INTERPRETATION

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.