Integrated multiplex PCR and metatranscriptomics reveal upper-lower airway microbial landscapes in pediatric...

Area of Science:

• Microbiology
• Pediatric Infectious Diseases
• Genomics

Background:

• A significant number of pediatric acute respiratory tract infections (ARTIs) are classified as unknown etiology despite multiplex PCR use.
• The microbial characteristics and clinical relevance of these unknown etiology ARTIs are not well understood.

Purpose of the Study:

• To compare airway microbiomes between PCR-positive and PCR-negative pediatric ARTIs.
• To investigate the relationship between microbial profiles, sampling site, and disease severity in ARTIs.
• To characterize the active microbial communities and antibiotic resistance genes in pediatric ARTIs.

Main Methods:

• Collected nasopharyngeal swabs (NS) and bronchoalveolar lavage fluid (BALF) from 514 hospitalized children with ARTIs.
• Utilized a 22-target multiplex PCR panel for initial pathogen identification.
• Performed pooled metatranscriptomic sequencing on samples stratified by pathogen status to profile microbial activity and antibiotic resistance genes.

Main Results:

• Multiplex PCR identified pathogens in 77.0% of NS and 54.1% of BALF samples.
• PCR-negative samples showed lower viral activity and higher bacterial transcript abundance, with enriched Pseudomonas.
• Microbial signatures varied by airway site and disease severity; severe cases had higher bacterial burden and Pseudomonas, while mild cases showed stronger viral signals.
• Antibiotic resistance genes were detected in patient samples but not in healthy controls.

Conclusions:

• PCR-negative pediatric ARTIs exhibit unique, bacteria-enriched microbial profiles.
• Integrating metatranscriptomics with PCR improves pathogen characterization in ARTIs.
• Identified microbial patterns related to sampling site and disease severity can aid in diagnostic evaluation and clinical management.