Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid

Miller, Steve; Naccache, Samia N.; Samayoa, Erik; Messacar, Kevin; Arevalo, Shaun; Federman, Scot; Stryke, Doug; Pham, Elizabeth; Fung, Becky; Bolosky, William J.; Ingebrigtsen, Danielle; Lorizio, Walter; Paff, Sandra M.; Leake, John A. D.; Pesano, Rick L.; DeBiasi, Roberta L.; Dominguez, Samuel R.; Chiu, Charles Y.

doi:10.1101/gr.238170.118

Cited by 341 publications

(252 citation statements)

References 43 publications

(48 reference statements)

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“…In our test set, 8% of samples showed insufficient sensitivity for Hazara virus; however, half of these contained a high titer of influenza virus, so only 4% were true sensitivity failures. This figure is in line with the reported 6% failure rate due to high background for RNA virus detection from a clinically validated metagenomic sequencing assay for pathogen detection in cerebrospinal fluid (50).…”

Section: Discussionsupporting

confidence: 89%

“…Further work is needed to investigate the sensitivity and specificity of our protocol for a wider array of respiratory sample types (also including bronchoalveolar lavage fluid, sputum, and saliva), which may contain different degrees of contaminating bacterial and/or human reads. Loss of assay sensitivity due to the presence of high-level background DNA from either the host or bacterial origin is a fundamental issue for metagenomic approaches, even in cell-free sample types such as cerebrospinal fluid (50). This challenge is exacerbated in throat swabs, as seen in our data.…”

Section: Discussionmentioning

confidence: 74%

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Metagenomic Nanopore Sequencing of Influenza Virus Direct from Clinical Respiratory Samples

et al. 2019

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Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential for a diagnostic test for influenza virus which also provides insights on transmission, evolution, and drug resistance and simultaneously detects other viruses. We therefore set out to apply the Oxford Nanopore Technologies sequencing method to metagenomic sequencing of respiratory samples. We generated influenza virus reads down to a limit of detection of 10 2 to 10 3 genome copies/ml in pooled samples, observing a strong relationship between the viral titer and the proportion of influenza virus reads (P ϭ 4.7 ϫ 10 Ϫ5 ). Applying our methods to clinical throat swabs, we generated influenza virus reads for 27/27 samples with mid-to-high viral titers (cycle threshold [C T ] values, Ͻ30) and 6/13 samples with low viral titers (C T values, 30 to 40). No false-positive reads were generated from 10 influenza virus-negative samples. Thus, Nanopore sequencing operated with 83% sensitivity (95% confidence interval [CI], 67 to 93%) and 100% specificity (95% CI, 69 to 100%) compared to the current diagnostic standard. Coverage of full-length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. However, at high influenza virus titers, we were able to reconstruct Ͼ99% complete sequences for all eight gene segments. We also detected a human coronavirus coinfection in one clinical sample. While further optimization is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza virus and other respiratory viruses.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 74%

Metagenomic Nanopore Sequencing of Influenza Virus Direct from Clinical Respiratory Samples

et al. 2019

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“…Blood and CSF bacterial cultures, as well as Rickettsia serology, returned negative. Metagenomic next‐generation sequencing testing of CSF (performed subsequently as a research test) using a clinically validated assay did not detect any viruses (RNA and DNA), bacteria, fungi, or parasites above preestablished reporting thresholds . His acute kidney injury improved rapidly with fluid administration.…”

Section: Patient Storymentioning

confidence: 98%

Severe Epididymo-Orchitis and Encephalitis Complicating Anti-PD-1 Therapy

et al. 2019

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Background Immune checkpoint inhibitors such as pembrolizumab and nivolumab have emerged as active treatment options for patients with many cancers, including metastatic melanoma, but can also cause symptomatic or life‐threatening immune‐related adverse events, including encephalitis. Epididymitis and orchitis are rare complications of these therapies. Case Presentation We describe herein a patient with metastatic melanoma who developed epididymo‐orchitis followed by encephalitis while receiving pembrolizumab. The patient developed testicular pain and fever after his third dose of pembrolizumab; ultrasound evaluation demonstrated bilateral epididymo‐orchitis. He then developed headaches, fever, and altered mental status over the next week and was admitted to the hospital. Lumbar puncture revealed inflammatory changes consistent with meningoencephalitis; he did not improve with broad‐spectrum antibiotics, and an extensive workup for infectious etiologies, including cerebrospinal fluid testing using a clinical metagenomic next‐generation sequencing assay, was negative. He received high‐dose steroids for suspected autoimmune encephalitis, and both his orchitis and meningoencephalitis improved rapidly after one dose. He fully recovered after a 5‐week taper of oral steroids. Discussion Here, we report a case of epididymo‐orchitis complicating immune checkpoint inhibitor therapy. This patient subsequently developed severe encephalitis but rapidly improved with steroids. Clinicians should be aware of rare complications of these agents. Key Points Epididymo‐orchitis is a rare and potentially life‐threatening complication of anti‐programmed death protein 1 (anti‐PD‐1) therapy. For patients on anti‐PD‐1 therapy who develop either epididymo‐orchitis or epididymitis without clear infectious cause, immune‐related adverse events should be considered in the differential diagnosis. If severe, epididymo‐orchitis related to anti‐PD‐1 therapy may be treated with high‐dose corticosteroids.

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“…The barcoded library then directly proceeds into a library recovery step. 5uL of the library is added to an amplification reaction mix (10uL Phusion 5X Buffer (ThermoFisher Scientific, Waltham, USA), 2.5uL of 10uM forward and reverse general primers, 1uL of 12.5mM dNTPs, 0.5uL Phusion enzyme (ThermoFisher Scientific, Waltham, USA), 31uL nuclease-free water) for 14 cycles, with cycling parameters as previously described (22). The amplicons were again purified using 0.9X volume of AMPure XP beads.…”

Section: Whole Genome Sequencing Of Sars-cov2mentioning

confidence: 99%

A Genomic Survey of SARS-CoV-2 Reveals Multiple Introductions into Northern California without a Predominant Lineage

Deng

Federman

et al. 2020

Preprint

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The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has spread globally, resulting in >300,000 reported cases worldwide as of March 21st, 2020. Here we investigate the genetic diversity and genomic epidemiology of SARS-CoV-2 in Northern California using samples from returning travelers, cruise ship passengers, and cases of community transmission with unclear infection sources. Virus genomes were sampled from 29 patients diagnosed with COVID-19 infection from Feb 3rd through Mar 15th. Phylogenetic analyses revealed at least 8 different SARS-CoV-2 lineages, suggesting multiple independent introductions of the virus into the state. Virus genomes from passengers on two consecutive excursions of the Grand Princess cruise ship clustered with those from an established epidemic in Washington State, including the WA1 genome representing the first reported case in the United States on January 19th. We also detected evidence for presumptive transmission of SARS-CoV-2 lineages from one community to another. These findings suggest that cryptic transmission of SARS-CoV-2 in Northern California to date is characterized by multiple transmission chains that originate via distinct introductions from international and interstate travel, rather than widespread community transmission of a single predominant lineage. Rapid testing and contact tracing, social distancing, and travel restrictions are measures that will help to slow SARS-CoV-2 spread in California and other regions of the USA.

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Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid

Cited by 341 publications

References 43 publications

Metagenomic Nanopore Sequencing of Influenza Virus Direct from Clinical Respiratory Samples

Metagenomic Nanopore Sequencing of Influenza Virus Direct from Clinical Respiratory Samples

Severe Epididymo-Orchitis and Encephalitis Complicating Anti-PD-1 Therapy

A Genomic Survey of SARS-CoV-2 Reveals Multiple Introductions into Northern California without a Predominant Lineage

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