To the Editor,
Rapid detection and effective isolation of symptomatic patients are essential to reduce the spread of SARS CoV-2 infection. Reverse-transcriptase polymerase chain reaction (RT-PCR), conducted using different specimens, is the diagnostic gold standard for the Coronavirus Disease 2019 (COVID-19) . However, specialized instruments, skilled personnel, and high operating time are required to conduct RT-PCR assays. Rapid antigen tests (RATs) are less laborious, do not require specific and expensive machinery, and improve turnaround times. However, the sensitivity of these tests has demonstrated variability compared with that of several kinds of RT-PCR [2, 3]. Self-collected anterior nasal swab specimens could greatly increase healthcare worker safety, patient comfort, and the number of persons tested . However, SARS-CoV-2 infection diagnosis by nasal collection and by RATs have yet to be fully evaluated. Thus, we determined the performance characteristics of Wondfo 2019-nCoV Antigen Test for detecting SARS-CoV-2 virus in supervised self-collected nasal and healthcare worker-collected nasopharyngeal samples and compared the results with RT-PCR as the standard diagnosis. To our knowledge, this is the first study in Brazil that evaluated self-testing using RATs.
The study was conducted with symptomatic individuals who visited the COVID-19 community testing centers of the Health Department of Campo Belo (Brazil) from April 6 to 16, 2021. A symptomatic individual was defined as a subject who reported having at least one COVID-19 compatible symptom (such as fever, headache, cough, loss of smell or taste), this being the criterion for inclusion in the study. Subjects with more than seven days since symptom onset were not included. This may be a limitation of our study, since we did not evaluate asymptomatic individuals, patients admitted to hospitals, or individuals with symptoms for more than seven days, however, our study objective was to determine the accuracy of RAT compared to RT-PCR for both self-collected nasal and professional-collected nasopharyngeal samples in early symptomatic patients. Under professional supervision, the patient was provided with a nasal swab and with written and verbal instructions on how to self-collect a sample from the right anterior nasal region (instructions were based on the CDC guidelines  for sample collection). Using another nasal swab, the patient was instructed to repeat the same procedure in the left anterior nasal region. These two nasal swabs were used for the RAT and RT-PCR, respectively. Immediately after the self-collection, a healthcare worker extensively trained with mannequins [4, 5] collected two nasopharyngeal swabs (one through the right nostril and another through the left nostril) and these two swabs were also used for the RAT and RT-PCR, respectively. Samples destined for RT-PCR were quickly frozen at −80 °C and transported on dry ice. The Wondfo 2019-nCoV Antigen Test (Guangzhou Wondfo Biotech Co., Ltd., China) was performed according to the manufacturer’s instructions. RT-PCR was conducted within 24 h of specimen collection at the Immunopathology Laboratory of Federal University of Ouro Preto (Brazil). Viral RNA was extracted using PureLink™ RNA Mini Kit (ThermoFisher Scientific, USA). For the detection of SARS-CoV-2, TaqPath™ COVID-19 RT-PCR kit (ThermoFisher Scientific, USA) was used, which has a positive cut-off of cycle threshold (CT) ≤ 37. The data analysis was performed using STATA software v. 14.0 (Stata Corp., USA). Specificity and sensitivity (95% confidence intervals) were calculated using the RT-PCR as a reference test. Sensitivity was evaluated globally and also according to CT (CT≤25, CT≤32, and CT≥32), and according to the time since symptom onset (≤4 days, or ≥5 days). Agreement between techniques was evaluated using Kappa statistic according to the following scale: 0.81–1.0 very good, 0.61–0.80 good, 0.41–0.60 moderate, 0.21–0.40 fair and ≤0.20 poor.
A total of 294 patients were evaluated. The mean time to onset of symptoms was 3.5 (SD 1.8) days. Through nasal collection, 103 (35.0%) cases of COVID-19 were detected by the RAT and 137 (46.6%) by RT-PCR. In nasopharyngeal samples, the RAT was reactive in 116 (39.5%) of the cases and in the RT-PCR the positivity increased to 152 (51.7%) (Figure 1). When evaluating the performance of RAT performed with a nasal swab, using the RT-PCR performed with a sample from the same location as a reference standard, it was observed that the rapid test had a sensitivity of 73.0%, specificity of 98.6%, accuracy of 86.3% and Kappa of 0.72. When the reference standard was nasopharyngeal RT-PCR, the sensitivity of the nasal antigen test decreased to 65.8%, the specificity increased to 99.3% and the accuracy and Kappa agreement decreased to 81.4% and 0.64 respectively. Assessing the performance of the RAT performed with nasopharyngeal swab and using RT-PCR from the same site as reference standard, there is an increase in RAT sensitivity to 75.6%, specificity to 100% and accuracy to 87.0%, with a kappa agreement of 0.74. Using the nasopharyngeal RT-PCR as a standard, there is a significant increase in the area under the Roc curve when we evaluate the performance of the rapid test using a nasopharyngeal sample compared to a nasal sample (Table 1). An agreement of 84.8% and Kappa of 0.70 was also observed between nasal RT-PCR and nasopharyngeal RT-PCR.
|Test evaluated/variables||TP||FP||FN||TN||Sensitivity % (CI 95%)||Specificity % (CI 95%)||Accuracy % (CI 95%)||AUC||Kappa|
|Nasal antigen test||100||2||37||145||73.0 (65.0–79.7)||98.6 (95.2–99.6)||86.3 (81.8–89.8)||0.858||0.72|
|Nasal antigen test||100||1||52||133||65.8 (57.9–72.9)||99.3 (95.9–99.9)||81.4 (76.6–85.5)||0.825||0.64|
|Nasopharyngeal antigen test||115||0||37||134||75.6 (68.3–81.8)||100 (97.2–100)||87.0 (82.7–90.5)||0.878b||0.74|
TP, true positive; FP, false positive; FN, false negative; TN, true negative; AUC, area under curve. aDays since onset of symptoms. bThere is a significant difference in the AUC between the nasal and nasopharyngeal antigen tests when the nasopharyngeal RT-PCR is used as a standard.
In nasal swab samples with CT>32, the sensitivity of the RAT was 4.5% and it increased to 86.1 and 94.1% for samples with CT≤32 and CT≤25, respectively. It was also observed that the test sensitivity for patients with four days or less of symptoms was 70.5% and that this increased to 77.6% when patients had five or more days since onset of symptoms. Using nasopharyngeal samples, the sensitivity of the RAT was 4.0% for samples with CT>32, and 89.8 and 95.5% for CTs less than 32 and 25, respectively. Regarding the time between the onset of symptoms and collection, the sensitivity was 71.0 and 84.6% when this period was less than or equal to four days and greater than or equal to five days respectively (Table 1).
Few studies have assessed the performance of nasal swabs using RATs for detection of SARS-CoV-2 infection compared to RT-PCR [6, 7]. Corroborating with our data, Takeuchi et al.  observed a lower sensitivity in nasal (72.5%) than nasopharyngeal samples (86.7%) using QuickNavi-COVID-19 Ag test (Denka Co., Ltd., Japan). The sensitivity of RAT is largely influenced by the viral load in collected samples. Thus, the lower sensitivity in nasal samples may be related to a lower viral load at this collection site . Although we observed the best performance of the Wondfo 2019-nCoV Antigen Test using professional-collected nasopharyngeal samples having as reference standard the RT-PCR from the same site, self-collected nasal samples reduce the risk of exposure of the healthcare professional to the virus. Besides, this form of collection is less inconvenient for the patient, preventing coughing, sneezing and vomiting.
In most individuals with symptomatic COVID-19 infection, viral RNA measured by RT-PCR becomes detectable as early as day 1 of symptoms and peaks within the first week of symptom onset when the viral load is higher . The sensitivity of Wondfo 2019-nCoV Antigen Test was largely influenced by the viral load in both nasal and nasopharyngeal samples. Although in most studies the RAT sensitivity is greater in individuals with ≤4 days since onset of symptoms, our study showed that sensitivity was greater between 5 and 7 days. Nonetheless, it is known that the pattern of viral load kinetics may vary among populations, sample types, viral strains, and intrinsic characteristics of RATs of each manufacturer, being the viral load peak within the first week of symptom onset . One important strength of our study is that healthcare workers were extensively trained with mannequins before performing nasopharyngeal collections, what is important to avoid low-quality nasopharyngeal samples that may resemble nasal samples [5, 11].
We believe that professional-collected nasopharyngeal RT-PCR is still the best option for diagnosing SARS-CoV-2 infection, however in its unavailability or infeasibility, other tests are also acceptable. Patients who undergo self-collected nasal antigen tests must be aware of the limitations: the diagnosis by RT-PCR and the collection of the nasopharyngeal region demonstrate more sensitive results, therefore better. Nonetheless, nasal self-tests are justified by the ability to screen more patients and enable serial diagnosis. In this sense, the CDC has encouraged the use of nasal self-tests before travel or meetings, even in vaccinated subjects, which is one of the many risk-reduction measures, along with vaccination, masking and physical distancing .
The authors would like to thank the Health Department of Campo Belo for their significant support in the logistics of this study.
Research funding: This study was financially supported by Celer Biotecnologia, a biotechnology company that represents Guangzhou Wondfo Biotech in Brazil. Celer Biotecnologia and Guangzhou Wondfo Biotech suggested the initial design of this study, but they had no role in the conduction of this study or in the preparation, review, or approval of this manuscript.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Competing interests: Authors state no conflict of interest.
Informed consent: Informed consent was obtained from all individuals included in this study.
Ethical approval: The study was approved by the Federal University of Ouro Preto Research Ethics Committee (Protocol Number 44424221.0.0000.5150).
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