When apple allergy is associated with birch pollinosis symptoms generally appears within 5-15 min and comprise local reactions of the mucosa of the upper aero-digestive tract with itching, inflammation and angioedema (called oral allergy syndrome, OAS). (Pastorello et al. 1999) [157]; (Anhøj et al. 2001) [591].

When apple allergy is not linked to birch pollinosis the most frequent clinical presentation is not OAS, but systemic symptoms such as generalised urticaria and anaphylaxis are more commonly observed, specially in those patients without an associated pollen allergy. (Fernandez-Rivas et al. 1997) [1060]. In these patients, clinical data also suggests that adverse reactions appear more frequently and are more severe when fruits are eaten with the peel (Fernandez-Rivas and Cuevas 1999) [833].

• Whole fruit (cvs. Golden Delicious, and/or Gloster): Rodriguez et al. (2000) [491]; Skamstrup Hansen et al. (2001) [589] and (2001) [590], Asero et al. 2001 [596], Crespo et al. (2002) [834]
• Pulp (cv. Golden Delicious): Fernandez-Rivas and Cuevas (1999) [157], Anhøj et al. (2001) [591], Osterballe et al. (2003) [581]
• Peel (cv. Golden Delicious): Fernandez-Rivas and Cuevas (1999) [157], Osterballe et al. (2003) [581]

Whole-apple extract:

• French cv. Golden Delicious and German cv. Gloster apples (Skamstrup Hansen et al. (2001) [590]
• cv. Golden delicious apple peel and pulp (Asero et al. 2001) [596]

Commercial apple extract: Anhøj et al. (2001) [591], Osterballe et al. (2003) [581].

Low temperature acetone powder extract of apple (A72): Osterballe et al. (2003) [581].

Recombinant Mal d 1: Osterballe et al. (2003) [581]

Purified Mal d 1: Skamstrup Hansen et al. (2001) [590]

Purified Mal d 3: Garcia-Selles et al. (2002) [594]

Authors agreed that although the diagnostic value of the prick-prick technique with fresh apple is useful, it is still very problematic that the expression of the major allergen Mal d1 varies between different apple strains and maturation stages (Osterballe et al. 2003) [581]. It is also of concern that fruit allergen extracts are often readily degradable or contain clinically irrelevant cross-reacting epitopes, resulting in diagnostic discrepancies (Anhøj et al. 2001) [591].

In most of the studies skin prick tests (SPTs) were considered positive when the wheal area was ≥7 mm² (diameter ≥3 mm).

Asero et al. (2001) [596] described negative SPTs wheals <2 (with diameters <50% of the diameter of the positive control which was 10 mg/ml histamine).

Osterballe et al. (2003) [581] also peformed scratch-chamber test (SCT) which was regarded as positive with a wheal-and-flare reaction.

33 patients showing clinical symptoms after the ingestion of apple, peach and/or pear (at least one of them) and a positive SPT response to the involved fruit/s. 22 patients had an associated grass pollinosis (fruit and pollinosis group) and 11 did not present any type of inhalant allergy (fruit group). Adverse reactions to apple were reported by 25 subjects, 15 of the fruit and pollinosis group and 10 patients of the fruit group. (Fernandez-Rivas and Cuevas, 1999) [833].

22 patients with adverse reactions to Rosaceae fruits is studied; 9 patients reported adverse reactions to apple which were confirmed by a DBPCFC in 6. Rodriguez et al. (2000) [491]

36 grass- and or birch-allergic patients based on a clinical history followed by an oral food challenge test and SPT with fresh fruits. The control group comprised five nonatopics and 12 birch and grass allergics with no symptoms of OAS and frequently eating fresh fruits. All controls had negative SPT to fresh fruits. Anhøj et al. (2001) [591]

36 patients with OAS after ingestion of fruits. 18 patients did not have birch pollen sensitivity and the other 18 had bich pollinosis (Asero et al. 2001) [596].

65 birch pollen-allergic patients with a history of rhinitis in the birch-pollen season, positive specific IgE to birch and a positive open oral challenge with apple. Skamstrup Hansen et al. (2001) [589].

26 patients based on a clinical history of rhinoconjunctivitis in the birch pollen season and OAS or other allergic manifestations upon ingestion of apple, together with positive specific IgE and a positive SPT to birch pollen. Skamstrup Hansen et al. (2001) [590]

10 adults diagnosed with IgE-mediated clinical allergy to apple from history and by skin tests (SPT), and/or food-specific IgE antibodies, and oral challenge. Crespo et al. (2002) [834].

10 patients with clinical allergy to birch pollen, a positive SPT to birch, and a positive case history of OAS to apple and 10 nonatopics without birch pollinosis, no symptoms of OAS and a negative SPT to birch as control subjects. Osterballe et al. (2003) [581]

Fernandez-Rivas and Cuevas (1999) [833] found that more than 40% of patients allergic to apple tolerated the ingestion of the pulp, and reactions were only elicited by the intake of the whole fruit. SPTs with peels of apple induced significantly higher responses than the pulps, both in fruit and pollinosis and in fruit allergic patients.

Anhøj et al. (2001) [591] found that the diagnostic sensitivity of the SPT with fresh apple fruit was 92% and specificity of 72%. Good concordance (test efficiency of >80%) was found between case history, SPT, and oral challenge tests. The negative predictive value of the SPT was >90%. A combination of case history of Oral Allergy syndrome with a SPT or basophil histamine release with fresh apple resulted in >90% positive predictive value.

In both patients groups with and without birch pollinosis, the SPTs with fresh apple and apple peel extract were positive and negative with apple pulp. In non birch pollen-allergic patients, the SPTs with fresh apple gave inconsistent results, therefore Asero et al. (2001) [596] suggests diagnosis of vegetable food hypersensitivity in birch pollen-allergic patients using fresh foods but fruit peel extracts for patients not allergic to birch pollen.

Skamstrup Hansen et al. (2001) [589] reported that groups I and II had higher skin wheal area to birch than group III (P=0.02/0.04). The freeze-dried apple powder proved to be useful for SPT but further investigation of the stability and the allergenic profile of the material is needed.

Skamstrup Hansen et al. (2001) [590] could not confirm an increase in reactivity during season by SPT.

Crespo et al. (2002) [834] SPT determinations were positive to apple in 23/30 (77%).

Osterballe et al. (2003) [581] demonstrated that the SPT had a high sensitivity to fresh apple peel (80%), apple pulp (70%), A72 (90%), birch (100%), and low to the commercial apple extract (10%). The authors concluded "In daily practice a detailed case history about symptoms of oral allergy syndrome combined with a SPT with fresh apple peel or low temperature acetone powder extract of apple will be useful"

• Whole fruit cv. Golden Delicious: Skamstrup Hansen et al. (2001) [589] and (2001) [590], Osterballe et al. (2003) [581], Rossi et al. (2003) [835].
• Pulp (cv. Golden Delicious): Fernandez-Rivas and Cuevas (1999) [833], Anhøj et al. (2001) [591], Osterballe et al. (2003) [581]
• Peel (cv. Golden Delicious): Fernandez-Rivas and Cuevas (1999) [833], Osterballe et al. (2003) [581]

• Rossi et al. (2003) [835] expressed the results in classes of positivity from 0 to 6, where class 0 corresponds to <0.35 kUA/l, class 1 to 0.35-0.7 kUA/l, class 2 to 0.7-3.5 kUA/l, class 3 to 3.5-17.5 kUA/l, class 4 to 17.5-50 kUA/l, class 5 to 50-100 kUA/l and class 6 to >100 kUA/l.
• Measurable specific IgE to apple was classified as a positive when CAP = 0.35 kU A /L by Osterballe et al. (2003) [581]

A histamine release test (HRT) was performed considered positive:

• A release greater than 10% (Fernandez-Rivas and Cuevas (1999)) [833].
• An AUC of >40 ng/ml and a release of >20 ng/ml histamine (Anhøj et al. (2001)) [591].
• Maximal release is ≥14 ng/ml (Skamstrup Hansen et al. (2001) [589]).
• A release greater than 10 ng/ml (Osterballe et al. (2003) [581])

33 patients showing clinical symptoms after the ingestion of apple, peach and/or pear (at least one of them) and a positive SPT response to the involved fruit/s. 22 patients had an associated grass pollinosis (fruit and pollinosis group) and 11 did not present any type of inhalant allergy (fruit group). Adverse reactions to apple were reported by 25 subjects, 15 of the fruit and pollinosis group and 10 patients of the fruit group. (Fernandez-Rivas and Cuevas, 1999) [833].

36 grass- and or birch-allergic patients based on a clinical history followed by an oral food challenge test and SPT with fresh fruits. The control group comprised five nonatopics and 12 birch and grass allergics with no symptoms of OAS and frequently eating fresh fruits. All controls had negative SPT to fresh fruits. Anhøj et al. (2001) [591]

65 birch pollen-allergic patients with a history of rhinitis in the birch-pollen season and positive specific IgE to birch. Skamstrup Hansen et al. (2001) [589].

26 patients based on a clinical history of rhinoconjunctivitis in the birch pollen season and OAS or other allergic manifestations upon ingestion of apple, together with positive specific IgE and a positive SPT to birch pollen. Skamstrup Hansen et al. (2001) [590]

10 patients with clinical allergy to birch pollen, a positive SPT to birch, and a positive case history of OAS to apple and 10 nonatopics without birch pollinosis, no symptoms of OAS and a negative SPT to birch as control subjects. Osterballe et al. (2003) [581]

372 Birch pollen allergic patients with a history of seasonal (February-April) pollen allergy with symptoms of rhinitis and/or asthma. A fraction of them suffered from oral allergy syndrome after eating apple, too. Rossi et al. (2003) [835]

Fernandez-Rivas and Cuevas (1999) [833] found that total IgE was significantly higher in the fruit and pollinosis group (mean 481.42 kU/L) than in fruit group (mean 152.73 kU/L). RAST to apple peel was significantly higher than RAST to pulps in the fruit patients, but no significant differences were observed in the fruit and pollinosis group. Fruit peel RAST was inhibited (from 60 to 97%) by itself and by its pulp, and viceversa. The area under the curve of histamine release was larger for peels of apple.

Histamine release tests with apple gave sensitivity of 71% and specificity of 93%. Anhøj et al. (2001) [591].

The maximum histamine release (ng/ml) against fresh Golden delicious apple was higher in group III than in group I (P=0.02). The freeze-dried apple powder proved to be useful for histamine release but further investigation of the stability and the allergenic profile of the material is needed. Skamstrup Hansen et al. (2001) [589]

Skamstrup Hansen et al. (2001) [590] found that specific IgE against apples cv. Golden Delicious increased during birch pollen season (P<0.05). None of the patients had positive specific IgE against apple measured by the Magic Lite system.

Similarly, Osterballe et al. (2003) [581] reported that the CAP test showed a higher diagnostic sensitivity (100%) than the Magic lite test (10%) for apple. The Histamine release test showed a high sensitivity to birch (90%), A72 (90%), Mal d1 (70%) and low to the commercial apple extract (25%).

Rossi et al. (2003) [835] found that 162 sera (43.55%) contained specific IgE to apple extract (75.35% of birch pollen Bet v 1 positive sera). The authors concluded "In this study we observed that three birch pollen recombinant allergens alone, could sufficiently identify 90% of birch pollen-sensitive patients".

Sodium dodecylsulfate-polyacrylamide gel electrophoresis was perdformed using 12% polyacrylamide gels (Vanek-Krebitz et al. 1995) [222], (Ebner et al. 1995) [46]

The apple extract was separated in a discontinuous buffer system in a SDS-PAGE gradient gel with 6% stacking and 7.5% to 20% separation gradient gel. The sample, at the protein concentration of 7 mg/mL, was diluted 1:2 in sample buffer containing TRIS adjusted to pH 6.1 with concentrated sulfuric acid, 10% SDS, 2-mercaptoethanol, 50% glycerol, and 1% bromphenol blue. The samples were denatured at 100°C for 5 minutes (Pastorello et al. 1999) [157]

The proteins were electroblotted to a nitrocellulose sheets with pore size 0.2 µm. The sheets were cut into 5 mm strips and blocked with phosphate-buffered saline pH 7.5 with 0.5% (v/v) Tween 20 and 0.5% (w/v) bovine serum albumin for 30 min. Strips were incubated with diluted (1:4) sera overnight. The IgE-binding components were detected with iodine 125–labeled rabbit anti-human IgE antiserum. Bots were visualised by autoradiography (Ebner et al. 1995) [46].

The proteins were electroblotted to a nitrocellulose membranes. The IgE-binding components were detected with iodine 125–labeled rabbit anti-human IgE antiserum. Blots were visualised by autoradiography (Vanek-Krebitz et al. 1995) [222]

Allergens separated by SDS-PAGE were electroblotted onto a nitrocellulose membrane (0.45 µm) with use of a transblot cell from Bio-Rad at 0.45 A, 100 V, for 4 hours at 4°C. The unoccupied protein binding sites in the nitrocellulose membrane were blocked by a 30 minute incubation at 37°C with PBS, pH 7.4, with 0.5% Tween 20. The nitrocellulose was then cut into strips, which were incubated overnight at room temperature with the serum of each single patient. IgE binding by specific antibodies was detected by incubation with iodine 125–labeled anti-human-IgE diluted 1:4 in blocking solution for 6 hours at room temperature and exposure on x-ray film at –70°C for 3 days (Pastorello et al. 1999) [157]

20/20 sera displayed IgE binding to apple extract. 20 sera reacted with a protein of 17 kDa, and 4 sera reacted with an polypeptide of 14 kDa. Sera from 4 patients recognized proteins in the higher MW range (Ebner et al. 1995) [46]

9/9 patients suffering from allergic reactions to birch pollen and apple displayed igE binding to nMal d1, rMal d 1 and rBet v1 (Vanek-Krebitz et al. 1995) [222]

Birch-positive patients had IgE binding to proteins with MWs of 9 kDa (9 patients, 28%), 15 kDa (16 patients, 50%), 18 kDa (29 patients, 91%), 28 kDa (2 patients, 6%), 31 kDa (13 patients, 41%), 43, 51, and 84 kDa (11 patients, 34%), and 60 kDa (7 patients, 22%). All birch-negative patients had IgE binding only to the 9-kDa polypeptide, and 6/7 patients with mugwort pollinosis (86%) (Pastorello et al. 1999) [157]

Anhøj et al. (2001) [591] and Pastorello et al. (1999) [157] used fresh apple.

A total of 17 g of dehydrated whole fruit was masked in a mix of orange (200 mL) and pineapple (200 mL) juices, sugar (16 g), wheat meal (13 g), and liquid coloring (McCormick). An open feeding of the whole fresh fruit in identical conditions and quantity as in the initial OFC followed all negative blinded challenges. The time between initial and final OFCs was not more than 10 days. Rodriguez et al. (2000) [491]

Skamstrup Hansen et al. (2001) [589].

• Model I: fresh apple juice and additional blackcurrant juice.
• Model II: freshly grated apple and additional grated cabbage and apple juice.
• Model III: freeze-dried apple powder and additional apple juice and blackcurrant juice

Skamstrup Hansen et al. (2001) [590] used grated cabbage, pasteurisated apple juice and grated apple (Golden delicious) for DBPCFC, and whole fresh apple (Golden delicious) for open challenge tests.

The meals contained a mixture of fresh shredded apple (Golden Delicious), yogurt, orange juice, apple juice, applesauce, oatmeal flakes and dry rasped coconut. The placebo doses consisted of the same ingredients except fresh apple. Apple juice and sauce did not contain any IgE-reactive Mal d 1 allergen due to processing steps and were added for optimal blinding of taste. Bolhaar et al. (2004) [1143]

Skamstrup Hansen et al. (2001) [589], Bolhaar et al. (2004) [1143]. Yes.

Skamstrup Hansen et al. (2001) [590]; Rodriguez et al. (2000) [491]. DBPCFC and open challenge.

Anhøj et al. (2001) [591] and Pastorello et al. (1999) [157]. Open challenge

43 patients with oral allergy syndrome for apple and positive open food challenge, skin prick test, and serum specific IgE antibodies to apple. 32 had birch pollinosis (documented by specific IgE for birch) and 11 were not allergic to birch. Pastorello et al. (1999) [157]

A total of 226 food challenges (including open food challenge and DBPCFC) were performed in the 28 patients with positive skin prick tests or CAP System FEIA. Rodriguez et al. (2000) [491]

65 birch pollen-allergic patients with a history of rhinitis in the birch-pollen season and positive specific IgE to birch, and positive open challenge with apple. For comparison of the DBPCFC models, patients with a positive open oral challenge ith apple were selected. Skamstrup Hansen et al. (2001) [589].

26 patients based on a clinical history of rhinoconjunctivitis in the birch pollen season and Oral allergy syndrome or other allergic manifestations upon ingestion of apple, together with positive specific IgE and a positive SPT to birch pollen. Skamstrup Hansen et al. (2001) [590]

36 grass- and or birch-pollen allergic patients based on a clinical history followed by an oral food challenge test and SPT with fresh fruits. The control group comprised five nonatopics and 12 birch and grass allergics with no symptoms of Oral allergy syndrome and frequently eating fresh fruits. All controls had negative SPT to fresh fruits. Anhøj et al. (2001) [591]

25 patients (nine males and 16 females, mean age 35.6 years) with a history of birch-pollen and apple allergy. Patients were randomized into two groups: one group to receive birch-pollen immunotheraphy (IT) (n=13) and the control group to receive only symptomatic treatment (n=12). Bolhaar et al. (2004) [1143]

From 4 to 64 g. Pastorello et al. (1999) [157].

Subjects were first challenged randomly with either food or placebo. After an interval of at least 24 hours, the second part of the DBPCFC took place with subjects receiving either food or placebo depending on what they had recieved in the first challenge. Confirmation by DBPCFC was accepted if the subject had symptoms after provocation with the active substance and no symptoms after the placebo challenge. Rodriguez et al. (2000) [491]

Model I: 5-50 ml. Model II: 20 g. Model III: 3 g. Skamstrup Hansen et al. (2001) [589].

20 g of grated apple. The authors note that only a low dose of apple could be used because of the flavour and suggest that more positives would occur if a higher dose were used. Skamstrup Hansen et al. (2001) [590].

A bite of an apple. Anhøj et al. (2001) [591].

The challenge meals were prepared within 5 min before administration and contained 4, 10, 40 or 120 g of fresh shredded apple (Golden Delicious). The patients of the immunotheraphy (IT) group received a standardized aluminium hydroxide adsorbed birch-pollen extract. All patients were given IT according to a modified cluster schedule, between November 2001 and January 2002, followed by a monthly maintenance injection of 100 000 standard quality units (SQ-U) for 1 year with a dose reduction of 50% during the pollen season. All patients were pre-treated with an antihistamine at least 1 h before injection, since this reduces side-effects and has been reported to possibly enhance efficacy. Bolhaar et al. (2004) [1143]

Oral allergy syndrome symptoms were classified in 4 grades of increasing severity: grade 1, only oral mucosa symptoms (22/32 and 9/11) ; grade 2, oral mucosa and gastrointestinal symptoms (1/11); grade 3, oral mucosa symptoms plus systemic symptoms (urticaria, rhinoconjunctivitis, or asthma) (2/32 and 1/11); grade 4, oral mucosa symptoms plus life-threatening symptoms (laryngeal edema, anaphylactic shock) (7/32). Pastorello et al. (1999) [157].

6/28 patients were positive after DBPCFC with apple. 2 patients had oral symptoms and 4 had generalised anaphylaxis (Rodriguez et al. 2000) [491]

Oral allergy syndrome: rhinitis, conjunctivitis, and local reactions at the oral cavity. Skamstrup Hansen et al. (2001) [590]; Anhøj et al. (2001).

Skamstrup Hansen et al. (2001) [589] found: Model I (17 positive, 13 negative, placebo 10 positive), Model II (14 positive, 5 negative, placebo 0 positive) and Model III (15 positive, 6 negative, placebo 4 positive). The authors concluded "It is highly desirable that the sensitivity of the DBPCFC with apple is increased further. Lyophilization of apples produces material useful for oral challenges and has some practical advantages over fresh apples, but an investigation of the long-time stability and the allergenic profile of the freeze-dried material is needed.

Skamstrup Hansen et al. (2001) [590] found that before the pollen season, 14/26 blinded challenges were positive, against 19/27 open challenges. During the pollen season, 16/26 blinded and 20/26 open challenges were positive. 1 positive placebo in each case with a different patient. None of the patients reacted to the blinded challenge without a subsequent reaction to the open challenge. The scores of the open challenge were significantly higher than the scores of the DBPCFC both before the season and during the in-season challenges (P<0.05).

Authors agreed that the diagnosis of oral allergy syndrome to apple has been complicated by unstable apple extracts and the changes of the allergenicity of apples during ripening, the test technique, the disease prevalence and the number of the patients included in studies.

Symptoms started within 5-10 min after ingestion of the challenge meal, as the oral allergy syndrome (OAS): itching or feeling of tightness in the oral cavity or tingling of the lips. Patient reactivity was expressed using VAS scores. After 1 year of immunotherapy (IT), both groups were tested with DBPCFC. In the IT group VAS scores were significantly decreased at t=12 (P<0.001) compared with those at the start of the study. This resulted in an increase of the amount of apple tolerated of a factor of 24. Nine of the 13 patients treated with IT improved significantly, whereas four patients showed VAS scores similar to t=0. In three of the nine patients who showed improvement, the provocation was completely negative after 1 year of birch-pollen IT, suggesting they had overcome their apple allergy. In the control group, nine out of 10 patients showed unchanged or even increased VAS scores relative to the year before, and no significant differences were found between t=0 and 12. One patient in the control group was not willing to undergo a challenge for the second time. No placebo reactions were observed in both groups. Bolhaar et al. (2004) [1143]