*2.1. Patients*

Defining the primary outcome as the relationship between the total PRIS score and the ΔMSS, a minimum sample size of 85 patients achieves 80% power assuming a medium effect size (d = 0.3; [18]). A two-tailed test on Pearson's correlation was considered with a significance level α = 0.05. Therefore, 110 patients (68 males and 42 females) diagnosed with allergic rhinitis with or without concomitant asthma at the Division of Allergy and Clinical Immunology of the University of Naples Federico II, Naples, Italy, were enrolled in this prospective cohort single-center study. All the patients presented a history of symptoms related to allergen exposure (rhinitis and/or asthma), documented positive SPT for pollen and/or perennial allergens, and allergen-specific IgE test. Spirometry was used to diagnose asthma [19].

We included both monosensitized and polysensitized patients with uncontrolled allergies despite optimal pharmacotherapy. Patients under six years of age were excluded. We also excluded patients with asthma not adequately controlled by pharmacotherapy [20–22] as assessed by Asthma Control Test (ACT) [23–25]. Finally, we excluded patients with nasal polyposis diagnosed by nasal endoscopy.

Demographic and clinical data were collected from patient medical charts and diaries. Data were available for all 110 patients.

Rhinitis and asthma symptoms were singularly measured using a Visual Analogue Scale (VAS) at baseline (T0) and after 12 (T12) and 24 months (T24) of SLIT treatment. In particular, patients were asked to place a mark on a 10 cm line for rating the severity and frequency of each symptom [26,27]. The symptoms evaluated for allergic rhinitis were sneezing, nasal congestion, rhinorrhea, and nasal, throat, eyes, and ears itching, while chest tightness, breathlessness, wheezing, and coughing were assessed for bronchial asthma. The VAS was anchored at 0 with "no symptoms" and 10 with "very severe symptoms". The VAS also included the assessment of the frequency of symptoms (0 with "no symptoms in the last 30 days" and at 10 with "I have experienced symptoms every day in the past 30 days"). In addition, we instructed patients to record monthly in a diary their symptoms, the number of asthma exacerbations, and on-demand therapy [28–30]. When the patients

were visited, they were asked to complete the VAS by checking their diaries. This helped patients take note of their clinical conditions both during and out of season. In particular, when the VAS was administrated for evaluating the asthma exacerbation, 0 corresponded to no exacerbation while 10 implied frequent exacerbations. All the patients enrolled were switched to the same on-demand therapy with second-generation oral antihistamines and intranasal corticosteroids. Patients with asthma were treated using inhaled corticosteroids (ICS) and long-acting β-agonists (LABAs) as the controller and the quick relief therapy. The patients were instructed to record monthly on the provided diary the use of on-demand therapy. Subsequently, when the patients were visited, their perception of on-demand therapy use was evaluated by the VAS (10 implied the highest medication use, while 0 corresponded to no medication use). The mark was then measured in millimeters for all the items explored to provide the VAS score and normalized to 100. For each patient, we assessed the mean symptom score (MSS) based on VAS results at T0 (MSS-0), T12 (MSS-12), and T24 (MSS-24). As efficacy index of SLIT, we calculated the percentage difference between the MSS-0 and MSS-12 [ΔMSS-12(%) = (MSS-0–MSS-12)/MSS-0\*100], and between MSS-0 and MSS-24 [ΔMSS-24(%) = (MSS-0–MSS-24)/MSS-0\*100]. Based on the ΔMSS-12(%) and ΔMSS-24(%) results, patients' SLIT outcome was stratified into quartiles (first quartile = ΔMSS ≥ 75% = very high symptom improvement; second quartile = 50% ≤ ΔMSS < 75% = high symptom improvement, third quartile = 25% ≤ ΔMSS < 50% = mild symptom improvement, fourth quartile = ΔMSS < 25% = low symptom improvement, Table 1).

**Table 1.** Classification of patients treated with sublingual immunotherapy (SLIT) based on ΔMSS. Patients were stratified based on ΔMSS to identify patients who had a better clinical response as compared to those with a poor response to SLIT. ΔMSS-12: evaluation after 12 months of SLIT treatment; ΔMSS-24: evaluation after 24 months of SLIT treatment.


All procedures performed in this study were in accordance with the ethical standards of the study center and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All the subjects enrolled gave informed consent to participate in the study.
