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Factors associated with a prolonged negative conversion of viral RNA in patients with COVID-19

  • Author Footnotes
    1 Cyrine Bennasrallah and Imen Zemni contributed equally to this work.
    Cyrine Bennasrallah
    Correspondence
    Corresponding author at: Cyrine Bennasrallah, Department of Epidemiology and Preventive Medicine, Fattouma Bourguiba University Hospital, University of Monastir, BP 99, République Monastir, 5060 Monastir, Tunisia.
    Footnotes
    1 Cyrine Bennasrallah and Imen Zemni contributed equally to this work.
    Affiliations
    Department of Epidemiology and Preventive Medicine, Fattouma Bourguiba University Hospital, University of Monastir, Monastir, Tunisia

    Department of Epidemiology, Faculty of Medicine of Monastir, University of Monastir, Monastir, Tunisia

    Technology and Medical Imaging Research Laboratory - LTIM - LR12ES06, Monastir, Tunisia
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  • Author Footnotes
    1 Cyrine Bennasrallah and Imen Zemni contributed equally to this work.
    Imen Zemni
    Footnotes
    1 Cyrine Bennasrallah and Imen Zemni contributed equally to this work.
    Affiliations
    Department of Epidemiology and Preventive Medicine, Fattouma Bourguiba University Hospital, University of Monastir, Monastir, Tunisia

    Department of Epidemiology, Faculty of Medicine of Monastir, University of Monastir, Monastir, Tunisia

    Technology and Medical Imaging Research Laboratory - LTIM - LR12ES06, Monastir, Tunisia
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  • Wafa Dhouib
    Affiliations
    Department of Epidemiology and Preventive Medicine, Fattouma Bourguiba University Hospital, University of Monastir, Monastir, Tunisia

    Department of Epidemiology, Faculty of Medicine of Monastir, University of Monastir, Monastir, Tunisia

    Technology and Medical Imaging Research Laboratory - LTIM - LR12ES06, Monastir, Tunisia
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  • Haythem Sriha
    Affiliations
    Department of Epidemiology and Preventive Medicine, Fattouma Bourguiba University Hospital, University of Monastir, Monastir, Tunisia
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  • Nourhene Mezhoud
    Affiliations
    Department of Epidemiology and Preventive Medicine, Fattouma Bourguiba University Hospital, University of Monastir, Monastir, Tunisia
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  • Samar Bouslama
    Affiliations
    Department of Epidemiology and Preventive Medicine, Fattouma Bourguiba University Hospital, University of Monastir, Monastir, Tunisia
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  • Wael Taboubi
    Affiliations
    Department of Epidemiology and Preventive Medicine, Fattouma Bourguiba University Hospital, University of Monastir, Monastir, Tunisia
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  • Meriem Oumaima Beji
    Affiliations
    Department of Epidemiology and Preventive Medicine, Fattouma Bourguiba University Hospital, University of Monastir, Monastir, Tunisia
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  • Meriem Kacem
    Affiliations
    Department of Epidemiology and Preventive Medicine, Fattouma Bourguiba University Hospital, University of Monastir, Monastir, Tunisia

    Department of Epidemiology, Faculty of Medicine of Monastir, University of Monastir, Monastir, Tunisia

    Technology and Medical Imaging Research Laboratory - LTIM - LR12ES06, Monastir, Tunisia
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  • Hela Abroug
    Affiliations
    Department of Epidemiology and Preventive Medicine, Fattouma Bourguiba University Hospital, University of Monastir, Monastir, Tunisia

    Department of Epidemiology, Faculty of Medicine of Monastir, University of Monastir, Monastir, Tunisia

    Technology and Medical Imaging Research Laboratory - LTIM - LR12ES06, Monastir, Tunisia
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  • Manel Ben Fredj
    Affiliations
    Department of Epidemiology and Preventive Medicine, Fattouma Bourguiba University Hospital, University of Monastir, Monastir, Tunisia

    Department of Epidemiology, Faculty of Medicine of Monastir, University of Monastir, Monastir, Tunisia

    Technology and Medical Imaging Research Laboratory - LTIM - LR12ES06, Monastir, Tunisia
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  • Chawki Loussaief
    Affiliations
    Department of Infectiology, Fattouma Bourguiba University Hospital, University of Monastir, Monastir, Tunisia
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  • Asma Sriha Belguith
    Affiliations
    Department of Epidemiology and Preventive Medicine, Fattouma Bourguiba University Hospital, University of Monastir, Monastir, Tunisia

    Department of Epidemiology, Faculty of Medicine of Monastir, University of Monastir, Monastir, Tunisia

    Technology and Medical Imaging Research Laboratory - LTIM - LR12ES06, Monastir, Tunisia
    Search for articles by this author
  • Author Footnotes
    1 Cyrine Bennasrallah and Imen Zemni contributed equally to this work.
Open AccessPublished:February 26, 2021DOI:https://doi.org/10.1016/j.ijid.2021.02.089

      Highlights

      • The median duration of viral shedding in this study was 20 days (interquartile range 17–32 days).
      • Face mask use was associated with accelerated RNA clearance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
      • The presence of symptoms was associated with delayed clearance of SARS-CoV-2 RNA.

      Abstract

      Objectives

      The aim of this study was to identify the factors influencing the delay in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA negative conversion.

      Methods

      A cohort study was conducted that included patients with coronavirus disease 2019 (COVID-19) admitted to the Tunisian national containment center. Follow-up consisted of a weekly RT-PCR test. Multivariate Cox regression analysis was performed to determine independent predictors associated with negative RNA conversion.

      Results

      Among the 264 patients included, the median duration of viral clearance was 20 days (interquartile range (IQR) 17–32 days). The shortest duration was 9 days and the longest was 58 days. Factors associated with negative conversion of viral RNA were symptoms such as fatigue, fever, and shortness of breath (hazard ratio (HR) 0.600, 95% confidence interval (CI) 0.401–0.897) and face mask use when exposed to COVID-19 cases (HR 2.006, 95% CI 1.247–3.228). The median time to RNA viral conversion was 18 days (IQR 16–21 days) when using masks versus 23 days (IQR 17–36 days) without wearing masks, and 24 days (IQR 18–36 days) for symptomatic patients versus 20 days (IQR 16–30 days) for asymptomatic patients.

      Conclusions

      The results of this study revealed that during SARS-CoV-2 infection, having symptoms delayed viral clearance, while wearing masks accelerated this conversion. These factors should be taken into consideration for the strategy of isolating infected patients.

      Keywords

      Introduction

      Cases of pneumonia of unknown etiology were first reported from Wuhan, Hubei Province, China in December 2019 (
      • Shi Ding
      • Wu Wenrui
      • Wang Qing
      • Xu Kaijin
      • Xie Jiaojiao
      • Wu Jingjing
      • et al.
      Clinical characteristics and factors associated with long-term viral excretion in patients with severe acute respiratory syndrome coronavirus 2 infection: a single-center 28-day study.
      ). In February 2020, the World Health Organization (WHO) named this emerging disease coronavirus disease 2019 (COVID-19) and the agent responsible was identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (
      • Lai Chih-Cheng
      • Shih Tzu-Ping
      • Ko Wen-Chien
      • Tang Hung-Jen
      • Hsueh Po-Ren
      Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): the epidemic and the challenges.
      ).
      The current COVID-19 pandemic has been spreading worldwide at an accelerated rate. According to the WHO, as of February 14, 2021, approximately 108 153 741 confirmed cases had been reported globally with more than 2 381 295 deaths across 216 countries (

      ‘WHO Coronavirus Disease (COVID-19) Dashboard’. n.d. Accessed 14 February 2021. https://covid19.who.int.

      )
      Tunisia reached a turning point on March 22, 2020, and general health containment was started. The strategy was based on testing, tracing, and isolation in accordance with the WHO guidelines. The government and health ministry of Tunisia have since launched a series of measures to screen, quarantine, diagnose, treat, and monitor suspected patients and their close contacts. The lifting of the compulsory confinement of SARS-CoV-2 carrying subjects in a dedicated center was announced on July 14, 2020.
      Tunisia is now experiencing a second wave of the pandemic and COVID-19 is spreading exponentially. Up until February 14, 2021 there had been 222 504 confirmed cases of COVID-19 with 7508 deaths (‘

      ‘Tunisia: WHO Coronavirus Disease (COVID-19) Dashboard’. n.d. Accessed 14 February 2021. https://covid19.who.int.

      .).
      Since the identification of the first cases of COVID-19, numerous studies focusing on the epidemiological, clinical, and radiological characteristics of infected patients and on treatment strategies have been reported in Tunisia. However, data regarding the potential factors associated with the delay in RNA negative conversion of patients with COVID-19 are limited (
      • Hu Xiaowen
      • Xing Yuhan
      • Jia Jing
      • Ni Wei
      • Liang Jiwei
      • Zhao Dan
      • et al.
      Factors associated with negative conversion of viral RNA in patients hospitalized with COVID-19.
      ).
      A patient’s infectivity is determined by the presence of the virus in body fluids, secretions, and excreta (
      • Ling Yun
      • Xu Shui-Bao
      • Lin Yi-Xiao
      • Di Tian
      • Zhu Zhao-Qin
      • Dai Fa-Hui
      • et al.
      Persistence and clearance of viral RNA in 2019 novel coronavirus disease rehabilitation patients.
      ). All patients with positive viral RNA detection need to be isolated, and isolated patients can only be discharged after the relief of symptoms and two successive negative viral nucleic acid results for respiratory specimens (
      • Qi Lin
      • Yang Yong
      • Jiang Dixuan
      • Tu Chao
      • Wan Lu
      • Chen Xiangyu
      • et al.
      Factors associated with the Duration of viral shedding in adults with COVID-19 outside of Wuhan, China: a retrospective cohort study.
      ). The predictors of persistence and clearance of viral RNA in different specimens from COVID-19 patients remain unclear. Knowledge has been accumulating on this topic for hospitalized and critically ill patients; however, information about patients with disease of mild severity is scarce (
      • Ling Yun
      • Xu Shui-Bao
      • Lin Yi-Xiao
      • Di Tian
      • Zhu Zhao-Qin
      • Dai Fa-Hui
      • et al.
      Persistence and clearance of viral RNA in 2019 novel coronavirus disease rehabilitation patients.
      ,
      • Qi Lin
      • Yang Yong
      • Jiang Dixuan
      • Tu Chao
      • Wan Lu
      • Chen Xiangyu
      • et al.
      Factors associated with the Duration of viral shedding in adults with COVID-19 outside of Wuhan, China: a retrospective cohort study.
      ,
      • Wang Kun
      • Zhang Xin
      • Sun Jiaxing
      • Ye Jia
      • Wang Feilong
      • Hua Jing
      • et al.
      Differences of severe acute respiratory syndrome coronavirus 2 shedding duration in sputum and nasopharyngeal swab specimens among adult inpatients with coronavirus disease 2019.
      ), and most COVID-19 patients have mild clinical symptoms (
      • Qi Lin
      • Yang Yong
      • Jiang Dixuan
      • Tu Chao
      • Wan Lu
      • Chen Xiangyu
      • et al.
      Factors associated with the Duration of viral shedding in adults with COVID-19 outside of Wuhan, China: a retrospective cohort study.
      ). Data on viral RNA shedding among patients with mild COVID-19 are of paramount importance to prevent transmission of this disease. Indeed, identifying people likely to be slow shedders is crucial to prolong their isolation, once infected, and avoid the virus spread, especially as it is not possible to confirm negative conversion by two reverse transcription PCR (RT-PCR) tests due to the international shortage and increasing numbers of cases. Thus, understanding factors associated with prolonged viral clearance among asymptomatic/mild cases is important to tailor prevention strategies.
      The aim of this study was to identify the potential predictors of RNA negative conversion delay among asymptomatic and mild symptoms COVID-19 patients.

      Patients and methods

      Study design

      A cohort study was conducted that included patients with confirmed COVID-19 confined in the national COVID-19 center from March to July 2020.

      Setting

      The national COVID-19 center (a hotel for COVID-19 patients) was located in Monastir governorate. This community facility was designated for the isolation of patients with or without symptoms of COVID-19 in Tunisia (from the 24 Tunisian governorates). The Tunisian government allocated individuals with asymptomatic or mild COVID-19 to dedicated isolation facilities and the remaining individuals with moderate-to-severe COVID-19 to hospitals. This allocation of individuals positive for COVID-19 was continued from March to July 29, 2020. Exposure to isolation and follow-up for each case covered the period from admission to the announcement of recovery. Data were collected prospectively on a daily basis.

      Participants

      Patients over 18 years of age with positive detection of SARS-CoV-2 RNA from nasopharyngeal/throat swabs by real-time RT-PCR were included. All cases progressing to a moderate or severe form requiring hospitalization were excluded from the analysis (Figure 1).
      Figure 1
      Figure 1Flow chart of inclusion and exclusion criteria.

      Methods of participant selection

      Cases were identified through the testing of individuals suspected to have COVID-19 and through contact-tracing involving close contacts of confirmed COVID-19 cases, tested within 24–48 h.

      Methods of follow-up

      The follow-up consisted of weekly RT-PCR testing for SARS-CoV-2 to check for viral clearance. Patients also had to have been symptom-free for at least 3 days before being considered for discharge. If the RT-PCR test result was positive, a swab was repeated after at least 7 days. If the RT-PCR test result was negative, a second swab was performed after at least 48 h to confirm viral clearance. Those individuals with two consecutive negative RT-PCR test results within 24 h were then considered virus-free and were discharged from the containment center.

      Variables and data collection

      Outcome

      Conversion of viral RNA was defined as the period between the day of the first RT-PCR positive result and the day of the second successive negative RNA SARS-CoV-2 test result, as proposed in previous studies dealing with a similar topic (
      • Hu Xiaowen
      • Xing Yuhan
      • Jia Jing
      • Ni Wei
      • Liang Jiwei
      • Zhao Dan
      • et al.
      Factors associated with negative conversion of viral RNA in patients hospitalized with COVID-19.
      ,
      • Gao W.J.
      • Li L.M.
      [Advances on presymptomatic or asymptomatic carrier transmission of COVID-19].
      ). A SARS-CoV-2 infection was confirmed by testing respiratory specimens based on RT-PCR assays from different agreed institutions, including the University of Monastir laboratory.

      Exposure

      Patients were required to stay in their hotel rooms during the isolation period. Their meals were served in their rooms. Telephone medical assistance was provided on a daily basis by the Monastir Preventive Medicine Department team. A toll-free number was given to each patient for any emergency to be resolved by the containment center staff.

      Predictors and diagnostic criteria

      Initially, demographic and clinical characteristics were collected. A daily telephone call was made to collect information on the clinical course and provide information on compliance with isolation measures. Patients were also asked if they had worn a mask when exposed to the index case and when they had contracted the virus, and whether they lived in a single room or not. The announcement of recovery was made after the second negative RT-PCR, reported by the laboratory within 24–48 h. After recovery, the patients were asked about their level of compliance in respecting hygiene and isolation measures.
      The SARS-CoV-2 infection was confirmed by testing respiratory specimens based on RT-PCR assays from different institutions, including the local laboratory of Fattouma Bourguiba University Hospital. Asymptomatic cases were defined as SARS-CoV-2-positive individuals without clinical signs. A symptomatic case was defined as any SARS-CoV-2-positive individual by RT-PCR with at least one symptom of COVID-19 since the admission, including but not limited to cough, fever, headache, muscle pain, shortness of breath, anosmia, and ageusia. Mild cases included patients positive for SARS-CoV-2 with upper airway symptoms and fever, myalgia and cough, with a normal pulmonary clinical examination. Patients were discharged based on respiratory samples consecutively negative for RNA on testing, with an interval of at least 1 day.

      Statistical analysis

      Continuous variables were presented as the mean and standard deviation, or as the median and interquartile range (IQR). Categorical variables were described as the frequency and percentage. The Mann–Whitney U-test was used to compare the differences between two groups for quantitative variables.
      In this study, negative conversion of viral RNA during the communicable period, as time-to-event data, was the outcome measure; this was illustrated with Kaplan–Meier curves. In order to detect the independent factors influencing the duration of RNA negative conversion, univariate and multivariate analyses were performed.
      For demographic, epidemiological, clinical, and virological variables, the log-rank test was first conducted.
      A multivariate Cox regression model was then performed with the significant factors selected by univariate analysis (P-value <0.2) to determine the independent predictors of RNA negative conversion.
      The association between independent factors and negative conversion was quantified by hazard ratio (HR), reported with the 95% confidence interval (CI). As negative conversion of viral RNA is a favorable event, an HR value >1 would indicate accelerated virological clearance, whereas an HR < 1 would mean that the independent predictor would delay negative conversion.
      A P-value of <0.05 was considered statistically significant. Analyses were performed using IBM SPSS Statistics version 21.0 software (IBM Corp., Armonk, NY, USA).

      Results

      Demographic and clinical characteristics of patients

      A total of 264 patients with laboratory-confirmed COVID-19 staying in the Monastir containment center were identified. Imported cases accounted for 30.6%. The median age of the patients was 42.5 years (IQR 30–55 years). Of the 264 confirmed cases, 133 were female (50.4%) and 131 were male (49.6%). The mean body mass index (BMI) of these patients was 26.3 ± 4.7 kg/m2.
      Nearly 31.7% of patients had at least one underlying disease such as diabetes (16.8%; n = 41), hypertension (15.6%; n = 38), and asthma (6.2%; n = 15). Almost 10.5% were current smoker. Symptoms such as anosmia, dry cough, and fatigue were reported by 34.4% (n = 75) of cases.
      Detailed demographic and clinical characteristics of the patients included in this study are presented in Table 1.
      Table 1Clinical characteristics of 264 patients with confirmed SARS-CoV-2 infection.
      CharacteristicsNumber%
      Sex
      Female13350.4
      Male13149.6
      Age (years)
      ≥604316.4
      <6021983.6
      Comorbidities
      Smoker2210.5
      Hypertension3815.6
      Diabetes4116.8
      Dyslipidemia52.1
      Obesity (BMI ≥ 30 kg/m2)2215.4
      Asthma156.2
      Symptoms
      Yes7534.4
      No14365.6
      Isolated room
      Yes16890.3
      No189.7
      Contact with face mask
      Yes2519.5
      No10380.5
      SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; BMI, body mass index.

      Duration of viral RNA conversion

      The median duration of viral conversion in this study was 20 days (IQR 17–32 days). The shortest duration was 9 days and the longest was 58 days. The patient with the shortest duration to RNA viral clearance was a 43-year-old male with no comorbidities. Regarding the longest duration of 58 days, the patient concerned was a 62-year-old male with hypertension. No symptoms were reported during RT-PCR positivity in either of these cases.
      The Kaplan–Meier survival curve representing the overall negative conversion of viral RNA in COVID-19 patients is illustrated in Figure 2. T he median time to viral clearance by age category is reported in Table 2.
      Figure 2
      Figure 2Overall negative conversion curve for COVID-19 patients.
      Table 2Median time to viral clearance according to age category, calculated using the Kaplan–Meier survival estimator.
      Age categories (years)Total (%) (N = 264)Time to viral clearance from first positive swab (days)
      MedianIQR (25%–75%)
      <3060 (22.7)2118–34
      30–4061(23.1)1816–27
      40–5048 (18.2)2316–34
      50–6052 (19.7)2016–34
      ≥6043 (16.3)2017–33
      IQR, interquartile range. Log-rank test P-value = 0.120.
      Ten patients (3.8%) had consecutive negative RT-PCR within 14 days, 136 patients (51.5 %) within 21 days, and 176 patients (66.7%) within 28 days.

      Factors related to SARS-CoV-2 RNA negative conversion duration

      The effect of each factor on negative conversion of COVID-19 patients was evaluated by log-rank test (Figure 3). It was found that SARS-CoV-2 RNA clearance was significantly delayed in symptomatic patients (log-rank test, P = 0.041; Figure 3C). The median duration was 24 days (IQR 18–36 days) for symptomatic cases and 20 days (IQR 16–30 days) for asymptomatic cases. Table 3 demonstrates the median time to SARS-CoV-2 RNA viral clearance according to the clinical characteristics of symptomatic and asymptomatic cases.
      Figure 3
      Figure 3Negative conversion curves for COVID-19 patients according to predictors, by day (d) after first positive RT-PCR: (A) sex; (B) age; (C) symptoms; (D) dyslipidemia; (E) isolated room; (F) was wearing masks when contracted the virus.
      Table 3Median time to SARS-CoV-2 RNA viral clearance according to clinical characteristics of symptomatic and asymptomatic cases.
      CharacteristicsSymptomatic casesAsymptomatic casesP-value
      (n = 75)(n = 143)
      Median (IQR)Median (IQR)
      Crude analysis
      24 (18–36)20 (16–30)0.017
      Stratified analysis
      Sex
      Female21 (17–36)20 (16–30)0.467
      Male31 (20–35)20 (16–30)0.009
      P-value0.150.45
      Age (years)
      <6024 (18–36)20 (16–30)0.016
      ≥6020 (17–36)21 (16–31)0.674
      P-value0.860.47
      Smoking
      No23 (17–36)20 (16–32)0.184
      Yes27 (21–38)18 (17–21)0.138
      P-value0.520.61
      Hypertension
      No24 (18–36)20 (17–30)0.040
      Yes25 (17–40)20 (15–28)0.180
      P-value0.860.27
      Diabetes
      No25 (18–36)20 (16–30)0.012
      Yes18 (15–30)20 (15–30)0.723
      P-value0.120.76
      Asthma
      No24 (18–36)20 (16–30)0.012
      Yes23 (15–33)16 (15–33)0.710
      P-value0.380.42
      Obesity
      BMI <3025 (19–37)20 (17–29)0.043
      BMI ≥ 3019 (14–37)17 (16–23)0.508
      P-value0.050.21
      Isolated room
      No31 (20–36)36.5 (29–39)0.147
      Yes24 (17–37)20 (17–30)0.101
      P-value0.470.01
      Contact with masks
      No29 (18–40)19 (16–30)0.007
      Yes19 (16–23)17 (16–19)0.198
      P-value0.020.24
      SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; IQR, interquartile range; BMI, body mass index (kg/m2).
      According to the study findings, viral RNA negative conversion was significantly faster in patients with dyslipidemia (log-rank test, P = 0.016; Figure 2D) and in patients who were wearing face masks when they contracted the virus (log-rank test, P = 0.003; Figure 2F). The median time to RNA viral conversion was 18 days (IQR 16–21 days) for patients using face masks versus 23 days (IQR 17–36 days) for patients who did not wear one.
      There was no significant difference in the duration of SARS-CoV-2 RNA negative conversion according to sex (Figure 2A), age over 60 years (Figure 2B), current smoking, comorbidities, and isolation in a single room (Figure 2E).
      Table 4 summarizes the results of the univariate and multivariate analyses.
      Table 4Risk factors associated with prolonged negative conversion of viral RNA.
      Univariate analysis Unadjusted HR (95% CI)Multivariate analysis Adjusted HR (95% CI)
      HR95% CIP-valueHR95% CIP-value
      Sex
      FemaleRef.
      Male0.965(0.756–1.232)0.776
      Age (years)
      <60Ref.
      ≥600.783(0.560–1.095)0.1531.480(0.834–2.624)0.180
      Smoking
      NoRef.
      Yes1.237(0.792–1.931)0.349
      Comorbidities
      Hypertension
      NoRef.
      Yes0.949(0.664–1.355)0.772
      Diabetes
      NoRef.
      Yes1.235(0.882–1.730)0.220
      Dyslipidemia
      NoRef.
      Yes2.742(1.119–6.717)0.0271.924(0.249–14.862)0.530
      Asthma
      NoRef.
      Yes1.157(0.685–1.954)0.586
      Symptoms
      NoRef.
      Yes0.756(0.570–1.002)0.0510.600(0.401–0.897)0.013
      Obesity
      BMI <30Ref.
      BMI ≥ 301.286(0.807–2.051)0.290
      Isolated room
      NoRef.
      Yes1.304(0.799–2.128)0.288
      Contact with masks
      NoRef.
      Yes1.924(1.218–3.039)0.0052.006(1.247–3.228)0.004
      HR, hazard ratio; CI, confidence interval; BMI, body mass index (kg/m2).
      Multivariate Cox regression was performed with the significant factors selected by univariate analysis. The presence of symptoms (HR 0.600, 95% CI 0.401–0.897) and the use of face masks when exposed to people diagnosed with COVID-19 (HR 2.006, 95% CI 1.247–3.228) were independently associated with negative conversion of viral RNA, suggesting that the presence of symptoms during SARS-CoV-2 infection delays virological clearance and wearing masks reduces the duration in COVID-19 patients.

      Discussion

      Key results

      Until now, there have been few studies on the predictors of the time to negative conversion of SARS-CoV-2 RNA. Viral shedding has been related to infectivity and transmissibility in influenza virus infections, and an understanding of this is crucial for the implementation of prevention strategies (
      • Ryoo Seung M.
      • Kim Won Y.
      • Sohn Chang H.
      • Seo Dong W.
      • Oh Bum J.
      • Lee Jae H.
      • et al.
      Factors promoting the prolonged shedding of the pandemic (H1N1) 2009 influenza virus in patients treated with Oseltamivir for 5 Days.
      ). Therefore, it is important to determine the duration of viral shedding and related factors in patients with COVID-19 (
      • Qi Lin
      • Yang Yong
      • Jiang Dixuan
      • Tu Chao
      • Wan Lu
      • Chen Xiangyu
      • et al.
      Factors associated with the Duration of viral shedding in adults with COVID-19 outside of Wuhan, China: a retrospective cohort study.
      ). The present study showed face mask wearing and having symptoms to be associated with the time to RNA conversion. The median time to RNA viral conversion was 18 days (IQR 16–21 days) for those who wore a mask when in contact with the index case versus 23 days (IQR 17–36 days) for those who did not wear a mask, and was 24 days (IQR 18–36 days) for symptomatic patients versus 20 days (IQR 16–30 days) for asymptomatic individuals.

      Interpretation

      The median time to viral conversion observed in the study cohort was 20 days (IQR 17–32 days) from the first positive RT-PCR test, similar to the results of the study by Zhou et al. (
      • Zhou Fei
      • Yu Ting
      • Du Ronghui
      • Fan Guohui
      • Liu Ying
      • Liu Zhibo
      • et al.
      Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study.
      ). Other studies have demonstrated a shorter median duration of viral shedding of 9.5 days (IQR 6.0–11.0 days) and 14 days (IQR 10–18 days) (
      • Hu Xiaowen
      • Xing Yuhan
      • Jia Jing
      • Ni Wei
      • Liang Jiwei
      • Zhao Dan
      • et al.
      Factors associated with negative conversion of viral RNA in patients hospitalized with COVID-19.
      ,
      • Ling Yun
      • Xu Shui-Bao
      • Lin Yi-Xiao
      • Di Tian
      • Zhu Zhao-Qin
      • Dai Fa-Hui
      • et al.
      Persistence and clearance of viral RNA in 2019 novel coronavirus disease rehabilitation patients.
      ). Another study in Italy reported a longer median duration of viral shedding of 31 days (IQR 24–41 days) (
      • Mancuso Pamela
      • Venturelli Francesco
      • Vicentini Massimo
      • Perilli Cinzia
      • Larosa Elisabetta
      • Bisaccia Eufemia
      • et al.
      Temporal profile and determinants of viral shedding and of viral clearance confirmation on nasopharyngeal swabs from SARS-CoV-2-positive subjects: a population-based prospective cohort study in Reggio Emilia, Italy.
      ). The discrepancies might be attributed to the heterogeneity of patients and disease severity in these studies. Differences may also have resulted from the different starting points for RNA viral clearance in previous reports. In the present study, the definition was in line with that used in other high-quality studies, as mentioned in the Methods section (
      • Hu Xiaowen
      • Xing Yuhan
      • Jia Jing
      • Ni Wei
      • Liang Jiwei
      • Zhao Dan
      • et al.
      Factors associated with negative conversion of viral RNA in patients hospitalized with COVID-19.
      ,
      • Gao W.J.
      • Li L.M.
      [Advances on presymptomatic or asymptomatic carrier transmission of COVID-19].
      ).
      The results of this study showed that sex was not related to the duration of viral shedding. However, a previous study showed that male sex was a predictor of prolonged SARS-CoV-2 RNA clearance (
      • Xu Kaijin
      • Chen Yanfei
      • Yuan Jing
      • Yi Ping
      • Ding Cheng
      • Wu Wenrui
      • et al.
      Factors associated with prolonged viral RNA shedding in patients with COVID-19.
      ). The mechanism of sex-related differences in SARS-CoV-2 infection is unclear. It might be related to sex hormones, which appear to influence the immune system. Females are reported to produce more cellular and humoral immune reactions, and so are more resistant to certain infections (
      • Bouman Annechien
      • Heineman Maas Jan
      • Faas Marijke M.
      Sex hormones and the immune response in humans.
      ).
      Smoking was found not to be a predictor of prolonged viral shedding in this study, in line with results reported in a previous study (
      • Xu Kaijin
      • Chen Yanfei
      • Yuan Jing
      • Yi Ping
      • Ding Cheng
      • Wu Wenrui
      • et al.
      Factors associated with prolonged viral RNA shedding in patients with COVID-19.
      ). A meta-analysis based on 19 peer-reviewed papers, demonstrated a significant association between smoking and the progression of COVID-19 (
      • Patanavanich Roengrudee
      • Glantz Stanton A.
      Smoking is associated with COVID-19 Progression: a meta-analysis.
      ). Indeed, the adverse effect of smoking on pulmonary immune function is a risk factor for serious outcomes among infected people (
      • Bauer Carla M.T.
      • Morissette Mathieu C.
      • Stämpfli Martin R.
      The influence of cigarette smoking on viral infections: translating bench science to impact COPD pathogenesis and acute exacerbations of COPD clinically.
      ). Health care providers should take active measures to slow the trend in smoking, and public health campaigns should underline the importance of smoking cessation during the pandemic.
      Furthermore, the current study does not support diabetes and hypertension as predisposing factors for late SARS-CoV-2 viral clearance, similar to previous studies (
      • Hu Xiaowen
      • Xing Yuhan
      • Jia Jing
      • Ni Wei
      • Liang Jiwei
      • Zhao Dan
      • et al.
      Factors associated with negative conversion of viral RNA in patients hospitalized with COVID-19.
      ).
      This study showed no increase in time to viral clearance with increasing age. Older age (over 60 years) was not found to be an independent predictive factor of prolonged viral RNA shedding. Similarly, a recent systematic review reported no significant age-related differences in the duration of both respiratory tract swab positivity and fecal sample positivity (
      • Morone Giovanni
      • Palomba Angela
      • Iosa Marco
      • Caporaso Teodorico
      • De Angelis Domenico
      • Venturiero Vincenzo
      • et al.
      Incidence and persistence of viral shedding in COVID-19 post-acute patients with negativized pharyngeal swab: a systematic review.
      ). However, it was demonstrated that older age was correlated to a later RT-PCR conversion (
      • Hu Xiaowen
      • Xing Yuhan
      • Jia Jing
      • Ni Wei
      • Liang Jiwei
      • Zhao Dan
      • et al.
      Factors associated with negative conversion of viral RNA in patients hospitalized with COVID-19.
      ,
      • Mori Hitoshi
      • Obinata Hirofumi
      • Murakami Wakana
      • Tatsuya Kodama
      • Sasaki Hisashi
      • Yu Miyake
      • et al.
      Comparison of COVID-19 disease between young and elderly patients: hidden viral shedding of COVID-19.
      ). COVID-19 is more likely to infect older patients due to their weaker immune system (
      • Liu Kai
      • Chen Ying
      • Lin Ruzheng
      • Kunyuan Han.
      Clinical features of COVID-19 in elderly patients: a comparison with young and middle-aged patients.
      ). Indeed, T-cell numbers and functions are highly affected by aging, leading to less controlled viral replication and host inflammatory responses (
      • Goronzy J.örg J.
      • Lee Won-Woo
      • Weyand Cornelia M.
      Aging and T-Cell Diversity.
      ). Moreover, age-related comorbidities may result in prolonged viral shedding among the elderly (
      • Liu Kai
      • Chen Ying
      • Lin Ruzheng
      • Kunyuan Han.
      Clinical features of COVID-19 in elderly patients: a comparison with young and middle-aged patients.
      ). However, no significant difference was noted concerning comorbidities in the current study. The effect of age on RNA viral clearance in patients with COVID-19 still needs further investigation.
      Interestingly, not wearing masks and having symptoms were associated with prolonged viral RNA clearance. Virological and epidemiological data have led to the hypothesis that face mask wearing may reduce the severity among infected people (
      • Gandhi Monica
      • Rutherford George W.
      Facial masking for covid-19 — potential for “Variolation” as we await a vaccine.
      ). In an outbreak on a closed Argentinian cruise ship, the rate of asymptomatic infection among passengers wearing surgical and N95 masks was 81% compared with 20% in those who did not wear a mask (
      • Gandhi Monica
      • Beyrer Chris
      • Goosby Eric
      Masks do more than protect others during covid-19: reducing the inoculum of SARS-CoV-2 to protect the wearer.
      ).
      Wearing a mask could reduce the virus dose received, leading to less severe manifestations of COVID-19. Long-lasting negative RNA conversion might also be proportionate to the viral SARS-CoV-2 inoculum received, explaining the association between facial mask wearing and viral shedding in the study findings. This indicates another possible advantage of population-wide mask-wearing for pandemic control regarding the viral inoculum, attenuating severe disease and accelerating SARS-CoV-2 recovery. Enforced population mask-wearing is the key strategy now as we await the results of vaccine trials. Therefore, strategic guidance should be established and a sufficient supply of masks should be guaranteed.
      Developing symptoms was a predictor of prolonged negative RNA conversion. Mild cases were found to have early clearance in comparison to severe cases, in which the mean viral load was 60 times higher than that in mild cases, suggesting that higher viral loads might be associated with a longer viral shedding period (
      • Liu Yang
      • Yan Li-Meng
      • Wan Lagen
      • Xiang Tian-Xin
      • Le Aiping
      • Liu Jia-Ming
      • et al.
      Viral dynamics in mild and severe cases of COVID-19.
      ). However, and unexpectedly, previous studies have reported that the viral load in asymptomatic SARS-CoV-2 patients was as high as that in symptomatic patients (
      • Lee Seungjae
      • Tark Kim
      • Eunjung Lee
      • Cheolgu Lee
      • Hojung Kim
      • Heejeong Rhee
      • et al.
      Clinical course and molecular viral shedding among asymptomatic and symptomatic patients with SARS-CoV-2 infection in a community treatment center in the Republic of Korea.
      ,
      • Zou Lirong
      • Ruan Feng
      • Huang Mingxing
      • Liang Lijun
      • Huang Huitao
      • Hong Zhongsi
      • et al.
      SARS-CoV-2 viral load in upper respiratory specimens of infected patients.
      ). The present study data indicate that patients with symptoms tend to have a prolonged viral clearance, and this might be a useful marker for assessing the disease prognosis.
      This study has some limitations. First, RNA was only analyzed in nasopharyngeal and not in patient excretions such as urine and feces. However, previous studies have shown that viral loads in patient excretions might be higher than those in respiratory specimens (

      Li, Tong-Zeng, Zhen-Huan Cao, Yu Chen, Miao-Tian Cai, Long-Yu Zhang, Hui Xu, Jia-Ying Zhang, et al. n.d. Duration of SARS-CoV-2 RNA Shedding and Factors Associated with Prolonged Viral Shedding in Patients with COVID-19. Journal of Medical Virology n/a (n/a). Accessed 11 August 2020. https://doi.org/10.1002/jmv.26280.

      ). Second, this study reports only qualitative results of RNA detection, but persistent positivity for SARS-CoV-2 RNA does not necessarily indicate persistent shedding of live virus. To date, it is unknown how the shedding of viral RNA correlates with the shedding of infectious virus and this needs further studies.

      Implications for practice and generalizability

      The study results highlight the point that asymptomatic and symptomatic COVID-19 patients will test positive for SARS-CoV-2 when released according to the latest WHO recommendation (). The study data indicate that asymptomatic patients recover more quickly than symptomatic patients. Future studies with viral load data are recommended to specify the containment periods for both symptomatic and asymptomatic cases.
      Finally, and since participants who were wearing masks at the time they contracted the virus had a shorter duration of viral clearance, masks should be encouraged not only to prevent the spread of the epidemic, but also to reduce the viral dose received when contracting the virus and to have rapid viral clearance after infection.

      Conclusions

      This study with a relatively large sample size is novel in focusing on the duration of RNA viral clearance and related factors in patients with COVID-19 in Tunisia. This study demonstrated that the presence of symptoms and not wearing face masks were associated with delayed negative RNA conversion in patients with mild COVID-19. The study results have relevant public health implications. It is hoped that these predictors could provide clues for the early identification of cases with prolonged viral shedding, in order avoid virus spread.

      Funding

      There was no external funding for this article

      Ethical approval

      The Ethics Committee of the Faculty of Medicine of Monastir approved the protocol of this study. To maintain the principle of confidentiality, the data used were anonymized.

      Consent for publication

      Not applicable.

      Conflict of interest

      We do not have any conflicts of interest associated with this publication. There was no significant financial support for this work that could have influenced its outcome.

      CRediT authorship contribution statement

      Cyrine Bennasrallah: Conceptualization, Formal analysis, Methodology, Writing - original draft. Imen Zemni: Formal analysis, Methodology, Supervision, Writing - review & editing. Wafa Dhouib: Validation. Haythem Sriha: Data curation. Nourhene Mezhoud: Data curation. Samar Bouslama: Data curation. Wael Taboubi: Data curation. Meriem Oumaima Beji: Data curation. Meriem Kacem: Validation. Hela Abroug: Validation. Manel Ben Fredj: Validation. Chawki Loussaief: Supervision, Validation. Asma Sriha Belguith: Conceptualization, Formal analysis, Writing - review & editing.

      Acknowledgements

      The authors wish to thank the following individuals and all of the staff of the national COVID-19 containment center for their help in data collection: Manel ben Belgacem, Mondher Rebhi, Kmar Mrad, Manel Mantassar, Mohamed Hammadi, Karawen Sakka, Aymen Nasri. We would also like to thank all of the team of the Microbiology Laboratory in Fattouma Bourguiba University Hospital for their tremendous effort during this pandemic. We take this opportunity to extend our gratitude to the team of the Department of Epidemiology and Preventive Medicine at Monastir University.

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