Comprehensive detection of pathogens in immunocompromised children with bloodstream infections by next-generation sequencing

Horiba, Kazuhiro; Kawada, Junji; Okuno, Yusuke; Tetsuka, Nobuyuki; Suzuki, Takehiko; Ando, Sugihiro; Kamiya, Yushi; Torii, Yuka; Yagi, Tetsuya; Takahashi, Yoshiyuki; Ito, Yoshinori

doi:10.1038/s41598-018-22133-y

Cited by 45 publications

(71 citation statements)

References 31 publications

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“…Recently, next‐generation sequencing (NGS) has enabled the comprehensive detection of pathogenic microorganisms, and this method has been applied in the field of infectious diseases . We previously analyzed sera of encephalitis, fulminant hepatitis, and bloodstream infection with unknown etiology and identified viral and bacterial genomes of presumptive pathogens of the diseases . In this study, we comprehensively analyzed microorganisms using NGS in sera of patients with myocarditis to identify the potential pathogenic viruses.…”

Section: Introductionmentioning

confidence: 99%

“…[14][15][16][17][18][19] We previously analyzed sera of encephalitis, fulminant hepatitis, and bloodstream infection with unknown etiology and identified viral and bacterial genomes of presumptive pathogens of the diseases. [20][21][22] In this study, we comprehensively analyzed microorganisms using NGS in sera of patients with myocarditis to identify the potential pathogenic viruses.…”

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confidence: 99%

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Identification of potential pathogenic viruses in patients with acute myocarditis using next‐generation sequencing

Takeuchi

Kawada

Horiba

et al. 2018

Journal of Medical Virology

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Myocarditis is an inflammatory disease of the myocardium and leads to cardiac dysfunction and heart failure. Although viral infections are considered to be the most common etiology of myocarditis, the identification of the causative virus is still challenging. Recently, next-generation sequencing (NGS) has been applied in the diagnosis of infectious diseases. The aim of the current study was to comprehensively analyze potential pathogenic microorganisms using NGS in the sera of patients with myocarditis. Twelve pediatric and five adult patients hospitalized for acute myocarditis were included. Serum samples in the acute phase were obtained and analyzed using NGS to detect pathogen-derived DNA and RNA. Viral sequence reads were detected in 7 (41%) of the 17 myocarditis patients by NGS. Among these patients, detection of Epstein-Barr virus, human parvovirus B19, torque teno virus, and respiratory syncytial virus reads by NGS was consistent with polymerase chain reaction or antigen test results in one patient each. A large number of human pegivirus reads were detected from one patient by RNA sequencing; however, its pathogenicity to human is unknown. Conversely, the number of detected virus-derived reads was small in most cases, and the pathophysiological role of these viruses remains to be clarified. No significant bacterial or fungal reads other than normal bacterial flora was detected. These data indicate that comprehensive detection of virus-derived DNA and RNA using NGS can be useful for the identification of potential pathogenic viruses in myocarditis.

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Section: Introductionmentioning

confidence: 99%

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confidence: 99%

Identification of potential pathogenic viruses in patients with acute myocarditis using next‐generation sequencing

Takeuchi

Kawada

Horiba

et al. 2018

Journal of Medical Virology

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“…PATHDET was used for taxonomic classification from sequences not mapped to the human reference genome following the removal of adapter sequences and low-quality sequences. Pathogen-derived sequences were reported based on previously established threshold criteria [8]. Results from PATHDET are shown according to taxonomic hierarchy; family, genus, and species.…”

Section: Resultsmentioning

confidence: 99%

Comprehensive pathogen detection in sera of Kawasaki disease patients by high-throughput sequencing: a retrospective exploratory study

et al. 2020

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Background Kawasaki disease (KD) is an idiopathic systemic vasculitis that predominantly damages coronary arteries in children. Various pathogens have been investigated as triggers for KD, but no definitive causative pathogen has been determined. As KD is diagnosed by symptoms, several days are needed for diagnosis. Therefore, at the time of diagnosis of KD, the pathogen of the trigger may already be diminished. The aim of this study was to explore comprehensive pathogens in the sera at the acute stage of KD using high-throughput sequencing (HTS). Methods Sera of 12 patients at an extremely early stage of KD and 12 controls were investigated. DNA and RNA sequences were read separately using HTS. Sequence data were imported into the home-brew meta-genomic analysis pipeline, PATHDET, to identify the pathogen sequences. Results No RNA virus reads were detected in any KD case except for that of equine infectious anemia, which is known as a contaminant of commercial reverse transcriptase. Concerning DNA viruses, human herpesvirus 6B (HHV-6B, two cases) and Anelloviridae (eight cases) were detected among KD cases as well as controls. Multiple bacterial reads were obtained from KD and controls. Bacteria of the genera Acinetobacter, Pseudomonas, Delfita, Roseomonas, and Rhodocyclaceae appeared to be more common in KD sera than in the controls. Conclusion No single pathogen was identified in serum samples of patients at the acute phase of KD. With multiple bacteria detected in the serum samples, it is difficult to exclude the possibility of contamination; however, it is possible that these bacteria might stimulate the immune system and induce KD.

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“…Direct sequencing from clinical samples could reduce time and improve diagnostic value and patient outcome. Challenges, however, include diagnostic sensitivity, optimized workflows and data analysis (Doughty et al, 2014;Hasman et al, 2014;Long et al, 2016;Horiba et al, 2018;Brown and Christiansen, 2019). The described workflow is currently offered as end-to-end isolate characterization service for research use only via our own NGS service laboratory (Vienna, Austria).…”

Section: Discussionmentioning

confidence: 99%

Analytical Performance Validation of Next-Generation Sequencing Based Clinical Microbiology Assays Using a K-mer Analysis Workflow

et al. 2020

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Next-generation sequencing (NGS) enables clinical microbiology assays such as molecular typing of bacterial isolates which is now routinely applied for infection control and epidemiology. Additionally, feasibility for NGS-based identification of antimicrobial resistance (AMR) markers as well as genetic prediction of antibiotic susceptibility testing results has been demonstrated. Various bioinformatics approaches enabling NGSbased clinical microbiology assays exist, but standardized, computationally efficient and scalable sample-to-results workflows including validated quality control parameters are still lacking. Bioinformatics analysis workflows based on k-mers have been shown to allow for fast and efficient analysis of large genomics data sets as obtained from microbial sequencing applications. We here demonstrate applicability of k-mer based clinical microbiology assays for whole-genome sequencing (WGS) including variant calling, taxonomic identification, bacterial typing as well as AMR marker detection. The wet-lab and dry-lab workflows were developed and validated in line with Clinical Laboratory Improvement Act (CLIA) guidelines for laboratory-developed tests (LDTs) on multi-drug resistant ESKAPE pathogens. The developed k-mer based workflow demonstrated ≥99.39% repeatability, ≥99.09% reproducibility and ≥99.76% accuracy for variant calling and applied assays as determined by intra-day and inter-day triplicate measurements. The limit of detection (LOD) across assays was found to be at 20× sequencing depth and 15× for AMR marker detection. Thorough benchmarking of the k-mer based workflow revealed analytical performance criteria are comparable to stateof-the-art alignment based workflows across clinical microbiology assays. Diagnostic sensitivity and specificity for multilocus sequence typing (MLST) and phylogenetic analysis were 100% for both approaches. For AMR marker detection, sensitivity and specificity were 95.29 and 99.78% for the k-mer based workflow as compared to 95.17 and 99.77% for the alignment-based approach. Summarizing, results illustrate that k-mer based analysis workflows enable a broad range of clinical microbiology assays, potentially not only for WGS-based typing and AMR gene detection but also genetic prediction of antibiotic susceptibility testing results.

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Comprehensive detection of pathogens in immunocompromised children with bloodstream infections by next-generation sequencing

Cited by 45 publications

References 31 publications

Identification of potential pathogenic viruses in patients with acute myocarditis using next‐generation sequencing

Identification of potential pathogenic viruses in patients with acute myocarditis using next‐generation sequencing

Comprehensive pathogen detection in sera of Kawasaki disease patients by high-throughput sequencing: a retrospective exploratory study

Analytical Performance Validation of Next-Generation Sequencing Based Clinical Microbiology Assays Using a K-mer Analysis Workflow

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