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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

Blog Overview

February 1st 2023
Immunoassays Infectious Diseases Swabs
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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 90–100% 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

Blog Overview

February 1st 2021
Immunoassays Infectious Diseases Swabs
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From the outset of the COVID-19 outbreak, testing for SARS-CoV-2 has been a priority. Initially just having a test for COVID-19 was a scientific success, enabled by the publication of the first viral genomic sequence in January 2020. As the pandemic has run its course many aspects of testing have been investigated. Here we highlight some of the key testing events that have taken place, and their outcomes.

Mass Covid Testing

–Vo’, Italy – Mass testing in this Italian town was triggered by the reporting of the first COVID-19 death. With a population of 3275, Vo managed to test 86% in a first testing round, and 72% in a second round 2 weeks later. 2.6% were RT-PCR positive in the first round of tests, reducing to 1.2% in the second round. The organiser stated that ‘early identification of clusters and timely isolation of people testing positive suppressed transmission and curbed the epidemic’.

–Wuhan – This COVID-19 mass testing event was orchestrated after the initial reactions to SARS-CoV-2 had been implemented and a stringent enforced lockdown had already successfully limited COVID-19 cases. In the last 2 weeks of May 2020 the nearly 10 million residents of Wuhan over 6 years old were tested using RT-PCR; pooled sensitivity was 73%, but no information provided for specificity. Asymptomatic cases reported numbered 300, with no new symptomatic cases found. Contact tracing of the 300 infected showed no cases of transmission. Pooling of five samples at a time were used to increase efficiency in some 23% of the samples. If a pooled sample was positive, all samples within the pool were re-tested as single samples.

–Luxembourg – Here, RT-PCR was performed on pooled samples. Reportedly, sensitivity and specificity of the test protocol was 100%. Overall, 49% of the residential population were tested as well as 22% of cross border workers, totalling over half a million people. 850 positive cases were found and a further 249 identified through contact tracing. The study indicated that asymptomatic carriers are at least as infectious as symptomatic patients. It was reported that containment of future outbreaks will critically depend on early testing in sectors and geographical regions. Higher participation rates must be achieved, using targeted incentives and invitations.

COVID-19 Testing Site

 

–Slovakia – 20000 medical and 40000 non-medical staff were used to administer 3 rounds of testing at the end of October 2020. The first round was a pilot and high prevalence areas were re-tested in the final round. In all, 5 million tests were performed, covering over 80% of the population. Samples taken by medical professionals used the SD Biosensor lateral flow rapid test, approved by WHO with a sensitivity of 95.5% (70-90% in the study) and specificity 99.2%. No confirmatory RT-PCR tests were perfomed. Over 50000 SARS-CoV-2 cases were positively identified, approximately 4% in the pilot, 1% in round 1 and 0.6% in round 2.

–Qingdao, China – The events leading to the mass testing in Qingdao came from three people testing positive using RT-PCR, leading back to 2 asymptomatic dock workers whose transmission path couldn’t be followed further. Over 3 weeks nearly 11 million individuals over 5 years old were tested. To cover this many people pooling was used. The result was the identification of 12 positive cases. Public transport was only available to those with evidence of a negative test. No information was published on the specificity and sensitivity of the RT-PCR test.

–Liverpool, UK – The objective was ‘to demonstrate massive asymptomatic cases can help identify far more cases and break the chain of transmission.’ Between the 6th and 26th November 108304 lateral flow tests (LFT) were carried out. Most tests were self-administered (military supervised) lateral flow tests from Innova. Some tests were repeated for quality assessment purposes with samples collected professionally and confirmed using PCR testing. Test sensitivity was substantially reduced depending on who carried out the tests; 78% when used by trained professionals, but only 58% when used by self-trained staff. Specificity was reportedly 99.93%, meaning false positives would be expected to be rare. Key workers, health and social care staff, school staff, and children aged 11 and over were targeted, but anyone could get tested, preferably at least twice within two weeks.

 

Table: Summary of SARS-CoV-2 mass testing events.

COVID Test Types

The variety of tests for SARS-CoV-2 are relatively well known thanks to media reporting.

The most sensitive is the RT-PCR test, we have described this in previous articles. RT-PCR uses engineered primers to amplify fragments of viral RNA present in the specimen and requires a laboratory. Sensitivity is high with figures ranging between 71-98%. However, this variation depends on multiple factors, including the diligence of sample collection, test type, swab type and time for which the sample is left in storage. The RT-PCR based mass testing events in China, taking place in a background of a small number of cases in the community, were seemingly effective in ending the outbreaks. 

Lateral flow tests to detect SARS-CoV-2 antigen came to the market more recently than RT-PCR tests, due to the fundamental timelines inherent in developing a new test of this type. However,  the technology has been around for years and is used in all manner of diagnostic tests, the most well known being pregnancy tests. Lateral flow tests  are relatively cheap to produce and can be tested on site with a result time varying between 5-30 minutes. This saving of time and money makes them an attractive option. Theoretically, they are extremely simple to perform and have the potential to be used by untrained individuals in any location, if the test protocol and instructions for use are simple enough. For SARS-CoV-2 specifically, some tests have now been approved by the US FDA for home-testing (Ellume and BINAX).

COVID-19 negative Lateral Flow test result

 

Lateral flow tests can also be used to identify human anti-SARS-CoV-2 antibodies present in samples, sometimes known as antibody tests or serology tests. However, these can not be used to determine if an individual has a current SARS-CoV-2 infection, only if the individual has ever had an infection. These tests have potential to be used to identify individuals who have already been infected and built up some immunity, and could therefore be a lower priority for vaccination. However, this would be a controversial use of such tests and no country has thus far decided to do this, despite the current context of a shortage of vaccine doses and an urgent drive to protect as many vulnerable people as possible. 

Mass testing analysis

The success of mass testing events is challenging to ascertain; the testing events are ambitious and complex operations featuring innumerable variables. In particular, where mass testing events are carried out alongside a “lockdown”, it is difficult to separate the results of the two and thus understand which element was responsible for any subsequent reduction in infections. It can be said that each mass testing event, in coordination with a lockdown, appeared to reduce numbers of positive covid tests, at least in the short-term.

Overall, RT-PCR is accurate but time consuming and costly. Pooling testing is a cost effective method of testing large numbers using RT-PCR. Lateral flow tests are cheap to produce and give fast results but lack accuracy. Multiple tests performed on the same individuals at different time points could help improve the accuracy. 

The experience of self-administered tests in Liverpool versus the professionally administered tests carried out China and Slovakia highlights the importance of medical expertise. Samples collected by trained professionals provide more accurate results than samples gathered by individuals. The new FDA approved Ellume and Abbott Binax tests are the first home test kits available, reporting sensitivity and specificity of 92% and 100%, respectively, and results within 20 minutes. But their use comes with a caveat in the form of a “Telehealth Proctor”. At present even the most user friendly tests available require supervision for best results; it remains to be seen whether they can be simplified to the extent that they can be performed effectively without this. 

The lateral flow tests used in Liverpool, were found to only confirm half of all COVID-19 cases identified by RT-PCR tests. It has been suggested this is understandable in low viral load cases, which may be less infectious, but it also missed 3 of 10 in higher viral load cases (based on Ct values below 25). These tests have been strongly criticised and called unfit for purpose by some,  but from a public health perspective it can be argued it is better to find 50% of cases rather than none. The UK government has announced more mass testing in COVID-19 high prevalence areas and it remains to be seen how it will modify its approach using the learning points from the Liverpool testing. 

Mass testing is an important tool in defeating SARS-CoV-2. The logistical challenges involved are exceedingly complex (and expensive) and few governments have thus far attempted them. If sensitive and user-friendly lateral flow tests can be developed we expect to see great demand for public and private sector test settings such as schools, airports, universities, care homes and cruise ships.  

Logical Biological provides swabs/serum/plasma for use in test development

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.

Swabs table
Table: Products available from Logical Biological

Blog Overview

January 11th 2021
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What are Ct values?  

In quantitative PCR (qPCR) and reverse transcriptase quantitative PCR (RT-qPCR), the cycle threshold (Ct) value is the number of nucleic acid amplification cycles required for the signal generated by the amplification of a specific target DNA to cross a set threshold. Labelling the DNA primers with a fluorescent tag is what makes this PCR ‘real time’; as the target genetic material increases with each amplification cycle so does the fluorescence signal.

The lower the amplification cycles required to meet the threshold, the higher the level of target nucleic acid (and thus virus in the case of SARS-CoV-2) in the patient sample. Therefore Ct values are inversely related to viral load. Within a specific test the Ct value result is compared to a value often known as a ‘cut-off’. A value lower than the cut-off is a positive result (for COVID-19 meaning the patient most likely has the infection), whereas a value higher than the cut-off is a negative result.

 

Many of Logical Biological’s COVID-19 swab samples are tested using Thermo-Fisher Taqpath, which has a positive cut-off of Ct ≤37. In that test, a sample with a Ct of 36 would be a low positive. At the other end of the range, Ct values below 20 would be considered a strong positive, and we have infrequently seen values below 15.

Ct values and COVID-19 

The course of SARS-CoV-2 infection shows viral levels vary throughout the duration of the disease, peaking a few days after symptom onset. Correspondingly RT-qPCR Ct values should alter depending on the timeline of an individuals infection. The lower the Ct values the increased probability the patient is near to peak-infection. However there are multiple practical factors unrelated to the actual infection that may also affect the Ct value achieved.

Correspondence between development of viral load during SARS-CoV-2 infection, clinical course and positivity of quantitative rRT-PCR assays (r = real-time). (Lippi et al, 2020).

qPCR/RT-qPCR Ct value variables include but are not limited to:

  1. Swabbing protocol
  2. Swabbing technique
  3. Swab type
  4. Swab brand
  5. Sample storage time
  6. Sample storage conditions
  7. UTM/VTM brand
  8. RNA extraction method
  9. PCR reagents used
  10. PCR test kit used
  11. Patient days since infection
  12. Patient viral load
  13. Environmental contamination
  14. Standard quantification curves

With so many variables any comparison of Ct values between individuals has obvious limitations.

Clinical Use of Covid-19 Ct data

Cycle threshold values are beginning to be used for COVID-19 patients as there is a suggested relationship between viral load and prognosis. Lower Ct values have been linked to a worse course of illness and poorer outcomes. Clinicians have been  encouraged to interpret the SARS-CoV-2 RT-qPCR test results and consider the Ct value where appropriate.

A positive PCR test alone does not correlate with infectivity (Tom and Mina, 2020); a positive test with high Ct value may indicate the patient is not infectious. If this information could be used to inform patient isolation decisions it could transform quarantine restrictions and take significant steps in reducing the already extensive economic consequences of the pandemic, not to mention the mental health implications of isolation. Conversely, low Ct values may indicate a high level of infectiousness, acting as a signal to healthcare professionals to ensure the patient remains isolated and avoid releasing them into the general population until their Ct value increases sufficiently. The challenge here is reliably using Ct values to determine infectiousness.

Contradictory research regarding viral load and shedding in asymptomatic cases require further investigation. Measuring asymptomatic Ct values provided evidence of little difference when compared to symptomatic cases, implying similar viral load. On the other hand, other researchers found asymptomatic cases tested had no viable SARS-CoV-2 virus and no evidence of viral transmission for these carriers. This particular study should be viewed in the context of the virus persisting undetected in a population (Wuhan) where non-pharmaceutical interventions had been used to almost eradicate the virus from the population over a relatively long period of time; selection for a weakened strain may have occurred.

Ct values and disease diagnosis test development

While the value of Ct values to clinicians is debatable, they definitely have utility in in vitro diagnostic (IVD) assay development. Assay sensitivity can be defined as the proportion of true positive samples that actually test positive. SARS-CoV-2 test makers are motivated to produce sensitive tests able to even detect virus in patient samples where the viral load is low. Lateral Flow SARS-CoV-2 antigen test manufacturers, for example, may use patient samples with known RT-qPCR Ct values to determine how the samples test in their assay. Samples with very low Ct counts (high viral loads) would be expected to test positive, but by testing samples with increasing Ct counts (lower viral loads) assay developers can assess the performance of their test, understand what viral loads correspond with a failure of their test to detect, and work to optimise assay performance where necessary.

Conclusions

Ct values have limited use in clinical settings. A multitude of variables impact on the viral load and a separate large set of variables impact on patient outcomes (e.g. age). Ultimately Ct values are not easily comparable between assays or patients. It is possible they can be used to assist patient prognosis in some circumstances where a full clinical history is available. 

However, in vitro diagnostic assay developers find Ct values useful as a way of quickly determining the lower limit of detection of the tests they are developing. In this way, Ct data has a great value in contributing to the development of sensitive tests. 

Available from Logical Biological

At Logical Biological we supply nasopharyngeal/oropharyngeal/nasal SARS-CoV-2, FluA and Flu B swabs available in Universal Transport Medium (UTM), Inactivating Transport Medium (ITM), saline or in dry frozen format. Negative swabs and COVID-19 / pre-COVID saliva are also available, as are serum and plasma. Typically, our swabs are provided together a Ct value measured from a ‘companion swab’ taken simultaneously. For UTM & ITM we can provide remnant samples at your preferred Ct values.

Swabs table

Table: Products available from Logical Biological

Blog Overview

December 14th 2020
Infectious Diseases Swabs
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Immunoassays Infectious Diseases Point of Care Testing

Dual HIV/ Syphilis POC Tests: Their Importance in Low to Middle Income Countries
March 30th 2023
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This article covers swab type and selection, transport products and medium enabling a smooth transfer of patient samples to the laboratory for analysis. Additionally, we discuss the implications of cycle thresholds and whether they should be assessed in patient prognosis.


Swabs

Swabs come in all shapes and sizes and some favourites have become well known in recent months:

  • Nasal Swab: needs to be inserted at least 1cm into the naris (nostril), directed upwards. May cause sneezing.
  • Nasopharyngeal Swab: one method is to measure the distance from the corner of the nose to the front of the ear, then half it. This is the approximate distance the nasopharyngeal swab should be entering the nasal cavity. Use the medial side of the septum as a guide and gently rotate to facilitate insertion. The swab should run parallel to the palate and needs to be left in place for ‘several seconds’. Taking a sample is a skill in itself. The complexity and invasive nature of this test paired with lack of discrepancy compared to nasal swab results has lead to a shift in favour of nasal swabs. May cause sneezing and watering eyes.
  • Oropharyngeal Swab: enters through the oral cavity (mouth). This swab aims to take material from the tonsils and the oropharyngeal wall (back of the throat), and avoids touching the tongue, teeth and gums. May cause retching.  

nasopharyngeal swab, oropharyngeal swab, nasal swab

Generally the shape of these swabs are incredibly similar, essentially long shafted cotton buds. The tips can be selected to best accommodate the chosen antigen or marker of interest and the shafts chosen for access purposes. Options include long-armed pharmaceutical grade cotton buds, or perhaps Rayon or Dacron swabs are better suited if considering a dry swab. Rayon swabs are made from synthetic fibres spun from wood pulp, whereas Dacron is made from polystyrene. Swabs destined for transport medium require different properties such as polyester or foam, they can be made less or more absorbent, have different porosities and have good ‘release’ factors. Canadian researchers investigated how specific these swabs need to be especially in the current pandemic with supplies at a premium. They determined swab type had limited impact on test results.


Transport Medium

Having collected samples with wisely chosen swabs, samples must be secured in transport medium to make their way to the lab. Again there are many choices. Generally used, Universal Transport Medium (aka UTM) will protect and conserve samples. This is a well know method and low risk regarding the preservation of the sample. Examples are the BD universal viral transport system, which can accommodate storage at room temperature or at +4°C, and Copan UTM, which contains proteins for virus stablisation, plus buffer, antibiotics and antimycotics. 

Other media can provide more specific care depending on sample consistency, for example Viral Transport Medium (VTM) has specific ingredients suited to the transport of viral content. However, UTMs are also suitable for virus sample maintenance and the ingredients list of VTMs is similar. Generon outline a mix including heat inactivated fetal bovine serum, gentamicin and amphotericin B, an antifungal agent. Some recipes suggest including additional calcium and magnesium. If you are making your own sterility can be ensured by filtration or using sterile ingredients and aseptic technique upon mixing. A newer alternative is the Inactivated Transport Medium (ITM), or Molecular Transport Medium.


Inactivation – Health & Safety

It is not a large leap to identify the advantages of using ITM, and in fact there has been a call for this to take place with SARS-CoV-2 from back in April. From a UK perspective, SARS-CoV-2 is classified as Hazard Group 3 (HG3) by the UK Advisory Committee for Dangerous Pathogens. The Containment Level 3 list of requirements for low-risk handling of active HG3 infectious organisms are long and require specialist facilities, including:

  • Maintenance of a negative air pressure
  • HEPA systemic exhaust air extraction and filtration 
  • Sealable laboratory to enable fumigation

For SARS-CoV-2 research and assay development, the ability to transport and analyse inactivated viral samples potentially negates the need for such stringent containment measures. This would assist COVID-19 test developers because they would no longer need to access Containment Level 3 / BSL 3 facilities as part of their test development. Such facilities are few and far between and potentially extremely expensive to access. The CDC advises transport of inactivated COVID-19 samples in a nucleic acid extraction buffer. The nucleic acids remain intact and identifiable, the medium inactivates the protein cap (using guanidine thiocyanate and N-Lauroylsarcosine (sodium) in ethanol) and renders the virus unable to reinfect. Of course, each user will need to abide by their local risk assessments and satisfy themselves that their choice of ITM truly inactivates the virus. Inactivation may denature the antigen of interest so users would need to be sure that the ITM would be compatible with their project objectives. Users should also seek specialist advice on the containment measures required when using any ITM. Logical Biological offers SARS-CoV-2 remnant swabs inactivated in NEST Scientific ITM.


Cycle Threshold Values

With an inactivated sample safely in the lab, the molecular components are ready to be amplified and analysed. Cycle Threshold (Ct) values are important as the quantitative unit of measurement in PCR methodologies and using them in diagnostic testing can be invaluable for patient prognosis. Essentially the lower the number of amplification cycles required to reach a threshold level for the target gene is, the higher the level of target nucleic acid (and thus virus in the case of SARS-CoV-2) in the patient sample; Ct values are inversely related to viral load. Generally speaking a very high Ct value indicates a low amount of target nucleic acid in the sample, and the possibility of environmental contamination should be borne in mind. Often a Ct value of around 34 is the cut-off point for a patient to be considered positive for the infection. Many of Logical Biological’s COVID-19 swab samples are tested using Thermo-Fisher Taqpath, which has a positive cut-off of Ct ≤37. At the other end of the range we have infrequently observed Ct values as low as 9, with Ct values below 15 considered an extremely strong positive. Some Logical Biological clients wish to access a selection of swabs with a range of different Ct values, to ensure that their assay is able to detect the various different ‘strengths’ of infection. This is something we have been able to accommodate readily

SARS-COV-2 PCR Ct values have been analysed and the scientific and clinical communities are assessing if indeed Ct values, that are not often reported in clinical results, should be included and used as a marker for future prognosis. As an example please refer to Rao et al, 2020. 

 

Available from Logical Biological

At Logical Biological we supply nasopharyngeal/oropharyngeal/nasal SARS-CoV-2, FluA and Flu B swabs available in UTM, Inactivating TM, saline or dry. Negatives, and COVID-19 / pre-COVID saliva is also available.

 

Swabs table
Table: Products available from Logical Biological

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November 6th 2020
Infectious Diseases Swabs
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Two types of tests for SARS-CoV-2 currently predominate. The first are molecular tests, based on PCR technology, that detect the presence of viral nucleic acid and therefore indicate a current infection. The second are serology tests that are designed to detect anti-SARS-CoV-2 antibodies (usually IgG) generated by the immune systems of individuals who have been infected in the past.

RT-PCR to detect SARS-CoV-2 virus is relatively slow and requires specialist laboratories. At the outset of the pandemic, testing capacity was too low to meet the needs of governments, and even 6 months later demand for tests can outstrip supply in areas where there is a surge of infections. At the end of June 2020 Quest Diagnostics, a key player in COVID-19 testing in the USA, announced that only the highest risk patients could get a test result faster than 3-5 days. Other companies reported similar issues.

Alternatives to slow and expensive procedures such as RT-PCR testing do exist. Rapid tests based on Lateral Flow technology have been put forward as a faster, cheaper, low technology alternative to RT-PCR testing. However, this technology is fundamentally of lower sensitivity than PCR-based testing; while PCR is able to exponentially increase the signal until it is detectable, Lateral Flow (also known as immunochromatography) tests have little scope for signal amplification.

An example of Lateral Flow rapid tests

There is also the question of what tissue type to use for the testing. While many rapid tests use serum or plasma derived from blood as the sample of choice, that would only be a suitable matrix for SARS-CoV-2 testing if the virus was present in samples from infected individuals. In the case of SARS-CoV-2 it would seem that blood (and therefore serum and plasma) is not suitable. In a March 2020 JAMA paper, Wang et al. reported that using RT-PCR, SARS-CoV-2 was only identified in 3 out of 307 blood samples (1%) collected from up to 205 patients in China.

Since the virus is thought to be spread by droplets in coughing and sneezing, saliva has been considered a high potential sample type for rapid tests. It does seem that in many cases the virus is present in saliva samples – a recent paper by Azzie et al. in the Journal of Infection has determined that in a cohort of 25 patients with severe COVID-19, virus was detectable in the saliva of all 25 of them via RT-PCR. The Cycle Threshold values ranged from 18 to 32 with a mean average of 27. The extent to which the virus is present in asymptomatic patients remains to be determined.

A further challenge around rapid antigen detection in saliva or other sample types is sensitivity. RT-PCR is highly sensitive and theoretically able to detect a very low number of viruses. However, in a recent study by Mizuno et al. on >100 patients, virus was only detected in 11.7% of them using a rapid antigen test (Fujirebio) compared to up to 82% using molecular diagnostic tests.

Intriguingly, faeces and rectal swabs have a high potential as a positive sample type; in a review article Bwire et al. reported positive rates of 32.8% and 87.8%, respectively. Rectal swabs have a vastly superior positive rate according to this review than both nasopharyngeal (45.5%) and oropharyngeal (7.6%) swabs. Although they are perhaps not so amenable to drive-through testing(!), they have some potential for self-testing at home.

Our surprising conclusion is that stool samples may be the best matrix for rapid SARS-CoV-2 virus testing. Serum and plasma are clearly not suitable and saliva, which is perhaps the most convenient alternative, may have a lower viral load than stools. A further challenge with both stools and saliva is that they are likely to need diluting into a suitable liquid prior to testing, to enable them to flow down the test strip. This would reduce the test sensitivity even further .

If rapid tests cannot reach the levels of sensitivity of RT-PCR tests, do they have any utility? Some believe that they do. If it was possible to make such a cheap and easy test that everyone in the country could be tested, say, 1 or 2 times every day, it could be that at some point in the infection cycle those carrying infections would have a high enough viral load to test positive using a low sensitivity test. In the context of this pandemic where a large proportion of those infected never find out due to being asymptomatic or not ill enough to get tested, this may be of value, and is presumably why companies continue to pursue the holy grail of SARS-CoV-2 rapid testing.

Logical Biological provides human patient material for SARS-CoV-2 relevant to both virus and serology testing. This includes nasal swabs with quantitative PCR results, saliva and serum/plasma.

Blog Overview

August 5th 2020
Antigens Immunoassays Infectious Diseases Saliva Swabs
Share this blog:
Infectious Diseases Contact Us

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