ALPHA HISTOPLASMA ANTIGEN EIA MODEL HAG102
Device Facts
| Record ID | K101407 |
|---|---|
| Device Name | ALPHA HISTOPLASMA ANTIGEN EIA MODEL HAG102 |
| Applicant | Immuno-Mycologics, Inc. |
| Product Code | MIZ · Microbiology |
| Decision Date | Jul 19, 2011 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3320 |
| Device Class | Class 2 |
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.
Device Story
The ALPHA Histoplasma Antigen EIA is an immunoenzymatic sandwich microplate assay used in clinical laboratories to detect Histoplasma antigens in urine. The device utilizes rabbit anti-Histoplasma IgG capture antibodies bound to 96-well microplates. Patient urine samples are added to the wells; if Histoplasma antigens are present, they bind to the capture antibodies. A biotinylated rabbit anti-Histoplasma detection antibody is added, followed by streptavidin-HRP. TMB substrate is added, producing a blue color proportional to the amount of bound antigen. The reaction is stopped, and optical density is measured at 450nm using a microplate reader. A four-parameter curve-fit, generated from kit-supplied standards, calculates EIA Units. Results assist clinicians in diagnosing histoplasmosis when used alongside culture, histology, and radiographic evidence. The assay provides a diagnostic aid, helping to identify patients with histoplasmosis, potentially facilitating timely clinical management.
Clinical Evidence
Clinical performance was evaluated using 278 culture- or histopathology-confirmed urine specimens (47 positive, 231 negative). The assay demonstrated 80.9% sensitivity (95% CI: 67.5-89.6%) and 98.7% specificity (95% CI: 96.3-99.6%) compared to the gold standard. Supplemental data from an independent study (n=177) reported 100% sensitivity and 99% specificity. Analytical specificity testing showed cross-reactivity with Blastomyces dermatitidis, Coccidioides immitis, and Paracoccidioides brasiliensis.
Technological Characteristics
Two-stage immunoenzymatic sandwich microplate assay. Components: 96-well microplate coated with rabbit polyclonal anti-Histoplasma IgG, biotinylated rabbit polyclonal anti-Histoplasma IgG detection antibody, streptavidin-HRP conjugate, TMB substrate, and stop solution. Requires microplate reader (450 nm or 450/630 nm filters). Quantitative calculation via four-parameter curve-fit. Standards/Guidelines: CLSI EP5-A2, EP12-A2, EP17-A.
Indications for Use
Indicated for the detection of Histoplasma antigens in urine samples as an aid in the diagnosis of histoplasmosis in patients suspected of active disease.
Regulatory Classification
Identification
Histoplasma capsulatum serological reagents are devices that consist of antigens and antisera used in serological tests to identify antibodies to Histoplasma capsulatum in serum. Additionally, some of these reagents consist of Histoplasma capsulatum antisera conjugated with a fluorescent dye (immunofluorescent reagents) used to identify Histoplasma 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 genus Histoplasma 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.
Special Controls
*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.
Predicate Devices
- Platelia Aspergillus EIA (K060641)
Related Devices
- K060641 — PLATELIA ASPERGILLUS EIA, MODEL 62793 · Bio-Rad · Jun 2, 2006
- K243496 — MycoMEIA Aspergillus Assay · Pearl Diagnostics, Inc. · Aug 1, 2025
- K120946 — ALPHA CRYPTOCOCCAL ANTIGEN EIA · Immuno-Mycologics, Inc. · Dec 17, 2012
Submission Summary (Full Text)
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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY ONLY TEMPLATE
A. 510(k) Number:
k101407
B. Purpose for Submission:
To obtain a substantial equivalence determination for ALPHA Histoplasma Antigen EIA from urine specimens
C. Measurand:
Histoplasma capsulatum antigens
D. Type of Test:
Qualitative Enzyme Immunoassay (EIA)
E. Applicant:
Immuno-Mycologics Inc.
F. Proprietary and Established Names:
ALPHA Histoplasma Antigen EIA
G. Regulatory Information:
1. Regulation section:
21 CFR Part 866.3320
2. Classification:
Class II
3. Product code:
MIZ
4. Panel:
83 - Microbiology
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H. Intended Use:
1. Intended use(s):
The ALPHA *Histoplasma* Antigen EIA is an immunoenzymatic sandwich microplate assay for the detection of *Histoplasma* antigens in urine samples.
2. Indication(s) for 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.
3. Special conditions for use statement(s):
Prescription use
4. Special instrument requirements:
Microplate reader equipped with 450 nm and/or 450/630 nm filters
I. Device Description:
The ALPHA *Histoplasma* Antigen EIA is a two-stage immunoenzymatic sandwich microplate assay which detects *Histoplasma* antigens in urine. Rabbit polyclonal anti-*Histoplasma* IgG antibodies bound to microwell plates are used as capture antibodies and biotinylated rabbit polyclonal anti-*Histoplasma* IgG antibodies are used as detect antibodies. Urine samples are run untreated and undiluted.
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The prepared samples are added to microwells coated with 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. 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 3,3',5,5' tetramethylbenzidine (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 450 nm alone or at 450nm and 630 nm. EIA Units for specimens are calculated using a four parameter curve-fit generated with the 4 standards supplied in the kit.
## J. Substantial Equivalence Information:
1. Predicate device name(s): Bio-Rad Platelia Aspergillus EIA
2. Predicate 510(k) number(s): k060641
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3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | Antigen Detection | Antigen Detection |
| Assay Principle | EIA | EIA |
| Assay components | 96-well microplate coated antibody, wash buffer, positive control, negative control, enzyme conjugate, TMB substrate, stop solution | 96-well microplate coated antibody, wash buffer, positive control, negative control, enzyme conjugate, TMB substrate, stop solution |
| Detection Chemistry | HRP + TMB | HRP + TMB |
| Controls/Standard | Antigen | Antigen |
| Instruments | Microplate reader equipped with 450 nm and 620/630 nm filters – none specified. | Microplate reader equipped with 450 nm and 620/630 nm filters – none specified. |
| Microplate | 96-well microplate coated with antibody | 96-well microplate coated with antibody |
| Differences | | |
| Item | Device | Predicate |
| Intended use | Detection of Histoplasma antigen in urine samples | Detection of Aspergillus galactomannan antigen in adult and pediatric serum samples |
| Indication for Use | Aid in the diagnosis of Histoplasmosis | Aid in the diagnosis of Aspergillosis |
| Sample Matrix | Urine | Serum |
| Controls | Histoplasma antigens | Aspergillus galactomannan |
| Detection Antibody | Biotinylated anti-Histoplasma polyclonal antibody | HRP-linked anti-Aspergillus monoclonal antibody |
| Output | “EIA Units” as determined from standard curve | “Index” as determined by OD of sample divided by Cut-Off Control OD |
| Microplate | 96-well microplate coated with anti-Histoplasma antibody | 96-well microplate coated with anti-Aspergillus antibody |
| Assay time | ~3 hours | ~2 hours |
| Shelf life | 1 year | Each kit component different |
| | | different |
| Sample | Antigen | Antigen |
| Instruments | Microplate reader equipped with 450 nm and 620/630 nm filters – none specified. | Microplate reader equipped with 450 nm and 620/630 nm filters – none specified. |
| Microplate | 96-well microplate coated with antibody | 96-well microplate coated with antibody |
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K. Standard/Guidance Document Referenced (if applicable):
- CLSI EP5-A2 Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline-Second Edition
- CLSI EP12-A2 User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline—Second Edition
- CLSI EP17-A Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline
L. Test Principle:
The ALPHA Histoplasma Antigen EIA is a two-stage immunoenzymatic sandwich microplate assay which detects Histoplasma antigens in urine. Rabbit polyclonal anti-Histoplasma IgG antibodies bound to microwell plates are used as capture antibodies and biotinylated rabbit polyclonal anti-Histoplasma IgG antibodies are used as detect antibodies. Urine samples are run untreated and undiluted. The prepared 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. 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 3,3',5,5' tetramethylbenzidine (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 450 nm alone or at 450 nm and 630 nm. EIA Units for specimens are calculated using a four parameter curve-fit generated with the 4 standards supplied in the kit.
M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
i. Study Design: 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, high negative (C₅), cut-off (C₅₀), low positive (C₉₅), and moderately positive. At the reference laboratory and at IMMY, each sample was tested in triplicate over the course of five
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days, using multiple operators. At the clinical laboratory, each sample was tested in triplicate over the course of three 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.
ii. Results/Acceptance Criteria: Variance was estimated by calculating the mean value of each sample, the standard deviation and percent CV, according to EP5-A2. 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 ( $\leq 20\%$ ), 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 below.
Intra-Run Reproducibility Analysis
| Intra-Run Analysis - Blanked OD Values | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Neg. Urine | C5 | C50 | C95 | Moderate Urine | Positive Control |
| Clinical Laboratory | Day 1 | Over 1 | Ave | 0.010 | 0.064 | 0.078 | 0.092 | 0.810 |
| | | | SD | 0.003 | 0.001 | 0.002 | 0.003 | 0.032 |
| | | | %CV | 26.5% | 1.8% | 2.0% | 3.3% | 3.9% |
| | Day 2 | Over 1 | Ave | 0.006 | 0.071 | 0.088 | 0.108 | 0.900 |
| | | | SD | 0.003 | 0.002 | 0.003 | 0.003 | 0.032 |
| | | | %CV | 50.0% | 2.9% | 3.4% | 2.8% | 3.6% |
| | Day 3 | Over 1 | Ave | 0.007 | 0.062 | 0.074 | 0.086 | 0.783 |
| | | | SD | 0.002 | 0.002 | 0.004 | 0.007 | 0.041 |
| | | | %CV | 31.2% | 3.4% | 5.1% | 8.1% | 5.3% |
| | | | | | | | | |
| Reference Laboratory | Day 1 | Over 1 | Ave | 0.006 | 0.035 | 0.054 | 0.057 | 0.523 |
| | | | SD | 0.003 | 0.002 | 0.001 | 0.007 | 0.011 |
| | | | %CV | 39.7% | 4.3% | 1.9% | 11.3% | 2.1% |
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Inter-Run Reproducibility Analysis
| | Inter-Run Analysis - Blanked OD Values | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | Neg. Urine | C5 | C50 | C95 | Moderate Urine | Positive Control |
| | Ave | 0.008 | 0.066 | 0.080 | 0.095 | 0.831 | 0.225 |
| | Std Dev | 0.003 | 0.005 | 0.007 | 0.011 | 0.061 | 0.017 |
| Clinical Laboratory | % CV | 38.7% | 7.0% | 8.6% | 11.0% | 7.4% | 7.6% |
| Reference Laboratory | Ave | 0.004 | 0.042 | 0.064 | 0.066 | 0.610 | 0.165 |
| | Std Dev | 0.007 | 0.005 | 0.009 | 0.009 | 0.059 | 0.025 |
| | % CV | 187.0% | 12.7% | 13.9% | 13.1% | 9.6% | 14.9% |
| IMMY | Ave | 0.006 | 0.043 | 0.068 | 0.074 | 0.670 | 0.215 |
| | Std Dev. | 0.004 | 0.006 | 0.009 | 0.009 | 0.048 | 0.020 |
| | % CV | 64.2% | 14.9% | 13.2% | 11.7% | 7.2% | 9.1% |
Inter-Site Reproducibility Analysis
| | Inter-Site Analysis - Blanked OD Values | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | Neg. Urine | C5 | C50 | C95 | Moderate Urine | Positive Control |
| All | Ave | 0.006 | 0.048 | 0.069 | 0.076 | 0.684 | 0.198 |
| | Std Dev | 0.005 | 0.011 | 0.010 | 0.014 | 0.101 | 0.034 |
| | % CV | 95.9% | 23.7% | 14.8% | 19.1% | 14.8% | 17.0% |
iii. Carry-Over Studies: To examine potential well-to-well carry-over, a positive sample (1x Wash Buffer spiked to 300 EIA Units) was used in series alternating with a negative sample (1x Wash Buffer) in a checkerboard pattern across 56 wells of plate. The test was repeated over the course of five days. Acceptance Criteria was defined as % CVs less than 20% and all 1x WB ODs less than 0.100.
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# Carry-Over Analysis Summary
| | | Negative Sample | Positive Sample |
| --- | --- | --- | --- |
| Day 1 | Ave | 0.052 | 1.243 |
| | Std Dev | 0.009 | 0.036 |
| | % CV | 18.3 | 2.9 |
| Day 2 | Ave | 0.054 | 1.097 |
| | Std Dev | 0.006 | 0.099 |
| | % CV | 11.0 | 9.0 |
| Day 3 | Ave | 0.049 | 1.202 |
| | Std Dev | 0.006 | 0.063 |
| | % CV | 12.2 | 5.2 |
| Day 4 | Ave | 0.035 | 1.223 |
| | Std Dev | 0.002 | 0.054 |
| | % CV | 7.1 | 4.4 |
iv. Prozone Studies: 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 (the top of the standard curve is 100 EIA units). Each sample was tested in duplicate. All samples remained strongly positive. The results are found in table below:
Prozone Study Results
| Sample | Blanked OD Rep 1 | Blanked OD Rep 2 |
| --- | --- | --- |
| 2000 EIA Units | 1.141 | 1.096 |
| 3000 EIA Units | 1.123 | 0.991 |
| 4000 EIA Units | 0.928 | 0.957 |
v. Single Versus Dual Wavelength Study: Eight positive urine samples and the four standards in the kit were tested in duplicate and according to the package insert. The microwells were read at 450/630 nm and then at 450 nm only. Line data is provided in the below. The average, standard deviation, and percent CV was calculated for each sample using all four data points (both replicates using both reading protocols). The study confirmed that the blanked OD of the samples is not affected by the read method, as indicated by the low percent CVs. Furthermore, 450/630 vs. 450 alone can be used with equal accuracy, and data from the two methods can be used interchangeably. Acceptance criteria were defined as % CV
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less than 20%.
Single Versus Dual Wavelength Line Data
| | Blanked OD | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Rep 1 | | Rep 2 | | | | |
| | 450/630 nm | 450 nm | 450/630 nm | 450 nm | Average | Std. Dev. | % CV |
| Sample 1 | 1.578 | 1.595 | 1.487 | 1.510 | 1.542 | 0.052 | 3.4% |
| Sample 2 | 1.271 | 1.286 | 1.261 | 1.282 | 1.275 | 0.012 | 0.9% |
| Sample 3 | 0.324 | 0.327 | 0.309 | 0.313 | 0.318 | 0.009 | 2.7% |
| Sample 4 | 0.253 | 0.257 | 0.251 | 0.255 | 0.254 | 0.003 | 1.1% |
| Sample 5 | 0.592 | 0.593 | 0.594 | 0.600 | 0.595 | 0.004 | 0.6% |
| Sample 6 | 0.198 | 0.198 | 0.192 | 0.195 | 0.196 | 0.003 | 1.5% |
| Sample 7 | 1.364 | 1.375 | 1.351 | 1.358 | 1.362 | 0.010 | 0.8% |
| Sample 8 | 1.077 | 1.077 | 1.085 | 1.097 | 1.084 | 0.010 | 0.9% |
| 100 Std | 1.040 | 1.065 | 0.936 | 0.940 | 0.995 | 0.067 | 6.7% |
| 30 Std | 0.566 | 0.564 | 0.483 | 0.484 | 0.524 | 0.047 | 9.0% |
| 10 Std | 0.253 | 0.255 | 0.234 | 0.235 | 0.244 | 0.011 | 4.6% |
| 2 Std | 0.062 | 0.061 | 0.051 | 0.050 | 0.056 | 0.006 | 11.4% |
| 1x WB | 0.005 | 0.005 | -0.005 | -0.005 | 0.000 | 0.005 | Na |
b. Linearity/assay reportable range:
Not applicable
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
i. Standard Curve Methods and Acceptance Criteria: The acceptable blanked OD ranges were determined by testing each standard on multiple lots for a total of 83 replicates. The acceptable OD was calculated by averaging all replicates and rounding up to an even number. The ranges were calculated by rounding 3x the standard deviations and adding to the rounded average. The table below summarizes the data.
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Acceptable Blanked OD Ranges for Standards
| | 100 Std | 30 Std | 10 Std | 2 Std |
| --- | --- | --- | --- | --- |
| | n=83 | n=83 | n=83 | n=83 |
| Ave | 0.775 | 0.387 | 0.169 | 0.038 |
| Std. Dev | 0.110 | 0.059 | 0.030 | 0.009 |
| 3x Std Dev | 0.330 | 0.176 | 0.091 | 0.027 |
| Acceptable OD | 0.800 | 0.400 | 0.200 | 0.040 |
| Acceptable OD Ranges +/- | 0.300 | 0.150 | 0.100 | 0.020 |
An assay is considered valid when the standards fall within the acceptable, blanked OD ranges as defined below and $\mathrm{R}^2 \geq 0.990$ .
| Standard | Acceptable Blanked OD |
| --- | --- |
| 100 | 0.80 ± 0.3 (1.1-0.5) |
| 30 | 0.40 ± 0.15 (0.55-0.25) |
| 10 | 0.20 ± 0.1 (0.3-0.1) |
| 2 | 0.04 ± 0.02 (0.06-0.02) |
| Positive Control | 0.20 ± 0.1 (0.3-0.1) |
| Negative Control | < 0.02 |
4 Parameter $(y = (A - D) / (1 + (x / C)^{*}\mathsf{B}) + D)$ $A = 0.9284$ B=-1.2141 C=39.6866 D=0.0095, R-Square = 0.9976
The users are instructed to run all specimens and controls in duplicate until the user becomes familiar with the kit performance. The Positive Control, Negative Control, and EIA standards must be assayed with each batch of patient specimens to provide quality assurance of the reagents. The Positive and Negative Controls are intended to monitor for substantial reagent failure. The Positive Control should not be used as an indicator of EIA standards precision and only ensures reagent functionality. The
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Positive Control EIA units should be between 7 and 13. The Negative Control EIA units should be less than 2.
If the EIA units of the Positive Control and/or Negative Control are not within these parameters, patient test results should be considered invalid, and the assay should be repeated.
ii. Specimen Stability Studies: In specimen stability studies, negative urine was spiked at four concentrations: Low-positive (C95), low-positive, and two moderate-positives. Specimens were divided and then stored at 4°C and -20°C. The set stored at 4°C was followed daily for stability over the course of one week. One set stored at -20°C went through multiple freeze/thaw cycles and was tested after each cycle. A second set was stored for one week without multiple freeze/thaw cycles, and a third set was stored for 2 weeks without multiple freeze/thaw cycles. When tested, each sample was run in duplicate. While there were reductions in EIA values after a 2-week storage at 4°C and after multiple freeze thaws, no specimen went from positive to negative. Since a reduction in EIA values was observed, it is possible that a fresh, very low-positive specimen (near 2.0 EIA units) could become negative if it is stored for several days. Results of the studies (averages of Blanked OD and EIA Units) are summarized below.
Blanked OD of Specimens Stored at 4°C
| Blanked OD (4°C) | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Day 0 (Fresh) | Day 1 | Day 2 | Day 3 | Day 6 | Day 7 | Day 14 |
| Sample 1 | 0.050 | 0.047 | 0.053 | 0.045 | 0.044 | 0.045 | 0.062 |
| Sample 2 | 0.103 | 0.092 | 0.119 | 0.096 | 0.091 | 0.073 | 0.095 |
| Sample 3 | 0.172 | 0.161 | 0.196 | 0.167 | 0.148 | 0.142 | 0.160 |
| Sample 4 | 0.260 | 0.198 | 0.303 | 0.242 | 0.242 | 0.229 | 0.274 |
EIA Units of Specimens Stored at 4°C
| EIA Units (4°C) | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Day 0 (Fresh) | Day 1 | Day 2 | Day 3 | Day 6 | Day 7 | Day 14 |
| Sample 1 | 2.70 | 2.68 | 2.61 | 2.47 | 2.55 | 2.65 | 2.96 |
| Sample 2 | 6.48 | 5.28 | 6.03 | 5.03 | 5.21 | 4.57 | 4.60 |
| Sample | 12.38 | 10.57 | 11.33 | 10.09 | 9.50 | 10.66 | 8.77 |
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| 3 | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sample 4 | 21.40 | 14.04 | 21.00 | 17.55 | 19.33 | 20.88 | 19.10 |
Blanked OD of Specimens Stored at -20°C
| Blanked OD (-20°) | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Freeze/Thaw | | | | | | Long Term Storage | |
| | Day 0 (Fresh) | Day 1 | Day 2 | Day 3 | Day 6 | Day 7 | Day 14 | 1 Week | 2 Weeks |
| Sample 1 | 0.050 | 0.043 | 0.053 | 0.040 | 0.042 | 0.044 | 0.060 | 0.042 | 0.051 |
| Sample 2 | 0.103 | 0.086 | 0.109 | 0.106 | 0.089 | 0.094 | 0.125 | 0.084 | 0.100 |
| Sample 3 | 0.172 | 0.171 | 0.206 | 0.177 | 0.170 | 0.173 | 0.185 | 0.150 | 0.174 |
| Sample 4 | 0.260 | 0.232 | 0.280 | 0.244 | 0.293 | 0.245 | 0.275 | 0.214 | 0.278 |
EIA Units of Specimens Stored at -20°C
| EIA Units (-20°) | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Freeze/Thaw | | | | | | Long Term Storage | |
| | Day 0 (Fresh) | Day 1 | Day 2 | Day 3 | Day 6 | Day 7 | Day 14 | 1 Week | 2 Weeks |
| Sample 1 | 2.70 | 2.48 | 2.61 | 2.25 | 2.46 | 2.58 | 2.85 | 2.43 | 2.47 |
| Sample 2 | 6.48 | 4.89 | 5.48 | 5.64 | 5.08 | 6.24 | 6.44 | 5.44 | 4.88 |
| Sample 3 | 12.38 | 11.41 | 12.06 | 11.03 | 11.59 | 14.00 | 10.70 | 11.61 | 9.80 |
| Sample 4 | 21.40 | 17.57 | 18.59 | 17.80 | 26.16 | 23.28 | 19.13 | 19.22 | 19.39 |
iii. Reagent/Kit Stability Studies: The following stability studies were performed to validate claims for the storage of reconstituted reagent.
| Stability of Opened / Reconstituted Reagents | Microplate stable for up to five weeks after opening when stored at 2-8°C.
Wash Solution stable for up to 15 days after dilution when stored at 2-8°C.
Controls stable for up to five weeks after reconstitution when stored at -20°C.
Chromogen / Substrate Buffer solution stable for up to six hours when stored at room temperature, in the dark. |
| --- | --- |
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# Real Time Kit Stability
The kit is stable for 1 year after date of manufacture.
iv. Time Parameter Studies for Reading the Test: To demonstrate that the test should be read within 15 minutes after the addition of the Stop Solution, all the standards were run in triplicate according to the package insert. The test was then read immediately after the addition of the Stop Solution, and then at 15, 30, 45, 60, 75, and 90 minutes. The signal began to decrease after 15 minutes and continued to decrease over the course of 90 minutes.
Blanked OD of Standards after Delayed Reading
Delayed Reading Line Data
| | Blanked OD | | | | | | | | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | T=0 | | | T=15 | | | T=30 | | | T=45 | | | T=60 | | | T=75 | | | T=90 | |
| | Rep 1 | Rep 2 | Rep 3 | Rep 1 | Rep 2 | Rep 3 | Rep 1 | Rep 2 | Rep 3 | Rep 1 | Rep 2 | Rep 3 | Rep 1 | Rep 2 | Rep 3 | Rep 1 | Rep 2 | Rep 3 | Rep 1 | Rep 2 |
| Block | 0.563 | 0.570 | 0.503 | 0.575 | 0.569 | 0.504 | 0.586 | 0.572 | 0.511 | 0.593 | 0.574 | 0.515 | 0.599 | 0.576 | 0.517 | 0.593 | 0.568 | 0.515 | 0.581 | 0.553 |
| Block | 0.310 | 0.279 | 0.275 | 0.302 | 0.278 | 0.265 | 0.302 | 0.279 | 0.264 | 0.302 | 0.277 | 0.262 | 0.305 | 0.277 | 0.263 | 0.299 | 0.274 | 0.259 | 0.295 | 0.285 |
| Block | 0.136 | 0.131 | 0.112 | 0.134 | 0.126 | 0.109 | 0.133 | 0.122 | 0.107 | 0.131 | 0.121 | 0.104 | 0.131 | 0.121 | 0.104 | 0.128 | 0.117 | 0.101 | 0.127 | 0.116 |
| 2std | 0.034 | 0.035 | 0.032 | 0.034 | 0.032 | 0.027 | 0.032 | 0.031 | 0.025 | 0.031 | 0.029 | 0.023 | 0.030 | 0.029 | 0.023 | 0.029 | 0.027 | 0.022 | 0.028 | 0.028 |
| LoWR | 0.001 | 0.001 | 0.000 | 0.001 | 0.001 | 0.000 | 0.001 | 0.001 | -0.001 | 0.001 | 0.000 | -0.001 | 0.002 | 0.001 | -0.002 | 0.002 | 0.001 | -0.001 | 0.003 | 0.001 |
| LoWR | 0.000 | 0.000 | -0.002 | 0.000 | 0.000 | -0.001 | 0.000 | -0.001 | -0.001 | 0.000 | -0.001 | -0.001 | 0.000 | -0.001 | -0.001 | 0.001 | 0.000 | -0.001 | 0.002 | 0.000 |
# d. Detection limit:
i. Analytical Sensitivity Methods: Analytical sensitivity was estimated at IMMY according to Clinical and Laboratory Standards Institute (CLSI) EP-17A. The limit of the blank (LoB) was estimated by running 82 blank measurements and the limit of detection (LoD) was estimated by running 30 measurements of 9 low standards (1.0 Std, 1.25 Std, 1.5 Std, 1.75 Std, 2.0 Std, 2.25 Std, 2.5 Std, 3.0 Std, 4.0 Std) all made by diluting the Histoplasma Antigen Standard (HAG100) in 1x Wash Buffer (HAGWB1).
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ii. Results: Line data for LoB and LoD can be found below.
LoB Line Data
| Rep 1 | 0.046 | Re 21 | 0.044 | Rep 41 | 0.058 | Rep 61 | 0.046 | Rep 81 | 0.048 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Rep 2 | 0.042 | Re 22 | 0.044 | Rep 42 | 0.043 | Rep 62 | 0.046 | Rep 82 | 0.047 |
| Rep 3 | 0.061 | Re 23 | 0.044 | Rep 43 | 0.042 | Rep 63 | 0.049 | | |
| Rep 4 | 0.044 | Re 24 | 0.044 | Rep 44 | 0.044 | Rep 64 | 0.048 | | |
| Rep 5 | 0.045 | Re 25 | 0.044 | Rep 45 | 0.053 | Rep 65 | 0.046 | | |
| Rep 6 | 0.043 | Re 26 | 0.043 | Rep 46 | 0.049 | Rep 66 | 0.049 | | |
| Rep 7 | 0.042 | Re 27 | 0.043 | Rep 47 | 0.047 | Rep 67 | 0.044 | | |
| Rep 8 | 0.042 | Re 28 | 0.046 | Rep 48 | 0.049 | Rep 68 | 0.048 | | |
| Rep 9 | 0.042 | Re 29 | 0.044 | Rep 49 | 0.046 | Rep 69 | 0.045 | | |
| Rep 10 | 0.044 | Re 30 | 0.044 | Rep 50 | 0.047 | Rep 70 | 0.045 | | |
| Rep 11 | 0.045 | Rep 31 | 0.044 | Rep 51 | 0.047 | Rep 71 | 0.046 | | |
| Rep 12 | 0.044 | Rep 32 | 0.044 | Rep 52 | 0.047 | Rep 72 | 0.044 | | |
| Rep 13 | 0.074 | Rep 33 | 0.045 | Rep 53 | 0.046 | Rep 73 | 0.047 | | |
| Rep 14 | 0.044 | Rep 34 | 0.043 | Rep 54 | 0.044 | Rep 74 | 0.043 | | |
| Rep 15 | 0.05 | Rep 35 | 0.042 | Rep 55 | 0.046 | Rep 75 | 0.045 | | |
| Rep 16 | 0.043 | Rep 36 | 0.041 | Rep 56 | 0.047 | Rep 76 | 0.045 | | |
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| Rep 17 | 0.043 | Rep 37 | 0.044 | Rep 57 | 0.048 | Rep 77 | 0.047 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Rep 18 | 0.042 | Rep 38 | 0.045 | Rep 58 | 0.048 | Rep 78 | 0.045 |
| Rep 19 | 0.044 | Rep 39 | 0.04 | Rep 59 | 0.048 | Rep 79 | 0.045 |
| Rep 20 | 0.045 | Rep 40 | 0.043 | Rep 60 | 0.051 | Rep 80 | 0.047 |
LoD Line Data
| Sample | RAW OD | Sample | RAW OD | Sample | RAW OD | Sample | RAW OD | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1std | 0.076 | 1.25std | 0.077 | 1.5 std | 0.084 | 1.75std | 0.100 | | |
| 1std | 0.082 | 1.25std | 0.077 | 1.5 std | 0.086 | 1.75std | 0.094 | | |
| 1std | 0.070 | 1.25std | 0.078 | 1.5 std | 0.084 | 1.75std | 0.094 | | |
| 1std | 0.073 | 1.25std | 0.073 | 1.5 std | 0.085 | 1.75std | 0.097 | | |
| 1std | 0.072 | 1.25std | 0.076 | 1.5 std | 0.080 | 1.75std | 0.100 | | |
| 1std | 0.072 | 1.25std | 0.075 | 1.5 std | 0.079 | 1.75std | 0.094 | | |
| 1std | 0.071 | 1.25std | 0.070 | 1.5 std | 0.082 | 1.75std | 0.094 | | |
| 1std | 0.072 | 1.25std | 0.074 | 1.5 std | 0.099 | 1.75std | 0.097 | | |
| 1std | 0.071 | 1.25std | 0.075 | 1.5 std | 0.076 | 1.75std | 0.093 | | |
| 1std | 0.072 | 1.25std | 0.073 | 1.5 std | 0.079 | 1.75std | 0.097 | | |
| 1std | 0.071 | 1.25std | 0.074 | 1.5 std | 0.086 | 1.75std | 0.100 | | |
| 1std | 0.071 | 1.25std | 0.074 | 1.5 std | 0.086 | 1.75std | 0.097 | | |
| 1std | 0.071 | 1.25std | 0.071 | 1.5 std | 0.079 | 1.75std | 0.104 | | |
| 1std | 0.071 | 1.25std | 0.077 | 1.5 std | 0.080 | 1.75std | 0.098 | | |
| 1std | 0.069 | 1.25std | 0.077 | 1.5 std | 0.078 | 1.75std | 0.106 | | |
| 1std | 0.073 | 1.25std | 0.075 | 1.5 std | 0.077 | 1.75std | 0.105 | | |
| 1std | 0.070 | 1.25std | 0.076 | 1.5 std | 0.081 | 1.75std | 0.096 | | |
| 1std | 0.071 | 1.25std | 0.081 | 1.5 std | 0.080 | 1.75std | 0.096 | | |
| 1std | 0.070 | 1.25std | 0.076 | 1.5 std | 0.102 | 1.75std | 0.102 | | |
| 1std | 0.070 | 1.25std | 0.076 | 1.5 std | 0.091 | 1.75std | 0.096 | | |
| 1std | 0.069 | 1.25std | 0.072 | 1.5 std | 0.095 | 1.75std | 0.092 | | |
| 1std | 0.070 | 1.25std | 0.077 | 1.5 std | 0.096 | 1.75std | 0.090 | | |
| 1std | 0.071 | 1.25std | 0.078 | 1.5 std | 0.095 | 1.75std | 0.087 | | |
| 1std | 0.076 | 1.25std | 0.075 | 1.5 std | 0.090 | 1.75std | 0.088 | | |
| 1std | 0.072 | 1.25std | 0.074 | 1.5 std | 0.091 | 1.75std | 0.105 | | |
| 1std | 0.069 | 1.25std | 0.074 | 1.5 std | 0.092 | 1.75std | 0.100 | | |
| 1std | 0.073 | 1.25std | 0.076 | 1.5 std | 0.095 | 1.75std | 0.098 | | |
| 1std | 0.084 | 1.25std | 0.070 | 1.5 std | 0.086 | 1.75std | 0.099 | | |
| 1std | 0.068 | 1.25std | 0.070 | 1.5 std | 0.096 | 1.75std | 0.097 | | |
| 1std | 0.074 | 1.25std | 0.070 | 1.5 std | 0.089 | 1.75std | 0.094 | | |
| | | | | | | | | | |
| 2.0std | 0.088 | 2.25std | 0.093 | 2.5std | 0.112 | 3.0std | 0.131 | 4.0std | 0.141 |
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| 2.0std | 0.093 | 2.25std | 0.095 | 2.5std | 0.106 | 3.0std | 0.118 | 4.0std | 0.135 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0std | 0.093 | 2.25std | 0.095 | 2.5std | 0.107 | 3.0std | 0.117 | 4.0std | 0.131 |
| 2.0std | 0.094 | 2.25std | 0.096 | 2.5std | 0.111 | 3.0std | 0.115 | 4.0std | 0.132 |
| 2.0std | 0.092 | 2.25std | 0.101 | 2.5std | 0.102 | 3.0std | 0.112 | 4.0std | 0.132 |
| 2.0std | 0.096 | 2.25std | 0.095 | 2.5std | 0.110 | 3.0std | 0.105 | 4.0std | 0.136 |
| 2.0std | 0.096 | 2.25std | 0.093 | 2.5std | 0.120 | 3.0std | 0.114 | 4.0std | 0.134 |
| 2.0std | 0.101 | 2.25std | 0.095 | 2.5std | 0.111 | 3.0std | 0.116 | 4.0std | 0.132 |
| 2.0std | 0.089 | 2.25std | 0.105 | 2.5std | 0.101 | 3.0std | 0.129 | 4.0std | 0.150 |
| 2.0std | 0.094 | 2.25std | 0.096 | 2.5std | 0.105 | 3.0std | 0.117 | 4.0std | 0.137 |
| 2.0std | 0.097 | 2.25std | 0.093 | 2.5std | 0.104 | 3.0std | 0.116 | 4.0std | 0.136 |
| 2.0std | 0.096 | 2.25std | 0.094 | 2.5std | 0.104 | 3.0std | 0.119 | 4.0std | 0.136 |
| 2.0std | 0.094 | 2.25std | 0.096 | 2.5std | 0.105 | 3.0std | 0.115 | 4.0std | 0.137 |
| 2.0std | 0.094 | 2.25std | 0.096 | 2.5std | 0.102 | 3.0std | 0.111 | 4.0std | 0.143 |
| 2.0std | 0.092 | 2.25std | 0.092 | 2.5std | 0.101 | 3.0std | 0.115 | 4.0std | 0.133 |
| 2.0std | 0.093 | 2.25std | 0.093 | 2.5std | 0.103 | 3.0std | 0.115 | 4.0std | 0.139 |
| 2.0std | 0.087 | 2.25std | 0.104 | 2.5std | 0.105 | 3.0std | 0.127 | 4.0std | 0.151 |
| 2.0std | 0.090 | 2.25std | 0.094 | 2.5std | 0.100 | 3.0std | 0.118 | 4.0std | 0.138 |
| 2.0std | 0.089 | 2.25std | 0.091 | 2.5std | 0.102 | 3.0std | 0.117 | 4.0std | 0.140 |
| 2.0std | 0.091 | 2.25std | 0.089 | 2.5std | 0.101 | 3.0std | 0.114 | 4.0std | 0.147 |
| 2.0std | 0.088 | 2.25std | 0.093 | 2.5std | 0.100 | 3.0std | 0.114 | 4.0std | 0.142 |
| 2.0std | 0.089 | 2.25std | 0.091 | 2.5std | 0.100 | 3.0std | 0.116 | 4.0std | 0.141 |
| 2.0std | 0.090 | 2.25std | 0.094 | 2.5std | 0.097 | 3.0std | 0.122 | 4.0std | 0.142 |
| 2.0std | 0.100 | 2.25std | 0.095 | 2.5std | 0.104 | 3.0std | 0.121 | 4.0std | 0.149 |
| 2.0std | 0.091 | 2.25std | 0.097 | 2.5std | 0.098 | 3.0std | 0.133 | 4.0std | 0.154 |
| 2.0std | 0.089 | 2.25std | 0.090 | 2.5std | 0.106 | 3.0std | 0.120 | 4.0std | 0.146 |
| 2.0std | 0.088 | 2.25std | 0.085 | 2.5std | 0.110 | 3.0std | 0.118 | 4.0std | 0.141 |
| 2.0std | 0.089 | 2.25std | 0.089 | 2.5std | 0.101 | 3.0std | 0.118 | 4.0std | 0.139 |
| 2.0std | 0.088 | 2.25std | 0.093 | 2.5std | 0.103 | 3.0std | 0.117 | 4.0std | 0.150 |
| 2.0std | 0.087 | 2.25std | 0.084 | 2.5std | 0.094 | 3.0std | 0.118 | 4.0std | 0.155 |
Analytical Sensitivity Analysis:
Limit of the Blank Calculations:
$$
\mathrm {L o B} = \mu_ {\beta} + 1. 6 4 5 \sigma_ {\beta}
$$
where $\mu_{\beta}$ is the mean of the blank measurements and $\sigma_{\beta}$ is the standard deviation of the blank measurements.
$$
\mathrm {L o B} = 0. 0 0 1 4 2 7 + 1. 6 4 5 (0. 0 0 4 4 6 9)
$$
$$
\mathrm {L o B} = 0. 0 0 9 \mathrm {O D}
$$
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Limit of Detection Calculations:
$\mathrm{LoD} = \mathrm{LoB} + \mathrm{c}_{\beta}\mathrm{SD}_{\mathrm{s}}$
where $\mathrm{SD}_{\mathrm{s}}$ is the standard deviation of the sample measurements and $c_{\beta}$ is derived from the $95^{\mathrm{th}}$ percentile of the standard Gaussian distribution:
$\mathrm{c}_{\beta} = 1.645 / (1 - 1 / (4 \times \mathrm{f}))$ , where $\mathrm{f}$ is the degrees of freedom from the $\mathrm{SD}_{\mathrm{s}}$ .
$\mathrm{f} = \mathrm{N}_{\mathrm{s}} - \mathrm{K}$ where $\mathrm{N_s}$ is the total number of measurements (9 x 30) and K is number of sources of measurements (9)
$\mathrm{LoD} = 0.009 + (1.645 / (1 - 1 / (4 \times (270 - 9))))(0.021)$
$\mathrm{LoD} = 0.044 \mathrm{OD}$
Analysis shows that the LoB is 0.009 RAW OD and LoD is 0.044 BLANKED OD. Our 2.0 EIA units calibrator is approximately equivalent to this Limit of Detection (0.044 OD).
# e. Analytical specificity:
i. Cross-Reactivity Studies: Urine specimens that tested negative for Histoplasma antigen were spiked with antigen from Blastomyces dermatitidis, Coccidioides immitis, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus terreus, Paracoccidioides brasiliensis and Candida albicans, individually, at $1\mu \mathrm{g / mL}$ . The ALPHA Histoplasma Antigen EIA was found to be cross-reactive with Blastomyces dermatitidis, Coccidioides immitis, and Paracoccidioides brasiliensis in urine. The assay was not cross-reactive with Candida albicans, Cryptococcus neoformans, or Aspergillus spp. in urine. Source/Strain information of culture filtrates used in cross-reactivity testing are shown in table below:
| Organism | Source and Strain |
| --- | --- |
| Blastomyces dermatitidis | Marano – Clinical Isolate from Marshfield Clinic |
| Coccidioides immitis | CDC B-475 |
| Paracoccidioides brasiliensis | CDC B-339 |
| Aspergillus spp. | ATCC 64026 (A. fumigatus) ATCC64028 (A. niger) ATCC 64025 (A. flavus) ATCC 28301 (A. terreus) |
| Candida albicans | ATCC 32354 |
| Cryptococcus neoformans | 184A – Clinical Isolate from New Orleans/Tulane* |
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* Infection and Immunity, June 1983, p. 1052-1059
Analytical Specificity Line Data and Analysis
| | Blanked OD | | | Statistical Analysis | | | EIA Units | | | Statistical Analysis | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Spiked at 1ug/ml | Rep 1 | Rep 2 | Rep 3 | Ave | Std. Dev. | % CV | Rep 1 | Rep 2 | Rep 3 | Ave | Std. Dev. | % CV |
| Blastomyces | 0.534 | 0.532 | 0.530 | 0.532 | 0.002 | 0.4% | 33.2 | 33.0 | 32.7 | 33.0 | 0.263 | 0.8% |
| Coccidioides | 1.136 | 1.158 | 1.198 | 1.164 | 0.031 | 2.7% | 170.1 | 180.4 | 200.4 | 183.6 | 15.396 | 8.4% |
| Paracoccidioides | 1.388 | 1.367 | 1.371 | 1.375 | 0.011 | 0.8% | 347.2 | 326.4 | 330.4 | 334.7 | 11.043 | 3.3% |
| Aspergillus sp. | 0.014 | 0.012 | 0.010 | 0.012 | 0.002 | 16.7% | <2 | <2 | <2 | <2 | n.a. | n.a. |
| Cryptococcus | 0.000 | 0.004 | 0.001 | 0.002 | 0.002 | 83.3% | <2 | <2 | <2 | <2 | n.a. | n.a. |
| Candida | 0.014 | 0.013 | 0.014 | 0.014 | 0.001 | 4.2% | <2 | <2 | <2 | <2 | n.a. | n.a. |
ii. Interfering Substances: Urine specimens containing various potential interfering substances were obtained from a national reference laboratory. Each specimen was tested in duplicate using the IMMY ALPHA Histoplasma Antigen EIA to assess the potential for false positive reactivity in the assay. Additionally, each specimen was spiked with Histoplasma antigen and tested in the assay. None of these substances were found to interfere with the ALPHA Histoplasma Antigen EIA. The data from this study is summarized in the table below. The "Interfering Value" is given as reported by the reference laboratory.
Interference Line Data
| | Unspiked EIA Units | | Spiked EIA Units | | Interfering Value |
| --- | --- | --- | --- | --- | --- |
| | Rep 1 | Rep 2 | Rep 1 | Rep 2 | |
| Protein | | | | | (mg/dl) |
| #4 | 0 | 0 | 3.856 | 3.574 | 30 |
| #6 | 0 | 0 | 3.735 | 3.979 | 100 |
| #7 | 0 | 0 | 3.109 | 3.694 | 30 |
| #8 | 0 | 0 | 2.848 | 2.959 | 30 |
| #9 | 0 | 0 | 6.685 | 6.786 | 30 |
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| Blood | | | | | Cells/High-Powered Field |
| --- | --- | --- | --- | --- | --- |
| #1 | 0 | 0 | 2.147 | 2.631 | Moderate |
| #2 | 0 | 0 | 3.574 | 4.021 | Moderate |
| #3 | 0 | 0 | 4.146 | 4.616 | Large |
| #4 (Blue)* | 0 | 0 | 8.491 | 6.736 | Large |
| #6 | 0 | 0 | 5.196 | 4.315 | Moderate |
| Epithelial Cells | | | | | Cells/High-Powered Field |
| #2 | 0 | 0 | 5.755 | 4.529 | 2-5 |
| #3 | 0 | 0 | 3.185 | 2.812 | 2 |
| #4 | 0 | 0 | 5.105 | 4.97 | 6 |
| #6 | 0 | 0 | 0 | 0 | 10-20 |
| #7 | 0 | 0 | 3.456 | 3.574 | 6 |
| Ketones | | | | | (mg/dl) |
| #1 | 0 | 0 | 6.939 | 6.189 | ≥80 |
| #3 | 0 | 0 | 4.146 | 4.357 | 60 |
| #4 | 0 | 0 | 0 | 0 | 10 |
| #5 | 0 | 0 | 0 | 2.015 | 40 |
| #6 | 0 | 0 | 3.034 | 3.378 | 10 |
| Mucus | | | | | Cells/High-Powered Field |
| #1 | 0 | 0 | 4.009 | 4.05 | Many |
| #2 (Blue)* | 0 | 0 | 8.004 | 7.322 | Moderate |
| #3 | 0 | 0 | 5.188 | 4.297 | Many |
| #4 | 0 | 0 | 5.893 | 5.626 | Many |
| #5 | 0 | 0 | 2.37 | 2.37 | Moderate |
| Cast | | | | | Cells/Low-Powered Field |
| #1 | 0 | 0 | 2.333 | 2.04 | 5-Hyal |
| #2 (Blue)* | 0 | 0 | 7.608 | 6.898 | Granular |
| #3 | 0 | 0 | 2.975 | 2.86 | 5-Hyal |
| #4 | 0 | 0 | 2.556 | 2.185 | 19-Hyal |
| #5 | 0 | 0 | 2.594 | 2.594 | 0-2 Hyal |
| Glucose | | | | | (mg/dl) |
| #1 | 0 | 0 | 3.525 | 3.725 | 250 |
| #2 | 0 | 0 | 2.898 | 2.86 | 250 |
| #3 | 0 | 0 | 5.538 | 5.319 | 500 |
| #4 | 0 | 0 | 4.548 | 5.714 | 100 |
| #5 | 0 | 0 | 4.132 | 3.846 | 500 |
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| Bilirubin | | | | | Cells/High-Powered Field |
| --- | --- | --- | --- | --- | --- |
| #1 | 0 | 0 | 5.362 | 5.45 | Moderate |
| #2 | 0 | 0 | 2.975 | 3.326 | Moderate |
| #3 | 0 | 0 | 2.86 | 3.366 | Moderate |
| #4 | 0 | 0 | 5.319 | 5.937 | Moderate |
| #5 | 0 | 0 | 5.275 | 5.102 | Moderate |
*Specimen was blue in color, reason unknown
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.
f. Assay cut-off:
i. Study Design: The assay cut-off was established following CLSI EP12-A2 and EP17-A. The 2 EIA units standard was created to be equivalent to the LoD. Using the 2 EIA units standard as the cut-off, the $\mathrm{C}_{5}-\mathrm{C}_{95}$ Interval and $\mathrm{C}_{50}$ were determined by running 30 replicates of 9 samples spiked with Histoplasma antigen around the 2 EIA units standard. Percent positive was determined by the number of samples that ran equal to or greater than the average blanked OD of the 2 Standard. The percentage of replicates that ran positive was calculated for each sample in the table below. The Receiver Operator Curve (ROC) analysis of culture-proven specimens indicated a lower cut-off of 1.3 EIA units (figure below). The ROC value is below the Limit of Detection, and therefore considered an inappropriate cutoff. As such, an equivocal zone was not created, as all data firmly supported a cut-off of 2.0 EIA units.
Cut-Off Analysis:
Percent Positives for 11 samples near the LoD
| Sample Concentration (EIA Units) | % Positive | |
| --- | --- | --- |
| 1.00 | 3.3% | |
| 1.25 | 0.0% | |
| 1.50 | 6.7% | C5 |
| 1.75 | 23.3% | |
| 2.00 | 53.3% | C50 |
| 2.25 | 62.5% | |
| 2.50 | 92.5% | C95 |
| 2.75 | 100% | |
| 3.00 | 100% | |
| 3.50 | 100% | |
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4.00 100%
ROC analysis of culture-proven specimens
The $C_5$ is 1.50 and the $C_{95}$ is 2.50 EIA Units while the $C_{50}$ is 2.00 EIA Units. $C_{50}$ is a good indicator of an assay's cut-off. However, ROC analysis indicated a lower cut-off of 1.3 EIA units. Since this is below the calculated LoD of the assay, the $C_{50}$ was used to establish the cut-off. Since analytical sensitivity (LoD) was more conservative than the clinical sensitivity (ROC analysis), an equivocal zone was not created.
The assay cut-offs are defined as follows:
Negative: $< 2.0$ EIA Units (Limit of Detection)
Positive: $\geq 2.0$ EIA Units
# 2. Comparison studies:
a. Method comparison with predicate device:
Not applicable
b. Matrix comparison:
Not applicable; urine only.
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# 3. Clinical studies:
# a. Clinical Sensitivity
i. Study Design: The clinical performance of the ALPHA Histoplasma Antigen EIA was evaluated using a total of 278 culture- or histopathology-confirmed urine specimens (47 culture/histopathology-positive and 231 culture/histopathology-negative). Specimens were obtained and tested at three geographically diverse hospitals/reference laboratories. All specimens were obtained from patients suspected of experiencing active disease by a physician and confirmed through culture or histopathology. Clinical sensitivity and specificity were calculated according to CLSI EP12-A2.
ii. Results: The data from this study demonstrated that the ALPHA Histoplasma Antigen EIA has an $80.9\%$ sensitivity (CI: 67.5-89.6%) and $98.7\%$ specificity (CI 96.3-99.6%), when compared to culture and/or histopathology, the current "gold standard" for diagnosis of histoplasmosis. The results are summarized in the tables below.
Histoplasma Ag EIA Versus Culture/Histopathology 2x2 Contingency Table
| | | Culture/Histopathology | |
| --- | --- | --- | --- |
| | | Positive | Negative |
| IMMY ALPHA | Positive | 38 | 3 |
| Histoplasma EIA | Negative | 9 | 228 |
Statistical Analysis Comparing Device to Culture
| | Point Estimate | 95% CI |
| --- | --- | --- |
| Sensitivity | 80.9% | 67.5-89.6% |
| Specificity | 98.7% | 96.3-99.6% |
iii. Supplemental Studies:
a) An independent comparison between culture/histopathology and the ALPHA Histoplasma Antigen EIA Kit was presented at the College of American Pathologists 2008 Annual meeting. A poster titled "Utility of a Histoplasma capsulatum Enzyme Immunoassay for Diagnosis of Disseminated Histoplasmosis and Correlation with Disease Activity" was presented *. In the IRB approved study, Cox, et. al. reported that
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during the course of one year, they enrolled 177 patients with suspected histoplasmosis and tested their urine specimens with the ALPHA Histoplasma Antigen EIA. Of those, only six were actually diagnosed with histoplasmosis through culture and/or histopathology. Below are tables showing the results of the study, including a 2x2 contingency table and a table of relevant statistics.
* Cox, M., G. Pesek, D. Phan, and G. Woods. 2008. Utility of a Histoplasma capsulatum Enzyme Immunoassay for Diagnosis of Disseminated Histoplasmosis and Correlation with Disease Activity. Arch. Pathol. Lab Med 132:1512.
Cox, et. al. Disease vs. IMMY EIA 2x2 Contingency Table
| Disease (By Culture and/or Histopathology) | | |
| --- | --- | --- |
| | Positive | Negative |
| IMMY | 6 | 2 |
| Positive | | |
| IMMY | 0 | 171 |
| Negative | | |
Statistical Analysis
| | | 95% CI |
| --- | --- | --- |
| Sensitivity | 100% | 54%-100% |
| Specificity | 99% | 96%-100% |
| Prevalence | 3.3% | 1.3%-7.2% |
| Positive | 75% | 35%-96% |
| Predictive Value | | |
| Negative | 100% | 98%-100% |
| Predictive Value | | |
b) Additional studies were performed using a non-FDA cleared Histoplasma capsulatum quantitative antigen EIA and resulted in a positive percent agreement of $71.4\%$ (CI: 57.6-82.2%) and negative percent agreement of $94.0\%$ (CI: 83.8-97.9%) when the non-FDA cleared test's equivocals were assumed positive. When the equivocals were assumed negative, positive percent agreement was $92.1\%$ (CI: 79.2-97.3%) and negative percent agreement was $95.1\%$ (CI: 86.5-98.3%).
b. Clinical specificity:
Clinical specificity was tested using non-Histoplasma, fungal culture-positive urines. The results from this study are summarized in the table below:
# Specificity Testing Using Fungal Culture Positive Urines
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| | No. Positive | No. Negative | % Specificity | 95% CI |
| --- | --- | --- | --- | --- |
| Aspergillus | 0 | 20 | 0% (0/20) | 83.9-100% |
| Candida spp. | 0 | 12 | 0% (0/12) | 75.8-100% |
| Paracoccidioides | 1 | 11 | 9.1% (1/11) | 62.3-98.4% |
c. Other clinical supportive data (when a. and b. are not applicable): Not applicable
4. Clinical cut-off:
The clinical cut-off is the same as the assay cut-off. (Also see Section 3.f.)
Negative: EIA Units $x < 2.0$
Positive: EIA Units $x \geq 2.0$
The clinical performance of the ALPHA Histoplasma Antigen EIA was evaluated using a total of 278 culture- or histopathology-confirmed urine specimens (47 culture/histopathology-positive and 231 culture/histopathology-negative). Specimens were obtained and tested at three geographically diverse hospitals/reference laboratories. All specimens were obtained from patients suspected of experiencing active disease by a physician and confirmed through culture or histopathology.
The clinical cut-off values are interpreted as follows:
- Negative results do not rule out the diagnosis of disease as the specimen may be drawn before detectable antigen is present.
- A positive result implies the presence of antigen to Histoplasma; however, all test results should be reviewed in light of other clinical data by the physician.
- The magnitude of the measured result, above the cutoff, is not indicative of the total amount of antigen present.
- Specimens should be repeated if the results are low-positive and inconsistent with clinical findings.
- Specimens should be repeated if the results are indeterminate or low-positive and inconsistent with clinical findings.
5. Expected values/Reference range:
The frequency of histoplasmosis is dependent on several factors including: patient population, type of institution, and epidemiology. In this study, $80.9\%$ of true positives determined by culture and/or histopathology were detected.
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N. Proposed Labeling:
The proposed labeling is sufficient and satisfies the requirements of 21 CFR section 809.10
O. Conclusion:
The submitted information in the premarket notification is complete and supports substantial equivalence decision.
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