SIMPLEXA C. DIFFICILE UNIVERSAL DIRECT
Device Facts
| Record ID | K113433 |
|---|---|
| Device Name | SIMPLEXA C. DIFFICILE UNIVERSAL DIRECT |
| Applicant | Focus Diagnostics, Inc. |
| Product Code | OMN · Microbiology |
| Decision Date | Apr 4, 2012 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.2660 |
| Device Class | Class 1 |
| Attributes | Pediatric |
Indications for Use
The Focus Diagnostics Simplexa™ C. difficile Universal Direct is a real-time polymerase chain reaction (PCR) assay and is intended for use on the 3M Integrated Cycler instrument for the detection of toxigenic Clostridium difficile toxin B gene (tcdB) in liquid or unformed stool samples from individuals suspected of C. difficile infection. This test aids in the diagnosis of Clostridium difficile associated disease (CDAD).
Device Story
The Simplexa™ C. difficile Universal Direct is a real-time PCR assay for the detection of the C. difficile toxin B gene (tcdB). The device processes liquid or unformed stool samples via an off-board 10-minute heat treatment step. The assay utilizes bifunctional fluorescent probe-primers to amplify and detect the tcdB gene and an internal control DNA sequence on the 3M Integrated Cycler. The system is intended for use in clinical laboratory settings by trained personnel. The output is a qualitative detection of toxigenic C. difficile, which aids clinicians in diagnosing CDAD. By providing rapid molecular detection, the device facilitates timely clinical decision-making and patient management compared to traditional culture methods.
Clinical Evidence
Clinical performance was evaluated using 970 prospective stool specimens. Compared to direct toxigenic culture, sensitivity was 90.1% (95% CI: 83.8-94.1%) and specificity was 93.0% (95% CI: 91.0-94.5%). Compared to enriched toxigenic culture, sensitivity was 79.6% (95% CI: 73.1-84.8%) and specificity was 95.8% (95% CI: 94.2-97.0%). Analytical studies included LoD determination (560.7 CFU/mL and 76.3 CFU/mL for tested strains), cross-reactivity testing (119 organisms), and interference testing (21 substances).
Technological Characteristics
The device is a real-time PCR assay utilizing bifunctional fluorescent probe-primers. It is designed for use on the 3M Integrated Cycler. The process involves an off-board 10-minute heat treatment of stool samples. The assay targets the tcdB gene and includes an internal control to monitor for PCR inhibitors.
Indications for Use
Indicated for the detection of toxigenic Clostridium difficile toxin B gene (tcdB) in liquid or unformed stool samples from pediatric and adult patients suspected of C. difficile infection to aid in the diagnosis of Clostridium difficile associated disease (CDAD).
Regulatory Classification
Identification
A microorganism differentiation and identification device is a device intended for medical purposes that consists of one or more components, such as differential culture media, biochemical reagents, and paper discs or paper strips impregnated with test reagents, that are usually contained in individual compartments and used to differentiate and identify selected microorganisms. The device aids in the diagnosis of disease.
Predicate Devices
Related Devices
- K163085 — Simplexa C. difficile Direct; Simplexa C. difficile Positive Control Pack · Focus Diagnostics, Inc.:Dba Diasorin Molecular, LLC · Feb 14, 2017
- K123998 — QUIDEL MOLECULAR DIRECT C. DIFFICILE ASSAY · Quidel Corp. · Mar 8, 2013
Submission Summary (Full Text)
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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY ONLY TEMPLATE
A. 510(k) Number:
K113433
B. Purpose for Submission:
To obtain substantial equivalence for Simplexa™ C. difficile Universal Direct assay
C. Measurand:
Clostridium difficile toxin B gene (tcdB)
D. Type of Test:
Real time PCR
E. Applicant
Focus Diagnostics Inc.
F. Proprietary and Established Name:
Simplexa™ C. difficile Universal Direct Assay
G. Regulatory Information:
| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| OMN | I | 21 CFR 866.2660 | 83 - Microbiology |
H. Intended Use:
1. Intended use:
The Focus Diagnostics Simplexa™ C. difficile Universal Direct is a real-time polymerase chain reaction (PCR) assay and is intended for use on the 3M Integrated Cycler instrument for the detection of toxigenic Clostridium difficile toxin B gene (tcdB) in liquid or unformed stool samples from individuals suspected of C. difficile infection. This test aids in the diagnosis of Clostridium difficile associated disease (CDAD).
2. Indications for use:
The Focus Diagnostics Simplexa™ C. difficile Universal Direct is a real-time polymerase chain reaction (PCR) assay and is intended for use on the 3M Integrated Cycler instrument for the detection of toxigenic Clostridium difficile toxin B gene (tcdB) in liquid or unformed stool samples from individuals suspected of C. difficile infection. This test aids in the diagnosis of Clostridium difficile associated disease (CDAD).
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3. Special conditions for use statement:
For Prescription Use
4. Special instrument requirements:
3M Integrated Cycler Instrument
I. Device Description:
The test is a real-time polymerase chain reaction (PCR) amplification and detection system that utilizes bi-functional fluorescent probe-primers for the detection of C. difficile in liquid or unformed stool. The Focus Diagnostics Simplexa™ C. difficile Universal Direct assay contains sufficient reagents for 100 reactions. Kit components are listed below.
| Kit Component | Component Description | | | | |
| --- | --- | --- | --- | --- | --- |
| Simplexa™ C. difficile Primer Mix | Bi-functional fluorescent probe-primers specific for detection of C. difficile and for the DNA Internal Control including DNA Internal Control template | | | | |
| | Target | Probe Fluorophore | Excitation (nm) | Emission (nm) | Targeted Gene |
| | C. difficile | FAM | 495 | 520 | Toxin B |
| | IC | Q670 | 644 | 670 | N/A |
| Simplexa™ Master Mix | DNA polymerase, buffer and dNTPs | | | | |
| Simplexa™ C. difficile Positive Control (PC) | Clostridium difficile Genomic DNA | | | | |
| Simplexa™ C. difficile Barcode Card | Assay specific parameters | | | | |
J. Substantial Equivalence Information:
1. Predicate device names:
a. BD GeneOhm C.diff. Assay
b. illumigene C. difficile Assay
c. Bartel's cytotoxicity assay for Clostridium
2. Predicate K numbers:
a. K081920
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b. K110012
c. K833447
3. Comparison with predicate:
Comparison is to BD GeneOhm C. diff Assay (K081920)
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | Detection of C. difficile toxin B gene | Same |
| Matrix | Liquid or uniformed stool samples | Same |
| Technology | Assay based on real time PCR | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Instrumentation | Test is performed on the 3M Integrated Cycler | Test uses the Cepheid Smart Cycler |
| Detection techniques | PCR with bifunctional fluorescent primer-probes | PCR with molecular beacons |
| Reference Method | Toxigenic Culture | Cytotoxicity Assay |
K. Standard/Guidance Document Referenced:
Draft Guidance for Industry and Food and Drug Administration Staff - Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection of Clostridium difficile. November 2010.
L. Test Principle:
The test is a real-time polymerase chain reaction (PCR) amplification and detection system that utilizes bi-functional fluorescent probe-primers for the detection of C. difficile in liquid or unformed stool. The assay is composed of two principal steps: (1) Heat treatment of stool samples, (2) Amplification of the C. difficile DNA and internal control DNA using bi-functional fluorescent probe-primers together with reverse primers. The DNA internal control is used to monitor potential presence of PCR inhibitors. The assay targets a sequence which is in a well conserved region of C. difficile toxin B gene (tcdB).
The 3M Integrated Cycler is a rapid real-time Polymerase Chain Reaction thermocycler used for the identification of nucleic acid from prepared biological samples. The instrument utilizes disc media to contain and process samples. The instrument uses real time fluorometric detection to identify targets within the sample wells. The instrument is controlled by an external computer running the Integrated Cycler Studio Software.
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M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
Inter-laboratory reproducibility and inter/intra-assay reproducibility were assessed at three sites. Each of the three sites used the same panel, which consisted of contrived samples in stool-TE buffer matrix spiked with C. difficile bacterial stock. The panel included a high negative, low positive and medium positive sample. Each site utilized at least two testing operators and one lot of Simplexa™ C.dificile Universal Direct kit across five days. On each day two runs were performed, one by each operator. These results met the acceptance criteria and are acceptable. A summary of the reproducibility results are shown in the table below.
| Sample | Site 1 | | | Site 2 | | | Site 3 | | | Total Agreement with Expected Results | 95% CI |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Agreement with Expected Results | Avg. Ct | Total %CV | Agreement with Expected Results | Avg. Ct | Total %CV | Agreement with Expected Results | Avg. Ct | Total %CV | | |
| Low Positive | 30/30 | 35.20 | 1.64 | 30/30 | 35.26 | 1.30 | 30/30 | 35.30 | 2.04 | 100% (90/90) | 95.9% - 100.0% |
| Medium Positive | 29/29 | 32.71 | 0.82 | 30/30 | 32.60 | 1.07 | 30/30 | 32.65 | 0.77 | 100% (89/89) | 95.9% - 100.0% |
| Positive Control (PC) | 30/30 | 32.55 | 1.11 | 29/29 | 32.13 | 0.87 | 31/31 | 32.33 | 0.63 | 100% (90/90) | 95.9% - 100.0% |
| High Negative | 29/30 | | | 30/30 | | | 30/30 | | | 98.9% (89/90) | 94.0% - 99.8% |
| No Template Control (NTC) | 30/30 | | | 30/30 | | | 29/30 | | | 98.9% (89/90) | 94.0% - 99.8% |
| Total Agreement | 148/149 (99.3%) | | | 149/149 (100.0%) | | | 150/151 (99.3%) | | | 447/449 (99.6%) | 98.4% - 99.9% |
One replicate was declared "invalid" based on the site operator discretion. It was "Not Detected".
One replicate was "Invalid" at Site 2 and additional replicate was tested in Run-1, Day-1 at Site 3 because the site had thought that one of the three replicates had a 'bubble' and therefore as a precaution loaded an additional replicate at the end of one of the run.
One replicate of the NTC is "Detected" and may be attributed to possible contamination due to handling.
Note: Two samples – “NTC” and “High Negative” were excluded from reporting Quantitative Reproducibility Results.
b. Linearity/assay reportable range:
Not applicable
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Quality control ranges have been established as indicated in the table below. If the controls are not within these parameters, patient results should be considered invalid and the assay repeated. Each laboratory should establish its own Quality Control ranges and frequency of QC testing based on applicable local laws, regulations and standard good laboratory practice.
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Expected Control Ranges
| Control Type | SimplexaTM C. difficile Ct value |
| --- | --- |
| No Template Control (NTC) | Ct=0 |
| Positive Control (PC) | Ct≤40, ≠0 |
# d. Detection limit:
The Limit of Detection (LoD) was determined for the Simplexa™ C. difficile Universal Direct assay by performing limiting dilution studies using bacterial stocks of two strains of each C. difficile bacteria strain. The strains (ATCC 43255 and NAP 1A) were cultured and quantified. The LoD was determined using one lot of the Simplexa™ C. difficile Universal Direct Kit. Tentative LoD was determined using three replicates in screening followed by confirmation using twenty replicates. LoD was determined to be 560.7 CFU/mL or 1.12 CFU/PCR for strain ATCC 43255 and 76.3 CFU/mL or 0.15 CFU/PCR for strain NAP 1A. These results met the acceptance criteria and are acceptable.
# e. Analytical specificity:
# Cross-Reactivity
Analytical specificity for various possible cross-reactants was performed. A total of 119 potential cross-reactants were tested. No cross-reactivity was observed as shown in the Table below.
| Tested Cross-Reactants | | | |
| --- | --- | --- | --- |
| No. | Cross Reactant | Concentration | Result |
| 1 | Abiotrophia defective | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 2 | Acinetobacter baumanii | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 3 | Acinetobacter lwoffii | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 4 | Adenovirus 40 | 1.00 × 105TCID50/mL | No Cross Reactivity Observed |
| 5 | Aeromonas hydrophila | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 6 | Alcaligenes faecalis subsp. Faecalis | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 7 | Anaerococcus tetradius | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 8 | Bacillus cereus | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 9 | Bacteroides caccae | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 10 | Bacteroides merdae | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 11 | Bacteroides stercoris | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 12 | Bifidobacterium adolescentis | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 13 | Bifidobacterium longum | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 14 | Campylobacter coli | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 15 | Campylobacter jejuni | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 16 | Candida albicans | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 17 | Candida catenulate | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 18 | Cedecea davisae | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 19 | Chlamydia trachomatis | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
| 20 | Citrobacter amalonaticus | 1.00 × 106cfu/mL | No Cross Reactivity Observed |
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| Tested Cross-Reactants | | | |
| --- | --- | --- | --- |
| No. | Cross Reactant | Concentration | Result |
| 21 | Citrobacter freundii | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 22 | Citrobacter koseri | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 23 | Citrobacter sedlakii | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 24 | Clostridium beijerinckii | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 25 | Clostridium bifermentans | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 26 | Clostridium bolteae | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 27 | Clostridium butyricum | 6.80 × 10^{5} cfu/mL | No Cross Reactivity Observed |
| 28 | Clostridium chauvoei | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 29 | Clostridium difficile non-toxigenic ATCC 43593 | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 30 | Clostridium difficile non-toxigenic ATCC43601 | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 31 | Clostridium fallax | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 32 | Clostridium histolyticum | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 33 | Clostridium innocuum | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 34 | Clostridium methylpentosum | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 35 | Clostridium nexile | 6.90 × 10^{5} cfu/mL | No Cross Reactivity Observed |
| 36 | Clostridium novyi | 8.90 × 10^{5} cfu/mL | No Cross Reactivity Observed |
| 37 | Clostridium paraputrificum | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 38 | Clostridium perfringens | 6.70 × 10^{5} cfu/mL | No Cross Reactivity Observed |
| 39 | Clostridium ramosum | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 40 | Clostridium scindens | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 41 | Clostridium septicum | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 42 | Clostridium sordellii | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 43 | Clostridium sphenoides | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 44 | Clostridium sporogenes | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 45 | Clostridium symbiosum | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 46 | Clostridium terdium | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 47 | Clostridium tetani | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 48 | Collinsella aerofaciens | 8.60 × 10^{5} cfu/mL | No Cross Reactivity Observed |
| 49 | Corynebacterium genitalium | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 50 | Coxsackie virus A16 | 1.00 × 10^{5} TCID_{50}/mL | No Cross Reactivity Observed |
| 51 | Cytomegalovirus AD-169 | 1.00 × 10^{5} TCID_{50}/mL | No Cross Reactivity Observed |
| 52 | Desulfovibrio piger | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 53 | Echovirus 9 | 1.00 × 10^{5} TCID_{50}/mL | No Cross Reactivity Observed |
| 54 | Edwardsiella tarda | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 55 | Eggerthellalenta | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 56 | Enterobacter aerogenes | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 57 | Enterobacter cloacae | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 58 | Enterococcus raffinosus | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 59 | Enterococcus casseliflavus | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 60 | Enterococcus cecorum | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 61 | Enterococcus dispar | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 62 | Enterococcus hirae | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 63 | Enterococcus faecalis vanB | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 64 | Enterococcus faecium vanA | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 65 | Enterococcus gallinarum vanC | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
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| Tested Cross-Reactants | | | |
| --- | --- | --- | --- |
| No. | Cross Reactant | Concentration | Result |
| 66 | Enterovirus 71 | 5.01 × 10^{4} TCID_{50}/mL | No Cross Reactivity Observed |
| 67 | Escherichia coli | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 68 | Escherichia fergusonii | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 69 | Escherichia hermannii | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 70 | Fusobacterium varium | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 71 | Gardnerella vaginalis | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 72 | Gemella morbillorum | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 73 | Hafnia alvei | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 74 | Helicobacter pylori | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 75 | Homo sapiens | 3.07 pg/mL | No Cross Reactivity Observed |
| 76 | Klebsiella oxytoca | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 77 | Klebsiella pneumoniae subsp. Pneumoniae | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 78 | Lactobacillus acidophilus | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 79 | Lactobacillus reuteri | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 80 | Lactococcus lactis | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 81 | Leminorella grimontii | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 82 | Listeria grayi | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 83 | Listeria innocua | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 84 | Listeria monocytogenes | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 85 | Norovirus Group I (recombinant) | 8.13 × 10^{4} TCID_{50}/mL | No Cross Reactivity Observed |
| 86 | Peptoniphilus asaccharolyticus | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 87 | Peptostreptococcus anaerobius | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 88 | Plesiomonas shigelloides | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 89 | Porphyromonas asaccharolytica | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 90 | Prevotella melaninogenica | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 91 | Proteus mirabilis | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 92 | Proteus penneri | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 93 | Providencia alcalifaciens | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 94 | Providencia rettgeri | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 95 | Providencia stuartli | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 96 | Pseudomonas aeruginosa | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 97 | Pseudomonas putida | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 98 | Rotavirus, Strain Wa | 1.00 × 10^{5} TCID_{50}/mL | No Cross Reactivity Observed |
| 99 | Salmonella enterica subsp. Arizonae (formerly Choleraesuis arizonae) | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 100 | Salmonella enterica subsp. Choleraesuis | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 101 | Salmonella enterica subsp. Enterica serovar Typhimurium | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 102 | Serratia liquefaciens | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 103 | Serratia marcescens | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 104 | Shigella boydii | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 105 | Shigella dysenteriae | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 106 | Shigella sonnei | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 107 | Staphylococcus aureus | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 108 | Staphylococcus epidermidis | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 109 | Stenotrophomonas maltophilia | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 110 | Streptococcus agalactiae | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
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| Tested Cross-Reactants | | | |
| --- | --- | --- | --- |
| No. | Cross Reactant | Concentration | Result |
| 111 | Streptococcus dysgalactiae | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 112 | Streptococcus intermedius | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 113 | Streptococcus uberis | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 114 | Trabulsiella guamensis | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 115 | Veillonella parvula | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 116 | Vibrio cholerae | 4.10 × 10^{-3} pg/mL | No Cross Reactivity Observed |
| 117 | Vibrio parahaemolyticus | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 118 | Yersinia bercovieri | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| 119 | Yersinia rohdei | 1.00 × 10^{6} cfu/mL | No Cross Reactivity Observed |
| Note: 1. Total 10 runs were performed to test 119 cross reactants in triplicate. Additionally, each run included five replicates of baseline (un-spiked) sample. 2. Each replicate of all 119 cross-reactants and baseline samples were "Not Detected". | | | |
## Interference:
The performance of this assay was evaluated with potentially interfering substances that may be present in stool specimens at the concentrations indicated in the Table below. A total of 21 potentially interfering substances were tested. No Interference was observed.
| Interferents | Active Ingredient | Interferent Concentration | Date | |
| --- | --- | --- | --- | --- |
| | | | C. difficile Strain - ATCC4325 | C. difficile Strain - NAP 1 |
| 1% Hydrocortisone Cream | Hydrocortisone | 2% (w/v) | 3 | 3 |
| Alleve | Naproxen | 14 mg/ml | 3 | 3 |
| Antacid and Anti-gas generic | Aluminum Hydroxide, Magnesium Hydroxide | 0.1 mg/ml | 3 | 3 |
| Antacid Generic | Calcium Carbonate | 0.1 mg/ml | 3 | 3 |
| Barium sulfate | Barium sulfate | 5 mg/ml | 3 | 3 |
| Fleet | Mineral Oil | 2% (v/v) | 3 | 3 |
| Imodium AD | Loperamide | 0.005 mg/ml | 3 | 3 |
| KY Jelly | Glycerin | 2%(w/v) | 3 | 3 |
| Laxative generic | Sennosides | 0.1 mg/ml | 5 | 3 |
| Metronidazole | Metronidazole | 14 mg/ml | 3 | 3 |
| Milk of Magnesia | Magnesium Hydroxide | 0.2 mg/ml | 3 | 3 |
| Moist towelettes | Benzalkonium Chloride | 10%(v/v) | 3 | 3 |
| Mucin | Mucin | 3 mg/ml | 3 | 3 |
| Nystatin | Nystatin | 10000 USP units/ml | 3 | 3 |
| Palmitic acid | Palmitic acid | 2 mg/ml | 3 | 3 |
| Pepto-Bismol | Bismuth Subsalicylate | 0.175 mg/ml | 3 | 3 |
| Preparation H | Phenylephrine | 2% (w/v) | 3 | 3 |
| Stearic acid | Stearic Acid | 4 mg/ml | 3 | 3 |
| Trojan with nonoxynol- | Nonoxynol-9 | 1.4 mg/ml | 3 | 3 |
| Vancomycin | Vancomycin | 1.4 mg/ml | 5 | 3 |
| Whole blood | Whole blood | 3% | 3 | 7 |
*One replicate reported as "Invalid" due to IC failure in initial run of three replicates. All three replicates reported as "Detected" in repeat run.
**One replicate reported as "Not Detected" in initial run of three replicates. However all five replicates reported as "Detected" in repeat run.
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Analytical reactivity of additional strains of C. difficile was evaluated in negative stool-TE buffer matrix. Quantified bacterial material was spiked into the negative stool-TE buffer matrix at a single dilution. A total of 20 different strains were tested in triplicate. All of the tested strains were detected as shown in the table below
Analytical Reactivity Results for C. difficile strains
| No. | Strain | Concentration (cfu/mL) | Toxinotype | C.dificile Result #Detected / #Total |
| --- | --- | --- | --- | --- |
| 1 | ATCC 17857 (870) A+B+ | 1.12 x 10^{3} | 0 | 3/3 |
| 2 | ATCC 43594 (W1194) A+B+ | 1.12 x 10^{3} | 0 | 3/3 |
| 3 | ATCC 43596 (545) A+B+ | 1.12 x 10^{3} | 0 | 3/3 |
| 4 | ATCC 43597 A+B+ | 1.12 x 10^{3} | | 3/3 |
| 5 | ATCC 43598 (1470) A-B+ | 1.12 x 10^{3} | VIII | 3/3 |
| 6 | ATCC 43599 (2022) A+B+ | 1.12 x 10^{3} | 0 | 3/3 |
| 7 | ATCC 43600 (2149) A+B+ | 1.12 x 10^{3} | 0 | 3/3 |
| 8 | ATCC 51695 (BDMS 18 AN) A+B+ | 1.12 x 10^{3} | 0 | 3/3 |
| 9 | ATCC 700792 (14797-2) A+B+ | 1.12 x 10^{3} | 0 | 3/3 |
| 10 | ATCC 9689 (90556-M6S) A+B+ | 1.12 x 10^{3} | 0 | 3/3 |
| 11 | ATCC BAA-1382 (630) A+B+ | 1.12 x 10^{3} | 0 | 3/3 |
| 12 | ATCC BAA-1805 A+B+ | 1.12 x 10^{3} | III | 3/3 |
| 13 | BAA-1814 A+B+ | 1.12 x 10^{3} | XXII | 3/3 |
| 14 | BAA-1870 A+B+ | 1.12 x 10^{3} | III | 3/3 |
| 15 | BAA-1871 A+B+ | 1.12 x 10^{3} | 0 | 3/3 |
| 16 | BAA-1872 A+B+ | 1.12 x 10^{3} | 0 | 3/3 |
| 17 | BAA-1873 A+B+ | 1.12 x 10^{3} | 0 | 3/3 |
| 18 | BAA-1874 A+B+ | 1.12 x 10^{3} | 0 | 3/3 |
| 19 | BAA-1875 A+B+ | 1.12 x 10^{3} | V | 3/3 |
| 20 | CCUG 8864 A-B+ | 1.12 x 10^{3} | X | 3/3 |
f. Assay cut-off:
Initial reportable range (assay cut-off) was determined by an analysis of Limit of Detection (LoD) Studies and Method Comparison studies during the feasibility phase where 45 cycles of amplification were run. The cycling time was later reduced to 40 cycles as testing demonstrated that samples at LoD were approximately at 38 cycles. Reportable range (assay cut-off) was determined by analysis of a Limit of Detection study and the Method Comparison data generated during assay verification testing. Forty cycles of amplification were performed during assay development to allow for the confirmation of the assay cut off. The Limit of Detection for C difficile was defined as the lowest concentration with $\geq 95\%$ detection for at least 24 replicates as determined by probit analysis. Analysis of the Limit of Detection study shows that the range of Ct values for the C. difficile positive samples at or just above the Limit of Detection was $< 39$. The same LoD sample returned an average Ct value of 36.8. Review of the Method Comparison data showed that $95\%$ of the positive samples have a Ct value $< 38.7$. Samples which were positive for C. difficile had Ct values in the range of 23.3 to 39.7. Based on the available data, the assay cut-off was set at $\mathrm{Ct} = 40$.
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2. Comparison studies:
a. Method comparison with predicate device:
Clinical specimens were tested using the Simplexa™ C. difficile Universal Direct assay and two different FDA cleared assays. A total of 402 samples were assayed using one FDA cleared molecular assay, and 305 samples were assayed using another FDA cleared molecular assay. The testing was performed at two different clinical sites. These two FDA cleared molecular assays and the Simplexa™ C. difficile were compared to direct and enriched toxigenic cultures.
In comparison to direct toxigenic culture, the sensitivity and specificity of the Simplexa™ C. difficile Universal Direct Assay were 90.1% (95% CI:83.8-94.1%) and 93% (95% CI:91-94.5%), respectively. The sensitivities and specificities of the two FDA cleared molecular tests were 86.1% (95% CI:76.3-92.3%) and 94.8% (95% CI:91.9-96.8%) for the first molecular assay and 81.8% (95% CI:65.6-91.4%) and 93% (95% CI:89.3-95.5%), for the second assay.
In comparison to enriched toxigenic culture, the sensitivity and specificity of the Simplexa™ C. difficile Universal Direct Assay were 79.6% (95% CI:73.1-84.8%) and 95.8% (95% CI:94.2-97%), respectively. The sensitivities and specificities of the two FDA cleared molecular tests were 78.7% (95% CI:69.0-85.9%) and 97.1% (95% CI:94.6-98.5%) for the first molecular assay and 69.6% (95% CI:56.7-80.1%) and 97.2% (95% CI:94.3-98.6%), for the second assay. These results met the acceptance criteria and are acceptable.
b. Matrix comparison:
Not applicable
3. Clinical studies:
a. Clinical Sensitivity:
A total of 970 prospectively collected stool specimens were obtained from patients with signs and symptoms of C. difficile infection from varied geographic locations. Demographic information, including age, gender was noted. Testing was performed at three external testing sites located on the East Coast. Tables below show results comparing the device to direct toxigenic culture method and to enriched toxigenic culture method.
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| | Reference Method: (Direct Culture + Toxin Assay) | | |
| --- | --- | --- | --- |
| Simplexa™ C. difficile Universal Direct Kit | Detected | Not Detected | Total |
| Detected | 118 | 59 | 177 |
| Not Detected | 13 | 779 | 792 |
| Total | 131 | 838 | 969 |
| | | | |
| Sensitivity | 90.1%(118/131)
95% CI:83.8-94.1% | | |
| Specificity | 93.0%(779/838)
95% CI:91.0-94.5% | | |
| | Reference Method: (Enriched Culture + Toxin Assay) | | |
| --- | --- | --- | --- |
| Simplexa™ C. difficile Universal Direct Kit | Detected | Not Detected | Total |
| Detected | 144 | 33 | 177 |
| Not Detected | 37 | 755 | 792 |
| Total | 181 | 788 | 969 |
| | | | |
| Sensitivity | 79.6%(144/181)
95% CI:73.1-84.8% | | |
| Specificity | 95.8%(755/788)
95% CI:94.2-97.0% | | |
Note: One sample was inadvertently missed from being cultured.
The Simplexa C. difficile Universal direct Assay showed a sensitivity of 90.1% (83.8 – 94.1% CI) and a specificity of 93% (91 – 94.5% CI) when compared to direct toxigenic culture method.
b. Clinical specificity:
See 3a. above
c. Other clinical supportive data (when a. and b. are not applicable):
Not applicable
4. Clinical cut-off:
Not applicable
5. Expected values/Reference range:
The prevalence of C. difficile varies between institutions. The frequency is affected by patient population, type of institution, and epidemiology. In the
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SimplexaTM C. difficile Universal Direct prospective study, 177 of the 969 or 18.3% of the samples tested were positive. The percent positivity between the sites varied from 14.9 to 21.6.
**N. Proposed Labeling:**
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
**O. Conclusion:**
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
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