One‐step and sequential SARSCOV‐2 polymerase chain reaction tests would not work every time

Abstract Introduction RT‐PCR is widely used as a diagnostic test for the detection of SARS‐CoV‐2. In this study, we aim to describe the clinical utility of serial PCR testing in the final detection of COVID‐19. Method We collected multiple nasopharyngeal swab samples from patients who had negative RT‐PCR test on the first day after hospitalization. RT‐PCR tests were performed on the second day for all patients with initial negative result. For the patients with secondary negative results on day 2, tertiary RT‐PCR tests were performed on day 3 after hospitalization. Result Among 68 patients with initial negative test results, at the end of follow‐up, the mortality number was 20 (29.4%). About 33.8% of patients had subsequent positive PCR test results for the second time and 17.4% of the patients who performed third PCR test had positive result. Conclusion Based on this study, serial RT‐PCR testing is unlikely to yield additional information.

Some clinicians suggested that performing serial RT-PCR tests on suspected cases with initial negative RT-PCR test improves detection capability. To date, there is no recommendation on the efficacy of serial testing in patients with an initial negative PCR. In this study, our aim was to explore the clinical utility of serial PCR testing and the contribution of each RT-PCR test to the final detection of COVID-19.

| Patient population
In this study, confirmed COVID-19 patients with clinical manifestations, positive CT-scan results, and/or consecutive negative RT-PCR tests in a short period of time were included. All the participants were in intensive care unit and the symptoms of COVID-19 were classified as moderate to severe based on the guideline. 7 The study was confirmed by the Ethics in Medical Research Committee IR.SBMU.RETECH.REC.1399.033.

| Sample collection
Nasopharyngeal swab samples were collected on the second day after hospitalization from patients who had negative RT-PCR test on the first day after hospitalization. RT-PCR tests were performed on the second day for all patients with initial negative result. For the patients with secondary negative results on day 2, tertiary RT-PCR tests were performed on day 3 after hospitalization at Loghman Hakim hospital as a major referral center. The personnel collecting the samples and performing tests were the same in each of the tests.
Synthetic fiber swabs on a wire shaft and sterile tubes containing Viral Transport Medium (VTM) were used to collect nasopharyngeal specimens. Prior to specimen collection, tubes were labeled with patients' information. Nasopharyngeal Swabs were collected according to Centers for disease control and prevention (CDC) recommendations (https://www.cdc.gov/coron aviru s/2019-ncov/lab/guide lines -clini cal-speci mens.html). Swabs were placed into VTM and were kept at 2-8°C for less than 24 h.

| RNA extraction and RT-PCR
Extraction procedure was carried out using commercial extraction kit (ROJE) according to manufacturer's instructions. Liferiver realtime multiplex RT-PCR kit was used for qualitative detection of COVID-19. The kit contains super mix, enzyme mix, negative control (NC), positive control (PC), and internal control (IC

| Negative results for SARS-COV-2
Negative signals (Ct > 41) were found in FAM, HEX, and Cal Red 610 channels and positive signal (Ct < 41) was observed in Cy5 channel.

| Inconclusive results
Only one channel from FAM, HEX, and Cal Red 610 was detected with Ct ≤ 41; positive signals were detected in HEX and Cal red 610 and negative signal (Ct > 41) was found in FAM channel. Inconclusive results were repeated to obtain positive or negative results. Therefore, our final results just included positive or negative results.

| Invalid results
Values of Ct > 41 or no value was measured in the fluorescence channels FAM, HEX, and Cal Red and values of Ct ≥ 41 or no value was measured in the channel Cy5. Invalid results were also repeated.

| Statistical analysis
Continuous variables were described using mean, median, and interquartile range (IQR) values and categorical variables were described as frequency rates and percentages, and Chi-square test or Fisher exact test was used to compare the distribution of categorical data including demographic and clinical variables between patients with negative PCR test and positive PCR test. p-Value less than 0.05 was considered as statistically significant. All statistical analyses were performed using R version 3.6.1 software.  Table 1. The most frequent symptom observed among patients at the admission time was myalgia followed by cough and hypoxia, respectively. Hypertension, diabetes, and pulmonary disease were the most common comorbidities in these patients (Table 1).

| DISCUSS ION
In this study, performing serial RT-PCR tests could not significantly increase the detection capability of SARS-COV-2 after initial negative test results. Based on our findings in most cases, repeated RT-PCR tests in short time intervals for patients with initial negative results also remained negative. The lack of promising laboratory tests for timely detection of SARS-COV-2 is tangible. Eliciting false positive results can be seen even by employing various SARS-COV-2 molecular detection kits.
RT-PCR sensitivity is highly affected by improper performing of laboratory practice standards and personal skills. 4 Proper specimen collection is an essential step for virus detection; it minimizes false negative results. Therefore, specimen collection, sample preparation, and experiments should be performed by well-trained staffs. 8 Considering these limitations as an important issue, frequent false negative RT-PCR results are reviewed. In our study, in order to adjust the impact of specimen collection and laboratory practice, the personnel collecting the samples and performing tests were the same in each of the tests since our aim was to evaluate the contribution of each RT-PCR test to the final detection of COVID-19.
High false negative results can be expected because of the emergence of various variants of SARS-COV-2 in each population. 9 Mutations occur frequently in SARS-COV-2, and the RT-PCR method usually detects 2 or 3 genes of SARS-CoV2. 10 Therefore, false negative is a disadvantage of RT-PCR, and even performing serial RT-PCR tests could not improve its detection capability.
Due to some clinical factors, repeated RT-PCR tests are preferred in some cases with suspected COVID-19. The viral load appears to peak approximately 24 h before the onset of symptoms in the upper respiratory tract and then decreases over the next 5 days. 11 The severity of the COVID-19 infection also plays a major role, and severe cases have a higher viral load. 12  The limitation of our study was that we did not use a lower respiratory tract specimen in patients with signs of lower respiratory tract infection. Nevertheless, the decision to repeat testing must be more tactful.

ACK N OWLED G M ENTS
The authors thank the Clinical Research Development Unit (CRDU) of Loghman Hakim Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran for their help and support in conducting this clinical trial.

CO N FLI C T O F I NTE R E S T
We declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.