Clungene RSV Antigen Rapid Test

{0} FDA U.S. FOOD &amp; DRUG ADMINISTRATION # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY ## I Background Information: A 510(k) Number K253318 B Applicant Hangzhou Clongene Biotech Co., Ltd. C Proprietary and Established Names Clungene RSV Antigen Rapid Test D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | GQG | Class I | 21 CFR 866.3480 - Respiratory Syncytial Virus Serological Reagents | MI - Microbiology | ## II Submission/Device Overview: A Purpose for Submission: To obtain a substantial equivalence determination for the Clungene RSV Antigen Rapid Test B Measurand: Nucleoprotein antigen from Respiratory Syncytial Virus (RSV) C Type of Test: Qualitative lateral flow immunoassay ## III Intended Use/Indications for Use: A Intended Use(s): See Indications for Use below. B Indication(s) for Use: The Clungene RSV Antigen Rapid Test is a lateral flow immunoassay intended for the qualitative detection of respiratory syncytial virus (RSV) nucleoprotein antigen in Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov {1} nasopharyngeal swab specimens from patients with signs and symptoms of respiratory infection. The test is intended for in vitro diagnostic use as an aid in the diagnosis of RSV infections. Negative results do not preclude RSV infection and should not be used as the sole basis for treatment or patient management decisions. A negative test is presumptive. It is recommended that negative test results be confirmed by viral cell culture or an FDA-cleared molecular assay. ## C Special Conditions for Use Statement(s): Rx - For prescription use only IVD – For in vitro diagnostic use ## D Special Instrument Requirements: Not applicable ## IV Device/System Characteristics: ## A Device Description: The Clungene RSV Antigen Rapid Test is a lateral flow immunoassay intended for the qualitative detection of respiratory syncytial virus (RSV) nucleoprotein antigen in human nasopharyngeal swab specimens from individuals with signs and symptoms of respiratory infection, suspected of RSV by their healthcare provider. The test is intended for in vitro diagnostic use as an aid in the diagnosis of RSV infection. The Clungene RSV Antigen Rapid Test is validated for use with direct specimens without transport media. The Clungene RSV Antigen Rapid Test contains reagents sufficient to run 25 tests and includes: - 25 Test cassettes: each cassette with desiccant in individual foil pouch. - 25 Buffer tubes: tube sealed with foil film containing 0.3 mL of extraction buffer. - 25 Sterilized swabs: single use swab for specimen collection. - 1 Positive Control Swab: non-infectious recombinant RSV nucleocapsid protein is dried on the swab tip - 1 Negative Control Swab: non-infectious recombinant Group C Streptococci protein is dried on the swab tip - 1 Instructions for Use - 1 Quick Reference Card - 1 Workstation ## B Principle of Operation: The Clungene RSV Antigen Rapid Test is a lateral flow immunoassay that operates based on the double antibody sandwich technique principle. Nasopharyngeal swab specimens from individuals are processed using extraction buffer in a buffer tube, and the extracted sample is then added to the specimen well. When present in the sample, respiratory syncytial virus (RSV) nucleoprotein antigen reacts with anti-RSV antigen antibody that has been conjugated with colored micro particles, forming an antigen-antibody labeled complex. This complex migrates across the membrane through capillary action until it reaches the test line, where it is captured by pre-coated anti-RSV antigen antibody. If the specimen contains RSV nucleoprotein antigen, a colored line will appear in the test line (T) region, indicating a positive result, while the absence of RSV nucleoprotein antigen will result in no colored line appearing in this region, indicating a negative result. As a procedural control, a colored line will always appear at the control line (C) K253318- Page 2 of 16 {2} region, confirming that the proper volume of specimen has been added, and that membrane wicking has occurred. The test result should be read at 15-20 minutes after adding the extracted sample to the specimen well. One line in the control line region (C), and another line in the test line region (T) indicates a positive result, regardless of color intensity. When only one line is present in the control region (C) and no line is present in the test line region (T), results are negative result. If the control line(C) fails to appear, the result is invalid, and the operator should review the procedure and repeat the test with a specimen and new test cassette. V Substantial Equivalence Information: A Predicate Device Name(s): Nano-Check RSV Test B Predicate 510(k) Number(s): K240280 C Comparison with Predicate(s): | Device & Predicate Device(s): | K240280 | K253318 | | --- | --- | --- | | Device Trade Name | Nano-Check RSV Test | Clungene RSV Antigen Rapid Test | | General Device Characteristic Similarities | | | | Intended Use/Indications For Use | The Nano-Check RSV Test is a rapid immunochromatographic assay for the qualitative detection of respiratory syncytial virus (RSV) nucleoprotein antigen in anterior nasal swab specimens from patients with signs and symptoms of respiratory infections. This test is intended for in vitro diagnostic use to aid in the diagnosis of RSV infections in infants and pediatric patients aged 6 months to 6 years old, and adults over 60 years of age. Negative results do not preclude RSV infection and should not be used as the sole basis for treatment or other management decisions. A negative test is presumptive. It is recommended that negative test results be confirmed by viral cell culture or an FDA-cleared molecular assay. | The Clungene RSV Antigen Rapid Test is a lateral flow immunoassay intended for the qualitative detection of respiratory syncytial virus (RSV) nucleoprotein antigen in nasopharyngeal swab specimens from patients with signs and symptoms of respiratory infection. The test is intended for in vitro diagnostic use as an aid in the diagnosis of RSV infections. Negative results do not preclude RSV infection and should not be used as the sole basis for treatment or patient management decisions. A negative test is presumptive. It is recommended that negative test results be confirmed by viral cell culture or an FDA-cleared molecular assay. | K253318- Page 3 of 16 {3} VI Standards/Guidance Documents Referenced: | Document | Title | Publisher | Applicable study | | --- | --- | --- | --- | | 21 CFR 866.3480 | Respiratory syncytial virus serological reagents | FDA | All | | 11135:2014 | Sterilization of health care products - Ethylene oxide - Requirements for development, validation and routine control of a sterilization process for medical devices | ISO | Sterility | | 11737-2:2020 | Sterilization of health care products - Microbiological methods | ISO | Sterility | | 10993-7 | Biological Evaluation of Medical Devices – Part 7: Ethylene Oxide Sterilization Residuals | ISO | Sterility | | 10993-1 -11:2017 | Biological Evaluation of Medical Devices – Acute systemic toxicity | ISO | Biocompatibility | | 10993-5: Third Edition 2009-06-01 | Biological Evaluation of Medical Devices - Tests for in vitro cytotoxicity | ISO | Biocompatibility | K253318- Page 4 of 16 {4} K253318- Page 5 of 16 | Document | Title | Publisher | Applicable study | | --- | --- | --- | --- | | 10993-10: 2021 | Biological Evaluation of Medical Devices – Tests for irritation and skin sensitization | ISO | Biocompatibility | | 10993-23:2021 | Biological Evaluation of Medical Devices – Intracutaneous reactivity | ISO | Biocompatibility | | 10993-11:2017 USP-NF (151) Pyrogen test | Biological Evaluation of Medical Devices – Pyrogen test | ISO | Biocompatibility | # VII Performance Characteristics: ## A Analytical Performance: ### 1. Precision/Reproducibility: #### a. Multi-Lot, Multi-Site Precision and Reproducibility Study: A multi-site reproducibility study was conducted to assess the performance consistency of the candidate device across different testing environments and operators. The study used a contrived sample panel consisting of a true negative sample (no analyte), a high negative sample at 0.05x LoD, a low positive sample at 1x LoD, and a moderate positive sample at 3x LoD. The evaluation was performed by two (2) untrained operators at each of three (3) testing sites over a five (5)-day period, providing an assessment of device’s precision and reproducibility under varied operational conditions and critical concentration thresholds. Contrived swab samples were prepared by spiking an RSV-A strain into pooled negative nasal fluid (PNF), with each diluted sample (50 µL) applied directly onto the sample collection swab head, while true negative swab samples consisted of 50 µL of negative PNF applied to the swab head. The contrived sample swabs were randomized and blinded to operators across all three (3) sites, with 150 replicates tested per concentration level (3 sites x 2 operators/site x 5 days x 5 replicates per day) yielding 450 total results. All outcomes met the predefined acceptance criteria with no significant differences observed by operators, by days, and testing sites, confirming acceptable device performance reproducibility across the multi-site evaluation. The data in the Table 1 is presented for site-to-site reproducibility. Table 1. Summary of Multi-Site Reproducibility Study Results | Sample | # of positive result/# of total tested (% positive rate) | | | Total sample count (% positive rate) | | --- | --- | --- | --- | --- | | | Site 1 | Site 2 | Site 3 | | | True negative | 0/150 (0.0%) | 0/150 (0.0%) | 0/150 (0.0%) | 0/450 (0.0%) | | High negative (0.05x LoD) | 0/150 (0.0%) | 0/150 (0.0%) | 0/150 (0.0%) | 0/450 (0.0%) | | Low positive (1x LoD) | 150/150 (100.0%) | 150/150 (100.0%) | 150/150 (100.0%) | 450/450 (100.0%) | {5} K253318- Page 6 of 16 ## b. Multi-Lot, Single-Site Precision Study: To supplement the initial five-day study, a second evaluation was conducted to determine the lot-to-lot precision of the Clungene RSV Antigen Rapid Test over 12 non-consecutive days. This single-site study utilized three (3) distinct test kit lots and three (3) concentrations of RSV-A and a negative sample. Consistent with the first study, RSV-A (same strain/lot) was spiked into pooled negative nasal fluid (PNF). Samples were blinded and randomized before allotting them to the operators. 50μL of each sample was applied to dry nasal swabs and processed per the IFU of the candidate device. All panel members were tested in duplicate with 3 device lots per day and the study was conducted for 12 days (i.e., 1 site x 3 lots x 2 replicates per day x 12 days). A total of 72 results were obtained per concentration. All replicates prepared at each concentration, demonstrated 100% agreement with the expected results. The results from precision study are summarized below. Table 2. Summary of Precision Study Results | Sample | # of positive result/# of total tested (% positive rate) | | | Total sample count (% positive rate) | | --- | --- | --- | --- | --- | | | Lot 1 | Lot 2 | Lot 3 | | | True negative | 0/24 (0.0%) | 0/24 (0.0%) | 0/24 (0.0%) | 0/72 (0.0%) | | High negative (0.05x LoD) | 0/24 (0.0%) | 0/24 (0.0%) | 0/24 (0.0%) | 0/72 (0.0%) | | Low positive (1x LoD) | 24/24 (100.0%) | 24/24 (100.0%) | 24/24 (100.0%) | 72/72 (100.0%) | | Moderate positive (3x LoD) | 24/24 (100.0%) | 24/24 (100.0%) | 24/24 (100.0%) | 72/72 (100.0%) | ## 2. Linearity: Not applicable. This is a qualitative assay with binary, visually read results ## 3. Analytical Specificity/Interference: ### a. Cross Reactivity and Microbial Interference: Cross reactivity and microbial interference studies were conducted to determine potential assay interference from other respiratory pathogens/microbial flora that may be present in nasal samples. A comprehensive panel of viruses, bacteria, fungi, and PNF was evaluated. For the cross-reactivity study, organisms were diluted in PNF and tested in triplicate per lot using three (3) lots of devices in the absence of RSV. No cross-reactivity was observed with the organisms tested (Table 3). For the microbial interference study, organisms were diluted in PNF in the presence of low levels (2x LoD) of RSV-A and tested in triplicate (3) using three (3) lots of devices. No microbial interference was observed (Table 3). Table 3. Cross Reactivity and Microbial Interference Study Results {6} K253318- Page 7 of 16 | Microorganism | Concentration | Test Results | | | | | --- | --- | --- | --- | --- | --- | | | | Cross Reactivity | | Microbial Interference | | | | | n/N^{1} | Result Agreement (%)^{2} | n/N^{1} | Result Agreement (%)^{2} | | Adenovirus 1 | 1.6 X 10^{6} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Adenovirus 2 | 1.1 X 10^{7} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Adenovirus 3 | 8.9 X 10^{8} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Adenovirus B7 | 8.9 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | SARS-CoV-2, USA-WA1/2020 | 3.8 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Human coronavirus (OC43) | 1.6 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Human coronavirus (229E) | 2.8 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Human coronavirus (NL63) | 1.8 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Human herpesvirus | 1.2 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Parainfluenza virus type 1 | 8.9 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Parainfluenza virus type 2 | 8.9 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Parainfluenza virus type 3 | 8.5 X 10^{6} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Parainfluenza virus type 4 | 1.6 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Measles virus | 2.1 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Mumps virus | 1.2 X 10^{6} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Rhinovirus | 2.2 X 10^{6} PFU/mL | 0/9 | 100% | 9/9 | 100% | | Herpes simplex virus | 1.4 X 10^{6} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Varicella virus | 2.9 X 10^{6} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Epstein-Barr Virus | 3.2 X 10^{7} Copies/mL | 0/9 | 100% | 9/9 | 100% | | Rotavirus A | 5.0 X 10^{6} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Enterovirus 68 | 1.5 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Enterovirus 71 | 8.9 X 10^{6} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | {7} K253318- Page 8 of 16 | MERS-CoV | 8.0 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | --- | --- | --- | --- | --- | --- | | Influenza A (H1N1, Brisbane/59/07) | 3.6 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Influenza A (H1N1, California/7/09) | 8.0 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Influenza A (H3N2 Brisbane/10/07) | 5.3 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Influenza A (H3N2, Victoria/361/11) | 3.8 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Influenza A (H3N2, HongKong/8/68) | 1.3 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Influenza A (H3N2, Wisconsin/67/05 | 1.1 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Influenza B (Yamagata/16/88) | 1.3 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Influenza B (Yamagata, 504/00 | 6.3 X 10^{5} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Influenza B (Victoria, Malaysia/2506/04) | 1.9 X 10^{6} TCID_{50}/mL | 0/9 | 100% | 9/9 | 100% | | Influenza B (Victoria, 2/87) | 7.6×10^{5} TCID50/mL | 0/9 | 100% | 9/9 | 100% | | Influenza B (Victoria, /78/2015) | 2.8×10^{6} TCID50/mL | 0/9 | 100% | 9/9 | 100% | | Human Metapneumovirus | 6.3×10^{5} TCID50/mL | 0/9 | 100% | 9/9 | 100% | | Coxsackievirus | 1.6×10^{6} TCID50/mL | 0/9 | 100% | 9/9 | 100% | | Bordetella pertussis | 8.7 X 10^{7} CFU/ml | 0/9 | 100% | 9/9 | 100% | | Chlamydophila pneumoniae | 9.1 X 10^{6} CFU/ml | 0/9 | 100% | 9/9 | 100% | | Mycoplasma pneumoniae^{3} | 4.2 X 10^{5} CFU/ml | 0/9 | 100% | 9/9 | 100% | | Pseudomonas aeruginosa | 2.8 X 10^{8} CFU/ml | 0/9 | 100% | 9/9 | 100% | | Staphylococcus aureus | 9.5 X 10^{8} CFU/ml | 0/9 | 100% | 9/9 | 100% | | Haemophilus influenzae | 8.0 X 10^{7} CFU/ml | 0/9 | 100% | 9/9 | 100% | | Streptococcus pneumoniae | 1.0 X 10^{7} CFU/ml | 0/9 | 100% | 9/9 | 100% | | Streptococcus pyogenes | 1.3 X 10^{8} CFU/ml | 0/9 | 100% | 9/9 | 100% | | Streptococcus salivarius | 1.3 X 10^{7} CFU/ml | 0/9 | 100% | 9/9 | 100% | {8} K253318- Page 9 of 16 | Streptococcus mutans | 1.2 X 10^{6} CFU/ml | 0/9 | 100% | 9/9 | 100% | | --- | --- | --- | --- | --- | --- | | Legionella pneumophila | 1.6 X 10^{6} CFU/ml | 0/9 | 100% | 9/9 | 100% | | Klebsiella pneumoniae | 5.8 X 10^{7} CFU/ml | 0/9 | 100% | 9/9 | 100% | | Staphylococcus epidermidis | 1.2×10^{9} CFU/mL | 0/9 | 100% | 9/9 | 100% | | Mycobacterium tuberculosis | 1.2×10^{7} CFU/mL | 0/9 | 100% | 9/9 | 100% | | Candida albicans | 3.6×10^{6} CFU/mL | 0/9 | 100% | 9/9 | 100% | | Corynebacterium diphtheriae | 1.4×10^{6} CFU/mL | 0/9 | 100% | 9/9 | 100% | | Moraxella catarrhalis | 2.2×10^{8} CFU/mL | 0/9 | 100% | 9/9 | 100% | | Lactobacillus sp. | 5.0×10^{7} CFU/mL | 0/9 | 100% | 9/9 | 100% | | Neisseria meningitidis | 8.8×10^{7} CFU/mL | 0/9 | 100% | 9/9 | 100% | | Escherichia coli | 2.6 X 10^{7} CFU/ml | 0/9 | 100% | 9/9 | 100% | | Pooled human nasal wash | N/A | 0/9 | 100% | 9/9 | 100% | 1 # of positive results/# of replicates. 2 Agreement with the expected result. 3 Tested at lower than recommended concentration. ## b. Endogenous/Exogenous Substances Interference: The Clungene RSV Antigen Rapid Test was evaluated for performance in the presence of a panel of common interfering endogenous and exogenous substances. Potentially interfering substances were prepared in PNF to the recommended concentration. Virus negative PNF specimens were evaluated in triplicate/lot using three (3) lots of devices to confirm that the potentially interfering substances were not cross-reactive with the test. Positive samples were also prepared in PNF at 2x LoD (RSV-A) and were evaluated in the presence of interfering substances in triplicate/lot using three (3) lots of devices to confirm that these substances do not interfere with detection of RSV-A. No interference was observed (Table 4). Table 4. Interfering Substances Study Results | Potentially Interfering Substances | Concentration Tested | Test Results | | | | | --- | --- | --- | --- | --- | --- | | | | No analyte | | With Analyte | | | | | n/N^{1} | Result Agreement (%)^{2} | n/N^{1} | Result Agreement (%)^{2} | | Purified Mucin Powder | 2.5 mg/mL | 0/9 | 100% | 9/9 | 100% | | Whole Blood | 4% | 0/9 | 100% | 9/9 | 100% | | Phenylephrine HCl | 15% v/v | 0/9 | 100% | 9/9 | 100% | | Oxymetazoline HCl | 15% v/v | 0/9 | 100% | 9/9 | 100% | | Sodium Chloride with Preservatives | 15% v/v | 0/9 | 100% | 9/9 | 100% | {9} | Beclomethasone | 0.5 mg/mL | 0/9 | 100% | 9/9 | 100% | | --- | --- | --- | --- | --- | --- | | Budesonide Nasal Spray | 15% v/v | 0/9 | 100% | 9/9 | 100% | | Mometasone Furoate | 0.5 mg/mL | 0/9 | 100% | 9/9 | 100% | | Dexamethasone | 5 mg/mL | 0/9 | 100% | 9/9 | 100% | | Flunisolide | 5 mg/mL | 0/9 | 100% | 9/9 | 100% | | Triamcinolone Acetonide | 5 mg/mL | 0/9 | 100% | 9/9 | 100% | | Fluticasone Propionate | 5 mg/mL | 0/9 | 100% | 9/9 | 100% | | Histamine diHydrochloride | 10 mg/mL | 0/9 | 100% | 9/9 | 100% | | Benzocaine | 3 mg/mL | 0/9 | 100% | 9/9 | 100% | | Menthol | 3 mg/mL | 0/9 | 100% | 9/9 | 100% | | Zanamivir | 5 mg/mL | 0/9 | 100% | 9/9 | 100% | | Ribavirin | 5 mg/mL | 0/9 | 100% | 9/9 | 100% | | Oseltamivir Phosphate | 5 mg/mL | 0/9 | 100% | 9/9 | 100% | | Peramivir | 100 μg/mL | 0/9 | 100% | 9/9 | 100% | | Mupirocin | 10 mg/mL | 0/9 | 100% | 9/9 | 100% | | Tobramycin | 5 μg/mL | 0/9 | 100% | 9/9 | 100% | | D-Biotin | 3500 ng/mL | 0/9 | 100% | 9/9 | 100% | | Remdesivir | 0.2 mg/mL | 0/9 | 100% | 9/9 | 100% | | Molnupiravir | 2.5 mg/mL | 0/9 | 100% | 9/9 | 100% | | Paracetamol | 50 μg/mL | 0/9 | 100% | 9/9 | 100% | | Euphorbium Nasal Spray | 15% v/v | 0/9 | 100% | 9/9 | 100% | | Sinna Nasal Spray | 15% v/v | 0/9 | 100% | 9/9 | 100% | | Quantum HEALTH TheraZinc | 15% v/v | 0/9 | 100% | 9/9 | 100% | | Leukocytes | 5×10^6 cells/mL | 0/9 | 100% | 9/9 | 100% | $^\dagger$ # of positive results/# of replicates. $^2$ Agreement with the expected result. # 4. Detection Limit and Assay Reportable Range: # a. Limit of Detection (LoD): An LoD study was conducted to determine the lowest detectable concentration of two (2) strains of RSV-A (2006 isolate and ATCC-2012-10), and two (2) strains of RSV-B (ATCC-2012-11 and BWV/14617/85) at which at least $95\%$ of all true positive replicates return a positive result. Testing was conducted on three (3) lots of test devices. A preliminary LoD was first determined by testing serial 10-fold dilutions of virus stocks diluted in PNF in three (3) replicates per device lot for a total of 9 replicates per dilution. A $50~\mu \mathrm{L}$ sample of each virus diluted in PNF was pipetted onto the dry swab. The swab was then tested per the IFU. The preliminary LoD of each virus was confirmed by testing an additional twenty samples/lot for each viral stock at the preliminary LoD concentration. If the preliminary LoD yielded $100\%$ -positive test results/lot from each 1:10 dilution, it was further evaluated using a 2-fold dilution series, in 20 replicates/lot for each level, to refine the LoD. As per the acceptance criteria for confirmation of the LoD, at least $95\%$ of the replicates $(\geq 19/20/\mathrm{lot})$ should be positive to be considered as the confirmed LoD. The confirmed LoDs observed were identical for the three (3) lots of devices tested for each virus strain. Table 5. Limit of Detection- Clungene RSV Antigen Rapid Test K253318- Page 10 of 16 {10} | Virus strains | LoD | #Positive/#Total | % Positive | | --- | --- | --- | --- | | RSV-A (2006 isolate) | 1.5 x 10² TCID_{50}/mL | 60/60 | 100.0% | | RSV-A (ATCC-2012-10) | 6.4 x10³ TCID_{50}/mL | 60/60 | 100.0% | | RSV-B (ATCC-2012-11) | 1.9 x 10² TCID_{50}/mL | 60/60 | 100.0% | | RSV-B (B WV/14617/85) | 2.2 x 10³ TCID_{50}/mL | 60/60 | 100.0% | ## b. High Dose Hook Effect: A high-dose hook effect study was conducted to evaluate whether high concentrations of target analyte could yield false negative results. Each sample was prepared beginning at a high "stock" viral concentration and subsequently diluted in 1:10 serial dilutions down to 1x LoD. Following preparation, 50 µL of each sample was applied to swabs, which were then processed according to the Instructions for Use (IFU). Three (3) lots of devices were tested, and all samples were tested in triplicate per lot. The results demonstrated no evidence of a high-dose hook effect across the concentrations evaluated in this study. Table 6. High-Dose Hook Effect Study Results | RSV Strain | Concentration (TCID50/mL) | #Positive/#Total | % Positive | | --- | --- | --- | --- | | RSV-A (2006 Isolate) | 1.5×10⁶ (10000×LoD) | 9/9 | 100.0% | | | 1.5×10⁵ (1000×LoD) | 9/9 | 100.0% | | | 1.5×10⁴ (100×LoD) | 9/9 | 100.0% | | | 1.5×10³ (10×LoD) | 9/9 | 100.0% | | | 1.5×10² (1×LoD) | 9/9 | 100.0% | | RSV-A (ATCC-2012-10) | 6.4×10⁶ (1000×LoD) | 9/9 | 100.0% | | | 6.4×10⁵ (100×LoD) | 9/9 | 100.0% | | | 6.4×10⁴ (10×LoD) | 9/9 | 100.0% | | | 6.4×10³ (1×LoD) | 9/9 | 100.0% | | RSV-B (ATCC-2012-11) | 3.8×10⁴ (200×LoD) | 9/9 | 100.0% | | | 1.9×10⁴ (100×LoD) | 9/9 | 100.0% | | | 1.9×10³ (10×LoD) | 9/9 | 100.0% | | | 1.9×10² (1×LoD) | 9/9 | 100.0% | | RSV-B (B WV/14617/85) | 8.9×10⁵ (400×LoD) | 9/9 | 100.0% | | | 2.2×10⁵ (100×LoD) | 9/9 | 100.0% | | | 2.2×10⁴ (100×LoD) | 9/9 | 100.0% | | | 2.2×10³ (100×LoD) | 9/9 | 100.0% | ## c. Inclusivity: K253318- Page 11 of 16 {11} Analytical reactivity testing for the Clungene RSV Antigen Rapid Test was conducted to ensure that the device can adequately detect a variety of strains for RSV viruses. A selection of temporally, geographically, and genetically diverse RSV-A and RSV-B strains were tested for inclusivity. A series of ten-fold dilutions of each virus strain was spiked into PNF and tested to determine an approximate LoD of the test for each virus. The lowest concentration with 100% positive replicates was identified and additional 2-fold dilutions below that approximate LoD were tested to demonstrate inclusivity. Three (3) lots of devices were tested, and all samples were tested in triplicate per lot. Based on the dilution series, the minimum detectable concentration was defined as the lowest concentration for which all nine (9) replicates (3 replicates/lot X 3 lots) were detected. Results are summarized below and demonstrate that the test tests can detect the analytes across a range of viral strains. Table 7. Inclusivity Results | Strain | Lowest Concentration with 100% Detection | | --- | --- | | RSV-A (A2) | 1.6×10^{3} TCID_{50}/mL | | RSV-A(Long) | 8.0×10^{2} PFU/mL | | RSV-A(1/2015 Isolate #1) | 6.3×10^{1} TCID_{50}/mL | | RSV-A (2/2015 Isolate 2) | 1.17×10^{1} TCID_{50}/mL | | RSV-B(CH93(18)-18) | 3.15×10^{2} TCID_{50}/mL | | RSV-A (A2023/06 12NSMM) | 7.0×10^{2} TCID_{50}/mL | | RSV-B (B1) | 7.0×10^{2} TCID_{50}/mL | | RSV-B (18537) | 1.24×10^{3} TCID_{50}/mL | | RSV-B (9320) | 1.4×10^{3} TCID_{50}/mL | d. **Assay Reportable Range:** Not applicable; the device is a binary qualitative assay that is visually read. 5. **Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):** a. **Controls:** i. **Internal Controls:** The Clungene RSV Antigen Rapid Test contains a built-in internal procedural control. The appearance of the control line 'C'-Line on the test strip ensures that sufficient flow of the sample occurred and that the reagents are functioning properly during the assay. If no visible signal appears on the control line, the test result is invalid, and the operator is instructed in the labelling to test the sample again with another test device. ii. **External Controls:** The Clungene RSV Antigen Rapid Test contains one positive external control swab and one negative external control swab that allows for monitoring of the performance of the assay. The positive control swab contains non-infectious recombinant RSV nucleocapsid protein, and the negative control swab non-infectious recombinant Group C Streptococci protein dried on the swab tip. K253318- Page 12 of 16 {12} Lot-to-lot precision of the external positive and negative control swabs was evaluated by testing ten (10) replicates each of three (3) lots of the external controls. For each external control lot, all positive and negative controls produced 100% agreement with the expected results, as summarized below. Table 8. Summary of External Controls Precision Study Results | External Control | Control Lot No. | Test Result/Total Tests | | % Agreement to Expected Result | | --- | --- | --- | --- | --- | | | | # Negative | # Positive | | | External Positive Control | 1 | 0/10 | 10/10 | 100% | | | 2 | 0/10 | 10/10 | 100% | | | 3 | 0/10 | 10/10 | 100% | | External Negative Control | 1 | 10/10 | 0/10 | 100% | | | 2 | 10/10 | 0/10 | 100% | | | 3 | 10/10 | 0/10 | 100% | b. **Stability:** i. **Specimen Stability:** A specimen stability study was conducted to evaluate the stability the specimen on swabs provided with the test kit (i.e., Copan Flexible FLOQSwab) and when tested under various conditions. Two test samples were prepared: negative samples (pooled negative nasal fluid, PNF) and contrived positive samples (prepared by spiking RSV-A at 2x LoD into PNF) and tested using three (3) device lots. Fifty (50) µL of either three (3) positive or three (3) negative samples per lot were applied to dry Copan Flexible FLOQSwabs. Each swab was placed into a sterile, empty tube and stored under temperature conditions for specified durations as described below: - **Refrigerated (2-8 °C):** Samples were stored for 0, 6, 12, 24, and 48 hours. - **Ambient conditions (Room temperature, and 35 °C):** Samples were stored separately at room temperature, and 35 °C for 0, 1, 2, 3, and 4 hours. - **Frozen (below -20 °C):** Samples were stored at -20 °C for 0, 1, 3, and 5 days. - **Freeze-thaw stability:** Samples were stored at -20 °C for 24 hours, then transferred to room temperature for thawing, constituting one freeze-thaw cycle. Repeated to constitute to 2 freeze-thaw cycles. After each freeze-thaw cycle, tests were performed in triplicate using three device lots by three operators. Subsequently, all exposed sample swabs were tested at each storage condition and timepoint and the results were compared with time 0. All negative samples produced negative results at all conditions and timepoints tested. All 2x LoD positive samples yielded expected positive results at all conditions and timepoints tested. K253318- Page 13 of 16 {13} ii. Real Time Stability: A real-time stability study was conducted to evaluate stability and determine the shelf-life of the unopened kit. To validate the shelf-life at 2-30°C, three (3) unopened Clungene RSV Antigen Rapid Test lots with three (3) unopened lots of external controls were stored at 4°C and 30°C. At defined intervals, an assessment of each lot was conducted with the following panel of test samples: negative clinical matrix and contrived positive samples prepared at 1x LoD of RSV-A virus. Fifty (50) µL of each sample were applied to the swab and tested according to the IFU. Five replicates of each sample were tested per lot for each time point. Baseline testing was performed within one month of each manufactured lot. Subsequent time testing was conducted every month for up to 3 months and then at 6, 12, 18, 21, 24, 25, 26, and 27 months. At the time of clearance, all study data have met the protocol defined acceptance criteria, and support storage of the test kits at 2-30°C for up to 26 months. 6. Assay Cut-Off: Not applicable as this is a qualitative visually read assay without numeric data. B. Comparison Studies: 1. Method Comparison with Predicate Device: See section C (clinical studies) below. 2. Matrix Comparison: The Clungene RSV Antigen Rapid Test is intended for use with direct nasopharyngeal swab specimens. As the intended sample type is evaluated in the clinical study, a matrix comparison study is not applicable. C. Clinical Studies: A prospective clinical study was conducted to evaluate the performance of the Clungene RSV Antigen Rapid Test using nasopharyngeal (NP) swab specimens. A total of seven hundred and twenty-two (722) NP swab specimens were prospectively collected from patients with signs and symptoms consistent with respiratory tract infection from five (5) clinical sites between November 2024 and April 2025. Two NP swabs were collected sequentially from each subject by the study operator/s. The sample for the RT-PCR was collected first from one nostril and placed into transport media for comparator testing. The second swab for the RSV Antigen Rapid Test was collected from the other nostril and tested immediately with the Clungene RSV Antigen Rapid Test at the site. Results from the candidate device were compared to NP swab specimen results when tested with an FDA cleared RT-PCR assay for RSV to demonstrate performance. Patient Demographics Demographic information was collected for all seven hundred and twenty-two (722) patients included in the study. Table 9. Patient Demographics K253318- Page 14 of 16 {14} | Characteristics of the study population | | N=722 | Percent (%) | | --- | --- | --- | --- | | Sex | Male | 321 | 44.5% | | | Female | 401 | 55.5% | | Age | <6 | 146 | 20.2% | | | 6-21 | 232 | 32.1% | | | 21-59 | 239 | 33.1% | | | ≥60 | 105 | 14.5% | | Race | Asian | 134 | 18.6% | | | Black or African American | 92 | 12.7% | | | White or Caucasian | 362 | 50.1% | | | Native Hawaiian or Other Pacific Islander | 2 | 0.3% | | | Other (Mixed race) | 10 | 1.3% | | | American Indian or Alaskan native | 1 | 0.1% | | | Prefer not to say | 121 | 16.8% | Table 10. RSV Performance of the Clungene RSV Antigen Rapid Test with NP Swab Specimens Compared to RT-PCR | Clungene RSV Antigen Rapid Test | Molecular Comparator | | | | --- | --- | --- | --- | | | Positive | Negative | Total | | Positive | 98 | 5 | 103 | | Negative | 13 | 606 | 619 | | Total | 111 | 611 | 722 | | Positive Percent Agreement (PPA) | 88.3% (95% CI: 81.0%-93.0%) | | | | Negative Percent Agreement (NPA) | 99.2% (95% CI: 98.1%-99.6%) | | | 1. Clinical Sensitivity: Please refer to Section VI.C (Clinical Studies) above for the clinical validation. 2. Clinical Specificity: Please refer to Section VI.C (Clinical Studies) above for the clinical validation. 3. Clinical Cut-Off: A clinical cut-off study is not applicable, there is no clinical cut-off related to the presence of RSV in patient samples. D Expected Values/Reference Range: A patient sample is expected to be negative for RSV. VIII Proposed Labeling: The labeling supports the finding of substantial equivalence for this device. K253318- Page 15 of 16 {15} IX Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision to the predicate. K253318- Page 16 of 16