LogoFDA 510(k) Search
K Number
K123355
Device Name
AMPLIVUE C. DIFFICILE ASSAY
Manufacturer
QUIDEL CORPORATION
Date Cleared
2012-12-13

(43 days)

Product Code
OZN,DAT
Regulation Number
866.3130
Type
Traditional
Panel
MI
Reference & Predicate Devices
K113358
AI/MLSaMDIVD (In Vitro Diagnostic)TherapeuticDiagnosticis PCCP AuthorizedThirdpartyExpeditedreview
Intended Use

The AmpliVue™ C. difficile Assay is an in vitro diagnostic test for the direct, qualitative detection of the Clostridium difficile Toxin A gene (tcdA) in unformed stool specimens of patients suspected of having Clostridium difficile-associated disease (CDAD). The AmpliVue™ C. difficile Assay is intended for use as an aid in diagnosis of CDAD. The assay utilizes helicase-dependent amplification (HDA) for the amplification of a highly conserved fragment of the Toxin A gene sequence and a self-contained disposable amplicon device that allows for manual evaluation of assay results.

Device Description

The AmpliVue™ C. difficile Assay combines simple specimen processing, an isothermal amplification technology named helicase-dependent amplification (HDA), and a self- contained disposable amplicon detection device for the detection of Clostridium difficile directly from CDAD-suspected stool specimens. A swab is used to transfer a small amount of specimen into a dilution tube. The diluted sample is then transferred into a sample lysis buffer tube, and the cells are lysed by simple heat treatment. After heat treatment, an aliquot of the lysed sample is added to a reaction tube containing a lyophilized mix of HDA reagents, including primers specific for the amplification of a fragment from the conserved 5' end of the tcdA gene that are located within the PaLoc of toxigenic C. difficile strains. The assay also includes a process control to monitor specimen processing and to confirm the integrity of the assay reagents and cassette detection, as well as to assess for HDA-inhibitors that may be present within the stool specimen. Competitive amplification of the process control DNA also takes place unless inhibitory substances are present or the specimen processing fails. The HDA reaction is asymmetric so that an excess of single stranded DNA is formed from a biotinylated primer present within the reaction mix. The capture probes for each amplicon bind to the corresponding biotinylated single-stranded DNA forming dual labeled amplicons. After completion of the HDA reaction, the reaction tube is transferred to a single-use, disposable cassette for detection. The self-contained cassette is comprised of two individual components: an amplicon cartridge that holds the running buffer and the single 0.2-ml thin wall reaction tube containing the amplified product. The detection chamber houses the amplicon cartridge and a vertical-flow DNA detection strip embedded into the cassette. The DNA detection strip is coated with an anti-FITC antibody and an anti-DNP antibody that serve as the C. difficile test (T2) line and the control (C) line in the assay, respectively. A razor blade and a plastic pin located at the bottom of the detection chamber opens the HDA reaction tube and the running buffer bulb when the handle of the detection chamber is closed. The mixture flows through a fiberglass paper connected to the DNA detection strip that contains a fiberglass pad pre-loaded with streptavidin-conjugated color particles for color visualization. The dual-labeled probe-amplicon hybrid is then detected by the lateral flow strip within the cassette. The bottom line captures the FITC-labeled probe-amplicon and the top line captures the DNPlabeled probe-amplicon. The biotin label attracts the streptavidin-conjugated color particles for visualization and the test result is shown as colored lines visible to the naked eye. Detection of C. difficile DNA is reported when the T2 and C lines are visible through the detection window of the cassette. No detection of C. difficile DNA is reported when only the C line is displayed. The assay is regarded as unresolved when neither line is displayed. The whole procedure takes approximately 75 minutes (Note: The T1 line present on the self-contained cassette is for a triplex assay and is not applicable/used for the AmpliVue C. difficile Assay).

AI/ML Overview

The Quidel AmpliVue™ C. difficile Assay is a qualitative in vitro diagnostic test for the direct detection of the Clostridium difficile Toxin A gene (tcdA) in unformed stool specimens from patients suspected of having Clostridium difficile-associated disease (CDAD). It uses helicase-dependent amplification (HDA) for gene amplification and a manual read of assay results.

Acceptance Criteria and Device Performance:

The document does not explicitly present specific, quantitative acceptance criteria for each performance characteristic in a dedicated table format. However, it provides performance data against which an implicit set of criteria would have been assessed by the regulatory body.

Based on the provided data, the following table summarizes the device's performance, which was implicitly accepted as meeting the regulatory requirements for substantial equivalence. The "Acceptance Criteria" column reflects what would generally be expected for an IVD device of this type, especially given the "Overall Percent Agreement" and 95% Confidence Intervals reported.

Performance CharacteristicImplicit Acceptance Criteria (Expected/Common)Reported Device Performance
Precision (Within-Lab)High agreement (e.g., ≥95%) for positive and negative controls/samples. Specific high negative agreement often lower but still acceptable within CI.C. difficile High Negative: 0% (0/48) overall agreement (CI: 0% - 8%)
C. difficile Low Positive: 100% (48/48) overall agreement (CI: 91.2% - 100%)
C. difficile Moderate Positive: 100% (48/48) overall agreement (CI: 91.2% - 100%)
Negative: 100% (48/48) overall agreement (CI: 91.2% - 100%)
C. difficile Positive Control: 100% (48/48) overall agreement (CI: 91.2% - 100%)
Assay Negative Control: 100% (48/48) overall agreement (CI: 91.2% - 100%)
Reproducibility (Multi-Lab)High agreement (e.g., ≥95%) for positive and negative controls/samples. Specific high negative agreement often lower but still acceptable within CI.C. difficile High Negative: 80% (72/90) overall agreement (CI: 71% - 87%)
C. difficile Low Positive: 100% (89/89) overall agreement (CI: 95% - 100%)
C. difficile Moderate Positive: 100% (90/90) overall agreement (CI: 95% - 100%)
Negative: 100% (90/90) overall agreement (CI: 95% - 100%)
C. difficile Positive Control: 100% (90/90) overall agreement (CI: 95% - 100%)
Assay Negative Control: 100% (90/90) overall agreement (CI: 95% - 100%)
Analytical Sensitivity (LoD)LoD defined at 95% positivity for target strains.ATCC 43255 (Toxinotype 0): 4.2 CFU/Assay
CCUG 8864 (Toxinotype X): 0.7 CFU/Assay
Final Assay LoD: 4.2 CFU/Assay
Analytical Reactivity (Inclusivity)Detection of a broad range of toxigenic C. difficile strains at specified concentrations.Detected 24 toxigenic strains (2-3x LoD) across 7 toxinotypes (0, Illb, IIIc, IV, V, VIII, XXIII).
Analytical Specificity (Cross-Reactivity)No cross-reactivity with common enteric pathogens/flora/human DNA at medically relevant concentrations.No cross-reactivity with 66 bacterial, viral, and yeast microorganisms (at >1.0E+06 CFU/mL or >1.0E+05 PFU/mL) and human DNA. In silico analysis showed no predicted cross-reactivity for C. botulinum.
Analytical Specificity (Interfering Substances)No interference from common substances found in stool specimens at medically relevant concentrations.None of 34 tested substances (at medically relevant concentrations) interfered with the assay.
Method Comparison (vs. Tissue Culture Cytotoxicity Assay)Performance comparable to predicate/gold standard, with acceptable sensitivity and specificity.Sensitivity: 93.6% (95% CI: 87.3% - 96.9%)
Specificity: 94.1% (95% CI: 92.1% - 95.6%)
PPV: 70.3% (95% CI: 62.5% - 77.2%)
NPV: 99.0% (95% CI: 97.9% - 99.5%)

Study Information:

  1. Sample sizes used for the test set and data provenance:

    • Precision (Within-Lab): 4-member panel (C. difficile positive and negative) tested for 13 days, twice a day by 2 operators. Each category (e.g., C. difficile Low Positive) had 48 results (24 per operator). The samples were artificially prepared by spiking in C. difficile into negative fecal matrix (indicated by "C. difficile High Negative (T2-/C+; 20-80%)", "C. difficile Low Positive (T2+/C+, ≥95%)", etc.). Data provenance is implied to be laboratory-based (in-house).
    • Reproducibility (Multi-Lab): Blinded and randomized panel of Clostridium difficile negative and positive samples. Each site tested the panel and assay controls for 5 days in triplicate by 2 operators. Each category (e.g., C. difficile High Negative) had 90 results (30 per site). The samples were artificially prepared by spiking in C. difficile into negative fecal matrix. Data provenance involved 3 test sites, two of which were clinical sites, within the United States.
    • Analytical Sensitivity (LoD): Quantified cultures of two C. difficile strains (ATCC 43255, CCUG 8864) serially diluted in negative fecal matrix. Replicates were tested to determine 95% positivity. The exact number of replicates is not stated, but typically 20+ replicates are used per concentration level. Data provenance is implied to be laboratory-based (in-house).
    • Analytical Reactivity (Inclusivity): 24 toxigenic strains tested at 2 to 3x LoD. Data provenance: Strains originated from at least five states (USA) and four countries (USA, Belgium, France, Sweden).
    • Analytical Specificity (Cross-Reactivity & Interfering Substances): 66 microorganisms and human DNA for cross-reactivity; 34 substances for interference. Data provenance is laboratory-based (in-house).
    • Method Comparison: 840 unformed stool samples tested. Data provenance: Four geographically diverse locations within the United States, collected prospectively between January 2012 and October 2012.

  2. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:

    • For the analytical studies (precision, reproducibility, LoD, inclusivity, specificity), the ground truth was established by the preparation methods (e.g., known positive/negative samples, spiked samples, quantified cultures). No external experts were used for these "test sets".
    • For the Method Comparison study, the Tissue Culture Cytotoxicity Assay served as the comparator method. While interpretations of this assay might involve expert judgment, the document doesn't specify an expert panel for ground truth determination, as the cytotoxin assay itself is considered a gold standard or a highly reliable reference method for clinical samples in this context. The results from the FDA-cleared molecular device, used to resolve discordant cases, would also rely on the performance characteristics of that device rather than expert consensus on individual cases.

  3. Adjudication method for the test set:

    • Discordant Analysis (Method Comparison Study): For the 835 clinical specimens analyzed by the AmpliVue C. difficile Assay (device) and Tissue Culture Cytotoxicity Assay (comparator):
      • 43 discordant specimens (AmpliVue Positive/Tissue Culture Cytotoxin Negative): 37 were positive for C. difficile by an FDA-cleared molecular device, and 6 were negative.
      • 7 discordant specimens (AmpliVue Negative/Tissue Culture Cytotoxin positive): 2 were positive for C. difficile by an FDA-cleared molecular device, and 5 were negative.
    • This represents a form of 2+1 adjudication, where results from the two primary methods (device and comparator) are adjudicated by a third, FDA-cleared molecular method in cases of discordance.

  4. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

    • A MRMC comparative effectiveness study was not performed. This device is a standalone in vitro diagnostic assay with a manual read of results. It does not involve human readers interpreting an AI output or benefiting from AI assistance. The "manual read" refers to visualizing colored lines on a cassette, not a complex interpretation task that would typically involve radiologists or other medical imaging specialists.

  5. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:

    • Yes, the performance data presented (analytical and method comparison) represents the standalone performance of the AmpliVue™ C. difficile Assay. Although it has a "manual read" component (visual interpretation of lines), the assay itself is a test-tube-to-cassette system without a human interpretation loop in the way described for AI-assisted diagnostics. The reported sensitivity and specificity values are for the algorithm (the assay) only, reflecting its direct detection capability.

  6. The type of ground truth used (expert consensus, pathology, outcomes data, etc.):

    • Analytical Studies (Precision, Reproducibility, LoD, Inclusivity, Specificity): Ground truth was based on the known composition of spiked samples or characterized bacterial strains.
    • Method Comparison Study: The Tissue Culture Cytotoxicity Assay served as the primary reference standard (ground truth). For discordant results, an FDA-cleared molecular device was used as the adjudicator for confirmatory ground truth.

  7. The sample size for the training set:

    • The document does not explicitly mention a "training set" in the context of machine learning. This is a traditional in vitro diagnostic device, not an AI/ML-based device. Therefore, the concept of a training set for an algorithm is not directly applicable here. The assay's parameters (e.g., primer sequences, HDA conditions) would have been developed and optimized through iterative laboratory R&D, rather than a formal machine learning training approach on a large dataset.

  8. How the ground truth for the training set was established:

    • As noted above, there is no explicit "training set" as understood in AI/ML. The development and optimization of the assay's components, such as primer design and reaction conditions, would have been based on established scientific principles in molecular diagnostics and empirical testing against characterized strains and clinical samples to achieve desired performance, not via ground truth establishment for an AI training set.

§ 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.