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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 23  |  Issue : 2  |  Page : 201-204

Speed versus sensitivity: Performance of SARS-CoV-2 rapid antigen test in the hospital setting


1 Department of Microbiology, AFMC, Pune, India
2 Department of Community Medicine, AFMC, Pune, India
3 Department of Internal Medicine, AFMC, Pune, Maharashtra, India

Date of Submission03-Mar-2021
Date of Decision22-May-2021
Date of Acceptance30-May-2021
Date of Web Publication21-Sep-2021

Correspondence Address:
Lt Col (Dr) Santosh Karade
AFMC, Pune, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jmms.jmms_29_21

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  Abstract 


Background: As the world battles the coronavirus disease 2019 (COVID-19) pandemic, rapid detection is the key to contain the spread of the disease. Although real-time polymerase chain reaction (RT-PCR) remains the gold standard for the diagnosis of severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) infection, it demands specialized equipment and technical expertise. SARS-CoV-2 rapid antigen detection test (RAT) is the user-friendly alternative, which needs evaluation in the hospital setting. Materials and Methods: This cross-sectional study was carried out at the Indian Council of Medical Research approved COVID-19 diagnostic molecular laboratory between November and December 2020. Nasopharyngeal swabs in viral transport medium (VTM) were received from suspected SARS-CoV-2 infected individuals were processed for SARS-CoV-2 RT-PCR and a rapid antigen detection test, COVID-19 Ag Respi-Strip. Results: A total of 342 samples were assessed for this study by RT-PCR and Rapid Antigen Test. The sensitivity of RAT was found to be 41% at an RT-PCR cycle threshold of 35 or below. Sensitivity of RAT improved to 81.2% when a RT-PCR cycle threshold of 25 or below was considered as positive. Specificity of RAT was 99% to 100%. Conclusion: Rapid antigen test is a cost-effective alternative for screening of symptomatic individuals in the hospital setting. However, due to low sensitivity, the negative results need to be interpreted with caution.

Keywords: COVID-19, real-time polymerase chain reaction, respiratory infection, pandemic


How to cite this article:
Sharma A, Karade S, Anand KB, Lall M, Sen S, Patrikar S, Naithani N. Speed versus sensitivity: Performance of SARS-CoV-2 rapid antigen test in the hospital setting. J Mar Med Soc 2021;23:201-4

How to cite this URL:
Sharma A, Karade S, Anand KB, Lall M, Sen S, Patrikar S, Naithani N. Speed versus sensitivity: Performance of SARS-CoV-2 rapid antigen test in the hospital setting. J Mar Med Soc [serial online] 2021 [cited 2021 Dec 9];23:201-4. Available from: https://www.marinemedicalsociety.in/text.asp?2021/23/2/201/326276




  Introduction Top


The severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) is an enveloped positive-strand RNA virus belonging to the Beta coronavirus genus and the Sarbecovirus subgenus[1] On 11 Mar 2020, World Health Organization declared coronavirus disease 2019 (COVID-19) as a pandemic.[2] Rapid detection, effective isolation of symptomatic cases, and systematic tracing of close contacts are paramount to moderate the community spread of SARS-CoV-2 infection.

Real-time reverse transcription-based polymerase chain reaction (RT-PCR) on respiratory samples is the current recommended laboratory method to diagnose SARS-CoV-2 infection. The method is extremely sensitive and can detect less than five genome copies of target genes.[3] However, RT-PCR assay requires special equipment and skilled personnel in molecular techniques. The procedure requires an elaborate setup, and it is also time-consuming.

The rapid antigen test (RAT) detects SARS-CoV-2 specific antigen in nasopharyngeal or oro-pharyngeal swabs using lateral flow immune-chromatographic platform. Antigen detection tests are less labour intensive and deliver results in 30 min. However, these tests are less sensitive as compared to nucleic acid detection based RT-PCR assay.[4],[5] There is a need to explore the performance of RAT in the setting of the current pandemic. Thus, the aim of this study was to assess the performance of RAT versus RT-PCR for screening of SARS-CoV-2 infection.


  Materials and Methods Top


This cross-sectional study was carried out in a hospital setting of a tertiary care health centre. The study population included symptomatic and asymptomatic adults referred from the Flu Clinic for screening of SARS-CoV-2 infection between November and December 2020. Nasopharyngeal and oropharyngeal swabs (NP/OP swab) received in viral transport medium (VTM) were processed at the Indian Council of Medical Research (ICMR) approved COVID-19 diagnostic molecular laboratory. The NP/OP swabs were simultaneously processed for SARS-CoV-2 RT-PCR and RAT.

SARS-CoV-2 real-time polymerase chain reaction

RNA extraction from 400 μl of VTM was performed by Qiagen viral RNA extraction kit as per manufacturer's instructions. In vitro detection of SARS-CoV-2 genetic targets was done using the ICMR, New Delhi, approved multiplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR kit). The assay contains primers and probes directed against Hong Kong University open reading frame 1b (HKU_Orf1_b) and envelope (E) gene targets of the SARS-CoV-2 genome. It also includes the RPP30 human gene, which served as the internal control. A cycle threshold value of 35 of less for each SARS-CoV-2 specific target was considered positive.

COVID-19 antigen detection

Qualitative detection of SARS-CoV-2 antigen in nasopharyngeal swab received in VTM was performed using ICMR, approved kit, COVID-19 Ag Respi-Strip (Coris Bioconcept, Gembloux, Belgium). This immunochromatographic test is based on a membrane technology with colloidal gold nanoparticles, and uses monoclonal antibodies to detect highly conserved SARS-CoV-2 nucleoprotein antigen. The test was performed according to the manufacturer's instruction using 100 μL of VTM. The appearance of test band and control band 15 min after loading of the sample was considered positive for SARS-CoV-2 infection.

Characteristics of the study population were reported as median (interquartile range). Results obtained by RT-PCR and RAT for each sample were tabulated in an MS Excel spreadsheet. The sensitivity and specificity with 95% confidence intervals (CIs) of COVID-19 Ag Respi-Strip were evaluated, considering RT-PCR as the gold standard.


  Results Top


Nasopharyngeal swab from 350 individuals collected between Dec 2020 and Jan 2021 were subjected to SARS-CoV-2 RT-PCR and rapid antigen test. Of these 8 were rejected due improper collection (spillage or absence of internal control amplification), and data from 342 samples were analysed. The study population was male predominant (70.46%, n = 241) with a mean age of 38.08 year [Figure 1]. The indication for SARS-CoV-2 testing as per ICMR criteria is shown in [Table 1].[6]
Figure 1: Distribution of age (in years) of study participants

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Table 1: Distribution of study participants based on the Indian Council of Medical Research patient category for severe acute respiratory syndrome corona virus-2 diagnostic testing

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According to RT-PCR results, based on the Ct value of <35 (as per manufacturers instruction) for both “E” and “HKU” targets, 34 (9.79%) were detected positive for SARS-CoV-2 infection. The distribution of Ct values of SARS-CoV-2 positive samples is shown in [Figure 2]. Of these 34 RT-PCR specimens, 21 were positive by RAT also.
Figure 2: Scatter plot distribution of SARS-CoV-2 Envelope gene (E) and HKU open reading frame (HKU Orf 1b) cycle threshold values of real-time polymerase chain reaction positive samples (n = 34)

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The sensitivity and specificity of RAT were found to be 41% (95% CI: 24%–58%) and 100% (95% CI: 99%–100%), respectively, at an RT-PCR cycle threshold of 35 or less. Sensitivity of RAT improved to 59% (95% CI: 38%–80%) and 81.2% (95% CI: 62%–100%) when a RT-PCR cycle threshold to declare a SARS-CoV-2 positive result was reduced to < 30 and < 25, respectively [Table 2]. Real-time PCR positivity was significantly associated with symptomatic individuals (P < 0.005) as compared to those without any symptoms [Figure 3].
Table 2: Sensitivity and specificity of severe acute respiratory syndrome corona virus-2 rapid antigen test

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Figure 3: Comparison of SARS-CoV-2 positive results between symptomatic and asymptomatic individuals

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  Discussion Top


COVID-19 pandemic has placed healthcare resources under tremendous pressure over the past one year. Till mid-February over 106 million cases of SARS-CoV-2 infections and 2.3 million deaths were reported globally.[7] The laboratory plays a crucial role in diagnosing and managing COVID-19 cases. Prompt diagnosis allows the early institution of preventive or isolation measures to break the chain of infection. The SARS-CoV-2 real-time PCR based molecular diagnosis is considered the gold standard and typically takes 5-6 h from sample collection to final report, for a batch of 90–94 samples. Whereas turn-around-time for RAT based SARS-CoV-2 nucleoprotein detection is less than an hour. The average cost of a single antigen test is less than one-fourth of RT-PCR. However, antigen detection tests lack sensitivity. A recent study on evaluation of RAT for diagnosing COVID-19 positive sample shows RAT to be 100,000 times lesser sensitive than RT-PCR.[8] Thus, currently ICMR, New Delhi recommends retesting of RAT negative samples by from symptomatic individuals RT-PCR.[9]

COVID-19 Ag Respi-Strip test (Coris BioConcept) showed poor sensitivity of 41% as compared to RT-PCR in our study. Scohy et al. also reported low sensitivity (30.2%) of the COVID-19 Ag Respi-Strip test necessitating RT-PCR confirmation of all negative tests.[10] Due to sub-optimal performance, the rapid test would fail to detect active infection during community screening where the spectrum of clinical cases are expected. Prior study by Lambert-Niclot et al., using the same detection platform showed a sensitivity of 50%.[11] The variation can be explained by the heterogeneous study population, different specimen quality and type of VTM. The authors observed sensitivity of 82.2% at a Ct value cut-off of under 25.[11] The present study also found a similar sensitivity of 81.2% at Ct value of <25. The RT-PCR Ct value is indirect measure of patient's viral load. Individuals with low Ct values for SARS-CoV-RT-PCR have higher viral load and are potentially infectious to the community. RAT is faster, low cost, user-friendly and value-added tool for screening of such individuals. Martin Möckel et al. found this test useful in symptomatic patients in the emergency department for prompt diagnosis and screening purposes.[12] In the present study also the performance of RAT was better in symptomatic individuals (P = 0.04).

As the curve of COVID-19 cases flatten, there would be the return of social and economic normalcy. The educational institutes, workplaces, worship places, hospitals and clinics will function like the pre-COVID era. In such setting, there is a need for a rapid identification and isolation of infectious individuals, to break the chain of transmission. The antigen detection test has high specificity but low sensitivity. The sensitivity improves with drop in RT-PCR Ct value indicating better performance in potential viral shedders. In a resource-limited setting of the second wave of COVID-19, the RT-PCR percentage positivity has crossed 20%–25% in certain states. In such scenario, RAT can be used as a screening tool to minimize the number of RT-PCR tests performed by a laboratory. However, samples tested negative by RAT needs to be re-tested by RT-PCR.

The study does have certain limitations. First, the study results are influenced by the prevalence of SARS-CoV-2 infection in a geographical area. Secondly, the study population was heterogeneous and hospital based. It includes outpatients and In-patients with variety of indications. The test result may differ in the community setting and RAT has a limited role in screening asymptomatic individuals.


  Conclusion Top


To conclude the rapid antigen tests are influenced by viral load. In our study we found a sensitivity of 81.2% in samples with Ct values <25. In a previous study by Bullard et al.[13] SARS-CoV-2 Vero cell infectivity was only observed for RT-PCR Ct <24 and STT (Symptom to testing time) <8 days and hence the sensitivity of 81.2% in samples with Ct values less than 25 may merit it as a tool to screen the infectious individuals to a limited extent. In a study conducted by Anand et al.[14] subgenomic mRNA a marker of active replication was detected even at a Ct value of 31 in symptomatic individuals, hence prompting us to consider missing of infectious individuals using the rapid antigen tests which is inherent drawback of this modality.

Overall, though the rapid antigen tests are easy to perform and can be used at community and field settings with minimally trained individuals, without sophisticated equipment. One needs to consider the cost benefits, rapidity and simplicity of performance of test with its sensitivity in a pragmatic manner. The present sensitivity of available kits, only merit their role in settings where NAAT is not readily available, in suspected outbreak investigations and widespread community transmission where the positive predictive value and negative predictive value of the test help to achieve adequate infection control.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species severe acute respiratory syndrome-related coronavirus: Classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 2020;5:536-44.  Back to cited text no. 1
    
2.
WHO. Coronavirus Disease 2019 Situation Report 51-11th March 2020. Vol. 2019, WHO Bulletin; 2020. Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019. [Last accessed on 2021 Mar 15].  Back to cited text no. 2
    
3.
Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 2020;25. doi: 10.2807/1560-7917.ES.2020.25.3.2000045.  Back to cited text no. 3
    
4.
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. 4
    
5.
Yamayoshi S, Sakai-Tagawa Y, Koga M, Akasaka O, Nakachi I, Koh H, et al. Comparison of rapid antigen tests for COVID-19. Viruses 2020;12:1420.  Back to cited text no. 5
    
6.
Indian Council of Medical Research, New Delhi. ICMR Specimen Referral Form for COVID-19 (SARS-CoV2) dated 16 December, 2020. Available from: https://www.icmr.gov.in/pdf/covid/labs/Revised_SRF_Form_16122020_1.pdf. [Last accessed on 2021 Mar 15].  Back to cited text no. 6
    
7.
World Health Organization. Coronavirus disease (COVID-19) Weekly Epidemiological Update and Weekly Operational Update. Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports. [Last accessed on 2021 Mar 15].  Back to cited text no. 7
    
8.
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. 8
    
9.
Indian Council of Medical Research, New Delhi. Strategy for COVID-19 Testing in India (Version 6, dated 04 Sep 2020). Available from: https://www.icmr.gov.in/pdf/covid/strategy/Testing_Strategy_v6_04092020.pdf. [Last accessed on 2021 Mar 15].  Back to cited text no. 9
    
10.
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. 10
    
11.
Lambert-Niclot S, Cuffel A, Le Pape S, Vauloup-Fellous C, Morand-Joubert L, Roque-Afonso AM, et al. Evaluation of a rapid diagnostic assay for detection of SARS-CoV-2 antigen in nasopharyngeal swabs. J Clin Microbiol 2020;58:e00977-20.  Back to cited text no. 11
    
12.
Möckel M, Corman VM, Stegemann MS, Hofmann J, Stein A, Jones TC, et al. SARS-CoV-2 antigen rapid immunoassay for diagnosis of COVID-19 in the emergency department. Biomarkers 2021;26:213-20.  Back to cited text no. 12
    
13.
Bullard J, Dust K, Funk D, Strong JE, Alexander D, Garnett L, et al. Predicting infectious SARS-CoV-2 from diagnostic samples. Clin Infect Dis. 2020 May 22:ciaa638.  Back to cited text no. 13
    
14.
Anand KB, Karade S, Sen S, Ray S, Patil P, Thosani P, et al. Can subgenomic mRNA predict course of COVID-19? - An observational study from a tertiary care center. J Mar Med Soc 2020;22:216-9.  Back to cited text no. 14
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