Section 2. Traditional Methods of Diagnosing Infections (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 2. Traditional Methods of Diagnosing Infections
Before molecular and serological diagnostics became widely accessible, detection of pathogens primarily relied on conventional microbiological methods, e.g., cultures on growth media, in cells, and in laboratory animals with further microscopy, identification of antigenic and pathogenic characteristics and the metabolic profile.
ELISA has become one of the most widely used tests, owing to high sensitivity and specificity (both usually reported at >90%), low price per sample, and the simplicity of sample processing and result interpretation. Modern commercial kits are designed to cover a large variety of antigens, bacterial and viral, even at femto- and attomole concentrations. All these factors contribute to the popularity of ELISA, turning it into a universal tool for disease screening and prognosis. At the same time, strong limitations restrict its use. Because the test basically relies on antigen detection, cross-reactivity between heterologous antigens can lead to false positive results, like with influenza virus-specific CD8+ T cell and hepatitis C virus antigens. Other fundamental methodological flaws have been described by Hoofnagle and Wener (2009) and include the hook effect, possible presence of anti-reagent and autoantibodies and lack of concordance. It is also of note that ELISA does not provide any extra data beyond approximate viral burden and limited detection of a pathogen, rendering the identification of new viruses impossible.
The development of molecular cloning and commercial solutions for Sanger sequencing aided the identification of new pathogens. The core principle of this approach includes three steps: (1) amplification of a selected region via PCR, (2) molecular cloning of the amplicons, and (3) Sanger sequencing of the molecular clones. In some cases, hybridization is used to detect viral nucleic acids. This requires at least some knowledge of the target sequences, as it utilizes a microarray with oligonucleotides nested on its surface that are complementary to the conservative sequences in the genomes of the chosen viral species. The technique never grew to become popular in diagnostics, because (1) content of viral nucleic acids in the sample had to be high, and (2) high levels of the host's DNA and the DNA of the microbiome interfered with amplification.
A number of alternative approaches have been proposed to detect target sequences in the pathogen's genome. Among them are SISPA (sequence-independent single primer amplification) and its modifications, and VIDISCA (virus discovery based on cDNA-AFLP), which is based on the AFLP (amplified fragment length polymorphism) method that was specifically adapted for studying viromes.
In spite of a multitude of available technologies, real-time PCR remains, as of now, one of the most powerful and common, often regarded as a reference diagnostic test. RT-PCR has been extensively used for studying viral genera, for which a significant number of primers have been designed. Since the potential of this method became apparent, many enterprises have stepped in, developing commercial PCR kits for reliable fast-track diagnosis of infections like enterovirus, influenza, herpes simplex, etc. While effective and highly sensitive, these panels by design cannot identify new strains and types with altered target sequences. Because of possible genetic similarities between known and previously undiscovered viruses, primers might anneal incorrectly, resulting in false positives and taxonomic misidentification. Multiplexing of primers and probes can be challenging, as there is always a risk of cross-specificity and primer-dimer formation. While separating reactions solves this issue, it requires greater volumes of clinical samples, which are usually limited. Furthermore, in the case of probes, the number of targets is strictly limited to a handful of available fluorophores.
The choice of a diagnostic test is normally based on the circumstances. For instance, while popular and simple methods (PCR, ELISA) are quick, they are less informative than Sanger or next generation sequencing, and it is obvious that the latter could supply extra information about pathogens. (Figure 1)
Figure 1: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077230/bin/viruses-12-00211-g001.jpg
Figure 1 caption:
The prevalence of clinical or scientific application depends on the method and the type of data it yields. Classic approaches, like serology and PCR, are quick, but naturally limited to only the known pathogens. More advanced methods, such as HTS, could supply vital data for diagnostics (e.g., optimal target regions for PCR) and further clinical research.
