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QIAstat-Dx Respiratory Panel Plus:

The QIAstat-Dx Respiratory Panel Plus is a multiplexed nucleic acid test intended for use with the QIAstat-Dx system for the simultaneous in vitro qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals with clinical signs and symptoms of respiratory tract infections, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

The following organism types and subtypes are identified using the QIAstat-Dx Respiratory Panel Plus: Adenovirus, Human Coronavirus 229E, Human Coronavirus HKU1, Human Coronavirus NL63, Human Coronavirus OC43, Human Metapneumovirus, Influenza A, Influenza A H1, Influenza A H1N1 pdm09, Influenza A H3, Influenza B, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Respiratory Syncytial Virus, Human Rhinovirus/Enterovirus (not differentiated), SARS-CoV-2, Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae.

Nucleic acids from viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. Detecting and identifying specific viral and bacterial nucleic acids from individuals presenting with signs and symptoms of a respiratory infection aids in the diagnosis of respiratory infection, if used in conjunction with other clinical, epidemiological 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 presence of a respiratory illness may be due to infection with pathogens that are not detected by the test, or due to lower respiratory tract infection that is not detected by a NPS specimen.

Conversely, positive results are indicative of the presence of the identified microorganism, but do not rule out co-infection with other pathogens not detected by the QIAstat-Dx Respiratory Panel Plus. The agent(s) detected by the QIAstat-Dx Respiratory Panel Plus may not be the definite cause of disease.

The use of additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection.

QIAstat-Dx Respiratory Panel Mini:

The QIAstat-Dx Respiratory Panel Mini is a multiplexed nucleic acid test intended for use with the QIAstat-Dx system for the simultaneous in vitro qualitative detection and identification of multiple respiratory viral nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals with clinical signs and symptoms of respiratory tract infections, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

The following viruses are identified using the QIAstat-Dx Respiratory Panel Mini: Influenza A, Influenza B, Respiratory Syncytial Virus, Human Rhinovirus, and SARS-CoV-2.

Nucleic acids from viral organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. Detecting and identifying specific viral nucleic acids from individuals presenting with signs and symptoms of a respiratory infection aids in the diagnosis of respiratory infection, if used in conjunction with other clinical, epidemiological 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 presence of a respiratory illness may be due to infection with pathogens that are not detected by the test or due to lower respiratory tract infection that is not detected by a NPS specimen.

Conversely, positive results are indicative of the presence of the identified microorganism, but do not rule out co-infection with other pathogens not detected by the QIAstat-Dx Respiratory Panel Mini. The agent(s) detected by the QIAstat-Dx Respiratory Panel Mini may not be the definite cause of disease.

The use of additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection.

The QIAstat-Dx Respiratory Panel Plus and the QIAstat-Dx Respiratory Panel Mini are multiplexed nucleic acid tests which are designed for use with the QIAstat-Dx system (currently QIAstat-Dx Analyzer 1.0 and QIAstat-Dx Analyzer 2.0). The device modification is to add the QIAstat-Dx Rise as an additional instrument for use with the QIAstat-Dx Respiratory Panel Plus and the QIAstat-Dx Respiratory Panel Mini ("QIAstat-Dx Respiratory Panels"). The QIAstat-Dx Rise is a higher throughput platform, incorporating up to eight QIAstat-Dx Analytical Modules (AM) on a small footprint. The instrument allows queuing up to 18 cartridges, which are scheduled for processing and delivered to the appropriate AM by an integrated robotic handler. The AM used with the QIAstat-Dx Rise is the same AM that can be used with the QIAstat-Dx Analyzer 1.0 or 2.0.

The modified QIAstat-Dx Respiratory Panel Plus and QIAstat-Dx Respiratory Panel Mini are identical to the QIAstat-Dx Respiratory Panel Plus (K233100) and the QIAstat-Dx Respiratory Panel Mini (K242353), respectively, with the exception of the Instructions for Use which were updated to include the assay-specific procedure for the QIAstat-Dx Rise.

The QIAstat-Dx Respiratory Panels are intended to be used with one nasopharyngeal swab (NPS) eluted in Universal Transport Media (UTM), which is not provided with the QIAstat-Dx Respiratory Panels.

All the reagents required for the complete execution of the test are pre-loaded and self-contained in a QIAstat-Dx Respiratory Panel cartridge. The user does not need to manipulate any reagents. During the test, reagents are handled by pneumatically-operated microfluidics without any direct contact with the user or the analyzer actuators.

Within the cartridge, multiple steps are automatically performed in sequence by using pneumatic pressure and a multiport valve to transfer the sample and fluids via the Transfer Chamber (TC) to their intended destinations. Following the introduction of the sample from a disposable transfer pipette, the following assay steps occur automatically and sequentially:

• Resuspension of Internal Control
• Cell lysis using mechanical and/or chemical means
• Membrane-based nucleic acid purification
• Mixing of the purified nucleic acid with lyophilized master mix reagents
• Transfer of defined aliquots of eluate/master mix to different reaction chambers
• Performance of multiplex real-time RT-PCR testing within each reaction chamber

The QIAstat-Dx Respiratory Panel Assay Definition File (ADF) automatically interprets test results and displays a summary on the instrument display screen. The detected analytes are displayed in red. All other tested but not detected analytes are listed in green. The instrument will report if an error occurs during processing, in which case the test will fail and no results will be provided (screen will show "FAIL").

The provided text describes a 510(k) premarket notification for the QIAstat-Dx Respiratory Panel Plus and QIAstat-Dx Respiratory Panel Mini, with a modification to include the QIAstat-Dx Rise instrument. The key takeaway from this document is that the FDA determined the device is substantially equivalent to previously cleared devices. Therefore, the "acceptance criteria" discussed here refer to the demonstration of equivalence to a predicate device, rather than specific performance metrics against a clinical ground truth for a new device.

Here's an analysis based on your questions:

1. A table of acceptance criteria and the reported device performance

Since this is a submission for a modification to an already cleared device, the acceptance criteria are not explicitly stated in terms of clinical performance numbers (e.g., sensitivity, specificity). Instead, the acceptance criteria are focused on demonstrating that adding the new instrument (QIAstat-Dx Rise) does not negatively impact the performance, and that the new system is "substantially equivalent" to the predicate devices.

The "reported device performance" is essentially that the studies "successfully demonstrated the equivalent performance."

2. Sample size used for the test set and the data provenance (e.g. country of origin of the data, retrospective or prospective)

The document does not specify the sample size for the test set used in the "Equivalence at Low Analyte Concentration," "Carryover," or "Reproducibility" studies. It also does not mention the data provenance (country of origin, retrospective or prospective). These details would typically be found in the actual study reports, which are not included in this FDA clearance letter.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience)

This information is not applicable or provided in this document. For an in vitro diagnostic device like this, ground truth for clinical performance would typically be established by comparing against FDA-cleared or gold standard laboratory methods (e.g., culture, sequencing, or other highly sensitive PCR assays) rather than expert consensus on imaging or clinical findings. Since this submission focuses on establishing equivalence and not initial clinical performance, such details are not expected.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set

This information is not provided and would not typically be part of a 510(k) clearance letter for an IVD device unless specific clinical adjudication was required for complex diagnostic outcomes.

5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

This is not applicable. The QIAstat-Dx Respiratory Panels are automated in vitro diagnostic devices for detecting nucleic acids. They do not involve human readers' interpretation of images or other data in a way that would necessitate an MRMC study or AI assistance.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

This is an in vitro diagnostic test. It is inherently a "standalone" system in its operation, as the instrument performs the test and provides a result. There is no mention of a human-in-the-loop component beyond loading the sample and reading the final result from the display screen. The device's "algorithm" (i.e., the assay's detection mechanism and interpretation software) operates without human intervention once the run starts.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.)

The document does not explicitly state the "ground truth" used for the analytical studies (Equivalence at Low Analyte Concentration, Carryover, Reproducibility). For such studies, the ground truth would be established by controlled laboratory experiments, where samples with known concentrations of analytes are used, and the assay's results are compared against these known inputs. For initial clinical performance, the ground truth would typically be a highly sensitive and specific reference method, but those studies are for the predicate devices, not this modification.

8. The sample size for the training set

This information is not provided. Training sets are typically associated with machine learning or AI models. While instruments like the QIAstat-Dx have underlying algorithms, they are based on established PCR principles and assay design, not a machine learning training paradigm in the way AI image analysis would be.

9. How the ground truth for the training set was established

This information is not provided and is not applicable for this type of IVD device in the context of a 510(k) modification for instrument compatibility.

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The BIOFIRE SPOTFIRE Respiratory/Sore Throat (R/ST) Panel Mini is an automated multiplexed polymerase chain reaction (PCR) test intended for use with the BIOFIRE SPOTFIRE System for the simultaneous, qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swab (NPS) or anterior nasal swab (ANS) 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 BIOFIRE 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/ANS/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, ANS, or TS specimen. Positive results do not rule out co-infection with other organisms. The agent(s) detected by the BIOFIRE 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 BIOFIRE SPOTFIRE R/ST Panel Mini (SPOTFIRE R/ST Panel Mini) simultaneously identifies 5 different respiratory viral pathogens in nasopharyngeal swabs (NPS) or anterior nasal swabs (ANS), 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® SPOTFIRE® System, a polymerase chain reaction (PCR)-based in vitro diagnostic system for infectious disease testing. The BIOFIRE SPOTFIRE System Software 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 into the hydration solution injection port of the SPOTFIRE R/ST Panel Mini pouch and NPS, ANS, or TS specimen, mixed with the provided Sample Buffer, into the other sample injection port of the SPOTFIRE R/ST Panel Mini pouch and placing it in the SPOTFIRE System. The pouch contains all 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 pouch into the instrument, scanning the pouch barcode, entering the sample identification, and initiating the run.

Nucleic acid extraction occurs within the SPOTFIRE R/ST Panel Mini 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 stages. 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 double-stranded 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.

The FDA 510(k) clearance letter details the acceptance criteria and study that proves the BIOFIRE SPOTFIRE Respiratory/Sore Throat Panel Mini meets these criteria, specifically for the addition of Anterior Nasal Swabs (ANS) as a sample type for the Respiratory Menu.

Here's the breakdown:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implicitly defined by the reported performance metrics (Positive Percent Agreement - PPA and Negative Percent Agreement - NPA) in the clinical study. The device is deemed to meet these criteria if the lower bound of the 95% Confidence Interval (95% CI) for PPA and NPA is above acceptable thresholds (though specific numerical thresholds for "acceptable" are not explicitly stated as separate criteria, the observed high performance and clearance imply they were met).

Archived Specimen Performance for Influenza B virus:

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

• Test Set Sample Size:
  • Clinical Performance (Prospective Study): 797 specimens (out of 820 initially enrolled, 23 excluded).
  • Archived Specimen Testing: 241 specimens for Influenza B virus (35 positive, 206 negative).
• Data Provenance:
  • Country of Origin: US (prospective multi-center study at five geographically distinct urgent care or emergency department study sites).
  • Retrospective/Prospective: The study was primarily prospective, conducted from March 2024 to February 2025. This was supplemented with archived specimens for Influenza B due to low prevalence in the prospective study.

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

The document does not explicitly state the number or qualifications of experts used to establish the ground truth. It states that the performance was evaluated by comparing the test results with those from a "commercially available FDA-cleared multiplexed respiratory pathogen panel." This suggests that the ground truth was established by the results of this comparator method, which themselves would have been validated. No human expert interpretation of the comparator method is described.

4. Adjudication Method for the Test Set

The document mentions "Investigations of discrepant results are summarized in the footnotes." These footnotes indicate that for discrepant cases (e.g., false positives, false negatives), "additional molecular methods" were used to re-test the specimens. This implies a form of post-hoc adjudication using a more definitive or orthogonal molecular method to resolve discrepancies between the SPOTFIRE R/ST Panel Mini and the initial comparator.

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

No. This study is for a diagnostic PCR test, not an AI-assisted imaging or interpretation device. Therefore, an MRMC study and analysis of human reader improvement with AI assistance are not applicable.

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

Yes. The study evaluates the performance of the BIOFIRE SPOTFIRE R/ST Panel Mini as a standalone diagnostic device. The results are automatically interpreted and reported by the system software, with no human interpretation step in the primary analysis flow. The study compares the device's output directly to the comparator method.

7. The Type of Ground Truth Used

The primary ground truth was established by a commercially available FDA-cleared multiplexed respiratory pathogen panel. For discrepant results, "additional molecular methods" were used for confirmatory testing, indicating a molecular gold standard approach.

8. The Sample Size for the Training Set

The document does not provide details about a training set size. This notice is a 510(k) clearance for a PCR-based in vitro diagnostic test, not a machine learning or AI algorithm in the traditional sense that requires distinct training and test sets in the same manner. The "test set" described is the clinical validation cohort for demonstrating performance. PCR assays are generally developed and optimized through laboratory analytical studies, not typically "trained" on large datasets in the way an AI model would be.

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

As noted above, the concept of a "training set ground truth" is not applicable in this context, as the device is a PCR assay. The development and optimization of such assays involve different molecular and analytical validation processes to ensure specificity and sensitivity.

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The cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems is an automated, multiplex, nucleic acid test that utilizes real-time polymerase chain reaction (PCR) technology intended for simultaneous in vitro qualitative detection and differentiation of severe acute respiratory syndrome coronavirus (SARS-CoV-2), influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) in nasopharyngeal swab specimens obtained from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory viral infection due to SARS-CoV-2, influenza A, influenza B and RSV can be similar. This test is intended to aid in the differential diagnosis of SARS-CoV-2, influenza A, influenza B, and RSV infections in humans and is not intended to detect influenza C virus infections.

Nucleic acids from the viral organisms identified by this test are generally detectable in nasopharyngeal swab specimens during the acute phase of infection. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory tract infection are indicative of the presence of the identified virus, and aid 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 do not preclude SARS-CoV-2, influenza A virus, influenza B virus, or RSV infections. Conversely, positive results do not rule out coinfection with other organisms, and the agent(s) detected by the cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems may not be the definite cause of disease.

cobas® Respiratory 4-flex for use on the cobas® 5800/6800/8800 Systems (cobas® Respiratory 4-flex) is based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas® 5800 System is designed as one integrated instrument. The cobas® 6800/8800 Systems consist of the sample supply module, the transfer module, the processing module, and the analytic module. Automated data management is performed by the cobas® 5800 System or cobas® 6800/8800 Systems software(s), which assigns results for all tests. Results can be reviewed directly on the system screen and printed as a report.

Nucleic acid from patient samples and added Internal Control RNA (RNA IC) molecules are simultaneously extracted. Nucleic acid is released by addition of proteinase and lysis reagent to the sample. The released nucleic acid binds to the silica surface of the added magnetic glass particles. Unbound substances and impurities, such as denatured protein, cellular debris and potential PCR inhibitors, are removed with subsequent wash steps and purified nucleic acid is eluted from the magnetic glass particles with elution buffer at elevated temperature. External controls (positive and negative) are processed in the same way.

Selective amplification of target nucleic acid from the sample is achieved by the use of target-specific forward and reverse primers detecting conserved viral genome regions as shown in Table 1.

Selective amplification of RNA IC is achieved by the use of non-competitive, sequence specific forward and reverse primers, which have no homology with the viral-target specific genomes. Amplified target is detected by the cleavage of fluorescently labeled oligonucleotide probes. Roche's temperature assisted generation of signal (TAGS) technology, short TAGS technology, is introduced to differentiate up to three targets per fluorescence channel, enabling the detection of up to14 targets, and the Internal Control, per well. A thermostable DNA polymerase enzyme is used for amplification.

Multiplicity of target detection is enabled with temperature-dependent quenching of cleaved fluorescent target-specific probes. This is achieved by separating signals from probes into introduced thermal channels, where fluorescence is acquired at two additional fixed temperatures for each amplification cycle.

During the PCR amplification step, hybridization of the probes to the specific single-stranded DNA template results in cleavage of the probe by the 5' to 3' exonuclease activity of the DNA polymerase, resulting in separation of the reporter and quencher dyes, and the generation of a fluorescent signal. Conventional probes release fluorescence signal immediately upon separation of reporter from quencher. TAGS probes rely on temperature dependent fluorescence activation, requiring both nuclease cleavage during the extension phase, as well as an increase in reaction temperature, to activate the otherwise dormant fluorophore. For this reason, during each PCR cycle the TAGS technology captures fluorescence in five available fluorescence channels in combination with three thermal channels (detection of fluorescence at three defined temperatures T1, T2 and T3).

The cobas® Respiratory 4-flex master mix contains detection probes which are specific for influenza A virus, influenza B virus, RSV, SARS-CoV-2 and the RNA Internal Control (RNA IC) nucleic acid, which enables simultaneous detection and differentiation of influenza A virus, influenza B virus, RSV, and SARS-CoV-2 viral targets and the RNA IC.

The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythymidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon). Any contaminating amplicons from previous PCR runs are destroyed by the AmpErase enzyme [uracil-N-glycosylase], which is included in the PCR mix, when heated in the first thermal cycling step. However, newly formed amplicons are not destroyed since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.

The RESP-4FLEX ASAP enables the system to differentiate and report the qualitative results of the four targets influenza A virus, influenza B virus, RSV and SARS-CoV-2. For each specimen the customer can test for any combination of the four enabled virus targets. Also, additional target calculation (digital reflex) can be ordered for the four enabled virus targets (influenza A virus, influenza B virus, RSV and SARS-CoV-2) on the cobas® 5800 System.

Here's an analysis of the acceptance criteria and study detailed in the provided FDA clearance letter for the cobas Respiratory 4-flex, structured according to your request:

1. Table of Acceptance Criteria and Reported Device Performance

The FDA clearance letter does not explicitly state pre-defined acceptance criteria for the clinical performance. Instead, it reports the observed performance metrics (PPA and NPA) from the clinical studies. For the purpose of this table, I will present the reported clinical performance as the "met acceptance criteria," assuming these values were deemed acceptable by the FDA for clearance.

Study Proving Device Meets Criteria: The detailed clinical performance evaluation described in "5. CLINICAL PERFORMANCE EVALUATION" (pages 23-26) demonstrates that the cobas Respiratory 4-flex achieves the reported Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) values against a U.S. FDA-cleared molecular comparator assay. These reported percentage agreements, along with their 95% confidence intervals, serve as the evidence that the device meets the performance expectations for clinical use.

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

• Prospective Clinical Study:

  • Total NPS specimens enrolled: 4,475
  • Total NPS specimens tested: 4,378 (1,832 fresh, 2,546 frozen)
  • Total NPS specimens evaluable: 4,341 (1,827 fresh, 2,514 frozen)
  • Data Provenance: Fresh prospective specimens collected at eleven collection sites during the 2023-2024 respiratory viral season. Frozen prospective specimens collected during parts of the 2022-2023 respiratory viral season at seven sites and the 2023-2024 respiratory viral season from 14 collection sites. The specific country of origin is not explicitly stated but implies U.S. based on the "U.S. testing sites" mention for retrospective samples, and the FDA clearance context. The data is prospective.

• Retrospective Clinical Study:

  • Total NPS specimens enrolled: 770
  • Total NPS specimens evaluable:
    • Influenza A: 657
    • Influenza B: 647
    • RSV: 659
  • Data Provenance: Archived NPS specimens collected between 2014 and 2022 from individuals with signs and symptoms of respiratory viral infection. Tested at three (3) U.S. testing sites. The data is retrospective.

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

The document does not mention the use of human experts to establish ground truth for the test set. The ground truth was established by a "U.S. FDA-cleared molecular assay" as the comparator method.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method involving experts for discrepant results. Instead, it states that an "FDA 510(k) cleared comparator" method was used to establish the "ground truth." For the influenza A target in the prospective study, "three (3) additional specimens (two (2) fresh and one (1) frozen) were excluded from analysis due to inconclusive results obtained from the comparator test," implying a reliance on the comparator's definitive result rather than external adjudication. The same pattern is noted for retrospective samples, with exclusions for failed or invalid comparator test results.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and the Effect Size of Human Readers' Improvement with AI vs. Without AI Assistance

This section is Not Applicable to the provided document. The cobas Respiratory 4-flex is an automated, multiplex nucleic acid detection test, not an AI-powered diagnostic tool requiring human reader interpretation or assistance. Therefore, an MRMC study or analysis of human reader improvement with AI is not relevant.

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

Yes, this was a standalone performance evaluation. The device is an automated in vitro diagnostic (IVD) test. The clinical performance evaluation directly compares the cobas Respiratory 4-flex's results (algorithm only, as it's an automated system) against a predicate FDA-cleared molecular assay. There is no human interpretation or intervention in the generation of the primary test result from the cobas system itself.

7. The Type of Ground Truth Used (Expert Consensus, Pathology, Outcomes Data, etc.)

The ground truth for the clinical performance evaluation was established by a "U.S. FDA-cleared molecular assay." This means another legally marketed and validated molecular diagnostic test was used as the reference standard.

8. The Sample Size for the Training Set

The document does not specify a separate training set or its sample size. This is typical for in vitro diagnostic (IVD) devices that use established laboratory techniques (like PCR) rather than machine learning algorithms which require explicit training data. The development of such assays involves extensive analytical validation (LoD, precision, specificity, inclusivity, etc.) and then clinical validation with independent samples.

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

Since no explicit training set for a machine learning algorithm is mentioned, the concept of "ground truth for the training set" is not applicable in the context of this device's validation as described. The analytical studies (LoD, inclusivity, specificity, etc.) characterize the inherent performance of the assay's chemical and hardware components.

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The Xpert Xpress CoV-2/Flu/RSV plus test, performed on the GeneXpert Xpress System, is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT-PCR) test intended for use in the simultaneous in vitro qualitative detection and differentiation of severe acute respiratory syndrome coronavirus (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) viral RNA in nasopharyngeal swab and anterior nasal swab specimens collected from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2, influenza A, influenza B, and RSV can be similar.

The Xpert Xpress CoV-2/Flu/RSV plus test is intended for use in the differential detection of SARS-CoV-2, influenza A, influenza B and/or RSV RNA and aids in the diagnosis of COVID-19, influenza and/or RSV infections if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza A, influenza B, and RSV viral RNA are generally detectable in nasopharyngeal swab and anterior nasal swab specimens during the acute phase of infection.

Positive results are indicative of the presence of the identified virus, but do not rule out bacterial infection or co-infection with other pathogens not detected by the test. The agent (s) detected by the Xpert Xpress CoV-2/Flu/RSV plus test may not be the definite cause of the disease.

Negative results do not preclude SARS-CoV-2, influenza A, influenza B and/or RSV infection. The results of this test should not be used as the sole basis for diagnosis, treatment or other patient management decisions.

The Xpert Xpress CoV-2/Flu/RSV plus test is an automated in vitro diagnostic test for the simultaneous qualitative detection and differentiation of SARS-CoV-2, Flu A, Flu B, and RSV viral RNA in nasopharyngeal swab (NPS) and anterior nasal swab (NS) specimens collected from individuals showing signs and symptoms of respiratory viral infection.

The Xpert Xpress CoV-2/Flu/RSV plus test is performed on GeneXpert Xpress System, which consist of a GeneXpert IV instrument that executes sample preparation, nucleic acid amplification and real-time fluorescent signal detection for the tests, and a GeneXpert Hub with preloaded GeneXpert Xpress software for running the tests and viewing the test results. The GeneXpert Hub accessory integrates the computer, touchscreen monitor and barcode scanner. Each of the GeneXpert modules in the GeneXpert IV instrument can perform independent sample preparation and testing. The GeneXpert Xpress System requires the use of single-use disposable cartridges that hold the RT-PCR reagents and host sample purification, nucleic acid amplification, and detection of the target sequences. Because the cartridges are self-contained, cross-contamination between samples is minimized.

The Xpert Xpress CoV-2/Flu/RSV plus test includes reagents for the detection of SARS-CoV-2, Flu A, Flu B and RSV viral RNA from NPS and NS specimens. The primers and probes in the Xpert Xpress CoV-2/Flu/RSV plus test are designed to amplify and detect unique sequences in the genes that encode the following proteins: SARS-CoV-2 nucleocapsid (N), SARS-CoV-2 envelope (E), SARS-CoV-2 RNA-dependent RNA polymerase (RdRP), influenza A matrix (M), influenza A basic polymerase (PB2), influenza A acidic protein (PA), influenza B matrix (M), influenza B non-structural protein (NS), and the RSV A and RSV B nucleocapsid.

A Sample Processing Control (SPC) and a Probe Check Control (PCC) are also included in the cartridge utilized by the GeneXpert instrument. The SPC is present to control for adequate processing of the sample and to monitor for the presence of potential inhibitor(s) in the RT-PCR reaction. The SPC also ensures that the RT-PCR reaction conditions (temperature and time) are appropriate for the amplification reaction and that the RT-PCR reagents are functional. The PCC verifies reagent rehydration, PCR tube filling, and confirms that all reaction components are present in the cartridge including monitoring for probe integrity and dye stability.

The Xpert Xpress CoV-2/Flu/RSV plus test is designed for use with NPS or NS specimens collected with nylon flocked swabs and placed into viral transport medium (VTM), Universal Transport Medium (UTM), or eNAT®. The ancillary specimen collection kits, swabs and transport media validated for use with the Xpert Xpress CoV-2/Flu/RSV plus test included:

• Nasopharyngeal Sample Collection Kit for Viruses

• Copan UTM® 3C057N (Flexible Minitip Flocked Swab with UTM® Medium without Beads)
• Copan eNAT® Molecular Collection and Preservation Medium P/N 6U074S01 (Flexible Minitap Flocked Swab with eNAT® Medium)

• Nasal Sample Collection Kit for Viruses

• Copan UTM® 3C064N (Regular Flocked Swab with UTM® Medium without Beads)
• Copan eNAT® Molecular Collection and Preservation Medium P/N 6U073S01 (Regular Flocked Swab with eNAT® Medium)

• Alternatively, swabs and transport media can be obtained separately:

• Nylon flocked swab (Copan P/N 502CS01, 503CS01)
• Viral Transport Medium, 3 mL (Copan P/N 330C, 3C047N, BD Universal Transport Medium, Remel M4RT, or Remel M5)

The ancillary reagents allow NPS and NS specimens from patients to be collected, preserved and transported to laboratory prior to analysis with the Xpert Xpress CoV 2/Flu/RSV plus test.

Based on the provided FDA 510(k) clearance letter and summary, here's an analysis of the acceptance criteria and study that proves the device meets them:

Important Note: This document describes a "Special 510(k) submission." This type of submission is used when changes are made to a previously cleared device that do not affect its fundamental technology, intended use, or safety/effectiveness. In this specific case, the changes were to the Assay Definition File (ADF) – essentially software parameter settings. Therefore, the "study that proves the device meets the acceptance criteria" largely relies on re-analysis of existing studies from the original device clearance (K242071) rather than entirely new, large-scale clinical trials.

1. Table of Acceptance Criteria and Reported Device Performance

The provided document doesn't explicitly state quantitative acceptance criteria in a dedicated table. However, since this is a Special 510(k) for software parameter changes, the primary "acceptance criteria" appear to be demonstrating non-inferiority or equivalency to the previously cleared predicate device in terms of:

• Valid Test Runs: The number of tests that yield a valid result.
• Non-Determinate (ND) Test Results: The number of tests that do not yield a definitive positive or negative result (e.g., "NO RESULT").
• Performance Claims: That the modifications did not negatively impact the overall analytical and clinical performance claims established for the predicate device (sensitivity, specificity for detecting SARS-CoV-2, Flu A, Flu B, and RSV).

Inferred Acceptance Criteria and Reported Performance:

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

• Sample Size:
  • Main analysis: 16,264 test results. This number represents the re-analysis of "verification, validation and flex studies data from the original studies."
  • SARS-CoV-2 only test mode analysis: 25 test results.
  • Simulated conditions for revised algorithm: The document mentions "Test cartridges were simulated to generate SARS-CoV-2 INVALID and SPC FAIL conditions," but does not explicitly state the number of simulated tests.
• Data Provenance: The data used for re-analysis originated from the "original studies" (K242071 submission). The document does not specify the country of origin or whether these original studies were retrospective or prospective, though typical clinical validation studies for IVDs are often prospective. Given it references "flex study," those can sometimes include both retrospective and prospective elements.

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

The document does not provide information on the number or qualifications of experts used to establish the ground truth. This is likely because the ground truth was established during the original predicate device clearance (K242071) and this Special 510(k) focused on demonstrating equivalence in specific software-related metrics through re-analysis. For IVD devices like this, ground truth is typically established by comparing against a highly sensitive and specific reference method (e.g., an FDA-cleared laboratory developed test or a combination of clinical diagnosis and other accepted testing methods).

4. Adjudication Method for the Test Set

The document does not specify any adjudication method (e.g., 2+1, 3+1, none) for the test set. Again, this specific submission involved re-analysis of existing data rather than new clinical trials where such adjudication might be more explicitly detailed if human reads were involved. For an automated RT-PCR test, adjudication as typically understood for image-based AI would not be directly applicable.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not conducted or is not mentioned in this document. This is an in vitro diagnostic device (RT-PCR test), not an AI-assisted diagnostic imaging system that would typically involve human readers interpreting images with or without AI assistance. The device is fully automated.

6. Standalone Performance (Algorithm Only)

Yes, the performance evaluated here is inherently standalone (algorithm only). The Xpert Xpress CoV-2/Flu/RSV plus is an automated RT-PCR test system. Its output is directly generated by the instrument and its embedded software (ADF). The "performance data" detailed in section 18.5 describes how the device's algorithmic and software changes affected its ability to produce valid results and interpret them correctly, without human intervention in the result generation.

7. Type of Ground Truth Used

The document does not explicitly state the type of ground truth used for the original studies, beyond stating "analytical, clinical and flex studies data." For RT-PCR assays, the ground truth for clinical performance is typically established by:

• Clinical Reference Method: Comparison against a highly sensitive and specific laboratory reference method (e.g., another FDA-cleared or EUA-authorized RT-PCR test, or a composite reference standard using multiple methods).
• Clinical Diagnosis/Outcomes Data (less common for purely diagnostic tests): While the device aids in diagnosis ("aids in the diagnosis of COVID-19, influenza and/or RSV infections if used in conjunction with other clinical and epidemiological information, and laboratory findings"), the performance claims themselves are based on detection of viral RNA, rather than patient outcomes as primary ground truth.

Given the nature of a molecular diagnostic test for viral RNA, the ground truth for the performance claims (sensitivity, specificity) of the original device would have been established by comparing the device's results against a highly reliable reference molecular test, often considered the "gold standard" for pathogen detection.

8. Sample Size for the Training Set

The document does not provide information on the sample size for the training set. This is because:

• This is an RT-PCR test, not a deep learning AI model that undergoes "training" in the typical machine learning sense. The "algorithm" here refers to the pre-defined logical rules and parameters within the Assay Definition File (ADF) for interpreting RT-PCR signals.
• The re-analysis was performed on verification, validation, and flex studies data, which are typically considered test or validation sets in the context of device development, not training sets.

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

As noted above, a "training set" and associated ground truth establishment in the context of deep learning AI are not applicable to this RT-PCR device. The "ground truth" for the device's performance relies on the known characteristics of the viral targets and the performance of the RT-PCR chemistry against reference methods.

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The Xpert Xpress CoV-2/Flu/RSV plus test, performed on the GeneXpert Dx and GeneXpert Infinity Systems, is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT-PCR) test intended for use in the simultaneous in vitro qualitative detection and differentiation of severe acute respiratory syndrome coronavirus (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) viral RNA in nasopharyngeal swab and anterior nasal swab specimens collected from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2, influenza A, influenza B, and RSV can be similar.

The Xpert Xpress CoV-2/Flu/RSV plus test is intended for use in the differential detection of SARS-CoV-2, influenza A, influenza B and/or RSV RNA and aids in the diagnosis of COVID-19, influenza and/or RSV infections if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza A, influenza B, and RSV viral RNA are generally detectable in nasopharyngeal swab and anterior nasal swab specimens during the acute phase of infection.

Positive results are indicative of the presence of the identified virus, but do not rule out bacterial infection or co-infection with other pathogens not detected by the test. The agent (s) detected by the Xpert Xpress CoV-2/Flu/RSV plus test may not be the definite cause of the disease.

Negative results do not preclude SARS-CoV-2, influenza A, influenza B and/or RSV infection. The results of this test should not be used as the sole basis for diagnosis, treatment or other patient management decisions.

The Xpert Xpress CoV-2/Flu/RSV plus test is an automated in vitro diagnostic test for the simultaneous qualitative detection and differentiation of SARS-CoV-2, Flu A, Flu B, and RSV viral RNA in nasopharyngeal swab (NPS) and anterior nasal swab (NS) specimens collected from individuals showing signs and symptoms of respiratory viral infection.

The Xpert Xpress CoV-2/Flu/RSV plus test is performed on GeneXpert Instrument Systems (GeneXpert Dx, GeneXpert Infinity-48s and GeneXpert Infinity-80 systems, GeneXpert System with Touchscreen), which consist of an instrument, computer or touchscreen, and preloaded software for running tests and viewing the results. The GeneXpert Instrument Systems automate and integrate sample preparation, nucleic acid extraction and amplification, and detection of the target sequences in simple or complex samples using real-time reverse transcription (RT)-polymerase chain reaction (PCR) and PCR technology. Depending on the instrument, the GeneXpert Instrument Systems can have from 1 and up to 80 randomly accessible modules, each capable of performing separate sample preparation and real-time RT-PCR and PCR tests. Each module contains a syringe drive for dispensing fluids (i.e., the syringe drive activates the plunger that works in concert with the rotary valve in the cartridge to move fluids between chambers), an ultrasonic horn for lysing cells or spores, and a proprietary I-CORE® thermocycler for performing real-time RT-PCR and PCR as well as detection. The systems require the use of single-use disposable cartridges that hold the RT-PCR reagents and host sample purification, nucleic acid amplification, and detection of the target sequences. Because the cartridges are self-contained, cross-contamination between samples is minimized.

The Xpert Xpress CoV-2/Flu/RSV plus test includes reagents for the detection of SARS-CoV-2, Flu A, Flu B and RSV viral RNA from NPS and NS specimens. The primers and probes in the Xpert Xpress CoV-2/Flu/RSV plus test are designed to amplify and detect unique sequences in the genes that encode the following proteins: SARS-CoV-2 nucleocapsid (N), SARS-CoV-2 envelope (E), SARS-CoV-2 RNA-dependent RNA polymerase (RdRP), influenza A matrix (M), influenza A basic polymerase (PB2), influenza A acidic protein (PA), influenza B matrix (M), influenza B non-structural protein (NS), and the RSV A and RSV B nucleocapsid.

A Sample Processing Control (SPC) and a Probe Check Control (PCC) are also included in the cartridge utilized by the GeneXpert instrument. The SPC is present to control for adequate processing of the sample and to monitor for the presence of potential inhibitor(s) in the RT-PCR reaction. The SPC also ensures that the RT-PCR reaction conditions (temperature and time) are appropriate for the amplification reaction and that the RT-PCR reagents are functional. The PCC verifies reagent rehydration, PCR tube filling, and confirms that all reaction components are present in the cartridge including monitoring for probe integrity and dye stability.

The Xpert Xpress CoV-2/Flu/RSV plus test is designed for use with NPS or NS specimens collected with nylon flocked swabs and placed into viral transport medium (VTM), Universal Transport Medium (UTM), or eNAT®.

The FDA 510(k) Clearance Letter for the Xpert Xpress CoV-2/Flu/RSV plus device describes modifications to an existing PCR test. The information provided outlines the acceptance criteria implicitly through the modifications and performance data presented. However, it's important to note that this document is a 510(k) summary, which provides a high-level overview of the submission and does not contain the full details of all studies conducted. Therefore, some requested information may not be explicitly present in the provided text.

Based on the provided text, here's an analysis:

Summary of Acceptance Criteria and Device Performance

The core of this 510(k) submission is a "Special 510(k)" which means the device (Xpert Xpress CoV-2/Flu/RSV plus) is largely the same as a previously cleared predicate device (K231481), but with minor design changes. Therefore, the acceptance criteria are implicitly tied to demonstrating that these changes do not negatively impact the previously established performance claims of the predicate device.

Specifically, the modifications include:

1. Turning off Signal Loss Detection (SLD) for the Flu B channel.
2. Revising the result reporting algorithm for the SARS-CoV-2 only test mode.
   • Previously: SARS-CoV-2 NEGATIVE if SARS-CoV-2 analyte result is INVALID and SPC is FAIL.
   • Revised: INVALID GeneXpert test result if SARS-CoV-2 analyte result is INVALID and SPC is FAIL.

Table of Acceptance Criteria (Implicit) and Reported Device Performance:

Study Details Proving Acceptance:

1. Sample sizes used for the test set and the data provenance:

   • Test Set (Re-analysis Data):
     • Analytical Test Results: 15,645
     • Clinical, Reproducibility-Precision, and Single-Site Precision Test Results: 9,525 (comprising 8,535 specimens and 990 controls).
   • Data Provenance: The document does not explicitly state the country of origin or whether the data was retrospective or prospective for the re-analyzed verification and validation studies. However, since it refers to "original studies" (K231481), it implies data previously collected and used for the predicate device's clearance.

2. Number of experts used to establish the ground truth for the test set and their qualifications:

   • This information is not provided in the given text. For an in vitro diagnostic (IVD) device like this, ground truth is typically established by comparative methods (e.g., another FDA-cleared PCR test, or a consensus of multiple clinical/laboratory results), not by human expert readers in the way an imaging AI device might use radiologists. The "ground truth" for this device's performance would be the presence or absence of viral RNA, determined by well-established laboratory methods.

3. Adjudication method for the test set:

   • This information is not provided and is generally not applicable to the type of re-analysis done for an IVD device where ground truth is established by laboratory methods rather than human interpretation requiring adjudication.

4. If a multi-reader multi-case (MRMC) comparative effectiveness study was done:

   • No, an MRMC study was not done. This type of study is primarily relevant for imaging AI devices that assist human readers in interpretation. This device is an automated, standalone diagnostic test.

5. If a standalone (i.e. algorithm only without human-in-the loop performance) was done:

   • Yes, implicitly. The re-analysis of performance data, particularly the comparison between the original and updated Assay Definition Files (ADF) and the verification of the revised algorithm, represents the standalone performance of the device's software (algorithm) logic. The device itself is an automated system.

6. The type of ground truth used:

   • Implicitly, the ground truth was established by laboratory reference methods (e.g., RT-PCR) for the original clinical specimens. For the re-analysis, the "ground truth" for evaluating the impact of the changes was the consistency of results and proper operation of the updated algorithm against the expected behavior, confirmed by the original analytical and clinical study data. For the SARS-CoV-2 algorithm change, "test cartridges were simulated to generate SARS-CoV-2 INVALID and SPC FAIL conditions," meaning the ground truth for this specific verification was a controlled simulation designed to trigger the specific algorithm conditions.

7. The sample size for the training set:

   • Not explicitly stated in relation to this Special 510(k) submission. As this is a modification to an existing device, it's likely that the original training/development data for the predicate device were used, but the size of that dataset is not provided here. The 15,645 analytical test results and 9,525 clinical/precision test results mentioned are re-analyzed verification and validation data, not training data.

8. How the ground truth for the training set was established:

   • Not explicitly stated in the provided text. For an RT-PCR diagnostic, the ground truth for training/development would typically involve characterized clinical samples or contrived samples with known viral concentrations and presence/absence of targets, verified by highly sensitive and specific reference methods (e.g., sequencing, confirmatory PCRs, or sometimes clinical outcomes correlated with virology).

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The cobas liat SARS-CoV-2 & Influenza A/B v2 nucleic acid test is an automated rapid multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) test intended for the simultaneous qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus and influenza B virus nucleic acids in anterior nasal (nasal) and nasopharyngeal swab specimens from individuals exhibiting signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2 and influenza can be similar. This test is intended to aid in the differential diagnosis of SARS-CoV-2, influenza A and influenza B infections in humans and is not intended to detect influenza C virus infections.

Nucleic acids from the viral organisms identified by this test are generally detectable in nasopharyngeal and nasal swab specimens during the acute phase of infection. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory tract infection are indicative of the presence of the identified virus, and aid 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. Positive results do not rule out coinfection with other organisms. The organism(s) detected by the cobas liat SARS-CoV-2 & Influenza A/B v2 nucleic acid test may not be the definite cause of disease. Negative results do not preclude SARS-CoV-2, influenza A virus or influenza B virus infections.

The cobas liat SARS-CoV-2 & Influenza A/B v2 nucleic acid test is performed on the cobas liat analyzer which automates and integrates sample purification, nucleic acid amplification, and detection of the target sequence in biological samples using real-time PCR assays. The assay targets both the ORF1 a/b non-structural region and membrane protein gene that are unique to SARS-CoV-2, a well-conserved region of the matrix gene of influenza A (Flu A target), and the nonstructural protein 1 (NS1) gene of influenza B (Flu B target). An Internal Control (IC) is included to control for adequate processing of the target virus through all steps of the assay process and to monitor the presence of inhibitors in the RT-PCR processes.

This document describes the validation study for the cobas liat SARS-CoV-2 & Influenza A/B v2 nucleic acid test.

Here's an analysis of the acceptance criteria and the study proving the device meets them:

1. Table of Acceptance Criteria and Reported Device Performance

Since this is a diagnostic test, the primary acceptance criteria revolve around analytical and clinical performance metrics like Limit of Detection, Inclusivity, Cross-Reactivity, Reproducibility, and Clinical Agreement (Positive Percent Agreement and Negative Percent Agreement). The document doesn't explicitly state "acceptance criteria" values in a separate table, but these are implied by the performance metrics reported and the general standards for diagnostic device clearance. I will extract the reported device performance from the provided text.

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

• Prospective Clinical Study:

  • Sample Size: 1729 symptomatic subjects enrolled.
    • 1705 evaluable NPS specimens for analysis (19 non-evaluable due to missing/invalid results, 5 due to handling).
    • 1706 evaluable ANS specimens for SARS-CoV-2 and Influenza B analysis (22 non-evaluable due to missing/invalid results, 1 due to handling).
    • 1704 evaluable ANS specimens for Influenza A analysis (2 additional found inconclusive for comparator).
  • Data Provenance: Prospective, collected between September 2023 and March 2024 at 14 point-of-care testing sites in the United States (US).

• Retrospective Clinical Study (Influenza B Supplement):

  • Sample Size: 223 archived NPS specimens and 206 archived ANS specimens (total 429).
    • One NPS sample pre-characterized as positive for influenza B was non-evaluable.
  • Data Provenance: Retrospective, frozen archived (Category III) specimens collected between 2019 and 2023. Distributed to 6 sites for testing.

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

The document does not mention the use of experts to establish ground truth for the clinical test sets. For molecular diagnostic tests like this, the ground truth is typically established by comparing the investigational device's results against a highly accurate, accepted comparator method (another FDA-cleared Nucleic Acid Amplification Test specific for the target analytes). The expertise lies in the development and validation of these comparator methods, not in individual expert review of each sample for ground truth in this context.

4. Adjudication Method for the Test Set

The document describes discrepant result analysis for both prospective and retrospective clinical studies.

• For the prospective study, "discrepant NAAT results" are detailed for SARS-CoV-2 (NPS and ANS), Influenza A (NPS and ANS), and Influenza B (NPS and ANS).
• For the retrospective study, discrepant NAAT results are detailed for Influenza B (NPS and ANS).

The method appears to be:

• The cobas liat test result is compared to the FDA-cleared comparator NAAT result.
• When there's a discrepancy (e.g., cobas liat positive, comparator negative), it explicitly states how many were "positive" and "negative" upon further investigation or re-evaluation (e.g., with "discrepant NAAT results").
  • For example: "Of 12 specimens negative on cobas® liat and positive on the comparator, 8 were positive and 4 were negative." This implies some form of re-testing or deeper analysis (not specified as "adjudication by experts" but rather "discrepant NAAT results"). It's more of a re-confirmation of the comparator or a third method, rather than a human expert consensus process. Such re-evaluation often involves re-testing using the comparator or a reference method.

Therefore, while there's no "2+1" or "3+1" expert adjudication method described as would be seen in imaging studies, there is a discrepant resolution process based on further NAAT results. It's not "none" in the sense that discrepancies are just reported without follow-up; rather, they are further investigated using additional NAAT results to re-confirm the original comparator status if possible.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, If So, What Was the Effect Size of How Much Human Readers Improve with AI vs Without AI Assistance

No. This is a standalone diagnostic test (RT-PCR), not an AI-assisted imaging device or a test that involves human "readers" interpreting results. Therefore, an MRMC comparative effectiveness study involving human readers and AI assistance is not applicable and was not performed.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

Yes, implicitly. This is a fully automated RT-PCR test run on the cobas liat analyzer. The performance metrics (LoD, inclusivity, cross-reactivity, reproducibility, and clinical agreement) are measures of the device's performance on its own against established ground truth (comparator NAAT). While humans load samples and interpret the final digital result (positive/negative), the core detection and differentiation is algorithm-driven within the instrument, making its performance essentially "standalone" in the context of diagnostic accuracy.

7. The Type of Ground Truth Used

• Clinical Performance (Prospective and Retrospective): The ground truth for clinical sample testing was established by comparing the cobas liat results against an FDA-cleared Nucleic Acid Amplification Test (NAAT), which serves as the reference or "ground truth" method for molecular diagnostic assays. The document explicitly states: "PPA and NPA were determined by comparing the results of cobas® liat SARS-CoV-2 & Influenza A/B v2 to the results of an FDA-cleared Nucleic Acid Amplification Test (NAAT)." and "The comparator method was an acceptable FDA-cleared molecular assay."
• Analytical Studies (LoD, Inclusivity, Cross-Reactivity, Interference, Reproducibility): Ground truth was established by preparing precisely known concentrations of viral material (cultured or inactivated viruses) or specific microorganisms in controlled laboratory settings. For these studies, the "ground truth" is meticulously prepared and verified laboratory standards.

8. The Sample Size for the Training Set

The document does not specify a separate "training set" sample size. For an RT-PCR diagnostic platform, the "training" involves the fundamental biochemical and optical engineering, and the optimization of assay (reagent) design to achieve sensitivity and specificity. This is distinct from machine learning models that often require large, labeled datasets for "training." The analytical and clinical validation studies described here are verification and validation (V&V) studies, akin to a "test set" to prove the device's performance against its design specifications and clinical utility.

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

Since no explicit "training set" for a machine learning algorithm is mentioned (as this is a molecular diagnostic test), this question is not directly applicable. However, the ground truth for assay development and optimization (which can be considered analogous to "training" in a broader sense of device development) would have been established through extensive laboratory work using:

• Highly characterized viral cultures or purified nucleic acids: Used to define target sequences, optimize primer/probe design, and determine initial analytical sensitivity.
• Spiked samples: Adding known quantities of targets or interferents to negative clinical matrices to mimic real-world conditions during early development.
• Early clinical samples: Used to refine assay performance and resolve initial issues prior to formal validation studies.

These processes ensure the assay correctly identifies the target nucleic acids.

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The cobas liat SARS-CoV-2, Influenza A/B & RSV nucleic acid test is an automated rapid multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) test intended for the simultaneous qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, influenza B virus and respiratory syncytial virus (RSV) nucleic acids in anterior nasal (nasal) and nasopharyngeal swab specimens from individuals exhibiting signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory viral infection due to SARS-CoV-2, influenza and RSV can be similar. This test is intended to aid in the differential diagnosis of SARS-CoV-2, influenza A, influenza B, and RSV infections in humans and is not intended to detect influenza C virus infections.

Nucleic acids from the viral organisms identified by this test are generally detectable in nasopharyngeal and nasal swab specimens during the acute phase of infection. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory tract infection are indicative of the presence of the identified virus, and aid 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. Positive results do not rule out coinfection with other organisms. The organism(s) detected by the cobas liat SARS-CoV-2, Influenza A/B & RSV nucleic acid test may not be the definite cause of disease. Negative results do not preclude SARS-CoV-2, influenza A virus, influenza B virus, or RSV infections.

The cobas liat SARS-CoV-2, Influenza A/B & RSV nucleic acid test is performed on the cobas liat analyzer which automates and integrates sample purification, nucleic acid amplification, and detection of the target sequence in biological samples using real-time PCR assays. The assay targets both the ORF1 a/b non-structural region and membrane protein gene that are unique to SARS-CoV-2, a well-conserved region of the matrix gene of influenza A (Flu A target), the nonstructural protein 1 (NS1) gene of influenza B (Flu B target) and the matrix gene of RSV (RSV target). An Internal Control (IC) is included to control for adequate processing of the target virus through all steps of the assay process and to monitor the presence of inhibitors in the RT-PCR processes.

The provided text describes the analytical and clinical performance evaluation of the cobas® liat SARS-CoV-2, Influenza A/B & RSV nucleic acid test, which is a real-time RT-PCR assay. The information mainly focuses on the performance characteristics required for FDA clearance (510(k)).

Here's a breakdown of the requested information based on the provided document:

Acceptance Criteria and Device Performance

The document does not explicitly present a table of "acceptance criteria" in a pass/fail format for clinical performance. Instead, it demonstrates the device's performance through various analytical studies and clinical agreement percentages relative to a comparator method. The acceptance for a 510(k) submission is typically that the device is "substantially equivalent" to a legally marketed predicate device, which implies demonstrating comparable performance characteristics.

The key performance metrics are the Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) in clinical studies. While there aren't explicit numeric acceptance criteria stated, the achieved performance values are presented as evidence of substantial equivalence.

Here’s a table summarizing the reported clinical device performance based on the prospective study (Table 13) and the retrospective study (Table 15). These are the metrics by which the device's clinical performance would be "accepted" as substantially equivalent.

Table of Reported Device Performance (Clinical)

Note on "Acceptance Criteria" for Analytical Performance: The document describes detailed analytical studies (LoD, inclusivity, cross-reactivity, interference, reproducibility), and the reported hit rates and concentrations demonstrate that the device met the internal analytical performance specifications, which are implicitly the "acceptance criteria" for these aspects. For instance, for LoD, the acceptance criterion is implied to be ≥95% hit rate at the determined concentration. For inclusivity, it's detection at or near 3x LoD. For cross-reactivity and interference, the acceptance criterion is no cross-reactivity/interference observed. The document states that "none of the organisms tested cross reacted or interfered" and that "substances... did not interfere," indicating successful meeting of these criteria. For reproducibility, the agreement percentages for positive and negative samples are above 99% for most categories.

Study Details

1. Sample sizes used for the test set and the data provenance:

   • Prospective Clinical Study (Category I):
     • NPS specimens: 1729 enrolled subjects leading to 1704 evaluable specimens for SARS-CoV-2, Flu A, Flu B, and 1705 for RSV.
     • ANS specimens: 1729 enrolled subjects leading to 1705 evaluable specimens for SARS-CoV-2, Flu B, 1703 for Flu A, and 1706 for RSV.
     • Data Provenance: Fresh specimens, prospective, collected between September 2023 and March 2024 at 14 point-of-care testing sites in the United States (US).
   • Retrospective Clinical Study (Category III):
     • Specimens: Frozen archived clinical NPS (n=223) and ANS (n=206) specimens.
     • Data Provenance: Retrospective, collected between 2019 and 2023. Distributed to 6 sites for testing. Country of origin not explicitly stated but implied to be US given the overall context of a US FDA clearance (though not definitively stated for the retrospective part).

2. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
   The ground truth for the clinical test set was established by comparing the results of the cobas® liat test to the "results of an FDA-cleared Nucleic Acid Amplification Test (NAAT)." The document does not specify the number of human experts, their qualifications, or their role in establishing this ground truth. The "ground truth" here is the result from the comparator NAAT, not human expert interpretation of images or clinical data.

3. Adjudication method (e.g. 2+1, 3+1, none) for the test set:
   The document describes a discrepant analysis for cases where the cobas® liat test results differed from the comparator NAAT. For the prospective study, the discrepancy analysis showed how many of the discrepant results (e.g., cobas® liat negative, comparator positive) were ultimately confirmed as positive or negative by further investigation (implied to be by the comparator method or potentially a third method, though not explicitly detailed beyond "discrepant NAAT results"). The details provided are:

   • SARS-CoV-2 NPS: Of 12 negative cobas® liat/positive comparator, 8 were positive and 4 negative. Of 35 positive cobas® liat/negative comparator, 12 were positive and 23 negative.
   • Similar analyses are provided for other targets and specimen types.
     This implies an adjudication method where discrepant results were further investigated, likely with repeat testing or a confirmatory reference method, but the specific "2+1" or "3+1" reader/expert adjudication model (common in imaging studies) is not applicable or described for this in vitro diagnostic (IVD) device.

4. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:
   No, an MRMC comparative effectiveness study was not done. This type of study is primarily relevant for imaging devices that assist human readers (e.g., AI for radiology). The cobas® liat test is an automated molecular diagnostic test directly detecting nucleic acids, not an AI-assisted interpretation device for human "readers."

5. If a standalone (i.e. algorithm only without human-in-the loop performance) was done:
   Yes, the performance reported (PPA, NPA, and analytical studies) represents the standalone performance of the cobas® liat device. It is an automated system where the "algorithm" (the RT-PCR assay and its interpretation software) directly produces a qualitative result (Detected/Not Detected), without human "in-the-loop" interpretation for the primary result. Human operators load samples and review results, but the analytical detection and differentiation itself is automated.

6. The type of ground truth used (expert consensus, pathology, outcomes data, etc.):
   The ground truth for both prospective and retrospective clinical studies was based on the results of an FDA-cleared Nucleic Acid Amplification Test (NAAT), which served as the comparator method. For discrepant samples, further re-testing against the comparator or a reference method was performed for adjudication. This falls under a "reference standard" or "comparator method" type of ground truth.

7. The sample size for the training set:
   The document does not specify the sample size for a "training set." This type of molecular diagnostic device typically relies on analytical validation (LoD, inclusivity, specificity) and clinical validation through comparison to a reference method, rather than a machine learning model that requires explicit training data. The development process would involve iterative optimization of primers, probes, and assay conditions, but this is not typically referred to as a "training set" in the context of IVD submissions, especially for traditional PCR assays.

8. How the ground truth for the training set was established:
   As no explicit "training set" for a machine learning model is described, the question of how its ground truth was established is not applicable based on the provided text. The "ground truth" in the context of this traditional IVD development refers to the reliable identification of the target analytes in samples for analytical and clinical validation, often through established reference methods or characterized materials.

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The Visby Medical Respiratory Health Test is a single-use (disposable), fully integrated, automated Reverse Transcription Polymerase Chain Reaction (RT-PCR) in vitro diagnostic test intended for the simultaneous qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, and influenza B RNA in nasopharyngeal swab and anterior nasal swab specimens from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2, influenza A, and influenza B can be similar.

The Visby Medical Respiratory Health Test is intended for use as an aid in the differential diagnosis of SARS-CoV-2, influenza A, and influenza B infection if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza A, and influenza B viral RNA are generally detectable in nasopharyngeal swab and anterior nasal swab specimens during the acute phase of infection. This test is not intended to detect influenza C virus infections.

Positive results are indicative of the identified virus, but do not rule out bacterial infection or co-infection with other organisms not detected by the test. The agent(s) detected by the Visby Medical Respiratory Health Test may not be the definitive cause of disease. Negative results do not preclude SARS-CoV-2, influenza A, or influenza B infection. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

The Visby Medical Respiratory Health Test is a single-use (disposable), fully integrated, compact device containing a reverse transcription polymerase chain reaction (RT-PCR) based assay for qualitative detection of influenza B, and/or SARS-CoV-2 viral RNA in upper respiratory tract specimens. The device automatically performs all steps required to complete lysis, reverse transcription (RT), PCR amplification, and detection.

Specimen collected using nasopharyngeal (NP) or anterior nasal (AN) swabs (without transport media) are placed in the Visby Medical Respiratory Health Buffer and then transferred into the sample port of the device using the provided fixed volume pipette. The sample enters a lysis module and rehydrates the RT enzyme and RT primers. The mixture then moves through a sample preparation module where viruses and human cells are simultaneously lysed, and RNA is reverse transcribed. The resulting fluid (containing cDNA) is then mixed with lyophilized PCR reagents containing the DNA polymerase enzyme and PCR primers. The PCR mixture (containing cDNA template and reagents) is then thermal cycled to amplify the targets, including human beta-2 microglobulin (B2M) RNA, which serves as a process control. After PCR, the biotinylated product is hybridized to covalently bound capture probes at specific locations along a flow channel. The flow channel is configured to facilitate an enzymatic reaction that uses streptavidin bound horseradish peroxidase (HRP) and a colorimetric substrate that forms a purple precipitate. The operator observes a color change at the specific locations indicating the presence of an amplified target. Test results can be expected in approximately 30 minutes: illumination of a "DONE" status light on the front of the device and a purple color in the "RESULTS VALID" spot, indicate a successful test. A purple spot adjacent to "Flu A", "Flu B", and/or "COVID-19" signifies the presence of, influenza A, influenza B, and/or SARS-CoV-2 viral RNA.

Here's an analysis of the acceptance criteria and study detailed in the provided text:

Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state "acceptance criteria" in a separate table. However, based on the Summary of Performance Data, the inferred acceptance criteria are the achieved Positive Percentage Agreement (PPA) and Negative Percentage Agreement (NPA) values in the clinical evaluation. The study aims to demonstrate substantial equivalence to the predicate device, implying that the performance needs to be comparable or better.

Here's a table summarizing the reported device performance, which implicitly represents the met acceptance criteria:

Study Details:

• Sample size used for the test set and the data provenance:

  • Sample Size: A total of 1,501 subjects were included in the performance analysis after exclusions. 1,575 Visby tests were initially performed.
  • Data Provenance: Prospectively collected fresh specimens from subjects presenting with signs and symptoms of a viral respiratory infection at five CLIA Waived study sites in the US (urgent care and family care clinics). Specimens were collected and tested between May 2022 and Feb 2024.

• 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 or qualifications of experts used to establish ground truth for the test set. Instead, it states that the comparator assays define the ground truth.

• Adjudication method (e.g., 2+1, 3+1, none) for the test set:

  • The document describes comparing the Visby Medical Respiratory Health Test results against an "FDA-cleared influenza molecular test and an FDA-EUA authorized SARS-CoV-2 RT-PCR test as a comparator." It also mentions "alternate molecular assay" for discordant results (footnotes a, b, e, f, g, h, i, j in Table 2). This indicates that the ground truth was established by these comparator assays, potentially with some form of reference-standard-based comparison and possibly resolution of discrepancies with alternate assays, rather than a multi-expert adjudication method on the test set itself.

• If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

  • No, an MRMC comparative effectiveness study was not done. This device is an in vitro diagnostic (IVD) test for molecular detection of viruses, not an AI-assisted diagnostic imaging or human-read interpretation system. The "operators" in the reproducibility study were "non-laboratorians representing healthcare professionals," but their performance was evaluated against expected results for spiked samples, not in comparison to their own performance with and without an AI assistant on clinical cases.

• If a standalone (i.e., algorithm only without human-in-the-loop performance) was done:

  • Yes, a standalone performance evaluation was primarily done for the device. The clinical study evaluated the device's ability to detect viral RNA in specimens. While "typical CLIA Waived operators" performed the test, their role was to execute the device's protocol, and the device's detection accuracy was then compared against the comparator assays. The "device automatically performs all steps required to complete lysis, reverse transcription (RT), PCR amplification, and detection," implying it functions as a standalone diagnostic unit once the sample is loaded.

• The type of ground truth used (expert consensus, pathology, outcomes data, etc.):

  • The ground truth for the clinical evaluation was established by comparator molecular assays: an "FDA-cleared influenza molecular test" and an "FDA-EUA authorized SARS-CoV-2 RT-PCR test." Discordant results were sometimes further investigated with an "alternate molecular assay."

• The sample size for the training set:

  • The document does not specify a separate training set or its sample size. For IVD devices like this RT-PCR test, the "training" typically refers to the assay development and optimization process in the lab, rather than a distinct training set in the way AI/ML models are trained. The clinical performance study evaluates the final, optimized device.

• How the ground truth for the training set was established:

  • As no distinct "training set" in the context of an AI/ML model is described, this information is not applicable. The development and optimization of such a diagnostic test would involve analytical studies (e.g., LoD, inclusivity, cross-reactivity) where the "ground truth" for those specific experiments (e.g., known concentrations of viral targets) is established by careful spiking and molecular characterization in a laboratory setting.

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Alinity m Resp-4-Plex is a multiplexed real-time in vitro reverse transcription polymerase chain reaction (RT-PCR) assay for use with the automated Alinity m System for the qualitative detection and differentiation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), influenza A virus, influenza B virus and Respiratory Syncytial Virus (RSV) in nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2, influenza B, and RSV can be similar.

The Alinity m Resp-4-Plex assay is intended for use in the differential detection of SARS-CoV-2, influenza B and/or RSV RNA and aids in the diagnosis of COVID-19, influenza and/or RSV infections if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza B and RSV viral RNA are generally detectable in nasopharyngeal swab specimens during the acute phase of infection. This test is not intended to detect influenza C virus infections.

Positive results are indication of the identified virus, but do not rule out bacterial infection or co-infection with other pathogens not detected by the test. The agent(s) detected by the Alinity m Resp-4-Plex assay may not be the definite cause of disease.

Negative results do not preclude SARS-CoV-2, influenza B and/or RSV infections and should not be used as the sole basis for diagnosis, treatment or other patient management decisions.

The Alinity m Resp-4-Plex assay requires two separate assay-specific kits: Alinity m Resp-4-Plex AMP Kit and Alinity m Resp-4-Plex CTRL Kit. The assay utilizes real-time PCR to amplify and detect genomic RNA sequences of influenza A (flu A), influenza B (flu B), RSV, and/or SARS-CoV-2 from nasopharyngeal (NP) swab specimens. The assay targets 2 different genes within the SARS-CoV-2 genome. Fluorescently labeled probes allow for simultaneous detection and differentiation of amplified products of all 4 viruses and Internal Control (IC) in a single reaction vessel. All steps of the assay procedure are executed automatically by the Alinity m System, which is a continuous random-access analyzer. The system performs automated sample preparation using magnetic microparticle technology. The IC is introduced into each specimen at the beginning of sample preparation. Purified RNA is combined with activation and amplification/detection reagents and transferred to a reaction vessel for reverse transcription, PCR amplification, and real-time fluorescence detection. A positive and negative control are tested to ensure performance. Patient results are automatically reported. The assay also utilizes the Alinity m Resp-4-Plex Assay Application Specification File, Alinity m System and System Software, Alinity m Sample Prep Kit 2, Alinity m Tubes and Caps, and Alinity m System Solutions.

The provided text is a 510(k) Summary for the Abbott Molecular Inc. Alinity m Resp-4-Plex assay, a multiplexed real-time RT-PCR assay for the qualitative detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV.

Here's an analysis of the acceptance criteria and the study that proves the device meets them, based on the provided document:

Acceptance Criteria and Reported Device Performance

The acceptance criteria for this device are demonstrated through various analytical and clinical studies, primarily focusing on analytical sensitivity (Limit of Detection), inclusivity, precision, reproducibility, analytical specificity (interfering substances and cross-reactants), competitive interference, carryover, and clinical performance (Positive Percent Agreement - PPA, and Negative Percent Agreement - NPA).

The document implicitly defines the acceptance criteria as the successful demonstration of performance metrics that are typically expected for such in vitro diagnostic devices, often compared to highly sensitive FDA-cleared or EUA assays. While specific numerical acceptance criteria (e.g., PPA > X%, NPA > Y%) are not explicitly stated as "acceptance criteria," they are demonstrated by the reported results. The conclusion statement (Section 5.0) explicitly states that the "analytical and clinical study results demonstrate that the Alinity m Resp-4-Plex assay... performs comparably to the predicate device... the results support a substantial equivalence decision." This implies that the demonstrated performance values met the FDA's criteria for substantial equivalence to the predicate device.

Here's a table summarizing the reported device performance, which implicitly met the acceptance criteria:

Table 1: Acceptance Criteria (as Demonstrated Performance) and Reported Device Performance

Study Details

Here's the breakdown of the study details as requested:

1. A table of acceptance criteria and the reported device performance: Already provided above.

2. Sample sizes used for the test set and the data provenance:

   • Analytical Studies (Test Set):

     • LoD: For each virus (Flu A, Flu B, RSV, SARS-CoV-2), preliminary LoD involved testing a minimum of 3 levels, each in a minimum of 3 replicates. Final LoD confirmation involved testing a minimum of 3 panel members with target concentrations bracketing the preliminary LoD, each panel member in a minimum of 20 replicates. (Total specific sample numbers not provided per virus, but this describes the method and minimums). Specimens were pooled negative NP clinical specimens.
     • Inclusivity: Each individual virus isolate or strain was tested in replicates of 5. (Total specific sample numbers not provided per virus, but 16 Flu A strains, 9 Flu B, 6 RSV, 9 SARS-CoV-2). Specimens were pooled negative clinical NP swab matrix.
     • Precision: 5 panel members (1 negative, 4 positive) tested with 4 replicates twice each day for 5 days, on 3 Alinity m Systems operated by 3 operators using 3 reagent lots. This leads to:
       • Flu A: 120 positive replicates for each level, 360 negative replicates.
       • Flu B: 120 positive replicates for each level, 357 negative replicates.
       • RSV: 120 positive replicates for moderate, 117 for low, 360 negative.
       • SARS-CoV-2: 120 positive replicates for each level, 357 negative.
     • Reproducibility: 5 panel members tested at 3 external clinical testing sites. Each site tested 2 Alinity m Resp-4-Plex AMP Kit lots, on 5 non-consecutive days for each lot. Four replicates of each panel member were tested on each of 5 days. This leads to:
       • Flu A: 120 positive replicates for each level, 360 negative replicates.
       • Flu B: 120 positive replicates for each level, 359 negative replicates.
       • RSV: 120 positive replicates for moderate, 119 for low, 360 negative.
       • SARS-CoV-2: 120 positive replicates for moderate, 117 for low, 359 negative.
     • Analytical Specificity (Interfering Substances): 34 substances evaluated in 2 different positive panel members (PM1 & PM2), each containing multiple analytes at 3xLoD. (Replicate number not specified).
     • Analytical Specificity (Cross-Reactants): 74 microorganisms added to pooled negative clinical NP swab matrix (replicate number not specified) and also to positive samples (replicate number not specified).
     • Competitive Interference: 4 panel members, each containing 3 viruses at low concentrations and one at high concentration. (Replicate number not specified).
     • Carryover: Negative and high positive samples tested in alternating positions, across 3 Alinity m Systems. 360 negative samples total.

   • Clinical Performance (Test Set):

     • Prospective Clinical Study:
       • Flu A/B, RSV: 2,753 valid results initially, 2,504 (Flu A), 2,710 (Flu B), 2,700 (RSV) used in analysis.
         • Data Provenance: Multicenter study using prospectively collected nasopharyngeal swab specimens. 4 US clinical sites for testing. Specimens collected during 2021-2022 flu season at 7 geographically distributed locations in the US and during the 2020 flu season at 1 location in the Southern Hemisphere.
       • SARS-CoV-2: 826 valid results initially, 698 used in analysis.
         • Data Provenance: Specimens collected at 10 geographically distributed locations in the US over 2 time periods (Dec 2020 - Feb 2021 and May 2023).
     • Retrospective Clinical Study:
       • Flu A/B, RSV: 515 valid results initially, 506 (Flu A), 504 (Flu B), 505 (RSV) used in analysis.
         • Data Provenance: Preselected archived flu B positive NP swab specimens in UVT or UTM collected during the 2017-2018 and 2019-2020 flu seasons. Randomly mixed with known negative specimens.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:

   • The ground truth for the clinical test sets (both prospective and retrospective) was established using a Composite Comparator (CC). This CC was based on results from "2 to 3 FDA cleared assays for flu A, flu B, and RSV" and "2 to 3 highly sensitive EUA SARS-CoV-2 molecular assays."
   • The document does not specify the number or qualifications of human experts (e.g., radiologists, pathologists) involved in establishing this ground truth. The ground truth method described is entirely based on laboratory comparator assays, not expert human interpretation of results.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set:

   • The ground truth was established by a Composite Comparator (CC) method:
     • A specimen was categorized as CC positive if a minimum of 2 comparator positive results were reported.
     • A specimen was categorized as CC negative if a minimum of 2 comparator negative results were reported.
     • A specimen was categorized CC indeterminate if a CC could not be determined due to missing results from the comparator assays.
   • This functions as a type of "majority rule" adjudication or consensus, but strictly between other molecular assays, not human experts.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

   • No, an MRMC comparative effectiveness study was not done.
   • This device is an in vitro diagnostic (RT-PCR assay) that provides a qualitative (positive/negative) result directly. It does not involve human "readers" interpreting images or other complex data that would typically benefit from AI assistance or an MRMC study design. Therefore, there's no data on human reader improvement with or without AI assistance.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:

   • Yes, the primary performance evaluation is a standalone algorithm-only performance.
   • The Alinity m Resp-4-Plex assay is an automated RT-PCR system. Its performance (PPA, NPA) in both analytical and clinical studies is the performance of the "algorithm only" in generating positive/negative results from the sample. Human intervention is limited to sample collection and system operation, not interpretation of the primary diagnostic output.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc):

   • The ground truth for the clinical studies was established using a Composite Comparator (CC) method based on the results of other FDA-cleared molecular diagnostic assays (or EUA high-sensitivity molecular assays for SARS-CoV-2).
   • It was not based on expert consensus, pathology, or outcomes data directly.

8. The sample size for the training set:

   • The document describes a de novo device, or a device for which substantial equivalence is being sought, not an AI/ML device that requires a distinct "training set" and "test set" in the context of model development.
   • The studies presented are primarily verification and validation studies to demonstrate the device's analytical and clinical performance after its development.
   • Therefore, the concept of a separate "training set" as understood in machine learning (where data is used to train a model) is not applicable to this RT-PCR assay. The assay's "knowledge" is embedded in its reagents, primers, probes, and system parameters, which were likely optimized during development using various analytical samples, but these are not typically referred to as a "training set" in the context of a 510(k) for an RT-PCR assay.

9. How the ground truth for the training set was established:

   • As explained in point 8, there isn't a "training set" in the AI/ML sense for this RT-PCR assay. The ground truth for any samples used during the development or optimization phases would similarly be established using well-characterized samples (e.g., cultured viruses, positive clinical samples confirmed by reference methods, synthetic nucleic acids, negative clinical samples).

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The Aptima® SARS-CoV-2 Assay is a nucleic acid amplification in vitro diagnostic test intended for the qualitative detection of RNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated and purified from nasopharyngeal (NP) swab and anterior nasal (AN) swab specimens obtained from patients with signs and symptoms of COVID-19.

Positive results are indicative of the presence of SARS-CoV-2 RNA. The Aptima SARS-CoV-2 Assay is intended for use as an aid in the diagnosis of COVID-19 if used in conjunction with other clinical, epidemiological, and laboratory findings. Clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out bacterial infection or co-infection with other viruses.

Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.

The Aptima SARS-CoV-2 Assay is a nucleic acid amplification in vitro diagnostic test developed for use on the fully automated Panther/Panther Fusion system to detect RNA from SARS-CoV-2 isolated and purified from nasopharyngeal and anterior nasal swab specimens collected into UTM/VTM or with the RespDirect Collection Kit.

The Aptima SARS-CoV-2 Assay combines the technologies of target capture, Transcription Mediated Amplification (TMA), and Dual Kinetic Assay (DKA).

Specimens are collected and transferred into their respective specimen transport tubes. The transport solutions in these tubes release the RNA target and protect them from degradation during storage. When the Aptima SARS-CoV-2 Assay is performed in the laboratory, the target RNA molecules are isolated from specimens by use of capture oligomers via target capture that utilizes magnetic microparticles. The capture oligomers contain sequences complementary to specific regions of the target molecules as well as a string of deoxyadenosine residues. A separate capture oligomer is used for each target. During the hybridization step, the sequence specific regions of the capture oligomers bind to specific regions of the target molecules. The capture oligomer:target complex is then captured out of solution by decreasing the temperature of the reaction to room temperature. This temperature reduction allows hybridization to occur between the deoxyadenosine region on the capture oligomer and the poly-deoxythymidine molecules that are covalently attached to the magnetic particles. The microparticles, including the captured target molecules bound to them, are pulled to the side of the reaction vessel using magnets and the supernatant is aspirated. The particles are washed to remove residual specimen matrix that may contain amplification reaction inhibitors. After the target capture steps are completed, the specimens are ready for amplification.

Target amplification assays are based on the ability of complementary oligonucleotide primers to specifically anneal and allow enzymatic amplification of the target nucleic acid strands. The Aptima SARS-CoV-2 Assay replicates specific regions of the RNA from SARS-CoV-2 virus. Detection of the RNA amplification product sequences (amplicon) is achieved using nucleic acid hybridization. Single-stranded chemiluminescent nucleic acid probes, which are unique and complementary to a region of each target amplicon and Internal Control (IC) amplicon, are labeled with different acridinium ester (AE) molecules. The AE-labeled probes combine with the amplicon to form stable hybrids. The Selection Reagent differentiates hybridized from unhybridized probe, eliminating the generation of signal from the unhybridized probe. During the detection step, light emitted from the labeled hybrids is measured as photon signals in a luminometer and are reported as Relative Light Units (RLU). In DKA, differences in the kinetic profiles of the labeled probes allow for the differentiation of signal; kinetic profiles are derived from measurements of photon output during the detection read time.

The Aptima SARS-CoV-2 Assay amplifies and detects 2 conserved regions of the ORF1ab gene in the same reaction, using the "glower" kinetic type. The 2 regions are not differentiated and amplification of either or both regions lead to RLU signal. The assay results are determined by a cut-off based on the total RLU and the kinetic curve type.

Here's a breakdown of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Acceptance Criteria and Reported Device Performance

The acceptance criteria are implicitly defined by the performance metrics presented as evidence of substantial equivalence to the predicate device. The key metrics are Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA). While explicit numerical acceptance thresholds are not provided in the document, the reported performance demonstrates "comparable" results to the predicate device.

Study Details:

1. Sample Sizes Used for the Test Set and Data Provenance:

   • Clinical Study 1 (NP Swab Specimens):
     • Total Enrolled: 1646 specimens
     • Evaluable (Final Data Set): 1495 NP swab specimens (1195 fresh, 300 frozen)
     • Provenance: Prospective multicenter study from four participating US pediatric/adolescent, private and/or university hospitals. Specimens collected between June-July 2020 and January-April 2023. Remnant specimens.
   • Clinical Study 2 (Anterior Nasal Swab Specimens):
     • Total Enrolled Subjects: 2301
     • Evaluable Subjects: 2177 individuals (1159 with evaluable anterior nasal swab specimens in UTM/VTM, and 1018 with evaluable nasal swab specimens in eSTM).
     • Provenance: Prospective, multicenter clinical study at nine geographically and ethnically diverse US sites during the 2022-2023 respiratory season.

2. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications:

   • The document does not specify the number or qualifications of experts directly establishing the clinical ground truth. Instead, it relies on a Composite Comparator Algorithm (CCA). The CCA consists of "two highly sensitive US FDA EUA SARS-CoV-2 molecular tests" and a "validated PCR followed by bi-directional sequencing (PCR/BDS) assay." This implies that the 'expertise' comes from the validation and regulatory clearance of these comparator assays, rather than individual human experts adjudicating each case.

3. Adjudication Method for the Test Set:

   • Composite Comparator Algorithm (CCA): "A final CCA result was assigned when two of the three comparator assay results were in concordance." This serves as the adjudication method for determining the true positive/negative status of the clinical samples.

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

   • No. This type of study (MRMC) is typically performed for diagnostic imaging devices where human readers interpret images with and without AI assistance. The Aptima SARS-CoV-2 Assay is an in vitro diagnostic (IVD), a laboratory test that detects nucleic acids, not an imaging device requiring human interpretation of visual data. Therefore, an MRMC study is not applicable here.

5. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done:

   • Yes, this is effectively a standalone performance study. The Aptima SARS-CoV-2 Assay is an automated nucleic acid amplification test run on the Panther/Panther Fusion system. Its performance is directly compared to the CCA, without human intervention in the interpretation of the device's results.

6. The Type of Ground Truth Used:

   • Clinical Studies: Composite Comparator Algorithm (CCA) based on the concordance of results from two highly sensitive FDA EUA SARS-CoV-2 molecular tests and a validated PCR/BDS assay. This is a form of reference standard derived from established and highly sensitive laboratory methods.
   • Analytical Studies (e.g., LoD, Reactivity, Specificity): The ground truth was established by known concentrations of SARS-CoV-2 virus strains or other microorganisms/interfering substances, prepared in controlled laboratory settings (e.g., "spiked with inactivated cultured SARS-CoV-2 virus").

7. The Sample Size for the Training Set:

   • The document describes performance evaluation studies (analytical and clinical) for market clearance. It does not provide information on the specific training set size used for the development or training of the assay's internal algorithms (e.g., for the kinetic curve analysis or cut-off determination). This information would typically be part of the assay development and validation, not necessarily detailed in a 510(k) summary unless it significantly changed or impacted performance during the clearance process for the specific assay rather than the underlying platform. The focus here is on the performance of the final, already "trained" device.

8. How the Ground Truth for the Training Set Was Established:

   • As noted above, details regarding the training set's ground truth establishment are not provided in this 510(k) summary. For IVD devices, ground truth for training internal algorithms typically involves using characterized positive and negative clinical samples, spiked samples with known viral loads, and potentially synthetic data, all carefully confirmed by highly sensitive reference methods or gold standard assays during the R&D phase of the product.

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