key: cord-0266754-khhhok74 authors: Gutmann, D.; Scheuch, G.; Lehmkuhler, T.; Herrlich, L.-S.; Hutter, M.; Stephan, C.; Vehreschild, M.; Khodamoradi, Y.; Gossmann, A.-K.; King, F.; Weis, F.; Weiss, M.; Rabenau, H. F.; Graf, J.; Donath, H.; Schubert, R.; Zielen, S. title: Aerosol measurement identifies SARS-CoV 2 PCR positive adults compared with healthy controls date: 2022-01-21 journal: nan DOI: 10.1101/2022.01.21.22269423 sha: 082178d575605f8bb516bcd78cadfd4426c2a016 doc_id: 266754 cord_uid: khhhok74 Background: SARS-CoV-2 is spread primarily through droplets and aerosols. Exhaled aerosols are generated in the lung periphery by reopening of collapsed airways. Aerosol measuring may detect highly contagious individuals ("super spreaders or super-emitters") and discriminate between SARS-CoV-2 infected and non-infected individuals. This is the first study comparing exhaled aerosols in SARS-CoV-2 infected individuals and healthy controls. Design: A prospective observational cohort study in 288 adults, comprising 64 patients testing positive by SARS CoV-2 PCR before enrollment, and 224 healthy adults testing negative (matched control sample) at the University Hospital Frankfurt, Germany, from February to June 2021. Study objective was to evaluate the concentration of exhaled aerosols during physiologic breathing in SARS-CoV-2 PCR-positive and -negative subjects. Secondary outcome measures included correlation of aerosol concentration to SARS-CoV-2 PCR results, change in aerosol concentration due to confounders, and correlation between clinical symptoms and aerosol. Results: There was a highly significant difference in respiratory aerosol concentrations between SARS-CoV-2 PCR-positive (median 1490.5/L) and -negative subjects (median 252.0/L; p<0.0001). There were no significant differences due to age, sex, smoking status, or body mass index. ROC analysis showed an AUC of 0.8918. Conclusions: Measurements of respiratory aerosols were significantly elevated in SARS-CoV-2 positive individuals and may become a helpful tool in detecting highly infectious individuals via a noninvasive breath test. Clinical Trial Number: ClinicalTrials.gov Identifier: NCT04739020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. Background: SARS-CoV-2 is spread primarily through droplets and aerosols. Exhaled aerosols are generated in the lung periphery by 'reopening of collapsed airways'. Aerosol measuring may detect highly contagious individuals ("super spreaders or superemitters") and discriminate between SARS-CoV-2 infected and non-infected individuals. This is the first study comparing exhaled aerosols in SARS-CoV-2 infected individuals and healthy controls. Results: There was a highly significant difference in respiratory aerosol concentrations between SARS-CoV-2 PCR-positive (median 1490.5/L) and -negative subjects (median 252.0/L; p<0.0001). There were no significant differences due to age, sex, smoking status, or body mass index. ROC analysis showed an AUC of 0.8918. Measurements of respiratory aerosols were significantly elevated in SARS-CoV-2 positive individuals and may become a helpful tool in detecting highly infectious individuals via a noninvasive breath test. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. pandemic had caused more than 187 million confirmed cases and four million deaths. [1] [2] [3] As the prevalence of SARS-CoV-2 infection and associated pulmonary disease (coronavirus disease-2019 ) remain high across the globe, the pandemic has been one of the greatest threats to the global economy and social infrastructure. 4 Current research suggests that SARS-CoV-2 is spread primarily through droplets and aerosols. 5, 6 In addition to symptomatic carriers, asymptomatic infections and highly contagious carriers ('super spreaders') are key drivers of virus spread. [7] [8] [9] [10] Aerosols are defined as a suspension of solid or liquid particles within a gas mixture (e.g., air); 11-13 while droplets are defined as particles of a size approximately >100 µm. During normal breathing, small aerosol particles can be detected in the exhaled air. [14] [15] [16] Larger particles with different sizes (between 1-50 µm) and compositions are exhaled more frequently during speech, laughter, or singing. [17] [18] [19] In a recently published study from Singapore, it was shown that 85% of SARS-CoV-2 viruses were detected in the small size fraction of exhaled aerosols. 20 The spread of viruses and bacteria via aerosols has already been investigated previously; e.g., in Mycobacterium tuberculosis, influenza viruses, and respiratory syncytial viruses (RSV), 7,21-23 and aerosols are characterized as an important factor in the spread of related diseases. Aerosols are a vital transmission route for SARS-CoV-2 and play a major role in the viral spreading via asymptomatic individuals, contributing to the rapid spread of the SARS-CoV-2 pandemic. 7, 8, 10, 13 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. however, this test is unspecific because other airway infections may also be detectable. If highly contagious individuals could be rapidly identified by aerosol measurement, and subsequently managed, a significant portion of new infections may be prevented. The aim of this prospective study was to investigate the difference in aerosol concentration and particle size between SARS-CoV-2 PCR-positive and -negative adults. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We conducted a prospective observational cohort study to evaluate exhaled aerosol concentration and particle size in SARS-CoV-2 PCR-positive and -negative individuals Recruitment commenced on January 18th, 2021, and was completed on June 4th, 2021. In total, 288 adults were analyzed (64 subjects tested positive by a nasal or pharyngeal swab SARS-CoV-2 PCR and 224 healthy controls were SARS-CoV-2 PCR-negative). SARS-CoV-2 PCR-positive patients were recruited from the Division of Infectious Disease, Goethe-University Hospital, Frankfurt, Germany. Healthy controls were recruited from parents or caregivers of hospitalized children at the Department for All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Clinical and Medical History The electronic chart and International Classification of Disease (ICD) were used for diagnosis, estimation of BMI, oxygen supplementation, and to cluster risk factors including obesity, diabetes, hypertension, and chronic heart, respiratory, and kidney disease. Before measurement of aerosols, all participants (SARS-CoV-2 PCR-positive patients and healthy controls) were questioned about the presence of typical SARS-CoV-2 symptoms. The typical SARS-CoV-2 symptoms included the presence of fever, cough or dry cough, shortness of breath, loss of taste or smell, sore throat, muscle pain, diarrhea, and vomiting. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.21.22269423 doi: medRxiv preprint G u t m a n n e t a l : The instrument compromised a heated hose section upstream of the measurement cell to avoid condensation effects and enable evaporation of larger droplets. The temperature and relative humidity in the sampled air was also measured. Exhaled breath from subjects was collected via mouthpiece, connected to a t-adapter with HEPA filter and connection port to the Resp-Aer-Meter via a hose. To ensure effective hygiene, sterile sampling kits were used for each measurement. Participants performed normal tidal breathing through the mouthpiece while the nose was closed via a nose clip. In the first minute of tidal breathing, a sharp drop of particle concentration was detected due to inhalation of clean air via the HEPA filter. This is the washout effect, during which the ambient aerosol still present in the lungs is washed out. After a few breaths, a baseline concentration of particles generated and exhaled from the lungs was determined. Subsequently, the measurement (lasting 1-1.5 minutes) was taken to establish the quantity of particles emitted from the lungs. The results of the test were directly displayed as a graphical curve (Supplemental eFig. 1), enabling calculation of the mean exhaled particle count per liter, particle size distribution, and mean particle size (in µm). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.21.22269423 doi: medRxiv preprint G u t m a n n e t a l : The primary outcome of this study was the measurement of aerosol particle concentration in SARS-CoV-2 PCR-positive and -negative subjects, and the distinction between positive and negative subjects via aerosol measurement. Secondary outcome measures comprised the correlation of aerosol concentration to SARS-CoV-2 PCR results, change in aerosol concentration due to confounders (such as age, sex, lung function, height, weight, BMI, and smoking status), and the correlation between clinical symptoms and aerosol measurements in SARS-CoV-2 PCR-positive patients. GraphPad Prism 5.01 (GraphPad Software, Inc.) and R 4.0.4 were used for statistical analysis. The values were presented as median and range for numeric data and as percentage for count data. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.21.22269423 doi: medRxiv preprint G u t m a n n e t a l : The Wilcoxon-Mann-Whitney U-Test and Fisher's exact test were used to test for group differences in numeric and count data, respectively. A p-value of less than 0.05 was considered statistically significant. In addition, the sensitivity and specificity of the aerosol measurement was evaluated using Receiver Operating Characteristic (ROC) analysis and the correlation between Ct values and aerosol measurement was calculated via Spearman correlation. Table 1 . Of 64 hospitalized SARS-CoV-2 PCR-positive patients, 71.9% (46/64) were diagnosed with acute respiratory failure and/or pneumonia associated with SARS-CoV-2 infection: 12 patients were diagnosed with respiratory failure and 34 with COVID-19 pneumonia. In total, 28.1% (18/64) of patients had moderate symptoms and were considered immunocompromised with precautionary hospital admissions when found to be SARS-All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The symptoms recorded in the SARS-CoV-2 PCR-positive and -negative subjects are presented in Table 2 . Aerosol Concentration The median exhaled particle count was highly significantly elevated in SARS-CoV-2 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In addition, there was a significant, negative correlation for exhaled particle count and Ct value (Spearman correlation, r: -0.4926; p<0.0001). There were no significant differences in aerosol concentration due to sex, BMI, or In order to analyze the accuracy of the exhaled particle count as a test to detect SARS-CoV-2 PCR-positive infection, a ROC analysis was conducted (Fig. 2) . At an exhaled All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. respectively. In addition, there was a significant difference of FEV1%pred between the patient (respiratory failure, pneumonia, and immunocompromised) and healthy control groups (Table 1) . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Current research suggests that SARS-CoV-2 infection is spread primarily through exhalation of droplets and aerosols containing viable virus particles, which may linger in the air and survive for several hours. 6, 17, 30 Despite inter-individual differences, SARS-CoV-2 PCR-positive patients produced significantly increased exhaled particle counts compared with healthy controls. In addition, greater exhaled particle counts may be associated with more severe infection and higher infectivity. Whereas Edwards et al. reported a significant correlation between exhaled particle counts and BMI, 28 no such correlation was observed within either the SARS-CoV-2 PCR-positive or -negative groups in this study. In addition, no correlation in particle viruses into the human cells). Therefore, it seems likely that SARS-CoV-2 viruses are replicated in AT2 cells; these cells consecutively may release more surfactant into the All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Recent studies have demonstrated that increases in exhaled particle concentration with SARS-CoV-2 positive primates are dominated by very small particles, which might only be visible with a lower detection limit below 0.3 µm. 28 Thus, measurement with a lower detection limit may provide greater accuracy for detecting exhaled particles, particularly in the size ranges that are crucial for transmission of SARS-CoV-2. Jones et al. highlighted a large-sample analysis of RT-PCR results, which showed that a small subset of subjects (9%) had a very high viral load and were thus considered highly infectious. 36 The current study demonstrated that a very small group (3.5% of all participants) was responsible for over 50% of all exhaled aerosols. Furthermore, in the SARS-CoV-2 PCR-positive group, 15.6% of patients were responsible for almost 70% the of exhaled aerosols. To assess the validity of the aerosol measurement as a tool to test for SARS-CoV-2 infected patients, a ROC analysis was conducted and demonstrated good validity (AUC of 0.89). Our analysis suggests that aerosol particle measurements alone are not All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Our study has several limitations, including that the SARS-CoV-2 PCR and aerosol particle measurements were not performed simultaneously. A timeframe from PCR test to aerosol measurement of 72 hours was accepted for all patients; this may affect the results, as other studies have found peak viral loads around day four of infection, which might be present 1-3 days before the onset of symptoms and followed by a steady decline in viral load. 36 In addition, only hospitalized SARS-CoV-2 positive patients were included. And it seems reasonable that aerosol particle counts may be greater in patients with severe disease, reflecting a certain level of lung framework damage due to this viral infection. This might explain the lower exhaled particle counts reported in the (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. aerosol particles and serial SARS-CoV-2 PCR measurements. In addition, this study allows no statement concerning differences in viral load 36 and consecutive aerosol shedding by virus variants, although these were previously observed clinically. Lastly, only aerosols across a particular size range were measured. While current research suggests that small particles may play a critical role in aerosol transmission, future studies investigating different aerosol size ranges should be performed to verify this hypothesis. In conclusion, the concentration of exhaled aerosols particles was significantly different between SARS-CoV-2 PCR-positive and -negative individuals. Because the origin of these aerosol particles are the bases of the lung, alveolar epithelial cells type 2 may produce more surfactant when infected by viruses, generating more small droplets to carry the virus out of the lung. A better understanding of respiratory aerosol generation may lead to improved control of SARS-CoV-2 transmission. In the future, portable devices for aerosol measurement may be a valuable tool to detect potentially contagious individuals with a non-invasive breath test. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.21.22269423 doi: medRxiv preprint G u t m a n n e t a l : This work was supported by Palas GmbH, Partikel-und Lasermesstechnik, which provided aerosol measurement devices, as well necessary equipment, and limited sponsorship for conduction of the study. Additional resources were provided by the University Hospital Frankfurt, Goethe University. Palas GmbH reviewed the study data and final manuscript before submission, but the authors retained editorial control. Palas GmbH also provided funding for a medical writer to assist in manuscript preparation. All authors had full access to all data in the study and had final responsibility for the decision to submit for publication. We would like to thank Dr Allan Johnson of Medical Writing Limited for his assistance in preparation and editing of this manuscript. We also thank the volunteers who participated in this study. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. underlying data supporting the findings of this manuscript. All authors had full access to the full data set in this study and critically reviewed and approved the final version. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Anonymized participant data will be made available upon requests directed to the corresponding author. Proposals will be reviewed and approved by investigator and collaborators on the basis of scientific merit. After approval of a proposal, data can be shared through a secure online platform after signing a data access agreement. All data will be made available for a minimum of five years from the end of the trial. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. Vomiting 0 (0.0%) 2 (5.9%) 0 (0.0%) 2 (3.1%) 0 (0.0%) 0.064 Symptoms were recorded at time of aerosol measurement. SARS-CoV-2 PCR-positive group is displayed as whole collective and divided in three clinical categories (respiratory failure, pneumonia, immunocompromised). p-Values for differences in SARS-CoV-2 PCR-positive andnegative participants are derived from Fisher's exact test. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. with corresponding sensitivity and specificity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.21.22269423 doi: medRxiv preprint COVID-19) Weekly Epidemiological Update and Weekly Operational Update Statistics and research: coronavirus pandemic (COVID-19). 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The copyright holder for this preprint this version posted Aerosol transmission of influenza A virus: a review of new studies Aerosol and surface stability of HCoV-19 (SARS CoV-2) compared to SARS-CoV-1 Breathing Is Enough: For the Spread of Influenza Virus and SARS-CoV-2 by Breathing Only Characterization of exhaled particles from the healthy human lung--a systematic analysis in relation to pulmonary function variables Exhaled Particles After a Standardized Breathing Maneuver Breathing, speaking, coughing or sneezing: What drives transmission of SARS-CoV-2? Visualizing speech-generated oral fluid droplets with laser light scattering Transmission of SARS-CoV-2: Physical Principles and Implications Front. Public Health No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted Aerosol Dynamics Model for Estimating the Risk from Short-Range Airborne Transmission and Inhalation of Expiratory Droplets of SARS-CoV-2 Epub ahead of print Surfactant Protein A in Exhaled Endogenous Particles Is Decreased in Chronic Obstructive Pulmonary Disease (COPD) Patients: A Pilot Study Airway monitoring by collection and mass spectrometric analysis of exhaled particles Aerodynamics of droplet nuclei Modality of human expired aerosol size distributions The mechanism of breath aerosol formation Estimating infectiousness throughout SARS-CoV-2 infection course No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted