key: cord-0957183-25vb9d4f
authors: Uhm, Jae-Sun; Ahn, Jin Young; Hyun, Jong Hoon; Sohn, Yujin; Kim, Jung Ho; Jeong, Su Jin; Ku, Nam Su; Choi, Jun Yong; Park, Yu-Kyung; Yi, Ho-sung; Park, Sung Kyu; Kim, Bong-Ok; Kim, Hyewon; Choi, Jinwoo; Kang, Seung-mo; Choi, Yeong Ho; Yoon, Hae Kyoung; Jung, Sunkyung; Kim, Hyeong Nyeon; Yeom, Joon-Sup; Park, Yoon Soo
title: Patterns of Viral Clearance in the Natural Course of Asymptomatic Coronavirus Disease 2019 (COVID-19): Comparison with Symptomatic Nonsevere COVID-19
date: 2020-08-05
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2020.07.070
sha: 6ebf9aa7263c46aa3b2ceeed2166cff12380df19
doc_id: 957183
cord_uid: 25vb9d4f

OBJECTIVES: The aim of the study was to elucidate patterns of SARS-CoV-2 clearance in the natural course of asymptomatic coronavirus disease 2019 (COVID-19). METHODS: Consecutive patients with nonsevere COVID-19 were retrospectively included. Asymptomatic patients with normal body temperature and no evidence of pneumonia throughout the disease course were classified in the asymptomatic group. The reverse transcription polymerase chain reaction (RT-PCR) assay was repeated every two to five days after the first follow-up RT-PCR assay. Negative conversion was defined as two consecutive negative results of RT-PCR assays within a 24-h interval. Rebound of threshold cycle (Ct) value was defined as negative from the single RT-PCR assay and positive from the following assay. RESULTS: Among a total of 396 patients [age, 42.5 (25.0–55.0) years; males, 35.6%], 68 (17.2%) and 328 (82.8%) patients were in the asymptomatic and symptomatic groups, respectively. The time until negative conversion was significantly shorter in the asymptomatic group than in the symptomatic group [14.5 (11.0–21.0) days and 18.0 (15.0–22.0) days, respectively; p = 0.001]. Rebound of Ct values was observed in 78 patients (19.7%). CONCLUSIONS: Time until negative conversion is shorter in asymptomatic COVID-19 than in symptomatic COVID-19. Rebound of Ct values is not uncommon.

The first cases of coronavirus disease 2019 were reported in Wuhan, China in December 2019 Huang et al., 2020) . The pathogen has been identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Lu et al., 2020; Zhu et al., 2020) . COVID-19 has rapidly become widespread across the world. In March 2020, the World Health Organization declared the COVID-19 outbreak a pandemic. From the end of February through March 2020, Daegu-the fourth largest city in South Korea-was emerging as an epicenter of COVID-19 in South Korea. South Korean government recommended that all people who have contacted the patients with COVID-19 and visited the outbreak places should be tested, even though they did not have symptoms. In the early phase of outbreak, the patients who were diagnosed with COVID-19 should be hospitalized for quarantine, regardless of disease severity. As the number of the patients was increasing, hospitals were running out of rooms. For that reason, asymptomatic and mildly symptomatic patients were quarantined in the COVID-19-dedicated facilities and moderately to severely symptomatic patients were hospitalized in COVID-19-dedicated hospitals (Park et al., 2020; Choi et al., 2020) .

The clinical features of COVID-19 range from asymptomatic to critical illness. According to epidemiological surveys, evidence that SARS-CoV-2 can be contagious in the presymptomatic stages of COVID-19 has been found (Rothe et al., 2020; Ye et al., 2020) . Furthermore, it is known that persistently asymptomatic COVID-19 is transmissible (Bai et al., 2020) . Cases of asymptomatic COVID-19 pose an emerging and serious public health issue given their elusive and contagious characteristics. However, patterns of SARS-CoV-2 clearance in asymptomatic COVID-19 remain unclear. The present study aimed to elucidate changes in results of real-time reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 and the time until negative conversion in the natural course of asymptomatic COVID-19 compared with J o u r n a l P r e -p r o o f symptomatic nonsevere COVID-19.

This study was a retrospective cohort study. Consecutive patients of all ages who were hospitalized in one of two COVID-19-dedicated hospitals (Korea Workers' Compensation and Welfare Services Daegu Hospital and Chungju Medical Center) after diagnosis of nonsevere COVID-19 by real-time RT-PCR for SARS-CoV-2 performed on specimens from nasopharyngeal swab from February 24, 2020 to April 2, 2020 were retrospectively included.

Severity of COVID-19 was defined using the current guidelines for diagnosis and treatment of community-acquired pneumonia (Metlay et al., 2019) . The patients with severe COVID-19 and patients in whom negative conversion was not confirmed were excluded. Patients with no COVID-19-related symptoms, body temperature ≤37.4°C, and no evidence of pneumonia based on chest X-rays throughout the disease course were classified into the asymptomatic group. Patients with COVID-19-related symptoms, body temperature ≥37.5°C, or findings compatible with pneumonia based on chest X-rays or computed tomography (CT) were classified into the symptomatic group. COVID-19-related symptoms included newlydeveloped cough, sputum, sore throat, rhinorrhea, headache, chest pain, fever, chills, myalgia, dyspnea, anosmia, ageusia, and diarrhea. Laboratory tests including complete blood cell count and blood chemistry were performed upon admission. Supportive care was conducted for the asymptomatic patients while treatment for specific symptoms with or without anti-microbial therapy was provided for the symptomatic patients. Anti-microbial therapy included lopinavir/ritonavir, hydroxychloroquine, third-generation cephalosporin, and azithromycin.

During hospitalization, the first follow-up RT-PCR assay for SARS-CoV-2 was performed on specimens from nasopharyngeal swab seven days after diagnosis for the asymptomatic patients J o u r n a l P r e -p r o o f and immediately after the disappearance of symptoms or improvement in pneumonia stage for the symptomatic patients. When the follow-up RT-PCR assay was negative, the following RT-PCR assay was performed on specimens from nasopharyngeal swab after 24-48 hours. When the follow-up RT-PCR assay was positive, the following RT-PCR assay was performed on specimens from nasopharyngeal swab after two to five days. For the patients hospitalized in of Ct values for the E, RdRp, and N genes, respectively (Hong et al., 2020) . Negative conversion was defined as two consecutive negative results for RT-PCR assays with 24-hour interval. The patients with negative conversion were released from quarantine and discharged. The time until negative conversion was defined as the interval between diagnosis and the first RT-PCR 

Continuous data are expressed as median values (interquartile range), while categorical data are presented as numbers (%). To compare the clinical parameters between the two groups, we used the Mann-Whitney U test for continuous data and Fisher's exact test for categorical data, as all datasets were nonnormally distributed. A p-value <0.05 for a two-sided test was considered statistically significant. Data were analyzed using Statistical Package for the Social Sciences (SPSS) Version 25.0 (IBM Corporation, Armonk, NY, USA).

A total of 422 patients [age, 45.0 (26.0-55.0) years; males, 36.0%] were screened. Among them, 70 patients (16.6%) were asymptomatic throughout the disease course. Twenty-two patients were excluded because they were referred to the high-level hospitals due to aggravation of their conditions. Four patients were excluded because their negative conversion was not confirmed. Table 1 . Pulse rate, body temperature, white blood cell count, and C-reactive protein levels were significantly higher in the symptomatic group than in the asymptomatic group. All patients with obesity, asthma, chronic obstructive pulmonary disease, and coronary artery disease had COVID-19related symptoms and were therefore included in the symptomatic group. There were no significant differences in age, sex, body mass index, past histories, prior medications, blood pressure, respiration rate, hemoglobin, proportions of neutrophils and lymphocytes, platelet count, blood urea nitrogen, creatinine, aspartate transaminase, alanine transaminase, and lactate dehydrogenase between the two groups. All patients from both groups were discharged from the hospital without any sequelae.

The data on the follow-up RT-PCR assays for SARS-CoV-2 are shown in Table 2 . The interval between diagnosis and the first follow-up RT-PCR assay was significantly shorter in the asymptomatic group than in the symptomatic group. There were no significant differences in the number of follow-up RT-PCR assays and median interval between follow-up RT-PCR assays between the two groups. The time until negative conversion was significantly shorter in the asymptomatic group than in the symptomatic group [14.5 (11.0-21.0) (ranging from 6.0 to 40.0) days and 18.0 (15.0-22.0) (ranging from 4.0 to 46.0) days, respectively; p = 0.001]. The proportion of the patients with negative conversion according to time until negative conversion in the asymptomatic and symptomatic groups is shown in Fig. 1 . In the asymptomatic group, negative conversion was achieved within one week in 11.8%, two weeks in 50.0%, three weeks in 76.5%, four weeks in 92.6%, five weeks 98.5% of the patients. In the asymptomatic group, negative conversion was not achieved within 30 days in 5.9% of the patients. In contrast, in the symptomatic group, negative conversion was achieved within one week in 2.7%, two weeks in J o u r n a l P r e -p r o o f 21.6%, three weeks in 74.1%, four weeks in 89.9%, and five weeks in 97.9% of the patients.

In the symptomatic group, negative conversion was not achieved within 30 days in 6.7% of the patients. Changes in the proportion of patients without negative conversion according to hospitalization day are shown in Fig. 2 . The proportion of patients with negative conversion within 20 days was significantly higher in the asymptomatic group than in the symptomatic group, while the proportion of patients with negative conversion in 20 days was similar between the groups. There were no significant differences in the positive RT-PCR rates at the seventh, fourteenth, twenty-first, and twenty-eighth day since the diagnosis among the patients who underwent follow-up RT-PCR assays between the groups (Table 3) .

RT-PCR assays for the E and RdRP genes were performed in all patients while the RT-PCR assay for the N gene was performed in 107 patients. There were no significant differences in the first follow-up Ct values of E, RdRP, and N genes between the two groups (Supplementary   table 1 ). There were no significant differences in minimum follow-up Ct values of E, RdRP, and N genes between the two groups (Supplementary table 1). There was no significant difference in rebound of Ct values between the two groups (Table 2) . Serial changes in Ct values for the three genes in the asymptomatic and symptomatic groups are shown in Fig. 3 .

The main findings of this retrospective cohort study were as follows: (1) 

Given that the asymptomatic cases are known to play a role in disease transmission (Bai et al., 2020; Rothe et al., 2020; Ye et al., 2020) , it is essential to know their proportion relative to symptomatic cases and the characteristics of viral shedding in asymptomatic infection to establish the guidelines for the management of COVID-19. The proportion of asymptomatic infection was estimated to be 17.9-30.8% (Mizumoto et al., 2020; Nishiura et al. 2020 ). If patients have no symptoms at the time of diagnosis, it is difficult to distinguish persistently asymptomatic patients throughout the disease course from patients in presymptomatic periods. According to a report on 13 asymptomatic patients with COVID-19, 12 patients had radiological abnormalities and three patients developed symptoms (Zhou et al., 2020) . Approximately 9.6% of residents in Wuhan, China who had never been symptomatic yet were diagnosed with COVID-19 exhibited positive IgG-antibody test results for SARS-CoV-2 suggesting that they had had asymptomatic infection (Wu et al., 2020) . Our present results are consistent with prior findings that the Ct values in the asymptomatic patients were not significantly different from those in the symptomatic patients (Zou et al., 2020) . The prior study demonstrated that the time from diagnosis to negative conversion was 7.5 days in the persistently asymptomatic patients with normal or atypical chest CT findings and 12.5 days in the persistently asymptomatic patients with pneumonia . There was difference J o u r n a l P r e -p r o o f in time until negative conversion between this prior study and the present study. That might be because the interval from disease onset to diagnosis was longer in the prior study than in the present study. Other prior study showed that the asymptomatic patients had a longer duration of viral shedding than the symptomatic patients, contrary to the present study (Long et al., 2020) . It has been suggested that asymptomatic cases showed relatively smaller transmission rates than did symptomatic cases (He et al., 2020) .

In Daegu, South Korea, mass investigation, and mass tests, as well as active surveillance have been performed throughout the COVID-19 outbreak. As a result, a number of asymptomatic patients with COVID-19 were detected. Among the population in this study, 95.5% of the patients were residents of Daegu. While hospital beds were limited, some asymptomatic or mildly symptomatic patients with COVID-19 were quarantined at home or at COVID-19dedicated facilities. Therefore, the proportion of asymptomatic patients in this study might be underestimated.

In the present study, all COVID-19 patients with obesity, asthma, chronic obstructive pulmonary disease, and coronary artery disease had symptoms. This might be because they are susceptible to the development of COVID-19-related symptoms or because symptoms related to these underlying diseases might be confused with COVID-19-related symptoms. In the symptomatic patients, body temperature, white blood cell count, and C-reactive protein level were higher than in the asymptomatic patients. These findings suggest that the symptomatic patients might mount a stronger inflammatory reaction than do the asymptomatic patients. High pulse rate may be associated with high body temperature in the symptomatic patients.

Although it was unclear when exposure to SARS-CoV-2 may have occurred, the median disease duration of asymptomatic COVID-19 must be longer than 14.5 days. The reasons for the shorter time until negative conversion in the asymptomatic patients might be because of short disease duration or delayed diagnosis. The optimal time at which the first follow-up RT-PCR assays in asymptomatic patients should be performed can be decided based on the present results. Rebound of Ct values was observed in a considerable number of patients. Although the reasons for rebound of Ct values remain unclear, possible explanations include reactivation of SARS-CoV-2, inadequate specimen collection, and laboratory errors. In any case, negative results for two consecutive RT-PCR assays within a 24-hour interval can be considered a reasonable criterion for lifting the quarantine.

No studies have yet dealt with the clinical characteristics and viral kinetics of a larger number of asymptomatic patients and studies reporting comparisons of the characteristics and changes in the RT-PCR results in the asymptomatic patients with those in the symptomatic patients are also scarce. The results of our present study provide the rationale for a quarantine strategy for the asymptomatic people who have been exposed to the patients with COVID-19, including recommendations on when best to perform follow-up RT-PCR assays for the asymptomatic patients with COVID-19 in the midst of shortages of medical facilities and equipment during COVID-19 outbreak.

This study had several limitations. First, time of exposure to SARS-CoV-2 was unclear in a number of patients. Therefore, it was difficult to specify the onset of the disease in asymptomatic patients. Second, the only nasopharyngeal specimens were collected in all J o u r n a l P r e -p r o o f patients. RT-PCR positivity can be higher or more prolonged in the lower respiratory specimens (Sethuraman et al., 2020; Wang et al., 2020) . Third, the Ct values at diagnosis were unavailable.

Fourth, given that the present study was retrospective, the follow-up intervals of RT-PCR assays lacked uniformity and density. In particular, intervals between diagnosis and the first follow-up RT-PCR assay were different between the asymptomatic and symptomatic groups because existing recommendations on when to perform the first follow-up RT-PCR assay were different between the two groups. Fifth, the RT-PCR assay for the N gene was not performed in all patients. Sixth, Ct values may not be linearly correlated to viral load. Seventh, chest CT was not performed in all patients. Specifically, the patients with normal chest X-ray results did not undergo chest CT.

Despite these limitations, this study had several strengths. A significant number of asymptomatic patients were included. Serial RT-PCR results were analyzed throughout the disease course. These findings may contribute to establishing the guidelines for asymptomatic COVID-19 management in pandemic situations and shortages of medical resources.

Time until negative conversion is shorter in asymptomatic COVID-19 than in symptomatic COVID-19. Rebound of Ct values is not uncommon.

Concept and design of the study: J-S.U., J.Y.A., J-S.Y., and Y.S.P. J o u r n a l P r e -p r o o f 

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The authors thank Young-Jin Song, MD, PhD, the director of Chungju Medical Center for his help with data collection and Nak-Hoon Son, PhD, the biostatistician of Yongin Severance Hospital, Yonsei University College of Medicine for his statistical analysis.J o u r n a l P r e -p r o o f