(299 days)
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No
The summary describes a chemical solution for sample stabilization and inactivation, with performance studies focused on analytical characteristics like LoD, stability, and inactivation. There is no mention of AI, ML, or any computational analysis of data.
No
The device is described as a transport medium intended for the stabilization, transportation, and inactivation of samples for diagnostic testing, not for treating or diagnosing a disease or condition in a patient.
No
This device is designed to stabilize, transport, and inactivate samples for subsequent testing, not to perform a diagnostic test itself. It prepares the sample for a diagnostic assay (e.g., PCR).
No
The device description clearly states it consists of a storage tube and a stabilization solution, which are physical components, not software.
Based on the provided information, yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The intended use explicitly states that the device is for the "stabilization, transportation and inactivation of infectious unprocessed nasal washes suspected of containing Influenza A virus RNA" and "stabilization, transportation and inactivation of infectious unprocessed sputum samples suspected of containing Mycobacterium tuberculosis DNA from human samples." This clearly indicates that the device is intended to be used in vitro (outside the body) to examine specimens from the human body for the purpose of providing information about a disease or condition (the presence of Influenza A or M. tuberculosis).
- Device Description: The description details a solution designed to inactivate pathogens, lyse cells, and stabilize genetic material (RNA and DNA) from human samples. This is a typical function of a specimen collection and transport device used in IVD workflows.
- Performance Studies: The document describes analytical performance studies (Limit of Detection, Stability, Inactivation) and compatibility studies with extraction platforms and amplification instruments (like real-time PCR). These are standard types of studies performed to validate the performance of IVD devices. The results are presented in terms of recovery rates and Ct values, which are relevant metrics for molecular diagnostic assays.
- Intended User: The intended users are "laboratory personnel, laboratory technicians," which are the typical users of IVD devices in a clinical or diagnostic laboratory setting.
While the document doesn't provide traditional diagnostic accuracy metrics like sensitivity, specificity, PPV, and NPV, the focus on stabilizing and preserving genetic material for downstream molecular testing (implied by the mention of PCR instruments) firmly places this device within the realm of IVD. It's a crucial component in the pre-analytical phase of a molecular diagnostic test.
N/A
Intended Use / Indications for Use
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.
Special conditions for use statement(s): For in vitro diagnostic use only For prescription use only
Product codes (comma separated list FDA assigned to the subject device)
QBD
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:
- Guanidine thiocyanate
- TCEP
- Sodium Citrate
- N-Lauroylsarcosine sodium (NLS)
- Antifoam A, TRIS
- EDTA
- Ethanol (molecular grade)
- HCl
- Nuclease-free water
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
Not Found
Indicated Patient Age Range
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Intended User / Care Setting
Not Found
Description of the training set, sample size, data source, and annotation protocol
Not Found
Description of the test set, sample size, data source, and annotation protocol
Not Found
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
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)
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.
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.
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 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.
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 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.
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 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).
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
§ 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.
0
EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR PrimeStore MTM DECISION SUMMARY
A. DEN Number:
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:
-
- Regulation section:
21 CFR 866.2950
- Regulation section:
-
- Classification:
Class II (Special Controls)
- Classification:
-
- Product code(s):
QBD
- Product code(s):
-
- Panel:
83- Microbiology
- Panel:
1
H. Indications for Use:
1. Indications for use:
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.
2. Special conditions for use statement(s):
For in vitro diagnostic use only For prescription use only
-
- Special instrument requirements:
None
- Special instrument requirements:
I. 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 ●
J. Standard/Guidance Document Referenced (if applicable):
Not applicable
K. Test Principle:
Not applicable
2
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)
3
| MTB
Concentration
(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.
4
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.
| FLUA
Concentration
(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
5
| 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
6
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
7
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
8
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).
| Concentration
of
$M. tuberculosis$
spiked in
sputum | Ratio of Sputum to
PrimeStore MTM | Exposure to PrimeStore MTM Time
(Minutes)
Number of replicates which exhibited
growth after 42 days | | | | | | |
|-----------------------------------------------------------------|--------------------------------------|--------------------------------------------------------------------------------------------------------------|-----|-----|-----|-----|-----|-----|
| | | 1 | 5 | 10 | 15 | 30 | 60 | 180 |
| 1.5 x 106
CFU/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 108
CFU/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