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ORTHO T. cruzi ELISA Test System is an enzyme-linked immunosorbent assay for the in vitro qualitative detection of antibodies (Immunoglobulin G) to Trypanosoma cruzi (T. cruzi) in human adult serum (glass, plastic, or serum separator tubes) and plasma (EDTA, lithium heparin or citrate) using whole-cell lysate antigens. Reactive assay results are presumptive evidence of past infection, and in conjunction with other serological and clinical information, may be used for the laboratory diagnosis of individuals with Chagas' disease.

Definitive diagnosis of an acute phase of infection (including acute congenital infection) must be made by alternate methods, e.g., hemoculture, blood smear.

This test is not intended for use on samples of cord blood or screening blood or plasma donors.

The ORTHO T. cruzi ELISA Test System is an enzyme-linked immunosorbent assay (ELISA). ELISA technology utilizes the principle that antibodies bound to the solid phase can be detected by complementary antibodies or antigens labeled with an enzyme capable of acting on a chromogenic substrate. When substrate is applied, the presence of antigens or antibodies can be detected by development of a colored end product. The optical densities are read spectrophotometrically.

This ELISA was developed to detect human antibodies to T. cruzi in serum and plasma. The assay utilizes microwells coated with a whole-cell lysate containing T. cruzi antigens as the solid phase. The assay procedure is a three-stage test carried out in a microwell coated with lysate (antigens) prepared from T. cruzi. In the first stage, test specimen, Negative Control, and Positive Calibrator are diluted directly in the test well containing Specimen Diluent, and incubated for a specified length of time. If antibodies to T. cruzi are present, antigen-antibody complexes will form on the microwell surface. If antibodies to T. cruzi are absent, complexes will not form. Unbound antibodies in the sample will be removed during the subsequent wash step.

In the second stage, murine monoclonal antibody conjugated with Horseradish Peroxidase (Conjugate) is added to the test well. The Conjugate binds specifically to the antibody portion of the antigen-antibody complex. If complexes are not present, the unbound Conjugate is removed by the subsequent wash step.

In the third stage, an enzyme detection system composed of o-phenylenediamine (OPD) and hydrogen peroxide is added to the test well. If bound Conjugate is present, the OPD with be oxidized, resulting in a colored end product. Sulfuric acid is then added to stop the reaction. The color intensity depends on the amount of bound Conjugate and, therefore, is a function of the concentration of antibodies to T. cruzi present in the specimen. The intensity of color in the substrate solution is then determined with a microwell reader (spectrophotometer) designed to measure light absorbance in a microwell.

Here's an analysis of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Device: ORTHO® T. cruzi ELISA Test System

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state "acceptance criteria" in a separate section with specific numerical thresholds for performance metrics. However, the performance study data implicitly demonstrates the device's acceptable performance by showing strong agreement with established methods and probable T. cruzi antibody status. The key performance metrics are Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA).

Given the context of a 510(k) submission, the implicit acceptance criteria would be for the device to perform comparably to, or demonstrate a high level of agreement with, the established comparator methods and the "most probable T. cruzi antibody status."

2. Sample Size Used for the Test Set and Data Provenance

• Sample Size for Test Set:
  • High and Low Risk Subjects Study: N = 1074 subjects
  • Serological Presumed Positive Population Study: N = 810 specimens
• Data Provenance:
  • Country of Origin: The "Serological Presumed Positive Population" study explicitly states specimens were obtained from endemic countries: Bolivia (17.8%), Brazil (24.7%), Chile (10.6%), Guatemala (2.2%), Mexico (32.5%), and Nicaragua (12.2%). The provenance for the "High and Low Risk" subjects is not explicitly stated, but given T. cruzi is endemic to Latin America, it's highly likely they came from similar regions or populations with risk factors associated with these regions.
  • Retrospective or Prospective: Not explicitly stated, but the description of "specimens from 1074 subjects" and "specimens obtained from" suggests these were pre-collected samples, which typically implies a retrospective study design.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts

• The document does not specify the number of experts used to establish the ground truth.
• It refers to "a pre-specified testing algorithm" involving the ORTHO T. cruzi ELISA Test System, a comparator T. cruzi IFA (indirect immunofluorescence assay), and supplemental T. cruzi RIPA (radioimmunoprecipitation assay) testing to determine the "most probable T. cruzi antibody status." This implies a laboratory-based algorithm rather than subjective expert interpretation for each individual case ground truth.
• Qualifications of Experts: Not specified. However, the reliance on established serological methods (IFA, RIPA) suggests that interpretation would be done by qualified laboratory personnel following established protocols, though individual "experts" for ground truth adjudication are not mentioned.

4. Adjudication Method for the Test Set

The adjudication method used to establish the "most probable T. cruzi antibody status" for the test sets was a pre-specified testing algorithm rather than an expert consensus method like 2+1 or 3+1.

• For High and Low Risk Subjects:
  • Specimens negative with both the ORTHO T. cruzi ELISA and the T. cruzi IFA were assigned a "most probable T. cruzi antibody status of negative" (if not tested with RIPA).
  • Specimens tested with RIPA were assigned a most probable status of positive, negative, or indeterminate based on RIPA results.
• For Serological Presumed Positive Population:
  • Specimens that were ORTHO T. cruzi ELISA repeatedly reactive AND positive with the T. cruzi IFA were assigned a "most probable T. cruzi antibody status of positive" without further RIPA testing.
  • All specimens negative with both assays OR with discordant results between the two assays were tested with the T. cruzi RIPA, and their status was assigned "based upon the RIPA results."

This method essentially uses a hierarchical testing algorithm with RIPA as a confirmatory test for ambiguous cases, which acts as the adjudicator.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and the Effect Size of How Much Human Readers Improve with AI vs. Without AI Assistance

• No, an MRMC comparative effectiveness study was not done.
• This device is an ELISA diagnostic kit, which is an in vitro diagnostic (IVD) assay to detect antibodies in serum/plasma. It is not an AI-assisted diagnostic device that would involve human readers interpreting images or data with or without AI assistance. Therefore, the concept of "human readers improve with AI vs. without AI assistance" does not apply here.

6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) Was Done

• Yes, the performance presented in the report is for the device operating in a standalone capacity (algorithm only).
• The ORTHO T. cruzi ELISA Test System is an automated/semi-automated in vitro diagnostic assay. The results (optical densities) are read spectrophotometrically, and the interpretation (reactive/nonreactive) is based on a pre-defined cutoff. While human operators are involved in running the assay (pipetting, washing, loading), the diagnostic decision itself from the optical density reading is based on the device's internal algorithm/parameters, not human interpretation of a raw signal. The performance metrics (PPA, NPA) directly reflect this standalone assay performance against the established ground truth.

7. The Type of Ground Truth Used

The ground truth used was a "most probable T. cruzi antibody status" established by a pre-specified testing and interpretation algorithm involving:

• The ORTHO T. cruzi ELISA Test System itself
• A comparator T. cruzi IFA
• Supplemental T. cruzi RIPA testing (Radioimmunoprecipitation Assay) as a confirmatory assay.

This approach combines multiple serological methods, with RIPA acting as the highest tier for ambiguous results, to arrive at a "most probable" truth, which is a form of composite reference standard or multi-test algorithm ground truth.

8. The Sample Size for the Training Set

• The document does not specify any sample size for a training set.
• This is common for in vitro diagnostic (IVD) devices like ELISA kits, which are often developed and optimized using a variety of samples during their analytical validation phase, but the rigorous performance evaluation for regulatory submission typically focuses on a distinct clinical validation (test) set. The concepts of "training set" and "test set" are more explicitly separated for machine learning/AI models.

9. How the Ground Truth for the Training Set Was Established

• Since a "training set" is not explicitly mentioned or detailed in the provided document, there is no information on how its ground truth was established. If one were used in development, it would likely follow similar principles of using established serological methods and clinical information for sample characterization.

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