COVID in the lab – Staying ahead of the curve

Hello, everybody, welcome to the UTS Science in Focus, this afternoon. It's a great pleasure for me to welcome you and I hope you very much enjoy the presentations today. As I said, this is a University of Technology Sydney, Science in Focus session, and it's great for us to be able to have this session despite the lockdown and despite the vast majority of us joining today from home. And it's really a wonderful world where technology nevertheless allows us to connect with each other and bring to light the wonderful efforts of our researchers and the pathology labs. So the Science in Focus is a free public lecture series that showcases the latest research from prominent UTS scientists and researchers. My name is Professor Bradley Williams. I am Associate Dean, External and International Engagement in the Faculty of Science here at the University of Technology, Sydney. And I'll be the moderator for today's event. Our Science in Focus talks provide an outstanding platform for University of Technology Sydney academics and public participants to meet for the sharing of ideas and to shine a spotlight on some of the top of the line work that we are doing at the Faculty of Science at the University of Technology, Sydney. Not only are we pushing the boundaries of scientific knowledge by making new and substantial contributions to a fundamental understanding of the world around us, but we have a dedication and devotion to translate our work into real world impact. Today, we'll hear from some experts who are making major contributions to our knowledge and understanding of SARS-CoV-2, the virus that's causing COVID-19. We'll also learn about the heroic response from the health care sector to ensure maximum delivery of high quality information to patients from the state government.

And how topical is the subject when we're in the thick of a lockdown here in Sydney due to the highly infectious Delta variant of this virus. So before I introduce our speakers today, I'd like to perform an acknowledgement of country. And so on behalf of everyone here present, I would like to acknowledge the Gadigal people of the Eora nation on whose ancestral lands the UTS City campus now stands. I would also like to pay respect to the elders past and present, acknowledging them as the traditional custodians of knowledge for this land. I'd also like to recognise the Cammeraygal people of the Kuringgai nation from which I join today and the people and elders of the country from which you join today. So a little housekeeping before we get started. With this being an online event, please bear with us if there are any technical issues, and I'll say that we came in a little bit late because we were experiencing some technical issues already. So if there are technical issues, please just bear with us and we'll work quickly to resolve them. And if you're not, if you find that you're not able to access the talk any point, just log out of the webinar and log back in again. And that that usually resolves the issues. If you have any questions during today's webinar, you'll see I have a slide up there now that shows you how you can enter the questions into the Q&A box in the zoom control panel. And we'll get, we'll do our best to get to the questions at the end of the session today.

And if you do like a question that someone else has asked and you'd like it answered, then please use the adverting tool, which is the little thumbs up symbol next to the question itself. I will just highlight that the session is being recorded today, but will not will not use any of the recording video or input from the audience. It's simply the received video as you see it now. You may contact UTS at the email address given there to discuss any concerns that you might have regarding the recording. So at this stage, these are the, I'm just highlighting the speakers that we have today, we have Professor Brian Oliver, Dr. Alen Faiz and Anna Condylios from the state, state government, New South Wales Health Pathology. And at this stage, I'm going to introduce Professor Brian Oliver, who is our first speaker of the day. Professor Brian Oliver's research aims to identify and develop new ways of treating respiratory diseases where he's globally recognised as a leader in the field. He studied at the National Heart and Lung Institute at the University of Leeds and then at Imperial College all in the U.K. before commencing his PhD studies at the University of Sydney. He now leads the Respiratory, Cellular and molecular biology group at UTS. Also with laboratory facilities located at the Woolcock Institute. He regularly presents his work at leading global conferences. Professor Oliver is currently co-director of the Respiratory Sleep, Environmental and Occupational Health Clinic, academic group at Maridulu Budyari Gumal and the Sydney Partnership for Health, Education, Research and Enterprise. Brian, over to you.

Thank you very much Bradley. I'll just share my screen. So what I'm going to talk to you all today about is the aerosolization of COVID and whenever I give a virus talk, I always put this image on the screen. And this is my daughter. She's a little bit older now. She's now six. But this image is there to remind us that under normal circumstances, children are a great source of viruses and virus transmission. But fortunately, with COVID, that doesn't seem to have happened. But I still like the picture, which is why I use it. I. A lot's happened in the last couple of years. And if you cast your mind back to December 2019, that's when COVID emerged. And the first case in Australia was in January 2020. And when covid emerged, there was a lot of panic, but we didn't really understand what was likely to come. And sometimes there was a little bit of disbelief about COVID. One of the major reasons why I was so worried about COVID is that the virus that causes it is very similar to the virus that caused the initial SARS and the initial SARS had a 50 percent mortality rate. So when you had a predecessor that was so dangerous, of course, everyone was very nervous around what may be happening, but we didn't actually know a whole lot about COVID. So in that initial stage, there was a lot of conjecture and a lot of worry and a lot of people trying to do the right thing.

And sometimes the right thing was the wrong thing. A good example of the right thing being the wrong thing was J.K. Rowling, who suggested that if you had COVID a good thing to do was to take some deep breaths and to cough. And I was approached by a magazine called The Conversation to come in and write an article about whether or not that was a good idea. And at that point in time, I said, no, it's not, because if you have a respiratory virus and you start coughing, you're likely to spread it. Now, this taught me all sorts of things. It taught me that if you are going to write things for, essentially, social media platforms, you have to be prepared for what comes next. I wasn't quite prepared for the death threats that came and literally there were death threats. She has some very staunch followers, as it turns out. But, you know, it was an important message that coughing wasn't necessarily a good thing to do. No at the end of April, The World Health Organization then decided to change its rhetoric and say that, in fact, the spread of COVID was by via airborne means. So between COVID emerging and the end of April, there was an awful lot of confusion around whether it was airborne spread or not. And of course, now we take it for granted, that there's airborne spread for COVID and we all wear masks when we go out and we do practice physical distancing.

So so now we accept it but at the time we didn't. The best example I can think of of why COVID-19 is spread by the aerosol route comes from this unfortunate situation that happened in America. Now what happened was there was a choir practice and this choir weren't wearing masks, but they were physically distanced from one another. And in this particular choir group, one patient was COVID positive, that COVID positive patient managed to spread COVID to 87 percent of the people in the choir practice. And unfortunately, there were some really bad outcomes for those people that developed COVID and there was at least one death. So this was the observation that actually led the WHO to change its mind. And it's a really good illustration of what can happen if people don't actually take precautions against the airborne spread. So you may be asking why I would stick my neck out and say aerosol spread was likely, and the reason for this is that back in 2008, we did our very first study looking at airborne spread of viruses. And it was also in this particular study, and it's the young woman on the right hand side, this is where we first went into the business of making homemade masks. Now I don't, I don't want to go into homemade most today, but it was sort of struck me as something that I hadn't actually thought about until I was putting this talk together.

What we actually showed in this study was that simple activities like talking or breathing was sufficient to generate aerosols containing virus. And that was a very first time ever been shown in humans. So this was sort of groundbreaking study, that was a long time ago. We've done all sorts of studies like this, and this is our last study that we've published. It was conducted by Alicia Mitchell and Alicia actually won the chancellor's medal for her PhD work at UTS. In this particular study, we use a different type of filter. And once again looking to exhale viruses. But what was nice about our last study was that we showed that just about every well, in fact, there was no respiratory viruses that we couldn't find evidence of them being exhaled. So every respiratory virus was capable of being aerosolized. So if you take those two things together, it's pretty logical to think that the same thing would happen with COVID. But just because something's exhaled doesn't necessarily mean that it's infectious, so Anna's going to talk to us later on about what PCR detection means. But in all of my studies, I've also used PCR to detect virus. And the trouble is, I got really good at PCR so I could detect as few as one virion.

Now, one very virion is basically one virus particle, but that doesn't necessarily mean that one virus particle is sufficient to cause infection. And what we've done. Not me personally, but other people have done is that they've experimentally, infect, human volunteers with different respiratory viruses. So in the first study on the screen that they infected people with influenza, which gives you the flu, and they found that you need ten thousand variants to cause an active infection with the flu. And in the study below, which is using rhinovirus, you only need a couple of virions to actually cause the infection. So different viruses from different infectious loads, which makes that debate about aerosolization of virus and what that means is somewhat more challenging because we just don't know what the infectious dose is for COVID-19. What we do know, and this study was carried out in Spain, and what they were doing was not actually looking at spread of COVID-19, they were looking at potential treatment for COVID-19. But what they did was allow people that were COVID-19 positive to be treated with an experimental drug. And these were people in the community. And then they looked at the transmission of the virus to other people in the community. And perhaps it's no surprise if you looked at the bottom of the graph that people with the most virus that they could detect had the most spread of the virus. So that's sort of indicating that there probably is some threshold for infection, there's something about infectivity. And sometimes in the news you'll hear low and high instances of the virus or lower amounts of PCR detection. And this is sort of what it's inferring. But we don't actually know the answer.

The answer will come in a few months time because over in the U.K., what they're now doing, there are experimental studies, where they're infecting healthy people with COVID-19 and they're doing this so that they can understand things like the infectious dose. They can try new potential therapies. They can look at the time, course and infection and of transmission of COVID-19. So I'm not sure that everyone would necessarily want to volunteer for such studies. But these studies will be going on and hopefully the data from the studies will be very informative for everyone. Now, just to put all of this in some sort of context and tell you about what I'm currently doing, there's lots of publications now showing that SARS-CoV-2, the virus that cause of COVID-19 can be spread by the airborne route. And in this particular study, they did a, it was conducted in a hospital and they looked in the air condition in the hospital. So if you imagine where my mouse is, this is the air, the aircon skeleton, if you want, of a hospital. And the COVID ward was down here in the bottom, left and right at the top, you see all this big machinery and this is where the filters are, that filters the air before it goes out into the environment. What they did was that they looked in these filters up here and they were able to find that COVID-19 was on these filters. So it is demonstrable that COVID-19 travelled through all these tubes and all these pipes right up to these filters. So this is just a good example of how COVID-19 could spread through air conditioners in the hospital setting. Fortunately, these filters capture the virus so it doesn't go on to infect other people.

Now, I've obviously had a long standing history in aerosolization of virus and what goes on and with some work with some colleagues, both in Sydney and in Newcastle, we published this paper earlier on. And it's our view that in hospitals we're not really doing enough to help prevent the spread of COVID-19 and other viruses. So I think we still have a lot to learn about what we can do to help protect people. One of the studies that we're currently carrying out, and this is a study that involves both David Chapman and Hema Vedam, is we're trying to understand both the generation of respiratory aerosols and actually the spread of viruses. So when a patient comes into hospital with COVID-19, they may be just in a bed without any supplemental oxygen or any other form of breathing support. But when that breathing support is applied to the patient, often in the form of air that's being mechanically ventilated into the lungs, we don't know what that does to the spread of virus. So. Now, unfortunately, that there's more cases of COVID-19 we'll actually be able to go into the hospital and look at the airborne spread.

One of the other things I'm going to briefly touch on are masks. And look, there's lots of different types of masks available, there's surgical masks and N95s, RP2s. And I'm not sure if you can see me or not, but this is an example of an N95 and the big problem with it, the N95 masks, is that for them to work adequately, they've got to fit your face. So this particular mask, if I put it on, and it actually falls off. So this mask, even though it's quite expensive, is actually no good for me. In a health care setting, what happens is something called a fit test. And the fit test decides which mask is right for your face. But unfortunately, the testing protocols are not so standardised, so there's lots of different movements that can happen. So you put the mask on, you bend over as if you're bending over a patient. And sometimes if you fail one of those manoeuvre, but you pass all the rest, the mask is still said to be acceptable. So with Sheree Smith, we've been looking at how many people can fail various manoeuvres one way or another, with the N95 or RP2 mask within a hospital setting and what that may mean.

The other thing I'd like to point out with masks is that often you'll see people wearing this type of mask with a little valve the front. My advice is, if you see someone wearing one of these is run away as fast as you can, because that valve there is there to allow exhaled air not to go through the mask directly out through the valve so that the person wearing that mask is offering you no protection. So they're not advisable for anyone. The other thing that I'd also like to point out is that if you are going to wear a mask, please wear it properly. So this is just an example of what we all see every day. So this person believes that the mask will provide them protection if they wear it around the chin, and not over their mouth and nose. So the mouth, the mask actually does need to cover both your mouth and nose in order for you to be protected and for other people to be protected from you in the off chance that you are COVID positive. That's the end of my talk. But if you do have any questions, please post them in the Q&A part of zoom. Thank you very much.

Our next speaker is Dr. Alen Faiz, who's a molecular biologist and geneticist completing his PhD studies at the Woolcock Institute of Medical Research. And he went on to postdoctoral fellowship at the Experimental Pulmonology and Inflammation Research Laboratory at the University Medical Centre Groningen in the Netherlands in 2018. He was a visiting postdoctoral fellow at Imperial College in London. Alen's primary research interest is understanding the biology of respiratory systems at the genetic and epigenetic levels. And Alen I hope that you explain that if it needs explanation, including conditions of exposure to cigarette smoke and the microbiome. His multidisciplinary skills allow him to move beyond simple associations towards deeper identification of causal pathways and mechanisms in respiratory conditions. He's also president of the Thoracic Society of Australia and New Zealand's New South Wales branch. Alen, over to you.

All right, so today I'm going to be talking to you about the determinants of SARS-CoV-2 receptors, gene expression in upper and lower airways, and comparisons between coronaviruses in using in vitro models. So SARS' entry into cells is an important factor. We know SARS-C0V-2 is a virus so it can't replicate outside of a human host or host in general. So it needs our cellular machinery to actually replicate itself. The way it actually infects the cell is something that a lot of investigation has undergone over the past year and a half. Now, what we know is that SARS-CoV-2, which is here in the image, has a RNA genome that is encapsulated by a capsid. Now, what we have is a S protein or spike protein, which then facilitates its ability to infect ourselves. What happens is this spike protein binds to a receptor on our own cells. And this receptor one one we mostly think it is known as ACE2. Now, as it stands, a spike protein can't directly bind to ACE2. It requires some pre processing and this is done by proteases within our own cells. And one of these proteases is known as TMPRSS2. So it is a little bit more complicated than that because we know that TMPRSS2 is not the only protease which is able to preprocess this spike protein. There's also CTSL, FURIN and there's many others that have been coming out as we researched this virus. Additionally, ACE2 is not the only receptor this virus has been able to bind to. There has been work showing that it's also able to bind to BSG. This is a receptor for other coronaviruses and also in NRP1, which has been found using CRISPR screening libraries. So this is the way that coronavirus comes in and actually infects ourselves.

What we were interested in, was investigating the expression of these genes required for COVID entry and where they're expressed. So we're interested in the nose, upper otherwise and also the lower airways. So what we found, what we investigated was nasal brushings from the upper airways and also the lower airways. And this was done across two different cohorts. Now, you don't need to understand this too much, but this is a heat map and the red indicates higher expression or higher levels. And what we observed here is this is the nasal brushes. And what we observed was this much higher expression in the upper airways than compared to the lower airways, if we, if we compared samples. This was observed in both studies. This is of interest because we know coronavirus affects the upper airways first. So this may indicate that coronavirus is much, may more easily infect the other airways. This is these results are showing a slightly different way. And again, what we can see when we pair the samples, everyone has higher expression of these COVID entry genes im the nose compared to the lower airways. But what are the environmental factors that change the expression of these genes that are required for COVID entry? Now we know that there are many environmental factors, such as cigarette smoke, that have been thought to play a role in COVID infection rate.

So what we did, first of all, we looked in the nose. Does smoking or chronic smoking increase the expression of these genes? Now where you can see here there was basically no difference between never and chronic smokers. However, if we now look at the lower rate, this is where the infection happens when you have more severe disease, we saw higher levels of ACE2 the receptor required for infection and also TMPRSS2 across all patients. So really, we saw an increase in the lower airways, which we didn't see in the upper airways. Now, this is really interesting because there has been studies to show that smoking is a risk factor for coronavirus and this is, has higher odds of progressing to more severe disease that never smoked if you are a chronic smoker. But do you have to be a chronic smoker, is an acute smoking sufficient to cause this increase? So we had a study of party smokers that abstain from smoking for two days and then smoked three cigarettes. Three, twenty four hours later, we then took a bronchial brush and then observed whether ACE2 increased. What we observed was when they didn't smoke and when they did smoke, ACE2 was increased after only three cigarettes so that, the term every cigarette is doing you damage is true. Every cigarette is actually increasing your ACE2 expression. Now again, this is gene expression, it may not fully be reflective of protein levels, but this is a good indication that it may increase susceptibility to COVID infection.

The next thing, and this is a little bit more concerning, is second hand smoke, if you weren't directly smoking, but you're in the presence of someone smoking. Is this also able to increase the expression of ACE2 in your lower airways. This was, this study was done in infants with, from parents who smoked and who didn't smoke. We found that infants who had parents that smoked had higher levels of ACE2 compared to those. So. I've told you a lot about what increases these receptors and these genes but what decreases them? Well, something that many of you may already be taking inhaled corticosteroids. This is a common therapy for asthmatics and patients with chronic obstructive pulmonary disease. If you are on long term treatment, here, we looked at six months, this was sufficient to decrease your ACE2 expression. And this has been replicated across a number of studies around the world. And this is a difference to the placebo, which we did not see is true. So these are the effects that change the expression of these COVID genes. But how can we actually study the virus itself? Now, it's important to realise that coronaviruses arne't new. There's been coronaviruses around for a very long time. So in many situations, coronaviruses have a host and it's a natural host and it's a spillover effect where this goes for intermediate host and usually then it affects humans. So they can be really broken down into two groups, common cold coronaviruses. And this will just sniffles like a common cold or severe acute respiratory illness like SARS.

And this is where SARS-CoV-2 or COVID-19 sits. So for our study, we're interested in looking at, how to compare all these coronaviruses at an equal playing field and see what the effects are. So we took one coronavirus from the common cold directory and then SARS-CoV-1 which happened, which happened coronavirus that caused the outbreak many, many years ago, early 2000s. MERS, which is a much more severe coronavirus that has about a 20 percent fatality rate and SARS-CoV-2. Now, how do we go about testing these viruses in the lab? We can't just go around infecting people, especially for the coronaviruses that have very severe or had the high fatality rates. Well, what we can do is we can use what is samples from patients. We go in with a bronchoscope and take a small amount of samples. Now, this does look painful, but actually you have no nerve endings after the vocal cord, so you don't actually feel a thing. But obviously your gag reflex may make this uncomfortable. We then take these samples and grow them in the lab. Now, traditionally, what you would do, is you would grow these cells with a nutrient broth of neutral media on top. Now, this is not exactly how your airways are. Because we know, our airways are exposed to air. So what you can do, is you can take away the liquid and allow and feed the cells from the bottom of the neutral broth and therefore the cells can differentiate and have more of an environment similar to what we have in the airways.

Now, your model's ready to test your viruses. And this is known as air liquid interface. So for our experimental setup, we took all the four coronaviruses, we did this in four patient samples. We made sure we did equal MOI since the number of virions is the same for all the viruses and this was a MOI of 0.1. And we created these alleys, which takes about six weeks which is very long time to grow, and then infected for up to 72 hours. We're very interested on the early effects of this coronavirus. After the 72 hours we then did sequencing and RNA sec, which is a process of just looking at the overall response of, to the virus. So first of all, let's look at our viral replication. What we observed was that all the coronaviruses that we tested did replicate in our model and they were able to increase exponentially increase over this time. Interestingly enough, MERS, which is much more, a very severe coronavirus that has a 20 percent fatality rate, was able to replicate a little faster than the others. Now, we also have to look at the response to the virus. And the way you can do this is looking at what is known as an interferon signature. Now, interferon is a, is a mechanism or a pathway which is activated when we are exposed to viruses that helps fight off the infection. And what we found was that interferon signature was actually increased in all viruses and this was sort of maximal around 72 hours and probably would continue to increase if we took this experiment out, past 72 two hours.

Now, one thing that was also interesting is that we looked at inflammation and we look here at IL-6, a well known inflammatory cytokine that we know is increased by the number of coronaviruses. What we found was that all three of the previous coronaviruses increased inflammation at the 72 hour time point. Interestingly enough, so SARS-CoV-2 was unable to increase inflammation at the 72 hour time point. But we know that it definitely increases inflammation, but it probably takes longer. And this is probably got to do with its extended incubation period. This is a process where the virus is replicating, but you do not see the effects. So in summary, what we found was that SARS-CoV-2 entry genes are increased in the nose compared to lower airways. They are increased by smoke exposure regardless of the route. That means that your lower airways can have increased expression if you're exposed to smoke and the pro-inflammatory effects of SARS-CoV-2 are delayed compared to other coronaviruses which may be associated with the viral incubation times. Okay, well, the first part of the work that I talked about today has been published as a prep, as you can see here. And this was a huge effort of a number of groups from around the world, the experimental side of things is something that we're working on and we're hoping to publish. I'd like to thank you and happy for questions through the chat.

Thanks Alen, outstanding, and apart from the information, what that last slide of yours shows most definitely is that this type of work definitely requires a very significant effort on behalf of a number of teams around the world. So outstanding. Thank you very much. I'd now like to introduce Anna Condylios. So Anna has 25 years of experience in microbiology and primary virology. She gained her Bachelor of Science degrees from the University of New South Wales and has been working within the Department of Microbiology at the Prince of Wales Hospital in Randwick since 1995. She's a senior hospital scientist in the virology area of the serology and Virology Division in New South Wales pathology and is clearly one of the many heroes in the story when it comes to New South Wales Health pathology and the response to the COVID pandemic. Along with Anna is Dr Zin Naing, who's also a senior hospital scientist in serology and virology division at New South Wales Health Pathology, and he'll be joining us for the Q&A session in the end to assist Anna in any specific questions. So, Anna, if you could share your screen and come off. There you go. That's excellent. Thank you very much. If you can share your screen, that should kick mine off.

Thank you, Bradley, for that kind introduction. OK, so as Bradley said, I'm from the virology laboratory. We're based at the Prince of Wales Hospital in Randwick and we're the largest in New South Wales health pathology testing facility in New South Wales. And it's a great honour and responsibility to help keep the people of New South Wales safe. I'll just begin with a simplified timeline of the outbreak, as Brian briefly already mentioned. There was talk of a new virus causing pneumonia in Wuhan China in December 2019. And it's actually in December when I became the senior. Previously, I was working part time and all of a sudden I was not only full time, but literally twenty four hours a day in the laboratory at some point trying to deal with the situation and all the things we had to work out regarding testing for coronavirus. By January 7th, the World Health Organization had released information that confirmed that there was indeed an outbreak of a previously unknown coronavirus. And a few days later, the first death was recorded. On the same day, January 11th, an Australian professor, Edward Holmes from the University of Sydney, released the genomic sequence of the virus. And this information was very important for us because now that we knew what the virus was made of, we could develop tests that would enable us to detect it. And as cases spread and by January 27th, we were ready to start testing in our laboratory.

There were a lot of mixed emotions, and as I mentioned, I was sort of thrown into the thick of it right at the very beginning, we were very excited that there was something new that we needed to develop a test for. But at the same time, we realised this could be potentially catastrophic. So it was very, very scary. We needed to get together and brainstorm and think about what we were going to do, how we were going to begin our testing and all the other things that were involved with it to ensure that we were able to provide results in a quick and timely manner. And the first thing we did was, all the seniors got together and we met daily to discuss all of these things that you see here in this diagram. The main thing was research. We needed to research and start developing our own in-house PCR assay. We then had to think about capacity. How many tests could we do in a day? We had to think about pre analytical and post analytical issues and then, you know, things that flowed on from those things, like the vendors, our logarithm, how we would do our testing. What about all the other tests that we routinely perform in the laboratory, staff and equipment.

So the gold standard was, as mentioned before is by PCR. This technique allows us to replicate the virus so that there is enough for us to detect it. It's like following a cooking recipe where we put different reagents together with patient sample and we put it through a number of heating and cooling cycles. And if virus is there, it's repeatedly copied and we can make billions of copies of the virus if present in just a short period of time. And it's very useful because even if there's only a tiny amount of virus present, we'll be able to find it. One of the first institutions to publish a technique for SARS-CoV-2 was Hong Kong University. So we used the paper that they published together with our own research, to develop a method that suited our laboratory. And we decided that we would detect three specific genes. We would target three genes within coronavirus, the E, N and ORF1B, so we developed our PCR test. You'd be able to pick up those genes initially before any commercial assays were available. And in the very beginning we were just one of two laboratories that were ready to perform our own in-house testing methods before any commercial assays were available. And the young lady in the photo here was, is Melena Liang, and she helped develop our in-house PCR assay. And once we did that, we had to make sure, because it was in-house and not commercial, that it actually worked. And the way we do that is by repeating the test multiple times, including controls and testing the sensitivity and specificity of the test.

And once we were happy with all of those parameters, we officially started testing on the 27th January. So all up it took us about 20 days to be ready with a validated method to be able to test for SARS-CoV-2. Now, testing numbers in the first few days were twenty five to thirty tests a day, but by mid-March the numbers went to well over a thousand a day. So originally we had eight staff and all leave was cancelled. Everybody worked extra shifts. Everybody worked overtime, 13, 14 hour days just so that we could get through this enormous amount of samples. But fatigue set in pretty quickly. So we had to think of ways where we could quickly employ people that we could just bring in, train them up and get them to help us as quickly as possible. We started off with asking for staff to volunteer from other laboratories, which may have been a little bit under-utilised, you know, like part timers and things like that. So we had a few volunteers from different, not just within our campus, but from Kogarah and RPA who came in to assist. We also had students from UNSW who at this point were locked down so they didn't have any classes. And we buddied all the inexperienced staff with our routine experienced staff to help facilitate training and to make sure everything ran smoothly. Then, and as you can see, we went from eight staff to twenty eight within just a few months.

And then we had to think about what if somebody became sick within our team? Did that mean the whole team would go down and we just couldn't perform our tasks and we'd have to shut the lab? So the way we got around that was by splitting the staff into two distinct shifts. We named them the blues and the whites. Basically it was a day shift and then a night shift. And we tried to encourage limited interaction between the two. So, you know, when the day shift left, that's when the night shift would come in. And this served its purpose very well. And we still maintain this rostering structure. Our equipment also needed to increase in order for us to deal with the enormous amount of samples we were receiving on a daily basis. But the problem was that our laboratory size didn't change, but we had to bring in a lot more equipment. So we had to be a little bit creative with where we put instruments. We had to undertake a number of renovations that involved adding extra benches and just basically fill every nook that we possibly could with instrumentation. So it was during March that some commercial lab space started to become available, which meant it was really good because we could process more samples in a shorter period of time. But it also meant we needed more space for all the plastics and consumables that came with the commercial testing.

We needed to be able to predict numbers, how much we needed to order, and we had to make sure that we didn't run out of anything. And because we also we were expecting worldwide shortages, as we could see happening all around the world, all the testing numbers were going crazy everywhere. So we had to order a lot of things in advance. And as you can see from some of the photos here, we, this is still these are recent photos. So we have to put stock wherever we can find space. And we've even had to politely ask our I.T. person to move to another area. And we've taken up the whole office. We've just stock. The middle photo down the bottom there is where I'm sitting at the moment, which is our laboratory manager's office, and that's filled as well with stock. And this larger photo is just an example of about four days worth of work. You can get an idea visually of the enormous amount of samples that we deal with daily. So the other things we needed to think about were pre analytically and post analytically what we would do with results. Firstly, we needed to work out how would doctors be requesting the test? Would they want urgent testing, routine testing? In the beginning, a lot of doctors were in a bit of a panic and everything was marked urgent. But we just couldn't sustain testing everything urgently.

There had to be a little bit of an order of things. So we put out a memo that said, OK, if a person is from a nursing home, which this is deemed a little bit more important, then we could do those tests urgently. But if somebody just walked in off the street into the COVID clinic, which might be a bit of a sniffly nose, that would be tested in a routine manner. Either way, results would go out within twenty four hours. We also had to have help from I.T., which and what they did was improve our data entry process and streamlined it so that we could enter the data of the samples a bit faster than usual. We had to liaise with our COVID clinic because they were having people just lined up out the street. So we had to make sure we had people going down there and routinely bringing samples up to us as quickly as possible. And we also had to think about minimising the foot traffic within the laboratory. So normally our specimen reception staff would hand deliver samples to us in the laboratory, but we had to stop them again to limit contamination or possible contamination, and they would leave everything in the hallway for us and ring a bell. And then someone, when they'd move away, someone would come from the laboratory and pick up the samples. We also increased our couriers within all our local health district hospitals to bring samples to us quickly.

And we also had to do our own cleaning because we didn't want cleaners coming in and maybe inadvertently touching something that they shouldn't and possibly becoming infected. So we performed our own we did our own mopping and we cleaned multiple times throughout the day. All surfaces, particularly if we did find a positive sample, we would then clean all equipment that was involved in testing that sample. Post analytically, it was important to be able to distribute results as soon as they were available. The Ministry of Health and our local public health unit needed to have the ability to start contact tracing as soon as possible. So we set up an hourly electronic transfer of results. We employed more staff in our call centre and we also later New South Wales introduced the SMS notification system. So what happens when you attend a COVID clinic? Firstly, the nurse will swab the back of your throat and then they will swab each nostril. It's a very quick procedure, but can be a little bit uncomfortable. And it usually makes people tear up a little bit from the COVID clinic. The sample will come to our specimen reception where we will check to make sure that the sample itself, the details on it, match the request form. It's then sent to the laboratory where we perform our testing. We report the results to the public health unit and then the patient gets the results sent to them as well.

This whole process takes less than 24 hours. These are just some images from around the laboratory, you can see the staff members here, they're from our, part of our specimen reception and they are all actually UTS graduates. The viruses, the swabs when collected are placed directly into a tube, usually at the point of collection. There's a solution in there. And this helps to preserve virus, if there's any present until it's time for us to test, we then take small amounts of the virus and we put it into these plastic plates you can see in these last two bottom photos. And each of those is like a little well in each of those plates and a patient sample goes into each one of those. And then we can put them onto our instruments for the PCR process to stop. So it's important to remember, as I mentioned before, we don't just test for coronavirus here. We have more than 30 other PCR tests that we perform throughout the week. And so the instruments that we use for SARS testing is determined by what else we are doing at the time and also by the fact by whether it's an urgent or a rapid or a routine. And as you can see here, different instruments allow for different turnaround times. So how do we know when a result is really positive? This slide shows you an example of a finished commercial PCR test and how the results are presented to us.

And a number of criteria must be filled before a sample is deemed positive. Firstly we check the controls worked and we have strict controls in this instance. On the top right hand side, you can see column 12 has a known positive and negative control and then another known positive control. So these must work for us to be able to say that the test has worked. If these don't work, then we need to go back to the drawing board from scratch and repeat the whole PCR process again. This very rarely happens, but it can occur. The next thing we do is check each positive patient sample. And these are indicated by these red dots here. In this instance, there are two positive patient samples. And then we also look at a curve that we're provided with. Now the curve must be this particular shape. This is called a sigmoidal curve. If the curve is either straight or flat, it's not a positive. So again, it must be repeated or it's deemed invalid. And we do it again. In this instance, with this particular positive, you can see four curves. There are three which target the different genes within coronavirus and they have a beautiful, sigmoidal curve. And there's also an internal control which tells us that the PCR test itself has worked. So it's just another check on top of the positive and negative controls that we have within the test.

And the point at which the curve starts to rise up is the point within the PCR process where virus is starting to become detected. And it's it's determined like, this is the cycle because there are multiple cycles within the PCR test. This is the cycle at which we start to see virus and we get a numeric value as to what cycle number the virus was detected. So we have all this different information before we can say something is positive, but then this is just an interim positive. We then repeat these two particular samples in this instance on a whole different machine instrument. And if it's, if they come up positive again on a different platform, then we can definitely say that these are true positives. And this is what we do for every positive that we detect in a laboratory. We repeat them on a different platform to make sure they are truly positive. The next thing we have to think about is how did this person become infected and is their infection caused by a variant of concern? And we do this by a process called whole genome sequencing. Whole genome sequencing can detect the genetic code of the virus and it can detect mutations that can occur as the virus evolves.

We can then track how the virus is being transmitted in the community. For example, if two people have a viral sequence, that's the same. We can say that they caught the virus from each other or from a known source. But if there are two people where there are a lot of differences between these sequences, then they didn't catch the virus from each other. And this can be shown in a diagram like this. And this is called a phylogenetic tree. It's like a family tree, which shows how the virus samples are related. Each dot represents the positive and the lines from where it may have travelled and further away the dot is from the centre, the more mutations it has from the original and every positive that exists in Australia, we can link back to China in the original instance, as well as via an outbreak in another foreign country. And in this phylogenetic tree, you can see that in yellow we have the positives that, from the South African strain and the UK and the Brazilian strain. Now, currently, these are the variants of concern, the Alpha, Beta, Gamma Delta, at the moment, the Delta is the one that we are seeing predominantly in our community. There are also other variants, categorised as interest, variants of interest, and at this point the variants of interest, because we haven't seen as many cases as these compared to others.

And apart from PCR and genomic sequencing, we also have facilities that enable us to grow the virus for further research and to perform serological testing, which is very handy in determining if an infection is old and new.

The information we gather combined paints a greater picture for the public health unit and enables them to perform contact tracing investigations. Our ongoing role also involves performing hotel quarantine testing. And we also perform daily saliva screening on quarantine workers where rather than having a daily nose/throat swab collected, which is a little bit invasive, they suck on a stick, which you can see here has these spirals on it, almost like those sticks you put in a hone jar and they suck on that stick which collects saliva within the little grooves. And then we use that for testing. And we're also recently, we've been involved in wastewater analysis. We also routinely evaluate commercial assays that come onto the market. And we've published many papers. This graph gives you an idea of our sample numbers from when we began testing until the end of June, 2021, and you can say that whenever there's an outbreak, the testing numbers goes up, go up. And the busiest we've ever been to date was just before Christmas last year during the northern beaches outbreak where we tested over 7000 samples in a single day. In closing, I'd like to remind everybody of the importance of following the advice from New South Wales health so that maybe be in the near future we can all get back to some sort of normal and our lab stuff can finally take a decent break.

Thank you. Well.

It's certainly a Herculean task, uh, that you faced, so thank you to all of our speakers. I really appreciate your presentations today. We unfortunately don't have a lot of time for questions, but there are a couple of questions that I think are really useful to be answered. So one that someone sent in in advance of today's session is why are second vaccine doses and booster vaccinations needed. So perhaps one of our panelists could respond to that. And I think it's it's very topical relating to the rollout of the vaccines.

I can answer that if you like, Bradley.

Yeah, go ahead.

So the first time we're immunised with most vaccines, that initiates an immune response, but that immune response is not so good and usually doesn't induce any sort of memory. So the second time we've vaccinated, our immune system gets better respondence. And so the immune response is more refined. But we also then have what's called immunological memory, and that memory can be quite long lasting. So usually the second shot is needed to ensure that we have that long lasting memory response.

Ok, that's that's excellent. Thanks to the

Booster shots are often needed because sometimes, like with everything in life, the immunological memory fades. So the booster is there just to actually remind our immune system that it needs to respond when it sees this.

Excellent. Excellent. And then again, for, for any one of our panelists, how do we stay on top of all the variants and whether the specific diagnostic test can pick up on each new variant? So I don't know if if Anna would like to respond to that or one of the UTS colleagues.

I can respond to that.

Anna can you just turn your your video on so that we can see

I can respond to that. So we have commercial assays that detect variants and they are constantly researching and producing newer versions as time progresses. So we're always on top of the new variants commercially, but we also have our own sequencing that we do where if something different from the what we're seeing now comes up, we can detect it straight away.

Ok, outstanding. Thanks. Thanks Anna. And here's an interesting one, and I suppose it refers back to one of the comments that you made at the beginning when you introduced the scientific papers that spoke about the genetic sequencing of the virus. So there have been a lot of conspiracy theories. This is comes from our attendees today relating to where the virus came from. We know geographically where it came from, but how do we know where it's actually originated and has transmitted across to us? And Alen, you in your presentation showed some of the intermediate hosts and so on. So possibly you could you could respond to this one. Is there is there a consensus? Is there is there some scientific knowledge that allows us to determine the origins of the particular virus?

So, based on the sequences that its similarities to other viruses, we suspect that it has come from bats and there's been a lot of investigations into this and at this stage this is the most likely where its natural host. There has been some intermediates, and there has been some thoughts on the different intermediates that may also have come. But it is thought, and I think it's well established now that the original natural, thing came from bats, but Anna might have some comments on that as well.

And any further comment from you or Brian?

No, sorry.

Ok, no worries. Here's a here's another interesting one, and we we know that the virus that was in circulation in Australia last year was less infectious than the Delta virus, which is currently in circulation. So the question reads, I'm curious as to how long the particles stay in the air. Do we have much information about this? And are smaller do smaller particles linger more in the air and do they give us a lower dose of the virus? And Brian, you mentioned that a little bit earlier. So, Brian, perhaps you could you could respond to that particular question.

So the easy answer is, we don't know. What we do know is that when particles are generated, those particles are mainly water and that water evaporates. And so you go from a big particle to a small particle and sometimes those very small particles and stay in air for a long time. So, in fact, fog is just a suspension of water particles. So you need the right environmental conditions for that to occur. What the question didn't ask is whether it's safer to be inside or outside when it comes to spreading things. And look, as far as I know, there's no good documented cases of incidental transmission in an outdoor environment. There have been some examples of those, a couple of people talking to each other very close for 15 minutes, and that apparently transmitted SARS-CoV-2. But I don't know of any other good examples of where COVID-19 has been transmitted outside. And probably the reason for that is the outside we've got such good air exchange. So those particles are diluted rapidly and and to such an extent that the infection doesn't actually isn't able to occur.

Alright. Excellent. I understand that. And possibly the last question for this session is when a person becomes infected with coronavirus, some have some are asymptomatic, others have low level symptoms. Others have extreme and severe symptoms. So what are the factors that determine the individual response to the infection? Alen, I see you have a smile on your face that perhaps you would like to respond first and then others can can add to what you say.

I think that's the million dollar question, isn't it? Trying to work out who will be the susceptible population. We know there are factors that increase susceptibility, so pre-existing conditions and other factors can contribute to this. I did talk about cigarette smoke. Exposure to current smokers have increased risk of progression of the SARS-CoV-2, but and there are thought to be some genetic factors as well. As this is although this is not coronaviruses in general aren't new. This is a new virus. It's very difficult to really identify the key factors at this point in time there are many groups around the world that are feverishly trying to identify these factors to really protect these people at risk. But I think the best thing to do is to if you are, you believe you're at risk to get vaccinated and we'll see how we go from there. But Brian and Anna will probably have some comments on that as well.

So asymptomatic infection or infection without overt symptoms is really common with all respiratory viruses as a phenomenon. This isn't anything new. And the way in which an individual deals with that viral infection is often related to a whole range of factors, Alen's touched on pre-existing chronic diseases. So if you've got heart problems or if you're obese or you've got diabetes, you seem to do much worse when you have COVID-19. But that's also true for a whole range of normal respiratory viruses as well. So what we don't know yet, and this is also what Alen said, is who and why and where and what, because there's often no patterns to these things. You know, we've got some generalisation now, but it doesn't mean to say that everyone that's got chronic disease, if they get SARS-CoV-2 , would actually do badly. So we're still trying to work out.

Right, excellent. Thank you very much. So I think that, we've gone over time this there's certainly been a lot of interest in the, in the topic and also based on the questions that we can see coming through on our system. So as far as the questions are concerned for our viewers, we'll definitely try to respond to as many of them as we can and load those written responses onto the website, along with a copy of a video recording of today's session. So it does remain for me to thank our speakers today and our colleagues from New South Wales Health. Thank you very much for attending. It's been an excellent session today, Brian and Alan as well. Outstanding information that you provided to us and also to our participants today. It's been wonderful having you here with us. And I hope that this has been interesting and informative to you. And also just to let you know that we have a second Science in Focus webinar that will come up in a few weeks time. Also on COVID. And I invite you to join us at that time.