Identification of Methicillin-Resistant Staphylococcus aureus Carriage in Less than 1 Hour during a Hospital Surveillance Program

MAJOR ARTICLE Identification of Methicillin-Resistant Staphylococcus aureus Carriage in Less than 1 Hour during a Hospital Surveillance Program Ann Huletsky, 1,2 Pierre Lebel, 3 François J. Picard, 1,2 Marthe Bernier, 1 Martin Gagnon, 1 Nathalie Boucher, 1 and Michel G. Bergeron 1,2 1 Centre de Recherche en Infectiologie and 2 Division de Microbiologie, Université Laval, Sainte-Foy, and 3 Department of Microbiology, Montreal General Hospital, McGill University Health Center, Montreal, Canada Background. Methicillin-resistant Staphylococcus aureus (MRSA) has spread worldwide and is responsible for significant morbidity, mortality, and health care costs. Control strategies to limit the emergence and spread of this organism rely on rapid and sensitive tests for detection of MRSA carriage. However, the standard surveillance culture method for detecting MRSA is labor intensive and time-consuming (2 3 days per procedure). There is thus a need for a rapid and accurate method to screen for MRSA carriage. Methods. We recently developed an easy-to-use real-time polymerase chain reaction (PCR) assay suitable for specific detection of MRSA in nasal specimens in!1 h. We studied the efficacy of our new PCR assay in routine screening for nasal MRSA carriage during a hospital surveillance program. A total of 331 nasal specimens obtained from 162 patients at risk for colonization were tested by both the standard mannitol agar culture method and our PCR assay. Results. The PCR assay detected MRSA in all 81 samples that were culture positive for MRSA. The PCR assay detected 4 additional MRSA-positive specimens, for a specificity of 98.4%, a positive predictive value of 95.3%, and a sensitivity and negative predictive value of 100%. Conclusions. This novel PCR assay allows reliable identification of MRSA carriers in!1 h. This test should facilitate the efficacy of MRSA surveillance programs. Staphylococcus aureus is one of the most significant human pathogens that cause both nosocomial and community-acquired infections. Strains of S. aureus that are resistant to methicillin (and oxacillin) have spread worldwide, with a mean prevalence of 60% among patients in US intensive care units [1, 2]. Infection with methicillin-resistant S. aureus (MRSA) strains, which are resistant to a wide range of antibiotics, is associated with considerable morbidity and mortality [3 7]. The mortality rate for patients with MRSA infection is 2 2.5 higher than that for patients with methicillin-susceptible S. aureus (MSSA) infection [4, 7, 8]. The spread Received 9 September 2004; accepted 22 November 2004; electronically published 4 March 2005. Reprints or correspondence: Dr. Michel G. Bergeron, Centre de Recherche en Infectiologie de l Université Laval, Centre Hospitalier de l Université Laval, Pavillon CHUL, 2705 boul. Laurier, Sainte-Foy, G1V 4G2 Canada (michel.g.bergeron @crchul.ulaval.ca). Clinical Infectious Diseases 2005; 40:976 81 2005 by the Infectious Diseases Society of America. All rights reserved. 1058-4838/2005/4007-0011$15.00 of MRSA has a huge impact on patient management in health care settings and results in enormous increases in health care costs [3 7]. Studies performed in 1999 showed that the direct medical cost in US dollars for MRSA infections in US hospitals is $27,083 $34,000 per patient, which is 1.5 3 times greater than that for MSSA infections [3, 7]. Consequently, annual US health care costs associated with MRSA infections are estimated to be approximately $6 billion. Therefore, the prevention and control of multidrug-resistant MRSA spread remains a major challenge of infection-control practitioners worldwide [9]. With the emergence of glycopeptide-resistant S. aureus, control of MRSA is becoming even more essential [10 13]. Despite controversy among infection-control practitioners during the past several years about the control of MRSA spread [9, 14], many recent studies have shown that the spread of MRSA can be controlled by implementing effective preventive measures not only in hospitals in which the prevalence of MRSA is low but in those where MRSA is highly endemic [8, 15 17]. 976 CID 2005:40 (1 April) Huletsky et al.

Current control strategies to limit the emergence and rapid spread of this organism rely on rapid and sensitive tests for detection of MRSA carriers. However, standard culture methods for the identification of MRSA are labor-intensive and require at least 48 h to complete. A screening test for MRSA that could provide accurate results in 1 h to identify patients who are carrying the bacteria would enable earlier implementation of contact precautions, thereby limiting the spread of MRSA. Over the past several years, a variety of DNA-based tests have been developed to detect MRSA carriage more quickly [18 21]. Most of these assays are based on the detection of an S. aureus specific gene and the meca gene, which encodes methicillin (and oxacillin) resistance. However, none of these molecular tests are suitable for detection of MRSA directly in specimens obtained during nasal screening, because such specimens often contain a mixed flora of coagulase-negative staphylococci and S. aureus, both of which can carry meca. Consequently, previously developed molecular methods to screen for MRSA carriage require either isolation of the bacteria, a growth enrichment period of at least 18 h, or complex technical steps lasting at least 6 h to allow discrimination between MRSA and methicillin-resistant coagulase-negative staphylococci [18 21]. By targeting MRSA-specific chromosomal sequences, we were able to develop a real-time PCR assay for the detection of MRSA that allowed us to discriminate MRSA from methicillin-resistant coagulase-negative staphylococci [22]. This unique and rapid PCR test is the first nucleic acid based diagnostic test that is sufficiently sensitive, specific, and ubiquitous in coverage (in terms of its ability to detect most MRSA strains) for direct detection of MRSA in nasal swab specimens. The assay has been shown to be specific and sensitive in tests of purified DNA and tests of nasal specimens spiked with MRSA cultures [22]. As part of a hospital MRSA surveillance program, we compared the performance of this new MRSA-specific and rapid PCR test with that of the standard mannitol culture method that requires at least 48 h to identify nasal MRSA carriage. METHODS Study design. From February through June 2002, a total of 162 patients who were 18 years of age and had been screened for MRSA carriage on the basis of the presence of risk factors for colonization during a MRSA surveillance program at the Montreal General Hospital (MGH) (McGill University Health Center; Montreal, Canada) were invited to participate in the study. For 66 of these patients, several specimens were collected at different periods (at intervals of at least 1 week) on the basis of their risk factor status during hospitalization or at readmission to the hospital. Persons with risk factors for colonization included patients with known MRSA infection or colonization who were readmitted, patients who were exposed to MRSA, patients who had transferred from any health care institution inside or outside of the Province of Quebec, foreign visitors, patients living at home who had previously been hospitalized in any health care institution (including the MGH), persons living in areas of high risk for MRSA carriage, and other persons whose risk was determined at the discretion of the MGH Infection Control Service. Ethical approval for the study was obtained from the institutional review boards at the Centre Hospitalier Universitaire de Québec (Sainte-Foy, Canada), which received support for the study, and at the MGH study site. Nasal specimens were obtained from all participating patients after written informed consent was obtained. Data concerning antibiotic treatment received during 48 h before MRSA screening, previous receipt of MRSA-decolonization protocols, risk factors for colonization, and dates of previous MRSApositive screening results at the MGH were collected from medical records. Patients who had received a MRSA-decolonization protocol were excluded from the study. Collection and culture of specimens. All nasal specimens were obtained with a collection swab (Venturi Transystem; Copan Canada). The swab, previously moistened with nonbacteriostatic sterile saline, was carefully inserted a short distance in each nostril and gently rotated for 5 s. The swab was inserted into the transport medium immediately after the sample was obtained. All specimens were transported at room temperature to the Centre de Recherche en Infectiologie de l Université Laval (Sainte-Foy, Canada) and tested within 24 h after collection. The same specimen was used for standard culture and for PCR. For the identification of MRSA, the swabs were streaked directly onto mannitol salt agar. The agar media were examined after 1 and 2 days of incubation for typical S. aureus colonies. All suspected S. aureus colonies were plated onto blood agar. Identification of S. aureus in suspicious colonies was based on measurement of catalase production, results of a slide agglutination test (Staphaurex; Murex Biotech Limited), and results of a tube coagulase test. Oxacillin resistance for each S. aureus isolate was determined by measuring growth on Mueller-Hinton agar containing 4% NaCl and 6 mg/ml oxacillin, as recommended by the NCCLS [23]. S. aureus identification and resistance were confirmed by means of the MicroScan WalkAway Overnight Panel Type Positive Breakpoint Combo 13 (Dade Behring Canada). The MIC of oxacillin was determined with Etest strips (AB Biodisk). A minimum of 48 h was required to identify MRSA by these culture-based methods. Bacteria remaining on the mannitol agar were placed in Brucella broth containing 15% glycerol and were stored at 80 C if additional cultures were necessary to resolve discordances. Additional cultures were performed in an enrichment broth by incubating the frozen bacteria for 24 h in 5 ml of Staphylococcus broth (Becton Dickinson) containing 8 mg/ml of aztreonam Real-Time PCR Detection of MRSA Carriage CID 2005:40 (1 April) 977

Figure 1. Schematic representation of the methicillin-resistant Staphylococcus aureus (MRSA) specific chromosomal region targeted by MRSAspecific primers and probes. The forward primers (left-hand arrows) are specific to the different SCCmec right-extremity sequences. The reverse primer (right-hand arrow) is specific to the S. aureus chromosomal orfx sequence. The 3 S. aureus specific fluorescent probes (central arrows) allow detection of the MRSA-specific amplification products. The figure is not drawn to scale. (Bristol-Myers Squibb). A total of 30 ml of broth was then plated onto blood agar and examined after 1 and 2 days of incubation for typical S. aureus colonies. Preparation of samples for PCR. After the nasal swab specimens were streaked onto mannitol salt agar, DNA was extracted rapidly from the same swab by means of methods described elsewhere [24, 25]. Crude DNA extracts were stored at 80 C if a second PCR was necessary to resolve discordances. Purified MRSA genomic DNA (the positive control) was prepared by means of methods described elsewhere [24]. Rapid PCR test. The MRSA-specific PCR assay was performed using the rapid DNA amplification apparatus Smart- Cycler (Cepheid). This multiplex PCR test is comprised of 5 primers specific to the different staphylococcal cassette chromosome mec (SCCmec) right-extremity sequences (mecii574, meciii519, meciv511, mecv492, and mecvii512) [22]. These 5 primers are used in combination with a primer specific to the S. aureus chromosomal orfx gene sequence located to the right of the SCCmec integration site (Xsau325) (figure 1). Three fluorescent-reporter labeled molecular beacon probes (XsauB5-FAM, XsauB8-FAM, and XsauB9-FAM) hybridizing to the MRSA-specific amplicons and a fourth molecular beacon probe (PSARM-TET) hybridizing to the internal control amplicon and labeled with a different fluorescent-reporter molecule were used for detection [22]. The multiplex PCR test allows simultaneous amplification and detection of the genomic DNA of MRSA and the DNA of the internal control template. Amplification reactions were performed with each crude lysate, as described elsewhere [22]. Internal and positive controls were used. PCR was repeated using frozen crude DNA extracts when it was necessary to resolve discordant results or when results were unresolved because of PCR inhibition. PCR assays with unresolved results were performed again to calculate the sensitivity, specificity, and positive and negative predictive values of the assay. When results of additional PCR assays remained unresolved, the samples were excluded from the study. Statistical analysis. The sensitivity, specificity, and positive and negative predictive values of PCR were calculated by comparing the results of PCR with the results of standard mannitol agar culture. The 95% CIs for sensitivity, specificity, and positive and negative values were calculated according to the method of Blyth and Still [26], because this method yields 95% CIs that have greater diagnostic accuracy than 95% CIs calculated using other methods, when the proportion of results is close to0or1. RESULTS Of the 331 nasal swab specimens obtained from 162 patients who were screened for MRSA colonization, 76 (23%) were culture positive for MRSA. PCR results for all 76 culture-positive specimens were positive (figure 2). Among the 255 culturenegative nasal specimens, 241 were PCR negative, 9 were PCR positive, and 5 had unresolved results because of PCR inhibition. PCR results for frozen crude DNA extracts of these 5 specimens, which comprised 1.5% of the 331 specimens tested, Figure 2. Disposition of nasal specimens screened for methicillin-resistant Staphylococcus aureus (MRSA) from February to June 2002. +, Positive;, negative. 978 CID 2005:40 (1 April) Huletsky et al.

Table 1. Sensitivity, specificity, and predictive values of PCR for detection of methicillin-resistant Staphylococcus aureus. Parameter Uncorrected value, % (95% CI) Corrected value, % (95% CI) Sensitivity a 100 (94 100) 100 (94.4 100) Specificity b 96.5 (93.2 98.3) 98.4 (95.7 99.5) Positive predictive value c 89.4 (80.4 94.7) 95.3 (87.7 98.5) Negative predictive value d 100 (98.1 100) 100 (98.1 100) NOTE. Corrected values were calculated at the conclusion of resolution testing. a Data are no. of both culture- and PCR-positive results/no. of culture-positive b Data are no. of both culture- and PCR-negative results/no. of culture-negative c Data are no. of both culture- and PCR-positive results/no. of PCR-positive d Data are no. of both culture- and PCR-negative results/no. of PCR-negative were negative (figure 2). Compared with the standard culture method, the real-time PCR assay had a sensitivity of 100% (95% CI, 94% 100%), a specificity of 96.5% (95% CI, 93.2% 98.3%), a positive predictive value of 89.4% (95% CI, 80.4% 94.7%), and a negative predictive value of 100% (95% CI, 98.1% 100%) (table 1). Additional experiments were performed to resolve discordances between PCR results and culture Among the 9 specimens with positive PCR results but negative standard culture results, 5 were shown to be culture positive after a second culture in an enrichment broth (figure 2). These 9 specimens were retested by PCR and remained PCR positive. Therefore, at the conclusion of resolution testing, 81 specimens were culture and PCR positive for MRSA, 246 specimens were culture and PCR negative, and 4 specimens were culture negative but PCR positive (i.e., specimens with false-positive PCR results). On the basis of the results of resolution tests, the specificity and the positive predictive value of the PCR assay increased to 98.4% (95% CI, 95.7% 99.5%) and 95.3% (95% CI, 87.7% 98.5%), respectively (table 1). Overall, MRSA carriers were detected more often by PCR than by culture. The time required to obtain results varied between 30 and 54 min for the new PCR assay, depending on the bacterial load of the specimens (figure 3), but was at least 48 h for the standard culture method. DISCUSSION Molecular epidemiology data and the observation that MRSA spread has been successfully managed by use of rigorous infection-control practices clearly demonstrate that transmission is the major factor contributing to the increasing prevalence of MRSA infection or colonization among patients [27 29]. Success in controlling MRSA spread has been the greatest in countries that adhere to transmission-based control policies that include active surveillance cultures to identify patients colonized or infected with MRSA [29]. However, standard MRSA culture methods take at least 48 h to perform, with the possible consequences that patients might needlessly be placed under contact precautions for 2 3 days or that MRSA transmission risk might increase if patients are placed under contact precautions only after MRSA culture results become available. Most molecular methods developed to more rapidly detect MRSA carriage require 6 18 h [18 21]. We have recently developed a new real-time PCR assay for the detection of MRSA [22]. Our PCR test is comprised of 5 primers specific to the right-extremity primer sequences of SCCmec types I, II, III, IVa, IVb, and IVc, including those of the newly recognized community-acquired MRSA strain [30] and 3 new genotypes that we recently characterized, which ensures the detection of most MRSA strains. These 5 primers are used in combination with a primer specific to the S. aureus chromosomal orfx gene sequence located to the right of the SCCmec integration site, thus providing a link between meca and S. aureus. By linking meca to S. aureus, this PCR test allows direct detection of MRSA in clinical specimens containing a mixture of staphylococci, without previous isolation, capture, or enrichment of the bacteria, thereby reducing the number of sample preparation steps and the time required to obtain By combining this test with a rapid and easy-to-use DNA extraction method for analysis of MRSA in nasal specimens, results of PCR performed directly on nasal specimens can be obtained in!1 h. Figure 3. Results of PCR for detection of methicillin-resistant Staphylococcus aureus (MRSA) in nasal specimens. The product of the MRSAspecific amplification is measured in terms of increases in fluorescence during the amplification process. Sample 1 was obtained from a patient with substantial MRSA colonization; sample 2 was obtained from a patient with minimal MRSA colonization; sample 3 was obtained from a patient with no MRSA colonization; sample A was a positive control, to which 300 fg of purified MRSA genomic DNA had been added; and sample B was a negative control, to which no target DNA had been added. Real-Time PCR Detection of MRSA Carriage CID 2005:40 (1 April) 979

We have evaluated the performance of our PCR assay during a surveillance culture program to screen patients with risk factors for MRSA colonization. MRSA was detected more often by PCR than by the standard mannitol culture method. Indeed, 9 (3.5%) of 255 culture-negative specimens were PCR positive. Antimicrobial agents that are active against MRSA could theoretically have decreased the specificity of the molecular assay, but there was no significant difference between culture results and PCR results among patients receiving or not receiving systemic anti-mrsa antimicrobial agents before specimens were obtained. Of the 9 specimens with positive results of PCR but negative results of standard mannitol culture, 5 were shown to be culture-positive when samples recovered from the initial mannitol agar plates after 24 h of incubation were subcultured in an enrichment broth. With the addition of these 5 specimens that were positive for MRSA on subculture, the specificity and the positive predictive value increase to 98.4% and 95.3%, respectively. A sixth specimen was culture- and PCR-positive for an MSSA strain. Of interest, 1 month later, the patient from whom MSSA was recovered was shown to be colonized by a MRSA strain with the same genetic background as the MSSA isolate, as revealed by randomly amplified polymorphic DNA analysis (data not shown). Detection of the MSSA isolate by PCR could be explained by the presence of a residual SCCmec rightextremity fragment after deletion of a chromosomal region containing meca from the original MRSA strain; spontaneous loss of the meca region has been described during cultivation of MRSA strains [22]. The 3 other PCR-positive, culture-negative specimens may be attributable to the presence of nonviable or noncultivatable MRSA or to an MRSA load that was below the detection limit of the mannitol culture method. Indeed, 1 of these 3 PCR-positive specimens was collected from a patient who was found to be colonized by MRSA 6 weeks later on the basis of both cultures and PCR methods. Overall, the PCR assay detected 3.5% more MRSA-positive specimens than did the mannitol agar culture method. Therefore, the sensitivity of our PCR assay was greater than that of the standard mannitol agar culture method and may be more comparable to the sensitivity of the enrichment broth method, which has also been shown to detect more MRSA carriers than direct culture on mannitol agar [31]. The use of our new PCR assay has decreased the turnaround time required to identify MRSA carriers to!1 hour. Recently, it has been shown that use of a PCR-based test for rapid detection of vancomycin-resistant enterococci (VRE) in rectal specimens during a hospital outbreak resulted in complete elimination of VRE transmission in this hospital [32]. Because our PCR assay is easy to perform and is rapid, sensitive, specific, and ubiquitous [22], it should provide a useful alternative for infection-control practitioners to better control the spread of MRSA. In conclusion, we have found that MRSA carriers can be identified rapidly and reliably by direct molecular testing of nasal specimens. The use of this test should facilitate MRSA surveillance programs. Acknowledgments We are indebted to Louise Côté (Microbiology Laboratory, Centre Hospitalier de l Université Laval; Sainte-Foy, Canada). We thank Line Guay and Gisèle Chassé for culturing clinical specimens and Karen Rye and Elaine Sheldon for recruiting study subjects. We also thank Pierre Provencher for statistical analysis of the data and Antoine Bouchard Fortier for helpful technical assistance. Financial support. Canadian Institutes of Health Research (grant PA- 15586) and Infectio Diagnostic. Potential conflicts of interest. 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