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Immunoassay

RADI COVID-19 Ag Rapid Test(REF: IM002)

Introduction

RADI COVID-19 Ag Rapid Test is a rapid immuno-chromatographic assay for qualitative detection of specific antigens to SARS-COV-2 present in human nasopharynx. This test contains colloid gold conjugate pad and a membrane strip pre-coated with antibodies specific to SARS-COV-2 antigen on the test lines.

Product Specification

  • RADI COVID-19 Ag Rapid TEST
    Intended use A lateral flow immunochromatographic assay intended for the qualitative detection of the nucleocapsid protein antigen from SARS-CoV-2 in nasopharyngeal swab specimens
    Specimen type Nasopharyngeal swab
    Test / kit 25 tests / kit
    Storage Condition 2~30 ℃
    Shelf life 12 months

Test Procedure

  • 1

    Insert a sterile swab into the nostril of the patient, and swab over the surface of the posterior nasopharynx. Withdraw the swab from the nasal cavity.

  • 2

    Insert the swab into an extraction tube. While squeezing the buffer tube, stir the swab more than 5 times strongly. Remove the swab while squeezing the sides of the tube to extract the liquid from the swab and screw the filter cap tightly onto the tube.

  • 3

    Apply 3 ~ 4 drops of extracted specimen to the specimen well of the test device.

  • 4

    Read the test result at 30 minutes.

INTERPRETATION

Note C : Control line / T : Test line

CLINICAL PERFORMANCE

1) Clinical evaluation in USA The clinical performance validation of RADI COVID-19 Ag Rapid Test was evaluated in the high complexity laboratory certified to conduct both molecular and serological diagnosis in the United States(U.S.) with 111 de-identified positive and 120 deidentified negative nasopharyngeal swab sample in VTM, which were confirmed by DTPM COVID- 19 RT PCR test with an Emergency Use Authorization (EUA) granted by the FDA (Comparator method).

The leftover de-identified samples used had Ct values ranging from 15.081 to 33.696 and the cut-off values for the EUA authorized DTPM molecular RT-qPCR assay is 35 cycles.

As a result, the RADI COVID-19 Ag Rapid Test had 100% specificity and 97.3% sensitivity for nasopharyngeal samples when using Ct-values less than 35 cycles as a cut-off for comparator method (DTPM COVID- 19 RT PCR test).

CLINICAL PERFORMANCE DTPM COVID- 19 RT PCR TEST
(Comparator Method)
Positive Negative Total
RADI COVID-19 Ag Rapid Test Positive 108 0 108
Negative 3 120 123
Total 111 120 231
Sensitivity 97.30% (95 CI (%): 92.4 to 99.1%)
Specificity 100% (95 CI (%): 96.9 to 100%)
Total Agreement 98.70%

2) Clinical evaluation in Ghana The RADI COVID-19 Ag Rapid Test Kit was evaluated on 8th February 2021 by NMIMR(Noguchi Memorial Institute For Medical Research), University of Ghana.

Leftover nasopharyngeal clinical specimens after routine diagnosis were enrolled. (Specimen Collection Period : ( January -February 2021). A total of 94 nasopharyngeal clinical specimens in VTM were randomly selected with variation in their Ct values which is consist of 39 SARS Cov-2 positive and 55 SARS-CoV-2 negative confirmed by Q-PCR(Detection kit for 2019-nCoV RNA (PCR Fluorescence Probing)) as comparator method which is a product included in the World Health Organization Emergency Use List(EUL).

The cut-off values for the Q-PCR assay is 40 cycles. The range of Ct value regarding positive samples was 15.41 to 23.62 (ORF1ab gene) and 14.2 to 25.96 (N gene). The Evaluation of RADI COVID-19 Ag Rapid Test was conducted in blinded fashion. The performance of RADI COVID-19 Ag Rapid test was shown in comparison with Comparator method as below.

CLINICAL PERFORMANCE Detection kit for 2019-nCoV RNA (PCR Fluorescence Probing)
(WHO EUL test)
Positive Negative Total
RADI COVID-19 Ag Rapid Test Positive 37 3 40
Negative 2 52 54
Total 39 55 94
Sensitivity 94.9% (95%CI : 83.1 to 98.6)
Specificity 94.6% (95%CI : 85.1 to 98.1)

3) Clinical evaluation in Cameroon RADI COVID-19 Ag Rapid Test kit was evaluated between February 1st and March 31st, 2021 by Epicentre (the research arm of Médecins Sans Frontières) in Yaoundé, Cameroon using fresh samples collected from symptomatic patients indiscriminately with respect to time from symptom onset.

A total of 127 fresh nasopharyngeal specimens with varying Ct values consisting of 33 SARS-CoV-2 positive and 94 SARS-CoV-2 negatives confirmed by RT-PCR(Abbott RT-PCR) were used. Collection of all samples was done less than 5 days from symptom onset.
The performance of RADI COVID-19 Ag Rapid test was shown in comparison with Comparator method as below.

CLINICAL PERFORMANCE Abbott RT-PCR
(USFDA EUA test)
Positive Negative Total
RADI COVID-19 Ag Rapid Test Positive 28 2 30
Negative 5 92 97
Total 33 94 127
Sensitivity 84.8% (95% CI 69.1 to 93.3%)
Specificity 97.9% (95% CI 92.6 to 99.4%)

ANALYTICAL SENSITIVITY _ Limit of Detection (LoD)

The RADI COVID-19 Ag Rapid Test has established the limit of detection by two different methods (VTM method / direct swab method) with serial diluted concentration of heat inactivated SARS-CoV-2 Isolate in negative nasopharyngeal clinical matrix. (Source of Material used: Hong Kong/VM20001061/2020, concentration : 1.15 x 107 TCID50/mL, Manufacturer : Zeptometrix).

1) VTM method: the heat inactivated virus was diluted with negative clinical matrix in VTM to make different viral diluted concentration (initial test concentration : 1.15 x 107 TCID50/mL).

2) Direct swab method: Spiking 30 ul of the heat inactivated virus to a swab and dilution with negative clinical matrix (Initial test concentration : 9.86 x105 TCID50/mL)

Test for each method was done according to the instructions for use. Once tentative confirmed LoD was established from different viral diluted concentrations for both VTM and direct swab method, the final LoD of each method was determined at the lowest virus concentration level where the product can be detectable more than 95%(19/20) detection rate in 20 replicates.

METHOD LoD
VTM METHOD 1.43 x103 TCID50/mL
DIRECT SWAB METHOD 9.86 x101 TCID50/mL (≒2.96 x100 TCID50/Swab)

CROSS-REACTIVITY AND MICROBIAL INTERFERENCE

Totally, 35 kinds of microorganisms following FDA EUA guideline were evaluated in the study by wet-testing of 30 microorganisms and in-silico analysis of 5 microorganisms. No cross-reactivity or interference was seen in the study.

Wet testing

  • NO. PATHOGEN
    1 Coronavirus (Strain: 229E)
    2 Coronavirus (Strain: OC43)
    3 Coronavirus (Strain: NL63)
    4 Coronavirus MERS
    5 Adenovirus
    6 Influenza A (H1N1)
    7 Influenza A (H3N2)
    8 Influenza B
    9 Human Metapneumovirus (hMPV)
    10 Parainfluenza Virus Type 1
    11 Parainfluenza Virus Type 2
    12 Parainfluenza Virus Type 3
    13 Parainfluenza Virus Type 4
    14 Rhinovirus
    15 Respiratory syncytial virus (Type A)
    16 Respiratory syncytial virus (Type B)
  • NO. PATHOGEN
    17 SARS-coronavirus
    18 Enterovirus A
    19 Enterovirus B
    20 Enterovirus C
    21 Enterovirus D
    22 Haemophilus influenzae
    23 Streptococcus pneumoniae
    24 Streptococcus pyogenes
    25 Candida albicans
    26 Bordetella pertussis
    27 Legionella pneumophila
    28 Staphylococcus aureus
    29 Staphylococcus epidermidis
    30 Pooled human nasal wash – representative of normal respiratory microbial flora

In-silico analysis

  • NO. PATHOGEN
    31 Mycoplasma pneumoniae 1
    32 Chlamydia pneumoniae 1
    33 Mycobacterium tuberculosis 1
  • NO. PATHOGEN
    34 Pneumocystis jirovecii (PJP) 1
    35 Human coronavirus HKU1 2

1No significant similarity found. Homology-based cross-reactivity can be ruled out.
2The comparison analysis revealed a 23% homology across 82% of the sequences. the likelihood of cross reactivity of human coronavirus HKU1 cannot be ruled out.

INTERFERENCES

RADI COVID-19 Ag Rapid Test has been tested with 20 potentially interfering endogenous or exogenous substances in negative clinical matrix with the absence or presence of SARS-CoV-2 (3X LoD). No inference was seen in the study.

NO. SUBSTANCE ACTIVE INGREDIENT CONCENTRATION
1 Endogenous Mucin 0.5 %
2 Endogenous Whole blood 4 %
3 Nasal spray Cromolyn 15 %
4 Nasal drops Phenylephrine 15 %
5 Nasal gel Sodium Chloride 5 %
6 Respiratory Specimens Biotin 100 ug/ml
7 Antibiotic Chloramphenicol 100 mg/dL
8 Anti-inflammatory medication Acetaminophen 10 mg/ml
9 Throat lozenges Benzocaine 1.5 mg/ml
10 Anti-viral drugs Oseltamivir Phosphate 5 mg/ml
11 Antibiotic, Nasal Ointment Mupirocin 10 mg/ml
12 Antibacterial, Systemic Tobramycin 4 ug/ml
13 Therapeutics/Drug Dexamethasone 1 mg/ml
14 Endogenous Oxymetazoline 15 %
15 Endogenous Fluticasone Propionate 5 %
16 Endogenous Menthol 1.5 mg/ml
17 Naso GEL (NeilMed)* Sodium bicarbonate 5%
18 Zicam* zinc acetate 5 %
19 Homeopathic (Alkalol)* Eucalyptol 1:10 dilution
20 Sore Throat Phenol Spray* Phenol 15 %(v/v)

* : For the list of substances highlighted with “*”, due to difficulty to secure branded commercial products, we tested its active ingredients for likelihood of potential interference in the consideration of main active ingredient of the concerning products.