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Influenza Virus Testing Methods

Background

Influenza is also more frequently referred to as flu and it is known as a respiratory disease. The flu virus is regarded as a common disease which can affect approximately 10% of the population at any given point throughout the year. These infectious diseases can spread quite easily amongst people mainly via airborne droplets which can be a result of sneezing, coughing and even through talking.

Influenza viruses are more noticeable during the colder, winter months which can in some cases result in seasonal epidemics. A physical examination carried out by a professional clinician is usually the first step taken to diagnose influenza. This involves reviewing any active signs and symptoms of flu. Influenza virus can display a wide range of symptoms such as: cough, runny nose, fever, sore throat, congestion, fatigue, body and headaches. For the majority of people they recover quite quickly and only experience mild to short lived symptoms.

The flu virus shares many similarities to the coronavirus (COVID-19). They are both known as contagious respiratory diseases that are caused by a virus. Both share common symptoms and spread from one person to another in a very similar fashion. Also, both influenza and COVID-19 infections can result in many types of serious complications for example: organ failure (lung, liver, heart and brain), Pneumonia, renal agenesis, cardiovascular disease, hypotension, heart attacks, thrombocytopenia, acute respiratory distress syndrome, stroke, major trauma, brain inflammation and death.

Based on the different influenza virus stains found to be in circulation throughout the winter months, this aid in the development of a vaccine. This vaccine will help to minimise the impact of flu. For many vulnerable people it can be extremely important to be diagnosed and treated for influenza.

Types of influenza

Currently, 4 different types of influenza viruses exist: A, B, C and D. The specific category type for each virus is based on the material present within the nucleus of a particular virus.

  1. Influenza A virus: The subtypes of type A group are distinguished from each other based on two glycoproteins being present on the virus cell surface. One of the glycoprotein is known as hemagglutinin (HA) and the other one is known as neuraminidase (NA). There are around 18 hemagglutinin that have been discovered (H1, H2 and H3 are only discovered in humans). Whereas over 100 types have been identified for neuraminidase (in this case only N1 and N2 are associated to humans).
    Characteristics of type A: Affecting humans and many other animals, present in all age groups and can lead to either moderate or severe illness.
  2. Influenza B virus: This type is made up of either B/Victoria and B/Yamagata (only two genetic lineages present). This virus is very different from how type A are classified through different subtypes. Also, there has been evidence that both genetic lineages (Victoria and Yamagata) are able to co-circulate during flu seasons. In general, influenza B virus tends to be only present in humans and is linked to milder epidemic in comparison to type A. Influenza B viruses are also known to undergo less rapid genetic changes when compared to the influenza A.
    Characteristics of type B: Only present in humans, lead to milder diseases in comparison to type A and affects predominately children.
  3. Influenza C virus: This virus is less studied and less common to influenza A and B. It tends to cause illnesses mainly to humans and pigs. Majority of individuals that are exposed to type C occurs primarily during childhood. Type C has very limited ability to mutate due to a lack of multiple subtypes that are present in type A.
    Characteristics of type C: Unlikely to cause epidemics and rarely reported in adults.
  4. Influenza D virus: This virus is extremely rare when compared to type A, B and C. However, it is about 50% similar in amino acid composition to type C. Type D is also less severe in illness in comparison to the other antigenic types. Influenza D is known to infect cattle and pigs, there is no evidence to indicate that it can cause human infections.
    Characteristics of type D: Only infects pigs and cattle, no human infections and causes mild illnesses.

Influenza virus tests

Other tests are frequently required to further diagnose influenza, this is often a follow-up from the initial physical examination which only observes general signs and symptoms of the flu. The detection method used by the other tests is the main form of categorising them.

  • Molecular diagnostic methods: These procedures tend to measure trace amounts of DNA or genetic material that is from the influenza virus present within the sample that is being analysed. High specificity and sensitivity can be achieved using reverse transcription polymerase reaction (RT-PCR) and various other nucleic acid amplification tests (often called NAATs).
    Many molecular tests can detect and even distinguish infections between an influenza A from an influenza B. There are even some procedures that will be able to identify the different types of influenza A subtypes or even a new subtype for influenza A. Some of these tests are even designed to only detect a specific type A subtype for example A (H2N2) or A (H1N1).
    There are also available multiplex molecular tests, these are able to detect and distinguish influenza nucleic acids from infections from other forms of respiratory pathogens.
  • Antigen detection methods: These protocols rely on identifying antigens present within the given sample that are found in influenza virus. The specific influenza antigens are located on the surface of the virus and result in an immune response being triggered. The detection of influenza antigens can indicate the presence of an active flu infection.
    There are two types of methods that are usually available: rapid (point of care) and a more specific laboratory test. Rapid antigen tests (lateral flow test), do not need to be sent to the laboratory for analysis but instead use a small device where a sample can be added. This protocol provides a result within 20-30 minutes. The result is based on a visual colour coded change that can be analysed on the small device. It is widely accepted that the rapid antigen protocols are more likely to deliver a false negative result since they may not be specific or sensitive to pick up a true positive.
    Laboratory ELISA assays (immunoassay) are more detailed and take much longer to process. They are also more sensitive and specific when compared to the rapid test method. The laboratory tests will lead to more accurate results being generated and fewer false negative results being detected. The laboratory tests can determine if the infection is by influenza A or influenza B, they can also distinguish between the different subtypes of influenza A.
  • Viral culture methods: These protocols can take between 1-10 days to complete, they are carried out in the laboratory and involve growing a large number of copies of the virus within a vial. It is a not a popular method for undertaking the diagnosis and treatment procedure. However, it can be very important in the detection of a particular type or even subtypes of the virus that is being transmitted. This can provide vital information to public health officials and researchers so that they can carefully monitor epidemics. This process will also help  them in the design and development of a new yearly flu vaccine.
  • Serology methods: These procedures involve identifying antibodies for influenza viruses that are present within blood samples. As part of the immune response, the human body will produce antibodies to combat any infections by the virus. Serology protocols are predominately used for research purpose use only and not for diagnostics. They provide useful information for evaluating prior influenza viral infections that can be present in a particular age group or in a specific area.

Possible treatments for influenza

For majority of individuals simply having plenty of rest, paracetamols and drinking more fluids is enough to treat the flu. However, for some more vulnerable patients when the infections are more serious and the chances of further complications are much higher. This situation may lead to the doctor prescribing various antiviral drugs that may help in treatment process.

Some of the more common drugs which are available include: Relenza (zanamivir), Xofluza (baloxavir), Tamiflu (oseltamivir) and Rapivab (peramivir). These antiviral drugs will help to combat the illness and in majority of cases result in overcoming the flu infection quicker, therefore prevent any further serious complications happening.

These medications function by preventing the enzyme neuraminidase from working on the surface of the virus, this results in the viral particles not being release from infected host cells. There are many publications to confirm that these antiviral drugs can effectively treat both influenza A and influenza B when administered within 48 hours from the onset of flu symptoms. This further highlights the importance of early diagnosis of the virus so that effective treatment can be given.

Antiviral medications are known to have side effects for example vomiting and nausea. It is therefore highly recommended to take these drugs after eating and not on an empty stomach in order to lessen these side effects.

Monitoring and surveillance of influenza

The global standard that is recognised for flu surveillance has been set up by WHO’s Global Influenza Programme (GIP). It plays a critical role in collecting and analysing epidemiological or virological surveillance data from all countries throughout the world. The sharing of this data will allow WHO to:

  • Monitor global trends from different parts of the world (for example: virus evolution, prevalence and the geographic spread)
  • Rapidly identify, report and react to outbreaks of influenza
  • Detect, isolate, understand and study new influenza viruses – this will lead to the development of new diagnostic tests and in some cases new antiviral drugs, identify whether they pose any new risks when compared to already existing viruses and finally they will allow earlier steps to be taken to manufacturer new vaccines to respond to the next flu pandemic.

The influenza surveillance within the United Kingdom is joint collaborative effort amongst various organisations that include: the government, local and national health departments, vital statistics offices, clinics, healthcare providers, emergency departments, clinical and public health laboratories.

Public Health England (PHE) is regarded as one of the leading public health systems for flu surveillance throughout the world. PHE has multiple data sources available (UK wide weekly graphs and reports are generated) to establish an accurate understanding of influenza virus activity throughout the whole year. Many of these data sources are well established and have been around for several years.

Summary:

There are many different methods that are available for testing influenza virus. The specificity and sensitivity of these procedures will vary depending on many factors which includes: testing method used, collection procedure undertaken, quality of the collected viral sample, source of the respiratory sample, processing and handling of the sample and the time taken from the initial collection to the actual testing of the sample.

It is widely recommended to use the rapid tests over diagnostic tests when dealing with specimens of outpatients. Whereas for patients that have been hospitalised then it is recommended to use molecular diagnostics procedures such as RT-PCR, NAATs and various antigen detection tests such as rapid antigen, ELISA and immunofluorescence assays.

When performing any diagnostic test, the results generated from this assay should always be evaluated in context to other epidemiological and clinical information that is available to any professional healthcare provider.

It is worth mentioning that at present a combination of diagnostic approaches (i.e. antigen detection, molecular tests and viral isolation) have to be used in order to detect the various different types of viruses. There is no single diagnostic procedure that is adequate and suitable for all the different clinical situations. This is accepted by majority of virologists and they understand the limitations of one method over another. Virologists have to select the appropriate protocol that best fits their specific clinical situations, along with the one which will generate the most reliable results.

There is evidence to indicate that despite a gradual decline in the prevalence of diseases, there is a steady increase in the molecular diagnostics procedure being identified for the detection of infectious diseases. There is also a massive focus on improving the accuracy and the speed of these novel diagnostic tests.

References

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