DIAGNOSTIC HYBRIDS' D3 DFA CHLAMYDIAE CULTURE CONFIRMATION KIT by DIAGNOSTIC HYBRIDS, INC.

The Diagnostic Hybrids' D3 DFA Chlamydiae Culture Confirmation Kit is intended for the qualitative detection of Chlamydiae lipopolysaccharide (LPS) in inoculated cell cultures by immunofluorescence using fluoresceinated monoclonal antibodies (MAbs). Performance has not been established with direct patient specimens.

Device Description

The Diagnostic Hybrids' D3 DFA Chlamydiae Culture Confirmation Kit includes a Chlamydiae DFA Reagent that contains a blend of two murine MAbs directed against epitopes on the lipopolysaccharide of Chlamydiae. The kit is used for Chlamydiae detection in cell cultures of patient specimens. Kit Components: Chlamydiae DFA Reagent - one dropper bottle containing fluorescein labeled murine MAbs directed against Chlamydiae. The buffered, stabilized, aqueous solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative. Chlamydiae Antigen Control Slides - 10 slides. Five individually packaged control slides with wells containing cell culture-derived Chlamydia trachomatis positive cells and five individually packaged control slides with wells containing cell culture- derived negative cells. Each slide is intended to be stained only one time. PBS Concentrate - a 40X concentrate consisting of 4% sodium azide (after dilution to 1X in water, the concentration of sodium azide in the solution is 0.1%) in PBS Mounting Fluid - an aqueous, buffered, stabilized solution of glycerol and 0.1% sodium azide. The cell cultures to be tested are fixed in acetone. The Chlamydiae DFA Reagent is added to the cells to determine the presence of chlamydial antigens (LPS). After incubating at 35°C to 37°C, the stained cells are rinsed with the diluted PBS Concentrate, a drop of the supplied Mounting Fluid is added and the monolayer is examined for presence of fluorescent inclusions using a fluorescence microscope equipped with the correct filter combination for FITC at a magnification of 100-400X. Chlamydiae infected cells will be stained with bright apple-green fluorescence while uninfected cells will contain no apple-green fluorescence but will fluoresce red by the Evan's Blue counter-stain which is included in the Chlamydiae DFA Reagent.

AI/ML Overview

Here's an analysis of the provided text to extract the acceptance criteria and study details for the D3 DFA Chlamydiae Culture Confirmation Kit:

Acceptance Criteria and Device Performance

Acceptance Criteria	Reported Device Performance
Specificity (No Cross-Reactivity): The device should not react with common host cells and a wide variety of microorganisms (viruses, bacteria, yeast, protozoa).	Met:

No cross-reactivity observed for 20 host culture cell types.
No cross-reactivity observed for 57 virus strains.
No cross-reactivity observed for 24 of 25 bacterial cultures tested (Staphylococcus aureus showed small points of fluorescence due to protein-A binding to MAb Fc portion, but not a positive Chlamydiae reaction).
No cross-reactivity with one protozoan (Trichomonas vaginalis) and one yeast (Candida glabrata). |
| Detection of Chlamydiae Species: The device must specifically detect various Chlamydiae species and serovars. | Met:
Reacted positively with Chlamydophila pneumoniae, Chlamydophila psittaci, and 15 serovars of Chlamydia trachomatis (A, B, C, D, E, F, G, H, I, J, K, L1, L2, L3). |
| Overall Agreement with Comparison Device: The device's results should show high agreement with existing legally marketed comparison tests for Chlamydiae culture confirmation. | Met:
Positive Percent Agreement (PPA): 95.5% (95% CI: 84.5%-99.4%)
Negative Percent Agreement (NPA): 99.4% (95% CI: 98.3%-99.8%)
(Note: The exact target for PPA and NPA as a formal "acceptance criterion" is not explicitly stated, but these high percentages demonstrate good performance relative to predicate devices.) |
| Characteristic Staining Patterns: The FITC-conjugated MAbs should exhibit characteristic staining patterns on Chlamydiae infected cells. | Met:
The FITC-conjugated MAbs exhibited characteristic staining patterns on Chlamydiae infected cells, with fluorescent inclusions in infected cells.
Chlamydiae infected cells stained with bright apple-green fluorescence, while uninfected cells contained no apple-green fluorescence. |

Study Details

Sample Size Used for the Test Set and Data Provenance:
- Clinical Performance Test Set: 994 original specimens.
  - Provenance: Collected from three external laboratory sites and Diagnostic Hybrids' in-house laboratory in the United States.
    - Site 1: A reference laboratory in the southeastern US (396 specimens – 156 fresh prospective, 240 frozen prospective).
    - Site 2: A hospital laboratory in the mid-west US (200 specimens – 90 fresh prospective, 84 frozen prospective, 26 archived frozen).
    - Site 3: A hospital laboratory in the southwestern US (278 specimens – 23 fresh prospective, 68 frozen prospective, 187 archived frozen).
    - Site 4: Diagnostic Hybrids' in-house virology laboratory (120 specimens – 120 frozen prospective).
  - Nature: A mix of fresh prospective, frozen prospective, and archived (frozen) specimens.
- Non-Clinical Performance (Specificity/Cross-Reactivity/Detection Test Set):
  - 20 host culture cell types.
  - 57 virus strains.
  - 25 bacterial cultures.
  - 1 protozoan (Trichomonas vaginalis).
  - 1 yeast (Candida glabrata).
  - Chlamydophila pneumoniae, Chlamydophila psittaci, and 15 serovars of Chlamydia trachomatis.
Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of those Experts:
- The document describes comparison to "three currently marketed Culture Confirmation Kits ('Comparison' tests)" at various laboratory sites. It does not explicitly state the number or qualifications of human experts who established the ground truth for the clinical samples. The "ground truth" for the clinical study appears to be the results obtained from these predicate devices.
- For the non-clinical specificity/reactivity testing, "cells were examined for cross reactivity" and "stained cells are rinsed... and the monolayer is examined for presence of fluorescent inclusions using a fluorescence microscope." This implies human observation, but no specific number or qualifications of experts are given.
Adjudication Method for the Test Set:
- Not specified. The clinical performance section directly compares the "Subject Device" to "Comparison Devices" to calculate percent agreement. There is no mention of an adjudication process if the results differed between the subject device and the comparison test.
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:
- No, an MRMC comparative effectiveness study involving human readers with and without AI assistance was not done. The device is a diagnostic kit (reagents and controls) for laboratory use, not an AI-assisted diagnostic tool for image interpretation by human readers.
If a Standalone (i.e. algorithm only without human-in-the loop performance) was done:
- Yes, implicitly. The performance data presented (Positive Percent Agreement, Negative Percent Agreement, Specificity, Detection) reflect the performance of the "Subject Device" (the D3 DFA Chlamydiae Culture Confirmation Kit) as a standalone diagnostic assay. While human observation through a fluorescence microscope is part of the kit's intended use for result interpretation, the performance statistics are attributed directly to the kit's diagnostic capability in identifying the Chlamydiae LPS antigens. The "algorithm" here refers to the immunofluorescence assay itself, not an AI algorithm.
The type of ground truth used (expert consensus, pathology, outcomes data, etc.):
- For clinical performance, the ground truth was established by results from "three currently marketed Culture Confirmation Kits ('Comparison' tests)," which are themselves established diagnostic methods. This can be considered a form of comparison to predicate device results.
- For non-clinical performance (specificity and reactivity), the ground truth was based on the known presence or absence of specific Chlamydiae species, other microorganisms, and host cell types in the prepared cultures, often at a "3+ to 4+ infection" level for positive controls. This relies on known culture characteristics/identification (e.g., specific Chlamydia trachomatis serovars, identified bacterial or viral strains).
The Sample Size for the Training Set:
- The document does not explicitly delineate a "training set" in the context of a machine learning algorithm. This device is a diagnostic reagent kit, not an AI system. The development and validation of such a kit would involve internal research and development using various samples to formulate and refine the reagents, but this is not typically referred to as a "training set" in the same way as for AI. The detailed data presented are for the validation or test sets.
How the Ground Truth for the Training Set Was Established:
- As stated above, the concept of a "training set" with established ground truth as used in AI/machine learning does not directly apply to this type of diagnostic kit. The formulation and initial testing of the antibodies and reagents would be based on known laboratory strains and isolates, confirmed through established microbiological techniques.

Summary

Regulation Number and Section

§ 866.3120 Chlamydia serological reagents.

(a)
Identification. Chlamydia serological reagents are devices that consist of antigens and antisera used in serological tests to identify antibodies to chlamydia in serum. Additionally, some of these reagents consist of chlamydia antisera conjugated with a fluorescent dye used to identify chlamydia directly from clinical specimens or cultured isolates derived from clinical specimens. The identification aids in the diagnosis of disease caused by bacteria belonging to the genusChlamydia and provides epidemiological information on these diseases. Chlamydia are the causative agents of psittacosis (a form of pneumonia), lymphogranuloma venereum (a venereal disease), and trachoma (a chronic disease of the eye and eyelid).(b)
Classification. Class I (general controls).