key: cord-348065-0tkx7aas authors: Liu, Bing; Han, Junyan; Cheng, Xiaohuan; Yu, Long; Zhang, Li; Wang, Wei; Ni, Lan; Wei, Chaojie; Huang, Yafei; Cheng, Zhenshun title: Persistent SARS-CoV-2 presence is companied with defects in adaptive immune system in non-severe COVID-19 patients date: 2020-03-30 journal: nan DOI: 10.1101/2020.03.26.20044768 sha: doc_id: 348065 cord_uid: 0tkx7aas Background: COVID-19 has been widely spreading. We aim to examine adaptive immune cells in non-severe patients with persistent SARS-CoV-2 shedding. Methods 37 non-severe patients with persistent SARS-CoV-2 presence transferred to Zhongnan hospital of Wuhan University were retrospectively recruited to PP (persistently positive) group, which was further allocated to PPP group (n=19) and PPN group (n=18), according to their testing results after 7 days (N=negative). Epidemiological, demographic, clinical and laboratory data were collected and analyzed. Data from age- and sex-matched non-severe patients at disease onset (PA [positive on admission] patients, n=37), and lymphocyte subpopulation measurements from matched 54 healthy subjects were extracted for comparison. Results Compared with PA patients, PP patients had much improved laboratory findings, including WBCs, neutrophils, lymphocytes, neutrophil-to-lymphocyte ratio, albumin, AST, CRP, SAA, and IL-6. The absolute numbers of CD3+ T cells, CD4+ T cells, and NK cells were significantly higher in PP group than that in PA group, and were comparable to that in healthy controls. PPP subgroup had markedly reduced B cells and T cells compared to PPN group and healthy subjects. Finally, paired results of these lymphocyte subpopulations from 10 PPN patients demonstrated that the number of T cells and B cells significantly increased when the SARS-CoV-2 tests turned negative. Conclusion Persistent SARS-CoV-2 presence in non-severe COVID-19 patients is associated with reduced numbers of adaptive immune cells. Monitoring lymphocyte subpopulations could be clinically meaningful in identifying fully recovered COVID-19 patients. Abbreviations COVID-19: Coronavirus disease 2019; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2; HC: Healthy controls. (29.7%), expectoration (24.3%), and diarrhea (13.5%). The less common symptoms included pharyngalgia (2.7%), hemoptysis (2.7%) and weep tears (2.7%). Common complications included CVD (13.5%), followed by diabetes (5.4%) and hepatitis (5.4%). There were 3 current smokers. The baseline characteristics were summarized in Table 1 . Table 2 presented the laboratory testing results of these patients (PP group) on admission to our hospital. Unfortunately, the results of the same patients at disease onset were not available since these patients were first admitted to mobile cabin hospital and then transferred to our hospital, we therefore randomly selected another 37 age-and sex-matched COVID-19 patients confirmed with non-severe disease (PA group), who had their blood test at disease onset on admission to our hospital, for comparison. Compared with patients from the PA group, those from the PP group had significantly higher numbers of lymphocytes (1. 5 p<0.001), as compared to those from the PA group. In together, these results demonstrated that PP patients, upon treatment in mobile cabin hospital and transferred to our hospital, had much improved laboratory findings than PA patients at disease onset, even though they had persistent SARS-CoV-2 shedding. It has been reported that dysregulated immune response were correlated with the severity of COVID-19 8 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 March 30, 2020. . [16] . However, changes in adaptive immune cells in non-severe COVID-19 patients with persistent SARS-CoV-2 shedding has yet to be examined. For this purpose, peripheral blood samples from patients in the PA and PP group were collected, the absolute numbers and relative frequencies of each lymphocyte subpopulations were compared between these two groups. In addition, 54 age-and sex-matched healthy subjects were randomly selected as healthy control (the HC group). As shown in Table 3 , we failed to find any differences between the PP group and the HC group, but patients from both groups had increased numbers of CD3 + T cells, CD4 + T cells, and NK cells compared to those from the PA group. In addition, PA patients had significantly lower frequency of B cells compared with healthy subjects (Table 3 ). These results indicated that non-severe COVID-19 patients (PA group) have already dysregulated immune system at diasese onset, and those with persistent SARS-CoV-2 shedding could restore this abnormality to some level. Upon admission, PP patients received the same standard treatment in our hospital. After at least 7 days, 18 of them that were tested negative for SARS-CoV-2 in two consecutive examinations were retrospectively allocated to the PPN group, and 19 of them who remained positive at the same time point were designated as PPP patients. Of note, the PPP group have more males than the PPN group (86.4% [16 of 19] vs 50% [9 of 18]; p=0.02; Table 1 ). We did not find any differences in symptoms and laboratory findings for these two groups (supplementary Table 1 and 2). However, when lymphocyte subpopulations were examined, PPP patients were found to have significantly lower numbers of CD3 + T cells (p=0.001), CD4 + T cells (p=0.005), CD8 + T cells (p=0.003), and B cells (p=0.005), but higher proportion of NK cells (p=0.02) than PPN patients (Fig 1A and 1B) . Next, we determined the abnormalities for each parameters Lymphopenia was observed at illness onset in 72.8% of non-severe COVID-19 patients (the PA group) in our study, which is similar to those reported by Zhang et al [15] (75.4%), Mo et al [17] (73.5%), Wang et al [27] (70.3%), and Guan et al [2] (83.2%), suggesting the involvement of lymphocytes in the early phase of SARS-CoV-2 infection. Furthermore, lymphocyte count was reported to be correlated with disease severity. Significant higher numbers of lymphocytes were found in survivors versus non-survivors [4] , as well as critically ill versus severe [13, 14] , and severe versus non-severe cases [15, 16] . We focused on non-severe patients with persistent viral presence, and found that the PP group had markedly higher lymphocyte count (1.5 [1.3-1.8] vs 0.9 [0.7-1.3]; p<0.001) than the PA group, and were comparable to healthy subjects. This finding, together with alleviated symptoms and improvements of other laboratory findings, indicated that PP patients might be in the process of recovery, albeit their viral RNA were still tested positive. However, other parameters are required to determine if they were fully recovered. We therefore examined lymphocyte subsets and found that PPP patients had significantly lower numbers of CD3 + T cells (p=0.001), CD4 + T cells (p=0.005), CD8 + T cells (p=0.003), and B cells (p=0.005) than PPN patients (Fig 1A and 1B ). When compared with healthy subjects, PPP patients again exhibited much less CD3 + T cells (p=0.044), CD4 + T cells (p=0.034), and B cells (p=0.02) (Fig 2C and 2D ). Most strikingly, 10 PPN patients showed markedly increased CD3 + T cells (p=0.001), CD4 + T cells (p=0.002), CD8 + T cells (p=0.009), and B cells (p=0.008) after they turned negative for SARS-CoV-2. Together, these results suggest that measurement of these lymphocyte subpopulations could be used to distinguish non-severe patients with persistent viral presence from healthy subjects and those turned negative, and thus have clinical relevance for discharge management. The latter can induce the activation and differentiation of cognate B cells, and subsequently promote the production of virus-specific antibodies, including neutralizing antibodies [29] . In turn, neutralizing antibodies are able to mediate antibody-dependent cell-mediated cytotoxicity to kill virus-infected cells, 12 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 March 30, 2020. . and to block the entrance of extracellular virus [30] . Therefore, it's not surprising that changes in these cells could reflect the viral presence. Accordingly, T cell subsets were reported to be profoundly affected in severe cases with SARS-CoV-2 infection [16] . However, we could not determine from our data and the current knowledge whether SARS-CoV-2 can directly infect these lymphocytes, or indirectly caused these alterations. We did not find any difference in NK cells between the PPP group and healthy subjects, in terms of both absolute numbers and relative frequency (Fig 2A and 2D) . Instead, NK cells were even higher in the PPP group than in the PPN group (p=0.02, Fig 1A) . As an innate immune cells, NK cells is among the first cell types to combat virus infection [31] . However, PP patients in our study were likely to be in the late phase of SARS-CoV-2 infection, during which the role of NK cells remained to be defined. Several limitations to the present study warrant mention. First, this retrospective study was conducted in a single hospital, which may result in selection bias. Our conclusion could be further strengthened by a multicenter, prospective study in a randomized setting. Second, only 37 non-severe COVID-19 patients with persistent viral presence were included in this investigation, interpretation of our findings might be limited by the sample size. Third, these patients were transferred to our hospital, we do not have their laboratory results and lymphocyte measurements at disease onset, we therefore randomly selected ageand sex-matched PA patients for comparison. Fourth, quantitative viral RNA detection and isolation of live virus were not performed due to limited resources in our hospital, which prevent us from building connections between lymphocyte subpopulations and these parameters. Despite these limitations, the present study, to the best of our knowledge, is the first investigation to examine changes of lymphocyte subpopulations in non-severe COVID-19 patients with persistent viral presence. We found that CD4 + T cells, CD8 + T cells, and B cells were markedly decreased in these patients. Our findings suggest that monitoring lymphocyte subpopulations could be clinical meaningful in discharge management for non-severe COVID-19 patients with persistent viral presence. 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 March 30, 2020. . Data are median (IQR) or n (%). P values were obtained from χ² tests , Fisher's exact tests, T tests or Mann-Whitney U tests, when appropriate. P < 0.05 was considered statistically significant (in bold). Abbreviations: COVID-19, coronavirus disease 19; PA (positive on admission); PP (persistently positive); NLR: Neutrophil-to-lymphocyte ratio; PLTs, platelets; Hb, hemoglobin; ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CRP, C-reactive protein; SAA, serum amyloid 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 March 30, 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 March 30, 2020. . blood of PP patients were tested positive again at least 7 days after they were admitted to our hospital (PPP), and PP patients were tested negative in 7 days after they were admitted to our hospital (PPN). P < 0.05 was considered statistically significant (in bold). (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. World Health Organization. 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