Draft Guidance for Industry and Food and Drug Administration Staff

DRAFT GUIDANCE
This guidance document is being distributed for comment purposes only.
Document issued on: November 29, 2010

FDA is issuing this draft guidance to provide industry and agency staff with updated recommendations concerning 510(k) submissions for various types of in vitro diagnostic devices (IVDs)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).

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:

21 CFR 866.2660 Microorganism differentiation and identification device

(a) 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.

(b) Classification. Class I (general controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to § 866.9.

The following are the product codes for C. difficile devices cleared under 21 CFR 866.2660:

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.

*

Analytical Reactivity (Inclusivity)
We recommend that you demonstrate that the test can detect at least twenty additional strains of toxigenic C. difficile with varying toxinotypes that represent temporal and geographical diversity at concentrations two to three times that of the LoD. If it is difficult to obtain sufficient samples to demonstrate detection of a particular strain, you should contact the Division of Microbiology Devices to discuss your study. The diversity of the strains tested for reactivity should be supported by inclusion of at least five different toxinotypes in the study. We suggest that the strains for reactivity studies also be selected from those shown in Table 1.

Analytical Specificity

Cross-reactivity

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.

Genera and SpeciesStrainAbiotrophia defective ATCC 49176Acinetobacter baumannii ATCC 19606Acinetobacter Iwoffii CDCF 3697Aeromonas hydrophila ATCC 7966/ CCRI-10071Alcaligenes faecalis subsp. Faecalis ATCC 15554Anaerococcus tetradius ATCC 35098Bacillus cereus ATCC 13472Bacillus cereus HER 1414Bacteroidescaccae ATCC 43185Bacteroides merdae ATCC 43184Bacteroides stercoris ATCC 43183Bifidobacterium adolescentis ATCC 15703Bifidobacterium longum ATCC 15707Campylobacter coli ATCC 43479Campylobacter jejuni sub sp .jejuni ATCC 33292Candida albicans ATCC 10231Candida catenulate IDI-1729Cedecea davisae ATCC 33431Chlamydia trachomatis ABI 08-901-000Citrobacter amalonaticus ATCC 25405Citrobacter freundii ATCC 8090Citrobacter koseri ATCC 27028Citrobacter sedlakii ATCC 51115 (IDI-2178)Clostridium beijerinckii ATCC 8260Clostridium bifermentans ATCC 638Clostridium bolteae BAA-613Clostridium botulinum Hall AClostridium butyricum CCRI-11128Clostridium chauvoei ATCC 11957Clostridium fallax ATCC 19400Clostridium haemolyticum ATCC 9650Clostridium histolyticum ATCC 19401Clostridium innocuum CCRI-9927 / IDI 1986Clostridium methylpentosum ATCC 43829Clostridium nexile ATCC 27757Clostridium novyi ATCC 19402Clostridium orbiscindens ATCC 49531Clostridium paraputrificum ATCC 25780Clostridium perfringens ATCC 13124Clostridium ramosurn ATCC 25582Clostridium scindens ATCC 35704Clostridium septicum ATCC 12464Clostridium sordellii ATCC 9714Clostridium difficile (non-toxigenic ATCC 43593)Clostridium difficile (non-toxigenic ATCC 43601)Clostridium sphenoides ATCC 19403Clostridium spiroforme ATCC 29899Clostridium sporogenes ATCC 15579Clostridium symbiosum CCRI-9928 /IDI 1989Clostridium symbiosum ATCC 14940Clostridium terdium ATCC 14573Ciostridium tetani ATCC 19406Collinsella aerofaciens ATCC 25986Corynebacterium genitalium LSPQ 3583Desulfovibrio piger ATCC 29098Edwardsiella tarda ATCC 15947Eggerthellalenta CCRI-9926 /IDI 1990Enterobacter aerogenes ATCC 13048Enterobacter cloacae ATCC 13047Enterococcus casselif/avus (vanC2) CCRI-1 566 / IDI 1981Enterococcus cecorum ATCC 43198Enterococcus dispar ATCC 51266Enterococcusfaecalis vanB ATCC 51299Enterococcusfaecium vanA ATCC 700221Enterococcusga/linarum vanC CCRI-1561 /IDI 1982Enterococcus hirae ATCC 8043Enterococcu sraffinosus ATCC 49427Escherichia coli ATCC 23511Escherichia coli ToplO (IDI-266)Escherichia fergusonii ATCC 35469Escherichia hermannii ATCC 33650Fusobacterium varium ATCC 8501Gardnerella vagina/is ATCC 14019Gemella morbillorum ATCC 27824Hafnia alvei ATCC 13337Helicobacter fennelliae ATCC 35683 / IDI-2180Helicobacter pylori ATCC 43504Homo sapiens ATCC MGC-1 5492 / 2.16Klebsiella oxytoca ATCC 33496Klebsielia oxytoca ATCC 33497Klebsiella pneumoniae subsp. Pneumoniae ATCC 13883Lactobacillus acidophilus ATCC 4356Lactobacillus reuteri ATCC 23272Lactococcus iactis ATCC 11454Leminorela grimontii ATCC 33999Listeria grayi ATCC 19120Listeria innocua ATCC 33090Listeria monocytogenes L374Peptoniphilus asaccharolyticus ATCC 14963Peptostreptococcus anaerobius ATCC 27337Plesiomonas shigelloides ATCC 14029Porphyromonas asaccharolytica ATCC 25260Prevotella melaninogenica ATCC 25845Proteus mirabilis ATCC 25933Proteus penneri ATCC 35198Providencia alcalifaciens ATCC 9886Providencia rettgeri ATCC 9250Providencia stuartli ATCC 33672Pseudomonas aeruginosa ATCC 35554Pseudomonas putida LCDC D7172Ruminococcus bromii ATCC 27255Salmonella choleraesuis (typhimurium) ATCC 14028Salmonella enterica subsp. Arizonae (formerly
Choleraesuis arizonae) ATCC 13314Salmonella enteric asubsp. Enterica (formerly ATCC 7001Salmonella choleraesuis subsp. choleraesuis) ATCC 14028Serratia liquefaciens ATCC 27592Serratia marcescens2 ATCC 13880Shigella boydii ATCC 9207Shigella dysenteriae ATCC 11835Shigella sonnei ATCC 29930Staphylococcus aureus3 ATCC 43300Staphylococcus epidermidis ATCC 14990Stenotrophomonas maltophilia ATCC 13637Streptococcus agalactiae ATCC 12973Streptococcus dysgalactiae ATCC 43078Streptococcus intermedius ATCC 27335Streptococcus uberis ATCC 19436Trabulsiella guamensis ATCC 49490Veillonella parvula ATCC 10790Vibrio cholerae ATCC 25870Vibrio parahaemolyticus ATCC 17802Yersinia bercovieri ATCC 43970Yersinia rohdei ATCC 43380Adenovirus Rotavirus Norovirus Enterovirus Echovirus Coxsackie virus Cytomegalovirus 

Microbial Interference
We recommend that you evaluate your assay for interference by microorganisms that are not detected by your assay using clinically relevant concentrations of potentially interfering microorganisms (usually 10⁶ cfu/ml or higher for bacteria and 10⁵ pfu/ml or higher for viruses). Potentially interfering microorganisms should be the same as those recommended for testing in the cross-reactivity study. We recommend that you use more than one strain of toxigenic C. difficile in this study at a level close to the LoD for each strain.

Interference
We recommend that you conduct a comprehensive interference study using medically relevant concentrations of the interferent and more than one strain of toxigenic C. difficile to assess the potentially inhibitory effects of substances encountered in blood and stool specimens. Potentially interfering substances include, but are not limited to, biological and chemical substances occasionally used or found in peri-anal, rectal and/or stool specimens, blood, and mucus. Examples of potentially interfering substances are presented in Table 4. We recommend that you test interference at the assay cut-off determined for each C. difficile strain and for each of the interfering substances. We also recommend that you evaluate each interfering substance at its potentially highest concentration (“worst case”). If no significant clinical effect is observed, no further testing is necessary. Please refer to the CLSI document EP7-A2 [Ref. 6] for additional information.  

Table 3. Substances recommended for interference studies

Precision

Within-Laboratory Precision/Repeatability
We recommend that you conduct within-laboratory precision studies for devices that include instruments or automated components. You may perform these studies in-house, i.e., within your own company.

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:

• A “high negative” sample (C 5 concentration): a sample with an analyte concentration below the clinical cut-off such that results of repeated tests of this sample are negative approximately 95% of the time (and results are positive approximately 5% of the time).²
• A “low positive” sample (C 95 concentration): a sample with a concentration of analyte just above the clinical cut-off such that results of repeated tests of this sample are positive approximately 95% of the time.
• A “moderate positive” sample: a sample with a concentration at which one can anticipate positive results approximately 100% of the time (e.g., approximately two to three times the concentration of the clinical cut-off).

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.

Reproducibility

The protocol for the reproducibility study may vary slightly depending on the assay format. As a general guide, we recommend the following protocol:

• Evaluate the reproducibility of your test at three testing sites (for example, two external sites and one in-house site).
• Use a five-day testing protocol, including a minimum of two runs per day, (unless the assay design precludes multiple runs per day) and three replicates of each panel member per run.
• Each day, have at least two operators at each facility perform the test. In order to represent the settings in which the devices will be used, you should include a larger number of devices in your evaluation for rapid testing or point-of-care (POC) devices.
• Use the same sample panel as described in the repeatability study above.

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.  

Study Protocol
We strongly suggest that you develop a detailed study protocol that includes patient inclusion and exclusion criteria, type and number of specimens needed, directions for use, and a statistical analysis plan that accounts for variances to prevent data bias. You should include this and any other relevant protocol information in your premarket submission. We encourage sponsors to contact the Division of Microbiology Devices to request a review of their proposed studies and selection of specimen types. This is referred to as the pre-IDE process [Ref. 11].  

Specimen Type(s)
We recommend that you include a sufficient number of prospectively collected samples to generate a sensitivity result with a lower bound of the two-sided 95% confidence interval (CI) greater than 90%. Generally, we recommend testing a minimum of 100 samples, determined to be positive using the reference method. You should use fresh prospectively collected specimens. If a limited number of samples are available, both fresh and frozen samples may be used; however, you should analyze the data separately. Acceptable samples include patient fecal specimens submitted to the testing laboratory to rule out suspected CDAD. Such specimens consist of diarrheal stool from patients with or without a history of antibiotic us e.

We recommend that you contact the Division of Microbiology Devices to discuss such alternative proposals.

Study Sites
We recommend that you conduct your studies at a minimum of three separate geograph ically distinct facilities, one of which may be in-house. Clinical investigations of unapproved and uncleared in vitro diagnostic devices, including diagnostic devices for C. difficile, are subject to the Investigational Device Exemption (IDE) provisions of Section 520(g) of the Federal Food, Drug, and Cosmetic Act (21 U.S.C. 360j) and the implementing regulations. You should consider how 21 CFR part 812 (IDEs) applies to your particular study and refer to 21 CFR part 50 (informed consent) and 21 CFR part 56 (institutional review board review) for other applicable requirements.

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.

Study Population
We recommend that you conduct your studies on individuals presenting with symptoms of
C. difficile infection (e.g., significant diarrhea (“new onset of 3 partially formed or watery stools per 24 hour period”), exposure of antibiotics, abdominal pain, foul stool odor, etc.).

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:

• Negative Control to rule out contamination
• Positive Control to verify that PCR reagents and instrument are functioning
• Internal Control to verify that a negative result for sample is not caused by PCR inhibitors
• Extraction Control to verify that lysis and nucleic acid isolation processes are functioning correctly

Negative Controls

Blanks or no template control
The blank, or no-template control, should contain buffer or sample transport media and all of the assay components except target nucleic acid. These controls are used to rule out contamination with target nucleic acid or increased background in the amplification reaction. This may not apply for assays performed in single test disposable cartridges or tubes.

Negative sample control
The negative sample control contains non-target nucleic acid or, if used to evaluate extraction procedures, it contains the whole organism. It reveals non-specific priming or detection and indicates that signals are not obtained in the absence of target sequences. Examples of acceptable negative sample control materials include:

• Specimen from a non-infected patient
• Samples containing a non-target organism (e.g., non-toxigenic C. difficile)
• Surrogate negative control (e.g., alien encapsidated DNA)

Positive Controls

Positive control for complete assay
The positive control contains target nucleic acids and is used to control the entire assay process, including DNA extraction, amplification, and detection. It is designed to mimic a patient specimen and is run as a separate assay, concurrently with patient specimens, at a frequency determined by a laboratory’s Quality System (QS). Examples of acceptable positive assay control materials include:

• A toxigenic strain of C. difficile containing the target region
• Packaged DNA from a toxigenic strain of C. difficile containing the target region

Positive control for amplification/detection

• The positive control for amplification/detection contains purified target nucleic acid at or near the limit of detection for a qualitative assay and is not taken through the extraction process. It controls for the integrity of the PCR reagents and instrument when negative results are obtained. It indicates that the target will be detected if it is present in the extracted sample. An example of this type of control is non-infectious DNA plasmid containing a portion of the targeted gene.

Internal Control
The internal control is a non-target nucleic acid sequence that is co-extracted and co-amplified with the target nucleic acid. It controls for integrity of the reagents (polymerase, primers, etc.), equipment function (thermal cycler), and the presence of inhibitors in the samples. Examples of acceptable internal control materials include human nucleic acid co-extracted with C. difficile and primers amplifying human housekeeping genes (e.g., RNaseP, β-actin) or nucleic acid that is added to sample prior to extraction. It is preferable that the target region that is amplified in the internal control is at least equal in length to that of the C. difficile target(s). The need for this control is determined on a case-by-case basis [Ref. 10].

Extraction Control
The extraction control verifies that lysis of C. difficile and subsequent nucleic acid isolation have occurred efficiently. Examples of extraction controls include an organism that is lysed similarly to C. difficile, a strain of C. difficile containing the target region, or a known positive clinical specimen. It is possible that the positive control or internal control, depending on their specific compositions, may also function as an extraction control.

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.”

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