In Vitro

Contains Nonbinding Recommendations
Draft - Not for Implementation
Draft Guidance for Industry and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
DRAFT GUIDANCE
This guidance document is being distributed for comment purposes only.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of publication in the Federal Register of the notice announcing the availability of the draft
guidance.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 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
[email protected].
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
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Preface
Additional Copies
Additional copies are available from the Internet at:
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/uc
m234868.htm. You may also send an e-mail request to [email protected] 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.
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Table of Contents
I. Introduction......................................................................................................................................................4
II. Background.....................................................................................................................................................5
III. Scope ..............................................................................................................................................................5
IV. Risks to Health ..............................................................................................................................................6
V. Establishing Performance Characteristics ...................................................................................................7
A. General Recommendations ...........................................................................................................................8
B. Controls........................................................................................................................................................8
C. Analytical Studies.........................................................................................................................................8
D. Clinical Performance Studies ....................................................................................................................16
E. Carry-Over and Cross-contamination Studies (for multi-sample assays and devices that require
instrumentation)...............................................................................................................................................18
F. Nucleic Acid-based C. difficile Devices .....................................................................................................18
G. CLIA Waiver ..............................................................................................................................................20
VI. References....................................................................................................................................................21
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Contains Nonbinding Recommendations
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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
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.
I. Introduction
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.
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II. Background
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)”,
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/HowtoMarketYourDevi
ce/PremarketSubmissions/PremarketNotification510k/default.htm. 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,” which is available at
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/uc
m084365.htm.
III. Scope
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
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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.)
IV. Risks to Health
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.
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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.
V. Establishing Performance Characteristics
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.
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.
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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.
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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 ∗
Strain
ATCC 43255 (CCUG19126, VPI 10463)
ATCC 9689 (90556-M6S)
ATCC 700792 (14797-2)
ATCC 17858 (1253)
ATCC BAA-1805
ATCC BAA-1382 (630)
ATCC 51695 (BDMS 18 AN)
ATCC 43600 (2149)
ATCC 43599 (2022)
ATCC 43596 (545)
ATCC 43594 (W1194)
ATCC 17857 (870)
ATCC 43598 (1470)
CCUG 8864
Toxinotype
0 A+B+
0 A+B+
A+B+
A+B+
III A+B+
A+B+
A+B+
A+B+
A+B+
A+B+
A+B+
A+B+
VIII A-B+
X A-B+
∗
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
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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
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5
viruses and bacteria (usually 10 cfu/ml or higher for bacteria and 10 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.
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Table 2. Microorganisms recommended for analytical specificity (cross-reactivity) studies.
Genera and Species
Strain
Abiotrophia defective
Acinetobacter baumannii
Acinetobacter Iwoffii
Aeromonas hydrophila
Alcaligenes faecalis subsp. Faecalis
Anaerococcus tetradius
Bacillus cereus
Bacillus cereus
Bacteroidescaccae
Bacteroides merdae
Bacteroides stercoris
Bifidobacterium adolescentis
Bifidobacterium longum
Campylobacter coli
Campylobacter jejuni sub sp .jejuni
Candida albicans
Candida catenulate
Cedecea davisae
Chlamydia trachomatis
Citrobacter amalonaticus
Citrobacter freundii
Citrobacter koseri
Citrobacter sedlakii
Clostridium beijerinckii
Clostridium bifermentans
Clostridium bolteae
Clostridium botulinum
Clostridium butyricum
Clostridium chauvoei
Clostridium fallax
Clostridium haemolyticum
Clostridium histolyticum
Clostridium innocuum
Clostridium methylpentosum
Clostridium nexile
Clostridium novyi
Clostridium orbiscindens
Clostridium paraputrificum
Clostridium perfringens
Clostridium ramosurn
Clostridium scindens
Clostridium septicum
Clostridium sordellii
ATCC 49176
ATCC 19606
CDCF 3697
ATCC 7966/ CCRI-10071
ATCC 15554
ATCC 35098
ATCC 13472
HER 1414
ATCC 43185
ATCC 43184
ATCC 43183
ATCC 15703
ATCC 15707
ATCC 43479
ATCC 33292
ATCC 10231
IDI-1729
ATCC 33431
ABI 08-901-000
ATCC 25405
ATCC 8090
ATCC 27028
ATCC 51115 (IDI-2178)
ATCC 8260
ATCC 638
BAA-613
Hall A
CCRI-11128
ATCC 11957
ATCC 19400
ATCC 9650
ATCC 19401
CCRI-9927 / IDI 1986
ATCC 43829
ATCC 27757
ATCC 19402
ATCC 49531
ATCC 25780
ATCC 13124
ATCC 25582
ATCC 35704
ATCC 12464
ATCC 9714
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Genera and Species
Strain
Clostridium difficile
Clostridium difficile
Clostridium sphenoides
Clostridium spiroforme
Clostridium sporogenes
Clostridium symbiosum
Clostridium symbiosum
Clostridium terdium
Ciostridium tetani
Collinsella aerofaciens
Corynebacterium genitalium
Desulfovibrio piger
Edwardsiella tarda
Eggerthellalenta
Enterobacter aerogenes
Enterobacter cloacae
Enterococcus casselif/avus (vanC2)
Enterococcus cecorum
Enterococcus dispar
Enterococcusfaecalis vanB
Enterococcusfaecium vanA
Enterococcusga/linarum vanC
Enterococcus hirae
Enterococcu sraffinosus
Escherichia coli
Escherichia coli
Escherichia fergusonii
Escherichia hermannii
Fusobacterium varium
Gardnerella vagina/is
Gemella morbillorum
Hafnia alvei
Helicobacter fennelliae
Helicobacter pylori
Homo sapiens
Klebsiella oxytoca
Klebsielia oxytoca
Klebsiella pneumoniae subsp. Pneumoniae
Lactobacillus acidophilus
Lactobacillus reuteri
Lactococcus iactis
Leminorela grimontii
Listeria grayi
Listeria innocua
Listeria monocytogenes
(non-toxigenic ATCC 43593)
(non-toxigenic ATCC 43601)
ATCC 19403
ATCC 29899
ATCC 15579
CCRI-9928 /IDI 1989
ATCC 14940
ATCC 14573
ATCC 19406
ATCC 25986
LSPQ 3583
ATCC 29098
ATCC 15947
CCRI-9926 /IDI 1990
ATCC 13048
ATCC 13047
CCRI-1 566 / IDI 1981
ATCC 43198
ATCC 51266
ATCC 51299
ATCC 700221
CCRI-1561 /IDI 1982
ATCC 8043
ATCC 49427
ATCC 23511
ToplO (IDI-266)
ATCC 35469
ATCC 33650
ATCC 8501
ATCC 14019
ATCC 27824
ATCC 13337
ATCC 35683 / IDI-2180
ATCC 43504
ATCC MGC-1 5492 / 2.16
ATCC 33496
ATCC 33497
ATCC 13883
ATCC 4356
ATCC 23272
ATCC 11454
ATCC 33999
ATCC 19120
ATCC 33090
L374
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Genera and Species
Peptoniphilus asaccharolyticus
Peptostreptococcus anaerobius
Plesiomonas shigelloides
Porphyromonas asaccharolytica
Prevotella melaninogenica
Proteus mirabilis
Proteus penneri
Providencia alcalifaciens
Providencia rettgeri
Providencia stuartli
Pseudomonas aeruginosa
Pseudomonas putida
Ruminococcus bromii
Salmonella choleraesuis (typhimurium)
Salmonella enterica subsp. Arizonae (formerly
Choleraesuis arizonae)
Salmonella enteric asubsp. Enterica (formerly
Salmonella choleraesuis subsp. choleraesuis)
Serratia liquefaciens
Serratia marcescens2
Shigella boydii
Shigella dysenteriae
Shigella sonnei
Staphylococcus aureus3
Staphylococcus epidermidis
Stenotrophomonas maltophilia
Streptococcus agalactiae
Streptococcus dysgalactiae
Streptococcus intermedius
Streptococcus uberis
Trabulsiella guamensis
Veillonella parvula
Vibrio cholerae
Vibrio parahaemolyticus
Yersinia bercovieri
Yersinia rohdei
Adenovirus
Rotavirus
Norovirus
Enterovirus
Echovirus
Coxsackie virus
Cytomegalovirus
Strain
ATCC 14963
ATCC 27337
ATCC 14029
ATCC 25260
ATCC 25845
ATCC 25933
ATCC 35198
ATCC 9886
ATCC 9250
ATCC 33672
ATCC 35554
LCDC D7172
ATCC 27255
ATCC 14028
ATCC 13314
ATCC 7001
ATCC 14028
ATCC 27592
ATCC 13880
ATCC 9207
ATCC 11835
ATCC 29930
ATCC 43300
ATCC 14990
ATCC 13637
ATCC 12973
ATCC 43078
ATCC 27335
ATCC 19436
ATCC 49490
ATCC 10790
ATCC 25870
ATCC 17802
ATCC 43970
ATCC 43380
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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
Substance
Active Ingredient
Anti-Fungal /Anti-Itch Vaginal
Nystatin
Creams/Ointments/Suppositories
Hydrocortisone
Anti-Hemorrhoid Creams/Ointments
Antacids
Phenylephrine
Calcium Carbonate/ Aluminum Hydroxide/
Magnesium Hydroxide
Mesalazine/Mineral Oil
Nonoxynol-9
Loperamide Hydrochloride/ Bismuth
Subsalicylate
Sennosides
Antibiotic
Naproxen Sodium
Benzalkonium Chloride, Ethanol
Lipids etc
Glucose, Hormones, Enzymes, Ions, Iron, etc
Immunoglobulins, Lysozyme, Polymers, etc
Enemas
Condoms with Spermicidal Lubricant
Anti-Diarrheal Medication
Laxatives
Antibiotics (Oral and Topical)
Non-Steroidal Anti-Inflammatory Medications
Moist Towelettes
Fecal Fat
Blood
Mucus
Precision
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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 (C5 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 (C95 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 C95 is the same as the
limit of detection (LoD) and the zero concentration (no analyte present in sample) is C5 [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).
For an ultrasensitive test, such as a real-time PCR assay, it may not be possible to obtain a
C5 sample. For such a device, the C5 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 (C20 to C80 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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•
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].
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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 use.
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 geographically
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.
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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 carryover 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:
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„ 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
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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 coamplified 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 coextracted 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,” is available at
http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDo
cuments/ucm070890.pdf.
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VI. References
1. Ryan KJ; Ray CG (editors) (2004). Sherris Medical Microbiology, 4th ed., McGraw
Hill, pp. 322-4. ISBN 0-8385-8529-9
2. "Pseudomembranous Colitis". eMedicine. WebMD (2005-07-01). Retrieved on 200701-11.
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)
5. Clinical and Laboratory Standards Institute. 2004. Protocol for Determination of Limits
of Detection and Limits of Quantitation; Approved Guideline (EP17-A)
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
(M40-A)
11. Center for Devices and Radiological Health, Office of Device Evaluation.
January 20, 1998. Guidance on IDE Policies and Procedures
12. Haeckel R. Proposals for the description and measurement of carry-over effects in
clinical chemistry. Pure Appl. Chem. 1991; 63:302-306.
13. Clinical and Laboratory Standards Institute. 2006 Molecular Diagnostic Methods for
Infectious Disease; Proposed Guideline. MM3-A2. Clinical and Laboratory Standards
Institute, Wayne PA.
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