The Cryptococcal Antigen Lateral Flow Assay (CrAg LFA) is an immunochromatographic test system for the qualitative or semi-quantitative detection of capsular polysaccharide antigens of Cryptococcus species complex (Cryptococcus neoformans and Cryptococcus gattii) in serum and cerebral spinal fluid (CSF).

The CrAg Lateral Flow Assay is a prescription-use laboratory assay, which can aid in the diagnosis of cryptococcosis.

Device Description

The CrAg Lateral Flow Assay is a dipstick sandwich immunochromatographic assay, which detects cryptococcal antigen in serum and CSF. For the qualitative procedure, specimens are diluted 1:2 in 1x Specimen Diluent and analyzed. For the semi-quantitative procedure, specimens are diluted 1:5 in 1x Specimen Diluent followed by 1:2 serial dilutions. All dilutions are then analyzed as in the qualitative procedure. Specimens are placed into an appropriate reservoir, such as a test tube or microtiter plate, and the lateral flow device is then placed into the reservoir allowing the specimen to come into contact with the test membrane. The test uses specimen wicking to capture gold-conjugated, anti-cryptococcal monoclonal antibodies and gold-conjugated control antibodies that are deposited onto a membrane. If cryptococcal antigen is present in the specimen, it binds to the gold-conjugated, anti-cryptococcal antibodies. The gold-labeled antibody-antigen complex will continue to wick up the membrane where it will interact with the Test Line (T). The Test Line is immobilized anti-cryptococcal monoclonal antibodies. If the specimen contains cryptococcal antigen, a sandwich is created with the gold-labeled antibodies and the immobilized antibodies, causing a visible line to develop at the test line site (T). If proper flow occurs and the reagents are reactive at the time of use, the wicking of any specimen, positive or negative, will cause the gold-conjugated control goat IgG antibody to move to the Control Line (C) which is immobilized bovine anti-goat IgG antibody. The immobilized anti-goat antibody will bind to the gold-conjugated goat IgG Control antibody and will cause a visible line to develop (C). A positive test result will create two lines, while a negative test result will create one line (Figure 1). If the control line fails to develop a line, then the test is not valid.

AI/ML Overview

Here's an analysis of the provided text regarding the acceptance criteria and study for the CrAg Lateral Flow Assay:

1. Table of Acceptance Criteria and Reported Device Performance

Acceptance Criteria (Implied)	Reported Device Performance (CSF)
High Sensitivity	100% (95% CI: 94.4-100.0%)
High Specificity	100% (95% CI: 96.3-100%)
Repeatability (Overall)	• Med. Pos: 100% positive detected • Low Pos: 100% positive detected • High Neg: 96% negative detected • Neg: 100% negative detected
Reproducibility	(Same as above, as "overall percent positive and percent negative detected were calculated by combining the data from all three sites")
Analytical Sensitivity	1.25 ng/ml (defined as concentration where 50% positive, 50% negative)
No High Dose Hook Effect	High dose hook effect possible for concentrations > 200ug/ml

Note on Acceptance Criteria: The document does not explicitly state numerical acceptance criteria (e.g., "sensitivity must be >95%"). However, it implicitly aims for "very high sensitivity and specificity" based on the background information and the excellent results presented. The repeatability and analytical sensitivity are reported as measured, indicating the performance achieved. The high dose hook effect is identified as a limitation rather than a criterion met.

2. Sample Size Used for the Test Set and Data Provenance

Test Set Sample Size (CSF for Method Comparison):
- Positive (Culture/India Ink): 65 specimens
- Negative (Culture/India Ink): 99 specimens
- Total: 164 specimens
Data Provenance: The studies contained a mix of both prospective and retrospective specimens. The country of origin is not specified, but the device is indicated for use in the US (implied by FDA submission).

3. Number of Experts Used to Establish Ground Truth for the Test Set and Qualifications

The document does not specify the number of experts or their qualifications for establishing the ground truth (Culture/India Ink).
For the repeatability and reproducibility study (CSF), "five different operators" across "three different sites" were involved. Their specific qualifications are not detailed beyond "internal" and "US reference/hospital laboratory" personnel.

4. Adjudication Method for the Test Set

The document does not describe any adjudication method for the test set. The "gold standard" (culture and/or India Ink) is presented as the definitive truth.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and Effect Size

No, an MRMC comparative effectiveness study was not explicitly described in this 510(k) summary. The study focuses on the standalone performance of the CrAg LFA compared to a gold standard, not on the improvement of human readers with or without AI assistance (as this is a point-of-care type diagnostic, not an AI-assisted interpretation tool for images).

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

Yes, a standalone performance study was conducted. The CrAg Lateral Flow Assay is a rapid diagnostic test where the result (presence/absence of lines) is directly interpreted. The reported sensitivity and specificity values represent the performance of the device itself (algorithm only, in a sense, as it's a defined chemical reaction leading to a visible outcome) against the gold standard. While a human reads the lines, the core performance metrics are about the device's ability to detect the antigen.

7. The Type of Ground Truth Used

For the method comparison study (CSF), the ground truth used was "gold standard for the diagnosis of cryptococcosis (culture and/or India Ink)."

8. The Sample Size for the Training Set

The document does not specify a training set sample size. This is common for lateral flow assays, which are typically developed and optimized through laboratory analytical studies rather than machine learning models that require distinct training sets. The studies described are primarily performance validation studies.

9. How the Ground Truth for the Training Set Was Established

Since a distinct "training set" in the context of machine learning is not mentioned as such, the method of establishing ground truth for development/optimization would likely involve spiking known concentrations of cryptococcal antigen into mock specimens (as seen in the analytical sensitivity sections) and testing against other validated methods or known positive/negative samples. The document refers to "mock CSF that was negative by the IMMY Latex-Cryptococcal Antigen Detection System" for repeatability/reproducibility.

Summary

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KII2Y22

MAR 2 8 2012

10(k) Sumi CrAg Lateral Flow Assav

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

Owner:	Immuno-Mycologics, Inc.2700 Technology PlaceNorman, OK 73071Tel: 405-360-4669Fax: 405-364-1058Contact: Dr. Sean K. Bauman, President & CEOSean-Bauman@immy.com
Prepared:	March 26, 2012
Trade Name:	CrAg Lateral Flow Assay
Common Name:	Cryptococcal Antigen Lateral Flow Immunoassay
Regulation:	866.3165
Predicate Device:	Immuno-Mycologics' CrAg Lateral Flow Assay (K102286)
Intended Use:	The CrAg Lateral Flow Assay is an immunochromatographic test systemfor the qualitative or semi-quantitative detection of capsularpolysaccharide antigens of Cryptococcus species complex (Cryptococcusneoformans and Cryptococcus gatti) in serum and cerebral spinal fluid(CSF).

The CrAg Lateral Flow Assay is a prescription-use laboratory assay, which can aid in the diagnosis of Cryptococcosis.

Device Description:

Explanation:

Detection of cryptococcal antigen in serum and CSF has been used for over forty years to aid in the diagnosis of cryptococcosis with very high sensitivity and specificity (9,14,15). Current guidelines for the management of cryptococcal disease partially base treatment recommendations on cryptococcal antigen presence and more specifically on cryptococcal antigen titers (16).

Cryptococcosis is caused by both species of the Cryptococcus species complex (Cryptococcus neoformans and Cryptococcus gatti) (5,6,12,13). Individuals with impaired cell-mediated

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immune (CMI) function due to acquired immunodeficiency syndrome (AIDS) (19), lymphoproliferative disorders (18), steroid therapy (8), and organ transplantation (7) are at increased risk of cryptococcosis. AIDS accounts for 80-90% of cryptococcal infections (11). The incidence of cryptococcosis in AIDS patients in the United States is estimated to be 5-10% (11), while the incidence of cryptococcosis in other parts of the world, such as Africa, is as high as 30% (3). Cryptococcosis is the fourth most common opportunistic, life-threatening infection among AIDS patients (10).

Description:

Image /page/1/Figure/3 description: The image shows a diagram of a cryptococcal antigen test. The diagram illustrates the components of the test, including the plastic overlay, backer card, sample application area, gold-conjugated antibodies, test line, and control line. It also shows the results of a positive and negative test. The positive test result shows two lines, while the negative test result shows only one line.

Figure 1. CrAg Lateral Flow Assay Schematic

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Technological Characteristics Summary

A comparison between the CrAg LFA and the CrAg LFA (K102286 - Serum only) is presented in Table 1.

Table 1. Comparison with Predicate Device

	SIMILARITIES
Feature	CrAg LFA (New Device)	CrAg LFA (Serum Only) (K102286)
Intended Use	Immunochromatographic test system for thequalitative or semi-quantitative detection of thecapsular polysaccharide antigens of Cryptococcusspecies complex (Cryptococcus neoformans andCryptococcus gattii) in serum	Immunochromatographic test system for thequalitative or semi-quantitative detection of thecapsular polysaccharide antigens of Cryptococcusspecies complex (Cryptococcus neoformans andCryptococcus gattii) in serum
Indication ForUse	Prescription-use laboratory assay, which can aid inthe diagnosis of cryptococcosis	Prescription-use laboratory assay, which can aid inthe diagnosis of cryptococcosis
Technology	Lateral Flow Assay	Lateral Flow Assay
Sample Matrix	Serum	Serum
Instruments	None	None
AssayComponents	Specimen diluent, lateral flow strips, built-in control,gold conjugated antibodies	Positive control, negative control, latex cards, latexconjugated antibodies
Specimen Pre-Treatment	Dilution	Dilution
DetectionAntibody	Anti-cryptococcal monoclonal antibody	Anti-cryptococcal monoclonal antibody
StorageRequirements	20-25°C	20-25°C
	DIFFERENCES
Feature	Cryptococcal Antigen Lateral Flow Assay	Latex-Cryptococcus Antigen DetectionSystem
Intended Use	Test for the qualitative or semi-quantitativedetection of capsular polysaccharide antigens ofCryptococcus in serum	Test for the qualitative or semi-quantitativedetection of capsular polysaccharide antigens ofCryptococcus in serum and CSF
Indication ForUse	No differences	No differences

Performance Summary

A. Precision Studies (Repeatability & Reproducibility)

Serum repeatability and reproducibility results can be found in the predicate device 510(k) (K102286)

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Repeatability and reproducibility with CSF specimens were determined by spiking a mock CSF that was negative by the IMMY Latex-Cryptococcus Antigen Detection System with cryptococcal antigen at four concentrations: Negative, high negative (CJ), low positive (near C95), and medium positive. The samples were analyzed on the CrAg Lateral Flow Assay in triplicate on five different days, at three different sites with a total of five different operators, on one lot, according to EP5-A2. One site was internal (Site 1) and the remaining two were a US reference laboratory (Site 2) and a US hospital laboratory (Site 3). For repeatability, percent positive and percent negative detected were calculated for each site (Table 2). For reproducibility, overall percent positive and percent negative detected were calculated by combining the data from all three sites (last two rows of Table 2).

	CSF
Sample	1Med. Pos		2Low Pos		3High Neg		4Neg
Neg/Pos	-	+	-	+	-	+	-	+
Site 1	0	30	0	30	27	3	30	0
Percent %	0	100	0	100	90	10	100	0
Site 2	0	30	0	30	30	0	30	0
Percent %	0	100	0	100	100	0	100	0
Site 3	0	15	0	15	15	0	15	0
Percent %	0	100	0	100	100	0	100	0
Total No.	0	75	0	75	72	3	75	0
Percent %	0	100	0	100	96	4	100	0

	Table 2. Repeatability at 3 Different Sites
--	---------------------------------------------	--	--	--

B. Analytical Sensitivity (lower limits of the assay/analytical cut-off)
Serum analytical sensitivity can be found in the predicate device 510(k) (K102286)

Analytical sensitivity for the CrAg Lateral Flow Assay was estimated by running 24 replicates of varying concentrations of cryptococcal antigen diluted in mock CSF on one lot of kits, according to EP12-A2. The analytical cut-off was defined as the concentration where 50% of the results were positive and 50% of the results were negative. The analytical cut-off is 1.25ng/ml.

C. Analytical Specificity (cross-reactivity)
Serum analytical specificity can be found in the predicate device 510(k) (K102286)

Due to specimen availability, the following CSF conditions were not tested in the CrAg Lateral Flow Assay: S. pneumonia, Enterovirus, Enterobacteriaceae, Streptococcus spp., Staphylococcus spp., diphtheroid, H. influenzae type B, N. meningitidis, Enterococcus spp., Epstein Barr, Herpes simplex virus Type 1 and 2, Listeria monocytogenes, Trichosporon beigelii, and samples with syneresis fluid condensation.

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This assay was not evaluated for potential interference related to specimen pretreatment with 2-mercaptoethanol or with specimens including the following subastances or conditions: bloody CSF, cloudy CSF, white blood cells, xanthochromic CSF, bilirubin, protein, systemic lupus erythmatosus (SLE), sarcoidosis, or N. memingitides.

D. Linearity
N/A
E. High Dose Hook Effect
High dose hook effect concentrations with specimens were determined by spiking negative serum that was negative by the IMMY Latex-Cryptococcus Antigen Detection System and CrAg Lateral Flow Assay, with cryptococcal antigen at various concentrations between 20 and 500ug/ml. Each concentration was tested in triplicate at IMMY on one lot of CrAg Lateral Flow Assay, according to the package insert. It was determined that serum specimens with a cryptococcal antigen concentration higher than 200ug/ml can produce a high dose hook effect and therefore may produce a false negative result.
F. Method Comparisons

Predicate Device Method Comparison

Not Applicable

Other Method Comparison - Culture/India Ink (Gold Standards)

Serum method comparison to gold standards can be found in the predicate device 510(k) (K102286)

The CrAg Lateral Flow Assay was compared to the gold standard for the diagnosis of cryptococcosis (culture and/or India Ink) to evaluate the sensitivity and specificity of the assay in CSF. These studies contained a mix of both prospective and retrospective specimens. A summary of the data collected is included in Tables 3 and 4 below:

	Culture/India Ink
	Positive	Negative
CrAg LFAAssay	Positive	65	0
	Negative	0	99
Table 4. CSF Statistical Analysis: Culture/India Ink
	Calculated	95% CI
Sensitivity	100%	94.4-100.0%
Specificity	100%	96.3-100%

CSF 2x2 Contingency Table: Culture/India Ink

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Conclusion

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

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Image /page/6/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a stylized eagle with three stripes forming its body and wing. The eagle's head is facing left. Encircling the eagle is the text "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" in a circular arrangement.

10903 New Hampshire Avenue Silver Spring, MD 20993

MAR 2 8 2012

Immuno-Mycologics, Inc. c/o Sean K. Bauman, Ph.D. President and CEO 2700 Technology PL Norman, OK 73071

Re: K112422

Trade/Device Name: CrAg Lateral Flow Assay (CrAg LFA) Regulation Number: 21 CFR § 866.3165 Regulation Name: Cryptococcal antigen lateral flow assay Regulatory Class: II Product Code: GMD Dated: March 26, 2012 Received: March 27, 2012

Dear Dr. Bauman:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commence prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into class II (Special Controls), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); and good manufacturing practice

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Page 2 - Sean K. Bauman, Ph.D.

requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter requirences as be form arketing your device as described in your Section 510(k) premarket with anow you to organ mading of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Parts 801 and 11 you don't up office of In Vitro Diagnostic Device Evaluation and Safety at (301) 796-5450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours,

Vayathra

Sally A. Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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Indications for Use Sta

510{k} Number (if known): K112422

Device Name: CrAg Lateral Flow Assay

Indications for Use:

The CrAg Lateral Flow Assay is a prescription-use laboratory assay, which can aid in the diagnosis of cryptococcosis.

Prescription Use X (Part 21 CFR 801 Subpart D)

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

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

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

Freddie lu. Poole

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

510(k): K112422

www.immy.com | 2700 Technology Pl Norman, OK 73071 USA | 405.360.4669

Rev. 03/28/2012

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Regulation Number and Section

§ 866.3165

Cryptococcus neoformans serological reagents.(a)
Identification. Cryptococcus neoformans serological reagents are devices that consist of antigens used in serological tests to identify antibodies toCryptococcus neoformans in serum. Additionally, some of these reagents consist of antisera conjugated with a fluorescent dye (immunofluorescent reagents) and are used to identifyCryptococcus neoformans directly from clinical specimens or from cultured isolates derived from clinical specimens. The identification aids in the diagnosis of cryptococcosis and provides epidemiological information on this type of disease. Cryptococcosis infections are found most often as chronic meningitis (inflammation of brain membranes) and, if not treated, are usually fatal.(b)
Classification. Class II (special controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to § 866.9.