The flu season is well and truly upon us, but is it Influenza, RSV or Covid? And does it matter?

 

The ‘tripledemic’ season of Influenza, COVID-19, and RSV viruses

Influenza A/B, COVID-19 with its variants and respiratory syncytial virus (RSV) are the three largest contenders responsible for respiratory infections globally. The European Centre of Disease prevention and Control and the Pan American Health Organisation have reported unusual infection spikes this year, collectively calling it a ‘triple threat’, and providing a worldwide picture of increased infections.

Global numbers for annual mortality associated with respiratory infections stands at up to 650,000 for influenza and 80,000 for RSV. The relative newness of SARS-COV-2 and its fatalities mean its numbers cannot yet be directly used in comparisons here, needless to say cumulative deaths from COVID-19 are over 6 million and continue to be recorded in great detail. Infections themselves number in the tens to hundreds of millions, some sources even estimate flu infections reach a billion cases annually, leading to between 300,000 and 800,000 hospitalizations in the U.S. alone each year. Australia’s increased flu infections this year have been an important prediction for the Northern Hemisphere flu season to be ready, and that prediction is unfortunately proving accurate.

 

Influenza, Covid and RSV symptoms are very similar- Is there a need to define which respiratory disease you have?

Covid Flu A/B RSV A/B symptoms

Influenza, SARS-COV-2, and RSV have incredibly similar symptoms and without medical intervention, the same treatment – namely rest, fluids and over the counter relief medication. An average, healthy infected individual will suffer for a week or two, but otherwise make a full recovery.  People suffering moderate to severe symptoms tend to self-impose an element of isolation from others, with awareness of the effectiveness of this as a prevention highlighted in the recent SARS-COV-2 pandemic. Problems arise when the young, old, or immunologically challenged are infected and need medical assistance in their recovery.

Vaccines and antiviral treatments however vary, and there is an increased interest in being able to identify which virus is responsible for specific infections. A test is the only way to be certain of which antiviral medication should be administered as soon as possible after infection.

Table source: CDC, Mayo Clinic

 

Combined tests offer streamlined workflow for doctors allowing viral specific diagnosis in a single test

Calls for simple, low cost, differential diagnosis combination tests for the three viruses discussed here are growing. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) or rapid lateral flow tests (LFT) that combine more than one pathogen identifier would enable the testing process to be streamlined, allowing for multiple test results from a single sample. Patient sample collection from one nasopharyngeal or mid-turbinate swab would decrease patient discomfort but also provide a financial saving and optimise clinician’s time.

 

Different types of combination tests available

Real-time quantitative reverse transcription PCR (rRT-PCR) testing that combines Flu A and B with COVID, is used by the Centres for Disease Control and Prevention to track disease trends. Other clinical combination testing that includes RSV is available for general diagnostic use, are FDA approved and encourage home sample collection. Further available tests, have Emergency Use Authorisation (EUA) from the FDA, but with time and increased demand we may well find more of these tests being approved. Globally companies were swift to produce combination tests for these viral respiratory diseases using TEM-PCR technology. CE-IVD marked RT-PCR and multiplex rRT-PCR tests are already available in European countries and the Middle East.

Laboratory test results are generally available between 24 to 48 hours. As with all tests needing additional equipment for sample processing and analysis, transport and storage facilities have to be considered and can add outlying costs to testing.

A limited number of lateral flow tests combining the three viral components in one test have been developed, although results are available within a 10 minute window, there are separate sample requirements, meaning it may require more than one sample extraction. Other technologies for combination testing include microfluid immunofluorescence assays, combinations remain limited to two viral components. These provide quick results, within 12 minutes, using a specific reader.

Currently there is no reliable, over the counter, rapid lateral flow test that combines these three, allowing quick, cheap testing in a clinical setting or indeed a home test.

 

Logical Biological Products

At Logical Biological we supply nasopharyngeal/ oropharyngeal/ nasal SARS-CoV-2, Influenza A, Influenza B and RSV swabs available in UTM, Inactivating TM, saline or dry frozen. Negative swabs and COVID-19 / pre-COVID saliva are also available. Typically, our swabs are provided together with a Ct value measured from a ‘companion swab’ taken simultaneously. We also provide serum and plasma samples from individuals infected with SARS-CoV-2, Influenza A/ B, RSV, Streptococcus A, Adenovirus, Parainfluenza and other respiratory infectious diseases.

SARS-CoV-2 Flu A/B RSV A/B products

Table: Products available from Logical Biological

Global vaccine roll out is making progress but with most countries wanting priority status and with a limited supply, how can governments best prioritise those requiring vaccination?

Vaccination strategies

Overall two vaccination strategies are in place: the first concentrates on those at highest risk, the second focuses vaccinations on people most likely to transmit the virus, known as direct and indirect vaccination strategies, respectively. Regardless of vaccine type and manufacturer, there are significant availability issues. Cell division time is a limiting factor in production, and considerations such as vaccine transport, worker health and storage capacity all contribute towards availability issues. Limited stores of reagents and chemicals involved in production have become a problem in the massive upscaling of vaccine production required for the global population.

As of writing, Israel has vaccinated its 9 million population once and is over halfway through the second dosage. The UAE is also high on the list of countries who have nearly managed to complete vaccination and has started its own vaccine manufacture.  

 

Countries with larger populations have different hurdles. The UK has targeted vaccinations for the old-aged, healthcare workers and those with underlying health conditions, with Norway doing similar. Studies in vaccination strategy suggest prioritising the over 60’s to minimise mortality. However, Indonesia was one of the first countries breaking from this template, focusing on vaccinations to reduce transmissions, namely the working age group 18-59. Indonesia’s population consists of only 10% over 60, whereas the UK is nearer 20%. The UK has vaccinated approximately 60% of its population, with nearly 40 million doses given, whereas the US is making astounding progress with well over 50% of the population having had the first dose; a whopping 185 million doses given. However, the overall picture is one of limited vaccine availability, with only a small proportion of the global population having been vaccinated.

Length of vaccine protection

It is unknown how long the vaccines provides protection for. Research has suggested after an asymptomatic, mild or moderate infection specific T cell immunity may be persist for around 6 months. The CEO of Pfizer has speculated that booster vaccinations for SARS-CoV-2 may be required within 12 months. 

Factors affecting vaccine efficacy:

  • age 
  • underlying health conditions 

Factors affecting vaccination strategy:

  • country specific age structure 
  • infection fatality rate
  • vaccine availability 
  • vaccine efficacy 
  • social distancing/isolation measures
  • seroprevalence

Over a dozen COVID-19 vaccines are currently approved. Types used include inactivated virus, mRNA, non-replicating viral vector and Adenovirus vectors. Whether these contrasting mechanisms provide differing outcomes regarding longevity of protection remains to be seen.  

Serology status

Serology, the study of antibodies in the blood, is relevant to vaccinations as individuals may already carry the antibodies required to fight SARS-CoV-2 through previous infection. If this is the case, should they be lower down the priority list for vaccination?

 

 

Hepatitis B Virus (HBV) is a disease known to require susceptibility status prior to vaccination, and therefore serology documentation. However, most vaccinations do not require serology paperwork. With limited supply of SARS-CoV-2 vaccines available it stands to reason the most impactful use of each dose would be given to susceptible individuals and not immune individuals. Modelling studies have advocated prioritising COVID-19 vaccinations by serostatus as well as age. There are clearly additional logistics, complexity and expense involved, and few if any governments have deprioritised citizens for vaccination on the basis of serology or positive PCR tests. Another rational approach that has not caught on would be to provide only a single dose to those who have recovered from SARS-CoV-2 infection; in Ferbruary 2021 it was reported that France intends to give only a single dose to those citizens who have recovered from COVID-19.

A SARS-CoV-2 serology test, or antibody test, is typically a lateral flow test, lab based ELISA or Chemiluminescence Immunoassay designed to detect whether an individual has ever been infected with the virus. Generally SARS-CoV-2 serology tests can detect immunoglobulins: IgG, IgM or a combination of both. There is research to indicate IgA should be included in these serology tests as it may be a more accurate marker than IgM regarding tests taken shortly after an infection. There is much activity taking place within the IVD industry to tailor such tests to areas where they might be useful, for example to develop a test identifying those who have produced adequate antibodies from the vaccine and those who would require a booster.

Time kinetics of antibody response in coronavirus disease 2019 (COVID-19). The illustration demonstrates the relative levels of host immunoglobulins (IgM, IgG, IgA) and SARS-CoV-2 viral load at different stages of COVID-19. Antibody-specific seroconversion occurs when the antibody reaches a detectable level in blood. Disclaimer: This graphic is for illustrative purposes only and does not represent actual levels of each antibody.

Summary 

As scientists we support the use of serology tests and previous PCR status to inform SARS-CoV-2 vaccine prioritisation in the context of limited vaccine availability, particularly in the young and healthy who are least at risk from the virus .

Logical Biological Products

At Logical Biological we provide serum and plasma from individuals pre- and post-COVID-19 vaccination, as well as SARS-CoV-2 IgG, IgM and IgA positive serum and plasma samples. We also provide SARS-CoV-2, Influenza and RSV swabs and saliva.

Table- Products available from Logical Biological

 

 

As the pandemic has struck the predicted second wave, public struggles persist with lifestyle restrictions, but our knowledge to identify and fight the virus has significantly progressed.

With tests developed, distributed and administered satisfactorily for most symptomatic sufferers (PCR), and the vaccine roll out for the most vulnerable, science and medicine turn to consider the silent carriers of SARS-CoV-2…the asymptomatic vectors.

The case for rapid antigen testing using Lateral Flow

At present testing generally consists of Polymerase Chain Reaction (PCR ) or Lateral Flow Tests (LFT) also known as rapid or antigen tests. Writing on these previously highlighted the differences and nuances of each. Regarding asymptomatic carriers it would appear that PCR will test positive for a longer period of the infection, even if the individual is not contagious. It can also take a long time to get PCR test results, often days – making them non-suitable for detecting asymptomatic carriers in everyday social settings such as schools. Lateral flow tests are considerably quicker, providing a result in 10-30 minutes, and significantly cheaper as they do not require a laboratory.

Graph – High-Frequency Testing with Low Analytic Sensitivity versus Low-Frequency Testing with High Analytic Sensitivity

Analysis of UK mass lateral flow test events have been beneficial. It is suggested the 60% of people who had “false negative” tests in the Liverpool pilot were not contagious. As the graph shows, PCR sensitivity may be detrimental when considering the effects of extreme and unnecessary self-isolation requirements. Evaluations have shown that lateral flow tests identify 90100% of asymptomatic individuals whose samples go on to provide viable virus in cultured samples. 

Results from an American University mass testing event showed the ability to culture viable virus from a sample means the virus is capable of reinfecting, hence the individual is contagious. This study also used lateral flow tests and showed similar results to the UK. Virus samples collected were able to infect cell cultures in vitro, showing their viability. Good news indeed for identifying individuals who are asymptomatic and infectious.

Repeat testing using lateral flow tests, especially in high transition groups, medical staff, teachers and carers, with a short turn around time, will help reduce transmissions. The cost effectiveness and ease of a lateral flow test makes repeat testing an obvious option.  

Not to rapid test

Evidence from mass testing events (previous blog) has suggested lateral flow tests can miss up to approximately 60% of infected people. In the UK Liverpool study a third of people tested negative even with high viral loads, implying the individuals would be infectious, even if they were asymptomatic. A negative lateral flow test would potentially release this individual into the population to spread the disease, although this assumes positive individuals ignore the guidance to not treat a negative result as a definitive confirmation of COVID-negative status. This scenario shows how important other preventative measures are, such as social distancing and hand washing. Unfortunately Liverpool, whilst providing reams of data, is also, to date (18th Jan 2021), one of the few UK areas with increased cases of the disease, even after all the lockdown measures and mass testing. 

France has recently voiced its concerns over lateral flow tests and have insisted that any non-EU travellers must have a negative RT-PCR test; they will no longer accept a negative lateral flow test as sufficient for entry into France. This will inevitably feed into the public concern over lateral flow test results and cause many issues, not only because at present, certainly in the UK, you only qualify for an RT-PCR test if you are symptomatic. Medicines and Healthcare products Regulatory Agency (MHRA) have raised concerns about using lateral flow tests in UK schools as there is no ‘test-to-enable’, this had led to a pause on testing in UK schools. A test-to-enable would provide definitive results as to the infectiousness of an individual.

Mass testing has been touted as the way out of the crisis since it began. Now we are getting into a position to test everyone how might this affect human behaviour? People are encouraged to act as if they are positive for COVID-19, but the increase in testing means many will have a “negative” test and could decide to ignore social distancing guidance on the basis of it. This had been flagged as a major (hypothetical) problem by some highly-credentialed scientists taking issue with the UK’s lateral flow test testing program.

Most tests need a swab of some kind to collect their sample, exceptions being drooled saliva and blood samples. Sample collection can be uncomfortable but most willingly go through the procedure. Often sample collection is supervised with the individual taking the actual sample. This has implications for correct procedure sample collection, with reports from the Liverpool mass testing suggesting the test performed worse when civilians performed their own test without supervision. For others, who are unwilling or unable to undergo the stress or complexities of testing, the test may fail from the start with inadequate sample collection. As yet lateral flow tests are not readily available without symptoms at home.

Logical Biological’s View

Our view is that lateral flow tests are a cheap and easy test that when performed on a regular basis in certain settings, schools being a prime example, can identify a substantial proportion of infectious individuals, enabling them to be removed quickly from that setting. This would undoubtedly improve the safety of those associating with them in the same setting, allowing the setting to remain open for longer, to the benefit of society. However, in order to maximise the utility of such an approach a minimum test frequency guideline should be defined in each setting. This testing should be frequent, ideally daily.  

Other high risk settings are likely to include meat processing plants, university halls of residence and care homes (staff and visitors). Imagine how much safer you would be as a teacher, parent or care home resident if most asymptomatic people you or your loved ones associated with were identified and self-isolated. This would also cut chains of transmission and reduce strain on health systems. For these reasons many scientists have pushed back against the warnings from researchers with ‘unfounded criticism’ of the lateral flow test.

Currently no test can definitively assess for SARS-CoV-2 infectiousness, and to attain that ideal may take many months/years or may never happen. Heeding the prescient words of Mike Ryan at the outset of the pandemic “perfection is the enemy of the good. Speed trumps perfection”, we are frustrated at resistance from within the scientific community to the use of technologies that, despite their imperfections, are likely to have an enormous benefit to society. 

Logical Biological Products

At Logical Biological we supply nasopharyngeal/oropharyngeal/nasal SARS-CoV-2, FluA and Flu B swabs available in UTM, Inactivating TM, saline or dry frozen. Negative swabs and COVID-19 / pre-COVID saliva are also available. Typically, our swabs are provided together a Ct value measured from a ‘companion swab’ taken simultaneously. We also provide serum and plasma samples from individuals infected with SARS-CoV-2 and other respiratory infectious diseases. Samples from vaccinated individuals can be collected.

Table: Products available from Logical Biological

We are in the midst of the most significant global pandemic since the 1918-19 Influenza pandemic, over 100 years ago. The 2020 pandemic has been caused by a Coronavirus, named SARS-CoV-2, which confers a severe respiratory illness (COVID-19) on a proportion of those infected. The virus is readily transmissible from human to human with many of those infected showing no or mild symptoms, meaning it is hard to know who has been infected.

The virus has resulted in severe economic impacts because many countries have adopted “lockdown” policies in order to limit its spread by limiting interaction between individuals. One idea for mitigating some of the economic impact has been to identify and liberate from lockdown those individuals who have already been infected by the virus and may therefore be immune from future infections. As yet, it is not known for sure if previous infection by the virus renders individuals immune from future viral challenge, nor how long such immunity would last for. However, for this idea to be viable, diagnostic tests that can identify the SARS-CoV-2 antibodies in the human blood are required. Such tests have been made by innumerable manufacturers but they vary greatly in their performance.

 

At what stage are antibodies exhibited?

To understand the value of SARS-CoV-2 antibody tests we need to know who exhibits what antibodies, and when. It is assumed that the vast majority of individuals will have a detectable antibody response, regardless of whether or not they are symptomatic. The below data suggests IgG and IgM antibodies are detectable 1-3 weeks after SARS-CoV-2 symptom onset.


Detection of IgG, IgM and Neutralising (NT) antibodies over time since symptom onset.
Taken from a pre-print published online by Borremans et al., (2020)

 

Why 100% test specificity is essential for SARS-CoV-2

Logical Biological received some CE-marked SARS-CoV-2 IgG and IgM antibody tests a few weeks ago (we won’t name the manufacturer). In the pack insert these tests reported 97% specificity, which sounds high, but what does it mean in the context of the proportion of people who actually have been infected in the population? Specificity can be defined as the number of true negative cases that actually return a negative test result.

As can be seen in the tables below, if 1 person is selected at random and tests positive using a 97% specificity test when the prevalence within the population is low, it is much more likely that a positive test result is a false positive than a true positive.

Even with a 99.6% specificity test, such as those now available in a leading UK high street pharmacist, there is a clear threat of false positives, the proportion of false positives to actual positives reducing as the true number of infected within the population increases. One serology study, performed in Santa Clara, California, received criticism that the specificity of the test was too low, at only 99.5%. Only with 100% can we be fully confident that the positive test result is a true positive. Fortunately, such tests are now becoming available, such as one developed by Ortho Clinical Diagnostics.

The tables below consider a random selection of the population. Individuals may feel more confident about a positive result they receive if they have also shown the classical symptoms.

97% Specificity

% of population prev. infected Test Specificity True positives (per 1000) Expected False positives (per 1000)
0.1% 97.0% 1 30
1% 97.0% 10 30
10% 97.0% 100 30
100% 97.0% 1000 30

 

99.6% Specificity

% of population prev. infected Test Specificity True positives (per 1000) Expected False positives (per 1000)
0.1% 99.6% 1 4
1% 99.6% 10 4
10% 99.6% 100 4
100% 99.6% 1000 4

 

100% Specificity

% of population prev. infected Test Specificity True positives (per 1000) Expected False positives (per 1000)
0.1% 100.0% 1 0
1% 100.0% 10 0
10% 100.0% 100 0
100% 100.0% 1000 0

 

Sensitivity

Sensitivity of a test can be defined as the proportion of those genuinely bearing a marker (such as SARS-CoV-2 antibodies) who test positive for it. In the context of SARS-CoV-2 antibody tests, results from low sensitivity tests are less “dangerous” than results from low specificity tests. Low specificity tests could result in a situation where susceptible individuals who have wrongly tested positive, believing themselves to be both immune and non-infective, stop taking precautions to protect themselves and others, leading to further infections. On the other hand, low sensitivity tests would likely result in previously-infected individuals who have wrongly tested negative continuing to be cautious and since they have already had the infection would not be able to contribute to further spread in any case.


Table shows theoretical results of a 97% sensitivity test

% of population prev. infected Test sensitivity True positives (per 1000) Expected positives based on 97% sensitivity
0.1% 97.0% 1 0.97
1% 97.0% 10 9.7
10% 97.0% 100 97
100% 97.0% 1000 970

 

 

How many people have actually been infected by SARS-CoV-2?

One of the countries most affected by the pandemic has been Spain. The government of Spain has recently performed a serological study (results published on 13th May 2020) where they assessed the blood of 70,000 individuals. The most affected province showed 14.2% positive tests whereas the least affected regions were at less than 2%. The overall figure for previously-infected individuals in Spain was assessed to be approximately 5%. The test looked for both IgG and IgM antibodies. In the context of the above information, it should be noted that the test used was reported to show 100% specificity and 79% sensitivity for IgG. This means it would miss 21% of those previously infected and also may miss some people in the early stages of infection. It was wise of them to choose a 100% specificity test, and in the context of a serological survey to assess prevalence within a large population, the results can be adjusted to account for the low sensitivity of the test.

Data from other countries is in line with that from the Spanish study. For example, study results announced on April 23rd from another of the world’s major hotspots, New York State, USA, found 21% of people to be antibody positive in New York City. High figures (10-20%) were also seen in other areas while outside of the most-affected areas in the state the average prevalence was 3.6%.

 

Conclusion

The prevalence of SARS-CoV-2 in some locales is 2-20%. At the upper end of this range the % of positive tests that would be false when using a 97% specificity test would be significant and unacceptably high. At the low end of this range there would be more false positives returned than true positives, rendering such a test completely useless. Beware SARS-CoV-2 antibody tests with <100% specificity.

 

Available from Logical Biological

  • SARS-CoV-2 PCR positive serum/plasma/swabs
  • SARS-CoV-2 IgG positive serum/plasma – 1ml samples and bulk units
  • SARS-CoV-2 IgM positive serum/plasma – 1ml samples and bulk units
  • Pre-Covid19 serum/plasma from normal healthy donors – any quantity

Due to the dynamic nature of the SARS-CoV-2 pandemic this blog post will be out of date shortly after it is written.