Section 1. Introduction (from DOI: 10.3390/v12020211)

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Article	Current Trends in Diagnostics of Viral Infections of Unknown Etiology (DOI: 10.3390/v12020211)
Sections in this Publication
Section	Section 1. Introduction (from DOI: 10.3390/v12020211)
Section	Section 2. Traditional Methods of Diagnosing Infections (from DOI: 10.3390/v12020211)
Section	Section 3. Studying Viral Pathogens with High Throughput Sequencing (HTS) (from DOI: 10.3390/v12020211)
Section	Section 3.1. Metagenomic Approach (from DOI: 10.3390/v12020211)
Section	Section 3.2. Problems of Metagenomic Approach (from DOI: 10.3390/v12020211)
Section	Section 3.3. Methods for Improving Sequencing Output (from DOI: 10.3390/v12020211)
Section	Section 3.3.1. Nucleic Acids Depletion (from DOI: 10.3390/v12020211)
Section	Section 3.3.2. Hybridization-Based Enrichment (from DOI: 10.3390/v12020211)
Section	Section 3.3.3. Target Amplification (from DOI: 10.3390/v12020211)
Section	Section 3.4. Whole Viral Genome Sequencing (from DOI: 10.3390/v12020211)
Section	Section 3.5. Methods of Sequencing Data Analysis (from DOI: 10.3390/v12020211)
Section	Section 4. Long Read Sequencing (from DOI: 10.3390/v12020211)
Section	Section 5. Obstacles to Overcome in the Nearest Future (from DOI: 10.3390/v12020211)
Section	Section 6. Conclusions (from DOI: 10.3390/v12020211)
Section	Author Contributions (from DOI: 10.3390/v12020211)
Section	Funding (from DOI: 10.3390/v12020211)
Section	Conflicts of Interest (from DOI: 10.3390/v12020211)
Section	References (from DOI: 10.3390/v12020211)
Named Entities in this Section

From publication: "Current Trends in Diagnostics of Viral Infections of Unknown Etiology" published as Viruses; 2020 Feb 14 ; 12 (2); DOI: https://doi.org/10.3390/v12020211

Section 1. Introduction

As some statistical models suggest, there are over 320,000 mammalian viruses in existence, a little over 200 of which are known to infect humans, with the number increasing steadily over decades. The recent years have been marked by a rising frequency of emerging viral infections, e.g., SARS, Zika, or a more recent Coronavirus 2019-nCoV outbreak. Bearing that in mind, the range of available diagnostic tools seems disproportionate to the growing number of diseases, because they detect only known pathogens, which constitute ~0.07% of viral entities. Metaphorically, we are attempting to look at a vast sea of threats through the eye of a needle.

As of this day, a multitude of clinical tests are available for detection of viruses, e.g., FISH, ELISA (enzyme-linked immunosorbent assay), and PCR- and their numerous modifications. The latter has become a gold standard, for example, in cases of gastrointestinal and respiratory infections. Indeed, the presence of viral nucleic acids in biological media, particularly in blood plasma, while not definitive, is highly suspect of an ongoing infection, because it strongly implies viral replication. Furthermore, PCR not only enables us to detect pathogens, but also quantify them with great precision, based on the number of copies of a pathogen's DNA/RNA templates in a sample.

As effective as they are, PCR tests are sometimes prone to major pitfalls, chiefly owing to high mutability of a viral genome, which significantly perplexes primer design and requires researchers to update primer sequences continually. With the entirety of human-infecting viruses, most of which are yet to be revealed, it becomes increasingly difficult to maintain up-to-date primer panels while also ensuring the correct reaction conditions and high specificity for differentiating closely related viral species. This situation is aggravated by the low rates of correct pathogen identification.

Immunoassays have been extensively used in clinical laboratories for over 60 years now. Fast and relatively reliable screening tests for infectious diseases have been successfully introduced into healthcare practice, e.g., for HIV, HBV, HEV, HAV, HCV, and Rubella virus. Although ELISA does not quantify pathogens with great precision, it effectively detects pathogens based on their antigen structure. Useful as they are, immunoassays are also subject to biases and fundamental pitfalls, although they are often disregarded for the sake of low cost and convenience.

Problems with conventional diagnostic methods have been stated in several studies. For example, Arnold et al. found that only 62% of viral respiratory infections among children could be confidently attributed to known pathogens, mostly to human metapneumovirus (63%) and adenoviruses (45%), while almost a third of all cases remained undiagnosed. In another study by Vu et al., it was demonstrated that in 42.7% of cases of viral gastroenteritis the pathogen could not be identified with conventional methods. Kennedy et al. outline that up to 40% of viral encephalitis infections remain undiagnosed with modern clinical tests.

High throughput sequencing (HTS) represents a range of technologies based on sequencing by synthesis, which allow retrieving of multiple nucleotide sequences at once with high reliability for further pathogen identification. Thus, HTS may tackle the aforementioned diagnostic issues, adding an extra layer of data for identification and further insight into a pathogen.

In this paper, we assess modern clinical tests for viral infections and explore the ways that HTS provides detailed data about a pathogen's genome, allowing for a deeper analysis of its features, such as drug resistance and phylogeny. We also hope to demonstrate how modern clinical laboratories would benefit from adopting HTS as a routine diagnostic technique.