(4 days)
None
Not Found
No
The description focuses on RT-PCR technology and automated sample processing, with no mention of AI or ML algorithms for data analysis or interpretation.
No.
This device is an in vitro diagnostic (IVD) device used to detect enterovirus (EV) RNA in cerebrospinal fluid (CSF) to aid in the diagnosis of enterovirus infection. It does not provide any treatment or therapeutic benefit.
Yes
The device is intended for the "presumptive qualitative detection of enterovirus (EV) RNA... to be used as an aid in the laboratory diagnosis of enterovirus infection in patients with a clinical suspicion of meningitis or meningoencephalitis," which are all functions of a diagnostic device.
No
The device is a system that includes hardware (GeneXpert Dx System instrument and cartridges) and reagents, in addition to software. It is not solely software.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The intended use explicitly states it is for the "presumptive qualitative detection of enterovirus (EV) RNA in cerebrospinal fluid (CSF) specimens". This involves testing a sample taken from the human body.
- Purpose: The test is used "as an aid in the laboratory diagnosis of enterovirus infection in patients with a clinical suspicion of meningitis or meningoencephalitis." This is a diagnostic purpose.
- Method: The device uses "reverse transcription polymerase chain reaction (RT-PCR)" to detect the target RNA. This is a common method used in in vitro diagnostic tests.
- Specimen Type: The test is performed on "cerebrospinal fluid (CSF) specimens", which are samples taken from the human body.
- Setting: The device is used in a "laboratory setting".
All of these characteristics align with the definition of an In Vitro Diagnostic device, which is a medical device intended for use in vitro for the examination of specimens derived from the human body solely or principally for the purpose of providing information concerning a physiological or pathological state, or concerning a congenital abnormality, or to determine the compatibility of a transplant, blood or tissue with a potential recipient, or to monitor therapeutic measures.
N/A
Intended Use / Indications for Use
The Cepheid® Xpert EV assay is a reverse transcription polymerase chain reaction (RT-PCR) using the GeneXpert® Dx System for the presumptive qualitative detection of enterovirus (EV) RNA in cerebrospinal fluid (CSF) specimens from individuals with signs and symptoms of meningitis. This test, in conjunction with other laboratory results and clinical information, may be used as an aid in the laboratory diagnosis of enterovirus infection in patients with a clinical suspicion of meningitis or meningoencephalitis.
Assay performance characteristics have not been established for immunocompromised or immunosuppressed patients.
CAUTION: The results obtained with the Xpert EV assay should be used only as an adjunct to clinical observation and other information available to the physician. Positive Xpert EV results do not rule out other causes of meningitis, including bacteria, mycobacteria, other viruses (e.g. herpes family viruses, arboviruses, mumps virus, etc) and fungi.
Product codes (comma separated list FDA assigned to the subject device)
OAI
Device Description
The Xpert EV assay is designed to detect enterovirus (EV) RNA (enterovirus genome 5' untranslated region (UTR) between nucleotide 452 and 596) in CSF samples from patients exhibiting meningitis like symptoms. The assay includes reagents, primers, and probes for the simultaneous detection of nucleic acid from the target EV and the sample-processing control/internal control (SPC/IC). The assay includes the SPC/IC to verify adequate processing of the target virus and monitors the presence of inhibitors in the RT-PCR assay to avoid a false negative result. The assay also includes a probe check control to verify reagent rehydration, probe integrity, and reaction-tube filling in the cartridge.
The assay is run on the GeneXpert Dx System. The GeneXpert Dx System automates and integrates sample purification, nucleic acid amplification, and detection of the target sequence using real-time PCR and RT-PCR assays. The system consists of an instrument, personal computer, and preloaded software for running tests on collected samples and viewing the results. The system requires the use of single-use disposable GeneXpert cartridges that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated. The above described sample-processing control/internal control (SPC/IC) is named CIC in the GeneXpert Dx System software.
To run a test, the CSF sample and four reagents are transferred into designated chambers of the Xpert EV cartridge. The GeneXpert Dx System performs sample preparation by lysing the virus and SPC (armored pseudovirus RNA), binding the RNA to the capture matrix, and eluting the RNA. The RNA is mixed with dry RT reagents and transferred into the reaction tube for preparation of cDNA. The cDNA is then mixed with dry PCR reagents and transferred into the reaction tube for real-time PCR and detection. The EV primers and probe amplify and detect a consensus region of the enterovirus 5' untranslated region (UTR). The test takes approximately 2.5 hours.
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
Cerebrospinal fluid (CSF)
Indicated Patient Age Range
Not Found
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)
Precision/Reproducibility:
Study 1: Multi-center, blinded study using a precision panel of four specimens. Three sites tested each panel three times per day over 10 testing days, for a total of 90 results per panel specimen. The panel consisted of a negative sample and three positive samples (CVA6 (134), CVA9 (320), CVA17 (3)), each with a specific EV serotype spiked into synthetic CSF close to the limit of detection.
Results: Negative samples were 30/30 correctly classified at all sites. For positive samples: CVA6 (134) was 30/30, 30/30, 29/29 (one site had two samples with no result); CVA9 (320) was 30/30 at all sites; CVA17 (3) was 30/30, 30/30, 29/29 (one site had two samples with no result). Total agreement was 100% for Site 1 and 2, and 100% for Site 3 (118/118). Overall %CV for Ct values ranged from 1.22% to 3.15%.
Study 2: A reproducibility study conducted over four different days on 31 GeneXpert instruments and 121 ICORE modules. Two whole virus subtypes (Coxsackievirus CVA9 and Enterovirus EV70) were spiked into human negative CSF at 2 x LoD and 4 x LoD. Negative samples were tested 20 times each day, positive samples 5 times per day.
Results: Negative: 78/78 total agreement (100%). CA9 2X LOD: 18/20 total agreement (90%), average Ct 36.56, SD 0.48, %CV 1.31%. CA9 4X LOD: 19/19 total agreement (100%), average Ct 35.28, SD 0.6, %CV 1.70%. EV70 2X LOD: 20/20 total agreement (100%), average Ct 37.2, SD 1.3, %CV 3.49%. EV70 4X LOD: 20/20 total agreement (100%), average Ct 36.29, SD 0.72, %CV 1.98%. Of 157 reportable results, 155 were correctly scored.
Linearity/assay reportable range:
Study: Serial dilution of CVA9 (2e9 TCID50/mL) in EV Negative synthetic CSF, duplicates at each dilution were tested.
Results: Detects CVA9 over 8 logs. The assay is linear over this range with an R2 value of 0.9989.
Detection limit:
Study: Replicates of 20, along with 20 EV negative samples, were run for CVA6, CVA9, CVA17, EV70, and Poliovirus 1 (PV1) in pooled EV negative human sample.
Results: CVA9 LOD: 80.0 TCID50/mL; EV70 LOD: 1.3 TCID50/mL; PV1 LOD: 4.0 TCID50/mL; CVA17 LOD: 1.0 TCID50/mL; CVA6 LOD: 33.0 TCID50/mL.
Analytical Reactivity/Enterovirus Serotype testing:
Study: 60 enterovirus serotypes tested with the Xpert EV assay. Dilutions of viral stock run in replicates of 3 for each serotype at presumed LoD in pooled EV negative human sample.
Results: Estimated TCID50/mL for the 60 serotypes ranged from 0.0002 to 200.00.
Analytical specificity:
Study: Nucleic acids from 1-2 x 10^6 cells of various non-EV pathogens (EBV, HSV-1, HSV-2, HHV-6, HHV-7, Adenovirus-2, Measles, Mumps, Parainfluenza 1-3, Influenza A, Influenza B, VZV, CMV, Group B Streptococcus, Haemophilus influenzae B, H. influenzae non-B, Escherichia coli. Neisseria meningitides, Citrobacter freundii, and Citrobacter koseri) were isolated and assayed. Additionally, "Whole Organisms" of these pathogens were tested at high titers. High concentrations of intact white blood cells and total RNA isolated from white blood cells were also tested.
Results: The Xpert EV assay did not generate any detectable amplicons. No cross-hybridization with purified human genomic RNA from WBC.
Assay cut-off:
Study: SPR/IC Ct value verification using negative assays from lot specific parameters (n=128) and preclinical studies (n=141).
Results: SPC/IC valid Ct range
§ 866.3225 Enterovirus nucleic acid assay.
(a)
Identification. An enterovirus nucleic acid assay is a device that consists of primers, probes, enzymes, and controls for the amplification and detection of enterovirus ribonucleic acid (RNA) in cerebrospinal fluid (CSF) from individuals who have signs and symptoms consistent with meningitis or meningoencephalitis. The detection of enterovirus RNA, in conjunction with other laboratory tests, aids in the clinical laboratory diagnosis of viral meningitis caused by enterovirus.(b)
Classification. Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus RNA.” See § 866.1(e) for the availability of this guidance document.
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510(k) De Novo DECISION SUMMARY ASSAY AND INSTRUMENT COMBINATION TEMPLATE
A. 510(k) Number: K061062
- B. Purpose for Submission: Marketing authorization of new device
- C. Measurand: Enterovirus (EV) RNA (enterovirus genome 5' untranslated region (UTR) between nucleotide 452 and 596)
- D. Type of Test: Nucleic acid amplification assay system, automated
- E. Applicant: Cepheid
- F. Proprietary and Established Names: Xpert EVTM
G. Regulatory Information:
-
- Regulation section: 21 CFR 866.3225
-
- Classification: Class II (de novo)
-
- Product code: OAI (Nucleic acid amplification assay system, enterovirus (EV) RNA in cerebrospinal (CSF) specimens, direct specimen test
-
- Panel: 83
H. Intended Use:
-
- Intended use(s):
The Cepheid® Xpert EV assay is a reverse transcription polymerase chain reaction (RT-PCR) using the GeneXpert® Dx System for the presumptive qualitative detection of enterovirus (EV) RNA in cerebrospinal fluid (CSF) specimens from individuals with signs and symptoms of meningitis. This test, in conjunction with other laboratory results and clinical information, may be used as an aid in the laboratory diagnosis of enterovirus infection in patients with a clinical suspicion of meningitis or meningoencephalitis.
- Intended use(s):
Assay performance characteristics have not been established for immunocompromised or immunosuppressed patients.
CAUTION: The results obtained with the Xpert EV assay should be used only as an adjunct to clinical observation and other information available to the physician. Positive Xpert EV results do not rule out other causes of meningitis, including bacteria, mycobacteria, other viruses (e.g. herpes family viruses, arboviruses, mumps virus, etc) and fungi.
1
-
- Indications for use: Same as intended use
-
- Special conditions for use statement(s):
Prescription Use Only
-
- Special instrument requirements: GeneXpert Dx System (instrument, computer, barcode wand reader)
I. Device Description:
The Xpert EV assay is designed to detect enterovirus (EV) RNA (enterovirus genome 5' untranslated region (UTR) between nucleotide 452 and 596) in CSF samples from patients exhibiting meningitis like symptoms. The assay includes reagents, primers, and probes for the simultaneous detection of nucleic acid from the target EV and the sample-processing control/internal control (SPC/IC). The assay includes the SPC/IC to verify adequate processing of the target virus and monitors the presence of inhibitors in the RT-PCR assay to avoid a false negative result. The assay also includes a probe check control to verify reagent rehydration, probe integrity, and reaction-tube filling in the cartridge.
The assay is run on the GeneXpert Dx System. The GeneXpert Dx System automates and integrates sample purification, nucleic acid amplification, and detection of the target sequence using real-time PCR and RT-PCR assays. The system consists of an instrument, personal computer, and preloaded software for running tests on collected samples and viewing the results. The system requires the use of single-use disposable GeneXpert cartridges that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated. The above described sample-processing control/internal control (SPC/IC) is named CIC in the GeneXpert Dx System software.
To run a test, the CSF sample and four reagents are transferred into designated chambers of the Xpert EV cartridge. The GeneXpert Dx System performs sample preparation by lysing the virus and SPC (armored pseudovirus RNA), binding the RNA to the capture matrix, and eluting the RNA. The RNA is mixed with dry RT reagents and transferred into the reaction tube for preparation of cDNA. The cDNA is then mixed with dry PCR reagents and transferred into the reaction tube for real-time PCR and detection. The EV primers and probe amplify and detect a consensus region of the enterovirus 5' untranslated region (UTR). The test takes approximately 2.5 hours.
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Xpert EV™ Reagent Kit and Xpert EV™ Self-contained Cartridge
Image /page/2/Picture/1 description: The image shows a medical testing kit. The kit includes a sealed package, a tray with vials, and a cartridge. The cartridge has a label with the text "Xpert EV" and a QR code.
GeneXpert Dx System
Image /page/2/Picture/3 description: The image shows a GeneXpert machine, a Dell computer tower, a Dell monitor, and a Dell mouse. The GeneXpert machine is a molecular diagnostic system used for rapid and accurate testing of infectious diseases. The Dell computer tower is a desktop computer that is used to control the GeneXpert machine. The Dell monitor is a display screen that is used to view the results of the tests. The Dell mouse is a pointing device that is used to navigate the computer screen.
J. Substantial Equivalence Information:
-
- Predicate device name(s): None
-
- Predicate 510(k) number(s): None
-
- Comparison with predicate: Not applicable
K. Standard/Guidance Document Referenced (if applicable):
-
- A special control guidance document will be promulgated.
3
-
- CDRH Draft Guidance for Industry and FDA staff titled: Nucleic Acid Based In Vitro Diagnostic Devices for Detection of Microbial Pathogens, December 8, 2005.
L. Test Principle:
The Xpert EV Test uses real-time PCR adapted to an automated instrument system that integrates sample processing (lysis and RNA purification) in a cartridge that contains freeze-dried PCR reagents and process controls for real-time PCR detection. This integration of nucleic acid extraction reduces labor-intensive steps requiring specially trained personnel and also reduces the likelihood of cross-contamination and technician-dependent variability. The instrument moves the sample and reagents to and from different chambers within the Xpert EVTM cartridge, automatically performing sample preparation by lysing the virus and the SPC/IC, binding the RNA to the capture matrix, and eluting the RNA.
The Xpert EV Target region is a 5' untranslated (UTR) region of the enterovirus genome RNA between nucleotide 452 and 596). The primers and probe sequences used in the Xpert EV assay are designed to these consensus regions and are essentially similar to those developed by Rotbart (J. Clin. Microbiol., 28, 438-442, 1990; US patent 5075212). The resulting amplicon is 146 bp. Probes are labeled with FAM (5') and OSY7 quencher (3'). Note: Primers are 19 mer (Tm 67.6. 58% GC) and 20 mer (63.4 Tm. 45 %GC) while the EV probe is 25 mer (74.9 Tm. 52 %GC). The reverse transcribing parameters are 30 min at 50° followed by 5 min at 93° and the cycling parameters are 15 min at 95° (denaturation), followed by 45 cycles of 20s at 58°C, and 30s at 73°C.
The GenXpert® instrument has 4 modules, each with an I-Core for thermal cycling and real-time detection of PCR amplicons. Each of the 4 modules is controlled independently, allowing for specimens to be loaded and tested when received, rather than batching. Each of the 4 modules in addition to the I-Core, has a syringe drive for aspirating and dispensing fluids, a valve drive for chamber access, and an ultrasonic horn for lysis. Prior to operation, a self-test verifies heater, fan and optics functionality, while the syringe drive, valve and ultrasonic horn current are continuously checked. The GenXpert can simultaneously detect signal from up four different spectral bands.
Each I-core has two ceramic plates to assure temperature uniformity and rapid heat transfer. Firmware controls the temperature inside by moving ambient air across the heater plates. Optics consist of a 4-color excitor module (high intensity LEDs to excite reporter dye molecules) and a 4color detector module (silicon photodetectors and filters to detect the 4 spectral bands). Calibration and data analysis algorithms compensate for spectral overlap. Thermal reaction chamber thermistors are calibrated to +0.50℃ and calibration coefficients correct for errors in raw thermistor reads (stored in firmware). Individual dye-oligos signals are corrected using spectral characteristics of pure dye-oligos after subtracting raw signal produced by a reaction tube alone.
For the EV application, three spectral signals are processed (separate dyes tagged to oligonucleotide probes for the SPC, IC, and the EV). Two of the 3 signals are from fixed components within the reagents, with one from unknown. Output results are color coded (redpositive; green-negative; gray-invalid; gold-error); tabular and graphic formats are accessible by password.
4
M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
Reproducibility was assessed in a multi-center, blinded study using a precision panel consisting of four specimens. Three sites tested each panel three times per day over 10 testing days, for a total of 90 results per panel specimen. The precision panel consisted of a negative sample and three positive samples, each with a specific EV serotype spiked into synthetic CSF at a concentration close to the limit of detection.
| Table 1. Summary of first reproducibility study results. | |||||
|---|---|---|---|---|---|
| -- | -- | -- | -- | -- | ---------------------------------------------------------- |
| No. of specimens correctly classified | Between Day | Between Site | Total | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Mean | ||||||||||
| Serotype | ||||||||||
| (TCID50/mL) | Site 1 | Site 2 | Site 3 | EV Ct | SD | %CV | SD | %CV | SD | %CV |
| Negative | 30/30 | 30/30 | 30/30 | |||||||
| CVA6 (134) | 30/30 | 30/30 | 29/29* | 35.0 | 0.343 | 0.98% | 0.175 | 0.50% | 1.101 | 3.15% |
| CVA9 (320) | 30/30 | 30/30 | 30/30 | 34.4 | 0 | 0.00% | 0 | 0.00% | 0.61 | 1.77% |
| CVA17 (3) | 30/30 | 30/30 | 29/29* | 33.8 | 0 | 0.00% | 0 | 0.00% | 0.414 | 1.22% |
| Total | ||||||||||
| Agreement | 120/ | |||||||||
| 120 | 120/ | |||||||||
| 120 | 118/ | |||||||||
| 118 | ||||||||||
| % | ||||||||||
| Agreement | 100.00 | |||||||||
| % | 100.00 | |||||||||
| % | 100.00 | |||||||||
| % |
- Two samples at site 3 did not give any GeneXpert result
In order to further stress the system a second study was performed. A reproducibility study was conducted over four different days on 31 GeneXpert instruments and 121 ICORE modules. Two representative whole virus subtypes (i.e., Coxsackievirus CVA9 and Enterovirus EV70) were spiked into human negative CSF to create simulated specimens at both 2 x LoD and 4 x LoD. Negative samples were tested 20 times each day whereas 2 positive samples at 2 different concentrations were tested 5 times per day. Table 2 shows the average Ct values for each concentration and the associated standard deviations and coefficients of variation for each day.
5
Table 2. Summary of second reproducibility study results.
| Total Agreement - Ct Results | |||||||
|---|---|---|---|---|---|---|---|
| Specimen ID | Day 1 | Day 2 | Day 3 | Day 4 | All Days | % Total | |
| Agreement | |||||||
| Negative | Total Agreement | 20/20 | 18/18A | 20/20 | 20/20 | 78/78 | 100% |
| Average | NA | NA | NA | NA | NA | ||
| SD | NA | NA | NA | NA | NA | ||
| % CV | NA | NA | NA | NA | NA | ||
| CA9 2X LOD | Total Agreement | 4/5B | 5/5 | 4/5B | 5/5 | 18/20 | 90% |
| Average | 36.65 | 36.54 | 36.53 | 36.54 | 36.56 | ||
| SD | 0.56 | 0.46 | 0.21 | 0.69 | 0.48 | ||
| % CV | 1.53% | 1.26% | 0.57% | 1.89% | 1.31% | ||
| CA9 4X LOD | Total Agreement | 5/5 | 5/5 | 5/5 | 4/4C | 19/19 | 100% |
| Average | 34.98 | 35.56 | 35.52 | 35.03 | 35.28 | ||
| SD | 0.53 | 0.67 | 0.7 | 0.3 | 0.6 | ||
| % CV | 1.52% | 1.88% | 1.97% | 0.86% | 1.70% | ||
| EV70 2X LOD | Total Agreement | 5/5 | 5/5 | 5/5D | 5/5 | 20/20 | 100% |
| Average | 37.38 | 37.3 | 37.55 | 36.88 | 37.2 | ||
| SD | 1.78 | 0.74 | 2.01 | 0.81 | 1.3 | ||
| % CV | 4.76% | 1.98% | 5.35% | 2.20% | 3.49% | ||
| EV70 4X LOD | Total Agreement | 5/5 | 5/5 | 5/5 | 5/5 | 20/20 | 100% |
| Average | 36.50 | 36.60 | 36.12 | 35.94 | 36.29 | ||
| SD | 0.58 | 0.97 | 0.29 | 0.84 | 0.72 | ||
| % CV | 1.59% | 2.65% | 0.80% | 2.34% | 1.98% | ||
| No. of Instruments used | 10 | 11 | 10 | 10 | 31 | ||
| No of Modules used | 40 | 41 | 41 | 40 | 121 |
The level of agreement, the average Ct values for each concentration, the associated standard deviations and percent coefficient of variation for each day are shown in the table below.
A Total runs = 21, 2-No Result, 1-Invalid
B Total runs = 5, 1 negative instead of positive result
C Total runs = 5, 1-Invalid
0 Total runs = 6, 1-No Result
Of the total samples tested there were two samples with "Invalid" and three samples with "No Result" by instrument software control definitions. Of the 157 reportable results, 155 were correctly scored.
b. Linearity/assay reportable range:
A stock concentration of CVA9 (2e9 TCID50/mL) was serially diluted in EV Negative synthetic CSF sample and loaded directly into each cartridge with appropriate sample preparation reagents. Duplicates at each dilution were tested. The GeneXpert EV assay detects serotype CVA9 over a dynamic range of 8 logs. The assay is linear over this range with an R2 value of 0.9989. The reported Ct range is shown below.
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Image /page/6/Figure/1 description: The image shows a standard curve for Live CVA9. The x-axis represents GX Cts for Live CVA9, ranging from 10 to 40, while the y-axis represents Log (Conc) of live CVA9, ranging from 0.00 to 9.00. The plot includes data points for 'Log Conc' and a linear trendline for 'Linear (Log Conc)', with the equation y = -0.3027x + 12.655 and an R-squared value of 0.9989 displayed on the graph.
Traceability, Stability, Expected Values (controls, calibrators, or methods): Not C. applicable
- Detection limit: d.
The analytical sensitivity, or limit of detection (LoD), is defined as the lowest concentration, or amount of an analyte that has been demonstrated by laboratory analysis to be reproducibly distinguished from a negative sample at a 95% confidence level. The dilutions were made in pooled EV negative human sample. For statistical confidence determination of the LoD, replicates of 20. along with 20 EV negative samples, were run. The samples run were Coxsackievirus A6 (CVA6), Coxsackievirus A9 (CVA9), Coxsackievirus A17 (CVA17), Enterovirus 70 (EV70), and Poliovirus 1 (PV1). Not all 63 serotypes were run in statistically significant numbers, since the primer and probe binding sites are conserved across all serotypes and the amplicon length is the same for all serotypes, so it would be expected that the amplification efficiency is the same for all serotypes. The five serotypes indicated above were selected to represent each of the enterovirus species CVA6 (A), CVA9 (B), CVA17 (C), EV70 (D) and PV1 (poliovirus).
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Table 3. Limit of detection for 5 serotypes.
| Serotype | Limit of Detection
(TCID50/mL) |
|----------|-----------------------------------|
| CVA9 20: | 80.0 |
| EV70: | 1.3 |
| PV1: | 4.0 |
| CVA17: | 1.0 |
| CVA6: | 33.0 |
The LoD of the 5 serotypes, one from each of the enterovirus species are shown in the table below.
Analytical Reactivity/Enterovirus Serotype testing
A total of 60 enterovirus serotypes were tested with the Xpert EV assay. Dilutions of the viral stock were run in replicates of 3 for each serotype at the presumed LoD. The dilutions were made in pooled EV negative human sample.
Table 4. Estimated analytical sensitivity.
Sixty of the serotypes were tested and the estimated TCID50/ml that these serotypes can be detected were shown in Table 4 below.
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Table 4 Estimated Analytical Sensitivity
| Species | Serotype | Estimated TCID 50/mL | |||
|---|---|---|---|---|---|
| A | Coxsackie A3 | 5.01 | |||
| A | Coxsackie A5 | 12.59 | |||
| A | Coxsackie A6 | 12.59 | |||
| A | Coxsackie A7 | 3.33 | |||
| A | Coxsackie A10 | 2.81 | |||
| A | Coxsackie A12 | 19.95 | |||
| A | Coxsackie A14 | 0.10 | |||
| A | Coxsackie A16 | 0.002 | |||
| A | EV 71 | 0.16 | |||
| B | Coxsackie A9 | 20.00 | |||
| B | Coxsackie B1 | 4.00 | |||
| B | Coxsackie B2 | 0.20 | |||
| B | Coxsackie B3 | 0.028 | |||
| B | Coxsackie B4 | 0.40 | |||
| B | Coxsackie B5 | 0.04 | |||
| B | Coxsackie B6 | 0.01 | |||
| B | Echo 1 | 0.10 | |||
| B | Echo 2 | 0.032 | |||
| B | Echo 3 | 200.00 | |||
| B | Echo 4 | 0.00032 | |||
| B | Echo 5 | 0.032 | |||
| B | Echo 6 | 200.00 | |||
| B | Echo 7 | 2.00 | |||
| B | Echo 8 | 0.10 | |||
| B | Echo 9 | 2.00 | |||
| B | Echo 11 | 40.00 | |||
| B | Echo 12 | 1.58 | |||
| B | Echo 14 | 0.0005 | |||
| B | Echo 15 | 0.0032 | |||
| B | Echo 16 | 0.0005 | |||
| B | Echo 17 | 0.05 | |||
| B | Echo 18 | 0.0002 | |||
| B | Echo 19 | 2.51 | |||
| B | Echo 20 | 0.032 | |||
| B | Echo 21 | 1.00 | |||
| B | Echo 24 | 0.02 | |||
| B | Echo 25 | 0.50 | |||
| B | Echo 26 | 0.032 | |||
| B | Echo 27 | 0.00032 | |||
| B | Echo 29 | 5.01 | |||
| B | Echo 30 | 0.01 | |||
| B | Echo 31 | 0.0032 | |||
| B | Echo 32 | 0.10 | |||
| B | Echo 33 | 0.05 | |||
| B | EV 69 | 0.0002 | |||
| C | Echo 13 | 0.01 | |||
| C | Coxsackie A11 | 0.11 | |||
| C | Coxsackie A13 | 13.27 | |||
| C | Coxsackie A15 | 0.00 | |||
| C | Coxsackie A17 | 1.58 | |||
| C | Coxsackie A18 | 0.02 | |||
| C | Coxsackie A19 | 0.03 | |||
| C | Coxsackie A20 | 0.002 | |||
| C | Coxsackie A21 | 0.03 | |||
| C | C | Coxsackie A22 | Coxsackie A22 | 0.02 | 0.02 |
| C | Coxsackie A24 | 0.10 | |||
| D | EV 68 | 199.53 | |||
| D | EV 70 | 2.00 | |||
| Poliovirus | Poliovirus 1 | 2.00 | |||
| Poliovirus | Poliovirus 2 | 0.40 | |||
| Poliovirus | Poliovirus 3 | 20.00 |
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- e. Analytical specificity:
Nucleic acids from 1-2 x 10° cells of EBV, HSV-1, HSV-2, HHV-6, HHV-7, Adenovirus-2, Measles, Mumps, Parainfluenza 1-3, Influenza A, Influenza B, VZV, CMV, Group B Streptococcus, Haemophilus influenzae B, H. influenzae non-B, Escherichia coli. Neisseria meningitides, Citrobacter freundii, and Citrobacter koseri were isolated with commercially available isolation kits and assayed in the Xpert. In addition, the specificity evaluation was repeated with "Whole Organisms" at titer levels much greater than those found typically in CSF infected with the respective organisms. The Xpert EV assay did not generate any detectable amplicons when "Whole Organisms" of the above listed pathogens were processed through the Xpert EV cartridge.
Also, high concentrations of both intact white blood cells and total RNA isolated from white blood cells were used in assays with the Xpert EV assay. There was no crosshybridization of purified human genomic RNA from WBC with the primer and probe sequences used in the Xpert EV assay.
Table 5. Analytical specificity for the Xpert EV.
Whole organisms were tested for specificity in the Xpert EV assay and the concentrations of the organisms tested were shown in the table below.
| Organism | No. organisms/test |
|---|---|
| HHV6 | 3.1e6 particles |
| HHV7 | 1.4e7 particles |
| CMV | 700 TCID50 |
| EBV | 140 TCID50 |
| HSV 1 | 1.4e5 TCID50 |
| HSV 2 | 1.4e5 TCID50 |
| ADV 2 | 1.4e12 TCID50 |
| Measles | 700 TCID50 |
| Mumps | 1.4e4 TCID50 |
| Parainfluenza 1 | 1.4e3 TCID50 |
| Parainfluenza 2 | 7e3 TCID50 |
| Parainfluenza 3 | 1.4e4 TCID50 |
| Influenza A | 3.5e4 TCID50 |
| Influenza B | 3.5e4 TCID50 |
| VZV | 14 TCID50 |
| Group B Streptococcus | 7e6 cells |
| H. influenzae B | 7e6 cells |
| H. influenzae NonB | 7e5 cells |
| E.coli | 7e6 cells |
| N. meningitidis | 7e6 cells |
| C. freundii | 7e6 cells |
| C. koseri | 7e6 cells |
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f. Assay cut-off:
Image /page/10/Figure/1 description: The image is a histogram titled "Distrubition of EV Cts for Positive Clinical Cases". The x-axis is labeled "Xpert EV Cycle Threshold" and ranges from 28 to "More". The y-axis is labeled "No. of Subjects" and ranges from 0 to 18. The histogram shows the distribution of EV Cts for positive clinical cases, with the highest number of subjects at cycle thresholds of 30, 32, and 34.
Graph 1. Histogram for determination of the Xpert EV assay cut-off.
The SPC/IC Ct value at which the assay would be called invalid was verified using:
-
- Negative assays run during the generation of lot specific parameters
-
- Preclinical studies.
Table 6. SPC/IC Ct values verifying "invalid" criteria.
| | Valid Ct
Range | LSP
Negatives | Pre-
clinical
Negatives |
|--------|-------------------|------------------|-------------------------------|
| | | n = 128 | n = 141 |
| SPC/IC | 21 | M | 2 (10.0) | 18 (90.0) | 20 |
| | F | 3 (12.5) | 21 (87.5) | 24 |
| Total | | 107 (24.7) | 327 (75.3) | 434 |
Table 10: Expected values for Xpert EV in population with signs and symptoms consistent with meningitis.
N. Instrument Name: GeneXpert Dx system
O. System Descriptions:
-
- Modes of Operation: 4 randomly accessible modules that are each capable of performing separate sample preparation and real-time PCR tests.
-
- Software:
FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types:
Yes _x or No _________________________________________________________________________________________________________________________________________________________
-
- Specimen Identification: barcodes
16
4. Specimen Sampling and Handling: automated
| Module Hardware
| Components | Function |
|---|---|
| Valve Drive | Rotates the cartridge valve body to address the different cartridge |
| chambers. | |
| Syringe Pump Drive | Dispenses fluids to and from the different cartridge chambers. |
| Ultrasonic Horn | Lyses the bacterial cells and sample prep control. |
| I-CORE® Module | Performs PCR amplification and detection. As the user inserts the |
| cartridge into the system, the reaction tube component of the cartridge | |
| is inserted into the I-CORE module. After sample preparation within | |
| the cartridge, the sample and reagent mixture is transferred from the | |
| cartridge chamber into the reaction tube. During the amplification | |
| process, the I-CORE heater heats up and the fan cools down the | |
| reaction tube contents. Two optical blocks positioned within the I- | |
| CORE excite the dye molecules that make up the probes and detect the | |
| fluorescence emitted. The system uses calibration and data analysis | |
| algorithms to determine a relative fluorescence value for each reporter | |
| dye after each thermal cycle. |
GeneXpert Dx System Hardware Components for Automated Sample Processing
- న. Calibration: Optical and thermal calibration of the GeneXpert Dx System is performed by Cepheid at the time of manufacture prior to installation and once yearly or after 1000 runs per module. The user does not calibrate or perform any serviceable functions on the instrument. The normalization function compensates for any optical degradation between calibrations.
The thermal reaction chamber thermistors are calibrated to ± 0.50℃ using National Institute of Standards and Technology (NIST)-traceable standards. During the manufacturing process, the temperature of the heating system is measured at two temperatures: 60℃ and 95℃. Calibration coefficients that correct for small errors in the raw thermistor readings of the heaters are stored in the memory of each I-CORE module.
The optical system is calibrated using standard concentrations of individual unquenched fluorescent dye-oligos. For each optical channel, the signal produced by a tube alone (the blank signal) is subtracted from the raw signal produced by the dye-oligo standard to determine the spectral characteristics. Using the individual spectral characteristics of the pure dye-oligos, signals from an unknown mixture of dye-oligos can be resolved into corrected signals for the individual dye-oligos in the mixture.
-
- Quality Control:
Before the start of the PCR reaction, the GeneXpert Dx System is programmed to perform a probe check on the EV target and SPC/CIC. The Probe Check control verifies reagent
- Quality Control:
17
bead rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability. PC is considered to PASS if it meets the predetermined acceptance criteria. If the PC fails in EV target or SPC/CIC; the test will not continue.
External controls must be used for training, proficiency testing and external QC of the GeneXpert Dx System. External controls should be used in accordance with local, state, and federal accrediting organizations as applicable.
External Controls can be prepared by diluting Coxsackievirus A9 strain: Bozek or Coxsackievirus A6 strain C.G. (Gdula) with known negative patient CSF or Synthetic CSF (e.g. SeraCare Life Sciences Inc. Catalog number HSP-515) to approximately 10-1000 TCID50/mL that gives an EV Ct range of 32-35 for the Xpert EV assay.
P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:
Q. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
R. Conclusion:
The petition for Evaluation of Automatic Class III Designation for this device is accepted. The device is classified as Class II under regulation 21 CFR 866.3225 with special controls. The special control guidance document "Class II Special Control Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus RNA" will shortly be available.
S. Other Supportive Device and Instrument Information:
Image /page/17/Picture/9 description: The image shows a GeneXpert Dx System, which includes a processing unit, a computer tower, and a monitor. The processing unit is a silver box with multiple slots, while the computer tower is black. The monitor is displaying a graphical user interface, and a mouse and keyboard are placed in front of it. The GeneXpert Dx System is a molecular diagnostic system used for a variety of applications.