Identification of pathogen(s) in infectious diseases using shotgun metagenomic sequencing and conventional culture: a comparative study

Abstract

Background: Early and accurate diagnosis of microorganism(s) is important to optimize antimicrobial therapy. Shotgun metagenomic sequencing technology, an unbiased and comprehensive method for pathogen identification, seems to potentially assist or even replace conventional microbiological methodology in the diagnosis of infectious diseases. However, evidence in clinical application of this platform is relatively limited.

Methods: To evaluate the capability of shotgun metagenomic sequencing technology in clinical practice, both shotgun metagenomic sequencing and conventional culture were performed in the PCR-positive body fluid specimens of 20 patients with suspected infection. The sequenced data were then analyzed for taxonomic identification of microbes and antibiotic resistance gene prediction using bioinformatics pipeline.

Results: Shotgun metagenomic sequencing results showed a concordance of 17/20 compared with culture results in bacterial detection, and a concordance of 20/20 compared with culture results in fungal detection. Besides, drug-resistant types annotated from antibiotic resistance genes showed much similarity with antibiotic classes identified by susceptibility tests, and more than half of the specimens had consistent drug types between shotgun metagenomic sequencing and culture results.

Conclusions: Pathogen identification and antibiotic resistance gene prediction by shotgun metagenomic sequencing identification had the potential to diagnose microorganisms in infectious diseases, and it was especially helpful for multiple microbial co-infections and for the cases where standard culture approached failed to identify microorganisms.

Keywords: Antibiotic resistance; Culture; Infectious disease; Pathogen identification; Shotgun metagenomic sequencing.

Figure 1. Study workflow.

(A) Schematic of comparative study workflow. Patients’ samples of body fluids were collected for conventional culture and 16S/ITS region PCR test. PCR-positive specimens which passed the quality control of sequencing were selected to perform shotgun metagenomic sequencing and bioinformatics analyses, and further comparative study between culture results and sequencing results. (B) Schematic of paired-end library construction in accordance with Illumina’s instruction. (C) Bioinformatics pipeline for shotgun metagenomic sequencing.

Figure 2. Flowchart for enrollment.

Figure 3. Comparison and concordance analysis between shotgun metagenomic sequencing and culture in pathogen detection and drug resistance information.

Comparison and concordance analysis between shotgun metagenomic sequencing and culture in pathogen detection and drug resistance information. (A) The number of positive specimens (y-axis) for pairwise shotgun metagenomic sequencing and culture is plotted against bacterial detection and fungal detection (x-axis) (n = 20). (B, C and D) Pie chart demonstrating the positivity distribution of shotgun metagenomic sequencing and culture for all specimens from bacterial detection (B), fungal detection (C), and antibiotic resistance information (D).