(91 days)
The PrimeraDx ICEPlex® C. difficile Assay is for the qualitative detection of the Clostridium difficile toxin B gene (tcdB gene) in nucleic acids purified from unpreserved liquid or soft human stool specimens from patients suspected of having C. difficile infection (CDI).
The ICEPlex C. difficile Assay is intended to be used only on the ICEPlex® System, which integrates PCR-based amplification with capillary electrophoresis (CE) for the detection of amplification products. The assay is intended to aid in the diagnosis of CDI. Results should be considered in conjunction with patient clinical history.
PrimeraDx's ICEPlex C. difficile Assay kit is intended to be used only on the ICEPlex System. The ICEPlex C. difficile Assay kit incorporates several universal features and approaches developed at PrimeraDx for the ICEPlex System. The ICEPlex C. difficile Assay kit includes a PCR enzyme and the appropriate PCR buffer system developed, optimized, verified and validated for ideal performance in multiplex PCR with subsequent capillary electrophoresis. The ICEPlex C. difficile assay kit includes a primer mix for detection of the Clostridium difficile toxin B gene (tcdB gene) in human stool specimen from patients suspected of having C. difficile infection (CDI). The ICEPlex C. difficile assay kit is comprised of a PCR enzyme, primer mix, PCR buffer, calibrators mix, injection buffer, internal control, and a positive control.
The ICEPlex System combines two functional modules: an amplification module - PCR (Polymerase Chain Reaction) thermal cycler - and an analysis module -CE (Capillary Electrophoresis) system with fluorescent detection. Individual fluorescent PCR products from multiplexed PCR reactions are analyzed by CE through direct electrokinetic injection into the separating capillaries. The labeled amplicons are separated by size and the dyes are excited by two lasers within the system.
This document describes the ICEPlex C. difficile Assay Kit and its performance characteristics.
Here is the requested information:
1. Acceptance Criteria and Reported Device Performance
Clinical Performance (Comparison to Toxigenic C. difficile Direct Culture)
| Performance Metric | Acceptance Criteria (Implicit) | Reported Device Performance (95% CI) |
|---|---|---|
| Positive Percent Agreement (PPA) | High agreement with direct culture | 90.0% (84.5 - 94.1) |
| Negative Percent Agreement (NPA) | High agreement with direct culture | 97.4% (96.1 - 98.4) |
Analytical Performance
| Performance Metric | Acceptance Criteria (Implicit) | Reported Device Performance |
|---|---|---|
| Precision (Low Positive) | High percentage of correct results across different operators, lots, and instruments. (Implicitly >95% agreement where applicable) | 99% agreement |
| Precision (Moderate Positive) | High percentage of correct results across different operators, lots, and instruments. (Implicitly >95% agreement where applicable) | 100% agreement |
| Precision (Negative) | High percentage of correct results across different operators, lots, and instruments. (Implicitly >95% agreement where applicable) | 100% agreement |
| Reproducibility (Low Positive) | High percentage of correct results across different sites, days, and runs. (Implicitly >95% agreement where applicable) | 100% agreement |
| Reproducibility (Moderate Positive) | High percentage of correct results across different sites, days, and runs. (Implicitly >95% agreement where applicable) | 100% agreement |
| Reproducibility (Negative) | High percentage of correct results across different sites, days, and runs. (Implicitly >95% agreement where applicable) | 100% agreement |
| Analytical Sensitivity (LOD) | Lowest concentration at which at least 95% of replicates are positive. | C. difficile strain 43255 (Toxinotype 0): 8CFU/rxn C. difficile strain BAA-1805 (Toxinotype III): 2CFU/rxn |
| Analytical Reactivity | Detection of all tested toxigenic C. difficile strains. | All 20 tested toxigenic strains were positive. |
| Analytical Specificity (Cross Reactivity) | No cross-reactivity with common non-C. difficile microorganisms or healthy intestinal flora. | Clostridium sordellii cross-reacted; otherwise, no cross-reactivity. |
| Interfering Substances | No interference from common substances found in stool. | None of the tested substances interfered with detection or caused false positives. |
| Carryover/Contamination | No well-to-well or run-to-run contamination. | 0% False Positive rate (0 out of 141 valid negatives). |
2. Sample Size and Data Provenance
Test Set (Clinical Study):
- Sample Size: 969 compliant specimens were initially enrolled. 952 (98.2%) yielded reportable results and were included in the statistical analysis.
- Data Provenance: Clinical samples collected from patients suspected of C. difficile infection at three independent clinical sites. The data is prospective, as samples were collected for the investigational study. The country of origin is not explicitly stated but implied to be the US, given the 510(k) submission to the FDA.
Analytical Studies:
- Precision: 144 observations for each of the low positive, moderately positive, negative, and Hi/Low samples.
- Reproducibility: 90 observations for C20-80, moderate positive, low positive, and negative samples (across 3 sites * 5 days * 2 runs/day * 3 replicates). 60 observations for positive and negative controls.
- Limit of Detection (LOD): 20 replicates at each concentration level for two C. difficile strains.
- Analytical Reactivity: 20 additional toxigenic C. difficile strains tested.
- Cross Reactivity: Panel of 5 non-toxigenic C. difficile strains, 14 other Clostridium strains, and 54 other pathogens/intestinal flora.
- Interfering Substances: Multiple replicates for each of 14 interfering substances, tested with both negative and contrived positive samples (two C. difficile strains).
- Contamination: 6 runs, each with 24 high positive and 24 negative samples.
3. Number and Qualifications of Experts for Ground Truth
Clinical Study (Test Set):
The ground truth for the clinical study was established by toxigenic C. difficile direct culture. This is a laboratory-based method. The document does not specify the number of human experts involved in interpreting these cultures or their specific qualifications (e.g., medical microbiologists, lab technologists), as it's a standard laboratory procedure.
Discordant Analysis:
For samples with discordant results between the ICEPlex assay and direct culture, microbiological isolation and PCR targeting of 3 appropriate regions of the toxin B gene (different recognition sites than the ICEPlex assay) with bi-directional DNA sequencing were performed. This "truth standard" is a higher-level molecular characterization, implying expert interpretation of molecular results, but the specific number and qualifications of these experts are not provided.
4. Adjudication Method for the Test Set
The primary comparison for the clinical study was the ICEPlex C. difficile Assay versus toxigenic C. difficile direct culture.
For discordant results:
An adjudication method was used where samples showing disagreement between the ICEPlex assay and toxigenic C. difficile direct culture underwent discordant analysis. This analysis involved "microbiological isolation of and PCR targeting of 3 appropriate regions of the toxin B gene (Different recognition sites than the ones used in the ICEPlex C. difficile assay) with bi-directional DNA sequencing."
The results of this discordant analysis were then used to adjudicate the initial discrepancies. For example, of the 20 samples called positive by ICEPlex but negative by direct culture, 16 were confirmed positive by discordant analysis. Similarly, of the 17 samples called negative by ICEPlex but positive by direct culture, 14 were confirmed positive by discordant analysis. This suggests a multi-method adjudictation approach rather than a human expert consensus panel on the original results.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study
No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. This device is a diagnostic kit for laboratory use, and the study evaluates the performance of the device itself against a reference method (culture), not the impact of the device on human reader performance or a human-in-the-loop scenario. The "readers" in this context are the laboratory instruments and the analytical interpretation of their output.
6. Standalone (Algorithm Only) Performance Study
Yes, a standalone performance study was done. The entire clinical performance section evaluates the performance of the ICEPlex C. difficile Assay (algorithm/device only) against toxigenic C. difficile direct culture. There is no human interpretation component integrated into the listed performance metrics for the ICEPlex assay itself. The results presented (PPA, NPA) directly reflect the algorithm's diagnostic accuracy.
7. Type of Ground Truth Used for the Test Set
The primary ground truth used for the clinical test set was toxigenic C. difficile direct culture.
For discordant samples, an enhanced ground truth was established using an "adjudication" or "truth standard" method that involved:
- Microbiological isolation
- PCR targeting of 3 appropriate regions of the toxin B gene (different from the ICEPlex assay targets)
- Bi-directional DNA sequencing
8. Sample Size for the Training Set
The document does not explicitly state a sample size for a training set. For molecular diagnostic assays like this, the 'training' phase often refers to internal assay development, optimization, and analytical validation. The performance studies detailed (precision, reproducibility, LOD, clinical performance) represent the formal validation/test set. It is common for 510(k) submissions to focus on validation rather than explicitly detailing a distinct "training set" for the algorithm itself, as the assay design (primers, probes, CE interpretation rules) is pre-established during development.
9. How Ground Truth for the Training Set Was Established
As noted above, no explicit training set is detailed. However, during the development and optimization of such assays, ground truth for initial assay design and analytical studies would typically be established using:
- Known characterized C. difficile strains (ATCC strains, clinical isolates) at defined concentrations.
- Spiked samples where known amounts of the target (C. difficile) are added to negative stool matrices.
- Reference laboratory methods (e.g., culture, established PCR methods, sequencing) to confirm the presence or absence of the target.
These methods are evident in the analytical performance studies (e.g., ATCC strains for precision, spiked samples for LOD and inclusivity).
§ 866.3130 Clostridium difficile toxin gene amplification assay.
(a)
Identification. AClostridium difficile toxin gene amplification assay is a device that consists of reagents for the amplification and detection of target sequences inClostridium difficile toxin genes in fecal specimens from patients suspected of havingClostridium difficile infection (CDI). The detection of clostridial toxin genes, in conjunction with other laboratory tests, aids in the clinical laboratory diagnosis of CDI caused byClostridium difficile. (b)
Classification. Class II (special controls). The special controls are set forth in FDA's guideline document entitled: “Class II Special Controls Guideline: Toxin Gene Amplification Assays for the Detection ofClostridium difficile; Guideline for Industry and Food and Drug Administration Staff.” See § 866.1(e) for information on obtaining this document.