A Comparative Analysis of GeneXpert Real-Time PCRwith Culture for the Detection of Methicillin-Resistant Staphylococcus aureus Colonization in Selected Hospital Admissions

Contact isolation of patients with methicillin-resistant Staphylococcus aureus (MRSA) reduces transmission to other patients and to health care workers. PCR technology can provide rapid detection of these patients. We tested the utility of using PCR for MRSA detection in patients with a history ofMRSA infection or colonization or in a high risk group admitted to a general referral hospital. Nasal swabs from 342 patients were tested for MRSA on days one and three using the GeneXpert MRSA system. Swabs with a positive PCR result were cultured to identify staphylococcal species present in the nares. Fiy-six patients (38% of 147) with a history of MRSA colonization or infection were positive; forty-seven patients (24% of 195) in a high risk group were positive. Eighty-one percent of the patients with positive PCR swabs grew out MRSA on culture. Some cultures grew out only methicillinsensitive Staphylococcus aureus,methicillin-sensitive, coagulase negative Staphylococcus, ormethicillin-resistant, coagulase negative Staphylococcus.is study demonstrates thatmost patients at risk forMRSA colonization are not colonized and thatmicrobiological surveillance using PCR technology can facilitate contact isolation decisions. Not all PCR positive results represent the presence of MRSA, and hospitals need to consider policies for additional evaluation of positive PCR tests.


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) causes skin and so tissue infections, sepsis, osteomyelitis, meningitis, endocarditis, urinary tract infections, necrotizing pneumonia, and toxic shock syndrome.ese infections occur more frequently in men and in patients with signi�cant comorbidity and increase hospital stays, mortality rates, and total costs [1].In the early 1990s, hospitals started to use more stringent contact isolation guidelines for patients colonized or infected with MRSA to prevent the spread to other patients in the hospital [2].However, many patients are not permanent carriers aer infection or colonization and do not need full contact precautions and isolation upon readmission.Consequently, some hospitals now screen patients for MRSA infection upon admission, and these protocols range from patients in selected patient care units to universal screening of all patients [3,4].We studied nasal colonization rates with MRSA in selected groups of patients rather than in all admissions or in particular patient care units to determine how useful faster diagnostic tests based on polymerase chain reaction (PCR) identi�cation would be in screening these patients.

Methods
e Infection Prevention and Control service at University Medical Center in Lubbock, TX, USA conducted a pilot project on the use of PCR screening for MRSA in June through August, 2010.Adult patients screened included those with a previous history of MRSA colonization or infection and those in high risk groups, including those in longterm care facilities, patients scheduled for CABG surgery, burn patients, and patients with trauma requiring intubation [2].Nasal swab samples (Transystem LQ Stuart, COPAN Diagnostics, Inc., Murrieta, CA, USA) were taken from each patient upon admission and immediately sent to the laboratory for PCR testing using the real-time GeneXpert PCR assay (Cepheid, Sunnyvale, CA, USA).is assay tests for all the types of MRSA SCCmec A gene and has an average turnaround time of about seven hours [5].When available, patients were also tested on the third hospital day.One deidenti�ed sample from each patient with a positive PCR for MRSA was plated on tryptic soy agar (TSA) with 5% sheep blood (BA) and provided to the research laboratory (Jane for bacteriological characterization.ese specimens were incubated at 34 ∘ C for at least 48 hours.Typical staphylococcal colonies were subcultured on TSA without blood for pigmentation, BA for hemolysis, and mannitol salt agar and tested for the presence of clumping factor A and coagulase.Bacterial isolates with characteristic morphology which grew in 7.5% sodium chloride, used mannitol, and synthesized coagulase and clumping factor A were identi�ed as Staphylococcus aureus.Bacterial isolates with characteristic morphology which grew in 7.5% sodium chloride, could not use mannitol, and did not make clumping factor A and coagulase were identi�ed as coagulase negative Staphylococcus.All isolates identi�ed as Staphylococcus were tested for resistance to oxacillin by spotting 25 L of a bacterial suspension in mueller hinton (MH) broth to oxacillin screen agar (MH agar with 4% sodium chloride, 6 g/mL oxacillin).e IRB at Texas Tech University Health Sciences Center approved the retrospective use of the results in this study.

Results
e clinical microbiology laboratory at University Medical Center conducted a pilot study before it introduced PCR technology for the identi�cation of MRSA.is study included 107 specimens; 23 (21.5%) were positive for MRSA either by PCR or culture or both.PCR tests had a sensitivity of 95.7% for identifying MRSA, a speci�city of 100% for identifying MRSA negative specimens, and an overall accuracy of 99.1%.e screening project included 342 patients with 613 specimens (158 positive tests and 455 negative tests).One hundred and forty-seven patients had a history of MRSA colonization or infection, and 56 (38%) had positive nasal screening using PCR (Table 1).One hundred and ninety-�ve patients had no history of MRSA colonization or infection but fell into high risk groups, and 47 (24%) had a positive PCR screen.irty patients had two samples taken for testing, 18 had positive screens on both samples, and 12 had only one specimen positive.Nine patients had three samples taken for PCR testing.Seven were positive on all three samples, and 2 were positive on two of three samples.

Discussion
Our study demonstrates that 38% of patients with a history of MRSA colonization or infection were nasal carriers upon readmission to the hospital and required contact isolation and that 24% of patients in a risk group were current carriers and required contact isolation.erefore, these results demonstrate that microbiological surveillance with selective screening using rapid detection methods with PCR technology facilitates decisions regarding contact isolation.is approach should reduce transmission of MRSA from patients to healthcare workers and other patients and limit the use of unnecessary isolation.is approach should be more cost effective than using universal screening [4].
Our culture results indicate that not all patients with a positive PCR for MRSA had MRSA identi�ed in nasal swab cultures.Some patients had only MSSA, methicillinresistant, coagulase negative Staphylococcus or methicillinsensitive, coagulase negative Staphylococcus recovered from nasal cultures.erefore, these results indicate that PCR technology can result in misclassi�cation using bacterial cultures as the gold standard.ese false positive results may be explained by laboratory mistakes, the presence of nonviable bacteria on nasal swabs, and/or various genetic events which create discrepancies between PCR results and methicillin sensitivity [6][7][8][9][10].ese patients with a positive PCR test do need isolation.However, the next step in their management is uncertain.Sturenburg maintains that positive PCR results always require con�rmation [9].However, this approach would increase the cost and complexity of screening, and whether or not the correct identi�cation of some hospitalized patients with staphylococcal colonization (but not MRSA) is important to discontinue contact isolation is uncertain.e answer to this question will likely depend on the prevalence Percent positive signi�cantly higher in patients with a history of MRSA infection or colonization than in patients in high risk groups.P < 0.01 by Chi square test.