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      Recent advances in diagnostic testing for viral infections

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          Abstract

          Viral infectious diseases represent an important portion of global public health concerns with thousands of deaths annually. From serious pandemics and highly contagious infections to common influenza episodes, clinical prognosis often relies on early detection of the infectious agent. Thus, effective identification of viral pathogens is needed to help prevent transmission, set up appropriate therapy, monitor response to treatment and lead to efficient disease management and control. The aim of this review is to outline some of the recent technological advances in viral identification, including polymerase chain reaction, mass spectrometry and next-generation sequencing, and how they are applied in the diagnosis and management of viral infections. These powerful tools combine rapidity and efficiency in detecting viral pathogens and have revolutionized the field of clinical diagnostics. However, a number of drawbacks such as high cost have limited their use in many laboratories, particularly in resource-limited settings. On the contrary, the advent of microfluidic technology has attracted increasing interest from biomedical research groups, and could represent a challenging alternative to diagnose viral infections at lower cost.

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          Most cited references69

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          An integrated semiconductor device enabling non-optical genome sequencing.

          The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.
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            Performance comparison of benchtop high-throughput sequencing platforms.

            Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).
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              Multiple, linked human immunodeficiency virus type 1 drug resistance mutations in treatment-experienced patients are missed by standard genotype analysis.

              To investigate the extent to which drug resistance mutations are missed by standard genotyping methods, we analyzed the same plasma samples from 26 patients with suspected multidrug-resistant human immunodeficiency virus type 1 by using a newly developed single-genome sequencing technique and compared it to standard genotype analysis. Plasma samples were obtained from patients with prior exposure to at least two antiretroviral drug classes and who were on a failing antiretroviral regimen. Standard genotypes were obtained by reverse transcriptase (RT)-PCR and sequencing of the bulk PCR product. For single-genome sequencing, cDNA derived from plasma RNA was serially diluted to 1 copy per reaction, and a region encompassing p6, protease, and a portion of RT was amplified and sequenced. Sequences from 15 to 46 single viral genomes were obtained from each plasma sample. Drug resistance mutations identified by single-genome sequencing were not detected by standard genotype analysis in 24 of the 26 patients studied. Mutations present in less than 10% of single genomes were almost never detected in standard genotypes (1 of 86). Similarly, mutations present in 10 to 35% of single genomes were detected only 25% of the time in standard genotypes. For example, in one patient, 10 mutations identified by single-genome sequencing and conferring resistance to protease inhibitors (PIs), nucleoside analog reverse transcriptase inhibitors, and nonnucleoside reverse transcriptase inhibitors (NNRTIs) were not detected by standard genotyping methods. Each of these mutations was present in 5 to 20% of the 20 genomes analyzed; 15% of the genomes in this sample contained linked PI mutations, none of which were present in the standard genotype. In another patient sample, 33% of genomes contained five linked NNRTI resistance mutations, none of which were detected by standard genotype analysis. These findings illustrate the inadequacy of the standard genotype for detecting low-frequency drug resistance mutations. In addition to having greater sensitivity, single-genome sequencing identifies linked mutations that confer high-level drug resistance. Such linkage cannot be detected by standard genotype analysis.
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                Author and article information

                Contributors
                Journal
                biohorizons
                Bioscience Horizons: The International Journal of Student Research
                Oxford University Press
                1754-7431
                30 November 2016
                30 November 2016
                : 9
                : hzw010
                Author notes
                [* ] Corresponding author: College of Science and Technology, Nottingham Trent University, Clifton campus NG11 8NS, Nottingham, UK. Email: soufselma@ 123456gmail.com

                Supervisor: Dr Jody Winter, College of Science and Technology, Nottingham Trent University, Clifton campus NG11 8NS, Nottingham, UK.

                Article
                hzw010
                10.1093/biohorizons/hzw010
                7189836
                2de66659-4c8e-456a-9518-592e92965394
                © The Author 2016. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

                History
                : 22 February 2016
                Page count
                Pages: 11
                Categories
                Article

                viral infections,laboratory testing,immunoassay,polymerase chain reaction,next-generation sequencing,mass spectrometry

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