Field evaluation of the performance of seven Antigen Rapid diagnostic tests for the diagnosis of SARs-CoV-2 virus infection in Uganda

Josephine Bwogi; Tom Lutalo; Phionah Tushabe; Henry Bukenya; James Peter Eliku; Isaac Ssewanyana; Susan Nabadda; Christopher Nsereko; Matthew Cotten; Robert Downing; Julius Lutwama; Pontiano Kaleebu; EPI Laboratory team; UVRI -COVID 19 Technical team

doi:10.1371/journal.pone.0265334

. 2022 May 10;17(5):e0265334. doi: 10.1371/journal.pone.0265334

Field evaluation of the performance of seven Antigen Rapid diagnostic tests for the diagnosis of SARs-CoV-2 virus infection in Uganda

Josephine Bwogi ^1,^*,^#, Tom Lutalo ^1,^#, Phionah Tushabe ¹, Henry Bukenya ¹, James Peter Eliku ¹, Isaac Ssewanyana ², Susan Nabadda ², Christopher Nsereko ³, Matthew Cotten ⁴, Robert Downing ¹, Julius Lutwama ¹, Pontiano Kaleebu ^1,⁴; EPI Laboratory team^¶; UVRI -COVID 19 Technical team^¶

Editor: Martin Chtolongo Simuunza⁵

PMCID: PMC9089886 PMID: 35536792

Abstract

Objective

The objective of this study was to evaluate the performance of seven antigen rapid diagnostic tests (Ag RDTs) in a clinical setting to identify those that could be recommended for use in the diagnosis of SARS-CoV-2 infection in Uganda.

Methods

This was a cross-sectional prospective study. Nasopharyngeal swabs were collected consecutively from COVID-19 PCR positive and COVID-19 PCR negative participants at isolation centers and points of entry, and tested with the SARS-CoV-2 Ag RDTs. Test sensitivity and specificity were generated by comparing results against qRT-PCR results (Berlin Protocol) at a cycle threshold (Ct) cut-off of ≤39. Sensitivity was also calculated at Ct cut-offs ≤29 and ≤33.

Results

None of the Ag RDTs had a sensitivity of ≥80% at Ct cut-off values ≤33 and ≤39. Two kits, Panbio^™ COVID-19 Ag and VivaDiag^™ SARS-CoV-2 Ag had a sensitivity of ≥80% at a Ct cut-off value of ≤29. Four kits: BIOCREDIT COVID -19 Ag, COVID-19 Ag Respi-Strip, MEDsan^® SARS-CoV-2 Antigen Rapid Test and Panbio^™ COVID-19 Ag Rapid Test had a specificity of ≥97%.

Conclusions

This evaluation identified one Ag RDT, Panbio^™ COVID-19 Ag with a performance at high viral load (Ct value ≤29) reaching that recommended by WHO. This kit was recommended for screening of patients with COVID -19-like symptoms presenting at health facilities.

Introduction

Coronavirus disease 2019 (COVID-19) was first confirmed in Uganda in March 2020 using real-time PCR reverse transcription polymerase chain reaction (qRT-PCR) tests. Over time, the number of specimens tested and the number of COVID-19 cases have increased with a second wave of the epidemic starting in May 2021, and by 6^th October 2021, the estimated cumulative PCR tests performed was 1,680,863 while the cumulative number of COVID-19 cases in Uganda stood at 123,445 with 3152 cumulative deaths giving a Case Fatality Rate of 1.1% [1]. However, it is unlikely that all cases were detected since not all suspected cases were tested [1]. There is an increasing demand for testing and sometimes this leads to increased turn-around times (72 hours instead of the desired 24 hours). The qRT-PCR tests are expensive (50–65 USD per test) when personal protective equipment and labour costs are included. In a search for cheaper diagnostic tests and tests that decrease the turn-around time for reporting results compared to qRT-PCR tests [2], the Ministry of Health requested the Uganda Virus Research Institute (UVRI), a COVID-19 national, Africa CDC and WHO reference laboratory to evaluate the performance of COVID-19 Ag RDTs. UVRI had previously evaluated and reported on one Ag RDT, the STANDARD Q COVID-19 Ag Test [3]. Here we provide a performance report for an additional seven Ag RDTs.

The seven RDTs that were evaluated were; BIOCREDIT COVID -19 Ag (RapiGEN, INC, Gyeonggi-do, Korea), COVID-19 Ag Respi-Strip (Coris BioConcept, Gembloux, Belgium), PCL COVID 19 Ag Rapid FIA (Inc, Geumcheon-gu, Seoul, Korea)), MEDsan^® SARS-CoV-2 Antigen Rapid test (MEDsan^®, Hamburg, Germany), Panbio^™ COVID-19 Ag Rapid Test (Abbott Rapid Diagnostics Jena, Germany), Novegent COVID-19 Antigen Rapid Test Kit (colloidal gold) (Chongqing Novegent Biotech Co., Ltd, Chongqing, China) and VivaDiag^™ SARS-CoV-2 Ag Rapid Test kit (VivaCheck Biotech(Hangzhou) Co., Ltd. Hangzhou, China). According to the manufacturers’ ‘Information for Use’ (IFU) of the different kits, these Ag RDTs were designed to directly detect SARS-CoV-2 antigens in respiratory secretions. In addition, with the exception of PCL, the other six Ag RDTs evaluated were Conformité Européene (CE) marked (S1 Table).

Although manufacturer’s reported high sensitivity (60.0–100%) and specificity (97.8–100%) for their Ag RDTs (S1 Table), the limited peer reviewed reports on their performance in clinical settings [4–8] showed conflicting performance.

The objective of this study was to evaluate the performance of the above Ag RDTs in clinical settings as compared to qRT PCR for detecting SARS-CoV-2 virus in nasopharyngeal samples in order to recommend Ag RDTs that can be used for COVID-19 diagnosis in Uganda.

Materials and methods

Study sites, study design, and implementation

This was a prospective cross-sectional study from August 2020 to March 2021. The study was carried out at national and regional COVID-19 isolation centres, established by Ministry of Health, that received patients from all over the country and at points of entry into the country. The study enrolled travellers requiring testing at points-of-entry (POE) into the country and patients admitted at isolation centres.

At isolation centers we used results from previous PCR tests and records of symptoms in the files to identify participants for recruitment. While at points of entry, participants with and without COVID-19 symptoms were enrolled. Patients that had difficulty in breathing and were receiving oxygen therapy or were on ventilators were excluded from the evaluation.

Enrolment of participants was convenient and consecutive from the selected sites.

Data were collected from each of the participants and entered in an Excel sheet. Data collected included socio-demographic (age, sex, health unit of isolation or point-of-entry), presence or absence of symptoms, date of admission and date of first symptom(s).

Sample collection and testing

The evaluation of each Ag RDT was performed at different time periods using different sets of samples drawn from participants enrolled at that time. This was because RDTs were received at UVRI from suppliers at different times and in different quantities.

Two nasopharyngeal samples were collected: one from each nostril. One sample was tested on the Ag RDT under evaluation and the second sample was used for qRT- PCR test. The sequence of collection of the nasopharyngeal samples for the Ag RDT and qRT- PCR test was alternated. Samples for qRT-PCR and COVID-19 Ag Respi-Strip were collected in a tube containing virus transport media (Hanks balanced salt solution, Fetal bovine serum, gentamycin sulfate and amphotericin B). The samples for the other six Ag RDT (BIOCREDIT COVID -19 Ag, PCL COVID 19 Ag Rapid FIA, MEDsan^® SARS-CoV-2 Antigen Rapid test, Panbio^™ COVID-19 Ag Rapid Test, Novegent COVID-19 Antigen Rapid Test Kit (colloidal gold), and VivaDiag^™ SARS-CoV-2 Ag Rapid Test kit) under evaluation were placed in the buffer provided by the kit manufacturer.

Antigen RDT testing was done within one hour of specimen collection without any cooling, following the manufacturer’s instructions. The results of the six Ag RDTs: BIOCREDIT COVID -19 Ag, COVID-19 Ag Respi-Strip, MEDsan^® SARS-CoV-2 Antigen Rapid test, Panbio^™ COVID-19 Ag Rapid Test, Novegent COVID-19 Antigen Rapid Test Kit (colloidal gold), and VivaDiag^™ SARS-CoV-2 Ag Rapid Test kit were visually read by trained UVRI laboratory staff. While, results from the PCL COVID19 Ag Rapid FIA were not visible and thus only read using the PCLOK EZ instrument supplied with the test kit. Interpretation of Antigen RDT results were as per the respective manufacturer’s guidelines. Those reading/interpreting the RDT results sometimes had access to the clinical information of the participant.

Samples collected for qRT-PCR were stored at 2°-8°C and transported within one week of collection to UVRI. At UVRI, samples were stored at -80°C for 1 to 28 days (median of 4–7 days) for the six RDTs except for RESPI Strip which ranged 1–37 days (median of 21 days) before qRT-PCR testing. The personnel carrying out the qRT-PCR testing were blinded to the Antigen RDT results. qRT-PCR testing was carried out at the EPI laboratory, one of the UVRI laboratories that tests for SARS-CoV-2, as described below:

RNA extraction

Viral RNA was extracted from the samples using the QIAGEN QIAamp Viral RNA extraction kit (QIAGEN, Hilden, Germany) following the manufacturer’s guidelines.

Real-time reverse-transcription PCR

qRT-PCR testing was carried out using the Charite-Berlin Protocol [9]. This assay was selected as the Standard reference for the kit evaluation because the protocol has good sensitivity with a limit of detection (LoD) of 3.9 RNA copies per reaction for E-gene assay and 3.6 RNA copies per reaction for RdRp gene assay using invitro transcribed RNA identical to 2019 novel coronavirus sequences and specificity of 100% [9].

Screening for SARS-CoV viruses was done using the SuperScript^™ III Platinum^™ One-Step qRT-PCR kit (Invitrogen, Carlsbad, CA) and LightMix^® SarbecoV E-gene primer/probe mix (TIB MOLBIOL, Berlin, Germany) following the manufacturer’s guidelines. All samples that had Ct values <45 in the above assay were subjected to a SARS-CoV-2 confirmatory qRT-PCR using the SuperScript III real-time RT-PCR kit (Invitrogen, Carlsbad, CA) and LightMix^® Modular SARS-CoV-2 (COVID-19) RdRp primer/probe mix (TIB MOLBIOL, Berlin, Germany) following the manufacturer’s guidelines. A sample was considered positive if it had a Ct value ≤39 on the confirmatory qRT-PCR. A sample was considered negative if the Ct value was undetermined on the screening qRT-PCR or if it was positive on the screening qRT-PCR but undetermined or >39 on the confirmatory qRT-PCR.

The thermal cycling conditions consisted of a reverse transcription reaction at 50°C for 10 minutes, an activation step at 95°C for 10 minutes and 45 cycles at 95°C/15 seconds and 60°C /1 minute. The platform used for the PCR was Applied Biosystems:7500 Real-Time PCR System (Marsiling, Singapore).

Data management and analysis

Ct values were recorded against each sample’s identifier and data entered in an MS Excel sheet which was transformed into a STATA^® v15 (StataCorp LP, 4905 Lakeway Drive, College Station, TX, USA) file for analysis. Inconsistency checks were performed using STATA and data cleaning done in collaboration with the field and laboratory staff. Samples with missing Antigen RDT and qRT-PCR results were excluded from the analysis.

Univariate analysis was performed to generate descriptive summaries for demographic and sample source characteristics. Frequencies, means, confidence intervals and medians were generated as summary statistics.

Performance evaluation

Sensitivity

Sensitivity was calculated as the number of specimens determined as positive by the Ag RDT under evaluation divided by the number of specimens determined as positive by PCR and expressed as a percentage with confidence intervals.

Specificity

Specificity was calculated as the number of specimens determined as negative by the Ag RDT under evaluation divided by the number of specimens determined as negative by PCR and expressed as a percentage with confidence intervals.

Accuracy

The accuracy was calculated as the proportion of results determined by the Ag RDT under evaluation that agreed with the PCR results and expressed as a percentage with confidence intervals.

False positive rate

The false positive rate was calculated as False Positive/(False Positive + True Negative) and expressed as a percentage.

False negative rate

The false negative rate was calculated as False Negative/(False Negative + True Positive) and was expressed as a percentage.

Sensitivity, specificity, accuracy, false positive rate and false negative rate calculations were performed using the proportion command in STATA 15 and confidence intervals produced with the Wilson score method [10].

Sensitivity of the Ag RDTs was also determined for different Ct cut-off values of ≤29, ≤33 and ≤39.

Sensitivity and specificity for all the Ag RDTs were also determined according to whether symptom onset was within 7 days of the specimen collection or after 7 days.

A sub-analysis (for sensitivity) was also performed for each Ag RDT considering different Ct cut off values following discussions with relevant stakeholders at the Ministry of Health and basing on literature [11–13]; a strong positive was defined as Ct value ≤29, a moderate positive was defined as Ct value 30–37 and a low positive was defined as Ct value 38–39.

Ethical considerations

The study protocol [14] was approved by Uganda Virus Research Institute’s Research Ethics Committee (No. GC/127/20) and the Uganda National Council for Science and Technology (No HS637ES). Informed written consent was obtained from each of the participants before enrolment into the study.

Results

Participant demographics

A total of 1,533 participants were included in the evaluation of the seven Ag kits as detailed in Table 1 below. The mean age group was 36 years, and 1024(66.8%) were males, 493 (32.2%) were females while 16(1.0%) had missing information on gender.

Table 1. Demographic characteristics of participants.

Antigen RDT	BIOCREDIT COVID -19 Ag	COVID-19 Ag Respi-Strip	PCL COVID19 Ag Rapid FIA	MEDsan^® SARS-CoV-2 Antigen Rapid test	Panbio^™ COVID-19 Ag Rapid test	Novegent COVID-19 Antigen Rapid test kit (colloidal gold)	VivaDiag^™ SARS-CoV-2 Ag Rapid Test kit
N	247	194	172	243	185	229	263
Sex
M	165 (66.8)	156 (80.4)	142(82.6)	156 (64.2)	136(73.5)	132(57.6)	137 (52.1)
F	76 (30.8)	38 (19.6)	30 (17.4)	77 (31.7)	49(26.5)	97(42.4)	126 (47.9)
Missing	6 (2.4)			10 (4.1)
Age in years
<20	2(0.8)	4(2.1)	3(1.7)	5(2.1)	6(3.2)	1(0.4)	4(1.5)
20–29	68(27.5)	54(27.8)	49(28.5)	39(16.0)	53(28.7)	28(12.2)	77(29.3)
30–39	87(35.2)	62(32.0)	48(27.9)	39(16.0)	59(31.9)	52(22.7)	66(25.1)
40–49	29(11.8)	34(17.5)	22(12.8)	36(14.8)	33(17.8)	46(20.1)	41(15.6)
50 and above	61(24.7)	17(8.8)	50(29.1)	41(16.9)	34(18.4)	97(42.4)	71(27.0)
Missing		23(11.9)		83(34.2)		5 (2.2)	4 (1.5)
Duration of symptoms
0–7 Days	97(39.3)	No information	32(18.6)	68(28.0)	117(63.2)	137(59.8)	235(89.4)
>7 Days	42(17.0)		11(6.4)	49(20.2)	20(10.8)	88(38.4)	21(8.0)
No symptoms/missing	108(43.7)		129(75.0)	126(51.9)	48(26.0)	4(1.8)	7(2.7)
PCR (Berlin protocol)
Positive	135 (54.7)	67 (34.5)	93 (54.1)	123 (50.6)	85 (46.0)	100(43.7)	43 (16.4)
Negative	112 (45.3)	127 (65.5)	79 (45.9)	120 (49.4)	100(54.1)	129(56.3)	220 (83.6)

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BIOCREDIT COVID -19 Ag test

Participant characteristics

Samples for the evaluation of the BIOCREDIT COVID-19 Ag RDT were collected in August 2020, from 247 participants (S1 Fig) with 122 (49.4%) from Mulago National Referral Hospital (NRH), 27 (10.9%) from Entebbe Regional Referral Hospital (RRH) and 98 (39.7%) from Namboole National Stadium isolation centre.

One hundred thirty-five participants (54.7%) were PCR-positive with 48 (36.1%) of these being females. The median age of the participants was 33 years (IQR 28–39 years).

There were 97 participants with onset of symptoms ranging between 0–7 days prior to the evaluation, 42 with onset of symptoms more than 7 days prior to the evaluation and 108 participants who were asymptomatic before the Ag RDT test (Table 1).

BIOCREDIT COVID -19 Ag test performance

There were 39 (15.7%) specimens that were BIOCREDIT COVID -19 Ag test positive (S1 Fig). The sensitivity of the test was 27.4% (95% CI: 20.5%– 35.6%) at ≤39 Ct cut-off, 44.7% (95% CI: 33.8–56.3%) at Ct cut-off values ≤33 and 60.0% (95% CI: 45.5%-72.9%) at Ct cut-off values ≤29 (Table 2).

Table 2. Field performance of 7 antigen RDTs for the diagnosis of SARs-CoV-2 virus in Uganda.

Kit name	Total participants recruited	Test sensitivity (%) Ct ≤ 39 (95% CI)	Test sensitivity (%) Ct ≤ 33 (95% CI)	Test sensitivity (%) Ct ≤ 29 (95% CI)	Test specificity % (95% CI)	RDT accuracy (%) Ct ≤ 39 (95% CI)
BIOCREDIT COVID -19 Ag	247	27.4 (20.5%–35.6%)	44.7 (33.8%–56.3%)	60 (45.5%–72.9%)	98.2 (93.1%–99.6%)	59.8 (53.6%–65.8%)
COVID-19 Ag Respi-Strip	194	19.4 (11.5%–30.9%)	35.1 (21.1%–52.4%)	46.4 (28.2%–65.7%)	99.2 (94.5%–99.9%)	71.6 (64.8%–77.6%)
PCL COVID19 Ag Rapid FIA	172	37.6 (28.2%–48.1%)	71.0 (51.8%–84.8%)	77.8 (57.0%–90.2%)	89.9 (80.8%–94.9%)	61.6 (54.1%–68.7%).
MEDsan^® SARS-CoV-2 Antigen Rapid test	243	13.0 (8.1%–20.3%)	23.7 (44.7%–74.3%)	43.5 (24.0%–65.3%)	100 (96.9%–100%)	56.0 (49.6%–62.1%)
Panbio^™ COVID-19 Ag Rapid test	185	49.4 (38.7%–60.1%)	72 (57.6%–83.0%)	85.7 (66.0%–94.9%)	100 (96.4%–100%)	76.8 (70.1%–82.3%)
Novegent COVID-19 Antigen Rapid test kit (colloidal gold)	229	46 (36.3%–56.0%)	58.2 (44.5%–70.8%)	66.7 (46.0%–82.5%)	89.9 (83.3%–94.1%)	70.7 (64.5%–76.3%)
VivaDiag^™ COVID-19 Ag	263	30.2 (18.0%–46.1%)	31.6 (18.4%–48.6%)	80 (37.8%–96.3%)	94.1 (90.1%–96.6%)	83.7 (78.6%–87.7%)

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Among the participants with symptom onset to date of Ag RDT test between 0–7 days, the sensitivity of the test was 39.3% (95% CI: 27.7%– 52.4%). The sensitivity of the test was 11.1% (3.3%–31.1%) for participants with more than 7 days from symptom onset to date of Ag RDT test: and 21.3% (95% CI: 11.6%–35.8%) for asymptomatic participants.

The Ag RDT specificity was 98.2% (95% CI: 93.1%–99.6%). The accuracy of the test was 59.8% (95% CI: 53.6%–65.8%) (Table 2); The false positive rate (FPR) was 5.1% (95% CI: 1.2%– 19.3%) and the false negative rate (FNR) was 72.6% (95% CI: 64.4%– 79.5%) (Table 2).

The BIOCREDIT COVID -19 Ag test result was negative for a large proportion of strong, moderate, and low positive specimens by qRT-PCR: 40%, 90% and 100% respectively. This was observed especially for Ct cut-off values between 30–38 (Table 3).

Table 3. COVID-19 Antigen Rapid test results compared to the Ct values on RT-PCR.

COVID-19 Antigen kit results		qRT-PCR Ct Value
Kit	Results	Strong Positive (≤29) N (%)	Moderate Positive (30–37) N (%)	Low Positive (38–39) N (%)
BIOCREDIT COVID-19 Ag	Positive	30 (60)	7 (9.6)	0 (0.0)
BIOCREDIT COVID-19 Ag	Negative	20 (40)	66 (90.4)	12 (100)
PCL COVID-19	Positive	21 (77.8)	12 (20.7)	2 (25.0)
PCL COVID-19	Negative	6 (22.2)	46 (79.3)	6 (75.0)
Respi-Strip COVID-19 Ag	Positive	13 (46.4)	0 (0)	0 (0)
Respi-Strip COVID-19 Ag	Negative	15 (53.6)	37 (100)	2 (100)
MEDsan^® SARS-CoV-2 Antigen Rapid test	Positive	10 (43.5)	6 (6.5)	0 (0.0)
MEDsan^® SARS-CoV-2 Antigen Rapid test	Negative	13 (56.5)	87 (93.6)	7 (100)
Panbio^™ COVID-19 Ag Rapid test	Positive	24 (85.7)	17 (30.9)	1 (50.0)
Panbio^™ COVID-19 Ag Rapid test	Negative	4 (14.3)	38 (69.1)	1 (50.0)
Novegent COVID-19 Antigen Rapid test kit (colloidal gold)	Positive	18 (66.8)	27 (40.3)	1 (16.7)
Novegent COVID-19 Antigen Rapid test kit (colloidal gold)	Negative	9 (33.3)	40 (59.7)	5 (83.3)
VivaDiag^™ COVID-19 Ag	Positive	8 (80.0)	5 (15.6)
VivaDiag^™ COVID-19 Ag	Negative	2 (20.0)	27 (84.4)

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COVID 19 antigen respi-strip test (Respi-Strip)

Participant characteristics

Samples for the Respi-Strip evaluation were collected in August 2020, from 194 participants (S2 Fig) with 84 (43.3%) from Mulago NRH, 71 (36.6%) from Malaba POE, 33 (17.0%) from Entebbe RRH and 6 (3.1%) from Mbale RRH. Seventy- six (66) participants had COVID-19 Ag Respi-Strip and PCL COVID19 Ag Rapid FIA. While fifteen (15) participants had both COVID-19 Ag Respi-Strip and BIOCREDIT COVID -19 Ag Test and One hundred and three (103) had only COVID-19 Ag Respi-Strip tests.

Sixty-seven (34.5%) participants out of the 194 were positive on PCR with 6 (9.0%) of these positives being females. The median age was 33 years (IQR 28–41 years).

Respi-Strip test performance

There were 14 (7.2%) specimens that were Respi-Strip test positive (S2 Fig). The test showed a sensitivity of 19.4% (95% CI: 11.5%– 30.9%) at Ct values ≤39; 35.1% (95% CI: 21.1–52.4%) at Ct values ≤33 and 46.4% (95% CI: 28.2%-65.7%) at Ct values ≤29 (Table 2). The specificity of the test was 99.2% (95% CI: 94.5%–99.9%).

The accuracy of the Respi-Strip test was 71.6% (95% CI: 64.8%–77.6%) at Ct values ≤39 (Table 2); the FPR was 0.8% (95% CI: 0.1%– 5.5%); and the FNR was 80.6% (95% CI: 69.1%– 88.5%) (Table 2).

The Respi-Strip determined the largest proportion of strong positives by qRT-PCR as negative (53.6%) and all moderate and low positives by qRT-PCR as negative (100%) (Table 3).

PCL COVID19 Ag Rapid FIA (PCL)

Participant characteristics

Samples for the evaluation were collected between August and September 2020, from 172 participants (S3 Fig) with 155 (90.1%) from Mulago NRH and 17 (9.9%) from Entebbe RRH. Ninety-three participants (54.1%) were PCR positive with 12 (12.9%) of these being females. The median age was 32 years (IQR 28–39 years).

There were 32 participants with onset of symptoms to date of Ag RDT test between 0–7 days. Of these, 19 were PCR-positive. Another 15 participants reported symptom onset of more than 7 days prior to the Ag RDT test with 9 (60%) being qRT-PCR positive (Table 1).

PCL Ag Rapid FIA test performance

There were 43 (25%) specimens that were Ag RDT-positive (S3 Fig). Overall, the PCL Ag test had a sensitivity of 37.6% (95% CI: 28.2%– 48.1%) at Ct values ≤39: 71.0% (95% CI: 51.8%– 84.8%) at Ct values ≤33 and 77.8% (95% CI: 57.0%– 90.2) at Ct values ≤29 (Table 2).

The sensitivity of the Ag RDT was 26.3% (95% CI: 10.4%– 52.4%) for participants with symptom onset to date of test within 0–7 days and 11.1% (0.9%–62.6%) for those with symptom onset to date of test greater than 7 days. For asymptomatic participants, sensitivity of the Ag RDT was 46.0% (95% CI: 33.9%–58.7%).

The specificity of the test was 89.9% (95% CI: 80.8%–94.9%) (Table 2). The accuracy of the Ag RDT was 61.6% (95% CI: 54.1%–68.7%) (Table 2). The FPR was 10.1% (95% CI: 5.1%– 19.2%) and the FNR was 62.4% (95% CI: 51.9%– 71.8%) (Ct cut-off ≤39) (Table 2).

The PCL test determined a large proportion of samples, whether strong or moderate or low positive by qRT-PCR as negative: 22.2%, 79.3% and 75.0% respectively (Table 3).

MEDsan^® SARS-CoV-2 Antigen Rapid test

Participant characteristics

A total of 243 samples (S4 Fig) were collected in November and December 2020 (from 228 participants; some participants gave multiple samples) with 137 samples (56.4%) from Mulago NRH, 10 (4.1%) from Entebbe RRH and 96 (39.5%) from Namboole National Stadium isolation center.

Of the 243 samples, 123 (50.6%) were qRT-PCR positive: 37 (30.1%) of these positives were females. The median age of the participants was 37 years (IQR 28–49 years). A total of 75(30%) participants had onset of symptoms 0–7 days before the antigen test (Table 1).

There were 25 qRT-PCR positive participants with the period between symptom onset to the date when the antigen test was done ranging between 0–7 days. For twenty- three (23) participants the period was greater than 7 days.

MEDsan^® SARS-CoV-2 Antigen Rapid test performance

There were 16 (6.6%) specimens that were MEDsan^® SARS-CoV-2 Antigen Rapid test positive (S4 Fig). The MEDsan^® SARS-CoV-2 Antigen Rapid test showed a sensitivity of 13.0% (95% CI: 8.1%– 20.3%) at a Ct cut off ≤39; a sensitivity of 23.7% (44.7–74.3%) at Ct values ≤33 and sensitivity of 43.5% (24.0–65.3%) at Ct values ≤29 (Table 2). The Ag RDT had a sensitivity of 12.0% (95% CI: 3.96%– 33.3%) for participants with 0–7 days between symptom onset and the antigen test and 21.7% (6.7%–44.8%) for participants with more than 7 days between symptom onset and the antigen test.

The specificity of the test was 100% (95% CI: 96.9%–100%) (Table 2).

The accuracy of MEDsan^® SARS-CoV-2 Antigen Rapid test was 56.0% (95% CI: 49.6%–62.1%) (Table 2). The FPR was 0.0% (95% CI: 0.0%– 19.4%) and the FNR was 87.0% (95% CI: 79.7%– 91.9%).

The MEDsan^® SARS-CoV-2 Antigen Rapid test determined a big proportion of strong, moderate and weak positives by qRT-PCR as negative, i.e. 56.5%, 93.6% and 100% respectively (Table 3).

Panbio^™ COVID-19 Ag Rapid test

Participant characteristics

One hundred eight five (185) participants (S5 Fig) were recruited in October 2020, 133 (71.9%) from Namboole Stadium isolation center, 40 (21.6%) from Mulago NRH and 12 (6.5%) from Entebbe RRH. Out of the 185 participants, 85 were qRT-PCR positive and 100 were qRT-PCR negative. One hundred seventeen (117(63.2%)) participants had a range of 0–7 days from symptom onset to the date when the antigen test was done (Table 1). Of these 117, forty-five (45) were positive by qRT-PCR. Twenty (20) participants had symptom onset more than 7 days prior to the test, and 9 out of the 20 (45%) tested positive by qRT-PCR.

The majority of the participants were males (73.5%) and the median age was 34 years (IQR 28–44 years) (Table 1).

Panbio^™ COVID-19 Ag Rapid test performance

There were 42 (22.7%) specimens that were Panbio^™ COVID-19 Ag Rapid test positive (S5 Fig). The Panbio^™ COVID-19 Ag Rapid test showed a sensitivity of 49.4% (95% CI: 38.7%– 60.1%) (Table 2) when compared to qRT-PCR test results at a Ct cut off value ≤ 39; a sensitivity of 72% (95% CI: 57.6%–83.0%) at Ct values ≤33 and 85.7% (95% CI: 66.0%–94.9%) at Ct values ≤29 (Table 2). The sensitivity of the Panbio^™ COVID-19 Ag Rapid test was 51.1% (95% CI: 36.3%– 65.8%) for participants with symptom onset 0–7 days before the antigen test while the sensitivity of the antigen test was 44.4% (13.4%–80.5%) for participants with symptom onset greater than 7 days before the antigen test. In asymptomatic participants, the sensitivity of the test was 48.4% (95% CI: 30.8%–66.4%).

The test had a specificity of 100% (95% CI: 96.4%–100%) (Table 2).

The Panbio^™ COVID-19 Ag Rapid test showed an accuracy of 76.8% (95% CI: 70.1%–82.3%) (Table 2). The FPR was 0% (95% CI: 0%–8.4%) and the FNR was 50.6% (95% CI: 39.9%– 61.2%).

It was observed that the Panbio^™ COVID-19 Ag Rapid test is more likely to determine a sample as positive when a sample has abundant target nucleic acid with Ct ≤ 29 (85.7%). However, when a sample is determined as moderate positive, 69% were determined as negative by Panbio^™ COVID-19 Ag Rapid test (Table 3).

Novegent COVID-19 antigen rapid test kit (colloidal gold)

Participant characteristics

A total of 229 participants (S6 Fig) were included in the evaluation: 32 (14.0%) participants from Entebbe RRH, 111 (48.5%) from Namboole National Stadium isolation center, 85 (37.1%) from Mulago RRH and 1 (0.4%) from Kisubi Hospital. The samples for evaluation were collected between January and February 2021. Of these 229 partcipants, 100 were qRT-PCR positive and 129 were qRT-PCR negative (Table 1).

There were 137 participants with a period of 0–7 days between symptom onset and the date of the antigen test and 56 of these were qRT-PCR positive. Forty- two (42) qRT-PCR positive participants had symptom onset more than 7 days prior to the test (Table 1).

The majority of the participants were males (57.6%) and the median age was 47 years (IQR 34–58 years) (Table 1).

Novegent COVID-19 Antigen Rapid test kit (colloidal gold) performance

There were 59 (25.8%) participants that were Novegent COVID-19 Antigen Rapid test positive (S6 Fig).

The test showed a sensitivity of 46% (95% CI: 36.3%– 56.0%) when compared to qRT-PCR test results at a Ct cut off value of ≤ 39;a sensitivity of 58.2% (95% CI: 44.5%–70.8%) at a Ct cut off value of ≤33 and sensitivity of 66.7% (95% CI: 46.0%–82.5%) at a Ct cut-off value of ≤29 (Table 2).

Taking into consideration participants with 0–7 days between symptom onset and the date of the antigen test, the Antigen RDT sensitivity was 65.0% (95% CI: 38.6%– 71.4%). For those participants where the period between symptom onset and the date of the antigen test was greater than 7 days the RDT sensitivity was 42.9% (28.4%–58.7%).

The test had a specificity of 89.9% (95% CI:83.3%–94.1%) (Table 2).

The RDT accuracy was 70.7% (95% CI: 64.5%–76.3%) (Table 2): the FPR was 22.0% (95% CI:13.0%–34.8%) and the FNR was 31.8% (95% CI: 25.1%– 39.2%) (Table 2).

Novegent COVID-19 Antigen Rapid test (Colloidal) is more likely to determine a sample as positive (66.7%) if the qRT-PCR result is a strong positive. However, for those samples that were moderately or weakly qRT-PCR positive, this antigen test was more likely to determine them as negative, 59.7% and 83.3% respectively (Table 3).

VivaDiag^™ SARS-CoV-2 Antigen Rapid test

Participant characteristics

In January to March 2021, samples for the evaluation were collected from 263 participants (S7 Fig): 122 (46.4%) from Kiruddu NRH, 59 (22.4%) from CASE Hospital, 77 (29.3%) from Mulago NRH and 5 (1.9%) from Entebbe RRH. Forty- three (16.4%) were positive on qRT-PCR with 21 (48.8%) of these positives being females. The median age of the participants was 36 years, (IQR 28–51 years) (Table 1).

There were 235(89.3%) participants with a period ranging between 0–7 days from symptom onset to the date when the antigen test was done(Table 1). Of these 235, thirty-eight (38) were positive by qRT-PCR. Twenty-one participants reported symptom onset more than 7 days prior to the antigen test with 5 (23.8%) determined as qRT-PCR-positive.

VivaDiag^™ SARS-CoV-2 Antigen Rapid test performance

There were 26 (7.8%) participants that were VivaDiag^™ antigen test positive (S7 Fig).

The VivaDiag^™ Ag test showed a sensitivity of 30.2% (95% CI: 18.0%– 46.1%) at qRT-PCR Ct values ≤39; a sensitivity of 31.6% (95% CI: 18.4%–48.6%) at qRT-PCR Ct values ≤33 and a sensitivity of 80% (95% CI: 37.8%–96.3%) at qRT-PCR Ct values ≤29 (Table 2). Taking into consideration participants with symptom onset 0–7 days prior to the antigen test, the sensitivity of the antigen test was 26.3% (95% CI: 14.4%– 43.2%).

The specificity of the test was 94.1% (95% CI: 90.1%–96.6%) (Table 2).

The VivaDiag^™ Ag test showed an accuracy of 83.7% (95% CI: 78.6%–87.7%) (Table 2); The FPR was 50.0% (95% CI: 30.5%– 69.5%); and the FNR was 12.7% (95% CI: 9.0%– 17.6%).

The VivaDiag^™ determined a large proportion of qRT-PCR strong positive cases as positive (80%) though the number of cases in this category was only five. However, VivaDiag^™ determined a very large proportion of the qRT-PCR moderately-positive cases (Ct values 30–38) as negative (84.4%) (Table 3).

Discussion

WHO recommends the use of antigen RDTs for SARS-CoV-2 diagnosis because they provide results within 15–30 minutes compared to qRT-PCR tests where results may not be available for more than 24 hours. RDTs cost far less than PCR tests, and do not require specialized laboratories and highly trained staff [2]. The use of accurate COVID -19 antigen RDTs will enable faster identification and management of COVID-19 patients leading to better control of the pandemic. A large number of antigen RDTs have been developed with varying sensitivities and specificities reported by the manufacturer’s IFUs. However, WHO has recommended field evaluations of antigen RDTs before they can be adopted in a national setting [2]. WHO recommends the use of antigen RDTs with a sensitivity of ≥80% and a specificity of ≥ 97% [2].

In this study, none of the seven antigen RDTs evaluated reached the sensitivity reported by the manufacturers, even after considering samples strongly positive by qRT-PCR (Ct values ≤29). These RDTs however had good specificity, with only three showing a specificity slightly lower than that reported in the IFUs. Poor performance in real-world settings compared to that found in the manufacturer’s IFUs has also been reported by others [4,7,8,15–18], emphasizing the need for performance evaluations of antigen RDTs in-country before adoption for use. In our study, we noted that the sensitivity of all seven Ag RDTs improved at lower qRT-PCR Ct values and this has also been observed in other studies [6–8,18–20]. Lower qRT-PCR Ct values in most cases correlate with higher viral loads and hence with increased transmissibility of the virus, usually in the early phases of the infection [21].

There were two antigen RDTs that reached the WHO recommended sensitivity performance of ≥80% at a Ct value of ≤29. These were the Panbio^™ COVID-19 Ag Rapid test and the VivaDiag^™ SARS-CoV-2 Ag Rapid test with sensitivities of 85.7% and 80% respectively. However, for the VivaDiag, the sample size was very small with only 10 samples at this Ct value. Of the two kits, Panbio^™ COVID-19 Ag Rapid test and VivaDiag^™ SARS-CoV-2 Ag Rapid test, only the PanBio^™ COVID-19 Ag Rapid Test had the WHO recommended specificity performance of ≥ 97%.

There was no correlation of antigen RDT performance with the presence or absence of symptoms. High viral loads usually appear in the pre-symptomatic and early symptomatic phases of the illness (within the first 5–7 days of symptom onset), however asymptomatic individuals can also have high viral loads in the early days of infection [22].

The Panbio^™ COVID-19 Ag Rapid Test met the sensitivity and specificity performance levels recommended by WHO at qRT-PCR Ct values of ≤29. This antigen RDT together with the Standard Q (by SD Biosensor) antigen RDT we previously evaluated [3], have been recommended in Uganda for COVID-19 diagnostic intervention in a phased approach as more experience and confidence is gained in their use through continuous field evaluation and the generation of additional data. These antigen RDTs have been recommended for rapid screening of the following populations; symptomatic alerts and symptomatic contacts of confirmed cases, patients with COVID -19-like symptoms presenting at health facilities.

The Uganda MOH recommends that, any antigen RDT positive case from the above mentioned populations will be considered “COVID– 19 positive” and managed accordingly and will not require additional qRT-PCR confirmation except in special cases such as genomic sequencing or routine quality control monitoring. Furthermore, any antigen RDT negative case with highly suggestive symptoms will be considered “a suspect COVID– 19 case” until confirmed to be uninfected by qRT-PCR and should be managed with enhanced infection prevention control (IPC). Laboratories at health units are expected to obtain/collect an additional specimen from those symptomatic clients who are antigen RDT negative to be sent for PCR testing.

Study limitations

Since samples were collected from different nostrils for the antigen test (BIOCREDIT COVID -19 Ag, MEDsan^® SARS-CoV-2 Antigen Rapid test, PCL and Panbio^™ COVID-19 Ag Rapid test) and qRT-PCR tests, the two swabs can be considered as two different samples which may lead to variation in the virus content of the collected samples.

The Panbio^™ COVID-19 Ag Rapid Test IFU recommends using the test within 5 days of symptom onset. However, in the evaluation of this antigen RDT, specimens from some asymptomatic cases were included. In asymptomatic cases it is difficult to determine the stage of the infection, whether early or late, thus affecting the overall interpretation of the results i.e. whether the antigen test result is negative because the client was in the early stage of disease and thus had low levels of antigen or was due to the poor performance of the test kit.

Participants in the study were recruited at different time periods thus there was varying prevalence of SARS-CoV-2 which could have affected the specificity of the tests. There was also significant difference in the population mix of the participants for the different tests (male/female, mean age, number of PCR positive and PCR negative participants) which may affect the comparability of the tests. Most participants were tested with only one of the evaluated RDT, implying different populations were used for evaluation of each RDT, thus making it difficult to compare the different RDT performance.

Conclusions

This field evaluation of seven SARS CoV-2 RDTs: COVID-19 Ag Respi-Strip, BIOCREDIT COVID -19 Ag, MEDsan^® SARS-CoV-2 Antigen Rapid test, PCL COVID19 Ag Rapid FIA, Panbio^™ COVID-19 Ag Rapid test, Novegent COVID-19 Antigen Rapid test kit (colloidal gold), and VivaDiag^™ COVID-19 Ag showed poorer performance than that reported by the manufacturers. Only the Panbio^™ COVID-19 Ag Rapid Test reached the WHO recommended performance of a sensitivity of ≥ 80% and a specificity ≥ 97% at qRT-PCR Ct values ≤ 29. This RDT can be recommended for COVID-19 infection diagnosis among symptomatic patients in health facilities in order not to delay patient management while waiting for qRT-PCR results.

Supporting information

S1 Fig. Flowchart summarizing test results using the BIOCREDIT COVID-19 Ag RDT.

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S2 Fig. Flowchart summarizing test results using the COVID-19 Ag Respi-Strip.

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Click here for additional data file.^{(135.7KB, tif)}

S3 Fig. Flowchart summarizing test results using the PCL COVID 19 Ag Rapid FIA.

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Click here for additional data file.^{(131.9KB, tif)}

S4 Fig. Flowchart summarizing test results using the MEDsan^® SARS-CoV-2 Antigen Rapid test.

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Click here for additional data file.^{(140KB, tif)}

S5 Fig. Flowchart summarizing test results using the Panbio^™ COVID-19 Ag Rapid test.

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S6 Fig. Flowchart summarizing test results using the Novegent COVID-19 Antigen Rapid test kit.

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Click here for additional data file.^{(139.7KB, tif)}

S7 Fig. Flowchart summarizing test results using the VivaDiag^™ SARS-CoV-2 Ag Rapid test kit.

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Click here for additional data file.^{(139KB, tif)}

S1 Table. Principal and kit performance of the seven evaluated COVID -19 Rapid Antigen test kits.

(DOCX)

Click here for additional data file.^{(17KB, docx)}

Acknowledgments

We thank Thomas Nsibambi, CDC Uganda for the technical input. We thank the participants who provided samples for the evaluation. We thank all the health workers at Mulago NRH, Kiruddu NRH, Mbale and Entebbe RRHs, CASE and Kisubi Hospitals, Namboole National Stadium isolation center and Malaba POE that collected the samples from the participants.

EPI Laboratory team: ¹**Prossy Namuwulya, ¹Francis Aine, ¹Irene Turyahabwe, ¹Lucy Nakabazzi, ¹Molly Birungi, ¹Rajab Dhatemwa, ¹Arnold Mugagga, ¹Mayi Tibanagwa, ¹Mathias Ssenono, ¹Charles Okia, ¹Mary Nyachwo.

Author affiliations: ¹: Uganda Virus Research Institute, Plot 51–59 Nakiwogo Road, P.O.BOX 49, Entebbe, Uganda.

**Lead author and contacts: Prossy Namuwulya

pnamuwulya@uvri.go.ug

UVRI COVID-19 Technical team: *** ¹John Kayiwa, ^1,2Jennifer Serwanga, ¹Stephen Balinandi, ¹Christine Watera, ¹Aminah Nalumansi, ¹Jocelyn Kiconco and ¹Bernard Kikaire.

Author Affiliations:

¹ Uganda Virus Research Institute, Plot 51–59 Nakiwogo Road, P.O.Box 49, Entebbe

² Medical Research Council/Uganda Virus Research Institute & London School of Hygiene and Tropical Medicine, Uganda Research Unit, P.O. Box 49, Plot 51–59, Nakiwogo Road, Entebbe, Uganda

***Lead author and contacts: John Kayiwa

jkayiwa@yahoo.com

Data Availability

The data underlying the results presented in the study are available from: http://eaccr.org/sites/default/files/2021-11/Field%20Evaluation%20of%20the%20Performance%20of%20Seven%20Antigen%20Rapid%20Diagnostic%20Tests.xlsx.

Funding Statement

The studies were largely funded by the providers of these test kits. We acknowledge the Uganda Government funding to UVRI. The procurement of the SARS-CoV2 qRT-PCR kits was funded mainly through the WHO and Africa CDC by different donors, Jack Ma Foundation and the UK Medical Research Council (MRC)/UK Research and Innovation. Part of the funding from the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) is under the MRC/DFID Concordat Agreement and is also part of the EDCTP2 programme supported by the European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0265334.r001

Decision Letter 0

Martin Chtolongo Simuunza

10 Sep 2021

PONE-D-21-24051Field Evaluation of the Performance of Seven Antigen Rapid Diagnostic Tests for the Diagnosis of SARs-CoV-2 Virus Infection in UgandaPLOS ONE

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Reviewer #1: The manuscript is interesting and well written.

The authors should acknowledge more limitations of the study. The comparison between the tests is not fair, because the tests are not compared in a randomized way. Sensitivity and specificity can be different if samples come from populations with different disease spectrum/prevalence.

The STARD checklist should be used (https://www.equator-network.org/reporting-guidelines/stard).

Table 1 should completed with percentages for all the variables (e.g. age groups).

Tables should be formatted in a better way.

Is the time lag between Antigen Test and PCR a possible explanation for the difference observed with vendors diagnostic performance?

Reviewer #2: Reviewer Recommendation and Comments for Manuscript Number PONE-D-21-24051

The comparison of the panel of tests is key to improving the use of these tests in local situation. Such is very important and carrying out such investigations improve the performance as mitigation measures can be put in place in real time. The fact that such tests can be performed at the bedside therefore hasten the recovery process and facilitates a speedy intervention and therefore recovery.

Observation

1. There is need for the authors to revisit the figures (number/values) in the text as well as those in the tables in order to align them e.g. the Table 1 says 245 (instead of 249) and Table 2 says 66* while the text in Line No. 220 is referring to sixty seven.

2. The presentation of the Table is little complicated as it is not clarified as to what is presented in the Table and what is not. In would be helpful for the reader to cite the Table or Figure at the first mention. The way the results are presented, the reader will only know where the table being referred to is when they reach the end of the paragraph.

3. In addition, navigating through the text and tables is a little tedious as the data is not presented according to the tables order. In the process, making locating what is being referred to in the text a little hard for the reader. For instance, Lines 192-197 appears to be referring to both Table 1 (asymptomatic participants) and Table 2(sensitivity). It would also be helpful for the reader if the authors can state when the results are "not shown" or are found in the supplementary.

Reviewer #3: This is a nice evaluation of seven RDTs for covid. However, diagnostic accuracy depends a lot on the study sample and participant mix. This is information that is lacking in this study and it is what I have most concerns about.

1. Participants were selected from travellers and people in isolation. To what extent are they representative for the population in which the RDTs will be used? For example, the conclusion says: "This RDT can be recommended for use among symptomatic patients in health facilities in order not to delay patient management while waiting for qRT-PCR results". But these are not the participants included.

2. For the reason mentioned in the previous comment, I do not think that this conclusion is valid. So please rephrase.

3. How were particpants selected? In a consecutive way? On particular days? This needs to be clarified.

4. How were the different tests allocated to the right person? At random, or in different time-periods? Please explain.

5. How many samples/tests per participant were included? Did some participants receive more than two tests? Please explain.

6. There are distinct differences in population mix (males/females; cases/controls) between the different tests. This could have influenced the accuracy, but the authors say little about these differences. Maybe this can be added?

7. In general, I think this manuscript would benefit from a better reporting. Please refer to the STARD guidelines for reporting of diagnostic accuracy studies.

8. RT-PCR is the reference standard, but very little information is provided. For example, I can imagine that having only one negative RT-PCR is not very reliable in determining a 'control' participant. Would it be possible to explian a bit more about the reference standard? And could the reference standard have led to bias? (which should then be mentioned in a limitations section in the Discussion)

9. The authors have analyzed the data against different Ct thresholds. As far as I can see, the Ct thresholds are different from what I have seen in other RDT analyses. Would it be possible for the authors to comment on these Ct thresholds? And how do they relate to clinical practice? Why were those Ct's chosen?

**********

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: Ngonda Saasa

Reviewer #3: Yes: Mariska Leeflang

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Click here for additional data file.^{(12.8KB, docx)}

PLoS One. 2022 May 10;17(5):e0265334. doi: 10.1371/journal.pone.0265334.r002

Author response to Decision Letter 0

5 Nov 2021

Response to Reviewers

PONE-D-21-24051

Field Evaluation of the Performance of Seven Antigen Rapid Diagnostic Tests for the Diagnosis of SARs-CoV-2 Virus Infection in Uganda

Response:

The information in the tables and numbers in text have been reviewed and now tally. The order of the results in the text has been reorganised to follow the order in which they appear in the tables.

2. Provide more information about the participants in this study and how they were selected. Do they represent the population on which such the tests are intended to be used on? Was each test applied to every participant and if not, what impact would this have on the comparison of the test results.

Response

The participants were enrolled consecutively whenever RDT kits for evaluation were supplied to UVRI. The evaluation did not receive the RDTs at the same time and quantities supplied for the evaluation were different (depended on how many the suppliers would be able to supply). The majority of participants considered for the evaluation were those found admitted with symptoms known to be for COVID-19 patients. Since RDTs were not received at the same time and because of the urgency due to the pandemic, we were not able to evaluate the kits using the same participants as we would have wanted but rather to enrol those who consented to the evaluation. This is further complicated by the acute nature of the disease and rapid changes in viral loads in individuals. We observed that our sample had significantly more men than women and this was also observed in the centres used (more men in health facilities and border crossings). This means that if the RDTs are to be used at health facilities where individuals with COVID-19 like symptoms would go for health care, or at border crossings, then it is more likely that a similar profile of individuals will be the ones to be tested using recommended RDTs.

3. Provide more information about the validity of the RT-PCR that was used as a Gold Standard in this study.

Response

More information on the sensitivity and specificity of the RT-PCR that was used as the Gold Standard in this study has been included in the manuscript.

Line162-166: The protocol has good sensitivity with a limit of detection (LoD) of 3.9 RNA copies per reaction for E-gene assay and 3.6 RNA copies per reaction for RdRp gene assay using in-vitro transcribed RNA identical to 2019 novel coronavirus sequences and specificity of 100%

Response

Protocol used has been referenced. Not deposited in the protocols.io .

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

Response:

Plos One formatting requirements have been addressed

2.Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.

Response:

No questionnaire has been included. Because the survey form was in excel. A copy of the Excel dataset used for data collection and which was transformed to STATA for the analysis can be accessed using the link below;-

http://eaccr.org/sites/default/files/2021-11/Field%20Evaluation%20of%20the%20Performance%20of%20Seven%20Antigen%20Rapid%20Diagnostic%20Tests.xlsx

3. Thank you for stating the following in the Competing Interests section:

[RD is a consultant at Abbott. Therefore, he did not participate in the investigation of Panbio™ COVID-19 Ag Rapid test performance.].

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

Response

Updated conflict of interest statement:

RD is a consultant at Abbott. Therefore he did not participate in the investigation of Panbio™ COVID-19 Ag Rapid test performance. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Response: The minimum Dataset has been made available. It can be accessed using the link below;-

http://eaccr.org/sites/default/files/2021-11/Field%20Evaluation%20of%20the%20Performance%20of%20Seven%20Antigen%20Rapid%20Diagnostic%20Tests.xlsx

Response:

Line 651-670: The members in EPI Laboratory team and UVRI COVID 19 Technical team have been named in the acknowledgement section. The author affiliation has been included and the Lead authors in the group named.

Response: The statement with this reference has been deleted from the manuscript. It was not a core part of the research being presented.

[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?

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

Response: The data has been made available. It can be found using the following URL

http://eaccr.org/sites/default/files/2021-11/Field%20Evaluation%20of%20the%20Performance%20of%20Seven%20Antigen%20Rapid%20Diagnostic%20Tests.xlsx

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

5. Review Comments to the Author

Reviewer #1: The manuscript is interesting and well written.

The STARD checklist should be used (https://www.equator-network.org/reporting-guidelines/stard).

Response:

Line 625-631 : Limitations why we could not use the mentioned randomization design have been added in the manuscript. Basically these kits were evaluated as and when the suppliers brought them to the UVRI. This was at different times and in different quantities. Because of the urgency to identify an RDT that would help in the diagnosis of COVID-19 we evaluated as soon as we received the kits and used participant sources established by the Ugandan Ministry of Health (Isolation centres and points of entry)

-We have followed the STARD checklist when reporting in the manuscript.

Table 1 should be completed with percentages for all the variables (e.g. age groups).

Response

Table has been completed with percentages for all the variables (age groups and the grouping according to duration of symptoms now have percentages).

Tables should be formatted in a better way.

Responses

Table 1, 2 and 3 have been formatted.

Is the time lag between Antigen Test and PCR a possible explanation for the difference observed with vendors diagnostic performance?

Response:

There is a time lag between the Antigen test and PCR which range from 1 to 37 days with a median of 4-7 days except for RESPI Strip which had a median of 21 days. We think this did not affect the diagnostic performance of the kits since the samples were stored at -80oC during that time. Please see table below: This information has been included in the manuscript.

Table : Days between the Rapid Diagnostic Test and the PCR test

Ag RDT Median days Mean Min Max

Biocredit 4 7.3 1 21

Respi 21 22.7 1 37

PCL 7 9.7 1 24

MedSan 5 4.9 2 12

PanBio 4 3.7 2 7

Colloidal/Novegent 4 4.3 1 11

VivaDiag 5 7.5 2 28

Reviewer #2: Reviewer Recommendation and Comments for Manuscript Number PONE-D-21-24051

Observation

Response: We have aligned the figures in the tables and text.

Response

We have maintained reference to the table at the end since we did not report per table but per kit. Referring to the table at the beginning for each kit would make the presentation monotonous

Response

We have rearranged the text to follow the order of the tables presented for the specific kit being evaluated.

Response

In addition, the Isolation Centers had patients that were admitted from different parts of the country.

2. For the reason mentioned in the previous comment, I do not think that this conclusion is valid. So please rephrase.

Response

Line 593-604: The conclusion has been rephrased.

3. How were particapants selected? In a consecutive way? On particular days? This needs to be clarified.

Response

Participants were recruited consecutively, and these were found at the sites of recruitment. Also recruitment for the different kits was as when the kits were received from the suppliers.

We have clarified this in the manuscript.

4. How were the different tests allocated to the right person? At random, or in different time-periods? Please explain.

Response

The different kits were allocated at different time periods as the different tests were received from the suppliers.

The dates when the different participants for the different kits were recruited is mentioned in the manuscript

5. How many samples/tests per participant were included? Did some participants receive more than two tests? Please explain.

Most participants/samples had only one test. This was because of how we received the samples and also when samples were taken off, they were put in the buffer supplied with that kit. A few samples were used to evaluate more than one test however a comparison of the results has not been included in the manuscript. Additionally, one RDT (RESPI) did not have a buffer but shared a sample with real time PCR.

Seventy- six samples had both PCL COVID19 Ag Rapid FIA and COVID-19 Ag Respi-Strip. Fifteen samples had both BIOCREDIT COVID -19 Ag Test and COVID-19 Ag Respi-Strip used. As mentioned above, no comparison between results from these tests has been included in the manuscript.

Line 332-335: The information about numbers of samples used to evaluate more than one RDT has been included in the manuscript.

Response

The population mix (cases/controls ) between the different tests has been added as a study limitation.

However, we do not think the difference in male/female mix could have affected the accuracy of the different tests.

Response

7. In general, I think this manuscript would benefit from a better reporting. Please refer to the STARD guidelines for reporting of diagnostic accuracy studies.

Response

We have referred to the STARD guidelines for reporting diagnostic accuracy studies and have made some edits to the manuscripts.

Response

Line 162-166: We have added more information concerning the RT-PCR we used as the reference standard: This assay was selected as the Standard reference for the kit evaluation because the protocol has good sensitivity with a limit of detection (LoD) of 3.9 RNA copies per reaction for E-gene assay and 3.6 RNA copies per reaction for RdRp gene assay using invitro transcribed RNA identical to 2019 novel coronavirus sequences and specificity of 100%. It was also recommended by WHO.

Since the specificity of the test was 100%, we believe using only one negative RT-PCR result for the control participants did not cause any bias in our study.

Response

The authors chose the Ct-thresholds in reference to literature and after discussions in-country.

A Ct ≤ 39: This is the cut off for positivity provided by the Charite Berlin PCR Protocol that we used in the analysis (Corman VM et al., 2020).

A Ct≤29 was used because literature showed that patients with Ct value below 30 were found to be more infectious due to the high viral load and these Ct values were more likely to be found in the first days of the infection. Since we are looking for kits that will detect the infection early we decided to include this cut off in our analysis.

https://www.sciencemag.org/news/2020/09/one-number-could-help-reveal-how-infectious-covid-19-patient-should-test-results

(Bayat SA et al. 2021)

A Ct value of ≤ 33 for the E-gene were found not to be contagious. No viruses were cultured from the patients with Ct value greater than 34 in a study carried out in France. Therefore, these were taken to be of less clinical value (La Scola B et al. 2020). Since we still need to identify these patients that is why we had this cut off value although our final report is on RdRp gene Ct value.

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: Ngonda Saasa

Reviewer #3: Yes: Mariska Leeflang

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(36.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0265334.r003

Decision Letter 1

Martin Chtolongo Simuunza

22 Nov 2021

PONE-D-21-24051R1Field Evaluation of the Performance of Seven Antigen Rapid Diagnostic Tests for the Diagnosis of SARs-CoV-2 Virus Infection in UgandaPLOS ONE

Dear Dr. Bwogi,

The reviewers are of the view that you did not adequately atted to their concerns. Please pay partcular attention to those raised by reviewer No. 3. Attend to all the concerns raised and return a revised manuscript as advised in this letter.

Please submit your revised manuscript by Jan 06 2022 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'.

We look forward to receiving your revised manuscript.

Kind regards,

Martin Chtolongo Simuunza, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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

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: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

6. Review Comments to the Author

Reviewer #1: Thanks for having addressed all my comments. I think that the manuscript is improved but there is still one thing missed. STARD checklist should be uploaded as supplementary material and the authors should explicitly state in what page/section the checklist item has been covered.

https://www.equator-network.org/wp-content/uploads/2015/10/STARD-2015-Checklist.docx

Reviewer #2: The authors have adequately addressed the concerns. The tables have beed realigned and some of the inconsistencies in the numbers in the table and text have been attended to.

Reviewer #3: The authors have improved their manuscript. However, there are a few remaining comments that have not or insufficiently have been addressed.

1. The selection criteria have not been clearly described. The authors now describe that participants were enrolled consecutively, but it is not clear in what setting the study was done or whether the patients enrolled in this study were representative. Also, the authors write that they included cases and controls, but that is ambiguous language. Please check the STARD guidelines about what to report about the selection criteria and the selection process.

2. The authors state that they followed the STARD guidelines, but there is still information missing. Please check. Also, the STARD guidelines recommend a flow chart to describe visually how patients enroll in the study and the tests they undergo and what the test results are. Such a flow chart would be really helpful in this case.

3. From the response to my comment about the timing of the tests, I understand that every patient is tested by only one of the index tests (and the reference standard). So patients enrolled at the start of the study will have received a different test than patients enrolled at the end of the study. That makes a comparisons between tests a bit difficult. So actually, authors should have mentioned this as a limitation. (see also my previous comment 6)

4. The authors provided now more information about the reference standard, but not how it was really used to make a diagnosis (e.g. for a participant who does not have COVID-19, should the test have been negative twice?).

**********

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: No

Reviewer #3: Yes: Mariska Leeflang

PLoS One. 2022 May 10;17(5):e0265334. doi: 10.1371/journal.pone.0265334.r004

Author response to Decision Letter 1

20 Jan 2022

Responses to Reviewers

PONE-D-21-24051R1

Field Evaluation of the Performance of Seven Antigen Rapid Diagnostic Tests for the Diagnosis of SARs-CoV-2 Virus Infection in Uganda

1. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response:

References have been reviewed and changes have been made to reference No 6 and 7.

Reference no 6. Bulilete O, Lorente P, Leiva A, Carandell E, Oliver A, Rojo E, et al. Evaluation of the Panbio rapid antigen test for SARS-CoV-2 in primary health care centers and test sites. medRxiv. 2020.

Replaced with more up to date reference below:

Bulilete O, Lorente P, Leiva A, Carandell E, Oliver A, Rojo E, Pericas P, Llobera J, COVID-19 Primary Care Research Group. Panbio™ rapid antigen test for SARS-CoV-2 has acceptable accuracy in symptomatic patients in primary health care. Journal of Infection. 2021 Mar 1;82(3):391-8.

Reference no 7.Olearo F, Noerz D, Heinrich F, Sutter JP, Roedel K, Schultze A, et al. Handling and accuracy of four rapid antigen tests for the diagnosis of SARS-CoV-2 compared to RT-qPCR. medRxiv. 2020.

Replaced with the reference below: The reference above is no longer on the website. The reference available is the one below

Olearo F, Nörz D, Heinrich F, Sutter JP, Roedl K, Schultze A, Zur Wiesch JS, Braun P, Oestereich L, Kreuels B, Wichmann D. Handling and accuracy of four rapid antigen tests for the diagnosis of SARS-CoV-2 compared to RT-qPCR. Journal of Clinical Virology. 2021 Apr 1;137:104782.

2. Thanks for having addressed all my comments. I think that the manuscript is improved but there is still one thing missed. STARD checklist should be uploaded as supplementary material and the authors should explicitly state in what page/section the checklist item has been covered.

Response

The STARD checklist has been attached to the rebuttal letter. In the checklist we have indicated the line no where the checklist has been covered. We have not included the STARD checklist in the supplementary material since we could not reference it in the manuscript

3. The selection criteria have not been clearly described. The authors now describe that participants were enrolled consecutively, but it is not clear in what setting the study was done or whether the patients enrolled in this study were representative. Also, the authors write that they included cases and controls, but that is ambiguous language. Please check the STARD guidelines about what to report about the selection criteria and the selection process.

Response

Line 84-93. More information has been included in the section of the selection criteria and process.

Line 27-28. Cases and Controls have been replaced with PCR positive and PCR negative participants.

4. The authors state that they followed the STARD guidelines, but there is still information missing. Please check. Also, the STARD guidelines recommend a flow chart to describe visually how patients enroll in the study and the tests they undergo and what the test results are. Such a flow chart would be really helpful in this case.

Response

• We have reviewed the STARD guidelines to guide us on inclusion of missing information in the manuscript. The STARD checklist has been attached to indicate where the different sections of the STARD checklist are in the paper.

• Flow charts showing patient enrollment and tests have been placed as figures in the supplementary material.

5. From the response to my comment about the timing of the tests, I understand that every patient is tested by only one of the index tests (and the reference standard). So patients enrolled at the start of the study will have received a different test than patients enrolled at the end of the study. That makes a comparisons between tests a bit difficult. So actually, authors should have mentioned this as a limitation. (see also my previous comment 6)

Response

Line 491-493. “Difficult in comparison of the tests because of timing of the tests and thus every patient tested with one of the index tests” has been included in the study limitation.

6. The authors provided now more information about the reference standard, but not how it was really used to make a diagnosis (e.g. for a participant who does not have COVID-19, should the test have been negative twice?).

Response : Line 137-146:

This has now been described: A sample was considered positive if it had a Ct value ≤39 on the confirmatory qRT-PCR. A sample was considered negative if the Ct value was undetermined on the screening qRT-PCR or if it was positive on the screening qRT-PCR but undetermined or >39 on the confirmatory qRT-PCR.

Attachment

Submitted filename: Responses to Reviewers.docx

Click here for additional data file.^{(21.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0265334.r005

Decision Letter 2

Martin Chtolongo Simuunza

1 Mar 2022

Field Evaluation of the Performance of Seven Antigen Rapid Diagnostic Tests for the Diagnosis of SARs-CoV-2 Virus Infection in Uganda

PONE-D-21-24051R2

Dear Dr. Bwogi,

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,

Martin Chtolongo Simuunza, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: I have no further comments, because all my previous comments have been addressed in one way or another.

**********

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 #3: Yes: Mariska M.G. Leeflang

PLoS One. doi: 10.1371/journal.pone.0265334.r006

Acceptance letter

Martin Chtolongo Simuunza

23 Mar 2022

PONE-D-21-24051R2

Field Evaluation of the Performance of Seven Antigen Rapid Diagnostic Tests for the Diagnosis of SARs-CoV-2 Virus Infection in Uganda

Dear Dr. Bwogi:

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. Martin Chtolongo Simuunza

Academic Editor

PLOS ONE

Associated Data

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

Supplementary Materials

S1 Fig. Flowchart summarizing test results using the BIOCREDIT COVID-19 Ag RDT.

(TIF)

Click here for additional data file.^{(139.8KB, tif)}

S2 Fig. Flowchart summarizing test results using the COVID-19 Ag Respi-Strip.

(TIF)

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S3 Fig. Flowchart summarizing test results using the PCL COVID 19 Ag Rapid FIA.

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S4 Fig. Flowchart summarizing test results using the MEDsan^® SARS-CoV-2 Antigen Rapid test.

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S5 Fig. Flowchart summarizing test results using the Panbio^™ COVID-19 Ag Rapid test.

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S6 Fig. Flowchart summarizing test results using the Novegent COVID-19 Antigen Rapid test kit.

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S7 Fig. Flowchart summarizing test results using the VivaDiag^™ SARS-CoV-2 Ag Rapid test kit.

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S1 Table. Principal and kit performance of the seven evaluated COVID -19 Rapid Antigen test kits.

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Submitted filename: Reviewer Recommendation and Comments for Manuscript Number PONE.docx

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Data Availability Statement

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PERMALINK

Field evaluation of the performance of seven Antigen Rapid diagnostic tests for the diagnosis of SARs-CoV-2 virus infection in Uganda

Josephine Bwogi

Tom Lutalo

Phionah Tushabe

Henry Bukenya

James Peter Eliku

Isaac Ssewanyana

Susan Nabadda

Christopher Nsereko

Matthew Cotten

Robert Downing

Julius Lutwama

Pontiano Kaleebu

Roles

Abstract

Objective

Methods

Results

Conclusions

Introduction

Materials and methods

Study sites, study design, and implementation

Sample collection and testing

RNA extraction

Real-time reverse-transcription PCR

Data management and analysis

Performance evaluation

Sensitivity

Specificity

Accuracy

False positive rate

False negative rate

Ethical considerations

Results

Participant demographics

Table 1. Demographic characteristics of participants.

BIOCREDIT COVID -19 Ag test

Participant characteristics

BIOCREDIT COVID -19 Ag test performance

Table 2. Field performance of 7 antigen RDTs for the diagnosis of SARs-CoV-2 virus in Uganda.

Table 3. COVID-19 Antigen Rapid test results compared to the Ct values on RT-PCR.

COVID 19 antigen respi-strip test (Respi-Strip)

Participant characteristics

Respi-Strip test performance

PCL COVID19 Ag Rapid FIA (PCL)

Participant characteristics

PCL Ag Rapid FIA test performance

MEDsan® SARS-CoV-2 Antigen Rapid test

Participant characteristics

MEDsan® SARS-CoV-2 Antigen Rapid test performance

Panbio™ COVID-19 Ag Rapid test

Participant characteristics

Panbio™ COVID-19 Ag Rapid test performance

Novegent COVID-19 antigen rapid test kit (colloidal gold)

Participant characteristics

Novegent COVID-19 Antigen Rapid test kit (colloidal gold) performance

VivaDiag™ SARS-CoV-2 Antigen Rapid test

Participant characteristics

VivaDiag™ SARS-CoV-2 Antigen Rapid test performance

Discussion

Study limitations

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Martin Chtolongo Simuunza

Roles

Author response to Decision Letter 0

Decision Letter 1

Martin Chtolongo Simuunza

Roles

Author response to Decision Letter 1

Decision Letter 2

Martin Chtolongo Simuunza

Roles

Acceptance letter

MEDsan^® SARS-CoV-2 Antigen Rapid test

MEDsan^® SARS-CoV-2 Antigen Rapid test performance

Panbio^™ COVID-19 Ag Rapid test

Panbio^™ COVID-19 Ag Rapid test performance

VivaDiag^™ SARS-CoV-2 Antigen Rapid test

VivaDiag^™ SARS-CoV-2 Antigen Rapid test performance