key: cord-0997406-tqhwsoey
authors: Puchinger, Kerstin; Castelletti, Noemi; Rubio-Acero, Raquel; Geldmacher, Christof; Eser, Tabea M.; Deák, Flora; Paunovic, Ivana; Bakuli, Abhishek; Saathoff, Elmar; von Meyer, Alexander; Markgraf, Alisa; Falk, Philine; Reich, Jakob; Riess, Friedrich; Girl, Philipp; Müller, Katharina; Radon, Katja; Guggenbuehl Noller, Jessica Michelle; Wölfel, Roman; Hoelscher, Michael; Kroidl, Inge; Wieser, Andreas; Olbrich, Laura
title: The interplay of viral loads, clinical presentation, and serological responses in SARS-CoV-2 – Results from a prospective cohort of outpatient COVID-19 cases
date: 2022-02-18
journal: Virology
DOI: 10.1016/j.virol.2022.02.002
sha: fc0bc541ad9dd79565dfe49fa0941b702298405d
doc_id: 997406
cord_uid: tqhwsoey

Risk factors for disease progression and severity of SARS-CoV-2 infections require an understanding of acute and long-term virological and immunological dynamics. Fifty-one RT-PCR positive COVID-19 outpatients were recruited between May and December 2020 in Munich, Germany, and followed up at multiple defined timepoints for up to one year. RT-PCR and viral culture were performed and seroresponses measured. Participants were classified applying the WHO clinical progression scale. Short symptom to test time (median 5.0 days; p = 0.0016) and high viral loads (VL; median maximum VL: 3x108 copies/mL; p = 0.0015) were indicative for viral culture positivity. Participants with WHO grade 3 at baseline had significantly higher VLs compared to those with WHO 1 and 2 (p = 0.01). VLs dropped fast within 1 week of symptom onset. Maximum VLs were positively correlated with the magnitude of Ro-N-Ig seroresponse (p = 0.022). Our results describe the dynamics of VLs and antibodies to SARS-CoV-2 in mild to moderate cases that can support public health measures during the ongoing global pandemic.

KoCo-19 study group 24 Emad Alamoudi, Jared Anderson, Maximilian Baumann, Marieke Behlen, Jessica Beyerl, Rebecca Böhnlein, Anna Brauer, Vera Britz, Jan 25 Bruger, Friedrich Caroli, Lorenzo Contento, Jana Diekmannshemke, Anna Do, Gerhard Dobler, Ute Eberle, Judith Eckstein, Jonathan Frese, 26 Felix Forster, Turid Frahnow, Günter Fröschl, Otto Geisenberger, Kristina Gillig, Arlett Heiber, Christian Hinske, Janna Hoefflin, Tim Hofberger, 27 Michael Höfinger, Larissa Hofmann, Sacha Horn, Kristina Huber, Christian Janke, Ursula Kappl, Charlotte Kiani, Arne Kroidl, Michael Laxy, 28 Reiner Leidl, Felix Lindner, Rebecca Mayrhofer, Hannah Müller, Dafni Metaxa, Leonie Pattard, Michel Pletschette, 29 Stephan Prückner, Konstantin Pusl, Elba Raimúndez, Camila Rothe, Nicole Schäfer, Paul Schandelmaier, Lara Schneider, Sophie Schultz, 30 Mirjam Schunk, Lars Schwettmann, Heidi Seibold, Peter Sothmann, Paul Stapor, Fabian Theis, Verena Thiel, Sophie Thiesbrummel, Niklas 31 Thur, Julia Waibel, Claudia Wallrauch, Simon Winter, Julia Wolff, Pia Wullinger, Houda Yaqine, Sabine Zange, Eleftheria Zeggini, Thomas 32 Zimmermann, Anna Zielke, Mohamed Ibraheem Mohamed Ahmed, Marc Becker, Paulina Diepers, Yannik Schälte, Mercè Garí, Peter Pütz, 33 Michael Pritsch, Volker Fingerle, Ronan Le Gleut, Leonard Gilberg, Isabel Brand, Max Diefenbach, Tabea Eser, Franz Weinauer, Silke Martin, Jürgen Durner, Philipp Girl, Katharina Müller, Katja Radon, Christiane Fuchs, Jan Hasenauer Munich, Germany, and followed up at multiple defined timepoints for up to one year. and viral culture were performed and seroresponses measured. Participants were classified 46 applying the WHO clinical progression scale. Short symptom to test time (median 5.0 days; 47 p= 0.0016) and high viral loads (VL; median maximum VL: 3x108 copies/mL; p=0.0015) were 48

indicative for viral culture positivity. Participants with WHO grade 3 at baseline had significantly 49

higher VLs compared to those with WHO 1 and 2 (p=0.01). VLs dropped fast within 1 week of 50 symptom onset. Maximum VLs were positively correlated with the magnitude of Ro-N-Ig 51 seroresponse (p=0.022). Our results describe the dynamics of VLs and antibodies to SARS-52 public health emergency of international concern. 75 The current reference standard to diagnose acute SARS-CoV-2 infection is reverse 76 transcriptase-polymerase chain reaction (RT-PCR), from nasopharyngeal swabs or other 77 respiratory samples [5] . Active, replicating SARS-CoV-2 virus can also be detected by viral 78 culture in Biosafety Level 3 laboratories and correlates with infectivity. In patients with positive 79 viral culture results, viral loads (VLs) tend to be significantly higher than in patients with 80 negative culture results. In addition, patients with high VLs are described to have more active 81 virus replication and thus being more infectious than those with low VLs [5] [6] [7] . 82

Dynamics of viral shedding as well as their association with demographic and clinical 83 characteristics during the acute phase of SARS-CoV-2 infection have been described. These 84 indicate that VL decrease gradually after symptom onset and serological responses mostly 85 develop within the first two weeks after infection [8] [9] [10] [11] [12] . Studies also suggest that in some 86 cases, RT-PCR positivity can persist more than 30 days from symptom onset, which is 87 described as prolonged viral shedding [13, 14] . The probability of positive culture decreases 88 10-14 days after symptom onset jointly with declining viral loads [8, 15] . Most published studies 89 were conducted in inpatient settings, and data on the interplay of viral, clinical, and serological 90 characteristics in an outpatient cohort are limited. 91

In the here-presented study, we aimed to assess acute phase VL and shedding dynamics, 92 clinical information, and the SARS-CoV-2-specific antibody response within a long-term 93 prospective cohort in Munich, Germany. We investigated a group of 51 individuals with acute 94 SARS-CoV-2 infection who underwent an in-depth analysis during multiple early time points 95 and for up to week 52 after symptom onset. 96 responses against nucleocapsid as well as spike/RBD were determined at all timepoints 105 respectively. During time of recruitment, only the SARS-CoV-2 Wuhan strain (lineage A) was 106 circulating in Germany, hence no further viral sequencing was performed within the study. 107

Questionnaires on clinical and demographic information were collected as previously 108

published [16, 17] . Symptom to Test Time (STT) was defined as the number of days from the 109 onset of symptoms to documented positive SARS-CoV-2 RT-PCR to reflect the onset of 110 disease more accurately. The clinical presentation of the participants was classified using the 111 WHO clinical progression scale (WHO clinical scale), a scoring system for disease severity 112 during SARS-CoV-2 infection including the following codes: (0) uninfected, (1) asymptomatic 113 cases, (2) mild symptomatic, (3) moderate symptomatic cases who needed assistance, but 114 hospitalisation was not necessary, (4) hospitalised but no oxygen therapy, (5) hospitalised and 115 oxygen by mask or nasal prongs, (6) hospitalised and oxygen by NIV or high flow, (7) 116 intubation and mechanical ventilation, (8) mechanical ventilation or vasopressors, (9) 117 mechanical ventilation and vasopressors, dialysis or ECMO, (10) dead [19] . The participants 118 of the study were all outpatients, therefore patients all fell within WHO category 1-3. 119

The study was approved by the Ethics Committee of the Faculty of Medicine at LMU Munich 120 (20-371). Informed consent was obtained prior to any study procedure. two amplified targets into consideration. Viral cultures were attempted mostly for samples with 131 a VL above the RKI (Robert-Koch-Institute) defined threshold of 1 x 10 6 RNA copies/ml, as 132 chance for culture positivity is minimal in samples below this threshold [20] . However, for some 133 samples a viral culture was not possible due to limited lab-capacities. For some samples below 134 the threshold the viral culture was attempted to verify negativity. 135

Serologic testing methods/ Laboratory Assays. 136 J o u r n a l P r e -p r o o f Serological assays were performed as previously published [16, 17] . Commercially available 137 assays were conducted following the manufacturer's instructions. For all sample time-points, 138 the following assays were performed: Euroimmun Anti-SARS-CoV-2-ELISA anti-S1 IgG 139 (hereafter called EI-S1-IgG; Euroimmun, Lübeck, Germany), Roche anti-N and Elecsys Anti-140 SARS-CoV-2 S anti-S1 (hereafter called Ro-N-Ig and Ro-RBD-Ig-quant, respectively; Roche, 141

Mannheim, Germany). Elecsys Anti-SARS-CoV-2 is an immunoassay for the in vitro 142 qualitative detection of antibodies using a double-antigen sandwich format. In this format, the 143 capture as well as detection is performed by using respectively labelled antigens. Thus, the 144 assay is antibody subclass agnostic by design.. In addition, the SARS-CoV-2 surrogate virus 145 neutralisation test (GS-cPass; GenScript®, Piscataway, New Jersey, USA) was performed. 146

We chose serological assays based on the following criteria: availability in large quantities, 147 enabled for at least semi-automated workup, acceptable pricing, licenced for the use in 148

Europe, and well-described performance [18] . 149

Prior to analysis, data was cleaned and locked. Statistical analysis and visualisation were 151 performed using the software R, version 4.0.5 (https:// cloud.r-project.org/). For operational 152

replicates, the first measurement of EI-S1-IgG was used. In the case of Ro-N-Ig and GS-cPass 153 the latest measurement was included, while for Ro-RBD-Ig-quant the most diluted was 154 selected. We report Pearson's correlation coefficient R for association among continuous 155 variables. For multiple group comparisons, Kruskal-Wallis tests, followed by post-hoc Dunn 156 tests using the Benjamini Yekutieli adjustment for pairwise comparisons were applied [21] . 157

We recruited a total of 51 participants and analysed virological, serological, and clinical data. 159 Table 1 provides an overview of the main demographic and clinical cohort characteristics. 160 57% (29/51) of the cohort were female with a median age of 32 years (IQR 27-47), while 43% 161 (22/51) were male with median age of 35 years (IQR 26-51). 162

Out of 51 participants with an initially documented positive RT-PCR, 82% (42/51) tested RT-164 PCR positive after recruitment during at least one subsequent visit. Overall, a total of 78 (43%, 165 78/182) positive samples from different time-points were available for viral load assessment. 166

The highest measured viral load was 1x10 11 copies per mL. Figure 1 presents the VL 167 dynamics, demonstrating that in our cohort, peak SARS-CoV-2 RNA levels declined rapidly in 168 all but one patient in the first two weeks after symptom onset. One participant was classified 169 J o u r n a l P r e -p r o o f as false-positive, as all follow-up RT-PCR tests remained negative and no seroresponse was 170 detected. Five days after symptom onset, 71% (36/51) of the participants were RT-PCR 171 positive and after 10 days, 59% (30/51) were still above the detection limit (~10 copies per 172 mL). The mean viral load decreased three orders of magnitude between the first and the 173 second week and four orders of magnitude between the first and third week after symptom 174 onset. After three weeks of STT, 22% (11/51) of the participants were tested positive in the 175 RT-PCR while only 6% (3/38) tested positive after 45 days of STT. All participants tested 176 negative in the RT-PCR after day 61, except one who was still tested positive on day 252. 177

During the whole period, this specific participant had varying positive as well as negative PCRs; and viral sequencing revealed the same strain for the whole follow-up period. 179

Viral culture was attempted mostly on samples with a VL >10 6 RNA copies and could be 180 1/2 (median VLs: 7.45 and 3.78, respectively), demonstrating strong evidence of a significant 206 association between VLs and disease severity (p=0.01, figure 4) . 207

Alongside clinical and virological information, the seroresponse at all sample time points for 209 all participants was examined. As participants were included up to four days after their first 210 RT-PCR result, a number of participants were reactive in one or more of the assays at time of 211 study recruitment: 26% for Ro-N-Ig (13/51), 35% for Ro-RBD-Ig-quant (18/51), 18% for El-S1-212

IgG (9/51), and 59% (30/51) using GS-CPass. 213 with VL, without reaching statistical significance (p=0.15). The other assays did not indicate 217 any associations with maximum viral loads (Ro-RBD-Ig-quant p=0.55; El-S1-IgG p=0.97). 218

Correlation of seroresponse and disease severity were also assessed, however, as shown in 219 supplemental figure 2, no significant association was observed. 220 shortened spike, respectively, and all binding immunoglobulin isotypes, while El-S1-IgG 244 targets specifically IgG binding to spike S1. In contrast, GS-cPass is a neutralisation surrogate 245 test and assesses the ability to block the interaction between ACE2 and the RBD of spike, 246 regardless of antibody subclass. In our study, higher VLs correlated significantly with the 247 highest signal detected in the Ro-N-Ig assay, however, this was not the case for the other 248 tests evaluated. This might be due to the abundance of the nucleocapsid antigen in the early 249 phases of viral replication, while the appearance of the anti-S response leads to quick viral 250 control. Here, the functional neutralization seems to be more relevant than the titre. This is in 251 line with GS-cPass as functional assay showing a trend to correlation with viral load as well. 252

Significance might be reached with better statistical power (larger sample size). Our cohort 253 also includes only outpatients with mild disease severity. 254

The clinical presentation of SARS-CoV-2 has been described extensively, and it is assumed 255 that infection mostly causes symptoms of common cold such as cough and fever, while severe 256 cases of pneumonia occur in 1% of cases [25, 26] . In this cohort, most participants were 257 asymptomatic-or oligosymptomatic, reporting cough, loss of taste, and loss of smell as the 258 predominant symptoms. This is in accordance with the reports by the Robert Koch Institute 259 (RKI) [25] where most clinical manifestations resolve in the first two weeks after symptom 260 onset. In other studies, loss of smell and loss of taste persisted for up to 4 months and patients 261 with anosmia are likely to recover within 12 months [25, 27] . This was also observed in two 262 participants in this study. 263

In our cohort, there was a non-significant trend of higher VLs in male compared to female 264 participants, a finding also described in other studies [10, 23] . Age is reportedly another 265 demographic factor potentially impacting VL, with higher VLs found in older patients and 266 respectively lower VLs in younger individuals [28] [29] [30] . In our cohort, younger participants 267 tended to have lower VLs, although this observation was not significant. This could be 268 explained by the composition of the cohort, as the median age in years was 32 and only 6% 269

(3/51) of the participants were above 60 years of age. There are ambiguous descriptions 270 regarding an association between VL and severity of disease in published studies. Some 271 studies report a strong association between higher SARS-CoV-2 VL and increasing disease 272 severity [12, 31] , while others do not find this association [23, 32] . This might be explained by 273 the time point chosen for VL-assessment, Munker et al. described no difference at admission 274 but saw a significantly higher VL in severely ill patients two weeks following admission [33] . In 275 our analyses, we used the highest measured VL for each participant and observed a 276 significant correlation with disease severity classified by WHO grading. Munker et al. 277 demonstrated that the site of sampling may influence the magnitude of VLs and disease 278 severity. In their study, samples collected from the lower respiratory tract, especially in severe 279 cases, exhibited higher viral load [33] . However, we focused on upper respiratory samples 280 and bronchial tract samples were not analysed in this study. 281

Our study has important limitations: Firstly, we have a relatively small sample size of 51 282 individuals in our cohort. Secondly, due to the study design, participants were recruited at The study received funding from the Deutsche Forschungsgemeinschaft (reference number: 296 GE 2128/3-1 and HO 2228/12-1 

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Supplemental Figure 1: Overview of all collected symptoms. Out of 30 different symptoms we 501 detected 23 symptoms in the PCR-positive cohort

Supplemental Figure 2: Serology and WHO grading

GS-cPass and WHO grading

Supplemental Figure 3: Baseline VL and the five most detected symptoms

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work

LO reports non-financial support from Roche Diagnostics. AW, MH and LO report nonfinancial support from Euroimmun, non-financial support from Viramed, non-financial support from Mikrogen. AW, MH, LO report grants, non-financial support and other from German Center for Infection Research (DZIF), grants and non-financial support from Government of Bavaria, non-financial support from BMW, non-financial support from Munich Police, non-financial support and other from Accenture. MH and AW report non-financial support from Dr. Becker MVZ during the conduct of the study