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FDA is issuing this draft guidance to provide industry and agency staff with recommendations for studies to establish the analytical and clinical performance of in vitro diagnostic devices (IVDs) intended for the detection of methicillin-resistant Staphylococcus aureus (MRSA) using culture based methods . These devices are used to aid in the prevention and control of MRSA infections in healthcare settings.
This guidance provides detailed information on the types of studies FDA recommends in order to support Class II premarket submissions for these devices. The guidance includes examples of MRSA strains recommended for analytical sensitivity and inclusivity studies and examples of microorganisms recommended for analytical specificity studies.
This document is limited to studies intended to establish the performance characteristics of devices that detect MRSA by growth in culture media or those devices that test for the protein, penicillin-binding protein 2a (PBP2a or PBP2'), expressed by the mecA gene. This includes culture-based devices that use selective or chromogenic media. The document does not address the detection of serological response from the host to the MRSA antigens, nor does it address establishing the performance of non-MRSA components of multi-analyte or multiplex nucleic acid based devices.
FDA's guidance documents, including this guidance, do not establish legally enforceable responsibilities. Instead, guidances describe the Agency's current thinking on a topic and should be viewed only as recommendations, unless specific regulatory or statutory requirements are cited. The use of the word should in Agency guidances means that something is suggested or recommended, but not required.
This document recommends studies for establishing the performance characteristics of invitro diagnostic devices for the detection of MRSA, including those for the detection or detection and differentiation of MRSA versus Staphylococcus aureus (SA) in either human specimens or bacterial growth detected by continuous monitoring blood culture systems . FDA believes that these recommended studies will be relevant for Class II premarket submissions (e.g., 510(k) or de novo classification petition) that may be required for a particular test.
A manufacturer who intends to market an in vitro diagnostic device for detection of MRSA must conform to the general controls of the Federal Food, Drug, and Cosmetic Act (the FD&C Act) and, obtain premarket clearance or approval prior to marketing the device (sections 510(k), 513, 515 of the FD&C 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 CDRH's device advice webpage, " Premarket Notification 510(k) ." Guidance on the content and format for abbreviated and traditional 510(k) s is available in the guidance entitled " Format for Traditional and Abbreviated 510(k)s ."
Further information on device testing can be found in the guidance entitled " In Vitro Diagnostic (IVD) Device Studies – Frequently Asked Questions ," and the guidance entitled " Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable ."
As previously described, this document recommends studies for establishing the performance characteristics of in vitro diagnostic devices for the detection of MRSA, including those for the detection or detection and differentiation of MRSA versus SA from either human specimens or bacterial growth detected by continuous monitoring blood culture systems. This document is limited to studies intended to establish the performance characteristics of devices that detect either MRSA by growth in culture media or those devices that test for the protein, PBP2a or PBP2, expressed by the mecA gene. This document includes culture-based devices that use selective or chromogenic media. This guidance does not address the detection of serological response from the host to the MRSA antigens, nor does it address establishing the performance of non-MRSA components of multi-analyte or multiplex nucleic acid based devices.
The scope of this document is limited to the following devices as described in existing classifications, as indicated below. The recommendations contained in this guidance may also assist manufacturers of future MRSA diagnostic devices that may not fall within these existing classifications in complying with requirements that apply to their devices, including devices that will be subject to requests for initial classification under section 513(f)(2) of the FD&C Act ("de novo classification"), as well as subsequent devices that seek determinations of substantial equivalence to future de novo cleared devices. Manufacturers of such devices should contact FDA for clarification about how the recommendations contained in this guidance apply to their device.
The following are existing MRSA detection IVD classification regulations, which are assigned to product codes JSO and MYI respectively:
Your premarket notification must include a device description that meets the requirements of 21 CFR 807.87(a) and (f). You should also identify your device by regulation and product code and identify a legally marketed predicate device. In order to quickly view all aspects of your device compared with the predicate, you should include a table that outlines the similarities and differences between the predicate and your device.
MRSA is a type of bacteria that causes staphylococcal infections which are resistant to treatment with usual antibiotics. The infection occurs most frequently among patients who undergo invasive medical procedures or who have weakened immune systems and are being treated in hospitals and healthcare facilities such as nursing homes and dialysis centers. In healthcare settings, it commonly causes serious and potentially life threatening infections, such as bloodstream infections, surgical site infections, or pneumonia.
The incidence of infections caused by MRSA has increased over the last decade in healthcare settings in the United States . According to the Centers for Disease Control and Prevention (CDC) data, the proportion of infections that are antimicrobial resistant has been growing.
Failure of devices for detection and differentiation of MRSA and SA to perform as expected or failure to correctly interpret results may also lead to inappropriate infection control responses. In the context of infection control in healthcare settings, a false negative report could lead to delays in initiating (or failure to initiate) infection control and prevention measures. A false positive report could lead to 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 nucleic acid-based MRSA/SA detection and differentiation devices are the basis for determining the safety and effectiveness or substantial equivalence of these devices.
One of the challenges that culture methods face is the accurate detection of oxacillin/methicillin resistance due to a phenomenon called heteroresistance. This is the presence of two subpopulations (one susceptible and the other resistant) that may coexist within a culture of staphylococci (2). All cells in a culture may carry the genetic information for resistance, however, only a small number may express the resistance in vitro. This occurs in staphylococci resistant to penicillinase-stable penicillins, such as oxacillin. Cells expressing heteroresistance grow more slowly than the oxacillin-susceptible population and may be missed at temperatures above 35ºC or less than 24 hour incubation. 2
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 MRSA detection devices are the basis for determining the substantial equivalence of these devices.
When conducting the performance studies described below, we recommend that you run appropriate external controls each day of testing for the duration of the analytical and clinical studies. Specific information about controls is provided in the section below. You may contact the Division of Microbiology Devices within the Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD) at FDA for further information.
B. Performance Studies
In your 510(k) you should provide detailed descriptive information on the studies you conducted to establish each of the performance characteristics outlined below. We recommend that you conduct prospective clinical studies to determine performance of your device. In general, for the clinical studies and the analytical precision studies, we recommend that you conduct testing at three sites, representative of your intended user sites (e.g., clinical laboratory sites). You should evaluate your assay performance for each specimen type that you recommend for your assay.
In order to accurately interpret acceptance criteria and data summaries during the FDA review, we recommend that you provide appropriate specific information concerning testing protocols. This is also important to aid users in understanding the information in your labeling. For example, when referring to Clinical and Laboratory Standards Institute (CLSI) protocols or guidelines, we recommend that you indicate which specific aspects of the testing protocols or guidelines were followed.
We recommend that you contact the Division of Microbiology Devices in the Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD) to obtain feedback regarding your planned study and the clinical claims you intend to support.
1. Analytical Sensitivity
You should test a minimum of 50 selected MRSA/SA strains representing those commonly encountered in the community and worldwide. For this study, we recommend that you obtain isolates from the American Type Culture Collection (ATCC), Center for Disease Control and Prevention (CDC) or Network on Antimicrobial Resistance in Staph aureus (NARSA). Please include genotypic (i.e. pulse-field gel electrophoresis), or phenotypic information such as exact Minimum Inhibitory Concentration (MIC) values in µg/mL for each organism tested and the geographic origin of the strain. The bacterial concentrations tested should be at levels at or near the lowest percent recovery of your device. All MRSA isolates, subtypes, or group designations and concentrations should be confirmed.
You may also enhance your study using in-house isolates, but these isolates should not comprise more than 50% of the total number tested. In-house isolates should be well characterized and the information on characterization provided in your submission. When using in-house isolates, you should provide detailed characterization to include MIC results and pulsed-field gel electrophoresis ( e.g., USA100, USA200, USA300, USA400, and other types encountered in community or healthcare facilities).
We recommend that you include the virulent strain USA300-114.
The following table includes examples of organisms that should be included in an Analytical Reactivity study. The list may not include all known MRSA isolates.
Table 1. Examples of Organisms to be in Included in the Analytical Reactivity Study
Expression of Resistance
a) We recommend that you demonstrate the expression of resistance for at least 10 high level and low level resistant MRSA strains on your new device and compare to a nonselective sheep blood agar plate. Strains used in the study should be recommended by CLSI or be well recognized in peer reviewed publications. Your study should include MIC values, mecA, and class (homogenous or heterogenous) of each strain tested and you should also include positive and negative controls.
In a request by industry, we were asked how they might provide this information. Table 2 is a format that you can use as an aide.
Table 2. Table Format for Presentation of the Expression of Resistance Study.
b) Borderline Oxacillin Resistant Staphylococcus aureus (BORSA)
BORSA strains usually lack the mec A gene but express resistance to oxacillin by another resistance mechanism. The MIC for Oxacillin in such strains is just above the susceptible breakpoint.
We recommend that you evaluate the performance of your new device with at least five BORSA isolates. The results should be presented in a tabular format.
c) There are other mechanisms of methicillin resistance such as production of modified intrinsic PBPs (modified S. aureus (MOD-SA) strains) with altered affinity for oxacillin; however, the occurrence of MOD-SA is rare.5 If these strains are available for testing, they should also be included in the Challenge Panel. Alternatively, you may include a limitation statement in your labeling if MOD-SA strains are not available for testing.
Mixed Infection Study
You should also perform a mixed infection study to evaluate the performance of your device in recovering MRSA in the presence of high levels of non-target organisms by using one Gram positive and one Gram negative isolate (e.g., samples of both MRSA and methicillin susceptible SA, or MRSA and methicillin resistant coagulase negative Staphylococcus or MRSA and E. coli) . You should test your target organism, i.e. MRSA, at the percent recovery rate and non target organisms at increasing concentrations and present the data. In a request by industry, we were asked how they might provide this information. Table 3 is a format that you can use as an aide .
Table 3. Mixed Infection Study Table
We recommend the following protocol for your recovery study
The recovery study should include at least six serial dilutions to obtain organism concentrations approximately equivalent to 106, 105, 104, 103, 102 and 10 CFU/mL. Aliquots of each dilution should be plated on at least two plates of the new media and on the Sheep Blood Agar Plate (BAP), which is used as a control. Calculate percent recovery per media by averaging the number of CFUs obtained from the two plates per dilution.
In a request by industry, we were asked how they might provide this information. Table 4 is a format that you can use as an aide.
Table 4. Table Format for Presentation of Recovery Study.
2. Analytical Specificity
You should evaluate the performance of your device against strains which are phylogenetically related to SA or potentially encountered in the sample type that is being claimed under the intended use or in the hospital environment. Streak for isolation in order to observe colonial morphology and color intensity of isolated colonies. In addition, the bacterial concentrations tested should be at levels of 106 CFU/mL or higher. We recommend that you confirm organism identities and concentrations on a non-selective agar media plate.
We understand that there may be organisms that are difficult to obtain for testing. In this case, you may include as a limitation in your package insert that these organisms were not available for testing.
You should test potentially interfering substances, i.e. exogenous and endogenous that may be encountered in your sample type. We recommend that you include a BAP to serve as a control. In addition, you should also consider diminished growth as another indicator of interference.
Examples of commonly encountered interfering substances in sample types such as anterior nares, blood, and skin and soft tissue infections are found in Tables 5-7. These are not exclusive lists.
In addition, we recommend that you test various blood culture bottle types, swabs, and transport media to determine if they will support the growth of your target organism. Alternatively, you may provide a limitation in your package insert for those blood culture bottle types, swabs, and transport media that are not tested.
Table 5. Examples of Substances Recommended for an Interference Study for Nasal Swab Specimens
Table 6. Examples of Substances Recommended for an Interference Study for Skin, Soft Tissue Infections
Table 7. Examples of Substances Recommended for an Interference Study for Blood
You should also include a study to determine the effect of various incubation times on the performance of your media. In addition, your established incubation time should support the performance of your media by meeting an acceptance criterion of ≥95%. If your media has different incubation times, please stratify your results according to each time or time points in hours. In a request by industry, we were asked how they might provide this information. Table 8 is a format that you can use as an aide.
For target organisms, we recommend that the bacterial concentration should be tested at levels at or near the percent recovery. In addition, you should express the final concentrations of target in CFU/mL on a non-selective agar media plate.
Table 8. Table Format for Presentation of an Incubation Study.
3. Additional Tests from Chromogenic Media
If you recommend that colonies grown on your new device may be used for additional testing, you should demonstrate that the results of these tests are accurate by performing validation studies for each test. Testing should include but is not limited to catalase, coagulase, and latex agglutination.
4. Clinical Performance Studies
We recommend that you develop a detailed study protocol that includes, for example;
We recommend that you include this and any other relevant study protocol information in your premarket submission.
In order to assist you in designing your protocol, we encourage you to send your proposed studies and selection of specimen types for review to the Division of Microbiology Devices. This is referred to as the pre-IDE4 process.
The total number of sample types you include in your study for substantiating a claim for detection of MRSA or MRSA/SA will depend on the prevalence of the bacteria in your clinical study patient population and on your device's performance. We recommend that all MRSA devices demonstrate sensitivity/specificity results of at least 95% with a lower 95% (two-sided) confidence bound exceeding 90%. If your device detects MRSA/SA, we recommend that the sensitivity/specificity of results for MRSA should be at least 95% with a lower 95% (two-sided) confidence bound exceeding 90% and the sensitivity/specificity of SA should also be at least 95% with a lower 95% (two-sided) confidence bound exceeding 90%.
In general, we recommend testing a minimum of 50 samples, which are determined to be positive using the reference method, per clinical site. If your device is intended for use with positive blood culture bottles detected by the continuous monitoring blood culture systems and you intend to claim different blood culture bottle types, at least 20 MRSA positive samples per claimed blood culture bottle type should be included in the clinical study. If the data, when stratified by blood culture system and blood culture bottle type, indicate a significant difference in assay performance between the blood culture systems or the blood culture bottle types within a particular blood culture system, the data will not be considered equivalent and can not be pooled, but rather should be analyzed separately. If this occurs, additional prospectively collected clinical samples should be tested to support each claimed blood culture instrument system and blood culture bottle type separately. To mitigate this risk, we recommend that you conduct an analytical poolability study where at least two of the claimed blood culture instruments and their respective claimed blood culture bottle types are tested at a single site. The data should then be analyzed to determine poolability across the different blood culture bottle types and blood culture instrument systems. Performing this study prior to the clinical study would confirm the feasibility of the subsequent clinical study by demonstrating that sample data can be pooled.
We recommend that you conduct your studies at a minimum of three geographically diverse facilities of which two sites should be in the United States.
We recommend that you conduct your studies using samples from your proposed target population. In order to preserve the true prevalence of SA in your clinical study patient population, patients previously enrolled in the clinical study should not be allowed to re-enter the same clinical study. History of patients' antibiotic use should be collected and recorded if it is available and reliable.
We recommend that you conduct daily testing of all quality control organisms for both the reference method and the new device as well as with any procedural options ( i.e. different method of inoculation such as swab vs swab suspension in a liquid media given in the labeling of the new device). The organisms included in your QC should be recommended by CLSI.
We recommend a minimum of 20 test results per site for each method of inoculation stated in the package insert. You should present both initial and repeat QC results with an explanation of the action taken for all out-of-range test results. Q uality control testing should not be continued until the root cause of the out-of-range error has been identified.
Test results on the new device for the recommended quality control isolates should be within the expected range 95% of the time. The same QC organisms should be recommended in the device package insert. The QC section of the package insert should also include the following statement "QC procedures should be performed in conformance with applicable state and/or federal accreditation requirements".
The reference methods described below only refer to those devices that detect oxacillin resistance mechanisms associated with mecA.
We recommend that you compare results obtained with your device to the results obtained by using enrichment broth (i.e. Trypticase Soy Broth containing 6.5% sodium chloride) incubated for 48 hours if test results were negative at 24 hours. Turbid growth should be subcultured to sheep blood agar. Suspicious colonies of S. aureus should be Gram stained then identified by using appropriate laboratory methods (e.g., Catalase, Tube or Slide coagulase, latex agglutination tests). Colonies should be further identified using FDA cleared commercially available microbiology systems. mecA-mediated oxacillin resistance should be determined by using cefoxitin (disk diffusion or broth microdilution), as recommended by the current CLSI Standard M100. In order to pool results, your reference methods should be consistent for each testing site.
Use of enrichment broth increases the sensitivity of isolation and identification of MRSA. We recommend that you use enrichment broth as it is a more robust reference method for comparison of your new device. Enrichment broth should be used in accordance with the product labeling.
In addition to the cefoxitin (disk diffusion or broth microdilution) method described above, we also accept those devices that detect the protein expressed by mecA in MRSA e.g. PBP2a (PBP2') Latex agglutination test, as another reference method. An alternate method may be used (i.e. predicate as comparator) to claim percent agreement; however, all negatives should be confirmed as true negatives.
Evaluating the Results of your Study
All raw data from studies should be submitted for review. You should provide your calculated results in a 2 X 2 table. The sensitivity (positive percent agreement) of your clinical study should be at least 95% and the specificity (negative percent agreement) should be at least 95% of the two sided 95% CI with a lower bound of 90%.
The quality control and reproducibility results should also be considered when assessing comparative performance of the reference method and the new device.
In order to satisfy the requirements of 21 CFR 807.87(e), the device application should include labeling, e.g. a package insert. The final labeling must conform to the requirements of 21 CFR 809.10 before introducing an in vitro diagnostic device into interstate commerce. You should ensure that the final labeling include the following items relevant to testing for MRSA.
Intended Use Statement
As per 21 CFR 809.10(b)(2), the intended use statement must indicate organism group(s) the device is indicated for testing or identifying.
A typical example of an intended use statement is:
Chromogenic media A is a selective and differential chromogenic medium for the qualitative detection of nasal colonization methicillin-resistant S. aureus (MRSA) to aid in the prevention and control of MRSA infections in healthcare settings. The test is performed on anterior nares swab specimens from patients and healthcare workers to screen for MRSA colonization. Chromogenic media A is not intended to diagnose MRSA, guide or monitor treatment for infections or provide susceptibility results to methicillin. A negative result does not preclude MRSA nasal colonization. Concomitant cultures are necessary for organism identification, susceptibility testing or epidemiological typing.
The step by step outline of the procedure must include details of the kinds of quality control procedures and materials required. 21 CFR 809.10(b)(8). You should ensure that the specifics of quality control procedures you recommend to users are those necessary to ensure satisfactory limits of performance. You must list all recommended quality control strains and the expected results for each organism. 21 CFR 809.10(b)(8)(vi).
You must provide an adequate description of each expected result. 21 CFR 809.10(b)(9). Interpretation of results may be presented in a tabular format. Results should not be reported in instances where performance has not been established.
You must include a statement of limitations of the procedure. 21 CFR 809.10(b)(10). The following are examples of some limitation statements that may apply to your device:
C. CLIA Waiver
If you are seeking a waiver for your device under the Clinical Laboratory Improvement Amendments of 1988 (CLIA),1 we recommend that you consult with the Division of Microbiology Devices staff regarding the design of specific studies to support the CLIA waiver application for your device. The guidance for industry and FDA staff, " Recommendations for Clinical Laboratory Improvement Amendments of 1988 (CLIA) Waiver Applications for Manufacturers of In Vitro Diagnostic Devices ."
1 See 42 U.S.C. § 263a(d)(3).