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TwiceHorn

Corona Virus Testing

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Posted (edited)

I am really curious about this whole testing debacle.

It appears that we are using a conventional PCR (polymerase chain reaction) test.  PCR is a common biotechnical way of basically "growing" or amplifying DNA.  As I understand it, the test takes a sample of biological material, grows or amplifies the DNA material in it, and permits identification of the "grown" viral material.

Whether my understanding is accurate, it is fairly clear that a conventional PCR test requires a "thermal cycler," which is a standard, if not ubiquitous, lab instrument.  The instrument is not purpose-built for corona virus.  The amplifying operation takes several hours in the usual case, but you can apparently do multiple amplifying operations in parallel even on a single cycler (multiple samples).

So, the comparative rarity of thermal cyclers and skilled technicians to operate them and analyze their output for corona virus seems to be the limiting factor, as is the time to ship samples to a testing location, and the time to run the analysis.

In Texas, for example, you get swabbed and the material is shipped to one of the local or regional labs, for example Dallas County's at 2727 Stemmons, where it is tested as above.  The results are reported and also sent to CDC for confirmation.

I cannot find any explanation or elaboration of a test that is not as described above.  South Korea's testing operation used "drive throughs" for collection of samples, not analysis or the actual testing.  I read that it took three days to get results.  So that's consistent with the above.  Maybe SK has more thermal cyclers and technicians.  Or maybe they're just more accessible (closer) than those in the US, where they are spread out and more common in urban areas.

There are snippets of information about some kind of "quickie" test or some kind of testing apparatus that the WHO shipped out or something, but I can't find any detailed information about one, so I can't tell if that's rank speculation about some kind of vaporware or what.

Discuss, contradict, supplement with information, please.

 

 

Edited by TwiceHorn

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Current criteria, that I believe originated with the CDC, require both exposure and symptoms before testing.

I'm not sure if that's driven by scarcity of the tests or not.  I note that there are similar testing protocols posted for H1N1 and other past viral diseases, where presumably tests are not scarce.

I read another article that was a little more explicit about tests that don't seem to require a full-blown lab analysis, from the WHO, and even the CDC, that have proven to be flawed.  But I can't recite any details because there aren't any.

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What do know about Roche’s fully automated cobas 6800 and cobas 8800 Systems? 

It is claimed the turn around time is 3.5 hours.  I wouldn’t call it a game changer because there are apparently only 100 of them in the US. 

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Posted (edited)

Those are "generic" thermal cyclers with additional process automation features and capability to run other types of assays.

They apparently have a package of reagents and program to conduct CV testing.  One might think that it is adaptable to other company's machines.  But it seems to be basically the same PCR analysis described above.

Edited by TwiceHorn

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Posted (edited)

PCR is a widely available analytical tool that many hospitals and forensic labs are capable of running. The key seems to be in the choice of which viral genes are targeted and the availability of those needed reagents.

PCR uses a small amount of synthetic DNA, called primer, that's about 10 base pairs long. The primer is designed to only bind to a segment of viral complimentary DNA, usually at the start of the strand for one particular gene. The polymerase enzyme finds this primer-bound segment and begins replication. In this manner, only the DNA you want is amplified, and the much greater amounts of other DNA, such as from the patient, is ignored and eventually drowned out by the replicated viral DNA.

My guess is that the primer is the reagent in short supply that we keep hearing about. Not only that, but in an early CDC test a primer was chosen that targeted a gene non-specific to CoV-2, leading to ambiguous results or even false positives. The South Koreans and others appear to have chosen the correct gene to target.

The South Koreans also have an antibody test that works in minutes, much faster than PCR. These are much like the standard flu test.

Edited by berlinerbaer

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Posted (edited)
12 minutes ago, berlinerbaer said:

PCR is a widely available analytical tool that many hospitals and forensic labs are capable of running. The key seems to be in the choice of which viral genes are targeted and the availability of those needed reagents.

PCR uses a small amount of synthetic DNA, called primer, that's about 10 base pairs long. The primer is designed to only bind to a segment of viral complimentary DNA, usually at the start of the strand for one particular gene. The polymerase enzyme finds this primer-bound segment and begins replication. In this manner, only the DNA you want is amplified, and the much greater amounts of other DNA, such as from the patient, is ignored and eventually drowned out by the replicated viral DNA.

My guess is that the primer is the reagent in short supply that we keep hearing about. Not only that, but in an early CDC test a primer was chosen that targeted a gene non-specific to CoV-2, leading to ambiguous results or even false positives. The South Koreans and others appear to have chosen the correct gene to target.

The South Koreans also have an antibody test that works in minutes, much faster than PCR. These are much like the standard flu test.

Might some of the "test kits" we see referred to as being offered by WHO, for example, be "reagent packages" and associated stuff for a particular instrument?  As in the case of the Roche test for the cobas instrument?

I haven't seen anything that references the reagent as being in short supply, just the "tests" or "test kits."  I don't doubt you at all, I just can't find much that reports anything above the 8th grade level.

The "antibody test" I think is sort of the grail that everyone assumes exists.

By googling "antibody test," I hit on these two npr articles that state that SG and CN have antibody tests.  And the second mentions that "an ingredient" was faulty in the CDC test, probably confirming your hypothesis.  https://www.npr.org/sections/health-shots/2020/02/28/810131079/how-a-coronavirus-blood-test-could-solve-some-medical-mysteries

https://www.npr.org/sections/health-shots/2020/02/27/809936132/cdc-fixes-issue-delaying-coronavirus-testing-in-u-s

Also, the first article questions whether the Chinese and Singaporean tests have been validated for accuracy.

One of the governmental failures here is the FDAs failure to implement Emergency Use Authorization, which streamlines validation for approval.  I believe the Roche process has received this type of approval.

Edited by TwiceHorn

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5 minutes ago, TwiceHorn said:

Might some of the "test kits" we see referred to as being offered by WHO, for example, be "reagent packages" and associated stuff for a particular instrument?  As in the case of the Roche test for the cobas instrument?

I haven't seen anything that references the reagent as being in short supply, just the "tests" or "test kits."  I don't doubt you at all, I just can't find much that reports anything above the 8th grade level.

The "antibody test" I think is sort of the grail that everyone assumes exists.

A PCR kit contains two necessary reagents: primers and polymerase. In the case of CoV-2, a reverse transcriptase is also needed to first convert the viral RNA to DNA. Polymerase should be standardized for the PCR method regardless of the application and widely available, reverse transcriptase less so as it would be more application limited. My educated guess is that the primer is the scarce resource since it must be custom manufactured for CoV-2 genes.

Oligonucleotide synthesis, for making primers, probably can't be done on a large scale everywhere but it isn't rocket science. I'm still surprised at how slow things are going and have no good explanation why there should be a physical limitation to their manufacture.

Therefore, I chalk it up to poor planning and decision making. I'll now head over to the Cloak Room.

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33 minutes ago, berlinerbaer said:

A PCR kit contains two necessary reagents: primers and polymerase. In the case of CoV-2, a reverse transcriptase is also needed to first convert the viral RNA to DNA. Polymerase should be standardized for the PCR method regardless of the application and widely available, reverse transcriptase less so as it would be more application limited. My educated guess is that the primer is the scarce resource since it must be custom manufactured for CoV-2 genes.

Oligonucleotide synthesis, for making primers, probably can't be done on a large scale everywhere but it isn't rocket science. I'm still surprised at how slow things are going and have no good explanation why there should be a physical limitation to their manufacture.

Therefore, I chalk it up to poor planning and decision making. I'll now head over to the Cloak Room.

The RNA isolation seems to be a holdup, but a more complete answer is:

https://www.theatlantic.com/health/archive/2020/03/why-coronavirus-testing-us-so-delayed/607954/

The first 3 points are spot on. The 4th point is dubious. Through the last 30 years that RT-PCR and qRT-PCR have been around, you would think that someone, somewhere would have developed a system to test for emergent disease: I'm looking at the CDC and the FDA. 

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I would imagine we have loads of thermal cyclers the feds could requisition at a moment's notice. Besides if we ran out of them couldn't we macguyver one up using a sous vide immersion circulator?

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Posted (edited)
9 hours ago, Bevo said:

The RNA isolation seems to be a holdup, but a more complete answer is:

https://www.theatlantic.com/health/archive/2020/03/why-coronavirus-testing-us-so-delayed/607954/

The first 3 points are spot on. The 4th point is dubious. Through the last 30 years that RT-PCR and qRT-PCR have been around, you would think that someone, somewhere would have developed a system to test for emergent disease: I'm looking at the CDC and the FDA. 

Great article.  Thanks.  I was wondering about my google-fu (duckduckgo fu), which is usually scrong, until I noticed the date.

Gottlieb, Trump's first FDA director, actually seemed to be a pretty solid guy on multiple fronts.**  I was saddened to see him leave.  I note he promoted more testing in early February.    I know nothing about the current hack, but it appears s/he fucked the dog.

**Against the odds, Trump has made a couple of solid appointments.  Andrei Iancu has been a pretty good to excellent Director of the Patent Office and has actually depoliticized it compared to his predecessor.  And, although I am not an FDA professional, my sense and things that I read told me Gottlieb was pretty good too.

Edited by TwiceHorn

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48 minutes ago, gsoda3 said:

I would imagine we have loads of thermal cyclers the feds could requisition at a moment's notice. Besides if we ran out of them couldn't we macguyver one up using a sous vide immersion circulator?

There are instruments of various complication/sophistication, apparently.  With the right reagent, apparently you could amplify a sample and visually "test" it for coronavirus.  I imagine that would implicate the skill and training of the technician.

Some, maybe most, of these tests are apparently designed to run automatically on pretty sophisticated instruments, like the Roche.

Despite the recent cynicism toward capitalism and profit-taking in the pharma/bio sectors, it has been my sense from the past that most of those companies stand ready to help if they can and reap either publicity or monetary profit later.  The Atlantic article seems to confirm that, but that the government didn't prompt them to do so, and in some cases actually deterred them from acting.

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23 hours ago, Bevo said:

The RNA isolation seems to be a holdup, but a more complete answer is:

 

I've heard from a lot of my coworkers in the field that RNA isolation reagent kits (Qiagen) are becoming hard to get.

There are quite a few test and extraction kits/instruments available, but it's a matter of getting them in the lab and the people trained. From what I've seen the majority of labs use a Qiagen platform, so if Qiagen is having trouble keeping up with demand everyone is screwed. 

Could be time for the lab techs to kick it old school with some manual phenol chloroform extractions.

 

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7 hours ago, Seger78 said:

I've heard from a lot of my coworkers in the field that RNA isolation reagent kits (Qiagen) are becoming hard to get.

There are quite a few test and extraction kits/instruments available, but it's a matter of getting them in the lab and the people trained. From what I've seen the majority of labs use a Qiagen platform, so if Qiagen is having trouble keeping up with demand everyone is screwed. 

Could be time for the lab techs to kick it old school with some manual phenol chloroform extractions.

 

Macherey Nagel has the same technology as Qiagen (they jointly developed the original products and jointly held the patents). They were sold through Clontech (now Takara Bio). I've been to the plant in Duren, Germany and everything is made in-house. Scale-up for them should be easy. Anyway, there are many products available these days for RNA isolation and I know a few different ones have been validated for clinical use. If there is a problem clinically, I could personally help out. I doubt there is a problem though.

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I think there are multiple issues in play with initial testing strategy.  
 

- The supply will certainly be limited. Going from 0 supply produced to millions in a few days is not easy. Especially with the quality control needed to get something this serious, “right”.  Only doing the test at concentrated locations will improve efficiency.

- Putting the testing in locations that can have biggest impact. These 6800/8800 are big, expensive instruments. Connecting them to an IT system, to report back to as many ordering locations as possible to report data, etc... is already in place for Labcorp, Quest, etc...  Not at every hospital lab in country.  

- Having fewer locations to amass the data and send to CDC, etc...  will help the experts watch the trends, quality, various strand origins, etc...  

The testing is a game changer. Next is vaccine and/or treatment (think Tamiflu). Once all 3 are in place, we are back to normal managing this like many of other medical issues.

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Bump.  The medical thread has some peripheral info on testing.  Perhaps those knowledgeable can add to this.

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FInally, an explanation of why testing has been such a shitshow in the US of A.

https://www.reuters.com/article/us-health-coronavirus-testing-specialrep/special-report-how-korea-trounced-u-s-in-race-to-test-people-for-coronavirus-idUSKBN2153BW?utm_medium=Social&utm_source=twitter

The more I learn about the FDA, the more I believe it is an agency in serious need of reform.

 

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