scholarly journals Metagenomic next-generation sequencing reveals Miamiensis avidus (Ciliophora: Scuticociliatida) in the 2017 epizootic of leopard sharks (Triakis semifasciata) in San Francisco Bay, California

2018 ◽  
Author(s):  
Hanna Retallack ◽  
Mark S. Okihiro ◽  
Elliot Britton ◽  
Sean Van Sommeran ◽  
Joseph L. DeRisi
Keyword(s):  
Next Generation Sequencing ◽  
San Francisco ◽  
San Francisco Bay ◽  
Metagenomic Sequencing ◽  
Next Generation ◽  
Ciliated Protozoan ◽  
Miamiensis Avidus ◽  
Triakis Semifasciata ◽  
Leopard Sharks ◽  
Generation Sequencing

ABSTRACTDuring March to August of 2017, hundreds of leopard sharks (Triakis semifasciata) stranded and died on the shores of San Francisco Bay, California, USA. Similar mass stranding events occurred in 1967 and 2011, yet analysis of these epizootics was incomplete and no etiology was confirmed. Our investigation of the most recent epizootic revealed severe meningoencephalitis in stranded sharks, raising suspicion for infection. On this basis, we pursued a strategy for unbiased pathogen detection using metagenomic next-generation sequencing followed by orthogonal validation and further screening. We show that the ciliated protozoan pathogen, Miamiensis avidus, was present in the central nervous system of leopard (n=12) and other shark species (n=2) that stranded in San Francisco Bay, but absent in leopard sharks caught elsewhere. Whereas this protozoan has previously been implicated in devastating outbreaks in teleost marine fish, our findings represent the first report of a ciliated protozoan infection in wild elasmobranchs. This discovery highlights the benefits of adopting unbiased metagenomic sequencing in the study of wildlife health and disease.

scholarly journals Metagenomic Next-Generation Sequencing for Pathogen Detection and Transcriptomic Analysis in Pediatric Central Nervous System Infections

2021 ◽  
Author(s):  
Nanda Ramchandar ◽  
Nicole G Coufal ◽  
Anna S Warden ◽  
Benjamin Briggs ◽  
Toni Schwarz ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Next Generation Sequencing ◽  
Usual Care ◽  
Transcriptomic Analysis ◽  
Cns Infection ◽  
Metagenomic Sequencing ◽  
Next Generation ◽  
Cns Infections ◽  
Generation Sequencing

Abstract Background Pediatric central nervous system (CNS) infections are potentially life-threatening and may incur significant morbidity. Identifying a pathogen is important, both in terms of guiding therapeutic management, but also in characterizing prognosis. Usual care testing by culture and PCR is often unable to identify a pathogen. We examined the systematic application of metagenomic next-generation sequencing (mNGS) for detecting organisms and transcriptomic analysis of cerebrospinal fluid (CSF) in children with CNS infections. Methods We conducted a prospective multi-site study that aimed to enroll all children with a CSF pleocytosis and suspected CNS infection admitted to one of three tertiary pediatric hospitals during the study timeframe. After usual care testing had been performed, the remaining CSF was sent for mNGS and transcriptomic analysis. Results We screened 221 and enrolled 70 subjects over a 12-month recruitment period. A putative organism was isolated from CSF in 25 (35.7%) subjects by any diagnostic modality. mNGS of the CSF samples identified a pathogen in 20 (28.6%) subjects, which were also all identified by usual care testing. The median time to result was 38 hours. Conclusion Metagenomic sequencing of CSF has the potential to rapidly identify pathogens in children with CNS infections.

scholarly journals Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

2016 ◽  
Vol 54 (4) ◽  
pp. 919-927 ◽  
Author(s):  
Mohammad R. Hasan ◽  
Arun Rawat ◽  
Patrick Tang ◽  
Puthen V. Jithesh ◽  
Eva Thomas ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pathogen Detection ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Next Generation ◽  
Simple Method ◽  
Clinical Specimens ◽  
Human Dna ◽  
Pathogen Dna ◽  
Generation Sequencing

Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%;P< 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P< 0.05) and improved the sensitivity of pathogen detection (P< 0.01), with a 20- to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples.

scholarly journals Detection of Anaplasma Phagocytophilum in Horses With Suspected Tick-Borne Disease in Northeastern United States by Metagenomic Sequencing

2021 ◽  
Vol 8 ◽  
Author(s):  
Murugan Subbiah ◽  
Nagaraja Thirumalapura ◽  
David Thompson ◽  
Suresh V. Kuchipudi ◽  
Bhushan Jayarao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Anaplasma Phagocytophilum ◽  
Metagenomic Analysis ◽  
Metagenomic Sequencing ◽  
Next Generation ◽  
Emerging Pathogens ◽  
Metagenomic Sequence ◽  
Blood Samples ◽  
Tick Borne Disease ◽  
Generation Sequencing

Metagenomic sequencing of clinical diagnostic specimens has a potential for unbiased detection of infectious agents, diagnosis of polymicrobial infections and discovery of emerging pathogens. Herein, next generation sequencing (NGS)-based metagenomic approach was used to investigate the cause of illness in a subset of horses recruited for a tick-borne disease surveillance study during 2017–2019. Blood samples collected from 10 horses with suspected tick-borne infection and five apparently healthy horses were subjected to metagenomic analysis. Total genomic DNA extracted from the blood samples were enriched for microbial DNA and subjected to shotgun next generation sequencing using Nextera DNA Flex library preparation kit and V2 chemistry sequencing kit on the Illumina MiSeq sequencing platform. Overall, 0.4–0.6 million reads per sample were analyzed using Kraken metagenomic sequence classification program. The taxonomic classification of the reads indicated that bacterial genomes were overrepresented (0.5 to 1%) among the total microbial reads. Most of the bacterial reads (~91%) belonged to phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria and Tenericutes in both groups. Importantly, 10–42.5% of Alphaproteobacterial reads in 5 of 10 animals with suspected tick-borne infection were identified as Anaplasma phagocytophilum. Of the 5 animals positive for A. phagocytophilum sequence reads, four animals tested A. phagocytophilum positive by PCR. Two animals with suspected tick-borne infection and A. phagocytophilum positive by PCR were found negative for any tick-borne microbial reads by metagenomic analysis. The present study demonstrates the usefulness of the NGS-based metagenomic analysis approach for the detection of blood-borne microbes.

scholarly journals A Retrospective Paired Comparison Between Untargeted Next Generation Sequencing and Conventional Microbiology Tests With Wisely Chosen Metagenomic Sequencing Positive Criteria

2021 ◽  
Vol 8 ◽  
Author(s):  
Hanyu Qin ◽  
Jinmin Peng ◽  
Ling Liu ◽  
Jing Wu ◽  
Lingai Pan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Fungal Infections ◽  
Rna Virus ◽  
Paired Comparison ◽  
Final Analysis ◽  
Metagenomic Sequencing ◽  
Next Generation ◽  
Dna Virus ◽  
Virus Identification ◽  
Generation Sequencing

Objectives: To evaluate the performance of metagenomic next generation sequencing (mNGS) using adequate criteria for the detection of pathogens in lower respiratory tract (LRT) samples with a paired comparison to conventional microbiology tests (CMT).Methods: One hundred sixty-seven patients were reviewed from four different intensive care units (ICUs) in mainland China during 2018 with both mNGS and CMT results of LRT samples available. The reads per million ratio (RPMsample/RPMnon−template−control ratio) and standardized strictly mapped reads number (SDSMRN) were the two criteria chosen for identifying positive pathogens reported from mNGS. A McNemar test was used for a paired comparison analysis between mNGS and CMT.Results: One hundred forty-nine cases were counted into the final analysis. The RPMsample/RPMNTC ratio criterion performed better with a higher accuracy for bacteria, fungi, and virus than SDSMRN criterion [bacteria (RPMsample/RPMNTC ratio vs. SDSMRN), 65.1 vs. 55.7%; fungi, 75.8 vs. 71.1%; DNA virus, 86.3 vs. 74.5%; RNA virus, 90.9 vs. 81.8%]. The mNGS was also superior in bacteria detection only if an SDSMRN ≥3 was used as a positive criterion with a paired comparison to culture [SDSMRN positive, 92/149 (61.7%); culture positive, 54/149 (36.2%); p &lt; 0.001]; however, it was outperformed with significantly more fungi and DNA virus identification when choosing both criteria for positive outliers [fungi (RPMsample/RPMNTC ratio vs. SDSMRN vs. culture), 23.5 vs. 29.5 vs. 8.7%, p &lt; 0.001; DNA virus (RPMsample/RPMNTC ratio vs. SDSMRN vs. PCR), 14.1 vs. 20.8 vs. 11.8%, p &lt; 0.05].Conclusions: Metagenomic next generation sequencing may contribute to revealing the LRT infection etiology in hospitalized groups of potential fungal infections and in situations with less access to the multiplex PCR of LRT samples from the laboratory by choosing a wise criterion like the RPMsample/RPMNTC ratio.

scholarly journals Comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood

BMC Genomics ◽  
2014 ◽  
Vol 15 (1) ◽  
pp. 96 ◽  
Author(s):  
Kenneth G Frey ◽  
Jesus Herrera-Galeano ◽  
Cassie L Redden ◽  
Truong V Luu ◽  
Stephanie L Servetas ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Metagenomic Sequencing ◽  
Next Generation ◽  
Sequencing Platforms ◽  
Generation Sequencing

Detection of Neisseria gonorrhoeae from Joint Aspirate by Metagenomic Sequencing in Disseminated Gonococcal Infection

2020 ◽  
Author(s):  
Betial Asmerom ◽  
Ian Drobish ◽  
Britanny Winckler ◽  
Leslie Chiang ◽  
Lauge Farnaes ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Neisseria Gonorrhoeae ◽  
Diagnostic Tests ◽  
Metagenomic Sequencing ◽  
Next Generation ◽  
Significant Morbidity ◽  
Gonococcal Infection ◽  
Culture Negative ◽  
Generation Sequencing ◽  
Limited Sensitivity

Abstract Disseminated gonococcal infection (DGI) often manifests as gonococcal arthritis and may carry significant morbidity. However, diagnosis remains elusive due to limited sensitivity of available diagnostic tests. We used metagenomic next-generation sequencing to detect Neisseria gonorrhoeae from culture-negative joint aspirates of 2 patients with clinically diagnosed DGI.

scholarly journals Next-generation sequencing in the diagnosis of viral encephalitis: sensitivity and clinical limitations

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Karol Perlejewski ◽  
Iwona Bukowska-Ośko ◽  
Małgorzata Rydzanicz ◽  
Agnieszka Pawełczyk ◽  
Kamila Caraballo Cortѐs ◽  
...  
Keyword(s):  
Viral Load ◽  
Next Generation Sequencing ◽  
Viral Encephalitis ◽  
Etiologic Factor ◽  
Metagenomic Sequencing ◽  
Next Generation ◽  
Median Viral Load ◽  
Sensitivity Testing ◽  
Wide Range ◽  
Generation Sequencing

Abstract Identification of pathogens causing viral encephalitis remains challenging, and in over 50% of cases the etiologic factor remains undetermined. Next-generation sequencing (NGS) based metagenomics has been successfully used to detect novel and rare infections, but its value for routine diagnosis of encephalitis remains unclear. The aim of the present study was to determine the sensitivity of shotgun metagenomic sequencing protocols, which include preamplification, and testing it against cerebrospinal fluid (CSF) samples from encephalitis patients. For sensitivity testing HIV and HBV positive sera were serially diluted in CSF from an uninfected patient. NGS repeatedly detected HIV and HBV sequences present at concentrations from 105 to 102 and from 105 to 10 viral copies/reaction, respectively. However, when the same protocols were applied to RT-PCR/PCR positive CSF samples from 6 patients with enteroviral encephalitis (median viral load 47 copies/ml) and 15 patients with HSV, CMV or VZV encephalitis (median viral load 148 copies/ml), only 7 (28.6%) were identified as positive. In conclusions, while NGS has the advantage of being able to identify a wide range of potential pathogens it seems to be less sensitive compared to the standard amplification-based assays in the diagnosis of encephalitis, where low viral loads are common.

Whole metagenomic sequencing reveals different compositions of pathogenic bacteria in patients with appendicitis:Bacterial culture is not suitable as the only therapeutic guidance

2020 ◽  
Author(s):  
Jie Yuan ◽  
Enming Qiu ◽  
Shuai Han ◽  
Zhou Li
Keyword(s):  
Next Generation Sequencing ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Bacterial Culture ◽  
Treatment Guidelines ◽  
Case Series ◽  
Sequencing Analysis ◽  
Metagenomic Sequencing ◽  
Next Generation ◽  
Generation Sequencing

Abstract Background Appendicitis is the result of a combination of factors, including bacterial infection, anatomy, blood supply, and intestinal contents. Previous diagnosis and treatment guidelines have suggested that bacteria are associated with the severity of appendicitis, and the use of postoperative antibiotics should be guided according to the results of intraoperative sample culture. However, this approach has many limitations. Therefore, in the present study, the relationship between pathogenic bacteria and appendicitis was assessed. Result We conducted a nonconsecutive case series analysis from January to July 2017. Nineteen patients were divided into two groups according to their intraoperative gross pathology and postoperative histological and bacterial culture results. During appendectomy, the diseased appendices were collected, and whole metagenomic sequencing was performed to identify the pathogenic bacteria in the specimens. We identified 361 species in the appendix samples. Six species in the appendix samples had relative abundances > 5%. No significant differences were observed in the bacterial composition of the three assayed groups. In particular, according to the grouping of culture results, the sequencing analysis results were completely different from those of the culture-based method. Conclusion Bacterial culture results are not suitable for exclusively guiding the use of antimicrobial agents after appendicitis. Next-generation sequencing has numerous advantages, such as in precisely characterizing the profiles of microbiota and their antibiotic resistance in appendicitis patients. Based on the above results, we propose that a combination of bacterial culture and next-generation sequencing should be used to improve the efficacy of guiding antibiotic therapy.

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