key: cord-342660-xigv4u3f authors: Benotmane, I.; Gautier-Vargas, G.; Wendling, M.-J.; Perrin, P.; Velay, A.; Bassand, X.; Bedo, D.; Baldacini, C.; Sagnard, M.; Bozman, D.-F.; Della-Chiesa, M.; Solis, M.; Gallais, F.; Cognard, N.; Olagne, J.; Delagreverie, H.; Gontard, L.; Panaget, B.; Marx, D.; Heibel, F.; Braun-Parvez, L.; Moulin, B.; Caillard, S.; Fafi-Kremer, S. title: In-depth virological assessment of kidney transplant recipients with COVID-19 date: 2020-06-19 journal: nan DOI: 10.1101/2020.06.17.20132076 sha: doc_id: 342660 cord_uid: xigv4u3f Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread widely, causing coronavirus disease 2019 (COVID-19) and significant mortality. However, data on viral loads and antibody kinetics in immunocompromised populations are lacking. We aimed to determine nasopharyngeal and plasma viral loads via RT-PCR and SARS-CoV-2 serology via ELISA and study their association with severe forms of COVID-19 and death in kidney transplant recipients. In this study we examined hospitalized kidney transplant recipients with non-severe (n = 21) and severe (n =19) COVID-19. SARS-CoV-2 nasopharyngeal and plasma viral load and serological response were evaluated based on outcomes and disease severity. Ten recipients (25%) displayed persistent viral shedding 30 days after symptom onset. The SARS-CoV-2 viral load of the upper respiratory tract was not associated with severe COVID-19, whereas the plasma viral load was associated with COVID-19 severity (p=0.0087) and mortality (p=0.024). All patients harbored antibodies the second week after symptom onset that persisted for two months. We conclude that plasma viral load is associated with COVID-19 morbidity and mortality, whereas nasopharyngeal viral load is not. SARS-CoV-2 shedding is prolonged in kidney transplant recipients and the humoral response to SARS-CoV-2 does not show significant impairment in this series of transplant recipients. In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China, and it has since spread widely across the world. 1 The resulting coronavirus disease 2019 (COVID-19) has led to a high death toll. Scientific knowledge on SARS-CoV-2 has evolved rapidly since the outbreak, but little is known about responses to the virus in immunocompromised populations. Infection with respiratory viruses has been shown to be particularly concerning in transplant recipients due to prolonged viral shedding and a higher risk of complications.2,3 However, there have been no reports indicating whether SARS-CoV-2 infection presents the same risks for transplant recipients as other respiratory viruses. Determining viral loads and antibody kinetics in immunocompromised individuals is necessary to protect this highly vulnerable population. Because prolonged viral shedding and/or a lack of protective immunity could lead to significant viral spread in patients' environments, protective measures may need to be increased. We thus conducted a retrospective cohort study in kidney transplant recipients (KTR) in Alsace, Grand-Est France, to determine the dynamics of nasopharyngeal and plasma viral loads and SARS-CoV-2 serology and to study their association with mortality and severe forms of COVID-19. Continuous data are presented as medians and interquartile ranges (IQR) and were analyzed using the non-parametric Mann-Whitney U test. Categorical variables are expressed as counts and percentages and were compared using the Fisher's exact test. The associations between maximum nasopharyngeal SARS-CoV-2 viral load and clinical, demographic, and laboratory variables were determined using Spearman's correlation coefficient (ρ) values. Receiver operating characteristic (ROC) curves were generated to investigate the viral loads in the upper respiratory tract and plasma with respect to disease severity and mortality. Survival plots of severe COVID-19-free survival and COVID-19-specific survival were graphically represented with Kaplan-Meier curves according to RNAaemia (i.e., positive plasma viral load), using the log-rank test to compare differences in survival. Severe COVID-19 was defined as one or more of the following: an oxygen requirement of >6 L/min, the need for ICU admission, and patient death. Patients were censored at the time of last follow-up. Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Inc., San Diego, CA, USA). A p value <0.05 (two-tailed) was considered statistically significant. (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 June 19, 2020. . https://doi.org/10.1101/2020.06.17.20132076 doi: medRxiv preprint (MPA) was being taken by 34 patients (85%), mTOR inhibitors were being administered to 6 (15%) patients, and steroids were taken by 23 (57.5%) patients. The median interval between the onset of symptoms and COVID-19 diagnosis was 4 days (IQR: 3−7 days). All but two patients had a fever. Respiratory (n=34, 85%) and gastrointestinal (n=31, 77.5%) symptoms were the most common clinical manifestations. Twenty-one patients had a non-severe clinical presentation, whereas 19 had a severe clinical course. During follow-up, the mortality rate was found to be 22.5% (9/40). A total of 118 upper respiratory specimens were analyzed (median of three swabs per patient [IQR: 2−3 swabs]). The median viral load was 5.17 log10 copies/reaction (IQR: 3.80−6.69) at diagnosis (between day 0 and day 14 after symptom onset). A total of 29 patients (74.4%) had their peak viral load at admission, and two patients hospitalized at day 10 and day 11 after symptom onset had negative RT-PCR results at admission and during the follow-up. The viral load at admission was not statistically different between severe and non-severe patients (6.22 log10 copies/reaction versus 5.17 log10 copies/reaction, respectively p=0.29) and was not predictive of disease severity (area under the ROC curve = 0.595, p=0.31) at admission, at the peak of viral load (Figure 1a and 1b) , or during the course of the disease ( Figure 2 ). Recipient age (ρ=0.23, p=0.16, Figure 3 ) and sex (p=0.05) were marginally associated with the viral load. Notably, patients receiving steroid therapy and not presenting gastrointestinal symptoms displayed a higher viral load ( Table 2) . No correlation was evident between maximum viral load and inflammatory markers, including interleukin (IL)-6 (ρ=0.041, p=0.81) and C-reactive protein (CRP, ρ=0.001, p=0.99). Among patients with an initial positive RT-PCR test result (n=37), no patient showed a viral clearance before D21. Fifteen (38.5%) patients displayed a 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 June 19, 2020. . https://doi.org/10.1101/2020.06.17.20132076 doi: medRxiv preprint positive viral load greater than 3 log10 copies/reaction after D10, and ten patients (24.4 %) showed persistent viral shedding after D30 ( Figure 2 ). SARS-CoV-2 loads were measured in 73 plasma samples obtained from 32 patients (21 in the non-severe group and 11 in the severe group). Plasma viral loads ranged from 1 to 4.55 log10 copies/reaction. Ten patients had at least one positive RNAaemia. Severe patients showed a higher frequency of RNAaemia compared with non-severe patients (50% versus 26.3%, respectively, p=0.0087, Figure 4a ). Moreover, RNAaemia was found to be associated with mortality ( Figure 4b ). Accordingly, RNAaemia was positive in all three non-survivors tested; in contrast, only 7 of 29 (24%) tested survivors had positive RNAaemia (p=0.024). Furthermore, two non-survivors harbored high viral loads (4.55 log10 copies/reaction and 4.26 log10 copies /reaction), whereas other patients were characterized by low RNAaemia (< 2.16 log10 copies/reaction). With regard to immunosuppressive therapy, patients receiving CNI tended to have more positive RNAaemia (10/24 versus 0/5, respectively, p=0.13). A total of 116 samples from 35 patients were analyzed, with a median of three sera tested per patient (IQR: 2−4 sera). All survivors were seropositive at follow-up. Four non-survivors had negative serology at their time of death, which occurred on D7, D10, D15, and D16. The two patients with a negative SARS-CoV-2 RT-PCR result had positive serology, with one patient showing positive serology at the time of diagnosis. Among the 25 samples tested before D8, six (24%) were seropositive, whereas all samples tested after D14 were seropositive ( Figure 5 ). The kinetics of the antibodies showed that a stable titer of IgG antibodies was maintained until D59, suggesting persistence of immunity for at least until two months after infection 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 June 19, 2020. . https://doi.org/10.1101/2020.06.17.20132076 doi: medRxiv preprint ( Figure 6 ). Notably, IgM and IgG antibody level and delays in seroconversion were not correlated with COVID-19 severity (Figure 7a and 7b). In this retrospective study conducted in a sample of 40 immunocompromised KTR hospitalized for COVID-19, we precisely determined the temporal evolution of nasopharyngeal and plasma SARS-CoV-2 loads, as well as the serological response to the virus. All parameters were correlated with patient characteristics, disease severity, and clinical outcomes. In our study, the viral load in the respiratory specimens of most patients was at the peak at the time of diagnosis. This finding is in line with those previously reported for the general population.5-7 Based on the data we analyzed, the viral load at the time of diagnosis did not predict the severity of the disease. Moreover, viral loads were not related to inflammatory markers that have been previously associated with COVID-19 severity.8 Reports on the relationship between viral load and disease severity are contradictory; three studies showed no correlation between the severity of the disease and the viral load in respiratory specimens,6,7,9 whereas Liu et al.10 described a higher viral load in patients with more severe disease. In our immunocompromised population with a median follow-up of 53 days, the duration of viral detection in the respiratory tract was longer compared to that in the general population. More than a third of our patients displayed a high viral load after D10. Furthermore, almost a quarter of them still had viral shedding at D30; in contrast, in immunocompetent populations, the median duration of viral shedding was 20 days.12 Thus, even if a positive RT-PCR result does not indicate an infectious virus, we should be cautious about viral spread in vulnerable populations and extend isolation after a SARS-CoV-2 infection. 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. The copyright holder for this preprint this version posted June 19, 2020. . https://doi.org/10.1101/2020.06.17.20132076 doi: medRxiv preprint fourth week after symptom onset, which is concordant with our findings. These results suggest that the anti-SARS-CoV-2 humoral response is not significantly impaired in our immunocompromised population.16, 17, 18 Notably, the delay after transplantation was long in our cohort, and only one patient had undergone depleting induction therapy during the year preceding COVID-19. The level of IgG remained steady until two months after onset of symptoms. Although the neutralizing effect of the antibodies was not studied here, it is encouraging that IgG levels are correlated with a neutralizing effect in the general population.6,16 Despite some limitations of this study, including the small sample size and lack of data from some patients, this is the first report that provides a precise assessment of SARS-CoV-2 virological and antibody response kinetics in an immunocompromised population, with a follow-up for two months after symptom onset. Taken together, our data indicate that 1) (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. (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 June 19, 2020. 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 June 19, 2020. (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. Nasopharyngeal maximal viral load are presented as median (interquartile range) Abbreviations: BMI, body mass index; RAAS, renin-angiotensin-aldosterone system; MMF, mycophenolate mofetil; MPA, mycophenolic acid, mTOR: mammalian target of rapamycin. 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. Non-severe disease 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 June 19, 2020. (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 June 19, 2020. . https://doi.org/10.1101/2020.06.17.20132076 doi: medRxiv preprint 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. 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