Faculty on the frontline

By combining their expertise for a singular goal, health experts from NTU’s Lee Kong Chian School of Medicine are helping Singapore tackle COVID-19 on all fronts. Image design (virus particle) based on Virtual SARS-CoV-2, a 3D modelling tool developed by Fraunhofer Singapore at NTU.

In a matter of months, SARS-CoV-2, the virus that causes COVID-19, has wreaked havoc across the globe. Although its impact is still being tallied, it is becoming clear that this is an outbreak that is on a scale not seen in over a hundred years.

For many people living in Singapore, life has slowed down tremendously. Circuit breaker measures meant to curb the spread of COVID-19 forced a bulk of the population to temporarily retreat into their homes. As the economy gradually reopens, the silent war being waged against the coronavirus by the nation’s top doctors and researchers continues to be stepped up.

The battleground? Hospitals and research laboratories, including those at NTU’s Lee Kong Chian School of Medicine, or LKCMedicine, Singapore’s third medical school, set up with Imperial College London. Despite having only accepted its first cohort of students in 2013 and officially opened in 2017, LKCMedicine’s talented roster of clinician-scientists has ensured its capacity to contribute to the nationwide COVID-19 response.

From improving diagnostics to creating mini lungs-in-a-dish, LKCMedicine’s faculty and students are collectively wielding their expertise to take on the coronavirus on different fronts. Here, we explore three ongoing research efforts from faculty members at the school.

Reinventing diagnostics
The gold standard for COVID-19 diagnosis is the reverse transcription polymerase chain reaction (RT-PCR), which detects sequences specific to SARS-CoV-2 in genetic material from patient samples. But its accuracy comes with several caveats. First, it is slow. A single run can last up to six hours. Second, it requires specialised equipment and reagents, which is why samples are generally shipped to centralised laboratories. This adds to the turnaround time, lengthening the excruciating wait for a test result to 24 hours or more.

Assoc Prof Eric Yap, who heads the Medical Genomics Laboratory at LKCMedicine, wants to overhaul PCR testing. He and his team are doing this by integrating RT-PCR’s many steps, a key reason why it takes so much time. Currently, a big bottleneck in the workflow is the RNA extraction step, where RNA is separated from other components in the patient sample. Not only is the process laborious, but it also requires reagents that are now in short supply.

What Assoc Prof Yap and his team have done is eliminate this step entirely. Instead, sputum or nasal exudate from patients is directly placed into the RT-PCR set-up, after which it is ready to run. “We normally can’t just take a sample and put it in a PCR because there are reaction inhibitors present. If you do that, the PCR assay will fail,” explains Assoc Prof Yap. “We’ve made the RT-PCR resistant to inhibitors.” Using their method known as direct PCR, the team shortened the process into 36 minutes from start to finish.

To make PCR testing more accessible, Assoc Prof Yap and his team are also hoping to reinvent the PCR machine, also called a thermocycler. Their goal is to build a thermocycler that costs ten times less than commercially available counterparts. “Thermal cycling is essentially just heating a sample up to near boiling point and then cooling it down. If you think about it, this happens in every kitchen,” Assoc Prof Yap points out. “When I make coffee or tea, the kettle goes up to near 100°C, then cools down. It is effectively carrying out one PCR cycle.”

Technically, anything that boils water could be used for PCR, says Assoc Prof Yap. His team is therefore trying to convert household appliances into potential low-cost thermocyclers. Meanwhile, the costly optics that detect genetic material during PCR could be miniaturised into a tiny spectrometer chip, like the one in our smartphone. By combining these two innovations, Assoc Prof Yap hopes to take PCR testing out of conventional laboratories into the low-resource settings that need them the most.

Such efforts wouldn’t have been possible without NTU’s support. “A lot of what I’m doing is more engineering than biology. I have to credit my engineering colleagues who have taught me about optics and electronics,” he shares. “NTU provides a great milieu for this cross-disciplinary work.”

Mission: PROTECT
Biological samples from patients reflect the ongoing battle against the coronavirus raging within their bodies. Analysing these samples gives scientists and physicians alike valuable insights into the inner workings of the virus—and the potential keys to its defeat.

LKCMedicine’s Assoc Prof David Lye is at the forefront of nationwide efforts to better understand SARS-CoV-2. As Director of the Infectious Disease Research and Training Office at the National Centre for Infectious Diseases, Assoc Prof Lye coordinates an outbreak protocol that enables Singapore’s top scientific institutions to study the coronavirus in greater detail.

Known as PROTECT, the protocol was developed in 2012 and was applied during the Zika and monkeypox outbreaks in 2016 and 2019 respectively. The protocol was immediately activated after Singapore reported its first COVID-19 case on 23 January. As of mid-April, 381 patients have been enrolled in the research effort.

As the patients progress through COVID-19 infection, additional biological samples such as nose swabs, blood, urine, stool and even tears are continuously obtained and sent to approved biosafety level 3 (BSL3) laboratories at Duke-NUS Medical School and DSO National Laboratories, Singapore’s national defence research agency, for further analysis. The samples are then compared to the disease’s clinical progression, allowing researchers to map the body’s immune response to the virus over time. This could help clinicians identify the periods when life-saving interventions could be best applied.

Research at PROTECT has already resulted in several successful outcomes. One notable example is the world’s first antibody-based test that linked two of Singapore’s largest COVID-19 clusters back in February. Clinicians also now know how to better manage COVID-19, thanks to information gleaned from PROTECT. “We know that COVID-19 viral load is higher early in the illness and drops towards the end of the week, so a five-day medical certificate from the family doctor makes sense,” says Assoc Prof Lye. “The virus is present in stool more than 50% of the time, so toilet hygiene is vital.”

In addition, findings from PROTECT have helped guide treatment options. As seen in their March 2020 publication in the medical journal JAMA, Assoc Prof Lye and his colleagues witnessed early on the lack of clinical effect of the anti-HIV cocktail lopinavir-ritonavir on COVID-19 patients.

With the recent uptick in local cases, the PROTECT team is now working to improve the triage process in community isolation facilities. For instance, they’ve been able to preliminarily identify blood markers that could distinguish among different disease manifestations in patients. The team has also evaluated the usefulness of chest X-rays as a screening tool, finding that those with normal X-rays have a low risk of developing severe COVID-19.

“This is a new virus and a new disease. There are many urgent research questions,” says Assoc Prof Lye. From host-virus interactions to antiviral treatments, clearly both medicine and science still have more to learn about COVID-19. “Research is harder work than clinical work, but it is vital to drive good care,” he shares.

Lessons from the lungs
Despite the quickened pace of science triggered by COVID-19, many aspects of the disease still perplex the best researchers and doctors worldwide. Until now, we don’t know why some infected people are asymptomatic, while others develop severe symptoms and require intubation.

“If you have impaired immunity, you’re likely to get very sick because you have no immune system to fight the virus. But if you have a robust immune system, you may also be at risk of getting a very severe disease,” says Asst Prof Sanjay Chotirmall, Provost’s Chair in Molecular Medicine and Principal Investigator of the Translational Respiratory Research Laboratory at LKCMedicine.

The latter effect, known as a cytokine storm, occurs when the body mounts such a strong immune response to the virus that it becomes damaging in itself. This could be why some young, otherwise healthy, COVID-19 patients suddenly deteriorate—and even die.

To better understand the disease’s progression, Asst Prof Chotirmall is turning to tiny, three-dimensional versions of the lungs called lung organoids. All Asst Prof Chotirmall and his team need is a lung biopsy or nasal swab from the patient. From this, the team can recreate the diversity of cell types found in the individual’s lung.

Lung organoids allow the team to analyse viral progression within a patient’s lung in a more personalised and systematic manner. The team can even study an individual’s susceptibility to coronavirus infection based on his or her cell receptors. Take, for instance, the ACE-2 receptor that mediates the cellular entry of SARS-CoV-2. Individual variations in the number of ACE-2 receptors could influence a person’s likelihood of contracting COVID-19, he says.

Asst Prof Chotirmall is also using the organoids to identify key differences between the new coronavirus and its relative, SARS-CoV-1, the causative agent of severe acute respiratory syndrome (SARS) that emerged in 2003. Other aspects of his research include leveraging multi-omics approaches to investigate how viral infection changes genes on an individual cell level and to identify new diagnostic biomarkers for COVID-19. Asst Prof Chotirmall is now collaborating with Prof Stephan Schuster, Deputy Centre Director (Facilities & Capacities) and Research Director (Meta-’omics & Microbiomes) at the Singapore Centre for Environmental Life Sciences Engineering (SCELSE), to assess the impact of air quality on COVID-19 patients.

Examining air samples from a number of countries globally, the team found that pollution was associated with high case fatality rates. “Exposure to PM2.5 particulate matter is known in animal models to upregulate ACE-2 receptor expression in the airways. Populations that are primed by exposure to PM2.5 will have higher numbers of ACE-2 in the airways and be more susceptible to the virus and severe outcomes,” says Asst Prof Chotirmall.

Another reason for the poorer health outcomes, he adds, is that polluted environments tend to be dominated by harmful bacteria. COVID-19 patients from such areas therefore not only have increased ACE-2 receptor expression, but are also potentially more susceptible to secondary bacterial or even co-infection. These findings, in the process of being published, could be of enormous interest especially to those living in countries with the poorest air quality.

Filling in the gaps
Amid the global focus on COVID-19, there’s an understandable worry that it could result in research on other pressing health concerns being neglected. Assoc Prof Yap, however, believes otherwise.

“Some people may see this as a distraction, that it’s now taking money and time and attention away from other important diseases like cancers and chronic diseases,” he explains. “But I’d say, conversely, a disease like this helps us to identify gaps in technology. In filling those gaps, we would actually be improving the way science is practised.”

COVID-19 may be a trial by fire for scientists and clinicians around the globe, but with LKCMedicine’s dedicated researchers working together on new innovative technologies to quell the outbreak, there’s no question that NTU’s medical school—and Singapore as a whole—will emerge better positioned to face future viral threats.

The article appeared first in NTU’s research & innovation magazine Pushing Frontiers (issue #17, August 2020).

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