Hp Detect™ Stool Antigen ELISA
Device Facts
| Record ID | K232892 |
|---|---|
| Device Name | Hp Detect™ Stool Antigen ELISA |
| Applicant | Biomerica, Inc. |
| Product Code | LYR · Microbiology |
| Decision Date | Dec 15, 2023 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3110 |
| Device Class | Class 1 |
| Attributes | Real-World Evidence |
Real-World Evidence
| Submission | Device | Sponsor | RWD Sources | RWE Use Summary | Key Tags |
|---|---|---|---|---|---|
| K232892 · Dec 15, 2023 | Hp Detect™ Stool Antigen ELISA | Biomerica, Inc. | Retrospective de-identified clinical fecal specimens; Patient medical records/chart review (for discrepant analysis); Routine clinical diagnostic results (Rapid Urease Test and Histology) | Retrospective clinical samples were used to establish clinical performance (sensitivity/specificity) against an FDA-cleared comparator and to validate post-therapy diagnostic utility. | Retrospective clinical study; Chart review; Post-therapy diagnosis; Clinical performance |
Clinical Evidence
| Study Design | Population | Comparator | Key Endpoints |
|---|---|---|---|
| Frozen Specimen Clinical Study; Retrospective method comparison | Patients presenting with dyspepsia undergoing routine endoscopy and biopsy; Sample Size: 433; Number of Sites: 4 (3 US sites, 1 Italy site) | FDA cleared device | Positive Percent Agreement (PPA), Negative Percent Agreement (NPA) |
| Post-Therapy Performance Study; Retrospective cohort; Follow-up/Duration: Minimum of 4 weeks post-therapy | Subjects initially positive for H. pylori who completed eradication therapy; Sample Size: 14; Number of Sites: 1 (Italy) | Composite reference method (histology and Rapid Urease Test) | Sensitivity, Specificity |
Indications for 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 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 Story
Hp Detect Stool Antigen ELISA is an enzyme immunoassay (EIA) for qualitative detection of H. pylori antigens in human fecal samples. Kit includes a 96-well polystyrene microplate coated with rabbit anti-H. pylori polyclonal antibody, HRP-conjugated mouse monoclonal detection antibody, buffers, and controls. Procedure: patient sample added to microwells; incubated at 37°C; washed; HRP-conjugated antibody added to form sandwich complex; washed; substrate added to generate color; reaction stopped. Results read spectrophotometrically at 450 nm (single) or 450/620-630 nm (dual). Used in clinical laboratories by trained personnel. Output aids physicians in diagnosing H. pylori infection and monitoring eradication post-treatment.
Clinical Evidence
Clinical performance was evaluated using 433 frozen and 142 fresh fecal specimens. For frozen specimens, the device showed 99.11% PPA and 95.95% NPA (dual wavelength) compared to an FDA-cleared device. For fresh specimens, PPA was 100% and NPA was 98.36%. Post-therapy performance was assessed in 14 paired samples compared to a composite reference method (histology/RUT), showing 100% sensitivity and 100% specificity. Analytical studies included reproducibility, precision, inclusivity, cross-reactivity, and interference testing, all yielding acceptable results.
Technological Characteristics
Enzyme immunoassay (EIA) using a 96-well clear flat bottom polystyrene high bind microplate. Capture: affinity-purified rabbit anti-H. pylori polyclonal antibody. Detection: HRP-conjugated mouse monoclonal anti-H. pylori antibody. Energy source: spectrophotometric plate reader (450 nm or 450/620-630 nm). Storage: 2°C–8°C. Manual/semi-automated workflow. No software algorithm; results determined by absorbance thresholding.
Indications for Use
Indicated for persons suspected of having H. pylori infection. Used for initial diagnosis, post-therapy diagnosis, and assessing infection status after treatment (retesting at least 4 weeks post-therapy).
Regulatory Classification
Identification
Campylobacter fetus serological reagents are devices that consist of antisera conjugated with a fluorescent dye used to identify Campylobacter 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.
Predicate Devices
- PREMIER Platinum HpSA PLUS (K182559)
Submission Summary (Full Text)
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FDA U.S. FOOD & DRUG ADMINISTRATION
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
ASSAY ONLY
## I Background Information:
A 510(k) Number
K232892
B Applicant
Biomerica, Inc.
C Proprietary and Established Names
Hp Detect Stool Antigen ELISA
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| LYR | Class I, reserved | 21 CFR 866.3110 - Campylobacter Fetus Serological Reagents | MI - Microbiology |
## II Submission/Device Overview:
A Purpose for Submission:
To obtain a substantial equivalence determination for the detection of *Helicobacter pylori* antigen in human stool.
B Measurand:
*Helicobacter pylori* antigen
C Type of Test:
Qualitative enzyme immunoassay
## III Intended Use/Indications for Use:
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
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A Intended Use(s):
See Indications for Use below.
B Indication(s) for 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 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
C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
D Special Instrument Requirements:
An ELISA plate reader capable of reading absorbance at one or more of the following settings is required but not provided: 450 nm, 450/620 nm, or 450/630 nm.
IV Device/System Characteristics:
A 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. pylori 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.
B Principle of Operation:
The Hp Detect Stool Antigen ELISA is an enzyme immunoassay which detects the H. pylori antigen in human fecal samples. Polyclonal anti-H. pylori capture antibodies are immobilized on microwells. Patient samples prepared in sample diluent are added to the microwells and incubated for one hour at 37 ± 2°C. If the H. pylori 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
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microwells and incubated for 30 minutes at 37 ± 2°C. 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°C 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
V Substantial Equivalence Information:
A Predicate Device Name(s):
PREMIER Platinum HpSA PLUS
B Predicate 510(k) Number(s):
K182559
C Comparison with Predicate(s):
| Device & Predicate Device(s): | Device: K232892 | Predicate: K182559 |
| --- | --- | --- |
| Device Trade Name | Hp Detect Stool Antigen ELISA | PREMIER Platinum HpSA PLUS |
| General Device Characteristic Similarities | | |
| Intended Use/Indications For 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 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 | The PREMIER Platinum HpSA PLUS enzyme immunoassay (EIA) is an in vitro qualitative procedure for the detection of Helicobacter pylori antigens in human stool. Test results are intended to aid in the diagnosis of H. pylori infection and to monitor response during and post-therapy in patients. Accepted medical practice recommends that testing by any current method, to confirm eradication, be done at least four weeks following completion of therapy. |
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| Device & Predicate Device(s): | Device: K232892 | Predicate: K182559 |
| --- | --- | --- |
| | consideration by the physician in conjunction with patient’s clinical information (history and symptoms). For Prescription Use Only. | |
| Measured Analyte | Detection of H. pylori antigen | Same |
| Type of Test | Qualitative | Same |
| Specimen Type | Human fecal specimens | Same |
| Controls | Positive and negative control included in the kit | Same |
| Target Population | Persons suspected of having H. pylori infection | Same |
| Storage | Refrigerated (2°C – 8°C) | Same |
| General Device Characteristic Differences | | |
| Specimen Storage | Specimens may be held up to 120 hours at 2°C – 8°C or should be frozen at ≤ -12°C or ≤ -60°C | Specimens may be held up to 72 hours at 2°C – 8°C or at 20°C – 25°C prior to testing |
| Time to Result | Approximately 1 hour and 40 minutes | Approximately 1 hour and 10 minutes |
| Antibody Format | Polyclonal/Monoclonal | Monoclonal |
| Incubation Temp | 37°C ± 2°C | 19-27°C |
| Reading Method | Spectrophotometric | Visual, Spectrophotometric |
VI Standards/Guidance Documents Referenced:
Not Applicable
VII Performance Characteristics (if/when applicable):
A Analytical Performance:
1. Reproducibility:
To demonstrate the reproducibility of the Hp Detect Stool Antigen ELISA, a panel of four (4) specimens was 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 a 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
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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 1 below. Testing at the dual and single wavelength (450 nm) produced equivalent results.
Table 1. Reproducibility of the Hp Detect Stool Antigen ELISA measured in dual wavelength.
| Sample ID | H. pylori Antigen | Combined (3 sites) | |
| --- | --- | --- | --- |
| | | Positive/Tested | % Detection |
| High Negative | 0.42 × LoD | 7/180 | 3.9% |
| Low Positive | 1.60 × LoD | 180/180 | 100% |
| Low Positive | 2.43 × LoD | 180/180 | 100% |
| Moderate Positive | 3.93 × LoD | 180/180 | 100% |
2. Within-Lab precision/repeatability:
The Within-Lab precision/repeatability of the Hp Detect Stool Antigen ELISA was determined onsite at Biomerica using a panel of four (4) 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 2. Testing at the dual and single wavelength (450 nm) produced equivalent results.
Table 2. Within-Lab precision/repeatability of the Hp Detect Stool Antigen ELISA measured in dual wavelength.
| Sample ID | H. pylori Antigen | Combined (Two kit lots) | |
| --- | --- | --- | --- |
| | | Positive/ Tested | % Detection |
| High Negative | 0.42 × LoD | 13/288 | 5% |
| Low Positive | 1.60 × LoD | 278/288 | 97% |
| Low Positive | 2.43 × LoD | 288/288 | 100% |
| Moderate Positive | 3.93 × LoD | 288/288 | 100% |
The results for reproducibility and precision/repeatability studies are acceptable.
3. Linearity:
Not applicable.
4. Analytical Specificity/Interference:
A. Microbial 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 $2.2 \times$ LoD (5.61 ng/mL) into the negative fecal matrix. Bacteria and fungi were spiked at concentrations of $10^{6}-10^{7}$ CFU/mL and viruses at a range from $10^{4}-10^{5}$ TCID $_{50}$ units per mL into either the
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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 were assayed in duplicates. The list of microorganisms and concentrations tested for cross-reactivity and microbial interference studies are shown in Table 3 below. No cross-reactivity 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.
Table 3. 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 |
| Escherichia 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 | |
*Bacteria were tested at $1 \times 10^{7}$ CFU/mL (except for Clostridium perfringens at $1 \times 10^{6}$ CFU/mL) and Candida albicans at $1 \times 10^{6}$ CFU/mL. Viruses were tested at $1 \times 10^{5}$ TCID $_{50}$ /mL except for Echovirus and Rotavirus which were tested at TCID $_{50}$ /mL of $1 \times 10^{4}$ .
The results for cross-reactivity and microbial interference with the Hp Detect Stool Antigen ELISA were acceptable.
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B. 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 $2.2 \times \mathrm{LoD}$ (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 potential interfering substances and their concentrations tested for cross-reactivity and interference studies were shown in Table 4 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.
Table 4. Interfering substance concentrations tested.
| 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 & Magnesium Hydroxide 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 |
The results for interfering substances study with the Hp Detect Stool Antigen ELISA were acceptable.
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# C. Inclusivity:
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 $1 - 3 \times \mathrm{LoD}$ (using LoD of $1.69 \times 10^{3}$ CFU/mL whole cells for strain ATCC 43504 and $5.86\mathrm{ng / mL}$ 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 $450 / 630\mathrm{nm}$ ) and single wavelength ( $450\mathrm{nm}$ ). Results for inclusivity are shown in Table 5. All strains were detected with Hp Detect Stool Antigen ELISA.
Table 5. List of $H$ . pylori strains tested for inclusivity study.
| H. pylori Strain | Concentration | Positive/Tested (% Detection) |
| --- | --- | --- |
| ATCC 43526 | 3.70 × 103CFU/mL | 3/3 (100%) |
| ATCC 43579 | 3.48 × 103CFU/mL | 3/3 (100%) |
| ATCC 49396 | 3.42 × 103CFU/mL | 3/3 (100%) |
| ATCC 700392 | 3.85 × 103CFU/mL | 3/3 (100%) |
| ATCC 700824 | 3.96 × 103CFU/mL | 3/3 (100%) |
| ATCC BAA-945 | 3.78 × 103CFU/mL | 3/3 (100%) |
| ATCC 49503 | 6.33 ng/mL Antigen | 3/3 (100%) |
The Inclusivity results were acceptable.
# 5. Assay Reportable Range:
Not applicable.
# 6. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
# 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 $5.42 \times$ 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: $2^{\circ}\mathrm{C}$ to $8^{\circ}\mathrm{C}$ , $\leq -12^{\circ}\mathrm{C}$ , and $\leq -60^{\circ}\mathrm{C}$ and tested for each time point and temperature range (shown in Table 6A). All the stability tests were measured in both dual (450/620 or $450 / 630\mathrm{nm}$ ) and single wavelength ( $450\mathrm{nm}$ ) with equivalent results. Results shown in Table 6B, 6C, and 6D are for 10 panel sample members grouped into 3 high negatives ( $<1 \times$ LoD), 4 low positives ( $1-2 \times$ LoD), and 3 moderate positives ( $3-5 \times$ LoD) measured in dual wavelengths.
Table 6A. Stability Testing Temperatures and Time Points
| Stability Condition | Temperature Range (°C) | Time Point (Days) |
| --- | --- | --- |
| Refrigerated | 2°C to 8°C | 0, 3, 5 |
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Table 6B. Results for storage of specimens at refrigerated conditions $(2 - 8^{\circ}\mathrm{C})$
| 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 6C. Results for storage of specimens at frozen conditions $(\leq -12^{\circ}\mathrm{C})$
| 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 6D. Results for storage of specimens at deep frozen conditions $(\leq -60^{\circ}\mathrm{C})$
| 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) freeze-thaw 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 $\leq -12^{\circ}\mathrm{C}$ and $\leq -60^{\circ}\mathrm{C}$ 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 7.
Table 7. Recommended Storage Conditions
| 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 |
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# 7. Detection Limit:
The limit of detection (LoD) for the Hp Detect Stool Antigen ELISA was determined by using antigens from two strains of $H.$ pylori, ATCC43504 (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 9.
Table 9. LoD values for Hp Detect Stool Antigen ELISA
| 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 |
The Limit of Detection (LoD) for the Hp Detect Stool Antigen ELISA was established at $2.53\mathrm{ng / mL}$ $(0.38\mathrm{ng / test})$ for strain ATCC 43504 and $5.86\mathrm{ng / mL}$ $(0.88\mathrm{ng / test})$ for strain ATCC 49503, measured in dual wavelength.
The correlation between antigen amount $(\mathrm{ng / mL})$ 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 $1.69\times 10^{3}$ CFU/mL. Therefore, $H.$ pylori strain ATCC 43504 has a LoD of $2.53~\mathrm{ng / mL}$ of antigen concentration correlates to a LoD of $1.69\times 10^{3}$ CFU/mL of bacteria concentration when compared to side to side (data not shown).
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.
# 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 $20,000 \, \mathrm{ng/mL}$ to $0.25 \, \mathrm{ng/mL}$ . Each of the serial dilutions were tested in 8 replicates measuring by both dual (450/620 or $450/630 \, \mathrm{nm}$ ) 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 $800 \, \mathrm{ng/ml}$ of $H.$ pylori antigen and remained at maximum level up to $20,000 \, \mathrm{ng/ml}$ . No high-dose hook effect was observed for the assay at antigen concentrations of up to $20,000 \, \mathrm{ng/mL}$ .
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# 8. 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: $\geq 0.100$
Spectrophotometric Single Wavelength (450 nm)
Negative: $< 0.140$
Positive: $\geq 0.140$
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.
# B Comparison Studies:
1. Method Comparison with Predicate Device:
Not applicable.
2. Matrix Comparison:
Not applicable.
# C Clinical Studies:
1. Clinical Performance - Frozen Specimens:
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 de-identified fecal samples were tested in which 355 specimens were collected from Italy and 78 specimens collected from three geographically different regions of the USA. 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 |
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The discrepant results were further analyzed by chart review and determined to have a rapid urease test (RUT) or history result and observed as follow:
(a) Four of the six discrepant false positives were positive by RUT or histology.
(b) The discrepant false negative was negative by RUT or histology.
Table 11. Clinical performance and statistics measured in single wavelength.
| Hp Detect Stool Antigen ELISA | Comparator: FDA Cleared Device | | |
| --- | --- | --- | --- |
| | Positive | Negative | Total |
| Positive | 111 | 13^{a} | 124 |
| Negative | 1^{b} | 308 | 309 |
| Total | 112 | 321 | 433 |
| 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 % |
The discrepant results were further analyzed by chart review and determined to have a RUT or history and observed as follow:
(a) Four of the 13 discrepant false positives 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.
2. Clinical Performance - Fresh Specimens
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 13. Fresh Specimens - Clinical performance and statistics measured in dual and single wavelength with same result.
| 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% |
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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.
## 3. Post-Therapy Performance
The performance of the Hp Detect Stool Antigen ELISA for post-therapy diagnosis was evaluated by using 14 paired (pre-and post- therapy) retrospective specimens from Italy. All subjects initially tested positive for H. pylori by composite reference method (CRM) consisting of histology and Rapid Urease Test and all completed eradication therapy. Post-therapy fecal samples were collected at a minimum of 4 weeks post completion of eradication therapy. All 14 pre-therapy samples were shown to be positive and matched with the CRM diagnosis. The result for post-therapy was shown in Table 13.
Table 13. Post-Therapy Performance with composite reference method
| Hp Detect Stool Antigen ELISA | Post-Therapy CRM | | |
| --- | --- | --- | --- |
| | Positive | Negative | Total |
| Positive | 10 | 0 | 10 |
| Negative | 0 | 4 | 4 |
| Total | 10 | 4 | 14 |
| 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.
## D Clinical Cut-Off:
Not applicable.
## E 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 sites in different geographic regions in the US and one site outside the US - Italy. The observed prevalence of H. pylori in this study was 27.0% (117/433). H. pylori detection and prevalence stratified by specimen origin are shown in Table 14 and categorized by gender is shown in Table 15.
Table 14: H. pylori detection and prevalence by specimen origin
| 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% |
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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% |
VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.
IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
