KR and ES are minimal shareholders of SIRS-Lab GmbH. FB received a travel grant from SIRS-Lab GmbH. MB received consulting fees from SIRS-Lab GmbH. ML was employee of SIRS-Lab GmbH during the conduct of the study, where he had the scientific responsibility for VYOO. ML is now director of Moldiax GmbH, where he has no competing interest regarding this study. SIRS-Lab GmbH has supplied the VYOO®-Kits and personnel to run the PCR. SIRS-Lab GmbH has also financed a prospective study which took place after this study was completed. The study goals and feasibility issues have been discussed with SIRS-Lab GmbH during the preparation phase of the study. ES holds a patent for the LOOXTER technique where prokaryotic DNA can be enriched in a way that a simple PCR protocol (VYOO) can detect such DNA. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Conceived and designed the experiments: FB KR MB. Performed the experiments: SS. Analyzed the data: FB AK MP. Contributed reagents/materials/analysis tools: ML. Wrote the paper: FB AK NR MB. Interpretation of PCR and blood culture results: ES SS. Organization of PCR setup and interpretation of PCR results: ML. Patient recruitment: FB NR KR. Clinical arbitrators: AK MP.
Current address: Moldiax GmbH, Jena, Germany
Treatment of septic shock relies on appropriate antimicrobial therapy. Current culture based methods deliver final results after days, which may delay potentially lifesaving adjustments in antimicrobial therapy. This study was undertaken to compare PCR with blood culture results under routine conditions regarding 1. impact on antimicrobial therapy, and 2. time to result, in patients with presumed sepsis.
This was an observational study in a 50 beds ICU of a university hospital. In 245 patients with suspected sepsis, 311 concomitant blood cultures and blood for multiplex PCR (VYOO®) were obtained. 45 of 311 blood cultures (14.5%) and 94 of 311 PCRs (30.1%) were positive. However, blood culture or microbiological sampling from the presumed site of infection rarely confirmed PCR results and vice versa. Median time to positivity and interquartile range were 24.2 (18.0, 27.5) hours for the PCR and 68 (52.2, 88.5) hours for BC (p<0.01). PCR median time to result was dependent on technician availability (53.5 hours on Saturdays, 7.2 hours under optimal logistic conditions). PCR results showed good correlation with procalcitonin (p<0.001). In 34% of patients with positive PCRs antimicrobial therapy was considered inadequate according to assessment of clinical arbitrators including 5 patients with vancomycin-resistant enterococci (VRE), 3 cases with multiresistant staphylococci, and 4 patients with fungi.
The results of this observational study support the hypothesis that PCR results are available faster, are more frequently positive, and may result in earlier adjustment of antimicrobial therapy. However, shorter time to result can only be fully exploited when the laboratory is adequately staffed for a 24 hour/7 day service, or when point of care/automated assay systems become available.
Sepsis is a leading cause of death in hospitalized patients worldwide, with a continuous increase in incidence of approximately 5–10% per year
The current guideline-driven strategy for empirical antimicrobial therapy of underlying pathogens is broad spectrum antibiotics with anti-pseudomonal acitivity
Faster microbiological workup could reduce the possibly fatal consequences of inappropriate antimicrobial therapy
Culture-independent molecular biology based techniques, such as Polymerase Chain Reaction (PCR), may offer a solution to the problems associated with culture-dependent methods. Recent studies from various groups with a commercially available multiplex PCR assay for pathogen detection were promising, with the rate of positivity being about two-fold higher than with blood culture
The primary objective of the current study was to compare the performance of PCR to detect microbial DNA with pathogen detection by blood culture and conventional microbiological results in patients with presumed sepsis in daily clinical practice. Data regarding the impact of PCR-based microbial detection on the clinical course of patients are very limited. In one prospective randomized study, patients who had received allogeneic stem-cell transplantation showed a lower 30 day mortality rate when treated with liposomal amphotericin B according to a PCR-based algorithm than when treated empirically
In principle, PCR technology currently delivers results within 6 to 8 hours. However, such a short time requires optimal logistic and technical conditions and has never been challenged in the routine clinical setting. A further goal of this study was thus to compare the turn-around times of PCR and blood culture from blood sampling until release of final result to the treating physician.
The study was approved by the institutional ethics committee of the Friedrich-Schiller-University Jena, which waived the need for informed consent due to the observational nature of the study. Patient data were de-identified by consecutive numbers and analysed anonymously. Only study personnel bound to confidentiality were able to reidentify data entries for quality control.
This study was an observational trial comparing the results of PCR with blood culture and microbiological results from the presumed source of sepsis. In critically ill patients treated on an interdisciplinary ICU between May 2009 and June 2010, blood for PCR was obtained in parallel if a blood culture was taken for suspected sepsis. Patients less than 18 years old were excluded from the study.
20 ml blood were taken by sterile venous puncture and distributed equally for conventional blood cultures into aerobic and anaerobic media by the treating physician. In addition to each pair of cultures, 10 ml of EDTA blood were taken for PCR analysis. Consecutive samples were taken if further blood cultures were deemed necessary due to the clinical course of the patient.
The multiplex PCR-based assay used (VYOO®, SIRS-Lab GmbH, Jena) combines a bead-based mechanical lysis protocol, a DNA preparation that includes the separation and relative enrichment of microbial DNA from background human DNA, and a multiplex PCR protocol
Detailed procedures of the sample preparation for the VYOO® assay have been described in detail elsewhere
Pairs of BCs were incubated at 37°C and monitored for 8 days. Isolated micro-organisms and their susceptibilities were determined by standard methods and criteria
The host response was monitored in all patients by leukocyte count, procalcitonin (PCT), and C-reactive protein (CRP). Results of all microbiological specimens obtained three days before and after the PCR sample were analysed and correlated with BC and PCR results. An independent infectious diseases consultant and an ICU consultant served as clinical arbitrators and reviewed appropriateness of antimicrobial therapy in patients with a positive PCR and assessed the impact of this result. The classification of Cohen and Calandra was used to define microbiologically confirmed, probable and possible infection
Data are considered to be not normally distributed. Therefore, discrete variables are expressed as percentages and continuous variables as median, 25th and 75th percentiles, unless stated otherwise. Frequencies were compared by the chi-square test, and differences of medians by the Mann-Whitney test. Biomarker plasma levels according to blood culture and PCR results were compared by the Kruskal-Wallis rank sum test. The Mann-Whitney test with Bonferroni correction was applied as a post-hoc test when the Kruskal-Wallis test delivered a significant result. Duration until positive PCR or blood culture was analysed as a time to event analysis by using Kaplan-Meier Curves. The Log-test served to detect differences between curves. Data processing and statistical analysis was done with R version 2.10.0
245 patients were included into this study, all of whom were treated for presumed sepsis. 336 blood samples for PCR were obtained from these patients. For 25 of the PCRs, the concomitant blood was not obtained or did not reach the laboratory, resulting in 311 complete pairs of blood culture with concomitant PCR. Demographic data are shown in
Survivor | Non-survivor | All | p-value | |
(N = 161) | (N = 84) | (N = 245) | ||
|
60.9% (98) | 63.1% (53) | 61.6% (151) | 0.734 |
|
67.0 (55.0; 74.0) | 67.5 (55.0; 74.0) | 67.0 (55.0; 74.0) | 0.791 |
|
26.2 (23.5; 30.5) | 25.4 (23.4; 28.7) | 26.1 (23.4; 30.1) | 0.100 |
|
0.289 | |||
General surgery | 44.1% (71) | 46.4% (39) | 44.9% (110) | |
Heart/thoracic surgery | 29.2% (47) | 32.1% (27) | 30.2% (74) | |
Neurosurgery | 11.8% (19) | 6.0% (5) | 9.8% (24) | |
Other | 14.9% (24) | 15.5% (13) | 15.1% (37) | |
|
2 (1; 3) | 1 (3; 4) | 2 (1; 4) | 0.130 |
|
17.0 (12.0; 21.0) | 20.5 (15.8; 26.2) | 18.0 (13.0; 23.0) | <0.001 |
|
38.0 (31.0; 46.0) | 50.0 (40.8; 58.2) | 42.0 (33.0; 52.0) | <0.001 |
|
8.0 (3.0; 19.0) | 12.0 (4.0; 24.2) | 9.0 (3.0; 21.0) | 0.119 |
|
32.5 (21.8; 50.2) | 20.0 (12.8; 35.0) | 28.0 (17.0; 46.0) | <0.001 |
Data are given as frequencies or medians (25th; 75th percentiles); p-values describe statistical differences between survivors and non-survivors; BMI: Body Mass Index; SAPSII: Simplified Acute Physiology score; LOS: length of stay.
311 pairs of blood cultures and PCRs were obtained. 45 (14.5%) of the blood cultures (BC) were positive, compared with 94 (30.2%) positive PCRs (p<0.001). 27 (8.7%) samples showed a concordantly positive result in both tests, while 199 (64.0%) samples were negative in both tests. There were 67 (21.5%) positive PCRs despite negative BC, while 18 (5.8%) PCRs remained negative despite a positive BC. Thus, sensitivity of the PCR to detect culture positive bacteremia was 0.6 (95% confidence interval: 0.44–0.74) and a corresponding specificity of 0.75 (0.69–0.8).
Pathogen | BC | PCR | recovery rate |
|
16.0% (8) | 20.7% (24) | 5 |
|
6.0% (3) | 19.8% (23) | 3 |
CoNS | 30.6% (15) | 17.2% (20) | 4 |
|
14.0% (7) | 10.3% (12) | 4 |
|
0 | 5.2% (6) | – |
|
4.0% (2) | 4.3% (5) | 1 |
|
2.0% (1) | 4.3% (5) | 1 |
|
0 | 4.3% (5) | – |
12.0% (6) | 4.3% (5) | 1 | |
|
2.0% (1) | 4.3% (5) | 1 |
|
0 | 1.7% (2) | – |
|
0 | 0.9% (1) | – |
|
0 | 0.9% (1) | – |
|
0 | 0.9% (1) | – |
|
0 | 0.9% (1) | – |
Other Gram negative bacteria |
6.0% (3) | 0 | 0 |
Other Gram positive bacteria |
6.0% (3) | 0 | 0 |
Recovery rate shows number of microorganisms of the concomitant blood culture also detected by the PCR. Only blood cultures with a concomitant PCR and vice versa are enlisted. Overall recovery rate is 40%. CoNS: Coagulase negative staphylococci.
: The assay detects a conserved gene contained by all fungi, reported as ‘pan-fungi’ without further classification of the species.
: Pathogens not on the VYOO® target list; Gram negative: bacteroides ovatus: 2, enterococcus avium: 1; Gram positive: unspecified cocci: 2, streptococcus infantarius: 1.
PCR positive patients had higher procalcitonin (PCT) levels than PCR negative and blood culture negative patients (
PCR−/BC−: n = 199, PCR−/BC+: n = 18, PCR+/BC−: n = 67. PCR+/BC+: n = 27. Pathogens in the blood culture in the ‘PCR− BC+’ subgroup: CoNS: 8,
On the sampling day, patients positive for CoNS in the BC had median CRP levels of 156 (107; 204) mg/l and PCT levels of 1.4 (0.7; 6.0) ng/ml. These were not different from the levels of CRP 168 (114; 217, p = 0.794) mg/l and PCT 2.9 (1.6; 8.0; p = 0.397) ng/ml on days with positive PCRs for CoNS. Presence of a fungal amplicon compared to presence of a bacterial amplicon in the PCR was not associated with differences in leukocyte count, CRP, or PCT.
Irrespective of the BC taken concomitantly with the PCR, 312 pathogens were detected by the microbiological sample taken from the presumed site of infection (
Venn diagram showing the number of microorganisms found in the PCR, the concomitant blood culture, and the microbiological specimens from the presumed site of infection. Numbers in the overlapping areas reflect microorganisms detected with more than one technique simultaneously. Most of the pathogens were not confirmed with any other technique. Unconfirmed results from the culture based methods are partly not on the PCR target list. Results where the same pathogen was detected more than once in the same specimen were removed from this diagram.
Pathogen | BC | recovery rate |
|
15.6% (39) | 1 |
|
10.7% (30) | 1 |
|
9.3% (26) | 5 |
|
7.5% (21) | 0 |
|
6.8% (19) | 5 |
Yeasts (not further specified) |
6.0% (19) | 0 |
|
5.0% (14) | 3 |
|
4.3% (12) | 2 |
|
3.9% (11) | 1 |
|
3.6% (10) | 0 |
|
3.6% (10) | 2 |
|
3.2% (9) | 0 |
|
3.2% (9) | 0 |
|
2.1% (6) | 1 |
|
1.8% (5) | 0 |
|
1.4% (4) | 1 |
|
0.7% (2) | 0 |
|
0.7% (2) | 0 |
|
0.4% (1) | 0 |
|
0.4% (1) | 0 |
Other Gram positive bacteria |
6.4% (16) | 0 |
Other Gram negative bacteria |
4.6% (13) | 0 |
Other fungi |
1.8% (5) | 0 |
Recovery rate shows number of microorganisms of the microbiological samples from presumed site of infection within 3 days before and after the PCR sampling also detected by the PCR. Same pathogens found in different samples of the same patient are only counted once. The overall recovery rate is 7.9%. CoNS: Coagulase negative staphylococci.
: The assay detects a conserved gene contained by all fungi, reported as ‘pan-fungi’ without further classification of the species.
: Pathogens not on the VYOO® target list.
Median time to positivity was significantly shorter for PCR (24.2 [interquartile range 18.0–27.5] hours) than for blood culture (68 [52.2–88.5] hours, p<0.001) (
Blood culture and PCR, which remain negative, have been excluded from this analysis. Median time to result was 25.2 (interquartile range: 18.2–32.2) hours for the PCR versus 189.5 (178.6–197.4) hours for the blood culture.
In order to assess the impact of positive PCR results on the physician's decision to change antimicrobial therapy, all charts were reassessed by an independent infectious diseases consultant and an ICU consultant. In opinion of the arbitrators, 94% of the positive PCR results revealed an important finding which is not suggestive of contamination and should be considered as a true pathogen. In 34% of patients with positive PCR, retrospective evaluation revealed that initial antimicrobial therapy did not cover the retrospectively suspected causative. This included fungi in 4 cases, vancomycin resistant enterococci in 5 cases, and multiresistant staphylococci in 3 cases. The treating physician changed antimicrobial in 38% of the positive PCRs. In patients, where the treating physicians changed antimicrobial therapy because of the PCR result, serum c-reactive protein (CRP) dropped from 219.5 (135.2; 250.8) to 126.0 (104.5; 198.5) mg/l (p = 0.03) within 4 days after change of antimicrobial therapy, and serum procalcitonin (PCT) dropped from 5.5 (2.3; 24.5) to 2.9 (2.0; 8.7) ng/ml (p = 0.01). The arbitrators also assessed that 24.2% of the PCR results would suggest a possible de-escalation.
In this study, we assessed the performance of culture-independent PCR-based detection of pathogens compared with blood culture regarding impact on antimicrobial therapy and turnaround time. We confirmed a significantly higher rate of positivity in PCR than in blood culture but the concordance regarding positive results between culture based methods was poor. In about 34% of the positive PCRs, the initial antimicrobial treatment did not cover the organism detected in the PCR, and was adjusted by the treating physician. Time to positive result was significantly shorter for PCR (PCR 24.2 hours) compared to 68.8 hours for the blood culture but exceeded the frequently suggested 6–8 hours due to limitations in staff availability. The validity of positive PCR results not confirmed by BC was suggested by elevated biomarkers of infection, i.e. C-reactive protein (CRP) and procalcitonin (PCT). In particular PCT has been demonstrated lately to be a good marker for guidance of antibiotic therapy
Our data are consistent with the notion that PCR technology cannot replace blood culture but may add valuable diagnostic information in critically ill patients with suspected sepsis. In this respect, the currently used test (VYOO®, SIRS-Lab GmbH, Jena, Germany) yielded similar findings to those of another assay in a recent multicenter study
Early adequate antibiotic therapy is vital in these patients, since both a delay in therapy and inadequate treatment are associated with an increased risk of death
Although the study was not designed to evaluate the impact of early antimicrobial adjustment on patient outcome, our findings point to potential advantages of early adjustment. In 13 patients, the PCR detected a pathogen not covered by the initial antimicrobial therapy, earlier than blood culture or the microbiological specimen from the presumed site of infection. These included patients with multi-resistant pathogens, where even a treatment according to current guidelines is inadequate
Some of the PCR results yielded Gram-positive pathogens such as enterococci. Contribution of enterococci to infection is difficult to assess. This is especially true for ICU patients where little data about the involvement of these bacteria are available. Recently, the importance of the involvement of enterococci in severe intraabdominal infections has been documented
Changes in antimicrobial therapy also included the addition of antifungal therapy in some cases. The possible contribution of PCR to diagnosing invasive candidiasis has recently been reported
Blood cultures are a crucial step in the diagnostic work-up, and are seen as mandatory in septic shock patients
One of the advantages of PCR is the short time to result compared with culture-based methods. Theoretically, PCR results may be available within 6–8 hours, but this is highly dependent on staff availability. In this study, where PCR was applied in a real clinical setting, median time to result was 23.4 hours on weekdays. Ideal setup conditions could only be reached during weekdays when blood sampling was conducted in the early morning hours. No technician was available to run the PCR at weekends or during the nights. Thus, blood samples drawn on the weekend were analysed the following Monday, resulting in a time to result close to that of blood culture. Therefore, PCR results may be available within one working day only if the laboratory is staffed appropriately. These results need to be taken into account when considering the cost effectiveness and clinical application of PCR technology. In order to reap the full benefits of PCR-based pathogen detection technologies, adjustments to laboratory staffing and workflows might be necessary. Completely automated systems from extraction to read-out although currently not available may overcome this limitation.
Data addressing the clinical value of a PCR result are still scarce
Many microorganisms detected in the PCR remained unconfirmed when culture-based pathogen detection was completed. The biological plausibility of PCR results was addressed in a previous study of patients with septic shock. Positive PCR results were associated with higher serum concentrations of biomarkers of inflammation such as interleukin 6 and procalcitonin
Although PCR delivers a higher rate of positivity than blood culture, PCR often remains negative, even in severe sepsis. This is in line with previous studies in this patient population
Interpretation of the study is limited by its observational nature. Although it was possible to describe potential benefits of including PCR results in the treatment decisions, we cannot conclude that PCR driven treatment decisions improve antimicrobial therapy in a way that infection is controlled earlier or outcome is improved. Such a conclusion would only be possible after a prospective randomized trial.
In conclusion, the results of this observational study support the hypothesis that PCR results are available faster, are more frequently positive, and may result in earlier adjustment of antimicrobial therapy. However, our data point to the fact that this shorter time to result can only be fully exploited when the laboratory is adequately staffed for a 24 hour/7 day service, or when point of care systems become available. Due to its potential to improve outcome of patients with life-threatening infection, the clinical utility and cost effectiveness of PCR should now be tested in adequately powered randomized controlled clinical trials.
This study would not have been possible without the data entry and sample handling by the study nurses Petra Bloos, Anke Braune, Daniela Fergen, Almut Noack, and Ulrike Redlich, as well as the VYOO® technical processing by Dana Schmidt, Martina Pick, Viviane Sternkopf, Sandra Hufnagel, and Kristin Wessel.