(55 days)
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No
The description details a molecular assay using PCR amplification and nanoparticle-based array hybridization with automated imaging and light scatter analysis for result determination. There is no mention of AI or ML algorithms being used for data analysis, interpretation, or decision-making. The results are determined based on light scatter intensity from the microarray spots, which is a direct measurement, not an interpretation by an AI/ML model.
No.
The device is an in vitro diagnostic test used as an aid in the diagnosis of C. difficile infection (CDI) by detecting gene sequences. It does not directly treat or cure a disease or condition.
Yes
The device is described as a "qualitative multiplexed in vitro diagnostic test for the rapid detection of toxin A (tcdA), toxin B (tcdB), and tcdC gene sequences" and is "indicated for use as an aid in the diagnosis of CDI." These phrases explicitly state its diagnostic purpose.
No
The device description explicitly states it is a "molecular assay" and is performed on the "Verigene System," which consists of hardware components: the Verigene Reader and the Verigene Processor SP. These components are integral to the device's function, including automated specimen preparation, PCR amplification, hybridization-detection, and automated imaging.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The "Intended Use / Indications for Use" section explicitly states that the Verigene® Clostridium difficile Nucleic Acid Test (CDF) is a "qualitative multiplexed in vitro diagnostic test".
- Purpose: The test is designed to detect specific genetic sequences of Clostridium difficile in stool specimens to aid in the diagnosis of C. difficile infection (CDI). This is a diagnostic purpose performed outside of the body (in vitro).
- Specimen Type: It uses unformed stool specimens, which are biological samples taken from the human body.
- Methodology: The test utilizes molecular techniques (PCR amplification and hybridization) to analyze these biological samples for the presence of specific markers related to the disease.
All of these characteristics align with the definition of an In Vitro Diagnostic device.
N/A
Intended Use / Indications for Use
The Verigene® Clostridium difficile Nucleic Acid Test (CDF) is a qualitative multiplexed in vitro diagnostic test for the rapid detection of toxin B (tcdB), and tcdC gene sequences of toxigenic Clostridium difficile and for presumptive identification of PCR ribotype 027 strains from unformed (liquid or soft) stool specimens collected from patients suspected of having C. difficile infection (CDI). Presumptive identification of the PCR ribotype 027 strain of C. difficile is by detection of the binary toxin (cdt) gene sequence and the single base pair deletion at nucleotide 117 in the tedC gene encodes for a negative regulator in C. difficile toxin production. The test is performed on the Verigene System and utilizes automated specimen preparation and polymerase chain reaction (PCR) amplification, combined with a nanoparticle-based array hybridization assay to detect the toxin gene sequences associated with toxin-producing C. difficile.
The CDF Test is indicated for use as an aid in the diagnosis of CDI. Detection of PCR ribotype 027 strains of C. difficile by the CDF Test is solely for epidemiological purposes and is not intended to guide or monitor treatment for C. difficile infections. Concomitant culture is necessary only if further typing or organism recovery is required.
Product codes (comma separated list FDA assigned to the subject device)
OZN Clostridium difficile toxin gene amplification assay
Device Description
The Verigene C. difficile Nucleic Acid Test (CDF) is a molecular assay which relies on detection of specific nucleic acid targets in a microarray format. For each of the bacterial nucleic acid sequences detected by CDF, unique Capture and Mediator oligonucleotides are utilized, with gold nanoparticle probe-based endpoint detection. The Capture oligonucleotides are covalently bound to the microarray substrate and hybridize to a specific portion of the nucleic acid targets. The Mediator oligonucleotides have a region which bind to a different portion of the same nucleic acid targets and also have a sequence which allows binding of a gold nanoparticle probe. Specific silver enhancement of the bound gold nanoparticle probes at the capture sites results in gold-silver aggregates that scatter light with high efficiency and provide accurate detection of target capture.
The CDF Test is performed on the Verigene System, a "sample-to-result", fully automated, bench-top molecular diagnostics workstation. The System enables automated nucleic acid extraction from unformed stool specimens (liquid or soft) and detection of bacterial-specific target DNA. The Verigene System consists of two components: the Verigene Reader and the Verigene Processor SP.
The Reader is the Verigene System's user interface, which serves as the central control unit for all aspects of test processing, automated imaging, and result generation using a touch-screen control panel and a barcode scanner. The Verigene Processor SP executes the test procedure, automating the steps of (1) Sample Preparation and Target Amplification - cell lysis and magnetic bead-based bacterial DNA isolation and amplification, and (2) Hybridizationdetection and identification of bacterial-specific DNA in a microarray format by using gold nanoparticle probe-based technology. Once the specimen is loaded by the operator, all other fluid transfer steps are performed by an automated pipette that transfers reagents between wells of the travs and finally loads the specimen into the Test Cartridge for hybridization. Single-use disposable test consumables and a self-contained Verigene Test Cartridge are utilized for each sample tested with the CDF assay.
To obtain the test results after test processing is complete, the user removes the Test Cartridge from the Processor SP, and inserts the substrate holder into the Reader for analysis. Light scatter from the capture spots is imaged by the Reader and intensities from the microarray spots are used to make a determination regarding the presence (Detected) or absence (Not Detected) of a bacterial nucleic acid sequence/analyte. This determination is made by means of software-based decision algorithm resident in the Reader.
Mentions image processing
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Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
Not Found
Indicated Patient Age Range
Not Found
Intended User / Care Setting
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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
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Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Analytical Testing
- Analytical Sensitivity / Limit of Detection (LoD): Determined for seven strains of C. difficile. LoD defined as concentration yielding >95% positive result. Serial dilutions tested and confirmed with 20 replicates. LoD ranged from 63 to 1250 CFU/ml of stool. Overall LoD was 1250 CFU/ml of organism in stool.
- Analytical Reactivity (Inclusivity): Demonstrated with 63 independently-confirmed C. difficile strains, tested in triplicate at 3 times the LoD (3,750 CFU/mL). Panel included various toxinotypes and 6 PCR ribotype 027 strains. All tests correctly reported expected results except for strain CDC 2009048, where subsequent sequencing verified the presence of the A117 deletion despite classification as non-027.
- Analytical Specificity (Cross-reactivity): Tested 94 microorganisms, including non-toxigenic C. difficile, non C. difficile Clostridium species, and one human cell line. Clostridium botulinum evaluated by in silico analysis. Bacterial strains prepared in Negative Stool Matrix at 5×10^6 CFU/mL stool. Cryptosporidium parvum and Giardia lamblia tested using genomic DNA at 1×10^5 copies gDNA. Viruses tested at 5×10^6 PFU/mL stool or 1×10^5 copies per reaction (genomic DNA/RNA). Noroviruses tested as clinical samples. Analytical specificity observed to be 100%.
- Microbial Interference: Evaluated potential for microbial interference using the same 95 organisms/cell line as for analytical specificity, at the same medically relevant concentrations, with two toxigenic C. difficile strains (ATCC BAA-1805 and ATCC 43255) at 1.5x LoD and 3x LoD. No interference observed.
- Interference: Evaluated 34 products/exogenous substances commonly found in stool samples for inhibitory effects, tested at "worst case" concentration against two C. difficile strains (ATCC BAA-1805, ATCC 43255). Cary-Blair media also tested. No inhibitory effect observed.
- Carry-over / Cross-contamination: Assessed by alternately testing a high positive C. difficile sample (5x10^8 CFU/mL) followed by a negative sample, three times on three unique Verigene SP Processors (total 18 tests). No carry-over or cross-contamination observed.
- Cutoff Verification: Analytical testing of 59 C. difficile strains (range of toxinotypes and non-toxigenic), performed in duplicate. CDF Test correctly detected expected analytes using established cut-off levels.
Clinical Testing
- Precision: In-house study. Seven-member Precision Study Sample Panel tested in duplicate twice daily by two operators for 12 non-consecutive days. Total of 48 replicates per specimen, 336 data points. Panel included two strains (ATCC 43255 and ATCC BAA-1805) at three concentrations (High Negative, Low Positive, Moderate Positive) and one negative sample. Results summarized in two tables.
- Reproducibility: Inter-laboratory study at three external sites. Seven samples tested daily in triplicate by two operators for 5 non-consecutive days at each site. Total of 90 tests per sample (630 total samples). Panel was the same as the precision study. Results summarized in two tables.
- Method Comparison: Multi-site prospective investigation study at five U.S. institutions. Compared Verigene CDF Test results to reference culture followed by cell cytotoxicity testing and strain typing (PCR Ribotyping and Bi-Directional Sequencing). Subjects were individuals undergoing C. difficile testing. Leftover unformed stool specimens used.
- Reference Methods:
- Direct Culture and PCR Ribotyping
- Enriched Culture and PCR Ribotyping
- Direct Culture and Bidirectional Sequencing (BDS)
- Enriched Culture and Bidirectional Sequencing (BDS)
- Sample Size: n=1875 specimens evaluated for overall clinical performance. (Note: 6 specimens excluded for PCR-ribotyping due to inconclusiveness; 2 specimens excluded for BDS due to inconclusiveness).
- Key Results (Summary Table 1): Detailed sensitivity, specificity, accuracy, PPV, NPV, and agreement for both toxigenic C. difficile and toxigenic C. difficile / 027 Strain against the four reference metrics.
- Reference Methods:
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
| Metric | Sensitivity (95% CI) | Specificity (95% CI) | Accuracy (95% CI) | PPV (95% CI) | NPV (95% CI) |
|---|---|---|---|---|---|
| Toxigenic C. difficile | |||||
| Direct Culture & PCR Ribotyping | 98.7% (154/156) (95.5-99.8) | 87.6% (1500/1713) (85.9-89.1) | 88.5% (1654/1869) (87.0-89.9) | 42.0% (154/367) (36.9-47.2) | 99.9% (1500/1502) (99.5-99.9) |
| Enriched Culture & PCR Ribotyping | 91.8% (247/269) (87.9-94.8) | 92.5% (1480/1600) (91.1-93.7) | 92.4% (1727/1869) (91.1-93.6) | 67.3% (247/367) (62.2-72.1) | 98.5% (1480/1502) (97.8-99.1) |
| Direct Culture & BDS | 98.7% (156/158) (95.5-99.9) | 87.5% (1500/1715) (85.8-89.0) | 88.4% (1656/1873) (86.9-89.8) | 42.1% (156/371) (37.0-47.3) | 99.9% (1500/1502) (99.5-100) |
| Enriched Culture & BDS | 91.9% (251/273) (88.1-94.9) | 92.5% (1480/1600) (91.1-93.7) | 92.4% (1731/1873) (91.1-93.6) | 67.7% (251/371) (62.6-72.4) | 98.5% (1480/1502) (97.8-99.1) |
| Metric | Agreement POS (95% CI) | Agreement NEG (95% CI) | Total (95% CI) | PPV (95% CI) | NPV (95% CI) |
|---|---|---|---|---|---|
| Toxigenic C. difficile / 027 Strain | |||||
| Direct Culture & PCR Ribotyping | 97.5% (39/40) (86.8-99.9) | 97.8% (1787/1828) (97.0-98.4) | 97.7% (1826/1869) (96.9-98.3) | 48.2% (39/81) (36.9-59.5) | 99.9% (1787/1788) (99.7-100) |
| Enriched Culture & PCR Ribotyping | 91.4% (53/58) (81.0-97.1) | 98.5% (1783/1811) (97.8-99.0) | 98.2% (1836/1869) (97.5-98.8) | 65.4% (53/81) (54.0-75.7) | 99.7% (1783/1788) (99.4-99.9) |
| Direct Culture & BDS | 97.7% (42/43) (87.7-99.9) | 97.8% (1790/1830) (97.0-98.4) | 97.8% (1832/1873) (97.0-98.4) | 51.2% (42/82) (39.9-62.4) | 99.9% (1790/1791) (99.7-100) |
| Enriched Culture & BDS | 93.7% (59/63) (84.5-98.2) | 98.7% (1787/1810) (98.1-99.2) | 98.6% (1846/1873) (97.9-99.1) | 72.0% (59/82) (60.9-81.3) | 99.8% (1787/1791) (99.4-99.9) |
Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.
Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).
Not Found
§ 866.3130 Clostridium difficile toxin gene amplification assay.
(a)
Identification. AClostridium difficile toxin gene amplification assay is a device that consists of reagents for the amplification and detection of target sequences inClostridium difficile toxin genes in fecal specimens from patients suspected of havingClostridium difficile infection (CDI). The detection of clostridial toxin genes, in conjunction with other laboratory tests, aids in the clinical laboratory diagnosis of CDI caused byClostridium difficile. (b)
Classification. Class II (special controls). The special controls are set forth in FDA's guideline document entitled: “Class II Special Controls Guideline: Toxin Gene Amplification Assays for the Detection ofClostridium difficile; Guideline for Industry and Food and Drug Administration Staff.” See § 866.1(e) for information on obtaining this document.
0
510(K) Summary
510(k) Number:
K123197: Verigene® Clostridium difficile Nucleic Acid Test (CDF)
Summary Preparation Date:
November 27, 2012
Submitted by:
Nanosphere, Inc. 4088 Commercial Avenue Northbrook, IL 60062 Phone: 847-400-9000 Fax: 847-400-9176
Contact:
Mark A. Del Vecchio Vice President, Regulatory Affairs
Proprietary Names:
For the instrument: Verigene® System For the assay: Verigene® Clostridium difficile Nucleic Acid Test (CDF)
Common Names:
For the instrument:
Bench-top molecular diagnostics workstation
For the assay:
Clostridium difficile Nucleic Acid Test Clostridium difficile identification and differentiation system C. difficile assay C. diff test
Nanosphere, Inc.
Page 1 of 10
November 27, 2012
DEC. 5 2012
1
Regulatory Information:
Regulation section:
866.3130
Classification:
Class II
Panel:
Microbiology (83)
Product Code(s):
OZN Clostridium difficile toxin gene amplification assay
Other codes used by predicate devices:
OMN C. difficile nucleic acid test assay
Reagents, Clostridium Difficile toxin LLH
Respiratory Virus Panel Nucleic Acid System OCC
Instrumentation for clinical multiplex test systems NSU
Predicate Devices:
Portrait Toxigenic C. Difficile Assay (K113358) (Great Basin Scientific)
Xpert C. difficile/Epi Assay (K110203) (Cepheid)
Verigene RVNATSP Test (K092566) (Nanosphere)
Indications for Use:
The Verigene® Clostridium difficile Nucleic Acid Test (CDF) is a qualitative multiplexed in vitro diagnostic test for the rapid detection of toxin B (tcdB), and tcdC gene sequences of toxigenic Clostridium difficile and for presumptive identification of PCR ribotype 027 strains from unformed (liquid or soft) stool specimens collected from patients suspected of having C. difficile infection (CDI). Presumptive identification of the PCR ribotype 027 strain of C. difficile is by detection of the binary toxin (cdt) gene sequence and the single base pair deletion at nucleotide 117 in the tedC gene encodes for a negative regulator in C. difficile toxin production. The test is performed on the Verigene System and utilizes automated specimen preparation and polymerase chain reaction (PCR) amplification, combined with a nanoparticle-based array hybridization assay to detect the toxin gene sequences associated with toxin-producing C. difficile.
Nanosphere, Inc.
Page 2 of 10
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The CDF Test is indicated for use as an aid in the diagnosis of CDI. Detection of PCR ribotype 027 strains of C. difficile by the CDF Test is solely for epidemiological purposes and is not intended to guide or monitor treatment for C. difficile infections. Concomitant culture is necessary only if further typing or organism recovery is required.
Technological Characteristics:
The Verigene C. difficile Nucleic Acid Test (CDF) is a molecular assay which relies on detection of specific nucleic acid targets in a microarray format. For each of the bacterial nucleic acid sequences detected by CDF, unique Capture and Mediator oligonucleotides are utilized, with gold nanoparticle probe-based endpoint detection. The Capture oligonucleotides are covalently bound to the microarray substrate and hybridize to a specific portion of the nucleic acid targets. The Mediator oligonucleotides have a region which bind to a different portion of the same nucleic acid targets and also have a sequence which allows binding of a gold nanoparticle probe. Specific silver enhancement of the bound gold nanoparticle probes at the capture sites results in gold-silver aggregates that scatter light with high efficiency and provide accurate detection of target capture.
The CDF Test is performed on the Verigene System, a "sample-to-result", fully automated, bench-top molecular diagnostics workstation. The System enables automated nucleic acid extraction from unformed stool specimens (liquid or soft) and detection of bacterial-specific target DNA. The Verigene System consists of two components: the Verigene Reader and the Verigene Processor SP.
The Reader is the Verigene System's user interface, which serves as the central control unit for all aspects of test processing, automated imaging, and result generation using a touch-screen control panel and a barcode scanner. The Verigene Processor SP executes the test procedure, automating the steps of (1) Sample Preparation and Target Amplification - cell lysis and magnetic bead-based bacterial DNA isolation and amplification, and (2) Hybridizationdetection and identification of bacterial-specific DNA in a microarray format by using gold nanoparticle probe-based technology. Once the specimen is loaded by the operator, all other fluid transfer steps are performed by an automated pipette that transfers reagents between wells of the travs and finally loads the specimen into the Test Cartridge for hybridization. Single-use disposable test consumables and a self-contained Verigene Test Cartridge are utilized for each sample tested with the CDF assay.
To obtain the test results after test processing is complete, the user removes the Test Cartridge from the Processor SP, and inserts the substrate holder into the Reader for analysis. Light scatter from the capture spots is imaged by the Reader and intensities from the microarray spots are used to make a determination regarding the presence (Detected) or absence (Not Detected) of a bacterial nucleic acid sequence/analyte. This determination is made by means of software-based decision algorithm resident in the Reader.
Nanosphere, Inc.
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Performance Data - Analytical Testing
Analytical Sensitivity / Limit of Detection (LoD)
Analytical sensitivity (LoD) of the CDF Test was determined for seven strains of C. difficile, representing all major toxinotypes found in North America and including two PCR Ribotype 027 strains. The LoD was defined as the concentration at which the test produces a positive result greater than 95% of the time. Serial dilutions of the strains were tested and the putative LoD confirmed with 20 replicates. The LoDs for the seven strains are shown in the table below and ranged from 63 to 1250 CFU/ml of stool. Thus, the study established the overall limit of detection of the CDF Test to be 1250 CFU/ml of organism present in stool.
| Strain Designation
(Source ID) | Toxinotype | Calculated
CFU/mL Stool
at LoD | CFU per
CDF Test at
LoD | LoD
Confirmation
Results |
|-----------------------------------|------------|--------------------------------------|-------------------------------|--------------------------------|
| ATCC BAA-1805 | III | 250 | 5 | 20/20 |
| ATCC 43255 (VPI 10463) | 0 | 63 | 1.25 | 20/20 |
| ATCC BAA-1875 (5325) | V | 500 | 10 | 20/20 |
| CDC 2007858 | IX/XXIII | 1250 | 25 | 20/20 |
| CDC 2009087 | 0 | 1250 | 25 | 20/20 |
| CDC 2009292 | III | 1250 | 25 | 20/20 |
| ATCC 43598 (1470) | VIII | 250 | 5 | 20/20 |
Analytical Reactivity (Inclusivity)
Analytical reactivity of the CDF Test was demonstrated with a comprehensive panel of sixtythree (63) independently-confirmed C. difficile strains, tested in triplicate at three times the LoD (i.e. 3.750 CFU/mL). The panel was comprised of a wide range of toxinotypes, including toxinotypes 0, 1, IV, V, VIII, IX, X, XI, XII, XXI, XXII, IX/XXIII, XIV/XV, and six (6) PCR ribotype 027 strains (toxinotype III).
All tests correctly reported the expected results for the detection of gene sequences for toxigenic C. difficile and for presumptive PCR ribotype 027, with one exception. Strain CDC 2009048 strain, classified by the CDC as Toxinotype XIV/XV, is associated with non-027 strains (Ribotypes 111/122). However, the Verigene CDF Test reported detection of the tcdA, tedB, binary and tcdC-MUT targets as would be expected for a PCR ribotype 027 strain. Subsequent sequencing of the tcdC gene verified the presence of the A117 deletion.
Nanosphere, Inc.
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Analytical Specificity (Cross-reactivity)
Ninety-four (94) microorganisms, including two (2) non-toxigenic C. difficile strains and fourteen (14) non C. difficile Clostridium species, along with one human cell line, were tested with the CDF Test to determine analytical specificity. In addition, the cross-reactivity of Clostridium botulinum was evaluated by in silico analysis.
Each bacterial strain was prepared in Negative Stool Matrix and tested in triplicate in concentrations of 5×106 CFU/mL stool. Two (2) organisms. Cryptosporidium parvum and Giardia lamblia, were tested using genomic DNA at a concentration of 1×10° copies of gDNA. For the viruses. Echovirus 11 and Coxsackievirus were tested at 5×10° PFU/mL stool. Adenovirus, Enterovirus, Cytomegalovirus and Rotavirus were also tested using genomic DNA or RNA at a concentration of 1×10° copies per reaction. Noroviruses were tested as clinical samples.
Analytical specificity was observed to be 100%. including that determined by in silico analysis.
Microbial Interference
Microorganisms that may be encountered in clinical stool samples, but not detected by the CDF Test, were tested in this study to evaluate the potential for microbial interference. The CDF Test was tested against the same ninety-five (95) organisms/cell line that were used for analytical specificity, at the same medically relevant concentrations, using two strains of toxigenic C. difficile (ATCC BAA-1805 [toxinotype III] and ATCC 43255 [toxinotype 0]) at 1.5x LoD and 3x LoD, respectively.
No interference was observed with the CDF Test for any of the samples tested.
Interference
Thirty-four (34) products/exogenous substances (shown in the following table) that are possibly encountered in stool samples were evaluated for potential inhibitory effects with the CDF Test. Each interfering substance was evaluated at its "worst case" concentration, against two C. difficile strains (ATCC BAA-1805, ATCC 43255). Additionally, Carv-Blair media was tested, None of the thirty four (34) substances or the Cary-Blair media tested in this study showed any inhibitory effect on the detection of C. difficile using the CDF Test.
Nanosphere, Inc.
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| Stearic Acid | Preparation H® Hemorrhoidal Ointment | Aluminum Hydroxide, Reagent Grade |
|---|---|---|
| Palmitic Acid | Walgreens Enema Mineral Oil Laxative | Mesalazine |
| Whole Blood | Options Conceptrol® Vaginal Contraceptive Gel | Immodium® AD Anti-Diarrheal |
| Nasopharyngeal Swab Sample in Universal | ||
| Transport Media (UTM) | Dulcolax® Laxative Suppositories | Pepto-Bismol Max Strength |
| Nystatin Suspension | Dimenhydrinate | Ex-lax® Maximum Strength Stimulant |
| Laxative | ||
| Monistat® 3 | Neosporin® First Aid Antibiotic Ointment | Vancomycin |
| Preparation H® Medicated Wipes | Wet Ones® Antibacterial Hand Wipes | Metronidazole Topical Cream (0.75%) |
| Vagisil Anti-Itch Crème Maximum Strength . | K-Y® Personal Lubricant Jelly | Naproxen Sodium |
| Preparation H® Anti-Itch Hydrocortisone 1% | Vaseline Original 100% Pure Petroleum Jelly | Mucin from bovine submaxillary |
| glands, Type I-S (Dehydrated) | ||
| Desitin Maximum Strength Original Paste | Sarna Anti-Itch Lotion, Sensitive | Barium Sulfate |
| Gaviscon® Extra Strength Liquid Antacid | Bile, bovine, dried, unfractioned | Cary-Blair Medium |
| Phillips® Genuine Milk of Magnesia Saline | ||
| Laxative | Tums® Antacid with Calcium Extra Strength 750 |
Carry-over / Cross-contamination
The potential for carry-over and cross-contamination of the CDF Test on the Verigene system was assessed by alternately testing a high positive C. difficile sample (toxigenic & ribotype 027 Clostridium difficile strain BAA-1805) at 5x108 CFU/mL, followed by testing a negative sample, comprising only of CDF-Negative Stool Matrix. The high-titer sample was alternated with the negative sample three times on three unique Verigene SP Processors, for a total of eighteen individual tests. No carry-over or cross-contamination was observed.
Cutoff Verification
Analytical testing of 59 strains of C. difficile, comprising a range of toxinotypes and nontoxinogenic strains, were performed in duplicate with the CDF Test to verify the cut off values of the two-tiered filter algorithm. Using the established cut-off levels for the assay, the CDF Test correctly detected the expected analytes for all the verification samples.
Performance Data - Clinical Testing
Precision
The precision study was conducted in-house by Nanosphere, during which a seven-member Precision Study Sample Panel was tested in duplicate twice daily by two operators for twelve non-consecutive days. This testing regime generated a total of 48 replicates per specimen and an overall total of 336 data points.
The seven sample panel was comprised of two different strains (ATCC 43255 and ATCC BAA-1805) at three different concentrations (six positive samples) and one negative sample. This panel included for each strain, a "High Negative" sample, which would be expected to produce a negative result approximately 20% to 80% of the time, a "Low Positive" sample, which would
Nanosphere, Inc.
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be expected to produce a positive result approximately 95% of the time, and a "Moderate Positive" sample, which would be expected to yield a positive result approximately 100% of the time. Results are summarized below.
| Panel
Member | Strain | Level | Expected
Occurrence | Total Agreement
with Expected
Result* |
|-----------------|----------------------------------------------|------------------------|------------------------|---------------------------------------------|
| 1 | CDF Negative Stool
Matrix | Negative | ~100%
Negative | 100%
48/48
(92.6-100%) |
| 2 | Toxigenic Wild Type
C. difficile | Moderate Positive (MP) | ~100% Positive | 100%
48/48
(92.6-100%) |
| 3 | Toxigenic Wild Type
C. difficile | Low Positive (LP) | ~95% Positive | 97.9%
47/48
(88.9-100%) |
| 4 | Toxigenic Wild Type
C. difficile | High Negative (HN) | ~20-80%
Negative | 12.5%
6/48
(4.7-25.3%) |
| 5 | Toxigenic
Hypervirulent
C. difficile | Moderate Positive (MP) | ~100% Positive | 100%
48/48
(92.6-100%) |
| 6 | Toxigenic
Hypervirulent
C. difficile ' | Low Positive (LP) | ~95% Positive | 95.8%
46/48
(85.6-99.5%) |
| 7 | Toxigenic
Hypervirulent
C. difficile | High Negative (HN) | ~20-80%
Negative | 20.8%
10/48
(10.5-35.0%) |
Reproducibility
The inter-laboratory reproducibility of the CDF was determined by conducting a reproducibility study at three external sites. Seven samples were tested daily in triplicate by two (2) operators for five (5) non-consecutive days at three (3) sites for a total of ninety (90) tests per sample (3 sites x 2 operators / site x 3 replicates / operator x 5 days = 90 tests per sample). The study tested a total of 630 samples.
The seven (7) sample panel for the reproducibility study was the same panel described previously for the precision study, comprised of two (2) different strains at three (3) different concentrations (six positive samples) and one (1) negative sample. The results of the Reproducibility Study are provided in the table below.
Nanosphere, Inc.
7
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| Panel
Member | Strain | Level | Expected
Occurrence | Total Agreement with Expected Result*
(95% CI) | | | |
|-----------------|--------------------------------------------|------------------------------|------------------------|---------------------------------------------------|--------------------------------|-------------------------------|--------------------------------|
| | | | | Site 1 | Site 2 | Site 3 | Total |
| 1 | CDF
Negative
Stool
Matrix | Negative | ~100%
Negative | 100%
30/30
(88.4-100) | 100%
30/30
(88.4-100) | 100%
30/30
(88.4-100) | 100%
90/90
(96.0-100) |
| 2 | Toxigenic
Wild Type
C. difficile | Moderate
Positive
(MP) | ~100%
Positive | 100%
30/30
(88.4-100) | 100%
30/30
(88.4-100) | 100%
30/30
(88.4-100) | 100%
90/90
(96.0-100) |
| 3 | Toxigenic
Wild Type
C. difficile | Low
Positive
(LP) | ~95%
Positive | 100%
30/30
(88.4-100) | 100%
30/30
(88.4-100) | 100%
30/30
(88.4-100) | 100%
90/90
(96.0-100) |
| 4 | Toxigenic
Wild Type
C. difficile | High
Negative
(HN) | ~20-80%
Negative | 30%
9/30
(14.7-49.4) | 33.30%
10/30
(17.3-52.8) | 16.70%
5/30
(5.6-34.7) | 26.70%
24/90
(17.9-37.0) |
| 5 | Toxigenic
Hypervirulent
C. difficile | Moderate
Positive
(MP) | ~100%
Positive | 100%
30/30
(88.4-100) | 100%
30/30
(88.4-100) | 100%
30/30
(88.4-100) | 100%
90/90
(96.0-100) |
| 6 | Toxigenic
Hypervirulent
C. difficile | Low
Positive
(LP) | ~95%
Positive | 96.70%
29/30
(82.8-99.9) | 96.70%
29/30
(82.8-99.9) | 100%
30/30
(88.4-100) | 97.80%
88/90
(92.2-99.7) |
| 7 | Toxigenic
Hypervirulent
C. difficile | High
Negative
(HN) | ~20-80%
Negative | 36.70%
11/30
(19.9-56.1) | 40.00%
12/30
(22.7-59.4) | 30.00%
9/30
(14.7-49.4) | 35.60%
32/90
(25.7-46.4) |
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Page 8 of 10
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8
Method Comparison
The performance characteristics of the CDF Test were determined in a multi-site prospective investigation study at five U.S. institutions by comparing the Verigene CDF Test results to reference culture followed by cell cytotoxicity testing on the isolates and strain typing on the toxigenic strains by PCR Ribotyping and Bi-Directional Sequencing methods.
Subjects included individuals whose routine care called for C. difficile testing. A portion of each leftover unformed stool specimen was obtained for testing with the CDF Test. In parallel to Verigene CDF Testing, an aliquot of the same specimen was sent to a central laboratory for reference culture and cytotoxin B isolate testing. Each stool specimen was inoculated onto prereduced CCFA-D (cycloserine-cefoxitin-fructose agar-direct plate) and CCMB-Tal (cycloserine cefoxitin mannitol broth with taurocholate lysozyme cysteine). After 24 hours the CCMB-TAL was subcultured onto a second CCFA-E plate (CCFA-Enriched). The direct culture method is referred to hereafter as the "direct culture" and the enriched culture method is referred to hereafter as the "enriched culture".
If C. difficile was isolated from the CCFA-D plate and the isolate was positive by the cell cytotoxicity assay, the specimen was classified as "toxigenic C. difficile positive" and the CCFA-E plate was not further analyzed. If no C. difficile was isolated from the CCFA-D plate or if the isolate was negative by the cell cytoxicity assay, the CCFA-E plate was further analyzed.
If CCFA-E was positive for C. difficile and the isolate was positive for cell cytotoxicity assay, the specimen was classified as "toxigenic C. difficile positive". The specimen was reported as "negative" if CCFA-E was negative for C. difficile or the isolate was tested negative by the cell cytotoxicity assay.
Following central site culture testing, the toxigenic C. difficile positive isolates were sent for strain identification by PCR Ribotyping at an external third-party site. In addition to conventional agarose-gel electrophoresis, for added discrimination, PCR products were analyzed using the Agilent 2100 Bioanalyzer with the strain type assignment based on a comparison of isolate sizing profiles with known C. difficile reference strains were designated into two PCR Ribotyping categories, 027 and non-027.
In parallel, following central culture testing, CDF Test extracted DNA from the cultureconfirmed C. difficile positive isolates or the DNA extracted during the PCR Ribotyping were sent for tcdC Bi-Directional Sequencing. Sequencing templates were prepared by PCR amplification of the stored DNA using sequencing primers. The same set of primers was also used for sequencing the amplified material. The PCR primers were designed from conserved regions based on multiple sequence alignments of all available sequence entries for each target in GenBank at the time of this study.
The clinical performance of the CDF Test was evaluated against four basic metrics, consisting of the combination of two reference methods for the detection of toxigenic C. difficile and two reference methods for the detection of the 027 (hypervirulent) strain of C. difficile: 1) Direct Culture and PCR Ribotyping, 2) Enriched Culture and PCR Ribotyping, and 3) Direct Culture
Nanosphere, Inc.
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and Bidirectional Sequencing, and 4) Enriched Culture and Bidirectional Sequencing. The overall performance of the CDF Test evaluated against these metrics is summarized in Table 1.
| Toxigenic C. difficile | Toxigenic C. difficile / 027 Strain | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Sensitivity | ||||||||||
| (95% CI) | Specificity | |||||||||
| (95% CI) | Accuracy | |||||||||
| (95% CI) | PPV | |||||||||
| (95% CI) | NPV | |||||||||
| (95% CI) | Agreement | PPV | ||||||||
| (95% CI) | NPV | |||||||||
| (95% CI) | ||||||||||
| POS | ||||||||||
| (95% CI) | NEG | |||||||||
| (95% CI) | Total | |||||||||
| (95% CI) | ||||||||||
| Direct | ||||||||||
| Culture & | ||||||||||
| PCR | ||||||||||
| Ribotyping(a) | 98.7% | |||||||||
| 154/156 | ||||||||||
| (95.5-99.8) | 87.6% | |||||||||
| 1500/1713 | ||||||||||
| (85.9-89.1) | 88.5% | |||||||||
| 1654/1869 | ||||||||||
| (87.0-89.9) | 42.0% | |||||||||
| 154/367 | ||||||||||
| (36.9-47.2) | 99.9% | |||||||||
| 1500/1502 | ||||||||||
| (99.5-99.9) | 97.5% | |||||||||
| 39/40 | ||||||||||
| (86.8-99.9) | 97.8% | |||||||||
| 1787/1828 | ||||||||||
| (97.0-98.4) | 97.7% | |||||||||
| 1826/1869 | ||||||||||
| (96.9-98.3) | 48.2% | |||||||||
| 39/81 | ||||||||||
| (36.9-59.5) | 99.9% | |||||||||
| 1787/1788 | ||||||||||
| (99.7-100) | ||||||||||
| Enriched | ||||||||||
| Culture & | ||||||||||
| PCR | ||||||||||
| Ribotyping(a) | 91.8% | |||||||||
| 247/269 | ||||||||||
| (87.9-94.8) | 92.5% | |||||||||
| 1480/1600 | ||||||||||
| (91.1-93.7) | 92.4% | |||||||||
| 1727/1869 | ||||||||||
| (91.1-93.6) | 67.3% | |||||||||
| 247/367 | ||||||||||
| (62.2-72.1) | 98.5% | |||||||||
| 1480/1502 | ||||||||||
| (97.8-99.1) | 91.4% | |||||||||
| 53/58 | ||||||||||
| (81.0-97.1) | 98.5% | |||||||||
| 1783/1811 | ||||||||||
| (97.8-99.0) | 98.2% | |||||||||
| 1836/1869 | ||||||||||
| (97.5-98.8) | 65.4% | |||||||||
| 53/81 | ||||||||||
| (54.0-75.7) | 99.7% | |||||||||
| 1783/1788 | ||||||||||
| (99.4-99.9) | ||||||||||
| Direct | ||||||||||
| Culture & | ||||||||||
| BDS (b) | 98.7% | |||||||||
| 156/158 | ||||||||||
| (95.5-99.9) | 87.5% | |||||||||
| 1500/1715 | ||||||||||
| (85.8-89.0) | 88.4% | |||||||||
| 1656/1873 | ||||||||||
| (86.9-89.8) | 42.1% | |||||||||
| 156/371 | ||||||||||
| (37.0-47.3) | 99.9% | |||||||||
| 1500/1502 | ||||||||||
| (99.5-100) | 97.7% | |||||||||
| 42/43 | ||||||||||
| (87.7-99.9) | 97.8% | |||||||||
| 1790/1830 | ||||||||||
| (97.0-98.4) | 97.8% | |||||||||
| 1832/1873 | ||||||||||
| (97.0-98.4) | 51.2% | |||||||||
| 42/82 | ||||||||||
| (39.9-62.4) | 99.9% | |||||||||
| 1790/1791 | ||||||||||
| (99.7-100) | ||||||||||
| Enriched | ||||||||||
| Culture & | ||||||||||
| BDS (b) | 91.9% | |||||||||
| 251/273 | ||||||||||
| (88.1-94.9) | 92.5% | |||||||||
| 1480/1600 | ||||||||||
| (91.1-93.7) | 92.4% | |||||||||
| 1731/1873 | ||||||||||
| (91.1-93.6) | 67.7% | |||||||||
| 251/371 | ||||||||||
| (62.6-72.4) | 98.5% | |||||||||
| 1480/1502 | ||||||||||
| (97.8-99.1) | 93.7% | |||||||||
| 59/63 | ||||||||||
| (84.5-98.2) | 98.7% | |||||||||
| 1787/1810 | ||||||||||
| (98.1-99.2) | 98.6% | |||||||||
| 1846/1873 | ||||||||||
| (97.9-99.1) | 72.0% | |||||||||
| 59/82 | ||||||||||
| (60.9-81.3) | 99.8% | |||||||||
| 1787/1791 | ||||||||||
| (99.4-99.9) |
| Table 1: | Summary of Overall CDF Test Clinical Performance n=1875 | |||
|---|---|---|---|---|
| ---------- | -- | -- | -- | --------------------------------------------------------- |
(ਕ) Of the 1,875 specimens evaluated, six specimens were not PCR-ribotyped because the isolate was either not sent or the result was inconclusive. These six specimens were not included in the performance characteristics above.
(b) Of the 1,875 specimens evaluated, two specifive but were not sequenced because the isolate was either not sent or the result was inconclusive. These two specimens were not included in the performance characteristics above.
Substantial Equivalence
The Verigene C. difficile Nucleic Acid Test (CDF) is as safe and effective as the combination predicate consisting of the Cepheid Xpert C. difficile/Epi Assay, the Great Basin Scientific Portrait C. difficile assay and the Nanosphere Verigene RVNATsp. CDF has similar intended use and indications, performance characteristics and principles of operation as Cepheid's Xpert C. difficile/Epi Assay and Great Basin Scientific's Portrait C. difficile assay. CDF has similar technological characteristics as Nanosphere's Verigene RVNATsp. The minor differences between CDF and its predicate devices raise no new issues of safety or effectiveness. Performance data demonstrate that the CDF is as safe and effective as the predicate devices. Thus, CDF is substantially equivalent.
Nanosphere, Inc.
10
DEPARTMENT OF HEALTH & HUMAN SERVICES
Image /page/10/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo features a stylized caduceus, which is a symbol often associated with medicine and healthcare. The words "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" are arranged in a circular pattern around the caduceus.
Public Health Service
Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-002
DEC 5 2012
Nanosphere, Inc. Mr. Mark A. Del Vecchio 4088 Commercial Avenue Northbrook. IL 60062
Re: K123197
Trade/Device Name: Regulation Number: Regulation Name: Regulatory Class: Product Code: Dated: Received:
Verigene® Clostridium difficile Nucleic Acid Test (CDF) 21 CFR 866.3130 C. Difficile Nucleic Acid Amplification Test Assay Class II OZN October 10, 2012 October 11, 2012
Dear Mr. Del Vecchio:
We have reviewed your Section 510(k) premarket notification of intent to market the device we nave reviewed your becement of the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate for use stated in the encrosure) to regars actment date of the Medical Device Amendments, or to comments from to May 20, 1978, the encordance with the provisions of the Federal Food, Drug, devices that have been recuire approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. I ou may, increfore, manot the Act include requirements for annual registration, listing of I he general controls provisions and prohibitions against misbranding and adulteration.
If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it If your device is classified (See acove) into the major regulations affecting your device can be may be subject to additions, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.
Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act that I DA has made a determination administered by other Federal agencies. You must or any I edelar sunates and regaraments, including, but not limited to: registration and listing (21 Comply with an the Act 3 requirements on the more reporting (reporting (reporting of medical CrK Fatt 607); labornig (21 OFR 803); good manufacturing practice requirements as set device-related adverse events) (21 CFR Part 820); and if applicable, the electronic forth in the quality systems (Sections 531-542 of the Act); 21 CFR 1000-1050.
11
Page 2 - Mark A. Del Vecchio
If you desire specific advice for your device on our labeling regulation (21 CFR Parts 801 and n you desire specific arriver of In Vitro Diagnostics and Radiological Health at (301) 796-5450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to
under the MDR regulation (21 Of NY are ook) poolem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.
You may obtain other general information on your responsibilities under the Act from the Tou may obtain other general memational and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.
Sincerely yours,
Sally A. Hojvat
Sally A. Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health
Enclosure
12
4. Indications for Use Statement
K123197 510(k) Number (if known): _
Device Name: Verigene® Clostridium difficile Nucleic Acid Test (CDF) on the Verigene® System
The Verigene® Clostridium difficile Nucleic Acid Test (CDF) is a qualitative multiplexed in vitro diagnostic test for the rapid detection of toxin A (tcdA), toxin B (tcdB), and tedC gene sequences of toxigenic Clostridium difficile and for presumptive identification of PCR ribotype 027 strains from unformed (liquid or soft) stool specimens collected from patients suspected of having C. difficile infection (CDI). Presumptive identification of the PCR ribotype 027 strain of C. difficile is by detection of the binary toxin (edf) gene sequence and the single base pair deletion at nucleotide 117 in the tcdC gene. The tcdC gene encodes for a negative regulator in C. difficile toxin production. The test is performed on the Verigene System and utilizes automated specimen preparation and polymerase chain reaction (PCR) amplification, combined with a nanoparticle-based array hybridization assay to detect the toxin gene sequences associated with toxin-producing C. difficile.
The CDF Test is indicated for use as an aid in the diagnosis of CDI. Detection of PCR ribotype 027 strains of C. difficile by the CDF Test is solely for epidemiological purposes and is not intended to guide or monitor treatment for C. difficile infections. Concomitant culture is necessary only if further typing or organism recovery is required.
Prescription Use x (Part 21 CFR 801 Subpart D) and/or
Over-The-Counter Use (21 CFR 801 Subpart C)
(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED)
Concurrence of CDRH, Office of In Vitro Diagnostic Devices (OIVD)
Nac cot
Division Sign-Off
Office of In Vitro Diagnostic Device Evaluation and Safety
510(k) K123197