Clinical trial NCT04640038
Conditions: Covid19 Multisystem Inflammatory Syndrome in Children (MIS-C)
Keywords: Ultrasound Contrast-enhanced ultrasound
Sponsor: Children's Hospital of Philadelphia
Start date: 2020-12-18; Completion date: 2021-09-17
Enrollment: 4 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Initial data from COVID-19 patients suggests that one of the primary causes of death is significant endothelial injury leading to blood clotting and impaired multiorgan microvascular perfusion. The current study uses a safe, convenient bedside imaging tool called contrast-enhanced ultrasound (CEUS) to estimate the extent of microvascular perfusion impairment in the heart, kidneys and/or brain of COVID-19 pediatric patients in vivo and assess the significance of imaging findings by correlating to clinical outcomes.

This pilot study will be conducted at one site, The Children's Hospital of Philadelphia. The investigators plan to enroll and evaluate 30 patients.

Detailed description:
The current study uses a safe, convenient bedside imaging tool called contrast-enhanced ultrasound (CEUS) to measure the extent of microvascular perfusion impairment in the heart, kidneys and/or brain of COVID-19 pediatric patients and, as exploratory analysis, to assess the significance of imaging findings by correlating to clinical outcomes.

Sulfur hexafluoride lipid-type A microspheres (LumasonTM, Bracco Inc) is an FDA-approved ultrasound contrast agent. Contrast-enhanced ultrasound scan with a duration of approximately 15 minutes will be performed when a COVID-19 diagnosis has been made (or is highly suspected) according to established clinical procedures. One CEUS will be performed per patient, with up to 2 intravenous injections of the contrast agent. The dosing plan will be weight-adjusted, based on a dose of 0.03 mL/kg (with a maximum dose of 2.4 mL per injection). Organ perfusion will be evaluated in the heart, kidneys, and/or brain. Clinical outcomes during hospital stay will be collected for correlation to CEUS-based measures.

Arms: arm 1: Intravenous administration of contrast agent Sulfur hexafluoride lipid-type A microspheres before performing contrast-enhanced ultrasound (CEUS). In pediatric patients, after reconstitution 0.03 mL per kg is administered intravenously. The weight-based dose of 0.03 mL per kg will be repeated one time during a single examination. Following each injection, an intravenous flush of 0.9% Sodium Chloride is injected. The study duration per subject will be approximately 15 minutes including the time to prepare the contrast agent and perform the CEUS, as well as the 60 minute monitoring period after the first and second injection (if there are two injections of contrast) of the contrast agent.
Interventions: intervention 1: Sulfur hexafluoride lipid-type A microspheres
Intervention descriptions: intervention 1: Injection of Sulfur hexafluoride lipid-type A microspheres (Lumason) contrast agent will be performed via the existing peripheral intravenous line using the FDA-recommended dose of 0.03 mg/kg. Two bolus injections will be performed to evaluate for dynamic bowel perfusion and several 2-minute cine clips as well as static images will be acquired during the exam.

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
Title: 
Contrast 
Enhanced 
Ultrasound 
(CEUS) 
Detection 
of 
Microvascular Perfusion Impairment in COVID-19 Pediatric 
Patients 
Short Title 
CEUS in COVID-19 
Drug Name(s):                  Sulfur hexafluoride lipid-type A microspheres (LumasonTM) 
 
Regulatory Sponsor: 
Misun Hwang, MD 
eIRB Number:  
NCT Number 
20-017501 
NCT04640038 
Protocol Date:                    
 
August 10, 2020 
Amendment 1 Date: October 19, 2020 
Amendment 3 Date:  
Amendment 2 Date: 
Amendment 4 Date: 
 
 
 
 
 
Sponsor-Investigator 
Misun Hwang, MD 
The Children’s Hospital of Philadelphia 
Department of Radiology 
Philadelphia, PA, 19104 
Phone 267.425.7110 
email: Hwangm@email.chop.edu  
 
 
 
 


2 
 
 
 
 
 
 
 
 
 
 
TABLE OF CONTENTS 
1 
BACKGROUND INFORMATION AND RATIONALE ..................................................................... 1 
1.1 
INTRODUCTION .................................................................................................................................. 1 
1.2 
NAME AND DESCRIPTION OF INVESTIGATIONAL PRODUCT OR INTERVENTION .................................. 6 
1.3 
COMPLIANCE STATEMENT ................................................................................................................. 6 
2 
STUDY OBJECTIVES ............................................................................................................................ 7 
2.1 
PRIMARY OBJECTIVE ......................................................................................................................... 7 
2.2 
SECONDARY OBJECTIVES ................................................................................................................... 7 
3 
INVESTIGATIONAL PLAN.................................................................................................................. 7 
3.1 
GENERAL SCHEMA OF STUDY DESIGN ............................................................................................... 7 
3.1.1 
Study Procedures .......................................................................................................................... 7 
3.2 
STUDY DURATION, ENROLLMENT AND NUMBER OF SITES................................................................. 8 
3.2.1 
Duration of Study Participation.................................................................................................... 8 
3.2.2 
Total Number of Study Sites/Total Number of Subjects Projected ................................................ 8 
3.3 
STUDY POPULATION .......................................................................................................................... 8 
3.3.1 
Inclusion criteria .......................................................................................................................... 8 
3.3.2 
Exclusion Criteria ......................................................................................................................... 8 
4 
STUDY PROCEDURES ......................................................................................................................... 8 
4.1 
STUDY VISIT ...................................................................................................................................... 9 
4.2 
IMAGE INTERPRETATION .................................................................................................................... 9 
4.2.1 
Follow-up ...................................................................................................................................... 9 
4.3 
SUBJECT COMPLETION/WITHDRAWAL ............................................................................................... 9 
4.4 
DATA VARIABLES ............................................................................................................................ 10 
4.4.1 
EPIC ........................................................................................................................................... 10 
4.4.2 
PACS ........................................................................................................................................... 10 
5 
STATISTICAL CONSIDERATIONS ................................................................................................. 10 
5.1 
PRIMARY ENDPOINTS ....................................................................................................................... 10 
5.2 
SECONDARY ENDPOINTS .................................................................................................................. 10 
5.3 
SAMPLE SIZE AND STATISTICAL ANALYSIS PLAN ............................................................................ 10 
6 
STUDY DRUG ....................................................................................................................................... 11 
6.1 
DESCRIPTION ................................................................................................................................... 11 
6.1.1 
Drug Handling and Accountability ............................................................................................. 12 
7 
SAFETY MANAGEMENT ................................................................................................................... 12 
7.1 
CLINICAL ADVERSE EVENTS ............................................................................................................ 12 
7.2 
ADVERSE EVENT REPORTING .......................................................................................................... 12 
7.3 
DEFINITION OF AN ADVERSE EVENT ................................................................................................ 12 
7.4 
DEFINITION OF A SERIOUS ADVERSE EVENT (SAE) ......................................................................... 12 
7.4.1 
Relationship of SAE to study drug or other intervention ............................................................ 13 
7.5 
IRB/IEC NOTIFICATION OF SAES AND OTHER UNANTICIPATED PROBLEMS ................................... 13 
7.5.1 
Follow-up report ......................................................................................................................... 13 
7.6 
INVESTIGATOR REPORTING OF A SERIOUS ADVERSE EVENT TO SPONSOR ....................................... 13 
8 
STUDY ADMINISTRATION ............................................................................................................... 13 
8.1 
DATA COLLECTION AND MANAGEMENT .......................................................................................... 13 
8.2 
CONFIDENTIALITY ........................................................................................................................... 14 
8.3 
REGULATORY AND ETHICAL CONSIDERATIONS ............................................................................... 15 


3 
 
 
 
 
 
 
 
 
 
 
8.3.1 
Data and Safety Monitoring Plan ............................................................................................... 15 
8.3.2 
Risk Assessment .......................................................................................................................... 15 
8.3.3 
Potential Benefits of Trial Participation ..................................................................................... 16 
8.4 
RECRUITMENT STRATEGY ................................................................................................................ 16 
8.5 
INFORMED CONSENT/ASSENT AND HIPAA AUTHORIZATION .......................................................... 16 
8.6 
PAYMENT TO SUBJECTS/FAMILIES ................................................................................................... 18 
9    PUBLICATION…………………….………………………………………………………………16 
10    REFERENCES…………………….………………………………………………………………16 


4 
 
 
 
 
 
 
 
 
 
 
ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
 
AE 
Category B Drug 
 
 
CEUS 
CFR 
CHOP 
ED 
FDA 
HIPAA 
ICU 
IV 
IRB 
LumasonTM 
 
MRI 
ORC 
PI 
SAE 
US 
PACS 
 
 
 
 
Adverse Event 
Animal reproduction studies have failed to demonstrate a risk 
to the fetus and there are no adequate and well-controlled 
studies in pregnant women 
Contrast-Enhanced Ultrasound 
Code of Federal Regulations 
Children’s Hospital of Philadelphia 
Emergency Department 
Food and Drug Administration 
Health Insurance Portability and Accountability Act 
Intensive Care Unit 
Intravenous 
Institutional Review Board 
FDA-approved 
for 
contrast-enhanced 
ultrasound; 
the 
investigational drug 
Magnetic Resonance Imaging 
The CHOP Office of Research Compliance 
Principal Investigator 
Serious Adverse Event 
Conventional Ultrasound Scan 
Picture Archiving and Communication System 


5 
 
 
 
 
 
 
 
 
 
 
ABSTRACT 
Context:   
Initial data from COVID-19 patients in China and Italy suggests that one of the primary 
causes of death is significant endothelial injury leading to blood clotting and impaired 
multiorgan microvascular perfusion. This pilot study uses a safe, convenient bedside imaging 
tool called contrast-enhanced ultrasound (CEUS) to estimate the extent of microvascular 
perfusion impairment in the heart, kidneys, and brain of COVID-19 pediatric patients and 
assess the significance of imaging findings by correlating to clinical outcomes.  
Objectives:  
Primary: Assess microvascular perfusion of the heart, kidneys and/or brain using CEUS in 
patients with confirmed or probable diagnosis of COVID-19.  
Secondary: Correlate CEUS measures of microvascular perfusion of the heart, kidneys and/or 
brain to clinical outcomes. 
Study Design:  
Cross-sectional observational study. 
Setting/Participants:  
     Pediatric subjects at the Children’s Hospital of Philadelphia. This is a pilot study with a 
projected recruitment of 30 children with confirmed or probable diagnosis of COVID-19 at 
CHOP. 
Study Interventions and Measures:  
Contrast-enhanced ultrasound scan with a duration of approximately 15 minutes will be 
performed when a COVID-19 diagnosis has been made (or is highly suspected) according to 
established clinical procedures. One CEUS will be performed per patient, with up to 2 
intravenous injections of the contrast agent. The dosing plan will be weight-adjusted, based 
on a dose of 0.03 mL/kg  (with a maximum dose of 2.4 mL per injection). Organ perfusion 
will be evaluated in the heart, kidneys, and/or brain. Clinical outcomes during hospital stay 
will be collected for correlation to CEUS-based measures.  
 
 


6 
 
 
 
 
 
 
 
 
 
 
PROTOCOL SYNOPSIS 
Study Title 
Contrast 
Enhanced 
Ultrasound 
(CEUS) 
Detection 
of 
Microvascular Perfusion Impairment in COVID-19 Pediatric 
Patients 
Funder 
Children’s Hospital of Philadelphia 
Clinical Phase 
Pilot study. 
Study Rationale 
Initial data from COVID-19 patients in China and Italy suggests that 
one of the primary causes of death is significant endothelial injury 
leading to blood clotting and impaired multiorgan microvascular 
perfusion.  This pilot study uses a safe, convenient bedside imaging 
tool called contrast-enhanced ultrasound (CEUS) to estimate the 
extent of microvascular perfusion impairment in the heart, kidneys 
and brain of COVID-19 pediatric patients and assess the significance 
of imaging findings by correlating to clinical outcomes. 
Study objectives 
Primary: Assess microvascular perfusion of the heart, kidneys and/or 
brain using CEUS in patients with confirmed or probable diagnosis 
of COVID-19. 
Secondary: Correlate CEUS measures of microvascular perfusion of 
the heart, kidneys and/or brain to clinical outcomes. 
Test Article(s) 
(If Applicable) 
Sulfur hexafluoride lipid-type A microspheres (LumasonTM, Bracco 
Inc) is an FDA-approved ultrasound contrast agent which consists of 
active ingredients including Sulfur hexafluoride (strength 60.7 mg in 
1 mg), Distearoylphosphatidylcholine, DL- (strength 0.19 mg in 1 
mg), 1,2-Dipalmitoyl-Sn-Glycero-3-Phospho-(1’-Rac- Glycerol), 
Sodium Salt (0.19 mg in 1 mg). Inactive ingredients include 
Polyethylene Glycol 4000 (strength 24.56 mg in 1 mg) and Palmitic 
Acid (0.04 mg in 1 mg). The Sulfur hexafluoride lipid microspheres 
are composed of SF6 (molecular weight 145.9) gas in the core 
surrounded by an outer shell monolayer of phospholipids consisting 
of 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-
Dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt (DPPG-Na) 
with palmitic acid as stabilizer. Sulfur hexafluoride lipid-type A 
microspheres fall under Category B, that is, animal reproduction 
studies have failed to demonstrate a risk to the fetus and there are no 
adequate and well-controlled studies in pregnant women.1,2-
Distearoyl-sn-glycero-3-phosphocholine (DSPC), with empirical 
formula C44H88NO8P, has a molecular weight of 790.6. 1,2-
Dipalmitoyl-sn-glycero-3-phospho-rac-glycerol sodium (DPPG-Na), 


7 
 
 
 
 
 
 
 
 
 
 
with empirical formula C38H74 NaO10P, has a molecular weight of 
745. 
Study Design 
 
Cross-sectional observational study. 
Subject Population 
key criteria for Inclusion 
and Exclusion: 
Inclusion Criteria           
1. Patient (male/female) hospitalized at CHOP. 
2. Patient 17 years of age or younger. 
3. Diagnosis of COVID-19 or high clinical suspicion for 
COVID-19 despite negative tests (according to the definition 
of probable case by the ECDC). 
4. Patients have evidence of cardiovascular compromise, 
myocardial injury, acute kidney injury, and/or new-onset 
neurological symptoms. 
5. Parental/guardian permission (informed consent)  
Exclusion Criteria 
1. Medical history of Lumason hypersensitivity 
Number of Subjects  
 
Total recruitment: 30 patients with confirmed or probable diagnosis 
of COVID-19.  
Study Duration 
CEUS has a total duration of approximately 15 minutes including 
contrast agent injection and image completion. 
Study Visit 
Subjects will be imaged at bedside at CHOP for approximately 15 
minutes. 
Efficacy Evaluations 
CEUS scans of diagnostic quality, qualitative/quantitative evaluation 
of microvascular perfusion of heart, kidneys and/or brain in COVID-
19 patients, correlation between CEUS and clinical outcomes of 
COVID-19. 
Pharmacokinetic 
Evaluations 
There are no pharmacokinetic evaluations. 
 
Safety Evaluations 
All subjects receiving at least one injection of investigational drug 
will be included in the safety analysis. The frequencies of AEs by 
type, severity, and temporal relationship to the CEUS scan will be 
summarized. SAEs (if any) will be described in detail.  
Statistical and Analytic 
Plan 
This is a pilot study with a projected recruitment of 30 children with 
COVID-19. 
See Section 5.3 for further detail. 


8 
 
 
 
 
 
 
 
 
 
 
DATA 
AND 
SAFETY 
MONITORING PLAN 
The safety monitoring for this study is the primary responsibility of 
the study team. Regular meetings to discuss the outcomes of the 
study, and of the safety events, will be conducted by the study team. 
The occurrence of adverse events, serious adverse events and 
unanticipated events will be reported by the study team in accordance 
with federal and institutional guidelines, as outlined in Section 7 of 
this clinical study. 
 


1 
 
 
 
 
 
 
1 
BACKGROUND INFORMATION AND RATIONALE 
1.1 
Introduction 
Initial data from COVID-19 patients in China and Italy suggests that one of the primary 
causes of death is significant endothelial injury leading to blood clotting and impaired 
multiorgan microvascular perfusion. Cardiovascular complications can arise in both patients 
with or without prior cardiovascular history. High cardiac troponin levels are associated with 
severe COVID-19. It has been postulated that direct viral effects as well as secondary 
inflammatory response can predispose to thromboembolic diseases. Therefore, thrombotic 
events affecting target organs, such as the heart and kidneys, are common (1). One of the 
most recently reported complications of COVID-19 infection in children is known as 
Multisystem Inflammatory Syndrome in Children (MIS-C). Key findings of this syndrome 
include prominent cardiac dysfunction, enteropathy, and relative thrombocytopenia.  Those 
patients frequently present with cardiogenic shock or acute left ventricular dysfunction in the 
setting of a multisystem inflammatory state (1). Similarly, patients with COVID-19 also 
present with neurological symptoms. Anosmia and ageusia are very common in adults but 
not very well elucidated in children. Abdel-Mannan et al (2) reported 4 cases of children 
without respiratory symptoms and new-onset neurological symptoms including 
encephalopathy, headaches, brainstem and cerebellar signs, muscle weakness, and reduced 
reflexes. Those patients had signal changes in the corpus callosum. There have been other 
cases reporting encephalopathy, encephalitis and cerebrovascular disease in COVID-19 
patients (3).  
The current study uses a safe, convenient bedside imaging tool called contrast-enhanced 
ultrasound (CEUS) to measure the extent of microvascular perfusion impairment in the heart 
and kidneys of COVID-19 pediatric patients and, as exploratory analysis, to assess the 
significance of imaging findings by correlating to clinical outcomes. To the investigator’s 
knowledge, this is the first study evaluating microvascular perfusion of the heart and kidneys 
in COVID-19 pediatric patients.  
The completion of this initial study will be important to obtain preliminary descriptive 
statistics which includes means, standard deviations, medians, minimum maximum and the 
95% confidence intervals (CI) for continuous variables. Frequencies, proportions, and the 
95% CIs necessary for designing a future trial. In the future, validation of such microvascular 
perfusion based biomarker for prognostication of severe COVID-19 related complications 
such as death, mechanical ventilation, myocardial infarction, stroke or renal failure will be 
tremendously helpful. In addition, the presence of an easily attainable in vivo biomarker of 
disease will help with resource allocation in the ICU when faced with limited supplies and 
staff. Whereas computed tomography and magnetic resonance imaging contrast agents can 
potentially be nephrotoxic, ultrasound contrast agents are not cleared through the kidneys 
and can be used for patients with renal failure, which is prevalent in COVID-19. CEUS can 
also be performed at the bedside at low cost, without the need to transport patients to CT or 
MRI suite, which reduces infection risk and provides real-time, rapid decision-making for 
clinicians.  
According to the European Centre for Disease Prevention and Control (ECDC), COVID-19 
cases can be classified as possible, probable, or confirmed. A case is possible if the person 


2 
 
 
 
 
 
 
meets the clinical criteria (at least one of the following symptoms: cough, fever, shortness of 
breath, or sudden onset anosmia, ageusia/dysgeusia). A case is probable if the person meets 
the clinical criteria with an epidemiological link or if the person meets the diagnostic criteria 
(radiologic evidence showing lesions compatible with COVID-19. A case is confirmed if the 
person meets the laboratory criteria (detection of SARS-CoV-2 nucleic acid in a clinical 
specimen). It is noteworthy that test accuracy highly depends on specimen collection by 
swabs among other variables (including intrinsic test accuracy). For that reason, patients with 
high clinical suspicion (probable cases as per the ECDC definition) for COVID-19 despite 
negative tests will be considered as subjects in our study (4).  
Microvascular Injury in COVID-19 
Initial data from COVID-19 patients demonstrates that poor outcomes from the disease is 
primarily due to hypercoagulability and multiorgan microvascular perfusion impairment (5-
7). Whereas pulmonary symptoms of COVID-19 are well known, less publicized are 
widespread incidence of multiorgan failure due to blood clots which are as common as 
pulmonary disease in deceased patients (5-10). An international collaborative review was 
recently presented to outline the pathogenesis, epidemiology, and management of COVID-
19 patients with thrombotic events (9). Other studies have reported the biochemical 
mechanisms that trigger thromboembolic diseases (10, 11).  
The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory 
syndrome coronavirus (SARS-CoV)-2  utilizes angiotensin-converting enzyme 2 (ACE2) as 
the host cellular receptor for virus spike (S) protein according to structural analysis and in 
vivo experiments (11). It has been shown that the intestine displays the highest expression of 
ACE2, followed by testis and kidney which explains  viral detection in feces and urine. The 
expression of ACE2 in the heart is lower than that in the intestine and kidney, but higher than 
that in the lung which serves as a main target organ of this virus, indicating a potential 
infection susceptibility of the human heart. Epidemiological data indicates that, over two-
thirds of patients who died from COVID-19 had diabetes or cardiovascular disease (12), 
potentially due to the preexisting vascular disease with enhanced vulnerability to COVID-19 
related vascular insult.  
In blood samples of COVID-19 positive patients, increased coagulation factors are 
commonly observed. In a cohort of 799 patients from China, marked derangement in 
coagulation factors was commonly observed in 113 deceased patients (5). Median 
prothrombin time was significantly longer in deceased patients (15.5) than in recovered 
patients (13.9). D-dimer concentrations were markedly greater in deceased patients (4.6 
µg/mL) than in recovered patients (0.6 µg/mL). In addition, concentrations of procalcitonin, 
high sensitivity C-reactive protein, and ferritin, as well as erythrocyte sedimentation rate, 
were significantly higher in deceased patients than in recovered patients. A very recent study 
by Yao et al. (7) (published online ahead of print) confirms that SARS-CoV-2 infection, in 
addition to the lungs, damages vessels of the      kidney and other organs, and hyaline thrombi 
were found in small vessels in different organs. Interestingly, Middle East respiratory 
syndrome (MERS) which first emerged on the Saudi Arabian peninsula in 2012 caused by 
MERS coronavirus (MERS-CoV) was also shown to cause microvascular thrombosis (9), 
suggesting the possible generalizability of using microvascular perfusion as a marker of 
disease burden, prognosis, and therapy guidance in viral outbreaks/pandemics.  


3 
 
 
 
 
 
 
Beyond the pulmonary involvement, the most commonly reported co-morbidities in COVID-
19 include acute kidney injury (AKI), cardiac damage and abdominal pain likely due to the 
tropism of SARS-CoV-2 for these organs given the high affinity of SARS-CoV-2 to ACE2 
in the kidney (13). A recent study reported that the human kidney is a specific target for 
SARS-CoV-2 infection (9). Diao and colleagues (9, 10) after examining the viral 
nucleocapsid protein in situ in the kidney post-mortem found that SARS-CoV-2 antigens 
accumulated in kidney tubules, suggesting the possibility of direct viral infection of the 
kidney prior to inducing AKI. CT scan of the kidneys showed reduced density, suggestive of 
inflammation and edema (14). Cardiac involvement of the disease in COVID-19 patients of 
poor outcomes has also been documented, with findings ranging from angina, arrhythmia, to 
acute cardiac injury (6, 15). Note that in the heart, ACE2 was found to be highly expressed 
in pericytes, which are distributed on the abluminal sides of endothelial cell of capillaries and 
part of venules, and play a critical role in myocardial microcirculation (6). 
Recent histopathological evidence from COVID-19 autopsy cases suggests that vascular 
coagulopathy is the predominant finding in the lungs, heart, and kidneys. Worsening 
respiratory failure is increasingly believed to be caused by pulmonary microvascular 
thrombosis (16) (Figure 1). In the lungs, diffuse thickening and fibrin deposition are seen in 
alveolar capillaries and serving as the nidus for entrapment of inflammatory cells (17); this 
contrasts with the finding that large arteries such as the pulmonary artery lacked thrombi. 
Small vessel thrombi were seen in the periphery of the lungs in the majority of cases (in 
keeping with radiographic appearance of peripheral predominant infiltrates). The majority of 
cases, with the exception of the immunocompromised, do not demonstrate neutrophilic 
infiltrates or evidence of infection in the airways or the interstitium. Another report details 
that in severe COVID-19 patients who become critically ill, generalized thrombotic 
microvascular injury is seen (18). This article reports striking septal capillary injury with 
extensive deposits of the terminal complement components C5b-9 (membrane attack 
complex), C4d, and mannose binding lectin (MBL)-associated serine protease (MASP) 2; 
furthermore, it was found that COVID-19 spike glycoproteins co-localized with C4d and 
C5b-9 in the inter-alveolar septa and the cutaneous microvasculature.  
Figure 1. Diffuse alveolar damage in fatal COVID-19 (A). Fibrinous microthrombi in small 
sized pulmonary arterioles, observed in 8 out of 10 patients (B-D). Marisa D et al. Journal of 
Thrombosis and Hemostasis 2020 [Epub ahead of print] 


4 
 
 
 
 
 
 
It is also interesting to see the sporadic reports of previously healthy young adults suddenly 
dying of COVID-19 or developing strokes. In such cases, a sudden thrombosis-related event 
seems plausible. A previously healthy COVID-19 patient has presented with deep vein 
thrombosis (19). Sudden deaths of infants and pediatric patients, while rare, also raise the 
same concern. Recent reports detail infant death with underlying heart condition (20) and 
without pre-existing conditions (21), in neither of which the cause of death is clear. The 
question as to whether and how viral infection causes sudden infant death has previously 
been raised (22), and is still unknown. In discussion with a CHOP pathologist, we have a 
COVID-19 positive fetal demise case at HUP in which fibrin/thrombi deposition in the 
placental vessels is evident. This suggests that while there is paucity of literature on 
infants/pediatric patients, it is plausible that the virus causes death in a similar way to      
adults.  
Note that current imaging tools cannot detect micron scale microcirculatory dysfunction; 
here, we have a unique tool and expertise to assess microvascular dysfunctions that may be 
the key factor leading to significant mortality and morbidity in these patients.  
Contrast-Enhanced Ultrasound (CEUS) for Microvascular Flow Assessment 
CEUS uses intravascularly injected microbubbles of 2-3 µm in size, smaller than red blood 
cells, to depict perfusion abnormalities down to 10 µm resolution. Organ perfusion 
abnormalities including focal or diffuse infarct can readily be detected with CEUS but not 
with Doppler ultrasound. It is FDA-approved for pediatric and adult applications for the 
evaluation of the heart and liver and used clinically at CHOP. CEUS has previously been 
used to evaluate cardiac/renal microvascular perfusion (23-28).  
The PI has pioneered the application of CEUS in the pediatric population (29-34) and is 
conducting two Investigational New Drug-approved clinical trials in the most vulnerable 
patients including critically ill, unstable infants with hypoxic ischemic injury and necrotizing 
enterocolitis. CEUS can be performed conveniently at bedside without the need for patient 
transport, it is easily repeatable, and cost-effective, which are critical aspects of resource 
allocation during the COVID-19 pandemic. Furthermore, there is no current technology that 
can accurately assess organ microvascular perfusion. Color Doppler, also performed at the 
bedside, is limited to macrovascular flow (Figure 2). 
Figure 2. Doppler versus 
CEUS Assessment of 
Brain 
Microvascular 
Flow. 
Shown 
are 
Doppler (A) and CEUS 
(B) images of the porcine 
brain in coronal plane 
demonstrating the lack of 
microvessels 
depicted 
with 
Doppler 
as 
compared to delineation 
of < 10 µm microbubbles 
throughout the brain on 
CEUS. 
[Grab your reader’s attention with 
a great quote from the document or 
use this space to emphasize a key 
point. To place this text box 
anywhere on the page, just drag it.] 


5 
 
 
 
 
 
 
 
CEUS images can be assessed in different ways. The degree of perfusion defect can be 
categorized as mild, moderate, or severe. Advanced quantitative analysis of perfusion can be 
done either by intensity analysis or microbubble tracking methods. (Figure 3).  
 
 
 
 
 
 
Figure 3. Shown to the left is a 
post-processed CEUS image of 
the same porcine brain depicting 
microvasculature down to the < 
10 µm resolution. Microvascular 
flow trajectory and velocity can 
also be calculated from this data. 
 
 
 
Even upon visual examination, organ ischemia is readily evident on CEUS, whereas it is 
difficult to assess with grayscale or Doppler ultrasound alone (Figure 4). At CHOP, CEUS 
is routinely done as part of clinical practice, and up to 4-5 CEUS exams  are performed per 
day.  
 
 
Figure 3. CEUS of the kidneys. Shown are sagittal CEUS images of the kidneys, (A) demonstrating 
homogeneous perfusion of normal right kidney with high signal due to intravascular microbubbles and (B) 
demonstrating low signal regions (yellow arrows) consistent with ischemia.  
 


6 
 
 
 
 
 
 
CEUS Quantification: It should be noted that CEUS gives a wide range of quantifiable, 
continuous parameters reflective of tissue perfusion. Injection of contrast results in increased 
acoustic signal quantifiable as signal intensity in decibels (dB). Spatiotemporal changes in 
signal intensity are then measured to reflect tissue perfusion, flow rate, blood volume, and 
many other parameters that can be derived from the time intensity curve (TIC) analysis. In 
TIC analysis, signal intensity in a given region of interest is plotted over time and parameters 
are extracted from the curve (for e.g. time to peak or TTP, wash-in area under the curve or 
WiAUC, rise time or RT, wash-in rate or WiR, wash-in perfusion index or WiPi). This is a 
well-accepted conventional approach to CEUS quantification previously used by the PI in 
the report of the first clinical guidelines for application of brain CEUS in infants (29), 
quantitative detection of hypoxic ischemic injury using brain CEUS (30), and quantification 
of tumor microvessel density (35). More advanced analysis involves the particle tracking 
method, in which individual intravascular microbubbles are tracked across thousands of 
ultrasound frames to quantify microbubble velocity, spatial distribution, trajectory (and other 
sophisticated parameters such as shear stress, acceleration-deceleration, etc.) (36). The 
conventional time intensity curve and particle tracking method combined would yield more 
than >30 continuous CEUS microvascular perfusion parameters that can each, or in 
combination, be correlated to clinical outcomes in this proposal. 
1.2 
Name and Description of Investigational Product or Intervention 
Sulfur hexafluoride lipid-type A microspheres (LumasonTM, Bracco Inc) is FDA-approved 
ultrasound contrast agent which consist of active ingredients including Sulfur hexafluoride 
(strength 60.7 mg in 1 mg), Distearoylphosphatidylcholine, DL- (strength 0.19 mg in 1 mg), 
1,2-Dipalmitoyl-Sn-Glycero-3-Phospho-(1’-Rac-Glycerol), Sodium Salt (0.19 mg in 1 mg). 
Inactive ingredients include Polyethylene Glycol 4000 (strength 24.56 mg in 1 mg) and 
Palmitic Acid (0.04 mg in 1 mg). The sulfur hexafluoride lipid microspheres are composed 
of SF6 (molecular weight 145.9) gas in the core surrounded by an outer shell monolayer of 
phospholipids consisting of 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-
Dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt (DPPG-Na) with palmitic acid as 
stabilizer. Sulfur hexafluoride lipid-type A microspheres fall under Category B, that is, 
animal reproduction studies have failed to demonstrate a risk to the fetus and there are no 
adequate and well-controlled studies in pregnant women.1,2-Distearoyl-sn-glycero-3-
phosphocholine (DSPC), with empirical formula C44H88NO8P, has a molecular weight of 
790.6. 1,2-Dipalmitoyl-sn-glycero-3-phospho-rac-glycerol sodium (DPPG-Na), with 
empirical formula C38H74 NaO10P, has a molecular weight of 745. In pediatric patients, 
after reconstitution 0.03 mL per kg is administered intravenously. The weight-based dose of 
0.03 mL per kg will be repeated twice during a single examination. Following each injection, 
an intravenous flush of 0.9% Sodium Chloride is injected.  
1.3 
Compliance Statement 
This study will be conducted in full accordance with all applicable Children’s Hospital of 
Philadelphia Research Policies and Procedures and all applicable Federal and state laws and 
regulations including 45 CFR 46, 21 CFR Parts 50, 56, and 312, and the HIPAA Privacy 
Rule. Any episode of noncompliance will be documented. 


7 
 
 
 
 
 
 
The investigators will perform the study in accordance with this protocol, will obtain consent 
and assent (unless a waiver is granted), and will report unexpected problems in accordance 
with The Children’s Hospital of Philadelphia IRB Policies and Procedures and all federal 
requirements. Collection, recording, and reporting of data will be accurate and will ensure 
the privacy, health, and welfare of research subjects during and after the study.  
2 
STUDY OBJECTIVES 
2.1 
Primary Objective  
Assess microvascular perfusion of the heart, kidneys and/or brain using CEUS in patients 
with confirmed or probable diagnosis of COVID-19. 
2.2 
Secondary Objectives 
Correlate CEUS measures of microvascular perfusion of the heart, kidneys and/or brain to 
clinical outcomes. 
3 
INVESTIGATIONAL PLAN 
3.1 
General Schema of Study Design 
This is a pilot study in which 30 subjects with confirmed or probable COVID-19 will undergo 
CEUS of heart, kidneys and/or brain.  
The dosing plan will be weight-adjusted, based on a dose of 0.03 mL/kg. Each patient will 
receive up to two contrast injections as per the product label/FDA approved guidelines, with 
a maximum dose of 2.4 mL per injection. Examinations (heart, kidneys, and/or brain) will be 
made at the same time, as soon as the first product intravenous injection takes place. The 
examination will last approximately 15 minutes but no more than one hour.  
As exploratory analysis, CEUS findings on these patients will be correlated to clinical 
outcomes (death, mechanical ventilation, heart attack, stroke, and renal failure). 
3.1.1 Study Procedures 
The study team will screen hospitalized patients for COVID-19 probable or confirmed 
cases. Recruitment will occur via medical chart review and/or physician referral. 
Study Phase 
Screening 
(telephone
) 
CEUS Study Encounter  
Informed Consent 
X 
X 
Review Inclusion/Exclusion 
Criteria 
X 
X 
CEUS safety 
X 
X 
Demographics/Medical 
History 
X 
X 
Clinical Laboratory 
Evaluation  
 
X 


8 
 
 
 
 
 
 
Lumason IV administration 
 
X 
CEUS examination 
 
X 
Acute Adverse Event 
Assessment (<20 minutes 
after contrast administration) 
 
X 
 
3.2 
Study Duration, Enrollment and Number of Sites 
3.2.1 Duration of Study Participation 
Study participation will occur during the inpatient stay. Study duration per participant will 
include telephone screening lasting approximately 5-10 minutes to confirm eligibility for the 
study and CEUS safety.  
The CEUS exam will last approximately 15 minutes but no more than one hour. Only one 
CEUS scan will be performed per patient.  
Clinical outcomes during hospitalization will be documented via chart review. No outpatient 
follow-up will be performed. 
3.2.2 Total Number of Study Sites/Total Number of Subjects Projected 
This is a pilot study with a projected recruitment of 30 children with confirmed or probable 
diagnosis of COVID-19 at the Children’s Hospital of Philadelphia.  
3.3 
Study Population 
3.3.1 Inclusion criteria 
1. Patient (male/female) hospitalized at CHOP. 
2. Patient 17 years of age or younger. 
3. Diagnosis of COVID-19 or high clinical suspicion for COVID-19 despite negative 
tests (according to the definition of probable case by the ECDC). 
4. Patient has evidence of cardiovascular compromise, myocardial injury, acute kidney 
injury, and/or new-onset neurological symptoms). 
5. Parental/guardian permission (informed consent) 
3.3.2 Exclusion Criteria 
1. Medical history of Lumason hypersensitivity. 
 
4 
STUDY PROCEDURES 
Screening will be performed via medical chart review and/or clinical referral of pediatric 
subjects at the Children’s Hospital of Philadelphia. Subjects who fulfill eligibility criteria 
will be included.  


9 
 
 
 
 
 
 
CEUS scan will be performed by CHOP study team members and/or dedicated research 
ultrasonographers. Subjects will undergo one CEUS exam. The dosing plan will be weight-
adjusted, based on a dose of 0.03 mL/kg.  Up to two injections per study will be performed 
as per product label/FDA guidelines. Subjects’ and personnel safety will be handled 
according to clinical protocol for COVID-19.  
Organ perfusion will be evaluated in the heart, kidneys and/or brain, provided there is 
evidence of cardiovascular compromise, myocardial injury, acute kidney injury, and new-
onset neurological symptoms. Brain ultrasound will occur if there are new-onset neurological 
symptoms. We will use a low energy approach to minimize the risk of thermal injury or 
disruption of the blood-brain-barrier (mechanical index < 0.2 is safe).  
Analysis of CEUS imaging by the Principal Investigator will be performed, in addition to 
medical chart review for clinical outcomes (exploratory analysis). 
4.1 
Study Visit 
Upon patient entry into this study we will: 
● Review inclusion/exclusion criteria 
● Obtain written informed consent in a private setting.  
● Perform CEUS exam 
● Department policies concerning COVID19 safety will be followed 
4.2 Image interpretation 
Images will be assessed by the Principal Investigator (see Primary Endpoint in Section 5.1). 
Results of image interpretation will not be reported nor impact clinical management. No 
personnel beyond the PI and study personnel will have access to the master list with 
identifiable findings. 
4.2.1 Follow-up 
The medical chart of subjects who underwent CEUS examination will be reviewed to 
assess clinical outcomes (see Secondary Endpoint in Section 5.2). 
4.3 
Subject Completion/Withdrawal 
Subjects may withdraw from the study at any time without prejudice to their care.  They may 
also be discontinued from the study if they become anxious during this research examination. 
No sedation will be given. The Investigator may also withdraw subjects who violate the study 
plan, or to protect the subject for reasons of safety or for administrative reasons.  It will be 
documented whether each subject completes the research study. If the Investigator becomes 
aware of any serious, related adverse events after the subject completes or withdraws from 
the study, they will be recorded in the source documents. 


10 
 
 
 
 
 
 
4.4 
Data variables 
4.4.1 EPIC 
● Name 
● MRN 
● Date of birth 
● Sex 
● Chief complaint  
● Symptoms 
● Duration of symptoms 
● Hospital stay 
● Medications being used during hospitalization 
● Laboratory tests during hospitalization 
● Presence of myocardial infarction      during hospitalization 
● Presence of stroke during hospitalization 
● Presence of renal failure during hospitalization 
● Time to recovery 
● Need for oxygen 
● Need for mechanical ventilation 
● Vascular complications 
 
4.4.2 PACS 
● Accession number 
● All images obtained using CEUS 
5 
STATISTICAL CONSIDERATIONS 
5.1 
Primary Endpoint 
1. Estimate the proportion of patients with normal perfusion versus area(s) of 
hypoperfusion in heart, kidneys and/or brain. 
 
5.2 
Secondary Endpoints 
1. Evaluate heart, kidneys and/or brain perfusion using time to peak (TTP) and wash-
in area under the curve (WiAUC). 
2. Death within 28 days of imaging or mechanical ventilation within 14 days of 
imaging as reported in the medical chart.  
3. Myocardial infarction, stroke, or renal failure as reported in the medical chart. 
5.3 
Sample Size and Statistical Analysis Plan 
We will enroll 30 subjects (15 male/15 female) in this pilot study. As part of clinical care, 
CHOP Radiology also performs approximately 4-5 contrast-enhanced ultrasound cases in a 
given day, which is higher in volume than any other children’s hospital in the country.  


11 
 
 
 
 
 
 
A sample size of 30 should provide reasonable estimates of the perfusion metrics. For 
example, a sample size of 30 participants produces a two-sided 95% CI with a width equal 
to 0.3 when the estimated proportion is as small as 0.3 (95% CI=0.1, 0.5). Also, a two-sided 
95% CI with a width equal 0.6 when the estimate of Pearson’s product-moment correlation 
is 0.4 (95% CI= 0.05, 0.7). 
Based on microangiopathy/ischemia observed in COVID-19 patients (9-11), we expect our 
cohort to display delayed perfusion, hypoperfusion or no perfusion of the myocardium,  
kidneys, and/or brain. Results of the myocardial, renal and brain perfusion will be assessed 
by two experienced researchers in the field of CEUS and report it qualitatively as either 
normal or abnormal. In case of diagnosis disagreement between the two raters, a third 
experienced researcher will confirm the correct diagnosis. Hence, the proportion and 
frequency of myocardial, renal, and/or brain perfusion abnormalities will be documented. As 
additional primary analysis, we will also assess quantitative CEUS and derive time-to-peak 
(TTP) and wash-in area under the curve (WiAUC).  
Demographic characteristics and descriptive statistics (such as measures of frequency, 
central tendency, dispersion, and position) will be summarized for the sample and by male 
and female sex. The percentage of COVID-19 patients with an abnormal clinical exam will 
be computed. Time intensity curve analysis will provide further details on the nature of 
perfusion abnormalities. The following presents the variable names, output, and the reported 
statistics for the primary and secondary aims.  
  
Measured Variables 
Output and statistics 
Primary Analysis 
Perfusion defect (hypo- or 
absent perfusion) 
Proportion of COVID-19 
patients with perfusion 
defect 
Primary Analysis 
Perfusion quantified using 
time-to-peak (TTP) and 
wash-in area under the 
curve (WiAUC) from the 
time-intensity curve of 
contrast signal 
Summary statistics for TTP 
and WiAUC, for e.g. mean, 
standard deviation, 95% CI 
Secondary Analysis 
Clinical outcomes from 
chart review etc. 
Correlation of CEUS 
metrics to clinical outcomes 
 
6 
STUDY DRUG 
6.1 
Description 
LumasonTM is currently FDA approved for use in the evaluation of heart and liver in the 
pediatric and adult population. Recommended intravenous dose for LumasonTM is weight-
based, 0.03mL/kg as an intravenous injection, up to a maximum of 2.4mL per injection. The 
dosing plan will be weight adjusted, based on a dose of 0.03 mL/kg. Subjects will be 
monitored for potential side effects for 60 minutes following the final study drug dose. It is 
currently used clinically at CHOP radiology department for routine diagnostic ultrasound 
studies.  


12 
 
 
 
 
 
 
 
6.1.1 Drug Handling and Accountability 
LumasonTM, will be maintained as a separate supply from the Radiology Department for 
accountability. For each subject, the LumasonTM lot number, expiration date, number of vials 
used, disposition of unused LumasonTM, and discard procedure, will be recorded as part of 
the study record. 
7 
SAFETY MANAGEMENT 
As is standard clinical practice at CHOP, we will follow all current safety standards for CEUS 
established by the FDA and ACR to provide adequate protection from potential hazards or 
injuries to subjects.  To assure the safety of the participants, all subjects will be monitored 
for adverse events during the procedure by direct communication through an intercom system 
and by visual observance. Data will only be transferred between members of the study team, 
and no identifiable data will be shared with non-CHOP or CHOP individuals who are not on 
the study team. 
7.1 
Clinical Adverse Events 
Clinical adverse events (AEs) will be monitored throughout the study.  
7.2 
Adverse Event Reporting 
Unanticipated problems related to the research involving risks to subjects or others that occur 
during this study (including SAEs) will be reported to the IRB in accordance with CHOP 
IRB SOP 408: Unanticipated Problems Involving Risks to Subjects. AEs that are not serious 
but that are notable and could involve risks to subjects will be summarized in narrative or 
other format and submitted to the IRB at the time of continuing review.  
7.3 
Definition of an Adverse Event 
An adverse event is any untoward medical occurrence in a subject who has received an 
intervention (drug, biologic, or other intervention).  The occurrence does not necessarily have 
to have a causal relationship with the treatment.  An AE can therefore be any unfavorable or 
unintended sign (including an abnormal laboratory finding, for example), symptom, or 
disease temporally associated with the use of a medicinal product, whether or not considered 
related to the medicinal product. 
All AEs (including serious AEs) will be noted in the study records and on the case report 
form with a full description including the nature, date and time of onset, determination of 
non-serious versus serious, intensity (mild, moderate, severe), duration, causality, and 
outcome of the event. 
7.4 
Definition of a Serious Adverse Event (SAE) 
We will follow IRB SOP 802 for reporting SAEs, should one occur.  
 


13 
 
 
 
 
 
 
7.4.1 Relationship of SAE to study drug or other intervention 
The relationship of each SAE to the study intervention should be characterized using one of 
the following terms in accordance with CHOP IRB Guidelines: definitely, probably, 
possibly, unlikely or unrelated.  
7.5 
IRB/IEC Notification of SAEs and Other Unanticipated Problems 
The Investigator is responsible for promptly notifying the IRB of all on-site AEs that are (1) 
serious, (2) unexpected and (3) related to the research intervention or other study procedures 
and any other unanticipated problems involving risk to subjects or others using the CHOP 
Internal SAE reporting form and in accordance with the following timeline. External SAEs 
that are both unexpected and related to the study intervention should be reported as they are 
received using the External SAE form (if applicable). 
Type 
of 
Unanticipated 
Problem 
Initial 
Notification  
(Phone, Email, Fax) 
Written Report 
Internal 
(on-site) 
SAEs 
Death or Life Threatening  
24 hours 
Within 2 calendar days 
Internal 
(on-site) 
SAEs 
All other SAEs 
7 days 
Within 7 business days 
Unanticipated 
Problems 
Related to Research 
7 days  
Within 7 business days 
All other AEs 
N/A 
Brief Summary of important 
AEs may be reported at time of 
continuing review 
7.5.1 Follow-up report 
If an SAE has not resolved at the time of the initial report and new information arises that 
changes the investigator’s assessment of the event, a follow-up report including all relevant 
new or reassessed information (e.g., concomitant medication, medical history) should be 
submitted to the IRB. The investigator is responsible for ensuring that all SAE are followed 
until either resolved or stable.  
7.6 
Investigator Reporting of a Serious Adverse Event to Sponsor 
Reporting must be consistent with regulatory, sponsor or GCRC requirements. 
8 
STUDY ADMINISTRATION 
8.1 
Data Collection and Management 
Confidentiality:  All subjects will be assigned a number unrelated to their medical record 
number or name and this will be kept in a master list. The master list will contain patient 
identifiers and a link to the research-specific code. The master list will be kept in the PI and 
Study coordinator’s password protected computer at CHOP, on the secure storage network 


14 
 
 
 
 
 
 
on the secure Hospital server. Co-investigators will have access to patient identifier’s (name, 
MRN) during the optimization phase of their study. When the optimization finishes, or the 
article is published all the identification will be erase and only kept in the master list at the 
PI and Study coordinator computer. The collection of data from EPIC, ChartMaxx, and/or 
iSite Radiology will be using the MRN or name. Data will only be transferred between 
members of the study team; no identifiable data will be shared with non-CHOP or CHOP 
individuals who are not on the study team. 
Security:  All files (master list and coded data) will be password protected and will be stored 
on a password protected computer at CHOP, or in a password-protected hard drive. The hard 
drive will meet CHOP IT standards for encryption and password protection. The only way 
we will use to transfer internal information will be [send secure] emails using the study 
members’ @email.chop.edu accounts. Access to the medical records and diagnostic studies 
to collect the data requires patient identifiers such as: medical record number and name. 
Without these, we would be unable to retrieve and review any information previously 
collected. For that reason, during the data collection process, we could send identifiable data 
using the protected CHOP-email.  
Image data will be given a unique code and will have no information that can identify the 
subjects.  Information that can identify subjects or image data may be kept in the master list 
at the PI and study coordinator’s computer. Only the study team (co-investigators, PI, study 
coordinator, research assistants, etc) will be able to see information that can identify the 
subjects. If the subject leaves the study, he/she can ask to have the image data collected to be 
removed or destroyed. Imaging data will be archived to permanent storage media at the 
CHOP secure PACS system.   
The study team will share unique research data accrued from this pilot clinical trial to enhance 
the value and further the advancement of research. The study team will make available such 
data after study completion and the data will only be provided to those investigators who 
agree to adhere to a signed research data use agreement. The data and associated 
documentation will be available to users only under a data-sharing agreement that provides 
for: (1) a commitment to using the data only for research purposes; (2) a commitment to 
securing the data using appropriate computer technology; and (3) a commitment to 
destroying or returning the data after analyses are completed. The data obtained will be 
periodically reviewed (once every month) by the study team for discussion and improvement 
as well as planning for dissemination of results to the scientific community. 
8.2 
Confidentiality 
All information gained about the individual patients will remain confidential.  When data is 
used for reports on the usefulness of the studies it will be compiled into statistical summaries 
from many studies, and the data will not be identified with any patient. After study closure, 
all data, including identifiers, gathered from the study will be archived for 6 years in a 
password-protected folder on the primary computer of the PI, which complies with CHOP 
policy A-3-9 for data retention. As per the FDA regulations, all study data (including PHI) 
will be kept for 2 years after last marketing approval for the drug. In keeping with CHOP 
policy A-3-9 and FDA requirements, the data will be destroyed based on whichever period 
of time is longer. 


15 
 
 
 
 
 
 
All HIPAA and IRB guidelines will be followed.  The records generated during this study 
will be kept confidential and the data will only be used for the purpose of conducting the 
study.  Safeguards are described under Section 8.1 Data Collection and Management. 
8.3 
Regulatory and Ethical Considerations 
8.3.1 Data and Safety Monitoring Plan 
The safety monitoring for this study is the primary responsibility of the study team. The 
occurrence of adverse events, serious adverse events and unanticipated events will be 
reported by the study team in accordance with federal and institutional guidelines, as outlined 
in Section 7 of this clinical study. 
 
8.3.2 Risk Assessment 
The potential risks associated with contrast-enhanced ultrasonography have been extensively 
studied, and the risk associated with the technique is less than that of CT or MRI contrast 
agents. The risk of adverse events is the lowest of all contrast agents available, with CT 
contrast being the highest (0.6%), followed by MR contrast (0.0088%) and ultrasound 
contrast (0.0086%). The safety profile of LumasonTM has been documented in a large cohort 
including 23,188 adults, with no fatal event and only 29 (3 severe, 3 moderate, and 23 mild) 
adverse events. Further studies detail the safety profile of ultrasound contrast agents in 
children and have shown minor adverse events including nausea, tinnitus, lightheadedness, 
altered taste sensation. One documented severe reaction in a child documented symptoms of 
generalized pruritus, nausea, hypotension with tachycardia initially then bradycardia [6]. 
Management in this instance consisted of oxygen, intravenous epinephrine, and fluids (0.9% 
normal saline) with resolution of symptoms in two hours. Treatment of both minor, mild, and 
severe adverse reactions post LumasonTM administration are the same as that of CT or MRI 
contrast agents. In comparison to CT or MRI contrast agents, however, ultrasound contrast 
agents have proven to be much safer in children with only one serious adverse event over 
decades of its use to date (contrasting to approximately 15-20 adverse events per 2000 
children if CT contrast agent were to be used). No serious adverse event has been reported in 
a neonate since its clinical use in this population. Animal studies on its toxicity profile also 
validate no fetal toxicity and the ultrasound contrast agent belongs to category B. However, 
a few serious adverse reactions in older patients have been reported, such as 
anaphylactoid/hypersensitivity reactions, arrhythmias and hypertensive episodes, commonly 
within 30 minutes of Lumason administration, some of them causing death. 
Risks of the administration of the study drug are considered a minor increase above minimal 
risk, with no prospect of direct benefit for subjects. Adverse effects are not dose-dependent, 
thus risk is not increased by modifying the timing of interventions or increasing the number 
of contrast-agent injections. 
Another risk of the study includes the insertion of a peripheral IV line. This is a no greater 
than minimal risk procedure, with the main risks of discomfort, bruising, and infection which 
are generally self-limited. There is a no greater than minimal risk of breach of confidentiality, 
which is minimized by having all of the study personnel undergo HIPAA training.  


16 
 
 
 
 
 
 
8.3.3 Potential Benefits of Trial Participation 
There is no prospect for direct benefit. However, improved understanding of viral impact on 
microvascular perfusion in relation to outcomes will be tremendously helpful for scientific 
knowledge and therapeutic directions.  
The potential finding that CEUS can detect microvascular perfusion abnormalities in the 
heart/kidneys, and the proportion of those with microvascular perfusion abnormalities in the 
heart/kidneys is high in COVID-19 pediatric patients. This will lead to larger observational 
or interventional trials that can advance this tool for detection of microvascular perfusion 
dysfunction, prognostication, and guidance of therapies.  
To the investigator’s knowledge, there is no study to date that has correlated microcirculatory 
dysfunction in vivo to outcomes in COVID-19 despite the strong link between microvascular 
perfusion impairment and high mortality/morbidity of the disease. 
8.3.4    Risk-Benefit Assessment 
The benefit to society outweighs the risks of this study.   
8.4 
Recruitment Strategy 
Patients will be recruited via chart review and/or clinical referral. 
8.5 
Informed Consent/Assent and HIPAA Authorization 
Approved members of the study team will obtain parental/guardian consent (from both 
parents or legal guardians) prior to the proposed study in a private setting. The investigators 
will assure that parents/guardian comprehend the nature of the study, the study procedures 
and the risks and benefits of participation, steps that will be taken to avoid coercion and 
documentation of consent.  
This study requires two parent signatures documented on the consent form. After full 
explanation of the study, and if the two parents/legal guardians are present at the hospital, 
they will sign the informed consent form (ICF) in the presence of the approved member of 
the study team who did the disclosure.  
However, the study team anticipates this to be a challenge, as (either or) both 
parents/guardians are not always present at the bedside. Given the critically ill subject 
population and the need to image patients as early in the disease process as possible, we will 
send a copy of the ICF in advance to the parent(s)/legal guardian(s) and obtain in either one 
of the following ways: 
- Consent conferences with either or both parents may happen in person or over the telephone. 
- Either or both parents may sign the consent form in the physical presence of the study team, 
or remotely. 
- Study team may obtain original signed copy (wet signature) or a copy of the original that is 
scanned/photographed and e-mailed, texted, faxed or mailed. 


17 
 
 
 
 
 
 
After the disclosure, the parent(s)/legal guardian(s) will sign the page of the ICF as indicated 
by the study team member that is consenting. The parent(s)/legal guardian(s) will send a 
photograph of the signed page by facsimile, text message, or e-mail to the 
investigator/designee. 
If the parent cannot print the ICF, a special provision is made for them to sign on a blank 
page. This requires a consent conference where either i) a witness not related to the study is 
present or ii) a recording of the call (with permission from parent(s)) is made. A copy of the 
consent form will be sent (e.g. via e-mail, fax, etc.) to the parent(s)/legal guardian(s) in 
advance of the consent conference so they can read it. If they do not have access to e-mail, 
the informed consent will be read to them. 
A standard process will be used to ensure:  
i) 
Identification of call participants.  
ii) 
Review of informed consent document by study team, response to parents’ 
questions and parents’ verbal confirmation that their questions were 
answered. 
iii) 
Verbal confirmation by the parent/legal guardian that they have agreed to 
provide permission for the child’s participation and verbal confirmation by 
the parent/legal guardian that they signed and dated a blank piece of paper 
with a written statement that they voluntarily agree to provide permission for 
the child’s participation in the study, noting both the Protocol ‘NUMBER’ 
and brief protocol title.  
iv) 
The parent sends a photograph of the signed and dated statement by 
facsimile, text message, or e-mail to the investigator/designee; OR returns 
the document to the investigator by e-mail, facsimile, or mail at a later date, 
or at a future study visit that might occur in person. 
Per §46.408(b) / §50.55(e)(2), if the second parent is reasonably unavailable (deceased, 
incarcerated, in active military service, etc), the study team will enroll the subject with just 
one parental/legal guardian signature.  
No amendments will be done on the ICF once it is signed by both parents/legal guardians.  A 
combined HIPAA consent-authorization document will be used.  
We plan to enroll children 17 years or younger who will be seriously ill. In the event that 
they turn 18 while hospitalized, we anticipate that they will be unable to re-consent for 
themselves. In this case, we will re-consent through their Legally Authorized Representative. 
A waiver of assent was requested since subjects will likely be very sick and unable to provide 
assent. However, we will obtain verbal assent from children seven years old or older who are 
able to provide it. Assent confirmation (or inability to assent) will be documented by the 
study team on the consent form. 
A copy of the signed consent form will be provided to the parent(s)/legal guardian(s). 
Subjects with limited English proficiency (LEP) will be consented in person or remotely. 
When parents/guardians consent in person, a witness/interpreter through CHOP Language 


18 
 
 
 
 
 
 
Services will be present in person or remotely. If the interpreter is remote, their reference 
number will be noted on the signature line. When parents/guardians consent remotely, a 
witness/interpreter provided through CHOP Language Services will join the consent meeting 
in person or by video/phone call. If remote, the witness/interpreter’s reference number will 
be noted on the signature line. Apart from using a witness/interpreter, LEP subjects consented 
remotely will follow the same plan of English-fluent subjects consented remotely. 
8.6 
Payment to Subjects/Families 
Families will be compensated with a $25 gift card.  
9 
PUBLICATION 
We plan to publish the data collected in a scientific journal. Data may also be presented as 
abstract, podium presentation, or poster presentations at scientific meetings and conventions. 
No patient identifying information will be used in publication. 
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Z. Belhadjer et al., Acute heart failure in multisystem inflammatory syndrome in 
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M. A. Ellul et al., Neurological associations of COVID-19. Lancet Neurol 19, 767-
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X. Yang et al., Clinical course and outcomes of critically ill patients with SARS-
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P. Zhou et al., A pneumonia outbreak associated with a new coronavirus of probable 
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Y. Cheng et al., Kidney disease is associated with in-hospital death of patients with 
COVID-19. Kidney Int,  (2020). 


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D. Wang et al., Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel 
Coronavirus-Infected Pneumonia in Wuhan, China. JAMA,  (2020). 
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M. Dolhnikoff et al., Pathological evidence of pulmonary thrombotic phenomena in 
severe COVID-19. Journal of Thrombosis and Haemostasis,  (2020). 
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S. E. Fox et al. (medRixv, 2020). 
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C. Magro et al., Complement associated microvascular injury and thrombosis in the 
pathogenesis of severe COVID-19 infection: A report of five cases. Transl Res,  
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L. Davoodi, M. Taghavi, A. Razavi, in Infectious DIseases. (Research Square, 2020). 
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C. McCarthy. (New York Post, 2020),  chap. https://nypost.com/2020/04/23/infant-
killed-by-coronavirus-went-out-looking-like-a-princess/. 
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S. 
Haigh, 
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(Associated 
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chap. 
https://weartv.com/news/coronavirus/infant-in-connecticut-who-had-coronavirus-
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