key: cord-0743905-ws5zjnu5 authors: Song, Yangzi; Chen, Chen; Wang, Yu; Zhang, Ju; Chen, Meiling; Gao, Guiju; Wang, Sa; Yang, Di; Song, Rui; Wang, Linghang; Xie, Wen; Yu, Fengting; Yan, Liting; Wang, Yajie; Zeng, Hui; Zhang, Fujie title: Early and consecutive RT-PCR tests with both oropharyngeal swabs and sputum could improve testing yield for patients with COVID-19: An Observation Cohort Study in China date: 2021-04-28 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2021.04.076 sha: 58c709069b4ba560148ef57ef7203094154e6df9 doc_id: 743905 cord_uid: ws5zjnu5 OBJECTIVE: The real-time polymerase chain reaction (RT-PCR) test is recommended for diagnosis of COVID-19 and provides a powerful tool to identify new infections and contact tracing. In fact, as COVID-19 prevalence decreases, this remains the main preventive measure to avoid rebound. However, inconsistent results due to biological sample variability in collection timing post infection and sampling procedures, misleads our application in clinic. METHODS: We applied Kaplan-Meier method and multivariate Logistic regression on the RT-PCR results from 258 confirmed patients with COVID-19 to evaluate the factors associated with negative conversion. We also estimated the negative percentages among patients who had tested twice or more and compared the proportions by oropharyngeal swab, sputum and combined double testing, respectively. MAIN RESULTS: The proportion of negative conversion was 6.7% at the 4(th) day, 16.4% at the 7(th) day, 41.0% at two weeks and 61.0% at three-weeks post-admission. We also found 34.1% and 60.3% with at least one negative RT-PCR result at the 7(th) days and 14(th) days after symptom onset, respectively. The negative proportion in sputum was higher than that in oropharyngeal swab in the early stage but lowered after symptoms. CONCLUSION: In the absence of effective treatments or vaccines, efficient testing strategies are critical to control COVID-19 epidemic. According to this study, early, consecutive and combined double testing will be the key to identify infected patients, particularly for asymptomatic and mild symptomatic cases, and minimize misdiagnosis and ineffective isolation of infected patients. Health Organization (WHO) [1] . Despite over 10 million infections and more than 2,000,000 deaths globally as of Feb 1 st , 2021 [2] , the number of infected patients has dramatically decreased in some local areas and countries. In these regions, the prevention and disease control policy has shifted from the diagnosis of endemic infections to the identification of new imported COVID-19 cases, with stringent contact tracing to prevent follow-up transmission. Therefore, early diagnosis, with low false negative rates to prevent miss detection, are crucial for proper isolation of infected individuals and prevent the rebound epidemics [3] . Recent rebound epidemics in several hot-spots cities or provinces, remind us that the battle is far from over, and is likely to keep households cautious for a while longer. COVID-19, with 1.4-6.9 of R0 [4] , posts a great challenge to complete and stringent J o u r n a l P r e -p r o o f patient identification, especially asymptomatic patients who can shed virus and contribute to SARS-CoV-2 transmission. Therefore, identification of these viral carriers is extremely important, even if COVID-19 is well controlled in China with most businesses and schools reopened nationwide. Notably, different from influenza viruses and community-acquired human coronaviruses, SARS-CoV-2 showed a high virus load during early phases of illness, prior to efficient testing [5] [6] . The real-time polymerase chain reaction (RT-PCR) test in respiratory specimens is recommended as the virologic criterion for the diagnosis and the outcome evaluation for treatments [7] . However, RT-PCR cannot detect the virus among 30% of patients, and showed inconsistent test results due to biological sample variability in collection timing post infection and sampling procedures [8] [9] [10] . Thus, it becomes crucial to understand when and how patients should be tested by RT-PCR to identify the infection status with the SARS-COV-2. In the absence of effective specific antiviral therapies or vaccines, rapid and efficient identification of new infection cases, contact tracing, and quarantine are the most effective strategy against local rebound epidemics, such as in China, Singapore and Korea. In this study, we conducted a cohort study among confirmed patients in Beijing Ditan Hospital. Our goal is to demonstrate the impact of sample collection timing and procedure in RT-PCR testing and diagnosis of COVID-19 patients. J o u r n a l P r e -p r o o f Our study included all COVID-19 confirmed patients admitted with symptoms between January 20 and April 17, 2020 in Beijing Ditan Hospital. In Total, we enrolled 258 confirmed cases, corresponding to 44% confirmed patients in Beijing. Clinical characteristics and cohort durations were summarized in supplemental text. Clinical samples, including oropharyngeal swabs or sputum specimens were collected at multiple timepoints for RT-PCR test according to the instructions for infection prevention and control measures by the Chinese guidance on infection prevention and control in healthcare settings [11] . A cycle threshold value less than or equal to 38 in at least one gene was interpreted as positive [12] [13] . Continuous variables were expressed as median and interquartile ranges (IQR) as appropriate. Categorical variables were summarized as the numbers and corresponding percentages in each category. Kaplan-Meier method was applied to plot and estimate the proportion of negative results conversion during the study period. This study starts at date of the initial symptom per patient, and proceeds until the date of negative conversion, defined as the day when both oropharyngeal swab and sputum appeared negative and never reverted in subsequent tests until the patient is discharges or followed up date. We also applied multivariate Logistic regression to All analyses were conducted with R software version 3.6.2 (R Foundation for Statistical Computing). A total of 258 patients with confirmed COVID-19 were enrolled in our study The median duration of patients with SARS-COV-2 was 17 days (range: 1-60 days Of 258 study patients, 222 had at least two positive RT-PCR results on oropharyngeal samples before negative conversion. All these patients were clinical recovered and discharged. In total, 1674 oropharyngeal swabs tests were performed prior to negative In this study, we retrospectively analyzed RT-PCR results in 258 patients with Considering the same sample collection protocols and testing approach used in this study, we speculated that the decreasing trend in positive testing rates overtime might also be representative of virus infection dynamics. Particularly, increase viral load in the oropharyngeal areas has been observed with MERS virus, which belongs to the same coronavirus family as COVID-19 [14] . COVID-19 is a self-limiting disease in about 80% mild patients [15] , who might be reluctant to visit a doctor during early stages of the infection, and only present later at the clinics if symptoms worsen. In addition, asymptomatic patients with SARS-CoV-2 have been extensively reported J o u r n a l P r e -p r o o f [12, 16] . Under such circumstances, these patients might have already contributed to massive transmission in the community and could also be miss-diagnosed at clinics before oropharyngeal swabs-based RT-PCR test started being used for case detection. Recent models suggest that asymptomatic and pre-symptomatic transmission, and delays in case recognition can greatly reduce the effectiveness of contact tracing [17] . The present study also showed that a certain number of patients who could rapidly Here, 81.1% of patients presented at least one inconsistent negative test result before clinical recovery, negative conversion and discharge. Similarly, a study with confirmed COVID-19 patients in Singapore also observed these inconsistent RT-PCR result before complete clinical recovery [9] . Consequently, single negative results can not completely preclude SARS-CoV-2 infection and should not be used as the sole basis for treatment or patient management decisions. Patients still shed virus even with mild symptoms or even after becoming asymptomatic (afebrile after treatment) [18] . Furthermore, at the early stages of illness, we observed a number of patients with J o u r n a l P r e -p r o o f negative RT-PCR result. Thus, although RT-PCR is recommended as a powerful tool for early diagnosis, negative results should be followed by multiple testing and clinical observations, patient history, and epidemiological information. Finally, we compared the negative proportions of the RT-PCT testing by oropharyngeal swab, sputum specimen and combined double testing within 21 days since the symptom onset. In general, the negative proportions were lower in the combined double testing of oropharyngeal swabs and sputum than each test independently, indicating that multiple specimen-based testing might be more sensitive to detect the infection. In our previous study, we have showed some patients who were negative in oropharyngeal swabs but positive in sputum, suggesting that the virus might persisted in sputum for a longer time [19] . Interestingly, in the early stage (before 5 th Day), the oropharyngeal swabs show a higher performance to identify infections, which suggest many infected patients with COVID-19 require a lag time to produce virus in the sputum. Also, many infected patients with COVID-19 have no sputum during early stages. Dynamic changes in viral loads have also been reported in different biological samples [20] . However, there is still no golden standard on how and when these biological samples of COVID-19 should be tested. Thus, considering our findings, we strongly recommended to test multiple biological specimens collected from suspected cases to minimize miss identification of infectious patients. 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American journal of respiratory and critical care medicine SARS-CoV-2-Positive Sputum and Feces After Conversion of Oropharyngeal Samples in Patients With COVID-19 SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients We acknowledge all health-care workers involved in the diagnosis and treatment of patients in China. We also particularly thank Dr. Hao Zhu for contribution on revision of the article.