There has been a lot of mania surrounding COVID-19 testing since the beginning of the pandemic. First, it was “there is no test!” Then it was “we have the test but can’t get the supplies/reagents!” Then turnaround times were greatly extended. Finally, the grand finale was lack of trust in the results. 

It seemed like every week there was someone new taking to social media screaming about how their test results make no sense. First, they had a COVID test and it was negative. Then a few days later another test, but this time the result was positive. They take another test to confirm the positive and get a negative result. The person takes this as proof that there’s a conspiracy going on and posts on every possible social media website to “warn people.” It is safe to assume that these people have little to no understanding as to how hospital lab testing works. 

It seems strange, but it is possible to have a negative-positive-negative or positive-negative-positive series of COVID-19 tests. There are several reasons for this and hopefully, this blog post will be able to break it down and make it easier for the layperson to understand why.

Types of COVID-19 Tests

Before explaining how discrepant test results can happen. It is important to understand the principles behind some of the common COVID-19 testing methods. Differences in testing methods are a contributing factor to this phenomenon.

Antigen Tests

Principle

An antibody reacts to a protein on the surface of the virus and produces a reaction. Most antigen tests are lateral flow assays, or rapid tests, that detect the presence of a target substance (in this case, the SARS-CoV-2 antigen) in a patient sample. Material on the patient’s swab is eluted (extracted) in a buffer solution. The eluted sample wicks up the reaction vessel and reacts with antibodies embedded in the membrane of the vessel. If the SARS-CoV-2 antigen is present, a color change indicates that the patient has a positive COVID-19 antigen test.

Pros and Cons

Antigen tests offer many advantages over traditional PCR tests. Antigen tests are quick, cheap, and can be done at the point-of-care. These tests can be used pretty much anywhere and, because they are waived, laboratory personnel do not need to be the ones performing them.

The disadvantage to this type of test is that it is less sensitive than PCR testing and also less specific. This means that a patient would need a very high viral load in order for the test to generate a positive result and that there is a possibility of cross-reactivity with other coronaviruses. If negative, this test does need to be confirmed with PCR.

RT-PCR

Principle

PCR tests detect a specific sequence in the viral RNA. The RNA, if present, is converted to DNA, and the entire strand is amplified. The presence of viral RNA is detected by a fluorescent probe attached to the DNA primers used in the amplification steps. As the DNA is amplified, the fluorescent probe is released and an instrument detects the signal emitted from this probe.

Pros and Cons

This test is considered the gold standard because it is extremely sensitive and specific. It can detect very low levels of virus and can identify pre-symptomatic individuals.

The main downside to this type of testing is that it is very expensive. This test requires specialized instrumentation and specially trained personnel to perform the testing. PCR testing also has a longer turnaround time and results may not be available for a full 24 hours.

LAMP

Principle

Loop-mediated isothermal amplification is a method that amplifies a specific sequence of RNA or DNA. Four to six (4-6) primers are used to recognize six to eight (6-8) distinct target regions of the RNA. These regions are then amplified at a constant temperature. A DNA polymerase initiates synthesis and forms loop structures at the end of the DNA strands to facilitate synthesis by extension and annealing of more primers. The amplification is then detected by either fluorescent probe or turbidometry.

Pros and Cons

LAMP tests are very specific and are faster than traditional PCR tests. They also require less expensive equipment and can be done at the point of care in some instances.

The problem with this type of testing is that the current platforms that are on the market require fairly high viral loads. They are less expensive than traditional PCR but more expensive than your standard laboratory test.

Positive-Negative-Positive: No, big tech CEO…it’s NOT a Conspiracy!

Do you remember back in November 2020 when a certain big tech CEO had a Twitter meltdown over his COVID test? Apparently, he had 4 rapid antigen tests done. Two gave positive results and two gave negative results. The same nurse did all 4 tests and used the same machine. According to this CEO, this was proof that there must be something afoot. After all, if the tests are certified by the FDA, they must 100% accurate all of the time, correct?

Nope and now let me tell you why…

Sensitivity (Limit of Detection)

Sensitivity is the lowest level of an antigen a test can detect. Different testing platforms have different sensitivities or limits of detection. For example, let’s say a patient is in the very early stages of their COVID-19 infection. The patient goes to urgent care and has a rapid molecular test done and the result is negative. The patient then tells the clinician that they went to a maskless rave in a poorly ventilated warehouse over the weekend. This information prompts the clinician to collect a second swab for a RT-PCR test that will be performed at a reference laboratory. Three days later, the clinician contacts the patient to inform them that the second test is in fact positive. 

“How is this possible?”, the patient demands. The clinician proceeds to explain to the patient that the test performed in the office has a limit of detection of 125 copies per microliter, whereas the reference lab has a limit of detection of 30 copies per microliter. The reference lab test is much more sensitive and more likely to catch pre-symptomatic individuals or individuals in the early stages of infection. The doctor explains to the patient that their viral load was 105 copies per microliter and, therefore, would not be detected on the instrumentation used in the office.

Specificity

Specificity is how well a test detects only the particular antigen it is supposed to detect. To clarify, there are 4 different coronaviruses that can cause the common cold. Some antigen tests on the market for COVID-19 have cross-reactivity with these other coronaviruses. A person can test positive with an antigen test and then negative with the more sensitive and more specific RT-PCR test. That does not mean the first test was wrong; maybe that test was only 80% specific. That means that there is a 20% chance that another coronavirus can give a positive result.

Let’s look back at Mr. Musk. The antigen test he had performed may not have been that specific. It is possible that cross reactivity with other coronaviruses could have caused false positives. 

Collection Technique

This is probably the single biggest thing that will cause variation in results. Two nurses can collect a specimen on the same patient at the same time and end up with two different results. If proper procedure is not followed, a low-quality specimen will be obtained and could cause false-negative results. For example, specimens collected from the anterior nares need to be collected from both nostrils. If this does not occur, there is a possibility that a false negative could occur.

Circling back to big tech CEO, who had two positive and then two negative tests. What’s the deal? Performing four COVID-19 tests in a row is pretty uncomfortable. In the first two tests, the nurse may have been able to obtain a high-quality sample. In the second two tests, the patient may have been less cooperative, which made it harder for the nurse to obtain a high-quality specimen.

Specimen Source

The gold standard for COVID-19 testing is considered the nasopharyngeal swab. Most commercial tests on the market were originally tested using NP swabs. This is because the virus tends to travel down into the nasopharyngeal cavity and then eventually into the lungs. Specimens collected from different areas may not give exactly the same results. A specimen collected from the anterior nares at the same time as an NP swab could quite possibly generate a negative result, whereas the NP swab is positive.

Type of Test

There all several different kinds of COVID-19 tests currently on the market. Each one of these tests has different limits of detection, different ways they should be used, and different methods of detection. Some tests have very specific windows in which it is optimal to test a patient, such as two days after symptom onset, but no more than seven days after. Other tests are more flexible. If these tests are mixed and matched, discrepant results can occur.

Storage Requirements

Each test has optimal storage requirements to guarantee the best results. If the test is not run within the appropriate time frame, discrepant results can occur. Dry swabs need to be run within an hour of collection to ensure that RNA does not degrade and no false negatives occur. Samples collected in viral transport media have stability up to 24 hours at room temperature and longer if the specimen is refrigerated or frozen. If a patient has two COVID tests close together, discrepant results can occur if the storage requirements are not adhered to.

Training

It’s important that all personnel collecting specimens and performing tests have thorough training. Variations in training can account for testing discrepancies. A nurse may not be told that, when collecting direct nasal swabs, both nostrils need to be swabbed, unlike with an NP swab. A technologist performing the test on an off-shift may not have all the testing updates communicated to them. If the testing company updated a specific part of the procedure and this does not get communicated to the tech performing the test, this could inadvertently cause false negative/positive results. 

Timing

Let’s go back to our specificity example: A grocery store worker goes to the doctor because one of their co-workers recently tested positive for COVID-19. The worker wants to get tested just in case.

They go to the urgent care and have a rapid antigen test performed and it is positive. However, the clinician realizes that the particular test used is not that specific so they send out a PCR test to the reference lab.

Due to a high volume of tests being performed at the reference lab, turnaround time is greatly increased and the patient doesn’t get their results back until 8 days later. The test is negative, but now the patient is paranoid. Which test do they trust?

The patient decides the best two out of three and goes back to the doctor for a second PCR test. As the patient awaits the results, they start to get sick. The patient gets their results back and the second PCR test is positive.

Conclusion

In summary, yes it is possible to have COVID-19 test results that don’t correlate when it seems like they should. This does not mean that certain tests are better than others.

Tests need to be interpreted by trained personnel. The clinician ordering the test needs to know the limitations of a particular test, when they should order confirmatory testing, and what type of test should be used for confirmatory testing. It is also up to the clinician to properly explain to the patient the test results, what they mean, as well as the limitations of the test.

Executing proper and consistent collection and testing techniques are all important when performing a test on a patient.

Positive-negative-positive…Is this a possibility? Yes, it is.

Does this mean that COVID tests cannot be trusted? No, it does not.

Does this mean that medical personnel and “Big Health” are playing some sort of game with the public? Trust me, they are not.

Health care professionals all over would love for this to be over sooner rather than later.