By Gracie Canales August 10, 2010 Cepheid Xpert MRSA By Gracie Canales August 10, 2010

Objectives At the end of this presentation you will be able to: Identify and define one of the leading causes of secondary hospital acquired infections. List the primary locations that MRSA harbors in the body. - Identify the genetic region that the Xpert MRSA assay detects. Describe the detection method of the GeneXpert System that is used for the Xpert MRSA assay. Compare the Xpert MRSA molecular test with the golden standard.

What is the primary cause of hospital acquired infections? Methicillin-Resistant Staphylococcus aureus (MRSA) is the leading cause for these secondary infections that are acquired during hospital stays and in long term nursing facilities.

MRSA Leading Cause of HAIs The National Healthcare Safety network estimates that hospitalized patients in the US acquire 2,000,000 health care associated infections each year causing more than 90,000 deaths and $4.5 billion in excess health care costs.

MRSA It is primarily located in the nares, on skin, and on mucosal surfaces of the vagina and rectum. Colonized patients (carriers) have a high likelihood of developing and transmitting the infections. Routine laboratory methods for detecting MRSA do not support a rapid detection system so Molecular technology has found a way.

Cepheid Xpert MRSA Test

GeneXpert System Only system that combines on-board sample preparation with real-time PCR amplification and Detection of targeted nucleic acid sequences Thereby delivering answers from unprocessed samples

Xpert MRSA assay Most traditional MRSA screening methods focus on the phenotypic identification of the mecA gene, the structural gene responsible for methicillin resistance. The gene is present within a transposon-encoded genetic region known as the staphylococcal cassette chromosome mec (SCCmec) The Xpert MRSA assay detects the SCCmec-orfX junction within this region.

GeneXpert System The system requires the use of a single use disposable GeneXpert cartridge that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained, cross contamination between the samples in the analyzer are eliminated. The Xpert reagents include sample processing controls and probe check controls. There are other internal checks the system must pass for proper analysis. The final detection and interpretation of the results are measured by the fluorescent signals that are interpolated in the specific targeted nucleic acid sequence.

The Process The GeneXpert System completely automates sample preparation, performing all the complex steps of DNA extraction in its advanced “microfluidic” cartridge. The cartridges are designed to handle a variety of sample volumes, enabling them to obtain higher concentrations of starting target material. Concentration and purification of the target in the cartridge further increases their sensitivity of the resulting test. Once the sample nucleic acid is extracted, it is moved from the sample processing chamber in the cartridge into the cartridge reaction tube where amplification and detection takes place. The GeneXpert System modules performs rapid heating and cooling cycles required for real-time PCR in the reaction tube of the cartridge. The module continuously monitors the chemical reactions in each cartridge in order to quickly create enough copies of the sample nucleic acid for reliable measurement.

The Process Con’t. Each of the modules works independently and can be used to conduct different tests simultaneously. The detection of the target gene sequence comes when the system’s multiple optics detects the presence of the fluorescent signals from the multiple target nucleic acids in the same cartridge. With a special setting continuous optical monitoring is possible, allowing the system to automatically stop the reaction as soon as the target is detected. This feature allows for faster turn-around-times.

The Results

Limitations The Xpert MRSA test is a fast screening test and is not 100% accurate. Tests should be followed up with the “Golden Standard” laboratory testing which is directly streaking the swabs on CHROMagar MRSA medium and in addition placing the swab in Trypticase soy broth with 6.5% sodium chloride and incubate for 18 to 24 hours at 35+/- 2oC. Erroneous test results might occur from improper specimen collection, handling or storage, technical error, sample mix-up, or because the number of organisms in the specimen is not detected by the test. The test is marketed towards non-molecular lab professionals so the emphasis on contamination and cleanliness must be given. Even though the test is self contained negligence can shut down the whole lab from one spilled amplified specimen.

Summary MRSA stands for Methicillin Resistant Staphylococcus aureus. MRSA is the leading cause for secondary hospital acquired infections. MRSA is harbored primarily in the human nares, on skin, and on mucosal surfaces of the vagina and rectum. The Xpert MRSA assay is a second generation real-time PCR assay for the detection of the SCCmec-orfx junction within the transposon-encoded genetic region. The GeneXpert System uses a fluorescent signal that are detected by the optics on the targeted nucleic acid sequence. 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 sample preparation to detection all within one vessel and with a very fast turn-around-time (<2hrs). The Xpert MRSA assay is a great screening test that can help facilities isolate and implement treatment from the admission stage in hospital care. However it is not 100% accurate and should be followed up by the “golden standard” for confirmation. The “Golden Standard” uses routine laboratory microbiological practices using special medium specific for MRSA. The medium is CHROMagar and Trypticase soy broth with 6.5% sodium chloride. The incubation time takes as long as 1-2 days at 35 +/- 2oC. It is also a wonderful system to have for a small facility that may not have access to a microbiology department at their location.

Conclusion It is truly amazing how far Molecular testing has come and still changing right before our very eyes. We are Blessed to be a part of that future!