illumigene Mycoplasma Direct DNA Amplification Assay, illumigene Mycoplasma Direct External Controls, illumipro-10

K160829 · Meridian Bioscience, Inc. · OZX · Jun 13, 2016 · Microbiology

Device Facts

• Record ID: K160829
• Device Name: illumigene Mycoplasma Direct DNA Amplification Assay, illumigene Mycoplasma Direct External Controls, illumipro-10
• Applicant: Meridian Bioscience, Inc.
• Product Code: OZX · Microbiology
• Decision Date: Jun 13, 2016
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3980
• Device Class: Class 2
• Attributes: Pediatric

Indications for Use

The illumigene Mycoplasma Direct DNA amplification assay, performed on the illumipro-10™, is a qualitative in vitro diagnostic test for the direct detection of DNA from Mycoplasma pneumoniae in human throat swabs obtained from patients suspected of having Mycoplasma pneumoniae infection. The illumigene Mycoplasma Direct assay utilizes loop-mediated isothermal DNA amplification (LAMP) technology to detect Mycoplasma pneumoniae by targeting a segment of the Mycoplasma pneumoniae genome. Results from the illumigene Mycoplasma Direct DNA amplification assay should be used in conjunction with clinical presentation, other laboratory findings, and epidemiological risk factors as an aid in the diagnosis of Mycoplasma infection and should not be used as the sole basis for treatment or other patient management. Positive results do not rule out co-infection with other organisms and negative results in persons with respiratory tract infections may be due to pathogens not detected by this assay. Lower respiratory tract infections due to M. pneumoniae may not be detected by this assay. If lower respiratory tract infection due to M. pneumoniae is suspected, additional laboratory testing using methods other than the illumigene Mycoplasma Direct DNA Amplification Assay may be necessary.

Device Story

Device is an in vitro diagnostic test for Mycoplasma pneumoniae detection in human throat swabs. Input: throat swab specimen processed via Sample Preparation Apparatus III (heat treatment). Operation: Loop-mediated isothermal DNA amplification (LAMP) on illumipro-10 system. System monitors light transmission changes (absorbance) in reaction chambers during amplification of 208 bp target sequence. Output: Qualitative positive/negative result based on fixed cut-off ratios of light transmission. Used in clinical laboratory settings by professional staff. Output aids clinicians in diagnosing M. pneumoniae infection alongside clinical presentation and other lab findings. Benefits: rapid, automated, self-contained molecular detection without manual DNA extraction.

Clinical Evidence

Clinical study evaluated 456 prospective throat swab specimens from symptomatic patients (ages 3 weeks to 97 years). Compared to reference molecular method (predicate). Results: Positive Percent Agreement 96.0% (95% CI: 80.5-99.3%), Negative Percent Agreement 97.7% (95% CI: 95.8-98.7%), Overall Percent Agreement 97.6% (95% CI: 95.7-98.6%). Invalid rate 0.0%. No performance differences observed based on age or gender.

Technological Characteristics

LAMP-based isothermal amplification; targets 208 bp region of intracellular protease-like gene. Instrumentation: illumipro-10 Automated Isothermal Amplification and Detection System. Detection: visible light transmission (turbidimetry via magnesium pyrophosphate precipitation). Connectivity: standalone. Software: rule-based, fixed cut-off absorbance ratio. Specimen processing: integrated SMP PREP II apparatus.

Indications for Use

Indicated for qualitative detection of Mycoplasma pneumoniae DNA in human throat swabs from patients suspected of infection. Applicable to all ages (pediatric to geriatric). Not for use as sole basis for treatment or patient management.

Regulatory Classification

Identification

A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B; (4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus; (5) Human Metapneumovirus; (6) Rhinovirus; and (7) Adenovirus.

Special Controls

*Classification.* Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;” (2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.

Predicate Devices

• illumigene Mycoplasma DNA Amplification Assay (K123423, K152800)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
K160829

B. Purpose for Submission:
The purpose of the 510(k) submission is to obtain a substantial equivalence determination for the illumigene Mycoplasma Direct DNA Amplification Assay.

C. Measurand:
DNA target sequence is a 353-560 base pair (208 bp) region of an intracellular protease-like gene from Mycoplasma pneumoniae

D. Type of Test:
The illumigene® Mycoplasma Direct DNA amplification assay is a qualitative in vitro diagnostic device for the direct detection of Mycoplasma pneumoniae in human throat swabs collected from patients suspected of having Mycoplasma pneumoniae infection. The molecular assay uses isothermal loop-mediated amplification (LAMP) technology that targets an intracellular protease-like gene of the Mycoplasma pneumoniae genome.

E. Applicant:
Meridian Bioscience, Inc.

F. Proprietary and Established Names:
illumigene® Mycoplasma Direct DNA Amplification Assay
illumigene® Mycoplasma Direct External Controls

G. Regulatory Information:
1. Regulation section:
21 CFR 866.3980: Respiratory Viral Panel Multiplex Nucleic Acid Assay
2. Classification:
Class II

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3. Product code:
OZX - Mycoplasma pneumoniae DNA Assay System
OOI – Real Time Nucleic Acid Amplification System

4. Panel:
Microbiology (83)

H. Intended Use/Indications for Use:

1. Intended Use/Indications for Use:

The illumigene Mycoplasma Direct DNA amplification assay, performed on the illumipro-10™, is a qualitative in vitro diagnostic test for the direct detection of DNA from Mycoplasma pneumoniae in human throat swabs obtained from patients suspected of having Mycoplasma pneumoniae infection.

The illumigene Mycoplasma Direct assay utilizes loop-mediated isothermal DNA amplification (LAMP) technology to detect Mycoplasma pneumoniae by targeting a segment of the Mycoplasma pneumoniae genome.

Results from the illumigene Mycoplasma Direct DNA amplification assay should be used in conjunction with clinical presentation, other laboratory findings, and epidemiological risk factors as an aid in the diagnosis of Mycoplasma infection and should not be used as the sole basis for treatment or other patient management. Positive results do not rule out co-infection with other organisms and negative results in persons with respiratory tract infections may be due to pathogens not detected by this assay. Lower respiratory tract infections due to M. pneumoniae may not be detected by this assay. If lower respiratory tract infection due to M. pneumoniae is suspected, additional laboratory testing using methods other than the illumigene Mycoplasma Direct DNA Amplification Assay may be necessary.

2. Special conditions for use statement(s):

For Prescription Use Only

3. Special instrument requirements:

illumipro-10™ Automated Isothermal Amplification and Detection System from Meridian Bioscience, Inc.

I. Device Description:

The illumigene Molecular Diagnostic Test System incorporates the illumigene® Mycoplasma

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Direct DNA Amplification Assay Test kit, the illumigene Mycoplasma Direct External Controls kit, and the illumipro-10™ Automated Isothermal Amplification and Detection System.

The illumigene Mycoplasma Direct assay utilizes loop-mediated isothermal amplification (LAMP) technology to detect the presence of Mycoplasma pneumoniae in human throat swab specimens. The illumigene Mycoplasma Direct assay is performed using a negative control (illumigene Sample Preparation Apparatus II/Negative Control III [SMP PREP II]), illumigene Heat Treatment Tubes, and an illumigene Mycoplasma Test Device. Collected throat swabs are placed directly into the SMP PREP II that contains assay control buffer. The specimen/control samples are processed through SMP PREP II and extracted DNA is heat-treated. Therefore, heat-treatment produces target and control DNA for isothermal amplification. The heat-treated specimen/control samples are added to the illumigene Mycoplasma Test Device where DNA amplification occurs.

The illumipro-10™ (Incubator/Reader) incubates each illumigene Mycoplasma Test Device containing prepared sample and control material to 63°C that facilitates isothermal amplification of target DNA. When M. pneumoniae is present in the prepared specimen, a 208 base pair region of the M. pneumoniae genome is amplified and magnesium pyrophosphate is produced as a by-product of the reaction. Magnesium pyrophosphate forms a precipitate in the reaction mixture.

The illumipro-10™ monitors the absorbance characteristics (visible light transmission) of each reaction solution at the assay Run Start (Signalinitial; Si) and at the assay Run End (Signalfinal, Sf). The illumipro-10™ calculates the ratio of the Run End absorbance (Signalfinal or Sf) with the Run Start absorbance (Signalinitial, Si) and compares the ratio (Sf:Si) to an established cut-off value. These ratios are calculated by the illumipro-10™ for both the TEST and CONTROL chambers of the device.

The illumigene Mycoplasma External Control Kit contains a Positive control reagent for use in routine quality control testing and is provided separately from the Mycoplasma Direct Kit. The Sample Preparation Apparatus II/Negative Control III (SMP PREP) reagent is included with the Mycoplasma Direct Kit. The Sample Preparation Apparatus II contains a buffered solution with formalin treated E. coli containing Staphylococcus aureus DNA and is processed as the assay's internal control. The internal control monitors amplification inhibition, assay reagent performance and sample processing effectiveness. The Negative Control III component of SMP PREP functions as an External Negative Control. The Negative Control aids the user in detection of reagent deterioration, adverse environmental or test conditions, or variance in operator performance that may lead to test errors.

J. Substantial Equivalence Information:

1. Predicate device name(s): illumigene® Mycoplasma DNA Amplification Assay
2. Predicate 510(k) number(s):

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K123423, K152800

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device illumigene® Mycoplasma Direct DNA Amplification Assay (K160829) | Predicate illumigene® Mycoplasma DNA Amplification Assay (K123423, K152800) |
| Organisms Detected | Mycoplasma pneumoniae | Mycoplasma pneumoniae |
| Amplification and Detection | Self-contained and automated | Self-contained and automated |
| Analyte | DNA | DNA |
| Test Format | Automated; qualitative | Automated; qualitative |
| Assay Target | 208 bp DNA sequence | 208 bp DNA sequence |
| Instrumentation | illumipro-10TM Automated Isothermal Amplification and Detection System | illumipro-10TM Automated Isothermal Amplification and Detection System |
| Reading Method | Visible light transmission | Visible light transmission |
| Differences | | |
| --- | --- | --- |
| Item | Device illumigene® Mycoplasma Direct DNA Amplification Assay (K160829) | Predicate illumigene® Mycoplasma DNA Amplification Assay (K123423, K152800) |
| Intended Use/Indications for Use | The illumigene Mycoplasma Direct DNA amplification assay, performed on the illumipro-10TM, is a qualitative in vitro diagnostic test for the direct detection of DNA from Mycoplasma pneumoniae in human throat swabs obtained from patients suspected of having Mycoplasma pneumoniae infection.The illumigene Mycoplasma Direct assay utilizes loop-mediated isothermal DNA amplification (LAMP) technology to detect Mycoplasma pneumoniae by targeting a segment of the Mycoplasma pneumoniae genome. | The illumigene Mycoplasma DNA amplification assay, performed on the illumipro-10TM, is a qualitative in vitro diagnostic test for the direct detection of DNA from Mycoplasma pneumoniae in human throat and nasopharyngeal swabs obtained from patients suspected of having Mycoplasma pneumoniae infection.The illumigene Mycoplasma assay utilizes loop-mediated isothermal DNA amplification (LAMP) technology to detect Mycoplasma pneumoniae by targeting a segment of the Mycoplasma pneumoniae genome. |

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| Differences | | |
| --- | --- | --- |
| Item | Device
*illumigene*^{®} Mycoplasma
Direct DNA Amplification
Assay (K160829) | Predicate
*illumigene*^{®} Mycoplasma
DNA Amplification Assay
(K123423, K152800) |
| | Results from the *illumigene* Mycoplasma Direct DNA amplification assay should be used in conjunction with clinical presentation, other laboratory findings, and epidemiological risk factors as an aid in the diagnosis of Mycoplasma infection and should not be used as the sole basis for treatment or other patient management. Positive results do not rule out co-infection with other organisms and negative results in persons with respiratory tract infections may be due to pathogens not detected by this assay. Lower respiratory tract infections due to *M. pneumoniae* may not be detected by this assay. If lower respiratory tract infection due to *M. pneumoniae* is suspected, additional laboratory testing using methods other than the *illumigene* Mycoplasma Direct DNA Amplification Assay may be necessary. | genome.

Results from the *illumigene* Mycoplasma DNA amplification assay should be used in conjunction with clinical presentation, other laboratory findings, and epidemiological risk factors as an aid in the diagnosis of Mycoplasma infection and should not be used as the sole basis for treatment or other patient management. Positive results do not rule out co-infection with other organisms and negative results in persons with respiratory tract infections may be due to pathogens not detected by this assay. Lower respiratory tract infections due to *M. pneumoniae* may not be detected by this assay. If lower respiratory tract infection due to *M. pneumoniae* is suspected, additional laboratory testing using methods other than the *illumigene* Mycoplasma DNA Amplification Assay may be necessary.

*illumigene* Mycoplasma is intended for use in hospital, reference or state laboratory settings. The device is not intended for point-of-care use. |
| Specimen Types | Human throat swabs | Human throat and nasopharyngeal swabs |

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| Differences | | |
| --- | --- | --- |
| Item | Device illumigene® Mycoplasma Direct DNA Amplification Assay (K160829) | Predicate illumigene® Mycoplasma DNA Amplification Assay (K123423, K152800) |
| Swab Types | Rayon, Flocked Nylon, or Polyester | Cotton, Foam, Flocked Nylon, Polyester, or Rayon |
| Transport Media | Liquid Amies without charcoal or Liquid Stuart | 0.85% saline, M4, M4-RT, M5, UTM-RT, or Liquid Amies without charcoal |
| Sample Processing/Preparation | Extraction reagents included with kit | Manual extraction with Qiagen® DSP DNA Minikit |
| External Controls | Positive control is provided separately in the illumigene Mycoplasma Direct External Control Kit; External Negative Control is part of the Direct kit (Sample Preparation Apparatus II/Negative Control III)
External Positive Control is a tris-buffered solution with plasmid target DNA; External Negative Control is formalin-treated E. coli with S. aureus DNA | External Positive and Negative Controls are provided separately in the illumigene Mycoplasma External Control kit
External Positive Control is a Tris-buffered solution with plasmid target DNA; External Negative Control is a tris-buffered solution with plasmid DNA carrying S. aureus insert |
| Kit Storage | 2-27°C | 2-8°C |

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

None

## L. Test Principle:

The illumigene Mycoplasma Direct assay uses loop-mediated amplification (LAMP) technology that employs specially designed loop primers for specific and continuous isothermal amplification. When the target DNA is present in the specimen, the sequence is amplified and magnesium pyrophosphate is produced as a by-product. The magnesium pyrophosphate forms a white precipitate and causes the reaction solution to appear turbid. If there is a change in sample absorbance between Run End and Run Start due to magnesium pyrophosphate precipitation, then the target DNA is detected and the sample is considered positive. When the target DNA is absent, there is no detectable change in sample absorbance and the sample is considered negative. Sample readouts are determined by comparing the initial to final absorbance reading ratios to a fixed cut-off value.

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# M. Performance Characteristics (if/when applicable):

# 1. Analytical performance:

# a. Precision/Reproducibility:

Blind coded panels of 10 samples were provided to three participating clinical laboratory sites. Samples were randomly sorted within each panel to mask the sample identities. Each panel consisted of three moderate positive samples, three low positive samples, three high negative samples, and one negative sample. Contrived moderate positive, low positive, and high negative panel members were prepared using inoculated rayon swabs with simulated negative matrix (simulated throat swab matrix in Liquid Amies medium) using M. pneumoniae strain M129 at final concentrations provided in table below.

| Sample ID | Final Concentration (CFU/Test) | Final Concentration (CFU/mL) | Expected Result |
| --- | --- | --- | --- |
| Moderate Positive | 3.08 | 800 | Positive |
| Low Positive | 1.54 | 400 | Positive |
| High Negative | 0.002 | 0.519 | Negative |
| Negative | 0 | 0 | Negative |

Reported Product LoD (CFU/mL): 200 CFU/mL, which is equivalent to approximately 0.77CFU/Test

Three lots of illumigene Mycoplasma Direct reagents, three lots of illumigene Mycoplasma Direct External Controls, and eight illumipro-10TM instruments were used in reproducibility studies. Each clinical site tested two panels each day for five days (inter-assay variability) with testing performed by two operators at each site on the same day (intra-assay variability). Positive and Negative Controls were tested each day of testing. Testing results are provided in the table below:

| | Site 1 | | Site 2 | | Site 3 | | Total - All Sites | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample Type | Percent Agreement | | Percent agreement | | Percent agreement | | Percent agreement | | Average \( {\mathrm{S}}_{\mathrm{f}} : {\mathrm{S}}_{\mathrm{i}} \) | SD | %SC |
| High Negative | 30/30 | 100.0% | 30/30 | 100.0% | 30/30 | 100% | 90/90 | 100.0% | 101.45 | 2.17 | 2.14 |
| Low Positive | 30/30 | 100.0% | 30/30 | 100.0% | 29/30 | 96.7% | 89/90 | 98.9% | 62.35 | 5.21 | 8.36 |
| Moderate Positive | 30/30 | 100.0% | 30/30 | 100.0% | 30/30 | 100.0% | 90/90 | 100.0% | 61.47 | 3.88 | 3.88 |
| Negative | 9/10 | 90.0% | 10/10 | 100.0% | 10/10 | 100.0% | 29/30 | 96.7% | 99.70 | 7.67 | 7.69 |
| Negative Control | 10/10 | 100.0% | 10/10 | 100.0% | 10/10 | 100.0% | 30/30 | 100.0% | 101.7 | 1.80 | 1.77 |
| Positive Control | 10/10 | 100.0% | 10/10 | 100.0% | 10/10 | 100.0% | 30/30 | 100.0% | 60.85 | 2.36 | 3.88 |

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b. Linearity/assay reportable range:

Not applicable as the illumigene Mycoplasma DNA amplification assay is a qualitative assay.

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

Specimen Stability:

Specimen storage and hold time parameters were evaluated for the illumigene Mycoplasma Direct DNA amplification assay. Validation studies performed at Meridian were completed using rayon, polyester, or flocked nylon swabs with Liquid Amies without charcoal and Liquid Stuart. Samples were prepared using a simulated throat swab matrix spiked with *M. pneumoniae* strain M129 at high negative and low positive concentrations (200 CFU/mL).

Testing was performed in triplicate for each of the following storage conditions: (1) upper limit of room temperature (31 ± 2°C), (2) refrigerated (2-8°C), or (3) frozen (-28 to -16°C). Samples stored at room temperature were tested through 49+ hours and samples stored refrigerated or frozen were tested through 15 days. Results of the study supported that throat swabs (rayon, polyester, flocked nylon) in the above mentioned transport media can be held at the following claimed storage conditions: room temperature for up to 24 hours and refrigeration for up to 14 hours prior to sample preparation. Frozen storage is not part of the Mycoplasma Direct Package Insert and was only evaluated to support this mechanism of storage for analytical and clinical studies.

Extracted Specimen Stability:

Studies were performed to evaluate storage conditions of processed samples prior to testing with the illumigene Mycoplasma Direct DNA amplification assay. Samples consisting of rayon, polyester, or flocked nylon swabs with Liquid Amies (without charcoal) or Liquid Stuart in a simulated negative matrix were spiked with *M. pneumoniae* M129. The prepared samples were held at room temperature (31 ± 2°C) for up to 3 hours and then heat-treated again for 10 minutes preceding testing on the illumipro-10™. Results of the study support the claimed stability of heat-treated samples for up to 3 hours at room temperature (19-29°C). Heat treatment must be re-performed (95 ± 5°C for 10 ± 2 minutes) if heat-treated samples are stored prior to illumipro-10™ testing.

Controls:

External Controls: The illumigene Mycoplasma Direct Positive External Control Reagent includes an External Positive Control and is sold separately from the illumigene Mycoplasma Direct DNA amplification assay. The External Positive Control contains non-infectious plasmid DNA with a *M. pneumoniae* insert that is

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stored in a tris-buffered solution with azide. The Negative Control is a component of the Sample Preparation Apparatus II/Negative Control III reagent that is included in the illumigene Mycoplasma Direct kit. The Negative External Control contains formalin-treated *E. coli* with *S. aureus* DNA. External Controls are necessary for routine Quality Control that help the end-user detect reagent deterioration, adverse environmental or test conditions, or differences in operator performance that result in test errors. Testing of external controls was performed for all analytical and clinical studies with no discordant results. It is recommended that external controls are run with each new lot and new shipment of illumigene Mycoplasma Direct kits. External control testing should be performed thereafter in accordance with appropriate federal, state, and local guidelines. Results from the illumigene Mycoplasma Direct kit should not be reported if external controls do not produce the expected results.

**Internal Control:** Each test device contains lyophilized amplification reagents and primers specific to the Internal Control and is housed in the CONTROL chamber. Internal Control DNA (tris-buffered solution with formalin-treated *E. coli* containing a plasmid with a segment of the *S. aureus* genome) is in the Sample Preparation Apparatus II (SMP PREP II) reagent and is processed through all steps of the test procedure. SMP PREP II is a built-in component of the illumigene Mycoplasma Direct amplification assay. A functioning Internal Control is demonstrated by a valid assay result from the TEST chamber.

**d. Detection limit:**

Analytical sensitivity studies were designed to evaluate the analytical limit of detection (LoD) of the illumigene Mycoplasma Direct DNA amplification assay. The LoD is the lowest number of colony forming units (CFUs) per test aliquot that can be distinguished from negative samples with a high degree of probability (95%). *M. pneumoniae* strains M129 and FH were used to establish the LoD. These strains were diluted in a simulated negative throat swab matrix (Liquid Amies with a rayon swab inoculated with throat gargle screened negative for *M. pneumoniae*). A minimum of three dilutions near the expected LoD in 20 individually prepared replicates were evaluated for each *M. pneumoniae* strain. Testing was performed using three lots of reagents and eight *illumipro-10™* instruments over multiple days with two different technicians. External Positive and Negative Controls were tested each day throughout the study. These studies confirmed that the LoD for the assay was 2350 CFU/mL for the FH strain and 200 CFU/mL for the M129 strain.

**e. Assay Reactivity/Inclusivity:**

As the primer and probe sequences for the illumigene Mycoplasma Direct did not change from the originally cleared assay, a repeat assay reactivity/inclusivity study was not required. It is expected that both Type I and Type II strains will be detected with the Direct assay.

See predicate K123423 for original studies.

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# f. Analytical Specificity/Cross Reactivity:

A re-evaluation of cross-reactivity with the illumigene Mycoplasma Direct DNA amplification assay was not performed. However, an interference study for the Direct assay was completed and demonstrated that the new extraction and DNA processing step included in the SMP PREP II was sufficient and did not result in interference with other substances (both biological and chemical) that could be present in clinical samples. A cross-reactivity study was previously conducted on the predicate device (illumigene Mycoplasma DNA amplification assay) in submission K123423 for which primers and probes are identical to the Mycoplasma Direct assay. Therefore, because the primer/probe sequences remain unchanged from the predicate and the modified extraction process was successfully clinically evaluated, a cross-reactivity study on the Direct assay was not required. No cross-reactivity is expected with the illumigene Mycoplasma Direct assay.

See predicate K123423 for original studies.

# g. Interference:

An interference study using 17 potential interfering chemical and biological non-microbial substances was conducted to evaluate the accuracy of results generated by the illumigene Mycoplasma Direct DNA amplification assay. Contrived low positive samples P1 (M129) and P2 (FH) were spiked into a simulated negative matrix (Liquid Amies media with screened negative throat gargle and a rayon swab) at $300\mathrm{CFU / mL}$ and $1762.5\mathrm{CFU / mL}$ , respectively. Each potential interferent was diluted to specific concentrations in sterile saline and added to one simulated negative sample (N1) and two unique contrived low positive samples (P1, P2). Testing included three replicates for each interferent alone (negative sample) and three replicates for each interferent mixed with each of the two M. pneumoniae strains (six positive samples per interferent).

The following substances, at the saturated solvent/diluent concentrations indicated, do not interfere with illumigene Mycoplasma Direct assay (no false positive or false negative results):

| Substance | Saturated Solvent/Diluent Concentration |
| --- | --- |
| Acetaminophen | 18.1 mg/mL |
| Albuterol Sulfate | 20 mg/mL |
| Aspirin | 9.1 mg/mL |
| Azithromycin dehydrate | 2.0 mg/mL |
| Cepacol® Mouthwash | [Alcohol (1.4% v/v), Cetylpyridinium chloride (0.005% v/v)] |
| Contac® Cold + Flu Tablets | [Acetaminophen (14.8 mg/mL), Chlorpheniramine maleate (0.06 mg/mL), |
| Phenylephrine HCl | 0.15 mg/mL |
| Diphenhydramine HCl | 2.6 mg/mL |

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| Erythromycin | 20.0 mg/mL |
| --- | --- |
| HALLS® Cough Drops | [Menthol (0.06 mg/mL)] |
| Ibuprofen | 12.7 mg/mL |
| Phenylephrine HCl | 0.595 mg/mL |
| Prednisone | 20.0 mg/mL |
| Robitussin® Cough+Chest Congestion Cough Syrup | [Dextromethorphan HBr (0.20 mg/mL), Guaifenesin (2.0 mg/mL)] |
| Saline Nasal Spray | [Sodium chloride (0.65 mg/mL)] |
| Mucus | 5.0mg/mL |
| White Blood Cells | 0.5% v/v |

All potentially interfering substances that were evaluated yielded acceptable results except whole blood at concentrations $>2\%$ v/v. As with the predicate K123423, phenylephrine HCl (found in nasal decongestants and flu and cold tablets) at concentrations $>0.595\mathrm{mg / ml}$ interfered with the Mycoplasma Direct assay and caused false negative results with M. pneumoniae strain M129. Therefore, this information is provided in the Mycoplasma Direct assay package insert as limitations. During the interference study, Robitussin® Cough+Congestion Cough Syrup initially produced 1/3 invalid results with Positive Sample P2. Repeat testing produced acceptable results (1/1 positive) and therefore, Robitussin® Cough+Congestion Cough Syrup is not considered an interferent. This information is relayed in the package insert.

# Assay cut-off:

See predicate K123432 for original studies. Below is a general summary.

There are fixed cut-off values for the illumigene Mycoplasma Direct assay that allows sample results to be reported. The pre-selected cut-off values for the TEST and CONTROL chambers of the Mycoplasma Test Device were previously optimized and validated for K123423. Fixed cut-off values for the CONTROL chamber assess validity. CONTROL chamber $S_{f}:S_{i}$ ratios less than $90\%$ are considered valid and results from the TEST chamber can be reported (POSITIVE, NEGATIVE, INVALID). CONTROL chamber $S_{f}:S_{i}$ ratios greater than or equal to $90\%$ are considered invalid which prevents TEST chamber results from being reported. More stringent cut-off criteria are applied to the CONTROL chamber reaction in order to ensure that amplification is not inhibited, reagents are performing as intended, and sample processing was performed appropriately.

Fixed cut-off values for the TEST chamber are used to report sample results. TEST chamber $S_{f}:S_{i}$ ratios less than $82\%$ are reported as 'POSITIVE' and TEST chamber $S_{f}:S_{i}$ ratios greater than or equal to $82\%$ are reported as 'NEGATIVE'. Numerical values for the CONTROL and the TEST chamber results are not displayed for the user.

# 2. Comparison studies:

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a. Clinical Sensitivity and Clinical Specificity:

The illumigene Mycoplasma Direct DNA amplification assay was evaluated in 2015-2016 by three independent clinical test sites located in the Midwestern, Southern, and Central regions of the United States. A total of 458 throat swab samples were collected from male and female patients suspected of having a Mycoplasma pneumoniae infection. The study included testing of prospectively collected throat swabs from pediatric, adult, and geriatric patients that were presumed to be symptomatic with an upper respiratory tract infection caused by M. pneumoniae. Double throat swabs were collected per patient; one swab was placed in Liquid Amies transport media and one swab was placed in M4 viral transport media that was only used for discrepant analysis. These clinical throat swab specimens were evaluated with the test device to establish performance characteristics.

The performance of the Mycoplasma Direct assay was compared to a reference molecular in-vitro diagnostic method, the illumigene Mycoplasma DNA Amplification Assay that was previously cleared by FDA. illumigene Mycoplasma predicate device testing and discrepant testing was performed at Meridian Bioscience and not at the clinical study sites.

Two specimens were excluded from performance analysis due to instrument error and an unacceptable sample type. A total of 456 prospectively collected throat swabs were eligible for analysis. From this clinical study, the overall prevalence of $M$ pneumoniae in the prospective throat swab specimens was $7.5\%$ (34/456). No invalid results were produced by the Mycoplasma Direct assay; however, the reference molecular method yielded 28 invalid results. Repeat predicate testing for these 28 samples yielded results that were identical to the test device and presented in the table below. During clinical evaluation, 11 discordant results were produced. Repeat testing was performed according to the Clinical Trial Protocols and Clinical Trial Discrepant Analysis Plan.

The illumigene Mycoplasma Direct assay performance for prospective throat swab specimens as compared to the reference method (illumigene Mycoplasma) are shown in the tables below. There were no restrictions on age, gender, medications, or known pharmaceutical therapies.

Performance Characteristics of the illumigene Mycoplasma Direct Assay (All Sites Combined)

| | illumigene Mycoplasma Predicate Assay | | | |
| --- | --- | --- | --- | --- |
| illumigene Mycoplasma Direct Assay | Positive | Negative | Invalid | Totals |
| Positive | 24 | 10a | 0 | 34 |
| Negative | 1b | 421 | 0 | 422 |
| Invalid | 0 | 0 | 0 | 0 |
| Totals | 25 | 431 | 0 | 456 |

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| | | | 95% CI |
| --- | --- | --- | --- |
| Overall Percent Agreement | 445/456 | 97.6% | 95.7 - 98.6% |
| Positive Percent Agreement | 24/25 | 96.0% | 80.5 - 99.3% |
| Negative Percent Agreement | 421/431 | 97.7% | 95.8 - 98.7% |
| Invalid Rate | 0/456 | 0% | 0.0 - 0.8% |

a 4/10 samples were identified positive by illumigene Mycoplasma after testing with an additional frozen sample.
b Repeat testing by illumigene Mycoplasma with the original patient sample and an additional frozen sample produced negative results.

Age information was known for all patients included in the performance analysis. Patients ranged in age from 3 weeks to 97 years old.

Performance of the illumigene Mycoplasma Direct Based on Age

| Patient Age | Total Positive | Total Samples | Prevalence (%) |
| --- | --- | --- | --- |
| 0 to 1 Month | 0 | 7 | 0.0% |
| 2 Months to 2 Years | 5 | 157 | 3.2% |
| 3 Years to 12 Years | 22 | 147 | 15.0% |
| 13 Years to 21 Years | 5 | 38 | 13.2% |
| 22 Years to 65 Years | 2 | 86 | 2.3% |
| >65 Years | 0 | 21 | 0.0% |

No performance differences were noted based on chronological age.

The study population included 226 male and 230 female patients. The illumigene Mycoplasma Direct detected 18 positives samples from specimens collected from males and 16 positive samples from specimens collected from females. When comparing the illumigene Direct to the illumigene predicate device, no performance differences were noted based on gender.

Clinical performance of the Direct assay compared to the illumigene Mycoplasma predicate assay was also evaluated based on clinical site. From the data presented below, there were no differences in clinical performance between the assays based on clinical site.

Performance of the illumigene Mycoplasma Direct Based on Clinical Site

| Clinical Site ID | illumigene Direct/illumigene Predicate | Positive Percent Agreement | 95% CI | illumigene Direct/illumigene Predicate | Negative Percent Agreement | 95% CI |
| --- | --- | --- | --- | --- | --- | --- |
| Clinical Site 1 | 3/4 | 75.0% | 30.1 - 95.4% | 147/148 | 99.3% | 96.3 – 99.9% |
| Clinical Site 2 | 2/2 | 100.0% | 34.2 -100.0% | 45/47 | 95.7% | 85.8 – 98.8% |
| Clinical Site 3 | 19/19 | 100.0% | 83.2 - 100.0% | 229/236 | 97.0% | 94.0 – 98.6% |
| All Sites Combined | 24/25 | 96.0% | 80.5% - 99.3% | 421/431 | 97.7% | 95.8 – 98.7% |

b. Matrix comparison:

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Sample type equivalency studies were performed to determine the suitability of two non-nutritive transport mediums with three swab types for use with the illumigene Mycoplasma Direct DNA amplification assay. The following transport mediums were evaluated: Liquid Amies (without charcoal) and Liquid Stuart. The following swab types were evaluated: rayon, polyester, and flocked nylon.

Simulated negative matrix was prepared by inoculation of Liquid Amies or Liquid Stuart with screened negative throat gargle and one of the three abovementioned swabs. Contrived low positive samples were prepared by inoculating simulated negative matrix with Mycoplasma pneumoniae strain M129 to approximately 300 CFU/mL. All negative and positive samples were tested in triplicate for all possible combinations of swab and media type. All negative samples yielded expected results. One contrived positive sample containing a Liquid Stuart/flocked nylon swab combination yielded a negative result. Testing 20 additional contrived positive samples in Liquid Stuart media with flocked nylon swabs yielded no discordant results further demonstrating that media/swab combination can be used with the Mycoplasma Direct assay.

3. Clinical studies: See Comparison Studies under Section M 2 on pages 11-12.

4. Clinical cut-off:

Not applicable

5. Expected values/Reference range:

The overall incidence of Mycoplasma pneumoniae in prospectively collected throat swab specimens in the 2015-2016 clinical study was 7.5% (34/456).

N. Instrument Name

illumipro-10™ Automated Isothermal Amplification and Detection System

O. System Description

1. Modes of Operation:

The following principles of operation enable the illumigene Mycoplasma Direct assay to detect the presence of M. pneumoniae in throat swab specimens:

A Mycoplasma pneumoniae throat swab is collected from a patient following appropriate institutional procedures. The swabs are placed in either non-nutritive Liquid Amies (without charcoal) transport media or Liquid Stuart and delivered to the clinical laboratory for testing. The collected throat swab is added directly to the Sample Preparation Apparatus II/Negative Control III reagent that initiates specimen and internal control processing and DNA extraction. The sample/control mixture is passed through a filter on the SMP PREP II and

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into heat-treatment tubes to prepare the DNA for amplification and detection. The heat-treated sample/control mixture is added to an illumigene Mycoplasma Test Device that contains the TEST and CONTROL chambers.

The Mycoplasma Test Device containing prepared samples and control reagent is placed in the illumipro-10™ instrument (incubator/reader). Each test device is heated, facilitating amplification of target DNA. When M. pneumoniae is present in a throat swab specimen, a conserved M. pneumoniae DNA sequence is amplified and magnesium pyrophosphate is generated as a byproduct of the reaction. Magnesium pyrophosphate forms a precipitate in the reaction mixture causing the solution to become turbid. The illumipro-10™ instrument detects the change in light transmission through the reaction mixture created by precipitating magnesium pyrophosphate. The illumipro-10™ calculates the ratio between the initial and final absorbance values (Sᵢ:Sₜ) and compares this ratio to an established cut-off value. Sample results are reported as POSITIVE, NEGATIVE, or INVALID based on a comparison between the calculated absorbance ratio and the fixed cut-off.

2. Software:

The illumigene Mycoplasma Direct assay utilizes the same software as the previously cleared predicate device, the illumigene Mycoplasma Assay. Since there have been no changes to the internal assay cut-off or other software specifications, the original assay file used in the cleared device is used in the Direct assay. FDA has previously reviewed the applicant's Hazard Analysis and software development processes in K123423.

3. Specimen Identification:

Specimens are identified by position in the heat block wells of the illumipro-10 instrument. Default Sample Identification is based on Block and Well position (e.g., Block A, Well 1). The user can provide Sample Identification information using the keypad, illumipro-10 barcode scanner, or the optional external keyboard. Sample information is entered by following instructions displayed on the illumipro-10 screen.

4. Specimen Sampling and Handling:

Clinical specimens are prepared using the Sample Preparation Apparatus II/Negative Control III reagent included with the illumigene Mycoplasma Direct DNA amplification assay. Incubation, loop-mediated isothermal amplification, and detection are automated using the illumipro-10™ instrument.

5. Calibration:

Calibration of the illumipro-10™ is not required. However, verification of a properly functioning OPTICS SYSTEM must be performed monthly for both Block A and B. Stepwise instructions for Optics System Verification are provided on the illumipro-10 display, utilizing the SERVICE MODE menu. When the illumipro-10 is powered on, the instrument automatically conducts an internal Power-On Self Test (POST) to ensure that

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software and hardware components are performing as expected. A POST Test failure is signaled by an Error Code that can be addressed following instructions in the illumipro-10 Operator's Manual.

## 6. Quality Control:

The illumigene Mycoplasma Direct External Positive Control Kit contains only a positive control reagent and is not a component of the Mycoplasma Direct test system. The External Positive Control is processed using the same DNA extraction methodology as the clinical patient specimens. The illumigene Sample Preparation Apparatus II/Negative Control III reagent is built into the illumigene Mycoplasma Direct kit and serves as the External Negative Control. In general, External Controls are intended to aid the user in detection of reagent deterioration, adverse environmental or test conditions, or variance in operator performance that may lead to test errors. Therefore, the illumigene Mycoplasma External Controls are recommended for routine Quality Control. The performance of External Positive and Negative Controls were validated as part of the clinical study.

Each test device also includes an internal control chamber that houses S. aureus control DNA. The internal control monitors amplification inhibition, assay reagent performance, and sample processing effectiveness. If the internal control fails, an invalid result is generated and no read-out is given for the TEST chamber of the device.

## P. Proposed Labeling:

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

## Q. Conclusion:

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

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