Calprotectin is a heterodimer protein expressed by leucocytes and used as a biomarker for Inflammatory Bowel Disease (IBD). It is packed into granular neutrophils (up to 60% of cytoplasmic protein) and released by shedding, active secretion, or cell death. Calprotectin is present in IBD patients because it is expressed by leucocytes that have accumulated in the gastrointestinal tract as part of the inflammatory response and is therefore present in the faeces when intestinal inflammation occurs.

Why measure Calprotectin?

The use of Calprotectin screening has dramatically altered diagnosis of IBD diseases, such as Crohn’s disease and ulcerative colitis. It is considered a reliable marker because it is resistant to degradation and samples can be kept for up to 5 days without affecting tests or results. Calprotectin can be used as a screening tool to differentiate between IBD and Irritable Bowel Syndrome (IBS) and prevent unnecessary endoscopies for patients with low Calprotectin levels. This is a relief to most and makes significant cost savings for healthcare payors, however an endoscopy is still recommended to diagnose patients with suspected IBD based on the UK, Canadian, World and US Gastroenterology Organization guidelines. With an increasing amount of young people, and children being diagnosed with IBD alternatives to invasive tests are vital.

If a patient presents with symptoms of lower abdominal pain, it is important to ascertain between a range of illnesses. Early symptoms of IBD are very similar to IBS. A short list of associated symptoms include:

  • Abdominal pain and discomfort
  • Irregular bowel movements
  • Irregular bowel consistencies
  • Nausea
  • Bloating

If IBD progresses bleeding, anaemia, weight loss and fever can occur. The causes of IBD versus IBS are very different and differentiating between the two is important for patient care.

Calprotectin tests

Calprotectin levels can be measured through faecal sampling via a variety of testing methodologies. From rapid immunochromatographic (also known as lateral flow) tests, enzyme-linked immunosorbent assays (ELISA), and automated immunoassays, there are tests to suit all situations, from home testing to larger scale laboratory testing. All provide quantitative results, however measurements exhibit variability and no current method is universally accepted as a standard.

 

The reference ranges for calprotectin levels can vary slightly between different laboratories and assay methods and each manufacturer sets their own cut-off values for IBD and IBS differentiation. Calprotectin Stool samples In the UK, many hospitals are using the New Faecal Calprotectin Care Pathway for interpretation of the values obtained from a faecal calprotectin test with the following criteria:

  • <100µg/g = IBD is unlikely
  • 100 – 250µg/g = Intermediate result – Repeat the test in 2 weeks
  • >250µg/g = Likely IBD refer to secondary care

It is important to note that this pathway is based on testing using the Bühlmann assay, with some compatibility testing performed to understand the cut off differences for the Thermo Fisher assay. It is essential that users are aware of which testing option they are utilizing to ensure the most effective outcomes in their chosen care pathways.

Tracking Calprotectin levels throughout the course of an IBD suffered illness is increasingly aiding treatment decisions. The move from lab-based ELISA to home-based lateral flow tests initiated  a study comparing 3 lateral flow tests utilising a smart phone app with known ELISA tests to confirm Calprotectin levels. All three calprotectin home tests and companion ELISAs agreed sufficiently when concentrations were at the high end (≤500 μg/g).

Calprotectin Conclusion

Calprotectin testing has reduced the numbers of endoscopies and colonoscopies required, saving time, money, and patient discomfort. Used in isolation preliminary Calprotectin tests may well have limitations, but in conjunction with full medical history and other diagnostic procedures it is a useful tool to differentiate between IBD and IBS. Using Calprotectin tests throughout the course of a sufferers IBD could help the patient manage their lifestyle and treatments. Aided by the use of smart phone apps that facilitate test readings and can link directly with GP surgeries. International standardisation remains an issue and is something for manufacturer collaboration to resolve.

Logical Biological Products

Logical Biological offers stools/faeces from donors, including those diagnosed with IBD, containing known levels of Calprotectin measured using the Diasorin Liaison test or Bühlmann fCAL® ELISA. This material is suitable for many applications including assay development, assay validation, quality control testing and External Quality Assurance schemes.

Sexually transmitted infection (STI) cases across the world are an ever present and ever-increasing issue, not only to adults but to a foetus. Two diseases of particular importance are HIV and Syphilis (caused by the bacterium Treponema pallidum).

How big a threat are STIs in today’s world?

The most recent figures show that in the US, Syphilis cases have tripled between 2013 and 2018 , which includes 5,726 pregnant women. Between 2008 and 2018 Europe also saw a 50% increase in Syphilis and Canada’s cases have more than doubled. In these high-income countries men are disproportionately  affected, however, in low to middle income countries (LMIC), syphilis is endemic to the general population and makes up over 90% of worldwide case numbers. This also means high numbers of infections in pregnant women, leading to increased cases of congenital Syphilis.

Syphilis is classed as an ulcerative STI, which means it causes genital ulcers. These ulcers facilitate the acquisition of HIV during intercourse, increasing the chance of transmission five-fold. Coinfections of Syphilis and HIV can increase the HIV viral load and HIV can accelerate the natural history of Syphilis. This means that individuals suffering from a coinfection will more frequently develop neurosyphilis than those with syphilis alone. In LMIC countries such as Tanzania, Uganda, and Ethiopia, the number of HIV positive patients with Syphilis are nearly 10%, and in Ghana, it is as high as 14.8%.

Source: UNAIDS WHO

What does Syphilis mean for pregnant women?

Globally, Congenital syphilis is the second leading cause of preventable stillbirths and there are a plethora of other complications that can arise from untreated congenital Syphilis, such as severe anaemia, jaundice, and ultimately blindness and deafness. In 2016, the World Health Organisation (WHO) estimated that there were 661,000 infants born with congenital Syphilis, and that approximately 40% of babies born to untreated Syphilis, or an estimated 143,000 infants, will suffer early deaths or stillbirths. This is not to say that this is inevitable, and in fact syphilis can be effectively treated with penicillin if caught early enough. This is why testing pregnant women for these infections is so important.

The World Health Organisation (WHO) ‘Prevention of Mother-to-Child Transmission of HIV/AIDS Program’ (PMTCT) aims to eliminate mother-to-child transmission of HIV and syphilis by providing technical support to member states on the uptake of antenatal services like HIV and Syphilis testing, as well as collecting and analysing regional trends.

In higher income countries, high sensitivity and high specificity tests for HIV and Syphilis are performed in labs and protocols are defined for screening of these diseases. In LMIC’s, there are often limited centralised health services or appropriate lab availability for these kinds of tests and so a point of care (POC) approach is taken, which allows for the collecting of a sample, and testing to be conducted in a single visit. POC tests for HIV have proved successful in LMIC countries, with 70-100% of pregnant women being screened. However, there is a clear deficit for pregnant women being screened for Syphilis, falling short at 40-60%. The high prevalence of syphilis in HIV infected patients indicates that there is a need to increase syphilis testing efforts.

What can be done to bridge the gap between HIV and Syphilis testing rates?

There are several combined HIV/ Syphilis POC rapid diagnostic tests (RTD) which aim to increase the rate of syphilis testing by leveraging existing HIV testing programs. To be successful the combined tests must be affordable, easy to use, and appropriate for a POC scenario. The widespread distribution of these combined tests is cheaper and more efficient than two individual tests and can allow for the early detection and treatment of HIV and Syphilis, saving both pregnant women and their unborn children.

Source: World Health Organization

Tests designed with decentralised testing and POC scenarios in mind can be visually interpreted and are easily used. Storage and distribution are big concerns and so these HIV/ syphilis POC tests are compact, have a 2-year shelf life, and can be stored across a broad temperature range. It is important to understand that these tests are only for initial screening, and if positives come back more specific alternative diagnosis methods should be used.

The importance of dual HIV/ Syphilis POC tests in LMIC’s cannot be understated. These tests are projected to allow for an additional 4.4 million women to be tested, with at least 285,000 Syphilis infections in women to be identified. Ultimately this could lead to 38,000 fewer cases of congenital syphilis, and 51,500 child mortalities being avoided.

HIV/ Syphilis Serum and Plasma from Logical Biological

Logical Biological provide a large portfolio of HIV and syphilis serum and plasma products. Testing can be performed on syphilis serum and syphilis plasma using a wide selection of methodologies including TPHA, ELISA and EIA. Available testing for HIV serum and HIV plasma includes LIA, EIA, PCR (copies/ml), Nucleic Acid Amplification, Western Blot, Ratio CD4/CD8 profiling and Chemiluminescent Immunoassay (ChLIA) (S/CO units).

All serum and plasma specimens can be provided according to your custom specifications and are supplied with demographic information available.

Table: HIV and Syphilis serum and plasma available from Logical Biological

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

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

At the outset of the 2019 SARS-CoV-2 / COVID-19 epidemic, diagnostic immunoassay manufacturers focused on developing anti-SARS-CoV-2 IgM & IgG tests.
While the epidemic has progressed to become a global pandemic, diagnostic test developers’ efforts have also evolved. Recently, we have noticed an increase in interest for patient material positive for Human anti-SARS-CoV-2 IgA. This article will take a look at the difference between the different immunoglobulin sub-classes and seek to understand test developers’ interest in human IgA antibodies specific to SARS-CoV-2.

 

SARS-CoV-2 Antibody Tests

In a recent article we have explained the reason for interest in tests to detect SARS-CoV-2 specific antibodies. Previously infected individuals may be expected to express antibodies specific to SARS-CoV-2. Therefore antibody tests have utility for serology studies that seek to understand how many in the population have previously been infected, and may also have future use as companion tests for vaccinated populations to understand if satisfactory immune responses have been generated. Furthermore, one idea early in the pandemic which hasn’t gained much traction was to mitigate some of the economic impact by identifying and liberating from freedom of movement constraints, i.e. “lockdown”, those individuals who have already been infected by the virus and may therefore be immune from future infections.

 

Types of Immunoglobulin

There are 5 major classes of immunoglobulins. These are IgG, IgM, IgA, IgD, and IgE, each with its own structure based around the classic antibody “Y” shape consisting of Heavy and Light chains.

 

IgG

The most common class of immunoglobulin, present in the largest amounts in blood and tissue fluids, and the most commonly detected type in diagnostic infectious disease tests.

IgM

The initial class of Immunoglobulin made by B cells following exposure to an antigen, commonly present as a receptor on the B cell surface. Typically the earliest class of immunoglobulin detectable before levels wane. However, the situation can vary for different infections and different individuals, e.g. in Lyme Disease and Toxoplasmosis which we have covered in previous articles.

IgA

The main class of antibody found in many bodily secretions including tears and saliva, respiratory and intestinal secretions. Typically, IgA is not as stable as IgG despite being synthesized in large amounts

IgE

IgE is present in low concentrations in the blood. IgE antibodies stimulate a histamine response when binding allergens and play a crucial role in allergy testing; allergen specific IgEs produced in response to exposure to a given allergen can be readily detected in human serum or plasma and are diagnostic of specific allergies.

IgD

IgD is present on the surface of most B cells early in their development but only limited amounts are released into circulation

 

Why Test for IgA?

Whereas early studies of SARS-CoV-serological responses focused on IgG and IgM responses, some papers have suggested COVID-19 IgA may be the most readily detectable of the immunoglobulins in COVID-19 patients and detection of it can serve to increase test sensitivity.

Back in March 2020 Guo et al reported that IgM and IgA appeared earlier than IgG while IgG titres were highest followed by IgA and then IgM.

In a May 2020 article, Jääskeläinen et al. analysed sera from 39 patients and determined that IgA levels were higher than IgG in most cases. In many cases, the IgA level was high enough for the patient to test positive whereas the IgG level was below the threshold for a positive test result. Euroimmun SARS-CoV-2 IgG and IgA kits were used, which are suitable for detection in serum and plasma.

In August 2020 Beavis et al showed 68 out of 82 SARS-CoV-2 PCR positive patients were positive for SARS-CoV-2 IgA whereas 55 out of 82 were positive for SARS-CoV-2 IgG. For patients tested 0, 1 or 2 days after symptom onset the vast majority were negative for IgG whereas most were positive for IgA.

 

 

Timelines of IgG and IgA results from SARS-CoV-2 PCR positive patients (from Beavis et al.)

 

Infantino et al., have recommended the use of IgA tests in order to enhance diagnostic sensitivity of COVID-19 serology tests. They found that IgA levels reached concentrations higher than those observed for IgG and IgM and were often positive in IgM negative patients. Therefore, IgA could shorten the amount of time needed post-infection for virus positive patients to test antibody positive.

SARS-CoV-2 IgG, IgA and IgM in SARS-CoV-2 patients who were initially IgM negative

 

Saliva testing

Collecting blood and converting to serum or plasma in order to detect antibodies is a fairly straightforward process, but perhaps not as straightforward as collecting saliva. Since IgA tends to be present in saliva, detection of  SARS-CoV-2 specific human IgA could theoretically be performed on saliva. Recent studies have confirmed that SARS-CoV-2 IgA is detectable in the saliva of COVID-19 patients.  Another paper in pre-print suggests that antibody levels in serum and saliva do not correlate particularly well so testing in both matrices would enhance test sensitivity even further.

 

Closing the serology gap

If the time window between viral infection and antibody detectability is short enough (e.g. 0-2 days post-infection) the utility of SARS-CoV-2 serology tests could greatly increase. With many countries struggling to expand molecular PCR testing capacity to the levels needed, serology tests carried out using different technology and by different laboratories/personnel to PCR tests, would be additive to the existing COVID-19 test capacity. The price per test would also likely be significantly lower. Unfortunately, it would be hard to tell from the test results exactly when the patient had become infected, especially as antibody levels post-infection vary greatly between individuals, so the utility of such a test is still in doubt.

Interestingly, SARS-CoV-2 IgA also has potential as a prognostic marker, being associated with more severe disease.

 

Conclusion

Data from several sources suggests that the detection of SARS-CoV-2 IgA in addition to the other immunoglobulins (IgG and IgM) represented in SARS-CoV-2 antibody tests  can increase the sensitivity of COVID-19 tests when compared with tests to detect anti-SARS-CoV-2 IgG and/or IgM .

Logical Biological offers serum and plasma samples with measured positive levels of SARS-CoV-2 IgA, IgM and IgG as well as SARS-CoV-2 positive swabs.

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.

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.

Lyme disease is an infectious disease caused by Borrelia bacteria transmitted by tick bites from ticks of the Ixodes genus. Particularly if untreated, serious and long-term symptoms can occur such as neurological problems, facial palsy, lower limb impairment, heart complications, arthritis, encephalomyelitis and psychosis. It is estimated to affect 300,000 people per year in North America and 65,000 in Europe. Transmission can occur across the placenta during pregnancy.

The causative agent of Lyme disease is a group of bacteria called Borrelia burgdorferi sensu lato. The group comprises 21 closely related species but only 4 of them clearly cause Lyme disease:

  • B. mayonii
  • B. burgdorferi sensu stricto
  • B. afzelii
  • B. garinii

The incubation period for Lyme is usually 1 to 2 weeks but can be longer or shorter. Most of those infected did not realise they had been bitten by a tick as the causative ticks are very small in the nymphal stage.

You didn’t want to see a picture of a tick, did you?

Some people infected with Lyme develop a distinctive bullseye-shaped rash known as erythema migrans. These are perhaps the lucky ones as the presence of it aids Lyme diagnosis. However, the rash commonly fails to conform to the notion of what it is supposed to look like and this can result in Lyme being misdiagnosed as spider bites, cellulitis or shingles. The rash can even be absent entirely and this makes it particularly challenging to diagnose since the other early symptoms of Lyme disease (fever, fatigue, etc.) are ones which are also common to a multitude of other ailments.















The classic Lyme erythema migrans rash

Aside from the rash, Lyme diagnosis typically looks for the patient’s immune response. Anti-Lyme IgM antibodies can usually be detected at 2-4 weeks post-infection, while Anti-Lyme IgG antibodies are usually present 4-6 weeks post-infection. IgM antibodies diminish approximately 4-6 months after infection while IgG antibodies can persist for years. Confusing the issue, the IgM antibodies can persist for years in some individuals and Lyme IgG may never be produced in some “seronegative” individuals. A seronegative patient who doesn’t get the classic rash – that’s going to be difficult to diagnose.

The reason for IgM antibodies being present in chronic patients could be the bacterium reactivating and producing novel proteins, recognisable to the immune system, due to genome instability in the bacterium. The host’s immune system therefore may recognise the altered bacterium as a new one it hasn’t encountered before and develop IgM antibodies against it.

The US Centers for Disease Control recommends a two-tier protocol – first test with ELISA or Immunofluorescence Assay to detect anti-Lyme antibodies, and if it is positive or equivocal, a Western Blot should be performed, also detecting anti-Lyme antibodies. Since Lyme disease is caused by numerous strains of bacterial species with inherent genome instability, the Western Blot test looks for antibodies raised by the patient against a large number of host proteins. Multiple bands should be present for the test to be considered positive.

Lyme Western Blot result annotated with protein bands and molecular weights

 

 

 

 

 

 

 

 

 

False positives are rare although possible where anti-Lyme antibodies cross-react with other bacterial antigens present in the patient sample, but false negatives are common particularly early in testing, partly because the anti-Lyme antibodies take some time to develop following the infection.

PCR is not particularly useful for Lyme diagnosis because of low sensitivity in certain sample types and a poor ability to detect Borrelia DNA in patients with neuroborreliosis.

If test sensitivity is the ability to correctly identify those with a disease and test specificity is the ability to correctly identify those without the disease, Lyme tests sadly still have some way to go to reach 100% for either metric.

 

Bulk Lyme IgM plasma is available from Logical Biological.

Congratulations! Anyone reading this far qualifies for extra reading.

Extra Reading: The Accuracy of Diagnostic Tests for Lyme Disease in Humans, A Systematic Review and Meta-Analysis of North American Research.
Waddell. L. et al. PLoS One. 2016, 11 (12).