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Q&A: Hong Kong University's Leo Poon Discusses Diagnostics for SARS-CoV-2

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NEW YORK – An expert in emerging diseases and public health, Leo Poon has pioneered molecular characterization and diagnostics development for the zoonotic viruses that straddle the animal and human worlds, like pandemic influenzas and coronaviruses.

In 2003, Poon and his team at Hong Kong University managed to sequence the Severe Acute Respiratory Syndrome, or SARS, virus and develop a molecular diagnostic test within weeks of the outbreak's onset, a feat dubbed a "technologic tour de force" at the time. The group also compared the sequence to viruses from animals to establish that the SARS animal reservoir was a type of wild cat-like creature called the Asian palm civet. Keeping with the tradition some 17 years later, Poon and his team have also developed and published the first molecular test for SARS-CoV-2 validated in clinical samples.

Poon is currently a professor and head of the division of public health laboratory sciences at Hong Kong University. In a video interview from his lab on Tuesday, Poon commented on the challenges for controlling the current outbreak, and preventing future viral spillover events. He also emphasized that the pace and vigor of scientific dedication and discovery globally over the past two months has been remarkable. 

Below is an edited transcript of the interview. 


Can you describe your background, and your role in developing the SARS-CoV-2 assay that was published recently in Clinical Chemistry?

I am a biologist, and I have been involved in infectious disease for the last 20 years. I study SARS, Middle East Respiratory Syndrome, H5N1, H7N9, so basically coronaviruses and influenza viruses of animal origins. I was the one who decoded the first [SARS] coronavirus sequence from which we developed a diagnostic test.

For this particular SARS-CoV-2 event, once we had the first public sequence available, we designed primers and probes to try to detect this virus. In the beginning, we didn't know much about the sequence variation. We only had the one sequence, so we didn't know whether this sequence is actually real, whether there is any sequencing error, or whether there are other types of similar viruses circulating in the animal or human populations.

We decided to develop assays which can detect basically all the SARS-like viruses in animals, and also in humans, including SARS and SARS-CoV-2.

We don't have SARS in human cases anymore, so anything that is positive by this test we would assume that they have been infected by SARS-CoV-2.

We have had the assay quite a long time, as early as mid-January, but we did not publish it, we only shared the protocol with the World Health Organization. We only published it once we had used the assay to test real clinical specimens.

Was it the very first SARS-CoV-2 assay in the world?

No, it was the second. There is a German group under Christian Drosten. He published a test, but without using clinical specimens, just positive controls. This is a good thing because they tried to use other strategies to evaluate their assay. For us, we were reluctant, because we really wanted to have real clinical specimens before we published. And since we are in Hong Kong, closer to China, it is much easier for us to get samples from China, or at least to get some of my collaborators to test my assay in China. We have a geographical advantage, and that is why we wanted to wait until we tested clinical specimens.

Is your test being used now in Hong Kong, or anywhere else?

Once we had this assay, we developed a protocol and posted it with WHO, and I also shared it with my network. We have distributed this assay to over 40 labs all over the world. I cannot name them all, but some countries have been using my assay to detect patients. For example, [a lab in] Egypt used my assay to pick up the positive case — but this one is public, so I can talk about it.

We also serve as a reference lab for WHO. We receive samples from overseas and try to confirm their cases. The example that I can name is Nepal. They had their first case, they sent a sample to us, we used the assay and confirmed the case, and they published in Lancet: Infectious Diseases three weeks ago.

Did they send the testing to you because they don't have the lab capabilities to do a test in Nepal?

Not really. There are two aspects. First of all, they didn't have the primers and probes in the beginning. It takes time to order these, particularly in developing countries. What we did is, we prepared a big batch of these primers and probes and sent these to countries that requested them.

Also, having been diagnosed positive is one thing, but then it is essential to get a reference lab that actually has been trained to do this test properly to confirm some of the cases. At least the early cases. There are a lot of technical problems, such as the staff is not able to run the test properly, or they are using the wrong reagent, and so on. It is better to try to send some positive samples, or even negative ones, to a reference lab, so that they can confirm the results are correct and so we can say, 'Hey, this lab has the capacity to do the testing, and do it correctly.'

And, we can sequence the viral genomes. We sequenced the viral genome from Nepal, for example, and we shared this information in the Global Initiative on Sharing All Influenza Data (GISAID).

There have been some problems with a test developed by the US Centers for Disease Control and Prevention — issues with negative control specimens testing inconclusive in some regional public health labs. Do you have any insights into what might be happening?

No, I think CDC knows better than me about their assay, so I can't comment on that. But you know, things do happen. That is the reason we wanted to test our assay with real clinical specimens in the first place.

At CDC, they are highly experienced scientists, so they know these tests should be extensively evaluated. But now, we are in a contingency situation. They tried to make sure that the regional public health laboratories have the capacity to do the test. That is why they had to move very quickly. Sometimes problems do happen, and it is good to know that they know that they had that problem, and that they can try to correct the problem in a timely manner.

Representatives of US public health labs here said today that they would like to make their own reagents, or get them commercially, so they can start running testing immediately. But now we have a regulatory issue, because once Emergency Use Authorization was issued, labs are not supposed to run any lab-developed tests.

That is a major problem we see. You know, viruses don't wait for us. They don't respect borders. This is spreading. There are existing tools which are known to be working, but now because of an administrative issue, that holds things up.

I know that in normal situations you should respect all the administrative regulations, but now, we may have a pandemic. I think we should try to move things forward, and try to override some of these problems. Of course, I understand a poorly developed test may create problems, I absolutely agree with that. But there are some tests which have been used extensively. Why don't they just use those tests?

Was there ever a pan-SARS test developed in the past that could be quickly adapted now for screening?

Yes, the test that I developed is basically a pan-SARS. It cross-reacts with SARS and SARS-CoV-2. There is another assay which I published more than 15 years ago that can cross-react with all coronaviruses, including animal and human ones. The problem is, humans also have OC43, 229E — these are all coronaviruses [that circulate in the community], and these would be positive on this test, as well. So that one may not be a good test for identifying SARS-2 patients.

There are many companies that have announced they are making tests and reagents – including lab-based rRT-PCR tests, syndromic panels, and point-of-care diagnostics. Do you have any sense of how these can be validated, and who might be able to use them?

Personally, I don't care who is going to use them, but I do care about who is developing them. Are they developing these in an optimal manner, have they evaluated the assay in the proper manner? That is my concern.

In China, there are a lot of local companies that make tests without proper evaluation, and this has caused problems. Some of the tests are not sensitive enough — even in a patient with a very high viral load, the Ct value is extremely high. Using a test like this, we would miss positive cases.

The other problem is a cross-reactivity issue. Do the primers and probes cross react with other respiratory diseases? I know this is unlikely, but things like this do happen. When they do the evaluation, they should not only test positive samples, and say "We have 100 percent sensitivity." That is not good enough. The primer and probe set has to be evaluated with a large panel of negative controls coming from patients with respiratory disease, so that we can be sure that the test will not come up with false-positive cases.

In the beginning of the outbreak in China, there were a lot of companies in China making their own tests.

Is that still happening?

There is a recent publication in Chinese that my colleagues in China just forwarded to me. It compared commercial tests and found one assay had a Ct value of 35, and the other had a Ct that was basically beyond the detection limit. That is a problem. I mean, come on, real-time PCR is not a cutting-edge technology. If one can evaluate and design it properly, the sensitivity should not be that bad. But there are some people that cut corners without doing a proper evaluation.

Have those tests you describe been granted Emergency Use Authorization in China, or are they being used without authorization?

I think, without authorization.

Well, it is an emergency …

Yes, but that is the problem. If someone doesn't have the background, and they just want to develop something for labs to use, they still have to take the responsibility to make sure that the assay is functioning properly.

Are you aware of the current test landscape in China, in terms of lab-based testing, or of any point-of-care tests that are authorized or being used to diagnose patients?

The China CDC has approved three different assays, but I don't know the details. And there are other assays available. Every day I get emails from companies that they have such-and-such a test. Of course, those assays [that are] coming from the big guns, I will trust they have done it properly. But there are a lot of companies [who] claim they have their own assay, and that I don't believe.

You know, during the pandemic H1N1 in 2009, [the sequence] was published, and the next day I received an email from a Chinese company saying they have an assay already. That is impossible.

Could point-of-care nucleic acid amplification-based tests be helpful for SARS-CoV-2, and do you think there will be enough time to develop and evaluate them to be useful in the current outbreak?

Point-of-care is not only about the RT-PCR assay. A dipstick test would be even more useful because it could be done in a more timely manner. For the RT-PCR assays, if there is a bedside test that would be great, but then, think about the contamination issues. I think it is a great thing to do, but do we have the technology now? I'm not so sure.

Overall, I think it is a good thing. There are a lot of assays being developed — someone used CRISPR to make a diagnostic test, and people have used LAMP, which is basically an isothermal amplification assay, to use that as a bedside test. These are all great.

But, we are fighting a war right now.

These other tests are fantastic, and they will be great for future use. It is good to use the current scenario to facilitate the studies needed. But, whether they will be essential for controlling the current epidemic, I have my reservations. Most labs do not have the capabilities or skills to run these tests.

As a biologist studying these viruses, have you been anticipating that another major spillover event like this could happen someday?

Yes, there are a lot of viruses in animals and we just don’t know which one will be the next. We've all thought about influenzas, like H5 or H7, and coronaviruses are no surprise because SARS and MERS are precedents. But the new coronavirus reiterates the potential impact of these viruses from animals.

Having studied this so deeply, what have the past two months of this outbreak been like for you?

I have mixed feelings. First of all, we have been doing this for a long time. To be honest, once this virus was detected in humans, there were a lot of scientific discoveries. A lot of people have been able to do something to facilitate and understand how the virus behaves. Just go to BioRxiV — there have been many papers published. We have full genomes, diagnostic assays, we know much about the transmission mode, just within one month. And we know that it is coming from bats. This is fantastic.

We have also learned a lot in the past, and now we are trying to exercise this knowledge to try to control this current problem.

But the sad thing is, the virus is very smart. The things we learned in the past may not be fully applicable to the current situation. This particular virus sheds like flu, in the sense that a patient is very infectious during the first few days of disease onset. For SARS patients, they were more infectious in the second week of disease onset.

So, the pattern is somewhat different. And that is why it is so difficult to control the current outbreak.

The other sad thing is, we know that animals are actually carrying these viruses, and some of the viruses may be harmful to humans. We knew this a long time ago. We know in the case of SARS, civets were the problem and spread the disease to humans at a wet market. But we are still consuming unsafe foods in China and other countries.

Somehow, we have not been able to fully convince the general public to develop effective measures to prevent the next outbreak. The government in China announced they will ban the trading of wildlife, and the selling of wild animals in wet markets for consumption. I think this is a good move. They learned a lesson and are trying to correct. But humans are humans. We make errors. And nature always catches us by surprise.

We have been working very hard — WHO, and scientists globally are working very hard to try to prevent the next outbreak, but it is just a matter of time until a future outbreak.

Do you recommend a One Health approach, where animals and the ecosystem are also monitored?

Yes. we have been arguing and advocating for a One Health approach for the last 15 years.

It almost feels like that approach has not been embraced enough worldwide. Why is that?

I agree. But, there have been improvements. Look at the poultry industry. In Hong Kong, we have been doing a lot to try and control avian flu in poultry. We haven't had any human cases for about 15 years. So, this is doable.

The problem is that the effort is quite substantial. You have to change regulations, change the law, change logistics, so it is a serious effort to come up with a good One Health approach.

But, if you have a second thought about this issue, look at the current outbreak. How much money will be lost, how much [gross domestic product], not only in China, but around the globe?

The amount of investment to develop a One Health policy is nothing in comparison to the impact of a major epidemic.

What are your thoughts about the new cases outside of China?

The disease is spreading — in China, the number of cases is starting to go down, which is good news, but the bad news is, other countries have the problem now, like South Korea, Japan, Iran, and Italy. This is going to be a global problem. My concern is whether this will become a pandemic, or not. We have to come up with rational strategies to deal with this disease.

Do diagnostics play a critical role in a pandemic?

Yes. And they are especially important in flu season. The initial clinical presentation of COVID-19 patients is quite similar to flu patients. Dry cough, and then a fever — these clinical symptoms overlap. It would be good to have a diagnostic test to identify either flu patients or COVID-19 patients early, so we can develop better policies to prevent spread of the disease in the community.

When you say early diagnosis, when do you mean, and are the viral loads high enough early on to detect?

I just published a paper in Lancet: Infectious Diseases. The viral load is actually very high, even before symptom onset. We monitored individuals who had contact with COVID-19 patients. They actually shed virus before they developed symptoms. They shed the virus from day 0 to day 5, and then the levels started to go down. They could still shed the virus in the second week of infection, but whether it is infectious or not, we don't know.

I was told that some patients can shed viral RNA for more than three weeks. So, there is still a lot to be learned about COVID-19. We have to be very cautious about the shedding pattern.

Is it maybe promising for a screening test, that the viral load is high initially? 

Yes, if you get a sample properly. The other problem that I'm having is that there is still lack of a comprehensive study identifying the best clinical specimen for detection of SARS-2. Some people use nasal swabs, some use throat swabs, or sputum, or saliva. But there has never been a systematic analysis of this. I've been advocating for this for a long time, for the last couple of weeks, and trying to talk to people in different labs. Because of the practical limitations, no one can manage to do a comprehensive analysis. Identifying the best clinical specimen for making a diagnosis is very important, but we still don't have that data. I hope someone can take this message and try to do a thorough analysis.

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