Validation of the STANDARD Q COVID-19 antigen test in Vojvodina, Serbia

Mioljub Ristić; Nataša Nikolić; Velibor Čabarkapa; Vesna Turkulov; Vladimir Petrović

doi:10.1371/journal.pone.0247606

. 2021 Feb 22;16(2):e0247606. doi: 10.1371/journal.pone.0247606

Validation of the STANDARD Q COVID-19 antigen test in Vojvodina, Serbia

Mioljub Ristić ^1,^2,^*, Nataša Nikolić ^2,³, Velibor Čabarkapa ^4,⁵, Vesna Turkulov ^6,⁷, Vladimir Petrović ^1,²

Editor: John Schieffelin⁸

PMCID: PMC7899368 PMID: 33617597

Abstract

Background

Since COVID-19 pandemic is a global crisis, tests with high sensitivity and specificity are crucial for the identification and management of COVID-19 patients. There is an urgent need for low-cost rapid antigen COVID-19 test with a good diagnostic performance. Although various antigen rapid detection tests are widely available, strong evidence of their usefulness in clinical practice are still limited. Therefore, our aim was to evaluate clinical performance of STANDARD Q COVID-19 Ag Test (SD Biosensor, Gyeonggi-do, South Korea).

Methods

The performance of the STANDARD Q COVID-19 Ag Test for the detection of SARS-CoV-2 antigen was evaluated in comparison to RT-qPCR results in 120 symptomatic patients (median age 49, IQR 36–70) who presented to health care facility in Novi Sad, Vojvodina, Serbia.

Results

Twenty five out of 120 samples have been tested positive using STANDARD Q COVID-19 Ag Test, and all of them were also positive on RT-qPCR. Overall, the STANDARD Q COVID-19 Ag Test showed sensitivity of 58.1% (95% CI 42.1–73.0) but it was higher in the early days of disease, when the highest viral loads were detected. During the first five days after the symptom onset, the sensitivity ranged from 66.7% to 100% and the pooled accuracy and Kappa values were high (0.92 and 0.852).

Conclusions

A strong agreement between performance of STANDARD Q COVID-19 Ag Test and RT-qPCR was observed during the first five days of illness, suggesting that this rapid antigenic test can be very useful for COVID-19 diagnosis in the early phase of disease.

Introduction

On January 20th, China’s “National Infectious Diseases Law” was amended to make 2019-novel coronavirus diseases (COVID-19) a Class B notifiable disease and its “Frontier Health and Quarantine Law” was revised to support the COVID-19 outbreak response effort [1]. Shortly thereafter, the COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become an ongoing global health crisis. The severity of COVID-19 symptoms range from very mild to severe pneumonia with multi-organ dysfunction and death [2, 3]. Due to the rapid spread of the SARS-CoV-2, on January 30th, WHO declared COVID-19 outbreak a public health emergency of international concern [3, 4]. As of November 4th, 2020, a total of 47,582,064 cases were confirmed worldwide causing a total 1,217,540 deaths [5].

Diagnostic tests of high sensitivity and specificity are crucial for the identification and management of COVID-19 patients. In particular, high diagnostic accuracy tests applied in the early phase of the illness would enable identification of COVID-19 patients and promptly implementation of control measures in order to reduce household and community transmission [2, 6, 7]. To this end, “point-of-care” or “near patient” antigen and molecular tests for detection of a current SARS-CoV-2 infection have the potential to allow fast laboratory confirmation and timely isolation of COVID-19 cases [6].

The gold standard for laboratory confirmation of SARS-CoV-2 infection is the quantitative real-time reverse transcription polymerase chain reaction test (RT-qPCR) [8, 9], but this technique is expensive and usually takes at least 24 hours to produce the result, making its implementation challenging in many countries [6]. On the other hand, reliable but less expensive and faster diagnostic tests have been developed to specifically detect antigens of SARS-CoV-2 virus. These rapid diagnostic tests, or RDTs, are designed to directly detect SARS-CoV-2 proteins produced by replicating virus in respiratory secretions, and they are developed for both laboratory-based and “near patient”(“while you wait”) use [6, 10]. So far, strong evidence of the usefulness of these tests in clinical practice are still largely lacking [6].

Therefore, the aim of the present study was to evaluate the performance of a STANDARD Q COVID-19 Ag TestSD Biosensor, Gyeonggi-do, South Korea among symptomatic patients in the early and late phases of the disease [6, 11].

The findings of this study provide an insight into the usefulness of this test, with special attention given to the potential (at least partial) replacement of the RT-qPCR test for confirmation of current SARS-CoV-2 infection in the first days of infection.

Materials and methods

Study design

Analysis of prospectively collected data, at the triage ambulance of primary and tertiary outpatients health care facility in Novi Sad (the capital of the autonomous province of Vojvodina, Serbia), was conducted. The outpatients were enrolled by Health Centre Novi Sad (COVID ambulance of primary health care level) and by the ambulance (“red zone”) of Clinical Centre of Vojvodina, Department for Infectious Diseases, from 21st August to 1st September, 2020. The education about adequate sampling and the samples handling procedures of all included physicians and nurses was conducted before the start of research and lasted for 10 days.

Inclusion/Exclusion criteria

We included all symptomatic patients suspected of SARS-CoV-2 infections presenting with one or more of the following signs/symptoms: fever, malaise, cough, sore throat, myalgia, arthralgia, headache, coryza, diarrhoea, nausea, anosmia or ageusia, vomiting), regardless of their age and gender. Asymptomatic patients were excluded from the analysis. Physicians interviewed patients (who met the inclusion criteria) through face -to-face structured interviews on the day of the sampling. The questionnaire was comprised of questions about sociodemographic factors, date of the symptoms onset and the severity (mild or moderate) of clinical signs and symptoms related to SARS-CoV-2 infection.

Specimen collection and laboratory testing

Posterior nasopharyngeal (PNP) swabs were collected. Using STANDARD Q COVID-19 Ag Test, PNP samples were tested immediately after collection, on-site using STANDARD Q COVID-19 Ag Test, by previously trained medical staff, as previously described in study design section. The transport of clinical samples in a hand refrigerator (at +2- +8°C) from the spot of sampling to the laboratory of the Centre for Virusology of Institute of Public Health of Vojvodina, Novi Sad that performed analyses was organized on a daily basis. The samples were accompanied with the previously completed questionnaires.

For RT-qPCR laboratory confirmation, the PNP swabs were transported in sterile commercially available tubes containing a specific viral transport medium with antifungal and antibiotic supplements. Samples were, held refrigerated at 4°C and tested within 12 hours of collection. Before testing, in order to reduce the risk of accidental transmission of SARS-CoV-2 to laboratory staff, the PNP swabs were inactivated by heat in a water-bath, at 56°C for 35 ± 5 minutes [9].

Molecular detection by RT-PCR. Each sample was initially examined for detection of SARS-CoV-2 genes by Argene®, SARS-COV-2 R-GENE® assay (bioMérieux, Marcy-l’Ėtoile, France), after RNA extraction on QIAcube automated workstation (Qiagen, Hilden, Germany). Automated extraction of nucleic acid was done with QIAamp Viral RNA Mini Kit in adapters of up to 12 samples. A two-step approach was used, a qualitative RT-PCR followed by the quantitative one focused on viral loads. Firstly, samples were screened for SARS-CoV-2 RNA by qualitative RT-PCR, targeting three regions that had conserved sequences: the RdRP gene (RNA-dependent RNA polymerase gene) in the open reading frame ORF1ab region, the E gene (envelope protein gene), and the N gene (nucleocapsid protein gene). Reaction, amplification conditions, and results interpretation were performed according to the manufacturer’s instructions. A qualitative real time assay was performed on the Applied Biosystems 7500 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) with software version 2.3. Samples showing an exponential growth curve with any cycle threshold (Ct) value were considered positive.

Secondly, in order to detect viral load, SARS-CoV-2 positive samples were additionally analyzed by quantitative RT-PCR (RT-qPCR). COVID-19 Genesig Real-Time PCR Kit (Primerdesign Ltd, Chandler’s Ford, UK) was utilized for RT-qPCR assay. The experiment was designed according to the manufacturer’s instructions. The target viral gene is the RdRP and the limit of detection (LoD) reported by the manufacturer is 0.58 copies/μL. The reverse transcription and amplification were performed on the Applied Biosystems 7500 Real-Time PCR System with software version 2.3. Quantification of the number of RNA copies was done according to a scale ranging from 2 to 2×10⁵ copies per μL of positive control used as the manufacturer’s standards. The cycle threshold value (Ct) of the positive sample is compared with the standard curve to determine the viral load (viral copy number) in the sample, which was expressed as copy number per μL or mL. The PNP swabs with SARS-CoV-2 RT-qPCR Ct under 41 were considered positive.

STANDARD Q COVID-19 Ag Test

STANDARD Q COVID-19 Ag Test (SD Biosensor, Gyeonggi-do, South Korea) is a chromatographic immunoassay. It allows rapid (in 15–30 minutes) and qualitative detection of SARS-CoV-2 proteins in nasopharyngeal swabs. This test is intended to be used in patients with clinical symptoms of SARS-CoV-2 infection. Test has two pre-coated lines, namely “C” line (Control line), and “T” line (Test line) on the surface of the nitrocellulose membrane. No lines in the result window are visible prior to applying any specimens. This STANDARD Q COVID-19 Ag Test consists of the mouse monoclonal anti-SARS-CoV-2 antibody (coated in the test line region) and the mouse monoclonal anti-Chicken IgY antibody (coated in the control line region). During the test, SARS-CoV-2 antigen in the specimen interacts with monoclonal anti-SARS-CoV-2 antibody conjugated with colour particles, resulting in a visible coloured antigen-antibody complex, without possibility for interaction with any disruptive agents. This complex migrates to the membrane via capillary action towards the test line, where it is captured by the mouse monoclonal anti-SARS-CoV-2 antibody. Consequently, a coloured test line appears in the result window if SARS-CoV-2 antigens are present in the specimen. The intensity of the coloured test line varies depending on the amount of SARS-CoV-2 antigen present in the specimen. If SARS-CoV-2 antigens are not present in the specimen, then no colour will appear in the test line. The control line is used as a quality control and should always appear if the test procedure is performed properly and the test reagents are valid [11].

Data analysis

Parametric and non-parametric, correlative, linear and nonlinear regressive analyses were performed using SPSS software tool (version 22) MedCalc for Windows, version 12.3.0 (MedCalcSoftware, Mariakerke, Belgium). Different statistical analyses were performed for different variables, according to the type of variable: Kruskal-Wallis H test for continuous, non-parametric (ordinal scale) variables, and Fisher’s exacttest or chi-square for categorical data. To determine the predictive validity of the STANDARD Q COVID-19 Ag Test and the level of its agreement with the RT-qPCR test, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), accuracy, Kappa coefficient (Kappa) and their 95% CI were calculated. Results of the STANDARD Q COVID-19 Ag Test were compared to those of RT-qPCR, which was considered the gold standard for this evaluation (positive and negative results obtained by RT-qPCR were considered to be true positive and true negative results, respectively). Test with an accuracy value above 0.71 was deemed useful, and above 0.90 was considered as being of high diagnostic value [12, 13]. Value of Kappa of ≥ 0.600 was defined as moderate level of agreement between the two tests [14]. Statistical significance was set at the value of p<0.05.

Ethical considerations

This quality assurance study was conducted as a part of the daily clinical routine practice. Oral informed consent for the research was obtained from all patients at the moment of sampling that took place in the period from August, 21st to September, 1st, 2020, in accordance with national regulations. The ethical agreement for this research was obtained from the Ethics Committee of the Institute of Public Health of Vojvodina, Novi Sad and from the Ethical Committee of the Faculty of Medicine, University of Novi Sad. No authors of this study were involved in the treatment of the patients included in the analysis, and all data were anonymized before the authors accessed it.

Results

A total of 120 participants were included in the study. The prevalence of various signs and symptoms as well as the details about the time elapsed between disease onset and sample collection, by age of the participants are shown in Figs 1 and 2, respectively. The most common (103 of 120, 85.8%) sign/symptom among all participants was fever (not measured at the spot), while the least frequent one (8 of 120, 6.7%) was vomiting.

Median age of observed patients was 49 years (IQR 36–70), with the youngest participant being 14, and the oldest one 91 years of age. The average period between signs/symptoms onset and swab collection was 9.4 days (ranging between 1 and 45 days) and the median time was 5 days (IQR 3–15). Out of all participants, 52.5% (63/120) cases were tested within the first five days after symptoms onset.

Twenty five out of 120 samples have been tested positive using STANDARD Q COVID-19 Ag Test, and all of them were also positive on RT-qPCR. On the other hand, 35.8% (43/120) were tested positive by RT-qPCR for SARS-CoV-2 infection. Furthermore, we assessed association of RT-qPCR confirmation of SARS-CoV-2 with age, days elapsed from symptom onset, results of rapid test results and the frequency of various signs/symptoms (Table 1). As a result, RT-qPCR confirmation of SARS-CoV-2 in tested respondents was statistically significant (p<0.05) associated with middle age, use of the STANDARD Q COVID-19 Ag Test and certain (cough, malaise, anosmia/ageusia, myalgia, arthralgia and headache) signs/symptoms.

Table 1. Demographic characteristics, STANDARD Q COVID-19 Ag Test and signs/symptoms associated with RT-qPCR laboratory confirmed SARS-CoV-2 infections.

Characteristics		Overall (n = 120)		RT-qPCR positive (n = 43)		RT-qPCR negative (n = 77)		p value ^a
Characteristics		No	%	No	%	No	%
Gender	Male	63	52.5	28	65.1	35	45.5	0.056
Gender	Female	57	47.5	15	34.9	42	54.5	0.056
Age group	14–30	13	10.8	3	7.0	10	13.0	0.040 ^b
	31–64	71	59.2	32	74.4	39	50.6
	≥65	36	30.0	8	18.6	28	36.4
Days from symptom onset	1–5	63	52.5	24	55.8	39	50.6	0.544 ^b
	6–10	20	16.7	9	20.9	11	14.3
	11–15	8	6.7	2	4.7	6	7.8
	≥16	29	24.2	8	18.6	21	27.3
Results of STANDARD Q COVID-19 Ag Test	Positive	25	20.8	25	58.1	0	0.0	<0.001 ^b
Results of STANDARD Q COVID-19 Ag Test	Negative	95	79.2	18	41.9	77	100.0	<0.001 ^b
Signs/symptoms	Fever (not measured)	103	85.8	38	88.4	65	84.4	0.786
	Cough	56	46.7	26	60.5	30	39.0	0.035
	Sore throat	53	44.2	18	41.9	35	45.5	0.848
	Malaise	77	64.2	37	86.0	40	51.9	<0.001
	Coryza	30	25.0	6	14.0	24	31.2	0.048
	Anosmia/Ageusia	20	16.7	14	32.6	6	7.8	<0.001
	Myalgia	46	38.3	25	58.1	21	27.3	0.002
	Arthralgia	35	29.2	20	46.5	15	19.5	0.003
	Diarrhoea	24	20.0	9	20.9	15	19.5	1.000
	Nausea	23	19.2	8	18.6	15	19.5	1.000
	Vomiting	8	6.7	3	7.0	5	6.5	1.000 ^b
	Headache	41	34.2	21	48.8	20	26.0	0.016
	Anxiety	16	13.3	5	11.6	11	14.3	0.785 ^b

Open in a new tab

^a Chi-square tests.

^b Fisher’s Exact test.

Statistically significant differences (p < 0.05) are marked in bold.

We also stratified Se, Sp, PPV, NPV, accuracy and Kappa of the STANDARD Q COVID-19 Ag Test with regard to certain characteristics based on comparison with the laboratory confirmation of SARS-CoV-2 infection by RT-qPCR. The optimum values of 100% of Sp and PPV were observed regarding gender, age group, and days of signs/symptoms onset. Values of accuracy and Kappa were slightly higher in females (0.89 and 0.689) than in males (0.81 and 0.597), as well as in the participants aged 14–30 years (0.92 and 0.755), compared to the other two age groups, namely 31–64 year group (0.85 and 0.677) and the ≥65 year group (0.83 and 0.341). Concerning the time period between the onset of disease and the sampling, we registered the maximum values of observed performance for STANDARD Q COVID-19 Ag Test between the first and fifth day after symptom onset. Although the overall Se for, STANDARD Q COVID-19 Ag Test was 58.1% (95% CI 42.1–73.0), the values of Se were higher during the first five days following signs/symptoms onset (100%, 83.3%, 66.7%, 71.4% and 100%, respectively) than in later days (55.6% for period between 6^th and 10^th day, 50% for 11-15th day period, and 0% for more than 16 days after signs/symptoms onset). In the line with this, the pooled accuracy and Kappa values were higher in the first five days of disease (0.92 and 0.852, respectively) compared to the overall observed period-from 1 to 45 days between signs/symptoms onset and swab collection (0.85 and 0.641, respectively) (Table 2).

Table 2. Validation of the STANDARD Q COVID-19 Ag Test by gender, age groups and days of signs/symptoms onset.

Characteristics		Se ^a % (95% CI)	Sp ^b % (95% CI)	PPV % (95% CI)	NPV % (95% CI)	Accuracy (95% CI)	Kappa (95% CI)
Gender	Male	57.1 (37.2–75.5)	100 (-)	100 (-)	74.5 (65.5–81.7)	0.81 (0.69–0.89)	0.597 (0.409–0.785)
Gender	Female	60.0 (32.3–83.7)	100 (-)	100 (-)	87.5 (79.0–92.9)	0.89 (0.78–0.96)	0.689 (0.465–0.913)
Age group	14–30	66.7 (9.4–99.2)	100 (-)	100 (-)	90.9 (66.9–98.0)	0.92 (0.64–0.99)	0.755 (0.307–1.000)
	31–64	65.6 (46.8–81.4)	100 (-)	100 (-)	78.0 (68.7–85.1)	0.85 (0.74–0.92)	0.677 (0.511–0.843)
	≥65	25.0 (3.2–65.1)	100 (-)	100 (-)	82.4 (75.8–87.4)	0.83 (0.67–0.94)	0.341 (-0.021–0.703)
Day/s of signs/symptoms onset	First	100 (-)	100 (-)	100 (-)	100 (-)	1.00 (-)	1.000 (-)
	Second	83.3 (35.9–99.6)	100 (-)	100 (-)	90.0 (60.1–98.2)	0.93 (0.68–0.99)	0.857 (0.589–1.000)
	Third	66.7 (22.3–95.7)	100 (-)	100 (-)	85.7 (22.3–95.7)	0.89 (0.66–0.99)	0.727 (0.384–1.000)
	Fourth	71.4 (29.0–96.3)	100 (-)	100 (-)	71.4 (43.7–89.0)	0.83 (0.52–0.98)	0.676 (0.288–1.000)
	Fifth	100 (-)	100 (-)	100 (-)	100 (-)	1.00 (-)	1.00 (-)
	Subtotal (1–5)	79.2 (57.9–92.9)	100 (-)	100 (-)	88.6 (78.2–94.5)	0.92 (0.82–0.97)	0.825 (0.680–0.970)
	6–10	55.6 (21.2–86.3)	100 (-)	100 (-)	73.3 (57.0–85.1	0.80 (0.56–0.94)	0.579 (0.244–0.914)
	11–15	50.0 (1.3–98.7)	100 (-)	100 (-)	85.7 (60.0–96.0)	0.88 (0.47–0.99)	0.600 (-0.072–1.000)
	≥16	0 (-)	100 (-)	NA	72.4 (72.4–72.4)	0.72 (52.8–87.3)	NA
Overall		58.1 (42.1–73.0)	100 (-)	100 (-)	81.1 (75.1–85.9)	0.85 (0.78–0.91)	0.641 (0.498–0.783)

Open in a new tab

^a Sensitivity

^b Specificity; NA-not applicable (when the sensitivity is zero).

Comparing the two observed tests, confirmation of COVID-19 using the STANDARD Q COVID-19 Ag Test was significantly associated (0.028) with the shorter period (4.4±2.48) between the disease onset and the day of sampling compared to the RT-qPCR (8.09±61.57). On the other hand, differences between the two tests among laboratory-confirmed cases regarding age and gender were not statistically significant (p>0.05) (Table 3).

Table 3. Comparison between RT-qPCR and STANDARD Q COVID-19 Ag Test laboratory confirmed cases of SARS-CoV-2 infections.

Characteristics		RT-qPCR positive	STANDARD Q COVID-19 Ag Test positive	P value
Age (years)	Mean ± SD	47.11±14.61	44.29±11.57	0.453 ^a
Age (years)	Median (IQR)	48 (35–57)	44 (34–52)
Gender	Male	28	16	0.865 ^b
Gender	Female	15	9	0.865 ^b
Days of symptoms onset to testing (Mean ± SD)		8.09±61.57	4.4±2.48	0.028 ^a

Open in a new tab

^a ANOVA analysis of variance/Kruskal±Wallis H test.

^b Chi-square test.

Statistically significant difference (p < 0.05) is marked in bold.

Finally, we have also compared the rapid Ag test results with the results of the quantitative RT-qPCR. Based on 43 RT-qPCR positive results, a median Cycle threshold (Ct) value was 26.5 (range: 12–41). The 25 concordant positive samples (positive results obtained both with RT-qPCR and STANDARD Q COVID-19 Ag Test) had a median Ct of 25 (range: 12–36), equivalent to a median of 676,851 copies/mL, whereas the median Ct of the 18 discordant samples (positive RT-qPCR with negative Ag rapid test) was 33 (range: 21–41), corresponding to a median of 557 copies/mL. During the first five days of disease, when the STANDARD Q COVID-19 Ag Test had the best performance, median number (676,851 copy number/mL) of copies was 177 time higher than the median number (3833 copy number/mL) after this period of disease (Table 4).

Table 4. Median cycle threshold and quantification of PCR and STANDARD Q COVID-19 Ag Test results.

Characteristics	Median cycle threshold (Ct) value		Quantity (copy number/mL)
Characteristics	Median	Range	Median	Range
Positive PCR (n = 43)	26.5	12–41	110,298	42–163,790,516
Positive PCR and STANDARD Q COVID-19 Ag Test (n = 25)	25.0	12–36	676,851	196–163,790,516
Positive PCR with negative STANDARD Q COVID-19 Ag Test (n = 18)	33.0	21–41	557	42–766,740
First five days post symptom onset	25.0	12–38	676,851	42–163,790,516
≥ 6 days post symptom onset	32.0	21–41	3833	170–766,740

Open in a new tab

Discussion

It seems that the actual number of COVID-19 cases in many countries is much higher than reported due to the limited testing since the beginning of the COVID-19 pandemic [2, 15]. Use of RT-qPCR test kits for laboratory confirmation of SARS-CoV-2 is the gold-standard for the diagnosis of COVID-19. However, this technique requires accredited medical laboratories, with advanced analytical instruments and trained personnel [2, 3, 6–8, 16]. Bearing this in mind, the low-cost rapid antigen COVID-19 test with a good diagnostic performance represents a global healthcare necessity. According to WHO, “The rapid diagnostic test refers to decentralized testing that is performed by a minimally trained healthcare professional near a patient and outside of central laboratory testing” [17].

To the best of our knowledge, this is the first study on the validation of the STANDARD Q COVID-19 Ag Test tests for SARS-CoV-2 infection for the clinical use in Serbia. Additionally, unlike other studies [18–26], this is the first study that provides the evidence of STANDARD Q COVID-19 Ag Test accuracy considering the days from symptom onset among patients who did not require hospitalization.

We determined the diagnostic performance of the STANDARD Q COVID-19 Ag Test for detecting SARS-CoV-2 virus in the upper respiratory tract samples, and compared the results with RT-qPCR test. Only patients with mild or moderate clinical signs and symptoms of COVID-19 were included in the study.

Here we presented evidence that all patients with symptoms related to SARS-CoV-2 infection who had positive STANDARD Q COVID-19 Ag Test were adequately classified as true positive, i.e. there were no respondents with STANDARD Q COVID-19 Ag Test positive who had RT-qPCR negative test. Owning to its Sp and PPV value of 100% and accuracy of 0.85, subjects in our study with a positive STANDARD Q COVID-19 Ag Test could receive immediate care and be isolated while those with STANDARD Q COVID-19 Ag Test negative results needed further RT-qPCR examination. Bearing this in mind, it seems that this STANDARD Q COVID-19 Ag Test has the promising diagnostic capacity for replacement of the RT-qPCR, especially during the outbreak, when there is an increasing incidence trend of COVID-19 cases in the population. This could also accelerate clinical decision making in majority of suspected patients, which is in line with the strategy to stop the current spread of infection in the community. It is worth noticing that validation of this test demonstrated higher diagnostic performance if it was used during the early phase of the illness (preferably in the first five days following symptoms onset) when diagnostic accuracy ranged between 0.83 and 1 (average 0.92, 95% CI 0.82–0.97).

Our results also showed that laboratory confirmation of SARS-CoV-2 using this STANDARD Q COVID-19 Ag Test was slightly more precise in younger compared to the oldest subjects. Yet, this finding must be interpreted with caution, especially considering the time from the symptom onset. Namely, the sensitivity of the STANDARD Q COVID-19 Ag Test in patients aged ≥65 years was only 25%, contrary to 14–30 and 31–64 age groups where it was about 66%. These results indicate that only one out of four elderly patients with signs/symptoms related to COVID-19 had a laboratory confirmation of SARS-CoV-2 infection using the STANDARD Q COVID-19 Ag Test. Given that the highest diagnostic accuracy of the rapid test was observed in the first five days after symptom onset, the observed discrepancy between the age groups of participants could be ascribed to a different average time elapsed between disease onset and testing, as it was 12 days among elderly patients was, compared to only 8 and 6 days in those aged 31–64 and 14–30 years, respectively,.

On the other hand, our findings indicate that the STANDARD Q COVID-19 Ag Test was less sensitive than RT-qPCR. Thus, negative results from the STANDARD Q COVID-19 Ag Test cannot exclude SARS-CoV-2 virus infection confidently and such results should be verified by further RT-qPCR testing. For this reason, we believe that the STANDARD Q COVID-19 Ag Test have the potential to replace the RT-qPCR test, but only to some extent [27]. Nevertheless, it is worth noticing that the average time periods from symptom onset to the testing for RT-qPCR and STANDARD Q COVID-19 Ag Test were 8 and 4 days, respectively.

It has been clearly shown that the probability of laboratory confirmation of COVID-19 using rapid antigen tests depends on the viral load in specimens [7, 22, 27, 28], and that the high loads of SARS-CoV-2 virus correlates with the very early respiratory symptomatic stage of COVID-19 [29, 30]. Although the RT-qPCR for SARS-CoV-2 virus can yield positive results as late as 83 days after symptom onset, detection of viral RNA by RT-qPCR is not necessarily associated to infectiousness [31]. In fact, viral culture from PCR positive upper respiratory tract samples has been rarely positive after the ninth day of illness [31–33]. Consistent with the findings of other authors [34], we here presented strong evidence that the probability of positive STANDARD Q COVID-19 Ag Test matched with the period of the highest viral load obtained by RT-qPCR test. Among STANDARD Q COVID-19 Ag Test positive tests, 71.4% of them had a high viral load (Ct<25). Median Ct for concordant RT-qPCR and STANDARD Q COVID-19 Ag Test positive results was 25, i.e. median copy number/mL was 676,851, which coincided with the results in patients tested within the first five days of illness. In contrast, low viral load (557 copy number/mL) was associated with the negative STANDARD Q COVID-19 Ag Test despite a potentially positive RT-qPCR test. Taking into account all these findings, it seems reasonable to use the STANDARD Q COVID-19 Ag Test at the healthcare settings where patients are admitted in the first five days after the onset of symptoms, and probably also among close contacts of index cases in families and collectives. On the other hand, based on the results of this validation, we believe that this test is not to be recommended for clinical laboratory practice at inpatient facilities where patients are admitted in the late stage of the illness, which is consistent with the recommendation of WHO [10]. Moreover, due to low viral loads, results of nasopharyngeal specimens in those patients could be false negative not only after STANDARD Q COVID-19 Ag Test use, but also after the testing using RT-qPCR [10, 33, 34].

Our study had some limitations. First, this study was performed during the descendent phase of the epidemiological curve in Vojvodina when the prevalence of positivity in the population was ≤5% [35]. We also did not collect specimens from asymptomatic patients. However, considering the performance of the STANDARD Q COVID-19 Ag Test, we believe that this test should not be used in the asymptomatic population (low viral shedding) in a low prevalence setting. Additionally, we believe that further research conducted on a bigger scale, including larger population sample will provide more reliable values of the sensitivity of the STANDARD Q COVID-19 Ag Test. Second, as a result of this study being conducted during late summer in Serbia with a low circulation of other respiratory viruses, it is possible that the obtained performance of the STANDARD Q COVID-19 Ag Test could be different in other periods of the year. Third, although we had organized training among health care providers on adequate way of sampling and the samples handling procedures before the study, it is possible that some of the swabs were inadequately sampled, which could have potentially resulted in false negative results.

In conclusion, results of this study provide the evidence that the STANDARD Q COVID-19 Ag Test with specificity and PPV of 100% represents a promising tool for the rapid diagnostic (“near patient” use) of SARS-CoV-2 infection in the population with high prevalence of COVID-19. The STANDARD Q COVID-19 Ag Test is preferable to be used during the first five days of illness when the accuracy of this test reached at 0.92 and Kappa coefficient showed a strong level (0.825) of agreement [14] with RT-qPCR test. In addition to its high diagnostic accuracy for confirming COVID-19 infection, this easy-to-use antigen SARS-CoV-2 test can be used to test large number of samples in a short period of time. However, due to the possibility of false negative STANDARD Q COVID-19 Ag Test results, we also recommend that the decision on further testing should be based on the clinical presentation of the patient [36] and, if possible, completed by RT-qPCR or IgM serology testing [37].

Acknowledgments

We thank the all health care workers from Health Centre Novi Sad and Clinical Centre of Vojvodina, Department for Infectious Diseases, Novi Sad as well as the clinical laboratory technicians at the Centre for Virusology of Institute of Public Health of Vojvodina, Novi Sad who participated in this project.

A special thanks goes to Dr Jasmina Boban (Department for Radiology, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia), and prof. Miloš Marković (Department of Immunology, Institute of Microbiology and Immunology, Faculty of Medicine University of Belgrade, Belgrade, Serbia) for the revision of the manuscript. We gratefully acknowledge to prof. Edita Stokić for her support during the study.

Data Availability

All relevant data are within the manuscript.

Funding Statement

This work is a part of the research that was supported by Provincial Secretariat for Higher Education and Scientific Research grant number 142-451-3072/2020-03. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Chinese Center for Disease Control and Prevention. The Novel Coronavirus Pneumonia Emergency Response Epidemiology Team. The Epidemiological Characteristics of an Outbreak of 2019 Novel Coronavirus Diseases (COVID-19)—China, 2020. CCDC Weekly/Vol. 2 10.46234/ccdcw2020.097 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Grant MC, Geoghegan L, Arbyn M, Mohammed Z, McGuinness L, Clarke EL, et al. The prevalence of symptoms in 24,410 adults infected by the novel coronavirus (SARS-CoV-2; COVID-19): A systematic review and meta-analysis of 148 studies from 9 countries. PLoS One. 2020;15(6):e0234765 10.1371/journal.pone.0234765 ; PMCID: PMC7310678. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Chan JF, Yuan S, Kok KH, To KK, Chu H, Yang J, et al. A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet. 2020;395(10223):514–523. 10.1016/S0140-6736(20)30154-9 ; PMCID: PMC7159286. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.World Health Organization. Novel coronavirus–China. Geneva, Switzerland: World Health Organization; https://www.who.int/csr/don/12-january-2020-novel-coronavirus-china/en/. [2020-01-12]. [Google Scholar]
5.Center for Systems Science and Engineering. 2020. COVID-19 dashboard by the Center for Systems Science and Engineering at Johns Hopkins University. https://gisanddata.maps.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6. [Accessed 4 November 2020].
6.Dinnes J, Deeks JJ, Adriano A, Berhane S, Davenport C, Dittrich S, et al. (Cochrane COVID-19 Diagnostic Test Accuracy Group). Rapid, point-of-care antigen and molecular-based tests for diagnosis of SARS-CoV-2 infection. Cochrane Database Syst Rev. 2020;8:CD013705 10.1002/14651858.CD013705 . [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Scohy A, Anantharajah A, Bodéus M, Kabamba-Mukadi B, Verroken A, Rodriguez-Villalobos H. Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis. J Clin Virol. 2020;129:104455 10.1016/j.jcv.2020.104455 ; PMCID: PMC7240272. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25(3):2000045 10.2807/1560-7917.ES.2020.25.3.2000045 ; PMCID: PMC6988269. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.World Health Organization. Laboratory testing strategy recommendations for COVID-19: interim guidance. Interim guidance, 21 March (2020).
10.World Health Organization. Antigen-detection in the diagnosis of SARS-CoV-2 infection using rapid immunoassays. Interim guidance, 11 September (2020).
11.Manufacturer’s instruction. SD Biosensor, Inc. Site Map. Available from: http://sdbiosensor.com/xe/product/7672.
12.Swets JA. Measuring the accuracy of diagnostic systems. Science. 1988; 240(4857):1285±93. 10.1126/science.3287615 . [DOI] [PubMed] [Google Scholar]
13.Gutiérrez G, Gresh L, Pérez MÁ, Elizondo D, Avilés W, Kuan G, et al. Evaluation of the diagnostic utility of the traditional and revised WHO dengue case definitions. PLoS Negl Trop Dis. 2013;7(8):e2385 10.1371/journal.pntd.0002385 . [DOI] [PMC free article] [PubMed] [Google Scholar]
14.McHugh ML. Interrater reliability: the kappa statistic. Biochem Med (Zagreb). 2012;22(3):276–82. ; PMCID: PMC3900052. [PMC free article] [PubMed] [Google Scholar]
15.Verity R, Okell LC, Dorigatti I, Winskill P, Whittaker C, Imai N, et al. Estimates of the severity of coronavirus disease 2019: a model-based analysis. Lancet Infect Dis. 2020;20(6):669–677. 10.1016/S1473-3099(20)30243-7 Erratum in: Lancet Infect Dis. 2020: Erratum in: Lancet Infect Dis. 2020: ; PMCID: PMC7158570. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Castro R, Luz PM, Wakimoto MD, Veloso VG, Grinsztejn B, Perazzo H. COVID-19: a meta-analysis of diagnostic test accuracy of commercial assays registered in Brazil. Braz J Infect Dis. 2020;24(2):180–187. 10.1016/j.bjid.2020.04.003 Epub 2020 Apr 18. ; PMCID: PMC7165277. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.World Health Organization. Diagnostic assessment: in vitro diagnostic medical devices (IVDs) used for the detection of high-risk human papillomavirus (HPV) genotypes in cervical cancer screening. Licence: CC BY-NC-SA 3.0 IGO. Available from: https://apps.who.int/iris/handle/10665/272282.
18.Assennato SM, Ritchie AV, Nadala C, Goel N, Tie C, Nadala LM, et al. Performance evaluation of the SAMBA II SARS-CoV-2 Test for point-of-care detection of SARS-CoV-2. J Clin Microbiol. 2020:JCM.01262-20 10.1128/JCM.01262-20 Epub ahead of print. . [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Harrington A, Cox B, Snowdon J, Bakst J, Ley E, Grajales P, et al. Comparison of Abbott ID Now and Abbott m2000 Methods for the Detection of SARS-CoV-2 from Nasopharyngeal and Nasal Swabs from Symptomatic Patients. J Clin Microbiol. 2020;58(8):e00798–20. 10.1128/JCM.00798-20 ; PMCID: PMC7383519. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Hogan CA, Garamani N, Lee AS, Tung JK, Sahoo MK, Huang C, et al. Comparison of the Accula SARS-CoV-2 Test with a Laboratory-Developed Assay for Detection of SARS-CoV-2 RNA in Clinical Nasopharyngeal Specimens. J Clin Microbiol. 2020;58(8):e01072–20. 10.1128/JCM.01072-20 ; PMCID: PMC7383558. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Loeffelholz MJ, Alland D, Butler-Wu SM, Pandey U, Perno CF, Nava A, et al. Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test. J Clin Microbiol. 2020;58(8):e00926–20. 10.1128/JCM.00926-20 ; PMCID: PMC7383535. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Mertens P, De Vos N, Martiny D, Jassoy C, Mirazimi A, Cuypers L, et al. Development and Potential Usefulness of the COVID-19 Ag Respi-Strip Diagnostic Assay in a Pandemic Context. Front Med (Lausanne). 2020;7:225 10.3389/fmed.2020.00225 ; PMCID: PMC7227790. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Rhoads DD, Cherian SS, Roman K, Stempak LM, Schmotzer CL, Sadri N. Comparison of Abbott ID Now, DiaSorin Simplexa, and CDC FDA Emergency Use Authorization Methods for the Detection of SARS-CoV-2 from Nasopharyngeal and Nasal Swabs from Individuals Diagnosed with COVID-19. J Clin Microbiol. 2020;58(8):e00760–20. 10.1128/JCM.00760-20 ; PMCID: PMC7383529. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Smithgall MC, Scherberkova I, Whittier S, Green DA. Comparison of Cepheid Xpert Xpress and Abbott ID Now to Roche cobas for the Rapid Detection of SARS-CoV-2. J Clin Virol. 2020;128:104428 10.1016/j.jcv.2020.104428 Epub 2020 May 13. ; PMCID: PMC7217789. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Wolters F, van de Bovenkamp J, van den Bosch B, van den Brink S, Broeders M, Chung NH, et al. Multi-center evaluation of cepheid xpert® xpress SARS-CoV-2 point-of-care test during the SARS-CoV-2 pandemic. J Clin Virol. 2020;128:104426 10.1016/j.jcv.2020.104426 Epub 2020 May 11. ; PMCID: PMC7211760. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Zhen W, Smith E, Manji R, Schron D, Berry GJ. Clinical Evaluation of Three Sample-to-Answer Platforms for Detection of SARS-CoV-2. J Clin Microbiol. 2020;58(8):e00783–20. 10.1128/JCM.00783-20 ; PMCID: PMC7383520. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Mak GC, Cheng PK, Lau SS, Wong KK, Lau CS, Lam ET, et al. Evaluation of rapid antigen test for detection of SARS-CoV-2 virus. J Clin Virol. 2020;129:104500 10.1016/j.jcv.2020.104500 Epub 2020 Jun 8. ; PMCID: PMC7278630. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Nagura-Ikeda M, Imai K, Tabata S, Miyoshi K, Murahara N, Mizuno T, et al. Clinical Evaluation of Self-Collected Saliva by Quantitative Reverse Transcription-PCR (RT-qPCR), Direct RT-qPCR, Reverse Transcription-Loop-Mediated Isothermal Amplification, and a Rapid Antigen Test To Diagnose COVID-19. J Clin Microbiol. 2020;58(9):e01438–20. 10.1128/JCM.01438-20 ; PMCID: PMC7448663. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Shi Y, Wang Y, Shao C, Huang J, Gan J, Huang X, et al. COVID-19 infection: the perspectives on immune responses. Cell Death Differ. 2020;27(5):1451–1454. 10.1038/s41418-020-0530-3 Epub 2020 Mar 23. ; PMCID: PMC7091918. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Cao B, Wang Y, Wen D, Liu W, Wang J, Fan G, et al. A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19. N Engl J Med. 2020;382(19):1787–1799. 10.1056/NEJMoa2001282 Epub 2020 Mar 18. ; PMCID: PMC7121492. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Cevik M, Kuppalli K, Kindrachuk J, Peiris M. Virology, transmission, and pathogenesis of SARS-CoV-2. BMJ. 2020;371:m3862 10.1136/bmj.m3862 . [DOI] [PubMed] [Google Scholar]
32.Wölfel R, Corman VM, Guggemos W, Seilmaier M, Zange S, Müller MA, et al. Virological assessment of hospitalized patients with COVID-2019. Nature. 2020;581(7809):465–469. 10.1038/s41586-020-2196-x . [DOI] [PubMed] [Google Scholar]
33.Cevik M, Tate M, Lloyd O, Enrico Maraolo A, Schafers J, Ho A. SARS-CoV-2, SARS-CoV-1 and MERS-CoV viral load dynamics, duration of viral shedding and infectiousness: a living systematic review and meta-analysis. Lancet Microbe 2020; (forthcoming). 10.1101/2020.07.25.20162107. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Lan FY, Filler R, Mathew S, Buley J, Iliaki E, Bruno-Murtha LA, et al. COVID-19 symptoms predictive of healthcare workers’ SARS-CoV-2 PCR results. PLoS One. 2020;15(6):e0235460 10.1371/journal.pone.0235460 ; PMCID: PMC7319316. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Institute of Public Health of Vojvodina. Epidemiological report on COVID-19 in Vojvodina. Available from: http://izjzv.org.rs/?lng=lat&cir=&link=3-15-2035 [in Serbian].
36.Caruana G, Croxatto A, Coste AT, Opota O, Lamoth F, Jaton K, et al. Diagnostic strategies for SARS-CoV-2 infection and interpretation of microbiological results. Clin Microbiol Infect. 2020;26(9):1178–82. 10.1016/j.cmi.2020.06.019 ; PMCID: PMC7315992. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Tollånes MC, Bakken Kran AM, Abildsnes E, Jenum PA, Breivik AC, Sandberg S. Evaluation of eleven rapid tests for detection of antibodies against SARS-CoV-2. Clin Chem Lab Med. 2020;58(9):1595–1600. 10.1515/cclm-2020-0628 . [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0247606.r001

Decision Letter 0

John Schieffelin

16 Dec 2020

PONE-D-20-34883

Validation of the COVID-19 Rapid Antigen Test in Vojvodina, Serbia

PLOS ONE

Dear Dr. Ristić,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jan 30 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

John Schieffelin, MD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that patients provided oral consent to have a swab taken. Please state whether the IRB or ethics committee waived the requirement for informed consent to participate in your specific study. If patients provided informed written consent to have data/samples from their medical records used in research, please include this information.

3. In the ethics statement in the manuscript and in the online submission form, please provide additional information about the patient records/samples used in your retrospective study, including the date range (month and year) during which patients' medical records/samples were accessed.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"This work is a part of the research that was supported by Provincial Secretariat for Higher

Education and Scientific Research grant number 142-451-3072/2020-03."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a well presented manuscript. The authors do a good job of addressing study limitations and caveats. One point is that the viral transport medium (VTM) used for qPCR is likely not optimal for performing an antigen test. The authors should specify whether or not the any of the swabs were collected in VTM with guanidine or other disruptive agent as this could decrease antigen detection. The authors should also mention the manufacturer of the test in the abstract as there are several and performance will vary. The sample size is limited, which should also be mentioned in the abstract.

Reviewer #2: General Comments:

The authors evaluated the performance of the STANDARD Q COVID-19 Ag Test among symptomatic patients who presented to health care facilities in Novi Sad, Serbia. Performance of the rapid antigen test was compared with qRT-PCR (the gold-standard diagnostic test recommended by WHO). The authors observed a strong agreement between performance of the rapid antigen test and qRT-PCR.

1. The author acknowledged the importance and novelty of such a validation in their setting and globally; however, it would have been helpful for the authors to set out a well- structured study design, with a well-defined and described study population, and selection criteria. There were major methodological weaknesses making the study design prone to a lot of biases, and hence, interpretation of the results.

2. Overall, there were many grammatical and scientific-writing errors.

3. It would have been helpful for the authors to use the standard manufacturer’s name (STANDARD Q COVID-19 Ag Test) of the rapid antigen test used for this validation.

Specific Comments:

1. Abstract:

a. The result and conclusion could benefit from review of grammatical and scientific-writing errors.

2. Introduction:

a. It would be helpful for the authors to review grammatical and scientific-writing errors.

b. Line 88: It would be helpful for the authors to clearly explain or edit sentence stating “no strong evidence to determine the usefulness of these tests in clinical practice”. What is being referred to as ‘strong evidence’?

c. Lines 93-94: The use of the words “potential partial replacement of qRT-PCR” appears misleading. The relevance of such a statement could be spelt out, otherwise it is debatable. Most studies have shown that the use of COVID-19 rapid antigen tests, as with many other rapid antigen tests is complementary to qRT-PCR.

3. Study design:

a. Lines 97- 99: It would be helpful for the authors to clearly state a specific study design, and to explain what is meant by “retrospective analysis of prospective collected data”. Was it a prospective or retrospective study?

b. It would be helpful for the authors to justify reasons for the short duration of the study which is a possible limitation for the generalization of the study; an inclusion of the COVID-19 prevalence would have been an added advantage in interpreting results from PPV and NPV.

c. Lines 102-104: The relevance of these sentence should be spelt out or reviewed.

d. Lines 106 -107: Inclusion and exclusion criteria should be rigid, specific and clearly stated. Information on these lines appear contrary to Line 100; Including all symptomatic patients could mean both in-patients and outpatients; COVID-19 symptoms are non-specific; hence it would have been helpful for the authors to state the criteria used by these facilities to define who a case suspected of COVID-19 is; and also, using a non-specific age group makes such a study difficult to reproduce.

e. Lines 107-109: The authors alluded that “physicians through face-face structured interviews on the day of admission interviewed all included participants”. It would be helpful for the authors to clarify what they meant by ‘day of admission’, as this sentence appears contradictory to Line 100.

f. Lines 118-119: The authors made mention of “previously trained medical staff”; the text could benefit from an explanation.

g. Lines 120-122: The authors alluded to “available tubes containing a specific viral transport medium with antifungal and antibiotic supplements”; what was the role of the antifungal and antibiotic supplements being referred to here?

h. Lines 122-126: It would be helpful for the authors to cite a reference for the laboratory protocol used.

i. Lines 128-152: Cite reference(s); this text could be shortened.

j. Lines 156-157: Cite reference(s).

4. Data Analysis:

a. Lines 174-176: The relevance of the broad classification of statistical analyses used should be spelt out, otherwise it would be helpful for the authors to indicate specific statistical analyses used.

b. Lines 177-178: The authors should review the scientific-writing of statistical tests. For example, it is commonly written Kruskal Wallis test or Kruskal-Wallis ANOVA rather than ANOVA-Kruskal±Wallis H-test; and Fisher’s Exact test rather than Fisher exact test. It would be helpful for the authors to also indicate that Kruskal-Wallis ANOVA was used for ordinal scale variables.

c. Lines 184-186: It would be helpful for the authors to clarify whether the accuracy and Kappa values used were derived from validation done by the manufacturer.

d. How was sample size determined?

e. Any consideration for missing data?

5. Ethical Considerations:

a. Lines 190-191: Why was verbal informed consent obtained only at the moment of sample collection? It would be helpful for the authors to explain why informed consent was not sought for the overall conduct of the research which also include, but not limited to data and sample collection.

6. Results:

a. It would be helpful for the authors to review the scientific-writing in this section.

b. Line 196: The relevance of the information should be spelt out as this sentence appears contrary to Line 100 stating only outpatients were enrolled in the study.

c. Lines 196-198: The relevance of the information should be spelt out, otherwise its inclusion is questionable.

d. It would be helpful for the authors to explain why fever was not measured.

e. Lines 201: The relevance of the information in line should be spelt out.

f. Lines 203-204: It would be helpful for the authors to explain the relevance of both a mean period of 9.4 days and a median time of 5.

g. Lines 206-207: The relevance of the information in these lines should be spelt out.

h. Lines 225-227: It would be helpful for the authors to explain the relevance of the values in the brackets.

i. What is the overall observed period in Line 229 referring to?

7. Discussion:

a. It would be helpful for the authors to review the grammar and scientific-writing in the section, and by extension interpretation of the results.

b. Line 257: The relevance of the information could be spelt out, otherwise its inclusion in this section is debatable

c. Lines 266-268: These lines appear contradictory to Lines 107-109 and Line 196. Were patients who presented with symptoms requiring admission excluded because they it was an exclusion criterion, or no patient with symptoms needed admission?

d. Lines 271-272: These lines appear contradictory to Line 106 stating all symptomatic patients were included. It would be helpful for the authors to clarify.

e. Cite reference(s) for Lines 304-306.

f. Lines 324: Cite reference stating the prevalence of positivity

g. Was there an explanation for participants presenting to facilities and being tested after ≥ 16 days from onset of signs/symptoms?

8. Tables:

a. Table 1: It would be helpful for the authors to consider using figures for “days from symptoms onset”; for example, 0-5 days for the “first five days”.

b. Line 215: It would be helpful for the authors to differentiate P values derived from Fisher’s Exact test or chi-square test

c. Table 2: It would be helpful for the authors to explain the meaning of NA underneath the table;

d. Table 2: Lines 239-240: The title of the table does not reflect the findings; the authors should consider replacing “Similarities and differences” with “Comparison”.

9. Figures:

a. Figure 2: The authors should consider indicating what unit is the period between symptom onset and laboratory testing on the Y-axis measured in?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Robert J. Samuels

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: Reviewers Comments on the COVID-19 validation paper.docx

Click here for additional data file.^{(19.5KB, docx)}

PLoS One. 2021 Feb 22;16(2):e0247606. doi: 10.1371/journal.pone.0247606.r002

Author response to Decision Letter 0

19 Jan 2021

Response to Reviewers

IMPORTANT!

Comments to the Author are shown in regular fonts, while the comments to the academic Editor are in the italic fonts.

In accordance with your suggestion and the suggestions of the Reviewers, we added:

Fig 2 (We added (days) on the Y-axis as Reviewer #2 suggested).

Thank you very much for those suggestions. As we explained in the Materials and methods section of the paper, our analysis of STANDARD Q COVID-19 Ag Test was performed in the “while you wait” manner - without the need for transportation of the sample to the laboratory. Thus, VTM was not used for testing with STANDARD Q COVID-19 Ag Test. However, according to the suggestion of the Reviewer #1, we added a more precise description of the procedure in the appropriate section of the paper.

In the sample collection and laboratory testing section (STANDARD Q COVID-19 Ag Test subsection) we mentioned that the manufacturer of STANDARD Q COVID-19 Ag Test was SD Biosensor, Gyeonggi-do, South Korea. This information is now also added to the Abstract section as well as the remark on the study sample size.

Reviewer #2:

General Comments:

2. Overall, there were many grammatical and scientific-writing errors.

3. It would have been helpful for the authors to use the standard manufacturer’s name (STANDARD Q COVID-19 Ag Test) of the rapid antigen test used for this validation.

Thank you for these suggestions. We tried to properly correct all the errors you mentioned. Our methodology was similar to other research studies that we listed in the Reference section. In addition, we replaced “AG-RDT”, “COVID-19 Ag rapid diagnostic test” and “Rapid Ag-RDT for COVID-19” with “STANDARD Q COVID-19 Ag Test” throughout the manuscript body. Finally, in order to better clarfy our study design, the study population and selection criteria have been explained in more details in the revised version of the manuscript.

Finally, the copy editing and English language, spelling and grammar check were performed throughout the paper.

We want to highlight that copy editing and English language, spelling and grammar check were performed by Dr Jasmina Boban (Department for Radiology, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia). Dr Jasmina Boban previously wrote the following papers in correct English language (four of all listed articles previously published in the PLoS ONE):

- Performance of the new clinical case definitions of pertussis in pertussis suspected infection and other diagnoses similar to pertussis. PLoS ONE. 2018; 13(9): e0204103. doi: 10.1371/journal.pone.0204103.

- Sero-epidemiological study in prediction of the risk groups for measles outbreaks in Vojvodina, Serbia. PLoS ONE. 2019;14(5): e0216219.

- Declining seroprevalence of hepatitis A in Vojvodina, Serbia. PLoS ONE. 2019;14(6): e0217176.

- Seroepidemiological study of rubella in Vojvodina, Serbia: 24 years after the introduction of the MMR vaccine in the national immunization programme. PLoS ONE. 2020;15(1): e0227413.

- Differentiation of Breast Lesions and Distinguishing Their Histological Subtypes Using Diffusion-Weighted Imaging and ADC Values. FRONTIERS IN ONCOLOGY. 2020.

- Neurometabolic Remodeling in Chronic Hiv Infection: a Five-Year Follow-up Multi-Voxel Mrs Study. SCIENTIFIC REPORTS. 2019.

- Thalamic volume loss as an early sign of amnestic mild cognitive impairment. JOURNAL OF CLINICAL NEUROSCIENCE. 2019; 68:168-173.

- Susceptibility-Weighted MR Imaging Hypointense Rim in Progressive Multifocal Leukoencephalopathy: The End Point of Neuroinflammation and a Potential Outcome Predictor. AMERICAN JOURNAL OF NEURORADIOLOGY. 2019.

- The role of TNF-alpha superfamily members in immunopathogenesis of sepsis. CYTOKINE. 2018; 111:125-130.

- Apparent diffusion coefficient reproducibility in brain tumors measured on 1.5 and 3 T clinical scanners: A pilot study. EUROPEAN JOURNAL OF RADIOLOGY. 2018; 108:249-253.

- Early Introduction of cART Reverses Brain Aging Pattern in Well-Controlled HIV Infection: A Comparative MR Spectroscopy Study. FRONTIERS IN AGING NEUROSCIENCE. 2018.

- Executive Functions Rating Scale and Neurobiochemical Profile in HIV-Positive Individuals. FRONTIERS IN PSYCHOLOGY, (2018).

- Complement component consumption in sepsis correlates better with hemostatic system parameters than with inflammatory biomarkers. THROMBOSIS RESEARCH. 2018; 170:126-132.

-APRIL and sTACI could be predictors of multiorgan dysfunction syndrome in sepsis. Virulence. 2018; 9(1):946-953. doi: 10.1080/21505594.2018.1462636.

-Leptomeningeal form of Immunoglobulin G4-related hypertrophic meningitis with perivascular spread: a case report and review of the literature. Neuroradiology. 2018. doi: 10.1007/s00234-018-2028-y.

-Basal ganglia shrinkage without remarkable hippocampal atrophy in chronic aviremic HIV-positive patients. J Neurovirol. 2018. doi: 10.1007/s13365-018-0635-3.

-A prominent lactate peak as a potential key magnetic resonance spectroscopy (MRS) feature of progressive multifocal leukoencephalopathy (PML): Spectrum pattern observed in three patients. Bosn J Basic Med Sci. 2017; 17(4):349-354. doi: 10.17305/bjbms.2017.2092.

-HIV-associated neurodegeneration and neuroimmunity: multivoxel MR spectroscopy study in drug-naive and treated patients. Eur Radiol. 2017; 27(10):4218-4236. doi: 10.1007/s00330-017-4772-5.

-Proton Chemical Shift Imaging Study of the Combined Antiretroviral Therapy Impact on Neurometabolic Parameters in Chronic HIV Infection. AJNR Am J Neuroradiol. 2017; 38(6):1122-1129. doi: 10.3174/ajnr.A5160.

-Bilateral bloody nipple discharge in a male infant: sonographic findings and proposed diagnostic approach. J Pediatr Endocrinol Metab. 2012; 25(1-2):163-4. doi: 10.1515/jpem-2011-0460.

Specific Comments:

1. Abstract:

a. The result and conclusion could benefit from review of grammatical and scientific-writing errors.

The abstract has been substantially revised, and copy editing and English language, spelling and grammar check were performed

2. Introduction:

a. It would be helpful for the authors to review grammatical and scientific-writing errors.

The introduction section has also been revised and copy editing and English language, spelling and grammar check were performed.

Thank you for this suggestion. We stated that “So far, there is no strong evidence available to determine the usefulness of these tests in clinical practice” reflecting that at the time when the paper was drafted, there was a lack of evidence for the level of clinical performance of this rapid antigen test. Supporting this fact, , Cochrane Database of Systematic Reviews (Dinnes J, Deeks JJ, Adriano A, Berhane S, Davenport C, Dittrich S, et al. (Cochrane COVID-19 Diagnostic Test Accuracy Group). Rapid, point-of-care antigen and molecular-based tests for diagnosis of SARS-CoV-2 infection. Cochrane Database Syst Rev. 2020;8:CD013705. doi: 10.1002/14651858) highlighted that “Antigen tests Sensitivity varied considerably across studies (from 0% to 94%): the average sensitivity was 56.2% (95% CI 29.5 to 79.8%) and average specificity was 99.5% (95% CI 98.1% to 99.9%; based on 8 evaluations in 5 studies on 943 samples). Data for individual antigen tests were limited with no more than two studies for any test.” Additionally, the article: Mak GC, Cheng PK, Lau SS, Wong KK, Lau CS, Lam ET, et al. Evaluation of rapid antigen test for detection of SARS-CoV-2 virus. J Clin Virol. 2020;129:104500. doi: 10.1016/j.jcv.2020.104500. stated: “However, according to WHO, the role of RAD tests for antigen detection for SARS−COV-2 needs to be evaluated and is not recommended for clinical diagnosis”, and “Although our data indicated that RAD test was capable of detecting SARS-CoV-2 virus in NPA & TS, NPS & TS, sputum and throat saliva with different sensitivities, this method was less sensitive than RT-PCR. Consequently, the negative results from this RAD method cannot exclude SARS-CoV-2 virus infection confidently and thus results should be verified by further RT-PCR testing.” The sentence in our paper was based on abovementioned facts.

Although we value this remark, we can not absolutely agree with it. Namely, rapid antigen tests are not absolutely complementary with results of RT-qPCR, as mentioned above in the Cochrane Database of Systematic Reviews. Consistently, our results showed that patients who had positive results obtained on rapid antigen test afterwards presented with positive RT-qPCR test. However, some of the patients with negative rapid antigen test also afterwards presented with positive RT-qPCR. More precisely, during the first five days of illness, around 20% of patients who had a negative rapid antigen test presented with positive RT-qPCR test. Based on this observation, we stated the “potential partial replacement of qRT-PCR”. Again, we lean on the Cochrane Database of Systematic Reviews that highlighted: “Where people are asymptomatic but are being tested on the basis of epidemiological risk factors, such as exposure to someone with confirmed SARS‐CoV‐2, no prior tests will have been conducted.”; “Point‐of‐care tests potentially have a role either as a replacement for RT‐PCR (if sufficiently accurate), or as a means of triaging and rapid management (quarantine or treatment, or both), with confirmatory RT‐PCR testing for negative results”.

3. Study design:

Thank you for this interesting suggestion. Similar to methodology used in previously published paper in the PLoS ONE (Performance of the new clinical case definitions of pertussis in pertussis suspected infection and other diagnoses similar to pertussis. PLoS ONE. 2018; 13(9): e0204103. doi: 10.1371/journal.pone.0204103), the data was collected in the prospective manner, while the analysis was retrospective. However, in order to avoid any possible confusion, the word “retrospective” has been removed in the revised version of the manuscript.

Thank you for this helpful suggestion. However, we explained that the main reason for the short duration of the study was the previous deadline given by the project which we conducted. In addition, in the Limitations, we highlighted that this study was performed when the prevalence of the COVID-19 positive cases was ≥5% and that we are aware that a study with a bigger sample would probably improve the sensitivity of this rapid antigen test.

c. Lines 102-104: The relevance of these sentence should be spelt out or reviewed.

After consultations and literature review, we reviewed the sentence.

Thank you very much for this suggestion. Generally, due to the test characteristics which was not recommended for use in asymptomatic patients, we included only symptomatic patients. Also, we did not include already hospitalized patients, however, there were patients who were tested before admitting to the tertiary health care setting (the ambulance (“red zone”) of Clinical Centre of Vojvodina, Department for Infectious Diseases). In addition, in present version of the paper, we added the signs/symptoms that were inclusion criteria for the testing. At enrolment, we imposed no limits regrading the age of participants. But, after finishing of the study, we stratified participants into three age groups: 14-30, 31-64, and ≥65 years of age. That was the reason for the statement that it was “A retrospective analysis of prospectively collected data”. Finally, after analysis of the data, we realized that most of the participants were aged 31-64 years, while there were no participants younger than 14 years of age.

We added more precise information about the inclusion criteria in the paper and changed “day of admission” into “day of sampling”.

f. Lines 118-119: The authors made mention of “previously trained medical staff”; the text could benefit from an explanation.

Thank you for this suggestion. At the end of section “Study design” we stated “The education about adequate sampling and the samples handling procedures of all included physicians and nurses was conducted before the start of research and lasted for 10 days”. We believe that this explanation is clear and detailed. In addition, we have refered to “study design” subsection in the revised sentence in the present version of the text.

Thank you very much for this. According to the WHO guidance, for transport of samples for viral detection, the use of viral transport medium (VTM) containing antifungal and antibiotic supplements is recommended (ref. 9: World Health Organization. Laboratory testing strategy recommendations for COVID-19: interim guidance. Interim guidance, 21 March (2020)). Antibiotics and antifungals in the VTM reduce the risk of bacterial and fungal contamination during the swab collections as well as to maintain the viability and virulence of collected samples. It is known that bacteria and fungi from the respiratory tract and other sites can disrupt viral particles’ viability and/or degrade DNA and RNA if allowed to proliferate (ref. Bradley Ford, Felix Lam, Johnathan Wilson, Majd Moubarak. Role of Viral Transport Media in Sustaining COVID-19 Testing. Medical Lab Management. October 2020.). If the reviewer feels it is important for the paper, we are ready to include this explanation in the appropriate section.

h. Lines 122-126: It would be helpful for the authors to cite a reference for the laboratory protocol used.

We strongly agree with your suggestion and we added reference. Also, all laboratory procedures were performed according to the manufacturer's instructions.

i. Lines 128-152: Cite reference(s); this text could be shortened.

Thank you for this very useful suggestion. However, the authors feel that it is very important to present these references at one place for the readers. . In this part of the paper, we explained in detail the laboratory procedures in case that other readers/researches compare their results with ours. Nevertheless, if the reviewer insists on shortentning this section, we are ready to move this part to the supplementary material. Other comparable studies also explained laboratory procedures without citation of special reference, and many of them highlighted that procedures were performed according to manufacturer's instructions. Regarding this, we suggest to look for the references 1,7,8,17-19,22,24,26,27,36 (section at the end of the paper before corrections in the paper).

j. Lines 156-157: Cite reference(s).

Please refer to the previous explanation. .

4. Data Analysis:

Thank you for noticing that. In additional text, we clarified which specific statistical analyses were used.

We completely agree with your suggestions and, in accordance with this, we replaced the words.

c. Lines 184-186: It would be helpful for the authors to clarify whether the accuracy and Kappa values used were derived from validation done by the manufacturer.

Thresholds of Accuracy and Kappa values were determined according to the reference standards (ref. 12-14 before corection in the paper). Since the manufacturer did not derive these values, we conducted thevalidation of this test.

d. How was sample size determined?

As we explained in Materials and methods section of the paper, we included the first 120 participants. Consistent with the methodology in many other studies (see reference section of the paper), in this phase of COVID-19 pandemic, it was necessary to both start and finish the research promptly. Taking into account above-mentioned facts, we are aware of these limitations we suggested conduction of a further research with a bigger sample (see Limitation of the paper).

e. Any consideration for missing data?

Taking into account the relatively small set of information (questions about sociodemographic features, as well as of questions related to the date of symptoms onset and questions about all mild or moderate clinical signs and symptoms related to SARS-CoV-2 infection), we are convinced that data obtained from our participants was collected without missing data.

5. Ethical Considerations:

It is a very helpful suggestion. We agree that this section of our paper can potentially confusethe readers. So, we added the explanation according to the Reviewer #2 suggestion.

6. Results:

a. It would be helpful for the authors to review the scientific-writing in this section.

Review of the scientific-writing check was performed and the whole section was revised

b. Line 196: The relevance of the information should be spelt out as this sentence appears contrary to Line 100 stating only outpatients were enrolled in the study.

We agree with this, and we removed confusing part of mentioned sentence.

c. Lines 196-198: The relevance of the information should be spelt out, otherwise its inclusion is questionable.

This is a very interesting suggestion, but we believe that these data provided information about relevant characteristics of included participants regarding their clinical characteristics as well as the timing of swabs taking; therefore, we suggested keeping this in the paper.

d. It would be helpful for the authors to explain why fever was not measured.

Primarily because of the intention for the rapid action (as fast as possible) at the health care settings, we decided to collect only oral information about fever. So, we honestly stated that the measurement of the fever was not performed at the spot. As you can see, the goal of our research was not the validation of sensitivity, specificity or accuracy for the signs/symptoms of the participants but the determination of rapid antigen test performance regarding the age, gender of participants as well as the days from signs/symptoms onset . After all, we believe that the absence of measured fever did not discredit the main results obtained by our research.

e. Lines 201: The relevance of the information in line should be spelt out.

In line 201 we stated “Fig 1. Prevalence of signs and symptoms among participants tested for COVID-19.” according to the proposition of the PLOS One.

f. Lines 203-204: It would be helpful for the authors to explain the relevance of both a mean period of 9.4 days and a median time of 5.

According to the previous good publishing practice in the PLOS One (see: Ristić M, Radosavljević B, Stojanović VD, Đilas M, Petrović V. Performance of the new clinical case definitions of pertussis in pertussis suspected infection and other diagnoses similar to pertussis. PLoS ONE. 2018; 13(9): e0204103; Ristić M, Milošević V, Medić S, Djekić Malbaša J, Rajčević S, Boban J, Petrović V. Sero-epidemiological study in prediction of the risk groups for measles outbreaks in Vojvodina, Serbia. PLoS ONE. 2019;14(5): e0216219; Medić S, Anastassopoulou C, Milošević V, Nataša D, Rajčević S, Ristić M, Petrović V. Declining seroprevalence of hepatitis A in Vojvodina, Serbia. PLoS ONE 2019;14(6): e0217176.), we stated mean and median periods in the Results section. However, in order to avoid potential misunderstanding, we specified mean (average) and median period (in days) between signs/symptom onset and day of swabs taking more precisely.

g. Lines 206-207: The relevance of the information in these lines should be spelt out.

In lines 206-207 we stated “Fig 2. Scatter plot of period between signs and symptoms onset and swab collection for laboratory testing for COVID-19 by age.” according to the proposition of the PLOS One and similar to the Fig 1 in the our previous published research in the PLOS One: Ristić M, Milošević V, Medić S, Djekić Malbaša J, Rajčević S, Boban J, Petrović V. Sero-epidemiological study in prediction of the risk groups for measles outbreaks in Vojvodina, Serbia. PLoS ONE. 2019;14(5): e0216219.

h. Lines 225-227: It would be helpful for the authors to explain the relevance of the values in the brackets.

Thank you for this very interesting suggestion. We corrected this sentence according to the suggestion of the Reviewer #2.

i. What is the overall observed period in Line 229 referring to?

Thank you for this suggestion. We corrected this sentence according to the suggestion of the Reviewer #2.

7. Discussion:

a. It would be helpful for the authors to review the grammar and scientific-writing in the section, and by extension interpretation of the results.

Thank you for this suggestion. The corrections were performed.

b. Line 257: The relevance of the information could be spelt out, otherwise its inclusion in this section is debatable.

This sentence was stated in the relevant literature which we listed.This information was published in PLoS ONE (Grant MC, Geoghegan L, Arbyn M, Mohammed Z, McGuinness L, Clarke EL, et al. The prevalence of symptoms in 24,410 adults infected by the novel coronavirus (SARS-CoV-2; COVID-19): A systematic review and meta-analysis of 148 studies from 9 countries. PLoS One. 2020;15(6):e0234765. doi: 10.1371/journal.pone.0234765. PMID: 32574165; PMCID: PMC7310678.) as a systematic review and meta-analysis of 148 studies from 9 countries (“Since the patients in the included studies are likely to have moderate-severe disease warranting hospitalisation and thus testing, it is likely that we over-estimate the true prevalence of symptoms in the population. Consequently, the use of symptoms alone to screening adults for SARS-CoV-2 infection is likely to miss a substantial number of infected individuals.”). Similar findings were reported by other authors:” Because of a sudden increased demand for confirmatory diagnostic testing, mildly affected and asymptomatic individuals have limited access to laboratory testing. As a result of such circumstances, the number of confirmed SARS-CoV-2 infections can significantly underestimate the actual number of cases” or “Generally, mildly affected or asymptomatic individuals are not screened. As a result, the number of confirmed SARS-CoV-2 infections is largely underestimated.” in: Verity R, Okell LC, Dorigatti I, Winskill P, Whittaker C, Imai N, et al. Estimates of the severity of coronavirus disease 2019: a model-based analysis. Lancet Infect Dis. 2020;20(6):669-677. doi: 10.1016/S1473-3099(20)30243-7. Erratum in: Lancet Infect Dis. 2020: Erratum in: Lancet Infect Dis. 2020: PMID: 32240634; PMCID: PMC7158570. So, we added mentioned references (ref. No 2, and 15) in the reference section.

We agree that these facts may be potentially confusing. Due to the mild or moderate clinical signs and symptoms of COVID-19 disease, none of our patients required hospitalization (as we stated in the Discussion section). Regarding additional text which we put in the Materials and methods section (Inclusion/Exclusion criteria subsection), the first sentence in the Results section was corrected.

d. Lines 271-272: These lines appear contradictory to Line 106 stating all symptomatic patients were included. It would be helpful for the authors to clarify.

Similar to the above explanation, we added data to the Materials and methods section (Inclusion/Exclusion criteria subsection). In our research all symptomatic patients who had one or more of the following signs/symptoms: fever, malaise, cough, sore throat, myalgia, arthralgia, headache, coryza, diarrhoea, nausea, anosmia or ageusia, vomiting were included.

e. Cite reference(s) for Lines 304-306.

We added.

f. Lines 324: Cite reference stating the prevalence of positivity.

We added.

g. Was there an explanation for participants presenting to facilities and being tested after ≥ 16 days from onset of signs/symptoms?

Thank you for this question. No specific explanation exists for this fact, except that we wanted to perform validation of this rapid antigen test among patients in the early (first five days) as well as in the late phase of disease. In this way, we were able to compare performance of this test regarding the different phases of disease. We can confirm that in a total of 29 patients with onset of the symptoms ≥16 prior to swab collection a delayed medical examination was performed.

8. Tables:

a. Table 1: It would be helpful for the authors to consider using figures for “days from symptoms onset”; for example, 0-5 days for the “first five days”.

Thank you for this suggestion. We accepted it and corrected in the Table 1.

b. Line 215: It would be helpful for the authors to differentiate P values derived from Fisher’s Exact test or chi-square test.

The values were derived in accordance with biostatistics proposition (similar statistical consideration was presented in the Table 2 - article previously published in the PLOS ONE: Arau´jo LO, Nunes AMPB, Ferreira VM, Cardoso CW, Feitosa CA, Reis MG, et al. Clinical and epidemiological features of pertussis in Salvador, Brazil, 2011–2016. PLoS ONE. 2020; 15(9): e0238932. https://doi.org/10.1371/journal. pone.0238932

c. Table 2: It would be helpful for the authors to explain the meaning of NA underneath the table.

Thank you for this. We added the explanation what NA means. In brief, when sensitivity is zero, then it is not possible to calculate PPV and Kappa (please look at below listed references:

Altman DG, Machin D, Bryant TN, Gardner MJ (Eds) (2000) Statistics with confidence, 2nd ed. BMJ Books. Gardner IA, Greiner M (2006) Receiver-operating characteristic curves and likelihood ratios: improvements over traditional methods for the evaluation and application of veterinary clinical pathology tests. Veterinary Clinical Pathology 35:8-17.

Griner PF, Mayewski RJ, Mushlin AI, Greenland P (1981) Selection and interpretation of diagnostic tests and procedures. Annals of Internal Medicine 94:555-600.

Hanley JA, McNeil BJ (1982) The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology 143:29-36.

Mercaldo ND, Lau KF, Zhou XH (2007) Confidence intervals for predictive values with an emphasis to case-control studies. Statistics in Medicine 26:2170-2183.

Metz CE (1978) Basic principles of ROC analysis. Seminars in Nuclear Medicine 8:283-298.

Zhou XH, NA Obuchowski, DK McClish (2002) Statistical methods in diagnostic medicine. New York: Wiley.

Zweig MH, Campbell G (1993) Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clinical Chemistry 39:561-577.).

d. Table 2: Lines 239-240: The title of the table does not reflect the findings; the authors should consider replacing “Similarities and differences” with “Comparison”.

Since we feel the suggestion is actually associated with Table 3 (not Table 2), we agreed with this and corrected the name of Table 3.

9. Figures:

a. Figure 2: The authors should consider indicating what unit is the period between symptom onset and laboratory testing on the Y-axis measured in?

Thank you for this suggestion. We added “days” on the Y-axis.

The authors feel that the paper is overall of a better quality after including the well-intended and helpful remarks obtained from the reviewers.

Thank you for the consideration of our manuscript, we look forward to the next step of the paper revisions and hope for the positive outcome.

Sincerely,

Mioljub Ristić, MD, PhD

Centre for Disease Control and Prevention, Institute of Public Health of Vojvodina, Novi Sad, Serbia

Futoška 121, Novi Sad 21 000, Serbia

E-mail: mioljub.ristic@mf.uns.ac.rs

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(40.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0247606.r003

Decision Letter 1

John Schieffelin

10 Feb 2021

Validation of the COVID-19 Rapid Antigen Test in Vojvodina, Serbia

PONE-D-20-34883R1

Dear Dr. Ristić,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

John Schieffelin, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All reviewer comments have been adequately addressed in the revised manuscript in this editor's opinion.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: (No Response)

Reviewer #2: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: General Comments:

The authors evaluated the performance of the STANDARD Q COVID-19 Ag Test (a rapid antigen test) against qRT-PCR (the gold-standard laboratory diagnostic test for SARS-CoV2 as recommended by WHO) among symptomatic patients suspected of Covid-19, who presented to primary and tertiary health care facilities in Novi Sad, Serbia. The authors observed a strong agreement between performance of the rapid antigen test and qRT-PCR.

The authors acknowledged the importance, timing and novelty of such a validation in their setting and globally. Significant changes were made to the manuscript from previous review. However, this manuscript could benefit from further review for scientific-writing errors. Examples include, but not limited to:

1. Repetition of words with resulting long text – see Lines 85,117, 143, 284 etc.

2. Slightly confusing presentation of results and discussion section – See Lines 212 -214, 217 – 220, 238-241, 257 (which of the two statistical tests was used, or both were used and what is the possible explanation for that), 314 – 318 etc.

The study could also benefit from a rigid study design for ease of reproducibility, reduction of bias and generalizability. It would be helpful for the authors to briefly explain how the sample size of 120 was determined. Was this a pre-specified/ determined sample size? The statement in Line 109 should be clarified. Asymptomatic patients were excluded from which “analysis” or were they pre-specified to be excluded from the study as stated in Line 352. Based on what should close contacts (could be asymptomatic or symptomatic) of index cases in families and “collectives” as stated Lines 340- 343 be tested using the RDT, when the study population was only symptomatic patients? It would have been helpful for the authors to also cite reference(s), criteria or case definitions used for the determination of the study population (i.e., patients with mild and moderate signs and symptoms).

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Robert Samuels

Attachment

Submitted filename: General Comments 2nd Review.docx

Click here for additional data file.^{(13KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0247606.r004

Acceptance letter

John Schieffelin

12 Feb 2021

PONE-D-20-34883R1

Validation of the STANDARD Q COVID-19 Antigen Test in Vojvodina, Serbia

Dear Dr. Ristić:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr, John Schieffelin

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Attachment

Submitted filename: Reviewers Comments on the COVID-19 validation paper.docx

Click here for additional data file.^{(19.5KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(40.5KB, docx)}

Attachment

Submitted filename: General Comments 2nd Review.docx

Click here for additional data file.^{(13KB, docx)}

Data Availability Statement

All relevant data are within the manuscript.

[pone.0247606.ref001] 1.Chinese Center for Disease Control and Prevention. The Novel Coronavirus Pneumonia Emergency Response Epidemiology Team. The Epidemiological Characteristics of an Outbreak of 2019 Novel Coronavirus Diseases (COVID-19)—China, 2020. CCDC Weekly/Vol. 2 10.46234/ccdcw2020.097 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref002] 2.Grant MC, Geoghegan L, Arbyn M, Mohammed Z, McGuinness L, Clarke EL, et al. The prevalence of symptoms in 24,410 adults infected by the novel coronavirus (SARS-CoV-2; COVID-19): A systematic review and meta-analysis of 148 studies from 9 countries. PLoS One. 2020;15(6):e0234765 10.1371/journal.pone.0234765 ; PMCID: PMC7310678. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref003] 3.Chan JF, Yuan S, Kok KH, To KK, Chu H, Yang J, et al. A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet. 2020;395(10223):514–523. 10.1016/S0140-6736(20)30154-9 ; PMCID: PMC7159286. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref004] 4.World Health Organization. Novel coronavirus–China. Geneva, Switzerland: World Health Organization; https://www.who.int/csr/don/12-january-2020-novel-coronavirus-china/en/. [2020-01-12]. [Google Scholar]

[pone.0247606.ref005] 5.Center for Systems Science and Engineering. 2020. COVID-19 dashboard by the Center for Systems Science and Engineering at Johns Hopkins University. https://gisanddata.maps.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6. [Accessed 4 November 2020].

[pone.0247606.ref006] 6.Dinnes J, Deeks JJ, Adriano A, Berhane S, Davenport C, Dittrich S, et al. (Cochrane COVID-19 Diagnostic Test Accuracy Group). Rapid, point-of-care antigen and molecular-based tests for diagnosis of SARS-CoV-2 infection. Cochrane Database Syst Rev. 2020;8:CD013705 10.1002/14651858.CD013705 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref007] 7.Scohy A, Anantharajah A, Bodéus M, Kabamba-Mukadi B, Verroken A, Rodriguez-Villalobos H. Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis. J Clin Virol. 2020;129:104455 10.1016/j.jcv.2020.104455 ; PMCID: PMC7240272. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref008] 8.Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25(3):2000045 10.2807/1560-7917.ES.2020.25.3.2000045 ; PMCID: PMC6988269. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref009] 9.World Health Organization. Laboratory testing strategy recommendations for COVID-19: interim guidance. Interim guidance, 21 March (2020).

[pone.0247606.ref010] 10.World Health Organization. Antigen-detection in the diagnosis of SARS-CoV-2 infection using rapid immunoassays. Interim guidance, 11 September (2020).

[pone.0247606.ref011] 11.Manufacturer’s instruction. SD Biosensor, Inc. Site Map. Available from: http://sdbiosensor.com/xe/product/7672.

[pone.0247606.ref012] 12.Swets JA. Measuring the accuracy of diagnostic systems. Science. 1988; 240(4857):1285±93. 10.1126/science.3287615 . [DOI] [PubMed] [Google Scholar]

[pone.0247606.ref013] 13.Gutiérrez G, Gresh L, Pérez MÁ, Elizondo D, Avilés W, Kuan G, et al. Evaluation of the diagnostic utility of the traditional and revised WHO dengue case definitions. PLoS Negl Trop Dis. 2013;7(8):e2385 10.1371/journal.pntd.0002385 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref014] 14.McHugh ML. Interrater reliability: the kappa statistic. Biochem Med (Zagreb). 2012;22(3):276–82. ; PMCID: PMC3900052. [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref015] 15.Verity R, Okell LC, Dorigatti I, Winskill P, Whittaker C, Imai N, et al. Estimates of the severity of coronavirus disease 2019: a model-based analysis. Lancet Infect Dis. 2020;20(6):669–677. 10.1016/S1473-3099(20)30243-7 Erratum in: Lancet Infect Dis. 2020: Erratum in: Lancet Infect Dis. 2020: ; PMCID: PMC7158570. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref016] 16.Castro R, Luz PM, Wakimoto MD, Veloso VG, Grinsztejn B, Perazzo H. COVID-19: a meta-analysis of diagnostic test accuracy of commercial assays registered in Brazil. Braz J Infect Dis. 2020;24(2):180–187. 10.1016/j.bjid.2020.04.003 Epub 2020 Apr 18. ; PMCID: PMC7165277. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref017] 17.World Health Organization. Diagnostic assessment: in vitro diagnostic medical devices (IVDs) used for the detection of high-risk human papillomavirus (HPV) genotypes in cervical cancer screening. Licence: CC BY-NC-SA 3.0 IGO. Available from: https://apps.who.int/iris/handle/10665/272282.

[pone.0247606.ref018] 18.Assennato SM, Ritchie AV, Nadala C, Goel N, Tie C, Nadala LM, et al. Performance evaluation of the SAMBA II SARS-CoV-2 Test for point-of-care detection of SARS-CoV-2. J Clin Microbiol. 2020:JCM.01262-20 10.1128/JCM.01262-20 Epub ahead of print. . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref019] 19.Harrington A, Cox B, Snowdon J, Bakst J, Ley E, Grajales P, et al. Comparison of Abbott ID Now and Abbott m2000 Methods for the Detection of SARS-CoV-2 from Nasopharyngeal and Nasal Swabs from Symptomatic Patients. J Clin Microbiol. 2020;58(8):e00798–20. 10.1128/JCM.00798-20 ; PMCID: PMC7383519. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref020] 20.Hogan CA, Garamani N, Lee AS, Tung JK, Sahoo MK, Huang C, et al. Comparison of the Accula SARS-CoV-2 Test with a Laboratory-Developed Assay for Detection of SARS-CoV-2 RNA in Clinical Nasopharyngeal Specimens. J Clin Microbiol. 2020;58(8):e01072–20. 10.1128/JCM.01072-20 ; PMCID: PMC7383558. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref021] 21.Loeffelholz MJ, Alland D, Butler-Wu SM, Pandey U, Perno CF, Nava A, et al. Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test. J Clin Microbiol. 2020;58(8):e00926–20. 10.1128/JCM.00926-20 ; PMCID: PMC7383535. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref022] 22.Mertens P, De Vos N, Martiny D, Jassoy C, Mirazimi A, Cuypers L, et al. Development and Potential Usefulness of the COVID-19 Ag Respi-Strip Diagnostic Assay in a Pandemic Context. Front Med (Lausanne). 2020;7:225 10.3389/fmed.2020.00225 ; PMCID: PMC7227790. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref023] 23.Rhoads DD, Cherian SS, Roman K, Stempak LM, Schmotzer CL, Sadri N. Comparison of Abbott ID Now, DiaSorin Simplexa, and CDC FDA Emergency Use Authorization Methods for the Detection of SARS-CoV-2 from Nasopharyngeal and Nasal Swabs from Individuals Diagnosed with COVID-19. J Clin Microbiol. 2020;58(8):e00760–20. 10.1128/JCM.00760-20 ; PMCID: PMC7383529. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref024] 24.Smithgall MC, Scherberkova I, Whittier S, Green DA. Comparison of Cepheid Xpert Xpress and Abbott ID Now to Roche cobas for the Rapid Detection of SARS-CoV-2. J Clin Virol. 2020;128:104428 10.1016/j.jcv.2020.104428 Epub 2020 May 13. ; PMCID: PMC7217789. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref025] 25.Wolters F, van de Bovenkamp J, van den Bosch B, van den Brink S, Broeders M, Chung NH, et al. Multi-center evaluation of cepheid xpert® xpress SARS-CoV-2 point-of-care test during the SARS-CoV-2 pandemic. J Clin Virol. 2020;128:104426 10.1016/j.jcv.2020.104426 Epub 2020 May 11. ; PMCID: PMC7211760. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref026] 26.Zhen W, Smith E, Manji R, Schron D, Berry GJ. Clinical Evaluation of Three Sample-to-Answer Platforms for Detection of SARS-CoV-2. J Clin Microbiol. 2020;58(8):e00783–20. 10.1128/JCM.00783-20 ; PMCID: PMC7383520. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref027] 27.Mak GC, Cheng PK, Lau SS, Wong KK, Lau CS, Lam ET, et al. Evaluation of rapid antigen test for detection of SARS-CoV-2 virus. J Clin Virol. 2020;129:104500 10.1016/j.jcv.2020.104500 Epub 2020 Jun 8. ; PMCID: PMC7278630. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref028] 28.Nagura-Ikeda M, Imai K, Tabata S, Miyoshi K, Murahara N, Mizuno T, et al. Clinical Evaluation of Self-Collected Saliva by Quantitative Reverse Transcription-PCR (RT-qPCR), Direct RT-qPCR, Reverse Transcription-Loop-Mediated Isothermal Amplification, and a Rapid Antigen Test To Diagnose COVID-19. J Clin Microbiol. 2020;58(9):e01438–20. 10.1128/JCM.01438-20 ; PMCID: PMC7448663. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref029] 29.Shi Y, Wang Y, Shao C, Huang J, Gan J, Huang X, et al. COVID-19 infection: the perspectives on immune responses. Cell Death Differ. 2020;27(5):1451–1454. 10.1038/s41418-020-0530-3 Epub 2020 Mar 23. ; PMCID: PMC7091918. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref030] 30.Cao B, Wang Y, Wen D, Liu W, Wang J, Fan G, et al. A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19. N Engl J Med. 2020;382(19):1787–1799. 10.1056/NEJMoa2001282 Epub 2020 Mar 18. ; PMCID: PMC7121492. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref031] 31.Cevik M, Kuppalli K, Kindrachuk J, Peiris M. Virology, transmission, and pathogenesis of SARS-CoV-2. BMJ. 2020;371:m3862 10.1136/bmj.m3862 . [DOI] [PubMed] [Google Scholar]

[pone.0247606.ref032] 32.Wölfel R, Corman VM, Guggemos W, Seilmaier M, Zange S, Müller MA, et al. Virological assessment of hospitalized patients with COVID-2019. Nature. 2020;581(7809):465–469. 10.1038/s41586-020-2196-x . [DOI] [PubMed] [Google Scholar]

[pone.0247606.ref033] 33.Cevik M, Tate M, Lloyd O, Enrico Maraolo A, Schafers J, Ho A. SARS-CoV-2, SARS-CoV-1 and MERS-CoV viral load dynamics, duration of viral shedding and infectiousness: a living systematic review and meta-analysis. Lancet Microbe 2020; (forthcoming). 10.1101/2020.07.25.20162107. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref034] 34.Lan FY, Filler R, Mathew S, Buley J, Iliaki E, Bruno-Murtha LA, et al. COVID-19 symptoms predictive of healthcare workers’ SARS-CoV-2 PCR results. PLoS One. 2020;15(6):e0235460 10.1371/journal.pone.0235460 ; PMCID: PMC7319316. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref035] 35.Institute of Public Health of Vojvodina. Epidemiological report on COVID-19 in Vojvodina. Available from: http://izjzv.org.rs/?lng=lat&cir=&link=3-15-2035 [in Serbian].

[pone.0247606.ref036] 36.Caruana G, Croxatto A, Coste AT, Opota O, Lamoth F, Jaton K, et al. Diagnostic strategies for SARS-CoV-2 infection and interpretation of microbiological results. Clin Microbiol Infect. 2020;26(9):1178–82. 10.1016/j.cmi.2020.06.019 ; PMCID: PMC7315992. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0247606.ref037] 37.Tollånes MC, Bakken Kran AM, Abildsnes E, Jenum PA, Breivik AC, Sandberg S. Evaluation of eleven rapid tests for detection of antibodies against SARS-CoV-2. Clin Chem Lab Med. 2020;58(9):1595–1600. 10.1515/cclm-2020-0628 . [DOI] [PubMed] [Google Scholar]

PERMALINK

Validation of the STANDARD Q COVID-19 antigen test in Vojvodina, Serbia

Mioljub Ristić

Nataša Nikolić

Velibor Čabarkapa

Vesna Turkulov

Vladimir Petrović

Roles

Abstract

Background

Methods

Results

Conclusions

Introduction

Materials and methods

Study design

Inclusion/Exclusion criteria

Specimen collection and laboratory testing

STANDARD Q COVID-19 Ag Test

Data analysis

Ethical considerations

Results

Fig 1. Prevalence of signs and symptoms among participants tested for COVID-19.

Fig 2. Scatter plot of period between signs and symptoms onset and swab collection for laboratory testing for COVID-19 by age.

Table 1. Demographic characteristics, STANDARD Q COVID-19 Ag Test and signs/symptoms associated with RT-qPCR laboratory confirmed SARS-CoV-2 infections.

Table 2. Validation of the STANDARD Q COVID-19 Ag Test by gender, age groups and days of signs/symptoms onset.

Table 3. Comparison between RT-qPCR and STANDARD Q COVID-19 Ag Test laboratory confirmed cases of SARS-CoV-2 infections.

Table 4. Median cycle threshold and quantification of PCR and STANDARD Q COVID-19 Ag Test results.

Discussion

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

John Schieffelin

Roles

Author response to Decision Letter 0

Decision Letter 1

John Schieffelin

Roles

Acceptance letter

John Schieffelin

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases