Dustream collector

The sampling method for household dust collection was based on the protocol of Tsutsui et al. with slight modifications 16. Household dust was obtained from the living rooms and bedrooms where each patient spent the most time via a domestic vacuum cleaner with a 40‐µm pore‐size sampling filter (Dustream Collector, Indoor Biotechnologies, Inc., Charlottesville, VA).
Approximately 100 mg of dust from each sample was added to 4 ml of PBS with Tween 20 and was shaken for 2 h at room temperature. After centrifugation for 5 min at 500 × g, the supernatant solution was filtered through a 40 µm filter (Cell Strainer, BD Biosciences, San Jose, CA), then filtered through a 0.45 µm syringe filter (Stertile Acrodisc® Syringe Filters with Supor® Membrane, PALL Life Sciences, Ann Arbor, MI). The dust extract solutions were stored frozen at −20°C until analysis.
Monoclonal and polyclonal antibodies against pigeon dropping extract were generated according to the methods of previous studies 15, 16. A sandwich ELISA with monoclonal and polyclonal antibodies was used to measure the AAA as described previously 16. To increase the sensitivity of avian antigen detection, we used the ELAST amplification system (ELAST®, PerkinElmer Life Sciences, Inc., Waltham, MA) per the manufacturer's instructions. The AAA is expressed in micrograms of pigeon dropping extract per 1 gram of dust.

We will quantify indoor allergens and endotoxin by obtaining dust samples from the mother’s bedroom at two home visits, the first at 24–30 weeks of pregnancy and later at 3 months after delivery. The samples will be obtained with a domestic vacuum cleaner coupled to a dust collector provided with a filter (Dustream collector, Indoor biotechnologies). Immediately after being collected, the sample is stored with an ice-pack unit and later stored at − 20 °C until its use. The extraction of allergens and endotoxin from the dust samples will be done according to the previously published protocol [28 (link)].

A trained field technician vacuumed the sleeping surface and a two square yard area (1.68-m²) on the floor next to the bed for 4 minutes with a DUSTREAM Collector (Indoor Biotechnologies Inc.). A subset of 879 dust samples were sent for microbiome analysis, with sample selection described elsewhere.(18 , 19 , 25 (link)) DNA extraction followed standard protocols following manufactured kits and is described elsewhere.(18 , 19 ) Extracted DNA samples were sent to the University of California San Diego IGM Genomics Center for library preparation, multiplexing, and whole genome shotgun sequencing using standard techniques.(26 (link)) Details on the full library preparation, sequencing protocols, and quality control steps are described in Wang et. al.(19 ) After quality control, 781 samples remained with 6,528 taxa for downstream analysis. A taxonomy chart was created that assigned all taxa to a taxonomic classification across the seven phylogenetic levels - kingdom, phylum, class, order, family, genus, and species. We filtered out samples with a minimum library size of less than 1,003 base pairs.

Of the 3,301 ALHS participants, 2,871 received a home visit and had adequate levels of collected dust from the bedroom (Figure 1), as described in Carnes et al. (2017) (link). A trained field technician vacuumed two 1-yd² (0.84-m²) areas—one on participants’ sleeping surface and one on the floor next to the bed— for 2 min each with a DUSTREAM Collector (Indoor Biotechnologies Inc.). The samples were divided into aliquots of 50 mg and stored at −20°C until DNA processing.
During the home visit, information was obtained on environmental factors, including current (past 12 months) farming activities (living on a farm, working with crops, and working with animals), type of animals raised on the farm (beef or dairy cattle, swine, or poultry) and the presence of indoor pets (cats and dogs). Field technicians noted the presence of carpeting in the bedroom and ranked overall home cleanliness on a standardized five-point scale (Arbes et al., 2003 (link)). For our analysis, we created a binary variable comprising poor/lower (score of 1 or 2) or good/higher (score of 3–5) home condition. We categorized season of dust collection based on the date of the home visit: March 21–June 20 for spring, June 21–September 20 for summer, September 21–December 20 for fall, and December 21–March 20 for winter.

A sample of dust from the flooring of each lounge was also collected by vacuuming two A4 areas 30 min into the air sampling time using a DUSTREAM^® Collector containing nylon collection filters (pore size 40 µm) (Indoor Biotechnologies Ltd, Cardiff) [6 ].