(265 days)
The cobas® Cdiff Test on the cobas® 4800 system is an automated, qualitative in vitro diagnostic test, that utilizes realtime polymerase chain reaction (PCR), for the direct detection of the toxin B (tcdB) gene of toxigenic Clostridium difficile (C. difficile) in unformed (liquid or soft) stool specimens obtained from patients suspected of having C. difficile infection (CDI). The cobas® Cdiff Test is intended for use as an aid in the diagnosis of CDI in humans in conjunction with clinical and epidemiological risk factors.
The Roche Molecular Systems (RMS) cobas® Cdiff Test utilizes real-time polymerase chain reaction (PCR) for the detection of Clostridium difficile (C. difficile) DNA from unformed (liquid or soft) stool specimens to aid in the diagnosis of Clostridium difficile infections in humans. The cobas Cdiff Test contains two major processes: (1) automated sample preparation to extract nucleic acids from the unformed stool specimens; (2) PCR amplification of target DNA sequences using C. difficile specific primers, and real-time detection of cleaved fluorescentlabeled C. difficile specific oligonucleotide detection probes. An Internal Control (IC), containing unrelated randomized DNA sequence, is added to all samples prior to automated sample preparation and is amplified and detected simultaneously with each sample to monitor the entire process. The cobas® Cdiff Test utilizes six reagent kits and is used with the cobas® 4800 System, which includes the cobas x 480 Instrument for sample preparation and the cobas z 480 Analyzer for amplification and detection, controlled by the cobas® 4800 System Software.
Here's a summary of the acceptance criteria and study details for the cobas® Cdiff Test, based on the provided text:
1. Table of Acceptance Criteria and Reported Device Performance
The document doesn't explicitly state quantitative acceptance criteria in a dedicated section. However, performance targets are implicitly demonstrated and met by the reported study results. The clinical performance section compares the cobas® Cdiff Test to a "combined direct and enrichment culture" which serves as the reference standard. The reported performance metrics are:
| Performance Metric | Acceptance Criteria (Implied by Study Results) | Reported Device Performance (95% CI) |
|---|---|---|
| Sensitivity | High (e.g., >85-90%) | 92.9% (131/141; 87.4% to 96.1%) |
| Specificity | High (e.g., >95%) | 98.7% (534/541; 97.4% to 99.4%) |
| Positive Predictive Value (PPV) | High (e.g., >90%) | 94.9% (89.9% to 97.5%) |
| Negative Predictive Value (NPV) | High (e.g., >95%) | 98.2% (96.6% to 99.0%) |
| Analytical Sensitivity (LOD) | Low concentration for 95% positive rate | 225 CFU/swab (highest LOD among 7 strains) |
| Inclusivity | ≥ 95% positive rate at specified concentration | Achieved for 28 additional toxigenic strains |
| Analytical Specificity (Cross-reactivity) | No cross-reactivity with common organisms | 0% (expected negative results for 131 organisms) |
| Cross-Contamination | Very low rate | 0.24% (1/423 negative samples became positive) |
| Carry-over Contamination | 0% | 0.00% (0/282 negative samples became positive) |
| Reproducibility (Positive Agreement) | High at 1xLOD and 3xLOD | 100.0% |
| Reproducibility (Negative Agreement) | High for negative samples | 100.0% |
2. Sample Size Used for the Test Set and Data Provenance
- Sample Size for Clinical Performance (Test Set): 682 specimens were included in the statistical analysis for comparison with combined direct and enrichment culture. 683 subjects were initially collected from.
- Data Provenance: Prospective, multi-site investigation across five geographically diverse sites in the US. The samples were "leftover, de-identified, unformed stool samples from subjects suspected of having CDI."
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts
The document does not explicitly state the number or qualifications of experts used to establish the ground truth. However, the ground truth for the clinical study was based on toxigenic culture, which involved laboratory procedures conducted at a "single reference laboratory." This suggests trained laboratory personnel, but no specific professional qualifications (e.g., "radiologist with 10 years of experience") are provided.
4. Adjudication Method for the Test Set
The primary ground truth for the clinical performance was established by toxigenic culture (direct and enrichment culture followed by cytotoxicity testing). Discrepant analysis was performed on all samples with discordant results and a random subset of concordant results between the cobas® Cdiff Test and toxigenic culture using a second FDA-cleared nucleic acid amplification test (NAAT). This indicates an adjudication method involving a third, independent test to resolve discrepancies.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done
No, a multi-reader multi-case (MRMC) comparative effectiveness study was not reported. This study evaluates the impact of AI on human reader performance. The cobas® Cdiff Test is an automated diagnostic test that doesn't involve human interpretation of images or complex data in a way that an MRMC study would be applicable.
6. If a Standalone Study Was Done
Yes, a standalone study was done. The entire clinical performance evaluation directly assesses the performance of the cobas® Cdiff Test (the algorithm/device) against the established ground truth (toxigenic culture) without human intervention in the interpretation of the device's results.
7. The Type of Ground Truth Used
The ground truth used for the clinical performance evaluation was expert consensus informed by culture and cytotoxicity testing. Specifically, a specimen was considered positive for toxigenic C. difficile if C. difficile was recovered from stool by either direct or enriched toxigenic culture and if isolates recovered tested positive by cytotoxicity testing. Specimens were negative only if they tested negative by both direct, repeat direct, and enrichment culture. Additionally, a second FDA-cleared NAAT was used for discrepant analysis, further supporting the ground truth adjudication.
8. The Sample Size for the Training Set
The document does not provide information on the sample size for the training set. This K142422 submission focuses on the performance of a developed device, not on the details of its developmental training data.
9. How the Ground Truth for the Training Set Was Established
The document does not provide information on how the ground truth for the training set was established. The submission focuses on the clinical validation of the device, not its initial development or training process.
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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002 December 7, 2015
ROCHE MOLECULAR SYSTEMS, INC. DAVID W. GATES. PH.D. SENIOR DIRECTOR, REGULATORY AFFAIRS 4300 HACIENDA DRIVE PLEASANTON CA 94588-2722
Re: K142422
Trade/Device Name: cobas Cdiff Test Regulation Number: 21 CFR 866.3130 Regulation Name: Clostridium difficile Toxin Gene Amplification Assay Regulatory Class: II Product Code: OZN, OOI Dated: April 16, 2015 Received: April 17, 2015
Dear Dr. Gates:
This letter corrects our substantially equivalent letter of May 20, 2015.
We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.
If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.
Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of
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medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 8091 ), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. Also, please note the regulation entitled. "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to
http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.
You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.
Sincerely yours,
Ribhi Shawar -S
Uwe Scherf, M.Sc., Ph.D. For Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known) K142422
Device Name cobas® Cdiff Test
Indications for Use (Describe)
The cobas® Cdiff Test on the cobas® 4800 system is an automated, qualitative in vitro diagnostic test, that utilizes realtime polymerase chain reaction (PCR), for the direct detection of the toxin B (tcdB) gene of toxigenic Clostridium difficile (C. difficile) in unformed (liquid or soft) stool specimens obtained from patients suspected of having C. difficile infection (CDI). The cobas® Cdiff Test is intended for use as an aid in the diagnosis of CDI in humans in conjunction with clinical and epidemiological risk factors.
| Type of Use (Select one or both, as applicable) | |
|---|---|
| Prescription Use (Part 21 CFR 801 Subpart D) | Over-The-Counter Use (21 CFR 801 Subpart C) |
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510(k) Summary
| Submitter Name | Roche Molecular Systems, Inc. |
|---|---|
| Address | 4300 Hacienda DrivePleasanton, CA 94588-2722 |
| Contact | David GatesPhone: (925) 730-8237FAX: (925) 225-0207 |
| Date Prepared | April 16, 2015 |
| Proprietary Name | cobas® Cdiff Test |
| Common Name | Clostridium difficile Test |
| Classification Name | 21 CFR 866.3130 - Clostridium difficile toxin gene amplification assay21 CFR 862.2570 - Real Time Nucleic Acid Amplification System |
| Product Codes | OZN, OOI |
| Predicate Devices | BD MAX™ Cdiff Assay, BD MAX™ Instrument, K130470 |
| Establishment Registration | Branchburg: 2243471Pleasanton: 3004141078Indianapolis: 1823260 |
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1. DEVICE DESCRIPTION
The Roche Molecular Systems (RMS) cobas® Cdiff Test utilizes real-time polymerase chain reaction (PCR) for the detection of Clostridium difficile (C. difficile) DNA from unformed (liquid or soft) stool specimens to aid in the diagnosis of Clostridium difficile infections in humans.
The cobas Cdiff Test contains two major processes: (1) automated sample preparation to extract nucleic acids from the unformed stool specimens; (2) PCR amplification of target DNA sequences using C. difficile specific primers, and real-time detection of cleaved fluorescentlabeled C. difficile specific oligonucleotide detection probes. An Internal Control (IC), containing unrelated randomized DNA sequence, is added to all samples prior to automated sample preparation and is amplified and detected simultaneously with each sample to monitor the entire process.
The cobas® Cdiff Test utilizes six reagent kits:
-
- cobas® 4800 Cdiff Amplification/Detection Kit
-
- cobas® 4800 Cdiff Controls and Cofactor Kit
-
- cobas® 4800 System Wash Buffer Kit
-
- cobas® 4800 System Lysis Kit 1
-
- cobas® 4800 System Internal Control Kit 1
-
- cobas 4800 System Sample Preparation Kit
Required but not provided:
cobas® PCR Media and Swab Sample Kit
Sealing mat or deep well plate cover
Caps, neutral color (for recapping post-run specimens)
1.1. Target Selection
The cobas® Cdiff Test utilizes real-time PCR technology to detect the conserved regions of toxin B (tcdB) gene. Fluorogenic target specific probes are used for the detection of the amplified C.
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difficile DNA as well as IC. Primer and probe oligonucleotide sequences were designed to select C. difficile conserved sequences without cross reacting with other organisms commonly found in stool specimens.
1.2. Test Principle
Sample Preparation 1.1.1.
Sample preparation for the cobas Cdiff Test is automated with the use of the cobas x 480 instrument. Unformed stool specimens are transferred into cobas® PCR Media by using a swab. Organisms are lysed with chaotropic agent, proteinase K, and SDS reagents. Released nucleic acids, along with added Internal Control DNA, are bound by magnetic glass particles. They are washed and then eluted into a small volume of buffer. The instrument then takes an aliquot of the eluted material and sets up the PCR reaction with an activated Master Mix.
Note: The cobas® Cdiff Test has been validated for use with the cobas® PCR Media and Swab Sample Kit. Do not use other devices or media types.
PCR Amplification and TaqMan® Detection 1.1.2.
The PCR cycling steps and detection of target signal occurs in the cobas z 480 Analyzer. The Master Mix reagent contains primer pairs and probes for two targets: the C. difficile toxin B gene and Internal Control. If the target nucleic acid sequences are present, amplification with the corresponding primers will occur by a thermostable DNA polymerase, generating PCR products (amplicons). These products are detected by specific TagMan probes containing a fluorescent dye and a quencher. Normally, the quencher suppresses the fluorescence of the dye. However, if the PCR product is present, the probe hybridizes to the product and gets cleaved by the 5' to 3' nuclease activity of the polymerase. This reaction allows the fluorescence to be emitted from the dye, and the signal is recorded in real time during each PCR cycle by the cobas z 480 analyzer. The signal is interpreted by the cobas 4800 System Software and reported as final results.
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cobas® 4800 System Description 1.3.
The cobas 4800 System uses the cobas x 480 Instrument for sample preparation, and the cobas z 480 Analyzer for amplification and detection. Both the cobas x 480 Instrument and the cobas z 480 Analyzer are controlled by a computer workstation running the cobas® 4800 System Software.
The system hardware is unchanged from that originally approved for IVD use in PMA P100020 (cobas® HPV Test, April 19, 2011). The software version has been updated to software release 2.1 in order to support the expanded test menu. The updated software was cleared for other currently available tests on the cobas 4800 System per Special 510(K) 140887.
2. INTENDED FOR USE
The cobas® Cdiff Test on the cobas® 4800 System is an automated, qualitative in vitro diagnostic test, that utilizes real-time polymerase chain reaction (PCR), for the direct detection of the toxin B (tcdB) gene of toxigenic Clostridium difficile (C. difficile) in unformed (liquid or soft) stool specimens obtained from patients suspected of having C. difficile infection (CDI). The cobas " Cdiff Test is intended for use as an aid in the diagnosis of CDI in humans in conjunction with clinical and epidemiological risk factors.
3. TECHNOLOGICAL CHARACTERISTICS
The primary technological characteristics and intended use of the RMS cobas® Cdiff Test are substantially equivalent to other legally marketed nucleic acid amplification tests intended for the qualitative detection of toxigenic Clostridium difficile.
As indicated in Table 1, the RMS cobas® Cdiff Test is substantially equivalent to technological characteristics of the predicate device. Both assays have the same intended use, and utilize nucleic acid amplification and detection technology for the detection of C. difficile DNA in patient specimens.
Formatted: Font color: Blue Deleted: Table 1
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| Submitted Device:RMS cobas® Cdiff Test | Predicate Device:BD MAX™ Cdiff Assay, K130470 | |
|---|---|---|
| Intended Use | The cobas® Cdiff Test on the cobas®4800 system is an automated, qualitativein vitro diagnostic test, that utilizes real-time polymerase chain reaction (PCR), forthe direct detection of the toxin B (tcdB)gene of toxigenic Clostridium difficile inunformed (liquid or soft) stool specimensobtained from patients suspected ofhaving C. difficile infection (CDI). Thecobas® Cdiff Test is intended for use asan aid in the diagnosis of CDI in humansin conjunction with clinical andepidemiological risk factors. | The BD MAX™ Cdiff Assay performed onthe BD MAX™ System is an automated invitro diagnostic test for the direct,qualitative detection of the Clostridiumdifficile toxin B gene (tcdB) in human liquidor soft stool specimens from patientssuspected of having C. difficile infection(CDI). The test, performed directly on thespecimen, utilizes real-time polymerasechain reaction (PCR) for the amplificationof C. difficile toxin B gene DNA andfluorogenic target-specific hybridizationprobes for the detection of the amplifiedDNA. The BD MAX™ Cdiff Assay isintended to aid in the diagnosis of CDI. |
| Conditions for use | For prescription use | Same |
| Sample Types | Liquid and soft stool specimens | Same |
| Subject Status | Symptomatic | Same |
| Sample Collection Devices | cobas® PCR Media and Swab Sample Kit | BD MAX™ Cdiff Sample Buffer |
| Analyte Targets | Toxin B (tcdB) gene | Same |
| Sample Preparation Procedure | Automated by cobas® x480 | Automated by BD MAX™ System |
| Amplification Technology | Real-time PCR | Same |
| Detection Chemistry | Paired reporter and quencherfluorescence labeled probes (TaqManTechnology) using fluorescenceresonance energy transfer (FRET) | Same |
| Controls used | Sample processing control (IC)Positive and negative control | Sample processing controlPositive and negative control (optional) |
| Result Analysis | Based on PCR cycle threshold analysis | Same |
Similarities and Differences between the cobas® Cdiff Test Table 1: and the Predicate Device
In summary, the intended use, technology, and characteristics of the cobas® Cdiff Test as compared to the predicate device do not raise any new types of safety or effectiveness questions and are substantially equivalent.
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NON-CLINICAL PERFORMANCE EVALUATION 4.
Analytical Sensitivity 4.1.
The analytical sensitivity (Limit of Detection or LOD) for the cobas® Cdiff Test was determined by analyzing 7 toxigenic C. difficile strains ATCC 43255 (VPI 10463), ATCC BAA-1382 (630), CDC 204118, R12087 (CD196), 2748-06, ATCC 43598 (1470), and F15. CDC 204118 and R12087 (CD196) are BI/ NAP1/027 hyper-virulent epidemic strains. Quantified cultures were diluted into pooled negative stool specimen matrix to determine the LOD. All levels were analyzed using cobas® Cdiff Test with 3 unique lots of C. difficile specific reagents. The LOD of the test was determined as the lowest concentration exhibiting at least 95% positive rate for which all higher concentrations were greater than or equal to 95% positive rate.
The highest LOD among 3 reagent lots are shown in Table 2. The claimed LOD among the seven strains tested was 225 CFU/swab based on 95% positive rate.
Formatted: Font color: Blue Deleted: Table 3
Deleted: 3
| Strain ID | Toxinotype | REA*Type | PFG†Type | Ribotype | Phenotype | LOD (CFU/swab) | ||
|---|---|---|---|---|---|---|---|---|
| ByPositiveRate | By ProbitAnalysis(95% CI) | |||||||
| ATCC 43255 (VPI10463) | 0 | N/A | N/A | 087 | A+B+CDT- | 113 | 90 (66 – 311) | |
| ATCC BAA-1382 (630) | 0 | R 23 | N/A | 012 | A+B+CDT- | 81 | 83 (62 – 145) |
NAP1
NAP1
N/A
N/A
N/A
027
027
078
017
N/A
A+B+CDT+
A+B+CDT+
N/A
A-B+
N/A
54
54
54
225
54
BI 8
Bl
N/A
N/A
N/A
- Restriction endonuclease analysis; 1 Pulse Field Gel
=
111
<
VIII
XII
4.2. Inclusivity
CDC 204118
R12087 (CD196)
2748-06
ATCC 43598 (1470)
F15
The sensitivity of the cobas® Cdiff Test was determined for 28 additional toxigenic strains. The inclusivity panel consisted of at least three concentrations per strain in pooled negative stool specimen matrix. Forty replicates were tested for each concentration level. The LOD was
Page 6
42 (30 – 129)
54 (39 -126)
45 (33-113)
130 (96 - 228)
59 (43 - 117)
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calculated as the lowest concentration level with ≥ 95 % positive rate for which all higher concentration levels show ≥ 95 % positive rate. One lot of reagents was used for this study.
The inclusivity study results are listed in Table_3, _________________________________________________________________________________________________________________________
Table 3: _ Toxigenic C. difficile Inclusivity Results _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
| No. | Strain | Toxinotype | Ribotype | Toxin Production | LOD | Positive Rate |
|---|---|---|---|---|---|---|
| 1 | EX 623 | I | 102 | A+ B+ CDT- | 77.9 | 95.0% |
| 2 | AC 008 | II | 103 | A+ B+ CDT- | 77.9 | 95.0% |
| 3 | SE 844 | IIIa | 80 | A+ B+ CDT+ | 234 | 100.0% |
| 4 | 55767 | IV | 23 | A+ B+ CDT+ | 77.9 | 100.0% |
| 5 | SE 881 | V | 45 | A+ B+ CDT+ | 234 | 100.0% |
| 6 | 51377 | VI | N/A | A+ B+ CDT+ | 234 | 100.0% |
| 7 | 57267 | VII | 63 | A+ B+ CDT+ | 77.9 | 97.5% |
| 8 | 51680 | IX | 19 | A+ B+ CDT+ | 77.9 | 100.0% |
| 9 | 8864 | X | 36 | A- B+ CDT+ | 77.9 | 97.5% |
| 10 | R 9367 | XIII | 70 | A+ B+CDT- | 77.9 | 97.5% |
| 11 | R 10870 | XIV | 111 | A+ B+ CDT+ | 234 | 100.0% |
| 12 | R 9385 | XV | 122 | A+ B+ CDT+ | 234 | 100.0% |
| 13 | SUC36 | XVI | 78 | A- B+ CDT+ | 234 | 100.0% |
| 14 | J9965 | XVII | N/A | A- B+ CDT+ | 460 | 97.5% |
| 15 | K095 | XVIII | 14 | A+ B+ CDT- | 234 | 95.0% |
| 16 | TR13 | XIX | N/A | A+ B+ CDT- | 234 | 97.5% |
| 17 | TR14 | XX | N/A | A+ B+ CDT- | 77.9 | 100.0% |
| 18 | CH6223 | XXI | N/A | A+ B+ CDT- | 234 | 100.0% |
| 19 | CD07-468 | XXII | N/A | A+ B+ CDT+ | 234 | 100.0% |
| 20 | 8785 | XXIII | N/A | A+ B+ CDT+ | 234 | 95.0% |
| 21 | 597B | XXIV | 131 | A+ B+ CDT+ | 234 | 97.5% |
| 22 | 7325 | XXV | 27 | A+ B+ CDT+ | 234 | 100.0% |
| 23 | 7459 | XXVI | N/A | A+ B+ CDT- | 234 | 95.0% |
| 24 | KK2443-2006 | XXVII | N/A | A+ B+ CDT- | 234 | 100.0% |
| 25 | CD08-070 | XXVIII | 126 | A+ B+ CDT+ | 234 | 97.5% |
| 26 | CD07-140 | XXIX | 56 | A+ B+ CDT- | 234 | 97.5% |
| 27 | ES 130 | XXX | N/A | A- B+ CDT+ | 234 | 100.0% |
| 28 | WA 151 | XXXI | N/A | A- B+ CDT+ | 460 | 100.0% |
Image /page/9/Figure/4 description: The image shows text snippets indicating formatting and deletion actions. The first line states "Formatted: Font color: Blue", indicating that the font color was changed to blue. The second line says "Deleted: Table 4", suggesting that a table labeled as Table 4 was removed. The third line says "Deleted: 4", suggesting that the number 4 was removed.
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4.3. Precision
Level 1
Level 2
Level 3
20-80%
≥ 95%
≥ 99%
In-house precision study was conducted with C. difficile concentrations below Limit of Detection (LOD), near LOD and above LOD of the cobas® Cdiff Test including pooled negative stool specimen matrix. The study used three unique lots of cobas® Cdiff Test reagents and three instruments for a total of 36 runs over 12 days (3 runs per day). A description of the precision panels, the study results, and variance components are shown in Table 4. An analysis of the variance of the Ct values from valid tests was performed on positive panel members at LOD and above LOD concentrations (Table 5) and suggested that most variability of target Ct values is attributed to within run (random) and lot to lot factors (60.0% and 25.3%, respectively) for concentration level at or around LOD. For concentration level above LOD, most of the Ct value variability is attributed to within run (random) and instrument to instrument factors (72.5% and 24.7%, respectively).
Overall CV (%) at LOD and above LOD were 1.5 and 1.1%, respectively (Table 6).
| PanelMember | ExpectedPositivity rate | No. ofNegative | No. ofPositive | Total | PositiveRate | 95% LCL |
|---|---|---|---|---|---|---|
| Level 0 | 0% | 72 | 0 | 72 | 0.0% | 0.0% |
51
0
0
| Table 5: Variance Components Analysis for Precision Panel at or around and above | |
|---|---|
| LOD (Limit of Detection) |
21
72
72
72
72
72
29.2%
100.0%
100.0%
19.0%
95.0%
95.0%
| Panel Member | N | Mean | Variance Components by Factor Percent Contribution to Total | Total | ||||
|---|---|---|---|---|---|---|---|---|
| Lot | Instrument | Kit Size | Day | Random | ||||
| At or around LOD (Level 2) | 72 | 38.5 | 0.0789 | 0.0189 | 0.0001 | 0.0270 | 0.1875 | 0.3123 |
| 25.3% | 6.0% | 0.0% | 8.6% | 60.0% | 100.0% | |||
| Above LOD (Level 3) | 72 | 37.5 | 0.0047 | 0.0404 | 0.0000 | 0.0000 | 0.1188 | 0.1638 |
| 2.8% | 24.7% | 0.0% | 0.0% | 72.5% | 100.0% |
| Formatted: | Font color: Blue |
|---|---|
| Deleted: | Table 5 |
| Deleted: | Table 6 |
| Formatted: | Font color: Blue |
| A | Formatted: Font color: Blue | |||||||
|---|---|---|---|---|---|---|---|---|
| Deleted: Table 7 | ||||||||
| Deleted: 5 |
Deleted: 6
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95% UCL 5.0%
41.1%
100.0%
100.0%
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| Table 6: Standard Deviations and Coefficients of Variation (%) Analysis for Precision | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Panel at or around and above LOD (Limit of Detection) | |||||||||
| Panel Member | N | Mean | SD Components by FactorPercent CV | Total | ||||
|---|---|---|---|---|---|---|---|---|
| Lot | Instrument | Kit Size | Day | Random | ||||
| At or aroundLOD (Level 2) | 72 | 38.5 | 0.2808 | 0.1374 | 0.0098 | 0.1643 | 0.4330 | 0.5589 |
| 0.7% | 0.4% | 0.0% | 0.4% | 1.1% | 1.5% | |||
| Above LOD(Level 3) | 72 | 37.5 | 0.0682 | 0.2009 | 0.0000 | 0.0000 | 0.3446 | 0.4047 |
| 0.2% | 0.5% | 0.0% | 0.0% | 0.9% | 1.1% |
Analytical Specificity 4.4.
The cobas® Cdiff Test was examined for analytical specificity by testing non-toxigenic C. difficile, other Clostridium genus species, human DNA, and other organisms commonly found in digestive tract in the absence and the presence of toxigenic C. difficile strain at ~3xLOD concentration.
In addition, analytical specificity against C. botulinum, which is a highly regulated national select agent, was examined by computer based in-silico analysis.
The cobas® Cdiff Test gave expected negative results in the presence of 131 organisms and human DNA. Computer based in-silico analysis indicated that any cross reactivity against C. botulinum is highly unlikely.
| No. | Organism | TestingConcentration | Unit |
|---|---|---|---|
| 1 | Abiotrophia defective | 1E6 | CFU/mL |
| 2 | Acinetobacter baumannii | 1E6 | CFU/mL |
| 3 | Acinetobacter Iwoffii | 1E6 | CFU/mL |
| 4 | Aeromonas hydrophila | 1E6 | CFU/mL |
| 5 | Alcaligenes faecalis subsp. Faecalis | 1E6 | CFU/mL |
| 6 | Anaerococcus tetradius | 1E6 | CFU/mL |
| 7 | Bacillus cereus (ATCC 13472) | 1E6 | CFU/mL |
| 8 | Bacillus cereus (ATCC 11778) | 1E6 | CFU/mL |
| 9 | Bacteroides caccae | 1E6 | CFU/mL |
| 10 | Bacteroides merdae | 1E6 | CFU/mL |
| 11 | Bacteroides stercoris | 1E6 | CFU/mL |
| 12 | Bifidobacterium adolescentis | 1E6 | CFU/mL |
| No. | Organism | TestingConcentration | Unit |
| 13 | Bifidobacterium longum | 1E6 | CFU/mL |
| 14 | Campylobacter coli | 1E6 | CFU/mL |
| 15 | Campylobacter jejuni | 1E6 | CFU/mL |
| 16 | Candida albicans | 1E6 | CFU/mL |
| 17 | Candida catenulate | 1E6 | CFU/mL |
| 18 | Cedecea davisae | 1E6 | CFU/mL |
| 19 | Chlamydia Trachomatis Serovar L2 | 1E6 | EB/mL |
| 20 | Citrobacter amalonaticus | 1E6 | CFU/mL |
| 21 | Citrobacter freundii | 1E6 | CFU/mL |
| 22 | Citrobacter koseri | 1E6 | CFU/mL |
| 23 | Citrobacter sedlakii | 1E6 | CFU/mL |
| 24 | Clostridium beijerinckii | 1E6 | CFU/mL |
| 25 | Clostridium bifermentans | 1E6 | CFU/mL |
| 26 | Clostridium bolteae | 1E6 | CFU/mL |
| 27 | Clostridium botulinum* | N/A | N/A |
| 28 | Clostridium butyricum | 1E6 | CFU/mL |
| 29 | Clostridium chauvoei | 1E6 | CFU/mL |
| 30 | Clostridium difficile(Non-toxigenic, Serogroup B) | 1E6 | CFU/mL |
| 31 | Clostridium difficile(Non-toxigenic, Serogroup I) | 1E6 | CFU/mL |
| 32 | Clostridium fallax | 1E6 | CFU/mL |
| 33 | Clostridium haemolyticum | 1E6 | CFU/mL |
| 34 | Clostridium histolyticum | 1E6 | CFU/mL |
| 35 | Clostridium innocuum | 1E6 | CFU/mL |
| 36 | Clostridium methylpentosum | 1E6 | CFU/mL |
| 37 | Clostridium nexile | 1E6 | CFU/mL |
| 38 | Clostridium novyi | 1E6 | CFU/mL |
| 39 | Clostridium orbiscindens(re-named Flavonifractor plautii) | 1E6 | CFU/mL |
| 40 | Clostridium paraputrificum | 1E6 | CFU/mL |
| 41 | Clostridium perfringens | 1E6 | CFU/mL |
| 42 | Clostridium ramosum | 1E6 | CFU/mL |
| 43 | Clostridium scindens | 1E6 | CFU/mL |
| 44 | Clostridium septicum | 1E6 | CFU/mL |
| 45 | Clostridium sordellii | 1E6 | CFU/mL |
| 46 | Clostridium sphenoides | 1E6 | CFU/mL |
| 47 | Clostridium spiroforme | 1E6 | CFU/mL |
| 48 | Clostridium sporogenes | 1E6 | CFU/mL |
| 49 | Clostridium symbiosum | 1E6 | CFU/mL |
| 50 | Clostridium tertium | 1E6 | CFU/mL |
| 51 | Clostridium tetani | 1E6 | CFU/mL |
| 52 | Collinsella aerofaciens | 1E6 | CFU/mL |
| 53 | Corynebacterium genitalium | 1E6 | CFU/mL |
| 54 | Desulfovibrio piger | 1E6 | CFU/mL |
| No. | Organism | TestingConcentration | Unit |
| 55 | Edwardsiella tarda | 1E6 | CFU/mL |
| 56 | Eggerthella lenta | 1E6 | CFU/mL |
| 57 | Enterobacter aerogenes | 1E6 | CFU/mL |
| 58 | Enterobacter cloacae | 1E6 | CFU/mL |
| 59 | Enterococcus casseliflavus | 1E6 | CFU/mL |
| 60 | Enterococcus cecorum | 1E6 | CFU/mL |
| 61 | Enterococcus dispar | 1E6 | CFU/mL |
| 62 | Enterococcus faecalis | 1E6 | CFU/mL |
| 63 | Enterococcus faecium | 1E6 | CFU/mL |
| 64 | Enterococcus gallinarum | 1E6 | CFU/mL |
| 65 | Enterococcus hirae | 1E6 | CFU/mL |
| 66 | Enterococcus raffinosus | 1E6 | CFU/mL |
| 67 | Escherichia coli (ATCC 11775) | 1E6 | CFU/mL |
| 68 | Escherichia coli (ATCC 25922) | 1E6 | CFU/mL |
| 69 | Escherichia fergusonii | 1E6 | CFU/mL |
| 70 | Escherichia hermannii | 1E6 | CFU/mL |
| 71 | Fusobacterium varium | 1E6 | CFU/mL |
| 72 | Gardnerella vaginalis | 1E6 | CFU/mL |
| 73 | Gemella morbillorum | 1E6 | CFU/mL |
| 74 | Hafnia alvei | 1E6 | CFU/mL |
| 75 | Helicobacter fennelliae | 1E6 | CFU/mL |
| 76 | Helicobacter pylori | 1E6 | CFU/mL |
| 77 | HCT-15 Human Cells | 1E6 | Cells/mL |
| 78 | Klebsiella oxytoca | 1E6 | CFU/mL |
| 79 | Klebsiella pneumoniae subsp. pneumoniae | 1E6 | CFU/mL |
| 80 | Lactobacillus acidophilus | 1E6 | CFU/mL |
| 81 | Lactobacillus reuteri | 1E6 | CFU/mL |
| 82 | Lactococcus lactis | 1E6 | CFU/mL |
| 83 | Leminorella grimontii | 1E6 | CFU/mL |
| 84 | Listeria grayi | 1E6 | CFU/mL |
| 85 | Listeria innocua | 1E6 | CFU/mL |
| 86 | Listeria monocytogenes | 1E6 | CFU/mL |
| 87 | Mitsuokella multacida | 1E6 | CFU/mL |
| 88 | Mobiluncus curtisii | 1E6 | CFU/mL |
| 89 | Moellerella wisconsensis | 1E6 | CFU/mL |
| 90 | Morganella morganii | 1E6 | CFU/mL |
| 91 | Neisseria gonorrhoeae | 1E6 | CFU/mL |
| 92 | Peptoniphilus asaccharolyticus | 1E6 | CFU/mL |
| 93 | Peptostreptococcus anaerobius | 1E6 | CFU/mL |
| 94 | Plesiomonas shigelloides | 1E6 | CFU/mL |
| 95 | Porphyromonas asaccharolytica | 1E6 | CFU/mL |
| 96 | Prevotella melaninogenica | 1E6 | CFU/mL |
| 97 | Proteus mirabilis | 1E6 | CFU/mL |
| 98 | Proteus penneri | 1E6 | CFU/mL |
| No. | Organism | TestingConcentration | Unit |
| 99 | Providencia alcalifaciens | 1E6 | CFU/mL |
| 100 | Providencia rettgeri | 1E6 | CFU/mL |
| 101 | Providencia stuartii | 1E6 | CFU/mL |
| 102 | Pseudomonas aeruginosa | 1E6 | CFU/mL |
| 103 | Pseudomonas putida | 1E6 | CFU/mL |
| 104 | Ruminococcus bromii | 1E6 | CFU/mL |
| 105 | Salmonella choleraesuis subsp. choleraesuis | 1E6 | CFU/mL |
| 106 | Salmonella enterica subsp. arizonae(f.k.a. Salmonella choleraesuis ssp. arizonae) | 1E6 | CFU/mL |
| 107 | Salmonella enterica subsp. enterica serovar Choleraesuis | 1E6 | CFU/mL |
| 108 | Serratia liquefaciens | 1E6 | CFU/mL |
| 109 | Serratia marcescens | 1E6 | CFU/mL |
| 110 | Shigella boydii | 1E6 | CFU/mL |
| 111 | Shigella dysenteriae | 1E6 | CFU/mL |
| 112 | Shigella sonnei | 1E6 | CFU/mL |
| 113 | Staphylococcus aureus | 1E6 | CFU/mL |
| 114 | Staphylococcus epidermidis | 1E6 | CFU/mL |
| 115 | Stenotrophomonas maltophilia | 1E6 | CFU/mL |
| 116 | Streptococcus agalactiae | 1E6 | CFU/mL |
| 117 | Streptococcus dysgalactiae | 1E6 | CFU/mL |
| 118 | Streptococcus intermedius | 1E6 | CFU/mL |
| 119 | Streptococcus uberis | 1E6 | CFU/mL |
| 120 | Trabulsiella guamensis | 1E6 | CFU/mL |
| 121 | Veillonella parvula | 1E6 | CFU/mL |
| 122 | Vibrio cholera | 1E6 | CFU/mL |
| 123 | Vibrio parahaemolyticus | 1E6 | CFU/mL |
| 124 | Yersinia bercovieri | 1E6 | CFU/mL |
| 125 | Yersinia rohdei | 1E6 | CFU/mL |
| 126 | Cytomegalovirus (HHV5) | 2E3 | IU/mL |
| 127 | Human Adenovirus 40 | 2.19E3 | PFU/mL |
| 128 | Human Coxsackievirus A 10 | 1E5 | PFU/mL |
| 129 | Human Echovirus 11 | 1E5 | IU/mL |
| 130 | Human Enterovirus 71 | 1E5 | IU/mL |
| 131 | Human Rotavirus | 9.77E3 | PFU/mL |
| 132 | Norovirus GII | 1E5 | Viral Particles/mL |
Table 7: Organisms for Analytical Specificity
Deleted: 8
Deleted: 7
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{13}------------------------------------------------
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- By in silico analysis.
4.5. Interference
Twenty six commonly used OTC products and antibiotic medicines, as well as whole blood, mucin, fecal fat were tested for potential interference effects with the cobas® Cdiff Test. All
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OTC products were tested at or above the levels what could be reasonably expected to be in a fecal specimen. C. difficile was spiked to ~ 3 x LOD (Limit of Detection) of the cobase Cdiff Test and used as targets in the tests.
No interference was observed for OTC products and fecal fat. For whole blood and mucin, no interference was observed at 25% (w/v), but 50% of mucin exhibited interference (Lable 8).
| No. | Substance | Interpretation |
|---|---|---|
| 1 | Whole blood | No interference observed at 50% (v/v) |
| 2 | Mucin | No interference observed at 25% (w/v)* |
| 3 | Fecal Fat | No interference observed up to 28% (w/v) |
| 4 | Tums | No interference observed at 10% (w/v) |
| 5 | Vancomycin | No interference observed at 1% (w/v) |
| 6 | Metronidazole | No interference observed at 10% (w/v) |
| 7 | Imodium AD® | No interference observed at 10% (w/v) |
| 8 | Stool Softener | No interference observed at 10% (w/v) |
| 9 | Pepto-Bismol®(Procter & Gamble) | No interference observed at 10% (v/v) |
| 10 | Nystatin Ointment USP | No interference observed at 10% (w/v) |
| 11 | Preparation H® with Bio-Dyne® Cream (Wyeth) | No interference observed at 10% (w/v) |
| 12 | GYNOL II | No interference observed at 10% (w/v) |
| 13 | Vagisil® Anti-itch cream | No interference observed at 10% (w/v) |
| 14 | Anusol® Plus | No interference observed at 10% (w/v) |
| 15 | Sunscreen | No interference observed at 1% (w/v) |
| 16 | Monistat®7 | No interference observed at 10% (w/v) |
| 17 | VaselineTM | No interference observed at 10% (w/v) |
| 18 | SAB-Dimenhydrinate® Suppositories (SABEX®) | No interference observed at 10% (w/v) |
| 19 | Mineral Oil | No interference observed at 10% (v/v) |
| 20 | Equate Natural Vegetable Laxative | No interference observed at 10% (w/v) |
| 21 | Dulcolax® | No interference observed at 10% (w/v) |
| 22 | Fleet® (CB Fleet Company) | No interference observed at 10% (w/v) |
| 23 | K-Y Jelly/Gelée® (McNeil-PPC) | No interference observed at 1% (w/v) |
| 24 | Afrin Original Nasal Spray | No interference observed at 10% (v/v) |
| 25 | Witch hazel | No interference observed at Liquid from 1 wipe |
| 26 | E-Z-HD™ High Density Barium Sulfate for suspension (E-Z-EM Canada) | No interference observed at 20% (w/v) |
| 27 | Palmitic acid | No interference observed at 10% (w/v) |
| 28 | Steric acid | No interference observed at 10% (w/v) |
| 29 | Aleve | No interference observed at 10% (w/v) |
*Mucin at 50% (v/v) concentration interfered with the detection of toxigenic C. difficile isolates.
Table 8: _ Results from Interference Substances Testing
Formatted: Font color: Blue Deleted: Table 9 Deleted: 9
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Cross Contamination 4.6.
The "worst case use scenario" cross contamination rate for the cobas Cdiff Test was assessed by testing high titer C. difficile and negative samples that were processed in a checkerboard configuration on cobas 4800 system. High titer samples were prepared by adding C. difficile culture to pooled negative stool specimen matrix to generate a Ct of the 95 percentile of the clinical specimen population.
Five runs were performed on each of three cobas® 4800 systems (total: 15 runs). The first run on each system contained only the negative samples to confirm the instrument was clean. The subsequent three runs on each system had alternating the positive and negative samples in checkerboard configurations to assess the cross contamination rate. The last run on each system contained only the negative samples to assess the carry-over contamination rate.
Results from this study are summarized in Table 9. In the nine checkerboard runs, 1 out of 423 negative samples exhibited a positive result, for an observed cross contamination rate of 0.24%. All results in the last 3 runs containing only the negative samples were negative, suggesting that there was no carry-over run-to-run contamination.
Table 9; Cross Contamination and Carry-over Contamination Rate
| Run Type | No. ofRuns | Total NegativeSamples | Number of Positive Results inNegative Samples | ContaminationRate |
|---|---|---|---|---|
| Checkerboard run(Cross Contamination) | 9 | 423 | 1 | 0.24% |
| Last run with negative panel(Carryover Contamination) | 3 | 282 | 0 | 0.00% |
5. CLINICAL PERFORMANCE EVALUATION
Reproducibility 5.1.
The reproducibility of the cobas® Cdiff Test on the cobas® 4800 System was established in a multi-site investigation using simulated clinical samples evaluated across lot, site/instrument, operator, day and within-run.
Formatted: Font color: Blue Deleted: Table 10
Deleted: 10
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Reproducibility test panels consisting of 4 specimens, with 3 replicates each, were prepared by seeding pooled, C. difficile-negative, unformed stool in cobas PCR Media with varying concentrations of C. difficile strain ATCC 43255 (Negative, Below LOD, 1 x LOD, and 3 x LOD) and tested at 3 sites by 2 operators/day for 5 days/lot over 2 lots, for a total of 720 tests per panel member (4 specimens x 3 replicates x 3 sites x 2 operators/site x 5 days/lot x 2 lots).
The results are summarized in Table 9 and Table 10. Overall, 60 runs were performed; all were valid. Of the 720 test performed across 4 panel members (Negative, Below LOD, 1 x LOD, 3 x LOD), there were 712 (98.9%) valid results; 7 failed results were due to clot detection or pipetting errors, and 1 invalid result due to IC dropout. All valid test results were included in percent agreement analyses.
Table 10 summarizes the Ct values and the percent agreement (two-sided 95% exact CI) by site and panel member. The SD and CV (%) for Ct values across positive panel members ranged from 0.64 to 0.71 and 1.7 to 1.9%, respectively. The positive percent agreement for the C. difficile positive panel members "Below LOD," "1 x LOD," and "3 x LOD" were 66.1% (95% CI: 58.7% to 73.0%), 100.0% (95% CI: 98.0% to 100.0%), and 100.0% (95% CI: 97.9% to 100.0%), respectively. The negative percent agreement for negative panel members was 100.0% (95% CI: 97.9% to 100.0%).
| Panel | ValidTests | Ct | Percent Agreement by Site(n/N) | Total Agreement | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Member | Results(n) | Mean | SD | CV (%) | 1 | 2 | 3 | Percent(n/N) | (95% CI)b | |
| Negative | 174 | N/A | N/A | N/A | 100.0(60/60) | 100.0(60/60) | 100.0(54/54) | 100.0%(174/174) | (97.9%, 100.0%) | |
| BelowLOD | 180 | 39.7 | 0.71 | 1.8 | 71.7(43/60) | 68.3(41/60) | 58.3(35/60) | 66.1%(119/180) | (58.7%, 73.0%) | |
| 1 x LOD | 180 | 37.6 | 0.64 | 1.7 | 100.0(60/60) | 100.0(60/60) | 100.0(60/60) | 100.0%(180/180) | (98.0%, 100.0%) | |
| 3 x LOD | 178 | 36.6 | 0.70 | 1.9 | 100.0(60/60) | 100.0(60/60) | 100.0(58/58) | 100.0%(178/178) | (97.9%, 100.0%) |
Table 10; Summary of Reproducibility Results: Ct Values and Percent Agreement by Site and Panel Member
Formatted: Font color: Blue Deleted: Table 11
Deleted: 11
Page 15
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Table 11 presents the SD and CV (%) of Ct values for positive panel members overall and attributable to lot, site/instrument, operator, day, and within-run.
Table 11: Overall Mean, Standard Deviations, and Coefficient of Variation (%) for Ct
| Values from Valid Results for Positive Panel Members | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Panel | Lot | Site/Inst. | Operator | Day | Within-Run | Total |
.. . . . . . .
| Lot | Site/Inst. | Operator | Day | Within-Run | Total | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| PanelMember | N | Mean Ct | SD | CV | SD | CV | SD | CV | SD | CV | SD | CV | SD | CV |
| Below LOD | 119 | 39.7 | 0.33 | 0.8% | 0.00 | 0.0% | 0.12 | 0.3% | 0.21 | 0.5% | 0.58 | 1.5% | 0.71 | 1.8% |
| 1 x LOD | 180 | 37.6 | 0.54 | 1.4% | 0.08 | 0.2% | 0.00 | 0.0% | 0.06 | 0.1% | 0.33 | 0.9% | 0.64 | 1.7% |
| 3 x LOD | 178 | 36.6 | 0.60 | 1.7% | 0.13 | 0.4% | 0.10 | 0.3% | 0.09 | 0.3% | 0.29 | 0.8% | 0.70 | 1.9% |
Ct = cycle threshold; CV = coefficient of variation; Inst. = instrument; LOD = limit of detection; SD = standard deviation.
Clinical Performance 5.2.
The clinical performance of the cobas® Cdiff Test was established in an IRBapproved, prospective, multi-site, investigation comparing the results with toxigenic culture using leftover, de-identified, unformed stool samples from subjects suspected of having CDI. Specimens were collected at five geographically diverse sites across the US from symptomatic eligible male and female subjects. The toxigenic culture was performed at a single reference laboratory and the cobas® Cdiff Test was performed at one of three designated sites. The toxigenic culture included direct and repeat direct and enrichment culture of stool followed by cytotoxicity testing. The direct culture included the transfer of sample to pre-reduced selective anaerobic media, cycloserinecefoxitin-fructose agar with horse blood and taurocholate (CCFA-HT), followed by cytotoxicity testing on C. difficile recovered from stool. Briefly, suspected colonies obtained from direct cultures were identified as C. difficile by Gram stain, aerotolerance test, and by the Pro Disk test (Hardy Diagnostics, Santa Maria, CA) and then inoculated into anaerobic chopped meat broth and incubated for 5 to 7 days at 35°C for cytotoxicity testing. Supernatants obtained from anaerobic chopped meat broth were then processed for the detection of C. difficile toxin B using cell culture cytotoxicity testing (C. DIFFICILE TOX-B TEST, TECHLAB®). Enriched toxigenic
Formatted: Font color: Blue Deleted: Table 12
Deleted: 12
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culture included culture using cycloserine-cefoxitin-manitol broth with taurocholate, lysozyme and cysteine (CCMB-TAL), followed by subculture on Brucella agar plates, and with identification and cytotoxicity testing of C. difficile recovered from enrichment culture as described. A specimen was considered positive for toxigenic C. difficile if C. difficile was recovered from stool either by direct or enriched toxigenic culture and if isolates recovered tested positive by cytotoxicity testing (any positive rule). If C. difficile was isolated from the direct culture and the isolate tested positive by cell cytotoxicity assay, the enrichment culture was not further analyzed. Specimens were classified as negative for toxigenic C. difficile only if they tested negative by both direct, and repeat direct and enrichment culture. The sensitivity, specificity, and PPV and NPV values were calculated by comparing cobas® Cdiff Test results with the combined results of direct and enrichment toxigenic culture. Discrepant analysis was performed on all samples with discordant results, and a random subset of specimens with concordant results, between the cobas® Cdiff Test and toxigenic culture, using a second FDA-cleared nucleic acid amplification test (NAAT). In addition, the positive percent agreement (PPA) and negative percent agreement (NPA) was determined comparing the cobas® Cdiff Test with the initial direct culture results.
Results
Specimens were collected from 683 subjects; 306 males (44.8%) and 377 females (55.2%) with a mean age of 56 years (range 3 to 99). Specimens from all 683 subjects had valid results for both direct toxigenic culture and the cobas® Cdiff Test but one sample lacked sufficient volume for repeat direct and enrichment culture and was not included in the statistical analysis. Of the 683 specimens, 113 were positive for toxigenic C. difficile during the initial direct toxigenic culture and 141 of 682 were positive for toxigenic C. difficile using the combined results from the initial direct and repeat direct and enrichment toxigenic culture, for a prevalence rate of 20.7% for the study.
Comparison with combined direct and enrichment culture
The clinical performance of the cobas® Cdiff Test compared with the combined results of initial direct and repeat direct and enriched toxigenic culture are shown in Table 13.
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The sensitivity and specificity of the cobas® Cdiff Test was 92.9% (131/141; 95% CI: 87.4% to 96.1%) and 98.7% (534/541; 95% CI: 97.4% to 99.4%), respectively; and the PPV and NPV was 94.9% (95% CI: 89.9% to 97.5%) and 98.2% (95% CI: 96.6% to 99.0%), respectively. Of the 10 specimens with false-negative cobas® Cdiff Test results relative to combined direct culture and enrichment culture, all 10 were negative by a second NAAT method. Of the 7 specimens with false-positive cobas® Cdiff Test results relative to combined direct and enrichment culture, 3 were positive and 4 were negative by that second NAAT method.
Table 13: Comparison of cobas® Cdiff Test with combined direct culture and enrichment culture
| Combined Direct and Enrichment Culturea | ||||
|---|---|---|---|---|
| Positive | Negative | Total | ||
| cobas® CdiffTest | Positive | 131 | 7c | 138 |
| Negative | 10b | 534 | 544 | |
| Total | 141 | 541 | 682 | |
| Sensitivity: | 92.9% (131/141; 95% CI = 87.4% to 96.1%) | |||
| Specificity: | 98.7% (534/541; 95% CI = 97.4% to 99.4%) | |||
| PPV: | 94.9% (95% CI = 89.9% to 97.5%) | |||
| NPV: | 98.2% (95% CI = 96.6% to 99.0%) |
ª Includes combined results from an initial direct culture and a repeat direct and enrichment culture performed on all initial direct culture-negative samples. One specimen with an initial direct culturenegative result had insufficient specimen volume to perform repeat direct culture and enrichment culture and was excluded from the analysis. Thirty-six (36) specimens with initial direct culturenegative results had their combined direct and enrichment culture results based on repeat culture that used three culture plate media (CCFA, CCFA-VA) in combination with enrichment culture. Of these 36 specimens, 21 were culture positive.
b Of the 10 specimens with false-negative cobas® Cdiff Test results relative to combined direct and enrichment culture, all 10 were negative by a second NAAT method
C Of the 7 specimens with false-positive cobas® Cdiff Test results relative to combined direct and enrichment culture, 3 were positive and 4 were negative by that second NAAT method.
Comparison with direct culture
The performance of the cobas® Cdiff Test compared to initial direct culture is shown in 14. The PPA and NPA of the cobas® Cdiff Test compared to the initial direct culture for all 683 subjects was 97.3% (110/113) and 94.9% (541/570), respectively. Of the 3 specimens with false-negative cobas® Cdiff Test results relative to direct culture, all 3 were negative by a second NAAT method. Of the 29 specimens with false-positive cobas® Cdiff Test results relative to direct culture, 15 were positive and 13 were
{21}------------------------------------------------
negative by that second NAAT method; 1 sample was not tested because of insufficient specimen volume.
| Table 14: Comparison of cobas Cdiff Test with direct culture | |
|---|---|
| Direct Culture | ||||
|---|---|---|---|---|
| Positive | Negative | Total | ||
| cobas® Cdiff Test | Positive | 110 | 29b | 139 |
| Negative | 3a | 541 | 544 | |
| Total | 113 | 570 | 683 | |
| Positive Percent Agreement: 97.3% (110/113; 95% CI = 92.5% to 99.1%) | ||||
| Negative Percent Agreement: 94.9% (541/570; 95% CI = 92.8% to 96.4%) |
ª Of the 3 specimens with false negative cobas® Cdiff Test results relative to direct culture, all 3 were negative by a second NAAT method.
b Of the 29 specimens with false positive cobas Cdiff Test results relative to direct culture, 15 were positive, 13 were negative by that second NAAT method, and 1 sample was not tested because of insufficient specimen volume.
5.3. Summary
The clinical performance evaluation as documented in the reproducibility and clinical study support the conclusion that the cobas® Cdiff test is as safe and effective as the predicate device.
6. CONCLUSIONS
A comparison of the intended use, technological characteristics, and the results of non-clinical and clinical performance studies support the conclusion that the cobas® Cdiff Test is substantially equivalent to the predicate device.
§ 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.