I continue to see widespread confusion in the news media about just what a virus test is, as well as what a positive test result means. I see the same confusion among people I know. It’s a quiet, warm Saturday afternoon, Memorial Day weekend, and I had promised (or threatened) to ‘splain virus testing to interested readers here at Coronavirus Central at AH. So today is a good day for that.
Here are diagrams of an infectious SARS-CoV-2 virus particle. On the left is an external view, and on the right is a cut-away with labels showing the various S, M, HE, E, and N viral proteins, the envelope (in red), and the coiled RNA inside. The S, M, HE and E proteins are located in the envelope layer, and the N protein is packaged with the RNA on the inside. The RNA is what's infectious, but only if it gets inside a cell. The purpose of the envelope and it's collection of proteins, is to deliver the RNA inside a host cell, and to protect the RNA while it's still outside of a host cell.
Virus RNA
Viral RNA, the virus’s genetic material, is measured by a technique called Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). It’s a sensitive assay capable of measuring extremely low levels of viral RNA. And it’s highly specific for SARS-CoV-2; it does not confuse other coronaviruses for SARS-CoV-2. It’s easily automated, allowing large numbers of samples to be run. However, the RT-PCR assay cannot tell us if the RNA is packaged in infectious virus particles or not. During the assay process, all protein and envelope material is stripped away, leaving the RNA bare for the next steps in the assay. The RNA could be contained in various non-infectious forms such as neutralized virus particles coated with a patient’s antibodies, incompletely assembled virus particles, or other non-infectious fragments of viral RNA.
ELISA
Without going into details, it is possible to measure the different virus proteins by Enzyme Linked Immune Absorbent Assays (ELISA). These assays, also easily automated, are sensitive and specific for each of the various virus proteins. As with the virus RNA assay, the results only tell us how much protein is present. It does not tell us anything about how much infectious virus is in a sample.
Antibody Tests
These tests detect antibodies directed against a virus in a patient’s blood sample. They don’t directly measure the virus itself, but might be useful if they reliably show whether a patient has been previously exposed to a virus. The problem with SARS-CoV-2 virus is that it’s similar enough to other coronaviruses, that antibodies against one will cross-react with antibodies against another. A patient may have been previously exposed to one or several other coronaviruses, at least 4 of which are known to be relatively harmless, causing common cold-like symptoms. The results appear as positive tests, false positives, in samples from people who were never exposed to SARS-CoV-2. About 40% of positive antibody tests are false positives, said to be caused by cross reactions like this. To be useful, antibody tests must be developed with false positive results brought down to a manageable level. This can certainly be done, but it requires a diligent validation effort, something that the FDA should always require.
Plaque Formation Assay (
https://en.wikipedia.org/wiki/Virus_quantification)
Plaque-based assays are a standard method used to determine infectious virus concentration. It measures a biological function, not the physical presence of viral RNA or proteins. Viral plaque assays determine the number of plaque forming units (PFU) in a virus sample, a direct measure of infectious virus quantity. This assay is based on a microbiological method conducted on host cells grown in petri dishes or multi-well plates. Specifically, a confluent monolayer of host cells (a lawn of cells) is infected with the virus at varying dilutions and covered with a semi-solid medium to prevent the virus particles from spreading too far.
When a single virus particle infects a single cell within the lawn of cells, the infected cell lyses (bursts open) and allows the viral projeny to spread to adjacent cells, where the infection/lysis cycle is repeated. This infected cell area creates an empty area, a plaque, surrounded by the lawn of living uninfected cells. Plaques can be seen after adding crystal violet to dye the cytoplasm of living cells, but not the plaque area where the virally lysed cells had been.
Plaques can take 3–14 days to form, depending on the virus being analyzed. Plaques are generally counted manually and the results, taking into account the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per milliliter (mL) of sample (PFU/mL). The PFU/mL result is the number of infective particles within the original sample and is based on the assumption that each plaque formed is representative of one infective virus particle.
This whole process (see the diagram) is tedious and labor intensive. Compared to the automated RNA or viral protein assays, it is much more difficult to do, especially if there are many samples taken from Covid-19 patients at various times during the course of their illness.
The bottom line to this story is that there is no simple way to correlate the viral RNA, viral protein, or PFU assay results. To find out a fudge-factor to estimate one assay result from another requires doing them all. There are no short cuts.
I hope this helps you to see the source of confusion between these different types of virus tests. It becomes especially important if, for example the viral RNA result is positive after someone recovers from Covid-19. How much of that viral RNA is actually actually infectious?
