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Rapid detection of pathogens in blood culture bottles by real-time PCR in conjunction with the pre-analytic tool MolYsis
Journal of Infection (2008) 57, 307e316
www.elsevierhealth.com/journals/jinf
Rapid detection of pathogens in blood culture bottles by real-time PCR in conjunction with the pre-analytic tool MolYsis Susanne Gebert, Dunja Siegel, Nele Wellinghausen* Institute of Medical Microbiology and Hygiene, University Hospital of Ulm, Albert-Einstein-Allee 23, D-89081 Ulm, Germany Accepted 17 July 2008 Available online 29 August 2008
KEYWORDS Blood culture; Real-time PCR; Sepsis; Molecular diagnostics
Summary Objectives: Rapid detection of pathogens in blood from septic patients is essential for adequate antimicrobial therapy and prognosis of patients. Aim of this study is the acceleration of detection and identification of bacteria and fungi in blood cultures by molecular methods before positive signalling in an automated system. This would allow an earlier appropriate antimicrobial therapy and may improve the prognosis of septic patients. Methods: Samples were analysed with an eubacterial real-time PCR assay that enables detection of bacterial DNA and simultaneous differentiation of Gram-positive and Gram-negative bacteria. In addition, genus- and species-specific real-time PCR assays were used. DNA preparation was performed with the new tool MolYsis. Results: With the Gram-differentiating PCR assay bacteria were detectable in concentrations of 10e20 CFU per PCR reaction. A positive PCR result was achieved in samples taken from spiked blood culture bottles between 5.0 and 8.7 h prior to positive signalling of the BACTEC system. We were able to identify the causative organism in 11 out of 18 culture-positive blood cultures from patients with septicaemia with an average of 10.7 h prior to positive signalling. Out of 83 culture-negative bottles six samples showed a positive PCR result. Conclusion: PCR analysis in conjunction with MolYsis DNA preparation allows rapid detection of pathogens in blood culture samples. Thus, the approach may be a valuable supplemental tool for blood cultures in patients with suspicion of infection with slow-growing pathogens or serious clinical condition. ª 2008 The British Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Tel.: þ49 731 5006 5316; fax: þ49 731 5006 5304. E-mail address: [email protected] (N. Wellinghausen).
Bloodstream infections are life-threatening conditions with increasing significance in modern medicine. In 2000, the incidence of sepsis in the USA was 240.4 cases per 100,000 people with an annual increase of 8.7% within the last years.1
0163-4453/$34 ª 2008 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2008.07.013
308 The mortality rate of sepsis ranges between 21% and 55%2,3 and remained unchanged during the last decades. Gram-positive bacteria are the most frequent cause of bloodstream infection with 30e50% of all cases, followed by Gram-negative bacteria in 25e30%. Fungi represent 1e 3% of all cases. In at least 30% no causative pathogen can be identified.4 For prognosis of patients suffering from bloodstream infections the identification of the causative agent and early initiation of adequate antimicrobial therapy is essential since it has a positive impact on the outcome.5e8 Rapid identification of the causative pathogen may also allow a reduction in the use of broad-spectrum antimicrobials and an earlier switch to narrow-spectrum substances. Thereby, the emergence of resistances as well as the costs of therapy may be reduced. The standard procedure for microbiological diagnostics of bloodstream infections is the inoculation of blood culture (BC) bottles followed by incubation in an automated blood culture system.9,10 After positive signalling of the automated system (mainly within 1e2 days) the pathogen is identified by biochemical tests. Altogether, detection and final identification of the bacteria mostly takes 2e4 days. In order to decrease the time to result, molecular methods for rapid identification of pathogens in positive BC have been described, including DNA microarrays,11 RNAbased fluorescence in situ hybridisation probes,12,13 and PCR-assays like real-time PCR.14e16 Universal broad-range PCR assays can detect all pathogenic bacteria but sequencing of the PCR product is time-consuming.17 An alternative is the application of a real-time PCR algorithm involving species- and genus-specific assays for the most relevant bacterial causes of sepsis.16 Nevertheless, all these techniques aiming at a rapid detection of bacteria in bloodstream infections are based on positive growth of bacteria in BC bottles. In contrast, an approach that focuses on the detection of pathogenic bacteria in BC before positive signalling in the automated system may further reduce the time to result of bacterial detection. Since commercial BC media are known to contain PCR-inhibiting substances, like polyanetholesulfonate (SPS),18 the sensitivity of PCR assays is limited in BC. Therefore optimal preparation of bacterial DNA is crucial for reliable PCR results. The concept of the new pre-analytic tool MolYsis (Molzym GmbH & Co. KG Bremen, Germany) involves lysis of human blood cells prior to degradation of bacterial cell walls, allowing digestion of human DNA by using a DNAse and enrichment of bacterial DNA. In addition, PCR inhibitors may be efficiently removed by the MolYsis procedure. Aim of this study was to facilitate a more rapid detection of bacterial pathogens in BC of patients with suspected bloodstream infection. This would allow an earlier appropriate antimicrobial therapy and may improve the prognosis of septic patients. A universal broad-range real-time PCR assay that allows simultaneous differentiation of Grampositive and Gram-negative bacteria as well as genus- and species-specific PCR assays for subsequent identification of pathogen were used in conjunction with the pre-analytic tool MolYsis. Results were compared with conventional BC diagnostics.
S. Gebert et al.
Materials and methods Universal broad-range Gram-differentiating LightCycler PCR-assay The used 16S rDNA based PCR assay (Gram-diff PCR) enables detection of bacterial DNA of all relevant species and simultaneous differentiation of Gram-positive and Gramnegative bacteria by dual-colour multiplex fluorescence resonance energy transfer (FRET) probe design. The PCR was performed with the eubacterial primers DG74 and RW01 (Thermo, Ulm, Germany)19 and the probes Gram-Anchor (50 -CGG AGG AAG GTG GGG ATG ACG TCA A-FL), Grampositive (50 -LCRed705-TCA TCA TGC CCC TTA CG-P) and Gram-negative (50 -LCRed640-TCA TCA TGG CCC TTA CG-P) (TibMolBiol, Berlin, Germany)20 on the LightCycler 1.5 (Roche, Mannheim, Germany). The probes are complementary to a unique and conserved site within the 16S rRNA gene of bacteria. At one position there is a significant base difference between Gram-positive and Gram-negative bacteria (underlined nucleotide in the probe sequences). Amplification mixture for Gram-diff PCR consisted of 0.5 mM primer DG74, 0.25 mM primer RW01, 0.15 mM probe Gram-Anchor, 0.15 mM probe Gram-negative, 0.15 mM probe Gram-positive, 4 ml LightCycler FastStart DNA MasterPLUS HybProbe-Mix (Roche), and 5 ml template-DNA in a total volume of 20 ml. Samples were amplified with the following program: Initial denaturation at 95 C for 10 min followed by 37 cycles of denaturation for 10 s at 95 C, annealing for 10 s at 55 C, and elongation for 20 s at 72 C. All steps were performed with a temperature transition rate of 20 C/s. Melting curve analysis was done after amplification by heating the samples from 50 C to 95 C with a temperature transition rate of 0.1 C/s. The generation of target amplicons was observed at 640 nm (channel F2, Gramnegative bacteria) and at 705 nm (channel F3, Grampositive bacteria) and analysed with LightCycler Software 3.5. According to the information of the manufacturers only DNA-free materials were used for PCR. A no-template control was added for every PCR run to exclude positive results due to contamination of PCR materials.
Species- and genus-specific LightCycler PCR assays An algorithm of species- and genus-specific PCR assays was used to identify bacteria as fast as possible. The algorithm includes Staphylococcus spp. PCR, Enterococcus/Streptococcus spp. PCR (detection of Enterococcus spp.- and Streptococcus spp.-DNA with differentiation by melting curve analysis), Enterobacteriaceae PCR, Pseudomonas aeruginosa PCR and Staphylococcus epidermidis PCR. These assays were adapted from a former publication16 and were used as described there. For detection of Staphylococcus aureus a nuc gene-specific PCR was used.21 The amplification mixture consisted of 0.5 mM primer nuc274f, 0.5 mM primer nuc274r, 0.15 mM probe nuc274-FL, 0.15 mM probe nuc274-LC705, 4 ml LightCycler FastStart DNA MasterPLUS HybProbe-Mix, and 5 ml template-DNA in a total volume of 20 ml. Samples were amplified with the following program: Initial denaturation at 95 C for 10 min followed
Molecular identification of blood cultures by 50 cycles of denaturation for 10 s at 95 C, annealing for 10 s at 50 C, and elongation for 15 s at 72 C, followed by melting curve analysis. In all samples the Gram-diff PCR was done first. Depending on the result, further assays were applied as shown in Fig. 1. For detection of Candida spp. the amplification mix consisted of 0.25 mM primer FungL543R, 0.75 mM primer FungL1046R,22 0.15 mM probe Candida-FL, 0.15 mM Candida-LC640,23 4 ml LightCycler FastStart DNA MasterPLUS HybProbe-Mix, and 5 ml template-DNA in a total volume of 20 ml. Samples were amplified with the following program: Initial denaturation at 95 C for 10 min followed by 50 cycles of denaturation for 10 s at 95 C, annealing for 10 s at 58 C, and elongation for 20 s at 72 C, followed by melting curve analysis.
Determination of the sensitivity and specificity of PCR-assays To determine the sensitivity of the Gram-diff PCR assay, bacteria were dissolved in physiological saline. Bacterial solutions were serially diluted (1:10) and 100 ml of the samples were plated on blood agar plates to determine the colony forming units (CFU) in each dilution. DNA isolation of the dilutions was performed with MolYsis Complete (Molzym GmbH & Co. KG, Bremen, Germany) with an initial volume of 500 ml according to the recommendation of the manufacturers. Bacterial DNA was eluted in 100 ml of buffer EB and used for the PCR reactions. For determination of the specificity of the Gram-diff PCR chromosomal DNA isolation from bacterial cultures (Tables 1a,b) was performed with QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the recommendation of the manufacturer. DNA solutions with a concentration of 1 ng/ml were taken for PCR reactions. To determine the sensitivity of Candida spp. PCR 5 ml citrate blood from healthy subject were spiked with Candida albicans solutions in different concentrations.
Isolated DNA from blood culture
Gram-differentiating PCR Positive result F3 (Gram-pos) Staphylococcus spp. PCR Streptococcus/ Enterococcus spp. PCR
If Staphylococcus spp. PCR pos: S. aureus nuc-gene PCR S. epidermidis PCR
Positive result F2 (Gram-neg)
309 DNA was isolated with MolYsis Complete and analysed with Candida spp. PCR. Candida albicans solutions were plated in parallel on blood agar plates to determine CFU.
Determination of the sensitivity of the Gramdifferentiating PCR in spiked blood cultures BACTEC Aerobic Plus/F BC bottles (Becton Dickinson, Germany) were inoculated with 10 ml blood from healthy subjects. 5e20 CFU of either Staphylococcus aureus (ATCC 29213) or Escherichia coli (ATCC 25922) were added. Bottles were incubated in the BACTEC 9240 system. During incubation, every 1e2 h a sample of 1300 ml was taken until positive signalling of the bottle. A total of 500 ml of the sample were used immediately for preparation of DNA with MolYsis Plus (Molzym GmbH & Co. KG, Bremen, Germany), 500 ml for DNA preparation with QIAamp DNA Mini Kit and 200 ml for DNA preparation with Roche High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany) according to the recommendations of manufacturers. Bacterial DNA was eluted in 100 ml of the provided elution buffers, respectively. With the isolated DNA the Gramdiff PCR was performed as described above. 100 ml of the sample were plated in parallel on blood agar plates in order to determine the bacterial concentration in the bottle.
Investigation of patient blood cultures A total of 101 BACTEC Plus/F BC bottles from 69 randomly selected patients with suspected septicaemia or sepsis treated at different departments of the University Hospital of Ulm were examined including 91 Aerobic Plus/F bottles and 10 Peds Plus/F bottles between June 2007 and October 2007. A total of 500 ml BC medium were taken and analysed from each BC bottle two times within the first hours after starting incubation in the automated BACTEC system. The first reading point was 2.6 h (2.1) and the second 6.3 h (2.1) after starting incubation. Between the two reading points a time difference between 3.5 and 5 h was kept. Samples from BC bottles were taken under sterile conditions as fast as possible to avoid contamination of the bottles and to interrupt incubation time as short as necessary. DNA isolation of the samples was performed immediately with MolYsis Plus as recommended by the manufacturers. Samples were analysed by real-time PCR as described above. A PCR inhibition control was performed by adding 10 pg of chromosomal E. coli DNA to the isolated DNA.
Enterobacteriaceae PCR Pseudomonas aeruginosa PCR
Figure 1 Diagnostic PCR algorithm for the identification of bacteria in patient blood cultures. PCR assays shown in the same box can be run simultaneously.
Cultural identification of pathogens from positive blood cultures Identification of all cultured bacteria was done by standard tests including clumping factor and protein A detection in staphylococci (Slide Staph Plus, BioMerieux) and Api (Api 20 strep, Api 20E, Api 20 staph, Api 20 NE, BioMerieux, Nu ¨rtingen, Germany). Yeasts were identified by Api ID 32 C (BioMerieux).
310 Table 1a
S. Gebert et al. Gram-negative control strains used for evaluation of the specificity of Gram-diff PCR
Gram-negative species
Source
Result F2 channel (Gram-negative)
Result F3 channel (Gram-positive)
Achromobacter xylosoxidans Acinetobacter baumannii Acinetobacter junii Acinetobacter lwoffii Alcaligenes faecalis Bacteroides fragilis Bacteroides ovatus Burkholderia multivorans Campylobacter fetus Citrobacter koseri Citrobacter freundii Eggerthella lenta Enterobacter aerogenes Enterobacter cloacae Escherichia coli Haemophilus influenzae Haemophilus parainfluenzae Klebsiella oxytoca Klebsiella pneumoniae Moraxella catharralis Morganella morganii Neisseria meningitidis Pantoea agglomerans Prevotella buccalis Prevotella denticola Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa Salmonella Enteritidis Serratia marcescens Stenotrophomonas maltophilia
Clinical isolate ATCC 19606 ATCC 17908 ATCC 15309 DSM 30030 ATCC 25285 ATCC 8483 DSM 13243 DSM 5361 ATCC 27028 ATCC 8090 ATCC 43055 ATCC 13048 DSM 30054 ATCC 25922 ATCC 49247 ATCC 33392 ATCC 43863 ATCC 33495 ATCC 43617 Clinical isolate ATCC 13017 ATCC 27155 ATCC 35310 ATCC 35308 ATCC 29906 ATCC 29905 ATCC 27853 ATCC 13076 ATCC 13888 ATCC 13637
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
Results Analytical sensitivity and specificity of the universal Gram-differentiating PCR assay and the Candida spp. PCR assay The sensitivity of the Gram-differentiating (Gram-diff) PCR assay was determined by measuring serial dilutions of bacterial DNA in PCR grade water. The sensitivity obtained for Staphylococcus aureus was 5 pg DNA per PCR, for Escherichia coli 500 fg DNA per PCR, and for Streptococcus oralis 500 fg DNA per PCR reaction, respectively. In addition, the sensitivity of the PCR was determined with serial dilutions of bacteria in physiological saline in conjunction with MolYsis Complete DNA isolation. The sensitivity was 10 CFU per PCR reaction for E. coli and 20 CFU per PCR reaction for S. aureus. For investigation of the specificity a panel of control strains, representing more than 95% of the species commonly found in blood cultures, was tested (Tables 1a,b). DNA of all bacterial strains gave correct positive PCR signals in the respective LightCycler fluorescence channel, except the anaerobic species Bacteroides ovatus, B. fragilis, Prevotella buccalis, P. denticola, and Eggerthella lenta. These species were negative
in the Gram-diff PCR due to mismatches in the probe binding region (Table 2). A sequence alignment was performed between the probe sequences and the 16S rDNA sequences of the 92 bacterial species found most often in positive blood cultures (Ref. 3 and own unpublished data). Only the above mentioned anaerobic species escaped detection by mutations within the hybridisation region of the corresponding LightCycler probes. Burkholderia multivorans, Alcaligenens faecalis, Achromobacter xylosoxidans, and Neisseria meningitidis were detected by the PCR assay but the melting point was shifted to lower values (45 C versus 60 C), probably due to observed mismatches (two bases) in the target gene. With MolYsis DNA isolation of 5 ml spiked blood samples in conjunction with the Candida spp. PCR a sensitivity of 260e330 CFU Candida albicans per ml blood (50 CFU per PCR reaction) could be achieved.
Investigation of spiked blood cultures To investigate the sensitivity of the universal Gram-diff PCR in conjunction with MolYsis in BC, Aerobic Plus/F BC bottles were inoculated with blood from healthy subjects, spiked with either Escherichia coli (ATCC 25922) or Staphylococcus
Molecular identification of blood cultures Table 1b
311
Gram-positive control strains used for evaluation of the specificity of Gram-diff PCR
Gram-positive species
Source
Result F2 channel (Gram-negative)
Result F3 channel (Gram-positive)
Aerococcus viridans Bacillus cereus Clostridium perfringens Corynebacterium minutissimum Enterococcus avium Enterococcus casseliflavus Enterococcus durans Enterococcus faecalis Enterococcus faecium Enterococcus gallinarum Enterococcus raffinosus Listeria monocytogenes Micrococcus luteus Peptostreptococcus anaerobius Propionibacterium acnes Staphylococcus aureus Staphylococcus auricularis Staphylococcus capitis Staphylococcus cohnii Staphylococcus epidermidis Staphylococcus gallinarum Staphylococcus haemolyticus Staphylococcus hominis Staphylococcus lugdunensis Staphylococcus simulans Streptococcus agalactiae Streptococcus anginosus Streptococcus bovis Streptococcus constellatus Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus parasanguinis Streptococcus pneumoniae Streptococcus pyogenes Streptococcus salivarus Streptococcus sanguinis
Clinical isolate Clinical isolate ATCC 13124 ATCC 23348 DSM 20679 DSM 20680 DSM 20633 ATCC 29212 ATCC 29213 ATCC 700425 DSM 5633 ATCC 15313 Clinical isolate Clinical isolate ATCC 6919 ATCC 29213 Clinical isolate Clinical isolate Clinical isolate ATCC 12228 ATCC 700401 Clinical isolate Clinical isolate ATCC 49576 Clinical isolate ATCC 13813 ATCC 12395 ATCC 33317 ATCC 27823 ATCC 49456 ATCC 25175 ATCC 35037 ATCC 15912 ATCC 49619 ATCC 12344 ATCC 7073 ATCC 10556
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
aureus (ATCC 29213) and analysed intermittently until positive signalling of the BACTEC-system. DNA preparation with MolYsis Plus was compared to QIAamp DNA Mini Kit (Qiagen) and High Pure PCR Template Preparation Kit (Roche). By use of MolYsis DNA preparation, it was possible
Table 2
to detect Staphylococcus aureus DNA by PCR 8.0e8.7 h prior to positive signalling of the BC bottle and Escherichia coli DNA 5.0 h prior to positive signalling (Table 3 and Fig. 2). In samples with the first positive Gram-diff PCR result the CFU were 1700e3400 CFU/ml BC for Staphylococcus
Mismatches between Gram-diff PCR-probes and the relevant binding sites of non-recognised anaerobic species Gram-anchor (reverse sequence)
Probe sequence Eggerthella lenta Prevotella denticola Bacteroides ovatus Bacteroides fragilis Prevotella buccalis
0
5 -CGGAGGAAGGTGGGGATGACGTCAA 50 -CGGAGGAAGGTGGGGACGACGTCAA 50 -GCGAGGAAGGCGGGGATGACGTCAA 50 -GTGAGGAAGGTGGGGATGACGTCAA 50 -GTGAGGAAGGTGGGGATGACGTCAA 50 -GTGAGGAAGGTGGGGATGACGTCAA
Gram-negative (reverse sequence) 50 -TCATCATGGCCCTTACG 50 -TCATCATGCCCTTTATG 50 -TCAGCACGGCCCTTACG 50 -TCAGCACGGCCCTTACG 50 -TCAGCACGGCCCTTACG 50 -TCAGCACGGCCCTTACG
GenBank Association Nos: Eggerthella lenta: AY937380; Prevotella denticola: AY323524; Bacteroides ovatus: L16484; Bacteroides fragilis: X83935; Prevotella buccalis: L16476.
312
S. Gebert et al. negative staphylococci and a-haemolytic streptococci BC contamination rather than bloodstream infection was assumed if only one out of two or more BC were positive and if no other underlying disease like endocarditis was recorded. Thereby, three out of 18 culture-positive BC are considered as probably contaminated, including no. 1, 4 and 10 (Table 4). These three BC were negative in the Gram-diff PCR at both time points of sample taking. Regarding the clinically relevant culture-positive BC only, it was possible to confirm the result of culture by the PCRalgorithm in conjunction with MolYsis DNA isolation in 11 out of 15 cases. Concerning the 83 culture-negative BC, 77 were also negative in Gram-diff PCR. In three culture-negative BC, DNA from Staphylococcus epidermidis was found with the described PCR algorithm. DNA from Enterococcus faecium was found in one BC. In two BC, DNA from Gram-positive bacteria was found, that could not be identified any further with the described PCR algorithm. All positive PCR results for the culture-negative BC bottles were observed only in one of the two measuring points. In order to interpret the positive PCR results we reviewed the microbiological reports of these patients. In four out of six cases we did not find any cultural detection of the same bacterial species during a time period of at least 5 weeks around the BC sampling. Therefore, the PCR results remain questionable and may be caused by contamination. In one patient with negative BC but positive PCR result with DNA from Grampositive bacteria coagulase-negative staphylococci were grown in another BC bottle from the same day. In the patient in whom Enterococcus faecium alone was identified by the PCR-algorithm, Enterococcus faecium was cultured from an intra-operatively taken swab of the patient’s bile 1 day after taking the BC.
aureus and at least 5000 CFU/ml BC for Escherichia coli (Table 3). It was not possible to detect bacterial DNA in BC bottles isolated by High Pure PCR Template Preparation. E. coli DNA isolated with QIAamp DNA Mini Kit from BC bottles was detectable 5.0 h prior to positive signalling of the BC bottle as well. With QIAamp DNA Mini Kit isolated S. aureus DNA was detected 6.5 h prior to positive signalling of the bottle.
Investigation of patient blood cultures Out of the 101 analysed patient BC bottles, 17 bottles were culture-positive with bacterial pathogens, in one bottle Candida parapsilosis was cultured (Table 4), and 83 bottles remained sterile. For culture-positive BC bottles the results of identification by the PCR algorithm in conjunction with MolYsis Plus were compared to the results observed by biochemical identification. Out of 17 bottles with bacterial growth, it was possible to identify pathogens in 10 bottles prior to positive signalling of the BACTEC-system (Table 4). The Gram-diff PCR revealed a positive result in samples taken between 1.2 and 37.7 h before positive signalling of the BACTEC-system (Fig. 3). PCR inhibition was ruled out in all samples, apart from number 4 in which DNA was not available for inhibition control analysis. In one BC Candida parapsilosis was cultured after 38 h of incubation in the BACTEC-system. In this case, the DNA isolated for the Gram-diff PCR was retrospectively additionally analysed with a Candida spp.-specific LightCycler PCR. It revealed a clear positive result already after less than 3 h of incubation in the BACTEC-system. All positive BC were judged with regard to the clinical significance of the grown bacteria. In BC growing coagulase-
Table 3 kits
Results of spiked blood culture bottles analysed with Gram-diff PCR in conjunction with different DNA preparation
No Inoculated Sample taking CFU/ml species after starting blood culture BACTEC incubation (h)
Time until positive MolYsis QIAamp High Pure BACTEC DNA DNA DNA preparation preparation preparation signalling (h)
Time difference between first sample with positive PCR result and positive BACTEC signalling (h)
1
þ þ þ þ þ
20.0
8.7
17.8
8.0
14.0
5.0
14.0
5.0
2
3
4
S. aureus
6.8 8.3 9.8 11.3 S. aureus 6.8 8.3 9.8 11.3 E. coli 4.0 5.5 7.0 9.0 E. coli 4.0 5.5 7.0 9.0
10 100 820 3400 160 900 1700 5000 170 650 4040 >5000 10 130 1310 >5000
Gram-diff PCR result
þ þ þ
Molecular identification of blood cultures
Figure 2 LightCycler Gram-differentiating PCR results of blood culture bottle, spiked with Escherichia coli (ATCC 25922). Gram-negative DNA is detected in fluorescence channel F2 (640 nm). The fluorescence signal in the PCR is shown in relation to positive signalling of the BC bottle in the BACTEC system. Time data are given in time of sampling prior to positive signalling of the BACTEC system. M: MolYsis Plus DNA isolation. Q: QIAamp DNA Mini Kit DNA isolation.
Discussion Bloodstream infections are associated with a high morbidity and mortality.1,2 Since early detection of the causative pathogen in septic patients is crucial for adequate
Figure 3 LightCycler Gram-differentiating PCR results in the fluorescence channel F3 (720 nm) of samples from patient blood culture bottles. DNA from Gram-positive bacteria is detected in fluorescence channel F3. Numbers of the bottles are presented as shown in Table 4.
313 antimicrobial therapy, we aimed at the development of a real-time PCR assay for detection of bacteria and Candida in BC before positive signalling in an automated blood culture system. PCR analysis of human blood samples is hampered by the presence of PCR-inhibiting substances and it has been shown that human background DNA causes cross-reactions with primers and probes and inhibits PCR reactions.24e27 The preanalytic tool MolYsis Plus is a new DNA isolation kit which has the feature to eliminate human background DNA prior to selective enrichment of bacterial DNA. According to the manufacturer it also removes other PCR-inhibiting substances contained in BC bottles, like SPS. The MolYsis tool is easy to use. Isolation of DNA includes pretreatment of samples with different enzymes included in the kit and the use of mini-spin columns. Complete manual preparation of DNA takes about 3 h with 1.5e2 h hands on time. MolYsis can, however, also be combined with any DNA isolation method including automated systems. The reagent costs for one reaction are approx. V5.80. Apart from a tabletop centrifuge no special equipment is needed. Compared to other commercial DNA isolation kits MolYsis showed the best results for preparation of bacterial DNA from spiked BC bottles. In our study, pretreatment of patient BC with MolYsis prior to DNA isolation resulted in absence of PCR inhibition in all tested samples. In contrast to results showed for other kits28,29 we did not note general ‘‘background’’ contamination of materials and buffers with bacterial DNA in our eubacterial 16S rDNA-based PCR assay. The eubacterial real-time Gram-diff PCR with dualcolour probe design evaluated in this study reliable differentiates Gram-positive and Gram-negative bacteria. It is comparable in specificity to a former assay published by Klaschik et al.30 but a much broader spectrum of species was investigated in our study. The non-recognition of a few anaerobes is explained by mismatches within the sequence of the relevant binding-site of the probes as shown in Table 2. Nevertheless, anaerobes are responsible for only 2% of nosocomial bloodstream infections.3 By using the Gram-diff PCR algorithm and Candida spp. PCR in conjunction with the MolYsis tool it was possible to detect bacterial pathogens and Candida spp. in 11 out of 18 BC (11 out of 15 concerning only culture-positive BC which were judged as clinically relevant) prior to positive signalling in the automated BACTEC blood culture system. The mean time difference between sample taking for PCR and positive signalling of the BC was 10.7 h. Considering the time needed for isolation of DNA and performing of the PCR assays, which sums up to 5e6 h (including 3 h of working steps and 2e3 h of incubation), there is still a mean time difference of 5.7 h between result of PCR analysis and positive signalling in the automated blood culture system. The advantage of a positive PCR result is further stressed by the fact that at the time point of positive BACTEC signalling only the Gram stain characteristics but not the final identification of the causative pathogen is available. Gram-diff PCR showed a negative result in samples taken from seven BC with a positive bacterial growth result. This is probably caused by the lower sensitivity of the PCR assay in comparison to prolonged culture. Interestingly, all positive BC, that were clinically judged as contamination
314
S. Gebert et al.
Table 4
1b 2 3 4b 5 6 7 8 9 10b 11 12 13 14 15 16 17 18
Investigation of patient blood cultures by PCR
Identified species by culture
Incubation time Result of Gram-diff to positive PCR BACTEC signalling (h)
CNSc CNSc CNSc CNSc and Staphylococcus epidermidis Enterococcus faecalis Enterococcus faecalis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus aureus Streptococcus bovis Streptococcus mutans Pseudomonas aeruginosa Pseudomonas aeruginosa Candida parapsilosis
17.8 14.4 10.4 31.4
þ (F3, Gram-positive)
2.2 n. a. 12.7 6.3 14.3 16.2 22.5 15.0 4.0 42.4 71.8 17.2 11.5 38.0
þ þ þ þ þ þ þ þ þ
(F3, (F3, (F3, (F3, (F3,
(F3, (F3, (F3,
(F2,
Gram-positive) Gram-positive) Gram-positive) Gram-positive) Gram-positive)
Identification by PCR-algorithm
Gain of time (h)a 1
2
Staphylococcus spp.
5.0
0
Enterococcus faecalis Enterococcus faecalis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Gram-positive) Staphylococcus epidermidis Gram-positive) Staphylococcus aureus Gram-positive) Streptococcus spp. Gram-negative) Pseudomonas aeruginosa Candida spp.
1.2 0 at least 3.9 0 7.9 2.9 3.1 0 6.3 1.3 8.8 3.3 3.5 0 37.7 32.7 5.3 0.3 35.1 30.1
No. 5/6 and 8/9 are blood culture bottles from the same patients, respectively. n.a.: not available due to setting manually positive by technician. a Gain of time (1) is calculated by the difference of time between positive signalling of the BACTEC-system and positive PCR result concerning incubation time; gain of time (2) is calculated by the difference of time between positive signalling of the BACTEC-system and time until positive PCR result including all working steps b Judged as blood culture contamination (only one out of two or more BC taken at the same time point from one patient positive) c Coagulase-negative staphylococci other than Staphylococcus epidermidis
rather than true infection were negative in the Gram-diff PCR. Thus, by use of the Gram-diff PCR assay time- and cost-intense identification steps done on contaminants may possibly be avoided. By use of Gram-diff PCR bacterial DNA was also detected in six culture-negative BC. The significance of the detected DNA in four samples cannot be definitely determined. Although all working steps were performed in different PCR-safety cabinets and separated in different rooms, a contamination of samples is not fully excluded. Nevertheless, detection of Enterococcus faecium DNA in one patient with intra-abdominal E. feacium infection appears clinically significant and stresses the advantages of real-time PCR analysis using the described algorithm. By Candida-specific real-time PCR, one isolate of Candida parapsilosis has been detected in the BC 35 h prior to positive signalling of the BACTEC system. Due to the slow growth and low concentration of yeasts, sampling of several BC are needed in patients with fungemia and BC are mostly reported positive at day 2e4 of incubation (31,32 and own unpublished data). Therefore, real-time PCR detection of Candida in conjunction with the new DNA preparation tool MolYsis may significantly improve detection of fungemia. In conclusion, the real-time Gram-differentiating and Candida-specific PCR assays in conjunction with pretreatment of MolYsis enables earlier detection of bacteria and
yeasts in BC of patients with bacteremia and fungemia but the procedure seems to be less sensitive than BC diagnostic. With the described procedure we reached a sensitivity of at least 400 CFU per ml blood culture. In contrast, blood cultures have a theoretical detection limit of one viable cell inoculated in one blood culture bottle. Therefore and because of the fact that conventional cultivation of BC enables identification and susceptibility testing of all pathogens blood cultures are still indispensable. In addition, it has to be considered that the real-time PCR approach is more labour-intensive and more expensive than BC diagnostics. Altogether for DNA preparation and PCR analysis of one patient the costs for materials and reagent per sample sum up to V15. However, it may be a valuable supplemental tool for patients with suspicion of infection with slow-growing pathogens like Brucella spp. and Candida spp. and for patients with serious clinical condition aiming at a rapid identification of the pathogen, like suspicion of meningococcal meningitis or severely immunosuppressed patients. Nevertheless, by using BC media for eubacterial PCR analysis is has to be considered that BC media can contain residual bacterial DNA that may be a reason for false-positive PCR results. The new DNA preparation tool MolYsis proved effective in removing PCR-inhibiting substances in BC, and was superior in sensitivity compared to other commercial kits. Due to the use of DNAse prior to bacterial DNA isolation it
Molecular identification of blood cultures theoretically removes human DNA and DNA from damaged or broken bacteria completely. Thus, it has the same advantages or rather disadvantages like BC diagnostics since only viable bacteria can be identified by BC. MolYsis may also be used for pretreatment of other clinical samples like whole blood samples. Real-time PCR analysis of native blood samples instead of BC may further accelerate detection of pathogens in septic patients. Despite the very low concentration of bacterial and fungal DNA in human blood from patients suffering from bacteraemia or fungemia33 real-time PCR has already shown some promising results.23,34 Further studies are underway to determine whether pretreatment with the MolYsis tool also improves direct detection of pathogenic bacteria and fungi in native blood samples.
315
12.
13.
14.
15.
16.
Acknowledgement This work was supported by a ProInno II grant from the German Ministry of Economy and Labour.
17.
References 1. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 2003;348(16):1546e54. 2. Engel C, Brunkhorst FM, Bone HG, Brunkhorst R, Gerlach H, Grond S, et al. Epidemiology of sepsis in Germany: results from a national prospective multicenter study. Intensive Care Med 2007;33(4):606e18. 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):309e17. 4. Annane D, Bellissant E, Cavaillon JM. Septic shock. Lancet 2005;365(9453):63e78. doi:10.1016/S0140-6736(04)17667-8. 5. Harbarth S, Garbino J, Pugin J, Romand JA, Lew D, Pittet D. Inappropriate initial antimicrobial therapy and its effect on survival in a clinical trial of immunomodulating therapy for severe sepsis. Am J Med 2003;115(7):529e35. doi:10.1016/j.amjmed.2003.07.005. 6. Kang CI, Kim SH, Park WB, Lee KD, Kim HB, Kim EC, et al. Bloodstream infections caused by antibiotic-resistant gramnegative bacilli: risk factors for mortality and impact of inappropriate initial antimicrobial therapy on outcome. Antimicrob Agents Chemother 2005;49(2):760e6. 7. Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med 2006;34(6):1589e96. 8. Micek ST, Lloyd AE, Ritchie DJ, Reichley RM, Fraser VJ, Kollef MH. Pseudomonas aeruginosa bloodstream infection: importance of appropriate initial antimicrobial treatment. Antimicrob Agents Chemother 2005;49(4):1306e11. 9. Weinstein MP, Mirrett S, Reimer LG, Wilson ML, SmithElekes S, Chuard CR, et al. Controlled evaluation of BacT/Alert standard aerobic and FAN aerobic blood culture bottles for detection of bacteremia and fungemia. J Clin Microbiol 1995;33(4):978e81. 10. Wilson ML, Weinstein MP, Mirrett S, Reimer LG, Feldman RJ, Chuard CR, et al. Controlled evaluation of BacT/alert standard anaerobic and FAN anaerobic blood culture bottles for the detection of bacteremia and fungemia. J Clin Microbiol 1995; 33(9):2265e70. 11. Cleven BE, Palka-Santini M, Gielen J, Meembor S, Kronke M, Krut O. Identification and characterization of bacterial
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
pathogens causing bloodstream infections by DNA microarray. J Clin Microbiol 2006;44(7):2389e97. Jansen GJ, Mooibroek M, Idema J, Harmsen HJ, Welling GW, Degener JE. Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes. J Clin Microbiol 2000;38(2):814e7. Kempf VA, Trebesius K, Autenrieth IB. Fluorescent In situ hybridization allows rapid identification of microorganisms in blood cultures. J Clin Microbiol 2000;38(2):830e8. Carroll KC, Leonard RB, Newcomb-Gayman PL, Hillyard DR. Rapid detection of the staphylococcal mecA gene from BACTEC blood culture bottles by the polymerase chain reaction. Am J Clin Pathol 1996;106(5):600e5. Kurupati P, Chow C, Kumarasinghe G, Poh CL. Rapid detection of Klebsiella pneumoniae from blood culture bottles by realtime PCR. J Clin Microbiol 2004;42(3):1337e40. Wellinghausen N, Wirths B, Franz AR, Karolyi L, Marre R, Reischl U. Algorithm for the identification of bacterial pathogens in positive blood cultures by real-time LightCycler polymerase chain reaction (PCR) with sequence-specific probes. Diagn Microbiol Infect Dis 2004;48(4):229e41. doi: 10.1016/j.diagmicrobio.2003.11.005. Peters RP, van Agtmael MA, Danner SA, Savelkoul PH, Vandenbroucke-Grauls CM. New developments in the diagnosis of bloodstream infections. Lancet Infect Dis 2004;4(12):751e60. doi:10.1016/S1473-3099(04)01205-8. Fredricks DN, Relman DA. Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate. J Clin Microbiol 1998;36(10): 2810e6. Greisen K, Loeffelholz M, Purohit A, Leong D. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol 1994;32(2):335e51. Bestmann L. A micromachined total nucleic acid analysis system with integrated DNA isolation, mix preparation, on-chip real-time PCR and melting curve analysis. Doctoral thesis, Institute of Clinical Chemistry, University Hospital of Zurich; 2005. Costa AM, Kay I, Palladino S. Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction. Diagn Microbiol Infect Dis 2005;51(1):13e7. doi:10.1016/j.diagmicrobio.2004.08.014. Einsele H, Hebart H, Roller G, Loffler J, Rothenhofer I, Muller CA, et al. Detection and identification of fungal pathogens in blood by using molecular probes. J Clin Microbiol 1997; 35(6):1353e60. Klingspor L, Jalal S. Molecular detection and identification of Candida and Aspergillus spp. from clinical samples using real-time PCR. Clin Microbiol Infect 2006;12(8):745e53. Cogswell FB, Bantar CE, Hughes TG, Gu Y, Philipp MT. Host DNA can interfere with detection of Borrelia burgdorferi in skin biopsy specimens by PCR. J Clin Microbiol 1996;34(4):980e2. Navarro E, Escribano J, Fernandez J, Solera J. Comparison of three different PCR methods for detection of Brucella spp in human blood samples. FEMS Immunol Med Microbiol 2002; 34(2):147e51. Al Soud WA, Jonsson LJ, Radstrom P. Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol 2000;38(1):345e50. Al Soud WA, Radstrom P. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 2001;39(2):485e93. Queipo-Ortuno MI, Tena F, Colmenero JD, Morata P. Comparison of seven commercial DNA extraction kits for the recovery of Brucella DNA from spiked human serum samples using real-time PCR. Eur J Clin Microbiol Infect Dis 2008;27(2): 109e14.
316 29. van der Zee A, Peeters M, de Jong C, Verbakel H, Crielaard JW, Claas EC, et al. Qiagen DNA extraction kits for sample preparation for Legionella PCR are not suitable for diagnostic purposes. J Clin Microbiol 2002;40(3):1126. 30. Klaschik S, Lehmann LE, Raadts A, Book M, Hoeft A, Stuber F. Real-time PCR for detection and differentiation of gram-positive and gram-negative bacteria. J Clin Microbiol 2002; 40(11):4304e7. 31. Cockerill III FR, Wilson JW, Vetter EA, Goodman KM, Torgerson CA, Harmsen WS, et al. Optimal testing parameters for blood cultures. Clin Infect Dis 2004;38(12):1724e30.
S. Gebert et al. 32. Lee A, Mirrett S, Reller LB, Weinstein MP. Detection of bloodstream infections in adults: how many blood cultures are needed? J Clin Microbiol 2007;45(11):3546e8. 33. Kreger BE, Craven DE, Carling PCR, McCrabe R. Gram-negative bacteremia. III. Reassessment of etiology, epidemiology and ecology in 612 patients. Am J Med 1980;68(3):332e43. 34. Peters RP, van Agtmael MA, Gierveld S, Danner SA, Groeneveld AB, Vandenbroucke-Grauls CM, et al. Quantitative detection of Staphylococcus aureus and Enterococcus faecalis DNA in blood to diagnose bacteremia in patients in the intensive care unit. J Clin Microbiol 2007;45(11):3641e6.
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Rapid detection of pathogens in blood culture bottles by real-time PCR in conjunction with the pre-analytic tool MolYsis Susanne Gebert, Dunja Siegel, Nele Wellinghausen* Institute of Medical Microbiology and Hygiene, University Hospital of Ulm, Albert-Einstein-Allee 23, D-89081 Ulm, Germany Accepted 17 July 2008 Available online 29 August 2008
KEYWORDS Blood culture; Real-time PCR; Sepsis; Molecular diagnostics
Summary Objectives: Rapid detection of pathogens in blood from septic patients is essential for adequate antimicrobial therapy and prognosis of patients. Aim of this study is the acceleration of detection and identification of bacteria and fungi in blood cultures by molecular methods before positive signalling in an automated system. This would allow an earlier appropriate antimicrobial therapy and may improve the prognosis of septic patients. Methods: Samples were analysed with an eubacterial real-time PCR assay that enables detection of bacterial DNA and simultaneous differentiation of Gram-positive and Gram-negative bacteria. In addition, genus- and species-specific real-time PCR assays were used. DNA preparation was performed with the new tool MolYsis. Results: With the Gram-differentiating PCR assay bacteria were detectable in concentrations of 10e20 CFU per PCR reaction. A positive PCR result was achieved in samples taken from spiked blood culture bottles between 5.0 and 8.7 h prior to positive signalling of the BACTEC system. We were able to identify the causative organism in 11 out of 18 culture-positive blood cultures from patients with septicaemia with an average of 10.7 h prior to positive signalling. Out of 83 culture-negative bottles six samples showed a positive PCR result. Conclusion: PCR analysis in conjunction with MolYsis DNA preparation allows rapid detection of pathogens in blood culture samples. Thus, the approach may be a valuable supplemental tool for blood cultures in patients with suspicion of infection with slow-growing pathogens or serious clinical condition. ª 2008 The British Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Tel.: þ49 731 5006 5316; fax: þ49 731 5006 5304. E-mail address: [email protected] (N. Wellinghausen).
Bloodstream infections are life-threatening conditions with increasing significance in modern medicine. In 2000, the incidence of sepsis in the USA was 240.4 cases per 100,000 people with an annual increase of 8.7% within the last years.1
0163-4453/$34 ª 2008 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2008.07.013
308 The mortality rate of sepsis ranges between 21% and 55%2,3 and remained unchanged during the last decades. Gram-positive bacteria are the most frequent cause of bloodstream infection with 30e50% of all cases, followed by Gram-negative bacteria in 25e30%. Fungi represent 1e 3% of all cases. In at least 30% no causative pathogen can be identified.4 For prognosis of patients suffering from bloodstream infections the identification of the causative agent and early initiation of adequate antimicrobial therapy is essential since it has a positive impact on the outcome.5e8 Rapid identification of the causative pathogen may also allow a reduction in the use of broad-spectrum antimicrobials and an earlier switch to narrow-spectrum substances. Thereby, the emergence of resistances as well as the costs of therapy may be reduced. The standard procedure for microbiological diagnostics of bloodstream infections is the inoculation of blood culture (BC) bottles followed by incubation in an automated blood culture system.9,10 After positive signalling of the automated system (mainly within 1e2 days) the pathogen is identified by biochemical tests. Altogether, detection and final identification of the bacteria mostly takes 2e4 days. In order to decrease the time to result, molecular methods for rapid identification of pathogens in positive BC have been described, including DNA microarrays,11 RNAbased fluorescence in situ hybridisation probes,12,13 and PCR-assays like real-time PCR.14e16 Universal broad-range PCR assays can detect all pathogenic bacteria but sequencing of the PCR product is time-consuming.17 An alternative is the application of a real-time PCR algorithm involving species- and genus-specific assays for the most relevant bacterial causes of sepsis.16 Nevertheless, all these techniques aiming at a rapid detection of bacteria in bloodstream infections are based on positive growth of bacteria in BC bottles. In contrast, an approach that focuses on the detection of pathogenic bacteria in BC before positive signalling in the automated system may further reduce the time to result of bacterial detection. Since commercial BC media are known to contain PCR-inhibiting substances, like polyanetholesulfonate (SPS),18 the sensitivity of PCR assays is limited in BC. Therefore optimal preparation of bacterial DNA is crucial for reliable PCR results. The concept of the new pre-analytic tool MolYsis (Molzym GmbH & Co. KG Bremen, Germany) involves lysis of human blood cells prior to degradation of bacterial cell walls, allowing digestion of human DNA by using a DNAse and enrichment of bacterial DNA. In addition, PCR inhibitors may be efficiently removed by the MolYsis procedure. Aim of this study was to facilitate a more rapid detection of bacterial pathogens in BC of patients with suspected bloodstream infection. This would allow an earlier appropriate antimicrobial therapy and may improve the prognosis of septic patients. A universal broad-range real-time PCR assay that allows simultaneous differentiation of Grampositive and Gram-negative bacteria as well as genus- and species-specific PCR assays for subsequent identification of pathogen were used in conjunction with the pre-analytic tool MolYsis. Results were compared with conventional BC diagnostics.
S. Gebert et al.
Materials and methods Universal broad-range Gram-differentiating LightCycler PCR-assay The used 16S rDNA based PCR assay (Gram-diff PCR) enables detection of bacterial DNA of all relevant species and simultaneous differentiation of Gram-positive and Gramnegative bacteria by dual-colour multiplex fluorescence resonance energy transfer (FRET) probe design. The PCR was performed with the eubacterial primers DG74 and RW01 (Thermo, Ulm, Germany)19 and the probes Gram-Anchor (50 -CGG AGG AAG GTG GGG ATG ACG TCA A-FL), Grampositive (50 -LCRed705-TCA TCA TGC CCC TTA CG-P) and Gram-negative (50 -LCRed640-TCA TCA TGG CCC TTA CG-P) (TibMolBiol, Berlin, Germany)20 on the LightCycler 1.5 (Roche, Mannheim, Germany). The probes are complementary to a unique and conserved site within the 16S rRNA gene of bacteria. At one position there is a significant base difference between Gram-positive and Gram-negative bacteria (underlined nucleotide in the probe sequences). Amplification mixture for Gram-diff PCR consisted of 0.5 mM primer DG74, 0.25 mM primer RW01, 0.15 mM probe Gram-Anchor, 0.15 mM probe Gram-negative, 0.15 mM probe Gram-positive, 4 ml LightCycler FastStart DNA MasterPLUS HybProbe-Mix (Roche), and 5 ml template-DNA in a total volume of 20 ml. Samples were amplified with the following program: Initial denaturation at 95 C for 10 min followed by 37 cycles of denaturation for 10 s at 95 C, annealing for 10 s at 55 C, and elongation for 20 s at 72 C. All steps were performed with a temperature transition rate of 20 C/s. Melting curve analysis was done after amplification by heating the samples from 50 C to 95 C with a temperature transition rate of 0.1 C/s. The generation of target amplicons was observed at 640 nm (channel F2, Gramnegative bacteria) and at 705 nm (channel F3, Grampositive bacteria) and analysed with LightCycler Software 3.5. According to the information of the manufacturers only DNA-free materials were used for PCR. A no-template control was added for every PCR run to exclude positive results due to contamination of PCR materials.
Species- and genus-specific LightCycler PCR assays An algorithm of species- and genus-specific PCR assays was used to identify bacteria as fast as possible. The algorithm includes Staphylococcus spp. PCR, Enterococcus/Streptococcus spp. PCR (detection of Enterococcus spp.- and Streptococcus spp.-DNA with differentiation by melting curve analysis), Enterobacteriaceae PCR, Pseudomonas aeruginosa PCR and Staphylococcus epidermidis PCR. These assays were adapted from a former publication16 and were used as described there. For detection of Staphylococcus aureus a nuc gene-specific PCR was used.21 The amplification mixture consisted of 0.5 mM primer nuc274f, 0.5 mM primer nuc274r, 0.15 mM probe nuc274-FL, 0.15 mM probe nuc274-LC705, 4 ml LightCycler FastStart DNA MasterPLUS HybProbe-Mix, and 5 ml template-DNA in a total volume of 20 ml. Samples were amplified with the following program: Initial denaturation at 95 C for 10 min followed
Molecular identification of blood cultures by 50 cycles of denaturation for 10 s at 95 C, annealing for 10 s at 50 C, and elongation for 15 s at 72 C, followed by melting curve analysis. In all samples the Gram-diff PCR was done first. Depending on the result, further assays were applied as shown in Fig. 1. For detection of Candida spp. the amplification mix consisted of 0.25 mM primer FungL543R, 0.75 mM primer FungL1046R,22 0.15 mM probe Candida-FL, 0.15 mM Candida-LC640,23 4 ml LightCycler FastStart DNA MasterPLUS HybProbe-Mix, and 5 ml template-DNA in a total volume of 20 ml. Samples were amplified with the following program: Initial denaturation at 95 C for 10 min followed by 50 cycles of denaturation for 10 s at 95 C, annealing for 10 s at 58 C, and elongation for 20 s at 72 C, followed by melting curve analysis.
Determination of the sensitivity and specificity of PCR-assays To determine the sensitivity of the Gram-diff PCR assay, bacteria were dissolved in physiological saline. Bacterial solutions were serially diluted (1:10) and 100 ml of the samples were plated on blood agar plates to determine the colony forming units (CFU) in each dilution. DNA isolation of the dilutions was performed with MolYsis Complete (Molzym GmbH & Co. KG, Bremen, Germany) with an initial volume of 500 ml according to the recommendation of the manufacturers. Bacterial DNA was eluted in 100 ml of buffer EB and used for the PCR reactions. For determination of the specificity of the Gram-diff PCR chromosomal DNA isolation from bacterial cultures (Tables 1a,b) was performed with QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the recommendation of the manufacturer. DNA solutions with a concentration of 1 ng/ml were taken for PCR reactions. To determine the sensitivity of Candida spp. PCR 5 ml citrate blood from healthy subject were spiked with Candida albicans solutions in different concentrations.
Isolated DNA from blood culture
Gram-differentiating PCR Positive result F3 (Gram-pos) Staphylococcus spp. PCR Streptococcus/ Enterococcus spp. PCR
If Staphylococcus spp. PCR pos: S. aureus nuc-gene PCR S. epidermidis PCR
Positive result F2 (Gram-neg)
309 DNA was isolated with MolYsis Complete and analysed with Candida spp. PCR. Candida albicans solutions were plated in parallel on blood agar plates to determine CFU.
Determination of the sensitivity of the Gramdifferentiating PCR in spiked blood cultures BACTEC Aerobic Plus/F BC bottles (Becton Dickinson, Germany) were inoculated with 10 ml blood from healthy subjects. 5e20 CFU of either Staphylococcus aureus (ATCC 29213) or Escherichia coli (ATCC 25922) were added. Bottles were incubated in the BACTEC 9240 system. During incubation, every 1e2 h a sample of 1300 ml was taken until positive signalling of the bottle. A total of 500 ml of the sample were used immediately for preparation of DNA with MolYsis Plus (Molzym GmbH & Co. KG, Bremen, Germany), 500 ml for DNA preparation with QIAamp DNA Mini Kit and 200 ml for DNA preparation with Roche High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany) according to the recommendations of manufacturers. Bacterial DNA was eluted in 100 ml of the provided elution buffers, respectively. With the isolated DNA the Gramdiff PCR was performed as described above. 100 ml of the sample were plated in parallel on blood agar plates in order to determine the bacterial concentration in the bottle.
Investigation of patient blood cultures A total of 101 BACTEC Plus/F BC bottles from 69 randomly selected patients with suspected septicaemia or sepsis treated at different departments of the University Hospital of Ulm were examined including 91 Aerobic Plus/F bottles and 10 Peds Plus/F bottles between June 2007 and October 2007. A total of 500 ml BC medium were taken and analysed from each BC bottle two times within the first hours after starting incubation in the automated BACTEC system. The first reading point was 2.6 h (2.1) and the second 6.3 h (2.1) after starting incubation. Between the two reading points a time difference between 3.5 and 5 h was kept. Samples from BC bottles were taken under sterile conditions as fast as possible to avoid contamination of the bottles and to interrupt incubation time as short as necessary. DNA isolation of the samples was performed immediately with MolYsis Plus as recommended by the manufacturers. Samples were analysed by real-time PCR as described above. A PCR inhibition control was performed by adding 10 pg of chromosomal E. coli DNA to the isolated DNA.
Enterobacteriaceae PCR Pseudomonas aeruginosa PCR
Figure 1 Diagnostic PCR algorithm for the identification of bacteria in patient blood cultures. PCR assays shown in the same box can be run simultaneously.
Cultural identification of pathogens from positive blood cultures Identification of all cultured bacteria was done by standard tests including clumping factor and protein A detection in staphylococci (Slide Staph Plus, BioMerieux) and Api (Api 20 strep, Api 20E, Api 20 staph, Api 20 NE, BioMerieux, Nu ¨rtingen, Germany). Yeasts were identified by Api ID 32 C (BioMerieux).
310 Table 1a
S. Gebert et al. Gram-negative control strains used for evaluation of the specificity of Gram-diff PCR
Gram-negative species
Source
Result F2 channel (Gram-negative)
Result F3 channel (Gram-positive)
Achromobacter xylosoxidans Acinetobacter baumannii Acinetobacter junii Acinetobacter lwoffii Alcaligenes faecalis Bacteroides fragilis Bacteroides ovatus Burkholderia multivorans Campylobacter fetus Citrobacter koseri Citrobacter freundii Eggerthella lenta Enterobacter aerogenes Enterobacter cloacae Escherichia coli Haemophilus influenzae Haemophilus parainfluenzae Klebsiella oxytoca Klebsiella pneumoniae Moraxella catharralis Morganella morganii Neisseria meningitidis Pantoea agglomerans Prevotella buccalis Prevotella denticola Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa Salmonella Enteritidis Serratia marcescens Stenotrophomonas maltophilia
Clinical isolate ATCC 19606 ATCC 17908 ATCC 15309 DSM 30030 ATCC 25285 ATCC 8483 DSM 13243 DSM 5361 ATCC 27028 ATCC 8090 ATCC 43055 ATCC 13048 DSM 30054 ATCC 25922 ATCC 49247 ATCC 33392 ATCC 43863 ATCC 33495 ATCC 43617 Clinical isolate ATCC 13017 ATCC 27155 ATCC 35310 ATCC 35308 ATCC 29906 ATCC 29905 ATCC 27853 ATCC 13076 ATCC 13888 ATCC 13637
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
Results Analytical sensitivity and specificity of the universal Gram-differentiating PCR assay and the Candida spp. PCR assay The sensitivity of the Gram-differentiating (Gram-diff) PCR assay was determined by measuring serial dilutions of bacterial DNA in PCR grade water. The sensitivity obtained for Staphylococcus aureus was 5 pg DNA per PCR, for Escherichia coli 500 fg DNA per PCR, and for Streptococcus oralis 500 fg DNA per PCR reaction, respectively. In addition, the sensitivity of the PCR was determined with serial dilutions of bacteria in physiological saline in conjunction with MolYsis Complete DNA isolation. The sensitivity was 10 CFU per PCR reaction for E. coli and 20 CFU per PCR reaction for S. aureus. For investigation of the specificity a panel of control strains, representing more than 95% of the species commonly found in blood cultures, was tested (Tables 1a,b). DNA of all bacterial strains gave correct positive PCR signals in the respective LightCycler fluorescence channel, except the anaerobic species Bacteroides ovatus, B. fragilis, Prevotella buccalis, P. denticola, and Eggerthella lenta. These species were negative
in the Gram-diff PCR due to mismatches in the probe binding region (Table 2). A sequence alignment was performed between the probe sequences and the 16S rDNA sequences of the 92 bacterial species found most often in positive blood cultures (Ref. 3 and own unpublished data). Only the above mentioned anaerobic species escaped detection by mutations within the hybridisation region of the corresponding LightCycler probes. Burkholderia multivorans, Alcaligenens faecalis, Achromobacter xylosoxidans, and Neisseria meningitidis were detected by the PCR assay but the melting point was shifted to lower values (45 C versus 60 C), probably due to observed mismatches (two bases) in the target gene. With MolYsis DNA isolation of 5 ml spiked blood samples in conjunction with the Candida spp. PCR a sensitivity of 260e330 CFU Candida albicans per ml blood (50 CFU per PCR reaction) could be achieved.
Investigation of spiked blood cultures To investigate the sensitivity of the universal Gram-diff PCR in conjunction with MolYsis in BC, Aerobic Plus/F BC bottles were inoculated with blood from healthy subjects, spiked with either Escherichia coli (ATCC 25922) or Staphylococcus
Molecular identification of blood cultures Table 1b
311
Gram-positive control strains used for evaluation of the specificity of Gram-diff PCR
Gram-positive species
Source
Result F2 channel (Gram-negative)
Result F3 channel (Gram-positive)
Aerococcus viridans Bacillus cereus Clostridium perfringens Corynebacterium minutissimum Enterococcus avium Enterococcus casseliflavus Enterococcus durans Enterococcus faecalis Enterococcus faecium Enterococcus gallinarum Enterococcus raffinosus Listeria monocytogenes Micrococcus luteus Peptostreptococcus anaerobius Propionibacterium acnes Staphylococcus aureus Staphylococcus auricularis Staphylococcus capitis Staphylococcus cohnii Staphylococcus epidermidis Staphylococcus gallinarum Staphylococcus haemolyticus Staphylococcus hominis Staphylococcus lugdunensis Staphylococcus simulans Streptococcus agalactiae Streptococcus anginosus Streptococcus bovis Streptococcus constellatus Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus parasanguinis Streptococcus pneumoniae Streptococcus pyogenes Streptococcus salivarus Streptococcus sanguinis
Clinical isolate Clinical isolate ATCC 13124 ATCC 23348 DSM 20679 DSM 20680 DSM 20633 ATCC 29212 ATCC 29213 ATCC 700425 DSM 5633 ATCC 15313 Clinical isolate Clinical isolate ATCC 6919 ATCC 29213 Clinical isolate Clinical isolate Clinical isolate ATCC 12228 ATCC 700401 Clinical isolate Clinical isolate ATCC 49576 Clinical isolate ATCC 13813 ATCC 12395 ATCC 33317 ATCC 27823 ATCC 49456 ATCC 25175 ATCC 35037 ATCC 15912 ATCC 49619 ATCC 12344 ATCC 7073 ATCC 10556
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
aureus (ATCC 29213) and analysed intermittently until positive signalling of the BACTEC-system. DNA preparation with MolYsis Plus was compared to QIAamp DNA Mini Kit (Qiagen) and High Pure PCR Template Preparation Kit (Roche). By use of MolYsis DNA preparation, it was possible
Table 2
to detect Staphylococcus aureus DNA by PCR 8.0e8.7 h prior to positive signalling of the BC bottle and Escherichia coli DNA 5.0 h prior to positive signalling (Table 3 and Fig. 2). In samples with the first positive Gram-diff PCR result the CFU were 1700e3400 CFU/ml BC for Staphylococcus
Mismatches between Gram-diff PCR-probes and the relevant binding sites of non-recognised anaerobic species Gram-anchor (reverse sequence)
Probe sequence Eggerthella lenta Prevotella denticola Bacteroides ovatus Bacteroides fragilis Prevotella buccalis
0
5 -CGGAGGAAGGTGGGGATGACGTCAA 50 -CGGAGGAAGGTGGGGACGACGTCAA 50 -GCGAGGAAGGCGGGGATGACGTCAA 50 -GTGAGGAAGGTGGGGATGACGTCAA 50 -GTGAGGAAGGTGGGGATGACGTCAA 50 -GTGAGGAAGGTGGGGATGACGTCAA
Gram-negative (reverse sequence) 50 -TCATCATGGCCCTTACG 50 -TCATCATGCCCTTTATG 50 -TCAGCACGGCCCTTACG 50 -TCAGCACGGCCCTTACG 50 -TCAGCACGGCCCTTACG 50 -TCAGCACGGCCCTTACG
GenBank Association Nos: Eggerthella lenta: AY937380; Prevotella denticola: AY323524; Bacteroides ovatus: L16484; Bacteroides fragilis: X83935; Prevotella buccalis: L16476.
312
S. Gebert et al. negative staphylococci and a-haemolytic streptococci BC contamination rather than bloodstream infection was assumed if only one out of two or more BC were positive and if no other underlying disease like endocarditis was recorded. Thereby, three out of 18 culture-positive BC are considered as probably contaminated, including no. 1, 4 and 10 (Table 4). These three BC were negative in the Gram-diff PCR at both time points of sample taking. Regarding the clinically relevant culture-positive BC only, it was possible to confirm the result of culture by the PCRalgorithm in conjunction with MolYsis DNA isolation in 11 out of 15 cases. Concerning the 83 culture-negative BC, 77 were also negative in Gram-diff PCR. In three culture-negative BC, DNA from Staphylococcus epidermidis was found with the described PCR algorithm. DNA from Enterococcus faecium was found in one BC. In two BC, DNA from Gram-positive bacteria was found, that could not be identified any further with the described PCR algorithm. All positive PCR results for the culture-negative BC bottles were observed only in one of the two measuring points. In order to interpret the positive PCR results we reviewed the microbiological reports of these patients. In four out of six cases we did not find any cultural detection of the same bacterial species during a time period of at least 5 weeks around the BC sampling. Therefore, the PCR results remain questionable and may be caused by contamination. In one patient with negative BC but positive PCR result with DNA from Grampositive bacteria coagulase-negative staphylococci were grown in another BC bottle from the same day. In the patient in whom Enterococcus faecium alone was identified by the PCR-algorithm, Enterococcus faecium was cultured from an intra-operatively taken swab of the patient’s bile 1 day after taking the BC.
aureus and at least 5000 CFU/ml BC for Escherichia coli (Table 3). It was not possible to detect bacterial DNA in BC bottles isolated by High Pure PCR Template Preparation. E. coli DNA isolated with QIAamp DNA Mini Kit from BC bottles was detectable 5.0 h prior to positive signalling of the BC bottle as well. With QIAamp DNA Mini Kit isolated S. aureus DNA was detected 6.5 h prior to positive signalling of the bottle.
Investigation of patient blood cultures Out of the 101 analysed patient BC bottles, 17 bottles were culture-positive with bacterial pathogens, in one bottle Candida parapsilosis was cultured (Table 4), and 83 bottles remained sterile. For culture-positive BC bottles the results of identification by the PCR algorithm in conjunction with MolYsis Plus were compared to the results observed by biochemical identification. Out of 17 bottles with bacterial growth, it was possible to identify pathogens in 10 bottles prior to positive signalling of the BACTEC-system (Table 4). The Gram-diff PCR revealed a positive result in samples taken between 1.2 and 37.7 h before positive signalling of the BACTEC-system (Fig. 3). PCR inhibition was ruled out in all samples, apart from number 4 in which DNA was not available for inhibition control analysis. In one BC Candida parapsilosis was cultured after 38 h of incubation in the BACTEC-system. In this case, the DNA isolated for the Gram-diff PCR was retrospectively additionally analysed with a Candida spp.-specific LightCycler PCR. It revealed a clear positive result already after less than 3 h of incubation in the BACTEC-system. All positive BC were judged with regard to the clinical significance of the grown bacteria. In BC growing coagulase-
Table 3 kits
Results of spiked blood culture bottles analysed with Gram-diff PCR in conjunction with different DNA preparation
No Inoculated Sample taking CFU/ml species after starting blood culture BACTEC incubation (h)
Time until positive MolYsis QIAamp High Pure BACTEC DNA DNA DNA preparation preparation preparation signalling (h)
Time difference between first sample with positive PCR result and positive BACTEC signalling (h)
1
þ þ þ þ þ
20.0
8.7
17.8
8.0
14.0
5.0
14.0
5.0
2
3
4
S. aureus
6.8 8.3 9.8 11.3 S. aureus 6.8 8.3 9.8 11.3 E. coli 4.0 5.5 7.0 9.0 E. coli 4.0 5.5 7.0 9.0
10 100 820 3400 160 900 1700 5000 170 650 4040 >5000 10 130 1310 >5000
Gram-diff PCR result
þ þ þ
Molecular identification of blood cultures
Figure 2 LightCycler Gram-differentiating PCR results of blood culture bottle, spiked with Escherichia coli (ATCC 25922). Gram-negative DNA is detected in fluorescence channel F2 (640 nm). The fluorescence signal in the PCR is shown in relation to positive signalling of the BC bottle in the BACTEC system. Time data are given in time of sampling prior to positive signalling of the BACTEC system. M: MolYsis Plus DNA isolation. Q: QIAamp DNA Mini Kit DNA isolation.
Discussion Bloodstream infections are associated with a high morbidity and mortality.1,2 Since early detection of the causative pathogen in septic patients is crucial for adequate
Figure 3 LightCycler Gram-differentiating PCR results in the fluorescence channel F3 (720 nm) of samples from patient blood culture bottles. DNA from Gram-positive bacteria is detected in fluorescence channel F3. Numbers of the bottles are presented as shown in Table 4.
313 antimicrobial therapy, we aimed at the development of a real-time PCR assay for detection of bacteria and Candida in BC before positive signalling in an automated blood culture system. PCR analysis of human blood samples is hampered by the presence of PCR-inhibiting substances and it has been shown that human background DNA causes cross-reactions with primers and probes and inhibits PCR reactions.24e27 The preanalytic tool MolYsis Plus is a new DNA isolation kit which has the feature to eliminate human background DNA prior to selective enrichment of bacterial DNA. According to the manufacturer it also removes other PCR-inhibiting substances contained in BC bottles, like SPS. The MolYsis tool is easy to use. Isolation of DNA includes pretreatment of samples with different enzymes included in the kit and the use of mini-spin columns. Complete manual preparation of DNA takes about 3 h with 1.5e2 h hands on time. MolYsis can, however, also be combined with any DNA isolation method including automated systems. The reagent costs for one reaction are approx. V5.80. Apart from a tabletop centrifuge no special equipment is needed. Compared to other commercial DNA isolation kits MolYsis showed the best results for preparation of bacterial DNA from spiked BC bottles. In our study, pretreatment of patient BC with MolYsis prior to DNA isolation resulted in absence of PCR inhibition in all tested samples. In contrast to results showed for other kits28,29 we did not note general ‘‘background’’ contamination of materials and buffers with bacterial DNA in our eubacterial 16S rDNA-based PCR assay. The eubacterial real-time Gram-diff PCR with dualcolour probe design evaluated in this study reliable differentiates Gram-positive and Gram-negative bacteria. It is comparable in specificity to a former assay published by Klaschik et al.30 but a much broader spectrum of species was investigated in our study. The non-recognition of a few anaerobes is explained by mismatches within the sequence of the relevant binding-site of the probes as shown in Table 2. Nevertheless, anaerobes are responsible for only 2% of nosocomial bloodstream infections.3 By using the Gram-diff PCR algorithm and Candida spp. PCR in conjunction with the MolYsis tool it was possible to detect bacterial pathogens and Candida spp. in 11 out of 18 BC (11 out of 15 concerning only culture-positive BC which were judged as clinically relevant) prior to positive signalling in the automated BACTEC blood culture system. The mean time difference between sample taking for PCR and positive signalling of the BC was 10.7 h. Considering the time needed for isolation of DNA and performing of the PCR assays, which sums up to 5e6 h (including 3 h of working steps and 2e3 h of incubation), there is still a mean time difference of 5.7 h between result of PCR analysis and positive signalling in the automated blood culture system. The advantage of a positive PCR result is further stressed by the fact that at the time point of positive BACTEC signalling only the Gram stain characteristics but not the final identification of the causative pathogen is available. Gram-diff PCR showed a negative result in samples taken from seven BC with a positive bacterial growth result. This is probably caused by the lower sensitivity of the PCR assay in comparison to prolonged culture. Interestingly, all positive BC, that were clinically judged as contamination
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Table 4
1b 2 3 4b 5 6 7 8 9 10b 11 12 13 14 15 16 17 18
Investigation of patient blood cultures by PCR
Identified species by culture
Incubation time Result of Gram-diff to positive PCR BACTEC signalling (h)
CNSc CNSc CNSc CNSc and Staphylococcus epidermidis Enterococcus faecalis Enterococcus faecalis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus aureus Streptococcus bovis Streptococcus mutans Pseudomonas aeruginosa Pseudomonas aeruginosa Candida parapsilosis
17.8 14.4 10.4 31.4
þ (F3, Gram-positive)
2.2 n. a. 12.7 6.3 14.3 16.2 22.5 15.0 4.0 42.4 71.8 17.2 11.5 38.0
þ þ þ þ þ þ þ þ þ
(F3, (F3, (F3, (F3, (F3,
(F3, (F3, (F3,
(F2,
Gram-positive) Gram-positive) Gram-positive) Gram-positive) Gram-positive)
Identification by PCR-algorithm
Gain of time (h)a 1
2
Staphylococcus spp.
5.0
0
Enterococcus faecalis Enterococcus faecalis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Gram-positive) Staphylococcus epidermidis Gram-positive) Staphylococcus aureus Gram-positive) Streptococcus spp. Gram-negative) Pseudomonas aeruginosa Candida spp.
1.2 0 at least 3.9 0 7.9 2.9 3.1 0 6.3 1.3 8.8 3.3 3.5 0 37.7 32.7 5.3 0.3 35.1 30.1
No. 5/6 and 8/9 are blood culture bottles from the same patients, respectively. n.a.: not available due to setting manually positive by technician. a Gain of time (1) is calculated by the difference of time between positive signalling of the BACTEC-system and positive PCR result concerning incubation time; gain of time (2) is calculated by the difference of time between positive signalling of the BACTEC-system and time until positive PCR result including all working steps b Judged as blood culture contamination (only one out of two or more BC taken at the same time point from one patient positive) c Coagulase-negative staphylococci other than Staphylococcus epidermidis
rather than true infection were negative in the Gram-diff PCR. Thus, by use of the Gram-diff PCR assay time- and cost-intense identification steps done on contaminants may possibly be avoided. By use of Gram-diff PCR bacterial DNA was also detected in six culture-negative BC. The significance of the detected DNA in four samples cannot be definitely determined. Although all working steps were performed in different PCR-safety cabinets and separated in different rooms, a contamination of samples is not fully excluded. Nevertheless, detection of Enterococcus faecium DNA in one patient with intra-abdominal E. feacium infection appears clinically significant and stresses the advantages of real-time PCR analysis using the described algorithm. By Candida-specific real-time PCR, one isolate of Candida parapsilosis has been detected in the BC 35 h prior to positive signalling of the BACTEC system. Due to the slow growth and low concentration of yeasts, sampling of several BC are needed in patients with fungemia and BC are mostly reported positive at day 2e4 of incubation (31,32 and own unpublished data). Therefore, real-time PCR detection of Candida in conjunction with the new DNA preparation tool MolYsis may significantly improve detection of fungemia. In conclusion, the real-time Gram-differentiating and Candida-specific PCR assays in conjunction with pretreatment of MolYsis enables earlier detection of bacteria and
yeasts in BC of patients with bacteremia and fungemia but the procedure seems to be less sensitive than BC diagnostic. With the described procedure we reached a sensitivity of at least 400 CFU per ml blood culture. In contrast, blood cultures have a theoretical detection limit of one viable cell inoculated in one blood culture bottle. Therefore and because of the fact that conventional cultivation of BC enables identification and susceptibility testing of all pathogens blood cultures are still indispensable. In addition, it has to be considered that the real-time PCR approach is more labour-intensive and more expensive than BC diagnostics. Altogether for DNA preparation and PCR analysis of one patient the costs for materials and reagent per sample sum up to V15. However, it may be a valuable supplemental tool for patients with suspicion of infection with slow-growing pathogens like Brucella spp. and Candida spp. and for patients with serious clinical condition aiming at a rapid identification of the pathogen, like suspicion of meningococcal meningitis or severely immunosuppressed patients. Nevertheless, by using BC media for eubacterial PCR analysis is has to be considered that BC media can contain residual bacterial DNA that may be a reason for false-positive PCR results. The new DNA preparation tool MolYsis proved effective in removing PCR-inhibiting substances in BC, and was superior in sensitivity compared to other commercial kits. Due to the use of DNAse prior to bacterial DNA isolation it
Molecular identification of blood cultures theoretically removes human DNA and DNA from damaged or broken bacteria completely. Thus, it has the same advantages or rather disadvantages like BC diagnostics since only viable bacteria can be identified by BC. MolYsis may also be used for pretreatment of other clinical samples like whole blood samples. Real-time PCR analysis of native blood samples instead of BC may further accelerate detection of pathogens in septic patients. Despite the very low concentration of bacterial and fungal DNA in human blood from patients suffering from bacteraemia or fungemia33 real-time PCR has already shown some promising results.23,34 Further studies are underway to determine whether pretreatment with the MolYsis tool also improves direct detection of pathogenic bacteria and fungi in native blood samples.
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Acknowledgement This work was supported by a ProInno II grant from the German Ministry of Economy and Labour.
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References 1. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 2003;348(16):1546e54. 2. Engel C, Brunkhorst FM, Bone HG, Brunkhorst R, Gerlach H, Grond S, et al. Epidemiology of sepsis in Germany: results from a national prospective multicenter study. Intensive Care Med 2007;33(4):606e18. 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):309e17. 4. Annane D, Bellissant E, Cavaillon JM. Septic shock. Lancet 2005;365(9453):63e78. doi:10.1016/S0140-6736(04)17667-8. 5. Harbarth S, Garbino J, Pugin J, Romand JA, Lew D, Pittet D. Inappropriate initial antimicrobial therapy and its effect on survival in a clinical trial of immunomodulating therapy for severe sepsis. Am J Med 2003;115(7):529e35. doi:10.1016/j.amjmed.2003.07.005. 6. Kang CI, Kim SH, Park WB, Lee KD, Kim HB, Kim EC, et al. Bloodstream infections caused by antibiotic-resistant gramnegative bacilli: risk factors for mortality and impact of inappropriate initial antimicrobial therapy on outcome. Antimicrob Agents Chemother 2005;49(2):760e6. 7. Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med 2006;34(6):1589e96. 8. Micek ST, Lloyd AE, Ritchie DJ, Reichley RM, Fraser VJ, Kollef MH. Pseudomonas aeruginosa bloodstream infection: importance of appropriate initial antimicrobial treatment. Antimicrob Agents Chemother 2005;49(4):1306e11. 9. Weinstein MP, Mirrett S, Reimer LG, Wilson ML, SmithElekes S, Chuard CR, et al. Controlled evaluation of BacT/Alert standard aerobic and FAN aerobic blood culture bottles for detection of bacteremia and fungemia. J Clin Microbiol 1995;33(4):978e81. 10. Wilson ML, Weinstein MP, Mirrett S, Reimer LG, Feldman RJ, Chuard CR, et al. Controlled evaluation of BacT/alert standard anaerobic and FAN anaerobic blood culture bottles for the detection of bacteremia and fungemia. J Clin Microbiol 1995; 33(9):2265e70. 11. Cleven BE, Palka-Santini M, Gielen J, Meembor S, Kronke M, Krut O. Identification and characterization of bacterial
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
pathogens causing bloodstream infections by DNA microarray. J Clin Microbiol 2006;44(7):2389e97. Jansen GJ, Mooibroek M, Idema J, Harmsen HJ, Welling GW, Degener JE. Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes. J Clin Microbiol 2000;38(2):814e7. Kempf VA, Trebesius K, Autenrieth IB. Fluorescent In situ hybridization allows rapid identification of microorganisms in blood cultures. J Clin Microbiol 2000;38(2):830e8. Carroll KC, Leonard RB, Newcomb-Gayman PL, Hillyard DR. Rapid detection of the staphylococcal mecA gene from BACTEC blood culture bottles by the polymerase chain reaction. Am J Clin Pathol 1996;106(5):600e5. Kurupati P, Chow C, Kumarasinghe G, Poh CL. Rapid detection of Klebsiella pneumoniae from blood culture bottles by realtime PCR. J Clin Microbiol 2004;42(3):1337e40. Wellinghausen N, Wirths B, Franz AR, Karolyi L, Marre R, Reischl U. Algorithm for the identification of bacterial pathogens in positive blood cultures by real-time LightCycler polymerase chain reaction (PCR) with sequence-specific probes. Diagn Microbiol Infect Dis 2004;48(4):229e41. doi: 10.1016/j.diagmicrobio.2003.11.005. Peters RP, van Agtmael MA, Danner SA, Savelkoul PH, Vandenbroucke-Grauls CM. New developments in the diagnosis of bloodstream infections. Lancet Infect Dis 2004;4(12):751e60. doi:10.1016/S1473-3099(04)01205-8. Fredricks DN, Relman DA. Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate. J Clin Microbiol 1998;36(10): 2810e6. Greisen K, Loeffelholz M, Purohit A, Leong D. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol 1994;32(2):335e51. Bestmann L. A micromachined total nucleic acid analysis system with integrated DNA isolation, mix preparation, on-chip real-time PCR and melting curve analysis. Doctoral thesis, Institute of Clinical Chemistry, University Hospital of Zurich; 2005. Costa AM, Kay I, Palladino S. Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction. Diagn Microbiol Infect Dis 2005;51(1):13e7. doi:10.1016/j.diagmicrobio.2004.08.014. Einsele H, Hebart H, Roller G, Loffler J, Rothenhofer I, Muller CA, et al. Detection and identification of fungal pathogens in blood by using molecular probes. J Clin Microbiol 1997; 35(6):1353e60. Klingspor L, Jalal S. Molecular detection and identification of Candida and Aspergillus spp. from clinical samples using real-time PCR. Clin Microbiol Infect 2006;12(8):745e53. Cogswell FB, Bantar CE, Hughes TG, Gu Y, Philipp MT. Host DNA can interfere with detection of Borrelia burgdorferi in skin biopsy specimens by PCR. J Clin Microbiol 1996;34(4):980e2. Navarro E, Escribano J, Fernandez J, Solera J. Comparison of three different PCR methods for detection of Brucella spp in human blood samples. FEMS Immunol Med Microbiol 2002; 34(2):147e51. Al Soud WA, Jonsson LJ, Radstrom P. Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol 2000;38(1):345e50. Al Soud WA, Radstrom P. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 2001;39(2):485e93. Queipo-Ortuno MI, Tena F, Colmenero JD, Morata P. Comparison of seven commercial DNA extraction kits for the recovery of Brucella DNA from spiked human serum samples using real-time PCR. Eur J Clin Microbiol Infect Dis 2008;27(2): 109e14.
316 29. van der Zee A, Peeters M, de Jong C, Verbakel H, Crielaard JW, Claas EC, et al. Qiagen DNA extraction kits for sample preparation for Legionella PCR are not suitable for diagnostic purposes. J Clin Microbiol 2002;40(3):1126. 30. Klaschik S, Lehmann LE, Raadts A, Book M, Hoeft A, Stuber F. Real-time PCR for detection and differentiation of gram-positive and gram-negative bacteria. J Clin Microbiol 2002; 40(11):4304e7. 31. Cockerill III FR, Wilson JW, Vetter EA, Goodman KM, Torgerson CA, Harmsen WS, et al. Optimal testing parameters for blood cultures. Clin Infect Dis 2004;38(12):1724e30.
S. Gebert et al. 32. Lee A, Mirrett S, Reller LB, Weinstein MP. Detection of bloodstream infections in adults: how many blood cultures are needed? J Clin Microbiol 2007;45(11):3546e8. 33. Kreger BE, Craven DE, Carling PCR, McCrabe R. Gram-negative bacteremia. III. Reassessment of etiology, epidemiology and ecology in 612 patients. Am J Med 1980;68(3):332e43. 34. Peters RP, van Agtmael MA, Gierveld S, Danner SA, Groeneveld AB, Vandenbroucke-Grauls CM, et al. Quantitative detection of Staphylococcus aureus and Enterococcus faecalis DNA in blood to diagnose bacteremia in patients in the intensive care unit. J Clin Microbiol 2007;45(11):3641e6.