Sensitivity of Next-Generation Sequencing Metagenomic Analysis for Detection of RNA and DNA Viruses in Cerebrospinal Fluid: The Confounding Effect of Background Contamination

2016 ◽  
pp. 53-62 ◽  
Author(s):  
Iwona Bukowska-Ośko ◽  
Karol Perlejewski ◽  
Shota Nakamura ◽  
Daisuke Motooka ◽  
Tomasz Stokowy ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Metagenomic Analysis ◽  
Next Generation ◽  
Confounding Effect ◽  
Dna Viruses ◽  
Generation Sequencing ◽  
Rna And Dna

scholarly journals Enhanced Detection of DNA Viruses in the Cerebrospinal Fluid of Encephalitis Patients Using Metagenomic Next-Generation Sequencing

2020 ◽  
Vol 11 ◽  
Author(s):  
Carmen F. Manso ◽  
David F. Bibby ◽  
Hodan Mohamed ◽  
David W. G. Brown ◽  
Mark Zuckerman ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Next Generation ◽  
Dna Viruses ◽  
Generation Sequencing

scholarly journals Diagnosis and analysis of unexplained cases of childhood encephalitis in Australia using metagenomic next-generation sequencing

2021 ◽  
Author(s):  
Ci-Xiu Li ◽  
Rebecca Burrell ◽  
Russell C Dale ◽  
Alison Kesson ◽  
Christopher C Blyth ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Rna Sequencing ◽  
Diagnostic Yield ◽  
Human Infection ◽  
Clinical Samples ◽  
Next Generation ◽  
Dna Viruses ◽  
Immune Mediated ◽  
Generation Sequencing

Encephalitis is most often caused by a variety of infectious agents, the identity of which is commonly determined through diagnostic tests utilising cerebrospinal fluid (CSF). Immune-mediated disorders are also a differential in encephalitis cases. We investigated the clinical characteristics and potential aetiological agents of unexplained encephalitis through metagenomic next-generation sequencing of residual clinical samples of multiple tissue types and independent clinical review. A total of 43 specimens, from both sterile and non-sterile sites, were collected from 18 encephalitis cases with no cause identified by the Australian Childhood Encephalitis study. Samples were subjected to total RNA sequencing to determine the presence and abundance of potential pathogens, to reveal mixed infections, pathogen genotypes, and epidemiological origins, and to describe the possible aetiologies of unexplained encephalitis. From this, we identified five RNA and two DNA viruses associated with human infection from both non-sterile (nasopharyngeal aspirates, nose/throat swabs, urine, stool rectal swab) and sterile (cerebrospinal fluid, blood) sites. These comprised two human rhinoviruses, two human seasonal coronaviruses, two polyomaviruses and one picobirnavirus. With the exception of picobirnavirus all have been previously associated with respiratory disease. Human rhinovirus and seasonal coronaviruses may be responsible for five of the encephalitis cases reported here. Immune-mediated encephalitis was considered clinically likely in six cases and RNA sequencing did not identify a possible pathogen in these cases. The aetiology remained unknown in nine cases. Our study emphasises the importance of respiratory viruses in the aetiology of unexplained child encephalitis and suggests that the routine inclusion of non-CNS sampling in encephalitis clinical guidelines/protocols could improve the diagnostic yield.

scholarly journals Development and Optimization of Metagenomic Next-Generation Sequencing Methods for Cerebrospinal Fluid Diagnostics

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Patricia J. Simner ◽  
Heather B. Miller ◽  
Florian P. Breitwieser ◽  
Gabriel Pinilla Monsalve ◽  
Carlos A. Pardo ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Sample Pretreatment ◽  
Standard Of Care ◽  
Nucleic Acid Amplification ◽  
Next Generation ◽  
Bead Beating ◽  
Dna And Rna ◽  
Positive Threshold ◽  
Generation Sequencing

ABSTRACT The purpose of this study was to develop and optimize different processing, extraction, amplification, and sequencing methods for metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) specimens. We applied mNGS to 10 CSF samples with known standard-of-care testing (SoC) results (8 positive and 2 negative). Each sample was subjected to nine different methods by varying the sample processing protocols (supernatant, pellet, neat CSF), sample pretreatment (with or without bead beating), and the requirement of nucleic acid amplification steps using DNA sequencing (DNASeq) (with or without whole-genome amplification [WGA]) and RNA sequencing (RNASeq) methods. Negative extraction controls (NECs) were used for each method variation (4/CSF sample). Host depletion (HD) was performed on a subset of samples. We correctly determined the pathogen in 7 of 8 positive samples by mNGS compared to SoC. The two negative samples were correctly interpreted as negative. The processing protocol applied to neat CSF specimens was found to be the most successful technique for all pathogen types. While bead beating introduced bias, we found it increased the detection yield of certain organism groups. WGA prior to DNASeq was beneficial for defining pathogens at the positive threshold, and a combined DNA and RNA approach yielded results with a higher confidence when detected by both methods. HD was required for detection of a low-level-positive enterovirus sample. We demonstrate that NECs are required for interpretation of these complex results and that it is important to understand the common contaminants introduced during mNGS. Optimizing mNGS requires the use of a combination of techniques to achieve the most sensitive, agnostic approach that nonetheless may be less sensitive than SoC tools.

scholarly journals An improved experimental pipeline for preparing circular ssDNA viruses for next-generation sequencing

2020 ◽  
Author(s):  
Catherine D. Aimone ◽  
J. Steen Hoyer ◽  
Anna E. Dye ◽  
David O. Deppong ◽  
Siobain Duffy ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rolling Circle Amplification ◽  
Next Generation ◽  
Viral Dna ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Short Read ◽  
Rolling Circle ◽  
Optimized Protocol ◽  
Generation Sequencing

AbstractWe present an optimized protocol for enhanced amplification and enrichment of viral DNA for Next Generation Sequencing of begomovirus genomes. The rapid ability of these viruses to evolve threatens many crops and underscores the importance of using next generation sequencing efficiently to detect and understand the diversity of these viruses. We combined enhanced rolling circle amplification (RCA) with EquiPhi29 polymerase and size selection to generate a cost-effective, short-read sequencing method. This optimized protocol produced short-read sequencing with at least 50% of the reads mapping to the viral reference genome. We provide other insights into common misconceptions about RCA and lessons we have learned from sequencing single-stranded DNA viruses. Our protocol can be used to examine viral DNA as it moves through the entire pathosystem from host to vector, providing valuable information for viral DNA population studies, and would likely work well with other CRESS DNA viruses.HighlightsProtocol for short-read, high throughput sequencing of single-stranded DNA viruses using random primersComparison of the sequencing of total DNA versus size-selected DNAComparison of phi29 and Equiphi29 DNA polymerases for rolling circle amplification of viral single-stranded DNA genomes

scholarly journals Diagnosis of Enterococcus faecalis meningitis associated with long-term cerebrospinal fluid rhinorrhoea using metagenomics next-generation sequencing: a case report

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xiaobo Zhang ◽  
Chao Jiang ◽  
Chaojun Zhou
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Enterococcus Faecalis ◽  
Bone Defect ◽  
Next Generation ◽  
History Of ◽  
Diagnostic Approaches ◽  
Post Traumatic ◽  
Skull Bone Defect ◽  
Generation Sequencing

Abstract Background Enterococcus faecalis (E. faecalis) meningitis is a rare disease, and most of its occurrences are of post-operative origin. Its rapid diagnosis is critical for effective clinical management. Currently, the diagnosis is focused on cerebrospinal fluid (CSF) culture, but this is quite limited. By comparison, metagenomic next-generation sequencing (mNGS) can overcome the deficiencies of conventional diagnostic approaches. To our knowledge, mNGS analysis of the CSF in the diagnosis of E. faecalis meningitis has been not reported. Case presentation We report the case of E. faecalis meningitis in a 70-year-old female patient without a preceding history of head injury or surgery, but with an occult sphenoid sinus bone defect. Enterococcus faecalis meningitis was diagnosed using mNGS of CSF, and she recovered satisfactorily following treatment with appropriate antibiotics and surgical repair of the skull bone defect. Conclusions Non-post-traumatic or post-surgical E. faecalis meningitis can occur in the presence of occult defects in the cranium, and mNGS technology could be helpful in diagnosis in the absence of a positive CSF culture.

scholarly journals Metagenomic Next-Generation Sequencing of Cerebrospinal Fluid for the Diagnosis of External Ventricular and Lumbar Drainage-Associated Ventriculitis and Meningitis

2020 ◽  
Vol 11 ◽  
Author(s):  
Lingye Qian ◽  
Yijun Shi ◽  
Fangqiang Li ◽  
Yufei Wang ◽  
Miao Ma ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Staphylococcus Epidermidis ◽  
Coincidence Rate ◽  
Next Generation ◽  
Lumbar Drainage ◽  
Gram Positive Bacteria ◽  
Neurosurgical Patients ◽  
Generation Sequencing ◽  
Guided Therapy

Metagenomic next-generation sequencing (mNGS) has become a widely used technology that can accurately detect individual pathogens. This prospective study was performed between February 2019 and September 2019 in one of the largest clinical neurosurgery centers in China. The study aimed to evaluate the performance of mNGS on cerebrospinal fluid (CSF) from neurosurgical patients for the diagnosis of external ventricular and lumbar drainage (EVD/LD)-associated ventriculitis and meningitis (VM). We collected CSF specimens from neurosurgical patients with EVD/LD for more than 24 h to perform conventional microbiological studies and mNGS analyses in a pairwise manner. We also investigated the usefulness of mNGS of CSF for the diagnosis of EVD/LD-associated VM. In total, 102 patients were enrolled in this study and divided into three groups, including confirmed VM (cVM) (39), suspected VM (sVM) (49), and non-VM (nVM) (14) groups. Of all the patients, mNGS detected 21 Gram-positive bacteria, 20 Gram-negative bacteria, and five fungi. The three primary bacteria detected were Staphylococcus epidermidis (9), Acinetobacter baumannii (5), and Staphylococcus aureus (3). The mNGS-positive coincidence rate of confirmed EVD/LD-associated VM was 61.54% (24/39), and the negative coincidence rate of the nVM group was 100% (14/14). Of 15 VM pathogens not identified by mNGS in the cVM group, eight were negative with mNGS and seven were inconsistent with the conventional microbiological identification results. In addition, mNGS identified pathogens in 22 cases that were negative using conventional methods; of them, 10 patients received a favorable clinical treatment; thus, showing the benefit of mNGS-guided therapy.

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