The Hp Detect™ Stool Antigen ELISA is an in vitro diagnostic qualitative enzyme immunoassay for the detection of Helicobacter pylori (H. pylori) antigens in human stool or feces. The Hp Detect™ Stool Antigen ELISA is intended to aid in the initial diagnosis and post-therapy diagnosis of H. pylori infection. Additionally, the test may be used to assess H. pylori infection status after treatment. Retesting at a minimum of 4 weeks after the completion of treatment may be done to assess H. pylori status. Test results should always be taken into consideration by the physician in conjunction with patient's clinical information (history and symptoms). For Prescription Use Only.

Device Description

The Hp Detect Stool Antigen ELISA is an enzyme immunoassay which detects the H. pylori antigen in human fecal samples. The Hp Detect Stool Antigen ELISA comes in a kit that contains materials to assay a total of 92 samples. The device consists of a 96-well clear flat bottom polystyrene high bind microplate coated with affinity purified rabbit anti-human H. pvlori polyclonal antibody. The device is provided with detection antibody which is a purified mouse monoclonal antibody specific for H. pylori antigen and has been conjugated to horseradish peroxidase (HRP). The device kit is also provided with sample diluent buffer, wash buffer, substrate solution, stop solution along with negative and positive controls. Negative control is a phosphate buffered protein solution and positive control is composed of purified H. pylori antigen (ATCC strain 43504) from cell lysate. Polyclonal anti-H. pylori captures antibodies that are immobilized on microwells. Patient samples prepared in sample diluent are added to the microwells and incubated for one hour at 37 ± 2℃. If the H. pvlori antigen is present in the sample, it will bind to the immobilized antibody on the plate. Following this incubation, the plate is washed thoroughly. A peroxidase conjugated anti-H. pylori monoclonal antibody is then added to the microwells and incubated for 30 minutes at 37 ± 2℃. If H. pylori antigen is bound to the microwells in the first step, the detection antibody would now bind in this step to form a sandwich complex. Following this incubation, a thorough wash step is performed to remove non-specific and non-binding materials. Substrate is then added and incubated for 10 minutes at 37±2℃ to generate a color in the presence of the enzyme complex. Stop solution is then added to end the reaction. The results are read spectrophotometrically at the following wavelengths: 1. Single Wavelength Measurement at 450 nm 2. Dual Wavelength Measurement 450/620 nm or 450/630 nm

AI/ML Overview

The provided document is an FDA 510(k) Pre-Market Notification for the Biomerica, Inc. Hp Detect Stool Antigen ELISA for the detection of Helicobacter pylori (H. pylori) antigens in human stool or feces. It is a qualitative immunoassay intended to aid in the initial diagnosis and post-therapy diagnosis of H. pylori infection.

Based on the provided text, the device itself has acceptance criteria for its analytical performance (e.g., reproducibility, LoD, specificity, inclusivity) and clinical performance (Positive Percent Agreement - PPA, Negative Percent Agreement - NPA). The study detailed in the document serves to prove that the device meets these internal acceptance criteria set by the manufacturer for FDA clearance.

Here's a breakdown of the requested information based on the document:

1. A table of acceptance criteria and the reported device performance

The document does not explicitly state pre-defined acceptance criteria values in a formal table for PPA and NPA. However, it presents the achieved performance and concludes that the results are "acceptable." We can infer the implicit acceptance criteria from these reported values. For analytical performance, criteria are implied by the "acceptable" statement for reproducibility, precision, LoD, cross-reactivity, interference, and inclusivity.

Performance Metric	Acceptance Criteria (Inferred from "Acceptable")	Reported Device Performance (Dual Wavelength)	Reported Device Performance (Single Wavelength)
Analytical Performance
Reproducibility (Detection Rate)	High Negative: Low % detection	High Negative (0.42xLoD): 3.9% (7/180)	Equivalent to Dual Wavelength
	Low Positive (1.60xLoD): 95%+ detection	Low Positive (1.60xLoD): 100% (180/180)	Equivalent to Dual Wavelength
	Low Positive (2.43xLoD): 95%+ detection	Low Positive (2.43xLoD): 100% (180/180)	Equivalent to Dual Wavelength
	Moderate Positive: 95%+ detection	Moderate Positive (3.93xLoD): 100% (180/180)	Equivalent to Dual Wavelength
Within-Lab Precision (Detection Rate)	High Negative: Low % detection	High Negative (0.42xLoD): 5% (13/288)	Equivalent to Dual Wavelength
	Low Positive (1.60xLoD): 95%+ detection	Low Positive (1.60xLoD): 97% (278/288)	Equivalent to Dual Wavelength
	Low Positive (2.43xLoD): 95%+ detection	Low Positive (2.43xLoD): 100% (288/288)	Equivalent to Dual Wavelength
	Moderate Positive: 95%+ detection	Moderate Positive (3.93xLoD): 100% (288/288)	Equivalent to Dual Wavelength
Limit of Detection (LoD)	Quantified LoD	Strain ATCC 43504: 2.53 ng/mL (0.38 ng/test) or 1.69 x 10^3 CFU/mLStrain ATCC 49503: 5.86 ng/mL (0.88 ng/test)	Equivalent to Dual Wavelength
Cross-Reactivity & Microbial Interference	No interference expected	No cross-reactivity or microbial interference observed with listed microorganisms	Equivalent to Dual Wavelength
Interfering Substances	No interference expected	No interference observed with listed substances	Equivalent to Dual Wavelength
Inclusivity (Detection Rate)	100% detection of tested strains	100% detection for all 6 H. pylori strains (whole cells) and 1 purified H. pylori antigen tested	Equivalent to Dual Wavelength
Prozone / Hook Effect	No hook effect up to high antigen concentration	No high-dose hook effect observed up to 20,000 ng/mL	Equivalent to Dual Wavelength
Clinical Performance
Frozen Specimen PPA	High PPA	99.11% (111/112)	99.11% (111/112)
Frozen Specimen NPA	High NPA	98.13% (315/321)	95.95% (308/321)
Fresh Stool Specimen PPA	High PPA	100.00% (20/20)	100.00% (20/20)
Fresh Stool Specimen NPA	High NPA	98.36% (120/122)	98.36% (120/122)
Post-Therapy Sensitivity	High Sensitivity	100% (10/10)	Not explicitly stated whether single wavelength was assessed for post-therapy; likely same as dual for qualitative results.
Post-Therapy Specificity	High Specificity	100% (4/4)	Not explicitly stated whether single wavelength was assessed for post-therapy; likely same as dual for qualitative results.

2. Sample sizes used for the test set and the data provenance (e.g., country of origin of the data, retrospective or prospective)

Clinical Study - Frozen Specimen:
- Sample Size: 433 frozen and de-identified fecal samples.
- Provenance:
  - 355 specimens from Italy.
  - 78 specimens from three geographically different regions of the USA (west, southwest, and southeast).
- Nature: Retrospective (frozen, de-identified samples). Patients were presenting with dyspepsia, undergoing endoscopy/biopsy, not on certain medications, and no H. pylori treatment within 6 months.
Clinical Study - Fresh Stool Specimen:
- Sample Size: 142 fresh, de-identified fecal specimens.
- Provenance: Collected through multiple biospecimen vendors and clinical laboratories. Locations not specified beyond "collection centers" and "Biomerica (internal site)."
- Nature: Likely prospective (freshly collected and then immediately tested/shipped). Patients had symptoms of H. pylori infection.
Post-Therapy Diagnosis:
- Sample Size: 14 paired (pre- and post-therapy) frozen retrospective specimens.
- Provenance: Italy.
- Nature: Retrospective (frozen, paired samples). All subjects initially positive by CRM and completed eradication therapy. Post-therapy samples collected minimum 4 weeks after treatment.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g., radiologist with 10 years of experience)

The document states that the ground truth for the clinical studies (frozen and fresh specimens) was established by comparison with an "FDA cleared device" (predicate device). For the frozen specimen study, discrepant results were further analyzed by "chart review and determined to have a RUT or history result."

For the post-therapy study, the ground truth was a "composite reference method (CRM) consisting of histology and Rapid Urease Test."

The document does not specify the number of experts or their qualifications (e.g., pathologists, gastroenterologists) involved in establishing the ground truth via histology, RUT, or chart review. It implies laboratory testing capabilities for the predicate device.

4. Adjudication method (e.g., 2+1, 3+1, none) for the test set

The document describes adjudication for discrepant results in the frozen specimen clinical study:

Discrepant results were "further analyzed by chart review and determined to have a RUT or history result."
This suggests a form of adjudication after the initial comparison, using additional clinical information or laboratory results (RUT or histology) as a reference. This is not a multi-reader adjudication method but rather a method for determining the "true" status of discrepant samples.

There is no mention of multi-reader adjudication for the Hp Detect Stool Antigen ELISA results themselves. The device's results are read spectrophotometrically based on defined cut-offs.

5. 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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not conducted. This device is an in-vitro diagnostic (ELISA), not an AI-powered diagnostic imaging tool that would typically involve human readers and MRMC studies. The device provides a quantitative or qualitative output (positive/negative) that is read spectrophotometrically, not interpreted by human readers in the same way as medical images.

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

This refers to the performance of the device itself (the "algorithm" in this context being the ELISA assay) without human interpretation variables beyond the initial lab procedure. Yes, the performance characteristics (analytical and clinical studies) directly report the standalone performance of the Hp Detect Stool Antigen ELISA device against comparator methods or reference standards. The results (PPA, NPA, sensitivity, specificity) represent the device's accuracy in detecting H. pylori antigens.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc)

The ground truth for the test sets was primarily established through:

Comparison to an FDA cleared predicate device for the frozen and fresh specimen clinical studies.
Composite Reference Method (CRM) consisting of histology and Rapid Urease Test (RUT) for the initial H. pylori status and post-therapy evaluation.
Chart review combined with RUT or histology results for resolving discrepant cases in the frozen specimen study.

This is a form of "laboratory reference standard" and "clinical/pathology confirmation."

8. The sample size for the training set

The document describes the analytical and clinical performance studies, which are validation studies or test sets. It does not provide information on the sample size used for the training set of the Hp Detect Stool Antigen ELISA device. As an ELISA assay, it is a biochemical test, not a machine learning model that typically undergoes data-driven training. The "training" for such a device involves assay development, optimization, and establishment of reagents and procedures, rather than a machine learning training set of data.

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

As the Hp Detect Stool Antigen ELISA is a biochemical immunoassay (not an AI/ML device), the concept of a "training set" and establishing ground truth for it in the machine learning sense is not applicable. The "ground truth" for developing and optimizing such a device would implicitly rely on well-characterized H. pylori positive and negative samples, purified antigens, and potentially clinical samples with known infection status (established through methods like culture, PCR, histology, RUT) to guide the reagent selection, assay design, and cut-off determination. The document details the methodology for setting assay cut-off based on a panel of 227 specimens (83 positive, 144 negative by predicate device), which could be considered part of the "development" or "optimization" phase for establishing the device's operational parameters, but not a "training set" in the context of deep learning.

Summary

{0}------------------------------------------------

Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

December 15, 2023

Biomerica, Inc. Bonnie Qiu Senior Director, Quality Assurance and Regulatory Affairs 17571 Von Karman Avenue Irvine, California 92614

Re: K232892

Trade/Device Name: Hp Detect Stool Antigen ELISA Regulation Number: 21 CFR 866.3110 Regulation Name: Campylobacter Fetus Serological Reagents Regulatory Class: Class I. reserved Product Code: LYR Dated: September 15, 2023 Received: September 18, 2023

Dear Bonnie Oiu:

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 (the 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 available 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.

Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

{1}------------------------------------------------

Your device is also subject to, among other requirements, the Quality System (OS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30, Design controls; 21 CFR 820.90, Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

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 Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 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,

Natasha Griffin -S

0.B.O. Ribhi Shawar, Ph.D. (ABMM) Branch Chief General Bacteriology and Antimicrobial Susceptibility Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

{2}------------------------------------------------

Indications for Use

510(k) Number (if known) K232892

Device Name Hp Detect™ Stool Antigen ELISA

Indications for Use (Describe)

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)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff(@fda.hhs.gov

"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

{3}------------------------------------------------

510(k) Summary

A. SUBMITTER/510(K) HOLDER

Biomerica, Inc 17571 Von Karman Ave Irvine, CA 92614

B. DEVICE NAME

Proprietary Name:	Hp Detect Stool Antigen ELISA
Common/Usual Name:	Helicobacter pylori stool antigen ELISA
Classification Name:	Helicobacter pylori

REGULATORY INFORMATION ﺰ

Regulation Section:	21 CFR 866.3110 Campylobacter fetus serological reagents
Classification:	Class I
Product Code:	LYR
Panel:	83-Microbiology

D. PREDICATE DEVICE PREMIER Platinum HpSA PLUS (K182559)

E. DEVICE DESCRIPTION

Polyclonal anti-H. pylori captures antibodies that are immobilized on microwells. Patient samples prepared in sample diluent are added to the microwells and incubated for one hour at 37 ± 2℃. If the H. pvlori antigen is present in the sample, it will bind to the immobilized antibody on the plate. Following this incubation, the plate is washed thoroughly. A peroxidase conjugated anti-H. pylori monoclonal antibody is then added to the microwells and incubated for 30 minutes at 37 ± 2℃. If H. pylori antigen is bound to the microwells in the first step, the detection antibody would now bind in this step to form a sandwich complex. Following this incubation, a thorough wash step is performed to remove non-specific and non-binding materials. Substrate is then added and incubated for 10 minutes at 37±2℃ to generate a color in the presence of the enzyme complex. Stop solution is then added to end the reaction. The results are read spectrophotometrically at the following wavelengths:

{4}------------------------------------------------

1. Single Wavelength Measurement at 450 nm
1. Dual Wavelength Measurement 450/620 nm or 450/630 nm

F. INDICATION FOR USE/INTENDED USE

The Hp Detect Stool Antigen ELISA is an in vitro diagnostic qualitative enzyme immunoassay for the detection of Helicobacter pylori (H. pylori) antigens in human stool or feces. The Hp Detect Stool Antigen ELISA is intended to aid in the initial diagnosis and posttherapy diagnosis of H. pylori infection. Additionally, the test may be used to assess H. pylori infection status after treatment. Retesting at a minimum of 4 weeks after the completion of treatment may be done to assess H. pylori status. Test results should always be taken into consideration by the physician in conjunction with patient's clinical information (history and symptoms). For Prescription Use Only.

ﻥ SUMMARY OF TECHNOLOGICAL CHARACTERISTICS COMPARED TO THE PREDICATE DEVICE

A summary comparison of technological characteristics, including design and materials is provided in the following Table 1.

{5}------------------------------------------------

Item	Device: K232892	Predicate : K182559
DeviceTrade Name	Hp Detect Stool Antigen ELISA	PREMIER Platinum HpSA PLUS
General Device Characteristic Similarities
ProductCode	LYR	Same
IntendedUse	The Hp Detect Stool AntigenELISA is an in vitro diagnosticqualitative enzyme immunoassayfor the detection of Helicobacterpylori ( H. pylori ) antigens inhuman stool or feces. The HpDetect Stool Antigen ELISA isintended to aid in the initialdiagnosis and post therapydiagnosis of H. pylori infection.Additionally, the test may be usedto assess H. pylori infection statusafter treatment. Retesting at aminimum of 4 weeks after thecompletion of treatment may bedone to assess H. pylori status. Testresults should always be taken intoconsideration by the physician inconjunction with patient's clinicalinformation (history andsymptoms). For Prescription UseOnly.	The PREMIER Platinum HpSAPLUS enzyme immunoassay(EIA) is an in vitro qualitativeassay for the detection ofHelicobacter pylori antigens inhuman stool. Test results areintended to aid in the diagnosisof H. pylori infection and tomonitor response during andpost-therapy in patients.Accepted medical practicerecommends that testing by anycurrent method, to confirmeradication, be done at least fourweeks following completion oftherapy.
MeasuredAnalyte	Detection of H. pylori antigen	Same
Type of Test	Qualitative	Same
SpecimenType	Human fecal specimens	Same
Controls	Positive and negative controlincluded in the kit	Positive and negative controlincluded in the kit
TargetPopulation	Persons suspected of having H.pylori infection	Same
Storage	Refrigerated (2°C – 8°C)	Same
Item	Device: K232892	Predicate : K182559
General Device Characteristic Differences
SpecimenStorage	Specimens may be held up to 120hours at 2°C – 8°C or should befrozen at ≤ -12°C or ≤ -60°C	Specimens may be held up to 72hours at 2°C – 8°C or at-20°C to -25°C prior to testing
Time toResult	Approximately 1 hour and 40minutes	Approximately 1 hour and 10minutes
AntibodyFormat	Polyclonal/Monoclonal	Monoclonal mixture/Monoclonalmixture
IncubationTemp	37°C ± 2°C	19-27°C
ReadingMethod	Spectrophotometric	Visual, Spectrophotometric

Table 1. Comparison with Predicate Device

{6}------------------------------------------------

H. PERFORMANCE CHARACTERISTICS

1. Analytical Performance

a. Precision / Reproducibility

To demonstrate the reproducibility of the Hp Detect Stool Antigen ELISA, a four (4) panel of specimens were prepared at high negative (0.42 × LoD), low positive (1.60 × LoD), low positive (2.43 × LoD) and moderate positive (3.93 × LoD) by spiking H. pylori antigen (ATCC strain 43504) into a negative pooled fecal matrix. The reproducibility panel was blinded, and each run included positive and negative controls. Testing was performed at three sites, two independent laboratories and one in-house at Biomerica, Inc. The samples were tested in triplicate twice per day over a 5-day period by two technicians at each site using one kit lot (2 Technicians × 3 sites × 3 replicates × 2 runs × 5 days = 180 results/panel member). The results for the reproducibility study were measured using the dual wavelength (450/620 or 450/630 nm) and are summarized in Table 2 below. Testing at the dual and single wavelength (450 nm) produced equivalent results.

Sample ID	H. pylori Antigen	Combined (3 sites)
		Positive/Tested	% Detection
High Negative	$0.42 \times \text{LoD}$	7/180	3.9%
Low Positive	$1.60 \times \text{LoD}$	180/180	100%
Low Positive	$2.43 \times \text{LoD}$	180/180	100%
Moderate Positive	$3.93 \times \text{LoD}$	180/180	100%

Table 2. Reproducibility of the Hp Detect Stool Antigen ELISA measured in dual wavelength

{7}------------------------------------------------

The Within-Lab precision/repeatability of the Hp Detect Stool Antigen ELISA was determined onsite at Biomerica using a four (4) panel of specimens as prepared for the reproducibility study as described above. Two operators performed the test with two validation lots per run over the period of 12 non-consecutive days. Two runs per day with a minimum of 3 hours separation between runs. Three replicates of each sample per run were tested along with positive and negative controls. The results for the Within-Lab precision/repeatability study measured in dual wavelength (450/620 or 450/630 nm) are summarized in Table 3. Testing at the dual and single wavelength (450 nm) produced equivalent results.

Sample ID	H. pylori Antigen	Combined (Two kit lots)
		Positive/ Tested	% Detection
High Negative	$0.42 \times LoD$	13/288	5%
Low Positive	$1.60 \times LoD$	278/288	97%
Low Positive	$2.43 \times LoD$	288/288	100%
Moderate Positive	$3.93 \times LoD$	288/288	100%

Table 3. Within-Lab precision/repeatability of the Hp Detect Stool Antigen ELISA measured in dual wavelength.

The results for reproducibility and precision/repeatability studies were acceptable.

b. Linearity / Assay Reportable Range

Not Applicable

c. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods)

Traceability:

Calibrator traceability is not applicable (No calibrator is included in kit).

Sample Stability:

Sample Stability - Contrived Specimens

The sample stability for contrived specimens was evaluated for both refrigerated and frozen storage conditions including freeze and thaw. Samples were prepared by spiking H. pylori antigen (ATCC strain 43504) into a negative pooled fecal matrix. The sample panel was made up of 10 members of H. pylori antigen concentrations ranging from 0.47 to 5.42 × LoD tested in triplicates. After establishing the baseline measurement (time zero), each of the 10 panel members were aliquoted and stored at three storage conditions: 2℃ to 8℃. < 12℃. and <-60°C and tested for each time point and temperature range (shown in Table 4A). All the stability tests were measured in both dual (450/620 or 450/630 mm) and single wavelength (450 nm) with equivalent results. Results shown in Table 4B, 4C, and 4D are for 10 panel sample members grouped into 3 high negatives (<1 × LoD), 4 low positives (1-2 × LoD), and 3 moderate positives (3-5 × LoD) measured in dual wavelengths.

{8}------------------------------------------------

Table 4A. Stability Testing Temperatures and Time Points

Stability Condition	Temperature Range (°C)	Time Point (Days)
Refrigerated	2°C to 8°C	0, 3, 5
Frozen	≤-12°C	0, 8, 15
Deep Frozen	≤-60°C	0, 8, 15

Table 4B. Results for storage of specimens at refrigerated conditions (2 - 8°C)

Sample ID	Positive/Tested in refrigerated (2-8°C) storage period
	0 day	3 days	5 days
High Negative (<1 × LoD)	0/9	0/9	0/9
Low Positive (1-2 × LoD)	12/12	12/12	12/12
Moderate Positive (3-4 × LoD)	9/9	9/9	9/9

Table 4C. Results for storage of specimens at frozen conditions (≤ -12°C)

Sample ID	Positive/Tested in frozen (≤ -12°C) storage period
	0 day	8 days	15 days
High Negative (<1 × LoD)	0/9	0/9	0/9
Low Positive (1-2 × LoD)	12/12	12/12	12/12
Moderate Positive (3-4 × LoD)	9/9	9/9	9/9

Table 4D. Results for storage of specimens at deep frozen conditions (≤ -60°C)

Sample ID	Positive/Tested in frozen (≤ -60°C) storage period
	0 day	8 days	15 days
High Negative (<1 × LoD)	0/9	0/9	0/9
Low Positive (1-2 × LoD)	12/12	12/12	12/12
Moderate Positive (3-4 × LoD)	9/9	9/9	9/9

The results demonstrated no change in the performance interpretation from time zero throughout all the test points at each storage condition.

A freeze/thaw study was also conducted to evaluate the antigen stability after two (2) freezethaw cycles. The freeze/thaw study used the same 10 panels members as described in the specimen stability study. Following the baseline measurement (time zero) each sample was frozen at <-12°C and <-60°C and tested after one (1) and two (2) freeze/thaw cycle respectively. Test results demonstrate no change in the performance interpretation from time zero throughout each of the two freeze-thaw cycles. The recommended storage conditions for stool samples are summarized in Table 5.

{9}------------------------------------------------

Stability Condition	Temperature Range (°C)	Recommended Storage Time
Refrigerated	2°C to 8°C	5 Days
Frozen	≤-12°C	15 Days
Deep Frozen	≤-60°C	15 Days
Freeze-Thaw	≤-12°C and ≤-60°C	2 Freeze-Thaw Cycles

Table 5. Recommended Storage Conditions

d. Detection Limit (LoD)

The limit of detection (LoD) for the Hp Detect Stool Antigen ELISA was determined by using antigens from two strains of H. pylori, ATCC 43504 (Primary strain) and ATCC 49503 (Secondary strain). Detergent solubilized whole cell lysate of H. pylori was used to purify antigens. For each strain, three positive sample panels were prepared by spiking purified H. pylori antigen into a negative stool matrix. Each positive sample was then used to prepare its initial series of six dilutions. For Strain ATCC 43504 additional four dilutions were made (total 10 dilutions) and for Strain ATCC 43503 additional 5 dilutions were made (total 11 dilutions) to ensure that at least 3 dilutions are within 0.10 to 0.90 (10% to 90%) hit rate for Probit analysis. To determine LoD, each dilution was tested in seven replicates across three days producing 63 data points per dilution per strain (3 positive samples × 7 replicates × 3 days = 63 data points). Testing was conducted using two reagent lots and measuring by both dual (450/620 or 450/630 nm) and single wavelength (450 nm). A Probit analysis was performed to estimate LoD which is defined as the concentration of H. pylori antigen that yields a positive result 95% of the time. LoD values determined for the Hp Detect Stool Antigen ELISA test are shown in Table 6.

ATCC Strain	ng/mL (95%CI)	Quantity/Test
Strain 43504	2.53 (2.48 to 2.60)	0.38 ng
Strain 49503	5.86 (5.56 to 6.37)	0.88 ng

Table 6. LoD values for Hp Detect Stool Antigen ELISA

The Limit of Detection (LoD) for the Hp Detect Stool Antigen ELISA was established at 2.53 ng/mL (0.38 ng/test) for strain ATCC 43504 and 5.86 ng/mL (0.88 ng/test) for strain ATCC 49503, measured in dual wavelength.

The correlation between antigen amount (ng/mL) and bacterial count (CFU/mL) was established for strain ATCC 43504 by dermining the LoD in bacterial count (CFU/mL). The LoD of bacterial concentration determined for H. pylori Strain ATCC 43504 is 1.69 × 103 CFU/mL. Therefore, H. pylori strain ATCC 43504 has a LoD of 2.53 ng/mL of antigen concentration correlates to a LoD of 1.69 × 103 CFU/mL of bacteria concentration when compared side to side (data not shown).

{10}------------------------------------------------

The study to determine LoD for the Hp Detect Stool Antigen ELISA is acceptable and these values in CFU/mL and ng/mL were used to guide the analytical studies as appropriate.

e. Analytical Specificity

Cross-Reactivity and Microbial Interference

The Hp Detect Stool Antigen ELISA test was evaluated for cross reactivity and microbial interference with the bacteria, fungi and viruses listed below. A contrived positive matrix was prepared by spiking H. pylori purified antigen (ATCC strain 43504) at 2.2 × LoD (5.61 ng/mL) into the negative fecal matrix. Bacteria and fungi were spiked at concentrations of 10°-10′ CFU/mL and viruses at a range from 10-105 TCID50 units per mL into either the contrived positive sample (for microbial interference evaluation) or into the negative fecal matrix (for cross-reactivity evaluation). Positive and negative controls were included with each run. The sample panel was assayed in duplicates. The list of microorganisms and concentrations tested for cross-reactivity and microbial interference studies are shown in Table 7 below. No crossreactivity or microbial interference was observed with the listed microorganisms when tested at the given concentrations when spiked into the matrix. The study was conducted with both dual (450/620 or 450/630 nm) and single wavelength (450 nm). All three-wavelength measurements produced equivalent results.

{11}------------------------------------------------

Table 7. Microorganisms tested* for cross-reactivity and microbial interference.
Bacteria and Fungi
Aeromonas hydrophila	Listeria monocytogenes
Bacillus subtilis	Peptostreptococcus anaerobius
Campylobacter coli	Plesiomonas shigelloides
Campylobacter fetus	Proteus mirabilis
Campylobacter hyointestinalis	Pseudomonas aeruginosa
Campylobacter jejuni	Pseudomonas fluorescens
Campylobacter upsaliensis	Salmonella enterica (Group B)
Candida albicans	Salmonella enterica (Group C)
Citrobacter freundii	Salmonella enterica (Group D)
Clostridium difficile	Salmonella enterica (Group E)
Clostridium perfringens	Serratia liquefaciens
Clostridium sordellii	Shigella boydii
Enterobacter cloacae	Shigella flexneri
Enterococcus faecalis	Shigella sonnei
Escherichia coli	Staphylococcus aureus
Escherichia hermannii	Staphylococcus epidermidis
Eschericia fergusonii	Vibrio parahaemolyticus
Klebsiella pneumonia	Yersinia enterocolitica
Lactobacillum lactis
Viruses
Adenovirus Type 2	Coxsackievirus Type B5
Adenovirus Type 40	Echovirus Type 9
Adenovirus Type 41	Rotavirus
Coxsackievirus Type B3

Table 7. Microorganisms tested* for cross-reactivity and microbial interference.

*Bacteria were tested at 1 × 107 CFU/mL (except for Clostridium perfringens at 1 × 106 CFU/mL) and Candida albicans at 1 × 106 CFU/mL. Viruses were tested at 1 × 105 TCID50/mL except for Echovirus and Rotavirus which were tested at TCID50/mL of 1 × 104.

The results for cross-reactivity and microbial interference with the Hp Detect Stool Antigen ELISA were acceptable.

Interfering Substances

The Hp Detect Stool Antigen ELISA was evaluated for interference with the potential interfering substances. A contrived positive matrix was prepared by spiking H. pylori purified antigen (ATCC strain 43504) at 2.2 × LoD (5.61 ng/mL) into the negative fecal matrix. Potentially interfering substances were spiked into either the contrived positive sample or negative fecal matrix. Positive and negative controls were included in each run. List of

{12}------------------------------------------------

potential interfering substances and their concentrations tested for cross-reactivity and interference studies were shown in Table 8 below. No Interference was observed when the listed interfering substances were tested at the given concentrations. Assays were performed in both dual (450/620 or 450/630 nm) and single wavelength (450 nm). All three-wavelength measurements produced equivalent results.

Interfering Substances	Concentration tested
Barium Sulfate	5% m/v
Human Hemoglobin	3 mg/mL
Metronidazole	701 µmol/L
Mucin	13.4 mg/mL
Palmitic Acid (Fecal Fat)	15.8 mg/mL
Polyethylene Glycol 3350	14.3 g/L
Steric Acid (Fecal Fat)	15.8 mg/mL
Vancomycin Hydrochloride	69 µmol/L
Whole Blood	200 µL/mL
Over the Counter (OTC) Medications
Imodium AD (Loperamide)	5%v/v
Kaopectate (Bismuth Subsalicylate)	0.70 mg/mL
Mylanta (Aluminum Hydroxide & MagnesiumHydroxide or Calcium Carbonate)	1.68 mg/mL
Pepto Bismol (Bismuth Subsalicylate)	0.70 mg/mL
Prilosec (Omeprazole) OTC	5 µg/mL
Simethicone	25 mg/mL
Tagamet (Cimetidine)	5 µg/mL
TUMS (Calcium Carbonate)	50 µg/mL

Table 8. Interfering substance concentrations tested.

The results for interfering substances study with the Hp Detect Stool Antigen ELISA were acceptable.

Inclusivitv study

A total of six (6) H. pylori strains (whole cells) and one purified H. pylori antigen were evaluated for inclusivity study. Samples were prepared by diluting each strain (whole cells) and antigen in negative stool matrix at the level of 1-3 × LoD (using LoD of 1.69 × 103 CFU/mL whole cells for strain ATCC 43504 and 5.86 ng/ml antigen for strain ATCC 49503). Samples were tested for reactivity in triplicate with the Hp Detect Stool Antigen ELISA measuring by both dual (450/620 or 450/630 mm) and single wavelength (450 nm). Results for inclusivity are shown in Table 9. All strains were detected with Hp Detect Stool Antigen ELISA.

{13}------------------------------------------------

H. pylori Strain	Concentration	Positive/Tested(% Detection)
ATCC 43526	$3.70 \times 103$ CFU/mL	3/3 (100%)
ATCC 43579	$3.48 \times 103$ CFU/mL	3/3 (100%)
ATCC 49396	$3.42 \times 103$ CFU/mL	3/3 (100%)
ATCC 700392	$3.85 \times 103$ CFU/mL	3/3 (100%)
ATCC 700824	$3.96 \times 103$ CFU/mL	3/3 (100%)
ATCC BAA-945	$3.78 \times 103$ CFU/mL	3/3 (100%)
ATCC 49503	6.33 ng/mL Antigen	3/3 (100%)

Table 9. List of H. pylori strains tested for inclusivity study.

The Inclusivity results were acceptable.

f. Assay Cut-off

Assay cut-off for Hp Detect Stool Antigen ELISA was determined for both spectrophotometric measurement of Dual Wavelength (450/620 nm or 450/630 nm) and Single Wavelength (450 nm). A panel of 227 specimens, of which 83 were positive and 144 were negative by the predicate device were evaluated with the Hp Detect Stool Antigen ELISA. Receiver operating characteristic (ROC) curve was constructed and the cut-off values for spectrophotometric single and dual wavelength reading methods were selected to achieve optimum diagnostic sensitivity and specificity:

Spectrophotometric Dual Wavelength (450/620 nm or 450/630 nm) Negative: <0.100 Positive: ≥0.100 Spectrophotometric Single Wavelength (450 nm) Negative: <0.140 Positive: >0.140

A positive result indicates that H. pylori antigens were detected. A negative result indicates that no H. pylori antigens were detected, or that the antigen levels are below what can be detected by the assay.

g. Prozone / Hook Effect

To ensure that a high concentration of H. pylori antigen does not interfere with a positive reaction in the Hp Detect Stool Antigen ELISA, a high positive sample was prepared by spiking H. pylori antigen (ATCC strain 43504) into negative fecal matrix. The sample was serially diluted at concentrations ranging from 20,000 ng/mL to 0.25 ng/mL. Each of the serial dilutions were tested in 8 replicates measuring by both dual (450/620 or 450/630 nm) and single wavelength (450 nm) settings. Positive and negative controls were included in each run. The reading value of the plate reader reached to maximum level at 800 ng/ml of H. pylori antigen and remained at maximum level up to 20,000 ng/ml. No high-dose hook effect was observed for the assay at antigen concentrations of up to 20,000 ng/mL.

{14}------------------------------------------------

2. Clinical Study

Clinical Study - Frozen Specimen

The performance of the Hp Detect Stool Antigen ELISA for frozen specimens was evaluated by using method comparison study with an FDA cleared device. A total of 433 frozen and deidentified fecal samples were tested in which 355 specimens were collected from Italy and 78 specimens collected from three geographically different regions of the USA (west, southwest, and southeast). The clinical specimens were collected from the patients presenting with dyspepsia, and would be undergoing a routine endoscopy and biopsy, not on antibiotics, bismuth, or proton-pump inhibitors, and those excluded if they had been treated for H. pylori withing the past 6 months. Specimens were distributed into three testing sites located within the USA which include two external sites and one internal (Biomerica) site. Results were evaluated by reading absorbance at dual wavelength (450/620 or 450/630 nm) and single wavelength (450 nm). The performance and statistics are shown in Table 10 for dual wavelength and Table 11 for single wavelength.

Table 10. Frozen Specimen - Clinical performance and statistics measured in dual wavelength.

Hp Detect Stool Antigen ELISA	Comparator: FDA Cleared Device
	Positive	Negative	Total
Positive	111	6a	117
Negative	1b	315	316
Total	112	321	433

Statistics	Performance	95% Confidence Interval
Positive Percent Agreement (PPA)	99.11%	95.12 - 99.84 %
Negative Percent Agreement (NPA)	98.13 %	95.98 - 99.14 %

The discrepant results were further analyzed by chart review and determined to have a RUT or history result and observed as follow:

(a) Four of the six discrepant false positives were either comparator positive by RUT or histology.

(b) The discrepant false negative was negative by RUT or histology.

{15}------------------------------------------------

Hp Detect Stool Antigen ELISA	Comparator: FDA Cleared Device
	Positive	Negative	Total
Positive	111	13a	124
Negative	1b	308	309
Total	112	321	433
Statistics	Performance	95% Confidence Interval
Statistics	Performance	95% Confidence Interval
Positive Percent Agreement (PPA)	99.11%	95.12 – 99.84 %
Negative Percent Agreement (NPA)	95.95 %	93.20 – 97.62 %

Table 11. Clinical performance and statistics measured in single wavelength.

The discrepant results were further analyzed by chart review and determined to have a RUT or history result and observed as follow:

(a) Four of the 13 discrepant false positive were positive by RUT or histology.
(b) The discrepant false negative was negative by RUT or histology.

The performance results for frozen specimen clinical study are acceptable.

Clinical Studv - Fresh Stool Specimen

The performance of the Hp Detect Stool Antigen ELISA for fresh specimens (never frozen) was evaluated by using method comparison study with an FDA cleared device. A total of 142 fresh, de-identified, fecal specimens from patients with physician's medical evaluation for symptoms of H. pylori infection were collected through multiple biospecimen vendor and clinical laboratories. The specimens were tested in the collection centers with the comparator device and specimen aliquots were shipped to Biomerica (internal site) and tested using Hp Detect Stool Antigen ELISA using both dual wavelength (450/620 or 450/630 nm) and single wavelength (450 nm) settings.

Table 12. Fresh Specimens - Clinical performance and statistics measured in dual and single
wavelength with same results.
Hp Detect Stool Antigen ELISA	Comparator: FDA Cleared Device
	Positive	Negative	Total
Positive	20	2	22
Negative	0	120	120
Total	20	122	142

Statistics	Performance	95% Confidence Interval
Positive Percent Agreement (PPA)	100.00%	83.89 - 100%
Negative Percent Agreement (NPA)	98.36%	94.22 - 99.55%

The Hp Detect Stool Antigen ELISA demonstrated the clinical performance of 100% positive percent agreement and 98.4% negative percent agreement for fresh stool specimens using both dual and single wavelength configurations. The performance results for fresh specimen clinical study are acceptable.

{16}------------------------------------------------

Post -Therapy Diagnosis

The performance of the Hp Detect Stool Antigen ELISA for post-therapy diagnosis was evaluated by using 14 paired (pre-and post- therapy) frozen retrospective specimens from Italy. All subjects initially tested positive for H. vvlori by composite reference method (CRM) consisting of histology and Rapid Urease Test and all completed eradication therapy. Posttherapy fecal samples were collected at a minimum of 4 weeks post completion of eradication therapy. All 14 pre-therapy samples were previously shown to be positive and matched with the CRM diagnosis. The result for post-therapy was shown in Table 13.

Hp Detect Stool Antigen ELISA	Post-Therapy CRM
	Positive	Negative	Total
Positive	10	0	10
Negative	0	4	4
Total	10	4	14

Table 13. Post-Therapy Performance with composite reference method

Statistics	Performance	95% Confidence Interval
Sensitivity	100%	72.25 - 100 %
Specificity	100 %	51.02 - 100 %

The results for post-therapy performance show that the Hp Detect Stool Antigen ELISA exhibited a 100% sensitivity and 100% specificity when compared to the composite reference method. The performance results for post-therapy study are acceptable and support the claim for the test to be used in assessing H. pylori status after treatment.

Clinical Cut-Off

Not Applicable.

Expected Values – Reference Range

The Hp Detect Stool Antigen ELISA assay detects the presence of H. pylori antigen in human feces. The clinical study included in this submission had results from 433 frozen specimens collected from three independent sites in the US, each located in a different geographical region of the country - west, southwest, and southeast, and outside the US - Italy. The observed prevalence of H. pvlori in this study was 27.0% (117/433). H. pvlori detection and prevalence stratified by specimen origin are shown in Table 14 and categorized by gender is shown in Table 15.

{17}------------------------------------------------

Specimen Origin	Hp Detect Stool Antigen ELISA Result		Total	Prevalence(% Positive)
	Negative	Positive
US - Southeast	26	8	34	23.5%
US - Southwest	13	3	16	18.8%
US - West	19	9	28	32.1%
US (All Sites)	58	20	78	25.6%
Italy	258	97	355	27.3%
Grand Total	316	117	433	27.0%

Table 14: H. pylori detection and prevalence by specimen origin

Table 15: H. pylori detection and prevalence by gender

Gender	Hp Detect Stool Antigen ELISA Result		Total	Prevalence(% Positive)
	Negative	Positive
Females	205	76	281	27.0%
Males	111	41	152	26.9%
Total	316	117	433	27.0%

I. PROPOSED LABELING

The labeling supports the finding of substantial equivalence for this device.

J. CONCLUSIONS

The submitted information in this premarket notification is complete and supports substantial equivalence decision.

Regulation Number and Section

§ 866.3110

Campylobacter fetus serological reagents.(a)
Identification. Campylobacter fetus serological reagents are devices that consist of antisera conjugated with a fluorescent dye used to identifyCampylobacter fetus from clinical specimens or cultured isolates derived from clinical specimens. The identification aids in the diagnosis of diseases caused by this bacterium and provides epidemiological information on these diseases.Campylobacter fetus is a frequent cause of abortion in sheep and cattle and is sometimes responsible for endocarditis (inflammation of certain membranes of the heart) and enteritis (inflammation of the intestines) in humans.(b)
Classification. Class I (general controls).