Clinical Study On Plasma Biomarkers Of Burn Sepsis And Analysis Of Nursing Interventions

The key to treat severe burn patients successfully lies on accurate injury judgementand early fluid resuscitation, timely detection for burn sepsis followed by rational antibiotictherapy, as well as removal of necrotic tissue as soon as possible and accelerating healingand regeneration of burn wound. However, in clinical practice, the judgment of burn wounddepth can be inevitably influenced by subjective factors, and clinical applications ofauxiliary equipments always confront limitations. In terms of burn condition monitoring,there are some biomarkers specific for some certain kind of organ injury, however, therestill lack a direct indicator, which can tell the overall condition of various cells and organspost burn. When it comes to the diagnosis of burn sepsis, due to the features of the verydisease (which includes fast progress, high mortality rate, and insidious clinicalmanifestations) as well as the limitations of clinical microbiological culture, accurate andtimely diagnosis of burn sepsis remains a major challenge for clinicians nowadays.Procalcitonin (PCT) is once considered the most promising biomarker for sepsis diagnosis.However, since it can not distinguish between sepsis and non-infectious systemicinflammation reaction syndrome effectively, PCT is not sufficient to work as anindependent biomarker. Therefore, further study of biomarkers for burns, especially burnsepsis has important clinical significance.Since plasma circulating DNA is a direct reflection of tissue and cell injury in varity ofdisease conditions, it has the potential to become an effective diagnostic and prognosticbiomarker for burn patients. In some previous clinical studies, it has also been found thatthe degree for plasma DNA elevation had a close correlation with burn depth, burn surfacearea and the length of hospital stay. But these studies lack a consecutive observation fordynamic levels of circulating nucleic acids, and lack a combined comprehensive analysis ofburn injury, vital organs function, and burns prognosis. Moreover, dynamic changes of burnpatients’ plasma16S rDNA, a highly conserved bacterial ribosomal RNA gene, help to understand the pathophysiology of burn sepsis from a dynamic point of view. Acombination of plasma DNA and plasma16S rDNA, representative of patient tissue andcell damage degree and pathogens nucleic acids’ release respectively, can present thedevelopment of sepsis theoretically. In addition, the rapid development of protemomicstechnologies provides a solid experimental foundation for screening protein biomarkersmore extensively. Finding out differential expressed proteins before and after the onset ofburn sepsis with the application of iTRAQ technology, may lay a clinical and experimentalfoundation for screening more sensitive early diagnostic biomarkes and furtherimprovement for comprehensive early diagnostic criteria for burn sepsis.This study is divided into four parts:(1) dynamic changes and clinical significance ofplasma circulating DNA levels in burn patients;(2) clinical significance of plasma16SrDNA levels in diagnosis of burn sepsis;(3) preliminary screening of plasma proteinbiomarkers in burn sepsis patients;(4) analysis of risk factors in burn sepsis andcorresponding nursing interventions in the course of early detection.1MethodsSixty burn patients with burn surface area (BSA) greater than30%were recruited asthe case group, among which17cases had developed a complication of sepsis, and11caseshad ultimately died. Another49healthy subjects were recruited as the healthy control group.A total of200plasma samples were collected from the case group during post-burn day1-3,4-7,8-14, and15-21respectively. Another49plasm samples were from the healthy controlgroup.1.1Plasma circulating DNA was extracted with QIAamp DNA Blood Mini Kit andquantified with Quant-iT PicoGreen fluorescent dye reagent method for double-strand DNAand a real-time PCR method for β-actin gene, respectively. Combined with clinical data,plasma circulating DNA level was analyzed for its variation after burns and its clinicalsignificance.1.2Plasma16S rDNA of all the plasm samples was also quantified with a real-timePCR method. Corresponding with the17burn sepsis case, the clinical significance ofplasma16S rDNA in early diagnosis of burn sepsis was analysed.1.3Differentially expressed proteins were screened from eight plasma samples of fourburn sepsis patients with an application of iTRAQ technology. 1.4Burn sepsis associated risk factors and the corresponding nursing countermeasureswere analyzed.2Results2.1In terms of burn severity judgment, plasma circulating DNA levels on admissionhad a significantly positive correlation with Acute Physiology and Chronic HealthEvaluation Ⅱ (APACHE Ⅱ) score, BSA and burn index (P<0.01). Refering to burncondition monitoring, plasma circulating DNA levels in different BSA groups have showeddifferent dynamic changes. Meanwhile, plasma circulating DNA levels in each stage owneda significantly positive correlation with Multiple Organ Dysfunction Score (MODS)(P<0.01) and several clinical and laboratory indicators (P<0.05). When it comes to clinicalprognosis, a combination of admission plasma levels of β-actin gene, APACHE Ⅱ score,MODS, BSA, third degree burn area, as well as burn index had a combined area under thereceiver operator charaeteristic (ROC) curve of0.985, which is higher than any individualindicator. The sensitivity and specificity of this combination were92.7%and100%,respectively.2.2Plasma16S rDNA levels in post-burn day1-3and day4-7were significantlyhigher in burn sepsis group than in both healthy control group and the non-sepsis group. Inthe200plasma samples,16S rDNA levels were significantly positively correlated withMODS (P <0.01), and were significantly correlated with prothrombin time, thrombin time,serum sodium, blood urea nitrogen(P<0.05). However, in the69plasma samples collectedfrom the sepsis group, plasma16S rDNA level was testified being positive correlated withPCT (P <0.01). In the aspect of burn sepsis diagnosis, plasma16S rDNA combined withPCT had an AUC of0.857, with a sensitivity and specificity of85.7%and81.3%respectively. As for the prognosis of burn sepsis,16S rDNA, PCT and plasma β-actin had ajoint AUC of0.946, with sensitivity and specificity being100%and75%.2.3A total of509proteins were identified from eight plasma samples of four burnsepsis patients’ with an iTRAQ method. For each case, the number of differentiallyexpressed proteins was64,62,77, and48respectively. And those proteins showed a highenrichment tendency for complement and coagulation cascade pathway. Further clusteranalysed that there were two differentially expressed proteins in the intersection of fourpatients, namely Peroxiredoxin-2(Prx-2) and Peptidoglycan recognition protein2 (PGLYRP2).2.4Based on the clinical data of17burn sepsis cases, the results of the first threechapters of this thesis, as well as relevant literatures, we analysed that burn sepsis riskfactors may include severe burn injury, immunocompromised patient, invasive treatment.And3-6days post burn is the high risk period for developing sepsis. Correspondingly,nursing strategies include effective control of nosocomial infections, early detection ofsepsis through monitoring burn wound and the state of multiple organs function,standardization of clinical microbiology sampling process, and the establishment ofevidence-based care model for burn sepsis patients.3Conclusions3.1In the aspect of quantitative methods for plasma circulating DNA, results ofPicoGreen fluorescence are highly correlated with the results of real-time PCR quantitativemethods. As the former can provide direct and comprehensive results, and be moreeconomical and fast, it can be a better choice for fundamental plasma DNA quantitativeresearches. Plasma DNA levels, especially plasma β-actin gene level on admission, whichcan objectively reflect the degree of cell injury, can, to some extent, assist clinicians makingjudgement of burn severity. As for the clinical monitoring, dynamic changes of plasmacirculating DNA levels have the potential to become biomarkers for illness deterioration orrecovery. Although, plasma circulating DNA level is insufficient to work as an independentbiomarker for burn prognosis, the combination of plasma β-actin gene, APACHE Ⅱ score,MODS, BSA, third degree burn area, burn index provide a better performance.3.2The dynamic changes of plasma burn16S rDNA may reflect the difference of highrisk time for developing sepsis in different BSA groups. Besides, for some suspected sepsiscases whose blood culture results presents negative continuously, plasma16S rDNA, whichis an evidence of a transient bacteremia, may provide assistance in making sepsis diagnosis.A combination of plasma16S rDNA level and PCT may work as auxiliary biomarkers forearly diagnosis of burn sepsis. In the realm of burn sepsis prognosis, plasma16S rDNAlevel together with plasma β-actin gene and PCT provides a better performance.3.3Of the four burn sepsis cases, a total of509proteins were identified with aniTRAQ technology. The number of differentially expressed proteins before and after sepsisfor each case was64,62,77, and48respectively. With the result of GO enrichment analysis and the cluster analysis, we highly valued protein Prx-2and PGLYRP2for their potential tobe sepsis biomarkers. Further study based on a larger sample size and new progress inunderstanding of sepsis pathophysiology is still needed3.4Nurses can play a vital and unique role in the course of early detection andaccurate diagnosis of burn sepsis by tight nosocomial infections control, monitoring clinicalmanifestations closely, standardizing clinical microbiology specimen collection process,establishing evidence-based care model for burn sepsis patients.