Background
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused significant disease (COVID-19) and deaths worldwide [
1]. COVID-19 symptoms range from mild respiratory illness to severe pneumonia, encompassing a wide clinical differential of numerous respiratory pathogens. Therefore, when patients present with COVID-19-like symptoms, highly accurate laboratory tests are essential to distinguish true COVID-19 cases and initiate public health steps to limit further SARS-CoV-2 spread.
Since SARS-CoV-2 emerged, there has been little research on the concurrent circulation of these other respiratory viruses, which had been the subject of broad surveillance in the years prior [
2‐
5]: only a handful of case reports have described co-infections between SARS-CoV-2 and other respiratory viruses able to cause similar symptoms [
6‐
13]. With unprecedented demand on clinical diagnostics, re-prioritized surveillance is one of many ways that laboratories have had to prioritize and adapt throughout this pandemic [
14‐
17]. However, understanding current virus co-circulation could help to develop evidence-based strategies to better tackle the pandemic in future pandemic surges and the approaching influenza season, as exponentially increasing numbers of patients require testing for COVID-19-like symptoms.
Furthermore, co-circulation of SARS-CoV-2 and endemic coronaviruses (eCoVs) may pose a particular diagnostic challenge due to potential cross-reactivity of SARS-CoV-2 with pre-existing assays targeting eCoVs and vice versa. While laboratories must perform in-lab validations of the specificity of their assays, in situ studies are an essential complement, showing real-world data that may indicate necessary and opportune changes in diagnostic approaches.
Accordingly, we sought to conduct a retrospective analysis of all specimens submitted to the provincial public health laboratory in Alberta, Canada for SARS-CoV-2 simultaneously tested for 17 additional respiratory pathogens—including influenza viruses and endemic coronaviruses—to inform our diagnostic algorithms during the COVID-19 pandemic and assess for potential co-infection or cross-reactivity of SARS-CoV-2 and eCoVs in a clinical setting.
Methods
Multiplex respiratory testing
The NxTAG Respiratory Pathogen Panel (RPP; Luminex) was used to test respiratory specimens from symptomatic patients admitted to hospital, emergency departments, in long-term care, or amid a suspected respiratory outbreak in the community. Following collection, respiratory specimens were transported to the provincial Public Health Laboratories (ProvLab) for testing. The RPP was validated to detect nucleic acids from 17 respiratory pathogens: influenza viruses A-B, parainfluenza viruses 1–4, respiratory syncytial viruses A-B, rhinovirus/enterovirus, adenovirus, human metapneumovirus, four eCoVs (229E, NL63, OC43, HKU1), and Mycoplasma pneumoniae.
SARS-CoV-2 testing
SARS-CoV-2 testing was performed at ProvLab using multiple assays with different analytical sensitivities (Sn): singleplex and multiplex laboratory-developed tests using the TaqMan Fast Virus One-Step real-time reverse transcription (RT)-PCR Master Mix (ABI; Sn = 145 and 375 copies/mL, respectively [
18]), or the Roche cobas 6800 SARS-CoV-2 test (Sn = 25 copies/mL [
19]). The Seegene Allplex 2019-nCoV assay was also used outside of ProvLab (Sn = 100 copies/reaction [
20]) and two rapid tests were employed across the province: DiaSorin Simplexa COVID-19 Direct (Sn = 242 copies/mL [
21]) and Cepheid GeneXpert Xpress SARS-CoV-2 (Sn = 250 copies/mL [
22]). No assays reported cross-reactivity with other respiratory pathogens, with the exceptions of three tests that cross-reacted with SARS-CoV-1: the cobas, the multiplex RT-PCR assay, and the GeneXpert, as previously reported [
18].
Data collection and exclusion
SARS-CoV-2 and RPP tests performed between January 1–June 6, 2020, and RPPs between January 1–June 6 in 2018/2019 were compiled. Only tests from validated, respiratory tract specimen types and from patients with a primary address in Alberta were assessed. Analyses of coronavirus test positivity in 2020 focused on March 7–May 28, wherein RPP testing criteria were consistent with those used in prior years, except for providing RPP tests to symptomatic patients in long-term care in 2020.
Statistics
Differences between categorical variables were compared using Fisher’s exact test or Chi-square analysis. Continuous variables were compared by Kruskal–Wallis test or Student’s t-test.
Discussion
The COVID-19 pandemic has seen unprecedented levels of respiratory testing, driven by the need to identify cases to control further spread. In the three months after COVID-19 was first reported in Alberta, Canada, nearly 300,000 respiratory specimens were tested for SARS-CoV-2. In parallel, the provincial public health laboratory maintained broad, syndromic nucleic acid testing for additional viral and bacterial pathogens, rapidly amassing data on 18 respiratory pathogens from 52,285 respiratory specimens, including SARS-CoV-2. As a result, to our knowledge this study represents the largest single-year eCoV study [
2‐
5] and by far the largest eCoV study during the COVID-19 pandemic [
7‐
13]. This broad testing approach helps to address a pivotal diagnostic gap amidst the emergence of a novel pathogen: co-infection and possible cross-reactivity with other pathogens that can cause similar clinical presentations. Using a large number of clinical specimens from a real-world setting that may capture true, biological co-infections, this study complements the essential in-lab validations that initially establish test specificity using a small set of known controls. Here, less than 0.01% of specimens tested positive for both SARS-CoV-2 and an eCoV, indicating no significant co-infection or cross-reactivity between SARS-CoV-2 and our four most common eCoVs.
This study identified NL63 as the predominant eCoV in Alberta in both 2018 and 2020, both before and during the COVID-19 pandemic; this is consistent with Albertan findings between 2009 and 2012 [
2]. While NL63 was the predominant eCoV (both in specimens where SARS-CoV-2 was and was not identified), other pathogens like enterovirus/rhinovirus were identified more frequently. Altogether, our data supports no cross reactivity between SARS-CoV-2 (which emerged after the RPP manufacturer validation) and other respiratory pathogen targets on the RPP assay, including four other coronaviruses.
By examining concurrent respiratory virus circulation during the COVID-19 pandemic, this study demonstrates the low number of SARS-CoV-2 co-infections in this population and builds upon smaller studies from around the world that have also reported lower viral co-infection rates in SARS-CoV-2-positive vs. SARS-CoV-2-negative patients [
7,
10‐
12]. The results of this study corroborate those of Nowak et al
., wherein other respiratory pathogens were detected in < 3% and 13.1% of SARS-CoV-2-positive
vs. -negative specimens, respectively [
10], compared to 3.4% and 10.6% in this study. Notably, the rate of SARS-CoV-2 co-infection with
Mycoplasma pneumoniae was lower here than reported elsewhere in the literature [
6,
13,
24‐
27]. As such, the role of viral exclusion and cross-protection may be interesting topics for future research [
28]. Overall, in contrast to the diagnostic, operational, and surveillance benefits of multiplex syndromic testing in non-pandemic years, broad panels had limited clinical or surveillance value in this pandemic setting.
Importantly, this study illustrates the challenges in performing direct, year-to-year comparisons of broad surveillance data for respiratory viruses. The emergence of the COVID-19 pandemic and the ensuing public health interventions dramatically changed how populations interact, travel, and work, thereby impacting viral spread, the underlying demographics of those requiring testing, and testing volumes. Testing volumes in 2020 substantially increased the underlying statistical power, confounding year-to-year surveillance. For instance, although the eCoV positivity rate among respiratory specimens in 2020 was comparable with prior studies [
2,
29], the statistically significant differences in the demographics, location, and age of patients meeting the RPP testing criteria in 2020 compared with previous years was a natural limitation to this study. This highlights a challenge in continuing broad-range (i.e. non-COVID-19) surveillance through this or future pandemics.
The unprecedented testing volumes in Alberta have also highlighted the substantial cost and inflexibility of sustaining fixed, broad-range testing approaches used in prior (non-pandemic) years. More flexible test panel designs and algorithms could better capture local epidemiology and changing needs, whether that is to identify circulating pathogens in routine ‘surveillance mode’ or with panels focused on public health, infection prevention and control, and novel pathogens in ‘pandemic mode.’ Ultimately, more flexible panel designs may support clinical and operational effectiveness in diagnostic laboratories, both during ‘surveillance mode’ (when panels are appropriately informed and customized by periodic local audits), and critically in ‘pandemic mode’ (including when novel pathogens like SARS-CoV-2 emerge). Indeed, flexible syndromic panels that identify pathogens of public health and infection control concern (e.g. SARS-CoV-2, influenza viruses) would be more easily adaptable to novel pathogens and could play an important role in the current pandemic response and future pandemic preparedness.
During a pandemic, clinical laboratories require greater test capacity to support public health efforts to limit further spread [
14,
30]. With ongoing surges in COVID-19 cases, laboratories must focus finite resources on targeted pathogens with direct consequences on patient treatment or infection prevention and control.
This study highlights the importance of ongoing diagnostic stewardship to best align laboratory resources with public health efforts. Consequently, as of May 29, 2020, our laboratory pandemic response shifted from routine ‘surveillance mode’ to a prioritized ‘pandemic mode’ by no longer routinely performing the RPP multiplex test with every SARS-CoV-2 test. This further demonstrates how clinical laboratories have adapted throughout this pandemic, as laboratory-driven alternatives continue to mitigate the myriad of ongoing challenges such as test supply shortages [
16,
31,
32]. As test volumes increase with the upcoming influenza season, diagnostic stewardship is a key strategy to prioritize public health as the pandemic continues.
Conclusions
By maintaining broad respiratory pathogen testing from previous years through the emergence of SARS-CoV-2, this study reveals concurrent respiratory virus circulation during the COVID-19 pandemic. Due to the massive surge in test volumes, this became the largest single-year eCoV study and the largest eCoV study during the COVID-19 pandemic. There was no evidence of assay cross-reactivity in a clinical setting between SARS-CoV-2 and the 17 respiratory pathogen targets, and less than 0.01% of specimens tested positive for both SARS-CoV-2 and any eCoV. In fact, specimens containing SARS-CoV-2 had a significantly lower viral co-infections rate. Overall, broad panels had limited clinical or surveillance value and high cost in this pandemic, and more flexible panel designs may better support clinical and operational effectiveness in diagnostic laboratories.
As a consequence of this data, our provincial laboratory eliminated reflexive multiplex testing in a shift from routine ‘surveillance mode’ to a prioritized ‘pandemic mode.’ Focused diagnostic stewardship can help mitigate ongoing challenges to better address future case surges—including future waves of COVID-19—without compromising public health benefits of ongoing testing.
Acknowledgements
The authors thank the Alberta ProvLab for their sustained efforts in respiratory virus surveillance and essential role in the provincial COVID-19 response, including the laboratory assistants, technologists, supervisors, managers, microbiologists, and researchers. We further thank Kanti Pabbaraju for thoughtful review of this manuscript, Stephanie Murphy and Fiona Ko for data compilation, and Andrew Santos for assistance with statistics software.
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