How molecular diagnosis can change allergen-specific immunotherapy prescription in a complex pollen area
Joaquín Sastre, Allergy Department, Fundación Jiménez Díaz, Av. Reyes Católicos 2, 28040 Madrid, Spain.
The identification of disease-eliciting allergens is a prerequisite for accurate prescription of allergen-specific immunotherapy (SIT). The aim of this study was to determine whether molecular diagnosis (MD) may change indication and allergen prescription of SIT.
A total of 141 patients with allergic rhinoconjunctivitis and/or asthma sensitized to pollen with or without concomitant food allergy were included. Skin prick testing with a panel of aeroallergens and a microarray-based panel of allergens (ISAC®; Phadia, Sweden) was performed in all patients. Prior to learning the results of molecular diagnosis, three of the authors reached a consensus on the indication of SIT and use of allergens following EAACI recommendations, basing their judgment on clinical history and skin prick test results before and after obtaining the ISAC results. The agreement coefficient (kappa index) was used to analyze the results.
Fifty-nine percent of the patients were women with a mean age of 31 ± 13.63. Agreement in SIT indication before and after ISAC® results was found in only 62 (46%) patients (kappa = 0.1057 ± 0.0413). Concerning allergens used in the most common prescriptions before and after MD results, we obtained the following results: κ = 0.117 ± 0.0825 for grass; κ = 0.1624 ± 0.0639 for olive; κ = 0.0505 ± 0.0548 for olive and grass; κ = 0.1711 ± 0.0471 for grass and cypress; κ = 0.1897 ± 0.0493 for grass and London plane; κ = 1 ± 0.0842 for olive and cypress, and κ = 0.3586 ± 0.0798 for other combinations.
There was very low agreement concerning indication and use of allergens for SIT before and after performing MD. This discrepancy emphasizes the usefulness of MD, at least in areas of complex sensitization to pollen, in determining correct indication of SIT.
kappa coefficient of agreement
skin prick test
Allergen-specific immunotherapy (SIT) is the only allergen-specific treatment of its kind . It can prevent progression of the disease and has a long-lasting therapeutic effect. As SIT is allergen-specific, the identification of the disease-eliciting allergen is a prerequisite for accurate prescription of treatment. Diagnostic tests based on allergen extracts use mixtures of various allergens, some of which are specific for the allergen source, while others are cross-reactive allergens of various unrelated allergen sources. It may therefore be difficult or even impossible to identify the disease-causing allergen by performing such tests, particularly in patients who are sensitized to more than one allergen source. Sensitization to pollen from cypress, plane, olive, and grasses is common in our area , and these patients are frequently treated with SIT. Advances in molecular techniques applied to allergens have provided us with new tools such as recombinant allergens that can improve the accuracy of allergy diagnosis . The aim of this study was to assess whether molecular diagnosis of pollen allergy leads to changes in the indication and allergen prescription of SIT when compared to skin prick testing with commercial extracts.
A total of 141 patients with allergic rhinoconjunctivitis and/or asthma sensitized to pollen with or without concomitant food allergy were recruited from our outpatient clinic.
Skin prick tests (SPTs) with a panel of commercial common inhalants (all from ALK-Abelló, Madrid, Spain) were carried out. The panel included the following pollens: Olea e, Platanus a, Cupressus a, grass mix, Cynodon d, Phragmites c, Artemisia v, Salsola k, and Plantago l. Histamine (10 mg/ml) and glycerol–saline solution were used as controls. A skin test was considered positive if the wheal size was at least 3 mm greater than that of the negative control.
Specific IgE to molecular allergens
A microarray-based panel of 96 allergens (ISAC®, Phadia, Sweden) was used. The panel included the following pollen allergens: Ole e 1, Cup s 1, Cry j 1, Pla a 1, Pla a 2, Phl p 1, Phl p 5, Phl p 4, Phl p 6, rPhl p 11, Phl p 12, Cyn d 1Sal k 1, Aln g 1, Bet v 1, Cor a 1.0101, Amb a 1, Art v 1, Art v 3, and Par j 2. Positive values were assessed according to the manufacturer's instructions. Sensitization to London plane or grass was considered when positivity to Pla a 1 and/or Pla a 2 or positivity to Phl p 5 and/or Phl p 6, respectively.
Prescription of allergen immunotherapy
Prior to learning the results of molecular diagnosis, three of the authors reached a consensus on the indication of SIT and use of allergens, basing their judgment on clinical history. This initial assessment took into account the time of year in which respiratory symptoms developed according to the pollen calendar, and following the recommendations for immunotherapy established by the European Academy of Allergy and Clinical Immunology (EAACI) Guidelines SPT results . After obtaining the result of ISAC®, the same panel of doctors came up with a new indication and prescribed the use of allergens for immunotherapy. Determinations were considered to be in agreement if indication of SIT and allergen(s) used were the same before and after molecular diagnosis (MD) results were obtained. The agreement coefficient (κ index) was used to analyze results.
The mean age of patients was 31 ± 13.63 years, and 59% were women. Agreement and disagreement are summarized in Table 1. Agreement in indication of SIT before and after ISAC® results occurred in only 62 (46%) patients (κ = 0.1057 ± 0.0413).
Table 1. Agreement and disagreement in indication of immunotherapy based on skin prick test only and molecular diagnosis
|Grass||17||10||97 (68)||44 (32)|
0.117 ± 0.0825
P = 0.0781
|Olive||1||1||132 (93)||9 (7)|
0.1624 ± 0.0639
P = 0.0055
|Grass + olive||4||1||101 (71)||40 (29)|
0.0505 ± 0.0548
P = 0.1782
|Grass + cypress||0||1||132 (93)||9 (7)|
0.1711 ± 0.0471
P = 0.0001
|Grass + plane||0||1||133 (94)||8 (6)|
0.1897 ± 0.0493
P = 0.0001
|Olive + cypress||0||2||141 (100)||0 (0)|
1 ± 0.0842
P < 0.0001
|Other extracts||3||4||129 (91)||12 (9)|
0.3586 ± 0.0798
P < 0.0001
|Total||25||20||62 (46)||79 (54)||0.1057 ± 0.0413|
Table 2 summarizes instances of agreement between commercial extracts and recombinant or purified allergens.
Table 2. Agreement between commercial extracts and recombinant or purified allergens of the main pollens found in the area of Madrid (Spain)
|Olive/Ole e 1|
|111 Positive SPT||80||31||100 (70)|
|30 Negative SPT||10||20|
|Cypress/Cup s 1|
|87 Positive SPT||72||15||115 (81)|
|54 Negative SPT||11||43|
|Plane/Pla a 1|
|102 Positive SPT||24||81||63 (44)|
|39 Negative SPT||2||39|
|Plane/Pla a 2|
|102 Positive SPT||57||48||87 (61)|
|39 Negative SPT||11||30|
|Plane/Pla a 1 or Pla a 2|
|102 Positive SPT||56||46||84 (60)|
|39 Negative SPT||11||28|
|Grass/Phl p 1|
|121 Positive SPT||105||16||114 (81)|
|20 Negative SPT||11||9|
|121 Positive SPT||77||44||89 (62)|
|20 Negative SPT||8||12|
|Grass/Phl p5 or Phl p1|
|121 Positive SPT||110||11||119 (84)|
|20 Negative SPT||11||9|
|Cynodon/Cyn d 1|
|109 Positive SPT||96||13||115 (82)|
|32 Negative SPT||13||19|
Selection of patients for SIT must take into consideration clinical parameters and confirmation of the presence of an IgE-mediated allergic sensitization. One factor that can limit the application and success of SIT is the correct identification of the disease-eliciting allergen.
Diagnostic tests are based on natural allergen extracts that are composed of mixtures of nonallergenic materials, major allergens, and cross-reactive allergens, thus making it difficult and often impossible to precisely identify the disease-eliciting allergen, particularly in patients sensitized to more than one allergen source. In such cases, it is important for the clinician to know whether a patient is co-sensitized to several allergen sources and needs SIT for each, or whether the patient is sensitized to several sources because of sensitization to cross-reactive components in each of the suspected allergen sources. Molecular diagnosis using recombinants or purified allergens can partially solve this problem and improve the diagnosis of allergy . In this study, we found that in 54% of patients, there was a disagreement in indication of SIT before and after ISAC® results. With regard to the extracts used for immunotherapy, the κ agreement coefficient varied from 1 for olive and cypress to <0.2 for the rest of the extracts, which is considered very weak from a clinical point of view. The most common indication of SIT was an extract of grass pollen: in this case, κ coefficient was only 0.11. When analyzing concordance of SPT results with recombinants of purified allergens from the ISAC® test, we also found disagreement levels that varied from 40% with plane extract and positivity to Pla a1 and/or Pla a2 to 16% with grass extract and positive reaction to Phl p1 and/or Phl p 5. This fact may be responsible for the high percentage of disagreement found when SIT was prescribed only on the basis of SPT results. This can be explained by false-negative results of SPT used in single application even when performed by skilled technicians and by technical problems in patients with low skin sensitivity [5, 6]. However, it is also possible to find cases of false-positive and false-negative results when performing specific IgE to recombinants or purified allergens . Another factor that had a substantial influence and led to disagreement was the presence of sensitization to cross-reactive components such as profilin or polcalcin.
In summary, very low agreement was found concerning indication and use of allergens for SIT before and after performing MD. This great discrepancy makes the case for the usefulness of MD, at least in areas of complex sensitization to pollen, as a means of facilitating accurate prescription of pollen immunotherapy.
We thank Oliver Shaw for editorial assistance.
This study was funded by CIBERES (CIBER de Enfermedades Respiratorias), Instituto de Salud Carlos III of the Ministry of Science and Innovation, Spain.
Conflict of interest
Dr. Landívar, Dr. Ruiz-Garcia, Dr. Andregnette-Roscigno, and Dr Mahillo have no conflicts to disclose. Dr. Sastre reports having served as a consultant to Phadia, MSD, Novartis; FAES Farma and GSK, having been paid lecture fees by Novartis, GSK, Stallergenes, UCB and having received grant support from Phadia, GSK and ALK-Abello.