1 INTRODUCTION

Currently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The exhibitions of the disease varied from asymptomatic to serious and fatal.¹ Since the introduction of this disease, it has resulted in more than 500 million confirmed cases and more than 6 million cases of morbidity, worldwide.²

Up to now, a few antiviral drugs including Remdesivir, Molnupiravir, and Paxlovid (ritonavir-boosted nirmatrelvir drug) approved by FDA for COVID-19 treatment.³ Furthermore, different variants of the virus are emerging and spreading throughout the world. Hence, the main measure to control the disease globally is to surveil the virus circulation through laboratory testing to reduce the chance of virus transmission by public health approaches such as quarantine and physical distancing.⁴ Besides, the early diagnosis of COVID-19 is critical to improving the survival of the patients.⁵ In fact, laboratory testing is the key to both pandemic and patient management.⁶ To reach a precise and timely COVID-19 diagnosis, laboratory diagnostic techniques especially nucleic acid detection procedures have developed rapidly.⁷ Each testing method has remarkable powers and drawbacks and cautions required when interpreting its results.⁸ Among the lab-based methods, real-time reverse-transcriptase polymerase chain reaction (real-time RT-PCR) is the gold standard diagnostic test.

Most of the available RT-PCR commercial kits target two genes of the SARS-CoV-2 genome. However, sometimes only one target gene is detected by these assays.⁹ RT-PCR method may have occasional false results ⁵ due to inadequate sensitivity and specificity of the test, personnel skills, time and quality of sample collection, and probable variations in the virus genome.⁷ Furthermore, the risk of false results especially false positivity rises as the pretest possibility of SARS-CoV-2 detection declines, for instance when the prevalence is low. It should be noted that like any other real-time RT-PCR tests, when the test result is near the detection limit of the assay the possibility of getting a false positive result is high.⁶ However single target gene positive (STGP) results can be a real marker of COVID-19 when a person is in the convalescent stage or in the incubation period. Both false negative and false positive results have potential consequences. False positive result leads to unnecessary self-isolation which can bring financial, social, and psychological complications, while following false negative result an asymptomatic patient may infect others.¹⁰ In Iran, since the first SARS-CoV-2 detection by National Influenza Center (NIC) laboratory,¹¹ different diagnostic commercial kits from different companies were introduced. Considering the importance of accurate interpretation, COVID-19 laboratory assays and awareness of limitations in available commercial kits, the present study tracked STGP results of real-time RT-PCR tests when it was positive just for N (nucleocapsid protein) or RdRp (RNA-dependent RNA polymerase) to interpret the results clearly.

2 METHODS

3 RESULTS

In this study, using a one-step RT-PCR commercial kit for SARS-CoV-2 detection, among 29,962 clinical samples, there were 1016 (3.39%) samples which appeared to be STGP. Among these samples, 875 (86.12%) were positive only for the N gene with a mean Ct value of 36.6 and 141 (13.88%) just for the RdRp gene with a mean Ct value of 37.5. These specimens were retested using 12.5 μL of each extraction which was 2.5 μL more than the first test according to the kit recommendation. The result of the second real-time PCR reactions showed that of all 1016 retested specimens, 325 (31.99%) samples were positive for both N and RdRp which were reported positive for SARS-CoV-2, 301 (29.65%) were positive only for one gene (N or RdRp) which were considered as suspicious cases and resampling was suggested for them. Finally, 390 (38.385%) samples were negative for both genes which reported as negative cases for SARS-CoV-2.

The results of retesting for those with positive results only for N gene were presented in Table 1 and those which were positive just for RdRp gene was shown in Table 2.

Among 301 individuals who were recommended to send a second sample, only 132 patients (44%) did so. Analysis of the third real-time reactions, revealed 90 (68.18%) cases were negative after 2–5 days of the first sample collection in which the mean Ct value of these cases in the second real-time tests was 37.05. However, 17 (12.88%) cases were still positive just for one gene with high Ct values (mean Ct value of 37.1 in the third reaction compared to 37.2 in the second one), and 25 (18.94%) cases were positive for both genes with the mean Ct value of 31 compared to the 35.5 in the second reaction. The Ct values of almost all samples (except two samples) in the last group decreased when tested on the second samples which were collected during 2–8 days (with the mean of 4.3 days) after the first test. The amount of decline in Ct values ranged from 1 to 20 with the mean of 6.33.

4 DISCUSSION

The result of this cross-sectional study performed on 29,962 respiratory specimens during July and August 2020 by real-time RT-PCR showed that 3.39% of cases (1016 samples) were positive just for one gene of SARS-CoV-2. In fact, of these 1016 cases, 86.12% were positive only for the N gene with a mean Ct value of 36.6 and the rest were positive just for the RdRp gene with a mean Ct value of 37.5. It was defined that in the real-time PCR reaction, the Ct value presented the required number of cycles to amplify a part of the viral genome to be detectable.¹²

In viral genome detection by real-time PCR, divergence of Ct values is dependent on the type of specimen, time and quality of sample collection, its transportation and storage, differences in the sensitivity and specificity of the kits and competency of staff who performed the test.¹³ In most of the research especially on SARS-CoV-2 detection, Ct ≥ 35 in real-time RT-PCR was considered as weak positive.¹³

It has been suggested that positive results of real-time RT-PCR tests that are near the detection limit of the assay have a higher risk of false positivity. A simple explanation is that during the test performance, when a specimen becomes contaminated with a very little amount of test target, it may result in a weak positive with a high Ct value near the detection limit of the assay.⁶ In this study, the quality of sample collection for all specimens was checked through housekeeping gene detection at the same time with SARS-CoV-2 detection. Besides, all samples were transported and stored in cold chain conditions. It should be noted that all assays were performed by qualified technicians who were experts in the real-time assay. However, due to limitations, the same commercial kit was used for all tests. As mentioned, in this study during 2 months, 29,962 SARS-CoV-2 real-time RT-PCR tests were performed, among which 3.39% were STGP. Furthermore, nonspecific signals may also occur during ending amplification cycles that are misinterpreted as the target detection in the specimen. This happening also causes false positivity with high Ct values.⁶

Herein, to clarify whether samples with STGP results, were positive or not, a second reaction was performed on each sample with the mentioned method. This time, roughly 32% of cases were positive and 38% were negative. The second test thoroughly defined the final result of 70% of weakly positive specimens for COVID-19.

To find out the precise result of specimens that even in the second reactions had STGP results, resampling was recommended. Approximately 44% of individuals sent the second sample. Analysis of the third real-time reaction revealed 68.18% of cases were negative after 2–5 days of the first sample collection. The mean Ct value of these cases in the second real-time test was 37.05. Here, we could conclude that this group of individuals might pass the convalescent period and would not spread the virus anymore. However, 12.88% of cases were yet positive just for one gene with high Ct values and 18.94% of cases were positive for both genes with a mean Ct value of 31 compared to the 35.5 in the second reaction. The amount of decline in Ct values ranged from 1 to 20 with a mean of 6.33. It has been observed that the genome of SARS-CoV-2 was detected in people who were in the presymptomatic period.⁶ Some of these individuals who showed Ct decline in real-time RT PCR, have developed COVID-19 symptoms later on. Then it might show that the first sample was in the early stage of the disease.

It is worth noting that, a real-time RT-PCR test only detects viral RNA which is not necessarily correlated to the infectivity of the patient. Lots of patients retain positive in RT-PCR tests for more than 2 weeks.^{7, 9, 14} In fact, a probable correlation between infectious SARS-CoV-2 shedding by a patient and Ct value in real-time RT-PCR is still controversial.^{9, 15} However, this feasible correlation was defined by a French guideline as follows: a Ct value less than 33 as strong probability of shedding, in the range of 33–37 as moderate possibility of shedding and more than 37 as weak chance of shedding.⁹

5 CONCLUSION

In conclusion, tracking STGP results of COVID-19 real-time RT-PCR revealed that most individuals with STGP results would clear the infection completely in less than a week which showed they were in the convalescent phase of infection. However, some of them who were at the beginning of the infection showed a decrease in Ct value for around a week. So they could spread the virus in the society. Regarding the last group, it seemed rational to recommend the individuals who are STGP for SARS-CoV-2 to recheck their test if it is possible or to keep quarantine and social distance.

AUTHOR CONTRIBUTIONS

Nazanin-Zahra Shafiei-Jandaghi: Conceptualization; writing—original draft. Forough Tavakoli: Investigation; methodology; writing—original draft. Jila Yavarian: Methodology; writing—original draft. Vahid Salimi: Methodology; writing—review and editing. Kaveh Sadeghi: Investigation; writing—review and editing. Sevrin Zadheidar: Investigation; writing—review and editing. Ahmad Nejati: Investigation; writing—review and editing. Somayeh Shatizadeh Malekshahi: Writing—review and editing. Talat Mokhtari-Azad: Conceptualization.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ETHICS STATEMENT

This study was approved by the Ethics Committee of Tehran University of Medical Sciences (TUMS). All methods were performed in accordance with the relevant guidelines and regulations. All specimens included in this study were collected by the Iran Ministry of Health. No extra sample was collected for this study. We sought permission from the TUMS review board for using the samples and informed consent is exempted.

TRANSPARENCY STATEMENT

The lead author Talat Mokhtari-Azad affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.