Flowflex® Plus Strep A Rapid Test Cassette; Flowflex® Plus Strep A Rapid Test Strip

{0} FDA U.S. FOOD & DRUG ADMINISTRATION # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY ## I Background Information: A 510(k) Number K251697 B Applicant ACON Laboratories, Inc. C Proprietary and Established Names Flowflex® Plus Strep A Rapid Test Cassette; Flowflex® Plus Strep A Rapid Test Strip D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | GTY | Class I | 21 CFR 866.3740 - Streptococcus Spp. Serological Reagents | MI - Microbiology | ## II Submission/Device Overview: A Purpose for Submission: A Dual Submission to obtain 510(k) clearance and CLIA Waiver for the Flowflex Plus Strep A Rapid Test Cassette and Flowflex Plus Strep A Rapid Test Strip. B Measurand: Group A β-hemolytic Streptococcus (GAS; Streptococcus pyogenes) antigens in throat swab specimens. C Type of Test: Lateral flow chromatographic immunoassay Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov {1} K251697 - Page 2 of 15 ## III Intended Use/Indications for Use: ### A Intended Use(s): See Indications for Use below. ### B Indication(s) for Use: **Flowflex Plus Strep A Rapid Test Cassette** The Flowflex Plus Strep A Rapid Test Cassette is a rapid chromatographic immunoassay for the qualitative detection of Group A Streptococcus antigen from throat swab specimens from symptomatic patients. The test is used to aid in the diagnosis of Group A Streptococcal infection. All negative test results should be confirmed by bacterial culture because negative results do not preclude infection with Group A Streptococcus and should not be used as the sole basis for treatment. **Flowflex Plus Strep A Rapid Test Strip** The Flowflex Plus Strep A Rapid Test Strip is a rapid chromatographic immunoassay for the qualitative detection of Group A Streptococcus antigen from throat swab specimens from symptomatic patients. The test is used to aid in the diagnosis of Group A Streptococcal infection. All negative test results should be confirmed by bacterial culture because negative results do not preclude infection with Group A Streptococcus and should not be used as the sole basis for treatment. ### C Special Conditions for Use Statement(s): - Rx - For Prescription Use Only - For in vitro diagnostic use only - A negative result must be confirmed by culture. A negative result may be obtained if the concentration of the Group A Streptococcus antigen present in the throat swab is not adequate or is below the detectable level of the test. ### D Special Instrument Requirements: N/A ## IV Device/System Characteristics: ### A Device Description: There are two formats of the test—cassette and strip. A test strip is included individually in the Flowflex Plus Strep A Rapid Test Strip format. In the cassette format, the test strip is already assembled in plastic housing to form the Flowflex Plus Strep A Rapid Test Cassette. The ACON Flowflex Plus Strep A Rapid Test Strip and Flowflex Plus Strep A Rapid Test Cassette (also referred to here collectively as the ACON Flowflex Plus Strep A Rapid Test Strip and Cassette) are rapid chromatographic immunoassays for the qualitative detection of Group A Streptococcus antigen from throat swab specimens from symptomatic patients (i.e., suspected of bacterial pharyngitis). In this test, a throat swab is collected from a patient, and the Strep A antigen is extracted in an extraction tube. The test utilizes antibodies specific for whole cell {2} Lancefield Group A Streptococcus to selectively detect Strep A antigen in a throat swab specimen. For the Flowflex Plus Strep A Rapid Test Strip, the test strip is immediately placed in the extracted sample solution, which migrates up through the test strip. In the case of the Flowflex Plus Strep A Rapid Test Cassette, drops of the extracted sample are added to the sample well of the cassette. Each format contains a test line (T line) and a control line (C line). Results can be read after 5 minutes. ## B Principle of Operation: The ACON Flowflex Plus Strep A Rapid Test Strip and Cassette are qualitative, lateral flow immunoassays for the detection of Strep A carbohydrate antigen in a throat swab. In this test, an antibody specific to Strep A carbohydrate antigen is coated on the test line region of the test strip. During testing, the extracted throat swab specimen reacts with an antibody to Strep A that is coated onto particles. This mixture migrates up the membrane to react with the antibody to Strep A on the membrane, and a colored line (pink-red) is generated in the test line region. The presence of this colored line in the test line region indicates a positive result for detection of Strep A antigen, while its absence indicates a negative result. To serve as a procedural control, a colored line will always appear at the control line region, indicating that a proper volume of specimen has been added and membrane wicking has occurred. If the pink-red control line does not appear, the test result is invalid. Each format of the Flowflex Plus Strep A Rapid Test (i.e., strip or cassette) has a unique Quick Reference Instruction (QRI) document and package insert and are provided as separate unique kits. Slight differences in the workflow between the strip and cassette formats are described below. Table I includes the components of Flowflex Plus Strep A Rapid Test Kits. - **Cassette Kit** A dropper tip is attached firmly onto the tube containing the extracted sample, and the tube is mixed thoroughly by swirling or flicking the bottom. Three full drops of the extracted solution are added to the sample well (S) before starting the timer. Results are read at 5 minutes. - **Strip Kit** With arrows pointing down, the test strip is inserted vertically into the extracted specimen solution and the timer is started. If the procedure is followed correctly, the solution should be below the maximum line (MAX) on the test strip. The strip is left in the tube for 5 minutes before reading results. Table I. Cassette Kit and Strip Kit Components | Cassette Kit Components | Strip Kit Components | | --- | --- | | • Test Cassettes • Test Tubes • Dropper Tips • Sterile Throat Swabs • Reagent A (2M Sodium Nitrite) • Reagent B (0.2M Acetic Acid) • Positive Control (Heat-inactivated Group A Streptococcus) | • Test Strips • Test Tubes • Sterile Throat Swabs • Reagent A (2M Sodium Nitrite) • Reagent B (0.2M Acetic Acid) • Positive Control (Heat-inactivated Group A Streptococcus) | K251697 - Page 3 of 15 {3} K251697 - Page 4 of 15 | Cassette Kit Components | Strip Kit Components | | --- | --- | | • Negative Control (Heat-inactivated Group C Streptococcus) • Package Insert • Quick Reference Instructions | • Negative Control (Heat-inactivated Group C Streptococcus) • Package Insert • Quick Reference Instructions | V Substantial Equivalence Information: A Predicate Device Name(s): Wondfo Strep A Rapid Test B Predicate 510(k) Number(s): K133343 C Comparison with Predicate(s): | Device & Predicate Device(s): | K251697 (subject device) | K133343 (predicate device) | | --- | --- | --- | | Device Trade Name | Flowflex Plus Strep A Rapid Test Cassette; Flowflex Plus Strep A Rapid Test Strip | Wondfo One Step Strep A Swab Test | | General Device Characteristic Similarities | | | | Intended Use/Indications For Use | Cassette: The Flowflex Plus Strep A Rapid Test Cassette is a rapid chromatographic immunoassay for the qualitative detection of Group A Streptococcus antigen from throat swab specimens from symptomatic patients. The test is used to aid in the diagnosis of Group A Streptococcal infection. All negative test results should be confirmed by bacterial culture because negative results do not preclude infection with Group A Streptococcus and should not be used as the sole basis for treatment. Strip: The Flowflex Plus Strep A Rapid Test Strip is a rapid chromatographic immunoassay for the qualitative detection of Group A Streptococcus antigen from throat swab | The Wondfo Strep A Rapid Test is a chromatographic immunoassay for the qualitative detection of Strep A antigen from throat swab specimens from symptomatic patients to aid in the diagnosis of Group A Streptococcal infection. All negative test results should be confirmed by bacterial culture because negative results do not preclude Group A Strep infection and should not be used as the sole basis for treatment. This test is intended for professional and laboratory use, only. | {4} K251697 - Page 5 of 15 | Device & Predicate Device(s): | K251697 (subject device) | K133343 (predicate device) | | --- | --- | --- | | | specimens from symptomatic patients. The test is used to aid in the diagnosis of Group A Streptococcal infection. All negative test results should be confirmed by bacterial culture because negative results do not preclude infection with Group A Streptococcus and should not be used as the sole basis for treatment. | | | Specimen Type | Throat swab specimen | Same | | Test Technology | Immunochromatographic lateral flow assay | Same | | Indication for Use | Prescription Use | Same | | Single Use | Yes | Same | | Test Result | Qualitative | Same | | Sample Collection Method | Use the throat swab supplied in the kit | Same | | General Device Characteristic Differences | | | | Test Format | Cassette and Strip formats | Strip | | Wait Time for Results Read | 5 minutes | 10 minutes | | LoD | 7.4 x 10^{5} CFU/mL (for contrived Strep A swab samples); 1.0 x 10^{5} CFU/mL (in extraction buffer) | 1.5 x 10^{5} organisms/mL | | Clinical Sensitivity/Specificity | **Cassette:** Sensitivity: 98.2% (95% CI: 93.6% - 99.5%) Specificity: 95.1% (95% CI: 92.0% - 97.1%) | **Strip:** Sensitivity: 95% (95% CI: 88 - 98%) Specificity: 98% (95% CI: 96 - 99%) | | | **Strip:** Sensitivity: 97.2% (95% CI: 92.2% - 99.1%) Specificity: 95.8% (95% CI: 92.9% - 97.6%) | | VI Standards/Guidance Documents Referenced: N/A {5} VII Performance Characteristics (if/when applicable): # A Analytical Performance: # 1. Reproducibility/Precision: The precision/reproducibility of the ACON Flowflex Plus Strep A Rapid Test Strip and Cassette) were evaluated using contrived specimens prepared by spiking heat-inactivated Strep A bacteria (Streptococcus pyogenes ATCC 49399) into pooled negative clinical matrix from healthy volunteer throat swabs. Performance of the Flowflex Plus Strep A Rapid Test Strip and Cassette with weakly reactive samples was also performed by untrained users as part of reproducibility testing. Four specimen levels were prepared—negative, low negative $(0.4 \times \mathrm{LoD})$ , low positive $(1 \times \mathrm{LoD})$ , and medium positive $(3 \times \mathrm{LoD})$ —with each specimen aliquoted into individual tubes and randomly coded for blinded testing. The Reproducibility Study was conducted at three CLIA-waived sites across different geographic locations using a total of nine untrained operators with diverse educational backgrounds. Each operator tested negative and positive controls daily with each kit format. One panel of samples was tested every day for five days using three different lots per strip and cassette formats. A total of 135 data points were collected per sample level per test format (3 operators $\times$ 3 sites $\times$ 5 days $\times$ 3 replicates $= 135$ results per concentration). The study showed that sample results met predefined acceptance criteria for each concentration level tested, and both positive and negative controls were in $100\%$ agreement with expected results across all operators and sites. Results demonstrated that users untrained in the Flowflex Plus Strep A Rapid Test Strip and Cassette test procedure were able to perform the test correctly. The study results were acceptable (Table II-Table IV). Table II. Summary of Precision/Reproducibility of ACON Flowflex Plus Strep A Rapid Test Strip from all Sites | Sample Type | Positives / Total (Positive agreement %) -Strip | | | | | | --- | --- | --- | --- | --- | --- | | | Site-1 | Site-2 | Site-3 | All Sites | Overall; 95% CI | | Negative | 0/45 (0.0 %) | 0/45 (0.0 %) | 0/45 (0.0 %) | 0/135 (0.0%) | 0%-2.8% | | Low negative | 22/45 (48.9 %) | 21/45 (46.7 %) | 21/45 (46.7 %) | 64/135 (47.4 %) | 39.2%-55.8% | | Low Positive | 44/45 (97.8 %) | 44/45 (97.8 %) | 43/45 (95.6 %) | 131/135 (97.0%) | 92.6%-98.8% | | Medium Positive | 45/45 (100.0 %) | 45/45 (100.0 %) | 45/45 (100.0 %) | 135/135 (100.0%) | 97.2%-100% | Table III. Summary of Precision/Reproducibility of ACON Flowflex Plus Strep A Rapid Test Cassette from all Sites | Sample Type | Positives / Total (Positive agreement %) - Cassette | | | | | | --- | --- | --- | --- | --- | --- | | | Site-1 | Site-2 | Site-3 | All Sites | Overall; 95% CI | | Negative | 0/45 (0.0 %) | 0/45 (0.0 %) | 0/45 (0.0 %) | 0/135(0.0%) | 0%-2.8% | | Low negative | 22/45 (48.9 %) | 22/45 (48.9 %) | 21/45 (46.7 %) | 65/135(48.1%) | 39.9%-56.5% | | Low positive | 43/45 (95.6 %) | 44/45 (97.8 %) | 45/45 (100.0%) | 132/135(97.8%) | 93.7%-99.2% | | Medium positive | 45/45 (100.0 %) | 45/45 (100.0 %) | 45/45 (100.0 %) | 135/135(100.0%) | 97.2%-100% | K251697 - Page 6 of 15 {6} Table IV. Summary of the External Controls Testing with ACON Flowflex Plus Strep A Rapid Test Strip/Cassette from Different Sites | Control | Flowflex Plus Strep A Rapid Test Strip (Observed results/Expected results) | | Flowflex Plus Strep A Rapid Test Cassette (Observed results/Expected results) | | | --- | --- | --- | --- | --- | | | Negative Control | Positive Control | Negative Control | Positive Control | | Site 1 | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | | Site 2 | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | | Site 3 | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | | Overall | 45/45 (100%) | 45/45 (100%) | 45/45 (100%) | 45/45 (100%) | 2. Linearity: N/A 3. Analytical Specificity/Interference: To prepare positive samples for the remaining analytical studies, all microorganisms were spiked into pooled negative clinical matrix prepared from healthy volunteer throat swabs that was confirmed negative by testing with a commercial Strep A test and bacterial culture. Cross-Reactivity Study A Cross-Reactivity Study was conducted to evaluate the performance of the ACON Flowflex Plus Strep A Rapid Test Strip and Cassette in the presence of fifty-five (55) microorganisms commonly found in throat specimens that could potentially interfere with test results. The microorganisms tested included various streptococcal species (Groups B, C, D, F, and G), respiratory viruses (influenza, RSV, rhinovirus), bacterial pathogens (Staphylococcus, Neisseria, Haemophilus), and other throat flora. Each potentially cross-reacting microorganism was tested in replicates of five (5) at clinically relevant levels of viruses (from $4.86 \times 10^{4} \mathrm{TCID}_{50} / \mathrm{mL}$ to $1.0 \times 10^{6} \mathrm{TCID}_{50} / \mathrm{mL}$ , or at the highest achievable concentration) and bacteria ( $1.0 \times 10^{7} \mathrm{CFU} / \mathrm{mL}$ ). Samples were randomized, blinded, and tested according to the package insert instructions. There were no false positive reactions with any of the tested microorganisms at the specified concentrations, confirming that the ACON Flowflex Plus Strep A tests do not cross-react with the certain bacteria and viruses that may be present in throat specimens. See Table V below for the cross-reactivity panel tested. | Table V. Organism Panel for Cross-Reactivity Study | | | --- | --- | | Adeno virus Type 1 | Neisseria sicca Z043, T | | Arcanobacterium haemolyticum | Neisseria Subflava Biovar Flava Z119 | | Bordetella pertussis A639 | Proteus vulgaris Z129 | | Candida albicans Z006 | Pseudomonas aeruginosa Z139 | | Corynebacterium diphtheriae Z116 | Respiratory syncytial virus Type A (RSV-A) (1/2015 isolate) | | Cytomegalo-virus | Rhinovirus Type 1A culture fluid | | Echovirus Type 09 | Serratia marcescens Z053 | | Enterococcus faecalis Z346 VSE | Staphylococcus aureus MRSA | | Enterococcus faecium Z265 | Staphylococcus epidermidis MRSE, RP62A | K251697 - Page 7 of 15 {7} | Table V. Organism Panel for Cross-Reactivity Study | | | --- | --- | | Epstein-Barr virus | Staphylococcus haemolyticus | | Escherichia coli clinical isolate, | Strep B | | Fusobacterium necrophorum Z239 | Strep C | | Haemophilus parainfluenza Z492 | Strep D | | Human metapneumovirus (hMPV) 16 Type A1 | Strep F | | Influenza A | Strep G | | Influenza A H3N2 | Streptococcus agalactiae Z019 | | Klebsiella pneumoniae KPC-2 | Streptococcus anginosus Z199 | | Klebsiella pneumoniae Z026 | Streptococcus aureus CoNS | | Lactobacillus salivarius | Streptococcus bovis Z167 | | Measles virus culture fluid | Streptococcus constellatus Z124 | | Moraxella catarrhalis Ne 11 | Streptococcus dysgalactiae subso. Equisimilis Z068 | | Mumps virus | Streptococcus intermedius Z216 | | Mycobacterium tuberculosis | Streptococcus mitis Clinical Isolate | | Mycoplasma pneumoniae | Streptococcus mutans Z072 | | Neisseria gonorrhoeae Z017 | Streptococcus oralis Z307 | | Neisseria lactamica Z041 | Streptococcus salivarius Z127 | | Neisseria Meningitidis Serogroup A | Streptococcus sanguinis Z089 | | Neisseria mucosa | | ## Microbial Interference Study A Microbial Interference Study was conducted to evaluate the performance of the ACON Flowflex Plus Strep A Rapid Test Strip and Cassette in the presence of microorganisms listed in Table V above. Each organism was tested in replicates of five (5) with clinically relevant levels of viruses ($\geq 1.0 \times 10^{5} \mathrm{TCID}_{50} / \mathrm{mL}$ or the highest achievable concentration) and bacteria ($\geq 1.0 \times 10^{6} \mathrm{CFU} / \mathrm{mL}$) in the presence of $2 \times \mathrm{LoD}$ of *S. pyogenes* ATCC 49399 ($1.48 \times 10^{6} \mathrm{CFU} / \mathrm{mL}$). All strain combinations were spiked into pooled negative clinical matrix prepared from healthy volunteer throat swabs that was confirmed negative by commercial Strep A testing and blood culture. No microbial interference was observed for any of the organisms at the concentrations tested and the results are acceptable. ## Interfering Substances Study Thirty-two (32) endogenous and exogenous substances were evaluated for potential to interfere with the ACON Flowflex Plus Strep A Rapid Test Strip and Cassette, including fresh whole human blood, bovine submaxillary mucin, over-the-counter mouthwashes, throat sprays, cough syrups, throat lozenges, and active pharmaceutical ingredients commonly found in cold and allergy medications. Each substance was tested in replicates of five (5) both in the absence and presence of GAS [$2 \times \mathrm{LoD}$ of *S. pyogenes* ATCC 49399 ($1.48 \times 10^{6} \mathrm{CFU} / \mathrm{mL}$)]. Samples were prepared in pooled negative clinical matrix from healthy volunteer throat swabs. None of the thirty-two (32) substances tested demonstrated a potential to cause false positive, false negative, or invalid results at the concentrations tested. All samples without GAS generated negative results, and all samples with GAS generated positive results for both the strip and cassette formats. Results confirmed that the tested endogenous and exogenous substances did not interfere with test performance. See Table VI below for a list of substances and concentrations tested. K251697 - Page 8 of 15 {8} Table VI. List of Endogenous and Exogenous Substances Tested | Substance | Concentration | | --- | --- | | Mucin (Bovine Submaxillary Gland, type I-S) | 0.4 mg/ml | | Ethanol | 99.5% | | Blood (human), EDTA anticoagulated | 20% (v/v) | | Group A β-Strep Blood Agar | N/A | | OTC Mouthwashes: | | | Listerine Antiseptic | 25% (v/v) | | Listerine Cool Mint | 25% (v/v) | | Crest Pro-Health Clean Night Mint | 25% (v/v) | | OTC Throat Sprays: | | | Chloraseptic Max | 25% (v/v) | | OTC Cough Syrups: | | | Tylenol Cold, Cough and Sore Throat | 25% (v/v) | | Rite Aid Tussin CF | 25% (v/v) | | Children’s Dimetapp Cold & Cough | 25% (v/v) | | Children’s Dimetapp Cold & Allergy | 25% (v/v) | | Children’s Dimetapp Nighttime Cold & Congestion | 25% (v/v) | | Children’s Wal-Tap Elixir Cold & Allergy | 25% (v/v) | | Robitussin Cough &Chest Congestion DM | 25% (v/v) | | Robitussin Nighttime Cough DM | 25% (v/v) | | Robitussin Cough & Cold-CF Max | 25% (v/v) | | Halls Cherry Mentholyptus | 20% (w/v) | | Halls Plus Mentholyptus | 20% (w/v) | | Ricola LemonMint Herb Throat Drops-Sugar Free | 20% (w/v) | | Cepacol Cherry Sore Throat | 20% (w/v) | | BreathSavers 3 Hour Mint-Spearmint | 20% (w/v) | | Tic Tac wintergreen | 20% (w/v) | | Active Ingredients: | | | Acetaminophen (Tylenol) | 10 mg/ml | | Brompheniramine Maleate | 5 mg/ml | | Chlorpheniramine Maleate | 5 mg/ml | | Dextromethorphan HBr | 5 mg/ml | | Diphenhydramine HCl | 5 mg/ml | | Doxylamine Succinate | 5 mg/ml | | Guaifenesin (GuaiacolGlyceryl) | 20 mg/ml | | Ibuprofen (Advil) | 10 mg/ml | | Phenylephrine HCl | 5 mg/ml | OTC = over the counter ## High Dose Hook Effect A High-Dose Hook Effect Study was conducted to evaluate the performance of the ACON Flowflex Plus Strep A Rapid Test Strip and Cassette when exposed to high concentrations of GAS. Contrived samples were prepared by spiking heat-inactivated S. pyogenes (ATCC 49399) into pooled negative clinical matrix at various levels—negative sample (0 CFU/mL), intermediate concentrations (3.7 × 10⁵ CFU/mL and 7.4 × 10⁵ CFU/mL), and very high concentrations (1.5 × 10⁸ CFU/mL). Testing was performed on both strip and cassette formats of the ACON Flowflex Plus Strep A Rapid Test, where each sample was tested in five replicates following the manufacturer's package insert instructions. Results demonstrated K251697 - Page 9 of 15 {9} that no hook effect (false negative results) was observed at the highest tested concentration, $1.5 \times 10^{8} \mathrm{CFU/mL}$, using both the strip and cassette formats. While concentrations at/above $7.4 \times 10^{5} \mathrm{CFU/mL}$ showed $100\%$ detection of the target, the lowest concentration of GAS spiked, $3.7 \times 10^{5} \mathrm{CFU/mL}$, showed variable results (2/5 positive for strips, 3/5 positive for cassettes). This was acceptable since the lower concentration tested in the study was below the reported LoD for the test with that strain. 4. Assay Reportable Range: N/A 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods): **Quality Control** The ACON Flowflex Plus Strep A Rapid Test Strip and Cassette formats incorporate three controls: - Internal Positive Control: A red or pink line appearing in the control line region (C) is an internal procedural control. The appearance of the procedural control line indicates that proper volume of sample has been added and capillary flow occurred. If the procedural control line does not develop, the test result is considered invalid, and retesting with a new strip/cassette and sample is recommended. - External Controls: Positive and Negative control solutions are supplied with each kit. The positive control solution is non-infectious heat-inactivated Strep A bacteria with buffer and stabilizer solution. The negative control solution is heat-inactivated Strep C bacteria with buffer solution. The instructions for use (package insert) recommends that a positive and negative external control be tested once for each untrained operator and once for each new shipment of kits—provided that each different lot in the shipment is tested. These controls help to ensure that the test procedure is performed correctly. **Evaluation of External Control Performance** During the Clinical Study, each operator tested one positive control and one negative control according to the instructions recommended in the package insert and quick reference instructions of the product. All positive controls [100% (14/14) each format] and negative controls [100% (14/14) each format] were detected accurately with the ACON Flowflex Plus Strep A Rapid Test Strip and Cassette. In the analytical studies, External Quality Controls (Negative Control and Positive Control) were tested prior to performing each analytical study and results were recorded. All controls gave expected results. Quality Control performance during the Reproducibility Study is presented above in Table IV. The data from both clinical and analytical studies demonstrate acceptable results when testing positive and negative external controls. **Sample Stability** - Delay in Sample Testing Study Recommended: Testing should ideally be performed immediately after specimen collection K251697 - Page 10 of 15 {10} An initial study was conducted to determine whether delays in sample testing at various temperatures could impact the performance of the ACON Flowflex Plus Strep A Rapid Test Strip and Cassette. Negative and positive (2× LoD) sample swabs were stored at different temperatures (e.g., 10°C, 15°C, 30°C, and 35°C) for varying durations (e.g., 0.5, 1, and 2 hours) before testing with both test formats. Five replicates were tested for each sample under all conditions. All negative and positive samples produced the expected results for both the strip and cassette formats under each condition tested. The study demonstrated that swabbed samples stored at temperatures ranging from 10°C to 35°C for up to 2 hours can produce consistent test results. - Sample in Swab Study (Additional Storage Times) Recommended: Testing should ideally be performed immediately after specimen collection An additional study was conducted to evaluate the effect of different temperatures on swab samples prior to analysis. Both negative and positive (contrived at 2×LoD) samples were applied to swabs, and swabs were stored at 4°C, room temperature, and 30°C. Five replicates of each swab sample were stored at various time points (fresh, 24, 48, 72, and 96 hours) and tested with the ACON Flowflex Plus Strep A Rapid Test Strip and Cassette. All negative and positive samples produced the expected results for both strip and cassette formats across all temperature and time conditions tested throughout the 96-hour storage period, regardless of whether the sample was stored at 4°C, room temperature, or 30°C. Labeling recommends testing immediately, when possible. 6. Detection Limit: **Limit of Detection** The limit of detection (LoD) of the ACON Flowflex Plus Strep A Rapid Test Strip and Cassette was determined using contrived stocks of heat-inactivated *S. pyogenes* (ATCC 49399) prepared in pooled negative clinical matrix from healthy volunteer throat swabs. The LoD was defined as the lowest Strep A concentration detected ≥95% of the time (at least 19 out of 20 replicates testing positive). Preliminary testing was conducted with concentrations ranging from 1.98 × 10⁴ to 1.98 × 10⁷ CFU/mL using five replicates per concentration across three lots of each test format. Confirmation studies were then performed with concentrations around the preliminary LoD using 20 replicates per concentration across three lots. Based on the confirmation study results, the limit of detection was established as 7.4 × 10⁵ CFU/mL for contrived Strep A samples [equivalent to 1.0 × 10⁵ CFU/mL in extraction buffer or 3.7 × 10⁴ CFU/swab for both the strip and cassette kit formats]. **Inclusivity Study** Inclusivity studies were conducted with three *S. pyogenes* strains (ATCC 12344, 12370, and 19615) in replicates of five per strain using both the ACON Flowflex Plus Strep A Rapid Test Strip and Cassette formats. Each cultured strain was prepared by serial dilution into pooled negative clinical matrix from healthy volunteer throat swabs. Preliminary LoDs were determined followed by confirmation testing at concentrations two-fold above and below the preliminary LoD. The study results demonstrated that all three GAS strains were correctly K251697 - Page 11 of 15 {11} detected by both test formats with confirmed LoD concentrations ranging from $2.71 \times 10^{5}$ to $3.68 \times 10^{5}$ CFU/mL for contrived samples. See Table VII below. Table VII. Inclusivity Study Report | Item No. | Pathogens | ATCC # | LoD Conc. of prepared sample (CFU/mL) | Conc. in extraction buffer (CFU/mL) | | --- | --- | --- | --- | --- | | 1 | Streptococcus pyogenes typing strain T1 [NCIB 11841, SF 130] | 12344 | 2.85×105 | 3.85×104 | | 2 | Streptococcus pyogenes typing strain C94 | 12370 | 3.68×105 | 4.97×104 | | 3 | Streptococcus pyogenes strain Bruno [CIP 104226] | 19615 | 2.71×105 | 3.66×104 | 7. Assay Cut-Off: N/A # B Comparison Studies: 1. Method Comparison with Predicate Device: N/A 2. Matrix Comparison: N/A # C Clinical Studies: 1. Clinical Sensitivity: Performance characteristics of the ACON Flowflex Plus Strep A Rapid Test Strip and Cassette were established during a prospective clinical study conducted from August 2022 to March 2024. Subjects enrolled were patients with signs and symptoms of pharyngitis with completed informed consent prior to sample collection. Patient demographics included male and female patients across all ages. Three hundred and ninety-seven (397) fresh throat swab specimens were prospectively collected at five (5) sites across the United States. All testing was performed at sites holding CLIA certificates of waiver using untrained healthcare professionals. Three swab specimens were collected simultaneously from each patient using throat swabs provided with the assay—one swab for ACON Flowflex Plus Strep A Strip testing, one swab for ACON Flowflex Plus Strep A Cassette testing, and one swab for bacterial culture and organism ID confirmation. The swab collection order was randomized to avoid bias. Bacterial culture was performed at CLIA-certified central clinical laboratories near the collection sites. Specimens were plated on blood agar plates and incubated following routine laboratory culture procedures for identifying S. pyogenes. Beta-hemolytic colonies from the blood agar plates were confirmed for Group A streptococci using streptococcal latex agglutination testing. Of the 397 specimens tested, 109 (27.5%) were culture-positive and 288 (72.5%) were culture-negative. K251697 - Page 12 of 15 {12} Tables VIII-IX summarize the clinical performance of the ACON Flowflex Plus Strep A Rapid Test Strip and Cassette. For the ACON Flowflex Plus Strep A Rapid Test Strip (Table VIII), sensitivity and specificity performances were reported as $97.2\%$ [(106/109) with $95\%$ CI = 92.2% - 99.1%] and $95.8\%$ [(276/288) with $95\%$ CI = 92.9% - 97.6%], respectively, when compared to the reference culture method. The ACON Flowflex Plus Strep A Rapid Test Cassette had similar performance (Table IX) with a reported sensitivity and specificity of $98.2\%$ [(107/109) with $95\%$ CI = 93.6% - 99.5%] and $95.1\%$ [(273/287) with $95\%$ CI = 92.0% - 97.1%], respectively, when compared to the reference culture method. Both test formats met acceptance criteria of $\geq 95\%$ sensitivity and specificity with lower bounds of the $95\%$ confidence intervals exceeding $90\%$ . Sixteen (16) untrained healthcare professional operators from diverse educational backgrounds participated in the study. No operators had prior clinical laboratory testing experience. Post-study questionnaire results supported usability with $100\%$ of operators agreeing that the overall instructions and test procedures were easy to follow and sample processing was easy to perform correctly. The study supports that untrained operators could successfully perform both test formats following QRI and package insert instructions. The clinical performance provided was used for both the $510(\mathrm{k})$ and CLIA Waiver by Application. The sensitivity and specificity performance from the clinical study are summarized in Table VIII-Table IX below. Table VIII. ACON Flowflex Plus Strep A Rapid Test Strip Clinical Performance vs. Reference Culture Method | Site | Total | TP | FN | TN | FP | Sensitivity [95% CI] | Specificity [95% CI] | | --- | --- | --- | --- | --- | --- | --- | --- | | Site 1 | 149 | 48 | 1 | 96 | 4 | 98.0% (48/49) [89.3%-99.6%] | 96.0% (96/100) [90.2%-98.4%] | | Site 2 | 144 | 31 | 2 | 107 | 4 | 93.9% (31/33) [80.4%-98.3%] | 96.4% (107/111) [91.1%-98.6%] | | Site 3 | 15 | 2 | 0 | 13 | 0 | 100% (2/2) [34.2%-100%] | 100% (13/13) [77.2%-100%] | | Site 4 | 66 | 21 | 0 | 42 | 3 | 100% (21/21) [84.5%-100%] | 93.3% (42/45) [82.1%-97.7%] | | Site 5 | 23 | 4 | 0 | 18 | 1 | 100% (4/4) [51.0%-100%] | 94.7% (18/18) [75.4%-99.1%] | | All Sites | 397 | 106 | 3 | 276 | 12 | 97.2% (106/109) [92.2%-99.1%] | 95.8% (276/288) [92.9%-97.6%] | | Prevalence (Reference Method): 27.5% (109/397) | | | | | | | | TP = true positive, FN = false negative, TN = true negative, FP = false positive, CI = confidence interval K251697 - Page 13 of 15 {13} Table IX. ACON Flowflex Plus Strep A Rapid Test Cassette Clinical Performance vs. Reference Culture Method | Site | Total | TP | FN | TN | FP | Sensitivity [95% CI] | Specificity [95% CI] | | --- | --- | --- | --- | --- | --- | --- | --- | | Site 1 | 149 | 49 | 0 | 95 | 5 | 100% (49/49) [92.7%-100%] | 95.0% (95/100) [88.8%-97.8%] | | Site 2 | 144 | 31 | 2 | 107 | 4 | 93.9% (31/33) [80.4%-98.3%] | 96.4% (107/111) [91.1%-98.6%] | | Site 3 | 15 | 2 | 0 | 12 | 1 | 100% (2/2) [34.2%-100%] | 92.3% (12/13) [66.7%-98.6%] | | Site 4 | 66 | 21 | 0 | 42 | 3 | 100% (21/21) [84.5%-100%] | 93.3% (42/45) [82.1%-97.7%] | | Site 5 | 22 | 4 | 0 | 17 | 1 | 100% (4/4) [51.0%-100%] | 94.4% (17/18) [74.2%-99.0%] | | All Sites | 396 | 107 | 2 | 273 | 14 | 98.2% (107/109) [93.6%-99.5%] | 95.1% (273/287) [92.0%-97.1%] | | Prevalence (Reference Method): 27.5% (109/396) | | | | | | | | TP = true positive, FN = false negative, TN = true negative, FP = false positive, CI = confidence interval 2. Clinical Specificity: See above. 3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable): N/A D Clinical Cut-Off: N/A E Expected Values/Reference Range: In the multi-center clinical study conducted by ACON from August 2022 to March 2024, 27.5% (Table X) of patients presenting with pharyngitis were found to be culture positive for Strep A in the study to evaluate ACON Flowflex Plus Strep A Rapid Test Strip and Cassette performance. Table X. Clinical Performance Stratified by Age | Test Format | Age | Number Samples per Age Range (% of Total Samples) | Sensitivity [95% CI] | Specificity [95% CI] | Number of Positives by Reference Method (% Prevalence by Age Group) | | --- | --- | --- | --- | --- | --- | | Strip | 1-5 | 64 (16.1%) | 100% [77.2%-100%] | 96.1% [86.8%-98.9%] | 13 (20.3%) | | | 6-20 | 176 (44.3%) | 95.9% [86.3%-98.9%] | 95.3% [90.1%-97.8%] | 49 (27.8%) | | | 21+ | 157 (39.5%) | 97.9% [88.9%-99.6%] | 96.4% [91.0%-98.6%] | 47 (29.9%) | | | All Ages | 397 | 97.2% [92.2%-99.1%] | 95.8% [92.9%-97.6%] | 109 (27.5%) | | Cassette | 1-5 | 64 (16.2%) | 100% [77.2%-100%] | 96.1% [86.8%-99.0%] | See numbers above | | | 6-20 | 175 (44.2%) | 95.9% [86.3%-98.9%] | 95.2% [90.0%-97.8%] | | | | 21+ | 157 (39.6%) | 100% [92.5%-100%] | 94.5% [88.6%-97.5%] | | | | All Ages | 396 | 98.2% [93.6%-99.5%] | 95.1% [92.0%-97.1%] | | VIII Proposed Labeling: The labeling supports the finding of substantial equivalence for this device. K251697 - Page 14 of 15 {14} IX Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision. K251697 - Page 15 of 15