The PrimeraDx ICEPlex® C. difficile Assay is for the qualitative detection of the Clostridium difficile toxin B gene (tcdB gene) in nucleic acids purified from unpreserved liquid or soft human stool specimens from patients suspected of having C. difficile infection (CDI).

The ICEPlex C. difficile Assay is intended to be used only on the ICEPlex® System, which integrates PCR-based amplification with capillary electrophoresis (CE) for the detection of amplification products. The assay is intended to aid in the diagnosis of CDI. Results should be considered in conjunction with patient clinical history.

Device Description

PrimeraDx's ICEPlex C. difficile Assay kit is intended to be used only on the ICEPlex System. The ICEPlex C. difficile Assay kit incorporates several universal features and approaches developed at PrimeraDx for the ICEPlex System. The ICEPlex C. difficile Assay kit includes a PCR enzyme and the appropriate PCR buffer system developed, optimized, verified and validated for ideal performance in multiplex PCR with subsequent capillary electrophoresis. The ICEPlex C. difficile assay kit includes a primer mix for detection of the Clostridium difficile toxin B gene (tcdB gene) in human stool specimen from patients suspected of having C. difficile infection (CDI). The ICEPlex C. difficile assay kit is comprised of a PCR enzyme, primer mix, PCR buffer, calibrators mix, injection buffer, internal control, and a positive control.

The ICEPlex System combines two functional modules: an amplification module - PCR (Polymerase Chain Reaction) thermal cycler - and an analysis module -CE (Capillary Electrophoresis) system with fluorescent detection. Individual fluorescent PCR products from multiplexed PCR reactions are analyzed by CE through direct electrokinetic injection into the separating capillaries. The labeled amplicons are separated by size and the dyes are excited by two lasers within the system.

AI/ML Overview

This document describes the ICEPlex C. difficile Assay Kit and its performance characteristics.

Here is the requested information:

1. Acceptance Criteria and Reported Device Performance

Clinical Performance (Comparison to Toxigenic C. difficile Direct Culture)

Performance Metric	Acceptance Criteria (Implicit)	Reported Device Performance (95% CI)
Positive Percent Agreement (PPA)	High agreement with direct culture	90.0% (84.5 - 94.1)
Negative Percent Agreement (NPA)	High agreement with direct culture	97.4% (96.1 - 98.4)

Analytical Performance

Performance Metric	Acceptance Criteria (Implicit)	Reported Device Performance
Precision (Low Positive)	High percentage of correct results across different operators, lots, and instruments. (Implicitly >95% agreement where applicable)	99% agreement
Precision (Moderate Positive)	High percentage of correct results across different operators, lots, and instruments. (Implicitly >95% agreement where applicable)	100% agreement
Precision (Negative)	High percentage of correct results across different operators, lots, and instruments. (Implicitly >95% agreement where applicable)	100% agreement
Reproducibility (Low Positive)	High percentage of correct results across different sites, days, and runs. (Implicitly >95% agreement where applicable)	100% agreement
Reproducibility (Moderate Positive)	High percentage of correct results across different sites, days, and runs. (Implicitly >95% agreement where applicable)	100% agreement
Reproducibility (Negative)	High percentage of correct results across different sites, days, and runs. (Implicitly >95% agreement where applicable)	100% agreement
Analytical Sensitivity (LOD)	Lowest concentration at which at least 95% of replicates are positive.	C. difficile strain 43255 (Toxinotype 0): 8CFU/rxn C. difficile strain BAA-1805 (Toxinotype III): 2CFU/rxn
Analytical Reactivity	Detection of all tested toxigenic C. difficile strains.	All 20 tested toxigenic strains were positive.
Analytical Specificity (Cross Reactivity)	No cross-reactivity with common non-C. difficile microorganisms or healthy intestinal flora.	Clostridium sordellii cross-reacted; otherwise, no cross-reactivity.
Interfering Substances	No interference from common substances found in stool.	None of the tested substances interfered with detection or caused false positives.
Carryover/Contamination	No well-to-well or run-to-run contamination.	0% False Positive rate (0 out of 141 valid negatives).

2. Sample Size and Data Provenance

Test Set (Clinical Study):

Sample Size: 969 compliant specimens were initially enrolled. 952 (98.2%) yielded reportable results and were included in the statistical analysis.
Data Provenance: Clinical samples collected from patients suspected of C. difficile infection at three independent clinical sites. The data is prospective, as samples were collected for the investigational study. The country of origin is not explicitly stated but implied to be the US, given the 510(k) submission to the FDA.

Analytical Studies:

Precision: 144 observations for each of the low positive, moderately positive, negative, and Hi/Low samples.
Reproducibility: 90 observations for C20-80, moderate positive, low positive, and negative samples (across 3 sites * 5 days * 2 runs/day * 3 replicates). 60 observations for positive and negative controls.
Limit of Detection (LOD): 20 replicates at each concentration level for two C. difficile strains.
Analytical Reactivity: 20 additional toxigenic C. difficile strains tested.
Cross Reactivity: Panel of 5 non-toxigenic C. difficile strains, 14 other Clostridium strains, and 54 other pathogens/intestinal flora.
Interfering Substances: Multiple replicates for each of 14 interfering substances, tested with both negative and contrived positive samples (two C. difficile strains).
Contamination: 6 runs, each with 24 high positive and 24 negative samples.

3. Number and Qualifications of Experts for Ground Truth

Clinical Study (Test Set):
The ground truth for the clinical study was established by toxigenic C. difficile direct culture. This is a laboratory-based method. The document does not specify the number of human experts involved in interpreting these cultures or their specific qualifications (e.g., medical microbiologists, lab technologists), as it's a standard laboratory procedure.

Discordant Analysis:
For samples with discordant results between the ICEPlex assay and direct culture, microbiological isolation and PCR targeting of 3 appropriate regions of the toxin B gene (different recognition sites than the ICEPlex assay) with bi-directional DNA sequencing were performed. This "truth standard" is a higher-level molecular characterization, implying expert interpretation of molecular results, but the specific number and qualifications of these experts are not provided.

4. Adjudication Method for the Test Set

The primary comparison for the clinical study was the ICEPlex C. difficile Assay versus toxigenic C. difficile direct culture.

For discordant results:
An adjudication method was used where samples showing disagreement between the ICEPlex assay and toxigenic C. difficile direct culture underwent discordant analysis. This analysis involved "microbiological isolation of and PCR targeting of 3 appropriate regions of the toxin B gene (Different recognition sites than the ones used in the ICEPlex C. difficile assay) with bi-directional DNA sequencing."
The results of this discordant analysis were then used to adjudicate the initial discrepancies. For example, of the 20 samples called positive by ICEPlex but negative by direct culture, 16 were confirmed positive by discordant analysis. Similarly, of the 17 samples called negative by ICEPlex but positive by direct culture, 14 were confirmed positive by discordant analysis. This suggests a multi-method adjudictation approach rather than a human expert consensus panel on the original results.

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 a diagnostic kit for laboratory use, and the study evaluates the performance of the device itself against a reference method (culture), not the impact of the device on human reader performance or a human-in-the-loop scenario. The "readers" in this context are the laboratory instruments and the analytical interpretation of their output.

6. Standalone (Algorithm Only) Performance Study

Yes, a standalone performance study was done. The entire clinical performance section evaluates the performance of the ICEPlex C. difficile Assay (algorithm/device only) against toxigenic C. difficile direct culture. There is no human interpretation component integrated into the listed performance metrics for the ICEPlex assay itself. The results presented (PPA, NPA) directly reflect the algorithm's diagnostic accuracy.

7. Type of Ground Truth Used for the Test Set

The primary ground truth used for the clinical test set was toxigenic C. difficile direct culture.

For discordant samples, an enhanced ground truth was established using an "adjudication" or "truth standard" method that involved:

Microbiological isolation
PCR targeting of 3 appropriate regions of the toxin B gene (different from the ICEPlex assay targets)
Bi-directional DNA sequencing

8. Sample Size for the Training Set

The document does not explicitly state a sample size for a training set. For molecular diagnostic assays like this, the 'training' phase often refers to internal assay development, optimization, and analytical validation. The performance studies detailed (precision, reproducibility, LOD, clinical performance) represent the formal validation/test set. It is common for 510(k) submissions to focus on validation rather than explicitly detailing a distinct "training set" for the algorithm itself, as the assay design (primers, probes, CE interpretation rules) is pre-established during development.

9. How Ground Truth for the Training Set Was Established

As noted above, no explicit training set is detailed. However, during the development and optimization of such assays, ground truth for initial assay design and analytical studies would typically be established using:

Known characterized C. difficile strains (ATCC strains, clinical isolates) at defined concentrations.
Spiked samples where known amounts of the target (C. difficile) are added to negative stool matrices.
Reference laboratory methods (e.g., culture, established PCR methods, sequencing) to confirm the presence or absence of the target.

These methods are evident in the analytical performance studies (e.g., ATCC strains for precision, spiked samples for LOD and inclusivity).

Summary

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510(k) Summary

ICEPlex C. difficile Kit on the ICEPlex System

A. 510(K) NUMBER: K132726

Date Prepared: November 29, 2013

B. SUBMITTED BY (APPLICANT):

PrimeraDx

171 Forbes Blvd., Suite 1000 Mansfield, MA 02048

NOV 29 2013

Contact Person

Fayyaz Memon Vice President, Regulatory Affairs and Quality Assurance Tel: 301-221-0245 Fax: 508-339-0452 Email: fmemon@primeradx.com

C. PURPOSE OF SUBMISSION

Substantial equivalence determination of ICEPlex C. difficile Assay Kit on the ICEPlex® System for qualitative detection of the C. difficile toxin B gene (tcdB gene) in a human stool specimen in patients suspected of having C. difficile infection (CDI).

D. Measurand

Clostridium difficile toxin B gene (tcdB)

E. TYPE OF TEST:

Qualitative Nucleic Acid Amplification Test for C. difficile toxin B gene from liquid or soft tool specimen.

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F. PROPRIETARY AND ESTABLISHED NAMES (TRADE NAMES)

Assay: ICEPlex C. difficile Assay

System: ICEPlex® System

G. REGULATORY INFORMATION

a. Regulatory Section:

21 CFR 866.3130 - C. difficile Nucleic Acid Amplification Test Assay

b. Classification:

Class II

c. Product Code

OZN, NSU

d. Panel

Microbiology (83)

H. INTENDED USE

a. Intended Use

b. Indication(s) for use

Same as Intended Use

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c. Special Conditions for use statement(s)

For prescription use only

d. Special Instrument Requirement

ICEPlex® System

I. DEVICE DESCRIPTION

# of vials and volume
2 vials, 1.5ml/vial	2x PCR BufferP/N: 560-1001
1 vial, 45µl/vial	PCR EnzymeP/N: 560-1002
1 vial, 220µl/vial	25x Primer MixP/N: 560-1003
1 vial, 220 µl/vial	25x Calibrators MixP/N: 560-1004
1 vial, 100µl/vial	Positive ControlP/N: 560-1005
3 vials, 1.5 ml/vial	10x Injection BufferP/N: 560-1006
1 vial, 1.0 ml/vial	Internal ControlP/N: 560-1007

ICEPlex C. difficile Assay kit components

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ICEPlex System Features

. Bench top instrument with PCR thermal cycler and integrated Capillary Electrophoresis (CE) system
Two solid state lasers enabling fluorescent detection of dye-labeled PCR . amplification products
On-board reagents with liquid level sensing .
. Touch screen software for easy, step-by-step processing of samples
. Ability to run multiple assays in the same plate
. Automated data analysis and result reporting
Simultaneous detection and quantification of multiple DNA targets in the . same reaction well.

J. SUBSTANTIAL EQUIVALENCE COMPARISON

a. PREDICATE DEVICE NAME BD Max C. diff Assay

b. PREDICATE DEVICE 510(K) NUMBER K130470

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	PrimeraDx	Predicate:
		Similarities
IntendedUse	The PrimeraDx ICEPlex® C.difficile Assay is for thequalitative detection of theClostridium difficile toxin Bgene ( tcdB gene) in nucleicacids purified from unpreservedliquid or soft human stoolspecimens from patientssuspected of having C. difficileinfection (CDI). The ICEPlexC. difficile Assay is intended tobe used only on the ICEPlex®System, which integrates PCR-based amplification withcapillary electrophoresis (CE)for the detection ofamplification products. Theassay is intended to aid in thediagnosis of CDI. Resultsshould be considered inconjunction with patient clinicalhistory.	The BD MAX C. diff Assayperformed on the BD MAXSystem is an automated in vitrodiagnostic test for the direct,qualitative detection of theClostridium difficile toxin Bgene ( tcdB ) in human liquid orsoft stool specimens frompatients suspected of having C.difficile infection (CDI).The test, performed directly onthe specimen, utilizes real-timepolymerase chain reaction(PCR) for the amplification ofC. difficile toxin B gene DNAand fluorogenic target-specifichybridization probes for thedetection of the amplified DNA.The BD MAX C. diff Assay isintended to aid in the diagnosisof CDI. (Similar)
Measurand	Clostridium difficiletoxin B gene ( tcdB )	Same
SpecimenType	Unformed (liquid orsoft stool)	Same
Principle	DNA: real time PCR	Same
	PrimeraDx (ICEPlex)	Predicate:
	Differences
InstrumentSystem	ICEPlex System	BD Max System
Detection/Probes	Assays use real time PCRamplification with CapillaryElectrophoresis and directlaser-induced fluorescentdetection of target-specificlabeled primers in opticalchannels.	Assay use PCR andamplified DNA targets are detectedusing hydrolysis (TaqMan) probes.Probes labeled flourophores areusedto detect tcdB. Emitted fluorescentismeasured in optical channels.
SampleExtraction	bioMérieux NucliSENSeasyMAG	Integrated
TestCartridge	Disposable single-usePCR plate and micro titerplate, and ICEPlex Cartridgewith 48Capillaries.	Disposable tray with reagents,microfluidics PCR chamber
Process	Extraction, PCRamplification, capillaryelectrophoresis, size- basedtarget detection	Extraction, PCRamplification, in- sampleoptical detection based onhydrolysis probes.
Time toresult	Four hours for 48samples	Under 3 hours for 24 samples
AssayControls	Calibration Control,Internal control. PCR Positivecontrol provided. Negativecontrol and Positive/ExtractionControl provided by user	Sample processing control. User-provided Positive and negativecontrols are recommended
	Negative	Negative
Results	PositiveInvalid, with error code	PositiveUnresolved - specimenfailureIndeterminate - systemfailure with error codeIncomplete Run - witherror code

Comparison with Predicate Devices Showing Similarities

. .

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Comparison with Predicate Devices Showing Difference

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K. TEST PRINCIPLE

The ICEPlex C. difficile Assay on the ICEPlex System is a molecular diagnostic test for the qualitative detection of toxigenic C. difficile nucleic acids isolated and purified from liquid or soft stool specimens obtained from symptomatic patients. This test targets the C. difficile toxin B encoding gene (tcdB). The ICEPlex C. difficile Assay has been developed for use on the PrimeraDx ICEPlex System.

This instrument platform integrates PCR-based amplification with capillary electrophoresis (CE) based detection of amplification products. Oligonucleotide primers are designed to produce PCR products with unique CE mobility, enabling simultaneous measurement of multiple targets and controls in a single reaction. Each individual test includes three target sites of the tcdB gene (not reported separately), an Internal Control) and Calibrators. Calibrators of three sizes are used for aligning and assigning CE peaks, a procedure unique to the ICEPlex System. The ICEPlex C. difficile Assay is PCR based and is performed on nucleic acids purified from human stool sample. The ICEPlex C. difficile Assay is intended to be used only on the ICEPlex System.

To perform the test, liquid or soft stool is collected in a standard sterile container that can be sealed. Sample could be stored at 2-8 C for up to 48 hours prior to testing or frozen at -70°C or below if not processed within 48 hours. Sample extraction is performed using bioMérieux NucliSENS easyMAG™ System as per the ICEPlex C. difficile Assay instruction for use. PCR reagent master mix is prepared using a Primers mix, PCR enzyme, PCR buffer and calibrators. In a designated well of PCR plate, 40 uL of ICEPlex C. difficile Master Mix is aliquoted and 10 uL of extracted sample is added. Negative and positive controls are added in designated wells of the PCR plate. After the PCR plate is prepared, it is placed in the ICEPlex System to execute an instrument run for the detection of the Clostridium difficile toxin B gene (tcdB gene)per the user manual.

Assay Controls:

Controls provided with the ICEPlex C. difficile assay kit include:

Internal Control - Non-target nucleic acid that is co-extracted and coamplified with the tcdB target. It controls for nucleic acid extraction efficiency, for the integrity of the reagents and for the presence of PCR

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inhibitors in a given sample. The Internal Control needs to be spiked into each sample before extraction.

Calibration Control - A group of ICEPlex specific elements used to align electropherograms and assign identities of the target peaks. It also controls for the integrity of the kit reagents .

Positive Control - Non-infectious and non-contagious double stranded DNA containing fragment of the C. difficile tcdB gene.

Controls not provided with the ICEPlex C. difficile assay kit but required include:

Negative Control - Substitute S.T.A.R. buffer for the clinical specimen and process normally through the extraction system and on the ICEPlex Instrument.

Known Positive Sample - It is also required to include previously characterized positive sample or simulated sample with every easyMAG extraction run and include it in the subsequent ICEPlex Instrument run to verify successful lysis.

L. PERFORMANCE CHARACTERISTICS

a. ANALYTICAL PERFORMANCE

Precision

. . . . .

The ICEPlex C. difficile Assay Precision Study was performed on a panel of samples prepared by spiking an appropriate amount of C. difficile culture isolate from ATCC strain BAA-1805 into a pooled clinical negative stool matrix. The panel members included:

. Low positive
Moderately positive .
Negative sample .
(C20-80) sample. .

Runs for this study also include the following controls:

Negative Control .
Positive Control .

The precision study consisted of two operators; three lots of ICEPlex C. difficile Assay kits; and three ICEPlex instruments. The precision study was

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run over 12 non-consecutive days with two runs per day and two replicates of each sample per run. One negative sample (out of a total of 144 observations) and one low positive sample (out of a total of 144 observations) produced invalid results during the study.

Concentration	Overall Agreement	Ct Value
		Mean Ct	SD	%CV
Negative	143/143	100%	N/A	N/A	N/A
Low Positive	142/143	99%	30.6	1.4	4.5
Moderate Positive	144/144	100%	28.7	0.8	2.7
Hi/Low (C20-80)	105/144	73%	34.6	2.1	6.2

Precision Study Overall Results

Reproducibility

Reproducibility of the ICEPlex C. difficile Assay was evaluated at 3 independent laboratory sites. The reproducibility study panel included 4 simulated samples - moderate positive (expected positive 100% of the time), low positive (near assay limit of detection, expected positive >95% of the time), negative (expected negative 100% of the time) and C20-80, ( expected positive 20-80% of the time). The panel also included positive and negative controls. Panel samples were tested at each independent laboratory site for 5 days with 2 runs per day and 3 replicates of each panel member per run. One low positive sample (out of a total of 90 observations) and one moderate positive sample (out of a total of 90 observations) produced invalid results during the study Study results are summarized in the tables below:

Reproducibility Study Result - Overall Agreement

	Observed	Total	% Agreement	95% CI
C20-80*	62/90	90	69	58.26 to 78.23
Moderate Positive	89/89	89	100	95.98 to 100
Low Positive	89/89	89	100	95.98 to 100
Negative	90/90	90	100	95.98 to 100
Negative control	60/60	60	100	94.04 to 100
Positive control	60/60	60	100	94.04 to 100

NOTE: *For C20-80 samples % agreement is given as % positive results

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	Site A			Site B			Site C
	Observed	Total	% Agreement	Observed	Total	% Agreement	Observed	Total	% Agreement
C20-80*	23	30	77	23	30	77	16	30	53
ModeratePositive	30	30	100	30	30	100	29	29	100
LowPositive	30	30	100	29	29	100	30	30	100
Negative	30	30	100	30	30	100	30	30	100
NegativeControl	20	20	100	20	20	100	20	20	100
PositiveControl	20	20	100	20	20	100	20	20	100

Reproducibility Study, Site to Site % Agreement

Analytical Sensitivity (Limit of Detection)

Analytical sensitivity of the ICEPlex C. difficile Assay was determined using a 2-fold serial dilution of two C. difficile strains that were spiked into qualified negative stool and processed according to ICEPlex C. difficile Assay Instructions for Use. 20 replicates at each concentration level were tested on 3 different ICEPlex instruments. Analytical sensitivity of the assay was defined as the lowest concentration at which at least 95% of all replicates were reported positive.

LOD of the ICEPlex C. difficile assay determined for:

. C. difficile strain 43255 (630) (Toxinotype 0): 8CFU/rxn
. C. difficile strain BAA-1805 (Toxinotype III): 2CFU/rxn

Analytical Reactivity (Inclusivity)

To assess the analytical inclusivity of the ICEPlex C. difficile Assay, a set of 20 additional toxigenic strains of C. difficile were spiked into negative matrix (pool of negative clinical samples) at a level approximately three times above the assay LOD. Spiked samples were processed in accordance with the ICEPlex C. difficile Assay Instructions for Use. The results of the study are as follows:

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Strain	Toxinotype	Result
BAA-1382 (630)	A+B+	Positive
BAA-1871 (4111)	0, A+B+binary-, NAP5	Positive
9689 (90556-M6S)	0	Positive
700792 (14797-2)	A+B+	Positive
BAA-1875 (5325)	V, A+B+, NAP7	Positive
51695 (BDMS 18AN)	A+B+	Positive
43598 (1470)	VIII, A-B+	Positive
43600 (2149)	A+B+	Positive
43599(2022)	A+B+	Positive
43597	A+B+	Positive
43594 (W1194)	A+B+	Positive
43596 (545)	I, A+B+	Positive
17858 (1253)	A+B+	Positive
17857 (870)	A+B+	Positive
BAA-1808	A+B+	Positive
BAA-1806	A+B+	Positive
BAA-1803	III A+B+, NAP1	Positive
BAA-1870 (4118)	III, binary+, NAP1	Positive
BAA-1873 (5283)	0, A+,B+,binary-	Positive

: ・・・

۔ ﺑ

. --

Note: Strain BAA-1814 (Toxinotype XXII) was determined to be nonviable. PrimeraDx cannot claim inclusivity to this strain

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Analytical Snecificity

Cross Reactivity

A microbial cross reactivity study was performed with the ICEPlex C. difficile Assay using a panel of samples, consisting of pooled negative clinical matrix. spiked with 5 non-toxigenic C. difficile strains, 14 other Clostridium strains or 54 other pathogens or representatives of healthy intestinal flora. Bacteria and parasites were tested at concentrations 1x10' organisms/ml, viruses at concentrations1x10'-- 1x10° TCID50/ml. In this study, Clostridium sordellii was identified as cross-reacting, due to high toxin sequence similarity. Clostridium sordellii is not typically found in GI tract. Reactivity with this organism has little to no clinical significance. One of three replicates for Clostridium difficile strain 43601 was reported positive at 14 cps/rxn just above the assay cutoff set at 12 cps/rxn. PrimeraDx was not able to verify interference to this strain in follow-up analysis. None of other species resulted in positive result with ICEPlex C. difficile Assay.

Interfering Substances

A chemical interference study was performed using a panel of samples, consisting of pooled negative clinical matrix and contrived samples produced by supplementing pooled negative clinical matrix with culture stock of two C. difficile strains, ATCC BAA-1805 and 43255, added to produce samples resulting in 9 and 21 cfu/rxn, respectively (approximately three times the assay LOD). The table below shows potential interfering substances and concentrations used in this study.

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Substance్రామెన్ నేని వేసా ప్రసి	Active Ingredient	Concentration
Anti-Fungal /Anti-Itch Vaginal	Nystatin	1% (w/v)
Creams/Ointments/Suppositories	Hydrocortisone	1% (w/v)
Anti-Hemorrhoid Creams/Ointments	Phenylephrine	1% (w/v)
Antacids	Calcium Carbonate/AluminumHydroxide/MagnesiumHydroxide	10% (w/v)
Enemas	Mesalazine/MineralOil	10% (w/v)
Condoms with Spermicidal Lubricant	Nonoxynol-9	1% (w/v)
Anti-Diarrheal Medication	LoperamideHydrochloride/BismuthSubsalicylate	10% (w/v)
Laxatives	Sennosides	1% (w/v)
Antibiotic	Metronidazole	12.5mg/ml
Antibiotic	Vancomycin	12.5mg/ml
Non-Steroidal Anti-InflammatoryMedications	Naproxen Sodium	12.5mg/ml
Moist Towelettes	BenzalkoniumChloride, Ethanol	().1% (v/v),1%(v/v)
Fecal Fat	Lipids, etc.	40% w/v
Whole Blood	Glucose, Hormones,Enzymes, Ions, Iron,etc.	40% v/v
Mucus	Mucin protein	3.5% (w/v)

None of the substances interfered with detection of C. difficile from both tested strains or caused a false positive result in the negative samples.

Contamination and Well-to-well Cross Run-to-run Carryover Contamination

Samples for Well-to-well Cross Contamination and Run-to-run Carryover Contamination study were prepared by the extraction of a series of high positive samples (with analyte concentration exceeding the concentration found in 95% positive samples in the intended use population) alternating with negative samples. Location of positive and negative samples on the extraction instrument was altered

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run-to-run. On the ICEPlex system, high positive and negative samples were run in a checkerboard fashion. In the consecutive run, the platemap was inverted to allow wells and capillaries that were running negative samples to run positive samples. The study included 6 runs: each run had 24 high positive and 24 negative samples. The study identified no contamination in the negative samples run in the course of the study, reporting False Positive rate at 0% (0 False Positives out of 141 valid negatives). These data allowed PrimeraDx to conclude that there was no well-towell Cross Contamination or run-to-run Carryover Contamination observed in the course of this study.

b. CLINICAL PERFORMANCE STUDIES

A clinical study was conducted at three independent sites to compare performance of the ICEPlex C. difficile Assay Kit on the ICEPlex System to toxigenic C. difficile direct culture.

This study protocol directed the laboratory testing of specimens from patients suspected of gastrointestinal tract infection with a toxigenic strain of Clostridium difficile bacteria. Each of the sites performed testing in the ICEPlex C. difficile Assay on specimens collected at the site. These specimens collected from all three sites were tested by toxigenic C. difficile direct culture. A total of 1103 (806 fresh, 297 frozen) samples were collected and enrolled in the investigational study at 3 clinical sites. 97 frozen samples were excluded from the analysis due to improper storage conditions and temperature excursions. An additional 37 samples were excluded due to study protocol deviations.

969 specimens were compliant and met all protocol requirements. 952 (98.2%) out of 969 gave reportable results and were included in the statistical analysis. 17 samples remained invalid upon retest (1.8% of all analyzed samples). 3 out of the 17 unresolved invalids were reported positive by direct culture.

Performance characteristics determined in the course of this study were as follows. The Clopper-Pearson exact method was used to calculate confidence intervals.

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Overall Results

		Direct Culture
		Positive	Negative
ICEPlex C.difficile Assay	Positive	153	20a
	Negative	17b	762
	Total:	170	782

Positive Percent Agreement (95% CI): 90.0% ( 84.5 - 94.1) Negative Percent Agreement (95% CI): 97.4% ( 96.1 - 98.4)

Notes:

Discordant testing was performed for samples where ICEPlex C. difficile Assay and toxigenic C. difficile direct culture reported results in disagreement.

Discordant analysis included microbiological isolation of and PCR targeting of 3 appropriate regions of the toxin B gene (Different recognition sites than the ones used in the ICEPlex C. difficile assay) with bi-directional DNA sequencing.

46 of 20 reported positive by ICEPIex C. difficile Assay were reported positive by discordant analysis.

b14 of 17 reported negative by ICEPlex C. difficile Assay samples were reported positive by discordant analysis.

	SiteA	SiteB	Site C
PPA(95% CI)	49/55 = 89.1%(77.8 - 95.9)	56/64 = 87.5%(76.8 - 94.4)	48/51 = 94.1%(83.8 - 98.8)
NPA(95% CI)	281/288 = 97.6%(95.1 - 99.0)	256/262 = 97.7%(95.1 - 99.2)	225/232 = 97.0%(93.9 - 98.8)

Results by Site

M. CONCLUSION

PrimeraDx believes that based on the clinical and analytical performance comparison data, ICEPlex C. difficile Assay on the ICEPlex System is substantially equivalent to predicate devices.

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Image /page/15/Picture/0 description: The image shows a logo with a stylized bird-like figure composed of three curved lines, suggesting movement or flight. The logo is encircled by text, which appears to be part of the organization's name or a related phrase. The text is arranged in a circular fashion around the bird symbol, indicating a formal or official emblem.

DEPARTMENT OF HEALTH & HUMAN SERVICES

Public Health Service

Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring. MI) 20995-0002

PRIMERADX, INC. FAYYAZ MEMON VP. REGULATORY AFFAIRS AND QUALITY ASSURANCE 171 FORBES BLVD SUITE 1000 MANSFIELD MA 02048

November 29, 2013

Re: K132726

Trade/Device Name: ICEPlex C. difficile Assav Kit. ICEPlex System Regulation Number: 21 CFR § 866.3130 Regulation Name: Clostridium difficile toxin gene amplification assay Regulatory Class: II Product Code: OZN, NSU Dated: September 6, 2013 Received: September 9. 2013

Dear Mr. Memon:

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. Iisting of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warrantics. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA). it may be rise is easilitional 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 I Teast be advised mar 1971 3 lessantes over device complies with other requirements of the Act many Federal statutes and regulations administered by other Federal agencies. You must or and with all the Act's requirements. including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803): good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CHR Part 820); and if applicable. the electronic product radiation control provisions (Sections 531-542 of the Act): 21 CFR 1000-1050.

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Page 2—Mr. Memon

If you desire specific advice for your device on our labeling regulations (2) CFR Parts 801 and 809). please contact 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://yww.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. Also, please note the regulation entitled. "Misbranding by reference to premarket notification" (21CFR 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 CDRF' s Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on vour 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/MedicalDevices/Resourcesfor You/Industry/default.htm.

Sincerely yours.

Uwe Scherf -5 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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Indications for Use

510(k) Number: K132726

Device Name: ICEPlex®C. difficile Assay on the ICEPlex® System

Indications For Use:

Prescription Use (Part 21 CFR 801 Subpart D) AND/OR

Over-The-Counter Use (21 CFR 807 Subpart C)

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

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

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Regulation Number and Section

§ 866.3130 Clostridium difficile toxin gene amplification assay.

(a)
Identification. AClostridium difficile toxin gene amplification assay is a device that consists of reagents for the amplification and detection of target sequences inClostridium difficile toxin genes in fecal specimens from patients suspected of havingClostridium difficile infection (CDI). The detection of clostridial toxin genes, in conjunction with other laboratory tests, aids in the clinical laboratory diagnosis of CDI caused byClostridium difficile. (b)
Classification. Class II (special controls). The special controls are set forth in FDA's guideline document entitled: “Class II Special Controls Guideline: Toxin Gene Amplification Assays for the Detection ofClostridium difficile; Guideline for Industry and Food and Drug Administration Staff.” See § 866.1(e) for information on obtaining this document.