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The Sentosa® SA201 HSV-1/2 PCR Test is a real-time PCR-based qualitative in vitro diagnostic test for detection and differentiation of Herpes Simplex Virus (HSV-1 and HSV-2) DNA from male and female skin lesions from anogenital or oral sites. The test is intended for use as an aid in diagnosis of herpes infection in symptomatic patients.

Warning: The Sentosa® SA201 HSV-1/2 PCR Test is not FDA cleared for use with cerebrospinal fluid (CSF). The test is not intended to be used for prenatal screening.

The Sentosa® SA201 HSV-1/2 PCR Test is a (4x24) configuration contains reagents and enzymes for specific amplification of a 104 bp (base-pair) fragment of the UL30 gene common to both HSV1 and HSV2, and specific probes for the direct detection and differentiation of HSV1 and HSV2 amplicons, respectively. Pathogen detection by PCR is based on the amplification of specific regions of the pathogen genome. In real-time PCR, the amplified product is detected via fluorescent dyes, which are usually linked to oligonucleotide probes that bind specifically to the target sequences. Real-time monitoring of the fluorescence intensities during a PCR run allows the detection of the accumulating product. Amplification of the targets occurs in three channels: green, orange and red on the Sentosa® SA201. Output is recorded as the increase of fluorescence over time in comparison to background signal. Monitoring the fluorescence intensities during the PCR run allows the detection of the accumulating product without having to re-open the reaction tubes after the PCR run.

The Sentosa® SA201 HSV-1/2 PCR Test workflow starts with extraction of nucleic acids from samples (anogenital or oral swabs) using the Sentosa® SX Virus Total Nucleic Acid Kit on the Sentosa SX101 instrument. Following extraction, the instrument will automatically set up the PCR with the extracted nucleic acids in a 96-well PCR plate. Subsequently, the 96-well PCR plate is sealed and transferred to the Sentosa® SA201 for PCR amplification, followed by data analysis.

The Sentosa® Link facilitates data transfer between the Sentosa® SX101, the Sentosa® SA201 Reporter and existing LIS/LIMS (laboratory information systems) in the clinical lab. The Sentosa SX101 instrument communicates with Sentosa® SA201 thermocycler. This creates a user environment that links the SX101 and the Sentosa® SA201 to facilitate automated workflow to export results in a LIS/LIMS-compatible format.

The provided document describes the analytical and clinical performance of the Sentosa SA201 HSV-1/2 PCR Test, a qualitative in vitro diagnostic test for the detection and differentiation of Herpes Simplex Virus (HSV-1 and HSV-2) DNA. It is not an AI/ML-based device. Therefore, the questions related to AI/ML specific aspects (e.g., number of experts, adjudication, MRMC study, training set, ground truth for training set) are not applicable. The information focuses on the device's ability to accurately detect and differentiate HSV-1 and HSV-2 DNA in patient samples.

Here's an analysis of the provided information, focusing on the device's acceptance criteria and the studies proving it meets them:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state "acceptance criteria" in a single table, but the performance studies demonstrate implied criteria. For diagnostic devices like this, the key performance metrics are sensitivity, specificity, limit of detection (LoD), precision, and freedom from interference.

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The Aptima Herpes Simplex Viruses 1 & 2 assay (Aptima HSV 1 & 2 assay) is an in vitro diagnostic nucleic acid amplification test (NAAT), using real time transcription-mediated amplification (TMA), for the qualitative detection and differentiation of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) messenger RNA (mRNA) in clinician-collected swab specimens from anogenital skin lesions. The assay is intended for use with swab specimens placed in Aptima specimen transport medium (STM) or in viral transport media (VTM) that is immediately diluted into STM.

The Aptima HSV 1 & 2 assay is intended for use as an aid in the diagnosis of HSV-1 and/or HSV-2 infections in symptomatic male and female patients. The Aptima HSV 1 & 2 assay is indicated for use on the Panther® system.

The Aptima Herpes Simplex Virus 1 & 2 assay (Aptima HSV assay) is a nucleic acid amplification test (NAAT) developed for use on the fully automated Panther system that utilizes target capture, transcription mediated amplification (TMA), and real-time detection of HSV-1, HSV-2, and an internal control (IC). The Aptima HSV assay amplifies and detects mRNAs for HSV-1 and HSV-2. These RNAs are expressed from the viral genome during the infection cycle, and are packaged inside HSV-1 and HSV-2 viral particles prior to virus release from infected cells. The Aptima HSV assay therefore detects virus-infected cells and the mature virus particles themselves.

The Aptima HSV assay involves three main steps, which all take place in a single tube on the Panther® system: target capture, target amplification by TMA, and detection of the amplification products (amplicon) by the fluorescent labeled probes (torches). The assay incorporates an IC in every test to monitor targeted nucleic acid capture, amplification and detection.

When the Aptima HSV assay is performed, the targeted viral mRNA and IC are isolated using magnetic microparticles and target-specific capture oligomers, in a process called target capture. The capture oligomers contain sequences complementary to specific regions of the targeted RNA (HSV mRNA or IC) as well as a string of deoxyadenosine residues. During the hybridization step, the sequence-specific regions of the capture oligomers bind to specific regions of the RNA target molecules. The microparticles, including the captured RNA target molecules bound to them, are pulled to the side of the reaction tube using magnets and the supernatant is aspirated. The particles are washed to remove residual specimen matrix that may contain amplification inhibitors. After target capture steps are completed, the specimens are ready for amplification.

Target amplification occurs via TMA, which is a transcription-based nucleic acid amplification method that utilizes two enzymes, MMLV (Moloney murine leukemia virus) reverse transcriptase and T7 RNA polymerase. The reverse transcriptase is used to generate a DNA copy (containing a promoter sequence for T7 RNA polymerase) of the target sequence. T7 RNA polymerase produces multiple copies of RNA amplicon from the DNA copy template. Detection is achieved using single-stranded nucleic acid torches that are present during the amplification of the target and hybridize specifically to the amplicon in real time. Each torch has a fluorophore and a quencher. The quencher suppresses the fluorescence of the fluorophore as it is designed to be in close proximity when not hybridized to the amplicon. When the torch binds to the amplicon, the quencher is moved farther away from the fluorophore and it will emit a signal at a specific wavelength when excited by a light source. More torch hybridizes when more amplicon is present. The increase in fluorescent signal from progressive amplification is detected by fluorometers within the Panther system. The Panther system can detect and discriminate between the three fluorescent signals corresponding to HSV-1, HSV-2 and IC amplification products. The fluorescence (measured in relative fluorescence units [RFU]) is monitored over time to produce a real-time fluorescence emergence curve for each reporter dye. The Panther system software compares the fluorescence emergence curves to fixed cut off times to report results (TTime) for HSV-1, HSV-2 and IC.

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

Acceptance Criteria and Device Performance for Aptima Herpes Simplex Viruses 1 & 2 Assay

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implied by the clinical performance targets presented in the study. While explicit pre-defined acceptance thresholds (e.g., "Sensitivity must be >90%") are not directly stated as pass/fail criteria, the reported performance metrics demonstrate the device's capability. For this analysis, I will use the clinical performance study results as the reported device performance against generally expected high standards for diagnostic accuracy.

Note: The document does not explicitly state the pre-defined "acceptance criteria" numerical targets. The reported performance below represents the observed results of the clinical study, which presumably met the internal performance requirements for the manufacturer and FDA review.

Table 1: Acceptance Criteria (Implied) and Reported Device Performance

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

• Clinical Test Set Sample Size:
  • Total Subjects: 544 evaluable subjects (195 males and 349 females).
  • Evaluated for HSV-1: 528 VTM specimens and 531 STM specimens.
  • Evaluated for HSV-2: 533 VTM specimens and 535 STM specimens.
• Data Provenance:
  • Country of Origin: United States. The study was conducted at 17 US clinical sites.
  • Retrospective or Prospective: Prospective. The study is described as a "prospective, multicenter clinical study."

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

The document does not specify the number of experts used to establish the ground truth or their specific qualifications (e.g., "radiologist with 10 years of experience").

However, it describes the methods used for the composite reference method:

• ELVIS HSV ID and D3 Typing Test system viral culture
• A validated bidirectional PCR/sequencing procedure
• A third FDA-cleared assay for HSV-1 and HSV-2 was used for final composite reference interpretation when the initial methods disagreed or when PCR/sequencing detected both types.

This suggests that the ground truth was established through a combination of highly reliable laboratory tests, implying a rigorous approach to defining true positive/negative cases, rather than relying solely on individual expert interpretation without further clarification.

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

The document describes an adjudication method for the ground truth:

• "A third FDA-cleared assay for HSV-1 and HSV-2, was used to determine the final composite reference interpretation when the ELVIS D3 culture and PCR/sequencing results did not agree on the type of HSV detected or when PCR/sequencing detected both HSV-1 and HSV-2."

This indicates a hierarchical or tie-breaking system rather than a simple 2+1 or 3+1 consensus among human readers. It relies on a "composite reference method" combining results from multiple validated laboratory tests.

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 is a diagnostic assay for detecting viral RNA, not an imaging device that involves human readers interpreting images with or without AI assistance. Therefore, no MRMC comparative effectiveness study involving human readers with AI assistance was performed or reported in this submission. The device (Aptima HSV 1 & 2 Assay) is designed to provide a direct qualitative result (positive/negative for HSV-1 and/or HSV-2) from a processed specimen.

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

Yes, a standalone performance study was done in the sense that the Aptima HSV 1 & 2 Assay (the "algorithm" in this context) directly processes specimens and generates results without requiring human interpretation for its output. The clinical performance study directly evaluated the accuracy of the assay's results against a composite reference standard. The "human-in-the-loop" would be the clinician collecting the sample and laboratory technicians running the test and reporting the results, but the analytical output itself is determined by the assay system.

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

The ground truth used was a composite reference method combining:

• ELVIS HSV ID and D3 Typing Test system viral culture
• A validated bidirectional PCR/sequencing procedure
• A third FDA-cleared assay for HSV-1 and HSV-2 (used for tie-breaking/discrepancy resolution)

This is a robust form of ground truth based on multiple established laboratory diagnostic methods.

8. The sample size for the training set

The document does not report a sample size for a training set. This is expected for a diagnostic assay of this type, as it's not a machine learning or AI algorithm that requires a separate "training set" in the conventional sense. The assay's design and optimization (e.g., probe sequences, amplification conditions) would have been developed iteratively, but a distinct "training set" for performance evaluation is not applicable here. The analytical studies (LoD, cross-reactivity, etc.) and the clinical performance study represent the validation of the finalized assay.

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

As there is no explicit "training set" in the context of machine learning, this question is not directly applicable. The assay's components and parameters would have been optimized using internal development processes and validated through analytical studies. For these analytical studies (e.g., LoD, cross-reactivity), the "ground truth" (i.e., known-positive or known-negative samples, specific viral strains/concentrations) would have been established through well-characterized laboratory standards, spiked samples, and reference materials.

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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 cobas® HSV 1 and 2 Test on the cobas® 4800 system is an automated, qualitative in vitro diagnostic test, that utilizes real-time polymerase chain reaction (PCR), for the direct detection and differentiation of Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) DNA in clinician-collected, external anogenital lesion specimens from symptomatic male and female patients. The cobas® HSV 1 and 2 Test is intended for use as an aid in diagnosis of anogenital HSV-1 and HSV-2 infections in symptomatic patients.

Warning: The cobas® HSV 1 and 2 Test is not FDA cleared for use with cerebrospinal fluid (CSF) and is not intended to be used for prenatal screening or for individuals under the age of 18 years.

The Roche Molecular Systems (RMS) cobas® HSV 1 and 2 Test utilizes real-time polymerase chain reaction (PCR) for detection of HSV-1 and HSV-2 DNA in clinician-collected external anogenital lesion specimens, collected in MSwab medium from symptomatic patients.

The cobas® HSV 1 and HSV 2 Test contains two major processes: (1) automated sample preparation to extract nucleic acids from swab specimens; (2) PCR amplification of target DNA sequences using HSV-1 and HSV-2 specific primers, and real-time detection of cleaved fluorescent-labeled HSV-1 and HSV-2 specific oligonucleotide detection probes. An Internal Control (IC), containing unrelated randomized DNA sequence, is added to all samples prior to automated sample preparation and is amplified and detected simultaneously with each sample to monitor the entire process.

The MSwab Collection. Transport and Preservation System (Copan Flock Technologies) is used for specimen collection, transportation and storage of specimen for the cobas " HSV 1 and HSV 2 Test.

The cobas® HSV 1 and HSV 2 Test utilizes six reagent kits:

• cobas® 4800 HSV 1 and HSV 2 Amplification/Detection Kit 1)
• cobas® 4800 HSV 1 and HSV 2 Controls and Cofactor Kit 2)
• cobas® 4800 System Wash Buffer Kit 3)
• cobas® 4800 System Lysis Kit 1 4)
• cobas® 4800 System Internal Control Kit 1 5)
• cobas® 4800 System Sample Preparation Kit 6)

Here's an analysis of the acceptance criteria and study detailed in the provided document for the cobas® HSV 1 and 2 Test:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria in a dedicated table. Instead, it presents performance metrics from various studies, which imply the acceptance thresholds met for FDA clearance. Based on the provided clinical performance data, the implicit acceptance criteria and the reported performance are:

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

• Clinical Study Test Set Sample Size: A total of 408 specimens from 205 female and 203 male subjects.
• Data Provenance: The specimens were clinician-collected external anogenital lesion specimens from symptomatic male and female patients attending family planning, OB/GYN, and sexually transmitted disease clinics at eight geographically diverse sites (seven across the United States and one in the United Kingdom). The study was prospective.

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

The document does not specify the number of individual experts or their qualifications used to establish the ground truth. It states that the ground truth was "established compared to a composite Reference Method derived from the combined results of culture (ELVIS® HSV ID and D3 Typing Test) and Sanger sequencing using the 'any positive rule'." This implies laboratory testing by trained personnel rather than clinical expert consensus.

4. Adjudication Method for the Test Set

The adjudication method for the clinical test set ground truth was a "composite Reference Method derived from the combined results of culture (ELVIS® HSV ID and D3 Typing Test) and Sanger sequencing using the "any positive rule"." This means if either culture or Sanger sequencing was positive, the specimen was considered positive for the ground truth.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not conducted. This device is an in-vitro diagnostic (IVD) test, where the performance is assessed by comparing its output to a reference method, not by how human readers improve with or without AI assistance. The "reader" in this context is the instrument itself.

6. Standalone Performance Study

Yes, a standalone performance study was done. The entire document focuses on the performance of the algorithm only (the cobas® HSV 1 and 2 Test on the cobas® 4800 system) without human-in-the-loop performance being part of the primary evaluation metrics for FDA clearance in this context. The clinical performance tables (Table 14 and 15) directly compare the device's results to the reference methods.

7. Type of Ground Truth Used

The primary ground truth for the clinical study was a composite reference method combining:

• Culture: ELVIS® HSV ID and D3 Typing Test
• Sanger Sequencing: PCR followed by bi-directional Sanger sequencing for HSV-1 and HSV-2 DNA.
  This composite ground truth used an "any positive rule."

8. Sample Size for the Training Set

The document does not specify a sample size for a training set. This is because the submission primarily describes the validation studies for an IVD kit, not a machine learning algorithm that typically requires a distinct training phase. The development of the assay (primer/probe design, optimization, etc.) would historically involve various samples, but these are not explicitly termed a "training set" in the context of this regulatory filing.

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

As no specific "training set" is mentioned in the context of a machine learning algorithm, the method for establishing its ground truth is not applicable or described in this document. The assay's analytical characteristics (e.g., target selection, primer and probe design) imply bioinformatic and laboratory validation during its development, but not in the sense of establishing ground truth for a distinct training dataset for a learning algorithm.

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The AnyplexTM II HSV-1/2 Assay is a real-time polymerase chain reaction (PCR)-based in vitro diagnostic test intended for the qualitative detection and differentiation of Herpes Simplex Virus Type-1 (HSV-1) and Herpes Simplex Virus Type-2 (HSV-2) DNA from female skin lesions from anogenital sites. The test is intended for use as and in the diagnosis of anogenital HSV infection in symptomatic patients.

WARNING: The AnyplexTM II HSV-1/2 Assay is not indicated for use with cerebrospinal fluid (CSF). The assay is not intended to be used for prenatal screening.

The Anyplex™ II HSV-1/2 Assay uses PCR to generate amplified product from HSV-1 and HSV-2 present in clinical specimens. The presence of HSV-1 and/or HSV-2 target DNA is indicated by the fluorescent signal generated through the use of fluorescently-labeled oligonucleotide probes (duplex Catcher) on the Cepheid SmartCycler® II Dx instrument. The probes do not generate a signal unless they are specifically bound to the amplified product. A preparation of HSV-1 and HSV-2 plasmids is included as the positive control in the Anyplex™ II HSV-1/2 Assay. Run as a separate control, the positive control serves to demonstrate that the HSV-1/2 PCR reagents are functional, and discriminate the validity of the run. In addition, the positive control functions as a process control, demonstrating that sample preparation has proceeded correctly during the run. An internal control (IC) is also included in the assay kit. The IC is added to each sample specimen during sample preparation, and is also used to create a Blank Negative Control by adding a set amount to viral transport media to serve as an extraction control. In addition, the RNase-free water is used to create the Negative Control by adding a set volume to the prepared master mix. Users are instructed to include all three controls, Positive, Negative, and Blank Negative Control with each test run.

The document describes the performance of the Anyplex™ II HSV-1/2 Assay, a real-time PCR-based in vitro diagnostic test for the qualitative detection and differentiation of Herpes Simplex Virus Type-1 (HSV-1) and Herpes Simplex Virus Type-2 (HSV-2) DNA from female skin lesions from anogenital sites.

Here's an analysis of the acceptance criteria and study details based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria (e.g., "The device must achieve sensitivity of X% and specificity of Y%"). Instead, it presents the results of its performance studies, which are implicitly understood to demonstrate adequate performance for regulatory approval based on comparison to a predicate device. The clinical performance metrics are the most relevant in this context.

Implicit Acceptance Criteria (based on reported clinical performance): The device is expected to demonstrate high sensitivity and specificity for both HSV-1 and HSV-2 detection when compared to a reference method in symptomatic patients with anogenital lesions.

Analytical Performance (examples from the document):

• Precision/Repeatability: Achieved 100% agreement with expected results for HSV-1 Low Positive, HSV-1 High Positive, HSV-2 Low Positive, HSV-2 High Positive, and Negative samples in an in-house study. Ct %CV values were low (e.g., 1.34% - 2.56%).
• Reproducibility: Across three sites, overall agreement for positive controls (3X LoD and 1X LoD) was high (e.g., 100% for 3X LoD for both HSV-1 and HSV-2; 96.67% for HSV-1 1X LoD; 97.8% for HSV-2 1X LoD). Negative controls showed 99.4% agreement.
• Limit of Detection (LoD): Varied by strain (e.g., HSV-1 MacIntyre: 3.75x10^2 TCID50/mL; HSV-2 MS: 3.75x10^1 TCID50/mL).
• Cross-Reactivity/Microbial Interference: No cross-reactivity or interference observed with a panel of 50 organisms.
• Interfering Substances: None of 22 tested substances showed detectable interference.

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

• Sample Size: A total of 656 valid prospective specimens were included in the final data set for clinical performance analysis.
  • For HSV-1 performance calculation, 550 specimens were used (specimens positive for HSV-2 by the reference method were removed due to the reference method's inability to detect co-infections).
  • For HSV-2 performance calculation, all 656 specimens were used.
• Data Provenance: The study was conducted at three geographically diverse locations within the United States from 2013-2014. The data is prospective, as indicated by "656 prospective specimens included in the study."

3. Number of Experts and their Qualifications for Ground Truth

The document does not explicitly state the number of experts or their specific qualifications (e.g., "radiologist with 10 years of experience") used to establish the ground truth.

4. Adjudication Method for the Test Set

The primary adjudication method for discordant results in the clinical performance study was bidirectional sequencing.

• For HSV-1:
  • 29 samples positive by Anyplex but negative by reference method: HSV-1 was detected in 18 cases by bidirectional sequencing. The remaining 11 remained discordant.
  • 1 sample negative by Anyplex but positive by reference method: HSV-1 was detected in this sample by bidirectional sequencing.
• For HSV-2:
  • 35 samples positive by Anyplex but negative by reference method: HSV-2 was detected in 21 cases by bidirectional sequencing. The remaining 14 remained discordant.
  • 3 samples negative by Anyplex but positive by reference method: HSV-2 was not detected in any of these by bidirectional sequencing (meaning the reference method's positive call was likely a false positive, and Anyplex was correct).

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. This device is an in-vitro diagnostic assay (laboratory test), not an imaging or interpretation device that typically involves human readers. The clinical performance study compares the device's results against a laboratory-based reference method, not against human interpretation with or without AI assistance.

6. Standalone Performance

Yes, a standalone performance study was done. The entire clinical performance section (Tables 6 and 7) and the analytical performance sections (precision, reproducibility, LoD, cross-reactivity, interfering substances) describe the performance of the Anyplex™ II HSV-1/2 Assay algorithm/device only, without human intervention in the interpretation of the output. The assay provides a qualitative "POS" or "NEG" result for HSV-1 and HSV-2.

7. Type of Ground Truth Used

The ground truth for the clinical performance study was established using the ELVIS® (Enzyme Linked Virus Inducible System) HSV ID and D Typing Test System (Diagnostic Hybrids, Athens, OH), which is described as the "reference ELVIS viral culture method." For discordant results, bidirectional sequencing was used as a confirmatory method to resolve discrepancies.

8. Sample Size for the Training Set

The document does not specify the sample size used for a training set. This is typical for a traditional IVD device using PCR technology, where "training" in the machine learning sense isn't applicable. The development of such assays involves designing primers and probes, and then validating their performance across various analytical and clinical studies.

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

As noted above, the concept of a "training set" with ground truth in the AI/machine learning sense is not applicable to this type of device. The assay design (e.g., selection of primers and probes) is based on known genetic sequences of HSV-1 and HSV-2. The extensive analytical performance studies (precision, LoD, cross-reactivity) ensure the assay's fundamental ability to detect targets accurately, and the clinical performance study validates this in human samples against a recognized reference method.

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The artus HSV-1/2 QS-RGQ MDx Kit is an in vitro real-time PCR DNA amplification assay performed on the QIAsymphony RGQ MDx system for the direct qualitative detection and differentiation of herpes simplex virus (HSV-1 and HSV-2) DNA in genital or oral vesicular lesions from male and female patients suspected of HSV infection.

The assay is intended for use as an aid in diagnosis of HSV infection in symptomatic patients.

Warning: The artus HSV-1/2 QS-RGQ MDx Kit is not FDA-cleared for use with cerebrospinal fluid (CSF) or for prenatal screening.

The artus HSV-1/2 QS-RGQ MDx Kit is an in vitro PCR assay for the qualitative detection and differentiation of nucleic acids encoding the Glycoprotein D and UL30 genes isolated from HSV-1 and HSV-2 DNA present in genital or oral lesions from male and female patients. Samples are extracted and prepared for PCR using the QIAsymphony SP/AS instrument with the QIAsymphony DSP Virus/Pathogen Mini Kit. Amplification and detection are carried out using the artus HSV-1/2 QS-RGQ MDx Kit with the Rotor-Gene O MDx (RGO MDx) and Rotor-Gene AssayManager software. The presence of a HSV-1 or HSV-2 target sequence is indicated by the fluorescent signal generated through the use of fluorescently labeled oligonucleotide probes. The probes do not generate a signal unless they are specifically bound to the amplified product. The amplification cycle at which fluorescent signal is detected by the RGQ MDx is inversely proportional to the HSV-1 and/or HSV-2 target concentration present in the original specimen. A plasmid construct containing DNA unrelated to HSV-1 and HSV-2 is introduced into each specimen during sample preparation to serve as an internal control. Run as a separate control, the positive control serves to demonstrate that the HSV-1/2 PCR reagents are functional. In addition, the positive control functions as a process control, to demonstrate that sample preparation has proceeded correctly during the run.

The provided documentation describes the performance characteristics and clinical study results for the artus® HSV-1/2 QS-RGQ MDx Kit, an in vitro real-time PCR assay for the detection and differentiation of HSV-1 and HSV-2 DNA.

Here's an analysis of the acceptance criteria and the study that proves the device meets them:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally qualitative for this type of diagnostic device, revolving around achieving sufficient sensitivity and specificity compared to a predicate device or established methods. The reported device performance is detailed in the tables under "Performance Characteristics - Clinical Studies."

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The IMDx HSV-1/2 for Abbott m2000 assay is an in vitro diagnostic test for the direct, qualitative detection and differentiation of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNA from male and female skin lesions from anogenital or oral sites. The test is intended for use as an aid in the diagnosis of HSV infection in symptomatic patients. The assay is intended to be run on the Abbott m2000 instrument system.

Warning: The IMDx HSV-1/2 for Abbott m2000 assay is not FDA cleared for use with cerebrospinal fluid (CSF). The assay is not intended for prenatal screening.

The IMDx FISV-1/2 for Abbott m2000 assay uses PCR to generate amplified product from HSV-1 and HSV-2 present in clinical specimens. The presence of HSV-1 and/or HSV-2 target DNA is indicated by the fluorescent signal generated through the use of fluorescently labeled oligonucleotide probes on the Abbott m2000rt instrument. The probes do not generate a signal unless they are specifically bound to the amplified product. The amplification cycle at which fluorescent signal is detected by the Abbott m2000rt is inversely proportional to the HSV-1 and/or HSV-2 DNA target concentration present in the original specimen. A plasmid containing DNA unrelated to HSV-1 and HSV-2 is introduced into each specimen during sample preparation to serve as an internal control. The internal control is amplified in the same reaction as the HSV-1 and HSV-2 DNA targets, and serves to demonstrate that the sample preparation and amplification proceeded correctly for each specimen. A preparation of intact, inactivated HSV-1 and HSV-2 virus is included as the positive control in the IMDx HSV-1/2 for Abbott m2000 assay. Run as a separate control, the positive control serves to demonstrate that the HSV-1/2 PCR reagents are functional. In addition, the positive control functions as a process control to demonstrate that sample preparation has proceeded correctly during the run. A negative control consisting of M4RT viral transport medium is included in each run to independently verify the absence of contaminating target material in assay reagents.

Acceptance Criteria and Device Performance Study for IMDx HSV-1/2 for Abbott m2000

The IMDx HSV-1/2 for Abbott m2000 assay is an in vitro diagnostic test for the direct, qualitative detection and differentiation of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNA from male and female skin lesions from anogenital or oral sites.

The study that proves the device meets the acceptance criteria involved Prospective Clinical Performance Characteristics and Retrospective Clinical Performance Characteristics studies, as well as several analytical performance studies. The primary comparison for clinical performance was against the ELVIS® (Enzyme Linked Virus Inducible System) HSV ID and D3 Typing Test System.

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria in terms of target sensitivity and specificity percentages. However, the study results, particularly from the prospective clinical performance, would implicitly represent the performance deemed acceptable for FDA clearance through substantial equivalence. If acceptance criteria were required, they would typically be set at a high level given the diagnostic nature of the device. For this summary, we will present the reported performance from the primary prospective study as the "met acceptance criteria".

Note on HSV-2 Oral Sensitivity: The reported sensitivity of 0.0% in the prospective study for oral HSV-2 is based on only 2 reference method positive cases. This low sample size limits definitive conclusions, and a contrived specimen study was performed to provide additional data for HSV-2 oral detection. This contrived study showed 100% detection of HSV-2 in spiked oral samples across various concentrations.

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

• Prospective Studies (Clinical Performance Characteristics):
  • Test Set Size: 954 prospective specimens (807 anogenital and 147 oral).
  • Data Provenance: From four geographically diverse locations within the United States.
  • Retrospective Studies:
  • Test Set Size: 54 retrospective specimens (27 anogenital and 27 oral).
  • Data Provenance: Not explicitly stated, but "historical results" for the ELVIS system suggests previously collected specimens.

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

The ground truth was established by the ELVIS® (Enzyme Linked Virus Inducible System) HSV ID and D3 Typing Test System. This is a commercially available viral culture system, which is a recognized reference method for HSV detection and typing. The document does not specify the number of human experts or their qualifications involved in interpreting the ELVIS results for establishing ground truth. The ELVIS system itself is the "expert" or gold standard in this context.

4. Adjudication Method for the Test Set

The document mentions "Discordant analysis" was performed.

• For anogenital HSV-1, 20 initial IMDx positive / ELVIS negative results were analyzed; 6 were confirmed positive for HSV-1, 11 remained discordant.
• For anogenital HSV-2, 68 initial IMDx positive / ELVIS negative results were analyzed; 55 were confirmed positive for HSV-2, 7 remained discordant.
• For oral HSV-1, 24 initial IMDx positive / ELVIS negative results were analyzed; 14 were confirmed positive for HSV-1, 10 remained discordant.
• For oral HSV-2, 2 initial IMDx positive / ELVIS negative results were analyzed; both were confirmed positive for HSV-2.

The specific "adjudication method" beyond re-testing or further analysis of discordant samples with an unspecified method is not detailed (e.g., whether a third, independent gold standard was used, or if expert review of discrepant results was performed). However, the results presented in the tables incorporate the outcomes of this discordant analysis by adjusting the counts (e.g., "HSV-1 was detected in 6 of the 17 specimens" changes the true positive count for HSV-1).

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 done. This device is an in vitro diagnostic assay (a lab test), not an imaging or interpretation assistance device for human readers. Therefore, the concept of "human readers improve with AI vs without AI assistance" is not applicable here. The device provides a direct qualitative detection and differentiation of HSV-1/2 DNA.

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

Yes, a standalone performance study was done. The entire set of clinical performance characteristics (prospective and retrospective studies) evaluates the performance of the IMDx HSV-1/2 for Abbott m2000 assay directly against the reference method (ELVIS viral culture). This is an "algorithm only" or "device only" performance without human interpretation being the primary variable. The results are based on the device's output.

7. The Type of Ground Truth Used

The primary ground truth used for clinical performance studies was the ELVIS® (Enzyme Linked Virus Inducible System) HSV ID and D3 Typing Test System. This is a viral culture-based method, which is a widely accepted laboratory gold standard for direct detection of viable virus.

8. The Sample Size for the Training Set

The document does not explicitly describe a separate "training set" for the IMDx HSV-1/2 for Abbott m2000 assay. This is a PCR-based diagnostic test, not a machine learning model that typically requires a distinct training phase with a large dataset. The assay's design and optimization (e.g., primer and probe selection, reaction conditions) would have occurred during development, likely using various characterized samples, but these are not formally presented as a "training set" in the context of this 510(k) submission. Therefore, the concept of a separate "training set" as understood in machine learning is not directly applicable or reported for this type of IVD.

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

As mentioned above, no explicit "training set" is described for this PCR-based IVD. The ground truth for the analytical and clinical validation of the device relies on established laboratory culture methods (ELVIS system for clinical specimens) and characterized viral strains/isolates for analytical studies (e.g., limit of detection, analytical reactivity, cross-reactivity). These reference methods serve as the "ground truth" to evaluate the accuracy of the IMDx assay.

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The AmpliVue® HSV 1+2 Assay is an in vitro diagnostic test for the direct, qualitative detection and differentiation of Herpes Simplex Virus 1 (HSV-1) and Herpes Simplex Virus 2 (HSV-2) DNA in cutaneous or mucocutaneous lesion specimens from symptomatic patients. The test is intended for use as an aid in diagnosis of HSV infection in symptomatic patients.

Warning: The AmpliVue HSV 1+2 Assay is not FDA cleared for use with cerebrospinal fluid (CSF). The assay is not intended for prenatal screening.

The AmpliVue HSV 1+2 Assay consists of three major steps: 1) specimen preparation, 2) isothermal Helicase-Dependent Amplification (HDA) of target amplicons specific to HSV-1 and HSV-2, and 3) detection of the amplified DNA by target-specific hybridization probes via a colorimetric reaction on a lateral-flow strip which is embedded in a self-contained disposable cassette to prevent amplicon contamination.

Specimen preparation involves one simple dilution step in which specimens in viral transport medium are diluted 80-fold in Dilution Tubes.

The diluted samples are transferred into a 0.2 mL Amplification Tube containing lyophilized HDA reagents. Incubation at 64°C for 45 minutes results in the release of the HSV DNA and subsequent isothermal amplification of the target sequence. The amplified DNA is detected by a set of specific detection probes included in the Amplification Tube: HSV-1 target hybridizes to two specific probes labeled with Biotin (BioTEG) and Digoxigenin (DIG) and HSV-2 target hybridizes to two specific probes labeled with Biotin (BioTEG) and Fluorescein isothiocyanate (FITC). A competitive internal control (IC) is included in the Amplification Tube to monitor inhibitory substances in clinical samples, reagent failure or device failure. The IC target is amplified by HSV-2 specific primers and hybridizes to the biotin-labeled HSV-2 probe and a 1C specific probe labeled with 2,4-dinitrophenyl (DNP-TEG).

Detection of the amplified DNA with specific probes is achieved by Type III BEStTM cassettes. The self-contained Type III BESTM cassettes carry lateral-flow DNA detection strips coated with anti-DNP antibodies (C line), anti-DIG antibodies (T1 line) and anti-FITC antibodies (T2 line). HSV-1 amplicon with BioTEG and DIG-labeled probes is captured by anti-DIG antibodies at the TI-Line and HSV-2 amplicon with BioTEG and FITC-labeled probes is captured by anti-FITC antibodies at the T2-Line, while the IC amplicon with BioTEG and DNP-labeled probes is captured by anti-DNP antibodies at the C-Line. The amplicon-probe complexes captures the streptavidin-conjugated color particles for visualization and the test result is shown as colored lines that are visually read.

A positive result for HSV-1 (detection of HSV-1 DNA) is reported when the T1 line is visible through the detection window of the cassette, while a positive result for HSV-2 (detection of HSV-2 DNA) is reported when the T2 line is visible through the detection window of the cassette. A positive result for both HSV-2 (detection of both HSV-1 and HSV-2 DNA) is reported when both the T1 line and the T2 line are visible through the detection window of the cassette. A negative result (no detection of HSV-2 DNA) is reported when only the C line is displayed. The assay result is regarded as invalid when the T1 line, T2 line and C line are not present and the assay should be repeated.

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

Device Name: AmpliVue® HSV 1+2 Assay

1. Table of Acceptance Criteria and Reported Device Performance

The provided document primarily focuses on demonstrating the substantial equivalence of the AmpliVue® HSV 1+2 Assay to a predicate device and presenting its analytical and clinical performance. While explicit "acceptance criteria" (numerical thresholds that must be met) are not clearly defined as a separate section with specific targets, the performance studies implicitly set the standard for acceptable performance compared to the gold standard.

Here's a summary of the key performance metrics reported in the document:

Note on Acceptance Criteria: The document describes the results of the studies and states that they "demonstrate that the AmpliVue® HSV 1+2 Assay is substantially equivalent to the predicate device." This implies that the performance shown met the internal criteria for substantial equivalence, which often means demonstrating non-inferiority or comparable performance to a legally marketed predicate. Specific numerical thresholds for "acceptance" are not explicitly listed in an "Acceptance Criteria" section but are inherent in the successful outcomes of these analytical and clinical studies. For example, for LoD, a positivity rate of ≥95% was defined within the study itself as the criterion for determining the LoD.

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

The primary clinical performance study (test set) involved:

• Sample Size: A total of 1355 specimens from symptomatic male and female patients were tested. 19 tests were considered invalid and removed, resulting in 1336 specimens for the initial analysis. For HSV-1 performance calculation, 211 HSV-2 positive specimens were removed, leaving 1125 specimens.
• Data Provenance:
  • Country of Origin: United States.
  • Retrospective or Prospective: Not explicitly stated as retrospective or prospective, but the description "evaluated at five geographically diverse locations within the United States" and involving "symptomatic male and female patients" suggests a prospective collection and testing of clinical samples.

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

• Number of Experts: Not applicable in the context of this diagnostic assay.
• Qualifications of Experts: The ground truth was established by a "gold standard/reference method" which is the ELVIS® HSV ID and D³ Typing Test System (cell culture using an enzyme linked virus inducible system). This is a laboratory-based reference method, not an expert panel.

4. Adjudication Method for the Test Set

• Adjudication Method: Not applicable. The ground truth was determined by a single reference method (ELVIS® viral culture), not by a panel of human adjudicators. Discrepancies would likely be investigated by re-testing or deeper analysis against the reference method. The document notes that the ELVIS culture inability to detect co-infected specimens led to the removal of HSV-2 positive samples when calculating HSV-1 performance, which served as a form of handling limitations of the ground truth.

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

• There was no MRMC comparative effectiveness study done.
• This device is an in vitro diagnostic (IVD) assay for the direct detection of viral DNA, not an AI-assisted diagnostic tool that involves human readers interpreting images or data. Therefore, the concept of human readers improving with AI assistance is not relevant to this device.

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

• This question is partially applicable. The AmpliVue® HSV 1+2 Assay is an "algorithm only" in the sense that it is a chemical and molecular diagnostic test with a qualitative visual readout (colored lines). There is no "human-in-the-loop" interpretation beyond reading the positive/negative lines based on established rules.
• The entire clinical performance study (sensitivity and specificity tables) represents the standalone performance of the assay compared to the reference method.

7. The Type of Ground Truth Used

• Ground Truth Type: Expert reference method / Gold standard.
  • Specifically, the ground truth was established using the ELVIS® HSV ID and D³ Typing Test System, which is described as the "gold standard/reference method i.e., cell culture using an enzyme linked virus inducible system."

8. The Sample Size for the Training Set

• Sample Size for Training Set: The document does not explicitly mention a separate "training set" as commonly understood in machine learning contexts. This is an IVD assay, and the development process would involve analytical studies (LoD, cross-reactivity, interference, etc.) and then clinical validation. The samples used for initial assay development and optimization (analogous to a training set) are not detailed in terms of specific numbers.
• However, the analytical performance section describes studies that would have informed the assay's design and operational characteristics, such as:
  • Level of Detection (LoD): Used serially diluted viral stocks (30 replicates across 3 lots for initial LoD determination, then 20 replicates for confirmation). This involves a number of samples to establish analytical sensitivity.
  • Analytical Specificity/Cross-Reactivity: Tested 89 microorganisms (each in triplicate).
  • Interfering Substances: Tested 33 substances and 5 viral transport media (each in triplicate).

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

• As noted above, a distinct "training set" with its own ground truth establishment method isn't explicitly described. However, for the analytical studies that would inform assay development (like LoD, specificity, and interference), the ground truth was established by:
  • Quantified viral stocks: For LoD studies, Herpes Simplex Virus 1 (HSV-1) and Herpes Simplex Virus 2 (HSV-2) viral stocks were quantified (TCID50/mL). These quantified stocks served as the known "true" concentrations for determining the assay's detection limit.
  • Known microorganisms and substances: For analytical specificity and interfering substances studies, known microorganisms (purified genomic DNA, quantified cultures) and specific chemical/biological substances were introduced at known concentrations. The "ground truth" here is the precise identity and concentration of the added components, and the expectation is zero interference or cross-reactivity.

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The IsoAmp® HSV Assay is an in vitro diagnostic test for the direct, qualitative detection of herpes simplex virus (HSV-1 & HSV-2) DNA in male and female genital and oral lesions. The test is intended for use as an aid in diagnosis of HSV infection in symptomatic patients.

Warning: The IsoAmp® HSV Assay is not FDA cleared for use with cerebrospinal fluid (CSF). The assay does not provide specific typing information to differentiate HSV-1 and HSV-2. The assay is not intended to be used for prenatal screening.

The IsoAmp® HSV Assay consists of three major steps: 1) specimen preparation: 2) isothermal Helicase-Dependent Amplification (HDA) of the HSV glycoprotein B (gB) gene using biotinylated primers; and 3) detection of the amplified DNA by a target-specific hybridization probe via a colorimetric reaction on a lateral-flow strip which is embedded in a self-contained disposable cassette to prevent amplicon contamination.

Specimen preparation includes a simple dilution step in which specimens in viral transport medium are diluted 40-fold in dilution buffer. The diluted samples are mixed with HDA reagents. Incubation at 64°C results in the release of the HSV DNA and subsequent isothermal amplification of the target sequence. A competitive internal control (IC) is included in the Amplification Reagents to monitor inhibitory substances in negative samples, reagent failure or device failure.

After incubation for one hour, the amplified DNA is detected by two detection probes, one labeled with fluorescein isothiocyanate (FITC) for hybridizing to the HSV target and the other labeled with digoxigenin (DIG) for binding to the IC target. The hybrid of FITC-labeled probe and HSV amplicon is captured at the Test Line (T-Line) on the lateral-flow strip by anti-FITC antibodies, while the DIG-labeled IC amplicon is captured at the Control Line (C-Line) on the strip by anti-DIG antibodies. The biotin label in each amplicon captures the streptavidinconjugated color particles for visualization and the test result is shown as colored lines that are visually read.

The self-contained Type II BESt™ cassettes contain lateral-flow DNA detection strips coated with anti-FITC antibodies and anti-DIG antibodies that serve as T line and C line respectively in the assay. A positive result (detection of HSV DNA) is reported when the T line is visible through the detection window of the cassette. A negative result (no detection of HSV DNA) is reported when only the C line is displayed. The assay result is regarded as invalid when both the T line and C line are not present and the assay should be repeated.

Here's a breakdown of the acceptance criteria and the study details for the IsoAmp® HSV Assay, based on the provided document:

1. Table of Acceptance Criteria and Reported Device Performance

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

• Clinical Performance Test Set (Prospective and Retrospective combined): 994 swab samples.
  • Prospective Samples: 962 samples (803 genital, 159 oral).
  • Retrospective Samples: 32 samples (15 genital, 17 oral) tested at a single study site.
• Data Provenance:
  • Country of Origin: United States.
  • Retrospective or Prospective: A mix of both. The majority (962) were prospectively collected, and a smaller number (32) were retrospectively collected.

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

• The document does not specify the number of experts or their qualifications for establishing the ground truth using the reference method (Cell Culture based ELVIS® HSV ID/Typing Test System). The ELVIS system is a commercial diagnostic kit, and its results would be interpreted according to its own instructions, potentially by trained laboratory personnel rather than clinical "experts" in the sense of physicians or radiologists.
• For the discordant samples, "bidirectional sequence analysis" was used to resolve discrepancies, but the personnel performing and interpreting this analysis are not described.

4. Adjudication Method for the Test Set

• The primary method for establishing ground truth was comparison to a "gold standard/reference method," the Cell Culture based ELVIS® HSV ID/Typing Test System.
• For discordant results between the IsoAmp® HSV Assay and the ELVIS system, bidirectional sequence analysis was used as an adjudication method.
  • For genital samples: 35 samples discordant by the IsoAmp assay were tested by sequencing.
  • For oral samples: 14 samples discordant by the IsoAmp assay were tested by sequencing.
  • This suggests a process where sequencing served as a tie-breaker or a higher-fidelity reference for ambiguous cases.

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

• No, an MRMC comparative effectiveness study was not done.
• This study evaluated a diagnostic assay (a lab test), not an AI algorithm assisting human readers in interpreting images or other complex data. Therefore, the concept of "how much human readers improve with AI vs. without AI assistance" does not apply here.

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

• Yes, a standalone performance was done. The entire study tests the performance of the IsoAmp® HSV Assay as a complete system, which operates independently to produce a result, with visual reading of the lateral flow strip. There isn't a human interpreting images or data that the algorithm generates and then making a judgment. The visual reading of the T-line and C-line on the lateral flow strip could be considered the "human-in-the-loop" component for result interpretation, but the core performance data (sensitivity, specificity, LoD, etc.) reflects the assay's output as an isolated system. The results presented are for the device's output itself.

7. Type of Ground Truth Used

• Reference Method: Cell Culture based ELVIS® HSV ID/Typing Test System. This is a recognized laboratory "gold standard" for HSV detection.
• Adjudication for Discordant Results: Bidirectional sequence analysis. This is a highly accurate molecular method used to confirm the presence and type of viral DNA.

8. Sample Size for the Training Set

• The document does not explicitly mention a distinct "training set" for the IsoAmp® HSV Assay. This is common for molecular diagnostic assays like this, which are developed through iterative R&D processes (optimizing reagents, protocols, etc.) rather than supervised machine learning where a specific "training set" is used to teach an algorithm.
• The analytical performance studies (LoD, cross-reactivity, interference) use spiked samples and cultured organisms, which contribute to the development and validation of the assay but are not typically referred to as a "training set" in the AI/ML sense.

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

• As no explicit "training set" is described for an AI/ML context, this question is not directly applicable.
• For the analytical studies (e.g., LoD, cross-reactivity), the "ground truth" for the samples used was established by:
  • Quantified cultures: For LoD studies, HSV strains were precisely quantified (TCID₅₀/mL).
  • Known organisms/DNA: For cross-reactivity, purified DNA and cultured organisms of known identity and concentration were used.
  • HSV-negative matrix pools: Used as negative controls and for diluting samples.
  • These are standard methods in in vitro diagnostic assay development to create samples with a known status.

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The MultiCode® -RTx HSV 1&2 Kit is a polymerase chain reaction (PCR)-based qualitative in vitro diagnostic test for the detection and typing of herpes simplex virus (HSV1&2) DNA in vaginal lesions. It is indicated for use in the detection and typing of HSV-1 or HSV-2 in vaginal lesion swab specimens from symptomatic female patients as an aid in the diagnosis of genital herpes infection.

Warning: The device is not FDA cleared for the use with cerebral spinal fluid (CSF) or any lesions other than vaginal. This assay is not intended to be used for male penile specimens, for prenatal screening, or for females under the age of 18 years.

Not Found

I am sorry, but I cannot answer your request based on the document you provided. The document primarily consists of an FDA clearance letter for the MultiCode-RTx Herpes Simplex Virus 1 & 2 Kit, and it does not contain the detailed information about acceptance criteria, study methodologies, sample sizes, or ground truth establishment that you are requesting. It confirms the device's clearance and provides its indications for use, but it does not include the specifics of the performance study.

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