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The xTAG™ Respiratory Viral Panel (RVP) is a qualitative nucleic acid multiplex test intended for the simultaneous detection and identification of multiple respiratory virus nucleic acids in nasopharyngeal swabs from individuals suspected of respiratory tract infections. The following virus types and subtypes are identified using RVP: Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza B, Respiratory Syncytial Virus subtype A, Respiratory Syncytial Virus subtype B, Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus, Human Metapneumovirus, Rhinovirus, and Adenovirus. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection if used in conjunction with other clinical and laboratory findings. It is recommended that specimens found to be negative after examination using RVP be confirmed by cell culture. Negative results do not preclude respiratory virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.

Positive results do not rule out bacterial infection, or co-infection with other viruses. The agent detected may not be the definite cause of disease. The use of additional laboratory testing (e.g. bacterial culture, immunofluorescence, radiography) and clinical presentation must be taken into consideration in order to obtain the final diagnosis of respiratory viral infection.

Due to seasonal prevalence, performance characteristics for Influenza A/H1 were established primarily with retrospective specimens.

The RVP assay cannot adequately detect Adenovirus species C, or serotypes 7a and 41. The R VP primers for detection of rhinovirus cross-react with enterovirus. A rhinovirus reactive result should be confirmed by an alternate method (e.g. cell culture).

Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation. When other Influenza A viruses are emerging, performance characteristics may vary. If infections 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 a 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 xTAG™ RVP is a PCR-based system for detecting the presence / absence of viral DNA / RNA in clinical specimens. The oligonucleotide primer / probe components of the xTAGTM RVP have been designed to specifically target unique regions in the RNA / DNA of each molecular species listed in the following Table:

Respiratory viral targets: Influenza A (Matrix Gene), Influenza A H1 (Hemagglutinin Gene), Influenza A H3 (Hemagglutinin Gene), Influenza B, Respiratory Syncytial Virus Type A, Respiratory Syncytial Virus Type B, Parainfluenza virus 1, Parainfluenza virus 2, Parainfluenza virus 3, Human Metapneumovirus, Rhinovirus, and Adenovirus.

Amplified products are sorted and analyzed on the Luminex® xMAP instrument, which generates signals based on the acquisition of spectrofluorometric data. The raw signals are median fluorescence intensities (MFI) which are acquired in a Luminex® Output.csv file that is subsequently analyzed by the software component of the xTAG™ RVP to establish the presence or absence of all viral types / subtypes for which a Luminex® microsphere population has been dedicated. The xTAG™ RVP primary components are:

1. PCR Primer Mix.
2. Target Specific (TS) Primer Mix.
3. Coupled Bead Mix.
4. Data Analysis Software.

Other reagents required to perform testing with the device include ancillary reagents for which specific lots have been qualified by Luminex Molecular Diagnostics (LMD) and incorporated in the LMD quality system, for use with the xTAG™ RVP.

The xTAG™ RVP has been designed to generate unique PCR products for each of the targets described above with the exception of RSV targets. RSV subtypes detected by the xTAG™ RVP are discriminated at the TSPE step. The discrimination of Parainfluenza subtypes occurs at both the PCR and TSPE step. The detection of Influenza A subtypes is achieved by amplifying conserved regions of the matrix gene common to all subtypes and target specific regions of the hemagglutinin gene (2 sets of PCR primers for the 2 listed subtypes).

Here's a breakdown of the acceptance criteria and the study details for the xTAG™ RVP (Respiratory Viral Panel) Multiplex Nucleic Acid Detection Assay, extracted and organized from the provided text:

Acceptance Criteria and Device Performance

The acceptance criteria for this device are implicitly tied to the performance metrics shown in the clinical and analytical studies, primarily Sensitivity and Specificity against comparator methods. The "Call zones" (MFI ≥ 300 for positive, < 150 for negative, 150-299 for no call) define the device's operational threshold.

Table of Acceptance Criteria and Reported Device Performance (Clinical Sensitivity and Specificity from Prospectively Collected Specimens):

Note: For Adenovirus, the clinical study noted that low overall sensitivity was mainly due to poor detection of serotypes within the Adenovirus C species.
Note: For hMPV, performance against PCR followed by sequencing as a sole comparator yielded 100.0% PPA (22/22) and 98.2% NPA (321/327).

Study Details

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

   • Prospective Clinical Study: 544 prospectively collected nasopharyngeal (NP) swabs.
     • Provenance: Collected and tested during the 2005/06 influenza season at 4 North American clinical laboratories.
   • Supplemental Pre-selected Clinical Dataset: 164 clinical specimens (NP swabs) to supplement analytes with low prevalence (147 for H1, Parainfluenza 1, 2, 3; an additional 17 for Para2).
     • Provenance: Pre-selected and tested at 2 of the 4 clinical sites mentioned above.
   • Fresh vs. Frozen Performance Study: 163 human pediatric NP swabs.
     • Provenance: Collected and tested at a 5th clinical site (not enrolled in the main prospective study).

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

   • The document implies the use of standard laboratory methods and well-characterized techniques as ground truth, rather than relying on a specific number of human experts for adjudication in the traditional sense of image interpretation.
   • Comparator methods used for ground truth:
     • Viral culture and/or DFA: For Influenza A, Influenza B, RSV, Parainfluenza 1, 2, 3, and Adenovirus. These are established laboratory methods performed by qualified personnel.
     • Well-characterized RT-PCR amplification followed by bidirectional sequencing: For Influenza A subtyping, RSV subtyping, hMPV, and Rhinovirus. This indicates molecular biology experts.
     • Composite analysis using viral culture, DFA, and RT-PCR amplification/sequencing: For hMPV.
     • CDC assay (surveillance program): For 9 initially untypeable Flu A specimens. This refers to public health laboratory experts following established protocols.
   • The specific number and qualifications (e.g., years of experience as a radiologist) of the individuals performing these comparator methods are not explicitly detailed in the provided text.

3. Adjudication Method for the Test Set:

   • The primary method was a comparison against established, well-characterized laboratory methods (e.g., viral culture, DFA, RT-PCR with sequencing).
   • For hMPV, a "composite analysis" of multiple comparator methods was used, suggesting an adjudication process to arrive at a final ground truth.
   • For untypeable Flu A specimens, the CDC assay provided the comparator result, implying that this external reference served as the adjudicator.
   • No explicit "2+1" or "3+1" expert adjudication method is described for this type of diagnostic assay, as ground truth is typically by reference to established, independent laboratory tests rather than subjective expert consensus.

4. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study:

   • No. This type of study (MRMC) is typically performed for devices that involve human interpretation, such as imaging AI, to compare human reader performance with and without AI assistance. The xTAG™ RVP is a standalone laboratory assay without a human-in-the-loop component for result interpretation, thus an MRMC study is not applicable.

5. Standalone Performance (Algorithm Only without Human-in-the-Loop Performance):

   • Yes. The entire submission describes the standalone performance of the xTAG™ RVP assay. The device generates qualitative results (POS, NEG, No Call) based on Median Fluorescence Intensity (MFI) values and analysis algorithms, without requiring human interpretation of raw signals to determine the presence or absence of a virus. The output of the test is directly displayed by the Data Analysis Software (TDAS RVP-I).

6. Type of Ground Truth Used:

   • Established Laboratory Methods / Molecular Sequencing:
     • Viral culture
     • DFA (Direct Fluorescent Antibody)
     • Well-characterized RT-PCR amplification followed by bidirectional sequencing (for specific analytes including subtyping)
     • CDC assay for particular influenza cases.
   • This is a combination of direct detection (culture, DFA) and molecular confirmation (PCR & sequencing), considered highly reliable "gold standards" for viral identification.

7. Sample Size for the Training Set:

   • The document primarily describes analytical and clinical verification and validation studies. It does not explicitly state a "training set" sample size in the context of machine learning. The assay development and optimization (which can be considered analogous to training) would have used various laboratory-prepared materials and possibly internal pilot clinical samples.
   • The reproducibility section discusses studies using simulated samples and various viral strains to establish assay performance characteristics near cut-offs and at clinical concentrations. These studies use numbers like:
     • 54 replicates per viral analyte (for precision near cut-offs at 3 sites over 3 days, 2 extractions per day).
     • 6 replicates per extract (for reproducibility at clinical concentrations).
     • Numerous viral reference strains for analytical reactivity and cross-reactivity studies.
   • However, these are more akin to test sets for analytical validation rather than a distinct "training set" for an algorithm in the modern AI sense. The assay relies on predefined MFI cut-offs.

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

   • Since there isn't a "training set" in the typical AI sense, the ground truth for optimizing the assay's performance parameters (e.g., determining the MFI cut-offs of 150 and 300) would have been established through a combination of:
     • Analytical studies: Using characterized viral strains at known concentrations (e.g., TCID50/mL), often acquired from reputable sources like ATCC or in-house strains.
     • Empirical determination: The document states "The universal "Call zones" established in the RVP assay has been defined empirically using clinical specimens..." This implies an iterative process involving testing clinical and analytical samples and optimizing the MFI thresholds to achieve desired sensitivity and specificity.
     • ROC curve analyses: Mentioned as being used to demonstrate diagnostically relevant sensitivity and specificity for the established "Call zones." This is a statistical method to evaluate classifier performance and optimize thresholds.

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