Category: Testing

There is no test for COVID-19.  Instead, testing focuses on detection of SARS-CoV-2, the virus known to cause COVID-19 or the body’s response to infection.  Today we will discuss antibody tests, the blood tests used to detect past infections.

Part of a healthy immune response to infection by any virus is the formation of antibodies.  Antibodies are made in plasma cells, a special type of lymphocyte, in response to an assault by a foreign pathogen such as a virus.  The immune response is amazing.  Once plasma cells “learn” how to identify the virus, they crank out millions of these little molecules which coat the surface of the virus, flagging them as “enemy”, and directing other cells of the immune system to isolate and eliminate them.  Once the infection is cleared, the plasma cells “remember” the code for that virus so they can be quickly recruited to make more antibody if the same infection occurs in the future.  This memory eliminates the time-consuming “learning” step.  This simple process explains why most people only get chickenpox once and explains how vaccines can protect children from mumps and measles. 

The first antibodies produced after infection are known as IgM Antibodies.  These are large pentamers, basically 5 antibody units brought together like a snowflake.  IgM antibody production is replaced with IgG antibody production generally after about a week, but the time varies by individual and virus.  IgG antibodies are produced for weeks to years after infection, again depending on the individual and the virus.  Other antibodies are produced by the immune system, but IgM and IgG are most useful in viral serology.  These antibodies are found in the blood stream, so a simple blood test can detect a variety of antibodies, including antibodies to SARS-CoV-2.  It is thought that antibodies to SARS-CoV-2 is what makes convalescent plasma an effective treatment for severe COVID-19.

There are several facts to keep in mind when interpreting antibody test results.  First, antibody tests tell us about past infections, not active infections.  Second, the antibody form tells us whether the infection was recent or distant.  IgM antibodies are made first but go away, so detection of IgM antibodies means the infection was recent.  IgG antibodies are made last but stay around for a long time.  Detection of IgG without IgM means that the infection occurred in the more distant past, at least three weeks ago, and detection of both IgG and IgM means the infection occurred in the transition period.

Antibody tests use an immunoassay methodology, which basically uses antibodies to detect antibodies.  Many of these tests have good specificity and sensitivity, but they are not perfect.  Cross reactivity and interfering substances can cause false positive and false negative results in the best of circumstances.  But we are not in the best of circumstances.  All SARS-CoV-2 antibody tests are available in the U.S. by an emergency use agreement (EUA) with the FDA.  Rigorous studies demanded by the FDA approval process have not yet occurred.

So, if you are positive for SARS-CoV-2 antibody, will you always be?  Don’t know.  If you are positive for SARS-CoV-2 antibody are you immune to COVID?  Don’t know.  It is simply too soon to define the body’s usual response to infection and its implication for future infection.  

What can we learn from antibody tests?  If your antibody test is positive, you probably had a SARS-CoV-2 infection in the past, even if you do not remember being sick.  If you do not have symptoms, and you have not been around someone with COVID-19, then you are not likely to have a current infection.  Continue normal activities, but with vigilant precautions.  Wear a mask and social distance.  These precautions reduce the spread of the disease.

Elon Musk recently tweeted that he was tested four times in the same day.  Two tests were positive, and two tests were negative.  How can this be? 

Mr. Musk was tested with the BD Veritor Plus rapid antigen test for SARS-CoV-2.  BD Veritor is one of several SARS-CoV-2 antigen tests made available in the United States by an Emergency Use Authorization (EUA) with the FDA.  While PCR tests have some limitations, antigen tests are fraught with many more challenges that bring their results into question.  

First the good news.  Antigen tests are cheap, plentiful, and rapid, usually providing results in 15 minutes or less.  And, with one exception, all antigen tests can be performed in laboratories operating under a CLIA Certificate of Waiver, meaning that they can be performed by personnel with very little education in laboratory science.

So what’s the bad news?  I see three major problems with antigen tests: the test process, the lack of amplification, and poor sensitivity.

The antigen tests from different vendors all follow, with minor variations, the same process.  A sample is collected on a swab, usually from the back of the nose (nasopharynx), and the swab is placed in an incubation well on the test device.  A few drops of reagent containing antibodies against the target (in this case, the SARS-CoV-2 virus capsule) are added.  If the antibodies bind to the target, a signal is sent to the test system.  This signal is usually a color change.  The whole process is very much like a home urine pregnancy test.  

Well, what could go wrong with that?  Strong lines are easy to see, but what about faint lines?  Where is the cut-off between positive and negative?  Some vendors include a reader calibrated to take the guesswork out of reading results, but you begin to see the problem.  The test system introduces an element of subjectivity and operator technique that varies from tester to tester, and these variations impact test results.  Recall that most of these tests are intended to be performed by personnel with very little training in laboratory medicine.  

This process lacks the amplification step of PCR.  That means when the swab is scraped against the back of the nose, what rubs off is all that you have for the test.  If you happened to not scrap off enough virus in an infected person, too bad—the test will be negative.

And that brings me to the final problem with antigen tests: low sensitivity.  According to the FDA submissions by BD, the antigen test used on Mr. Musk has a sensitivity that may be as low as 67%.  What does that mean?  Sensitivity of 67% means that one out of every three infected patients tests negative with the system. That’s right: wrong answers one-third of the time! And that is in the best of circumstances, using data that the manufacturer chose to submit to the FDA. These tests are not always used in the best of circumstances; remember, the test is approved for use by personnel with very little education in laboratory science.

BD makes the following statement in its submission to the FDA: “Negative test results do not preclude infection and should not be used as the sole basis for treatment or other patient management decisions, including infection control decisions, particularly in the presence of clinical signs and symptoms consistent with COVID-19, or in those who have been in contact with the virus. It is recommended that these results be confirmed by a molecular testing method, if necessary, for patient management.” Mr. Musk’s experience is not surprising.

My colleague says she has a Magic 8–Ball on her desk that is a lot like an antigen test: it’s cheap, plentiful and rapid. And, she says, it gives about as many correct answers. I agree.

We want a test to tell us whether someone has COVID-19, but that test does not exist.  Instead, laboratory tests look for the presence of SARS-CoV-2, the virus known to cause COVID-19.  A positive test does not necessarily mean that someone has COVID-19 and a negative test does not always mean that someone does not have COVID-19.  How can this be?  And if this is true, what is all the fuss about testing?

Let me be clear.  Testing is the most important tool available to determine who has COVID-19 and who does not.  But it is not quite as simple as “positive” equals COVID-19 and “negative” does not.  At least not quite.  The test must be interpreted, and to do so, a few simple nuances must be considered.

The most reliable test uses PCR methodology, which includes both amplification and detection steps, making it highly sensitive and specific for the detection of virus.

A negative PCR test means that an individual is not currently infected and has not been recently.  But a negative PCR test cannot be used to predict the future.  In other words, a negative test last week does not prove someone is virus-free today.  Instead, a negative test simply tells us the last time a person was known to be negative for virus.  The significance of a negative test result diminishes rapidly as the collection time fades into the past.  

A positive PCR test means that the person tested is or has been infected by the virus.  If the individual is also symptomatic, we can say the person has COVID-19.  That patient is also infectious (i.e., can spread the virus to others), and should be considered infectious for at least ten days after the first positive test, or for 24-hours after symptoms resolve, whichever is longer.

Our best understanding is that a person does not need to have COVID-19 to spread the virus to others.  Most people test positive five to seven days after exposure, but they can infect others for ten days from time of exposure, longer in exceptional cases.  That is why a person is considered infectious and instructed to quarantine for at least ten days after the first positive test, even without symptoms.  A PCR test may continue to be positive for weeks to months after infection, making it impossible to know whether asymptomatic people with a first-time positive test are newly infected or were infected in the past.  Because we cannot know when the person became infected, the asymptomatic individual should be considered infectious for at least ten days after the first positive test.

Another consequence of the persistence of positive PCR tests after the infection has cleared is that there is no need to require a negative test to prove an individual is no longer infectious.  After the appropriate amount of time has passed since the first positive test and/or resolution of symptoms, an asymptomatic individual should be considered virus-free for at least three months. 

If you may have been exposed, when should you test?  A test for SARS-CoV-2 becomes positive 2 to 14 days after infection, with most patients turning positive five to seven days later.  Most authorities suggest testing no earlier than five days after possible exposure, unless you have symptoms earlier.  But if you have to wait five days, should you even test at all? Whether you test or not may be a decision that addresses your peace of mind more than anything else.  The most important thing you can do if you think you may have been exposed is to self-quarantine for two weeks.  That is how you keep others safe and stop the virus spread. 

There is no test for COVID-19, the disease of the pandemic.  Instead, tests look for the presence of SARS-CoV-2, the virus known to cause COVID-19, or the body’s response to SARS-CoV-2.  This last group, known as “antibody” tests, have found very little utility during the pandemic. There are different kinds of tests in the first group, the tests for the virus, and the reliability of these tests vary widely based on methodology.  Today, we will discuss the gold-standard method: polymerase chain reaction, known as “PCR”.

PCR is a several step process.  It begins by exposing the sample to the components necessary for replication of nucleotide segments.  Nucleotide segments are pieces of DNA or RNA which carry the genetic material of cells.  By exposing the sample in this environment to a series of 30 to 40 heating and cooling cycles, all of the DNA or RNA sequences contained in the sample are amplified—and not just 30 to 40 times, but one to 2³⁰ to 2⁴⁰ times.  That’s one billion to one hundred billion! This produces a highly sensitive test able to detect minute quantities of genetic material.

But, you might ask, if all genetic material is being amplified, won’t other genetic material in the sample not related to the virus be amplified also?  Yes, it will, but that’s where the second part of the test becomes important.

Probes are incubated with the amplified sample that bind to a specific nucleic acid sequence that is known to be unique to the target—in this case the SARS-CoV-2 virus.  If that sequence exists, then the probe binds, and a signal is sent to the test system.  If the sequence doesn’t exist, then there is no signal.  It’s like looking for a specific password to a website among all the passwords that could possibly be entered.

But, you might ask again, couldn’t that nucleic acid sequence exist in the sample just by chance?  That would be like you guessing the password to a billionaire’s bank account; unlikely, but not entirely out of the question.  

That is why PCR tests for SARS-CoV-2 include a second set of probes, specific a different nucleic acid sequence that is also unique to the virus.  Only if both signals are detected is the test positive.  Some PCR tests even use a third set of probes.  The result is a highly sensitive (meaning detection of tiny amounts of viral RNA) and a highly specific (meaning false positive rates close to zero) test system.  

But like any human endeavor, PCR is not perfect.  False negative results can occur if the sample is improperly collected, resulting in no viral RNA to amplify.  Sloppy analytic practices can lead to false positive results by a process referred to as “carry-over”—literally, the genetic material from one sample is carried-over to another sample.  

There is another source of apparent false positives: a patient who is not sick and not infectious but has a positive test.  I call this pseudo-false positive, because it’s not a false positive result at all.  Instead, it’s a failure to interpret the result correctly.  Remember that laboratories do not test for the COVID-19 disease; they test for the SARS CoV-2 virus itself, and in the case of PCR tests, for viral RNA.  Viral RNA is known to persist in detectable quantities for weeks, even months, after the patient has recovered from the disease.  How is this possible?  Because to be infectious, the virus must have the genetic code wrapped in a capsule that binds to human cells.  Without the capsule, the RNA is inert.  When the body clears an infection, it denatures the capsule.  Remnants of the viral RNA persist for a long time afterwards, just like a bombed-out city after a war.

It gets even more complicated.  There are tests which have unfortunately received Emergency Use Authorization (EUA) from the FDA which are not as sensitive or specific as PCR tests. These are many of the super rapid tests (<15 minutes), some of which have sensitivity rates as low as 67%.  These tests are known as “antigen” tests, and we will address these tests soon.  Next, we will discuss the meaning of positive and negative tests.

It may surprise you to learn that there is no test for COVID-19, the disease of the pandemic.  Instead, tests look for SARS-CoV-2, the virus known to cause COVID-19, or the body’s response to SARS-CoV-2.  These different tests are all commonly (but inaccurately) called “COVID Tests”, probably because it is easy to say.

Tests for SARS-CoV-2, like all laboratory tests, have two phases: pre-analytic and analytic.  The analytic phase occurs within the walls of the laboratory at the testing bench.  The pre-analytic phase includes all activities that occur up to the time that the specimen is placed on the analyzer in the laboratory, including specimen collection and transportation.

A direct test for the virus requires collection of a sample by swab.  The greatest concentration of virus is found in the back of the nose (“nasopharynx”), so that is where most swab collections are taken, but virus may also be found in other places in the body, including the middle portion of the nose and in saliva.  Collection can be uncomfortable.  There is no blood test that can directly detect the virus.  Most drive-through “testing” centers are actually drive-through collection centers since very few actually analyze samples on-site.  Most of these sites send their collections to a large central laboratory for analysis. 

A test for the body’s response to viral infection is known as an antibody test.  The antibody is present in the bloodstream, so a blood sample is all that is needed for this test.  There is a time lag between infection and detectable antibody in blood, so an antibody test is not useful to detect patients that might infect others.  Therefore, antibody tests have had limited utility during the present pandemic. 

The most accurate test for SARS-CoV-2 is the PCR test, which will be the subject of the next blog.

Before we can talk about testing, we must first agree on terminology.  The official name of the disease of the pandemic is COVID-19, short for Coronavirus Disease of 2019.  The official name of the virus that causes COVID-19 is SARS-CoV-2, so named because it is a coronavirus (“CoV”) that produces Severe Acute Respiratory Syndrome (“SARS”).  The “2” is tacked on the end to distinguish this virus from the SARS-producing coronavirus that caused the outbreak in 2003; that virus is now called SARS-CoV-1.  I am just reporting here—no one asked for my help to come up with these names.  

The relationship of SARS-CoV-2 to COVID-19 is the same as the relationship of HIV to AIDS.  The first is the name of the virus; the second is the disease that may be caused when infected by the virus.  I said “may” because being infected by the virus is not the same as having the disease.  In order to have the disease, you must test positive for the disease-causing virus, and you must also have the disease’s defining symptoms.  In the case of AIDS, the defining symptoms include co-infection by at least one of a long list of “opportunistic organisms”, organisms that take advantage of a weakened immune system.   In the case of COVID-19, criteria for disease requires symptoms of respiratory illness which may range from “cold symptoms” to pneumonia.  A person infected by SARS-CoV-2 who does not have those symptoms does not have COVID-19.

It is important to note that there is no laboratory test for COVID-19.  Instead, the laboratory tests for the presence of the virus that causes COVID-19 (i.e., SARS-CoV-2) or the body’s response to infection by that virus.  Next time, we will discuss the various tests available for detection of the virus or the body’s response to infection.

Much attention has been given to testing this year, so much attention that the subject has become confused and misunderstood by many.  What is testing?  Who should be tested and when?  What kinds of tests are there?  What is the meaning of positive and negative results?  These and other questions should and do have clear, simple answers. But in the noise that accompanies the pandemic, clarity is lost.  The result is widespread and profound misunderstanding of the utility and application of diagnostic testing in our fight against COVID-19, the disease of the pandemic.

A clear understanding of testing is important.  Diagnostic excellence is the foundation of excellent treatment outcomes.  Clarity of understanding must precede planning and execution.  Tragic accidents are more likely in the fog of distorted perceptions.  Using my perspective and experience as a clinical pathologist, my only purpose here is to make the use of laboratory testing as clear and understandable as possible.

During the nine years I spent in medical school and pathology training at the UT Southwestern Medical Center at Dallas, I had the good fortune to encounter some of the most renowned researchers and practitioners of my profession, men and women whose mind-power far exceeds my own, but whose common sense approach to solving diagnostic problems had a profound and enduring impact on my career in pathology.  Added to the formal education and training, I have spent more than 25 years in practice at a mid-sized community hospital as pathologist and laboratory medical director.  My career has driven home fundamental lessons of laboratory medicine: when it is important to test, when it is better not to test, and how misunderstanding of test results can lead well-meaning doctors astray, to the detriment of their patients.

In the blogs that will follow, I will sacrifice scientific rigor for clarity and understandability.  I am a practitioner, not a scientist.  Accordingly, my emphasis is on the use and pitfalls of diagnostic tools, not the precise science that makes the tools possible.  The science is fascinating, and, at a certain level, necessary for the appropriate use of the clinical laboratory.  But a rigorous understanding of science is not the same as mastery of use of the diagnostic tools created by science any more than the knowledge of piano construction confers the ability to play.  Incidentally, I firmly believe that even our most fundamental scientific theories should never be considered true and unchangeable representations of reality, but rather artificial constructions that make certain phenomena understandable and predictable.  However, as I am not a scientist, I am neither a philosopher, so I leave to the philosophers the further contemplation of these compelling yet impractical notions.  For now.