(116 days)
The illumigene® Pertussis DNA Amplification Assay, performed on the illumipro-10™, is a qualitative in vitro diagnostic test for the direct detection of Bordetella pertussis in human nasopharyngeal swab samples taken from patients suspected of having respiratory tract infection attributable to Bordetella pertussis.
The illumigene Pertussis assay utilizes loop-mediated isothermal DNA amplification (LAMP) technology to detect Bordetella pertussis by targeting the IS481 insertional element of the Bordetella pertussis genome. The IS481 insertional element can also be found in Bordetella holmesii and Bordetella bronchiseptica strains. Respiratory infection with Bordetella pertussis, Bordetella bronchiseptica may yield positive test results in IS481 assays. B. holmesii infection may cause clinical illness similar to B. pertussis, and mixed outbreaks involving both B. pertussis and B. holmesii infection have been reported. Additional testing should be performed if necessary to differentiate B. holmesii and B. pertussis. B. bronchiseptica is a rare cause of infection in humans. When clinical factors suggest that B. pertussis may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines.
Negative results for the illumigene Pertussis DNA Amplification Assay do not preclude Bordetella pertussis infection and positive results do not rule out co-infection with other respiratory pathogens. Results from the illumigene Pertussis assay should be used in conjunction obtained during the patient's clinical evaluation as an aid in diagnosis of Bordetella pertussis infection and should not be used as for treatment or other patient management decisions.
illumigene Pertussis is intended for use in hospital, reference or state laboratory settings. The device is not intended for point-of-care use.
The illumigene Molecular Diagnostic Test System is comprised of the illumigene® Pertussis DNA Amplification Assay Test Kit, the illumigene® Pertussis External Control Kit and the illumipro-10™ Automated Isothermal Amplification and Detection System.
The illumigene Pertussis assay utilizes loop-mediated isothermal amplification (LAMP) technology to detect the presence of Bordetella pertussis in human nasopharyngeal swab specimens. Each illumigene Pertussis assay is completed using an illumigene Assay Control Reagent containing Control material, an illumigene Sample Buffer, an illumigene Pertussis Test Device and Mineral Oil. Nasopharyngeal swab specimens are eluted with illumigene Sample Buffer. The illumigene Assay Control Reagent is added to the eluted sample and heat-treated. Target and Control DNA are made available for isothermal amplification via heat-treatment. The heat-treated Specimen/Control sample is added to the illumigene Test Device. Mineral oil is added to the illumigene Test Device to prevent evaporation. DNA amplification occurs in the illumigene Test Device.
The illumipro-10 heats each illumigene Pertussis Test Device containing prepared Sample and Control material, facilitating amplification of target DNA. When B. pertussis is present in the specimen, a 198 base pair sequence located within the IS481 insertional element of the B. pertussis genome is amplified and magnesium pyrophosphate is generated. Magnesium pyrophosphate forms a precipitate in the reaction mixture.
The illumipro-10 monitors the absorbance characteristics of the reaction solutions at the assay Run Start (Signal Initial or Si) and at the assay Run End (Signal final or Sf). The illumipro-10 calculates the ratio of the Run End (Signal final or Sf) reads with the Run Start (Signal Initial or Si) reads and compares the ratio to an established cut-off value. The illumipro-10 performs this ratio calculation to both the TEST chamber and the CONTROL chamber.
Fixed cut-off values for the CONTROL chamber are used to determine validity. CONTROL chamber Sf:Si ratios less than 90% are considered valid and allow for reporting of TEST chamber results (POSITIVE, NEGATIVE). CONTROL chamber Sf:Si ratios greater than or equal to 90% are considered invalid. Results are reported as 'INVALID'; Test chamber results are not reported. More stringent cut-off criteria are applied to the Control chamber reaction to ensure amplification is not inhibited, reagents are performing as intended and that sample processing was performed appropriately.
Fixed cut-off values for the TEST chamber are used to report sample results. TEST chamber Sf:Si ratios less than 82% are reported as 'POSITIVE'; TEST chamber Sf:Si ratios greater than or equal to 82% are reported as 'NEGATIVE'. Numerical values are not reported.
The illumigene Pertussis External Control Kit contains a Positive Control Reagent. The illumigene Assay Control/Negative Control Reagent provided in the illumigene Pertussis kit serves as the External Negative Control Reagent. External Control reagents are provided to aid the user in detection of reagent deterioration, adverse environmental or test conditions, or variance in operator performance that may lead to test errors. External Control reagents are provided for use in routine Quality Control testing.
illumigene® Pertussis DNA Amplification Assay - Acceptance Criteria and Study Summary
Here's an analysis of the illumigene® Pertussis DNA Amplification Assay's acceptance criteria and the study data presented:
1. Table of Acceptance Criteria and Reported Device Performance
The document does not explicitly state formal "acceptance criteria" with numerical thresholds for Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA). However, the device's performance is compared against a Composite Comparator Reference Method. Based on the clinical study results, the reported performance is:
| Acceptance Criteria (Implied) | Reported Device Performance (illumigene® Pertussis) |
|---|---|
| High PPA (Overall) | 87.8% (95% CI: 75.8 - 94.3%) |
| High NPA (Overall) | 97.8% (95% CI: 96.3 - 98.7%) |
Note: The document presents separate performance for prospective, retrospective (all comers), and retrospective (selected) samples. The "Overall" values for PPA and NPA (based on "Composite Method Comparator, All Comers" Total) are used here as the primary performance metrics from the clinical study.
2. Sample Size Used for the Test Set and Data Provenance
The test set refers to the clinical study samples used for performance evaluation.
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Sample Size: A total of 729 qualified nasopharyngeal (NP) swab specimens.
- Prospective: 508 specimens (69.7%)
- Retrospective: 221 specimens (30.3%) (includes "all comers" and "selected" retrospective samples)
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Data Provenance:
- Country of Origin: The samples were collected from four independent clinical test sites located in the Midwestern, Northern, and Eastern regions of the United States.
- Retrospective or Prospective: Both retrospective and prospective specimens were included in the clinical study.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications
The document does not specify a number of "experts" in the traditional sense (e.g., radiologists) for establishing ground truth. Instead, the ground truth was established using a Composite Comparator Reference Method:
- This method involved two manufacturer-validated, real-time PCR Assays (PCR1 and PCR2).
- These were followed by bi-directional sequencing.
- Qualifications (Implied): The PCR assays were "manufacturer validated," implying they were developed and verified by experts in molecular diagnostics. Bi-directional sequencing is a standard molecular biology technique, performed and interpreted by trained personnel.
4. Adjudication Method for the Test Set
The adjudication method for the ground truth was as follows:
- Sequential Adjudication (Implicit):
- Initial screening by two manufacturer-validated, real-time PCR Assays (PCR1 and PCR2), both targeting unique sequences within the IS481 insertional element.
- Specimens producing positive Bordetella pertussis results from either comparator PCR assay were then sent for bi-directional sequencing.
- Final Ground Truth Definition: Only samples that matched sequences within the Bordetella pertussis genome with pre-defined quality scores (PHRED20 and E-values) were considered true positives. Samples were considered negative when neither comparator PCR assay returned positive Bordetella pertussis results.
This can be seen as a form of "consensus with confirmation" or a multi-step adjudication process.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study
No. A Multi-Reader Multi-Case (MRMC) comparative effectiveness study, as typically understood for evaluating human reader performance with and without AI assistance, was not performed. This study evaluated the automated device's performance against a reference method, not against human readers.
6. Standalone Performance
Yes. The study primarily evaluated the standalone performance of the illumigene® Pertussis DNA Amplification Assay (algorithm only, with instrumentation for detection) without human-in-the-loop adjustments, beyond the initial sample preparation and loading. The results reported (PPA, NPA) directly reflect the device's accuracy in identifying Bordetella pertussis from nasopharyngeal swab samples.
7. Type of Ground Truth Used
The type of ground truth used was expert consensus / reference method based on molecular testing. Specifically:
- A Composite Comparator Reference Method was used.
- This involved two manufacturer-validated, real-time PCR Assays (PCR1 and PCR2).
- Confirmation of positive results was done via bi-directional sequencing with pre-defined quality scores.
This is a robust method leveraging highly sensitive and specific molecular techniques, which is considered a strong proxy for definitive diagnosis in this context.
8. Sample Size for the Training Set
The document does not specify a sample size for a "training set." This type of diagnostic assay, particularly for molecular tests, often relies on wet-lab optimization and development using characterized samples, rather than a distinct "training set" in the machine learning sense. The "Development optimization" section mentions "evaluation of characterized positive and negative clinical specimens" but without quantifying these for a training set.
9. How the Ground Truth for the Training Set Was Established
As no specific "training set" is described, the method for establishing its ground truth is also not explicitly stated. However, the "Assay cut-off" section mentions:
- "Development optimization includes evaluation of characterized positive and negative clinical specimens."
- "Amplification reagent concentrations are adjusted during design as needed to ensure illumigene results are aligned with clinical specimen reported results."
This implies that the assay's parameters (like cut-off values) were refined using characterized clinical specimens, presumably with their Bordetella pertussis status determined by established laboratory methods, potentially similar to or derivative of the Composite Comparator Reference Method used for the main clinical study, or other well-accepted diagnostic techniques.
§ 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.