Received date: June 23, 2015 Accepted date: July 27, 2015 Published date: August 03, 2015
Citation: Yadzir ZHM, Misnan R, Bakhtiar F, Abdullah N, Abdullah H, et al. (2015) Production of Allergen Extracts for Bivalve Allergy Diagnosis Using Skin Prick Test. J Allergy Ther 6:216. doi: 10.4172/2155-6121.1000216
Copyright: © 2015 Yadzir ZHM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Background: Whilst the consumption of bivalves is reasonably high in Malaysia, the frequency of allergy to this group of shellfish in the local population is largely unknown. The aim of this preliminary study was to produce bivalve allergen extracts and to investigate the frequency of bivalve sensitization among the local atopic population. Methods: Raw allergen extracts were prepared from 5 different species of bivalves. Their protein profiles were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results: In SDS-PAGE, the bivalve extracts demonstrated 10 to 23 protein bands. The five protein profiles varied considerably but most visible protein bands lay within 25-100 kDa. At the same time, fifty patients with a history of atopy were skin prick tested (SPT) with these raw extracts of bivalves. Of the 50 subjects, 13 (26%) had a positive SPT to at least one of the 5 bivalve extracts tested, 8 (61%) reacted to one bivalve extract while only one (8%) reacted to all 5 bivalve extracts. The frequency of skin test reactivity to Malaysian cockle was the highest at 22%, followed by Carpet clam, 12% and 4% to the other 3 bivalve extracts; Tropical oyster, Asian clam and Asian green mussel. Conclusion: This study showed that of the five different bivalve species, cockle was found to have the highest frequency in skin test reactivity. It appears that an individual, who is sensitized to a mite, is also likely to be sensitized to bivalves.
Mollusc; Bivalve; Allergy; Atopy; Skin prick testing; Allergen extracts; Mites
Molluscs are frequently grouped together with crustaceans under the term shellfish. However, molluscs represent a completely separate phylum (Mollusca), whilst crustacean shellfish are classified under the phylum Arthropoda. The most important divisions of the phylum Mollusca are the classes Bivalvia (clam, cockle, mussel, scallop and oyster), Cephalopods (octopus, cuttlefish and squid) and Gastropods (abalone and snail) .
Molluscs play an ever-increasing role in the human diet and the world economy . Molluscan shellfish meat is recommended in several dietary regimes for their high protein content, low fat/cholesterol profile, the presence of good lipids, significant amounts of omega-3-fatty acids, dietary essential amino acids, vitamin B12 and several important minerals such as zinc, iron and copper [3,4]. However, molluscan shellfish allergy is receiving attention . Molluscan shellfish including bivalves are considered to be among a group of allergenic foods. The Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO)  have identified shellfish, including molluscs, as one of the eight major sources of food allergens. The European Union has also added molluscan shellfish to the list of most commonly allergenic foods in Europe .
The prevalence of allergy to bivalves varies throughout the world. It is likely to parallel consumption patterns being more frequent in locales where consumption is high . IgE-mediated bivalve allergy has been described in several countries including France , Spain , South Africa , Japan [12,13], Canada , Italy [15,16], Germany , Sweden, Denmark, Estonia Lithuania and Russia .
Symptoms of bivalve’s allergy are largely similar to those of allergic reactions reported for other foods. Common symptoms include mild oral allergy (itching of the lips, mouth and throat and swelling of the lips, tongue, throat and palate), cutaneous reactions (urticaria, eczema), respiratory symptoms (asthma, rhinitis), gastrointestinal symptoms (diarrhoes, vomiting) and systemic reactions (anaphylactic shock) [8,19]. Allergies to bivalves have also been documented in work places that handle bivalves. These have been found to occur primarily through inhalation of aerosols generated during cutting, scrubbing or cleaning, cooking or boiling and drying [20-22]. Reactions can also occur through the skin as a result of directly handling the bivalves [23,24].
Allergies to bivalves are diagnosed similarly to other food allergies. Double-blind, placebo-controlled food challenge (DBPCFC) is the gold standard to establish a food allergy but is a time-consuming and expensive technique not widely practised outside research centers. Skin prick tests (SPTs) used in conjunction with history and physical plus clinical judgment is acceptable practice for diagnosing IgE-mediated food allergy .
In Malaysia, bivalves have long been part of the diet of the local population . The major edible bivalves that are frequently consumed include clams, cockles, mussels and oysters. Although bivalves are likely the most frequently ingested class of molluscan shellfish, local data on allergy to bivalves is not available. In this preliminary study, we investigated the frequency of bivalve sensitization among the local atopic population by skin prick tests (SPT) using in house allergen extracts prepared from local bivalves.
Five commercially important and commonly consumed bivalve species were used in this study: Anadara granosa (Malaysian cockle/ Kerang), Crassostrea belcheri (Tropical oyster/Tiram tropika), Corbicula fluminea (Asian clam/Kepah Asia), Perna viridis (Asian green mussel/Kupang hijau Asia) and Paphia textile (Carpet clam/Lala). All bivalve samples were purchased directly from the local market. The bivalve samples were stored in the laboratory freezer at -20°C to reduce biological deterioration prior to analysis.
Study subjects include 50 out-patients and in-patients with atopic disease referred to the Allergy Clinic, Hospital Kuala Lumpur (HKL). Subjects were enrolled according to the predetermined inclusion and exclusion criteria as stated:
Inclusion criteria: Subjects aged ≥ 18 years old.
Subjects had a history of allergy-related diseases such as allergic rhinitis, asthma, sinusitis or allergic conjunctivitis.
Skin prick test (SPT) results showed positive reactions to at least one type of allergen on the allergen panels tested.
Exclusion criteria: Pregnant women.
Received anti-histamine within 72 hours of consultation.
Subjects have chronic skin disease (dermographism).
This study received ethics approval from the Medical Research and Ethics Committee (MREC), Ministry of Health Malaysia and patients gave their informed consent before participating in the study.
Preparation of bivalve allergen extracts
In the preparation of the bivalve extracts, the shell was split open and the inner muscle tissue was used for protein extraction. About 20 g of the muscle mass was homogenized in 200 ml of 0.1 M phosphate buffered saline (PBS), pH 7.2 for 10 min using a Waring blender. This homogenate was then agitated overnight at 4°C, followed by centrifugation at 4,500 and 14,000 rpm, for 30 and 15 min, respectively. The recovered, clear supernatant was sterilized by passage through a 0.22 μm syringe filter, frozen and lyophilized. The lyophilized extracts were stored at -20°C until further use. The protein content of the extracts was estimated using the Total Protein Kit (Sigma, USA).
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE was carried out under denaturing conditions with the stacking and separating gels containing 5% and 12% of acrylamide, respectively. Bivalve extracts were dissolved in Laemmli sample buffer in the presence of 5% 2-mercaptoethanol and heated at 97°C for 4 min before being loaded onto the gel. Each lane was loaded with 10 μl of sample, containing 10 μg of protein or protein standards before the proteins were separated for 45 min at 120 mA and 200 V using the Mini-Protean 3 system (Biorad, USA). The separated proteins were then visualized by staining with Coomassie brilliant blue R-250. Molecular weights of the protein bands were determined by comparison with molecular weight markers using an Imaging Densitometer GS800 and Quantity One Software (Biorad, USA).
Skin prick test (SPT)
The SPTs were carried out on the forearm using sterile lancets. A drop of the allergen extract was placed on the forearm after which the skin under the drop was pricked with a sterile lancet. The size of the wheal was recorded after 15 minutes: a wheal 3 mm or greater than the negative control was regarded as positive. Saline solution and histamine hydrochloride 1% served as negative and positive controls, respectively (Alk Abello, Spain).
Data recorded included demographic data, clinical history and symptoms of allergy and SPT results. All data were analyzed using the Statistical Package for Social Sciences (SPSS) software. Simple associations were assessed with frequency tables and Fisher’s exact test for two independent proportions. P-values less than 0.05 were considered statistically significant.
Total protein content of bivalve extracts
Table 1 shows the total protein content of bivalve allergen extracts. Overall, the total protein content of the five species of bivalves fell in the range between 3.5 mg/ml to 5.2 mg/ml. Corbicula fluminea (Asian clam/Kepah Asia) showed the highest protein content at 5.2 mg/ml while Paphia textile (Carpet clam/Lala) was found to have the lowest at 3.5 mg/ml.
|Species||Total Protein (mg/ml)|
|Anadaragranosa (Malaysian cockle/ Kerang)||4|
|Crassostreabelcheri(Tropical oyster/ Tiramtropika)||3.8|
|Corbiculafluminea (Asian clam/ Kepah Asia)||5.2|
|Pernaviridis (Asian green mussel/ Kupanghijau Asia)||4.8|
|Paphia textile (Carpet clam/ Lala)||3.5|
Table 1: Total protein content of bivalve allergen extracts.
Protein profile of bivalve extracts
The SDS-PAGE of bivalve extracts demonstrated 10 to 23 protein bands (Figure 1). The protein bands were in the wide molecular weight range of 13-250 kDa. The protein profiles of the 5 bivalve extracts varied considerably, although majority of the visible protein bands ranged between within 25-100 kDa.
Fifty subjects, 20 males, 30 females, with a history of atopy were studied. Table 2 shows the demographic data of these subjects. The mean age was 29.9 years with ages ranging between 18 to 58 years. Of the 50 subjects, 13 (26%) had a positive SPT to at least one of the 5 bivalve extracts tested.
|Demographic Data||Subjects||Subjects sensitized to bivalves|
|(n = 50), % (n)||(n = 13), % (n)|
|Male||40 (20)||77 (10)|
|Female||60 (30)||23 (3)|
|Malay||68 (34)||76 (10)|
|Chinese||8 (4)||8 (1)|
|Indian||18 (9)||8 (1)|
|Others||6 (3)||8 (1)|
|18-24||46 (23)||54 (7)|
|25-34||32 (16)||38 (5)|
|35-44||10 (5)||8 (1)|
|45-54||2 (1)||0 (0)|
|≥55||10 (5)||0 (0)|
Table 2: Demographic data of the subjects.
In the group of subjects that were found to be sensitized to bivalves, males appeared to be more prevalent than females. Most subjects were in the 18-24 year age group. In terms of the subject’s ethnicity, Malays comprised the majority.
Clinical history and symptoms of allergy
Table 3 summarizes the clinical histories of these subjects. Most of the subjects were allergic rhinitis patients, 78%, followed by allergic conjuctivitis patients, 46%. Clinical symptoms were shown in Table 4. The most common presenting symptom was rhinorrhoea, 64%.
|Clinical History||Subjects||Subjects sensitized to bivalves|
|(n = 50), % (n)||(n = 13), % (n)|
|Allergic rhinitis||78 (39)||85 (11)|
|Allergic conjuctivitis||46 (23)||46 (6)|
|Eczema||18 (9)||23 (3)|
|Asthma||16 (8)||23 (3)|
|Sinusitis||12 (6)||8 (1)|
|Urticaria||2 (1)||0 (0)|
Table 3: Clinical history of the subjects.
|Clinical Symptoms||Subjects||Subjects sensitized to bivalves|
|(n = 50), % (n)||(n = 13), % (n)|
|Nasal||Running nose||64 (32)||77 (10)|
|Blocked nose||58 (29)||77 (10)|
|Sneezing||50 (25)||62 (8)|
|Itchy nose||34 (17)||54 (7)|
|Loss/decrease of sense of smell||2 (1)||8 (1)|
|Mouth breathing/snoring||2 (1)||8 (1)|
|Sniffling||2 (1)||8 (1)|
|Eye||Redness||40 (20)||62 (8)|
|Itching||52 (26)||62 (8)|
|Watery||26 (13)||23 (3)|
|Dark circles||2 (1)||0 (0)|
|Puffiness||8 (4)||15 (2)|
|Eye discharge||12 (6)||8 (1)|
|Sinus||Headaches||6 (3)||8 (1)|
|Sore throats||2 (1)||8 (1)|
|Post nasal drip||2 (1)||0 (0)|
|Bad breath||4 (2)||8 (1)|
|Hoarseness||2 (1)||0 (0)|
|Throat itchiness||2 (1)||8 (1)|
|Skin||Rash||8 (4)||15 (2)|
|Hives||10 (5)||8 (1)|
|Eczema||12 (6)||15 (2)|
|Pruritus||6 (3)||0 (0)|
|Blisters||2 (1)||0 (0)|
|Ear||Ear discharge||2 (1)||0 (0)|
|Painful||2 (1)||0 (0)|
|Ringing||2 (1)||0 (0)|
|Itching||2 (1)||0 (0)|
|Chest||Wheezing||2 (1)||0 (0)|
|Coughing||6 (3)||8 (1)|
|Tightness||2 (1)||0 (0)|
|Shortness of breath||2 (1)||8 (1)|
Table 4: Clinical symptoms of the subjects.
Skin prick test (SPT) reactivity
Table 5 shows the SPT reactivity to various allergens tested. Most of the subjects showed positive reactions to aeroallergens and seafood allergens. Mites were among the aeroallergens that elicited the highest sensitivity in 76% (n=38) of the subjects while amongst the seafood allergens, prawn was found to be the most common allergen in 48% (=24) subjects.
(n = 50), % (n)
|Malaysian cockle||22 (11)|
|Carpet clam||12 (6)|
|Tropical oyster||4 (2)|
|Seafood||Asian clam||4 (2)|
|Asian green mussel||4 (2)|
|Royal jelly||12 (6)|
|Black sesame||12 (6)|
|Cat dander||30 (15)|
|Anisakis simplex||8 (4)|
Table 5: SPT positivity to various allergens.
Amongst the bivalve extracts tested, Malaysian cockle elicited the highest frequency of positive skin test reactivity in 22% (n=11) of the subjects. Carpet clam was the second most common bivalve to elicit a positive reaction in the SPT, 12%, followed by Asian clam, 4%, Tropical oyster, 4% and Asian green mussel, 4%.
In the group of subjects that were sensitized to bivalves, more than half of the subjects, 61% (n=8) displayed a positive skin reaction to a single bivalve extract (monosensitive) and 23% (n=3) were in the oligosensitive group with positive skin reactions to two bivalve extracts. Only one patient had positive skin reaction to all the bivalve allergens tested (Table 6).
|Number of bivalve allergens||Number of subjects positive SPT|
|(n = 13), % (n)|
Table 6: Distribution of positive SPT in the group of subjects that were sensitized to bivalves.
Among subjects positive to bivalves, significant cross sensitization was found between bivalves and different types of allergens; specifically crustacean shellfish (prawn and crab), arachnids (mites), insects (cockroach) and other molluscan shellfish (squid) (Table 7).
|Crab||< 0.001 *|
Table 7: Association of bivalve allergens SPT results with other allergens.
SPT is a safe, sensitive and rapid method for screening patients with suspected allergy to shellfish [27,28]. However, the limited availability of commercial SPT reagents for this group  indicates a need for the production of in-house bivalve allergen extracts for SPT.
The SDS-PAGE of the 5 bivalve extracts revealed individual protein profiles that varied considerably although the majority of visible protein bands lie within the 25-100 kDa range. These differences might be influenced by endogenous and exogenous factors [4,30]. The endogenous factors consist of genetic differences, physiological status, reproductive cycle and feeding habits. The exogenous factors include habitat, abundance of food available, temperature, size, dissolved organic matter/debris, soil composition, starvation and time available for feeding [4,30].
Allergic reactions to bivalves occur not only after ingestion of cooked bivalves, but they may also be triggered by raw bivalves, particularly after direct contact [23,24] or inhalation of bivalve odours or fumes [20-22]. Therefore, in this study, the raw extract was used in skin prick tests, predominantly to avoid missing IgE-binding proteins. Additionally, raw extract contains both thermostable and thermolabile proteins, whereas the cooked extract comprises only thermostable proteins.
In our study, it appears that the majority of our subjects were allergic rhinitis patients. Allergic rhinitis is a highly prevalent disease affecting 20% of the general population in developed countries . The most common cause of allergic rhinitis is allergic sensitization to aeroallergens with most persistent aeroallergens being found indoors . Mites are the best described persistent indoor allergens and the warm and humid tropical climates offer favourable conditions for them to thrive. In our skin prick test, house dust mites elicited the highest positive reactions of all allergens studied.
Furthermore, the present study was carried out in the Allergy Clinic, Hospital Kuala Lumpur, which caters for patients who are mainly urban dwellers. Recent epidemiological studies suggest that urban living and exposure to air pollution at home and work are risk factors for allergic rhinitis [32,33]. This may be due to the exposure to ozone, nitrogen dioxide and especially particulate matter related to traffic and industrial activities . In contrast, those who live in rural areas showed a considerably lower prevalence of allergic rhinitis [33,34].
In this study, amongst the bivalve extracts tested, Malaysian cockle was found to elicit the highest frequency of skin test reactivity. The reason for this is not clear. However, it could be that this is due to the low price of the Malaysian cockle in the local markets and its consequent higher consumption as compared to other bivalve species.
It is apparent that an individual sensitized to a bivalve, is also likely to be sensitized to crustacean shellfish (prawn and crab), arachnids (mites), insects (cockroach) and other molluscan shellfish (squid). This is possibly due to the IgE cross-reactivity of proteins which could be derived amongst the crustaceans itself, molluscs itself, between crustaceans and molluscs, and between crustaceans and terrestrial arthropods, such as cockroaches and mites . Cross-reactivity is caused by homologous proteins containing conserved sequence motifs, which are IgE-binding epitopes .
The principal allergen in many molluscs is the protein tropomyosin, which is also the major allergen in many crustaceans [8,36-42]. Tropomyosin is also considered to be responsible for cross-reactivity between other arthropods such as mites or cockroach [43,44]. Tropomyosin showed very high homologies of up to 98% among crustacean species, including crawfish, crab and lobster . Crustacean allergic subjects also often react to species of the mollusc group. Leung et al.  demonstrated in vitro that sera from nine crustacean allergic patients had IgE binding to antigens from all ten mollusc species tested. Furthermore, Reese et al.  reported that the amino acid sequence identity between mollusc tropomyosins varies from 68 to 88% and between crustaceans and molluscs is 56-68%. Reese et al.  also demonstrated that tropomyosin from house dust mite has 75-80% homology to shrimp and fruitfly tropomyosin and 65% homology to mollusc tropomyosin. Tropomyosins from house dust mite and cockroach have high sequence identities to shellfish tropomyosin of around 80%.
In conclusion, this study showed that of the five different bivalve species, cockle was found to have the highest frequency in skin test reactivity. It appears that an individual who is sensitized to a mite, is also likely to be sensitized to bivalves. However, these preliminary findings are derived from a relatively small group of subjects and extension of this study with a larger study group is clearly warranted.
The authors thank the Director General of Health, Malaysia for permission to publish this paper. This work was supported by a grant UPSI 2013-0197-102-01.
We declare that we have no conflicts of interest.