Saliva versus nasopharyngeal swab specimens for the detection of SARS-CoV-2
Aligned with our mission to advance the understanding and impact of microbiology, the Society reached out to our community of microbiologists to share their experiences in responding to SARS-CoV-2. We aim to showcase the perspective of scientists during the COVID-19 pandemic and the variety of roles adopted to mitigate the global crisis.
This case study comes to you from Dr Anne Wyllie, a Research Scientist at the Yale School of Public Health. She gives us a unique perspective on the pandemic from our neighbours over the water in the US. Her case study highlights the bottlenecks that were present in mass scale testing at the start of the pandemic and how she contributed to overcoming these problems.
How did you respond to SARS-CoV-2 during the COVID-19 pandemic?
Working at the intersection of epidemiology and medical microbiology meant that right from the start of the pandemic, I was witness to or participated in many balanced discussions regarding the emergence of this new virus. Initially, I was simply relaying this information to family and friends, helping to point them in the right direction and alleviate their concerns, where possible. Like many other scientists, I wanted to do more but didn’t see a clear role for my involvement. However, Yale had the foresight to establish the IMPACT Biorepository, which had the goal of collecting samples from SARS-CoV-2 positive individuals to allow researchers and clinicians across the university to study the virus and better understand COVID-19 disease progression. Due to my prior work in the molecular detection of upper respiratory tract pathogens, I was invited to join the effort and co-lead the testing division.
Since March 2020, I have been working on the pandemic response. We started by testing an array of clinical specimen types as they arrived at the biorepository. However, from the outset, challenges were faced on a daily basis – many of which revolved around the ‘gold-standard’ nasopharyngeal swab. From shortages, to patients’ aversion to its invasive nature, to healthcare workers’ hesitation due to the close proximity to patients required for obtaining swabs, these challenges caused hiccups and delays and I started to wonder whether the self-collection of saliva could help to overcome these. The team was supportive and agreed to build up a dataset of paired saliva and nasopharyngeal swab samples from SARS-CoV-2 positive individuals. By the end of April we had established that saliva was at least as good as nasopharyngeal swabs for SARS-CoV-2 detection.
In April, we also realised that there was going to be a need for frequent, repeat testing and that nasopharyngeal swabs were in no way suitable for that. We recognised that those who could get tested – other than those who had symptoms – were those who could afford it. Many laboratories were charging premium prices as demand was high, but this left testing inaccessible to many. We were therefore determined to develop a test that was low-cost and could quickly be implemented to scale up community access to tests. We therefore developed SalivaDirect – an open-source, streamlined RT-qPCR test for SARS-CoV-2. Our goal was to make testing available to all, and we have worked to openly distribute our protocol to labs around the country. We validated each step with multiple instruments and reagents from multiple vendors to circumvent supply chain disruptions while permitting labs to utilise their existing infrastructure, which demands less investment for rapid scale-up. Importantly, we charge no licensing or agreement fees – our protocol is freely available.
We have designated 160 laboratory sites in 40 states across the US to test under our FDA Emergency Use Authorisation. We continued to adapt the protocol and validated additional workflows in response to varied laboratory-specific needs to further aid the implementation of saliva testing to local communities. We also worked to develop guidance for laboratories transiting from traditional diagnostic testing to screening or surveillance testing programs. Across the US, we have established a network where laboratories can come together to discuss challenges and provide suggestions for further protocol amendments. Internationally, SalivaDirect has also been validated in multiple countries overseas, including Ireland, Mexico, New Zealand, Portugal, The Philippines and the United Arab Emirates.
Was this response related to your work prior to the outbreak?
Prior to the pandemic, my academic research had been focused on characterising the upper respiratory tract microbiome in health and disease. Identifying the true prevalence of pathogens within a population can help better inform public health strategies and maximise efficient resource allocation. The main aim of this work was to identify reservoirs of Streptococcus pneumoniae in the healthy population to better understand transmission and disease patterns. By unveiling the true rates of acquisition and transmission of respiratory viruses and bacterial infections, we can improve our understanding of the effects of co-infection and better inform vaccine development and policy.
At the core of this work was the validation and optimisation of molecular methods for sensitive pneumococcal detection in alternative sample types, particularly focused on, though not limited to, saliva. For over a century, due to the polymicrobial nature of saliva, culture-based methods for pneumococcal detection (as used for the gold-standard nasopharyngeal swab) proved near impossible on this more complex sample type. My work provided new molecular strategies to reliably detect and serotype pneumococcus in saliva, transforming surveillance for bacterial presence. With this experience, I was unexpectedly well-positioned to expand these methods for SARS-CoV-2 detection. Being able to quickly validate saliva as an accessible sample type for COVID-19 diagnostics was key to the rapid development of our now widely-used clinical diagnostic assay with FDA Emergency Use Authorisation – SalivaDirect.
What were some of the challenges that you faced during this time?
Although my previous research had allowed me to perfect the use of saliva as a clinical sample type, at the start of 2020 most labs neither had the same experience nor the skillset that comes from nearly a decade of working with saliva. This meant that many clinical laboratories didn’t have the go-to methods or protocols in place to effectively and reliably process saliva for pathogen detection. It has therefore been a major challenge to convince these labs – and at times, associated departments or ministries of health – that saliva as a sample holds so much potential for SARS-CoV-2 detection. Throughout the pandemic, my team have been working to highlight how the methods, the protocol, the combinations of reagents and PCR technology are all important for ensuring a sensitive and reliable saliva-based test.
Additionally, sourcing saliva samples positive for either SARS-CoV-2 or other target diseases that we may want to detect has been challenging; primarily because, for robust investigations, we need paired nasopharyngeal swabs. As the pandemic progressed, fewer clinics collected the deep, nasopharyngeal swabs. But at the same time, few testing programs are collecting multiple sample types, as that’s certainly not a sustainable approach. Moreover, throughout the pandemic there have been few cases of other respiratory disease infections circulating in our communities, such as influenza A and B, for example. While that has of course been great news for public health in general, for research it has been challenging and limited our ability to further evaluate saliva as a sample type.
What can we learn about the importance of microbiology from the COVID-19 pandemic?
First, we need to be prepared to re-evaluate our beliefs and approaches as new information comes to light. What has worked best previously for other viruses or bacteria won’t always be the most robust approach for others. What we’re currently seeing with Omicron – earlier detection in the oral cavity as compared to the nasal cavity – exemplifies that this even applies to variants of existing species.
Second, I hope that we can take away the potential of saliva as a sample type for future applications. How easy it is to collect samples and how much more affordable it can be, both to collect and process, really opens up options for more streamlined testing paradigms. I see great value in its use in clinical research, which so often suffers from limited funds; this could help to make limited research funds go further and help us learn even more about how common infectious diseases circulate within our communities.
About the author
Dr Anne Wyllie is a Research Scientist at the Yale School of Public Health. Prior to the COVID-19 pandemic, her work has previously focused on characterising the upper respiratory tract microbiome in health and disease. However, her research area pivoted in response to the COVID-19 pandemic to improving sampling techniques for mass testing strategies for SARS-CoV-2.
