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The BIOFIRE® SPOTFIRE® Respiratory/Sore Throat (R/ST) Panel Mini is a multiplexed polymerase chain reaction (PCR) test intended for use with the BIOFIRE® System for the simultaneous, qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swab (NPS) specimens obtained from individuals with signs and symptoms of respiratory tract infection, including COVID-19; (Respiratory menu) or in throat swab (TS) specimens from individuals with signs and symptoms of pharyngitis (Sore Throat menu).

The following analytes are identified and differentiated using the SPOTFIRE R/ST Panel Mini:

Respiratory Menu:
Viruses
Coronavirus SARS-CoV-2
Human rhinovirus
Influenza A virus
Influenza B virus
Respiratory syncytial virus

Sore Throat Menu:
Viruses
Human rhinovirus
Influenza A virus
Influenza B virus
Respiratory syncytial virus
Bacteria
Streptococcus pyogenes (group A Strep)

Nucleic acids from the viral and bacterial organisms identified by this test are generally detectable in NPS/TS specimens during the acute phase of infection. The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and symptoms of respiratory infection and/or pharyngitis is indicative of the presence of the identified microorganism and aids in diagnosis if used in conjunction with other clinical and epidemiological information, and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Negative results in the setting of a respiratory illness and/or pharyngitis may be due to infection with pathogens that are not detected by this test, or a respiratory tract infection that may not be detected by an NPS or TS specimen. Positive results do not rule out co-infection with other organisms. The agent(s) detected by the SPOTFIRE R/ST Panel Mini may not be the definite cause of disease.

Additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection and/or pharyngitis.

The SPOTFIRE R/ST Panel Mini simultaneously identifies 5 different respiratory viral pathogens in nasopharyngeal swabs (NPS) or 5 different viral and bacterial pharyngitis pathogens in throat swabs (TS) from individuals with signs and symptoms of respiratory tract infections or pharyngitis, respectively, (see Table 1) The SPOTFIRE R/ST Panel Mini is compatible with the BIOFIRE® System, a polymerase chain reaction (PCR)-based in vitro diagnostic system for infectious disease testing. The BIOFIRE System Sottware executes the SPOTFIRE R/ST Panel Mini test and interprets and reports the test results. The SPOTFIRE R/ST Panel Mini was designed to be used in CLIA-waived environments.

A test is initiated by loading Hydration Solution injection solution injection port of the SPOTFIRE R/ST Panel Mini pouch and NPS or TS specimen, mixed with the provided Sample injection port of the SPOTFIRE R/ST Panel Mini pouch and placing it in the SPOTFIRE System. The reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration and Sample/Buffer Mix rehydrates the reagents. After the pouch is prepared, the SPOTFIRE System Software guides the user through the steps of placing the pouch into the instrument, scanning the sample identification, and initiating the run.

The SPOTFIRE System contains coordinated systems of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically-controlled pneumatic pistons are positioned over multiple plungers in order to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the pouch to drive the PCR reactions and the melt curve analysis.

Nucleic acid extraction occurs within the SPOTFIRE R/ST Panel Mini pouch using mechanical Ivsis followed by purfication using standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, the SPOTFIRE System performs a nested multiplex PCR that is executed in two stage. During the first stage, the SPOTFIRE System performs a single, large volume, highly multiplexed reverse transcription PCR (rt-PCR) reaction. The products from first stage PCR are then diluted and combined with a fresh, primer-free master mix and a fluorescent doublestranded DNA binding dye (LC Green® Plus, BioFire Diagnostics). The solution is then distributed to each well of the array. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR, or nested PCR, is performed in singleplex fashion in each well of the array. At the conclusion of the 2nd stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the 2nd stage PCR captures fluorescent images of the PCR reactions and software interprets the data.

The SPOTFIRE System Software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the SPOTFIRE R/ST Panel Mini.

This document describes the BIOFIRE® SPOTFIRE® Respiratory/Sore Throat (R/ST) Panel Mini, a multiplex PCR test.

1. Table of Acceptance Criteria and Reported Device Performance

The document provides extensive analytical performance data rather than a direct comparison of acceptance criteria to reported clinical performance metrics (like PPA and NPA). However, the "Summary of Performance Data" for clinical studies does present sensitivity/PPA and specificity/NPA, which can be interpreted as the reported device performance against implied clinical acceptance criteria.

Clinical Performance Summary (NPS Specimens - Respiratory Menu)

Clinical Performance Summary (TS Specimens - Sore Throat Menu)

Analytical Acceptance Criteria and Results for key studies:

2. Sample Sizes and Data Provenance

• Clinical Performance (Test Set):
  • NPS Specimens (Respiratory Menu - Prospective): Total of 1115 specimens. The document doesn't explicitly state the country of origin but implies clinical sites (e.g., "as tested by intended users"). This is prospective data.
  • NPS Specimens (Respiratory Menu - Archived): Used for some analytes, e.g., Human Rhinovirus (30 positive, 454 negative), Influenza A (59 positive, 423 negative), Influenza B (30 positive, 28 negative), RSV (37 positive, 447 negative). This is retrospective data.
  • TS Specimens (Sore Throat Menu - Prospective): Total of 876 specimens for most viral targets. Streptococcus pyogenes had 217 positive (PCR) / 177 positive (Culture) and 660 negative (PCR) / 692 negative (Culture). This is prospective data.
  • TS Specimens (Sore Throat Menu - Archived): Used for some analytes, e.g., Human Rhinovirus (2 positive, 57 negative), Influenza A (11 positive, 44 negative), Influenza B (20 positive, 0 negative), RSV (2 positive, 57 negative), Streptococcus pyogenes (39 positive, 10 negative). This is retrospective data.
  • TS Specimens (Sore Throat Menu - Contrived): Used for some analytes, e.g., Influenza A (93 positive, 332 negative), Influenza B (49 positive, 333 negative), RSV (50 positive, 381 negative). This would be laboratory-generated data.

3. Number of Experts and Qualifications for Ground Truth

The document does not specify the number or qualifications of experts used to establish the ground truth for the clinical test set. It mentions using "molecular assays or known specimen composition" as comparator methods for most analytes, and "culture" as the reference method for Streptococcus pyogenes.

4. Adjudication Method

The document does not describe any specific adjudication method (e.g., 2+1, 3+1) for establishing the ground truth or resolving discrepancies in the clinical test set.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No MRMC comparative effectiveness study is mentioned or implied, as this device is an in vitro diagnostic (IVD) PCR test for direct pathogen detection, not an AI-assisted diagnostic imaging or interpretation tool for human readers. Therefore, the concept of "human readers improve with AI vs without AI assistance" does not apply.

6. Standalone Performance

Yes, the studies described are for standalone performance. The BIOFIRE® SPOTFIRE® R/ST Panel Mini provides automated interpretation and reporting of test results based on the PCR assay. It is designed to be used independently to generate a qualitative detection and identification of microbial nucleic acids.

7. Type of Ground Truth Used

• Clinical Performance (Prospective/Archived): The ground truth for most analytes was established using molecular assays or, in some cases, known specimen composition. For Streptococcus pyogenes, culture was also used as a reference method for some comparisons.
• Analytical Performance (LoD, Inclusivity, Exclusivity, Interference, Reproducibility, Matrix Validation, Transport Media Validation, Carry Over): The ground truth was established through known specimen composition (e.g., contrived samples with known concentrations of organisms, presence of interfering substances, specific transport media).

8. Sample Size for the Training Set

The document does not explicitly mention a "training set" in the context of machine learning or AI algorithm development. This device is a PCR-based test, and its performance is validated through analytical and clinical studies, not typically through a machine learning training phase with a distinct dataset. The "training" in this context refers to the development and optimization of the PCR primers, probes, and reaction conditions.

9. How the Ground Truth for the Training Set Was Established

Given that this is a PCR diagnostic device, not an AI algorithm in the typical sense of needing a "training set" for model learning, this question isn't directly applicable. The "ground truth" for developing and optimizing the PCR assays themselves would have been established through:

• Careful selection and validation of synthetic nucleic acid targets.
• Testing with characterized microbial isolates and clinical samples whose status was confirmed by established reference methods (e.g., sequencing, culture, validated molecular tests).
• In silico analysis of genetic sequences to design primers and probes with high specificity and inclusivity.

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The BIOFIRE® SPOTFIRE® Respiratory/Sore Throat (R/ST) Panel is a multiplexed polymerase chain reaction (PCR) test intended for use with the BIOFIRE® System for the simultaneous, qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swab (NPS) specimens obtained from individuals with signs and symptoms of respiratory tract infection, including COVID-19; (Respiratory menu) or in throat swab (TS) specimens from individuals with signs and symptoms of pharyngitis (Sore Throat menu).

The following organism types and subtypes are identified and differentiated using the SPOTFIRE R/ST Panel:

Respiratory Menu Viruses Adenovirus Coronavirus SARS-CoV-2 Coronavirus (seasonal) Human metapneumovirus Human rhinovirus/enterovirus Influenza A virus Influenza A virus/H1-2009 Influenza A virus/ H3 Influenza B virus Parainfluenza virus Respiratory syncytial virus

• Bacteria Bordetella parapertussis Bordetella pertussis Chlamydia pneumoniae Mycoplasma pneumoniae
  Sore Throat Menu Viruses Adenovirus Coronavirus (seasonal) Human metapneumovirus Human rhinovirus/enterovirus Influenza A virus Influenza A virus/H1-2009 Influenza A virus/H3 Influenza B virus Parainfluenza virus Respiratory syncytial virus

Bacteria Chlamydia pneumoniae Mycoplasma pneumoniae Streptococcus dysgalactiae (Group C/G Strep) Streptococcus pyogenes (Group A Strep)

Nucleic acids from the viral and bacterial organisms identified by this test are generally detectable in NPS/TS specimens during the acute phase of infection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and symptoms of respiratory infection and/or pharyngtis are indicative of the identified microorganism and aids in diagnosis if used in conjunction with other clinical and epidemiological information, and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Negative results in the setting of a respiratory illness and/or pharyngitis may be due to infection with pathogens that are not detected by this test, or a respiratory tract infection that may not be detected by an NPS or TS specimen. Positive results do not rule out coinfection with other organisms. The agent(s) detected by the SPOTFIRE R/ST Panel may not be the definite cause of disease.

Additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection and/or pharyngitis.

The SPOTFIRE R/ST Panel simultaneously identifies 15 different respiratory viral and bacterial pathogens in nasopharyngeal swabs (NPS) or 14 viral and bacterial pharyngitis pathogens in throat swabs (TS) from individuals with signs and symptoms of respiratory tract infections or pharyngitis, respectively, (see Intended Use:). The SPOTFIRE R/ST Panel is compatible with the SPOTFIRE System, a polymerase chain reaction (PCR)-based in vitro diagnostic system for infectious disease testing. The SPOTFIRE System Software executes the SPOTFIRE R/ST Panel test and interprets and reports the test results. The SPOTFIRE RIST Panel was designed to be used in CLIA-waived environments. A test is initiated by loading Hydration Solution into one port of the SPOTFIRE R/ST Panel pouch and NPS or TS specimen, mixed with the provided Sample Buffer, into the port of the SPOTFIRE R/ST Panel pouch and placing it in the SPOTFIRE System. The pouch contains all of the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and Sample/Buffer Mix rehydrates the reagents. After the pouch is prepared, the SPOTFIRE System Software guides the user through the steps of placing the instrument, scanning the pouch barcode, entering the sample identification, and initiating the run.

The SPOTFIRE System contains coordinated systems of inflatable bladders and seal points, which act on the pouch to control the movement of liguid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liguid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically controlled pneumatic pistons are positioned over multiple plungers in order to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier and cooling of the pouch to drive the PCR reactions and the melt curve analysis.

Nucleic acid extraction occurs within the SPOTFIRE R/ST Panel pouch using mechanical and chemical lysis followed by purification using standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, the SPOTFIRE System performs a nested multiplex PCR that is executed in two stage, the SPOTFIRE System performs a single, large volume, highly multiplexed reverse transcription PCR (rt-PCR) reaction. The products from first stage PCR are then diluted and combined with a fresh, primer-free master mix and a fluorescent doublestranded DNA binding dye (LC Green® Plus, BioFire Diagnostics). The solution is then distributed to each well of the array. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR, is performed in singleplex fashion in each well of the array. At the conclusion of the 2nd stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the 2nd stage PCR captures fluorescent images of the PCR reactions and software interprets the data.

The SPOTFIRE System Software automatically interprets the results of each DNA met curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the SPOTFIRE R/ST Panel.

Here's a breakdown of the acceptance criteria and study proving the device meets those criteria, based on the provided FDA 510(k) summary for the BIOFIRE® SPOTFIRE® Respiratory/Sore Throat (R/ST) Panel:

The document describes the BIOFIRE® SPOTFIRE® Respiratory/Sore Throat (R/ST) Panel, a multiplexed PCR test for simultaneous, qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids. The data presented primarily focuses on analytical performance and clinical performance to demonstrate substantial equivalence to a predicate device.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally implied by the performance metrics reported, specifically the Positive Percent Agreement (PPA) / Sensitivity and Negative Percent Agreement (NPA) / Specificity. For analytical studies, acceptance criteria related to percentages of positive results and agreement levels are explicitly stated.

Clinical Performance Acceptance Criteria (Implied by Data Reported):
For each analyte, the acceptance criteria are generally to achieve high PPA and NPA. While specific numerical thresholds for PPA/NPA are not explicitly listed as "acceptance criteria" in the clinical tables themselves, the FDA's clearance implies that the presented performance met their internal requirements for substantial equivalence. For diagnostic tests, generally, PPA and NPA values in the high 90s are expected.

Analytical Performance Acceptance Criteria and Reported Performance:

2. Sample Sizes Used for the Test Set and Data Provenance

The sample sizes vary by analyte and study type (prospective, archived, contrived).

• Clinical Performance (Test Set):

  • NPS Specimens (Respiratory Menu):
    • Prospective Data: The total number of NPS specimens tested across all viruses and bacteria is not explicitly stated as a single number but can be aggregated from the "Positive" and "Negative" counts for each analyte. For example, for Adenovirus, there were 33 positives and 1082 negatives, totaling 1115 prospective NPS specimens where Adenovirus was assessed. This appears to be the total number of clinical samples evaluated in the prospective study.
    • Archived Data: Similarly, for Adenovirus, there were 31 positives and 439 negatives, totaling 470 archived NPS specimens.
    • Contrived Data: Indicated as 0/0 for all NPS analytes, suggesting contrived samples were not used for clinical performance evaluation of NPS specimens.
  • TS Specimens (Sore Throat Menu):
    • Prospective Data: For Adenovirus, 65 positives and 810 negatives (total 875). This seems to be the total number of clinical throat swab specimens assessed.
    • Archived Data: For Adenovirus, 11 positives and 48 negatives (total 59).
    • Contrived Data: For Adenovirus, 50 positives and 381 negatives (total 431). Contrived samples were used for TS clinical performance studies.
  • Data Provenance: The document states "as tested by intended users," implying clinical sites. No specific country of origin is mentioned, but typically, these studies for FDA clearance involve sites within the US. The "Prospective" studies indicate prospective collection, while "Archived" refers to retrospective samples. "Contrived" samples are laboratory-prepared.

• Analytical Performance (Test Set):

  • Limit of Detection (LoD): At least 20 replicates (e.g., 19/20) tested at LoD and 10-fold below LoD for each analyte.
  • Analytical Reactivity (Inclusivity): 3/3 or 4/5 replicates tested within 10x LoD for each isolate.
  • Near-LoD/Reproducibility: Not explicitly stated but mentions "multiple days," "multiple operators," "three unique SPOTFIRE Systems," "three distinct clinical sites holding a CLIA waiver." The reported positive and negative agreement totals are 2070 (BioFire) and 1380 (clinical sites) tests.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and the Qualifications of Those Experts

The document does not specify the number of experts or their qualifications (e.g., radiologist with 10 years of experience) used to establish the ground truth. This is a common characteristic of in vitro diagnostic (IVD) submissions focusing on molecular diagnostic tests.

• For molecular tests, the "ground truth" is typically established by comparator methods, often laboratory-developed tests (LDTs) or other cleared/validated molecular diagnostic assays (e.g., PCR followed by sequencing, or highly sensitive and specific reference PCR methods).
• For bacterial culture (used for Streptococcus dysgalactiae and Streptococcus pyogenes), the ground truth is established by standard microbiological culture and identification techniques, performed by trained laboratory personnel.

4. Adjudication Method for the Test Set

The document does not describe a formal adjudication method (e.g., 2+1, 3+1) for the clinical test sets. This is expected given that the ground truth is established by laboratory reference methods (molecular or culture), which typically do not involve human reader adjudication in the same way imaging studies might. Any discrepancies between the investigational device and the reference method would be investigated by the manufacturer, rather than through an expert consensus adjudication.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No, an MRMC comparative effectiveness study was not done. This type of study is primarily relevant for imaging-based AI devices where human readers interpret medical images, and the AI assists or augments their performance. The BIOFIRE SPOTFIRE R/ST Panel is a molecular diagnostic test where the output is an automated detection of nucleic acids, not an interpretation by a human reader that could be augmented by AI.

6. If a Standalone (i.e. Algorithm Only Without Human-in-the Loop Performance) Was Done

Yes, the performance data presented (Tables 2 and 3 for Clinical Performance, and Table 4 for Analytical Performance) represents the standalone performance of the BIOFIRE SPOTFIRE R/ST Panel, which includes the instrument and its automated software interpretation. The device itself is designed to provide automated results without human interpretive input for the detection of targets. The "as tested by intended users" for clinical performance indicates that the device was used in a realistic setting, but the performance metrics reflect the direct output of the system.

7. The Type of Ground Truth Used

The ground truth for the clinical performance evaluation was established using:

• Comparator molecular assays or known specimen composition for most analytes (referred to as "molecular assays or known specimen composition were used as comparator methods" for PPA/NPA). This suggests a combination of validated PCR assays, and potentially sequencing for confirmation.
• Culture for Streptococcus dysgalactiae (Group C/G Strep) and Streptococcus pyogenes (Group A Strep). This is explicitly stated: "Performance measures of sensitivity and specificity refer to the prospective and archived Streptococcus and archived Streptococcus analytes for which culture was used as the reference method."

For analytical studies, the ground truth was established by:

• Known concentrations of organisms (e.g., viable or infectious units, nucleic acid copies/mL) for LoD and Inclusivity studies.
• Defined panels of organisms or substances for Exclusivity and Interference studies.

8. The Sample Size for the Training Set

This document does not provide information on the training set size directly. The presented studies are for validation/testing of the device's performance. For molecular diagnostic assays like this, the 'training' of the algorithms (e.g., primer design, melt curve analysis interpretation) typically happens during the assay development phase, often using synthetic targets, cultured organisms, and preliminary clinical samples. However, this is not detailed in a 510(k) summary, which focuses on validation data against a defined product. The "software was verified and validated" statement implies that developmental data was used, but details on sample size for that specific phase are not provided in this regulatory summary.

9. How the Ground Truth for the Training Set Was Established

Since the training set details are not provided, the method for establishing its ground truth is also not explicitly stated in this document. However, based on typical IVD development practices:

• Ground truth for assay development (which informs the 'training' of a molecular diagnostic system) would involve well-characterized positive and negative controls, reference strains, and potentially sequenced clinical isolates.
• Molecular target sequences (in silico analysis)
• Analytical dilution series where the exact concentration of the pathogen is known.
• Samples confirmed by multiple, orthogonal laboratory methods.

The rigorous analytical and clinical studies described in the results section serve as the validation of the final trained/developed system.

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