Methods for Diagnosing Viral Infections

Immunoassay-based Tests:

Antibodies produced immediately after the invasion of a foreign substance can inform on primary infection, reinfection or a deactivation state. Therefore, measuring the level of immunoglobulin’s (Ig) is a widely considered approach for the diagnosis of viral infections. Immunoassays use labels conjugated to synthetic antibodies or antigens which are linked to a solid phase, and used to capture corresponding antigens or antibodies present in sera samples. These labels could be radioactive isotopes, enzymes that cause a change in colour or light-generating substances. Consequently, this principle has generated several methodologies for the testing.

Radio-immunoassay (RIA) is probably the initiating method; it uses radioisotopes to label antigen or antibody. The amount of substance to analyse is determined by the amount of the generated radioactivity. RIA is a highly sensitive method but the main drawback is the handling and disposal of hazardous radioactive substances.

 

 The enzymatic labelling alternative using alkaline phosphatase or horseradish peroxidase as markers is, however, the most widely used and was long considered a reference method. These enzymes induce emission of signals or change in colour respectively and allow the amount of analyst of interest to be measured. This enzyme-linked immunoassay (EIA) has numerous variants, including ELISA, and they differ in the enzyme used and the signal detection principle.

Automated immunoassay techniques for virus detection overcome some of the limitations encountered with the conventional tests, particularly the delay to respond.

One of the limitations is that the Immunoassays are more prone to interferences than any other assay, which leads to false-positive or false-negative results.

Amplification-based Assays:

Nucleic acid amplification by polymerase chain reaction (PCR) has revolutionized the field of molecular diagnosis. The basic PCR assay relies on extraction and purification of the nucleic acid, then exponential amplification of the target sequence, using a thermostable polymerase enzyme and specific primers. The resulting amplicons are then identified using a fluorescence-based detection system, and the result is reported in international units IU/ml.

Soon after its invention, modifications in PCR were tested and patented, with the aim of improving the assay capabilities. The term nucleic acid amplification tests (NAAT) was applied to this range of new variants. NAAT is very popular in the diagnosis and management of viral infections because they allow determination of the viral load. In other terms, quantitation of the viral nucleic acid by amplifying the target sequence thousands-fold. The most widely used variants of conventional amplification are real-time PCR (quantitative PCR) and reverse transcription-PCR (RT-PCR). Both are nowadays becoming benchmarks in assessing the viral load, and while the first method quantifies DNA throughout the reactions in real time the second performs RT of the mRNA (RNA messenger) and amplifies the resulting cDNA (complementary DNA). It also quantifies RNA. The combination of both techniques increases sensitivity in detecting viruses, particularly influenza viruses. Other amplification-based tests such as nucleic acid sequence-based amplification (NASBA) and transcription-mediated amplification (TMA) are suited for detection of RNA viruses by amplification of the mRNA instead of conversion to cDNA.

The limitations of PCR are an important parameter to consider, despite the cost-effectiveness and reliability in the diagnosis of viral infections. The risk of contamination is very high while handling, especially during the sample preparation step, in addition, real-time PCR has a longer run-time (2–5 h) by comparison to other techniques. The high mutation rate of some viruses could trigger mutation within PCR primer regions of the viral genome, which would lead the virus to escape the detection by this assay.

 Next Generation Sequencing:

Next-generation sequencing (NGS) is one of the greatest achievements of the modern era. Beyond genome sequencing from known organisms, it allowed discovery of novel viruses responsible for unknown human diseases, and tracking of outbreaks and pandemics such as influenza to understand their emergence and transmission profiles. Improvements and automation have dramatically increased the speed and accuracy in delivering maximum volume of data comparing to dideoxynucleotide sequencing. Technically, NGS is inclusive of three main steps: sample preparation, sequencing and data analysis. Efficient and accurate clinical diagnosis of viral infections using NGS is increasingly aiming to provide accurate longer read-length in the shortest time and at a lower cost. Bioinformatics platforms are key components of the sequencing process. They allow interpretation of the sequencing output through computational analysis and then convert it into useful information on species, genotypes and the occurrence of mutations conferring virulence or resistance to antivirals.

NGS is undeniably a key technology in specialized clinical laboratories, but its implementation is still a challenge in many countries, where not only their resource-limited settings cannot afford a sequence analyser, sample and library preparation, but the vast majority of the population cannot afford the cost of the test. 

                                         

Mass Spectrometry:

Mass spectrometry (MS) is nowadays a benchmark of laboratory qualitative and quantitative investigation, particularly in bacteriology. The principle of MS relies on converting the sample into charged particles (ions) by ionization process. These ions are separated according to their mass-to-charge ratio (m/z) and analysed by a detector. The result obtained is compared to a reference database (library), existing within the system and delivered as an interpretive spectrum.

In clinical laboratories, matrix-assisted laser desorption ionization (MALDI) and electrospray (ES) are the most used ionization methods because they allow processing of considerable amounts of analyte. The combination (RT-PCR/ESI-MS) was able to detect viral pathogens usually undetected by regular testing methods and provided rapid and detailed data (types and subtypes) within a short time. The blend of two powerful types of machinery (PCR-MS) can detect drug resistance to antiviral therapy as well as the presence of multiple viruses within the same sample and diagnose for co-infections.

Mass spectrometric-based methods are versatile, sensitive, rapid and cost-effective, and do not require interpretation software for data analysis. The automated machinery necessitates easy sample preparation and fewer operators. The analysis capacity can reach up to 960 specimens/day, which makes it suitable for routine diagnosis in high-volume laboratories and large-scale studies. Tests can also be performed efficiently on the archived specimen.

The main limitation of MS is the high cost, particularly in high pandemic areas, which are usually the poorest; not all laboratories can afford a mass analyser for their activities. The second major drawback is within the reference library. The identification is limited by known data from well-identified organisms only; therefore, rare mutations cannot be detected if they do not exist within the reading platform, but there is hope that MS database libraries will rapidly expand.