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

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.