A viral metagenomic approach on a non-metagenomic experiment: Mining next generation sequencing datasets from pig DNA identified several porcine parvoviruses for a retrospective evaluation of viral infections

Abstract Background Viral infections are common complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Allo-HSCT recipients with steroid-refractory/dependent graft-versus-host disease (GvHD) are highly immunosuppressed and are more vulnerable to infections with weakly pathogenic or commensal viruses. Here, twenty-five adult allo-HSCT recipients from 2016 to 2019 with acute or chronic steroid-refractory/dependent GvHD were enrolled in a prospective cohort at Geneva University Hospitals. We performed metagenomics next-generation sequencing (mNGS) analysis using a validated pipeline and de novo analysis on pooled routine plasma samples collected throughout the period of intensive steroid treatment or second-line GvHD therapy to identify weakly pathogenic, commensal, and unexpected viruses. Results Median duration of intensive immunosuppression was 5.1 months (IQR 5.5). GvHD-related mortality rate was 36%. mNGS analysis detected viral nucleotide sequences in 24/25 patients. Sequences of ≥ 3 distinct viruses were detected in 16/25 patients; Anelloviridae (24/25) and human pegivirus-1 (9/25) were the most prevalent. In 7 patients with fatal outcomes, viral sequences not assessed by routine investigations were identified with mNGS and confirmed by RT-PCR. These cases included Usutu virus (1), rubella virus (1 vaccine strain and 1 wild-type), novel human astrovirus (HAstV) MLB2 (1), classic HAstV (1), human polyomavirus 6 and 7 (2), cutavirus (1), and bufavirus (1). Conclusions Clinically unrecognized viral infections were identified in 28% of highly immunocompromised allo-HSCT recipients with steroid-refractory/dependent GvHD in consecutive samples. These identified viruses have all been previously described in humans, but have poorly understood clinical significance. Rubella virus identification raises the possibility of re-emergence from past infections or vaccinations, or re-infection.

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Detection of severe fever with thrombocytopenia syndrome virus and other viruses in cats via unbiased next-generation sequencing

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638720967506 ◽

2020 ◽

pp. 104063872096750

Author(s):

Yasuyuki Momoi ◽

Aya Matsuu

Keyword(s):

Next Generation Sequencing ◽

Viral Infections ◽

Nucleotide Sequencing ◽

Plasma Samples ◽

Next Generation ◽

Dna Library ◽

Immunodeficiency Virus ◽

Next Generation Sequencing Ngs ◽

Severe Fever ◽

Generation Sequencing

We used unbiased next-generation sequencing (NGS) to detect unknown viruses in cats. Serum or plasma samples were obtained from clinically ill cats with suspected acute viral infections. Nucleic acid was extracted from serum or plasma samples to construct a complementary DNA library for NGS. Comprehensive nucleotide sequencing analyses enabled detection of the genomes of various viruses, including the severe fever with thrombocytopenia syndrome virus, feline immunodeficiency virus, feline morbillivirus, parvovirus, and Torque teno felis virus. Our findings indicate that comprehensive nucleotide analyses of serum or plasma samples can be used to detect infections with unknown viruses in cats.

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Metagenomic Next Generation Sequencing in the Detection of Pathogens in Cerebrospinal Fluid of Patients After Alternative Donor Transplantation: A Feasibility Analysis

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.720132 ◽

2021 ◽

Vol 11 ◽

Author(s):

Binglei Zhang ◽

Jian Zhou ◽

Ruirui Gui ◽

Zhen Li ◽

Yingling Zu ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Next Generation Sequencing ◽

Viral Infections ◽

Fungal Infections ◽

Clinical Manifestations ◽

Next Generation ◽

Hematopoietic Stem ◽

Cns Infections ◽

Alternative Donor ◽

Generation Sequencing

Central nervous system (CNS) complications can occur in 9%–15% of patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The clinical manifestations of the CNS complications are non-specific, with most of them being disturbances of consciousness, convulsions, headaches, fever, and epilepsy, making it difficult to infer the cause of the complications based on clinical manifestations. We retrospectively analyzed the sensitivity and feasibility of metagenomic next generation sequencing (mNGS) in the diagnosis of CNS infections after allo-HSCT. Lumbar punctures were performed on 20 patients with CNS symptoms after receiving alternative donor HSCT(AD-HSCT) at the Affiliated Cancer Hospital of Zhengzhou University from February 2019 to December 2020, and their cerebrospinal fluid (CSF) was collected. The mNGS technique was used to detect pathogens in the CSF. Routine CSF testing, biochemical analyses, G experiments, GM experiments, ink staining, acid-fast staining, and bacterial cultures were carried out, and quantitative PCR (qPCR) tests were used to detect cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK polyomavirus (BKPyV), and human alphaherpesvirus (HHV). A total of 29 tests were performed with 21 of them being positive. Of the five negative patients, three were diagnosed with a posterior reversible encephalopathy syndrome, one as having transplantation-associated thrombotic microangiopathy, and one with transient seizure caused by hypertension. Fifteen patients tested positive, of which four had single infections and eleven had mixed infections. Five cases of fungal infections, six cases of bacterial infections, and 13 cases of viral infections were detected. Among the 13 cases of viral infections, ten cases were CMV(HHV-5); three were BKPyV; two were Torque teno virus (TTV); Two were HHV-1,two were EBV(HHV4), and one each of HpyV5 and HHV-6B. Thirteen patients tested positive for virus while the qPCR detection method of 6 identical specimens were below the minimum detection limit(<1×103 U/ml). The mNGS technique is highly sensitive, and it can be used to diagnose CNS infections after allo-HSCT.

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Metagenomic Next-Generation Sequencing in the Diagnosis of HHV-1 Reactivation in a Critically Ill COVID-19 Patient: A Case Report

Frontiers in Medicine ◽

10.3389/fmed.2021.715519 ◽

2021 ◽

Vol 8 ◽

Author(s):

Lei Shi ◽

Han Xia ◽

Matthew D. Moore ◽

Chao Deng ◽

Na Li ◽

...

Keyword(s):

Case Report ◽

Next Generation Sequencing ◽

Critically Ill ◽

Viral Infections ◽

X Rays ◽

Next Generation ◽

Reverse Transcription Pcr ◽

Negative Results ◽

Low Sensitivity ◽

Generation Sequencing

Background: Secondary infections pose tremendous challenges in Coronavirus disease 2019 (COVID-19) treatment and are associated with higher mortality rates. Clinicians face of the challenge of diagnosing viral infections because of low sensitivity of available laboratory tests.Case Presentation: A 66-year-old woman initially manifested fever and shortness of breath. She was diagnosed as critically ill with COVID-19 using quantitative reverse transcription PCR (RT-qPCR) and treated with antiviral therapy, ventilator and extracorporeal membrane oxygenation (ECMO). However, after the condition was relatively stabled for a few days, the patient deteriorated with fever, frequent cough, increased airway secretions, and increased exudative lesions in the lower right lung on chest X-rays, showing the possibility of a newly acquired infection, though sputum bacterial and fungal cultures and smears showed negative results. Using metagenomic next-generation sequencing (mNGS), we identified a reactivation of latent human herpes virus type 1 (HHV-1) in the respiratory tract, blood and gastrointestinal tract, resulting in a worsened clinical course in a critically ill COVID-19 patient on ECMO. Anti-HHV-1 therapy guided by these sequencing results effectively decreased HHV-1 levels, and improved the patient's clinical condition. After 49 days on ECMO and 67 days on the ventilator, the 66-year-old patient recovered and was discharged.Conclusions: This case report demonstrates the potential value of mNGS for evidence-based treatment, and suggests that potential reactivation of latent viruses should be considered in critically ill COVID-19 patients.

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Quality control implementation for universal characterization of DNA and RNA viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow

10.1101/367367 ◽

2018 ◽

Author(s):

A. Bal ◽

M. Pichon ◽

C. Picard ◽

JS. Casalegno ◽

M. Valette ◽

...

Keyword(s):

Next Generation Sequencing ◽

Real Time ◽

Real Time Pcr ◽

Viral Genome ◽

Viral Infections ◽

Rna Viruses ◽

Next Generation ◽

Dna And Rna ◽

Generation Sequencing

AbstractBackgroundIn recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on clinical respiratory samples and implementation of quality controls (QCs) is still lacking.MethodsA total of 3 QCs were implemented and processed through the whole mNGS workflow: a notemplate-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples including 6 multiple viral infections; 9 negative samples). Selected specimens included nasopharyngeal swabs (n = 20), aspirates (n = 10), or sputums (n = 7).ResultsThe optimal spiking level of the internal QC was first determined in order to be sufficiently detected without overconsumption of sequencing reads. According to QC validation criteria, mNGS results were validated for 34/37 selected samples. For valid samples, viral genotypes were accurately determined for 36/36 viruses detected with PCR (viral genome coverage ranged from 0.6% to 100%, median = 67.7%). This mNGS workflow allowed the detection of DNA and RNA viruses up to a semi-quantitative PCR Ct value of 36. The six multiple viral infections involving 2 to 4 viruses were also fully characterized. A strong correlation between results of mNGS and real-time PCR was obtained for each type of viral genome (R2 ranged from 0.72 for linear single-stranded (ss) RNA viruses to 0.98 for linear ssDNA viruses).ConclusionsAlthough the potential of mNGS technology is very promising, further evaluation studies are urgently needed for its routine clinical use within a reasonable timeframe. The approach described herein is crucial to bring standardization and to ensure the quality of the generated sequences in clinical setting. We provide an easy-to-use single protocol successfully evaluated for the characterization of a broad and representative panel of DNA and RNA respiratory viruses in various types of clinical samples.

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Evaluation of Next-Generation Sequencing for the Diagnosis of Infections of the Central Nervous System Caused by the Neurotropic Herpes Viruses: A Pilot Study

European Neurology ◽

10.1159/000497319 ◽

2018 ◽

Vol 80 (5-6) ◽

pp. 283-288 ◽

Cited By ~ 3

Author(s):

Xiao-Wei Xing ◽

Jia-Tang Zhang ◽

Yu-Bao Ma ◽

Xiao-Yan Chen ◽

Lei Wu ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Next Generation Sequencing ◽

Viral Infections ◽

Herpes Viruses ◽

Next Generation ◽

Viral Dna ◽

Cns Infections ◽

Generation Sequencing ◽

Hsv 1

Background: There are sparse and limited studies on small sample size reporting the application of next-generation sequencing (NGS) in the detection of central nervous system (CNS) viral infections. We assessed the diagnostic performance of NGS of cerebrospinal fluid (CSF) for predicting viral infections of the CNS caused by the neurotropic herpes viruses in a pilot population. Materials and Methods: We prospectively collected CSF samples from 24 patients with CNS viral infection from April 2017 to October 2018. Of the 24 patients, 19 patients were infected with herpes simplex virus 1 (HSV-1), 1 patient with HSV-2, and 4 patients with varicella-zoster virus (VZV). All CSF samples were screened for viral DNA using NGS technologies to detect viral CNS infections. Results: Of the 24 patients with confirmed viral CNS infection caused by the neurotropic herpes viruses, 10 (10/24, 41.67%) patients exhibited positive NGS results. With the help of NGS, HSV-1 DNA was detected in the CSF of 6 patients (6/19; 31.58%). HSV-2 DNA was detected in 1 patient (1/1; 100%) and VZV DNA was detected in 3 patients (3/4; 75%). The positive rate of virus detected by NGS decreased with time. The positive rates of NGS of CSF in the first, second, and third weeks were 54.5% (6/11), 44.4% (4/9), and 0% (0/4), respectively. Conclusions: NGS method is a promising pathogen detection tool for identifying viral CNS infections. It should be recommended to sequence viral DNA of CSF in the early stage of CNS viral infections.

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Metagenomic next-generation sequencing aids the diagnosis of viral infections in febrile returning travellers

Journal of Infection ◽

10.1016/j.jinf.2019.08.003 ◽

2019 ◽

Vol 79 (4) ◽

pp. 383-388 ◽

Cited By ~ 8

Author(s):

Hanna Jerome ◽

Callum Taylor ◽

Vattipally B. Sreenu ◽

Tanya Klymenko ◽

Ana Da Silva Filipe ◽

...

Keyword(s):

Next Generation Sequencing ◽

Viral Infections ◽

Next Generation ◽

Returning Travellers ◽

Generation Sequencing

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Metagenomic sequencing to detect respiratory viruses in persons under investigation for COVID-19

10.1101/2020.09.09.20178764 ◽

2020 ◽

Author(s):

Ahmed Babiker ◽

Heath Bradley ◽

Victoria Stittleburg ◽

Autum Key ◽

Colleen Suzanne Kraft ◽

...

Keyword(s):

Next Generation Sequencing ◽

Viral Infections ◽

Respiratory Viruses ◽

Metagenomic Sequencing ◽

Next Generation ◽

Rt Pcr ◽

Generation Sequencing

We used metagenomic next-generation sequencing (mNGS) to assess the frequencies of alternative viral infections in SARS-CoV-2 RT-PCR negative persons under investigations (PUIs) (n=30) and viral co-infections in SARS-CoV-2 RT-PCR positive PUIs (n=45). mNGS identified both co-infections and alternative viral infections that were not detected by routine clinical workup

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