K Number

Device Name

AMPLIVUE GAS ASSAY

Manufacturer

QUIDEL CORPORATION

Date Cleared

2014-07-24

(79 days)

Product Code

PGX

Regulation Number

866.2680

Intended Use

The AmpliVue® GAS Assay is an in vitro diagnostic test for the qualitative detection of Group A 8hemolytic Streptococcus (Streptococcus pyogenes) nucleic acids isolated from throat swab specimens obtained from patients with signs and symptoms of pharyngitis, such as sore throat. The AmpliVue® GAS Assay is intended for use in hospital, reference or state laboratory settings. The device is not intended for point-of-care use.

Device Description

The AmpliVue® GAS Assay combines simple processing, an isothermal amplification technology named helicase-dependent amplification (HDA), and a self-contained disposable amplicon device, for the detection of GAS from throat swab specimens obtained from patients with signs and symptoms of pharyngitis, such as sore throat. Patient specimen on a throat swab is transferred to a Lysis Tube and subjected to heat treatment at 95℃ for 10 minutes. The heat-treated sample is diluted 10-fold in a Dilution Tube, and then transferred to a Reaction Tube. The reaction tube contains a lyophilized mix of HDA reagents including primers specific for the amplification of the DNase B (sdaB) gene sequence. The assay also includes a process control that monitors sample processing, confirms the integrity of the assay reagents and cassette detection, and assays for HDA inhibitors that may be present within a specimen. After completion of the HDA reaction tube is transferred to a cassette for detection with the test result displayed as test and/or control lines in the window of the cassette.

More Information

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Center

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Device Name

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Third Party Reviewed

Expedited Review

Predicate Devices

K133883

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description details a chemical and biological assay using isothermal amplification and a disposable cassette for detection, with no mention of AI or ML in the process or results interpretation.

Therapeutic

No
The device is an in vitro diagnostic test designed to detect the presence of Group A Streptococcus nucleic acids, which is a diagnostic function, not a therapeutic one.

Diagnostic

Yes

Explanation: The "Intended Use / Indications for Use" section explicitly states that the AmpliVue® GAS Assay is an "in vitro diagnostic test" for the detection of Group A β-hemolytic Streptococcus nucleic acids. This indicates its purpose is to diagnose an infection.

SaMD (Software as a Medical Device)

The device description clearly outlines physical components like Lysis Tubes, Dilution Tubes, Reaction Tubes, and a cassette for detection, indicating it is a hardware-based in vitro diagnostic test, not software-only.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the AmpliVue® GAS Assay is an "in vitro diagnostic test".
Purpose: The test is designed to detect the presence of Group A β-hemolytic Streptococcus nucleic acids in a sample (throat swab) taken from a patient to aid in the diagnosis of pharyngitis. This is a classic function of an in vitro diagnostic device.
Sample Type: It uses a biological sample (throat swab specimen) obtained from the human body.
Testing Location: It is intended for use in laboratory settings (hospital, reference, or state laboratories), which is typical for many IVDs.
Device Description: The description details the process of analyzing the sample outside of the body (in vitro) using chemical and biological reagents and a detection system.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The AmpliVue® GAS Assay is intended for use in hospital, reference or state laboratory settings. The device is not intended for point-of-care use.

Product codes (comma separated list FDA assigned to the subject device)

PGX

Device Description

Patient specimen on a throat swab is transferred to a Lysis Tube and subjected to heat treatment at 95℃ for 10 minutes. The heat-treated sample is diluted 10-fold in a Dilution Tube, and then transferred to a Reaction Tube. The reaction tube contains a lyophilized mix of HDA reagents including primers specific for the amplification of the DNase B (sdaB) gene sequence. The assay also includes a process control that monitors sample processing, confirms the integrity of the assay reagents and cassette detection, and assays for HDA inhibitors that may be present within a specimen. After completion of the HDA reaction tube is transferred to a cassette for detection with the test result displayed as test and/or control lines in the window of the cassette.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

throat swab specimens

Indicated Patient Age Range

Not Found

Intended User / Care Setting

hospital, reference or state laboratory settings

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Analytical Performance:

Precision/Reproducibility:
- Precision Study: Conducted by two operators, three times per day for twelve days, using a panel of four simulated samples (moderate positive, low positive, high negative, and negative GAS).
  - Results for High Neg: 38/72 detected pos (52.8%)
  - Results for Low Pos: 72/72 detected pos (100%)
  - Results for Mod Pos: 72/72 detected pos (100%)
  - Results for Neg: 0/72 detected pos (0%)
- Reproducibility Study: Evaluated at three laboratory sites (two external, one in-house) using the same panel of four simulated samples. 2 operators x 3 replicates x 5 days x 3 sites = 90 results per concentration.
  - Results for High Neg: 50/90 detected pos (55.6%)
  - Results for Low Pos: 90/90 detected pos (100%)
  - Results for Mod Pos: 90/90 detected pos (100%)
  - Results for Neg: 0/90 detected pos (0%)
Detection Limit (LoD): Determined using quantified (CFU/mL) contrived stocks of two strains of group A streptococci serially diluted in a negative matrix. 20 replicates tested for each of three dilutions. LoD is the lowest concentration at which >= 95% of replicates tested positive.
- Strain ATCC 19615: 1.9 x 10^4 CFU/ml
- Strain ATCC 12344: 2.74 x 10^4 CFU/ml
Analytical Sensitivity (Inclusivity): Conducted with seven Group A ß-hemolytic Streptococcus strains (in addition to two from LoD studies) against 3 different reagent lots. All strains tested positive.
Analytical Specificity (Cross-reactivity):
- In silico BLAST analysis: Primers against NCBI database against sixty-one (61) potential interfering organisms showed no evidence of cross-reactivity.
- Microorganism Study: Evaluated in the presence of forty-seven (47) other microorganisms commonly found in throat specimens. Each tested in triplicate with 2 x LoD Group A Streptococcus. None of the microorganisms cross-reacted.
- Interfering Substances: Twenty-eight (28) chemical and biological substances (including blood and human saliva) were evaluated. None found to interfere.

Comparison Studies:

Matrix Comparison: A study comparing negative clinical matrix and a contrived negative matrix (Porcine Gastric Mucin (PGM), Phosphate Buffered Saline (PBS), Bovine Serum Albumin and sodium azide) was conducted to validate the contrived matrix.
- Contrived Negative Matrix (1 x LoD): 20/20 detected (100%)
- Pooled Negative Clinical Matrix (1 x LoD): 20/20 detected (100%)

Clinical Studies:

Prospective Study: Conducted from February to March 2014 at five distinct geographical sites across the United States.
- Sample Size: 1192 fresh throat swab specimens from female and male patients. One specimen collected per patient.
- Method: Specimens cultured for Group A ß-hemolytic Streptococcus (GAS) and tested with AmpliVue® GAS Assay. Considered positive if culture from swab or transport fluid was positive (Composite Culture).
- Invalid Results: 1 invalid result determined, which was retested.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Overall Clinical Performance (All Sites):

Sensitivity: 98.4% (189/192) 95% CI (95.5%-99.5%)
Specificity: 95.0% (949/999) 95% CI (93.5%-96.2%)
NPV at 16.1% prevalence: 99.7% with lower bound of 95% CI of 99.0%
Extrapolated NPV to 30% prevalence: 99.3% with lower bound of 95% Cl of 97.9%

Site 1 Clinical Performance:

Sensitivity: 98.8% (82/83) 95% CI (93.5%-99.8%)
Specificity: 96.4% (402/417) 95% CI (94.2%-97.8%)

Site 2 Clinical Performance:

Sensitivity: 100.0% (45/45) 95% CI (92.1%-100.0%)
Specificity: 88.6% (132/149) 95% CI (82.5%-92.8%)

Site 3 Clinical Performance:

Sensitivity: 100.0% (16/16) 95% CI (80.6%-100.0%)
Specificity: 95.1% (174/183) 95% CI (90.9%-97.4%)

Site 4 Clinical Performance:

Sensitivity: 100.0% (8/8) 95% CI (67.6%-100.0%)
Specificity: 96.7% (89/92) 95% CI (90.8%-98.9%)

Site 5 Clinical Performance:

Sensitivity: 95.0% (38/40) 95% CI (83.5%-98.6%)
Specificity: 96.2% (152/158) 95% CI (92.0%-98.2%)

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

K133883

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 866.2680

Streptococcus spp. nucleic acid-based assay.(a)
Identification. AStreptococcus spp. nucleic acid-based assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify variousStreptococcus spp. nucleic acids extracted directly from clinical specimens. The device detects specific nucleic acid sequences for organism identification. The identification aids in the diagnosis of diseases caused by bacteria belonging to the genusStreptococcus 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.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include detailed device description documentation, including the device components, ancillary reagents required but not provided, and a detailed explanation of the methodology including primer/probe sequence, design, and rationale for sequence selection.
(2) Premarket notification submissions must include detailed documentation from the following analytical and clinical performance studies: Analytical sensitivity (Limit of Detection), reactivity, inclusivity, precision, reproducibility, interference, cross reactivity, carry-over, and cross contamination.
(3) Premarket notification submissions must include detailed documentation from a clinical study. The study, performed on a study population consistent with the intended use population, must compare the device performance to results obtained from well-accepted reference methods.
(4) Premarket notification submissions must include detailed documentation for device software, including, but not limited to, software applications and hardware-based devices that incorporate software.
(5) Premarket notification submissions must include database implementation methodology, construction parameters, and quality assurance protocols, as appropriate.
(6) The device labeling must include limitations regarding the need for culture confirmation of negative specimens, as appropriate.
(7) A detailed explanation of the interpretation of results and acceptance criteria must be included in the device's 21 CFR 809.10(b)(9) compliant labeling.
(8) Premarket notification submissions must include details on an end user device training program that will be offered while marketing the device, as appropriate.

Summary

510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE

A. 510(k) Number:

K141173

B. Purpose for Submission:

To obtain a substantial equivalence determination for the Amplivue® GAS Assay.

C. Measurand:

Group A ß-hemolytic Streptococcus (GAS; Streptococcus pyogenes) nucleic acids.

D. Type of Test:

The Amplivue® GAS Assay is a helicase-dependent amplification in vitro diagnostic test for the qualitative detection and differentiation of GAS nucleic acids isolated from throat swab specimens obtained from symptomatic patients.

E. Applicant:

Quidel Corporation

F. Proprietary and Established Names:

Amplivue® GAS Assay

G. Regulatory Information:

1. Regulation section:
  21 CFR 866.2680 - Streptococcus spp. Nucleic Acid-Based Assay
1. Classification:
  Class II
1. Product code:
  PGX – Groups A, C and G Beta-Hemolytic Streptococcus Nucleic Acid Amplification System
1. Panel:
- 83- Microbiology

H. Intended Use:

1. Intended use(s):
  The AmpliVue® GAS Assay is an in vitro diagnostic test for the qualitative detection of Group A 8hemolytic Streptococcus (Streptococcus pyogenes) nucleic acids isolated from throat swab specimens obtained from patients with signs and symptoms of pharyngitis, such as sore throat.

The AmpliVue® GAS Assay is intended for use in hospital, reference or state laboratory settings. The device is not intended for point-of-care use.

1. Indication(s) for use:
  Same as intended use.
1. Special conditions for use statement(s):
  For prescription use only.

The device is not intended for point-of-care use.

1. Special instrument requirements:
  Heat blocks capable of 95℃ ± 2℃ and 64℃ ± 2℃.

I. Device Description:

J. Substantial Equivalence Information:

1. Predicate device name(s):
  LyraTM Direct Strep (K133883)
1. Predicate 510(k) number(s):
  K133883

3. Comparison with predicate:

Similarities
Item	Device	Predicate
	Amplivue® GAS Assay
(K141173)	Lyra™ Direct Strep Assay
(K133883)
Intended Use	The AmpliVue® GAS Assay is
an in vitro diagnostic test for the
qualitative detection of Group A
β-hemolytic Streptococcus
(Streptococcus pyogenes)
nucleic acids isolated from
throat swab specimens obtained
from patients with signs and
symptoms of pharyngitis, such
as sore throat.
The AmpliVue® GAS Assay is
intended for use in hospital,
reference or state laboratory
settings. The device is not
intended for point-of-care use.	The Lyra Direct Strep Assay is
a Real-Time PCR in vitro
diagnostic test for the
qualitative detection and
differentiation of Group A ß-
hemolytic Streptococcus
(Streptococcus pyogenes) and
pyogenic Group C and G ß-
hemolytic Streptococcus
nucleic acids isolated from
throat swab specimens
obtained from patients with
signs and symptoms of
pharyngitis, such as sore throat.
The assay does not
differentiate between pyogenic
Groups C and G ß-hemolytic
Streptococcus.
All negative test results should
be confirmed by bacterial
culture, because negative
results do not preclude Group
A, C or G Strep infection and
should not be used as the sole
basis for treatment.
The assay is intended for use in
hospital, reference, or state
laboratory settings. The device
is not intended for point-of-care
use.
Sample Type	Throat swab	Same
Heat Lysis
Extraction	Manual	Same
Testing Time	55 - 70 minutes	60 - 70 minutes

Differences
Item	Device	Predicate
	Amplivue® GAS Assay
(K141173)	Lyra™ Direct Strep Assay
(K133883)
Target Sequence
Detected	78 bp sequence S. pyogenes
genome, resident in the DNase
B ( sdaB ) gene	GAS* – 99bp product in the
putative competence ( comX1.1 )
gene
Pyo GCS/GGS* – 188bp product
in the tagatose-6-phosphate
kinase ( lacC ) gene
DNA Amplification
Technology	Helicase-dependent
amplification (HDA); self-
contained	Real-time polymerase chain
reaction
Detection
Techniques	Manual; visually read after a
biotinylated and 6-
carboxyfluorescein (6-FAM)
labeled amplicon is captured by
anti-6-FAM on an
immunoreactive strip and
streptavidin-conjugated red
particles attach to the captured
amplicon.	Automatically detects
fluorescence after dissociation
of fluorophore from quencher
during amplification
Reagents/
Components	Dry heating blocks, Dilution
Buffer, Lysis Buffer, Reaction
Tubes, Amplicon Cartridges	Lyra™ Direct Strep Master
Mix, Process Buffer, and
Rehydration Solution
		ABI 7500 Fast Dx 96-well
PCR Plate, optical plate films
and plate centrifuge
Instrument	None	Dry heating block
ABI 7500 Fast DX
Thermocycler
Performance
Characteristics	Sensitivity:
98.4%[95% CI: 95.5% - 99.5%]
Specificity:
95.0%[95% CI: 93.5% - 96.2%]	GAS* Sensitivity:
96.5%[95% CI: 91.3% - 98.6%]
GAS* Specificity:
98.0%[95% CI: 97.0% - 98.6%]
Pyo GCS/GGS* Sensitivity:
95.7%[95% CI: 88.1% - 98.5%]
Pyo GCS/GGS* Specificity:
98.3%[95% CI: 97.4% - 98.9%]

*GAS = Group A Streptococcus; Pyo GCS/GGS = Pyogenic Group C/G Streptococcus

K. Standard/Guidance Document Referenced (if applicable):

Not applicable.

L. Test Principle:

The AmpliVue® GAS Assay detects GAS DNA isolated from throat swab specimens obtained from symptomatic patients. The assay consists of three major steps: 1) specimen preparation, 2) isothermal Helicase-Dependent Amplification (HDA) of a target sequence specific to GAS, and 3) detection of the amplified DNA by target-specific hybridization probes via a colorimetric reaction on a lateral-flow strip which is embedded in a self-contained disposable cassette to prevent amplicon contamination.

Patient specimen on a throat swab is transferred to a Lysis Tube and subjected to heat treatment at 95° C for 10 minutes. The heat-treated sample is diluted 10-fold in a Dilution Tube, and then transferred to a Reaction Tube. A HDA reaction is carried out in the Reaction Tube which contains lyophilized HDA reagents, dNTPs, primers and probes. Incubation at 64°C for 35 minutes results in isothermal amplification of the target sequence by GAS specific primers. The amplified DNA is detected by a set of specific detection probes included in the Reaction Tube: GAS target hybridizes to two specific probes labeled with biotin-triethylene glycol (Bio-TEG) and 6-carboxyfluorescein (6-FAM). A competitive process control (PRC) is included in the Lysis Tube to monitor specimen processing and inhibitory substances in clinical samples, reagent hybridizes to the PRC specific probes labeled with Bio-TEG and 2,4dinitrophenyl (DNP-TEG).

Detection of the amplified DNA with specific probes is achieved by AmpliVue cassettes. The AmpliVue cassettes carry lateral-flow DNA detection strips with an internal control stripe of anti-DNP antibodies (C-line) and a test stripe anti-FAM antibodies (T2-line). GAS amplicon with Bio-TEG and 6-FAM-labeled probes is captured by anti-FAM antibodies at the T2-line, while the internal control amplicon with Bio-TEG and DNP-labeled probes is captured by anti-DNP antibodies at the C-line. The biotin in the amplicon-probe complexes captures the streptavidin-conjugated color particles for visualization and the test result is shown as colored lines that are visually read.

A positive result for GAS (detection of GAS DNA) is reported when the T2-line is visible through the detection window of the cassette. A negative result (no detection of GAS DNA) is reported when only the C-line is displayed. The assay result is regarded as invalid when the T2-line and C-line are not present and the assay should be repeated. The total time to run this assay is 55-70 minutes.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:
- a. Precision/Reproducibility:

Precision studies for the AmpliVue® GAS Assay were conducted by two (2) operators three times (3x) per day for twelve (12) days with a panel of four (4) simulated samples that include moderate positive (3 x LoD) and low positive (LoD), high negative (0.3 x LoD) and negative GAS. The study results are acceptable. The results are shown in the Table I below.

		Operator 1		Operator 2		Combined
Table I: Precision
Group
A Strep	Panel ID	Detected
Pos/Total	% Pos	Detected
Pos/Total	% Pos	Detected
Pos/Total	% Pos
	High Neg	20/36	55.6%	18/36	50.0%	38/72	52.8%
	Low Pos	36/36	100%	36/36	100%	72/72	100%
	Mod Pos	36/36	100%	36/36	100%	72/72	100%
	Neg	0/36	0%	0/36	0%	0/72	0%

The reproducibility of the AmpliVue® GAS Assay was evaluated at three (3) laboratory sites (two external, one in-house). Reproducibility was assessed using a panel of four (4) simulated samples that include moderate positive and low positive, high negative GAS. The panels and controls were processed and tested on the AmpliVue® GAS Assay at each site by 2 operators for 5 non-consecutive days (2 operators x 3 replicates x 5 days x 3 sites = 90 results per concentration). The LoD values were based on the values obtained in the LoD study. The reproducibility study results are acceptable. The results are shown in the Table II below.

Table II: Reproducibility
		Site 1		Site 2		Site 3		Combined
	Panel ID	Detected
Pos/Total	% Pos	Detected
Pos/Total	% Pos	Detected
Pos/Total	% Pos	Detected
Pos/Total	% Pos
Group
A
Strep	High Neg	18/30	60%	19/30	63.3%	13/30	43.3%	50/90	55.6%
	Low Pos	30/30	100%	30/30	100%	30/30	100%	90/90	100%
	Mod Pos	30/30	100%	30/30	100%	30/30	100%	90/90	100%
	Neg	0/30	0%	0/30	0%	0/30	0%	0/90	0%

These study results are acceptable.

b. Linearity/assay reportable range:
Not applicable.
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
AmpliVue® GAS Assay incorporates a process control in the lysis buffer tube that is used to monitor sample processing and evaluate the presence of inhibitory substances and to confirm the integrity of assay reagents and cassette detection. The Quidel Molecular A + G Streptococci Control Set #M111, which contains positive and negative controls, serves as an external processing and extraction controls for the AmpliVue® GAS Assay and were run each day of testing.

Studies were performed to determine the stability of specimens collected using the following routinely used swab systems: flocked nylon, rayon and polyester swabs in Amies media, and rayon and polyester swabs in Stuart media, and rayon swabs in Amies gel. Contrived negative matrix was spiked with fresh GAS (ATCC 12344) at 1 x LoD was used to inoculate the swabs listed above. The spiked samples were tested with the AmpliVue® GAS Assay. Triplicate testing for each condition with each collection/transport system listed above demonstrated that specimens can be stored at 25°C ± 2°C for 2 days and then at 2 to 8°C for up to 8 more days before testing or at 99%, with the lower bound of the 95% CI of >97% extrapolated based on 30% prevalence (recent studies presented to the FDA indicated that it is unlikely that the prevalence for GAS will exceed 30% during the peak season for GAS pharyngitis), and

3) Each testing site demonstrates an NPV of ≥98% and an approximately even distribution of samples is observed among the sites.

The overall sensitivity of the AmpliVue® GAS Assay is 98.4% with the lower bound of the 95% CI of 95.5%, the overall NPV is 99.7% with the lower bound of the 95% CI of 99.0% at a prevalence of

16.1% encountered in this study, with the lowest site NPV of 98.7% (Site 5). When extrapolated to a prevalence of 30%, the overall NPV is 99.3% with a lower bound of the 95% Cl of 97.9%. Based on these values and the criteria noted above, there is no need to add a statement requiring follow-up culture for negative results in the Intended Use for this assay.

However, to further mitigate the risks of a false negative result, the following limitation is added to the limitation section of the package insert: "Additional follow-up testing using the culture method is required if the result is negative and clinical symptoms persist, or in the event of an acute rheumatic fever (ARF) outbreak."

Clinical specificity: b.
See table above.
c. Other clinical supportive data (when a. and b. are not applicable):
Not applicable.
1. Clinical cut-off:
  Not Applicable.
1. Expected values/Reference range:
  The overall incidence of Group A B-hemolytic Streptococcus in patients tested during this study was 16.1% (192/1191) based on composite bacterial culture and 20.1% (239/1191) based on the Amplivue® GAS Assay. All clinical specimens collected during this study were collected between February, 2014 and March 2014.

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:

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