Summary
Introduction: Using the rapid antigen test (RAT) before exhausting the reverse transcriptase-polymerase chain reaction (RT-PCR) test’s capacity is crucial to enhance suitable detection of patients and timely reception of results. Therefore, this study was done to evaluate the sensitivity and specificity of RAT and compare it with the RT-PCR method in the diagnosis of Coronavirus disease-2019 (COVID-19).
Materials and Methods: This study was performed on 634 individuals referred to public sampling centers performing the COVID-19 test in Shiraz City, Fars Province, Southern Iran. The sampling process was done following a multi-stage stratified protocol. The COVITECH® one-step real-time RT-PCR kit method as the reference standard test was compared with the RAT using E-Health Barakat Company® rapid antigen kit in the pharyngeal specimens. The trained personnel collected the data. Sensitivity, specificity, positive predictive value (PPV), negative predictive value, and the
accuracy of the RAT were calculated using the MedCalc software. Moreover, the Kappa value was used to assess the level of agreement between RT-PCR and RAT.
Results: According to the results of the RT-PCR method as a reference test; sensitivity, specificity, Kappa value, and accuracy of the RAT were 81.82%, 92.28%, 63.8%, and 91.01%, respectively in the diagnosis of COVID-19. In asymptomatic individuals, the specificity and the PPV of the RAT were 100%.
Conclusion: In conclusion, our results suggested that a positive RAT provides considerable information for diagnosing COVID-19 in adults. As a result, in general, due to the high sensitivity, specificity, and accuracy of the RAT, it can be replaced with the RT-PCR test considering factors such as time, cost, and speed.
Introduction
Severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2) has recently caused a widespread pandemic with severe consequences for healthcare systems worldwide. In many countries, access to diagnostic tests has faced challenges. Many studies are being conducted to make reliable, inexpensive, and rapid diagnostic tests (RDTs) to detect specific antigens for Coronavirus disease-2019 (COVID-19). Many areas in the United States seek to rapidly expand their testing capabilities for the virus because they believe that diagnostic tests could be a crucial tool in fighting against COVID-19[1, 2]. The reverse transcription-polymerase chain reaction (RT-PCR) test used a RT-PCR to detect viral ribonucleic acid[3]. The RT-PCR test has been recently considered as the benchmark for SARS-CoV-2 detection, but no comprehensive evaluation has been done on the potential of using antibody and antigen testing procedures yet[2].
Rapid diagnostic tests are tests that detect direct antigens of SARS-CoV-2 proteins that are found in respiratory secretions. Despite the availability of RDTs, before their application, they need to be thoroughly assessed and compared in terms of their ability to detect SARS CoV-2 antigens[4, 5]. Rapid antigen testing (RAT) can enhance the overall functional capacity of the COVID-19 testing centers. It offers several advantages, such as the shorter time needed to obtain results and the reduced costs, especially with a limitation on the capacity of the RT-PCR method. Rapid antigen test reduces disease transmission by more efficiently detecting highly infectious cases and enabling more effective contact tracing[6, 7].
So far, COVID-19 has been mostly diagnosed by the RT-PCR method applied on samples from the upper part of the throat[7]. There are various advantages in using RATs compared to RT-PCR for COVID-19 diagnosis. These tests have been designed for both laboratory and clinical use, and their results are usually presented at a low cost within 10-30 min post-analysis. Notably, they have sufficient sensitivity for detecting individuals having high amounts of viral antigens, such as those with symptoms related to the pre-phase of the disease and initial symptoms (until five days after symptom manifestations), and probably greatly contribute to the spread of disease[<6>,
Methods
This study was performed on 634 individuals who are referred to the public sampling centers that perform COVID-19 tests in Shiraz city, Fars province, and southern Iran on December 13-18, 2020. The sampling method was done following a multi-stage stratified protocol. First, 10 public sampling centers that perform both RAT and RT-PCR tests were selected. According to the statistics provided by the Deputy of Development, in the next stage, the share proportionate to each of the centers was calculated. Finally, within those centers, patients and their close contacts were randomly selected. The inclusion criterion was the residence of individuals in Shiraz city. The exclusion criteria included incomplete information and conscious disagreement to participate in the study. The required sample size for this study was calculated by the formula used for determining the sample size in agreement studies[2]. A sample size of 300 people was determined to be appropriate for the study. The sample size was multiplied by 1.5 due to the effect of study design on cluster sampling and stratification; therefore, the final sample size was calculated as 500 people (b=0.20, a=0.01).
This study used a checklist for data collection, which included the following variables: age, sex, contact to any positive cases of COVID-19, pregnancy, travel history in the last two weeks before the positive result, symptoms like fever, sore throat, myalgia, general weakness, diarrhea, nausea, and shortness of breath (dyspnea), and history of underlying diseases including diabetes mellitus, cardiovascular disease, chronic pulmonary disease, hypertension, malignancy, obesity, and smoking status. These variables were assessed using the subgroup analysis. Additionally, the RT-PCR test results were considered as the gold standard and compared with the RAT results. In a center that operates between 8 AM and 10 PM, we conducted PCR and RATs at around 3 PM to 5 PM. The trained personnel collected study data. Quality assurance was performed by the supervision of gathering, extracting, and introducing data into the software and data analysis processes.
Rapid antigen tests were performed on nasopharyngeal specimens by E-Health Barakat Company® rapid antigen kit as a SARS-CoV-2 antigen test, detecting the presence of virus S protein by immunochromatography method[9].
RT-PCR Test
RT-PCR tests were performed on the pharyngeal specimens using COVITECH® one-step real-time RT-PCR kit containing primers and probes of S and E genes for SARS-CoV-2 gene and RNase P detection as the internal control. The resulting cycle threshold values of <35 were considered positive. This test was considered the gold standard for SARS-CoV-2 detection[10].
Statistical Analysis
Descriptive analyses, including frequency and percentage, were used for qualitative variables. According to the RT-PCR outcomes in the role of a standard test, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and precision of fast testing were calculated by the MedCalc software. Moreover, the Kappa value was used to assess the level of agreement between RT-PCR and RAT. A 95% confidence interval (CI) was provided by the Wilson score method. All analyses were performed in the MedCalc software version 11.6.0.0 and Statistical Package for the Social Sciences software version 26.0. P values of <0.05 were considered statistical significance. This study was approved by the Ethics Committee of the Shiraz University of Medical Sciences with the ethics code of IR.SUMS.REC. 1399.1253.
Results
Overall Findings
This study analyzed 634 patients [mean age of 37.98±15.90 years; 61.4% (389/634) were males]. Table 1 shows the demographic information of the participants. Among 634 patients, 77 had positive and 557 had negative RT-PCR results with COVID-19 prevalence of 12.15% (95% CI: 9.70-14.94) (Figure 1). Among 77 patients with positive RT-PCR results, 86.30% (63/77) had positive RAT results. Of 557 individuals with non-positive RT-PCR outcomes, 92.28% (514/557) had negative rapid test results.
Performance of RAT in SARS-CoV-2 Diagnosis
Positive RAT results were determined in 106 patients (<60 years old, n=69; ≥60 years old, n=8; 48 males and 29 females). According to RT-PCR results as the reference test, the sensitivity, specificity, Kappa value, and accuracy of the RAT were equal to 81.82% (95 CI%: 71.38-89.69), 92.28% (95 CI%: 89.74-94.36), 63.8% (95 CI%: 55.2-72.3), and 91.01% (95 CI%: 88.51-93.12) in COVID-19 diagnosis, respectively (Table 2). Patients aged ≥60 years had greater PPV and RAT precision for detecting SARS-CoV-2 compared to individuals aged <60 years. Females showed greater specificity and NPV in the RAT for detecting SARS-CoV-2 than males. For asymptomatic people, the RAT has 100% specificity and 100% PPV, whereas 96.51% specificity and 81.08% PPV in symptomatic individuals. Performance of the RAT in COVID-19 diagnosis in terms of various demographic and clinical characteristics is presented in Table 3. Additionally, Figure 2 shows the receiver operating characteristic curve of the RAT performance in detecting SARS-CoV-2 compared to the RT-PCR as a standard test (area under the curve of 0.87).
red line: Random classifier, black line: better line of RAT for performance for COVID-19 diagnosis compared to the RT-PCR as a reference test, blue line: CI of better line
Discussion
This study compared the RAT with RT-PCR. First, RAT was found to have a great function for clinic-related purposes, with 81.82% sensitivity and 92.28% specificity. Second, based on our findings, its specificity was very high in asymptomatic individuals (100%).
With the spread of SARS-CoV-2, there will be an increasing inconsistency between the count of required testing procedures and the capability of labs or major health care clinics[11]. The RAT is performed on-site and is interpreted by minimally trained health workers and does not require special equipment. It has low cost and fast delivery of outcomes compared to the RT-PCR. The previous reports on RATs for SARS-CoV-2 diagnosis have shown their weak function. Therefore, they are not generally applied[12-15]. Some RATs have been indicated to have remarkably higher reliability[16-19]. Despite the promising use of RAT as a part of a larger strategy to detect and control COVID-19[20], preliminary research is needed to confirm their application in different contexts.
Results of the two recent investigations in Spain that consist of 412 (54 RT-PCR positive cases)[16] and 255 patients (60 RT-PCR positive cases)[17] have reported 79.3% and 76.3% sensitivity, respectively for RAT. However, the second research revealed an 86.5% RAT sensitivity in individuals whose symptoms had been manifested in <7 days[17]. The World Health Organization (WHO) guidelines require the RATs to show a sensitivity of >80% and a specificity of >97% compared to the RT-PCR as a standard test[20]. A study[21] revealed the sensitivity of RAT as remarkably greater in specimens related to patient detection (92.6%) and followed the people in contact with patients (94.2%) than monitoring asymptomatic cases (79.5%). Recently a study[17]revealed a greater sensitivity in symptomatic individuals (85.3%) compared to those asymptomatic (54.5%), which is in line with the recommendation by the WHO in using RATs to screen asymptomatic individuals in communities with a low COVID-19 prevalence because of higher risk of false-positive results[20]. Our study revealed a high specificity in asymptomatic individuals, indicating that the negative results of the RAT test in asymptomatic individuals can be trusted. This could be due to the differences in the type of PCR and RAT performed, as well as differences in the kits used and the time of the two tests.
The function of RATs may be influenced by the epidemiological characteristics of the participants. Thus, epidemiological characteristics in a specific community would determine the type of test application and the procedure of interpreting the outcomes[20]. In communities that face a great disease incidence and number of symptomatic individuals, a positive RAT is considered to confirm the infection; however, if the symptoms are consistent with the disease, non-positive outcome results need more evaluation to confirm ventilatory disease-causing agents, such as the RT-PCR for SARS-CoV-2 detection. In communities that face a low disease incidence and asymptomatic individuals, a non-positive result is acceptable. Otherwise, the result may require RT-PCR test confirmation as it may be incorrect.
Using RATs for diagnosis-related purposes highly decreases the load of testing procedures seen in diagnostic labs. Changes within the major health care units are necessary; however, experiencing overloads of patients for COVID-19 testing and diagnosis to equip them with RAT right at the place. This testing process is conducted in health care centers, thus it simplifies the procedure and provides fast results to the physician and patient, thereby improving the decision-making process and reducing the working pressure imposed on healthcare providers. RAT centers are required to completely follow the proper steps needed for biosecurity considerations.
Our findings have instant implications for clinic settings because they revealed that RATs are a valid diagnostic test for timely diagnosis, monitoring, and rein of SARS-CoV-2 transmission.
Therefore, our results revealed that the RAT offers excellent function in clinic settings as a testing procedure for patient care and it yields greater outcomes in asymptomatic individuals. However, the results should be interpreted in the light of the local epidemiological context. The simplicity and speed offered by RAT and its good clinical performance can help prevent higher working loads imposed on the health care system since it is expected to witness the increased number of individuals with ventilatory diseases in labs in winter.
This study was a historical analysis that focused on the probabilistic experience of diagnostic errors, which was the study’s primary limitation. Additionally, this research has insufficient data on the severity of infections in the participants and measurements of viral load in the samples, such as a period threshold. Probably, various RT-PCR standards were also a limitation of the study. However, they are all used for routine hospital diagnosis and are all validated and widely used worldwide.
Conclusion
Finally, the current study results revealed that the non-negative outcome of RAT would present remarkable data for detecting COVID-19 in adults. A high total consistency was observed between the RAT and RT-PCR test [Kappa=0.638 (95% CI: 0.552-0.723)]. The RAT can be a confirmation that individuals are healthy and that the false-positive value is 0% due to its high specificity, especially in asymptomatic individuals (whose RAT is negative and are not sick). Therefore, in general, the RAT could be replaced with the RT-PCR test considering factors such as time, cost, and speed, due to its high sensitivity, specificity, and accuracy. Therefore, the RAT test is very reliable if it is negative in asymptomatic individuals.
Acknowledgment
The authors would like to thank the staff of the laboratories in the Shiraz University of Medical Sciences, Shiraz City, Fars Province, Iran.
Ethics
Ethics Committee Approval: The study were approved by the Shiraz University of Medical Sciences of Ethics Committee (protocol number: IR.SUMS.REC.1399.1253, date: 24.02.2021).
Informed Consent: Consent form was filled out by all participants.
Peer-review: Externally peer-reviewed.
Authorship Contributions
Concept: A.M., H.G., Design: J.H., Data Collection or Processing: B.S.Y., L.B., Analysis or Interpretation: M.V., Z.M., Literature Search: A.A., Writing: M.V., T.P., H.G.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The present study received financial support from the Shiraz University of Medical Sciences (grant number: 22608).
