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Potential utility of targeted Nanopore sequencing for improving etiologic diagnosis of bacterial and fungal respiratory infection.

ABSTRACT: BACKGROUND:Diversified etiology of lower respiratory tract infection renders diagnosis challenging. The mainstay microbial culture is time-consuming and constrained by variable growth requirements. In this study, we explored the use of Nanopore sequencing as a supplementary tool to alleviate this diagnostic bottleneck. METHODS:We developed a targeted Nanopore method based on amplification of bacterial 16S rRNA gene and fungal internal transcribed spacer region. The performance was compared with routine infectious disease workups on 43 respiratory specimens. RESULTS:Nanopore successfully identified majority of microbes (47/54, 87.04%) and 7 possible pathogens not detected by routine workups, which were attributable to the content of microbiological investigations (n?=?5) and negative culture (n?=?2). The average sequencing time for first target reads was 7?min (1-43?min) plus 5?h of pre-sequencing preparation. CONCLUSIONS:The Nanopore method described here was rapid, economical and hypothesis-free, which might provide valuable hints to further microbiological follow-up for opportunistic pathogens missed or not detectable by conventional tests.

SUBMITTER: Chan WS

PROVIDER: S-EPMC7184685 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Potential utility of targeted Nanopore sequencing for improving etiologic diagnosis of bacterial and fungal respiratory infection.

Chan Wai Sing WS Au Chun Hang CH Leung Sau Man SM Ho Dona N DN Wong Elaine Yue Ling EYL To Man Yan MY Ng Man Kin MK Chan Tsun Leung TL Ma Edmond Shiu Kwan ESK Tang Bone Siu Fai BSF

Diagnostic pathology 20200427 1

<h4>Background</h4>Diversified etiology of lower respiratory tract infection renders diagnosis challenging. The mainstay microbial culture is time-consuming and constrained by variable growth requirements. In this study, we explored the use of Nanopore sequencing as a supplementary tool to alleviate this diagnostic bottleneck.<h4>Methods</h4>We developed a targeted Nanopore method based on amplification of bacterial 16S rRNA gene and fungal internal transcribed spacer region. The performance was ...[more]

PMID: 32340617

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Project description:BACKGROUND:High-throughput sequencing techniques are used to analyse the diversity of the respiratory microbiota in health and disease. Although extensive data are available regarding bacterial respiratory microbiota, its fungal component remains poorly studied. This is partly due to the technical issues associated with fungal metagenomics analyses. In this study, we compared two DNA extraction protocols and two fungal amplification targets for combined bacterial and fungal targeted amplicon sequencing analyses of the respiratory microbiota. METHODS:Six sputa, randomly selected from routine samples in Mondor Hospital (Creteil, France) and treated anonymously, were tested after bacterial and fungal routine culture. Two of which were spiked with Aspergillus Fumigati and Aspergillus Nigri (105 conidia/mL). After mechanical lysis, DNA was extracted using automated QIAsymphony® extraction (AQE) or manual PowerSoil® MoBio extraction (MPE). DNA yield and purity were compared. DNA extracted from spiked sputa was subjected to (i) real-time PCR for Aspergillus DNA detection and (ii) combined metagenomic analyses targeting barcoded primers for fungal ITS1 and ITS2, and bacterial V1-V2 and V3-V4 16S regions. Amplicon libraries were prepared using MiSeq Reagent V3 kit on Illumina platform. Data were analysed using PyroMIC© and SHAMAN software, and compared with culture results. RESULTS:AQE extraction provided a higher yield of DNA (AQE/MPE DNA ratio = 4.5 [1.3-11]) in a shorter time. The yield of Aspergillus DNA detected by qPCR was similar for spiked sputa regardless of extraction protocol. The extraction moderately impacted the diversity or relative abundances of bacterial communities using targeted amplicon sequencing (2/43 taxa impacted). For fungi, the relative abundances of 4/11 major taxa were impacted and AQE results were closer to culture results. The V1-V2 or V3-V4 and ITS1 or ITS2 targets assessed similarly the diversity of bacterial and fungal major taxa, but ITS2 and V3-V4 detected more minor taxa. CONCLUSION:Our results showed the importance of DNA extraction for combined bacterial and fungal targeted metagenomics of respiratory samples. The extraction protocol can affect DNA yield and the relative abundances of few bacterial but more fungal taxa. For fungal analysis, ITS2 allowed the detection of a greater number of minor taxa compared with ITS1.

Project description:Central nervous system (CNS) infections are a leading cause of death in patients. Nanopore-targeted sequencing (NTS) has begun to be used for pathogenic microbial detection. This study aims to evaluate the ability of NTS in the detection of pathogens in cerebrospinal fluid (CSF) through a prospective study. Fifty CSF specimens collected from 50 patients with suspected CNS infections went through three methods including NTS, metagenomic next-generation sequencing (mNGS), and microbial culture in parallel. When there was an inconsistency between NTS results and the results of the mNGS, the 16S rDNA gene was amplified followed by Sanger sequencing to further verify pathogens detected by NTS. Among 50 CSF specimens, 76% were NTS-positive, which is lower than mNGS (94.0%), yet higher than microbial culture (16.0%). The overall validation rate, diagnostic accordance rate (DAR), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of NTS were 86.7%, 50.0%, 71.0%, 15.8%, 57.9%, and 25.0%, respectively. In the CSF total nucleated cell (TNC) number ≤10 cells/µL, DAR, specificity, and PPV were 20%, 11.1%, and 11.1%, whereas in that with CSF TNC number >10 cells/µL, DAR, sensitivity, specificity, PPV, and NPV were 57.5%, 70.0%, 20.0%, 72.4%, and 18.2%, respectively. Although NTS has a higher microbial detection rate than microbial culture, it should combine CSF TNC result to evaluate the value of NTS for the diagnosis of CNS infections.ImportanceThis study aims to prospectively evaluate the ability of nanopore-targeted sequencing (NTS) in the detection of pathogens in cerebrospinal fluid (CSF). It was the first time combining mNGS and microbial culture to verify the NTS-positive results also using 16S rDNA amplification with Sanger sequencing. Although microbial culture was thought to be the gold standard for pathogens detection and diagnosis of infectious diseases, this study suggested that microbial culture of CSF is not the most appropriate way for diagnosing central nervous system (CNS) infection. NTS should be recommended to be used in CSF for diagnosing CNS infection. When evaluating the value of NTS for diagnosis of CNS infections, the results of CSF TNC should be combined, and NTS-positive result is observed to be more reliable in patients with CSF TNC level >10 cells/μL.

Project description:Etiologic diagnosis of bacterial pneumonia relies on identification of causative pathogens by cultures, which require extended incubation periods and have limited sensitivity. Next-generation sequencing of microbial DNA directly from patient samples may improve diagnostic accuracy for guiding antibiotic prescriptions. In this study, we hypothesized that enhanced pathogen detection using sequencing can improve upon culture-based diagnosis and that certain sequencing profiles correlate with host response. We prospectively collected endotracheal aspirates and plasma within 72 h of intubation from patients with acute respiratory failure. We performed 16S rRNA gene sequencing to determine pathogen abundance in lung samples and measured plasma biomarkers to assess host responses to detected pathogens. Among 56 patients, 12 patients (21%) had positive respiratory cultures. Sequencing revealed lung communities with low diversity (p < 0.02) dominated by taxa (>50% relative abundance) corresponding to clinically isolated pathogens (concordance p = 0.009). Importantly, sequencing detected dominant pathogens in 20% of the culture-negative patients exposed to broad-spectrum empiric antibiotics. Regardless of culture results, pathogen dominance correlated with increased plasma markers of host injury (receptor of advanced glycation end-products-RAGE) and inflammation (interleukin-6, tumor necrosis factor receptor 1-TNFR1) (p < 0.05), compared to subjects without dominant pathogens in their lung communities. Machine-learning algorithms identified pathogen abundance by sequencing as the most informative predictor of culture positivity. Thus, enhanced detection of pathogenic bacteria by sequencing improves etiologic diagnosis of pneumonia, correlates with host responses, and offers substantial opportunity for individualized therapeutic targeting and antimicrobial stewardship. Clinical translation will require validation with rapid whole meta-genome sequencing approaches to guide real-time antibiotic prescriptions.

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Potential utility of targeted Nanopore sequencing for improving etiologic diagnosis of bacterial and fungal respiratory infection.

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Potential utility of targeted Nanopore sequencing for improving etiologic diagnosis of bacterial and fungal respiratory infection.

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