This guidance document is being distributed for comment purposes only.
Document issued on: January 5, 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draft document within 90 days of publication in the Federal Register of the notice announcing the availability of the draft guidance.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 e lectronic comments to http://www.regulations.gov. Identify a ll comments with the docket number listed in the notice of availability that publishes in the Federal Register.
For questions regarding this document contact Li Li at 301-796-6200, or by email at firstname.lastname@example.org.
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Devices and Radiological Health
Office of In vitro Diagnostic Device Evaluation and Safety
Division of Microbiology Devices
Additional copies are available from the Internet. You may also send an e-mail request to CDRH-Guidance@fda.hhs.gov to receive a copy of the guidance. Please use the document number 1722 to identify the guidance you are requesting.
Table of Contents
- RISKS TO HEALTH
- DEVICE DESCRIPTION
- INTENDED USE
- TEST METHODOLOGY
- INSTRUMENTS - HARDWARE AND SOFTWARE
- ANCILLARY REAGENTS
- INTERPRETING AND REPORTING TEST RESULTS
- ESTABLISHING PERFORMANCE CHARACTERISTICS
- ANALYTICAL PERFORMANCE STUDIES
- Analytical Sensitivity
- Analytical Specificity
- Cut-off and Equivocal Zone
- Specimen Collection, Storage, and Shipping Conditions
- Carry-Over and Cross-contamination Studies (for multi-sample assays and devices that require instrumentation.)
- CLINICAL PERFORMANCE STUDIES
- Study Protocol
- Specimen Type(s)
- Study Sites
- Study Population
- Reference Methods
- CLIA WAIVER
- ANALYTICAL PERFORMANCE STUDIES
Draft Guidance for Industry and Food and Drug Administration Staff
Establishing the Performance Characteristics of Nucleic Acid-Based In vitro Diagnostic Devices for the Detection and Differentiation of Methicillin-Resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA)
This draft guidance, when finalized, will represent the Food and Drug Administration's (FDA's) current thinking on this topic. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public. You can use an alternative approach if the approach satisfies the requirements of the applicable statutes and regulations. If you want to discuss an alternative approach, contact the FDA staff responsible for implementing this guidance. If you cannot identify the appropriate FDA staff, call the appropriate number listed on the title page of this guidance.
FDA is issuing this draft guidance to provide industry and agency staff with recommendations for studies to establish the analytical and clinical performance of nucleic acid-based in vitro diagnostic devices (IVDs) intended for the detection and differentiation of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA). These devices are used to aid in the prevention and control of MRSA/SA infections in healthcare settings.
This guidance provides detailed information on the types of studies FDA recommends using to support Class II premarket submissions for these devices. The guidance includes examples of MRSA and SA 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 the MRSA/SA genome (nucleic acid). It does not address detection of MRSA/SA antigens or serological response from the host to the MRSA/SA antigens, nor does it address establishing performance of non-MRSA/SA components of multi-analyte or multiplex devices.
FDA’s guidance documents, including this guidance, do not establish legally enforceable responsibilities. Instead, guidance documents 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 guidance documents means that something is suggested or recommended, but not required.
This document recommends studies for establishing the performance characteristics of nucleic acid-based in vitro diagnostic devices for the detection and differentiation of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA) in either human specimens or culture growths. In an attempt to limit the spread of these infections, several governmental, public health, and professional organizations have published evidence-based guidelines and policies for the prevention and control of MRSA transmission in health care settings [Ref. 1] . Currently, the standard surveillance method for detecting MRSA is traditional culture. Faster nucleic acid-based in vitro diagnostic devices for the detection and differentiation of MRSA and SA represent an important tool for infection control programs to aid in the prevention and control of MRSA infections in healthcare settings. They are also useful for aiding patient management, such as to aid in the diagnosis of MRSA/SA from skin and soft tissue infections when used in conjunction with other laboratory tests such as microbial growth culture and clinical data available to the clinician, or to detect MRSA/SA from blood culture bottles that are detected as positive for microbial growths and shown to contain Gram Positive Cocci (GPC) by Gram stain.
FDA believes that the studies recommended in this guidance will be relevant for Class II premarket submissions (e.g., 510(k) or a de novo classification petition) that may be required for a particular test.
A manufacturer who intends to market a nucleic acid-based in vitro diagnostic device for the detection or the detection and differentiation of MRSA and SA 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 “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.”
Information regarding the use of standards can be found in section 514(c)(1)(B) of the Act (21 U.S.C. 360d(c)(1)(B)), and in the FDA guidance entitled “Use of Standards in Substantial Equivalence Determinations.” The Special 510(k) is an option for manufacturers considering modifications to their own cleared devices. Information on how to prepare a Special 510(k) is available.
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 nucleic acid-based in vitro diagnostic devices for the detection and differentiation of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA) either directly from human specimens or microbial growth in cultures. This document is limited to studies intended to establish the performance characteristics of devices that detect the MRSA/SA genome (nucleic acid). This guidance does not address detection of MRSA/SA antigens or serological response from the host to the MRSA/SA antigens, nor does it address establishing performance of non-MRSA components of multi-analyte or multiplex devices.
The scope of this document includes the devices described in the existing classification, as indicated below, and may also be applicable to future nucleic acid-based MRSA/SA diagnostic devices that may not fall within this existing classification. Those future 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 is the existing nucleic acid-based MRSA/SA detection and differentiation IVD classification regulation:
21 CFR 866.1640 Antimicrobial susceptibility test powder:
(a) Identification. An antimicrobial susceptibility test powder is a device that consists of an antimicrobial drug powder packaged in vials in specified amounts and intended for use in clinical laboratories for determining in vitro susceptibility of bacterial pathogens to these therapeutic agents. Test results are used to determine the antimicrobial agent of choice in the treatment of bacterial diseases.
(b) Classification. Class II (performance standards).
The product code for these nucleic acid-based MRSA/SA detection and differentiation IVD devices is NQX.
IV. Risks to Health
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial and life threatening staphylococcal infections. Infections with MRSA have been associated with a significantly higher morbidity, mortality and costs than methicillin susceptible S. aureus(MSSA) [Ref. 2, 3]. The incidence of infections caused by MRSA has increased dramatically over the last decade in the United States, despite widespread measures aimed at preventing the spread [Ref. 4, 5] . In addition, community-associated MRSA (CA-MRSA) has become a major problem in US hospitals already dealing with high levels of healthcare-associated MRSA (HA-MRSA) [Ref. 6].
Therefore, FDA has identified potential risks to health, i.e., issues that may impact safety or effectiveness of a nucleic acid-based assay that detects and differentiates methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA). These risks include failure of the device to perform as indicated, leading to inaccurate results or lack of results, and incorrect interpretation of results; these potential risks may lead to incorrect patient management decisions. A false positive MRSA result could lead to unnecessary or inappropriate treatment for MRSA. A false negative MRSA result, could lead to failure to provide a diagnosis and the correct treatment for MRSA. A lack of result could also lead to delayed diagnosis and inadequate treatment.
Failure of devices for detection and differentiation of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (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.
V. Device Description
In your 510(k) submission, you should identify the regulation, the product code, and a legally marketed predicate device. We recommend that you include a table that outlines the similarities and differences between the predicate and your device. You should include the following descriptive information to adequately characterize your device that is intended to detect and differentiate methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA).
A. Intended Use
The intended use should specify the genomic target(s), test platform, specimen types for which testing will be indicated, the clinical indications for which the test is to be used, and the specific population(s) for which the test is intended. The intended use should state that the test is qualitative, whether analyte detection is presumptive and any specific conditions of use.
B. Test Methodology
You should describe in detail the methodology used by your device. For example, you should describe the following elements, as applicable to your device:
- Test platform (e.g., real-time PCR, bead array).
- Information and rationale for selection of specific targets and the methods used to design primers and probes.
- Specimen type (e.g., swabs, blood culture bottles, etc.) including collection and handling methods.
- Assay components provided or recommended for use, and their function within the system (e.g., buffers, enzymes, fluorescent dyes and other reagents, instrumentation and software).
- Internal controls and a description of their specific function in the system.
- External controls that you recommend or provide to users.
- Types of output generated by the device and system parameters (e.g., measurement ranges, when applicable)
- The computational path from raw data to the reported result (please see section C below “Instruments - Hardware and Software” for details).
- Illustrations or photographs of non-standard equipment or methods.
In your 510(k), you should provide performance information that supports the conclusion that your design requirements have been met.
C. Instruments - Hardware and Software
For instruments and systems that measure multiple signals, and other complex laboratory instrumentation that has not been previously cleared, refer to the FDA guidance document entitled "Class II Special Controls Guidance Document: Instrumentation for Clinical Multiplex Test Systems" [Ref. 8], for details on the types of data you should provide to support instrument clearance.
If your system includes software, you should submit software information in accordance with the level of concern described in the FDA guidance document entitled “Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices” [Ref. 9]. You should determine the level of concern prior to the mitigation of hazards. In vitro diagnostic devices of this type are typically considered a moderate level of concern; software flaws could indirectly affect the patient and potentially result in injury because inaccurate information may be given to the healthcare provider and the patient.
A clear description of how raw signals are converted into a result, including adjustment to the background signal for normalization, if applicable, should be included in the regulatory application. The following documents for software development and implementation should also be included in the regulatory submission:
- System and Software Requirements
- Hazard Analysis
- Architecture Design Chart
- Software Design Specification
- Software Development Environment Description
- Verification and Validation
- Traceability Analysis
- Unresolved Anomalies (do the anomalies influence safety and effectiveness?)
Configuration of the hardware and software components should be very similar or identical to the final version of the device before beginning clinical studies. A risk assessment should be performed if any significant changes are made to the hardware or software after the completion of the clinical studies and before the clearance and distribution of the device.
Below are additional references to help you develop and maintain your device under good software life cycle practices consistent with FDA regulations.
- General Principles of Software Validation; Final Guidance for Industry and FDA Staff;
- Guidance for Off-the-Shelf Software Use in Medical Devices; Final;
- 21 CFR 820.30 Subpart C – Design Controls of the Quality System Regulation.
- ISO 14971-1; Medical devices - Risk Management - Part 1: Application of risk analysis.
- AAMI SW68:2001; Medical Device Software - Software life cycle processes.
D. Ancillary Reagents
Ancillary reagents are those reagents that an assay manufacturer specifies in device labeling as “required but not provided” in order to carry out the assay as indicated in its instructions for use and to achieve the test performance claimed in labeling for the assay. For the purposes of this document, ancillary reagents of concern are those that must be specified according to the manufacturer and the catalog or product number, or other specific designation, in order for your device to achieve its labeled performance characteristics. For example, if your device labeling specifies the use of Brand X DNA amplification enzyme, and use of any other DNA amplification enzyme may alter the performance characteristics of your device from that reported in your labeling, then Brand X DNA amplification enzyme is an ancillary reagent of concern for the purposes of this document. In contrast, if your device requires the use of 95% ethanol, and any brand of 95% ethanol will allow your device to achieve the performance characteristics provided in your labeling, then 95% ethanol is not an ancillary reagent of concern for the purposes of this document.
If the instructions for use of your device specify ancillary reagents of concern, you should address how you will ensure that the results of testing with your device and these ancillary reagents, in accordance with your instructions, will be consistent with the performance established in your premarket submission. Your plan may include application of quality systems approaches, product labeling, and other measures. Your submission should address the elements described below. FDA will evaluate whether your plan will sufficiently mitigate the risks presented by the device to offer reasonable assurance of the safety and effectiveness of the device and establish its substantial equivalence.
1. The use of ancillary reagents should be addressed in your risk assessment, including risks associated with the management of reagent quality and variability, risks associated with any inconsistency between instructions for use provided directly with the ancillary reagent and those supplied by you with your device, and any other issues that could present a risk of obtaining incorrect results with your device.
2. Using your risk assessment, you should describe how you intend to mitigate risks through implementation of any necessary controls over ancillary reagents. These may include, where applicable:
- Plans for assessing user compliance with labeling instructions regarding ancillary reagents
- Material specifications for ancillary reagents
- Identification of reagent lots that will allow appropriate performance of your device
- Stability testing
- Complaint handling
- Corrective and preventive actions
- Plans for alerting users in the event of an issue involving ancillary reagents that would impact the performance of your device, or
- Any other issues that must be addressed in order to assure safe and effective use of your test in combination with named ancillary reagents, in accordance with your device’s instructions for use.
In addition, you should provide testing data to establish that the quality controls you supply or recommend are adequate to detect performance or stability problems with the ancillary reagents.
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. We recommend that you consult with FDA when designing specific controls for your device. For nucleic acid-based devices, we generally recommend that you include the following types of 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. It may not be applicable 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:
- Patient specimen from a MRSA/SA DNA negative individual
- Samples containing a non-target organism (e.g., MRSA/SA DNA negative specimen spiked with Staphylococcus epidermidis)
- Surrogate negative control, e.g., alien encapsidated DNA
Positive control for complete assay
The positive control contains target nucleic acids, and is used to control the entire assay process, including sample lysis or 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 a cceptable positive assay control materials include:
- MRSA/SA strains/isolates containing the target sequences
- Packaged DNA from a MRSA/SA strain containing the target sequences
- Purified full length double stranded genomic DNA from a MRSA isolate
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 sample lysis or DNA extraction process . It controls the integrity of the reaction components and instrument when negative results are obtained. It also indicates that the target can be detected if it is present in the sample lysates or extracted sample. An example of this type of control is a non-infectious DNA plasmid containing the target sequence.
The internal control is a non-target nucleic acid sequence that is co-extracted and/or co-amplified with the target nucleic acid. It controls for integrity of the reagents (polymerase, primers, etc.), equipment function (thermal cycler), and the presence of inhibitors in the samples. Examples of acceptable internal control materials include (1) human nucleic acid co-extracted with MRSA/SA and primers amplifying human housekeeping genes (e.g., RNaseP, β-actin) and (2) a non-infectious DNA plasmid containing the non-target nucleic acid that is added to the sample either prior or after sample lysis/extraction . The need for this control is determined on a device case-by-case basis [Ref. 10].
The extraction control verifies that lysis of MRSA/SA and/or subsequent nucleic acid isolation has occurred efficiently. Examples of extraction controls include a strain of MRSA containing the target sequences, or a known MRSA positive clinical specimen. It is possible that a negative sample control, a positive control for complete assay, or an internal control, as described earlier, depending on their specific compositions, may also function as an extraction control.
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 regarding controls.
F. Interpreting and Reporting Test Results
A detailed description of how positive, negative, equivocal (if applicable), or invalid results are determined and how their interpretations should be included in the regulatory submission. We recommend that you indicate the cut-off values for all outputs of the assay including the cut-off value for defining a negative or positive result of the assay. If the assay has an equivocal zone, the cut-off values (limits) for the equivocal zone should also be defined. If your interpretation of an initial equivocal result requires re-testing, you should 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 confirms the significance of the cut-offs.
If the assay has an invalid result, you should describe how an invalid result is defined. If controls are part of the determination of invalid results, you should describe each possible combination of control results for defining the invalid result. Additionally, recommendations on how to follow up any invalid result, i.e., whether the result should be reported as invalid or if the specimen should be re-tested, should be provided. If re-testing is recommended, you should provide information similar to the information for re-testing of equivocal results, which is, whether re-testing should be repeated from the same nucleic acid preparation, a new extraction, or a new patient specimen. Additional information on result evaluation and reporting can be found in CLSI document ILA 18-A2 [Ref. 11], Specifications for Immunological Testing for Infectious Diseases.
VI. Establishing Performance Characteristics
A. Analytical Performance Studies
We recommend you perform the following analytical studies:
1. Analytical Sensitivity
Limit of Detection
We recommend that you determine the limit of detection (LoD) using serial dilutions of well characterized MRSA/SA isolates obtained from public and private collections or clinical isolates. Cover MRSA/SA genetic diversity by using SCCmec types, MERJ types, spa types, geographically diverse SA and MRSA strains selected to broadly span the range of genetic diversity found in the species Staphylococcus aureus based on its phylogenetic structure, and representative strains of various pulse- field gel electrophoresis (PFGE) types currently found in the healthcare and community settings to the extent that they are relevant to the particular choice of MRSA/SA specific DNA targets of the device. These factors should all be taken into consideration when choosing well characterized MRSA/SA strains for the LoD study. The initial LoD estimation study should include serial dilutions of multiple MRSA/SA strains representative of relevant genotypes or subtypes of MRSA/SA (please see Table 1, Table 2, and Table 3 for examples of recommended MRSA/SA strains) and 3-5 replicates for each dilution. You should report the LoD as the level of MRSA/SA that gives a 95% detection rate. The estimated LoD should then be confirmed by preparing at least 20 additional replicates at the LoD concentration and demonstrating that the MRSA/SA was detected 95% of the time. The reference method we recommend for LoD determination is plating and counting bacteria colony forming units (CFU). CFU should be reserved only for colony counts from actual plating and counting of bacteria, not for a theoretical calculation deduced from an estimated cells/mL number (i.e. McFarland units). In addition, presentation of LoDs in genomic DNA copy numbers is also helpful.
We recommend that you determine the LoD for each analyte (i.e., MRSA and SA) and each specimen type tested by the device.
For devices that test nasal swab specimens or skin and soft tissue infection swab specimens, a recommended way of performing the bacterial LoD study is to spike a pair of negative nasal swab samples or a pair of negative skin and soft tissue swab samples, or simulated nasal swab or skin and soft tissue swab samples, with serial dilutions of each MRSA and SA strain. A simulated nasal swab sample matrix can be contrived using saline solution, mucin, and human genomic DNA. A simulated skin and soft tissue swab sample matrix can be made of white blood cells (WBC), red blood cells (RBCs), and plasma. If you choose to use a simulated nasal swab sample matrix or simulated skin and soft tissue swab sample matrix in the LoD study, an analytical sample matrix equivalency study should be carried out to demonstrate that your assay will generate equivalent results using both the natural sample matrix and the simulated sample matrix. Use one spiked nasal swab sample or skin and soft tissue swab sample for the determination of viable bacteria count in CFU/mL or CFU/swab, and use the other paired spiked nasal swab sample or skin and soft tissue swab sample for the investigational device testing.
For devices that test samples from blood culture bottles that are detected as positive for microbial growth and shown to contain Gram Positive Cocci (GPC) by Gram stain, we recommend that you determine the LoD of your device using quantified (CFU/mL) cultures of MRSA/SA isolates, serially diluted in a simulated sample matrix consisting of a mixture of human blood and the most challenging blood culture media for your particular device (e.g. bottles with resin or charcoal). Methicillin-sensitive Staphylococcus epidermidis (MSSE) at a minimum of 10 7 CFU/mL should also be added to the simulated sample matrix to simulate the most common skin contaminant organism. The dilutions in the simulated sample matrix should be incubated in an environment, which simulates the real microbial growth conditions, on the continuous monitoring blood culture instruments or other manual or semi-automated systems for a period of time that can be pre-determined in an analytical study to achieve the desired concentrations of each dilution before you test the dilutions in the LoD study. Due to the enrichment process involved in blood cultures, almost all positive blood culture specimens contain very high bacterial loads resulting in CFUs per test that may be well above the estimated LoD of the device. Therefore, it may not be necessary to confirm the estimated LoD of the device using a large number of replicates for this specific specimen type.
The LoD for multiplex devices that detect multiple targets simultaneously starting from a single sample should be established for each target independently, as well as verified with other targets that could potentially be present in the same sample. If one target of a multiplex assay could be present at a high level, the simultaneous detection of another target present at low levels could potentially be impaired. Consequently, it is useful to test samples prepared with one target at the LoD concentration and another target at a very high concentration. We recommend that you conduct an analytical study for three to four consecutive days to determine the potential competitive inhibitory effect of increasing concentrations of methicillin-sensitive Staphylococcus aureus (MSSA) or methicillin-resistant coagulase negative Staphylococci ( MRCoNS) to the device LoD detecting MRSA. Specimens prepared with MRSA at the established LoD concentration and MSSA or MRCoNS at increasing concentrations (i.e. MRSA to MSSA or MRCoNS ratios of 1:1 to 1:10 6) should be tested in this study, and an appropriate sample matrix, as recommended above, should always be used.
We suggest that you refer to Clinical Laboratory Standards Institute (CLSI) document EP17-A [Ref. 12], Protocols for Determination of Limits of Detection and Limits of Quantitation when designing your LoD studies.
Analytical Reactivity (Inclusivity)
We recommend that you demonstrate that the device can detect an extensive (at least 75) list of MRSA/SA strains representing the global genetic diversity present in S. aureus. We recommend that you take into consideration the phylogenetic structure of S. aureus, as well as the key clonal complexes and sequence types, in selecting MRSA strains for the Inclusivity Study. We also recommend that you test additional MRSA strains representing the various Pulse-Field Gel Electrophoresis (PFGE) types (e.g. USA 100, 200, 300 and 400), with emphasis on the USA 300 strains. The bacterial concentrations tested in the Inclusivity Study should be at levels at or near the assay cut-off. All MRSA isolates identities and concentrations should be confirmed.
Examples of MRSA and SA strains for LoD and analytical reactivity studies are shown in Table 1 thru Table 3.
Table 1. Example #1: MRSA/SA strains for analytical sensitivity (LoD) and inclusivity studies obtained from the CDC.
SCCmec Type PFGE Type Source Origin IV USA1000 Skin lesion VT IV USA500 Blood CT MSSA USA900 Stool NM II USA200 Blood NC II USA600 Blood NY IVa USA400 Unknown MN II USA100 Blood OH IVa USA700 Nasal MS IVa USA300 Skin/Soft Tissue MS IV USA800 Wound WA MSSA USA900 Nasal NHANES II USA100 Unknown GA-ABC IVb USA300 Unknown GA-ABC IV USA500 Unknown GA-ABC IVa USA1100 Abscess AK MSSA USA1200 Nasal CA IVc USA1100 Abscess Uruguay IVc USA800 Wound HI IV USA1000 Blood CA IVb USA1000 Wound HI IVb USA800 Nasal NHANES
Table 2. Example #2: MRSA strains (known clinical associated strains) obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for analytical sensitivity (LoD) and inclusivity studies.
Strain SCCmec Type /PFGE Type Origin NRS384 IVa/USA300-0114 Mississippi NRS642 II/USA100 California NRS643 IV/USA300 California NRS645 IV/IBERIAN California NRS648 II/USA600 California NRS651 II/USA200 California NRS652 IV/USA1000 California NRS653 IV/USA800 California NRS661 II/USA100 Colorado NRS662 IV/USA300 Colorado NRS666 II/USA100 Colorado NRS668 IV/USA800 Colorado NRS669 II/USA100 Colorado NRS670 II/USA100 Connecticut NRS673 II/USA100 Connecticut NRS675 IV/USA800 Connecticut NRS676 IV/USA1000 Connecticut NRS678 IV/USA500 Connecticut NRS681 II/USA100 Georgia NRS683 IV/USA300 Georgia NRS685 IV/USA500 Georgia NRS686 IV/IBERIAN Georgia NRS689 IV/USA700 Georgia NRS691 IV/USA500 Georgia NRS692 IV/USA800 Georgia NRS694 IV/USA300 Georgia NRS696 II/USA100 Minnesota NRS701 II/USA200 Minnesota NRS703 IV/USA300 Minnesota NRS705 II/USA100 New York NRS707 IV/USA300 New York NRS708 IV/USA500 New York NRS714 IV/USA800 New York NRS715 II/USA600 New York NRS719 II/USA100 New York NRS721 II/USA100 Oregon NRS722 II/USA200 Oregon NRS724 IV/USA300 Oregon NRS730 IV/USA1000 Oregon NRS732 IV/USA300 Tennessee NRS734 IV/USA800 Tennessee NRS735 II/USA100 Tennessee NRS738 II/USA100 Tennessee NRS740 II/USA200 Tennessee NRS745 V/USA1000 California
Table 3. Example #3: MRSA strains (clinical isolates and isolates obtained from public and private collections) for analytical sensitivity (LoD) and inclusivity studies.
Geographic Origin MREJ Type SCCmec Type
Year of the strain's isolation
/ year of freezing
Denmark (Gentofte) i I 1960’s Hungary (Dunaújváros) iii III 1993 Portugal (Lisbon) iv III 1994 USA (NYC) ii II 1994 Portugal (Lisbon) ii VI 1996 Portugal (Lisbon) iii III 1998 Portugal (Lisbon) ii I 1992 USA (NYC) iv III Between 1979 and 1981 Canada vi II 2001 Canada ii II 2001 Canada iii III 2001 Canada ii II 2001 Canada ii IV 2001 Belgium (Brussels) ii mut25 IV 1992 Belgium (Oostende) ii mut25 IV 1995 Finland (NK) ii mut16 IV 1990 Germany (Bas Saarow) ii IV 1996 Germany (Frankfurt) ii mut25 IV 1996 United Kingdom ii mut16 IV 2001 United Kingdom ii mut16 IV 2001 United Kingdom ii mut16 IV 2001 United Kingdom ii mut16 IV 2001 Greece (Athens) iii III 1994 Greece (Athens) iii III 1994 USA (Ohio) ii II/USA 100 2009 USA (N. Carolina) ii II/USA 200 2009 USA (Mississippi) ii IV/USA 300 1998 USA (N. Dakota) ii IV/USA 400 1998 USA (Connecticut) ii IV/USA 500 2009 USA (NYC) ii ND/USA 600 1999 USA ( Louisiana ) ii IV/USA 700 2009 USA (Washington) ii IV/USA 800 2009 USA (Vermont) ii IV/USA 1000 2009 USA (Alaska) ii IV/USA 1100 2009 Brazil i I 2002 Poland i I 2002 NA i ND/USA500 2009 USA (New Jersey) ii IV 2007 USA (New Jersey) ii IV 2007 USA (New Jersey) ii II 2007 Slovenia (Ljubljana) ii I 2007 Slovenia (Ljubljana) ii IV 2007 Belgium (Brugge) ii IV 2007 Sweden ( Solna ) ii III 2008 Sweden ( Solna ) ii II/USA100 2008 NA ii II/USA100 1993 NA ii II/USA100 2002 NA ii II/USA100 2003 NA ii ND/USA100 2009 USA (Connecticut) ii IV/USA 300 2007 Chile iii III 2002 Israel iii Not Typed 2002 Netherlands (Den Bosch) iii V 2007 USA (New Jersey) iii V 2007 Denmark (Copenhague) iv III 2000 Canada (Toronto) v IV 1996 Canada v IV 1997 Canada (Québec) v IV 2002 Canada (Québec) v IV 1999 USA (Philadelphia) v IV 2008 Canada (Toronto) vii II 2001
Evaluation of a Well Characterized Challenge Strain Panel
We recommend that you test a well characterized MRSA/SA challenge strain panel containing MRSA strains with high and low oxacillin MIC values, borderline oxacillin-resistant S. aureus (BORSA) strains (BORSA strains are mecA negative, but usually exhibit an oxacillin minimum inhibitory concentration (MIC) >= 2 and <= 8 ug/ml), methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) strains. The MRSA strains in the challenge strain panel should represent the various Pulse-Field Gel Electrophoresis (PFGE) types (e.g. USA 100, 200, 300 and 400), with emphasis on the USA 300 strains. FDA cleared Broth Dilution Susceptibility Tests using Oxacillin should be carried out in determining MIC values. MRSA strains contained in the challenge strain panel should be tested at concentrations close to the assay cut-off (i.e. 2 to 3 X LoD). MSSA, BORSA, and MRSE strains contained in the challenge strain panel should be tested at concentrations of 10 6 CFU/mL or higher.
2. Analytical Specificity
For devices that are intended for testing nasal swab specimens collected from patients at risk for MRSA colonization, we recommend that you test for potential cross-reactivity with organisms that are phylogenetically related to Staphylococcus aureus and members of the nasal commensal. In particular, methicillin-sensitive Staphylococcus aureus (MSSA), coagulase negative Staphylococci (CNS), methicillin-resistant Staphylococcus epidermidis (MRSE), Gram-negative rod (GNR) and Gram-negative cocci (GNC) to include Neisseria spp. and Moraxella spp., yeast, Gram-positive rod (GPR), Gram-positive cocci (GPC), and other pathogenic and commensal flora found in the nares should all be tested in the cross-reactivity study. In addition, coagulase positive staphylococci strains that are not S. aureus, such as S. lutrae, S. pseudointermedius, S. delphini, S. schleiferi, S. schleiferi subsp. coagulans, and S. intermedius, should also be tested in the cross-reactivity study. We recommend that you test high levels of organisms (usually 10 6 CFU/mL or higher for bacteria and yeast and 10 5 PFU/mL or higher for viruses). We recommend that you confirm the organism identities and titers.
For devices that are intended for testing skin and soft tissue infections specimens, or for testing samples from blood culture bottles that are detected as positive for microbial growth and shown to contain Gram Positive Cocci (GPC) by Gram stain, we recommend that you include additional organisms, including skin flora, which are commonly isolated from the respective sample source (i.e. skin and soft tissue infections or blood culture bottles). Testing potential cross-reactivity with viruses may not be necessary for devices that are indicated for testing samples from blood culture bottles that are flagged as positive for microbial growth and shown to contain Gram Positive Cocci (GPC) by Gram stain.
Examples of microorganisms recommended for cross-reactivity studies are listed in Table 4 and Table 5.
Table 4. Examples of microorganisms (non-stap hylococci str ains of various bacterial and yeast species and viruses) recommended for analytical specificity (cross-reactivity) studies for devices that are indicated for testing nasal swab specimens collected from patients at risk for MRSA colonization
Organism Type Acinetobacter baumannii Acinetobacter haemolyticus Bacillus cereus Bordetella pertussis Chlamydia pneumoniae Citrobacter freundii Citrobacter koseri Corynebacterium aquaticus Corynebacterium bovis Corynebacterium flavescens Corynebacterium genitalium Enterobacter aerogenes Enterobacter cloacae Enterococcus faecium Enterococcus faecalis Enterococcus flavescens Enterococcus gallinarum Enterococcus hirae Escherichia coli ESBL producer Haemophilus influenzae Homo sapiens Human Cells Klebsiella oxytoca Klebsiella pneumoniae ESBL producer Lactobacillus crispatus Lactobacillus sp. Legionella spp Listeria monocytogenes Micrococcus luteus Moraxella catarrhalis Mycobacterium tuberculosis avirulent Mycoplasma pneumoniae Neisseria sp. Neisseria meningitidis Pasteurella aerogenes Proteus mirabilis Proteus vulgaris Providenciae stuartii Pseudomonas aeruginosa Pseudomonas fluorescens Salmonella typhimurium Serratia marcescens Shigella sonnei Streptococcus agalactiae Streptococcus anginosus Streptococcus mitis Streptococcus mutans Streptococcus pneumoniae Streptococcus pyogenes Streptococcus salivarius Streptococcus sanguinis Streptococcus suis Yersinia enterocolitica Candida albicans Candida glabrata Cryptococcus neoformans Adenovirus Type 1 Adenovirus Type 7 Human coronavirus* Cytomegalovirus Enterovirus Epstein Barr Virus Human influenza virus A Human influenza virus B Human parainfluenza Type 1 Human parainfluenza Type 2 Human parainfluenza Type 3 Human metapneumovirus Measles Mumps virus Respiratory syncytial virus Type B Rhinovirus Type 1A
* We recommend that you include the OC43 and 229E strains of Human coronavirus in your cross-reactivity study.
Table 5. Examples of coagulase negative Staphylococci (CNS) from public and private collections (including methicillin-resistant Staphylococcus epidermidis (MRSE), methicillin-resistant coagulase negative Staphylococci (MRCoNS) and methicillin-sensitive coagulase negative Staphylococci (MSCoNS)) recommended for analytical specificity (cross-reactivity) studies for devices that are indicated for testing nasal swab specimens collected from patients at risk for MRSA colonization
Genera and species Strain mecA Country of origin Staphylococcus arlettae CCRI-9265 - Unknown Staphylococcus auricularis CCRI-1252 + Argentina Staphylococcus capitis CCRI-9572 + Canada Staphylococcus caprae CCRI-9117 + Unknown Staphylococcus carnosus CCRI-1341 - Unknown Staphylococcus chromogenes ATCC 43764 - Unknown Staphylococcus cohnii subsp.
CCRI-1286 + China Staphylococcus delphini ATCC 49171 - Unknown Staphylococcus epidermidis ATCC 35983 + USA Staphylococcus epidermidis ATCC 29887 + Unknown Staphylococcus epidermidis ATCC 51625 + USA Staphylococcus epidermidis ATCC 35984 + USA Staphylococcus epidermidis ATCC 51624 + USA Staphylococcus equorum ATCC 43958 - Unknown Staphylococcus felis ATCC 49168 - Japan Staphylococcus gallinarum ATCC 35539 - Belgium Staphylococcus hominis CCRI-1347 - Canada Staphylococcus hominis CCRI-1369 - Canada Staphylococcus intermedius ATCC 29663 - Unknown Staphylococcus kloosii ATCC 43959 - Unknown Staphylococcus lentus ATCC 29070 - France Staphylococcus pasteuri ATCC 51129 - France Staphylococcus pulvereri ATCC 51698 - Sweden Staphylococcus xylosus CCRI-8823 - Unknown Staphylococcus haemolyticus CCRI-9749 + Denmark Staphylococcus epidermidis CCRI-1257 (R418) + Argentina Staphylococcus hominis CCRI-9760 + Denmark Staphylococcus haemolyticus CCRI-1274 + China Staphylococcus hominis subsp. hominis ATCC 27844 Unknown USA Staphylococcus simulans CCRI-1370 + China Staphylococcus xylosus CCRI-231 - Canada Staphylococcus warneri CCRI-9551 + UK Staphylococcus warneri CCRI-9105 + Unknown Staphylococcus saprophyticus ATCC 43867 + Canada Staphylococcus sciuri CCRI-1289 + China Staphylococcus haemolyticus CCRI-1251 - Argentina Staphylococcus epidermidis CCRI-8946 + Argentina Staphylococcus epidermidis ATCC 14990 - USA
We recommend that you conduct a microbial interference study using clinically relevant concentrations of the microorganisms that are normally present in the respective specimen types (i.e., nasal swab, skin and soft tissue infection, and blood culture specimens) and at least two strains of MRSA that represent different MRSA genotypes (e.g., SCCmec type, PFGE type, or MREJ type) to evaluate your device for potential microbial interference . We recommend that you evaluate potential microbial interference at the device cut-off determined for each MRSA strain. Potentially interfering microorganisms utilized in the microbial interference study should be the same as those recommended for testing in the cross-reactivity study.
We recommend that you conduct a comprehensive interference study using medically relevant concentrations of the interferent and at least two strains of MRSA that represent different MRSA genotypes (e.g., SCCmec type, PFGE type, or MREJ type) to assess the potentially inhibitory effects of substances that are normally encountered in the respective specimen types (i.e., nasal swab, skin and soft tissue infection, and blood culture specimens).
Potentially interfering substances for nasal swab specimens include, but are not limited to, the following: blood, nasal secretions or mucus, and nasal and throat medications used to relieve congestion, nasal dryness, irritation, or asthma and allergy symptoms. Examples of potentially interfering substances for nasal swab specimens are presented in Table 7.
Table 7. Examples of substances recommended for an interference study for nasal swab specimens
Substance Active Ingredient Mucin: bovine submaxillary gland, type I-S Purified mucin protein Blood (human) Nasal sprays or drops Phenylephrine, Oxymetazoline, Sodium chloride with preservatives, Benzalkonium chloride, Sodium Phosphate, Phenylcarbinol, Propylene glycol, Sorbitol, benzyl alcohol, disodium edetate, hypromellose, phosphoric acid Nasal corticosteroids Beclomethasone, Dexamethasone, Flunisolide, Triamcinolone, Budesonide, Mometasone, Fluticasone Nasal gel Luffa opperculata, sulfur Homeopathic allergy relief medicine Galphimia glauca, Histaminum hydrochloricum FluMist© Live intranasal influenza virus vaccine Throat lozenges, oral anesthetic and analgesic Benzocaine, Menthol Anti-viral drugs Zanamivir Antibiotic, nasal ointment Mupirocin Antibacterial, systemic Tobramycin
Potentially interfering substances for skin and soft tissue infections specimens include, but are not limited to, the following: blood, pus, plasma, topical ointments (antibiotic/antiseptic/pain relieving), debriding agents, and tinctures. Examples of potentially interfering substances for skin and soft tissue infections specimens are presented in Table 8.
Table 8. Examples of substances recommended for an interference study for skin and soft tissue infection swab specimens
Substance Active Ingredient Buffy Coat (wound stimulant) WBC (1.5 x 10 9/mL) Whole Blood (MRSA/SA Free) N/A Plasma Neosporin Bacitracin
StaphA +Septic Benzethonium Chloride, Lidocaine HCl Hyrdocortisone Hyrdocortisone Biol-Ease Benzocaine Iodine Tincture Iodine Mupirocin Benzethonium Chloride Lidocaine HCl Saline Sodium Chloride Antibacterial hand sanitizer Ethyl alcohol 70% Isopropyl alcohol 70% Isopropyl alcohol
Potentially interfering substances for blood culture specimens include, but are not limited to, the following: anticoagulated whole blood and blood culture media components containing the anticoagulant sodium polyanetholesulfonate (SPS) or ion exchange and nonionic adsorbent resins. You should also test additional potentially interfering substances that may be relevant to the specific blood culture bottle types you intend to claim for your device in the interference study.
We recommend that you test interference at the device cut-off determined for each MRSA strain and for each of the interfering substances. We also recommend that you evaluate each interfering substance at its potentially highest concentration (“the worst case”). If no significant clinical effect is observed, no further testing is necessary. Please refer to the CLSI document EP7-A2 [Ref. 13], Interference Testing in Clinical Chemistry, for additional information.
Evaluation of Empty Cassette Variants
For nucleic acid-based devices, especially for those devices that do not directly detect the mecA gene that confers methicillin-resistant in Staphylococcus aureus, we recommend that you test at least 15 strains of well characterized “Empty Cassette Variants”1 of Staphylococcus aureus at concentrations of 10 6 CFU/mL or higher to determine whether your device will generate false positive MRSA results when testing these isolates. FDA cleared Broth Dilution Susceptibility Tests using Oxacillin should be carried out in determining MIC values for the “Empty Cassette Variants” of Staphylococcus aureus isolates, and appropriate molecular methods (e.g., bi-directional sequencing) should be utilized in the characterization of these isolates.
3. Cut-off and Equivocal Zone
In your submission, you should explain how the assay cut-off was determined and how the cut-off values were validated (see also the section entitled “Interpreting and Reporting Test Results”). The cut-off should be determined using appropriate statistical methods. For example, you may provide a result distribution, 95 th and 99 th percentiles, percents of the non-negative (positive or equivocal) results, and other statistics, for the clinical samples without any MRSA/SA 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. 14], Assessment of the Clinical Accuracy of Laboratory Tests Using Receiver Operating Characteristics (ROC) Plots). 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.
We recommend that you conduct within-laboratory precision studies for nucleic acid-based 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 2 runs per day, and at least 2 replicates of each sample per run. These test days should span at least two calibration cycles, if it is applicable to your device. The test panel should consist of 2-4 MRSA 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 nature sample matrix and the simulated sample matrix) at four concentration levels that include:
- A negative sample: a sample with no analyte such that results of repeated tests of this sample are negative 100% of the time.
- A “high negative/low positive” sample ( C 20 to C 80 concentration) : a sample with an analyte concentration below the clinical cut-off such that results of repeated tests of this sample are negative approximately 20% to 80% of the time (i.e., positive approximately 20% to 80% of the time).
- A “low positive” sample (C 95 concentration) : a sample with a concentration of analyte just above the clinical cut-off such that results of repeated tests of this sample are positive approximately 95% of the time.
- A “moderate positive” sample: a sample with a concentration at which one can anticipate positive results approximately 100% of the time (e.g., approximately two to three times the concentration of the clinical cut-off).
CLSI documents EP5-A2 [Ref. 15], Evaluation of Precision Performance of Quantitative Measurement Methods, and EP12-A [Ref. 16], User Protocol for Evaluation of Qualitative Test Performance, contain further information about designing and performing precision studies.
- The protocol for the reproducibility study may vary slightly depending on the device format. As a general guide, we recommend the following protocol:
- Evaluate the reproducibility of your device at 3 testing sites (for example, two external sites and one in-house site).
- Use a five day testing protocol, including a minimum of two runs per day, (unless the assay design precludes multiple runs per day) and three replicates of each panel member per run.
- Each day, have at least two operators at each facility to perform the test. We recommend that, for rapid testing or point-of-care (POC)2 devices, you include a larger number of devices in your evaluation, in order to best represent the settings in which the devices will be used.
- Use the same sample panel as described in the repeatability study above.
The CLSI document EP15-A2 [Ref. 17], User Verification of Performance for Precision and Trueness, contains additional information on reproducibility study design.
5. Specimen Collection, 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 ancillary specimen collection devices (e.g., Liquid Stuart, Liquid Amies, and Amies Gel without charcoal, etc.) are recommended for specimen collection, storage or shipping, you should conduct appropriate studies to demonstrate that the device will perform as described when the specimen is collected by and preserved in such ancillary specimen collection devices. We also recommend that you evaluate the performance of your device after exposing the device to various shipping and storage conditions that are identical to storage and shipping conditions that the end users will encounter in the intended use sites for your device.
6. 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 negative samples in patterns dependent on the operational function of the device. At least 5 runs with alternating high positive and negative samples should be performed. 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 infected patients in the intended use population. We recommend that the negative samples contain the analyte concentration below the cut-off such that repeat testing of this sample is negative 100% of the time. The carry-over and cross-contamination effect can then be estimated by the percent of negative results for the negative sample in the carry-over study.
B. 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’s performance to the established reference methods of culture and mecA-mediated oxacillin resistance determination tests using cefoxitin.3
We recommend that you assess the ability of your device to detect MRSA/SA in fresh specimens from the intended use patient population.
1. Study Protocol
We recommend that you develop a detailed study protocol that includes the specific patient inclusion and exclusion criteria, the type and number of specimens needed, the directions for use, and a statistical analysis plan that accounts for variances to prevent data bias. We recommend that you 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 prior to study initiation. This is referred to as the pre-IDE [Ref. 18] process.
2. Specimen Type(s)
- The total number of samples you should include in your study for substantiating a claim for detection and differentiation of MRSA and SA will depend on the prevalence of the bacteria in your clinical study patient population and on device performance. We recommend that a ll MRSA/SA devices demonstrate specificity results of at least 95% with a lower 95% (two-sided) confidence bound exceeding 90%.
- For devices that are intended to be used to aid in the prevention and control of MRSA infections in healthcare settings by testing nasal swab specimens from patients that are eligible for MRSA screening according to institutional policies, and for devices that are intended to be used to aid in the diagnosis of MRSA/SA from skin and soft tissue infections when they are used in conjunction with other laboratory tests such as microbiology culture and clinical data available to the clinician, we recommend that you include a sufficient number of prospectively collected samples to generate a sensitivity result of at least 90% with a lower bound of the two-sided 95% confidence interval (CI) greater than 85% (all clinical sites combined, provided that appropriate statistical analysis demonstrates that the stratified data by clinical site can be pooled). Generally, we recommend testing a minimum of 50 samples determined to be positive using the reference method per clinical site.
- For devices that are intended to detect MRSA/SA by evaluating Gram Positive Cocci (GPC) from blood culture bottles when they are used in conjunction with Gram stain, we recommend that you include a sufficient number of prospectively collected samples to generate a sensitivity result of at least 95% with a lower bound of the two-sided 95% confidence interval (CI) greater than 92.5% (all clinical sites and all blood culture bottle types combined, provided that appropriate statistical analysis demonstrates that the stratified data by clinical site and bottle type can be pooled). In general, at least 20 MRSA positives per claimed blood culture bottle type should be included in the clinical study. If the data, 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 are not considered equivalent and cannot be pooled, but rather should be analyzed separately. If this occurs, additional prospective clinical samples will need to 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 all the claimed blood culture bottle types would be tested at a single site. The study should consist of spiking samples into each of the claimed blood culture bottle types at the LoD level previously determined in the analytical LoD study described in the “Limit of Detection” section of this guidance. 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.
If you have questions regarding the choice of appropriate specimen numbers for the clinical study, please contact the Division of Microbiology Devices.
3. Study Sites
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. One of the three study sites may be in-house. Clinical investigations of unapproved and uncleared in vitro diagnostic devices, including diagnostic devices for MRSA/SA, 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 a clinical laboratory with more experienced and trained personnel, in addition to testing in 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.
4. Study Population
For devices that are intended to be used to aid in the prevention and control of MRSA infections in healthcare settings by testing nasal swab specimens, we recommend that you conduct your studies on individuals at risk for MRSA colonization in healthcare settings, and therefore are eligible for MRSA screening according to institutional policies that include, but are not limited to, all patients admitted into a particular healthcare system; patients admitted to an Intensive Care Unit; patients transferred to an Intensive Care Unit; pre-elective surgery patients; and patients being admitted from long term care facilities.
For devices that are intended to be used to aid in the diagnosis of MRSA/SA from skin and soft tissue infections when they are used in conjunction with other laboratory tests such as microbiology culture and clinical data available to the clinician, we recommend that you conduct your studies on patients whose routine care called for collection of a swab from the patient's skin and soft tissue infection for culture testing.
For devices that are intended to detect MRSA/SA by evaluating Gram Positive Cocci (GPC) from blood culture bottles when they are used in conjunction with Gram stain, we recommend that you conduct your clinical studies on patients on whom healthcare providers requested blood culture testing be performed for routine patient care. Patient inclusion criteria may include, but are not limited to, a positive blood culture specimen taken from a specific type or types of blood culture bottle of the commonly marketed blood culture systems (e.g., BD BACTEC blood culture system, bioMérieux BacT/ALERT blood culture system, or Trek Diagnostic Systems VersaTREK Automated Microbial Detection System), and Gram stain contained Gram Positive Cocci (GPC).
In order to preserve the true prevalence of MRSA/SA in your clinical study patient population, patients previously enrolled in the clinical study should be denied repeat entry into the same clinical study. History of study patient’s antibiotic use should be collected and recorded if it is available and reliable.
5. Reference Methods
For devices that test nasal swab specimens, we recommend that you compare results obtained with your device to the results obtained by using one or more of the following established comparative reference methods to claim sensitivity and specificity: (1) Culture method consisting of an initial enrichment process in Trypticase Soy Broth (TSB) with 6.5% Sodium Chloride followed by subculture on Blood Agar (BA) of turbid growth from TSB. Identification and confirmation of presumptive S. aureus colonies recovered from the subcultured BA plate should be performed using appropriate FDA cleared tests. Susceptibility testing should be performed on all confirmed Staphylococcus aureus colonies in accordance with the CLSI documents M2-A10 [Ref. 19] and M100-S19 (or the most current M100) [Ref. 20], using the Cefoxitin disc testing to detect methicillin/oxacillin resistance. (2) Culture method consisting of an initial analysis on FDA cleared Staphylococcus aureus selective chromogenic media followed by subculture on Blood Agar (BA) of presumptive S. aureus colonies. In all instances when methicillin resistant S. aureus is not recovered using this method, enrichment of the original specimen in TSB with 6.5% Sodium Chloride should be employed, followed by subculture to Blood Agar (BA). Identification and confirmation of presumptive S. aureus colonies recovered from the subcultured BA plate should be performed using appropriate FDA cleared tests. Susceptibility testing should be performed on all confirmed Staphylococcus aureus colonies as described previously in method (1). If the original swab is not available for enrichment in TSB, then this alternate method (2) should not be used.
For devices that test skin and soft tissue infections (SSTI) specimens, we recommend that you compare results obtained with your device to the results obtained by using the following established comparative reference method to claim sensitivity and specificity: Culture method consisting of an initial enrichment process in Trypticase Soy Broth (TSB) with 6.5% Sodium Chloride followed by subculture on Blood Agar (BA) of turbid growth from TSB. Identification and confirmation of presumptive S. aureus colonies recovered from the subcultured BA plate should be performed using appropriate FDA cleared tests. Susceptibility testing should be performed on all confirmed Staphylococcus aureus colonies in accordance with the CLSI documents M2-A10 [Ref. 19], Performance Standards for Antimicrobial Disk Susceptibility Tests, and M100-S19 (or the most current M100) [Ref. 20], Performance Standards for Antimicrobial Susceptibility Testing, using the Cefoxitin disc testing to detect methicillin/oxacillin resistance.
For devices that test samples from blood culture bottles that are detected as positive for microbial growth and shown to contain Gram Positive Cocci (GPC) by Gram stain, we recommend that you compare results obtained with your device to the results obtained by using the following established comparative reference method to claim positive and negative percent agreement: Subculture the positive blood culture specimen (Gram stain showing GPC) onto a non-selective Blood Agar Plate (BAP), and incubate the plate following the product package insert recommendations for S. aureus. Suspicious colonies of S. aureus should be identified by using appropriate laboratory methods (e.g., Catalase, Tube or Slide coagulase and Gram stain). Confirmed S. aureus colonies should be tested for methicillin resistance using the CLSI recommended reference method (e.g., using cefoxitin, disk diffusion or broth microdilution). Refer to CLSI M35-A2 [Ref. 21], Abbreviated Identification of Bacteria and Yeast, for information on identification testing, and M100-S19 (or the most current M100) [Ref. 20] , Performance Standards for Antimicrobial Susceptibility Testing, for detailed information on Staphylococcus spp. susceptibility testing.
You can also compare results obtained with your device to the results obtained by using an FDA cleared MRSA/SA predicate device to present your device performance as positive and negative percent agreements against such an FDA cleared predicate device in addition to presenting your device performance as sensitivity and specificity (or positive and negative percent agreement) against the comparative reference methods described above.
C. CLIA Waiver
If you are seeking a waiver for your device under the Clinical Laboratory Improvement Amendments of 1988 (CLIA),4 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.”
- Calfee DP, Salgado CD, Classen D, et al. Strategies to prevent transmission of methicillin-resistant Staphylococcus aureus in acute care hospitals. Infect Control Hosp Epidemiol. 2008 Oct;29 (Suppl 1):S62-80.
- Muto, C. A.. J. A. Jernigan, B. E. Ostrowosky, H. M. Richet, W. R. Jarvis, J. M. Boyce, B. M. Farr. SHEA guideline for preventing nosocomial transmission of multidrugresistant strains of Staphylococcus aureusand Enterococcus.Infect. Control and Hospital Epidemiol. 2003;May;24(5):362-386.
- Cosgrove SE, Qi Y, Kaye KS, Harbarth S, Karchmer AW, Carmeli Y. The impact of methicillin resistance in Staphylococcus aureus bacteremia on patient outcomes: mortality, length of stay, and hospital charges. Infect. Control and Hospital Epidemiol. 2005; Feb;26(2):166-174.
- Klevens RM, Edwards JR, Tenover FC, et al. Changes in the epidemiology of methicillin-resistant Staphylococcus aureus in intensive care units in US hospitals, 1992-2003. Clin Infect Dis. 2006;42(3):389-391.
- Noskin GA, Rubin RJ, Schentag JJ, et al. National trends in Staphylococcus aureus infection rates: impact on economic burden and mortality over a 6-year period (1998-2003). Clin Infect Dis. 2007;45(9):1132-1140.
- Klein E, Smith DL, Laxminarayan R. Community-associated Methicillin-Resistant Staphylococcus aureus in Outpatients, United States, 1999-2006. Emerging Infectious Diseases. 2009;15(12):1925-1930.
- Hardy K, Price C, Szczepura A, et al. Reduction in the rate of methicillin-resistant Staphylococcus aureus acquisition in surgical wards by rapid screening for colonization: a prospective, cross-over study. Clin Microbiol Infect. 2010 Apr;16(4):333-339. Epub 2009 Jul 20.
- Class II Special Controls Guidance Document: Instrumentation for Clinical Multiplex Test Systems.
- Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices.
- Clinical and Laboratory Standards Institute. 2006 Molecular Diagnostic Methods for Infectious Disease; Proposed Guideline-Second Edition. MM3-A2. Clinical and Laboratory Standards Institute, Wayne PA.
- Clinical and Laboratory Standards Institute. 2001. Specifications for Immunological Testing for Infectious Diseases; Approved Guideline-Second Edition. ILA18-A2. Clinical and Laboratory Standards Institute, Wayne PA.
- Clinical and Laboratory Standards Institute. 2004. Protocol for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline. EP17-A. Clinical and Laboratory Standards Institute, Wayne PA.
- Clinical and Laboratory Standards Institute. 2005. Interference Testing in Clinical Chemistry; Approved Guideline-Second Edition. EP7-A2. Clinical and Laboratory Standards Institute, Wayne PA.
- Clinical and Laboratory Standards Institute. 1995. Assessment of the Clinical Accuracy of Laboratory Tests Using Receiver Operating Characteristics (ROC) Plots; Approved Guideline. GP10-A. Clinical and Laboratory Standards Institute, Wayne PA.
- Clinical and Laboratory Standards Institute. 2004. Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline-Second Edition. EP5-A2. Clinical and Laboratory Standards Institute, Wayne PA.
- Clinical and Laboratory Standards Institute. 2002. User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline. EP12-A. Clinical and Laboratory Standards Institute, Wayne PA.
- Clinical and Laboratory Standards Institute. 2006. User Verification of Performance for Precision and Trueness; Approved Guideline-Second Edition. EP15-A2. Clinical and Laboratory Standards Institute, Wayne PA.
- Center for Devices and Radiological Health, Office of Device Evaluation.
January 20, 1998. Guidance on IDE Policies and Procedures.
- Clinical and Laboratory Standards Institute. 2009 . Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Tenth Edition. M2-A10. Clinical and Laboratory Standards Institute, Wayne PA.
- Clinical and Laboratory Standards Institute. 2009. Performance Standards for Antimicrobial Susceptibility Testing; Nineteenth Informational Supplement. M100-S19. Clinical and Laboratory Standards Institute, Wayne PA.
- Clinical and Laboratory Standards Institute. 2008. Abbreviated Identification of Bacteria and Yeast; Approved Guideline-Second Edition. M35-A2. Clinical and Laboratory Standards Institute, Wayne PA.
1 “Empty Cassette Variants” are MRSA strains that are of the methicillin-sensitive phenotype due to genetic excisions within the SCCmec region of the MRSA the genome.
2 Point-of-care tests, also known as bedside or near-patient tests, is a term that encompasses any diagnostic testing near the site of patient care regardless of whether the device is intended for use by a trained medical professional or by a lay user. The person conducting the diagnostic testing near the point of care, whether a trained medical professional or a lay user, is the caregiver in that instance as that person is the person providing care.
3 Comparing performance of a new assay against an established reference method creates a frame of reference for evaluating the device that is useful whether the data is to be considered in an initial classification action or to facilitate comparison with the performance of a predicate device, in the case of a premarket notification and evaluation of substantial equivalence.
4 See 42 U.S.C. § 263a(d)(3).