We in the US are flying blind through the current epidemic, with cases popping up all over, testing done on very few people, and the rest ranging between nervousness and panic. What is the death rate? We still do not know. Did China contain its outbreak by draconian measures, or by wide-spread infection and natural burnout? How about South Korea, or Taiwan? Everyone claims the former, but it far from certain what actually happened. We need more testing, and particularly scientifically sampled population testing, and post-infection exposure testing. The basics of epidemiology, in other words.
SARS-CoV-2 is the virus, and COVID-19 is the disease. Most people do not seem to have mortality risk from infection, other than the elderly and infirm. In these respects, and in its great infectiousness, this disease resembles influenza. Testing from patient samples is done by RT-PCR, which stands for reverse-transcription polymerase chain reaction. The reverse transcription part employs specialized enzymes to copy the viral genomes, which are RNA, from the patient sample, into DNA, the more stable molecule that can be used in PCR. And PCR is the revolutionary method that won a Nobel prize in 1993, which uses a DNA polymerizing enzyme, and short segments of DNA (primers), to repetitively (and exponentially) replicate a chosen stretch of DNA. In this way, a minuscule amount of a pathogen can be processed to an easily detectable amount of DNA. The FDA mandates using three target regions of the new Coronavirus N protein encoding gene for its tests, but will accept one target, if the test is otherwise properly validated. They point test makers to the NAID resource that provides positive control material- RNA genomes from SARS-CoV-2.
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| Just the primers, Ma'am. These tubes contained dried DNA- the short primers with specific sequences needed to amplify specific portions of the SARS-CoV-2 viral genome. Using these requires quite of bit of other laboratory equipment and expertise. |
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| Schematic of PCR, the exponential amplification of small amounts of DNA to huge amounts. Primers are in green, nucleotides are light blue, and the target template is dark blue. |
So far, so good. But there are a range of test technologies and ways to do this testing, from the bare-bones set of primers, to a roboticized, fully automated system, each appropriate to different institutions and settings. To use the basic primer set, the lab would have to have RNA extraction kits or methods to purify the viral genomes from patient samples, then a reverse transcription kit or method, then a PCR machine and the other materials (nucleotides, high-temperature DNA polymerase, purified water and other proper solution ingredients). The PCR machine is basically a heater that cycles rapidly between the low temperature required for polymerizing and primer annealing, and the higher temperature required to melt all the DNA strands apart so that another round of primer annealing can take place. And all this needs to happen in very clean conditions, since PCR is exceedingly sensitive (of course) to small amounts of contamination. Lastly, the DNA product is typically detected by trace fluorescent markers that light up only double-stranded DNA, and can generally be detected right in the tube, with an advanced PCR machine.
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| Automated sample handling machines are used in clinical labs. |
Virtually all of this can be mustered by any competent molecular biology lab. Results would take a few days, due to the work involved in all the setup steps. The PCR itself and analysis of its results would take a few hours. But such labs do not operate at the requisite scale, or for this purpose. That is the province of clinical testing labs, which come in various sizes, from a small hospital in-house operation to a multinational behemoth. The latter run these tests on a vast, mechanized scale. They might manufacture the DNA primers themselves, or buy them in bulk, and have the proper logistical structures to do these tests from scratch in a reproducible way, to a high standard. Providers at these scales need different kinds of materials for their testing. A small provider may need a turn-key solution that comes with pre-packaged cassettes that just need the sample added before plugging into the machine, while a larger provider would save costs by using bulk reagents and massively robotized sample handling and PCR machines.
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| A one-hour test in a turn-key package. But at relatively high cost. |
So who are the players and what is the status? The CDC did not, for some reason, use the WHO test, or tests already developed in China, whose capacity for such manufacturing and testing is prodigious. The CDC at first didn't allow anyone else to run the tests, and when they did, they did not work correctly. It has been a bad scene and much valuable time has been lost- time that resulted in the US losing any chance of containment. Now, the FDA is authorizing others to run these tests, with detailed instructions about sampling, extraction, and machinery to be used, and is slowly granting authorization to selected manufacturers and kit makers for more kinds of tests.
Large suppliers like Roche and ThermoFisher have just been approved to supply clinical labs with testing systems. Most significant is Roche, whose tests are pre-positioned and ready to go already at clinical labs around the country. The biggest clinical lab, ominously named LabCorp, offers a home-made test, but only "several thousand tests per day", which is not yet the capacity needed. So capacity for testing will rise very rapidly, and soon enable the diagnostic and surveillance testing that is so important, and has been missing to date.
- Notes on previous pandemics.
Post script:
An aspect I forgot to include is how to select the portions of the viral genome sequence to include in testing kits. Different institutions have clearly come up with primers to different genes, few as they are, and regions within those genes. For example, "The primers currently target the N1, N2, and RP genes of the virus, but these are subject to change."; "In particular, the test detects the presence of SARS-CoV-2’s E gene, which codes for the envelope that surrounds the viral shell, and the gene for the enzyme RNA-dependent RNA polymerase." There is a balance between finding regions and primer sites that are unique to the particular virus you are interested in, so cross-reaction to other viruses is 100% eliminated, and the problem of viral drift and mutation. Some regions of viral genomes mutate much more rapidly than others, but these viruses tend to mutate at pretty high rates overall, so keeping a test current from one year to the next can be challenging. That is also what our immune systems have to deal with, as cold and flu viruses change continually to evade our defenses. So the specific DNA primer targets of a test need to be relatively highly conserved, but not too highly conserved, to put it in evolutionary terms, and the regulating agencies have to keep a close eye on this issue as they approve various test versions, to find a proper balance of high specificity and long-term usability.
Post-Post script:
Yet more significant testing solutions have emerged by late March, including a rapid (~10 minute) system from Abbot, and rapid antigen testing kits that also render results in the ~10 minute range. This speed is enormously helpful, obviously, from the patient, provider, and health system perspectives. The Abbot system is based on something called isothermal PCR, which gets rid of the temperature cycling described above. It is run at an intermediate temperature (~60 degrees C) where the DNA is somewhat loose, and primers can invade duplex strands, and also used a DNA polymerase that can displace duplex DNA as it plows ahead. This plus some other clever tricks allows the DNA amplification process to happen continuously in the reaction tube, going to completion in the rapid time quoted for these tests. These tests also tend to be tough- relatively robust to junk in the samples, and variations in temperature and other conditions.
The antigen tests that are coming on line are particularly significant, since they can be used for wide-spread population surveillance, to figure out what proportion of the population has been exposed, even if no active infection is present. Due to what seems like a complete or virtually complete lack of contact tracing + quarantine, the current pandemic will only stop once most of the population has been exposed, providing herd immunity. Before that point, anytime we give up self-isolation, it will start over again, due to the relatively high rate of low- or asymptomatic cases, and their lengthy course. Health care workers that have been exposed and recovered will have a special role before then by being able to freely staff hospitals that otherwise may be in dire straights.
Post-Post script:
Yet more significant testing solutions have emerged by late March, including a rapid (~10 minute) system from Abbot, and rapid antigen testing kits that also render results in the ~10 minute range. This speed is enormously helpful, obviously, from the patient, provider, and health system perspectives. The Abbot system is based on something called isothermal PCR, which gets rid of the temperature cycling described above. It is run at an intermediate temperature (~60 degrees C) where the DNA is somewhat loose, and primers can invade duplex strands, and also used a DNA polymerase that can displace duplex DNA as it plows ahead. This plus some other clever tricks allows the DNA amplification process to happen continuously in the reaction tube, going to completion in the rapid time quoted for these tests. These tests also tend to be tough- relatively robust to junk in the samples, and variations in temperature and other conditions.
The antigen tests that are coming on line are particularly significant, since they can be used for wide-spread population surveillance, to figure out what proportion of the population has been exposed, even if no active infection is present. Due to what seems like a complete or virtually complete lack of contact tracing + quarantine, the current pandemic will only stop once most of the population has been exposed, providing herd immunity. Before that point, anytime we give up self-isolation, it will start over again, due to the relatively high rate of low- or asymptomatic cases, and their lengthy course. Health care workers that have been exposed and recovered will have a special role before then by being able to freely staff hospitals that otherwise may be in dire straights.
