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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 24  |  Issue : 1  |  Page : 16-20

Evaluation of rapid antigen test against reverse transcription-polymerase chain reaction for the diagnosis of severe acute respiratory syndrome coronavirus 2 in a tertiary care centre in South Kerala


Department of Microbiology, Sree Gokulam Medical College and Research Foundation, Venjaramoodu, Kerala, India

Date of Submission22-Dec-2021
Date of Acceptance24-Feb-2022
Date of Web Publication11-Jul-2022

Correspondence Address:
O R Reshma
Department of Microbiology, Sree Gokulam Medical College and Research Foundation, Venjaramoodu - 695 607, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jacm.jacm_70_21

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  Abstract 


BACKGROUND AND OBJECTIVES: The objective of this study was to evaluate the performance of a rapid antigen test against reverse transcription– polymerase chainreaction (RT-PCR) for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) in a tertiary care centre in South Kerala. In this study, we compared the performance of a rapid antigen test, coronavirus disease-2019 (COVID-19) Ag Respi-Strip, with that of a real-time PCR assay using PerkinElmer Reagent kit.
MATERIALS AND METHODS: A descriptive (diagnostic evaluation) study was conducted at a tertiary care teaching hospital in South Kerala. Samples were taken for RAT and real-time reversetranscriptase–polymerase chain reaction (rRT-PCR) (reference standard) from patients visiting fever clinic in Sree Gokulam Medical College and Research Foundation to calculate the sensitivity, specificity and accuracy of the rapid antigen test. Samples were collected by the following protocols approved by the Institutional Research Committee, Sree Gokulam Medical College and Research Foundation, Trivandrum. Signed informed consent was obtained from all participants. Tests were performed in biosafety level 3 laboratories at the Molecular Laboratory, Department of Microbiology, Sree Gokulam Medical College and Research Foundation, Trivandrum.
RESULTS: Of the 241 symptomatic participants, 50 were rRT-PCR positive for SARS-CoV-2. Thirty-nine of these patients also tested positive for SARS-CoV-2 by RAT. The overall sensitivity and specificity were 76% and 99.47%, respectively. We got an accuracy of 94.6%.
INTERPRETATION AND CONCLUSIONS: With excellent specificity and moderate sensitivity, a rapid antigen test may be used to rule out COVID-19 in patients. The comparison data obtained in this study indicate that this can be used for rapid screening of patients with high SARS-CoV-2 viral load, but the rate of sensitivity is highly dependent on computed tomography value. This method also enables instrument-free and low-cost point-of-care testing. However, it depends on various factors such as viral loads and clinical status, which could significantly influence the final performance of the antigen test. Hence, negative results from an antigen test may need to be confirmed with a molecular test before treatment.

Keywords: Coronavirus disease-2019, rapid antigen test, reverse transcription–polymerase chain reaction


How to cite this article:
Reshma O R, Ajimsha A, Krishna GR, Deepthi K N, Subhash K, Jitendranath A, Ramani Bai J T. Evaluation of rapid antigen test against reverse transcription-polymerase chain reaction for the diagnosis of severe acute respiratory syndrome coronavirus 2 in a tertiary care centre in South Kerala. J Acad Clin Microbiol 2022;24:16-20

How to cite this URL:
Reshma O R, Ajimsha A, Krishna GR, Deepthi K N, Subhash K, Jitendranath A, Ramani Bai J T. Evaluation of rapid antigen test against reverse transcription-polymerase chain reaction for the diagnosis of severe acute respiratory syndrome coronavirus 2 in a tertiary care centre in South Kerala. J Acad Clin Microbiol [serial online] 2022 [cited 2022 Aug 11];24:16-20. Available from: https://www.jacmjournal.org/text.asp?2022/24/1/16/350322




  Introduction Top


On 11 March 2020, World Health Organisation (WHO) declared coronavirus disease-2019 (COVID-19) as a pandemic.[2] Already early in the pandemic, the WHO highlighted the critical importance of testing for epidemic control.[3] The global demand for testing has put a substantial strain on governments and institutions. The gold-standard diagnostic test recommended by WHO (WHO 2020a) is the real-time, quantitative reverse transcription–polymerase chain reaction (qRT-PCR), a nucleic acid amplification test (NAAT), aimed at the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA.[4] Such testing has excellent sensitivity and specificity. However, it takes hours to detect the nucleic acid and days to isolate the virus, it is expensive and specialised instruments and expertise are required resulting in long delays in the reporting of results, compromising their utility in epidemiologic investigation and high frequency of testing approaches.[2],[5],[6]

The aim is to develop rapid, reliable, less expensive, qualitative diagnostic tests which are designed to directly detect SARS-CoV-2 proteins produced by replicating virus in respiratory secretions and have been developed as both laboratory-based tests and, for near-patient use, so-called rapid diagnostic tests or RDTs.[2],[6] RAT tests detect viral antigen by the immobilised coated SARS-CoV-2 antibody on the device. The test results of RAT can be interpreted without specialised instruments and available within 30 min. Hence, RAT tests can relieve the workload in diagnostic hospitals and laboratories and improve the turnaround time.[2]

Detection of pathogen-derived antigens on lateral flow assays is a proven concept with excellent performance. This method also enables rapid, instrument-free and low-cost point-of-care testing. Since the onset of the pandemic, numerous companies have been working on bringing a rapid SARS-CoV-2 antigen-detection test (Ag-RDT) to the market. WHO has released interim guidance that the use of Ag-RDTs that meet at least 80% sensitivity and 97% specificity may be considered to diagnose active SARS-CoV-2 infection 'where nucleic acid amplification technology (NAAT) is unavailable or where prolonged turnaround times preclude clinical testing'.[3] These lateral flow tests are based on immunochromatography and show SARS-CoV-2 antigen presence by a coloured test line.[7]

In this study, we compared the performance of a rapid antigen test, COVID-19 Ag Respi-Strip, with that of a real-time PCR assay using PerkinElmer Reagent kit.

Relevance of the study

Most of the data generated on diagnosing SARS-CoV-2 have been institute specific; hence, we would like to evaluate and analyse data generated from our institute.

Aims and objectives

  • This study aimed to find out the diagnostic accuracy of the rapid antigen test in a tertiary care centre in Kerala.



  Materials and Methods Top


A descriptive (diagnostic evaluation) study was conducted in a tertiary care hospital in South Kerala to evaluate the performance of the rapid antigen test compared with a clinical reference standard (Real-time reversetranscriptase–polymerase chain reaction [rRT-PCR]).

After obtaining informed consent from patients attending fever clinic in Sree Gokulam Medical College and Research Foundation, Venjaramoodu, from 1 April 2021 to 31 May 2021, 241 samples from symptomatic patients were collected. Nasopharyngeal and throat swabs were collected from each patient. Both the swabs were placed in a 2 ml VTM tube.

Study procedure

Samples received in the molecular microbiology laboratory of SGMCRF were subjected to rRT-PCR as per the manufacturer's instructions.

Viral transport kit – Q-line Molecular.

RT-PCR Extraction Kit – Qiagen Extraction kit.

RT-PCR Master Mix and Template addition kit – PerkinElmer Reagent kit.

Rapid antigen test

  • Name of kit-CORIS COVID-19 Ag Respi-Strip, CORIS BioConcept, Gembloux, Belgium (Science Park CCREALY, Rue Jean Sonet 4A B-5032 Gembloux – Belgium).


Kit was allowed to reach room temperature (15°C–30°C) before performing the test.

About 100 ul of the sample was transferred in the tube, and 100 ul or four drops of the LY-S dilution buffer was added. The tube was closed with the stopper and stirred thoroughly.

The tube was opened, and the strip was immersed in the direction indicated and closed with the stopper. It was allowed to react for 30 min. The swab was discarded according to biosecurity requirements.

Interpretation of results

After 30 min, the results were read. Two bands developed in the test device in two different positions. As per the manufacturer's instructions,

C – Control line.

T – Test line

  • Positive – A visible reddish-purple (even weak) band appears at the test line position
  • Negative – A reddish-purple line appears at the control line (C) position (upper line).


Real-time reversetranscriptase–polymerase chain reaction

Tests were conducted on nasopharyngeal swabs collected for rRT-PCR, received at molecular microbiology laboratory. Nucleic acid extraction was done with Qiagen extraction kit. RT-PCR Master Mix and Template addition were carried out with PerkinElmer SARS-CoV-2 RT-qPCR Reagent kits, respectively. The interpretation was done as per the manufacturer's instructions.

Name of kit: PerkinElmer SARS-CoV-2 RT-qPCR REAGENT KIT.

The PerkinElmer® New Coronavirus Nucleic Acid Detection kit uses TaqMan-based real-time PCR technique to conduct in vitro reverse transcription of SARS-CoV-2 RNA, DNA amplification and fluorescence detection. The assay targets specific genomic regions of SARS-CoV-2: nucleocapsid (N) gene and open reading frame 1ab (ORF1ab) gene. The TaqMan probes for the two amplicons are labelled with FAM and ROX fluorescent dyes, respectively, to generate a target-specific signal. The assay includes an RNA internal control (IC, bacteriophage MS2) to monitor the processes from nucleic acid extraction to fluorescence detection. The IC probe is labelled with VIC fluorescent dye to differentiate its fluorescent signal from SARS-CoV-2 targets.

Microsoft Office Excel for handling and preparing data and SPSS-2 software for analysis of diagnostic test accuracy.

Interpretation of results

Detection of RdRp gene (RNA dependent RNA Polymerase) and SARS-CoV-2 ORF 1a/b gene (Open Reading Frame) for confirmation.

Computed tomography (CT) values ≤35 were considered as positive results.

Positive predictive value is the probability that symptomatic patients with a positive rapid antigen test truly are COVID-19 positive.

Negative predictive value (NPV) is the probability that symptomatic patients with a negative rapid antigen test truly are COVID-19 negative.

Analysis

Overall mean age group belonged to adolescents (19–59 years) [Table 1].
Table 1: Age-wise distribution of samples

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Majority were females (60.6%) [Table 2].
Table 2: Gender-wise distribution of samples

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Nasal discharge was the most common symptom, followed by fever, breathlessness, diarrhoea, cough, myalgia, sore throat and headache [Table 3].
Table 3: Symptoms present among the study population

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  Results Top


Performance characteristics

The distribution of COVID-19 Ag Respi-Strip test results against the qRT-PCR results is presented in [Table 4].
Table 4: Distribution of coronavirus disease-2019 Ag Respi-Strip test results against the quantitative reverse transcription-polymerase chain reaction

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Sensitivity and specificity

Sensitivity

Sensitivity was calculated as the number of specimens identified as positive by the COVID-19 Ag Respi-Strip test divided by the number of specimens identified as positive by the qRT-PCR reference assay, expressed as a percentage.

Specificity

Specificity was calculated as the number of specimens identified as negative by the COVID-19 Ag Respi-Strip test divided by the number of specimens identified as negative by the qRT-PCR reference assay, expressed as a percentage.

Accuracy

This was calculated as the proportion of COVID-19 Ag Respi-Strip test results that were in agreement with the qRT-PCR results (positive and negative), expressed as a percentage.

Sensitivity = (a/[a + c]) × 100

Specificity = (d/[b + d]) × 100

Positive predictive value (PPV) = (a/[a + b]) ×100

NPV = (d/[c + d]) ×100

The COVID-19 Ag Respi-Strip test showed a sensitivity of 76% and a specificity of 99.47%.

Accuracy = 94.6%.

Positive predictive value = 97.4%.

NPV = 94.05%.

False positive rate = 0.5%.

False negative Rate = 24%.

The 50 concordant cases had CT values ≤35, whereas the 12 discordant cases had CT values >35.


  Discussion Top


In this study, it was found that the CORIS COVID-19 Ag Respi-Strip antigen test had a sensitivity and specificity of 76% and 99.47%, respectively. The accuracy of the test was 94.6%. The majority of the discordant cases had cycle threshold (CT) values >28. The antigen test performs well in the presence of high viral loads, whereas lower levels are missed. Twelve false negative test results were from samples with high real-time RT-PCR cycle threshold (CT). We got one false positive result, which may be due to test interference from patient-specific factors, such as the presence of human antibodies or highly viscous specimens [Table 5].
Table 5: Comparison with other similar studies

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In a study conducted in AIIMS, New Delhi, out of 330 participants, 77 were detected to be SARS-CoV-2 positive. Sixty-four of these were positive for RDT. The overall sensitivity and specificity were 81.8% and 99.6%, respectively. In this study, follow-up testing of symptomatic patients was done, and rapid antigen test showed an excellent specificity to “rule-in” COVID-19 patients within the first five days of illness and had a moderate sensitivity. Therefore, patients showing positive results need to be immediately triaged and those with negative tests should be reconfirmed by an rRT-PCR.[7]

In a study done in Uganda Virus Research Institute, 262 samples were collected, including 90 qRT-PCR positives. Sensitivity and specificity of the antigen test were 70.0% and 92%, respectively.[4] Our study got almost similar sensitivity and specificity, compared with this study.

In a study done in the Netherlands, specificity of 100% and sensitivity of 80.7% were found for rapid antigen test compared to qRT-PCR.[8] In this study, the results are comparable with our study.

In a study done by, Department of Microbiology, Cliniques Universitaires Saint-Luc-University Catholique de Louvain, Avenue Hippocrate, Brussels, Belgium, 148 nasopharyngeal swabs were tested. Amongst the 106 positive RT-qPCR samples, 32 were detected by the rapid antigen test, given an overall sensitivity of 30.2%. All the samples detected positive with the antigen rapid test were also positive with RT-qPCR.[9] In this study, we can see that the antigen test has low performance.


  Conclusion Top


COVID-19 Ag Respi-Strip Antigen test has shown a good performance which aids in the SARS-CoV-2 screening and diagnosing, achieving an excellent percentage of specificity and moderate sensitivity. However, it depends on various factors such as viral loads and clinical status, which could significantly influence the final performance of the antigen test.

Antigen tests have been indicated as the tests that have the potential of quickly identifying individuals that can transmit infectious viral particles. Although less sensitive than molecular tests that detect viral RNA, antigen tests perform well on individuals with high viral load in their upper respiratory tract. Antigen test is easy to perform, it does not require skilled personnel and gives results in about 15 min. The probability of a successful isolation is more if the samples are processed immediately after collection and the viral load in the sample is more. Viral load is maximum if the samples are collected immediately after the onset of clinical symptoms and before the administration of antiviral medications.

Hence, negative results from an antigen test may need to be confirmed with a molecular test before treatment.[1]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



[10]



 
  References Top

1.
Ciotti M, Maurici M, Pieri M, Andreoni M, Bernardini S. Performance of a rapid antigen test in the diagnosis of SARS-CoV-2 infection. J Med Virol 2021;93:2988-91.  Back to cited text no. 1
    
2.
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.  Back to cited text no. 2
    
3.
World Health Organisation. Advice on the use of point-of-care immunodiagnostic tests for COVID-19. 2020 https://www.who.int/news-room/commentaries/detail/advice-on-theuse-of-point-of-care-immunodiagnostic-tests-for-covid-19. [Last accessed on 26.08. 2020 Aug 26].  Back to cited text no. 3
    
4.
Nalumansi A, Lutalo T, Kayiwa J, Watera C, Balinandi S, Kiconco J, et al. Field evaluation of the performance of a SARS-CoV-2 antigen rapid diagnostic test in Uganda using nasopharyngeal samples. Int J Infect Dis 2021;104:282-6.  Back to cited text no. 4
    
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Pilarowski G, Lebel P, Sunshine S, Liu J, Crawford E, Marquez C, et al. Performance characteristics of a rapid SARS-CoV-2 antigen detection assay at a public plaza testing site in San Francisco. J Infect Dis 2021;223:1139-44. Available from: http://medrxiv.org/lookup/doi/10.1101/2020.11.02.20223891. [Last accessed on 2021 Jun 16].  Back to cited text no. 5
    
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WHO Antigen-Detection in the Diagnosis of SARS-CoV-2 Infection Using Rapid Immunoassays Interim Guidance 11 September 2020 COVID-19: Laboratory and Diagnosis; 2020. Available from:https://www.who.int/publications/i/item/antigen-detection-in-the-diagnosis-of-sars-cov-2infection-using-rapid-immunoassays. [Last accessed on 2022 Mar 06].  Back to cited text no. 6
    
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Gupta A, Khurana S, Das R, Srigyan D, Singh A, Mittal A, et al. Rapid chromatographic immunoassay-based evaluation of COVID-19: A cross-sectional, diagnostic test accuracy study & its implications for COVID-19 management in India. Indian J Med Res 2021;153:126-31.  Back to cited text no. 7
[PUBMED]  [Full text]  
8.
Performance Evaluation of a SARS-COV-2 Rapid Antigentest: Test Performance in the Community in the Netherlands | medRxiv. Available from: https://www.medrxiv.org/content/100.1101/2020.10.19.20215202v1. [Last accessed on 2020 Nov 26].  Back to cited text no. 8
    
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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.  Back to cited text no. 9
    
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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.  Back to cited text no. 10
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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