Healgen Strep A Rapid Test Strip (Throat Swab)

K212623 · Healgen Scientific,, LLC · GTY · Mar 16, 2022 · Microbiology

Device Facts

• Record ID: K212623
• Device Name: Healgen Strep A Rapid Test Strip (Throat Swab)
• Applicant: Healgen Scientific,, LLC
• Product Code: GTY · Microbiology
• Decision Date: Mar 16, 2022
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3740
• Device Class: Class 1
• Attributes: Pediatric

Indications for Use

The Strep A Rapid Test Strip (Throat Swab) is a rapid chromatographic immunoassay for the qualitative detection of Streptococcus pyogenes (Group A β-hemolytic Streptococcus, Strep A) antigen from throat swab specimens of symptomatic patients to aid in the diagnosis of Group A Streptococcus bacterial 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.

Device Story

Lateral flow immunoassay for qualitative detection of Streptococcus pyogenes antigen in throat swab specimens. Principle: extracted specimen reacts with antibody-coated gold particles; complex migrates to membrane test line coated with anti-Strep A antibody; visible red line indicates positive result. Procedural control line confirms proper wicking. Used in clinical settings by professional operators. Output is visual color line; aids diagnosis of Group A Strep infection. Negative results require culture confirmation.

Clinical Evidence

Clinical study of 368 symptomatic subjects compared to culture. Results: 97.1% sensitivity (95% CI: 93.7-98.8%) and 99.4% specificity (95% CI: 96.2-100.0%). Performance consistent across age groups (0-5, 5-21, 21+).

Technological Characteristics

Lateral flow immunochromatographic assay. Materials: Rabbit Polyclonal Anti-Strep A (test line), Goat polyclonal anti-Rabbit IgG (control line). Gold particle conjugate. Form factor: Test strip. Storage: 4-30°C. No electronic components or software.

Indications for Use

Indicated for symptomatic patients of all ages to aid in the diagnosis of Group A Streptococcus bacterial infection via qualitative detection of Strep A antigen from throat swab specimens. Negative results require confirmation by bacterial culture.

Regulatory Classification

Identification

Streptococcus spp. serological reagents are devices that consist of antigens and antisera (excluding streptococcal exoenzyme reagents made from enzymes secreted by streptococci) used in serological tests to identify Streptococcus spp. from cultured isolates derived from clinical specimens. The identification aids in the diagnosis of diseases caused by bacteria belonging to the genus Streptococcus and provides epidemiological information on these diseases. Pathogenic streptococci are associated with infections, such as sore throat, impetigo (an infection characterized by small pustules on the skin), urinary tract infections, rheumatic fever, and kidney disease.

Predicate Devices

• Wondfo Strep A Rapid Test (K133343)

Submission Summary (Full Text)

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FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY ONLY

## I Background Information:

A 510(k) Number

K212623

B Applicant

Healgen Scientific, LLC

C Proprietary and Established Names

Healgen Strep A Rapid Test Strip (Throat Swab)

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:

This is a new 510(k) submission to obtain substantial equivalence for the Healgen StrepA Rapid Test.

B Measurand:

Group A Streptococcus Antigen

C Type of Test:

Lateral flow chromatographic immunoassay.

## III Intended Use/Indications for Use:

A Intended Use(s):

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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See Indications for Use below.

## B Indication(s) for Use:

The Strep A Rapid Test Strip (Throat Swab) is a rapid chromatographic immunoassay for the qualitative detection of Streptococcus pyogenes (Group A β-hemolytic Streptococcus, Strep A) antigen from throat swab specimens of symptomatic patients to aid in the diagnosis of Group A Streptococcus bacterial 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:

None

## IV Device/System Characteristics:

### A Device Description:

Healgen Strep A Rapid Test Strip (Throat Sdfwab) is a qualitative, lateral flow immunoassay for the detection of Strep A carbohydrate antigen from a throat swab. In this test, a throat swab is collected from the patient and the Strep A antigen is extracted in the extraction tube. This test uses a strip with antibody specific to Strep A carbohydrate antigen coated on the test line region of the test. The test strip is immediately placed in the sample solution that migrates up through the test strip to generate a color line in the test line region. The presence of a 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 red line should always appear in the control line region, indicating that proper volume of specimen has been added and membrane wicking has occurred. If the red control line does not appear, or remains blue, the test result is invalid.

### Device Contents:

- 25 Test strips
- 25 Sterile swabs
- 25 Disposable extraction tubes (one tube for each test strip)
- 1 Reagent A (10mL; 2M Sodium Nitrite)
- 1 Reagent B (10mL; 0.2M Acetic Acid)
- 1 Positive control (1mL; Non-viable Strep A; 0.05% Proclin300)
- 1 Negative control (1mL; Non-viable Strep C; 0.05% Proclin300)
- 1 Package insert

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B Principle of Operation:

Group A Streptococcus antigen reacts with the anti-Strep A antibody conjugated to the gold particle. The complex is then bound by the anti-Strep A capture antibody and a visible red test line appears, indicating a positive result. To serve as an onboard procedural control, a control line observed at the control site prior to running the assay will turn red, indicating that the test has been performed properly.

V Substantial Equivalence Information:

A Predicate Device Name(s):

Wondfo Rapid Strep A Test

B Predicate 510(k) Number(s):

K133343

C Comparison with Predicate(s):

| Device & Predicate Device(s): | K212623 | K133343 |
| --- | --- | --- |
| Device Trade Name | Healgen Strep A Rapid Test Strip (Throat Swab) | Wondfo One Step Strep A Swab Test |
| General Device Characteristic Similarities | | |
| Intended Use | The Strep A Rapid Test Strip (Throat Swab) is a rapid chromatographic immunoassay for the qualitative detection of Streptococcus pyogenes (Group A β-hemolytic Streptococcus, Strep A) antigen from throat swab specimens of symptomatic patients to aid in the diagnosis of Group A Streptococcus bacterial 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. | 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. |

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| Device & Predicate Device(s): | K212623 | K133343 |
| --- | --- | --- |
| Specimen | Throat swab | Same |
| Assay technical | Immunochromatographic | Same |
| Control Antibodies | Goat polyclonal anti-Rabbit IgG | Same |
| Test Antibody | Rabbit Polyclonal Anti-Strep A | Same |
| Indication for Use | Prescription Use | Same |
| | | |
| General Device Characteristic Differences | | |
| Test format | Strip | Cassette |
| Analytical sensitivity | 7.2×103CFU/mL (360 organisms/test) | 1.5 x 105organisms/mL |
| Clinical Sensitivity | 97.1%: 95% CI (93.7-98.8%) | 95%: 95% CI (88-98%) |
| Clinical Specificity | 99.4%: 95% CI (96.2-100.0%) | 98%: 95% CI (96-99%) |
| Results Reading Time | 5 minutes | 10 minutes |

# VI Standards/Guidance Documents Referenced:

Not applicable

# VII Performance Characteristics (if/when applicable):

# A Analytical Performance:

# Precision/Reproducibility:

To demonstrate the reproducibility of the Healgen StrepA Rapid Test, a four-member test panel consisting of Streptococcus pyogenes ATCC 19615 prepared at $2 \times \mathrm{LoD}$ , $1 \times \mathrm{LoD}$ and $0.5 \times \mathrm{LoD}$ concentrations, as well as a negative (diluent only) sample, was tested. The panel was tested at three sites by two operators at each site. Each operator tested each panel member, in duplicate, over 5 days using three lots of the device (2 operators $\times$ 3 sites $\times$ 2 replicates $\times$ 3 lots $\times$ 5 days = 180 results/panel member). The results are summarized in Table 1.

Table 1. Reproducibility of Healgen Strep A Rapid Test.

| Target Level | Site A %Detection (Positive/Tested) | Site B %Detection (Positive/Tested) | Site C %Detection (Positive/Tested) | Combined %Detection (Positive/Tested) |
| --- | --- | --- | --- | --- |
| Negative | 0% (0/60) | 0% (0/60) | 0% (0/60) | 0% (0/180) |
| 2 × LoD | 100% (60/60) | 100% (60/60) | 100% (60/60) | 100% (180/180) |
| 1 × LoD | 98.3% (59/60) | 93.3% (56/60) | 95% (57/60) | 95.6% (172/180) |
| 0.5 × LoD | 48.3% (29/60) | 43.3% (26/60) | 41.7% (25/60) | 44.4% (80/180) |

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There were no significant differences in the test results obtained between different users, different sites, and different lots on different days. The Healgen StrepA Rapid Test reproducibility is acceptable.

# 1. Linearity:

Not applicable. This is a qualitative detection assay.

# 2. Analytical Specificity/Interference:

A. Analytical specificity (Cross-reactivity and Microbial interference) To evaluate the potential for cross-reactivity with the Healgen Strep A Rapid Test Strip, microorganisms likely to be found in the respiratory tract were tested by three operators using three lots of the device for a total of nine results for each microorganism. The test organisms, concentrations and cross-reactivity results are shown in Table 2.

To evaluate the potential for microbial interference of Healgen Strep A Rapid Test Strip performance, potential interfering organisms likely to be found in the respiratory tract were tested in the presence of Streptococcus pyogenes at $2 \times$ LoD. Samples were randomized and blinded for single replicate testing by three (3) operators with three (3) device lots. The test organisms, concentrations and interference results are shown in Table 2.

Table 2. Analytical specificity (Cross-reactivity & Microbial interference) for Healgen Strep A Rapid Test

| Test Microorganisms | Concentrations | Test Result (Positive/Tested) | |
| --- | --- | --- | --- |
| | | Absence of S. pyogenes | Presence of S. pyogenes |
| Arcanobacterium haemolyticum | 2.6×108CFU/mL | 0/9 | 3/3 |
| Bordetella pertussis | 7.5×108CFU/mL | 0/9 | 3/3 |
| Candida albicans | 9.5×108CFU/mL | 0/9 | 3/3 |
| Corynebacterium diphtheria | 5.37×108CFU/mL | 0/9 | 3/3 |
| Enterococcus faecalis | 2.3×108CFU/mL | 0/9 | 3/3 |
| Enterococcus faecium | 4.4×108CFU/mL | 0/9 | 3/3 |
| Escherichia coli | 1.1×108CFU/mL | 0/9 | 3/3 |
| Fusobacterium necrophorum | 7.3×108CFU/mL | 0/9 | 3/3 |
| Haemophilus parahaemolyticus | 1.3×108CFU/mL | 0/9 | 3/3 |
| Haemophilus parainfluenzae | 1.6×108CFU/mL | 0/9 | 3/3 |
| Haemophilus influenzae | 4.5×108CFU/mL | 0/9 | 3/3 |
| Klebsiella pneumoniae | 3.1×108CFU/mL | 0/9 | 3/3 |
| Legionella pneumophila | 1×104bacteria/mL | 0/9 | 3/3 |
| Lactobacillus sp. (Lactobacillus casei) | 6.5×108CFU/mL | 0/9 | 3/3 |

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| Test Microorganisms | Concentrations | Test Result (Positive/Tested) | |
| --- | --- | --- | --- |
| | | Absence of S. pyogenes | Presence of S. pyogenes |
| Moraxella lacunata | 1.95×10^{8} CFU/mL | 0/9 | 3/3 |
| Moraxella catarrhalis | 4.8×10^{8} CFU/mL | 0/9 | 3/3 |
| Mycobacterium tuberculosis | 1×10^{3} bacteria/mL | 0/9 | 3/3 |
| Mycobacterium tuberculosis (avirulent strain) | 2.3×10^{8} CFU/mL | 0/9 | 3/3 |
| Neisseria gonorrhoeae | 3.8×10^{8} CFU/mL | 0/9 | 3/3 |
| Neisseria lactamica | 1.19×10^{8} CFU/mL | 0/9 | 3/3 |
| Neisseria meningitides | 7.5×10^{8} CFU/mL | 0/9 | 3/3 |
| Neisseria mucosa | 3.25×10^{8} CFU/mL | 0/9 | 3/3 |
| Neisseria sicca | 8.5×10^{8} CFU/mL | 0/9 | 3/3 |
| Neisseria subflava | 3.27×10^{8} CFU/mL | 0/9 | 3/3 |
| Proteus vulgaris | 2.9×10^{8} CFU/mL | 0/9 | 3/3 |
| Pseudomonas aeruginosa | 5.1×10^{8} CFU/mL | 0/9 | 3/3 |
| Serratia marcescens | 2.1×10^{8} CFU/mL | 0/9 | 3/3 |
| Staphylococcus aureus | 3.2×10^{8} CFU/mL | 0/9 | 3/3 |
| Staphylococcus epidermidis | 2.1×10^{8} CFU/mL | 0/9 | 3/3 |
| Staphylococcus marcescens | 1.5×10^{8} CFU/mL | 0/9 | 3/3 |
| Staphylococcus haemolyticus | 1.58×10^{8} CFU/mL | 0/9 | 3/3 |
| Streptococcus agalactiae (Group B) | 7.9×10^{7} CFU/mL | 0/9 | 3/3 |
| Streptococcus dysgalactiae (Group C) | 1.43×10^{5}CFU/mL | 0/9 | 3/3 |
| Streptococcus sp. Strain H60R (Group F) | 1×10^{6} CFU/mL | 0/9 | 3/3 |
| Streptococcus anginosus (Group G) | 4.2×10^{7} CFU/mL | 0/9 | 3/3 |
| Streptococcus sp. (bovis II) Group D | 5.6×10^{8} CFU/mL | 0/9 | 3/3 |
| Streptococcus pneumoniae | 4.2×10^{6} CFU/mL | 0/9 | 3/3 |
| Streptococcus salivarius | 8.7×10^{8} CFU/mL | 0/9 | 3/3 |
| Streptococcus mitis | 5.9×10^{8} CFU/mL | 0/9 | 3/3 |
| Streptococcus mutans | 4.7×10^{8} CFU/mL | 0/9 | 3/3 |
| Streptococcus oralis | 6.4×10^{8} CFU/mL | 0/9 | 3/3 |
| Streptococcus sanguis | 1.5×10^{8} CFU/mL | 0/9 | 3/3 |
| Yersinia enterocolitica | 2.0×10^{8} CFU/mL | 0/9 | 3/3 |
| Adenovirus Type I | 3.09×10^{8} TCID_{50}/mL | 0/9 | 3/3 |
| Adenovirus Type II | 3.9×10^{7} TCID_{50}/mL | 0/9 | 3/3 |

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| Test Microorganisms | Concentrations | Test Result (Positive/Tested) | |
| --- | --- | --- | --- |
| | | Absence of S. pyogenes | Presence of S. pyogenes |
| Adenovirus 3 | 1.5×108TCID50/mL | 0/9 | 3/3 |
| Adenovirus 7 | 2.8×106TCID50/mL | 0/9 | 3/3 |
| Cytomegalovirus | 1.6×105TCID50/mL | 0/9 | 3/3 |
| Enterovirus (VR-28 Human Coxsackievirus) | 1.6×108TCID50/mL | 0/9 | 3/3 |
| Epstein Barr Virus | 7.85×107copies/mL | 0/9 | 3/3 |
| HSV Type 1 MacIntyre strain | 1.6×105TCID50/mL | 0/9 | 3/3 |
| Human coronavirus OC43 | 1.7×105TCID50/mL | 0/9 | 3/3 |
| Human metapneumovirus (HMPV-27 A2) | 3.55×105TCID50/mL | 0/9 | 3/3 |
| Human parainfluenza Type 1 | 1.6×105TCID50/mL | 0/9 | 3/3 |
| Human parainfluenza Type 2 | 1.6×105TCID50/mL | 0/9 | 3/3 |
| Human parainfluenza Type 3 | 1.6×105TCID50/mL | 0/9 | 3/3 |
| Human rhinovirus 26 | 5×106TCID50/mL | 0/9 | 3/3 |
| Measles Virus | 8.9×105TCID50/mL | 0/9 | 3/3 |
| Mumps Virus | 1.38×107TCID50/mL | 0/9 | 3/3 |
| Respiratory syncytial virus Type A | 5.5×107PFU/mL | 0/9 | 3/3 |
| Respiratory syncytial virus Type B | 2.8×105TCID50/mL | 0/9 | 3/3 |

No cross reactivity or microbial interference were found for the above organisms at the concentrations tested and the results are acceptable.

# B. Interfering Substances:

To evaluate the potential for interference of Healgen Strep A Rapid Test performance, potentially interfering substances were evaluated in samples spiked with S. pyogenes ATCC 19615. Each sample was tested using three lots of Healgen Strep A Rapid Test Strip. The list of interfering substances and tested concentrations are provided in Table 3.

Table 3. Interfering substances evaluated for the performance of Healgen Strep A Rapid Test

| Interfering Substances | |
| --- | --- |
| Blood (human) | 20% (vol/vol) |
| Mucin | 1 mg/mL |
| Listerine Antiseptic | 20% (vol/vol) |
| Cool Mint | 20% (vol/vol) |

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| Interfering Substances | Concentration Tested |
| --- | --- |
| Crest Pro-Health Clean Mint | 20% (vol/vol) |
| Crest Pro Health Multi Protection Clean Mint | 20% (vol/vol) |
| Colgate Total Pro-Shield, | 20% (vol/vol) |
| Spearmint | 20% (vol/vol) |
| Sucrets Sore Throat & Cough Lozenges, Honey Lemon, | 5 mg/mL |
| Sucrets Sore Throat Lozenges Cherry | 5 mg/mL |
| Halls Mentho-Lyptus Drops Cherry | 5 mg/mL |
| Halls Cough Suppressant Cherry Triple Soothing Action | 5 mg/mL |
| Cepacol Extra Strength Sore Throat & Cough Drop Lozenges, Cherry | 5 mg/mL |
| Cepacol Dual Relief | 20% (vol/vol) |
| Chloraseptic Max | 20% (vol/vol) |
| Tylenol Cough and Sore Throat | 10% (vol/vol) |
| Basic Care Tussin DM, Cough Suppressant & Expectorant | 10% (vol/vol) |
| Robitussin (Guaifenesin Syrup) | 10% (vol/vol) |
| Robitussin Nighttime Cough | 10% (vol/vol) |
| Children’s Dimetapp Cold & Flu | 10% (vol/vol) |
| Children’s Dimetapp Cold & Cough | 10% (vol/vol) |
| Acetaminophen (Tylenol) | 5 mg/mL |
| Brompheniramine Maleate | 5 mg/mL |
| Chlorpheniramine Maleate | 5 mg/mL |
| Dextromethorphan HBr | 5 mg/mL |
| Diphenhydramine HCI | 5 mg/mL |
| Doxylamine Succinate | 5 mg/mL |
| Guaifenesin (Guaiacol Glyceryl) | 5 mg/mL |
| Ibuprofen (Advil) | 5 mg/mL |
| Phenylephrine HCI | 5 mg/mL |

Each of listed interfering substances at the given concentrations when spiked with $2 \times \mathrm{LoD}$ of $S.$ pyogenes ATCC 19615 tested $100\%$ positive for Strep A using Healgen Strep A Rapid Test. As a control, the listed interfering substances at the given concentrations tested for Strep A results into no detection using Healgen Strep A Rapid Test. No interference with any of the interfering substances tested were found at the tested concentrations and the results are acceptable.

3. Assay Reportable Range:

Not applicable

4. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

A. Quality Control:

The Healgen Strep A Rapid Test incorporates three controls:

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- Internal Positive Control: A color line appearing in the control region (C) is an internal positive procedural control. It confirms sufficient specimen volume, adequate membrane wicking and correct procedural technique. If a color band is not visible in the control region (C), the test is invalid.
- External Positive Control: The external positive control is provided with the Healgen Strep A Rapid Test. It contains non-viable Group A Streptococcus in 0.05% Proclin 300.
- External Negative Control: The external negative control is provided with the Healgen Strep A Rapid Test. It contains non-viable Streptococcus Group C in 0.05% Proclin 300.

The instructions for use (package insert) recommends that a positive and negative external control be tested at least once within each test kit and by each operator performing testing within a kit. This will verify that the reagents and test strips are working properly, and the operator is able to correctly perform the test procedure.

B. Evaluation of Control Performance:

During the Clinical Study to evaluate performance of the Healgen Strep A Rapid Test, each operator tested five positive and five negative controls. All these control samples were blinded and randomized. All controls gave the expected results as shown in Table 4.

Table 4. Evaluation of Control Performance of Healgen Strep A Rapid Test.

| Sites | Operators | Test Results (Positive/Tested) | |
| --- | --- | --- | --- |
| | | Negative Control | Positive Control |
| Site 1 | Operator 1 | 0/5 | 5/5 |
| | Operator 2 | 0/5 | 5/5 |
| | Operator 3 | 0/5 | 5/5 |
| | Operator 4 | 0/5 | 5/5 |
| Site 2 | Operator 1 | 0/5 | 5/5 |
| | Operator 2 | 0/5 | 5/5 |
| Site 3 | Operator 1 | 0/5 | 5/5 |
| | Operator 2 | 0/5 | 5/5 |
| | Operator 3 | 0/5 | 5/5 |

The performance when testing the positive and negative control was acceptable.

C. Specimen Storage Stability:

The instructions for use (package insert) for the Healgen Strep A Rapid Test states that swab specimens may be stored in a clean, dry plastic tube for up to 48 hours at room temperature or 72 hours at 2-8°C. To evaluate sample storage, negative clinical matrix swab spiked with S. pyogenes at 2× LoD and 4× LoD were placed in a clean plastic tube and capped tightly. These swab samples were divided into two groups: one stored at refrigerated (2-8°C) and other at room temperature (15-30°C). The results for these samples are in Table 5.

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Table 5. Specimen Storage Stability for Healgen Strep A Rapid Test.

| Storage Temperature | Results (Positive/Tested) in time points | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | 0 hr | | 8 hr | | 24 hr | | 48 hr | | 72 hr | |
| | 2 × LoD | 4 × LoD | 2 × LoD | 4 × LoD | 2 × LoD | 4 × LoD | 2 × LoD | 4 × LoD | 2 × LoD | 4 × LoD |
| Refrigerated (2-8°C) | 20/20 | 20/20 | 20/20 | 20/20 | 20/20 | 20/20 | 20/20 | 20/20 | 20/20 | 20/20 |
| Room Temperature (15-30°C) | 20/20 | 20/20 | 20/20 | 20/20 | 20/20 | 20/20 | 20/20 | 20/20 | NA | NA |

Healgen Strep A Rapid Test demonstrates that the swab samples can be stored at refrigerated condition $(2 - 8^{\circ}\mathrm{C})$ for 72 hours or at room temperature $(15 - 30^{\circ}\mathrm{C})$ for 48 hours. Specimen Storage Stability is acceptable.

# 5. Detection of Limit:

The limit of detection (LoD) for the Healgen Strep A Rapid Test Strip was established by testing limiting dilutions of Streptococcus pyogenes spiked onto a negative throat swab (clinical matrix). A concentrated stock $(3.6 \times 10^{7} \mathrm{CFU/mL})$ of inactivated S. pyogenes ATCC 19615 was serially diluted in saline solution. $5 \mu \mathrm{L}$ of each dilution was pipetted onto a negative throat swab for testing by seven operators with three lots of Healgen Strep A Rapid Test Strip, for a total of 21 results for each dilution. The test results are shown in Table 6.

Table 6. Determining Limit of Detection (LoD) for Healgen Strep A Rapid Test in clinical matrix

| Dilutions | Number of bacteria loaded* | | |
| --- | --- | --- | --- |
| 1.8×105CFU/mL | 900 | 21/21 | 100% |
| 7.2×104CFU/mL | 360 | 20/21 | 95.2% |
| 3.6×104CFU/mL | 180 | 12/21 | 57.6% |
| 1.8×104CFU/mL | 90 | 1/21 | 4.8% |
| 4.5×103CFU/mL | 23 | 0/21 | 0% |

*Calculated values based on dilution and volume of sample loaded on the swab per test.

The LoD was determined to be $7.2 \times 10^{4} \mathrm{CFU/mL}$ when $5 \mu \mathrm{L}$ of sample was pipetted onto a negative clinical matrix (equivalent to 360 bacteria on the swab).

The LoD for the Healgen Strep A Rapid Test Strip was also examined by using limiting dilutions of S. pyogenes ATCC 19615 in saline and pipetting $50~\mu \mathrm{L}$ of each dilution with equivalent bacterial amounts as those used with the clinical matrix onto a swab. The study results are shown in Table 7.

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Table 7. Determining Detection of Limit (LoD) for Healgen Strep A Rapid Test in Saline Solution.

| Dilutions | Number of bacteria loaded* (50 μL per swab) | Positive/Tested | % Detection |
| --- | --- | --- | --- |
| 1.8×104CFU/mL | 900 | 21/21 | 100% |
| 7.2×103CFU/mL | 360 | 20/21 | 95.2% |
| 3.6×103CFU/mL | 180 | 11/21 | 47.6% |
| 1.8×103CFU/mL | 90 | 1/21 | 4.8% |
| 4.5×103CFU/mL | 23 | 0/21 | 0% |

*Calculated values based on dilution and volume of sample loaded on the swab per test.

The LoD was determined to be $7.2 \times 10^{3} \mathrm{CFU/mL}$ when $50~\mu \mathrm{L}$ of sample was pipetted onto a swab (equivalent to 360 bacteria on the swab) which is consistent with the LoD established using negative throat swab clinical matrix.

# Inclusivity:

Two strains of S. pyogenes ATCC 19615 and ATCC 49399 were tested at the level of $2 \times$ LoD with 30 replicates in three lots of test kits (see Matrix Comparisons/Equivalency). Both the strains result into $100\%$ detection to indicate the coverage of phenotypic diversity.

# 6. Assay Cut-Off:

Not Applicable

# B Comparison Studies:

# 1. Method Comparison with Predicate Device:

Not applicable

# 2. Matrix Comparison:

A Matrix Equivalency Study was performed to evaluate the equivalency between saline solution and natural clinical throat matrix. A collection of 360 swabs from healthy subjects were screened for GAS using the Healgen Strep A Rapid Test Strip to produce a negative clinical matrix. Streptococcus pyogenes ATCC 19615 and ATCC 49399 were serially diluted in saline solution to achieve final LoD levels of $5 \times$ , $2 \times$ , $0.5 \times$ and negative. $5 \mu \mathrm{L}$ of each sample was added to negative clinical matrix swabs or swabs without clinical matrix (saline solution). Three lots of the devices with 10 replicates were tested for target levels $5 \times$ , $0.5 \times$ and negative LoD samples producing 30 test results whereas 30 replicates were tested for the target level $2 \times$ LoD sample generating 90 test results. The results are summarized in Table 8.

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Table 8. Determining matrix equivalency for Healgen Strep A Rapid Test.

| LoD Level | ATCC 19615 | | | | ATCC 49399 | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Saline solution | | Clinical matrix | | Saline solution | | Clinical matrix | |
| | Positive /Tested | % Detection | Positive /Tested | % Detection | Positive /Tested | % Detection | Positive /Tested | % Detection |
| 5 × | 30/30 | 100% | 30/30 | 100% | 30/30 | 100% | 30/30 | 100% |
| 2 × | 90/90 | 100% | 90/90 | 100% | 90/90 | 100% | 90/90 | 100% |
| 0.5 × | 13/30 | 43.3% | 14/30 | 46.7% | 10/30 | 33.3% | 11/30 | 36.7% |
| Negative | 0/30 | 0% | 0/30 | 0% | 0/30 | 0% | 0/30 | 0% |

Healgen demonstrates the matrix equivalency between saline solution and natural clinical matrix. The matrix equivalency study is acceptable.

## C Clinical Studies:

### 1. Clinical Sensitivity and Specificity:

The performance of the Healgen Strep A Rapid Test Strip was evaluated at Point of Care (POC) in Physicians' offices located at three different geographical sites. Two throat swabs were collected from a total of 368 patients exhibiting symptoms of pharyngitis. One swab was used for the Healgen Strep A Rapid Test Strip and tested on site right after sample collection. The other swab was transported to a reference laboratory for culture confirmation. The combined performance of the Healgen Strep A Rapid Test compared to culture results is summarized in Table 9A. The performance stratified by each site is shown in Tables 9-B-D.

Table 9A. Clinical performance of Healgen Strep A Rapid Test.

| | Culture Results | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| Healgen Strep A Rapid Test | Positive | 200 | 1 | 201 |
| | Negative | 6 | 161 | 167 |
| Total | | 206 | 162 | 368 |

Sensitivity: 97.1% (200/206) with 95% CI = 93.7% - 98.8%
Specificity: 99.4% (161/162) with 95% CI = 96.2% - 100.0%

Table 9B. Site 1: Clinical Performance:

| Site 1 | Culture Results | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| | Positive | 48 | 0 | 48 |

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| Healgen Strep A Rapid Test | Negative | 3 | 51 | 54 |
| --- | --- | --- | --- | --- |
| Total | | 51 | 51 | 102 |

Sensitivity: 94.1% (48/51) with 95% CI = 83.5% - 98.6%

Specificity: 100% (51/51) with 95% CI = 91.6% - 100.0%

Table 9C. Site 2: Clinical Performance:

| Site 2 | Culture Results | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| Healgen Strep A Rapid Test | Positive | 26 | 0 | 26 |
| | Negative | 1 | 39 | 40 |
| Total | | 27 | 39 | 66 |

Sensitivity: 96.3% (26/27) with 95% CI = 80.2% - 100.0%

Specificity: 100% (39/39) with 95% CI = 89.3% - 100.0%

Table 9D. Site 3: Clinical Performance:

| Site 3 | Culture Results | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| Healgen Strep A Rapid Test | Positive | 126 | 1 | 127 |
| | Negative | 2 | 71 | 73 |
| Total | | 128 | 72 | 200 |

Sensitivity: 98.4% (126/128) with 95% CI = 94.1% - 99.9%

Specificity: 98.6% (71/72) with 95% CI = 91.8% - 100.0%

Healgen Strep A Rapid Test demonstrated the ability to detect Strep A antigen as compared to the culture conducted at three different point of care testing sites with overall Sensitivity 97.1% and Specificity 99.4%. The clinical results are acceptable.

2. Clinical Specificity:

See Section 1A.

3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):

D Clinical Cut-Off:

Not applicable

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# E Expected Values/Reference Range:

The clinical study analysis included results from 368 throat swab specimens collected from three point of care sites in the U.S. between February to May of 2017 to 2019. The prevalence of S. pyogenes for this period in this clinical study was $55.9\%$ (206/368) as determined by culture and the positivity was $54.6\%$ (201/368) as determined by the Healgen Strep A Rapid Test. No specific reasons could be identified based on the study protocols that could explain the high prevalence observed in this study. However, the higher-than-expected prevalence was possibly due to prior determination to conduct the study during a peak of S. pyogenes infection season covering Feb-May each given calendar year.

The clinical outcome stratified by age of the participants are shown in Table 10.

Table 10. Clinical outcomes stratified by age.

| Age (in years) | | | | |
| --- | --- | --- | --- | --- |
| 0 to 5 | 97.4% (74/76) | 90.4% - 99.8% | 98.1% (52/53) | 89.1% - 100.0% |
| 5 to 21 | 96.7% (119/123) | 91.7% - 99.0% | 100% (88/88) | 95.0% - 100.0% |
| 21 & older | 100% (7/7) | 59.6% - 100.0% | 100% (21/21) | 81.8% - 100.0% |
| All | 97.1% (200/206) | 93.7% - 98.8% | 99.4% (161/162) | 96.2% - 100.0% |

# VIII Proposed Labeling:

The labeling supports the finding of substantial equivalence for this device.

# IX Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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