MycoMEIA Aspergillus Assay

K243496 · Pearl Diagnostics, Inc. · NOM · Aug 1, 2025 · Microbiology

Device Facts

• Record ID: K243496
• Device Name: MycoMEIA Aspergillus Assay
• Applicant: Pearl Diagnostics, Inc.
• Product Code: NOM · Microbiology
• Decision Date: Aug 1, 2025
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3040
• Device Class: Class 1

Indications for Use

The MycoMEIA® Aspergillus Assay (MycoMEIA) is an enzyme immunoassay (EIA) for the in vitro qualitative detection of Aspergillus antigens in human urine from adults (>= 18 years old) with suspected invasive aspergillosis (IA). The assay is not intended for use in lung transplant recipients. The results should be interpreted by trained healthcare professionals, incorporating other diagnostic procedures such as microbiological culture, histological examination of biopsy samples, and radiographic evidence to support the diagnosis of IA.

Device Story

MycoMEIA is a sandwich enzyme immunoassay (EIA) for detecting Aspergillus galactofuranose-containing antigens in human urine. Urine samples are processed through columns to remove inhibitors, then added to 96-well microplates coated with two mouse monoclonal antibodies (mAb476 and mAb501). Bound antigen is detected using horseradish-peroxidase-conjugated antibodies. A colorimetric reaction (blue to yellow upon acid stop) is measured via spectrophotometer at 450/620 nm. Results are calculated as an index value relative to a threshold control. Used in clinical laboratories by trained professionals. Output aids diagnosis of invasive aspergillosis when combined with culture, histology, and radiography. Benefits include non-invasive urine-based testing for high-risk patients.

Clinical Evidence

Clinical performance evaluated via retrospective (475 samples, 290 subjects) and prospective studies (166 subjects, 169 episodes). Per-subject sensitivity for proven/probable IA was 92.4% (95% CI 82.1-97.0%). Per-sample sensitivity was 58.2% (95% CI 54.3-61.9%). Specificity in prospective fresh samples was 86.1% (95% CI 75.7-92.5%). Cross-reactivity observed with other Ascomycetes fungi (e.g., Fusarium).

Technological Characteristics

Sandwich EIA; 96-well microtiter plates coated with mouse monoclonal antibodies; HRP-conjugated reporter antibodies; TMB substrate; spectrophotometric detection (450/620 nm). Requires Sample Processing Columns and 25X Concentrated Wash Solution. Manual calculation via Excel worksheet. Standards: CLSI EP05-A3 (precision), EP07 (interference), EP12 (qualitative output), EP17-A2 (LoD), EP25-A (stability).

Indications for Use

Indicated for adults (>= 18 years) with suspected invasive aspergillosis (IA). Not for use in lung transplant recipients. Contraindicated for patients where other diagnostic procedures are not available.

Regulatory Classification

Identification

Aspergillus spp. serological reagents are devices that consist of antigens and antisera used in various serological tests to identify antibodies to Aspergillus spp. in serum. The identification aids in the diagnosis of aspergillosis caused by fungi belonging to the genus Aspergillus. Aspergillosis is a disease marked by inflammatory granulomatous (tumor-like) lessions in the skin, ear, eyeball cavity, nasal sinuses, lungs, and occasionally the bones.

Predicate Devices

• Platelia Aspergillus EIA Model 62793 (K093678)

Submission Summary (Full Text)

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FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY AND INSTRUMENT

## I Background Information:

A 510(k) Number

K243496

B Applicant

Pearl Diagnostics, Inc.

C Proprietary and Established Names

MycoMEIA Aspergillus Assay

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| NOM | Class I | 21 CFR 866.3040 - Aspergillus Spp. Serological Reagents | MI - Microbiology |

## II Submission/Device Overview:

A Purpose for Submission:

To obtain substantial equivalence determination for the MycoMEIA Aspergillus Assay.

B Measurand:

Aspergillus galactofuranose (galf)-containing antigens

C Type of Test:

Enzyme immunoassay (EIA)

## III Intended Use/Indications for Use:

A Intended Use(s):

See Indications for Use below.

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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B Indication(s) for Use:

The MycoMEIA Aspergillus Assay (MycoMEIA) is an enzyme immunoassay (EIA) for the in vitro qualitative detection of Aspergillus antigens in human urine from adults (>= 18 years old) with suspected invasive aspergillosis (IA). The assay is not intended for use in lung transplant recipients.

The results should be interpreted by trained healthcare professionals, incorporating other diagnostic procedures such as microbiological culture, histological examination of biopsy samples, and radiographic evidence to support the diagnosis of IA.

C Special Conditions for Use Statement(s):

Rx - For Prescription Use Only

There is a risk of false positive results due to the presence of other Ascomycetes fungi in urine specimens. Cross-reactivity has been observed with the MycoMEIA Aspergillus Assay with infections caused by other Ascomycetes fungi (Histoplasma, Blastomyces, Fusarium).

There is a risk of false positive results in the presence of some interferents in urine specimens. Cross-reactivity has been observed at high concentrations of betanin, bilirubin, caffeine, Gyne-Lotrimin cream (clotrimazole), hemoglobin, Lotrimin Ultra cream (butenifine), Monistat 7 cream (miconazole), Plasma-Lyte, Vaginal contraceptive gel, Vagisil cream (benzocaine), and Vagistat 1 cream (ticonazole).

D Special Instrument Requirements:

The assay is for use with the MycoMEIA Calculation Worksheet, an Excel spreadsheet to manually enter sample values to determine test results.

IV Device/System Characteristics:

A Device Description:

The MycoMEIA Aspergillus Assay (MycoMEIA) is an enzyme immunoassay (EIA) that detects Aspergillus galactofuranose (galf)-containing antigens in urine. The urine sample is first processed through Sample Processing Columns to eliminate an inhibitor of antibody-antigen recognition. The processed urine samples eluted from the columns are added to the assay plate wells coated with the antibodies and incubated. The bound antigen is then incubated with antibodies linked to horseradish peroxidase (HRP) conjugates. Wash steps between each reaction remove unbound material. Next, the peroxidase substrate solution is added, which reacts with the complexes bound to the well to form a blue color reaction. The enzyme reaction is stopped by the addition of acid, which also changes the blue color to yellow. The absorbance (optical density) of specimens and controls is determined with a spectrophotometer set at 450 and 620 nm wavelengths.

External quality controls are required and provided in the kit, to validate assay results and determine the index values that are calculated to determine test results. The amount of antigen in the clinical sample is determined by optical density (OD) using a spectrophotometer and interpreted as an OD index relative to the mean OD of the threshold control. The index values are determined manually or by entering the test sample and control spectrophotometer readings

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into the MycoMEIA Calculation Worksheet, an Excel spreadsheet that is provided to users and calculates the index values to determine the qualitative test result, positive or negative. If any of the controls are outside of the acceptable range, the test is invalid. For positive results with index values of $\geq 0.6$ to $< 0.7$ and invalid results, the user is instructed to repeat the testing.

The assay kit includes 96-well microtiter plates with pre-coated mAbs, column washing solution, HRP-conjugated mAbs, conjugate diluent, a peroxidase TMB substrate (chromogen), a stopping solution, and three controls, Negative, Positive and Threshold. 25X Concentrated Wash Solution and Sample Processing Columns are required to perform the assay and are provided separately from the kit.

# B Principle of Operation:

The MycoMEIA Aspergillus Assay (MycoMEIA) is based on a sandwich enzyme immunoassay (EIA) technology. The assay uses two mouse monoclonal antibodies (mAbs) that recognize galactofuranose (galf) epitopes and are coated to 96-well microplates. Both mAbs are used as capture and reporter antibodies that bind the antigen, and the bound antigen is detected with the same two antibodies conjugated to horseradish peroxidase.

# V Substantial Equivalence Information:

# A Predicate Device Name(s):

Platelia Aspergillus EIA Model 62793

# B Predicate 510(k) Number(s):

K093678

# C Comparison with Predicate(s):

| Device & Predicate Device(s): | K243496 (This device) | K093678 (Predicate) |
| --- | --- | --- |
| Device Trade Name | MycoMEIA Aspergillus Assay | Platelia Aspergillus EIA |
| Intended Use/Indications For Use | The MycoMEIA Aspergillus Assay (MycoMEIA) is an enzyme immunoassay (EIA) for the in vitro qualitative detection of Aspergillus antigens in human urine from adults (>= 18 years old) with suspected invasive aspergillosis (IA). The assay is not intended for use in lung transplant recipients. The results should be interpreted by trained healthcare professionals, incorporating other diagnostic procedures such as microbiological culture, histological examination of biopsy samples, and radiographic evidence to | The Platelia Aspergillus Ag EIA is an immunoenzymatic sandwich microplate assay for the detection of Aspergillus galactomannan antigen in adult and pediatric serum and Bronchoalveolar Lavage (BAL) fluid samples. The Platelia Aspergillus 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 Invasive Aspergillosis. |

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VI Standards/Guidance Documents Referenced:

| Document # | Title |
| --- | --- |
| CLSI EP05-A3 | Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline —Third Edition |
| CLSI EP07 3rd Edition | Interference Testing in Clinical Chemistry |
| CLSI EP12 3rd Edition | Evaluation of Qualitative Binary Output Examination Performance |
| CLSI EP17-A2 | Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline - Second Edition |
| CLSI EP25-A | Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline |

VII Performance Characteristics (if/when applicable):

Analytical Performance:

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# 1. Precision/Reproducibility:

Precision/reproducibility was conducted according to CLSI EP05-A3 $^{1}$ to assess the performance of the MycoMEIA Aspergillus Assay (MycoMEIA).

# Multi-Site Precision

A multisite precision study was conducted at three sites (one internal and two external) on a panel of eight samples: three assay controls, one negative sample consisting of pooled human urine, two samples of pooled human urine spiked at 1x and 2x the limit of detection (LoD) with Aspergillus antigen, and two positive clinical samples consisting of pooled human urine. Testing was conducted on one lot of the assay and accessories and one set of instruments. Ninety (90) data points were generated for each sample: 3 replicates per run $\times$ 2 runs/operators per day $\times$ 5 days $\times$ 3 sites $= 90$ data points. Acceptance criteria are $\leq 0.025$ SD for negative samples and $\leq 25\%$ CV for positive samples, and results were as expected. Results are summarized in Table 1 below.

Table 1. Summary Results of Multisite Precision Study for All Sites (N=90)

| Sample | Optical Density Precision | | | MycoMEIA Index Precision | | |
| --- | --- | --- | --- | --- | --- | --- |
| | Mean OD | SD | %CV | Mean MI | SD | %CV |
| 1 Positive Control | 1.580 | 0.213 | 13 | 19.0 | 2.470 | 13 |
| 2 Threshold Control | 0.515 | 0.104 | 20 | 6.2 | 1.077 | 17 |
| 3 Negative Control | 0.024 | 0.011 | NA | 0.3 | 0.123 | NA |
| 4 Negative Sample | 0.031 | 0.012 | NA | 0.4 | 0.138 | NA |
| 5 Contrived Low Positive Sample | 0.080 | 0.019 | 24 | 1.0 | 0.210 | 22 |
| 6 Contrived Positive Sample | 0.142 | 0.031 | 22 | 1.7 | 0.318 | 19 |
| 7 Pooled Positive Sample | 0.084 | 0.016 | 19 | 1.0 | 0.186 | 18 |
| 8 Pooled Positive Sample | 0.207 | 0.029 | 14 | 2.5 | 0.297 | 12 |

Sample 1= Assay Positive Control, Sample 2= Assay Threshold Control, Sample 3= Assay Negative Control, Sample 4= Pooled Negative Sample, Sample 5= Contrived Low Positive Sample containing $2.5\mathrm{ng / mL}$ Aspergillus antigen ( $\sim 1x$ LoD), Sample 6= Contrived Positive Sample containing $5\mathrm{ng / mL}$ Aspergillus antigen ( $\sim 2x$ LoD), Sample 7= Pooled Positive Sample, Sample 8= Pooled Positive Sample, SD= Standard Deviation, $\% \mathrm{CV} =$ Percent Coefficient of Variation.

# Repeatability and Within-Laboratory Precision

A single-site precision study was conducted at one internal site on a panel of eight samples including three assay controls, one negative sample consisting of pooled human urine, two samples of pooled human urine spiked at 1x and 2x the limit of detection (LoD) with Aspergillus antigen, and two positive clinical samples consisting of pooled human urine. Testing was conducted on three lots of the assay. Eighty (80) data points were generated per lot: 2 replicates per run $\times$ 2 runs/operators per day $\times$ 20 days $= 80$ data points for a total of 240 data points. Acceptance criteria are $\leq 0.010$ SD for negative samples and $\leq 10\%$ CV for positive samples for Repeatability, and $\leq 0.025$ SD for negative samples and $\leq 25\%$ CV for

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positive samples for Within-Lab Precision. Results were as expected except for Sample 3 in Lot 2, which was outside the acceptance criteria for both Repeatability and Within-Lab Precision and Sample 5 in Lot 2, which was outside the acceptance criteria for Repeatability. The data are acceptable. Results are summarized in Tables 2 and 3 below.

Table 2. Optical Density (OD) Values and Repeatability Results by Lot (N=80)

| Sample | Lot 1 | | | Lot 2 | | | Lot 3 | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Mean OD | SD | %CV | Mean OD | SD | %CV | Mean OD | SD | %CV |
| 1 PC | 1.801 | 0.0603 | 3 | 1.645 | 0.0447 | 3 | 1.909 | 0.0899 | 5 |
| 2 TC | 0.620 | 0.0547 | 9 | 0.493 | 0.0454 | 9 | 0.603 | 0.0484 | 8 |
| 3 NC | 0.018 | 0.0016 | NA | 0.025 | 0.0316 | NA | 0.019 | 0.0019 | NA |
| 4 Negative Sample | 0.025 | 0.0029 | NA | 0.024 | 0.0014 | NA | 0.022 | 0.0020 | NA |
| 5 Contrived Low Positive Sample | 0.080 | 0.0056 | 7 | 0.069 | 0.0118 | 17 | 0.065 | 0.0066 | 10 |
| 6 Contrived Positive Sample | 0.158 | 0.0131 | 8 | 0.128 | 0.0096 | 8 | 0.118 | 0.0058 | 5 |
| 7 Pooled Positive Sample | 0.116 | 0.0069 | 6 | 0.126 | 0.0081 | 7 | 0.135 | 0.0081 | 6 |
| 8 Pooled Positive Sample | 0.256 | 0.0109 | 4 | 0.225 | 0.0135 | 7 | 0.238 | 0.0095 | 4 |

Sample 1= Assay Positive Control, Sample 2= Assay Threshold Control, Sample 3= Assay Negative Control, Sample 4= Pooled Negative Sample, Sample 5= Contrived Low Positive Sample containing $2.5\mathrm{ng / mL}$ Aspergillus antigen $(\sim 1\times$ LoD), Sample 6= Contrived Positive Sample containing $5\mathrm{ng / mL}$ Aspergillus antigen $(\sim 2\times$ LoD), Sample 7= Pooled Positive Sample, Sample 8= Pooled Positive Sample, SD= Standard Deviation, $\% \mathrm{CV} =$ Percent Coefficient of Variation.

Table 3. Optical Density (OD) Values and Within-Lab Precision Results by Lot (N=80)

| Sample | Lot 1 | | | Lot 2 | | | Lot 3 | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Mean OD | SD | %CV | Mean OD | SD | %CV | Mean OD | SD | %CV |
| 1 PC | 1.801 | 0.1597 | 9 | 1.645 | 0.1143 | 7 | 1.909 | 0.1625 | 9 |
| 2 TC | 0.620 | 0.0965 | 16 | 0.493 | 0.0747 | 15 | 0.603 | 0.0798 | 13 |
| 3 NC | 0.018 | 0.0039 | NA | 0.025 | 0.0318 | NA | 0.019 | 0.0035 | NA |
| 4 Negative Sample | 0.025 | 0.0051 | NA | 0.024 | 0.0023 | NA | 0.022 | 0.0031 | NA |
| 5 Contrived Low Positive Sample | 0.080 | 0.0125 | 16 | 0.069 | 0.0140 | 20 | 0.065 | 0.0085 | 13 |
| 6 Contrived Positive Sample | 0.158 | 0.0214 | 14 | 0.128 | 0.0166 | 13 | 0.118 | 0.0130 | 11 |
| 7 Pooled Positive Sample | 0.116 | 0.0109 | 9 | 0.126 | 0.0137 | 11 | 0.135 | 0.0133 | 10 |
| 8 Pooled Positive Sample | 0.256 | 0.0430 | 17 | 0.225 | 0.0494 | 22 | 0.238 | 0.0382 | 16 |

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Sample 1= Assay Positive Control, Sample 2= Assay Threshold Control, Sample 3= Assay Negative Control, Sample 4= Pooled Negative Sample, Sample 5= Contrived Low Positive Sample containing $2.5\mathrm{ng / mL}$ Aspergillus antigen ( $\sim 1x$ LoD), Sample 6= Contrived Positive Sample containing $5\mathrm{ng / mL}$ Aspergillus antigen ( $\sim 2x$ LoD), Sample 7= Pooled Positive Sample, Sample 8= Pooled Positive Sample, SD= Standard Deviation, $\% \mathrm{CV} =$ Percent Coefficient of Variation.

# 2. Linearity:

This study is not applicable as the test device is a qualitative assay.

# 3. Analytical Specificity/Interference:

The MycoMEIA Aspergillus Assay (MycoMEIA) was evaluated for analytical specificity/ cross-reactivity and interference in accordance with CLSI EP07².

# 1. Cross-Reactivity and Microbial Interference (Analytical Specificity)

The MycoMEIA was evaluated for microbial cross-reactivity. Clinical urine samples were obtained from individuals diagnosed with infectious diseases but known to be free of Aspergillus infection. Additional samples were contrived from pooled human urine that was negative for Aspergillus infection and spiked with other infectious agents.

Acceptance criteria are that all samples should yield the expected negative results, with MycoMEIA index values $< 0.6$ . Table 4 shows the results for microbial cross-reactivity from replicate testing. Cross-reacting positive results are shown in red font. Cross-reactivity is observed with Ascomycetes fungi (Histoplasma, Blastomyces, Fusarium). The observed cross-reactivity is noted in the device labeling.

Table 4. Summary Results of Microbial Cross-reactivity Study

| Sample Description | Organism / Concentration Tested | MycoMEIA Index Value | | Interpretation | |
| --- | --- | --- | --- | --- | --- |
| | | Replicate 1 | Replicate 2 | Replicate 1 | Replicate 2 |
| Clinical urine sample | UTI Enterococcus spp., Enterobacter lung / blood | 0.5 | 0.5 | Negative | Negative |
| Clinical urine sample | UTI Streptococcus spp. | 0.3 | 0.3 | Negative | Negative |
| Clinical urine sample | UTI Staphylococcus spp. | 0.3 | 0.4 | Negative | Negative |
| Clinical urine sample | Klebsiella lung / blood | 0.3 | 0.2 | Negative | Negative |
| Clinical urine sample | E. coli lung / blood | 0.3 | 0.3 | Negative | Negative |
| Clinical urine sample | Possible IA + UTI Enterococcus, Enterococcus lung / blood | 0.6* | 0.6* | Positive* | Positive* |
| Clinical urine sample | Enterobacter lung / blood, Streptococcus lung / blood | 0.2 | 0.2 | Negative | Negative |
| Clinical urine sample | Streptococcus lung / blood | 0.3 | 0.2 | Negative | Negative |
| Clinical urine sample | UTI Mixed gram positive | 0.3 | 0.2 | Negative | Negative |
| Clinical urine sample | UTI Staphylococcus spp. | 0.5 | 0.5 | Negative | Negative |
| Clinical urine sample | UTI Streptococcus spp. | 3.9* | 3.8* | Positive* | Positive* |
| Clinical urine sample | Acinetobacter junii lung / blood, URI - Rhinovirus, Parainfluenzavirus | 0.2 | 0.3 | Negative | Negative |
| Clinical urine sample | Streptococcus lung / blood | 0.4 | 0.4 | Negative | Negative |
| Clinical urine sample | MSSA, Staphylococcus lung / blood; URI - Influenza | 0.3 | 0.3 | Negative | Negative |
| Clinical urine sample | Staphylococcus lung / blood | 0.2 | 0.3 | Negative | Negative |
| Clinical urine sample | P. aeruginosa lung / blood | 0.3 | 0.3 | Negative | Negative |
| Clinical urine sample | MRSA, Staphylococcus lung / blood | 0.2 | 0.3 | Negative | Negative |
| Clinical urine sample | Candida krusei | 0.2 | 0.2 | Negative | Negative |
| Clinical urine sample | Candida krusei | 0.2 | 0.2 | Negative | Negative |
| Clinical urine sample | MRSA, Staphylococcus lung / blood | 0.2 | 0.2 | Negative | Negative |
| Clinical urine sample | URI - Rhinovirus, Parainfluenzavirus | 0.2 | 0.3 | Negative | Negative |
| Clinical urine sample | E. coli lung / blood | 0.1 | 0.1 | Negative | Negative |

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| Sample Description | Organism / Concentration Tested | MycoMEL4 Index Value | | Interpretation | |
| --- | --- | --- | --- | --- | --- |
| | | Replicate 1 | Replicate 2 | Replicate 1 | Replicate 2 |
| Clinical urine sample | Other IFI lung / blood | 0.2 | 0.2 | Negative | Negative |
| Clinical urine sample | MRSA, Staphylococcus lung / blood | 0.3 | 0.3 | Negative | Negative |
| Clinical urine sample | MRSA, Staphylococcus lung / blood | 0.3 | 0.5 | Negative | Negative |
| Clinical urine sample | Histoplasmosis | 0.2 | 0.2 | Negative | Negative |
| Clinical urine sample | Candida albicans (candidiasis) | 0.2 | 0.2 | Negative | Negative |
| Clinical urine sample | Pneumocystis jirovecii (pneumonia) | 0.2 | 0.3 | Negative | Negative |
| Clinical urine sample | Other IFI lung / blood (cryptococcosis) | 0.5 | 0.5 | Negative | Negative |
| Clinical urine sample | Histoplasmosis | 0.3 | 0.3 | Negative | Negative |
| Clinical urine sample | Histoplasmosis | 0.3 | 0.3 | Negative | Negative |
| Clinical urine sample | Histoplasmosis | 0.5 | 0.4 | Negative | Negative |
| Clinical urine sample | Pneumocystis jirovecii (pneumonia) | 0.3 | 0.3 | Negative | Negative |
| Clinical urine sample | Candidiasis | 0.2 | 0.2 | Negative | Negative |
| Clinical urine sample | Staphylococcus lung / blood | 0.3 | 0.3 | Negative | Negative |
| Clinical urine sample | P. aeruginosa lung / blood | 0.2 | 0.2 | Negative | Negative |
| Clinical urine sample | Pneumocystis jirovecii (pneumonia) | 0.3 | 0.3 | Negative | Negative |
| Clinical urine sample | MRSA, Stenotrophomonas lung / blood | 0.4 | 0.4 | Negative | Negative |
| Clinical urine sample | P. aeruginosa lung / blood | 0.4 | 0.3 | Negative | Negative |
| Clinical urine sample | Fusariosis lung / blood | 0.2 | 0.2 | Negative | Negative |
| Clinical urine sample | P. aeruginosa lung / blood | 0.2 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Acinetobacter baumanii 4.00 x 105 CFU/mL | 0.3 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Bacteroides fragilis 1.01 x 106 CFU/mL | 0.3 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Candida albicans 1.73 x 106 CFU/mL | 0.3 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Candida glabrata 1.91 x 106 CFU/mL | 0.3 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Candida parapsilosis 3.06 x 106 CFU/mL | 0.3 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Candida tropicalis 1.57 x 106 CFU/mL | 0.2 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Chlamydia trachomatis 7.00 x 105 CFU/mL | 0.3 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Citrobacter freundii 6.70 x 105 CFU/mL | 0.2 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Clostridia 5.28 x 105 CFU/mL | 0.2 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Corynebacterium amycolatum 2.80 x 105 CFU/mL | 0.1 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Cryptococcus neoformans 1.25 x 106 CFU/mL | 0.2 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Enterobacter cloacae 1.65 x 106 CFU/mL | 0.1 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Enterococcus faecalis 1.40 x 106 CFU/mL | 0.3 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Escherichia coli 1.15 x 106 CFU/mL | 0.2 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Geotrichum 2.80 x 105 CFU/mL | 0.2 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Klebsiella pneumoniae 6.90 x 105 CFU/mL | 0.2 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Lactobacillus crispatus 2.80 x 105 CFU/mL | 0.2 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Neisseria gonorrhoeae 3.90 x 105 CFU/mL | 0.4 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Parainfluenza virus Type 1 1.00 x 106 virus/mL | 0.2 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Parainfluenza virus Type 2 5.00 x 105 virus/mL | 0.2 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Parainfluenza virus Type 3 | 0.2 | 0.2 | Negative | Negative |

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| Sample Description | Organism / Concentration Tested | MycoMEIA Index Value | | Interpretation | |
| --- | --- | --- | --- | --- | --- |
| | | Replicate 1 | Replicate 2 | Replicate 1 | Replicate 2 |
| | 1.00 x 10^6 virus/mL | | | | |
| Pooled, spiked urine | Peptostreptococci 6.84 x 10^5 CFU/mL | 0.3 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Prevotella bivia 1.84 x 10^6 CFU/mL | 0.2 | 0.1 | Negative | Negative |
| Pooled, spiked urine | Proteus mirabilis 1.35 x 10^6 CFU/mL | 0.3 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Pseudomonas aeruginosa 1.10 x 10^6 CFU/mL | 0.2 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Rhinovirus 2 2.00 x 10^6 virus/mL | 0.1 | 0.1 | Negative | Negative |
| Pooled, spiked urine | Rhinovirus 83 4.50 x 10^5 virus/mL | 0.2 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Serratia marcescens 1.10 x 10^6 CFU/mL | 0.3 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Staphylococcus aureus 1.37 x 10^6 CFU/mL | 0.3 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Staphylococcus epidermidis 5.80 x 10^5 CFU/mL | 0.1 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Staphylococcus saprophyticus subsp. saprophyticus 2.60 x 10^5 CFU/mL | 0.2 | 0.2 | Negative | Negative |
| Pooled, spiked urine | Stenotrophomonas maltophilia 2.00 x 10^6 CFU/mL | 0.2 | 0.3 | Negative | Negative |
| Pooled, spiked urine | Streptococcus agalactiae 5.80 x 10^5 CFU/mL | 0.2 | 0.3 | Negative | Negative |
| Cross-reactants for which cross-reactivity was observed | | | | | |
| Clinical urine sample | URI - Rhinovirus, Parainfluenzavirus | 0.6 | 0.5 | Positive | Negative |
| Clinical urine sample | Streptococcus lung / blood | 0.6 | 0.67 | Positive | Positive |
| Clinical urine sample | Histoplasmosis | 18.3 | 18.9 | Positive | Positive |
| Clinical urine sample | Blastomyces lung / blood | 0.6 | 0.6 | Positive | Positive |
| Clinical urine sample | Candidiasis | 0.6 | 0.6 | Positive | Positive |
| Pooled, spiked urine | Fusarium 8.80 x 10^5 CFU/mL | 1.0 | 1.0 | Positive | Positive |

* Two clinical samples were identified as possible invasive aspergillosis.

Microbial interference was also evaluated. Samples were prepared using pooled negative urine that was spiked with Aspergillus to a final concentration of 8 ng/mL (prepared from a stock of ethanol precipitate (EP) of whole organism) and spiked with other infectious agents. Acceptance criteria are that all samples should yield the expected positive results, with MycoMEIA index values ≥ 0.6. No microbial interference was observed.

2. Endogenous/Exogenous Interference and Cross-reactivity

An interfering substances study was conducted to assess the performance of the MycoMEIA in the presence of potentially interfering substances. Samples were contrived from pooled human urine that was negative for Aspergillus and spiked with endogenous and exogenous materials. In addition, clinical urine samples were obtained from individuals with other disorders but known to be free of Aspergillus infection. Both cross-reactivity and interference were evaluated in the presence or absence of Aspergillus antigen. Acceptance criteria are that all samples should yield the expected results. Negative samples should yield MycoMEIA index values of < 0.6, which is the expected result for samples where Aspergillus antigen is absent. Positive samples should yield index values of ≥ 0.6 which is the expected result for samples where Aspergillus antigen is present. Table 5 lists the interferents and concentrations for which no cross-reactivity or interference was observed.

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Table 5. Summary Results of Interfering Substances Testing

| Potential Interferent | Concentration Tested | Aspergillus Ag Added | MycoMEIA Index Value | | Interpretation | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Replicate 1 | Replicate 2 | Replicate 1 | Replicate 2 |
| 1-Methylnicotinamide (Vitamin B3 metabolite) | 0.130 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 1.8 | 1.5 | Positive | Positive |
| Acetaminophen | 2.5 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 3.0 | 2.7 | Positive | Positive |
| Acetone (Ketone) | 2.4 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 1.9 | 1.9 | Positive | Positive |
| Albumin | 10 mg/mL | No | 0.2 | 0.2 | Negative | Negative |
| | | Yes | 1.7 | 1.7 | Positive | Positive |
| Amoxicillin | 1.59 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 1.9 | 2.1 | Positive | Positive |
| Amphotericin B | 0.22 mg/mL | No | 0.4 | 0.3 | Negative | Negative |
| | | Yes | 3.0 | 3.0 | Positive | Positive |
| Ascorbic acid 1 (Vitamin C) | 0.5 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 1.5 | 1.5 | Positive | Positive |
| Ascorbic acid 2 (Vitamin C) | 1 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 1.6 | 1.8 | Positive | Positive |
| Azithromycin | 0.15 mg/mL | No | 0.4 | 0.5 | Negative | Negative |
| | | Yes | 1.7 | 1.8 | Positive | Positive |
| Biotin (Vitamin B) | 0.4 μg/mL | No | 0.4 | 0.3 | Negative | Negative |
| | | Yes | 1.6 | 1.7 | Positive | Positive |
| Boric Acid | 7.89 mg/mL | No | 0.4 | 0.3 | Negative | Negative |
| | | Yes | 2.0 | 1.8 | Positive | Positive |
| CD14 (contrived for RA) | 3 μg/mL | No | 0.2 | 0.2 | Negative | Negative |
| | | Yes | 2.0 | 1.8 | Positive | Positive |
| Cimetidine | 0.9 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 1.8 | 1.6 | Positive | Positive |
| Ciprofloxacin | 1.2 mg/mL | No | 0.4 | 0.4 | Negative | Negative |
| | | Yes | 2.3 | 2.0 | Positive | Positive |
| Erythromycin | 0.675 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 6.5 | 7.0 | Positive | Positive |
| Ethanol | 30 mg/mL | No | 0.5 | 0.5 | Negative | Negative |
| | | Yes | 2.3 | 2.4 | Positive | Positive |
| Ganciclovir hydrate | 0.9 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 2.0 | 2.0 | Positive | Positive |
| Glucose | 6 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 1.5 | 1.5 | Positive | Positive |
| Human chorionic gonadotropin (HCG) | 0.3 μg/mL | No | 0.2 | 0.2 | Negative | Negative |
| | | Yes | 1.7 | 1.8 | Positive | Positive |
| Ibuprofen | 0.27 mg/mL | No | 0.4 | 0.3 | Negative | Negative |
| | | Yes | 2.4 | 2.3 | Positive | Positive |
| Immunoglobulin G (contrived for hypergammaglobulinemia) | 2 mg/mL | No | 0.4 | 0.4 | Negative | Negative |
| | | Yes | 1.4 | 1.4 | Positive | Positive |
| Itraconazole | 0.22 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 2.1 | 2.2 | Positive | Positive |
| Metronidazole | 1 mg/mL | No | 0.2 | 0.3 | Negative | Negative |
| | | Yes | 2.0 | 2.0 | Positive | Positive |
| Mucin | 5% w/v | No | 0.4 | 0.4 | Negative | Negative |
| | | Yes | 0.7 | 0.7 | Positive | Positive |
| Mucin-5B | 0.1% w/v | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 2.4 | 2.7 | Positive | Positive |
| Naproxen sodium | 0.02 mg/mL | No | 0.4 | 0.3 | Negative | Negative |
| | | Yes | 2.0 | 1.9 | Positive | Positive |
| Nicotine (contrived for tobacco) | 0.015 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 2.4 | 2.4 | Positive | Positive |
| Orosomucoid 2 (contrived for RA) | 0.2 μg/mL | No | 0.3 | 0.2 | Negative | Negative |
| | | Yes | 2.1 | 2.2 | Positive | Positive |
| Oxalic acid 1 (contrived for | 0.1 mg/mL | No | 0.3 | 0.2 | Negative | Negative |

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K243496 - Page 11 of 20
| Potential Interferent | Concentration Tested | Aspergillus Ag Added | MycoMEIA Index Value | | Interpretation | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Replicate 1 | Replicate 2 | Replicate 1 | Replicate 2 |
| spinach) | | Yes | 1.4 | 1.3 | Positive | Positive |
| Oxalic acid 2 (contrived for spinach) | 1 mg/mL | No | 0.2 | 0.2 | Negative | Negative |
| | | Yes | 1.4 | 1.6 | Positive | Positive |
| Penicillin G sodium salt | 3.2 mg/mL | No | 0.3 | 0.2 | Negative | Negative |
| | | Yes | 1.4 | 1.5 | Positive | Positive |
| Phenazopyridine HCl (Pyridium) | 1.2 µg/mL | No | 0.2 | 0.2 | Negative | Negative |
| | | Yes | 1.7 | 1.5 | Positive | Positive |
| Phylloquinone (Vitamin K1) | 0.05 µg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 2.1 | 2.0 | Positive | Positive |
| Piperacillin sodium salt | 19 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 2.2 | 2.8 | Positive | Positive |
| Potassium clavulanate (contrived for Augmentin) | 0.65 mg/mL | No | 0.2 | 0.2 | Negative | Negative |
| | | Yes | 1.6 | 1.6 | Positive | Positive |
| RBC (Red Blood Cells / Erythrocytes) | 2% v/v | No | 0.5 | 0.5 | Negative | Negative |
| | | Yes | 2.7 | 3.2 | Positive | Positive |
| Riboflavin (Vitamin B2) | 0.24 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 2.3 | 2.2 | Positive | Positive |
| Rifampicin | 10 mg/mL | No | 0.4 | 0.3 | Negative | Negative |
| | | Yes | 2.1 | 1.8 | Positive | Positive |
| Sodium salicylate (metabolite of acetylsalicylic acid) | 4.73 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 1.4 | 1.4 | Positive | Positive |
| Tazobactam sodium salt | 17 mg/mL | No | 0.4 | 0.4 | Negative | Negative |
| | | Yes | 2.2 | 1.8 | Positive | Positive |
| Triglycerides 1 | 1 mg/mL | No | 0.2 | 0.2 | Negative | Negative |
| | | Yes | 1.5 | 1.6 | Positive | Positive |
| Triglycerides 2 | 0.3 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 1.4 | 1.3 | Positive | Positive |
| Urobilinogen | 0.06 mg/mL | No | 0.2 | 0.2 | Negative | Negative |
| | | Yes | 1.4 | 1.7 | Positive | Positive |
| Vancomycin HCl | 3.1 mg/mL | No | 0.4 | 0.3 | Negative | Negative |
| | | Yes | 1.8 | 1.4 | Positive | Positive |
| Voriconazole N-oxide | 0.022 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 2.4 | 2.4 | Positive | Positive |
| Vanillylmandelic acid | 0.03 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 1.8 | 1.8 | Positive | Positive |
| Water-based personal lubricant | 50 mg/mL | No | 0.4 | 0.3 | Negative | Negative |
| | | Yes | 1.6 | 1.4 | Positive | Positive |
| WBC (White Blood Cells / Leukocytes) | 300,000 cells/mL | No | 0.2 | 0.3 | Negative | Negative |
| | | Yes | 1.4 | 1.4 | Positive | Positive |
| α1-Acid glycoprotein (contrived for RA) | 0.6 µg/mL | No | 0.2 | 0.2 | Negative | Negative |
| | | Yes | 2.2 | 2.0 | Positive | Positive |
| α-CEHC (metabolite of Vitamin E) | 5 µg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 2.0 | 2.0 | Positive | Positive |
| β-carotene (parent compound of Vitamin A) | 3 mg/mL | No | 0.3 | 0.3 | Negative | Negative |
| | | Yes | 2.1 | 2.1 | Positive | Positive |
| High pH urine | Contrived by adding NaOH to normal urine (pH 10.01) | No | 0.2 | 0.2 | Negative | Negative |
| | | Yes | 1.4 | 1.5 | Positive | Positive |
| Low pH urine | Contrived by adding HCl to normal urine (pH 3.82) | No | 0.2 | 0.2 | Negative | Negative |
| | | Yes | 1.7 | 1.8 | Positive | Positive |
| Clinical urine | Protein 30 mg/dL, pH 8.0, Hemolyzed blood 200 Ery/µL, Ketones > 160mg /dL, Bilirubin +++, Glucose 250mg/dL | No | 0.4 | 0.3 | Negative | Negative |
| | | Yes | 1.6 | 1.5 | Positive | Positive |
| Clinical urine | pH 7.51 Bilirubin +++ | No | 0.4 | 0.4 | Negative | Negative |
| | | Yes | 1.9 | 1.8 | Positive | Positive |
| Clinical urine | pH 5.16 | No | 0.4 | 0.4 | Negative | Negative |
| | | Yes | 2.3 | 2.6 | Positive | Positive |
| Clinical urine | Protein 100 mg/dL | No | 0.2 | 0.2 | Negative | Negative |

{11}

Potential cross-reacting interferents that gave positive results in the absence of Aspergillus antigen at the initial concentrations tested were further investigated, and additional testing determined that the assay yields the expected negative result when the substance was tested at lower concentrations. Results are shown in Table 6. Results for concentrations that still show cross-reactivity at lower dilutions are noted in red font indicating the expected negative result was not obtained. The observed interference at high concentrations is noted in the device labeling.

Table 6. Results of Additional Interfering Substances Testing

| Potential Interferent / Sample | Concentration Tested | Mean MycoMEIA Index Value | Interpretation |
| --- | --- | --- | --- |
| Betanin | 0.00% w/v | 0.2 | Negative |
| | 0.05 mg/mL | 0.2 | Negative |
| | 0.25% w/v | 1.1 | Positive |
| | 0.50% w/v | 2.0 | Positive |
| | 0.75% w/v | 2.9 | Positive |
| | 1.00% w/v | 4.0 | Positive |
| Bilirubin | 0.00 mg/mL | 0.2 | Negative |
| | 0.03 mg/mL | 0.2 | Negative |
| | 0.06 mg/mL | 0.6 | Positive |
| | 0.09 mg/mL | 0.8 | Positive |
| | 0.12 mg/mL | 1.1 | Positive |
| Caffeine (in urine) | 0.00 mg/mL | 0.2 | Negative |
| | 0.16 mg/mL | 0.2 | Negative |
| | 3.375 mg/mL | 0.5 | Negative |
| | 6.750 mg/mL | 0.8 | Positive |
| | 10.125 mg/mL | 1.3 | Positive |
| | 13.50 mg/mL | 1.7 | Positive |
| Gyne-Lotrimin cream (clotrimazole) | 0.00% w/v | 0.2 | Negative |
| | 0.14% w/v | 0.2 | Negative |
| | 1.25% w/v | 0.5 | Negative |
| | 2.50% w/v | 0.8 | Positive |
| | 3.75% w/v | 1.3 | Positive |
| | 5.00% w/v | 1.9 | Positive |
| Hemoglobin | 0.00 mg/mL | 0.2 | Negative |
| | 0.15 mg/mL | 0.4 | Negative |
| | 1.05 mg/mL | 0.4 | Negative |
| | 2.10 mg/mL | 0.5 | Negative |
| | 3.15 mg/mL | 0.5 | Negative |
| | 4.20 mg/mL | 0.7 | Positive |
| Lotrimin Ultra cream (butenifine) | 0.00% w/v | 0.3 | Negative |
| | 0.14% w/v | 0.2 | Negative |
| | 1.25% w/v | 0.8 | Positive |
| | 2.50% w/v | 0.9 | Positive |
| | 3.75% w/v | 1.3 | Positive |
| | 5.00% w/v | 1.8 | Positive |
| Monistat 7 cream (miconazole) | 0.00% w/v | 0.3 | Negative |
| | 0.14% w/v | 0.2 | Negative |

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| Potential Interferent / Sample | Concentration Tested | Mean MycoMEIA Index Value | Interpretation |
| --- | --- | --- | --- |
| | 1.25% w/v | 0.7 | Positive |
| | 2.50% w/v | 1.0 | Positive |
| | 3.75% w/v | 1.3 | Positive |
| | 5.00% w/v | 1.9 | Positive |
| Nitrites | 0.00 mg/mL | 0.3 | Negative |
| | 0.04 mg/mL | 0.4 | Negative |
| | 0.0750 mg/mL | 0.8 | Positive |
| | 0.1125 mg/mL | 1.0 | Positive |
| | 0.1500 mg/mL | 1.3 | Positive |
| Plasma-lyte A | 0.0% v/v | 0.3 | Negative |
| | 7.5% v/v | 0.5 | Negative |
| | 10% v/v | 0.7 | Positive |
| | 22.5% v/v | 2.3 | Positive |
| | 45.0% v/v | 5.4 | Positive |
| | 67.5% v/v | 9.3 | Positive |
| | 90.0% v/v | 14.0 | Positive |
| Vaginal contraceptive gel | 0.00% w/v | 0.3 | Negative |
| | 0.14% w/v | 0.2 | Negative |
| | 1.25% w/v | 0.9 | Positive |
| | 2.50% w/v | 1.5 | Positive |
| | 3.75% w/v | 1.5 | Positive |
| | 5.00% w/v | 2.1 | Positive |
| Vaginal cream (benzocaine) | 0.00% w/v | 0.2 | Negative |
| | 0.14% w/v | 0.2 | Negative |
| | 1.25% w/v | 0.6 | Positive |
| | 2.50% w/v | 1.1 | Positive |
| | 3.75% w/v | 2.1 | Positive |
| | 5.00% w/v | 2.8 | Positive |
| Vaginal 1 cream (ticonazole) | 0.00% w/v | 0.3 | Negative |
| | 0.14% w/v | 0.2 | Negative |
| | 1.25% w/v | 1.0 | Positive |
| | 2.50% w/v | 1.8 | Positive |
| | 3.75% w/v | 3.3 | Positive |
| | 5.00% w/v | 5.0 | Positive |
| Clinical urine (High Nitrites) | NA | 0.2 | Negative |

3. High-dose Hook Effect

A high-dose hook effect study was conducted to evaluate if a hook effect occurs for the MycoMEIA Aspergillus Assay (MycoMEIA) when high levels of the target analyte are present in the test sample. Samples were prepared from pooled human urine spiked with Aspergillus antigen (Ethanol Precipitate (EP)) at 0 ng/mL, 125 ng/mL, 250 ng/mL, 500 ng/mL, and 1000 ng/mL; the LoD for the MycoMEIA is 3 ng/mL. No high-dose hook effect was observed.

4. Assay Reportable Range:

This study is not applicable as the test device is a qualitative assay.

5. Traceability, Stability and Expected Values (Controls, Calibrators, or Methods):

Sample Stability

Sample stability studies were conducted to demonstrate the stability of urine after refrigerated storage and after freezing and serial freeze-thaw cycles. One hundred twenty-four (124) hospitalized subjects with suspected aspergillosis were consented to provide urine,

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and 148 fresh samples were prospectively collected, aliquoted and tested in duplicate. Urines received in the clinical lab were tested fresh, within 6 hours of receipt, refrigerated for 5 days, and retested to compare results with that of fresh samples. Of 129 evaluable samples from 104 subjects that underwent paired testing of refrigerated storage over 5 days, 118 gave concordant positive or negative test results (118/129, 91%). From these samples, 13 positive samples (confirmed by MycoMEIA testing) were frozen at -70°C and underwent 5 freeze-thaw cycles. All remained positive through each freeze-thaw cycle (13/13, 100%). Additional testing was performed with 2 positive samples and 10 negative samples each from the different urine collection methods (clean-catch, catheter-collected, and non-sterile collection devices (e.g., bedpans, urine hats)). Samples were assessed at timepoints after 6 days of refrigeration followed by 4 freeze-thaw cycles. All samples showed ≥ 90% agreement compared to Day 0 results. These results demonstrate that freezing does not impact results and supports the use of refrigerated and frozen samples in analytical and clinical validations.

## Quality Controls

The MycoMEIA Aspergillus Assay (MycoMEIA) contains three external quality controls, Negative, Positive and Threshold Control. The Threshold Control (TC) mean optical density (OD) is calculated and should fall between 0.331 and 0.770. A TC mean OD outside the range defined above indicates that the assay results are invalid, and the user is instructed to repeat the assay. The TC determines the Index calculation used to normalize the assay results by utilizing a multiplication factor based on the TC mean OD values.

| Multiplication Factor | Mean Threshold OD |
| --- | --- |
| 5 | 0.331 – 0.440 |
| 6 | 1.441 – 0.550 |
| 7 | 0.551 – 0.660 |
| 8 | 0.661 – 0.770 |

The MycoMEIA Index values for Positive and Negative Controls are calculated using the equation shown below.

$$
\text{MycoMEIA Index} = \left( \frac{\text{Mean Control OD}}{\text{Mean TC OD}} \right) \times \text{Multiplication Factor}
$$

The MycoMEIA Index value of the Negative Control must be less than 0.6. The MycoMEIA Index of the Positive Control must be greater than 10.0. Negative and Positive Controls outside of the boundaries defined above indicate that the assay results are invalid, and the user is instructed to repeat the assay. Failure of any of the controls to meet the validity criteria described above upon repeat testing renders the assay invalid, and patient specimen results should not be reported. The controls were run as part of the analytical and clinical validations according to the Instructions for Use.

## 6. Detection Limit:

A study was conducted according to the CLSI EP17-A2³ to evaluate the limit of blank (LoB) and limit of detection (LoD) of the MycoMEIA Aspergillus Assay (MycoMEIA).

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LoB refers to the highest measurement result that is expected to be observed for a blank sample containing no analyte with a probability of 95%. LoB was confirmed by testing 30 clinical samples known to be free of Aspergillus infection (by prior testing with MycoMEIA and by clinical determination). Samples were pre-tested, aliquoted into single-use aliquots, and frozen. Each sample was tested on two different days with each of three assay lots, generating 60 results for each assay lot. Results were interpreted independently per assay lot and judged based on positive or negative clinical interpretations according to the MycoMEIA Index (Negative: MycoMEIA Index < 0.6, Positive: ≥ 0.6). One sample (Sample 26, Lot 2, Day 1) gave a false positive result. The LoB of 0 is confirmed.

LoD

LoD refers to the lowest concentration of target analyte that can be detected with a probability of 95%. A 7-dilution series contrived of pooled urine spiked with Aspergillus antigen (Ethanol Precipitate (EP)) were used for the testing. Dilutions were tested with three lots of MycoMEIA over 5 days. Each day of testing, 4 replicates of each dilution were tested generating a total of 20 results for each sample dilution with each assay lot per day. Results were interpreted independently per assay lot and judged based on positive or negative clinical interpretations according to the MycoMEIA Index (Negative: MycoMEIA Index < 0.6, Positive: ≥ 0.6). LoD was verified using the Probit approach with an assigned Type II (β) error of 0.05 (5%). The maximum experimentally determined LoD is 2.869 ng/mL; the claimed LoD is 3 ng/mL.

7. Assay Cut-Off:

An MycoMEIA Index result assay cut-off of 0.6 for positivity was established by the receiver operating characteristic (ROC) curve based on the results of urine samples obtained from a cohort of 21 cases with proven or probable Invasive Aspergillosis (IA) and 161 controls with no infectious diagnosis. The ROC AUC of 0.96 (95% CI 0.92–1.00) demonstrated good performance and predicted an optimal index result cutoff of 0.6 to define positivity, where per subject sensitivity of 90.48% (95% CI 71.09–98.31%) and specificity 92.55% (95% CI 87.42–95.02%) were determined at index cutoff of 0.6. Sensitivity of approximately 85.71% (95% CI 65.36–95.02%) and specificity 96.27% (95% CI 92.11–98.28%) at index cut-off of 0.8 was also observed. Index results ranging from ≥ 0.6 to < 0.7 will be recommended to be retested.

Comparison Studies:

1. Method Comparison with Predicate Device:

Please refer to Section VI.C (Clinical Studies) below for the clinical validation, regarding the method comparison studies.

2. Matrix Comparison:

A matrix equivalency study was conducted according to CLSI EP35⁴ to evaluate equivalence of the MycoMEIA Aspergillus Assay (MycoMEIA) with the different types of urine specimen sources in the clinical study. Negative matrices were prepared from pooled human urine

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obtained from the different specimen sources (clean-catch, catheter-collected, and non-sterile collection devices (e.g., bedpans, urine hats)) and served as negative samples (0x LoD). Samples were prepared by spiking negative matrix with Aspergillus antigen (Ethanol Precipitate (EP)) at high negative (0.3x LoD), low positive (1x LoD), and positive (3x LoD) analyte levels. Acceptance criteria are that all samples should yield the expected results, with ≥ 95% agreement (PPA and NPA) of catheter and dirty collection methods compared to clean-catch. Results showed 100% agreement of samples.

## Clinical Studies:

The clinical performance of the MycoMEIA Aspergillus Assay (MycoMEIA) was evaluated in a multi-site clinical evaluation.

## Study Sites

Archived, retrospective samples were sourced from patients enrolled in three separate clinical studies at a total of four geographically diverse locations in the United States and one in Belgium. Fresh, prospective samples were obtained and tested from one study site.

Study 1: Sites 1, 2 and 3 (Retrospective, Archived). Urine samples from patients with hematological malignancies at risk for invasive aspergillosis (IA) were collected from adults (> 18 years) at three, geographically diverse locations within the United States. Patients were enrolled and samples were collected into sterile containers and aliquoted into cryovials within 12 hours of collection and stored frozen at -70°C. Samples were collected twice weekly, providing multiple samples per subject. Clinical data were collected, and independent clinical reviewers adjudicated cases as proven IA, probable IA, possible IA, or no infection.

Study 2: Site 4 (Retrospective, Archived). Urine samples were collected from adults being treated for hematological malignancies at one hospital in Belgium. These samples were aliquoted and stored frozen. Twice weekly serial samples were collected, and clinical data were evaluated to adjudicate diagnoses, by independent clinical reviewers.

Study 3: Site 5 (Retrospective, Archived, Prospective). Urine samples were collected from adults and children with suspected and confirmed invasive fungal disease (IFD) and multiple underlying immunosuppressive conditions, including hematological malignancies, organ transplantation (excluding lung transplantation) and receipt of immunosuppressive biologic therapy for autoimmune conditions at one site within the US. This study was performed in two stages:

- Stage 1: Prospective Collection and Retrospective Testing. Patients suspected of having an IFD were consented for collection of urine samples and clinical data. Consenting subjects at high-risk for IFD with hematologic malignancies and/or allogeneic blood or marrow transplantation (BMT) had urine samples collected twice weekly. Urine samples were collected in sterile containers, frozen immediately after collection at -70°C. Samples were stored frozen in aliquots. Clinical data were collected, and an independent clinical reviewer adjudicated cases as proven IA, probable IA, possible IA, or no infection.
- Stage 2: Prospective Collection and Testing. Samples were collected as part of the MycoMEIA fresh, prospective testing to evaluate specificity. Subjects recognized as having suspected IA were identified after lab receipt of clinical specimens (e.g., serum or

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{16}

BAL) for BioRad Platelia galactomannan (GM) enzyme immunoassay (EIA) testing, and urine was collected from those who provided consent. An independent clinical reviewer adjudicated cases as proven IA, probable IA, possible IA, or no infection.

## Enrollment Criteria

Selection and Inclusion: Subjects were selected who were at high risk of IA due to hematologic malignancies, and/or receipt of allogenic BMT, solid organ transplant (kidney, heart, liver; excluding lung), having current or recent neutropenia (e.g., due to cytotoxic therapy), or immunosuppressed people with suspected IA (e.g., those given immunosuppressants for autoimmune disease, inherited severe immunodeficiency or another underlying disease).

Subjects were inpatients that had at least two weeks of clinical follow-up to determine clinical diagnosis, with at least four urine samples obtained during the clinical evaluation period or at least one sample obtained within one week of diagnosis.

## Exclusion:

1. Lung transplant subjects were excluded.
2. Subjects were excluded if radiographic evaluation was not available.
3. Subjects who had received more than three days of exposure to mold-active antifungal therapy at the time of collection were excluded, unless there was mycologic and clinical evidence of antifungal failure (e.g., radiographic and microbiologic test positivity).

## Subjects and Samples (Archived Samples)

In some cases, subjects provided more than one sample over time for analysis. A total of 475 samples were collected from 290 different subjects: 226 samples were tested from 50 subjects with IA that was proven (n = 3) or probable (n = 47) per 2020 EORTC/MSG consensus definitions. Most subjects had received therapy for hematologic malignancies or an allogeneic BMT and were neutropenic, with or without other biologic immunosuppression. Three subjects (6.0%) had proven IA, with a biopsy of the lung (n=1), skin (n=1), or bone (n=1). Positive subject demographics, including clinical characteristics and IA diagnostic evidence, are shown in Table 7.

Table 7. Demographic Distribution of Positive Subjects (Retrospective, Archived)

| | Number (%) |
| --- | --- |
| Age, mean (range) | 53.4 years (14-74 years) |
| Gender, male (%)^{1} | 27 (55.1) |
| Underlying risk for IA | |
| Hematologic malignancy, no BMT^{2} | 27 (54.0) |
| Allogeneic BMT | 19 (38.0) |
| Solid organ transplant^{3} | 2 (4.0) |
| Other^{4} | 2 (4.0) |
| Biologic immunosuppression^{5} | |
| Neutropenia | 24 (48.0) |
| Corticosteroids or other immunosuppression | 15 (30.0) |
| Both neutropenia and other immunosuppression | 11 (22.0) |
| Infection diagnosis | |
| Probable IA | 47 (94.0) |
| Proven IA | 3 (6.0) |

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| Microbiologic evidence^{6} | |
| --- | --- |
| GM EIA+, BAL | 29 (58.0) |
| GM EIA+, serum | 25 (50.0) |
| GM EIA+, both BAL & serum | 8 (16.0) |
| Aspergillus culture positive | 10 (20.0) |

1 Gender was not available for one subject.
2 Diagnoses included acute myelogenous leukemia or myelodysplastic syndrome (n=20), acute lymphocytic leukemia (n=3), chronic myelomonocytic leukemia (n=1), lymphoma (n=1), hematologic malignancy NOS (n=2).
3 Solid organ transplants included heart (n=1) and kidney + liver (n=1).
4 Diagnoses included severe malnutrition (n=1) and autoimmune disease (n=1).
5 Of 49 people, 2 people who had received allogeneic BMT and 1 person with severe malnutrition were not listed as currently receiving biologic immunosuppression.
6 Percentage is based on 50 cases. Multiple diagnostics can be positive in subjects. Culture positivity included 7 BAL A. fumigatus, 1 sputum A. fumigatus, 1 sputum A. ustus and 1 bone biopsy A. fumigatus.

## Subjects and Samples (Prospective Study)

A prospective study was performed (Study 3, Site 5, Stage 2 as described above) to analyze specificity of the assay using fresh samples. All samples were tested within 6 hours of collection. A total of 210 subjects were consented and provide 254 urine samples for testing. Clinical adjudication was performed by an independent reviewer who was blinded to MycoMEIA test results based on pre-established acceptance criteria, i.e., per 2020 EORTC/MSG consensus definitions. Thirty-four (34) subjects had lung transplants and were excluded from analysis. One sample was excluded due to likely contamination prior to testing. Three subjects that did not have CT scans performed were excluded. Four subjects were discontinued because samples were collected after receipt of >3 days of mold-active antifungal therapy. Six (6) subjects (13 samples) with probable IA were obtained. Of the enrolled negative cohort, 171 samples from 168 subjects were evaluable, 39 no IA, 102 possible IA, and 23 with mixed or other (non-IA) infections. Negative subject demographics are shown in Table 8 below.

Table 8. Demographic Distribution of Negative Subjects (Prospective)

| | Number (%) |
| --- | --- |
| Age, median (range) | 64 years (5-89 years) |
| Gender, male (%) | 112 (66.7) |
| Underlying risk for IA^{1} | |
| Hematologic malignancy | 132 (78.6) |
| Solid organ transplant (SOT) | 28 (16.7) |
| Solid tumor | 18 (10.7) |
| Other | 8 (4.7) |
| Biologic immunosuppression | |
| Neutropenia | 54 (32.1) |
| Corticosteroids | 39 (23.2) |
| Infection diagnosis^{2} | |
| Possible IA | 102 (59.6) |
| No infection | 39 (22.8) |
| Proven or Probable IA (Cases) | 7 (4.1) |
| Mixed or other infections | 23 (13.4) |
| Microbiologic evidence of IA^{3} | |
| GM EIA+, BAL | 4 (14.8) |
| GM EIA+, serum | 3 (1.5) |
| Aspergillus culture positive | 0 |
| β-D glucan serum | 14 (7.4) |

{18}

$^{1}$ Of 168 subjects. 8 had multiple risks: hematologic malignancy + solid tumor (n=8); hematologic malignancy + HIV/AIDS (n=2); hematologic malignancy + kidney transplant (n=2); hematologic malignancy + solid tumor + rheumatologic disease (n=2); rheumatologic disease + kidney transplant (n=1); hematologic malignancy + liver transplant (n=1); hematologic malignancy + rheumatologic disease (n=1); solid tumor + rheumatologic disease (n=1). SOTs include liver (n=14), kidney (n=7), heart (n=2) and kidney + liver (n=3), kidney + heart (n=1), kidney + pancreas (n=1). Other include rheumatologic or connective tissue disease (n=5), HIV/AIDS (n=2) and Idiopathic CD4 T cell deficiency (n=1).
2 Positive tests from 171 episodes shown. Possible IA included 6 episodes with positive $\beta$ -D glucan assays and 1 with positive Karius cfDNA assay.
3 Positive tests shown from 27 BAL performed, 203 sera sent for GM EIA, 187 sent for $\beta$ -D glucan assays, and 2 Karius cfDNA assays.

# Sample Testing and Reference Method

Criteria for the determination of proven, probable, and possible IA follow the consensus definitions of invasive fungal disease from the EORTC/MSGERC Consortium as described in Donnelly et al. Information regarding clinical diagnoses were blinded to the operators performing the testing. True positive subjects include those with proven or probable IA. Negative subjects include a true negative group consisting of randomly selected subjects at risk for IA but who did not have proven, probable or possible IA, or other infections, or receipt of antifungal therapy, and those with other infections but who did not have proven, probable or possible IA. Subjects with "possible" IA were not included in the performance analysis.

# Performance and Data Analysis

Each sample obtained at study sites were tested at three CLIA-certified clinical laboratories within the U.S. according to the MycoMEIA Instructions for Use (IFU). An additional 6 pediatric subjects were excluded from the clinical performance analysis since the MycoMEIA is only for use in adults. Sensitivity was established with 47 evaluable subjects who provided 623 archived, retrospective samples from the three studies and 6 evaluable subjects who provided 13 fresh, prospective samples from a single site. Sensitivity was calculated by sample and by subject (diagnoses of proven or probable IA) as not all samples from patients are expected to be positive. High sensitivity was observed compared to patient diagnosis based on established criteria. Specificity was analyzed by sample from 65 fresh, prospective samples obtained from a single study site. Possible IA samples $(n = 106)$ were excluded from the analysis; however, mixed infections adjudicated as no IA were included into the analysis as negative samples. Overall sensitivity of the MycoMEIA was determined as $92.4\%$ (95%CI: 82.1-97.0) by subject and $58.1\%$ (95%CI: 54.2-61.9) by sample, and specificity was determined to be $86.1\%$ (95%CI: 75.7-92.5) by sample. A summary of results is shown in Table 9 below.

Table 9. Clinical Performance of the MycoMEIA

| | MycoMEIA Positive | MycoMEIA Negative | Sensitivity (95% CI) |
| --- | --- | --- | --- |
| Positive Subjects | | | |
| Cases, proven and probable IA (N=53) | 49 | 4 | 92.4% (82.1-97.0) |
| Cases, proven IA, archived (n=3) | 3 | 0 | 100% (43.8-100) |
| Cases, probable IA, archived (n=44) | 41 | 3 | 93.2% (81.8-97.7) |
| Cases, probable IA, prospective (n=6) | 5 | 1 | 83.3% (43.6-97.0) |

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| Positive Samples | | | |
| --- | --- | --- | --- |
| Samples, proven and probable IA (N=636) | 370 | 266 | 58.2% (54.3-61.9) |
| Samples, proven IA, archived (n = 72) | 46 | 26 | 63.9% (52.3-74.0) |
| Samples, probable IA, archived (n=551) | 318 | 233 | 57.7% (53.5-61.8) |
| Samples, probable IA, prospective (n=13) | 6 | 7 | 46.1% (23.2-70.9) |
| | MycoMEIA Positive | MycoMEIA Negative | Specificity (95% CI) |
| Negative Samples (Prospective Study) | | | |
| Prospective (N=65) | 9 | 56 | 86.1% (75.7-92.5) |
| No infection (n=39) | 4 | 34 | 89.5% (75.9-95.8) |
| Mixed or other infections (n=26) | 5 | 21 | 80.8% (62.1-91.5) |

Clinical Cut-Off:
Not applicable.

Expected Values/Reference Range:
Invasive aspergillosis, caused by *Aspergillus* species, is an infection that develops in patients who have impaired immune defenses or other underlying conditions. The occurrence of the disease occurs in groups at risk due to factors such as hematologic malignancy, hematopoietic or solid organ transplant, granulocytopenia, immunosuppressive medications, lung disease or other infections, and the stage of infection.

F Other Supportive Performance Characteristics Data:
Not applicable.

VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.

IX Conclusion:
The premarket notification requires additional information to complete the substantive review and support a substantial equivalence decision.

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