In this study, we established a diagnostic workflow for the longitudinal surveillance of acute SARS-CoV-2 infections and potentially other respiratory viruses by quantitative PCR in large groups of individuals. Due to its sample and data management, as well as due to the use of sample pooling, this workflow is highly cost- and labor-efficient. Simultaneously, its data architecture allows for easy access for participants and physicians without compromising the safety of personal data.

We performed a longitudinal cohort study with our newly established diagnostic workflow. Over a period of more than 2 years, medical and dental students were invited to submit self-sampled tongue swabs for pooled PCR analysis. The attendance to the study was high and the testing behavior among participants differed considerably from that of the general population. However, high COVID-19 incidences in the study cohort were often concomitant with high COVID-19 case numbers in the public. The rates of newly detected COVID-19 cases per tests performed were considerably higher from 2022 onwards compared to pre-2022, coinciding with the emergence of SARS-CoV-2 VoC omicron. Time-resolved analysis of virus variants revealed a great overlap between COVID-19 cases among study participants and the Bavarian population.

The combination of self-sampling, pooled PCR testing and online result communication led to a cost reduction of SARS-CoV-2 testing that we estimated to be more than 10-fold compared to PCR testing in routine diagnostics. Thus, our approach is similarly priced as screening for COVID-19 using rapid antigen tests but, presumably, substantially more sensitive. The time to result in our method was, in most cases, comparable to that of PCR testing in routine diagnostics.

Our diagnostic workflow was built on pre-existing infrastructure: All students had a student ID card with an ID number that could be read by an appropriate device. Moreover, a server with restricted access through personalized authentication that was already being used for coordinating education was employed to communicate test results between physicians and participants. Thus, we anticipate it to be challenging to implement a comparable diagnostic workflow on a populational level. Nonetheless, we expect our approach to be feasible as a screening method in universities, hospitals, and companies, where similar infrastructure as described herein is readily available.

Instead of medical professionals sampling nasopharyngeal swabs from study participants, they self-sampled a tongue swab and submitted it for analysis. On one hand, this considerably decreased the cost and labor intensity of the study, but on the other hand, the diagnostic sensitivity of tongue swabs in PCR testing was shown to be somewhat lower than those of nasopharyngeal swabs [21,22,23]. Moreover, self-sampling is vulnerable to manipulation by participants, e.g., by submitting tubes without the appropriate sample to be tested for acute COVID-19. In addition, sample pooling might impair the diagnostic sensitivity of the PCR measurement. This effect, however, was shown to be marginal [24,25,26]. To counteract these potential caveats, our diagnostic workflow can easily be modified, e.g. it may be performed in a setup with nasopharyngeal sampling by healthcare professionals. Furthermore, our workflow can be re-adjusted for the measurement and communication of other diagnostic parameters. This includes infection with other viral pathogens with high transmissibility and substantial risk of causing severe respiratory diseases, e.g., influenza and respiratory syncytial virus. Our workflow can be adapted for the surveillance of these RNA viruses simply by modifying the PCR primers.

We recommended participants to submit samples either every two weeks or in weekly intervals. However, a large number of participants contributed swabs fewer than 10 times. We also observed that participation was more frequent at the beginning of each term than towards its end. Of note, a recently acquired, negative PCR test was obligatory for students to participate in bedside courses. Students who preferred to attend such courses in person, instead of completing them online, thus might have participated in our study sporadically and on demand, rather than self-organizing their own COVID-19 tests, potentially explaining the aforementioned testing patterns.

High numbers of PCR tests in the general population were mirrored by the outbreak of novel virus variants causing considerable waves of the ongoing pandemic. This suggests that the testing behavior of the public differed from that of the study cohort. Conceivably, more individuals got tested at times of high COVID-19 incidence because there were more infected persons with typical symptoms. Further, more persons might have tested positive for an acute SARS-CoV-2 infection using rapid antigen tests, when pandemic waves were at their peak, prompting them to officially verify their antigen test result by PCR.

Regardless of differences in the testing behavior, periods of high COVID-19 incidence in the general population often coincided with high numbers of newly detected SARS-CoV-2 infections in our study cohort. On top of that, we registered one short period of high infection numbers in the study cohort in May 2022 that was not matched by high COVID-19 incidences in the public. This potentially indicates the value of screening efforts to detect larger COVID-19 outbreaks in defined cohorts that are not seen on a populational level.

The observation of higher COVID-19 rates per number of tests being performed in 2022 and 2023, after the emergence of SARS-CoV-2 omicron, possibly points towards the often-reported increased transmissibility of this virus variant that is, partially, due to its pronounced immune escape [27,28,29,30]. On July 1, 2021, PCR-testing on demand without the need to provide a reason was replaced in Bavaria by the requirement of a positive rapid antigen test for asymptomatic persons to become PCR tested [19, 20], potentially contributing to the high rates of positive individuals per PCR tests performed in the general population.

After changing the recommendations for sample submission in our study from once every two weeks to once per week in October 2021, we observed a considerable increase in the amount of submitted tongue swab specimens. This may have increased the sensitivity to detect COVID-19 cases in the study cohort. Shortly after changing the submission recommendations, however, COVID-19 incidences in the general population surged dramatically. Therefore, we assume that the increase in newly detected SARS-CoV-2 infections among the study participants after changing the sample submission recommendations was rather due to higher COVID-19 incidences at that time than due to increased sensitivity for the detection of COVID-19 in the cohort.

A limitation of the study is that the attendance of participants dropped considerably during the holiday periods. However, comparing the outbreaks detected in the study with those in the general population showed that no major SARS-CoV-2 outbreak was missed in the study cohort.

There was a considerable overlap between the SARS-CoV-2 variants longitudinally detected in the study cohort and those registered in the Bavarian population. This finding demonstrates that only those virus variants circulated in the study population that were, at that time, also highly prevalent in the public. Discrepancies in the virus variant distribution between the participants and the general population, e.g. in the beginning of 2023, might be due to low case numbers in the study cohort.

Another limitation that may have contributed to the different results comparing the study cohort and the general population is demographic disparity. Being medical and dental students, study participants likely were by average considerably younger and had a higher percentage of females compared to the Bavarian public [31, 32]. We anticipated these medical and dental students to be highly motivated to participate in the study, submit specimens regularly and not cheat during self-sampling. The adherence of individuals from the general population to participate in a similar study may be lower [33].

In summary, our study introduces a new tool for cost- and labor-efficient surveillance of SARS-CoV-2 infections. We show that it is feasible and cost effective to longitudinally screen for acute COVID-19 over several years in a large cohort of individuals utilizing our method. Our screening approach enables the detection of COVID-19 outbreaks in a surveilled group that are independent of high incidences in the population. In certain settings and during times when infection surveillance on a populational level is less vigorous, tools like ours might be especially valuable to monitor SARS-CoV-2 infections in groups of vulnerable individuals and healthcare professionals. Furthermore, our method can be swiftly adapted for screening of other diagnostic parameters, including newly emerging infectious diseases.