Keywords:
used paper tissue (UPT); acute respiratory infection (ARI); respiratory
pathogens; SARS-CoV-2; non-invasive sampling method
Introduction
Acute respiratory tract infections (ARTIs), including pneumonia,
constitute a major disease burden worldwide, especially in young
children and the elderly [1][2]. Diagnostic testing for
respiratory pathogens is usually performed on samples collected by
invasive methods, such as nasopharyngeal swabs, nasopharyngeal aspirates
or bronchoalveolar lavages, obtained in hospital or medical practice
settings. For some respiratory viruses such as severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2), influenzavirus and respiratory
syncytial virus (RSV), fast diagnosis using antigen-detection rapid
diagnostic test (Ag-RDTs) can be performed on self-collected nasal
swabs. Although these nasal swabs are less invasive than nasopharyngeal
swabs, they can still cause some discomfort, which can pose a barrier to
testing [3]. Furthermore, since some of the most vulnerable
populations for ARTI outbreaks, such as residents of long-term care
facilities for the elderly or mentally impaired, or infants and toddlers
attending day-nurseries, are difficult to sample using these invasive
methods, there is a need for less to non-invasive methods for
respiratory sampling. We assessed whether paper tissues, used for nose
blowing, can be used for the identification of respiratory pathogens, on
an individual as well as on a community level.
materials and methods
Used paper tissues (UPT) were self-collected by individuals with
clinical symptoms indicative of ARTI, including rhinorrhea, sneezing,
coughing, fever, hoarseness, sore throat, loss of smell, and shortness
of breath, and who had nasal discharge which could be collected by
blowing or wiping the nose with a facial tissue. Collective UPT samples
were gathered anonymously in a childcare facility, 3 kindergartens and 2
primary schools. Tissues that were used in a classroom or childcare
group for nose blowing or wiping during the course of one day were
pooled in a plastic container and delivered to the lab. UPT were stored
at room temperature prior to analysis. For retrieval of respiratory
pathogens from the UPT, tissues were transferred with sterile tweezers
into a 100 mL disposable syringe (~4 tissues per
individual or the maximal amount of tissues that could be fitted into
the syringe in case of the pooled samples). Phosphate buffered saline
(PBS) was added until tissues were soaked (~25 mL).
After incubation at room temperature for 5 minutes, the plunger of the
syringe was pressed to recover the eluate (~ 10 mL) into
a 15 mL Falcon tube.
Nasal swabs were self-collected in parallel with UPT by 20 individuals
with symptoms of ARTI. After collection, the nasal swabs were inserted
in 1 mL Universal Transport Medium (UTM) (Copan) and stored at 4°C until
extraction. Nasal swabs in UTM were rigorously vortexed prior to
extraction.
Nucleic acids were extracted using the MagMAX™ Viral/Pathogen Nucleic
Acid Isolation Kit on Kingfisher Flex-96 (ThermoFisher Scientific,
Europe), using 400 µL of eluate of UPT or of nasal swab UTM.
Screening for respiratory pathogens was done using the in-house
developed respiratory panel (RP) of the University Hospitals Leuven for
simultaneous detection of 22 respiratory viruses (influenza A, influenza
B, RSV, human metapneumovirus , parainfluenzavirus (PIV) -1 to -4,
adenovirus , human bocavirus , enterovirus/rhinovirus (EV/RV), EV D-68 ,
human parechovirus, human coronavirus (HCoV)-NL63, -229E, -OC43, -HKU-1,
-SARS and - MERS, cytomegalovirus (CMV), herpes simplex virus (HSV) -1
and -2) and 7 non-viral pathogens (Mycoplasma pneumoniae ,Coxiella burnetii , Chlamydia pneumoniae , Chlamydia
psittaci , Streptococcus pneumoniae , Legionella
pneumophila and Pneumocystis jirovecii ), as described previously
[4]. This RP was developed for testing of clinical samples of
hospitalized patients in specific clinical indications (lower
respiratory tract infections in immunocompromised patients or in
immunocompetent patients requiring intensive care admission or not
responding to initial therapy), and therefore includes HSV-1, HSV-2 and
CMV which are not typical respiratory pathogens but can be the cause of
severe pneumonitis in immunocompromised patients. Since the current
study was not focused on immunocompromised populations, these parameters
were not included in our analysis. An additional SARS-CoV-2 specific
RT-qPCR was carried out on samples positive for HCoV-SARS using the
2019-nCoV CDC EUA kit N1 primer probe set [5].
Typing of respiratory pathogens from UPT based on partial sequencing was
done as described previously for EV/RV [6], SARS-CoV-2 [7], and
influenza [8][9]. Complete genome sequencing of SARS-CoV-2 on
RNA extracted from UPT was done with the nanopore technique using the
ARTIC protocol as described in Wawina-Bokalanga et al. [10].
Ag-RDTs were carried out as described in the test instruction manual.
Results
As a proof of concept, used UPT of 9 individuals with symptoms of ARTI
were investigated for the presence of respiratory pathogens. In 2 cases,
EV/RV was detected, which could be further typed as RV C in one case and
as co-infection of RV B and coxsackievirus A19 in the other case. Three
samples tested positive for RSV, all three with concomitant detection of
an additional pathogen (HCoV-OC43, adenovirus and Streptococcus
pneumoniae respectively). In one sample, HCoV-OC43 was detected in
combination with Streptococcus pneumoniae . One sample was
positive for PIV-4 and one sample tested positive for SARS-CoV-2 (Cq
28.1), further typed by complete genome sequencing as variant BA.5.2.1
with a genome coverage of 99.3%. In one sample, no respiratory
pathogens were detected.
Combined UPTs from 6 collectivities (one childcare center, 3
kindergartens and 2 primary schools) were collected by anonymously
gathering tissues used in a classroom or childcare group over the course
of one day. The presence of multiple respiratory pathogens was detected
in these combined samples (Table 1).
Table 1. Identification of respiratory pathogens in combined
used paper tissues from collectivities (n = 6 samples)