Introduction
Coronaviruses are a large viral family that can cause upper respiratory infections and more serious diseases such as severe acute respiratory syndrome-coronavirus (SARS-CoV) (1). Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes coronavirus disease-2019 (COVID-19). The COVID-19 pandemic caused by SARS-CoV-2 has had global effects (2). While its burden is decreasing, it continues to spread worldwide and is expected to remain a public health problem for some time to come.
Direct person-to-person viral spread via respiratory secretions is the main SARS-CoV-2 transmission route (3,4). SARS-CoV-2 transmission begins before symptoms develop and is highest in the early disease stages. Thereafter, the infection risk decreases. Transmission is unlikely after seven to ten days of illness (5). Infected cases are more likely to be contagious during the early disease stages, when viral ribonucleic acid (RNA) levels in the upper respiratory tract are highest (5).
SARS-CoV-2 affects children less than adults, whose clinical course is more severe. While children typically have a lower exposure risk and are less frequently tested than adults, the incidence of adenocarcinoma in children is close to that in adults (6,7). In a SARS-CoV-2 childhood study, the infection rates of children ≥5 years old were similar to those of adults, regardless of symptoms (8). A case series conducted early in the pandemic showed that most children were infected by household exposure, usually from infected adults. Later, they were more frequently infected through peer contact than through household exposure due to reduced protective measures and school reopening (6,9).
The duration of viral RNA excretion is variable and may increase with age and disease severity (10,11). The detection of viral RNA in respiratory tract samples was demonstrated for 18 days after the onset of disease symptoms. In some cases, this situation can last up to several months (10,12). This study evaluated factors associated with the duration of SARS-CoV-2 presence in upper respiratory swabs from infected children.
Methods
Compliance with Ethical Standards
This study was approved by the University of Health Sciences Turkey, Istanbul Haseki Training and Research Hospital Clinical Research Ethics Committee (approval number: 23-2021, dated: 05.05.2021). Informed consent was obtained from the parents of all the children enrolled in this study.
Setting Design
This retrospective cohort study included children and adolescents (1 month-18 years) diagnosed with SARS-CoV-2 infections and admitted to the University of Health Sciences Turkey, Istanbul Haseki Training and Research Hospital between March 31 and July 1, 2020. In total, 931 patients were consecutively included in this study.
Diagnostic Criteria
The SARS-CoV-2 infection diagnosis was made on the basis of a positive polymerase chain reaction (PCR) test
result from upper respiratory swabs taken from children with suspected SARS-CoV-2 infections, according to the Ministry of Health COVID-19 guidelines (13).
Group Definitions
The SARS-CoV-2 presence duration was defined on the basis of consecutive SARS-CoV-2 PCR test results from upper respiratory swabs during clinical follow-up in hospital records. The duration was calculated as the period between the initial positive and negative test result dates. Cases whose inter-test interval was more than 14 days were excluded from this study. In addition, cases that had not received a negative test result were excluded from this study. Cases with additional morbidity that interfered with the SARS-CoV-2 positivity duration were excluded from this study.
Cases were divided into two groups based on their duration of SARS-CoV-2 PCR test positivity: (i) those who had at least two samples taken <14 days apart and who tested negative before day 14 were assigned to the regular positivity duration group (Group 1); (ii) those who had at least two positive test results within a 14-day interval (without a negative test result in between) and who tested negative after day 14 were assigned to the prolonged positivity duration group (Group 2; Figure 1).
Clinical SARS-CoV-2 infection courses were divided into three groups according to the World Health Organization guidelines: asymptomatic, mild, and moderate-severe (14). Those with pneumonia or signs of sepsis or in need of oxygen support were evaluated in the moderate-severe group.
Data Collection
The clinical features and laboratory and radiologic findings for each patient were retrospectively obtained from their medical records. Complete blood count, biochemistry, C-reactive protein (CRP), erythrocyte sedimentation rate, procalcitonin, D-dimer, fibrinogen, troponin, creatine kinase myocardial band, fibrinogen/albumin ratio (FAR), thorax computed tomography or anterior-posterior chest X-ray, and index cases were recorded.
The SARS-CoV-2 PCR assay was performed on oro-nasopharyngeal swabs (Bioksen ArGe Teknik Co. Ltd., Turkey) using the Biospeedy reverse transcriptase quantitative PCR detection kit.
Demographic, clinical, laboratory, and radiological characteristics were compared between the groups. The secondary objective of this study was to identify predictors of prolonged positivity.
Statistical Analysis
Statistical analyses were performed using SPSS 22.0 for Windows (IBM Corp.; Armonk, NY, USA). The Shapiro-Wilk test was used to assess the normality of each variable’s distribution. Numbers and percentages are used to represent categorical variables. The mean ± standard deviation or the median with interquartile range [(IQR); 25th-75th percentiles] were used to present continuous variables depending on whether they had a parametric or non-parametric distribution, respectively. Categorical variables were compared using the chi-square test. Median or mean values were compared between the two groups using the Mann-Whitney U test and Student’s t-test, depending on sample distribution. A p-value of <0.05 was considered the alpha significance level. Multivariate analyses included variables with significant univariate associations between groups and no collinearity within a logistic regression model to identify independent predictors of prolonged SARS-CoV-2 PCR positivity. The Hosmer-Lemeshow test was used to evaluate the model’s goodness of fit. A 5% type I error level was used to assess statistical significance.
Results
This study screened 931 patients, and 518 cases meeting the inclusion criteria were included. Among them, 432 cases (83.4%) met the group 1 criteria, and 86 (16.6%) met the group 2 criteria. The median SARS-CoV-2 PCR positivity duration of all included cases (n=518) was 11.1 days (IQR: 2-35). We balanced the number of patients in Groups 1 and 2 by excluding 260 patients from Group 1 via randomization according to age, enabling appropriate comparisons between groups (172 vs. 86 patients). Of these 258 cases, 134 (52%) were female and 124 (48%) were male. Their median age was 132.6 months (IQR: 53-187).
Intergroup Comparisons
The median age of group 1 was 121 (57-182) and the median age of group 2 was 156 (46-198) months, and there was no statistical difference between them (p=0.172). There were 87 males in Group 1 and 37 males in Group 2. There was no statistical difference between the groups in terms of sex (p=0.252). Comparisons of the age distribution of Groups 1 and 2 are shown in Figure 2.
The median SARS-CoV-2 PCR positivity durations in Group 1 for each clinical course were: 5.50 (2.75-8.00) days for asymptomatic, 6.00 (4.00-8.00) days for mild, and 7.00 (5.00-8.00) days for moderate-severe courses. Although the median SARS-CoV-2 PCR positivity durations gradually increased with disease severity, they did not differ significantly (p=0.737). The median SARS-CoV-2 PCR positivity durations in Group 2 for each clinical course were: 22.5 (19.5-25.0) days for asymptomatic, 22.0 (16.0-28.0) days for mild, and 20.0 (19.0-23.0) days for moderate-severe courses. Again, durations did not differ significantly among courses (p=0.481). In addition, the hospitalization ratio did not significantly differ between the groups (5.8% vs. 5.8%; p=0.100).
Contact with a SARS-CoV-2 positive case occurred in 138 (53.5%) cases, of which 119 (86%) were exposed to household contact. Contact history did not significantly differ between the groups (58.1% vs. 44.1%; p=0.140).
Among the 213 (82.5%) symptomatic cases, fever was significantly more common in Group 1 (68.1%) than in Group 2 (55.1%; p=0.020). However, the frequencies of other symptoms did not significantly differ between the groups (Table 1).
The laboratory findings of each group are compared in Table 2.
We used a multivariate logistic regression model to predict prolonged SARS-CoV-2 PCR positivity. This model comprised demographic features, fever, D-dimer value >0.55 mg/L, lymphopenia, and CRP level at admission. Lymphopenia was identified as an independent predictor of prolonged SARS-CoV-2 PCR positivity (odds ratio=2.96; 95% confidence interval: 1.08-8.13; p=0.035).
Discussion
As the primary outcome of this study, the SARS-CoV-2 PCR positivity duration was more than 14 days in 86 (16.6%) of 518 cases. Our secondary outcomes were the associations of high CRP, D-dimer >0.55 mg/L, fever, and lymphopenia with factors affecting prolonged SARS-CoV-2 PCR positivity. In addition, we identified lymphopenia as an independent risk factor.
The sex and age distributions did not significantly differ between the groups. Although no statistically significant age-related differences were identified in this study, several studies have shown that symptoms and PCR positivity depend on age (15). In addition, it has been reported that there are, on average, 10- to 100-fold greater viral loads in children aged <5 years, and COVID-19 symptoms appear in 7.4-fold more children aged <1 year (15-17). In these studies, the risk of severe symptoms was elevated in children <5 years old compared to other age groups (15-17). Hua et al. (18) found that the incubation period was longer in children than in adults. Symptoms may begin, on average, 3-7 days after a positive PCR test in some patients. Therefore, the duration of SARS-CoV-2 presence in the upper respiratory mucosa may be longer than that detected (19,20). Because studies on children and their data are limited, studies on large pediatric populations are needed.
The median duration of SARS-CoV-2 presence was 11.1 days (2-35 days) in all 518 patients included in this study. In addition, no virus was detected in 83.4% of the patients after day 14. The median duration of SARS-CoV-2 presence in our study is consistent with the meta-analysis conducted by Li et al. (21), who found that the mean time required for SARS-CoV-2 RNA to become undetectable in nasopharyngeal/throat swabs was 11.43 days. Another study found the median duration of viral shedding and PCR positivity in children to be seven days (5-10), with 96.3% of patients becoming negative within 14 days (22). Different studies have reported that the average duration of viral spread may be longer than 14 days, similar to our study (23,24). The longer SARS-CoV-2 presence in this study compared with others may reflect our comprehensive duration evaluation with close follow-up by performing tests at frequent intervals.
This study found no significant differences in the duration of SARS-CoV-2 presence, disease severity, or hospitalization need. We could not find published studies that determined the significance of the relationships between clinical severity and the duration of SARS-CoV-2 presence. In this study, we found that hospitalization rates for critically ill patients were low, and the rate of patients with mild symptoms was high because PCR testing was performed on all cases with complaints or contact histories. The reasons for the relatively mild disease course in children remain incompletely understood. A weaker inflammatory response and differences in the expression and regulation of angiotensin-converting enzyme 2 receptors in the airway epithelium have been suggested as causes (25,26).
In this study, the most common symptoms were fever (56.3%) and cough (58.6%); fever with cough was 34.2%, while concomitant fever, cough, and shortness of breath were 35.6%. Previous studies have reported fever (51-68%) and cough (41-61%) as the most common symptoms in children diagnosed with COVID-19 (27-29). In this study, we found that SARS-CoV-2 presence was longer in patients with fever and lymphopenia. A retrospective study in Wuhan, China, found that the median viral shedding period during COVID-19 hospitalization was longer in children with symptoms (especially fever, pneumonia, and lymphopenia), consistent with this study (30). Moreover, Lu et al. (30) found that symptomatic children had a longer viral shedding period (17 days) than asymptomatic children (11 days). However, Korean and Kuwaiti studies found no correlation between viral shedding and symptoms (31,32). The relationship between symptoms and prolonged SARS-CoV-2 presence was not explored in this study. It should be considered that the presence of SARS-CoV-2 may last more than 14 days, especially in patients with fever signs.
In this study, high CRP levels and lymphopenia were significantly associated with prolonged SARS-CoV-2 presence. Although elevated D-dimer levels did not significantly differ between the groups, D-dimer levels >0.55 mg/L were significantly associated with prolonged SARS-CoV-2 presence. Similarly, we found that lymphopenia increased the prolonged SARS-CoV-2 presence by 2.96-fold. However, there was no relationship between age and SARS-CoV-2 presence, whereas elevated CRP levels and lymphopenia were associated with prolonged SARS-CoV-2 presence. Lu et al. (30) in Wuhan, China, found that high CRP and low lymphocyte levels correlated with the duration of SARS-CoV-2 presence in patients aged <5 years, especially those <1 year. In this study, D-dimer and fibrinogen levels were not significantly associated with prolonged SARS-CoV-2 presence (30). However, lymphopenia should be considered as an independent risk factor. We hypothesize that lymphopenia weakens the body’s ability to fight the virus, thus prolonging viral persistence. In addition, there is an urgent need for more detailed studies on the relationship between D-dimer levels and prolonged SARS-CoV-2 presence.
FAR is a proportional parameter recently defined as an inflammation indicator. Studies have shown that it predicts mortality in COVID-19 patients and can be used as a marker for serious disease (33,34). In this study, FAR was marginally significant. However, we could not find any studies exploring the association between the duration of SARS-CoV-2 presence and FAR in children. Although there is no statistically significant association, we believe that FAR should be considered in these patients and requires further study.
Study Limitations
Our study was limited by its retrospective design. In addition, because this study included all children regardless of their symptoms, hospital admissions were generally late due to the difficulties children experience in expressing their complaints compared with adults. Finally, the findings of this study were impacted by the unknown number of positive days before admission. Despite these limitations, our number of cases was high compared with a pediatric study. Our study group was homogeneously distributed. We were able to show what we wanted to show about the severity of the disease and the prolongation of PCR positivity.
Conclusion
The risk of SARS-CoV-2 transmission from children should not be ignored. Understanding the duration of the SARS-CoV-2 presence is important for determining suitable isolation periods for the pediatric population. In this study, the duration of SARS-CoV-2 presence was associated with the disease’s clinical findings and laboratory results. Our findings recommended more careful follow-up in pediatric patients due to prolonged SARS-CoV-2 presence for more than 14 days, especially those with fever, elevated CRP and D-dimer levels, and lymphopenia.
Ethics
Ethics Committee Approval: This study was approved by the University of Health Sciences Turkey, Istanbul Haseki Training and Research Hospital Clinical Research Ethics Committee (approval number: 23-2021, dated: May 5, 2021).
Informed Consent: Informed consent was obtained from the parents of all the children enrolled in this study.
Peer-review: Externally and internally peer-reviewed.
Authorship Contributions
Concept: B.O., G.A., Design: B.O., G.A., Data Collection or Processing: F.C.Y., Z.U.O., Analysis or Interpretation: B.O., F.C.Y., Z.U.O., G.A., Literature Search: B.O., F.C.Y., Z.U.O., G.A.,Writing: B.O., G.A.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.