key: cord-0983310-ev1bhofo
authors: Donato, M.; Park, S.; Baker, M.; Korngold, R.; Morawski, A.; Geng, X.; Tan, M. T.; Rowley, S.; Chow, K.; Brown, E.; Zenreich, J.; McKiernan, P.; Buttner, K.; Ullrich, A.; Long, L.; Kemp, M.; Vendivil, M.; Ricourt, A.; Feinman, R.; Suh, H.; Bindu, B.; Cicogna, C.; Sebti, R.; Al-Khan, A.; Sperber, S.; Desai, S.; Fanning, S.; Arad, D.; Go, R.; Tam, E.; Rose, K.; Sadikot, S.; Siegel, D. S.; Gutierrez, M.; Ip, A.; Goldberg, S.; Feldman, T.; Goy, A.; Pecora, A.; Biran, N.; Leslie, L.; Gillio, A.; Timmapuri, S.; Boonstra, M.; Singer, S.; Kaur, S.; Richards, E.; Perlin, D.
title: Clinical and laboratory evaluation of patients with SARS-CoV-2 pneumonia treated with high-titer convalescent plasma: a prospective study
date: 2020-07-26
journal: nan
DOI: 10.1101/2020.07.20.20156398
sha: f4f1af197676fb1446ab24311143d2846602fda9
doc_id: 983310
cord_uid: ev1bhofo

Background Effective antiviral therapy against the severe acute respiratory syndrome virus 2 (SARS-CoV-2) remains elusive. Convalescent plasma is an anti-viral approach currently under investigation. We aimed to assess the laboratory and clinical parameters of patients with COVID-19 pneumonia treated with convalescent plasma containing high levels of neutralizing anti-SARS-CoV-2 antibodies.

As of July 11, 2020, over twelve million people around the world have been infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and over 550,000 have died. 1 The human and economic impact, unprecedented in our generation, has mobilized the medical community in search of effective treatment strategies. The angiotensin-converting enzyme 2 (ACE2) is necessary for SARS-CoV-2 to enter human cells. 2 The initial phase of the disease occurs when SARS-CoV-2 infects the respiratory epithelial cells, but in addition to lung tissue, expression of ACE2 in found broadly, including in renal, intestinal, and adipose cells, leading to a wide viral impact on the host. 3 Moreover, ACE2 has been shown to be upregulated by SARS-CoV-2 infections. 4 The innate immune response to the viral infection leads to the release of cytokines, and the ensuing cytokine storm results in acute respiratory distress syndrome and multiorgan failure. 5 The natural response to viral infections including coronaviruses, is the production of high affinity immunoglobulin G (IgG) during the adaptive immune response. 6 SARS-CoV-2 has been associated with the suppression of this T cell-mediated immune response, which bring into question the quality of the adaptive immunity in severely ill patients. 7 A therapeutic intervention focused on viral neutralization is therefore a priority.

Convalescent plasma as a method of passive immunity transfer has a long history dating to the Spanish flu of 1918. 8 More recently, convalescent plasma was deployed in the management of SARS 9 and MERS 10 , with evidence of viral neutralization. Convalescent plasma therapy in the setting of SARS-CoV-2 infection is currently an active field of investigation, but information on immune transfer, the subsequent endogenous response, and the clinical course of patients at different stages of the disease remains incomplete. Furthermore, since the development of neutralizing antibody titers varies among COVID-19 recovered patients, convalescent plasma is a heterogeneous product of varying potency. In this study, we investigated both the clinical and laboratory parameters characterizing patients treated with high-titer anti-SARS-CoV-2 neutralizing convalescent plasma.

We conducted a single institution prospective phase IIa clinical trial, registered with ClinicalTrials.gov NTC04343755, FDA IND approval obtained 4/4/2020 and approved by our Institutional Review Board. The study was performed at Hackensack University Medical Center, a tertiary medical center and home to the John Theurer Cancer Center. Patients were included if they were aged 18 years or older and were hospitalized for the

The rapid progression of SARS-CoV-2 infections around the world has led to the expedient reporting of management strategies. In March 2020, the first report of 5 patients with severe COVID-19 disease treated with convalescent plasma was published. 11 This initial report, and previous data on the use of convalescent plasma for the management of other viral infections, renewed interest in this approach. We searched PubMed using the terms "COVID-19" or "SARS-CoV-2" and "convalescent plasma" on July 11, 2020 and found 193 articles covering this topic. We identified 16 publications reporting on the clinical outcome of patients receiving convalescent plasma, excluding single case reports. The largest study focused on the safety of this modality, 12 and a single randomized study reported on the time to improvement of symptoms. 13 Other studies involved a small number of patients (25 patients or less). 14, 23 Added value of this study We prospectively treated patients with high-titer convalescent plasma, clinically characterized in two groups based on severity. Novel aspects of our study include the comparison of fresh and frozen plasma, the evaluation of immunoglobulin subset dosing and the plasma antiviral titer levels. Importantly, we also evaluated the effectiveness of antiviral immunity transfer and the late impact on the recipients' antiviral immunity. We also analyzed the outcome parameters of patients with no or minimal pre-treatment immunity. Strengths of this study include the prospective and complete collection of clinical and basic science information on recipients and plasma components. This study offers the most comprehensive analysis aiming at understanding the clinical and laboratory impact of this therapeutic approach.

As the pandemic continues to expand, a clear understanding of the use and effects of convalescent plasma is critical for patient management. Clinicians will be able to apply this knowledge when managing patients with COVID-19 disease. Furthermore, this information will assist in the design of future research.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 26, 2020. . https://doi.org/10.1101/2020.07.20.20156398 doi: medRxiv preprint management of symptoms associated with a documented infection with SARS-CoV-2. Patients were excluded for a history of severe transfusion reactions, infusion of immunoglobulins with 30 days, AST or ALT greater than 10 times the upper limit of normal, requirement for vasopressors and dialysis. Patients requiring intermittent vasopressors for sedation management were treated. Prospective plasma donors were included if they were aged 18 to 60 years, had a history of a positive nasopharyngeal swab for COVID-19 or a positive antibody test, were at least 14 days from resolution of symptoms, had one subsequent negative swab, were found to have high-titers of neutralizing antibodies against SARS-CoV-2 (>1:500), and met institutional and FDA regulations for donation of blood products.

Volunteer donors were recruited through advertising in the local community. Individuals who agreed to participate and gave informed consent were evaluated at the John Theurer Cancer Center where they underwent a physical examination, completed a donor health questionnaire, had a nasopharyngeal swab for SARS-CoV-2 and blood drawn for complete blood count and chemistry, infectious disease markers, and HLA antibodies for female donors.

The presence of SARS-CoV-2 neutralizing antibodies was evaluated using the previously described COVID-19 ELISA protocol with recombinant spike receptor binding domain (RBD) as capture antigen. 24 High-titer sera was evaluated for virus neutralization in a viral cytopathic assay performed with Vero E6 cells at 100 x the TCID50 value. The assay using SARS-CoV-2 in Vero E6 cells was established under biosafety level 3 (BSL-3) containment to assess intracellular inhibitory potencies of small molecules. Final assay conditions were 30,000 Vero E6 cells per well and virus at a MOI of 0.01-0.05 in 200 ul. The plates were incubated for 48 or 72 hours at 37 o C and 5% CO 2 . Viral ToxGlo™ Luminescent Cell Viability Kit (Promega Corp, Madison, WI, USA) was used to provide a semiquantitative measure of virus infected cell viability. To assess the distribution of the different antibody isotypes/subclasses in the plasma samples, another ELISA was performed with different secondary antibodies. Donors found to have neutralizing IgG Spike RBD greater than 1:500 were selected for plasma donation, with a preference for titers 1:1000-10,000 and >1:10,000. Donors underwent plasmapheresis using the Trima Accel® system for either a planned fresh infusion of 500 mL or for cryopreservation in aliquots of 200 mL.

Recipients were referred by the clinical teams through the institutional COVID-19 research request process and were treated if eligible. A single infusion of convalescent plasma was administered at a rate less than 250 mL per hour. Premedication with diphenhydramine 25 mg IV and hydrocortisone 100 mg IV with or without acetaminophen was given. The use of fresh versus frozen plasma was based solely on the availability of product at the time of request. Exploratory blood work including serology for anti-SARS CoV-2 titers was performed immediately preinfusion and on day+3, +10, +30 and +60 post treatment. SARS CoV-2 testing by RT-PCR from nasopharyngeal or endotracheal tube secretions was done on day+10 and if positive again on day+30. A 10 mL sample of plasma was collected at the bedside from the donor plasma bag immediately pre-infusion for analysis.

The primary endpoint for patients hospitalized for SARS-CoV-2 infection but not receiving invasive or non-invasive positive pressure mechanical ventilation, was to evaluate the efficacy of convalescent plasma in reducing the rate of intubation. The primary objective for patients already receiving invasive or non-invasive positive pressure ventilation was to evaluate the efficacy of convalescent plasma in reducing the mortality rate at day +30. The safety of convalescent plasma was also a primary objective. Secondary objectives for both groups included duration of hospitalization, overall survival, rate of virologic clearance by nasopharyngeal swab RT-PCR at day +10 and +30, impact of donor neutralizing antibody titer levels on the primary objectives, and recipient anti-SARS-CoV-2 titer levels pre-infusion and on days +3, +10, +30 and +60.

It is important to note that at the time of the study's statistical design in late-March 2020, the availability of outcomes data was more limited. For research purposes across studies, patients with COVID-19 at our institution were divided in three tracks based on acuity, track 1 being attributed to outpatients, track 2 for patients hospitalized but not requiring positive pressure mechanical ventilation, and track 3 for patients receiving positive pressure mechanical ventilation. Our statistical plan for this study included only patients ascribed to tracks 2 and 3. We used a multistage design based on the sequential conditional probability ratio test. 25 The statistical design was based on the following hypothesis: for track 2 the null hypothesis assumed an intubation rate of 30% and the alternative hypothesis was an intubation rate < 15%. For track 3 the null hypothesis assumed a mortality rate of at least 49% with an alternative hypothesis of < 25%. The design for each track had a type I error rate of 0.1 with statistical power of at least 0.8. The decision to accept or reject the null hypothesis was made based on interim data analysis in . CC-BY-NC-ND 4.0 International license It is made available under a 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 July 26, 2020. . https://doi.org/10.1101/2020.07.20.20156398 doi: medRxiv preprint a three-stage process. Descriptive statistics were used to characterize the baseline profile of the subjects and exploratory outcomes. Frequency and percentages were used for categorical variables; mean (SD) and median (IQR) were used for the continuous variables. Confidence intervals for the intubation and mortality rates, and virologic clearance at day+10 and+30 were calculated using exact binomial. Kaplan-Meier method was used for overall survival (OS). Log-rank statistics was used to compare the OS between product types, donor titers, and pretreatment immunity. Cox proportional hazards model was utilized to assess the effect of infused plasma neutralizing titers on OS. Univariate test was performed to explore associations between exploratory outcomes and interested groups. Fisher's exact test was used for categorical variables, and t-test/ANOVA, or its non-parametric version, for the continuous variables based on the normalized of the data. P-value less than 0.05 was considered significant. All statistical analyses were performed using SAS (Version 9.4) and RStudio (Version 0.99.902).

The funder of this study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Between April 15 and June 16, 2020, 48 patients were enrolled, one had a negative SARS-CoV-2 nasopharyngeal swab RT-PCR and was ineligible. Forty-seven patients were treated, 32 met criteria for track 2, and 15 patients met criteria for track 3. All 47 patients had radiographic evidence of pneumonia. A significant proportion of patients in track 2 were either immunocompromised (22%) or had active cancer (19%), as our hospital harbors the largest cancer center and stem cell transplant program in the State of New Jersey. Demographic and baseline characteristics of patients in track 2 and 3 are summarized in table 1.

Among the 32 patients in track 2, 24 (75%) were infused with 500 mL of liquid fresh irradiated plasma and 8 (25%) received 400 mL of fresh frozen plasma. The median dose of plasma IgG 1-4 infused was 27,537 ug/kg (IQR 21,550-61,408; n=23); 10/32 (31%) received plasma with viral neutralizing titers >1:10,000 and 20/32 (62.5%) with titers 1:1000-10,000. The primary endpoint analysis for track 2 showed that patients had an intubation rate of 15.6% (95% CI: 5.3%-32.8%), this is enough evidence to reject the null hypothesis (p=0.038). Univariate analysis of numerous parameters was performed and is described in table 2. Older age was highly associated with an increased risk of intubation. The false discovery rate (FDR) adjusted p-value for the donor antibody level was not significant. The univariate analysis significance of tocilizumab cannot be ascertained as it was administered to patients with more severe disease.

Among the 15 patients in track 3, 12 (80%) were infused with 500 mL of liquid fresh irradiated plasma, 3 patients received fresh frozen plasma either 200 mL (1 patient) or 400 mL. The median dose of infused plasma IgG 1-4 ug/kg was 38,260 (IQR 33,3076-50,426; n=12); 5/15 (33.3%) received plasma with neutralizing titers >1:10,000 and 9/15 (60%) with titers 1:1000-10,000. The primary endpoint analysis for track 3 showed that patients had a day-30 mortality of 46.7% (95% CI:21.3%-73.4%). Univariate analysis of numerous parameters was performed and is described in table 3. The overall survival plots for each track is shown in figures 1 and 2. There was a single adverse event for all 47 patients, one patient developed a grade 2 rash (CTCAE v4.0) for which hydrocortisone 100 mg IV was administered once with resolution.

Secondary endpoints analysis for track 2 demonstrated a day-30 discharged alive rate of 87.1% (95% CI: 70.17%-96.37%). The rates of negative nasopharyngeal swab by PR-PCR at day+10 and +30 post treatment were 42.9% (95% CI: 24%-63%; n=28) and 78% (95% CI: 56%-63%; n=23) respectively, in the context of a median time from symptom onset to treatment of 8 days (IQR [4] [5] [6] [7] [8] [9] [10] [11] [12] . There was only one COVID-19-related readmission and the patient was subsequently discharged. Secondary endpoints analysis for track 3 demonstrated rates of negative nasopharyngeal swab or endotracheal secretion analysis by PR-PCR at day+10 and +30 of 85.7% (95% CI: 42-100%; n=7) and 100% (95% CI: 63-100%; n=8) respectively, with a median time from symptom onset to treatment of 15 days (IQR 9-19). The day-30 discharged alive rate was 46.7% (7/15) with one patient extubated but not yet discharged. There were no readmissions.

For either tracks, there was no statistically significant difference in survival, duration of hospitalization, post infusion anti-viral titers, and post infusion inflammatory markers (CRP, ferritin, IL-6 and D-dimers) between fresh and frozen plasma, infused plasma immune globulin subtype (IgA, IgM, IgG 1-4 ) content, or concomitant medications . CC-BY-NC-ND 4.0 International license It is made available under a 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 July 26, 2020. . https://doi.org/10.1101/2020.07.20.20156398 doi: medRxiv preprint (listed in table 1 ). There was also no difference in these endpoints within the ranges of donor IgG anti-viral titers used, which were all above >1:500 (2 donors) and predominantly >1:1000 (tables 2 and 3).

Transfer of immunity was evaluated by measuring the recipients' anti-SARS-CoV-2 neutralizing titer levels immediately pre-infusion and again on day+3. Seven patients (15%), all in track 2 had no pre-infusion titers, and subsequently all 7 were found to have anti-SARS-CoV-2 neutralizing titers on day+3. One transplant patient on immunosuppression was found to have undetectable titers on day+10. Patients in track 3 all had anti-SARS-CoV-2 titers pre-infusion, 4/15 (27%) >1:10,000, 10/15 (67%) 1:1000-10,000, and 1/15 (7%) 1:500-1000. However, we observed an increase on day+3 with 12/15 (80%) >1:10,000 and 3/15 (20%) 1:1000-10,000. All evaluated patients on study were found to have neutralizing titers on day+30 (n= 30) and on day+60 (n=12) (figure 3).

We performed a post hoc analysis of the patients who received convalescent plasma while either non-immune or minimally immune defined as titers 1:100-500. Fourteen patients met these criteria, all in track 2. The intubation rate was 14.3% (95% CI: 1.8%-42.8%). Day+10 and +30 viral clearance by nasopharyngeal swab was 50% (95% CI: 21%-79%) and 78% (95% CI 40-98%) respectively. Immunity over time is shown in figure 4 . There was a trend but no statistically significant difference in overall survival between the non-or minimally immune and the immune group ( figure 5 ). Of note, the only 2 deaths in the non-immune group were attributed to patients with active lymphoma on chemotherapy, one peripheral T cell lymphoma and one with refractory relapsed chronic lymphocytic leukemia.

In this prospective study investigating the therapeutic use of convalescent plasma in patients with COVID-19 disease, we showed that the administration of high-titer donor plasma is safe and effectively transfers anti-viral titers, while preserving the endogenous development of immunity. The study was conducted at the height of the epidemic in New Jersey, when most patients were hospitalized only if requiring oxygen supplementation. In congruence with this fact, all patients treated had pneumonia. Only 13% of patients concomitantly received remdesivir, allowing for the evaluation of convalescent plasma as the sole antiviral agent administered for most patients. Our results showed an intubation rate of 15.6% and for the ventilated patients a day-30 mortality of 46.7%. Within the ranges of plasma anti-viral titers above 1:1000, we were not able to see a difference in outcome based on titer levels. Frozen plasma was not inferior to fresh plasma. Plasma was infused without adverse events, except for one mild rash, to a wide spectrum of recipients including those who were ventilated, elderly, pregnant and immunocompromised.

In the search for anti-viral therapy, our findings clearly demonstrate the safety of convalescent plasma and the passive immunity transfer. As the original data from China used fresh liquid plasma 11 and most centers in the United States make use of fresh frozen plasma, the lack of a significant difference between these products is important information. Frozen plasma allows the flexibility of use, as it can be accumulated and rapidly deployed during a viral surge. Since most of the plasma were from donors with titers above 1:1000, we cannot determine a lowest level acceptable. However, we can ascertain within the statistical limits of this study that we need not limit our donor pool to those with the higher titers >1:10,000, and a cut-off of 1:1000 will be used for our subsequent studies.

Early viral neutralization, with the ensuing prevention of the catastrophic immune response to viral damage, forms the basis for the infusion of high-titer convalescent plasma. Our expectation at protocol inception was to have access to patients early in the course of their disease. The reality, however, of conducting a clinical trial in the setting of an overwhelming influx of cases meant that most patients were not hospitalized until later in their course, during the inflammatory phase. We therefore conducted an ad hoc analysis of the non-immune patients which included patients early in their course and patients unable to mount an immunity, such as immunocompromised patients.

Understanding the kinetics of immune response to the virus is important and has been recently elegantly described. In a series of 23 patients with mild or severe disease, 26 IgG antibodies emerged at 10-15 days post onset of symptoms, were sustained for at least 6 weeks and with a similar IgG response for both the mild and severe groups. Based on these kinetic descriptions, we can confirm that the presence of antibodies on day+3 was from passive transfer and not time related. Interestingly, the same authors reported that most patients with severe disease still had viral shedding 30-40 days post onset of disease, bringing into question the neutralizing capability of those endogenous antibodies. 27 In our study, recipients demonstrated a high level of viral clearance at post infusion day+10 and +30.

Track 3 represents a group of severely ill patients, either non-invasively or invasively ventilated, all with endogenous immune titers. Our management of patients with COVID-19 reserved invasive ventilation almost exclusively for patients failing non-invasive positive pressure ventilation measures. The clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia has been previously reported. 28, 29 In a series of 52 . CC-BY-NC-ND 4.0 International license It is made available under a 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 July 26, 2020. . https://doi.org/10.1101/2020.07.20.20156398 doi: medRxiv preprint patients similar to our track 3 patients, receiving either invasive or non-invasive mechanical ventilation, 32 (61.5%) had died by day 28, and of the remaining 20 patients, only 8 (15.4%) were discharged. 28 In our current study, track 3 patients had a day-30 discharge alive rate of 46.7% and a viral clearance of 86.7% at day+10 post treatment. This may support the position that passive transfer of anti-viral titers may be of benefit even in patients with immunity.

The focus of most anti-viral therapy has been early in the course of the disease. In comparison to patients in track 3, patients in track 2 had a shorter time from symptoms onset to treatment. The track 2 day-30 discharge alive rate was 87.1%, even though 22% of patients were immunocompromised either from cancer or transplantation, 100% had pneumonia, and 91% required oxygen supplementation. The performance of the non-immune group is of most interest, as the only fatalities in this group were attributed to the two patients with advanced lymphoma on active chemotherapy. Our day-30 discharge alive rate for this patient population was 85.7% (12/14) . A recent randomized study evaluated the effect of convalescent plasma on the time to symptom improvement in severe COVID-19 disease. 11 Patients were excluded if they had high titers of S-protein-RBD-specific IgG antibodies (> 1:640), leaving a similar patient population to our non-immune or minimally immune patients. The median volume infused was 200 mL compared to 400-500 mL in our study. In this randomized study the day-28 mortality was 15.7% for the patients in the plasma group, with a discharge rate of 51%. Details of the plasma content or immunity transfer was not provided. There was a statistically significant increase in the rate of viral negativity by PCR in the plasma group, but no difference in the primary outcome of time to clinical improvement. This study was unfortunately limited by the small sample size.

Our study was limited by the lack of a control group and the access to patients early in the disease course, where anti-viral interventions is presumed to be of greatest impact. Our study was also not powered or designed to evaluate the optimal donor antiviral titer level, or the optimal dose of IgM and IgG to be infused. We are conducting a randomized study of convalescent plasma in high-risk patients with early onset disease with the aim of reducing hospitalizations.

In conclusion, we aimed at gaining a better understanding of the clinical and laboratory effects of high-titer convalescent plasma in hospitalized patients with severe COVID-19 pneumonia. We found that the infusion of convalescent plasma is safe, effectively transfers of anti-viral immunity, leads to a high incidence of viral clearance, and does not preclude the development of endogenous immunity. The low rate of intubation and the survival at day 30 are encouraging and warrant further evaluation within the context of a randomized study.

. CC-BY-NC-ND 4.0 International license It is made available under a 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 July 26, 2020. . https://doi.org/10.1101/2020.07.20.20156398 doi: medRxiv preprint Acknowledgments:

To Dr. Florian Krammer PhD and his group at Mt. Sinai that developed the Spike-RBD ELISA . CC-BY-NC-ND 4.0 International license It is made available under a 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 July 26, 2020. CC-BY-NC-ND 4.0 International license It is made available under a 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 July 26, 2020. . https://doi.org/10.1101/2020.07.20.20156398 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a 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 July 26, 2020. . https://doi.org/10.1101/2020.07.20.20156398 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a 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 July 26, 2020. . https://doi.org/10.1101/2020.07.20.20156398 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 26, 2020. . https://doi.org/10.1101/2020.07.20.20156398 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 26, 2020. . https://doi.org/10.1101/2020.07.20.20156398 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 26, 2020. . https://doi.org/10.1101/2020.07.20.20156398 doi: medRxiv preprint

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