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Comparison of Culture Screening Protocols for Methicillin-Resistant Staphylococcus aureus (MRSA) Using a Chromogenic Agar (MRSA-Select)

Address correspondence to Yeon-Joon Park, M.D., Department of Laboratory Medicine, College of Medicine, Catholic University of Korea, Kangnam St. Mary’s Hospital, 505 Banpo-dong, Seocho-ku, Seoul 137-701, Korea; tel 82 2 590 1604; fax 82 2 590 2547; e-mail yjpk{at}catholic.ac.kr.

	Abstract

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To compare the culture screening protocols for methicillin-resistant Staphylococcus aureus (MRSA), a total of 300 duplicate nasal swabs (233 initial cultures and 67 weekly follow-up cultures) were collected consecutively from 233 patients in the Intensive Care Unit (ICU). One swab was plated directly on MRSA-Select agar (D-MRSA-Select) and observed at 24 hr. The duplicate swab was incubated in tryptic soy broth (TSB) with 6.5% NaCl for 24 hr, and then subcultured on MRSA-Select (B-MRSA-Select), BAP (B-BAP), and mannitol salt agar with 4 mg/L oxacillin (B-MSA_OXA), and observed at 24 hr. MRSA was detected in 13.7% (32/233) of the initial and 22.4% (15/67) of the follow-up specimens. A patient was classified as MRSA-positive if any of the media grew colonies that were tested and confirmed to be MRSA. In the initial screening samples, the sensitivities of D-MRSA-Select, B-MRSA-Select, B-BAP, and B-MSA_OXA were 78.1%, 84.4%, 78.1%, and 65.6%, respectively, and the specificities were 100%, 98.0%, 83.1%, and 93.5%, respectively. The sensitivities of all but the B-MRSA-Select protocol were significantly lower (p <0.05). In follow-up screening, the sensitivities of D-MRSA-Select, B-MRSA-Select, B-BAP, and B-MSA_OXA were 66.7%, 86.7%, 66.7%, and 53.3%, respectively, and the specificities were 100%, 98.1%, 90.4%, and 90.4%, respectively. D-MRSA-Select protocol was considered useful in screening for MRSA because it was fast, highly specific, and showed sensitivity comparable to B-BAP. Salt-containing enrichment broth in conjunction with MRSA-Select (B-MRSA-Select) provides a promising way to increase sensitivity in initial and follow-up screening for MRSA.

Keywords: methicillin-resistant Staphylococcus aureus (MRSA), MRSA-Select agar

	Introduction

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Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen, which shows a higher prevalence in ICUs than in general wards [1]. Rapid and accurate screening of MRSA carriers among high-risk patients is essential for MRSA control. Various screening protocols introduced for MRSA detection such as simple direct agar media including blood agar, mannitol-salt agar with oxacillin, and chromogenic agar, pre-enrichment broth media, or molecular methods [2]. The use of salt-containing enrichment broth in conjunction with a blood agar plate and a mannitol-salt agar plate containing 4 to 6 mg/L of oxacillin has been recommended to increase sensitivity [3].

In a previous study, we observed that pre-enrichment was an essential step to increase detection of MRSA in the nasal surveillance of ICU patients [4]. Recently, MRSA-Select agar (Bio-Rad, France), a chromogenic selective agar for detecting MRSA, has been introduced. We undertook this study to compare the MRSA detection rates obtained by the use of various culture screening protocols including MRSA-Select agar for nasal swabs from ICU patients.

	Methods and Materials

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This study was performed prospectively from March to April 2007. A total of 300 specimens from 233 ICU patients were screened consecutively. For 233 newly admitted ICU patients, the screening for MRSA was carried out within the first 48 hr in the ICU and repeated weekly in 67 cases of prolonged hospitalization. Duplicate nasal swab specimens were transported in Amie’s medium (Micromedia Co., Korea) and were processed within 2 hr of collection in the following manner: one was streaked directly on MRSA-Select agar plate (Bio-Rad, France) (D-MRSA-Select), incubated at 35°C, and then examined at 24 hr; another swab was inoculated into tryptic soy broth (TSB) (BBL, Cockeysville, MD, USA) containing 6.5% NaCl, and after 24 hr subcultured in MRSA-Select agar (B-MRSA-Select), BAP (B-BAP), and mannitol salt agar containing 4 mg/L of oxacillin (B-MSA_OXA) (Shinyang Chemical Co., Korea). The subcultures were examined at 24 hr. Small and pink colonies on MRSA-Select agar medium or colonies showing characteristics of S. aureus on BAP or MSA were picked up and then identified as S. aureus by catalase production, positive slide coagulase, or positive tube coagulase tests. MRSA was confirmed by the cefoxitin disk diffusion susceptibility test according to the CLSI guideline [5].

A patient was classified as MRSA-positive if any of the media grew colonies that were tested and confirmed to be MRSA. For follow-up weekly testing, whether a patient was initially negative, but converted to a true carrier, or a patient was initially positive and still carried MRSA, the patient was counted as a new positive case. The sensitivity and specificity of each agar or agar-broth combination was judged against the patient classification. The report time was defined as the average time from receiving the sample in the laboratory to reporting the result to the clinician.

Statistical analysis was carried out with SPSS program 9.0 for Windows (SPSS Inc., Chicago, IL, USA). Pearson’s Chi-square test was applied to compare the detection rate of MRSA between initial and follow-up screening specimens. McNemar test was applied to compare the culture screening protocols. A p value of <0.05 was considered significant.

	Results

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Of the initial 233 patients and follow-up 67 patients, 32 (13.7%) and 15 (22.4%) were classified as MRSA-positive, respectively. There was no significant difference in the detection rate of MRSA between initial and follow-up screening specimens (p = 0.086). The report time was fastest (24 hr) in D-MRSA-Select, the same (48 hr) in B-MRSA-Select and B-MSA_OXA, and slowest (72 hr) in B-BAP. In the initial screening samples, the sensitivity was highest (84.4%) in B-MRSA-Select, the same (78.1%) in both D-MRSA-Select and B-BAP, and lowest (65.6%) in B-MSA_OXA (Table 1). The sensitivities of all but the B-MRSA-Select were significantly lower (p <0.05). The specificity was the highest (100%) in D-MRSA-Select, followed in order by B-MRSA-Select (98.0%), B-MSA_OXA (93.5%), and B-BAP (83.1%). In the follow-up screening samples, sensitivity was highest (86.7%) in B-MRSA-Select, the same (66.7%) in both D-MRSA-Select and B-BAP, and lowest (53.3%) in B-MSA_OXA, but only the sensitivity of B-MSA_OXA was significantly lower (p = 0.016). The specificity was highest (100%) in D-MRSA-Select, followed by B-MRSA-Select (98.0%) and the same in B-MSA_OXA and B-BAP (90.4%).

Five cases that were considered to be false-positive in B-MRSA-Select in initial and follow-up screening were all identified as methicillin-susceptible S. aureus. Atypical, tinted pink colonies, observed mainly on the first quadrant of the agar plate in both D-MRSA-Select (10%, 30/300) and B-MRSA-Select (15%, 45/300), were identified as coagulase-negative Staphylococcus or Gram-negative bacilli.

	Discussion

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D-MRSA-Select was the most rapid (24 hr) and the most specific (100%) protocol for initial and follow-up screening samples. It has been reported that the sensitivity of D-MRSA-Select varied from 68.0–97.3% according to the study design, ie, the selection of media compared with one another [6,7]. In the present study, we compared MRSA-Select agar with the media of salt-containing enrichment broth in conjunction with BAP and MSA_OXA [3]. Its sensitivity was 78.1%, which was comparable to that of B-BAP. By adding a broth pre-enrichment step, the sensitivity increased (6.3–20%) with a slight loss of specificity (2%).

MRSA-detecting selective agar media contain indicators to distinguish S. aureus from other organisms and agents to select methicillin-resistant strains. The most common selective agar medium has been MSA containing the indicators of mannitol/phenol red and the selective agents consisting of oxacillin (2–6 mg/L) and NaCl (3–7.5%), but its sensitivity was very low [2]. The sensitivity of MSA_OXA in this study showed corroborative data (61.7%). A new MRSA-Select agar contains cefoxitin, which shows better selectivity than oxacillin because it also inhibits S. aureus isolates that hyper-produce penicillinase [8,9]. This selectivity was remarkable in D-MRSA-Select, and as recommended by the manufacturer, pink colonies on D-MRSA-Select agar media could be regarded as MRSA. However, the selectivity decreased slightly in B-MRSA-Select according to the result that all 5 false-positive cases showing typical pink colonies in B-MRSA-Select were MSSA. It is likely that the pre-enrichment step enhances the growth of not only MRSA but also some S. aureus with low-level resistance to cefoxitin, and not all isolates can be inhibited on screening agar media because of the inoculum effect. However, atypical, tinted pink colonies should be carefully examined using complementary tests such as the Gram stain and slide coagulase test [9].

Since MRSA screening is the basic tool to prevent the nosocomial spread of MRSA, the most useful method should be selected. Although the D-MRSA-Select protocol showed limited sensitivity in both initial and follow-up screening, the combination with B-BAP increased the sensitivity (from 25/32 to 29/32 and from 10/15 to 14/15, respectively). In particular, the combination of B-MRSA-Select and B-BAP showed the highest sensitivity in both initial and follow-up screening samples (93.8%, 93.3%, respectively), though the report time could be delayed as much as 72 hr.

Molecular methods may be an alternative as their sensitivity was reported to be superior or comparable to those of selective agar media [10,11]. However, their disadvantage is in the follow-up screening after medication because a non-viable MRSA remnant could be detected, not to mention the high running costs and the possibility of amplification failure.

In conclusion, no single medium could recover all MRSA strains. The D-MRSA-Select protocol was considered useful for initial screening of MRSA because it can report within 24 hr with a sensitivity comparable to B-BAP and high specificity. The combination of B-MRSA-Select and B-BAP achieves an increase in sensitivity in both the initial and follow-up screening for MRSA.

	Acknowledgement

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This work was partially supported by a grant from the Clinical Medical Research Institute, Holy Family Hospital.

	References

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