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Journal of Clinical Microbiology, December 2001, p. 4529-4531, Vol. 39, No. 12
Department of Microbiology, Public Health
Laboratory Service,1 and Department of
Virology, Public Health Laboratory Service,2
Cardiff, and Laboratory of Hospital Infections, Central Public
Health Laboratory, London,3 United Kingdom
Received 7 June 2001/Returned for modification 23 July
2001/Accepted 13 September 2001
Methicillin-resistant Staphylococcus aureus
septicemia is associated with significant morbidity and mortality and
requires treatment with intravenous glycopeptides. For blood cultures
positive for gram-positive cocci, 24 to 48 h is required for the
detection of S. aureus bacteremia and the provision of
antibiotic susceptibility testing results. We describe a molecular
biology-based assay that requires 2 h from the time of initial
positivity of blood cultures. The assay correctly detected 96%
of the S. aureus isolates including all
methicillin-resistant S. aureus isolates. Clinical data
collected during the study suggest that 28% of patients with S.
aureus bacteremia do not receive early and appropriate
treatment and that 10% of patients may initially be receiving
inappropriate glycopeptide treatment.
Staphylococcus aureus
septicemia is associated with a mortality rate of 15 to 30%
(17). The increasing proportion of infections caused by
methicillin-resistant S. aureus (MRSA) (8) has
resulted in the widespread empirical use of glycopeptides, which
increases the pressure for selection of vancomycin resistance
(19, 24). The use of vancomycin also requires therapeutic
monitoring (15) to reduce the potential for use of a
suboptimal dosage (5, 10) and side effects. From the time
that a blood culture is positive, conventional methods of culture and
antibiotic susceptibility testing require 48 h for the detection
of S. aureus bacteremia and the provision of antibiotic
susceptibility testing results. Rapid identification of MRSA
from blood cultures would accelerate the diagnosis of S. aureus bacteremia and reduce the level of empirical use of vancomycin.
This paper reports on a method for the detection of S. aureus and MRSA directly from positive blood cultures by rapid
real-time fluorescence PCR and also attempts to measure the potential
clinical impact of the more rapid provision of test results.
BACTEC 9240 (Becton Dickinson, Le Pont de Claix, France) blood culture
bottles were inoculated with 5 to 10 ml of blood from patients with
suspected bacteremia and were incubated in the BACTEC 9240 automated
continuous monitoring system. When a positive growth index was
achieved, aliquots of blood were taken from 141 positive blood cultures
showing gram-positive cocci in clusters for culture and PCR.
Clinical data were requested from the clinicians directly involved in
the care of the patient. All isolates of S. aureus were considered clinically significant. Coagulase-negative staphylococci (CoNS) were considered clinically significant if all of the following factors were present: (i) the patient had had multiple episodes of
bacteremia (26); (ii) intravascular catheters, prosthetic heart valves, or other risk factors were present in situ (13, 25); or (iii) the patient had pyrexia, peripheral leukocytosis, or hypotension. Therapy was considered effective if the patient was
receiving an antibiotic that was recognized to have activity against
the organism isolated from blood cultures.
Lysis and DNA extraction were achieved for each positive blood culture
with the Generation DNA Purification Capture Column kit (Gentra
Corporation, Minneapolis, Minn.). Oligonucleotide primers and
fluorescence-labeled probes were designed for amplification and
sequence-specific detection of a 179-bp fragment within the S. aureus sequence (16) and a 98-bp fragment within the
mecA gene (Table 1). Two
separate PCRs were performed with each sample; the first one detected
the Sa442 fragment, and the second one detected the
mecA gene. PCR was performed with the LightCycler device
(Biogene, Kimbleton, United Kingdom), which combines rapid thermal
cycling and probe-specific detection of the amplified product.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.12.4529-4531.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Rapid Identification of Methicillin-Resistant
Staphylococcus aureus from Positive Blood Cultures
by Real-Time Fluorescence PCR
ABSTRACT
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TABLE 1.
Oligonucleotide primers and probes used in the study
The amplification mixture for the Sa442 fragment consisted of 5 µl of a 2× Taq-based master mixture containing 2 mM MgCl2, 0.5 µl each of primers Sa442-F and Sa442-R (final concentrations, 0.4 µM), 1 µl of cyanine 5 (Cy5)-labeled probe Sa442-P (final concentration, 0.3 µM), 1 µl of 1× SYBR Green I, and 2 µl of template DNA. The amplification mixture for the mecA fragment consisted of 5 µl of a 2× Taq-based master mixture containing 3 mM MgCl2, 0.5 µl each of primers mecA-F and mecA-R (final concentrations, 0.4 µM), 1 µl of Cy5-labeled probe MecA-P (final concentration, 0.2 µM), 0.5 µl of 1× SYBR Green I, and 2 µl of template DNA.
Details of the 50-cycle amplification profiles are listed in Table
2.
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Overnight culture of the 141 blood cultures on Columbia agar (Oxoid, Basingstoke, England) isolated 20 strains of methicillin-susceptible S. aureus (MSSA), 30 strains of MRSA, and 93 strains of CoNS. Two blood cultures were positive for a mixture of methicillin-resistant CoNS and MRSA. Methicillin susceptibility was determined by oxacillin antibiotic disk testing, in line with the guidelines issued by the British Society for Antimicrobial Chemotherapy (3, 4). Identification of S. aureus was achieved by colonial morphology, with a latex agglutination kit (Staphaurex; Murex BioTech Ltd., Dartford, England), and by tube coagulase tests.
Of the 50 blood cultures positive for S. aureus, 48 were positive for the Sa442 gene fragment. PCR failed to identify a recurrent isolate of S. aureus from a patient with persistent bacteremia. The Sa442 fragment was not detected in any of the blood cultures positive for CoNS. Two blood cultures were positive for a mixture of methicillin-resistant CoNS and MRSA and were positive by PCR for both the Sa442 and the mecA genes. Identification of S. aureus in blood cultures by PCR showed a sensitivity of 96% and a specificity of 100%.
Detection of methicillin resistance in S. aureus isolates by PCR showed a sensitivity and a specificity of 100% each compared with the results of conventional susceptibility testing. In comparison, identification of mecA in CoNS showed a sensitivity of 97% and a specificity of 95%.
Fifty-two blood cultures positive for clinically significant organisms
were available for evaluation. MSSA accounted for 13% (n = 16) of all bacteremias, and MRSA accounted for
another 19% (n = 23). Eleven patients with S. aureus bacteremia remained on ineffective antibiotic therapy in
the first 24 h following notification that the blood cultures were
positive (Table 3). Patients with MRSA
bacteremia were at a higher risk of initially receiving ineffective antibiotics. A total of 28% of the patients with S. aureus
bacteremia remained on ineffective treatment, despite active clinical
microbiological involvement.
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Prescribed antibiotic therapy was evaluated for all patients with clinically significant bacteremia. Four patients (25%) with MSSA bacteremia were initially treated with vancomycin. Following notification of culture results, only one patient was continued on vancomycin therapy. Two patients (12.5%) with MSSA bacteremia were initially receiving cephalosporins. Seven patients with nonclinically significant pseudobacteremia were initially receiving vancomycin, but therapy was discontinued for only three patients.
S. aureus is a frequent cause of bacteremia (26) and is associated with increased rates of mortality and secondary complications (14, 18). The detection of the mecA gene from positive blood cultures by the conventional PCR process can take 4 to 5 h (6; Y. C. Lee, J. Wu, and P. Della-Latta, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 896, 2000). PCR with the LightCyler device provides species identification and methicillin susceptibility results within 2 h of detection of a positive blood culture, which allows faster clinical decision making.
Although the numbers of patients investigated in the present study was small, the study reveals that a significant proportion of cases of MRSA bacteremia remain untreated in the first 24 h following notification that a blood culture is positive. A PCR assay would allow early detection and treatment of such cases. Early and appropriate treatment of septicemia has been associated with a reduction in the rate of mortality (1, 9, 11). However, one study reported that although patients with MRSA bacteremia were less likely to receive effective antibiotic therapy, this was not significantly linked to an increased rate of mortality (22).
Rapid susceptibility testing may also reduce the rate of empirical use of glycopeptides. Although 10% of patients infected with methicillin-susceptible isolates in the present study were initially receiving vancomycin therapy, only half of these patients subsequently discontinued vancomycin therapy. In principle, rapid provision of antibiotic susceptibility testing results should reduce the rates of empirical use of vancomycin and cephalosporins, which are associated with additional side effects and costs (2, 23).
The use of PCR technology is likely to be significantly more expensive than the use of conventional culture methods. An assay such as the one described here may be useful in patients with known risk factors for S. aureus bacteremia (20). However, the clinical and economic benefits to the provision of this information remain to be elucidated. Direct detection of S. aureus from blood (7, 12) may provide an even earlier diagnosis, but such techniques require further evaluation.
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ACKNOWLEDGMENTS |
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This study was funded by grants from the Pathological Society of Great Britain and Ireland and the Small Scientific Initiatives Fund, Public Health Laboratory Service, London, United Kingdom.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Cardiff Public Health Laboratory, University Hospital Wales, Cardiff CF14 4XW, United Kingdom. Phone: 44-029-20742718. Fax: 44-029-20742161. E-mail: theanyen.tan{at}phls.wales.nhs.uk.
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