The Prodesse® ProGastro SSCS Assav is a multiplex real time PCR in vitro diagnostic test for the qualitative detection and differentiation of Salmonella, Shigella, and Campylobacter (C. jejimi and C. coli only, undifferentiated) nucleic acids and Shiga Toxin 1 (stxl) and Shiga Toxin 2 (stx2) genes. Shiga toxin producing E. coli (STEC) typically harbor one or both genes that encode for Shiga Toxins I and 2. Nucleic acids are isolated and purified from preserved stool specimens obtained from symptomatic patients exhibiting signs and symptoms of gastroenteritis. This test is intended for use, in conjunction with clinical presentation and epidemiological risk factors, as an aid in the differential diagnosis of Salmonella, Shigella, Campylobacter jejuni/Campylobacter coli, and STEC infections in humans.

The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Positive results do not rule out co-infection with other organisms that are are not detected by this test, and may not be the sole or definitive cause of patient illness. Negative ProGastro SSCS Assay results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

Device Description

The ProGastro SSCS Assay enables detection and differentiation of Salmonella, Shigella, Campylobacter (C. jejuni and C. coli only, undifferentiated) and an Internal Control in the SSC Mix and Shiga Toxin Producing E. coli (STEC, stx1 and stx2 differentiated) and an Internal Control in the STEC Mix.

An overview of the procedure is as follows:

1. Collect raw stool specimens from symptomatic patients and place into Cary Blair Transport Medium or ParaPak C&S (C&S) Transport Medium .
1. Add the Gastro RNA/DNA Internal Control (GIC) to every sample to monitor for inhibitors present in the specimens.
1. Perform isolation and purification of nucleic acids using a NucliSENS easyMAG System and the Automated Magnetic Extraction Reagents (bioMérieux).
1. Add purified nucleic acids to the SSC Mix included in the ProGastro SSCS Assay Kit. The SSC Mix contains target-specific oligonucleotide primers and probes for detection of Salmonella, Shigella, and Campylobacter (C. jejuni and C. coli only). The primers and probes are complementary to highly conserved regions of genetic sequences for these organisms. The probes are dual-labeled with a reporter dye and a quencher (see table below).
1. Add purified nucleic acids to the STEC Mix included in the ProGastro SSCS Assay Kit. The STEC Mix contains target-specific oligonucleotide primers and probes for detection of Shiga Toxin 1 and 2 genes (stxl and stx2). The primers and probes are complementary to highly conserved regions of these genes. The probes are dual-labeled with a reporter dye and a quencher (see table below).
1. Perform amplification of DNA in a Cepheid SmartCycler II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProGastro SSCS Assay is based on Tagman reagent chemistry, which utilizes the 5' - 3' exonuclease activity of Taq polymerase to cleave the probe thus separating the reporter dye from the quencher. This generates an increase in fluorescent signal upon excitation from a light source. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification products present at that time. Fluorescent intensity is monitored during each PCR cycle by the real-time instrument.

AI/ML Overview

Here's a breakdown of the acceptance criteria and study information for the ProGastro SSCS Assay:

The document provided details the clinical performance and analytical performance (reproducibility and precision) of the ProGastro SSCS Assay. The primary acceptance criteria for clinical performance are presented as Sensitivity, Specificity, Positive Percent Agreement (PPA), and Negative Percent Agreement (NPA). For analytical performance, it focuses on the agreement with expected results and the Coefficient of Variation (CV) for Ct values.

1. Table of Acceptance Criteria and Reported Device Performance

Clinical Performance (Prospective Study):

Target	Acceptance Criteria (Implicit from Context)	Reported Device Performance (95% CI)
Campylobacter	High Sensitivity & Specificity	Sensitivity 100.0% (83.9% - 100.0%)
(C. jejuni / C. coli)		Specificity 98.8% (98.0% - 99.3%)
Salmonella	High Sensitivity & Specificity	Sensitivity 95.2% (77.3% - 99.2%)
		Specificity 99.1% (98.4% - 99.5%)
Shigella	High Sensitivity & Specificity	Sensitivity 100.0% (79.6% - 100.0%)
		Specificity 99.5% (98.8% - 99.8%)
STEC	High Sensitivity & Specificity	Sensitivity 100.0% (70.1% - 100.0%)
		Specificity 99.2% (98.5% - 99.6%)
*stx1* (Shiga Toxin 1)	High PPA & NPA	PPA 100.0% (67.6% - 100.0%)
		NPA 40.0% (16.8% - 68.7%)
*stx2* (Shiga Toxin 2)	High PPA & NPA	PPA 100.0% (43.9% - 100.0%)
		NPA 80.0% (54.8% - 93.0%)

Clinical Performance (Retrospective Study):

Target	Acceptance Criteria (Implicit from Context)	Reported Device Performance (95% CI)
Campylobacter	High PPA & NPA	PPA 96.4% (82.3% - 99.4%)
		NPA 93.5% (85.7% - 97.2%)
Salmonella	High PPA & NPA	PPA 100.0% (43.4% - 100.0%)
		NPA 100.0% (96.4% - 100.0%)
Shigella	High PPA & NPA	PPA 100.0% (51.0% - 100.0%)
		NPA 100.0% (96.3% - 100.0%)
STEC	High PPA & NPA	PPA 100.0% (83.2% - 100.0%)
(Overall)		NPA 100.0% (95.7% - 100.0%)
*stx1*	High PPA & NPA	PPA 100.0% (78.5% - 100.0%)
		NPA 100.0% (56.6% - 100.0%)
*stx2*	High PPA & NPA	PPA 100.0% (78.5% - 100.0%)
		NPA 100.0% (56.6% - 100.0%)

Reproducibility (Across 3 Sites, 2 Operators/Site, 5 Days - Total 90 runs per condition):

Target Condition	Acceptance Criteria (Implicit: High agreement, Low CV)	Reported Device Performance (Overall Agreement)	Overall Mean Ct Value	Overall % CV
C. jejuni Low Positive	Agreement > 95%, CV < 5% (Example)	100% (95.9%-100.0%)	37.3	3.1
C. jejuni Medium Positive	Agreement > 95%, CV < 5% (Example)	100% (95.9%-100.0%)	34.8	2.8
C. coli Low Positive	Agreement > 95%, CV < 5% (Example)	100% (95.9%-100.0%)	35.6	3.2
C. coli Medium Positive	Agreement > 95%, CV < 5% (Example)	100% (95.9%-100.0%)	33.3	3.1
Salmonella Low Positive	Agreement > 95%, CV < 5% (Example)	100% (95.9%-100.0%)	35.9	1.6
Salmonella Medium Positive	Agreement > 95%, CV < 5% (Example)	100% (95.9%-100.0%)	33.0	1.5
Shigella Low Positive	Agreement > 95%, CV < 5% (Example)	98.9% (94.0%-99.8%)	35.5	2.0
Shigella Medium Positive	Agreement > 95%, CV < 5% (Example)	100% (95.9%-100.0%)	33.1	1.6
STEC (stx1) Low Positive	Agreement > 95%, CV < 5% (Example)	100% (95.9%-100.0%)	35.9	2.2
STEC (stx1) Medium Positive	Agreement > 95%, CV < 5% (Example)	100% (95.9%-100.0%)	33.4	1.6
STEC (stx2) Low Positive	Agreement > 95%, CV < 5% (Example)	100% (95.9%-100.0%)	36.7	1.8
STEC (stx2) Medium Positive	Agreement > 95%, CV < 5% (Example)	100% (95.9%-100.0%)	34.5	1.5
High Negative (IC Ct Value) SSC	Agreement > 95%, CV < 5% (Example)	100% (98.6%-100.0%)	33.2	2.1
High Negative (IC Ct Value) STEC	Agreement > 95%, CV < 5% (Example)	100% (98.6%-100.0%)	32.9	1.5

Precision (Internal, 2 Operators, 12 Days - Total 72 runs per condition):

Target Condition	Acceptance Criteria (Implicit: High agreement, Low CV)	Reported Device Performance (Total Agreement)	Overall Mean Ct Value	Overall % CV
C. jejuni Low Positive	Agreement > 95%, CV < 5% (Example)	100% (72/72)	36.7	2.6
C. jejuni Medium Positive	Agreement > 95%, CV < 5% (Example)	100% (72/72)	34.2	2.4
C. coli Low Positive	Agreement > 95%, CV < 5% (Example)	100% (72/72)	35.0	2.1
C. coli Medium Positive	Agreement > 95%, CV < 5% (Example)	100% (72/72)	32.7	2.3
Salmonella Low Positive	Agreement > 95%, CV < 5% (Example)	100% (72/72)	35.6	1.5
Salmonella Medium Positive	Agreement > 95%, CV < 5% (Example)	100% (72/72)	32.7	1.1
Shigella Low Positive	Agreement > 95%, CV < 5% (Example)	100% (72/72)	35.1	1.6
Shigella Medium Positive	Agreement > 95%, CV < 5% (Example)	100% (72/72)	32.7	1.3
STEC (stx1) Low Positive	Agreement > 95%, CV < 5% (Example)	100% (72/72)	35.6	1.8
STEC (stx1) Medium Positive	Agreement > 95%, CV < 5% (Example)	100% (72/72)	33.0	1.3
STEC (stx2) Low Positive	Agreement > 95%, CV < 5% (Example)	100% (72/72)	36.5	1.8
STEC (stx2) Medium Positive	Agreement > 95%, CV < 5% (Example)	100% (72/72)	34.2	1.2
High Negative (IC Ct Value) SSC	Agreement > 95%, CV < 5% (Example)	98.6% (71/72)	32.9	0.9
High Negative (IC Ct Value) STEC	Agreement > 95%, CV < 5% (Example)	100% (72/72)	32.5	1.0

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

Prospective Clinical Study:
- Initial samples: 1214 patients.
- Eligible samples for analysis: 1139 (after excluding 61 for protocol deviations and 14 for the SSC Mix and 14 for the STEC Mix due to "Unresolved" results and no successful retest).
- Data Provenance: Prospective, collected from July 2011 - November 2011 and May 2012 - July 2012, and tested November 2011 - August 2012. Conducted at four U.S. clinical laboratories. Samples were "excess remnants of stool specimens that were prospectively collected from symptomatic individuals suspected of gastrointestinal infection, and were submitted for routine care or analysis by each site, and that otherwise would have been discarded."
Retrospective Study:
- Sample size: 105 stool samples.
- Data Provenance: Retrospective, collected from 2007 - 2011. Conducted at two clinical sites. These samples were previously determined positive or negative by culture and/or Broth Enrichment/EIA.

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

The document does not explicitly state the number or qualifications of individual "experts" used to establish ground truth for the test set.

Instead, the ground truth was established by:

Reference Methods:
- Culture: For Campylobacter, Salmonella, and Shigella. This typically involves trained microbiologists following established laboratory protocols, but specific expert qualifications are not detailed.
- Broth enrichment followed by FDA cleared EIA test: For Shiga Toxin producing E. coli (STEC). Similar to culture, this relies on standard laboratory procedures and trained personnel.
Confirmatory Methods for Discrepancies:
- PCR followed by bi-directional sequencing: Used to confirm the presence of stx1 and/or stx2 genes in samples positive for STEC by broth/EIA and/or the ProGastro SSCS Assay.
- Analytically validated PCR/sequencing assays: Used to evaluate discrepant results between the ProGastro SSCS Assay and the initial reference methods. This suggests a rigorous molecular biology approach.

4. Adjudication Method for the Test Set

The adjudication method involved confirmatory testing for discrepant results:

For STEC: Samples positive by either the broth/EIA method or the ProGastro SSCS Assay underwent PCR followed by bi-directional sequencing to confirm stx1 and/or stx2 genes. "True" STEC positives were considered any sample testing positive by broth/EIA, while "True" stx2 positives were defined as positive by broth/EIA and PCR/sequencing. This suggests a hierarchical adjudication focused on confirming the genes.
For all targets: Discrepant results between the ProGastro SSCS Assay and the initial reference methods were evaluated using analytically validated PCR/sequencing assays. The results of these confirmatory assays are footnoted in the performance tables, indicating they informed the final determination of "true" status for discrepant samples.

There is no mention of a traditional expert panel consensus (e.g., 2+1, 3+1) for initial ground truth establishment; rather, it relies on established laboratory methodologies and subsequent molecular confirmation for discrepancies.

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 study evaluates the performance of an in vitro diagnostic test (a PCR assay), not a device that is read or interpreted by human readers in a diagnostic imaging or similar context. Therefore, the concept of "how much human readers improve with AI vs without AI assistance" is not applicable here. The assay provides a qualitative result (positive/negative) for specific nucleic acids.

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

Yes, the study primarily represents standalone performance.

The ProGastro SSCS Assay is an in vitro diagnostic test. Its performance (detection of nucleic acids) is assessed as an algorithm only, without direct human cognitive input for interpretation of the primary result (e.g., "reading" a PCR curve to decide positive/negative beyond a set threshold). The technical procedure is performed by trained laboratory personnel, but the "performance" data (Sensitivity, Specificity, PPA, NPA) presented are solely based on the assay's output compared to the ground truth, reflecting the diagnostic capability of the test itself. The intended use states it is "an aid in the differential diagnosis" and "should not be used as the sole basis for diagnosis," implying it contributes to a physician's overall assessment, but its technical performance is standalone.

7. The Type of Ground Truth Used

The ground truth for the clinical studies was established using a combination of:

Culture: For bacterial pathogens (Campylobacter, Salmonella, Shigella). This is a widely accepted gold standard for bacterial identification.
FDA Cleared EIA Test (with broth enrichment): For Shiga Toxin producing E. coli (STEC). This is a recognized method for STEC detection.
PCR followed by bi-directional sequencing: This molecular method served as a confirmatory gold standard for discrepant results and for the specific identification of stx1 and stx2 genes. This represents a highly specific and sensitive molecular confirmation.

So, the ground truth is a hybrid of established microbiological culture/EIA methods and molecular sequencing confirmation.

8. The Sample Size for the Training Set

The document does not report a separate training set or details on how the training of any underlying algorithm (if present, which is unlikely for a PCR assay) was performed.

For in vitro diagnostic assays like PCR tests, the "training" equivalent typically involves extensive analytical validation (e.g., limit of detection, inclusivity, exclusivity, interference studies) to establish the assay's performance characteristics, rather than a machine learning training set as understood for AI algorithms. The clinical studies (prospective and retrospective) are primarily for validation of performance.

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

As noted above, a distinct "training set" is not mentioned in the context of this PCR assay. The establishment of ground truth for the validation/test sets is detailed in point 7 (Culture, FDA cleared EIA, and PCR/sequencing).

Summary

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123274

Page 1 of 11

01/15/2013

Gen-Probe Prodesse, Inc. ProGastro Assay 510(k) Submission

Attachment D 510(k) SUMMARY

CONTACT

Karen Harrington, PhD Manager, Clinical Affairs Gen-Probe Prodesse. Inc. 20925 Crossroads Circle Waukesha, WI 53186

NAME OF DEVICE

Trade Name: Regulation Number: Product Code:

ProGastro SSCS Assay 21 CFR 866.3990 PCH and PCI

PREDICATE DEVICE

K121454 Luminex GPP Test

INTENDED USE

PRODUCT DESCRIPTION

An overview of the procedure is as follows:

1. Collect raw stool specimens from symptomatic patients and place into Cary Blair Transport Medium or ParaPak C&S (C&S) Transport Medium .
1. Add the Gastro RNA/DNA Internal Control (GIC) to every sample to monitor for inhibitors present in the specimens.
1. Perform isolation and purification of nucleic acids using a NucliSENS easyMAG System and the

Gen-Probe Prodesse, Inc.

JAN 1 6 2013

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Automated Magnetic Extraction Reagents (bioMérieux).

1. Add purified nucleic acids to the SSC Mix included in the ProGastro SSCS Assay Kit. The SSC Mix contains target-specific oligonucleotide primers and probes for detection of Salmonella, Shigella, and Campylobacter (C. jejuni and C. coli only). The primers and probes are complementary to highly conserved regions of genetic sequences for these organisms. The probes are dual-labeled with a reporter dye and a quencher (see table below).
1. Add purified nucleic acids to the STEC Mix included in the ProGastro SSCS Assay Kit. The STEC Mix contains target-specific oligonucleotide primers and probes for detection of Shiga Toxin 1 and 2 genes (stxl and stx2). The primers and probes are complementary to highly conserved regions of these genes. The probes are dual-labeled with a reporter dye and a quencher (see table below).
1. Perform amplification of DNA in a Cepheid SmartCycler II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProGastro SSCS Assay is based on Tagman reagent chemistry, which utilizes the 5' - 3' exonuclease activity of Taq polymerase to cleave the probe thus separating the reporter dye from the quencher. This generates an increase in fluorescent signal upon excitation from a light source. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification products present at that time. Fluorescent intensity is monitored during each PCR cycle by the real-time instrument.

Supermix	Analyte	Gene Targeted	Probe Fluorophore	Absorbance Peak	Emission Peak	Instrument Channel
SSC Mix	Campylobacter(C. jejuni and C.coli only)	C. jejuniglyAC. colicadF	FAM	495 nm	520 nm	FAM
SSC Mix	Salmonella spp.	orgC	CAL FluorOrange 560	538 nm	559 nm	TET
SSC Mix	Shigella spp.	ipaH	CAL FluorRed 610	590 nm	610 nm	Texas Red
STEC Mix	Shiga Toxin 1	stx1	CAL FluorOrange 560	538 nm	559 nm	TET
STEC Mix	Shiga Toxin 2	stx2	FAM	495 nm	520 nm	FAM
SSC Mixand STECMix	Internal Control	NA	Quasar 670	647 nm	670 nm	Cy5

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SUBSTANTIAL EQUIVALENCE

Similarities
Element	Prodesse ProGastro SSCS (K123274)	Luminex xTAG GPP (K121454)
OrganismsDetected	Salmonella spp., Shigella spp.,Campylobacter ( C. jejuni and C. coli ),and STEC ( stx1 and stx2 genes)	Same(See below for differences)
Analyte	DNA	Same(See below for differences)
TechnologicalPrinciples	Multiplex nucleic acid	Same(See below for differences)
SpecimenTypes	Stool specimens	Same
User Complexity	High	Same
SamplePreparationMethod	Up front sample processing is requiredto extract nucleic acids	Same
Controls	Internal control in each sample.External control processed with eachbatch of samples.	Same
Differences
Element	Prodesse ProGastro SSCS (K123274)	Luminex xTAG GPP (K121454)
OrganismsDetected	(See above for similarities)	Can also detect and distinguish C. lari .In addition, can detect and distinguishClostridium difficile toxin A/B,Cryptosporidium ( C. parvum and C.hominis only), Escherichia coli ( E.coli ) O157, Enterotoxigenic E. coli(ETEC) LT/ST, Giardia ( G. lambliaonly), Norovirus GI/GII, andRotavirus A.
Analyte	DNA	RNA/DNA
TechnologicalPrinciples	Real time multiplex PCR based on theTaqman reagent chemistry	Multiplex RT-PCR and beadhybridization followed byFluorescence-activated sorting oflabeled beads coupled to streptavidin-conjugated biotinylated products
Instrumentation	Cepheid SmartCycler II	PCR Thermocycler and Luminex100/200 system

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Clinical Performance

Prospective Study

The clinical performance of the ProGastro SSCS Assay was established during prospective studies at four U.S. clinical laboratories. Leftover stool samples were collected during July 2011 - November 2011 and May 2012 - July 2012 and tested during November 2011 thru August 2012. All specimens used in the study meeting the inclusion criteria represented excess remnants of stool specimens that were prospectively collected from symptomatic individuals suspected of gastrointestimal infection, and were submitted for routine care or analysis by each site, and that otherwise would have been discarded.

Demographic details for the patient population included in the prospective study are summarized in the following table.

Sex	Number of Samples SSC Mix	Number of Samples STEC Mix
Male	615/1214 (50.6%)	615/1214 (50.6%)
Female	581/1214 (47.9%)	581/1214 (47.9%)
Unknown	18/1214 (1.5%)	18/1214 (1.5%)
Age (yrs)
≤ 5 years	378/1214 (31.1%)	378/1214 (31.1%)
6 - 18 years	296/1214 (24.4%)	296/1214 (24.4%)
19 - 64 years	357/1214 (29.4%)	357/1214 (29.4%)
≥ 65 years	164/1214 (13.5%)	164/1214 (13.5%)
Unknown	19/1214 (1.6%)	19/1214 (1.6%)

Performance of the ProGastro SSCS Assay was assessed and compared to the reference method of culture (Campylobacter, Salmonella, and Shigella) or broth enrichment followed by FDA cleared EIA test (Shiga Toxin producing E. coli). Samples positive for STEC by broth/EIA and/or the ProGastro SSCS Assay underwent PCR followed by bi-directional sequencing to confirm the presence of the stx 1 and/or stx2 genes. Two PCR/sequencing assays were used that each targeted different regions of the stxl or stx2 gene than the ProGastro SSCS Assay. "True" STEC positives were considered as any sample that tested positive for STEC by the broth/ElA method, and "True" STEC negatives were considered as any sample that tested negative for STEC by the broth/EIA method. "True" stx2 positives were considered as any sample that tested positive for STEC by the broth/E/A method and by PCR/sequencing. Bi-directional sequencing data was required to meet pre-defined quality acceptance criteria for both the forward and the reverse sequences that matched stx1 or stx2 sequences deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.gov), respectively, with acceptable E-values. The E-Value from NCBI BLAST Alignment indicates the statistical significance of a given pair-wise alignment and reflects the size of the database and the scoring system used. The lower the E-Value, the more significant the hit is. A sequence alignment that has an E-Value of le-3 means that this similarity has a 1 in 1000 chance of occurring by chance alone. (http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=handbook.section.614).

Discrepant results between the ProGastro SSCS Assav and the reference methods were also evaluated using analytically validated PCR/sequencing assays and results are footnoted in the performance tables below.

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A total of 1214 patients were initially enrolled in the prospective clinical trial. Prospective stool specimens were initially included in the prospective clinical trial. Sixty-one (61) patient/specimens were excluded from the performance calculations due to deviations from the clinical study protocol. Fourteen (14) specimens were excluded for the SSC Mix and 14 were excluded for the STEC Mix from the prospective clinical study data analysis because they remained "Unresolved" after repeat testing with the respective ProGastro SSCS Assay Mix. Unresolved results occur when the sample is negative for all target detections and the Internal Control, indicating potentially PCR-inhibited samples, This resulted in a total of 1139 eligible prospective specimens to be included in the prospective clinical study data analysis.

	Culture
	Positive	Negative	Total
ProGastroSSCSAssay	Positive	20	13a	33	Sensitivity 100.0%(83.9% - 100.0%) 95% CI
	Negative	0	1106	1106	Specificity 98.8%(98.0% - 99.3%) 95% CI
	Total	20	1119	1139

Campylobacter (C. jejuni / C. coli) Comparison Results

a Six (6) samples were positive for Campylobacter (C. coli or C. jejuni) by bi-directional sequence analysis.

Salmonella Comparison Results

	Culture
	Positive	Negative	Total
ProGastroSSCSAssay	20	10a	30	Sensitivity 95.2%(77.3% - 99.2%) 95% CI
	1b	1108	1109	Specificity 99.1%(98.4% - 99.5%) 95% CI
Total	21	1118	1139

a Ten (10) samples were positive for Salmonella by bi-directional sequence analysis.

Sample was positive for Salmonella by bi-directional sequence analysis.

Shigella Comparison Results

	Culture
	Positive	Negative	Total
ProGastroSSCSAssay	Positive	15	6a	21	Sensitivity 100.0%(79.6% - 100.0%) 95% CI
	Negative	0	1118	1118	Specificity 99.5%(98.8% - 99.8%) 95% CI
	Total	15	1124	1139

a Six (6) samples were positive for Shigella by bi-directional sequence analysis.

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STEC Comparison Results

	Broth Enrichment/EIA
	Positive	Negative	Total
ProGastroSSCSAssay	Positive	9a	9b	18	Sensitivity 100.0%(70.1% - 100.0%) 95% CI
	Negative	0	1121	1121	Specificity 99.2%(98.5% - 99.6%) 95% CI
	Total	9	1130	1139

a Six (6) samples positive for stx1, one (1) sample positive for stx2, and two (2) samples positive for stx1 and stx2 by bi-directional sequence analysis.

b Six (6) samples positive for stx1 and three (3) samples positive for stx2 by bi-directional sequence analysis.

stx1 Comparison Results

	Broth Enrichment/EIA andsequencing for stx1
	Positive	Negative	Total
ProGastroSSCS Assay	Positive	8	6a	14	Positive Percent Agreement100.0%(67.6% - 100.0%) 95% CI
	Negative	0	4	4	Negative Percent Agreement 40.0%(16.8% - 68.7%) 95% CI
	Total	8	10	18

4 Six (6) samples negative by broth/EIA, five (5) were positive for stx/by bi-directional sequencing, but were negative by Broth Enrichment/EIA.

stx2 Comparison Results

	Broth Enrichment/EIAand sequencing for stx2
	Positive	Negative	Total
ProGastroSSCS Assay	Positive	3	3a	6	Positive Percent Agreement100.0%(43.9% - 100.0%) 95% CI
	Negative	0	12	12	Negative Percent Agreement 80.0%(54.8% - 93.0%) 95% CI
	Total	3	15	18

4 Three (3) samples were positive for stx2 by bi-directional sequence analysis, but were negative by Broth Enrichment/EIA.

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The ProGastro SSCS Assay detected one mixed infections in the prospective clinical evaluation. This represents 0.98% of the total positive specimens (1/102). The one mixed infections sample was double infections and was confirmed by the reference methods.

Distinct Co-infection Combinations Detected by the ProGastro SSCS Assay in the Prospective Clinical Trial

Distinct Co-infection CombinationsDetected by ProGastro SSCS Assay
Analyte 1	Analyte 2	Analyte 3	TotalCo-infections	Number ofDiscrepantCo-infectionsa	Discrepant Analyte(s)a
Salmonella	Campylobacter	N/A	0	0
Salmonella	Shigella	N/A	0	0
Salmonella	STEC	N/A	0	0
Campylobacter	Shigella	N/A	0	0
Campylobacter	STEC	N/A	1	0
STEC	Shigella	N/A	0	0
Salmonella	Campylobacter	STEC	0	0
Total Co-infections			1	0
Total Double Infections			1	0
Total Triple Infections			0	0

ª A discrepant co-infection or discrepant analyte was defined as one that was detected by the ProGastro SSCS Assay but not detected by the reference methods.

There were no co-infections that were detected by the reference method and not detected by the ProGastro SSCS Assay.

Retrospective Study

In addition to the prospective clinical study, two clinical sites also performed testing using retrospective samples that were collected from 2007 - 2011. A total of 105 stool samples were included in the retrospective study. These samples had been previously determined to be positive or negative by culture and/or Broth Enrichment/ElA. The ProGastro SSCS Assay was compared to the same reference method that was employed for the prospective study to determine positive and negative percent agreement.

Demographic details for this patient population are summarized in the table below.

Sex*	Number of Subjects
Female	24/55 (43.6%)
Male	31/55 (56.4%)
Age	Number of Subjects
≤ 5 years	12/105 (11.4%)
6 - 18 years	24/105 (22.9%)
19 – 64 years	51/105 (48.6%)
≥ 65 years	18/105 (17.1%)

*For all of the 50 specimens tested from one site the gender was unknown

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Campylobacter Comparison Results

	Culture
		Positive	Negative	Total
ProGastroSSCS Assay	Positive	27	5	32	Positive Percent Agreement 96.4%(82.3% - 99.4%) 95% CI
	Negative	1	72	73	Negative Percent Agreement93.5%(85.7% - 97.2%) 95% CI
	Total	28	77	105

Salmonella Comparison Results

			Culture
			Positive	Negative	Total
ProGastroSSCS Assay	Positive	3	0	3		Positive Percent Agreement100.0%(43.4% - 100.0%) 95% CI
	Negative	0	102	102		Negative Percent Agreement100.0%(96.4% - 100.0%) 95% CI
Total		3	102	105

Shigella Comparison Results

		Culture
		Positive	Negative	Total
ProGastroSSCS Assay	Positive	4	0	4	Positive Percent Agreement100.0%(51.0% - 100.0%) 95% CI
	Negative	0	101	101	Negative Percent Agreement100.0%(96.3% - 100.0%) 95% CI
Total		4	101	105

STEC Comparison Results

			Culture or Broth Enrichment/EIA
			Positive	Negative	Total
ProGastroSSCSAssay	Positive		19a	0	19	Positive Percent Agreement 100.0%(83.2% - 100.0%) 95% CI
	Negative		0	86	86	Negative Percent Agreement 100.0%(95.7% - 100.0%) 95% CI
		Total	19	86	105

4 Five (5) samples positive for stx1, 5 samples positive for stx2, and 9 samples positive for stx1 and stx2.

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stx1 Comparison Results

	Culture or Broth Enrichment/EIAand sequencing for stx1
ProGastroSSCSAssay		Positive	Negative	Total
	Positive	14	0	14	Positive Percent Agreement 100.0%(78.5% - 100.0%) 95% CI
	Negative	0	5	5	Negative Percent Agreement 100.0%(56.6% - 100.0%) 95% CI
	Total	14	5	19

stx2 Comparison Results

		Culture or Broth Enrichment/EIAand sequencing for stx2
		Positive	Negative	Total
ProGastroSSCSAssay	Positive	14	0	14	Positive Percent Agreement 100.0%(78.5% - 100.0%) 95% CI
	Negative	0	5	5	Negative Percent Agreement 100.0%(56.6% - 100.0%) 95% CI
	Total	14	5	19

Of the prospective and retrospective specimens run using the SSC Mix Assay, 98.0% (1233/1258) of these specimens were successful on the first attempt. The remaining 25 (25/1258 = 2.0%) gave "Unresolved" results on the first attempt. An "Unresolved" result is generated when the Gastro Internal Control (GIC) fails to be detected in a clinical specimen. A failure of the GIC to be detected can occur if inhibitors are present in a sample or due to technical error (e.g., GIC not added prior to nucleic acid extraction). Of the 25 "Unresolved" specimens on the first attempt with sufficient nucleic acid for retest, 44.0% (11/25) gave a valid result on the second attempt. The remaining 14 were "Unresolved" on the second attempt.

Of the prospective and retrospective specimens run using the STEC Mix Assay, 97.9% (1232/1258) of these specimens were successful on the first attempt. The remaining 26 (26/1258 = 2.1%) gave "Unresolved" results on the first attempt. Of the 26 "Unresolved" specimens on the first attempt with sufficient nucleic acid for retest, 48.0% (12/25) gave a valid result on the second attempt. The remaining 14 were "Unresolved" on the second attempt.

Reproducibility

The reproducibility of the ProGastro SSCS Assay was evaluated at three laboratory sites. Reproducibility was assessed using a panel of 15 simulated samples that included medium positive, low positive (near the assav limit of

detection, ≥ 95% positive), and high negative (below the assay limit of detection, ≤ 95% positive) samples for each of the assay targets. The reproducibility panel and controls were run with the ProGastro SSCS Assay (SSC and STEC Mixes) at three sites by each of two operators per site for five days.

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Reproducibility Panel Member Results

	SSC Mix								STEC Mix						SSC STECMix Mix
	PanelMember ID	C. jejuni Low Positive	C. jejuni Medium Positive	C. coli Low Positive	C. coli Medium Positive	Salmonella Low Positive	Salmonella MediumPositive	Shigella Low Positive	Shigella Medium Positive	STEC (stx 1) Low Positive	STEC (stx 1) MediumPositive	STEC (stx 2) Low Positive	STEC (stx 2) MediumPositive	High Negative (IC CtValue)	High Negative (IC CtValue)
	Concentration	20X*LoD	100X*LoD	6X*LoD	30X*LoD	2XLoD	10XLoD	2XLoD	10XLoD	2XLoD	10XLoD	2XLoD	10XLoD	0.0001XLoD
	Agreementwith ExpectedResult	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	90/90100%	90/90100%
Site 1	Mean Ct Value	37.6	35.2	35.9	33.5	35.9	33.2	35.8	33.2	36.1	33.4	36.7	34.5	33.6	33.2
	% CV	3.5	3.0	3.9	3.7	1.4	1.2	1.7	1.4	2.0	1.6	1.8	1.3	3.0	1.7
	Agreementwith ExpectedResult	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	29/3096.7%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	90/90100%	90/90100%
Site 2	Mean Ct Value	37.3	34.8	35.8	33.4	36.0	33.1	35.6	33.2	36.2	33.6	36.8	34.7	33.3	33.0
	% CV	3.0	2.8	3.1	3.2	1.7	1.6	1.9	1.8	2.5	1.6	1.9	1.9	0.9	0.9
	Agreementwith ExpectedResult	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	90/90100%	90/90100%
Site 3	Mean Ct Value	37.0	34.4	35.2	32.9	35.7	32.7	35.0	32.8	35.6	33.1	36.5	34.2	32.9	32.6
	% CV	2.6	2.2	2.1	1.9	1.4	1.2	1.8	1.3	1.7	1.3	1.6	1.0	0.8	1.1
	TotalAgreementwith ExpectedResult	90/90100%	90/90100%	90/90100%	90/90100%	90/90100%	90/90100%	89/9089.9%	90/90100%	90/90100%	90/90100%	90/90100%	90/90100%	270/270100%	270/270100%
	95% CI	95.9%-100.0%	95.9%-100.0%	95.9%-100.0%	95.9%-100.0%	95.9%-100.0%	95.9%-100.0%	94.0%-99.8%	95.9%-100.0%	95.9%-100.0%	95.9%-100.0%	95.9%-100.0%	95.9%-100.0%	98.6%-100.0%	98.6%-100.0%
	Overall MeanCt Value	37.3	34.8	35.6	33.3	35.9	33.0	35.5	33.1	35.9	33.4	36.7	34.5	33.2	32.9
	Overall% CV	3.1	2.8	3.2	3.1	1.6	1.5	2.0	1.6	2.2	1.6	1.8	1.5	2.1	1.5

Note: The Campylobacter strains were tested at higher concentrations because Campylobacter is sensitive to environmental stressors including freezing where it loses viability. However, the average Ct value for the C. jejuni (ATCC 33291) Low Positives was 37.3, which is very close to the average Ct value of 38.8 for the same C. jejuni strain tested at the estimated LoD level in the Analytical Reactivity Study. The average Ct value for the C. coli (ATCC BAA-371) Low Positives was 35.6, which is very close to the average Ct value of 34.4 for the same C. coli strain tested at the estimated LoD level in the Analytical Reactivity Study. Therefore, the effective DNA concentrations of the C. jejuni (ATCC 33291) and C. codi (ATCC BAA-371) low positive samples tested in the Reproducibility Study are very close to the estimated LoD DNA concentrations for these two strains tested in the Analytical Reactivity Study.

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Precision

The precision of the ProGastro SSCS Assay was evaluated internally using a panel of 15 simulated samples that included medium positive (near the assay limit of detection, ≥ 95% positive), and high negative (below the assay limit of detection, ≤ 95% positive) samples for each of the assay targets. A panel of 15 contrived samples including the necessary controls was run with the ProGastro SSCS Assay (SSC and STEC Mixes) by each of two operators for twelve days.

	SSC Mix								STEC Mix				SSCMix	STECMix
PanelMember ID	C. jejuni Low Positive	C. jejuni Medium Positive	C. coli Low Positive	C. coli Medium Positive	Salmonella Low Positive	Salmonella Medium Positive	Shigella Low Positive	Shigella Medium Positive	STEC ( stx 1 ) Low Positive	STEC ( stx 1 ) Medium Positive	STEC ( stx 2 ) Low Positive	STEC ( stx 2 ) Medium Positive	High Negative (IC Ct Value)	High Negative (IC Ct Value)
Concentration	20X*LoD	100X*LoD	6X*LoD	30X*LoD	2XLoD	10XLoD	2XLoD	10XLoD	2XLoD	10XLoD	2XLoD	10XLoD	0.0001X LoD	0.0001X LoD
TotalAgreementwith ExpectedResult	72/72100%	72/72100%	72/72100%	72/72100%	72/72100%	72/72100%	72/72100%	72/72100%	72/72100%	72/72100%	72/72100%	72/72100%	71/7298.6%	72/72100%
Overall MeanCt Value	36.7	34.2	35.0	32.7	35.6	32.7	35.1	32.7	35.6	33.0	36.5	34.2	32.9	32.5
Overall % CV	2.6	2.4	2.1	2.3	1.5	1.1	1.6	1.3	1.8	1.3	1.8	1.2	0.9	1.0

Precision Panel Member Results

Note: The Campylobacter strains were tested at higher concentrations because Campylobacter is sensitive to environmental stressors including freezing where it loses viability. However, the average Ct value for the C. jejuni (ATCC 33291) Low Positives was 37.3, which is very close to the average Ct value of 38.8 for the same C. jejuni strain tested at the estimated LoD level in the Analytical Reactivity Study. The average Ct value for the C. coli (ATCC BAA-371) Low Positives was 35.6, which is very close to the average Ct value of 34.4 for the same C. coli strain tested at the estimated LoD level in the Analytical Reactivity Study. Therefore, the effective DNA concentrations of the C. jejuni (ATCC 33291) and C. codi (ATCC BAA-371) low positive samples tested in the Precision Study are very close to the estimated LoD DNA concentrations for these two strains tested in the Analytical Reactivity Study.

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DEPARTMENT OF HEALTH & HUMAN SERVICES

Public Health Service

Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-002

Gen-Probe Prodesse, Inc. c/o Karen Harrington, Ph.D. Manager, Clinical Affairs 20925 Crossroads Circle Waukesha, WI 53186

JAN 1 6 2013

Re: K123274

Trade/Device Name: ProGastro SSCS Assay Regulation Number: 21 CFR 866.3990 Regulation Name: Gastrointestinal Microorganism Multiplex Nucleic Acid-Based Assay Regulatory Class: Class II Product Code: PCH, PCI, OOI Dated: October 17. 2012 Received: October 19, 2012

Dear Dr. Harrington:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set

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Page 2 -- Karen Harrington

forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulation (21 CFR Parts 801 and 809), please contact the Office of In Vitro Diagnostics and Radiological Health at (301) 796-5450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours,

Uwe Scherf for

Sally A. Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indication for Use

510(k) Number: K123274

Device Name: ProGastro SSCS Assay

Indication For Use:

The Prodesse® ProGastro SSCS Assay is a multiplex real time PCR in vitro diagnostic test for the qualitative detection and differentiation of Salmonella, Shigella, and Campylobacter (C. jejuni and C. coli only, undifferentiated) nucleic acids and Shiga Toxin 1 (stxl) and Shiga Toxin 2 (stx2) genes. Shiga toxin producing E. coli (STEC) typically harbor one or both genes that encode for Shiga Toxins 1 and 2. Nucleic acids are isolated and purified from preserved stool specimens obtained from symptomatic patients exhibiting signs and symptoms of gastroenteritis. This test is intended for use, in conjunction with clinical presentation and epidemiological risk factors, as an aid in the differential diagnosis of Salmonella, Shigella, Campylobacter jejuni/Campylobacter coli, and STEC infections in humans.

The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Positive results do not rule out co-infection with other organisms that are not detected by this test, and may not be the sole or definitive cause of patient illness. Negative ProGastro SSCS Assay results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

Prescription Use X (21 CFR Part 801 Subpart D) And/Or

Over the Counter Use _ (21 CFR Part 801 Subpart C)

(PLEASE DO NOT WRITE BELOW THIS LINE; CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of In Vitro Diagnostics and Radiological Health (OIR)

R. H. Hitz

ion Sign-Off Office of In Vitro Diagnostics and Radiological Health k 23274

Regulation Number and Section

§ 866.3990 Gastrointestinal microorganism multiplex nucleic acid-based assay.

(a)
Identification. A gastrointestinal microorganism multiplex nucleic acid-based assay is a qualitativein vitro diagnostic device intended to simultaneously detect and identify multiple gastrointestinal microbial nucleic acids extracted from human stool specimens. The device detects specific nucleic acid sequences for organism identification as well as for determining the presence of toxin genes. The detection and identification of a specific gastrointestinal microbial nucleic acid from individuals exhibiting signs and symptoms of gastrointestinal infection aids in the diagnosis of gastrointestinal infection when used in conjunction with clinical evaluation and other laboratory findings. A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.(b)
Classification. Class II (special controls). The special controls are set forth in FDA's guideline document entitled: “Class II Special Controls Guideline: Gastrointestinal Microorganism Multiplex Nucleic Acid-Based Assays for Detection and Identification of Microorganisms and Toxin Genes from Human Stool Specimens.” For availability of the guideline document, see § 866.1(e).