Section 5. Obstacles to Overcome in the Nearest Future (from DOI: 10.3390/v12020211)
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 5. Obstacles to Overcome in the Nearest Future
The risk of contamination and the issues with sensitivity are important to sequencing viral nucleic acids, because they cause false positive and false negative results, respectively. High-sensitivity sequencing (be it metagenomic, PCR-based or probe-enriched) allows for detection of minimal quantities of contaminating viral nucleic acids. For example, murine leukemia virus (MLV) and Parvovirus-like sequences are only two kinds of the vast range of common contaminants found even in commercial laboratory supplies.
Like with other highly sensitive technologies, only robust validated protocols and their strict implementation can effectively reduce the probability of contamination. This principle has been observed in action for quite a long time in paleogenetics, although methods in this field might prove somewhat exorbitant in everyday NGS research with samples not as valuable and unique. Nevertheless, some practices common in this field surely will find application in clinical NGS, when human wellbeing and life are at stake, compared to an abstract scientific interest of pure research.
It is also notable that detection of viral nucleic acids does not necessarily imply an ongoing infection, and therefore, positive results should be validated using supplementary methods targeted at pathogens suggested by the NGS tests. For instance, in the case of idiopathic encephalitis, positive results of NGS can be corroborated either using immunohistochemical assay, electron microscopy or microbiological methods, i.e., cultivation. At any rate, setting validation thresholds and developing a method for NGS standardization is a significant challenge that has to be addressed prior to introducing NGS into clinical routine.
Undoubtedly, standardization of NGS-based methods, including data processing, will lay a foundation for their widespread integration into clinical routine. Existing algorithms for analyzing sequence output, given user-friendly graphical interfaces, should become easy to handle even without expert knowledge of Linux commands. Another important step is the creation of reliable, comprehensive drug-resistance databases that will enable detection of clinically significant strains required for the special treatment. Such databases are already available for HIV, HBV and HCV.
Proper quality control is another problem. It is difficult to set a valid standard when the method is aimed at discovering new types of organisms. To confirm the NGS data, the pathogen in question has to be extracted, cultured as a pure strain and genetically characterized. Perhaps, the samples subject to NGS assays should be analyzed at several facilities to confirm the reproducibility of the results. The use of different sequencing platforms would ensure even better reproducibility and help prevent biases and typical mistakes that various sequencing technologies are prone to, while also excluding the human factor.
Despite multiple reasons clearly pointing to the necessity of NGS-based tests in virology, most importantly WGS, it is crucial to convince clinical laboratories that it is absolutely worth the effort. For this, clinicians should receive indisputable evidence of the advantages of this approach for the patient, institution budget and long-term prospects for the healthcare system. As for the laboratory staff, the workflow should be clearly structured, time-efficient, scalable and, preferably, automatable, rendering WGS a competitive approach with a highly convenient, reliable and useful outcome.
