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Simplexa C. difficile Direct: The Focus Diagnostics Simplexa™ C. difficile Direct assay is intended for use on the Integrated Cycler instrument for the detection of Clostridium difficile toxin B gene (tcdB) present in liquid or unformed stool samples from individuals suspected of C. difficile infection (CDI). This test aids in the diagnosis of illness resulting from CDI. The assay is for professional and prescription use only.

Simplexa C. difficile Positive Control Pack: The Simplexa™ C. difficile Positive Control Pack is intended to be used as a control with the Simplexa™ C. difficile Direct kit. This control is not intended for use with other assays or systems.

The Simplexa™ C. difficile Direct assay system is a real-time PCR system that enables the direct amplification and detection of toxigenic C. difficile DNA from unprocessed liquid or unformed stool specimens that have not undergone nucleic acid extraction. The system consists of the Simplexa™ C. difficile Direct assay, the Integrated Cycler (with Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories. In the Simplexa™ C. difficile Direct assay, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify C. difficile and DNA internal control targets. The assay targets a sequence which is in a well conserved region of C. difficile toxin B gene (todB). A DNA internal control is used to detect PCR failure and/or inhibition

Acceptance Criteria and Device Performance for Simplexa C. difficile Direct

1. Table of Acceptance Criteria and Reported Device Performance

The provided document describes the performance of the Simplexa C. difficile Direct assay in comparison to various gold standards and other NAATs. The acceptance criteria are implicit in the presented tables.

Acceptance Criteria for Simplexa C. difficile Direct (versus Combined Culture + Toxin Assay):

Additional Performance (versus other FDA-cleared NAATs for reference):

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

The primary clinical performance study (method comparison) used 2351 samples prospectively collected.

• Data Provenance: Samples were prospectively collected from five (5) geographically diverse sites in the USA between December 3, 2015, and June 10, 2016.
• Evaluable Samples: 2330 samples were evaluable on Simplexa C. difficile Direct & the Direct Culture method, and 2336 were evaluable on Simplexa C. difficile Direct & the Combination of Direct/Enriched Culture methods.
• Invalid Rate: The initial invalid rate was 0.64% (15/2351), reduced to a final invalid rate of 0.13% (3/2351) after repeat testing.

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

The document does not explicitly state the "number of experts" or their "qualifications" involved in establishing the ground truth for the clinical sample test set. However, the ground truth was based on Direct Culture + Toxin Assay and Combined Culture (Direct & Enriched) + Toxin Assay, with confirmatory testing using FDA cleared nucleic acid amplification test (NAAT) results for discrepant analysis. This implies that the ground truth was established by laboratory methods and not by individual expert interpretation.

4. Adjudication Method for the Test Set

For discrepant samples in the clinical study, discrepant analysis was performed using FDA cleared nucleic acid amplification test (NAAT) results provided by the sites.

• For the "Simplexa C. difficile Direct versus Direct Toxigenic Culture Method" table:
  • 116/163 discrepant Simplexa-positive/Culture-negative samples were positive, and 46/163 were indeterminate for C. difficile Toxin B gene DNA when tested using an FDA cleared molecular test.
  • 8/12 discrepant Simplexa-negative/Culture-positive samples were negative, and 4/12 were positive for C. difficile Toxin B gene DNA when tested using an FDA cleared molecular test.
• For the "Simplexa C. difficile Direct versus Combined Direct and Enriched Toxigenic Culture Methods" table:
  • 59/96 discrepant Simplexa-positive/Culture-negative samples were positive, and 37/96 were negative for C. difficile Toxin B gene DNA when tested using an FDA cleared molecular test.
  • 43/55 discrepant Simplexa-negative/Culture-positive samples were negative for C. difficile Toxin B gene DNA when tested using an FDA cleared molecular test.

This indicates an adjudication process involving a third, independent, FDA-cleared NAAT for resolving discrepancies between the investigational device and the culture methods.

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

No, an MRMC comparative effectiveness study was not explicitly described in the provided text. The study focuses on the performance of the analytical device rather than human reader interpretation with or without AI assistance.

6. If a Standalone Study (Algorithm Only Without Human-in-the-loop Performance) Was Done

Yes, the studies presented are standalone performance evaluations of the Simplexa C. difficile Direct assay. The assay is an automated real-time PCR system designed for direct detection of C. difficile toxin B gene, meaning its performance is measured independently of human interpretation of its results, although trained lab personnel would operate the instrument.

7. The Type of Ground Truth Used

The ground truth for the clinical performance study was established using:

• Direct Culture + Toxin Assay
• Combined Culture (Direct & Enriched) + Toxin Assay
• FDA cleared molecular tests (NAATs) for discrepant analysis.

This represents a composite reference standard based on microbiological culture techniques and molecular detection for toxigenic C. difficile, with additional molecular confirmation for discordant results.

8. The Sample Size for the Training Set

The document does not specify a separate "training set" sample size. The studies described are performance evaluations of the device, implying that the device's development and internal optimization would have occurred prior to these validation studies. Therefore, information regarding a training set is not provided as this is a diagnostic assay, not a machine learning algorithm that requires a separate training and test set in the same way.

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

As no specific training set is mentioned for the device (which is a molecular diagnostic assay), the method for establishing ground truth for such a set is not applicable or described in this document. The focus is on the analytical and clinical validation of the completed product.

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The Focus Diagnostics Simplexa™ Flu A/B & RSV Direct assay is intended for use on the 3M Integrated Cycler instrument for the in vitro qualitative detection and differentiation of influenza B virus, and respiratory syncytial virus (RSV) RNA in nasopharyngeal swabs (NPS) from human patients with signs and symptoms of respiratory tract infection in conjunction with clinical and epidemiological risk factors. This test is intended for use as an aid in the differential diagnosis of influenza B, and RSV viral infections in humans and is not intended to detect influenza C.

Negative results do not preclude influenza virus or RSV infection and should not be used as the sole basis for treatment or other patient management decisions.

Performance characteristics for influenza A were established with clinical specimens collected during the 2010/2011 influenza season when 2009 H1N1 influenza and H3N2 were the predominant influenza A viruses in circulation. When other influenza A viruses are emerging, performance characteristics may vary.

If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to the state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

Focus Diagnostics' Simplexa™ Flu A/B & RSV Positive Control Pack is intended to be used as a control with the Simplexa™ Flu A/B & RSV Direct kit. This control is not intended for use with other assays or systems.

The Simplexa™ Flu AB & RSV Direct assay system is a real-time RT-PCR system that enables the direct amplification, detection and differentiation of human influenza A (Flu A) virus RNA, human influenza B (Flu B) virus RNA and RSV RNA from unprocessed nasopharyngeal swabs that have not undergone nucleic acid extraction. The system consists of the Simplexa™ Flu A/B & RSV Direct assay, the 3M Integrated Cycler (with Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories.

In the Simplexa™ Flu A/B & RSV Direct assay, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify Flu A, Flu B, RSV and internal control RNA. The assay provides three results; conserved regions of influenza A viruses (matrix gene), influenza B viruses (matrix gene) and RSV (M gene) are targeted to identify these viruses in the specimen. An RNA internal control is used to detect RT-PCR failure and/or inhibition.

Here's a summary of the acceptance criteria and the study information for the Simplexa™ Flu A/B & RSV Direct device, based on the provided document:

Acceptance Criteria and Device Performance Study

The purpose of this Special 510(k) (K152408) is to expand the analytical reactivity of the Simplexa™ Flu A/B & RSV Direct assay to include 53 additional strains. Therefore, the primary acceptance criteria for this specific submission relate to the successful detection of these new strains.

1. Table of Acceptance Criteria and Reported Device Performance

The core acceptance criterion for the additional strains is detection by the Simplexa™ Flu A/B & RSV Direct assay. All tested strains were successfully detected.

Note: The document explicitly states: "Fifty-three strains met the established acceptance criteria and passed validation testing. Analytical Reactivity will be expanded to add 53 additional strains." This confirms that the acceptance criteria for these specific strains was successful detection.

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

• Test Set Sample Size: For each of the 53 additional strains, the testing was performed in triplicate. This means a total of 53 strains * 3 replicates = 159 individual tests were performed for this specific analytical reactivity study.
• Data Provenance: The strains were sourced from various locations and historical periods, indicating a focus on analytical validity and strain diversity rather than clinical performance from a specific population or region. Examples include strains like "A/California/4/2009", "A/Massachusetts/15/2013", "A/Japan/305/57", "B/Brisbane/33/2008", and "ATCC-2012-10". The study itself is an analytical study conducted in a lab setting, not a direct clinical study. The document doesn't specify the country of origin where the testing took place, but it's likely a controlled lab environment.

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

This study focuses on analytical reactivity, where the "ground truth" is the presence and identification of a specific viral strain at a known concentration. This type of ground truth does not typically involve expert clinical adjudication. The ground truth (i.e., confirmation of the viral strain and its concentration) would have been established by standard laboratory methods for viral culture, quantification (e.g., TCID50/mL, CEID50/mL, PFU/mL), and characterization, prior to being used in the Simplexa™ assay. The document does not specify the number or qualifications of experts involved in establishing this initial ground truth, as it's a standard process in virology.

4. Adjudication Method for the Test Set

No adjudication method (e.g., 2+1, 3+1) is described, as this is an analytical study evaluating device performance against known quantities of target analytes, not a clinical study requiring human interpretation or consensus for diagnosis. The "result" is the output of the RT-PCR system ("Flu A Detected," "Flu B Detected," "RSV Detected").

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

No MRMC comparative effectiveness study was done for this particular submission. This 510(k) is a "Special 510(k)" to expand analytical reactivity, not to re-evaluate or compare clinical effectiveness with human readers. The clinical performance characteristics were established in a previous submission (K142365).

6. Standalone Performance Study (Algorithm Only Without Human-in-the-Loop)

Yes, a standalone performance study was done for the analytical reactivity. The study evaluated the ability of the Simplexa™ Flu A/B & RSV Direct assay system (algorithm/device only) to detect the specified viral strains. The results presented ("Flu A Detected," "Flu B Detected," "RSV Detected") are direct outputs from the device, with no human intervention in the detection process itself.

7. Type of Ground Truth Used

The ground truth used for this analytical reactivity study was based on known quantified viral material. This means:

• Viral Culture/Quantification: Viral samples were quantified using methods like TCID50/mL (Tissue Culture Infectious Dose 50%), CEID50/mL (Chicken Embryo Infectious Dose 50%), PFU/mL (Plaque Forming Units/mL), or using purified RNA at known concentrations (e.g., ng/µL, IU/mL).
• Strain Identification: The specific identity and subtype of each viral strain were confirmed through standard virological methods.
• These known viral materials were then spiked into a negative swab matrix to simulate clinical samples for testing.

8. Sample Size for the Training Set

The document does not provide details about the training set sample size. This submission (K152408) is an expansion of analytical reactivity and refers to the predicate device K142365 for most other performance characteristics, including clinical studies. RT-PCR assays typically do not have a "training set" in the machine learning sense, but rather a development and validation process during which assay parameters are optimized and locked.

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

Similar to point 8, the document does not specifically detail how a "training set ground truth" was established, as it's not a machine learning algorithm in the typical sense that would require a distinct training phase with labeled data in the way, for example, an image recognition AI would. The development of an RT-PCR assay involves optimization and validation studies using characterized viral isolates and clinical samples, where the "ground truth" for these samples is derived from:

• Reference Methods: Such as viral culture, sequencing, or other FDA-cleared assays.
• Expert Consensus: For clinical samples, expert microbiologists or virologists would confirm the presence/absence of target viruses using gold standard methods.

The current document focuses singularly on the analytical reactivity towards an expanded panel of viral strains, relying on the already established methodologies from the predicate device (K142365) for other aspects of performance.

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The Focus Diagnostics Simplexa™ HSV 1 & 2 Direct assay is intended for use on the 3M Integrated Cycler instrument for the qualitative detection and differentiation of herpes simples virus (HSV-1 and HSV-2) DNA present in genital lesion swabs samples from patients with signs and symptoms of HSV-1 or HSV-2 infection of the genitalia. This test is an aid in the differential diagnosis of HSV-1 and HSV-2 genital infections.

The assay is not intended for use as a screening test for the presence of HSV-1 and HSV-2 in blood or blood products. The assay is for professional use only.

Simplexa™ HSV 1 & 2 Positive Control Pack

The Simplexa™ HSV 1 & 2 Positive Control Pack is intended to be used as a control with the Simplexa™ HSV 1 & 2 Direct kit. This control is not intended for use with other assays or systems.

The Simplexa™ HSV 1 & 2 Direct assay system is a real-time PCR that enables the direct amplification, detection and differentiation of HSV-1 and/or HSV-2 DNA from unprocessed genital swab specimens without nucleic acid extraction. The system consists of the Simplexa™ HSV 1 & 2 Direct assay, the 3M Integrated Cycler (with 3M Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories.

In the Simplexa™ HSV 1 & 2 Direct assay, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify HSV-1, HSV-2 and internal control targets. Well conserved regions of the HSV-1 and HSV-2 DNA polymerase genes are targeted to identify HSV-1 and HSV-2 DNA respectively in the specimen. An internal control is used to detect PCR failure and/or inhibition.

The provided document describes the Simplexa™ HSV 1 & 2 Direct assay and its performance evaluation. Here's a breakdown of the acceptance criteria and study details:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are not explicitly stated as distinct "acceptance criteria" but can be inferred from the reported performance results and the common thresholds for such diagnostic tests (e.g., typically ≥ 95% agreement/sensitivity/specificity). The document presents the performance in terms of percent agreement with expected results for reproducibility and sensitivity/specificity for clinical agreement.

Reproducibility (Inter-site, Inter-day, Inter/Intra-assay)

Analytical Sensitivity/Limit of Detection (LoD)

Clinical Agreement (Prospective Study vs. Composite Comparator)

Clinical Agreement (Retrospective Study vs. Validated Bi-Directional Sequencing Assay)

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

• Reproducibility Study:

  • Test Set Size: For each of the six sample pools (HSV-1 Low Positive, HSV-1 Medium Positive, HSV-2 Low Positive, HSV-2 Medium Positive, High Negative, Positive Control), they were tested in triplicate on five different days by two operators at each of three sites. This amounts to: 6 samples * 3 replicates * 5 days * 2 operators * 3 sites = 540 data points (assuming complete datasets). The tables consolidate this, showing "Total Agreement" out of 539 samples for HSV-1, and 539 for HSV-2 and DNA IC across all categories (e.g., 531/539 for HSV-1 Total Agreement, 528/539 for HSV-2 Total Agreement, 539/539 for DNA IC Total Agreement).
  • Data Provenance: The study was conducted at "Three investigative sites," implying a multi-center study within the U.S. (typical for FDA submissions). It's a prospective design as samples were "contrived" (prepared specifically for the study) and tests were performed over different days.

• Clinical Agreement (Prospective Study):

  • Test Set Size:
    • Initially, 718 genital swab samples were prospectively collected.
    • 9 samples were removed (not tested or invalid results on 3 assays).
    • 13 samples were removed (not tested on comparator method sufficiently).
    • 696 samples were used for the primary analysis.
    • Further exclusions for specific analyses: 6 samples excluded from discordant analysis due to insufficient volume for FDA cleared NAAT; 2 samples excluded for HSV-1/HSV-2 discrepancy due to insufficient volume for FDA cleared NAAT.
    • Final analysis for HSV-1: 684 samples.
    • Final analysis for HSV-2: 688 samples.
  • Data Provenance: "Prospectively collected from patients with signs and symptoms of genital herpes simplex virus (HSV) infection from 6 geographically diverse locations." This indicates the data is from the U.S. and is prospective.

• Clinical Agreement (Retrospective Study):

  • Test Set Size: 28 genital swab samples (14 positive HSV-1 and 14 positive HSV-2).
  • Data Provenance: "Retrospectively collected from male patients with signs and symptoms of genital herpes simplex virus (HSV) infection." Specific country of origin is not stated but implies within the U.S. given the FDA submission.

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

• For the Reproducibility Study, the ground truth for contrived samples (low positive, medium positive, high negative, positive control) was established based on the known concentrations of HSV-1 and HSV-2 spiked into the matrix. This is an analytical study, not one requiring clinical experts for ground truth.

• For the Clinical Agreement Studies (Prospective and Retrospective), the ground truth was established by a "composite comparator algorithm" and "validated bi-directional sequencing assay." This ground truth relies on laboratory results rather than expert interpretation of clinical data or images. Therefore, the document does not specify human experts for establishing ground truth, but rather relies on the interpretative methods of the comparator assays. Those interpreting the culture results, sequencing data, and FDA-cleared NAAT results would typically be trained laboratory personnel or clinical laboratorians.

4. Adjudication Method for the Test Set

• Reproducibility Study: No explicit adjudication method is mentioned as the ground truth was "expected results" for contrived samples based on known concentrations. Adjudication wasn't necessary for these controlled samples.

• Clinical Agreement (Prospective Study): An adjudication method was used for discordant results in the composite comparator algorithm:

  • "A positive result for HSV-1 and/or HSV-2 was determined by a positive test result in either the culture or the bi-directional sequencing."
  • "If both the culture and the bi-directional sequencing yielded positive results but disagreed in the differentiation of HSV-1 versus HSV-2, the results of the FDA cleared NAAT were used and a 2 out of 3 rule was followed to determine the type of the virus (e.g. if two of the methods were positive for HSV-1, the final comparator result was HSV-1 positive)." This is a form of 2-out-of-3 or majority rule adjudication.

• Clinical Agreement (Retrospective Study): The ground truth was established by a "validated bi-directional sequencing assay." No specific adjudication process is described beyond the sequencing assay itself.

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, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This study focuses on an in vitro diagnostic (IVD) PCR assay for the detection of viral DNA, not on image interpretation or other tasks typically performed by human readers that could be augmented by AI. Therefore, there's no mention of human readers or AI assistance.

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

• Yes, the performance presented for the Simplexa™ HSV 1 & 2 Direct assay is standalone performance. It describes the diagnostic accuracy of the assay itself (device only) against a comparator method. The device is an automated, real-time PCR system, and its output (qualitative detection and differentiation of HSV-1 and HSV-2 DNA) is directly compared. The assay operates without human "in-the-loop" interpretation for the final diagnostic call.

7. The Type of Ground Truth Used

• Reproducibility Study: Known concentrations of spiked viral material (contrived samples) were used as ground truth.
• Clinical Agreement (Prospective Study): A composite comparator algorithm was used as ground truth, consisting of:
  • Culture
  • Bi-directional sequencing
  • An FDA cleared NAAT (used for discordant resolution)
• Clinical Agreement (Retrospective Study): A validated bi-directional sequencing assay was used as ground truth.

8. The Sample Size for the Training Set

• The document describes performance evaluation studies (reproducibility and clinical agreement) for the Simplexa™ HSV 1 & 2 Direct assay. It does not explicitly mention a "training set" or "validation set" in the context of machine learning model development. This is typical for IVD assays which are developed and then verified/validated rather than 'trained'. The data presented is for validation of the a priori defined assay.

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

• As no "training set" in the machine learning sense is described, this question is not applicable based on the provided document. The ground truth for the validation and reproducibility sets is detailed in point 7.

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The Focus Diagnostics Simplexa™ Group A Strep Direct assay is intended for use on the 3M Integrated Cycler for the in vitro qualitative detection of Group A Streptococcus (GAS) from throat swabs collected from human patients with signs and symptoms of pharyngitis, such as sore throat. This test is intended for use as an aid in the diagnosis of GAS infection. The assay is intended for use in hospital, reference, or state laboratory settings. The device is not intended for point-of-care use.

The Simplexa™ Group A Strep Direct assay system is a real-time PCR system that enables the direct amplification and qualitative detection of Group A Strep bacterial DNA from throat swabs that have not undergone a nucleic acid extraction. The system consists of the Simplexa™ Group A Strep Direct assay, the 3M Integrated Cycler (with Integrated Cycler Studio Software), the Direct Amplification Disc (DAD) and associated accessories.

In the Simplexa™ Group A Strep Direct assay, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify Group A Strep bacterial DNA and the Internal Control (DNA IC). The assay targets a conserved region of Group A Strep (pyroqenic exotoxin B gene) to identify this bacteria in the specimen. The DNA IC is used to detect PCR failure and/or inhibition.

Here is a summary of the acceptance criteria and study information for the Simplexa™ Group A Strep Direct device, based on the provided text:

Acceptance Criteria and Device Performance for Simplexa™ Group A Strep Direct

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria for the clinical study's sensitivity and specificity. However, the performance characteristics of the device and its predicate device are compared. For the purpose of this response, we will consider the reported "Performance Characteristics" of the predicate device as a benchmark or implicit acceptance criteria, though the device has slightly lower specificity than the predicate.

Note: The device's sensitivity (97.4%) is numerically higher than the predicate's (96.5%), and its specificity (95.2%) is numerically lower than the predicate's (98.0%). The FDA's substantial equivalence determination implies these results were acceptable.

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

• Sample Size for Test Set: 1352 evaluable samples.
• Data Provenance: Prospectively collected from four geographically diverse sites. The country of origin is not explicitly stated but can be inferred to be the USA given the FDA submission document.

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

The document does not mention the use of "experts" in the traditional sense (e.g., radiologists) for establishing ground truth. Instead, for the clinical prospective study, the comparator culture method was used as the primary reference for ground truth, and discrepant results were further analyzed.

4. Adjudication Method for the Test Set

The primary comparison was against a "comparator culture method" performed at one central laboratory.
For discrepant analysis, a "validated bidirectional sequencing assay" was performed.

• 46 out of 57 samples where Simplexa™ was Positive and Culture was Not Detected were confirmed as Group A Strep Positive by sequencing.
• 2 out of 4 samples where Simplexa™ was Not Detected and Culture was Detected were confirmed as Group A Strep Positive by sequencing.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not performed. This device is a molecular diagnostic assay (PCR-based system) for the qualitative detection of Group A Streptococcus, not an imaging device requiring human reader interpretation. Therefore, the concept of improving human readers with AI assistance is not applicable here.

6. Standalone Performance Study

Yes, a standalone performance study (algorithm only without human-in-the-loop performance) was performed. The entire clinical prospective study and analytical studies evaluate the device's performance independently. The device's results are compared directly against a culture method and a sequencing assay, not against human interpretation of the device's output.

7. Type of Ground Truth Used

• Primary Ground Truth: Comparator culture method.
• Confirmatory Ground Truth for Discrepancies: Validated bidirectional sequencing assay.

8. Sample Size for the Training Set

The document describes the performance of the device and does not explicitly mention a "training set" in the context of machine learning. The studies described are validation studies (e.g., Limit of Detection, Analytical Reactivity, Cross Reactivity, Interference, Clinical Prospective Study) that assess the device's performance characteristics. If the device's internal algorithms underwent a "training" phase during its development, the details are not provided in this regulatory summary.

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

As no specific "training set" is mentioned for an algorithm, the method for establishing its ground truth is not provided. The document focuses on the validation of the finalized device.

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The Focus Diagnostics Simplexa™ Flu A/B & RSV Direct assay is intended for use on the 3M Integrated Cycler instrument for the in vitro qualitative detection and differentiation of influenza B virus, and respiratory syncytial virus (RSV) RNA in nasopharyngeal swabs (NPS) from human patients with signs and symptoms of respiratory tract infection in conjunction with clinical and epidemiological risk factors. This test is intended for use as an aid in the differential diagnosis of influenza B, and RSV viral infections in humans and is not intended to detect influenza C.

Negative results do not preclude influenza virus or RSV infection and should not be used as the sole basis for treatment or other patient management decisions.

Performance characteristics for influenza A were established with clinical specimens collected during the 2010/2011 influenza season when 2009 H1N1 influenza and H3N2 were the predominant influenza A viruses in circulation. When other influenza A viruses are emerging, performance characteristics may vary.

If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent Influenza viruses and sent to the state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

Simplexa™ Flu A/B & RSV Positive Control Pack REF MOL2660

Focus Diagnostics' Simplexa™ Flu A/B & RSV Positive Control Pack is intended to be used as a control with the SimplexaTM Flu A/B & RSV Direct kit. This control is not intended for use with other assays or systems.

The Simplexa™ Flu A/B & RSV Direct assay system is a real-time RT-PCR system that enables the direct amplification, detection and differentiation of human influenza A (Flu A) virus RNA, human influenza B (Flu B) virus RNA and RSV RNA from unprocessed nasopharyngeal swabs that have not undergone nucleic acid extraction. The system consists of the Simplexa™ Flu A/B & RSV Direct assay, the 3M Integrated Cycler (with Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories.

In the Simplexa™ Flu A/B & RSV Direct assay, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify Flu A, Flu B, RSV and internal control RNA. The assay provides three results; conserved regions of influenza A viruses (matrix gene), influenza B viruses (matrix gene) and RSV (M gene) are targeted to identify these viruses in the specimen. An RNA internal control is used to detect RT-PCR failure and/or inhibition.

The provided text describes a 510(k) summary for the Simplexa™ Flu A/B & RSV Direct and Simplexa™ Flu A/B & RSV Positive Control Pack. This submission is intended to add eight additional influenza strains to the analytical reactivity of the device and addresses modifications made to the device from an earlier version (K120413).

Here's a breakdown of the requested information based on the document:

1. Table of acceptance criteria and the reported device performance

The document does not explicitly state "acceptance criteria" in the format of pass/fail thresholds for clinical performance. Instead, it presents "Positive Percent Agreement (PPA)" and "Negative Percent Agreement (NPA)" with a predicate device (Gen 1.0) and between device versions (Gen 2.0 and Gen 2.1) as part of method comparison studies, along with analytical reactivity and specificity data.

Method Comparison Results (Performance in relation to K120413 and between versions)

Analytical Reactivity (Gen 2.1): All tested influenza A, influenza B, and RSV strains at specified concentrations were detected (100% detection for all, assayed in triplicate). These include:

• 18 Influenza A strains (H1, H3, H7N9)
• 10 Influenza B strains
• 4 RSV strains (A and B)

Cross Reactivity (Analytical Specificity) (Gen 2.1): No cross-reactivity was observed with 32 tested organisms (bacteria and other viruses) at clinically relevant concentrations. All results showed 0% detection for Flu A, Flu B, and RSV, and 100% detection for the Internal Control.

Interference (Gen 2.1): No evidence of interference was observed from potentially interfering substances (e.g., nasal sprays, antiviral drugs, blood, mucin protein) tested in contrived samples. All showed 100% detection for Flu A, Flu B, RSV, and RNA IC.

Limit of Detection (LoD) (Gen 2.1): The LoD for various strains across Gen 1.0, Gen 2.0, and Gen 2.1 are provided (e.g., Influenza A/Hong Kong/8/68 (H3N2) Gen 2.1 LoD: 0.1 TCID50/mL). The criteria for LoD determination was ≥95.0% detection (at least 31/32 replicates).

Precision (Gen 2.1): High reproducibility (low %CV) was observed for Ct values across inter-day, inter-run, inter-lot, and intra-run/lot variations for low and moderate positive samples of Flu A, Flu B, and RSV, as well as positive and negative controls. Qualitatively, all expected positive samples were detected at 100%, and negative samples were not detected for the target analytes.

2. Sample size used for the test set and the data provenance

• Sample Size (Method Comparison): For each comparison (Gen 1.0 vs Gen 2.0, and Gen 2.0 vs Gen 2.1), 265 archived clinical samples were used.

  • Composition: 55 positive for influenza A, 55 positive for influenza B, 55 positive for RSV, and 100 negative for all tested viruses.
  • Data Provenance: The samples were "archived clinical samples" in Universal Transport Medium (UTM) or Viral Transport Medium (VTM).
    • 131 of these samples for the Gen 1.0 vs Gen 2.0 comparison were originally tested in support of K120413. The remaining 134 included 33 from the 2010-2011 flu season and 101 from the 2013-2014 flu season.
    • For the Gen 2.0 vs Gen 2.1 comparison, 125 samples were from K120413 study. The remaining 140 included 48 from the 2010-2011 flu season, 9 from 2012-2013, and 83 from 2013-2014.
    • The country of origin is not specified, but the context implies data likely from the USA (given FDA submission). The data is retrospective as it uses archived samples.

• Sample Size (Analytical Reactivity/Cross Reactivity/Interference):

  • Analytical Reactivity: Each viral strain was assayed in triplicate.
  • Cross Reactivity: Each organism was tested in triplicate (3 replicates). Baseline negative matrix was tested in five (5) replicates.
  • Interference: Each interfering substance was tested in triplicate (3 replicates). Baseline was tested in 15 replicates.
  • Limit of Detection: Initially, 4 concentrations per virus tested in triplicate. Confirmatory testing involved 32 replicates for the lowest concentration.
  • Precision: Each sample panel member tested in duplicate for each Reaction Mix lot in each run, two runs per day for a total of three days, yielding at least 36 replicates per panel member (41 for one Flu B sample).

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

The document does not explicitly state the use of "experts" to establish ground truth for the test set in the method comparison studies. The ground truth for these studies appears to be based on results from the predicate device (Simplexa™ Flu A/B & RSV Direct Gen 1.0) and/or other FDA cleared Nucleic Acid Tests (NATs) for discrepant samples. For analytical studies (reactivity, cross-reactivity, interference, LoD, precision), the ground truth is established by the known concentration/presence of the spiked organisms or substances.

4. Adjudication method for the test set

For the method comparison studies, discrepancies between the modified device (Gen 2.0 or Gen 2.1) and the predicate device (Gen 1.0 or Gen 2.0 respectively) were sometimes resolved using another FDA cleared NAT. For example, for Flu A discrepancies in the Gen 1.0 vs Gen 2.0 comparison, "7/9 discrepant (K120413 – Negative and K142365 – Positive) samples were positive for Flu A on another FDA cleared NAT." This suggests a form of adjudication using a third, independent, cleared method.

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

This section is not applicable. The device described is an in vitro diagnostic (IVD) assay for the detection of viruses using real-time RT-PCR, not an AI-powered diagnostic imaging device involving human readers or interpretation of medical images. Therefore, MRMC studies and the concept of human reader improvement with AI assistance do not apply.

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

This concept is not directly applicable in the context of an IVD assay like the Simplexa™ Flu A/B & RSV Direct. The device is essentially a "standalone" algorithm/assay from the perspective of direct human interpretation providing a qualitative result (detected/not detected). The performance metrics (PPA, NPA, analytical reactivity, LoD, etc.) represent the standalone performance of the assay system. There is no human "in the loop" for interpreting the raw assay output; the instrument's software interprets the Ct values to provide a qualitative result.

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

• Clinical Studies (Method Comparison): The ground truth for the clinical sample comparisons was based on:
  • The performance of the predicate device (Simplexa™ Flu A/B & RSV Direct Gen 1.0) for direct comparison between versions.
  • Other FDA cleared Nucleic Acid Tests (NATs) for resolving discrepant results between the device versions.
• Analytical Studies (Reactivity, Cross-Reactivity, Interference, LoD, Precision): The ground truth was established by known spiked concentrations of characterized viral strains, bacterial organisms, or potentially interfering substances into negative matrix.

8. The sample size for the training set

The document does not explicitly mention a "training set" in the context of machine learning or AI models, as this is an IVD assay. The development and optimization ("changes to the reaction mix formulation and cycling conditions," "changes to the manufacturing process and materials") that led to Gen 2.0 and Gen 2.1 would have involved internal validation and optimization data, which could be considered analogous to training data in a broad sense for assay development. However, specific "training set sizes" are not provided.

9. How the ground truth for the training set was established

As described in point 8, a formal "training set" for an AI model is not applicable here. For the assay development and optimization, ground truth would have been established through controlled laboratory experiments using well-characterized viral isolates and defined concentrations, analogous to how ground truth for the analytical studies (reactivity, LoD) was established.

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The Focus Diagnostics Simplexa™ HSV 1 & 2 Direct is intended for use on the 3M Integrated Cycler instrument for the qualitative detection and differentiation of HSV-1 and HSV-2 DNA in cerebrospinal fluid (CSF) samples from patients suspected of herpes simplex virus (HSV) infections of the central nervous system (CNS). This test is intended as an aid in the diagnosis of HSV-1 and HSV-2 infections of the CNS.

Negative results do not preclude HSV-1 or HSV-2 infection and should not be used as the sole basis for treatment or other patient management decisions.

The assay is not intended for use as a donor screening test. The assay is for professional use only.

The Simplexa™ HSV 1 & 2 Positive Control Pack is intended to be used as a control with the Simplexa™ HSV 1 & 2 Direct kit. This control is not intended for use with other assays or systems.

The Simplexa™ HSV 1 & 2 Direct assay system is a real-time PCR that enables the direct amplification, detection and differentiation of HSV-1 and/or HSV-2 DNA from unprocessed CSF specimens without nucleic acid extraction. The system consists of the Simplexa™ HSV 1 & 2 Direct assay, the 3M Integrated Cycler (with 3M Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories.

In the Simplexa™ HSV 1 & 2 Direct assay, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify HSV-1, HSV-2 and internal control targets. Well conserved regions of the HSV-1 and HSV-2 DNA polymerase genes are targeted to identify HSV-1 and HSV-2 DNA respectively in the specimen. An internal control is used to detect PCR failure and/or inhibition.

The provided text describes a 510(k) summary for the Simplexa™ HSV 1 & 2 Direct and Simplexa™ HSV 1 & 2 Positive Control Pack, which is a modification to a previously cleared device (K133621). The changes are specifically related to the Integrated Cycler Studio Software version 6.0.

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

1. A table of acceptance criteria and the reported device performance

The document mentions that "Verification activities for the Integrated Cycler Studio software version 6.0 included development of verification test plans with defined acceptance criteria (design inputs), conducting and documenting verification testing and review of the verification results as they compared to the verification test plans predetermined acceptance criteria (design outputs)." It also states that "Integrated Cycler Studio software version 6.0 was validated in a similar fashion with the development of validation test plans with defined acceptance criteria (design inputs), conducting validation testing and review of the validation results as they compared to the validation test plans predetermined acceptance criteria (design outputs)."

However, the specific acceptance criteria (e.g., percentage agreement, sensitivity, specificity, or specific values for these metrics) for the software's performance with the assay, and the reported device performance against these criteria, are not explicitly detailed in the provided text. The text only states that "The results of verification and validation of Integrated Cycler Studio software version 6.0 show the results met the predetermined acceptance criteria." and "The results of assays ran with the Integrated Cycler Studio software version 6.0 demonstrate that the results obtained with the previously released versions of Integrated Cycler Studio software were equivalent to the results obtained using Integrated Cycler Studio software version 6.0."

Without the actual defined acceptance criteria and the quantitative or qualitative results against them, a detailed table cannot be created from this document.

2. Sample size used for the test set and the data provenance (e.g. country of origin of the data, retrospective or prospective)

The document focuses on software changes and comparison to a predicate device. It indicates that "The results of assays ran with the Integrated Cycler Studio software version 6.0 demonstrate that the results obtained with the previously released versions of Integrated Cycler Studio software were equivalent to the results obtained using Integrated Cycler Studio software version 6.0." This suggests that a comparison study was performed, likely using existing assay data or running new assays with the modified software.

However, the sample size for any test set, the specific data provenance (country of origin), and whether the data was retrospective or prospective are not mentioned in the provided text.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience)

This information is not provided in the text. The device is an in vitro diagnostic for detecting HSV-1 and HSV-2 DNA. The "ground truth" for such devices is typically established through molecular reference methods or clinical diagnosis, not usually expert consensus in the same way as imaging analysis.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set

This information is not applicable or provided in the text. Adjudication methods are typically used in studies involving human interpretation (e.g., radiologists reviewing images), where disagreement among experts needs resolution. For a PCR-based diagnostic device, the assessment of results against a ground truth would follow established laboratory protocols for molecular diagnostics, not human adjudication.

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

An MRMC study is not applicable here as the device is a direct molecular diagnostic test, not an AI-assisted human reader interpretation system. The device directly detects viral DNA.

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

The device is inherently a "standalone" algorithm (PCR assay interpreted by software) in the sense that it performs the detection and differentiation of HSV DNA without a human interpreting the primary assay signal directly for diagnosis. The software (Integrated Cycler Studio) processes the raw data from the PCR reaction to deliver a qualitative result (presence/absence of HSV-1/HSV-2 DNA). The statement that "The results of assays ran with the Integrated Cycler Studio software version 6.0 demonstrate that the results obtained with the previously released versions of Integrated Cycler Studio software were equivalent to the results obtained using Integrated Cycler Studio software version 6.0" implies an assessment of the software's performance on its own.

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

Given that this is a molecular diagnostic for HSV DNA, the ground truth would typically be established by a highly sensitive and specific reference molecular method (e.g., an FDA-cleared or a well-validated in-house laboratory developed test with stringent analytical performance characteristics, or potentially by clinical diagnosis combined with other laboratory findings). The specific method for establishing ground truth is not explicitly stated in the provided text.

8. The sample size for the training set

The document describes software version changes and verification/validation activities. There is no mention of a "training set" in the context of machine learning or AI. The software is designed to interpret PCR assay results.

9. How the ground truth for the training set was established

As there is no mention of a "training set" or AI/machine learning development, this question is not applicable based on the provided text.

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The Focus Diagnostics Simplexa™ HSV 1 & 2 Direct is intended for use on the 3M Integrated Cycler instrument for the qualitative detection and differentiation of HSV-1 and HSV-2 DNA in cerebrospinal fluid (CSF) samples from patients suspected of Herpes Simplex Virus (HSV) infections of the central nervous system (CNS). This test is intended as an aid in the diagnosis of HSV-1 and HSV-2 infections of the CNS.

Negative results do not preclude HSV-1 or HSV-2 infection and should not be used as the sole basis for treatment or other patient management decisions.

The assay is not intended for use as a donor screening test. The assay is for professional use only.

The Positive Control is intended to be used as a control with the Simplexa™ HSV 1 & 2 Direct. This control is not intended for use with other assays or systems.

The Simplexa™ HSV 1 & 2 Direct system is a real-time PCR that enables the direct amplification, detection and differentiation of HSV-2 DNA from unprocessed CSF samples without nucleic acid extraction. The system consists of the Simplexa™ HSV 1 & 2 Direct, the 3M Integrated Cycler (with 3M Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories.

In the SimplexaTM HSV 1 & 2 Direct, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify HSV-1, HSV-2 and DNA internal control (IC) targets. Well conserved regions of the HSV-1 and HSV-2 DNA polymerase genes are targeted to identify HSV-1 and HSV-2 DNA respectively in the specimen. The IC is used to detect PCR failure and/or inhibition.

The 3M Integrated Cycler is a real-time PCR thermocycler which uses real-time fluorometric detection to identify targets within the sample wells. The instrument is controlled by an external computer running the Integrated Cycler Studio Software.

The Simplexa™ HSV 1 & 2 Direct assay reaction takes place in the DAD consumable. The DAD consumable is compartmentalized into 8 separate wedges and up to 8 separate samples or controls may be processed on each disc. Each wedge can be used only once, however, the disc may be reused until all wedges have been utilized. Each wedge contains sample and reagent input wells, microfluidic channels and laser activated valves to control the fluid flow. and a reaction chamber. The disc is specifically designed to meter the amount of reagent (Reaction Mix) and sample that are placed into specific wells in the disc. To start processing a patient sample, a foil seal is lifted and the user adds 50 µL of Reaction Mix to the reagent input well using a fixed volume pipette. Next, the user adds 50 uL of unextracted specimen to the sample input well.

The SimplexaTM HSV 1 & 2 Direct kit contains reagents for 24 reactions. Each vial contains sufficient material for a single reaction. The kit contains the Reaction Mix, the Simplexa™ HSV 1 & 2 Direct Barcode Card with assay specific information, and the Package Insert.

This document describes the evaluation of the Simplexa™ HSV 1 & 2 Direct device, a real-time PCR test for detecting and differentiating HSV-1 and HSV-2 DNA in cerebrospinal fluid (CSF). The evaluation supports its classification as a Class II device with special controls.

Here's an analysis of the acceptance criteria and the studies that prove the device meets them:

1. Table of Acceptance Criteria and Reported Device Performance:

The document does not explicitly state "acceptance criteria" in a singular table for all performance measures. Instead, performance is demonstrated through various analytical and clinical studies, with implicit acceptance criteria being met if the results are favorable (e.g., high agreement rates, low variability, no cross-reactivity). For the clinical study, the performance is measured by Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) compared to a comparator method.

Let's summarize the key performance aspects that serve as de facto acceptance criteria based on the presented data:

2. Sample Sizes Used for the Test Set and Data Provenance:

• Reproducibility Test Set:

  • 6 samples (high negative, low positive, moderately positive for HSV-1 and HSV-2, and a positive control)
  • Total of 90 replicates per sample panel member (3 sites x 5 days x 2 runs x 3 replicates) = 540 replicates per analyte group (HSV-1, HSV-2, IC).
  • Data Provenance: Not explicitly stated, but implies multi-site, laboratory-based testing for analytical performance validation.

• Detection Limit (LoD) Test Set:

  • Two primary strains (HSV-1 McIntyre, HSV-2 G): Each dilution assayed in 32 replicates across 32 DAD runs.
  • Two additional strains (HSV-1 HF, HSV-2 MS): 3-32 replicates per dilution/concentration for screening and confirmation.
  • Data Provenance: Laboratory-generated samples (viral stock spiked into negative human CSF matrix).

• Analytical Reactivity Test Set:

  • 2 additional HSV-1 strains. Each spiked into negative CSF, assayed in triplicate. (3 replicates per strain, with 2 repeated; total 12 technical replicates for HSV-1 strains).
  • Data Provenance: Laboratory-generated samples.

• Analytical Specificity (Cross-Reactivity) Test Set:

  • 51 potential cross-reactants.
  • Each assayed in triplicate. (3 replicates per cross-reactant).
  • Data Provenance: Laboratory-generated samples (organisms spiked into negative CSF).

• Interference Test Set:

  • Interfering Substances: 7 substances tested. Each in low positive HSV-1 and HSV-2 sample, assayed in triplicate (3 replicates per interferent).
  • Competitive Interference: Samples contrived with low and high concentrations of HSV-1 and HSV-2. Replicates ranged from 3 to 8 depending on the condition.
  • Inhibition by Other Microorganisms: 51 microorganisms. Initially tested in triplicate, then 5 additional replicates if any "Not Detected" (total 8 replicates if retested).
  • Data Provenance: Laboratory-generated samples (substances/organisms spiked into CSF).

• Sample Stability Test Set:

  • A panel of 120 pairs of contrived samples (60 for HSV-1, 60 for HSV-2). Each pair consisted of a fresh and a frozen aliquot.
  • Data Provenance: Laboratory-contrived samples (spiked in negative human CSF matrix).

• Carry-over Contamination Test Set (from K120413 study):

  • 60 negative samples and 60 high positive samples tested across 17 runs.
  • Data Provenance: Not specified, but likely laboratory-contrived positive and negative samples for the previous device clearance.

• Clinical Studies Test Set:

  • Prospective Samples: 164 CSF samples.
  • Retrospective/Preselected Positive Samples: 55 CSF samples.
  • Total Clinical Samples: 219 CSF samples.
  • Data Provenance: Collected from eight external sites. Patients with signs and symptoms of Herpes Simplex Virus (HSV) central nervous system (CNS) infection. Submitted to Focus Diagnostics (frozen). Tested at five external sites (Simplexa™) and Focus Diagnostics (comparator). Retrospective samples were collected between 2004 and 2013, indicating retrospective data collection for these specific samples.

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

• Analytical Studies (Reproducibility, LoD, Reactivity, Specificity, Interference, Stability, Carry-over): The ground truth for these studies was established by the precise formulation and characterization of the contrived samples (e.g., known viral strains at specific concentrations, known presence/absence of interferents). No "experts" in the clinical sense were used to establish ground truth for these analytical tests.

• Clinical Studies:

  • The ground truth for the clinical samples was established using a "comparator" method: two PCR/bi-directional sequencing assays targeting two distinct regions of the HSV genome.
  • The interpretation of clinical diagnosis (e.g., "Final Diagnosis positive for HSV infection") used "the patient's attending physician and other clinical information such as chemistries, bacterial culture, MRI/CT scans and in-house PCR results." This implies expertise by the treating physicians, but the ground truth for comparison with the device was the defined PCR/sequencing method.
  • Number of experts and specific qualifications (e.g., radiologist with 10 years of experience) for establishing the comparator ground truth are not specified. It's implicitly handled by the laboratory performing the two PCR/bi-directional sequencing assays following established protocols.

4. Adjudication Method for the Test Set:

• Analytical Studies: Adjudication method is not applicable as the ground truth is pre-defined by the experimental setup. Results are evaluated based on agreement with expected outcomes.

• Clinical Studies:

  • The "comparator" method itself involved two PCR/bi-directional sequencing assays. A positive result was defined as positive by at least one of these two assays. A negative result required both assays to be negative.
  • This is a form of "rule-based adjudication" to derive a single ground truth from a multi-component comparator. It's not a consensus of human readers, but rather a defined algorithm for combining results from two independent laboratory assays.

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, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done.
• This device is an in vitro diagnostic (IVD) assay (a laboratory test) for direct detection of viral DNA, not an artificial intelligence (AI) solution intended to assist human readers in interpreting images or other data. Therefore, the concept of human readers improving with AI assistance is not applicable to this submission.

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

• Yes, the primary performance evaluation of the Simplexa™ HSV 1 & 2 Direct assay is a standalone, algorithm-only performance.
• The device performs the amplification, detection, and differentiation of HSV-1 and HSV-2 DNA automatically based on the real-time PCR method. The results are calculated and displayed by the Integrated Cycler Studio Software. The performance characteristics (e.g., sensitivity, specificity, reproducibility, LoD) are determined for the device operating independently without subjective human interpretation of the primary signal generation. Human involvement is limited to sample preparation, loading, and interpreting the final "Detected," "Not Detected," or "Invalid" report from the software.

7. The Type of Ground Truth Used:

• Analytical Studies: The ground truth used for analytical studies (LoD, reactivity, specificity, interference) was laboratory-contrived samples with known concentrations of viral targets or known presence/absence of interferents/cross-reactants. For reproducibility, the expected result for each sample was known.
• Clinical Studies: The ground truth for clinical studies was established by a comparator method consisting of two PCR/bi-directional sequencing assays. This is a molecular/laboratory-based ground truth, not pathology or direct outcomes data, although clinical information was collected to characterize the patient population.

8. The Sample Size for the Training Set:

• This submission describes the evaluation and approval of an in vitro diagnostic (IVD) assay, not a machine learning or AI algorithm. Therefore, there is no explicit "training set" in the context of machine learning.
• The development of the assay (e.g., primer design, probe selection, cut-off determination) would have involved internal optimization and verification studies, but these are not typically referred to as a "training set" in the same way as for AI. The document mentions "feasibility and verification studies" for determining the assay cut-off.

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

• As noted above, there is no explicit "training set" for a machine learning algorithm.
• For the development and optimization of the PCR assay itself, the "ground truth" (e.g., optimal primer/probe sequences, reaction conditions, Ct cut-offs) would have been established through iterative laboratory experimentation, validation against characterized viral stocks, and empirical performance testing to achieve desired sensitivity and specificity. The document mentions that the cut-off was determined during "feasibility and verification studies" using LoD verification study replicates.

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The Focus Diagnostics' STRATIFY JCV® DxSelect™ assay is intended for the qualitative detection of antibodies to JC virus in human serum or plasma. The assay is intended for use in conjunction with other clinical data, in multiple sclerosis patients receiving or considering natalizumab therapy, as an aid in risk stratification for progressive multifocal leukoencephalopathy development. The assay is for professional use only.

The assay is not intended for donor screening. The performance of this assay has not been established for use in other immunocompromised patient populations or patients with different disease conditions or undergoing other treatments or in neonates and pediatric patient populations.

The Focus Diagnostics' STRATIFY JCV® DxSelect™ test is an ELISA assay. JC virus-like particles (VLP) are pre-coated onto 96-well microiter plates. Diluted serum or plasma specimens and controls are incubated in the wells to allow JCV-specific antibodies present in the specimens to react with the JC VLP antigen. Nonspecific reactants are removed by washing. Peroxidase-conjugated anti-human antibodies are added to react with JCV-specific antibodies. Excess conjugate is removed by washing. Enzyme substrate and chromogen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is quantified by a spectrophotometric reading of optical density (OD). Specimen OD readings are compared with Cut-Off Calibrator OD readings to determine results. Each specimen result is reported as an index value. A specimen with an index value that is greater than a specified upper cut-off is reported as positive for detectable JCV-specific antibodies, whereas a specimen with an index value less than the specified lower cut-off is reported as negative for detectable JCV-specific antibodies. A specimen with an index value that is equal to or between the upper and lower cut-off values is reported as indeterminate. An indeterminate result requires further evaluation in the confirmation (inhibition) assay.

In the confirmation assay, soluble JC VLP antigen will compete with plate bound JC VLP antigen for the JCV-specific antibodies present in the serum or plasma specimens. After washing away the unbound antibodies, peroxidase-conjugated anti-human antibodies are added and bind to any captured JCVspecific antibodies. Excess conjugate is removed by washing. Enzyme substrate and chromagen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is quantified by a spectrophotometric reading of OD. The percent inhibition is calculated to confirm presence of JCV-specific antibodies in the specimens with a percent inhibition value that is greater than the specified cut-off are reported as positive for detectable JCV-specific antibodies, whereas specimens with percent inhibition values less than or equal to the cut-off are reported as negative for detectable JCV-specific antibodies.

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

Acceptance Criteria and Device Performance for STRATIFY JCV® DxSelect™

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly present a "table of acceptance criteria" in the traditional sense, but rather demonstrates performance against the predicate device and established clinical guidelines. The key performance metrics evaluated are Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) with a validated laboratory methodology (the predicate device) and JCV antibody positivity rate in certain patient populations.

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The Focus Diagnostics Simplexa™ Flu A/B & RSV Direct assay is intended for use on the 3M Integrated Cycler instrument for the in vitro qualitative detection and differentiation of influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) RNA in nasopharyngeal swabs (NPS) from human patients with signs and symptoms of respiratory tract infection in conjunction with clinical and epidemiological risk factors. This test is intended for use as an aid in the differential diagnosis of influenza A, influenza B, and RSV viral infections in humans and is not intended to detect influenza C.

Negative results do not preclude influenza virus or RSV infection and should not be used as the sole basis for treatment or other patient management decisions.

Performance characteristics for influenza A were established with clinical specimens collected during the 2010/2011 influenza season when 2009 H1N1 influenza and H3N2 were the predominant influenza A viruses in circulation. When other influenza A viruses are emerging, performance characteristics may vary.

If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent Influenza viruses and sent to the state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

Focus Diagnostics' Simplexa™ Flu A/B & RSV Positive Control Pack is intended to be used as a control with the Simplexa™ Flu A/B & RSV Direct kit. This control is not intended for use with other assays or systems.

The Simplexa™ Flu A/B & RSV Direct assay system is a real-time RT-PCR system that enables the direct amplification, delection and differentiation of human influenza A (Flu A) virus RNA, human influenza B (Flu B) virus RNA and RSV RNA from unprocessed nasopharyngeal swabs that have not undergone nucleic acid extraction. The system consists of the Simplexa™ Flu A/B & RSV Direct assay, the 3M Integrated Cycler (with Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories.

In the Simplexa™ Flu A/B & RSV Direct assay, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify Flu A, Flu B, RSV and internal control RNA. The assay provides three results; conserved regions of influenza A viruses (matrix gene) influenza B viruses (matrix gene) and RSV (M gene) are targeted to identify these viruses in the specimen. An RNA internal control is used to detect RT-PCR failure and/or inhibition.

The 3M Integrated Cycler is a rapid real-time Polymerase Chain Reaction thermocycler used for the identification of nucleic acid from prepared biological samples. The instrument utilizes disk media to contain and to process samples. The instrument uses real time flourometric detection to identify targets within the sample wells. The instrument is controlled by an external computer running the Integrated Cycler Studio software.

Here's a breakdown of the acceptance criteria and study details for the Simplexa™ Flu A/B & RSV Direct assay, based on the provided 510(k) summary:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are not explicitly stated as numerical targets in the document. However, the reported performance data from the clinical studies serve as the basis for demonstrating the device's acceptable performance. For clarity, I've listed the reported performance as if those were the implicit acceptance targets for the study.

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The Focus Diagnostics Simplexa™ C. difficile Universal Direct is a real-time polymerase chain reaction (PCR) assay and is intended for use on the 3M Integrated Cycler instrument for the detection of toxigenic Clostridium difficile toxin B gene (tcdB) in liquid or unformed stool samples from individuals suspected of C. difficile infection. This test aids in the diagnosis of Clostridium difficile associated disease (CDAD).

The test is a real-time polymerase chain reaction (PCR) amplification system that utilizes bifunctional fluorescent probe-primers for the detection of C. difficile in liquid or unformed stool. The Simplexa™ C. difficile Universal Direct kit contains primes, buffers and controls. The assay is composed of two principal steps: (1) Heat treatment of stool samples, (2) Amplification of the C. difficile DNA and internal control DNA using bi-functional fluorescent probe-primers together with reverse primers. The DNA internal control is used to monitor potential presence of PCR inhibitors. The assay targets a sequence which is in a well conserved region of C. difficile toxin B gene (tcdB).

1. Acceptance Criteria and Reported Device Performance

Note: For clinical performance, the acceptance criteria are not explicitly stated as numerical targets within the document provided. Instead, the performance is reported and implicitly compared to predicate devices or considered acceptable for the intended use. The reproducibility acceptance criteria are inferred from the 100% or >90% agreement shown in the predicate device data section of the comparison table.

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

• Reproducibility: A "panel" of contrived samples (high negative, medium positive) spiked with C. difficile bacterial stock was used. For each of the three sites, the Low Positive, Positive Control, High Negative, and No Template Control samples were tested in 30 replicates each (with 29 replicates for one medium positive sample at one site).
• Limit of Detection (LoD): The LoD was determined using three replicates in an initial screening phase, followed by confirmation using twenty replicates for two C. difficile bacterial strains.
• Analytical Reactivity: 20 different C. difficile strains were tested, each in triplicate.
• Cross-Reactivity: A total of 119 potential cross-reactants were tested. Each cross-reactant and baseline sample was tested in multiple replicates (implied at least 3, as mentioned in the interference section that "One replicate reported as "Invalid"... in initial run of three replicates").
• Interference: 21 potentially interfering substances were spiked into low positive C. difficile samples and tested. The results typically show 3/3 replicates detected for each substance and strain, with some exceptions tested in 5/5 or with repeat runs for invalid/not detected results.
• Clinical Studies: A total of 970 prospectively collected stool specimens were obtained.
  • Data Provenance:
    • Site 1: Prospectively collected fresh specimens from the East Coast of the US.
    • Site 2: Prospectively collected fresh specimens (and performed toxigenic culture for all specimens, including those from other sites) from the East Coast of the US.
    • Site 3: Prospectively collected clinical specimens from the West Coast of the US and Upper Mid-West of the US.

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

• Reproducibility, LoD, Analytical Reactivity, Cross-Reactivity, Interference: Ground truth for these analytical studies was established by preparing bacterial stocks and contrived samples with known concentrations and identities. This does not typically involve human experts in the same way clinical ground truth does. The experiments were likely designed and performed by trained laboratory personnel. The document does not specify the number or qualifications of these individuals.
• Clinical Studies:
  • Ground Truth Method: "Toxigenic Culture" (Direct Culture + Toxin Assay and Enriched Culture + Toxin Assay) was used as the reference method. This is a laboratory-based method.
  • Experts: The document does not specify the number of experts or their qualifications for establishing the toxigenic culture results. It states that "Site 2 conducted all direct and enriched toxigenic culture testing for all specimens," implying trained laboratory personnel rather than a panel of clinical experts for interpretation.

4. Adjudication Method for the Test Set

• Analytical Studies (Reproducibility, LoD, Analytical Reactivity, Cross-Reactivity, Interference): Adjudication methods are not explicitly described for these laboratory experiments. The results are typically quantitative or categorical (detected/not detected) based on the assay's output. Any "invalid" results (e.g., in reproducibility and interference sections) led to retesting or were noted.
• Clinical Studies: The reference method for clinical studies was "Toxigenic Culture." The document does not describe any specific adjudication process involving multiple experts for the toxigenic culture results. "Site 2 conducted all direct and enriched toxigenic culture testing."

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

• No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not performed. This study focuses on an in vitro diagnostic (IVD) assay (PCR) for detecting a pathogen, not on human readers interpreting images or data with or without AI assistance. Therefore, there is no effect size of human readers improving with AI assistance.

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

• Yes, this study primarily assesses the standalone performance of the Simplexa™ C. difficile Universal Direct assay (an algorithm-based PCR method) without human interpretation as part of the primary diagnostic output. The device itself is an automated real-time PCR system. While human operators are involved in sample preparation and running the instrument, the result (detected/not detected) is generated automatically by the "detection techniques" of "Real time PCR with bi-functional fluorescent probe-primers using the 3M Integrated Cycler."

7. The Type of Ground Truth Used

• Analytical Studies: Ground truth was established through known concentrations of bacterial strains and contrived samples for LoD, analytical reactivity, cross-reactivity, and interference studies.
• Clinical Studies: Ground truth for the clinical agreement study was established using Toxigenic Culture (Direct Culture + Toxin Assay and Enriched Culture + Toxin Assay). This is a laboratory-based gold standard for detecting toxigenic C. difficile.

8. The Sample Size for the Training Set

• The document describes premarket notification (510(k)) studies for a diagnostic device. It does not mention a "training set" in the context of machine learning. The studies described are validation studies (analytical and clinical) performed on the final device. Therefore, a specific sample size for a training set is not applicable in this context.

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

• As a "training set" for machine learning is not applicable in this context, the method for establishing its ground truth is also not applicable.

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