JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2007, p. 3641–3646 0095-1137/07/$08.00⫹0 doi:10.1128/JCM.01056-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 45, No. 11
Quantitative Detection of Staphylococcus aureus and Enterococcus faecalis DNA in Blood To Diagnose Bacteremia in Patients in the Intensive Care Unit䌤 Remco P. H. Peters,1,2* Michiel A. van Agtmael,2 Sonja Gierveld,1 Sven A. Danner,2 A. B. Johan Groeneveld,3 Christina M. J. E. Vandenbroucke-Grauls,1 and Paul H. M. Savelkoul1 Departments of Medical Microbiology and Infection Control,1 Internal Medicine,2 and Intensive Care,3 VU University Medical Center, Amsterdam, The Netherlands Received 23 May 2007/Returned for modification 31 August 2007/Accepted 12 September 2007
Direct detection of bacterial DNA in blood offers a fast alternative to blood culture and is presumably unaffected by the prior use of antibiotics. We evaluated the performance of two real-time PCR assays for the quantitative detection of Staphylococcus aureus bacteremia and for Enterococcus faecalis bacteremia directly in blood samples, without prior cultivation. Whole-blood samples for PCR were obtained simultaneously with blood cultures from patients admitted to the intensive care unit of our hospital. After the extraction of DNA from 200 l of blood, real-time PCR was performed for the specific detection and quantification of S. aureus and E. faecalis DNA. The sensitivity for bacteremia of the S. aureus PCR was 75% and that of the E. faecalis PCR was 73%, and both tests had high specificity values (93 and 96%, respectively). PCR amplification reactions were positive for S. aureus for 10 (7%) blood samples with negative blood cultures, and 7 (4%) PCR reactions were positive for E. faecalis. The majority of these PCR results were likely (50%) or possibly (42%) related to infection with the specific microorganism, based on clinical data and radiological and microbiological investigations. PCR results were concordant for 95% of paired whole-blood samples, and blood culture results were concordant for 97% of the paired samples. We conclude that the detection of S. aureus and E. faecalis DNA in blood by real-time PCR enables a rapid diagnosis of bacteremia and that a positive DNAemia is related to proven or possible infection with the specific microorganism in the majority of patients with negative blood cultures.
Gram-positive microorganisms, especially Staphylococcus aureus and Enterococcus faecalis, account for the majority of episodes of bacteremia in critically ill patients in the intensive care unit (ICU) (16, 25). The occurrence of bacteremia is an ominous prognostic factor for these patients, with an attributable mortality rate of 16 to 25% (9, 17). As such, the early recognition of bacteremia is important for instituting adequate antimicrobial therapy (15, 28). At present, blood culture is the gold standard for diagnosing bacteremia, but it may take more than 2 days before results are available. Furthermore, the sensitivity of blood cultures is markedly reduced if blood samples are obtained during antimicrobial treatment (10, 19). Therefore, the clinical impact of blood cultures is often limited (20, 24). PCR detection of bacteremia directly in blood samples, without prior cultivation, offers a fast alternative to the blood culture method and is presumably unaffected by the prior use of antibiotics (5, 11, 13, 18). PCR assays can be aimed at universally detecting bacterial DNA in blood or at targeting a specific microorganism (5, 6, 27). An additional advantage of the use of a real-time PCR
assay is the possibility of quantifying the amount of bacterial DNA. In this study, we evaluated the performance of two real-time PCR assays, one assay for the direct, quantitative detection of S. aureus bacteremia and the other assay for E. faecalis bacteremia in patients admitted to the ICU.
MATERIALS AND METHODS Blood samples. Blood samples for PCR assay and samples collected simultaneously for blood culture were taken because of suspected infection or bacteremia from patients admitted to the ICU of our university hospital between November 2003 and December 2005. A total of 2,000 blood samples from 552 patients was collected and stored at ⫺20°C. From these stored samples, blood samples were selected for S. aureus PCR (the S. aureus cohort) and E. faecalis PCR (the E. faecalis cohort), and each cohort included all blood samples from 80 individual patients. For the S. aureus cohort, blood samples from patients (n ⫽ 16) in whom S. aureus was isolated from simultaneous blood cultures were selected and combined with blood samples from 64 randomly selected patients in whom no growth of S. aureus was detected. The same principle was applied to blood samples selected from patients in the E. faecalis cohort, i.e., 13 cases with blood cultures positive for E. faecalis and 67 controls in whom no growth of E. faecalis was detected. This study was approved by the medical ethical committee of our hospital. Clinical data of this cohort are described elsewhere (R. P. Peters, M. A. van Agtmael, S. Gierveld, S. A. Danner, C. M. Vandenbroucke-Grauls, P. H. Savelkoul, and A. B. J. Groeneveld, unpublished data). Blood cultures. Blood was collected for routine culture in blood culture bottles (BACTEC Aerobic/F and Anaerobic/F; Becton Dickinson) and incubated in an automated blood culture system (BACTEC 9240; Becton Dickinson). After microorganisms were subjected to automated detection of growth, they were identified by standard microbiological techniques.
* Corresponding author. Mailing address: VU University Medical Center, Department of Medical Microbiology and Infection Control, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. Phone: 31 20 4442780. Fax: 31 20 4444313. E-mail:
[email protected]. 䌤 Published ahead of print on 19 September 2007. 3641
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J. CLIN. MICROBIOL. tions. Positive PCRs for samples with negative simultaneous blood cultures were defined as “likely”, “possible”, or “unlikely” to be related to a clinically relevant infection with the specific microorganism. These results were defined as “likely” if S. aureus or E. faecalis was isolated from any microbial culture within 24 h around the time point of blood sampling, “unlikely” if other microorganisms were isolated from those cultures, and “possible” if no microorganisms were isolated from any microbial culture and infection by S. aureus or E. faecalis was possible on clinical grounds, including laboratory and radiological investigations. Statistical analysis. Data are described as numbers (percentage) and medians (range). The 2 test or, if appropriate, Fisher’s exact test was used to compare categorical variables; the Mann-Whitney U test was used for continuous variables. The BDLs were compared between paired blood samples with the Wilcoxon test.
RESULTS
FIG. 1. Flowchart of study. SA, S. aureus; EF, E. faecalis.
DNA isolation. Whole-blood samples for PCR were collected in 3-ml EDTA tubes (Becton Dickinson), and bacterial DNA was isolated from 200-l blood samples. Prior to DNA isolation, 250 CFU of Escherichia coli (50 CFU equivalents/PCR; ATCC strain 25922) was spiked to each blood sample as the extraction control. To remove hemoglobin, 1,000 l of Triton-Tris-EDTA (20 mM Tris-HCl, pH 8.3, 1 mM EDTA, 1% Triton X-100) was added to the blood sample and centrifuged at 12,000 rpm for 10 min. The supernatant fraction was removed, and the pellet was resuspended in 1,000 l of Triton-Tris-EDTA. This step was repeated but with the pellet resuspended in 200 l of Tris-EDTA (10 mM Tris-HCl, pH 8.3, 1 mM EDTA). Bacterial DNA was then extracted from this 200-l sample with a QIAamp DNA minikit (QIAGEN, Germany) according to the manufacturer’s instructions. The extract was eluted in 100 l of elution buffer. PCR assays. DNA amplification and detection were performed with a TaqMan 7000 system (Applied Biosystems, Foster City, CA). The reaction mixture (50 l) used in the PCR assay was as follows: 25 l TaqMan Universal PCR Mastermix (Applied Biosystems) and 20 l of extracted DNA and 5 l of specific primers and probes as described below. Standard PCR cycling conditions were applied, as follows: heating at 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. We designed a set of primers and probes for specific amplification of the species-specific Sa442 DNA fragment of S. aureus. The sequences were 5⬘-CATCGGAAACATTGTGTTCTGTATG-3⬘ and 5⬘-TTTGG CTGGAAAATATAACTCTCGTA-3⬘ for the forward and reverse primers, respectively; the probe sequence was 5⬘-FAM-AAGCCGTCTTGATAATCTTTA GTAGTACCGAAGCTGGT-3⬘ (where FAM is 6-carboxyfluorescein). The primers and probes used to amplify and detect the 16S rRNA genes of E. faecalis and E. coli are described elsewhere (14, 26). A standard curve of S. aureus or E. faecalis DNA consisting of four 10-fold dilutions of a stock solution of 5,000 CFU equivalents/PCR was included in each run for automatic calculation of the bacterial DNA load (BDL). The reproducibility of the BDL was determined by spiking 20 pooled culture-negative whole-blood samples with 250 CFU (equivalent to 50 CFU/PCR) of S. aureus or E. faecalis, followed by DNA isolation. Analysis of PCR results. The DNA in each blood sample was tested with three separate reactions: (i) a diagnostic PCR to detect either S. aureus or E. faecalis DNA, (ii) a PCR to detect E. coli DNA to evaluate extraction efficiency, and (iii) an assay to detect 50 CFU equivalents of S. aureus DNA or E. faecalis DNA added to the PCR mixture to test for possible specific template inhibition. Blood samples were retested (including DNA isolation and PCR) in cases of discrepant results between blood culture and PCR and when the blood sample did not pass the process or inhibition control. If the control reactions failed twice, the sample was considered inhibited or not isolated with sufficient efficacy and was excluded from further analysis. Definitions. Bacteremia was defined as the isolation of a pathogen from blood culture. PCR results were defined as positive when amplification signals were observed or as negative if such a signal was absent with adequate control reac-
A total of 175 blood samples was included in the S. aureus cohort and 180 in the E. faecalis cohort. Briefly, the median age of the study patients was 60 years (range, 15 to 95 years), and 57% of patients were male. The majority of patients were admitted for cardiovascular disease (29%), followed by respiratory tract disease (16%) and suspected sepsis (16%); 47% of patients had received antibiotics less than 48 h prior to blood sampling. S. aureus was isolated from 30 (17%) simultaneous blood cultures in the S. aureus cohort, including a methicillinresistant strain from two blood samples from one patient, and E. faecalis was present in 16 (9%) of the blood cultures in the E. faecalis cohort. No differences were noted for sex, age, reason of admission, and severity of illness between patients with and without S. aureus or E. faecalis bacteremia in the S. aureus and E. faecalis cohorts. PCR assays. The analytical sensitivity of both PCR assays, as determined with pooled blood culture-negative samples spiked with S. aureus (ATCC 25923) and E. faecalis (ATCC 29212), was below 5 CFU equivalents/PCR. No false-positive amplification reactions were observed when the PCR assays were evaluated against a wide variety of bacterial strains, including Staphylococcus epidermidis (ATCC 12228), Staphylococcus capitis (ATCC 35661), Enterococcus faecium (ATCC 35667), Streptococcus pyogenes (ATCC 19615), Streptococcus agalactiae (ATCC 12386), Streptococcus pneumoniae (ATCC 6306), Streptococcus sanguis (ATCC 10556), Pseudomonas aeruginosa (ATCC 27853), E. coli (ATCC 25922), and Bacteroides fragilis (ATCC 25285). We did not observe false-positive PCR amplification results when a microorganism other than the target microorganism was isolated from the blood culture. This ensured adequate specificity of the PCR assays. The reproducibility of the S. aureus and E. faecalis PCRs was evaluated by spiking 250 CFU bacteria (equivalent to 50 CFU in PCR) in 20
TABLE 1. Detection of S. aureus and E. faecalis bacteremia by PCR versus blood culture No. of blood culture samples
PCR blood samples
Positive
Negative
Total
S. aureus
Positive Negative Total
21 7 28
10 125 135
31 132 163
E. faecalis
Positive Negative Total
11 4 15
7 155 162
18 159 177
Organism
3643 PCR TO DETECT S. AUREUS AND E. FAECALIS BACTEREMIA VOL. 45, 2007
Miscellaneous observation(s)
Likely
Positive PCR result
BAL fluid
Likely
Microbial culture result
Pneumonia
Tracheal aspirate
Possible
Suspected clinical infection
TABLE 2. Clinical characteristics of patients with positive PCR results and no growth in simultaneous blood culturesa Antibiotics received ⬍48 h
Gender
Reason for admission to ICU
Age (yr)
Microorganism
Pneumonia
Negative
Possible
Possible
Negative
Negative
Possible
Catheter-related infection Wound infection
Negative
Likely
Catheter-related infection Pneumonia
Wound infection
Serratia marcescens cultured from pus and catheter tip Blood culture one day earlier Pus
Possible
Likely
Unlikely Extensive wounds of the leg
Wound infection
Negative
Likely
Wound infection Yes
Extensive wounds of the leg
Peritoneal fluid
Likely
CXR showing pulmonary infiltrates CXR showing pulmonary infiltrates Vancomycin induced leukopenia CABG 1 wk prior to admission Catheters inserted for more than 7 days Alcohol abuse; CXR showing interstitial infiltrates Pus collection in sternal wound
Yes
Catheter-related infection Fecal peritonitis
Peritoneal fluid and pus wound
Yes
Yes
Peritonitis and wound infection
Yes
Yes
Chemotherapy for multiple myeloma CT suggestive of anastomotic leakage Fluid collections around the pancreas and in the skin
Yes
No
Yes
Number of samples
Patient
S. aureus 2b 84 F
Yes
A S. aureus 2 80 F
Pneumonia with RF
B S. aureus 1 75 F
Multitrauma with open cruris facture Multitrauma with open femur fracture Pneumonia in pancytopenia Acute abdomen after sigmoid resection Sepsis after resection of pancreas tail tumor
Cardiogenic shock after PTCA RF after nearly drowning Cardiogenic shock after CABG
F
C S. aureus 1 61 M
74
D S. aureus 1 74 F
2b
E S. aureus 1 61 M
Yes
F S. aureus 3 44 F
RF after CABG
G E. faecalis 1 52
M
M
H E. faecalis 1 46
F
78
I E. faecalis 1 73
Yes
J E. faecalis 1
M
K E. faecalis
Septic pneumonia cardiac ischemia Pneumonia with RF
L
a The number of samples is the number of blood samples that were PCR positive and blood culture negative. Suspected clinical infection is the focus of infection as judged by the clinician at the moment blood is drawn for culture. Culture results represent the positive isolation of S. aureus or E. faecalis in any microbiological culture, unless indicated otherwise. F, female; M, male; RF, respiratory failure; CXR, chest X ray; BAL, bronchoalveolar lavage; CABG, coronary artery bypass grafting; PTCA, percutaneous transluminal coronary angioplasty; CT, computed tomography. b The two blood samples from these patients were obtained with an interval of more than 3 weeks between samples. One patient had a CXR showing pulmonary infiltrates and a positive culture of tracheal aspirate with S. aureus in both episodes.
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TABLE 3. Number of patients with indicated PCR and blood culture results for paired blood samplesa PCR results
⫹/⫹ ⫹/⫺ ⫺/⫺
No. of patients with indicated results for paired blood cultures ⫹/⫹
⫹/⫺
⫺/⫺
7 1 1
1 2b 0
3 2 76
a The result of each paired blood sample is indicated as positive (⫹) or negative (⫺). b Individual PCR and blood culture results were concordant between these samples.
pooled culture-negative blood samples. The mean cycle threshold value (⫾ standard deviation) for the S. aureus PCR was 33.09 ⫾ 0.67; the median cycle threshold value for the E. faecalis PCR was 33.26 ⫾ 0.59. PCR results compared to blood cultures. PCR amplification results for the detection of S. aureus and E. faecalis DNA in blood were available for 163 (93%) and 177 (98%) blood samples, respectively, including 49 (14%) blood samples that were tested twice because the control reaction values were below the requested value at the first test. Fifteen (4%) blood samples were excluded because of repeated inadequate extraction (n ⫽ 10) or inhibition (n ⫽ 5) controls (Fig. 1). Two of the excluded samples grew S. aureus in simultaneous blood cultures, and one grew E. faecalis. The initial PCR amplifications were positive for 64% and 53% of blood samples with S. aureus and E. faecalis isolates from simultaneous blood cultures, respectively. The second tests of blood samples with discrepant results of PCR and blood culture yielded three extra positive PCR amplifications with the S. aureus PCR and three with the E. faecalis PCR. As such, the sensitivity of the S. aureus PCR increased to 75% and to 73% for the E. faecalis PCR when samples were tested twice (Table 1). The S. aureus PCR was positive in 10/135 (7%) and the E. faecalis PCR in 7/162 (4%) of blood samples with no growth in blood culture, resulting in specificities of the PCR assays of 93% and 96%, respectively. The detection of bacterial DNA by PCR in these samples was likely related to infection with these microorganisms in 50% of the patients, possibly related in 42%, and unlikely in one patient (Table 2). The origin of the positive PCR result in the latter patient remains unclear, because the S. aureus PCR was repeatedly positive for this blood sample, while no amplification reaction was observed when the cultured strain of Serratia marcescens was tested. This makes a technical failure (crossreaction of S. marcescens DNA in the S. aureus PCR) unlikely. S. aureus PCR was negative for 7 (25%) blood samples with positive blood cultures and E. faecalis PCR for 4 (27%). In 8/11 (73%) of these samples, only a single blood sample was obtained from the patient. Paired blood samples. There were 101 paired whole-blood samples, i.e., blood samples that were drawn at the same time. PCR results were available for 93 of these paired samples (39 in the S. aureus and 52 in the E. faecalis cohort); in the other 8 samples, at least one PCR showed inhibition. PCR results were concordant for 88/93 (95%) of paired blood samples, while simultaneous paired blood cultures were concordant in 90/93 (97%) (Table 3). Two patients each had a positive and a
negative PCR result for the two blood samples, which was identical to the positive and negative results of the simultaneous paired blood cultures. No differences were observed for BDL between paired PCR positive blood samples (P ⫽ 0.67). Determination of BDL. Quantification of the amount of DNA was possible for 27/31 (87%) samples with positive S. aureus and 17/18 (94%) with positive E. faecalis PCR amplifications. The median S. aureus BDL in PCR positive samples was 10.6 (range, 0.3 to 84) ⫻ 103 CFU equivalents/ml, and the median E. faecalis BDL was 2.7 (range, 0.3 to 165) ⫻ 103 CFU equivalents/ml. Longitudinal positive blood samples were available for one patient with S. aureus bacteremia. This patient was a 46-year-old man who was admitted to the ICU of our hospital with cardiogenic shock after receiving a coronary stent because of acute myocardial infarction. He developed fever on day 6 after admission, and intravascular lines and devices (including a subclavian line and intra-aortic balloon pump) were either replaced or removed because an intravascular source of infection was suspected. Flucloxacillin therapy was initiated when S. aureus growth was detected from the first blood culture, which was available 30 h after the patient’s sera had been obtained. Subsequent blood cultures and culture of the subclavian line also grew a S. aureus strain resistant to penicillin only. Although the fever initially subsided, it soon rose again and, despite the addition of ceftriaxone to the treatment regimen, the patient died of septic shock without apparent focus. Figure 2 illustrates the course of BDL and evolution of temperature over time for this patient. DISCUSSION In this study, we show that real-time PCR can be used for the direct detection of the majority of blood culture-proven cases of S. aureus and E. faecalis bacteremia in patients admitted to the ICU. With current PCR techniques, results can be available within a few hours and thus provide a fast complement to blood culture. The possibility of making early adjustments to antimicrobial therapy based on these PCR results might improve the patient’s prognosis (8, 15). The sensitivities of our PCR assays were comparable to those found in other studies of detection specific for Neisseria meningitidis and S. pneumoniae DNA in blood samples (3, 5, 11, 22, 29). However, sensitivity may still be too low to warrant implementation of the assays in routine practice. The main issue in this regard is the small volume of 200 l of blood used in DNA isolation (which results in an equivalent of 40 l of blood tested in the PCR) compared to the much larger volume of blood (10 ml) in blood cultures. The use of such a small volume of blood could cause a sampling error, especially in the case of low-grade bacteremia, which may be solved by processing a larger volume of blood for PCR. The clear increase in sensitivity observed after the second DNA isolation and PCR of a blood sample supports this hypothesis. Developments aimed at increasing the input volume of blood that is possible in DNA isolation have to overcome the increasing risk of inhibition in the PCR that is associated with larger amounts of human DNA and immunoglobulins (1, 2, 4). The presence of these inhibitive components in blood may also explain the relatively high number of blood samples that were tested twice in our study because of failing control reactions for the initial
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FIG. 2. Courses of temperature and S. aureus BDL in a patient with S. aureus bacteremia. S. aureus was isolated from all corresponding blood cultures except from those obtained on day 2. The mean BDL was calculated in case BDL was available for two simultaneously obtained blood samples. Antimicrobial treatment was administered as indicated.
test. An alternative option for the increase in input volume of blood would be to test multiple samples from one patient, which might also have the advantage of a better detection of intermittent bacteremia. PCRs were positive for several blood samples with no growth in simultaneous blood cultures. The detection of cases of bacteremia by PCR only has been recognized previously, especially when blood samples are obtained after the initiation of antimicrobial treatment (6, 31). In our study, the detection of S. aureus and E. faecalis DNA by PCR in the blood of these patients is related to proven or possible infection with these microorganisms in the majority of patients, based on clinical grounds and microbiological and radiological investigations. A proportion of these episodes of positive “DNAemia” can probably be attributed to the presence of bacteria in the bloodstream that did not grow in culture because of the use of antibiotics, while other episodes may be the result of leakage of bacterial DNA into the bloodstream from a localized infection or colonization. Although we did not discriminate between infection and colonization, the role of colonization in falsepositive PCR results may be limited (7). Therefore, a systemic or localized infection with S. aureus or E. faecalis should be suspected in the case of a positive PCR result in combination with a suggestive clinical presentation. The S. aureus and E. faecalis BDLs in our study were comparable to the amount of DNA reported during S. pneumoniae bacteremia but lower than that of invasive Salmonella enterica serovar Typhi or N. meningitidis infection (12, 18, 23, 29). This suggests that the BDL in blood during bacteremia may vary depending on the type of microorganism (33). The median S. aureus BDL was 100-fold higher than the number of circulating bacteria reported during S. aureus endocarditis, suggesting that
both bacterial DNA fragments and viable bacteria are detected by PCR (30, 32). The clinical interpretation of a positive DNAemia is different from that of culture-positive bacteremia. Not only may the origin of bacterial DNA be unclear but the kinetics may also be different. For example, the amount of living bacteria is substantially reduced within hours after the administration of antibiotics, while bacterial DNA may persist in blood during antimicrobial treatment and may still be detected over several days (12, 21, 23). As a result of the possible detection of bacterial DNA during antimicrobial treatment, the BDL might be used to classify patients into prognostic risk groups or to serve as a surrogate marker for comparison of different antimicrobial agents in future clinical trials (12, 21, 23). A major advantage of PCR is the short turnaround time of 4 to 6 h under the present conditions. As a result, PCR determination of the BDL could lead to same-day initiation or optimization of antimicrobial treatment. For example, adequate therapy could have been started more than 24 h earlier for the patient that we reported. The possible correlation between BDL and temperature that we observed for this patient indicates the potential value of BDL in blood; future studies will be aimed at gaining insight into the kinetics of BDL during infection. In conclusion, quantitative detection of S. aureus and E. faecalis DNA in blood by real-time PCR enables rapid detection of bacteremia. The clinical value of a positive bacterial DNAemia, including determination of the BDL, will be the subject of future studies. REFERENCES 1. Al-Soud, W. A., and P. Radstrom. 2001. Purification and characterization of PCR-inhibitory components in blood cells. J. Clin. Microbiol. 39:485–493.
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