Molecular rapid diagnostic testing for bloodstream infections: Nanopore targeted sequencing with pathogen-specific primers

Dongsheng Han; Fei Yu; Dan Zhang; Juan Hu; Xuan Zhang; Dairong Xiang; Bin Lou; Yu Chen; Shufa Zheng

doi:10.1016/j.jinf.2024.106166

Molecular rapid diagnostic testing for bloodstream infections: Nanopore targeted sequencing with pathogen-specific primers

J Infect. 2024 Apr 24;88(6):106166. doi: 10.1016/j.jinf.2024.106166. Online ahead of print.

Authors

Dongsheng Han¹, Fei Yu¹, Dan Zhang¹, Juan Hu², Xuan Zhang³, Dairong Xiang³, Bin Lou¹, Yu Chen⁴, Shufa Zheng⁵

Affiliations

¹ Department of Laboratory Medicine, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, China; Zhejiang Key Laboratory of Clinical In Vitro Diagnostic Techniques, Hangzhou, Zhejiang 310003, China; Institute of Laboratory Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China.
² Department of Critical Care Units, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China.
³ State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, National Medical Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.
⁴ Department of Laboratory Medicine, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, China; Zhejiang Key Laboratory of Clinical In Vitro Diagnostic Techniques, Hangzhou, Zhejiang 310003, China; Institute of Laboratory Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China. Electronic address: chenyuzy@zju.edu.cn.
⁵ Department of Laboratory Medicine, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, China; Zhejiang Key Laboratory of Clinical In Vitro Diagnostic Techniques, Hangzhou, Zhejiang 310003, China; Institute of Laboratory Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China. Electronic address: zsfzheng@zju.edu.cn.

PMID: 38670268
DOI: 10.1016/j.jinf.2024.106166

Abstract

Background: Nanopore sequencing, known for real-time analysis, shows promise for rapid clinical infection diagnosis but lacks effective assays for bloodstream infections (BSIs).

Methods: We prospectively assessed the performance of a novel nanopore targeted sequencing (NTS) assay in identifying pathogens and predicting antibiotic resistance in BSIs, analyzing 387 blood samples from December 2021 to April 2023.

Results: The positivity rate for NTS (69.5 %, 269/387) nearly matches that of metagenomic next-generation sequencing (mNGS) (74.7 %, 289/387; p = 0.128) and surpasses the positivity rate of conventional blood culture (BC) (33.9 %, 131/387; p < 0.01). Frequent pathogens detected by NTS included Klebsiella pneumoniae (n = 54), Pseudomonas aeruginosa (n = 36), Escherichia coli (n = 36), Enterococcus faecium(n = 30), Acinetobacter baumannii(n = 26), Staphylococcus aureus(n = 23), and Human cytomegalovirus (n = 37). Against a composite BSI diagnostic standard, NTS demonstrated a sensitivity and specificity of 84.0 % (95 % CI 79.5 %-87.7 %) and 90.1 % (95 % CI 81.7 %-88.5 %), respectively. The concordance between NTS and mNGS results (the percentage of total cases where both either detected BSI-related pathogens or were both negative) was 90.2 % (359/387), whereas the consistency between NTS and BC was only 60.2 % (233/387). In 80.6 % (50/62) of the samples with identical pathogens identified by both NTS tests and BCs, the genotypic resistance identified by NTS correlated with culture-confirmed phenotypic resistance. Using NTS, 95 % of samples can be tested and analyzed in approximately 7 h, allowing for early patient diagnosis.

Conclusions: NTS is rapid, sensitive, and efficient for detecting BSIs and drug-resistant genes, making it a potential preferred diagnostic tool for early infection identification in critically ill patients.

Keywords: Antibiotic susceptibility testing; Blood culture; Bloodstream infections; Metagenomic next-generation sequencing; Targeted nanopore sequencing.