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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