Indoor air pollution (IAP) or environmental tobacco smoke (ETS) exposure may influence nasopharyngeal carriage of bacterial species and development of lower respiratory tract infection (LRTI). The aim of this study was to longitudinally investigate the impact of antenatal or postnatal IAP/ETS exposure on nasopharyngeal bacteria in mothers and infants. A South African cohort study followed mother–infant pairs from birth through the first year. Nasopharyngeal swabs were taken at birth, 6 and 12 months for bacterial culture. Multivariable and multivariate Poisson regression investigated associations between nasopharyngeal bacterial species and IAP/ETS. IAP exposures (particulate matter, carbon monoxide, nitrogen dioxide, volatile organic compounds) were measured at home visits. ETS exposure was measured through maternal and infant urine cotinine. Infants received the 13-valent pneumococcal and Haemophilus influenzae B conjugate vaccines. There were 881 maternal and 2605 infant nasopharyngeal swabs. Antenatal ETS exposure was associated with Streptococcus pneumoniae carriage in mothers (adjusted risk ratio (aRR) 1.73 (95% CI 1.03–2.92)) while postnatal ETS exposure was associated with carriage in infants (aRR 1.14 (95% CI 1.00–1.30)) Postnatal particulate matter exposure was associated with the nasopharyngeal carriage of H. influenzae (aRR 1.68 (95% CI 1.10– 2.57)) or Moraxella catarrhalis (aRR 1.42 (95% CI 1.03–1.97)) in infants. Early-life environmental exposures are associated with an increased prevalence of specific nasopharyngeal bacteria during infancy, which may predispose to LRTI.
This study was nested within the Drakenstein Child Health Study (DCHS), a birth cohort in a peri-urban area of South Africa [12]. Consenting pregnant women were enrolled at 20–28 weeks’ gestation at two public primary health clinics: Mbekweni (serving a predominantly black African population) and Newman (serving a predominantly mixed ancestry population) from March 2012 to July 2015. All births occurred at a single, central hospital, Paarl hospital. Thereafter, mother–infant pairs were followed at 6, 10 and 14 weeks, 6, 9 and 12 months, for healthcare and immunisations including the 13-valent pneumococcal conjugate vaccine (PCV-13) given at 6, 14 weeks and 9 months and Haemophilus influenzae type b conjugate vaccine at 6, 10, 14 weeks according to the South African Expanded Programme on Immunisation schedule [13]. Study questionnaires and clinical data were collected at enrolment and follow-up visits. A validated socioeconomic score (SES) was used to categorise participants into quartiles as lowest, low-moderate, moderate-high or highest SES [5]. Clinical data collected at each follow-up visit included details on recent respiratory tract infections, including respiratory symptoms, otitis media, wheeze or LRTIs in the preceding month and any antibiotic use in the prior 6 months. The participant’s home environment was assessed and dwellings categorised based on having two or more defined household dimensions (type of home, building material, water supply, toilet facilities, kitchen type, ventilation in kitchen areas) [5]. IAP was measured at home visits conducted antenatally (within 4 weeks of enrolment) and postnatally (between 4–6 months of the infant’s life) [5]. Home visits were conducted over 3 years with sampling occurring throughout the year covering all seasons and weather conditions. PM10 and CO were measured by separate monitors (AirChek 52; SKC, Eighty-Four, PA, USA for PM10 and Altair; Troy, MI, USA for CO) left in homes over 24 h. NO2 and VOCs, benzene and toluene, were measured using diffusion tubes (Radiello absorbent filters in polyethylene diffusive body; Sigma-Aldrich, St Louis, MO, USA) and (Markes thermal desorption tubes; Llantrisant, UK) left in homes for 2 weeks [5]. These monitors were internally calibrated for temperature and humidity as per the manufacturer information, whereas diffusion tubes were corrected for humidity during laboratory analysis [5, 14]. The South African National Ambient Air Quality Standards were used to define expected exposure levels for each pollutant based on an averaging period of 1 year for each measure [15, 16]. Maternal, paternal and household tobacco smoking and exposure were assessed using questionnaires administered at enrolment, and antenatal and postnatal visits. These were validated using maternal and infant urine cotinine measures. Maternal cotinine was measured antenatally and at birth, and infant cotinine at birth and 6–10 week with the highest result used to assign smoking status and exposure. Cotinine levels were classified as <10 ng·mL−1 (nonsmoker), 10–499 ng·mL−1 (passive smoker/exposed), or ≥500 ng·mL−1 (active smoker) [7]. Nasopharyngeal swabs were obtained from mothers (at the time of delivery) and infants at birth, 6 and 12 months by trained study staff according to World Health Organization recommendations [17]. The swabs were immediately stored in 1 mL of skimmed milk, tryptone, glucose and glycerol transport medium (STGG), transported on ice to the laboratory and frozen at −80°C for later batch processing. After thawing at room temperature (22°C), samples were vortexed for 15 s before plating out a 10 µL aliquot onto four different solid media (National Health Laboratory Services, Green Point Media Laboratory Cape Town, South Africa). Standard laboratory protocols were used for the phenotypic and biochemical identification of common bacterial species that asymptomatically colonise the upper respiratory tract. For S. pneumoniae culture, Columbia blood agar base with 2% agar, 5% horse blood and 4 mg·mL−1 gentamicin (CAG) was incubated at 37°C in 5% CO2 overnight. Presumptive S. pneumoniae isolates were identified by colony morphology, α-haemolysis, optochin disk susceptibility (Oxoid, Basingstoke, UK) and confirmed using lytA PCR [18]. For H. influenzae, bacitracin heated blood agar plates were incubated at 37°C with 5% carbon dioxide (CO2). Suspected H. influenzae colonies were inoculated onto Columbia agar and identified using Factors X, V and XV discs and by observing growth in the haemolytic zone of Staphylococcus aureus on blood agar plates. S. aureus isolates were identified by culturing on mannitol salt agar, and DNase testing whereas Moraxella catarrhalis isolates were identified by culturing on 2% blood agar and incubated overnight at 37°C. Isolates were presumptively identified by push test and confirmed by copB PCR [19]. Gram-negative bacteria were subcultured onto MacConkey agar and identified on Vitek 2® (bioMérieux, Marcy I'Etoile, France). The study was approved by the Human Research Ethics Committees of the Faculties of Health Sciences, University of Cape Town and Stellenbosch University, and by the Western Cape Provincial Health Research committee (HREC 149/2013). Mothers provided written informed consent at enrolment. Study data were captured in a relational Microsoft Access® database or collected and managed using REDCap electronic data capture tools hosted at the University of Cape Town [20]. Statistical analyses were conducted in Stata version 14.2 for Windows (Stata Corp, College Station, TX, USA). Statistical tests were considered significant if the p-value was <0.05 or if p-value cut-offs were derived using the Benjamini–Hochberg procedure for assessing the association between IAP and the pathogens [21]. Categorical variables were summarised using frequency counts and percentages, n (%). Normally and non-normally distributed continuous variables were described using mean (sd) and median (interquartile range (IQR)) values, respectively. Mann–Whitney or Wilcoxon signed-rank tests, as appropriate, were used to compare medians as well as their spread. Cross tabulations with Fishers’ exact or Chi-squared tests were used to describe and compare the prevalence of pathogen carriage between the infants (at all time points) and their mothers or between different time points for infants. Multivariable modified Poisson regression analyses with robust error variance [22] were performed to estimate adjusted risk ratios (aRRs) between each bacterial pathogen and IAP measures (individually (adjusted) or together (adjusted 2)). The association between antenatal exposures or maternal cotinine and maternal carriage was explored as was the association between postnatal exposure or infant cotinine and infant carriage. The multivariable Poisson regressions adjusted for demographic and clinical factors (weight-for-age z-score at birth, preterm, ethnicity, sex, HIV exposure, time on exclusive breastfeeding, average number of people per sleeping room, dwelling category, recent respiratory infection, day care attendance, vaccination, number of other children under 5 years in the household, antibiotic use) that have been associated with pathogen acquisition. Multivariable regressions were then further performed for each site [23, 24]. Further, we explored the possible confounding effects of bacterial co-carriage by including indicator variables for each pathogen.
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