PrimeStore MTM is intended for the stabilization, transportation and inactivation of infectious unprocessed nasal washes suspected of containing Influenza A virus RNA. PrimeStore MTM is also intended for the stabilization, transportation and inactivation of infectious unprocessed sputum samples suspected of containing Mycobacterium tuberculosis DNA from human samples.

Device Description

The PrimeStore MTM device consists of a storage tube with an O-ring and lip seal containing 1.5 mL of the stabilization solution. These components are intended to inactivate Influenza A and Mycoplasma tuberculosis, lyse cells, disrupt/lyse lipid membranes, denatures proteins, inactivates enzymes, and stabilize Influenza A RNA and M. tuberculosis DNA. The transport device is designed for storage of specimens between 36-77 °F (2-25 °C).

The media contains the following reagents:

o Guanidine thiocyanate
o TCEP
Sodium Citrate ●
N-Lauroylsarcosine sodium (NLS) ●
Antifoam A, TRIS ●
EDTA ●
Ethanol (molecular grade)
HCl ●
Nuclease-free water ●

AI/ML Overview

The PrimeStore MTM device is intended for the stabilization, transportation, and inactivation of infectious unprocessed nasal washes suspected of containing Influenza A virus RNA, and infectious unprocessed sputum samples suspected of containing Mycobacterium tuberculosis DNA. The acceptance criteria and the studies proving the device meets these criteria are detailed below.

1. Table of Acceptance Criteria and Reported Device Performance

Criteria Category	Specific Criteria	Reported Device Performance
Limit of Detection (LoD)	Mycobacterium Tuberculosis (MTB): - Recoverable concentration where at least 95% of replicates are within a 3 Ct range.	MTB: - At 10¹ CFU/mL, 25 of 25 replicates had recoverable concentrations, and all fell within the 3 Ct range. Average CT = 34.0, S.D. = 0.98. - At 10⁴ CFU/mL and 10² CFU/mL, all 25 replicates met the acceptance criteria.
	Influenza A (Flu A): - Recoverable concentration where at least 95% of replicates are within a 3 Ct range.	Flu A: - At 10² TCID50/mL, 25 of 25 replicates had recoverable concentrations; one replicate had a high Ct value outside the 3 Ct range, but the overall concentration still met the criterion. Average CT = 34.5, S.D. = 0.88.
Stability	MTB: - +/- 3.0 Ct from time zero for 36 days at 4°C and 27°C, without loss of detection signal.	MTB: - DNA from MTB in PrimeStore MTM showed a variation of 1.6 Ct or less over 36 days at both 4°C and 27°C. Positive samples not stored in PrimeStore MTM demonstrated degraded DNA as time and temperature increased.
	Flu A: - +/- 3.0 Ct from time zero for 29 days at 4°C and for 8 days at 27°C, without loss of detection signal.	Flu A: - RNA from Influenza A in PrimeStore MTM showed a variation of 2.7 Ct over 29 days at 4°C and a variation of 2.0 Ct over 8 days at 27°C. Positive samples not stored in PrimeStore MTM demonstrated degraded RNA as time and temperature increased.
Inactivation	MTB: - No growth in all samples after 42 days of incubation, requiring a specific sputum-to-PrimeStore MTM ratio and minimum exposure time.	MTB: - At 1.5 x 10⁶ CFU/mL, no growth was observed across all ratios (1:3, 1:2, 1:1) and all time points (1 to 180 minutes). - At 1.5 x 10⁸ CFU/mL, inactivation was successful (no growth) with a ratio of at least 1:3 sputum to PrimeStore MTM and a minimum of 60 minutes exposure time. Intermittent growth was observed at other ratios and shorter exposure times.
	Flu A: - Demonstrate rapid inactivation (e.g., log reduction) within specified timelines, while noting potential cytotoxicity of the media at high concentrations.	Flu A: - PrimeStore MTM showed cytotoxicity on MDCK cells when diluted 1:100 but not at 1:1,000. - Rapidly inactivated Influenza A virus with a >4.0 log reduction in concentration at 10 seconds. - Viral CPE could not be observed at < 3.0 logs due to cellular destruction by PrimeStore MTM. - Requires a ratio of at least 1:3 nasal to PrimeStore MTM and a minimum of 10 seconds exposure time to demonstrate inactivation.
Extraction/Amplification Compatibility	MTB: - Show similar Limit of Detection for MTB DNA in PrimeStore MTM compared to PBS control using various nucleic acid extraction and PCR amplification systems.	MTB: - For QIAamp, MagNA Pure 96, and NucliSENS easyMAG, the LoDs for MTB DNA in PrimeStore MTM were mostly similar to or better than the PBS control using an FDA cleared assay (except for QIAamp at lower concentrations). - The ABI 7500 and LightCycler 2.0 PCR platforms showed similar results, with variation within each instrument averaging within 1 Ct, and between instruments ranging from 1 to 1.7 Ct. The QIAamp extraction method increased the LoD by two logs compared to others.

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

Mycobacterium Tuberculosis (LoD & Stability):
- LoD (preliminary): 7 concentrations tested, each in quadruplicate.
- LoD (confirmatory): 25 replicates at 10¹ CFU/mL.
- Stability: 25 replicates extracted at each time point (Day 0, 1, 8, 15, 22, 29, 36) for both 4°C and 27°C.
- Provenance: Spiked pooled sputum known to be negative for MTB. The M. tuberculosis strain used for LoD studies was HN878 (BEI Resources, Catalog No. NR-13647).
Influenza A (LoD & Stability):
- LoD (preliminary): Multiple concentrations, each in triplicate.
- LoD (confirmatory): 25 replicates at 10² TCID50/mL.
- Stability: 25 replicates extracted at each time point (Day 0, 1, 8, 15, 22, 29 for 4°C; Day 0, 1, 8 for 27°C).
- Provenance: Spiked pooled, clinically negative nasal washes. Influenza A strain used was A/Texas/78209/2008 (H3N2).
Mycobacterium Tuberculosis (Inactivation):
- Sample Source: Sputum samples not submitted for MTB investigation obtained from the diagnostic laboratory at the University of Pretoria (Pretoria, South Africa).
- Test Set: Good quality purulent sputum specimens (Bartlett test score of 2+) were split and spiked with MTB H37Rv strain (1.5 x 10⁶ and 1.5 x 10⁸ CFU/mL). Inoculated into PrimeStore MTM, incubated in triplicate for each time point (1, 5, 10, 30, 60, and 180 minutes) and ratio (1:3, 1:2, 1:1 sputum to PrimeStore MTM).
Influenza A (Inactivation):
- Test Set: Influenza/A/Wuhan/359/95 (10⁰ TCID50/ml) incubated with PrimeStore MTM for 10, 30, and 60 seconds.
- Provenance: Not specified as clinical or laboratory samples, but refers to "Influenza/A/Wuhan/359/95" which is a known laboratory strain.
Extraction/Amplification Compatibility:
- MTB: Sputum spiked with MTB at final concentrations ranging from 3 to 250,000 CFU/mL. For each extraction method, 20 samples were tested at various concentrations. For amplification comparison, three 10-fold concentrations (1 X 10, 1 X 10³, and 1 x 10⁴ CFU/mL) of MTB extracted from sputum stored in PrimeStore MTM were tested in duplicate on each instrument.
- Provenance: Spiked sputum samples.

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

The document does not mention the use of human experts to establish ground truth. The ground truth for the analytical performance studies (LoD, stability, inactivation, compatibility) was established through laboratory-controlled spiking experiments using known concentrations of bacterial or viral strains into clinically negative matrices, followed by molecular detection methods (real-time PCR) or culture methods.

4. Adjudication Method for the Test Set

Not applicable. The studies are analytical performance studies based on objective measurements (Ct values, growth/no growth) and not subjective interpretation requiring adjudication among experts.

5. If a Multi Reader Multi Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

Not applicable. This device is a transport medium for nucleic acids, not an AI-assisted diagnostic tool. Therefore, MRMC studies and assessment of AI assistance for human readers are not relevant.

6. If a Standalone (i.e. algorithm only without human-in-the loop performance) was done

Not applicable. This device is not an algorithm or a software-based diagnostic tool. It is a physical transport medium. The "standalone" performance here refers to the device's ability to preserve and inactivate without human intervention during the storage and transport phase, which is what the analytical studies (stability, inactivation) directly assess.

7. The Type of Ground Truth Used

The ground truth used in these analytical studies is primarily:

Known concentrations of specific microbial strains: For LoD and stability, quantified CFU/mL (for MTB) or TCID50/mL (for Influenza A) were spiked into relevant matrices.
Absence of target nucleic acid/organism: Clinically negative pooled sputum or nasal washes were used as matrices for spiking experiments, serving as negative controls.
Absence of microbial growth: For inactivation studies, the absence of growth after incubation in culture media (MGIT 960 system) served as the ground truth for successful inactivation.
Real-time PCR Ct values: These values serve as a quantitative measure of nucleic acid presence and concentration, forming the basis for assessing stability and LoD within predefined acceptance ranges.

8. The Sample Size for the Training Set

Not applicable. This device is a physical transport medium and does not involve machine learning or AI, and therefore does not utilize a "training set" in the context of algorithm development. The studies described are analytical validation studies for device performance.

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

Not applicable, as there is no training set for this type of device.

Summary

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EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR PrimeStore MTM DECISION SUMMARY

A. DEN Number:

DEN170029

B. Purpose for Submission:

De Novo request for evaluation of automatic class III designation for the PrimeStore MTM

C. Measurands:

Storage and stability of nucleic acids from Mycobacterium Tuberculosis and Influenza A virus.

D. Type of Device:

Transport device for the stabilization of microbial nucleic acids

E. Applicant:

Longhorn Vaccines and Diagnostics, LLC

F. Proprietary and Established Names:

PrimeStore MTM

G. Regulatory Information:

1. Regulation section:
  21 CFR 866.2950
1. Classification:
  Class II (Special Controls)
1. Product code(s):
  QBD
1. Panel:
  83- Microbiology

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H. Indications for Use:

1. Indications for use:

2. Special conditions for use statement(s):

For in vitro diagnostic use only For prescription use only

1. Special instrument requirements:
  None

I. Device Description:

The media contains the following reagents:

o Guanidine thiocyanate
o TCEP
Sodium Citrate ●
N-Lauroylsarcosine sodium (NLS) ●
Antifoam A, TRIS ●
EDTA ●
Ethanol (molecular grade)
HCl ●
Nuclease-free water ●

J. Standard/Guidance Document Referenced (if applicable):

Not applicable

K. Test Principle:

Not applicable

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L. Performance Characteristics:

1. Analytical performance:

a. Limit of detection

LoD testing was conducted to determine the lowest concentration of organisms that contains measurable nucleic acids that can be repeatedly recovered from the transport media with a greater than 95% accuracy. The LoD studies for M. tuberculosis and Influenza A were designed using a specific extraction platforms and amplification instrument to establish a concentration of organisms which will form the basis of additional testing. Testing near the LoD for the additional studies will challenge PrimeStore MTM ability to preserver nucleic acids from degradation under a variety of test conditions.

Mycobacterium Tuberculosis Limit of Detection:

LoD testing was initially performed by spiking seven concentrations of MTB diluted 1:10 into pooled sputum which was known to be negative for MTB. The spiked sputum was then added at a concentration of 1:3 into the PrimeStore MTM for recovery using the PrimeXtract extraction kit and amplification using the ABI 7500 realtime PCR instrument. The study was conducted in quadruplicate to determine a recoverable concentration of MTB after being spiked into sputum and subsequently added to PrimeStore MTM. No detection (Ct = 40) was observed for one of the replicates at the 10% CFU/mL concentration. All other concentrations demonstrated recovery of MTB DNA from all the replicates. Table 1 shows the results of MTB organisms diluted 1:10 at multiple concentrations, extraction of DNA and amplification at each concentration. Additional LoD testing was provided at a concentration of 101 CFU/mL with 25 replicates. An acceptance criteria with a range of 3 C; was used to determine the concentration that yielded at least a 95% of the replicates were recoverable within this range. At a concentration of 101 CFU/mL 25 of 25 replicates had recoverable concentrations and all replicates fell within the range of 3 Ct values, Table 2. The MTB DNA extracted from PrimeStore MTM had an average CT = 34.0; S.D. = 0.98 at a concentration of 101 CFU/mL.

The M. tuberculosis strain used for LoD studies was strain HN878 (BEI Resources, Catalog No. NR-13647)

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MTBConcentration(CFU/mL)	Rep 1(Ct)	Rep 2(Ct)	Rep 3(Ct)	Rep 4(Ct)	Average(Ct)	SD(Ct)
106	17.8	17.7	17.6	17.6	17.7	0.1
105	22.1	22.6	21.9	22.1	22.2	0.4
104	23.5	23.4	23.2	23.5	23.4	0.2
103	26.3	26.7	26.6	26.8	26.6	0.2
102	29.4	29.3	29.6	28.7	29.3	0.2
101	32.8	32.6	33.4	32.2	32.8	0.4
10	34.4	40.0	34.6	35.6	36.2	3.2

Table 1. MTB preliminary Limit of Detection

Table 2. MTB LoD

10¹ CFU/mL
Replicates	Ct Value
1	33.3
2	33.2
3	33.0
4	32.7
5	33.0
6	34.1
7	33.6
8	33.4
9	35.4
10	35.1
11	35.3
12	35.4
13	36.0
14	35.1
15	34.1
16	33.4
17	33.2
18	32.3
19	34.3
20	34.3
21	33.3
22	33.1
23	33.9
24	34.0
25	34.6
AVG:	34.0
SD:	1.0

LoD testing at both 104 CFU/mL and 102 CFU/mL resulted in all 25 replicates for each concentration to meet the pre-defined acceptance criteria.

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Influenza A Limit of Detection:

LoD testing was initially performed by spiking multiple concentrations of influenza A diluted 1:10 into pooled, clinically negative, nasal washes. The final concentration of each spiked nasal was then added to achieve a final ratio of 1:3 specimen to PrimeStore MTM for recovery using the PrimeXtract extraction kit and amplification in conjunction with the ABI 7500 realtime PCR instrument. The study objective was to determine the detectable concentration of influenza A (A/Texas/78209/2008 (H3N2)) after spiking into nasal wash and added to PrimeStore MTM. No detection (C: = 40) was observed at the 101 TCID50/mL concentration. All other concentrations demonstrated recovery of Influenza A. Table 3 shows the results of the 1:10 dilutions of Influenza A. Additional LoD testing was provided at a concentration of 102 TCID50/mL. 102 TCID50/mL was replicated 25 times. An acceptance criteria with a range of 3 Ct was used to determine the concentration that yielded at least a 95% of the replicates were recoverable within this range. At a concentration of 102 TCID50/mL 25 of 25 replicates had recoverable concentrations; however, one replicate had a high C value and fell outside the range of 3 Ct values, Table 4. The Influenza A RNA extracted from PrimeStore MTM had the following performance average CT = 34.5; S.D. = 0.88 at a concentration of 102 TCIDso/mL.

FLUAConcentration(TCID50/mL)	Rep 1(Ct)	Rep 2(Ct)	Rep 3(Ct)	Average(Ct)	SD(Ct)
105	25.5	25	25.1	25.2	0.26
104	28.1	28	27.9	28	0.10
103	31.4	31.3	32.1	31.6	0.44
102	35.3	35.5	34.9	35.2	0.31
101	40	40	40	40	0

Table 3. Influenza A Preliminary Limit of Detection

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102 TCID50/mL
Replicates	CT Value
1	35.8
2	33.7
3	34.6
4	33.3
5	34.2
6	33.9
7	34.1
8	36.3
9	34.9
10	33.9
11	34.6
12	34
13	34.2
14	33.8
15	34.7
16	34.4
17	33.6
18	34.4
19	34.5
20	34.4
21	34.9
22	36.1
23	35.5
24	32.6
25	35.6
AVG:	34.5
SD:	0.88

Table 4. Influenza A LoD

LoD testing at 102 TCID50 resulted in all 25 replicates for the concentration to meet the pre-defined acceptance criteria.

b. Stability

Mycobacterium Tuberculosis stability:

Stability studies evaluated the stability of DNA from whole organism Mycobacterium tuberculosis (MTB; 1x103 CFU/mL) spiked into clinically negative sputum samples incubated in PrimeStore MTM at: refrigerated temperature (4°C, 39.2°F) for 36 days, Table 5, and ambient temperature (27°C, 80.6°F) for 36 days, Table 6. DNA from MTB (Strain HN878) in PrimeStore MTM was extracted using PrimeXtract spin columns. Nucleic acid stability was measured and analyzed by real-time PCR using the ABI 7500 (Thermo Fisher Scientific) instrument. The study analyzed 25 replicates extracted at each time point and at each temperature range. An initial time point designated as Day 0 for each temperature was included as the initial Cr average from

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which all other time points were compared to. Time points for sample extractions were performed at Day 0, 1, 8, 15, 22, 29, 36, with an embedded Internal Negative Control (INC) included at both temperatures. The INC consisted of a test that used negative clinical samples in PrimeStore MTM, i.e., sputum without MTB. Additionally, a control that contained clinical specimen plus target, i.e., sputum plus MTB, was incubated in PBS, without PrimeStore MTM ((POS-(PS)), and included in the evaluation. A pre-defined acceptance criteria of (+/-) 3.0 C; from time zero was used to establish stability and preservation of nucleic acids (DNA from MTB) as determined by real-time PCR, for 4 and 27°C without loss of detection signal using statistical analysis.

Day (4°C)	0	1	8	15	22	29	36
AVG (Ct):	29.8	29.5	29.4	29.3	30.8	29.5	29.3
SD (Ct):	0.8	0.4	0.4	0.3	0.3	0.5	0.7
INC:	NEG	NEG	NEG	NEG	NEG	NEG	NEG
POS (-PS) (Ct):	28.9	29.7	29.8	29.9	32.4	32.2	30.8

Table 5 MTB (1 x 103 CFU/mI ) stability at 4°C

Table 6 MTR (1 x 103 CFU/mI ) stability at 27°C

Day (27°C)	0	1	8	15	22	29	36
AVG (Ct):	29.4	29.4	29.4	29.4	31.0	29.8	29.4
SD (Ct):	0.2	0.2	0.3	0.3	0.3	0.4	0.3
INC:	NEG	NEG	NEG	NEG	NEG	NEG	NEG
POS (-PS) (Ct):	29.3	34.6	34.9	34.7	34.3	34.8	34.7

Stability testing of DNA from M. tuberculosis whole organism spiked into sputum and stored in PrimeStore MTM resulted in a variation of 1.6 C; or less over 36 days at both 4°C and 27°C. The Positive samples not stored in Primestore MTM demonstrated degraded DNA as time and temperature increased.

Influenza A stability:

Stability studies evaluated the stability of Influenza A virus (Flu A: 1 x 102 TCID50/mL) RNA from clinical samples incubated in PrimeStore MTM at: refrigerated temperature (4°C, 39.2°F) for 29 days, Table 7, and ambient temperature (27°C, 80.6°F) for 8 days, Table 8. The clinical matrix for the flu A stability study was nasal washes. The RNA was stabilized under the predefined conditions and was then extracted using PrimeXtract spin columns. Nucleic acid stability was measured and analyzed with the ABI 7500 (Thermo Fisher Scientific) instrument. The experiment analyzed 25 replicates extracted at each time point and for each temperature range. An initial time point designated as Day 0 and was included as the initial Ct average for each of the two temperature ranges tested. Time points for sample extractions were performed at Day 0, 1, 8, 15, 22 and 29 for refrigerated temperature (4°C, 39.2°F) and Day 0, 1 and 8 for ambient temperature (27°C, 80.6°F). An embedded Internal Negative Control (INC) was included in the study at both temperatures. The INC consisted of a test using negative clinical sample in PrimeStore MTM, i.e., nasal washing without Flu A virus. Additionally, a control that contained clinical specimen plus target, i.e., nasal washing plus Flu A was incubated

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in PBS (POS (-PS)) and included in the evaluation. Pre-defined acceptance criteria of (+/-) 3.0 C; from time zero was used to establish stability and preservation of nucleic acids (RNA from Flu A virus) as determined by real-time PCR, for 4°C and 27°C without loss of detection signal using statistical analysis.

Day (4°C)	0	1	8	15	22	29
AVG (Ct):	31.9	32.3	34.4	34.3	34.1	34.6
SD (Ct):	0.6	0.8	1.3	0.9	0.8	0.8
INC:	NEG	NEG	NEG	NEG	NEG	NEG
POS (-PS) (Ct):	34.4	33.6	35.8	NEG	36.5	NEG

Table 7. Flu A (1 x 103 TCID50 / mL) stability at 4°C

Table 8. Flu A (1 x 103 TCID50 / mL) stability at 27°C

Day (27°C)	0	1	8
AVG (Ct):	32.3	33.0	34.3
SD (Ct):	0.4	0.9	1.6
INC:	NEG	NEG	NEG
POS (-PS) (Ct):	34.4	34.9	37.6

Stability testing of RNA from Influenza A whole virus spiked into nasal wash and stored in PrimeStore MTM resulted in a variation of 2.7 Cr over 29 days at 4°C and a variation of 2.0 Ct over 8 days at 27℃. The Positive samples not stored in Primestore MTM demonstrated degraded RNA as time and temperature increased.

c. Inactivation

Mycobacterium tuberculosis inactivation:

Sputum samples not submitted for MTB investigation were obtained from the diagnostic laboratory at the University of Pretoria (Pretoria, South Africa) and assessed for the presence of acid-fast bacilli by smear microscopy, cultured by MGIT 960 system to confirm the absence of MTB followed by quality assessment using the Bartlett Scoring System. Good quality purulent sputum specimens (Bartlett test score of 2+) were included for use in the spiking matrix experiments. These sputa were split and spiked with MTB H37Rv strain with concentrations of 1.5 x106 and 1.5 x 108 CFU/mL followed by inoculation into PrimeStore MTM (without decontamination or other pre-culture steps). A matrix assessment to determine effect of concentration was performed by adding to 1 mL of spiked sputum to 3, 2, and 1 mL of PrimeStore MTM. Samples were incubated in triplicate at ambient temperature for 1, 5, 10, 30, 60 and 180 minutes, including two positive and negative controls, and analyzed using the MGIT 960 system. Effective inactivation at each concentration and time point is defined as no growth in all samples after 42 days.

Transport media inactivation results:

No growth of MTB was observed when the concertation of MTB in sputum was 1.5 x 100 CFU/mL and incubated for a time of greater than 5 minutes with PrimeStore

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MTM. Intermittent growth was observed at each time point when MTB concentration was1.5 x 10° CFU/ml and added at a ratio of 1:1, 1:2 and 1:3 sample to PrimeStore MTM. Growth was determined after 42 days of incubation (Table 9. below).

Concentrationof$M. tuberculosis$spiked insputum	Ratio of Sputum toPrimeStore MTM	Exposure to PrimeStore MTM Time(Minutes)Number of replicates which exhibitedgrowth after 42 days
		1	5	10	15	30	60	180
1.5 x 106CFU/mL	1-3	0/3	0/3	0/3	0/3	0/3	0/3	0/3
	1-2	1/3	0/3	0/3	0/3	0/3	0/3	0/3
	1-1	0/3	0/3	0/3	0/3	0/3	0/3	0/3
1.5 x 108CFU/mL	1-3	0/3	1/3	0/3	1/3	0/3	0/3	0/3
	1-2	3/3	0/3	0/3	0/3	2/3	0/3	0/3
	1-1	3/3	1/3	2/3	0/3	0/3	1/3	2/3

Table 9. MTB Growth at 42 days after exposure to PrimeStore MTM at different concentrations and ratios

The data shows that PrimeStore MTM must be used at a ratio of at least 1:3 sputum to PrimeStore MTM and a minimum of 60 minutes exposure time to demonstrate MTB inactivation.

Influenza A inactivation:

Influenza/A/Wuhan/359/95 (10° TCID50/ml) were incubated with PrimeStore MTM for 10, 30 and 60 seconds. Influenza only and PrimeStore MTM only were also incubated accordingly to serve as internal controls. Four days after inoculation, the cells were fixed and stained with 0.06% crystal violet in 1% glutaraldehyde. Wells that were not stained demonstrated the cytopathic effect (CPE) of the virus. The titer of the virus was recorded as the TCID50.

Inactivation rate:

PrimeStore MTM showed cytotoxicity on MDCK cells when diluted 1:100 but not cytotoxicity at 1:1.000. The mixture of PrimeStore MTM and Influenza had similar profiles as PrimeStore MTM alone with 10, 30 and 60 second incubations, while Influenza only samples had viral loads of 1 x 107.75, 1 x 10 7.5 and 1 x 107.75 TCIDs0 respectively.

Transport media inactivation:

PrimeStore MTM rapidly inactivated Influenza virus with a >4.0 log reduction in concentration at 10 seconds. Viral CPE could not be observed at < 3.0 logs due to cellular destruction by PrimeStore MTM, Table 10 below.

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	10s incubation1 X 10x TCID50	30s incubation1 X 10x TCID50	60s incubation1 X 10x TCID50
Flu A only	7.75	7.5	7.75
Flu A andPrimeStoreMTM	< 3.0	< 3.0	< 3.0
PrimeStoreMTM only*	< 3.0	< 3.0	< 3.0

Table 10 MTB inactivation in PrimeStore MTM

*PrimeStore shows cytotoxicity on MDCK cells when diluted to 1:1,000.

The PrimeStore MTM must be used at a ratio of at least 1:3 nasal to PrimeStore MTM and a minimum of 10 seconds exposure time to demonstrate inactivation of Influenza A. Measuring Influenza inactivation below 1 X 103 was not possible because of the cytotoxic affects PrimeStore MTM has on the cell culture based testing.

2. Comparison Studies:

Clinical Comparison: a.

Method comparison is not applicable for a nucleic acid transport device. The device itself does not provide a result that can be used in making a clinical decision. Bench testing studies were done to determine the ability of PrimeStore MTM to stabilize the nucleic acids from MTB and Flu A samples. Nucleic Acid stability was performed using clinically negative matrix pooled and a minimum of 50 distinct matrices were used for each analyte for the stability study.

b. Extraction platform and amplification instrument compatibility

Mycobacterium Tuberculosis:

A study to establish that the PrimeStore MTM media is compatible with commercially available nucleic acid isolation reagents was performed using MTB spiked into sputum with final concentrations ranging from 3 to 250,000 CFU/mL. Aliquots of sputum in PrimeStore MTM were prepared and processed using the following nucleic acid extraction systems as per manufacturers' instructions:

The QIAamp DNA mini kit (Qiagen, Hilden, Germany)
MagNA Pure 96 System (Roche Diagnostics, Mannheim, Germany) using the DNA Bacterial/Viral small volume kit
NucliSENS easyMAG (bioMerieux, Marcy I'Etoile, France) and using the generic protocol.
An FDA cleared assay was performed as a control. ●

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An aliquot of a sputum sample was tested using the control assay prior to inoculating in PrimeStore MTM, as instructed by the manufacturer. An input volume of 200 µL PrimeStore MTM sample was used along with the manufacturer recommended output volume of 50 uL (QIAamp and NucliSENS) and 100 uL (MagNA Pure 96) for further analysis. Real-time PCR targeting the MTB specific insertion sequence element 6110 (IS6110) was performed on the LightCycler 480II platform (Roche Diagnostics, Mannheim, Germany) to identify MTB in the DNA extracts. For each extraction method, the limit of detection for MTB DNA in PrimeStore MTM was similar to the PBS control as noted in Table 11.

	MagNA Pure(PrimeStoreMTM)1,2	EasyMAG(PrimeStoreMTM)1,3	QIAamp(PrimeStoreMTM)1,4	FDA Clearedcontrol (PBSControl)
MTBConcentration(CFU/mL)	Number of positive replicates out of 20 samples
2.5 X 105	20	20	20	20
2.5 X 104	20	20	20	20
2.5 X 103	20	20	16	13
2.5 X 102	20	20	18	5
2.5 X 101	13	10	7	3

Table 11. MTB extraction comparison

1 LightCycler 480II platform (Roche Diagnostics, Mannheim, Germany).

2 MagNA Pure LoD, 2.5 X 102 CFU/mL

3 EasyMAG LoD, 2.5 X 102 CFU/mL

4 QIAamp LoD, 2.5 X 104 CFU/mL

A study was performed to establish the performance of PrimeStore MTM with the same amplification assay performed using two PCR amplification instruments. The study evaluated the detection of three 10-fold concentrations (1 X 10, 1 X 104, and 1x103 CFU/mL) of MTB (Strain HN878, 1x108 CFU/mL) extracted from sputum stored in PrimeStore MTM. Amplification was performed using two commercial real-time PCR platforms: 1) The ABI 7500 (Thermo Fisher Scientific) and 2) LightCycler 2.0 (Roche Diagnostics). The results using the two platforms are similar as shown in Table 12.

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			Average CT Value
ABI 7500 Instrument	CT Value Rep 1	CT Value Rep 2	(High Concentration)
105 Extraction 1	19.2	18.8	19.0
105 Extraction 2	19.4	18.9	19.1
104 Extraction 1	22.0	22.1	22
104 Extraction 2	21.6	22.0	21.8
103 Extraction 1	24.4	24.7	24.5
103 Extraction 2	24.8	24.7	24.7
NTC	NEG	NEG
NTC	NEG	NEG
Positive Control	POS	POS

			Average CT Value
Roche LightCycler	CT Value Rep 1	CT Value Rep 2	(Low Concentration)
105 Extraction 1	19.9	20.3	20.1
105 Extraction 2	20.3	20.0	20.1
104 Extraction 1	23.4	22.8	23.1
104 Extraction 2	22.6	23.1	22.8
103 Extraction 1	25.6	25.9	25.7
103 Extraction 2	26.9	26.7	26.8
NTC	NEG	NEG
NTC	NEG	NEG

Table 12: Amplification comparison for two amplification instruments

The test results indicate that the extraction method used may affect the LoD of the downstream assay. The extraction methods listed above all indicate they are compatible with PrimeStore MTM but the QIAamp increases the LoD by two logs. A similar result is observed when the ABI 7500 and Roche LightCycler are compared to each other. The variation within each instrument is on average within 1 C. The variation between instruments is between 1 to 1.7 Cc depending on the concentration of M. tuberculosis used for testing. The results indicate that the extraction platform and amplification instrument may need additional validation to ensure robust results are provided.

c. Matrix Equivalence Study
Not applicable
1. Clinical Studies:
  Not applicable
1. Clinical cut-off:
  Not applicable

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1. Expected values/Reference range:
  Not applicable.

M. Instrument Name

Not applicable

N. System Descriptions:

1. Modes of Operation:
  Does the applicant's device contain the ability to transmit data to a computer, webserver, or mobile device?

Yes ___________ or No ________________________________________________________________________________________________________________________________________________________

Does the applicant's device transmit data to a computer, webserver, or mobile device using wireless transmission?

Yes ___________ or No ____X

1. Software:
  FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types:

Yes ___________ or No X

Hazard Analysis and software development are not applicable to this class of device:

1. Specimen Identification:
  Transport device are not intended to identify specimens. The device itself does not provide a result that can be used in making a clinical decision. Transport devices are intended to preserve and stabilize nucleic acids.
1. Specimen Sampling and Handling: See section L.1.b regarding specimen stability.
1. Calibration: Not applicable.
1. Quality Control: Not applicable.

O. Other Supportive Instrument Performance Characteristics Data Not Covered in the "Performance Characteristics" Section above:

Not applicable

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P. Proposed Labeling:

The labeling is sufficient and satisfies the requirements of 21 CFR parts 801 and 809 as well as the Special Controls for this type of device.

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Q. Identified Risks to Health and Mitigation Measures:

Identified Risks to Health	Mitigation Measures
Failure to stabilize pathogen nucleic acidresulting in a false negative result	General Controls and Special Controls (1),(2), and (3)
Failure to inactivate the specimen	General Controls and Special Controls (1),(2)(i), (2)(ii), (3)(i), (3)(ii) and (3)(iv)

R. Benefit/Risk Analysis:

Summary
Summary ofthe Benefit(s)	0PrimeStore MTM is a transport media that inactivates and stabilizes sputum specimensfrom patients suspected of influenza A or M. tuberculosis infection.
	PrimeStore MTM facilitates downstream diagnostic testing by stabilizing pathogen●nucleic acids to allow storage and transport of specimens between facilities or frompatient care areas to the laboratory.
	PrimeStore MTM may help prevent occupational transmission of infection to●laboratory personnel by inactivating infectious agents.
Summary ofthe Risk(s)	· False negative results or failure to inactivate infectious pathogens are the primary risksassociated with use of the PrimeStore MTM transport media.
	· If PrimeStore MTM does not stabilize pathogen nucleic acids, a false negative resultcould occur and may result in a delay of antimicrobial therapy, with subsequentworsening of infection and associated increase in morbidity or mortality.
	· Failure to inactivate pathogen nucleic acids may increase risk of transmission tolaboratory personnel.
Summary ofOtherFactors	None
ConclusionsDo theprobablebenefitsoutweigh theprobablerisks?	The probable benefits of the PrimeStore MTM device outweigh the potential risks in lightof the listed special controls and applicable general controls. The PrimeStore MTMinactivates and stabilizes influenza A and M. tuberculosis nucleic acids, which may protectlaboratory workers from occupational exposures and assist downstream molecular assaysto detect Influenza A and M. tuberculosis. The proposed special controls will ensure thaterrors will be uncommon, and potential errors are further mitigated by current laboratorypractices, which include universal precautions to protect laboratory technicians. ThePrimeStore MTM performance characteristics suggest that the device will be safe andeffective, if used as directed by the package insert, and could provide potential benefits topatients and laboratory personnel by facilitating the safe transport of clinical specimens.

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S. Patient Perspectives

This submission did not include specific information on patient perspectives for this device.

T. Conclusion:

The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 866.2950. FDA believes that the stated special controls, and applicable general controls provide reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following:

Product Code:	QBD
Device Type:	Microbial nucleic acid storage and stabilization device
Class:	II (special controls)
Regulation:	21 CFR 866.2950

(a) Identification. A microbial nucleic acid storage and stabilization device is a device that consists of a container and reagents intended to stabilize microbial nucleic acids in human specimens for subsequent isolation and purification of nucleic acids for further molecular testing. The device in not intended for preserving morphology or viability of microorganisms.
(b) Classification. Class II (special controls). the special controls for this device are:
The intended use for the 21 CFR 809.10 labeling must include a detailed description (1) of microorganisms and types of human specimens intended to be preserved.
(2) The 21 CFR 809.10(b) labeling must include:
- (i) A detailed device description, including all device components.
- (ii) Performance characteristics from applicable analytical studies, including but not limited to, nucleic acid stability and microorganism inactivation.
- (iii) A limiting statement that erroneous results may occur when the transport device is not compatible with molecular testing.
- (iv) A limiting statement that the device has only been validated to preserve the representative microorganisms used in the analytical studies.
(3) Design verification and validation must include the following:
- (i) Overall device design including all device components and all control elements incorporated into the analytical validation procedures.
- (ii) Thorough description of the microorganisms and methodology used in the

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validation of the device including, but not limited to, extraction platforms and assays used for the detection of preserved nucleic acids.

(iii) The limit of detection (LoD) of the molecular test used to establish microorganism nucleic acid stability.

Regulation Number and Section

§ 866.2950 Microbial nucleic acid storage and stabilization device.

(a)
Identification. A microbial nucleic acid storage and stabilization device is a device that consists of a container and reagents intended to stabilize microbial nucleic acids in human specimens for subsequent isolation and purification of nucleic acids for further molecular testing. The device is not intended for preserving morphology or viability of microorganisms.(b)
Classification. Class II (special controls). The special controls for this device are:(1) The intended use for the labeling required under § 809.10 of this chapter must include a detailed description of microorganisms and types of human specimens intended to be preserved.
(2) The labeling required under § 809.10(b) of this chapter must include the following:
(i) A detailed device description, including all device components;
(ii) Performance characteristics from applicable analytical studies, including nucleic acid stability and microorganism inactivation;
(iii) A limiting statement that erroneous results may occur when the transport device is not compatible with molecular testing; and
(iv) A limiting statement that the device has only been validated to preserve the representative microorganisms used in the analytical studies.
(3) Design verification and validation must include the following:
(i) Overall device design, including all device components and all control elements incorporated into the analytical validation procedures;
(ii) Thorough description of the microorganisms and methodology used in the validation of the device including, extraction platforms and assays used for the detection of preserved nucleic acids; and
(iii) The limit of detection (LoD) of the molecular test used to establish microorganism nucleic acid stability.