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. 2023 Feb 15:16:923-936.
doi: 10.2147/IDR.S397755. eCollection 2023.

Detection of Pathogens and Antimicrobial Resistance Genes in Ventilator-Associated Pneumonia by Metagenomic Next-Generation Sequencing Approach

Ting Chen #  1   2 , Lei Zhang #  3 , Wenhua Huang #  3 , Huijun Zong  1   2 , Qian Li  3 , Yuling Zheng  3 , Qingyu Lv  3 , Decong Kong  3 , Yuhao Ren  3 , Yongqiang Jiang  3 , Yan Li  1   2 , Peng Liu  3
Affiliations

Detection of Pathogens and Antimicrobial Resistance Genes in Ventilator-Associated Pneumonia by Metagenomic Next-Generation Sequencing Approach

Ting Chen et al. Infect Drug Resist. .

Abstract

Background: The early identification of pathogens and their antibiotic resistance are essential for the management and treatment of patients affected by ventilator-associated pneumonia (VAP). However, microbiological culture may be time-consuming and has a limited culturability of many potential pathogens. In this study, we developed a rapid nanopore-based metagenomic next-generation sequencing (mNGS) diagnostic assay for detection of VAP pathogens and antimicrobial resistance genes (ARGs).

Patients and methods: Endotracheal aspirate (ETA) samples from 63 patients with suspected VAP were collected between November 2021 and July 2022. Receiver operating characteristic (ROC) curves were established to compare the pathogen identification performance of the target pathogen reads, reads percent of microbes (RPM) and relative abundance (RA). The evaluation of the accuracy of mNGS was performed comparing with the gold standard and the composite standard, respectively. Then, the ARGs were analyzed by mNGS.

Results: ROC curves showed that RA has the highest diagnostic value and the corresponding threshold was 9.93%. The sensitivity and specificity of mNGS test were 91.3% and 78.3%, respectively, based on the gold standard, while the sensitivity and specificity of mNGS test were 97.4% and 100%, respectively, based on the composite standard. A total of 13 patients were virus-positive based on mNGS results, while the coinfection rate increased from 27% to 46% compared to the rate obtained based on clinical findings. The mNGS test also performed well at predicting antimicrobial resistance phenotypes. Patients with a late-onset VAP had a significantly greater proportion of ARGs in their respiratory microbiome compared to those with early-onset VAP (P = 0.041). Moreover, the median turnaround time of mNGS was 4.43 h, while routine culture was 72.00 h.

Conclusion: In this study, we developed a workflow that can accurately detect VAP pathogens and enable prediction of antimicrobial resistance phenotypes within 5 h of sample receipt by mNGS.

Keywords: NGS; antibiotic resistance; endotracheal aspirate; mechanical ventilation; pathogen diagnosis.

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Conflict of interest statement

Ting Chen, Wenhua Huang, Huijun Zong, Qingyu Lv, Yongqiang Jiang, Yan Li, and Peng Liu report grants from National Natural Science Foundation of China, during the conduct of the study. The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Schematic of samples inclusion and exclusion.
Figure 2
Figure 2
Schematic representation of the MinION-based mNGS workflow.
Figure 3
Figure 3
(A) Evaluation of the performance of three indicators (reads, RPM and RA) to detect VAP pathogens using ROC curves. (B and C) Accuracy evaluation by 2×2 contingency tables based on the gold standard culture test and composite standard, respectively.
Figure 4
Figure 4
Comparison of the pathogen detection rates between clinical laboratory findings and mNGS results.
Figure 5
Figure 5
ARGs profile in the mNGS-positive samples. The heatmap strip at the right with different colors corresponds to different antimicrobial class. The heatmap strip at the bottom represents different pathogen species. The bar chart indicates the number of ARGs per sample.
Figure 6
Figure 6
Boxplots demonstrating the statistical analyses for ARGs detected from different types of VAP clinical cases. Median values are indicated by the line within the boxplot. The box extends from the 25th to 75th percentile, and whiskers indicate the minimum and maximum values. *P < 0.05; ns, nonsignificant. (A) Lg (total reads). (B) Lg (ARGs proportion).
Figure 7
Figure 7
Turnaround time: mNGS vs routine microbial culture.
See this image and copyright information in PMC

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