Allergen sensitization linked to climate and age, not to intermittent-persistent rhinitis in a cross-sectional cohort study in the (sub)tropics

Twenty-six study centers were selected, distributed over the six climate zones across Mexico. The division of the climate zones was according to the official INEGI assignation (http://​www.​inegi.​org.​mx/​geo/​contenidos/​recnat/​clima/​default.​aspx and related charts), see Figure 1. Consecutive patients (2-70 years) with rhinitis symptoms were pre-selected if they had symptoms compatible with allergic rhinitis, e.g. symptoms exacerbating during a specific season and/or on exposure to certain allergen(s). After testing positively with the routine SPT of the center, subjects were asked to participate in the study. Thus, recruited patients were those routinely diagnosed as having allergic rhinitis by the allergists. The patients (and for the pediatric patients the parents/legal guardian) gave written informed consent. The study was evaluated and approved by an independent Ethics Committee. Subjects were excluded if they had any contra-indication for undergoing skin prick testing or if they had had immunotherapy within the previous five years. The elimination criteria were an invalid study SPT or if the criteria for the study diagnosis of rhinitis –see below- were not met.

Eligible subjects were invited to fill out a validated questionnaire on rhino-conjunctivitis symptoms [12], which allows classification of allergic rhinitis (AR) as seasonal-perennial (SAR-PAR) on one hand and according to the ARIA classification as intermittent (IAR) versus persistent (PER) and mild versus moderate-severe on the other hand [13]. For children below 12 years, the carer completed the questionnaire. A subject was defined as suffering from rhinitis if at least two of the four enquired rhinitis symptoms (congestion, rhinorrhea, pruritus, sneezing) were positive and conjunctivitis if one of three conjunctivitis symptoms (erythema, pruritus, lacrimation) were present. AR was classified according to the questionnaire data as completed by the patients and following the ARIA guidelines as intermittent (symptoms appearing <4 days per week or <4 wks.) or persistent.

Once the presence of rhinitis/rhinoconjunctivitis was confirmed, subjects underwent the study skin prick test with a blinded set of sixteen of the most common [14] allergens in Mexico (Dermatophagoides mix^stand, cat^stand, Poa pratensis^stand, Phleum pratense^stand, Cynodon dactylon^stand, Ambrosia^stand, profilin, peach: ALK-Abelló, Madrid, Spain,^stand = standardized; Blomia tropicalis^stand: Biocen, La Havana, Cuba; Blatella germanica/Periplaneta americana mix, Fraxinus excelsior, Quercus ilex, Prosopis, Cupressus sempervirens, Chenopodium album, Heliantus, Alternaria alternata, Aspergillus fumigatus: Allerquim, Mexicocity, Mexico). Dermatophagoides mix is a 50-50% mix of Dermatophagoides pteronyssinus and farinae. The English names of the allergens are: house dust mite, cat, Kentucky bluegrass, Timothy, Bermuda grass, Ragweed, Blomia, cockroach mix, Ash, oak, mesquite, cypress, lamb’s quarter, sunflower, Alternaria, Aspergillus. Blinding of SPT extracts was carried out at a central site, not involved in patient recruitment. Technicians applying the SPT, physicians and patients were all blinded to the content of the vials, as was the statistical team. The skin testing was done in duplicate. With an ALK-lancet (ALK-Abelló, Madrid, Spain), the skin was penetrated, passing through each drop of allergen extract at 90°. Personnel that applied the test in the centers passed a previously described proficiency test [15] (coefficient of variation <20%) before recruiting the first patient. The results were read at 15 min by drawing around the perimeter of the wheal with a ballpoint pen, placing transparent tape against the skin and transferring the prints to the data collection sheet. A test was considered positive if the wheal surface ≥ 5 mm [2] and if it met the criteria of a valid test (negative control <5 mm², positive control ≥5 mm²) [16].

The programmed sample was 90 patients per climate zone. Sample size calculation [17] was based on an estimated population size of 500,000 AR patients (500 practicing allergists seeing approximately 1,000 allergic rhinitis patients/year), an estimated frequency of SPT positivity for HDM of 55% with a confidence level or 95% and a confidence interval of 10% (standard error 0.05). For the allergens with an estimated frequency of SPT positivity close to 25% (or 75%), 73 patients per climate zone would have been sufficient to achieve the identical confidence interval. Pearson’s χ squared tests were used to compare the frequency of the SPT positivity between the climate zones, the age-groups and the phenotypes of rhinitis (if necessary Yates’ correction was applied). Differences in wheal size were compared with the Mann-Whitney rank test.

Nationally, SPT positivity was mostly found for mites (proportion 0.599, 95% confidence interval (CI) 0.55-0.64), followed by tree pollens (0.442, 95% CI: 0.40-0.48), grass pollens (0.338, 95% CI: 0.30-0.38), weed pollens (0.265, 95% CI: 0.23-0.30) and finally molds (0.113, 95% CI: 0.09-0.14). As such the frequency of SPT positivity for individual allergens could be divided into three groups (Figure 2). Firstly, the most frequent allergen, Dermatophagoides mix (pteronyssinus + farinae), with a positive SPT in more than 56% of the patients. Secondly, the ‘frequent’ allergens, with percentages of SPT positivity between 20-26% -cat allergen is in this group- and thirdly, the less frequent ones (SPT positivity from 6 to 14%). As for the mean wheal size in SPT positive patients, there was large variation. The largest wheals were produced by Cynodon dactylon, followed by profilin, peach and Aspergillus. Dermatophagoides mix produced a mean wheal size in the middle range (Additional file 1: Figure S1). As such, the most frequent allergen was not the one producing the largest wheals.

Figure 3 shows the skin prick test positivity according to the age-groups. Adults (age 18+ years) had more SPT positivity for pollens of trees (p < 0.0001) and weeds (p < 0.0005), as compared to the younger age groups. However, SPT positivity for mites was statistical significantly more frequent in the 2-11 and 12-17 year age-groups, as compared to the adult subjects. These differences were maintained even if only subjects from the subtropic zones 1-5 were taken into account (HDM sensitization 2-11 years: 68%; 12-17 years 71%; 18 + years 46%; p < 0.0005 for comparisons of children-adults and adolescents-adults). For subjects from the tropical zone 6 the same trend could be seen, but because of small numbers the differences did not reach statistical significance (HDM sensitization 91%, 85% and 67%, respectively for children-adolescents-adults). We also compared the mean wheal size of SPT positive subjects between age-groups (see Additional file 2: Table S1 and S2). Mean wheal size of the 2-11 year age-group were generally smaller than the other two age-groups, reaching statistical significance for several allergens. In general there was no difference between the 12-17 year and 18+ year subjects, with even larger wheals in the adolescents as compared to the adult subjects for both mites (Dermatophagoides: p = 0.023; blomia: p = 0.026).

There was no difference between intermittent (IAR) and persistent (PER) allergic rhinitis in the frequency of SPT positivity for any allergen tested, except for trees. Patients with PER had more frequent SPT positivity to trees as compared to patients with IAR (p = 0.012, Figure 4). For mild versus moderate-severe AR no difference could be found for any allergen. Comparing SAR with PAR patients, grass, tree and cat SPT positivity was more frequent in SAR patients (Figure 5).

As for wheal size comparing IAR versus PER and mild versus moderate-severe patients, no differences could be found, with one exception: PER patients presented a larger wheal surface for Alternaria. PAR patients had larger wheals than SAR patients for Prosopis, Chenopodium, cockroach mix and profilin.

Statistically significant differences of positive SPT in a specific zone as compared to the rest of the country are documented in Figure 6 and Table 2 compares sensitization between the climate zones. In the dry-hot and the (sub)humid zones HDM was the most frequent allergen, followed by trees. In the dry North of Mexico sensitization to molds and weeds (and consequently also to pan-allergens such as peach –rich in lipid transfer protein- and profilin) are high. In the tropical zone 6, warm with rainfall all year long, almost all patients are sensitized to HDM, leaving only 20% of the patients with some additional pollen allergy. However, for the patients in the middle of the country, living in the agricultural zone with a (semi)-dry temperate climate, both tree and grass pollen sensitization is more common than HDM sensitization. Cockroach sensitization is important all over the country.

This study was approved by Comité de Ética del Instituto Jalisciense de investigación Clínica SA de CV on the 12^th of January 2010.