High-throughput Pathogen Sequencing Technology

The development and clinical application of nucleic acid detection technology has significantly improved the diagnostic capability for respiratory pathogens. How to appropriately select nucleic acid testing technology based on patients' underlying conditions, types of respiratory infections, and pathogen spectrum, and correctly understand its clinical application value has become an important clinical issue.

To address this, the Chinese Laboratory Medicine Training Project Expert Committee organized multidisciplinary experts to jointly write the "Expert Consensus on Clinical Application of Nucleic Acid Detection Technology for Adult Respiratory Infection Pathogen Diagnosis (2023)." Professor Xu Yingchun from the Laboratory Department of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, and Professor Qu Jieming from the Department of Respiratory and Critical Care Medicine, Ruijin Hospital Affiliated to Shanghai Jiao Tong University served as corresponding authors. This article introduces high-throughout pathogen sequencing technology based on the expert consensus.

✧ Recommendation: High-throughput pathogen sequencing should be selectively used when traditional microbiological testing fails to identify pathogens or when rapid identification of infection and its causative agents is urgently needed. It is primarily indicated for severe respiratory infections, suspected special pathogen infections, and cluster-transmitted respiratory infections.

✧ Expert consensus: 98.8%

High-throughput pathogen sequencing technology utilizes high-throughput sequencing to sequence nucleic acids in clinical specimens, identifying microbial species composition and abundance through comparison with microbial reference genomes. Currently, this technology comprises three categories: unbiased metagenomic next-generation sequencing (mNGS), targeted next-generation sequencing (tNGS) for detecting multiple pathogens and their resistance/virulence genes, and whole-genome sequencing (WGS) for obtaining complete genomic data of one or multiple pathogens.

Applications

Given the complexity, interpretative challenges, and high cost of high-throughput pathogen sequencing, it is recommended to use selectively in addition to traditional microbiological testing based on clinical needs, such as when rapid pathogenic diagnosis is required, when available microbiological testing methods cannot provide definitive diagnosis, or when source tracing of pathogen is needed for clusters of respiratory infectious cases.

Technical Characteristics

High-throughput pathogen sequencing can identify species from nucleic acid fragments in specimens. However, the accuracy of species, resistance gene, and virulence gene identification is influenced by multiple factors including specimen sampling, experimental procedures, pathogen reference genome databases, and bioinformatic analysis. When interpreting clinical reports, besides in-depth analysis of sequencing data, it is crucial to comprehensively evaluate and differentiate between colonizing organisms and responsible pathogens by incorporating patient clinical information and other laboratory data.

Performance Validation Requirements

Before implementing clinical testing using high-throughput pathogen sequencing, laboratories need to complete method establishment and performance verification according to laboratory-developed test requirements, establishing expected performance indicators based on intended clinical use, including:
☑️ Bioinformatics pipeline evaluation: minimum sequencing data volume, accuracy, recall, precision, F1-Score, etc.;
☑️ Complete process performance verification: background nucleic acid data model, positive threshold, repeatability, limit of detection, interference resistance, stability, etc.;
☑️ Clinical evaluation: testing of a sufficient number of real clinical specimens to determine negative and positive predictive values.

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