Dataset Information

Performance of Nanopore and Illumina Metagenomic Sequencing for Pathogen Detection and Transcriptome Analysis in Infantile Central Nervous System Infections.

ABSTRACT:

Background

Infantile central nervous system infections (CNSIs) can be life-threatening and cause severe sequelae. However, the causative microorganism remains unknown in >40% of patients with aseptic infections. This study aimed to analyze the metagenome for detection of pathogens and the transcriptome for host immune responses during infection in a single cerebrospinal fluid (CSF) sample using 2 different next-generation sequencing (NGS) platforms, Nanopore and Illumina.

Methods

Twenty-eight CNSIs patients (<12 months) were enrolled, and 49 clinical samples (28 CSF and 21 blood) were collected. The DNA extracted from all 49 samples was sequenced using the Illumina sequencer for the detection of pathogens. Extracted RNA was obtained in sufficient quantities from 23 CSF samples and subjected to sequencing on both Nanopore and Illumina platforms. Human-derived reads subtracted during pathogen detection were used for host transcriptomic analysis from both Nanopore and Illumina sequencing.

Results

RNA metagenomic sequencing using both sequencing platforms revealed putative viral pathogens in 10 cases. DNA sequencing using the Illumina sequencer detected 2 pathogens. The results of Nanopore and Illumina RNA sequencing were consistent; however, the mapping coverage and depth to the detected pathogen genome of Nanopore RNA sequencing were greater than those of Illumina. Host transcriptomic analysis of Nanopore sequencing revealed highly expressed genes related to the antiviral roles of innate immunity from pathogen-identified cases.

Conclusions

The use of Nanopore RNA sequencing for metagenomic diagnostics of CSF samples should help to elucidate both pathogens and host immune responses of CNSI and could shed light on the pathogenesis of these infections.

SUBMITTER: Horiba K

PROVIDER: S-EPMC9587384 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Publications

Performance of Nanopore and Illumina Metagenomic Sequencing for Pathogen Detection and Transcriptome Analysis in Infantile Central Nervous System Infections.

Horiba Kazuhiro K Torii Yuka Y Aizawa Yuta Y Yamaguchi Makoto M Haruta Kazunori K Okumura Toshihiko T Suzuki Takako T Kawano Yoshihiko Y Kawada Jun-Ichi JI Hara Shinya S Saitoh Akihiko A Giske Christian G CG Ogi Tomoo T Ito Yoshinori Y

Open forum infectious diseases 20220930 10

<h4>Background</h4>Infantile central nervous system infections (CNSIs) can be life-threatening and cause severe sequelae. However, the causative microorganism remains unknown in >40% of patients with aseptic infections. This study aimed to analyze the metagenome for detection of pathogens and the transcriptome for host immune responses during infection in a single cerebrospinal fluid (CSF) sample using 2 different next-generation sequencing (NGS) platforms, Nanopore and Illumina.<h4>Methods</h4> ...[more]

PMID: 36299531

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Project description:BackgroundEarly and accurate etiological diagnosis is very important for improving the prognosis of central nervous system (CNS) infections in human immunodeficiency virus (HIV)-infected patients. The goal is not easily achieved by conventional microbiological tests. We developed a nanopore targeted sequencing (NTS) platform and evaluated the diagnostic performance for CNS infections in HIV-infected patients, with special focus on cryptococcal meningitis (CM). We compared the CM diagnostic performance of NTS with conventional methods and cryptococcal polymerase chain reaction (PCR).MethodsThis study included 57 hospitalized HIV-infected patients with suspected CNS infections from September 2018 to March 2022. The diagnosis established during hospitalization includes 27 cases of CM, 13 CNS tuberculosis, 5 toxoplasma encephalitis, 2 cytomegalovirus (CMV) encephalitis and 1 Varicella-zoster virus (VZV) encephalitis. The 2 cases of CMV encephalitis also have co-existing CM. Target-specific PCR amplification was used to enrich pathogen sequences before nanopore sequencing. NTS was performed on stored cerebrospinal fluid (CSF) samples and the results were compared with the diagnosis during hospitalization.Results53 (93.0%) of the patients were male. The median CD4 cell count was 25.0 (IQR: 14.0-63.0) cells/uL. The sensitivities of CSF culture, India ink staining, cryptococcal PCR and NTS for CM were 70.4% (95%CI: 51.5 - 84.1%), 76.0% (95%CI: 56.6 - 88.5%), 77.8% (59.2 - 89.4%) and 85.2% (95%CI: 67.5 - 94.1%), respectively. All those methods had 100% specificity for CM. Our NTS platform could identify Cryptococcus at species level. Moreover, NTS was also able to identify all the 5 cases of toxoplasma encephalitis, 2 cases of CMV encephalitis and 1 VZV encephalitis. However, only 1 of 13 CNS tuberculosis cases was diagnosed by NTS, and so did Xpert MTB/RIF assay.ConclusionsNTS has a good diagnostic performance for CM in HIV-infected patients and may have the ability of simultaneously detecting other pathogens, including mixed infections. With continuing improving of the NTS platform, it may be a promising alterative microbiological test for assisting with the diagnosis of CNS infections.

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.

Dataset Information

Performance of Nanopore and Illumina Metagenomic Sequencing for Pathogen Detection and Transcriptome Analysis in Infantile Central Nervous System Infections.

Background

Methods

Results

Conclusions

Publications

Performance of Nanopore and Illumina Metagenomic Sequencing for Pathogen Detection and Transcriptome Analysis in Infantile Central Nervous System Infections.

Similar Datasets

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