Real-time whole genome sequencing direct diagnosis of Streptococcus pneumoniae meningitis: A case report

Morsli, Madjid; Kerharo, Quentin; Amrane, Sophie; Parola, Philippe; Fournier, Pierre‐Edouard; Drancourt, Michel

doi:10.1016/j.jinf.2021.10.002

Cited by 3 publications

(21 citation statements)

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“…DNA and RNA were manually extracted in 44% of studies, automatic extraction was performed in 20%, and 36% of studies did not report on the nucleic acid extraction method. The sample pre-treatment reported in 18/51 (35%) studies included centrifugation (∼15,000 g ), filtration and vortexing in 12 studies, DNase treatment in four studies ( Piantadosi et al, 2018 ; Edridge et al, 2019 ; Manso et al, 2020 ; Morsli et al, 2021d ) and proteinase K treatment in five studies ( Table 1 ; Guan et al, 2015 ; Miller et al, 2019 ; Carbo et al, 2020 ; Manso et al, 2020 ; Morsli et al, 2021a , c ; Piantadosi et al, 2021 ), while no pre-treatment was performed in 15 studies and no such information was provided in 16 studies ( Table 1 ). Extracted nucleic acids (retro-transcribed RNA and DNA) were always quantified using either a Qubit fluorometer and a Qubit DNA and RNA High Sensitivity Assay Kit (Life Technology, United States) ( Table 1 ; Guan et al, 2015 ; Miller et al, 2019 ; Carbo et al, 2020 ; Manso et al, 2020 ; Morsli et al, 2021a , c ; Piantadosi et al, 2021 ), or NanoDrop spectrophotometer (Thermo Fisher Scientific, United States) ( Li et al, 2021 ).…”

Section: Resultsmentioning

confidence: 99%

“…Optimisation in the human-to-microbial ratio of DNA was achieved by intermediate microbial genome enrichment or human genome depletion ( Table 1 ). DNase treatment was used to reduce human DNA in extracted RNA for mNGS investigations of RNA-viruses ( Table 1 ; Guan et al, 2015 ; Miller et al, 2019 ; Carbo et al, 2020 ; Manso et al, 2020 ; Morsli et al, 2021a , c ; Piantadosi et al, 2021 ), and bead-based capture kits were also used to remove human DNA ( Miller et al, 2019 ; Leon et al, 2020 ; Gao et al, 2021 ; Piantadosi et al, 2021 ). Further, microbial genomes could be enriched by specific primer amplification for whole genome investigations ( Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…Two studies used the PACEseq mNGS test (Hugobiotech, Beijing, China) ( Mao et al, 2021 ; Zeng et al, 2021 ), two studies used VIDISCA and Ion Torrent end-repair library ( Edridge et al, 2019 ; Gao et al, 2021 ), and one study each used the NEBNext Ultra II Directional RNA Library prep kit (New England Biolabs, Ipswich, MA, United States) ( Carbo et al, 2020 ; Leon et al, 2020 ), the KAPA Hyper Prep Kit (Kapa Biosystem, Potters Bar, United Kingdom) ( Yin et al, 2021 ), the NuGEN Ovation Ultralow Library System V2 kit (NuGEN Technologies, United States) ( Guan et al, 2015 ) the Trio RNA-Seq kit (NuGEN Technologies) ( Li et al, 2021 ), and the Roche 454 GS FLX single-end library (Roche Diagnostics, Branford, CT, United States) ( Joanna María et al, 2016 ). In three studies, the mNGS library was constructed following the single-end Oxford Nanopore library preparation protocol ( Morsli et al, 2021a , b , 2022 ). Finally, the library preparation protocol and kits were not reported in 14 studies ( Table 1 and Figure 3 ).…”

Section: Resultsmentioning

confidence: 99%

“…The basis for such an evaluation includes the flow of samples and cost of mNGS relative to multiplex PCR. Indeed, mNGS also provides an antimicrobial susceptibility profile to guide medical management of the patient (Morsli et al, 2021a(Morsli et al, ,b, 2022 and genotyping to detect outbreaks and to guide source tracing compared to multiplex PCRs (Broberg et al, 2018) Finally, a few current limitations of the mNGS approach are opportunities for improvements from the perspective of routine use. Most enzymes used in the above-described sequencing protocols were issued from cloning in competent bacteria such as Escherichia coli (E. coli) (Morsli et al, 2021a), meaning potential contamination by E. coli DNA and a risk of a false-positive diagnosis of E. coli meningitis.…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, mNGS also provides an antimicrobial susceptibility profile to guide medical management of the patient (Morsli et al, 2021a(Morsli et al, ,b, 2022 and genotyping to detect outbreaks and to guide source tracing compared to multiplex PCRs (Broberg et al, 2018) Finally, a few current limitations of the mNGS approach are opportunities for improvements from the perspective of routine use. Most enzymes used in the above-described sequencing protocols were issued from cloning in competent bacteria such as Escherichia coli (E. coli) (Morsli et al, 2021a), meaning potential contamination by E. coli DNA and a risk of a false-positive diagnosis of E. coli meningitis. As an illustration, Nanopore sequencing results regularly consisted of E. coli DNA resulting from the recommended internal library control, and Shigella and T4 phage reads issued from repair and ligation enzymes (Morsli et al, 2021a(Morsli et al, ,b, 2022.…”

Section: Discussionmentioning

confidence: 99%

See 4 more Smart Citations

Direct Metagenomic Diagnosis of Community-Acquired Meningitis: State of the Art

2022

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Current routine diagnosis of community-acquired meningitis (CAM) by multiplex real-time polymerase chain reaction (RT-PCR) is limited in the number of tested pathogens and their full characterisation, requiring additional in vitro investigations to disclose genotype and antimicrobial susceptibility. We reviewed 51 studies published through December 2021 reporting metagenomic next generation sequencing (mNGS) directly applied to the cerebrospinal fluid (CSF). This approach, potentially circumventing the above-mentioned limitations, indicated 1,248 investigated patients, and 617 patients dually investigated by routine diagnosis and mNGS, in whom 116 microbes were detected, including 50 by mNGS only, nine by routine methods only, and 57 by both routine methods and mNGS. Of 217 discordant CSF findings, 103 CSF samples were documented by mNGS only, 87 CSF samples by routine methods only, and 27 CSF samples in which the pathogen identified by mNGS was different than that found using routine methods. Overall, mNGS allowed for diagnosis and genomic surveillance of CAM causative pathogens in real-time, with a cost which is competitive with current routine multiplex RT-PCR. mNGS could be implemented at point-of-care (POC) laboratories as a part of routine investigations to improve the diagnosis and molecular epidemiology of CAM, particularly in the event of failure of routine assays.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

Direct Metagenomic Diagnosis of Community-Acquired Meningitis: State of the Art

2022

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Real-time metagenomics-based diagnosis of community-acquired meningitis: A prospective series, southern France

Morsli¹,

Boudet²,

Kerharo³

et al. 2022

eBioMedicine

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Clinical Diagnostics of Bacterial Infections and Their Resistance to Antibiotics—Current State and Whole Genome Sequencing Implementation Perspectives

2023

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Antimicrobial resistance (AMR), defined as the ability of microorganisms to withstand antimicrobial treatment, is responsible for millions of deaths annually. The rapid spread of AMR across continents warrants systematic changes in healthcare routines and protocols. One of the fundamental issues with AMR spread is the lack of rapid diagnostic tools for pathogen identification and AMR detection. Resistance profile identification often depends on pathogen culturing and thus may last up to several days. This contributes to the misuse of antibiotics for viral infection, the use of inappropriate antibiotics, the overuse of broad-spectrum antibiotics, or delayed infection treatment. Current DNA sequencing technologies offer the potential to develop rapid infection and AMR diagnostic tools that can provide information in a few hours rather than days. However, these techniques commonly require advanced bioinformatics knowledge and, at present, are not suited for routine lab use. In this review, we give an overview of the AMR burden on healthcare, describe current pathogen identification and AMR screening methods, and provide perspectives on how DNA sequencing may be used for rapid diagnostics. Additionally, we discuss the common steps used for DNA data analysis, currently available pipelines, and tools for analysis. Direct, culture-independent sequencing has the potential to complement current culture-based methods in routine clinical settings. However, there is a need for a minimum set of standards in terms of evaluating the results generated. Additionally, we discuss the use of machine learning algorithms regarding pathogen phenotype detection (resistance/susceptibility to an antibiotic).

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Real-time whole genome sequencing direct diagnosis of Streptococcus pneumoniae meningitis: A case report

Cited by 3 publications

References 7 publications

Direct Metagenomic Diagnosis of Community-Acquired Meningitis: State of the Art

Direct Metagenomic Diagnosis of Community-Acquired Meningitis: State of the Art

Real-time metagenomics-based diagnosis of community-acquired meningitis: A prospective series, southern France

Clinical Diagnostics of Bacterial Infections and Their Resistance to Antibiotics—Current State and Whole Genome Sequencing Implementation Perspectives

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