Rapid Metagenomic Diagnostics for Suspected Outbreak of Severe Pneumonia

Fischer, Nicole; Rohde, Holger; Indenbirken, Daniela; Guenther, Thomas; Reumann, Kerstin; Lütgehetmann, Marc; Meyer, Thomas; Kluge, Stefan; Aepfelbacher, Martin; Alawi, Malik; Grundhoff, Adam

doi:10.3201/eid2006.131526

Cited by 59 publications

(71 citation statements)

References 8 publications

(8 reference statements)

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“…Screening for HVs and genomic characterizations were done using broadly reactive and highly sensitive nested reverse transcription-PCR (RT-PCR) assays as described previously (8). Illumina NGS was done on a HiSeq2500 as described previously (15). Genome ends were determined following a 5=/3=-rapid amplification of cDNA ends (RACE) strategy using a 5=/3=-RACE kit (Roche, Penzberg, Germany).…”

Section: Methodsmentioning

confidence: 99%

Highly Divergent Hepaciviruses from African Cattle

Corman

Grundhoff

Baechlein

et al. 2015

J Virol

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The hepatitis C virus (HCV; genus Hepacivirus) is a highly relevant human pathogen. Unique hepaciviruses (HV) were discovered recently in animal hosts. The direct ancestor of HCV has not been found, but the genetically most closely related animal HVs exist in horses. To investigate whether other peridomestic animals also carry HVs, we analyzed sera from Ghanaian cattle for HVs by reverse transcription-PCR (RT-PCR). Nine of 106 specimens from different sampling sites contained HV RNA (8.5%) at median viral loads of 1.6 ؋ 10 5 copies/ml. Infection seemed unrelated to cattle age and gender. Near-full-genome sequencing of five representative viruses confirmed taxonomic classifications. Cattle HVs formed two distinct phylogenetic lineages that differed by up to 17.7% on the nucleotide level in the polyprotein-encoding region, suggesting cocirculation of different virus subtypes. A conserved microRNA122-binding site in the 5= internal ribosomal entry site suggested liver tropism of cattle HVs. Phylogenetic analyses suggested the circulation of HVs in cattle for several centuries. Cattle HVs were genetically highly divergent from all other HVs, including HCV. HVs from genetically related equine and bovine hosts were not monophyletic, corroborating host shifts during the evolution of the genus Hepacivirus. Similar to equine HVs, the genetic diversity of cattle HVs was low compared to that of HCV genotypes. This suggests an influence of the human-modified ecology of peridomestic animals on virus diversity. Further studies should investigate the occurrence of cattle HVs in other geographic areas and breeds, virus pathogenicity in cattle, and the potential exposure of human risk groups, such as farmers, butchers, and abattoir workers. IMPORTANCEHCV (genus Hepacivirus) is a major human pathogen, causing liver failure and cancer. Unique hepaciviruses (HVs) were discovered over the last few years in animals, but the direct ancestor of HCV has not been found. The animal HV most closely related to HCV so far originated from horses, suggesting that other livestock animals also harbor HVs. Therefore, we investigated African cattle and discovered previously unknown HVs at high prevalence and viral loads. Because of the agricultural importance of cattle, it may be relevant to investigate HV pathogenicity. The frequent exposure of humans to cattle also may warrant investigations of the zoonotic potential of these viruses. Evolutionary analyses suggested that cattle HVs have existed for centuries. Despite the genetic relatedness of their animal hosts, HVs from cattle and horses were not phylogenetically related, corroborating frequent host shifts during the evolution of the genus Hepacivirus.

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Section: Methodsmentioning

confidence: 99%

Highly Divergent Hepaciviruses from African Cattle

Corman

Grundhoff

Baechlein

et al. 2015

J Virol

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“…La metagenómica es independiente del cultivo, lo que la hace un técnica muy atractiva para identificar patógenos virales o bacterianos que no crecen fácilmente en el laboratorio 25 . Recientemente se utilizó la metagenómica en un hospital en Alemania para identificar el agente causal de neumonía grave en pacientes que dieron negativos a todos los patógenos que comúnmente se asocian a neumonía 26 . En este estudio, el ADN fue purificado de muestras de lavado broncoalveolar, el que fue posteriormente secuenciado con el MiSeq TM de Illumina; en un total de 50 h (incluyendo los análisis bio-informáticos), basándose en los datos obtenidos, los investigadores fueron capaces de identificar a Chlamydophila psittacii como agente causal de las neumonías por lo cual los médicos a cargo pudieron modificar el tratamiento, lo que se tradujo en la recuperación de uno de los pacientes 26 .…”

Section: Aplicaciones De Metagenómica En El Diagnóstico De Enfermedadunclassified

“…Recientemente se utilizó la metagenómica en un hospital en Alemania para identificar el agente causal de neumonía grave en pacientes que dieron negativos a todos los patógenos que comúnmente se asocian a neumonía 26 . En este estudio, el ADN fue purificado de muestras de lavado broncoalveolar, el que fue posteriormente secuenciado con el MiSeq TM de Illumina; en un total de 50 h (incluyendo los análisis bio-informáticos), basándose en los datos obtenidos, los investigadores fueron capaces de identificar a Chlamydophila psittacii como agente causal de las neumonías por lo cual los médicos a cargo pudieron modificar el tratamiento, lo que se tradujo en la recuperación de uno de los pacientes 26 . De la misma manera, la metagenómica se ha utilizado para diagnosticar la presencia de Campylobacter jejuni 27 , norovirus 17 , inclusive la presencia de parásitos muy extraños (Kudoa septempunctata) 28 , en pacientes con sintomatología de diarrea.…”

Section: Aplicaciones De Metagenómica En El Diagnóstico De Enfermedadunclassified

Infectología en la era de la genómica

Switt

Toledo

2015

Rev. chil. infectol.

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“…Further, in a proof-of-principle study, the same process was used to identify and characterize pathogenic mycobacteria in modern sputum samples (48)(49)(50). There have been several other recent proof-of-principle studies that demonstrate the utility of this diagnostic approach (13,15,51,52).…”

Section: The Role Of Clinicogenomics In Public Health Microbiologymentioning

confidence: 99%

“…While a number of studies have proven the technique's ability to identify diverse pathogens directly from clinical material and, in some instances, in a clinically actionable time frame (11)(12)(13)(14)(15)(16), substantially more empirical data will have to be collected to address a number of open questions. For example, given that shotgun sequencing usually only recovers snippets of genomic information rather than whole genomes, what are the requirements to call the presence of a specific infectious agent to a given taxonomic level?…”

Section: Unbiased Infectious Disease Diagnosticsmentioning

confidence: 99%

Role of Clinicogenomics in Infectious Disease Diagnostics and Public Health Microbiology

et al. 2016

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Clinicogenomics is the exploitation of genome sequence data for diagnostic, therapeutic, and public health purposes. Central to this field is the high-throughput DNA sequencing of genomes and metagenomes. The role of clinicogenomics in infectious disease diagnostics and public health microbiology was the topic of discussion during a recent symposium (session 161) presented at the 115th general meeting of the American Society for Microbiology that was held in New Orleans, LA. What follows is a collection of the most salient and promising aspects from each presentation at the symposium. The explosion of microbiome research is driven by highthroughput DNA sequencing, so-called next-generation sequencing (NGS), technologies that allow the genomic content of entire microbial communities (bacterial, viral, and eukaryotic organisms) to be described. Although much of this work is aimed at describing the structure of "commensal" communities, the methodology works equally well to identify pathogens in clinical samples. The key concept in using NGS methodology is that detection of microbes is independent of culture and is not limited to targets used for PCR assays. Rather, it is a process of generating large-scale sequence data sets that adequately sample a specimen for microbial content and then of applying computational methods to resolve the sequences into individual species, genes, pathways, or other features.Most microbiome analyses have focused on describing bacterial content, and this is usually performed by sequencing the 16S rRNA gene. PCR primers with degenerative sequences are used to amplify all or part of the 16S rRNA gene from a broad range of species in the sample. The mix of amplicons generated from different organisms in the community is then sequenced, and the abundance of each species is determined by the number of sequences found for its respective 16S rRNA gene. Although this is useful for defining communities, it also affords the identification of pathogens with unique 16S rRNA sequences.The sensitivity and specificity of this method are determined in large part by the NGS technology. Before NGS, the full-length 16S rRNA gene was sequenced with high-quality, 700-base-long reads of Sanger, or chain termination, sequencing (sometimes referred to as "first-generation" sequencing technology). This was laborious and expensive, and deep sampling was not possible. When NGS became available, most work was done on the FLX sequencing instrument (a second-generation sequencing technology) from 454 Life Sciences (Roche Diagnostics, Indianapolis, IN, USA). This only permitted 400-base-long sequencing reads, and only a portion of the 16S rRNA gene was sequenced. The 16S rRNA gene has nine hypervariable regions that provide much of the specificity in species identification. With 454 sequencing, typically only three of these regions can be sequenced. Nevertheless, this allowed detection to the genus level of most taxa. This methodology can correctly identify pathogens in stool samples from patients with diarrhea comp...

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Rapid Metagenomic Diagnostics for Suspected Outbreak of Severe Pneumonia

Cited by 59 publications

References 8 publications

Highly Divergent Hepaciviruses from African Cattle

Highly Divergent Hepaciviruses from African Cattle

Infectología en la era de la genómica

Role of Clinicogenomics in Infectious Disease Diagnostics and Public Health Microbiology

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