Combined RT-qPCR and pyrosequencing of a Spike glycoprotein polybasic cleavage motif can uncover pediatric SARS-CoV-2 infections associated with heterogeneous presentation.

BACKGROUND: Reverse transcription of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (+)RNA genome and subgenomic RNAs (sgRNAs) and subsequent quantitative polymerase chain reaction (RT-qPCR) is the reliable diagnostic gold standard for COVID-19 diagnosis and the identification of potential spreaders. Apart from clinical relevance and containment, for specific questions, it might be of interest to (re)investigate cases with low SARS-CoV-2 load, where RT-qPCR alone can deliver conflicting results, even though these cases might neither be clinically relevant nor significant for containment measures, because they might probably not be infectious. In order to expand the diagnostic bandwidth for non-routine questions, particularly for the reliable discrimination between negative and false-negative specimens associated with high CT values, we combined the RT-qPCR workflow with subsequent pyrosequencing of a S-gene amplicon. This expansion can help to confirm SARS-CoV-2 infections without the demand of confirmative antibody testing, which requires to summon patients again for blood sampling few to several weeks after symptom onset. RESULTS: We successfully established a combined RT-qPCR and S-gene pyrosequencing method which can be optionally exploited after routine diagnostics. This allows a reliable interpretation of RT-qPCR results in specimens with relatively low viral loads and close to the detection limits of qPCR. After laboratory implementation, we tested the combined method in a large pediatric cohort from two German medical centers (n=769). Pyrosequencing after RT-qPCR enabled us to uncover 5 previously unrecognized cases of pediatric SARS-CoV-2-associated diseases, mainly exhibiting mild and heterogeneous presentation-apart from a single case of multisystem inflammatory syndrome in children (MIS-C) associated with SARS-CoV-2, who was hospitalized in the course of the study. CONCLUSIONS: The proposed protocol allows a specific and sensitive confirmation of SARS-CoV-2 infections close to the detection limits of RT-qPCR. The tested biotinylated primers do not negatively affect the RT-qPCR pipeline and thus can be optionally applied to enable deeper inspection of RT-qPCR results by subsequent pyrosequencing. Moreover, due to the incremental transmission of SARS-CoV-2 variants of concern, we note that the used strategy can uncover (Spike) P681H allowing the pre-selection of SARS-CoV-2 B.1.1.7 candidate specimens for deep sequencing.

Speckle tracking stress echocardiography in children: interobserver and intraobserver reproducibility and the impact of echocardiographic image quality.

Speckle tracking echocardiography (STE) is increasingly used during functional assessments. However, reproducibility and dependence on echocardiographic image quality for speckle tracking stress echocardiography in pediatric patients have not been studied to date. 127 consecutive normotensive children without structural heart disease (mean age 13.4 ± 3.0 years, 50.4% female) underwent a stepwise semisupine cycle ergometric protocol. Left ventricular (LV) myocardial peak strain and strain rate were assessed at rest and during exercise. Interobserver and intraobserver assessments were performed and analyzed regarding echocardiographic image quality. LV peak global strain and strain rate were well reproducible with narrow limits of agreement without any significant bias both at rest and during all stages of exercise testing. Moreover, strain rate reproducibility slightly deteriorated in values between -1.5 and -3 s-1. Surprisingly, there was no significant difference in reproducibility between optimal, intermediate and poor quality of echocardiographic images. STE derived strain and strain rate measurements in children are feasible and highly reproducible during semisupine cycle ergometric stress echocardiography. Echocardiographic image quality does not seem to influence strain (rate) reproducibility. Myocardial deformation measurements in images with suboptimal visualization quality must be interpreted with caution.

Novel functional advanced echocardiography for the assessment of myocardial mechanics in children with neurocardiogenic syncope - a blinded prospective speckle tracking head-up tilt-table challenge study.

BACKGROUND: Data on left ventricular (LV) function in patients with neurocardiogenic syncope (NS) is conflicting in adults and lacking in children. The aim of this study was to analyze LV myocardial performance in children with NS at rest and during head-up tilt-table (HUTT) testing. METHODS: This is the first study to combine HUTT and speckle-tracking echocardiography (STE) in children with NS. 43 consecutive normotensive pediatric patients with NS (mean age 13.9 ± 2.6 years, 51% female) and 41 sex- and age-matched healthy controls were included in the study. The study groups consisted of 21 patients with a positive HUTT reaction (HUTT+) and 22 with a negative HUTT reaction (HUTT-). STE was used to analyze peak systolic LV myocardial strain and strain rate. RESULTS: Conventional echocardiographic parameters were similar in all analyzed groups. When compared to healthy controls, children with NS had depressed levels of circumferential strain rate (p = 0.032) and significantly depressed longitudinal strain rate (p <  0.001) at rest. Interestingly, during HUTT testing LV global strain and strain rate were similar in both groups. LV strain rate was lowest in HUTT+ followed by HUTT- and control subjects both at rest and during HUTT. CONCLUSIONS: Resting LV longitudinal strain rate is attenuated in children with NS, especially in those with a positive HUTT response. This is further evidence that NS patients feature altered cardiac mechanics rendering them prone to vasovagal perturbations that can ultimately result in collapse. TRIAL REGISTRATION: Witten/Herdecke University ethics committee clinical study number: UWH-73-2014.

Intraobserver and interobserver reproducibility of M-mode and B-mode acquired mitral annular plane systolic excursion (MAPSE) and its dependency on echocardiographic image quality in children.

BACKGROUND: Mitral annular plane systolic excursion (MAPSE) is an increasingly used echocardiography technique to assess left ventricular (LV) function. However, reproducibility and dependence on echocardiographic image quality for MAPSE in pediatric patients have not been studied to date. METHODS: We analyzed 284 transthoracic echocardiograms performed on consecutive normotensive children without structural heart disease (mean age 12.6±3.1 years, 50.4% female). B-mode and M-mode derived MAPSE measurements were performed and analyzed regarding inter- and intraobserver reliability and the influence of echocardiographic image quality. RESULTS: Overall, MAPSE measurements were highly reproducible with only minor bias. Both inter- and intraobserver reliability were significantly better for M-mode derived MAPSE (p<0.001). Echocardiographic image quality did not significantly influence M-mode MAPSE reproducibility (p>0.235). In contrast, B-mode lateral MAPSE was significantly better reproducible in optimal image quality (-0.07±1.04) when compared to suboptimal echocardiographic images (0.42±1.59, p<0.001). Moreover, poor quality images yielded significantly lower M-mode MAPSE values (14.3±2 mm) than near-optimal (15.2±1.9 mm, p<0.001) or optimal images (15.1±2.2 mm, p = 0.006). CONCLUSION: Echocardiographic image quality essentially has a negligible effect on MAPSE reproducibility and measurements. Consequently, MAPSE is a robust echocardiographic parameter with convincing reproducibility for the assessment of LV function in children-even in patients with substandard imaging conditions.

Speckle Tracking Stress Echocardiography Uncovers Early Subclinical Cardiac Involvement in Pediatric Patients with Inflammatory Bowel Diseases.

Inflammatory bowel disease (IBD) is an established risk factor for cardiovascular disease (CVD). However, whether cardiac consequences present early in IBD is unknown. This is the first study in children aiming to unmask altered myocardial mechanics in IBD. We enrolled 50 consecutive normotensive children with Crohn's disease (CD) (n = 28) or ulcerative colitis (UC) (n = 22). The study groups consisted of 18 patients with active inflammatory disease (mean age 14.6 ± 2.5 years) and 32 children with IBD in remission (14.3 ± 2.3 years). 60 age- and gender-matched children served as healthy controls. Speckle tracking stress echocardiography (STE) was used to assess left ventricular (LV) myocardial strain and strain rate. Circumferential strain rate was significantly decreased in children with active IBD (-1.55 ± 0.26 s-1) and IBD in remission (-1.49 ± 0.26 s-1) versus healthy controls (1.8 ± 0.4 s-1) both at rest (p < 0.001) and during exercise (p = 0.021). Moreover, longitudinal strain rate, circumferential strain and E/E' ratio were significantly impaired in IBD. Pediatric patients with IBD feature subclinical signs of LV systolic and diastolic myocardial impairment early in the course of CD and UC. This may not be reversible even when IBD is clinically controlled. Patients with IBD should be regularly screened for signs of CVD.

Transthoracic Speckle Tracking Echocardiography for the Quantitative Assessment of Left Ventricular Myocardial Deformation.

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the examiners' expertise. Speckle tracking Echocardiography (STE) is a promising but technically challenging method that can be used to quantitatively assess regional and global systolic and diastolic myocardial performance. Myocardial strain and strain rate can be measured in all three dimensions - radial, circumferential, longitudinal - of myocardial deformation. Standard cross-sectional two-dimensional B-mode images are recorded and subsequently postprocessed by automated continuous frame-by-frame tracking and motion analysis of speckles within the myocardium. Images are recorded as digital loops and synchronized to a 3-lead EKG for timing purposes. Longitudinal deformation is assessed in the apical 4-, 3-, and 2-chamber views. Circumferential and radial deformation are measured in the parasternal short axis plane. Optimal image quality and accurate tissue tracking are paramount for the correct determination of myocardial performance parameters. Utilizing transthoracic STE in a healthy volunteer, the present article is a detailed outline of the essential steps and potential pitfalls of quantitative echocardiographic myocardial deformation analysis.

Subclinical Alterations of Cardiac Mechanics Present Early in the Course of Pediatric Type 1 Diabetes Mellitus: A Prospective Blinded Speckle Tracking Stress Echocardiography Study.

Diabetic cardiomyopathy substantially accounts for mortality in diabetes mellitus. The pathophysiological mechanism underlying diabetes-associated nonischemic heart failure is poorly understood and clinical data on myocardial mechanics in early stages of diabetes are lacking. In this study we utilize speckle tracking echocardiography combined with physical stress testing in order to evaluate whether left ventricular (LV) myocardial performance is altered early in the course of uncomplicated type 1 diabetes mellitus (T1DM). 40 consecutive asymptomatic normotensive children and adolescents with T1DM (mean age 11.5 ± 3.1 years and mean disease duration 4.3 ± 3.5 years) and 44 age- and gender-matched healthy controls were assessed using conventional and quantitative echocardiography (strain and strain rate) during bicycle ergometer stress testing. Strikingly, T1DM patients had increased LV longitudinal (p = 0.019) and circumferential (p = 0.016) strain rate both at rest and during exercise (p = 0.021). This was more pronounced in T1DM patients with a longer disease duration (p = 0.038). T1DM patients with serum HbA1c > 9% showed impaired longitudinal (p = 0.008) and circumferential strain (p = 0.005) and a reduced E/A-ratio (p = 0.018). In conclusion, asymptomatic T1DM patients have signs of hyperdynamic LV contractility early in the course of the disease. Moreover, poor glycemic control is associated with early subclinical LV systolic and diastolic impairment.

The influence of real-time blood glucose levels on left ventricular myocardial strain and strain rate in pediatric patients with type 1 diabetes mellitus - a speckle tracking echocardiography study.

BACKGROUND: Echocardiographic myocardial performance parameters such as strain and strain rate are increasingly used to assess systolic and diastolic function in patients with diabetes mellitus and several other clinical and scientific scenarios. While long-term metabolic marks such as HbA1C are inherently assessed in diabetic patients, the actual blood glucose level at the very moment of the echocardiographic study has not yet been taken into account for the assessment of cardiac mechanics. The aim of this study was to investigate the influence of real-time blood glucose levels on left ventricular (LV) myocardial strain and strain rate in pediatric patients with type 1 diabetes mellitus (T1DM). METHODS: We performed speckle tracking echocardiography on 39 normotensive pediatric patients with uncomplicated type 1 diabetes mellitus (mean age 11.5 ± 3.5 years, 40 % female) and 44 sex- and age-matched healthy controls (mean age 11.4 ± 2.9 years, 45 % female). T1DM patients were sub-categorized according to their blood sugar levels (with a cutoff of 150 mg/dL) at the moment of the echocardiographic exam. Investigators were blinded to the participants' study group status. RESULTS: Interestingly, diabetic patients with higher blood sugar levels demonstrated significantly increased LV circumferential strain (p = 0.003) and strain rate (p = 0.005) as well as global longitudinal strain rate (p = 0.002) in comparison to T1DM patients with lower blood sugar levels or healthy controls. CONCLUSIONS: For the investigation of myocardial performance with sensitive methods such as speckle tracking echocardiography in diabetic study populations real-time blood sugar levels should be taken into account. Further studies are needed to verify these findings in large-scale patient cohorts and serial intra-individual measurements in different metabolic states.

Duplex ultrasound findings before and after surgery in children and adolescents with renovascular hypertension.

We report our experience with duplex ultrasound in young patients with renal artery stenosis (RAS) or middle aortic syndrome (MAS) before and after surgery (1995 and 2009). Of 36 patients (mean age: 13 ± 7 y), 21 had RAS and 15 had MAS. For patients with RAS, the Vmax in the affected artery was 350 ± 111 cm/s before surgery and 145 ± 55 cm/s after surgery. The resistance index was 0.46 ± 0.1 in the post-stenotic kidney and increased to 0.60 ± 0.08 after revascularization. Determination of the flow profile in the iliac artery revealed triphasic flow. In individuals with MAS, Vmax in the aorta was 323 ± 98 and the resistance index in both kidneys was low, even in the absence of RAS. The flow profile in the iliac arteries was monophasic before surgery and became triphasic after surgery. Duplex ultrasound is useful for the evaluation of children and young adults both pre- and post-surgery. Duplex ultrasound criteria for RAS in adults appear to be applicable in children and young adults also. The diagnostic evaluation of suspected renal vascular disease should include assessment of the aorta and the flow profile in the iliac arteries, as this could help differentiate between aortic and isolated renal artery stenosis.

Expression of human beta-defensins in children with chronic inflammatory bowel disease.

BACKGROUND: Human beta-defensins (hBDs) are antimicrobial peptides known to play a major role in intestinal innate host defence. Altered mucosal expression of hBDs has been suggested to be implicated in chronic inflammatory bowel disease pathogenesis. However, little is known about expression of these peptides in children. METHODS: Intestinal biopsies were obtained from the duodenum (n = 88), terminal ileum (n = 90) and ascending colon (n = 105) of children with Crohn's disease (n = 26), ulcerative colitis (n = 11) and healthy controls (n = 16). Quantitative real-time (RT) PCR was performed and absolute mRNA copy numbers analyzed for hBD1-3 as well as inflammatory cytokines IL-8 and TNF-alpha. RESULTS: Significant induction of hBD2 and hBD3 was observed in the inflamed terminal ileum and ascending colon of IBD children. In the ascending colon induction of hBD2 was found to be significantly lower in children with Crohn's disease compared to ulcerative colitis. A strong correlation was found between inducible defensins hBD2 and 3 and the inflammatory cytokines IL-8 and TNF-alpha, both in the terminal ileum and ascending colon. CONCLUSION: Our study demonstrates distinct changes in hBD expression throughout the intestinal tract of children with IBD, lending further support for their potential role in disease pathogenesis.

Off-label drug use among hospitalised children: identifying areas with the highest need for research.

OBJECTIVE OF THE STUDY: To analyse the off-label use of drugs on a paediatric ward in Germany, and to identify domains of pharmacotherapy with the highest need for research concerning off-label use in children. SETTING: A prospective observational study was conducted on a paediatric ward in Duesseldorf in Germany between January and June 2006. METHOD: Data about patients, diagnoses and prescribed drugs were collected from the prescription records and the discharge letters. Diagnoses were classified in groups by means of the International Classification of Diseases. Drugs were grouped according to the Anatomical Therapeutic Chemical Classification system. We compared the off-label prescriptions with those on the list of paediatric needs and priority list established by the European Medicines Agency (EMEA). MAIN OUTCOME MEASURE: Off-label use was defined due to age, indication, route of application and dose. RESULTS: The study included 417 patients. We analysed 1,812 prescriptions representing 211 different drugs. In total, 253 patients (61%) received at least one off-label prescription. Of all analysed prescriptions, 553 (31%) were off-label. The percentage of off-label prescriptions among the five most frequently prescribed drug groups were as follows: 60% cardiovascular drugs (CV: 129/216), 42% anti-infectives (AI: 190/449), 30% drugs for respiratory system (RS: 100/335), 25% drugs for alimentary tract and metabolism (AM: 67/269) and 3% analgesics and antipyretics (AA: 8/264); with 17 drugs, the cardiovascular drugs also showed the highest number of different off-label prescribed drugs due to age: AI: 14; AM: 11; RS: 5; AA: 1. In addition, there was a nearly complete overlap between the identified off-label prescriptions in cardiovascular drugs and those listed by the EMEA to be prioritized for urgent research in Europe. CONCLUSION: Cardiovascular drugs are a domain of pharmacotherapy, with a large need for research in paediatrics. The results of our study can guide the researcher to future trials on off-label prescriptions such as cardiovascular drugs, especially due to the fact that the identified off-label prescribed drugs in this group are also mentioned by the EMEA to be prioritized for paediatric research.

Transesophageal 3-dimensional versus cross-sectional echocardiographic assessment of the volume of the right ventricle in children with atrial septal defects.

The study was designed to investigate the value of assessing right ventricular volume by transoesophageal 3-dimensional echocardiographic techniques compared with the standard transoesophageal cross-sectional approach. Echocardiography was performed using a multiplane probe. The 3-dimensional data sets were reconstructed after electrocardiographic and respiratory gated scanning, calculating the 3-dimensional volumes by the method of multiple slices. Cross-sectional determination of volume was performed using a modified area-length method, and the biplane multiple slice method following Simpson's rule. We studied 15 patients, with ages ranging from 6 to 19 years, and body surface areas from 1.1 to 1.67 square metres. It proved possible top determine volumes with both methods in all patients. As determined by 3-dimensional echo, volumes were greater, being 113.0 plus or minus 61.2 millilitres at end-systole, and 61.7 plus or minus 36 millilitres at end-diastole, than those calculated from cross-sectional images using Simpson's rule, which gave values of 92.5 plus or minus 52 millilitres, and 41.3 plus or minus 22 millilitres. Compared to the values obtained using the area-length method, at 116.9 plus or minus 61 millilitres, and 60.3 plus or minus 30 millilitres, there were only small differences at end-systole, with a bias of 1.4, and limits of agreement of 20.9 millilitres, as well as at end-diastole, when bias was minus 3.8, and limits of agreement 22.3 millilitres. Correlation was also good, with coefficients of 0.93, and 0.91, respectively. The mean difference between the volumes by 3-dimensional acquisition and the multiple slice method was larger, with higher limits of agreement, at end-diastole showing bias of 20.5, and limits of agreement of 30.1 millilitres, and for end-systole bias of 20.4, and limits of agreement of 32.2 millilitres. Our data confirm that cross-sectional echocardiographic assessment of right ventricular volumes in children with atrial septal defects is quick, and reasonably reliable in clinical practice when employing the area-length method.

Evidence for palliative enlargement of the right ventricular outflow tract in severe tetralogy of Fallot.

OBJECTIVE: If the pulmonary artery (PA) tree in patients with Fallot's tetralogy (TOF) is extremely hypoplastic, a shunt procedure may be difficult and not desirable because of side-effects. Moreover, the failing catch-up growth of the pulmonary annulus is well known. In patients with a severe form of TOF, we performed palliative transannular patching of the right ventricular outflow tract. The early and long-term follow-up was evaluated. METHODS: Eleven patients (93 days (10-245 days); 3.5+/-0.7 kg (2.5-4.3 kg)) had highly symptomatic TOF (Hb: 18+/-2g/dl, SO2: 68+/-11%); angiographic diameters: RPA: 4.1 mm (2.5-6.4 mm), LPA: 3.4 mm (1.6-7.0 mm), PA trunc: 4.4 mm (2.5-7.0 mm). All 11 underwent transannular enlargement of the right ventricular outflow tract without closure of the ventricular septum defect. A PA index (cross-sectional area of the pulmonary arteries to BSA) was used to compare pre- and postoperative data. For follow-up, the patients were repetitively examined clinically and echocardiographically. RESULTS: Preoperative PA index was 87+/-40 mm2/m2 (normal: 330+/-35 mm2/m2). Postpalliation angiograms (age: 10-14 months) demonstrated a significant catch-up growth in nine patients (PA index from 99+/-40 to 310+/-54 mm2/m2) and inadequate growth in two patients (PA index 63 and 115 mm2/m2). Perioperative mortality was zero. Ten patients (43 months; 6-105 months) underwent elective repair. Six patients received pulmonary homograft valves (6-15 years after repair) because of severe pulmonary valve insufficiency and severe RV dilation. COMPLICATIONS: One patient died 10 months postpalliation due to pneumonia, one patient received a pacemaker after repair and died (2 months post-repair) due to pacemaker failure, a 5-year-old patient died 1 month after repair due to sepsis. All eight long-term survivors (12-17 years) are in excellent clinical condition. Echocardiography revealed good RV function and near normal diameters at peak systolic pressures between 25 and 50 mmHg. Only one patient developed brady-arrhythmia; a pacemaker was implanted 8 years after repair and 2 years after homograft implantation. CONCLUSIONS: In a very severe form of TOF, palliative right ventricular outflow tract construction may provide the potential for complete repair. In the presented high-risk patient group, mortality was not related to the hypoplastic pulmonary arteries. Obviously, all patients need pulmonary valve implantation in the long run.

Normative angiographic data relating to the dimensions of the aorta and pulmonary trunk in children and adolescents.

BACKGROUND: Definition of normative data of the great arteries from neonatal to adult ages may aid in assessment of the growth of cardiovascular structures, thus guiding the timing and type of intervention in patients with congenital cardiac disease. METHODS: We calculated the cross-sectional areas of the arterial roots at the basal attachment of the valvar leaflets, the sinuses, and standardized distal sites using cineangiograms of 59 normal children and adolescents with mean age of 5.4 plus or minus 4.7 years and a range from 0.1 to 16 years, the children having a mean weight of 21.2 plus or minus 15.7 kilograms, with a range from 2.2 to 68 kilograms, and mean height of 108 plus or minus 35 centimetres, with a range from 43 to 184 centimetres. Values at each site were calculated averaging end-diastolic and end-systolic measurements, and indexed to body surface area. Results are expressed as the mean plus or minus the standard deviation. RESULTS: The diameter of the aortic root at the basal attachment of the leaflets was 249 plus or minus 26, the midpoint of the sinuses 379 plus or minus 59, the sinutubular junction 290 plus or minus 58, the isthmus 158 plus or minus 36, the postisthmic region 152 plus or minus 33, and the descending aorta at the level of diaphragm 130 plus or minus 18 millimetres squared per metre squared. The pulmonary root measured at the basal attachment of the leaflets was 253 plus or minus 28, the midpoint of the sinuses 352 plus or minus 58, the sinutubular junction 293 plus or minus 58, the right pulmonary artery 176 plus or minus 25, the left pulmonary artery 153 plus or minus 20, and sum of right and left pulmonary arteries 330 plus or minus 37 millimetres squared per metre squared. All indexes were consistent over a wide range for body surface areas. CONCLUSIONS: Definition of normative data of the great vessels may aid in the evaluation of congenital or acquired abnormalities, serving as guidelines for intervention during medical or surgical management and follow-up.

Evans syndrome in a patient with chromosome 22q11.2 deletion syndrome: a case report.

One patient with a chromosome 22q11.2 deletion and Evans syndrome is reported in this paper. Microdeletions of 22q11.2 are the main etiology for DiGeorge syndrome, a disorder characterized by heart defects, immune deficiencies due to aplasia or hypoplasia of the thymus, and hypocalcemia. Evans syndrome refers to a hematological autoimmune disorder with autoimmune hemolytic anemia accompanied by immune thrombocytopenia. A wide range of autoimmune disorders have been described in DiGeorge syndrome and velocardiofacial syndrome, including one prior report of autoimmune hemolytic anemia and immune thrombocytopenia. The patient reported herein strengthens the association between the 22q11.2 deletion spectrum and Evans syndrome.