Study design
The primary goal of this study was to investigate the association between exposure to airborne ragweed pollen and daily ocular, nasal and asthmatic symptoms in two sub-cohorts of patients throughout one whole ragweed season. We followed individuals sensitized to ragweed and suffering from seasonal ragweed oculo-rhinitis with/or without asthma, from July 16 to September 15, 2014. Study subjects weredivided in two cohorts: one consisted of individuals who had never received a ragweed AIT, hence “non-AIT”,while subjects in the other cohort had been treated with ragweed AIT, hence “AIT treated”. Patients were treated with AIT prior to, and independent of, their enrollment in the study. These patients received AIT treatment either in the same year as the study or in the three years immediately preceding the study.
Originally, we actually envisioned a different study design. Ragweed and mugwort coexist in the study area, and since mugwort and ragweed blooms are partly overlapping, at firstwe also wanted to investigate the influence of mugwort exposure on allergy symptoms attributed to ragweed. However, since the number of patients with dual sensitization (i.e. ragweed and mugwort) was very small (n=25), the original study design was modified to include only subjects sensitized to ragweed.
From July 16, 2014 to September 15, 2014, all patients filled in a daily Clinical Diary of Symptoms and Drugs (CDSD). During the same period, pollen counts were measured from three pollen traps located in the study area (see the ‘Pollen concentration’ section for exact locations). The daily averages of ragweed and mugwort pollen concentration obtained from the three pollen traps were used for statistical calculations against the daily mean of the symptom/drug scores for each of the two sub-cohorts.
Setting
Patients were recruited and followed in the areas of the ASST Ovest Milanese and ASST Rhodense Districts, both of which are under the milanese health protection agency ATS (Fig. 1). Both districts are located in the North West area of the Metropolitan Area of Milan, covering an area of approximately 827 sq. km (geographical coordinates: to the East: Lat 8 ° 39’ 59” E, to the North: Long 45° 35' 54” E, to the West: Lat 9 °11' 47” E, to the South: long 45°16' 46” N (with the exclusion of the Municipality of Milan)). Patients within the two districts were enrolled in the Allergy Units of the following hospitals: Legnano Hospital (Long 45° 35' 44” N, Lat 8° 55' 23” E), Abbiategrasso Hospital (Long 45° 23' 40” N, Lat 8° 54' 49” E), Garbagnate Milanese Hospital, Pneumology and Pediatric Allergy Units (Long 45° 34' 53” N, Lat l9° 05'14” E), Cesano Boscone Ambrosiana Clinic (Long 45°26' 57” N, Lat 9° 05' 33” E). Each one of these five Allergy Units was located in proximity of one of the three pollen traps (i.e. min-max distance: 0.2 -19.6 km) (Fig. 1).
Participants
Participants in the study were a random sample of citizens residing in the designated area, with a confirmed diagnosis of ragweed seasonal rhino-conjunctivitis (with or without asthma), some of which were treated with AIT. 66 subjects (male: 32, female: 34) were enrolled in the study. The Eligibility Criteria to participate in the study were: 1) subjects were sensitized to ragweed pollen, demonstrated by a positive SPT with commercial ragweed extracts (Lofarma SpA, Milan; ALK-Abellò SpA, Milan) and IgEs for the total ragweed extract and for rAmb a 1 (pectate lysase) (Immuno -CAP Thermo Fisher Scientific Inc., Monza, Italy). In case of discordant test results, ragweed sensitization was confirmed based on positive IgEs for rAmb a 1. 2) Subjects had an established and documented history of clinical manifestations of ragweed allergy (i.e. symptoms of oculo-rhinitis, associated or not with asthma), coinciding with the ragweed flowering period, in the years immediately preceding enrollment. 3) Subjects had given their informed consent. 4) Subjects were able to adhere to the study protocol, and 5) subjects would remain in their residence throughout the observation period.
All eligiblestudy subjects also performed SPT with 12 other inhalant allergens, namely pollens of grass, birch, hazel, alder, mugwort, D.pteronyssinus and D. farinae, Aspergillus, Cladosporium, Alternaria, cat and dog dandruff and, moreover, IgEs for the total mugwort allergen, rArt v 1 (defensin-like protein linked to the polyproline-rich region) and rArt v 3 (NS LTP type 1).
At the end of the recruitment period, from February 01, 2014 to July 15, 2014, 71 subjects were assessed to the Eligibility Criteria. One subject was excluded because he did not perform the IgE test and four because the IgEs for rAmb a 1 were negative. The remaining 66 patients were confirmed eligible and were included in the study. The mean age was 36.5 years (range: 8 - 69 years). None of the subjects were pregnant, nor suffering from chronic diseases. All subjects resided in the study area, and in the period from February 01, 2014 to July 15, 2014, they had an allergic visit for respiratory symptoms in one of the five Allergy Units participating in the study. Moreover, 42 subjects had never been treated with AIT for ragweed, while 24 subjects had previously received ragweed AIT.Thus, patients were divided into two sub-cohorts: non-AIT and AIT treated. All recruited subjects (100%) completed the follow-up and were subjected to statistical analysis. The characteristics of the study base at enrollment are shown in Table 1.
Table 1
Characteristics of the study base at enrollment
|
non-AIT
n= 42
|
AIT treated
n= 24
|
Male
Female
|
23 (55%)
19 (45%)
|
9 (37,5%)
15 (62.5%)
|
Mean age at enrolment (range)
|
34.47 (8-69)
|
39.58 (11-62)
|
Allergy / Sensitization
|
rAmb a 1
|
42/42 (100%)
|
24/ 24 (100%)
|
rArt v 1
|
14/ 42 (33%)
|
7/24 (29%)
|
rArt v 3
|
4/42 (9.5%)
|
3/24 (12.5%)
|
Asthma
|
17/42 (40%)
|
11/24 (46%)
|
Pollen concentration data
Ragweed and mugwort’s pollenwas sampled daily from three Hirst-type pollen traps located respectively in Legnano (Lat 45° 35' 44” N, Long 8° 55' 23” E), Magenta (Lat 45° 28' 16” N, Long 8° 53' 33” E) and Rho (Lat 45° 32' 51” N, Long 9° 02' 42” E). The Hirst volumetric trap continuously draws 10 liters of air per minute onto an adhesive coated tape. Particles in the air stick to the tape, which moves at 2mm per hour to provide a time sample. The pollen collected from the traps was identified and quantified by a specialized technician and then correlated to the average air volume over 24 hours. The reference standard adopted for the sampling and counting of pollen was the UNI 11108 of 2004, valid at the time of the study.
The following definitions were adopted from the Quality Control Working Group of the European Society of Aerobiology (EAS) and the International Association of Aerobiology (IAA) (31). Pollen count: result of the analysis on the slide; this quantity is not comparable and needs to be converted into concentration. Pollen grain: male gametophyte of the seed plant. Pollen concentration: expressed as pollen grains/m3, i.e. the number of pollen grains dispersed in the air per unit of air volume. Main Pollen Season (MPS): the length of time that pollen is present in the atmosphere in significant concentrations in a place. The MPS in our study was based on the average of the three monitoring stations using the Nillson & Persson criterion (32): “the period from when the sum of the average daily pollen concentrations reaches 5% of the total sum up to at the time when the sum reaches 95%; i.e. the main pollen season with 90% of the entire quantity of pollen”.
Clinical Diary of Symptoms and Drugs (CDSD)
All study participants were asked to complete a CDSD during the follow up period. Patients were asked to record their daily symptoms and medication use every evening from July 16th to September 15th, 2014. Each symptom was rated on a 4-point scale: 0: no symptoms; 1: mild symptoms; 2: moderate symptoms; 3: severe symptoms. These ratings were used to score each one of the following 9 distinct symptoms: Nasal: 1. nasal congestion/nasal difficulty of breathing, 2. runny nose, 3. itchy nose, 4. itchy throat/ears; Ocular: 5. itching and/or burning in the eyes, 6. tearing and/or wet eyes; Bronchial: 7. cough, 8. breathing difficulty while moving, 9. breathing difficulty at rest. Therefore, the total symptom score ranged from 0 to 24, while the nasal score ranged from 0 to 9, the ocular from 0 to 6 and the bronchial from 0 to 9. Furthermore, patients were also asked to record every single unit of drug taken, i.e. number of tablets/day of oral antihistamines, number of spray/day of inhaled topical nasal corticosteroids or topical bronchial corticosteroids or topical bronchial corticosteroids + Long Acting Bronchodilator Agents (LABA) or + Short Acting Bronchodilator Agents (SABA), and number of drops/day of antihistamine eye drops.
Patient follow up during the exposure period
All patients enrolled in the study went to their reference Allergy Unit immediately before the exposure period began. During the visit, their clinical history was collected, a medical examination was performed and patients gave their informed consent for participation in the study. All patients were also provided with a CDSD, which was returned compiled for all dates during the second visit after September 15, 2014.
Comparability of evaluation methods.
Patients in both cohorts, non-AIT and AIT treated, were recruited for the study in the same way, underwent the same diagnostic tests, received the same CDSD, were visited in the follow up on the same dates, and received the same Informative Consent.
Bias
The known overlap in the initial time period of ragweed MPS with mugwort MPS was a possible cause of bias with respect to the symptom score attributed to ragweed, given that the symptoms possibly caused by exposure to mugwort pollen are indistinguishable from those caused by ragweed pollen in subjects sensitive to both pollen species. However, the concentration of mugwort pollen detected during the study period was very low (see below) and therefore, unlikely to have influenced in any significant way the respiratory symptoms of those patients also allergic to mugwort.
Other possible causes of bias on the symptom/drug score may have been: climatic variables, air pollution, as well as any intercurrent respiratory infectious diseases (which were not considered in orderto simplify the analyses).
Ethics
The study protocol complies with the Declaration of Helsinki and all its subsequent amendments of Tokyo 1975, Venice, 1983, Hong Kong, 1989, as well as with the current regulations for good clinical practice (GCP). The protocol also complies with all national and community regulations applicable to observational studies and all ethical and deontological principles that inspire the medical profession. The study was approved by the Ethics Committee of Milan Area C (No 80_122013).
Quantitative variables.
Only ragweed pollen concentration was considered as a feature in the regression models,since the correlation between symptom/drug score and mugwort pollen concentration was overall low and not significant.
Statistical analyses
Spearman's rank ρ correlation coefficient (33) and Kendall's rank τ correlation coefficient (34) nonparametric statistical tests were used to examine the correlation between daily ragweed pollen concentrations and symptoms’ intensity, measured as the total daily number of symptoms observed in study patients. We adopted a non-parametric statistical approach as the target variables were not normally distributed.
The non-parametric Kruskal-Wallis test (35) and the Wilcoxon rank sum test (36) were used to compare mean symptom level scores between the two patient cohorts: non-AIT and AIT treated.
Time series analysis was used to analyze the influence of the daily pollen concentrations cycle on the onset of allergic symptoms. It is reasonable to assume that the total number of symptoms currently observed depends on the daily pollen concentrations, as well as on the previous day's (lag=1) symptom values. For this reason, we applied a first-order autoregressive distributed lag (ARDL) model to explore these short-run relationships (37).
Let \({x}_{t}\)(t=1, …,T) be the daily pollen concentrations and \({y}_{t}\)the total number of symptoms observed in the study patients. The ARDL model is defined by
\({y}_{t}=\alpha +{\varphi }_{1}{y}_{t-1}+{\theta }_{0}{x}_{t}+{\epsilon }_{t},\) (t=1, …,T) (1)
where 𝛆t is a white noise process, independent of \({x}_{t}\), \({y}_{t}\) and \({y}_{t-1}\), so that the model (1) can be estimated using ordinary least squares (OLS). The regression coefficients can be interpreted as measures of the influence of the feature on the target variable.
We also considered a first-order autoregressive distributed lag model similar to the model formulated in (1) with the addition of a lagged \({x}_{t-1}\) as a further explanatory variable,
\({y}_{t}=\alpha +{\varphi }_{1}{y}_{t-1}+{\theta }_{0}{x}_{t}+{\theta }_{1}{x}_{t-1}+{\epsilon }_{t},\) (t=1, …,T) (2)
Furthermore, the Akaike information criterion (AIC) and the Bayesian information criterion (BIC) were used for model selection. Models with the lowest AIC and BIC values were considered to be the ‘best’. Finally, since the pollen concentration data showed high variance, we used a square root transformation to stabilize the variance.
All statistical analyses were conducted using R 4.0.2 (38).