The B. anthracis Real-Time PCR Assay is an in vitro diagnostic test for the qualitative detection of plasmid and chromosomal DNA sequences from B. anthracis. The assay can be used to test human respiratory samples, whole blood, serum, plasma, swabs from lesions, cerebrospinal fluid, and bacterial culture isolates from individuals suspected of having anthrax.

Results generated from direct specimentesting are presumptive for the identification of B. anthracis. Results generated from culture isolate testing should be used in conventional methods for identification of Bacillus anthracis isolates as part of the LRN Bacillus anthracis Testing Algorithm. The diagnosis of anthrax infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidences, in addition to the identification of B. anthracis from cultures or detection directly in clinical specimens.

Use is limited to Laboratory Response Network (LRN) designated laboratories.

The B. anthracis Real-time PCR Assay is also intended for environmental specimen testing for biothreat detection and response. FDA has not evaluated claims related to the use of this assay on environmental specimens.

Device Description

The B. anthracis Real-time PCR Assay uses a fluorogenic probe, consisting of an oligonucleotide with a reporter dve (FAM) attached to the 5' end and a quencher dye (BHQ1) attached at or near the 3′ end. The probe anneals to a specific target sequence located between the forward and reverse primers. During the extension phase of the PCR cycle, the 5′ nuclease activity of Taq polymerase degrades the probe causing the reporter dye to separate from the quencher dye and a fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes and the fluorescence intensity is monitored during the PCR. The Tag polymerase used in this assay is inactive at room temperature. It must be activated by incubation at 95°C, which also minimizes the production of nonspecific amplification products.

Each extracted DNA sample is tested with three B. anthracis primer and probe sets run as individual reactions. The primer and probe sets target genes encoding virulence factors as well as conserved regions of DNA from the B. anthracis chromosome. All primer and probe sets must be positive for the overall result of the B. anthracis Real-time PCR Assay to be interpreted as positive. Any result that is positive for some, but not all target regions, is still considered equivocal and follow-up laboratory investigation should be performed per the LRN Bacillus anthracis testing algorithm.

AI/ML Overview

The provided document is a 510(k) premarket notification for the B. anthracis Real-time PCR Assay. It indicates substantial equivalence to a predicate device and provides information on its intended use, device description, and a comparison with the predicate. However, it explicitly directs inquiries regarding performance characteristics (including analytical limit of detection, analytical sensitivity and specificity, and clinical performance) to the Centers for Disease Control and Prevention. Therefore, the acceptance criteria and the study that proves the device meets those criteria are not detailed within this document.

Here is a summary of the information that is available in the document, and what is explicitly stated as not available:

1. Table of Acceptance Criteria and Reported Device Performance

Not available in the provided document. The document directs inquiries regarding performance characteristics to the Centers for Disease Control and Prevention.

2. Sample size used for the test set and the data provenance

Not available in the provided document. The document states that inquiries regarding analytical LoD, analytical sensitivity and specificity, and clinical performance should be directed to the Centers for Disease Control and Prevention. This would include information about sample sizes and data provenance for validation studies.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

Not applicable / Not available in the provided document. For a PCR assay, the "ground truth" is typically established by definitive laboratory methods (e.g., bacterial culture, sequencing, or a reference method) rather than expert opinion on images or clinical assessments. The details of how ground truth was established for the analytical and clinical performance studies are not present.

4. Adjudication method for the test set

Not applicable / Not available in the provided document. See point 3.

5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, and the effect size of how much human readers improve with AI vs without AI assistance

Not applicable. This device is a diagnostic PCR assay, not an AI-assisted imaging or diagnostic tool that involves human readers in the traditional sense of an MRMC study.

6. If a standalone (i.e., algorithm only without human-in-the-loop performance) was done

Yes, implicitly. The device is a Real-time PCR Assay, which is an in vitro diagnostic test. Its performance inherently refers to its standalone analytical and clinical performance in detecting target DNA sequences. While human operators are involved in running the assay and interpreting results, the "algorithm" (the PCR chemistry and detection) itself functions stand-alone. However, specific details of "standalone performance" metrics (like sensitivity, specificity, accuracy against a reference standard) are not provided and are referred to the CDC.

7. The type of ground truth used

For a PCR assay detecting DNA sequences from B. anthracis, the ground truth for performance studies would typically be established by a "gold standard" laboratory method, such as:
- Bacterial culture and identification: Confirmation of B. anthracis growth and subsequent identification using conventional microbiological methods (e.g., phenotypic differences, susceptibility testing).
- Reference molecular methods: Other validated PCR assays or DNA sequencing for definitive identification.
Specific details of how ground truth was established for the validation studies are not available in the provided document.

8. The sample size for the training set

Not applicable. For a PCR assay, there isn't a "training set" in the machine learning sense. The assay is developed based on known genetic targets of B. anthracis. Development involves optimizing primer/probe design and assay conditions, but not "training" an algorithm on a dataset.

9. How the ground truth for the training set was established

Not applicable. See point 8.

Summary

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November 7, 2019

Centers for Disease Control and Prevention Julie Villanueva Chief, Laboratory Preparedness and Response Branch 1600 Clifton Road NE, MS: H24-11 Atlanta, Georgia 30329

Re: K192871

Trade/Device Name: B. anthracis Real-time PCR Assay Regulation Number: 21 CFR 866.3045 Regulation Name: In vitro diagnostic device for Bacillus spp. detection Regulatory Class: Class II Product Code: NHT Dated: October 7, 2019 Received: October 8, 2019

Dear Julie Villanueva:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's

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requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

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 Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K192871

Device Name B. anthracis Real-time PCR Assay

Indications for Use (Describe)

Use is limited to Laboratory Response Network (LRN) designated laboratories.

Type of Use (Select one or both, as applicable)
☑ Prescription Use (Part 21 CFR 801 Subpart D)	☐ Over-The-Counter Use (21 CFR 801 Subpart C)

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5. 510(k) Summary

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of 21 CFR 807.92.

Assigned 510(k) number:	K192871
Submitted by:	Centers for Disease Control and Prevention1600 Clifton Road, MS: H24-11Atlanta, GA 30329-4027
Contact Person:	Julie Villanueva, Ph.D.Laboratory Preparedness and Response Branch ChiefDivision of Preparedness and Emerging InfectionsNational Center for Emerging and Zoonotic Infectious DiseaseCenters for Disease Control and Prevention(Registration number: 1050190)1600 Clifton Road, NE, MS: H24-11Atlanta, GA 30329-4027(404) 639-3851 (office)fkb8@cdc.gov
Date prepared:	October 31, 2019
Device trade name:	B. anthracis Real-time PCR Assay
Classification name and regulation: (if applicable)	In vitro diagnostic device for Bacillus spp. detection; 21 CFR 866.3045
Predicate device(s):	B. anthracis Real-time PCR Assay(K140426)

Background

Anthrax is a zoonotic disease caused by B. anthracis that is transmissible to humans through handling or consumption of contaminated animal products. Infection can also occur through inhalation of B. anthracis spores from contaminated animal products such as wool or hides. Infection caused by human-to-human contact has been reported only rarely, and only via the cutaneous route (Versalovic, 2011). There have been 3 major presentations of anthrax in humans: cutaneous, ingestion, and inhalation. In cases of cutaneous anthrax, patients typically present with a painless blister or skin ulcer with a black area in the center. Inhalation anthrax is typically associated with cold or flu-like symptoms, cough, chest discomfort, shortness of breath, fatigue, and muscle aches. Symptoms of gastrointestinal anthrax typically include nausea, loss of appetite, bloody diarrhea, fever and severe stomach pain.

Prior to the development of the LRN B. anthracis Real-time PCR Assay, identification of B. anthracis was determined by using phenotypic differences between B. anthracis and the rest of the B. cereus group. (i.e. lack of motility and hemolysis, susceptibility to penicillin, colony morphology, susceptibility to

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lysis by gamma phage) (Hoffmaster, 2002). However, these methods require growth of the microorganism and can take at least 24 hours incubation to obtain a result. Due to the prevalence of B. anthracis in the environment, and its past use as a biological weapon, it has long been an organism of concern. The use of B. anthracis in the bioterrorism attacks of 2001 resulting in cases of inhalation and cutaneous anthrax increased public health concern and reinforced the worry that it would be used in the same way again. For these reasons, there was a need for rapid testing to aid in the identification of B. anthracis. The Laboratory Response Network (LRN) is part of a national bioterrorism preparedness initiative and one of the major goals of this initiative is the development and validation of rapid and specific assays for agents likely to be used in a bioterrorism event. Accordingly, scientists at the Centers for Disease Control and Prevention have developed several real-time PCR based assays to detect B. anthracis and other potential aqents of bioterrorism in an effort to meet the need for rapid detection.

Device Description

Intended Use

The B. anthracis Real-Time PCR Assay is an in vitro diagnostic test for the qualitative detection of plasmid and chromosomal DNA sequences from B. anthracis. The assay can be used to test human respiratory samples, whole blood, serum, plasma, swabs from lesions, CSF, pleural fluid, and bacterial culture isolates from individuals suspected of having anthrax.

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Results generated from direct specimen testing are presumptive for the identification of B. anthracis. Results generated from culture isolate testing should be used in conjunction with other conventional methods for identification of Bacillus anthracis isolates as part of the LRN Bacillus anthracis Testing Algorithm. The diagnosis of anthrax infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidences, in addition to the identification of B. anthracis from cultures or detection directly in clinical specimens.

Use is limited to Laboratory Response Network (LRN) designated laboratories.

Device Comparison

The following table summarizes the similarities and differences between the cleared assay and the new submission for this device.

	PredicateB. anthracis Real-timePCR Assay (K140426)	DeviceB. anthracis Real-timePCR Assay (new)
Intended Use	The B. anthracis Real-Time PCR Assay is an invitro diagnostic test forthe qualitative detectionof plasmid andchromosomal DNAsequences from B.anthracis . The assay canbe used to test humanrespiratory samples,whole blood, serum,plasma, swabs fromlesions, CSF, pleural fluid,and bacterial cultureisolates from individualssuspected of havinganthrax.Results generated fromdirect specimen testingare presumptive for theidentification of B.anthracis . Resultsgenerated from cultureisolate testing should beused in conjunction withother conventionalmethods for identificationof Bacillus anthracisisolates as part of the	Unchanged
	LRN Bacillus anthracisTesting Algorithm. Thediagnosis of anthraxinfection must be madebased on history, signs,symptoms, exposurelikelihood, and otherlaboratory evidences, inaddition to theidentification of B.anthracis from cultures ordetection directly inclinical specimens.
	Use is limited toLaboratoryResponse Network(LRN) designatedlaboratories.
	The B. anthracis Real-time PCR Assay is alsointended forenvironmental specimentesting for biothreatdetection and response.FDA has not evaluatedclaims related to the useof this assay onenvironmentalspecimens.
Principle ofOperation	Nucleic acid amplificationand fluorescent probedetection	Unchanged
Targets	BA1 - pXO2 DNABA2 - pXO1 DNABA3 - B. anthracischromosomal region DNA	Unchanged
Sample Types	• Swabs from lesionsand vesicular material• Whole Blood (EDTA orsodium citrate)• Serum/Plasma• Respiratory specimens(transtrachealaspirates, bronchiallavage, and sputum)• Cerebrospinal fluid• Pleural Fluid• Bacterial cultureisolates• Environmental samplescollected forinvestigational orsurveillance use	Unchanged
Instrumentation	• Applied Biosystems7500 Fast Dx Real-TimePCR• Cepheid SmartCycler I• Cepheid SmartCycler II	• Applied Biosystems 7500Fast Dx Real-Time PCRInstrument• Cepheid SmartCycler I• Cepheid SmartCycler II• QuantStudio Dx Real-TimePCR Instrument
Software/Hardware	Software/Hardware notincluded as part ofdevice; may be run onthe manufacturerinstalled and validatedsoftware from the AB7500 Fast Dx,SmartCycler I, orSmartCycler II	Software/Hardware notincluded as part of device;may be run on themanufacturer installed andvalidated software from theAB 7500 Fast Dx,SmartCycler I, SmartCyclerII, or QuantStudio Dx real-time PCR instruments
Master mix	• Quanta PerfeCTaMultiPlex qPCRSuperMix, Low ROX• Roche LightCyclerFastStart DNA MasterHybProbe	Unchanged

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Establishment of Performance Characteristics

Inquiries regarding performance characteristics for the B. anthracis Real-time PCR Assay should be directed to the Centers for Disease Control and Prevention.

Analytical Limit of Detection (LoD)

The limit of detection for the B. anthracis Real-time PCR Assay was determined through an analytical sensitivity study.

Analytical Sensitivity and Specificity

Inquiries regarding performance characteristics for the B. anthracis Real-time PCR Assay should be directed to the Centers for Disease Control and Prevention.

Clinical Performance

Inquiries regarding clinical performance characteristics for the B. anthracis Real-time PCR Assay should be directed to the Centers for Disease Control and Prevention.

Regulation Number and Section

§ 866.3045 In vitro diagnostic device for

Bacillus spp. detection.(a)
Identification. An in vitro diagnostic device forBacillus species (spp.) detection is a prescription device used to detect and differentiate amongBacillus spp. and presumptively identifyB. anthracis and otherBacillus spp. from cultured isolates or clinical specimens as an aid in the diagnosis of anthrax and other diseases caused byBacillus spp. This device may consist ofBacillus spp. antisera conjugated with a fluorescent dye (immunofluorescent reagents) used to presumptively identify bacillus-like organisms in clinical specimens; bacteriophage used for differentiatingB. anthracis from otherBacillus spp. based on susceptibility to lysis by the phage; or antigens used to identify antibodies toB. anthracis (anti-toxin and anti-capsular) in serum. Bacillus infections include anthrax (cutaneous, inhalational, or gastrointestinal) caused byB. anthracis, and gastrointestinal disease and non-gastrointestinal infections caused byB. cereus. (b)
Classification. Class II (special controls). The special controls are set forth in FDA's special controls guideline document entitled “In Vitro Diagnostic Devices forBacillus spp. Detection; Class II Special Controls Guideline for Industry and Food and Drug Administration Staff.” For availability of the guideline document, see § 866.1(e).(c)
Restriction on Distribution. The distribution of these devices 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.(d)
Restriction on Use. The use of this device is restricted to prescription use and must comply with the following:(1) The device must be in the possession of:
(i)(A) A person, or his agents or employees, regularly and lawfully engaged in the manufacture, transportation, storage, or wholesale or retail distribution of such device; or
(B) A practitioner, such as a physician, licensed by law to use or order the use of such device; and
(ii) The device must be sold only to or on the prescription or other order of such practitioner for use in the course of his professional practice.
(2) The label of the device shall bear the statement “Caution: Federal law restricts this device to sale by or on the order of a ____”, the blank to be filled with the word “physician” or with the descriptive designation of any other practitioner licensed by the law of the State in which he practices to use or order the use of the device.
(3) Any labeling, as defined in section 201(m) of the Federal Food, Drug, and Cosmetic Act, whether or not it is on or within a package from which the device is to be dispensed, distributed by, or on behalf of the manufacturer, packer, or distributor of the device, that furnishes or purports to furnish information for use of the device contains adequate information for such use, including indications, effects, routes, methods, and frequency and duration of administration and any relevant hazards, contraindications, side effects, and precautions, under which practitioners licensed by law to employ the device can use the device safely and for the purposes for which it is intended, including all purposes for which it is advertised or represented. This information will not be required on so-called reminder-piece labeling which calls attention to the name of the device but does not include indications or other use information.
(4) All labeling, except labels and cartons, bearing information for use of the device also bears the date of the issuance or the date of the latest revision of such labeling.