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Sensitivity of prick test with walnut commercial extracts and of prick by prick with raw walnut compared with open food challenge in walnut allergy

Background and aim: Diagnosis of walnut allergy includes the evaluation of IgE sensitization by skin prick tests (SPT) with standardized commercial extracts. When assuming the loss of relevant allergens due to extract preparation and storage, it is possible to perform SPT with fresh foods, i.e., prick by prick (PbP). To our knowledge, there is no published comparison between SPT with commercial extracts and PbP with fresh food about their sensitivity to the diagnosis of walnut allergy. Therefore, we describe our experience. Methods: We observed seven children (mean age ± SD 6.8 years ± 5.2, range 2 - 15 years; male 85%) with an history of immediate adverse reaction following walnut ingestion. All but one the patients underwent SPT with at least two out of three walnut commercial extracts (Lofarma, Milan, Italy; ALK-Abellò, Milan, Italy; Allergopharma, Rome, Italy). It has also been performed PbP with raw walnut. IgE-mediated walnut allergy was diagnosed based on suggestive history, positivity of PbP and failed open food challenge with walnut. Results: The SPT with Lofarma extract was never positive (sensitivity = 0%), that performed with ALK extract was positive in 2/5 cases (sensitivity 40%) and that of Allergopharma extract was positive in 1/5 cases (sensitivity 20%). PbP was positive in 7/7 cases (sensitivity 100%). Conclusions: In the specific case of walnut allergy in pediatric age, the execution of SPT alone with commercial extract may not be sufficient and clarifying in the diagnostic iter. We suggest to always associate the execution of PbP test. (www.actabiomedica.it)


Introduction
Walnut allergy is an increasing public health problem. Crucial to the diagnostic process is the collection of clinical history and the evaluation of IgE sensitization by skin prick tests (SPT) with standardized commercial extracts (1). When assuming the loss of relevant allergens due to extract preparation and storage techniques, it is possible to perform SPT with fresh foods, i.e. prick by prick (PbP). To our knowledge, there is no published comparison between SPT with commercial extracts and PbP with fresh food with regard to their sensitivity to the diagnosis of walnut allergy made by food challenge in pediatric age. Therefore, we describe our experience.
Written and oral consents from the patients and their parents were obtained, and the study was approved by the local ethical committee.

Methods
Between September 2017 to March 2019, we observed seven children (mean age + SD = 6.8 years + 5.2, range = 2 -15 years; male 85%) with an history of immediate adverse reaction (cutaneous, respiratory, gastrointestinal, anaphylactic) following walnut ingestion, evaluated in the pediatric allergy unit of the Policlinico Gemelli Universitary Foundation IRCCS of Rome. The diagnosis of anaphylaxis was made according to the definition of Sampson et al (2). All but one the patients underwent SPT with at least two out of three walnut commercial extracts (Lofarma, Milan, Italy; ALK-Abellò, Milan, Italy; Allergopharma, Rome, Italy) according to the standard international methodology (3). It has also been performed PbP with raw walnut, pricked with ALK lancet (at least 20 times in different areas of walnut); then, the patients forearm skin was pricked and raw walnut was pressed and rubbed on patients forearm skin spot. Moreover, PbP were performed twice on both forearms and the mean of the largest wheal diameters was reported. Regardless of the results of SPT and PbP, children were subjected to open food challenges (OFC) with raw walnut (4). We performed an incremental OFC, starting with a very small fragment of walnut, approximately a 68th walnut kernel, and continuing, in the absence of adverse reactions, with doubling the dose every 20 minutes. We considered passed the OFC if the patient tolerated an overall dose of walnut equal to 30 grams (5). When food dependent exercise induced anaphylaxis (FDEIA) was suspected, an OFC followed by physical exercise was performed (6). In addition, SPT have been performed with commercial extract of birch tree (Lofarma, Milan, Italy), peach and palm oil profilin (ALK, Milan, Italy) as paradigms of the panallergens respectively pathogenesis-related protein (PR-10), lipid transfer protein (LTP) and profilin. The sensitivity of SPT with commercial extracts and PbP was calculated using the 2 x 2 table and the formula "sensitivity = a / (a + c)".

Results
The results concerning SPT with commercial extracts of walnut, PbP and OFC with raw walnut are listed in table 1. The case n. 1 did not carry out the SPT with Allergopharma extract, the case n. 2 did not carry out the SPT with ALK extract, and the case n. 5 did not carry out the SPT neither with Allergopharma nor with ALK extracts. Table 1 also shows the results of SPT performed with birch tree, peach and palm oil profilin commercial extract. For the case n. 7 these last SPT were not performed. OFC failed in all seven cases. In particular, case n. 6 completed an incremental OFC up to a dose compatible with its age (20 grams) without adverse reactions. Then, she performed an exercise (free running for 10 minutes) and presented with hives and bronchospasm. The initial suspicion was then confirmed and FDEIA was diagnosed.
The SPT with Lofarma extract was never positive (sensitivity = 0%), that performed with ALK extract was positive in 2/5 cases (sensitivity = 40%) and that of Allergopharma extract was positive in 1/5 cases (sensitivity = 20%). PbP was always positive (sensitivity = 100%). Only in the case n. 6 the positivity to an allergen typically resistant to extractive processes, such as the peach LTP ALK extract, was found.

Discussion
In the 7 cases described, IgE-mediated walnut allergy was diagnosed based on suggestive history, positivity of PbP and failed OFC. The sensitivity of SPT with 3 different commercial walnut extracts was insufficient.
There are no guidelines that prohibits the use of natural food in the execution of the SPT but, at the same time, none explicitly encourages its use in normal clinical practice. Instead, in our experience, the use of PbP was definitely more effective and predictive of the presence of specific walnut IgE compared to SPT with commercial extract. This made it possible to avoid misdiagnosis with possible risks of serious reactions for the patients. This could happen mostly in cases with not very suggestive history, as in case n. 2 described below. The child in question, in fact, had presented only slight perioral erythema in a single occasion when he had ingested a small piece of walnut. The history, therefore, were not sufficient to raise suspicion in the presence of negative SPT with commercial extract.
The walnut allergens known until now, are classified into three main families called respectively prolamine ( Jug r 1, Jug n 1, Jug r 3), cupine ( Jug r 2, Jug r 6  , and a second non-specific lipid transfer protein (nsLTP2, Jug r 8) (4 7, 8). The major allergen is Jug r 1: its specific IgE have been identified in 50% of the sera of patients with walnut allergy and it is considered the potential responsible for severe allergic reactions and possible cause of anaphylaxis and angioedema as it is particularly resistant to the gastric juices action (9). In the Italian population the main walnut allergen is Jug r 3 and it is associated with serious reactions such as anaphylaxis and glottis edema (10). The profiline family ( Jug r 7), instead, is considered responsible for more moderate reactions and relegated to the oral cavity (oral allergy syndrome) (11). Therefore, in the specific case of walnut, the negativity of SPT with commercial extract against the positivity of PbP with fresh food, could be partially related to the degradation of labile allergens in the process of the extract preparation. As evidence of this, of the 2 patients with anaphylaxis after OFC, case 6 was positive for LTP (and therefore the OFC outcome could be so justified), while for case 4 we cannot offer an explanation other than a hypothetical sensitization to oleosins or to seed storage proteins (although the latter should be contained in commercial extracts). We have not performed a component resolved diagnosis (CRD), is a weakness in our study. A better characterization of our patients through CRD would have been appropriate.

Conclusions
From our experience, it could be deduced that, at least in the specific case of walnut and in pediatric age, the execution of SPT alone with commercial extract may not be sufficient and clarifying in the diagnostic iter. We suggest to always associate the execution of PbP test which, instead, showed greater accuracy and allowed us to reinforce the diagnostic suspicion, then confirmed by OFC.
Funding. The authors declare that the study did not receive funding.

Potential conflict of interests related to the manuscript content:
Each author declares that he or she has no commercial associa-tions (e.g., consultancies, stock ownership, equity interest, patent/ licensing arrangement etc.) that might pose a conflict of interest in connection with the submitted article.