(266 days)
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.
| Analyte | Acceptance Criteria (Reported PPA CI) | Device Performance (PPA %) | Acceptance Criteria (Reported NPA CI) | Device Performance (NPA %) |
|---|---|---|---|---|
| Influenza A | ||||
| Fresh Pros. | (90.5%, 98.5%) | 96.2 | (99.3%, 99.9%) | 99.7 |
| Frozen Pros. | (89.0%, 99.1%) | 96.8 | (99.4%, 99.8%) | 99.7 |
| Combined Pros. | (92.3%, 98.3%) | 96.4 | (99.5%, 99.8%) | 99.7 |
| Retrospective | (94.1%, 100.0%) | 100.0 | (97.6%, 99.4%) | 98.8 |
| Influenza B | ||||
| Fresh Pros. | (94.5%, 100.0%) | 100.0 | (99.6%, 100.0%) | 99.9 |
| Frozen Pros. | (79.8%, 99.3%) | 95.8 | (99.8%, 100.0%) | 100.0 |
| Combined Pros. | (94.0%, 99.8%) | 98.9 | (99.8%, 100.0%) | 99.9 |
| Retrospective | (87.1%, 99.6%) | 97.5 | (98.3%, 99.7%) | 99.3 |
| RSV | ||||
| Fresh Pros. | (77.4%, 94.7%) | 88.7 | (99.8%, 100.0%) | 100.0 |
| Frozen Pros. | (81.5%, 95.3%) | 90.4 | (99.8%, 100.0%) | 100.0 |
| Combined Pros. | (83.1%, 93.9%) | 89.7 | (99.9%, 100.0%) | 100.0 |
| Retrospective | (96.4%, 100.0%) | 100.0 | (98.2%, 99.7%) | 99.3 |
| SARS-CoV-2 | ||||
| Fresh Pros. | (92.4%, 98.6%) | 96.7 | (98.0%, 99.1%) | 98.6 |
| Frozen Pros. | (95.3%, 98.9%) | 97.7 | (97.8%, 98.8%) | 98.4 |
| Combined Pros. | (95.4%, 98.5%) | 97.3 | (98.0%, 98.8%) | 98.5 |
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.
§ 866.3981 Device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test.
(a)
Identification. A device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test is an in vitro diagnostic device intended for the detection and identification of SARS-CoV-2 and other microbial agents when in a multi-target test in human clinical respiratory specimens from patients suspected of respiratory infection who are at risk for exposure or who may have been exposed to these agents. The device is intended to aid in the diagnosis of respiratory infection in conjunction with other clinical, epidemiologic, and laboratory data or other risk factors.(b)
Classification. Class II (special controls). The special controls for this device are:(1) The intended use in the labeling required under § 809.10 of this chapter must include a description of the following: Analytes and targets the device detects and identifies, the specimen types tested, the results provided to the user, the clinical indications for which the test is to be used, the specific intended population(s), the intended use locations including testing location(s) where the device is to be used (if applicable), and other conditions of use as appropriate.
(2) Any sample collection device used must be FDA-cleared, -approved, or -classified as 510(k) exempt (standalone or as part of a test system) for the collection of specimen types claimed by this device; alternatively, the sample collection device must be cleared in a premarket submission as a part of this device.
(3) The labeling required under § 809.10(b) of this chapter must include:
(i) A detailed device description, including reagents, instruments, ancillary materials, all control elements, and a detailed explanation of the methodology, including all pre-analytical methods for processing of specimens;
(ii) Detailed descriptions of the performance characteristics of the device for each specimen type claimed in the intended use based on analytical studies including the following, as applicable: Limit of Detection, inclusivity, cross-reactivity, interfering substances, competitive inhibition, carryover/cross contamination, specimen stability, precision, reproducibility, and clinical studies;
(iii) Detailed descriptions of the test procedure(s), the interpretation of test results for clinical specimens, and acceptance criteria for any quality control testing;
(iv) A warning statement that viral culture should not be attempted in cases of positive results for SARS-CoV-2 and/or any similar microbial agents unless a facility with an appropriate level of laboratory biosafety (
e.g., BSL 3 and BSL 3+, etc.) is available to receive and culture specimens; and(v) A prominent statement that device performance has not been established for specimens collected from individuals not identified in the intended use population (
e.g., when applicable, that device performance has not been established in individuals without signs or symptoms of respiratory infection).(vi) Limiting statements that indicate that:
(A) A negative test result does not preclude the possibility of infection;
(B) The test results should be interpreted in conjunction with other clinical and laboratory data available to the clinician;
(C) There is a risk of incorrect results due to the presence of nucleic acid sequence variants in the targeted pathogens;
(D) That positive and negative predictive values are highly dependent on prevalence;
(E) Accurate results are dependent on adequate specimen collection, transport, storage, and processing. Failure to observe proper procedures in any one of these steps can lead to incorrect results; and
(F) When applicable (
e.g., recommended by the Centers for Disease Control and Prevention, by current well-accepted clinical guidelines, or by published peer-reviewed literature), that the clinical performance may be affected by testing a specific clinical subpopulation or for a specific claimed specimen type.(4) Design verification and validation must include:
(i) Detailed documentation, including performance results, from a clinical study that includes prospective (sequential) samples for each claimed specimen type and, as appropriate, additional characterized clinical samples. The clinical study must be performed on a study population consistent with the intended use population and compare the device performance to results obtained using a comparator that FDA has determined is appropriate. Detailed documentation must include the clinical study protocol (including a predefined statistical analysis plan), study report, testing results, and results of all statistical analyses.
(ii) Risk analysis and documentation demonstrating how risk control measures are implemented to address device system hazards, such as Failure Modes Effects Analysis and/or Hazard Analysis. This documentation must include a detailed description of a protocol (including all procedures and methods) for the continuous monitoring, identification, and handling of genetic mutations and/or novel respiratory pathogen isolates or strains (
e.g., regular review of published literature and periodic in silico analysis of target sequences to detect possible mismatches). All results of this protocol, including any findings, must be documented and must include any additional data analysis that is requested by FDA in response to any performance concerns identified under this section or identified by FDA during routine evaluation. Additionally, if requested by FDA, these evaluations must be submitted to FDA for FDA review within 48 hours of the request. Results that are reasonably interpreted to support the conclusion that novel respiratory pathogen strains or isolates impact the stated expected performance of the device must be sent to FDA immediately.(iii) A detailed description of the identity, phylogenetic relationship, and other recognized characterization of the respiratory pathogen(s) that the device is designed to detect. In addition, detailed documentation describing how to interpret the device results and other measures that might be needed for a laboratory diagnosis of respiratory infection.
(iv) A detailed device description, including device components, ancillary reagents required but not provided, and a detailed explanation of the methodology, including molecular target(s) for each analyte, design of target detection reagents, rationale for target selection, limiting factors of the device (
e.g., saturation level of hybridization and maximum amplification and detection cycle number, etc.), internal and external controls, and computational path from collected raw data to reported result (e.g., how collected raw signals are converted into a reported signal and result), as applicable.(v) A detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review.
(vi) For devices intended for the detection and identification of microbial agents for which an FDA recommended reference panel is available, design verification and validation must include the performance results of an analytical study testing the FDA recommended reference panel of characterized samples. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(vii) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens, the design verification and validation must include a detailed description of the identity, phylogenetic relationship, or other recognized characterization of the Influenza A and B viruses that the device is designed to detect, a description of how the device results might be used in a diagnostic algorithm and other measures that might be needed for a laboratory identification of Influenza A or B virus and of specific Influenza A virus subtypes, and a description of the clinical and epidemiological parameters that are relevant to a patient case diagnosis of Influenza A or B and of specific Influenza A virus subtypes. An evaluation of the device compared to a currently appropriate and FDA accepted comparator method. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(5) When applicable, performance results of the analytical study testing the FDA recommended reference panel described in paragraph (b)(4)(vi) of this section must be included in the device's labeling under § 809.10(b) of this chapter.
(6) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens in addition to detection of SARS-CoV-2 and similar microbial agents, the required labeling under § 809.10(b) of this chapter must include the following:
(i) Where applicable, a limiting statement that performance characteristics for Influenza A were established when Influenza A/H3 and A/H1-2009 (or other pertinent Influenza A subtypes) were the predominant Influenza A viruses in circulation.
(ii) Where applicable, a warning statement that reads if infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to State or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
(iii) Where the device results interpretation involves combining the outputs of several targets to get the final results, such as a device that both detects Influenza A and differentiates all known Influenza A subtypes that are currently circulating, the device's labeling must include a clear interpretation instruction for all valid and invalid output combinations, and recommendations for any required followup actions or retesting in the case of an unusual or unexpected device result.
(iv) A limiting statement that if a specimen yields a positive result for Influenza A, but produces negative test results for all specific influenza A subtypes intended to be differentiated (
i.e., H1-2009 and H3), this result requires notification of appropriate local, State, or Federal public health authorities to determine necessary measures for verification and to further determine whether the specimen represents a novel strain of Influenza A.(7) If one of the actions listed at section 564(b)(1)(A) through (D) of the Federal Food, Drug, and Cosmetic Act occurs with respect to an influenza viral strain, or if the Secretary of Health and Human Services determines, under section 319(a) of the Public Health Service Act, that a disease or disorder presents a public health emergency, or that a public health emergency otherwise exists, with respect to an influenza viral strain:
(i) Within 30 days from the date that FDA notifies manufacturers that characterized viral samples are available for test evaluation, the manufacturer must have testing performed on the device with those influenza viral samples in accordance with a standardized protocol considered and determined by FDA to be acceptable and appropriate.
(ii) Within 60 days from the date that FDA notifies manufacturers that characterized influenza viral samples are available for test evaluation and continuing until 3 years from that date, the results of the influenza emergency analytical reactivity testing, including the detailed information for the virus tested as described in the certificate of authentication, must be included as part of the device's labeling in a tabular format, either by:
(A) Placing the results directly in the device's labeling required under § 809.10(b) of this chapter that accompanies the device in a separate section of the labeling where analytical reactivity testing data can be found, but separate from the annual analytical reactivity testing results; or
(B) In a section of the device's label or in other labeling that accompanies the device, prominently providing a hyperlink to the manufacturer's public website where the analytical reactivity testing data can be found. The manufacturer's website, as well as the primary part of the manufacturer's website that discusses the device, must provide a prominently placed hyperlink to the website containing this information and must allow unrestricted viewing access.