key: cord-0695456-2pjjz45i authors: Munker, Dieter; Osterman, Andreas; Stubbe, Hans; Muenchhoff, Maximilian; Veit, Tobias; Weinberger, Tobias; Barnikel, Michaela; Mumm, Jan-Niclas; Milger, Katrin; Khatamzas, Elham; Klauss, Sarah; Scherer, Clemens; Hellmuth, Johannes C.; Giessen-Jung, Clemens; Zoller, Michael; Herold, Tobias; Stecher, Stephanie; de Toni, Enrico N.; Schulz, Christian; Kneidinger, Nikolaus; Keppler, Oliver T.; Behr, Jürgen; Mayerle, Julia; Munker, Stefan title: Dynamics of SARS-CoV-2 shedding in the respiratory depends on the severity of disease in COVID-19 patients date: 2021-02-18 journal: Eur Respir J DOI: 10.1183/13993003.02724-2020 sha: 9e52ff0132ff0316df16746a82f14f3d2baa1e38 doc_id: 695456 cord_uid: 2pjjz45i A fraction of COVID-19 patients progress to a severe disease manifestation with respiratory failure and the necessity of mechanical ventilation. Identifying patients at risk is critical for optimized care and early therapeutic interventions. We investigated the dynamics of SARS-CoV-2 shedding relative to disease severity. We analyzed nasopharyngeal and tracheal shedding of SARS-CoV-2 in 92 patients with diagnosed COVID-19. Upon admission, standardized nasopharyngeal swabs or sputum were collected. If patients were mechanically ventilated, tracheal aspirates were additionally obtained. Viral shedding was quantified by real-time PCR detection of SARS-CoV-2 RNA. 45% (41 of 92) of COVID-19 had a severe disease course with the need for mechanical ventilation (severe group). At week 1, the initial viral shedding determined from nasopharyngeal swabs showed no significant difference between non-severe and severe cases. At week 2, a difference could be observed as the viral shedding remained elevated in severely ill patients. A time course of C-reactive-Protein (CRP), Interleukin-6 (Il-6), and Procalcitonin (PCT) revealed an even more protracted inflammatory response following the delayed drop of virus shedding load in severely ill patients. A significant proportion (47.8%) of patients showed evidence of prolonged viral shedding (>17 days), which was associated with severe disease courses (73.2%). We report that viral shedding does not differ significantly between severe and non-severe cases upon admission to the hospital. Elevated SARS-CoV-2 shedding in the second week of hospitalisation, a systemic inflammatory reaction peaking between second and third week and prolonged viral shedding are associated with a more severe disease course. In COVID-19, rapid pulmonary worsening is frequently observed after an initial period of symptom stability. Clinical features of SARS-CoV-2 infections or COVID-19 were previously reported [1] [2] [3] . Several reports have described viral shedding to occur for extended periods 4, 5 . Complete assessment of viral shedding can give valuable insight into the underlying immunological mechanisms 6 . Detection of viral RNA by PCR is not necessarily associated with an infectious virus since infectivity was shown to be significantly reduced at later time points despite the presence of SARS-CoV-2 RNA 7-10 . Pneumonia represents the most important clinical manifestation of COVID-19 infection and is the primary determinant of prognosis in severely ill patients. There is a remarkable heterogeneity in the individual course and severity of the disease. Therefore pulmonary clearance of the virus is of particular interest 10 . An exaggerated response or reduced immunedependent viral clearance in some patients may aggravate the pulmonary manifestation 11 . Individual differences in viral tropism, viral shedding load, duration of viral shedding and viral tissue distribution may play a role therein. Data about the tissue distribution and temporal dynamics of viral shedding are scarce, and further clinical characterization is necessary. Recent investigations shed light on the longitudinal inflammatory response associated to Covid-19 11 , it remains of high interest connecting clinically viable inflammatory parameters to virus shedding. In our hospital, patients diagnosed with COVID-19 were repeatedly tested for evidence of SARS-CoV-2 RNA in material from the respiratory tract, including repeated endotracheal aspirates (ETA), sputum, and nasopharyngeal swabs (NPS). Here we report the clinical and virological findings describing the dynamic of viral shedding in the cohort of 92 consecutive patients admitted to our hospital due to COVID-19 between 29 th February and 17 th May 2020. Methods: This study is a retrospective cohort study of all laboratory-confirmed COVID-19 patients admitted consecutively to the University Hospital of LMU Munich from 29 th February 2020 to 17 th May 2020. All consecutive patients were either referred to or walked into the emergency care unit of our University hospital, a major academic center in southern Germany, with suspected COVID-19. These patients were retrospectively identified as confirmed COVID-19 cases by positive SARS-CoV-2 PCR. Only adults (age≥18 years) were included. We used a simple classification for disease severity: severe cases were defined as patients with the need for mechanical ventilation as it was used before 12 . Moderate disease in our patients was defined by the absence of mechanical ventilation and the need for oxygen insufflation, while the absence of both defined mild disease courses. Non-severe disease includes mild to moderate disease. Samples NPS, sputum, or ETA (in 7 patients with intubation at admission) were routinely obtained on admission and were performed according to local guidelines. NPS samples were taken on clinical suspicion of COVID-19. In addition, sputum samples were obtained when CT scanning showed COVID-19 typical infiltrates and NPS were negative or for clinical monitoring purposes. At admission, up to two NPS samples (with at least 12h distance) and one sputum sample (if necessary) were obtained. Repeated collection of either sample (NPS, sputum, and ETA) was performed for clinical monitoring. When COVID-19 symptoms receded and two consecutive NPS (at least with a day distance) showed a negative result, testing was stopped. Viral loads are expressed as SARS-CoV-2-RNA copy numbers per ml sputum, ETA, or transport medium of the swab sample. The standard swabs used in our hospital contain 1 ml liquid Amies transport medium (eSwab™, COPAN Diagnostics). The following PCR assays were used for quantification in the accredited routine diagnostics laboratory of the Max von Pettenkofer-Institute: The nucleocapsid (N1) reaction of the CDC protocol 13 , the envelope amplification of the Charité protocol 14, 15 , the nucleocapsid amplification of the Seegene Allplex 2019-nCoV Assay and the Roche Cobas SARS-CoV-2 nucleocapsid reaction. Standard curves were generated in multiple diluted replicates using either a plasmid containing the nucleocapsid gene (2019-nCoV-N-PositiveControl, IDT) or a clinical sample with copy numbers based on digital droplet PCR results as described previously 16 being the most common comorbidities. Additional patient characteristics are shown in Table S1 . A total of 473 respiratory samples (245 NPS, 228 tracheal aspirates, and 9 sputum samples) were examined. On average, 5.3 samples were collected per patient, and the testing frequency was similar among both groups Table S2 . Table S2 . In nasopharyngeal swabs of patients with non-severe disease, SARS-CoV-2 viral shedding showed a significant drop at week 2 (p=0.0098), week 3 (p=0.0003), and week 4 (p=0.0004) when compared to week 1 Figure 2d . In patients with severe disease, viral shedding was not different at week 2 (p=0.3089) but decreased at week 3 (p=0.0056) and week 4 (p<0.0001), as depicted in Figure 2d . In ETA of patients with severe disease, viral shedding dropped significantly at week 3 (p=0.0358) and week 4 (p=0.0022) compared to week 1 Figure 2e . To further characterize the longitudinal inflammatory response to viral shedding and disease severity, we characterized the time course of Interleukin-6, Procalcitonin, and CRP. We Table 1 ; this is illustrated in Figure 4 . To correct for influences by other variables, prolonged viral shedding was investigated by uniand multivariate Cox-Regression analysis. To validate the definition "duration of viral shedding" the Cox-Regression analysis was also performed with "duration of viral shedding" defined as the time from onset of symptoms to the first negative test result. The significance and interpretation of the results are basically unchanged between both definitions (see supplementary Figure S1 ). Multivariable analysis confirmed the association of prolonged virus shedding with severe disease. Further, no correlations between viral shedding and immunosuppression were found in Table 2 . Discussion: Our study shows that viral shedding remains elevated the first two weeks in COVID-19 patients with severe disease, whereas it drops earlier in the non-severe patient group in NPS. Several studies investigating SARS-CoV-2 viral shedding and disease severity subsumed bronchial and nasopharyngeal tract samples as respiratory tract samples 8, 21, 22 . As discussed before, a separate analysis of lower respiratory tract samples and upper respiratory tract samples may prevent a sampling bias. When analyzed separately, we found that SARS-CoV-2 nasopharyngeal viral shedding remained high at week 2 in the severe patient group, whereas it dropped at week 2 of the non-severe group. When comparing absolute viral shedding at admission, we did not find significant differences according to disease severity. The persistent elevation at week 2 in the severe group indicates a lack of virus clearance as a causative mechanism for pulmonary worsening. Initial viral loads did not differ according to disease severity, which may further suggest a replication ceiling as a consequence of the saturation of ACE-II receptor binding 23 21 . In this study, respiratory tract samples were not differentiated between sputum or saliva, which may explain the observed differences in viral shedding since elevated levels may also be caused by the inclusion of more sputum samples in the severe group. When analyzing systemic markers of inflammation, we observed a protracted systemic inflammatory response of Il-6 and Procalcitonin at week 2-3 after an initial elevation and decrease of CRP (week 2). These results support previously published data characterizing the immunological response in severely diseased patients 11, 24, 25 . Interestingly a small but relevant proportion of COVID-19 patients develop hyperinflammatory severe disease courses, while initial viral loads do not differ between severely and non-severely diseased patients but stay elevated in patients with severe disease at week 2. These findings are in line with the reported efficacy of the RECOVERY trial, which showed immune suppression by steroid therapy led to a highly significant reduction of 28-day mortality 26 . Analogies can be drawn to other imbalanced hyperinflammatory syndromes, e.g., only a small fraction of patients after EBV infection develop haemophagocytic lymphohistiocytosis 27 . The absence of efficacy of IL-6 receptor blockade by tocilizumab in moderately ill COVID-19 patients indicates other underlying pathways involved in this inflammatory process 28 . The discordant movement of Il-6 and CRP is suggestive of innate factors dominating the early immune response. It was recently shown that Il-6 does not exclusively correspond to CRP (which is commonly produced by hepatocytes in response to IL-6) despite a certain (low) threshold of Il-6 being necessary for CRP production [29] [30] [31] . Two larger studies have shown that serum IL-6 is superior to CRP, ferritin, liver enzymes, and other simple clinical laboratory markers for predicting COVID-19 clinical outcomes, such as respiratory failure and death, with an optimal cutoff of 80 and 86 pg/L, respectively 32, 33 . A procalcitonin value of 0.2-0.5 ng/ml is recognized to be sensitive and specific for bacterial pneumonia in patients with lower respiratory tract symptoms, and pulmonary infiltrates 34, 35 . Interestingly, we observed in the group with and without coinfection or superinfection highly elevated PCT levels, which may indicate the presence of a subclinical bacterial coinfection ( Figure S2b ). It has to be emphasized that timing of sputum/ETA culture may be preceded by antibacterial therapy, therefore the proportion of patients with positive sputum might be underestimated. Further investigations addressing the importance of bacterial coinfection, subclinical coinfection and colonization in COVID-19 patients are warranted [36] [37] [38] . The duration of viral shedding of SARS-CoV-2 has been investigated in several selected patient groups so far. In an early comprehensive study of clinical characteristics of 191 Chinese COVID-19 inpatients, prolonged viral shedding was evident. However, data on absolute copy numbers or sampling sites (sputum, NPS, or ENTA) are not available 4 . Another study investigated viral shedding and transmissibility, and the temporal pattern of viral shedding was stratified according to patient subgroups 7 . Increased duration of viral shedding was not shown in any of the investigated subgroups. However, in this analysis, only a few patients in the non-severe and severe subgroup were included, and a definition of these subgroups was not available. Our study demonstrates the persistence of viral shedding in our hospitalized patients (n=44; 44.8% patients had viral shedding at least 17 days after onset of symptoms) occurs more frequently in patients with severe disease Table 2 . Persistently elevated SARS-CoV-2 viral shedding in respiratory specimens suggests a decreased immune clearance in patients with severe courses. Whereas in individuals of young age and few comorbidities, viral clearance was swift, but prolonged viral shedding was observed among a few oligosymptomatic patients 11 . Important underlying factors responsible for this phenomenon might be differences in host factors or immune response. Interestingly male gender was associated with prolonged viral shedding in Table 2 . The delayed viral clearance of male patients may be explained by immunological and epidemiological gender-specific differences 39, 40 . Further, viral RNA's presence more than 50 days after onset of symptoms may be suggestive for ongoing viral replication, which gives rise to a chronic local inflammatory response. These findings can explain the often difficult and protracted recovery of COVID-19 patients, accompanied by an ongoing local immune reaction with detrimental effects on the respiratory system and other organs 41, 42 . In SARS, similar viral shedding patterns were observed 43 . As in SARS, the slow decrease in SARS-CoV-2 viral shedding despite seroconversion suggests an ongoing cellular clearance with an ineffective antibody-mediated clearance in COVID-19 10, 43 . Table S2 shows corresponding statistical data. Student's T-Test determined differences of means (Table S3) . Table 2 Cox-Regression analysis of factors associated with prolonged SARS-Cov-2 positivity. 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