(299 days)
The Aptima HCV Quant Dx assay is a real-time transcription-mediated amplification (TMA) test used for both detection and quantitation of hepatitis C virus (HCV) RNA in fresh and frozen human serum and plasma from HCV-infected individuals.
Plasma may be prepared in ethylenediaminetetraacetic acid (EDTA), anticoagulant citrate dextrose (ACD) solution, and plasma preparation tubes (PPT). Serum may be prepared in serum separator tubes (SST). Specimens are tested using the Panther system for automated specimen processing, and quantitation. Specimens containing HCV genotypes 1 to 6 are validated for detection and quantitation in the assay.
The Aptima HCV Quant Dx assay is indicated for use as an aid in the diagnosis of active HCV infection in the following populations: individuals with antibody evidence of HCV infection with evidence of liver disease of to be actively infected with HCV antibody evidence, and individuals at risk for HCV infection with antibodies to HCV. Detection of HCV RNA indicates that the virus is replicating and, therefore, is evidence of active infection of HCV RNA does not discriminate between acute and chronic states of infection.
The Aptima HCV Quant Dx assay is also indicated for use as an aid in the management of HCV infected patients undergoing HCV antiviral drug therapy. The assay can be used to measure HCV RNA levels periodically prior to, during, and after treatment to determine sustained virological response (SVR) or nonsustained virological response (NSVR), Assay performance characteristics have been established for individuals infected with HCV and treated with certain direct-acting antiviral agents (DAA) regimens. No information is available on the assay's predictive value when other therapies are used. The results from the Aptima HCV Quant Dx assay must be interpreted within the context of all relevant clinical and laboratory findings.
The Aptima HCV Quant Dx assay is not approved for use as a screening test for the presence of HCV RNA in blood or blood products.
Clearance of this pre-market application will 1) enable additional reagent on-board / off-board cycling, allowing operators to load reagents onto the Panther System 8 times instead of 5 times; and 2) update the Assay Definition Module (ADM) to detect, flag and invalidate results impacted by a faulty or flickering LED, using estimated background minimum limits. These changes do not introduce any changes to the original design, method of manufacture, assay procedure, principle of operation, mechanism of action, conditions of use or hardware of the Panther instrument, or to the results interpretation for the cleared assay.
The Aptima HCV Quant Dx assay is a nucleic acid amplification test that uses real-time TMA technology to detect and quantitate HCV RNA for aiding diagnosis or to establish baseline viral load, as well as to measure on-treatment and post-treatment responses. The assay targets a conserved region of the HCV genome, detecting and quantitating genotypes 1, 2, 3, 4, 5, and 6. The assay is standardized against the 2nd WHO International Standard for Hepatitis C Virus (NIBSC Code 96/798).
The Aptima HCV Quant Dx assay involves three main steps, which all take place in a single tube on the Panther system: target capture, target amplification by TMA, and detection of the amplification products (amplicon) by the fluorescent labeled probes (torches).
During target capture, viral RNA is isolated from specimens. The specimen is treated with a detergent to solubilize the viral envelope, denature proteins, and release viral genomic RNA. Capture oligonucleotides hybridize to highly conserved regions of HCV RNA, if present, in the test specimen. The hybridized target is then captured onto magnetic microparticles that are separated from the specimen in a magnetic field. Wash steps remove extraneous components from the reaction tube.
Target amplification occurs via TMA, which is a transcription-mediated nucleic acid amplification method that utilizes two enzymes, Moloney murine leukemia virus (MMLV) reverse transcriptase and T7 RNA polymerase. The reverse transcriptase is used to generate a DNA copy (containing a promoter sequence for T7 RNA polymerase) of the target sequence. T7 RNA polymerase produces multiple copies of RNA amplicon from the DNA copy template. The Aptima HCV Quant Dx assay utilizes the TMA method to amplify a portion of the 5' UTR of the HCV genome. Amplification of this region is achieved using specific primers which are designed to amplify HCV genotypes 1, 2, 3, 4, 5, and 6.
Detection is achieved using single-stranded nucleic acid torches that are present during the amplification of the target and that hybridize specifically to the amplicon in real time. Each torch has a fluorophore and a quencher. When the torch is not hybridized to the amplicon, the quencher is in close proximity of the fluorophore and suppresses the fluorescence. When the torch binds to the amplicon, the quencher is moved farther away from the fluorophore and it will emit a signal at a specific wavelength when excited by a light source. As more torches hybridize to amplicon a higher fluorescent signal is generated. The time taken for the fluorescent signal to reach a specified threshold is proportional to the starting HCV concentration. Each reaction has an internal calibrator/internal control (IC) that controls for variations in specimen processing, amplification, and detection. The concentration of a sample is determined by the Panther system software using the HCV and IC signals for each reaction and comparing them to calibration information.
The Aptima HCV Quant Dx assay has been designed for and validated on the Panther system. The Panther system is an integrated hardware and software system that together with the Aptima HCV Quant Dx assay fully automates all the steps necessary to perform the assay from sample preparation through amplification of nucleic acid, detection, data reduction and amplicon inactivation.
The provided document describes the acceptance criteria and a study for the Aptima HCV Quant Dx Assay.
Here's a breakdown of the requested information:
1. Table of Acceptance Criteria and Reported Device Performance
| Performance Metric | Acceptance Criteria | Reported Device Performance | Met? |
|---|---|---|---|
| Intended Use Testing (Software 7.2.9 vs 7.2.7) | For software 7.2.9 vs 7.2.7 (reported in PMA annual report for P160023/R004): Assay performance is equivalent to prior comparators. | Yes | |
| QC Control A (Negative) | Not Detected. Max 1 false positive. | 1 false positive. | Yes |
| QC Control B (2.16 log IU/mL) | ±0.5 logs from Value Assignment (or Target Conc if Value). Max 1/30 outliers. Std Dev ≤ 0.25 log copy. | 100% Positivity, Ave diff between expected and recovered is -0.05 log IU/mL, largest SD is 0.07 log IU/mL. | Yes |
| QC Control D (6.52 log IU/mL) | ±0.5 logs from Value Assignment (or Target Conc if Value). Max 0/25 outliers. Std Dev ≤ 0.25 log copy. | 100% Positivity, Ave diff between expected and recovered is -0.12 log IU/mL, largest SD is 0.03 log IU/mL. | Yes |
| QC Panel S (1.13 log IU/mL) | 95% Positivity | 100% Positivity. | Yes |
| Assay Verification (5 to 8 reagent reload change) | |||
| HCV 3.3 log IU/mL (5 Cycles) | Not explicitly stated for each concentration, but implicitly refers to maintaining performance within acceptable ranges. | Cycle Avg LogIU/mL: 3.22, Cycle Std Dev: 0.05, Baseline Avg LogIU/mL: 3.20, Diff (Cycle-Baseline): 0.02. | Yes |
| HCV 3.3 log IU/mL (10 Cycles) | Not explicitly stated for each concentration, but implicitly refers to maintaining performance within acceptable ranges. | Cycle Avg LogIU/mL: 3.28, Cycle Std Dev: 0.06, Baseline Avg LogIU/mL: 3.10, Diff (Cycle-Baseline): 0.18. | Yes |
| HCV 7.0 log IU/mL (5 Cycles) | Not explicitly stated for each concentration, but implicitly refers to maintaining performance within acceptable ranges. | Cycle Avg LogIU/mL: 7.45, Cycle Std Dev: 0.15, Baseline Avg LogIU/mL: 7.56, Diff (Cycle-Baseline): -0.11. | Yes |
| HCV 7.0 log IU/mL (10 Cycles) | Not explicitly stated for each concentration, but implicitly refers to maintaining performance within acceptable ranges. | Cycle Avg LogIU/mL: 7.30, Cycle Std Dev: 0.07, Baseline Avg LogIU/mL: 7.56, Diff (Cycle-Baseline): -0.26. | Yes |
| Assay Verification (Flickering LED) | New estimated background limits will increase fault detection for these assays. | One test order flagged by FAM estimated background limit was also flagged by QNS and considered a true fault. This analysis supports the implementation of proposed Estimated Background minimum limits. | Yes |
| Software V&V (PRM, PRD, SRS, PSD) | All requirements pass. | PRM: 23 Pass, 0 Fail; PRD: 29 Pass, 0 Fail; SRS: 210 Pass, 0 Fail; PSD: 4 Pass, 0 Fail. | Yes |
| Software V&V (Regression Testing) | All requirements pass. | 12 Pass, 0 Fail. | Yes |
| Software V&V (Anomalies) | No known software anomalies exceeding "Negligible" severity. | 12 anomalies, all rated "Negligible." | Yes |
| Additional Intended Use Testing (HCV Assay Software 5.2.5.1 vs 5.3.5.1) | All acceptance criteria met for low level clinical panel. | Aptima HCV Assay Software versions 5.2.5.1 and 5.3.5.1 both resulted in 100% not detected for the negative clinical matrix panel. Both resulted in 100% positivity for the 30 IU/mL clinical positive panel. | Yes |
| Additional On-Board Stability (OBS) and Reagent Cycling | All acceptance criteria met (for 5, 8, and 10 cycles with 72 hours on board stability). | Aptima reagents successfully completed five, eight, and ten on and off on-board cycles with a cumulative 72 hours on board stability and met all acceptance criteria. | Yes |
(*) Note: The document states "may be removed from calculation of Standard Deviatio logs from Target Concentration if Value Assignm" and "±0.50 logs from Target Concentration if Value", which appear to be incomplete sentences or truncated notes in the original document.
2. Sample Sizes Used for the Test Set and Data Provenance
- Intended Use Testing (Software 7.2.9 vs 7.2.7):
- QC Control A (Negative): 120 samples.
- QC Control B (2.16 log IU/mL): 40 samples.
- QC Control D (6.52 log IU/mL): 40 samples.
- QC Panel S (1.13 log IU/mL): 80 samples.
- Data Provenance: Not explicitly stated beyond "previous software version, 7.2.7 (reported to the FDA in the 2022 PMA annual report for the Aptima HCV Quant Dx Assay, P160023/R004)".
- Assay Verification (5 to 8 reagent reload change):
- HCV 3.3 log IU/mL: Not explicitly stated, but "5-10 cycles of reagent storage" and performance evaluated.
- HCV 7.0 log IU/mL: Not explicitly stated, but "5-10 cycles of reagent storage" and performance evaluated.
- Data Provenance: "prepared virus and armored RNA (aRNA) spiked into negative human serum matrix (BI0052)".
- Assay Verification (Flickering LED process control change):
- 381 field instruments and 445,910 unique test orders were analyzed from March 2018 to March 2022.
- Data Provenance: "field instruments" indicates real-world data, likely retrospective. Country of origin not specified.
- Additional Intended Use Testing (HCV Assay Software 5.2.5.1 vs 5.3.5.1):
- Negative clinical specimen: 1 (tested with 10 replicates per run).
- HCV RNA concentration around 3xLLoQ (30 IU/ml): 3 specimens (tested with 10 replicates of each per run).
- Total replicates per software version: (1 negative specimen * 10 replicates) + (3 positive specimens * 10 replicates) = 40 replicates. This was done for both software versions.
- Data Provenance: "clinical specimens", "negative clinical matrix panel", "low positive clinical positive panel". Samples prepared by diluting high positive sample in negative clinical matrix.
- Additional On-Board Stability (OBS) and Reagent Cycling:
- Minimum of one negative sample, a sample around 3xLLoQ (30 IU/ml), and a moderate positive or high positive sample.
- Minimum of 5 replicates per sample for each timepoint, with more replicates at baseline.
- Data Provenance: Not explicitly stated, but implies laboratory-controlled testing.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts
Not applicable. This device is a diagnostic assay for HCV RNA detection and quantitation, not an imaging device that requires interpretation by human experts. The ground truth would be based on the quantitative/qualitative results of the assay itself and reference standards/controls.
4. Adjudication Method for the Test Set
Not applicable. As noted above, this refers to a diagnostic assay. The "results" are numerical or qualitative (detected/not detected) from an automated system compared against established performance characteristics and controls.
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
Not applicable. This information is for an in-vitro diagnostic (IVD) assay, not an AI-powered medical imaging or decision support system that typically involves human readers.
6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done
Yes, the studies described are essentially standalone performance evaluations of the assay system (Aptima HCV Quant Dx Assay on the Panther system software). The device operates automatically from sample preparation through amplification, detection, and quantitation. The performance checks compare the output of the system against expected values, referent methods, or established criteria.
7. The Type of Ground Truth Used
The ground truth for the performance evaluations appears to be based on:
- Reference standards and controls: For quantitative and qualitative measurements (e.g., QC Control A, B, D, S, prepared virus and armored RNA, 3xLLoQ and negative samples).
- Previous established performance: Comparison of new software/hardware changes against the predicate device's established performance (PMA P160023).
- Root-cause analysis: For the flickering LED study, specific root-cause analysis was used to identify true faults.
8. The Sample Size for the Training Set
No training set information is provided as this is a diagnostic assay product, not a machine learning algorithm that requires a distinct training phase. The "development" or "calibration" of the assay would typically involve internal studies not detailed here, but these are not referred to as "training sets" in the context of conventional IVD development.
9. How the Ground Truth for the Training Set Was Established
Not applicable, as no training set is described.
§ 866.3170 Nucleic acid-based hepatitis C virus ribonucleic acid tests.
(a)
Identification. A nucleic acid-based hepatitis C virus (HCV) ribonucleic acid (RNA) test is identified as an in vitro diagnostic device intended for prescription use as an aid in the diagnosis of HCV infection in specified populations, and/or as an aid in the management of HCV-infected patients including guiding the selection of genotype-specific treatment in individuals with chronic HCV infection. The test is intended for use with human serum or plasma. The test is not intended for use as a donor screening test for the presence of HCV antibodies in blood, blood products, or tissue donors.(b)
Classification. Class II (special controls). The special controls for this device are:(1) For all nucleic acid-based HCV RNA tests, the labeling required under § 809.10(b) of this chapter must include:
(i) A prominent statement that the test is not intended for use as a donor screening test for the presence of HCV RNA from human cells, tissues, and cellular and tissue-based products.
(ii) A detailed explanation of the principles of operation and procedures for performing the assay.
(iii) A detailed explanation of the interpretation of results.
(iv) Limitations, which must be updated to reflect current clinical practice and disease presentation and management. These limitations must include, but are not limited to, statements that indicate:
(A) The specimen types for which the device has been cleared and that use of this test kit with specimen types other than those specifically cleared for this device may result in inaccurate test results.
(B) When applicable, that assay performance characteristics have not been established in populations of immunocompromised or immunosuppressed patients or, other populations where test performance may be affected.
(C) Test results are to be interpreted by qualified licensed healthcare professionals in conjunction with the individual's clinical presentation, history, and other laboratory results.
(2) For all nucleic acid-based HCV RNA tests, the design verification and validation must include:
(i) Detailed device description, including the device components, ancillary reagents required but not provided, and an explanation of the device methodology. Additional information appropriate to the technology must be included such as design of primers and probes, rationale for the selected gene targets, specifications for amplicon size, and degree of nucleic acid sequence conservation.
(ii) For devices with assay calibrators, the design and nature of all primary, secondary, and subsequent quantitation standards used for calibration as well as their traceability to a standardized reference material that FDA has determined is appropriate (
e.g., a recognized consensus standard). In addition, analytical testing must be performed following the release of a new lot of the standard material that was used for device clearance or approval, or when there is a transition to a new calibration standard.(iii) Documentation and characterization (
e.g., determination of the identity, supplier, purity, and stability) of all critical reagents (including nucleic acid sequences for primers and probes) and protocols for maintaining product integrity.(iv) Detailed documentation of analytical performance studies conducted as appropriate to the technology, specimen types tested, and intended use of the device, including, but not limited to, limit of detection (LoD), upper and lower limits of quantitation (ULoQ and LLoQ, respectively), linearity, precision, endogenous and exogenous interferences, cross reactivity, carryover, matrix equivalency, and sample and reagent stability. Samples selected for use in analytical studies or used to prepare samples for use in analytical studies must be from subjects with clinically relevant circulating genotypes in the United States. Cross-reactivity studies must include samples from HCV RNA negative subjects with other causes of liver disease, including autoimmune hepatitis, alcoholic liver disease, chronic hepatitis B virus, primary biliary cirrhosis, and nonalcoholic steatohepatitis, when applicable. The effect of each claimed nucleic-acid isolation and purification procedure on detection must be evaluated.
(v) Risk analysis and management strategies, such as Failure Modes Effects Analysis and/or Hazard Analysis and Critical Control Points summaries and their impact on test performance.
(vi) Final release criteria to be used for manufactured test lots with appropriate evidence that lots released at the extremes of the specifications will meet the claimed analytical and clinical performance characteristics as well as the stability claims.
(vii) Multisite reproducibility study that includes the testing of three independent production lots.
(viii) All stability protocols, including acceptance criteria.
(ix) Final release test results for each lot used in clinical studies.
(x) Analytical sensitivity and specificity of the test must be the same or better than that of other cleared or approved tests.
(xi) Lot-to-lot precision studies, as appropriate.
(3) For devices intended for the qualitative detection of HCV RNA, in addition to the special controls listed in paragraphs (b)(1) and (2) of this section, the design verification and validation must include detailed documentation of performance from a multisite clinical study. Performance must be analyzed relative to an FDA cleared or approved qualitative HCV RNA test, or a comparator that FDA has determined is appropriate. This study must be conducted using appropriate patient samples, with appropriate numbers of HCV positive and negative samples in applicable risk categories. Additional genotypes must be validated using appropriate numbers and types of samples. The samples may be a combination of fresh and repository samples, sourced from within and outside the United States, as appropriate. The study designs, including number of samples tested, must be sufficient to meet the following criteria:
(i) Clinical sensitivity of the test must have a lower bound of the 95 percent confidence interval of greater than or equal to 95 percent.
(ii) Clinical specificity of the test must have a lower bound of the 95 percent confidence interval of greater than or equal to 96 percent.
(4) For devices intended for the quantitative detection of HCV RNA, the following special controls, in addition to those listed in paragraphs (b)(1) and (2) of this section, apply:
(i) Labeling required under § 809.10(b) of this chapter must include a prominent statement that the test is not intended as a diagnostic test to confirm the presence of active HCV infection, when applicable.
(ii) Design verification and validation must include the following:
(A) Detailed documentation of the following analytical performance studies conducted as appropriate to the technology, specimen types tested, and intended use of the device, including but not limited to: LoD, ULoQ and LLoQ. LoD, LLoQ, and linearity studies must demonstrate acceptable device performance with all HCV genotypes detected by the device.
(B) Detailed documentation of clinical performance testing from either:
(
1 ) A multisite clinical study with an appropriate number of clinical samples from chronically HCV infected patients in which the results are compared to an FDA-cleared or approved quantitative HCV RNA test, or a comparator that FDA has determined is appropriate. This study must include a sufficient number of HCV positive samples containing an analyte concentration near the LLoQ to describe performance at this level. Clinical samples must cover the full range of the device output and must be consistent with the distribution of these genotypes in the U.S. population. Clinical samples may be supplemented with diluted clinical samples for those viral load concentrations that are not sufficiently covered by natural clinical specimens, or(
2 ) A clinical study with prospectively collected samples demonstrating clinical validity of the device.(C) Detailed documentation of a qualitative analysis near the lower end of the measuring range demonstrating acceptable performance when used as an aid in diagnosis.
(5) For devices intended for HCV RNA genotyping, in addition to the special controls listed in paragraphs (b)(1) and (2) of this section, design verification and validation must include the following:
(i) Detailed documentation of an analytical performance study demonstrating the LoD for all HCV genotypes detected by the device.
(ii) Detailed documentation, including results, of a multisite clinical study that assesses genotyping accuracy (
i.e., the proportion of interpretable results that match with the reference method result) and the genotyping rate (i.e., the proportion of results that were interpretable).(6) For any nucleic acid-based HCV RNA test intended for Point of Care (PoC) use, the following special controls, in addition to those listed in paragraphs (b)(1) and (2) of this section, apply:
(i) Clinical studies must be conducted at PoC sites.
(ii) Additional labeling must include a brief summary of the instructions for use that are appropriate for use in a PoC environment.