Monday, June 15, 2020

PCR, serotesting, population level detection - what does this mean for me?! (covid19)

After more then three months of living in a pandemic, everyone is tired of it. It doesn't matter if it's living in a lockdown where people weren't allowed outside, or a country that sort of didn't mandated isolation for more than risks groups but every country has seen either extreme lockdown, massive death or lockdown and some death.

A few buzz words (describing mitigation techniques) have been floated during this time. I think that there are a fair amount of misunderstanding when using a lot of these words/techniques - compared to what they mean in a epidemiological setting. And since I have spent a fair amount of my time last three months discussing, explaining and listening to my friends and family telling me what limitations are involved with "testing", I would like to write a post about them (might have to be several).

First, a difference between tests. Two tests are mentioned a lot; PCR and serological (antibody). PCR test will indicate if there is RNA from the virus inside of a person.
Serological (antibody) will show if the person has developed antibodies against the virus (and therefore can not be sick again from the same virus). That is "the person has already 'seen' the virus".

Both tests have details attached to them that most people wouldn't know or care about. My primitive opinion is that people shouldn't really need know these details since FDA/EMA/country's each agency that approves test should know these details and only approve according to high standards. It might be obvious that I have an issue with how tests have been approved since I made this sentence in italics.

To evaluate biological tests for diagnostics there are three things to remember (there are a lot of others but these three are fundamental for how to view the test accuracy and validity - big words to say "does the test work well"):

  • Sensitivity (how likely is a negative test to be really negative)
  • Specificity (does the test detect the right thing that it's looking for)
  • Limit of Detection (LOD) (related to sensitivity however slightly different and especially when talking about a whole chain of things together)

Before I go through these parameters, let's throw in another caveat with tests in general - especially since it goes hand in glove with these three fundaments. A test result should for all intent and purposes be seen as a probability and not a certainty. The golden and easy example for everyone to talk about is pregnancy tests. Those are, even if they are really really good, still a question of probability and certainty. The easy thing - the great thing - with pregnancy tests are that they measure one hormone (hCG) that only exists if the woman has a growing fetus inside. Then the test becomes only the question of "how low of a level of hCG can the test detect" (LOD). That's usually made into an equivalent of an average level of hCG at certain number of days after implantation, which is why tests have "early detecting, 5 days before period" (very low amounts of hormone can be detected) or "detection as early as first day of missed period" (a decent amount of hormone can be detected). If only all tests could be this clear cut.

With virus and PCR there is another issue that isn't much talked about outside the world of virus and microbiology. The fact that "just because RNA is detected doesn't mean that there is an infectious virus present". Dr Raciniello has described this on his blog a long time ago (here). The main take home message is "just because you find RNA, doesn't mean you find a virus particle that can infect." Further making it complicated, it will also depend where in the infection phase the person is, and what was investigated". It's been clear looking at published aggregated case studies from hospitals in regards to Sars-cov-2  virus is the likelihood of a positive test at different times in the infection within a person. The amount of virus, more specifically the amount of virus loose in the body available for detection and reinfection, varies a lot during the infection. There is a lot of virus day before symptoms show, there's evidence that 4 days after first symptoms there is more likelihood of testing positive. Combining the points (existing RNA but not active virion) seem to support the findings that 30 days after first symptoms, even if a positive PCR test, doesn't mean shedding (spreading) infectious virus. Brings back to the idea "it's a probablity, not a certainty". Also why hospitals where patients test negative on PCR but have distinctive 'glass lung' on CT are treated as covid19 positive.

(Side note for extra points; there is an assay that virologists use to evaluate virus load based on "plaque forming units" pfu which is dead cells that a virus have infected and killed, and that would give a "how many viruses who can infect exist in a sample" <- .="" p="" virions="">

All this is more than a 5 min simple read, and I apologize for even trying to cram a lot into a hopefully half-interesting blog post, I'm trying to explain why it's more complicated than "positive serotest" and "negative PCR and you're not sick" while not getting caught in the weeds.

So, back to the Sensitivity, Specificity and LOD.

When you take a PCR test - especially for an RNA virus - there is alway an overall chance/risk it will end up as a negative if you fail running the test itself. Why? Because the test is looking for a detection of something that will have to be going through at least 3 steps where the actual RNA might disappear before showing up in the test results. That's why there's always a control sample run together will all the real samples to make sure that all the steps worked out.

  • Sensitivity is generally descried as how good the test is to find the true positives of what the test is looking for. That is to say, only the people who really have RNA from the virus should test positive when using the PCR test. Similar for the serotest (antibody), only people who have encountered Sars-cov-2 and developed those specific antibodies should test positive.
It also tells about the false negatives, which would the least good outcome. This since someone who tests with a PCR test, to see if they have virus and therefore might spread to other people, that comes back as negative might not keep safe distancing to people but move along closer so a false negative brings people safety when they really shouldn't have thought so.
  • Specificity is generally described as how good the test is to show true negatives of what the test is saying it looks for. To simplify, if you test for sars-cov-2 RNA it's important that anyone with any old cold doesn't show up as positive (the common cold is part of the coronavirus family and share some parts of the RNA with sars-cov-2). If you don't have the virus, you are negative when testing.
There are repercussions of this part failing as well since looking at serotests (looking for presence of antibody in the blood) and if this shows antibody titers but it's the wrong type of antibody (say, from the common cold) then the test will have a low specificity.
  • Limit of Detection (LOD) is the lowest concentration/level of test subject that can be detected in a sample. The reason for my addition to the list is that when it comes to certain tests, the LOD will depend on a number of different processes than "just the level in the blood/saliva" as is the case for a lot of samples. Again the pregnancy test as an example, pee on a stick. The hormone is in urine. The test is designed for levels that are found in urine (side note, the blood test is more sensitive and have a lower limit of detection). There is no part of the test that requires any manipulation/handling of the urine (or blood). However for a PCR test of an RNA virus - there are a lot of details involved. First of all, there is the base level of "amount of viral RNA present in the nose swab" that has to be present to be picked up on the swab. Then there are different ways to get the RNA (virus) off the swab, extracted into liquid and being able to even "run the PCR on" (PCR=polymerase chain reaction, a mix of stuff in a little tube to amplify what is in the tube from the start). And the limitations on how big of a volume was the RNA extracted into and how little was brought on into the PCR reaction etc..... 
I'm aware I didn't do a great job on the LOD discussion if nothing else I hope that I made it clear that it is complicated and also super important. Even if you don't know how to calculate this step - it should be super clear in the FDA approval process since the LOD will determine the limits of the usefulness of the test. 

All three of these parameters will decide if the test is useful. It will further determine if the test is useful on an individual or population view. Why this difference? Because of statistics. If the test is only to be roughly an estimate on where the population - a higher lever of uncertainty (higher possible wrong results) are more acceptable since stats work that many tests together with a fairly high certainty turns into an ok stats.

The same test can have a detrimental effect on an individual basis. As previously stated, imagine going for a PCR test to determine you don't have the virus before going to visit your grandmother. You get a false negative (you think it is negative), and then go visit grand mother. That's a huge risk. Similar with testing if you have antibodies (the serotest) and it comes back positive (you have antibodies). You then might start doing relaxing and not wash your hands as much as before, and then you get sick.

All of these parameters are things that I (we) anticipate and expect that the FDA (EMA/country of your living) have evaluated and deemed acceptable. However, it's important to remember that in this case of the pandemic - there were a lot of changes in the acceptance criteria for a lot of tests. In order  to get an Emergency Use Authorization (EUA) the documentation from companies were relaxed a lot and most of all, not required to clearly state the limits on the test themselves.

You can argue that there isn't time to go through rigorous testing and documentation in a pandemic with an unknown virus - and I would agree whole hearted that time is of the essence. However, I would say that these details, the basis of the validity of the entire test, are extremely important to clearly state. If nothing else exactly to use when calculating the results on a population basis since the errors will compound/become very different based on these base numbers.

From an individual point of view - I wouldn't take a serotest without wanting to know the exact specifications since I wouldn't know what to do with a positive test unless I knew the cross-reactivity number. And that's not even mentioning the issue what kind of levels (or types) of antibodies that might be protective against sars-cov-2. 

Friday, June 05, 2020

know it all - intentional or not?!

"She's so knowledgeable about all these things. I mean, I'm American and I didn't know that DC doesn't have a congress person or a Senator but she did and she's not even from here."

Welcome to "my musings when I take things as a backhanded comment but maybe it really isn't but I had a rough childhood and end up in a defense situation".

Obviously at this time - internal self reflections are a clear sign of something not great. I feel that I should write something about BlackLivesMatters, the protests, the killings by the police, the shooting of teargas, the riots, the looting, the general upheaval of the constitution of the United States of America if the Active military are patrolling or surveilling regular US cities.

However, encountering another one of these comments as written in the first paragraph brought me back to my childhood. The comment this time was voices after we had discussed the current situation in the country and mentioned the yellow paint of BlackLivesMatter on the DC street. And I had seen a video where that street close to the White House was renamed "Black Lives Matter Plaza" 

I had a small smile since I find these things (non violent yet clear statements for the public) very on point and something to point the finger at something that isn't quite right. It's easy to prove when someone is upset about something like this, to object they have to state their case and that means they have to state WHY it's upsetting. Case in point; in Iran the British Embassy was on Bobby Sands street and there was short period time that the Saudi embassy in DC was located on Khashoggi way.

Anyway, this is my long way into a shorter comment that DC is a District of Columbia and doesn't have state hood and not right as the "other states" of the USA (since it isn't a state). I pointed out that there is a person who can talk in the House of Rep but doesn't have a vote, and that they don't have a senator. Briefly touched on the idea that DC wasn't originally planned to have people living there to vote since they were either IN congress or worked FOR a person in congress but in any event they were signed up in their state they represented, and then there were "non persons" who built the White house etc.... (african americans 3/5 of a person deal)

And I understand that this is part of my issue - that I get uncomfortable when people drop those kind of comments since I don't know if it's to point out that I make them uncomfortable, or if I push too much or if it's simply a "wow you know a lot". I know that I automatically say "well, I don't have any kids so I have a lot of time to read" as an excuse.

Part of my time in school as a younger child was a lot about making fun of me for knowing a lot. And I know that I at that time was less smart on how I said things so I probably came off as a know-it-all. However, as a grown up - there are so many times when I've decided not to rock the boat and not offer an explanation or a fact on the topic of discussion. Why? Because it's really not liked a lot of times. And, like when dating or getting a report with people in power at work or networking, it's just best to be a woman with "just enough" brain and wit.

My issues are those times are more obvious when discussing something that might have a more complex background and leading to some faulty conclusion. And when these things arrive - I've made a decision to state my knowledge and see what happens. If nothing else, I can feel ok with having said something, and not argue away. That's not the key point.

However, it would be great to not feel like a freak knowing things that apparently other people don't know. I guess I have to work on how I say things (yet again). Luxury issues - that's what this is. Real issues - outside our windows every day. #blacklivesmatter