LogoFDA 510(k) Search
K Number
K071799
Device Name
MODIFICATION TO BIOSTAR OIA SHIGATOX
Manufacturer
IVERNESS MEDICAL PROFESSIONAL DIAGNOSTICS
Date Cleared
2007-10-02

(92 days)

Product Code
GNA,GMZ
Regulation Number
866.3255
Type
Traditional
Panel
Microbiology
Reference & Predicate Devices
K061889
Predicate For
N/A
AI/MLSaMDIVD (In Vitro Diagnostic)TherapeuticDiagnosticis PCCP AuthorizedThirdpartyExpeditedreview
Intended Use

The BioStar® OIA® SHIGATOX assay is an Optical Immunoassay (OIA) test for the qualitative, rapid detection of the presence of Shiga toxins in human diarrheal fecal specimens, broth cultures, fecal specimens in Cary Blair Transport Media, or swab sampling of colonies from a culture plate. This test is intended for in vitro diaqnostic use as an aid in the diagnosis of infection by Shiga toxin-producing Escherichia coli (STEC) both O157 and all non - O157 Shiga toxin-producing strains.

Device Description

The OIA SHIGATOX test involves the qualitative detection of Shiga toxins 1 and 2 (Stx1 and Stx 2) produced by certain strains of Escherichia coli and other organisms. The Optical ImmunoAssay technology enables the direct visual detection of a physical change in the optical thickness of molecular thin films. This change is the result of antigen - antibody binding on an optical surface (silicon wafer). After a specimen potentially containing Shiga toxin is mixed with conjugates and placed directly on the optical surface, the immobilized surface antibodies capture the antigen/conjugate complex. After washing, the substrate is added, increasing the thickness (mass enhancement) of the molecular thin film. This change in thickness alters the reflected light path, and this alteration is visually perceived as a color change. Slight changes in the optical thickness produce a distinct visible color change. A positive result appears as a purple spot on the gold background. When antigen is not present in the specimen, no binding takes place. Therefore, the optical thickness remains unchanged, and the surface retains the original gold color indicating a negative result.

More specifically, the BioStar OIA SHIGATOX device is based on a novel thin film optical detection technology that relies on the interaction of white light with thin films to create a destructive interference phenomenon. Characteristic of this phenomenon is the generation of a reflective surface that changes color as a function of the change in optical thickness (refractive index x thickness) of the films on the surface of the device. To take advantage of this phenomenon for monitoring biological binding events, the optical surface with a special background color is coated with a capture reagent specific to the analyte of interest. In the OIA SHIGATOX device, the biological capture film is a combination of affinity-purified polyclonal antibodies to Shiga toxins 1 and 2 (Stx 1 and Stx 2). Samples suspected of containing either or both of the toxins are mixed with cocktail containing polyclonal antibodies to Stx 1 and Stx 2 that have been covalently conjugated to horseradish peroxidase (HRP). Once a sample containing toxins or either toxin is applied to the surface, the immune complex of toxin(s) and the anti-toxin-HRP conjugate(s) are bound to the surface antibodies. Following a wash step, a precipitating substrate for HRP is added, and a thin film generated by the immobilized immune complex is enhanced by the precipitation of the HRP product. Once washed and dried, a simple color change relative to the gold background color is observed as an indication of the presence of Stx 1 or Stx 2 in the original specimen.

The OIA SHIGATOX device produces a qualitative result for the presence or absence of Shiga toxin as the device output. Input to the device is the simple addition of an aliquot of fecal material (direct or in transport media) or broth culture to the reagents contained in the kit. Fecal samples are routinely collected and no special collection requirements exist. Test devices within the kit are single use devices, and disposal instructions are provided in the Package Insert. The kit contains all components necessary for analysis of the range of samples approved for use in this product, with the exception of a timer.

AI/ML Overview

Here's a summary of the acceptance criteria and study details for the BioStar® OIA® SHIGATOX device based on the provided text:

Acceptance Criteria and Reported Device Performance

The acceptance criteria for each study type are implicitly defined by the reported performance, which consistently achieved high accuracy. For the analytical studies, the key criteria were demonstrating sensitivity and specificity, and the reproducibility studies aimed for 100% agreement. For clinical studies, the percentage agreements with comparator methods (EIA, SMAC, CTA, or reference OIA) were the primary metrics.

Metric / Test TypeAcceptance Criteria (Implicit from Results)Reported Device Performance (OIA SHIGATOX)
Analytical Sensitivity (LOD)Lowest toxin concentration producing at least 50% positive results.Stx 1: 1 ng/mL (in both buffer and liquid stool)Stx 2: 0.5 ng/mL (in antigen diluent); 1 ng/mL (in liquid stool)
Analytical Strain RecognitionDetect all Shiga toxin-producing strains.100% agreement (70/70 clinical isolates and Shigella dysenteriae)
Analytical Specificity (Cross-Reactivity)Expected negative without toxin spike, expected positive with toxin spike.All members of the cross-reactivity panel produced expected negative results without toxin spike and expected positive results with toxin spike. Demonstrated no cross-reactivity with various bacteria, fungi, and parasites, nor with commercial Rotavirus EIA positive stools.
Interfering SubstancesNo false positive or false negative results in presence of interferents.None of the tested substances (Barium Sulfate, Bovine Mucin, Kaopectate®, Pepto Bismol®, Imodium®, Whole Blood) caused false positives or false negatives in antigen diluent or in liquid/semi-solid stool, up to specified concentrations.
Reproducibility100% inter-site and intra-site reproducibility.Overall Score for the Study: 100% (486/486 samples across 3 clinical sites, 3 POL sites, and 3 days). Achieved 100% agreement for all operators, all days, within runs, between runs, and between sites.
Clinical Sensitivity/Specificity (Colony Sweep)100% agreement with previous specimen result.100% agreement (21/21 colony sweeps) with previous specimen results.
Clinical Performance (Direct Stool vs. EIA - Prospective)High agreement with commercial EIA; positive samples confirmed by CTA reference.Positive Agreement: 100% (95% CI: 73.5 - 100%)Negative Agreement: 98.1% (95% CI: 95.6 - 99.4%)Overall Percent Agreement: 98.2% (95% CI: 95.8 - 99.4%)1/5 OIA positive/EIA negative samples confirmed by CTA.
Clinical Performance (Direct Stool vs. EIA - Frozen)High agreement with commercial EIA.Positive Agreement: 87.5% (95% CI: 67.6 – 97.3%)Negative Agreement: 97.4% (95% CI: 86.2 – 99.9%)Overall Percent Agreement: 93.6% (95% CI: 84.3 – 98.2%)
Clinical Performance (Broth Culture vs. EIA - Fresh)High agreement with commercial EIA; positive samples confirmed by CTA reference.Positive Agreement: 100% (95% CI: 73.5 - 100%)Negative Agreement: 99.6% (95% CI: 97.9 - 100%)Overall Percent Agreement: 99.6% (95% CI: 98.0 - 100%)1 OIA positive/EIA negative sample confirmed by CTA.
Clinical Performance (Broth Culture vs. EIA - Frozen)High agreement with commercial EIA.Positive Agreement: 100% (95% CI: 84.6-100%)Negative Agreement: 95.6% (95% CI: 81.7-99.9%)Overall Percent Agreement: 98% (95% CI: 89.4 - 100%)
Clinical Performance (Direct Stool vs. SMAC - Fresh)High agreement, acknowledging SMAC limitations.Positive Agreement: 90% (95% CI: 55.5 – 99.8%)Negative Agreement: 96.9% (95% CI: 94.0 – 98.7%)Overall Percent Agreement: 96.7% (95% CI: 93.7 – 98.5%)4/8 OIA+/SMAC- samples confirmed by CTA.
Clinical Performance (Direct Stool vs. SMAC - Frozen)High agreement, acknowledging SMAC limitations.Positive Agreement: 100% (95% CI: 66.4-100%)Negative Agreement: 75.5% (95% CI: 61.7 - 86.2%)Overall Percent Agreement: 79% (95% CI: 66.8 - 88.3%)All 13 OIA+/SMAC- samples were previously positive for STEC.
Clinical Performance (Direct Stool vs. CTA)High detection rate of CTA positives.OIA SHIGATOX detected 12/13 CTA positive direct stool samples. EIA detected 11/13.
Clinical Performance (Broth Culture vs. CTA)100% detection rate of CTA positives.OIA SHIGATOX detected 12/12 CTA positive broth aliquots. EIA also detected 12/12.
Clinical Performance (1 Hour Cary Blair vs. Direct OIA)High agreement with direct OIA reference.Positive Agreement: 88% (95%CI: 71 - 96.5%)Negative Agreement: 100% (95%CI: 94.6 - 100%)Overall Percent Agreement: 96% (95%CI: 89.9 - 98.9 %)
Clinical Performance (24 Hour Cary Blair vs. Direct OIA)High agreement with direct OIA reference.Positive Agreement: 91% (95%CI: 75 - 98%)Negative Agreement: 100% (95%CI: 94.6 - 100%)Overall Percent Agreement: 97% (95%CI: 91.3 – 99.4 %)
Clinical Performance (GN Broth vs. MAC Broth - Direct Fecal)High agreement with MAC broth reference.Positive Agreement: 100% (95%Cl: 86.8 - 100%)Negative Agreement: 95% (95%Cl: 84.5 - 99.4%)Overall Percent Agreement: 97.1% (95%CI: 90.1 - 99.7%)
Clinical Performance (GN Broth from Cary Blair vs. Direct MAC Broth)High agreement with MAC broth reference.Positive Agreement: 100% (95%CI: 86.8 - 100%)Negative Agreement: 98% (95%CI: 87.4 - 99.9%)Overall Percent Agreement: 98.5% (95%CI: 92.1 - 100%)
Clinical Performance (MAC Broth from Cary Blair vs. Direct MAC Broth)High agreement with MAC broth reference.Positive Agreement: 93% (95%CI: 76.5 – 99.1%)Negative Agreement: 93% (95%CI: 80.9 – 98.5%)Overall Percent Agreement: 93% (95%CI: 84.3 – 97.7%)

Study Details:

  1. Sample sizes used for the test set and data provenance:

    • Analytical Sensitivity: No specific sample size for a "test set" in the traditional sense, but tested in triplicate for LOD determination (5 samples at each concentration analyzed on 2 lots of devices).
    • Analytical Strain Recognition: 70 clinical isolates (49 from a Department of Public Health, 21 from a university laboratory) + Shigella dysenteriae (ATCC 13313). Data provenance appears to be from US public health and university laboratories.
    • Analytical Specificity (Cross-Reactivity): 43 different organisms and Rotavirus-positive stools.
    • Reproducibility Studies: 9 sample types (Negative, Low Stx 1, Low Stx 2, Moderate Stx 1, Moderate Stx 2, various combinations). Each sample tested in triplicate by each of 6 operators over 3 consecutive days for a total of 486 tests (9 samples * 3 replicates * 6 operators * 3 days). Data provenance: Not explicitly stated beyond "clinical trial sites" and "Physician Office Laboratories (POL) sites." Samples were prepared by spiking stools from a healthy individual.
    • Clinical Specimen Testing (Unmodified Device):
      • Colony Sweep: 21 positive samples (from 22 frozen fecal specimens, one failed to grow). Data provenance: Not explicitly stated, samples were "previously found to contain Shiga toxin producing E. coli."
      • Direct Stool (Prospective): 272 prospective diarrheal fecal specimens. Data provenance: Collected from three clinical trial sites in the Eastern and Western regions of the United States.
      • Direct Stool (Frozen): 62 additional frozen specimens. Data provenance: Not explicitly stated, but "two of the clinical sites" performed this study.
      • Broth Culture (Fresh): 269 prospective diarrheal fecal specimens (from original 272, three failed to grow). Data provenance: Same as Direct Stool (Prospective).
      • Broth Culture (Frozen): 50 frozen specimens (from 62, 12 were not tested or failed to grow). Data provenance: Same as Direct Stool (Frozen).
      • SMAC Culture Comparison (Fresh Stool): 269 direct stool samples. Data provenance: Same as Direct Stool (Prospective).
      • SMAC Culture Comparison (Frozen Stool): 62 frozen samples. Data provenance: "two of the clinical sites."
      • CTA Comparison: 19 specimens (direct stool aliquot and broth culture aliquot) from the clinical study (one excluded as inconclusive).
    • Clinical Specimen Testing (Modified Device - New Sample Types):
      • Cary Blair & Broth Inoculation Validation: 98 frozen fecal specimens. Data provenance: Collected from 2 laboratory sites (University of Utah & Primary Children's, Fairfax Hospital).
      • 1 and 24 Hour Cary Blair (vs. Direct OIA): 98 frozen fecal specimens.
      • GN/MAC Broth Culture (Direct Fecal vs. MAC Broth OIA): 98 fecal specimens.
      • GN Broth from Cary Blair (vs. Direct MAC Broth OIA): 98 fecal specimens.
      • MAC Broth from Cary Blair (vs. Direct MAC Broth OIA): 98 fecal specimens.

  2. Number of experts used to establish the ground truth for the test set and their qualifications:

    • Analytical Strain Recognition: Ground truth based on "previously analyzed for the presence of Shiga toxin genes and serotyped" by the Department of Public Health and a university laboratory. No specific number or qualifications of experts are given, but implies laboratory expertise in molecular and serological typing.

    • Clinical Studies (Unmodified Device):

      • Comparator Methods: A commercial EIA test, SMAC culture, and Cytotoxicity Testing Assay (CTA) were used as comparators or ground truth methods.
      • CTA Confirmation: "All positive results from either immunoassay method were confirmed by cytotoxicity testing, CTA." CTA itself is a reference method for Shiga toxin activity. No specific number or qualifications of "experts" involved in CTA interpretation are provided, but it's an established laboratory method.
      • Clinical sites were staffed by MT ASCP (Medical Technologists certified by the American Society for Clinical Pathology).

    • Reproducibility Studies: Expected results were based on the known spiked concentrations of toxins.

    • Clinical Studies (Modified Device): Direct fecal testing in the OIA method or direct fecal inoculation of MAC broth were used as reference methods.

  3. Adjudication method for the test set:

    • For the prospective clinical studies, "All positive results from either immunoassay method were confirmed by cytotoxicity testing, CTA." This implies a form of adjudication where discrepant positive immunoassay results were further investigated by CTA, serving as a higher-level reference.
    • For reproducibility studies, there was no adjudication method mentioned, as results were compared to known spiked concentrations.

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

    • This is not an AI-assisted device. The device is a rapid diagnostic test (Optical Immunoassay) for direct detection of Shiga toxins, generating a visible color change for interpretation. Therefore, an MRMC comparative effectiveness study comparing human readers with and without AI assistance is not applicable.
    • The "Reproducibility Studies" did involve multiple operators (6 operators across 6 sites) reading 27 blinded and randomized samples in triplicate over 3 days, demonstrating consistency in human interpretation of the device's visual output. This is a multi-reader, multi-case study for reproducibility, but not for AI-assisted human improvement.

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

    • Yes, the performance data presented (e.g., sensitivity, specificity, agreement percentages) is the standalone performance of the OIA SHIGATOX device. The device produces a "qualitative result for the presence or absence of Shiga toxin as the device output" based on a visual color change. While a human interprets this visual change, the performance metrics reported represent the device's ability to correctly identify the toxin compared to reference methods, independent of human variability in the interpretation step. The high reproducibility across multiple readers attests to the clarity and consistency of this visual output for human interpretation.

  6. The type of ground truth used (expert concensus, pathology, outcomes data, etc):

    • Analytical Strain Recognition: Shiga toxin gene presence and serotyping (presumably by molecular and serological methods).
    • Clinical Studies (Unmodified Device):
      • Primary Comparator: Commercial EIA test.
      • Confirmatory Reference: Cytotoxicity Testing Assay (CTA).
      • Additional Comparator: SMAC culture (acknowledged for its limitations as a direct comparison method for all STEC).
    • Clinical Studies (Modified Device):
      • Primary Reference: Direct fecal testing in the OIA method (for Cary Blair comparisons) or direct fecal inoculation of MAC broth (for broth culture comparisons). These are essentially using the device itself or a similar culture method as the internal reference for evaluating new sample types with the device.

  7. The sample size for the training set:

    • The document does not explicitly describe a "training set" in the context of a machine learning algorithm. This is a medical device (Optical Immunoassay), not an AI/ML algorithm. The development process involved various analytical studies and clinical trials to establish performance, but these are not referred to as "training sets." The 70 clinical isolates used for Analytical Strain Recognition and the samples used for analytical sensitivity and specificity contribute to establishing the device's operational characteristics, which is analogous to a development set in traditional terms.

  8. How the ground truth for the training set was established:

    • As noted above, there isn't a "training set" in the context of an AI/ML device. However, the ground truth for the various analytical and clinical studies were established using methods such as:
      • Known spiked concentrations of purified Stx 1 and Stx 2 toxins (for analytical sensitivity).
      • Clinical isolates "previously analyzed for the presence of Shiga toxin genes and serotyped" (for analytical strain recognition).
      • Reference tests like commercial EIA, SMAC culture, and crucially, Cytotoxicity Testing Assay (CTA) for clinical samples (for general device performance validation).

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This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92(c).

The assigned 510(k) number is: K071799

OCT 2 2007

Information required per [§807.92(a)(1)]: A.

Submitter's Name:Inverness Medical-BioStar, Inc.
Address:331 South 104th StreetLouisville, CO 80027
Telephone:(303) 530 – 6612
Fax:(303) 530 – 6601
Email:robin.hart@invmed.com
Contact Name:Robin C. Hart, Ph.D.
Date 510(k) Summary Prepared:August 21, 2007

Information required per [§807.92(a)(2)]: в.

Name of the Device:BioStar® OIA® SHIGATOX
Common Name:Direct Antigen Detection, Shiga Toxins,Escherichia coli, other enterohemorrhagicorganisms
Product Code:GNA -- Antisera, all types, Escherichia coliGMZ - Antigens, all types, Escherichia coli
Regulation Section:21 CFR Part 866.3255Escherichia coli Serological Reagents
Classification:Class I
Panel:83 Microbiology

். Identification of legally marketed device to which we are claiming equivalence {§807.92(a)(3)]:

BioStar OIA SHIGATOX; K061889; Inverness Medical-BioStar Inc.

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Device Description [§807.92(a)(4)]: D.

There has been no change in the device description from the previously cleared device. However, a description of the device is provided below.

Summary and Explanation a.

Shiga toxin-producing Escherichia coli (STEC) strains are an important cause of epidemic and endemic diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). The most commonly reported serotype associated with outbreaks in the United States has been 0157:H7. However, as many as 50 other serotypes of E. coli have also been shown to produce Shiga toxins and have been reported to be associated with outbreaks and sporadic disease both within and outside the United States'. Due to the morbidity and mortality associated with outbreaks and sporadic cases of STEC diseases, these pathogens are now considered major public health problems of worldwide importance. STEC strains share the potential to produce a variety of virulence factors, including two Shiga toxins (Stx 1 and Stx 2). Human STEC strains can produce Stx 1 or Stx 2 alone or in combination2.

The ability to control the spread of outbreaks associated with STEC depends upon the rapid detection of these pathogens. Laboratory diagnosis of diarrhea has been recommended as it may alert public health officials to a common source of illness in a community. For the affected patient, laboratory confirmation of STEC may preclude costly additional tests, allow earlier implementation of supportive treatment in impending HUS, and, while controversial, exclude administration of potentially harmful antimicrobial agents . The method used in most clinical microbiology laboratories is based on sorbitol MacConkey agar culture (SMAC), coupled with specific detection of the 0157 antigen. This approach neglects other STEC serotypes and also other Shiga toxin-producing bacteria". The cytotoxin assay described by Karmali is used to screen stool samples for evidence of STEC infections. However, this test is slow, labor-intensive, difficult to standardize, and it requires cell culture facilities, making it impractical for routine diagnostic laboratories 4.

References cited may be found in the draft package insert in the Proposed Labeling section on page 49 of this submission.

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Principle of the Test b.

The OIA SHIGATOX test involves the qualitative detection of Shiga toxins 1 and 2 (Stx1 and Stx 2) produced by certain strains of Escherichia coli and other organisms. The Optical ImmunoAssay technology enables the direct visual detection of a physical change in the optical thickness of molecular thin films. This change is the result of antigen - antibody binding on an optical surface (silicon wafer). After a specimen potentially containing Shiga toxin is mixed with conjugates and placed directly on the optical surface, the immobilized surface antibodies capture the antigen/conjugate complex. After washing, the substrate is added, increasing the thickness (mass enhancement) of the molecular thin film. This change in thickness alters the reflected light path, and this alteration is visually perceived as a color change. Slight changes in the optical thickness produce a distinct visible color change. A positive result appears as a purple spot on the gold background. When antigen is not present in the specimen, no binding takes place. Therefore, the optical thickness remains unchanged, and the surface retains the original gold color indicating a negative result.

More specifically, the BioStar OIA SHIGATOX device is based on a novel thin film optical detection technology that relies on the interaction of white light with thin films to create a destructive interference phenomenon. Characteristic of this phenomenon is the generation of a reflective surface that changes color as a function of the change in optical thickness (refractive index x thickness) of the films on the surface of the device. To take advantage of this phenomenon for monitoring biological binding events, the optical surface with a special background color is coated with a capture reagent specific to the analyte of interest. In the OIA SHIGATOX device, the biological capture film is a combination of affinity-purified polyclonal antibodies to Shiga toxins 1 and 2 (Stx 1 and Stx 2). Samples suspected of containing either or both of the toxins are mixed with cocktail containing polyclonal antibodies to Stx 1 and Stx 2 that have been covalently conjugated to horseradish peroxidase (HRP). Once a sample containing toxins or either toxin is applied to the surface, the immune complex of toxin(s) and the anti-toxin-HRP conjugate(s) are bound to the surface antibodies. Following a wash step, a precipitating substrate for HRP is added, and a thin film generated by the immobilized immune complex is enhanced by the precipitation of the HRP product. Once washed and dried, a simple color change relative to the gold background color is observed as an indication of the presence of Stx 1 or Stx 2 in the original specimen.

The OIA SHIGATOX device produces a qualitative result for the presence or absence of Shiga toxin as the device output. Input to the device is the simple addition of an aliquot of fecal material (direct or in transport media) or broth culture to the reagents contained in the kit. Fecal samples are routinely collected and no special collection requirements exist. Test devices within the

INVERNESS MEDICAL-BIOSTAR INC. OIA SHIGATOX 510(k) NOTIFICATION

REVISION 8/21/2007 Page 15 of 69

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kit are single use devices, and disposal instructions are provided in the Package Insert. The kit contains all components necessary for analysis of the range of samples approved for use in this product, with the exception of a timer.

Device Description C.

Reagent 1 - Conjugate 1: Contains anti-Shiga Toxin 1 antibodies (rabbit) conjugated to Horseradish Peroxidase (HRP) in a buffered protein solution, preserved with 0.5% ProClin® 300 preservative.

Reagent 2 - Conjugate 2: Contains anti-Shiga Toxin 2 antibodies (rabbit) conjugated to Horseradish Peroxidase (HRP) in a buffered protein solution, preserved with 0.5% ProClin 300 preservative.

Wash - Contains buffered detergent solution preserved with 0.1% ProClin 300 preservative.

Substrate - Tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2).

Test Devices - Surfaces coated with anti-Stx 1 and anti-Stx-2 affinity purified rabbit polyclonal antibodies.

Positive Control - Inactivated purified Shiga toxin preserved with 0.01% Microcide II and 0.005% gentamycin preservative in a buffered protein solution.

Diluent / Negative Control - Buffered protein solution preserved with 0.01% Microcide II and 0.005% gentamycin.

Reaction Tubes

Transfer Pipettes

Rayon Swabs

Materials Required But Not Provided - timer

Assay Protocol

Liquid Specimens: The Transfer Pipette provided should be . used for liquid fecal specimens.

Semi-Solid Specimens: Dip the swab into the specimen, . rotating gently to absorb sample. Rotate the swab gently against the wall of

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the sample container to remove excess fecal material. Add 2 drops of Diluent / Negative Control to the reaction tube.

. Cary Blair Specimens: The Transfer Pipette provided should be used for Cary Blair Specimens.

. Colony Collection: Wipe a swab across the first and second quadrants of the growth area on the plate and then immerse the swab into a reaction tube containing 3 drops each of Reagents 1 and 2 and follow the Assay Procedure.

Remove reagents from refrigerated storage and allow to warm to room temperature (15° to 30°C). Store Wash Solution at room temperature (15° to 30°C) after opening. If stored refrigerated, Wash Solution will take up to 2 hours to warm to room temperature.

Remove one Reaction Tube for each specimen to be tested and place it upright in a rack or holder. Label Reaction Tubes and Test Devices with appropriate patient information. Place Test Devices on a level surface while the assay is being performed. Mix all fecal specimens thoroughly before sampling.

    1. Add 3 drops of Reagent 1 to the Reaction Tube.
  • Add 3 drops of Reagent 2 to the Reaction Tube. 2.

Note: The combined Reagents 1 and 2 in the Reaction Tube should have a blue color.

Thoroughly mix the fecal specimen with the Transfer Pipette. Add 2 ന drops of the specimen to the Reaction Tube and mix by squeezing the sides of the tube. For semi-solid specimens, an enclosed swab may be used to add specimen into the tube. Refer to "Semi-Solid Specimens" under the Test Procedure for additional information.

Within 1 minute of mixing, use the Transfer Pipette to place 1 to 2 4. drops (to cover the surface) of the sample mixture directly onto the center of the test surface. Wait 10 minutes.

Wash the surface vigorously with a hard squirt of Wash Solution, 5. taking care not to exceed the capacity of the absorbent material surrounding the test surface.

Note: Vigorous washing will aid in obtaining a clean test surface. Insufficient washing of the test surface may leave debris that may result in the appearance of a ring or spots surrounding the positive Internal Control dot. These effects should not be interpreted as a positive result due to the lack of color shading within the ring area.

Confirm that the blotter in the Test Device lid is in Position I. Close the 6. Test Device at the corners. Leave closed for 10 seconds to remove residual moisture from the test surface.

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  1. Open the lid, change the blotter to Position II, and apply 1 drop of Substrate directly to the center of each Test Device. Wait 5 minutes.

Note: Do not cover the entire surface of the Test Device with Substrate. The gold, unreacted areas surrounding the reaction circle serves as a negative Internal Control and reference for comparing signal intensity.

Repeat Step 5, washing the test surface vigorously with Wash 8. Solution. Close the Test Device at the corners. Leave closed for 10 seconds. Open and examine the test surfaces for color changes (See Interpretation of Test Results).

ய் Intended Use [§807.92(a)(5)]:

The Inverness Medical BioStar OIA SHIGATOX assav is an Optical Immunoassay (OIA) test for the qualitative, rapid detection of the presence of Shiga toxins in human diarrheal fecal specimens, broth cultures, fecal specimens in Cary Blair Transport Media, or swab sampling of colonies from a culture plate. This test is intended for in vitro diaqnostic use as an aid in the diagnosis of infection by Shiga toxin-producing Escherichia coli (STEC) both O157 and all non - O157 Shiga toxin-producing strains.

ய Summary of non - clinical testing [§807.92(b)(1)]

  • Analytical Sensitivity a.
    To determine the analytical sensitivity, two-fold serial dilutions of purified Stx 1 or Stx 2 toxin were prepared. These dilutions were then spiked into buffer and the spiked samples were tested in triplicate. The Limit of Detection (LOD) was defined as the lowest toxin concentration producing at least two positive results of the three tests or at least 50% of the total number of samples tested. In an antigen diluent formulation, the limits of detection were 1 ng/mL and 0.5 ng/mL for Stx 1 and Stx 2, respectively. The same levels were tested in a liquid stool and the LOD for each toxin was determined to be 1 ng/mL. Five samples at each concentration were analyzed on 2 lots of devices.

  • b. Analytical Strain Recognition

Toxigenic Strain Testing

To establish that the OIA SHIGATOX assay detects all Shiga toxin producing strains and organisms, the following studies were conducted. Clinical isolates, previously analyzed for the presence of Shiga toxin genes and serotyped, were obtained from a Department of Public Health (49 isolate strains) and a university laboratory (21 isolate strains). Lists of the isolates from each source are presented in the tables. Tubes of MacConkey broth

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were inoculated with each isolate and cultured overnight at 37° C under aerobic conditions. A 60-µL sample of the broth was then tested in the OIA SHIGATOX assay without further processing using the same protocol as was used with clinical samples after broth enrichment.

Somatic AntigenFlagellar AntigenNumber of strains
Stx-1 onlyStx-2 onlyStx-1 + Stx-2
O26H1193
O28H251
O76H191
O103H21
O103H251
O111Non-motile41
O121H192
O121Non-motile1
O145Non-motile31
O146H211
O157H7314
O165Non-motile1
UndeterminedH341
Non-motile1

Department of Public Health Isolate List

University Laboratory Isolate List

Number of strains
SomaticAntigenFlagellarAntigenStx-1onlyStx-2onlyStx-1 +Stx-2
O26Not Available5
O103Not Available5
O128Not Available21
O157H753

In addition, Shigella dysenteriae (ATCC 13313) was tested and produced a positive result as expected. All 70 clinical isolates produced the expected positive assay result. The OIA SHIGATOX assay detects the Shiga toxin from a wide range of toxin-producing E. coli. Similar results were observed with multiple lots of material throughout the development process.

REVISION 8/21/2007 Page 19 of 69

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C. Analytical Specificity (Cross Reactivity)

Bacteria were grown on appropriate media and suspended in Antigen Diluent, a buffered protein solution, to stock concentrations of 1 x 10° organisms/mL or higher. The organisms were tested with and without spiking with 2.5 ng/mL Stx 1 and 2.5 ng/mL Stx 2. The exceptions were that Cryptosporidium and Giardia were tested at 1 x 10° cysts/mL, and Candida albicans was tested at 9.3 x 107 cells/mL. All members of the cross reactivity panel produced the expected negative result without the toxin spike and the expected positive result with the toxin spike.

Aeromonas hydrophila Bacillus cereus Bacillus subtilis Bacteroides fragilis Bifidobacterium adolescentis Campylobacter fetus Campvlobacter ieiuni Candida albicans Citrobacter freundii Clostridium botulinum Type A Clostridium butyricum Clostridium histolyticum Clostridium innocuum Clostridium novvi Clostridium perfringens Clostridium septicum Clostridium sordellii Clostridium subterminale Clostridium tetani Cryptosporidium parvum Enterobacter aerogenes Enterobacter cloacae Enterococcus faecalis Escherichia coli (non-STEC)

Giardia lamblia Klebsiella pneumoniae Peptostreptococcus anaerobius Porphyromonas asaccharolytica Proteus vulgaris Providencia rettgeri Pseudomonas aeruginosa Salmonella diarizonae Salmonella enteriditis Salmonella typhi Salmonella typhimurium Serratia liquefaciens Serratia marcescens Shigella flexneri Serotype 1A Shigella sonnei Staphylococcus aureus Staphylococcus aureus Cowan I Staphylococcus epidermidis Staphylococcus saprophyticus Veillonella parvula Vibrio cholerae Vibrio parahaemolyticus Yersinia enterocolitica

In addition, one clinical trial site tested three stools in the OIA SHIGATOX assay that were positive in a commercial lateral flow, EIA assay for Rotavirus. All 3 samples were negative in the OIA SHIGATOX assay as expected.

ರ. Interfering Substances

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The OIA SHIGATOX assay was tested with whole blood, mucin, liquid Imodium® AD, Pepto Bismol®, Kaopectate®, and Barium Sulfate for the potential of these materials to interfere with assay results. Clinical trial lots of product were tested, and the conjugate and test surface were specific to each lot. To test for potential nonspecific signal generation, a 30 µL aliquot of each a potential interferent stock was mixed with 30 µL of Antigen Diluent or a l'ouid or semi – solid stool specimen and run as the sample with the standard assay protocol. To test for potential interference with the positive signal, the 30 มป. aliquot was mixed with 30 µL Antigen Diluent or a liquid or semi – solid stool specimen containing 5 ng/mL of each toxin (final concentrations of Stx 1 and Stx 2 were 2.5 ng/mL). Each sample was tested in duplicate.

Test MaterialStockConcentration(used 1:1 inthe sample)Interferent+ NegativeSampleInterferent+ PositiveSample
Lot1Lot2Lot1Lot2
Barium Sulfate58% (Neat)-,--,-+,++,+
Bovine Mucin25 mg/g-,--,-+,++,+
Kaopectate®Neat-,--,-+,++,+
Pepto Bismol®Neat-,--,-+,++,+
Imodium®Liquid (Neat)-,--,-+,++,+
Whole BloodNeat-,--,-+,++,+

Interferents diluted into antigen diluent

None of the substances, tested at the stated concentration, caused a false positive or false negative result in the assay. The OIA SHIGATOX assay is tolerant to:

29% Barium sulfate 12.5 mg/g mucin 50% Kaopectate 50% Pepto Bismol 50% Liquid Imodium 50% Whole blood

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Interferents tested in liquid and semi – solid stool

TestMaterialStockConcentration(used 1:1 inthe sample)Interferent +NegativeSampleInterferent +PositiveSample
LiquidStoolSemi-SolidLiquidStoolSemi-Solid
BariumSulfate58% (Neat)-,--,-+,++,+
BovineMucin25 mg/g-,--,-+,++,+
Kaopectate®Neat-,--,-+,++,+
PeptoBismol®Neat-,--,-+,++,+
Imodium®Liquid (Neat)-,--,-+,++,+
Whole BloodNeat-,--,-+,++,+

Reproducibility Studies e.

Objective:

The objective of the Reprodubility Studies was to demonstrate the inter - site and intra – site reproducibility of results obtained with the OIA SHIGATOX test. Testing at the Physician Office Laboratories (POL) sites was conducted to verify the ease of use of the product and is not included as an indication that testing should be performed at a POL site.

Materials & Methods:

The reproducibility of the OIA SHIGATOX assay was determined for six operators, one operator at each of the three clinical trial sites and three operators at two Physician Office Laboratory (POL) sites. Samples were blinded and randomized throughout the study. Samples were made by spiking stools from a healthy individual that were screened and confirmed to be negative for Shiga toxin. Two levels of each toxin (8 and 16 ng/mL) were tested both alone and in combinations with the other toxin. This scheme led to the preparation of 9 sample types: Negative, Low Stx 1 only, Low Stx 2 only, Moderate Stx 1 only, Moderate Stx 2 only, Low Stx 1/Low Stx 2, Low Stx 1/Moderate Stx 2, Moderate Stx 1/Low Stx 2, and Moderate Stx 1/Moderate Stx 2. By testing each sample in triplicate on each day, each operator evaluated a total of twenty-seven fecal samples on 3 consecutive days. Sites were instructed to analyze the samples as normal patient samples according

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to the Package Insert instructions. The Sample Key is provided in Table 15, and the individual results by day are presented in Tables 16 to 18.

Abbreviations Used:

No toxin added Neg:

8 ng/mL Stx 1 L1:

L2: 8 ng/mL Stx 2

M1: 16 ng/mL Stx 1

M2: 16 ng/mL Stx 2

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.. .. .. ........ .. .. .

SampleIDDay 1ToxinExpectedResultsDay 2ToxinExpectedResultsDay 3ToxinExpectedResultsSampleIDExpectedResultsClinicalSite 1ClinicalSite 2ClinicalSite 3POL 1Operator1POL 2Operator2POL 3Operator3
1L2PositiveM1PositiveL1Positive1PositivePositivePositivePositivePositivePositivePositive
2M2PositiveNegNegativeM1 +M2Positive2PositivePositivePositivePositivePositivePositivePositive
3L1PositiveL1 +M2PositiveL2 +M1Positive3PositivePositivePositivePositivePositivePositivePositive
4L2 +M1PositiveL2 +M1PositiveL1 +L2Positive4PositivePositivePositivePositivePositivePositivePositive
5L1 +L2PositiveL1 +L2PositiveL2Positive5PositivePositivePositivePositivePositivePositivePositive
6L1PositiveL1 +L2PositiveL1 +L2Positive6PositivePositivePositivePositivePositivePositivePositive
7L2 +M1PositiveL1PositiveM1Positive7PositivePositivePositivePositivePositivePositivePositive
8M1 +M2PositiveL1 +M2PositiveL2 +M1Positive8PositivePositivePositivePositivePositivePositivePositive
9NegNegativeNegNegativeM2Positive9NegativeNegativeNegativeNegativeNegativeNegativeNegative
10L2PositiveM1 +M2PositiveL1 +L2Positive10PositivePositivePositivePositivePositivePositivePositive
11M1PositiveM1PositiveL1 +M2Positive11PositivePositivePositivePositivePositivePositivePositive
12M1PositiveL2PositiveNegNegative12PositivePositivePositivePositivePositivePositivePositive
13L2 +M1PositiveL1 +L2PositiveM1Positive13PositivePositivePositivePositivePositivePositivePositive
14L1 +M2PositiveM2PositiveL1 +M2Positive14PositivePositivePositivePositivePositivePositivePositive
15L1 +M2PositiveL2PositiveM1 +M2Positive15PositivePositivePositivePositivePositivePositivePositive
16NegNegativeL1PositiveNegNegative16NegativeNegativeNegativeNegativeNegativeNegativeNegative
17M1 +M2PositiveM1PositiveL2Positive17PositivePositivePositivePositivePositivePositivePositive
18L1PositiveNegNegativeL2Positive18PositivePositivePositivePositivePositivePositivePositive
19M1PositiveM2PositiveL1Positive19PositivePositivePositivePositivePositivePositivePositive
20M1 +M2PositiveM2PositiveNegNegative20PositivePositivePositivePositivePositivePositivePositive
21M2PositiveL1PositiveL1 +M2Positive21PositivePositivePositivePositivePositivePositivePositive
22NegNegativeM1 +M2PositiveM1 +M2Positive22NegativeNegativeNegativeNegativeNegativeNegativeNegative
23L1 +M2PositiveL2 +M1PositiveM2Positive23PositivePositivePositivePositivePositivePositivePositive
24L2PositiveL2 +M1PositiveL1Positive24PositivePositivePositivePositivePositivePositivePositive
25M2PositiveM1 +M2PositiveM2Positive25PositivePositivePositivePositivePositivePositivePositive
26L1 +L2PositiveL1 +M2PositiveM1Positive26PositivePositivePositivePositivePositivePositivePositive
27L1 +L2PositiveL2PositiveL2 +M1Positive27PositivePositivePositivePositivePositivePositivePositive
Score100%100%100%100%100%100%

. . . . . .

... ..........

Reproducibility Study Key

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Study Results, Dav 1

. ...

......

. ------

.

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SampleIDExpectedResultsClinicalSite 1ClinicalSite 2ClinicalSite 3POL 1Operator1POL 2Operator2POL 3Operator3
1PositivePositivePositivePositivePositivePositivePositive
2NegativeNegativeNegativeNegativeNegativeNegativeNegative
3PositivePositivePositivePositivePositivePositivePositive
4PositivePositivePositivePositivePositivePositivePositive
5PositivePositivePositivePositivePositivePositivePositive
6PositivePositivePositivePositivePositivePositivePositive
7PositivePositivePositivePositivePositivePositivePositive
8PositivePositivePositivePositivePositivePositivePositive
9NegativeNegativeNegativeNegativeNegativeNegativeNegative
10PositivePositivePositivePositivePositivePositivePositive
11PositivePositivePositivePositivePositivePositivePositive
12PositivePositivePositivePositivePositivePositivePositive
13PositivePositivePositivePositivePositivePositivePositive
14PositivePositivePositivePositivePositivePositivePositive
15PositivePositivePositivePositivePositivePositivePositive
16PositivePositivePositivePositivePositivePositivePositive
17PositivePositivePositivePositivePositivePositivePositive
18NegativeNegativeNegativeNegativeNegativeNegativeNegative
19PositivePositivePositivePositivePositivePositivePositive
20PositivePositivePositivePositivePositivePositivePositive
21PositivePositivePositivePositivePositivePositivePositive
22PositivePositivePositivePositivePositivePositivePositive
23PositivePositivePositivePositivePositivePositivePositive
24PositivePositivePositivePositivePositivePositivePositive
25PositivePositivePositivePositivePositivePositivePositive
26PositivePositivePositivePositivePositivePositivePositive
27PositivePositivePositivePositivePositivePositivePositive
Score100%100%100%100%100%100%

Study Results, Day 2

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.. .....

:

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SampleIDExpectedResultsClinicalSite 1ClinicalSite 2ClinicalSite 3POL 1Operator1POL 2Operator2POL 3Operator3
1PositivePositivePositivePositivePositivePositivePositive
2NegativeNegativeNegativeNegativeNegativeNegativeNegative
3PositivePositivePositivePositivePositivePositivePositive
4PositivePositivePositivePositivePositivePositivePositive
5PositivePositivePositivePositivePositivePositivePositive
6PositivePositivePositivePositivePositivePositivePositive
7PositivePositivePositivePositivePositivePositivePositive
8PositivePositivePositivePositivePositivePositivePositive
9NegativeNegativeNegativeNegativeNegativeNegativeNegative
10PositivePositivePositivePositivePositivePositivePositive
11PositivePositivePositivePositivePositivePositivePositive
12PositivePositivePositivePositivePositivePositivePositive
13PositivePositivePositivePositivePositivePositivePositive
14PositivePositivePositivePositivePositivePositivePositive
15PositivePositivePositivePositivePositivePositivePositive
16PositivePositivePositivePositivePositivePositivePositive
17PositivePositivePositivePositivePositivePositivePositive
18NegativeNegativeNegativeNegativeNegativeNegativeNegative
19PositivePositivePositivePositivePositivePositivePositive
20PositivePositivePositivePositivePositivePositivePositive
21PositivePositivePositivePositivePositivePositivePositive
22PositivePositivePositivePositivePositivePositivePositive
23PositivePositivePositivePositivePositivePositivePositive
24PositivePositivePositivePositivePositivePositivePositive
25PositivePositivePositivePositivePositivePositivePositive
26PositivePositivePositivePositivePositivePositivePositive
27PositivePositivePositivePositivePositivePositivePositive
Score100%100%100%100%100%100%

Study Results, Day 3

Overall Score for the Study: 100%

Conclusions:

Overall inter - site and intra - site reproducibility was 100% (486/486 samples) for all operators and all days. There were no differences within run (replicates tested by one operator), between run (different days), between sites (6 sites), or between operators (6 operators).

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Summary of clinical specimen testing: G.

Summary of Performance Data for Unmodified Device:

Clinical Trial Sites:

SiteGeographicAreaInstitution TypeInstitutionSizeSiteTesters
Universityof UtahandPrimaryChildrens(UT)Western USChildren'sHospital280 bedsMTASCP
FairfaxHospital(VA)EasternSouthernUSTertiary CareCommunityHospital850 bedsMTASCP
Children'sHealthcareof Atlanta(GA)EasternSouthernUSHospitalPediatricSystem/Universityand Community-based417 bedsMTASCP

Clinical Sensitivity and Specificity

  • Swab Sampling of Colonies from a Culture Plate (Colony Sweep 1. Method)
    One clinical site evaluated twenty two frozen fecal specimens in a colony sweep procedure. These samples were previously found to contain Shiga toxin producing E. coli. All samples were streaked onto Sorbito! MacConkey (SMAC) and XLD (xylose lysine deoxycholate) plates and incubated overnight at 37°C. One sample failed to produce any growth. A sterile rayon swab was used to sweep the first and second quadrants of the growth area and was then immersed into a reaction tube containing 3 drops each of Reagents 1 and 2 and the standard assay protocol followed. The OIA SHIGATOX assay detected 21/21 of the colony sweeps that produced growth for 100% agreement with the previous specimen result.

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2. Direct Stool

A prospective study was conducted at three clinical trial sites in the Eastern and Western regions of the United States to compare the performance of the BioStar OIA SHIGATOX to a commercial EIA test. Sites analyzed the stool specimens collected for direct testing from the stool sample by both assays and then placed an aliquot of the stool in MacConkey broth within 48 hours of specimen collection. Broth cultures were incubated for 20 – 30 hours and then tested by both immunoassays. A SMAC culture (Sorbitol MacConkey plates) was also plated within 48 hours of the specimen collection for the determination of E. coli O157. All positive results from either immunoassay method were confirmed by cytotoxicity testing, CTA.

A total of 272 prospective specimens from diarrheal patients were tested in the OIA SHIGATOX and the EIA method.

Comparison of OIA SHIGATOX to EIA for Direct Stool Samples

EIA
OIA SHIGATOX12
255
ve Agreement: 100% (95%Cl: 73.5 -100%)

Positiv Negative Agreement: 98.1% (95%CI: 95.6 - 99.4%) Overall Percent Agreement: 98.2% (95% Cl: 95.8 - 99.4%)

Of the five OIA+/EIA - specimens, one was positive by CTA. One of the samples that was negative by direct stool testing in both the EIA and the OIA methods was positive in the OIA broth culture sample and by CTA from the direct stool.

Two of the clinical sites also performed a study in which sixty-two additional frozen specimens were prospectively tested by OIA SHIGATOX and EIA without the operator's knowledge of the original Shiga toxin result.

Comparison of OIA SHIGATOX to EIA for Frozen Direct Stools

EIA
+-
OIA SHIGATOX +211
-337

Positive Agreement: 87.5% (95% CI: 67.6 – 97.3%)

Negative Agreement: 97.4% (95% CI: 86.2 – 99.9%)

Overall Percent Agreement: 93.6% (95% CI: 84.3 – 98.2%)

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3. Broth Culture

A total of 269 prospective specimens from diarrheal patients were tested by OIA SHIGATOX and the EIA method from the broth culture. Three fecal specimens failed to produce any growth upon broth culture.

Comparison of OIA SHIGATOX to EIA for Broth Enriched Culture from Fresh Stools

EIA
+-
OIA SHIGATOX121
0256
Positive Agreement: 100% (95% CI: 73.5 -100%)Negative Agreement: 99.6% (95% CI: 97.9 - 100%)

Posit Negative Agreement: 99.6% (95% Cl: 97.9 - 100%), Overall Percent Agreement: 99.6% (95% CI: 98.0 - 100%)

The single OIA SHIGATOX +/EIA – result was confirmed to be a true positive by the CTA analysis of the direct stool.

In the prospective frozen sample study, ten of the frozen specimens were not tested in overnight MacConkey broth culture. Two of the remaining specimens failed to exhibit growth after overnight MacConkey broth culture. The percent positive agreement was 100% and the percent negative agreement was 96.4%. The overall percent agreement in the study was 98%.

Comparison of OIA SHIGATOX to EIA for Broth Enriched Culture from Frozen Stools

Image /page/17/Figure/9 description: The image shows a 2x2 table comparing two tests, EIA and OIA SHIGATOX. The table shows the number of concordant and discordant results between the two tests. The table shows that there were 22 positive results for both tests, 1 discordant result, 0 discordant results, and 27 negative results for both tests. The positive agreement is 100% (95% CI: 84.6-100%) and the negative agreement is 95.6% (95% CI: 81.7-99.9%).

Overall Percent Agreement: 98% (95% Cl: 89.4 - 100%)

SMAC Culture 4.

Two hundred and sixty nine (269) of the direct stool samples were analyzed by SMAC culture. The OIA SHIGATOX and EIA assays were compared to the SMAC culture results. Interpretation of the comparison between the OIA SHIGATOX or the EIA test and SMAC is confounded by the fact that, as a metabolic test, SMAC is specific for E. coli O157:H7, while OIA SHIGATOX reacts with all Shiga toxin-producing E. coli (STEC). Also, SMAC requires the presence of live cells in the sample, while the OIA SHIGATOX test does not

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have that limitation. Based on these differences, it was anticipated that a number of samples could be SMAC-negative and OIA SHIGATOX positive.

OIA SHIGATOX Direct Fresh Stool Results compared to SMAC Culture of Direct Stools

SMAC

+-
OIA SHIGATOX +98
-1251
Positive Agreement: 90% (95% CI: 55.5 – 99.8%)
Negative Agreement: 96.9% (95% CI: 94.0 – 98.7%)
Overall Percent Agreement: 96.7% (95% CI: 93.7 – 98.5%)

EIA Direct Fresh Stool Results compared to SMAC Culture of Direct Stools

SMAC
+-
EIA+84
-2255
Positive Agreement: 80% (95% CI: 44.4 - 97.5%)
Negative Agreement: 98.5% (95% CI: 96.1 - 99.6%)
Overall Percent Agreement: 97.8% (95% CI: 95.2 - 99.2%)

The one apparent OIA false negative result compared to the SMAC result was not confirmed by CTA and was not positive by EIA. Of the 8 apparent false positives by the OlA method, 4 of the samples were positive by CTA, The second EIA false negative result was positive by CTA and the OIA method. One of the OIA +/SMAC + samples was negative by CTA. The 2 EIA -/SMAC + samples were negative by CTA and one of the samples was negative by the OIA method as well. Three of the EIA +/SMAC - samples were positive by CTA and the OlA method. The remaining EIA +/SMAC sample was negative by CTA but positive by the OIA method.

In addition, two of the clinical sites conducted a prospective comparison of the OIA method to SMAC culture using frozen samples. Thawed aliquots of all samples were tested in the OIA and SMAC methods for this comparison.

Frozen Stool samples comparing OIA SHIGATOX to SMAC

Image /page/18/Figure/10 description: The image shows a table comparing OIA SHIGATOX and SMAC. The table has four values: 9, 13, 0, and 40. The positive agreement between the two is 100% with a 95% confidence interval of 66.4-100%.

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Negative Agreement: 75.5% (95%Cl: 61.7 - 86.2%) Overall Percent Agreement: 79% (95%C1: 66.8 - 88.3%)

All thirteen of the OIA+/SMAC- samples were positive for STEC in previous testing.

    1. CTA
      In the clinical study there were 19 specimens positive by OIA. EIA, or both methods. An aliquot of the stool specimen for each of these 19 specimens was submitted for CTA alonq with an aliguot of the broth culture media. One sample produced an inconclusive result and was excluded from this analysis. Thirteen of the samples were positive by CTA. The OIA SHIGATOX detected 12 of these 13 samples while the EIA method detected 11. Twelve of the broth aliquots were positive by CTA. The OIA SHIGATOX assay detected all 12 of these samples as did the ElA method. Of the 13 CTA positives, SMAC was positive for only 8 samples.

Comparison of OIA SHIGATOX, EIA, and SMAC to CTA for Direct Stool and Broth Culture Samples

CTA DirectStoolCTA BrothCulture
OIA SHIGATOX12/1312/12
EIA11/1312/12
SMAC8/13N/A

Direct Stool:

The following additions were made as a change to the previously cleared device.

Ninety-eight frozen fecal specimens were collected from 2 laboratory sites. Fecal specimens were thawed and tested directly in the BioStar OIA SHIGATOX assay and used to inoculate Cary Blair transport media. Cary Blair diluted fecal samples were tested within one and 24 hours of inoculation in the BioStar OIA SHIGATOX test. For the Cary Blair analysis, the direct fecal testing in the OIA method was used as the reference method.

Broth Culture:

Ninety eight fecal specimens were thawed and inoculated into MacConkey (MAC) broth and Gram Negative (GN) broth. The broth tubes were incubated for 24 hours at 37°C. An aliquot from each sample was tested in the OIA

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SHIGATOX assay. In addition, the Cary Blair samples that were held for 24 hours at room temperature were used to inoculate both GN and MAC broths. These samples were incubated for 24 hours at 37°C, and then an aliquot was tested in the OIA SHIGATOX method. The reference method used in this evaluation was the direct fecal inoculation of the MAC broth.

Conclusions:

Based on the results obtained during these studies, the instructions for use of the OIA SHIGATOX assay have been amended to reflect the following changes in acceptable sample type:

  • Cary Blair transport media inoculated with a fecal specimen for � direct testing
  • GN enrichment broth inoculated with direct fecal specimens �
  • GN enrichment broth inoculated with Cary Blair samples �
  • MAC enrichment broth inoculated with Cary Blair samples �

Summary of Performance Data After Device Modification:

SiteGeographicAreaInstitutionTypeInstitutionSizeSiteTesters
Universityof Utah andPrimaryChildrens(UT)Western USChildren'sHospital280 bedsMT ASCP
FairfaxHospital(VA)EasternSouthern USTertiary CareCommunityHospital850 bedsMT ASCP

Fecal Specimen Collection Laboratories:

Analytical Site:

Inverness Medical-BioStar Inc. 331 South 104th Street Louisville, CO 80027

    1. Colony Sweep
      No change.

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2. Direct Stool

The following additions were made as a change to the previously cleared device. All of the above data remains the same.

Ninety eight frozen fecal specimens were collected from 2 laboratory sites. Fecal specimens were thawed and tested directly in the BioStar OIA SHIGATOX assay and used to inoculate Cary Blair transport media. Carv Blair diluted fecal samples were tested at 2 time points, within one hour and then 24 hours of inoculation, with the BioStar OIA SHIGATOX test. In the direct fecal testing with the OIA SHIGATOX test, 32 specimens were determined to be positive and 66 specimens were negative. For the Cary Blair analysis, the direct fecal testing in the OIA method was used as the reference method.

Frozen Stool specimens comparing 1 Hour Cary Blair-diluted fecal specimens to direct OIA test results

OIA SHIGATOX

1 Hour Cary Blair-Diluted Sample in the OIA SHIGATOX method

+280
-466

Positive Agreement: 88% (95%Cl: 71 - 96.5%) Negative Agreement: 100% (95%CI: 94.6 - 100%) Overall Percent Agreement: 96% (95%C1: 89.9 - 98.9 %)

Frozen Stool specimens comparing 24 Hour Cary Blair-diluted fecal specimens to direct OIA test results

OIA SHIGATOX

24 Hour Cary Blair-Diluted Sample in the OIA SHIGATOX method

+-
290
366

Positive Agreement: 91% (95%CI: 75 - 98%) Negative Agreement: 100% (95%CI: 94.6 - 100%) Overall Percent Agreement: 97% (95%Cl: 91.3 – 99.4 %)

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3. Broth Culture

Ninety eight fecal specimens were thawed and inoculated into MacConkey (MAC) broth and Gram Negative (GN) broth. The broth tubes were incubated for 24 hours at 37°C. An aliquot from each sample was tested in the OIA SHIGATOX assay. In addition, the Cary Blair samples that were held for 24 hours at room temperature were used to inoculate both GN and MAC broths. These samples were incubated for 24 hours at 37°C and aliquot tested in the OIA SHIGATOX method. The reference method used in this evaluation was inoculation of the direct fecal specimen into MAC broth. Variation in results was due to the failure of a fecal specimen to grow in one or more of the broths.

Frozen Stool specimens comparing direct fecal specimens cultured in GN to MAC broth performance in the OIA test

MAC OIASHIGATOX
+
GN OIA26
SHIGATOXO42
Positive Agreement: 100% (95%Cl: 86.8 - 100%)Negative Agreement: 95% (95%Cl: 84.5 - 99.4%)Overall Percent Agreement: 97.1% (95%CI: 90.1 - 99.7%)

Frozen Stool specimens comparing 24 hour Cary Blair samples cultured in GN broth to Direct MAC broth performance in the OIA test

Direct MAC OIASHIGATOX
+-
Cary Blairsample in GNbroth OIASHIGATOX+261
-041
Positive Agreement: 100% (95%CI: 86.8 - 100%)
Negative Agreement: 98% (95%CI: 87.4 - 99.9%)

Overall Percent Agreement: 98.5% (95%CI: 92.1 - 100%)

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Frozen Stool specimens comparing 24 hour Cary Blair samples cultured in MAC broth to Direct MAC broth performance in the OIA test

Direct MAC OIASHIGATOX
+-
Cary Blairsample in MACbroth OIASHIGATOX263
240
Positive Agreement: 93% (95%CI: 76.5 – 99.1%)Negative Agreement: 93% (95%CI: 80.9 – 98.5%)Overall Percent Agreement: 93% (95%CI: 84.3 – 97.7%)

H. Conclusions from the nonclinical / clinical testing [§807.92(b)(3)]:

The results of the above described internal and external studies demonstrate that the BioStar OIA SHIGATOX is as safe and effective as the cleared predicate device.

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Image /page/24/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a stylized caduceus symbol, which is a staff with two snakes coiled around it, and the text "DEPARTMENT OF HEALTH & HUMAN SERVICES USA" arranged in a circular fashion around the symbol. The text is in all caps and is evenly spaced around the circle.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Robin C. Hart, Ph.D. Director, Regulatory Affairs Inverness Medical Professional Diagnostics 331 South 104th Street Louisville, Colorado 80027

QCT 2 2 2007

Re: K071799

Trade/Device Name: BioStar® OIA® SHIGATOX Regulation Number: 21 CFR 866.3255 Regulation Name: Escherichia coli Serological Reagents Regulatory Class: Class I Product Code: GNA, GMZ Dated: August 21, 2007 Received: August 23, 2007

Dear Dr. Hart:

We have reviewed vour 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. 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.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. 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 requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820).

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Page 2 -

This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at 240-276-0450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). For questions regarding postmarket surveillance, please contact CDRH's Office of Surveillance and Biometric's (OSB's) Division of Postmarket Surveillance at 240-276-3474. For questions regarding the reporting of device adverse events (Medical Device Reporting (MDR)), please contact the Division of Surveillance Systems at 240-276-3464. You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (240) 276-3150 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours.

Sally attaym

Sally A. Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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INDICATIONS FOR USE

510(k) Number (if known): K071799

BioStar® OIA® SHIGATOX Device Name:

Indications For Use:

The BioStar® OIA® SHIGATOX assay is an Optical Immunoassay (OIA) test for the qualitative, rapid detection of the presence of Shiga Toxins in human diarrheal fecal specimens, broth cultures, fecal specimens in Cary Blair Transport Media, or swab sampling of colonies from a culture plate. This test is intended for in vitro diagnostic use as an aid in the diagnosis of infection by Shiga Toxin-producing Escherichia coli (STEC) both 0157 and all non ~ O157 Shiga toxin producing strains.

Prescription Use X OR (Part 21 CFR 801 Subpart D) Subpart C)

Over-The-Counter Use (Part

CFR 801 21

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Concurence of CDRH, Office of Device Evaluation (ODE)

Freddie L. Poole

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Office of In Vitro Diagnostic " Evaluation and Safety

10(k) K071799

§ 866.3255

Escherichia coli serological reagents.(a)
Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identifyEscherichia coli from cultured isolates derived from clinical specimens. Additionally, some of these reagents consist ofEscherichia coli antisera conjugated with a fluorescent dye used to identifyEscherichia coli directly from clinical specimens or cultured isolates derived from clinical specimens. The identification aids in the diagnosis of diseases caused by this bacterium belonging to the genusEscherichia, and provides epidemiological information on diseases caused by this microorganism. AlthoughEscherichia coli constitutes the greater part of the microorganisms found in the intestinal tract in humans and is usually nonpathogenic, those strains which are pathogenic may cause urinary tract infections or epidemic diarrheal disease, especially in children.(b)
Classification. Class I (general controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 866.9.