NG-Test® CTX-M MULTI

K243499 · Ng Biotech · PTJ · Jun 4, 2025 · Microbiology

Device Facts

• Record ID: K243499
• Device Name: NG-Test® CTX-M MULTI
• Applicant: Ng Biotech
• Product Code: PTJ · Microbiology
• Decision Date: Jun 4, 2025
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.1640
• Device Class: Class 2

Indications for Use

NG-Test® CTX-M MULTI is an in vitro rapid and visual immunochromatographic assay for the qualitative detection of CTX-M enzymes (groups 1, 2, 8, 9, and 25) from pure colonies of Enterobacterales suspected of ESBL production when grown on the following media: - 5% sheep blood agar or MacConkey agar (16-24 hours) - HardyCHROM™ ESBL agar (18-24 hours) The NG-Test® CTX-M MULTI is intended as an aid for infection control in the detection of CTX-M enzymes-producing organisms (Enterobacterales) in healthcare settings. NG-Test® CTX-M MULTI is not intended to guide or monitor treatment. A positive or negative NG-Test® CTX-M MULTI test result does not rule out the presence of other mechanisms of antibiotic resistance. NG-Test® CTX-M MULTI should be used in conjunction with other laboratory tests including phenotypic antimicrobial susceptibility testing.

Device Story

Rapid, visual immunochromatographic assay; detects CTX-M enzymes (groups 1, 2, 8, 9, 25) in pure Enterobacterales colonies. Input: bacterial colonies from agar plates (blood, MacConkey, or HardyCHROM ESBL) mixed with extraction buffer. Operation: sample added to cassette port; capillary action draws sample through conjugate pad (colloidal gold-labeled monoclonal antibodies) and nitrocellulose membrane (immobilized monoclonal antibodies). Output: visual result at 15 minutes; two red lines (control and test) indicate positive; one red line (control) indicates negative. Used in clinical laboratories by technicians/microbiologists. Results aid infection control decisions; do not replace phenotypic antimicrobial susceptibility testing or rule out other resistance mechanisms.

Clinical Evidence

Clinical study evaluated 309 Enterobacterales isolates across three sites compared to PCR and phenotypic AST. Sensitivity 100% (95% CI: 97.5-100.0%); specificity 99.4% (95% CI: 96.5-99.9%). Seeded study of 193 isolates on HardyCHROM ESBL agar showed 100% sensitivity and 97.7% specificity. Analytical reactivity confirmed 100% detection across 57 strains. Analytical specificity study showed no cross-reactivity with 55 non-target resistance mechanisms (TEM, SHV, VEB, KPC, NDM, etc.).

Technological Characteristics

Immunochromatographic lateral flow assay; nitrocellulose membrane with immobilized monoclonal antibodies; colloidal gold-labeled conjugate. Form factor: plastic cassette with sample port. Manual visual readout. No instrumentation required. Sterilization: not specified. Connectivity: none (standalone).

Indications for Use

Indicated for qualitative detection of CTX-M enzymes (groups 1, 2, 8, 9, and 25) in pure colonies of Enterobacterales suspected of ESBL production. For use in healthcare settings as an aid for infection control. Not for treatment guidance or monitoring. Requires prior organism identification and elevated third-generation cephalosporin MIC determination. Performance established only for 5% sheep blood agar, MacConkey agar, and HardyCHROM ESBL agar.

Regulatory Classification

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.

Predicate Devices

• NG-Test CARBA 5 (K191889)

Submission Summary (Full Text)

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FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY ONLY

## I Background Information:

A 510(k) Number

K243499

B Applicant

NG Biotech

C Proprietary and Established Names

NG-Test CTX-M MULTI

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| PTJ | Class II | 21 CFR 866.1640 - Antimicrobial Susceptibility Test Powder | MI - Microbiology |

## II Submission/Device Overview:

A Purpose for Submission:

To obtain a substantial equivalence determination for the qualitative detection of CTX-M enzymes from pure colonies of Enterobacterales.

B Measurand:

CTX-M enzymes (groups 1, 2, 8, 9, and 25)

C Type of Test:

Qualitative immunochromatographic assay (lateral flow)

## III Intended Use/Indications for Use:

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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A Intended Use(s):

See Indications for Use below.

B Indication(s) for Use:

NG-Test CTX-M MULTI is an in vitro rapid and visual immunochromatographic assay for the qualitative detection of CTX-M enzymes (groups 1, 2, 8, 9, and 25) from pure colonies of Enterobacterales suspected of ESBL production when grown on the following media:

- 5% sheep blood agar or MacConkey agar (16-24 hours)
- HardyCHROM ESBL agar (18-24 hours)

The NG-Test CTX-M MULTI is intended as an aid for infection control in the detection of CTX-M enzymes-producing organisms (Enterobacterales) in healthcare settings. NG-Test CTX-M MULTI is not intended to guide or monitor treatment. A positive or negative NG-Test CTX-M MULTI test result does not rule out the presence of other mechanisms of antibiotic resistance. NG-Test CTX-M MULTI should be used in conjunction with other laboratory tests including phenotypic antimicrobial susceptibility testing.

C Special Conditions for Use Statement(s):

- Rx - For Prescription Use Only
- The performance of NG-Test CTX-M MULTI was established with colonies from blood agar, MacConkey agar, and HardyCHROM ESBL agar. Performance with other culture media has not been evaluated and is therefore unknown.
- The performance of NG-Test CTX-M MULTI with bacteria other than Enterobacterales has not been evaluated.
- Organism identification and elevated third-generation cephalosporin MICs should be determined prior to testing with NG-Test CTX-M MULTI.

D Special Instrument Requirements:

None (results are read manually/visually)

IV Device/System Characteristics:

A Device Description:

NG-Test CTX-M MULTI is an in vitro rapid and visual immunochromatographic assay for the qualitative detection of CTX-M enzymes (groups 1, 2, 8, 9, and 25) from pure colonies of Enterobacterales suspected of extended spectrum beta-lactamase (ESBL) production after culturing on agar and processing in an extraction buffer. The device consists of a sample port, sample and conjugate pad, and nitrocellulose test strip, which are contained within a plastic cassette, in addition to reagents for liquid extraction.

B Principle of Operation:

Monoclonal antibodies that recognize the five major CTX-M groups (groups 1, 2, 8, 9, and 25) are immobilized on a nitrocellulose membrane. Free monoclonal antibodies are present in the conjugate pad and labeled with colloidal gold. Colonies are mixed with extraction buffer to lyse

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the bacteria then added to the sample pad. Upon addition of the processed sample to the sample pad, the capillary action of the nitrocellulose draws the sample through the mobile antibodies in the conjugate pad and the immobile antibodies on the test strip. The immobilized control antibodies capture any mobile antibodies that do not bind to the test line. The result can be read 15 minutes after adding the sample to the sample well. A positive result on the NG-Test CTX-M MULTI occurs when two red lines appear, one on the control (C) region and one on the test (T) region. A negative result occurs when only the control line is observed and indicates the sample does not contain any target CTX-M enzymes, or the CTX-M enzymes are present at a non-detectable level. If the control line does not appear, the test result is invalid.

V Substantial Equivalence Information:

A Predicate Device Name(s): NG-Test CARBA 5

B Predicate 510(k) Number(s): K191889

C Comparison with Predicate(s):

| Device & Predicate Device(s): | Device: K243499 | Predicate: K191889 |
| --- | --- | --- |
| Device Trade Name | NG-Test CTX-M MULTI | NG-Test CARBA 5 |
| General Device Characteristic Similarities | | |
| Intended Use/Indications for Use | NG-Test CTX-M MULTI is an in vitro rapid and visual immunochromatographic assay for the qualitative detection of CTX-M enzymes (groups 1, 2, 8, 9, and 25) from pure colonies of Enterobacterales suspected of ESBL production when grown on the following media:
• 5% sheep blood agar or MacConkey agar (16-24 hours).
• HardyCHROM ESBL agar (18-24 hours).
The NG-Test CTX-M MULTI is intended as an aid for infection control in the detection of CTX-M enzymes-producing organisms (Enterobacterales) in healthcare settings. NG-Test CTX-M MULTI is not intended to guide or monitor treatment. A positive or negative NG-Test | 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 Enterobacterales (formerly Enterobacteriaceae) and Pseudomonas aeruginosa.
• HardyCHROM CRE agar (18-24 hours) for testing E. coli and KES |

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VI Standards/Guidance Documents Referenced:

- CLSI M100, 34th ed., “Performance Standards for Antimicrobial Susceptibility Testing”.
- CLSI M02, 13th ed., “Performance Standards for Antimicrobial Disk Susceptibility Tests”.

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# VII Performance Characteristics (if/when applicable):

# A Analytical Performance:

# 1. Precision/Reproducibility:

The reproducibility of NG-Test CTX-M MULTI was evaluated in a study conducted with three lots of tests at three sites including one internal site. A panel of 20 well-characterized strains grown on $5\%$ sheep blood agar (blood agar) and MacConkey agar was tested over five separate days. The panel included an equal mix of isolates that were determined prior to the study to be positive (i.e., harboring CTX-M) or negative (i.e., not harboring CTX-M) (Table 1). The CTX-M status determination was based on molecular characterization and AST disk diffusion. Two independent readers, blinded to the panel contents and each other's results, conducted testing following the package insert and reviewed the test results from both types of agar media. A total of 300 positive results were expected (3 sites x 2 operators x 5 days x 10 strains that harbor CTX-M genes), and a total of 300 negative results were expected (3 sites x 2 operators x 5 days x 10 strains that do not harbor CTX-M genes).

Table 1: List of Strains Used in NG-Test CTX-M MULTI Reproducibility Study

| Organism | Strain ID | Molecular Summary | CTX-M Group | Expected Results |
| --- | --- | --- | --- | --- |
| Enterobacter cloacae | CDC 0501 | CTX-M-9, TEM-1B, VIM-1 | 9 | Positive |
| Enterobacter cloacae | IHMA 871550 | CTX-M-9 | 9 | Positive |
| Enterobacter cloacae | CDC 0502 | IMP-8, SHV-12, TEM-1B | N/A | Negative |
| Enterobacter cloacae | IHMA 958153 | ACT-32, IMP-8, SHV-12(2be), TEM-OSBL(2b) | N/A | Negative |
| Escherichia coli | IHMA 959898 | CTX-M-15, CTX-M-40 | 1, 8 | Positive |
| Escherichia coli | IHMA 958085 | CTX-M-116 | 1 | Positive |
| Escherichia coli | LSI 971 | NDM-1, SHV-12, TEM-1 | N/A | Negative |
| Escherichia coli | IHMA 951011 | CMY-42, NDM-5, TEM-OSBL(2b) | N/A | Negative |
| Klebsiella oxytoca | IHMA 870581 | CTX-M-30, CTX-M-75 | 1, 2 | Positive |
| Klebsiella oxytoca | LSI 4744 | CTX-M-14, DHA-1, OXA, tet(A) | 9 | Positive |
| Klebsiella oxytoca | CDC 0375 | SHV-5 | N/A | Negative |
| Klebsiella oxytoca | IHMA 949841 | ACC-1, NDM-1, TEM-OSBL(2b) | N/A | Negative |
| Klebsiella pneumoniae | IHMA 984357 | CTX-M-124, SHV-OSBL(2b) | 2 | Positive |
| Klebsiella pneumoniae | IHMA 869028 | CTX-M-14, TEM-129(2be) | 9 | Positive |
| Klebsiella pneumoniae | IHMA 845661 | KPC-3, SHV-11(2b), TEM-1(2b) | N/A | Negative |
| Klebsiella pneumoniae | IHMA 928329 | KPC-3, SHV-12(2be), TEM-OSBL(2b) | N/A | Negative |
| Proteus mirabilis | IHMA 930043 | CTX-M-32 | 1 | Positive |
| Proteus mirabilis | IHMA 874812 | CTX-M-79, TEM-1(2b) | 1 | Positive |
| Proteus mirabilis | IHMA 966438 | DHA-1, IMP-26 | N/A | Negative |
| Proteus mirabilis | IHMA 1104843 | CMY-16, TEM-OSBL(u), VIM-1 | N/A | Negative |

Across all five days of the study, $100\%$ of the isolates yielded results in agreement with the expected positive and negative outcomes for NG-Test CTX-M MULTI from both blood agar and MacConkey agar at all three sites (Tables 2 and 3). The reproducibility of NG-Test CTX-M MULTI was determined to be acceptable.

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Table 2: Summary of Reproducibility Results from Blood Agar

| Day | N | NG-Test CTX-M MULTI Positive | %* | N | NG-Test CTX-M MULTI Negative | %* |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 60 | 60 | 100.0 | 60 | 60 | 100.0 |
| 2 | 60 | 60 | 100.0 | 60 | 60 | 100.0 |
| 3 | 60 | 60 | 100.0 | 60 | 60 | 100.0 |
| 4 | 60 | 60 | 100.0 | 60 | 60 | 100.0 |
| 5 | 60 | 60 | 100.0 | 60 | 60 | 100.0 |
| Overall | 300 | 300 | 100.0 | 300 | 300 | 100.0 |

* % Calculated as percent of observed/expected

Table 3: Summary of Reproducibility Results from MacConkey Agar

| Day | N | NG-Test CTX-M MULTI Positive | %* | N | NG-Test CTX-M MULTI Negative | %* |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 60 | 60 | 100.0 | 60 | 60 | 100.0 |
| 2 | 60 | 60 | 100.0 | 60 | 60 | 100.0 |
| 3 | 60 | 60 | 100.0 | 60 | 60 | 100.0 |
| 4 | 60 | 60 | 100.0 | 60 | 60 | 100.0 |
| 5 | 60 | 60 | 100.0 | 60 | 60 | 100.0 |
| Overall | 300 | 300 | 100.0 | 300 | 300 | 100.0 |

* % Calculated as percent of observed/expected

2. Linearity:
Not applicable

3. Analytical Specificity/Cross Reactivity:
The analytical specificity of the NG-Test CTX-M MULTI was evaluated using organisms that possess antimicrobial resistance mechanisms other than CTX-M or are not susceptible to third-generation cephalosporins (cefotaxime, ceftazidime, ceftriaxone, cefpodoxime) and other agents (aztreonam and cefoxitin). Some of the antimicrobial resistance mechanisms included were TEM, SHV, VEB, ACT, CMY, DHA, FOX, MIR, ACC, VIM, KPC, IMP, NDM, and OXA. A panel of 55 organisms was tested after growth on blood agar and MacConkey agar (including 2 Citrobacter freundii, 3 Enterobacter asburiae, 7 Enterobacter cloacae, 13 Escherichia coli, 4 Klebsiella oxytoca, 15 Klebsiella pneumoniae, 1 Morganella morganii, 7 Proteus mirabilis, and 3 Serratia marcescens).

For evaluation of colonies grown on HardyCHROM ESBL agar, a panel of 50 strains intended for use with this agar medium were seeded in raw stool or stool in C&S Cary Blair Transport Media, followed by sub-culture and testing (including 1 Citrobacter freundii, 3 Enterobacter asburiae, 7 Enterobacter cloacae, 12 Escherichia coli, 4 Klebsiella oxytoca, 14 Klebsiella pneumoniae, 1 Morganella morganii, 5 Proteus mirabilis, and 3 Serratia marcescens; 5 strains included for testing with blood and MacConkey were susceptible to the antimicrobial agents in the agar, thus were not evaluated from the HardyCHROM ESBL agar).

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The organism groups and their relevant molecular characteristics used in the analytical cross-reactivity study is provided in Table 4. All non-target organisms evaluated from blood (55/55, 100%), MacConkey (55/55, 100%), and HardyCHROM ESBL (50/50, 100%) agar yielded a negative NG-Test CTX-M MULTI result. The cross-reactivity of the NG-Test CTX-M MULTI was determined to be acceptable.

Table 4: Resistant mechanisms evaluated for Cross-Reactivity

| Organism | Resistance Mechanisms Evaluated | |
| --- | --- | --- |
| | Blood & MacConkey Agar | HardyCHROM ESBL Agar |
| Citrobacter freundii | CMY-41, CMY-49, IMP-8, KPC-2, TEM-OSBL(2b) | CMY-41, IMP-8, TEM-OSBL(2b) |
| Enterobacter asburiae | ACT-2, IMP-14, MIR-8, NDM-1, OXA-48(c), TEM-OSBL(2b), VEB-2 | ACT-2, IMP-14, MIR-8, NDM-1, OXA-48(c), TEM-OSBL(2b), VEB-2 |
| Enterobacter cloacae | ACT-17, ACT-32, ACT-45, ACT-type, DHA-1, FOX-5, IMP-1, IMP-8, KPC-2, KPC-3, OXA-1, OXA-9, SHV-12, SHV-12(2be), TEM-1A, TEM-1B, TEM-OSBL(2b) | ACT-17, ACT-32, ACT-45, ACT-type, DHA-1, FOX-5, IMP-1, IMP-8, KPC-2, KPC-3, OXA-1, OXA-9, SHV-12, SHV-12(2be), TEM-1A, TEM-1B, TEM-OSBL(2b) |
| Escherichia coli | CMY-2, CMY-42, KPC-3, NDM-1, NDM-5, OXA-1, OXA-30, OXA-48(c), SHV-12, TEM-1, TEM-1(2b), TEM-6(2be), TEM-10, TEM-12(2be), TEM-20(2be), TEM-OSBL(2b) | CMY-2, CMY-42, KPC-3, NDM-1, NDM-5, OXA-1, OXA-30, SHV-12, TEM-1, TEM-6(2be), TEM-10, TEM-12(2be), TEM-20(2be), TEM-OSBL(2b) |
| Klebsiella oxytoca | ACC-1, NDM-1, SHV-5, SHV-7(2be), TEM-52(2be), TEM-OSBL(2b) | ACC-1, NDM-1, SHV-5, SHV-7(2be), TEM-52(2be), TEM-OSBL(2b) |
| Klebsiella pneumoniae | CMY-2, IMP-8, KPC-2, KPC-3, KPC-12, NDM-1, OXA-9, OXA-48(c), SHV-2A(2be), SHV-7, SHV-11, SHV-11(2b), SHV-12(2be), SHV-27(2b), SHV-55(2be), SHV-83(2b), SHV-90(2be), SHV-133(u), SHV-136(u), SHV-154, TEM-1, TEM-1(2b), TEM-4(2be), TEM-11(2be), TEM-141, TEM-OSBL(2b), VEB-1 | CMY-2, IMP-8, KPC-2, KPC-3, KPC-12, NDM-1, OXA-9, SHV-2A(2be), SHV-7, SHV-11, SHV-11(2b), SHV-12(2be), SHV-27(2b), SHV-55(2be), SHV-83(2b), SHV-90(2be), SHV-133(u), SHV-136(u), SHV-154, TEM-1, TEM-1(2b), TEM-4(2be), TEM-11(2be), TEM-141, TEM-OSBL(2b), VEB-1 |
| Morganella morganii | DHA-1 | DHA-1 |
| Proteus mirabilis | CMY-16, DHA-1, IMP-26, KPC-3, TEM-1A, TEM-15(2be), TEM-24(2be), TEM-92(2be), TEM-OSBL(u), VIM-1 | CMY-16, DHA-1, IMP-26, TEM-15(2be), TEM-92(2be), TEM-OSBL(u), VIM-1 |
| Serratia marcescens | CMY-16, FOX-5, IMP-47, OXA-2, OXA-10, TEM-1(2b) | CMY-16, FOX-5, IMP-47, OXA-2, OXA-10, TEM-1(2b) |

4. Assay Reportable Range: Not applicable

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5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

a) Agar Incubation Study

The ability of the NG-Test CTX-M MULTI to consistently provide correct results over the range of recommended agar incubation times was evaluated using five strains previously characterized to have a target CTX-M. The five strains were tested after growth on blood and MacConkey agar every two hours from 16 to 24 hours of incubation. The same five strains were also tested after growth on HardyCHROM ESBL agar every two hours from 18 to 24 hours of incubation. All organisms tested produced the expected result on NG-Test CTX-M MULTI at each time point tested.

b) Refrigerated Storage Study

To evaluate whether organism cultures (plated on agar media) stored in the refrigerator could be used with NG-Test CTX-M MULTI, strains were evaluated each day for three days after refrigeration. Five strains were tested after growth on blood agar, MacConkey agar, and HardyCHROM ESBL agar. All organisms tested produced the expected result on NG-Test CTX-M MULTI for each day of refrigeration.

c) Quality Control

Quality control testing was performed each day of the clinical study. The QC panel consisted of one positive control organism and one negative control organism (Table 5). The QC testing gave the expected results each day of testing.

Table 5: Organisms for NG-Test CTX-M MULTI QC Testing

| QC Organism | Expected Test Line Result |
| --- | --- |
| Escherichia coli, NCTC 13353^{1} | Positive |
| Escherichia coli, ATCC 25922 | Negative |

¹QC organism shall be maintained on blood agar with a ceftriaxone (CRO) disk to ensure the phenotype does not change, i.e. no ESBL plasmid loss occurs.

6. Detection Limit:

Analytical Reactivity

The analytical reactivity (inclusivity) of NG-Test CTX-M MULTI was evaluated using a panel of 57 strains of Enterobacterales characterized to have at least one target CTX-M enzyme (Table 6). Each organism was inoculated onto 5% sheep blood agar (blood agar) and MacConkey agar plates. Additionally, 56 of these strains were inoculated on HardyCHROM ESBL (HC ESBL) agar after being seeded in raw/unpreserved stool or stool preserved in C&S Cary Blair transport medium. Blood and MacConkey agar plates were incubated at 35°C for 16-24 hours, and HC ESBL agar plates were incubated for 18-24 hours. Each isolate was tested with the NG-Test CTX-M MULTI test in triplicate from each media type in line with the instructions for use.

The percent agreement for all target organisms evaluated was 100% (57/57), 100% (57/57) and 100% (56/56) from blood, MacConkey, and HC ESBL agar, respectively (Table 6).

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Table 6: Summary of Analytical Reactivity Study

| Organism | Number of Strains Tested on Blood/MacConkey | Number of Strains Tested on HardyCHROM ESBL | CTX-M Groups and Number of Variants Tested | CTX-M Variants Tested | NG-Test CTX-M MULTI Percentage Agreement Among All Tested Media |
| --- | --- | --- | --- | --- | --- |
| Citrobacter species | 1 | 1 | Group 1 (n=1) | CTX-M-15 | 1/1 (100 %) |
| Enterobacter cloacae | 9 | 9 | Group 1 (n=4) | CTX-M-15, -22, -30 | 9/9 (100 %) |
| | | | Group 9 (n=4) | CTX-M-9, -9-like | |
| | | | Group 25 (n=1) | CTX-M-100 | |
| Escherichia coli | 19 | 19 | Group 1 (n=11) | CTX-M-1, -3, -15, -55, -79, -116 | 19/19 (100 %) |
| | | | Group 2 (n=1) | CTX-M -2 | |
| | | | Group 8 (n=1) | CTX-M-40 | |
| | | | Group 9 (n=7) | CTX-M-14, -24, -27 | |
| | | | Group 25 (n=1) | CTX-M-100 | |
| Klebsiella oxytoca | 5 | 5 | Group 1 (n=3) | CTX-M -15, -22, -30 | 5/5 (100 %) |
| | | | Group 2 (n=1) | CTX-M -75 | |
| | | | Group 9 (n=1) | CTX-M-14 | |
| | | | Group 25 (n=1) | CTX-M -100 | |
| Klebsiella pneumoniae | 14 | 14 | Group 1 (n=6) | CTX-M-3, -12, -15, -22, -64 | 14/14 (100 %) |
| | | | Group 2 (n=2) | CTX-M-74, -124 | |
| | | | Group 8 (n=1) | CTX-M-40 | |
| | | | Group 9 (n=6) | CTX-M-14, -14b, -38 | |
| | | | Group 25 (n=1) | CTX-M-25 | |
| Klebsiella variicola | 1 | 1 | Group 25 (n=1) | CTX-M-152 | 1/1 (100 %) |
| Kluyvera ascorbata | 1 | 1 | Group 2 (n=1) | CTX-M-124 | 1/1 (100 %) |
| Proteus mirabilis | 6 | 5^{1} | Group 1 (n=5) | CTX-M-3, -15, -32, -79 | 6/6 on Blood and MC, 5/5 on HC ESBL (100 %)^{1} |
| | | | Group 25 (n=1) | CTX-M-160 | |
| Serratia marcescens | 1 | 1 | Group 1 (n=1) | CTX-M-15 | 1/1 (100 %) |

1 One P. mirabilis strain with a characterized CTX-M-3 variant was susceptible to an antimicrobial agent in the HC ESBL formula thus was not evaluated with NG-Test CTX-M MULTI from HC ESBL.

Organisms were further analyzed by the CLSI ESBL Disk Test for comparison. Per the CLSI M100 document, 34th edition, the eligible isolates included K. pneumoniae, K. oxytoca, E. coli, and P. mirabilis with the following antimicrobial susceptibility profile:

- For K. pneumoniae, K. oxytoca, and E. coli: Cefpodoxime ≤17mm; Ceftazidime ≤22mm; Aztreonam ≤27mm; Cefotaxime ≤27mm; Ceftriaxone ≤25mm.
- For P. mirabilis: Cefpodoxime ≤22mm; Ceftazidime ≤22mm; Cefotaxime ≤27mm. A summary of results is provided in Table 7.

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Table 7: CLSI ESBL Disk Test Results Summary

| Organism | Number of Strains Tested | CTX-M Groups Tested | CTX-M Variants Tested | Positive CLSI ESBL Disk Test Results | Indeterminate CLSI ESBL Disk Test Results^{1} | Number of Ineligible Isolates for the CLSI ESBL Disk Test^{2} | Percentage of Isolates with Detected ESBL-Production via the CLSI ESBL Disk Test |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Citrobacter species | 1 | 1 | CTX-M-15 | N/A | N/A | 1 | N/A |
| Enterobacter cloacae | 9 | 1, 9, 25 | CTX-M-15, -22, -30, -9, -9-like, -100 | N/A | N/A | 9 | N/A |
| Escherichia coli | 19 | 1, 2, 8, 9, 25 | CTX-M-1, -2, -3, -14, -15, -24, -27, -40, -55, -79, -100, -116 | 16 | 3 | 0 | 84% |
| Klebsiella oxytoca | 5 | 1, 2, 9, 25 | CTX-M-14, -15, -22, -30, -75, -100 | 5 | 0 | 0 | 100% |
| Klebsiella pneumoniae | 14 | 1, 2, 8, 9, 25 | CTX-M-3, -12, -14, -14b, -38, -40, -64, -74, -124, -15, -22, -25 | 13 | 1 | 0 | 93% |
| Klebsiella variicola | 1 | 25 | CTX-M-152 | N/A | N/A | 1 | N/A |
| Kluyvera ascorbata | 1 | 2 | CTX-M-124 | N/A | N/A | 1 | N/A |
| Proteus mirabilis | 6 | 1, 25 | CTX-M-3, -15, -32, -79, -160 | 6 | 0 | 0 | 100% |
| Serratia marcescens | 1 | 1 | CTX-M-15 | N/A | N/A | 1 | N/A |

¹Indeterminate results refer to cases where an increase of ≥ 5 mm in the zone diameter for either antimicrobial agent tested in combination with clavulanate, compared to the zone diameter of the agent tested alone (CTX vs. CTX-CLA and CAZ vs. CAZ-CLA), was not observed.
²Per the CLSI M100, the Enterobacterales eligible for the CLSI ESBL Disk Test are limited to K. pneumoniae, K. oxytoca, E. coli, and P. mirabilis with the following antimicrobial susceptibility profile: For K. pneumoniae, K. oxytoca, and E. coli: Cefpodoxime ≤17mm; Ceftazidime ≤22mm; Aztreonam ≤27mm; Cefotaxime ≤27mm; Ceftriaxone ≤25mm. For P. mirabilis: Cefpodoxime ≤22mm; Ceftazidime ≤22mm; Cefotaxime ≤27mm

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The analytical reactivity of the NG-Test CTX-M MULTI for detection of CTX-M enzymes in Enterobacterales was determined to be acceptable.

7. Assay Cut-Off:

Not applicable

B Comparison Studies:

1. Method Comparison with Predicate Device:

Not applicable

2. Matrix Comparison:

Not applicable

C Clinical Studies:

1. Clinical Sensitivity:

The performance of the NG-Test CTX-M MULTI was evaluated in a clinical study that was conducted at three U.S. sites using a total of 309 Enterobacterales isolates, including 226 prospectively collected isolates (recovered fresh or less than 6 months prior to testing) and 83 retrospectively collected isolates (stock, recovered more than 6 months prior to testing). The NG-Test CTX-M MULTI performance was evaluated using bacterial colonies from these isolates grown on blood and MacConkey agar.

To be enrolled in the study and included in the analysis of performance, retrospective isolates must have been (i) identified to the species level and (ii) determined to be non-susceptible to one or more 3rd generation cephalosporins by an FDA-cleared methodology, or contain a previously determined mechanism of resistance (CTX-M, TEM, SHV, AmpC, etc.). The isolates were from various geographical origins, including from both the United States and outside the United States. All prospective isolates of Enterobacterales were enrolled regardless of susceptibility to 3rd generation cephalosporins; a minimum of 50 prospective isolates were enrolled per site.

The NG-Test CTX-M MULTI results for each isolate were interpreted using the combined reference algorithm as depicted in Table 8. The combined reference test comprised an FDA-cleared PCR test for detecting CTX-M-encoding genes and AST determination of resistance to at least one of the tested antimicrobial agents. Ceftriaxone disks were routinely used to maintain selective pressure for isolated colonies of retrospective Enterobacterales isolates.

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Table 8: Composite Reference Method Algorithm for Interpretation of NG-Test CTX-M MULTI Results

| PCR Result | AST1 | Combined Reference (PCR & AST) Result | NG-Test CTX-M MULTI Result | NG-Test CTX-M MULTI Interpretation |
| --- | --- | --- | --- | --- |
| + | NS | Positive | + | True Positive |
| + | S | Negative | + | False Positive |
| - | NS | Negative | + | False Positive |
| - | S | Negative | + | False Positive |
| + | NS | Positive | - | False Negative |
| + | S | Negative | - | True Negative – Lack of gene expression |
| - | NS | Negative | - | True Negative – Other mechanism of resistance is present |
| - | S | Negative | - | True Negative |

${}^{1}\mathrm{{NS}} =$ Not susceptible (intermediate or resistant results to one or more antimicrobial agents tested per CLSI M100 Ed 34 breakpoints); $\mathrm{S} =$ Susceptible to all antimicrobial agents tested per CLSI M100 Ed 34 breakpoints.

A total of 311 Enterobacterales isolates were initially enrolled. One organism was excluded because it was unable to be recovered for testing by the clinical site. Additionally, one organism that was initially enrolled was excluded from the analysis because it was identified as $E$ faecalis and not an Enterobacterales upon subculture. Thus, a total of 309 isolates were included in the analysis from blood and MacConkey agar. A total of 308 isolates showed concordant results between the NG-Test CTX-M MULTI and comparator PCR testing combined with susceptibility results (Composite Reference Method). One false-positive result was observed. The analysis for NG-Test CTX-M MULTI is summarized in Table 9.

Table 9: Comparison of NG-Test CTX-M MULTI (Blood and MacConkey Agar) to Composite Reference Method

| | Composite Reference Method | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| NG-Test CTX-M | Positive | 151 | 11 | 152 |
| | Negative | 0 | 157 | 157 |
| | Total | 151 | 158 | 309 |
| Positive Percent Agreement (PPA) | | 151/151 = 100% (95% CI: 97.5-100%) | | |
| Negative Percent Agreement (NPA) | | 157/158 = 99.4% (95% CI: 96.5-99.9%) | | |

1 One isolate of Citrobacter amalonaticus was positive on NG-Test CTX-M MULTI, not susceptible to at least one antimicrobial tested, and negative for blaCTX-M by PCR.

The CTX-M variants detected during the NG-Test CTX-M MULTI clinical study are outlined in Table 10.

Table 10: Overall CTX-M Variants Detected by NG-Test CTX-M MULTI in Clinical Study (Blood Agar and MacConkey Agar)

| CTX-M Group | Overall Variants Detected in US Clinical Trial |
| --- | --- |
| 1 | CTX-M-1, -3, 15, -55, -178, -232, -V236A1 |
| 2 | CTX-M-2 |
| 8 | CTX-M-8 |
| 9 | CTX-M-14, -24, -27, -65 |

1 The CTX-M variant in this isolate features a V263A substitution (valine to alanine at position 263).

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The performance of NG-Test CTX-M MULTI was further evaluated at a single internal site using bacterial growth on HardyCHROM ESBL in compliance with the intended use of this medium. A subset of 193 isolates were selected based on the intended use of the medium. The isolates comprised of recent isolates from the prospective and retrospective isolates collected during the CTX-M MULTI clinical evaluation that were available for testing and fall under HardyCHROM ESBL organism claims (i.e., ceftazidime and cefpodoxime non-susceptible Enterobacterales).

Isolates were seeded in both raw/unpreserved stool and stool preserved in C&S Cary Blair transport medium and plated on HardyCHROM ESBL agar. Colonies were then tested with the NG-Test CTX-M MULTI. Among the tested Enterobacterales, 192 showed concordant results between the NG-Test CTX-M MULTI and the composite reference method. One false-positive result was identified, corresponding to the same discrepant isolate from the testing with blood agar and MacConkey agar. The analysis for NG-Test CTX-M MULTI is summarized in Table 11.

Table 11: Seeded Study. Comparison of NG-Test CTX-M MULTI (HC ESBL Agar – Raw and C&S Stool) to Composite Reference Method

| | Composite Reference Method | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| NG-Test CTX-M | Positive | 150 | 1^{1} | 151 |
| | Negative | 0 | 42 | 42 |
| | Total | 150 | 43 | 193 |
| Positive Percent Agreement (PPA) | | 150/150 = 100% (95% CI: 97.5-100%) | | |
| Negative Percent Agreement (NPA) | | 42/43= 97.7% (95% CI: 87.9^{2}-99.9%) | | |

1 One isolate of Citrobacter amalonaticus was positive on NG-Test CTX-M MULTI, not susceptible to at least one antimicrobial tested, and negative for blaCTX-M by PCR.
2 Lower bound is below 90% due to lower quantity of CTX-M negative clinical isolates that meet HardyCHROM ESBL agar claims. Additional CTX-M negative isolates were evaluated in the cross-reactivity study.

Table 12 shows the false-positive result of the NG-Test CTX-M MULTI compared to the reference method PCR combined with AST from all agar media evaluated. The clinical isolate of Citrobacter amalonaticus was resistant to cefotaxime, ceftriaxone, cefpodoxime, and aztreonam, according to the breakpoints described in the CLSI M100, 34th edition. The isolate was re-tested with NG-Test CTX-M MULTI and yielded positive results. A PCR re-test confirmed the negative PCR result.

Table 12: False-Positive Result of NG-Test CTX-M MULTI (Blood, MacConkey, and HC ESBL Agar) vs. Reference Method

| Organism ID | NG-Test CTX-M MULTI | PCR | AST (mm)^{1} | | | | | | Combined Reference (PCR & AST) Result | Interpretation |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | CTX | CAZ | CRO | CPD | ATM | FOX | | |
| Citrobacter amalonaticus | Positive | Negative | 21 | 27 | 16 | 15 | 13 | 26 | Negative | False Positive |

1 CTX= cefotaxime; CAZ= ceftazidime; CRO= ceftriaxone; CPD= cefpodoxime; ATM= aztreonam; FOX= cefoxitin

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The results were determined to be acceptable.

To address the indications for use, the following statements are included in the Limitations section of the device labeling:

The performance of NG-Test CTX-M MULTI was established with colonies from blood agar, MacConkey agar, and HardyCHROM ESBL agar. Performance with other culture media has not been evaluated and is therefore unknown.

The performance of NG-Test CTX-M MULTI with bacteria other than Enterobacterales has not been evaluated.

Organism identification and elevated third-generation cephalosporin MICs should be determined prior to testing with NG-Test CTX-M MULTI.

2. Clinical Specificity:
Refer to Section VII C(1) above.

3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):
Not applicable

D Clinical Cut-Off:
Not applicable

E Expected Values/Reference Range:
Not applicable

VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.

IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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