LIGHTCYCLER MRSA ADVANCED TEST FOR USE WITH THE LIGHTCYCLER 2.0 INSTRUMENT
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LightCycler® MRSA Advanced Test
For Use With The LightCycler® 2.0 Instrument
FOR IN VITRO DIAGNOSTIC USE.
®
LightCycler MRSA Advanced Test LC MRSA 96 Tests P/N: 05352894 190
LightCycler® Advanced Lysis Kit 96 Tests P/N: 05205743 190
LYS ADV
INTENDED USE
The LightCycler® MRSA Advanced Test is a qualitative in vitro diagnostic test for the direct detection of
nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) to aid in the prevention and
®
control of MRSA infections in healthcare settings. The test is performed on the LightCycler 2.0
Instrument with nasal swab specimens from patients suspected of colonization, uses swab extraction and
mechanical lysis for specimen preparation followed by polymerase chain reaction (PCR) for the
amplification of MRSA DNA, and fluorogenic target specific hybridization probes for the detection of the
amplified DNA.
The LightCycler® MRSA Advanced Test is not intended to diagnose, guide or monitor treatment for MRSA
infections. Concomitant cultures are necessary to recover organisms for epidemiology typing or for further
susceptibility testing.
SUMMARY AND EXPLANATION OF THE TEST
Staphylococcus aureus is an opportunistic pathogen carried as a commensal organism on the skin and
nares of approximately 30% of the normal population, with the potential to cause a broad spectrum of
diseases, fatal within 30 days of infection in approximately 34% of cases1,2. S. aureus can rapidly adapt to
the selective pressure from antibiotic treatments, which has resulted in the emergence and spread of
methicillin-resistant S. aureus (MRSA) strains. The mecA gene is located on a large mobile genetic
element called the Staphylococcal Cassette Chromosome mec (SCCmec), and is responsible for resistance
to methicillin and to other β-lactam antibiotics. The mecA gene encodes for an altered penicillin binding
protein, PBP2a or PBP2’, which has a very low affinity for all β-lactam antibiotics allowing normal synthesis
of the peptidoglycan layer to occur, even in the presence of these antibiotics3. Numerous MRSA strains
have emerged and spread worldwide, and SCCmec has been acquired by different lineages of methicillin-
sensitive S. aureus4. To date, five SCCmec types (I–V) have been distinguished, and several variants of
these SCCmec types have been described5.
Healthcare associated infections (HAIs) caused by MRSA have recently become an important issue for
healthcare facilities worldwide due to high rates of infection, mortality, and high costs of treatment6-9. In
addition, community-associated MRSA (CA-MRSA) has spread in the past few years10, feeding the
pipeline of infection in hospitals,11, 12 and underscoring the need for comprehensive infection control
programs. In 2008, recommendations from published guidelines of several governmental, professional, and
public health organizations were summarized to help institutions prioritize and implement their MRSA
transmission prevention efforts13. Any program to prevent MRSA transmission must comprise a number of
key strategies, including active surveillance screening, hand hygiene, patient isolation, and other
transmission prevention activities. Real-time PCR detection of MRSA significantly reduces time to
The Document Revision Information section is located at the end of this document.
06369120001-01EN 1 Doc Rev. 1.0detection (under 2 hours of laboratory time) compared with standard culture tests and, therefore,
improves the efficiency of control and management strategies.
PRINCIPLES OF THE PROCEDURE
Detection of MRSA with the LightCycler® MRSA Advanced Test relies on 3 major processes:
• Specimen preparation by mechanical lysis of the bacterial cell walls
• PCR amplification of target DNA and detection by specific hybridization probes
• Automated result generation after melting peak analysis
Specimen Preparation
®
The mechanical lysis of the nasal swab specimens is performed by using the LightCycler Advanced Lysis
Kit and the MagNA Lyser Instrument. Swab heads are cut into lysis tubes and subjected to heating to
inactivate all bacteria. Subsequently lysis tubes are transferred in the MagNA Lyser Instrument where
mechanical lysis of bacterial cell walls occurs, resulting in crude lysate preparations. After a brief
centrifugation step to spin down glass beads and swab fibers, the processed specimen are subjected to
PCR analysis using the LightCycler® MRSA Advanced Test.
PCR Amplification
Target Selection
The primers and probes in the LightCycler® MRSA Advanced Test detect a proprietary sequence indicative
of the integration of the SCCmec cassette into the S. aureus chromosome, indicating the presence of
MRSA DNA.
Amplification
The processed samples and the amplification mixture containing hot-start Taq polymerase are placed in
LightCycler® Capillaries (20μL) in which PCR amplification will occur. Each LightCycler® MRSA Advanced
Test reaction contains an internal control, which is designed to control for specimen inhibition, and to
®
monitor reagent integrity. The AmpErase (uracil-N-glycosylase) enzyme included in the LightCycler
MRSA Advanced Test recognizes and catalyzes the destruction of DNA strands containing deoxyuridine,
but not DNA containing deoxythymidine. Since amplicons produced with the LightCycler® MRSA
Advanced Test contain deoxyuridine, potential amplicon contaminants are eliminated during a prolonged
heating step performed prior to the start of PCR amplification.
A target sequence in a plasmid is simultaneously amplified in the Positive Control. The Positive Control is
intended to monitor for reagent failure and is included into each run. Each run also includes a Negative
Control used to detect reagent or environmental contamination by MRSA DNA.
Specific detection of PCR products by Hybridization Probes
MRSA and Internal Control amplicons are detected by fluorescence using a specific pair of hybridization
probes. The probes attach to a specific internal sequence in the amplified fragment and are positioned in
a closed proximity to one another. Upon excitation, these bound probes emit a fluorescence signal of a
specific wavelength using a process called Fluorescence Resonance Energy Transfer (FRET). The emitted
light is measured by the LightCycler® 2.0 Instrument. MRSA or internal control specific amplicons are
detected in parallel in two different detection channels and thus can be differentiated.
After completion of the real-time PCR process, a melting peak analysis is performed automatically by the
®
LightCycler 2.0 Instrument. Single stranded DNA amplicons with bound hybridization probes are
subjected to increasing temperatures. When the PCR products reach a specific temperature one of the
two bound hybridization probes melts off, resulting in a loss of fluorescence signal. The decrease in the
fluorescence signal occurs at a specific temperature and results in melting peaks which are used to
identify and distinguish MRSA- and IC-specific amplicons.
Automated result generation after melting peak analysis
After a visual identification of the melting peaks is performed using TM Bars in the LightCycler® Software,
test results are transferred to a dedicated interpretation tool (the Micro Analysis Software) and a report is
generated.
06369120001-01EN 2 Doc Rev. 1.0REAGENTS
LightCycler® Advanced Lysis Kit LYS ADV 96 Tests
(P/N: 05205743 190)
LYS 96 x 0.6 mL
(Lysis Reagent)
Tris buffer
EDTA
< 15% Silica beads
0.09% Sodium azide
LightCycler® MRSA Advanced Test LC MRSA 96 Tests
(P/N: 05352894 190)
MRSA RXNMX 3 x 0.352 mL
(MRSA Reaction Mix)
Tris bufferWARNINGS AND PRECAUTIONS
A. FOR IN VITRO DIAGNOSTIC USE. Use of this product should be limited to personnel trained in
the techniques of PCR.
B. This test is for use with nasal swab specimens only.
C. Do not pipette by mouth.
D. Do not eat, drink or smoke in laboratory work areas. Wear protective disposable gloves, laboratory
coats and eye protection when handling specimens and kit reagents. Wash hands thoroughly after
handling specimens and test reagents.
E. Avoid microbial contamination of reagents when removing aliquots from reagent bottles. The use of
sterile disposable pipette tips is recommended.
F. Do not pool reagents from different lots or from different bottles of the same lot.
G. Do not mix reagents from different kit lots.
H. Dispose unused reagents, waste and specimens in accordance with country, federal, state and local
regulations.
I. Do not use a kit after its expiration date.
J. Material Safety Data Sheets (MSDS) are available on request from your local Roche office.
K. Specimens and controls should be handled as if infectious using safe laboratory procedures such
as those outlined in Biosafety in Microbiological and Biomedical Laboratories14 and in the CLSI
Document M29-A315. Thoroughly clean and disinfect all work surfaces with a freshly prepared
solution of 0.5% sodium hypochlorite in deionized or distilled water.
Note: Commercial liquid household bleach typically contains sodium hypochlorite at a
concentration of 5.25%. A 1:10 dilution of household bleach will produce a 0.5% sodium
hypochlorite solution.
L. LYS, MRSA RXNMX, MRSA DETMX and MRSA (+) C contain sodium azide. Sodium azide may
react with lead and copper plumbing to form highly explosive metal azides. While disposing of
sodium azide-containing solutions down laboratory sinks, flush the drains with a large volume of
water to prevent azide buildup.
M. Wear eye protection, laboratory coats and disposable gloves when handling any reagent. Avoid
contact of these materials with the skin, eyes or mucous membranes. If contact does occur,
immediately wash with large amounts of water. Burns can occur if left untreated. If spills of these
reagents occur, dilute with water before wiping.
N. Contamination from positive controls and positive specimens can be avoided by good laboratory
practices and careful adherence to the procedures specified in this package insert.
STORAGE AND HANDLING REQUIREMENTS
®
A. Store the LightCycler Advanced Lysis Kit (LYS) upright at 15 to 25ºC upon arrival.
B. Store the LightCycler® MRSA Advanced Test Kit (MRSA RXNMX, MRSA DETMX and
MRSA (+) C) at -15 to -25ºC upon arrival. Unopened, these reagents are stable until the expiration
date indicated. Once opened, store the remaining MRSA RXNMX and MRSA DETMX at -15 to
-25ºC for up to 30 days. MRSA RXNMX and MRSA DETMX can sustain up to 5 freeze-thaw
cycles.
C. Once opened, store the remaining MRSA (+) C at -15 to -25ºC or 2 to 8ºC for up to 30 days. The
MRSA (+) C can sustain up to 22 freeze-thaw cycles.
D. Protect the MRSA DETMX from light exposure.
E. Working Master Mix (prepared by addition of MRSA RXNMX and MRSA DETMX) must be
prepared fresh and utilized within 24 hours of preparation. If the Working Master Mix is not used
06369120001-01EN 4 Doc Rev. 1.0immediately following preparation, the Working Master Mix must be stored away from light at 2 to
8°C for up to 24 hours.
F. Processed specimens and negative controls prepared from Liquid Stuart swabs are stable for up to
1 day at 15 to 25ºC or 2 to 8°C or up to 8 months at -15 to -25°C. Processed specimens and
negative controls prepared from Amies gel swabs (with or without charcoal) are stable for up to 5
days at 15 to 25ºC or 2 to 8°C or up to 3 months at -15 to -25°C.
G. Amplification should be started immediately after addition of the processed specimens and controls
to the Working Master Mix. If this is not possible, store the filled capillaries away from light for a
maximum 3 hours at 2 to 8°C.
MATERIALS PROVIDED
®
A. LightCycler Advanced Lysis Kit LYS ADV
(P/N: 05205743 190)
LYS
(Lysis Reagent)
®
B. LightCycler MRSA Advanced Test LC MRSA
(P/N: 05352894 190)
MRSA RXNMX
(MRSA Reaction Mix)
MRSA DETMX
(MRSA Detection Mix)
MRSA (+) C
(MRSA Positive Control)
MATERIALS REQUIRED BUT NOT PROVIDED
Instrumentation and Software
• ®
LightCycler 2.0 Instrument (P/N: 03 531 414 201)
Note: For the USA, different catalog numbers do apply. Please contact the US sales
office for details. (Serial numbers 1415001 and higher are approved for IVD use and use
with this kit.)
• LightCycler® Software 4.1 (P/N: 04 898 915 001) or higher
• ®
LightCycler MRSA Advanced Software Set v1.0 CE (P/N: 05 414 326 190) or higher
• LC Carousel Centrifuge 2.0 [P/N: 03 709 582 001 (230 V) or P/N: 03 709 507 001 (110V)]
Note: If you already have purchased a LC Carousel Centrifuge [P/N 12 189 682 001 (230
V) or P/N 03 030 512 001 (115V)], a LightCycler® Carousel 2.0 rotor set [P/N 03 724 697
001] is also required
• Or standard bench top microcentrifuge containing a rotor for 2.0 mL tubes, and LC
centrifuge adapters [P/N: 11 909 312 001]
• MagNA Lyser Instrument [P/N: 03 358 976 001 (230 V) or 03 358 968 001 (110 V)]
• Optional barcode reader [P/N: 05 536 421 001]
Reagents and Disposables
• LightCycler® Multicolor Compensation Set (P/N: 04 484 355 001)
• ®
LightCycler Capillaries, 20 μL (P/N: 04 929 292 001)
06369120001-01EN 5 Doc Rev. 1.0OTHER MATERIALS REQUIRED BUT NOT PROVIDED
• BD CultureSwab™ Liquid Stuart (BD P/N: 220099 or 220109), or Copan Venturi
Transystem™ Liquid Stuart (Copan Italia International P/N: 141C or 139C);
• Or BD CultureSwab™ plus Amies gel without charcoal (BD P/N: 220116 or 220117), or
Copan Venturi Transystem™ plus Amies gel without charcoal (Copan Italia International
P/N: 108C or 134C);
• Or BD CultureSwab™ plus Amies gel with charcoal (BD P/N: 220121 or 220122), or
Copan Venturi Transystem™ plus Amies gel with charcoal (Copan Italia International
P/N: 114C or 136C)
• Disposable gloves, powderless
• Gauze
• Ethanol (70 %)
• Adjustable Pipettors*: ( capacities: 10 μL, 100 μL and 1000 μL) with sterile, DNase-free,
filter-blocked or positive displacement micropipettor tips
• Dry heating block set at 95 ºC (± 2 ºC): VWR (P/N: 12621-088) or equivalent with 2.0mL
tube block : VWR (P/N: 12985-050)
• Microcentrifuge capable of delivering 8,000 - 20,000 x g centrifugal force with rotor
appropriate for 2.0 mL screw cap tubes: (P/N: Eppendorf model 5417C) or equivalent
• Sterile 1.5 mL Siliconized polypropylene microcentrifuge tubes: (P/N: Eppendorf order
Nr. 0030 108.051) or equivalent
• Vortex mixer: Fisher (P/N: 12-812) or equivalent
• Optional microtube tool: VWR (P/N: 20170-688) or Rainin (P/N: PJ-4A) or equivalent
* Pipettors should be accurate within 3% of stated volume. Aerosol barrier or positive displacement
DNase-free tips must be used where specified to prevent specimen and amplicon cross-contamination.
SPECIMEN COLLECTION, TRANSPORT AND STORAGE
Note: Handle all specimens and controls as if they are capable of transmitting infectious agents.
A. Specimen Collection
1. Moisten the swab with two drops (approximately 50 μL) of sterile saline or use dry.
2. Gently insert the swab inside the nostril approximately 0.5 inch or 1.3 cm (both swab heads at once
in the case of a double headed swab) and rotate against the mucosa five times while applying light
pressure on the outside of the nose (to help insure contact of the swab heads with the inside of the
nose).
3. Insert the same swab into the other nostril and repeat the procedure.
4. Return the swab to its container and label appropriately.
B. Specimen Transport and Storage
Transportation of specimens must comply with country, federal, state and local regulations for the
transport of etiologic agents16. Specimens can be transported at 15 to 25 ºC.
Specimens may be stored before processing (including time needed for transport) as defined below.
06369120001-01EN 6 Doc Rev. 1.0• Specimen taken with Liquid Stuart swabs is stable
– at 15 to 25 ºC for up to 4 days,
– at 2 to 8 ºC for up to 5 days,
– at -15 to -25°C for up to 1 month.
• Specimen taken with Amies gel swabs with or without charcoal is stable
– at 15 to 25°C for up to 4 days.
INSTRUCTIONS FOR USE
Instrument Preparation
A. Installation of the Micro Analysis Software (MAS)
For installation of updates, check whether additional information is provided with the product.
®
1. Login as an Administrator on the LightCycler Control Unit
®
2. Insert the LightCycler MRSA Advanced Software Set v1.0 CE CD (or higher) into the CD-drive of
the LightCycler® Control Unit.
Note If the program setup does not start automatically, start the installation of MAS manually by
double clicking on the setup.exe file provided on the LightCycler® MRSA Advanced
Software Set v1.0 CE CD (or higher).
3. For first time installation:
a. if the Microsoft Data Access Components 2.8 is not already installed, you will be prompted
to install it at the beginning of the MAS setup. The Microsoft Data Access Components 2.8
must be installed, otherwise MAS will not run properly.
b. if the Microsoft .NET Framework 2.0 (x86) is not already installed, you will be prompted to
install it at the beginning of the MAS setup. The Microsoft .NET Framework 2.0 (x86) must
be installed, otherwise MAS will not run properly.
c. if the Microsoft Windows Installer 3.1 is not already installed, you will be prompted to install
it at the beginning of the MAS setup. The Microsoft Windows Installer 3.1 must be installed,
otherwise MAS will not run properly.
d. if the Microsoft Visual Studio 2008 Report Viewer is not already installed, you will be
prompted to install it at the beginning of the MAS setup. The Microsoft Visual Studio 2008
Report Viewer must be installed, otherwise MAS will not run properly.
e. if the Microsoft SQL Server 2005 Express SP2 (x86) is not already installed, you will be
prompted to install it at the beginning of the MAS setup. The Microsoft SQL Server 2005
Express SP2 (x86) must be installed, otherwise MAS will not run properly.
4. If you are requested to reboot, confirm with .
®
Note: Do not remove the LightCycler MRSA Advanced Software Set CD during the rebooting
process.
5. After rebooting, follow the instructions of the installation wizard. Press to start installation
of MAS.
6. Select if only one user will work on the LightCycler® Control Unit, select
if more than one user will work on the LightCycler® Control Unit.
7. Acknowledge that the MAS installation was successful by clicking on in the
window.
8. Launch the application using the MAS icon on the computer’s desktop.
06369120001-01EN 7 Doc Rev. 1.09. The data folder for *.ixo and *.pdf files is automatically created upon start of
the MAS.
B. Verifying the installation of the Micro Analysis Software (MAS)
® ®
1. Copy the proof files (LightCycler MRSA Advanced Proof File v1.0 CE.ixo and LightCycler MRSA
Advanced Proof File v1.0 CE.pdf, or higher) from the LightCycler® MRSA Advanced Software Set
v1.0 CE CD (or higher) into the folder.
2. If the MAS is not already open, double click on the MAS icon on desktop, and press .
3. Select the file MRSA Advanced Proof File v1.0 CE.ixo (or higher) in the folder.
®
4. Click on , indicating that the LightCycler report does not contain the flag .
5. Enter Lot No. Expiration Date and Lab Info into the appropriate fields of the MRSA Configuration
Settings window.
Advanced Lysis Kit
Lot No 11A00000
Expiration Date 12201812
MRSA Advanced
Test
Lot No 23A00000
Expiration Date 24201812
Personalized Info
Lab Info Roche Lab
6. Select the report type and proceed with . The file will be analyzed
automatically and the data will be displayed.
7. Print the report.
8. Navigate to the folder, open and print the MRSA Advanced Proof File v1.0
CE.pdf (or higher).
9. Insure that the printed MAS report and the printed PDF provide the same results. If not, please
contact your local Roche office for technical assistance.
C. Removal of the Micro Analysis Software (MAS)
1. Login as an administrator on the LightCycler® Control Unit.
®
2. Click on in the taskbar of the LightCycler Control Unit.
3. Choose / / in the bottom left corner of
Windows desktop.
4. A list of all currently installed programs will appear, click on .
5. Choose and confirm by clicking on .
06369120001-01EN 8 Doc Rev. 1.0Note: The MRSA Advanced Software Set CD provides an uninstall and a repair feature. The
uninstall feature can be used optional to the described process. The repair feature can be
used to recover corrupted or missing files of the Micro Analysis Software.
D. Installation of the LightCycler® MRSA Advanced Macros
1. Install the *.lckit macros LightCycler® MRSA Advanced MCC Macro v1.0 (MRSA
®
MCC_04484355001A_1.0.Ickit) or higher and LightCycler MRSA Advanced Macro v1.0 CE
(MRSA_05352894190_1.0. Ickit) or higher from the LightCycler® MRSA Advanced Software Set v1.0
CE CD (or higher).
2. Please refer to the LightCycler® 2.0 Instrument Operator’s Manual A, “Executing a Roche Macro”
chapter.
®
E. Multicolor Compensation Experiment using the LightCycler Multicolor Compensation Set
®
For Use with the LightCycler 2.0 Instrument
Use the following steps to create a new or to replace an expired Color Compensation file.
1. Thaw vials 1,2, 3 and 5; vortex gently and spin down briefly.
2. Prepare a 3 fold dilution of vial 2 by mixing together:
a. 10 μL of solution contained in vial 2
b. 20 μL of buffer from a tube of LYS without transferring glass beads
3. Pipette 10 μL of each component into a separate LightCycler® capillary (20 μL).
a. Capillary #1: Vial 1
b. Capillary #2: dilution step E2
c. Capillary #3: Vial 3
d. Capillary #4: Vial 5
Note: Discard dilution of vial 2 after run is completed.
4. Pipette 10 μL of buffer from a tube of LYS (without transferring glass beads) into each capillary to
reach a total volume of 20 μL.
5. Seal each capillary with a stopper using the Capping Tool.
6. Centrifuge the capillaries using the LC Carousel Centrifuge 2.0 or a standard centrifuge and
centrifuge adapters. The tubes must be in the following positions in the Carousel:
a. Carousel position 1: CCB (vial 1)
b. Carousel position 2: CC530 (vial 2)
c. Carousel position 3: CC610 (vial 3)
d. Carousel position 4: CC670 (vial 5)
Note: Do not reduce the volume used for each capillary.
® ®
7. Transfer the LightCycler Sample Carousel to the LightCycler 2.0 Instrument. If a regular
centrifuge was used, transfer the capillaries into the LightCycler® Sample Carousel in the order
described in step E6.
Note: Do not change the order of the reagents in the Carousel.
8. Start and save the Color Compensation Experiment (CCE)
06369120001-01EN 9 Doc Rev. 1.0®
a. For use of the Light Cycler MRSA Advanced MCC Macro please refer to the
®
LightCycler 2.0 Instrument Operator’s Manual A, “Executing a Roche Macro”
chapter.
Note: Do not use the field . If you want to add the assay lot number to the
experiment, insert information into the appropriate field in the sample editor.
b. Save the run using a short unique name (e.g. MRSA_CCE_YYMMDD-xx, Y=year,
M=month, D=day, xx=run number).
c. After the run is finished, a report is automatically created.
d. Print the report.
9. Run Validation
The report will not indicate, if the Color Compensation Experiment was successful. The success of
the Color Compensation Experiment is only established upon applying the Color Compensation
Object generated from the CCE to the initial MRSA Advanced run.
® ®
a. If the LightCycler Software (LCS) report for the LightCycler Color Compensation
Experiment is flagged (e.g., ), then the
CCE is invalid. Repeat the Color Compensation Experiment (from step E1).
b. If the report of the LightCycler® Color Compensation Experiment does not contain a
flag, proceed to the next step.
10. Create a Color Compensation Object (CCO)
a. Activate Color Compensation analysis module, and press button .
b. Create a short unique name using the mandatory naming convention for the Color
Compensation Object: MRSA_CCO_YYMMDD-xx (Y=year, M=month, D=day,
xx=run number).
®
c. This object is used for further analysis of assays with the LightCycler MRSA
Advanced Test.
Specimen and Control Preparation
A. Specimen Preparation
1. Label one LYS tube appropriately for each patient specimen.
Note: Do not mark tubes on the top of the cap.
2. Remove the swab from the transport container.
3. Place a single swab head in a LYS tube.
Note: If a single headed swab is used, a direct culture may be performed by streaking the swab
onto the plate before placing it into the LYS tube.
4. Hold the swab by the stem near the rim of the tube, lift the swab a few millimeters from the bottom
and bend the stem against the rim of the tube to break it.
Note: Use a gauze to minimize risk of contamination when breaking the swab. Use a new gauze
for each swab.
5. Close the LYS cap tightly.
06369120001-01EN 10 Doc Rev. 1.0B. Negative Control Preparation
Note: The NC should be prepared at the same time as the samples to be tested.
1. Label one LYS tube appropriately for negative control.
Note: Do not mark tubes on the top of the cap.
2. Place a sterile swab into its transport container. Refer to Other Materials Required But Not Provided
for a list of recommended swabs.
3. Incubate at room temperature for at least 1 minute.
4. Remove the swab from the transport container.
5. Place a single swab head in a LYS tube.
6. Hold the swab by the stem near the rim of the tube, lift the swab a few millimeters from the bottom
and bend the stem against the rim of the tube to break it.
Note: Use a new gauze to minimize risk of contamination when breaking the swab.
7. Close the LYS cap tightly.
C. Specimen and Control Processing
1. Heat specimens and negative control at 95 ± 2 ºC for 2 minutes in a dry heating block.
2. Lyse specimen and negative control in the MagNA Lyser instrument for 70 seconds at a speed of
5,000 rpm. Refer to the MagNA Lyser Operator’s Manual for details on how to use.
3. Centrifuge specimen and negative control at 8,000 – 20,000 x g at room temperature for 1 minute.
Note: Processed specimens and negative controls prepared with Liquid Stuart swabs are stable
for up to 1 day at 15 to 25ºC or 2 to 8°C or up to 8 months at -15 to -25°C. Processed
specimens and negative controls prepared with Amies gel swabs (with or without charcoal)
are stable for up to 5 days at 15 to 25ºC or 2 to 8°C or up to 3 months at -15 to -25°C.
Reagent Preparation
1. Thaw MRSA RXNMX and MRSA DETMX and MRSA (+) C at room temperature. Ensure the
reagents are completely thawed before use.
2. Vortex MRSA RXNMX and MRSA DETMX and MRSA (+) C for 3-5 seconds and centrifuge
briefly to collect material at the base of the tube.
3. Prepare the Working Master Mix for the number of specimens and controls being tested into a
sterile siliconized polypropylene microcentrifuge tube. (Use Table 1 as a guide). Each run must
contain a minimum of one specimen, one PC, and one NC.
Note: The recommended volumes include 2 reactions for the negative control and the positive
control and also an additional volume to account for dead volume and facilitate pipetting
out of the mixing tube and into LC capillaries.
Note: Use Microtube Tool (VWR P/N 20170-688 or Rainin P/N PJ-4A) or equivalent to aid in
opening MRSA RXNMX and MRSA DETMX if necessary.
06369120001-01EN 11 Doc Rev. 1.0Table 1
Preparation of Working Master Mix
Volume of Volume of
Number of Samples MRSA RXNMX MRSA DETMX Total Volume (μL)*
(μL) (μL)
1 40 20 60
2 50 25 75
3 60 30 90
4 70 35 105
5 80 40 120
6 90 45 135
7 100 50 150
8 110 55 165
9 120 60 180
10 130 65 195
11 150 75 225
12 160 80 240
13 170 85 255
14 180 90 270
15 190 95 285
16 200 100 300
17 210 105 315
18 220 110 330
19 230 115 345
20 240 120 360
21 260 130 390
22 270 135 405
23 280 140 420
24 290 145 435
25 300 150 450
26 310 155 465
27 320 160 480
28 330 165 495
29 340 170 510
30 350 175 525
*Includes volume for NC, MRSA (+) C, and dead volume.
06369120001-01EN 12 Doc Rev. 1.04. Vortex Working Master Mix for 3-5 seconds and centrifuge briefly to collect material at the base of
the tube. Working Master Mix must be prepared fresh daily. Discard any unused Working Master
Mix within 24 hours of preparation.
Processed Specimen and Control Preparation
1. Place the required number of LightCycler® capillaries into the LC Carousel or in the centrifuge
adapters. One (1) capillary for the PC (position 1), 1 capillary for the processed NC (position 2), and
1 capillary for each processed specimen (positions 3 and higher) are needed.
®
Note: Handle the LightCycler capillaries by the reservoir portion of the capillary
2. Pipette 15 μL of Working Master Mix into each capillary reservoir.
3. Pipette 5 μL of clear supernatant from each processed specimen into capillaries # 3 and higher.
Change the pipette tip after each sample.
4. Pipette 5 μL of the processed negative control into the capillary # 2.
Note: Processed specimens and negative controls prepared with Liquid Stuart swabs are stable
for up to 1 day at 15 to 25ºC or 2 to 8°C or up to 8 months at -15 to -25°C. Processed
specimens and negative controls prepared with Amies gel swabs (with or without charcoal)
are stable for up to 5 days at 15 to 25ºC or 2 to 8°C or up to 3 months at -15 to -25°C. If
frozen processed specimens are used, thaw at room temperature, vortex for 10 sec, then
centrifuge at 8,000 – 20,000 x g at room temperature for 1 minute.
5. Pipette 5 μL of the thawed MRSA (+) C into capillary # 1.
6. Seal each capillary with a stopper using the Capping Tool.
7. Centrifuge the capillaries in the LC Carousel Centrifuge 2.0 or using a standard centrifuge and
centrifuge adapters.
8. Transfer the LightCycler® Sample Carousel to the LightCycler® 2.0 Instrument.
®
Note: If a regular centrifuge was used, transfer the capillaries into the LightCycler Sample
Carousel in the order described in step 1.
Note: Amplification should be started immediately after addition of the processed specimens and
controls to the Working Master Mix. In case this is not possible, the filled capillaries can be
stored for up to 3 hours, protected from light, at 2 to 8°C.
LightCycler® 2.0 Instrument Loading and Operation
1. Log into the LightCycler® Software.
2. Start the LightCycler® MRSA Advanced Macro (refer to the LightCycler® 2.0 Instrument Operator’s
Manual A, chapter “Executing a Roche Macro” for details).
Note: Do not use the field . MAS will request reagent information prior to
launching the result analysis process (the assay lot number can also be inserted into the
appropriate field in the sample editor in the LightCycler® Software).
3. Name the run using a unique identifier.
4. Reduce the default loading list to the number of samples to be tested. If too many sample positions
are accidentally deleted, start the procedure from step 2.
5. Scan specimen barcode labels or manually enter specimen names in the sample name field, or
leave the default settings. The field can be used to enter data if required.
Note: Do not change the default name for Positive and Negative Controls.
6. Follow the onscreen instructions and start the LightCycler® MRSA Advanced Test run.
06369120001-01EN 13 Doc Rev. 1.0RESULTS
Peak Identification and Automatic Result Analysis:
®
Note: Do not start data analysis while a LightCycler run is in process.
• Manual TM Calling requires manual operation in two TM Calling modules automatically
selected.
• The Absolute Quantification modules are not used for the analysis of this test.
• In the melting peak window, all curves of the selected graphs are shown.
• The TM bar for each analysis module is predefined in the LightCycler® MRSA Advanced
Macro.
• It is possible to select or deselect a TM bar without creating a flag.
®
• The baseline of each analysis module is predefined in the LightCycler MRSA Advanced
Macro according to the following table and must not be deselected or changed. If
accidentally changed, type in the correct values from Table 2.
Table 2
Baseline values
CH 610 CH 670
Baseline value 0.085 0.100
• Peaks are defined as maxima above the baseline. Shoulders are not peaks.
• Examples for typical positive control (Graph 1, Channel 610) and Internal Control (Graph 2,
Channel 670) peaks and peak with shoulder (Graph 3):
Graph 1
Graph 2
06369120001-01EN 14 Doc Rev. 1.0Graph 3
Adjusting the TM bars:
1. After the run has finished, the experiment wizard shows .
2. Move the message window to the bottom of the screen in order to see the TM and Baseline values
(do not press ).
3. Click on the TM Calling 610 analysis module; an overview of all positions is displayed.
4. Reduce the size of the graph display window by sliding the left handle bar of that window to the
right; the “Baseline”, “TM1”, and “Height1” columns should now be visible.
Note: If more than one position is selected, the settings of the first selected capillary are
displayed.
Note: Shifting of bars affects all activated and selected capillaries.
5. Click on the positive control (#PC#) position to select it.
6. If the TM bar is not positioned at the peak maxima (above the baseline), move it to the peak maxima,
focusing on the specified TM ranges (see Table 3).
7. If there are no peaks above the baseline, leave the TM bar at the default position. Ensure that bars
do not mark slopes of any present peaks (e.g. outside the specified TM range).
8. Ensure that values are displayed in the “TM1” and “Height1” columns for that sample (any values).
If no values are observed (blank space), reposition the TM bar to the peak maxima; values should
now be displayed in the “TM1” and “Height1” columns.
Table 3
Specified TM ranges
Low TM (°C) High TM (°C)
MRSA
57.00 62.00
(CH 610)
Internal Control
57.00 62.00
(CH 670)
Note: In samples without peaks (e.g. in the Negative Control) auto scaling will adjust to the
highest background value. The predefined baseline may not be visible in this case. If you
want to make the baseline visible select this capillary position together with the positive
control.
Note: Marked peaks outside the specified TM range will be reported in the MAS report.
9. Proceed with peak detection as described in steps 5 to 8 above for all remaining positions (controls
and specimens).
10. Click on the TM Calling 670 analysis module.
06369120001-01EN 15 Doc Rev. 1.011. Proceed with peak detection as described in steps 4 to 8 above for all positions (controls and
specimens).
Finish TM calling:
1. Move the experiment wizard window back to the center of the screen and press .
®
2. A LightCycler report is automatically generated and must be printed.
3. Check the LightCycler® report for flags and whether is displayed. If flags
or other error messages are displayed, refer to the quality control and interpretation of
results sections.
4. The run is automatically saved.
®
Note: If changes are made to any sample name fields of the LightCycler Software after saving,
the operator must refresh the database by selecting and clicking on the “Abs Quant 610”
®
and “Abs Quant 670” icons and save the file again. Reprint LightCycler report.
5. Export the *.ixo file by clicking on \ to the folder .
Automatic result calling using the Micro Analysis Software (MAS):
1. Double click on the MAS icon on the computer’s desktop, and press .
2. Select the run to be analyzed from the folder and load *.ixo file.
®
3. Answer the question “Does the LightCycler report contain the flag ?” appropriately, i.e. consistent with the LightCycler® report.
4. Scan or manually enter the Lot Number and Expiration Date information supplied with the
Advanced Lysis Kit and LightCycler® MRSA Advanced Test.
5. Enter personalized information (e.g. operator's name) if desired.
Note: The latest information entered in MAS will be used from this point forward.
6. Select an appropriate report format (tabular, grouped or combined).
7. The MRSA Advanced experiment *.ixo file will be analyzed automatically, and the data will be
displayed in the selected report format.
8. Print the MAS report and/or export as *.pdf.
INTERPRETATION OF RESULTS
®
1. The results are calculated by the LightCycler Software from measured fluorescent signals and
embedded calculation algorithms and presented with the Micro Analysis Software (MAS).
2. Run, controls and sample validation is automatically done by the MAS.
Note: Flags and comments may be generated for RUN STATUS, CONTROLS STATUS and SAMPLE
STATUS. The operator must check the MAS report printouts for flags or comments in all
fields.
®
Note: A negative LightCycler MRSA Advanced Test result does not preclude MRSA nasal
colonization.
06369120001-01EN 16 Doc Rev. 1.03. For a valid run, the results are interpreted as follows:
CH 610 CH 670
MRSA
Result Result Comment Interpretation
Result
(MRSA) (IC)
Specimen is positive for
POSITIVE or
POSITIVE DETECTED - MRSA DNA. Presumed MRSA
NEGATIVE
nasal colonization.
Invalid result. Repeat PCR
procedure with thawed
IC processed specimen or new
NEGATIVE NEGATIVE INVALID
not detected specimen. If still invalid,
report invalid result. Contact
local Roche office.
MRSA DNA not detected.
NEGATIVE POSITIVE NOT DETECTED - MRSA nasal colonization
unlikely.
Additional comments for Positive Control, Negative Control and Specimen Result may be reported by the
MAS:
Comment Interpretation
Peak(s) outside Target TM range Amplicons detected outside specified MRSA TM range
Peak(s) outside IC TM range Amplicons detected outside specified IC TM range
If the comment “Peak(s) outside Target TM range” is reported by MAS for a Specimen Result which is
either “Not Detected / Valid” or “Invalid / Invalid” (“MRSA Result” and “Status” columns on the MAS report,
respectively), the specimen shall be suspected of being positive for MRSA DNA. Collect another specimen
(or use leftover swab from a double-headed swab) and re-test using an alternate procedure such as
culture to determine the patient’s nasal colonization status.
Specimen Results which are “Detected / Valid” (“MRSA Result” and “Status” columns on the MAS report,
respectively), and for which the “Peak(s) outside Target TM range” is displayed by MAS shall be interpreted
as being positive for MRSA DNA (presumed MRSA nasal colonization).
The presence of these comments displayed in the Controls Status bar of the MAS report does not change
the Controls Status or the Run Status interpretation generated by MAS and displayed on the MAS report.
The following flags may be displayed by the MAS:
Flag Comments Interpretation Status Corrective Action
Repeat LightCycler® run.
Check for correct macro in
Invalid macro File could not Run ®
Wrong Macro applied the LightCycler Software
version be loaded INVALID
and/or in the MRSA
Advanced Software Set.
Critical parameters in the If additional flags are
CCE and / or CCO and / displayed refer to the
User or MRSA Advanced *.ixo corrective action given for
Developed or files are different from the Run the respective flag.
-
Modified reference *.ixo files of the INVALID If no additional flags are
Test Method MRSA Advanced Macros. displayed repeat
Possible causes are experiment.
described in the If this flag is repeatedly set,
06369120001-01EN 17 Doc Rev. 1.0Flag Comments Interpretation Status Corrective Action
Operator's Manual of the please call your local Roche
®
LightCycler 2.0 Service.
Instrument (e.g. modifying
default settings of the
Macro)
Lysis Kit LightCycler® Advanced Run Repeat LightCycler® run
-
expired Lysis Kit expired INVALID with non expired reagents.
MRSA Test LightCycler® MRSA Run Repeat LightCycler® run
-
expired Advanced Test expired INVALID with non expired reagents.
Prepare a new Color
Compensation run and
CCO expired The Color Compensation create a new Color
Run
on - file is older than six Compensation Object.
1 INVALID ®
YYMMDD months Repeat LightCycler run
which had been performed
with the expired CCO.
Prepare a new Color
Compensation run and
create a new Color
Compensation Object
INVALID Color Compensation Run following the naming
-
CCO NAME Object incorrectly named INVALID convention stated in this
document. Repeat
®
LightCycler run which had
been performed with the
incorrectly named CCO.
Open MRSA experiment
®
*.ixo file in the LightCycler
Different Color
Software, select the correct
MULTIPLE Compensation files Run
- Color Compensation file.
CCO USED applied in the analysis INVALID
Save the experiment, print
modules ®
LightCycler report and
export again.
Open MRSA experiment
*.ixo file in the LightCycler®
One or more Color
Software, select the correct
NO CCO Compensation files are Run
- Color Compensation file.
USED missing in the analysis INVALID
Save the experiment, print
modules ®
LightCycler report and
export again.
Wrong LightCycler® SW Open LightCycler®
INVALID LCS Run
- version applied for Software and check for
VERSION INVALID
procedure correct version number.
Open MRSA experiment
*.ixo file in the LightCycler®
Software and correct
The baseline values might
INVALID Run baseline values according
- have been changed
BASELINE INVALID to Table 2. Save the
accidentally
experiment, print
®
LightCycler report and
export again.
• NC: • Amplicon(s)
Target detected in the Controls
INVALID NC See section Quality Control.
peak(s) MRSA TM range of INVALID
detected the Negative Control
06369120001-01EN 18 Doc Rev. 1.0Flag Comments Interpretation Status Corrective Action
• NC: • No IC detected in
IC not Negative Control
detected
PC: PC not No MRSA target detected Controls
INVALID PC See section Quality Control.
detected in the Positive Control INVALID
Open MRSA experiment
®
Default name for *.ixo file in the LightCycler
INVALID NC Controls
- Negative Control has Software and enter #NC#.
NAME INVALID ®
been changed Save, print LightCycler
report and export again.
Open MRSA experiment
®
*.ixo file in the LightCycler
Default name for Positive
INVALID PC Controls Software and enter #PC#.
- Control has been
NAME INVALID Save the experiment, print
changed ®
LightCycler report and
export again.
1
The MAS provides a warning 30 days prior to reagent expiration.
QUALITY CONTROL
®
1. Each LightCycler MRSA Advanced reaction contains an Internal Control (IC). The IC is designed to
control for specimen inhibition, and to monitor reagent integrity.
a. Internal Control (IC)
The IC must be detected positive in all MRSA negative specimens as well as in the Negative
Control (NC). If the IC is negative in a negative specimen, the specimen is invalid and PCR
procedure with freshly thawed processed specimen has to be repeated. If the IC is negative
in the NC, the entire run is invalid and must be repeated from frozen processed specimens
or new specimens. If the repeat frozen processed specimen run is still invalid, contact local
Roche office. The IC result is ignored in MRSA positive specimens and in a valid Positive
Control (PC) since MRSA amplification may compete with this control.
2. At least 1 PC and 1 NC must be processed with each run. The PC is intended to monitor for reagent
failure. The NC is used to detect reagent or environment contamination by MRSA DNA.
Amplification reagents contain an enzyme to prevent contamination with MRSA amplicons. The
®
LightCycler Software automatically assigns the position of both controls in the instrument.
a. Negative Control (NC)
The NC must be negative while its internal control must be positive. If the NC does not meet
these criteria, the entire run is invalid and must be repeated from frozen processed
specimens or new specimens. If the repeat run is still invalid, contact your local Roche office.
b. Positive Control (PC)
The PC must be positive (with IC either positive or negative). If the PC does not meet this
criterion, the entire run is invalid and must be repeated from frozen processed specimen or
new specimen. If the repeat run is still invalid, contact your local Roche office.
Flags and comments may be generated by the Micro Analysis Software (MAS). The Operator
must check the run printouts for flags and comments to verify that the run is valid.
06369120001-01EN 19 Doc Rev. 1.0CH 610 CH 670
Control Result Result Interpretation
(MRSA) (IC)
NEGATIVE POSITIVE Run valid, No action
POSITIVE or Run invalid, Run new specimens
POSITIVE
NEGATIVE
Negative
Control Run invalid, Repeat run from frozen
processed specimens (or new specimens).
NEGATIVE NEGATIVE
If the repeat frozen processed specimen run
is still invalid, contact local Roche office.
POSITIVE or Run valid, No action
POSITIVE
NEGATIVE
Positive Run Invalid, Repeat run from frozen
Control POSITIVE or processed specimens (or new specimens).
NEGATIVE
NEGATIVE If the repeat frozen processed specimen run
is still invalid, contact local Roche office.
®
Note: If the Negative Control (NC) or the MRSA (+) C of the LightCycler MRSA Advanced Test
is consistently invalid, the LightCycler® Multicolor Compensation file might be incorrect.
Repeat the Multicolor Compensation run or contact your local Roche office for technical
assistance.
External Positive Control (EPC)
®
The MicroBioLogics KWIK-STIK™ MRSA positive control (P/N: 0158ROCHE) may be used for training,
proficiency testing and external Quality Control of the LightCycler® MRSA Advanced Test. An MRSA strain
representative of RE2 types is used for this external positive control. MRSA strains representing RE3 and
RE7 types, if available may be used as additional external positive controls to monitor assay primers and
probes not directly controlled in the assay. External controls may be used in accordance with local, state,
and federal accrediting organization, as applicable. Follow the steps in the KWIK-STIK™ package insert for
swab preparation, but do not place the swab onto culture medium. After saturation of the swab with the
hydrated material, follow the LightCycler® MRSA Advanced Test package insert instructions for specimen
preparation and testing of the swab.
The EPC must be positive (with IC either positive or negative). If the EPC does not meet this criterion, the
entire MagNa Lyser run is invalid and must be repeated from new specimen. If the repeat run is still invalid,
contact your local Roche office.
Note: To date, seven SCCmec types (I–VII) have been distinguished, and several variants of these
5 ®
SCCmec types have been described . The LightCycler MRSA Advanced Test is based on a
proprietary detection system which is able to detect MRSA strains with different molecular
sequences in the vicinity surrounding the right extremity (RE) junction of the SCCmec
cassette with the orfX gene. The different RE types which are detected by the LightCycler®
MRSA Advanced Test are referred to as RE2, RE3 and RE7 (proprietary nomenclature). In
addition, sequence analysis of type RE1 has shown 100% homology to the primers and
probes used in the LightCycler® 2.0 MRSA Advanced Test.
PROCEDURAL PRECAUTIONS
1. As with any test procedure, good laboratory technique is essential to the proper performance of this
assay.
PROCEDURAL LIMITATIONS
®
1. This product can only be used with the LightCycler 2.0 Instrument.
2. This test has been validated for use with only human nasal specimens.
06369120001-01EN 20 Doc Rev. 1.03. Reliable results are dependent on adequate specimen collection (number of organisms in the
sample), transport, storage and processing procedures.
4. Use of this product should be limited to trained personnel.
®
5. A positive LightCycler MRSA Advanced Test result does not indicate the presence of viable MRSA.
It is however, presumptive for the presence of MRSA. Therefore, a positive result does not
necessarily indicate intervention eradication failure since non-viable DNA may persist. However, a
negative result following a previously positive test result may or may not indicate eradication
success.
6. Due to inherent differences between technologies, it is recommended that, prior to switching from
one technology to the next; users perform method correlation studies in their laboratory to qualify
technology differences.
7. The presence of AmpErase enzyme in the LightCycler® MRSA Advanced Reaction Mix reduces the
risk of amplicon contamination. However, contamination from MRSA (+) C and clinical specimens
can be avoided only by good laboratory practices and careful adherence to the procedures
specified in this Package Insert.
8. Though rare, mutations or polymorphisms within the region of the bacterial genome covered by the
LightCycler® MRSA Advanced Test primers and/or probe may impair detection. Rare forms of
MSSA strains carrying a genetic variant of SCCmec cassette may lead to positive results with the
®
LightCycler MRSA Advanced Test.
9. As with all PCR based in vitro diagnostic tests, extremely low levels of target below the LOD of the
assay may be detected, but results may not be reproducible (refer to “Reproducibility” section for
further details).
INTERFERING SUBSTANCES
Substances that may interfere with the detection of MRSA by the LightCycler® MRSA Advanced Test and
potentially generate invalid results include blood, excessive amounts of nasal secretions/mucus.
The following exogenous substances (see Table 4), which are components of decongestants and
substances used to relieve nasal dryness and/or irritation, have been shown not to interfere with the
®
detection of MRSA by the LightCycler MRSA Advanced Test.
Table 4
®
Exogenous Substances Tested For Interference With The LightCycler MRSA Advanced Test
Trade name Active ingredient
®
InfectoPyoderm Mupirocin 20 mg/g (antibiotic)
®
Turixin Mupirocin calcium 21.5 mg/g (antibiotic)
®
Bepanthen nasal ointment Dexpanthenol 50 mg/g (provitamin B5)
Accumed 12-hour Nasal Spray Oxymetazoline hydrochloride 0.05% (decongestant)
®
Otriven Xylometazoline 0.1 % (decongestant)
1,624 specimens were tested with the LightCycler MRSA Advanced Test. Overall, 10/1,624 (0.6%)
specimens had invalid results because of internal control (IC) failure following initial testing, and 7/1,624
(0.4%) specimens remained invalid because of IC failure following retesting. Blood was reported on the
nasal swab for 20/1,624 (1.2%) specimens, none of which had invalid results because of IC failure.
06369120001-01EN 21 Doc Rev. 1.0EXPECTED VALUES
®
In the LightCycler MRSA Advanced Test clinical trial, a total of 1,406 nasal swab specimens were
collected from 1,406 subjects at 5 sites across the United States. The study population was grouped into
subjects in hospital non-intensive care units (1,011; 71.9%), hospital intensive care units (67; 4.8%) nursing
homes/extended care facilities (255; 18.1%), and medical facility staff (73; 5.2%). The overall number of
positive MRSA samples by direct culture was 183 (13.0%).
PERFORMANCE CHARACTERISTICS
CLINICAL PERFORMANCE
®
Performance characteristics of the LightCycler MRSA Advanced Test were determined in a multi-site
®
prospective investigational study at 5 institutions by comparing the LightCycler MRSA Advanced Test
with a second FDA-cleared nucleic acid amplification test (NAAT), direct culture, and enrichment culture
(the most sensitive culture method). Subjects included individuals and medical staff at risk for nasal
colonization. Each subject was enrolled in the study only one time. Subjects who had received systemic or
topical-nasal antibiotics used to treat nasal colonization with MRSA from the day of sample collection and
up to one week prior to study enrollment, under 2 years of age, and/or had contraindication to nasal swab
collection were excluded from the study. Only those subjects meeting the inclusion and exclusion criteria
were enrolled.
A double-headed nasal swab was collected from each subject. One swab head was directly streaked onto
a quadrant of a chromogenic agar plate with cefoxitin, and then processed according to the package insert
®
for testing with the LightCycler MRSA Advanced Test. The remaining quadrants of the chromogenic agar
were streaked using a sterile loop. The second swab head was directly streaked onto a separate
chromogenic plate with cefoxitin, and then processed for testing with the second FDA-cleared NAAT test
(according to the package insert). Thereafter, the second swab head was transferred into Trypticase Soy
Broth and incubated for 48 hours at 35-37ºC and then subcultured onto a chromogenic plate with cefoxitin
(enrichment culture). Chromogenic culture plates were incubated at 35-37ºC for 20–52 hours. Presumptive
MRSA colonies from all culture plates were confirmed by coagulase testing and Gram staining if found
after 44-52 hours of incubation. Each participating site performed all tests.
Overall, 99.3% of specimens had concordant culture results for the two swab heads directly streaked onto
the chromogenic plates with cefoxitin. Only specimens with concordant results between the two swab
heads were considered in the analysis.
®
Performance of the LightCycler MRSA Advanced Test and the second FDA-cleared NAAT test were
calculated relative to the directly plated culture, and the enrichment culture results.
A summary of results for 1,389 eligible subjects (one nasal swab per patient) tested for MRSA by the
®
LightCycler MRSA Advanced test, a second FDA-cleared NAAT test, and directly culture is shown in
Table 5. For subjects tested, this resulted in a positive predictive value of 80.1% and a negative predictive
®
value of 99.6 %. The LightCycler MRSA Advanced Test correctly identified 173 of 178 of specimens
positive for MRSA by direct culture (positive percent agreement of 97.2%) and 1,172 of 1,215 of specimens
negative for MRSA relative to direct culture (negative percent agreement of 96.5%).
06369120001-01EN 22 Doc Rev. 1.0Table 5
®
Comparison Of The LightCycler MRSA Advanced Test,
The Second FDA-Cleared NAAT, And Direct Culture
Direct culture
® nd
LightCycler MRSA Advanced Test 2 NAAT Positive Negative Total
Positive Positive 172 24 196
Positive Negative 1 19 20
Negative Positive 1 76 77
Negative Negative 4 1,092 1,096
Total Total 178 1,211 1,389
® nd
Note: Only evaluable specimens contributing both valid results in the LightCycler MRSA Advanced Test and the 2 NAAT
test results and concordant direct culture results are included in this summary table.
Further investigation (i.e., testing for methicillin resistance using CLSI disk diffusion methodology, fem- and
mecA-specific PCR, and sequencing of SCCmec regions) confirmed the presence of MRSA in 30 of 43
samples in which the LightCycler® MRSA Advanced Test gave a positive result but direct culture was
®
negative. The presence of MRSA was confirmed in 4 of 5 samples in which the LightCycler MRSA
Advanced Test gave a negative result but direct culture was positive. The presence of MRSA was
confirmed in 13 of 76 samples in which the 2nd FDA-cleared NAAT gave a positive result but the
® 17
LightCycler MRSA Advanced Test and the direct culture gave a negative result .
®
Overall, 2 (1.7%) of 117 LightCycler runs performed during the investigational study were invalid because
of protocol deviations.
Performances of the LightCycler® MRSA Advanced Test, the second FDA-cleared NAAT, and the
enrichment culture relative to the direct culture are shown in Tables 6 and 7.
Table 6
®
Positive Percent Agreement Of The LightCycler MRSA Advanced Test, The Second FDA-Cleared
NAAT, and Enrichment Culture For Specimens Positive for MRSA by Direct Culture
Positive Percent Agreement (95% exact CI)a
®
LightCycler MRSA
Site Advanced Test 2nd NAAT Enrichment Culture
1 96.8% 96.8% 93.5%
(30/31; 83.3% to 99.9%) (30/31; 83.3% to 99.9%) (29/31; 78.6% to 99.2%)
2 97.7% 97.7% 97.7%
(42/43; 87.7% to 99.9%) (42/43; 87.7% to 99.9%) (42/43; 87.7% to 99.9%)
3 100.0% 100.0% 100.0%
(14/14; 76.8% to 100.0%) (14/14; 76.8% to 100.0%) (14/14; 76.8% to 100.0%)
4 96.2% 96.2% 97.5%
(76/79; 89.3% to 99.2%) (76/79; 89.3% to 99.2%) (77/79; 91.2% to 99.7%)
5 100.0% 100.0% 100.0%
(11/11; 71.5% to 100.0%) (11/11; 71.5% to 100.0%) (11/11; 71.5% to 100.0%)
Total 97.2% 97.2% 97.2%
(173/178; 93.6% to 99.1%) (173/178; 93.6% to 99.1%) (173/178; 93.6% to 99.1%)
a
CI = confidence interval.
06369120001-01EN 23 Doc Rev. 1.0Table 7
Negative Percent Agreement Of The LightCycler® MRSA Advanced Test, The Second FDA-
Cleared Test, and Enrichment Culture For Specimens Negative for MRSA by Direct Culture
a
Negative Percent Agreement (95% exact CI)
®
LightCycler MRSA
Site Advanced Test 2nd NAAT Enrichment Culture
97.5% (197/202; 94.3% to 92.0% (184/200; 87.3% to 97.5% (197/202; 94.3% to
1
99.2%) 95.4%) 99.2%)
96.2% (300/312; 93.4% to 82.1% (256/312; 77.3% to 96.7% (295/305; 94.1% to
2
98.0%) 86.1%) 98.4%)
97.6% (247/253; 94.9% to 96.8% (245/253; 93.9% to 98.8% (250/253; 96.6% to
3
99.1%) 98.6%) 99.8%)
93.9% (185/197; 89.6% to 93.4% (183/196; 88.9% to 98.0% (193/197; 94.9% to
4
96.8%) 96.4%) 99.4%)
96.8% (243/251; 93.8% to 97.2% (243/250; 94.3% to 98.8% (248/251; 96.5% to
5
98.6%) 98.9%) 99.8%)
96.5% 91.7% 97.9%
Total
(1,172/1,215; 95.3% to 97.4%) (1,111/1,211; 90.0% to 93.2%) (1,183/1,208; 97.0% to 98.7%)
a
CI = confidence interval.
®
Performances of the LightCycler MRSA Advanced Test, the second FDA-cleared NAAT, and the direct
culture relative to the enrichment culture are shown in Tables 8 and 9.
Table 8
Positive Percent Agreement Of The LightCycler® MRSA Advanced Test, The Second FDA-Cleared
NAAT, and Direct Culture For Specimens Positive for MRSA by Enrichment Culture
a
Positive Percent Agreement (95% exact CI)
LightCycler® MRSA
nd
Site Advanced Test 2 NAAT Direct Culture
1 85.3% (29/34; 68.9% to 95.0%) 88.2% (30/34; 72.5% to 96.7%) 85.3% (29/34; 68.9% to 95.0%)
2 88.5% (46/52; 76.6% to 95.6%) 86.5% (45/52; 74.2% to 94.4%) 80.8% (42/52; 67.5% to 90.4%)
3 82.4% (14/17; 56.6% to 96.2%) 82.4% (14/17; 56.6% to 96.2%) 82.4% (14/17; 56.6% to 96.2%)
4 97.5% (79/81; 91.4% to 99.7%) 96.3% (78/81; 89.6% to 99.2%) 95.1% (77/81; 87.8% to 98.6%)
5 78.6% (11/14; 49.2% to 95.3%) 78.6% (11/14; 49.2% to 95.3%) 78.6% (11/14; 49.2% to 95.3%)
90.4% (179/198; 85.4% to 89.9% (178/198; 84.8% to 87.4% (173/198; 81.9% to
Total
94.1%) 93.7%) 91.7%)
a
CI = confidence interval.
06369120001-01EN 24 Doc Rev. 1.0You can also read
