Draft Guidance for Industry and Food and Drug Administration Staff - Class II Special Controls Guidance Document: In Vitro Diagnostic Devices for Bacillus spp. Detection
This guidance document is being distributed for comment purposes only.
Document issued on: May 18, 2011.
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draft document within 90 days of publication in the Federal Register of the notice announcing the availability of the draft guidance.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 Beena Puri, Ph.D., Division of Microbiology Devices at 301-796-6202 or by email: email@example.com.
U.S. Department of Health and Human Services
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Table of Contents
- Bacillus Spp. - Background
- Premarket Notifications - Background
- Issues of Safety and Effectiveness Requiring Special Controls
- Device Description
- Performance Studies
- Specific Considerations for Bacillus spp. Devices Using Nucleic Acid Amplification
Draft Guidance for Industry and Food and Drug Administration Staff
Class II Special Controls Guidance Document: In Vitro Diagnostic Devices for Bacillus spp. Detection
This draft special controls guidance document was developed to support the proposed classification of in vitro diagnostic device for Bacillus spp. detection, a previously unclassified type of preamendments device, into class II, as recommended by the Microbiology Devices Advisory Panel on March 7, 2002.
When finalized, designation of a guidance document as a special control means that any firm currently marketing, or intending to market, in vitro diagnostic devices for Bacillus spp. detection will need to address the issues covered in the special controls guidance. The firm will need to show that its device addresses the issues of safety and effectiveness identified in the guidance, either by meeting the recommendations of the guidance or by some other means that provides equivalent assurances of safety and effectiveness.
After a final rule is made effective, the device must comply with the limitation on distribution specified as one special control in the classification regulation. (See proposed 21 CFR 866.3045(b)(2) and sections 3 and 8 of this document.)
An in vitro diagnostic device for Bacillus spp. detection is used to detect and differentiate among Bacillus spp. and presumptively identify Bacillusanthracis and other Bacillus spp. from cultured isolates or clinical specimens, as an aid in the diagnosis of anthrax and other diseases caused by Bacillus spp. This device may consist of Bacillus spp. antisera conjugated with a fluorescent dye (immunofluorescent reagents) used to presumptively identify Bacillus-like organisms in clinical specimens; or bacteriophage used to differentiate B. anthracis from other Bacillus spp. based on susceptibility to lysis by the phage; or antigens used to identify antibodies to B. anthracis in serum. This draft guidance includes recommendations for satisfying the proposed requirement of special controls for all devices of this type, including both the preamendments technologies described above and nucleic acid amplification-based B.anthracis assays, several of which FDA has determined to be substantially equivalent to other devices within this type through the 510(k) process, and thus would also be classified as class II devices and subject to special controls under the proposed classification.
Anthrax caused by B. anthracis is a disease of humans and animals. Human infections differ in clinical presentation depending on the portal of entry. The most common form is cutaneous anthrax, which results from entry through cuts or abrasions in the skin. Gastrointestinal anthrax is caused by ingestion of contaminated meat or other food products. Inhalation anthrax results from inhalation of spores and is fatal if untreated. B. cereus is a causative agent of gastrointestinal disease that is generally self-limiting; non-gastrointestinal infections also occur and are usually associated with trauma or surgery, implants, catheters and shunts.
FDA believes that special controls, when combined with general controls, will be sufficient to provide reasonable assurance of the safety and effectiveness of in vitro diagnostic devices for Bacillus spp. detection. Designation of this guidance document as a special control means that a manufacturer who intends to market a device of this type should (1) conform to the general controls of the Federal Food, Drug & Cosmetic Act (the FD&C Act), including the premarket notification requirements described in 21 CFR 807 Subpart E, (2) address the specific issues of safety and effectiveness identified in this guidance document, (3) satisfy the other special control designated in 21 CFR 866.3045(b), and (4) obtain a substantial equivalence determination from FDA prior to marketing the device.
This draft guidance document identifies the proposed classification regulation and associated product codes for in vitro diagnostic devices for Bacillus spp. detection (refer to Section 3 - Scope). In addition, other sections of this draft guidance document list the issues of safety and effectiveness and describe measures that, if followed by manufacturers and combined with the general controls, will generally address the issues associated with these devices and lead to a timely premarket notification [510(k)] review and clearance. This draft document, when final, will supplement other FDA documents regarding the specific content requirements of a premarket notification submission. You should also refer to 21 CFR 807.87 and CDRH’s Device Advice.
The scope of this document is limited to devices as described in proposed 21 CFR 866.3045, which have the following product codes:
NVQ [Bacteriophage and controls, B. anthracis lysis]
NPO [ Kit, Immunochromatographic, Bacillus anthracis differential antibody]
NRL [Enzyme linked immunoabsorbent assay, antibody, B. anthracis]
NHT [Assay, Nucleic Acid Amplification, B. anthracis]
NWZ [Gas chromatography, Bacillus anthracis membrane fatty acids]
This draft guidance is not intended to address specific issues for testing environmental samples, or for samples collected to assess exposure by the presence/absence of spores on mucosal, skin or other surfaces. Methods and approaches for specific detection of spores alone are beyond the scope of this guidance.
In the companion proposed rule, FDA identifies these devices as follows, and proposes to classify them into class II:
§ 866.3045 In vitro diagnostic device for Bacillus spp. detection.
(a) Identification. An in vitro diagnostic device for Bacillus spp. detection is used to detect and differentiate among Bacillus spp. and presumptively identify Bacillus anthracis and other Bacillus spp. from cultured isolates or clinical specimens as an aid in the diagnosis of anthrax and other diseases caused by Bacillus spp. This device may consist of Bacillus spp. antisera conjugated with a fluorescent dye (immunofluorescent reagents) used to presumptively identify Bacillus-like organisms in clinical specimens; or bacteriophage used for differentiating B. anthracis from other Bacillus spp. based on susceptibility to lysis by the phage; or antigens used to identify antibodies to B. anthracis (anti-toxin and anti-capsular) in serum. Bacillus infections include anthrax (cutaneous, inhalational, or gastrointestinal) caused by B. anthracis, and gastrointestinal disease and non-gastrointestinal infections caused by B. cereus.
In addition to this guidance document, FDA proposes that the special controls for this device include limiting distribution to laboratories with experienced personnel who have training in principles and use of microbiological culture identification methods and infectious disease diagnostics, and with appropriate biosafety equipment and containment. (See proposed 21 CFR § 866.3045(b).)
FDA has identified the risk of a false negative test result, and the risk of a false positive test result, which can lead to individual and public health consequences as issues of safety and effectiveness associated with this device that require special controls. In addition, FDA has identified as requiring special controls the health risks to laboratory workers that may be associated with handling specimens and control materials. These issues and the location of recommendations for addressing them are summarized in the table below.
To elaborate, failure of Bacillus spp. devices to perform as indicated or an error in interpretation of results may lead to misdiagnosis and improper patient management or to inaccurate epidemiological information that may contribute to inappropriate public health responses. A false positive result may lead to a medical decision causing a patient to undergo unnecessary or ineffective treatment, as well as inaccurate epidemiological information on the presence of anthrax disease in a community. A false negative result may lead to delayed recognition by the physician of the presence or progression of disease and inaccurate epidemiological information to control and prevent additional infections. A false negative result could also potentially delay diagnosis and treatment of infection caused by Bacillusanthracis or other Bacillus spp.
Exposure to organisms potentially present in test specimens and those used as control materials poses a risk of infection to laboratory workers. Consequently, FDA includes in the proposed rule as a special control that use of Bacillus spp. detection devices be restricted to laboratories with experienced personnel who have training in principles and use of microbiological culture identification methods and infectious disease diagnostics, and with appropriate biosafety equipment and containment.
In the table below, FDA has identified the issues requiring special controls generally associated with the use of in vitro diagnostic devices for Bacillus spp. detection. The measures recommended to mitigate these identified issues are given in this guidance document, as shown in the table below, and one requirement in proposed 21 CFR 866.3045(b)(2). We recommend that you also conduct a risk analysis, prior to submitting your premarket notification, to identify any other risks specific to your device. The premarket notification should describe the risk analysis method. If you elect to use an alternative approach to address a particular risk identified in this guidance document, or have identified risks additional to those in this document, you should provide sufficient detail to support the approach you have used to address that risk.
|Identified issue||Recommended / Required mitigation measures|
|A false negative test result may lead to delay of therapy and progression of disease and epidemiological failure to promptly recognize disease in the community||Sections 5- 8|
|A false positive test result may lead to unnecessary treatment and incorrect epidemiological information that leads to unnecessary prophylaxis and management of others||Sections 5-8|
|Biosafety and risks to laboratory workers handling test specimens and control materials||Sections 8 ;21 CFR 866.3045(b)(2)|
Key elements of a 510(k) submission are the intended use, the type of specimens tested, and the technological characteristics of your device. Additionally, you should also identify the regulation, the product code, and a legally marketed predicate device that you will compare with your device. In order to help FDA review your submission efficiently, we recommend that you include a table outlining similarities and differences between the predicate and your device. We encourage you to reference appropriate peer-reviewed articles that support the use of your device for its intended diagnostic use and the specific test principles incorporated into the device design. We recommend that you describe each of these device elements in detail.
Furthermore, we recommend that you include the following descriptive information to adequately characterize your device for the detection of Bacillus spp.:
Your 510(k) must include labeling that describes the intended use of your product (see 21 CFR 807.87(e)). Your 510(k) should specify what the assay measures, for example, Bacillus anthracis cell surface protein or target DNA sequences from specific Bacillus anthracis plasmids (See 21 CFR 807.87(e) and 21 CFR 808.92(a)). The clinical indications for which the test is to be used, and the specific population for which the test is intended should be clearly stated. You should include clinical and demographic description of patients (e.g. gender, age, symptoms) for whom clinical performance has been demonstrated. The intended use should specify whether the test is qualitative or quantitative (see 21 CFR 807(e) and 21 CFR 807.92(a)). You should ensure that all elements of the intended use are clearly stated, including specific conditions of use such as type of specimens to be tested, for example, whole blood collected in sodium citrate from individuals suspected of having anthrax, positive blood cultures, or cultured organisms grown on blood agar
You must also prominently provide the following statement immediately below your intended use: “For use in laboratories with experienced personnel who have training in principles and use of microbiological culture identification methods and infectious disease diagnostics, and with appropriate biosafety equipment and containment.” 21 CFR 866.3045(b)(2)
Reagents and other device components
You must describe reagents and other device components in your 510(k) (see 21 CFR 807.87 (e) and 809.10). We recommend that you follow general guidance provided in other FDA guidance documents.
Testing Procedures using your device
In your 510(k), you should describe, in detail, the principles of operation applicable to your device for its intended use. We recommend that you specifically describe testing conditions, procedures and controls designed to provide safeguards for conditions that can cause false positive and false negative results, or present a biosafety hazard. These include, but are not limited to, procedures, methods, and practices incorporated into your directions for use (see Section 8 - Labeling) to mitigate risks associated with testing [Ref. 1].
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 a transport medium 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 the transport medium.
Interpreting Test Results/Reporting
In your 510(k), we recommend that you describe how presumptive positive, equivocal, and negative results are determined and how they should be interpreted, if applicable. You should provide clear explanations for how interpretative algorithms have been determined.
We recommend that you evaluate the following performance characteristics, in order to document performance and properly label your device in conformance with 21 CFR 809.10(b)(12). You should describe the studies in your 510(k) and clearly summarize results, preferably in tabular form where applicable. Relevant findings in published literature may be cited. Specific additional guidance for devices used in molecular diagnostic test methods and for devices used in immunological test methods can be found in CLSI I/LA 18-A2 Section11 [Ref. 2].
In order to review data from your studies, we recommend that you provide specific details of the study protocols used to generate the data. These specifics are also helpful to aid users in interpreting performance data in your labeling. When referring to Clinical Laboratory Standards Institute (CLSI) protocols or guidelines, we recommend that you indicate which specific aspects of the protocols or guidelines you followed.
A. Analytical/Laboratory Performance Studies
The appropriate types of analytical studies will depend on the applied technology, principles of operation and scientific evidence available. The following are some pertinent examples. These are not intended as an all-inclusive list. Additional types of analytical data may be appropriate, depending on the device type.
(1) Determination of assay/reagent specificity
For devices used to identify Bacillus anthracis from cultures, we recommend that you characterize assay performance for bacteriophage and immunofluorescent antibody reagents using at least 25 different strains of Bacillus anthracis (representing geographic and temporal diversity, including known genotypic and phenotypic variants), 10 strains of Bacillus thuringiensis, and 10 strains of Bacillus cereus, along with other representative Bacillus spp. and non-Bacillus Gram positive rods. If your device is indicated for direct specimen testing methods, you should also include organisms that could be expected to be found at the sampling site (e.g., both normal flora and other potential pathogens). Strains used for characterization of assay/reagent specificity can be selected from well-characterized archives or repositories. Definitive species identification of Bacillus spp. may call for a combination of phenotypic and genotypic methods (e.g., biochemical, antigenic, morphology, plasmid characterization, genotyping).
We recommend that you evaluate antigens for anti-Bacillus anthracis antibody testing using human sera from naturally infected humans and those immunized with anthrax vaccine. Sera from at least 200 human samples, including those from individuals with compatible diseases and conditions, should also be tested for specificity.
We recommend that you characterize within-day, day-to-day, intra-laboratory, and inter-laboratory reproducibility. The reproducibility panel should consist of samples around the cutoff of the assay, at low and moderate positive concentrations, and at high negative concentrations. If results of your device are interpreted visually, you should also conduct precision studies with all instruments recommended for use with your device. [Ref. 3].
(3) Interfering/inhibitory substances
We recommend that you provide information and data to demonstrate that potentially interfering or inhibitory substances encountered in specific specimen types do not affect results. See Table 1 for a list of potentially endogenous and exogenous interfering substances that could be present in clinical specimens, e.g. blood, sputum, culture, etc. If interference or inhibition has been reported in the literature or is evident in your studies, you should provide validated procedures or methods that can be used to avoid erroneous results.
Table 1. List of Evaluated Potentially Interfering Substances
- Endogenous Substances
- Cholesterol (total)
- Exogenous Substances
- Ascorbic acid
- Gentamicin sulfate
- Naproxen sodium
- Citrate (sodium)
- Sodium polyanethol
- sulfonate (SPS)
- Albuterol (Salbutamol)
- Cromolyn sodium
- Flunisolide (Flovent®)
- Blood culture media
- Sheep blood agar media
- Technique-specific Substances
- Snap n’ Digest
- IT 1-2-3™ kit Buffer 1
- IT 1-2-3™ kit Buffer 1A
- IT 1-2-3™ kit Buffer 1B
- IT 1-2-3™ kit Buffer 1C
- IT 1-2-3™ kit Buffer 2
- QIAGEN Buffer AL
- QIAGEN Buffer AW1
- QIAGEN Buffer AW1 (w/o EtOH)
- QIAGEN Buffer AW2
- QIAGEN Buffer AW2 (w/o EtOH)
*These are solvents used to dissolve potentially interfering substances in preparation for testing.
Your studies should include the effect of culture age and growth media with specific antibody and nucleic acid reagent testing from cultures (solid or liquid). The results should be included in the package insert in the Performance Characteristics section and also as a Limitation statement to inform the user that cultures older than a specified number of days may result in a false negative result.
(4) Effect of culture inoculation density on results with bacteriophage reagents (bacteriophage assays using culture plating methods)
We recommend that you conduct studies to demonstrate whether there is a risk of a false negative test result due to heavy culture inoculation when using bacteriophage-specific assays. One technique may be to streak a suspect culture for isolation and add bacteriophage to areas of the plate with varying amounts of inoculum. You should also assess phage titer and stability of bacteriophage reagents.
B. Clinical Information
This guidance addresses clinical specimens collected from individuals with suspected infection, for the detection of vegetative forms and spores. However, it is unlikely that only spores would be present in clinical specimens (with the possible exception of cutaneous lesions).
You should provide information to demonstrate the reliability of your device for detecting Bacillus spp. in each type of clinical specimen that you indicate as suitable for testing with your device. In general, when the number of human clinical samples available for clinical testing is very low or non-existent, the available evidence for FDA's premarket review may, of necessity, be obtained from analytical rather than clinical studies; spiked human samples and/or animal samples may be adequate. In this circumstance, it is particularly critical to have well designed analytical studies. Animal studies are optional and can be used to supplement analytical studies where appropriate. Performance assessments should be relative to the known presence or absence of a characterized Bacillus spp., or to the definitive identification of culture growth. Multiple tests and methods may be needed to appropriately identify/characterize a Bacillus spp. recovered from human specimens by culture methods or detected directly in human specimens.
We recommend that you also provide data from testing specimens from the intended use population (e.g., patients with febrile illnesses or skin lesions). Because anthrax would not be expected in a prospective evaluation, these data should not be represented as specificity, but rather as agreement with an expected negative result.
For devices used to identify culture isolates or growth, clinical evaluations are not applicable, as long as studies with culture stocks reasonably represent fresh culture growth and conditions for testing (e.g., 12-18 h growth from 5% sheep blood agar plates).
IVD devices for Bacillus spp. detection, like other devices, are subject to statutory requirements for labeling (the FD&C Act, Sections 502(a), 201(n); 21 USC §§ 352(a), 321(n)). Labeling for these devices must provide adequate directions for use and adequate warnings and precautions. (Section 502(f); 21 USC § 352(f)). Specific labeling requirements for all IVD devices are set forth in 21 CFR 809.10.
The premarket notification must include labeling in sufficient detail to satisfy the requirements of 21 CFR 807.87(e). Final labeling for an in vitro diagnostic device must comply with the requirements of 21 CFR 809.10 before being introduced into interstate commerce; however, final labeling is not required for 510(k) clearance.
To ensure compliance with section 502 of the FD&C Act and 21 C.F.R. 809.10, FDA recommends that labeling for IVD devices for Bacillus spp. detection address the items identified below. These labeling recommendations also help to mitigate the risks identified previously in this guidance, and thus help to ensure safe and effective use of these devices.
A. Intended Use
A clear intended use statement is critical. We recommend that you incorporate into the intended use statement the intended specimen type(s), whether the testing to be performed is qualitative, semi-quantitative, or quantitative, the testing methodology, along with the indicated patient population and other conditions for use.
- You should also prominently provide the following statement immediately below your intended use:
“For use in laboratories with experienced personnel who have training in principles and use of microbiological culture identification methods and infectious disease diagnostics, and with appropriate biosafety equipment and containment.”
B. Directions for Use
We recommend that you address the following in the directions for use:
- Emphasize appropriate storage conditions for reagents, and identify reagents that are temperature, humidity, and/or light sensitive.
- When testing requires the culture of Bacillus spp., specify the appropriate type of culture media from which growth is to be tested, incubation conditions, and length of incubation (including minimum and maximum incubation times).
- Provide directions for using control reagents provided with the product, as well as required or recommended control materials that may be used but are not provided. Describe each aspect of the testing procedure that is controlled. Provide acceptable values for the control reagent testing and justification for your selected values.
- Provide guidance for biosafety precautions with specimen handling and testing procedures. Specify at which procedural step the test sample is non-infectious.
- For products that rely on antigen/antibody reactions in the testing procedure, include recommendations for reducing the risk of a false negative test result due to prozone or Hook effect.
We recommend that you include the following type of statement in the Precaution Section of the package insert: “The interpretation of test results requires experienced clinical personnel who have training in principles and use of microbiological culture identification methods and infectious disease diagnostics and have the necessary awareness to report an identification of B. anthracis and coordinate with local or state public health directors.”
D. Interpretation and Reporting of Assay Results
We recommend that you include the following:
- We recommend that you define each of the possible testing results: e.g., positive, equivocal or indeterminate, and negative. You should also describe how the operator should interpret these test results, and give acceptance/rejection criteria for controls. Include suggestions for how to proceed if control results are not acceptable.
- We recommend that you provide clear and exact criteria for evaluating a test result as positive, negative, or equivocal/indeterminate. We recommend using photographs and/or diagrams to indicate how to interpret results for tests that give a qualitative result. Additionally, we recommend that you provide the likelihood of a B. anthracis identification (for culture identification reagents), the likelihood of infection (for reagents detecting specific anti-B. anthracis antibodies), or the likelihood of B. anthracis presence (for reagents detecting B. anthracis directly in patient specimens), based on reliable information available from the literature or other sources.
- For tests that rely on antigen/antibody interactions to detect bacterial cell components or bacterial products, you should include recommendations for reducing the risk of a false negative test result due to non-optimal initial inoculum density.
- Testing to detect intact bacteria or bacterial products may give different results with samples that contain spores versus those that contain vegetative cells only, or a mixture of spores and vegetative cells. Your labeling should describe results, and use photographs and diagrams as needed, to show results that can be expected if a sample contains spores.
- For tests to detect an antibody response to Bacillus anthracis, you should include a warning statement concerning the interpretation of a positive test and how it does not by itself conclusively establish recent infection, as persons immunized with an anthrax vaccine may test positive with this test in the absence of any natural exposure to Bacillus anthracis. Labeling should also indicate that antibiotic treatment early in natural infection with Bacillus anthracis may decrease the antibody response and therefore may give a negative result with this test.
Your labeling should include a statement that Bacillus anthracis is a Nationally Notifiable Disease that must be reported to public health authorities in accordance with state and local law. Users should verify reporting requirements in their area, and notify their state or local public health laboratory, the Centers for Disease Control and Prevention, and any other Agency specified by their accreditation guidelines, if Bacillus anthracis or anthrax is suspected.
E. Performance Characteristics
We recommend that you include in the package insert a summary of the study designs and the results of the studies described in Section 6 that would aid users in interpreting test results. This includes clinical and analytical performance characteristics. If the assay was not evaluated using specimens from individuals presenting with signs and symptoms of anthrax, users should be instructed to establish the clinical sensitivity of this test on prospectively collected clinical specimens as these specimens become available. Data for negative agreement/clinical specificity should be included and indicated clearly to the users.
Bacillus spp. detection devices that employ Nucleic Acid Amplification are used to determine the presence of pathogenic Bacillus spp. directly in human specimens and/or blood or colony cultures derived from clinical specimens by detecting nucleic acid sequences or regions that are unique to Bacillus spp. and which discriminate Bacillus spp. pathogen from other microbial organisms. These devices include primers, probes, enzymes and specific controls for amplification and are designed for use in specific instrument systems. Detection of B. anthracis by a n ucleic acid amplification detection system aids in the definitive identification of B. anthracis in infected patients in conjunction with other laboratory results and clinical presentation. The following are specific considerations applicable to this type of IVD for Bacillus spp. detection.
A. Reagents and other device components
For Nucleic Acid Amplification- based devices that are intended for presumptive detection of B.anthracis DNA in human specimens or blood or colony cultures or liquid culture derived from clinical specimens, we recommend that you describe design requirements for your device that address or mitigate risks of false negative or false positive results associated with primers, probes, instruments, and controls used in a nucleic-acid test procedure to detect targeted DNA segments from B. anthracis. Some examples are given below:
- Designing your freeze-dried sets of reagents, or any other closed tube test system (e.g., self containing cartridge), to minimize false positives due to contamination or carryovers.
- Designing one or more than one assay for targeting different DNA sequences unique to B.anthracis .
- Developing positive, negative, and inhibition controls to ensure accurate test results.
- Developing methods for extraction and purification that yield suitable quality and quantity of DNA from human specimens or in blood or colony cultures or liquid culture derived from clinical specimens for use in the test system with your reagents.
- Optimizing your reagents and test procedures for recommended instruments.
- Illustrations or photographs of any non-standard equipment or methods, if applicable.
In your 510(k), you should provide performance information supporting the conclusion that your design requirements have been met. We recommend that you provide the rationale for selection of specific DNA target sequences and selection of primers and probes (See Section 6 – Performance Studies)
The specific extraction method recommended for each specimen type should be listed by name and catalog number in the package insert of your device (See Section 7 – Labeling).
B. Testing Procedures
We recommend that you include the following information in your 510(k) application:
- Overall design of the testing procedure, including control elements incorporated into the recommended testing procedures. These controls should approximate the lower range of clinically relevant Bacillus DNA levels and should be extracted as a clinical sample.
- Development, description, or recommendations for additional external or internal positive and negative controls that monitor for contamination and extraction efficiency (e.g., an internal control, as a control for nucleic acid extraction and inhibition). Features and additional controls that reduce failure to recognize procedural errors or factors (e.g., degradation of master mix) that adversely affect amplification and detection conditions.
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. You may contact OIVD’s Division of Microbiology Devices at FDA for further information regarding controls. For devices based on nucleic acid technology, we generally recommend that you include the following types of controls:
(1). Negative Controls
Blank or no template control
The blank, or no-template, control contains buffer or sample transport media and all of the assay components except nucleic acid. This control is used to rule out contamination with target nucleic acid or increased background in the amplification reaction. It may not be needed 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 (other than B. anthracis). 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:
- Patient specimen from a non - B. anthracis infected individual
- Samples containing a non-target organism
(2) 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:
- Patient specimen from a B. anthracis infected individual or spiked matrices with live B. anthracis
- B. anthracis culture isolates
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. It controls the integrity of the sample and the reaction components when negative results are obtained. It indicates that the target is detected if it is present in the sample.
(3) 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 amplification inhibitors in the samples. Examples of acceptable internal control materials include human nucleic acid co-extracted with the B. anthracis DNA and primers amplifying human housekeeping genes (e.g., RNaseP, β-actin). The need for this control is determined on a device case-by-case basis [Ref. 4].
D. Performance Studies
For studies intended to determine the performance characteristics of a nucleic acid amplification-based device, we recommend that you include in the 510(k) application the information described below. This section complements the recommendations for performance studies described earlier in this document.
(1) Nucleic acid extraction
Different extraction methods may yield B. anthracis DNA of varying quantity and quality, and therefore the extraction method can be crucial to a successful result. Purification of B. anthracis DNA from the clinical specimens or liquid blood culture or colony culture specimens can be challenging because biological samples may contain low bacterial loads in the background of human genomic DNA, as well as high levels of proteins and other contaminants.
For these reasons, we recommend that you evaluate the effect of your chosen extraction methods on the performance of the assay with respect to satisfactory B. anthracis DNA quantity and quality for the intended use of the assay. In addition, you should evaluate your assay’s analytical and clinical performance characteristics using the entire analytical process (including extraction procedures) that you recommend for use with your assay. This should include demonstrating the Limit of Detection (LoD) and reproducibility of your assay with each extraction procedure. Recommendations for conducting the LoD study are provided under “ Limit of Detection” (See Section V below – Analytical Sensitivity). In addition, external site studies (including reproducibility and clinical studies) should include the extraction procedures prescribed in your labeling.
We recommend that you perform these evaluations whether you intend to actually provide reagents in your test kit for extraction and preparation of nucleic acid, or whether you simply instruct users concerning appropriate reagents.
If you recommend or include multiple extraction methods, you should demonstrate the LoD and reproducibility for each method. With the assumption that the extraction method introduces minimum variability to the overall assay performance you may be able to combine the extraction method variable with each site performance variable. For example, if you recommend three different extraction methods, you can design a reproducibility study by evaluating one of the three extraction methods at each testing site: test extraction method A at site 1, method B at site 2, and method C at site 3. If the results generated from the test panel mentioned above do not show significant differences, no further reproducibility studies are needed. However, if the initial extraction equivalency studies from the three sites indicate statistically significant differences in assay performance, the reproducibility study should be expanded to include testing each extraction method at three study sites (e.g., site 1 extraction method A, B, and C, site 2 extraction method A, B, and C, and site 3 extraction method A, B and C).
In addition to the analytical studies (LoD and Reproducibility), each extraction method should be utilized in at least one clinical site during the clinical trials to generate clinical performance data. If results from the expanded reproducibility testing indicate a significant difference in efficiency among the extraction methods, the data from each clinical testing site (using a different nucleic acid extraction method) are not considered equivalent and should not be pooled, but rather analyzed separately. As a result, additional prospective clinical samples may be included in order to support the claimed extraction method.
(2) Assay Cut-off
We recommend that you explain how the cut-off(s) was determined (see also Section IV below Interpreting Test Results/Reporting) as well as how it was validated. The cut-off should be determined using appropriate statistical methods. To support the cutoff you determined you may provide for example, a result distribution, 95 th and 99 th percentiles, percents of the non-negative (positive or equivocal) results, and so on, for the clinical samples without any B. anthracis DNA in your pilot studies. Selection of the appropriate cut-off can be justified by the relevant levels of sensitivity and specificity based on Receiver Operating Curve (ROC) analysis of the pilot studies with clinical samples (for details about ROC analysis, see CLSI document GP10-A [Ref. 5]). If the assay has an equivocal zone, you should explain how you determined the limits of the equivocal zone. The performance of your device using the pre-determined cut-off (and equivocal zone, if applicable) should be validated in an independent population consistent with the defined intended use of your device.
(3) Interpreting Test Results/Reporting
We recommend that you describe how presumptive positive, negative, equivocal, or invalid results are determined and how they should be interpreted, if applicable. We recommend that you clearly explain how interpretative algorithms have been determined.
- We recommend that you provide the cut-off value for defining a negative result of the assay. If the assay has only two output results (negative/positive), this cut-off also defines a positive result of the assay.
- If the assay has an equivocal zone, we recommend that you provide cut-off values (limits) for the equivocal zone.
- If your interpretation of the initial equivocal results requires re-testing, we recommend that you provide (1) a recommendation whether re-testing should be repeated from the same nucleic acid preparation, a new extraction, or a new patient specimen, and (2) an algorithm for defining a final result by combining the initial equivocal result and the results after re-testing (note that this algorithm should be developed before the pivotal clinical study that evaluates the clinical performance of the assay).
- If one of the reported outputs of your assay can be an equivocal result, we recommend that you provide the interpretation and recommendation for how the user should follow up the equivocal results.
- If the assay has an invalid result, we recommend that you describe how an invalid result is defined. If internal controls are part of the determination of invalid results, we recommend that you provide the interpretation of each possible combination of control results for defining the invalid result. You should provide recommendations for how to follow up any invalid result, i.e., whether the result should be reported as invalid or whether re-testing is recommended. If re-testing is recommended, you should provide information similar to that for re-testing of equivocal results (i.e., whether re-testing should be repeated from the same nucleic acid preparation, a new extraction, or a new patient specimen).
(4) Analytical Sensitivity (Limit of Detection)
We recommend that you determine the limit of detection (LoD) of your assay at the preclinical stage using approaches described in CLSI EP17-A [Ref. 6]. You should determine the lowest level of B. anthracis detection in appropriate specimen types using your device. The study should include testing serial dilutions of viable (live) B. anthracis in replicates of 3-5. Each dilution should be made using B. anthracis negative pooled human samples such as blood or sputum or an equivalent matrix. We recommend that you report the LoD as the level of B. anthracis that gives a 95% detection rate. Based on the titration results, the LoD may be further confirmed by preparing at least 50 additional replicates at the LoD concentration and demonstrating that B. anthracis was detected 95% of the time. The LoD should be correlated to CFU/ml and DNA copy numbers present in B. anthracis . The lowest level of B. anthracis DNA specimen should be close to the LoD concentration and correlated to CFU/mL and DNA copy numbers.
(5) Carry-Over and Cross-contamination Studies (for multi-sample assays and devices that require instrumentation.)
We recommend that you 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. We recommend that you perform at least 5 runs with alternating high positive and high negative 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. We recommend that the high negative samples contain the analyte concentration below the cut-off such that repeat testing of this sample is negative approximately 95% of the time. 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%.
We recommend that you test the effects of potentially endogenous interfering substances encountered in blood, or human specimens or colony cultures or liquid culture derived from clinical specimens and exogenous interfering substances that could be introduced during sample purification or reaction set-up. These interfering substances may interfere with assay performance. The tabulated data for the evaluated endogenous and exogenous interfering substances for your device should be included in the submission. The concentration of each substance tested should be represented at a relevant concentration in accordance with CLSI EP7-A2 [Ref. 7].
We recommend that you conduct within-laboratory precision studies for devices that include instruments or automated components. You should characterize within-day, day-to-day, intra-laboratory, and inter-laboratory precision. As a general guide, we recommend the following protocol for a nucleic acid amplification assay:
- We recommend that you perform reproducibility studies at three sites (two external, one in-house site).
- We recommend that you use a five day testing protocol, including at a minimum two runs per day, unless the assay design precludes multiple runs per day, and three replicates of each panel member per run.
- We recommend that you have at least two operators each day at each facility perform the test.
We recommend that you prepare reproducibility panels by spiking each matrix (e.g., blood, or human specimens or colony cultures or liquid culture derived from clinical specimens ) with B. anthracis at low (near LOD), medium, or high level. We recommend that negative sample panels be unspiked specimens for each matrix.
Each panel should consist of 6-9 samples that include three levels of analyte as described below:
- A “high negative” sample with a concentration of analyte below the clinical cut-off so that retesting of this sample should be negative approximately 95% of the time.
- A “low positive” sample with a concentration of analyte just above the clinical cut-off so that repeated test results should be positive approximately 95% of the time.
- A “moderate positive” sample with a concentration that one can anticipate positive results approximately 100% of the time.
You may refer to the CLSI document EP15-A2 [Ref. 8], EP5-A2 [Ref. 3], and EP12-A2 [Ref. 9] for guidance on reproducibility study design.
- Clinical and Laboratory Standards Institute (CLSI). Procedures for the Handling and Processing of Blood Specimens; Approved Guideline—Third Edition. CLSI document H18-A3 (ISBN 1-56238-555-0). CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.
- Clinical Laboratory Standards Institute (CLSI). Specifications for Immunological Testing for Infectious Diseases; Approved Guideline – Second Edition. CLSI document I/LA18-A2 (ISBN 1-56238-445-7) CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2001.
- Clinical and Laboratory Standards Institute (CLSI). Evaluation of Precision Performance of Quantitative Methods; Approved Guideline-Second Edition. CLSI document EP5-A2 (ISBN 1-56238-000-0). CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.
- Clinical and Laboratory Standards Institute (CLSI). Molecular Diagnostic Methods for Infectious Disease; Approved Guideline. CLSI document MM3-A2 [ISBN 1-56238-596-8] Clinical and Laboratory Standards Institute, Wayne, Pennsylvania 19087-1898 USA , 2006.
- Clinical and Laboratory Standards Institute (CLSI). Assessment of the Clinical Accuracy of Laboratory Tests Using Receiver Operating Characteristics (ROC) Plots; CLSI document GP10-A Approved Guideline: Clinical and Laboratory Standards Institute; Wayne, Pennsylvania 19087-1898 USA, 1995).
- Clinical Laboratory Standards Institute (CLSI). Protocols for Determination of Limits of Detection and limits of Quantitation; Approved guideline – CLSI document EP17-A (ISBN 1-56238-551-8) CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.
- Clinical Laboratory Standards Institute (CLSI). Interference Testing In Clinical Chemistry Approved Guidelines – Second Edition. CLSI document EP7-A2 (ISBN 1-56238-000-0) CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2005.
- Clinical Laboratory Standards Institute (CLSI). User Verification Of Performance For Precision And Trueness Approved Guidelines – Second Edition. CLSI document EP15-A2 (ISBN 1-56238-000-0) CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2005.
- Clinical and Laboratory Standards Institute (CLSI). User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline. CLSI document EP12-A (ISBN 1-56238-468-6). CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2002.