Metagenomics next-generation sequencing tests take the stage in the diagnosis of lower respiratory tract infections.

Metagenomic next-generation sequencing (mNGS) has changed the diagnosis landscape of lower respiratory tract infections (LRIs). With the development of newer sequencing assays, it is now possible to assess all microorganisms in a sample using a single mNGS analysis. The applications of mNGS for LRIs span a wide range of areas including LRI diagnosis, airway microbiome analyses, human host response analyses, and prediction of drug resistance. mNGS is currently in an exciting transitional period; however, before implementation in a clinical setting, there are several barriers to overcome, such as the depletion of human nucleic acid, discrimination between colonization and infection, high costs, and so on. Aim of Review: In this review, we summarize the potential applications and challenges of mNGS in the diagnosis of LRIs to promote the integration of mNGS into the management of patients with respiratory tract infections in a clinical setting. Key Scientific Concepts of Review: Once its analytical validation, clinical validation and clinical utility been demonstrated, mNGS will become an important tool in the field of infectious disease diagnosis.

Host DNA depletion efficiency of microbiome DNA enrichment methods in infected tissue samples.

Shotgun metagenomic sequencing or metagenomic whole genome sequencing is a genome-wide sequencing approach to explore bacterial communities directly from their habitat or sites of infection. However, host DNA contamination in metagenomic sequencing overwhelm low biomass of microbial signals and decrease sensitivity for microbial detection. In this study, we evaluated the host DNA depletion efficiency of four different microbiome DNA enrichment methods (NEBNext Microbiome DNA Enrichment kit, Molzym Ultra-Deep Microbiome Prep, QIAamp DNA Microbiome kit and Zymo HostZERO microbial DNA kit) in diabetic foot infection (DFI) tissue samples using quantitative real-time PCR and their effect on bacterial community composition by 16S ribosomal RNA amplicon sequencing. The host DNA depletion ratio (18S/16S rRNA), the percentage of bacterial DNA component and the microbial community profile of DFI were compared before (control) and after each microbiome DNA enrichment method. There was a significant difference in the 18S/16S rRNA ratio among different microbiome DNA enrichment methods (p <.001). QIAamp and HostZERO method reduced 18S/16S rRNA ratio by 32 and 57 fold than control method respectively. The percentage of bacterial DNA component increased more than ten-fold in QiaAmp (71.0 ± 2.7%) and HostZERO (79.9 ± 3.1%) method respectively than those in control method without host DNA depletion (6.7 ± 0.1%). It demonstrated the host DNA contamination was efficiently depleted and bacterial DNA was effectively enriched in HostZERO and QIAamp methods, attesting to the efficacy of these two methods in shotgun metagenomic sequencing studies. Overall, bacterial community composition of DFI samples was similar between control and microbiome enriched DNA samples.