Dataset Information

Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples.

ABSTRACT: High throughput sequencing is currently revolutionizing the genomics field and providing new approaches to the detection and characterization of microorganisms. The objective of this study was to assess the detection of influenza D virus (IDV) in bovine respiratory tract samples using two sequencing platforms (MiSeq and Nanopore (GridION)), and species-specific qPCR. An IDV-specific qPCR was performed on 232 samples (116 nasal swabs and 116 tracheal washes) that had been previously subject to virome sequencing using MiSeq. Nanopore sequencing was performed on 19 samples positive for IDV by either MiSeq or qPCR. Nanopore sequence data was analyzed by two bioinformatics methods: What's In My Pot (WIMP, on the EPI2ME platform), and an in-house developed analysis pipeline. The agreement of IDV detection between qPCR and MiSeq was 82.3%, between qPCR and Nanopore was 57.9% (in-house) and 84.2% (WIMP), and between MiSeq and Nanopore was 89.5% (in-house) and 73.7% (WIMP). IDV was detected by MiSeq in 14 of 17 IDV qPCR-positive samples with Cq (cycle quantification) values below 31, despite multiplexing 50 samples for sequencing. When qPCR was regarded as the gold standard, the sensitivity and specificity of MiSeq sequence detection were 28.3% and 98.9%, respectively. We conclude that both MiSeq and Nanopore sequencing are capable of detecting IDV in clinical specimens with a range of Cq values. Sensitivity may be further improved by optimizing sequence data analysis, improving virus enrichment, or reducing the degree of multiplexing.

SUBMITTER: Zhang M

PROVIDER: S-EPMC7472010 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples.

Zhang Maodong M Huang Yanyun Y Godson Dale L DL Fernando Champika C Alexander Trevor W TW Hill Janet E JE

Viruses 20200728 8

High throughput sequencing is currently revolutionizing the genomics field and providing new approaches to the detection and characterization of microorganisms. The objective of this study was to assess the detection of influenza D virus (IDV) in bovine respiratory tract samples using two sequencing platforms (MiSeq and Nanopore (GridION)), and species-specific qPCR. An IDV-specific qPCR was performed on 232 samples (116 nasal swabs and 116 tracheal washes) that had been previously subject to vi ...[more]

PMID: 32731471

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Project description:BackgroundLower respiratory tract infections (LRTI)are known to be diagnosed late or inaccurately. This has fueled the unscrupulous use of antibiotics, as they are often used empirically and clinically, leading to antibiotic abuse and multidrug resistance in patients. Metagenomic next-generation sequencing (mNGS), now widely used in clinical studies, could be a potential intervention to revolutionize microbiology by rapidly identifying unknown species.MethodsThis review and meta-analysis were conducted on eligible studies with respect to metagenomic sequencing on clinical LRTI diagnostics up to May 01, 2022. QUADAS-2 was employed to assess the methodological bias as well as applicability. After that, a meta-analysis was conducted to analyze the accuracy of mNGS, compared with the composite reference standard (CRS), among the enrolled studies.ResultsThis work collected 1248 samples in 13/21 qualified articles to factor in the accuracy of the diagnostic test. Typically, methods like molecular testing, culture, composite measures, and clinical decision-making were adopted as the reference criteria. With regard to Bronchoalveolar Lavage Samples, their sensitivity was 89% (82-93%) while their specificity was 90% (66-98%), with obvious heterogeneities in these two factors as demonstrated by different studies. The summary receiver operating characteristic (SROC) curve was plotted for mNGS as a function of LRTI, and the area under the curve (AUC) was 0.94. A Funnel plot with a p-value greater than 0.05 indicated the absence of publication bias. Positive and negative likelihood ratios (PLR and NLR) were >10 and > 0.1, respectively. In this pre-test probability-post-probability-likelihood ratio relationship graph, the values were Prior prob (%) = 20, Post-prob-Pos (%) = 77 and Post-prob-Neg (%) = 4.ConclusionThe AUC value of SROC suggested a high accuracy of mNGS in diagnosis, with no publication bias and high reliability. The application of mNGS exhibits notable diagnostic efficacy in discerning pathogens present in bronchoalveolar lavage fluid (BALF) among patients afflicted with LRTI. However, mNGS is more meaningful for the definitive diagnosis of the disease rather than the exclusion of the disease. This post-test probability is significantly higher than the pre-test probability.

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Dataset Information

Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples.

Publications

Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples.

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