HardyCHROM CRE

K190553 · Hardy Diagnostics · JSO · Apr 29, 2019 · Microbiology

Device Facts

• Record ID: K190553
• Device Name: HardyCHROM CRE
• Applicant: Hardy Diagnostics
• Product Code: JSO · Microbiology
• Decision Date: Apr 29, 2019
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.1700
• Device Class: Class 2

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. 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 Story

HardyCHROM™ CRE is a selective/differential chromogenic agar medium for screening fecal specimens for carbapenem non-susceptible E. coli and KES (Klebsiella aerogenes, Klebsiella pneumoniae, Enterobacter cloacae complex, Serratia marcescens). Selective components inhibit yeast, Gram-positive bacteria, and carbapenem-susceptible Gram-negative bacteria. Principle of operation: enzymatic chromogenic reaction produces distinct colony colors (pink for E. coli, blue for KES) after 18-24 hours incubation at 35°C. Used in clinical laboratories; operated by technicians/microbiologists. Visual inspection of plates identifies target colonies. Output is presumptive identification; requires subculture to non-selective media for confirmation, AST, and epidemiological typing. Benefits include rapid screening for colonization to support hospital infection control programs.

Clinical Evidence

Prospective clinical study at three hospitals (n=1,484 valid samples) compared HardyCHROM™ CRE to routine culture (TSB enrichment + MacConkey subculture). Overall sensitivity for E. coli was 100% (67.6-100% CI) and specificity 99.3% (98.8-99.6% CI). Overall sensitivity for KES was 95.8% (86.0-98.9% CI) and specificity 98.2% (97.4-98.7% CI). Contrived specimen testing (n=203) confirmed performance at 2x LoD. Bench testing established LoD at 1.5x10^2 CFU/mL.

Technological Characteristics

Selective and differential chromogenic agar medium. Employs enzymatic metabolism of chromogenic substrates for differentiation. Inoculation is direct; incubation is aerobic at 35-37°C. Manual visual interpretation. Requires subculture to non-selective media for confirmation. No electronic components or software algorithms.

Indications for Use

Indicated for the qualitative and presumptive detection of carbapenem non-susceptible Escherichia coli and KES (Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae complex, and Serratia marcescens) from stool specimens in a healthcare setting. For prescription use only.

Regulatory Classification

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.

Predicate Devices

• Bio-Rad VRESelect (K122187)

Submission Summary (Full Text)

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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 ([e-mail]) or phone [phone] 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: [phone] 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

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.

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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.

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.

| Attribute | Device | Comparator | Substantially<br>Equivalent? |
|-----------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------|
| Name | HardyCHROM™ CRE | Bio Rad VRESelect | |
| 510(k) | 510(k) number | 510(k) number | Yes |
| Details | N/A Product Code<br>ISO<br>21 CFR 866.1700<br>"66.1700umberCS for anti-microbial<br>susceptibility testsp<br>Class II<br>Panel 83 Microbiology | K122187 Product<br>Code JSO<br>21 CFR 866.1700<br>"Culture medium for anti-<br>microbial susceptibility tests"<br>Class II<br>Panel 83 Microbiology | |
| Attribute | Device | Comparator | Substantially |
| Name | HardyCHROM™ CRE | Bio Rad VRESelect | Equivalent? |
| Intended Use | HardyCHROM™ CRE is a selective and<br>differential chromogenic agar medium<br>intended for the qualitative and presumptive<br>detection from stool specimens of Escherichia<br>coli that are non-susceptible to carbapenems as<br>pink colonies and KES (Klebsiella aerogenes,<br>Klebsiella oxytoca, Klebsiella pneumoniae,<br>Enterobacter cloacae complex, and Serratia<br>marcescens.) that are non-susceptible to<br>carbapenems as blue colonies.<br>HardyCHROM CRE is intended as an aid in<br>the detection, identification of colonization and<br>control of these bacteria in a healthcare setting.<br>HardyCHROM™ CRE is not intended to<br>diagnose infection or guide therapy. Results<br>can be interpreted after incubation for 18-24<br>hours. Subculture to non-selective medium is<br>required for confirming identification,<br>antimicrobial susceptibility testing and<br>epidemiological typing.<br>A lack of growth or the absence of pink or blue<br>colonies on HardyCHROM™ CRE does not<br>preclude the presence of Escherichia coli and<br>KES that are non-susceptible to carbapenems. | VRESelect™ is a selective and<br>differential chromogenic medium,<br>containing 8 ug/mL of vancomycin,<br>for the qualitative detection of<br>gastrointestinal colonization of<br>vancomycin resistant Enterococcus<br>faecium (VREfm) and vancomycin<br>resistant Enterococcus faecalis<br>(VREfs) and to aid in the<br>prevention and control of<br>vancomycin resistant Enterococcus<br>(VRE) in healthcare settings. The<br>test is performed on rectal swabs or<br>fecal specimens from patients to be<br>screened for VRE colonization.<br>VRESelect™ is not intended to<br>diagnose VRE infection nor to<br>guide or monitor treatment of<br>infection. Results can be interpreted<br>after 24 to 28 hours incubation.<br>Subculture to non-selective media<br>(e.g., trypticase soy agar with 5%<br>sheep blood) is needed for<br>susceptibility testing and<br>epidemiological 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 and<br>antimicrobial susceptibility testing | Required for confirmation of identity<br>and antimicrobial susceptibility testing | Yes |
| Organisms<br>Detected | Carbapenem non-susceptible Escherichia coli,<br>and KES (Klebsiella aerogenes, Klebsiella<br>oxytoca, Klebsiella pneumoniae, Enterobacter<br>cloacae complex, and Serratia marcescens) | Vancomycin resistant E. faecalis and<br>E. 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 |
| HardyCHROM<br>CRE | 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 |
| HardyCHROM<br>CRE | 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 |
| HardyCHROM<br>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 | | Reference Method | | |
| | | Positive | Negative | Total |
| | Pink (Positive) | 8 | 112 | 19 |
| HardyCHROM<br>CRE | 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 |
| HardyCHROM<br>CRE | 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 |
| HardyCHROM<br>CRE | 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 |
| HardyCHROM<br>CRE | 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 |
| HardyCHROM<br>CRE | 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 |
| HardyCHROM<br>CRE | 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 |
| HardyCHROM<br>CRE 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 |
| HardyCHROM<br>CRE 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 |
| HardyCHROM<br>CRE 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 |
| HardyCHROM<br>CRE 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 | ETP<br>MIC | IMP<br>MIC | MEM<br>MIC | LoD at 24 hours |
|---------------------------------|----|------------------------------------------------------------------------------------------|------------|------------|------------|--------------------------------------------------------|
| E. coli | 17 | OXA-48, NDM (1,5,6)<br>KPC (3, KPC-type) | 1->32 | 4->32 | 2->32 | 16 at 102 CFU/mL<br>1 at 103 CFU/mL |
| E. cloacae complex | 15 | OXA-48, MIR-8, KPC -4,<br>ACT (6, ACT type),<br>NDM-1, VIM (6, 23, 31),<br>IMP(1, 8) | 1->32 | 1->32 | 0.5->32 | 13 at 102 CFU/mL<br>1 at 103 CFU/mL<br>1 at 104 CFU/mL |
| K. oxytoca and<br>K. pneumoniae | 39 | OXA (48, 163, 181, 232),<br>KPC (2, 3, 12, KPC-type),<br>NDM-1, IMP (1, 4, 8),<br>VIM-27 | 0.5->32 | 2->32 | 0.5->32 | 37 at 102 CFU/mL<br>1 at 103 CFU/mL<br>1 at 104 CFU/mL |
| S. marcescens | 5 | IMP-8, NDM-1, VIM-4,<br>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<br>(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 | Concentration<br>in Sample<br>Matrix 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/potassium<br>carbonate) | 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.