You should submit comments and suggestions regarding this draft document within 90 days of publication in the Federal Register of the notice announcing the availability of the draft guidance. Submit written comments to the Division of Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD 20852. Submit electronic comments to http://www.regulations.gov . Identify all comments with the docket number listed in the notice of availability that publishes in the Federal Register.
For questions regarding this document, contact Stephen Lovell at 301-796-6968 or by e-mail at Stephen.Lovell@fda.hhs.gov .
When final, this document will supersede “Review Criteria for Assessment of Laboratory Tests Directed at Assisting in the Diagnosis of C. difficile associated Disease” dated May 31, 1990.
Additional copies are available from the Internet . You may also send an e-mail request to email@example.com to receive an electronic copy of the guidance or send a fax request to 301-827-8149 to receive a hard copy. Please use the document number 1715 to identify the guidance you are requesting.
This draft guidance, when finalized, will represent the Food and Drug Administration's (FDA's) current thinking on this topic. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public. You can use an alternative approach if the approach satisfies the requirements of the applicable statutes and regulations. If you want to discuss an alternative approach, contact the FDA staff responsible for implementing this guidance. If you cannot identify the appropriate FDA staff, call the appropriate number listed on the title page of this guidance.
FDA is issuing this draft guidance to provide industry and agency staff with updated recommendations concerning 510(k) submissions for various types of in vitro diagnostic devices (IVDs) intended to be used for detecting Clostridium difficile (C. difficile). The document is a revision of “Review Criteria for Assessment of Laboratory Tests Directed at Assisting in the Diagnosis of C. difficile Associated Disease” issued on May 31, 1990. It is updated to include new issues and technologies identified since the 1990 guidance. Such methods include detection of C. difficile nucleic acids (e.g., C. difficile toxin B gene by nucleic acid amplification (NAAT) methods such as the Real-Time Polymerase Chain Reaction (RT-PCR) technique).
FDA’s guidance documents, including this guidance, do not establish legally enforceable responsibilities. Instead, guidances describe the Agency’s current thinking on a topic and should be viewed only as recommendations, unless specific regulatory or statutory requirements are cited. The use of the word should in Agency guidances means that something is suggested or recommended, but not required.
This document recommends studies for establishing the performance characteristics of in vitro diagnostic devices for the detection of C. difficile bacteria in human specimens. FDA believes that these recommended studies will be relevant for Class I and Class II premarket submissions (e.g., 510(k)s or de novo classification petitions) that may be required for a particular test.
A manufacturer who intends to market an in vitro diagnostic device for detecting C. difficile bacteria in human specimens should conform to the general controls of the Federal Food, Drug, and Cosmetic Act (the FD & C Act). In addition, unless exempt, they must obtain premarket clearance or approval prior to marketing the device (sections 510(k), 513, 515 of the Act; 21 U.S.C. 360(k), 360c, 360e).
This document is intended to supplement 21 CFR 807.87 (information required in a premarket notification) and other FDA resources such as “ Premarket Notification (510k) ”. Guidance on the content and format for abbreviated and traditional 510(k)s can be found in the guidance entitled “ Format for Traditional and Abbreviated 510(k)s. ”
Detection methods subject to this guidance include antigen, antibody, and nucleic acid tests using stool samples. The scope of this document is limited to the devices described in existing classifications, as indicated below, and may be applicable to other C. difficile diagnostic devices that do not fall within these existing classifications. These other devices may include devices that will be subject to requests for initial classification under section 513(f)(2) of the act ("de novo classification"), as well as subsequent devices that seek determinations of substantial equivalence to future de novo cleared devices.
The following are existing C. difficile IVD classification regulations:
The following are the product codes for C. difficile devices cleared under 21 CFR 866.2660:
Therefore the following information should be included in your submission:
C. difficile is a Gram-positive, anaerobic, spore-forming rod (bacillus) bacterium [Ref. 1] that is a common cause of antibiotic-associated diarrhea (AAD). C. difficile colonization is the most significant cause of pseudomembranous colitis [Ref. 2], which is a severe infection of the colon, often occurring after normal gut flora is eradicated by use of antibiotics. This decrease in intestinal flora causes overgrowth of C. difficile bacteria due to the lack of any competitive inhibition, from other microorganisms, for nutrients. Overgrowth of C. difficile is harmful because pathogenic strains release toxin (enterotoxin (toxin A) and cytotoxin (toxin B)) [Ref. 3]. These toxins are responsible for the diarrhea and inflammation seen in infected patients although their relative contributions have been debated by researchers. Another toxin, binary toxin, has also been described, but its role in disease is not yet fully understood. C. difficile is resistant to most antibiotics and treatment is performed by stopping unresponsive therapy and commencing specific anti-clostridial antibiotics, (e.g. metronidazole or vancomycin).
It is transmitted from person to person by the fecal-oral route. Because the organism forms heat-resistant spores, it can remain in the hospital or nursing home environment for long periods. It can be cultured from almost any surface in the hospital. Once spores are ingested, they pass through the stomach unscathed because of their acid-resistance. They change to their active form in the colon and multiply.
Several disinfectants commonly used for infection control in hospitals are ineffective in killing the bacteria, and may actually promote spore formation. However, disinfectants containing bleach are successful in killing the organisms [Ref. 4].
Methods for detection of C. difficile include Cytotoxicity Assays, Enzyme- Linked Immunoabsorbent Assays (ELISA) for Toxin, and Nucleic Acid Assays. 1 Failure of devices for detection of C. difficile to perform as expected or failure to interpret results correctly may lead to incorrect patient management decisions. In the context of individual patient management, a false negative report could lead to delays in providing (or failure to provide) a definitive diagnosis, appropriate treatment, infection control and prevention measures. A false positive report could lead to unnecessary or inappropriate treatment or unnecessary control and prevention actions. Therefore, establishing the performance of these devices and understanding the risks that might be associated with the use of these devices is critical to their safe and effective use.
The studies conducted by manufacturers to establish the performance of C. difficile detection devices are the basis for determining the safety and effectiveness or substantial equivalence of these devices. We recommend use of the cytotoxicity assay as a confirmatory test. This assay detects C. difficile toxin due to the toxin’s cytopathic effect in cell culture that can be neutralized with specific anti-sera.
We recommend that you provide a copy of your study protocols. These protocols should include information regarding exclusion and inclusion criteria, comparative methods used in the study, type and number of specimens, directions for use, and a scientifically sound statistical analysis plan. These protocols will enable us to better interpret your data and thus expedite review of your submission.
When referring to Clinical Laboratory Standards Institute (CLSI) standards or guidelines, we recommend that you indicate which specific aspects of the standards or guidelines you followed. In addition, you should specify whether you modified any part(s) of the standard and describe these modifications.
We encourage sponsors to contact the Division of Microbiology Devices to discuss their proposed studies and selection of specimen types. This is referred to as the pre-IDE process. We particularly encourage manufacturers to seek this type of discussion if they have difficulty obtaining samples.
A. General Recommendations
We recommend the use of a cytotoxicity assay to confirm or exclude the diagnosis of C. difficile infection. The number of positive or negative tests required depends on whether these tests are to be used for the initial diagnosis (before treatment, to confirm infection) or used to document eradication (after the completion of therapy). The definitions of baseline infection and eradication following therapy need to be considered separately (in terms of the number and type of endoscopic tests used) since at baseline it is important to achieve high specificity (low false positives) to confirm infection while at the test-of-cure time point, sensitivity is more important to exclude infection (low false negatives). Definitions of infection (or no infection) have been developed to assist sponsors in deciding which patients should be considered infected, not infected, or not evaluable based on endoscopic tests. It is important to note that the correct definition of these terms depends upon the quality and quantity of cytotoxicity assay C. difficile diagnostic tests.
When conducting the performance studies described below, we recommend that you run appropriate external controls every day of testing for the duration of the analytical and clinical studies. Examples of appropriate external controls include clinical specimens previously characterized as being positive or negative for C. difficile or commercially available positive and negative controls.
C. Analytical Studies
You should describe the antigen used in the device as a substrate. Briefly describe the production of antigen, strain of organism, purification process, etc. (You may label this as "Proprietary Information”). If the antigen you employ is a native antigen, you should identify its source. In addition, we recommend you provide a rationale for the selection of the antigen.
Validation of Reactive Cut-off
We recommend that you describe and explain the rationale for how you determined the reactive cut-off value for your device. If you included clinical data, you should identify the number of patients enrolled and treated in the study, the patient population, and methods used to determine the presence of C. difficile for diagnosis in these patients. The data should be presented graphically.
Limit of Detection
We recommend that you refer to Clinical Laboratory Standards Institute (CLSI) document EP17-A [Ref. 5], when designing your studies. Some examples of strains for inclusion in your LoD studies are shown in Table 1.
Table 1. Toxigenic strains of C. difficile recommended for analytical sensitivity (reactivity and LoD) studies *
Analytical Reactivity (Inclusivity)
We recommend that you test for potential cross-reactivity with medically relevant levels of viruses and bacteria (usually 106 cfu/ml or higher for bacteria and 105 pfu/ml or higher for viruses). You should confirm the virus and bacteria identities and titers. Examples of microorganisms recommended for cross-reactivity studies are listed in Table 2.
Table 2. Microorganisms recommended for analytical specificity (cross-reactivity) studies.
Table 3. Substances recommended for interference studies
We recommend that you test sources of variability (such as operators, days, assay runs, etc.) for a minimum of 12 days (not necessarily consecutive) with two runs per day and two replicates of each sample per run. These test days should span at least two calibration cycles. The test panel should consist of 3-6 samples (1-2 strains) spiked in relevant sample matrix or simulated sample matrix (provided that you can demonstrate that your device will generate equivalent results using both the actual sample matrix and the simulated sample matrix) at three concentrations that include:
When the limit of blank (LoB) is used as a cut-off, then the concentration C 95 is the same as the limit of detection (LoD) and the zero concentration (no analyte present in sample) is C 5 [Ref. 5]. CLSI documents EP5-A2 [Ref. 7] and EP12-A2 [Ref. 8] contain further information about designing and performing precision studies.
The protocol for the reproducibility study may vary slightly depending on the assay format. As a general guide, we recommend the following protocol:
The CLSI document, EP15-A2 [Ref. 9], contains additional information on reproducibility study design.
Specimen Storage and Shipping Conditions
If you recommend specimen storage conditions, you should demonstrate that your device generates equivalent results for the stored specimens at several time points throughout the duration of the recommended storage and at both ends of your recommended temperature range. If special selective/transport medium (viz., Cary Blair) is recommended for storage or shipping, you should conduct appropriate studies to demonstrate that the device will perform as described when the specimen is preserved in such media. [Ref. 10]
D. Clinical Performance Studies
We recommend that you conduct prospective clinical studies to determine the performance of your device for all the specimen types you claim in your labeling. We recommend that you compare your device to an established method or “gold standard,” dependent on the analyte being detected for C. difficile assays. A tissue culture assay for the detection of toxin is considered the gold standard for studies investigating new C. difficile Associated Diarrhea (CDAD) diagnostic techniques.
As with studies to evaluate performance characteristics, for any new technologies, you may contact the Division of Microbiology Devices for input on your study plan prior to initiating comparison studies.
We recommend that you contact the Division of Microbiology Devices to discuss such alternative proposals.
We recommend that the performance evaluation for devices intended for point-of-care (POC) use or rapid testing include, at a minimum, one site at a clinical laboratory as well as sites representative of non-laboratory settings where the device is intended to be used (e.g., physician’s office, emergency department). Conducting testing with the device in (1) a clinical laboratory with more experienced and trained personnel and (2) non-laboratory sites where the device is intended to be used but operators are likely to have less laboratory training will help to determine whether training of the person conducting the test is likely to affect the performance of the device.
If your device is intended for screening individuals for C. difficile infection, you should also include asymptomatic individuals in your study population. We recommend that you include a meaningful number of samples from each age group. We recommend that you present the data stratified by age (e.g., less than 5, 6- 21, 22-59, and greater than 60 years old) in addition to the overall data summary table.
E. Carry-over and Cross-contamination Studies (for Multi-sample Assays and Devices that Require Instrumentation)
You should demonstrate that carry-over and cross-contamination do not occur with your device. In a carry-over and cross-contamination study, we recommend that high positive samples be used in series alternating with high negative samples in patterns dependent on the operational function of the device. You should perform a minimum of five extraction runs with alternating high positive and high negative (C5) samples. We recommend that the high positive samples in the study be high enough to exceed 95% or more of the results obtained from specimens of diseased patients in the intended use population. The high negative samples should contain the analyte concentration below the cut-off such that repeat testing of these samples is negative approximately 95% of the time. (For an ultrasensitive test, such as a real-time PCR assay, it may not be possible to obtain high negative (C5) samples and, for such a device, the high negative samples may be replaced by negative samples). The carry-over and cross-contamination effect can then be estimated by the percent of negative results for the high negative sample in the carry-over study compared with 95% [Ref. 12]
F. Nucleic Acid-based C. difficile Devices
Nucleic acid tests (NAT) for pathogenic C. difficile generally target a specific region(s) of the pathogenicity locus (PaLoc) and should correlate closely with cytoxicity assays. Primers and probes for the selected target region should be carefully designed because mutations and/or deletions in the target could result in diminished sensitivity of the NAT but have minimal or no effect on cytotoxicity. Such mutations and/or deletions could result in an increase in false negative results for the NAT. If the target region is not closely associated with cytotoxicity, the sensitivity of the NAT may be unaffected by changes in the PaLoc that eliminate cytotoxicity, resulting in false positive results for the NAT. We recommend that you collate or develop bioinformatic data to support the choice of target and its correlation with cytotoxicity. Carry-over and cross-contamination studies are especially important in determining the performance of nucleic acid-based C. difficile assays. This section complements the recommendations for performance studies described previously in this document [Ref. 13].
Controls for Nucleic Acid-based C. difficile Assays
We recommend that you use quality control material for verification of assay performance in analytical and clinical studies. In addition, you should identify the acceptable ranges for each type of control and explain how you established acceptance criteria for these controls.
We recommend that you consult with FDA when designing specific controls for your device. We generally recommend that you include the following four types of controls. Some of these controls may be combined depending on the specific composition of the control and assay workflow:
Blanks or no template control
Negative sample control
Positive control for complete assay
Positive control for amplification/detection
G. CLIA Waiver
If you wish to pursue CLIA waiver status for your device under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), we recommend that you consult with the Division of Microbiology Devices staff regarding the design of specific studies to support the CLIA waiver application for your device. The guidance for industry and FDA staff, “ Recommendations for Clinical Laboratory Improvement Amendments of 1988 (CLIA) Waiver Applications for Manufacturers of In Vitro Diagnostic Devices. ”
1 Other detection methods include: a. microbial culture and b. immunoassays that detect common antigen(s) of C. difficile such as glutamate dehydrogenase and do not distinguish between toxigenic and nontoxigenic strains of C. difficile. These methods are not included in this draft guidance document.
* Examples of nontoxigenic strains of C. difficile that can also be used in LoD studies for devices that detect common antigen such as glutamate dehydrogenase include: ATCC 700057 (VPI 11186); ATCC 43593; C. difficile Xla (A-B-tox bin+) IS58; C. difficile Xlb (A-B-tox bin+) R1 1402
2 For an ultrasensitive test, such as a real-time PCR assay, it may not be possible to obtain a C 5 sample. For such a device, the C 5 sample may be replaced by the following two samples: