key: cord-0897899-qrztj94r authors: Hu, Xiaowen; Xing, Yuhan; Jia, Jing; Ni, Wei; Liang, Jiwei; Zhao, Dan; Song, Xin; Gao, Ruqin; Jiang, Fachun title: Factors associated with negative conversion of viral RNA in patients hospitalized with COVID-19 date: 2020-04-22 journal: Sci Total Environ DOI: 10.1016/j.scitotenv.2020.138812 sha: 20643f4794141b851e6c265bce1001e36724a980 doc_id: 897899 cord_uid: qrztj94r Abstract Factors associated with negative conversion of SARS-CoV-2 RNA in hospitalized patients have not yet been systematically determined. We conducted a retrospective cohort study of COVID-19 patients in Qingdao, China. Both univariate and multivariate analysis were performed to identify independent factors for time to viral RNA negative conversion. Data on patients with re-detectable viral RNA after showing negative on RT-PCR test (intermittent negative status) were also analyzed. A total of 59 patients confirmed with COVID-19 were included in this study, with a median duration of 1 (interquartile range, IQR: 0–2) day from symptom onset to hospital admission. Median communicable period (from first day of positive nucleic acid test to first day of consecutive negative results) was 14 (IQR: 10–18) days, and 7 (IQR: 6–10) days for 10 patients with intermittent negative results. Age older than 45 years (hazard ratio, HR: 0.378; 95% confidence interval, CI: 0.205–0.698) and chest tightness (HR: 0.290; 95%CI: 0.091–0.919) were factors indecently affecting negative conversion of SARS-CoV-2 RNA. Headache (odds ratio: 7.553; 95%CI: 1.011–28.253) was significantly associated with intermittent negative status, with a predicted probability of 60%. Older age and chest tightness were independently associated with delayed clearance of SARS-CoV-2 RNA in hospitalized patients. These predictors would provide a new perspective on early identification of patients with prolonged viral shedding and facilitate optimal isolation protocols and treatment strategies. J o u r n a l P r e -p r o o f On December 12, 2019, 27 cases of pneumonia of unknown causes were reported in Wuhan, Hubei province, China (WMHC, 2020). On February 11, 2020, the world health organization (WHO) officially named this emerging disease as coronavirus disease 2019 , and the etiological agent was subsequently identified and renamed as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the Coronavirus Study Group (CSG) of the International Committee on Taxonomy of Viruses (CSGICTV, 2020; WHO, 2020) . With the evolving pandemic, COVID-19 has wreaked havoc in China and spread rapidly to around 210 countries and regions all over the world, constituting a global threat with the highest risk impact (Our World in Data, 2020). Since the identification of the first few cases of COVID-19, numerous studies have mushroomed with a focus on the epidemiological, clinical, laboratory, and radiological characteristics of infected patients, prompting the development of diagnosis and treatment strategies (Ai et al., 2020; Chen et al., 2020; Cortegiani et al., 2020; Huang et al., 2020; Xing et al., 2020; . However, there is limited data regarding the potential predictors of negative conversion with COVID-19 patients. Possible reasons underlying this research gap may be due to the lack of detailed sequential virology profiles at the early stage of the disease. Another possible J o u r n a l P r e -p r o o f epidemic center, took a rapid response in the face of COVID-19 outbreak. The local government and municipal center for disease control and prevention in Qingdao has launched series of aggressive measures to screen, quarantine, diagnose, treat and monitor suspected patients and their close contacts (Jia et al., 2020; Xing et al., 2020) . COVID-19 cases in Qingdao were identified and admitted to designated hospitals at an early stage, providing a strong foundation for our research. This study aimed to ascertain viral clearance time and factors associated with SARS-CoV-2 RNA negative-conversion in upper respiratory specimens. We described the dynamic changes of viral shedding in laboratory-confirmed COVID-19 patients on presentation till hospital discharge. This retrospective cohort study included patients with confirmed COVID-19 hospitalized in Qingdao, Shandong Province, China. All patients were diagnosed with COVID-19 based on New Coronavirus Pneumonia Diagnosis and Treatment Plan J o u r n a l P r e -p r o o f 6 detailed field investigations and collect respiratory specimens for centralized testing at Qingdao CDC. Data were collected onto standardized forms through interviews of infected persons. Epidemiological, demographic, clinical, laboratory, treatment, and outcome data were extracted from the interview as well as field reports. This study was approved by the Ethics Commission of Municipal Centre of Disease Control and Prevention of Qingdao and written informed consent was waived considering the emergency of infectious disease. A case was confirmed by a positive result of real-time reverse-transcription-polymerase-chain-reaction (RT-PCR) analysis for SARS-CoV-2 in nasopharyngeal swabs. A severe case is defined as a patient who fulfills one of the following criteria: respiratory rate ≥30 breaths/min; oxygen saturation ≤93% at a rest state; arterial partial pressure of oxygen (PaO 2 )/oxygen concentration (FiO 2 ) ≤300mmHg. Patients were discharged based on the clinical guideline such as respiratory samples consecutively negative for nucleic acid testing with an interval of at least one day (NHCPRC, 2020a; NHCPRC, 2020b; NHCPRC, 2020c SARS-CoV-2 detection in respiratory specimens was conducted by Qingdao CDC using real-time RT-PCR targeted at open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) genes. Tests were carried out in biosafety level 2 facilities, using a commercial kit approved by the China Food and Drug Administration. Nasopharyngeal swabs were obtained from patients on a daily basis within 3 days after illness onset. RNA was extracted from the samples and then underwent real-time RT-PCR with SARS-CoV-2-specific primers and probes. A cycle threshold (Ct) value were used to approximately reflect the viral loads (inversely related to Ct-value) in the respiratory tract, a method as suggested by previous studies . A Ct value less than 37 was defined as a positive test, and a Ct value of 40 or more was considered as a negative test. An equivocal result, defined as a Ct value between 37 to 40, required confirmation by retesting. If the repeated Ct value was less than 40 and an obvious peak was observed, or if the repeated Ct value was less than 37, the result was deemed positive. J o u r n a l P r e -p r o o f 8 univariate and multivariate analysis were performed. The log-rank test was first conducted with a total of 25 factors, such as virological, demographic, epidemiological, clinical, radiological and laboratory variables. Multivariate Cox regression model was then performed with the significant factors selected by univariate analysis to tease out the independent predictors for RNA negative conversion. The association between independent predictors and negative conversion was quantified by hazard ratio (HR). As negative conversion of viral RNA is a beneficial event, HR value greater than 1 would promote virological clearance, whereas HR value less than 1 means the independent predictor would delay negative conversion. Additionally, we investigated factors associated with intermittent negative status. Mann-Whitney U test, χ² test, or Fisher's exact test was first conducted to compare diff erences between groups with and without intermittent negative status. Next, factors with statistically significance (P<0.05) were further analyzed using the logistic regression model, and odds ratio (OR) and predicted probability were calculated. Continuous and categorical variables were presented as median (interquartile range, IQR) and n (%), respectively. A P value less than 0.05 (two-tailed) was considered statistically significant. Analyses were performed using SPSS software J o u r n a l P r e -p r o o f whom 43 were cluster cases from eleven outbreaks, including 33 family members and 10 close contacts. Eighteen patients had a travel history to Hubei Province before illness onset, and 11 patients had been to Wuhan in January, 2020. Female accounted for 52.5% (31) of confirmed cases and 47.5% (28) were male. The majority of patients (48) were local residents in Qingdao, and 11 patients were floating population. The median age of patients was 46 years (IQR: 33-57). More than a quarter of patients (17) had underlying diseases, such as cardiovascular and cerebrovascular diseases (9), endocrine disease (3), gastrointestinal disease (3), respiratory disease (1) and neurological disorder (1). Detailed demographical and epidemiological information was represented in Table 1 . The median duration from disease onset to hospital admission was 1 (IQR: 0-2) day, with a median of 19 days from illness onset to hospital discharge (IQR: 16-25). Table 1 , 83.1% of the 59 COVID-19 cases in Qingdao were non-severe (mild to moderate cases), and 16.9% were severe cases. Most common symptoms at onset of the disease were fever and dry cough. Other symptoms included fatigue, headache, chest tightness, myalgia, and sputum production. All patients received standard care including supportive and anti-viral treatment according to the latest clinical guidelines (NHCPRC, 2020a; NHCPRC, 2020b; NHCPRC, 2020c) . Antibiotics were only prescribed to patients who were at risk of or presented with bacterial infection. Patients with symptoms of respiratory distress received oxygen Journal Pre-proof J o u r n a l P r e -p r o o f 10 therapy. Corticosteroids were used shortly to inhibit inflammatory cascade in patients with progressing disease. Critically ill patients were transferred to intensive care unit. On admission, 7 patients had leucopenia (white blood cell count <4×10 9 /L), 16 patients had increased proportion of neutrophils (>70%), and 13 patients showed elevated lymphocyte percentage (>40%). Abnormalities in chest computed tomograms (CT) were detected in 46 patients (80.0%). The most common pattern of CT changes was high-density shadow over bilateral lungs (54.2%). Viral loads in nasopharyngeal swabs were estimated with Ct values by RT-PCR. Median Ct values for RT-PCR on ORF 1ab gene was 26 (IQR: 24-31), and median Ct value for N gene was 28 (IQR: 26-31). All patients were discharged base on two consecutive negative tests at least 24h apart. Median duration of communicable period was 14 (IQR: 10-18) days, ranging from 4 to 25 days. Moreover, 10 patients were in intermittent negative status after first showing negative for RT-PCR, with a median duration in between of 7 days (IQR: 6-10, Fig. 1 ). The rate of RNA negative conversion within 7 days, 14 days and 21 days among all patients were 10.2% (95% confidence interval, 95%CI: 2.1%-17.5%), 62.7% (48.1%-73.2%), and 91.2% (80.4%-96.4%), respectively (Fig. 2) . Fig. 3 showed that age older than 45 years (HR: 0.378; 95%CI: 0.205-0.698) and chest tightness (HR: 0.290; 95%CI: 0.091-0.919) were independently associated with negative conversion of viral RNA, suggesting that older age and presentation of chest tightness would delay virological clearance in COVID-19 patients (Fig. 4) . Table 2) . After analyzing the variables by Mann-Whitney U test, χ² test, or Fisher's exact test, there were two variables including headache, days from illness onset to hospital admission, which were significant different (P< 0.05) between groups with and without intermittent negative status. Further logistic regression with the two variables indicated that headache (OR: 7.553; 95%CI: 1.011-28.253) was positively associated with intermittent negative status, rather than days from illness onset to hospital admission (per day increase OR: 1.133; 95%CI: 0.977-1.343). After controlling the effect of days from illness onset to hospital admission, the predicted probability of intermittent negative status in response to headache was 60% as revealed by logistic J o u r n a l P r e -p r o o f regression analysis. This study retrospectively analyzed demographical, epidemiological, laboratory, clinical and radiological data in a cohort of patients hospitalized with COVID-19 in To the best of our knowledge, this is the first study focused on factors associated with SARS-CoV-2 RNA negative conversion. We found that negative conversion of SARS-CoV-2 RNA occurred 14 (IQR: 10-18) days from the first positive RT-PCR test. It has been reported that the viral RNA existed for a median duration of 20.0 (IQR: 17.0-24.0) days in patients survived from COVID-19 (Zhou et al., 2020), while another study demonstrated a median duration of viral shedding of 9.5 (IQR: 6.0-11.0) days (Ling et al., 2020) . The discrepancies between studies might attribute to differences in disease severity as well as sampling methods. Cox regression multivariate analysis revealed that growth of age and chest tightness were correlated to later RT-PCR conversion. Older age has been constantly J o u r n a l P r e -p r o o f 13 linked to poor clinical outcomes in COVID-19 patients (Zhou et al., 2020) . T-cell numbers and functions are largely compromised with aging, resulting in less controlled viral replication and host inflammatory responses (Webster, 2000; Goronzy et al., 2007) . Besides, older patients had more underlying diseases as compared with younger patients infected with SARS- CoV-2 (Liu et al., 2020) . Although disease severity and comorbidities were not significant predictors from univariate and multivariate analysis, meaning the two factors had no direct effect on time to RT-PCR conversion, they may indirectly influence viral nucleic acid clearance via effect of age. Further spearman correlation analysis showed that disease severity (r= 0.283, P= 0.03) and comorbidities (r= 0.450, P< 0.001) were positively correlated with age. These age-related defects may make it more difficult for the host cell immunity to eradicate the invasive pathogen, resulting in prolonged viral shedding in the elderly. However, when we compared the 5 patients presented with chest tightness with the other patients, no obvious differences could be found in terms of epidemiological, laboratory and radiological characteristics. A previous study on SARS-CoV (2002 -2003 demonstrated that urine positive for RT-PCR analysis on presentation was associated with delayed RNA negative conversion, while treatment with ribavirin and corticosteroid was related to a sooner virological clearance (Chu et al., 2005) . In our study, ribavirin was empirically prescribed to all patients confirmed with COVID-19, whereas corticosteroids was rarely given. We could not detect any relationship between treatment strategies and time to RT-PCR conversion in our patients. The clinical significance of these predictors is still unknown and further J o u r n a l P r e -p r o o f 14 studies would be warranted. The potential risk factors for a delayed viral nucleic acid clearance would shed light on early recognition of patients with poor prognosis. In the current study, 10 patients had re-detectable positive results for SARS-CoV-2 RNA test during hospitalization. We found that headache was correlated with intermittent negative results in these patients. Previous studies have reported a proportion of recovered patients who met criteria for hospital discharge showed re-detectable positive results for RT-PCR testing Lan et al., 2020; Ling et al., 2020) . In general, patients with intermittent negative RT-PCR were younger and had milder symptoms (r=0.220, P= 0.041; r=0.242, P=0.033, respectively). Cautionary measures should be considered when dealing with patients with recurrence of positive RT-PCR results as SARS-CoV could be transmitted from convalescent patients to others via close contact (Chan et al., 2003) . It might be advisable to extend the duration of hospitalization and isolation of patients with intermittent negative RT-PCR results. These findings provide important implications for decision making on quarantine protocols and facilitating optimal antiviral interventions. Additionally, we analyzed Ct values of RT-PCR test (inversely related to viral load) on admission as accumulating evidence supported the notion that viral loads of SARS-CoV-2 are among the highest in COVID-19 patients early after onset (Pan et al., 2020; Zou et al., 2020) . A high viral load on presentation was an independent factor contributing to worse prognosis in patients with SARS (Chu, et al. 2004 ). Although we failed to find significant relationship between Ct values and J o u r n a l P r e -p r o o f on admission (as reflected by lower Ct values, r=-0.345 P= 0.007). Our findings verified the results from a recent study showing that older age, rather than disease severity, was positively associated with viral RNA copy numbers . Studies for SARS-CoV also indicated that increased age was independently associated with higher viral load (Chen et al., 2006) . There are several limitations of this study. First, our sample size is relatively small in comparison with other studies. There were 59 COVID-19 patients in Qingdao during the study period. However, we believe our population was a good preventative of patients outside of the epidemic center. Second, we did not detect the existence of viral RNA in patients' excretions such as urine and fecal specimens. Previous studies indicated the possibility of viral transmission via fomite and viral loads in the stools might be greater than that in the respiratory specimens (Wölfel et al., 2020; Xu et al., 2020) . Third, we did not record the dynamic profiles of viral loads (as reflected by Ct values). How the chronological changes in viral loads correlate with disease prognosis merits further investigation. This study identified factors of older age and chest tightness were associated with delayed clearance of viral RNA in patients hospitalized with COVID-19. We hope these predictors could provide clues for early identification of patients with prolonged viral shedding, paving the way for development of optimal isolation protocols and treatment strategies. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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