ABSTRACT
BACKGROUND: Conventional blood culture is still the gold standard for sepsis diagnosis but results are not immediately available and pathogens are only detected in approximately 25% of cases. New molecular assays for the detection of blood stream pathogens are promising diagnostic tools. OBJECTIVES: The aim of the study was to adapt and evaluate a multiplex PCR system using 100 µl blood. - METHODS: 46 blood specimens of very low birth weight infants (818 ± 242 g) with suspected sepsis were analyzed using the Roche SeptiFast MGRADE PCR with a modified DNA extraction protocol and software handling tool for decreased blood volume requirements. RESULTS: In the non-infected group, 5/21 infants had a positive PCR result with coagulase-negative staphylococci. All pathogens detected in the blood culture positive group (n = 15) were also detected by PCR. In addition, 4/6 patients had a positive PCR result in the clinical sepsis group (clinical and laboratory signs of sepsis but negative blood culture). Overall, the PCR was demonstrated to have a higher sensitivity (90.5%; 95%CI 68.2-98.3%) in comparison to blood culture (71.4%; 95%CI 47.7-87.8%) including clinical sepsis cases, even though it had a lower specificity (80.0%; 95%CI 58.7-92.4% versus 100.0%; 95%CI 83.4-100.0%). CONCLUSIONS: These first data demonstrate the usability and potential benefit of this multiplex PCR using a modified DNA extraction for the rapid detection of nosocomial sepsis in preterm infants in addition to blood culture.
Subject(s)
Bacteriological Techniques , Blood Specimen Collection , Cross Infection/diagnosis , DNA, Bacterial/blood , DNA, Fungal/blood , Infant, Premature, Diseases/diagnosis , Infant, Premature/blood , Multiplex Polymerase Chain Reaction , Sepsis/diagnosis , Automation, Laboratory , Biomarkers/blood , Cross Infection/blood , Cross Infection/microbiology , Gestational Age , Humans , Infant, Newborn , Infant, Premature, Diseases/blood , Infant, Premature, Diseases/microbiology , Infant, Very Low Birth Weight/blood , Predictive Value of Tests , Real-Time Polymerase Chain Reaction , Sepsis/blood , Sepsis/microbiology , SoftwareABSTRACT
BACKGROUND: Concise regulation of the Toll signaling pathway is mandatory in neonatal innate immunity. The microRNA-146 family (miR-146a/b) was recently reported to be a regulator of Toll-like receptor 4 (TLR4) through a negative feedback loop mechanism. Acting as a potent regulator, miRNA helps to protect the organism from developing overwhelming proinflammatory immune responses leading to septic shock or chronic inflammatory diseases. OBJECTIVE: We investigated for the first time whether miRNA-146a/b plays a regulatory role in human monocytes derived from infant cord or adult blood, and whether differences in miRNA-146 expression exist. METHODS: Expression profiles of miR-146a/b and TLR4 were studied by real-time PCR upon stimulation with lipopolysaccharide. RESULTS: Both members of the miRNA-146 family showed a time-dependent upregulation. For miR-146a, a statistically higher significant increase was found after 24 h of stimulation in monocytes from cord blood compared to those derived from adults. In contrast, no differences were found for miR-146b and TLR4, respectively. CONCLUSION: We conclude that differences between the negative regulatory role for miR-146a obviously exist in neonatal and adult TLR4 signaling, and suggest that more intense research in the involvement of miRNA in immune regulation will facilitate the understanding of the development and function of the innate immune system of neonates.
