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The performance of the BD geneOhm MRSA™ assay for MRSA isolated from clinical patients in Japan, including the effects of specimen contamination and ways to improve it
J Infect Chemother (2011) 17:214–218 DOI 10.1007/s10156-010-0104-2
ORIGINAL ARTICLE
The performance of the BD geneOhm MRSATM assay for MRSA isolated from clinical patients in Japan, including the effects of specimen contamination and ways to improve it Hiroki Ishikawa • Etsuko Kutsukake • Katsumi Chiba • Toshie Fukui • Tetsuya Matsumoto
Received: 2 April 2010 / Accepted: 23 July 2010 / Published online: 17 August 2010 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2010
Abstract The BD geneOhm MRSATM assay has been increasingly used in recent years, and it is possible to use it to screen and detect methicillin-resistant Staphylococcus aureus (MRSA) from a specimen within 2 h. The purpose of the present study was to evaluate the performance, i.e., the specificity and sensitivity, of the BD geneOhm MRSATM assay to detect MRSA. Its specificity was assessed to be 100% compared to bacterial culture methods, which are commonly used in medical laboratories. Its bacterial limit of detection was over 10 colony-forming units (cfu) per reaction, although MRSA was detected at a cfu below 10 per reaction in a few samples. Additionally, the effect of MRSA isolate contamination was examined. While contamination with protein or other bacteria did not affect the outcome, contamination with a high concentration of blood resulted in an unresolved outcome. To inactivate polymerase chain reaction (PCR) inhibitors, the DNA samples were freeze–thawed prior to the BD geneOhm MRSATM assay, which led to the sensitivity of the assay increasing. In summary, the BD geneOhm MRSATM assay is rapid and shows high specificity and sensitivity of cultured MRSA isolates. It will, therefore, be a valuable diagnostic tool for detecting MRSA in specimens from clinical patients.
H. Ishikawa and E. Kutsukake contributed equally to this work. H. Ishikawa (&) E. Kutsukake T. Fukui T. Matsumoto Department of Microbiology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan e-mail: [email protected] K. Chiba Central Clinical Laboratory Division, Tokyo Medical University Hospital, Tokyo, Japan
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Keywords Methicillin-resistant Staphylococcus aureus BD geneOhm MRSATM assay Real-time PCR SCCmec/orfX
Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens responsible for nosocomial infection. In Japan, it was reported that MRSA was isolated from 59.7% of patients who were diagnosed with respiratory tract infections involving Staphylococcus aureus in 2007 [1]. When MRSA is the cause of bacteremia among compromised patients, the risks of morbidity and mortality are dramatically increased compared to those for cases of bacteremia caused by methicillin-susceptible Staphylococcus aureus (MSSA) infection [2, 3]. Additionally, compared to a patient with bacteremia caused by MSSA, a patient with bacteremia caused by MRSA has a higher hospital cost and will require a longer period of hospitalization [4]. The presence of MRSA in a hospital is very detrimental to patients and to hospital management. Therefore, early detection and breaking the infection route of MRSA play important roles in preventing infection and reducing the prevalence of MRSA in hospitals. Clinical microbiology laboratories usually use the Gram staining and bacteria culture methods to detect MRSA in patient specimens. However, it takes at least 24 h to culture MRSA on a selective agar plates and requires an additional antibiotic susceptibility test to produce a final result. On the other hand, a polymerase chain reaction (PCR) assay targeting the mecA gene of MRSA can yield results within 3–5 h. Although the PCR assay is rapid, a few reports have suggested that it may lead to a false-positive diagnosis in specimens containing both
J Infect Chemother (2011) 17:214–218
methicillin-resistant coagulase-negative Staphylococcus spp. (MRCNS), which possesses the mecA gene, and MSSA [5]. It has been reported that 96.7% of MRCNS strains carry the mecA gene in their genome [6]. Recently, the BD geneOhm MRSATM assay (BD geneOhm, USA) was developed to allow more reliable MRSA identification, the results of which are received within 2 h [7]. This process involves a multiplex real-time PCR method that amplifies specific target sequences of MRSA. The specific target sequences are the right-extremity sequence of the staphylococcal cassette chromosome (SCC), the mec insertion site found in MRSA, and the S. aureus chromosomal orfX gene, but not the methicillin-resistant Staphylococcus epidermidis (MRSE) chromosomal DNA that is located to the right of the SCCmec insertion site in MRSA. This PCR assay, therefore, provides distinct results for each target and differentiates between MSSA and MRSA in addition to preventing the occurrence of false-positives from specimens containing MRCNS and MSSA. This study investigated the ability of the BD geneOhm MRSATM assay to detect MRSA isolated from clinical patients in Japan, with particular attention paid to the effects of sample contamination and ways to improve the assay.
Materials and methods Bacteria isolates MRSA, MSSA, and MRSE were originally isolated from clinical patients by the Central Clinical Laboratory of Tokyo Medical University Hospital in 2007, and their strains and characteristics were identified by Gram staining, the MicroScan autoSCAN-4 system using the PC6.1J panel (Siemens, Germany), and the coagulase test. Staphylococcus epidermidis ATCC12228 (methicillin-susceptible S. epidermidis; MSSE) was purchased from the American Type Culture Collection (Manassas, VA, USA). Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Enterobacter cloacae, and Salmonella enterica were also previously isolated from clinical patients by the Central Clinical Laboratory of Tokyo Medical University Hospital. BD geneOhm MRSATM assay The primers and fluorescent hybridization probe used in the BD geneOhm MRSATM assay were described in detail previously by Huletsky et al. [8]. The assay was performed according to the manufacturer’s instructions. In brief, the bacteria were lysed using glass beads in 50 ll of sample buffer, and the suspension was heated at 95°C for 2 min to inactivate PCR inhibitors. A 2.8-ll sample of the
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supernatant was added as a DNA sample to reconstituted master mix, and real-time PCR was performed using a SmartCycler. The detection of MRSA-specific genes was analyzed using the geneOhm MRSATM assay with SmartCycler software, which uses a decisional algorithm to interpret the assay result as negative, no MRSA DNA detected; positive, MRSA DNA detected; or unresolved, internal control failure. Analytical specificity and sensitivity of the BD geneOhm MRSATM assay To determine whether an MRSA-specific reaction occurred, rather than a reaction to MSSA, which possess the orfX gene, or MRSE, which possess the SCCmecA gene, by the BD geneOhm MRSATM assay, ten isolates each of MRSA, MSSA, and MRSE were assayed. In addition, one isolate each of Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Enterobacter cloacae, and Salmonella enterica were also tested. In brief, the bacteria were cultured in brain heart infusion broth for 18 h at 37°C, and the number of bacteria was adjusted to 1 9 106 colony-forming units (cfu) based on their absorbance at O.D. 600 nm prior to the BD geneOhm MRSATM assay. To assess the analytical sensitivity of the BD geneOhm MRSATM assay, nine of the cultured MRSA isolates were serially diluted 10 times with saline until they contained \10 cfu per reaction. Three samples each of diluted MRSA suspensions containing \10 cfu per reaction, between 10 and 100 cfu per reaction, or between 100 and 1000 cfu per reaction, respectively, were then prepared, and bacterial DNA were extracted independently. At the same time, diluted MRSA suspensions containing \10 cfu per reaction were cultured on brain heart infusion agar plates to confirm the number of MRSA at 37°C for 24 h. The BD geneOhm MRSATM assay was then performed to confirm the limit of detection. Effects of sample contamination We next investigated whether specimen contamination affects the results of the BD geneOhm MRSATM assay. MRSA isolate number 1 of Table 2 was used in this experiment. The MRSA suspensions were contaminated with S. epidermidis ATCC12228 (MSSE), protein, or blood before the bacterial DNA was extracted. In brief, in the test of the effects of MSSE contamination, an MRSA suspension of 2 cfu per reaction was mixed with 2,000, 20,000, or 200,000 cfu of MSSE based on their absorbance at O.D. 600 nm. For the protein and blood contamination tests, bovine serum albumin (BSA, Sigma, MO, USA) and defibrinated horse blood (Nippon Biotest Laboratories Inc., Tokyo, Japan), respectively, was added to the MRSA
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suspension at 2 cfu per reaction, i.e., 6.25, 12.5, 25, 50, or 100 mg/ml and 3.125, 6.25, 12.5, 25, or 50%, respectively. Two samples of each contaminated MRSA suspension were prepared. The bacterial DNA was then extracted, and the BD geneOhm MRSATM assay was performed in accordance with the manufacturer’s instructions. In some experiments, the DNA samples were freeze–thawed and centrifuged at 15,000g for 5 min before the addition of the master mix.
shown). However, as shown in Table 2, when the bacterial count was \10 cfu per reaction, the percentage of positive outcomes was below 50%. While the same results were obtained from three DNA samples of eight MRSA isolates, MRSA isolate number 7 was found to be negative in one of three DNA samples at a cfu of \10 per reaction.
Results
As clinical samples obtained from nasal swabs, pus swabs, or blood are frequently contaminated with other bacteria, especially coagulase-negative staphylococci, including MSSE, proteins, and blood components, we investigated the effects of specimen contamination on the effectiveness of the BD geneOhm MRSATM assay. As a consequence, no effect of MSSE or BSA contamination was found (Table 3 or data not shown). On the other hand, blood contamination of over 6.25% in the specimen produced an unresolved result, i.e., the BD geneOhm MRSATM assay was unable to make a correct assessment (Table 4). Therefore, the DNA samples were freeze–thawed and centrifuged prior to the BD geneOhm MRSATM assay. As a result, the BD geneOhm MRSATM assay produced correct results in samples with 25% or less blood contamination, but not in those with over 50% blood contamination. The same results were
Specificity of MRSA detection by the BD geneOhm MRSATM assay MRSA, MSSA, or MRSE isolates were tested using the BD geneOhm MRSATM assay. As shown in Table 1, all of the MRSA isolates were detected as positive by the BD geneOhm MRSATM assay. On the other hand, all of the MSSA and MRSE isolates were certified as negative. The BD geneOhm MRSATM assay was also performed to detect other species that cause nosocomial infection, such as Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Enterobacter cloacae, and Salmonella enterica. All of them were identified as negative (data not shown). Sensitivity of MRSA detection by the BD geneOhm MRSATM assay To estimate the sensitivity of MRSA detection by the BD geneOhm MRSATM assay, nine cultured MRSA isolates were assayed. When the bacterial count for a reaction was more than 10 cfu, all MRSA isolates were determined as positive by the BD geneOhm MRSATM assay (data not Table 1 Methicillin-resistant Staphylococcus aureus (MRSA) detection by the BD geneOhm MRSATM assay No.
Clinical isolates MRSA
MSSA
MRSE
1 2
Positive Positive
Negative Negative
Negative Negative
3
Positive
Negative
Negative
4
Positive
Negative
Negative
5
Positive
Negative
Negative
6
Positive
Negative
Negative
7
Positive
Negative
8
Positive
Negative
9
Positive
10
Positive
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Effects of contamination with other bacteria, protein, or blood on the efficacy of the BD geneOhm MRSATM assay
Table 2 Analytical sensitivity of the BD geneOhm MRSATM assay MRSA isolate no.
cfu/reaction
DNA
1
2
Positive
Positive
Positive
2
3
Negative
Negative
Negative
3
2
Negative
Negative
Negative
4
8
Negative
Negative
Negative
5
4
Positive
Positive
Positive
6
2
Negative
Negative
Negative
7 8
5 5
Negative Negative
Positive Negative
Positive Negative
9
8
Positive
Positive
Positive
Sample 1
Sample 2
Sample 3
Table 3 Effect of methicillin-susceptible Staphylococcus epidermidis (MSSE) specimen contamination Mixed suspension no.
MRSA (cfu/reaction)
MSSE (cfu/reaction)
Result
Negative
1
2
2000
Positive
Negative
2
2
20000
Positive
Negative
Negative
3
2
200000
Positive
Negative
Negative
4
2
0
Positive
J Infect Chemother (2011) 17:214–218 Table 4 Effect of blood specimen contamination
Treatment of DNA sample
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% of defibrinated horse blood in specimen 50
25
12.5
6.25
3.125
Untreated
Unresolved
Unresolved
Unresolved
Positive
Positive
Freeze–thawed
Unresolved
Positive
Positive
Positive
Positive
obtained in the contamination tests of two independent samples. In addition, in cases of blood contamination, when MRSA was added at a cfu of 50,000 per reaction and the BD geneOhm MRSATM assay was performed, samples containing blood contamination of over 6.25% were determined as unresolved, and those with blood contamination of 25% or less were determined as positive after being freeze–thawed and centrifuged (data not shown).
Discussion The prevalence of MRSA is an important problem worldwide. In hospitals and nursing homes, outbreaks of MRSA occur as a result of clonal spread by MRSA being transferred from patient to patient. The infectious diseases caused by MRSA, such as bacteremia and pneumonia, are more severe and carry a more significant risk of mortality in compromised patients than those caused by MSSA [2, 3]. As well as the prevalence of hospital-acquired MRSA, the prevalence of community-acquired MRSA is also now increasing. Community-acquired MRSA infection is commonly observed in children and young adults, and is a potentially life-threatening invasive infection involving necrotizing pneumonia and severe bacteremia [9]. Therefore, the rapid identification of MRSA in patient specimens is essential for allowing appropriate antibiotic drug treatment to be administered and preventing hospital outbreaks through the active control of infection. In fact, active surveillance for MRSA is now recommended in Europe and the United States [10]. In the present study, the BD geneOhm MRSATM assay performance, i.e., its sensitivity and specificity to MRSA isolates and the effect of sample contamination, was examined. The analytical sensitivity (limit of detection) of the BD geneOhm MRSATM assay was over 10 cfu per reaction for MRSA, and the bacteria in some samples could not be detected at a cfu below 10 per reaction. The sensitivity of the BD geneOhm MRSATM assay might be attributed to the MRSA strain itself, as a low number of bacteria in reactions involving\10 cfu could cause ambiguous results, such as false-negative diagnoses, although this has not been confirmed. On the other hand, the specificity of the assay for MRSA was 100% concordant with the results of bacterial culture methods. However, it is unknown whether these results accurately reflect the clinical situation that arises
when direct clinical specimens, such as nasal swabs, pus swabs, or blood, are used because the MRSA isolates tested in this experiment were purified by in vitro culture. Stamper et al. [7] demonstrated the high specificity of the BD geneOhm MRSATM assay for MRSA in bacteria-positive blood samples from blood culture bottles. When 211 bacteriapositive blood samples from blood culture bottles containing coagulase-negative staphylococci, Micrococcus species, Streptococci, Gram-positive rods, Gram-negative rods, or yeast were tested using the BD geneOhm MRSATM assay, two of the coagulase-negative staphylococci were identified as MRSA. Moreover, they showed a 98.4% specificity with a positive predictive value of 92.6% and a negative predictive value of 100% compared to bacterial culture methods, together with a sensitivity of 100% [7]. Discrepancies between the results of the BD geneOhm MRSATM assay and bacterial culture methods have also been reported by other groups [6, 7, 11]. False-positive diagnoses are possibly caused in the BD geneOhm MRSATM assay by some MSSA lineages that carry SCCmec without possessing the functional region containing mecA [12]. In fact, approximately 5% of bacterial isolates identified as MRSA by the BD geneOhm MRSATM assay are phenotypically defined as MSSA [8, 13, 14]. False-negative diagnoses can be attributed to a bacterial count below the limit of detection or the existence of DNase or PCR inhibitors in the sample. Moreover, recently, the failure of the BD geneOhm MRSATM assay to detect MRSA carrying SCCmec V, but not type IVa–d or VT, was reported in Ireland [15]. In the present study, the outcome of the BD geneOhm MRSATM assay was not affected by severe contamination with other bacteria or proteins. On the other hand, contamination of the specimen with a high concentration of blood produced an unresolved result due to internal control failure. However, we showed that the sensitivity of the assay was dramatically improved when freeze–thawed DNA samples were used. Several components of blood are PCR inhibitors or fluorescence signal inhibitors. Heme A, leukemia DNA, coagulant, and immunoglobulin G in plasma, and hemoglobin and lactoferrin in erythrocytes and leukocytes, respectively, were found to be major PCR inhibitors [16–18]. In addition, bilirubin and hemin act as fluorescent signal inhibitors in real-time PCR reactions [16]. Although we could not identify any inhibitors of the BD geneOhm MRSATM assay in blood, we hypothesized that, if any inhibitors were present, they might be
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susceptible to heat and/or freezing, therefore, we attempted to inactivate any such inhibitors using a freeze–thaw procedure, which improved the sensitivity of the BD geneOhm MRSATM assay. In conclusion, the BD geneOhm MRSATM assay is a reliable, rapid, and efficient method for identifying MRSA isolates. The BD geneOhm MRSATM assay will, therefore, be an effective tool for controlling nosocomial infection.
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8.
9. 10.
Acknowledgments This study was supported by the donation of BD geneOhm MRSATM assay reagent kits and the lease of a SmartCycler from Nippon Becton Dickinson Company, Ltd., Tokyo, Japan.
11.
References 1. Niki Y, Hanaki H, Matsumoto T, Yagisawa M, Kohno S, Aoki N, et al. Nationwide surveillance of bacterial respiratory pathogens conducted by the Japanese Society of Chemotherapy in 2007: general view of the pathogens’ antibacterial susceptibility. J Infect Chemother. 2009;15(3):156–67. 2. Weinstein MP, Towns ML, Quartey SM, Mirrett S, Reimer LG, Parmigiani G, et al. The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults. Clin Infect Dis. 1997;24(4):584–602. 3. Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis. 2004;39(3):309–17. 4. Ben-David D, Novikov I, Mermel LA. Are there differences in hospital cost between patients with nosocomial methicillinresistant Staphylococcus aureus bloodstream infection and those with methicillin-susceptible S. aureus bloodstream infection? Infect Control Hosp Epidemiol. 2009;30(5):453–60. 5. Becker K, Pagnier I, Schuhen B, Wenzelburger F, Friedrich AW, Kipp F, et al. Does nasal cocolonization by methicillin-resistant coagulase-negative staphylococci and methicillin-susceptible Staphylococcus aureus strains occur frequently enough to represent a risk of false-positive methicillin-resistant S. aureus determinations by molecular methods? J Clin Microbiol. 2006;44(1):229–31. 6. Thomas L, van Hal S, O’Sullivan M, Kyme P, Iredell J. Failure of the BD GeneOhm StaphS/R assay for identification of Australian methicillin-resistant Staphylococcus aureus strains: duplex assays as the ‘‘gold standard’’ in settings of unknown SCCmec epidemiology. J Clin Microbiol. 2008;46(12):4116–7. 7. Stamper PD, Cai M, Howard T, Speser S, Carroll KC. Clinical validation of the molecular BD GeneOhm StaphSR assay for
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direct detection of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in positive blood cultures. J Clin Microbiol. 2007;45(7):2191–6. Huletsky A, Giroux R, Rossbach V, Gagnon M, Vaillancourt M, Bernier M, et al. New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci. J Clin Microbiol. 2004;42(5):1875–84. Rice LB. Antimicrobial resistance in gram-positive bacteria. Am J Med. 2006;119(6 Suppl 1):S11–9; discussion S62–70. Nijssen S, Bonten MJ, Weinstein RA. Are active microbiological surveillance and subsequent isolation needed to prevent the spread of methicillin-resistant Staphylococcus aureus? Clin Infect Dis. 2005;40(3):405–9. 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(2):743–6. Donnio PY, Oliveira DC, Faria NA, Wilhelm N, Le Coustumier A, de Lencastre H. Partial excision of the chromosomal cassette containing the methicillin resistance determinant results in methicillin-susceptible Staphylococcus aureus. J Clin Microbiol. 2005;43(8):4191–3. Desjardins M, Guibord C, Lalonde B, Toye B, Ramotar K. Evaluation of the IDI-MRSA assay for detection of methicillinresistant Staphylococcus aureus from nasal and rectal specimens pooled in a selective broth. J Clin Microbiol. 2006;44(4):1219–23. Oberdorfer K, Pohl S, Frey M, Heeg K, Wendt C. Evaluation of a single-locus real-time polymerase chain reaction as a screening test for specific detection of methicillin-resistant Staphylococcus aureus in ICU patients. Eur J Clin Microbiol Infect Dis. 2006; 25(10):657–63. Rossney AS, Herra CM, Fitzgibbon MM, Morgan PM, Lawrence MJ, O’Connell B. Evaluation of the IDI-MRSA assay on the SmartCycler real-time PCR platform for rapid detection of MRSA from screening specimens. Eur J Clin Microbiol Infect Dis. 2007;26(7):459–66. Al-Soud WA, Ra˚dstro¨m P. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol. 2001;39(2):485–93. Al-Soud WA, Jo¨nsson LJ, Ra˚dstro¨m P. Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol. 2000;38(1):345–50. Morata P, Queipo-Ortun˜o MI, de Dios Colmenero J. Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis. J Clin Microbiol. 1998; 36(9):2443–6.
ORIGINAL ARTICLE
The performance of the BD geneOhm MRSATM assay for MRSA isolated from clinical patients in Japan, including the effects of specimen contamination and ways to improve it Hiroki Ishikawa • Etsuko Kutsukake • Katsumi Chiba • Toshie Fukui • Tetsuya Matsumoto
Received: 2 April 2010 / Accepted: 23 July 2010 / Published online: 17 August 2010 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2010
Abstract The BD geneOhm MRSATM assay has been increasingly used in recent years, and it is possible to use it to screen and detect methicillin-resistant Staphylococcus aureus (MRSA) from a specimen within 2 h. The purpose of the present study was to evaluate the performance, i.e., the specificity and sensitivity, of the BD geneOhm MRSATM assay to detect MRSA. Its specificity was assessed to be 100% compared to bacterial culture methods, which are commonly used in medical laboratories. Its bacterial limit of detection was over 10 colony-forming units (cfu) per reaction, although MRSA was detected at a cfu below 10 per reaction in a few samples. Additionally, the effect of MRSA isolate contamination was examined. While contamination with protein or other bacteria did not affect the outcome, contamination with a high concentration of blood resulted in an unresolved outcome. To inactivate polymerase chain reaction (PCR) inhibitors, the DNA samples were freeze–thawed prior to the BD geneOhm MRSATM assay, which led to the sensitivity of the assay increasing. In summary, the BD geneOhm MRSATM assay is rapid and shows high specificity and sensitivity of cultured MRSA isolates. It will, therefore, be a valuable diagnostic tool for detecting MRSA in specimens from clinical patients.
H. Ishikawa and E. Kutsukake contributed equally to this work. H. Ishikawa (&) E. Kutsukake T. Fukui T. Matsumoto Department of Microbiology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan e-mail: [email protected] K. Chiba Central Clinical Laboratory Division, Tokyo Medical University Hospital, Tokyo, Japan
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Keywords Methicillin-resistant Staphylococcus aureus BD geneOhm MRSATM assay Real-time PCR SCCmec/orfX
Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens responsible for nosocomial infection. In Japan, it was reported that MRSA was isolated from 59.7% of patients who were diagnosed with respiratory tract infections involving Staphylococcus aureus in 2007 [1]. When MRSA is the cause of bacteremia among compromised patients, the risks of morbidity and mortality are dramatically increased compared to those for cases of bacteremia caused by methicillin-susceptible Staphylococcus aureus (MSSA) infection [2, 3]. Additionally, compared to a patient with bacteremia caused by MSSA, a patient with bacteremia caused by MRSA has a higher hospital cost and will require a longer period of hospitalization [4]. The presence of MRSA in a hospital is very detrimental to patients and to hospital management. Therefore, early detection and breaking the infection route of MRSA play important roles in preventing infection and reducing the prevalence of MRSA in hospitals. Clinical microbiology laboratories usually use the Gram staining and bacteria culture methods to detect MRSA in patient specimens. However, it takes at least 24 h to culture MRSA on a selective agar plates and requires an additional antibiotic susceptibility test to produce a final result. On the other hand, a polymerase chain reaction (PCR) assay targeting the mecA gene of MRSA can yield results within 3–5 h. Although the PCR assay is rapid, a few reports have suggested that it may lead to a false-positive diagnosis in specimens containing both
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methicillin-resistant coagulase-negative Staphylococcus spp. (MRCNS), which possesses the mecA gene, and MSSA [5]. It has been reported that 96.7% of MRCNS strains carry the mecA gene in their genome [6]. Recently, the BD geneOhm MRSATM assay (BD geneOhm, USA) was developed to allow more reliable MRSA identification, the results of which are received within 2 h [7]. This process involves a multiplex real-time PCR method that amplifies specific target sequences of MRSA. The specific target sequences are the right-extremity sequence of the staphylococcal cassette chromosome (SCC), the mec insertion site found in MRSA, and the S. aureus chromosomal orfX gene, but not the methicillin-resistant Staphylococcus epidermidis (MRSE) chromosomal DNA that is located to the right of the SCCmec insertion site in MRSA. This PCR assay, therefore, provides distinct results for each target and differentiates between MSSA and MRSA in addition to preventing the occurrence of false-positives from specimens containing MRCNS and MSSA. This study investigated the ability of the BD geneOhm MRSATM assay to detect MRSA isolated from clinical patients in Japan, with particular attention paid to the effects of sample contamination and ways to improve the assay.
Materials and methods Bacteria isolates MRSA, MSSA, and MRSE were originally isolated from clinical patients by the Central Clinical Laboratory of Tokyo Medical University Hospital in 2007, and their strains and characteristics were identified by Gram staining, the MicroScan autoSCAN-4 system using the PC6.1J panel (Siemens, Germany), and the coagulase test. Staphylococcus epidermidis ATCC12228 (methicillin-susceptible S. epidermidis; MSSE) was purchased from the American Type Culture Collection (Manassas, VA, USA). Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Enterobacter cloacae, and Salmonella enterica were also previously isolated from clinical patients by the Central Clinical Laboratory of Tokyo Medical University Hospital. BD geneOhm MRSATM assay The primers and fluorescent hybridization probe used in the BD geneOhm MRSATM assay were described in detail previously by Huletsky et al. [8]. The assay was performed according to the manufacturer’s instructions. In brief, the bacteria were lysed using glass beads in 50 ll of sample buffer, and the suspension was heated at 95°C for 2 min to inactivate PCR inhibitors. A 2.8-ll sample of the
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supernatant was added as a DNA sample to reconstituted master mix, and real-time PCR was performed using a SmartCycler. The detection of MRSA-specific genes was analyzed using the geneOhm MRSATM assay with SmartCycler software, which uses a decisional algorithm to interpret the assay result as negative, no MRSA DNA detected; positive, MRSA DNA detected; or unresolved, internal control failure. Analytical specificity and sensitivity of the BD geneOhm MRSATM assay To determine whether an MRSA-specific reaction occurred, rather than a reaction to MSSA, which possess the orfX gene, or MRSE, which possess the SCCmecA gene, by the BD geneOhm MRSATM assay, ten isolates each of MRSA, MSSA, and MRSE were assayed. In addition, one isolate each of Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Enterobacter cloacae, and Salmonella enterica were also tested. In brief, the bacteria were cultured in brain heart infusion broth for 18 h at 37°C, and the number of bacteria was adjusted to 1 9 106 colony-forming units (cfu) based on their absorbance at O.D. 600 nm prior to the BD geneOhm MRSATM assay. To assess the analytical sensitivity of the BD geneOhm MRSATM assay, nine of the cultured MRSA isolates were serially diluted 10 times with saline until they contained \10 cfu per reaction. Three samples each of diluted MRSA suspensions containing \10 cfu per reaction, between 10 and 100 cfu per reaction, or between 100 and 1000 cfu per reaction, respectively, were then prepared, and bacterial DNA were extracted independently. At the same time, diluted MRSA suspensions containing \10 cfu per reaction were cultured on brain heart infusion agar plates to confirm the number of MRSA at 37°C for 24 h. The BD geneOhm MRSATM assay was then performed to confirm the limit of detection. Effects of sample contamination We next investigated whether specimen contamination affects the results of the BD geneOhm MRSATM assay. MRSA isolate number 1 of Table 2 was used in this experiment. The MRSA suspensions were contaminated with S. epidermidis ATCC12228 (MSSE), protein, or blood before the bacterial DNA was extracted. In brief, in the test of the effects of MSSE contamination, an MRSA suspension of 2 cfu per reaction was mixed with 2,000, 20,000, or 200,000 cfu of MSSE based on their absorbance at O.D. 600 nm. For the protein and blood contamination tests, bovine serum albumin (BSA, Sigma, MO, USA) and defibrinated horse blood (Nippon Biotest Laboratories Inc., Tokyo, Japan), respectively, was added to the MRSA
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suspension at 2 cfu per reaction, i.e., 6.25, 12.5, 25, 50, or 100 mg/ml and 3.125, 6.25, 12.5, 25, or 50%, respectively. Two samples of each contaminated MRSA suspension were prepared. The bacterial DNA was then extracted, and the BD geneOhm MRSATM assay was performed in accordance with the manufacturer’s instructions. In some experiments, the DNA samples were freeze–thawed and centrifuged at 15,000g for 5 min before the addition of the master mix.
shown). However, as shown in Table 2, when the bacterial count was \10 cfu per reaction, the percentage of positive outcomes was below 50%. While the same results were obtained from three DNA samples of eight MRSA isolates, MRSA isolate number 7 was found to be negative in one of three DNA samples at a cfu of \10 per reaction.
Results
As clinical samples obtained from nasal swabs, pus swabs, or blood are frequently contaminated with other bacteria, especially coagulase-negative staphylococci, including MSSE, proteins, and blood components, we investigated the effects of specimen contamination on the effectiveness of the BD geneOhm MRSATM assay. As a consequence, no effect of MSSE or BSA contamination was found (Table 3 or data not shown). On the other hand, blood contamination of over 6.25% in the specimen produced an unresolved result, i.e., the BD geneOhm MRSATM assay was unable to make a correct assessment (Table 4). Therefore, the DNA samples were freeze–thawed and centrifuged prior to the BD geneOhm MRSATM assay. As a result, the BD geneOhm MRSATM assay produced correct results in samples with 25% or less blood contamination, but not in those with over 50% blood contamination. The same results were
Specificity of MRSA detection by the BD geneOhm MRSATM assay MRSA, MSSA, or MRSE isolates were tested using the BD geneOhm MRSATM assay. As shown in Table 1, all of the MRSA isolates were detected as positive by the BD geneOhm MRSATM assay. On the other hand, all of the MSSA and MRSE isolates were certified as negative. The BD geneOhm MRSATM assay was also performed to detect other species that cause nosocomial infection, such as Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Enterobacter cloacae, and Salmonella enterica. All of them were identified as negative (data not shown). Sensitivity of MRSA detection by the BD geneOhm MRSATM assay To estimate the sensitivity of MRSA detection by the BD geneOhm MRSATM assay, nine cultured MRSA isolates were assayed. When the bacterial count for a reaction was more than 10 cfu, all MRSA isolates were determined as positive by the BD geneOhm MRSATM assay (data not Table 1 Methicillin-resistant Staphylococcus aureus (MRSA) detection by the BD geneOhm MRSATM assay No.
Clinical isolates MRSA
MSSA
MRSE
1 2
Positive Positive
Negative Negative
Negative Negative
3
Positive
Negative
Negative
4
Positive
Negative
Negative
5
Positive
Negative
Negative
6
Positive
Negative
Negative
7
Positive
Negative
8
Positive
Negative
9
Positive
10
Positive
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Effects of contamination with other bacteria, protein, or blood on the efficacy of the BD geneOhm MRSATM assay
Table 2 Analytical sensitivity of the BD geneOhm MRSATM assay MRSA isolate no.
cfu/reaction
DNA
1
2
Positive
Positive
Positive
2
3
Negative
Negative
Negative
3
2
Negative
Negative
Negative
4
8
Negative
Negative
Negative
5
4
Positive
Positive
Positive
6
2
Negative
Negative
Negative
7 8
5 5
Negative Negative
Positive Negative
Positive Negative
9
8
Positive
Positive
Positive
Sample 1
Sample 2
Sample 3
Table 3 Effect of methicillin-susceptible Staphylococcus epidermidis (MSSE) specimen contamination Mixed suspension no.
MRSA (cfu/reaction)
MSSE (cfu/reaction)
Result
Negative
1
2
2000
Positive
Negative
2
2
20000
Positive
Negative
Negative
3
2
200000
Positive
Negative
Negative
4
2
0
Positive
J Infect Chemother (2011) 17:214–218 Table 4 Effect of blood specimen contamination
Treatment of DNA sample
217
% of defibrinated horse blood in specimen 50
25
12.5
6.25
3.125
Untreated
Unresolved
Unresolved
Unresolved
Positive
Positive
Freeze–thawed
Unresolved
Positive
Positive
Positive
Positive
obtained in the contamination tests of two independent samples. In addition, in cases of blood contamination, when MRSA was added at a cfu of 50,000 per reaction and the BD geneOhm MRSATM assay was performed, samples containing blood contamination of over 6.25% were determined as unresolved, and those with blood contamination of 25% or less were determined as positive after being freeze–thawed and centrifuged (data not shown).
Discussion The prevalence of MRSA is an important problem worldwide. In hospitals and nursing homes, outbreaks of MRSA occur as a result of clonal spread by MRSA being transferred from patient to patient. The infectious diseases caused by MRSA, such as bacteremia and pneumonia, are more severe and carry a more significant risk of mortality in compromised patients than those caused by MSSA [2, 3]. As well as the prevalence of hospital-acquired MRSA, the prevalence of community-acquired MRSA is also now increasing. Community-acquired MRSA infection is commonly observed in children and young adults, and is a potentially life-threatening invasive infection involving necrotizing pneumonia and severe bacteremia [9]. Therefore, the rapid identification of MRSA in patient specimens is essential for allowing appropriate antibiotic drug treatment to be administered and preventing hospital outbreaks through the active control of infection. In fact, active surveillance for MRSA is now recommended in Europe and the United States [10]. In the present study, the BD geneOhm MRSATM assay performance, i.e., its sensitivity and specificity to MRSA isolates and the effect of sample contamination, was examined. The analytical sensitivity (limit of detection) of the BD geneOhm MRSATM assay was over 10 cfu per reaction for MRSA, and the bacteria in some samples could not be detected at a cfu below 10 per reaction. The sensitivity of the BD geneOhm MRSATM assay might be attributed to the MRSA strain itself, as a low number of bacteria in reactions involving\10 cfu could cause ambiguous results, such as false-negative diagnoses, although this has not been confirmed. On the other hand, the specificity of the assay for MRSA was 100% concordant with the results of bacterial culture methods. However, it is unknown whether these results accurately reflect the clinical situation that arises
when direct clinical specimens, such as nasal swabs, pus swabs, or blood, are used because the MRSA isolates tested in this experiment were purified by in vitro culture. Stamper et al. [7] demonstrated the high specificity of the BD geneOhm MRSATM assay for MRSA in bacteria-positive blood samples from blood culture bottles. When 211 bacteriapositive blood samples from blood culture bottles containing coagulase-negative staphylococci, Micrococcus species, Streptococci, Gram-positive rods, Gram-negative rods, or yeast were tested using the BD geneOhm MRSATM assay, two of the coagulase-negative staphylococci were identified as MRSA. Moreover, they showed a 98.4% specificity with a positive predictive value of 92.6% and a negative predictive value of 100% compared to bacterial culture methods, together with a sensitivity of 100% [7]. Discrepancies between the results of the BD geneOhm MRSATM assay and bacterial culture methods have also been reported by other groups [6, 7, 11]. False-positive diagnoses are possibly caused in the BD geneOhm MRSATM assay by some MSSA lineages that carry SCCmec without possessing the functional region containing mecA [12]. In fact, approximately 5% of bacterial isolates identified as MRSA by the BD geneOhm MRSATM assay are phenotypically defined as MSSA [8, 13, 14]. False-negative diagnoses can be attributed to a bacterial count below the limit of detection or the existence of DNase or PCR inhibitors in the sample. Moreover, recently, the failure of the BD geneOhm MRSATM assay to detect MRSA carrying SCCmec V, but not type IVa–d or VT, was reported in Ireland [15]. In the present study, the outcome of the BD geneOhm MRSATM assay was not affected by severe contamination with other bacteria or proteins. On the other hand, contamination of the specimen with a high concentration of blood produced an unresolved result due to internal control failure. However, we showed that the sensitivity of the assay was dramatically improved when freeze–thawed DNA samples were used. Several components of blood are PCR inhibitors or fluorescence signal inhibitors. Heme A, leukemia DNA, coagulant, and immunoglobulin G in plasma, and hemoglobin and lactoferrin in erythrocytes and leukocytes, respectively, were found to be major PCR inhibitors [16–18]. In addition, bilirubin and hemin act as fluorescent signal inhibitors in real-time PCR reactions [16]. Although we could not identify any inhibitors of the BD geneOhm MRSATM assay in blood, we hypothesized that, if any inhibitors were present, they might be
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218
susceptible to heat and/or freezing, therefore, we attempted to inactivate any such inhibitors using a freeze–thaw procedure, which improved the sensitivity of the BD geneOhm MRSATM assay. In conclusion, the BD geneOhm MRSATM assay is a reliable, rapid, and efficient method for identifying MRSA isolates. The BD geneOhm MRSATM assay will, therefore, be an effective tool for controlling nosocomial infection.
J Infect Chemother (2011) 17:214–218
8.
9. 10.
Acknowledgments This study was supported by the donation of BD geneOhm MRSATM assay reagent kits and the lease of a SmartCycler from Nippon Becton Dickinson Company, Ltd., Tokyo, Japan.
11.
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