In December 2019, during the early stages of the coronavirus disease 2019 (COVID-19) outbreak, discharge guidelines for hospitalized patients were not clear, leading to many patients remaining hospitalized until their real-time reverse transcription-polymerase chain reaction (rRT-PCR; hereinafter referred to as PCR) tests returned negative results. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PCR test is crucial for diagnosing COVID-19 [1, 2], monitoring the disease progression [3, 4], and determining whether hospitalized patients can be discharged [5, 6]. However, interpreting the PCR result can be more challenging during follow-up than at the time of diagnosis. During the initial diagnosis, a large amount of virus particles is present in the test specimen, making it easy to determine positive or negative results. However, during follow-up testing, especially at the time of discharge, the virus concentration is low, rendering interpretation of the results difficult.

Since the emergence of COVID-19 in 2019, numerous studies have reported on the diagnosis [7, 8], clinical features [9, 10], and treatment of the disease [11, 12]. Additionally, the implementation and clinical application of the PCR-based SARS-CoV-2 detection assay have been well reported [13-15]. However, few studies have conducted follow-up PCR testing on SARS-CoV-2-positive patients and analyzed the results. Therefore, the aim of our present study was to analyze changes in the follow-up PCR results obtained from the repeated testing of hospitalized COVID-19 patients.

The diagnostic and follow-up data used in this study were from COVID-19 patients in Daegu, the site of the first large-scale infection in Korea. During the study period, COVID-19 diagnosis was conducted using the SARS-CoV-2 rRT-PCR assay, making analysis of the results challenging. There were instances where it was difficult to determine the results at the time of monitoring and discharge. By analyzing the follow-up PCR results of SARS-CoV-2-positive patients in detail, we aim to provide useful information that can be used to improve the diagnosis and monitoring of COVID-19 or any new infectious diseases.

This study was approved by the Institutional Review Board of Daegu Catholic University Medical Center. We analyzed the PCR results of patients diagnosed with COVID-19 at Daegu Catholic University Medical Center from March to May 2020. In total, 84 patients who underwent four or more PCR tests and had their medical records confirmed were selected for the study. The PowerCheck 2019-nCoV Kit (Kogene Biotech, Seoul, Korea) and CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) were used for the SARS-CoV-2 PCR tests, and delta cycle threshold (ΔCt) values and clinical features were retrospectively analyzed.

A Ct value of 39 was used as the cut-off for determining positive and negative PCR results. PCR negativity was defined as the first days when negative results were received twice in a row [16]. If either the E or RdRp gene alone was positive, the results were considered positive when the positive results persisted and negative when the negative results persisted. For patients who tested negative, a subsequent positive and then negative result was determined on the basis of both the E and RdRp genes being positive and negative, respectively. To observe changes in the Ct values (i.e., ΔCt ≥8.0), the results for 20 patients with a PCR Ct value of 30 or less at the time of initial diagnosis were evaluated for changes based on the duration of hospitalization.

Statistical analyses were performed using MedCalc software (version 20.218; MedCalc Software Ltd., Ostend, Belgium). Oneway analysis of variance was used to analyze the differences in ΔCt values among the follow-up PCR subgroups. The Wilcoxon signed-rank test was used to compare the Ct values of the E and RdRp genes. The Mann–Whitney test was used to compare time intervals for negative conversion of the SARS-CoV-2 PCR results. A P-value of less than 0.05 was considered statistically significant.

To address the lack of data on the ideal frequency and duration of PCR testing for COVID-19 patients, we analyzed the follow-up PCR records of 84 SARS-CoV-2-positive patients to evaluate the optimal duration and frequency of repeat testing. Challenges include discrepancies between the PCR results of the two genes tested and difficulties in adhering to the cut-off Ct values for positive results as suggested by manufacturers.

In this study, in which a total of nine tests were performed at intervals of 4.3 days over a period of 38.5 days, the median duration required for negative PCR conversion was 28.0 days. The median duration of SARS-CoV-2 detection in the upper respiratory tract has been reported to be 14.5–34.0 days [16-19]. Generally, the viral load of SARS-CoV-2 is very high on the day of symptom development and then gradually decreases, with the virus typically being undetectable within 3 weeks of symptom onset [16, 17, 20]. The duration of virus detection also depends on the chosen cutoff Ct value [19].

In the ΔCt analysis based on the duration of hospitalization, the viral concentration decreased rapidly until the second week of hospitalization and then decreased gradually thereafter, but it then surprisingly increased again after the fourth week. In clinically stable patients, the increase in viral concentrations after the fourth week does not indicate active viral replication but rather the presence of residual nucleic acid or persistent inactive viral particles. Therefore, repeated PCR testing for SARS-CoV-2 after 4 weeks may not accurately reflect the actual viral load in the patient.

Among the 77 patients who tested negative for SARS-CoV-2, 19 (22.6%) experienced PCR re-positivity (Fig. 1). Moreover, the median duration of the first virus-negative period was significantly longer in the re-positive group than in the consistently negative group (37.0 vs. 27.5 days). Wong et al. [21] reported that 34.3% of 35 patients showed SARS-CoV-2 re-positivity, without COVID-19 symptoms, likely as a result of residual viral nucleic acid. However, Hossain et al. [18] found that two of 12 patients with symptom recurrence also had a re-positive PCR result. Intermittent viral shedding has also been identified as a major cause of PCR re-positivity [22]. Hu et al. [23] reported that delayed clearance of SARSCoV-2 RNA was associated with an older age and chest tightness in hospitalized patients, whereas Abrahim et al. [24] found that patients with comorbidities, such as cancer and HIV infection, had a prolonged viral clearance period. Lim et al. [25] suggested that the duration of viral shedding was longer in individuals with more clinical symptoms, indicating that the time required for negative conversion might be related to the severity of clinical symptoms.

Compared with the 58 consistently virus-negative patients, the 19 re-positive patients were significantly older (median age 61.5 vs. 81.0 years), whereas other clinical symptoms were generally unremarkable. Among the 19 re-positive patients, 12 had CRP levels within the normal range, and two showed slightly increased levels (5.0–12.0 mg/dL). Seventeen of the re-positive patients recovered from COVID-19, whereas the remaining two patients experienced severe respiratory symptoms and died from respiratory failure. The re-positivity in the 17 recovered patients was likely due to residual nucleic acid or intermittent viral shedding. By contrast, the re-positivity in the two deceased patients may have been due to delayed viral clearance or persistent viral presence, indicating the potential ongoing complications of COVID-19.

The Ct values of the RdRp gene were higher than those of the E gene, aligning with the findings from another study that used the same PCR reagent [13]. This discrepancy in Ct values is attributed to variations in PCR conditions rather than differences in target gene concentration. Furthermore, the PCR concordance rate between the two genes was 82.4%, reflecting a mismatch rate of 17.6% (Table 3). Sung et al. [13] reported that in follow-up PCRs of confirmed infected patients, only one of the target genes might test positive and the detected gene may vary.

Of the seven patients who remained persistently virus positive, three experienced symptom improvement, whereas the other four individuals either died or required intensive care. In a study comparing 67 patients, Chang et al. [4] found that patients who tested persistently positive for SARS-CoV-2 had higher incidences of intensive care requirement and pulmonary infiltration. Conversely, Ikegami et al. [20] reported that six out of 272 recovered patients continued to test positive for SARS-CoV-2 in nasopharyngeal swab PCR assays. This indicates that persistent viral detection can occur in both recovered and deteriorating patients. Therefore, the clinical significance of PCR results has to be interpreted in conjunction with CRP levels or ΔCt values.

In summary, it took a median of 28.0 days for 77 patients to test negative in the SARS-CoV-2 PCR assay, with the re-positive group taking a median of 37.0 days to achieve this. An 82.4% concordance rate between the E and RdRp genes was observed. We conclude that for clinically stable patients, continued follow-up PCR testing for up to 4 weeks is advisable to assess negative PCR conversion.