Read instruction leaflet carefully prior to testing. For professional use, only.

COVID-19 Antigen Card Test

CIPtest

Rapid Immunochromatographic Card Test for the detection of SARS-CoV-2 Antigen in Human Nasopharyngeal discharge.

Intended Use

CIPTEST COVID-19 Antigen Card Test is a lateral flow immunochromatographic assay for qualitative detection of nucleocapsid protein antigen from SARS-CoV-2 in nasopharyngeal (NP) swab specimens directly collected from individuals who are suspected of COVID-19 by their healthcare provider.

CIPTEST COVID-19 Antigen Card Test is for in vitro diagnostic use and intended as an aid to detection of nucleocapsid protein antigen in patient with clinical symptoms of SARS-CoV-2 infection. It provides only an initial screening test results and more specific alternative diagnosis method should be performed in order to obtain the confirmation of SARS-CoV-2 infections. The test is not automated and does not require any additional instrument. Test is designed to be performed by Laboratory professionals/trained users only.

Introduction

The novel coronavirus 2019-nCoV has recently emerged as a human pathogen in the city of Wuhan in China’s Hubei province, causing fever, severe respiratory illness, and pneumonia—a disease recently named COVID-19. According to the World Health Organization (WHO), as of 24 July 2020, there have been 15,296,926 confirmed cases of COVID-19, including 628,903 deaths, reported to WHO. The emerging pathogen was rapidly characterized as a new member of the beta-coronavirus genus, closely related to several bat coronaviruses and to severe acute respiratory syndrome coronavirus (SARS-CoV). Compared with SARS-CoV, 2019-nCoV appears to be more readily transmitted from human to human, spreading to multiple continents and leading to the WHO’s declaration of a Public Health Emergency of International Concern (PHEIC) on 30 January 2020.

Common signs of a person infected with a coronavirus include respiratory symptoms, fever, cough, shortness of breath and dyspnea. In more severe cases infection can cause pneumonia, severe acute respiratory syndrome (SARS), kidney failure and even death. Corona virus can be extracted through respiratory secretions or transmitted through oral fluid, sneezing, physical contact, and other air droplets.

There are 2 main protein involved and necessary for the infection of humans by corona virus. Which includes Spike protein (which is a trimeric protein containing 3 different sub-units, among three subunits subunit S2 is highly conserved and subunit S1 is essential to make and entry into host cell) and nucleocapsid protein which is essential for viral assembly. Thus these, 2 protein play an important role in the infection and viral life cycle in human beings and are the potential target molecules for development of rapid test for detection of corona virus infection in humans. CIPTEST COVID-19 Antigen Card Test uses nucleocapsid protein as a measurand and particularly, used for the identification of subclinical or asymptomatic cases. It also plays a role in reducing or preventing the transmission of infection as these individuals may transmit the virus.

As a Rapid Diagnostic Test with a 15-20 min testing time, CIPTEST COVID-19 Antigen Card Test could be effectively used at a large scale during population screening of COVID-19 infection.

Assay Principle

The detection kit uses the principle of immunochromatography: separation of components in a mixture through a medium using capillary force and the specific and rapid binding of an antibody to its antigen. Each cassette is a dry medium that has been coated separately with anti-coronavirus (anti Nucleocapsid) antibody molecule, this can be a monoclonal or a poly clonal antibody directed against nucleocapsid of 2019-nCoV and goat anti-chicken IgY antibody (control line). 2 free colloidal gold labeled antibodies, i.e. anti-nucleocapsid antibody as well as chicken IgY, will be sprayed on the conjugate pad. Once specimen as a nasopharyngeal swab is collected and diluted in extraction buffer, this diluted specimen will be applied on sample pad. The specimen in buffer will pass through the conjugate pad and bind with anti-nucleocapsid protein antibody conjugated with colloidal gold on the conjugate pad will form a complex of antigen-antibody colloidal gold and will migrate towards test and control lines. Application of diluted specimen will also rehydrate the colloidal gold conjugates sprayed in conjugate pads. Thus, the formed complex of antigen-antibody-colloidal gold will migrate through capillary action and binds with the coated antibody molecules at test line, thus providing reactive result. If there is no formation of antigen-antibody colloidal gold complex, it will not bind with the test line and there will not be any development of test lines. Chicken IgY conjugate with colloidal gold will bind with control line antibody irrespective of reactive/non-reactive specimens.

MATERIALS PROVIDED	PACK SIZE: 25 TESTS
Test device pouch containing: test device and desiccant	25 Nos.
Nasopharyngeal swab	25 Nos.
Reaction buffer vial with nozzle	25 Nos.
Extraction Buffer Bottle	3 No.
Instructions for use	1 No.

Materials required but not provided

New pair of disposable gloves & facemask
Pen
Timer
Extra Nasopharyngeal swab, if needed
PPE Kits
Biohazardous waste container

Storage and Stability

CIPTEST COVID-19 Antigen Card Test kit should be stored at 4-30°C, out of direct sunlight.
Do not freeze the kit or components.
The kit is sensitive to humidity and heat. Do not store the kit at temperatures above 30°C and in humid conditions.
Extraction Buffer Bottle (unopened) & the unopened test device are stable until the expiry date printed on the label, when stored at 4-30°C.
Perform the test immediately after removing the test device from the aluminium pouch. If a desiccant color has changed from orange to green, do not use the test device.
The test device is stable until the printed expiry date on the pouch/external secondary packaging.

Precautions

Wear protective clothing, masks, gloves and take other appropriate safety precautions to avoid or reduce the risk of infection while handling of specimen.
Dispose of used gloves as biohazard waste. Wash hands thoroughly afterward.
Avoid splashing or aerosol formation.
Clean up spills thoroughly using an appropriate disinfectant.
Decontaminate and dispose of all used specimens, test devices, nasopharyngeal swab, Extraction buffer bottle and Reaction buffer vial with nozzle, in a biohazardous waste container.
CIPTEST COVID-19 Antigen Card Test kit requires the handling of human specimens. It is recommended that all human-sourced materials and all consumables contaminated with potentially infectious materials be considered potentially infectious and handled in accordance with the OSHA Standard on Bloodborne Pathogens. Biosafety Level 2 or other appropriate regional, national, and institutional biosafety practices should be used for materials that contain, are suspected of containing, or are contaminated with infectious agents.

Warnings

For in vitro diagnostic use only.
The test has been authorized only for the presence of specific antigen to SARS-CoV-2 and not for any other viruses or pathogens.
Read the instructions carefully before performing the test, any deviation will invalidate the test results.
Apply standard biosafety precautions for handling and disposal of potentially infective materials including human biological specimens irrespective of disease state.
Do not drink the reaction tube/extraction buffer vial solution. It contains a preservative which may be toxic if ingested. When disposed of through sink, flush with a large quantity of water.
Devices and Extraction buffer bottle from different lot must not be used.
Do not use the test device if the pouch is not intact.
Do not use the nasopharyngeal swab, if found opened.
Do not use the test device if the desiccant color has changed from orange to green.
Do not smoke, eat or drink while handling specimens and performing a test.
Do not re-use the test device, nasopharyngeal swab and reaction buffer bottle with nozzle as these are for single use only.
Perform the test by using kit extraction buffer solution, any other solution or fluid will invalidate the test results.

Specimen Collection

Procedural Notes

Process the test sample immediately after collection.
Use only provided or recommended nasopharyngeal swab for specimen collection.
Collect the specimen wearing safety gloves to avoid contamination.
Do not touch the tip (specimen collection area) of the swab.

Nasopharyngeal (NP) swab collection: Insert minitip swab through the nostril parallel to the palate (not upwards) until resistance is encountered or the distance is equivalent to that from the ear to the nostril of the patient, indicating contact with the nasopharynx. Swab should reach depth equal to distance from nostrils to outer opening of the ear. Gently rub and roll the swab. Leave swab in place for several seconds to absorb secretions. Slowly remove swab while rotating it. Specimens can be collected from both sides using the same swab, but it is not necessary to collect specimens from both sides if the minitip is saturated with fluid from the first collection. If a deviated septum or blockage create difficulty in obtaining the specimen from one nostril, use the same swab to obtain the specimen from the other nostril.

Note: Specimen should be tested as soon as possible after collection. Specimens may be stored at room temperature for up to 1 hours prior to testing.

Pictorial Presentation

Nasopharyngeal Discharge Collection

Step 1

Tilt patient’s head back 70 degrees. Insert a (Minitip) sterile swab into the nostril of the patient, swab over the surface of the posterior nasopharynx. (Swab should reach depth equal to distance from nostrils to outer opening of the ear).

Step 2

Slowly rotate swab (right and left) in nostril to absorb secretions. Slowly remove swab after rotating it.

Instructions for Use

Follow the WHO/US CDC Universal Precautions for the safety against Novel Corona Virus (SARS-CoV-2).

Test Procedure

Bring the CIPTEST COVID-19 Antigen Card Test kit components to room temperature prior to testing.
Remove the test device and the other kit components from kit and place them on a flat, clean and dry surface.
Take extraction buffer bottle provided, twist open the cap and add 12 drops (approx 300 ?l) of extraction buffer into reaction tube. Collect the Nasopharyngeal discharge specimen with the help of Nasopharyngeal Swab provided inside the kit. Note: Please refer the Pictorial Presentation for Specimen collection.
Insert the Swab Specimen in the reaction tube and swirl the swab 5-10 times. Dispose of the used Nasopharyngeal Swab as biohazardous waste.
Remove the swab while squeezing the sides of the tube to extract the liquid from the swab. Close the nozzle cap tightly onto the reaction tube by pressing.
Invert the reaction tube and gently squeeze it to draw 3 drops of specimens into a sample well on the device and wait for 15-20 minutes for result. Do not interpret after 20 minutes.

Note: If test window (Background) is not clear at 15 minutes then read the result at 20 minutes.

After recording the results, dispose of the test device and remaining reaction tube solution as biohazardous waste.

Internal Quality Control

The visualization of the Red colored control line in CIPTEST COVID-19 Antigen Card Test indicates that the active ingredient of the strips are functional and the migration is successful. The control line is a procedural control, serves to demonstrate functional reagents and correct migration of fluid. If the procedural control line does not develop in 15 minutes, the test result is considered invalid and retesting with a new device is recommended.

Test Procedure Pictorial Presentation

Step 3

Take extraction buffer bottle provided, twist open the cap and add 12 drops (Approx 300 ?l) of extraction buffer into reaction tube.

Step 4

Insert the swab into an reaction tube filled with extraction buffer. swirl the swab 5-10 times.

Step 5

Remove the swab while squeezing the sides of the tube to extract the liquid from the swab.

Step 6

Close the nozzle cap tightly onto the reaction tube by pressing.

Step 7

Invert the reaction tube and gently squeeze it to draw 3 drops of specimens into a sample well of the device and wait for 15-20 minutes for result. Do not interpret after 20 minutes.

Results

Do not interpret after 20 minutes.

How to Interpret Test Results

Positive Results

In addition to the presence of the C line, if the T line develops, the test indicates the presence of SARS-CoV-2 Antigen. The result is positive or reactive.

Note: Interprete faint line as reactive line.

Negative Results

If only a single line appears, at control line “C” as in the figure, the test indicates the absence of SARS-CoV-2 Antigen.

The result is Negative or non-reactive.

Invalid Results

No presence of control line ’C’ in the results window (irrespective of presence of test lines) indicates an invalid result. The directions may not be followed correctly or the test may have detoriorated.

The Invalid test results should be retested with new test device.

Performance Characteristics

Clinical Performance

Clinical performance characteristics of CIPTEST COVID-19 Antigen Card Test was evaluated in the U.S. The study was carried out at a single site. Testing was performed by qualified operators having laboratory experience. In this study, testing was conducted by two trained personnel. Clinical samples were collected from suspected COVID-19 patients who presented symptoms within the last 7 days. Only those clinical samples were included in the analysis. Nasopharyngeal swabs were collected from patients and were tested using the CIPTEST COVID-19 Antigen Card Test, simultaneously nasal and oral swabs were collected in viral transport medium.

An FDA Emergency Use Authorized Real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) assay for the detection of SARS-CoV-2 was used as the comparator method for this study. In the study, the nasopharyngeal swabs were tested directly using the CIPTEST COVID-19 Antigen Card Test according to product instructions. Other two swabs (i.e. nasal and oral) were eluted in viral transport media (VTM) and sent to the clinical laboratory for rRT-PCR for the comparator method study.

CIPTEST COVID-19 Antigen Card Test Performance against the Allplex 2019-nCoV Assay method - 2 x 2 Contingency Table of the test results

CIPTEST COVID-19 Antigen Card Test Performance against the Comparator Method (Rrt-Pcr)-By threshold Cycle (Ct) Counts

The performance of CIPTEST COVID-19 Antigen Card Test with positive nasopharyngeal swab specimens was validated using an FDA Emergency Use Authorized Real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) assay. We correlated the results obtained for the thirty six (36) positive nasopharyngeal swab specimens with the comparator method (rRT-PCR) against CIPTEST COVID-19 Antigen Card Test. As presented in the table below, out of the thirty six (36) positive nasopharyngeal swab specimens, thirty (30) samples had a Ct value <30. Six (6) out of thirty six (36) had a Ct value ≥ 30. Our test was able to detect all 30 specimens having Ct value < 30. Out of the 6 specimens having Ct value ≥ 30, we correctly detected 4 specimens. This shows that our test is able to accurately detect positive nasopharyngeal swab specimens having a low viral count.

Analytical Performance

Limit of Detection (Analytical Sensitivity)

Limit of Detection (LOD) studies determine the lowest detectable concentration of SARS-CoV-2 at which 100% of all (true positive) replicates test positive. The LOD for the CIPTEST COVID-19 Antigen rapid test was established using limiting dilutions of gamma-irradiated SARS-CoV-2 (BEI Resources; NR-52287, Lot# 70033322).

The NR-52287 is a preparation of SARS-Related Coronavirus 2 (SARS-CoV2), isolate USA WA1/2020, that has been inactivated by gamma-irradiation at 5 x 10⁶ RADs. The material was supplied frozen at a concentration of 2.8 x 105 TCID50/ml.

Presumed negative nasopharyngeal samples were diluted in PBS (supplemented with 0.05% Tween, pH=7.4) collected and combined to create a clinical nasal swab matrix.

SARS-Related Coronavirus 2, Isolate USA WA1/2020, gamma irradiated was diluted in this natural nasal swab matrix pool to obtain the different viral dilutions required for the LOD study. Contrived nasal swab samples were prepared by absorbing 20 ?l of each of the viral dilutions onto the swab. The swabs were tested according the testing procedure provided in the package insert.

Based on this study we found the tentative limit of detection (LOD) to be 22.4 TCID₅₀/ swab. The Final LOD was determined as the lowest virus concentration that was detected ≥95% of the time the concentration of 22.4 TCID₅₀ / swab was tested (20) times. Twenty (20) of twenty (20) tests were found to be positive for the concentration 22.4 TCID₅₀ / swab. Based on this testing the final limit of detection (LOD) was confirmed as 22.4 TCID₅₀ / swab.

High Dose Hook Effect

High Dose Hook Effect studies determine the level at which false-negative results can be seen when very high levels of a target are present in a tested sample. To determine if the CIPTEST COVID-19 Antigen Test suffers from any high dose hook effect, increasing concentrations of gamma-irradiated SAR-SCoV-2 virus (BEI Resources NR-52287) were tested up to a concentration of 2.8 X 105 TCID₅₀/ml. In this study, the starting material was spiked into a volume of a pooled human nasal matrix obtained from healthy donors and confirmed negative for SARS-CoV-2. At each dilution, 20 ?L samples were added to swabs and the swabs processed for testing on the CIPTEST COVID-19 Antigen Test as per the Product Insert using the procedure appropriate for patient nasal swab specimens.

No impact on test performance or high dose hook effect was observed up to 2.8 X 10⁵TCID₅₀ / mL of gamma-irradiated SARS-CoV-2 with the CIPTEST COVID-19 Antigen Test.

Endogenous Interfering Substance Studies

A study was performed to demonstrate the potentially interfering substances that may be found in the upper respiratory tract in symptomatic subjects (including over the counter medications) do not cross-react or interfere with the detection of SARS-CoV-2 in the CIPTEST COVID-19 Antigen Test. Each substance was tested in triplicate in the absence or presence of SARS-CoV-2 at 67.2 TCID₅₀ / swab (3X LOD).

No cross-reactivity or interference was seen with the following endogenous interfering substances when tested at the concentration presented in the table below

Cross-Reactivity and Microbial Interference Study (Analytical Specificity)

Cross-reactivity of the CIPTEST COVID-19 Antigen Card Test was evaluated by testing a panel of related pathogens, high prevalence disease agents, and normal or pathogenic flora that are reasonably likely to be encountered in the clinical specimen and could potentially cross-react with the CIPTEST COVID-19 Antigen test including various microorganisms, viruses, and negative matrix.

Cross-Reactivity and potential interference of CIPTEST COVID-19 Antigen Card Test was evaluated by testing sixteen (16) viruses, twelve (12) micro-organisms and two (2) negative matrixes. Each organism and virus were tested in triplicate in the absence or presence of gamma irradiated SARS-CoV-2 at 3 X the concentration of LOD (67.2 TCID₅₀/swab). The final concentration of the organisms and viruses used in the study are documented in the table below (106 CFU/mL or higher for bacteria and 105 PFU/mL or higher for viruses was studied).

No cross-reactivity or interference was seen with the following microorganisms when tested at the concentration presented in the table below

Limitations

Negative results, should be treated as presumptive and confirmation with a molecular assay, if necessary for patient management, may be performed.
Failure to follow the instructions for use may adversely affect test performance and/or invalidate the test result.
If the differentiation of specific SARS viruses and strains is needed, additional testing, in consultation with state or local public health departments, is required.
Collected specimens may be frozen at -20°C and used up to 10 days after freezing and it is stable for 1 hour in extraction buffer at room temperature (15-30°C).
Results from antigen testing should not be used as the sole basis to diagnose or exclude SARS-CoV-2 infection or to determine infection status.
This test will indicate the presence of SARS-CoV-2 nucleocapsid protein antigen in the specimen from both viable and non-viable SARS-CoV-2 virus.
Test performance depends on the amount of virus (antigen) in the sample and may or may not correlate with viral culture results performed on the same sample.
The detection of SARS-CoV-2 nucleocapsid antigen is dependent upon proper specimen collection, handling, storage, and preparation. Failure to observe proper procedures in any one of these steps can lead to incorrect results.
Results from the device should be correlated with the clinical history, epidemiological data and other data available to the clinician evaluating the patient.
This device has been evaluated for use with human specimen material only. False-negative results may occur if the concentration of the target antigen in the clinical specimen is below the detection limits of the device.
This device is a qualitative test and does not provide information on the viral concentration present in the specimen.
This test cannot rule out diseases caused by other bacterial or viral pathogens.

Symbol Legends

Product Disclaimer & Warnings

Every warnings and precaution should be taken into consideration before using the test. Failure to consider “Precaution, Warning, and Limitations” may not ensure the diagnostic ability and accuracy of this product. The test result may accordingly be affected by environmental factors and/or user error outside of the control of the Manufacturer and Distributor.

A definitive clinical diagnosis should not be based on the results of a single test, but it should be made by the physician after all clinical and laboratory findings have been evaluated.

“In no event shall our company or its distributor is liable for any direct, indirect, punitive, unforeseen, incidental, special consequential damages, to property or life, whatsoever arising out of or connected with an incorrect diagnosis, whether positive or negative, in the use or misuse of this product”.

In the event of performance changes or product malfunction, please contact manufacturer and distributor.

Manufacturing Address

Premier Medical Corporation Private Limited

A1-302, GIDC, Sarigam 396155. Dist. Valsad, Gujarat, INDIA.

Customer support E-mail: info@premiermedcorp.com

Tel.: +91 2602780112/113.

Website: www.premiermedcorp.com

Marketed By

Cipla Ltd

Tower C, Peninsula Business Park,

Ganpatrao Kadam Marg, Lower Parel, Mumbai 400 013, India.

Date of Revision

24/11/2020