الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Sepsis is a life-threatening organ dysfunction due to a dysregulated host response to infection. It is a growing problem worldwide, associated with a mortality rate as high as
60%. It is a highly detrimental problem for infants and the elderly, as well as immune-compromised and critically ill patients. The aim of present work was to validate the diagnostic and prognostic value of miRNA-150 and 146-a as biomarkers for patients with sepsis in different intensive care units in hospitals in Alexandria. The study was carried out in the department of Microbiology in the period of June 2017 until June 2018.
After the approval of the Ethical Committee of the Medical Research Institute, Alexandria University, fifty patients suffering from sepsis selected from different intensive care units (Surgical Intensive Care unit, post-operative I.C.U, internal medicine department of continuous renal therapy and critical cases and intermediate care unit I.C.U) from Alexandria University hospitals and Hepatology intensive care unit in Alexandria fever hospital were included in this study. Twenty control healthy volunteers matched by age and sex were also included in the study.
Blood samples (15 mL) were collected from each patient. 10 mL were inoculated into an aerobic culture bottle for culture and sensitivity and samples were processed in Bact/ALERT 3D automatic system at 35°C for at least seven days. Samples were monitored for bacterial growth by detection of fluorescent change. Samples from positive blood culture bottles were withdrawn, sub-cultured on blood agar, MacConkey agar and Sabouraud Dextrose agar (SDA) plates which were incubated for 24 hours up to 48 hours. The isolated colonies were then further identified by Gram staining and biochemical tests and antimicrobial susceptibility testing was carried out by Kirby-Bauer method.
Sera separated from the remaining 5 ml blood were used for detection and quantification of miRNA-146a and 150 using real-time PCR and the relative expression of miRNA 150 and 146-a was calculated using the comparative CT method.
In the present study, the ratio of male to female was 3:1 and the age of the patients ranged from (34.0-79.0) years with a median of 69. Sepsis cases included in this study stayed in the hospital for at least 4 days and a maximum of 16 with a mean of 9 days. As regarded to the patients‘ temperature the mean was 38.84. Mean of heart rate was 110.90 and respiratory rate was 25.30.
The mean WBCs count of the 50 cases included in the study was 35380 with a minimum of 20000 and a maximum of 55000 cells/mm3
Patients admitted to the ICU for medical reasons were 88%, while 12% were admitted after surgical interventions. 18 (36%) of cases suffered from a gastrointestinal tract infection and 16 (32%) suffered from a blood stream infection. The least source was skin and soft tissue infection detected in only 5 (10%) of cases.
Summary, Conclusion and Recommendations
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The most common microorganism isolated using blood culture was E.coli in 13(26%) of cases, followed by Candida in 9 (18%) of cases then Staphylococcus aureus, Pseudomonas, Klebsiella each one of them in 6 (12%) cases.
The most active agents against Gram positive bacteria were linezolid (100%), Teicoplanin (100%) and vancomycin (100%) followed by imipenem (93.8%) and Rifampicin (93.8%). On the other hand, the isolates exhibited high level of resistance to Cefotaxime and Ceftazidime (93.8%) each, Trimethoprim (87.5%) and moderate resistance to Cefepime and Levofloxacin (68.7) each, then Ciprofloxacin (62.5%), amikacin and amoxicillin/clavulanic acid (43.8%) each.
The most active agents against Gram negative bacteria were colistin (96%), imipenem (96%) followed by Gentamicin (80%). On the other hand, the isolates exhibited high level of resistance to Ceftazidime (88%) and Cefotaxime and trimethoprim/sulphamethoxazole (80%) each. As regard the antifungal sensitivity pattern among the 9 cases diagnosed with Candida; all (100%) of isolates were sensitive to the 3 antifungal agents used.
As regards to the relative expression of miRNA 146a, 56% (28/50) of the cases showed overexpression in sepsis patients, while 44% (22/50) of the cases showed downregulated expression with a range between 0.08 – 10.30, a mean and S.D. of 1.52 ± 1.71.
As regards to the relative expression of miRNA 150, 52% (26/50) of the cases showed upregulated expression in sepsis patients, while 48% (24/50) of the cases showed downregulated expression with a range between 0.06 – 11.10, a mean and S.D. of 1.70 ± 2.46.
The co-expression of miRNA-146a and miRNA 150 showed a moderate positive correlation in the studied group which was found to be statistically significant (r = 0.489, p=0.001). Moreover, there was a statistically significant correlation between the relative expression of microRNA-146a and 150.
The association between the type of organism (G +ve, G -ve) and the expression of microRNA 146a and 150 showed no statistically significant difference between the type of and the level of expression of microRNA-146a and 150.
6.2. Conclusion:
The present study revealed that:
1. Serum microRNAs might be used as biomarkers for sepsis.
2. The co-expression of microRNA 146a and microRNA 150 showed a moderate positive correlation in the studied group which was found to be statistically significant
3. Up to our Knowledge, this is the first study to associate between the type of organism (G +ve, G -ve) and the expression of microRNA 146a and 150 showing no statistically significant difference between the type of organism and the level of expression of microRNA 146-a and 150.
Summary, Conclusion and Recommendations
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6.3. Recommendations
1. miRNA expression is a strong candidate for the future of intensive care because of the early diagnosis opportunity and because of its capacity to interact with certain key points of the biochemical pathways
2. miRNA species can be determined in different body fluids, such as serum, plasma, and blood, widening the range of options for the determination of sepsis in critically ill patients.
3. micRNA 146-a and micRNA 150 are useful as prognostic and diagnostic biomarkers.
4. The use of miRNAs as diagnostic biomarkers may represent a new perspective in the differential diagnosis between Gram-positive and Gram-negative bacteria. (The presence of microRNA in significant concentrations in plasma or other body fluids makes the ideal as biomarkers in clinical practice and their expression may be pathogen-specific.)
5. miRNA are helpful in the diagnosis and prognosis of sepsis as well as monitoring therapeutic responses in clinical settings.
6. Different miRNAs are differentially expressed in sepsis. Therefore, further larger studies on microRNA expression in sepsis patients should be performed. 7. Using miRNAs as circulating biomarker for sepsis is still in its infancy and additional studies are required to increase the specificity and selectivity of this method. Further larger scale studies are necessary to identify the correct context for miRNA expression strengthening a broader range of specific miRNAs for sepsis
8. miRNAs other than miRNA 146-a and 150 such as miRNA-122,-223, -133 and -16 may also be used for diagnosis of sepsis. 9. In conclusion, we can affirm that it is necessary to improve detection and validation methods of specific miRNAs for sepsis.