Metagenomic sequencing and evaluation of the host response in the pediatric aerodigestive population

Abstract

Objectives: To assess the diagnostic utility of metagenomic sequencing in pediatric aerodigestive clinic patients being evaluated for chronic aspiration. We hypothesize that using a metagenomics platform will aid in the identification of microbes not found on standard culture. Study Design and Methods: Twenty-four children referred to an aerodigestive clinic were enrolled in a prospective, single-site, cross-sectional cohort study. At the time of clinical evaluation under anesthesia, two samples were obtained: an upper airway sample and a sample from bronchoalveolar lavage (BAL). Samples were sent for routine culture and analyzed using Explify® Respiratory, a CLIA Laboratory Developed Test which identifies respiratory commensals and pathogens through RNA and DNA sequencing. Since RNA was sequenced in the course of the metagenomic analysis to identify organisms (RNA viruses and bacteria), the sequencing approach also captured host derived messenger RNA during sample analysis. This incidentally obtained host transcriptomic data were analyzed to evaluate the host immune response. The results of these studies were correlated with the clinical presentation of the research subjects. Results: In 10 patients, organisms primarily associated with oral flora were identified in the BAL. Standard culture was negative in three patients where clinical metagenomics led to a result with potential clinical significance. Transcriptomic data correlated with the presence or absence of dysphagia as identified on prior videofluoroscopic evaluation of swallowing. Conclusions: Clinical metagenomics allows for simultaneous analysis of the microbiota and the host immune response from BAL samples. As the technologies in this field continue to advance, such testing may improve the diagnostic evaluation of patients with suspected chronic aspiration.

Enhancing Clinical Utility: Utilization of International Standards and Guidelines for Metagenomic Sequencing in Infectious Disease Diagnosis

“…However, RNA sequencing in metagenomics poses challenges, such as the inherent instability of RNA molecules, which results in transcript degradation and biases in transcript representation [ 53 ]. This instability can lead to inaccurate gene expression quantifications and data analysis artifacts [ 53 , 54 ]. Moreover, microbial RNA samples containing host-derived RNA can complicate the analysis and interpretation of metagenomic data; therefore, robust bioinformatics pipelines are required for precise taxonomic and functional analysis [ 54 ].…”

“…This instability can lead to inaccurate gene expression quantifications and data analysis artifacts [ 53 , 54 ]. Moreover, microbial RNA samples containing host-derived RNA can complicate the analysis and interpretation of metagenomic data; therefore, robust bioinformatics pipelines are required for precise taxonomic and functional analysis [ 54 ].…”