scholarly journals Analysis Comparison for Rapid Identification of Pathogenic Virus from Infected Tissue Samples

Diagnostics ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 196
Author(s):  
Junji Hosokawa-Muto ◽  
Yukiko Sassa-O’Brien ◽  
Yoshihito Fujinami ◽  
Hiroaki Nakahara
Keyword(s):  
Influenza Virus ◽  
Next Generation Sequencing ◽  
Rapid Determination ◽  
Rapid Identification ◽  
Infected Tissue ◽  
Mouse Lung ◽  
Next Generation ◽  
Tissue Samples ◽  
Time Required ◽  
Generation Sequencing

When examining infectious samples, rapid identification of the pathogenic agent is required for diagnosis and treatment or for investigating the cause of death. In our previous study, we applied exhaustive amplification using non-specific primers (the rapid determination system of viral genome sequences, the RDV method) to identify the causative virus via swab samples from a cat with a suspected viral infection. The purpose of the current study is to investigate suitable methods for the rapid identification of causative pathogens from infected tissue samples. First, the influenza virus was inoculated into mice to prepare infected tissue samples. RNA extracted from the mouse lung homogenates was transcribed into cDNA and then analyzed using the RDV method and next-generation sequencing, using MiSeq and MinION sequencers. The RDV method was unable to detect the influenza virus in the infected tissue samples. However, influenza virus reads were detected using next-generation sequencing. Comparing MiSeq and MinION, the time required for library and sequence preparation was shorter for MinION sequencing than for MiSeq sequencing. We conclude that when a causative virus needs to be rapidly identified from an infectious sample, MinION sequencing is currently the method of choice.

A Novel Method for Microsatellite Instability Detection by Liquid Biopsy Based on Next- Generation Sequencing

2020 ◽  
Vol 15 ◽  
Author(s):  
Zheng Jiang ◽  
Hui Liu ◽  
Siwen Zhang ◽  
Jia Liu ◽  
Weitao Wang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Microsatellite Instability ◽  
Liquid Biopsy ◽  
Tumor Tissue ◽  
High Sensitivity ◽  
Next Generation ◽  
Tissue Samples ◽  
Next Generation Sequencing Technology ◽  
Medication Selection ◽  
Generation Sequencing

Background: Microsatellite instability (MSI) is a prognostic biomarker used to guide medication selection in multiple cancers, such as colorectal cancer. Traditional PCR with capillary electrophoresis and next-generation sequencing using paired tumor tissue and leukocyte samples are the main approaches for MSI detection due to their high sensitivity and specificity. Currently, patient tissue samples are obtained through puncture or surgery, which causes injury and risk of concurrent disease, further illustrating the need for MSI detection by liquid biopsy. Methods: We propose an analytic method using paired plasma/leukocyte samples and MSI detection using next-generation sequencing technology. Based on the theoretical progress of oncogenesis, we hypothesized that the microsatellite site length in plasma equals the combination of the distribution of tumor tissue and leukocytes. Thus, we defined a window-judgement method to identify whether biomarkers were stable. Results: Compared to traditional PCR as the standard, we evaluated three methods in 20 samples (MSI-H:3/MSS:17): peak shifting method using tissue vs. leukocytes, peak shifting method using plasma vs. leukocytes, and our method using plasma vs. leukocytes. Compared to traditional PCR, we observed a sensitivity of 100%, 0%, and 100%, and a specificity of 100.00%, 94.12%, and 88.24%, respectively. Conclusion: Our method has the advantage of possibly detecting MSI in a liquid biopsy and provides a novel direction for future studies to increase the specificity of the method.

scholarly journals Panel of serum miRNAs as potential non-invasive biomarkers for pancreatic ductal adenocarcinoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Imteyaz Ahmad Khan ◽  
Safoora Rashid ◽  
Nidhi Singh ◽  
Sumaira Rashid ◽  
Vishwajeet Singh ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Early Stage ◽  
Ductal Adenocarcinoma ◽  
Next Generation ◽  
Serum Samples ◽  
Tissue Samples ◽  
Non Invasive ◽  
Specific Symptoms ◽  
Generation Sequencing

AbstractEarly-stage diagnosis of pancreatic ductal adenocarcinoma (PDAC) is difficult due to non-specific symptoms. Circulating miRNAs in body fluids have been emerging as potential non-invasive biomarkers for diagnosis of many cancers. Thus, this study aimed to assess a panel of miRNAs for their ability to differentiate PDAC from chronic pancreatitis (CP), a benign inflammatory condition of the pancreas. Next-generation sequencing was performed to identify miRNAs present in 60 FFPE tissue samples (27 PDAC, 23 CP and 10 normal pancreatic tissues). Four up-regulated miRNAs (miR-215-5p, miR-122-5p, miR-192-5p, and miR-181a-2-3p) and four down-regulated miRNAs (miR-30b-5p, miR-216b-5p, miR-320b, and miR-214-5p) in PDAC compared to CP were selected based on next-generation sequencing results. The levels of these 8 differentially expressed miRNAs were measured by qRT-PCR in 125 serum samples (50 PDAC, 50 CP, and 25 healthy controls (HC)). The results showed significant upregulation of miR-215-5p, miR-122-5p, and miR-192-5p in PDAC serum samples. In contrast, levels of miR-30b-5p and miR-320b were significantly lower in PDAC as compared to CP and HC. ROC analysis showed that these 5 miRNAs can distinguish PDAC from both CP and HC. Hence, this panel can serve as a non-invasive biomarker for the early detection of PDAC.

Rapid Somatic Mutation Testing in Colorectal Cancer by Use of a Fully Automated System and Single-Use Cartridge: A Comparison with Next-Generation Sequencing

2018 ◽  
Vol 3 (2) ◽  
pp. 178-184 ◽  
Author(s):  
M Rabie Al-Turkmani ◽  
Kelley N Godwin ◽  
Jason D Peterson ◽  
Gregory J Tsongalis
Keyword(s):  
Colorectal Cancer ◽  
Next Generation Sequencing ◽  
Ffpe Tissue ◽  
Braf V600e ◽  
Automated System ◽  
Next Generation ◽  
Tissue Samples ◽  
Tissue Sections ◽  
Single Use ◽  
Generation Sequencing

AbstractBackgroundMolecular tests have been increasingly used in the management of various cancers as more targeted therapies are becoming available as treatment options. The Idylla™ system is a fully integrated, cartridge-based platform that provides automated sample processing (deparaffinization, tissue digestion, and DNA extraction) and real-time PCR-based mutation detection with all reagents included in a single-use cartridge. This retrospective study aimed at evaluating both the Idylla KRAS and NRAS-BRAF-EGFR492 Mutation Assay cartridges (research use only) against next-generation sequencing (NGS) by using colorectal cancer (CRC) tissue samples.MethodsForty-four archived formalin-fixed paraffin-embedded (FFPE) CRC tissue samples previously analyzed by targeted NGS were tested on the Idylla system. Among these samples, 17 had a mutation in KRAS proto-oncogene, GTPase (KRAS), 5 in NRAS proto-oncogene, GTPase (NRAS), and 12 in B-Raf proto-oncogene, serine/threonine kinase (BRAF) as determined using the Ion AmpliSeq 50-gene Cancer Hotspot Panel v2. The remaining 10 samples were wild-type for KRAS, NRAS, and BRAF. Two 10-μm FFPE tissue sections were used for each Idylla run, 1 for the KRAS cartridge, and 1 for the NRAS-BRAF-EGFR492 cartridge. All cases met the Idylla minimum tumor content requirement for KRAS, NRAS, and BRAF (≥10%). Assay reproducibility was evaluated by testing commercial controls derived from human cell lines, which had an allelic frequency of 50% and were run in triplicate.ResultsThe Idylla system successfully detected all mutations previously identified by NGS in KRAS (G12C, G12D, G12V, G13D, Q61K, Q61R, A146T), NRAS (G12V, G13R, Q61H), and BRAF (V600E). Compared with NGS, Idylla had a sensitivity of 100%. Analysis of the mutated commercial controls demonstrated agreement with the expected result for all samples and 100% reproducibility. The Idylla system produced results quickly with a turnaround time of approximately 2 h.ConclusionThe Idylla system offers reliable and sensitive testing of clinically actionable mutations in KRAS, NRAS, and BRAF directly from FFPE tissue sections.

scholarly journals Teaching NeuroImage: Rapid Identification of Infectious Optic Neuritis by Next-Generation Sequencing

Neurology ◽  
2021 ◽  
pp. 10.1212/WNL.0000000000013213
Author(s):  
Ying Huang ◽  
Yulu Liu ◽  
Yongguang Liu ◽  
Qiang Li ◽  
Xuejun Fu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Optic Neuritis ◽  
Rapid Identification ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals No Metagenomic Evidence of Causative Viral Pathogens in Postencephalitic Parkinsonism Following Encephalitis Lethargica

2021 ◽  
Vol 9 (8) ◽  
pp. 1716
Author(s):  
Dániel Cadar ◽  
Kurt A. Jellinger ◽  
Peter Riederer ◽  
Sabrina Strobel ◽  
Camelia-Maria Monoranu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Neuronal Loss ◽  
Unknown Etiology ◽  
Next Generation ◽  
Encephalitis Lethargica ◽  
Tissue Samples ◽  
Viral Pathogen ◽  
Postencephalitic Parkinsonism ◽  
Generation Sequencing ◽  
Post Infectious

Postencephalitic parkinsonism (PEP) is a disease of unknown etiology and pathophysiology following encephalitis lethargica (EL), an acute-onset polioencephalitis of cryptic cause in the 1920s. PEP is a tauopathy with multisystem neuronal loss and gliosis, clinically characterized by bradykinesia, rigidity, rest tremor, and oculogyric crises. Though a viral cause of EL is likely, past polymerase chain reaction-based investigations in the etiology of both PEP and EL were negative. PEP might be caused directly by an unknown viral pathogen or the consequence of a post-infectious immunopathology. The development of metagenomic next-generation sequencing in conjunction with bioinformatic techniques has generated a broad-range tool for the detection of unknown pathogens in the recent past. Retrospective identification and characterization of pathogens responsible for past infectious diseases can be successfully performed with formalin-fixed paraffin-embedded (FFPE) tissue samples. In this study, we analyzed 24 FFPE brain samples from six patients with PEP by unbiased metagenomic next-generation sequencing. Our results show that no evidence for the presence of a specific or putative (novel) viral pathogen was found, suggesting a likely post-infectious immune-mediated etiology of PEP.

Balamuthia mandrillaris-Related Primary Amoebic Encephalitis in China Diagnosed by Next Generation Sequencing and a Review of the Literature

2019 ◽  
Author(s):  
Yinan Yang ◽  
Xiaobin Hu ◽  
Li Min ◽  
Xiangyu Dong ◽  
Yuanlin Guan
Keyword(s):  
Next Generation Sequencing ◽  
Inflammatory Diseases ◽  
Clinical Manifestations ◽  
Infectious Agent ◽  
Disease Diagnosis ◽  
Rapid Identification ◽  
Next Generation ◽  
Balamuthia Mandrillaris ◽  
Granulomatous Amoebic Encephalitis ◽  
Generation Sequencing

Abstract Background Encephalitis is caused by infection, immune mediated diseases, or primary inflammatory diseases. Of all the causative infectious pathogens, 90% are viruses or bacteria. Granulomatous amoebic encephalitis (GAE), caused by Balamuthia mandrillaris, is a rare but life-threatening disease. Diagnosis and therapy are frequently delayed due to the lack of specific clinical manifestations. Method A healthy 2 year old Chinese male patient initially presented with a nearly 2 month history of irregular fever. We present this case of granulomatous amoebic encephalitis caused by B. mandrillaris. Next generation sequencing of the patient’s cerebrospinal fluid (CSF) was performed to identify an infectious agent. Result The results of next generation sequencing of the CSF showed that most of the mapped reads belonged to Balamuthia mandrillaris. Conclusion Next generation sequencing (NGS) is an unbiased and rapid diagnostic tool. The NGS method can be used for the rapid identification of causative pathogens. The NGS method should be widely applied in clinical practice and help clinicians provide direction for the diagnosis of diseases, especially for rare and difficult cases.

scholarly journals Propionibacterium acnes: Disease-Causing Agent or Common Contaminant? Detection in Diverse Patient Samples by Next-Generation Sequencing

2016 ◽  
Vol 54 (4) ◽  
pp. 980-987 ◽  
Author(s):  
Sarah Mollerup ◽  
Jens Friis-Nielsen ◽  
Lasse Vinner ◽  
Thomas Arn Hansen ◽  
Stine Raith Richter ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Propionibacterium Acnes ◽  
Opportunistic Pathogen ◽  
Next Generation Sequencing Data ◽  
Clinical Samples ◽  
Next Generation ◽  
Sequencing Data ◽  
Tissue Samples ◽  
Content Type ◽  
Generation Sequencing

Propionibacterium acnesis the most abundant bacterium on human skin, particularly in sebaceous areas.P. acnesis suggested to be an opportunistic pathogen involved in the development of diverse medical conditions but is also a proven contaminant of human clinical samples and surgical wounds. Its significance as a pathogen is consequently a matter of debate. In the present study, we investigated the presence ofP. acnesDNA in 250 next-generation sequencing data sets generated from 180 samples of 20 different sample types, mostly of cancerous origin. The samples were subjected to either microbial enrichment, involving nuclease treatment to reduce the amount of host nucleic acids, or shotgun sequencing. We detected high proportions ofP. acnesDNA in enriched samples, particularly skin tissue-derived and other tissue samples, with the levels being higher in enriched samples than in shotgun-sequenced samples.P. acnesreads were detected in most samples analyzed, though the proportions in most shotgun-sequenced samples were low. Our results show thatP. acnescan be detected in practically all sample types when molecular methods, such as next-generation sequencing, are employed. The possibility of contamination from the patient or other sources, including laboratory reagents or environment, should therefore always be considered carefully whenP. acnesis detected in clinical samples. We advocate that detection ofP. acnesalways be accompanied by experiments validating the association between this bacterium and any clinical condition.

scholarly journals Next Generation Sequencing Analysis of Biofilms from Three Dogs with Postoperative Surgical Site Infection

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
L. M. König ◽  
R. Klopfleisch ◽  
D. Höper ◽  
A. D. Gruber
Keyword(s):  
Next Generation Sequencing ◽  
Sequencing Analysis ◽  
Next Generation ◽  
Bacterial Composition ◽  
Tissue Samples ◽  
Next Generation Sequencing Analysis ◽  
Total Dna ◽  
Chronic Wound Infections ◽  
Generation Sequencing ◽  
Formalin Fixed

The composition of biofilms in chronic wound infections of dogs is unclear. In the present study, histologically identified biofilms attached to sutures in chronically infected wounds of three dogs were examined by next generation sequencing of total DNA extracted from formalin-fixed and paraffin-embedded tissue samples. The analysis identified an inhomogeneous bacterial composition in three tissues containing biofilms. Some of the identified bacterial families such as Staphylococci and Streptococci have been found before in biofilms associated with human and canine wounds but in this study were quantitatively in the minority. The majority of the reads classified as bacterial sequences had the highest identity with sequences belonging to the Porphyromonadaceae, Deinococcaceae, Methylococcaceae, Nocardiaceae, Alteromonadaceae, and Propionibacteriaceae and thus taxons of so far minor relevance in veterinary medicine.

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