Rapid Detection of Bacterial Pathogens and Antimicrobial Resistance Genes in Clinical Urine Samples With Urinary Tract Infection by Metagenomic Nanopore Sequencing

Abstract

Urinary tract infections (UTIs) are among the most common acquired bacterial infections in humans. The current gold standard method for identification of uropathogens in clinical laboratories is cultivation. However, culture-based assays have substantial drawbacks, including long turnaround time and limited culturability of many potential pathogens. Nanopore sequencing technology can overcome these limitations and detect pathogens while also providing reliable predictions of drug susceptibility in clinical samples. Here, we optimized a metagenomic nanopore sequencing (mNPS) test for pathogen detection and identification in urine samples of 76 patients with acute uncomplicated UTIs. We first used twenty of these samples to show that library preparation by the PCR Barcoding Kit (PBK) led to the highest agreement of positive results with gold standard clinical culture tests, and enabled antibiotic resistance detection in downstream analyses. We then compared the detection results of mNPS with those of culture-based diagnostics and found that mNPS sensitivity and specificity of detection were 86.7% [95% confidence interval (CI), 73.5–94.1%] and 96.8% (95% CI, 82.4–99.9%), respectively, indicating that the mNPS method is a valid approach for rapid and specific detection of UTI pathogens. The mNPS results also performed well at predicting antibiotic susceptibility phenotypes. These results demonstrate that our workflow can accurately diagnose UTI-causative pathogens and enable successful prediction of drug-resistant phenotypes within 6 h of sample receipt. Rapid mNPS testing is thus a promising clinical diagnostic tool for infectious diseases, based on clinical urine samples from UTI patients, and shows considerable potential for application in other clinical infections.

A culture-, amplification-independent, and rapid method for identification of pathogens and antibiotic resistance profile in bovine mastitis milk

“…Furthermore, our data showed how MinION sequencing could provide extra diagnostic confidence when there is a disagreement between colony morphology and PCR results. Although similar promising results were previously reported for blood (Taxt et al, 2020) and urine (Zhang et al, 2022) specimens, this is the first study that reported how to handle mastitis milk samples for effective and rapid pathogen and AMR detection.…”

Enhancing urinary tract infection diagnosis for negative culture patients with metagenomic next-generation sequencing (mNGS)

“…The applications of mNGS on non-sterile materials (e.g., urine) remains are still scarce and controversial. In one of the few recent studies on urine (Zhang et al, 2021;Zhang et al, 2022), a prospective proof-ofconcept study by Janes, V. A and colleagues confirmed the feasibility of metagenomic sequencing in detecting of urinary pathogens and drug resistance (Janes et al, 2022). We conducted a mNGS study in patients with suspected UTI and compared it with conventional test in terms of diagnostic performance, pathogenic findings to demonstrate its significance.…”

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

“…In addition, the respective libraries were pooled and multiplex sequencing was performed to improve cost-effectiveness, making the sequencing cost drop to $92. 17 Although the cost for a microbial culture is $60, more than one test and/or combinations with other ones (such as staining, immunology-related tests and/or PCR test) are often needed, the sequencing-based method developed here is more feasible and cost-effective.…”

“…13 In contrast, the real-time analysis and ultra-long reads (>100kb)-based approaches, such as the nanopore sequencing is more suitable for clinical laboratories. 14,15 In this regard, the feasibility of the nanopore sequencing application for clinical diagnosis has been demonstrated in clinical cases of meningitis, 16 peritonitis, 12 urinary tract infection, 17 lower respiratory infection, 18 and sepsis. 19 However, due to several combinations of the pathogens isolated in different clinical cases, suitable mNGS approaches for comprehensive pathogens detection and drug resistance prediction of VAP in ICU are still limited.…”