(34 days)
The Pro hMPV™+ Assay is a Real-Time PCR (RT-PCR) in vitro diagnostic test for the qualitative detection of human Metapneumovirus (hMPV) nucleic acid isolated and purified from nasopharyngeal swab (NP) specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. This assay targets a highly conserved region of the Nucleocapsid gene of hMPV. The detection of hMPVnucleic acid from symptomatic patients aids in the diagnosis of human respiratory hMPV infection if used in conjunction with other clinical and laboratory findings. This test is not intended to differentiate the four genetic sub-lineages of hMPV.
Negative results do not preclude hMPV infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.
The Pro hMPV+ Assay enables detection human Metapneumovirus and Internal Control nucleic acid. Nasopharyngeal swab specimens are collected from patients with signs and symptoms of a respiratory infection using a polyester, rayon or nylon tipped swab and placed into viral transport medium.
An Internal Control (IC) is added to each sample prior to nucleic acid isolation to monitor for inhibitors present in the specimens. The isolation and purification of the nucleic acids is performed using either a MagNA Pure LC Instrument (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS easyMAGTM System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).
The purified nucleic acids are added to Pro hMPV+ Supermix along with enzymes included in the Pro hMPV+ Assay Kit. The Pro hMPV+ Supermix contains oligonucleotide primers and target-specific oligonucleotide probes. The primers are complementary to highly conserved regions of genetic sequences for these respiratory viruses. The probes are dual-labeled with a reporter dye attached to the 51-end and a quencher dye attached to the 3'-end.
Reverse transcription of the RNA in the sample into complementary DNA (cDNA) and subsequent amplification of DNA is performed in a Cepheid SmartCycler® II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The Pro hMPV+ 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. 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 SmartCyclerII instrument.
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1. Table of Acceptance Criteria and Reported Device Performance
The document compares the "New Pro hMPV+ Assay" (reformulated) to the "Current Pro hMPV+ Assay" (predicate device). The acceptance criteria are implied by the "Percent Positive Agreement" and "Percent Negative Agreement" with confidence intervals. While specific numerical acceptance criteria (e.g., "must be >90%") are not explicitly stated, the reported performance is presented as demonstrating substantial equivalence.
| Metric | Acceptance Criteria (Implied) | Reported Device Performance (New Pro hMPV+ Assay vs. Current Pro hMPV+ Assay) |
|---|---|---|
| Percent Positive Agreement | High agreement with predicate device for positive samples. | 100% (91.80%-100% 95% CI) |
| Percent Negative Agreement | High agreement with predicate device for negative samples. | 98.6% (94.91%-99.61% 95% CI) |
| Limit of Detection (LoD) | Comparable or improved LoD for hMPV strains. | Identical for hMPV A2 (10^2^ TCID50/mL), 0.5 log lower for hMPV B2 (10^0.5^ TCID50/mL). |
| Positive Control | Effective in detecting procedural errors (e.g., reagent absence). | Effective (no PC replicates detected in defective mixes). |
2. Sample Size Used for the Test Set and Data Provenance
- Sample Size (Clinical Comparison): 183 nasopharyngeal swab samples (one sample was excluded from the final analysis, resulting in 182).
- Data Provenance: Retrospective, collected during 2011-2012 from two sites: Milwaukee, WI, and Chicago, IL, USA.
3. Number of Experts Used to Establish Ground Truth for the Test Set and Qualifications of Those Experts
The document does not mention the use of experts to establish the ground truth for the clinical comparison study. Instead, the "ground truth" was established by:
- "True" hMPV positives: Defined as any sample that tested positive by the original Pro hMPV+ Assay.
- "True" hMPV negatives: Defined as any sample that tested negative by the original Pro hMPV+ Assay.
- Discrepant Analysis: For samples where the new and original assays disagreed, RT-PCR with hMPV specific primers targeting the hMPV phosphoprotein gene followed by bi-directional genetic sequencing was performed. The document does not specify who performed this analysis or their qualifications, but this would be a laboratory-based method.
4. Adjudication Method for the Test Set
The primary comparison was against the predicate device's results. For discrepancies, a molecular method (RT-PCR followed by bi-directional genetic sequencing) was used to resolve disagreements. This acts as a form of "adjudication" based on a more definitive molecular test, rather than human expert consensus.
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 reported. This study evaluates human reader performance, with or without AI assistance. The described study is a comparison of two in vitro diagnostic (IVD) assays.
6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done
Yes, the described clinical comparison study is a standalone assessment of the new IVD assay's performance against the predicate IVD assay. There is no human-in-the-loop component mentioned; it evaluates the assay's ability to detect hMPV directly from samples.
7. The Type of Ground Truth Used
The ground truth for the clinical comparison study was multi-faceted:
- Reference standard (initial): The results of the predicate device (original Pro hMPV+ Assay).
- Adjudication/Confirmatory method: For discrepant results, RT-PCR with hMPV specific primers targeting the hMPV phosphoprotein gene followed by bi-directional genetic sequencing was used, which can be considered a more definitive molecular ground truth.
8. The Sample Size for the Training Set
The document does not specify a separate training set. The study describes the re-formulation of an existing assay and its performance evaluation. Diagnostic assays like this typically undergo development and optimization phases (which might involve various "training" or optimization samples), but the clinical comparison details the final performance validation using a test set.
9. How the Ground Truth for the Training Set Was Established
As no specific training set is outlined in this document, the method for establishing its ground truth is not provided. The information focuses on the validation of the reformulated assay.
§ 866.3980 Respiratory viral panel multiplex nucleic acid assay.
(a)
Identification. A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B;
(2) Influenza A subtype H1 and Influenza A subtype H3;
(3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B;
(4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus;
(5) Human Metapneumovirus;
(6) Rhinovirus; and
(7) Adenovirus.
(b)
Classification. Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;”
(2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and
(3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.