(291 days)
No
The document describes a PCR test kit and associated interpretive software, but there is no mention of AI or ML being used in the analysis or interpretation process. The software is described as "automated interpretive software" but this does not necessarily imply AI/ML.
No.
The device is an in vitro diagnostic (IVD) test kit intended for the qualitative detection of target DNA sequences for Bacillus anthracis, used as an aid in diagnosis, not for therapy or treatment.
Yes
Explanation: The "Intended Use/Indications for Use" section explicitly states that the kit is an "in vitro diagnostic (IVD) detection" kit and is "intended for use as an aid in the diagnosis of anthrax infection."
No
The device description explicitly states that the kit includes reagents (lyophilized in a 96-well plate format) and is intended for use with a specific hardware instrument (ABI 7500 Fast Dx Real-Time PCR Instrument). While it includes interpretive software, it is not solely software.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Explicit Statement: The "Intended Use / Indications for Use" section explicitly states: "The Applied Biosystems Bacillus anthracis Detection Kit is a real-time polymerase chain reaction (PCR) test kit intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences for Bacillus anthracis, or BA)."
- Device Description: The "Device Description" section also reiterates this: "The Applied Biosystems™ Bacillus anthracis Detection Kit is a multiplexed real-time polymerase chain reaction (PCR) test kit intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences for B. anthracis."
- Intended Use: The intended use is to detect target DNA sequences for Bacillus anthracis in human specimens (whole blood and blood culture) to aid in the diagnosis of anthrax infection. This is a classic definition of an in vitro diagnostic device.
- Specimen Type: It is designed to test human biological specimens (blood).
- Laboratory Setting: It is intended for use in CLIA-certified high-complexity laboratories, which are settings where IVD tests are typically performed.
- Regulatory Context: The mention of being used in response to a confirmed Bacillus anthracis event and in accordance with public health authorities' guidelines further indicates its role in a diagnostic context.
- Comparison to Predicate Device: The mention of a predicate device (K170883; BioFire Defense, Inc. FilmArray NGDS Warrior Panel) which is also an IVD, supports the classification of this device as an IVD.
Therefore, based on the provided text, the Applied Biosystems Bacillus anthracis Detection Kit is clearly identified and intended as an In Vitro Diagnostic device.
N/A
Intended Use / Indications for Use
The Applied Biosystems Bacillus anthracis Detection Kit is a real-time polymerase chain reaction (PCR) test kit intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences for Bacillus anthracis, or BA). The Applied Biosystems Bacillus anthracis Detection Kit is intended to test human whole blood (EDTA) specimens and blood culture specimens with growth detected by a continuous monitoring blood culture system. Blood culture specimens must be determined to contain gram-positive bacilli by Gram stain prior to testing of whole blood specimens must be performed concomitantly with standard of care blood culture.
The Applied Biosystems Bacillus anthracis Detection Kit is indicated for use in CLIA-certified high-complexity laboratories in response to a confirmed Bacillus anthracis event only in accordance with the guidelines provided by public health authorities prior to or during a public health emergency. Testing with the Applied Biosystems Bacillus anthracis Detection Kit must only be performed when public health authorities have determined the need for this test must only be used with specimens from individuals with clinical signs and symptoms of B. anthracis infection and who have either been exposed to B. anthracis or may have been exposed to B. anthracis.
The Applied Biosystems Bacillus anthracis Detection Kit is intended for use as an aid in the diagnosis of anthrax infection and results are for the presumptive identification of Bacillus anthracis. The diagnosis of B. anthracis infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidence, in addition to the identification of B. anthracis from cultures or directly from clinical specimens. The definitive identification of B. anthracis requires additional testing and confirmation procedures in consultation with the appropriate public health authorities for whom reports may be required.
The Applied Biosystems Bacillus anthracis Detection Kit has not been clinically evaluated with specimens collected from individuals with B. anthracis infection or those presumed to B. anthracis. 'B. anthracis Not detected' results do not preclude infection with Bacillus anthracis and should not be used as for diagnosis, treatment, or other patient management decisions.
Laboratories implementing this test must have the appropriate biosafety equipment, personal protective equipment (PPE), containment facilities and personnel trained in the safe handling of diagnostic clinical specimens potentially containing B. anthracis. Anthrax is a nationally notifiable disease caused by a biothreat microbial agent and must be reported to public health authorities.
The distribution of in vitro diagnostic devices for Bacillus spp. detection is limited to laboratories that follow public health guidelines that address appropriate biosafety conditions, interpretation of test results, and coordination of findings with public health authorities.
The Applied Biosystems Bacillus anthracis Detection Kit is intended for use with the AB1 7500 Fast Dx Real-Time PCR Instrument with analysis using the Applied Biosystems Bacillus anthracis Interpretive Software (BaIS).
Product codes (comma separated list FDA assigned to the subject device)
QIF, OOI
Device Description
The Applied Biosystems™ Bacillus anthracis Detection Kit is a multiplexed real-time polymerase chain reaction (PCR) test kit intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences for B. anthracis. Reagents are lyophilized in a 96-well plate format as a fully formulated Mastermix and are stable at room temperature for up to one year. The kit is specifically designed for performing real-time PCR using the Applied Biosystems (ABI) 7500 Fast Dx instrument and software, with nucleic acids extracted from clinical specimens using a Qiagen manual extraction method or Roche MagNA Pure automated extraction methods. An automated interpretative software component (BalS) is included in the kit but supplied separately and operates on a computer(s) that is separate from the ABI 7500 Fast Dx computer.
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
Not Found
Indicated Patient Age Range
Not Found
Intended User / Care Setting
CLIA-certified high-complexity laboratories (commercial diagnostic laboratories, hospital-based laboratories and clinical institutions) in the event of a public health emergency.
Description of the training set, sample size, data source, and annotation protocol
Not Found
Description of the test set, sample size, data source, and annotation protocol
Not Found
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Analytical Sensitivity:
- Limit of Detection (LOD) determined for Whole Blood, Blood Culture (Aerobic and Anaerobic) for Qiagen DSP DNA Blood Mini Kit and Roche MagNA Pure.
- 1612 technical replicates tested to determine LOD.
- LOD values ranged from 50 CFU/mL (Roche MagNA Pure, Whole Blood) to 10,000 CFU/mL (Qiagen DSP DNA Blood Mini Kit, Blood Culture Aerobic and Anaerobic).
Analytical Inclusivity:
- Tested 24 different Bacillus anthracis strains.
- Each strain spiked into whole blood (K2 EDTA) at 3X LOD and processed using both Qiagen and Roche extraction methods in triplicate.
- Detection rate for both extraction methods was 100% for B. anthracis strains with both plasmid targets.
- For three strains with only one plasmid target, the assay generated 'Bacillus anthracis suspected' results.
Inclusivity in silico Analysis Summary:
- 144 Bacillus anthracis genome assemblies from NCBI.
- Local BLAST performed with imported assemblies against assay amplicon sequence.
- Results indicated assemblies without hits contained only chromosome sequences or alternate plasmids, or low plasmid coverage.
Analytical Exclusivity:
- Specificity evaluated by testing 154 non-target organisms (near-neighbors, other bacteria, viruses, fungi).
- Initial no detection rate was 95.68%. Repeat testing conducted for three organisms. Final no detection rate for exclusivity testing was 100%.
- Some known Bacillus cereus strains containing a pXO1-like plasmid resulted in 'Bacillus anthracis suspected'.
Exclusivity in silico Analysis Summary:
- BLAST search against NCBI nr, wgs, and genome databases for assay primers and probes.
- Results filtered for exclusion of B. anthracis hits, >=80% identity, and >=80% HSP length.
- One organism (Bacillus cereus m1550) identified with 100% identity to pXO1 assay primer and probe sequences.
- Two organisms (B. cereus AH1272 and AH1273) identified with 100% identity to pXO2 assay probe, but with SNPs in primer binding sequences.
Interfering Substances:
- 50 potentially interfering substances (endogenous, exogenous, technique-specific) tested.
- Technique-specific substances found to interfere: MagNA Pure Wash Buffer I, MagNA Pure Lysis/Binding Buffer, MagNA Pure Proteinase K, 10% Bleach, Fisherbrand™ DNase Displace, QIAGEN Protease, QIAGEN Buffer AL, QIAGEN Buffer AW1 (with and without EtOH), EtOH at greater than 5%.
Microbial Interference:
- Detection of B. anthracis in presence of other clinically-relevant organisms.
- Detection rate of B. anthracis was 100% in the presence of potentially interfering organisms in whole blood and blood culture.
Reproducibility:
- Seven panel members tested twice a day by three teams of two operators on five non-consecutive days using two ABI 7500 Fast Dx instruments across three reagent lots.
- One low positive sample returned negative results (sample prep error).
- One high positive sample returned 'Bacillus anthracis suspected' (pXO1 assay failed to amplify), but repeated PCR was 'Bacillus anthracis detected'.
- No false positive events out of 270 PCR tests of negative samples.
- 24 initial indeterminate results, all determined negative by supervisor review. No invalid results.
Carry-Over/Cross-contamination:
- Automated method (Roche MagNA Pure): 279 samples in nine runs. Total of two false positive events out of 135 negative samples analyzed.
- Manual method (Qiagen DSP): 279 samples in twelve batches. No false positive events out of 139 negative samples analyzed.
- Surface monitoring control swabs identified contamination events on one BSC surface and five MagNA Pure surfaces (automated method) and one BSC location (manual method).
Clinical Specificity:
- Study used 401 blood culture specimens and 439 whole blood specimens.
- All samples assumed to be negative for Bacillus anthracis.
- Generated 'Bacillus anthracis not detected' results for all samples.
- Demonstrating 100% negative percent agreement when compared to the expected negative result.
Clinical Sensitivity:
- Used febrile whole blood specimens, spiked with Bacillus anthracis at or near LOD.
- 87 low-positive whole blood specimens.
- 84 of 87 samples generated 'Bacillus anthracis detected' results.
- Three specimens initially generated 'Bacillus anthracis suspected' due to detection of only one plasmid target.
- Final positive percent agreement was 96% (Cl: 90.4-96,5%).
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
Analytical Exclusivity: Final no detection rate for exclusivity testing was 100%.
Carry-Over/Cross-contamination (Automated): specificity rate of 98.5%.
Carry-Over/Cross-contamination (Manual): specificity rate of 100%.
Clinical Specificity: 100% negative percent agreement.
Clinical Sensitivity: Final positive percent agreement was 96% (Cl: 90.4-96,5%).
Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.
Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.
Predetermined Change Control Plan (PCCP) - All Relevant Information
Not Found
N/A
0
Image /page/0/Picture/0 description: The image contains the logos of the Department of Health and Human Services and the Food and Drug Administration (FDA). The Department of Health and Human Services logo is on the left, and the FDA logo is on the right. The FDA logo includes the letters "FDA" in a blue square, followed by the words "U.S. FOOD & DRUG ADMINISTRATION" in blue text.
October 1, 2019
MRIGlobal % Fran White President MDC Associates, LLC 180 Cabot Street Beverly, Massachusetts 01915
Re: K183462
Trade/Device Name: Applied Biosystems Bacillus anthracis Detection Kit Regulation Number: 21 CFR 866.4000 Regulation Name: Device to detect and identify biothreat microbial agents in human clinical specimens Regulatory Class: Class II Product Code: QIF, OOI Dated: December 13, 2018 Received: December 14, 2018
Dear Fran White:
We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.
If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.
Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's
1
requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.
For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).
Sincerely,
Kristian Roth, Ph.D. Branch Chief Bacterial Multiplex and Medical Countermeasures Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Ouality Center for Devices and Radiological Health
Enclosure
2
Indications for Use
510(k) Number (if known) K183462
Device Name
Applied Biosystems(TM) Bacillus anthracis Detection Kit
Indications for Use (Describe)
The Applied Biosystems Bacillus anthracis Detection Kit is a real-time polymerase chain reaction (PCR) test kit intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences for Bacillus anthracis, or BA). The Applied Biosystems Bacillus anthracis Detection Kit is intended to test human whole blood (EDTA) specimens and blood culture specimens with growth detected by a continuous monitoring blood culture system. Blood culture specimens must be determined to contain gram-positive bacilli by Gram stain prior to testing of whole blood specimens must be performed concomitantly with standard of care blood culture.
The Applied Biosystems Bacillus anthracis Detection Kit is indicated for use in CLIA-certified high-complexity laboratories in response to a confirmed Bacillus anthracis event only in accordance with the guidelines provided by public health authorities prior to or during a public health emergency. Testing with the Applied Biosystems Bacillus anthracis Detection Kit must only be performed when public health authorities have determined the need for this test must only be used with specimens from individuals with clinical signs and symptoms of B. anthracis infection and who have either been exposed to B. anthracis or may have been exposed to B. anthracis.
The Applied Biosystems Bacillus anthracis Detection Kit is intended for use as an aid in the diagnosis of anthrax infection and results are for the presumptive identification of Bacillus anthracis. The diagnosis of B. anthracis infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidence, in addition to the identification of B. anthracis from cultures or directly from clinical specimens. The definitive identification of B. anthracis requires additional testing and confirmation procedures in consultation with the appropriate public health authorities for whom reports may be required.
The Applied Biosystems Bacillus anthracis Detection Kit has not been clinically evaluated with specimens collected from individuals with B. anthracis infection or those presumed to B. anthracis. 'B. anthracis Not detected' results do not preclude infection with Bacillus anthracis and should not be used as for diagnosis, treatment, or other patient management decisions.
Laboratories implementing this test must have the appropriate biosafety equipment, personal protective equipment (PPE), containment facilities and personnel trained in the safe handling of diagnostic clinical specimens potentially containing B. anthracis. Anthrax is a nationally notifiable disease caused by a biothreat microbial agent and must be reported to public health authorities.
The distribution of in vitro diagnostic devices for Bacillus spp. detection is limited to laboratories that follow public health guidelines that address appropriate biosafety conditions, interpretation of test results, and coordination of findings with public health authorities.
The Applied Biosystems Bacillus anthracis Detection Kit is intended for use with the AB1 7500 Fast Dx Real-Time PCR Instrument with analysis using the Applied Biosystems Bacillus anthracis Interpretive Software (BaIS).
Type of Use (Select one or both, as applicable)
X Prescription Use (Part 21 CFR 801 Subpart D)
Over-The-Counter Use (21 CFR 801 Subpart C)
CONTINUE ON A SEPARATE PAGE IF NEEDED.
3
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4
510(k) SUMMARY
Date of Summary:
September 27, 2019
Product Name
Applied Biosystems™ Bacillus anthracis Detection Kit
Sponsor
MRIGlobal 425 Volker Boulevard Kansas City, Missouri 64110
Correspondent
MDC Associates, Inc. Fran White, President 180 Cabot Street Beverly, MA 01915 Phone: (978) 705 5011 Fax: (866) 540 3448 Email: regulatory@mdcassoc.com
Common Name
Device to detect and identify biothreat microbial agents in human clinical specimens
Product Classification
866.4000
Classification
QIF, Class II
Substantial Equivalency
| Characteristic | MRIGlobal
Applied Biosystems™ Bacillus anthracis
Detection Kit
(New Device) | BioFire Defense, Inc.
FilmArray® NGDS Warrior Panel
K170883
(Predicate Device) |
|--------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Similarities | | |
| Product Code | QIF | PRD |
| Intended Use | The Applied Biosystems Bacillus anthracis
Detection Kit is a real-time polymerase
chain reaction (PCR) test kit intended for
the qualitative in vitro diagnostic (IVD)
detection of target DNA sequences for
Bacillus anthracis ( B. anthracis , or BA). The
Applied Biosystems Bacillus anthracis
Detection Kit is intended to test human
whole blood (EDTA) specimens and blood
culture specimens with growth detected by
a continuous monitoring blood culture
system. Blood culture specimens must be
determined to contain gram-positive bacilli | The FilmArray® NGDS Warrior Panel is
a qualitative, multiplexed, nucleic acid-
based in vitro diagnostic test intended
for use with the FilmArray® 2.0 system.
The FilmArray® NGDS Warrior Panel
detects and identifies Bacillus
anthracis, Yersinia pestis, Francisella
tularensis, Coxiella burnetii, Ebola
virus, and Marburg virus nucleic acids
directly from human whole blood
(EDTA).
The FilmArray® NGDS Warrior Panel is
also intended to be used to test for |
| Characteristic | MRIGlobal
Applied Biosystems™ Bacillus anthracis
Detection Kit
(New Device) | BioFire Defense, Inc.
FilmArray NGDS Warrior Panel
K170883
(Predicate Device) |
| | by Gram stain prior to testing. Testing of
whole blood specimens must be performed
concomitantly with standard of care blood
culture. | Bacillus anthracis or Yersinia pestis
nucleic acids in blood cultures that are
determined to be positive either by an
automated system, by turbidity, or by
daily Gram stain. In addition, the
FilmArray® NGDS Warrior Panel may
also be used to detect and identify
Yersinia pestis and Francisella
tularensis nucleic acids directly from
sputum specimens. |
| | The Applied Biosystems Bacillus anthracis
Detection Kit is indicated for use in CLIA-
certified high-complexity laboratories in
response to a confirmed Bacillus anthracis
event only in accordance with the
guidelines provided by public health
authorities prior to or during a public health
emergency. Testing with the Applied
Biosystems Bacillus anthracis Detection Kit
must only be performed when public health
authorities have determined the need for
this test. The test must only be used with
specimens from individuals with clinical
signs and symptoms of B. anthracis
infection and who have either been
exposed to B. anthracis or may have been
exposed to B anthracis.
The Applied Biosystems Bacillus anthracis
Detection Kit is intended for use as an aid in
the diagnosis of anthrax infection and
results are for the presumptive
identification of Bacillus anthracis. The
diagnosis of B. anthracis infection must be
made based on history, signs, symptoms,
exposure likelihood, and other laboratory
evidence, in addition to the identification of
B. anthracis from cultures or directly from
clinical specimens. The definitive
identification of B. anthracis requires
additional testing and confirmation | The FilmArray® NGDS Warrior Panel is
intended to test individuals with signs
and symptoms of infection from
biothreat agents and/or individuals
who are at risk for exposure or may
have been exposed to these agents.
The FilmArray® NGDS Warrior Panel is
indicated as an aid in the diagnosis of
anthrax, plague, tularemia, Q fever,
and the hemorrhagic fevers caused by
Ebola and Marburg viruses, in
response to a suspected or confirmed
bioterrorism event or outbreaks. It is
for diagnostic use in conjunction with
other clinical, epidemiologic, and
laboratory data, in accordance with
the guidelines provided by the
appropriate Department of Defense
and public health authorities.
Results are for the presumptive
identification of Bacillus anthracis,
Yersinia pestis, Francisella tularensis,
Coxiella burnetii, Ebola virus, and
Marburg virus. The definitive
identification of Bacillus anthracis,
Yersinia pestis, Francisella tularensis, |
| | procedures in consultation with the
appropriate public health authorities for
whom reports may be required. | Coxiella burnetii, Ebola virus, and
Marburg virus requires additional
testing and confirmation procedures in
consultation with the appropriate |
| | The Applied Biosystems Bacillus anthracis
Detection Kit has not been clinically
evaluated with specimens collected from
individuals with B. anthracis infection or
those presumed to be exposed to B.
anthracis. 'Bacillus anthracis not detected'
results do not preclude infection with
Bacillus anthracis and should not be used as
the sole basis for diagnosis, treatment, or
other patient management decisions. | Department of Defense and public
health authorities for whom reports
may be necessary. |
| Characteristic | MRIGlobal
Applied Biosystems™ Bacillus anthracis
Detection Kit
(New Device) | BioFire Defense, Inc.
FilmArray NGDS Warrior Panel
K170883
(Predicate Device) |
| Methodology/
Technological
Principle | Real-time-PCR | Nested PCR with melt curve analysis |
| Specimen types | Whole Blood or Blood Culture | Whole Blood; Blood Culture (for
Bacillus anthracis and Yersinia pestis),
Sputum (for Yersinia pestis and
Francisella tularensis) |
| Assay Targets | DNA sequences unique to B. anthracis | Nucleic acid sequences unique to
Bacillus anthracis, Yersinia pestis,
Francisella tularensis, Coxiella
burnetii, Ebola virus, and Marburg
virus |
| Result Output | Qualitative detection of B. anthracis DNA | Qualitative detection of Bacillus
anthracis, Yersinia pestis, Francisella
tularensis, Coxiella burnetii, Ebola
virus, and Marburg virus nucleic acids |
| Test Interpretation | Automated test, interpretation, and report
generation | Automated test, interpretation, and
report generation |
| Differences | | |
| Multiplex
Capability | Multiplexed assay. Two gene targets and
internal process control run in single
reaction. | Multiplexed assay for detection of
Bacillus anthracis, Yersinia pestis,
Francisella tularensis, Coxiella |
| Characteristic | MRIGlobal
Applied Biosystems™ Bacillus anthracis
Detection Kit
(New Device) | BioFire Defense, Inc.
FilmArray NGDS Warrior Panel
K170883
(Predicate Device) |
| Sample Extraction | Qiagen QIAamp™ DNA Mini Blood Kit
(manual) and Roche MagNA Pure
Automated Nucleic acid Extraction
(automated) | Automated in FilmArray pouch |
| PCR Reagents | Lyophilized reagents are formulated as a
complete Mastermix and are reconstituted
upon sample addition with no requirement
to add water or buffer. | Reagents contained within FilmArray
pouch |
| Instrumentation | Applied BioSystems® 7500 Fast Dx
[K082562] | FilmArray 2.0 |
| Throughput | 96-well format allows for simultaneous
analysis of up to 93 patient samples (+
controls) for detection of two Bacillus
anthracis targets and an internal process
control on a single plate | One sample at a time. |
| User Complexity
End User | High Complexity
Targeted CLIA-certified, sentinel
laboratories (commercial diagnostic
laboratories, hospital-based laboratories
and clinical institutions) in the event of a
public health emergency | Low/Moderate Complexity
The FilmArray® NGDS Warrior Panel is
solely for use by United States
Department of Defense laboratories,
and laboratories designated by the
Department of Defense. |
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Page 5 of 12
Intended Use
The Applied Biosystems Bacillus anthracis Detection Kit is a real-time polymerase chain reaction (PCR) test kit intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences for Bacillus anthracis, or BA). The Applied Biosystems Bacillus anthracis Detection Kit is intended to test human whole blood (EDTA) specimens and blood culture specimens with growth detected by a continuous monitoring blood culture system. Blood culture specimens must be determined to contain gram-positive bacilli by Gram stain prior to testing. Testing of whole blood specimens must be performed concomitantly with standard of care blood culture.
The Applied Biosystems Bacillus anthracis Detection Kit is indicated for use in CLIA-certified highcomplexity laboratories in response to a confirmed Bacillus anthracis event only in accordance with the guidelines provided by public health authorities prior to or during a public health emergency. Testing with the Applied Biosystems Bacillus anthracis Detection Kit must only be performed when public health authorities have determined the need for this test. The test must only be used with specimens from individuals with clinical signs and symptoms of B. anthracis infection and who have either been exposed to B. anthracis or may have been exposed to B anthracis.
The Applied Biosystems Bacillus anthracis Detection Kit is intended for use as an aid in the diagnosis of anthrax infection and results are for the presumptive identification of Bacillus anthracis. The diagnosis of B. anthracis infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidence, in addition to the identification of B. anthracis from cultures or directly from clinical specimens. The definitive identification of B. anthracis requires additional testing and confirmation procedures in consultation with the appropriate public health authorities for whom reports may be required.
The Applied Biosystems Bacillus anthracis Detection Kit has not been clinically evaluated with specimens collected from individuals with B. anthracis infection or those presumed to be exposed to B. anthracis. 'Bacillus anthracis not detected' results do not preclude infection with Bacillus anthracis and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.
Laboratories implementing this test must have the appropriate biosafety equipment, personal protective equipment (PPE), containment facilities and personnel trained in the safe handling of diagnostic clinical specimens potentially containing B. anthrax is a nationally notifiable disease caused by a biothreat microbial agent and must be reported to public health authorities.
The distribution of in vitro diagnostic devices for Bacillus spp. detection is limited to laboratories that follow public health guidelines that address appropriate biosafety conditions, interpretation of test results, and coordination of findings with public health authorities.
9
Page 6 of 12
The Applied Biosystems Bacillus anthracis Detection Kit is intended for use with the ABI 7500 Fast Dx Real-Time PCR Instrument with analysis using the Applied Biosystems Bacillus anthracis Interpretive Software (BaIS).
Methodology
The Applied Biosystems™ Bacillus anthracis Detection Kit is a multiplexed real-time polymerase chain reaction (PCR) test kit intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences for B. anthracis. Reagents are lyophilized in a 96-well plate format as a fully formulated Mastermix and are stable at room temperature for up to one year. The kit is specifically designed for performing real-time PCR using the Applied Biosystems (ABI) 7500 Fast Dx instrument and software, with nucleic acids extracted from clinical specimens using a Qiagen manual extraction method or Roche MagNA Pure automated extraction methods. An automated interpretative software component (BalS) is included in the kit but supplied separately and operates on a computer(s) that is separate from the ABI 7500 Fast Dx computer.
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10
Performance Data
Analytical Sensitivity
Limit of Detection (expressed as CFU/mL) was determined using Bacillus anthracis Ames, tested with twenty technical replicates per reagent lot of the Applied Biosystems™ Bacillus anthracis Detection Kit to achieve >95% detection when averaged across three reagent lots. Limit of Detection (LOD) was independently confirmed for whole blood, aerobic blood culture, and anaerobic blood culture for each extraction method, Qiagen DSP DNA Blood Mini Kit and Roche MagNA Pure using the Roche MagNA Pure LC 2.0 Robot. One thousand six hundred and twelve technical replicates were tested to determine LOD. See Table 1 below.
Table 1. Limit of Detection (expressed as CFU/mL) determined for Whole Blood, Blood Culture, Aerobic and Anaerobic per Extraction Method
| Extraction Method | Matrix | Limit of Detection
(CFU/mL) |
|----------------------------------|-----------------------------|--------------------------------|
| Qiagen DSP DNA
Blood Mini Kit | Whole Blood | 150 |
| | Blood Culture,
Aerobic | 10,000 |
| | Blood Culture,
Anaerobic | 10,000 |
| Roche MagNA Pure | Whole Blood | 50 |
| | Blood Culture,
Aerobic | 2270 |
| | Blood Culture,
Anaerobic | 3040 |
Analytical Inclusivity
Quantified stocks of 24 different Bacillus anthracis strains representing geographic, temporal, genotypic, and phenotypic diversity of the species were prepared in culture under ATCCrecommended conditions and quantitated by enumeration on agar media to evaluate inclusivity using the Applied Biosystems™ Bacillus anthracis Detection Kit. Each BA inclusivity panel member was spiked into whole blood (K2 EDTA), at 3X the established LoD (expressed as CFU/mL) and processed using both extraction methods in triplicate – Roche MagNa Pure (MNP) extraction robot and Qiagen DSP DNA Blood Mini Kit per the kit instructions. The detection rate for both extraction methods was 100% for B. anthracis strains that carry both plasmid targets. For three strains that are known to carry only one plasmid target, the assay generated 'Bacillus anthracis suspected' results as expected.
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Inclusivity in silico Analysis Summary
All available Bacillus anthracis genome assemblies were downloaded from NCBI and imported into the CLC Genomics Workbench (Qiagen) for a total of 144 panel members. A local BLAST within the software was then performed with imported assemblies against the amplicon sequence for each assay. A list of assemblies without amplicon hits was generated for each assay to further investigate assemblies not producing hits. The results indicated that assemblies that did not produce a hit contained only chromosome sequences or only the alternate plasmid. Several assemblies showed very low coverage of plasmids, likely due to absence of the plasmid but presence of conserved regions in the genome, which have homology to small regions of plasmid sequences.
Analytical Exclusivity
Specificity of the Applied Biosystems™ Bacillus anthracis Detection Kit was evaluated by testing a panel of 154 non-target organisms which included near-neighbors and other bacteria, virus and fungi that might present with similar clinical presentation to those patients suspected of anthrax. For instances where bacteria, virus or fungi were not available as isolates, purified nucleic acid was used. Some known Bacillus cereus strains (BAG1X1-1 NR28575, 03BB102, and G-9241 NR-9564) contain a pXO1-like plasmid. Detection of these strains was indicated by a test result of 'Bacillus anthracis suspected' with amplification of the plasmid manually confirmed by the supervisor. Initial no detection rate for exclusivity testing was 95.68%. Repeat testing was conducted for three organisms. Final no detection rate for exclusivity testing was 100%.
In addition, in silico exclusivity analysis was conducted with assay primers and probe positions aligned against additional non-target organisms. For in silico analysis, the results were filtered for: Exclusion of Bacillus anthracis hits; ≥80% for % Identity; and ≥80% for high-scoring segment pair (HSP) length (of the sequence for that primer or probe BLAST query).
Exclusivity in silico Analysis Summary
In silico exclusivity analysis was conducted with each of the assay primers and probes to identify non-target organisms with the potential to cross react with the assays. A BLAST search was performed against the NCBI nr, wgs, and genome databases. Organisms identified with high and reduced risk of amplification by in silico analysis were then evaluated by wet laboratory testing. Additionally, a specific set of organisms were screened by in silico analysis. A BLAST search of the primer and probe sequences was conducted. A primer and probe list was created and a BLAST search against each of the three databases performed for "all organisms" within the CLC Genomics Workbench software. For each primer or probe BLAST, the results were filtered for: (1) exclusion of Bacillus anthracis hits; (2) ≥80% for % identity; and (3)≥80% for HSP length (of the sequence for that primer or probe BLAST query). The results of each BLAST were transferred to a Microsoft Excel spreadsheet and reviewed to identify organisms with "hits" for all three primer and probe sequences for each assay.
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For the pXO1 assay, one organism (Bacillus cereus m1550) was identified as a match with 100% identity to the primer and probe sequences as high potential to support amplification and was evaluated by wet laboratory testing. For the pXO2 assay, two organisms (B. cereus AH1272 and AH1273) were identified as matches with 100% identity to the probe sequence. However, through a secondary NCBI Primer BLAST, each strain also contains three SNPs in both primer binding sequences, reducing the likelihood to support amplification.
Interfering Substances
The Applied Biosystems™ Bacillus anthracis Detection Kit's potential to detect Bacillus anthracis in the presence of potentially interfering substances was evaluated. Fifty potentially interfering substances consisting of endogenous, exogenous, and technique-specific substances were tested for potential interference with the assay. Each substance was tested in whole blood with triplicate-paired samples in the presence and absence of B. anthracis Ames, spiked at 3X LoD. Endogenous substances were spiked at concentrations typically found in human whole blood or at the highest achievable concentration.
Technique-specific substances were spiked directly into PCR reactions at 2.5%, 5% and 10% v/v test concentrations.
The following technique-specific substances were found to interfere with the assay and appropriate limitation was added to the product labeling.
- MagNA Pure Wash Buffer I MagNA Pure Lysis/Binding Buffer MagNA Pure Proteinase K 10% Bleach Fisherbrand™ DNase Displace QIAGEN Protease QIAGEN Buffer AL QIAGEN Buffer AW1 (with and without EtOH) EtOH at greater than 5%
Microbial Interference
Detection of Bacillus anthracis in the presence of other clinically-relevant organisms, e.g. pathogens or normal flora that may be present in whole blood or positive blood culture specimens, was tested with the Applied Biosystems™ Bacillus anthracis Detection Kit. The detection rate of B. anthracis was 100% in the presence of potentially interfering organisms in whole blood and blood culture.
Reproducibility
Seven panel members were tested twice a day by three teams of two different operators on five non-consecutive days using two Applied Biosystems™ 7500 Fast Dx Real-Time PCR Instruments across three different reagent lots to determine the reproducibility rate of detecting Bacillus
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anthracis Ames with the Applied Biosystems™ Bacillus anthracis Detection Kit. Two teams performed extraction using the Qiagen QlAamp DSP DNA Blood Mini Kit and one team performed extraction using the Roche MagNA Pure LC. A breakdown of the concentrations tested is below:
HP = High Positive: organism spiked at 10 × LOD; 1.5e3 CFU/mL for DSP and 5e2 CFU/mL for MNP
LP = Low Positive: organism spiked at 3 × LOD; 4.5e2 CFU/mL for DSP and 1.5e2 CFU/mL for MNP
NEG = Negative: non-Ba organism spiked at 3 x Ba LOD; 4.5e2 CFU/mL for DSP and 1.5e2 CFU/mL for MNP
One low positive sample extracted by one operator returned negative results when tested against three reagent lots indicating an error during the sample preparation procedure. In a separate instance a high positive sample returned 'Bacillus anthracis suspected' results when the pXO1 assay failed to amplify. The PCR for this sample was repeated and returned 'Bocillus anthracis detected' results as expected. No false positive events occurred out of the 270 PCR tests of negative samples.
There were a total of 24 sample replicates (across all three reagent lots) that returned initial indeterminate results. All were determined to be negative by supervisor review. There were no invalid results for this study.
Carry-Over/Cross-contamination
The Applied Biosystems™ Bacillus anthracis Detection Kit workflow was evaluated for the potential for cross-contamination or sample carry-over during extraction using the automated system (Roche MagNA Pure) and the manual DSP (Qiagen) methods. For the automated method nine full MagNA Pure runs were performed with each run of 31 samples placed in a checkerboard fashion alternating high positive, spiked whole blood (K2 EDTA), blood culture aerobic and blood culture anaerobic and negative samples throughout the sample cartridge. Blood culture spiked samples were spiked with Bacillus anthracis Ames at 1 x 10° CFU/mL. Whole blood spiked samples were spiked with Bacillus anthracis Ames at 500 CFU/mL (10X LOD). For the manual method 12 batches of 15-24 samples each were performed using the DSP method with samples batched such that high positive samples were placed next to negative whole blood, aerobic blood culture and anaerobic blood culture. Blood culture spiked samples were spiked with Bacillus anthracis Ames at 1 x 10° CFU/mL. Whole blood spiked samples were spiked with Bacillus anthracis Ames at 1500 CFU/mL (10X LOD).
Two hundred and seventy-nine samples were extracted on the MagNA Pure in nine separate runs and resulting extracts were analyzed in singlet PCR reactions. A total of two false positive events occurred out of a total of 135 negative samples analyzed, resulting in a specificity rate of 98.5%.
Two hundred and seventy-nine samples were manually extracted using the Qiagen DSP blood mini kit in twelve separate extraction batches and resulting extracts were analyzed in singlet PCR
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reactions. No false positive events occurred out of a total of 139 negative samples analyzed, resulting in a specificity rate of 100%.
Test area surface monitoring control swabs were collected at various steps in the workflow during execution of the Carryover/Cross-Contamination study to support a detailed evaluation of sample handling and processing steps that have the potential to introduce contamination.
One hundred and sixty-two test area surface monitoring control swabs were collected during nine separate MagNA Pure runs for the Carryover/Cross-Contamination study. Swabs were processed and analyzed in singlet PCR. Results were used to determine areas of the instrument and steps in the process that are at high risk of cross-contamination. A total of nine swab locations were tested to include three swab locations in the BSC and six surfaces inside the MagNA Pure work deck. Contamination events occurred on one BSC surface location and five surfaces of the MagNA Pure during testing.
Two hundred and sixteen test area surface monitoring control swabs were collected during twelve separate DSP extraction batches for the Carryover/Cross-Contamination study. Swabs were processed and analyzed in singlet PCR. Results were used to determine areas of the BSC and steps in the process that are at high risk of cross-contamination. One location (BSC - left surface - after loading DSP columns) was identified as having a higher rate of contamination during the workflow.
Clinical Specificity
To demonstrate clinical specificity, the Applied Biosystems™ Bacillus anthracis Detection Test System was tested using left-over fresh and frozen blood culture samples, (confirmed by Gram stain to be positive for any bacterial species and selectively positive for Gram Positive rods), and blood culture samples spiked with any bacterial species and a subset spiked with Gram positive rod bacteria. Randomly accessed, residual blood - blood collected in K2EDTA (e.g., CBC, tested in hematology) and with no correlation to positive blood culture specimens, for which standard of care testing was performed (patients may or may not have fever, but specimens were obtained for other laboratory testing) was collected from Hematology laboratories for immediate testing with the Applied Biosystems™ Bacillus anthracis Detection Kit. All samples (blood culture and whole blood) were collected prospectively and serially without further selection criteria. Additionally, febrile whole blood samples were collected from consented patients presenting with fever and flu-like symptoms. Samples were collected from three (3) point-of-care collection sites including physician offices, urgent care centers or hospital clinics/ER. Clinical performance testing was conducted at three laboratories within the US. Febrile whole blood samples were shipped to one laboratory on the day of collection and were processed within 24 hours of receipt.
No reference method testing was performed.
All samples were assumed to be negative for Bacillus anthracis.
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The study generated 'Bacillus anthracis not detected' results for the 401 blood culture specimens and 439 whole blood specimens included in the performance analyses, demonstrating 100% negative percent agreement when compared to the expected negative result.
Clinical Sensitivity
To demonstrate product sensitivity, the Applied Biosystems™ Bacillus anthracis Detection Test System was tested using whole blood specimens collected from febrile patients presenting with fever of unknown origin and flu-like symptoms. Following testing of febrile whole blood specimens determined to be negative for Bacillus anthracis, simulated samples positive for Bacillus anthracis were contrived using unique febrile whole blood specimens each spiked with Bacillus anthracis at or near LOD. Simulated positive samples were prepared and tested in BSL-3 laboratory facilities. Each febrile whole blood aliquot was spiked with 1 of 18 strains of Bacillus anthracis. Contrived samples were randomized with negative, unspiked whole blood samples and blinded to the operators. Two different operators processed samples using either the Qiagen DSP manual method or with the Roche MagNA Pure automated robot. Two different instruments were used to analyze samples. A minimum of 25 samples were spiked with Bacillus anthracis Ames at LOD and additional samples were spiked at 3× LOD with 1 of 17 other Bacillus anthracis strains.
The study generated 'Bacillus anthracis detected' results for 84 of 87 low-positive whole blood specimens. Three specimens initially generated 'Bacillus anthracis suspected' results due to detection only one plasmid target. Final positive percent agreement was 96% (Cl: 90.4-96,5%).