Table 3.

Specimen types for pediatric airway microbiome studies: strengths and limitations

Airway compartment	Specimen type	How obtained	Strengths	Key limitations and bias	Typical use in pediatric NMD studies
Upper airway	Nasal swab	Swab of the anterior nares or nasopharynx	Easy, low-risk and repeatable. Samples the nasal reservoir and immigration source	Poor proxy for distal airways and captures upper-airway flora	Noninvasive source-community measure and longitudinal surveillance
	Nasal lavage	Instillation and recovery of saline in the nasal cavity	Noninvasive with higher biomass than a dry swab	Upper airway only, with dilution effects and no distal surrogate	Comparator and source-community sampling across ages
	Oropharyngeal or cough swab	Swab of the oropharynx or a cough swab	Easy and low-risk. May approximate a source community for microaspiration	Overestimates oral taxa and is a poor distal proxy	Comparator, not a sole surrogate for the lower airway
	Exhaled breath (condensate)	Collection of exhaled air or condensate with a cooled collector	Fully noninvasive, feasible even in young or ventilated children	Very low biomass, high contamination risk and limited standardization	Emerging noninvasive option, needs strict low-biomass controls
Lower airway	Bronchoalveolar lavage (BAL)	Bronchoscopy with instillation and aspiration of saline	Closest approximation of the distal community, pairs with cytology	Invasive, limited feasibility, dilution and contamination risk	Mechanistic studies during clinically indicated bronchoscopy
	Protected brush or protected BAL	Bronchoscopy with a protected catheter or brush	Improves anatomical specificity and reduces upper-airway carryover	Invasive, limited availability and very low biomass	Selected research protocols when bronchoscopy is indicated
	Tracheal aspirate or endotracheal aspirate	Suction through a tracheostomy or endotracheal tube	Scalable and directly relevant to ventilated patients	Device biofilm contribution and upper-airway carryover. Colonization versus infection is hard to adjudicate	Longitudinal surveillance in ventilated or tracheostomy-dependent children
	Sputum (expectorated or induced)	Expectoration or induction, for example with hypertonic saline	Less invasive and repeatable, may reflect lower-airway processes	Strict adherence to Standard Operating Procedures, for example from ECFS**, is needed to limit upper-airway contamination	Feasible in milder NMD or older children with quality controls
Device	Device biofilm (tracheostomy tube, suction catheter, humidifier)	Sampling of a removed device or circuit component	Interrogates biofilm reservoirs and device-associated resistance genes	May not reflect the mucosal community, with timing and handling confounds	Adjunct sampling to link device care with persistence and resistance

For infants (0 to 1 year) and toddlers (2 to 3 years), noninvasive upper-airway specimens and, where a tracheostomy is present, tracheal aspirates are most appropriate. Induced or expectorated sputum becomes feasible mainly in older children (from about 4 to 6 years and above) who can cooperate. Bronchoalveolar lavage is generally limited to clinically indicated bronchoscopy at any age. **ECFS Clinical Trials Network SOP, Sputum induction for expectorating children and adults, V2.0