key: cord-0719794-xhsmfjt5 authors: Hu, Q.; Cui, X.; Liu, X.; Peng, B.; Jiang, J.; Wang, X.; Li, Y.; Hu, W.; Ao, Z.; Duan, J.; Zhu, L.; Guo, S.; Wu, G. title: The production of antibodies for SARS-CoV-2 and its clinical implication date: 2020-04-24 journal: nan DOI: 10.1101/2020.04.20.20065953 sha: 68c9c407f9ff722f6b28052910f915cad6eeca44 doc_id: 719794 cord_uid: xhsmfjt5 Background: Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), a novel betacoronavirus, has caused an outburst of pneumonia cases in Wuhan, China. We report the production of specific IgM and IgG antibodies after the infection of SARS-CoV-2 and its implication for the diagnosis, pathology and the course of the disease as well as the recurrence of positive nucleic acid tests after discharge. Methods: Test results for SARS-CoV-2 IgM and IgG antibodies of 221 confirmed COVID-19 patients were retrospectively examined, and their clinical data were collected and analyzed based on various subgroups. SARS-CoV-2 IgM and IgG antibodies were determined with the chemiluminescence method. Findings: The concentration (S/CO) of SARS-CoV-2 IgM and IgG antibodies peaked on day 19-21 after symptom onset, with a median of 17.38 (IQR 4.39-36.4) for IgM and 5.59 (IQR 0.73-13.65) for IgG. Detection rates reached highest on day 16-18 and day 19-21 for IgM and IgG, which were 73.6% and 98.6%, respectively, with significantly higher concentration of IgG in critically ill patients than in those with mild to moderate disease (P=0.027). The concentration of the antibodies on day 16-21 is not correlated with the course or outcome of the disease (Spearman r < 0.20, P > 0.05). Nasopharyngeal swabs revealed positive SARS-CoV-2 RNA in up to 52.7% of recovered patients after discharge, whose IgG proved to be significantly lower than that of those with negative RNA results (P = 0.009). IgG and IgM were tested twice within 14 days after discharge with a 7-day interval, and the second testing of these antibodies displayed a decrease in concentration of 21.2% (IQR, 11.2%34.48%) for IgG and 23.05% (IQR, -27.96%46.13%) for IgM, without statistical significance between the patients with re-detectable positive RNA results and those with negative RNA results after discharge. However, those with positive results experienced a count decrease in lymphocyte subsets. Interpretation: The concentration of SARS-CoV-2 IgM and IgG antibodies peaked on day 19-21 after symptom onset, and antibody testing on day 16-21 is associated with increased detection rates, but the antibody concentration does not affect the course and outcome of the infection. Recovering patients with re-detectable positive SARS-CoV-2 RNA displayed lower concentration of IgG, but the downward trend of IgG during recovery indicated its limited duration of protection, and the protective effect of IgG remains to be investigated. Corona virus disease 2019 (COVID- 19) , which is the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally since its first appearance in Wuhan, China in December, 2019, 1 accounting for 638146 cases and 30039 deaths in 203 countries, areas or territories. 2 SARS-CoV-2 is the seventh coronavirus known to infect humans; SARS-CoV, MERS-CoV and SARS-CoV-2 can cause severe disease, whereas HKU1, NL63, OC43 and 229E are associated with mild symptoms. 3 The infection of SARS-CoV-2 provokes immunological defense and production of specific antibodies, among which IgM antibodies are indicators of current or recent infection as the earliest to develop after exposure to the pathogen, while IgG antibodies, being the most common antibodies of the immunological response, indicates recovery of the disease or past infection. Therefore, the testing of SARS-CoV-2 IgG and IgM antibodies facilitates not only the diagnosis of COVID-19, but the evaluation of infection status as well. In fact, the testing of SARS-CoV-2 IgG and IgM antibodies has been investigated with regard to its value in early diagnosis of the disease and has been adopted as one of the diagnosis criteria in the Chinese guideline. 4,5 However, the production of the antibodies and their protective effect, implication on the outcome and association with re-detectable positive RNA remain to be clarified. This work represents a retrospective analysis of 211 confirmed COVID-19 patients in Chongqing, China with their antibody testing to throw light on the development of SARS-CoV-2 IgG and IgM antibodies and their association with the course and outcome of the disease and re-detectable positive RNA testing after discharge. The study included 211 confirmed COVID-19 patients in Chongqing Three Gorges Central Hospital from January 23 rd to March 3 rd , 2020, among which were 181 mild and moderate cases (the mild group) and 40 severe and critical cases (the severe group). There were 86 female and 135 male patients, with an average age of 47· 8 (47· 8±15· 1) years. This study was approved by the Medical Ethical Committee of the First Affiliated Hospital of Chongqing Medical University (approval number 20200601). Due to the special reasons of the epidemic, the patients' informed consent was not obtained. Clinical data including epidemiology, clinical manifestations, laboratory, course, outcome and follow-up were collected. Testing of SARS-CoV-2 IgG and IgM antibodies was performed every 3 days post symptom onset (PSO) and the results were analyzed for production, detection rates and difference of concentration between the mild group and the severe group. The 145 patients with initial antibody testing results upon admission were divided according to positive and negative IgM and positive and negative calculated according to Jiong Wu et al. 6 The relation of antibody concentration with the duration of positive virus detection (the duration between first and last positive nucleic acid testing), duration of fever (the duration between first and last detection of fever as defined by temperature > 37· 3°C), length hospital stay, the progression of PII (PII value on day 7-10 after admission minus that within 3 days after admission) and outcome of the disease was investigated with the antibody testing result of 78 patients on day 16-21 after symptom onset. The predictive value of SARS-CoV-2 IgG and IgM antibody concentration for re-detectable positive nucleic acid testing was investigated with antibody testing results of 74 recovered patients within 7 days after discharge. The difference in antibody concentration between the patients with and without re-detectable positive nucleic acid was studied with two repeated antibody tests performed on day 1-7 and day 8-14 with a 7-day interval in 40 patients, and the lymphocyte subsets of the 20 patients with re-detectable positive nucleic acid were examined. Diagnosis and discharge decisions were made according to Guidelines for the Diagnosis and Treatment of Novel Coronavirus Infection by the National Health Commission (Trial Version 6). Suspected cases were subjected to reverse-transcription polymerase chain reaction (RT-PCR), and documented cases were categorized into mild, moderate, severe and critical types by clinical manifestations. Discharge criteria included: 1) normal temperature lasting over 3 days; 2) significant improvement of respiratory symptoms; 3) significant improvement of chest radiology; 4) negative nucleic acid testing in two consecutive respiratory specimens collected with an interval of at least 1 day. Testing of SARS-CoV-2 IgG and IgM was performed with serum samples which had been kept at 56 °C for 30 min, using a CFDA approved Magnetic Chemiluminescence Enzyme Immunoassay (MCLIA) kit supplied by Bioscience Co., Ltd (Chongqing, China) following the manufacturer's instructions. The MCLIA for IgG or IgM detection was developed based on the double-antibodies sandwich immunoassay. The recombinant antigens containing the nucleoprotein and a peptide from the SARS-CoV-2 spike protein were conjugated with fluorescein isothiocyanate (FITC) and immobilized on the anti-FITC antibody-conjugated magnetic particles. Alkaline phosphatase-conjugated human IgG/IgM antibody was used as the detection antibody. The tests were conducted on an automated magnetic chemiluminescence analyzer (Axceed 260, Bioscience, China) according to the manufacturer's instructions. Antibody levels were expressed as the ratio of the chemiluminescence signal to the cutoff value (S/CO). S/CO < 1· 0 was designated as negative and otherwise was positive. Statistical analysis was performed using the SAS 9· 4 software. The dynamic changes of the antibodies were analyzed with the logarithm of the IgG and IgM value using a generalized linear mixed model and mapped by locally weighted polynomial regression. Count data were presented as frequency with percentage and compared using χ² test or Fisher's exact test. Measurement data were presented as mean with standard deviation or median with interquartile range and comparison was made using Student's t test or Wilcoxon rank sum test according to the normality of the data. Correlation between variables were examined with Spearman's rank correlation. 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 April 24, 2020. . https://doi.org/10.1101/2020.04. 20.20065953 doi: medRxiv preprint The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. The analysis included 993 test results of 221 patients, whose characteristics were listed in Supplementary Table S1 . The patients in the severe group were older than those in the mild group and the difference is statistically significant (P=0· 0001). Patients with diabetes, cardiovascular diseases and chronic obstructive pulmonary disease were more prone to severe and critical disease (P=0· 0007, P=0· 01 and 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 April 24, 2020. . Positive rate (%) Days after symptoms onset 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 April 24, 2020. Similarly, there were no correlation between the outcome (exacerbation or improvement) and the IgG (P=0· 126) or IgM (P=0· 172) level. 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. There were 74 recovered patients who met the discharge criteria and were discharged to isolation with medical observation for 14 days, and up to 39 (52· 7%) of them presented with re-detectable positive virus nucleic acid during this period. These patients had significantly lower IgG concentration within 7 days after discharge (P=0· 009), but the difference in IgM concentration was not significant (P=0· 06). The decrease of SARS-CoV-2 IgG and IgM antibodies in 40 recovered patients between two tests within 14 days after discharge with a 7-day interval was not statistically significant (P=0· 16 and P=0· 265, respectively), but the decrease of IgG and IgM each reached 21· 2% (IQR, 11· 2%, 34· 48%) and 23· 05% (IQR, -27· 96%，46· 13%) ( Table 1 & 2). In addition, decrease was seen in the count of total lymphocytes, total T lymphocytes, CD4 lymphocytes, CD8 lymphocytes, total B lymphocytes and natural killer lymphocytes to various extents, accounting for 90%, 55%, 65%, 25%, 55% and 20% of the 20 patients with re-detectable positive virus nucleic acid when readmitted, respectively (Supplementary Table S4 ) 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. Data are median (IQR) or n (%). * The difference of two antibody tests within 14 days after discharge with a 7-day interval. # The decrease of antibody level detected within 14 days after discharge with a 7-day interval, as calculated by (difference of the results of the two antibody tests/result of the first antibody test) x100. Positive cases, recovered COVID-19 patients with re-detectable SARS-CoV-2 nucleic acid of nasopharyngeal swabs within 14 days after discharge; negative cases, recovered COVID-19 patients with negative SARS-CoV-2 nucleic acid of nasopharyngeal swabs within 14 days after discharge. As the epidemic escalates, antibody testing has been included in the diagnostic procedure according to Therefore, SARS-CoV-2 IgG and IgM antibodies testing should be combined with RT-PCR as an early diagnosis method, 8 and 173 COVID-19 patients also revealed worse prognosis associated with higher IgG concentration. 18, 19 Therefore, the protective effect of IgG and its association with the deterioration of lung injury remain to be explored with further researches, and these findings have also raised questions on validity of the practice of treating the disease with the serum of recovering patients despite encouraging results of clinical improvement in all 5 critically ill patients after transfusion of convalescent plasma containing 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 April 24, 2020. This study demonstrated a rate of re-detectable positive nucleic acid as high as 52.7% (39/74) despite strict adherence to domestic guidelines on discharge criteria, which may be attributed to the fact that SARS-CoV-2 mainly resides in the lower respiratory system. 21, 22 In addition, possible deviation in specimen collection may lead to false negativity of nucleic testing and consequently inappropriate discharge decisions, 23, 24 resulting in recurrence and even deterioration of the disease. The 20 patients with re-detectable positive nucleic acid displayed decline in lymphocyte subsets on the second admission, which was similar to the changes of initial infection. [25] [26] [27] [28] [29] These patients with SARS-CoV-2 nucleic acid detected in nasopharyngeal swabs within 14 days after discharge had significantly lower IgG concentration compared to those with negative nucleic acid after discharge, but the difference in IgM concentration was not significant. Meanwhile, the changes in IgG and IgM tests with a 7-day interval did not display significant differences. Therefore, repeated nucleic acid testing should be conducted using specimens collected at multiple sites before discharge, 30 and the level of IgG may contribute to the discharge criteria. The results also suggested the possibility that IgG not only serves as a protective antibody, but also bears the potential to deteriorate the injury of multiple organs, which, if proven, will raise concern on treatment with serum antibody of recovered patients, and may indicate IgG or its immune complex as indicators to evaluate organ injury and disease severity. QF H, SL G, made substantial contributions to the study concept and design. QF H, XZ L,was in charge of the manuscript draft. WH H, X W and LX Z ,Z A collecting and confirming data accuracy. JY J，J D, applied for the ethical approval, B P,QF H participated in statistical analysis. XP C,QF H were in charge of the laboratory tasks, including sample processing , detection , data acquisition and interpretation. Y L,GC W were the clinical expert in charge of the treatment of the patients. SL G,GC W,made substantial revisions to the manuscript.All authors contributed to data acquisition, data analysis, or data interpretation, and reviewed and approved the final version. The authors declare no competing interests. With the permission of the corresponding authors, we can provide participant data without names and identifiers, but not the study protocol, statistical analysis plan, or informed consent form. Data can be provided after the Article is published. 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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity This work is funded by Chongqing Education Board "new coronavirus infection and prevention"