SUMMARY A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.
BACKGROUND Metagenomic next-generation sequencing (NGS) of cerebrospinal fluid (CSF) has the potential to identify a broad range of pathogens in a single test. METHODS In a 1-year, multicenter, prospective study, we investigated the usefulness of metagenomic NGS of CSF for the diagnosis of infectious meningitis and encephalitis in hospitalized patients. All positive tests for pathogens on metagenomic NGS were confirmed by orthogonal laboratory testing. Physician feedback was elicited by teleconferences with a clinical microbial sequencing board and by surveys. Clinical effect was evaluated by retrospective chart review. RESULTS We enrolled 204 pediatric and adult patients at eight hospitals. Patients were severely ill: 48.5% had been admitted to the intensive care unit, and the 30-day mortality among all study patients was 11.3%. A total of 58 infections of the nervous system were diagnosed in 57 patients (27.9%). Among these 58 infections, metagenomic NGS identified 13 (22%) that were not identified by clinical testing at the source hospital. Among the remaining 45 infections (78%), metagenomic NGS made concurrent diagnoses in 19. Of the 26 infections not identified by metagenomic NGS, 11 were diagnosed by serologic testing only, 7 were diagnosed from tissue samples other than CSF, and 8 were negative on metagenomic NGS owing to low titers of pathogens in CSF. A total of 8 of 13 diagnoses made solely by metagenomic NGS had a likely clinical effect, with 7 of 13 guiding treatment. CONCLUSIONS Routine microbiologic testing is often insufficient to detect all neuroinvasive pathogens. In this study, metagenomic NGS of CSF obtained from patients with meningitis or encephalitis improved diagnosis of neurologic infections and provided actionable information in some cases. (Funded by the National Institutes of Health and others; PDAID ClinicalTrials.gov number, .)
SUMMARY Background Enterovirus D68 (EV-D68) is implicated in a widespread 2014 outbreak of severe respiratory illness across the United States, and has also been sporadically reported in patients with acute flaccid myelitis (AFM). The association between EV-D68 infection and AFM remains unclear. Methods Here we report metagenomic and molecular epidemiological analyses of 25 AFM cases in California and Colorado from 2012−2014. Findings EV-D68 was detected in respiratory secretions from 7 of 11 (64%) patients comprising two temporally and geographically linked AFM clusters at the height of the 2014 outbreak, and from 12 of 25 (48%) investigated AFM cases overall. Phylogenetic analysis revealed that all AFM-associated EV-D68 sequences grouped into a single novel clade B1 strain that originally emerged in 2010. Out of six observed coding polymorphisms in the clade B1 EV-D68 polyprotein, 5 of 6 polymorphisms were shared between neuropathogenic poliovirus and/or EV-D70. One child with AFM and a sibling with only upper respiratory illness were both infected by identical EV-D68 strains, suggesting a potential role for host-specific factors in differential responses to EV-D68 infection. Notably, EV-D68 viremia was identified in a child experiencing acute neurologic progression of his paralytic illness. Deep metagenomic sequencing of CSF from 14 AFM cases failed to reveal evidence of an alternative infectious etiology to EV-D68. Interpretation Taken together, these findings strengthen the putative association between EV-D68 and AFM, as well as the contention that AFM is a rare yet severe clinical manifestation of EV-D68 infection in susceptible hosts.
Unbiased next-generation sequencing (NGS) approaches enable comprehensive pathogen detection in the clinical microbiology laboratory and have numerous applications for public health surveillance, outbreak investigation, and the diagnosis of infectious diseases. However, practical deployment of the technology is hindered by the bioinformatics challenge of analyzing results accurately and in a clinically relevant timeframe. Here we describe SURPI (''sequence-based ultrarapid pathogen identification''), a computational pipeline for pathogen identification from complex metagenomic NGS data generated from clinical samples, and demonstrate use of the pipeline in the analysis of 237 clinical samples comprising more than 1.1 billion sequences. Deployable on both cloud-based and standalone servers, SURPI leverages two state-of-the-art aligners for accelerated analyses, SNAP and RAPSearch, which are as accurate as existing bioinformatics tools but orders of magnitude faster in performance. In fast mode, SURPI detects viruses and bacteria by scanning data sets of 7-500 million reads in 11 min to 5 h, while in comprehensive mode, all known microorganisms are identified, followed by de novo assembly and protein homology searches for divergent viruses in 50 min to 16 h. SURPI has also directly contributed to real-time microbial diagnosis in acutely ill patients, underscoring its potential key role in the development of unbiased NGS-based clinical assays in infectious diseases that demand rapid turnaround times.
Because of the paucity of available tissue, little information has previously been available regarding the gene expression profiles of primary melanomas. To understand the molecular basis of melanoma progression, we compared the gene expression profiles of a series of nevi, primary melanomas, and melanoma metastases. We found that metastatic melanomas exhibit two dichotomous patterns of gene expression, which unexpectedly reflect gene expression differences already apparent in comparing laser-capture microdissected radial and vertical phases of a large primary melanoma. Unsupervised hierarchical clustering accurately separated nevi and primary melanomas. Multiclass significance analysis of microarrays comparing normal skin, nevi, primary melanomas, and the two types of metastatic melanoma identified 2,602 transcripts that significantly correlated with sample class. These results suggest that melanoma pathogenesis can be understood as a series of distinct molecular events. The gene expression signatures identified here provide the basis for developing new diagnostics and targeting therapies for patients with malignant melanoma.bioinformatics ͉ human ͉ microarray ͉ metastasis ͉ laser capture I n the current staging system for cutaneous melanoma, vertical thickness of the primary tumor is the dominant prognostic factor, belying the fact that a subset of thin tumors metastasize, whereas some thick tumors do not undergo metastasis (1). The original melanoma tumor progression model is characterized by an initial radial growth phase, encompassing in situ and minimally invasive tumors (2). This phase is followed by the development of vertical growth phase, which has been postulated to be the first point at which the tumor gains metastatic capacity. However, metastasis occurs, although with decreased frequency, in patients whose primary melanoma pathology exhibits only a radial growth pattern (3). Previous transcriptome analysis in melanoma defined a cluster of genes expressed in a majority of metastatic melanomas (4); however, this cluster was not related to radial or vertical growth, and precursor nevi (moles) and primary melanomas were not examined. Likewise, mutations in B-RAF occur commonly in both nevi (5) and melanoma (6), and, thus, do not distinguish progressive stages in melanoma progression. In this study, we used cDNA expression array profiling to characterize the global patterns of transcript modulation that underlie the various phases in the known tumor progression pathway of melanoma. MethodsStudy Subjects. Samples from melanoma patients and nevus volunteers presenting to the Melanoma Center were obtained with informed consent under a protocol approved by the UCSF Institutional Review Board. After biopsy, all samples were frozen in OCT freezing medium over dry ice. Subsequently, samples were processed for hematoxylin͞eosin staining and confirmed by pathologic review. Only samples comprised of Ͼ95% tumor cells were analyzed.
We report unbiased metagenomic detection of chikungunya virus (CHIKV), Ebola virus (EBOV), and hepatitis C virus (HCV) from four human blood samples by MinION nanopore sequencing coupled to a newly developed, web-based pipeline for real-time bioinformatics analysis on a computational server or laptop (MetaPORE). At titers ranging from 107–108 copies per milliliter, reads to EBOV from two patients with acute hemorrhagic fever and CHIKV from an asymptomatic blood donor were detected within 4 to 10 min of data acquisition, while lower titer HCV virus (1 × 105 copies per milliliter) was detected within 40 min. Analysis of mapped nanopore reads alone, despite an average individual error rate of 24 % (range 8–49 %), permitted identification of the correct viral strain in all four isolates, and 90 % of the genome of CHIKV was recovered with 97–99 % accuracy. Using nanopore sequencing, metagenomic detection of viral pathogens directly from clinical samples was performed within an unprecedented <6 hr sample-to-answer turnaround time, and in a timeframe amenable to actionable clinical and public health diagnostics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0220-9) contains supplementary material, which is available to authorized users.
We identified an emerging SARS-CoV-2 variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the Western United States. Named B.1.427/B.1.429 to denote its 2 lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6-24% increased transmissibility relative to wild-type circulating strains. The variant carries 3 mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0 to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation.
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