CHROMID CARBA agar (CARB)

{0} 1 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE A. 510(k) Number: K181092 B. Purpose for Submission: To obtain a substantial equivalence determination for the CHROMID CARBA Agar C. Measurand: Colonies of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae D. Type of Test: Selective and differential culture medium E. Applicant: bioMérieux SA F. Proprietary and Established Names: CHROMID CARBA agar (CARB) G. Regulatory Information: 1. Regulation section: 21 CFR 866.1700: Culture medium for antimicrobial susceptibility tests 2. Classification: Class II 3. Product code: JSO: Culture Media, Antimicrobial Susceptibility Test, Excluding Mueller Hinton Agar 4. Panel: 83: Microbiology {1} H. Intended Use: 1. Intended use(s): CHROMID CARBA agar is a selective and differential chromogenic medium that is intended for the qualitative detection and presumptive identification of carbapenemase-producing *Escherichia coli* and *Klebsiella pneumoniae* in rectal swab specimens from patients at risk of colonization. CHROMID CARBA agar is intended as an aid in the detection, identification of colonization and control of these bacteria in a healthcare setting. Rectal swabs are inoculated directly onto CHROMID CARBA agar without enrichment and results can be interpreted after incubation for 18-24 hours. Presumptive carbapenemase-producing colonies of *E. coli* appear pink to burgundy and those of *K. pneumoniae* appear blue-green or blue-grey. Other organisms besides carbapenemase-producing *E. coli* and *K. pneumoniae* can also grow on CHROMID CARBA agar with colonies that appear pink to burgundy or blue-green to blue-grey. Sub-culture to non-selective medium is required to confirm organism identity, for antimicrobial susceptibility testing, confirmation of carbapenemase production and epidemiological typing. A lack of growth or the absence of pink to burgundy or blue-green to blue-grey colonies does not preclude the carriage of carbapenemase producing organisms. 2. Indication(s) for use: Same as Intended Use. 3. Special conditions for use statement(s): For prescription use only. A lack of growth or the absence of pink-burgundy or blue-green/blue-grey colonies does not preclude the carriage of carbapenemase producing organisms. Based on analytical studies, high concentrations (≥10⁸ CFU/mL) of carbapenemase-producing *E. cloacae*, *M. morganii* and *P. aeruginosa* may inhibit or mask the growth of low levels of carbapenemase-producing *E. coli*. In addition, high concentrations (≥10⁸ CFU/mL) of carbapenemase-producing *E. coli* and *M. morganii* may inhibit or mask the growth of low levels of carbapenemase-producing *K. pneumoniae*. Additional testing by an alternative method (e.g., enrichment broth culture) is required if a negative result is obtained with CHROMID CARBA agar and carriage of carbapenemase-producing *E. coli* or *K. pneumoniae* is suspected. Performance of CHROMID CARBA agar was established for carbapenemase-producing *E. coli* and *K. pneumoniae* that carry *K. pneumoniae* Carbapenemase (KPC). 2 {2} Performance with Carbapenemase-producing Enterobacteriaceae (CPE) other than E. coli and K. pneumoniae and other types of carbapenemase enzyme has not been established. Organisms producing the following non-KPC carbapenemase enzymes may grow and produce pink to burgundy or blue-green to blue-grey pigmented colonies after 18 to 24 hours on CHROMID CARBA agar: IMP, NDM, OXA-48, SME and VIM. Some carbapenem non-susceptible organisms that do not produce carbapenemase but which exhibit other resistance mechanisms may grow on CHROMID CARBA agar and produce blue-green to blue-grey or pink to burgundy pigmented colonies after 18 to 24 hours. Sub-culture to non-selective medium is required to for organism identification, antimicrobial susceptibility testing and confirmation of carbapenemase production. 4. Special instrument requirements: None I. Device Description: CHROMID CARBA agar is a selective and differential culture medium that contains a combination of antimicrobial agents and chromogenic substrates. The medium is intended for use in screening of rectal swab specimens for the presence of carbapenemase-producing E. coli and K. pneumoniae. Presumptive carbapenemase-producing colonies of E. coli appear pink-burgundy whereas those of K. pneumoniae appear blue-green/blue-grey. All isolates must be sub-cultured for identification, antimicrobial susceptibility testing and confirmation of carbapenemase production, as well as epidemiological typing (if required). J. Substantial Equivalence Information: 1. Predicate device name(s): bioMérieux CHROMID VRE agar 2. Predicate 510(k) number(s): K091025 {3} 3. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | | CHROMID CARBA agar (K181092) | CHROMID VRE agar (K091025) | | Intended Use | CHROMID CARBA agar is a selective and differential chromogenic medium that is intended for the qualitative detection and presumptive identification of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in rectal swab specimens from patients at risk of colonization. CHROMID CARBA agar is intended as an aid in the detection, identification of colonization and control of these bacteria in a healthcare setting. Rectal swabs are inoculated directly onto CHROMID CARBA agar without enrichment and results can be interpreted after incubation for 18-24 hours. Presumptive carbapenemase-producing colonies of E. coli appear pink to burgundy and those of K. pneumoniae appear blue-green or blue-grey. Other organisms besides carbapenemase-producing E. coli and K. pneumoniae can also grow on CHROMID CARBA agar with colonies that appear pink to burgundy or blue-green to blue-grey. Sub- | CHROMID VRE agar is a selective and differential chromogenic medium containing 8 μg/mL vancomycin, for the qualitative detection of Enterococcus faecium and E. faecalis showing acquired vancomycin resistance (VRE) in stool specimens. CHROMID VRE agar can be used as an aid to identify, prevent and control VRE colonization in healthcare settings. CHROMID VRE agar is not intended to diagnose VRE infection nor to guide or monitor treatment for infections. Subculture to non-selective media (e.g. trypticase soy agar with 5% sheep blood) is needed for further identification, susceptibility testing and epidemiological typing. | {4} 5 | Differences | | | | --- | --- | --- | | Item | Device | Predicate | | Item | CHROMID CARBA agar (K181092) | CHROMID VRE agar (K091025) | | Specimen Type | Rectal swab | Stool | | Target Species, Resistance Mechanisms and Associated Colony Colors | Carbapenemase-producing E. coli (pink-burgundy) and K. pneumoniae (blue-green/blue-grey) | Vancomycin-resistant E. faecium (violet) and E. faecalis (blue-green) | | Incubation Time | 18-24 hours | 24-48 hours | K. Standard/Guidance Document Referenced (if applicable): Guidance for Industry and FDA: Class II Special Controls Guidance Document: Antimicrobial Susceptibility Test (AST) Systems; August 28, 2009. {5} CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 27th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2017. L. Test Principle: CHROMID CARBA agar consists of nutritive medium containing peptones and a mixture of antimicrobial agents that select for growth of carbapenemase-producing Enterobacteriaceae (CPE), and chromogenic substrates that enable presumptive identification of the most common carbapenemase-producing species. Colonies of carbapenemase-producing E. coli appear pink-burgundy due to the production of β-glucuronidase or β-galactosidase, while colonies of K. pneumoniae produce β-glucosidase and appear blue-green/blue-grey. The performance of CHROMID CARBA agar was established with strains of E. coli and K. pneumoniae that produce K. pneumoniae Carbapenemase (KPC). Performance with other species or other types of carbapenemase has not been evaluated. Testing is performed by direct inoculation of a rectal swab onto the surface of culture medium. The plates are incubated for 18-24 hours at 35±2°C in an aerobic atmosphere. All presumptive isolates of carbapenemase-producing E. coli and K. pneumoniae must be subcultured for identification, antimicrobial susceptibility testing and confirmation of carbapenemase production according to established methods. M. Performance Characteristics (if/when applicable): 1. Analytical performance: a. Precision/Reproducibility: The reproducibility of results obtained with CHROMID CARBA agar was evaluated in a study conducted at three sites using blinded panels of samples comprised of 10 confirmed KPC-positive, carbapenem non-susceptible strains of E. coli (4) and K. pneumoniae (6). The samples were prepared in saline at approximately the Limit of Detection (LoD) (Section M(1)(d)) and tested in triplicate on each of five days. Testing of bacterial suspensions in saline was deemed acceptable because the LoD was shown to be similar with organisms suspended in saline and rectal swab matrix (Section M(2)(b)). On each day at each site, one operator prepared three independent suspensions of each strain and used these to inoculate duplicate CHROMID CARBA agar plates (50μL per plate) that were then incubated for 24 hours and read independently by two different operators (3 sites x 5 days x 3 suspensions x 1 plate x 2 operators = 90 results per strain). A summary of the results of the study is presented in Table 1. The day-to-day, site-to-site, operator-to-operator and overall reproducibility of the CHROMID CARBA agar was determined to be acceptable. 6 {6} Table 1. Results from the CHROMID CARBA agar Reproducibility Study, stratified by site | # | Species | Resistance Marker(s) | Site 1 | Site 2 | Site 3 | Total | | --- | --- | --- | --- | --- | --- | --- | | 1 | K. pneumoniae | KPC | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | | 2 | E. coli | KPC-3, TEM-1 | 100% (30/30) | 80%1 (24/30) | 100% (30/30) | 93.3% (84/90) | | 3 | K. pneumoniae | KPC | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | | 4 | E. coli | KPC-3 | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | | 5 | K. pneumoniae | KPC-2 | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | | 6 | K. pneumoniae | KPC-2 | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | | 7 | K. pneumoniae | KPC-3 | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | | 8 | E. coli | KPC | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | | 9 | E. coli | KPC | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) | | 10 | K. pneumoniae | KPC-2 | 100% (30/30)) | 100% (30/30) | 100% (30/30) | 100% (90/90) | | Overall | | | 100% (300/300) | 98.0% (294/300) | 100% (300/300) | 99.3% (894/900) | 1 Both operators at Site 2 on Day 4 of testing observed no growth with Sample #2 b. Linearity/assay reportable range: Not applicable. c. Traceability, Stability, Expected values (controls, calibrators, or methods): Quality Control Quality Control (QC) testing must be performed in accordance with applicable local, state and/or federal regulations or accreditation requirements and the testing laboratory's internal procedures. Recommended strains for use as Positive and Negative External Controls are included in the device labeling. QC was performed on each day of testing during the Clinical Studies (Prospective, Contrived and Challenge) described in Section M(3)(a) by preparing a 1:100 dilution of a $0.5\mathrm{McFarland}$ suspension of each recommended QC strain, streaking for isolation using a $10\mu \mathrm{L}$ loop and incubating for 18-24 hours at $35\pm 2^{\circ}\mathrm{C}$ . A summary of the results from QC testing at the four external sites involved in the Clinical Studies is shown in Table 2. {7} Table 2. Summary of Quality Control test results using CHROMID CARBA agar | Species | Strain | Carbapenemase | Expected Result | Agreement (%) | | --- | --- | --- | --- | --- | | K. pneumoniae | ATCC BAA-1705 | blaKPC Positive | Blue-green/blue-grey colonies | 165/165 (100) | | E. coli | ATCC BAA-2340 | blaKPC Positive | Pink-burgundy colonies | 165/165 (100) | | K. pneumoniae | ATCC 700603 | Negative | No growth | 164/165 (99.4) | Reagent Stability CHROMID CARBA agar should be stored at $2 - 8^{\circ}\mathrm{C}$ until the expiration date shown on the outer packaging. Specimen Stability Rectal swab specimens should be collected using the Copan Liquid Amies Elution (Eswab) Collection and Transport System (K061301), or equivalent, and may be stored for up to 24 hours at $2 - 8^{\circ}\mathrm{C}$ prior to culture on CHROMID CARBA agar. Performance with other specimen collection and transport devices has not been demonstrated. d. Detection limit: Limit of Detection The analytical sensitivity of CHROMID CARBA agar was determined for one strain each of KPC-producing $E.$ coli (ATCC BAA-2340) and $K.$ pneumoniae (ATCC BAA-1705). To estimate the Limit of Detection (LoD), a standardized 0.5 McFarland suspension of each strain was prepared and diluted in simulated rectal swab matrix (stool in saline solution) prior to plating on CHROMID CARBA agar (50μL per plate). Trypticase Soy Agar (TSA) was also inoculated in parallel as a growth control. All the plates were incubated for 18-24 hours at $35 \pm 2^{\circ} \mathrm{C}$ and the LoD was estimated as the lowest concentration at which $\geq 10$ CFU exhibiting the expected colony color were obtained on each CHROMID CARBA agar plate. The estimated LoD for each strain was $1.5 \times 10^{3}$ CFU/mL and this value was confirmed by observation of appropriate colony counts on three additional plates that were inoculated at the LoD target level. Analytical Reactivity/Inclusivity A study was performed using a panel of well-characterized isolates to evaluate the ability of CHROMID CARBA agar to recover carbapenemase-producing strains of $E.$ coli and $K.$ pneumoniae. Strains were characterized as carbapenemase producing based the presence of $bla_{\mathrm{KPC}}$ and non-susceptibility to at least one carbapenem (ertapenem, imipenem or meropenem). Included in the panel were strains with known $bla_{\mathrm{KPC}}$ variants: KPC-2 (7), KPC-3 (3), KPC-4 (1). Of 11 strains of $bla_{\mathrm{KPC}}$ positive $E.$ coli that were included in the study, nine (9) grew {8} on CHROMID CARBA agar with the expected pink-burgundy colony color and all were carbapenem non-susceptible. Of the two strains that failed to grow, one (KPC-4) had an MIC in the susceptible range for ertapenem and was intermediate for imipenem and meropenem, and the other was susceptible to all three carbapenems and its failure to grow on CHROMID CARBA agar was therefore expected. All (100%) of 41 blaKPC-positive, carbapenem non-susceptible strains of K. pneumoniae grew on CHROMID CARBA agar with the expected blue-green/blue-grey colony color. These results are acceptable. The absence of data to demonstrate reliable detection of carbapenemase-producing strains of E. coli and K. pneumoniae that carry other types of carbapenemase enzyme besides KPC is noted in the Intended Use and as a Limitation in the device labeling. ## Incubation Time A study was performed to evaluate the effect of incubation time on the ability to recover KPC carbapenemase-producing strains of E. coli and K. pneumoniae on CHROMID CARBA agar. Ten blaKPC-positive, carbapenemase non-susceptible strains were included in the study (four (4) E. coli and six (6) K. pneumoniae). Each strain was diluted in saline to 4.5 x 10³ CFU/mL (3X LoD) and inoculated onto CHROMID CARBA agar (50μL per plate). The plates were incubated aerobically at 35±2°C. Colonies were counted and their color noted after 16 hours and every two (2) hours thereafter up to 28 hours. Colony counts remained stable over the course of the study and each of the strains exhibited their characteristic colony color. The results of this study support the ability to recover carbapenemase-producing E. coli and K. pneumoniae on CHROMID CARBA agar after incubation for 18-24 hours as stipulated in the device labeling. ## e. Analytical specificity: A study was performed to evaluate the analytical specificity of CHROMID CARBA agar for carbapenemase-producing strains of E. coli and K. pneumoniae by inoculating the medium with high concentrations of target and non-target organisms that may be present in stool specimens. Included in the study were CPE other than E. coli and K. pneumoniae as well as strains of these two species that produce other carbapenemases besides KPC type enzyme. Fifty microliters of a 0.5 McFarland suspension of each non-target organism (approximately 1-2 x 10⁸ CFU/mL for bacteria and 1-5 x 10⁶ CFU/mL for yeast) was used to inoculate each plate of CHROMID CARBA agar. The plates were incubated for 18-24 hours at 35±2°C in aerobic conditions after which the presence/absence of growth was recorded together with the characteristics of any colonies present. A list of the organisms tested and the associated results is shown in Table 3. 9 {9} Table 3. Organisms used to evaluate the analytical specificity of CHROMID CARBA agar | Species1 | CHROMID CARBA | | Known Resistance Marker | Carbapenem Susceptibility2 | | --- | --- | --- | --- | --- | | | Growth | Colony Color | | | | Acinetobacter baumannii | Yes | Colorless | NDM | | | Aeromonas hydrophila | No | | | | | Bacillus cereus | No | | | | | Bacillus subtilis | No | | | | | Candida albicans | No | | | | | Candida glabrata | No | | | | | Candida krusei | No | | | | | Candida tropicalis | No | | | | | Citrobacter freundii (3) | Yes | Blue-grey | KPC | Non-susceptible | | Corynebacterium diphtheriae | No | | | | | Corynebacterium jeikium | No | | | | | Klebsiella (Enterobacter) aerogenes | No | | CASE | | | Klebsiella (Enterobacter) aerogenes | Yes | Blue-green | KPC | Non-susceptible | | Enterobacter cloacae | Yes | Blue-green | KPC | Non-susceptible | | Enterococcus durans | No | | | | | Enterococcus faecalis (2) | No | | | | | Enterococcus faecium (2) | No | | | | | Escherichia coli | No | | | | | Escherichia coli | Yes | Pink-burgundy | NDM | Non-susceptible | | Escherichia coli | Yes | Pink-burgundy | VIM | Non-susceptible | | Escherichia coli | Yes | Pink-burgundy | IMP | Non-susceptible | | Escherichia coli | No | | ESBL | | | Hafnia alvei | No | | | | | Klebsiella pneumoniae | No | | | | | Klebsiella pneumoniae (2) | No | | ESBL | | | Klebsiella pneumoniae | Yes | Blue-green | NDM | Non-susceptible | | Klebsiella pneumoniae | Yes | Blue-green | VIM | Non-susceptible | | Klebsiella pneumoniae | Yes | Blue-green | IMP | Non-susceptible | | Klebsiella pneumoniae | Yes | Blue-green | OXA-48 | Non-susceptible | | Lactobacillus acidophilus | No | | | | | Leuconostoc mesenteroides | No | | | | | Morganella morganii | Yes | Blue-green | NDM | Susceptible | | Morganella morganii | Yes | Blue-green | NDM | Non-susceptible | | Proteus mirabilis | No | | | | | Proteus mirabilis | No | | CASE | | | Proteus vulgaris | No | | | | | Providencia rettgerii | Yes | Blue-green | KPC | Non-susceptible | | Pseudomonas aeruginosa | No | | | | | Pseudomonas aeruginosa | Yes | Colorless | VIM | Non-susceptible | {10} | Species1 | CHROMID CARBA | | Known Resistance Marker | Carbapenem Susceptibility2 | | --- | --- | --- | --- | --- | | | Growth | Colony Color | | | | Pseudomonas putida | Yes | Colorless | | No breakpoints3 | | Saccharomyces cerevisiae | No | | | | | Salmonella enteritidis | No | | | | | Serratia marcescens | No | | | | | Serratia marcescens | Yes | Blue-green | SME | Non-susceptible | | Shigella sonnei | No | | | | | Staphylococcus aureus | No | | | | | Staphylococcus aureus | No | | mecA | | | Staphylococcus epidermidis (2) | No | | | | | Staphylococcus haemolyticus | No | | | | | Stenotrophomonas maltophilia | Yes | Colorless | VIM | No breakpoints2 | | Streptococcus agalactiae | No | | | | | Streptococcus group C | No | | | | | Streptococcus group O | No | | | | Shaded cells: Not applicable CASE: Cephalosporinase; ESBL: Extended Spectrum $\beta$ -Lactamase; IMP: Imipenem hydrolyzing $\beta$ -lactamase; KPC: Klebsiella pneumoniae Carbapenemase; NDM: New Delhi Metallo- $\beta$ -lactamase; SME: Serratia marcescens enzyme; VIM: Verona Integron-encoded Metallo- $\beta$ -lactamase 1 The number of strains is noted in parenthesis if $>1$ 2 Carbapenem susceptibility using ertapenem, imipenem and meropenem was only determined for those organisms that grew on CHROMID CARBA agar 3 No recognized breakpoints exist for carbapenems with $P$ putida or S. maltophilia (intrinsically resistant) Of the 59 organisms tested, 20 grew of CHROMID CARBA agar after 18-24 hours. Of these, four (4) non-lactose fermenting species (A. baumannii, P. aeruginosa, P. putida and S. maltophilia) exhibited colorless colonies that could readily be distinguished from the characteristic pink-burgundy and blue-green/blue-grey colonies of carbapenemase-producing E. coli and K. pneumoniae. Three (3) of these strains were known to harbor a carbapenem resistance marker ( $bla_{\mathrm{VIM}}$ or $bla_{\mathrm{NDM}}$ ). The remaining 16 organisms that grew on CHROMID CARBA agar included three (3) strains of E. coli (pink-burgundy colonies) and four (4) strains of K. pneumoniae (blue-green/blue-grey colonies), all of which carried a carbapenemase resistance marker other than $bla_{\mathrm{KPC}}$ ( $bla_{\mathrm{IMP}}$ , $bla_{\mathrm{NDM}}$ , $bla_{\mathrm{OXA-48}}$ or $bla_{\mathrm{VIM}}$ ) and were phenotypically carbapenem non-susceptible. In addition, there were nine (9) other strains of Enterobacteriaceae (C. freundii (3), M. morganii (2), K. aerogenes [E. aerogenes] (1), E. cloacae (1), P. rettgerii (1) and S. marcescens (1)) that exhibited blue-green/blue-grey colonies and which carried a carbapenemase gene ( $bla_{\mathrm{KPC}}$ , $bla_{\mathrm{NDM}}$ or $bla_{\mathrm{SME}}$ ), eight (8) of which were non-susceptible to carbapenems. The results of the Analytical Specificity Study demonstrate that $E.$ coli and $K.$ pneumoniae with carbapenemases other than KPC-type carbapenemase may exhibit pink-burgundy or blue-green/blue-grey colonies on CHROMID CARBA agar, although performance with such isolates has not been established. Similarly, CPE other than $E.$ coli and $K.$ pneumoniae may also grow on CHROMID CARBA agar {11} with pink-burgundy or blue-green/blue-grey colonies, although insufficient isolates were tested to demonstrate performance. These Limitations are noted in the device labeling. f. Assay cut-off: Not applicable. g. Assay interference: ## Microbial Interference A study was conducted to evaluate the ability to recover the targeted carbapenemase-producing *E. coli* and *K. pneumoniae* in the presence of other carbapenem susceptible and non-susceptible organisms. Testing was performed by plating four (4) representative KPC-producing target organisms at low levels (3X LoD) in the presence of $10^{4}-10^{8}$ CFU/mL of seven (7) carbapenem non-susceptible competitor organisms that exhibited different resistance mechanisms. In addition, one (1) carbapenem susceptible strain of *K. pneumoniae* was also included as a competitor and each competitor strain was also inoculated onto CHROMID CARBA agar in the absence of the target species in order to evaluate its growth characteristics. As observed in the Analytical Specificity Study (Section M(1)(e)), both carbapenemase-producing *Enterobacter cloacae* and *Providencia rettgeri* exhibited blue-green colonies on CHROMID CARBA agar, while carbapenemase-producing *M. morganii* and *P. aeruginosa* produced blue and colorless colonies, respectively. In all cases, when the competitive species was present at $<10^{8}$ CFU/mL, colonies of the target organisms grew with the characteristic color (either pink-burgundy for *E. coli* or blue-green/blue-grey for *K. pneumoniae*). However, when the competitive species were present at $10^{8}$ CFU/mL, in some cases, characteristic colonies of the target organisms were not observed, either due to no growth or the inability to discern the characteristic colored colonies of the target species from colonies of the competing organism (i.e., masking) (Table 4). The potential for high concentrations of carbapenemase-producing non-target organisms to interfere with recovery of carbapenemase-producing *E. coli* and *K. pneumoniae* is noted as a Limitation in the device labeling. 12 {12} Table 4. Summary of results with obtained with mixtures of organisms on CHROMID CARBA agar | Competitor Organism | | | | | Carbapenemase-producing Target Organism1 | | | | --- | --- | --- | --- | --- | --- | --- | --- | | Species | Strain | Carbapenem Phenotype | Resistance Mechanism | CHROMID CARBA agar | Species | Strain2 | CHROMID CARBA agar | | E. cloacae | bMx 1012298 | CPE | KPC | Blue-green | E. coli | bMx 0911025 | No growth/Masked | | K. pneumoniae | bMx 0911175 | Resistant | Impermeability | No Growth | | ATCC BAA-2340 | Pink-burgundy | | | | | | | | bMx 0911025 | Pink-burgundy | | K. pneumoniae | ATCC 13883 | Non-CPE (susceptible) | Not applicable | No Growth | | ATCC BAA-2340 | Pink-burgundy | | M. morganii | bMx 1411132 | CPE | NDM | Blue | | bMx 0911025 | No growth/Masked | | P. aeruginosa | bMx 1008028 | CPE | VIM | Colorless | | ATCC BAA-2340 | No growth/Masked | | | | | | | | bMx 0911025 | Pink-burgundy | | P. mirabilis | ATCC BAA-856 | Non-CPE (susceptible) | ESBL | No Growth | ATCC BAA-2340 | Pink-burgundy | | | E. coli | bMx 1011190 | CPE | NDM | Pink-burgundy | K. pneumoniae | ATCC BAA-1705 | No growth/Masked | | | | | | | | bMx 0505034 | No growth/Masked | | M. morganii | bMx 1411132 | CPE | NDM | Blue | | bMx 0505034 | No growth/Masked | | P. aeruginosa | bMx 1008028 | CPE | VIM | Colorless | | ATCC BAA-1705 | Blue-green/Blue-grey | | | | | | | | bMx 0505034 | Blue-green/Blue-grey | | P. mirabilis | ATCC BAA-856 | Non-CPE (susceptible) | ESBL | No Growth | | ATCC BAA-1705 | Blue-green/Blue-grey | | P. rettgeri | bMx 1411134 | CPE | KPC | Blue-green | | ATCC BAA-1705 | Blue-green/Blue-grey | CPE: Carbapenemase-producing Enterobacteriaceae; ESBL: Extended Spectrum $\beta$ -Lactamase; KPC: Klebsiella pneumoniae-carbapenemase; NDM: New Delhi Metallo- $\beta$ -lactamase; VIM: Verona Integron-encoded $\beta$ -lactamase; ATCC: American Type Culture collection; bMx: bioMérieux Results shown are in the presence of $10^{8}$ CFU/mL of the Competitor Organism. In all cases when the Competitor Organism was present at $< 10^{8}$ CFU/mL, growth and appearance of colonies of the Carbapenemase-producing Target Organism were as expected. 2 Both strains of each of the two Carbapenemase-producing target species were positive for $bla_{\mathrm{KPC}}$ {13} # Interfering Substances In addition to investigating the potential for microbial interference, a study was also conducted to evaluate the potential for interference with growth on CHROMID CARBA agar from endogenous and exogenous substances that may be present in rectal swab specimens. Testing was performed with each substance in the presence of two (2) representative carbapenemase-producing target organisms: $E.$ coli strain ATCC BAA-2340 and $K.$ pneumoniae ATCC BAA-1705 (both $bla_{\mathrm{KPC}}$ -positive). Each target organism was tested at the LoD target level ( $1.5 \times 10^{3} \mathrm{CFU/mL}$ ) in the presence of 22 potentially interfering substances at the highest concentration likely to be encountered in a clinical specimen. The number of colonies of characteristic appearance obtained on CHROMID CARBA agar was compared to the number obtained on MacConkey agar that was inoculated in parallel. For there to be no evidence of interference, the number of colonies present on the CHROMID CARBA agar after incubation for 18-24 hours had to equal or exceed the number observed on the MacConkey agar. No interference on CHROMID CARBA agar was observed in the presence of any of the substances tested (Table 5). These results are acceptable. Table 5. Substances evaluated for potential interference with growth on CHROMID CARBA agar | Category | Substance | Concentration or Quantity Tested | | --- | --- | --- | | Antidiarrhea Medications | Loperamide hydrochloride (Loperamide) | 1 capsule/mL | | | Diosmectite (Smecta) | 50% v/v1 | | Dietary Supplement | Ispaghula husk (Transilane) | 50% v/v2 | | Diluent | Physiological saline water | 50% v/v | | Endogenous Substance | Human Blood | 50% v/v | | Personal Hygiene Products | Playtex Personal wipes | 50% v/v3 | | | Talc | 4.5% w/w | | | Trojan Enz condoms | 50% v/v3 | | Laxatives | Glycerol (Bebegel) | 50% w/w | | | Bisacodyl (Dulcolax) | 1 tablet/mL | | Lubricants | K-Y Jelly | 0.5% w/w | | | K-Y Liquibeads vaginal moisturizer | 0.5 ovules/mL | | | Vaseline | 50% w/w | | Topical Medications | Zinc Oxide (Oxyplastine) | 50% w/w | | | Pramocaine hydrochloride (Tronothane) | 33% w/w | | | Miconazole nitrate (Daktarin) | 2.4% w/w | | | Witch Hazel (Tucks Pads) | 50% v/v3 | | | Phenylephrine HCI (Preparation H Ointment) | 50% w/w | | | Benzocaine + Resorcinol (Vagisil anti-itch creme) | 33% w/w | | | Ruscoside + Lidocaine hydrochloride + Prednacinolone acetonide (Cirkan Suppositories) | 50% v/v4 | | | Hydrocortisone (CortiSedermyl) | 8.3% w/w | 1 1 sachet in $100\mathrm{mL}$ water mixed 1:1 with bacterial suspension 2 1 sachet in $200\mathrm{mL}$ water mixed 1:1 with bacterial suspension 3 1 pad, wipe or condom in $10\mathrm{mL}$ water mixed 1:1 with bacterial suspension 4 1 suppository in $5\mathrm{mL}$ water mixed 1:1 with bacterial suspension {14} # 2. Comparison studies: a. Method comparison with predicate device: Not applicable. b. Matrix comparison: A study was conducted to compare the analytical sensitivity of CHROMID CARBA agar for the recovery of carbapenemase-producing strains of $E.$ coli and $K.$ pneumoniae from simulated rectal swab matrix (stool diluted in saline) and saline solution alone. Testing was performed by diluting standardized suspensions of $E.$ coli ATCC BAA-2340 and $K.$ pneumoniae ATCC BAA-1705 (both $bla_{\mathrm{KPC}}$ -positive) in the appropriate matrix to different concentrations close to the claimed LoD of CHROMID CARBA agar. Each suspension was used to inoculate multiple CHROMID CARBA agar plates that were incubated for 18-24 hours at $35 \pm 2^{\circ}\mathrm{C}$ , after which colonies exhibiting the characteristic pink-burgundy of $E.$ coli or blue-green/blue-grey of $K.$ pneumoniae were counted. The mean colony count was calculated using data form all inoculated plates. Similar colony counts were obtained with suspensions prepared in saline or simulated rectal swab matrix (Table 6). Use of organisms diluted in saline for selected Analytical Studies was therefore deemed acceptable. Table 6. Comparison of colony counts obtained on CHROMID CARBA agar from culture of bacterial suspensions prepared in simulated rectal swab matrix or saline | Target Species/Strain | Level | | | Mean Colony Count (CFU/plate)1 | | | --- | --- | --- | --- | --- | --- | | | Multiple of LoD | CFU/mL | Number of plates | Rectal Swab Matrix2 | Saline | | E. coliATCC-2340 | 5X | 7.5 x 103 | 10 | >200 | >200 | | | 3X | 4.5 x 103 | 30 | 86 | 94 | | | 1X | 1.5 x 103 | 10 | 31 | 31 | | | Negative | 0 | 10 | 0 | 0 | | K. pneumoniaeATCC-1705 | 5X | 7.5 x 103 | 10 | >300 | >300 | | | 3X | 4.5 x 103 | 30 | >200 | >200 | | | 1X | 1.5 x 103 | 10 | 74 | 71 | | | Negative | 0 | 10 | 0 | 0 | 1 Only colonies exhibiting the characteristic colony color associated with the carbapenemase-producing target species were counted 2 Simulated rectal swab matrix prepared by diluting liquid stool in saline # 3. Clinical studies: a. Clinical Sensitivity: Prospective Clinical Study The performance of CHROMID CARBA agar was evaluated in a prospective Clinical {15} Study that was performed at three sites (two US and one ex-US). Recovery of presumptive carbapenemase-producing colonies of $E.$ coli and $K.$ pneumoniae from rectal swabs on CHROMID CARBA agar was compared to enrichment broth Reference Culture, followed by subculture to MacConkey agar and phenotypic and genetic characterization of isolated lactose fermenting colonies. Isolates of $E.$ coli and $K.$ pneumoniae obtained by the Reference Culture method and on CHROMID CARBA agar were identified biochemically and tested for susceptibility to ertapenem, imipenem and meropenem using an FDA-cleared method. In addition, each isolate was also evaluated for carbapenem susceptibility using an FDA-cleared Carba NP test $^{1}$ and for the presence of carbapenemase resistance markers ( $bla_{\mathrm{IMP}}$ , $bla_{\mathrm{KPC}}$ , $bla_{\mathrm{NDM}}$ , $bla_{\mathrm{OXA-48}}$ and $bla_{\mathrm{VIM}}$ ) using a validated multiplex PCR assay. Isolates were defined as carbapenemase-producing using the composite algorithm depicted in Table 7. Table 7. Composite algorithm for determination of carbapenemase status of isolates obtained by the Reference Culture method or recovered on CHROMID CARBA agar | Carbapenem MIC1 | Carba NP Test | Carbapenemase PCR2 | Carbapenemase Status | | --- | --- | --- | --- | | Non-susceptible | Positive | Positive | Positive | | Non-susceptible | Positive | Negative | Negative | | Non-susceptible | Negative | Positive | Positive | | Non-susceptible | Negative | Negative | Negative | | Susceptible | Positive | Positive | Positive | | Susceptible | Positive | Negative | Negative | | Susceptible | Negative | Positive | \(Negative^3\) | | Susceptible | Negative | Negative | Negative | 1 Non-susceptible: Intermediate or Resistant to one or more of the carbapenem antimicrobial agents tested (ertapenem, imipenem and meropenem) based on FDA recognized breakpoints 2 Multiplex PCR for $bla_{\mathrm{IMP}}$ , $bla_{\mathrm{KPC}}$ , $bla_{\mathrm{NDM}}$ , $bla_{\mathrm{OXA-48}}$ or $bla_{\mathrm{VIM}}$ 3 Phenotypic evidence of non-susceptibility to carbapenems in addition to a positive PCR result was required to establish the Carbapenemase Status as positive A total of 1099 residual rectal swab specimens from unique individuals that were leftover following standard of care testing were initially enrolled in the study. All the specimens were collected using ESwabs (flocked swab in liquid Amies transport medium). Three hundred and ninety (390) specimens were excluded from the analysis of performance for the following reasons: Quality Control failure with either the Reference Culture (318) or both the Reference Culture and the CHROMID CARBA agar (25); missing data (42); delay in data entry resulting in questionable data reliability (5). Isolates from a total of 709 rectal swabs specimens were therefore included in the analysis of performance. The results of the Prospective Clinical Study are summarized in Tables 8 (a) & (b) and 9. Reference Culture yielded no Carbapenemase Status positive strains of $E.$ coli over the course of the study, precluding estimation of Positive Percent Agreement (PPA), {16} although the Negative Percent Agreement (NPA) was 99.7% (95% confidence interval: 99.0-99.9%). For Carbapenemase Status-positive K. pneumoniae, PPA was 84.3% (95% CI 72.0-91.8%) and NPA was 97.7% (95% CI 96.3-98.6%). All the specimens from which Carbapenemase Status-positive K. pneumoniae was recovered by Reference Culture were obtained from the ex-US clinical site (Table 9). Each of the strains of K. pneumoniae that was recovered by Reference Culture but which failed to grow when the rectal swabs were inoculated directly on CHROMID CARBA agar during the prospective study was shown to grow with the characteristic blue-green/blue-grey colony color when tested analytically in stool matrix at the LoD target level (Section M(1)(d)). Failure to recover these strains on CHROMID CARBA agar directly from rectal swabs in the prospective study may have been due to their presence at levels below the LoD of the device. A lack of growth on CHROMID CARBA agar or the absence of pink to burgundy or blue-green to blue-grey colonies does not preclude the carriage of carbapenemase producing organisms. This is noted in the device labeling. 17 {17} Tables 8 (a) & (b). Prospective Clinical Study with rectal swab specimens: CHROMID CARBA agar Colony Color vs Reference Culture (Confirmed Organism Identity and Carbapenemase Status) | Table 8(a): Carbapenemase-producing E. coli | | | | | | --- | --- | --- | --- | --- | | | Reference Culture | | | | | | | Positive | Negative | Total | | CHROMID CARBA agar | Positive1 | 0 | 23 | 2 | | | Negative2 | 0 | 707 | 707 | | | Total | 0 | 709 | 709 | | Positive Percent Agreement | | Not applicable | | | | Negative Percent Agreement | | 99.7% (707/709); 95% CI 99.0-99.9% | | | | Positive Predictive Value | | Not applicable | | | | Negative Predictive Value | | 100% (707/707) | | | | Prevalence | | 0.0% (0/709) | | | 95% CI: 95% score confidence interval 1 Pink-burgundy colonies 2 No growth or colonies not pink-burgundy 3 Colonies from 1/2 specimens confirmed as carbapenem non-susceptible, carbapenemase negative K. pneumoniae; colonies from 1/2 specimens identified as Enterococcus faecalis | Table 8(b): Carbapenemase-producing K. pneumoniae | | | | | | --- | --- | --- | --- | --- | | | Reference Culture | | | | | | | Positive | Negative | Total | | CHROMID CARBA agar | Positive1 | 43 | 155,6 | 58 | | | Negative2 | 83,4 | 643 | 651 | | | Total | 51 | 658 | 709 | | Positive Percent Agreement | | 84.3% (43/51); 95% CI 72.0-91.8% | | | | Negative Percent Agreement | | 97.7% (643/658); 95% CI 96.3-98.6% | | | | Positive Predictive Value | | 74.1% (43/58) | | | | Negative Predictive Value | | 98.8% (643/651) | | | | Prevalence | | 7.2% (51/709) | | | 95% CI: 95% score confidence interval 1 Blue-green/blue-grey colonies 2 No growth or colonies not blue-green/blue-grey 3 Includes 1 specimen from which carbapenemase-positive K. pneumoniae was recovered by the Reference Method but which yielded blue-green/blue-grey colonies on CHROMID CARBA agar that were identified biochemically as P. aeruginosa 4 8/8 isolates grew on CHROMID CARBA agar when inoculated at approximately the LoD target level (10³ CFU/mL in stool); all 8 carried blaKPC, were Carba NP Test positive and carbapenem non-susceptible (resistant to ertapenem, imipenem and meropenem) 5 5/15 isolates on CHROMID CARBA agar were confirmed as carbapenem non-susceptible K. pneumoniae that carried: 4/5 isolates were Carbapenemase Status-positive and carried blaKPC and 1/5 isolates was Carbapenemase Status-negative 6 10/15 specimens produced blue-green/blue-grey colonies that were identified as Enterobacteriaceae other than K. pneumoniae (Serratia marcescens (6), Citrobacter freundii (2) and Enterobacter cloacae (2)) {18} Table 9. Prospective Clinical Study: agreement between CHROMID CARBA agar and Reference Culture, stratified by study site | Target Organism | Study Site | Percent Agreement1 | | | --- | --- | --- | --- | | | | Positive | Negative | | Carbapenemase Status-positive E. coli | 1 | Not applicable | 100%; 98.0-100% (187/187) | | | 2 | Not applicable | 99.5%; 98.1-99.9% (379/381) | | | 32 | Not applicable | 100%; 97.3-100% (141/141) | | | Total | Not applicable | 99.7; 99.0-99.9% (707/709) | | Carbapenemase Status-positive K. pneumoniae | 1 | Not applicable | 99.5%; 97.0-99.9% (186/187) | | | 2 | Not applicable | 97.6%; 95.6-98.8% (372/381) | | | 32 | 84.3%; 72.0-91.8% (43/51) | 94.4%; 87.6-97.6% (85/90) | | | Total | 84.3%; 72.0-91.8% (43/51) | 97.7%; 96.3-98.6% (643/658) | 1 With $95\%$ score confidence interval 2 Ex-US site # Contrived Specimen Study Because of the anticipated low prevalence of Carbapenemase Status-positive specimens in the Prospective Clinical Study, additional testing was also performed with contrived specimens. These were prepared by spiking simulated rectal swab matrix (stool diluted in saline) with isolates of $E$ coli, $K$ pneumoniae and other Enterobacteriaceae of known Carbapenemase Status as defined using the algorithm depicted in Table 7. A summary of the isolates tested is shown in Table 10. Each contrived specimen was spiked with a unique bacterial strain at a concentration equivalent to 1-10X the analytical LoD of the CHROMID CARBA agar (Section M(1)(d)). Testing was performed in a blinded fashion at four different sites. A summary of the results obtained on CHROMID CARBA agar in comparison to the expected organism identity and Carbapenemase Status is provided in Tables 11 (a) & (b). {19} Table 10. Isolates included in the Contrived Specimen Study | Species | Number of Isolates Tested | Carbapenemase Status | | | --- | --- | --- | --- | | | | Positive | Negative | | Citrobacter freundii | 1 | 1 | 0 | | Enterobacter cloacae | 4 | 4 | 0 | | Escherichia coli | 38 | 201 | 18 | | Klebsiella oxytoca | 1 | 1 | 0 | | Klebsiella ozaenae | 1 | 1 | 0 | | Klebsiella pneumoniae | 160 | 882 | 72 | | Kluyvera ascorbata | 1 | 1 | 0 | | Morganella morganii | 1 | 1 | 0 | | Proteus mirabilis | 2 | 2 | 0 | | Raoultella ornithinolytica | 1 | 1 | 0 | | Total | 210 | 1203 | 90 | ${}^{1}$ blaKPC: 11 (KPC-2: 9, KPC-3: 2); blaNDM: 4; blaOXA-48: 2; blaVIM: 2; blaIMP: 1 ${}^{2}$ blaKPC: 77 (45 KPC-3, 30 KPC-2, 2 other variants); blaVIM: 4; blaNDM: 3; blaIMP: 2; blaOXA-48: 2 3 Overall, 110/120 isolates (92%) were positive for $bla_{\mathrm{KPC}}$ , including 12/12 isolates (100%) of species other than $E.$ coli and $K.$ pneumoniae {20} Tables 11(a) & (b). Contrived Specimen Study: CHROMID CARBA agar Colony Color vs Expected Organism Identity and Carbapenemase Status | Table 11(a): Carbapenemase-producing E. coli | | | | | | --- | --- | --- | --- | --- | | | Organism Identity & Carbapenemase Status | | | | | | | Positive | Negative | Total | | CHROMID CARBA agar | Positive1 | 16 | 23 | 18 | | | Negative2 | 44 | 188 | 192 | | | Total | 20 | 190 | 210 | | Positive Percent Agreement | | 80.0% (16/20); 58.4-91.9% | | | | Negative Percent Agreement | | 98.9% (188/190); 95% CI 96.2-99.7% | | | 95% CI: 95% score confidence interval 1 Pink-burgundy colonies 2 No growth or colonies not pink-burgundy 3 2/2 isolates were carbapenem non-susceptible E. coli (resistant to meropenem and ertapenem and an intermediate MIC for imipenem); both isolates were positive for Extended Spectrum $\beta$ -Lactamase (ESBL) 4 False negative results were obtained with 4 strains of carbapenem non-susceptible E. coli that harbored $bla_{\mathrm{OXA - 48}}$ (2), $bla_{\mathrm{NDM}}$ (1) and $bla_{\mathrm{VIM}}$ (1) and which were spiked at target levels ranging from 1X to 10X LoD; 2/4 strains had an intermediate MIC to at least one carbapenem including 1 strain that was also susceptible to ertapenem | Table 11(b): Carbapenemase-producing K. pneumoniae | | | | | | --- | --- | --- | --- | --- | | | Organism Identity & Carbapenemase Status | | | | | | | Positive | Negative | Total | | CHROMID CARBA agar | Positive1 | 84 | 113,4 | 95 | | | Negative2 | 35 | 1126 | 115 | | | Total | 87 | 123 | 210 | | Positive Percent Agreement | | 96.6% (84/87); 95% CI % 90.3-98.8% | | | | Negative Percent Agreement | | 91.1% (112/123); 95% CI 84.7-94.9% | | | 95% CI: 95% score confidence interval 1 Blue-green/blue-grey colonies 2 No growth or colonies not blue-green/blue-grey 3 5/11 false positive results were obtained with carbapenem non-susceptible $K$ pneumoniae, all of which were positive for ESBL (3) or ESBL and AmpC (2) 4 6/11 false positive results were due to Carbapenemase Status-positive species of Enterobacteriaceae other than $K$ pneumoniae that harbored $bla_{\mathrm{KPC}}$ : E. cloacae (4), K. oxytoca (1) and K. ozaenae (1) 5 3/3 false negative results were obtained with Carbapenemase Status positive isolates at the LoD target level; each strain carried a different carbapenemase resistance marker ( $bla_{\mathrm{IMP}}$ (1), $bla_{\mathrm{KPC}}$ (1) or $bla_{\mathrm{OXA-48}}$ (1)); 2/3 strains had a susceptible or intermediate MIC to at least one of the three carbapenems tested (ertapenem, imipenem or meropenem) 6 18/112 samples contained carbapenem non-susceptible strains of $K$ pneumoniae of which 17 were phenotypically carbapenemase negative; 11/18 were positive for ESBL and 6/18 were positive for AmpC # Challenge Study To supplement the results of the Prospective Clinical Study and Contrived Specimen Study, additional testing was performed using well characterized Carbapenemase Status-positive and -negative isolates of $E.$ coli and $K.$ pneumoniae that were suspended in saline at $3 \times 10^{3}$ CFU/mL (equivalent to 2X LoD [Section M(1)(d)]). Included in the study were strains with known $bla_{\mathrm{KPC}}$ variants (KPC-2 [12], KPC-3 {21} [18] and KPC-4 [1]). Samples were blinded and tested at one study site. A summary of the results is provided in Tables 12 (a) & (b). Table 12 (a) & (b). Challenge Study: CHROMID CARBA agar Colony Color vs Expected Organism Identity and Carbapenemase Status | Table 12(a): Carbapenemase-producing E. coli | | | | | | --- | --- | --- | --- | --- | | | Organism Identity & Carbapenemase Status | | | | | | | Positive | Negative | Total | | CHROMID CARBA agar | Positive1 | 123 | 0 | 12 | | | Negative2 | 0 | 38 | 38 | | | Total | 12 | 38 | 50 | | Positive Percent Agreement | | 100% (12/12); 75.8-100% | | | | Negative Percent Agreement | | 100% (38/38); 95% CI 90.8-100% | | | 95% CI: 95% score confidence interval 1 Pink-burgundy colonies 2 No growth or colonies not pink-burgundy 3 Includes strains of $E.$ coli known to carry the following $bla_{\mathrm{KPC}}$ variants: KPC-2: 1; KPC-3: 3; KPC-4: 1 | Table 12(b): Carbapenemase-producing K. pneumoniae | | | | | | --- | --- | --- | --- | --- | | | Organism Identity & Carbapenemase Status | | | | | | | Positive | Negative | Total | | CHROMID CARBA agar | Positive1 | 273 | 0 | 27 | | | Negative2 | 0 | 23 | 23 | | | Total | 27 | 23 | 50 | | Positive Percent Agreement | | 100% (27/27); 95% CI % 87.5-100% | | | | Negative Percent Agreement | | 100% (23/23); 95% CI 85.7-100% | | | 95% CI: 95% score confidence interval 1 Blue-green/blue-grey colonies 2 No growth or colonies not blue-green/blue-grey 3 Includes strains of $K$ . pneumoniae known to carry the following $bla_{\mathrm{KPC}}$ variants: KPC-2: 11; KPC-3: 15 # b. Clinical specificity: Refer to Section M(3)(a), above. c. Other clinical supportive data (when a. and b. are not applicable): Not applicable. # 4. Clinical cut-off: Not applicable. # 5. Expected values/Reference range: In the Prospective Clinical Study described in Section M(3)(a), the prevalence of {22} carbapenemase-producing *E. coli* and *K. pneumoniae* based on the reference enrichment culture method was 0.0% (0/709) and 7.2% (51/709), respectively. In contrast, the prevalence of presumptive carbapenemase-producing isolates of *E. coli* and *K. pneumoniae* observed on CHROMID CARBA agar was 0.3% (2/709) and 8.2% (58/709), respectively. N. Proposed Labeling: The labeling supports the finding of substantial equivalence for this device. O. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision. 23