Clinical and Prognostic Utility of Cycle Threshold (Ct) Value of SARS-CoV-2 in Pediatric Population: Single-Center Experience

Abstract

The cycle threshold (Ct) value of PCR, used to detect SARS-CoV-2 viral load, has been associated with disease severity in adults, but few studies have examined these relationships in pediatric patients. The aim of this study was to evaluate the relationship between the Ct value at hospital admission and the duration of fever, length of hospital stay, and the need for medical interventions in pediatric patients with COVID-19. We conducted a retrospective study of 124 pediatric patients with a positive nasopharyngeal swab for SARS-CoV-2 between April 2020 and March 2022. A negative correlation was observed between the Ct value and the duration of fever (p = 0.012), with a similar trend for length of hospital stay (p = 0.25). A low Ct value was also associated with the development of MIS-C (p = 0.026) and the need for respiratory support and steroid therapy (p = 0.036). The Ct value may be useful in stratifying pediatric patients with COVID-19, helping to predict the duration of fever and hospital stay, as well as the need for medical interventions.

1. Introduction

Spreading from China since December 2019, coronavirus 2 caused a severe acute respiratory syndrome (SARS-CoV-2) [1,2]. Coronavirus disease 2019 (COVID-19) is a global health threat commonly spread through respiratory droplets and aerosol transmission. The causative agent for COVID-19 is an enveloped positive single-stranded RNA virus, whose infection may affect the lungs primarily, leading to respiratory failure, but simultaneously involves several organs, from kidneys to the heart, blood vessels, liver, pancreas, and immune system [3]. Children have shown to be affected by SARS-CoV-2 with a milder clinical presentation and a more favorable prognosis [4,5] if compared with adult patients. Most common symptoms include cough, fever, and fatigue [4,6,7,8], followed by other upper respiratory symptoms, such as nasal congestion and runny nose, involvement, and neurological and psychiatric disorders [4,9,10]. Severe cases with lower respiratory tract infection and severe acute symptoms have also been reported [5,11,12]. In addition, a post-infectious clinical entity with hyper-inflammatory multisystem involvement, commonly named MIS-C, has been described with gastrointestinal symptoms and cardiovascular dysfunction due to multisystem involvement with a potentially fatal course [5]. In fact, SARS-CoV-2 has a genome with a 5′ terminal rich in open reading frames, which encodes proteins essential for virus replication. Instead, the 3′ terminal includes five structural proteins: spike protein (S), membrane protein (M), nucleocapsid protein (N), envelope protein (E), and hemagglutinin-esterase protein (HE). Specifically, the spike protein is responsible for the pathogenesis in the human species since its receptor-binding domain (RBD) links to the human cell surface receptor protein angiotensin-converting enzyme-2 (ACE-2), encoded by the ACE2 gene, which has a ubiquitous distribution in the organs [13]. The gold standard for SARS-CoV-2 infection’s diagnosis is a molecular nasopharyngeal swab, which is usually also performed to ensure the end of infection and contagiousness. While plenty of studies have been carried out about COVID disease in adults, a fewer number is available for pediatric care [14]. Studies performed on adult patients’ data have shown the clinical utility of PCR amplification cycles (cycle threshold value), which seems to be related to clinical features such as disease severity and disease duration, enlightening the Ct value’s predictive role [14]. The primary objective of our study was to evaluate, in pediatric patients, the correlation between the number of PCR amplification cycles obtained from molecularly positive nasopharyngeal swabs at the time of hospital admission and the duration of fever, as well as the correlation between amplification cycles and the length of hospital stay. As secondary endpoints, we analyzed the correlations between the Ct value at admission and the need for respiratory support, the use of steroid therapy, and the development of multisystem inflammatory syndrome (MIS-C). Through these analyses, we aim to understand whether the Ct value can be used clinically to stratify patients and predict disease evolution. Measuring viral and immunological markers over time could be useful to understand the evolution of the infectious response and to stratify patients according to disease severity. Recent studies on SARS-CoV-2 antibody tests have highlighted the importance of the “personal timeline” of antibody levels, where the response varies individually, peaking and then decreasing after infection or vaccination. This temporal dynamic has direct implications for patient classification since two samples collected at different times, even with the same antibody value, may reflect very different phases of the infection [15]. Similarly, cycle threshold (Ct) values in SARS-CoV-2-positive molecular swabs reflect the amount of viral load at a specific phase of the disease. However, as suggested for antibodies, the Ct value could also vary over time with different clinical meanings depending on the disease phase in which it was measured. Our study explores the hypothesis that an adaptive approach to Ct values may allow for better stratification of pediatric patients, helping to predict the duration of symptoms such as fever and clinical course. Interpreting Ct as a dynamic variable, rather than an absolute value, may therefore provide a more precise estimate of clinical risk and guide treatment.

2. Materials and Methods

2.1. Sampling Strategy

We performed a retrospective single-center cohort study from April 2020 to March 2022. For the aim of the present study, we used the database of the laboratory of San Marco University Hospital (Catania, Italy) and collected all positive molecular nasopharyngeal swabs performed from April 2020 to March 2022 in patients between 0 days of life and 18 years of age. For everyone, we recorded Ct values at the admission and at the discharge and any medical data available. We investigated symptoms and their duration, hospitalization’s duration, need for respiratory support, use of steroid therapy, and occurrence of MIS-C. Written informed consent was obtained from all the parents of the patients. We wish to thank Research Project “Pandemicamente. Le narrazioni come risorsa in tempo di pandemia” for this research. The project was entrusted by the Higher Institute of Health (ISS, code 0015978-26/04/2021).

2.2. Inclusion and Exclusion Criteria

The inclusion criteria of our study were a positive swab for SARS-CoV-2 with an available PCR Ct value for amplification of the N2 gene. We excluded patients with incomplete clinical information and patients with no available PCR Ct gene amplification results.

2.3. Laboratory Analysis of Swab

Our laboratory uses the “Xpert Xpress SARS-CoV-2 test” developed by Cepheid, which is a real-time RT-PCR test for qualitative detection of nucleic acid from the SARS-CoV-2 in upper respiratory samples [16]. Ct values greater than or equal to 40 were considered negative; values less than 40 were positive. The analysis was made using primers and probes in relation to 110,206 SARS-CoV-2 sequences available at the date of 21 October 2020 in the GISAID gene database for only two genetic targets of the virus, E and N2 [16]. To note, unfortunately, many other genes from the SARS-CoV-2 genome were not tested by our laboratory.

2.4. Statistical Analysis

All statistical analyses were conducted using R (v.4.2.1). To compare qualitative variables, we used Pearson correlation analysis, and coefficients were considered reliable when the p-value was less than 0.05. Pearson correlation was chosen to assess the linear relationship between continuous variables, such as Ct values and fever duration, since both variables are measured on a continuous scale. Each correlation was plotted via the package ggpubr with a scatter plot. The number of cycles at admission and discharge were compared with the occurrence of MIS-C, the need for respiratory support, the need for use of steroid therapy employing the Kruskal–Wallis test. Since our data do not follow a normal distribution and do not meet the assumptions for parametric tests, the Kruskal–Wallis test is an appropriate choice. This test compares the median distribution of the groups and thus provides a robust analysis of differences even in the presence of skewed distributions and non-homogeneous variances. Internal contrast of these tests was performed using the Wilcoxon test, which is suitable for comparing two related samples or matched groups when normality cannot be assumed. Only deviations with p-value < 0.05 were considered significant.

2.5. Endpoints

2.5.1. Primary Endpoints

The primary endpoints were:

• The correlation between Ct value at admission and the duration of fever (in days).
• The correlation between Ct value at admission and hospital stay (in days).

2.5.2. Secondary Endpoints

The secondary endpoints were:

• The correlation between Ct value at admission and use of steroid therapy.
• The correlation between Ct value at admission and need for respiratory support.
• The correlation between Ct value at admission and occurrence of MIS-C.

3. Results

3.1. Cohort Characterization

The cohort of our study consists of 124 subjects, selected based on a positive molecular nasopharyngeal swab for SARS-CoV-2 with an available number of amplifications of the N2 gene, and the availability of complete clinical data: 49% of patients (61) were male and 51% (63) were female, aged from 3 days to 15 years, with average age 4.42 years. Overall, from April 2020 to March 2022, in the laboratory of the San Marco University Hospital, 5165 molecular swabs for SARS-CoV-2 were performed, 612 proved to be positive, and in 267 cases, PCR amplification of genes targeting N2 of SARS-CoV-2 was available. Out of these 267 cases, 124 fulfilled inclusion and exclusion criteria. Data collection was achieved through analysis of medical records, focused on the main elements that we defined as the object of study, meaning the number of days with positive swabs, symptoms of COVID-19, and duration of the symptoms. Furthermore, we extrapolated from medical records any available clinical information, including severity at admission, symptoms’ occurrence, medical acts during hospitalization, requirement for respiratory support, and for drugs.

3.2. Duration of Fever, Hospitalization, and Molecular Swab Positivity

In our cohort, fever lasted from 0 to 15 days, with an average of 3.98 days (DS ± 2.77) (Figure 1). Molecular swab positivity persisted in a range from 5 to 42 days, 14.5 days being the average (DS ± 9.45) (Figure 2). Patients stayed in hospital in a period that ranged from 0 to 40 days, with an average of 7–8 days (DS ± 7.04) (Figure 3).

3.3. Clinical Events and Medical Acts

Multisystemic inflammatory syndrome occurred in 10 of our patients (8.1%). It was needed to recourse to respiratory support in 7 patients (5.65%); in particular, in 3 patients, invasive ventilation (IV) was necessary, and in 4 patients, non-invasive ventilation (NIV) was used. Steroid therapy was required in 15 patients (12.1%). Results are illustrated in Figure 4.

3.4. Statistical Correlations

The primary endpoint of our study was to investigate a possible correlation between Ct value at the onset of symptoms, days of hospital stay, and duration of fever. Values of Ct obtained by RT-PCR analysis of nasopharyngeal molecular swabs performed at clinical onset ranged from 13 to 40.8 (average 23.52 ± 6.93 DS). In our cohort, a negative correlation emerged between Ct value at onset and fever duration. Lower Ct values were observed in swabs from patients who underwent more sustained fever (p = 0.012). A tendency to a negative correlation between Ct value at onset and days of hospitalization has also been observed; although in our sample, that correlation did not reach statistical significance (p = 0.25) (Figure 5).

According to secondary endpoints, we analyzed the correlation between Ct value at onset of symptoms and clinical events, in particular occurrence of MIS-C, requirement for respiratory support, and requirement for steroid therapy. Our sample demonstrates the existence of a negative correlation between Ct value at onset and evolution into a multisystemic inflammatory syndrome. Patients with a lower number of cycles of amplification at admission revealed a greater tendency to develop MIS-C (p = 0.026). In Figure 6, a comparison is illustrated between the population of patients who underwent MIS-C and the population of patients who did not, based on the Ct value at clinical onset.

Similarly, patients with lower Ct values at onset proved to be more prone to require respiratory support and/or steroid therapy, enlightening a negative correlation the between number of cycles and the necessity of these medical acts (p = 0.036). Figure 7 and Figure 8 illustrate these findings.

4. Discussion

SARS-CoV-2 infection usually presents in children with milder clinical features and a more favorable prognosis than in adult patients [5]. In a smaller number of cases, infection progresses into lower respiratory tract infection with severe acute symptoms [11,12]. Furthermore, pediatric patients can develop a post-infective multisystemic hyper-inflammatory syndrome (MIS-C) [5]. RT-PCR is widely considered the gold standard for diagnosis of SARS-CoV-2 infection [17]. Real-time PCR cycle threshold value is the number of amplification cycles needed to exceed a threshold level of the target gene, and it is inversely related to viral load [14]. Serial RT-PCR performed on a nasopharyngeal swab of infected patients proved that viral load usually reaches a peak within the first few days after hospital admission [18]. Knowledge about COVID-19 is constantly evolving, and whilst plenty of studies are now available for SARS-CoV-2 in the adult population, fewer have been performed in pediatric patients. Based on results that emerged from the analysis of our sample, a negative correlation between Ct values at onset of symptoms and fever duration seems to exist; therefore, those who undergo longer fever duration presented at the admission time have lower Ct values in the nasopharyngeal swab. An analogous negative correlation between Ct values at the onset time and duration of hospitalization was detected. To date, little correlation between Ct value and timing features has been investigated in other studies; in fact, no data are now available about the correlation between Ct value evaluated during hospital admission with the duration of fever and hospital stay. The association between Ct values and period until test negativity was investigated by Anastasiou et al. for the purpose of optimizing retesting regimens. According to their results, lower initial Ct values are associated with a longer period of RT-PCR positivity, intended as a Ct value < 30. Their cohort included both adult and pediatric patients, and a tendency to have a longer period of positivity has emerged in adults than in children [19]. Moreover, statistical analysis of our cohort showed an inversely proportional correlation between Ct value and requirement for medical acts including steroid therapy and respiratory support, as well as between Ct value and evolution into MIS-C. To date, these correlations have not been described in other analyses. Therefore, our results reveal that RT-PCR Ct value is inversely related to fever duration, duration of hospitalization, occurrence of MIS-C, requirement for steroid therapy, and for respiratory support. These specific correlations have not yet been described in the literature. Meanwhile, according to the updated literature, Ct value is established to be correlated to severity of disease and risk of worsening. Xia et al. in 2020 suggested that lower RT-PCR-Ct values may predict the severity of the disease at an early stage and may also provide information about the infectivity of the patient [17]. Similarly, other studies performed on hospitalized patients reported an association between Ct values or viral load and clinical outcomes, enlightening a more severe course of disease in patients with lower Ct values from respiratory samples [14,18,20,21,22]. Yu et al. proved that patients presenting at admission with mild-to-moderate clinical manifestations demonstrated a higher risk of progression to severe type when their Ct value at admission was significantly lower than average [23]. Ct value and viral load also seem to be related to clinical and biochemical index [24]. In contrast with the above-mentioned results, some authors conclude that Ct value alone does not have a role in severity stratification due to the variability in viral load of variants of SARS-CoV-2 in different waves of the pandemic [25]. To change the clinical course of COVID-19, vaccination plays an important role at all ages and during pregnancy. Indeed, through vaccination it is possible to see a less severe course of the disease with reduced need for intensive care and assistance [26,27,28]. Based on this evidence, the availability of Ct values data from nasopharyngeal molecular swabs may have a useful role to stratify patients at the admission time to predict course of illness, duration of hospitalization, and probability of use of more intensive care. During the same period and on the same sample of patients, other studies were conducted at the San Marco University Hospital, whose results can be usefully integrated with those presented here to draw broader reflections. In one of these studies, the characteristics of patients affected by MIS-C, who represented 8% of the total COVID-19 cases recorded in the period under examination, were examined [29]. It emerged that vaccination of the patients’ parents was negatively correlated with the onset of MIS-C. Only 4% of patients with both parents vaccinated developed the syndrome, while this percentage rose to 20% in patients whose parents were not vaccinated. Furthermore, the study showed that complete vaccination of both parents reduced the probability of the child developing MIS-C by 83%. This protective effect of vaccination aligns with the hypothesis that a reduced viral load in household contacts, measured by PCR, may contribute to mitigate the severity of the infection and its post-infectious complications, such as MIS-C. Consequently, the relationship between vaccination and the severity of cases observed by PCR highlights how the effective management of a pandemic requires an integrated approach. This approach must consider both the accuracy of detection by molecular tests and the importance of preventive measures, such as vaccination, in reducing serious complications associated with the infection. Another study performed at San Marco University Hospital in the same period evaluated pediatric patients affected by respiratory infections, comparing cases infected only with RSV (respiratory syncytial virus) with those co-infected with RSV and other viruses [30]. Viral positivity was detected by a rapid test capable of demonstrating only the presence or absence of the pathogen but not the viral load. The main objective was to determine whether co-infection influenced the severity of the disease, the duration of hospitalization, and the need for ventilation. The results showed that the presence of co-infections did not determine significant differences in the severity of the disease or the need for hospital care compared to patients infected only with RSV. These results contrast with the common hypothesis according to which the presence of multiple pathogens would amplify the clinical severity. This highlights a limitation of the use of non-quantitative tests, which are not able to discriminate the viral load. Indeed, the positivity of the swab did not provide information on the quantity of virus present, making it difficult to establish the actual impact of co-infection and negatively influencing clinical decisions, especially in situations where it is crucial to distinguish between severe and moderate diseases. Therefore, the need for molecular tests capable of detecting not only the presence but also the quantity of the virus emerges, which would significantly improve clinical management and therapeutic decisions, especially in cases of co-infection or suspected severity of the disease.

5. Conclusions

Our study was performed in a small cohort of patients through a retrospective analysis. In addition, the cohort studied was very variable because the study period included vaccinated and non-vaccinated children and both children who required hospitalization and those that did not, including patients of a neonatal age (0–28 days). RT-PCR on a nasopharyngeal swab is to date the gold standard to detect SARS-CoV-2 infection and provides an estimate of viral load, through Ct values. Our study points out a negative correlation between this value and several features, including fever duration, duration of hospitalization, requirement for steroid therapy, requirement for wave, and occurrence of multisystemic inflammatory syndrome, although not all these correlations reached statistical significance on our sample. Therefore, it is necessary to conduct large-scale studies to define the exact clinical role of gene amplification of SARS-CoV-2.