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The ALPHA Cryptococcal Antigen enzyme immunoassay (CrAg EIA) is a qualitative or semi-quantitative (titration) test system for the detection of capsular polysaccharide antigens of Cryptococcus species complex (Cryptococcus neoformans and Cryptococcus gattii) in serum and cerebrospinal fluid (CSF). The ALPHA Cryptococcal Antigen Enzyme Immunoassay is an assay which can be used as an aid in the diagnosis of cryptococcosis. Test results are to be used in conjunction with information available from the patient clinical evaluation and other diagnostic procedures.

The ALPHA Cryptococcal Antigen Enzyme Immunoassay (EIA) is a direct immunoenzymatic sandwich microplate assay which detects Cryptococcus antigens in serum and CSF. Anti-Cryptococcus antibodies bound to microwell plates are used as capture antibodies, and horseradish peroxidase (HRP)-conjugated anti-Cryptococcus antibodies are used as detect antibodies. The positive control and standard curve material are composed of cryptococcal capsular polysaccharide antigen in a buffered protein solution with a preservative. In the qualitative procedure, specimens are analyzed undiluted. In the titration procedure, specimens are analyzed after serial dilution in specimen diluent. Either serum or CSF is added to the microwells coated with the capture antibodies and incubated. If the patient specimen contains cryptococcal antigens that are recognized by the capture antibodies, those antigens will become bound to the microwells. The microwells are washed to remove unbound patient material, and HRP-conjugated detect antibody is added to the wells. If Cryptococcus antigens are bound to the microwells by the capture antibodies, the detect antibody will also become bound to the microwells. The wells are then washed to remove any unbound detect antibody. Next, tetramethylbenzidine (TMB) substrate is added to the microwells, and in the presence of HRP, a blue color will develop. The reaction is stopped by the addition of a stop solution. The optical density (OD) is determined with a microplate reader at 450 nm with reference at 630 nm (reference is optional).

Here's an analysis of the ALPHA Cryptococcal Antigen EIA based on the provided 510(k) summary, structured to address your specific points:

Acceptance Criteria and Device Performance Study

The document describes several analytical performance studies rather than clinical studies with patient outcomes or a specific "acceptance criteria" table for diagnostic accuracy. The primary criteria for device approval appear to be substantial equivalence to the predicate device (Meridian's Premier™ Cryptococcal Antigen EIA, K904393) and meeting internal laboratory performance benchmarks for precision, sensitivity, specificity, and non-interference.

However, based on the ROC analysis and method comparison studies, we can infer some performance expectations for sensitivity and specificity.

1. Table of Acceptance Criteria and Reported Device Performance

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

The "test set" for performance evaluation consists of several cohorts:

• Precision Studies: Spiked serum and mock CSF samples. The number of unique samples is not given, but they were tested twice per day over five days across three sites. Each panel consisted of negative, high negative, low positive, and moderate positive samples for both serum and CSF.
• Analytical Sensitivity (LoD): 80 replicates of normal human serum, 80 replicates of artificial CSF for LoB. For LoD, initially 20 replicates of 4 concentrations, then 24 replicates of serum and CSF spiked with antigen near initial LoD.
• Analytical Specificity (Cross-Reactivity): 118 serum specimens and 15 fungal culture filtrates.
• Interference Studies: Five icteric, five hemolyzed, and five lipemic serum specimens.
• High Dose Hook Effect: One negative serum specimen pool spiked with antigen.
• Freeze-Thaw Study: Three CrAg-Negative human CSF specimens and four CrAg-Negative human serum specimens, spiked.
• ROC Analysis (for Cutoff Determination): 995 combined serum and CSF specimens.
• Method Comparison (vs. Commercial EIA and IMMY LFA): 1782 specimens (CSF n=426; serum n=1356).
  • Data Provenance: The studies were conducted at Immuno-Mycologics, Inc. (IMMY) and two national reference laboratories, as well as a clinical lab for precision. The specimens for the method comparison were "submitted for cryptococcal antigen EIA testing," implying they were clinical samples, likely retrospective or leftover clinical samples. The country of origin is not explicitly stated, but the locations (Norman, OK; national reference labs) suggest United States origin. The nature of the samples (spiked or "submitted for testing") suggests retrospective use of clinical samples or artificially prepared samples.

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

The ground truth for the test sets was primarily established through:

• Spiking: For precision, LoD, interference, high dose hook, and freeze-thaw studies, samples were spiked with known concentrations of cryptococcal antigen, establishing a de facto ground truth.
• Reference Methods:
  • For ROC analysis and comparison to LFA, the IMMY CrAg Lateral Flow Assay (LFA) was used as the reference method to classify specimens as true positive or negative. The expertise behind the LFA's accuracy would pertain to its own validation.
  • For comparison to another manufacturer's device, the commercial Cryptococcal antigen EIA (the predicate device, Meridian's Premier™ Cryptococcal Antigen EIA) was used as the comparator.
  • For analytical specificity, known positive/negative states for cross-reactants (e.g., Aspergillus galactomannan positive confirmed by Bio-Rad's Platelia™ Aspergillus EIA) were used.

The document does not mention the use of human experts (e.g., radiologists, pathologists) to establish ground truth for the test sets in the traditional sense of medical image analysis or complex diagnostic interpretation. The focus is on analytical performance and agreement with established laboratory tests.

4. Adjudication Method for the Test Set

No explicit adjudication method (e.g., 2+1, 3+1) is mentioned for the test sets. The ground truth was primarily defined by:

• Known concentrations (for spiked samples).
• Results from a single reference laboratory test (IMMY CrAg LFA or the commercial predicate EIA).
• Known status for cross-reactivity samples (e.g., confirmed Aspergillus positive).

There is no indication of multiple human readers or a consensus process for determining the ground truth for any of these studies.

5. 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 was not done. This device is an in vitro diagnostic (IVD) immunoassay that provides a quantitative or qualitative result. It is not an AI-powered diagnostic tool designed to assist human readers in interpreting images or complex data, nor does it involve "human readers" in the sense of medical specialists interpreting results that the device then supports or enhances.

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

This device is a standalone diagnostic test. It functions as an "algorithm only" in the sense that the assay procedure and subsequent optical density (OD) readings lead to a quantitative value or a positive/negative determination based on a defined cutoff. The performance studies described (precision, LoD, cross-reactivity, method comparison) are reflections of its standalone performance. There isn't a "human-in-the-loop" component in interpreting the EIA's direct output beyond standard laboratory practice of running the assay and reporting the result.

7. The Type of Ground Truth Used

The ground truth used for these studies can be categorized as:

• Known Spiking Concentrations: For analytical sensitivity, precision, interference, high-dose hook, and freeze-thaw studies.
• Reference Laboratory Assay Results:
  • IMMY CrAg Lateral Flow Assay (LFA): Used as the reference for ROC analysis and comparison studies against the LFA.
  • Commercial Cryptococcal Antigen EIA (Predicate Device): Used as the reference for method comparison studies to demonstrate substantial equivalence.
  • External reference assays: For confirming known states of potentially cross-reacting substances (e.g., Bio-Rad's Platelia™ Aspergillus EIA).

8. The Sample Size for the Training Set

No explicit "training set" for an AI algorithm is mentioned as this is a traditional immunoassay.

However, if we interpret "training set" in the context of establishing assay parameters or a cutoff:

• The ROC analysis to determine the cutoff (0.265 blanked OD) was performed on a dataset of 995 combined serum and CSF specimens. This dataset could be considered analogous to a "training set" for defining the assay's threshold for positivity.

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

For the 995 specimens used in the ROC analysis to establish the cutoff:

• The ground truth was established by comparing the ALPHA Cryptococcal Antigen EIA results to the IMMY CrAg Lateral Flow Assay (LFA). The LFA was used to classify a specimen as a "true positive or negative" for the purpose of matching the sensitivity and specificity of the new EIA to the LFA. This means the LFA's results were accepted as the ground truth for this particular analysis aiming for concordance.

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The Cryptococcal Antigen Lateral Flow Assay (CrAg LFA) is an immunochromatographic test system for the qualitative or semi-quantitative detection of capsular polysaccharide antigens of Cryptococcus species complex (Cryptococcus neoformans and Cryptococcus gattii) in serum and cerebral spinal fluid (CSF).

The CrAg Lateral Flow Assay is a prescription-use laboratory assay, which can aid in the diagnosis of cryptococcosis.

The CrAg Lateral Flow Assay is a dipstick sandwich immunochromatographic assay, which detects cryptococcal antigen in serum and CSF. For the qualitative procedure, specimens are diluted 1:2 in 1x Specimen Diluent and analyzed. For the semi-quantitative procedure, specimens are diluted 1:5 in 1x Specimen Diluent followed by 1:2 serial dilutions. All dilutions are then analyzed as in the qualitative procedure. Specimens are placed into an appropriate reservoir, such as a test tube or microtiter plate, and the lateral flow device is then placed into the reservoir allowing the specimen to come into contact with the test membrane. The test uses specimen wicking to capture gold-conjugated, anti-cryptococcal monoclonal antibodies and gold-conjugated control antibodies that are deposited onto a membrane. If cryptococcal antigen is present in the specimen, it binds to the gold-conjugated, anti-cryptococcal antibodies. The gold-labeled antibody-antigen complex will continue to wick up the membrane where it will interact with the Test Line (T). The Test Line is immobilized anti-cryptococcal monoclonal antibodies. If the specimen contains cryptococcal antigen, a sandwich is created with the gold-labeled antibodies and the immobilized antibodies, causing a visible line to develop at the test line site (T). If proper flow occurs and the reagents are reactive at the time of use, the wicking of any specimen, positive or negative, will cause the gold-conjugated control goat IgG antibody to move to the Control Line (C) which is immobilized bovine anti-goat IgG antibody. The immobilized anti-goat antibody will bind to the gold-conjugated goat IgG Control antibody and will cause a visible line to develop (C). A positive test result will create two lines, while a negative test result will create one line (Figure 1). If the control line fails to develop a line, then the test is not valid.

Here's an analysis of the provided text regarding the acceptance criteria and study for the CrAg Lateral Flow Assay:

1. Table of Acceptance Criteria and Reported Device Performance

Note on Acceptance Criteria: The document does not explicitly state numerical acceptance criteria (e.g., "sensitivity must be >95%"). However, it implicitly aims for "very high sensitivity and specificity" based on the background information and the excellent results presented. The repeatability and analytical sensitivity are reported as measured, indicating the performance achieved. The high dose hook effect is identified as a limitation rather than a criterion met.

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

• Test Set Sample Size (CSF for Method Comparison):
  • Positive (Culture/India Ink): 65 specimens
  • Negative (Culture/India Ink): 99 specimens
  • Total: 164 specimens
• Data Provenance: The studies contained a mix of both prospective and retrospective specimens. The country of origin is not specified, but the device is indicated for use in the US (implied by FDA submission).

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

• The document does not specify the number of experts or their qualifications for establishing the ground truth (Culture/India Ink).
• For the repeatability and reproducibility study (CSF), "five different operators" across "three different sites" were involved. Their specific qualifications are not detailed beyond "internal" and "US reference/hospital laboratory" personnel.

4. Adjudication Method for the Test Set

• The document does not describe any adjudication method for the test set. The "gold standard" (culture and/or India Ink) is presented as the definitive truth.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and Effect Size

• No, an MRMC comparative effectiveness study was not explicitly described in this 510(k) summary. The study focuses on the standalone performance of the CrAg LFA compared to a gold standard, not on the improvement of human readers with or without AI assistance (as this is a point-of-care type diagnostic, not an AI-assisted interpretation tool for images).

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

• Yes, a standalone performance study was conducted. The CrAg Lateral Flow Assay is a rapid diagnostic test where the result (presence/absence of lines) is directly interpreted. The reported sensitivity and specificity values represent the performance of the device itself (algorithm only, in a sense, as it's a defined chemical reaction leading to a visible outcome) against the gold standard. While a human reads the lines, the core performance metrics are about the device's ability to detect the antigen.

7. The Type of Ground Truth Used

• For the method comparison study (CSF), the ground truth used was "gold standard for the diagnosis of cryptococcosis (culture and/or India Ink)."

8. The Sample Size for the Training Set

• The document does not specify a training set sample size. This is common for lateral flow assays, which are typically developed and optimized through laboratory analytical studies rather than machine learning models that require distinct training sets. The studies described are primarily performance validation studies.

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

• Since a distinct "training set" in the context of machine learning is not mentioned as such, the method of establishing ground truth for development/optimization would likely involve spiking known concentrations of cryptococcal antigen into mock specimens (as seen in the analytical sensitivity sections) and testing against other validated methods or known positive/negative samples. The document refers to "mock CSF that was negative by the IMMY Latex-Cryptococcal Antigen Detection System" for repeatability/reproducibility.

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The CrAg Lateral Flow Assay is an immunochromatographic test system for the qualitative or semi-quantitative detection of capsular polysaccharide antigens of Cryptococcus species complex (Cryptococcus neoformans and Cryptococcus gatti) in serum.
The CrAg Lateral Flow Assay is a prescription-use laboratory assay, which can aid in the diagnosis of Cryptococcosis.

The CrAg Lateral Flow Assay is a dipstick sandwich immunochromatographic assay, which detects cryptococcal antigen in serum. For the qualitative procedure, specimens are diluted 1:2 in 1x Specimen Diluent and analyzed. For the semi-quantitative procedure, specimens are diluted 1:5 in 1x Specimen Diluent followed by 1:2 serial dilutions. All dilutions are then analyzed as in the qualitative procedure. Specimens are placed into an appropriate reservoir, such as a test tube or microtiter plate, and the lateral flow device is then placed into the reservoir allowing the specimen to come into contact with the test membrane. The test uses specimen wicking to capture gold-conjugated, anti-cryptococcal monoclonal antibodies and gold-conjugated control antibodies that are deposited onto a membrane. If cryptococcal antigen is present in the specimen, it binds to the gold-conjugated, anti-cryptococcal antibodies. The gold-labeled antibody-antigen complex will continue to wick up the membrane where it will interact with the Test Line (T). The Test Line is immobilized anti-cryptococcal monoclonal antibodies. If the specimen contains cryptococcal antigen, a sandwich is created with the gold-labeled antibodies and the immobilized antibodies, causing a visible line to develop at the test line site (T). If proper flow occurs and the reagents are reactive at the time of use, the wicking of any specimen, positive or negative, will cause the gold-conjugated control goat IgG antibody to move to the Control Line (C) which is immobilized bovine anti-goat IgG antibody. The immobilized anti-goat antibody will bind to the gold-conjugated goat IgG Control antibody and will cause a visible line to develop (C). A positive test result will create two lines, while a negative test result will create one line (Figure 1). If the control line fails to develop a line, then the test is not valid.

The CrAg Lateral Flow Assay is an immunochromatographic test system for the qualitative or semi-quantitative detection of capsular polysaccharide antigens of Cryptococcus species complex in serum. The study provided compares the CrAg Lateral Flow Assay to a Cryptococcal Antigen EIA and to culture/India Ink as gold standards.

1. Table of Acceptance Criteria and Reported Device Performance:

The document does not explicitly state pre-defined acceptance criteria in terms of specific sensitivity and specificity thresholds for the de novo approval. However, the performance is reported as follows:

Comparison with Cryptococcal Antigen EIA (Serum):

Comparison with Culture/India Ink (Gold Standard for Serum):

Semi-Quantitative Serum Correlation (CrAg LFA vs. Latex Agglutination):

• Correlation (R²): Not explicitly stated | 0.905 |

2. Sample Size and Data Provenance:

• Test Set (Method Comparison with Cryptococcal Antigen EIA): 197 serum specimens. The data provenance is from a "US reference laboratory" and the study was retrospective (collected and stored frozen until studies were performed).
• Test Set (Method Comparison with Culture/India Ink): The exact number of specimens is not clearly enumerated, but the table indicates a 2x2 contingency table for "Culture/India Ink" with "NO ANDRE & FOR A LE SE SE SE SE SE SE SERVED AN A SE" in one row and other unclear annotations. However, the sample size can be inferred from the 95% Confidence Intervals for Sensitivity and Specificity, which suggest a reasonable sample size for a diagnostic test. The document states "These studies contained a mix of both prospective and retrospective specimens."
• Test Set (Predicate Device Comparison - Latex-Cryptococcal Antigen Detection System): 197 serum specimens. The data provenance is from a "US reference laboratory" and the study was retrospective (collected and stored frozen until studies were performed).

3. Number of Experts and Qualifications for Ground Truth:

The document does not specify the number or qualifications of experts used to establish the ground truth for the test set.

4. Adjudication Method for the Test Set:

The document does not describe an adjudication method for the test set results. The ground truth (EIA or Culture/India Ink) was likely established by the reference laboratory or standard clinical procedures without explicit mention of an additional adjudication process for this study.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study:

No multi-reader multi-case (MRMC) comparative effectiveness study was done. The CrAg Lateral Flow Assay is a diagnostic device that provides an objective result (presence/absence of line, or semi-quantitative titer), and its performance is assessed against established laboratory methods or clinical gold standards, not by comparing the performance of human readers with and without AI assistance.

6. Standalone Performance Study:

Yes, a standalone performance study was done. The method comparison studies (against Cryptococcal Antigen EIA and Culture/India Ink) and the precision, analytical sensitivity, and analytical specificity studies all evaluate the intrinsic performance of the CrAg Lateral Flow Assay device without human-in-the-loop assistance.

7. Type of Ground Truth Used:

• For Method Comparison: Cryptococcal Antigen EIA results from a US reference laboratory.
• For Gold Standard Comparison: Culture and/or India Ink results. These are considered the "gold standard for the diagnosis of cryptococcosis."
• For Training Set: Not applicable as this is a diagnostic assay, not a machine learning model requiring a separate training set. The performance studies evaluate the assay itself.

8. Sample Size for the Training Set:

Not applicable. This is a conventional in vitro diagnostic assay, not an AI/ML-based device that typically requires a distinct training set for model development. The described studies are all for performance evaluation of the developed assay.

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

Not applicable, as there is no mention of a training set for an AI/ML model. The "ground truth" in the context of this device refers to the established diagnostic methods used to evaluate the CrAg Lateral Flow Assay's accuracy.

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The ALPHA Histoplasma Antigen EIA is an immunoenzymatic sandwich microplate assay for the detection of Histoplasma antigens in urine samples. The ALPHA Histoplasma Antigen EIA is a test which, when used in conjunction with other diagnostic procedures such as microbiological culture, histological examination of biopsy samples and radiographic evidence, can be used as an aid in the diagnosis of histoplasmosis.

The ALPHA Histoplasma Antigen EIA is an immunoenzymatic sandwich microplate assay which detects Histoplasma antigens in urine. Rabbit anti-Histoplasma IgG antibodies bound to microwell plates are used as capture antibodies and biotinylated rabbit anti-Histoplasma IgG antibodies are used as detect antibodies. The positive control is made of buffer spiked with Histoplasma capsulatum antigens and the negative control is buffer only. Standards are made of Histoplasma capsulatum antigens from culture filtrate. The kit contains the 100 Standard, 30 Standard, and 2 Standard that are used to generate a sigmoid calibration curve using a four-parameter fit of the blanked absorbance values versus the assigned EIA Values. R-squared values must be greater than or equal to 0.990. Urine samples are run untreated and undiluted. The samples are added to the microwells coated with the capture antibody and incubated. If the patient specimen contains Histoplasma antigens that are recognized by the capture antibody, those antigens will become bound to the microwell. The wells are washed to remove unbound patient material and biotinylated detection antibody is added to the wells. If Histoplasma antigens are bound to the microwell by the capture antibody, then the detect antibody will also become bound to the microwell. The wells are then washed to remove any unbound detect antibody. For detection, biotin-streptavidin chemistry reagents including 3,3′,5′,5′ tetramethybenzadine (TMB) and stop solution are used. Streptavidin conjugated to horseradish peroxidase (HRP) is added to the microwells. In the presence of the biotinylated detect antibody, streptavidin-HRP will become bound to the plate. The plate is then washed to remove any unbound streptavidin-HRP, and TMB substrate solution is added to the microwells. A blue color develops in the presence of the HRP enzyme. The reaction is stopped by the addition of a stop solution. The optical density (absorbance) is determined with a microplate reader at 450nm or 450nm or 450nm alone. EIA Units for specimens are calculated using a four-parameter curve-fit generated with the 4 standards supplied in the kit.

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

ALPHA Histoplasma Antigen EIA Performance Study Analysis

The ALPHA Histoplasma Antigen EIA is an immunoenzymatic sandwich microplate assay designed for the detection of Histoplasma antigens in urine samples, serving as an aid in the diagnosis of histoplasmosis when combined with other diagnostic procedures.

1. Acceptance Criteria and Reported Device Performance

The direct acceptance criteria for this device are not explicitly stated in a consolidated manner as pass/fail thresholds in the provided document. However, the studies conducted demonstrate acceptable performance characteristics. Based on the "Comparison to Culture/Histopathology" section, two key performance metrics were evaluated: Sensitivity and Specificity.

Note: The document implies "acceptable" performance for these metrics rather than specifying strict numerical thresholds for acceptance prior to the study.

2. Sample Size and Data Provenance

• Test Set Sample Size: A total of 278 urine specimens were used for the clinical performance evaluation (comparison to culture/histopathology).
• Data Provenance: The document does not explicitly state the country of origin for the clinical samples. It does mention that "urine specimens containing various substances were obtained from a national reference laboratory" for analytical interference studies, suggesting some US-based data. However, for the main clinical performance, the geographic origin is not specified. The data is retrospective, as specimens were "culture- or histopathology-confirmed" prior to testing with the device.

3. Number of Experts and Qualifications for Ground Truth

The document does not explicitly state the number of experts used to establish the ground truth or their specific qualifications (e.g., "radiologist with 10 years of experience"). However, the ground truth was established by "culture- or histopathology-confirmed" results. This implies that the diagnoses were made by medical professionals, likely pathologists and/or microbiologists, following standard diagnostic procedures.

4. Adjudication Method for the Test Set

The document does not describe a specific adjudication method (e.g., 2+1, 3+1) for establishing the ground truth of the clinical test set. The ground truth was based on "culture- or histopathology-confirmed" results, which are considered definitive diagnostic methods. This suggests that individual confirmation by these methods was sufficient, and a separate adjudications process was not deemed necessary for the diagnostic truth.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not performed. The study evaluates the performance of the device against established diagnostic methods (culture/histopathology) and against another, non-FDA-cleared Histoplasma antigen EIA, but it does not assess human reader improvement with or without AI assistance.

6. Standalone Performance Study

Yes, a standalone performance study was done. The "Comparison to Culture/Histopathology" section directly assesses the algorithm's (device's) ability to detect Histoplasma antigens in urine samples against established clinical diagnostic methods, without human interpretation as part of the primary outcome.

7. Type of Ground Truth Used

The type of ground truth used was expert consensus, pathology, and microbiological culture results. Specifically, the clinical performance was evaluated against "culture- or histopathology-confirmed urine specimens."

8. Sample Size for the Training Set

The document does not provide information regarding the sample size used for the training set. This is a 510(k) submission, typically focusing on validation rather than development details.

9. How Ground Truth for the Training Set Was Established

The document does not provide information on how the ground truth for any potential training set was established. Given this is a 510(k) for an immunoassay, the device itself is a diagnostic test, and the development process likely involved internal validation and optimization, but specific training set details are not included in this regulatory summary.

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