Development and first evaluation of a novel multiplex real-time PCR on whole blood samples for rapid pathogen identification in critically ill patients with sepsis (original) (raw)
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Journal of Clinical Microbiology, 2010
Sepsis is caused by a heterogeneous group of infectious etiologies. Early diagnosis and the provision of appropriate antimicrobial therapy correlate with positive clinical outcomes. Current microbiological techniques are limited in their diagnostic capacities and timeliness. Multiplex PCR has the potential to rapidly identify bloodstream infections and fill this diagnostic gap. We identified patients from two large academic hospital emergency departments with suspected sepsis. The results of a multiplex PCR that could detect 25 bacterial and fungal pathogens were compared to those of blood culture. The results were analyzed with respect to the likelihood of infection, sepsis severity, the site of infection, and the effect of prior antibiotic therapy. We enrolled 306 subjects with suspected sepsis. Of these, 43 were later determined not to have infectious etiologies. Of the remaining 263 subjects, 70% had sepsis, 16% had severe sepsis, and 14% had septic shock. The majority had a definite infection (41.5%) or a probable infection (30.7%). Blood culture and PCR performed similarly with samples from patients with clinically defined infections (areas under the receiver operating characteristic curves, 0.64 and 0.60, respectively). However, blood culture identified more cases of septicemia than PCR among patients with an identified infectious etiology (66 and 46, respectively; P ؍ 0.0004). The two tests performed similarly when the results were stratified by sepsis severity or infection site. Blood culture tended to detect infections more frequently among patients who had previously received antibiotics (P ؍ 0.06). Conversely, PCR identified an additional 24 organisms that blood culture failed to detect. Real-time multiplex PCR has the potential to serve as an adjunct to conventional blood culture, adding diagnostic yield and shortening the time to pathogen identification.
Improved detection of blood stream pathogens by real-time PCR in severe sepsis
Intensive Care Medicine, 2010
Objective Evaluation of the technical and diagnostic feasibility of commercial multiplex real-time polymerase chain reaction (PCR) for detection of blood stream infections in a cohort of intensive care unit (ICU) patients with severe sepsis, performed in addition to conventional blood cultures. Design Dual-center cohort study. Setting Surgical ICU of two university hospitals. Patients and participants One hundred eight critically ill patients fulfilling the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) severe sepsis criteria were included. Interventions None. Measurements and results PCR results obtained in 453 blood samples from 108 patients were compared with corresponding blood culture results. PCR resulted in a twofold higher positivity rate when compared with conventional blood culture (BC) testing (114 versus 58 positive samples). In 40 out of 58 PCR positive assays the results of the corresponding blood cultures were identical to microorganisms detected by PCR. In 18 samples PCR and BC yielded discrepant results. Compared with conventional blood culture the sensitivity and specificity of PCR was 0.69 and 0.81, respectively. Further evaluation of PCR results against a constructed gold standard including conventional microbiological test results from other significant patient specimen (such as bronchio-alveolar lavage fluid, urine, swabs) and additionally generated clinical and laboratory information yielded sensitivity of 0.83 and specificity of 0.93. Conclusions Our cohort study demonstrates improved pathogen detection using PCR findings in addition to conventional blood culture testing. PCR testing provides increased sensitivity of blood stream infection. Studies addressing utility including therapeutic decision-making, outcome, and cost-benefit following diagnostic application of PCR tests are needed to further assess its value in the clinical setting.
Molecular detection of bloodstream pathogens in critical illness
2012
Background: Critically ill patients are at particular risk of developing bloodstream infection. Such infections are associated with the development of sepsis, leading to a marked increase in mortality rate. Early detection of the causative organism and appropriate antibiotic treatment are therefore critical for optimum outcome of patients with nosocomial infection. Current infection diagnosis is based on standard blood culture techniques. However, microbiological culture has a number of limitations, not least that it takes several days to confirm infection and is therefore not useful in directing the early treatment with antibiotics. New techniques based on the detection of pathogen DNA using real-time polymerase chain reaction (PCR) technology have the potential to address these limitations but their clinical utility is still to be proved.Objectives: Develop and evaluate novel PCR-based approaches to bloodstream infection diagnosis in critical illness based on detection and identif...
Clinical Microbiology and Infection, 2009
Severe sepsis is increasingly a cause of death. Rapid and correct initial antimicrobial treatment reduces mortality. The aetiological agent(s) cannot always be found in blood cultures (BCs). A novel multiplex PCR test (SeptiFast (alpha version)) that allows identification of 20 bacterial and fungal species directly from blood was used, comparatively with BC, in a multicentre trial of patients with suspected bacterial or fungal sepsis. Five hundred and fifty-eight paired samples from 359 patients were evaluated. The rate of positivity was 17% for BC and 26% for SeptiFast. Ninety-six microorganisms were isolated with BC, and 186 microorganisms were identified with SeptiFast; 231 microrganisms were found by combining the two tests. Of the 96 isolates identified with BC, 22 isolates were considered to be contaminants. Of the remaining 74 non-contaminant BC isolates available for comparison with SeptiFast, 50 were identified as a species identical to the species identified with SeptiFast in the paired sample. Of the remaining 24 BC isolates for which the species, identified in the BC, could not be detected in the paired SeptiFast sample, 18 BC isolates were identified as a species included in the SeptiFast master list, and six BC isolates were identified as a species not included in the SeptiFast master list. With SeptiFast, 186 microorganisms were identified, 12 of which were considered to be contaminants. Of the 174 clinically relevant microorganisms identified with SeptiFast, 50 (29%) were detected by BC. More than half of the remaining microorganisms identified with SeptiFast (but not isolated after BC) were also found in routine cultures of other relevant samples taken from the patients. Future clinical studies should assess whether the use of SeptiFast is of significant advantage in the detection of bloodstream pathogens.
Quantitative multiplexed-tandem PCR for direct detection of bacteraemia in critically ill patients
Pathology, 2017
Culture remains the gold standard for diagnosis of blood stream infections (BSI), but its clinical utility is limited by slow turnaround times. Here we describe a method for rapid quantitative detection of bacterial DNA directly extracted from whole blood using a multiplexed tandem real-time PCR (MT-PCR) assay targeting Staphylococcus, Streptococcus, Pseudomonas, Enterococcus and Enterobacteriaceae 16S rDNA genes. Results were available less than 3.5 hours after blood collection with all five bacterial targets having limits of detection between 10 1 and 10 3 CFU/mL. A small-scale clinical evaluation of the assay using blood samples collected from 15 patients admitted to the Intensive Care Unit at our institution demonstrated 93.3% (14/15) concordance between MT-PCR and blood culture when detection of persistent bacterial DNAemia by MT-PCR was considered a true result. Further evaluation with clinical samples is needed; however, this method has potential as an effective rule-in diagnostic tool for bacteraemic sepsis and septic shock.
The Journal of Infection in Developing Countries, 2014
Introduction: Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus spp., Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumanii have been found to be the most prevalent bacteremia-causing bacteria in patients with septicemia. Early detection of bloodstream infection (BSI) is crucial in the clinical setting. A multiplex PCR method for identification of these agents in clinical samples has been developed in parallel by conventional microbiological methods. Methodology: The target genes selected for each of the organisms were very specific for designing primers. Design of primers was done using Mega4, Allel ID6, Oligo6, and Oligo analyzer software. The test comprises a universal PCR from the 16S rDNA gene and multiplex PCR from the rpoB, gyrA, sss, and chromosome X (as an internal control). Results: The sensitivity and specificity for universal PCR and multiplex PCR in comparison with BC were 83.87% and 91.58%, and 74.19% and 91.58%, respectively. The positive predictive value (PPV) and the negative predictive value (NPV) for these two PCRs were 76.47% and 94.57%, and 74.19% and 91.58%, respectively. PCR failed to identify bacteria which were found conventionally in only 3.96% and 6.34% of the cases by universal and multiplex PCR (mostly bacteria not included in the PCR cassette). In 6.34% of the cases, multiplex PCR afforded identification of bacteria, but BC showed no bacteria in the sample. Conclusions: The multiplex PCR approach facilitates the detection of bacteremia in blood samples within a few hours. Rapid detection of bacteria by multiplex PCR appears to be a valuable tool, allowing earlier pathogen-adopted antimicrobial therapy in critically ill patients.
PLOS ONE, 2016
The Magicplex Sepsis Real-time Test (MST) is a commercial multiplex PCR that can detect more than 90 different pathogens in blood, with an analysis time of six hours. The aim of the present study was to evaluate this method for the detection of bloodstream infection (BSI). An EDTA whole blood sample for MST was collected together with blood cultures (BC) from patients with suspected sepsis at the Emergency Department of a university hospital. Among 696 study patients, 322 (46%) patients were positive with at least one method; 128 (18%) were BC positive and 268 (38%) were MST positive. Considering BC to be the gold standard, MST had an overall sensitivity of 47%, specificity of 66%, positive predictive value (PPV) of 23%, and a negative predictive value of 87%. Among the MST positive samples with a negative BC, coagulase-negative staphylococci (CoNS) and species that rarely cause community-acquired BSI were frequently noted. However, the quantification cycle (Cq) values of the MST+/BC-results were often high. We thus hypothesized that the performance of the MST test could be improved if the Cq cutoff level was adjusted downwards. With a lower Cq cutoff value, i.e. 6.0 for Staphylococcus species and 9.0 for all other species, the number of MST positive cases decreased to 83 (12%) and the overall sensitivity decreased to 38%. However, the PPV increased to 59% and the specificity increased to 96%, as many MST positive results for CoNS and bacteria that rarely cause community-acquired BSI turned MST negative. In conclusion, our study shows that with a lower Cq cutoff value, the MST will detect less contaminants and findings with unclear relevance, but to the cost of a lower sensitivity. Consequently, we consider that a positive MST results with a Cq value above the adjusted cutoff should be interpreted with caution, as the result might be clinically irrelevant. In a correspondent way, quantitative results could probably be useful in the interpretation of positive results from other molecular assays for the detection of BSI.
BMC Infectious Diseases, 2009
Background: Timely identification of pathogens is crucial to minimize mortality in patients with severe infections. Detection of bacterial and fungal pathogens in blood by nucleic acid amplification promises to yield results faster than blood cultures (BC). We analyzed the clinical impact of a commercially available multiplex PCR system in patients with suspected sepsis. Methods: Blood samples from patients with presumed sepsis were cultured with the Bactec 9240™ system (Becton Dickinson, Heidelberg, Germany) and aliquots subjected to analysis with the LightCycler ® SeptiFast ® (SF) Test (Roche Diagnostics, Mannheim, Germany) at a tertiary care centre. For samples with PCR-detected pathogens, the actual impact on clinical management was determined by chart review. Furthermore a comparison between the time to a positive blood culture result and the SF result, based on a fictive assumption that it was done either on a once or twice daily basis, was made. Results: Of 101 blood samples from 77 patients, 63 (62%) yielded concordant negative results, 14 (13%) concordant positive and 9 (9%) were BC positive only. In 14 (13%) samples pathogens were detected by SF only, resulting in adjustment of antibiotic therapy in 5 patients (7,7% of patients). In 3 samples a treatment adjustment would have been made earlier resulting in a total of 8 adjustments in all 101 samples (8%). Conclusion: The addition of multiplex PCR to conventional blood cultures had a relevant impact on clinical management for a subset of patients with presumed sepsis. Background Early adequate antibiotic treatment improves the outcome of patients with sepsis [1-5]. Even if broad spectrum antibiotics are used empirically, adjustments of antimi-crobial therapy may be necessary. Generally adjustments are based on the results of positive blood or other cultures that are available after 8 to 48 hours [6].
Medical Microbiology and Immunology, 2008
is crucial in the clinical setting. Blood culture remains the gold standard for diagnosing BSI. Molecular diagnostic tools can contribute to a more rapid diagnosis in septic patients. Here, a multiplex real-time PCR-based assay for rapid detection of 25 clinically important pathogens directly from whole blood in <6 h is presented. Minimal analytical sensitivity was determined by hit rate analysis from 20 independent experiments. At a concentration of 3 CFU/ml a hit rate of 50% was obtained for E. aerogenes and 100% for S. marcescens, E. coli, P. mirabilis, P. aeruginosa, and A. fumigatus. The hit rate for C. glabrata was 75% at 30 CFU/ml. Comparing PCR identiWcation results with conventional microbiology for 1,548 clinical isolates yielded an overall speciWcity of 98.8%. The analytical speciWcity in 102 healthy blood donors was 100%. Although further evaluation is warranted, our assay holds promise for more rapid pathogen identiWcation in clinical sepsis.
BMJ Open, 2011
Background: There is growing interest in the potential utility of real-time PCR in diagnosing bloodstream infection by detecting pathogen DNA in blood samples within a few hours. SeptiFast is a multipathogen probe-based real-time PCR system targeting ribosomal DNA sequences of bacteria and fungi. It detects and identifies the commonest pathogens causing bloodstream infection and has European regulatory approval. The SeptiFast pathogen panel is suited to identifying healthcare-associated bloodstream infection acquired during complex healthcare, and the authors report here the protocol for the first detailed healthtechnology assessment of multiplex real-time PCR in this setting.