DNAemia Detection by Multiplex PCR and Biomarkers for Infection in Systemic Inflammatory Response Syndrome Patients

University of São Paulo, Brazil
PLoS ONE (Impact Factor: 3.23). 06/2012; 7(6):e38916. DOI: 10.1371/journal.pone.0038916
Source: PubMed


Fast and reliable assays to precisely define the nature of the infectious agents causing sepsis are eagerly anticipated. New molecular biology techniques are now available to define the presence of bacterial or fungal DNA within the bloodstream of sepsis patients. We have used a new technique (VYOO®) that allows the enrichment of microbial DNA before a multiplex polymerase chain reaction (PCR) for pathogen detection provided by SIRS-Lab (Jena, Germany). We analyzed 72 sepsis patients and 14 non-infectious systemic inflammatory response syndrome (SIRS) patients. Among the sepsis patients, 20 had a positive blood culture and 35 had a positive microbiology in other biological samples. Of these, 51.4% were positive using the VYOO® test. Among the sepsis patients with a negative microbiology and the non-infectious SIRS, 29.4% and 14.2% were positive with the VYOO® test, respectively. The concordance in bacterial identification between microbiology and the VYOO® test was 46.2%. This study demonstrates that these new technologies offer great hopes, but improvements are still needed.

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    • "Moreover, given the good specificity and NPV and the shorter turnaround time of the negative results, SeptiFast data might be combined with C-reactive protein, procalcitonin, and the results of previous microbiological findings in other compartments to take decisions (Lodes et al., 2012; Loonen et al., 2014). This will need specifically designed studies (Fitting et al., 2012) and might be most useful in clinical contexts in which the SeptiFast panel of pathogens is representative of the vast majority of findings, as is the case in kidney patients (Li and Chow, 2012). "
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    ABSTRACT: The LightCycler® SeptiFast Test was prospectively compared with the standard blood culture technique in a series of 86 kidney patients. The sensitivity of the PCR compared with the culture was 71% and the specificity was 88%. All the species identified by culture in these patients were in the SeptiFast panel. The median time to results was 1 day for the PCR, 3 days for positive cultures and 5 days for negative cultures.
    Full-text · Article · Oct 2014 · Diagnostic Microbiology and Infectious Disease
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    • "Recently, several molecular diagnostic tests for whole blood became commercially available (SepsiTest (Molzym), MagicPlex Sepsis Real-Time Test (Seegene), VYOO (SIRS Lab), and SeptiFAST (Roche)) and were evaluated by several independent research groups [7], [8], [9], [10], [11], [12], [13]. However, none of the abovementioned tests combines pathogen DNA enrichment with fast identification, they provide either pathogen DNA enrichment or fast sensitive detection. "
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    ABSTRACT: For patients suffering from bloodstream infections (BSI) molecular diagnostics from whole blood holds promise to provide fast and adequate treatment. However, this approach is hampered by the need of large blood volumes. Three methods for pathogen DNA isolation from whole blood were compared, i.e. an enzymatic method (MolYsis, 1-5 ml), the novel non-enzymatic procedure (Polaris, 1-5 ml), and a method that does not entail removal of human DNA (Triton-Tris-EDTA EasyMAG, 200 µl). These methods were evaluated by processing blood spiked with 0-1000 CFU/ml of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Downstream detection was performed with real-time PCR assays. Polaris and MolYsis processing followed by real-time PCRs enabled pathogen detection at clinically relevant concentrations of 1-10 CFU/ml blood. By increasing sample volumes, concurrent lower cycle threshold (Ct) values were obtained at clinically relevant pathogen concentrations, demonstrating the benefit of using larger blood volumes. A 100% detection rate at a concentration of 10 CFU/ml for all tested pathogens was obtained with the Polaris enrichment, whereas comparatively lower detection rates were measured for MolYsis (50-67%) and EasyMAG (58-79%). For the samples with a concentration of 1 CFU/ml Polaris resulted in most optimal detection rates of 70-75% (MolYsis 17-50% and TTE-EasyMAG 20-36%). The Polaris method was more reproducible, less labour intensive, and faster (45 minutes (including Qiagen DNA extraction) vs. 2 hours (MolYsis)). In conclusion, Polaris and MolYsis enrichment followed by DNA isolation and real-time PCR enables reliable and sensitive detection of bacteria and fungi from 5 ml blood. With Polaris results are available within 3 hours, showing potential for improved BSI diagnostics.
    Full-text · Article · Aug 2013 · PLoS ONE
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    • "Accordingly, the hypothesis of a low bacterial load can be ruled out. In this study, the blood volume used for the MST was smaller than in other studies (Bloos et al., 2012; Fitting et al., 2012; Tsalik et al., 2010; Wallet et al., 2010). The significant difference in the sample volumes used for the MST and BC testing is likely a major contributing factor to the poor sensitivity of PCR. "
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    ABSTRACT: Purpose: Mortality from bloodstream infections (BSI) correlates with diagnostic delay and the use of inappropriate empirical treatment. Early PCR-based diagnosis could decrease inappropriate treatment, improving patient outcome. The aim of the present study was to assess the clinical utility of this molecular technology to diagnose BSI. Methods: We assessed a new dual priming oligonucleotide-based multiplex PCR assay, Magicplex™ Sepsis-Test (MST) (Seegene), along with blood culture (BC). A total of 267 patients from the intensive care unit and haematology and emergency departments were enrolled. Clinical data were also used by physicians to determine the likelihood of infection. Results: Ninety-eight (37 %) specimens were positive: 29 (11 %) by both MST and BC, 29 (11 %) by MST only, and 40 (15 %) by BC only. The proportion of agreement between the two methods was 73 % (Cohen's kappa coefficient: 0.45; 0.28-0.6; indicating fair to moderate agreement). According to clinical assessment, 63 (64 %) positive specimens were considered bloodstream infections: 23 (36 %) were positive by both MST and BC, 22 (35 %) were positive only by BC, and 18 (29 %) were positive only by MST. Thirty-eight (14 %) positive specimens by MST and/or BC were considered as contaminants. Of 101 specimens collected from patients receiving antibiotics, 20 (20 %) were positive by MST and 32 (32 %) by BC. Sensitivity and specificity were 65 % and 92 %, respectively, for MST and 71 % and 88 % for BC. Conclusion: Magicplex™ Sepsis-Test shows a high specificity but changes in design are needed to increase bacteraemia detection. For viability in clinical laboratories, technical improvements are also required to further automate the process.
    Full-text · Article · Aug 2013 · Journal of Medical Microbiology
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