Evaluation of the performance of the COVID-19 rapid antigen tests in a tertiary level hospital in Bangladesh

The tests providing high sensitivity and specificity are essential to identify and manage COVID-19 patients.There is aheavy demand for low-cost rapid antigen tests (RATs) for COVID-19 with a decent diagnostic value. Globally,several RATs for COVID-19 have been developed, but their clinical efficacy has not been well recognized.Thepurpose of the study was to evaluate the performance of RATs. We conducted this prospective observational study atShaheed Suhrawardy Medical College hospital from February 2021 to April 2021 in Dhaka, Bangladesh. This studyincluded the patients admitted in this hospital at the COVID-19 isolation unit or referred from the triage facility of theoutdoor department of this hospital suspected as COVID-19 case. Two nasopharyngeal samples were collectedsimultaneously. We used one sample on the spot for the RATs and the other was sent to the adjacent ShaheedSuhrawardy Medical College COVID-19 RT-PCR laboratory for real-time reverse transcription PCR (RT-PCR). Theperformance of the RATs was evaluated by consuming the real-time RT-PCR results as a reference. In thisinvestigation, SARS-CoV-2 was identified in 84 (30.7%) of the 223 patients who participated. Of these 84 patients, 9(10.7%) were asymptomatic. The overall sensitivity and specificity of RATs were 78.6% and 99.3%, respectively. Thesensitivity was 81.3% in symptomatic cases and 55.6% in asymptomatic cases. There were 18 false-negativespatients,including 3 asymptomatic cases, who had a low viral load (cycle threshold(Ct) > 30). When the Ct value was up to24, the detection rate of RATs was 100%. The detection rate was 42.3% when the Ct was >29. When symptoms beganwithin three days, the detection rate was 92.3%. When the start of symptoms was over 7 days, the detection rate was33.3%. RATs for COVID-19 used in this study delivered an acceptable performance in patients with high viral loadand within the first week of the onset of symptoms. This method can be a supplementary method to RT- PCR for thediagnosis of symptomatic COVID-19 patients.
Bangabandhu Sheikh Mujib Med. Coll. J. 2022;1(2):58-64


Introduction
Since SARS-CoV-2 surfaced in China, it has become a major public health issue worldwide.
There are 233,251,109 confirmed COVID-19 cases detected worldwide with 4,772,794 deaths, and in Bangladesh, there are 1,553,873 cases with 27,470 deaths to date (1). The incubation period of SARS-CoV-2 infection is approximately 2-5 days, and fever, cough, and fatigue are the most common primary symptoms (2). Diagnosis of COVID-19 becomes more complicated by the high overlap between the clinical symptoms of SARS-CoV-2 infection and those of other respiratory diseases and by the asymptomatic carriers (3). Rapid and effective diagnostic methods are required for the isolation and early management of SARS-CoV-2 infected patients (4). The mainstay for identifying infected individuals has been nucleic acid amplification tests (NAATs) for upper respiratory samples (5). The highest viral load is found in the upper respiratory tract early in the infection (6). While these assays are regarded as gold-standard evaluations, their clinical utility has been limited due to limited availability, significant turnaround time, and the requirement of highly trained personnel (7). In outbreak situations, the number of patients eligible for these tests may outnumber the testing capacity.
Several rapid antigen tests (RATs) for COVID-19 are currently available on the market. They could be used to detect acute infection instead of RT-PCR. RATs are less expensive, more accessible point-of-care tests that take less time to obtain findings; as long as they consistently detect SARS-CoV-2, they can be more effective in low-resource situations.
The RATs' accuracy, on the other hand, is questioned (6). The World Health Organization (WHO) issued interim recommendations on RATs for patient management where they must have a minimum of 80% sensitivity and 97-100% specificity as a minimum requirement (8).
They could be used as triage tests to quickly identify people who are very likely to have COVID-19, decreasing or eliminating the need for costly nucleic acid amplification testing. In Bangladesh, most COVID-19 RT-PCR laboratories remain in urban areas. Rural people sometimes don't get a chance to perform RT-PCR tests or get the result timely. It delays the isolation of the patient and contact tracing; as a result, the virus spreads rapidly. Issues also remain regarding reagent supply, cost, and inadequate testing capacity. The government of Bangladesh already sets up RAT facilities for COVID-19 from tertiary to thana level hospitals.
The few field tests of SARS-CoV-2 RATs that have been published previously have yielded conflicting results. The COVID-19 Ag Respi-Strip (Coris Bioconcept, Gembloux, Belgium) showed high specificity (99.5-100%) but low sensitivity (30.2-57.6%) when compared to qRT-PCR in three different studies (9) (10). The BIOCREDIT COVID-19 Ag test (RapiGEN Inc., Gyeonggido, 14119, Korea) also had low sensitivity (11.1-45.7 percent with specimens from the nasopharynx, throat, saliva, and sputum) (11). The authors concluded that this test should only be used to supplement the qRT-PCR test due to the risk of false-negative results. However, two other investigations found that another antigen test, the Fluorescence Immunochromatographic SARS-CoV-2 Antigen Test (Bioeasy Biotechnology Co., Shenzhen, China), performed well. The overall sensitivity and specificity were 93.9 percent and 100 percent, respectively, in a trial of 127 participants in Chile (12), while the accuracy was 96.1 percent. The total sensitivity and specificity were 67.8% and 100%, respectively, in the second investigation of 239 people in China (13). The rapid antigen test can be performed in triage (with later selective testing by qRT-PCR) in scenarios requiring quick isolation of cases, such as symptomatic individuals or "high-risk" truck drivers at border crossings people from abroad in airports or seaports. Because a few antigen false positives are not infected, all individuals classified as COVID-19 positive would require customized isolation until the qRT-PCR results are available and prevent contact with true positives until the qRT-PCR results are available.
Suspected individuals who become negative in the COVID-19 rapid antigen test should be tested sequentially by RT-PCR to rule out infection. In contrast, a positive test should be considered as a true positive for high-risk groups in containment zones or hotspots and healthcare settings". In this study, the performance of several SARS-CoV-2 rapid antigen test kits was evaluated compared to the RT-PCR as the gold standard method.

Study population
This prospective observational study was conducted at Shaheed Suhrawardy Medical College hospital from February 2021 to April 2021 in Dhaka, Bangladesh. This study included the patients admitted in this hospital at the COVID-19 isolation unit or referred from the triage facility of the outdoor department of this hospital suspected as COVID-19 case.

Specimen collection
Paired nasopharyngeal swabs were taken from each patient by trained medical technologists.
One swab sample was used for the rapid antigen test. Another was used for RT-PCR, which was transferred to Shaheed Suhrawardy Medical College COVID-19 RT-PCR laboratory in viral transport media by maintaining the cold chain. A rapid antigen test was performed onsite, and the RT-PCR was done within 24 hours of sample collection for the case of every participant. A trained medical technologist filled a data collection sheet before sample collection.

Rapid antigen test
In this study, there are eight manufacturers' Rapid antigen tests are used (Table 1). Each test was performed according to each manufacturer's information for use (IFU), and the visual interpretation obtained the results within 30 minutes. They are membrane-based immunochromatography assays that detect SARS-CoV-2 nucleocapsid antigen (N-protein) in nasopharyngeal samples over monoclonal antibodies. The manufacturers provide all necessary reagents to perform the assay, and no assay-specific, specialized equipment is needed.
According to the IFU, the assay kits were stable when stored at room temperature.

Results
In total, 223 COVID-19 cases were included in this evaluation. The majority were males (60%), and the overall mean age was 34 years (95% CI: 32-35 years; Table 2). 37% of cases were positive among the total cases. Among them, 64% were male, and 36% were female. Positive cases belong to the 18-64 years age group. Maximum people were symptomatic (89%). All demographic data of the study population are shown in Table 2. Among the symptomatic SARS-CoV-2 RT-PCR positive cases, fever was the most common symptom (90.7%), followed by loss of smell (72.0%) and cough (72.0%). The study subjects were also present with sore throat, breathlessness, fatigue, and diarrhea (  (Figure 1). Sensitivities of rapid antigen tests by the onset of symptoms were different according to time. When symptoms were within three days, the detection rate was 92.3%, 4-7 days. The detection rate was 86.8%. But when symptoms were >7 days, the detection rate was 33.3% ( Figure 2).   Sensitivity, specificity, positive, and negative predictive values are provided with 95% confidence intervals.

Discussion
From the beginning of the COVID-19 pandemic, it appears that the actual number of COVID-19 cases in many countries is much higher than reported due to the limited testing (14), (15).
Though RT-PCR is the gold standard for diagnosing COVID-19, this technique needs biosafety level-2 laboratories with sophisticated analytical instruments and trained staff personnel (16), (17). The low-cost RATs for rapid COVID-19 with good diagnostic performance have become a global healthcare requirement. The rapid diagnostic test performed by a minimally trained healthcare worker close to a patient and outside of central laboratory testing thus widen the testing facility (18).
Laboratory confirmation of COVID-19 using rapid antigen tests depends on the viral load in samples (19), (20), (21). High viral loads (low Ct value) of SARS-CoV-2 is linked with the early respiratory symptomatic stage of COVID-19 (22), (23). SARS-CoV-2 viral RNA can be detected by RT-PCR as late as 83 days after the symptom onset, though detection of viral RNA by RT-PCR is not essentially related to infectiousness (24). When the SARS-CoV-2 virus was cultured from the upper respiratory tract samples PCR positive patients, growth was rarely positive after the ninth day of illness (25). This study revealed similar data which was found in previous studies. The RATs showed 100% sensitivity when the Ct value was <24.
The sensitivity was highest (92.3%) when the onset of symptoms was within three days.
Symptomatic patients showed the highest sensitivity, which was 81.3%. On the other hand, low viral load (high Ct value) was correlated with the negative RATs despite a positive RT-PCR test. Based on these findings, RATs can be used as a screening test for the patients who developed symptoms COVID-19 within 3-5 days. RATs are less effective when patients are in the late stage of the illness, which is consistent with WHO (26). Besides RATs and RT-PCR, both test results could be false negative due to low viral load in nasopharyngeal specimens (26), (25), (27).
Previous studies have shown that, besides the lower sensitivity of RATs compared with PCR, they increase the turnaround time for results, which is vital to disrupt transmission chains to control the spread of this SARS-CoV-2 infection (28), (29), (30). Therefore, several diagnostic algorithms recommend the use of RATs as the first step for symptomatic patients within the first 5-7 days of symptom onset (31), (30), (32). swab. The sensitivity found in this study is much lower than that claimed by the manufacturers.
The overall (both symptomatic and asymptomatic patients) sensitivity was 78.6%, and specificity was 99.3%. The PPV and NPV were 98.5% and 88.5%, respectively, and accuracy was 86.15%. Cohen's kappa value was 0.14, which indicates a poor agreement between the two assays. These data were similar to another recently published study (34

Conclusion
The RATs evaluated in this study showed an acceptable sensitivity in respiratory samples when the patient came in early days of infection or samples with low Ct values related to higher viral loads. Thus, it has the potential to become supplementary to qRT-PCR for the early diagnosis of SARS-CoV-2, especially in situations with inadequate access to molecular methods. But the results obtained from RATs should be confirmed by qRT-PCR.

Consent for publication:
Written informed consent was taken from each participant.

Availability of data and materials:
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.