Comments in answer to some questions from journalists about the different types of testing for COVID-19.
Dr Zania Stamataki, Viral Immunologist, University of Birmingham, said:
“There are two types of tests that we carry out for COVID-19, one is to confirm if you currently have the virus (PCR) and one is to ask if you have made antibodies against the virus (serology). Both kinds of tests help us make public health decisions about measures to contain the outbreak. Information from these tests is important to help us return to work, reopen schools and relax restrictions.
“The PCR test detects the virus, and it is important to determine if someone who is very ill has COVID-19. The test uses swabs from the nose and throat and has a high accuracy rate, but it doesn’t pick up all infections. Some countries test as many people as possible, others test those that develop serious symptoms of the disease. This has to do with logistics, it is very difficult to scale up testing for large proportions of the population.
“PCR testing is important early in infection because it can help us isolate infected people and contain the outbreak. PCR testing is also important later in the outbreak because it shows us how the virus is transmitting through the population and we can then calculate the percentage of people that develop serious complications, which reveals how dangerous this virus is for different people (young and old, men and women, people with other health problems). If we don’t test for the virus, we will be unable to decide if our measures to contain transmission are working. For example, have the school closures made any difference?
“Serology testing reveals if you have been exposed to the virus, and your body has developed antibodies. IgM antibodies are present early in infection, IgG antibodies (seroconversion) arise a few days later. COVID-19 is new for humanity and you will not have antibodies against it if you have not been infected. We don’t know yet if your antibodies are able to protect you against re-infection, and we don’t know how long they will last in your blood. It is likely, however, that if you have recovered, you should be able to return back to work. Some countries like Germany are investigating using serology results as an “immunity passport” to advise those that recovered to return to work.
“Antibodies can reduce infection by blocking virus attachment to target cells (neutralising antibodies). If antibodies are protective, a vaccine will generate immunity to the virus and help us return to normality. There are dangers to antibodies too in COVID-19, and it was reported that antibodies can activate immune cells which leads to collateral damage in the lungs https://insight.jci.org/articles/view/123158. A study in five critically ill patients that received antibodies from recovered individuals together with anti-inflammatory drugs showed an encouraging reduction in mortality https://jamanetwork.com/journals/jama/fullarticle/2763983.”
Dr James Gill, Locum GP & Honorary Clinical Lecturer, Warwick Medical School, said:
“There has been a lot of swirl about the timelines with regard to antibody and PCR tests. Let’s clear up what is being looked for. As with everything about coronavirus and science in general, the more you simplify, the more get’s missed. So let’s try to add a little detail back in, so people can understand the PCR and antibody investigations as more than black and white tests.
“With PCR testing – here you are look for DNA or RNA evidence of an infectious agent – in the case of Coronavirus, we’re looking for RNA. If you take a test-tube filed with a virus – no patient here – and run a PCR test on it, the test will come back indicating the RNA sequence of what is in the tube. We match that sequence to known sequences of viruses, which then tells us “Yes you’ve found a virus and the sequence matches to this virus”. That is obviously a pure lab test example. When used in practice, we are taking a swab from a patient, hoping that if they have the corona virus infection, that enough of the virus is picked up on the swab for the PCR test to be able to detect the virus. As the swab needs to pick up enough of the virus to work, this is one of the reasons the coronavirus the PCR test can have a higher false negative test rate – i.e. missing the virus even though someone has the infection.
“On the other side of the fence we have the newer antibody blood test. The antibody test needs to be run on a sample of blood from a patient. If you were to put a sample of the virus directly on the antibody test you will get no result. The antibody test is not looking for the virus, but evidence of the virus being in the body, and the body reacting to it by producing antibodies to help fight it off. Not all antibodies are the same, nor created at the same time over the course of an infection – and here is the complicated bit with the antibody test – WHEN do you do the test? The antibody IgM is the bodies first response to an infection – normally within 5-10 days of an infection taking hold, peaking at 21 days after the infection. That time frame is crucial, if you have just developed symptoms you think are coronavirus, it will take approximately a week for your body to raise IgM antibodies to the virus. Now whilst with coronavirus we do have evidence of IgM being presenting the blood within 1 day of symptoms, but that isn’t going to be a reliable test at that stage, as the likely won’t be large amounts of IgM for use to detect. The best test for an early infection is combining the antibody test AND the PCR swab taken from the patient. Then we have a 98.6% detection rate within the first 5.5 days of infection – https://www.jwatch.org/na51255/2020/03/31/serologic-tests-sars-cov-2-first-steps-long-road
“When tested alone, the PCR test has a 66.7% detection rate within the first week, whilst the antibody test has a lower 38.3% detection rate.
“But what about the other antibody, IgG, also detected by the new Coronavirus antibody test? IgG shows that someone has had the virus and is now protected from the virus, this can be detected in a patients blood ~10-14 days after infection. Perhaps one way of looking at this is the IgM is the fast reaction force, whereas IgG is the main, slower, but more powerful army, which acts to keep the body safe afterwards as well. What if a patient were to be exposed to coronavirus again – not infected? Once a patient has cleared the infection, the IgG, the army now knows it’s target and can be quickly mobilised again. A huge volume of IgG is raised to combat the virus within 24-48hrs of reexposure and will then hopefully prevent a new infection. Thus if a patient is found to have IgM, that suggests that they may be within the first week of an infection. If they have BOTH IgM and IgG, that suggests they will be within the first month of infection, and should hopefully be protected from repeat infection.”
Prof Robert Dingwall, Professor of Sociology, Nottingham Trent University, said:
“Basically, you should never test unless you have accurate tests and know what you are going to do with the information. In the present circumstances, there are only three reasons to use the current PCR test or an antigen test, if and when available, which will tell you, whether someone actually has the infection at the time:
“1. You want to know how many people are infected on a given day. This would be between 1,500 and 10,000 tests, depending upon the degree of accuracy you need, from a random sample of the population – like election polls. This would also give you some clue as to whether we were approaching a level of population immunity that would cause the virus to decline because it was running out of people to infect. You don’t need to do this every day – maybe every couple of weeks.
“2. You want to get the correct treatment of people who are seriously ill. If they have a bacterial pneumonia, you give them antibiotics. If they have a viral pneumonia, you give them supportive care. You avoid the risk of over-prescribing antibiotics.
“3. You want to get NHS and social care staff (and possibly other sectors on a case-by-case basis) back to work more quickly by reducing the scale of precautionary self-isolation for mild symptoms. If they have a significant respiratory infection, they shouldn’t be coming back to work anyway – and there is emerging evidence that the virus stops shedding in infectious amounts 7 days after symptoms appear anyway. That finding is not yet robust but it is plausible.
“If an effective and affordable antibody test becomes available, which would tell you who has had the disease, there would be other reasons to test. Bear in mind that antibody tests should only be done about 3-4 weeks after symptoms disappear, because it takes time for the antibody levels to be detectable.
“1. You want to know how long immunity levels persist. In which case, you would go back to a group that had been antigen tested and retest them. If you went back to the first group above, it would also help with estimates of population immunity and the risks of flare-ups.
“2. You want to get NHS and social care staff (and possibly other sectors on a case-by-case basis) back to work, especially in areas where they may be exposed to infection – but you need to do the previous exercise first and by the time you have done that, the information may not be particularly relevant, given the level of population immunity that has been established. Moreover, the wait until antibodies reach a stable and measurable level means that this test is unlikely to play a major role in clearing people for work.
“3. You want to know who should be prioritized for vaccinations, when they become available, because they have no detectable antibodies, either because they were never infected or because their antibody levels have declined to a level where they are not protective. There is no point in doing this before the vaccine is becoming available because there is no public or patient benefit. which is likely to consume resources that could more effectively be used elsewhere in the NHS at the present time.”
Prof Robin May, Professor of Infectious Diseases & Director, Institute of Microbiology and Infection, University of Birmingham, said:
“PCR testing detects specific pieces of DNA – so, for instance, this can be used to check whether a ‘lamb stew’ is really lamb, etc. In the case of COVID19, there is a slightly complication in that the virus encodes its genome in the related molecule RNA, rather than DNA (many viruses do this), so the viral RNA needs to be ‘copied’ into DNA and then checked by PCR to test for the presence of the virus. However, the process to do this is straightforward and fast and can be automated fairly well – hence the ability to roll out large scale viral testing. Consequently, PCR testing (or, more accurately, ‘quantitative RT-PCR testing’) measures the amount of virus in a sample (typically a nasal or throat swab) and a threshold is set, above which a person is deemed to be “positive” for the virus.
“Antibody testing looks for the presence of antibodies against the virus in a person’s blood. This is usually done by taking a protein that the virus makes and then testing whether any antibodies in blood bind to that protein. The problem with this kind of test is identifying a specific protein (or combination of proteins) that are unique to this virus (and not in other coronaviruses, for instance, which would lead to false positives) but for which the antibody/protein interaction is still strong enough to allow sensitive detection. Optimising and validating this part of the test is what is currently taking time. Once such a test is developed, initially the testing process is indeed slow. However, it is often possible to adapt such laboratory tests into simpler ‘point of care’ devices – rather like a pregnancy test. If that is achievable for a SARS-COV-2 antibody test, then rapid testing may well become very feasible on a large scale. Once rolled out, such a test would tell us who has HAD the virus, but not necessarily who HAS it at the moment (since it takes several days for antibodies to be made, and thus people can have an active infection but no detectable antibodies.
“Antigen testing looks for the presence of a molecule that is specific to the pathogen – for instance, a protein that is on the virus surface. It usually works by having a specific antibody that recognises that specific antigen – if the antibody binds that antigen, then the test records a ‘positive’. Like PCR tests, antigen tests reveal the presence of the virus, but don’t tell us anything about whether the person is mounting a robust immune response. Once the infection has gone, the antigen also disappears (since it is produced by the pathogen, not the host). In principle, the PCR test referred to above is also a sort of antigen test, since the viral genome is an ‘antigen’ in this sense. However, usually people don’t refer to the PCR test as an antigen test, because it doesn’t work in the same way (it is based on copying the virus genome, rather than on detecting a specific protein or other molecule that is released by the virus).”
Comments sent out on Wednesday 1st April 2020
Prof Eleanor Riley, Professor of Immunology and Infectious Disease, University of Edinburgh, said:
“Tests for the virus (current infection) are often called “antigen” tests – where antigen refers to some component of the virus, typically the external (coat) protein of the virus. However, the test being used for COVID-19 is actually looking for viral RNA (which is technically not a viral antigen). So when people talk of “antigen” tests and others talk of tests for viral RNA or “PCR tests” they are actually talking about the same thing. I would prefer if people would simply talk about tests for the presence of the virus – which covers all eventualities – and clearly differentiates it from tests for antibodies (which we don’t have yet).”
Prof Nicola Stonehouse, Professor in Molecular Virology, University of Leeds, said:
“There are basically two kinds of tests – one evaluated whether you are currently infected and the other evaluates whether you have been. We need both.
“For SARS CoV2, the former test detects viral genetic material – RNA. This is a PCR based test. The latter detects antibodies to the virus.
“I think the confusion has arisen from the viral RNA incorrectly being termed ‘antigen’.”
Dr Andrew Preston, Reader in Microbial Pathogenesis, University of Bath, said:
“Virus tests. Detect the presence of virus, so diagnoses active infection. Either:
“(a) RT-PCR (PCR test) detects the viral RNA, its genetic material. An advantage of PCR is that it involves amplification of signal, so can be very sensitive (detect very low levels of viral RNA). Uses swab sample or fluid from lower respiratory tract (really only feasible with sedated, hospitalised patients). In theory, results within a few hours of sampling (depending on whether sampling is close to the testing lab). Samples are batched so hundreds run in a batch. Laboratory test. Requires expert personnel. Other viral RNA detection based assays available and in development (e.g. Abbot’s ID Now).
“(b) Antigen test. Detects viral proteins in samples. The ones I have seen appear to be devised for use with swab samples, sampling the respiratory tract. Both laboratory and POC (point of care) assays in development. POC assays, in theory, provide use-at-home capacity. These are the ‘dipstick’ style tests, similar to the home use at home blood glucose devices/pregnancy tests. Give a yes/no answer. I’m not aware of any that have received formal approval, many are showing as in development on company websites.
“These, as the name suggests, detect antibodies against viral proteins. Antibodies are one of the key immune response components. They start to be detectable around a week after initial infection. Some types of antibody (IgM) are produced first before the response switches to an IgG dominated one. Many tests can distinguish between IgM and IgG and thus give information on the phase of infection (early/current vs later stage/previous infection). In a vast majority of infectious diseases, recovery from disease and evidence of a strong immune response would lead to a period of immunity from re-occurrence. Thus there is great interest in the use of an antibody test to indicate immunity against disease for use in the lifting of lock down restrictions. Both lab based versions and POC versions, similar to the antigen test.
“Important to articulate the question being asked to decide on which test. Diagnosis of someone presenting with disease favours use of a virus test. Determining the number of people who might have experienced COVID-19, either symptomatically or asymptomatically favours the antibody test. Determining immunity – antibody test (with the caveat that we don’t know for sure that a positive antibody test does imply someone is immune). Logistics- POCs for use at home allow for mass testing without the need for massive lab capacity, but how would an at-home test result be validated?
“When e.g. press conferences are talking about tests, it is important that the person is very careful over the type of test being discussed. It’s not been clear of late whether spokespersons are referring to virus, antigen or antibody tests in some cases.”
Dr Michael Skinner, Reader in Virology, Imperial College London, said:
“Some confusion has, of late, crept into discussions about virus (and antibody testing). This is understandable because of some overlapping terminology.
“Headlines such as: “NHS frontline staff become first to get coronavirus ‘antigen test’” and “Frontline NHS staff first to receive new Covid-19 antigen tests” to 27 & 28 March were unfortunately victims of this confusion.
“1) Test available now (and already for a couple of months) – qPCR – identifies a virus component (nucleic acid, RNA genome) in swabs and other samples from a few days after infection (even pre-symptomatic) to a couple of weeks after first symptoms (longer for acutely ill). Performed in laboratories, taking few hours from sample delivery (faster point, point-of-care alternatives will become available in time).
“2) Test available shortly – Antibody test – identifies virus-specific antibodies in blood. Point-of-care, fast. Indicates likelihood of immunity to COVID-19 but does not exclude the presence of the virus so, if used too early, the individual might still be shedding virus.
“3) Under development – Antigen test – identifies protein component of the virus (probably in swabs not blood). Could be used as point-of-care, earlier than antibody test. Will offer alternative to nucleic acid tests.
“As soon as the genome sequence of SARS-COV-2 was published with amazing rapidity by the Chinese, scientists around the world could design a specific and highly sensitive test to detect the presence of the virus’s genetic material, its nucleic acid (in this case, RNA) genome (or rather, a small part of its genome), using a technique called polymerase chain reaction (PCR) [because it is RNA not DNA, they actually use a modification called reverse transcription PCR, or RT-PCR].
“Some will have performed PCR at college or school, and viewed the products (amplicons; a couple of dozen at a time) by agarose gel electrophoresis, and that’s still used in molecular biology labs for some purposes.
“However, processing of 100 to 300 samples at a time in research or diagnostic labs is now performed using machines called (confusingly) Real-Time or Quantitative PCR (qPCR). Once the samples are loaded, a run takes 1 to 2 hours and the output is digital, ready for data analysis and reporting.
“The sensitivity of the qPCR assay means that it can pick up small amounts of virus shed (in this case) into nasal or pharyngeal mucous a few days after infection, even before symptoms arise.
“The government has spoken about the need for and its desire to employ tests for ‘virus-specific antibodies’ in our blood. We only make these antibodies about a week or so after the earliest symptoms, so these tests are not as good as PCR for revealing infection. They’ve also taken longer to develop because they rely on us having produced one of the virus proteins (which could be done when we saw the genome sequence) for inclusion in the kit so that the antibodies can attach to it and give a positive result. The test itself can however be very quick (15 mins). The value of these tests is to indicate whether someone has had the infection and, if recovered, may be now immune to it. They have to be used carefully. Used too early, the convalescent patient may still be carrying and shedding the virus, they may still be a risk to others. Such kits have been developed (by e.g. BD and BioMedomics).
“The confusion about testing terminology comes because the antibody test is one form of “serological test”. Another form looks not for antibodies against the virus but for proteins of the virus, so called “antigens”. It uses similar technology to the antibody test but it works in reverse. We have had to identify then produce in the lab or factory antibodies specific for the virus. These will be included in the kit where they will bind virus antigens from the sample, returning a positive result. They would be equally quick to run and could be used at point-of-care, detecting the presence of the virus (earlier than the presence of antibodies). However, the complexity of identifying and producing the required antibodies for the kit means that development of the tests lags well behind that for antibodies. Another issue for antigen tests is the nature of the sample – qPCR tests indicate that there is little SARS-COV-2 in blood, so the test would probably have to work on swab samples.”
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