High resolution metagenomic characterization of complex infectomes in paediatric acute respiratory infection

Abstract

the diversity of pathogens associated with acute respiratory infection (ARi) makes diagnosis challenging. traditional pathogen screening tests have a limited detection range and provide little additional information. We used total RNA sequencing ("meta-transcriptomics") to reveal the full spectrum of microbes associated with paediatric ARI. Throat swabs were collected from 48 paediatric ARI patients and 7 healthy controls. Samples were subjected to meta-transcriptomics to determine the presence and abundance of viral, bacterial, and eukaryotic pathogens, and to reveal mixed infections, pathogen genotypes/subtypes, evolutionary origins, epidemiological history, and antimicrobial resistance. We identified 11 RNA viruses, 4 DNA viruses, 4 species of bacteria, and 1 fungus. While most are known to cause ARIs, others, such as echovirus 6, are rarely associated with respiratory disease. Co-infection of viruses and bacteria and of multiple viruses were commonplace (9/48), with one patient harboring 5 different pathogens, and genome sequence data revealed large intra-species diversity. Expressed resistance against eight classes of antibiotic was detected, with those for MLS, Bla, Tet, Phe at relatively high abundance. In summary, we used a simple total RNA sequencing approach to reveal the complex polymicrobial infectome in ARi. this provided comprehensive and clinically informative information relevant to understanding respiratory disease.Acute respiratory infections (ARI) are a leading cause of morbidity and mortality in newborns and young children, who experience an average of 3 to 6 ARIs annually 1-3 . Identifying the diversity of pathogens responsible for ARIs remains challenging because they involve a diverse set of viruses, bacteria, and fungal pathogens, with co-infection among them commonplace 4,5 . Traditional testing methods such as PCR, serological typing, bacterial culture and antibody detection, are regarded as the "gold standard" and widely used in ARI diagnosis 6,7 . However, despite an ongoing effort to include multiple pathogens in a single assay 8,9 , it remains difficult to simultaneously identify all potential ARI pathogens and capture new or uncommon respiratory pathogens 10 .Metagenomic next-generation sequencing (mNGS) is an unbiased way of discovering a broad range of infectious agents 11-13 , and has been recently introduced into clinical research to investigate the microbial cause of unusual disease cases 14 , perform broad-scale surveys for pathogens in undiagnosed diseases 15,16 , and understand the role of opportunistic infections 17,18 . For example, a study of severe pneumonia revealed that mNGS is both efficient and reliable 19,20 . Importantly, the utility of mNGS goes beyond pathogen identification. In particular, total RNA sequencing ("meta-transcriptomics") has successfully revealed the entire "infectome" (viruses, bacteria and eukaryotes) present within an organism and provided relevant data on genome sequence, gene expression, open Scientific RepoRtS | (2020) 10:3963 | https://doi.o...

Epidemic and pandemic viral infections: impact on tuberculosis and the lung

“…Clinical presentation of these infections in the elderly can be subtle, with atypical manifestation such as delirium, and may present with complications. SARS, and COVID-19 respiratory failure are welldocumented in the elderly [138,139] resistance mutations and clusters using phylogenetic approaches [155,156]. Specific antibody detection remains useful for seroprevalence studies in selected populations and in vaccine studies.…”

“…Using metagenomic next-generation sequencing, pathogens not included in the tests can be detected including both known and novel viruses. Genomic data provides information on virulence genes [ 154 ], resistance mutations and clusters using phylogenetic approaches [ 155 , 156 ]. Specific antibody detection remains useful for seroprevalence studies in selected populations and in vaccine studies.…”

Diagnosis and analysis of unexplained cases of childhood encephalitis in Australia using metagenomic next-generation sequencing

“…The concentration and quality of final extractions were examined using a NanoDrop spectrophotometer (ThermoFisher Scientific, USA). The Trio RNA-Seq kit (NuGEN Technologies, USA) was used for library preparation in all cases as it targets low concentration RNA samples (as low as 500 pg) [55]. Paired-end (150 bp) sequencing of these RNA library was performed using the Illumina NovaSeq platform.…”

“…Virus abundance were estimated as Reads Per Million (RPM) [55], calculated by using the relation "mapped reads / total reads * one million". For picobirnavirus, open reading frames were predicted using orffinder (https://www.ncbi.nlm.nih.gov/orffinder/) and gene annotated against the conserved domain database (CDD) [60].…”

High Heterogeneity of Echoviruses in Brazilian Children with Acute Gastroenteritis

“…However, detection and identification of echovirus genotypes in clinical samples is challenging due to its high genetic diversity [ 8 ]. Recently, next-generation sequencing (NGS) has been providing a new method for identifying echovirus [ 21 , 22 ].…”