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)