REDLINE ANTHRAX ALERT TEST

K030370 · Tetracore, Inc. · NPO · Dec 9, 2003 · Microbiology

Device Facts

• Record ID: K030370
• Device Name: REDLINE ANTHRAX ALERT TEST
• Applicant: Tetracore, Inc.
• Product Code: NPO · Microbiology
• Decision Date: Dec 9, 2003
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3045
• Device Class: Class 2

Indications for Use

The Tetracore RedLine Anthrax Alert™ Test is intended for the rapid, in vitro qualitative detection of Bacillus anthracis from non-hemolytic colonies obtained from sheep blood agar plates. The test is intended for use in clinical, public health, and hospital laboratories in conjunction with other markers and testing for the identification of Bacillus anthracis.

Device Story

RedLine Anthrax Alert™ Test is an immunochromatographic assay for presumptive identification of Bacillus anthracis. Input: non-hemolytic colonies from sheep blood agar plates suspended in Colony Isolation Buffer. Operation: reagent strip utilizes monoclonal and polyclonal antibodies to detect B. anthracis antigen in aqueous specimens. Output: qualitative visual result on test cassette. Used in clinical, public health, and hospital laboratories by trained personnel. Results used in conjunction with other markers to support identification of B. anthracis. Benefits: rapid presumptive identification to assist laboratory workflows.

Clinical Evidence

No clinical trials performed. Evidence consists of analytical performance and a field evaluation. Inclusivity testing included 145 B. anthracis strains (98.6% positive). Exclusivity testing included 49 non-hemolytic Bacillus/Bacillus-like species, 52 hemolytic Bacillus species, and 26 non-Bacillus pathogens. Field evaluation of 633 non-hemolytic isolates compared the device to gamma phage susceptibility testing, showing 100% agreement (410 B. anthracis, 223 non-B. anthracis).

Technological Characteristics

Immunochromatographic test cassette; utilizes monoclonal and polyclonal antibodies for antigen detection. Includes Colony Isolation Buffer and Positive Control reagent. Stable for 12 months at 15-30°C.

Indications for Use

Indicated for rapid, in vitro qualitative presumptive identification of Bacillus anthracis from non-hemolytic colonies cultured on sheep blood agar plates. For use in clinical, public health, and hospital laboratories. Not for use with spore preparations, suspicious powders, or samples other than colonies from culture growth.

Regulatory Classification

Identification

An in vitro diagnostic device for Bacillus species (spp.) detection is a prescription device used to detect and differentiate among Bacillus spp. and presumptively identify B. anthracis and other Bacillus spp. from cultured isolates or clinical specimens as an aid in the diagnosis of anthrax and other diseases caused by Bacillus spp. This device may consist of Bacillus spp. antisera conjugated with a fluorescent dye (immunofluorescent reagents) used to presumptively identify bacillus-like organisms in clinical specimens; bacteriophage used for differentiating B. anthracis from other Bacillus spp. based on susceptibility to lysis by the phage; or antigens used to identify antibodies to B. anthracis (anti-toxin and anti-capsular) in serum. Bacillus infections include anthrax (cutaneous, inhalational, or gastrointestinal) caused by B. anthracis, and gastrointestinal disease and non-gastrointestinal infections caused by B. cereus.

Special Controls

*Classification.* Class II (special controls). The special controls are set forth in FDA's special controls guideline document entitled “In Vitro Diagnostic Devices for*Bacillus* 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.

Predicate Devices

• preamendments

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY DEVICE ONLY TEMPLATE

A. 510(k) Number: k030370
B. Analyte: *Bacillus anthracis* cell surface protein
C. Type of Product: Assay kit, Immunochromatographic, Bacillus Anthracis Differential Antibody
D. Applicant: Tetracore, Inc.
E. Proprietary and Established Names of the Product: Redline Alert™ Test

F. Regulatory Information:

1. Regulation section: Unclassified
2. Classification: Unclassified
3. Product code: NPO
4. Panel: 83

G. Intended Use:

Intended use(s): The Tetracore RedLine Alert™ Test is intended for the rapid, *in vitro* qualitative presumptive identification of *Bacillus anthracis* from non-hemolytic *Bacillus* colonies cultured on sheep blood agar plates. The test is intended for use in clinical, public health, and hospital laboratories in conjunction with other markers and testing for the presumptive identification of *Bacillus anthracis*.

Warning: The RedLine Alert™ Test has not been evaluated for use with spore preparations, suspicious powders, or samples other than colonies from culture growth.

1. Indication(s) for use: non-hemolytic Bacillus colonies cultured on sheep blood agar plates
2. Special conditions for use statement(s): used in conjunction with other markers and testing for the presumptive identification of B. anthracis
3. Special instrument requirements: none

H. Device Description:

RedLine Alert™ Test is an immunochromatographic lateral flow cassette-type device. It has a nitrocellulose membrane (backed with a vinyl adhesive support) that is striped with a polyclonal rabbit IgG (*B. anthracis* Sterne vegetative and spore mix used as immunogen) for the T line and a 2nd polyclonal antibody for the C (control) line. The T-line polyclonal is obtained from rabbits immunized with *B. anthracis* Sterne, purified by Protein-G chromatography. The membrane is encased in a plastic cassette-type casing.

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A mouse monoclonal antibody reagent is purified by Protein A chromatography, sterile filtered, preserved in 0.1% NaAzide and conjugated to colloidal gold, and then added to a glass fiber pad that is assembled in the cassette under the cellulose sample pad. An additional cellulose pad is affixed in the cassette assembly at the distal end of the membrane strip to facilitate migration. To use the device, colony isolation buffer is also needed. The kit contains tubes with 7.5 mL of TRIS, EDTA, KCl, Triton-X100 and NaAzide formulated buffer (pH 8.0). Aliquots of 200 μL are used for performing a test.

I. Substantial Equivalence Information (if known):

1. Predicate device name(s): preamendments
2. Predicate 510(k) number(s): preamendments
3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended use | Identification and differentiation of B. anthracis from other non-hemolytic Bacillus-like species | Identification and differentiation of B. anthracis from other Bacillus-like species |
| Indications | Adjunct to microbiological culture procedures | Same |
| Differences | | |
| Item | Device | Predicate |
| Antibody type | Monoclonal | Polyclonal |
| Conjugate tag | Colloidal gold | FITC |
| Format | Lateral flow single unit | Fluorescent microscopy |

J. Standard/Guidance Document Referenced (if applicable): NA

K. Test Principle: Alert™ is an immunochromatographic test format with manual preparation of test suspension and visual interpretation of endpoint (presence/absence of a colored line in the T and C stripe regions. A visible colored line appears in the cassette window if antigen (a cell surface protein) homologous with the monoclonal conjugate (murine MAB-colloidal gold) is present in the colony tested. This cell surface protein is also found in some B. cereus and B. thuringiensis strains. However, those strains are invariably hemolytic when cultured on sheep blood agar.

L. Performance Characteristics (provide the information described below as appropriate to support the substantial equivalence determination):

1. Analytical performance:

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a. Precision/Reproducibility): During inclusivity, exclusivity, and other analytical testing, test replicates of the same organism were done. These were done from different preparations of the same culture and were read by 2 different observers. Intensity of colored lines was also documented by using a densitometric reader (a reflected light optical reader) giving sample values (SV), a semiquantitative scoring. Three of 52 colonies tested were scored as either indeterminate or a faint band by one or more observers.

b. Linearity/Assay reportable range: Variations in cultural growth conditions were assessed. These included 8-48 h incubation times, temperature incubation at both 30° and 37° C, ambient air and CO₂ incubation, and density of test inoculum. Conclusions from these studies: 12-24 h cultures, incubated either ambient or CO₂, and with incubation temperature as low as 30°C provide reliable results.

c. Traceability (controls, calibrators, or method): A positive control material is included in the kit: individual polypropylene tubes of lyophilized supernatant from liquid broth cultures of B. anthracis Sterne. Each cassette contains an internal procedural control, antibody (anti-murine) striped onto the test membrane that captures excess and unbound conjugate. The monoclonal reactive with the cell surface protein is proprietary and there is no standard reference material available.

d. Detection limit: NA – the assay is intended for use with colonies, using a prescribed amount of inoculum. Inclusivity studies were performed with 145 different B. anthracis strains (organisms from 28 different countries on 6 continents and from 12 different states within the US), representing each of the 5 VNTR (variable-number tandem repeats) groups and 53 of the 89 described genotypes. One strain had unusually small atypical colonies. The Alert™ showed positive results for 143/145 (98.6%).

e. Analytical specificity: Exclusivity studies were done with 49 nonhemolytic Bacillus and Bacillus-like spp., 52 hemolytic Bacillus and Bacillus-like spp. and 26 non-Bacillus common pathogens. All non-hemolytic colony types were negative when tested with Alert™; 12 of 52 hemolytic Bacillus spp. were positive with Alert™.

f. Assay cut-off: NA

2. Comparison studies:

a. Method comparison with predicate device: There is no predicate device to use for comparison testing. There are established methods for identification of B. anthracis and for differentiating Bacillus spp. by evaluating different phenotypic characteristics of culture growth, and microscopic and colony morphology. A field evaluation of the Alert™ was done in a laboratory performing testing for B. anthracis surveillance of a contaminated worksite. At this laboratory, 633 nonhemolytic isolates were tested with gamma phage and Alert™. Of these, 410 were identified as B. anthracis and 223 as non-B. anthracis isolates. Results for Alert™ and gamma phage susceptibility agreed for all of these culture isolates. [Note: all B. anthracis in this

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evaluation were Ames strain so sensitivity across a spectrum of different strains cannot be inferred].

b. Matrix description and comparison: NA – the Alert™ is only indicated for use with colonies recovered on sheep blood agar culture plates.

3. Clinical Studies:
a. Clinical sensitivity: NA
b. Clinical specificity: NA
b. Other clinical supportive data (when a and b are not applicable): NA

4. Clinical cut-off (where applicable): NA
5. Expected values/Reference range: NA

M. Conclusion: Substantially equivalent

Page 4, SE Decision Summary