(22 days)
The ProFAST™+ Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the qualitative detection and discrimination of seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza viral nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens from human patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. This assay targets conserved regions of the Hemagglutinin (HA) gene for seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza Virus, respectively. This assay is not intended to detect Influenza B or Influenza C Viruses.
A negative ProFAST+ Assay result is a presumptive negative result for Influenza A. These results should be confirmed by an FDA cleared nucleic acid-based test (NAT) detecting Influenza A.
Negative results do not preclude Influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.
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 department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
The ProFAST+ Assay enables detection and discrimination of Influenza A Virus subtypes: seasonal H1, seasonal H3, and 2009 H1N1.
An overview of the procedure is as follows:
- Collect nasopharyngeal swab specimens from symptomatic patients using a polyester, ravon, or nylon tipped swab and place into viral transport medium (refer to Materials Required but not Provided section of this Instruction for Use).
- Add an Internal Control (IC) to every sample to monitor for inhibitors present in the specimens.
- Perform isolation and purification of nucleic acids using a MagNA Pure LC Instrument (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS easyMAG System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).
- Add purified nucleic acids to the ProFAST+ Supermix along with enzymes included in the ProFAST+ kit. The ProFAST+ Supermix contains target-specific oligonucleotide primers and probes. The primers are complementary to conserved regions of the Hemagglutinin (HA) gene for seasonal influenza A/H1, seasonal influenza A/H3 and 2009 H1N1 Influenza Virus (swine-origin), respectively. The probes are dual-labeled with a reporter dye and a quencher dye (see table below).
- Perform reverse transcription of RNA into complementary DNA (cDNA) and subsequent amplification of DNA in a Cepheid SmartCycler II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProFAST+ Assay is based on Taqman chemistry, which utilizes the 5' - 3' exonuclease activity of the Taq polymerase to cleave the probe thus separating the reporter dye from the quencher. This generates an increase in fluorescent signal upon excitation from a light source. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification products present at that time. Fluorescent intensity is monitored during each PCR cycle by the real-time instrument.
Here's a summary of the acceptance criteria and study details for the ProFAST+ Assay based on the provided document:
1. Table of Acceptance Criteria and Reported Device Performance
The acceptance criteria are implied by the "Positive Percent Agreement" and "Negative Percent Agreement" values, along with their 95% Confidence Intervals. For the reproducibility study, the acceptance criterion is an "overall percent agreement with the expected result" of 99.7%.
| Analyte (Clinical Performance - Prospective Study) | Acceptance Criteria (Implied) | Reported Device Performance (95% CI) |
|---|---|---|
| Seasonal Influenza A/H1 (Positive) | High Positive Percent Agreement | 100.0% (93.2% - 100.0%) |
| Seasonal Influenza A/H1 (Negative) | High Negative Percent Agreement | 99.0% (98.0% - 99.5%) |
| Seasonal Influenza A/H3 (Positive) | High Positive Percent Agreement | 100.0% (86.7% - 100.0%) |
| Seasonal Influenza A/H3 (Negative) | High Negative Percent Agreement | 99.6% (98.9% - 99.9%) |
| 2009 H1N1 Influenza (Positive) | High Positive Percent Agreement | 95.4% (87.3% - 98.4%) |
| 2009 H1N1 Influenza (Negative) | High Negative Percent Agreement | 100.0% (99.5% - 100.0%) |
| Analyte (Clinical Performance - Retrospective Study) | Acceptance Criteria (Implied) | Reported Device Performance (95% CI) |
| Seasonal Influenza A/H1 (Positive) | High Positive Percent Agreement | 94.4% (74.3% - 99.0%) |
| Seasonal Influenza A/H1 (Negative) | High Negative Percent Agreement | 99.3% (96.1% - 99.9%) |
| Seasonal Influenza A/H3 (Positive) | High Positive Percent Agreement | 100.0% (94.9% - 100.0%) |
| Seasonal Influenza A/H3 (Negative) | High Negative Percent Agreement | 100.0% (95.8% - 100.0%) |
| 2009 H1N1 Influenza (Positive) | High Positive Percent Agreement | 100.0% (86.7% - 100.0%) |
| 2009 H1N1 Influenza (Negative) | High Negative Percent Agreement | 100.0% (97.2% - 100.0%) |
| Reproducibility | Overall Percent Agreement ≥ 99.7% with expected result | 99.7% (628/630) (95% CI: 98.8% - 99.9%) |
2. Sample Size Used for the Test Set and Data Provenance
- Prospective Study:
- Sample Size: 842 eligible prospective NP swab specimens (initially 874, but 32 were excluded).
- Data Provenance: U.S. clinical laboratories (4 sites), collected between December 2009 and May 2010. Also included prospectively collected archived samples (from January-March, 2008, February-March, 2009, and October-November, 2009) due to absence of seasonal influenza during the 2009-2010 winter season. These represented "excess, remnants of nasopharyngeal (NP) swab specimens that were prospectively collected from symptomatic individuals suspected of respiratory infection, and were submitted for routine care or analysis by each site, and that otherwise would have been discarded."
- Retrospective Study:
- Sample Size: 160 retrospective nasopharyngeal (NP) swab samples.
- Data Provenance: Two clinical sites, collected from January-March, 2008, January-November 2009, and March 2010.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts
The document does not specify the number or qualifications of experts involved in establishing the ground truth. The ground truth was established by a "composite comparator/reference method."
4. Adjudication Method for the Test Set
The document does not describe an explicit adjudication method between human experts, as the ground truth was established by a composite of laboratory tests rather than expert consensus on interpretation. It mentions that samples were "considered as any sample that was tested positive for Influenza A by the ProFlu+ Assay, and had bidirectional sequencing data meeting pre-defined quality acceptance criteria."
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done
No, a multi-reader multi-case (MRMC) comparative effectiveness study was not performed. This is a diagnostic test (RT-PCR assay) intended for standalone use, not an imaging device requiring human reader interpretation, so this type of study would not be applicable.
6. If a Standalone Study Was Done
Yes, a standalone study (algorithm/device-only performance without human-in-the-loop performance) was done. The ProFAST+ Assay's performance was compared directly against a composite reference method. The clinical performance data (Positive and Negative Percent Agreements) reflect the standalone performance of the device.
7. The Type of Ground Truth Used
The ground truth used was a composite comparator/reference method consisting of:
- FDA cleared ProFlu+ Assay (another nucleic acid-based test for Influenza A).
- Individual well-characterized Influenza A subtype-specific RT-PCR assays.
- Bi-directional sequencing (targeting different regions of the hemagglutinin gene than the ProFAST+ Assay).
"True" positives required:
- Positive for Influenza A by ProFlu+ Assay
- And bidirectional sequencing data meeting pre-defined quality acceptance criteria, matching seasonal A/H1, A/H3, or A/2009 H1N1 sequences in the NCBI GenBank database with acceptable E-values.
"True" negatives required:
- Negative for Influenza A by ProFlu+ Assay
- OR positive for Influenza A by ProFlu+ Assay but negative by the respective Influenza A subtype-specific RT-PCR assay followed by bi-directional sequencing.
8. The Sample Size for the Training Set
The document does not explicitly state the sample size used for a training set. The clinical performance studies describe the evaluation of the device using prospective and retrospective samples, implying these are test sets rather than training sets. Typically for an in vitro diagnostic (IVD) assay like this, development and optimization (which might use internal "training-like" datasets) are usually completed before the formal clinical validation described in the 510(k) summary.
9. How the Ground Truth for the Training Set Was Established
Since a distinct "training set" is not explicitly mentioned as part of this premarket notification validation, the method for establishing its ground truth is not detailed. For typical IVD development, internal positive and negative controls, spiked samples, and characterized clinical samples would be used during development and optimization phases.
§ 866.3332 Reagents for detection of specific novel influenza A viruses.
(a)
Identification. Reagents for detection of specific novel influenza A viruses are devices that are intended for use in a nucleic acid amplification test to directly detect specific virus RNA in human respiratory specimens or viral cultures. Detection of specific virus RNA aids in the diagnosis of influenza caused by specific novel influenza A viruses in patients with clinical risk of infection with these viruses, and also aids in the presumptive laboratory identification of specific novel influenza A viruses to provide epidemiological information on influenza. These reagents include primers, probes, and specific influenza A virus controls.(b)
Classification. Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Reagents for Detection of Specific Novel Influenza A Viruses.” See § 866.1(e) for information on obtaining this document.
(2) The distribution of these devices is limited to laboratories with experienced personnel who have training in standardized molecular testing procedures and expertise in viral diagnosis, and appropriate biosafety equipment and containment.