Cepheid Xpert MRSACepheid Xpert MRSAByByGracie CanalesGracie CanalesAugust 10, 2010August 10, 2010

ObjectivesObjectivesAt the end of this presentation you will be able to:At the end of this presentation you will be able to:- Identify and define one of the leading causes of Identify and define one of the leading causes of

secondary hospital acquired infections.secondary hospital acquired infections.- List the primary locations that MRSA harbors in List the primary locations that MRSA harbors in

the body.the body.- Identify the genetic region that the Xpert MRSA - Identify the genetic region that the Xpert MRSA

assay detects.assay detects.- Describe the detection method of the GeneXpert Describe the detection method of the GeneXpert

System that is used for the Xpert MRSA assay.System that is used for the Xpert MRSA assay.- Compare the Xpert MRSA molecular test with Compare the Xpert MRSA molecular test with

the golden standard.the golden standard.

What is the primary What is the primary cause of hospital cause of hospital acquired infections?acquired infections?Methicillin-Resistant Methicillin-Resistant

Staphylococcus aureus Staphylococcus aureus (MRSA) is (MRSA) is the leading cause for these the leading cause for these secondary infections that are secondary infections that are acquired during hospital stays acquired during hospital stays and in long term nursing facilities.and in long term nursing facilities.

MRSA Leading Cause MRSA Leading Cause of HAIsof HAIsThe National Healthcare Safety The National Healthcare Safety

network estimates that network estimates that hospitalized patients in the US hospitalized patients in the US acquire 2,000,000 health care acquire 2,000,000 health care associated infections each year associated infections each year causing more than 90,000 deaths causing more than 90,000 deaths and $4.5 billion in excess health and $4.5 billion in excess health care costs.care costs.

MRSAMRSAIt is primarily located in the nares, on It is primarily located in the nares, on

skin, and on mucosal surfaces of the skin, and on mucosal surfaces of the vagina and rectum.vagina and rectum.

Colonized patients (carriers) have a high Colonized patients (carriers) have a high likelihood of developing and likelihood of developing and transmitting the infections. transmitting the infections.

Routine laboratory methods for Routine laboratory methods for detecting MRSA do not support a rapid detecting MRSA do not support a rapid detection system so Molecular detection system so Molecular technology has found a way.technology has found a way.

Cepheid Xpert MRSA Cepheid Xpert MRSA TestTest

GeneXpert SystemGeneXpert System Only system that Only system that

combines on-board combines on-board sample preparation sample preparation

with real-time PCR with real-time PCR amplification andamplification and

Detection of targeted Detection of targeted nucleic acid nucleic acid sequences sequences

Thereby delivering Thereby delivering answers from answers from unprocessed samplesunprocessed samples

Xpert MRSA assayXpert MRSA assayMost traditional MRSA screening methods Most traditional MRSA screening methods

focus on the phenotypic identification of focus on the phenotypic identification of the the mecmecA gene, the structural gene A gene, the structural gene responsible for methicillin resistance. responsible for methicillin resistance.

The gene is present within a transposon-The gene is present within a transposon-encoded genetic region known as the encoded genetic region known as the staphylococcal cassette chromosome staphylococcal cassette chromosome mec mec (SCC(SCCmecmec))

The Xpert MRSA assay detects the The Xpert MRSA assay detects the SCCSCCmec-orfXmec-orfX junction within this region. junction within this region.

GeneXpert SystemGeneXpert System The system requires the use of a The system requires the use of a

single use disposable GeneXpert single use disposable GeneXpert cartridge that hold the PCR cartridge that hold the PCR reagents and host the PCR reagents and host the PCR process. process.

Because the cartridges are self-Because the cartridges are self-contained, cross contamination contained, cross contamination between the samples in the between the samples in the analyzer are eliminated.analyzer are eliminated.

The Xpert reagents include The Xpert reagents include sample processing controls and sample processing controls and probe check controls. There are probe check controls. There are other internal checks the system other internal checks the system must pass for proper analysis.must pass for proper analysis.

The final detection and The final detection and interpretation of the results are interpretation of the results are measured by the fluorescent measured by the fluorescent signals that are interpolated in signals that are interpolated in the specific targeted nucleic the specific targeted nucleic acid sequence.acid sequence.

The ProcessThe Process The GeneXpert System completely automates sample The GeneXpert System completely automates sample

preparation, performing all the complex steps of DNA preparation, performing all the complex steps of DNA extraction in its advanced “microfluidic” cartridge.extraction in its advanced “microfluidic” cartridge.

The cartridges are designed to handle a variety of sample The cartridges are designed to handle a variety of sample volumes, enabling them to obtain higher concentrations of volumes, enabling them to obtain higher concentrations of starting target material.starting target material.

Concentration and purification of the target in the cartridge Concentration and purification of the target in the cartridge further increases their sensitivity of the resulting test.further increases their sensitivity of the resulting test.

Once the sample nucleic acid is extracted, it is moved from the Once the sample nucleic acid is extracted, it is moved from the sample processing chamber in the cartridge into the cartridge sample processing chamber in the cartridge into the cartridge reaction tube where amplification and detection takes place. reaction tube where amplification and detection takes place.

The GeneXpert System modules performs rapid heating and The GeneXpert System modules performs rapid heating and cooling cycles required for real-time PCR in the reaction tube cooling cycles required for real-time PCR in the reaction tube of the cartridge.of the cartridge.

The module continuously monitors the chemical reactions in The module continuously monitors the chemical reactions in each cartridge in order to quickly create enough copies of the each cartridge in order to quickly create enough copies of the sample nucleic acid for reliable measurement.sample nucleic acid for reliable measurement.

The Process Con’t.The Process Con’t.

Each of the modules works independently and can be Each of the modules works independently and can be used to conduct different tests simultaneously.used to conduct different tests simultaneously.

The detection of the target gene sequence comes The detection of the target gene sequence comes when the system’s multiple optics detects the when the system’s multiple optics detects the presence of the fluorescent signals from the multiple presence of the fluorescent signals from the multiple target nucleic acids in the same cartridge.target nucleic acids in the same cartridge.

With a special setting continuous optical monitoring is With a special setting continuous optical monitoring is possible, allowing the system to automatically stop possible, allowing the system to automatically stop the reaction as soon as the target is detected.the reaction as soon as the target is detected.

This feature allows for faster turn-around-times.This feature allows for faster turn-around-times.

The ResultsThe Results

LimitationsLimitations The Xpert MRSA test is a fast The Xpert MRSA test is a fast screeningscreening test and is not test and is not

100% accurate. 100% accurate. Tests should be followed up with the “Golden Standard” Tests should be followed up with the “Golden Standard”

laboratory testing which is directly streaking the swabs laboratory testing which is directly streaking the swabs on CHROMagar MRSA medium and in addition placing on CHROMagar MRSA medium and in addition placing the swab in Trypticase soy broth with 6.5% sodium the swab in Trypticase soy broth with 6.5% sodium chloride and incubate for 18 to 24 hours at 35+/- 2chloride and incubate for 18 to 24 hours at 35+/- 2ooC.C.

Erroneous test results might occur from improper Erroneous test results might occur from improper specimen collection, handling or storage, technical specimen collection, handling or storage, technical error, sample mix-up, or because the number of error, sample mix-up, or because the number of organisms in the specimen is not detected by the test.organisms in the specimen is not detected by the test.

The test is marketed towards non-molecular lab The test is marketed towards non-molecular lab professionals so the emphasis on contamination and professionals so the emphasis on contamination and cleanliness must be given. Even though the test is self cleanliness must be given. Even though the test is self contained negligence can shut down the whole lab from contained negligence can shut down the whole lab from one spilled amplified specimen.one spilled amplified specimen.

SummarySummary MRSA stands for Methicillin Resistant MRSA stands for Methicillin Resistant Staphylococcus aureus.Staphylococcus aureus. MRSA is the leading cause for secondary hospital acquired infections.MRSA is the leading cause for secondary hospital acquired infections. MRSA is harbored primarily in the human nares, on skin, and on MRSA is harbored primarily in the human nares, on skin, and on

mucosal surfaces of the vagina and rectum.mucosal surfaces of the vagina and rectum. The Xpert MRSA assay is a second generation real-time PCR assay for The Xpert MRSA assay is a second generation real-time PCR assay for

the detection of the SCCthe detection of the SCCmec-orfxmec-orfx junction within the transposon- junction within the transposon-encoded genetic region.encoded genetic region.

The GeneXpert System uses a fluorescent signal that are detected by The GeneXpert System uses a fluorescent signal that are detected by the optics on the targeted nucleic acid sequence.the optics on the targeted nucleic acid sequence.

The Xpert MRSA assay is a test that is tested from start to finish in a The Xpert MRSA assay is a test that is tested from start to finish in a self-contained cartridge. With the GeneXpert System it goes from self-contained cartridge. With the GeneXpert System it goes from sample preparation to detection all within one vessel and with a very sample preparation to detection all within one vessel and with a very fast turn-around-time (<2hrs). fast turn-around-time (<2hrs).

The Xpert MRSA assay is a great screening test that can help facilities The Xpert MRSA assay is a great screening test that can help facilities isolate and implement treatment from the admission stage in hospital isolate and implement treatment from the admission stage in hospital care. However it is not 100% accurate and should be followed up by care. However it is not 100% accurate and should be followed up by the “golden standard” for confirmation.the “golden standard” for confirmation.

The “Golden Standard” uses routine laboratory microbiological The “Golden Standard” uses routine laboratory microbiological practices using special medium specific for MRSA. The medium is practices using special medium specific for MRSA. The medium is CHROMagar and Trypticase soy broth with 6.5% sodium chloride. The CHROMagar and Trypticase soy broth with 6.5% sodium chloride. The incubation time takes as long as 1-2 days at 35 +/- 2incubation time takes as long as 1-2 days at 35 +/- 2ooC.C.

It is also a wonderful system to have for a small facility that may not It is also a wonderful system to have for a small facility that may not have access to a microbiology department at their location. have access to a microbiology department at their location.

ConclusionConclusion It is truly amazing how far It is truly amazing how far

Molecular testing has come and Molecular testing has come and still changing right before our still changing right before our very eyes. We are Blessed to be very eyes. We are Blessed to be a part of that future!a part of that future!