnosing fish allergy. However, there is currently no regulatory requirement for
standardisation of protein and allergen content, potentially impacting the diagnostic reliability
of SPTs. We therefore sought to analyse commercial fish extracts for the presence and
concentration of fish proteins and in vitro IgE reactivity using serum from fish-allergic
patients.
Methods: Twenty-six commercial fish extracts from 5 different manufacturers were
examined. The protein concentrations were determined, protein compositions analysed by
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mass spectrometry, followed by SDS-PAGE and subsequent immunoblotting with antibodies
detecting 4 fish allergens (parvalbumin, tropomyosin, aldolase, collagen). IgE-reactive
proteins were identified using serum from 16 children with confirmed IgE-mediated fish-
allergy, with focus on cod, tuna, and salmon extracts.
Results: The total protein, allergen concentration and IgE reactivity of the commercial
extracts varied over 10-fold between different manufacturers and fish species. The major fish
allergen parvalbumin was not detected by immunoblotting in 6/26 extracts. In 7/12 extracts
5 known fish allergens were detected by mass spectrometry. For cod and tuna almost 70% of
patients demonstrated the strongest IgE reactivity to collagen, tropomyosin, aldolase A, or β-
enolase but not parvalbumin.
Conclusions: Commercial fish extracts often contain insufficient amounts of important
allergens including parvalbumin and collagen, resulting in low IgE reactivity. A
comprehensive proteomic approach for the evaluation of SPT extracts for their utility in
allergy diagnostics is presented. There is an urgent need for standardised allergen extracts,
which will improve the diagnosis and management of fish allergy.
Keywords: allergy diagnostics, fish allergy, IgE reactivity, parvalbumin, skin prick test
1 Background
IgE-mediated fish allergy is typically a life-long disease 1, with sensitisation rates of up to 3%
in the general population 2, and up to 8% of fish processing workers 3. The diagnosis of fish
allergy is often confirmed by in vivo allergy skin prick tests (SPT) 4,5, a relatively non-
invasive point of care testing, providing immediate results. Commercial fish extracts for SPT
for approximately 30 (mostly European species) are available, while over 1,000 different fish
species are consumed worldwide. Allergic sensitisation to fish seems to be region- and
species-specific, leading to study bias when utilising SPT extracts from European species 6 in
other regions. Furthermore, commercially available extracts do not appear to be standardised
regarding protein and allergen content, thereby impacting the reproducibility and diagnostic
value of SPTs.
Previous studies have demonstrated significant variation in allergen content of commercial
SPT extracts for birch 7 and grass pollen 8, mould 9,10 and occupational allergens 11-13. A large
heterogenicity in in vivo and in vitro reactivity and allergen content among 5 commercial
crustacean extracts was recently demonstrated 14. Some commercial fish extracts have
previously been analysed for their protein concentration and parvalbumin (PV) content 15.
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The current study aimed to examine a more complete fish allergen repertoire including PV 16,
tropomyosin 17,18, aldolase A 19, β-enolase 19, vitellogenin 20, and collagen 21 in a large range
of commercially available fish extracts for SPT as well as the molecular IgE reactivity using
serum from fish-allergic children in a southern hemisphere setting.
2 Methods
2.1 Skin prick test and in-house extracts
Twenty-six commercial extracts for SPT from 5 different manufacturers, covering 11 fish
families/groups including mixes of species, were obtained (details are listed in Table S1).
Extracts from different manufactures for the same species were assigned a number according
to table S 1, i.e. cod-1. In-house protein extracts and purified PVs were generated as controls
for this study, using frozen muscle tissue from Atlantic cod (Gadus morhua), yellowfin tuna
(Thunnus albacares), and Atlantic salmon (Salmo salar). Tissue was homogenised with a
rotor-stator homogeniser (5 min at 13,000 rpm on ice) in phosphate buffered saline (PBS,
10 mM phosphate; pH 7.2; 2 ml/g tissue). After gentle agitation overnight at 4°C, subsequent
centrifugation (20,000 x g) and filtration, extracts were stored at -20°C until further use,
referred as raw protein extracts. For the heated extracts, tissue was heated in PBS (95-100°C)
for 20 min before homogenisation. PV from these 3 species were purified from heated
extracts by ammonium sulfate precipitation with subsequent dialyses against 100 mM
ammonium bicarbonate as previously described 6.
2.2 Patients
Sera were obtained from 16 Australian children (1-18 years) with allergist confirmed clinical
history of IgE-mediated symptoms after ingesting fish. The median sIgE levels, determined
by ImmunoCAP (Thermo Fisher Scientific), were 7.0 kU/l for cod (F3), 4.7 for tuna (F40),
and 9.8 for salmon (F41), with a interquatile range of 1.6-75.0, 0.8-17.3, and 1.6-38.9,
respectively (Stata v14). The sIgE level for patient 16 was <0.01 kU/l for all 3 species and
has been excluded for the statical analyses (Table 1). The fish implicated in the allergic
reaction was in 8 cases white fish/cod, in 7 salmon, and in 3 tuna. A pool of serum from
3 non-atopic fish-tolerant donors was used as a negative control. Parents of all participants
gave written informed consent, and patient anonymity was preserved. Ethical approval was
obtained from the Sydney Children's Hospitals Network (LNR-14/SCHN/185).
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2.3 Protein concentration and SDS gel-electrophoresis
The protein concentration for all fish extracts was estimated using the Pierce™ 660nm
Protein Assay (Thermo Scientific) with bovine serum albumin as standard. In-house extracts
were diluted to 1 mg/ml, while commercial extracts were analysed undiluted. Proteins
(2.5 μl) were separated according to their molecular weights using a Criterion™ SDS-PAGE
system (Bio-Rad) as described by Laemmli [18]. The acrylamide content varied between 10
and 16% depending on the molecular weight of the proteins of interest. Proteins were
visualised by Coomassie Brilliant Blue R-250 (CBB) staining and identified by subsequent
immunoblotting with allergen-specific antibodies or patient serum IgE.
2.4 Immunoblotting
The separated proteins were transferred onto nitrocellulose membrane. After drying,
rehydrating in PBS and blocking for 1 h with 1xCasein (Thermo Scientific) in PBS at room
temperature, the membranes were incubated with 4 antibodies detecting fish allergens for 1 h
at room temperature or overnight with patient serum at 4°C (1:15 with 0.2x Casein in PBS-T
(PBS with 0.5% Tween-20®)). The allergens, PV and tropomyosin, were detected using in-
house generated polyclonal antibodies raised in rabbits against PV from Asian seabass and
Atlantic salmon 22, and tropomyosin from prawn 23. Aldolase A and collagen were identified
utilising commercial polyclonal antibodies (goat anti-rabbit aldolase 100-1141 by Rockland
Immunochemicals 19 and rabbit anti-tuna collagen ab23730 by Abcam, respectively). No
antibody against β-enolase or vitellogenin were available.
After washing with PBS-T, the patient blots were incubated with monoclonal mouse anti-
human IgE antibody (sc-53346 by Santa Cruz, 1:1,000 with 0.2xCasein in PBS-T for 1 h at
room temperature) and subsequently washed. All blots were developed with a corresponding
IR-labelled antibody (DyLight anti-mouse/rabbit 4xPEG by Thermo Scientific or IR-Dye
anti-goat by LI-COR®). The Odyssey® Imaging Systems (LI-COR®) was used for imaging
membranes and CBB-stained gels. Densitometric analyses were conducted utilising Image
Studio Version 5.2 (LI-COR®) allowing sensitive but semi-quantitative evaluation of signals.
The colours presented in heatmaps arise from individual protein bands or the sum of all
signal intensities for each separate sample. The densitometric analyses utilising this system
are independent of background, contrast or other settings used for best visualisation of the
immunoblot.
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2.5 Mass spectrometry analysis
All commercial cod, tuna, and salmon extracts (diluted to 200 µg/ml), as well as all
prominent IgE-reactive bands of the respective extracts, were subjected to mass spectrometric
analysis after tryptic digestion (see 24 for details). In brief, the extracted peptides were
analysed using an LTQ Orbitrap Elite (Thermo Scientific) coupled to an Ultimate 3000
RSLC nanosystem (Dionex). The nanoLC system was equipped with an Acclaim Pepmap
nano-trap column and an Acclaim Pepmap analytical column. The peptide mix was loaded
onto the trap column before the enrichment column was switched in-line with the analytical
column. The LTQ Orbitrap Elite mass spectrometer was operated in the data-dependent
mode, spectra acquired first in positive mode at 240k resolution followed by collision-
induced dissociation (CID) fragmentation. Twenty of the most intense peptide ions with
charge states ≥2 were isolated and fragmented using normalized collision energy of 35 and
activation Q of 0.25 (CID). All results were analysed with Mascot search engine and cross-
referenced against NCBI protein database for all bony fish (February 2018). Variable
modifications of carbamidomethyl-C and N-terminus, deamidation N, deamidation Q and
oxidation of M were selected. The search results were further processed using Proteome
Discoverer (Thermo Scientific) for false discovery rate control (1% at peptide spectral match,
peptide and protein level) and protein grouping. A protein group was considered to be
identified with at least 1 significant peptide. Perseus was used for generation of the Venn
diagram 25.
3 Results
3.1 Protein contents of extracts
The total protein content varied between the 26 commercial fish extracts more than 17-fold,
ranging from 0.17 to 2.94 mg/ml (Figure 1A).
3.2 SDS-PAGE and parvalbumin detection
The protein composition showed large differences in number and abundance of proteins of
different molecular weight in all analysed commercial fish extracts (Figure 1B). In 29% of
the commercial fish extracts, no protein bands were visible in the 10 to 15 kDa range, the
molecular weight of the major allergen, PV.
Utilising a PV-specific antibody, PV was detected with similar intensities in all commercial
cod extracts (Figure 1C). Among the commercial tuna extracts, PV was only detected in tuna-
1. For salmon-1 the signal for PV was 7-times higher compared to salmon-2, while no PV
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was detected in salmon-3. In all other commercial extracts, except flounder-2 and halibut-2,
PV was identified. The highest PV-specific antibody reactivity was observed for the catfish1
extract, while the reactivity was very weak for extracts mackerel-5, perch-1, and mix-2.
3.3 Protein composition evaluated by mass spectrometric analyses
All analysed commercial cod, tuna, and salmon extracts revealed large differences in the
presence of fish proteins and allergens. Across all 12 extracts, an average of 251 protein
groups were identified, ranging from 217 to 381 for cod, 146 to 236 for tuna, and 209 to 279
for salmon (see Figure 2 A-C). In all commercial cod extracts, 155 common protein groups
were identified, while only 94 and 125 were found in all tuna and salmon extracts,
respectively. The cod-3, tuna-2, and salmon-2 extracts differed the most from other extracts
of the same species with 29, 51, and 103 unique proteins identified, respectively.
Four fish proteins registered with the WHO/IUIS, as well as allergenic collagen, were
investigated for all 5 cod, 4 tuna, and 3 salmon commercial extracts (Figure 2D). PV,
tropomyosin, aldolase A, β-enolase, and collagen were detected in 2/5 cod (-3 and -5), 2/4
tuna (-1 and -2), and all 3 salmon extracts. In the other 3 cod extracts, no collagen was
detected but the other allergens. No detectable traces of tropomyosin and collagen were found
in the other 2 tuna extracts, as well as no PV was detected in tuna-4. The fish allergen
vitellogenin was not detected in any of the commercial fish extracts.
3.4 Allergen profiles of commercial cod, tuna, and salmon extracts by SDS-PAGE and
mass spectrometric analyses
The subsequent analysis focused on the 2 commercial extracts with the lowest and highest
protein concentrations each for cod, tuna and salmon - cod-2 (0.3 mg/ml) and cod-5 (1.1
mg/ml), tuna-3 (0.7 mg/ml) and tuna-1 (2.9 mg/ml), and salmon-2 (0.2 mg/ml) and salmon-1
(2.2 mg/ml) (Figure 3F). The selected extracts were compared to the respective in-house
generated raw and heated extract by SDS-PAGE and subsequent immunoblotting utilising
allergen-specific antibodies (Figure 3).
The protein profiles (Figure 3A) of cod-5, tuna-1, tuna-3 and salmon-1 were similar to the
corresponding raw extract, while cod-2 and salmon-2 showed even fewer protein bands than
the corresponding heated extract. Salmon raw and heated extract, as well as salmon-2,
contained 2 prominent bands in the molecular weight region of PV monomers (10–15 kDa),
but only one was observed in salmon-1.
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Bands identified as allergens by mass spectrometric analyses were quantified by
densitometric analyses (Figure 3G). The abundance of the PV monomer varied maximal 2-
fold between all cod extracts, salmon-1, and salmon raw and heated extract. In salmon-1 the
PV monomer was 3-times more abundant as compared to salmon-2, while among all tuna
extracts only in tuna-1 a PV monomer could be identified. The fish allergens tropomyosin
and aldolase A have a similar molecular weight as many other allergenic proteins such as
glyceraldehyde-3-phosphate dehydrogenase, making a clear distinction not possible. Notable
differences between the extracts were found in the abundance of proteins in this molecular
weight range (35–40 kDa).
In cod-5, tuna-1, and salmon-1 a 4-, 6-, and 13-times higher abundance was found as
compared to cod-2, tuna-3, and salmon-2, respectively. The heat-sensitive allergen β-enolase
could not be identified in any of the commercial cod extracts or heated extract of any of the
3 species. The highest abundance of β-enolase was detected in tuna-1, being 7- and 4-times
more abundant than in tuna-3 and raw tuna extract, respectively. In salmon-1 β-enolase was
twice as abundant as compared to the raw salmon extract. No β-enolase was identified in
salmon-2. Collagen could only be identified in the heated extract from all 3 species, however,
myosin heavy chain was also identified in addition to collagen in the high molecular weight
region. In cod heated extract, collagen was 4-times and 9-times more abundant compared to
tuna and salmon heated extract, respectively.
3.5 Allergen profiles of commercial cod, tuna, and salmon extracts using specific
antibodies
The presence and relative abundance of the 4 fish allergens PV, tropomyosin, aldolase A, and
collagen was further evaluated by immunoblotting (Figure 3B-E) with subsequent
comparative densitometric analyses (Figure 3H). Using the anti-salmon PV antibody, PV
monomers (11-13 kDa) were detected in all cod and salmon extracts, but in only 1 tuna
extract (tuna-1). The antibody reactivity was 5- and 3-times stronger for cod-5 and salmon-1
as compared to cod-2 and salmon-2, respectively. A PV dimer (22-26 kDa) was identified
with a strong signal in salmon-1, while the corresponding signal was 6-times weaker in raw
and heated extract, and 44-times weaker for salmon-3. In cod and tuna extracts, no PV dimers
were detected. In the molecular weight range of PV trimers (33-39 kDa), faint signals were
observed in all extracts, however, their intensity varied greatly.
The signal for the tropomyosin monomer (35-40 kDa) was 11-, 6-, and 7-times stronger for
the heated compared to raw extract from cod, tuna, and salmon, respectively, and stronger
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than the signal for any of the corresponding commercial extracts. Cod-5, tuna-1, and salmon-
1 showed a 4-6-times stronger signal intensity as compared to cod-2, tuna-3, and salmon-2.
Faint signals below 35 kDa were observed in all extracts when using the anti-tropomyosin
antibody. However, only for tuna-1 a very strong signal was observed at 25 kDa, indicating a
fragment of this allergen as recently documented 26.
Aldolase A was detected in all raw but none of the heated extracts, and neither in cod-2, tuna-
3, and salmon-2. The signal for cod-3 was 3-times stronger as for the respective raw extract,
while the signal for tuna-1 and salmon-1 was about 2-fold weaker as for the respective raw
extract.
Collagen was detected in all heated extracts, but none of the raw extracts or commercial
extracts. The signal for cod heated extract was 3- and 5-times stronger than for the tuna and
salmon heated extract, respectively.
3.6 Patient IgE reactivity to specific allergens
The 12 extracts were further analysed for IgE reactivity using serum from 16 patients with
confirmed IgE-mediated fish allergy (Figure S1) and further evaluated for the total IgE
reactivity (Figure 4) and reactivity to specific allergens (Figure 5). Subsequent densitometric
analyses demonstrated large differences in total IgE reactivity among tuna and salmon, but
less between commercial cod extracts (Figure 4). A stronger IgE reactivity was observed,
using densitometric analysis, for cod-5 compared to cod-2 for 11/16 patients, while the IgE
reactivity differed in average 1.4-times. Salmon-2 showed a weak to moderate IgE reactivity
with 8/16 patients, while tuna-3 showed a relatively weak IgE reactivity only with three
patients (3, 4, 11). In average tuna-1 and salmon-1 showed a 22- and 8-times stronger IgE
reactivity, respectively, with an up to 65-fold difference between the 2 commercial salmon
extracts. For 11 and 3 patients, the IgE reactivity was more than 10-fold different between the
2 commercial tuna and salmon extracts, respectively.
Among the 6 analysed commercial SPT extracts, cod demonstrated the lowest IgE reactivity;
while 2/16 patients showed the strongest IgE reactivity to cod, whereas 6 and 8 patients
showed the strongest reactivity to tuna or salmon, respectively.
The most prominent IgE-reactive bands were further analysed by mass spectrometry and the
signal intensities compared between all patients (Figure 5). IgE from all, but 2 patients (11
and 16), detected a PV monomer (11-13 kDa) in at least one of the 12 extracts. However, the
PV monomer was the most and exclusive IgE-reactive band for only 5/16 patients for cod and
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tuna, but 12/16 patients for salmon. Most patients with no or little IgE reactivity to the PV
monomer as well as a level low sIgE level for the respective fish species identified collagen
(100–250 kDa) as the most prominent IgE-reactive protein. IgE-reactive collagen was
detected in all heated, but none of the raw extracts. Collagen was the most IgE-reactive
protein in cod, tuna, and salmon extracts for 6/16, 4/16, and 3/16 patients, respectively.
Tropomyosin and aldolase A were identified in the 35-40 kDa region, while PV and
triosephosphate isomerase were in the 20-30 kDa region. Additional proteins detected in
these regions include glyceraldehyde-3-phosphate dehydrogenase and myosin light chain.
Patients with weak or no IgE reactivity to the PV monomer and collagen, showed the
strongest binding to one of these 2 regions, applicable for 5/16, 6/16, and 1/16 patients for
cod, tuna, and salmon respectively. Half of the patients showed weak to moderate IgE
reactivity to β-enolase (50 kDa) from tuna (10/16) and salmon (8/16) extracts. No β-enolase
band was detected in any of the cod extracts.
4 Conclusions
This study demonstrates a considerably high heterogenicity in protein and allergen content in
SPT extracts used in routine diagnosis of fish allergy. Twenty-six commercial fish extracts
were evaluated through a comprehensive biochemical and immunological analysis of the
complete protein and allergen content, using immunoblotting and advanced mass
spectrometric analysis. We found the total protein concentration between 26 fish extracts
varying up to 17-fold, from the lowest in flounder-2 (0.17 mg/ml) to the highest in tuna-1
(2.94 mg/ml). In contrast, comparable small variations in total protein content (up to 7-fold)
were previously reported for 14 commercial fish SPT extracts 15. Subsequently using a PV-
specific antibody, PV was detected in all 4 analysed salmon extracts and none of 3 tuna
extracts 15. In our study, using an in-house antibody, PV was detected in most of the 26
extracts except in 3 of 4 tuna, 1 of 3 salmon and 1 of 3 flounder/halibut extracts.
The difference in PV content and antibody reactivity among tuna extracts is likely explained
by the different species utilised, as none of the manufacturers provided the details of the exact
species. A comparative analysis of in-house raw and heated extracts from yellowfin tuna
(Thunnus albacares) and albacore tuna (T. alalunga) demonstrated over 100-times stronger
antibody reactivity for the latter (unpublished data). We therefore conclude that the analysed
extracts are likely derived from very different tunas, highlighting the importance of choosing
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the appropriate region-specific species when preparing SPT extracts or performing prick-to-
prick tests using fresh fish.
Subsequent analysis of all soluble proteins by mass spectrometry demonstrated that protein
diversity varied greatly between the extracts. PV, tropomyosin, and/or collagen were not
detected in 3/5 cod and 2/4 tuna extracts, while aldolase A and β-enolase were detected in all
12 commercial cod, tuna, and salmon extracts. For example, 155 protein groups were
identified in 5 cod extracts, while the total number of identified proteins groups varied from
217 to 349, indicating very different methods of generating the commercial extracts between
manufacturers.
A recent study by Asero et al. demonstrated the unsuitability of 3 commercial prawn extracts,
using SDS-PAGE and antibody-based approaches, due to the absence of important shellfish
allergens 27. In our study, we present for the first time a comprehensive proteomic approach
using mass spectrometry for a fast and reliable evaluation of commercial SPT extracts for
their utility in allergy diagnostics. We demonstrate that 5/9 commercial cod and tuna extracts,
examined by mass spectrometry, lack important fish allergens.
Among the 6 cod, tuna and salmon extract analysed in detail, using 4 antibodies detecting
fish allergens, we confirmed the large heterogenicity in allergen content. Heat-labile
aldolase A was detected in all in-house raw extracts as well as the 3 commercial extracts with
a high total protein concentration. The same 3 extracts also showed a comparable high PV
and tropomyosin content. In contrast, collagen, poorly extracted without heat treatment, was
only detected in in-house heated extracts, but not in the raw or any SPT extracts. Therefore,
we can assume that these 6 extracts were not heat-treated.
The total in vitro IgE reactivity to commercial fish extracts depend on antigen content and
differed between patients and fish species. In the Asia-Pacific region, clinicians often
consider reactivity to cod as a marker for allergy to any white fish. According to our data,
even the cod SPT extract with the highest antigen amount showed less reactivity than salmon
and tuna extracts with higher antigen content. This study highlights region- and species-
specific IgE reactivity to fish, leading possible to false-negative diagnostics 6. Species-
specific sensitisation was also demonstrated in other studies for salmonids, tilapia, catfish,
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and angler 28-30. However, the patient- and fish species-dependant reactivity requires further
investigations with a larger patient cohort and region-specific species.”
Comparing the IgE reactivity of serum from patients with clinically confirmed fish allergy to
cod and tuna, 11/16 patients showed the strongest IgE reactivity to collagen, tropomyosin,
aldolase A, and β-enolase and 5/16 to PV. In contrast, for salmon PV was the most IgE-
reactive in 12/16 (75%) patients. Several studies have previously indicated that the PV
content is species-specific affecting allergenicity, however no additional fish allergens were
investigated 15,31-33. While it is generally accepted that PV is the major fish allergen
accounting for 70-95% of allergic reactions, here we demonstrate the importance of
additional fish allergens in different fish species 4.
Analysing the IgE reactivity for in-house heated extracts, collagen was the second most IgE-
reactive fish protein after PV, with up to 9/16 Australian fish allergic patients showing strong
reactivity to this allergen. Initial protein analysis confirmed the differential abundance of
collagen, which was 4- and 9-times higher in cod as compared to tuna and salmon,
respectively. However, in all analysed commercial extracts collagen was underrepresented,
resulting in low IgE reactivity in serum from patients sensitised to the fish allergen collagen.
In a recent study, prick-to-prick testing with raw fish resulted in false-negative results due to
lack of collagen 34. Several studies from Japan confirmed the importance of collagen as a fish
allergen and highlighted the high collagen content in the skin 21,35. According to the
ImmunoCAP results, one patient in our cohort was not sensitised to fish, but showed strong
IgE reactivity to only collagen. The next generation of improved SPT extracts should
consider the utilisation of fish skin and heat treatment. The possible introduction of purified
collagens in in vitro fish allergy diagnostics should also be evaluated in future studies.
In conclusion, many commercial fish extracts contain insufficient amounts of allergens such
as PV and collagen, resulting in low IgE reactivity and limited suitability for reliable in vivo
allergy testing. We recommend either standardisation and regulation of protein concentration
and profiles in commercial allergen extracts or the use of improved in vitro diagnostics
implementing a large variety of fish species and purified allergens. Further research must be
conducted to meet the urgent need of region- and species-specific diagnostics, allowing
improved management of this life-threatening disease.
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Table 1: Clinical characteristics and relevant sIgE profiles of 16 fish-allergic patients
recruited for the study.
Patients ImmunoCAP results Implicated
fish* Symptoms # Sex Age
(yr) Cod Tuna Salmon
(kU/L) Class (kU/L) Class (kU/L) Class
1 M 11 88.50 5 4.83 3 25.30 4 Tuna and white fish
AE, GIS, OAS, U
2 F 1 5.47 3 5.14 3 5.58 3 Asian
seabass AE, RD, U
3 M 11 92.10 5 39.50 4 66.10 5 Salmon AE
4 F 2 90.40 5 31.00 4 73.40 5 Cod fish AE, U
5 M 8 75.00 5 21.00 4 70.50 5 White fish GIS, RD, U
6 F 7 2.87 2 1.19 2 3.08 2 White fish U
7 M 4 25.50 4 17.30 3 21.00 4 White fish U,
abdominal pain
8 F 4 1.54 2 0.71 2 0.47 1 Basa fish AE, E, U
9 F 12 55.00 5 14.90 3 38.90 4 Flathead AE
10 M 12 7.01 3 3.90 3 31.80 4 Salmon, tuna
E, U
11 M 14 0.10 1 0.28 1 0.21 1 Salmon AE
12 F 2 0.14 1 0.78 2 0.60 1 Salmon AE, U
13 M 8 11.50 3 4.67 3 9.80 3 Salmon OAS
14 M 14 1.42 2 0.71 2 1.60 2 Salmon OAS, U
15 M 18 3.36 2 1.06 2 3.21 2 Tuna RD, U
16 M 1 <0.01 0 <0.01 0 <0.01 0 Tinned salmon
AE, U
Note: AE, angioedema (lip swelling); E, eczema; GIS, gastrointestinal syndrome (vomiting); OAS, oral allergy syndrome (itchy mouth or swelling throat); RD, respiratory distress; U, urticaria (erythema, hives, rash). *Fish species which was associated with the clinical reaction upon ingestion.
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Figure legends
Figure 1: Protein concentration, SDS-PAGE profile, and PV-specific antibody reactivity
of 26 different fish SPT extracts from 5 different manufacturers.
Protein concentrations were determined and the mean values from 3 replicates with the
corresponding standard deviation are shown (A). The extracts were further analysed by SDS-
PAGE (B) and immunoblot with anti-PV antibody (C). Purified PV from Atlantic cod,
yellowfin tuna, and Atlantic salmon was used as positive control for the cod, tuna, and
salmon SPT extracts. The numbers in superscript on the common species names represent the
different manufacturers listed in Table S1.
Figure 2: Protein groups and fish allergens detected in cod, tuna, and salmon SPT
extracts.
Mass spectrometric analyses were performed after in solution tryptic digestion. Proteins were
identified by mascot and groups were defined using Proteome Discoverer (Thermo
Scientific). For each SPT extract the total number of identified protein groups is given in
brackets. The Venn diagram (InteractiVenn) shows the number of shared protein groups
between the 5 cod (A), 4 tuna (B), and 3 salmon (C) SPT extracts. Detected fish allergens are
indicated by solid field (D). The numbers after species name in A-C represent the different
manufacturers listed in Table S1.
Figure 3: Protein profile and allergen-specific reactivity of selected cod, tuna, and
salmon extracts.
The two SPTs with the lowest protein concentration (cod-2/2, tuna-3/3, salmon-2/2) and the
highest (cod-5/5, tuna-1/1, salmon-1/1) were compared with in-house prepared raw (RE) and
heated (HE) protein extracts from the corresponding species. Proteins were separated by
SDS-PAGE (A) and allergens identified using specific antibodies for PV (B), tropomyosin
(C), aldolase A (D), and collagen (E). Heatmaps were generated for the protein concentration
of each SPT extracts (F), the allergen abundance on the gel (G), and reactivity of the
allergen-specific antibodies (H). *The identity of proteins extracted from bands was
confirmed by mass spectrometric analyses. The superscript numbers on the fish species
represent the different manufacturers listed in Table S1. Note: The colouring of the boxes
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represent intensity, with red being the highest, orange 50% and green 0% of the highest
band/signal intensity. The colour scheme is valid for each row only and not comparible with
shading in other figures of the manuscript. TM, tropomyosin; G3PD, glyceraldehyde-3-
phosphate dehydrogenase; MHC, myosin heavy chain; PV, parvalbumin; n.d., not
determined.
Figure 4: Patients’ total IgE reactivity to proteins to selected cod, tuna, and salmon SPT
extracts.
The IgE reactivity of 16 patients to the two SPTs with the lowest protein concentration (cod-
2, tuna-3, salmon-2) and the highest (cod-5, tuna-1, salmon-1) was compared. The numbers
after the species name represent the different manufacturers listed in Table S1. The patient
number corresponds to Table 1. Note: The colouring of the boxes represent intensity, with red
being the highest, orange 50% and green 0% of the highest total IgE reactivity of the patient.
The colour scheme is valid for each row only and not comparible with shading in other
figures of the manuscript.
Figure 5: Allergogram analysis of patients’ IgE reactivity to allergens in different cod,
tuna, and salmon extracts.
The IgE reactivity of 16 patients to the two SPTs with the lowest protein concentration (cod-
2, tuna-3, salmon-2) and the highest (cod-5, tuna-1, salmon-1) was compared with in-house
prepared raw (R) and heated (H) protein extracts from the corresponding species. The identity
of proteins extracted from IgE-reactive bands (Figure S1) was confirmed by mass
spectrometric analyses. The numbers after the species name represent the different
manufacturers listed in Table S1. The patient number corresponds to Table 1. Note: The
colouring of the boxes represent intensity, with red being the highest, orange 50% and green
0% of the highest IgE reactivity of the patient to extracts. The colour scheme is valid for each
patient and species only and not comparible with shading in other figures of the manuscript..
PV, parvalbumin; MLC, myosin light chain; TPI, triosephosphate isomerase; TM,
tropomyosin; G3PD, glyceraldehyde-3-phosphate dehydrogenase; MHC, myosin heavy
chain.
Supporting information
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Table S1: Details of analysed 26 Skin Prick Test fish extracts from 5 different
manufacturers.
Figure S1: IgE reactivity of 16 fish-allergic patients to cod, tuna, and salmon extracts.
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