HardyCHROM™ CRE is a selective and differential chromogenic agar medium intended for the qualitative and presumptive detection from stool specimens of Escherichia coli that are non- susceptible to carbapenems as pink colonies and KES (Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae complex, and Serratia marcescens) that are non-susceptible to carbapenems as blue colonies.

HardyCHROM CRE is intended as an aid in the detection, identification of colonization and control of these bacteria in a healthcare setting. HardyCHROM™ CRE is not intended to diagnose infection or guide therapy. Results can be interpreted after incubation for 18-24 hours. Subculture to non-selective medium is required for confirming identification, antimicrobial susceptibility testing and epidemiological typing.

A lack of growth or the absence of pink or blue colonies on HardyCHROM™ CRE does not preclude the presence of Escherichia coli and KES that are non-susceptible to carbapenems.

Device Description

HardyCHROM™ CRE is a selective and differential chromogenic medium designed to screen for carbapenem non-susceptible Escherichia coli and KES (Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae complex, and Serratia marcescens.) from fecal specimens. The selective components in HardyCHROM™ CRE inhibit the growth of yeast, Gram-positive bacteria, and Gram-negative bacteria sensitive to ertapenem. HardyCHROM™ CRE differentiates Escherichia coli (pink colonies) from KES (blue colonies).

The CLSI guidelines recommend screening for CREs with an MIC greater than or equal to an established limit to one of several selected agents. Phenotypic confirmation of non-susceptibility to carbapenems is done by demonstrating non-susceptibility to one of the selected agents using MIC or disk diffusion. Further biochemical analysis is required to confirm the production of carbapenemase or any other mechanism of resistance.

AI/ML Overview

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

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state "acceptance criteria" in discrete numerical targets. Instead, it presents performance data (Sensitivity, Specificity, PPV, NPV) and concludes with a statement that the data "support the safety of the device verification and validation" and "demonstrate that the HardyCHROM™ CRE device is substantially equivalent to the predicate device." Therefore, the reported performance metrics serve as the de facto demonstration of meeting the required standards for substantial equivalence.

Performance of HardyCHROM™ CRE for detection of Carbapenem non-susceptible E. coli (Pink Colonies)

Metric	Overall Performance (18 hours)	Overall Performance (24 hours)
Sensitivity	100% (67.6-100%)	100% (67.6-100%)
Specificity	99.3% (98.8-99.6%)	99.3% (98.8-99.6%)
PPV	42.1% (23.1-63.7%)	42.1% (23.1-63.7%)
NPV	100% (99.7-100%)	100% (99.8-100%)

Performance of HardyCHROM™ CRE for detection of Carbapenem non-susceptible KES (Blue Colonies)

Metric	Overall Performance (18 hours)	Overall Performance (24 hours)
Sensitivity	95.8% (86.0-98.9%)	95.8% (86.0-98.9%)
Specificity	98.2% (97.4-98.7%)	97.9% (97.0-98.5%)
PPV	61.3% (50.0-71.5%)	57.5% (46.6-67.7%)
NPV	99.9% (99.5%-100%)	99.9% (99.5%-100%)

Agreement of Carbapenem NS Target Species with color on HardyCHROM CRE (at 24 hours)

Metric	Performance
Positive Percent Agreement	100% (95.6-100%)
Negative Percent Agreement	99.4% (98.9-99.7%)

Performance on Contrived Specimens (E. coli Pink Morphology)

Metric	Performance (Raw Stool)	Performance (Cary Blair)
Positive Percent Agreement	100% (90.8%-100%)	100% (90.8%-100%)
Negative Percent Agreement	100% (97.7%-100%)	100% (97.7%-100%)

Performance on Contrived Specimens (KES Blue Morphology)

Metric	Performance (Raw Stool)	Performance (Cary Blair)
Positive Percent Agreement	95.7% (90.2%-98.1%)	95.7% (90.1%-98.1%)
Negative Percent Agreement	100% (95.8%-100%)	100% (95.8%-100%)

Analytical Sensitivity (LoD) and Reactivity:

LoD: 1.5x10^2 CFU/mL in stool specimen (for all 10 target strains evaluated)
Analytical Reactivity: Recovered 72 of 77 (93.5%) carbapenem non-susceptible strains at 1.5x10^2 CFU/mL.

Analytical Specificity:

Majority of organisms tested did not produce target morphology or were inhibited.
Certain non-target organisms developed blue pigmentation after 24-48 hours or had distinct morphology, allowing differentiation. None developed pink color.

Microbial Interference:

HardyCHROM™ CRE was able to recover all target organisms from mixed suspensions in the presence of high concentrations of non-target organisms.

Incubation Study:

All organisms recovered with expected color by 18 hours.

Stool Specimen Stability:

100% recovery from raw stool and stool in Cary Blair at room temperature for up to 24 hours.
100% recovery from raw stool and stool in Cary Blair at 2-8℃ for up to 7 days.

Reproducibility:

95% agreement with known test results. All CRE-positive isolates (100%) detected.

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

Test Set Sample Size (Clinical Study): A total of 1,628 samples were tested. 144 specimens were excluded, resulting in 1,484 valid samples for analysis.
Contrived Test Set Sample Size: A total of 203 contrived specimens were evaluated.
Data Provenance: The clinical study used freshly collected stool specimens from "three geographically diverse hospitals." This indicates a prospective study collected from clinical settings in the United States (implied by FDA submission).

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

The document does not explicitly state the number of experts or their specific qualifications (e.g., radiologist with 10 years of experience). However, it mentions:

"Identity and susceptibility of organisms that grew on both HardyCHROM™ CRE and MacConkey Agar were confirmed using FDA-cleared ID and AST systems."
For the reproducibility study: "The testing was done with at least one operator and two readers, blinded to each other's results, per site."
This implies that trained laboratory personnel and validated laboratory methods were used for ground truth determination, which is standard for in vitro diagnostic devices.

4. Adjudication method for the test set

Clinical Study: The "routine culture" method served as the reference method, followed by confirmation using "FDA-cleared ID and AST systems." This acts as the primary ground truth. There is no explicit mention of an adjudication process (e.g., 2+1, 3+1) involving multiple human readers reviewing results for initial discrepancies to establish a ground truth. Rather, the "reference method" and "FDA-cleared systems" for confirmation established the ground truth.
Reproducibility Study: "at least one operator and two readers, blinded to each other's results, per site" suggests a form of consensus or independent reading for the reproducibility of the device's visual interpretation, but not for establishing the ultimate ground truth of the organisms' identity and susceptibility, which was pre-determined.

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

Not applicable. This document describes the performance of a culture medium (HardyCHROM™ CRE), which is a device for bacterial detection and differentiation based on colony color. It is not an AI-assisted diagnostic tool. Therefore, an MRMC study comparing human readers with and without AI assistance was not conducted and is not relevant to this type of device.

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

Yes, in essence. The HardyCHROM™ CRE functions as a standalone diagnostic medium. Its performance metrics (Sensitivity, Specificity, PPV, NPV) were calculated based on the observable color reaction on the agar medium compared to the reference method (culture with confirmed ID and AST). While human observation is required to read the colony color, the "performance" as presented (e.g., "Pink (Positive)" vs. "Negative 1") reflects the intrinsic capability of the medium itself to produce the correct visual signal for Carbapenem non-susceptible E.coli or KES. The study directly evaluates the medium's performance in identifying target organisms based on their chromogenic reaction.

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

The ground truth for the clinical study was established by a reference culture method (selective enrichment in Tryptic Soy Broth with meropenem and vancomycin, followed by subculture to MacConkey Agar), with subsequent identification (ID) and antimicrobial susceptibility testing (AST) using FDA-cleared systems. This is considered a laboratory-based reference standard, which is highly objective for microbiological assays.

8. The sample size for the training set

The document does not explicitly mention a "training set" in the context of machine learning or AI, as the device is a chromogenic culture medium. The studies described are for performance evaluation, not for training an algorithm.

However, if "training set" is broadly interpreted as any data used to refine or develop the product before final validation, then:

The "Analytical Reactivity" study used 77 well-characterized carbapenem non-susceptible strains.
The "Analytical Specificity" study used 110 strains (carbapenem-susceptible target species and non-target species).
The "Microbial Interference" study used the organisms from the Analytical Specificity study plus target organisms.
The "Reproducibility" study used a panel of 10 blinded isolates (5 positive, 5 negative) at three sites.

These analytical studies and reproducibility tests likely serve as internal development and verification steps before the large-scale clinical validation.

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

As noted above, there isn't a "training set" in the common AI sense. For the analytical studies, the ground truth was established by:

Known strains and concentrations: For LoD and Analytical Reactivity, "well-characterized carbapenem non-susceptible strains" at known concentrations were used.
Known strains (susceptible/non-susceptible) and non-target species: For Analytical Specificity and Microbial Interference, the identity and susceptibility of the tested strains were pre-determined and "well-characterized."
Known test results: For the Reproducibility study, the panel of 10 isolates had "known test results," meaning their identity and carbapenem resistance status were previously confirmed by standard laboratory methods.

Summary

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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

April 29, 2019

Hardy Diagnostics Rianna Malherbe Performance Studies Coordinator 1430 West McCoy Lane Santa Maria, California 93455

Re: K190553

Trade/Device Name: HardyCHROM CRE Regulation Number: 21 CFR 866.1700 Regulation Name: Culture medium for antimicrobial susceptibility tests Regulatory Class: Class II Product Code: JSO Dated: March 4, 2019 Received: March 5, 2019

Dear Rianna Malherbe:

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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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. Please note: CDRH does not evaluate information related to contract liability warranties. 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 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

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requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/CombinationProducts/GuidanceRegulatoryInformation/ucm597488.htm); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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 comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely.

for

Uwe Scherf, 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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510(k) Summary

I. SUBMITTER

Rianna Malherbe Performance Studies Coordinator Hardy Diagnostics 1430 W. McCoy Lane Santa Maria, CA 93455 Phone: 805-346-2766 x5714 E-mail: MalherbeR @hardydiagnostics.com

II. DEVICE

Name of Device: HardyCHROM™ CRE Classification Name: Culture medium for anti-microbial susceptibility tests Regulatory Class: II Product Code: JSO

III. PREDICATE DEVICE Bio Rad VRESelect, K122187

IV. DEVICE DESCRIPTION

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V. INDICATIONS FOR USE / INTENDED USE

HardyCHROM™ CRE is a selective and differential chromogenic agar medium intended for the qualitative and presumptive detection from stool specimens of Escherichia coli that are non- susceptible to carbapenems as pink colonies and KES (Klebsiella aerogenes, Klebsiella pneumoniae, Enterobacter cloacae complex, and Serratia marcescens) that are non-susceptible to carbapenems as blue colonies.

A lack of growth or the absence of pink or blue colonies on HardyCHROM™ CRE does not preclude the presence of Escherichia coli and KES that are non-susceptible to carbapenems.

Attribute	Device	Comparator	SubstantiallyEquivalent?
Name	HardyCHROM™ CRE	Bio Rad VRESelect
510(k)	510(k) number	510(k) number	Yes
Details	N/A Product CodeISO21 CFR 866.1700"66.1700umberCS for anti-microbialsusceptibility testspClass IIPanel 83 Microbiology	K122187 ProductCode JSO21 CFR 866.1700"Culture medium for anti-microbial susceptibility tests"Class IIPanel 83 Microbiology
Attribute	Device	Comparator	Substantially
Name	HardyCHROM™ CRE	Bio Rad VRESelect	Equivalent?
Intended Use	HardyCHROM™ CRE is a selective anddifferential chromogenic agar mediumintended for the qualitative and presumptivedetection from stool specimens of Escherichiacoli that are non-susceptible to carbapenems aspink colonies and KES (Klebsiella aerogenes,Klebsiella oxytoca, Klebsiella pneumoniae,Enterobacter cloacae complex, and Serratiamarcescens.) that are non-susceptible tocarbapenems as blue colonies.HardyCHROM CRE is intended as an aid inthe detection, identification of colonization andcontrol of these bacteria in a healthcare setting.HardyCHROM™ CRE is not intended todiagnose infection or guide therapy. Resultscan be interpreted after incubation for 18-24hours. Subculture to non-selective medium isrequired for confirming identification,antimicrobial susceptibility testing andepidemiological typing.A lack of growth or the absence of pink or bluecolonies on HardyCHROM™ CRE does notpreclude the presence of Escherichia coli andKES that are non-susceptible to carbapenems.	VRESelect™ is a selective anddifferential chromogenic medium,containing 8 ug/mL of vancomycin,for the qualitative detection ofgastrointestinal colonization ofvancomycin resistant Enterococcusfaecium (VREfm) and vancomycinresistant Enterococcus faecalis(VREfs) and to aid in theprevention and control ofvancomycin resistant Enterococcus(VRE) in healthcare settings. Thetest is performed on rectal swabs orfecal specimens from patients to bescreened for VRE colonization.VRESelect™ is not intended todiagnose VRE infection nor toguide or monitor treatment ofinfection. Results can be interpretedafter 24 to 28 hours incubation.Subculture to non-selective media(e.g., trypticase soy agar with 5%sheep blood) is needed forsusceptibility testing andepidemiological typing.	Yes
Methodology	Enzymatic - Chromogenic	Enzymatic - Chromogenic	Yes
Inoculation	Direct	Direct	Yes
Sample Type	Fecal Specimen	Rectal Swab or Fecal Specimen	Yes
Interpretation	Manual, Visual	Manual, Visual	Yes
Subculture	Required for confirmation of identity andantimicrobial susceptibility testing	Required for confirmation of identityand antimicrobial susceptibility testing	Yes
OrganismsDetected	Carbapenem non-susceptible Escherichia coli,and KES (Klebsiella aerogenes, Klebsiellaoxytoca, Klebsiella pneumoniae, Enterobactercloacae complex, and Serratia marcescens)	Vancomycin resistant E. faecalis andE. faecium	Yes

VI. COMPARISON OF TECHNOLOGICAL CHARACTERISTICS WITH THE PREDICATE DEVICE

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VII. PERFORMANCE DATA

Performance of HardyCHROM™ CRE was evaluated at three geographically diverse hospitals. Freshly collected stool specimens were used in this study. The recovery of carbapenem nonsusceptible Escherichia coli and KES (K. aerogenes, K. oxytoca, K. pneumoniae, E. cloacae complex, and S. marcescens) on HardyCHROM™ CRE was compared to routine culture, defined as selective enrichment in Tryptic Soy Broth (TSB) containing 1 ug/mL meropenem and 3 ug/mL vancomycin, followed by a subculture to MacConkey Agar.

Identity and susceptibility of organisms that grew on both HardyCHROM™ CRE and MacConkev Agar were confirmed using FDA-cleared ID and AST systems. Quality control was performed in parallel every day of testing.

A total of 1,628 samples were tested against routine culture. A total of 144 specimens did not meet enrollment criteria, and were therefore excluded from the analysis. Of the remaining 1,484 valid samples tested, a total of 8 carbapenem non-susceptible Escherichia coli and 46 carbapenem non- susceptible KES (K. aerogenes, K. oxytoca, K. pneumoniae, E. cloacae complex, and S. marcescens) isolates were recovered by expected morphology on HardyCHROM™ CRE with concordant results obtained by the reference method and confirmed by ID and AST.

Product performance is summarized below:

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reference method (stratified by clinical site)
		Reference Method: Non-Susceptible E. coli
	Site 1	Positive	Negative	Total
HardyCHROM	Pink (Positive)	0	5	5
CRE	Negative 1	0	523	523
	Total	0	528	528
	Sensitivity	Not applicable
	Specificity	523/528 = 99.1% (97.8-99.6%)
	Site 2	Positive	Negative	Total
HardyCHROM	Pink (Positive)	2	2	4
CRE	Negative 1	0	575	575
	Total	2	577	579
	Sensitivity	2/2 = 100% (34.2%-100%)
	Specificity	575/577 = 99.7% (98.7-99.9%)
	Site 3	Positive	Negative	Total
HardyCHROM	Pink (Positive)	6	4	10
CRE	Negative 1	0	507	507
	Total	6	511	517
	Sensitivity	6/6 = 100% (61.0-100%)
	Specificity	507/511 = 99.2% (98.0-99.7%)
	Overall	Positive	Negative	Total
HardyCHROM	Pink (Positive)	8	112	19
CRE	Negative 1	0	1605	1605
	Total	8	1616	1624
	Sensitivity	8/8 = 100% (67.6-100%)
	Specificity	1605/1616 = 99.3% (98.8-99.6%)
	PPV	8/19 = 42.1% (23.1-63.7%)
	NPV	1605/1605 = 100% (99.7-100%)

Performance of HardyCHROM CRE Pink Morphology at 18 hours in comparison to the reference method (stratified by clinical site)

PPV: Positive Predictive Value; NPV: Negative Predictive Value

1 Negative: No growth, or growth of any color other than pink (Blue, yellow, others)

2 There were 11 FP isolates observed at 18 hours: 5/11 were confirmed to be carbapenem non-susceptible E. coli, but were not recovered from the reference method, 5/11 produced pink colonies but were not the target organism for that color (2 Enterobacter cloacae complex, 2 Citrobacter freundii and 1 Citrobacter youngae), 1/11 Citrobacter freundii was confirmed to be susceptible but grew pink.

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reference method (stratified by clinical site)
Site 1		Reference Method: Non-Susceptible E. coli
		Positive	Negative	Total
	Pink (Positive)	0	5	5
HardyCHROMCRE	Negative 1	0	530	530
	Total	0	535	535
	Sensitivity	Not applicable
	Specificity	530/535 = 99.1% (97.8-99.6%)
Site 2		Reference Method
		Positive	Negative	Total
	Pink (Positive)	2	2	4
HardyCHROMCRE	Negative 1	0	582	582
	Total	2	584	586
	Sensitivity	2/2 = 100% (34.2%-100%)
	Specificity	582/584 = 99.7% (98.7-99.9%)
Site 3		Reference Method
		Positive	Negative	Total
	Pink (Positive)	6	4	10
HardyCHROMCRE	Negative 1	0	507	507
	Total	6	511	517
	Sensitivity	6/6 = 100% (61.0-100%)
	Specificity	507/511 = 99.2% (98.0-99.7%)
Overall		Reference Method
		Positive	Negative	Total
	Pink (Positive)	8	112	19
HardyCHROMCRE	Negative 1	0	1619	1605
	Total	8	1630	1638
	Sensitivity	8/8 = 100% (67.6-100%)
	Specificity	1619/1630 = 99.3% (98.8-99.6%)
	PPV	8/19 = 42.1% (23.1-63.7%)
	NPV	1619/1619 = 100% (99.8-100%)

Performance of HardyCHROM CRE Pink Morphology at 24 hours in comparison to the reference method (stratified by clinical site)

PPV: Positive Predictive Value; NPV: Negative Predictive Value

1 Negative: No growth, or growth of any color other than pink (Blue, yellow, others)

2 There were 11 FP isolates observed at 24 hours: 5/11 were confirmed to be carbapenem non-susceptible E. coli, but were not recovered from the reference method, 5/11 produced pink colonies but were not the target organism for that color (2 Enterobacter cloacae complex, 2 Citrobacter freundii and 1 Citrobacter youngae), 1/11 Citrobacter freundii was confirmed to be susceptible but grew pink.

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Reference Method, (stratified by clinical site)
Site 1		Reference Method: Non-Susceptible KES2
		Positive	Negative	Total
HardyCHROMCRE	Blue (Positive)	7	10	17
	Negative 1	1	510	511
	Total	8	520	528
		Sensitivity $7/8= 87.5% (52.9%-97.8%)$
		Specificity $510/520 = 98.1% (96.5-99.0%)$
Site 2		Reference Method
		Positive	Negative	Total
HardyCHROMCRE	Blue (Positive)	5	8	13
	Negative 1	1	565	566
	Total	6	573	579
		Sensitivity $5/6 = 83.3% (43.7%-97%)$
		Specificity $565/573 = 98.6% (97.3%-99.3%)$
Site 3		Reference Method
		Positive	Negative	Total
HardyCHROMCRE	Blue (Positive)	34	11	45
	Negative 1	0	472	472
	Total	34	483	517
		Sensitivity $34/34 = 100% (89.9-100%)$
		Specificity $472/483 = 97.7% (96.0-98.7%)$
Overall		Reference Method
		Positive	Negative	Total
HardyCHROMCRE	Blue (Positive)	46	293	75
	Negative 1	24	1547	1549
	Total	48	1576	1624
	Sensitivity	$46/48 = 95.8% (86.0-98.9%)$
	Specificity	$1547/1576 = 98.2% (97.4-98.7%)$
PPV		$46/75 = 61.3% (50.0-71.5%)$
NPV		$1547/1576 = 99.9% (99.5%-100%)$

Performance of HardyCHROM CRE Blue Morphology at 18 hours in Comparison to the Reference Method (stratified by clinical site)

PPV: Positive Predictive value; NPV: Negative Predictive Value

1 Negative: No growth, or growth of any color other than blue

2 Detection of carbapenem non-susceptible Klebsiella pneumoniae, Klebsiella aerogenes, Enterobacter cloacae complex, or Serratia marcescens.

3 There were 29 False Positive observed at 18 hours: 23/29 were confirmed as carbapenem non-susceptible KES (11 Klebsiella pneumoniae, 12 Enterobacter cloacae complex, 2 Serratia marcescens, 1 Klebsiella oxytoca) that were recovered as blue isolated colonies on HardyCHROM CRE but were not recovered by the reference method; 1/29 was a carbapenem non-susceptible S. liquefaciens; 2/29 isolates were non-target Gram positive organisms.

4 There were 2 False Negative observed at 18 hours: 2/2 carbapenem non-susceptible Enterobacter cloacae

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Reference Method, (stratified by clinical site)
		Reference Method: Non-Susceptible KES2
	Site 1	Positive	Negative	Total
	Blue (Positive)	7	12	19
HardyCHROM	Negative1	1	515	516
CRE	Total	8	527	535
	Sensitivity	$7/8 = 87.5% (52.9-97.8%)$
	Specificity	$515/527 = 97.7% (96.1-98.7%)$
		Reference Method
	Site 2	Positive	Negative	Total
	Blue (Positive)	5	10	15
HardyCHROM	Negative1	1	570	571
CRE	Total	6	580	586
	Sensitivity	$5/6 = 83.3% (43.7-97%)$
	Specificity	$570/580 = 98.3% (96.9-99.1%)$
		Reference Method
	Site 3	Positive	Negative	Total
	Blue (Positive)	34	12	46
HardyCHROM	Negative1	0	471	471
CRE	Total	34	483	517
	Sensitivity	$34/34 = 100% (89.9-100%)$
	Specificity	$471/483 = 97.5% (95.7-98.6%)$
		Reference Method
	Overall	Positive	Negative	Total
	Blue (Positive)	46	343	80
	Negative1	24	1556	1558
HardyCHROM	Total	48	1590	1638
CRE	Sensitivity	$46/48 = 95.8% (86.0-98.9%)$
	Specificity	$1556/1590 = 97.9% (97.0-98.5%)$
	PPV	$46/80 = 57.5% (46.6-67.7%)$
	NPV	$1556/1558 = 99.9% (99.5%-100%)$

Performance of HardyCHROM CRE Blue Morphology at 24 hours in Comparison to the Reference Method (stratified by clinical site)

PPV: Positive Predictive value; NPV: Negative Predictive Value

1 Negative: No growth, or growth of any color other than blue

2 Detection of carbapenem non-susceptible Klebsiella pneumoniae, Klebsiella aerogenes, Enterobacter cloacae complex, or Serratia marcescens.

3 There were 34 False Positive observed at 24 hours: 23/34 were confirmed as carbapenem non-susceptible KES (11 Klebsiella pneumoniae, 12 Enterobacter cloacae complex, 2 Serratia marcescens, 1 Klebsiella oxytoca) that were recovered as blue isolated colonies on HardyCHROM CRE but were not recovered by the reference method; 1/29 was a carbapenem non-susceptible S. liquefaciens; 2/29 isolates were non-target Gram positive organisms. 7/34 isolates were non-target Gram positive organisms.
4 There were 2 False Negative observed at 24 hours: 2/2 carbapenem non-susceptible Enterobacter cloacae

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The agreement between colony color observed on HardyCHROM CRE with prospectively collected clinical specimens at 24 hours and the identity and carbapenem susceptibility of the isolates recovered from the HardyCHROM CRE culture medium was also analyzed. Results of this data analysis are summarized below.

		Carbapenem Non-susceptible Target Species
		Positive	Negative	Total
HardyCHROMCRE	Positive 1	84 3	15 4	99
		Negative2	0	740
		Total	84	755
Positive Percent Agreement		100% (91/91); 95.6-100%
Negative Percent Agreement		99.4% (740/755); 98.9-99.7%

Agreement of Carbapenem NS Target Species with color on HardyCHROM CRE

1 Pink or Blue colonies

2 Colonies other than pink or blue

3 Includes the following species: K. pneumoniae (41), E. cloacae complex (23), E. coli (13), S. marcescens (3), K. aerogenes (2), K. oxytoca (2).

4 Includes Gram positive cocci (7), C. freundii (3), E. youngae (1), S. liquefaciens (1)

To supplement testing of prospectively collected clinical specimens, a total of 203 contrived specimens were also evaluated at Hardy Diagnostics. CRE-negative patient specimens were inoculated with known carbapenem non-susceptible organisms at 2x LoD and tested on HardyCHROM™ CRE. Results are summarized below.

Raw Stool		Expected Result: Non-Susceptible E. coli
		Positive	Negative	Total
HardyCHROMCRE Color	Pink	38	0	38
	Negative/Other	0	165	165
	Total	38	165	203
	Positive Percent Agreement¹	38/38 = 100% (90.8%-100%)
Negative Percent Agreement¹		165/165 = 100% (97.7%-100%)
Cary Blair		Expected Result: Non-Susceptible E. coli
		Positive	Negative	Total
HardyCHROMCRE Color	Pink	38	0	38
	Negative/Other	0	165	164
	Total	38	165	202
	Positive Percent Agreement¹	38/38 = 100% (90.8%-100%)
Negative Percent Agreement¹		165/165 = 100% (97.7%-100%)

E. coli Pink Morphology on HardyCHROM™ CRE at 18 or 24 hours vs Expected

1The Positive Percent Agreement and Negative Percent Agreement were the same for both 18 and 24 hours.

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Raw Stool		Expected Result: Non-Susceptible KES1
		Positive	Negative	Total
HardyCHROMCRE Color	Blue	110	0	110
	Negative/Other	53	88	93
	Total	115	88	203
Positive Percent Agreement2		110/115=95.7 %(90.2%-98.1%)
Negative Percent Agreement2		88/88=100% (95.8%-100%)
Cary Blair		Expected Result: Non-Susceptible KES2
		Positive	Negative	Total
HardyCHROMCRE Color	Blue	110	0	110
	Negative/Other	53	88	93
	Total	115	88	203
Positive Percent Agreement2		110/115=95.7 %(90.1%-98.1%)

KES Blue Morphology on HardyCHROM™ CRE at 18 and 24 hours vs Expected

1 KES = K. aerogenes, K. oxytoca, K. pneumoniae, E. cloacae complex, and S. marcescens

2The Positive Percent Agreement and Negative Percent Agreement were the same for both 18 and 24 hours.

3There were 10 False Negative samples for both Raw and Cary Blair samples at 18 and 24 hours. 2/10 (20%) of the organisms were Klebsiella variicola. While these organisms are carbapenem non-susceptible, it is not a claimed species. Upon retest, 4/10 (40%) of the organisms were confirmed to have sub populations more susceptible to carbapenems than expected and were not recovered at 2x LoD, as tested in the contrived study. 4/10 (40%) of the organisms were retested and the reason for failure is unknown.

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RECOVERY RATE

To determine the recovery (Limit of Detection (LoD)) of HardyCHROM™ CRE, the media was challenged with 10 strains of target carbapenem non-susceptible organisms. Two well-characterized strains each of E. coli, K. oxytoca, K. pneumoniae, E. cloacae complex and S. marcescens were evaluated for recovery on HardyCHROM™ CRE. Note: The LoD of K. (Enterobacter) aerogenes was not evaluated. Each organism was tested at 10-fold decreasing concentrations and evaluated for colony growth and color development. The lowest concentration at which a positive color reaction was seen, indicated by a pink (E. coli) or blue (KES) colony color, was determined to be the LoD. The determined LoD was confirmed by testing HardyCHROM™ CRE with five replicate dilutions of the determined LoD concentrations. HardyCHROM™ CRE was able to recover all strains tested at a LoD of 1.5x102 CFU/mL in stool specimen with 100 uL inoculum (spread-plate).

ANALYTICAL REACTIVITY

HardyCHROM™ CRE was evaluated for the recovery of seventy-seven well-characterized carbapenem non-susceptible strains at the limit of detection of 1.5x102 CFU/mL. The strains were tested using a clean suspension in the absence of stool matrix. HardyCHROM™ CRE was able to recover 72 of 77 (93.5%) of the carbapenem non-susceptible strains at 1.5x102 CFU/mL (100 uL inoculum) after 24 hours of incubation. Some strains were either more susceptible to the selective agent or may have struggled to maintain resistance once the organism was serially diluted and inoculated to the media. After 24 hours of incubation, three strains were recovered at 1.5x10 CFU/mL and two strains were recovered at 1.5x10d CFU/mL. All target strains tested were recovered with the expected color development.

Species	n	Mechanism	ETPMIC	IMPMIC	MEMMIC	LoD at 24 hours
E. coli	17	OXA-48, NDM (1,5,6)KPC (3, KPC-type)	1->32	4->32	2->32	16 at 102 CFU/mL1 at 103 CFU/mL
E. cloacae complex	15	OXA-48, MIR-8, KPC -4,ACT (6, ACT type),NDM-1, VIM (6, 23, 31),IMP(1, 8)	1->32	1->32	0.5->32	13 at 102 CFU/mL1 at 103 CFU/mL1 at 104 CFU/mL
K. oxytoca andK. pneumoniae	39	OXA (48, 163, 181, 232),KPC (2, 3, 12, KPC-type),NDM-1, IMP (1, 4, 8),VIM-27	0.5->32	2->32	0.5->32	37 at 102 CFU/mL1 at 103 CFU/mL1 at 104 CFU/mL
S. marcescens	5	IMP-8, NDM-1, VIM-4,SME (2, SME-type)	0.5->32	8->32	1->32	5 at 102 CFU/mL

Summary of Analytical Sensitivity testing

1 K. (Enterobacter) aerogenes was not evaluated in this study.

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ANALYTICAL SPECIFICTY

To determine the potential for cross-reactivity on HardyCHROM™ CRE, internal testing was conducted with carbapenem-susceptible target species as well as non-target species commonly found in stool samples. A total of 110 strains were tested on HardyCHROM™ CRE by streaking 10µL of a 1.5x10° CFU/mL suspension of each organism onto HardyCHROM™ CRE. After 24 hours of incubation, all HardyCHROM™ CRE plates were evaluated for growth and color reaction. The

majority of organisms tested either did not produce a target organism morphology or were inhibited on HardyCHROM™ CRE at 24 hours. One organism (Aspergillus brasiliensis) developed blue pigmentation at 24 hours. Three organisms (Pediococcus, E. faecalis (vanB), E. faecium (vanA)) developed blue pigmentation at 48 hours of incubation. Organisms that developed a blue color either took 48 hours of incubation (Pediococcus, Enterococcus with vancomycin resistance) to develop or had distinct morphology (Aspergillus) that allowed differentiation from the target species. None of the non-target organisms tested developed a pink color.

List of non-target organisms tested in Analytical Specificity
Organism	Organism	Organism
Acinetobacter baumannii	Enterococcus hirae	Proteus mirabilis
Aeromonas hydrophila	Enterococcus raffinosus	Proteus vulgaris
Aspergillus brasiliensis	Enterococcus saccharolyticus	Providencia alcalifaciens
Bacillus cereus	Escherichia coli	Providencia rettgeri
Campylobacter coli	Geotrichum candidum	Providencia stuartii
Campylobacter jejuni subsp jejuni	Hafnia alvei	Pseudomonas aeruginosa
Candia glabrata	Klebsiella oxytoca	Pseudomonas fluorescens
Candida albicans	Klebsiella pneumoniae	Saccharomyces cerevisiae
Candida guilliermondii	Klebsiella pneumoniae(ESBL)	Salmonella enterica
Candida tropicalis	Lactobacillus acidophilus	Serratia marcescens
Citrobacter braakii	Lactobacillus gasseri	Shigella boydii
Citrobacter freundii	Lactococcus lactis	Shigella flexneri
Citrobacter koseri	Listeria grayi	Shigella sonnei
Corynebacterium jeikeium	Listeria monocytogenes	Staphylococcus aureus
Corynebacterium pseudodiptherium	Micrococcus luteus	Staphylococcus epidermidis
Enterobacter aerogenes	Morganella morganii	Stenotrophomonas maltophilia
Enterobacter cloacae	Pediococcus acidilactici	Streptococcus agalactiae
Enterobacter cloacae (AmpC)	Penicillium aurantiogriseum	Streptococcus mitis
Enterococcus casseliflavus (vanC)	Penicillium chrysogenum	Streptococcus pyogenes
Enterococcus durans	Penicillium rubens	Vibrio cholera
Enterococcus faecalis (vanB)	Plesiomonas shigelloides	Yersinia enterocolitica
Enterococcus faecium (vanA)	Pantoea agglomerans	Weisella confusa

List of non-target organisms tested in Analytical Specificity

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MICROBIAL INTERFERENCE

HardyCHROM™ CRE was challenged to determine if target organisms at low concentration could be recovered in the presence of non-target organisms at a high concentration. All organisms that were recovered on HardyCHROM™ CRE in the Analytical Specificity study were used in this Microbial Interference study. Non-target organisms at a high concentration (1.5x10° CFU/mL) were mixed with each target organism at a concentration of 7.5x103 CFU/mL and inoculated to HardyCHROM™ CRE using a 10uL loop. If the target organism was not recovered, the concentration of the non-target organism was lowered 10-fold until the target organism was recovered.

HardyCHROM™ CRE was able to recover all target organisms from the mixed suspension when in the presence of high concentrations of all non-target organisms used in this study. For most of the target organisms, colony color was as expected. The following non-target organisms had an effect on colony size of all target strains tested except Serratia marcescens: Acinetobacter baumanii, Aeromonas hydrophila, Pseudomonas fluorescens, and Stenotrophomonas maltophilia. Pseudomonas aeruginosa had an effect on colony size of all target strains tested except Serratia marcescens and Enterobacter asburiae. One of the target strains, E. cloacae, was recovered as purple colonies when mixed with Stenotrophomonas maltophilia. Pigmentation of other target organisms tested was not affected by any of the other non-target organisms tested.

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INTERFERENCE

Commonly used or encountered endogenous and exogenous substances that may be present in stool specimen were evaluated for potential interference of growth or chromogenic reaction on HardyCHROM™ CRE. The substances tested are listed in the table below. No interference was observed with any substance at the highest clinically relevant concentration in the CRE-negative specimen matrix.

Category	Substance	Concentrationin SampleMatrix 1
Antifungal	Nystop (Nystatin)	5% w/v
Antifungal	Lotrimin (Clotrimazole)	5% v/v
Antifungal	Lotrimin Ultra (Butenafine Hydrochloride)	5% v/v
Antifungal	Lamisil (Terbinafine Hydrochloride 1%)	5% v/v
Antiseptic	Bactine (Benzalkonium Chloride)	1% v/v
Antiseptic	Ethanol	1% v/v
Biologic	Whole blood	5% v/v
Contraceptive	Nonoxynol-9	5% w/v
GI Medication	Pepto-Bismol (Bismuth Subsalicylate)	5% v/v
GI Medication	Prilosec OTC (Omeprazole)	5% v/v
GI Medication	Alka-Seltzer (Sodium carbonate/potassiumcarbonate)	5% v/v
GI Medication	Mylanta (Al(OH)3)	5% v/v
GI Medication	Tums (CaCO3)	5% v/v
GI Medication	Rolaids (Mg(OH)2)	5% w/v
GI Medication	Milk of Magnesia (Mg(OH)2)	5% v/v
GI Medication	Dulcolax (Sodium picosulfate solution)	5% w/v
GI Medication	Immodium AD (Loperamide)	5% v/v
Lubricant	Mineral oil	10% v/v
Lubricant	Petroleum jelly	10% v/v
Lubricant	Fleet (Glycerin)	10% v/v
Lubricant	KY Jelly	10% v/v
Other	C&S Transport Medium	75% v/v
Other	Physiological Saline	10% v/v
Other	Tween80 (Polysorbate80)	10% v/v
Topical Medication	Preparation H (Hemorrhoid Cream)	10% v/v
Topical Medication	Cortizone 10 (Hydrocortizone)	10% v/v

Interfering Substances
------------------------	--

'Specific amounts of substance added to stool specimen matrix calculated using C; V=C≥V2 with the assumption that 1g=1mL.

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INCUBATION STUDY

In order to determine a recommended incubation time range, the performance of HardyCHROM™ CRE was evaluated using seven carbapenem non-susceptible target organisms at various incubation times. Each target organism was inoculated to HardyCHROM™ CRE at the limit of detection and incubated at 35°C. Plates were evaluated for growth and chromogenic performance every 2 hours from 18-26 hours and 42-50 hours of incubation. All organisms were recovered from HardyCHROM™ CRE with expected color development as early as 18 hours.

STOOL SPECIMEN STABILITY

Stool specimen with and without Cary Blair Transport Media was evaluated to determine the acceptable storage conditions required to recover carbapenem non-susceptible target organisms. Stool specimens were spiked with carbapenem non-susceptible target organisms near LoD and kept at both room temperature and refrigerated conditions. Specimens were inoculated to HardyCHROM™ CRE at 0. 1, 4, 8, 12, 24, 48, 72, 96, 120, 144. and 168 hours after inoculating the stool with target organism.

HardyCHROM™ CRE was able to recover 6/6 (100%) of the carbapenem non-susceptible target strains from raw stool specimen and stool specimen in Cary Blair Transport Media when stored at room temperature for up to 24 hours. HardyCHROM™ CRE was able to recover 6/6 (100%) of the carbapenem non-susceptible target strains from raw stool specimen and stool specimen in Cary Blair Transport Media when stored at 2-8℃ for up to 7 days.

REPRODUCIBILITY

Prior to initiating the prospective clinical study, a panel of 10 blinded isolates provided by Hardy Diagnostics was tested at three distinct study sites in duplicate on five work days to demonstrate reproducibility and to document proficiency in the performance of the test. Each panel included five CRE-positive [Enterobacter cloacae complex (one isolate), Escherichia coli (one isolate) and Klebsiella pneumoniae (three isolates) at 103 CFU/mL] , and 5 CRE negative [Shigella flexneri (one isolate), Shigella sonnei (one isolate), Staphylococcus aureus (one isolate), Staphylococcus epidermidis (one isolate) and Escherichia coli (one isolate) at 107 CFU/ml]. Agreement of >95% with known test results was required before proceeding with the study. The testing was done with at least one operator and two readers, blinded to each other's results, per site. All CRE-positive isolates tested (100%) were detected by HardyCHROM™ CRE on all days of the reproducibility study.

CONCLUSIONS

The non-clinical data support the safety of the device verification and validation demonstrate that the HardyCHROM™ CRE device should perform as intended in the specified use conditions. The analytical and clinical data demonstrate that the HardyCHROM™ CRE device is substantially equivalent to the predicate device.

Regulation Number and Section

§ 866.1700 Culture medium for antimicrobial susceptibility tests.

(a)
Identification. A culture medium for antimicrobial susceptibility tests is a device intended for medical purposes that consists of any medium capable of supporting the growth of many of the bacterial pathogens that are subject to antimicrobial susceptibility tests. The medium should be free of components known to be antagonistic to the common agents for which susceptibility tests are performed in the treatment of disease.(b)
Classification. Class II (performance standards).