Discussion
Our method for detection of respiratory pathogens from UPT samples allowed the identification of respiratory pathogens responsible for ARTI, with adequate sample quality to allow further genetic characterization. We were able to detect respiratory viruses as well as bacteria, and a putative causative pathogen could be identified in used paper tissues of all symptomatic patients who had a positive nasal swab. All pathogens that were detected in nasal swabs were also found in concurrent UPTs from the same patient, except for one EV/RV co-infection in an influenza B positive sample. In only four of the symptomatic individuals no pathogen could be detected in UPT nor in the corresponding nasal swab. Although reported positivity rates in ARTI patients are highly variable, depending on the range of viruses and bacteria tested, the epidemiological situation and the population under investigation, our positivity rate of 80% is comparable to other studies in which both viral and bacterial respiratory pathogens were tested in nasal or nasopharyngeal swabs [11], [12]. Negative samples could be due to sampling being performed too early or too late in the course of the infection (when pathogens load is below the detection limit), or symptoms originating from other microbial infections or non-microbial causes, such as allergies.
Bacterial pathogens have already been shown to be reliably detectable from paper tissues of patients with upper respiratory tract infections [13]. In a recent study, Lagathu et al. were able to identify multiple respiratory viruses in pooled facial tissues obtained in communities of children. They compared SARS-CoV-2 Cq values between nasopharyngeal swabs and facial tissues of individual COVID-19 patients and found a higher signal from the tissues in 11 out of in 15 cases [14]. In our study, we compared Cq values for SARS-CoV-2 but also for other common respiratory pathogens such as EV/RV, influenzaviruses and S. pneumoniae , obtained from 20 UPT and nasal swabs, and found a high variety in Cq difference between both sample types. We also were able to detect the presence of multiple respiratory pathogens in pooled UPT samples of collectivities, confirming its applicability for community testing. This would especially be useful in schools and preschool daycare centers, since taking nasal samples from (young) children is an invasive method and requires training, or in elderly homes and homes for disabled people, in whom taking nasal samples is less well tolerated. Because sequencing a complete genome is possible from UPT this method can also be applied for epidemiological surveillance. We demonstrated that UPTs can be stored at room temperature for up to 8 weeks prior to analysis. This implies that UPT samples can be transported to diagnostic laboratories at low cost, even from distant locations. We did measure fluctuations in viral load between samples analyzed at different timepoints, which we hypothesize to be the result of the non-homogenous nature of the sample rather than a decline in sample quality.
Since our sample contains eluted material from entire paper tissues, the pathogen load in the sample is not only dependent on the amount of virus shedding but also on the amount of nasal discharge collected in the tissue. This makes the method less suited for (semi-) quantitative analyses. It also implies that the method cannot be used when there is very little to no nasal discharge, or when nasal discharge is difficult to collect by nose blowing or wiping with a tissue.
We were able to detect the corresponding virus in UPT of all Ag-RDT positive cases, indicating that UPTs are sufficiently sensitive to detect individuals with high virus shedding, who are most likely to be infectious. As such, UPT could provide an interesting non-invasive sampling method for screening of individuals. In the patient that was followed over the course of a COVID infection, UPT tested positive as of the start of symptoms, whereas Ag-RDTs turned positive only on day 4. This is in accordance with the notion that SARS-CoV-2 viral loads in persons with pre-existing immunity (by previous infection or by vaccination) may only rise to Ag-RDT detectable levels after several days of symptoms. Although only based on a single observation, UPT testing seems to be sensitive enough to allow detection as of the start of infection, reducing the amount of false negative test results.
Since pathogen detection was possible from combined UPTs obtained in collectivities, it can also provide a good alternative to sampling of sewage water of buildings or aircraft wastewater to obtain a community sample for pathogen screening. This would be very useful to complement the current strategy of wastewater testing of incoming aircraft for SARS-CoV-2 variant screening [15], [16].
Ethics statement
Informed consent, approved by the UZ Leuven Ethics Committee, was obtained from all individuals providing self-collected swab samples.
Acknowledgements
UZ Leuven, as national reference center, is supported by Sciensano.
Conflict of interest
The authors declare no conflict of interest.
author contributions
Annabel Rector: Conception and design of the study, data collection, data analysis and interpretation, writing of the manuscript, final approval of the version to be published.
Mandy Bloemen: Conception and design of the study, data collection, data analysis and interpretation, final approval of the version to be published.
Marc Van Ranst: Conception and design of the study, final approval of the version to be published.
Elke Wollants: Conception and design of the study, data collection, data analysis and interpretation, final approval of the version to be published.
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