Report on the Research Paper: Comprehensive Pathogen Identification and Antimicrobial Resistance Prediction from Positive Blood Cultures Using Nanopore Sequencing Technology

Academic Context

Bloodstream infections (BSIs) are severe clinical conditions typically diagnosed through positive blood culture results and systemic signs of infection. BSIs can be caused by various pathogens including bacteria, fungi, and viruses, and their incidence is rising globally. With the widespread use of antibiotics, the emergence of multidrug-resistant (MDR) microorganisms has significantly complicated treatment strategies for blood infections. Traditional methods for pathogen identification and antimicrobial susceptibility testing (AST) generally take 2-5 days, which can delay timely treatment for patients.

Recently, rapid AST devices such as the Accelerate PhenoTest BC system and the Q-linea ASTar system have reduced testing times to within 6-7 hours. However, these devices are still limited in their ability to provide detailed epidemiological information. Genomic sequencing technology, especially nanopore sequencing, has shown increasing potential for real-time analysis of long DNA or RNA fragments, making it a powerful tool for pathogen identification and antimicrobial resistance (AMR) genotyping. However, due to the high abundance of human DNA in blood culture samples, the low coverage of microbial pathogens has limited its applicability. To address this, Oxford Nanopore Technologies (ONT) introduced adaptive sampling technology, which enables real-time depletion of human DNA during sequencing, enhancing microbial detection efficiency.

This study aimed to evaluate the feasibility and performance of nanopore sequencing combined with adaptive sampling for pathogen identification and AMR prediction in bloodstream infections, exploring its potential for clinical applications.

Paper Overview

The research was conducted by Po-Yu Liu, Han-Chieh Wu, Ying-Lan Li, Hung-Wei Cheng, Ci-Hong Liou, Feng-Jui Chen, and Yu-Chieh Liao, from institutions including Taichung Veterans General Hospital, National Health Research Institutes, and National Yang Ming Chiao Tung University in Taiwan. The paper was published in Genome Medicine in 2024, titled Comprehensive pathogen identification and antimicrobial resistance prediction from positive blood cultures using nanopore sequencing technology.

Research Workflow and Methods

Sample Collection and Conventional Testing

The study analyzed 458 positive blood culture samples obtained from bloodstream infection patients in central Taiwan. Samples were cultivated using the BD BACTEC™ FX system and identified with MALDI-TOF VITEK MS (BioMérieux). AST was conducted with the VITEK 2 system. Six negative control samples were included to assess the background levels of DNA contamination.

DNA Extraction and Nanopore Sequencing Library Preparation

DNA was extracted from each positive blood culture sample using the QIAamp Biostic Bacteremia DNA Kit (Qiagen) on the QIAcube Connect automated platform. Extracted DNA was prepared for sequencing using the ONT Rapid Barcoding Kit 96 and sequenced on a GridION SpotON flow cell (R9.4.1). During sequencing, adaptive sampling was activated to deplete human DNA.

Bioinformatics Analysis

A custom bioinformatics pipeline was developed. Centrifuge 1.0.4 was used for species-level classification of sequencing reads, and Flye 2.9.2 was used for genome assembly. AMR prediction was performed with ResFinder 4.3.2 and PointFinder. Full-length 16S rRNA sequencing using the PacBio Sequel IIe system further validated polymicrobial infections.

Key Findings

Pathogen Identification

A total of 76 species were identified, including: - 88 Escherichia coli samples, - 74 Klebsiella pneumoniae samples, - 43 Staphylococcus aureus samples, and - 9 Candida samples.

Nanopore sequencing enabled accurate identification of both monomicrobial infections and polymicrobial infections. Notably, 23 polymicrobial infections were detected, validated further by full-length 16S rRNA sequencing.

Antimicrobial Resistance Prediction

With a modified ResFinder database, the study achieved over 90% categorical agreement for AMR predictions among monomicrobial infection samples (³⁷⁹⁹⁄₄₁₉₅), with minimal very major errors observed for key pathogens such as: - K. pneumoniae – 1.1% - S. aureus – 1.1%

Conclusions

The study demonstrated that nanopore sequencing with adaptive sampling can directly identify pathogens and predict AMR from positive blood culture samples. This technology offers rapid (1-hour species identification, 15-hour complete AMR profile) and accurate diagnostic results. Its integration into clinical workflows represents a groundbreaking approach to managing bloodstream infections by enabling earlier and targeted antimicrobial therapies.

Highlights of the Study

1. Rapid Pathogen Identification: Nanopore sequencing could achieve species-level identification within 1 hour and comprehensive AMR profiling within 15 hours.
2. Detection of Polymicrobial Infections: The technique successfully identified polymicrobial infections, something challenging for conventional methods.
3. High-Accuracy AMR Predictions: The modified ResFinder database provided highly accurate resistance predictions for key pathogens, with very low major error rates.
4. Clinical Applicability: The technology’s speed and accuracy underscore its potential to transform diagnostic practices in bloodstream infections, especially in addressing MDR pathogens.

Significance and Impact

This groundbreaking research illustrates the capability of nanopore sequencing to enhance the detection efficiency of bloodstream infection pathogens by removing human DNA in real time. The results provide faster and more precise diagnostic information to clinicians, allowing optimized antibiotic use, reduced resistance development, and improved patient outcomes. With decreasing costs and increasing implementation of user-friendly workflows, nanopore sequencing could become a mainstay in clinical diagnostics.

Additional Considerations

The study compared nanopore sequencing with rapid AST devices like the Accelerate PhenoTest BC and Q-linea ASTar systems. While these devices offer quicker results in some cases, they are generally limited in species and antibiotic panels. Nanopore sequencing surpasses these limitations by enabling the identification of a wider range of pathogens, including novel species, and by delivering valuable epidemiological and genomic insights.

Practical considerations for clinical implementation include: - Cost: Flow cells ($700 each), library preparation kits ($100 per run), DNA extraction ($5 per sample). - Training: Laboratory staff must be trained in DNA extraction, library preparation, sequencing operations, and data analysis. - Regulatory Approval: Rigorous validation studies will be needed to secure regulatory clearances (e.g., FDA).

The study raises the potential for reduced hospital stays, risk mitigation, and overall healthcare cost savings by providing timely antimicrobial guidance. Further research is needed to quantify these benefits.

Limitations

1. Single-hospital study: Results were limited to one facility and relied solely on the VITEK 2 system for AST.
2. Restricted analyses: Examinations were limited to species identification and resistance predictions, with no subtyping or virulence determinant analyses included.
3. Database limitations: While the ResFinder database was updated, continual inclusion of new resistance genes is necessary.

Summary

Nanopore sequencing, combined with adaptive sampling, provides a rapid, accurate, and comprehensive solution for pathogen identification and AMR prediction from positive blood cultures. This approach enhances patient care by delivering actionable results within 24 hours, revolutionizing the management of bloodstream infections and supporting optimized antimicrobial stewardship strategies.