Rapid Diagnosis of Lower Respiratory Infection using Nanopore-based Clinical Metagenomics

Abstract

Lower respiratory infections (LRIs) accounted for three million deaths worldwide in 2016, the leading infectious cause of mortality. The "gold standard" for investigation of bacterial LRIs is culture, which has poor sensitivity and is too slow to guide early antibiotic therapy.Metagenomic sequencing potentially could replace culture, providing rapid, sensitive and comprehensive results. We developed a metagenomics pipeline for the investigation of bacterial LRIs using saponin-based host DNA depletion combined with rapid nanopore sequencing. The first iteration of the pipeline was tested on respiratory samples from 40 patients. It was then refined to reduce turnaround and increase sensitivity, before testing a further 41 samples. The refined method was 96.6% concordant with culture for detection of pathogens and could accurately detect resistance genes with a turnaround time of six hours.This study demonstrates that nanopore metagenomics can rapidly and accurately characterise bacterial LRIs when combined with efficient human DNA depletion.

Clinical metagenomics

“…Differential lysis of human cells followed by degrada tion of background DNA with DNase I -thus retain ing and enriching for nucleic acid from organisms with cell walls, which include some bacteria and fungi -has been shown to provide substantial microbial enrichment of up to 1,000 times 94,99,100 . However, the performance of differential lysis methods can be limited by a number of factors.…”

“…In a published research study 126 , mNGS-based detection of Ebola and chikungunya virus infections on a nanopore sequencer was possible in <10 minutes of sequencing time and in <6 hours of sample-to-answer turnaround time overall. Research studies have also demonstrated the clinical potential of nanopore sequencing in targeted universal 16S ribosomal RNA (rRNA) bacterial detection 127 , microbiome analyses 128 , whole-genome sequencing of bacteria 129 and outbreak viruses 44,45,47 , RNA sequencing (RNA-seq) using standardized controls 130 and diagnosis of prosthetic joint 131 and lower respiratory infections 99 . Untargeted approaches such as mNGS or whole-transcriptome RNA-seq, however, may be limited by the lower throughput of nanopore sequencing relative to short-read sequencing such as with an Illumina instrument.…”

Applications of sequencing technology in clinical microbial infection

“…74 Moon et al 75 77 A team led by Justin O'Grady in the UK used Nanopore sequencing technology to rapidly identify bacterial genera and species and bacterial resistance genes in patients with lower respiratory tract infections. 78 Runtuwene et al 79 used Nanopore sequencing to genotype the malaria parasite-Plasmodium falciparum-and infer its drug resistance status. The clinical development and application of Nanopore sequencing technology have become a new milestone in precise pathogen detection.…”

Portable sequencer in the fight against infectious disease

“…For metagenomic sequencing, there are several methods can be employed to overcome the difficulties. Host DNA depletion with saponin [45], negative CpG selection [46], or physical disruption of bacterial wall [47,48] may be used to remove or reduce the background DNA and to increase sensitivity. Filtration, nuclease digestion, and random amplification allow reliable recovery of viral genomes [49].…”

“…Pathogens causing lower tract infections can be identified by sequencing bronchial lavage with or without host depletion [45,53]. This allowed the finding of bacteria composition and drug resistance phenotype in lower respiratory infection, which led to early antibacterial therapy within 6 h [45].…”