Int Arch Allergy Immunol 2012;158:232–240 DOI: 10.1159/000331581
Anisakis simplex Recombinant Allergens Increase Diagnosis Specificity Preserving High Sensitivity
María Luisa Caballero a Ana Umpierrez b Teresa Perez-Piñar a Ignacio Moneo a
Carmen de Burgos c Juan A. Asturias d Rosa Rodríguez-Pérez a
Departments of a Immunology, b Allergy and c Clinical Epidemiology, Hospital Carlos III, Madrid , and d Bial-Arístegui Research and Development Department, Bilbao , Spain
Anisakis simplex infects humans accidentally when raw or undercooked fish contaminated with larvae is consumed. Symptoms of acute gastric infection include sudden epigastric pain, nausea and vomiting and can oc-
Background: So far, the frequency of Anisakis simplex- spe-cific IgE antibodies has been determined by skin prick tests (SPTs) and the ImmunoCAP system. These commercial meth-ods have good sensitivity, but their specificity is poor be-cause they use complete parasite extracts. Our aim was to determine the frequency of sensitization to A. simplex using recombinant Ani s 1, Ani s 3, Ani s 5, Ani s 9 and Ani s 10 and to evaluate these allergens for diagnosis, comparing their performance with the commercial methods. Patients and
Methods: We conducted a descriptive, cross-sectional vali-dation study performed in an allergy outpatient hospital clinic. Patients without fish-related allergy (tolerant patients, n = 99), and A. simplex- allergic patients (n = 35) were studied by SPTs, ImmunoCAP assays and detection of specific IgE to A. simplex recombinant allergens by dot blotting. Results: SPTs and ImmunoCAP assays were positive in 18 and 17%of tolerant patients, respectively. All A. simplex- allergic pa-tients had positive SPTs and ImmunoCAP assays. Specific
Received: April 16, 2011 Accepted after revision: August 5, 2011 Published online: March 2, 2012
Correspondence to: Dr. Rosa Rodríguez-Pérez Immunology Department, Hospital Carlos III C/Sinesio Delgado, 10 ES–28029 Madrid (Spain) Tel. +34 91 453 2656, E-Mail mrosa_ro @ hotmail.com
cur 1–12 h after the infected meal [1] . Anisakiasis is often associated with a strong allergic response, with clinical symptoms ranging from isolated swelling to urticaria, angioedema and life-threatening anaphylactic shock [2, 3] .
Diagnosis of A. simplex allergy is currently based on a compatible medical history in addition to skin prick tests (SPTs) and specific IgE measured by the ImmunoCAP system. These two methods have good sensitivity, but their specificity is poor perhaps because they use com-plete parasite extracts.
The presence of allergen-specific IgE without clinical allergy has been defined as subclinical sensitization, pre-dicting the onset of allergic symptoms several years in advance [4] . Twenty-three percent of 87 subjects attend-ing an allergy outpatient clinic [5] and 13.1% among 434 asymptomatic subjects in a multicenter study performed by the Spanish Society of Allergy and Clinical Immunol-ogy (SEAIC) [6] had A. simplex- specific IgE. These stud-ies were performed with A. simplex complete parasite ex-tracts, so their results could be reflecting cross-reactions with other nematodes, crustaceans or dust mites [7, 8] or the detection of carbohydrate-type determinants of para-site antigens [9] .
One way to overcome this problem is using the most important and specific A. simplex allergens in diagnostic tests. To date, the following 12 proteins have been identi-fied as A. simplex allergens. Ani s 1 (21 kDa) [10] belongs to the serin-type endopeptidase inhibitor family since it contains a Kunitz domain. Ani s 2 (paramyosin, 100 kDa) [11] and Ani s 3 (tropomyosin, 41 kDa) [12] are muscular proteins. Ani s 4 (9 kDa) [13] and Ani s 6 (7 kDa) [14] are protease inhibitors. Ani s 5 (15 kDa) [15] , Ani s 8 (15 kDa) [16] and Ani s 9 [17] are members of the SXP/RAL-2 nem-atode family protein. Ani s10 (21 kDa) [18] is composed of 7 almost identical repetitions of 29 amino acids. Ani s 11 (30 kDa) [19] is characterized by having 6 and 5 types of short repetitive sequences and shares 45% identity with Ani s 10. Last, Ani s 7 (139 kDa) [20] and Ani s 12 (33 kDa) [19] include 5 and 19 tandem repeats of a CX(13–25)CX(9)CX(7,8)CX(6) sequence, respectively.
Our aim was to determine the prevalence of sensitiza-tion to A. simplex using the recombinant allergens: Ani s 1, Ani s 3, Ani s 5, Ani s 9 and Ani s 10 in adult patients without fish-related allergy, attending in an allergy out-patient clinic, and to assess the diagnostic yield of these allergens among patients with allergy to A. simplex as standard reference.
Materials and Methods
Patient Sera We analyzed sera from two groups of patients diagnosed in
Carlos III Hospital: a group of 99 patients who attended the con-sultation for suspected allergy without fish-related allergy (group of tolerant patients) and a group of 35 retrospectively recruited patients with allergy to A. simplex, selected based on the presence of anaphylaxis, angioedema, urticaria or gastrointestinal symp-toms few hours after eating raw or undercooked fish (group of allergic patients). Both tolerant and A. simplex- allergic patients belonged to the same geographical area. Informed consent was obtained from the participants.
Patients were first studied by skin prick tests (SPTs) with com-mercial A. simplex extract (ALK-Abelló, Madrid, Spain). Hista-mine dihydrochloride (1 mg/ml) and saline solution were used as positive and negative controls, respectively. SPT response was considered positive if the largest wheal diameter was at least 3 mm greater than that produced by the negative control.
All the patients (tolerant and allergic) were further studied by specific IgE determinations to A. simplex by the ImmunoCAP system (Phadia, Uppsala, Sweden), followed by detection of spe-cific IgE to A. simplex recombinant allergens by dot blotting.
A. simplex Recombinant Allergens Ani s 1 and Ani s 5 were cloned in Pichia pastoris and purified
by ion exchange chromatography and RP-HPLC [10] . Ani s 9 and Ani s 10 were expressed with His-tag in Escherichia coli and puri-fied by affinity chromatography [17, 18] . The recombinant aller-gen Ani s 3 was expressed in E. coli and purified by isoelectric precipitation and gel permeation chromatography [12] .
Dot-Blotting Assay Recombinant Ani s 1, Ani s 3, Ani s 5, Ani s 9 and Ani s 10
(5 � g) were dotted onto a nitrocellulose membrane (Nitro-Pure supported, 0.45 � m, GE Osmonics Labstore, Minnetonka, Minn., USA) using a dot blot manifold Bio-Dot TM (Bio-Rad, Hercules, Calif., USA). The membrane was dried and blocked with 3% Nonidet NP-40 (Amresco, Solon, Ohio, USA) in PBS for 30 min. Membranes were incubated overnight at room temperature with individual patient sera (1: 10 dilution). Specific IgE determination was performed with a monoclonal anti-human-IgE (1: 1,000) (Ingenasa, Madrid, Spain) followed by an alkaline phosphatase-labeled goat anti-mouse antibody (1: 5,000) (Biosource Int., Ca-marillo, Calif., USA). Finally, the signal was visualized with the alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate/4-nitroblue tetrazolium system (Amresco) for 30 min [21] . Dot blotting was considered positive when the signal was clearly vis-ible.
ImmunoCAP Inhibition Assay Inhibition with the glycoreporter bromelain was performed
according to Malandain et al. [22] . Briefly, individual sera from the tolerant patients with a positive A. simplex ImmunoCAP were inhibited with heat-inactivated bromelain (100 mg/ml; Sigma, St. Louis, Mo., USA). Bromelain is a cysteine protease and the heat-inactivation (100 ° C, 30 min) was performed in order to avoid serum IgE proteolysis [15] . Patient serum (80 � l) was mixed with 40 � l of heat-inactivated bromelain or with 40 � l of PBS as nega-tive control, and incubated for 2 h at 25 ° C. Then, determination
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of specific IgE to A. simplex by the ImmunoCAP system was per-formed. An inhibition bromelain immunoCAP assay was per-formed with 3 bromelain-positive sera in order to assess that 99% inhibition was reached with the amount of heat-inactivated bro-melain used.
Statistical Analysis A descriptive analysis of the main demographic characteris-
tics of samples was carried out. Quantitative variables were ex-pressed by means and standard deviations (SDs), and the qualita-tive variables wereexpressed with their relative frequency.
For the bivariate comparison of proportions, the Pearson � 2 method was applied. When the application conditions of the � 2 test were not met (any expected cell count under 5), Fisher’s exact bilateral test was used. Student’s t test was applied for the bivariate comparison of means. p values ! 0.05 were considered statistical-ly significant.
Sensitivity was defined as the percentage of allergic patients with A. simplex ImmunoCAP and A. simplex recombinant aller-gen (at least one of them)-positive reactions. Specificity was de-fined as the percentage of tolerant patients with A. simplex Im-munoCAP and A. simplex recombinant allergen-negative reac-tions. Sensitivity and specificity were calculated with their 95% confidence intervals (95% CIs).
Positive (PPV) and negative (NPV) predictive values were cal-culated for an A. simplex allergy prevalence of 5.7%, also with their 95% CIs. This prevalence was obtained in a group of indi-viduals with suspected allergy [5] .
Data analysis was performed using SPSS 15.0 software (SPSS Inc., Chicago, Ill., USA). The 95% CIs of the predictive values were calculated with the method developed by Monsour et al. [23] .
Results
The average age of the tolerant group was 36.21 years, which is significantly lower than the average age of the A. simplex- allergic patients (52.5 years). Positive SPTs to A. simplex were found in 18 of the 99 tolerant patients. The further study by ImmunoCAP was positive in 17 of these 18 patients. Surprisingly, ImmunoCAP class 3 was sig-nificantly more frequent among tolerant patients. As ex-pected, ImmunoCAP classes 4–6 were only present in al-lergic patients. On the other hand, there was a small per-centage (8.6%) of A. simplex- allergic patients with CAP class 2 ( table 1 ).
The patients from the tolerant group were allergic mainly to pollen (78%) and cat/dog dander (33%). Al-lergy was ruled out in 2 patients ( table 2 ). Dot blotting performed with A. simplex recombinant allergens was positive (at least one) in 15 of the 99 tolerant patients (15.2%) ( fig. 1 ) and all of the 35 A. simplex-allergic pa-tients detected at least one A. simplex recombinant aller-gen ( fig. 2 ).
A. simplex Complete Extract (ImmunoCAP) Compared with Recombinant Allergens (Dot Blotting) Among the 18 positive SPT tolerant patients, 6 (33.33%)
had specific IgE directed exclusively against 1 of the al-lergens tested, 5 (27.77%) patients detected two allergens, and 1 (0.06%) patient detected 3 allergens. The most fre-quently recognised allergen was Ani s 1 ( table 2 ). In the A. simplex-allergic patients, 10 of 35 (29%) had specific IgE directed against 1 of the allergens tested, predomi-nantly against Ani s 1 (7 patients), followed by Ani s 9 (2 patients) and Ani s 5 (1 patient). Seven patients (20%) de-tected 2 allergens, 9 (26%) detected 3 allergens, 8 (23%) detected 4 allergens and 1 (3%) detected the 5 allergens ( table 3 ).
The measurement of specific IgE levels to A. simplex using the ImmunoCAP system yielded positive results in 18% of the tolerant patients and in 100% of the A. simplex-allergic patients. Specific IgE was detected against at least 1 of the 5 A. simplex allergens tested in 15% sera from tol-erant patients and in 100% sera from A. simplex-allergic patients. Sera from A. simplex-allergic patients showed higher specific IgE levels (ImmunoCAP) and contained specific IgE to Ani s 1, Ani s 3, Ani s 5, Ani s 9 or Ani s 10 more frequently than sera from the tolerant patients(p ! 0.001) ( table 4 ).
Diagnostic sensitivity, specificity, PPV and NPV of the test were calculated to evaluate the potential of the indi-vidual A. simplex allergens as in vitro diagnostic reagents ( table 4 ). The detection of at least one A. simplex allergen
Table 1. Demographic characteristics and results of total IgE, spe-cific IgE (CAP classes) of the patients studied
Fig. 1. Dot-blotting assay performed with the sera from 18 tolerant patients with positive SPTs to A. simplex to detect specific IgE to the 5 recombinant allergens tested.
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of the panel of allergens studied and the specific IgE against a complete extract measured by ImmunoCAP showed a diagnostic sensitivity of 100%. Nevertheless, the specificity of the ImmunoCAP assay was lower (82.83%) than the specificity of dot blotting with the A. simplex recombinant allergens (84.85%) ( table 4 ).
Influence of Cross-Reactive Carbohydrates on IgE Reactivity It has been published that cross-reactive carbohy-
drates (CCD) could influence the A. simplex- specific IgE result [9] . We compared the A. simplex ImmunoCAP val-ues before and after CCD inhibition in order to quantify the influence of N-glycan structures on IgE determina-tion in the sera from the 18 tolerant patients. The result was an inhibition of 27.44% on average (range 0–48.62%)
( fig. 3 ). Although the specific IgE values decreased after CCD inhibition, none of the ImmunoCAP-positive re-sults were negative after inhibition; therefore, we cannot affirm that CCD interference is the main cause of false A. simplex- positive ImmunoCAP results.
Discussion
The diagnosis in allergy is changing to systems that use isolated recombinant allergens (termed ‘component-resolved diagnosis’) [24] . These systems allow identifica-tion of the allergen recognition profile of each patient. To date, only Ani s 1 and Ani s 3 are available in component-resolved diagnosis systems for A. simplex allergy [25] . Our aim was to evaluate 5 A. simplex recombinant aller-
1 171615141312111098765432 18
19 343332313029282726252423222120 35
rAni s 1
rAni s 3
rAni s 5
rAni s 9
rAni s 10
rAni s 1
rAni s 3
rAni s 5
rAni s 9
rAni s 10
* * * * * * * * * * * * * * * * * w
*
w
** * * * * * * * * * * * * * *w
*
Fig. 2. Dot-blotting assay performed with the sera from 35 A. simplex-allergic patients to detect specific IgE to the 5 recombinant allergens tested.
gens for diagnosis and compare their performance with the commercial SPT and ImmunoCAP methods, which are both based on complete parasite extracts.
Our results showed that detection by dot blotting of at least one of the A. simplex recombinant allergen tested
(Ani s 1, Ani s 3, Ani s 5, Ani s 9 and Ani s 10), has the same diagnostic sensitivity as A. simplex ImmunoCAP for the diagnosis of A. simplex-allergic patients. However, the use of the recombinant allergens increases diagnostic specificity compared with the determination of specific
Table 3. Clinical data of the 35 patients allergic to A. simplex
Pa-tient
Age/sex
Symptomsafter fishingestion
Time ofreaction
Fish dish A. simplexSPT whealmm
TotalIgEkU/l
A. simplexIgE Immu-noCAPkU/l
D ot blotting
rAni s1
rAni s3
rAni s5
rAni s9
rAni s10
1 56/M A, U, GI 1.5 h Pickled anchovies 17 ! 20 >3,000 >100 + w+ + +2 65/F A, GI 4 h Pickled anchovies 6 ! 14 24 10.30 + +3 57/F GI 6 h Ceviche n.d. >3,000 88.90 + w+4 58/F AE, GI 4 h Fried whiting 12 ! 16 2,355 >100 + + + +5 60/M A, U 30 min Fried whiting n.d. 230 17.40 + + + +6 57/F U, GI 4 h Pickled anchovies n.d. 903 >100 + + + +7 33/M GI 24 h Pickled anchovies n.d. 43 0.72 + +8 58/F U 4 h Pickled anchovies n.d. 2,958 >100 + + + +9 67/F U, AE, GI 2 h Pickled anchovies n.d. 206 54.90 + + + +
10 49/F A, U, GI 3 h Fried hake 15 ! 10 >3,000 >100 + + +11 62/F U, GI 6 h Fried whiting and hake 13 ! 15 120 24.30 + +12 26/M U 1 h Fried hake n.d. 567 13.73 +13 55/F A, U, AE 30 min Pickled anchovies n.d. >3,000 >100 + + + + +14 30/F U, GI 6 h Pickled anchovies 10 ! 12 265 37.30 + + + +15 58/F A, U 5 h Pickled anchovies n.d. 155 23.30 + + + +16 47/F GI 3 h Fried whiting and hake 13 ! 15 294 17.10 + + +17 51/F GI 2 h Fried whiting and hake n.d. 61 11.50 +18 65/F U 6 h Fried sole 11 ! 15 49 3.30 w+ w+19 50/M U 3 h Fried whiting and hake n.d. 526 13.70 w+20 50/F U 30 min Pickled anchovies n.d. 868 91.70 + + +21 35/M U 1.5 h Pickled anchovies 12 ! 9 139 22.30 +22 46/M A, U, AE 1 h Grilled swordfish n.d. >3,000 >100 + + +23 50/F U, AE 2 h Fried whiting n.d. 121 2.77 + + +24 52/M A, U 3.5 h Rare grilled hake 15 ! 10 36 6.68 w+25 49/M U, GI 2 h Pickled anchovies 7 ! 5 78 12.10 + + +26 53/M U, AE 30 min Pickled anchovies n.d. >3,000 >100 + + +27 63/F U 2 h Fried whiting and hake 6 ! 7 297 6.70 +28 83/F U 4 h Fried sole 13 ! 11 32 11.60 +29 48/M A 30 min Grilled tuna 5 ! 4 28 5.30 +30 25/M U, GI 5 h Pickled anchovies 10 ! 12 425 83.70 +31 49/F U, AE 2 h Fried whiting and hake 6 ! 12 57 4.30 +32 64/M GI 3 h Oil-packed anchovies n.d. 541 >100 + + +33 78/F U, AE 3.5 h Pickled anchovies 18 ! 20 >3,000 >100 + +34 52/M U, GI 6 h Pickled anchovies n.d. 1,253 67.20 + +35 54/M A, U 3 h Pickled anchovies n.d. 2,334 >100 + + +
A = Anaphylaxis; AE = angioedema; GI = gastrointestinal symptoms (pain, vomiting, diarrhea); U = urticaria. ImmunoCAP assay, reported as classes of specific IgE (kU/l) assigned: (1) 0.35–0.70; (2) 0.70–3.50; (3) 3.50–17.50; (4) 17.5–50; (5) 50–100; (6) >100. n.d. = Not done; + = positive detection by dot blotting; w+ = weak detection by dot blotting.
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IgE against a compete extract measured by ImmunoCAP (84.85 vs. 82.83%). The allergen that showed the highest specificity was Ani s 9 (98.99%) although its sensitivity is only 42.86%. The major allergen, Ani s 1, had the highest sensitivity (85.71%) with a good specificity (90.91%).
The PPV is directly proportional to the prevalence of the disease and the NPV is inversely proportional to it. The values of these indicators have been calculated using the prevalence data from the only study found in the lit-erature although it was conducted in 1999 and was based
on a population with suspected allergy [5] . The preva-lence of A. simplex allergy could be increasing since it depends on the habit of eating raw fish. If the prevalence were higher, the PPV would be higher and the NPV would be lower than that calculated in this study.
We were interested in distinguishing between clini-cally irrelevant IgE and specific IgE as an early marker of allergy. As shown in this research, the percentages of sub-clinical sensitization obtained by techniques that use complete extract decreased when measured by dot blot-
Table 4. Diagnostic sensitivity and specificity of the 5 A. simplex recombinant allergens tested by dot blotting and ImmunoCAP assays using complete extract
Allergens Tolerant(n = 99)
A. simplexallergic(n = 35)
�2
p valueSensitivity a, % Specificity a, % PPV a, % NPV a, %
Ani s 1 9 (9.1%) 30 (85.7%) <0.001 85.71 (70.62–93.74) 90.91 (83.62–95.14) 36.3 (23.15–51.88) 99.06 ( 97.90–99.59)Ani s 3 6 (6.1%) 19 (54.3%) <0.001 54.29 (38.19–69.53) 93.94 (87.40–97.19) 35.13 (19.05–55.46) 97.14 (95.94–98.04)Ani s 5 2 (2%) 7 (20%) 0.001b 20 (10.04 –35.89) 97.98 (92.93–99.44) 37.44 (11.54–73.30) 95.30 (94.48–96.00)Ani s 9 1 (1%) 15 (42.9%) <0.001b 42.86 (27.99–59.14) 98.99 (94.50–99.82) 71.95 (26.01–94.93) 96.63 (95.56–97.45)Ani s 10 4 (4%) 17 (48.6%) <0.001 48.57 (32.99–64.43) 95.96 (90.07–98.42) 42.08 (20.78–66.81) 96.86 (95.71–97.73)Ani s 1 or Ani s 5 9 (9.1%) 32 (91.4%) <0.001 91.43 (77.62–97.04) 90.91 (83.62–95.14) 37.81 (24.44–53.33) 99.43 (98.34–99.81)Ani s 1 or Ani s 9 10 (10.1%) 33 (94.3%) <0.001 94.29 (81.39–98.42) 89.90 (82.40–94.42) 36.07 (23.77–50.52) 99.62 (98.54–99.90)At least one positive 15 (15.1%) 35 (100%) <0.001 100 (90.11–100) 84.85 (76.50–90.60) 28.52 (20.02–38.87) 100 (100–100)InmunoCAP 17 (17.2%) 35 (100%) <0.001 100 (90.11–100) 82.83 (74.21–88.99) 26.04 (18.59–35.17) 100 (100–100)
a Figures in parentheses are 95% CI. b Fisher’s exact bilateral test.
0 2 4 6IgE (kU/l)
CAP kU/lCAP after CCD inhibition
8 10 12
Sera
num
ber
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
0.00%
26.40%
27.12%
47.17%
22.99%
20.80%
37.10%
0.00%
45.18%
15.36%
20.13%
20.59%
48.62%
26.83%
23.54%
17.39%
32.16%
35.04%
Fig. 3. Quantification of the influence of cross-reactive carbohydrates on IgE reac-tivity. Comparison of the ImmunoCAP values (kU/l) before and after CCD inhibi-tion performed with the sera from the 18 tolerant patients positive for A. simplex .
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2 Del Pozo MD, Audícana M, Diez JM, Munoz D, Ansotegui IJ, Fernández E, García M, Et-xenagusia M, Moneo I, Fernández de Corres L: Anisakis simplex , a relevant etiologic fac-tor in acute urticaria. Allergy 1997; 52: 576–579.
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ting with A. simplex recombinant allergens. This result agrees with previous studies in which the application of recombinant allergens to measure clinically relevant IgE had priority over classical extracts owing to a better spec-ificity [26] . In fact, the results obtained with a test using complete extract (ImmunoCAP) surprisingly revealed that IgE class 3 was more frequent among tolerant pa-tients than among A. simplex-allergic patients ( table 1 ). On the other hand, there is a small percentage (8.6%) of truly A. simplex-allergic patients with CAP IgE class 2. These results make clear that the in vitro A. simplex di-agnosis should be improved.
In order to seek an explanation for the positive SPT and ImmunoCAP results in tolerant patients, we per-formed CAP inhibition with bromelain as a source of CCDs. Our results showed that CCDs are not the main cause of false-positive ImmunoCAP results. Another plausible explanation could be the presence of a panaller-gen like tropomiosyn (Ani s 3) among invertebrates. To test this hypothesis, Ani s 3 was included in our panel of recombinant allergens; however, Ani s 3 was not recog-nized more frequently by tolerant patients allergic to mites and its reactivity in dot blotting was the weakest among the recombinant allergens tested.
There is a strong need for a reliable assay that exclu-sively measures clinically relevant IgE since subclinical sensitization could precede full-blown allergy as already pointed out in cat allergy [27] .
Therefore, a relevant fact of A. simplex subclinical sen-sitization could be the risk of the patients to become al-
lergic. It is possible that at the time of diagnosis patients do not yet suffer from clinical symptoms despite showing in vitro reactions because the levels and/or affinity of spe-cific IgE antibodies might be too low to trigger a clinical reaction but the sensitization process may already be in progress [4, 27] . Prophylactic strategies should thus be implemented to avoid the appearance of symptoms. We are following the evolution of these 18 A. simplex- sensi-tized patients without fish-related allergy, in order to de-termine how many of them will eventually develop aller-gic symptoms upon exposure to A. simplex . Moreover, the analysis of recombinant allergens detected by these patients during the subclinical phase and their evolution over time could determine whether any of the allergens could have prognostic value [28] .
To conclude, there were 15% of tolerant patients with specific IgE against important A. simplex allergens. The recombinant allergens studied here have increased A. simplex diagnositic specificity. As in other models of al-lergy, A. simplex recombinant allergens should be intro-duced among the A. simplex allergy diagnostic tests.
Acknowledgements
Financial support was provided by FIS from the Ministry of Health (grant No. FIS-CS09/01501), and R.R.-P. was supported by the Miguel Servet Program from the Ministerio de Ciencia y Tec-nologia.
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