Granada Medium is a selective and differential agar which is intended for the qualitative detection of Group B Streptococus (GBS) from LIM Broth enrichment cultures of vaginal/rectal swabs from antepartum women following 18-24 hours of incubation.

Recovery of orange colored colonies on Granada Medium is a positive result for presence of β-hemolytic GBS. Results can be interpreted after 18-24 hours of anaerobic incubation. Due to the properties of Granada Medium, white colonies recovered on Granada Medium must undergo additional testing to confirm absence of GBS colonies must be performed for conducting susceptibility testing as recommended for penicillin- allergic women. A lack of growth or the absence of orange colonies on Granada Medium does not presence of GBS. Granada Medium is not intended to diagnose infection, or to guide or monitor treatment for infections.

Device Description

AI/ML Overview

Here's a breakdown of the acceptance criteria and study details for the Granada Medium device:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are not explicitly stated as numerical targets in the provided document, but rather implied by the overall performance comparison to the reference method and the conclusion of substantial equivalence. The key performance metrics reported are sensitivity and specificity for GBS detection.

Performance Metric	Acceptance Criteria (Implied)	Reported Device Performance (Overall)
Sensitivity (GBS, Table 1)	High, comparable to reference method for detecting GBS (β-hemolytic and non-hemolytic)	94.5% (95% CI: 89.8 - 97.1)
Specificity (GBS, Table 1)	High, comparable to reference method for detecting GBS (β-hemolytic and non-hemolytic)	98.0% (95% CI: 96.6 - 98.9)
Sensitivity (β-hemolytic GBS, Table 2)	High, comparable to reference method for detecting β-hemolytic GBS	98.1% (95% CI: 94.5 - 99.3)
Specificity (β-hemolytic GBS, Table 2)	High, comparable to reference method for detecting β-hemolytic GBS	97.9% (95% CI: 96.4 - 98.8)
Recovery Rate (LoD) for S. agalactiae ATCC® 12386 & ATCC® 12403 (direct inoculation)	Low concentration for positive reaction	1.5x10² CFU/mL (15 CFU)
Recovery Rate (LoD) for S. agalactiae ATCC® 12386 & ATCC® 12403 (after LIM Broth enrichment)	Low concentration for positive reaction after enrichment	1.5x10² CFU/mL (4.5 CFU)
Analytical Reactivity (GBS Strains)	All tested GBS strains produce expected color at LoD	100% of 54 GBS strains (48 β-hemolytic, 6 non-hemolytic) produced expected color reaction
Analytical Specificity (Non-target Organisms)	Negative color reaction or no recovery	100% (39/84 negative color, 45/84 no recovery)
Microbial Interference	Recovery of target GBS in presence of high concentrations of non-target organisms	Expected orange color and recovery for all but one non-target organism; E. faecalis (ATCC 29212) inhibited at high concentration, but GBS recovered when E. faecalis concentration reduced
Interference (Exogenous/Endogenous)	No interference with growth or color reaction	No interference observed for any tested substance at highest clinically relevant concentration
Incubation Time	All organisms grow with visible orange colonies within the range	All organisms grew with visible orange colonies by 18 hours (set range 18-24 hours)
Specimen Stability	Recovery of GBS for all time points and storage conditions	100% recovery from specified swabs at room temp (up to 24 hours) and 2-8°C (up to 120 hours)
Reproducibility	>95% agreement with known test results	>95% agreement; 100% of β-hemolytic GBS isolates recovered with expected orange color

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

Sample Size (Clinical Study): A total of 771 valid clinical specimens were tested (out of 884 initially collected, with 113 excluded for not meeting enrollment criteria).
Data Provenance: The study was conducted at four geographically diverse hospitals with routine GBS specimens in the form of vaginal/rectal swabs. This suggests a multi-center, potentially prospective (implied by "routine GBS specimen") collection of real-world clinical samples. It does not explicitly state the country of origin, but "US Food & Drug Administration" context usually refers to studies conducted in the USA or accepted for the US market. The nature of the study (comparing to a reference method) indicates it's a diagnostic accuracy study.

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

The document does not specify the number of experts or their qualifications for establishing the ground truth. It describes the "reference method" as:

Selective enrichment of specimen in LIM Broth
Followed by subculture to blood agar
Confirmation of Group B Streptococci by: hemolytic reaction on blood agar, gram-stain, catalase test, and StrepPRO™ latex agglutination.

This indicates a standard microbiology laboratory workflow, where trained laboratory personnel (e.g., medical technologists, microbiologists) would be performing and interpreting these established diagnostic tests. The discrepant analysis involved isolates being returned to Hardy Diagnostics for re-testing and confirmation of identity, implying expertise at the manufacturer's facility as well.

4. Adjudication Method for the Test Set

The document primarily describes a comparison against a "reference method" and subsequent "discrepant analysis."

For the initial comparison (Table 1 and 2), the "reference method" was considered the ground truth.
For Discrepant Analysis: All discrepant isolates (False Positives and False Negatives) were re-tested and confirmed using the reference method described above (hemolytic reaction, gram-stain, catalase, StrepPRO™ latex agglutination). This serves as an adjudication method where initial discrepancies are resolved by re-examination using definitive laboratory tests.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, an MRMC comparative effectiveness study was not explicitly described in the provided text. The study focuses on the standalone performance of the Granada Medium compared to a traditional reference laboratory method.
A brief mention under Reproducibility states: "The testing was done with at least one operator and two readers, blinded to each other's results, per site." This element incorporated some "multi-reader" aspects for reproducibility checks but this was for concordance of interpretations of Granada Medium itself, not a comparative effectiveness study of human readers with vs. without AI assistance. The device is a culture medium, not an AI-assisted diagnostic tool for interpretation.

6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study

Yes, a standalone study was performed. The clinical performance data (Tables 1 and 2) directly compares the results of the Granada Medium (interpreted visually for orange colonies, which is a human interpretation step, but without assistance from another diagnostic tool) against the established laboratory reference method. The device's "performance" is its ability to produce a visual indicator (orange colonies) corresponding to GBS presence. This is its "standalone" performance in its intended use. There is no AI algorithm involved.

7. The Type of Ground Truth Used

The ground truth used was expert consensus / established laboratory methods which identified specific bacterial species. Specifically:

The "reference method" involved selective enrichment in LIM Broth, subculture to blood agar, and confirmation based on hemolytic reaction, gram-stain, catalase test, and StrepPRO™ latex agglutination. This is a highly accurate and accepted method for identifying Group B Streptococci in clinical microbiology.
For analytical studies (reactivity, specificity, LoD), well-characterized ATCC, NCIMB, NCTC reference strains and clinical GBS isolates were used, which represent a known ground truth.

8. The Sample Size for the Training Set

The document does not explicitly mention a separate "training set" as would be typical for machine learning or AI models.
For a diagnostic device like a culture medium, the development and initial optimization (which parallels a "training" phase) would typically involve internal laboratory testing with known bacterial strains and challenging conditions. While not quantified as a "training set" in the context of this document, the Analytical Reactivity, Specificity, Recovery Rate, Microbial Interference, and Incubation studies collectively represent the robust characterization and "training" of the medium's performance during development. These studies involved specific numbers of ATCC/reference strains and non-target organisms (e.g., 54 GBS strains for reactivity, 84 non-target organisms for specificity).

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

Given that a specific "training set" with separate ground truth establishment wasn't explicitly defined for an AI model, for the developmental and analytical studies (which function similarly to training data for the medium's design):

The ground truth was established by using well-characterized, identified bacterial strains (ATCC, NCIMB, NCTC reference strains, and clinical isolates).
For these strains, their identity (e.g., S. agalactiae, specific serotypes, or other bacterial species) was already established through standard microbiological identification techniques.
The "expected color development" (orange for β-hemolytic GBS, white for non-hemolytic GBS, negative for non-GBS organisms) served as the pre-defined target for the medium's differentiation capabilities, based on the known Granada reaction.

Summary

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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

March 22, 2018

Hardy Diagnostics Rianna Malherbe Performance Studies Coordinator 1430 West McCov Lane Santa Maria, California 93455

Re: K173903

Trade/Device Name: Granada Medium Regulation Number: 21 CFR 866.2360 Regulation Name: Selective culture medium Regulatory Class: Class I Product Code: PQZ Dated: December 21, 2017 Received: December 26, 2017

Dear Rianna Malherbe:

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 commerce 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. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. 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 Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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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.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Ribhi Shawar -S
For

Uwe Scherf, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K 1 73903

Device Name Granada Medium

Indications for Use (Describe)

X Prescription Use (Part 21 CFR 801 Subpart D)

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

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510(k) Summary

I. SUBMITTER

Rianna Malherbe Performance Studies Coordinator Hardy Diagnostics 1430 W. McCoy Lane Santa Maria, Ca. 93455 Phone: 805-346-2766 x5714 E-mail: MalherbeR @hardydiagnostics.com

II. DEVICE

Name of Device: Granada Medium Classification Name: 21 CFR 866.2360 Selective Culture Medium Regulatory Class: I Product Code: POZ

III. PREDICATE DEVICE

chromID Strepto B Agar, K163042

IV. DEVICE DESCRIPTION

Currently, Group B Streptococci (GBS) remains the primary cause of early-onset neonatal sepsis in the United States. Infection of the newborn baby is typically preceded by previous colonization of GBS in the maternal genitourinary or gastrointestinal tract. The Centers for Disease Control and Prevention (CDC), in their 11/19/2010 MMWR "Guidelines for the Prevention of Perinatal Group B Streptococcal Disease", recommends the screening of all pregnant women for vaginal and rectal GBS colonization between 35 and 37 weeks of gestation, using an enrichment broth, followed by additional testing to identify GBS.

Hardy Diagnostics Granada Medium agar utilizes the Granada reaction and contains the necessary components for pigment detection of beta-hemolytic GBS. The production of a light orange to dark orange pigmented colony is a unique characteristic of hemolytic GBS on Granada Medium due to a reaction with substrates such as starch, peptone, serum, and folate pathway inhibitors. Since the original description of starch serum agar by Islam in 1977, there have been many improvements to the original formula. GBS detection (orange colonies) with Granada Medium is only possible with beta-hemolytic GBS, which also provides evidence of a direct genetic linkage between pigment production on Granada Medium and hemolysin production. Beta-hemolytic, pigment producing GBS occurs with 95.3 to 99.5% of all GBS strains isolated from clinical specimens (Young, et. al. Korean J. Clin. Path, 1998; Merrit and Jacobs, J. Clin. Microbiol, 1978; Noble, Bent and West, J. Clin. Path. 1983).

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V. INTENDED USE/INDICATIONS FOR USE

Granada Medium is a selective and differential agar which is intended for the qualitative detection of Group B Streptococcus (GBS) from LIM Broth enrichment cultures of vaginal/rectal swabs from antepartum women following 18-24 hours of incubation.

Recovery of orange colored colonies on Granada Medium is a positive result for presence of B-hemolytic GBS. Results can be interpreted after 18-24 hours of anaerobic incubation. Due to the properties of Granada Medium, white colonies recovered on Granada Medium must undergo additional testing to confirm absence of GBS. Subculture of GBS colonies must be performed for conducting susceptibility testing as recommended for penicillin allergic women. A lack of growth or the absence of orange colonies on Granada Medium does not preclude the presence of GBS. Granada Medium is not intended to diagnose infection, or to guide or monitor treatment for infections.

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VI. COMPARISON OF TECHNOLOGICAL CHARACTERISTICS WITH THE PREDICATE DEVICE

Attribute	Device	Comparator	SubstantiallyEquivalent?
Name	Granada Medium	chromID Strepto B Agar	Yes
510(k)Details	510(k) number K173903Product Code PQZ21 CFR 866.2360GBS culture media, selective anddifferentialClass IPanel 83 Microbiology	510(k) number K163042Product Code PQZ21 CFR 866.2360GBS culture media, selective anddifferentialClass IPanel 83 Microbiology	Yes
Intended Use	Granada Medium is a selective anddifferential agar which is intended forthe qualitative detection of Group BStreptococcus (GBS) from LIM Brothenrichment cultures of vaginal/rectalswabs from antepartum womenfollowing 18-24 hours of incubation.Recovery of orange colored colonies onGranada Medium is a positive resultfor presence of β-hemolytic GBS.Results can be interpreted after18-24hours of anaerobic incubation. Due tothe properties of Granada Medium,white colonies recovered on GranadaMedium must undergo additionaltesting to confirm absence of GBS.Subculture of GBS colonies must beperformed for conducting susceptibilitytesting as recommended for penicillinallergic women. A lack of growth orthe absence of orange colonies onGranada Medium does not preclude thepresence of GBS. Granada Medium isnot intended to diagnose infection, or toguide or monitor treatment forinfections.	chromID® Strepto B agar is aselective chromogenic medium that isintended to aid in the qualitativedetermination of Group BStreptococcus (GBS) colonization inpregnant women. This mediumsupports the growth of, but does notdifferentiate between, hemolytic andnon-hemolytic GBS strains. The testis performed on 18-24 hour LIMbroth enrichments of vaginal/rectalswabs obtained from pregnantwomen. chromID® Strepto B agarresults can be interpreted after 24hours incubation with confirmation ofcharacteristic GBS colonies from themedia. chromID® Strepto B agar isnot intended to diagnose infection norto guide or monitor treatment forinfections. chromID® Strepto B agardoes not provide susceptibility results.Subculture to non-selective mediashould be performed as needed forsusceptibility testing. chromID®Strepto B agar is intended for use bylaboratory health practitioners in aclinical laboratory.	Yes
Methodology	Selective, Chromogenic	Selective, Chromogenic	Yes
Inoculation	LIM Broth Enrichment ofVaginal/Rectal Swabs	LIM Broth Enrichment ofVaginal/Rectal Swabs	Yes
Interpretation	Manual, Visual	Manual, Visual	Yes

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VII. PERFORMANCE DATA

The performance of Granada Medium was evaluated at four geographically diverse hospitals with routine GBS specimen in the form of vaginal/rectal swabs. In Table 1, the detection of Group B Streptococci (B-hemolytic GBS and non-hemolytic GBS) by the reference method was compared to the recovery of orange colonies on Granada Medium. In Table 2, the detection of 8hemolytic GBS by the reference method was compared to the recovery of orange colonies on Granada Medium. The reference method was defined as the selective enrichment of specimen in LIM Broth followed by subculture to blood agar. Organisms that grew on Granada Medium or blood agar were confirmed to be Group B Streptococci by looking at hemolytic reaction on blood agar, gram-stain, catalase test, and StrepPRO™ latex agglutination. For the clinical study, vaginal/rectal swab specimens were collected in ESwab™ Liquid Amies, sponge based Liquid Amies and Sponge based Stuart's liquid.

All discrepant isolates were frozen in CryoSavers™ with Brucella Broth and returned to Hardy Diagnostics for testing. As part of discrepant analysis, the identity of each isolate was confirmed (ß Group B Streptococci, non-hemolytic (NH) Group B Streptococci, or non-Group B Streptococci). Once the identity was confirmed. positive organisms (ß Group B Streptococci or NH Group B Streptococci) were tested at 1.5x102 CFU/mL in donated negative-vaginal/rectal matrix (equivalent to an inoculum of 4.5 CFU (1 - 9 CFU tested) and evaluated for their recovery from LIM Broth reference method and color development on Granada Medium.

A total of 884 specimens were tested against routine culture, 113 specimens did not meet enrollment criteria, and were therefore excluded from the analysis. Of the remaining 771 valid samples tested, a total of 154 specimens showed orange color development on Granada Medium after 18-24 hours of incubation at 35°C that were positive for Group B Streptococci by the LIM reference method. Results are shown in Table 1.

Site	TP	FP1	FN2	TN	Sensitivity	95% CI		Specificity	95% CI
1	46	1	5	141	90.2	79.0	95.7	99.3	96.1	99.9
2	41	7	2	133	95.3	84.5	98.7	95.0	90.0	97.6
3	27	1	1	82	96.4	82.3	99.4	98.8	93.5	99.8
4	40	3	1	240	97.6	87.4	99.6	98.8	96.4	99.6
Overall	154	12	9	596	94.5	89.8	97.1	98.0	96.6	98.9

Table 1. LIM Reference Method vs. Granada Medium Color reaction

'There were 12 False Positives observed after 18 to 24 hours of incubation. All isolates were re-tested and confirmed by the discrepant analysis protocol above. Ten isolates recovered from Granada Medium were confirmed to be ß-Group B Streptococci. Two isolates were recorded as pale orange on Granada Medium, but were not identified as Group B Strep. During discrepant analysis, both isolates were confirmed to be Streptococcus sqlivarius subsp. salivarius. One Streptococcus salivarious isolate exhibited yellow colonies on Granada Medium and the other isolate grew as pale orange colonies.

"There were 9 False Negatives observed after 18 to 24 hours of incubation. All isolates were re-tested and confirmed by the discrepant analysis protocol above. Of these nine GBS isolates recovered by the reference method, three were B-hemolytic and six were non-hemolytic. For the B GBS isolates identified by the reference method and evaluated by discordant analysis, all three isolates grew as orange colonies on Granada Medium where originally the colony color was white. Samples where the six NH Group B Streptococci were identified by the reference method showed white colonies on Granada Medium as expected (no discordant analysis was performed for these isolates).

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Because NH GBS cannot be detected by colony color, the performance of Granada Medium (orange color development) was compared to the recovery of only B-hemolytic GBS by the reference method. In Table 2, the overall sensitivity value increased to 98.1% (95% CI: 94.5-99.3%) while the overall specificity value, at 97.9% (95% CI: 96.4-98.8%), did not significantly change.

Site	TP	FP1	FN2	TN	Sensitivity	95% CI	Specificity	95% CI
1	45	2	2	144	95.7	85.8 98.8	98.6	95.1 99.6
2	41	7	0	135	100.0	91.4 100.0	95.1	90.2 97.6
3	27	1	1	82	96.4	82.3 99.4	98.8	93.5 99.8
4	40	3	0	241	100.0	91.2 100.0	98.8	96.4 99.6
Overall	153	13	3	602	98.1	94.5 99.3	97.9	96.4 98.8

Table 2. LIM Reference Method (beta hemolytic Group B Streptococcus) vs. Granada Medium Color reaction

12 of the 13 False Positives are identical to the False Positives listed in Table 1. The additional False Positive was originally recorded as non-hemolytic Group B Strep on blood agar and orange on Granada Medium. During discrepant analysis, the isolate was later confirmed as beta-hemolytic on blood agar.

2The 3 False Negatives are the 3 ß-hemolytic GBS described in Table 1.

RECOVERY RATE

To determine the recovery [Limit of Detection (LoD)] of Granada Medium, the media was challenged with two beta-hemolytic ATCC® strains of Group B Streptococci at 10-fold decreasing concentrations and evaluated for color reaction. The lowest concentration at which a positive reaction was seen, indicated by orange colonies, was determined to be the LoD. The LoD was confirmed by testing Granada Medium with five replicate dilutions of the determined LoD concentration. Granada Medium, by direct inoculation, was able to recover both S. agalactiae ATCC® 12386 and S. agalactiae ATCC® 12403 at a LoD of 1.5x102 CFU/mL, or 15 CFU (3-30 CFU) inoculated directly to Granada Medium. Blood agar plates were used to determine the concentrations of organisms present in each dilution.

To evaluate the performance of Granada Medium with a sample of overnight enriched culture, LIM Broth was challenged with two beta-hemolytic ATCC® strains of Group B Streptococci at 10-fold decreasing concentrations in GBS-negative specimen matrix. After overnight enrichment, LIM Broth culture was subcultured to Granada Medium and evaluated for color reaction. The lowest GBS concentration (previously spiked in LIM Broth and incubated overnight) yielding expected results on Granada Medium was then confirmed with five replicate dilutions of the lowest concentration. Following overnight enrichment in LIM Broth, Granada Medium was able to recover both S. agalactiae ATCC® 12386 and S. agalactiae ATCC® 12403 from a flocked vaginal/rectal swab specimen with 1.5x102 CFU/mL, corresponding to an inoculum of 4.5 CFU (1 - 9 CFU). Blood agar plates were used to determine the concentrations of organisms present in each dilution.

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ANALYTICAL REACTIVITY

Fifty-four selected ATCC, NCIMB, NCTC reference and clinical GBS strains representing seven of the nine different serotypes were directly inoculated to Granada Medium at the determined LoD of 1.5x10- CFU/mL that corresponded to an inoculum of 15 CFU (3-30 CFU). Granada Medium was able to recover all GBS strains tested at the LoD with the expected color development. The GBS serotypes included in this study were serotypes Ia, Ib, III, IV, V, and VI. Serotypes VII and VIII are rare and were not available for testing. Four strains that were nontypable against the nine known serotypes were also included. Of the 54 GBS strains tested, 48 were beta-hemolytic strains (88.9%) and six were non-hemolytic strains (11.1%). All betahemolytic GBS strains produced the expected orange colony color reaction and all nonhemolytic GBS strains produced the expected white colony color reaction on Granada Medium after 24 hours of incubation.

ANALYTICAL SPECIFICITY

Eighty-four organisms that are phylogenetically-related to Group B Streptococci or potentially encountered in a vaginal-rectal swab were tested on Granada Medium following overnight enrichment in LIM Broth. A 1.5x10° CFU/mL suspension of each organism was inoculated to a LIM Broth tube, corresponding to an inoculum of 1.5x106 CFU. After incubation, each LIM broth tube was subcultured to a Granada Medium plate and evaluated for growth and color reaction after 24 hours of anaerobic incubation. Organisms tested either produced a negative color reaction (39/84, 46.4%) or were not recovered (45/84, 53.6%) on Granada Medium after LIM Broth enrichment.

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Non-target Organisms Tested in Analytical Specificity
Acinetobacter baumannii	Enterococcus hirae	Providencia alcalifaciens
Aeromonas hydrophila	Enterococcus malordoratus	Pseudomonas aeruginosa
Bacillus cereus	Enterococcus mundtii	Pseudomonas fluorescens
Bacillus subtilis	Enterococcus pseudoavium	Salmonella enterica (typhii)
Bacteroides fragilis	Enterococcus raffinosus	Salmonella entericaarizonae
Bifidobacterium breve	Enterococcus saccharolyticus	Serratia marcescens
Campylobacter jejuni	Enterococcus sulfureus	Shigella boydii
Candida albicans	Escherichia coli	Shigella flexneri
Candida glabrata	Gardnerella vaginalis	Shigella sonnei
Candida parapsilosis	Geotrichum candidum	Staphyloccocus aureus
Candida tropicalis	Klebsiella oxytoca	Staphylococcus epidermidis
Citrobacter freundii	Klebsiella pneumoniae	Staphylococcussaprophyticus
Clostridium difficile	Lactobacillus acidophilus	Stenotrophomonasmaltophilia
Clostridium novyi	Lactobacillus fermentum	Streptococcus anginosus
Clostridium perfringens	Lactobacillus gasseri	Streptococcus bovis
Clostridium sporogenes	Lactobacillus leichmannii	Streptococcus dysgalactiae
Enterobacter aerogenes	Lactococcus lactis	Streptococcus equi subsp.zooepidemicus
Enterobacter cloacae	Leuconostoc mesenteroides	Streptococcus gallolyticus
Enterococcus avium	Listeria monocytogenes	Streptococcus mitis
Enterococcus casseliflavus	Moraxella cartarrhalis	Streptococcus mutans
Enterococcus cecorum	Morganella morganii	Streptococcus pneumoniae
Enterococcus columbae	Neisseria gonorrhoeae	Streptococcus pyogenes
Enterococcus dispar	Pediococcus acidilacti	Streptococcus salivariussubsp. salivarius
Enterococcus durans	Pediococcus damnosus	Streptococcus salivariussubsp. thermophilus
Enterococcus faecalis	Peptostreptococcus anaerobius	Streptococcus uberis
Enterococcus faecium	Plesiomonas shigelloides	Vibrio parahaemolyticus
Enterococcus flavescens	Proteus mirabilis	Yersinia enterocolitica
Enterococcus gallinarum

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MICROBIAL INTERFERENCE

Granada Medium was challenged to determine if target organism at low concentration could be recovered in the presence of non-target organisms at a high concentration. All organisms that were recovered on Granada Medium in the Analytical Specificity study were tested in the Microbial Interference study. Non-target organisms at a high concentration (1.5x10° CFU/mL) were mixed 1:1 with each target organism (1.5x10 CFU/mL) and inoculated directly to Granada Medium with a 10uL loop. If the target organism was not recovered on Granada Medium, the concentration of the non-target organism was lowered 10-fold until the target organism was recovered.

Granada Medium was able to produce the expected orange color reaction and recover target organisms when in the presence of high concentrations of all but one of the non-target organisms used in this study. The only organism found to affect recovery of GBS was Enterococcus faecalis (ATCC 29212). At 1.5x108 CFU/mL, this organism inhibited recovery of GBS on Granada Medium; however when reduced to 1.5x10° CFU/mL, target GBS was recovered.

Several organisms did not affect recovery, but did affect colony size of target organisms when inoculated to Granada Medium at 1.5x108 CFU/mL: E. faecalis (ATCC 51299), E. avium (ATCC 14025), E. gallinarum (ATCC 49573), E. saccharolyticus (ATCC 43076), Lactococcus lactis (ATCC 19435), Morganella Morganii (ATCC 25830), Proteus mirabilis (ATCC 43071), and Serratia marcescens (ATCC 13880). However, when these non-target organisms were reduced to 1.5x10 CFU/mL, GBS colony size was larger and more clearly visible on Granada Medium. Colony size and color, but not recovery, of GBS was affected by Vibrio parahaemolyticus (ATCC 17802) when present at a high concentration (1.5x108 CFU/mL). When V. parahaemolyticus concentration was reduced to 1.5x10' CFU/mL, the colony morphology of target GBS was as expected.

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INTERFERENCE

Commonly used or encountered endogenous and exogenous substances that may be present in vaginal/rectal specimens were evaluated for potential interference of growth or color reaction on Granada Medium. The substances tested are listed in the table below. No interference was observed with any substance at the highest clinically relevant concentration in the GBS-negative specimen matrix.

Interfering Substances
Category	Substance/Supplier	Concentrationin SampleMatrix1
Anti-diarrhealMedication	Pepto-Bismol® (Bismuth subsalicylatesolution)Imodium A-D® (Loperamide HCl)	2% w/v
Body Oil	Neutrogena Body Oil	2% v/v
Body Powder	Gold Bond Body Powder	1% w/v
Contraceptive Gel	Options Gynol II® (Nonoxynol-9)	0.59% w/v
Enema Solution	Physiological saline	0.25% v/v
Lubricating Gel	K-Y® Jelly	0.57% w/v
Oral Laxative	Milk of MagnesiaDulcolax® (Sodium picosulfate solution)	1.78% v/v1% w/v
Polysorbate 80	Tween®80	10% v/v
Rectal Laxative	Fleet® Glycerin Suppositories	10% v/v
TopicalHemorrhoidOintment	Preparation-H®	0.26% w/v
Vaginal Anti-ItchMedication	Vagisil® Cream	0.41% w/v
Vaginal Anti-FungalMedication	Monistat® (Miconazole nitrate)Lotrimin® (Clotrimazole)	0.29% w/v0.29% w/v
Endogenous Substances
Human AmnioticFluid	Medfusion	2% v/v
Human Feces	Central Coast Pathology	2% v/v
Human Meconium	LEE Biosolutions	2% v/v
Human Urine	Central Coast Pathology	2% v/v
Human WholeBlood	In-house	2% v/v
Mucin	Sigma, M2378	0.05% w/v

1 Specific amounts of substance added to vaginal/rectal specimen matrix calculated using C1V1=C2V2 with the assumption that 1g=1mL.

{12}------------------------------------------------

INCUBATION STUDY

In order to determine a recommended incubation time range, Granada Medium was evaluated using nine beta-hemolytic strains of GBS by direct inoculation from a 1.5x102 CFU/mL suspension, corresponding to an inoculum of 15 CFU (3-30 CFU). Growth and color reaction were recorded every 2 hours from 18 to 24 hours. All organisms tested grew with visible orange colonies by 18 hours. The incubation range for Granada Medium was set from 18-24 hours.

SPECIMEN STABILITY

Various types of specimen transport swabs were evaluated to determine the storage conditions that allowed for recovery of GBS on Granada Medium after LIM Broth enrichment. Swabs were spiked with beta-hemolytic Group B Streptococci strains in vaginal/rectal matrix and were kept under both room temperature and refrigerated conditions. The swabs were inoculated to LIM Broth at 0, 24, 48, 72, 96, and 120 hours. TransPRO™ swabs with Liquid Amies (liquid-based transport system) and five types of Healthlink swabs were used in this study: Liquid Amies, Amies Gel, Liquid Stuart's, Stuart's Gel, and Amies Charcoal. After an overnight incubation at 35°C, LIM Broth cultures were subcultured to Granada Medium.

Granada Medium was able to recover 2/2 (100%) of GBS strains after LIM Broth enrichment from TransPROTM Liquid Amies and Healthlink swabs in Liquid Amies Gel, Liquid Stuart's, Stuart's Gel, and Amies Charcoal when stored at room temperature up to 24 hours and at 2-8°C for up to 120 hours. GBS was recovered from Granada Medium for all time points and storage conditions tested.

REPRODUCIBILITY

Prior to initiating the study, a panel of 22 blinded isolates (set of 11 organisms tested in duplicate) provided by Hardy Diagnostics was tested at three distinct study sites on five work days to demonstrate reproducibility and to document proficiency in the performance of the test. Agreement of >95% with known test results was required before proceeding with the study. The testing was done with at least one operator and two readers, blinded to each other's results, per site. Strains in the reproducibility panel produced the expected color results with Granada Medium > 95% of the time after 24 hours. All beta-hemolytic GBS isolates tested (100%) were recovered by Granada Medium with the expected orange color reaction for isolated colonies on all days of the reproducibility study.

CONCLUSIONS

The clinical and analytical data summarized above demonstrate that Granada Medium is substantially equivalent to the predicate device.

Regulation Number and Section

§ 866.2360 Selective culture medium.

(a)
Identification. A selective culture medium is a device that consists primarily of liquid or solid biological materials intended for medical purposes to cultivate and identify certain pathogenic microorganisms. The device contains one or more components that suppress the growth of certain microorganisms while either promoting or not affecting the growth of other microorganisms. The device aids in the diagnosis of disease caused by pathogenic microorganisms and also provides epidemiological information on these diseases.(b)
Classification. Class I (general controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 866.9.