K Number
K101407
Device Name
ALPHA HISTOPLASMA ANTIGEN EIA MODEL HAG102
Date Cleared
2011-07-19

(426 days)

Product Code
Regulation Number
866.3320
AI/MLSaMDIVD (In Vitro Diagnostic)TherapeuticDiagnosticis PCCP AuthorizedThirdparty
Intended Use
The ALPHA Histoplasma Antigen EIA is an immunoenzymatic sandwich microplate assay for the detection of Histoplasma antigens in urine samples. The ALPHA Histoplasma Antigen EIA is a test which, when used in conjunction with other diagnostic procedures such as microbiological culture, histological examination of biopsy samples and radiographic evidence, can be used as an aid in the diagnosis of histoplasmosis.
Device Description
The ALPHA Histoplasma Antigen EIA is an immunoenzymatic sandwich microplate assay which detects Histoplasma antigens in urine. Rabbit anti-Histoplasma IgG antibodies bound to microwell plates are used as capture antibodies and biotinylated rabbit anti-Histoplasma IgG antibodies are used as detect antibodies. The positive control is made of buffer spiked with Histoplasma capsulatum antigens and the negative control is buffer only. Standards are made of Histoplasma capsulatum antigens from culture filtrate. The kit contains the 100 Standard, 30 Standard, and 2 Standard that are used to generate a sigmoid calibration curve using a four-parameter fit of the blanked absorbance values versus the assigned EIA Values. R-squared values must be greater than or equal to 0.990. Urine samples are run untreated and undiluted. The samples are added to the microwells coated with the capture antibody and incubated. If the patient specimen contains Histoplasma antigens that are recognized by the capture antibody, those antigens will become bound to the microwell. The wells are washed to remove unbound patient material and biotinylated detection antibody is added to the wells. If Histoplasma antigens are bound to the microwell by the capture antibody, then the detect antibody will also become bound to the microwell. The wells are then washed to remove any unbound detect antibody. For detection, biotin-streptavidin chemistry reagents including 3,3′,5′,5′ tetramethybenzadine (TMB) and stop solution are used. Streptavidin conjugated to horseradish peroxidase (HRP) is added to the microwells. In the presence of the biotinylated detect antibody, streptavidin-HRP will become bound to the plate. The plate is then washed to remove any unbound streptavidin-HRP, and TMB substrate solution is added to the microwells. A blue color develops in the presence of the HRP enzyme. The reaction is stopped by the addition of a stop solution. The optical density (absorbance) is determined with a microplate reader at 450nm or 450nm or 450nm alone. EIA Units for specimens are calculated using a four-parameter curve-fit generated with the 4 standards supplied in the kit.
More Information

Not Found

No
The device description details a standard immunoassay with optical density measurement and curve fitting, which does not involve AI/ML. The document explicitly states "Mentions AI, DNN, or ML: Not Found".

No
Explanation: This device is an in-vitro diagnostic (IVD) test used to aid in the diagnosis of histoplasmosis by detecting antigens. It does not provide therapy or treatment for the condition.

Yes

The "Intended Use / Indications for Use" section explicitly states that the device "can be used as an aid in the diagnosis of histoplasmosis." This phrasing clearly indicates its role in the diagnostic process.

No

The device is a laboratory assay kit that includes physical reagents and microwell plates, not solely software.

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

  • Intended Use: The intended use explicitly states it's for the "detection of Histoplasma antigens in urine samples" and is used "as an aid in the diagnosis of histoplasmosis." This clearly indicates it's a test performed on a sample taken from the human body to provide information for diagnostic purposes.
  • Device Description: The description details a laboratory-based assay (immunoenzymatic sandwich microplate assay) that uses reagents to detect a specific analyte (Histoplasma antigens) in a biological sample (urine). This is characteristic of an in vitro diagnostic test.
  • Performance Studies: The document describes clinical performance evaluations using human urine specimens and provides metrics like sensitivity and specificity, which are standard for evaluating the performance of IVD devices.
  • Predicate Device: The mention of a predicate device (Bio-Rad's Platelia™ Aspergillus EIA) is common in regulatory submissions for IVDs, indicating a comparison to an already cleared IVD.

All these elements align with the definition and characteristics of an In Vitro Diagnostic device.

N/A

Intended Use / Indications for Use

The ALPHA Histoplasma Antigen ElA is an immunoenzymatic sandwich microplate assay for the detection of Histoplasma antigens in urine samples.

The ALPHA Histoplasma Antigen EIA is a test which, when used in conjunction with other diagnostic procedures such as microbiological culture, histological examination of biopsy samples and radiographic evidence, can be used as an aid in the diagnosis of histoplasmosis.

Product codes

MIZ

Device Description

The ALPHA Histoplasma Antigen EIA is an immunoenzymatic sandwich microplate assay which detects Histoplasma antigens in urine. Rabbit anti-Histoplasma IgG antibodies bound to microwell plates are used as capture antibodies and biotinylated rabbit anti-Histoplasma IgG antibodies are used as detect antibodies.

The positive control is made of buffer spiked with Histoplasma capsulatum antigens and the negative control is buffer only. Standards are made of Histoplasma capsulatum antigens from culture filtrate. The kit contains the 100 Standard, 30 Standard, and 2 Standard that are used to generate a sigmoid calibration curve using a four-parameter fit of the blanked absorbance values versus the assigned EIA Values. R-squared values must be greater than or equal to 0.990.

Urine samples are run untreated and undiluted. The samples are added to the microwells coated with the capture antibody and incubated. If the patient specimen contains Histoplasma antigens that are recognized by the capture antibody, those antigens will become bound to the microwell. The wells are washed to remove unbound patient material and biotinylated detection antibody is added to the wells. If Histoplasma antigens are bound to the microwell by the capture antibody, then the detect antibody will also become bound to the microwell. The wells are then washed to remove any unbound detect antibody. For detection, biotinstreptavidin chemistry reagents including 3,3′,5′,5′ tetramethybenzadine (TMB) and stop solution are used. Streptavidin conjugated to horseradish peroxidase (HRP) is added to the microwells. In the presence of the biotinylated detect antibody, streptavidin-HRP will become bound to the plate. The plate is then washed to remove any unbound streptavidin-HRP, and TMB substrate solution is added to the microwells. A blue color develops in the presence of the HRP enzyme. The reaction is stopped by the addition of a stop solution. The optical density (absorbance) is determined with a microplate reader at 450nm or 450nm or 450nm alone. ElA Units for specimens are calculated using a four-parameter curve-fit generated with the 4 standards supplied in the kit.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies

The clinical performance of the ALPHA Histoplasma Antigen EIA was evaluated using a total of 278 culture- or histopathology-confirmed urine specimens.

Comparison to Other Histoplasma Antigen EIA: Supplemental comparison studies were performed using a non-FDA-cleared Histoplasma capsulatum quantitative antigen EIA.

Key Metrics

Comparison to Culture/Histopathology:
Sensitivity: 80.9% (95% CI: 67.5-89.6%)
Specificity: 98.7% (95% CI: 96.3-99.6%)

Comparison to Other Histoplasma Antigen EIA:
Equivocals Assumed Positive:
% Agree Positive: 71.4% (95% CI: 57.6-82.2%)
% Agree Negative: 94.0% (95% CI: 83.8-97.9%)

Equivocals Assumed Negative:
% Agree Positive: 92.1% (95% CI: 79.2-97.3%)
% Agree Negative: 95.1% (95% CI: 86.5-98.3%)

Predicate Device(s)

K060641

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

§ 866.3320

Histoplasma capsulatum serological reagents.(a)
Identification. Histoplasma capsulatum serological reagents are devices that consist of antigens and antisera used in serological tests to identify antibodies toHistoplasma capsulatum in serum. Additionally, some of these reagents consist ofHistoplasma capsulatum antisera conjugated with a fluorescent dye (immunofluorescent reagents) used to identifyHistoplasma capsulatum from clinical specimens or cultured isolates derived from clinical specimens. The identification aids in the diagnosis of histoplasmosis caused by this fungus belonging to the genusHistoplasma and provides epidemiological information on the diseases caused by this fungus. Histoplasmosis usually is a mild and often asymptomatic respiratory infection, but in a small number of infected individuals the lesions may spread to practically all tissues and organs.(b)
Classification. Class II (special controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to § 866.9.

0

JUL 19 2011

510(k) Premarket Notification ALPHA Histoplasma Antigen EIA

510(k) Summary ALPHA Histoplasma Antigen EIA

This 510(k) summary is submitted in accordance with 21 CFR §807.92

Assigned 510(k) No .: K101407

Owner: Immuno-Mycologics, Inc. 2700 Technology Place Norman, OK 73071 Tel: 405-360-4669 Fax: 405-364-1058 Contact: Dr. Sean K. Bauman, President & CEO July 7, 2011 Prepared:

Trade Name: ALPHA Histoplasma Antigen EIA

Histoplasma Antigen EIA Common Name:

Classification Name: None

Regulation: 866.3320

Bio-Rad's Platelia™ Aspergillus EIA, K060641 Predicate Device:

Intended Use: The ALPHA Histoplasma Antigen ElA is an immunoenzymatic sandwich microplate assay for the detection of Histoplasma antigens in urine samples.

The ALPHA Histoplasma Antigen EIA is a test which, when used in conjunction with other diagnostic procedures such as microbiological culture, histological examination of biopsy samples and radiographic evidence, can be used as an aid in the diagnosis of histoplasmosis.

Device Description:

The ALPHA Histoplasma Antigen EIA is an immunoenzymatic sandwich microplate assay which detects Histoplasma antigens in urine. Rabbit anti-Histoplasma IgG antibodies bound to microwell plates are used as capture antibodies and biotinylated rabbit anti-Histoplasma IgG antibodies are used as detect antibodies.

The positive control is made of buffer spiked with Histoplasma capsulatum antigens and the negative control is buffer only. Standards are made of Histoplasma capsulatum antigens from culture filtrate. The kit contains the 100 Standard, 30 Standard, and 2 Standard

Rev. 07/11/2011

Page 5-1

1

that are used to generate a sigmoid calibration curve using a four-parameter fit of the blanked absorbance values versus the assigned EIA Values. R-squared values must be greater than or equal to 0.990.

Urine samples are run untreated and undiluted. The samples are added to the microwells coated with the capture antibody and incubated. If the patient specimen contains Histoplasma antigens that are recognized by the capture antibody, those antigens will become bound to the microwell. The wells are washed to remove unbound patient material and biotinylated detection antibody is added to the wells. If Histoplasma antigens are bound to the microwell by the capture antibody, then the detect antibody will also become bound to the microwell. The wells are then washed to remove any unbound detect antibody. For detection, biotinstreptavidin chemistry reagents including 3,3′,5′,5′ tetramethybenzadine (TMB) and stop solution are used. Streptavidin conjugated to horseradish peroxidase (HRP) is added to the microwells. In the presence of the biotinylated detect antibody, streptavidin-HRP will become bound to the plate. The plate is then washed to remove any unbound streptavidin-HRP, and TMB substrate solution is added to the microwells. A blue color develops in the presence of the HRP enzyme. The reaction is stopped by the addition of a stop solution. The optical density (absorbance) is determined with a microplate reader at 450nm or 450nm or 450nm alone. ElA Units for specimens are calculated using a four-parameter curve-fit generated with the 4 standards supplied in the kit.

Comparison with Predicate:

A comparison of the similarities and differences between the ALPHA Histoplasma Antigen ElA and the predicate device is presented in the table below (Table 1).

FeatureALPHA Histoplasma Ag EIA DevicePlatelia Aspergillus EIA K060641
Intended UseAntigen detectionAntigen detection
Indication For UseAid in the diagnosisAid in the diagnosis
Device Description
Assay PrincipleEIAEIA
Assay components96-well microplate coated antibody, wash buffer, positive control, negative control, enzyme conjugate, TMB substrate, stop solution96-well microplate coated antibody, wash buffer, positive control, negative control, enzyme conjugate, TMB substrate, stop solution
Detection ChemistryHRP + TMBHRP + TMB
Controls/StandardAntigenAntigen
Instrumentsnonenone
Microplate96-well microplate coated with antibody96-well microplate coated with antibody
Performance Characteristics
PrecisionAcceptable % CVsAcceptable % CVs
Table 1. Similarities and Differences between ALPHA Histoplasma EIA and Platelia Aspergillus ElA
----------------------------------------------------------------------------------------------------

2

LinearityNot applicableNot applicable
SpecificityCross-reacts with other fungal organismsCross-reacts with other fungal organisms
FeatureALPHA Histoplasma Ag EIA DevicePlatelia Aspergillus EIA K060641
Intended UseDetection of Histoplasma antigen in urine samplesDetection of Aspergillus galactomannan antigen in
adult and pediatric serum samples
Indication For UseAid in the diagnosis of histoplasmosis.Aid in the diagnosis of invasive aspergillosis.
Sample MatrixUrineSerum
ControlsHistoplasma antigensAspergillus galactomannan
Output"EIA Units" as determined from standard curve"Index" as determined by OD of sample divided by
Cut-Off Control OD
Detection
AntibodyBiotinylated anti-Histoplasma polyclonal antibodyHRP-linked anti-Aspergillus monoclonal antibody
Microplate96-well microplate coated with anti-Histoplasma
polyclonal antibody96-well microplate coated with anti-Aspergillus
monoclonal antibody
Assay Time~3 hours~2 hours
Performance
Characteristics
Shelf life1 yearEach kit component different

Analytical Performance Summary

  • A. Urine Precision Studies
    Three sites, a reference laboratory (RL) (Western US), a clinical laboratory (CL) (Upper Mid-Western US), and IMMY (Central US), were used to assess the assay's reproducibility. The panel consisted of urine samples at five levels: negative (روم), cut-off (دول), low positive (C35), and moderately positive. At the reference laboratory and at IMMY, each sample was tested in triplicate over the course of 5 days, using multiple operators. At the clinical laboratory, each sample was tested in triplicate over the course of 3 days, using a single operator. Throughout the study, reagent lots and instrument calibrations were held constant. No runs were removed from analysis due to failed runs.

Variance was estimated by calculating the mean value of each sample, the standard deviation and percent CV. The data was analyzed separately to evaluate any inter-assay, intra-assay, and inter-site variation. Overall, no major source of variability was identified. Intra-run, Inter-Run, and Inter-site percent CVs are within acceptable limits (≤ 20%), with the exception of the negative urine and low negative urine samples, which is expected when testing beyond the limit of detection. A summary of the data is reported in the Tables 2-4 below.

3

: :

Table 2. Intra-run Reproducibility Analysis
Intra-run Analysis Blanked OD Values
Neg.
UrineC5C50C95Moderate
UrinePositive
Control
Clinical LaboratoryDay 1Operator 1Ave0.0100.0640.078
SD0.0030.0010.0020.0030.0320.008
%CV26.5%1.8%2.0%3.3%3.9%3.4%
Day 2Operator 1Ave0.0060.0710.0880.1080.9000.246
SD0.0030.0020.0030.0030.0320.004
%CV50.0%2.9%3.4%2.8%3.6%1.5%
Day 3Operator 1Ave0.0070.0620.0740.0860.7830.211
SD0.0020.0020.0040.0070.0410.009
%CV31.2%3.4%5.1%8.1%5.3%4.3%
Reference LaboratoryDay 1Operator 1Ave0.0060.0350.0540.0570.5230.136
SD0.0030.0020.0010.0070.0110.008
%CV39.7%4.3%1.9%11.3%2.1%5.6%
Day 2Operator 2Ave-0.0030.0390.0650.0610.6120.172
SD0.0060.0020.0010.0020.0240.008
%CV183%4.0%0.9%2.5%3.9%4.9%
Day 3Operator 3Ave0.0110.0480.0720.0640.6420.182
SD0.0010.0030.0040.0090.0780.007
%CV9.1%5.2%4.9%13.5%12.2%3.9%
Day 4Operator 4Ave-0.0010.0450.0710.0770.6540.195
SD0.0100.0050.0030.0070.0180.005
%CV766%11.8%4.3%9.1%2.7%2.4%
Day 5Operator 4Ave0.0070.0420.0610.0690.6200.142
SD0.0010.0020.0150.0020.0310.011
%CV17.3%3.7%24.5%2.2%5.0%8.0%
IMMYDay-1Operator 1Ave0.0120.0490.0700.0810.6860.204
SD0.0030.0100.0140.0110.0390.027
%CV24.2%20.6%19.4%13.6%5.7%13.0%
Day 2Operator,1Ave0.0040.0390.0680.0660.6230.226
SD0.0020.0020.0100.0050.0430.028
%CV57.1%4.5%15.1%7.6%6.8%12.5%

Tahle 2 Intra-run Reproducibility Analysis

・一

4

| Day 3

Operator 1Ave0.0030.0450.0670.0760.7230.216
SD0.0010.0060.0090.0040.0510.004
%CV36.4%13.6%13.3%5.3%7.0%1.6%
Day 4
Operator 2Ave0.0080.0430.0640.0680.6530.202
SD0.0030.0060.0090.0060.0250.017
%CV38.6%15.1%14.7%8.1%3.8%8.4%
Day 5
Operator 2Ave0.0050.0420.0720.0800.6630.228
SD0.0020.0050.0080.0080.0340.006
%CV31.6%12.2%11.3%10.3%5.1%2.7%

Table 3. Inter-Run Reproducibility Analysis

Inter-Run Analysis - Blanked OD Values
Neg.
UrineC5C50C95Moderate
UrinePositive
Control
Ave0.0080.0660.0800.0950.8310.225
Std. Dev.0.0030.0050.0070.0110.0610.017
% CV38.7%7.0%8.6%11.0%7.4%7.6%
Reference
LaboratoryAve0.0040.0420.0640.0660.6100.165
Std. Dev.0.0070.0050.0090.0090.0590.025
% CV187.0%12.7%13.9%13.1%9.6%14.9%
IMMYAve0.0060.0430.0680.0740.6700.215
Std. Dev.0.0040.0060.0090.0090.0480.020
% CV64.2%14.9%13.2%11.7%7.2%9.1%

Table 4. Inter-Site Reproducibility Analysis

Inter-Site Analysis - Blanked OD Values
Neg.
UrineC5C50C95Moderate
UrinePositive
Control
Ave0.0060.0480.0690.0760.6840.198
Std. Dev.0.0050.0110.0100.0140.1010.034
% CV95.9%23.7%14.8%19.1%14.8%17.0%
  • B. Analytical Sensitivity (Lower limits of the assay)
    Analytical sensitivity was determined (according to CLSI EP17-A) by determining the assay's Limit of the Blank and Limit of Detection. Wash buffer was spiked with Histoplasma antigens at a concentration range of 1 – 4 EIA Units. To determine the LoB, 82 replicates of wash buffer were assayed. To determine the LoD, 30 replicates of the spiked wash buffer were assayed. The Limit of the Blank is 0.009 OD and the Limit of Detection is 2 EIA Units (0.044 Blanked OD).

5

C. Analytical Specificity (Cross-Reactivity)

Urine specimens that tested negative for Histoplasma antigen were spiked with antigen from Blastomyces dermatitidis, Coccidioides immitis, Aspergillus sp., Paracoccidioides brasilliensis, Candida albicans, and Cryptococcus neoformans, individually at 1ug/ml. The ALPHA Histoplasma Antigen EIA is found to be cross-reactive with Blastomyces dermatitidis, Coccidioides immitis, and Paracoccidioides brasilliensis. The assay is not cross-reactive with Candida albicans, Cryptococcus neoformans, or Aspergillus sp. in urine. Furthermore, Aspergillus, Candida, Paracoccidioides, and Penicillium culture-positive urine specimens were tested in the ALPHA Histoplasma Antigen EIA. The results are summarized in Table 5.

Table 5. Specificity Using Culture-Confirmed Urine Specimens

Percent Positive
Aspergillus0% (0/20)
Candida spp.0% (0/12)
Paracoccidioides9.1% (1/11)
Penicillium0% (0/2)
  • D. Analytical Interference
    To evaluate substances that could potentially interfere with the ALPHA Histoplasma Antigen EIA, urine specimens containing various substances were obtained from a national reference laboratory. Substances included protein, blood, epithelial cells, ketones, mucus, casts, glucose, and bilirubin. Each specimen was spiked with Histoplasma antigen and tested. None of these substances were found to interfere with the ALPHA Histoplasma Antigen EIA. Vaginal cream urines and foods which produce color in urine were not tested. Additionally, drugs, such as itraconazole, amphotericin B, acetaminophen, acetylsalicylic acid, ascorbic acid, and caffeine were not tested for interference.

  • E. Carry-over
    To examine potential well-to-well carry-over, a positive sample was used in series alternating with a negative sample in a checkerboard pattern. Samples were found not to carry-over when methods described in the package insert were followed.

  • F. Prozone Effect
    To detect the prozone effect in the ALPHA Histoplasma Antigen EIA, negative urine was spiked with Histoplasma antigen to 2000, 3000, and 4000 EIA Units and tested. All samples remained positive, therefore, prozoning is not seen in the ALPHA Histoplasma Antigen EIA.

  • G. Linearity
    N/A

6

H. Assay cut-off

-------. ..

The assay's cut-off of 2.0 EIA Units was established by Limit of Detection (Section B) experiments and confirmed with the determination of the C50. Receiver Operator Curve (ROC) analysis of culture-proven specimens indicated a 1.3 ElA Unit cutoff. However, this is below the Limit of Detection (2.0 EIA units), and therefore an inappropriate cutoff.

Negative: ✓ |
|-----------------------------|-----------------------------------------------------------------------------------------|
| (Part 21 CFR 801 Subpart D) | |

Over-The-Counter Use AND/OR (21 CFR 801 Subpart C)

(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of In Vitro Diagnostic Devices (OIVD)

Freddie K. Poole

Division Sign-Off Office of In Vitro Diagnostic Device Evaluation and Safety

510(k): K101407