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Prevalence of methicillin-resistant Staphylococcus aureus based on culture and PCR in inpatients at a tertiary care center in Tokyo, Japan
J Infect Chemother (2012) 18:630–636 DOI 10.1007/s10156-012-0385-8
ORIGINAL ARTICLE
Prevalence of methicillin-resistant Staphylococcus aureus based on culture and PCR in inpatients at a tertiary care center in Tokyo, Japan Hirokazu Taguchi • Tetsuya Matsumoto • Hiroki Ishikawa • Shoichi Ohta • Tetsuo Yukioka
Received: 5 March 2010 / Accepted: 26 January 2012 / Published online: 23 February 2012 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2012
Abstract We investigated active screening for colonization with methicillin-resistant Staphylococcus aureus (MRSA) on admission and weekly follow-up surveillance after admission to a tertiary care center (TCC) between June 2007 and 31 December 2007. Eleven percent (30/267) of patients were found to be positive for MRSA by polymerase chain reaction (PCR) and/or culture on admission; 5% (12/267) became positive during the TCC stay. The major primary diagnoses in MRSA-positive patients were pneumonia and cerebrovascular diseases. Twenty-two (52%) of 42 patients were found to be MRSA positive by both PCR and culture, compared with 19 (45%) of 42 who were PCR positive and culture negative. These findings suggest that active surveillance with PCR is highly sensitive and useful for the detection of MRSA colonization. To our knowledge, this is the first report of active surveillance of MRSA by PCR and bacterial culture in critically ill inpatients in Japan. Keywords Methicillin-resistant Staphylococcus aureus Real-time PCR Tertiary care center Active surveillance testing
H. Taguchi (&) S. Ohta T. Yukioka Department of Emergency and Critical Care Medicine, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Sinjuku, Tokyo 160-0023, Japan e-mail: [email protected] T. Matsumoto H. Ishikawa Department of Microbiology, Tokyo Medical University, Tokyo, Japan
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Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen that causes infections among critically ill patients admitted to tertiary care centers. The incidence of MRSA infection continues to increase in hospitals worldwide, in some studies accounting for 20–60% of all Staphylococcus aureus isolates. Countries with the highest prevalence of MRSA include the United States, Australia, parts of Europe, and Asia, including Japan [1–5]. The most recent report from the National Nosocomial Infection Surveillance System noted that approximately 60% of all S. aureus nosocomial infections in intensive care units in 2003 were methicillin resistant, representing an 11% increase in resistance compared to the preceding 5-year period [6]. However, the role of polymerase chain reaction (PCR) and culture screening in MRSA detection in tertiary care centers in Japan has not been investigated. Screening for MRSA is a key component of successful infection control. Active surveillance culture (ASC) and subsequent isolation of colonized patients are effective and frequently used strategies for reducing the transmission of MRSA in intensive care units (ICUs) [7]. ASC for MRSA in the nares of at-risk populations is an important component of the Society for Healthcare Epidemiology of America (SHEA) recommendations for control of nosocomial transmission of MRSA [7]. Conventional culture detection methods for MRSA are time consuming and often lead to delayed or unnecessary isolation precautions [8]. Although recently developed rapid detection polymerase chain reaction (PCR) assays detect MRSA within a few hours directly from screening swabs, these are expensive and their efficacy has not been convincingly established [9]. To allow outbreaks of MRSA to be identified more quickly and to facilitate treatment for MRSA
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infection in tertiary care centers of Japan, improvements in the efficiency and timeliness of ASC have been sought. Here, to characterize patients positive for either or both PCR screening or culture, we investigated the prevalence of MRSA infection among inpatients presenting to a tertiary care center in a metropolitan area of Tokyo, Japan.
Patients and methods Study design and setting The study was conducted in a prospective cohort of adults patients admitted to the Tertiary Care Center of Tokyo University School of Medicine Hospital between June 2007 and 31 December 2007. The hospital is a 1,000-bed tertiary teaching hospital situated in the Tokyo metropolitan area that includes a 20-bed tertiary care center. Tertiary care centers in Japan differ from those in the United States and Europe in that they provide acute intensive care and interventions for patients with serious illnesses and trauma only. During the 7-month study period, 267 admissions to the tertiary care center were recorded, representing 502 samples. Mean age was 65.9 years (range, 12–94 years) and 98 (36.7%) were female. The most common primary diagnoses at the time of admission were infectious disease (72 cases, 27%), trauma (60 cases, 22%), cerebrovascular disease (58 cases, 22%), postresuscitation (42 cases, 16%), drug intoxication (26 cases, 10%), and circulatory disease (9 cases, 3%). Written informed consent (IC) concerning this study was obtained in accordance with the requirements of the hospital’s Institutional Review Board. Microbiological procedures All patients underwent MRSA screening culture of nasal swabs and PCR using the GeneOhm MRSA assay (Becton Dickinson, Franklin Lakes, NJ, USA) on admission. We attempted to perform unilateral anterior nares swab (CultureSwab Liquid Stuart Single Swab; Becton Dickinson). Samples of nasal swabs for bacterial culture and PCR were obtained on admission and every 7 days thereafter. Bacterial culture methods After sampling, nasal swabs were immediately transported to the laboratory, plated directly onto mannitol salt agar, incubated at 35°C with 5% CO2 for 48 h, and then maintained at 25°C for 5 days. Colonies that were mannitol fermenting, catalase positive, and coagulase positive were then screened for methicillin resistance on Mueller–Hinton agar supplemented with sodium chloride cations and oxacillin at
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4 lg/ml according to the Clinical Laboratory Standards Institute (CLSI) guidelines. Antimicrobial susceptibility testing for agents was conducted by broth microdilution using MicroScan (WalkAway). PCR assay The swabs were also processed with the real-time PCRbased BD GeneOhm MRSA Assay (Becton Dickinson Diagnostics) in accordance with the manufacturer’s instructions. Our exclusive use of nasal swabs was determined by the manufacturer’s recommendation and by the fact that the nostrils are the main site of S. aureus colonization. The nasal swab was placed in a tube with the buffer (7% NaCl), and the resulting suspension was transferred to a lysis tube for DNA extraction. Subsequent addition of the kit’s molecular reagents was followed by real-time PCR. The entire process run-time was about 2 h. In the GeneOhm assay, the use of primer sequences for the SCCmec and orfX regions generates an MRSA-specific amplicon, which is in turn detected by a complementary molecular beacon probe. A positive control supplied with the kit and negative control (reaction without template) were included in each run. Data analysis GeneOhm MRSA results were compared with those obtained from culture. Samples that were positive under both GeneOhm assay and culture were expressed as PCRand culture positive, those that were negative under both methods were expressed as PCR- and culture negative, and those that were positive under GeneOhm assay and negative under culture were expressed as PCR positive and culture negative. Data regarding patient characteristics and culture results were obtained by prospective chart review. Additional background information was obtained regarding hospitalization in the preceding year, whether the patient was admitted from the community or another hospital, and previous antibiotic use. Therapeutic antimicrobial use was defined as treatment of a microbiologically or clinically documented infection. Data are presented as the mean ± SEM. Statistical analysis Data were analyzed with the general linear models of the SPSS v. 15 statistical package (SPSS, Chicago, IL, USA). Differences in antimicrobial treatment for MRSA between positive and negative cultures were tested using the chisquare test. The distribution of positive and negative results
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only. After admission, 6 of 12 patients were positive by both PCR and culture and 6 by PCR only (Fig. 2). Among all MRSA-positive patients, 71% (30/42) were detected on admission and 29% (12/40) within 4 weeks after admission. Nine (10%) of 90 patients who were MRSA negative on admission became MRSA positive within a week (Fig. 3).
between PCR and culture was evaluated using the chisquare test. Characteristics of the 42 MRSA-positive patients were compared using Student’s t test. P values less than 0.05 were considered statistically significant.
Results
Characteristics of MRSA-positive patients by detection method
Study patients A total of 502 samples were obtained from 267 patients admitted during the study period. Mean duration of stay in the tertiary care center was 18.2 days (range, 1–98 days), although 147 patients were hospitalized for less than 7 days and underwent a single nasal swab only. Forty-two patients (16%) were found to be positive by PCR, culture, or both. Of these, 11% (30/267) were positive on admission whereas 5% (12/267) became positive during their stay (Fig. 1). On admission, 16 of 30 patients were positive by both PCR and culture, 13 by PCR only, and 1 by culture
Fig. 1 Distribution of study population. Data are number (%) of patients. MRSA methicillin-resistant Staphylococcus aureus
Characteristics of the 42 MRSA-positive patients (22 males and 19 females) are summarized in Table 1. No significant differences were seen in mean total Glasgow Coma Scale, sepsis-related organ failure assessment (SOFA) score, or APACHE II score between those who were PCR- and culture positive versus those who were PCR positive and culture negative on admission. Among patients with a history of visiting or being admitted to a hospital in the past 5 years, rate of detection by both PCR and culture was
Admissions to the Tertiary Care Center (TCC) N = 267
Sampling of nasal swab on admission 267 (100%)
MRSA positive on admission 30 (11%)
MRSA negative on admission 237 (89%) Leave TCC within 7 days 147 (62%)
Swabbed during the TCC stay 90 (38%)
MRSA positive 12 (5%)
Fig. 2 Distribution of study population: polymerase chain reaction (PCR)- and culture positive, PCR positive and culture negative, and PCR negative and culture positive
MRSA negative 78 (29%)
All MRSA Positive Cases N = 42
Positive on Admission 30 (71%)
PCR+ / culture+ 16 (38%)
PCR+ / culture13 (31%)
Positive after admission 12 (29%)
PCR- / culture+ 1 (2%)
PCR+ / culture+ 6 (14.5%)
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PCR+ / culture6 (14.5%)
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Fig. 3 Classification of cases of methicillin-resistant Staphylococcus aureus positivity according to whether they were imported or acquired, and whether they represented infection or colonization
n=267 237
No. of cases
MRSA negative MRSA positive
n=90 81 n=40 38
30
n=13 12
n=6 6
9
2
1 week
2 week
3 week
4 week
5 week
1
PCR
Culture
+
+
16
4
1
0
1
0
+
-
13
5
1
0
0
0
-
+
1
0
0
0
0
0
30
9
2
0
1
0
Total
Table 1 Demographic characteristics and risk factors of methicillin-resistant Staphylococcus aureus (MRSA)-positive patients
Admission
n=25 25
PCR? culture? (n = 22)
PCR? culture(n = 19)
Female
13
6
Male
9
13
73 (±21)
67 (±20)
Length of stay in tertiary care center, days (mean ± SD)
11 (±54)
9 (±20)
Existence of a past history (%)
21 (96)
18 (95)
Clinic visit history (%)
10 (46)#
3 (16)
Variable Gender
Age, years (mean ± SD)
Total Glasgow coma scale on admission
7.1
7.7
SIRSa
16
16
SOFAb score
11
11
Significant difference (#P \ 0.05)
APACHE II score Administration of antimicrobial drug for MRSA (%)
28 6 (27)#
31 2 (11)
a
Outcome Survival
18
17
Death
4
2
Systemic inflammatory response syndrome
b
Sepsis-related Organ Failure Assessment
significantly higher than that of PCR-positive and culturenegative patients (P value \0.05). Primary diagnoses in MRSA-positive inpatients are shown in Table 2. Infectious disease was the most common primary diagnosis in PCR- and culture-positive patients, mostly caused by pneumonia 82% (9/11), followed by pyelonephritis 18% (2/11). Cerebrovascular disease was the most common primary diagnosis in PCR-positive and culture-negative patients, consisting of four with heart attack (21%) and three with gastrointestinal hemorrhage (16%). The three trauma patients had spinal cord injury and were positive by both methods. Among the ten patients with cerebrovascular disease, Activity of Daily Living
(ADL) scores before admission were satisfactory in nine. Results in the seven postresuscitation patients were similar to those of patients with cerebrovascular disease, with satisfactory ADL in all but one patient. MRSA finding by PCR assay and comparison with bacterial culture method Table 3 shows the results of PCR assay and culture method with total 502 swab samples tested in this study. Of these, 41 (8.2%), and 23 (4.6%) nasal swabs were positive for MRSA colonization by PCR and culture method, respectively. Among these, 19 (3.8%) were diagnosed by PCR
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Table 2 Mode of admittance of MRSA-positive patients ?
?
PCR culture?
PCR culture-
Total (%)
Infectious diseases
10a
1
11 (26.8)
Cerebrovascular diseases
5
5
10 (24.4)
Post-resuscitation
3
4
7 (17.1)
Trauma
3
0
3 (7.3)
Gastrointestinal hemorrhage
0
3
3 (7.3)
Other diseases
1
6b
10 (17.1)
Total
22
19
41 (100)
a
Eight under the influence of pneumonia
b
Heart attack, four cases
Table 3 Positive and negative predictive values of culture and polymerase chain reaction (PCR) PCR
Culture Positive
Positive Negative Total
Negative
Total
22
19
41
1
460
461
23
479
502
alone whereas 1 (0.2%) was diagnosed by culture alone. The sensitivity and the specificity of PCR were 95.6% (22/ 23) and 96.0% (460/479) if culture alone is considered to be the positive standard.
Discussion In this study, we identified a relatively high rate of MRSA colonization and infection in patients admitted to a tertiary care center in Japan. With regard to the infection versus colonization rate, 22 patients were positive under both methods. Sixteen were carriers on admission whereas 6 were colonized in the tertiary care center. The colonization rate was 6% (16/267) but increased to 11% (30/267) if the 14 PCR-positive and culture-negative patients who were MRSA carriers on admission were included. Nine of the 90 patients acquiring MRSA after admission became positive within a week. The 11% prevalence found in this study is consistent with the high rates at ICU admission seen in some earlier studies in Europe [10, 11], which ranged from 8% to 10%, but contrast with lower rates at admission in more recent studies in the United States, Canada, and France of 4–9% [12–14]. The value of MRSA screening at ICU admission remains contentious. Although most MRSA control recommendations include screening at hospital admission [15, 16] in all
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patients or in those selected according to epidemiological conditions, others [9] do not include screening at admission, arguing that it is costly and has not been proven in controlled studies to curb the incidence of MRSA. Other authors [17, 18] suggest that MRSA screening may be crucial to the successful control of MRSA outbreaks. The high prevalence of MRSA infection in our series of high-risk patients supports the value of PCR screening in such patients. The proportion of patients in our study positive under both methodologies, 5%, was closely similar to the 4% on culture reported by Girou et al. [11], all of whom had been transferred from other wards within the hospital or had a history of visiting other clinics. This similarity can be ascribed to their restriction of screening to patients considered at high risk of MRSA carriage, namely those transferred from other wards with a known high prevalence of MRSA; those with a prolonged stay in another ward before ICU admission; and those with a history of hospitalization in high-risk areas. Patients with these risk factors for MRSA carriage may be more likely to have an MRSA-positive clinical specimen at ICU admission [13]. Of interest, PCR successfully detected MRSA even in the nasal samples of patients with a negative nasal culture. In contrast, only one patient was PCR negative and culture positive. This outcome may have been the result of errors in obtaining the culture specimen. These findings provide support for the greater efficacy of PCR over culture. In our study, 67% (8/12) of those acquiring MRSA during admission to our tertiary care center had risk factors for infection, such as intubation, diabetes mellitus, and central venous catheter placement. Further, 100% (4/4) of those acquiring MRSA colonization during the center stay developed MRSA infection. Davis et al. reported that risk factors for MRSA colonization included hemodialysis; illicit intravenous drug use; dermatitis, diabetes mellitus; admission to a burns unit; previous exposure to antimicrobial agents, particularly cephalosporins, aminoglycosides, and fluoroquinolones; prolonged hospitalization; and severe underlying illness. Further, 25% of individuals who acquired MRSA colonization during a hospital stay subsequently developed MRSA infection [19]. Identifying MRSA colonization at or after admission might target a high-risk population that may benefit from interventions to decrease the risk for subsequent MRSA infection. In the present study, 45% of infected or colonized patients were PCR positive and culture negative. Of patients requiring treatment, 25% were PCR positive and culture negative. In their study of ICU patients, Lucet et al. [13] reported that MRSA in 54% of colonized patients would have remained undetected had comprehensive multisite screening for MRSA not been performed at the time of admission. Although the use of nasal cultures alone for the detection of MRSA colonization has a sensitivity of
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78–85% [13], our findings suggest that active surveillance requires the performance of PCR, and that doing so will lead to an increase in MRSA detection rate. PCR assay in the present study was markedly effective in the detection of MRSA in critically ill patients. With regard to the BD GeneOhm MRSA assay, the present and previous studies have confirmed that this assay is both specific and sensitive [20–22]. Similar results for the sensitivity of this assay for single throat swabs have been reported by Rossney et al. (89.0% sensitivity and 99.0% specificity) [23] and by van Hal et al. [24] (90.0% sensitivity and 96.0% specificity). In a recent study by Svent-Kucina et al. [25], the GeneOhm MRSA assay protocol was modified during the specimen preparation step by the addition of an extra washing step, followed by the pooling of up to three samples per patient (nose, skin, with or without throat) at the lysis step. These methods provided sensitivity and specificity rates of 94.3% and 99.2%, respectively. Moreover, Boyce and Havill [26] compared the GeneOhm MRSA assay with the CHROMagar MRSA assay (BD Diagnostics) for the detection of MRSA in 286 nasal surveillance specimens, and found the GeneOhm assay to be both faster and more sensitive. These findings agree with our present results and confirm the sensitivity and specificity of this method. Interestingly, 9 of our present patients who were MRSA negative by both PCR and culture at admission subsequently became positive by both methods after admission; this may represent ‘selective pressure’ for MRSA. If so, this may suggest that some of or all our false-positive cases may have in fact been true positive. By type of antimicrobial, 4 of 12 patients (33%) were administered cepahalosporin while 3 received sulbactam/ampicillin (25%). In total, 92% (11/12) of positive patients were treated with an antibiotic. Several limitations of this study warrant mention. First, various variables of the culture method have not been evaluated, including the optimal frequency of culture and anatomical sites for screening. Here, we screened by nasal culture only, on the basis that the nose is the main ecological niche of this bacterium in humans [27]. Second, our study population was from a single tertiary university teaching hospital. Rates of MRSA in the community may differ in other settings, however, and thus our results may not be applicable to other populations. Third, the study was conducted under a single-center design and the number of infected patients was small. Patients infected with MRSA in our tertiary care center but not detected until after transfer to other wards would also have been missed, underestimating the rate of ICU-acquired MRSA infection. Fourth, we were unable to account for infections that occurred at other medical facilities, likely causing our reported risk of MRSA infection to be underestimated.
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In conclusion, we found that a significant proportion of patients in our tertiary care center were colonized with MRSA at the time of admission. These findings confirm that colonization is a high risk factor for MRSA infection, and that MRSA surveillance is effective in preventing MRSA infection in Japan. Further research into the costeffectiveness of molecular testing is required. Acknowledgments We appreciate the advice and expertise of Drs. Shiro Mishima and Kimihito Koshihara. We also thank Etsuko Kutsukake of the Microbiology Department of Tokyo Medical University for her PCR analysis of MRSA.
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636 13. Lucet JC, Chevret S, Durand-Zaleski I, Chastang C, Regnier B. Prevalence and risk factors for carriage of methicillin-resistant Staphylococcus aureus at admission to the intensive care unit: results of a multicenter study. Arch Intern Med. 2003;163:181–8. 14. Warren DK, Guth RM, Coopersmith CM, Merz LR, Zack JE, Fraser VJ. Epidemiology of methicillin-resistant Staphylococcus aureus colonization in a surgical intensive care unit. Infect Control Hosp Epidemiol. 2006;27:1032–40. 15. Boyce JM. Methicillin-resistant Staphylococcus aureus in hospitals and long-term care facilities: microbiology, epidemiology, and preventive measures. Infect Control Hosp Epidemiol. 1992; 13:725–37. 16. British Society for Antimicrobial Chemotherapy. Hospital Infection Society and the Infection Control Nurses Association. Revised guidelines for the control of methicillin-resistant Staphylococcus aureus infection in hospitals. J Hosp Infect. 1998;39:253–90. 17. Coello R, Jimenez J, Garcia M, Arroyo P, Minguez D, Fernandez C, et al. Prospective study of infection, colonization and carriage of methicillin-resistant Staphylococcus aureus in an outbreak affecting 990 patients. Eur J Clin Microbiol Infect Dis. 1994;13: 74–81. 18. Cox RA, Conquest C, Mallaghan C, Marples RR. A major outbreak of methicillin-resistant Staphylococcus aureus caused by a new phage-type (EMRSA-16). J Hosp Infect. 1995;29:87–106. 19. Davis KA, Stewart JJ, Crouch HK, Florez CE, Hospenthal DR. Methicillin-resistant Staphylococcus aureus (MRSA) nares colonization at hospital admission and its effect on subsequent MRSA infection. Clin Infect Dis. 2004;39(6):776–82. 20. Bishop EJ, Grabsch EA, Ballard SA, Mayall B, Xie S, Martin R, et al. Concurrent analysis of nose and groin swab specimens by the IDI-MRSA PCR assay is comparable to analysis by individual-specimen PCR and routine culture assays for detection of colonization by methicillin-resistant Staphylococcus aureus. J Clin Microbiol. 2006;44:2904–8.
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J Infect Chemother (2012) 18:630–636 21. Farley JE, Stamper PD, Ross T, Cai M, Speser S, Carroll KC. 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 an at-risk community population. J Clin Microbiol. 2008;46:743–6. 22. Sumitani Y, Kobayashi Y. Comparative evaluation of a rapid MRSA detection assay based on multiple real-time PCR versus MRSA screening cultures containing egg yolk. J Infect Chemother. 2009;15:262–5. 23. Rossney AS, Herra CM, Fitzgibbon MM, Morgan PM, Lawrence MJ, O’Connell B. Evaluation of the IDI-MRSA assay on the Smart Cycler real-time PCR platform for rapid detection of MRSA from screening specimens. Eur J Clin Microbiol Infect Dis. 2007;26:459–66. 24. van Hal SJ, Stark D, Lockwood B, Marriott D, Harkness J. Methicillin-resistant Staphylococcus aureus (MRSA) detection: comparison of two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) with three selective MRSA agars (MRSA ID, MRSA Select, and CHROMagar MRSA) for use with infection-control swabs. J Clin Microbiol. 2007;45:2486–90. 25. Svent-Kucina N, Pirs M, Mueller-Premru M, Cvitkovic-Spik V, Kofol R, Seme K. One-year experience with modified BD GeneOhm MRSA assay for detection of methicillin-resistant Staphylococcus aureus from pooled nasal, skin, and throat samples. Diagn Microbiol Infect Dis. 2009;63:132–9. 26. Boyce JM, Havill NL. Comparison of BD GeneOhm methicillinresistant Staphylococcus aureus (MRSA) PCR versus the CHROMagar MRSA assay for screening patients for the presence of MRSA strains. J Clin Microbiol. 2008;46:350–1. 27. Wertheim HF, Melles DC, Vos MC, van Leeuwen W, van Belkum A, Verbrugh HA, et al. The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect Dis. 2005;5:751–62.
ORIGINAL ARTICLE
Prevalence of methicillin-resistant Staphylococcus aureus based on culture and PCR in inpatients at a tertiary care center in Tokyo, Japan Hirokazu Taguchi • Tetsuya Matsumoto • Hiroki Ishikawa • Shoichi Ohta • Tetsuo Yukioka
Received: 5 March 2010 / Accepted: 26 January 2012 / Published online: 23 February 2012 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2012
Abstract We investigated active screening for colonization with methicillin-resistant Staphylococcus aureus (MRSA) on admission and weekly follow-up surveillance after admission to a tertiary care center (TCC) between June 2007 and 31 December 2007. Eleven percent (30/267) of patients were found to be positive for MRSA by polymerase chain reaction (PCR) and/or culture on admission; 5% (12/267) became positive during the TCC stay. The major primary diagnoses in MRSA-positive patients were pneumonia and cerebrovascular diseases. Twenty-two (52%) of 42 patients were found to be MRSA positive by both PCR and culture, compared with 19 (45%) of 42 who were PCR positive and culture negative. These findings suggest that active surveillance with PCR is highly sensitive and useful for the detection of MRSA colonization. To our knowledge, this is the first report of active surveillance of MRSA by PCR and bacterial culture in critically ill inpatients in Japan. Keywords Methicillin-resistant Staphylococcus aureus Real-time PCR Tertiary care center Active surveillance testing
H. Taguchi (&) S. Ohta T. Yukioka Department of Emergency and Critical Care Medicine, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Sinjuku, Tokyo 160-0023, Japan e-mail: [email protected] T. Matsumoto H. Ishikawa Department of Microbiology, Tokyo Medical University, Tokyo, Japan
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Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen that causes infections among critically ill patients admitted to tertiary care centers. The incidence of MRSA infection continues to increase in hospitals worldwide, in some studies accounting for 20–60% of all Staphylococcus aureus isolates. Countries with the highest prevalence of MRSA include the United States, Australia, parts of Europe, and Asia, including Japan [1–5]. The most recent report from the National Nosocomial Infection Surveillance System noted that approximately 60% of all S. aureus nosocomial infections in intensive care units in 2003 were methicillin resistant, representing an 11% increase in resistance compared to the preceding 5-year period [6]. However, the role of polymerase chain reaction (PCR) and culture screening in MRSA detection in tertiary care centers in Japan has not been investigated. Screening for MRSA is a key component of successful infection control. Active surveillance culture (ASC) and subsequent isolation of colonized patients are effective and frequently used strategies for reducing the transmission of MRSA in intensive care units (ICUs) [7]. ASC for MRSA in the nares of at-risk populations is an important component of the Society for Healthcare Epidemiology of America (SHEA) recommendations for control of nosocomial transmission of MRSA [7]. Conventional culture detection methods for MRSA are time consuming and often lead to delayed or unnecessary isolation precautions [8]. Although recently developed rapid detection polymerase chain reaction (PCR) assays detect MRSA within a few hours directly from screening swabs, these are expensive and their efficacy has not been convincingly established [9]. To allow outbreaks of MRSA to be identified more quickly and to facilitate treatment for MRSA
J Infect Chemother (2012) 18:630–636
infection in tertiary care centers of Japan, improvements in the efficiency and timeliness of ASC have been sought. Here, to characterize patients positive for either or both PCR screening or culture, we investigated the prevalence of MRSA infection among inpatients presenting to a tertiary care center in a metropolitan area of Tokyo, Japan.
Patients and methods Study design and setting The study was conducted in a prospective cohort of adults patients admitted to the Tertiary Care Center of Tokyo University School of Medicine Hospital between June 2007 and 31 December 2007. The hospital is a 1,000-bed tertiary teaching hospital situated in the Tokyo metropolitan area that includes a 20-bed tertiary care center. Tertiary care centers in Japan differ from those in the United States and Europe in that they provide acute intensive care and interventions for patients with serious illnesses and trauma only. During the 7-month study period, 267 admissions to the tertiary care center were recorded, representing 502 samples. Mean age was 65.9 years (range, 12–94 years) and 98 (36.7%) were female. The most common primary diagnoses at the time of admission were infectious disease (72 cases, 27%), trauma (60 cases, 22%), cerebrovascular disease (58 cases, 22%), postresuscitation (42 cases, 16%), drug intoxication (26 cases, 10%), and circulatory disease (9 cases, 3%). Written informed consent (IC) concerning this study was obtained in accordance with the requirements of the hospital’s Institutional Review Board. Microbiological procedures All patients underwent MRSA screening culture of nasal swabs and PCR using the GeneOhm MRSA assay (Becton Dickinson, Franklin Lakes, NJ, USA) on admission. We attempted to perform unilateral anterior nares swab (CultureSwab Liquid Stuart Single Swab; Becton Dickinson). Samples of nasal swabs for bacterial culture and PCR were obtained on admission and every 7 days thereafter. Bacterial culture methods After sampling, nasal swabs were immediately transported to the laboratory, plated directly onto mannitol salt agar, incubated at 35°C with 5% CO2 for 48 h, and then maintained at 25°C for 5 days. Colonies that were mannitol fermenting, catalase positive, and coagulase positive were then screened for methicillin resistance on Mueller–Hinton agar supplemented with sodium chloride cations and oxacillin at
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4 lg/ml according to the Clinical Laboratory Standards Institute (CLSI) guidelines. Antimicrobial susceptibility testing for agents was conducted by broth microdilution using MicroScan (WalkAway). PCR assay The swabs were also processed with the real-time PCRbased BD GeneOhm MRSA Assay (Becton Dickinson Diagnostics) in accordance with the manufacturer’s instructions. Our exclusive use of nasal swabs was determined by the manufacturer’s recommendation and by the fact that the nostrils are the main site of S. aureus colonization. The nasal swab was placed in a tube with the buffer (7% NaCl), and the resulting suspension was transferred to a lysis tube for DNA extraction. Subsequent addition of the kit’s molecular reagents was followed by real-time PCR. The entire process run-time was about 2 h. In the GeneOhm assay, the use of primer sequences for the SCCmec and orfX regions generates an MRSA-specific amplicon, which is in turn detected by a complementary molecular beacon probe. A positive control supplied with the kit and negative control (reaction without template) were included in each run. Data analysis GeneOhm MRSA results were compared with those obtained from culture. Samples that were positive under both GeneOhm assay and culture were expressed as PCRand culture positive, those that were negative under both methods were expressed as PCR- and culture negative, and those that were positive under GeneOhm assay and negative under culture were expressed as PCR positive and culture negative. Data regarding patient characteristics and culture results were obtained by prospective chart review. Additional background information was obtained regarding hospitalization in the preceding year, whether the patient was admitted from the community or another hospital, and previous antibiotic use. Therapeutic antimicrobial use was defined as treatment of a microbiologically or clinically documented infection. Data are presented as the mean ± SEM. Statistical analysis Data were analyzed with the general linear models of the SPSS v. 15 statistical package (SPSS, Chicago, IL, USA). Differences in antimicrobial treatment for MRSA between positive and negative cultures were tested using the chisquare test. The distribution of positive and negative results
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only. After admission, 6 of 12 patients were positive by both PCR and culture and 6 by PCR only (Fig. 2). Among all MRSA-positive patients, 71% (30/42) were detected on admission and 29% (12/40) within 4 weeks after admission. Nine (10%) of 90 patients who were MRSA negative on admission became MRSA positive within a week (Fig. 3).
between PCR and culture was evaluated using the chisquare test. Characteristics of the 42 MRSA-positive patients were compared using Student’s t test. P values less than 0.05 were considered statistically significant.
Results
Characteristics of MRSA-positive patients by detection method
Study patients A total of 502 samples were obtained from 267 patients admitted during the study period. Mean duration of stay in the tertiary care center was 18.2 days (range, 1–98 days), although 147 patients were hospitalized for less than 7 days and underwent a single nasal swab only. Forty-two patients (16%) were found to be positive by PCR, culture, or both. Of these, 11% (30/267) were positive on admission whereas 5% (12/267) became positive during their stay (Fig. 1). On admission, 16 of 30 patients were positive by both PCR and culture, 13 by PCR only, and 1 by culture
Fig. 1 Distribution of study population. Data are number (%) of patients. MRSA methicillin-resistant Staphylococcus aureus
Characteristics of the 42 MRSA-positive patients (22 males and 19 females) are summarized in Table 1. No significant differences were seen in mean total Glasgow Coma Scale, sepsis-related organ failure assessment (SOFA) score, or APACHE II score between those who were PCR- and culture positive versus those who were PCR positive and culture negative on admission. Among patients with a history of visiting or being admitted to a hospital in the past 5 years, rate of detection by both PCR and culture was
Admissions to the Tertiary Care Center (TCC) N = 267
Sampling of nasal swab on admission 267 (100%)
MRSA positive on admission 30 (11%)
MRSA negative on admission 237 (89%) Leave TCC within 7 days 147 (62%)
Swabbed during the TCC stay 90 (38%)
MRSA positive 12 (5%)
Fig. 2 Distribution of study population: polymerase chain reaction (PCR)- and culture positive, PCR positive and culture negative, and PCR negative and culture positive
MRSA negative 78 (29%)
All MRSA Positive Cases N = 42
Positive on Admission 30 (71%)
PCR+ / culture+ 16 (38%)
PCR+ / culture13 (31%)
Positive after admission 12 (29%)
PCR- / culture+ 1 (2%)
PCR+ / culture+ 6 (14.5%)
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PCR+ / culture6 (14.5%)
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Fig. 3 Classification of cases of methicillin-resistant Staphylococcus aureus positivity according to whether they were imported or acquired, and whether they represented infection or colonization
n=267 237
No. of cases
MRSA negative MRSA positive
n=90 81 n=40 38
30
n=13 12
n=6 6
9
2
1 week
2 week
3 week
4 week
5 week
1
PCR
Culture
+
+
16
4
1
0
1
0
+
-
13
5
1
0
0
0
-
+
1
0
0
0
0
0
30
9
2
0
1
0
Total
Table 1 Demographic characteristics and risk factors of methicillin-resistant Staphylococcus aureus (MRSA)-positive patients
Admission
n=25 25
PCR? culture? (n = 22)
PCR? culture(n = 19)
Female
13
6
Male
9
13
73 (±21)
67 (±20)
Length of stay in tertiary care center, days (mean ± SD)
11 (±54)
9 (±20)
Existence of a past history (%)
21 (96)
18 (95)
Clinic visit history (%)
10 (46)#
3 (16)
Variable Gender
Age, years (mean ± SD)
Total Glasgow coma scale on admission
7.1
7.7
SIRSa
16
16
SOFAb score
11
11
Significant difference (#P \ 0.05)
APACHE II score Administration of antimicrobial drug for MRSA (%)
28 6 (27)#
31 2 (11)
a
Outcome Survival
18
17
Death
4
2
Systemic inflammatory response syndrome
b
Sepsis-related Organ Failure Assessment
significantly higher than that of PCR-positive and culturenegative patients (P value \0.05). Primary diagnoses in MRSA-positive inpatients are shown in Table 2. Infectious disease was the most common primary diagnosis in PCR- and culture-positive patients, mostly caused by pneumonia 82% (9/11), followed by pyelonephritis 18% (2/11). Cerebrovascular disease was the most common primary diagnosis in PCR-positive and culture-negative patients, consisting of four with heart attack (21%) and three with gastrointestinal hemorrhage (16%). The three trauma patients had spinal cord injury and were positive by both methods. Among the ten patients with cerebrovascular disease, Activity of Daily Living
(ADL) scores before admission were satisfactory in nine. Results in the seven postresuscitation patients were similar to those of patients with cerebrovascular disease, with satisfactory ADL in all but one patient. MRSA finding by PCR assay and comparison with bacterial culture method Table 3 shows the results of PCR assay and culture method with total 502 swab samples tested in this study. Of these, 41 (8.2%), and 23 (4.6%) nasal swabs were positive for MRSA colonization by PCR and culture method, respectively. Among these, 19 (3.8%) were diagnosed by PCR
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Table 2 Mode of admittance of MRSA-positive patients ?
?
PCR culture?
PCR culture-
Total (%)
Infectious diseases
10a
1
11 (26.8)
Cerebrovascular diseases
5
5
10 (24.4)
Post-resuscitation
3
4
7 (17.1)
Trauma
3
0
3 (7.3)
Gastrointestinal hemorrhage
0
3
3 (7.3)
Other diseases
1
6b
10 (17.1)
Total
22
19
41 (100)
a
Eight under the influence of pneumonia
b
Heart attack, four cases
Table 3 Positive and negative predictive values of culture and polymerase chain reaction (PCR) PCR
Culture Positive
Positive Negative Total
Negative
Total
22
19
41
1
460
461
23
479
502
alone whereas 1 (0.2%) was diagnosed by culture alone. The sensitivity and the specificity of PCR were 95.6% (22/ 23) and 96.0% (460/479) if culture alone is considered to be the positive standard.
Discussion In this study, we identified a relatively high rate of MRSA colonization and infection in patients admitted to a tertiary care center in Japan. With regard to the infection versus colonization rate, 22 patients were positive under both methods. Sixteen were carriers on admission whereas 6 were colonized in the tertiary care center. The colonization rate was 6% (16/267) but increased to 11% (30/267) if the 14 PCR-positive and culture-negative patients who were MRSA carriers on admission were included. Nine of the 90 patients acquiring MRSA after admission became positive within a week. The 11% prevalence found in this study is consistent with the high rates at ICU admission seen in some earlier studies in Europe [10, 11], which ranged from 8% to 10%, but contrast with lower rates at admission in more recent studies in the United States, Canada, and France of 4–9% [12–14]. The value of MRSA screening at ICU admission remains contentious. Although most MRSA control recommendations include screening at hospital admission [15, 16] in all
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patients or in those selected according to epidemiological conditions, others [9] do not include screening at admission, arguing that it is costly and has not been proven in controlled studies to curb the incidence of MRSA. Other authors [17, 18] suggest that MRSA screening may be crucial to the successful control of MRSA outbreaks. The high prevalence of MRSA infection in our series of high-risk patients supports the value of PCR screening in such patients. The proportion of patients in our study positive under both methodologies, 5%, was closely similar to the 4% on culture reported by Girou et al. [11], all of whom had been transferred from other wards within the hospital or had a history of visiting other clinics. This similarity can be ascribed to their restriction of screening to patients considered at high risk of MRSA carriage, namely those transferred from other wards with a known high prevalence of MRSA; those with a prolonged stay in another ward before ICU admission; and those with a history of hospitalization in high-risk areas. Patients with these risk factors for MRSA carriage may be more likely to have an MRSA-positive clinical specimen at ICU admission [13]. Of interest, PCR successfully detected MRSA even in the nasal samples of patients with a negative nasal culture. In contrast, only one patient was PCR negative and culture positive. This outcome may have been the result of errors in obtaining the culture specimen. These findings provide support for the greater efficacy of PCR over culture. In our study, 67% (8/12) of those acquiring MRSA during admission to our tertiary care center had risk factors for infection, such as intubation, diabetes mellitus, and central venous catheter placement. Further, 100% (4/4) of those acquiring MRSA colonization during the center stay developed MRSA infection. Davis et al. reported that risk factors for MRSA colonization included hemodialysis; illicit intravenous drug use; dermatitis, diabetes mellitus; admission to a burns unit; previous exposure to antimicrobial agents, particularly cephalosporins, aminoglycosides, and fluoroquinolones; prolonged hospitalization; and severe underlying illness. Further, 25% of individuals who acquired MRSA colonization during a hospital stay subsequently developed MRSA infection [19]. Identifying MRSA colonization at or after admission might target a high-risk population that may benefit from interventions to decrease the risk for subsequent MRSA infection. In the present study, 45% of infected or colonized patients were PCR positive and culture negative. Of patients requiring treatment, 25% were PCR positive and culture negative. In their study of ICU patients, Lucet et al. [13] reported that MRSA in 54% of colonized patients would have remained undetected had comprehensive multisite screening for MRSA not been performed at the time of admission. Although the use of nasal cultures alone for the detection of MRSA colonization has a sensitivity of
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78–85% [13], our findings suggest that active surveillance requires the performance of PCR, and that doing so will lead to an increase in MRSA detection rate. PCR assay in the present study was markedly effective in the detection of MRSA in critically ill patients. With regard to the BD GeneOhm MRSA assay, the present and previous studies have confirmed that this assay is both specific and sensitive [20–22]. Similar results for the sensitivity of this assay for single throat swabs have been reported by Rossney et al. (89.0% sensitivity and 99.0% specificity) [23] and by van Hal et al. [24] (90.0% sensitivity and 96.0% specificity). In a recent study by Svent-Kucina et al. [25], the GeneOhm MRSA assay protocol was modified during the specimen preparation step by the addition of an extra washing step, followed by the pooling of up to three samples per patient (nose, skin, with or without throat) at the lysis step. These methods provided sensitivity and specificity rates of 94.3% and 99.2%, respectively. Moreover, Boyce and Havill [26] compared the GeneOhm MRSA assay with the CHROMagar MRSA assay (BD Diagnostics) for the detection of MRSA in 286 nasal surveillance specimens, and found the GeneOhm assay to be both faster and more sensitive. These findings agree with our present results and confirm the sensitivity and specificity of this method. Interestingly, 9 of our present patients who were MRSA negative by both PCR and culture at admission subsequently became positive by both methods after admission; this may represent ‘selective pressure’ for MRSA. If so, this may suggest that some of or all our false-positive cases may have in fact been true positive. By type of antimicrobial, 4 of 12 patients (33%) were administered cepahalosporin while 3 received sulbactam/ampicillin (25%). In total, 92% (11/12) of positive patients were treated with an antibiotic. Several limitations of this study warrant mention. First, various variables of the culture method have not been evaluated, including the optimal frequency of culture and anatomical sites for screening. Here, we screened by nasal culture only, on the basis that the nose is the main ecological niche of this bacterium in humans [27]. Second, our study population was from a single tertiary university teaching hospital. Rates of MRSA in the community may differ in other settings, however, and thus our results may not be applicable to other populations. Third, the study was conducted under a single-center design and the number of infected patients was small. Patients infected with MRSA in our tertiary care center but not detected until after transfer to other wards would also have been missed, underestimating the rate of ICU-acquired MRSA infection. Fourth, we were unable to account for infections that occurred at other medical facilities, likely causing our reported risk of MRSA infection to be underestimated.
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In conclusion, we found that a significant proportion of patients in our tertiary care center were colonized with MRSA at the time of admission. These findings confirm that colonization is a high risk factor for MRSA infection, and that MRSA surveillance is effective in preventing MRSA infection in Japan. Further research into the costeffectiveness of molecular testing is required. Acknowledgments We appreciate the advice and expertise of Drs. Shiro Mishima and Kimihito Koshihara. We also thank Etsuko Kutsukake of the Microbiology Department of Tokyo Medical University for her PCR analysis of MRSA.
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