Yves here. Ignacio describes some of the promising methods for Covid-19 diagnosis, and the considerations involved in scaling up their use. He explains why different countries may wind up opting for different tests, or different sequencing and frequency of tests. Sadly, I would assume those variations will complicate, and perhaps impede, aggregating findings across nations.
By Ignacio Moreno Echanove
I will try to focus on Covid-19 diagnosis not as a clinical tool but as an epidemiological surveillance tool badly needed If we are one day to return to whatever can be the new normal. This means that besides being accurate, the methodology used must be suitable for extensive geographical deployment (particularly sampling), early detection (even before symptoms appear), scalable (automation), and avoid delays in the flow of information.
Regarding accuracy the requisites are the same as for clinical diagnosis: The method must be precise (reproducible), sensitive (positive/infected > 85%) and specific (negative/non-infected closest to 100%). All those measures of diagnostic efficacy are relative, obtained by comparison to what is considered the gold standard for a particular pathogen. The gold standard for Covid-19 detection is based on the amplification of viral nucleic acids by RT-PCR tests (NAATs). In the case of NAATs for virus diseases, sensitivity can be measured as the number of viral genome copies per test required for 100% positive results. Real Time quantitative RT-PCR methods are quite sensitive detecting as few as about 3 genome copies in a single reaction (1). Well-designed NAATs kits are also highly specific and spurious amplification (reaction artifacts) or amplification of genomes other than SARS CoV 2 can be ruled out in best lab-conditions (1).
Being true that NAATs are quite sensitive and specific, and can be automated, there are problems with the “A” for amplification. Being so sensitive, you can amplify virus genomes obtained by sampling infected individuals and/or present in the reaction by environmental contamination anywhere in the process. This results in false negatives, lower specificity. Thus, precautions are needed in the whole testing process, particularly during the sampling because lab conditions are better controlled than sampling sets and internal controls can detect or rule out lab contamination.
Besides, such high sensitivity doesn’t necessarily translate into an optimal value for sensitivity in the real world (positives/infected). Problems with sampling or transport/storage, as well as the highly variable extent/state of the infection in each subject tested may reduce sensitivity very much. Sampling is probably one of the most important factors reducing the sensitivity of the method and should be done by trained people. Sampling is usually done by swabbing the nasopharyngeal and/or the oropharyngeal tracts (Upper Resp. Tract, URT) because it is convenient and fast. Some studies indicate that the combination of both (nasal+oral) gives best results (2). However, Covid-19 infections in the URT may not be very extensive and constant in time because there have been several reports indicating, for instance, that symptomatic individuals showed negative only to test positive in a second test.
Also, it looks like there is, in the population at large, wide variability in URT infection development that will be matched by similar variability in sensitivity. Information on infection dynamics in the URT is scant probably because this is not that important from the clinical point of view. Notwithstanding, this is critical from the epidemiological point of view and the ability of SARS CoV 2 to infect and replicate in the URT is what distinguishes it from the not so infectious SARS1.0 (3). This paper shows that the window for accurate Covid-19 detection in URT swabs in symptomatic subjects by NAATs is between 6-14 days after symptom appearance, with best results between 7-11 d.a.s.a. This is a narrow opportunity for test accuracy when you are testing many individuals.
Very little is known about symptomless or nearly symptomless individuals but it has been shown that they can prove positive in tests and be readily infected (3). All this translates into lower sensitivity than expected and some puzzlement amongst physicians. Besides, there are mistakes: Labelling and sample tracing mistakes aren’t that rare particularly in stressing situations. To complicate the picture symptoms reported during the first week are quite variable but anyone feeling fever, cough, cephalgia, rhinitis or sinusitis should be tested. Widespread allergies compound to make symptoms more unreliable.
Even with the methodological limitations exposed, NAATs that have been well designed, validated, and performed by properly trained (and protected) personnel are quite a useful tool for epidemiological surveillance as South Korea has demonstrated. To get an idea on what is needed, how the sampling is done, and how the HC system must be protected I recommend watching this video: https://www.youtube.com/watch?v=b0D8b1WZJTM, particularly go to minute 14 to see correct sampling procedures in a drive-through testing set. You will see the nasopharyngeal swab is much nastier than that from the oropharynx.
And an important conclusion one can draw from this video is that this is not all about the diagnostic method. Effective surveillance and containment require additional measures such as social distancing, greased reporting, fever surveillance and the population at large must collaborate in the surveillance and containment. Remarks by Prof. Schaffner at about min 3:30 of the video are quite correct. My conclusion is that the limitations of the method can be overcome with numerous, frequent and repeated tests that span well beyond reported symptomatic cases. Also, isolation of positives preventing home contagions might be necessary or at least desirable in those cases where self-isolation is difficult. In any case, the known difficulties with NAATs has sparked research on alternative methods.
I will skip immunological assays (detection of antigens or antibodies) because more time is needed for kit production and validation and many of these have drawbacks for deployment in epidemiological surveillance (5). Some are not sensitive enough while others are sensitive but not easily scalable etc. Detecting antibodies is important from a clinical point of view and also from the epidemiological point of view to have an idea on the state of immunity throughout the population but there are issues to discuss that would make this too long.
Yet, there are some other methods based on NAT. There are NATs that avoid amplification and are highly sensitive, for instance CRISPR based methods (a couple mentioned in 5). Other set of methods are based on RNA sequencing. Next generation sequencing, HiSeq, Single-Cell RNA sequencing, and/or single-cell RNA sequencing are rapidly evolving methods that have been developed to study and diagnose an array of non-infectious and infectious diseases. These are based on the sequencing of all cellular mRNAs, the “transcriptome”, that will include viral RNAs if infected. The sequences obtained are in silico filtered to remove human sequences and then you may obtain specific viral RNA sequences. You can run a ‘blast’ (tool for massive comparison of sequences with existing databases) with the sequences obtained and get unbiased results: this is certainly Covid-19, or no, this is flu H5N1.
These kinds of tests, though complex and requiring further training, can be easily automated, scaled up (“multiplexable” in the jargon) and some of them do not depend on the availability of processing kits. Investment in costly equipment is needed but it is already available in some research centres.
These methods, compared with real-time qPCR offer several advantages: the pathogen is simultaneously detected and genotyped, internal controls ensure unambiguity better that with qPCR and there isn’t amplification associated risks. I cannot show links but according to personal communications the sensitivity can be higher by an order of magnitude in terms of number of infected cells required for virus detection so these methods may allow for earlier contagion detection compared with NAATs. Regarding speed, results can be obtained in a couple of hours, similar to NAATs in this sense.
The problems are that standardization of the procedures is needed, followed by validation against the gold standard, and in many regions of the world there is not equipment neither trained personnel available. There are already some commercial systems based on these techniques using slightly different platforms only for research purposes. To my knowledge, some are in the validation process for Covid-19 in at least four countries. This epidemic could see the rise of these techniques as the new gold standard for clinical diagnostic of virus diseases.
Anyway, the South Korean experience, as well as some other and more negative experiences –for instance Spain is running out of imported processing kits for NAATS– suggests that each country will make decisions to ensure they have the necessary resources to keep their surveillance programs going on without depending on technologic platforms developed by possibly unreliable providers. This suggests that we may end with a mosaic of diagnostics methods developed and validated in each region.
- Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988269/
- Which sampling method for the upper respiratory tract specimen should be taken to diagnose patient with COVID-19?https://www.ncbi.nlm.nih.gov/pubmed/32166939 (abstract in English)
- Virological assessment of hospitalized patients with COVID-2019. https://www.nature.com/articles/s41586-020-2196-x_reference.pdf
- Alert for non-respiratory symptoms of Coronavirus Disease 2019 (COVID-19) patients in epidemic period: A case report of familial cluster with three asymptomatic COVID-19 patients. https://onlinelibrary.wiley.com/doi/epdf/10.1002/jmv.25776
- Coronavirus tests: researchers chase new diagnostics to fight the pandemic. https://www.nature.com/articles/d41586-020-00827-6
Best regards,
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Thank you Ignacio – it does seem to me that its quite likely that when all this finishes, we will not have any scientific consensus on the number of deaths or infections. There are an enormous range of estimates for Spanish Flu – its possible I think that a hundred years later, we are still no better at what seems at first sight to be a straightforward task.
Incidentally, Elaine (Lainey) Doyle, who wrote the twitter thread featured in todays Water Cooler, has an article based on it in the Guardian today where she addresses some of the arguments that were put to her on twitter about comparing Irish and UK rates.
The management of the epidemic in the UK seems to be awful. My friend living somewhere in the English countryside, and was infected with symptoms matching Covid-19 probably via school contagion, was told by the NHS to calm down and watch long movies (this is exactly what she has told me). This sparked lots of ironic comments on NHS treatments in our chat. Some days later the NHS phoned her to say that she was clear of Covid-19, no test provided, no matter she was still coughing. I don’t know if this is standard NHS procedure but, wow!
The delay in isolation measures in the UK is almost certainly a reason for fast epidemic development. So far, only Spain in the worst moment saw faster development that the UK (may be New York but I haven’t made the curve). Direct comparisons between Ireland and the UK in the very same dates might not be correct and one should “synchronize” the epidemics starting for instance the day when 10-20 cumulative deaths had been reported and see differences onwards. Yet because differences in overall population are big comparisons are difficult. The shape of the exponential curve but not the slope of the curve would say something about the management. I have “syncroniced” the curve for Italy, UK and France (similar population) and Spain (25% lower population). Spain was the fastest in the initial exponential phase (Initial R0 was highest in Spain) followed by the UK and then France and Italy at par. Then the slopes are more or less similar but the “champion” here is (so far) France with the biggest slope followed by the UK and then Italy and Spain at par. Now, to complete the picture we need to see how episode 1 unravels. Spain seems to be doing a bit better than Italy suggesting bolder reaction but it is too soon to say about the UK and France.
One thing that is striking about the UK is that the NHS and media seem very much on board with a very obvious and deliberate attempt to downplay the number of deaths in the UK. Its very clear that the number of deaths are far above the official levels – even private nursing home operators are saying this – but there seems little interest in the media in giving this anything but a passing nod. I can only attribute it to a Brexit hangover.
Add in the complicating factor of political backside-covering and nationalistic evasions, and this disease which challenges the whole of humanity, while it provides a chance to do something better and more “pro-social,” across the planet, the parasites will do their damndest to keep that from happening.
You were saying that Spain is running out of imported processing kits for NAATS which sounds bad. But isn’t Spain going to relax some of their restrictions now? What does Spain do if that virus comes roaring back again and they need those kits in quantity? I had assumed that testing would be a relatively exercise but after reading your article, I can see the layers of complexity involved. There seems to be no easy answers with this virus – not its diagnosis nor its treatment.
The lack of processing kits has solution but it is not immediate. There are labs now validating tests with new processing kits made in Spain, so this is being solved. This week we have started some relaxing with the restart of part of the construction activity. Quarantine is still in place and the streets are as empty as usual. Also, for those who go to work in public transport, masks are now provided for free (only this week) and there are controls to ensure distancing. I don’t fear this will result in a new outburst anytime soon. Yet, apart from the caution measures cited, testing will be necessary, as well as cooperation, to detect any new cluster.
I’ve been reading about ‘pool testing’ because it reminded me of computing optimization techniques. I read that its being used in India. Seems like an interesting optimization:
https://healthcare-in-europe.com/en/news/corona-pool-testing-increases-worldwide-capacities-many-times-over.html
I guess this is useful when the incidence is very low so that few pooled samples are positive and repeating tests from positive pools yet result in many less total tests. Testing resources but not sampling units are limiting. The sensitivity must be somehow lower but yet acceptable. Thank you for the link!
. . . to return to whatever can be the new normal
Useful: Merkel’s go-to experts
https://www.spiegel.de/international/germany/scientific-experts-release-proposals-for-loosening-the-lockdown-a-f6247ecc-0583-45b5-a269-ebf385c1e698
I very much appreciate this link. This are the studies I am looking for now. Thank you SOS.
In Madrid it has been estimated that about 600.000 have been readily infected. Nearly 10%. But yet we still ignore what percentage of the population has developed neutralizing antibodies or whether these are protective enough.
That is.
and well etc…
Ignacio Moreno Echanove, what an excellent job, thank you. A couple of points, I have been a NIH/NIS fellow(s) since 1991 ( mostly, extramural ) and have to say the ability to act on what you have presented, and sad as it is, has never been lower. trump and his minions, none of which believe in science and in fact hate it, have driven off most of directors, best researchers, and destroyed most of the extramural relationships. People have said people Biden and trump are the same, but as Biden may have dementia, he is not homicidally delusional. It’s not so much the feds won’t help but they can’t. Ok, small rant, ended.
You say “My conclusion is that the limitations of the method can be overcome with numerous, frequent and repeated tests that span well beyond reported symptomatic cases.” Absolutely, the repeated part especially until we know can people get re-infected and infect others, if one can be reinfected then… Covid-19, I’m have a waking nightmare thinking about it. Not that you want the job, put I’d, put you in charge of real task force tomorrow. Stay safe.
I feel sorry about what you wrote and hope the necessary infrastructure, equipment training etc is set up ASAP. The US has for many years and in many occasions showed a capacity to organize and deploy emergency measures in quite an efficient and possibly unparalleled way. I hope that there is still some leadership able to do so.
Ignacio, thank you for your work and wisdom! I just read this (my Italian is good enough to get the basic idea) from an Italian doctor in Bologna (Giannini) who believes that corvid19 might be primarily a cardiac event not respiratory and that anti-inflammatory meds are the best defense. This seems contradictory since there have been warnings against using powerful anti-infam drugs, like cortisone. Do you have an informed opinion about Giannini’s claim?
https://buongiornonews.it/coronavirus-una-speranza-dalla-scoperta-del-prof-giannini/
No. I can’t claim to have enough knowledge about the various clinical developments of the disease. For what I have read there are many variations, being the most common in severe cases a hyper-inflammatory response that “cements” the lungs but I don’t know the details. Yes I have read about it but not tried to comprehend it all. There are commenters at NC more knowledgeable on this subject.
My informed opinion on Giannini’s piece is that nobody, no doctor and no non-doctor, knows exactly what’s happening in a Covid19-infected body. And nobody will know in the foreseeable future.
Dr. Mary Helen Hensley documented on her FaceBook page her experiences to date, while staying in LA and contracting the disease. When her symptoms were serious she went to the hospital (Hollywood area). The surgeon who saw her (in an empty ER room, no one else waiting) told her all her symptoms definitely indicated she had the virus, and that to imagine it might be anything else would be ridiculous.
However at that hospital they had been instructed that morning that they could not use a test on anyone unless they were over 70 and in respiratory failure. She was told to go home and do her best on her own. If things got really serious (respiratory failure) she should come back.
The doctor also told her that what was being portrayed as the typical symptom profile was inaccurate. People were presenting with a wide range of symptoms (and sometimes lacking the most expected respiratory symptoms). Symptoms he’d seen (presumably including people he’d tested previously?) might have just some symptoms like sore throat, no sore throat, diarrhea, vomiting, no digestive issues, productive cough, non-productive cough, various kinds of rashes/pox, no rash/pox, high fever, no fever, chest pressure/pain, no chest pain, etc.
Fortunately with her professional medical background (chiropractor) Dr. Hensley had her own “toolbox” of ways of working with the numerous severe symptoms she experienced. She recorded her experiences in detail on her FaceBook page, in part to help reduce the fear level and to show that there were many ways of experiencing this virus, and also to show that getting accurate statistics was apparently NOT important to those regulating such things. (In the first week she was ill she and her temporary roommate called about 20 places trying to get a test, with no success.) She wanted to be tested and contribute to accurate statistics, but no luck.
Her roommate never got any symptoms at all.
Test availability (and thus any hope for accurate data) is still problematic.
Ignacio, thank you so much for your reporting on Covid 19, and thank you Yves for publishing it. I always study your comments.