NG-Test CARBA 5 is an in vitro rapid and visual multiplex immunochromatographic assay for the qualitative detection and differentiation of five common carbapenemases (KPC, OXA-48-like, VIM, IMP and NDM) from carbapenem non-susceptible pure bacterial colonies when grown on the following media:

5% sheep blood agar or MacConkey agar (16-24 hours) for testing Enterobacteriaceae and Pseudomonas aeruginosa
HardyCHROM™ CRE agar (18-24 hours) for testing E. coli and KES (Klebsiella aerogenes, . Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae complex, and Serratia marcescens).
The NG-Test CARBA 5 is intended as an aid for infection control in the detection of carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa in healthcare settings. NG-Test CARBA 5 is not intended to guide or monitor treatment for carbapenem non-susceptible bacterial infections. A positive or negative NG-Test CARBA 5 test result does not rule out the presence of other mechanisms of antibiotic resistance. NG-Test CARBA 5 should be used in conjunction with other laboratory tests including phenotypic antimicrobial susceptibility testing.

Device Description

NG-Test CARBA 5 is an in vitro rapid and visual multiplex immunochromatographic assay that detects one or more of the five common types of carbapenemase enzymes (KPC (K), OXA-48-like (O), IMP (I), VIM (V), NDM (N)) in bacterial colonies. Liquid extraction buffer is used as a cell lysing solution when mixed with colonies. Monoclonal antibodies that individually recognize each of the five carbapenemases are immobilized on a nitrocellulose membrane. Free monoclonal antibodies are present in the sample pad and labelled with colloidal gold. Upon addition of colonies mixed with extraction buffer to the sample pad, the capillary action of the nitrocellulose draws the sample the mobile antibodies and immobile antibodies on the test strip. The immobilized control antibodies capture any mobile antibodies that run through the sample pad and nitrocellulose without binding to other test lines. A positive result occurs when a red line appears on the control region and one or more lines appear in the test regions (K. O. V. I. or N) and indicates that the sample contains one or more carbapenemases. A negative result occurs when only the control line is observed and indicates that the sample does not contain any of the five carbapenemases. If the control line does not appear, the test result is invalid. The sample pad, and nitrocellulose strip are contained within a plastic cassette. After addition of colonies in extraction buffer to the sample port, a result can be read after 15 minutes.

AI/ML Overview

The NG-Test CARBA 5 is an in vitro rapid and visual multiplex immunochromatographic assay designed to detect five common carbapenemase enzymes (KPC, OXA-48-like, IMP, VIM, NDM) in bacterial colonies. The device's performance was evaluated through a multi-center study to establish its acceptance criteria.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for the NG-Test CARBA 5 are based on Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) compared to a composite reference method. The reported device performance met or exceeded these criteria for both Enterobacteriaceae and P. aeruginosa across different culture media.

Table 1: Acceptance Criteria and Reported Device Performance

Category / Organism Group	Metric	Acceptance Criteria (Implicit)	Reported Device Performance (95% CI)
Enterobacteriaceae (Agar: 5% Sheep Blood or MacConkey)	Overall PPA	High agreement (generally ≥90%)	100.0% (97.6% - 100.0%)
	Overall NPA	High agreement (generally ≥90%)	95.5% (88.9% - 98.2%)
After Discrepant Analysis	Overall PPA		100.0% (97.7% - 100.0%)
	Overall NPA		100.0% (95.6% - 100.0%)
P. aeruginosa (Agar: 5% Sheep Blood or MacConkey)	Overall PPA	High agreement (generally ≥90%)	100.0% (77.2% - 100.0%)
	Overall NPA	High agreement (generally ≥90%)	94.6% (85.4% - 98.2%)
After Discrepant Analysis	Overall PPA		100.0% (80.6% - 100%)
	Overall NPA		100.0% (93.2% - 100%)
E. coli, KES (Agar: HardyCHROM™ CRE, Raw Stool)	Overall PPA	High agreement (generally ≥90%)	100.0% (97.4% - 100.0%)
	Overall NPA	High agreement (generally ≥90%)	90.2% (77.5% - 96.1%)
After Discrepant Analysis	Overall PPA		100.0% (Likely same as without disp. analysis)
	Overall NPA		100.0% (90.6% - 100.0%)
E. coli, KES (Agar: HardyCHROM™ CRE, C&S Cary Blair Stool)	Overall PPA	High agreement (generally ≥90%)	100.0% (97.3% - 100.0%)
	Overall NPA	High agreement (generally ≥90%)	90.2% (77.5% - 96.1%)
After Discrepant Analysis	Overall PPA		100.0% (97.4% - 100.0%)
	Overall NPA		100.0% (Likely same as without disp. analysis)

(Note: "Implicit" acceptance criteria are derived from the fact that the device was found substantially equivalent, meaning it met FDA's expectations for performance for this class of device. Exact pre-defined numerical thresholds for acceptance were not explicitly stated in the provided text, but the reported performance generally reflects high agreement to the reference methods.)

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

Agar: 5% Sheep Blood or MacConkey agar:
- Total organisms initially tested: 310
- Organisms included in analysis: 309 (1 Pseudomonas species other than P. aeruginosa excluded)
- Enterobacteriaceae: 240 isolates (yielding 244 results due to co-production of two carbapenemases in four isolates).
- P. aeruginosa: 69 isolates.
- Provenance: Prospectively-collected and stock bacterial isolates from three geographically diverse hospitals.
Agar: HardyCHROM™ CRE agar (from stool specimens):
- Total organisms initially enrolled: 186
- Organisms included in analysis: 185 (1 organism unavailable)
- Raw Stool: 180 organisms (184 target results) for E. coli and KES (Klebsiella aerogenes, K. oxytoca, K. pneumoniae, Enterobacter cloacae complex, and Serratia marcescens).
- C&S Cary Blair Stool: 178 organisms (182 target results) for E. coli and KES.
- Provenance: This data was collected internally using the same bacterial isolates (from blood and MacConkey agar study) cultured on HardyCHROM™ CRE after being seeded in raw and C&S Cary Blair stool.

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

The document does not explicitly state the number or qualifications of experts (e.g., radiologists, microbiologists) who established the ground truth. However, the ground truth was established through a "composite reference method" combining:

An FDA-cleared device: Xpert Carba-R by Cepheid (PCR for KPC, OXA-48 or 181, IMP, VIM, NDM). This is a molecular method, so human expertise here would primarily be in running the test and interpreting its output according to established protocols.
Modified carbapenem inactivation method (mCIM) and EDTA carbapenemase inactivation method (eCIM) as described by CLSI M100, S29. These are phenotypic tests requiring skilled microbiologists for execution and interpretation.
Antibiotic susceptibility testing results to ertapenem, imipenem, and meropenem. This also requires expertise in microbiology and adherence to CLSI guidelines.
Identity and susceptibility of organisms were confirmed using FDA-cleared ID and AST systems. This implies expert oversight or use of automated systems with established performance.

For discrepant analysis, alternative PCR assays and bidirectional sequencing were used, which would involve highly specialized molecular microbiologists.

4. Adjudication Method for the Test Set

The study employed discrepant analysis to resolve inconsistencies between the NG-Test CARBA 5 results and the initial composite reference method results. This means that when the device result did not agree with the initial ground truth, further, more definitive tests (like alternative PCR assays and bidirectional sequencing) were performed. The results from these additional, often more gold-standard methods, were then used to adjudicate the true status of the isolate, effectively updating the ground truth for those specific cases. This "2+1" or "3+1" type of adjudication (NG-Test vs. Composite Reference, then a tie-breaker/definitive test) was used to ensure the most accurate ground truth for performance calculation.

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

The provided text does not describe a multi-reader multi-case (MRMC) comparative effectiveness study involving human readers with and without AI assistance (as the NG-Test CARBA 5 is an immunochromatographic assay, not an AI-driven image analysis tool requiring human interpretation assistance). The device provides a visual, rapid result, and its performance is evaluated against laboratory gold standards, not through human reader performance improvement.

6. Standalone Performance Study

Yes, the performance study directly evaluates the standalone performance of the NG-Test CARBA 5 device. The provided tables and metrics (PPA, NPA) reflect the algorithm's performance (in this case, the immunochromatographic assay's ability to detect carbapenemases) without human intervention in the result determination process beyond the initial preparation and visual reading of the test strip. The operators were blinded to the expected result during setup and interpretation, indicating an objective assessment of the device's inherent performance.

7. Type of Ground Truth Used

The ground truth used was a composite reference method consisting of:

Molecular (PCR): Xpert Carba-R (FDA-cleared device).
Phenotypic (Enzymatic Activity): Modified Carbapenem Inactivation Method (mCIM) and EDTA Carbapenemase Inactivation Method (eCIM) as per CLSI guidelines.
Phenotypic (Susceptibility Testing): Antibiotic susceptibility testing results (ertapenem, imipenem, meropenem).
Discrepant Analysis: For conflicting results, more definitive molecular techniques (alternative PCR and bidirectional sequencing) were employed to establish the definitive truth.

This multifaceted approach to ground truth ensures robust and accurate classification of carbapenemase production.

8. Sample Size for the Training Set

The provided document does not mention a training set or its sample size. This is typical for in vitro diagnostic (IVD) devices like the NG-Test CARBA 5 which are based on biochemical or immunological principles, rather than machine learning algorithms that require explicit training data. The "analytical reactivity" section describes testing against a panel of 92 characterized strains, which could be considered an internal validation or analytical performance assessment, but not a "training set" in the machine learning sense.

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

As no training set (in the context of machine learning) is described, there is no information on how its ground truth was established. The "Analytical Reactivity" study used 92 strains "characterized to have a target carbapenemase," implying that their carbapenemase status was already established by other reference methods prior to this specific evaluation. This characterization would have been done using methods similar to the composite reference described for the main performance study (e.g., PCR, sequencing, phenotypic tests).

Summary

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October 2, 2019

NG Biotech % Anna Klavins Lead Performance Studies Microbiologist Hardy Diagnostics 1430 West McCoy Lane Santa Maria, California 93455

Re: K191889

Trade/Device Name: NG-Test CARBA 5 Regulation Number: 21 CFR 866.1640 Regulation Name: Antimicrobial Susceptibility Test Powder Regulatory Class: Class II Product Code: PTJ Dated: July 11, 2019 Received: July 15, 2019

Dear Anna Klavins:

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/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); 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 https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). 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 (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Ribhi Shawar, Ph.D. (ABMM) Chief, General Bacteriology and Antimicrobial Susceptibility Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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510(k) Summary

I. SUBMITTER

July 11th, 2019 Correspondent: Anna Klavins Lead Performance Studies Microbiologist Hardy Diagnostics 1430 W. McCoy Lane Santa Maria, CA 93455 Phone: 805-346-2766 x5752 E-mail: ClinicalTrials@hardydiagnostics.com

Applicant: Milovan Stankov-Puges CEO NG Biotech Z.A. Courbouton, secteur 1, Atelier relais le Tremplin Guipry 35480 France Phone: :+33 (0) 2 23 30 17 83 Fax: +33 (0) 9 71 70 53 10 E-mail: msp@ngbiotech.com

II. DEVICE

Name of Device: NG-Test CARBA 5 Classification Name: Antimicrobial Susceptibility Test Powder Regulatory Class: II Product Code: PTJ

III. PREDICATE DEVICE RAPIDEC CARBA NP, K162385

IV. DEVICE DESCRIPTION

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a red line appears on the control region and one or more lines appear in the test regions (K. O. V. I. or N) and indicates that the sample contains one or more carbapenemases. A negative result occurs when only the control line is observed and indicates that the sample does not contain any of the five carbapenemases. If the control line does not appear, the test result is invalid. The sample pad, and nitrocellulose strip are contained within a plastic cassette. After addition of colonies in extraction buffer to the sample port, a result can be read after 15 minutes.

V. INDICATIONS FOR USE

5% sheep blood agar or MacConkey agar (16-24 hours) for testing Enterobacteriaceae and Pseudomonas aeruginosa
HardyCHROM™ CRE agar (18-24 hours) for testing E. coli and KES (Klebsiella aerogenes, . Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae complex, and Serratia marcescens).

The NG-Test CARBA 5 is intended as an aid for infection control in the detection of carbapenemaseproducing Enterobacteriaceae and Pseudomonas aeruginosa in healthcare settings. NG-Test CARBA 5 is not intended to guide or monitor treatment for carbapenem non-susceptible bacterial infections. A positive or negative NG-Test CARBA 5 test result does not rule out the presence of other mechanisms of antibiotic resistance. NG-Test CARBA 5 should be used in conjunction with other laboratory tests including phenotypic antimicrobial susceptibility testing.

VI. PERFORMANCE DATA

Performance of NG-Test CARBA 5 was evaluated at three geographically diverse hospitals with prospectively-collected and stock bacterial isolates. The identification of carbapenemase production on NG-Test CARBA 5 was compared to another FDA-cleared device, Xpert Carba-R by Cepheid (PCR for KPC, OXA-48 or 181, IMP, VIM, NDM), modified carbapenem inactivation method (mCIM) and EDTA carbapenemase inactivation method (eCIM) as described by CLSI M100, S29, and antibiotic susceptibility testing results to ertapenem, imipenem, and meropenem. Identity and susceptibility of organisms were confirmed using FDA-cleared ID and AST systems. NG-Test CARBA 5 quality control was performed in parallel every day of testing.

A total of 310 organisms were tested against PCR (Xpert Carba-R. Cepheid) and phenotypic tests (mCIM, eCIM, and disk diffusion). One organism did not meet enrollment criteria because it was a species of Pseudomonas (Cornell 50) other than P. aeruginosa and was therefore excluded from the analysis. Of the remaining 309 organisms tested, a total of 240 Enterobacteriaceae (which provided 244 results since four isolates co-produced two carbapenemases) and 69 P. aeruginosa isolates were tested on NG-Test CARBA 5 with concordant results obtained by phenotypic testing paired with Xpert Carba-R results.

Performance was equivalent between blood and MacConkey agar. Table 1 indicates the PPA and NPA for each individual target separated out by organism group. The overall PPA for Enterobacteriaceae was 100.0% (97.6% - 100.0%) and the overall NPA was 95.5% (88.9% - 98.2%) (Table 2).

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The overall PPA for P. aeruginosa was 100.0% (77.2% - 100.0%) and the overall NPA was 94.6% (85.4% - 98.2%) (Table 3). P. aeruginosa with NDM (n=2) were evaluated analytically in the bench testing.

Plate	Organism Group6	Total Targets	Target	TP1	FP3	TN	PPA	low95%2	high95%	NPA	low95%	high95%
5% Sheep Blood orMacConkey agar	Enterobacteriaceae(ENT)	244	KPC	84	0	160	100.0	95.6	100.0	100.0	97.7	100.0
			OXA-48-like	20	0	224	100.0	83.9	100.0	100.0	98.3	100.0
			VIM	11	0	233	100.0	74.1	100.0	100.0	98.4	100.0
			IMP	4	34	237	100.0	51.0	100.0	98.8	96.4	99.6
			NDM	37	14	206	100.0	90.6	100.0	99.5	97.3	99.9
	Pseudomonasaeruginosa	69	KPC	2	0	67	100.0	34.2	100.0	100.0	94.6	100.0
			OXA-48-like	0	0	69	n/a	n/a	n/a	100.0	94.7	100.0
			VIM	9	0	60	100.0	70.1	100.0	100.0	94.0	100.0
			IMP	2	35	64	100.0	34.2	100.0	95.5	87.6	98.5
			NDM	0	0	69	n/a	n/a	n/a	100.0	94.7	100.0

Table 1. Performance of the Comparator Method vs. CARBA 5 for all sites combined - Analysis by Target

1No True Positive results for OXA-48-like and NDM for the P. aeruginosa organism group in multicentric clinical testing.

4 ower bounds are below 90% due to the OXA, IMP, and VIM carbapenemases. The claim of NC-Test CARBA 5 detection of OXA, IMP, and VIM carbapenemases is supported by analytical reactivity data.

For Enterobacteriaceae, three isolates were for IMP on NG-Test CARBA 5 (positive MP on NG-Test CARBA 5, positive mCM, and negative Xpert Carba-R result). One isolate was a false positive for NDM on NG-Test CARBA 5, positive mCM, negative Xpet Carba-R result). For P. aeruginou, three isolates were false positive IMP on NG-Test CARBA 5. positive InCM, and negative Xpert Carba-R result).

*All three isolates were confirmed to have the MP-3 gene, making these true positives for IMP after discrepant analysis. (IMP-8 is predicted to be detected by Xpert Carbased on in silico analysis but has not been demonstrated analytically.) One isolate was confirmed to have an NDM-1 gene making this isolate true positive for NDM after discrepant analysis. (NDM-1 is predicted to be detected by Xpert Carba-R based on in silico analysis and has been tested analytically.) After discrepant analysis, the Enterobacteriaceae overall PPA increased to 100.0% (97.7% - 100.0%) and the vereall NPA increased to 100.0% (95.6% - 100.0%).

5 MI three isolates were confirmed to have 7, IMP-15, and IMP-19) making these true positives for IMP after discrepant analysis. (IMP-7 is a known limitation of Xpert Carba R. IMP-19 is predicted by Xpert Carbed on in silico analysis but has not been tested analytically. The ability of Xpert Carba-R to detect IMP-15 is unknown.) After discrepant analysis, the P. aerwinosa overall PPA increased to 100% (80.6% - 100%) and the overall NPA increased to 100% (93.2% - 100%).

"Ertapenen disks were routinely used to maintain solated colonies of retrospective Enterobacteriaceae isolates. No selective pressure was used for isolated colonies of retrospective P. aeruginosa isolates.

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	Enterobacteriaceae	Composite Reference Method
		Positive	Negative	Total
NG-Test CARBA 5	Positive	156	41,2	160
	Negative	0	84	84
	Total	156	88	244
Positive Percent Agreement (PPA)		156/156 = 100% (95% CI: 97.6-100%)
Negative Percent Agreement (NPA)		84/88 = 95.5% (95% CI: 88.9-98.2%)

Table 2. Agreement of NG-Test CARBA 5 with the composite reference method when testing Enterobacteriaceae

1 An alternative PCR assay showed that the NDM false positive isolate harbored a blayDM-1 variant. Isolate was positive by mCIM.

2 An alternative PCR assay and bidirectional sequencing showed that the three IMP false positive isolates harbored blamp -8/-47 variant that is predicted by in silico analysis but not analytically demonstrated to be detected by the assay. Isolates were positive by mCIM.

Table 3. Agreement of NG-Test CARBA 5 with the composite reference method when testing P. aeruginosa

		Composite Reference Method
P. aeruginosa		Positive	Negative	Total
NG-Test CARBA 5	Positive	13	31	16
	Negative	0	53	53
	Total	13	56	69
Positive Percent Agreement (PPA)		13/13 = 100% (95% CI: 77.2-100%)
Negative Percent Agreement (NPA)		53/56 = 94.6% (95% CI: 85.4-98.2%)

1 An alternative PCR assay and bidirectional sequencing showed that the three IMP false positive isolates harbored blamp variants that (i) are not detected by the FDA-cleared PCR assay (blamp variant -7), (ii) are predicted by in silico analysis but not analytically demonstrated to be detected by the assay (blamp -19), or (ii) the reactivity of the assay is unknown (bla]MP variant -15). Isolates were positive by mCIM.

The bacterial isolates used to evaluate NG-Test CARBA 5 from blood and MacConkey agar were also used internally to evaluate the performance of NG-Test CARBA 5 from HardyCHROM™ CRE agar. These results were compared to Xpert Carba-R, mCIM, and eCIM as described by CLSI M100, S29, and antibiotic susceptibility testing results to ertapenem, and meropenem. Identity and susceptibility of organisms were confirmed using FDA-cleared ID and AST systems. NG-Test CARBA 5 quality control was performed in parallel every day of testing.

Of the 186 organisms enrolled, one organism was not available for testing and was excluded from the analysis. Of the 185 organisms that fell under HardyCHROM™ CRE claims, 180/185 (97.3%) organisms (184 target results) were recovered from Raw stool, and 178/185 (96.2%) organisms (182 target results) were recovered from C&S Cary Blair stool onto HardyCHROM™ CRE. Table 4 indicates the PPA and NPA for each individual target separated out by organism group. The overall PPA from raw stool specimen inoculated to HardyCHROM™ CRE was 100.0% (97.4% - 100.0%) and the overall NPA was 90.2% (77.5% - 96.1%) (Table 5). The overall PPA from C&S Cary Blair stool specimen inoculated to HardyCHROM™ CRE was 100.0% (97.3% - 100.0%) (Table 6) and the overall NPA was the same as the raw stool specimen.

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Plate	SpecimenType	OrganismGroup	Target	TP	FP2	TN	PPA	low95%1	high95%	NPA	low95%	high95%
HardyCHROM™ CRE agar	RawStool	E. coli ,KES	KPC	76	0	108	100.0	95.2	100.0	100.0	96.6	100.0
			OXA-48-like	18	0	166	100.0	82.4	100.0	100.0	97.7	100.0
			VIM	9	0	175	100.0	70.1	100.0	100.0	97.9	100.0
			IMP	4	3	177	100.0	51.0	100.0	98.3	95.2	99.4
			NDM	36	1	147	100.0	90.4	100.0	99.3	96.3	99.9
	C&S CaryBlairStool	E. coli ,KES	KPC	75	0	107	100.0	95.1	100.0	100.0	96.5	100.0
			OXA-48-like	18	0	164	100.0	82.4	100.0	100.0	97.7	100.0
			VIM	8	0	174	100.0	67.6	100.0	100.0	97.8	100.0
			IMP	4	3	175	100.0	51.0	100.0	98.3	95.2	99.4
			NDM	36	1	145	100.0	90.4	100.0	99.3	96.2	99.9

Table 4. Performance of NG-Test CARBA 5 vs. the comparator method - Analysis by Target

'Lower bounds are below 90% due to the low prevalence of the OXA, IMP, and VIM carbapenemases. The claim of NG-Test CARBA 5 detection of OXA, IMP, and VIM carbapenemases is supported by analytical reactivity data.

"Three isolates were false positive for IMP on NG-Test CARBA 5, positive mCM, and negative Xpert Carbe-R result). All three isolates were confirmed to have the positives for IMP after discrepant analysis. (IMP-3 is predicted to be deceded by Xpert Carba-R based on in silico analysis but has not been demonstrated analytically, ) One isolate was a false positive for NDM on NG-Test CARBA 5 (positive NDM on NG-Text CARBA 5, positive mCM, negative Xpert Carba-R result). This isolate was confirmed to have an NDM-1 gene making this for NDM after discrepant analysis. (NDM-1 is predicted by Xpert Carba-R based on in silico analysis and has been tested analytically.) After discrepant analysis, the overall PPA increased to 100.0%) and the overall NPA increased to 100.0% (90.6% - 100.0%) for raw stool specimen. The overall PPA increased to 100.0% (97.4% - 100.0%) and the overall NPA increased to 100.0%) for C&S Cary Blair stool specimen.

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Raw Stool		Composite Reference Method
		Positive	Negative	Total
NG-Test CARBA 5	Positive	143	41,2	147
	Negative	0	37	37
	Total	143	41	184
Positive Percent Agreement (PPA)		143/143 = 100% (95% CI: 97.4-100%)
Negative Percent Agreement (NPA)		37/41 = 90.2% (95% CI: 77.5-96.1%)

Table 5. Agreement of NG-Test CARBA 5 with the composite reference method when testing bacterial growth on HardyCHROM™ CRE agar after seeded in Raw Stool

1 An alternative PCR assay showed that the NDM false positive isolate harbored a blayDM -1 variant. Isolate was positive by mCIM.

Table 6. Agreement of NG-Test CARBA 5 with the composite reference method when testing bacterial growth on HardyCHROM™ CRE agar after seeded in C&S Cary Blair Stool

C&S Cary Blair Stool		Composite Reference Method
		Positive	Negative	Total
	Positive	141	41,2	145
NG-Test CARBA 5	Negative	0	37	37
	Total	141	41	182
Positive Percent Agreement (PPA)		141/141 = 100% (95% CI: 97.3-100%)
Negative Percent Agreement (NPA)		37/41 = 90.2% (95% CI: 77.5-96.1%)

1 An alternative PCR assay showed that the NDM false positive isolate harbored a blayDM -1 variant. Isolate was positive by mCIM.

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VII. COMPARISON OF TECHNOLOGICAL CHARACTERISTICS WITH THE PREDICATE DEVICE

Attribute	Device	Predicate	SubstantiallyEquivalent?
Name	NG-Test CARBA 5	RAPIDEC CARBA NP	Yes
510(k) Details	Product Code PTJ21 CFR 866.1640"Antimicrobial Susceptibility Test Powder"Class IIPanel 83 Microbiology	Product Code PTJ21 CFR 866.1640"Antimicrobial Susceptibility TestPowder"Class IIPanel 83 Microbiology	Yes
Intended Use	NG-Test CARBA 5 is an in vitro rapid andvisual multiplex immunochromatographicassay for the qualitative detection anddifferentiation of five commoncarbapenemases (KPC, OXA-48-like, VIM,IMP and NDM) from carbapenem non-susceptible pure bacterial colonies whengrown on the following media:• 5% sheep blood agar or MacConkey agar(16-24 hours) for testingEnterobacteriaceae and Pseudomonasaeruginosa• HardyCHROM™ CRE agar (18-24 hours)for testing E. coli and KES ( Klebsiellaaerogenes, Klebsiella oxytoca, Klebsiellapneumoniae, Enterobacter cloacaecomplex, and Serratia marcescens ).The NG-Test CARBA 5 is intended as an aidfor infection control in the detection ofcarbapenemase-producingEnterobacteriaceae and Pseudomonasaeruginosa in healthcare settings. NG-TestCARBA 5 is not intended to guide or monitortreatment for carbapenem non-susceptiblebacterial infections. A positive or negativeNG-Test CARBA 5 test result does not ruleout the presence of other mechanisms ofantibiotic resistance. NG-Test CARBA 5should be used in conjunction with otherlaboratory tests including phenotypicantimicrobial susceptibility testing.	RAPIDEC CARBA NP is aphenotypic (colorimetric) in vitrodiagnostic test for the qualitativedetection of carbapenemase enzymesin Enterobacteriaceae andPseudomonas aeruginosa coloniesthat have elevated MIC values to anycarbapenem. RAPIDEC CARBA NPis performed on pure colonies grownon non-selective sheep blood agarculture media.RAPIDEC CARBA NP is intended asan aid in the prevention and control ofinfection caused by carbapenemase-producing Enterobacteriaceae andPseudomonas aeruginosa .RAPIDEC CARBA NP is notintended to guide or monitor thetreatment for these bacterialinfections. A negative result does notpreclude the presence ofcarbapenemase enzymes. The abilityof RAPIDEC CARBA NP to detectcarbapenemase enzymes encoded bygenetic markers other than KPC,NDM, OXA-48, VIM, and IMP hasnot been established. RAPIDECCARBA NP testing should be used inconjunction with other laboratorytests including antimicrobialsusceptibility testing.	Yes
InoculumPreparation	By touching well-isolated colonies with aloop	By touching well-isolated colonieswith an applicator stick	Yes
Sample Type	Bacterial isolates/colonial growth	Bacterial isolates/colonial growth	Yes
Interpretation	Visual	Visual	Yes
Controls	Build-in procedural control in every test strip	Build-in procedural control in everytest strip	Yes

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ANALYTICAL REACTIVITY

NG-Test CARBA 5 was evaluated with ninety-two strains characterized to have a target carbapenemase. Each organism was incubated aerobically for 16 hours on sheep's blood agar and MacConkey agar at 35°C or 18 hours on HardyCHROM™ CRE agar at 35°C. Each test was performed in triplicate from each type of media. NG-Test CARBA 5 test result was read 15 minutes after inoculating the buffer mixed with bacteria into the sample port. The operator was blinded to the expected result while setting up and interpreting the test. All organisms that yielded a negative NG-Test CARBA 5 result were further analyzed by modified carbapenemase inactivation method (mCIM, CLSI M100, S29). After the mCIM analysis, the final sensitivity for all target organisms evaluated was 88/92 (95.7%) from blood agar and 90/92 (97.8%) from MacConkey agar. After the mCIM analysis, the final sensitivity for all target organisms evaluated was 41/41 (100%) from HardyCHROM™ CRE agar. Two IMP-producing P. aeruginosa isolates (IMP-14 and IMP-18) were negative on NG-Test CARBA 5 but positive by mCIM. On blood agar only, two Proteus mirabilis strains resulted in false negative results.

Organism Group	Number ofstrainstested onBlood/MacConkey	Detected Target	Number oftargetstested onBlood/MacConkeyagar	NumberoftargetsTestedHCCRE	Variants Tested	Variants NotDetected
Enterobacteriacae	66	KPC	17	8	2, 3, 4, 6, 12
		OXA-48-like	12	7	48, 181, 163, 232 (48 type)
		VIM	11	9	1, 4, 5, 6, 23, 27, 31
		IMP	8	7	4, 8/472, 261
		NDM	15	11	11, 5, 6, 7
		None	5	2
		Total	68	44
Pseudomonasaeruginosa	26	KPC	5		2, 5
		OXA-48-like	0
		VIM	13		2, 11
		IMP	6		1, 7, 14, 18, 19, 26	14, 18
		NDM	2		1
		None	0
		Total	26

Table 7. Analytical Reactivity Summary for Carbapenemase Producing Organisms (CPO)

'NDM-1 and IMP-26 not detected in P. mirabilis growth from blood agar, but yielded positive results from MacConkey agar.

2IMP-8 and IMP-47 were determined to be the same protein based on sequence analysis by the Beta-Lactamase Database (http://www.bldb.eu/BLDB.php?prot=B1#IMP).

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ANALYTICAL SPECIFICTY

81 organisms that exhibit antibiotic resistance mechanisms other than the targets NG-Test CARBA 5 lateral flow assay detects, are carbapenem-susceptible, or are carbapenem nonsusceptible were tested on NG-Test CARBA 5 from blood agar and MacConkey agar. Organisms tested included Enterobacteriaceae (n=54) and P. aeruginosa (n=20), as well as other phylogenetically related organisms (n=7). HardyCHROM™ CRE was inoculated with 16 cross reactive organisms that were tested on NG-Test CARBA 5 and showed no cross reactivity. These organisms were included in the list of claimed organisms for HardyCHROM™ CRE but which do not produce one of the 5 target carbapenemases. Each organism was incubated aerobically at 35°C on sheep's blood agar and MacConkey agar for 16 hours. HardyCHROM™ CRE was incubated for 18 hours prior to testing. NG-Test CARBA 5 test result was read 15 minutes after inoculating the buffer mixed with bacteria into the sample port. The operator was blinded to the expected result while setting up and interpreting the test. 81/81 (100%) of organisms tested from blood and MacConkey agar produced a negative NG-Test CARBA 5 result. 16/16 (100%) of organisms tested from HardyCHROM™ CRE produced a negative NG-Test CARBA 5 result. Organisms with NMC-A, FRI, and GIM mechanisms were tested by the National Reference Center in France and did not cross react.

	Resistant mechanisms evaluated
Organism Group	Blood & MacConkey agar	HardyCHROM™CRE agar
Enterobacteriaceae	ACT-type, ACT-2, AmpC, CTX-M [1, 3, 8, 9, 14, 15, 22, 24, 30, 40, 55, 74, 75, 79, 124], DHA-1, ESBL, IMI, mrc-1, OmpK35, OmpK37, OXA [1, 2, 30], SHV [11(2b), 12(2be), 18, 28, 31, 89(2b), 108(u), 154, 179(u), 180(u), 182(u), OSBL(2b)], SME, SME-2, TEM [1, 1(2b), 11(2be), 63(2be), 93(2be), 210(u), OSBL(2b)], tet(A), tet(B)	ACT-2, AmpC, CTX-M [9, 14, 30], DHA-1, IMI, MIR-8, OXA, SME, TEM-129(2be), tet(A)
Pseudomonasaeruginosa	aadA6, aadB, aph(3')-IIb, catB7, GES-1, GES-5(c), OXA [10, 50], PAO, PDC [1, 5, 19, 35], PER-1, strA, strB, sull, tet(c), VEB-1, inducible AmpC	N/A
Other	VanA	N/A

Table 8. Resistance Mechanisms Evaluated with NG-Test CARBA 5 for Specificity

INCUBATION STUDY

In order to confirm that NG-Test CARBA 5 delivered consistent results over a range of incubation time, twenty-two strains were tested from blood and MacConkey agar every two hours from 16 to 24 hours of incubation. Fifteen of the twenty-two organisms were also tested from HardyCHROM™ CRE every two hours from 18 to 24 hours. All organisms tested produced the expected result on NG-Test CARBA 5 at every time point tested. NG-Test CARBA 5 test result was read 15 minutes after inoculating the buffer mixed with bacteria into the sample port. The operator was blinded to the expected result while setting up and interpreting the test.

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REFRIGERATION STORAGE STUDY

In order to determine if agar media that has been stored in the refrigerator can be used with NG-Test CARBA 5, twelve strains were cultured and evaluated over time from refrigerated storage. Blood and MacConkey agar plates were inoculated directly with organism (colonies) for the fresh culture, streaking for isolation. HardyCHROM™ CRE was inoculated with organisms at 3x104 CFU/mL in raw stool and stool in C&S Cary Blair Transport Media, streaking for isolation with a 1 µL loop. Each strain was incubated aerobically on sheep's blood agar and MacConkey agar at 35°C and tests were performed after 16 to 24 hours of incubation (Day 0). Ten of the twelve organisms were also tested from HardyCHROM™ CRE after 18 to 24 hours of incubation. All organisms tested produced the expected result on NG-Test CARBA 5 for each day of refrigeration for up to 3 days. The operator was blinded to the expected result while setting up and interpreting the test.

REPRODUCIBILITY

Prior to initiating the clinical study, a panel of 20 blinded isolates provided by Hardy Diagnostics was tested at three distinct study sites on five work days to demonstrate reproducibility and to document proficiency in the performance of the test. 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 target carbapenemase positive isolates tested (100%) were detected by NG-Test CARBA 5 on all days of the reproducibility study.

CONCLUSIONS

The analytical data presented in this submission demonstrates that NG-Test CARBA 5 is substantially equivalent to the predicate device.

Regulation Number and Section

§ 866.1640 Antimicrobial susceptibility test powder.

(a)
Identification. An antimicrobial susceptibility test powder is a device that consists of an antimicrobial drug powder packaged in vials in specified amounts and intended for use in clinical laboratories for determining in vitro susceptibility of bacterial pathogens to these therapeutic agents. Test results are used to determine the antimicrobial agent of choice in the treatment of bacterial diseases.(b)
Classification. Class II (performance standards).