Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
N Engl J Med ; 380(24): 2327-2340, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31189036

RESUMO

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, NCT02910037.).


Assuntos
Líquido Cefalorraquidiano/microbiologia , Encefalite/microbiologia , Genoma Microbiano , Meningite/microbiologia , Metagenômica , Adolescente , Adulto , Líquido Cefalorraquidiano/virologia , Criança , Pré-Escolar , Encefalite/diagnóstico , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Infecções/diagnóstico , Tempo de Internação , Masculino , Meningite/diagnóstico , Meningoencefalite/diagnóstico , Meningoencefalite/microbiologia , Pessoa de Meia-Idade , Mielite/diagnóstico , Mielite/microbiologia , Estudos Prospectivos , Análise de Sequência de DNA , Análise de Sequência de RNA , Adulto Jovem
2.
Genome Res ; 29(5): 831-842, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30992304

RESUMO

Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been successfully tested in proof-of-concept case studies in patients with acute illness of unknown etiology but to date has been largely confined to research settings. Here, we developed and validated a clinical mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from cerebrospinal fluid (CSF) in a licensed microbiology laboratory. A customized bioinformatics pipeline, SURPI+, was developed to rapidly analyze mNGS data, generate an automated summary of detected pathogens, and provide a graphical user interface for evaluating and interpreting results. We established quality metrics, threshold values, and limits of detection of 0.2-313 genomic copies or colony forming units per milliliter for each representative organism type. Gross hemolysis and excess host nucleic acid reduced assay sensitivity; however, spiked phages used as internal controls were reliable indicators of sensitivity loss. Diagnostic test accuracy was evaluated by blinded mNGS testing of 95 patient samples, revealing 73% sensitivity and 99% specificity compared to original clinical test results, and 81% positive percent agreement and 99% negative percent agreement after discrepancy analysis. Subsequent mNGS challenge testing of 20 positive CSF samples prospectively collected from a cohort of pediatric patients hospitalized with meningitis, encephalitis, and/or myelitis showed 92% sensitivity and 96% specificity relative to conventional microbiological testing of CSF in identifying the causative pathogen. These results demonstrate the analytic performance of a laboratory-validated mNGS assay for pan-pathogen detection, to be used clinically for diagnosis of neurological infections from CSF.


Assuntos
Encefalite/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Meningite Asséptica/diagnóstico , Metagenômica/métodos , Mielite/diagnóstico , Criança , Biologia Computacional , Encefalite/líquido cefalorraquidiano , Humanos , Meningite Asséptica/líquido cefalorraquidiano , Mielite/líquido cefalorraquidiano , Sensibilidade e Especificidade , Vírus/isolamento & purificação
3.
J Clin Microbiol ; 58(9)2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32651238

RESUMO

Analytical sensitivity for SARS-CoV-2 detection is a key performance metric for the evaluation of viral detection assays. We determined analytical limits of detection for seven SARS-CoV-2 assays using serial dilutions of pooled patient material quantified with droplet digital PCR. Limits of detection ranged from ≤10 to 74 copies/ml for commercial high-throughput laboratory analyzers (Roche Cobas, Abbott m2000, and Hologic Panther Fusion) and 167 to 511 copies/ml for sample-to-answer (DiaSorin Simplexa, GenMark ePlex) and point-of-care instruments (Abbott ID NOW). The CDC assay yielded limits of detection ranging from 85 to 499 copies/ml, depending on the extraction method and thermocycler used. These results can help to inform the assay choice for testing approaches to manage the current COVID-19 outbreak.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular , Pneumonia Viral/diagnóstico , COVID-19 , Teste para COVID-19 , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Pandemias , Pneumonia Viral/epidemiologia , RNA Viral/análise , RNA Viral/genética , SARS-CoV-2
6.
Genome Med ; 13(1): 98, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074327

RESUMO

BACKGROUND: Metagenomic next-generation sequencing (mNGS) of body fluids is an emerging approach to identify occult pathogens in undiagnosed patients. We hypothesized that metagenomic testing can be simultaneously used to detect malignant neoplasms in addition to infectious pathogens. METHODS: From two independent studies (n = 205), we used human data generated from a metagenomic sequencing pipeline to simultaneously screen for malignancies by copy number variation (CNV) detection. In the first case-control study, we analyzed body fluid samples (n = 124) from patients with a clinical diagnosis of either malignancy (positive cases, n = 65) or infection (negative controls, n = 59). In a second verification cohort, we analyzed a series of consecutive cases (n = 81) sent to cytology for malignancy workup that included malignant positives (n = 32), negatives (n = 18), or cases with an unclear gold standard (n = 31). RESULTS: The overall CNV test sensitivity across all studies was 87% (55 of 63) in patients with malignancies confirmed by conventional cytology and/or flow cytometry testing and 68% (23 of 34) in patients who were ultimately diagnosed with cancer but negative by conventional testing. Specificity was 100% (95% CI 95-100%) with no false positives detected in 77 negative controls. In one example, a patient hospitalized with an unknown pulmonary illness had non-diagnostic lung biopsies, while CNVs implicating a malignancy were detectable from bronchoalveolar fluid. CONCLUSIONS: Metagenomic sequencing of body fluids can be used to identify undetected malignant neoplasms through copy number variation detection. This study illustrates the potential clinical utility of a single metagenomic test to uncover the cause of undiagnosed acute illnesses due to cancer or infection using the same specimen.


Assuntos
Líquidos Corporais , Biópsia Líquida/métodos , Metagenoma , Metagenômica/métodos , Neoplasias/diagnóstico , Neoplasias/etiologia , Líquidos Corporais/microbiologia , Estudos de Casos e Controles , Biologia Computacional/métodos , Análise Citogenética , Gerenciamento Clínico , Suscetibilidade a Doenças , Citometria de Fluxo , Histocitoquímica , Humanos , Hibridização in Situ Fluorescente , Biópsia Líquida/normas , Metagenômica/normas , Neoplasias/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
7.
Nat Med ; 27(1): 115-124, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33169017

RESUMO

We developed a metagenomic next-generation sequencing (mNGS) test using cell-free DNA from body fluids to identify pathogens. The performance of mNGS testing of 182 body fluids from 160 patients with acute illness was evaluated using two sequencing platforms in comparison to microbiological testing using culture, 16S bacterial PCR and/or 28S-internal transcribed ribosomal gene spacer (28S-ITS) fungal PCR. Test sensitivity and specificity of detection were 79 and 91% for bacteria and 91 and 89% for fungi, respectively, by Illumina sequencing; and 75 and 81% for bacteria and 91 and 100% for fungi, respectively, by nanopore sequencing. In a case series of 12 patients with culture/PCR-negative body fluids but for whom an infectious diagnosis was ultimately established, seven (58%) were mNGS positive. Real-time computational analysis enabled pathogen identification by nanopore sequencing in a median 50-min sequencing and 6-h sample-to-answer time. Rapid mNGS testing is a promising tool for diagnosis of unknown infections from body fluids.


Assuntos
Bactérias/isolamento & purificação , Líquidos Corporais/microbiologia , Fungos/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica , Adulto , Idoso , Bactérias/genética , Ácidos Nucleicos Livres/análise , Ácidos Nucleicos Livres/genética , Feminino , Fungos/genética , Humanos , Masculino , Pessoa de Meia-Idade
8.
JAMA Neurol ; 78(11): 1355-1366, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34515766

RESUMO

Importance: Cerebrospinal fluid (CSF) cytologic testing and flow cytometry are insensitive for diagnosing neoplasms of the central nervous system (CNS). Such clinical phenotypes can mimic infectious and autoimmune causes of meningoencephalitis. Objective: To ascertain whether CSF metagenomic next-generation sequencing (mNGS) can identify aneuploidy, a hallmark of malignant neoplasms, in difficult-to-diagnose cases of CNS malignant neoplasm. Design, Setting, and Participants: Two case-control studies were performed at the University of California, San Francisco (UCSF). The first study used CSF specimens collected at the UCSF Clinical Laboratories between July 1, 2017, and December 31, 2019, and evaluated test performance in specimens from patients with a CNS malignant neoplasm (positive controls) or without (negative controls). The results were compared with those from CSF cytologic testing and/or flow cytometry. The second study evaluated patients who were enrolled in an ongoing prospective study between April 1, 2014, and July 31, 2019, with presentations that were suggestive of neuroinflammatory disease but who were ultimately diagnosed with a CNS malignant neoplasm. Cases of individuals whose tumors could have been detected earlier without additional invasive testing are discussed. Main Outcomes and Measures: The primary outcome measures were the sensitivity and specificity of aneuploidy detection by CSF mNGS. Secondary subset analyses included a comparison of CSF and tumor tissue chromosomal abnormalities and the identification of neuroimaging characteristics that were associated with test performance. Results: Across both studies, 130 participants were included (median [interquartile range] age, 57.5 [43.3-68.0] years; 72 men [55.4%]). The test performance study used 125 residual laboratory CSF specimens from 47 patients with a CNS malignant neoplasm and 56 patients with other neurological diseases. The neuroinflammatory disease study enrolled 12 patients and 17 matched control participants. The sensitivity of the CSF mNGS assay was 75% (95% CI, 63%-85%), and the specificity was 100% (95% CI, 96%-100%). Aneuploidy was detected in 64% (95% CI, 41%-83%) of the patients in the test performance study with nondiagnostic cytologic testing and/or flow cytometry, and in 55% (95% CI, 23%-83%) of patients in the neuroinflammatory disease study who were ultimately diagnosed with a CNS malignant neoplasm. Of the patients in whom aneuploidy was detected, 38 (90.5%) had multiple copy number variations with tumor fractions ranging from 31% to 49%. Conclusions and Relevance: This case-control study showed that CSF mNGS, which has low specimen volume requirements, does not require the preservation of cell integrity, and was orginally developed to diagnose neurologic infections, can also detect genetic evidence of a CNS malignant neoplasm in patients in whom CSF cytologic testing and/or flow cytometry yielded negative results with a low risk of false-positive results.


Assuntos
Biomarcadores Tumorais/líquido cefalorraquidiano , Neoplasias do Sistema Nervoso Central/líquido cefalorraquidiano , Neoplasias do Sistema Nervoso Central/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Metagenômica , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos
10.
Nat Microbiol ; 5(3): 443-454, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31932713

RESUMO

Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use.


Assuntos
Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenoma , Metagenômica/métodos , Vírus/genética , Vírus/isolamento & purificação , Vírus Chikungunya/genética , Vírus Chikungunya/isolamento & purificação , Biologia Computacional , DNA Viral/genética , Dengue/diagnóstico , Vírus da Dengue/genética , Vírus da Dengue/isolamento & purificação , Ebolavirus/genética , Ebolavirus/isolamento & purificação , Doença pelo Vírus Ebola/diagnóstico , Humanos , Reação em Cadeia da Polimerase , RNA Viral/genética , RNA Viral/isolamento & purificação , Viroses/diagnóstico , Febre Amarela/diagnóstico , Zika virus/genética , Infecção por Zika virus/diagnóstico
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA