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Draft Guidance for Industry and Food and Drug Administration Staff - Establishing the Performance Characteristics of In Vitro Di

Posted Nov 29 2010 12:00am

This guidance document is being distributed for comment purposes only.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

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draft document within 90 days of publication in the Federal Register of the notice announcing the availability of the draft guidance.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 . 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 .

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.


U.S. Department of Health and Human Services
Food and Drug Administration
Center for Devices and Radiological Health
Office of In Vitro Diagnostic Device Evaluation and Safety
Division of Microbiology Devices

Additional copies are available from the Internet . You may also send an e-mail request to 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.

  1. Introduction
  2. Background
  3. Scope
  4. Risks to Health
  5. Establishing Performance Characteristics
    1. General Recommendations
    2. Controls
    3. Analytical Studies
    4. Clinical Performance Studies
    5. Carry-Over and Cross-contamination Studies (for multi-sample assays and devices that require instrumentation)
    6. Nucleic Acid-based C. difficile Devices
    7. CLIA Waiver
  6. References

This draft guidance, when finalized, will represent the Food and Drug Administration's (FDA's) current thinking on this topic.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:

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:

LLH – C. difficile, Antigenic Components (Class I)

MCB - Antigen, C. difficile (Class I)

OMN - C. difficile nucleic acid amplification test assay (Class I)

Therefore the following information should be included in your submission:

  • The diagnostic marker for the device (antigens, antibodies, or nucleic acids)
  • Methodology or test principle of the device - (e.g., Immunoassay, RT-PCR, etc.)
  • Sample preparation methods
  • Length of time taken to report results, (e.g., within 6-24 hours of the beginning the test, etc.)

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.

B. Controls

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

Antigen Characterization

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.

Analytical Sensitivity

Limit of Detection
We recommend that you determine the limit of detection (LoD) for C. difficile to calculate the analytical sensitivity of your device. The study should include serial dilutions of at least two strains and should include a variety of toxinotypes, one of which should be toxinotype 0, and 3-5 replicates for each dilution. The LoD is defined as the level of C. difficile in a specimen that gives a 95% detection rate. The LoD should be confirmed by preparing at least 20 additional replicates at the LoD and demonstrating that C. difficile was detected 95% of the time. Each of these additional replicates should be extracted separately prior to detection with the device. Confirmation of the LoD in CFU/mL should be carried out by actual colony counting. You should generate a colony count corresponding to each of the twenty or more replicates and not rely on estimates based on the titer of the stock suspension.

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 *

ATCC 43255 (CCUG19126, VPI 10463)0 A+B+
ATCC 9689 (90556-M6S)0 A+B+
ATCC 700792 (14797-2)A+B+
ATCC 17858 (1253)A+B+
ATCC BAA-1382 (630)A+B+
ATCC 51695 (BDMS 18 AN)A+B+
ATCC 43600 (2149)A+B+
ATCC 43599 (2022)A+B+
ATCC 43596 (545)A+B+
ATCC 43594 (W1194)A+B+
ATCC 17857 (870)A+B+
ATCC 43598 (1470)VIII A-B+
CCUG 8864X A-B+

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


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 SpeciesStrain
Abiotrophia defective ATCC 49176
Acinetobacter baumannii ATCC 19606
Acinetobacter Iwoffii CDCF 3697
Aeromonas hydrophila ATCC 7966/ CCRI-10071
Alcaligenes faecalis subsp. Faecalis ATCC 15554
Anaerococcus tetradius ATCC 35098
Bacillus cereus ATCC 13472
Bacillus cereus HER 1414
Bacteroidescaccae ATCC 43185
Bacteroides merdae ATCC 43184
Bacteroides stercoris ATCC 43183
Bifidobacterium adolescentis ATCC 15703
Bifidobacterium longum ATCC 15707
Campylobacter coli ATCC 43479
Campylobacter jejuni sub sp .jejuni ATCC 33292
Candida albicans ATCC 10231
Candida catenulate IDI-1729
Cedecea davisae ATCC 33431
Chlamydia trachomatis ABI 08-901-000
Citrobacter amalonaticus ATCC 25405
Citrobacter freundii ATCC 8090
Citrobacter koseri ATCC 27028
Citrobacter sedlakii ATCC 51115 (IDI-2178)
Clostridium beijerinckii ATCC 8260
Clostridium bifermentans ATCC 638
Clostridium bolteae BAA-613
Clostridium botulinum Hall A
Clostridium butyricum CCRI-11128
Clostridium chauvoei ATCC 11957
Clostridium fallax ATCC 19400
Clostridium haemolyticum ATCC 9650
Clostridium histolyticum ATCC 19401
Clostridium innocuum CCRI-9927 / IDI 1986
Clostridium methylpentosum ATCC 43829
Clostridium nexile ATCC 27757
Clostridium novyi ATCC 19402
Clostridium orbiscindens ATCC 49531
Clostridium paraputrificum ATCC 25780
Clostridium perfringens ATCC 13124
Clostridium ramosurn ATCC 25582
Clostridium scindens ATCC 35704
Clostridium septicum ATCC 12464
Clostridium sordellii ATCC 9714
Clostridium difficile (non-toxigenic ATCC 43593)
Clostridium difficile (non-toxigenic ATCC 43601)
Clostridium sphenoides ATCC 19403
Clostridium spiroforme ATCC 29899
Clostridium sporogenes ATCC 15579
Clostridium symbiosum CCRI-9928 /IDI 1989
Clostridium symbiosum ATCC 14940
Clostridium terdium ATCC 14573
Ciostridium tetani ATCC 19406
Collinsella aerofaciens ATCC 25986
Corynebacterium genitalium LSPQ 3583
Desulfovibrio piger ATCC 29098
Edwardsiella tarda ATCC 15947
Eggerthellalenta CCRI-9926 /IDI 1990
Enterobacter aerogenes ATCC 13048
Enterobacter cloacae ATCC 13047
Enterococcus casselif/avus (vanC2) CCRI-1 566 / IDI 1981
Enterococcus cecorum ATCC 43198
Enterococcus dispar ATCC 51266
Enterococcusfaecalis vanB ATCC 51299
Enterococcusfaecium vanA ATCC 700221
Enterococcusga/linarum vanC CCRI-1561 /IDI 1982
Enterococcus hirae ATCC 8043
Enterococcu sraffinosus ATCC 49427
Escherichia coli ATCC 23511
Escherichia coli ToplO (IDI-266)
Escherichia fergusonii ATCC 35469
Escherichia hermannii ATCC 33650
Fusobacterium varium ATCC 8501
Gardnerella vagina/is ATCC 14019
Gemella morbillorum ATCC 27824
Hafnia alvei ATCC 13337
Helicobacter fennelliae ATCC 35683 / IDI-2180
Helicobacter pylori ATCC 43504
Homo sapiens ATCC MGC-1 5492 / 2.16
Klebsiella oxytoca ATCC 33496
Klebsielia oxytoca ATCC 33497
Klebsiella pneumoniae subsp. Pneumoniae ATCC 13883
Lactobacillus acidophilus ATCC 4356
Lactobacillus reuteri ATCC 23272
Lactococcus iactis ATCC 11454
Leminorela grimontii ATCC 33999
Listeria grayi ATCC 19120
Listeria innocua ATCC 33090
Listeria monocytogenes L374
Peptoniphilus asaccharolyticus ATCC 14963
Peptostreptococcus anaerobius ATCC 27337
Plesiomonas shigelloides ATCC 14029
Porphyromonas asaccharolytica ATCC 25260
Prevotella melaninogenica ATCC 25845
Proteus mirabilis ATCC 25933
Proteus penneri ATCC 35198
Providencia alcalifaciens ATCC 9886
Providencia rettgeri ATCC 9250
Providencia stuartli ATCC 33672
Pseudomonas aeruginosa ATCC 35554
Pseudomonas putida LCDC D7172
Ruminococcus bromii ATCC 27255
Salmonella choleraesuis (typhimurium) ATCC 14028
Salmonella enterica subsp. Arizonae (formerly
Choleraesuis arizonae)
ATCC 13314
Salmonella enteric asubsp. Enterica (formerly ATCC 7001
Salmonella choleraesuis subsp. choleraesuis) ATCC 14028
Serratia liquefaciens ATCC 27592
Serratia marcescens2 ATCC 13880
Shigella boydii ATCC 9207
Shigella dysenteriae ATCC 11835
Shigella sonnei ATCC 29930
Staphylococcus aureus3 ATCC 43300
Staphylococcus epidermidis ATCC 14990
Stenotrophomonas maltophilia ATCC 13637
Streptococcus agalactiae ATCC 12973
Streptococcus dysgalactiae ATCC 43078
Streptococcus intermedius ATCC 27335
Streptococcus uberis ATCC 19436
Trabulsiella guamensis ATCC 49490
Veillonella parvula ATCC 10790
Vibrio cholerae ATCC 25870
Vibrio parahaemolyticus ATCC 17802
Yersinia bercovieri ATCC 43970
Yersinia rohdei ATCC 43380
Coxsackie virus 

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.

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

Substance Active Ingredient
Anti-Fungal /Anti-Itch VaginalNystatin
Anti-Hemorrhoid Creams/OintmentsP henylephrine
Antacids  Calcium Carbonate/ Aluminum Hydroxide/ Magnesium Hydroxide
EnemasMesalazine/Mineral Oil
Condoms with Spermicidal LubricantNonoxynol-9
Anti-Diarrheal MedicationLoperamide Hydrochloride/ Bismuth Subsalicylate
Antibiotics (Oral and Topical)Antibiotic
Non-Steroidal Anti-Inflammatory MedicationsNaproxen Sodium
Moist TowelettesBenzalkonium Chloride, Ethanol
Fecal FatLipids etc
BloodGlucose, Hormones, Enzymes, Ions, Iron, etc
MucusImmunoglobulins, Lysozyme, Polymers, etc

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.


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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  2. "Pseudomembranous Colitis". eMedicine. WebMD (2005-07-01). Retrieved on 2007-01-11.
  3. Hall I, O'Toole E (1935). "Intestinal flora in newborn infants with a description of a new pathogenic anaerobe, Bacillus difficilis". Am J Dis Child 49: 390.
  4. "Cleaning agents 'make bug strong'", BBC News Online (2006-04-03)
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  6. Clinical and Laboratory Standards Institute. 2005. Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition (EP7-A2)
  7. Clinical and Laboratory Standards Institute. 2004. Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline—Second Edition (EP5-A2)
  8. Clinical and Laboratory Standards Institute. 2008. User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline— Second Edition (EP12-A2)
  9. Clinical and Laboratory Standards Institute. 2005. User Verification of Performance for Precision and Trueness; Approved Guideline—Second Edition (EP15-A2)
  10. Clinical and Laboratory Standards Institute. 2003. Quality Control for Microbiological Transport Systems; Approved Standard
  11. Center for Devices and Radiological Health, Office of Device Evaluation.
    January 20, 1998. Guidance on IDE Policies and Procedures
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  13. Clinical and Laboratory Standards Institute. 2006 Molecular Diagnostic Methods for Infectious Disease; Proposed Guideline. MM3-A2. Clinical and Laboratory Standards Institute, Wayne PA.

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:

  • A negative sample: a sample with no analyte such that results of repeated tests of this sample are negative 100% of the time.
  • A “high negative/low positive” sample (C 20 to C 80 concentration): a sample with a concentration below the clinical cut-off such that results of repeated tests of this sample are negative approximately 20% to 80% of the time.
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