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Friday, December 23, 2016 Written by Ellen Jo Baron, Ph.D., D(ABMM), Prof. Emerita, Stanford University Director of Medical Affairs, Cepheid

Decisions, decisions: Which Staphylococcus Surveillance Assay Should You Use?

QUIZ: How should this result be reported to your clinicians?

No two healthcare systems are alike, nor are the patient-care needs of your clinicians, so Cepheid offers choices for detecting both staphylococcal infections and staphylococcal colonization. In fact, there are more FDA-cleared Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) assays available for GeneXpert® System than for any other diagnostic platform. Although it is more obvious in which situations the Xpert® MRSA/SA Blood Culture Assay or the skin and soft tissue infection (SSTI) assay (Xpert® MRSA/SA SSTI) should be used, the choice between the two nasal surveillance assays is less clear. The performance characteristics of each assay are slightly different, so each assay has a unique place in an overall surveillance program. The original surveillance tool, the Xpert® MRSA assay, is the most utilized molecular MRSA surveillance method in the U.S. This assay detects only a single staphylococcal target, which is the junction where the staphylococcal cassette chromosome mec (SCCmec) [i.e., the genetic element that contains the mecA gene, which confers resistance to the extended-spectrum penicillins and cephalosporins], integrates into the S. aureus chromosome. Published data suggest that decades ago, a wild-type S. aureus strain [i.e., one susceptible to oxacillin, methicillin, and other extended-spectrum beta-lactam agents] acquired a large DNA segment containing mecA and the genes that enable it to integrate into another DNA molecule, from another organism (probably Staphylococcus sciuri). Once the DNA cassette was integrated into the chromosome, the mecA gene proved to be fully functional and the newly resistant organism began to spread.1 The integration site of the cassette into the staphylococcal genome is located in the middle of open reading frame X (orfX), a genetic region of unknown function, but one that is unique to S. aureus strains. SCCmec, with its internal mecA gene, inserts into the orfX and in so doing, splits orfX into two parts. The Xpert MRSA surveillance assay targets one set of primers that bind to orfX in the wild type staphylococcal chromosome and additional primers that bind to the outer edge of the newly incorporated SCCmec element, bridging the integrated DNA and chromosomal DNA, insuring that the SCCmec element is present in an S. aureus strain and not some other staphylococcal species. This target is called the SCCmec/orfX junction region (Figure 1).

The region targeted by the Xpert MRSA assay is similar to those used in the GeneOhm® MRSA assay, the Roche assay, and the bioMérieux EasyQ assay. Although the regions of SCCmec and orfX that are targeted are usually highly conserved, some isolates of S. aureus that have SCCmec may lose just the mecA portion of the cassette through excision. While the basic structure of the element remains intact, the mecA gene (i.e., one kilobase out of the 23 to 56 kb of the DNA that constitutes SCCmec) is lost Thus, the mecA portion of the cassette is now "empty," at least as far as the ability to confer methicillin resistance.2 To be clear, the separated halves of orfX and the outer regions of the SCCmec are still there, but mecA is missing. Such strains are called "empty cassette" variants. So, although they will be identified as MRSA by molecular tests targeting the presence of the orfX/SCCmec junction fragment, phenotypically the organisms are methicillin-susceptible because they lack mecA. The prevalence of such strains varies by community within the United States and Europe, with some localized areas reporting as many as 13% of strains identified as MRSA by a nucleic acid amplification tests as empty cassettes, although the accuracy of this figure has been questioned.3,4 More common percentages range from 1–5% based on local surveillance data. A number of strains from various geographic regions in the United States and Canada have been characterized and two different mechanisms for false-positive MRSA PCR results were described.5 Missing mecA sequences, i.e., empty cassettes, accounted for most of the false positive observed although in rare cases the false-positive result was due to prevalence of a SCCmec-like cassette in an isolate of coagulase-negative staphylococci.

What are the potential consequences of an inaccurate report of MRSA for a nasal surveillance test? First, a patient could be placed in contact precautions in the hospital unnecessarily. In addition, if the patient is scheduled for a surgical procedure, the patient may be placed on a staphylococcal decolonization regimen of mupirocin and chlorhexidine baths. The patient may also receive vancomycin for surgical prophylaxis instead of a cephalosporin.6 Over-utilization of vancomycin can contribute to development of vancomycin-resistant enterococci (VRE), which is a major cause of healthcare-associated infections in the US.7 The actual impact of these potential problems, if any, has never been elucidated.

Scientists from the U.S. Veterans Administration (VA) recently published results of the largest systematic program of MRSA screening in a healthcare system to date. This was part of a nationwide VA initiative to reduce healthcare-associated infections (HAI), particularly those due to MRSA.8 The initiative began in 2007 and Cepheid's Xpert® MRSA assay was selected for use by 91% of participating Veterans Administration hospitals across all 22 veterans' integrated service networks (VISNs) in the U.S. and Puerto Rico. Almost 2 million admissions occurred between the full implementation of the infection control strategy and June, 2010, of which 1,712,537 patients had their nares tested for MRSA. A few sites chose to screen patients with chromogenic agar or another culture-based method, but the majority used the Xpert MRSA PCR assay. Surveillance was only one of the steps in the infection control "bundle", which also included contact precautions for patients colonized or infected with MRSA, enhanced emphasis on hand hygiene, and a change in the institutional culture whereby infection control would become the responsibility of everyone who had contact with patients. The results of the study were dramatic. In this healthcare system where previous infection control mandates had failed to lower infection rates, the rate of MRSA infections per 1000 patient days dropped from 1.5 for the previous 2 years, to a low of around 0.6 per 1000 patient days, a statistically significant 59% decrease (Figure 2).8 The types of MRSA infections that declined included, but were not limited to bacteremia and pneumonia. Some types of infections, including ventilator-associated pneumonia, skin and soft tissue infections, and urinary tract infections declined even more, showing a highly significant 75% decrease. These results mirror those of previous, smaller interventions, as discussed in a previous edition of On Demand (Volume 3, Issue 1, Winter 2010). A side benefit of the intervention, possibly due to a decrease in empiric use of vancomycin, was a drop in rates of both Clostridium difficile and vancomycin-resistant enterococcal (VRE) infections, as reported in a subset of VA facilities. C. difficile infections declined 61% in non-ICU settings, and there was a significant decrease in the rates of health-care-associated VRE infection in the ICUs, from 1.51 to 0.00 per 1000 patient days (P<0.001), and in non-ICUs, from 0.33 to 0.09 per 1000 patient days, a decrease of 73% (P<0.001).8

The success of the VA program must be attributed in part to the rapid availability of nasal surveillance PCR results and the inclusion of all patients in the surveillance cohort. The importance of rapid results, and perhaps the effect of testing only a limited subset of patients, can be appreciated by examining the contrary results of a similar controlled interventional study undertaken at several medical sites encompassing 18 intensive care units, also published recently in the New England Journal of Medicine.9 A key difference from the VA experience was that the nasal surveillance was performed by culture and the results were not available for a mean time of 5.2 days. The interventions appeared to be similar to those of the VA. Strikingly, the results of this intervention did not differ from those in the control patient care units. Numbers of both colonized and infected patients were statistically the same.

Despite the success of universal MRSA surveillance programs in VA hospitals and others like it in healthcare institutions around the world, it was recognized that expanding nasal surveillance programs to include all S. aureus isolates and not just MRSA was needed, particularly to support surveillance of patients who were going to have surgery and were at risk for autoinfection with the patient's own strain of S. aureus.10 In response to this need, Cepheid developed the Xpert® SA Nasal Complete assay, which correctly detects both S. aureus and MRSA and appropriately reports empty cassette strains as S. aureus and not MRSA.

Developing a S. aureus infection, whether caused by MRSA or MSSA, can be debilitating or even fatal, for some patients. Those patients undergoing orthopedic or open-heart surgery are particularly prone to getting post-surgical site infections with S. aureus. Of course, MRSA is more difficult and expensive to treat and extends hospitalization even more than MSSA, but both organisms can wreak havoc on a patient's health.11 Knowing that a patient is colonized with either MRSA or MSSA indicates that some sort of antimicrobial prophylaxis and/or decolonization should be undertaken to prevent infection.10 However, decolonization with mupirocin ointments and chlorhexidine baths can cause side effects, so this should not be undertaken in patients who are not colonized with S. aureus. Knowing a patient had MSSA instead of MRSA would also allow use of cefazolin or other more effective drugs for surgical prophylaxis rather than vancomycin. Tests that contain specific markers that enable empty cassette strains to be designated as S. aureus can help optimize infection control efforts.

Monitoring for both MRSA and MSSA nasal colonization to prevent surgical site infections and correctly classifying empty cassette strains as MSSA are reasons for selecting the Xpert SA Nasal Complete assay for surveillance instead of Xpert MRSA. The Xpert SA Nasal Complete identifies the presence of either MRSA or S. aureus individually. This test format (and coincidently both the Xpert Blood Culture and SSTI assays share the same format) avoids the problem of reporting false-positive MRSA results for empty cassette variants because it tests for the spa gene (a gene unique to S. aureus) and the mecA determinant in addition to the orfX/SCCmec junction fragment. The target used to indicate the presence of S. aureus is the staphylococcal protein A gene, called spa, which is present in virtually all S. aureus isolates. In fact, until quite recently, it was assumed that spa was both specific and necessary for a microbe to be identified as S. aureus. Recent studies have shown that there are rare spa-negative variants of S. aureus and a few rare staphylococci other than S. aureus that carry genes with DNA sequence similarity to spa.12,13 It is necessary to include a gene unique to S. aureus in an MRSA assay, especially given the sample sites: blood, wounds, and the mucosa lining the nostrils (nares), to avoid calling false-positive results due to mecA-carrying coagulase-negative staphylococci. Coagulase-negative staphylococci, many of which carry mecA-mediated methicillin-resistant genes, are common colonizers of the skin and the mucosa of human nares (and other mammals, too). So if your assay tests for mecA only, it will likely detect a number of methicillin-resistant coagulase-negative staphylococci, which pose no greater than normal risk for subsequent infection to the patient or spread within the environment. If you test for a S. aureus determinant (spa) and mecA both, you may generate a false-positive MRSA result when there are both mecA-containing coagulase-negative staphylococci (CoNS) and a S. aureus in the same site. This is because the CoNS can contribute the mecA signal and the MSSA can contribute the spa signal. So you must in addition add the orfX/SCCmec junction region target, which indicates the presence of the SCCmec cassette in a S. aureus chromosome. When all three targets are detected, the test result is positive for MRSA.

Of course, that MRSA is by definition also a S. aureus. Cepheid was asked by FDA to format the results screen so that users see two sets of results i.e., results for S. aureus (SA) and results for MRSA. This double result (SA positive; MRSA positive) has resulted in confusion among users. How would you interpret the result? Let's explore this.

Clearly if spa is positive but the mecA gene and the SCCmec junction region are not detected, the result will be reported as "SA detected; MRSA not detected." New sequence variants of SCCmec and spa can occur; thus, Dr. Fred Tenover and the Healthcare Associated Infections Consortium (see article in On Demand Volume 3, Issue 3, Fall 2010) keep abreast of new genetic variants of MRSA as they appear, such as the recent detection of SCCmec type XI in Europe, and continually make changes to our assay that will be incorporated into the next generation assays.14

It is time for a quiz. Now that you understand the principle of the three targets in the GeneXpert SA Nasal Complete, how would you report the result shown on the screen shot from the quiz at the top of the page? Does this result indicate the presence of two different strains of staphylococci or just one? Click here to view the answer.

In summary, for MRSA surveillance, particularly when there is a specific infection control intervention that is time-sensitive, such as placing patients in isolation to reduce transmission, the Xpert® MRSA is likely to be your primary choice. It is clearly faster than culture when performed on demand and ~20% more sensitive than direct culture without enrichment.15 False-positive MRSA results due to empty cassette strains will cause a rare patient to be placed in contact isolation unnecessarily. For hospitals conducting surveillance that are particularly concerned about overcalling MRSA colonization due to empty cassette strains, and especially for patients being admitted for hemodialysis, cardiothoracic or orthopedic surgery, or those with immunocompromising conditions for which any staphylococcal infection would be more difficult to eradicate, the Xpert® SA Nasal Complete should be considered. This will identify those patients colonized with either S. aureus or MRSA and will not overcall empty cassette strains. Laboratories should work with their Infection Prevention and Infectious Diseases specialists to arrive at the best algorithm for surveillance testing in their facility. Whichever way the laboratory chooses to perform surveillance, they will be taking steps in the direction of providing better care for individual patients and lowering the risks of HAIs even beyond those caused by S. aureus for all patients in the institution, as proven by the experience of VA hospitals across our country.

REFERENCES

1. Wu, S., et al. 1996. Tracking the evolutionary origin of the methicillin resistance gene: cloning and sequencing of a homologue of mecA from a methicillin susceptible strain of Staphylococcussciuri. Microb Drug Resist. 2: 435-441.
2. Chlebowicz, M. A., et al. Recombination between ccrC genes in a type V (5C2&5) staphylococcal cassette chromosome mec (SCCmec) of Staphylococcus aureus ST398 leads to conversion from methicillin resistance to methicillin susceptibility in vivo. Antimicrob Agents Chemother. 54: 783-791.
3. Snyder, J. W., G. K. Munier & C. L. Johnson. Comparison of the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) PCR assay to culture by use of BBL CHROMagar MRSA for detection of MRSA in nasal surveillance cultures from intensive care unit patients. J Clin Microbiol. 48: 1305-1309.
4. Blanc, D. S., et al. High proportion of wrongly identified methicillin-resistant Staphylococcus aureus carriers by use of a rapid commercial PCR assay due to presence of staphylococcal cassette chromosome element lacking the mecA gene. J Clin Microbiol. 49: 722-724.
5. Wong, H., et al. Characterization of Staphylococcus aureus isolates with a partial or complete absence of staphylococcal cassette chromosome elements. J Clin Microbiol. 48: 3525-3531.
6. Patel, N., et al. Vancomycin: we can't get there from here. Clin Infect Dis. 52: 969-974.
7. Hidron, A. I., et al. 2008. NHSN annual update: antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006-2007. Infect Control Hosp Epidemiol. 29: 996-1011.
8. Jain, R., et al. Veterans Affairs initiative to prevent methicillin-resistant Staphylococcus aureusinfections. N Engl J Med. 364: 1419-1430.
9. Huskins, W. C., et al. Intervention to reduce transmission of resistant bacteria in intensive care. N Engl J Med. 364: 1407-1418.
10. Bode, L. G., et al. 2010. Preventing surgical-site infections in nasal carriers of Staphylococcus aureus. N Engl J Med. 362: 9-17.
11. Anderson, D. J., et al. 2009. Clinical and financial outcomes due to methicillin resistantStaphylococcus aureus surgical site infection: a multi-center matched outcomes study. PLoS One. 4: e8305.
12. Rosander, A., B. Guss & M. Pringle. An IgG-binding protein A homolog in Staphylococcus hyicus.Vet Microbiol. 149: 273-276.
13. Baum, C., et al. 2009. Non-spa-typeable clinical Staphylococcus aureus strains are naturally occurring protein A mutants. J Clin Microbiol. 47: 3624-3629.
14. Shore, A. C., et al. Detection of Staphylococcal Cassette Chromosome mec Type XI Encoding Highly Divergent mecA, mecI, mecR1, blaZ and ccr Genes in Human Clinical Clonal Complex 130 Methicillin-Resistant Staphylococcus aureus. Antimicrob Agents Chemother.
15. Wolk, D. M., et al. 2009. Comparison of MRSASelect Agar, CHROMagar Methicillin-ResistantStaphylococcus aureus (MRSA) Medium, and Xpert MRSA PCR for detection of MRSA in Nares: diagnostic accuracy for surveillance samples with various bacterial densities. J Clin Microbiol.47: 3933-3936.