key: cord-0682702-dilwk3xq authors: Malik, Madiha; Kunze, Ann-Cathrin; Bahmer, Thomas; Herget-Rosenthal, Stefan; Kunze, Thomas title: SARS-CoV-2: Viral Loads of Exhaled Breath and Oronasopharyngeal Specimens in Hospitalized Patients with COVID-19 date: 2021-07-07 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2021.07.012 sha: 0ef40221a6c6a5a25edc42e0d7cfa7053fb36b90 doc_id: 682702 cord_uid: dilwk3xq BACKGROUND: : SARS-CoV-2 seems to be mainly transmissible via respiratory droplets. We compared the time-dependent SARS-CoV-2 viral load in serial pharyngeal swab and exhaled breath (EB) samples of hospitalized COVID-19 patients. METHODS: : In this prospective proof of concept study, we examined hospitalized patients initially tested positive for SARS-CoV-2. The screening consisted of collecting paired oronasopharyngeal swab and EB specimens taken at different days of hospitalization. The EB collection was performed through a noninvasive simple method using an electret air filter-based device. SARS-CoV-2 RNA detection was determined with qRT-PCR. RESULTS: : Of 187 serial samples taken from 15 hospitalized patients, 87 oronasopharyngeal swabs and 70 of the 100 EB specimens tested positive. Comparing the number of SARS-CoV-2 copies, the viral load of the oronasopharyngeal swabs (n=87) was significantly higher (CI 99%, p<<0,001) than the viral load of the EB samples (n=70). The mean viral load per swab was 7.97 × 10(6) (1.65 × 10(2)-1.4 × 10(8)), whereas EB samples showed 2.47 × 10(3) (7.19 × 10(1)-2.94 × 10(4)) copies per 20 times exhaling. Viral loads of paired oronasopharyngeal swab and EB samples showed no correlation. CONCLUSIONS: : Assessing the infectiousness of COVID-19 patients merely through pharyngeal swabs might not be accurate. Exhaled breath could represent a more suitable matrix for evaluating the infectiousness and might allow screening for superspreader individuals and widespread variants such as the Delta variant. The continuous dissemination of SARS-CoV-2 has emerged as a global health threat due to its high risk of transmissibility. The challenge in managing COVID-19 remains difficult due to unrestrained viable virus shedding, with both symptomatic and asymptomatic patients being capable of spreading COVID-19 [1] [2] [3] . Understanding the dynamics of viable virus shedding and its transmission is essential to propose infection prevention and control precautions to de-escalate the pandemic. Commonly, the detection of SARS-CoV-2 RNA is carried out through routinely implemented real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) from nasopharyngeal or oropharyngeal swabs [4] . Alternatively, SARS-CoV-2 has been detected in several other biological materials, including saliva, sputum, olfactory mucosa, urine, feces and plasma or serum samples of COVID-19 patients [5] [6] [7] [8] . Although SARS-CoV-2 RNA was detected, there have been ambiguous published reports discussing the relevance and potential of these biological materials for transmission of the virus. Thus, viral transmission through these specimen types remains uncertain. However, recent studies presume a correlation between the viral load in samples taken from the upper respiratory tract and the infectiousness of viable virus [6, 9, 10] . According to current knowledge, SARS-CoV-2 seems to be mainly transmissible via droplets and/or aerosols, i.e., respiratory droplets contain replication and infection-competent viruses that can reach susceptible individuals, causing an infection [11] [12] [13] [14] . Alternative sampling of COVID-19 patients, especially those representing the lower respiratory tract, is urgently needed. Nevertheless, invasive sampling methods such as bronchoalveolar lavage and tracheal aspirates are described in the literature but might be impractical in routine COVID-19 diagnosis [6, 15] . In contrast, the detection of SARS-CoV-2 in exhaled breath condensate (EBC) samples has been reported, implying exhaled breath (EB) as a promising biological matrix to examine the transmission of SARS-CoV-2 as well as its risk of contagion [16] [17] [18] . The objective of this experimental proof of concept study was to evaluate the SARS-CoV-2 viral load and its progression in paired serial oronasopharyngeal swab and exhaled breath samples of COVID-19 patients. The viral load in EB samples was detected through a noninvasive and simple method using an air filter-based device, followed by routine qRT-PCR. The main purpose of this study was to resolve the question of whether the viral load of samples from the upper respiratory tract allows an accurate prediction of the viral load in EB, which may represent a more appropriate biological material for assessing the contagiousness of infected COVID-19 patients. In this prospective proof of concept study, we examined patients who initially tested positive for SARS-CoV-2 on admission to the hospital. In total, 187 specimens from 15 hospitalized patients diagnosed with COVID-19 were collected between July 18 th and November 16 th 2020. The screening consisted of collecting swabs of the upper respiratory tract (oronasopharyngeal swabs) and EB specimens and testing for the presence of SARS-CoV-2. In the statistical analysis, paired results were considered to compare the rate of the two sampling methods and to describe differences in the viral loads during the progression of COVID-19. If the disease progression of patients was classified according to the NIH classification of severity of illness [19] as asymptomatic or presymptomatic, mild and moderate patients were recruited to participate in the study. Furthermore, participants were informed and required to sign a written consent form. Patients who developed a critical condition during hospitalization that led to admission to the intensive care unit and those who were discharged from the hospital due to recovery were considered dropouts. The Ethics Committee of the Faculty of Medicine, Kiel University, Germany, approved this study (D527/20). Informed consent for COVID-19 research was waived by the data protection office of the Faculty of Medicine, Kiel University. The investigators were not blinded to allocation during experiments and outcome assessment. After initial positive COVID-19 diagnosis, a simultaneous paired collection of oronasopharyngeal and EB specimens was performed. The day after the initial diagnosis of COVID-19 was set as day 1, and specimen collection was repeated every 1-3 days during hospitalization, particularly on days 3, 5, 7, 10 and 14. Patients were instructed to avoid eating, drinking, smoking, chewing gum or brushing teeth 30 minutes prior to sample collection. EB specimens were collected using a filter-based device (SensAbues®, Stockholm, Sweden) consisting of a mouthpiece, a polymeric electret filter enclosed in a plastic collection chamber, and an attached clear plastic bag. The mouthpiece is designed in such a way to avoid oral fluid contamination during sampling, allowing only microparticles to pass through and to be collected on the filter inside the device. A clear plastic bag indicates adequate individual use and a sufficient volume of exhaled breath passing the electret filter [20] [21] [22] . The patients were instructed to inhale through the nose and tidally exhale without 20 times through the mouthpiece onto the filter inside the collection device. A new device was used for each EB specimen collection. EB specimen collection was performed under the supervision of an investigator. Next, specimens from the upper respiratory tract were collected. Oro-nasopharyngeal sampling was performed using sterile swabs (Nerbe Plus GmbH & Co. KG, Winsen/Luhe, Germany) following the standard recommended procedures [4] . Both swabs and the EB samples were stored at -80 °C until extraction. Paired nasopharyngeal throat swabs and EB specimens were analysed simultaneously and under identical conditions. Viral RNA was extracted using the QIAamp viral RNA mini kit (QIAGEN GmbH, Hilden, Germany). Nasopharyngeal throat swabs were extracted in 0.5 mL buffer. Two hundred microliters of the extract was taken and further diluted (1:1). For the extraction of viral RNA from the filter of the EB collection device, the manufacturer's instructions were modified. The electret air filter was first wetted by frequently adding 1 mL of buffer every 5 minutes to a total volume of 3 mL. Then, the collection device was gently agitated and vortexed for 2 min, and an additional 0.5 mL of buffer was pipetted onto the filter. To elute the solvent from the prewetted filter, the EB collection device was placed into plastic test tubes, and 400 µL of the extracted EB samples were taken for RNA isolation. After concentration, the RNA suspension was eluted in 50 μL of buffer. Ten microliters of the sample eluate was added to 15 µL master mix for each PCR. Ct values of the targeted E gene were converted to log 10 SARS-CoV-2 RNA copies/µL using calibration curves based on an in vitro transcribed (IVT)-quantified coronavirus 2019 E gene control (European Virus Archive GLOBAL, Charité University Berlin). The IVT stock solution at a concentration of 10 6 copies/10 µL was serially diluted to 10 5 ,10 4 , 10 3 , 10 2 , 10 1 and 10 0 copies/10 µL, and 10 µL of each was added to the master mix. qRT-PCR of the calibration was performed under the conditions described above. The results are presented as the means or medians, SDs and interquartile ranges (IQRs), unless stated otherwise. Statistical details for each analysis are described in each figure legend or in the respective part of the text. Student's t-test was used to assess group differences for continuous numerical variables, and a one-tailed p-value was calculated. Additionally, a Welch t-test correction was applied because of unequal sample distribution variance. P-values were considered to be significant at p <0.05. No data points were excluded. Statistical analyses were carried out using R 2020 version 1.3.1093 and Origin A comparison of oronasopharyngeal swabs and EB samples is required to analyse the correlation between the respective viral loads. Here, we tested 187 specimens of 15 hospitalized patients with a confirmed SARS-CoV-2 infection. Of these, 87 were of the upper respiratory tract (oronasopharyngeal swabs), and 100 EBs were collected with a filter-based device (Supplementary Table 1 Finally, the respective viral loads of 70 simultaneously collected paired samples of oronasopharyngeal swabs and EB were not found to correlate (correlation coefficient R 2 < 0.01), as presented in Figure 4 . The evaluation and analysis of exhaled breath is important to extend existing knowledge, providing further proof for SARS-CoV-2 quantification using this biological matrix. Although EB sampling seems challenging, it is a promising biological matrix to explore, especially as it is a noninvasive and more comfortable sampling method than oronasopharyngeal swab sampling and can be easily repeated. [27] , an infected patient would shed approximately 3.89x10 3 -1.59x10 6 per hour simply via regular breathing. This amount of viral load could possibly remain in the air at least for several minutes [14] . While analysing the difference in the viral load of EB and swab samples, the distinctive sampling method as well as the biological material should be considered. Cells of the oropharyngeal or nasopharyngeal mucosa containing the viral RNA were mechanically abraded using a swab, which subsequently led to an artificially generated higher viral load in swabs. In comparison, the collection of respiratory droplets in exhaled breath is noninvasive and consequently does not undergo such mechanical stress. Ljungkvist et al. and Beck at al. describe the collection efficiency as well as the recovery from the filter to be more than 90% [28, 29] . Moreover, the collection efficiency seems to be 99% in the particle diameter range of 0.5-20 μm. As respiratory droplets are typically 5-10 µm in diameter, it can be assumed that the collection efficiency of the system lies between 90 and 99%. Hence, the sampled viral load measured from EB samples almost completely translate to the actual viral emission. Although the mean viral load in EB samples was low, it represents the minimal viral load that would have been exhaled into the environment by the patient just while tidal breathing. Consequently, a non-infected close contact would be exposed at least to this viral load in a non-ventilated room. Hence, talking, singing or even coughing and sneezing might cause higher aerosol emissions [14, 30] . In contrast, patients swallow the nasopharyngeal and oropharyngeal mucus with cells containing viral RNA. Therefore, there is a high possibility that even though the viral load of swab samples is significantly higher, the viral amount found in swabs is not fully shed into the environment, as is the case when exhaling. Furthermore, each patient enrolled in this study exhaled at least once SARS-COV-2 RNA. Nevertheless, the emitted amount of virus copies is extremely heterogeneous as some patients Lastly, no correlation between the viral load of swab samples and EB samples was found ( Figure 4 ). In contrast, Pan et al. showed that sputum and swab samples seem to correlate [31] . This is most likely due to both specimen types being collected from the upper respiratory tract, whereas exhaled breath mainly originates from the lower respiratory tract. As mentioned earlier, SARS-CoV-2 is mainly transmitted via respiratory droplets [11] [12] [13] [14] 24] . Hence, assessing the infectiousness merely through swabs of the upper respiratory tract might not be suitable in all cases. It appears that even though swabs from the upper respiratory tract might serve as an efficient tool in COVID-19 diagnostics, the oropharyngeal or nasopharyngeal mucosa does not grant an accurate prediction of how many virus copies are actually emitted by infected patients. In conclusion, exhaled breath could serve as a more suitable biological matrix for evaluating the infectiousness of COVID-19 patients. Using a simple filter-based device is a promising method for the detection of SARS-CoV-2 viral load in exhaled breath. The EB viral shedding can last during the course of illness for up to 12 days after the first diagnosis. We strongly believe that exhaled breath testing is of tremendous importance to overcome the challenges in managing COVID-19 and should be included in infection prevention and control precautions. Correspondence and request for materials should be addressed to T. Kunze. 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We gratefully acknowledge SensAbues®, Stockholm, Sweden, and JohnTrainor for providing us with the filter-based devices that were used for EB specimen collection. We thank the MVZ Medizinisches Labor Nord, Kiel, Germany, especially Ulrich Klostermeier and Andrea Kruck for their analytical and technical contributions. AngelikaOfft from the data protection office of the Faculty of Medicine, Kiel University, is acknowledged for her helpful advice for the study protocol on data protection. We thank the Interdisciplinary Centre for Statistics and Anna Titova, Institute for Statistics and Econometrics, Kiel University, for the fruitful discussions about statistical analysis and study conception. We would like to give our sincere thanks to the hospital staff that supported us in recruiting the patients and specimen collection. The authors are most grateful to all COVID-19 patients for participating in this study. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.