Nitrogen dioxide is not associated with respiratory infection during the first year of life

Jordi Sunyer1,2, Carme Puig3, Maties Torrent4, Oscar Garcia-Algar3, Ignasi Calicó5, Laura Muñoz-Ortiz1, Maria Barnes6 and Paul Cullinan6 on behalf of the AMICS study*

1 Unitat Recerca Respiratria i Ambietal, Institut Municipal Investigació Mèdica (IMIM), Barcelona, Catalonia, Spain
2 Universitat Pompeu Fabra, Barcelona, Spain
3 Pediatrics Service, Hospital del Mar, Barcelona, Spain
4 Area de Salud de Menorca, INSALUD, Menorca, Spain
5 Microbiological Department, Hospital Vall d'Hebron, Barcelona, Spain
6 Department of Occupational and Environmental Medicine, Imperial College, London, UK

Correspondence: Jordi Sunyer, IMIM, C/ Dr. Aiguader 80, 08003-Barcelona, Spain. E-mail: jsunyer{at}imim.es


    Abstract
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Background Nitrogen dioxide (NO2) has been related to respiratory infections in experimental studies but its role remains controversial in general population studies of children. We aim to assess the association between indoor NO2 and lower respiratory tract infections (LRTI) during the first year of life in a multicentre prospective cohort study.

Methods Children (n = 1611) were recruited prior to birth for the Asthma Multicentre Infants Cohort Study (AMICS). Three concurrent cohorts (Ashford, Kent [UK]; Barcelona city, and Menorca Island [Spain]) followed the same research protocol. NO2 was measured with passive diffusion tubes placed in the living room for 2 weeks when infants were approximately 3 months old. Doctor-diagnosed LRTI during the first year of life (as well as antibiotic use) were measured by questionnaire, and in Ashford validated by the examination of clinical records. In Barcelona, direct measurements using nasopharyngeal lavage and cultures within a continuous surveillance system were done.

Results The cumulative rates of LRTI (39% in Ashford, 28% in Barcelona, and 45% in Menorca) were unrelated to NO2 levels (corresponding medians 6, 46, and 12 ppb, respectively) in all three centres (all odds ratios being around 1). Similarly, the rates of LRTI in Barcelona measured with the continuous record showed no association with NO2 (all rate ratios being below 1). In addition, there was no association between rate of antibiotics courses per year per child (2.4 in Ashford, 1.7 in Barcelona, 0.9 in Menorca) and NO2 levels.

Conclusions Indoor NO2 at current levels does not seem to be involved in increasing respiratory infections by itself in infants, suggesting that the effects observed in studies on outdoor air are probably due to other copollutants.


Accepted 2 October 2003

Nitrogen dioxide (NO2) is a common air pollutant both in homes and in the urban outdoor atmosphere. It is not highly soluble and most inhaled NO2 is retained in the small airways. Experimental studies have suggested that NO2 increases the effect of respiratory pathogens by reducing the efficacy of lung defence mechanisms (due to effects on mucocilliary clearance1 and the alveolar macrophage2), or by activating pro-inflammatory cells.3 The toxicological evidence suggests that NO2 at the low concentrations found in everyday life may play a role in increasing the incidence and severity of respiratory infections.3,4 In epidemiological studies, however, the evidence is controversial. In a follow-up study of a birth cohort in Albuquerque (New Mexico, USA) with a very comprehensive surveillance method to detect respiratory illness during the first year of life, indoor NO2 measurements were not associated with illness incidence.4 However, levels of NO2 were very low (median around 10 ppb) consistent with the low frequency of cooking with gas stoves in that community. By contrast, in a Dutch birth cohort, upper respiratory tract infections and doctor-diagnosed serious colds were related to NO2 outdoor levels.5 In two German birth cohorts, outdoor NO2 was associated with cough during the first year of life,6 and similarly in a panel study on infants with personal samplers in Finland.7 However, NO2 in outdoor air is mainly generated by traffic and the authors could not differentiate the role of NO2 from that of fine particles or other traffic pollutants.5,6 In indoor environments, NO2 is strongly related with indoor combustion sources associated with cooking or heating,8 and confounding by traffic pollutants is less of a limitation.

We aimed to assess the association between indoor NO2 and LRTI in a multicentre cohort study of newborn children with a broad range of indoor NO2 exposures.


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Study population
Birth cohorts were recruited during pregnancy in Ashford (UK), Barcelona and Menorca (Spain) to join the international AMICS study (Asthma Multicentre Infant Cohort Study). All cohorts followed the same basic research protocol. Two of the cohorts (Ashford and Menorca) were population-based (including all women presenting for prenatal care in Ashford during 1995–1996 and in Menorca over 12 months starting in mid 1997). The third cohort was hospital-based (mothers were enrolled during last trimester of pregnancy in the Obstetric Department of the Hospital del Mar in Barcelona during the years 1996–1997. Mothers were city residents without plans to move, literate, and possessed telephones). Among the selected mothers, 615 full-term live children born in Ashford, 487 in Barcelona and 485 in Menorca were enrolled in the cohort. The study was approved by the corresponding research committees and mothers signed a written consent form.

Exposure assessment
During the first 3 months of life a field technician made a home visit to complete a questionnaire on household characteristics (cooking appliances, heating and cooling systems, hours of ventilation, size of house), smoking and occupation, to collect dust samples, and to measure NO2. Average 2-week NO2 concentrations were measured by passive diffusion tubes9 installed in the living room wall at a height of 2 m and away from any window or air conditioner. NO2 concentrations were measured in a single laboratory by colorimetric reaction. Social class was defined using the UK Registrar General's 1990 classification of the current paternal occupation.

Respiratory infections
The frequency (and nature) of LRTI during infancy was obtained indirectly in all cohorts using maternal reports of upper and lower respiratory tract symptoms; as well as by systematic scrutiny of general practice medical and prescription records in Ashford. In all three centres, at the end of the first year, mothers were asked to record, retrospectively, the approximate frequency of symptoms consistent with upper or lower respiratory tract infection. Occurrence of an LRTI was defined as a positive answer to the question ‘Has a doctor ever told you that your son/daughter has had a chest infection?’. Information on antibiotic use was gathered using the question ‘Has your son/daughter had any antibiotics?’ and ‘if yes, how many times?’.

In the Barcelona cohort, mothers were asked to contact a local 24-hour co-ordinating centre if their child should develop, during the first year of life, symptoms of an LRTI: wheezing, wet cough, or troubled breathing for at least one day. A standard symptoms form was then completed and a home visit made at which nasal and throat swabs were collected from the child after examination by a physician. Samples were cultured within the first 6 hours. A system of telephoned monthly reminders to all mothers in this cohort was instituted. Among 487 children, 462 (96%) provided data by the active method or the monthly recall.

Other measurements
At the enrollment mothers completed a questionnaire on previous diseases, allergic and respiratory symptoms, smoking, and occupation. A prick test to common allergens was carried out following standardized methods within AMICS, and classified as positive if it produced a skin wheal >=3 mm (mean of perpendicular measures) in the presence of a positive histamine control and a negative uncoated control.

Data analysis
The cumulative incidence of LRTI during the first year of life was obtained from the one-year questionnaire. Similarly, LRTI data ascertained via the Barcelona surveillance system were treated as cumulative incidence. The association between LRTI and NO2 levels was measured with the odds ratio (OR), using logistic regression methods. It is assumed that the odds is an approximation of the cumulative incidence. Adjustment, then, could be evaluated using multivariate logistic regression models. Rates of antibiotic prescriptions, as well as incidence rate of LRTI in Barcelona were modelled with Poisson regression. Confounding variables selected were those showing a statistically significant association (P < 0.1) with LRTI in our study (i.e. ever breastfeeding, social class, sex, family size, and maternal smoking among a set of variables including parental asthma or atopy).


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A description of reported doctor-diagnosed LRTI is shown in Table 1. The cumulative incidence was highest in Menorca while reported antibiotic courses were highest in Ashford. NO2 levels and gas stove use were highest in Barcelona. Family size was largest in Ashford where social class was unevenly distributed with higher prevalence in both the two extreme categories.


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Table 1 Descriptives (%) by site

 
The rate of LRTI and antibiotics courses decreased with increasing levels of NO2 in Ashford and Barcelona and slightly increased in Menorca, though neither trend was statistically significant (Table 2). Differences according to appliance type were also non-significant, except decreasing antibiotic use among those having a gas stove in Ashford. In Barcelona, the analysis by stove use has no relevance given that only 4% of subjects did not have a gas stove. A lack of association was also observed with any respiratory symptom (i.e. wheeze, cough, chestiness) reported by parents at age one in the three centres (data not shown). Some of the parents reported infants with wheeze but without a diagnosis of LRTI (17.2% in London, 8.7% in Barcelona, and 3.8% in Menorca). It was not related to NO2 levels in any of the three centres (P > 0.5).


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Table 2 Distribution of lower respiratory tract infections (LRTI) during first year of life and rate of antibiotics prescription courses according to nitrogen dioxide (NO2) levels, gas appliances, and site

 
When all centres were pooled, the only positive significant association was between antibiotics and gas heating, though the association disappeared after adjusting for variables related to LRTI, such as social class (Table 3). It is important to note that the measures of association were consistently near to one (i.e. no increased/decreased relative risk) whatever the outcome or the exposure. These findings were homogeneous among the three centres (P for heterogeneity = 0.7). Stratification by maternal atopy did not show any effect modification (P for interaction = 0.8). When NO2 was treated as a continuous variable (logarithmically transformed) results were very similar.


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Table 3 Adjusted association between lower respiratory tract infections (LRTI) during first year of life and rate of antibiotic prescription per child and nitrogen dioxide (NO2) and gas appliances

 
The incidence of LRTI assessed by the surveillance system in Barcelona was not associated with NO2 or the use of gas appliances either in the crude or the adjusted models (Table 4). Limiting LRTI to a positive culture from a nasal swab did not change these results.


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Table 4 Incidence rate of lower respiratory tract infections (LRTI) per child during first year of life using a surveillance method of detection in Barcelona according to nitrogen dioxide (NO2) quartiles and having gas appliances

 

    Discussion
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At high concentrations in animals and humans, NO2 damages the epithelial cells by oxidant injury, reduces the clearance of infecting organisms, depresses alveolar macrophages, and releases pro-inflammatory mediators.10 However, we did not find any association of indoor NO2 with any of the different indicators of LRTI in three birth cohorts of infants using individual passive samplers for measuring NO2. These results confirm the findings observed by Samet et al.4 in a previous large birth cohort with lower indoor levels, and the discrepancies between experiments and results with general populations in current environments.11 Smaller studies also using individual measurement of indoor NO2 among infants, a cross-sectional study in the UK,12 and a Norwegian case-control study13 also did not find any relation with respiratory symptoms. A different issue arises from studies measuring outdoor NO2. Both a recent Dutch birth cohort5 and German birth cohorts6 showed an association with outdoor NO2 measurements, which has also been observed in studies at school age.14,15 Similarly, in the Swiss study, SAPALDIA, the duration of lower respiratory symptoms in children under 5 years of age was related to an individual outdoor measurement, but not with indoor levels.16 The data presented here, however, suggest that the NO2 effects on respiratory infections in outdoor air at current levels are probably due to other pollutants that have the same source and environmental dynamics as NO2, since NO2 measured with indoor samplers (even at high concentrations such as in Barcelona) was unrelated to LRTI in infants.

Misclassification of the outcome is unlikely to explain this lack of association, given the efforts made to ensure the quality of the diagnosis, in Ashford by checking the maternal reports with family practitioner clinical records, and in Barcelona by using a surveillance system and a direct method of evaluation of LRTI. Similarly no association was found with antibiotic use which may be an indicator of LRTI. However, the number of antibiotic courses is strongly related to social class and may indicate medical contact rather than actual LRTI or also other infections such as upper respiratory tract infections. Furthermore, no association was observed with any respiratory symptom at age one. Only data on the first year were examined, given that most incident cases occur in this period and further ascertainment is more complex. During this period, however, protection by breastfeeding should be taken into account. When we adjusted for length of breastfeeding (with a median length of 8 weeks among the 73% having breastfed) the results did not change.

Another potential bias could be a misclassification of exposure given that samples were collected only once. However, the results were very consistent with the use of gas appliances and a sub-analysis including only children home-bound throughout most of the year did not modify any of the results. In addition, indoor NO2 levels in this study have been strongly related to the type of gas appliance (i.e. higher levels with gas stove and heating)8 suggesting that the measurement error, if it exists, is small.

Overall, indoor NO2 at current levels does not seem to be involved in increasing respiratory infections by itself in infants, suggesting that the effects observed in studies on outdoor air are probably due to other pollutants.


    Notes
 
* Members of the AMICS (Asthma Multicentre Infants Cohort Study) Group: Ashford: Paul Cullinan, Jessica Harris, Warwick Atkinson, Pamela Mills, Susan Moffat, Carol White, Meinir Jones, Anthony Newman-Taylor; Barcelona: Goncal Figueras, Oriol Vall, Oscar Garcia, Cecilia Figueroa, Carme Puig, Eva Sanchez, Josep M Antó, Jordi Sunyer, Xavier Basagaña, Laura Muñoz-Ortiz; Menorca: Maties Torrent, M Victoria Estraña, Mireia García. The AMICS study is co-ordinated by Maria Barnes. Back


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