key: cord-0712909-653viyaq authors: Huang, Jing-Tao; Ran, Ruo-Xi; Lv, Zhi-Hua; Feng, Li-Na; Ran, Chen-Yang; Tong, Yong-Qing; Li, Dong; Su, Han-Wen; Zhu, Cheng-Liang; Qiu, Shi-Li; Yang, Jie; Xiao, Meng-Yao; Liu, Ming-Jun; Yang, Yu-Ting; Liu, Song-Mei; Li, Yan title: Chronological Changes of Viral Shedding in Adult Inpatients with COVID-19 in Wuhan, China date: 2020-05-23 journal: Clin Infect Dis DOI: 10.1093/cid/ciaa631 sha: a388bb0f505ada63f9e25ceecd3da44038f626a7 doc_id: 712909 cord_uid: 653viyaq BACKGROUND: In December 2019, the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) broke out in Wuhan. Epidemiological and clinical characteristics of patients with COVID-19 have been reported, but the relationships between laboratory features and viral load has not been comprehensively described. METHODS: Adult inpatients (≥18 years old) with COVID-19 who underwent multiple (≥ 5 times) nucleic acid tests with nasal and pharyngeal swabs were recruited from Renmin Hospital of Wuhan University, including general patients (n=70), severe patients (n=195) and critical patients (n=43). Laboratory data, demographic and clinical data were extracted from electronic medical records. The fitted polynomial curve was used to explore the association between serial viral loads and illness severity. RESULTS: Viral load of SARS-CoV-2 peaked within the first few days (2-4 days) after admission, then decreased rapidly along with virus rebound under treatment. Critical patients had the highest viral loads, in contrast to the general patients showing the lowest viral loads. The viral loads were higher in sputum compared with nasal and pharyngeal swab (p=0.026). The positive rate of respiratory tract samples was significantly higher than that of gastrointestinal tract samples (p<0.001). The SARS-CoV-2 viral load was negatively correlated with portion parameters of blood routine and lymphocyte subsets, and was positively associated with laboratory features of cardiovascular system. CONCLUSIONS: The serial viral loads of patients revealed whole viral shedding during hospitalization and the resurgence of virus during the treatment, which could be used for early warning of illness severity, thus improve antiviral interventions. In early December 2019, the type of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) broke out in Wuhan, China [1, 2] . The World Health Organization (WHO) has characterized the coronavirus disease 2019 (COVID-19) as a pandemic [3] . As of May 11, 2020, over 4.3 million confirmed cases and more than 280,000 deaths were reported globally, including 82919 cases and 4633 deaths in China, including 54214 cases and 3869 deaths in Wuhan [4] . Recently, researchers have described the clinical characteristics and risk factors of COVID-19 [5, 6] . Details of the laboratory test and virological course of COVID- 19 have not yet been comprehensively investigated. Here, we report findings from a comprehensive analysis of viral load and laboratory findings to reveal viral replication and shedding during hospitalization. Chronological changes in viral shedding of SARS-CoV-2 after admission in individual patients, overall patients and patients grouping by illness severity (general, severe and critical) were shown by polynomial fitting curves. Meanwhile, serum albumin was decreased in high viral load patients, and serum glucose, corrected calcium were increased in high viral load patients. Of particular note, serum CKMB, MYO, ultra-TnI and NT-proBNP were significantly increased in high viral load patients, indicating that high viral loads were more likely to damage cardiovascular system. We also investigated the link between viral load and laboratory findings. Our data will provide general tendency of viral replication and shedding, which could help better guiding the clinical treatment. A c c e p t e d M a n u s c r i p t 6 A total of 308 adult inpatients (≥18 years old) from Renmin hospital of Wuhan University between January 11 to February 21, 2020 were enrolled in this retrospective study. Each patient was diagnosed as COVID-19 according to the WHO interim guidance [7] and underwent multiple (at least 5 times with nasal and pharyngeal swab) nucleus acid testing. A confirmed COVID-19 case was defined as a positive result from the real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assay according to the manufacturer's protocol (Shanghai Geneodx Biotechnology, Shanghai, China) of nasal and pharyngeal swab specimens [8] . The clinical outcomes, such as, discharges, mortality, were followed up to March 6, 2020. The study was approved by the Research Ethics Commission of Renmin Hospital (Approval No. WDRY2020-K066). Data were extracted from electronic medical records and laboratory information system using a standardized form. All data were checked by two researchers (M-Y X and M-J L) independently for accuracy. qRT-PCR [8] . The cycle threshold (Ct) values of qRT-PCR were collected, which were inversely proportional and exponential correlated with copies number of the virus. We defined the degree of severity of COVID-19 (general, severe and critical) according to the Chinese management guideline for COVID-19 (version 7.0) [9, 10] . Common cases defined as showing fever and respiratory symptoms with radiological findings of pneumonia. Severe cases meet any of the following criteria: respiratory distress ( ≥30 breaths/min), or oxygen saturation ≤93% at rest, or arterial partial pressure of oxygen (PaO2)/ fraction of inspired oxygen (FiO2) ≤300 mmHg, or chest imaging showing an obvious lesion progression within 24-48 hours >50%. Critical cases met any of the following criteria: respiratory failure and requiring mechanical ventilation, shock, or exhibiting other organ failure that requires ICU care [9, 10] . According to the Chinese management guideline for COVID-19 (version 7.0), patients were received antiviral therapy, respiratory support, circulatory support, blood purification treatment, immunotherapy and traditional Chinese medicine treatment. Antiviral therapy included α- Continuous variables were expressed as medians and interquartile ranges, categorical variables were summarized as counts and percentages. A two-sided α of less than 0.05 was considered statistically significant. All analyses were performed with SPSS software (version 19 .0). The polynomial fitting models and curves were performed by Python (version 3.6), with R-square as criterion to evaluate these models. The viral load testing of SARS-CoV-2 was performed for each patient during hospitalization, and those patients (n=308) who underwent at least 5 times nucleus acid tests with nasal and pharyngeal swab were enrolled in the study. At the conclusion of the study period, 16 patients died of COVID-19, 82 patients were discharged, and 210 patients remained in the hospital. The median age of 308 patients was 63.0 years (IQR 52.0-71.0), and male ratio was 49.0% (Table 1 ). Fever (87.0%) and cough (67.2%) were the most common symptoms, followed by shortness of breath (42.5%) and fatigue (30.2%) ( (Table.S1 ). Notably, significant elevated IL-6, lactate dehydrogenase (LDH), and significant reduced lymphocyte and lymphocyte subsets were another features of deceased patients, which were agreement with previous study (Table.S1) [5] . There were 2475 specimens collected from 308 patients with COVID-19 during hospitalization. Although bronchoalveolar lavage fluid specimens showed the highest positive rate, 50% ( No SARS-CoV-2 was detected for blood, eye secretion, cerebrospinal fluid, milk and vaginal secretions specimens. To identify the optimal specimen for SARS-CoV-2 testing, we compared the positive rate We observed the viral excretion from respiratory and gastrointestinal tracts in all patients by a chronological series during hospitalization. The changes of Ct values in the serial nasal and pharyngeal swab, sputum and stool samples could approximately reflect the changes of viral load in these patients. We analyzed the viral load in specimens (include nasal and pharyngeal swab, sputum and stool) obtained from the 308 inpatients after admission by polynomial fitting models using Python. The R-square serves as the criterion to evaluate the best models. Our polynomial fitting curves suggested that the highest viral loads (inversely related to Ct value) A c c e p t e d M a n u s c r i p t 11 were detected a few days after admission and then decreased accompanied with treatment ( Fig.2B) . Although the overall viral load was reduced, the viral load could rebound within several days (day7, 16, 22 after admission) (Fig.2B) . The chronological series Ct values of hospitalized patients showed similar change, viral loads decreased rapidly after admission, but rebounded in day 7-10, day 15-18 and day 20-22 ( Fig. 2A) . The viral load of critical patients was much higher than general patients and severe patients in the early stage of hospitalization, and then decreased rapidly under treatment (Fig.2C) . The overall viral load of general patients was lower than severe patients (Fig.2C ), but the rebound of viral was more common. On day 30 of treatment, the Ct values approximated to 40, indicating that the virus was molecularly undetectable in most patients. The viral shedding in different types of specimens appeared to be diverse. The average viral load of sputum was the highest, followed by nasal and pharyngeal swab and stool (Fig.2D) . Interestingly, different specimens from same patients were various (Fig.S1A-L) . Sixteen of thirty-one patients showed higher viral loads in sputum than that in nasal and pharyngeal swab A c c e p t e d M a n u s c r i p t 12 Next, we analyzed the associations of viral load with clinical features. High viral load cases had higher neutrophil counts (3.62×10 9 ; p=0.0001), lower lymphocytes counts (1.33×10 9 ; p=0.0034), as well as eosinophils counts (0.09×10 9 ; p=0.0004), and basophils counts (0.40×10 9 ; p=0.0053) ( Table 2 ). The number of T cells were significantly decreased in patients with COVID-19, which was more evident in the high viral load cases (783/μL; p<0.0001) compared with the low viral load group (Table. 2). The decline of Th cells and Ts cells were more pronounced in high viral load cases, suggested T cells were shown to be more affected by SARS-CoV-2. Notably, the myocardial enzymes, include creative kinase MB (CKMB), myoglobin (MYO), ultra-troponin I (ultra-TnI) and N-terminal pro-brain natriuretic peptide (NT-proBNP), were significantly increased in high viral group, confirmed that cardiovascular was an important target of SARS-CoV-2. Besides, elevated serum albumin, hypophosphatemia and hypercalcemia were observed in high viral load group. Elevated LDH was another feature of high viral load cases, which indicated poor prognosis in COVID-19 patients [5] . This retrospective study reported the series viral loads of the 308 hospitalized patients with COVID-19. The respiratory tract samples tested positive more frequent for SARS-CoV-2 than the gastrointestinal tract samples. The lower respiratory tract samples showed higher viral load than the upper respiratory tract samples (Fig.1A) . Human ACE2 protein was the receptor to facilitate viral into target cells [12] , and alveolar epithelial type II cells (AECII) were the major ACE2-expressing cells [13] , suggesting that these cells can serve as the major target cells for In this study, the viral load in patients with COVID-19 was found to have peaked within the first few days after admission, and then decreased rapidly along with virus rebound after treatment. The serials copy numbers of SARS-CoV-2 were associated with disease severity, the critically ill patients had the highest viral load peak and the general patients showed the lowest average viral load (Fig.2C) , which is similar to SARS [14] . Toward the end of this period virus was intermittently detectable from clinical samples, and the viral shedding in stools persisted longer than respiratory samples in portion patients, suggesting a strict discharge criterion should be performed to avoid transition by the fecal-oral route, i.e., their respiratory tract samples and gastrointestinal tract samples should be monitored till testing for negative SARS-CoV-2 at least two times continuously. In a previous study, SARS-CoV-2 was detected in 1% (3/307) blood samples [15] . Though limited by a small sample size of 31 blood samples, we were not able to detect SARS-CoV-2 in blood by qRT-PCR, suggesting that the virus probably did not cause viremia. The average viral load decreased rapidly under treatment, but could rebound within 1-3 weeks (i.e., day7, 16, 22 after admission) (Fig. 2B) , and a similar rebound pattern was observed in individual patients ( Fig. 2A) . The phenomenon may be due to that AECII cells were the major ACE2-expressing cells. Therefore, clear coronavirus particles were Abnormal complete blood count was a notable feature of COVID-19 patients, with decreased basophil, eosinophils and lymphocyte in peripheral blood. Besides, the count of basophil, eosinophils, lymphocyte, CD3 T lymphocyte, CD4 T lymphocyte, CD8 T lymphocyte was significantly negatively associated with viral load in our study. A pathological result of three COVID-19 cases reported that cell degeneration and necrosis were observed in spleen and all three cell lines decreased in bone marrow [17] . Recently, we detected high viral load of SARS-CoV-2 in bone marrow from an 84-year-old woman with sever leukopenia (data not published) in Renmin Hospital of Wuhan University. We reasoned that immune cells or organs were candidate targets of SARS-CoV-2, which needed more studies to confirm. In addition to the lung, ACE2 is also highly expressed in the heart, playing vital role in the cardiovascular system [18] . Although SARS-CoV-2 mainly invades the lung, it can also cause myocardial injury. Cardiac complications, including heart failure, arrhythmia and myocardial infarction, were major comorbidities in COVID-19 patients [5, 8] . Our findings also supported that Red means nasal and pharyngeal swab samples, blue means sputum samples and yellow means stool samples. The Novel Coronavirus Originating in Wuhan, China: Challenges for Global Health Governance Coronaviridae Study Group of the International Committee on Taxonomy of V. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2 World Health Organization. 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The high viral load and low A c c e p t e d A c c e p t e d