Blood Samples Of Pediatric Patients Research Articles

Abstract P209: The novel angiotensin Alamandine-(1-5) is increased in plasma of pediatric patients with primary nephrotic syndrome

Background and Aim: The Renin Angiotensin System (RAS) has an important role in the pathophysiology of primary nephrotic syndrome (NS). It was previously found that urinary levels of angiotensin converting enzyme 2 (ACE2) are reduced in patients with NS in comparison to health controls. The aim of this study was to measure the novel angiotensin peptide, Alamandine-(1-5), in plasma samples of patients with primary NS and to compare with sex and age-matched controls. Methods: Blood samples of pediatric patients with primary NS (n=11) and of sex and age-matched health controls (n=11) were collected in sterile tubes with 7.5% (w/v) EDTA. After plasma separation and purification by solid phase extraction, the circulating levels of Alamandine and of Alamandine-(1-5) were measured by Mass Spectrometry using multiple reaction monitoring on an ACQUITY I-Class UPLC system coupled to electrospray ionization (ESI) tandem mass spectrometry (LC-ESI-MS/MS Xevo TQ-S). Data were compared between patients and controls and associated with laboratory findings. Continuous variables were presented as mean ± standard error of the mean. Results: A total of 11 patients (7 males) with NS with mean age of 10.65±3.66 were compared to 11 controls (6 males) and with 10.86±4.35 years. In terms of disease response, 5 patients were in partial remission, 5 patients in total remission and only 1 patient was in disease relapse. All patients have normal renal function. Plasma levels of Alamandine-(1-5) were significantly higher in patients when compared to controls (28.03±7.54 vs. 10.68±3.02 pg.mL-1 in controls). Despite a mild decrease in patients, plasma levels of Alamandine did not differ between groups (19.54±6.33 vs 26.09±6.12 pg.mL-1 in controls). No correlations were found between RAS molecules and proteinuria and with the medications in use. However, Alamandine-(1-5) negatively correlated with plasma levels of albumin (r= -0.695; p=0.038). In addition, Alamandine negatively correlated with total cholesterol (r=-0.700; p = 0.036), LDL (r=-0.800; p=0.004), and HDL (r = -0.762; p = 0.038). Conclusion: The novel RAS peptide Alamandine-(1-5) is significantly increased in primary NS and inversely correlated with plasma albumin levels. These findings suggest a potential role of Alamandine-(1-5) in NS

A novel NTRK1 splice site variant causing congenital insensitivity to pain with anhidrosis in a Chinese family.

Congenital insensitivity to pain with anhidrosis (CIPA, OMIM #256800), also known as hereditary sensory and autonomic neuropathy type Ⅳ (HSAN-IV), is a rare autosomal recessive disorder characterized by recurrent episodic fevers, anhidrosis, insensitivity to noxious stimuli, self-mutilating behavior and intellectual disability. CIPA can be caused by the variants in NTRK1 gene, which encodes a high-affinity tyrosine kinase receptor for nerve growth factor. To ascertain the hereditary cause of a patient with CIPA accompanied by the additional symptoms of mild growth retardation, prone to fracture, underdeveloped nails of fingers and toes, irregular tooth alignment, enamel hypoplasia, postoperative wound healing difficulty, hand and limb deformity, and dislocation of hip joint, whole exome sequencing was used and revealed a compound heterozygous variant in NTRK1. DNA was extracted from peripheral blood samples of pediatric patients and their parents, and subjected to comprehensive analysis using whole-exome sequencing (WES), followed by verification of variant sites in the NTRK1 gene through Sanger sequencing. To elucidate the functional impact of the newly discovered variants, an in vitro experimental system was established. Splicing analysis was conducted using PCR and Sanger sequencing, while expression levels were assessed through qPCR and Western blot techniques. One hotspot variant c.851-33T>A(ClinVar ID: 21308) and a novel variant c.850 + 5G>A(ClinVar ID:3069176) was inherited from her father and mother, respectively, identified in the affected individuals. The c.850 + 5G>A variant in NTRK1 resulted in two forms of aberrant mRNA splicing: 13bp deletion (c.838_850del13, p. Val280Ser fs180) and 25bp deletion (826_850del25, p. Val276Ser fs180) in exon 7, both leading to a translational termination at a premature stop codon and forming a C-terminal truncated protein. The expression of two abnormal splicing isoforms was decreased both in the level of mRNA and protein. In conclusion, this study elucidated the genetic cause of a patient with CIPA and identified a novel variant c.850 + 5G>A in NTRK1, which broadened the and enriched the NTRK1 mutation spectrum.

Trend of Antimicrobial Resistance Among Salmonella Typhi Isolated from Pediatric Population Presenting at a Tertiary Care Hospital in Lahore

<p><strong>Background and Objective:</strong> Typhoid fever caused by Salmonella typhi (S. typhi) is an important cause of morbidity and mortality worldwide. The emergence of resistance against the first and second line of drugs has limited the range of drug choices for its treatment. Therefore, the present study was designed to assess the status of drug resistance in S. typhi isolated from the blood samples of pediatric patients reporting at a leading tertiary care hospital in Lahore.</p>
 <p><strong>Methods:</strong> This descriptive study was conducted at the Pathology Department of Allama Iqbal Medical College, Lahore, Pakistan, over a period of 2 years. A total of 1,306 blood samples were aseptically collected from the pediatric population of Jinnah Hospital, Lahore, Pakistan, after obtaining informed assent from the parents/guardians. The samples were transferred into a Bact/Alert blood culture bottle. After the detection of microbial growth by the Bact/Alert 3-D system, bacterial isolates were identified by standard microbiological procedures. VITEK 2 compact automated identification and antimicrobial susceptibility testing instrument (bioMerieux) was used for the antimicrobial sensitivity testing. Data were entered and analyzed by using Statistical Package for the Social Sciences version 21.0.</p>
 <p><strong>Results:</strong> A total of 235 out of 1,306 (17.9%) blood culture samples yielded bacterial growth, among which 62.5% (n = 147) were S. typhi. Among 147 S. typhi isolates, 70.6% were multidrug resistant, while 63% were extensively drug resistant (XDR). Maximum resistance against first-line drugs observed was for ampicillin (79.4) and co-trimoxazole (67.2).</p>
 <p><strong>Conclusion: </strong>The emergence and spread of XDR S. typhi with a high level of resistance are quite alarming, leaving limited treatment options for the pediatric population. Moreover, increasing resistance to antibiotics demonstrates the quick waning of the efficacy of available first and second-line drugs in the treatment of typhoid fever.</p>

5610325 VARIABILITY OF OXYGENSCAN PARAMETERS ACCORDING TO THE TYPE OF THERAPY IN CHILDREN WITH SICKLE CELL DISEASE: PRELIMINARY DATA FROM A FEASIBILITY STUDY

Background: Sickle Cell Disease (SCD) is characterized by significant phenotypic variability even when the same causative mutation is present (1). Validated biomarkers for routine use in SCD, to evaluate phenotypic severity and predict the evolution of organ damage and response to different therapies, are currently lacking. Oxygenscan (function of the instrument Laser-assisted Optical Rotational Red Cell Analyser) is a novel physiological assay of red blood cells’ deformability at different oxygen tensions; it allows the measurement of parameters such as maximum/minimum elongation index e point of sickling (2,3). Aims: This feasibility study aims at preliminarily evaluating the variability of the point of sickling, as a possible biomarker in SCD, with respect to different clinical conditions and types of treatment. Methods: We’ve prospectively gathered peripheral blood samples of pediatric patients with SCD followed in the Veneto Region Pediatric Sickle Cell Disease Reference Center of Padua University Hospital. The Oxygenscan test has been repeated twice for each sample. Results: We’ve analysed 67 blood samples of 48 pediatric patients with SCD (36 HbSS, 5 HbSbeta0, 7 HbSC). Patients were not receiving a disease-modifying therapy (n.11) or were on hydroxyurea (HU) (n.32), on HU and chronic transfusion regimen (n.2), HU and crizanlizumab (n.2), or after HSCT (n.2, one of them had been evaluated also before HSCT). Parameters obtained by Oxygenscan seem to vary according to the type of treatment received by patients (Figure 1).Conclusion: A higher number of samples and further analyses will be necessary to investigate a possible relationship between the point of sickling and clinical data such as the evolution of organ damage and response to different types of treatment.

Long Noncoding RNA THAP9-AS1 and TSPOAP1-AS1 Provide Potential Diagnostic Signatures for Pediatric Septic Shock.

Background Sepsis is a systemic inflammatory syndrome caused by infection with a high incidence and mortality. Although long noncoding RNAs have been identified to be closely involved in many inflammatory diseases, little is known about the role of lncRNAs in pediatric septic shock. Methods We downloaded the mRNA profiles GSE13904 and GSE4607, of which GSE13904 includes 106 blood samples of pediatric patients with septic shock and 18 health control samples; GSE4607 includes 69 blood samples of pediatric patients with septic shock and 15 health control samples. The differentially expressed lncRNAs were identified through the limma R package; meanwhile, GO terms and KEGG pathway enrichment analysis was performed via the clusterProfiler R package. The protein-protein interaction (PPI) network was constructed based on the STRING database using the targets of differently expressed lncRNAs. The MCODE plug-in of Cytoscape was used to screen significant clustering modules composed of key genes. Finally, stepwise regression analysis was performed to screen the optimal lncRNAs and construct the logistic regression model, and the ROC curve was applied to evaluate the accuracy of the model. Results A total of 13 lncRNAs which simultaneously exhibited significant differences in the septic shock group compared with the control group from two sets were identified. According to the 18 targets of differentially expressed lncRNAs, we identified some inflammatory and immune response-related pathways. In addition, several target mRNAs were predicted to be potentially involved in the occurrence of septic shock. The logistic regression model constructed based on two optimal lncRNAs THAP9-AS1 and TSPOAP1-AS1 could efficiently separate samples with septic shock from normal controls. Conclusion In summary, a predictive model based on the lncRNAs THAP9-AS1 and TSPOAP1-AS1 provided novel lightings on diagnostic research of septic shock.

Incidence of BKV in the urine and blood samples of pediatric patients undergoing HSCT.

The aims were to investigate the incidence of BKV infection and the presence of HC in pediatric patients undergoing HSCT. Twenty-four children patients (M/F: 17/7) undergoing HSCT in a single center over a period of 1year were included in the study. The presence of BKV DNA was determined by quantitative real-time PCR in plasma and urine samples at the following times: before transplantation, twice a week until engraftment time, and weekly for+100days. The mean age of the patients was 7.79±5.03years, the mean follow-up time was 95.6±25.9days, and the average number of samples per patient was 15.8±3.2. BKV DNA was detected in at least one urine sample in 91.6% (n: 22) and at least one plasma sample in 75% (n:18) of the patients. The median time to the first BKV DNA positivity in urine and plasma samples was 11 (range: 1-80) and 32days (range: 2-79), respectively. The median value of BKV DNA copies in urine and plasma were 1.7×106 (range: 2.8×101 -1.2×1014 ) and 1.9×103 copies/mL (range: 3-2.1×106 ), respectively. Thirteen patients (54.2%) had hematuria with BKV viruria; 8 (33.3%) patients had viremia. The median value of the BKV DNA copies in urine and plasma was 4.4×107 (range: 65-1×1011 ) and 2.9×103 (range: 7-7.8×104 ) copies/mL in these patients. Two (15.4%) of the 13 patients with BKV viruria and hematuria were diagnosed with BKV-related HC. BKV DNA viral load monitoring of urine and plasma in pediatric HSCT patients with a high risk for viral infections is valuable for understanding the development of BKV-related HC.

Blood-Derived DNA Methylation Signatures of Crohn's Disease and Severity of Intestinal Inflammation

Crohn's disease is a relapsing and remitting inflammatory disorder with a variable clinical course. Although most patients present with an inflammatory phenotype (B1), approximately 20% of patients rapidly progress to complicated disease, which includes stricturing (B2), within 5 years. We analyzed DNA methylation patterns in blood samples of pediatric patients with Crohn's disease at diagnosis and later time points to identify changes that associate with and might contribute to disease development and progression. We obtained blood samples from 164 pediatric patients (1-17 years old) with Crohn's disease (B1 or B2) who participated in a North American study and were followed for 5 years. Participants without intestinal inflammation or symptoms served as controls (n= 74). DNA methylation patterns were analyzed in samples collected at time of diagnosis and 1-3 years later at approximately 850,000 sites. We used genetic association and the concept of Mendelian randomization to identify changes in DNA methylation patterns that might contribute to the development of or result from Crohn's disease. We identified 1189 5'-cytosine-phosphate-guanosine-3' (CpG) sites that were differentially methylated between patients with Crohn's disease (at diagnosis) and controls. Methylation changes at these sites correlated with plasma levels of C-reactive protein. A comparison of methylation profiles of DNA collected at diagnosis of Crohn's disease vs during the follow-up period showed that, during treatment, alterations identified in methylation profiles at the time of diagnosis of Crohn's disease more closely resembled patterns observed in controls, irrespective of disease progression to B2. We identified methylation changes at 3 CpG sites that might contribute to the development of Crohn's disease. Most CpG methylation changes associated with Crohn's disease disappeared with treatment of inflammation and might be a result of Crohn's disease. Methylation patterns observed in blood samples from patients with Crohn's disease accompany acute inflammation; with treatment, these change to resemble methylation patterns observed in patients without intestinal inflammation. These findings indicate that Crohn's disease-associated patterns of DNA methylation observed in blood samples are a result of the inflammatory features of the disease and are less likely to contribute to disease development or progression.