MALDI BIOTYPER CA (MBT-CA) SYSTEM

K130831 · Bruker Daltonics, Inc. · PEX · Nov 21, 2013 · Microbiology

Device Facts

• Record ID: K130831
• Device Name: MALDI BIOTYPER CA (MBT-CA) SYSTEM
• Applicant: Bruker Daltonics, Inc.
• Product Code: PEX · Microbiology
• Decision Date: Nov 21, 2013
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3361
• Device Class: Class 2

Indications for Use

The Bruker Daltonics, Inc. MALDI Biotyper CA System is a qualitative in vitro diagnostic mass spectrometer system for the identification of Gram-negative bacterial colonies cultured from human specimens using matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF) mass spectrometry technology. The MALDI Biotyper CA System is indicated for use in conjunction with other clinical and laboratory findings to aid in the diagnosis of Gram-negative bacterial infections.

Device Story

The MBT-CA System is a benchtop MALDI-TOF mass spectrometer used in clinical microbiology laboratories to identify Gram-negative bacterial colonies. The operator transfers an isolated colony from culture media (e.g., blood, chocolate, or MacConkey agar) onto a reusable steel target plate, adds matrix (α-Cyano-4-hydroxycinnamic acid), and allows it to crystallize. The system uses a laser to ionize microbial proteins (primarily ribosomal proteins). These ions are accelerated through a flight tube; the time-of-flight is measured to determine the mass-to-charge ratio. The resulting mass spectrum acts as a molecular fingerprint. The system software compares this spectrum against a reference library to generate a log(score) (0.00–3.00). A score ≥2.00 indicates high-confidence identification; 1.70–1.99 indicates low-confidence. If the initial direct transfer analysis yields a low score, an ethanol/formic acid extraction procedure is performed. Microbiologists review the output alongside Gram stain and morphology to confirm identification. This process provides faster, more accurate identification than traditional biochemical methods, aiding in timely clinical decision-making for bacterial infections.

Clinical Evidence

Performance validated via method comparison study of 2,263 fresh and stored isolates against 16s bi-directional sequencing and biochemical methods. High-confidence identification (log score ≥2.0) achieved 96.07% accuracy; combined high/low confidence identification achieved 98.19% accuracy. Precision/repeatability studies across 12 days, 3 operators, 3 instruments, and multiple reagent lots showed consistent performance. Specificity studies confirmed no false identifications for non-claimed organisms (anaerobes, mycobacteria, yeast). Stability studies confirmed sample/matrix integrity for up to 24 hours and 12 months respectively.

Technological Characteristics

MALDI-TOF mass spectrometer (microflex LT/SH); 337 nm nitrogen laser; polished steel 48-spot target plates; HCCA matrix; US IVD Bacterial Test Standard (BTS) for calibration; mass range 2,000–20,000 m/z. Software performs automated spectral acquisition and peak list comparison against a reference library. Connectivity via local client-server architecture. Sterilization: N/A (reusable steel plates cleaned per protocol).

Indications for Use

Indicated for the identification of Gram-negative bacterial colonies cultured from human specimens to aid in the diagnosis of Gram-negative bacterial infections. For prescription use only.

Regulatory Classification

Identification

A mass spectrometer system for clinical use for the identification of microorganisms is a qualitative in vitro diagnostic device intended for the identification of microorganisms cultured from human specimens. The device is comprised of an ionization source, a mass analyzer, and a spectral database. The device is indicated for use in conjunction with other clinical and laboratory findings to aid in the diagnosis of bacterial and fungal infections.

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include detailed documentation for device software, including, but not limited to, standalone software applications and hardware-based devices that incorporate software. (2) Premarket notification submissions must include database implementation methodology, construction parameters, and quality assurance protocols. (3) A detailed explanation of the interpretation of results and acceptance criteria must be included in the device's 21 CFR 809.10(b)(9) compliant labeling. (4) As part of the risk management activities performed under 21 CFR 820.10(c) design and development, you must document an appropriate end user device training program that will be offered as part of your efforts to mitigate the risk of failure to correctly operate the instrument. (5) Premarket notification submissions must include details on the appropriate end user device training program that will be offered while marketing the device.

Predicate Devices

• Vitek® MS (K124067)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k)

K130831

B. Purpose for Submission:

To obtain a substantial equivalent determination for a premarket notification for the MALDI Biotyper CA System

C. Measurand:

| Achromobacter xylosoxidans | Klebsiella pneumoniae |
| --- | --- |
| Acinetobacter baumannii complex | Klebsiella oxytoca/Raoultella ornithinolytica |
| Acinetobacter lwoffii | Moraxella (Branhamella) catarrhalis |
| Acinetobacter radioresistens | Moraxella sg Moraxella osloensis |
| Acinetobacter ursingii | Morganella morganii |
| Aeromonas sp | Pantoea agglomerans |
| Alcaligenes faecalis | Pasteurella multocida |
| Burkholderia gladioli | Proteus mirabilis |
| Burkholderia multivorans | Proteus vulgaris group |
| Burkholderia cepacia complex | Providencia rettgeri |
| Citrobacter amalonaticus complex | Providencia stuartii |
| Citrobacter koseri | Pseudomonas aeruginosa |
| Citrobacter freundii complex | Pseudomonas fluorescens group |
| Eikenella corrodens | Pseudomonas putida group |
| Enterobacter aerogenes | Salmonella sp |
| Enterobacter cloacae complex | Serratia liquefaciens |
| Escherichia coli | Serratia marcescens |
| Haemophilus influenzae | Stenotrophomonas maltophilia |
| Haemophilus parainfluenzae | Yersinia enterocolitica |
| Hafnia alvei | Yersinia pseudotuberculosis |

D. Type of Test:

The MALDI Biotyper CA System is a qualitative *in vitro* diagnostic device intended for the identification of Gram-negative bacterial colonies cultured from human specimens. The device is comprised of an ionization source, a mass analyzer and a spectral database. The device is indicated for use in conjunction with other clinical and laboratory findings to aid in the diagnosis of Gram-negative bacterial infections.

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E. Applicant:

Bruker Daltonics, Inc

F. Proprietary and Established Names:

Trade Name: MALDI Biotyper CA System

Common Name: MBT-CA

G. Regulatory Information:

1. Regulation section: 21 CFR 866.3361
2. Classification: Class II (special controls)
3. Product code: PEX
4. Panel: Microbiology (83)

H. Intended Use:

1. Intended use(s):

The Bruker Daltonics, Inc. MALDI Biotyper CA System is a qualitative *in vitro* diagnostic mass spectrometer system for the identification of Gram-negative bacterial colonies cultured from human specimens using matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF) mass spectrometry technology.

The MALDI Biotyper CA System is indicated for use in conjunction with other clinical and laboratory findings to aid in the diagnosis of Gram-negative bacterial infections.

The following organisms are claimed:

Achromobacter xylosoxidans
Acinetobacter baumannii complex
Acinetobacter lwoffii
Acinetobacter radioresistens
Acinetobacter ursingii
Aeromonas species
Alcaligenes faecalis
Burkholderia gladioli
Burkholderia multivorans
Burkholderia cepacia complex
Klebsiella pneumoniae
Klebsiella oxytoca Raoultella ornithinolytica
Moraxella sg Branhamella catarrhalis
Moraxella sg Moraxella osloensis
Morganella morganii
Pantoea agglomerans
Pasteurella multocida
Proteus mirabilis
Proteus vulgaris group
Providencia rettgeri

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| Citrobacter amalonaticus_complex | Providencia stuartii |
| --- | --- |
| Citrobacter koseri | Pseudomonas aeruginosa |
| Citrobacter freundii complex | Pseudomonas fluorescens group |
| Eikenella corrodens | Pseudomonas putida group |
| Enterobacter aerogenes | Salmonella species |
| Enterobacter cloacae complex | Serratia liquefaciens |
| Escherichia coli | Serratia marcescens |
| Haemophilus influenzae | Stenotrophomonas maltophilia |
| Haemophilus parainfluenzae | Yersinia enterocolitica |
| Hafnia alvei | Yersinia pseudotuberculosis |

2. **Indication(s) for use**: Same as intended use.

3. **Special conditions for use statement(s)**:

The MALDI Biotyper CA System is for prescription use only in accordance with 21 CFR 801.109.

4. **Special instrument requirements**:

Mass Spectrometer: microflex LT/SH mass spectrometer

Target Plates: US IVD 48 Spot Target

Reagents:
- US IVD Bacterial Test Standard (BTS)
- US IVD HCCA portioned (α-Cyano-4-hydroxycinnamic acid)

Database: MALDI Biotyper for Clinical Applications (MBT-CA)

Software:
- MBT-CA System Software Package:
- MBT-CA System client software displaying the user interface
- MBT-CA System Server
- MBT-CA System DB Server
- flexControl Software Package
- GTPS firmware
- flexControl acquisition software

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# I. Device Description:

The MBT-CA System consists of the microflex LT/SH mass spectrometer, reference library, kit reagents (US IVD HCCA, US IVD Bacterial Test Standard), US IVD 48 Spot Target slides, and software. The MALDI Biotyper CA System with closed safety covers is a Class 1 Laser product. With the safety cover opened it becomes a Class 4 Laser product. The laser is a 337 nm fixed focus, nitrogen laser. The MALDI Biotyper CA System also referred to as the MBT-CA System.

The reference library, MALDI Biotyper for Clinical Applications, includes type strains, clinical strains and culture collection strains. The MALDI Biotyper CA System reference library was established by analyzing the type strain from each claimed species combined with 5 to 10 additional strains from the same species provided by clinical laboratories or different commercial strain collections for a total of 528 strains. Library mass spectra used for matching contain up to 70 peaks. The MALDI Biotyper for Clinical Applications is also referred to as the MBT-CA.

US IVD HCCA portioned (α-Cyano-4-hydroxycinnamic acid) is a solution that is used when processing test organisms for identification on the MALDI Biotyper CA System. US IVD HCCA is reconstituted in accordance with instructions provided using recommended solvent. 1.0 μL of the matrix is added to the spot with the sample and allowed to dry.

US IVD Bacterial Test Standard (BTS) is an in-vitro-diagnostic product used for quality control and validation of the microflex LT/SH mass spectrometers. US IVD BTS contains a manufactured extract of *Escherichia coli* DH5 alpha that demonstrates a characteristic peptide and protein profile mass spectrum, when tested on the MALDI Biotyper CA System. US IVD BTS is spiked with two additional proteins that extend the upper boundary of the mass range of the US IVD BTS. The overall mass range covered by US IVD BTS is 3.6 to 17 kDa.

US IVD 48 Spot Target plates are reusable steel plates which have been developed for the preparation and identification of test organisms using the MALDI Biotyper CA System. The target allows for the identification of 48 test organisms. The target has five cross-joint positions which should be used for US IVD BTS control. Target plate cleaning is performed after each run.

MALDI Biotyper CA System client software displays a user-interface which guides the user through the MALDI Biotyper CA System workflow. The MALDI Biotyper CA System client also interfaces to the flexControl software for automated acquisition of mass spectra on the microflex LT/SH instrument.

The MALDI Biotyper CA System server communicates with the MALDI Biotyper CA System client and the MBT-DB server. It performs preprocessing on acquired spectra, and matches peaks lists against the Main Spectrum (reference pattern, (MSP)) for matching and calculates the score value (log (score)).

The MBT-DB server stores all information for the MALDI Biotyper CA System. The MBT-DB maintains spectra data (creation information and mass/intensity lists), project data (results of defined and executed runs), method data (parameter lists for spectra preprocessing and identification), user management data, reference patterns and other peak lists plus additional

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maintenance data.

GTPS firmware communicates with the flexControl PC software, controls and monitors the vacuum, moves the sample carrier and performs the docking of the target plate, controls and monitors high voltages in the ion source, generates trigger signals, and monitors instrument status.

The flexControl acquisition software communicates with the MALDI Biotyper CA System client, loads automatic run jobs, communicates with the GTPS firmware, communicates with the laser in the microflex LT/SH instrument, sets the acquisition parameters in the digitizer and reads the acquired data from the digitizer, performs automated data acquisition, evaluates acquired spectra, adjusts the laser power during automatic data acquisition, performs a re-calibration of the time-of-flight to mass transformation, stored acquired spectra on disk and performs source cleaning. The flexControl software does not display a user interface.

## J. Substantial Equivalence Information:

1. Predicate device name(s):

Vitek® MS

2. Predicate 510(k) number(s):

K124067

3. Comparison with predicate:

| DIFFERENCES | | |
| --- | --- | --- |
| Characteristic | NEW DEVICE
MALDI Biotyper CA System
(K130831) | PREDICATE DEVICE
Vitek® MS
(K124067) |
| Culture Age | Bacteria growth should be between 18h to 36h. | Bacteria and yeast growth should be between 24 to 72 hours. |
| Calibration | Bruker US IVD Bacterial Test Standard (BTS) | E. coli ATCC 8739 |
| MALDI Target Plate | US IVD 48 Spot Target
48 positions reusable steel targets | VITEK MS DS Target Slides
48 positions disposable plastic targets |
| MALDI TOF MS Instrument | Bruker microflex LT/SH (benchtop) | Shimadzu AXIMA® Assurance MS (floor standing) |
| Database | MALDI Biotyper for Clinical Applications (MBT CA) | VITEK® MS V2.0 Knowledge Base |

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These differences do not affect substantial equivalence of the MBT CA System and Vitek MS system. Both systems are mass spectrometer systems using matrix-assisted laser desorption/ionization time of flight (MALDI TOF) for the identification of microorganisms cultured from human specimens.

| SIMILARITIES | | |
| --- | --- | --- |
| Characteristic | NEW DEVICE
MALDI Biotyper CA System
(K130831) | PREDICATE DEVICE
Vitek® MS
(K124067) |
| Product Codes | PEX | PEX |
| Intended use | The MALDI Biotyper CA System is a qualitative in vitro diagnostic mass spectrometer system for the identification of Gram negative bacterial colonies cultured from human specimens using matrix assisted laser desorption/ ionization-time of flight (MALDI-TOF) mass spectrometry technology.

The MALDI Biotyper CA System is indicated for use in conjunction with other clinical and laboratory findings to aid in the diagnosis of Gram negative bacterial infections. | The Vitek® MS is a mass spectrometer system using matrix assisted laser desorption/ionization-time to flight (MALDI-TOF) for the identification of microorganisms cultured from human specimen.

The VITEK MS is a qualitative in vitro diagnostic device indicated for use in conjunction with other clinical and laboratory findings to aid in the diagnosis of bacterial and yeast infections. |
| Sample type | Isolated colony from any patient sample source.

**Acceptable media:**
• Columbia blood agar with 5% sheep blood
• Trypticase soy agar with 5% sheep
• Blood
• Chocolate agar
• MacConkey Agar | Isolated colony from any patient sample source.

**Acceptable media:**
• Columbia blood agar with 5% sheep blood
• Trypticase soy agar with 5% sheep
• Blood
• Chocolate polyvitex agar
• Campylosel agar
• MacConkey Agar
• Modified Sabouraud dextrose Agar
• ChromID CPS |
| Type of Test | Automated Mass Spectrometry System | Automated Mass Spectrometry System |
| Matrix | α–Cyano-4-hydroxycinnamic acid | α-Cyano-4-hydroxycinnamic acid |

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| SIMILARITIES | | |
| --- | --- | --- |
| Characteristic | NEW DEVICE
MALDI Biotyper CA System
(K130831) | PREDICATE DEVICE
Vitek® MS
(K124067) |
| Method of Testing | Bacteria: Direct testing

If after initial analysis the log(score) is reported at < 2.00, organisms are processed using the extraction procedure. | Bacteria: Direct testing |
| Result Reporting | Organism identification is reported with high confidence if the log(score) is ≧ 2.00.

An organism identification is reported with low confidence if the log(score) is between 1.70 and <2.00. | A single identification is displayed, with a confidence value from 60.0 to 99.9, when one significant organism or organism group is retained.

“Low-discrimination” identifications are displayed when more than one but not more than four significant organisms or organism groups are retained.

When more than four organisms or organism groups are found, or when no match is found, the organism is considered unidentified. |
| Matching Algorithm | Calculates matches by comparing a new spectrum against each single reference entry of a reference database. | Uses a proprietary process called "mass binning." In this process, the spectrum between 3,000 and 17,000 Daltons are divided into 1300 pre-defined intervals called “bins”. Next, an algorithm based on supervised machine learning known as the “Advanced Spectrum Classifier”, is used to determine how informative each bin was in differentiating that species from all other species in the database. |
| Recorded mass range | 2,000 - 20,000 m/z | 2,000 - 20,000 m/z |

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# K. Standard/Guidance Document Referenced (if applicable):

| | Standards No. | Recognition No.(FDA) | Standards Title | Date |
| --- | --- | --- | --- | --- |
| 1 | CLSI MM-18A | 7-192 | Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing; Approved Guideline, 1^{st} Edition | 4/28/2008 |
| 2 | CLSI EP09-A2-IR | 7-92 | Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline--Second Edition (Interim Revision), | 7/30/2010 |

# L. Test Principle:

Organisms to be identified with the MALDI Biotyper CA System are isolated for on appropriate isolation media. Users are instructed to first test the organism using the direct transfer technique; if results are less than &lt;2.0 log(score), users are then directed to perform extraction procedure.

**Direct Transfer (DT):** An individual colony from an overnight subculture plate is transferred to a selected position on an US IVD 48 Spot Target (target). The target is air dried and US IVD HCCA portioned (matrix) is added. The standard solvent (50% acetonitrile / 47.5% H₂O / 2.5% trifluoroacetic acid) in the matrix solution extracts proteins (mainly ribosomal proteins, which are present in high concentration) from the microorganisms. When dried matrix crystallizes, the inoculated target is ready to be analyzed on the MALDI Biotyper CA System.

**Extraction Procedure (Ext):** For this purpose, isolated colonies from the overnight subculture plate are extracted using an ethanol/formic acid procedure. Afterwards they are transferred to the target and treated as described above.

Samples are analyzed using MALDI (matrix-assisted laser desorption/ionization) TOF (time-of-flight) mass spectrometry. The matrix transfers protons onto the extracted proteins and absorbs UV light. After complete drying, the mixture is exposed to laser pulses, resulting in energy transfer from the matrix causing evaporation and release of positively charged intact proteins and peptides ("soft" ionization technique). The ionized molecules are accelerated by electrical potentials through a flight tube to the mass spectrometer, with separation of the particles determined by their mass/charge ratio (m/z). As different proteins/peptides have different masses, ions arrive at the detector at different times (time of flight). The system measures the time (in the nanosecond range) between pulsed acceleration and the corresponding detector signal, the speed is converted into an exact molecular mass. The mass-to-charge ratio of an ion is proportional to the square of its drift time. Highly abundant microbial proteins (mainly ribosomal proteins) result in a mass spectrum with characteristic mass and intensity distribution. It is species-specific for many bacteria and is interpreted as a molecular fingerprint to identify the test organism. Data acquisition is controlled with MALDI Biotyper CA System Software. The spectrum of the unknown organism is first transformed into a peak list. This peak list is compared to the reference peak list of each organism found in the reference library (database) and a log(score) is generated. A higher log(score) indicates a higher degree of similarity to the organism in the reference library. Organism identification is

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reported with high confidence if the log(score) is $\geq 2.00$. An organism identification is reported with low confidence if the log(score) is between 1.70 and $&lt; 2.00$.

## M. Performance Characteristics (if/when applicable):

### 1. Analytical performance:

#### a. Precision/Reproducibility:

**Precision/Repeatability:**

Validation of the complete MALDI Biotyper CA System was performed on twelve working days with two runs/day following manufacturer’s instructions for use. Ten test organisms were tested in triplicate in each run. The study also tested multiple sources of system variability including three test operators, three microflex LT/SH instruments, three target lots, three BTS lots and three Matrix lots. As triplicates of each test organism were prepared and tested in each run, a total of 72 MALDI Biotyper CA System identifications were carried out for each test organism. Overall results from the precision/repeatability study are presented below.

**Overall Precision per Test Organism**

| Test Organism | # samples measured | # samples passed (DT) | # samples passed (DT+Ext) |
| --- | --- | --- | --- |
| Acinetobacter baumannii | 72 | 62 (86.1%) | 72 (100%) |
| Pseudomonas aeruginosa | 72 | 70 (97.2%) | 72 (100%) |
| Stenotrophomonas maltophilia | 72 | 70 (97.2%) | 72 (100%) |
| Enterobacter cloacae | 72 | 70 (97.2%) | 72 (100%) |
| Escherichia coli | 72 | 71 (98.6%) | 72 (100%) |
| Hafnia alvei | 72 | 72 (100%) | 72 (100%) |
| Proteus mirabilis | 72 | 72 (100%) | 72 (100%) |
| Brevundimonas diminuta* | 72 | 72 (100%) | 72 (100%) |
| Haemophilus influenzae | 72 | 72 (100%) | 72 (100%) |
| Moraxella catarrhalis | 72 | 72 (100%) | 72 (100%) |

* Brevundimonas diminuta was tested but is not included in the claim.

**Table 3: Overall Precision per Test Organism Average log(score)**

| Test Organism | # samples measured | Average log(score) [DT+Ext] |
| --- | --- | --- |
| Acinetobacter baumannii | 72 | 2.172 ± 0.113 |
| Pseudomonas aeruginosa | 72 | 2.368 ± 0.121 |
| Stenotrophomonas maltophilia | 72 | 2.364 ± 0.078 |
| Enterobacter cloacae | 72 | 2.138 ± 0.064 |
| Escherichia coli | 72 | 2.385 ± 0.097 |
| Hafnia alvei | 72 | 2.452 ± 0.095 |

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| Test Organism | # samples measured | Average log(score) [DT+Ext] |
| --- | --- | --- |
| Proteus mirabilis | 72 | 2.587 ± 0.079 |
| Brevundimonas diminuta | 72 | 2.489 ± 0.045 |
| Haemophilus influenzae | 72 | 2.344 ± 0.118 |
| Moraxella catarrhalis | 72 | 2.523 ± 0.080 |

Results confirmed the repeatability and precision of the MALDI Biotyper CA System independent from System Operators, microflex LT/SH instruments, Production Lots, Matrix Lots, and BTS Lots.

## Reproducibility

Reproducibility testing was performed at four sites. Sites were provided with a test panel containing 10 organisms. Testing was performed for five days by two operators. Testing via direct transfer and extraction procedure was performed in accordance with MALDI Biotyper CA System instruction for use. Results are summarized below:

| Reproducibility Panel | Gram-negative bacteria | MBT-CA System ID of samples (DT only) | MBT-CA System ID of samples (DT+Ext) |
| --- | --- | --- | --- |
| REPRO-1 | Stenotrophomonas maltophilia | 77/80 (96%) | 80/80 (100%) |
| REPRO-2 | Citrobacter koseri | 80/80 (100%) | 80/80 (100%) |
| REPRO-3 | Enterobacter aerogenes | 77/80 (96%) | 80/80 (100%) |
| REPRO-4 | Escherichia coli | 78/80 (98%) | 80/80 (100%) |
| REPRO-5 | Klebsiella pneumoniae | 76/80 (95%) | 80/80 (100%) |
| REPRO-6 | Morganella morganii | 80/80 (100%) | 80/80 (100%) |
| REPRO-7 | Pasteurella multocida | 79/80 (99%) | 80/80 (100%) |
| REPRO-8 | Proteus mirabilis | 80/80 (100%) | 80/80 (100%) |
| REPRO-9 | Pseudomonas aeruginosa | 78/80 (98%) | 80/80 (100%) |
| REPRO-10 | Salmonella sp | 78/80 (98%) | 80/80 (100%) |

100% of all test organisms were correctly identified on the species level at each test site after final extraction testing confirming reproducibility of the MALDI Biotyper CA System.

b. Linearity/assay reportable range:

Not applicable, qualitative assay.

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

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# Calibrator:

US IVD BTS is used for mass spectrum calibration and optimization as well as a performance control for the identification of microorganisms with the MALDI Biotyper CA System. US IVD BTS contains a manufactured extract of *Escherichia coli* DH5 alpha that demonstrates a characteristic peptide and protein profile mass spectrum, when tested on the MALDI Biotyper CA System. US IVD BTS is spiked with two additional proteins that extend the upper boundary of the mass range of the US IVD BTS. The overall mass range covered by US IVD BTS is 3.6 to 17 kDa. Two US IVD BTS control positions on a US IVD 48 Spot Target are selected and inoculated with US IVD BTS solution. The US IVD BTS solution is allowed to dry at room temperature and then overlayed with reconstituted US IVD HCCA portioned solution. If US IVD BTS does not meet all required performance specifications, the test run will be invalid. If US IVD BTS is not inoculated onto a target prior to processing, the test run will be invalid.

# Controls:

*Klebsiella pneumoniae*, *Haemophilus influenzae*, *Proteus vulgaris*, *Pseudomonas aeruginosa*, and *Escherichia coli*, was used as controls. Of the 193 Quality Control runs conducted during the course of the method comparison study, there were only five instances where a Quality Control organism failed to yield an expected result. As a result, all isolates included in that plate run were repeated using a fresh QC organism transfer. The overall plate repeat rate was 2.6% (5/193).

## d. Assay cut-off:

Using statistical analysis, a probability ranking of the organism identification is generated. The probability ranking is represented as a log(score) between 0.00 and 3.00.

Organism identification (direct or extracted) is reported with high confidence if the log(score) is ≥ 2.00. If a direct transfer organism identification log(score) is &lt;2.00; the user is instructed to follow the extraction procedure.

After extraction:

- If the organism identification log(score) is between 1.7 and &lt;2.0, the identification is reported as low confidence.
- If the organism identification log(score) is &lt;1.7, it is reported as ‘No Identification’.

## e. Detection limit:

The Limit of Detection study was designed to establish the estimated dynamic range of sample size for both the Direct Transfer and Extraction method procedure. Seven frequently occurring clinically relevant test organisms were chosen for this study. Cell density and cell concentration were estimated by measuring the optical density of the suspension at a wavelength of 600 nm. Approximately 3×10⁸ cells/mL were reported to correspond to an optical density of OD₆₀₀ = 1 according to the McFarland Standard. All suspensions were tested in duplicate. Each cell stock was diluted to a minimum five (5) concentrations and tested in duplicate. A cell concentration was considered within the dynamic range if the

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MALDI Biotyper CA System correctly identified the organism for both replicates with a log(score) of $\geq 2.00$.

Study results concluded that the estimated dynamic range for the direct and extracted method is as follows:

| Technique | Lower limit [cells/uL] | Upper limit [cells/uL] |
| --- | --- | --- |
| Direct Transfer | 6.3×10^{3}–1.4×10^{4} | 1.4×10^{6}–$\geq$6.5×10^{7} |
| Extraction | 9.0×10^{3}–1.3×10^{5} | 1.1×10^{7}–$\geq$6.9×10^{7} |

## f. Analytical specificity:

**Database development:** The MALDI Biotyper CA System reference library was established by analyzing the type strain from each claimed species combined with 5 to 10 additional strains from the same species provided by clinical laboratories or different commercial strain collections for a total of 528 strains. Matches are calculated by comparing a new spectrum against each single reference entry of a reference database library mass spectra used for matching contain up to 70 peaks. The spectrum of the unknown test organism, acquired through the MALDI Biotyper CA System Software, is electronically transformed into the peak list. Using a biostatistical algorithm, this peak list is compared to reference peak lists of all organisms in the reference library (database) and a log(score) value between 0.00 and 3.00 is calculated.

**Analytical Specificity Study:** The specificity study was designed to validate the performance of the proposed MALDI Biotyper CA System reference library by ensuring that organisms not included in the reference library would not yield an incorrect identification and would be reported as “No Identification.” Additionally, the study was designed to further demonstrate that the MALDI Biotyper CA System identification is not impacted when closely related species not included in the reference library are run on the system.

The study was conducted in two phases. In phase one, organisms currently not included in the initial system reference library were tested via Direct Transfer and extraction method to ensure that the organisms would not be falsely identified by the MALDI Biotyper CA system. Organisms tested fell into the following five groupings:

- Anaerobe bacteria
- Mycobacteria
- Gram-Negative bacteria (not currently claimed within the library)
- Gram-Positive bacteria
- Yeast species

Results from this phase are presented below:

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Phase 1: Summary Results

| Organism | Strain | # of “No Identification” | | # of false identification |
| --- | --- | --- | --- | --- |
| | | DT | Ext | |
| Bacteroides fragilis | DSM 2151 | 2 / 2 | 2 / 2 | 0 |
| Bacteroides fragilis | DSM 9669 | 2 / 2 | 2 / 2 | 0 |
| Prevotella copri | DSM 18205^{T} | 2 / 2 | 2 / 2 | 0 |
| Prevotella buccae | DSM 19025^{T} | 2 / 2 | 2 / 2 | 0 |
| Mycobacterium fortuitum ssp. fortuitum | DSM 43477 | 2 / 2 | 2 / 2 | 0 |
| Mycobacterium fortuitum ssp. Fortuitum | DSM 46621^{T} | 2 / 2 | 2 / 2 | 0 |
| Neisseria gonorrhoeae | DSM 9188^{T} | 2 / 2 | 2 / 2 | 0 |
| Neisseria gonorrhoeae | DSM 15130 | 2 / 2 | 2 / 2 | 0 |
| Erwinia tasmaniensis | DSM 17949 | 2 / 2 | 2 / 2 | 0 |
| Erwinia tasmaniensis | DSM 17950 | 2 / 2 | 2 / 2 | 0 |
| Vagococcus fluvialis | DSM 5731^{T} | 2 / 2 | 2 / 2 | 0 |
| Vagococcus fluvialis | DSM 21402 | 2 / 2 | 2 / 2 | 0 |
| Facklamia hominis | CCUG 59179 | 2 / 2 | 2 / 2 | 0 |
| Facklamia hominis | CCUG 49614 | 2 / 2 | 2 / 2 | 0 |
| Guehomyces pullulans | CBS 2532^{T} | 2 / 2 | 2 / 2 | 0 |
| Guehomyces pullulans | CBS 2542 | 2 / 2 | 2 / 2 | 0 |
| Cyberlindnera mississippiensis | CBS 7023^{T} | 2 / 2 | 2 / 2 | 0 |
| Cyberlindnera mississippiensis | CBS 7027 | 2 / 2 | 2 / 2 | 0 |

In phase two of the study testing, Burkholderia cepacia/multivorans/gladioli were investigated via direct transfer and extraction method to ensure that closely related organisms can be differentiated when tested on the MALDI Biotyper CA System. Results from this phase of testing are reported below:

Phase 2: Summary Results

| Organism | Strain | # of Correct Identifications | | # of false identification |
| --- | --- | --- | --- | --- |
| | | DT | Ext | |
| Burkholderia cepacia | DSM 9241 | 2 / 2 | 2 / 2 | 0 |
| Burkholderia cepacia | DSM 50181 | 2 / 2 | 2 / 2 | 0 |
| Burkholderia multivorans | 1A11237234 4v MVD | 2 / 2 | 2 / 2 | 0 |
| Burkholderia multivorans | H480 MCRF | 2 / 2 | 2 / 2 | 0 |
| Burkholderia gladioli | DSM 8361 | 2 / 2 | 2 / 2 | 0 |
| Burkholderia gladioli | LMG 6956 | 2 / 2 | 2 / 2 | 0 |

Phase 1 data demonstrates with high confidence that Anaerobes, Mycobacteria, Gram-negative, Gram-positive and Yeast organisms not included in the MALDI Biotyper CA System database are not identified confirming the specificity of the MALDI Biotyper CA System reference library when following product instructions for use for both DT and extraction method. Phase 2 data confirms that closely related species can be unambiguously identified by the MALDI Biotyper CA System.

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14

### g. Sample stability studies:

**Sample Stability after Matrix Overlay:** This study was conducted to prove the stability of test organisms on the spotted target plate following matrix addition at various temperature and relative humidity conditions. In addition, the study served to prove that matrix alone will not influence MALDI Biotyper CA System identification. Three gram negative target organisms were cultured on Columbia Blood Agar (CBA) and aging experiments were done at two different temperature and relative humidity testing conditions. For each condition, two target plates were inoculated and each contained four target spots of directly transferred test organism, four spots of extracted test organism and eight spots of matrix solution alone. All spots containing test organism were then overlaid with matrix in accordance with product instructions for use and tested immediately and then stored at one of the testing conditions and retested at 4±1 hour, 8±1 hour and 24±1 hour.

**Summary of Sample Stability overlaid with Matrix**

| Test Condition | Test Age | Test Organism Correct Identification | Matrix “No Peaks Found” |
| --- | --- | --- | --- |
| DT
20 ± 1°C, 40 ± 5% | 0 hour | 24/24 | 8/8 |
| | 4 hours | 24/24 | 8/8 |
| | 8 hours | 24/24 | 8/8 |
| | 24 hours | 24/24 | 8/8 |
| | | | |
| Ext
20 ± 1°C, 40 ± 5% | 0 hour | 24/24 | 8/8 |
| | 4 hours | 24/24 | 8/8 |
| | 8 hours | 24/24 | 8/8 |
| | 24 hours | 24/24 | 8/8 |
| | | | |
| DT
20 ± 1°C, 70 ± 5% | 0 hour | 24/24 | 8/8 |
| | 4 hours | 24/24 | 8/8 |
| | 8 hours | 23/24 | 8/8 |
| | 24 hours | 24/24 | 8/8 |
| | | | |
| Ext
20 ± 1°C, 70 ± 5% | 0 hour | 24/24 | 8/8 |
| | 4 hours | 24/24 | 8/8 |
| | 8 hours | 24/24 | 8/8 |
| | 24 hours | 24/24 | 8/8 |
| | | | |
| DT
25 ± 1°C, 30 ± 5% | 0 hour | 24/24 | 8/8 |
| | 4 hours | 24/24 | 8/8 |
| | 8 hours | 24/24 | 8/8 |
| | 24 hours | 24/24 | 8/8 |
| | | | |
| Ext
25 ± 1°C, 30 ± 5% | 0 hour | 24/24 | 8/8 |
| | 4 hours | 24/24 | 8/8 |
| | 8 hours | 24/24 | 8/8 |
| | 24 hours | 24/24 | 8/8 |
| | | | |
| DT
25 ± 1°C, 70 ± 5% | 0 hour | 24/24 | 8/8 |
| | 4 hours | 24/24 | 8/8 |
| | 8 hours | 18/24 | 8/8 |
| | 24 hours | 24/24 | 8/8 |

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| Test Condition | Test Age | Test Organism Correct Identification | Matrix “No Peaks Found” |
| --- | --- | --- | --- |
| Ext
25 ± 1°C, 70 ± 5% | 0 hour | 24/24 | 8/8 |
| | 4 hours | 24/24 | 8/8 |
| | 8 hours | 23/24 | 8/8 |
| | 24 hours | 24/24 | 8/8 |

All study results confirmed that inoculated test organisms once overlaid with matrix are stable for up to 24 hours at room temperature. In addition, matrix alone does not interfere or influence MALDI Biotyper CA System identification.

h. Stability studies (reagent/target plates):

US IVD Bacterial Test Standard (BTS)

BTS was assessed using three lots of BTS material. A shipping/accelerated stability study confirmed that BTS is stable for three weeks at temperatures up to $37 \pm 2^{\circ} \mathrm{C}$. (Two of these weeks account for the shipping and one for the long-term storage.) Real-time stability testing confirms that BTS is stable for up to 12 months. In-use stability confirms that reconstituted BTS is stable for five (5) months when stored in accordance with product claims.

HCCA portioned (Matrix) Stability

HCCA portioned (Matrix) is used when processing test organisms for identification on the MALDI Biotyper CA System. Matrix must be reconstituted prior to use. Studies were conducted to determine the stability of un-reconstituted matrix as well as in-use (reconstituted) matrix. Shipping/accelerated stability studies confirm that matrix is stable for sixteen weeks at temperatures up to $37 \pm 2^{\circ} \mathrm{C}$. Real-time stability studies confirm that matrix is stable for up to 18 months when stored in accordance with product instructions for use. In-use (reconstituted) stability testing confirmed that reconstituted matrix is stable for one week when stored at controlled room temperature and for 12 hours when stressed by temperatures up to $15^{\circ} \mathrm{C}$ or $30^{\circ} \mathrm{C}$.

Target plates stability: Four representative MSP target polished steel plates were tested over a two year period with approximately two applications per week. Target plates were used in accordance with instructions included in the user manual. No damage to the target plate was noted. No bleeding of spots was observed. The target plates do not have a shelf-life; but rather can be used until physical damage is observed.

i. Carry-Over and Cross Contamination:

This study was conducted to determine the effect of carry over and cross-contamination, defined as microbial sample convergence between adjacent blank target spots. Two targets and two frequently occurring Gram negative bacteria were chosen for this testing. Each target was inoculated with test organism four times via direct transfer and extraction method in an alternating pattern. All sample positions were overlaid with matrix solution including the remaining unused target positions to serve as blank measurements. Testing was performed in accordance with product instructions for use. Targets were then cleaned in accordance with the Target Cleaning procedure and organism prepared in a similar fashion but in the reverse pattern. The test cycle described above was repeated four times on each

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target plate.

No cross-contamination or carry-over effects were seen in the automated MALDI Biotyper CA System identification process.

## j. Influence of Agar Media

This study was completed in order to demonstrate that impurities such as salts, peptides or carbohydrates introduced from culture media do not interfere with MALDI Biotyper CA System identification. In addition, the study set-out to prove that isolation media alone would not generate mass spectra leading to false identification on the MALDI Biotyper CA System. TSA, CBA, MAC and CHOC agars were tested by the following methods:

- Each agar media was inoculated using the direct transfer and extraction method alone six times each.
- Three frequently occurring Enterobacteriaceae and non-fermenting Gram Negative Bacteria were transferred to the target plate in duplicate via DT and Ext method to serve as a control.
- Each target organism was then inoculated in duplicate via DT and Ext method such that a sample agar media was included with the isolate.

Summary of Influence of Agar Media Study

| Media | Agar Alone | | Target Organism Alone | | Target Organism + Agar | |
| --- | --- | --- | --- | --- | --- | --- |
| | # replicates | % No ID | # replicates | % False ID | # replicates | % False ID |
| TSA | 12/12 | 100 | 12/12 | 0 | 10/12 | 0 |
| CBA | 12/12 | 100 | 12/12 | 0 | 12/12 | 0 |
| MAC | 12/12 | 100 | 12/12 | 0 | 12/12 | 0 |
| CHOC | 12/12 | 100 | 12/12 | 0 | 12/12 | 0 |

The study confirms that the media recommended for use on the MALDI MALDI Biotyper CA System do not interfere with MALDI Biotyper CA System performance or organism identification.

## k. Organism Stability

### Media and Colony Stability

In accordance with device instructions for use, primary or secondary isolation plates of recommended media [Trypticase Soy Agar with 5% sheep blood (TSA), Columbia Blood Agar with 5% sheep blood (CBA), MacConkey Agar (MAC), and Chocolate Agar (Choc)] may be held for up to 12 hours at room temperature prior to testing on the MALDI Biotyper CA System.

Testing was conducted using seven gram-negative organisms at two different incubation time points (18h, 24h). After initial incubation, isolates were further tested at two temperatures (18°C, 25°C) for 12 hours post-incubation.

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17

Summary of Media and Colony Stability Study

| Media | ≥2.0 Identification (DT) | False Identification (DT) | ≥2.0 Identification (Ext) | False Identification (Ext) |
| --- | --- | --- | --- | --- |
| TSA | 288/288 | 0/288 | 288/288 | 0/288 |
| CBA | 284/288 | 0/288 | 288/288 | 0/288 |
| MAC | 263/288 | 0/288 | 288/288 | 0/288 |
| CHOC | 288/288 | 0/288 | 288/288 | 0/288 |

The study results confirm that the following culture media can be used on the MALDI Biotyper CA System:
- Trypticase Soy Agar with 5% sheep blood (TSA)
- Columbia Blood Agar with 5% sheep blood (CBA)
- MacConkey Agar (MAC)
- Chocolate Agar (CHOC)

Study results conclude that sample colony is stable for up to 18-36 hours.

Organism Stability prior to MALDI Biotyper CA System Analysis

This study was conducted to assess isolate stability on the target plate prior to matrix overlay via direct transfer and extraction method. In addition, the study set out to confirm the stability of extracted material prior to target plate inoculation.

To test for isolate stability on the target plate prior to matrix overlay via DT, three common gram negative bacteria were inoculated eight times and overlaid with matrix at five different time points. After matrix overlay, isolates were tested in accordance with product instructions. For the Ext method, colonies were prepared following the extraction technique per product instructions for use. Extracts were overlaid with matrix at five different time points and tested per product instructions. For the third phase of testing, the three gram negative isolates were extracted twice. The extracts were stored at controlled room temperature for up to 24 hours and tested at five time points in replicates of eight.

Summary of Organism Stability Prior to MALDI Biotyper CA System Analysis Study

| Test Phase | Testing Condition | Measurands | Correct Identification | False Identification |
| --- | --- | --- | --- | --- |
| Direct Transfer (DT) | 0 min | 24 | 24/24 | 0/24 |
| | 15 min | 24 | 24/24 | 0/24 |
| | 30 min | 24 | 24/24 | 0/24 |
| | 60 min | 24 | 24/24 | 0/24 |
| | 120 min | 24 | 24/24 | 0/24 |
| | | | | |
| Extraction Method (Ext) | 0 min | 6 | 6/6 | 0/6 |
| | 10 min | 6 | 6/6 | 0/6 |
| | 20 min | 6 | 6/6 | 0/6 |
| | 30 min | 6 | 6/6 | 0/6 |
| | 60 min | 6 | 6/6 | 0/6 |
| | | | | |

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18
| Test Phase | Testing Condition | Measurands | Correct Identification | False Identification |
| --- | --- | --- | --- | --- |
| Extract #1 | 0 hour | 24 | 24/24 | 0/24 |
| | 1 hour | 24 | 24/24 | 0/24 |
| | 4 hours | 24 | 24/24 | 0/24 |
| | 8 hours | 24 | 24/24 | 0/24 |
| | 24 hours | 24 | 24/24 | 0/24 |
| | | | | |
| Extract #2 | 0 hour | 24 | 24/24 | 0/24 |
| | 1 hour | 24 | 24/24 | 0/24 |
| | 4 hours | 24 | 24/24 | 0/24 |
| | 8 hours | 24 | 24/24 | 0/24 |
| | 24 hours | 24 | 24/24 | 0/24 |

Study results confirm that samples are stable on the target plate when tested via DT or Ext method for up to sixty minutes prior to analysis. In addition, extracts are stable for up to 24 hours when stored at room temperature.

## 1. Other supportive Instrument Performance Characteristics

### Mixed Culture:

To assess the effect of testing a mixed culture on MALDI Biotyper CA System identification, *Pseudomonas aeruginosa* was used as the target; four non-target organisms consisting of gram-negative and gram-positive bacteria were introduced with the target organisms at varying concentrations to determine the affect a mixed culture would have on MALDI Biotyper CA System identification.

| Condition | Target Organism Amount | Non-Target Organism Amount | # of MBT-CA System False Identifications |
| --- | --- | --- | --- |
| A | 100% | 0% | 0/32 |
| B | 75% | 25% | 0/32 |
| C | 50% | 50% | 0/32 |
| D | 25% | 75% | 0/32 |

Although system users are instructed to test a single isolated colony on the MALDI Biotyper CA System, this study demonstrated that analyzing a mixed culture on the system, no false results are obtained.

### Viability Study

Viability studies with gram negative rods mixed with matrix on the target plate were not performed. The user is advised to consider all samples, microbial cultures and inoculated products as infectious. Aseptic techniques and usual precautions for handling organisms should be observed throughout the MALDI Biotyper CA System workflow according to "CLSI M29-A, Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline - Current revision". For additional handling precautions, refer to

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"Biosafety in Microbiological and Biomedical Laboratories - CDC/NIH -Latest edition".

## Run Failures from Clinical Trial:

Of the 193 Quality Control runs conducted during the course of the method comparison study, there were only five instances where a Quality Control organism failed to yield an expected result. As a result, all isolates included in that plate run were repeated using a fresh QC organism transfer. The overall plate repeat rate was 2.6% (5/193).

## 2. Comparison studies:

a. Method comparison with predicate device:

Not applicable. Refer to the Clinical Studies section of this document.

b. Matrix comparison:

Not applicable

## 3. Clinical studies:

a. Clinical Sensitivity:

### Proficiency/Familiarity

Prior to method comparison study initiation, each study operator from the four US Study sites underwent a proficiency/familiarity period to ensure that each operator was familiar with all aspects of instrument operation. Each operator was asked to test five QC organisms using both the Direct Transfer (DT) and Extraction (Ext) method following product instructions for use.

### Challenge Panel

To demonstrate intra-laboratory performance, a challenge panel of 100 organisms was provided to five test sites. Each site tested the challenge panel in accordance with MALDI Biotyper CA System instructions for use. The following is a summary of results:

| Test procedure | Site 1 | Site 2 | Site 3 | *Site 4 | **Site 5 |
| --- | --- | --- | --- | --- | --- |
| DT only | 99/100
(99%) | 98/100
(98%) | 99/100
(99%) | 96/100
(96%) | 86/87 (99%) |
| MBT-CA System
workflow (+ Ext) | 99/100
(99%) | 99/100
(99%) | 99/100
(99%) | 97/100
(97%) | 86/87 (99%) |

* One sample was incorrectly identified due to a site error and another sample was a mixed culture.
** The number of test organisms was reduced as thirteen (13) samples were not received.

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20

# Prospective Clinical Study:

The following is a summary of a clinical study performed at five sites to confirm product performance. Testing was performed using direct transfer of organism to target plate. If the MALDI Biotyper CA System identification of the test organism did not result in a bacterial identification with a log(score) value of $\geq 2.0$, repeat testing was performed using the extraction procedure. Only $6.8\%$ of all clinical results needed extraction. MALDI Biotyper CA System identifications were compared to sequencing in accordance with MM18-A using GenBank and/or EzTaxon. If no match was observed, biochemical testing and protein target sequencing was applied. The following tables summarize MALDI Biotyper CA System results for all organisms included in the current reference library. In total, 2263 fresh and stored isolates were tested to support the initial reference library claims. Site summaries and an overall performance table is presented below:

The following comments apply to site performance summaries below:

1) THE MBT-CA System reported 5 times *E.cloacae*, 8 times *H.influenzae*, and once *Y.enterocolitica* with high confidence [log(score)$\geq$2.0] whereas the reference method reported something else. (See Table 1a, 1b, and 2)
2) The MBT-CA System reported 2 times *P.stuartii*, 20 times *H.influenzae* with low confidence [log(score)$\geq$1.7; $&lt;$2.0] whereas the reference method reported something else. (See Table 1a and 1b)
3) THE MBT-CA System reported 2 times *C.amalonaticus* with high confidence [log(score)$\geq$2.0] whereas the reference method did not report a reliable result. (See Table 1a and 1b)
4) The MBT-CA System reported once *A.xylosoxidans* with low confidence [log(score)$\geq$1.7; $&lt;$2.0] whereas the reference method reported something else. (See Table 1a and 1b)
5) The MBT-CA System reported "no result" [log(score)$&lt;$1.7] for one *P.putida* and one *S.maltophilia* identified by the reference method. (See Table 2)
6) n/a - Not Applicable. There are no "correct negatives" in the performance table for the whole study data. There are no samples where the reference method is negative and the MBT-CA System is negative, too. Samples with negative reference identification and negative MBT-CA System identification were excluded from the study.
7) MBT-CA System identifications were compared to sequencing in accordance with MM18-A using GenBank and/or EzTaxon. If no match was observed, biochemical testing and protein target sequencing was applied.
8) 18 strains of Haemophilus haemolyticus were collected and soley tested at Site 5 to determine whether H. haemolyticus could be differentiated from Haemophilus influenzae. As these species could not be differentiated, the performance of this site was significantly decreased

| All Isolates - ALL SITES | REFERENCE ALGORITHM^{7)} | | |
| --- | --- | --- | --- |
| | Positive | Negative | Total |
| Positive Organism ID; (High Confidence); log(score) ≥2.0 | 2174 | 16^{1) + 3)} | 2190 |
| Positive Organism ID; (Low Confidence); log(score) ≥1.7; <2.0 | 48 | 23^{2) + 4)} | 71 |
| Negative | 2^{5)} | n/a^{6)} | 2 |
| Total | 2224 | 39 | 2263 |

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| Gram negatives
ALL SITES | Correct: Genus ID
Correct: Species ID or Group ID or Complex ID | | Correct: Genus ID
Incorrect: Species ID or Group ID or Complex ID | | Incorrect Genus ID | | no ID |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | log(score) <2.0 |
| IDs | 2174 / 2263
96.07% | 48 / 2263
2.12% | 14^{1)} / 2263
0.62% | 22^{2)} / 2263
0.97% | 2^{3)} / 2263
0.09% | 1^{4)} / 2263
0.04% | 2^{5)} / 2263
0.09% |
| Combined IDs | 2222 / 2263
98.19%
95% CI [95.04 ; 100.00] | | 36 / 2263
1.59%
95% CI [0.00 ; 4.81] | | 3 / 2263
0.13%
95% CI [0.01 ; 0.25] | | n/a |
| All Isolates – Site 1 | REFERENCE ALGORITHM^{7)} | | |
| --- | --- | --- | --- |
| | Positive | Negative | Total |
| Positive Organism ID; (High Confidence); log(score) ≥2.0 | 801 | 5^{1) + 3)} | 806 |
| Positive Organism ID; (Low Confidence); log(score) ≥1.7; <2.0 | 11 | 1^{2) + 4)} | 12 |
| Negative | 0 | n/a^{6)} | 0 |
| Total | 812 | 6 | 818 |
| Gram negatives
Site 1 | Correct: Genus ID
Correct: Species ID or Group ID or Complex ID | | Correct: Genus ID
Incorrect: Species ID or Group ID or Complex ID | | Incorrect: Genus ID | | no ID |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | log(score) <2.0 |
| IDs | 801 / 818
97.92% | 11 / 818
1.34% | 3 / 818
0.37% | 1 / 818
0.12% | 2 / 818
0.24% | 0 | 0 |
| Combined IDs | 812 / 818
99.27% | | 4 / 818
0.49% | | 2 / 818
0.24% | | n/a |

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| All Isolates – Site 2 | REFERENCE ALGORITHM^{7)} | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Positive | Negative | | Total |
| Positive Organism ID; (High Confidence); log(score) ≥2.0 | | | | 432 | 2^{1) + 3)} | | 434 |
| Positive Organism ID; (Low Confidence); log(score) ≥1.7; <2.0 | | | | 9 | 2^{2) + 4)} | | 11 |
| Negative | | | | 0 | n/a^{6)} | | 0 |
| Total | | | | 441 | 4 | | 445 |
| Gram negatives
Site 2 | Correct: Genus ID
Correct: Species ID or Group ID or Complex ID | | Correct: Genus ID
Incorrect: Species ID or Group ID or Complex ID | | Incorrect: Genus ID | | no ID |
| | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | log(score) <2.0 |
| IDs | 432 / 445
97.08% | 9 / 445
2.02% | 2 / 445
0.45% | 2 / 445
0.45% | 0 | 0 | 0 |
| Combined IDs | 441 / 445
99.10% | | 4 / 445
0.90% | | 0 | | n/a |
| All Isolates – Site 3 | REFERENCE ALGORITHM^{7)} | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Positive | Negative | | Total |
| Positive Organism ID; (High Confidence); log(score) ≥2.0 | | | | 355 | 1^{1) + 3)} | | 356 |
| Positive Organism ID; (Low Confidence); log(score) ≥1.7; <2.0 | | | | 7 | 1^{2) + 4)} | | 8 |
| Negative | | | | 0 | n/a^{6)} | | 0 |
| Total | | | | 362 | 2 | | 364 |
| Gram negatives
Site 3 | Correct: Genus ID
Correct: Species ID or Group ID or Complex ID | | Correct: Genus ID
Incorrect: Species ID or Group ID or Complex ID | | Incorrect: Genus ID | | no ID |
| | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | log(score) <2.0 |
| IDs | 355 / 364
97.53% | 7 / 364
1.92% | 1 / 364
0.27% | 0 | 0 | 1 / 364
0.27% | 0 |
| Combined IDs | 362 / 364
99.45% | | 1 / 364
0.27% | | 1 / 364
0.27% | | n/a |

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23

| All Isolates – Site 4 | REFERENCE ALGORITHM^{7)} | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Positive | Negative | | Total |
| Positive Organism ID; (High Confidence); log(score) ≥2.0 | | | | 403 | 1^{1)} + 3) | | 404 |
| Positive Organism ID; (Low Confidence); log(score) ≥1.7; <2.0 | | | | 17 | 1^{2)} + 4) | | 18 |
| Negative | | | | 2 | n/a^{6)} | | 2 |
| Total | | | | 422 | 2 | | 424 |
| Gram negatives
Site 4 | Correct: Genus ID
Correct: Species ID or Group ID or Complex ID | | Correct: Genus ID
Incorrect: Species ID or Group ID or Complex ID | | Incorrect: Genus ID | | no ID |
| | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | log(score) <2.0 |
| IDs | 403 / 424
95.05% | 17 / 424
4.01% | 1 / 424
0.24% | 1 / 424
0.24% | 0 | 0 | 2 / 424
0.47% |
| Combined IDs | 420 / 424
99.06% | | 2 / 424
0.48% | | 0 | | n/a |
| All Isolates – Site 5^{8)} | REFERENCE ALGORITHM^{7)} | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Positive | Negative | | Total |
| Positive Organism ID; (High Confidence); log(score) ≥2.0 | | | | 183 | 7^{1)} + 3) | | 190 |
| Positive Organism ID; (Low Confidence); log(score) ≥1.7; <2.0 | | | | 4 | 15^{2)} + 4) | | 22 |
| Negative | | | | 0 | n/a^{6)} | | 0 |
| Total | | | | 187 | 25 | | 212 |
| Gram negatives
Site 5 | Correct: Genus ID
Correct: Species ID or Group ID or Complex ID | | Correct: Genus ID
Incorrect: Species ID or Group ID or Complex ID | | Incorrect: Genus ID | | no ID |
| | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | high confidence log(score) ≥2.0 | low confidence log(score) ≥1.7 ... <2.0 | log(score) <2.0 |
| IDs | 183 / 212
86.32% | 4 / 212
1.89% | 7 / 212
3.30% | 18 / 212
8.49% | 0 | 0 | 0 |
| Combined IDs | 187 / 212
88.21% | | 25 / 212
11.79% | | 0 | | n/a |

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Performance and Species Claimed in the MALDI Biotyper CA System Reference Library

| Matching Hint4 | Species | # of Isolates | Correct Identification | | | | | | | | | No ID | Discordant | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Species Confirmation high confidence log(score) ≥2.0 | | | Species Confirmation low confidence log(score) ≥1.7 ... <2.0 | | | Combined Performance | | | | Species Confirmation high confidence log(score) ≥2.0 | Species Confirmation low confidence log(score) ≥1.7 ... <2.0 |
| a | Achromobacter xylosoxidans | 75 | 70 | 70/75 | 93% | 4 | 4/75 | 5% | 74 | 74/75 | 99% | | | 1^{1)} |
| b | Acinetobacter baumannii complex | 70 | 67 | 67/70 | 96% | 3 | 3/70 | 4% | 70 | 70/70 | 100% | | | |
| | Acinetobacter lwoffii | 69 | 69 | 69/69 | 100% | | | | 69 | 69/69 | 100% | | | |
| | Acinetobacter radioresistens | 27 | 27 | 27/27 | 100% | | | | 27 | 27/27 | 100% | | | |
| | Acinetobacter ursingii | 50 | 49 | 49/50 | 98% | 1 | 1/50 | 2% | 50 | 50/50 | 100% | | | |
| c | Aeromonas sp | 56 | 56 | 56/56 | 100% | | | | 56 | 56/56 | 100% | | | |
| | Alcaligenes faecalis | 26 | 26 | 26/26 | 100% | | | | 26 | 26/26 | 100% | | | |
| d | Burkholderia gladioli | 6 | 6 | 6/6 | 100% | | | | 6 | 6/6 | 100% | | | |
| | Burkholderia multivorans | 19 | 19 | 19/19 | 100% | | | | 19 | 19/19 | 100% | | | |
| e | Burkholderia cepacia complex | 29 | 29 | 29/29 | 100% | | | | 29 | 29/29 | 100% | | | |
| f | Citrobacter amalonaticus complex | 64 | 62 | 62/64 | 97% | | | | 62 | 62/64 | 97% | | 2^{1)} | |
| | Citrobacter koseri | 89 | 89 | 89/89 | 100% | | | | 89 | 89/89 | 100% | | | |
| g | Citrobacter freundii complex | 89 | 89 | 89/89 | 100% | | | | 89 | 89/89 | 100% | | | |
| | Eikenella corrodens | 16 | 16 | 16/16 | 100% | | | | 16 | 16/16 | 100% | | | |
| | Enterobacter aerogenes | 80 | 80 | 80/80 | 100% | | | | 80 | 80/80 | 100% | | | |
| h | Enterobacter cloacae complex | 95 | 74 | 74/95 | 78% | 16 | 16/95 | 17% | 90 | 90/95 | 95% | | 5^{1)} | |
| i | Escherichia coli | 122 | 122 | 122/122 | 100% | | | | 122 | 122/122 | 100% | | | |
| j | Haemophilus influenzae | 95 | 63 | 63/95 | 66% | 4 | 4/95 | 4% | 67 | 67/95 | 71% | | 8^{2)} | 20^{2)} |
| | Haemophilus parainfluenzae | 34 | 32 | 32/34 | 94% | 2 | 2/34 | 6% | 34 | 34/34 | 100% | | | |
| k | Hafnia alvei | 45 | 45 | 45/45 | 100% | | | | 45 | 45/45 | 100% | | | |
| l | Klebsiella | 101 | 101 | 101/101 | 100% | | | | 101 | 101/ | 100% | | | |

{24}

25
| Matching Hint4 | Species | # of Isolates | Correct Identification | | | | | | | | | No ID | Discordant | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Species Confirmation high confidence log(score) ≥2.0 | | | Species Confirmation low confidence log(score) ≥1.7 ... <2.0 | | | Combined Performance | | | | Species Confirmation high confidence log(score) ≥2.0 | Species Confirmation low confidence log(score) ≥1.7 ... <2.0 |
| | pneumoniae | | | | | | | | | 101 | | | | |
| n | Klebsiella oxytoca Raoultella ornithinolytica | 68 | 68 | 68/68 | 100% | | | | 68 | 68/68 | 100% | | | |
| | Moraxella_sg Branhamella catarrhalis | 66 | 66 | 66/66 | 100% | | | | 66 | 66/66 | 100% | | | |
| n | Moraxella_sg Moraxella osloensis | 28 | 28 | 28/28 | 100% | | | | 28 | 28/28 | 100% | | | |
| o | Morganella morganii | 80 | 80 | 80/80 | 100% | | | | 80 | 80/80 | 100% | | | |
| p | Pantoea agglomerans | 27 | 27 | 27/27 | 100% | | | | 27 | 27/27 | 100% | | | |
| | Pasteurella multocida | 46 | 46 | 46/46 | 100% | | | | 46 | 46/46 | 100% | | | |
| | Proteus mirabilis | 67 | 67 | 67/67 | 100% | | | | 67 | 67/67 | 100% | | | |
| q | Proteus vulgaris group | 48 | 48 | 48/48 | 100% | | | | 48 | 48/48 | 100% | | | |
| r | Providencia rettgeri | 55 | 50 | 50/55 | 91% | 5 | 5/55 | 9% | 55 | 55/55 | 100% | | | |
| | Providencia stuartii | 56 | 54 | 54/56 | 96% | | | | 54 | 54/56 | 96% | | | 2^{1)} |
| | Pseudomonas aeruginosa | 78 | 78 | 78/78 | 100% | | | | 78 | 78/78 | 100% | | | |
| s | Pseudomonas fluorescens group | 19 | 19 | 19/19 | 100% | | | | 19 | 19/19 | 100% | | | |
| t | Pseudomonas putida group | 61 | 47 | 47/61 | 77% | 13 | 13/61 | 21% | 60 | 60/61 | 98% | 1^{3)} | | |
| u | Salmonella sp | 86 | 86 | 86/86 | 100% | | | | 86 | 86/86 | 100% | | | |
| v | Serratia liquefaciens | 28 | 28 | 28/28 | 100% | | | | 28 | 28/28 | 100% | | | |
| w | Serratia marcescens | 69 | 69 | 69/69 | 100% | | | | 69 | 69/69 | 100% | | | |
| x | Stenotrophomonas maltophilia | 76 | 75 | 75/76 | 99% | | | | 75 | 75/76 | 99% | 1^{3)} | | |
| | Yersinia enterocolitica | 44 | 43 | 43/44 | 98% | | | | 43 | 43/44 | 98% | | 1^{1)} | |
| y | Yersinia pseudotuberculosis | 4 | 4 | 4/4 | 100% | | | | 4 | 4/4 | 100% | | | |
| | All Isolates | 2263 | 2174 | | | 48 | | | 2222 | | | 2 | 16 | 23 |

{25}

1) For detailed information please refer to table 1A and 1B; below.
2) For detailed information please refer to table 2; below.
3) For detailed information please refer to table 3; below.
4) For detailed information please refer to table 4; below.

Table 1A: Summary of Incorrect Identifications

| Species | noID | Discordant | |
| --- | --- | --- | --- |
| | | Species Confirmation high confidence log(score) ≥2.0 | Species Confirmation low confidence log(score) ≥1.7 ... <2.0 |
| Achromobacter xylosoxidans | | | 1 |
| Citrobacter amalonaticus complex | | 2 | |
| Enterobacter cloacae complex | | 5 | |
| Providencia rettgeri | 2 | | |
| Providencia stuartii | | | 2 |
| Yersinia enterocolitica | | 1 | |

Table 1B: Details of Incorrect Identifications

| | MBT-CA System result | Log (score) | Reference Method |
| --- | --- | --- | --- |
| Achromobacter xylosoxidans | Achromobacter xylosoxidans | 1.810 | Bordetella bronchiseptica |
| C. amalonaticus complex | C. amalonaticus complex | 2.584 | Ref. Method did not confirm any species |
| | C. amalonaticus complex | 2.513 | Ref. Method did not confirm any species |
| E. cloacae complex | E. cloacae complex | 2.240 | Enterobacter amnigenus |
| | E. cloacae complex | 2.461 | Enterobacter amnigenus |
| | E. cloacae complex | 2.490 | Enterobacter amnigenus |
| | E. cloacae complex | 2.447 | Enterobacter amnigenus |
| | E. cloacae complex | 2.415 | Enterobacter amnigenus |
| Providencia stuartii / rettgeri | P.stuartii | 1.806 | Providencia rettgeri |
| | P.stuartii | 1.899 | Providencia rettgeri |
| Yersinia enterocolitica | Yersinia enterocolitica | 2.043 | Yersinia aldovae* |

* Y.aldovae was formerly reported as a member of the "Y.enterocolitica like group X2".

{26}

27

Table 2: Summary of Incorrect Identifications (Haemophilus haemolyticus / influenzae)

| Species | # of Isolates | Correct Identification | | | | | | | | | No ID | Discordant | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Species Confirmation high confidence log(score) ≥2.0 | | | Species Confirmation low confidence log(score) ≥1.7 ... <2.0 | | | Combined Performance | | | | Species Confirmation high confidence log(score) ≥2.0 | Species Confirmation low confidence log(score) ≥1.7 ... <2.0 |
| Haemophilus haemolyticus | 20 | | | | | | | 0 | 0/20 | 0% | 20 | | |
| Haemophilus influenzae | 95 | 63 | 63/95 | 66% | 4 | 4/95 | 4% | 67 | 67/95 | 71% | | 8 | 20 |

Haemophilus haemolyticus was removed from the claimed organisms during the clinical study. Therefore, it has to be considered to be falsely identified as H. influenza; see matching hint j.

Table 3: Details of not Identified strains

| | MBT-CA System result | log(score) | Reference Method |
| --- | --- | --- | --- |
| Pseudomonas putida_group | no ID | 1.530 | Pseudomonas putida_group |
| Stenotrophomonas maltophilia | no ID | 1.638 | Stenotrophomonas maltophilia |

{27}

Table 4: Matching Hint Table

| Matching Hint | Species / Group / Complex | Strains Included in Database | Different species are potentially associated with the displayed identification. Based on 16S rRNA gene sequencing a secure species differentiation between the displayed species is difficult. Confirmatory tests are required to differentiate between listed organisms. | Matching Hint Table |
| --- | --- | --- | --- | --- |
| a | Achromobacter xylosoxidans | A. xylosoxidans | A.xylosoxidans, A.denitrificans, A.insolitus, A.marplatensis, A.ruhlandii, A.spanius | All species associated with the displayed identification have been reported as isolated from human specimens. |
| b | Acinetobacter_ baumannii complex [4] | A. baumannii A. calcoaceticus A. pittii A. nosocomialis | A.baumannii, A.calcoaceticus, A.pittii, A.nosocomialis | Differentiation between members of the displayed groups or complexes based on 16S rRNA gene sequencing is difficult, whereas they are grouped together. All species associated with the displayed identification have been reported as isolated from human specimens. |
| | Acinetobacter lwoffii | A. lwoffii | | No matching hint required. |
| | Acinetobacter radioresistens | A. radioresistens | | No matching hint required. |
| | Acinetobacter ursingii | A. ursingii | | No matching hint required. |
| c | Aeromonas sp | A. allosaccharophila A. caviae A. culicicola A. hydrophila A. ichthiosmia A. sobria A. veronii | A.allosaccharophila, A.aquariorum, A.caviae, A.culicicola, A.enteropelogenes, A.fluvialis, A.hydrophila, A.ichthiosmia, A.jandaei, A.media, A.punctata, A.rivuli, A.sanarelli, A.sobria, A.taiwanensis, A.veronii. | Differentiation between members of the displayed groups or complexes based on 16S rRNA gene sequencing is difficult, whereas they are grouped together. A.hydrophila, A. caviae and A.sobria (A.veronii bv sobria) are the most frequently reported species associated with human infection. A.ichthiosmia is considered a synonym of A.veronii and A.punctata is considered a synonym of A.caviae. |
| | Alcaligenes faecalis | A. faecalis | | No matching hint required. |
| d | Burkholderia gladioli | B. gladioli | B.gladioli, B.glumae, B.caryopylii | B. glumae and B. caryopylii have not been reported as isolated with human specimens. |

{28}

| Matching Hint | Species / Group / Complex | Strains Included in Database | Different species are potentially associated with the displayed identification. Based on 16S rRNA gene sequencing a secure species differentiation between the displayed species is difficult. Confirmatory tests are required to differentiate between listed organisms. | Matching Hint Table |
| --- | --- | --- | --- | --- |
| | Burkholderia multivorans | B. multivorans | B.multivorans | No matching hint required. |
| e | Burkholderia_cepacia complex | B. ambifaria
B. anthina
B. cenocepacia
B. cepacia
B. diffusa
B. dolosa
B. lata
B. latens
B. metallica
B. pyrrocinia
B. seminalis
B. stabilis
B. vietnamiensis | B.ambifaria, B.anthina, B.cenocepacia, B.cepacia, B.diffusa, B.dolosa, B.lata, B.latens, B.metallica, B.pyrrocinia, B.seminalis, B.stabilis, B.vietnamiensis | Differentiation between members of the displayed groups or complexes based on 16S rRNA gene sequencing is difficult, whereas they are grouped together.
All species associated with the displayed identification have been reported as isolated from human specimens. |
| f | Citrobacter amalonaticus_complex | C. amalonaticus
C. farmeri | C.amalonaticus, C.farmeri | Differentiation between members of the displayed groups or complexes based on 16S rRNA gene sequencing is difficult, whereas they are grouped together. Both species associated with the displayed identification have been reported as isolated from human specimens. |
| | Citrobacter koseri | C. koseri | | No matching hint required. |
| g | Citrobacter_freundii complex | C. braakii
C. freundii
C. gillenii
C. murliniae
C. rodentium
C. sedlakii
C. werkmannii
C. youngae | C.braakii, C.freundii, C.gillenii, C.murliniae, C.rodentium, C.sedlakii, C.werkmannii, C.youngae | Differentiation between members of the displayed groups or complexes based on 16S rRNA gene sequencing is difficult, whereas they are grouped together.
C. rodentium has not been reported as isolated from human specimens. |
| | Eikenella corrodens | E. corrodens | | No matching hint required. |
| | Enterobacter aerogenes | E. aerogenes | | No matching hint required. |

29

{29}

| Matching Hint | Species / Group / Complex | Strains Included in Database | Different species are potentially associated with the displayed identification. Based on 16S rRNA gene sequencing a secure species differentiation between the displayed species is difficult. Confirmatory tests are required to differentiate between listed organisms. | Matching Hint Table |
| --- | --- | --- | --- | --- |
| h | Enterobacter cloacae_complex | E. asburiae
E. cancerogenus
E. cloacae
E. hormaechei
E. kobei
E. ludwigii | E.asburiae, E.cancerogenus, E.cloacae, E.cowanii, E.hormaechei, E.kobei, E.ludwigii, E.mori, E.nimipressuralis, E.soli | Differentiation between members of the displayed groups or complexes based on 16S rRNA gene sequencing is difficult, whereas they are grouped together.
E. mori and E. soli have not been reported as isolated from human specimens. |
| i | Escherichia coli | E. coli | E.albertii, E.coli, E.fergusonii, Shigella spp. | All species associated with the displayed identification have been reported as isolated from human specimens. |
| j | Haemophilus influenzae | H. influenzae | H.aegyptius, H.influenzae, H.haemolyticus | H. haemolyticus is reported as a commensal of the oropharynx and is generally considered nonpathogenic; it can be a rare pathogen. H. aegyptius has been reported as a causative agent of conjunctivitis and a rare cause of systemic disease.
Further, some rare isolates that belong to the genus Haemophilus and cannot be assigned to a validated species may be identified by the H. influenzae reference spectrum. |
| | Haemophilus parainfluenzae | H. parainfluenzae | | No matching hint required. |
| k | Hafnia alvei | Hafnia alvei | H.alvei, H.paralvei, Obesumbacterium proteus | Obesumbacterium proteus is most commonly associated with brewery spoilage and has not reported as isolated from human specimens |

30

{30}

| Matching Hint | Species / Group / Complex | Strains Included in Database | Different species are potentially associated with the displayed identification. Based on 16S rRNA gene sequencing a secure species differentiation between the displayed species is difficult.
Confirmatory tests are required to differentiate between listed organisms. | Matching Hint Table |
| --- | --- | --- | --- | --- |
| l | Klebsiella pneumoniae | Klebsiella pneumoniae | K.pneumoniae, K.granulomatis, K.singaporensis, K.variicola | All species associated with the displayed identification have been isolated from human specimens; K. pneumoniae is the most common species reported as isolated from human specimens. |
| m | Klebsiella_oxytoca
Raoultella_ornithinolytica | K. oxytoca
R. ornithinolytica | K.oxytoca, R.ornithinolytica, R.planticola | Differentiation between members of the displayed groups or complexes based on 16S rRNA gene sequencing is difficult, whereas they are grouped together. All species associated with the displayed identification have been reported as isolated from human specimens. |
| | Moraxella_sg_Branhamella
catarrhalis | M. catarrhalis | | No matching hint required. |
| n | Moraxella_sg_Moraxella
osloensis | M. osloensis | Enhydrobacter aerosaccus, Moraxella osloensis | The rare species Enhydrobacter aerosaccus is closely related to Moraxella osloensis, and has not reported as isolated from human specimens. |
| o | Morganella morganii | Morganella morganii | M.morganii, M.psychrotolerans | Both species associated with the displayed identification have been reported as isolated from human specimens. |
| p | Pantoea agglomerans | Pantoea agglomerans | P.agglomerans, P.anthophila, P.brenneri, P.conspicua, P.eucalypti, P.vagans | All species associated with the displayed identification have been reported as isolated from human specimens. Pantoea agglomerans, is the most commonly reported Pantoea species isolated from human specimens. |
| | Pasteurella multocida | P. multocida | | No matching hint required. |
| | Proteus mirabilis | P. mirabilis | | No matching hint required. |

31

{31}

| Matching Hint | Species / Group / Complex | Strains Included in Database | Different species are potentially associated with the displayed identification. Based on 16S rRNA gene sequencing a secure species differentiation between the displayed species is difficult. Confirmatory tests are required to differentiate between listed organisms. | Matching Hint Table |
| --- | --- | --- | --- | --- |
| q | Proteus vulgaris_group | P. hauseri
P. penneri
P. vulgaris | P.hauseri, P.penneri, P.vulgaris | Differentiation between members of the displayed groups or complexes based on 16S rRNA gene sequencing is difficult, whereas they are grouped together. All species have been reported as isolated from human specimens. |
| r | Providencia rettgeri | Providencia rettgeri | P.rettgeri, P.alcalifaciens, P.burhodogranariea, P.heimbachae, P.rustigianii, P.vermicola | P. burhodogranariea and P. vermicola have not been reported as isolated from human specimens |
| | Providencia stuartii | P. stuartii | | No matching hint required. |
| | Pseudomonas aeruginosa | P. aeruginosa | | No matching hint required. |
| s | Pseudomonas fluorescens_group | P. congelans
P. corrugata
P. extremorientalis
P. fluorescens
P. gessardii
P. libanensis
P. mandelii
P. marginalis
P. migulae
P. mucidolens
P. orientalis
P. poae
P. rhodesiae
P. synxantha
P. tolaasii
P. trivialis
P. veronii | P.congelans, P.corrugata, P.extremorientalis, P.fluorescens, P.gessardii, P.libanensis, P.mandelii, P.marginalis, P.migulae, P.mucidolens, P.orientalis, P.poae, P.rhodesiae, P.synxantha, P.tolaasii, P.trivialis, P.veronii | Differentiation between members of the displayed groups or complexes based on 16S rRNA gene sequencing is difficult, whereas they are grouped together. Members of the P. fluorescens group are environmental organisms. P. fluorescens is the most commonly isolated species reported as isolated from human specimens. |

32

{32}

| Matching Hint | Species / Group / Complex | Strains Included in Database | Different species are potentially associated with the displayed identification. Based on 16S rRNA gene sequencing a secure species differentiation between the displayed species is difficult.
Confirmatory tests are required to differentiate between listed organisms. | Matching Hint Table |
| --- | --- | --- | --- | --- |
| t | Pseudomonas putida_group | P. fulva
P. monteilii
P. mosselii
P. plecoglossicida
P. putida | P.fulva, P.monteilii, P.mosselii, P.plecoglossicida, P.putida | Differentiation between members of the displayed groups or complexes based on 16S rRNA gene sequencing is difficult, whereas they are grouped together.
Members of the P. putida group are environmental organisms. P. putida is the most commonly isolated species reported as isolated from human specimens. |
| u | Salmonella sp | Salmonella sp | | Identification is possible on genus level only. |
| v | Serratia liquefaciens | Serratia liquefaciens | S.liquefaciens,
S.proteamaculans,
S.grimesii, S.plymutica,
S.ficaria | All species associated with the displayed identification have been reported as isolated from human specimens. |
| w | Serratia marcescens | Serratia marcescens | S.marcescens,
S.nematodiphila, S.ureilytica | S. nematodiphila and S. ureilytica have not been reported as isolated from human specimens. |
| x | Stenotrophomonas maltophilia | S. maltophilia
Pseudomonas beteli
Pseudomonas hibiscola
Pseudomonas geniculata | S.maltophilia, Pseudomonas beteli, Pseudomonas hibiscola, Pseudomonas geniculata | S. maltophilia, P. beteli, P. hibiscola, P. geniculata are synonymously used taxonomical names. |
| | Yersinia enterocolitica | Y. enterocolitica | | No matching hint required. |
| y | Yersinia pseudotuberculosis | Y. pseudotuberculosis | Y.pestis,
Y.pseudotuberculosis,
Y.similis | Y. pestis is a select agent and should be ruled out; handle isolate with extreme caution and handle in accordance with local, state, and federal accrediting organizations’ requirements as applicable. All species associated with the displayed identification have been reported as isolated from human specimens. |

33

{33}

b. Clinical specificity: See clinical sensitivity results
c. Other clinical supportive data (when a. and b. are not applicable):

4. Clinical cut-off:
See Assay cut-off

5. Expected values/Reference range:
See Assay cut-off

N. Instrument Name:
MBT CA System

O. System Descriptions:
1. Modes of Operation:

After the sample has been applied to the target plate and the spots have been manually identified in the software, organism identification is completely automated. A mass spectrum is acquired by the MALDI Biotyper CA System from the unknown organisms and is transformed into a numerical list of peak intensity and mass to charge ratio. Using an algorithm, the peak list is compared to reference peak lists of organisms in the reference library (database) and a log(score) value between 0.00 and 3.00 is calculated. The log(score) value ranges reflect the probability of organism identification. Results should be reviewed by a trained microbiologist and final organism identification should be based on all relevant information available. This information includes, but is not limited to, Gram staining, colony morphology, growth characteristics, sample matrix, or other factors that might impact organism identification.

The workflow to perform the test is summarized in the table below. The User is responsible for all sample preparation steps prior to inserting the target plate into the mass spectrometer. Sample preparation steps are described in the "MALDI Biotyper CA System System Package Insert Reference Library." After the user has prepared the target plate according to the package insert the Sample-IDs are manually entered into the system software which links a target plate spot position with a sample ID. After these steps are complete the sample analysis proceeds automatically through Step 14 (below) when the results report is presented to the user.

{34}


Before any MALDI Biotyper CA System analysis is started a quality control (QC) step is mandatory. This QC is executed using a sample preparation of an IVD Bacterial Test Standard (IVD BTS). The respective IVD BTS mass spectrum contains eight well known peaks which are used for an automatic calibration and for checking whether the instrument acquires spectra of sufficient quality.


## 2. Software:

FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types:

Yes ☐ X ☐ or No ☐

Level of Concern:

Moderate

## Software Description:

The MALDI Biotyper CA System software runs together with the flexControl software on the same computer which is referred to as the instrument control computer. Accordingly the link between the MALDI Biotyper CA System and the flexControl software occurs as a communication of processes running on the same computer. The applied communication

{35}

protocol is COM where flexControl provides the COM server while MALDI Biotyper CA System acts as COM client. As shown in the figure below:


The MALDI Biotyper CA System software has a three layer architecture. The components of the single layers run independently on the same computer and exchange information via various interfaces. The appropriate protocols used in the interfaces are selected according to special requirements of the participating sides.

The interface between the MALDI Biotyper CA System application and the MALDI Biotyper CA System server uses language independent protocols. The interface between the MALDI Biotyper CA System server and the MBT-DB server uses a standard protocol for accessing relational databases.


In the MALDI Biotyper CA System software the user creates a run by assigning the identifier of the prepared test organisms to the appropriate target plate positions. The run name automatically contains the unique target plate ID. After acquisition and analysis, the MALDI Biotyper CA System client outputs a report with the analysis results.

The flexControl software is used to control the microflex LT/SH mass spectrometer and the automated acquisition of mass spectra and includes the embedded firmware for the electronics in the microflex LT/SH instrument. Control involves setting and monitoring parameters such as high voltages, moving the sample carrier, docking and loading and saving acquisition methods. Automated tasks include loading the specified acquisition method, moving the sample carrier to the defined position and acquiring and storing a spectrum from that position on the target. In the MALDI Biotyper CA System workflow, the flexControl software is controlled by the MBT software and does not display a user interface. A functional diagram of the flexControl software is shown below:

{36}


## Architecture Design Chart: Acceptable

The MALDI Biotyper CA System consists of the following main components and the functional relationship between these components is illustrated in the architecture design chart below:

- microflex LT/SH L T/SH mass spectrometer
- Computer running the MBT -CA software
- MSP 48 target polished steel plates (Target)
- US IVD matrix HCCA (on prepared target spots)
- US IVD Bacterial Test Standard (US IVD BTS) (on a portion of the prepared target spots)

{37}


# Software Requirements Specifications (SRS): Acceptable

## MALDI Biotyper CA System SRS

The MALDI Biotyper CA System SRS defines the requirements for the MALDI Biotyper CA System software package. This package consists of several components designed according to a client / server structure. Only the client component is directly accessible by the end user via a graphical user interface (GUI). The MALDI Biotyper CA System client communicates with the mass spectrometer control and acquisition software (flexControl) as well as with its server components. Product requirements, interface requirements, performance requirements, design constraints, and a number of system attributes are included in the SRS.

## Flexcontrol SRS

This document defines the requirements for the flexControl software from the MALDI Biotyper CA System perspective. The flexControl software is used to control the microflex LT/SH mass spectrometer and the automated acquisition of mass spectra. Product requirements, interface requirements, performance…