Clinical trial NCT03300648
Conditions: Malaria, Cerebral
Keywords: coma pediatrics
Sponsor: Michigan State University
Start date: 2018-01-08; Completion date: 2023-12-31
Enrollment: 57 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This study evaluates the effectiveness of two interventions in Malawian children with cerebral malaria at high risk of death. One-third of the participants will receive treatment as usual, one-third will receive treatment as usual and be placed on a mechanical ventilator, and one-third will receive treatment as usual plus intravenous hypertonic saline.

Detailed description:
An important mechanism of death in children with cerebral malaria is diffuse cerebral swelling, cerebral herniation, compression of the brainstem respiratory center, and respiratory arrest. In those who survive their illness without specific interventions, reversal of diffuse cerebral swelling is rapid.

Mechanical ventilation may help to preserve life while diffuse brain swelling diminishes. Intravenous hypertonic saline may work as an osmotic diuretic, directly decreasing brain swelling.

Arms: arm 1: Hospitalization in a high dependency pediatric unit with skilled nursing, intravenous artesunate followed by oral artemisinin combination therapy, intravenous fluids, nasogastric feeding, elevation of the head of the bed by 30 degrees arm 2: Hospitalization in a high dependency pediatric unit with skilled nursing, intravenous artesunate followed by oral artemisinin combination therapy, intravenous fluids, nasogastric feeding, elevation of the head of the bed by 30 degrees, along with intubation and mechanical ventilation for a maximum of 7 days arm 3: Hospitalization in a high dependency pediatric unit with skilled nursing, intravenous artesunate followed by oral artemisinin combination therapy, intravenous fluids, nasogastric feeding, elevation of the head of the bed by 30 degrees, along with intravenous 3% hypertonic saline for a maximum of 7 days
Interventions: intervention 1: Mechanical ventilation intervention 2: Hypertonic saline
Intervention descriptions: intervention 1: Intubation and mechanical ventilation for a maximum of 7 days intervention 2: Intravenous 3 percent hypertonic saline for a maximum of 7 days

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
1 
Treating Brain Swelling 
in Pediatric Cerebral Malaria (TBS)  
DMID Protocol Number:  #16-0087 
DMID Funding Mechanism: PAR 15-040 
Pharmaceutical Support Provided by: NA 
Other Identifying Numbers: NA 
IND Sponsor: NA 
Principal Investigator:  Terrie E. Taylor, DO 
DMID Program Officer: Greg Deye, MD 
DMID Medical Monitor:  Mo Elsafy, MD, MSc 
DMID Clinical Project Manager: Walter Jones, PhD 
DMID Regulatory Affairs Specialist: Blossom Smith, MS 
Draft or Version Number: 4.1 
14 June 2023 
 
 
 
 
 
NCT03300648 
 
 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
2 
STATEMENT OF COMPLIANCE 
 
The study will be carried out in accordance with Good Clinical Practice (GCP) as required by the 
following:  
x 
NIH Clinical Terms of Award 
x 
Malawi Pharmacy and Medicines Regulatory Authority 
 
All key personnel have completed Human Subjects Protection Training. 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
3 
SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable US federal regulations and ICH guidelines. 
 
Site Investigator: 
 
Signed: 
 
Date: 
 
 
Name 
Title 
 
 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
4 
TABLE OF CONTENTS 
 
 
Page 
Statement of Compliance 
2 
Signature Page 
3 
Table of Contents 
4 
List of Abbreviations 
7 
Protocol Summary 
10 
1 
Key Roles 
13 
2 
Background Information and Scientific Rationale 
19 
2.1 
Background Information ........................................................................................ 19 
         2.2       Description of the Study Agent(s)/Intervention(s)... …………………………………33 
2.3 
Rationale .............................................................................................................. 46 
2.4 
Potential Risks and Benefits ................................................................................. 54 
2.4.1 
Potential Risks ...................................................................................................... 54 
2.4.2 
Known Potential Benefits ...................................................................................... 55 
3 
Objectives 
56 
3.1 
Study Objectives ................................................................................................... 56 
3.2 
Study Outcome Measures .................................................................................... 56 
3.2.1 
Primary Outcome Measures ................................................................................. 56 
3.2.2 
Secondary Outcome Measures ............................................................................. 57 
4 
Study Design 
58 
5 
Study Enrollment and Withdrawal 
66 
5.1 
Subject Inclusion Criteria ...................................................................................... 66 
5.2 
Subject Exclusion Criteria ..................................................................................... 66 
5.3 
Treatment Assignment Procedures ....................................................................... 67 
5.3.1 
Randomization Procedures ................................................................................... 67 
5.3.2 
Masking Procedures ............................................................................................. 68 
5.3.3 
Reasons for Withdrawal ........................................................................................ 68 
5.3.4 
Handling of Withdrawals ....................................................................................... 68 
5.3.5 
Termination of Study ............................................................................................. 69 
6 
Study Intervention/Investigational Product 
70 
6.1 
Study Product Description - HPS .......................................................................... 70 
6.1.1 
Acquisition ............................................................................................................ 70 
6.1.2 
Formulation, Packaging, and Labeling .................................................................. 70 
6.1.3 
Product Storage and Stability ................................................................................ 70 
6.2 
Dosage, Preparation and Administration of Study Intervention/Investigational 
Product - HPS ....................................................................................................... 70 
6.3 
Modification of Study Intervention/Investigational Product for a Participant - 
HPS ...................................................................................................................... 68 
6.4 
Accountability Procedures for the Study Intervention/Investigational Product(s) 
HPS ...................................................................................................................... 69 
6.5 
Assessment of Subject Compliance with Study Intervention/Investigational 
Product - HPS ....................................................................................................... 69 
6.6 
Study Product Description - VENT ........................................................................ 72 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
5 
6.6.1 
Acquisition ............................................................................................................ 72 
6.6.2 
Formulation, Packaging, and Labeling .................................................................. 72 
6.6.3 
Product Storage and Stability ................................................................................ 72 
6.7 
Dosage, Preparation and Administration of Study Intervention/Investigational 
Product - VENT ..................................................................................................... 73 
6.8 
Modification of Study Intervention/Investigational Product for a Participant - 
VENT .................................................................................................................... 74 
6.9 
Accountability Procedures for the Study Intervention/Investigational Product(s) 
- VENT .................................................................................................................. 74 
6.10 
Assessment of Subject Compliance with Study Intervention/Investigational 
Product - VENT ..................................................................................................... 74 
6.11 
Concomitant Medications/Treatments ................................................................... 74 
7 
Study Schedule 
73 
7.1 
Screening ............................................................................................................. 74 
7.2 
Enrollment/Baseline .............................................................................................. 74 
7.3 
Follow-up .............................................................................................................. 75 
7.4 
Final Study Visit .................................................................................................... 75 
7.5 
Early Termination Visit .......................................................................................... 75 
7.6 
Unscheduled Visit ................................................................................................. 75 
8 
Study Procedures/Evaluations 
76 
8.1 
Clinical Evaluations ............................................................................................... 76 
8.2 
Laboratory Evaluations ......................................................................................... 78 
8.3 
Special Assays or Procedures .............................................................................. 83 
8.4 
Speciimen Preparation, Handling, and Shipping ................................................... 84 
 9      Assessment of Safety  
9.1 
Specification of Safety Parameters ....................................................................... 85 
9.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters ........................................................................................................... 85 
9.2.1 
Adverse Events ..................................................................................................... 85 
9.2.2 
Reactogenicity (for Vaccine Studies and Some Therapeutic Trials) ...................... 87 
9.2.3 
Serious Adverse Events ........................................................................................ 90 
9.2.4 
Procedures to be Followed in the Event of Abnormal Laboratory Test alues or 
Abnormal Clinical Findings.................................................................................... 91 
9.3 
Reporting Procedures ........................................................................................... 91 
9.3.1 
Serious Adverse Events ........................................................................................ 92 
9.3.2 
Regulatory Reporting for Studies Conducted Under DMID-Sponsored IND .......... 92 
9.3.3 
Regulatory Reporting for Studies Not Conducted Under DMID-Sponsored IND .... 92 
9.3.4 
Other Adverse Events (if applicable) ..................................................................... 93 
9.3.5 
Reporting of Pregnancy ........................................................................................ 93 
9.4 
Type and Duration of Follow-up of Subjects after Adverse Events ........................ 93 
9.5 
Halting Rules ........................................................................................................ 93 
9.6 
Safety Oversight (ISM plus SMC or DSMB) .......................................................... 93 
10 
Clinical Monitoring 
94 
10.1 
Site Monitoring Plan .............................................................................................. 94 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
6 
11 
Statistical Considerations 
95 
 
11.1  General Design and Objectives ...……………………………………………………..95 
 
11.2  Stratification and Randomization …...…………………………………………………95 
 
11.3  Testing the adequacy of randomization ………………………………………………95 
 
11.4  Outcomes ………………………………………………………………………………. 95 
            11.5  Sample size and power………………………………………………………………... 95 
            11.6 Interim Analysis  …………………………………………………………………………96 
 
 
11.6.1  Futility Analysis  ………………………………………………………………97 
 
11.7 Statistical Analyses ………………………………………………………………………97 
 
 
11.7.1  Missing data   …………………………………………………………………98 
 
 
11.7.2  Additional exploratory modeling …………………………………………….99 
12 
Source Documents and Access to Source Data/Documents 
100 
13 
Quality Control and Quality Assurance 
101 
14 
Ethics/Protection of Human Subjects 
102 
14.1 
Ethical Standard ................................................................................................. 102 
14.2 
Institutional Review Board .................................................................................. 102 
14.3 
Informed Consent Process ................................................................................. 102 
14.4 
Detailed Informed Consent Process .................................................................... 102 
14.5 
Subject Confidentiality ........................................................................................ 103 
14.6 
Study Discontinuation ......................................................................................... 104 
14.7 
Future Use of Stored Specimens ........................................................................ 104 
15 
Data Handling and Record Keeping 
105 
15.1 
Data Management Responsibilities ..................................................................... 105 
15.2 
Data Capture Methods ........................................................................................ 105 
15.3 
Types of Data ..................................................................................................... 107 
15.4 
Timing/Reports ................................................................................................... 107 
15.5 
Study Records Retention .................................................................................... 107 
15.6 
Protocol Deviations ............................................................................................. 108 
16 
Publication Policy 
109 
17 
Literature References 
107 
18     Protocol Appendices 
125 
 
 
 
 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
7 
LIST OF ABBREVIATIONS 
AE 
Adverse Event/Adverse Experience 
AQP4 
Aquaporin 4 
BBB 
Blood-Brain Barrier 
BCS 
Blantyre Coma Score 
BIO 
Binocular Indirect Ophthalmoscopic (eye examination) 
BMP                                       
 
Blantyre Malaria Project (a research affiliate within the 
University of Malawi College of Medicine) 
BRIC 
Biomedical Research and Informatics Core (at Michigan State 
University) 
CM 
Cerebral Malaria 
COMREC 
College of  Medicine Research and Ethics Committee (in the 
University of Malawi) 
CQMP 
Clinical Quality Management Plan 
CRF 
Case Report Form 
CRI™ 
Compensatory Reserve Index 
CROMS 
Clinical Research Operations and Management Support 
CSF 
Cerebral Spinal Fluid 
CT 
Computed Tomography 
DMID 
Division of Microbiology and Infectious Diseases 
DMU 
Data Management Unit 
DSMB 
Data and Safety Monitoring Board 
DWI 
Diffusion-Weighted Imaging 
eCRF 
Electronic Case Report Form 
EKG 
Electrocardiogram 
GOS 
Glasgow Outcome Scale 
HIV 
Human Immunodeficiency Virus 
HOB 
Head of the Bed 
HPS 
Hypertonic saline arm of the clinical trial 
HTTPS 
Secure hypertext transfer protocol 
IB 
Investigator’s Brochure 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
ICMJE 
International Committee of Medical Journal Editors 
ICP 
Intra-Cranial Pressure 
IM 
Intramuscular (injection) 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
IV 
Intravenous (lines, infusions) 
JAMA 
Journal of the American Medical Association 
KABC-2 
Kaufman Assessment Battery for Children 2 
LUS 
Protocolized lung ultrasound 
MBP 
Myelin basic protein 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
8 
MDAT 
Malawi Developmental Assessment Tool 
MOP 
Manual of Procedures 
MP 
Malaria Parasitemia 
MR 
Malarial Retinopathy 
MRI 
Magnetic Resonance Imaging 
MSU 
Michigan State University 
N 
Number (typically refers to subjects) 
NDA 
New Drug Application 
NSE 
Neuron-specific enolase 
NIAID 
National Institute of Allergy and Infectious Diseases,  
NIH 
National Institutes of Health 
NIRS 
Near infrared spectroscopy 
OCRA 
Office of Clinical Research Affairs,  
OHRP 
Office for Human Research Protections 
OHSR 
Office for Human Subjects Research 
ONSD 
Optic Nerve Sheath Diameter 
OR 
Odds Ratio 
ORA 
Office of Regulatory Affairs,  
PHI 
Protected Health Information 
PCV 
Packed Cell Volume 
PNGT 
Per Naso-Gastric Tube 
PO 
Per Os (by mouth) 
PR 
Per Rectum 
PRW 
Paediatric Research Ward (Queen Elizabeth Central Hospital) 
PI 
Principal Investigator 
PICU 
Paediatric Intensive Care Unit 
PK 
Pharmacokinetics 
QA 
Quality Assurance 
QECH 
Queen Elizabeth Central Hospital 
QC 
RBC 
Quality Control 
Red Blood Cell 
REDCap 
Research Electronic Data Capture 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
SSL 
Secure sockets layer 
TBI 
Traumatic Brain Injury 
TNF 
Tumor Necrosis Factor 
UC 
Usual care arm of the clinical trial 
US 
United States 
VENT 
Mechanical ventilation arm of the clinical trial 
WHO 
World Health Organization 
 
 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
9 
 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
10 
PROTOCOL SUMMARY 
Title: 
 
Treating Brain Swelling in Pediatric Cerebral Malaria  
Phase: 
3 
Population: 
Malawian children aged 6 months to 12 years, with cerebral 
malaria and MRI evidence of increased brain volume (brain 
volume score  6).  Total of 128 children. 
Number of Sites: 
Single site:  Paediatric Research Ward, Queen Elizabeth 
Central Hospital, Blantyre Malawi 
Study Duration: 
Six years participant accrual; one-year data analysis 
Subject Participation 
Duration: 
55 weeks:  duration of hospital stay, and follow-up visits at 1, 6, 
and 12-months post-randomization. 
Description of Agent or 
Intervention: 
Arm 1:  Usual care + immediate ventilatory support for a 
maximum of 7 days.  Brain death or death will be considered a 
failure of the primary intervention.   
Arm 2:   Usual care + infusion of 3% hypertonic saline to 
achieve a blood sodium concentration of 150-160 mmol/L.  
Ventilatory support will be provided to patients who experience a 
respiratory arrest, and this will be considered a failure of the 
primary intervention. 
Arm 3:  Usual care (elevation of the head of the bed 30 degrees 
and intravenous antimalarial drugs).  Ventilatory support will be 
provided to patients who experience a respiratory arrest, and 
this will be considered a failure of the primary intervention. 
 
Final outcomes will be determined at 7 days post-randomization:  
The primary study outcome will be recovery vs treatment failure, 
where treatment failure is a composite of death, ventilatory 
rescue (Arms 2 and 3), and brain death  
 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
11 
Objectives: 
Primary Objective 
Compare final outcomes in pediatric CM patients receiving usual 
care (Arm 3) with both of two interventions (Arm 1 and Arm 2).  
Secondary Objectives:  
1. Among survivors of this interventional study, compare 
rates of adverse neurological outcomes in those 
assigned to the two interventions and those assigned to 
usual care. 
2. Among all children admitted to the Paediatric Research 
Ward and screened for eligibility for randomization, 
evaluate and validate biomarkers of increased brain 
volume. 
3. Assess the safety of early mechanical ventilation as well 
as intravenous hypertonic saline in children with CM. 
4. Establish association between specific pathogenic 
mechanisms and TCD-derived phenotypes.   
 
Description of Study 
Design: 
Prospective, randomized, controlled non-blinded clinical trial 
Estimated Time to 
Complete Enrollment:    
Six years 
 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
12 
Cerebral Malaria patient is sent to Paediatric Research 
Ward, stabilized clinically, and started on anti-malarial 
therapy
Ophthalmoscopy
Retinopathy +
Retinopathy -
Standard (non-research) nursing 
care on Paediatric Research 
Ward (PRW)
Research protocol on PRW
Q6h, Q12h, Q24 hrs as below
1, 6 and 12 month f/u
As below
Randomized?
Arm 1 (PICU)
Usual care +
Immediate ventilation
Arm 22 (PICU)
Usual care +
Hypertonic saline
Arm 33 (PICU)*
Usual care 
Arm 1 (PICU)
Usual care +
Immediate ventilation
Arm 22 (PICU)
Usual care +
Hypertonic saline
Arm 33 (PICU)
Usual care 
Q6hr: electrolytes, blood glucose, lactate, hematocrit and parasitemia checks
(electrolyte checks will be Q3h initially in Arm 2)
BD: neuro checks 
Q24hr: optic nerve sheath diameter, TCD, ophthalmoscopy, MRI until improved
First 24 hrs: monitoring for heart rate variability analysis
Assess final outcome on/by Day 7
Final outcome: treatment success or failure3
Discharge survivors when fully conscious and clear 
of parasites
Follow-up visits at 1, 6, and 12 months:
MDAT or KABC-2, MPs and PCV at each visit
Repeat MRI at 1 month
PICU1: will be on continuous monitoring 24hrs, then transferred to PRW
2Pts in Arms 2 and 3 who experience respiratory arrest will be placed on ventilators.
3Treatment failure is a composite of death, ventilatory rescue or brain death
Consent to 
screen?
Yes
No
Increased brain 
volume?
Obtain MRI
Yes
Consent for 
randomized clinical 
trial?
Yes
No
No
 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
13 
KEY ROLES
Individuals: 
Principal Investigator:   
Terrie E. Taylor, DO 
University Distinguished Professor 
MSU Foundation Professor  
Department of Osteopathic Medical Specialties 
College of Osteopathic Medicine 
909 Wilson Road 
B309-B W. Fee Hall 
E. Lansing MI  48824-1315 USA 
Tel : 517 353 3211 
In Malawi : +265 999 969 220 
FAX: 517 432 1062 
Email ttmalawi@msu.edu  
 
Co-Investigators: 
Pediatrician 
Yamikani Chimalizeni FCPaed (SA) 
Senior Lecturer 
Department of Pediatrics 
Kamuzu University of Health Sciences 
Private Bag 360 
Chichiri, Blantyre 3 
Malawi 
Tel: +265 992 233 221 
Fax: +265 1 875 774 
Email: gaveta3@yahoo.com 
 
 
Pediatric Neurologists  
Douglas G. Postels, MD, MS 
Associate Professor 
George Washington University 
Children’s National Medical Center 
111 Michigan Avenue NW 
Department of Neurology 
Washington DC 
20011 
Tel: 202 476 6231 
FAX 202 476 2864 
Email :  dpostels@childrensnational.org 
 
Macpherson Mallewa, MD,MRCPCH, PhD 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
14 
Consultant Pediatric Neurologist 
Associate Professor  
Department of Paediatrics 
Kamuzu University of Health Sciences 
P/Bag 360 
Blantyre 3 
Malawi 
Tel: +265 995 83 50 83 
FAX: +265 1 87 5774 
Email: macphers@liverpool.ac.uk or  
mmallewa@mlw.medcol.mw 
 
Pediatric Intensivists 
Adnan Bhutta, MBBS 
Clinical Professor of Pediatrics 
Division Chief, Pediatric Critical Care Medicine 
Department of Pediatrics 
Affiliate Faculty Member, Center for Global Health Equity 
Indiana University School of Medicine/ Riley Children's Health 
705 Riley Hospital Drive, Suite 4900 phase II 
Indianapolis, IN 46202 
Email-Adbhutta@iu.edu 
 
 
 
Allan Doctor, MD 
Professor of Pediatrics, Biochemistry (UMB) and 
Bioengineering (UMCP) 
University of Maryland Schools of Medicine and Engineering 
Director, Center for Blood Oxygen Transport and Hemostasis 
HSF III, 8th Floor 
670 West Baltimore Street  
Baltimore, MD 21201 
Phone: 410-706-7084 
Fax: 410-706-6417 
adoctor@som.umaryland.edu 
 
Nicole O’Brien, MD 
Associate Professor of Clinical Medicine 
Division of Critical Care Medicine 
Chief, Neurocritical Care 
Nationwide Children’s Hospital  
700 Children’s Drive, FOB 2C2 
Columbus, OH 43205 
Tel: 614-722-2000 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
15 
FAX: 614-722-3443 
Email: Nicole.obrien@nationwidechildrens.org 
 
Radiologists 
Manu S. Goyal, MD, MSc 
Assistant Professor of Radiology, Neurology and Neuroscience 
Neuroimaging Laboratories 
Mallinckrodt Institute of Radiology 
Washington University School of Medicine 
510 S Kings Highway Blvd 
St. Louis, MO 63110-1076 
Phone: (314) 362-5950 
Fax: (314) 362-4886 
Email: goyalm@wustl.edu 
 
Lorenna L. M. Vidal, MD, DABR 
Attending Physician 
  Pediatric Neuroradiology 
  Children’s Hospital of Philadelphia 
  Department of Radiology, Neuroradiology Division 
Clinical Instructor, Radiology 
  Perelman School of Medicine  
  University of Pennsylvania 
3401 Civic Center Blvd., Suite W225 (Neuroradiology Suite) 
Philadelphia PA 19104 
Phone: (267) 426-3883 
Email: vidall@email.chop.edu 
 
Ophthalmologists : 
Simon Harding 
Professor of Clinical Ophthalmology 
Department of Eye and Vision Science 
William Henry Duncan Building 
Royal Liverpool University Hospital 
Liverpool L7 8TX 
UK 
 
Petros Kayange, MBBS, MMed(Ophth), FCOphth.(EDSA), 
FICO(Hons) 
Head of Ophthalmology, Senior Lecturer 
Kamuzu University of Health Sciences 
Ophthalmology Unit, Dept of Surgery 
P/B 360 
Blantyre 3 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
16 
Malawi 
 
Nicholas Beare  
Senior Lecturer and Consultant Ophthalmologist 
Department of Eye and Vision Science 
William Henry Duncan Building 
University of Liverpool 
Liverpool L7 8TX 
UK 
Tel +44 (0)7855 019379 
Email: n.beare@liv.ac.uk  
 
Senior Study Clinician 
Karl Seydel, MD, PhD 
Assistant Professor 
Department of Osteopathic Medical Specialties 
College of Osteopathic Medicine 
909 Wilson Road 
B309 W. Fee Hall 
East Lansing MI 48824 
USA 
Tel :  +1 517 353 3211 
Email:  seydel@msu.edu 
 
Heart rate variability expert : 
Peter Hu, Ph.D 
Associate Professor of Anesthesiology, Surgery and 
Epidemiology 
University of Maryland School of Medicine 
11 S. Paca Street, Suite LL01 
Baltimore MD 20219 
USA 
Tel : 410 328 7264 
Fax : 410 328 7230 
Email : phu@som.umaryland.edu 
 
Vascular physiology collaborator: 
Hans C. Ackerman, MD DPhil MSc 
NIH-Lasker Clinical Research Scholar 
Investigator and Chief, Physiology Unit 
Laboratory of Malaria and Vector Research 
Division of Intramural Research 
National Institute of Allergy and Infectious Diseases 
12735 Twinbrook Parkway, Lab 3E-20 
Rockville, MD 20853 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
17 
USA 
Tel: (301) 761-5646 
Email: hans.ackerman@nih.gov 
Statistician: 
Joseph C. Gardiner, PhD 
University Distinguished Professor  
Michigan State University 
909 Wilson Road,  
Room B629 West Fee 
East Lansing, MI 48824-6410 USA 
Tel: 517-884-3967 
Fax: 517-432-1130 
Email gardine3@ msu.edu  
 
 
Administrative Support: 
University of Malawi College of Medicine: 
Mrs. Esther Gondwe 
Blantyre Malaria Project (BMP) 
PO Box 32256 
Blantyre 3 
MALAWI 
Tele: +265-(0)999-969222 
Email: egondwe@bmp.medcol.mw 
 
Michigan State University: 
Ms. Karen Hassankhani 
Department of Osteopathic Medical Specialties 
College of Osteopathic Medicine 
909 Wilson Road 
B309-B W. Fee Hall 
E. Lansing MI  48824-1315 USA 
Tel : +1 517 353 3211 
Fax: +1-517-432-1062 
Email: Karen.Hassankhani@hc.msu.edu 
 
University of Maryland: 
Tori Boyer, DNP, BSN-RN 
Research Associate | Departments of Pediatrics 
Director, Biospecimen Repository and Clinical Research Core   
Center for Blood Oxygen Transport and Hemostasis 
University of Maryland School of Medicine 
HSF III, 8th Floor 
670 West Baltimore Street  


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
18 
Baltimore, MD 21201 
Tori.boyer@som.umaryland.edu   
Office: (410) 706-7093 
Cell (preferred): (618) 977-7952 
Fax: (410) 706-4148 
 
 
Medical Monitor:  
Mo Elsafy, MD, MSc.  
Medical Monitor 
Office of Clinical Research Affairs 
Division of Microbiology and Infectious Diseases 
5601 Fishers Lane, 7E56 MSC 9826 
Bethesda, Maryland 20892-9826 
For courier service use Rockville, MD 20852 
Phone: 240-292-4241   
Cell:    301-204-3735   
Email: elsafymm@nih.gov  
Institutions: 
 
Michigan State University (USA) 
Nationwide Children’s Hospital (Columbus OH, USA) 
Children’s National Hospital (Washington DC, USA) 
Children’s Hospital of Philadelphia (Philadelphia PA, USA) 
Kamuzu University of Health Sciences (Malawi) 
Washington University (St. Louis MO, USA) 
University of Maryland (Baltimore MD, USA) 
University of Liverpool (UK) 
National Institute of Allergy and Infectious Diseases (USA) 
 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
19 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
Background Information 
Recent advances in treatment, prevention and control notwithstanding, malaria remains a major 
scourge, claiming more than 429,000 lives each year1. Cerebral malaria (CM) is a severe 
complication characterized by coma and seizures.  The case fatality rate is 15-50% and the 
annual incidence ranges from 1-12 cases per 1,000 children in malaria-endemic regions 2, 3.   
Most (85%) of the morbidity and mortality occurs in young African children 4. Of the five species 
of malaria parasite which can infect humans, Plasmodium falciparum is the primary cause of 
severe disease, and is responsible for the vast majority of malaria associated mortality 4.   
 
2.1.1 Life cycle  
Transmitted by the bite of a female Anopheles mosquito, the malaria parasite undergoes 
asexual replication in hepatocytes before invading red blood cells (RBC). The erythrocytic cycle 
occurs entirely within the circulatory system. The parasite consumes hemoglobin as it grows 
and replicates within the RBC; one merozoite develops into a schizont over the course of 
approximately 48 hours in P. falciparum infections. The infected RBC eventually ruptures, 
releasing 16-32 daughter merozoites. The diagnosis of malaria infection is usually made by 
detecting the intra-erythrocytic parasites in peripheral blood samples, examined microscopically.  
The life cycle of P. falciparum is distinguished histopathologically from the four other species of 
Plasmodia which infect humans by the capacity of RBCs containing mature forms of the 
parasite (generally 32 hours or more into the life cycle) to adhere to endothelial cells in the 
microvasculature of various organs.  This phenomenon is known as sequestration, and P. 
falciparum does this so effectively that the mature trophozoites and schizonts are only very 
rarely seen in the peripheral blood. The life cycle stages most commonly seen in diagnostic 
blood films are the younger, ring-form stages. 
 
2.1.2 Epidemiology  
CM is a disease primarily of young African children as older individuals living in malaria-endemic 
areas have acquired immunity to severe disease 5.  The semi-immune population remains 
susceptible to infection, but rarely do those infections progress to severe and complicated 
malaria.  Asymptomatic parasitemic individuals are commonly identified during population-
based surveys in malaria-endemic areas.  The biological basis for antimalarial immunity remains 
largely unknown, but the epidemiology is consistent: in areas where malaria transmission is 
intense and regular, the burden of severe disease falls on younger children.  
 
For reasons yet to be fully elucidated, even within the vulnerable population of non-immune 
young children, only a relatively small proportion of those infected with P. falciparum develop a 
life-threatening illness.  Why some non-immunes are more susceptible than others to severe 
disease is not known; the relative contributions of host resistance and parasite virulence are 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
20 
unclear.  Only one percent of symptomatic infections progresses to severe disease 6.  Most 
falciparum infections result in a mild flu-like febrile illness and asymptomatic infections are 
common, even in young children 7. 
 
Because the level of antimalarial immunity in individuals cannot be measured, and because 
non-immunes are differently susceptible to severe disease, the diagnosis of a malaria illness is 
not as straightforward as merely identifying malaria infection by detecting parasites in a blood 
film.   Peripheral parasitemia is necessary in order to identify an infected individual, but, in a 
symptomatic individual, the presence of parasitemia alone does not establish a causal 
connection to the symptoms.  
 
 
2.1.3 Pathogenesis 
There are many hypotheses regarding the pathogenesis of CM, but the unifying feature is the 
sequestration of parasitized RBCs in the microvasculature of various organs, including the brain 
8.  During the latter half of the 48-hour life cycle, parasite proteins stud the surface of the host 
RBC, and mediate the adherence of infected RBCs to host endothelial cells, primarily in 
capillaries and post-capillary venules (Fig.1) 9.  This interaction is hypothesized to cause 
localized impaired perfusion, leading to hypoxia 9 and acidosis 10.  It is also thought to activate 
endothelial cells 11, 12, leading to subsequent blood-brain barrier (BBB) breakdown 13 and 
increased brain volume as a result of the sequestered mass and increased cerebral blood flow 
related to anemia, seizures and fever 14. Endothelial cell activation may also lead to a systemic 
inflammatory response 15, 16 and a pro-thrombotic state 17-20.    
  
  Figure 1. (A, B, C) Histologic evidence of parasite sequestration in small blood vessels of the brain.  
2.1.4 Antimalarial treatment 
Intravenous quinine has been the mainstay of treatment for CM for two hundred years,  but 
recently completed clinical trials established the superiority of artesunate over quinine as the 
primary antimalarial treatment in both adults 21 and children 22 with severe malaria.  The benefits 
of artemisinin treatment are thought to be related to the more rapid parasite clearance times, 
and to the fact that the artemisinin drugs exert their activity over a broader period of the parasite 
life cycle 23, thus diminishing ongoing sequestration. Even under the ”best case scenario” of 
prompt treatment with artesunate, the case fatality rate in the subgroup of African children with 
CM in the AQUAMAT study was 18% 22.  Additional impact on the case fatality rate will likely 
require adjunctive therapies targeting key pathogenic mechanisms. 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
21 
2.1.5 Adjunctive therapy 
A number of clinical trials targeting various putative pathogenic mechanisms have been carried 
out 24: 
x 
Modulating the immune response 
o Dexamethasone to decrease cerebral edema 25, 26 
o Intravenous immune globulin to reverse sequestration 27 
o Monoclonal antibodies to tumor necrosis factor (TNF) to decrease immune 
activation 28 
o Pentoxifylline to decrease TNF production and improve RBC deformability 29, 30 
x 
Countering a pro-thrombotic state, using heparin or aspirin 31 
x 
Decreasing cerebral edema and reducing raised intracranial pressure, using osmotic 
agents 14, 32, 33 
x 
Ameliorating acidosis by expanding intravascular volume 34, 35 
 
None of these trials improved clinical outcomes and in fact, recently published data suggest that 
intravascular volume expansion in pediatric malaria participants with impaired perfusion may be 
counterproductive 36. 
 
There are several possible explanations for these failed attempts: 
 
1. The trials were underpowered.  In a recent review of the quality of randomized clinical 
trials of adjunctive therapy for the treatment of CM, sample sizes were recalculated to 
determine the study power to detect 25% and 50% reductions in mortality.  Of nine trials, 
only two had sufficient power to detect a 50% reduction in mortality and none could 
detect a 25% reduction 24. None of the trials carried out to date have included an ocular 
funduscopic examination for the detection of malarial retinopathy (see 2.1.1.7); the loss 
of the added specificity such an examination provides would further decrease the power 
of these studies.  
 
2. The putative mechanism is not critical to the pathogenesis of fatal malaria. Hypotheses 
often emerge from clinical studies in which an association is noted between a factor 
(e.g., TNF concentration) and disease severity or outcome 28, 37.  The hypothesis may be 
bolstered by data from an animal model in which an intervention has an impact on the 
exposure (in this case TNF) in question (e.g., intravenous immune globulin in Saimiri 
monkeys)38.  The clinical trial may be successful in terms of modifying the exposure,  but 
the impact on the outcome of the malaria participant is not clinically significant because 
the mechanism itself was not on the causal pathway between malaria infection and 
death or neurological morbidity28. 
 
3. The mechanism is relevant, but the intervention was timed ineffectively.  In the murine 
and non-human primate malaria worlds, interventions are often initiated simultaneously 
with the induced malaria infection and, in effect, prevent the evolution of severe malaria 
39.  In the world of human malaria, CM can develop very rapidly, and although the child is 
often rushed to the hospital, they may have been comatose for several hours before 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
22 
treatment can be started.  Interventions in this setting need to be able to reverse the key 
process in order to rescue the participant. 
 
 
2.1.6   Fatal pediatric malaria  
Case fatality rates for children meeting the standard clinical case definition of CM, and with 
evidence of at least one feature of malarial retinopathy (see 2.1.1.7) are approximately 15% 40.  
Risk factors for a poor outcome include a Blantyre Coma Score (BCS) < 2, an elevated white 
blood cell count, pre-treatment hypoglycemia, hyperlactatemia, and papilledema 41-43.  The most 
commonly observed mode of death is respiratory arrest with subsequent bradycardia and 
eventually asystole 44. 
A case/control study of fatal CM has been ongoing in Blantyre, Malawi since 1996. Cases are 
children who meet the standard clinical case definition of CM, P. falciparum parasitemia, BCS < 
2 with no other obvious cause of coma; controls are aparasitemic children with non-malarial 
causes of coma.  An early finding of this study was that 24% of children meeting the standard 
clinical case definition of malaria had no evidence of the post-mortem histological sine qua non 
of CM, the sequestration of parasitized RBCs in the cerebral microvasculature; all of these 
children had a non-malaria cause of death identified at autopsy. Two different histological 
patterns were observed in the majority of children with cerebral sequestration: sequestration 
alone (known as “CM1”); or sequestration with intra- and peri-vascular pathology (“CM2”)      
(Fig 2) 8.   
Figure 2. Different histological patterns in children meeting the standard clinical case definition of  
     cerebral malaria.  A: CM1, sequestration alone.  B: CM2, sequestration with intra- and peri-vascular  
     pathology. C: CM3, no sequestration. 
2.1.7 Linking ocular funduscopic findings to autopsy results  
In conjunction with the autopsy study described above, ophthalmologists observed a 
constellation of ocular funduscopic features in many Malawian children with classically defined 
CM.  Four components were described:  white-centered hemorrhages (Fig 3A), vessel color 
changes (Fig 3D), retinal whitening (peri- and extra-macular)(Fig 3B,3C), and papilledema. 
These were observed individually and in various combinations 42, 45-48.  
 
Post mortem examinations of participants with CM1 and CM2 revealed histologic parallels 
between the eye findings and the cerebrum. Retinal hemorrhages (Fig 3B) resembled the 
classically described cerebral ring hemorrhages, where endothelial disruption leads to the 
extravasation of non-parasitized RBCs 49.  Retinal vessel color changes similar to those 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
23 
previously seen in the cerebral microvasculature were evident and are caused by the 
sequestration of parasitized RBCs.  As the parasite consumes hemoglobin, the cytoplasm of the 
parasitized RBC becomes less red, thus during funduscopy the color of the column of blood in 
the eye appears yellow or orange (Fig 3C). 
 
 
Figure 3.  Fundus photographs illustrating features of malarial retinopathy.  A: Extensive macular 
whitening (black arrow) and extra-macular whitening (white arrow).  B: Multiple retinal hemorrhages; white 
areas are reflection artifacts. C: A single hemorrhage and vessel changes; lower image is the 
corresponding fluorescein angiogram showing a mottled appearance, likely due to sequestered 
parasitized red cells. D: Peri-macular whitening (black arrows) and fluorescein angiography of the same 
area, showing impaired perfusion which corresponds to the areas of macular whitening (white arrows) 
The etiology of the macular and extra-macular whitening was revealed by fluorescein 
angiography to reflect areas of non-perfusion beyond vascular obstruction, caused by the 
sequestration of parasitized RBCs (Fig 3D) 50.   
 
Papilledema is not a feature of malarial retinopathy per se, but the finding is associated with 
worse clinical outcomes when it is found in conjunction with any of the other three retinal 
features 48. 
Among participants in the aforementioned ongoing autopsy study, there was a very strong 
association between the presence of any of the three features of malarial retinopathy during life 
(hemorrhages, vessel changes, whitening)  and the presence of cerebral sequestration (CM1 or 
CM2) identified post-mortem (Table 1, and 8) 
 
 
 
 
 
                  
Table 1. Malarial retinopathy and histological evidence of  
sequestration in postmortem samples 
 
Malarial retinopathy 
present 
Malarial retinopathy 
absent 
Sequestration 
present 
40 
1 
No cerebral 
sequestration
2 
14 
A 
B 
C 
D 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
24 
Sensitivity: 98%          Positive predictive value:  95% 
                 Specificity  88%          Negative predictive value: 93% 
 
 
The ongoing autopsy-based study of the clinicopathological correlates of fatal CM in Blantyre, 
using the gold standard of sequestered parasites in the cerebral microvasculature of fatal cases 
to diagnose CM, has established that the standard clinical case definition misclassifies 25-30% 
of participants and that the added presence of malarial retinopathy increases the specificity of 
the case definition from 61% to 95%.   Studies conducted prior to the recognition of malarial 
retinopathy are likely compromised by this misclassification bias 46. 
 
A biomarker for malarial retinopathy:  Histidine-rich protein 2 (HRP2) is a P. falciparum protein 
released primarily (but not exclusively) at the time of schizont rupture51.  Plasma concentrations 
are significantly higher in patients with CM1/CM2 on histology, compared to autopsy subjects 
with the CM3 (no sequestration) pattern; plasma HRP2 concentrations are also significantly 
higher in patients with evidence of malarial retinopathy, compared to those with retinopathy-
negative CM52.  
 
 
2.1.8 Postmortem features of fatal pediatric CM  
There are only a few systematic, standardized autopsy-based studies of fatal pediatric malaria 
53-63 and only one 61 has classified participants on the basis of the presence or absence of 
malarial retinopathy.  There, 37 participants with retinopathy-positive CM were compared with 
13 comatose, retinopathy-negative participants (individuals who were parasitemic, but in whom 
the autopsies identified non-malarial etiologies for coma and death), and with 18 participants 
who were aparasitemic and comatose.   
x 
Brain weights (adjusted for age) were increased in all three groups, and differences 
between groups were not statistically significant. 
x 
8 of 37 retinopathy-positive CM participants had evidence of mild tonsillar and/or 
uncal herniation, compared to one of 13 retinopathy-negative participants and one of 
18 participants with non-malarial coma 
x 
Sequestration of parasitized red cells was present in all cases of retinopathy-positive 
CM, and in none of the retinopathy-negative parasitemic participants or in the 
aparasitemic controls. In the retinopathy-positive CM cases, 25% were CM1, 75% 
were CM2 (evidence of intravascular thrombin, ring hemorrhages and the 
intravascular accumulation of hemozoin-laden macrophages). 
x 
Diffuse myelin and axonal damage were seen in association with ring hemorrhages 
and thrombi, and in areas of intense sequestration. 
x 
The permeability of the blood-brain barrier (BBB) was assessed by immunostaining 
for fibrinogen, and BBB breaches were seen in association with ring hemorrhages, 
fibrin thrombi, and areas of intense sequestration. 
x 
There was no evidence of an extravascular localized immune response to the 
vascular, myelin, and axonal pathology.  Microvessels were often distended by the 
accumulation of CD45/CD68-positive monocytes, which appeared to occupy most of 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
25 
the lumen of capillaries and venules but did not migrate out of the vessels into the 
brain tissue. 
x 
Endothelial cells of parasitized vessels often displayed large vesicular nuclei, 
suggestive of activation. 
   
These findings do not definitively establish causation, or even a sequence of events, but are 
consistent with pathogenic mechanisms involving thrombotic events, BBB disruption, endothelial 
cell activation and edema formation.  Additional studies in pre-fatal and non-fatal cases of CM 
were suggested to identify mechanisms by which malaria infections cause coma, brain damage 
and death 61. 
 
2.1.9 Imaging studies 
Owing to the resource-limited settings in which pediatric CM occurs, research involving imaging 
studies has been limited to computed tomography (CT) case series and occasional magnetic 
resonance imaging (MRI) case reports 64-67. Until recently the largest published imaging case 
series on pediatric CM was a computed tomography (CT) study from Malawi that included 14 
participants, 3 of whom were studied in the acute illness and 11 of whom were studied up to 18 
months after discharge 68.  
 
In June 2008, MRI technology became available in Blantyre, Malawi. We were able to describe 
the acute brain MRI findings in children with CM admitted to the Paediatric Research Ward 
(PRW) of Blantyre’s Queen Elizabeth Central Hospital (QECH) between 2009 and 2011. 
Findings from 120 children with retinopathy-positive CM were compared to the more 
heterogeneous group of comatose, parasitemic children who met the standard clinical case 
definition of CM but lacked evidence of retinopathy and thus were presumed to have a non-
malarial cause of coma (n=32) 69 . 
 
None of the children with retinopathy-positive CM had a normal MRI.  Findings which were more 
commonly seen in the retinopathy-positive participants include the following: 
 
x 
Increased brain volume (odds ratio (OR) 9.9; 95% confidence interval (CI) 4.0-24.4):  
Moderate to severe increases in brain volume were seen in 47% of participants.  Ten 
percent had severely increased volume, i.e. their scans exhibited loss of all sulcal 
markings and had cisternal effacement.  Diffuse volume increases were the predominant 
pattern and were observed in 79% of participants with increased volume. Given the 
limitations of the 0.35T MRI in Malawi (low field strength, abandoned platforms), it is not 
possible to delineate the pathogenic mechanism underlying the volume changes (e.g., 
vasogenic or cytotoxic edema) at this time. 
 
x 
Basal ganglia involvement (OR 8.9; CI 3.7-21.1) including diffusion-weighted imaging 
(DWI) changes (OR 4.7; CI 1.5-14.2):  The most common finding was abnormal T2 
signal in the basal ganglia, present in 84% of the children with retinopathy-positive CM. 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
26 
x 
Cortical abnormalities on T2 (OR 4.1; CI 1.7-9.7):  Supratentorial cortical thickening and 
T2 signal abnormalities were present in 62% of children. This generally did not follow a 
typical vascular distribution. Cortical DWI abnormalities were seen in 21% of children. 
These also often failed to correspond to a typical vascular distribution. Specifically, there 
were no cases of watershed ischemia  
 
x 
Periventricular white matter changes (OR 3.7; CI 1.6-8.3):  Increased T2 signal in the 
white matter was present in 72% of children and was often associated with DWI 
abnormalities, which were present in 45%. White matter DWI abnormalities tended to 
occur in the absence of cortical DWI abnormalities. 
 
x 
Corpus callosum changes on T2 and DWI (OR 4.2; CI 1.6-10.9 and OR 3.3; CI 1.3-8.6, 
respectively):  T2 signal changes in the corpus callosum were present in 49%, and often 
occurred in the absence of other white matter changes.  Involvement of the corpus 
callosum was closely associated with corresponding positive DWI changes, seen in 43% 
of total cases.  Although some cases of corpus callosum abnormality had diffuse 
involvement, the splenium was predominantly affected in a majority (38/52, 73.1%) of 
cases. 
 
x 
Cerebellum (posterior fossa) changes (OR 3.5; CI 1.4-8.6):  Increased T2 signal was 
identified in the cerebellum in nearly half of children.  This ranged from diffuse 
involvement with associated edema to multifocal areas of deep involvement with 
localized mass effect.  
 
2.1.10 Imaging features associated with fatal outcomes in pediatric CM 
Of the MRI features associated with retinopathy-positive CM described above, only one was 
statistically significantly associated with outcome (survival vs death): severely increased brain 
volume52 (Table 2). This sample included 168 participants, enrolled from 2009-2011.  Twenty-
one of the 25 fatalities had severely increased brain volume on the initial MRI (OR 7.5).  All the 
retinopathy-positive children with lesser degrees of increased volume survived.   These findings 
are consistent with the brain swelling observed at autopsy 61 and with earlier clinical 
observations suggesting raised intracranial pressure as a step on the pathway to death in CM 14, 
70 
 
 
    Table 2.    MRI features associated with fatal outcomes in Malawian children with 
                 retinopathy-positive CM (multiple regression analyses) 
 
Ret-pos CM 
survivors 
(n=143) 
Ret-pos CM 
fatalities 
(n=25) 
OR 
(CI) 
Pre-pontine CSF (mm) 
10.0 
 
8.5 
 
0.5 
(0.3-0.8) 
Posterior 
predominance 
16 
 
8 
 
1.6 
(0.7-3.8) 
Severely increased 
brain volume  
39 
 
21 
 
3.4 
(0.9-12.7) 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
27 
A neuroimaging study performed in children with retinopathy-negative CM revealed that 
severe neurological morbidity in survivors is associated with increased brain volume on 
admission brain MRI 71.  In this study there were only three deaths and one of these three 
children had greatly increased brain volume on admission scan.  Brain MRI factors 
associated with death in children with retinopathy negative CM are still unclear due to the 
low number of scans interpreted in children with this condition.  Therefore, in the clinical trial 
proposed here, sample size and power calculations were performed on children with 
retinopathy-positive CM.  We postulate that the same association between increased brain 
volume and death will be valid in children with retinopathy-negative CM.   
 
2.1.11 The natural history of malaria-associated increased brain volume 
The evolution of increased brain volume in pediatric CM has been captured in sequential 
MRIs from 35 of the 168 retinopathy-positive CM participants scanned in Blantyre between 
2009 and 2011.  These participants received antimalarial drugs and (as needed), 
antipyretics, anticonvulsants and antibiotics 8, but were not treated specifically for the 
volume changes observed on MRI scans. The post-admission changes in the mean pre-
pontine CSF measurement of the 30 patients who survived (dashed black line) was 
significantly different from that in the 5 patients who died (solid yellow line) (P=0.02) (Fig 4).   
 
 
 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
28 
Figure 4. Evolution of increased brain volume in pediatric CM. 
The line graph (a) shows the distribution of pre-pontine CSF measures on first and second MRIs. 
Second MRIs were obtained 16 to 30 hours after the first. Dashed black lines reflect subjects who 
survived; solid orange lines are subjects who died.  The post-admission changes in mean pre-pontine 
CSF measures in retinopathy-positive CM patients who recovered and were discharged (N = 30, 
dashed blue line) was statistically significantly different from those patients who died (N = 5, solid blue 
line) (P = 0.02). The corresponding MR images (b, c) show rapid resolution of the increased volume 
over 48 hours in one patient.   
 
2.1.12 Quantitative vascular physiology measurements 
Transcranial Doppler Ultrasound (TCD)  
Transcranial Doppler Ultrasound (TCD) is a portable, non-invasive, easy to use tool that 
quantifies cerebral blood flow velocities (CBFV)72.  TCD has been widely used to assess 
cerebrovascular physiology in number of neurologic illnesses in children73-75. Specific alterations 
to TCD derived flow velocities and waveform morphologies enable characterization of specific 
pathologic changes to the neurovasculature during illness. In 160 children with retinopathy 
positive cerebral malaria, five distinct changes to expected flow velocity patterns were noted 
(Figure 5).76     
 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
29 
 
 
Figure 5. A. Normal middle cerebral artery (MCA) transcranial Doppler ultrasound (TCD) flow velocities 
and waveform for a 3-year-old child. Peak= systolic, EDV= end diastolic velocity, PI= pulsatility index  .  
TCD with normal systolic flow velocity, reduced diastolic flow velocity, increased pulsatility index.  These 
findings represent a child categorized as having microvascular obstruction.  C.  TCD with increased 
systolic flow velocity, increased diastolic flow velocity, loss of dichrotic notch. Lindegaard ratio (LR) <3.  
These findings represent a child categorized as having hyperemia. D. TCD with increased systolic flow 
velocity, increased diastolic flow velocity, presence of dichrotic notch. LR >3.  These findings represent a 
child categorized as having cerebral vasospasm. E. TCD with decreased systolic flow velocity, decreased 
diastolic flow velocity, decreased mean flow velocity.  These findings represent a child categorized as 
having low flow. F. TCD with increased systolic flow velocity, increased diastolic flow velocity, increased 
mean flow velocity in the basilar artery.  At the same time, all measurements in the middle cerebral 
arteries were normal. These findings represent a child categorized as having isolated posterior 
hyperemia.  
Each flow velocity phenotype was identified with different frequency (microvascular obstruction 
22%, hyperemia 26%, vasospasm 13%, low flow 26%, isolated posterior hyperemia 4%) and 
was associated with different neurologic outcomes.   Children found to have low flow were most 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
30 
likely to die (Table 3).  Children diagnosed with vasospasm were most likely to survive, but with 
neurologic sequelae.   
Table 3:  Predicted probabilities (with 95% confidence intervals) of neurologic sequelae 
or death in children with cerebral malaria in each TCD diagnostic group. 
  
Probability of Neurosequelae, % 
Probability of Death, % 
Microvascular 
obstruction 
16 
(5, 26.) 
22 
(10, 34.) 
Hyperemia 
17 
(6, 29) 
27 
(14, 41) 
Vasospasm 
45 
(25, 66) 
18 
(2, 34) 
Low Flow 
24.62 
(14, 35) 
321 
(21, 44) 
IPH 
20 
(0, 45) 
20 
(0, 45) 
IPH; isolated posterior hyperemia 
These findings suggest that multiple different neurovascular changes may contribute to 
neurologic injury across a cohort of pediatric patients with CM.  However, 96% were able to be 
placed into a single phenotypic group and remained in that group until normalization of flow or 
progression to death. This suggests that a predominant neurovascular change may occur on an 
individual level in most cases. 
Near-infrared spectroscopy 
Near-infrared spectroscopy (NIRS) is an established technology for measuring the oxygen 
saturation of hemoglobin in tissues.  Near-infrared light can penetrate through biological tissues, 
including skin, bone, and muscle.  To interrogate a tissue, light is applied to the region of 
interest and undergoes scattering and absorption before being detected by a photosensor77. 
Using different wavelengths, NIRS can differentiate between oxygenated and deoxygenated 
hemoglobin in blood and can measure changes in total hemoglobin concentration using the sum 
of oxygenated and deoxygenated hemoglobin78. NIRS can assess oxygen availability and 
consumption in living tissues. 
NIRS can provide continuous assessment of the oxygen saturation of hemoglobin in cerebral 
tissue via a sensor placed against the scalp. One study used NIRS to monitor cerebral 
oxygenation in twenty adults with malaria79.  They found that the oxygen saturation of 
hemoglobin was generally low (56-60%) across all clinical groups of malaria patients: cerebral 
malaria (Glasgow coma score < 11), non-cerebral severe malaria, and uncomplicated malaria.  
However, when they applied spectral analysis to calculate the amplitude of cerebral blood flow 
oscillations, they discovered that patients with cerebral malaria had severely diminished 
amplitude of oscillations in the 0.02 – 0.04 Hz band.  The amplitude of this oscillation increased 
after recovery from coma.  This may reflect an impairment of the ability cerebral vessels to 
vasodilate and vasoconstrict. 
In a pilot study carried out on the Pediatric Research Ward in 2013-2014, we used NIRS to 
measure total hemoglobin concentration (the sum of oxy- and deoxy-hemoglobin) and 
hemoglobin oxygen saturation using a NIRS probe placed on the forehead where it interrogated 
the frontal cortex. We found that patients with retinopathy-positive cerebral malaria had higher 
total hemoglobin concentrations in the brain than healthy Malawian children (p < 0.0001), but 
the oxygen saturations did not differ (Figure 6). Higher tissue densities of total hemoglobin could 
reflect increased vessel diameter (e.g, hyperemia) or increased venous congestion. 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
31 
 
Figure 6.  Cerebral total hemoglobin concentration (THC) (a) and cerebral hemoglobin oxygen saturation (b). 
THC and oxygen saturation were determined from a 30-minute recording obtained from a NIRS probe applied to the 
forehead of each patient. Horizontal lines represent median values for each group. 
To investigate the frequencies and patterns of periodic oscillations in cerebral hemoglobin 
oxygen saturations, we applied detrended fluctuation analysis (DFA) to the continuous 
recordings. DFA is a mathematical method that analyzes the long-range correlation and long-
range memory of a signal. It has been used to analyze diverse signals from heart-rate dynamics 
to neural spiking to intercranial pressure. DFA analysis determines a single exponent, called Į, 
which reflects the level of self-correlation and randomness in the signal80. When Į is greater 
than 0.5, the signal is correlated, i.e., has repeating patterns; an Į of 0.5 represents random 
fluctuations. Increased randomness in cerebral blood-flow indicates a failure of cerebral 
autoregulation. We applied DFA to both the THC (which reflect fluctuations in hemoglobin tissue 
density, e.g., pulsatility of vessels) and the hemoglobin oxygen saturation (which reflects 
fluctuations changes in oxygen supply and demand). 
Patients with retinopathy-positive cerebral malaria had lower ĮTHC than healthy Malawian 
children (p < 0.001) or children with uncomplicated malaria (p < 0.0001) (Figure 7). Moreover, 
the pattern of fluctuations clustered around Į = 0.5, indicating that the fluctuations in THC were 
random. The Įsat revealed a similar pattern whereby patients with cerebral malaria again had 
nearly random fluctuations in cerebral hemoglobin oxygen saturation, much lower than healthy 
Malawian children (p < 0.001) or children with uncomplicated malaria (p < 0.01). Together, the 
results of detrended fluctuation analysis of the total hemoglobin concentration and the cerebral 
hemoglobin oxygen saturation suggest a loss of cerebrovascular autoregulation. 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
32 
Figure 7.  Detrended fluctuation analysis of the instantaneous changes in the total hemoglobin concentration 
(THC) (a) and cerebral hemoglobin oxygen saturation (b). Horizontal lines represent median values for each 
group. 
NIRS can also be applied to measure arterial vasoreactivity in the peripheral circulation. This is 
accomplished by inflating a pneumatic cuff around the upper arm or thigh while monitoring 
muscle oxygenation distal to the occlusive cuff.  During occlusion, blood flow stops and 
oxygenation decreases in the tissue distal to the occlusion.  After release of the occlusion (3 
minutes duration), both blood flow and tissue oxygenation increase rapidly and exceed their 
baseline resting value.  The rate of increase is a quantitative measure of endothelium-
dependent vasodilation. 
In a pilot study carried out on the Pediatric Research Ward during 2013-2104, we measured 
skeletal muscle reperfusion dynamics in children with retinopathy-positive cerebral malaria and 
compared them against reperfusion dynamics in healthy Malawian children and children with 
uncomplicated malaria. Measurements were performed within six hours of admission to the 
Pediatric Research Ward. Peak muscle reoxygenation rate was lower in patients with cerebral 
malaria compared to healthy Malawian children (p < 0.0001) or compared to children with 
uncomplicated malaria (p < 0.01) (Figure 8). The muscle reoxygenation rate improved on 
hospital day 2 (p < 0.01), and at follow up on day 28 (p < 0.05) among children with cerebral 
malaria (Figure 9). 
Together, these studies of skeletal muscle reperfusion in children with retinopathy-positive 
cerebral malaria identify a defect in muscle reperfusion. This may be attributable to impaired 
endothelium dependent vasodilation, a physiological hallmark of endothelial injury or 
dysfunction, changes in blood rheology, or changes in autonomic control of blood flow. Like the 
cerebral NIRS oxygenation fluctuations described above, decreased skeletal muscle 
reoxygenation is a feature that distinguishes uncomplicated malaria from cerebral malaria. In 
the current study, we plan to measure both cerebral oxygenation fluctuations and skeletal 
muscle reperfusion rates as a vascular physiological biomarker of cerebral malaria; in addition, 
we will examine correlations between NIRS and TCD measurements to further characterize the 
4 major neurovascular abnormalities identified by TCD. 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
33 
2.1.13 Hypothesis  
 
Figure 8. Peak muscle reoxygenation rates on the day of admission to hospital. Peak 
muscle reoxygenation rate was identified as the maximal rate of change in muscle hemoglobin 
oxygen saturation (%) during reperfusion following a 3-minute occlusion with a pneumatic cuff. 
Figure 9. Peak muscle reoxygenation rates among children with retinopathy-positive 
cerebral malaria on hospital days 0-2 and at follow on day 28. Peak reoxygenation rate 
increased by hospital day 2 and at day 28 follow-up analyzing paired data using Wilcoxon’s test. 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
34 
Description of the Study Refining the standard clinical case definition of CM to include malarial 
retinopathy may improve specificity and focusing on retinopathy-positive CM participants 
with moderately severe increased brain volume identifies the group at highest risk of dying. 
 
Our data suggest that these children experience a disruption of the respiratory control center 
manifesting as a respiratory arrest.  The final cause of death appears to be respiratory arrest 
secondary to intracranial hypertension. 
 
Hypothesis:   Death in children with CM occurs when raised intracranial pressure 
from severely increased brain volume impairs the function of the medullary 
respiratory center; interventions which reduce brain volume or support ventilation 
will improve survival rates in CM patients. 
 
Using numbers derived from the ongoing studies in Malawi, of 100 children who meet the 
standard clinical case definition of CM, approximately 70-75 will be retinopathy-positive.  Of 
these, ~ 47% (34-35 children) will have severely increased brain volume on MRI, and, with 
the observed mortality rate of 37% in this clinical subset, about 12 of these children will die.   
The majority recover quickly and completely, suggesting that the adverse effects of 
increased brain volume on respiratory status are reversible and short-lived52.     
 
All CM patients enrolled in the study will contribute to the evaluation of surrogate markers of 
brain volume. 
 
CM patients who are retinopathy-negative and who have evidence of increased brain 
volume on MRI will be eligible to be enrolled in an identical interventional study (of assisted 
ventilation or hypertonic saline).  This group, when combined with the retinopathy-positive 
CM patients described above, represents children who satisfy the less stringent but 
standard WHO definition of cerebral malaria81.  In order to increase generalizability of our 
study results, we will analyze data from this combined group, but perform a sensitivity 
analysis to determine if treatment effects vary in children of different retinopathy status. 
Agent(s)/Intervention(s) 
The goal of this clinical trial is to compare outcomes in children treated with the current standard 
of care (antimalarial drugs and elevation of the head of the bed 30 degrees) to outcomes in 
children randomized to receive one of two different approaches to ameliorating the effects of 
increased brain volume: 
x 
Arm 1: Usual care + immediate mechanical ventilatory support during the initial high risk 
period, with ventilator withdrawal once brain volume has decreased (brain volume score 
 5), and the patient has improved clinically (Blantyre Coma Score > 3). 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
35 
x 
Arm 2: Usual care + intravenous hypertonic saline, withdrawn once brain volume has 
decreased (brain volume score 5) and the patient has improved clinically (Blantyre 
Coma Score > 3). 
x 
Arm 3:  Usual care (antimalarial drugs plus elevation of the head of the bed by 30 
degrees) 
 
Background 
The study design was informed by (a) the strong association between increased brain volume 
and a fatal outcome in pediatric CM, (b) the rapid reversibility of increased brain volume in CM 
survivors, and (c) the importance of identifying the most feasible intervention for use in malaria-
endemic settings. 
One challenge is that direct monitoring of intracranial pressure (via intraventricular catheters, or 
subarachnoid or intraparenchymal bolts) in this setting is relatively contra-indicated --- the risk of 
infection is too high 82.  Indirect measurements of brain volume are required; the data to date 
have been derived via MRI.  Though the MRI findings are useful and reliable, they are not scale-
able across sub-Saharan Africa.  Consequently, efforts to identify more user-friendly 
approaches to identifying patients with severely increased brain volume will be evaluated at the 
same time as this clinical trial is conducted. 
In developed countries, current neurocritical care of children with raised ICP from increased 
brain volume incorporates multiple strategies. Some of the measures commonly used to 
decrease ICP are already standard of care on the QECH Pediatric Research Ward, and as 
such, are not appropriate for inclusion in a randomized controlled trial. These include: elevation 
of the head, adequate oxygenation (including use of supplemental oxygen via nasal prongs and 
mask), prompt blood transfusions to optimize oxygen delivery, antipyretics for fever, 
anticonvulsants for clinically evident seizures, EEG to identify sub-clinical seizures, and glucose 
monitoring to avoid hypo- and hyperglycemia. Sedation is often recommended for agitated 
patients with raised ICP, but CM patients are, by definition, deeply comatose and very rarely 
require additional sedation 83. 
 
Potential etiologies of increased brain volume in pediatric CM 
Increased brain volume can result from a number of molecular, cellular, structural or functional 
changes in the blood brain barrier (BBB), the microcirculation, and various autoregulatory 
systems 84.  Because intracranial volume is fixed, any additional increase in brain parenchyma, 
blood or cerebrospinal fluid can cause a non-linear increase in ICP, aggravating and 
accelerating the original insults and injuries.   
The etiology of increased brain volume in CM patients has not been established, but autopsy 
and clinical information provide evidence to support four possibilities:  
1. Vasogenic (extracellular) edema:  Vasogenic edema results from breakdown of the BBB, 
and primarily involves the white matter 85.  Plasma leaks into the brain parenchyma as a 
result of the increased permeability.  There is abundant evidence of BBB breakdown in 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
36 
CM, from the ring hemorrhages observed in the cortex and cerebellum 8, 61 to fibrinogen 
leakage from apparently intact vessels 13, 61. 
2. Cytotoxic (cellular) edema:  This type of edema is often the result of cellular injury 
following cerebral ischemia, and may play a role in situations where the microcirculation 
is impeded 85.  It is characterized by sustained intracellular water accumulation involving 
both astrocytes and neurons 86.  Fluorescein angiography has shown impaired perfusion 
in the eyes of children with CM 50, and there is immunohistologic evidence of axonal 
damage and myelin loss associated with sequestered parasitized RBCs in fatal cases of 
pediatric CM 61. 
3. Increased blood flow volume:  The anemia, fever and seizures87 that are commonly seen 
in CM patients may lead to increased cerebral blood flow volume (hyperemia) and 
dysfunction of autoregulatory systems88. 
4. Vascular congestion: Intense sequestration of parasitized red cells in postcapillary 
venules may lead to venous obstruction and vascular congestion 89, 90.
The current standard of care on the Paediatric Research Ward in Blantyre addresses the likely 
determinants of increased blood flow volume (seizures, fever and anemia). Potential 
interventions were reviewed to identify those most likely to address the other three putative 
etiologies:  vasogenic edema, cytotoxic (cellular) edema and vascular congestion. 
 
  Summary of Previous Pre-clinical Studies 
 
 
None 
 
  Summary of Relevant Clinical Studies 
2.2.2.1  Mechanical ventilation 
There is only one published study addressing endotracheal intubation in children with life-
threatening malaria, and it is a retrospective cohort study 91.  Intubations were performed by 
intensivists in the ICU and by pediatricians in the emergency rooms.  Intensivists tuned the 
ventilators, and the nurse to patient ratio in the ICU was 1:5 during the day and 1:10 during the 
night.  Children were managed according to WHO guidelines, which included prompt blood 
transfusions, management of hypoglycemia, and an anticonvulsant protocol for seizures.  
Raised intracranial pressure (protracted episodes of decorticate or decerebrate rigidity in 
addition to dilated sluggish pupils) was treated with 10-20 ml/kg of 20% mannitol and short-time 
induced hypocapnia (pCO2 adjusted to 25-35 Torr). Parenteral antibiotics were used only for 
concomitant community-acquired and nosocomial infections (which were defined as occurring 
after 48 hours of stay). 
During the study period of 5 years, a total of 502 children were hospitalized with severe malaria.  
Of these, 83 patients required intubation. Indications for intubation included deep coma (BCS < 
2; n=16), overt cortical or diencephalic injury (i.e., status epilepticus or decorticate posturing; 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
37 
n=20), severe brainstem involvement (decerebrate posturing or opisthotonos; n=15), shock 
(systolic BP < 50 in children under five, systolic BP <80 in children over five; n=15), cardiac 
arrest (n=13) and severe respiratory distress (acute lung injury with pulmonary infiltrates and a 
PiO2/Fio2<300 Torr; n=4).   
The median age was 8.6 years, the median duration of ventilation was 36 hours, and 50 (60%) 
patients died.  The lowest mortality rate (12.5%) was in the group intubated for deep coma only. 
All of those with cardiac arrest subsequently died, and mortality rates for the other subsets are 
shown below (Fig. 10). 
 
 
Sixty-one children survived more than two days and of these, 20 (33%) had evidence of hospital 
acquired infections (twelve had pneumonia, 8 had septicemia).  The cumulative incidence rates 
were 2.3% after 48 hours, 15.3% after 72 hours, 26.6% after 92 hours and 63.4% after 12 
hours.   
The authors concluded that mechanical ventilation in the setting of life-threatening childhood 
malaria was required for a short time, and that overall outcome depended more on clinical 
presentation than on critical care complications.  Without retinopathy data, or any other clinical 
indicators of raised intracranial pressure (e.g., opening CSF pressure, evidence of papilledema, 
neuro-imaging), it is difficult to extrapolate from this patient population to those in the proposed 
clinical trial – but the relatively good outcomes in the group with “coma only”, and the low rates 
of nosocomial infection are encouraging. 
Figure 10.  Kaplan-Meier curves related to reasons for intubation among 83 
children with severe malaria who required intubation.


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
38 
2.2.2.2  Head elevation  
For decades, routine treatment of raised ICP has included maintaining the patient’s head in a 
neutral position with elevation of the head of the bed to 30 degrees 92. This approach has been 
evaluated and supported in adults 93-99 and recently in children 93. The benefits of elevating the 
head, relative to the heart, are attributed to gravity-induced changes in cerebral blood and 
cerebrospinal fluid volume.  In most clinical situations, head elevation contributes positively 
towards the usual therapeutic goal of maintaining ICP below 20 mm Hg while maintaining 
cerebral perfusion pressures between 40-65 mm Hg 100. This approach is standard practice in 
neuro-intensive care units for patients with increased intracranial hypertension 101. 
2.2.2.3  Corticosteroids  
Glucocorticoids were first used for the control of cerebral edema associated with cerebral 
neoplasms 102 and their introduction coincided with a significant decline in peri-operative 
mortality rates 103. Their effectiveness was eventually traced to actions on the BBB, a highly 
selective interface composed of brain endothelial cells. These endothelial cells, unlike those in 
extracerebral capillaries, are connected by tight junctions; opening of these tight junctions plays 
a key role in the formation of vasogenic cerebral edema 104 and corticosteroids ameliorate this 
disruption.  Dexamethasone is the most commonly used corticosteroid in this setting. It is 
approximately six times as potent as prednisolone and reaches full effect within 24-72 hours 104.  
Doses vary between 4 to 100 mg/day, but in patients with brain tumors, side effects begin to 
outweigh therapeutic effects at doses above 16 mg/day 105. If the tumor is causing impaired 
consciousness or signs of increased ICP, a standard approach includes a loading dose of 10 
mg (intravenously) followed by doses of 16 mg/day, in four divided doses.  
Prolonged use of high dose steroids can produce serious side effects including adrenal 
suppression, steroid myopathy, Cushingoid facies, hyperglycemia, and peptic ulcers, and 
requires a tapering regimen following treatment of the underlying etiology of increased ICP (e.g., 
tumor resection). Side-effects are similar with short-term use, but tapering is unnecessary for 
treatments less than three weeks 106.  
Corticosteroids have also been used as adjunctive therapy for bacterial meningitis, primarily to 
reduce inflammation in the subarachnoid space.  Several large randomized trials have had 
conflicting results in terms of overall outcome, but authors of a recently published meta-analysis 
of individual participant data from five major trials concluded that in bacterial meningitis, 
adjunctive dexamethasone does not significantly reduce rates of death or neurological disability 
107.  Dexamethasone doses of up to 0.4 mg/kg twice daily were used for 2-4 days in the pediatric 
participants; in adults, the dosages ranged from 16 mg twice daily to 10 mg four times daily for 4 
days. No participants were “weaned” from the corticosteroids; tapered doses were not required 
for regimens ranging from 2-4 days in length.  Gastrointestinal bleeding was reported in all 
studies (1.3% of 1,021 participants on dexamethasone, 1.9% of 1,014 participants on placebo), 
but the difference between the two groups was not statistically significant (p=0.14).  
Hyperglycemia was more common in the adults receiving dexamethasone than among the 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
39 
placebo recipients (20.3% vs. 16.0%, p = 0.02); no data on glucose concentrations after 
treatment were collected in the pediatric study populations. 
2.2.2.3.1 Corticosteroids in malaria  
There have been two randomized controlled trials of dexamethasone as adjunctive therapy for 
CM 25, 26. Warrell et al.25, used a total of 2 mg/kg dexamethasone for the first 48 hours (in 
children, 0.6 mg/kg loading, followed by 0.2 mg/kg q 6h;  in adults, 0.5 mg/kg initially, followed 
by 10 mg q 6h), in conjunction with intravenous quinine.  One hundred patents with CM were 
included, and the mortality rates did not differ between dexamethasone and placebo recipients.  
Coma recovery times were prolonged in the dexamethasone cohort, and they experienced more 
complications (pneumonia, gastrointestinal bleeding) than did those who received placebo. 
The second study 26 used a much higher dose of dexamethasone (11.4 mg/kg in total over 48 
hours, administered as 3 mg/kg initially, and then 1.4 mg/kg q 8h for 48 hours). Thirty-eight 
stuporous or comatose participants infected with P. falciparum were randomized to receive 
either dexamethasone or placebo.  All participants were treated with intravenous quinine. 
Overall mortality rates were the same in the two study arms; four participants in each group 
died. Although there was not a statistically significant difference in the rate of complications, 3 of 
19 dexamethasone recipients developed gastrointestinal bleeding, compared to 0 of 19 placebo 
recipients. In contrast to the results observed in the Warrell study 25, 3 of 19 placebo recipients 
developed pneumonia, compared to 0 of 19 dexamethasone recipients. 
A Cochrane review combined results from these two trials to assess the effects of 
corticosteroids in participants with CM on death, life-threatening complications and residual 
disability in survivors 108.  The meta-analysis showed that deaths were distributed evenly 
between the corticosteroid and control groups in both trials (RR 0.89, 95% CI 0.48-1.68, 143 
participants).  Gastrointestinal bleeding and seizures were more common in the corticosteroid 
group (RR 8.17, 95% CI 1.05-63.6 for bleeding; RR 3.32, 95% CI 1.05-10.47 for seizures).  For 
pneumonia, the confidence intervals were very wide, and no statistically significant difference 
was observed between the two groups. For pulmonary edema, urinary tract infections and 
bacteremia/septicemia, there were no differences between groups.    
The authors of the Cochrane review concluded that there is insufficient evidence to support the 
use of corticosteroids in the routine management of CM but recognized that the two trials did not 
have sufficient power to exclude a beneficial effect. Given the unclear efficacy and consistent 
evidence of adverse effects of adjunctive steroid therapy, we have chosen two non-steroid 
interventions for testing in this clinical trial. 
At present, corticosteroids are recommended to treat intracranial hypertension in only two 
settings: acute mountain sickness109  and brain tumors110; in all other circumstances studied 
(e.g. stroke111, traumatic brain injury112, brain swelling following cardiac arrest113, sub-arachnoid 
hemorrhage114), and in the majority of CNS infections (with niche exceptions, and in those 
exceptions steroids are not believed to act by reducing intracranial hypertension)115, 116, the risk 
benefit ratio is unambiguously unfavorable and not considered a component of standard care, 
nor worthy of further investigation.  


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
40 
2.2.2.4   Osmotic agents (mannitol and hypertonic saline) 
Osmotic agents play an important role in reducing raised ICP and treating cerebral edema 85, 117-
119.  The salutary effects of osmotherapy on ICP are thought to result from brain shrinkage after 
the shift of water out of the brain parenchyma. The ideal osmotic agent remains in the 
intravascular compartment, thus promoting movement of water from the intracellular to the 
extracellular compartment. As fluid moves into the vascular space, the tissue shrinks. The ideal 
agent is inert, nontoxic, and has minimal systemic side effects. The two main agents in clinical 
use today are mannitol and hypertonic saline 85, 117-119.  
2.2.2.4.1 Mannitol  
Mannitol is a freely filtered, non-metabolized solute that decreases the reabsorption of water 
across the renal tubules, creating diuresis.  It has a biphasic effect on ICP. Initially it affects 
RBCs, and reduces blood viscosity 120. At the same time, intravascular volume increases 
because mannitol increases plasma osmolality.  The combined effect is an increase in cardiac 
output which, if autoregulatory pathways are intact, leads to compensatory cerebral 
vasoconstriction, and a drop in ICP 121.  This effect typically wanes 4 hours after administration 
120. 
The second phase of ICP reduction occurs as mannitol extracts water from the cerebral 
extracellular space into the intravascular compartment. This occurs due to an induced osmotic 
gradient.  An intact BBB is required for this to occur 85.     
In traumatic brain injury (TBI), bolus dosing is preferred. Mannitol becomes less effective with 
repeat dosing and works best when cerebral vascular autoregulation is intact. 
Mannitol administration decreases cerebrospinal fluid production by up to 50%, which, via the 
Monro-Kellie Doctrine, can lead to prolonged ICP decreases 122, 123. 
In combination with hyperventilation, mannitol was shown to reverse transtentorial herniation in 
a heterogeneous cohort of 28 participants with cerebral edema related to neoplasms, 
intracerebral hemorrhage, TBI and ischemic stroke 124. 
Side-effects:   Mannitol can cause significant diuresis, necessitating close observation of fluid 
balance to avoid hypovolemia.  Target osmolality in mannitol recipients is generally between 
300 and 320 mOsm 125.  Hyperosmolality can cause acute renal failure via dose-dependent 
vasoconstriction of the renal arteries 126 or by massive diuresis 127.  Accumulation of mannitol in 
cerebral tissue may lead to a rebound phenomenon of increased ICP, but this is controversial 
117, 119.  
2.2.2.4.2 Mannitol in malaria 
The earliest use of osmotherapy in malaria involved urea in invert sugar (i.e., a mixture of 
glucose and fructose derived from sucrose) 128, 129, but these studies lacked controls. More 
recently, mannitol has been used in a study of intracranial hypertension in Kenyan children with 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
41 
CM14 and in two randomized clinical trials in CM, one involving adults 33 and the other involving 
children14. 
Newton, et al 14 found that mannitol controlled the ICP in children with moderate intracranial 
hypertension (ICP above 20 mm Hg and cerebral perfusion pressure less than 50 mm Hg, 
lasting longer than 15 minutes continuously), but it did not prevent the development of 
intractable intracranial hypertension in children with severe elevations of intracranial pressure 
(ICP about 40 mm Hg and cerebral perfusion pressure less than 40 mm Hg lasting longer than 
15 minutes continuously). 
A large, randomized, double-blind controlled trial comparing mannitol to placebo was conducted 
in Uganda, involving 156 pediatric participants with CM 32. The mannitol recipients were given a 
single dose (1g/kg) of mannitol, and those in the placebo group received an equal volume (5 
ml/kg) of normal saline.  All children were treated with intravenous quinine.  The trial was 
powered to detect a difference in coma resolution time in mannitol recipients (66% were 
predicted to regain full consciousness in 24 hours) compared to those receiving placebo (where 
41% were predicted to regain full consciousness in 24 hours).  Mortality rates in the two groups 
were similar (13.2% in those receiving mannitol, 16.3% in the placebo group).  The median time 
to regain consciousness was shorter in the mannitol group (18.9 hours) than in the placebo 
group (20.5 hours) but the difference was not statistically significant.    Intracranial pressure was 
not measured in the participants, as CSF opening pressures were not reported in participants 
who had lumbar punctures. 
Mohanty, et al 33 used CT scan evidence of cerebral edema to identify adult CM participants for 
an open, randomized trial comparing intravenous mannitol (1.5 g/kg, followed by 0.5 g/kg every 
8 hours) to controls receiving standard of care..  Thirty-one participants received mannitol and 
30 received standard care; all received intravenous quinine. There was no difference between 
the two groups in terms of survival, and the median coma resolution time was longer in the 
mannitol recipients than in those receiving quinine only (90 hours vs 32 hours, p = 0.02). 
Analysis of the CT scan findings from the participants enrolled in this study suggested that mild 
to moderate cerebral swelling was common in adult participants with CM but did not correlate 
with coma depth or disease outcome. 
A Cochrane review of mannitol and other osmotic diuretics as adjuncts for treating CM was 
published in 2011130, before Mohanty et al 33.  Only one trial, the Uganda study described 
above, met the inclusion criteria, and the conclusion of the review was that there are insufficient 
data on the effects of osmotic diuretics in children with CM. 
We did not select mannitol for our osmotherapy agent given the aforementioned weak support 
for efficacy, the challenging management of intravascular volume required when using mannitol 
(which may result in hypotension – and thereby exacerbate brain injury associated with 
increased intracranial pressure)131, 132 and the mounting body of evidence demonstrating 
superiority of hypertonic saline as first line osmotherapy for cerebral edema in numerous 
settings133.  
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
42 
 
 
2.2.2.4.3 Hypertonic saline 
Hypertonic saline gained popularity initially as a volume expander in acute resuscitation 134 and 
investigators noted improved survival rates in participants with traumatic brain injuries who had 
received hypertonic saline 135.  Like mannitol, hypertonic saline has a two-phase effect on ICP.  
The first is via rheology, and the second by osmotic activity across the BBB 136, 137.  The BBB is 
less permeable to hypertonic saline than to mannitol, and as a result, the potential for water to 
“follow” the solute into the brain, thus worsening cerebral edema, is less 138.  
Hypertonic saline has been effective in reversing transtentorial herniation.  In a retrospective 
analysis involving 75 herniation events in 68 participants, boluses (30-60 mL) of 23.4% 
hypertonic saline reversed 75% of the clinical herniation events 139.  These participants had a 
variety of neurologic illnesses including intracerebral hemorrhage, subarachnoid hemorrhage, 
stroke, tumor, subdural and epidural hematomas, and meningitis. 
 
Hypertonic saline has been demonstrated to act via salutary effects on aquaporin water 
channels, which are newly recognized as therapeutic targets in cerebral edema 140-142.   
Abundant in brain, the aquaporin 4 (AQP4) water channels play multiple roles regulating brain 
water homeostasis, astrocyte migration, neuronal excitability, and neuroinflammation 143. AQP4 
is highly expressed in ependymal cells and astrocytes with polarized distribution in end-feet 
membranes facing cerebral capillaries and pia mater 144. The strategic location at the blood–
brain interface and the cerebrospinal fluid–brain interface makes AQP4 a major factor in 
regulation of water movement into and out of the brain. AQP4 has been investigated in brain 
edema associated with multiple forms of brain injury such as stroke, meningitis, neuromyelitis 
optica, and brain tumor145. 
 
 A role for AQP4 in CM has recently been suggested and AQP4 has been demonstrated to be 
protective in murine models of CM. In addition to its effect on AQP4, hypertonic saline has been 
demonstrated to have significant salutary effects upon neuro-inflammation146-150 and endothelial 
injury (a putative mechanistic basis for vasogenic edema in cerebral malaria)151-154.  
 
Side-effects:  A theoretical danger of hypertonic saline administration is that rapid increases in 
sodium concentration may induce central pontine myelinolysis.  This devastating neurological 
abnormality is a rare consequence of overly rapid correction of chronic hyponatremia.  To date, 
there are no reports of central pontine myelinolysis occurring in participants receiving hypertonic 
saline for the treatment of increased ICP 139, 155, 156.  Additionally, there are no data clearly linking 
the use of hypertonic saline to the development of acute renal failure or to rebound increases in 
ICP in humans 85, 117, 119. 
 
The hypernatremia induced by hypertonic saline has been associated with coagulopathies and 
excessive intravascular volume 85, 119.  Electrolyte abnormalities (hyperchloremic metabolic 
acidosis and hypokalemia) can develop, necessitating careful monitoring 85, 118, 119.  
 
 
 
2.2.2.4.4. Mannitol vs. hypertonic saline 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
43 
There are numerous biochemical and physiologic considerations when choosing between 
hypertonic saline and mannitol for the treatment of elevated ICP.  Hypertonic saline infusions 
result in significant volume expansion, along with improved cardiac output, regional cerebral 
blood flow, and increased cerebrospinal fluid absorption 123.  Mannitol’s advantages include 
improved microvascular cerebral blood flow 121, 157 and blood rheology 117, 158, reduced blood 
viscosity 120 and cerebrospinal fluid production 158, free-radical scavenging 159, and inhibition of 
apoptosis 160. 
Hyperosmolar therapy has been studied most extensively in the setting of TBI 161.  The first 
head to head comparison of mannitol and hypertonic saline in TBI was a randomized trial 
comparing 20% mannitol to 7.5% hypertonic saline (both as boluses) 162.  The volumes 
delivered were identical, but the osmolar loads were different; the mannitol group received 4.8 
mOsm/kg and the hypertonic saline group received 2.3 mOsm/kg.  The mean number and 
duration of episodes of intracranial hypertension were significantly lower in the hypertonic saline 
group, but overall outcomes of the two treatment groups were similar. 
The effects of sustained equimolar doses of mannitol and hypertonic saline on cerebral blood 
flow and metabolism following TBI were compared in 47 adult participants 163.  Both 
interventions effectively and equally reduced increased ICP while improving cerebral perfusion 
pressure and cerebral blood flow, but the effect was significantly stronger and of longer duration 
after hypertonic saline.  The effect of hypertonic saline on raised ICP appeared to be more 
robust in participants with diffuse brain injury. This study was published after the meta-analysis 
described immediately below (2.2.2.4.6) 
 
 
2.2.2.4.5 Meta-analysis of mannitol vs. hypertonic saline in adults 
Five trials enrolling a total of 112 adults participants, comparing equimolar doses of mannitol 
and hypertonic saline for the treatment of elevated ICP in settings where quantitative ICP 
measurements were feasible were identified 157, 164-167. None of the studies was blinded, and all 
were small; the largest enrolled 40 participants. Many studies amplified the sample size by 
including multiple episodes of raised ICP per participant. Most studies included a 
heterogeneous group of participants (TBI, stroke, intracerebral hemorrhage, subarachnoid 
hemorrhage). 
Mannitol was effective in controlling elevated ICP in 69 of 89 episodes (78%, 95% CI 67-86%), 
whereas hypertonic saline was effective in 88 of 95 episodes (93%, 95% CI 85%-97%). The 
pooled relative risk of ICP control using hypertonic saline compared to mannitol was 1.16 (95% 
CI 1.00-1.33, p=0.046), and the weighted mean difference in ICP reduction using hypertonic 
saline compared to mannitol was 2.0 mm Hg (95% CI -1.6 – 5.7 mm Hg, p=0.276).  When a 
fixed-effects model was applied, the point estimate for the relative risk did not change, but the 
confidence intervals narrowed (95% CI 1.05-1.35, p = 0.007). With the fixed effects model, the 
weighted mean difference in ICP reduction using hypertonic saline compared to mannitol 
remained 2.0 mm Hg, but again, the confidence interval narrowed (95% CI 0.1 – 3.8 mm Hg, 
p=0.036). 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
44 
Conclusions:  This meta-analysis found that hypertonic saline was more effective than mannitol 
in controlling episodes of elevated ICP and identified a trend toward greater quantitative ICP 
reduction in the hypertonic saline group.  These findings are corroborated by the results of 
seven observational studies demonstrating the effectiveness of hypertonic saline in lowering 
ICP after failure of standard mannitol therapy 139, 168-173. 
 
 
2.2.2.4.6. Systematic review of mannitol vs hypertonic saline in children 
Gwer et al 174 recently completed a very thorough review of the use of osmotic agents in 
children with acute encephalopathies.  The goal of this review was to examine the effectiveness 
of osmotic agents in reducing ICP in children with acute encephalopathies, and to determine the 
effect of osmotic agents on clinical outcome in the same population. 
They identified four randomized trials 32, 175-177, three prospective observational studies 14, 155, 178, 
two retrospective studies 156, 179 and one case report 180.  Of these ten studies, four involved non-
traumatic encephalopathies 14, 32, 176, 179 and ICP was monitored in seven. Both the observational 
study of mannitol in pediatric CM 14, and the Ugandan clinical trial of mannitol in pediatric CM 32 
described above were included in this systematic review 
Effects on ICP:  One study 175 was a crossover trial involving 18 children with TBI; there was a 
significant drop from the initial ICP following treatment with hypertonic saline (p = 0.003) 
compared to normal saline (p = 0.32). Another randomized, controlled trial 177 compared 
hypertonic saline with Ringer’s lactate; more interventions for raised ICP were used in the 
Ringer’s lactate group compared to the hypertonic saline group (p < 0.001). Hypertonic saline 
was administered as a continuous infusion to ten children with TBI with raised ICP that was 
refractory to mannitol 155; it produced a sustained reduction of ICP that was maintained over 72 
hours. As noted above, bolus therapy with mannitol reduced moderately raised ICP in children 
with CM, but did not impact those with severely increased ICP 14. Oral glycerol exerted a short-
term effect on ICP in 3 children with TBI;  ICP decreased by at least 50% within the first half 
hour of administration, but the reduction was not maintained beyond 90 minutes 178.  Another 
case report describing two children with TBI showed a dose response relationship between both 
hypertonic saline and mannitol and ICP 180, but mannitol appeared to cause a reduction in 
cerebral perfusion pressure. 
Effects on outcome:  Clinical outcome was evaluated only in the four randomized, controlled 
trials as these were the only studies with comparison groups.  In one trial of children with 
bacterial meningitis that compared glycerol alone versus glycerol plus dexamethasone versus 
placebo, the mortality was lower in those given glycerol compared to placebo (RR 0.64, 95% CI 
0.54 – 0.76), and in those given glycerol and dexamethasone, compared to placebo (RR 0.79, 
95% CI 0.68 - 0.92) 176. There were only two deaths in the trial comparing hypertonic saline and 
Ringer’s lactate, both in the Ringer’s lactate group 177.  As noted earlier, the clinical trial of 
mannitol in pediatric CM was not powered to detect an impact on outcome, and it did not 
demonstrate any differences between the mannitol recipients and those receiving the normal 
saline placebo. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
45 
Conclusions:  Hypertonic saline appeared to achieve greater reduction in ICP than other 
osmotic agents, and all examined agents demonstrated a dose response in the reduction of 
ICP. When compared to mannitol, hypertonic saline maintained or improved cerebral perfusion 
pressure, an important determinant of neurological outcome 180.The duration of the effect on ICP 
was transient in a number of cases, and continuous infusions of hypertonic saline appeared to 
achieve sustained reductions in ICP more commonly than other osmotic agents.  The review 
supports the use of oral glycerol in acute bacterial meningitis, and the use of hypertonic saline in 
acute traumatic and non-traumatic encephalopathies.  To date, the data are not felt to be 
sufficient to generate specific guidelines, and clinical trials were recommended to establish the 
safest and most efficacious concentrations, and to determine optimum routes and rates of 
administration.  
As described in detail above, as a pharmacologic rescue for severe CM, there is a (1) plausible 
mechanistic rationale, (2) empiric efficacy and safety data as well as (3) a pragmatic basis for 
selecting osmotherapy over corticosteroid therapy and more specifically, for selecting hypertonic 
saline over mannitol.  
As stated in the package insert for hypertonic saline: Safety and effectiveness of sodium 
chloride injections in pediatric patients have not been established by adequate and well 
controlled trials, however, the use of electrolyte solutions in the pediatric population is 
referenced in the medical literature. The warnings, precautions and adverse reactions identified 
in the label copy should be observed in the pediatric population. 
 
 
 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
46 
2.2.3 
Summary of Epidemiological Data 
NA 
Rationale 
This clinical trial of adjuvant therapy (immediate ventilatory support, or hypertonic saline) for 
Malawian children with CM is directed toward the supportive treatment of increased brain 
volume, and it avoids the pitfalls that have bedeviled previous trials because:   
 
x 
The interventions target a mechanism recently recognized as being strongly 
associated with a fatal outcome, severely increased brain volume; this mechanism is 
self-limited in CM survivors, suggesting that supportive care or rescue therapy may 
be effective.   
 
The clinical trial setting in Malawi has been transformed because, beginning in July 2017, 
mechanical ventilation became available in the new Paediatric Intensive Care Unit (PICU) at the 
Queen Elizabeth Central Hospital.  
 
This introduces new ethical issues:  mechanical ventilation cannot be withheld in patients who 
suffer a respiratory arrest, and ventilatory support may result in a cadre of survivors with severe 
neurological sequelae.  Therefore, “treatment failure” (failure of the primary intervention) is 
defined differently in the different therapeutic arms. In Arms 2 and 3, in which participants are 
not randomized to immediate ventilatory support, any participants who suffer respiratory arrest 
and are placed on ventilators or who die or are brain dead will be classified as treatment 
failures.  In Arm 1, the patients who are randomized to receive immediate ventilatory support, 
“treatment failure” is defined as brain death at any time, or death at 7 days, with ventilatory 
support withdrawn.  
 
The hypothesis underlying this prospective, randomized clinical trial is that death in children with 
CM occurs when raised intracranial pressure from severely increased brain volume impairs the 
function of the medullary respiratory center and that interventions which reduce ICP or support 
ventilation will improve survival rates in CM patients.   
 
Support for this mechanism of death is based on MRI evidence of severely increased volume in 
all fatal cases and on clinical observations consistent with pre-morbid brainstem compression 
and cerebral herniation 14, 44, 52, 181. Neuroimaging was required to identify this mechanism: full 
autopsies with detailed neuropathological studies did not reveal the cause of death in 
participants with CM.  Cerebral edema was a constant feature of all fatal CM cases at autopsy, 
but gross findings consistent with transtentorial or uncal herniation were less common. This 
suggests that medullary impingement is a transient event, or that the autopsy itself, performed 
within 12 hours of death, became effectively a decompressive craniotomy, and may have 
obscured physical evidence of herniation.   
 
The proposed trial will simultaneously test the hypothesis that increased ICP is the cause of 
death in children with CM and evaluate two therapeutic approaches:  one treatment regimen 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
47 
targets raised ICP directly and the other targets the effect of ICP (respiratory arrest). If the trial 
is successful, at least one effective treatment for children with CM at high risk of death will be 
identified. 
 
The study population  
 
The mortality rate in children with CM who have highly increased brain volume (37%) is high 
enough to support a single-site three-armed clinical trial over six years.   
 
The survival rate in this subset (63% survive, and nearly all are discharged within 72 hours of 
admission) suggests that in survivors, severe edema rapidly subsides spontaneously, and that 
interventions which (a) reduce edema enough to prevent cerebral herniation or (b) support 
ventilation during the vulnerable period, might be sufficient to save lives. 
 
Using malarial retinopathy to identify the high risk group:  Recognizing the importance of being 
able to scale up the findings of this study, and appreciating the operational difficulties of 
identifying patients with malarial retinopathy, children with retinopathy-negative CM will also be 
enrolled and randomized to one of the three study arms.  Randomization for the retinopathy-
positive patients will be carried out independently of randomization for the retinopathy-negative 
patients.  Should the clinical trial results reveal a positive therapeutic effect of either intervention 
(Arm 1 or Arm 2), uptake of study results across Africa will be enhanced if treatment decisions 
do not depend on the results of an ocular funduscopic examination, which requires expensive 
equipment and extensive training.  This trial represents an opportunity to assess efficacy in the 
entire CM population (combined retinopathy-positive and retinopathy-negative). If efficacy can 
be demonstrated in the entire CM population, scalability will be enhanced, as indirect 
ophthalmoscopy will not be necessary before instituting an intervention which will have been 
shown to be beneficial.    
 
Using MRI to identify the high-risk group: The data driving this study were collected using the 
0.35T MRI in Malawi52.  Evidence of severely increased brain volume is readily discernible via 
MRI, but clinically reliable surrogate markers of increased brain volume have yet to be identified. 
A secondary outcome of this study will be an evaluation of these potential surrogate markers so 
that, if a successful intervention is identified, its implementation will not require MRI technology 
to identify subjects at high risk of death.  
 
During the first enrollment season (January-June 2018) 57 participants had MRI images 
interpreted in real time by a single neuroradiologist.  When the season was completed, all 
images were independently re-interpreted by two different neuroradiologists.  If there was a 
discrepancy in the assigned brain volume scores determined by the radiologists interpreting the 
images post-season, a consensus score was obtained. 
 
Comparisons between the brain volume scores assigned in real time and the consensus score 
revealed an inability for neuroradiologists to differentiate a score of 6 or 7 in real time, leading to 
non-differential misclassification.  Four subjects who had real-time assignment of brain volume 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
48 
scores of 7 (and therefore were eligible for randomization in real time) had consensus scores of 
6.  Three subjects with real-time assignment of brain volume scores of 6 had a consensus score 
of 7.  
 
Comparisons between brain volume scores assigned in real time and the consensus score 
showed that for scores of 6 or less, neuroradiologists interpreting images in real time generally 
assigned brain volume scores higher than the consensus score.  During the first enrollment 
season, a score of 5 or 6 was assigned 17 times in real-time.  Adjudicated scores were the 
same (4), lower (12), or higher (1).  One subject assigned a brain volume score of 5 had an 
adjudicated score of 7. 
 
To include all participants who should be randomized (e.g. that have an adjudicated brain 
volume score of 7 or 8) UHTXLUHVWKDWDOOSDUWLFLSDQWVZLWKEUDLQYROXPHVFRUHVRIDUHHOLJLEOH
for randomization.  To try to avoid including participants who will eventually have an adjudicated 
score of 5 or less, beginning in the 2019 enrollment season, all participants will have 2 
independent MRI interpretations for brain volume score performed in real-time.  If both real-time 
interpretations have a brain volume VFRUHRIWKHSDUWLFLSDQWZLOOEHHOLJLEOHIRUUDQGRPL]DWLRQ
If either of the independent real-time neuroradiologists assigns a brain volume score of < 6 the 
participant will not be eligible for randomization.   
 
Beginning in 2021, the 0.35T fixed magnet General Electric MRI will be replaced with a portable 
MRI scanner manufactured by Hyperfine.  Clinical trial participants may be scanned with the 
0.35 T General Electric MRI machine, the Hyperfine scanner, or both. 
 
Interventions  
Our site, by virtue of its ready access to MRI and pediatric intensive care, is uniquely qualified to 
identify the study population and carry out the clinical trial. For the findings to be useful in other 
malaria-endemic settings, it will be important to identify and validate more user-friendly 
approaches to identifying children with life-threatening increases in brain volume, and to 
determine the simplest approach to treat them.  These dual objectives are the basis of the 
primary and secondary aims of the study.   
 
Criteria for selecting experimental interventions  
The pathophysiology of intracranial hypertension is complex and depends on the mechanism of 
cerebral edema, the volume of intracranial contents, the integrity of the BBB, and cerebral 
perfusion pressure. The balance of Starling forces (the transcapillary hydrostatic pressure 
gradient that is counterbalanced by an osmotic pressure gradient) determines the magnitude of 
extracellular fluid flow into the brain substance. 
 
The following issues were considered: 
 
x 
The etiology of increased brain volume in this patient population has not been 
established, and the relative contribution of any of the four putative mechanisms, at any 
single point in the disease process, is unknown.  Furthermore, the relative contributions 
may change over the course of the illness. Since no animal model of CM exists, it is 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
49 
unlikely that insights into the mechanism of increased brain volume in children with CM 
will be rapidly forthcoming.  Although ongoing research may eventually illuminate the 
specific mechanisms contributing to the elevated ICP of these patients, our previous 
work has identified a group of children at high-risk for mortality and broadly effective 
interventions exist. 
x 
The treatment for increased brain volume that is most likely to be effective in this 
setting is hypertonic saline. 
x 
Given the rapid reversibility of the increased brain volume, that its etiology is 
unclear, and that respiratory arrest is the likely cause of death, the other trial arm 
is ventilatory support.   
x 
Given the high mortality rate in children with CM with highly increased brain volume and 
the now ready availability of ventilatory support in the ICU in Malawi, those randomized 
to “usual care” or “hypertonic saline” will receive ‘rescue ventilation’ if they experience a 
respiratory arrest.  
  
 
Experimental regimens 
Duration of therapy: Serial MRIs have shown that, in survivors, the increased brain volume 
generally resolves within 48 hours.  A summary of data from CM participants on the research 
ward shows that 85% have either died or been discharged within 72 hours (data not shown).  To 
maximize the chance of observing a positive result, the experimental regimens will continue for 
7 days, or until the child experiences death, brain death, or clinical improvement (brain volume 
score < 5 and Blantyre Coma Score > 3). 
 
Elevation of the head of the bed:  In the PICU and PRW, the head of the bed will be elevated to 
30o, and the head maintained in the midline with towels. 
 
Hyperosmolar therapy: The choice of hypertonic saline (over mannitol) as the hyperosmolar 
agent was made on the basis of data demonstrating the efficacy of hypertonic saline in a wide 
variety of clinical settings 138, 155, 156, 162, 163, 174, 182-187, especially in reversing herniation133, and its 
volume expansion effects (which may increase its efficacy in CM, where sequestration of 
parasitized erythrocytes impairs cerebral microcirculation).  A recent review, focusing on 
pediatric encephalopathies, recommended hypertonic saline over mannitol 174.  Participants 
receiving mannitol are often subject to diuresis and maintaining the appropriate intravascular 
volume in a resource-challenged setting such as ours is complicated. 
 
The hypertonic saline regimen will be “goal directed”, aiming to achieve serum sodium (Na) 
concentrations between 150-160 mmol/L188. This range is safe and has been shown to be 
effective in reducing ICP in pediatric participants with a variety of underlying pathologies155, 182. 
Sodium balance is rarely deranged in malaria 189, but doses of hypertonic saline will be titrated 
to serum sodium levels, measured with a bedside instrument (I-Stat, Abbot Labs).  Adjustments 
for hyper- and hyponatremia will be made (see Section 9.2.4.1).   
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
50 
3% saline was chosen as the specific hypertonic saline product for this trial because: (1) the 
target range of serum sodium can be effectively achieved 155, 156, 175, 190, (2) it can be 
administered through peripheral intravenous lines191, 192 therefore mitigating the risk associated 
with placing and maintaining central venous catheters in our setting. We will employ a 
continuous infusion to maintain elevated sodium levels as is common practice for other 
therapeutic indications.  
 
Estimating intracranial pressure 
We are in the unusual position of being able to obtain magnetic resonance images of 
participants without being able to measure intracranial pressure directly. We have clear cut 
evidence of severely increased brain volume on MRI, and a clinical syndrome compatible with 
raised intracranial pressure. However, the risks of infection with placement of an intracranial 
pressure monitoring device, in both the short- and long-term, outweigh the benefits of the direct 
measurements 193. Additionally, there is no evidence to suggest that monitoring of intracranial 
pressures in cases of cerebral malaria or other forms of meningitis/encephalitis, are of any 
clinical benefit 194. 
 
Concurrent with our clinical trial, we will test and validate other surrogate measures of increased 
brain volume, an approach that is gaining ground in more sophisticated intensive care settings 
195.  These can all be carried out at the bedside and include optic nerve sheath diameter 
(ONSD) by ultrasound, cerebral flow velocities by transcranial Doppler ultrasound, NIRS 
monitoring of both cerebral oxygenation and skeletal muscle reperfusion dynamics, heart rate 
variability from the EKG, and clinical neurological examination.  
 
Ultrasound measures of the optic nerve sheath diameter (ONSD):  The optic nerve sheath is 
continuous with the dura mater of the brain and expands in the presence of raised ICP.  The 
distention can be measured non-invasively by ultrasound. Several studies suggest that ONSD 
has potential as a screening test for elevated ICP 196-200. ONSD was a sensitive and specific 
predictor of CT scan signs of raised ICP and was elevated in 25 of 27 children with papilledema 
201, 202.  A small study in Uganda found increased ONSD in all patients meeting a broad case 
definition of CM 203. We used this approach on the Research Ward in two different studies, and 
serial data have been collected on all patients since 2013, providing reassurance that this 
approach is feasible.  
 
Ultrasound measures of the cerebral blood flow velocities: In both adults and children TCD-
derived parameters have been shown to have utility to non-invasively estimate an absolute 
value for ICP.204-206 As ICP rises, distal resistance to cerebral flow increases and results in a 
progressive reduction in diastolic flow velocity (Vd) and an increase in pulsatility index (PI= 
Systolic flow velocity-Vd / Mean flow velocity).207  In a prospective cohort of adult patients, 
6SHDUPDQFRUUHODWLRQEHWZHHQ¨,&3PHDVXUHGLQYDVLYHO\DQGFKDQJHVWR3,ZHUHDYHUDJHG
across time during induced ICP increase.208 At the 0.05 level, baseline and plateau distributions 
of ICP and PI were significantly correlDWHGU ௗௗ. In children with hydrocephalus, TCD 
has been shown to be a valid measure of ICP changes during cerebrospinal fluid drainage.209-211 
In 7 patients, TCD was performed with simultaneous invasive ICP measurement through a 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
51 
ventricular reservoir during ventricular taps and ventriculoperitoneal shunting.  Resistive index 
ecreased significantly from a mean of 0.9 pre-CSF removal to 0.75 post-CSF removal.209  In 
another 22 infants with hydrocephalus and a ventricular reservoir, a volume of 5ml/kg body 
weight was removed twice daily and ICP and TCD flow velocities were determined before and 
after CSF tapping.7  A significant decrease in ICP after CSF removal was accompanied by a 
concomitant increase in mean flow velocity (p < 0.05) and Vd (p < 0.01).  A significant decrease 
in resistive index was also noted (p < 0.01). The current expert consensus is WKDWD
change in Vd or PI represented a clinically meaningful change to ICP (NOB personal 
communication). 
 
Heart rate variability (HRV) patterns: Heart rate is the net result of complex interactions at every 
level of the organism and these interactions are severely impacted by critical illness.  Ultimately, 
heart rate, strength of cardiac contraction and vascular resistance are regulated on a beat-by-
beat basis in order to optimize cardiac output to the needs of the body (resulting in healthy 
HRV).  A primary mediator of heart rate is the autonomic nervous system which directly 
innervates the sino-atrial node (the heart’s pacemaker). In healthy individuals, the autonomic 
nervous system is responsive to the needs of the body, i.e., heart rate accelerates with 
inhalation and decelerates with respiration (respiratory sinus arrhythmia) and also changes at a 
slower rate in association with oscillations in the baroreflex and other physiologic rhythms, as 
well as in response to external stimuli.  It has been clearly shown that there is a loss of normal 
HRV in the presence of critical illness and that it returns in association with clinical 
improvement212. Although nothing is known about the evolution of HRV in CM, we postulate that 
HRV will be diminished and possibly disorganized (lacking the expected intrinsic rhythms), and 
that potentially HRV will be significantly worse in CM with brain swelling compared to CM 
without it.  Furthermore, it would be expected that HRV will improve in association with 
underlying clinical improvement.  In order to test these hypotheses, we will use monitors to 
record the EKG in all children for at least 24 hours.  These data will be downloaded for off-line 
analysis.    HRV will be obtained from a beat-to-beat file generated by research scanning of the 
EKG signal and then examined in multiple ways: 
 
1. A tachogram of instantaneous heart rates vs. time will be plotted.  That will permit 
identification of the presence of any HRV and the evolution of regular respiratory sinus 
arrhythmia and other trends in heart rate patterns as the patient’s condition evolves or 
interventions are undertaken.   
2. Heart rate and multiple HRV parameters will be calculated for every 2 minutes of the 
recording period and their evolution plotted over time.  They will reflect how much HRV 
there is, the underlying oscillatory rhythms in the HRV pattern and the relative 
organization vs. randomness of heart rate patterns.  These parameters can also be 
averaged over longer periods of time for statistical purposes. 
 
Biobanking:  Admission samples of plasma, urine and CSF will be bio banked for later analysis, 
via a separate funding source.  The most widely studied of biomarkers in pediatric brain injury 
include S100B, neuron specific enolase (NSE), and myelin basic protein (MBP) 213.  Although 
the neuropathological role of these biomarkers is incompletely understood 214, 215, they have 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
52 
shown utility in stratifying patients with mild traumatic brain injury (TBI), identifying TBI patients 
on admission, and modestly predicting outcomes in patients with TBI 216-218. Despite these 
advantages, S100B, NSE, and MBP all possess significant limitations that preclude their 
selection for further clinical testing in this proposal.  The most significant limitations include a 
large range of age-dependent variability for S100B 219, an inadequately high presence in 
erythrocytes for NSE 213, and latent rise (peaking around 48-72 hours after injury) for MBP 213. 
UCH-L1 is a highly abundant neuronal protein thought to play a critical role in cellular protein 
degradation during both normal and pathological conditions 220. It constitutes up to 10% of 
cytoplasmic protein in neurons, is elevated in cerebrospinal fluid (CSF) and serum following TBI, 
and is significantly associated with measures of injury severity and outcome in adults 221-228 and 
children 225. UCH-L1 is localized to neurons in the cerebral cortex, and has demonstrated 
remarkable accuracy in stratifying patients with varying degrees of TBI within an hour of injury in 
addition to predicting outcomes 220-222, 225. GFAP, a marker of astroglial injury, is a type III 
intermediate filament that forms part of the cytoskeleton of mature astrocytes and other glial 
cells. Importantly, GFAP is only found in the central nervous system 229.  Central nervous 
system (CNS) injury causes gliosis and subsequently up-regulates GFAP, making GFAP an 
attractive candidate biomarker for evaluation of acute brain injury. Data suggest that GFAP, 
which is released after TBI but not after trauma without brain injury, is an indicator of cell 
destruction, while S100B is an indicator of glial activation but not necessarily of cell destruction 
230, 231. GFAP is a product of astrocyte cytoskeleton degradation by calpain protease activation 
and therefore considered specific to the CNS 232. Recent studies the investigators and others 
have documented elevated GFAP levels in both CSF and serum after TBI in adult 222, 225, 233-237
and pediatric patients 238-240. GFAP is useful in stratifying patients with mild to severe TBI, may 
remain elevated for several days post injury, and shows better specificity than NSE and S100B 
in detecting TBI and predicting outcome 241-243. Moreover, elevated concentrations of GFAP two 
days post injury portend a poor prognosis and, given the short half-life of GFAP in the blood 
stream, are thought to reflect the presence of a secondary insult (i.e. ICH and PbtO2) 244, 245.  
 
 
Potential Risks and Benefits 
 Potential Risks 
2.4.1.1 
Risk from mydriatics 
Instillation of dilating eye drops is required prior to the indirect and direct ophthalmoscopy.  The 
side effects of blurred vision and photophobia are not expected to trouble these particular 
participants (who will be unconscious), and the risk of acute narrow angle closure glaucoma (a 
rare side-effect of mydriatics) is very low in the pediatric population.  Cardiovascular effects 
have been reported in premature infants, who are not candidates for enrollment.  The dilating 
effect of mydriatics generally dissipates within 2 hours of instillation. 
 
2.4.1.2 
Hypertonic saline 
The known side-effects of hypertonic saline include: 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
53 
x 
Hypernatremia 
x 
Hypokalemia 
x 
Rebound cerebral edema upon termination of therapy 
These conditions are all a direct result of the increased sodium and osmolarity that are also the 
goal of the intervention. Assiduous monitoring of electrolytes (as described in 9.2.4) will 
therefore be undertaken to balance desired effects with possible side effects.  
 
2.4.1.3 
Assisted ventilation 
Mechanical ventilation via an endotracheal tube has a number of associated risks which can be 
broadly classified into following categories246  
x 
Adverse effects of positive pressure, volume and flow: 
o Pneumothorax 
o Pneumomediastinum 
o Hypotension 
o Bronchospasm 
o Pulmonary hemorrhage 
x 
Adverse effects of endotracheal intubation 
o Endotracheal tube obstruction 
o Inadvertent endotracheal tube dislodgment 
o Bradycardia induced by vagal response 
o Mucosal inflammation 
o Stridor 
o Subglottic stenosis 
o Respiratory nosocomial infections 
These events may be persistent and severe, necessitating tracheostomy. 
 Known Potential Benefits 
Participants will have the benefit of the increased level of nursing care provided on the PICU 
and PRW.  Were they not to be enrolled in this study, they would be hospitalized in the general 
pediatrics ward at QECH. The general ward is staffed by government employed nurses. The 
patient to nurse ratio is often > 50:1. In contrast, the PRW patient to nurse ratio is 2:1 at 
maximum and is 1:1 in the PICU. The diligent observations and monitoring that are provided on 
the PRW and in the PICU are impossible in the general ward. For a disease such as CM that 
demonstrates rapid progression, extreme clinical vigilance and the ability and resources to 
respond to clinical needs are crucially important. These can only be provided on the PRW/PICU 
and serve as the major benefit of participants to this study – independent of any benefit 
provided by the investigational efforts.  When the guardians of potential study participants 
decide against providing consent, the patients are eligible to receive the “usual care” (sans 
study-specific investigations) on the PRW. 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
54 
OBJECTIVES
Study Objectives 
Primary Objective 
Compare final outcomes in pediatric CM patients receiving usual care (Arm 3) with both 
of two interventions (Arm 1 and Arm 2).  
Secondary Objectives: 
1. Among survivors of this interventional study, compare rates of adverse neurological 
outcomes in those assigned to the two interventions and those assigned to usual care. 
2. Among all children admitted to the PRW with CM whose parents consent to screening, 
evaluate and validate biomarkers of increased brain volume.  
3. Assess the safety of early intubation and mechanical ventilation as well as intravenous 
hypertonic saline in children with CM. 
4. Establish association between specific pathogenic mechanisms and TCD-derived 
phenotypes.   
a. microvascular obstruction phenotype with MRI findings 
b. hyperemia phenotype with hypercapnia on blood gas analysis, acidosis, and 
seizures on EEG 
c. vasospasm phenotype with regional EEG findings of loss of fast frequencies and 
increased slow frequencies in territories of affected vessels and MRI findings with 
localized ischemic changes 
d. low flow phenotype with compromised hemodynamics, diminished compensatory 
reserve and increased brain volume per MRI 
Study Outcome Measures 
 Primary Outcome Measures 
Final outcomes will be determined within 7 days: treatment success or failure, where failure is a 
composite outcome of death, ventilatory rescue, and brain death.  Final outcomes within 7 days 
in children who meet the standard WHO definition of cerebral malaria treatment success or 
failure, where failure is a composite outcome of death, ventilatory rescue, and brain death.  
  
3.2.2 Secondary Outcome Measures
1. Neurological sequelae in survivors, assessed at 1, 6, and 12 months after 
randomization via the Malawi Developmental Assessment Tool (MDAT) for 
participants up to the age of 59 months old, and the Kaufman Assessment Battery 
for Children, Version II (KABC II) for participants over the age of 59 months.  All 
survivors will also be assessed using the Glasgow Outcome Scale (GOS)247.  The 
GOS is a global scale for functional outcome that rates patient status into one of five 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
55 
categories:  dead, vegetative state, severe disability, moderate disability or good 
recovery. 
2. Surrogate measures of increased brain volume:  At the time of admission (only):  
opening CSF pressure, archived samples of blood, urine and CSF. Daily while 
comatose:  ophthalmoscopy for papilledema (present/absent), optic nerve sheath 
diameter in mm, NIRS measurements of cerebral oxygenation and muscle 
reperfusion, standardized neurologic observations.   
3.  Standard TCD phenotypes: microvascular obstruction, hyperemia, vasospasm, low 
flow, isolated posterior hyperemia).  Potential mechanisms:  increased brain volume 
per MRI/ischemic changes, hypercapnia/acidosis/seizures, localized ischemia on 
MRI, compromised hemodynamics/diminished compensatory reserve/increased 
brain volume per MRI 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
56 
STUDY DESIGN
This clinical trial is a single site, prospective, open, randomized controlled study in which the 
impact on final outcome of two different, adjunctive therapeutic approaches aimed at reducing 
increased brain volume or ameliorating its effect on ventilatory status in Malawian children with 
CM and severely increased brain volume per MRI will be evaluated.   
Children who meet the World Health Organization (WHO) clinical case definition of CM 
(peripheral P. falciparum parasitemia of any density, Blantyre Coma Score (BCS) 2, and no 
other discernible cause of coma) will be admitted to the Pediatric Research Ward (PRW) at 
Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi, and will be eligible to be 
screened for participation in the trial. All children whose parents/guardians’ consent for 
screening will undergo ophthalmoscopy and MRI. 
x 
Those without MRI evidence of increased brain volume will be cared for on the PRW, 
and will contribute to Secondary Objective #2, identifying surrogate markers of increased 
brain volume and to Secondary Objective #4, establishing association between specific 
pathogenic mechanisms and each TCD-derived phenotype.   
x 
Children with clinically defined CM and increased brain volume are eligible to be 
randomized; those whose caregivers consent to screening but decline to participate in 
randomization will undergo continuous monitoring on the PRW as required for HRV 
analysis, and will contribute to Secondary Objective #2, identifying surrogate markers of 
increased brain volume.  They would be eligible for rescue ventilation in the event of a 
respiratory arrest. 
x 
Children with or without evidence of malarial retinopathy (see Section 2.1.1.7) and with 
evidence of increased brain volume per MRI (see Section 8.2.2.2 and 2.3.3) will be 
eligible to be randomized and participate in the clinical trial.  If the parents/guardians 
grant informed consent, each participant will be randomized to one of three arms: 
Arm 1:  Usual care plus immediate ventilatory support (VENT) 
Arm 2:  Usual care plus hypertonic saline (HPS) 
Arm 3:  Usual care (UC) 
Participants will be monitored as follows:  
x 
PICU-specific monitoring 
o Continuous monitoring of heart rate and rhythm from EKG, respiratory rate, 
and pulse oximetry via monitor, and blood pressure via arterial line or cuff.  
Signals will be archived for offline analyses aimed at identifying heart rate 
variability patterns over time associated with CM and with clinical 
improvement or decline in association with treatment.  


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
57 
o Electrolytes and acid-base balance 3-hourly during the first 24 hours and 6-
hourly thereafter (in those randomized to hypertonic saline) 
x 
PRW and PICU 
o 2–hourly assessments of vital signs (including temperature) and Blantyre 
Coma Score until BCS = 5 and temperature is <37.5oC for 24 hours, then 6-
hourly. 
o 6-hourly assessments of malaria parasitemia and hematocrit, until two 
consecutive parasitemia counts are negative. 
o 12-hourly neurological examinations,  
o Daily ophthalmoscopy while unconscious 
o Daily ONSD measurements (while unconscious) 
o Daily TCD examinations  
o Daily MRI examinations (while unconscious if participant is clinically stable for 
transportation to undergo neuroimaging) 
The primary objective (treatment success or failure) will be determined within the first 7 days of 
hospitalization (Figure 8) 
Secondary objectives (Figure 8) 
1. The secondary objective involving neurological sequelae at 1, 6, and 12 months after 
randomization will be determined in survivors randomized in the clinical trial. 
2. The secondary objective of identifying surrogate measures of increased brain volume 
will include participants in the clinical trial as well as study subjects who are screened 
but do not meet inclusion criteria for clinical trial enrollment e.g. their brain volume is not 
increased enough to quality for enrollment in the trial and are therefore not randomized. 
3. The secondary objective of assessing the safety of early intubation and mechanical 
ventilation will include participants in the clinical trial as well as study subjects with CM 
who are screened but do not meet inclusion criteria for clinical trial enrollment e.g. their 
brain volume is not increased enough to quality for enrollment in the trial and are 
therefore not randomized.as well as intravenous hypertonic saline in children with CM. 
4. TCD endpoints will be determined on all participants (see section 7.2.2.4)] 
 
 
Choice of participant population and sample size considerations 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
58 
By focusing on children with CM who have severely increased brain volume per MRI, the study 
population represents those at highest risk of dying of CM.  The mortality rate of retinopathy-
positive children with severely increased brain volume is 37%. 
Prior to study commencement and based on recruitment patterns over the previous five years 
(the years that the MRI has been available for characterizing brain volume) we expected to 
enroll approximately 30-35 retinopathy-positive and 10-12 retinopathy-negative participants with 
severely increased brain volume per year.  
The rate of participant enrollments has decreased, and challenges have been faced in almost all 
study years: 
• 2017:  the pediatric intensive care unit remained under construction.  Given a steady 
decline in enrollment rates for pediatric cerebral malaria on the Research Ward, a 
referral network of 6 hospitals within an hours’ drive of the Queen Elizabeth Central 
Hospital was established.  No participants were recruited or randomized. 
• 2018:  The Malawi Pharmacy, Medicines and Poisons Board unexpectedly delayed the 
clearance needed to import 3% hypertonic saline (HPS).  Protocol amendments were 
made quickly. Fifty-seven participants were recruited, and 8 were randomized: 4 to usual 
care and 4 to ventilatory support. 
• 2019:  The inability to import 3% HPS to Malawi continued.  A total of 42 participants 
were screened, and 24 were randomized (12 to ventilatory support and 12 to usual 
care).  
• 2020:  The MRI scanner became non-functional and repair efforts were stymied by the 
adverse impact of the COVID-19 pandemic on international travel. (The repair technician 
could not travel from South Africa to Malawi after all commercial flights to Malawi 
ceased.) A total of 48 patients was screened, but none were randomized because brain 
volume could not be assessed.  We will have a new, low field MRI machine 
(manufactured by Hyperfine) capable of assessing brain volume in place for the 2021 
season. 
x 
2021:  A low field MRI machine (manufactured by Hyperfine) capable of assessing 
brain volume was placed in service in late March 2021.The possibility of 
randomization was delayed until after the portable MRI became functional. 
a. Given these successes and challenges and the potential beneficial effects on 
mortality described in Section 11.2, a study comparing two interventions to Usual 
Care that will answer our original research questions prior to study closure will be 
challenging but remains feasible.  Answering the question whether mechanical 
ventilation or intravenous hypertonic saline decreases mortality risk in children 
with CM requires sing participants enrolled in the BMP observational study prior 
to clinical trial commencement as Historical Controls (obviating the need for 
randomizing high numbers of participants into the Usual Care arm) 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
59 
Historical Controls: 
We identified potential Historical Controls from the electronic database of the completed 
observational study of cerebral malaria pathogenesis.  In order to minimize temporal differences 
between participants randomized in the clinical trial, we began our search with children enrolled 
in the observational study in 2017 and moved backwards in time. 
 
Neuroradiologists affiliated with the Treating Brain Swelling clinical trial assessed the brain MRI 
scans on over 200 children enrolled in the observational study.  These images dated from 2014-
2017.   
 
Once consensus brain volume scores on these MRI scans were assigned by the 
neuroradiologists, we retrieved demographic and clinical data on children who had assigned 
brain volume scores of 6-8 from the master Blantyre Malaria Project database.  Demographic, 
clinical, and outcome data were compared between children enrolled in the observational study 
who had brain volume scores of 6-8, and children randomized in the Treating Brain Swelling 
clinical trial and were randomized to Usual Care with the same range of brain volume scores.   
Comparisons were made between the following variables:  age (in months), sex, Blantyre coma 
score, admission glucose, admission hematocrit, admission platelet count, and outcome.   
Statistical analysis:  Comparisons between groups were made using t-tests or Wilcoxon test 
(continuous variables), ANOVA (categorical variables), or chi squared tests (dichotomous 
variables), as appropriate. 
 
We found no difference in the variables in question between children enrolled in the 
observational study who had MRI brain volume scores of 6-8, and participants randomized in 
the clinical trial who had MRI brain volume scores of 6-8.   
 
Table  4.  Continuous variables compared between historical controls and randomized study 
participants 
 
Variable 
OS mean 
OS SD 
TBS mean 
TBS SD 
P value 
Age (months) 
48.25 
27.54 
59.63 
27.88 
0.168 
Glucose 
5.87 
2.94 
5.63 
3.13 
0.914d 
Hematocrit (%) 
23.36a 
6.29 
25.54 
6.97 
0.555d 
Platelets 
(*1000/MicroL) 
148.9b 
373.7 
94.3c 
176.2 
0.993d 
OS= observational study (n=48), SD= standard deviation, TBS= treating brain swelling clinical trial (n=16) 
a n=45, b n=47, c n=15. d nonparametric test. 
 
There was no significant difference in admission Blantyre Coma score (p=0.948), sex (p=0.885) 
or outcome (death/survival, p=0.551) between the 48 children in the observational study and the 
16 randomized participants in the Treating Brain Swelling clinical trial. 
 
We therefore conclude that participants enrolled in the observational study (n=48) are 
comparable to participants randomized to Usual Care (n=16) in the Treating Brain Swelling 
clinical trial.   
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
60 
We anticipate enrollment in the Usual Care arm in the 2023 seasos (see below) at a reduced 
ratio compared to the intervention arms.   
 
Possible future declaration of futility of the ventilation arm:  
If we combine the mortality risk in Historical Controls and participants already randomized to 
Usual Care, the mortality risk of the combined historical control group and children randomized 
to Usual Care is 36%.  At the end of the 2021 season, the mortality risk in the VENT arm was 
26.3%. (Five of 19 participants randomized to VENT died or were brain dead.) With only these 
data, the test of the null hypothesis of a null difference in mortality is not significant (p-value 
0.818, 2-sided test). 
We believe it likely that VENT will, in the future, be declared futile.  If so, randomization of  
participants enrolled in the Treating Brain Swelling clinical trial after the declaration of futility of 
VENT would be to HPS or UC.  A formal approach to futility assessment for VENT is provided 
below. 
 
      Table 4.  Projected enrollment  
 
2018 – 2021 
2022 
2023 
Total 
*Usual care (UC) 
64 
 0 
 4  
68 
Ventilatory support (VENT) 
19 
 7 
 6 
32 
Hypertonic saline (HPS) 
  3 
14 
15 
32 
 
 *historical controls + study participants enrolled in 2018 and 2019. 
 
   
 
Assuming that enrollment in 2023 follows the same trend seen since the beginning of the clinical 
trial, we will be able to detect a 50% or more risk reduction in either the VENT or HPS arms, 
compared to UC.      
 
 
Choice of interventions 
The three study arms of the study will be:  
1. Arm 1: Usual care + immediate mechanical ventilatory support (VENT) for a 
maximum of 7 days, with ventilator withdrawal once brain volume has decreased 
(brain volume score < 5) or the patient has improved clinically (modified Blantyre 
Coma Score > 3) or the patient dies or meets criteria for severe irreversible 
neurologic failure. 
2. Arm 2: Usual care + intravenous hypertonic saline (HPS) for a maximum of 7 days, 
withdrawn once brain volume has decreased (brain volume score <5) and the patient 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
61 
has improved clinically (Blantyre Coma Score > 3) or the patient dies or meets 
criteria for severe irreversible neurologic failure. 
3. Arm 3:  Usual care (UC; intravenous fluids, intravenous artesunate; antipyretics, 
anticonvulsants and blood transfusions as needed; nutritional support) for a 
maximum of 7 days, or until brain volume has decreased (brain volume score <5) 
and the patient has improved clinically (Blantyre Coma Score > 3) or the patient dies 
or  meets criteria for severe irreversible neurologic failures  
Participants will remain in the study throughout their hospitalization and, in survivors, through 
the 12-month post-randomization follow-up visit. Children are discharged when two sequential 
6-hourly blood films are free of malaria parasites, when they have been afebrile for 24 
consecutive hours, and when they are able to take medications and food by mouth. Final data 
collection will be at 12 months post-randomization.  
 
Stratification:  
No stratification is required. The ages of retinopathy-positive CM with severely increased brain 
volume per MRI who survived were compared to those of participants from the same population 
that died. There was no difference in the age structure (data not shown, p=0.95).  
Primary and secondary outcomes.  
The primary outcome is treatment success or failure, determined within the first 7 days.  
Treatment success is recovery.  Treatment failure is death, ventilatory rescue (for 
participants in Arms 2 and 3) or meeting criteria for severe irreversible neurologic failure.  
The secondary outcomes include rates of neurological sequelae in all 3 groups; potential 
surrogate measures of increased intracranial pressure (opening CSF pressure, ONSD 
measurements from serial ultrasound examinations, NIRS measurements of cerebral 
oxygenation and muscle reperfusion, serial examinations for papilledema, decreased 
diastolic flow velocity or increased pulsatility index on TCD, heart rate variability patterns 
from continuous cardiac monitoring measured from enrollment through initial MRI scan, 
and standardized clinical observations) and development of hypotension following 
induction/intubation, defined by proportional deviation from the initial systolic blood 
pressure at enrollment. 
Data collection methods.  
The primary outcome will be determined by study clinicians. 
For secondary objectives, the following methods will be used: 
x 
ONSD measurements will be collected at the bedside. Data will be transferred to the 
electronic case report form (CRF).  


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
62 
x 
TCD measurements will be collected at the bedside.  Data will be transferred to the 
electronic case report form (CRF).   
x 
NIRS will be used to record cerebral oxygenation fluctuations and to measure muscle 
reperfusion rates after transient occlusion. 
x 
The evolution of heart rate variability and its relationship to clinical status and outcomes 
will be determined by extracting the archived and time-stamped continuous ECG signal.   
x 
The neurological findings will be captured by clinicians on an electronic case report form 
(CRF) at the bedside.  
x 
Neurologic status at the 1, 6, and 12-month post-randomization follow-up will be 
assessed by one of two pediatric neurologists or clinicians trained by them. The 
Glasgow Outcome Scale (GOS), a global scale for functional outcome that rates patient 
status into one of five categories (dead, vegetative state, severe disability, moderate 
disability or good recovery)247 will be used..  Study nurses will administer standardized 
assessments: The Malawi Developmental Assessment Tool (MDAT) for participants up 
to the age of 59 months; the Kaufman Assessment Battery for Children, Version II 
(KABC-II), for participants over the age of 59 months. Both have been validated for use 
in African children 248, 249.   
The details of the techniques involved for the specialized assays (funduscopy, MRI, ONSD, 
TCD, NIRS, heart rate variability) are included in Section 8 of this Protocol.  
Centralization of evaluations: MRI, ophthalmoscopy, ONSD, TCD, EEG, and neuro-checks 
will be carried out either at the bedside in the PRW/PICU or in the MRI suite.  
 
Safety monitoring:  A local independent safety monitoring team (ISM) will be appointed for the 
study. The team will consist of two local physicians with knowledge of the clinical setting and 
practices, but without direct involvement with this study. They will be notified of all SAEs and 
asked to comment on the relatedness of these events to the interventions. They will also be 
asked to review AEs at the discretion of the local investigators, IRBs, or the Data Safety and 
Monitoring Board (DSMB).  
 
A DSMB is justified for this study for two reasons: (1) the anticipated mortality in this group of 
gravely ill children, without adjunctive treatment, is over 30% (2), and serious adverse events 
have been reported for HPS (Section 2.3.1.2) and assisted ventilation (Section 2.3.1.3). 
Interventions may result in survival of children who would have otherwise died, and some of these 
children may be severely neurologically impaired. In a country like Malawi where there is no social 
support for such children and their families, their survival could create a burden for the family 
which might worsen stress and social problems.  
 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
63 
STUDY ENROLLMENT AND WITHDRAWAL
Subject Inclusion Criteria 
For screening: 
x 
Peripheral P. falciparum parasitemia of any density  
x 
Blantyre Coma Score 2 
x 
No evidence of meningitis on lumbar puncture 
x 
Consciousness not regained after correction of hypoglycemia (if hypoglycemia is 
present)  
x 
Male or female whose age on the day of screening is between 6 months and 12 years 
old 
If the parents/guardians agree to screening, antimalarial treatment will begin (see Section 6.6), 
and participants will undergo an ocular funduscopic examination and a brain MRI scan.   
Inclusion criteria for randomization: 
x 
Severely increased brain volume on MRI:  brain volume score  6 (see Section 8.2.2.2 
for details)  
x 
Willingness to return for follow up visits 1, 6- and 12-months post-randomization 
If these criteria are met, guardians will be approached for possible enrollment by a dedicated 
study nurse or clinician, speaking in the local language (Chichewa).  
Antimalarial therapy (standard-of-care) will have already been initiated prior to either of these 
consent processes 
Subject Exclusion Criteria 
Subjects showing evidence of the following at baseline will be excluded from the study: 
x 
Gross malnutrition as evidenced by peripheral edema, hair color changes, or severe 
wasting 
x 
Advanced Human Immunodeficiency Virus (HIV) disease – defined as known HIV 
positive status and evidence of severe wasting   
x 
Evidence of recent head trauma by history or physical examination 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
64 
x 
Evidence of severe co-morbidity at time of initial evaluation 
 
 
- Pneumonia as evidenced by oxygen saturation on room air of <85% 
 
 
- Gastroenteritis and shock as evidenced by capillary refill >3 seconds or skin  
 
 
 
tenting  
 
Treatment Assignment Procedures 
 Randomization Procedures 
The study will be randomized, but not blinded. Participants will be randomized after completing 
the eligibility screening procedures.  A block randomization scheme will be used to ensure 
groups of equal size.   Blocks sizes of six and nine will be rotated to decrease the predictability 
of group allocation within a block for data collectors. Children with retinopathy-positive CM will 
be randomized separately from those with retinopathy-negative CM. 
Randomization is based on obtaining a consensus interpretation of brain volume per MRI.  If 
this is not possible for any reason, study participants will be screened but not randomized, and 
will remain on the PRW.  All other study procedures will be followed as described in this 
protocol. 
Randomization is predicated on the risks and benefits as described in this protocol.  
Endotracheal intubation is required for participants randomized to the “intubation + mechanical 
ventilation” arm.  The current coronavirus pandemic affects the risk/benefit ratio of this arm of 
the study because endotracheal intubation increases the risk of aerosol spread of the 
coronavirus (US CDC COVID19 guidance, 4.4.20, www.cdc.gov/coronavirus/2019-
ncov/hcp/guidance-risk-assesment-hcp.html). Randomization to the mechanical ventilation arm 
will be adjusted in accordance with local IRB recommendations during the coronavirus 
pandemic. 
All possible blocks with balanced combinations of group assignment will be documented.  Next 
computer-generated random numbers will be used to select from the balanced 
blocks.  Assignment of study subjects to randomization arm will be performed electronically, 
through the online data capture mechanism.  If Internet service is unavailable at the time of 
randomization, back up sequentially numbered opaque sealed envelopes prepared before the 
trial begins will be available.  The envelopes will be stored in a secure location with access 
limited to key study personnel.  After a caregiver provides consent for a patient to participate in 
the study, study arm assignment will first be attempted electronically.  If Internet service is 
unavailable, study personnel will open the next unopened sequentially numbered envelope to 
reveal the group assignment and dosing calculation sheet.  


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
65 
A master log of all screened and enrolled subjects will be maintained. A subject is considered 
randomized upon online assignment or opening the envelope to indicate treatment assignment. 
Additional subjects may be enrolled into the study at the discretion of the sponsor in the case of 
any subject who: 
x 
Is randomized but does not receive treatment for any reason 
x 
Has consent for participation in the study withdrawn for reasons other than an adverse 
event 
 Masking Procedures 
Treatment arms will not be masked. 
Reasons for Withdrawal
A study subject will be withdrawn from the study if: 
x 
A screening error is discovered that resulted in incorrect enrollment (subject did 
not meet inclusion or exclusion criteria)  
x 
CSF from admission lumbar puncture results in growth of pathogenic bacteria 
x 
Parental consent is withdrawn at any stage for any of the procedures or 
treatment 
x 
It is discovered that the participant has an intercurrent illness that, when 
combined with the study interventions, might compromise the recovery of the 
patient 
x 
Safety reasons as judged by the investigator, ISM, or DSMB  
 Handling of Withdrawals 
Participants withdrawn from the study will remain on the PICU/PRW and will continue to benefit 
from the increased level of clinical care provided. If the participant is withdrawn due to an 
adverse event (AE; see section 9 below for more details), laboratory monitoring will continue 
until the event resolves. Participants will continue to have brain volumes evaluated by multiple 
modalities as long as this is deemed non-injurious. Participants will still be scheduled for a 28-
day post randomization follow up visit and measures of neurologic sequelae and brain volume 
will be collected. If the participant is voluntarily withdrawn from the study by the parent/guardian, 
all efforts will be made to encourage the participant to return for 28-day post-randomization 
follow up.  


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
66 
Attempts will be made to recruit additional participants to replace any participants that withdraw 
prior to reaching the 12-month post-randomization visit.  At the conclusion of the study separate 
analyses will be run on per-protocol and intention-to-treat populations.  
 Termination of Study  
One interim analysis will be carried out as detailed in Section 11.3. Stopping rules related to 
toxicities attributed to the study agents are delineated in this section. Early study termination is 
at the discretion of DMID, either of the participating institutional review boards (IRBs), or the 
DSMB. The investigator will notify the IRB(s) when the study has been completed. 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
67 
STUDY INTERVENTIONS/INVESTIGATIONAL 
PRODUCTS 
Study Product Description – Hypertonic Saline 
 Acquisition 
Hypertonic saline (HPS) (3%) will be obtained in the United States and shipped to Blantyre. 
 Formulation, Packaging, and Labeling 
Product is packaged in 500ml bags. Storage is recommended at room temperature (25°C). 
Product Storage and Stability
Bags of HS will be stored in a cabinet in the PRW pharmacy adjacent to the ward. It will be 
protected from direct light. The pharmacy is equipped with an air conditioning unit and is 
provided with back up electricity by a diesel operated generator in case of power outages. 
Minimum and maximum ambient temperatures will be recorded daily. 
 
6.2     Dosage, Preparation and Administration of Study 
Intervention (HPS) 250, 251 
HPS will be administered through a cannula placed in a large peripheral vein. All participants 
will already have a peripheral cannula in place for maintenance fluid replacement.  Dosage and 
adjustments in infusion rates will be administered per Standard Operating Procedures. 
The HPS saline bag will be connected to a volumetric infusion set. Based on the weight of the 
subject, and the current sodium concentration, the appropriate amount of HPS will be placed in 
the chamber and the tubing attached to the secondary port of the intravenous line already in use 
for delivery of maintenance fluids.  Only the amount of fluid to be installed in the current 
intervention will be delivered to the chamber, to avoid the possibility of overdose.   
The HPS loading dose will take place over a 30-minute period, with duration regulated by 
adjustment of the drop rate. Maintenance fluids will continue during the infusion of the HPS 
loading dose to minimize the possibility of phlebitis.  
After the loading dose is complete, the infusion rate will be reduced to the maintenance infusion 
rate (see Standard Operating Procedure).  


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
68 
6.3    Modification of Study Intervention for a Participant  
(HPS) 
Although sodium abnormalities are rare in CM, in the event that a participant has a (Na+) level of 
> 150 mmol/L on admission, the initial bolus will be deferred and the sliding scale dosing 
detailed in Table 3 will instead be followed.   
The subsequent dosing of HPS is planned on a sliding scale, with the goal of maintaining serum 
sodium levels between 150 and 160 mmol/L.  Serum sodium levels will be checked every 3 
hours for the first 12 hours of the study, and then every six hours, unless designated otherwise 
(Table 3).  
               Table 5.  Dosing schedule for hypertonic saline 
Serum Sodium 
Dose of 3% HPS 
Sodium 
recheck 
< 145 mmol/L 
5 mL/kg (bolus)* 
3 hours 
145 – 150 mmol/L 
2 mL/kg (bolus)* 
3 hours 
150 - 160 mmol/L 
None 
6 hours 
>160 mmol/L 
Hold infusion until 
VHUXPVRGLXPLV 155, 
restart at 0.5 mL/kg/hr 
1 hour 
*If > 3 boluses are required to maintain serum sodium between 150-160, then increase infusion by 0.25 mL/kg until 
no further boluses are required.  
Further modifications to the HPS dose will be made in the event of the following laboratory 
abnormalities (electrolyte abnormality grading detailed in Appendix B): 
x 
Grade 4 hypokalemia (see Toxicity Table in Appendix B) 
x 
A pH drop of more than 0.2 within 6 hours 
x 
Chloride > 125 mmol/L 
x 
Serum osmolality > 340 mOsm 
In these instances, planned HPS doses will be held, and corrective actions (detailed in Section 
9.2.4) will be taken. Electrolytes will be rechecked in 3 hours and the normal dosing schedule 
will be reinstated if electrolytes are no longer in the Grade 4 toxicity range.  
If the pH < 7.25 or falls by 0.2 within 6 hours or if the serum chloride is > 125 mmol/L, the 3% 
saline infusion will be stopped and converted to a 1:1 ratio of 3% sodium chloride/sodium 
acetate solution. 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
69 
6. 4  Accountability Procedures for the Study 
(Intervention/Investigational Product(s) (HPS) 
Based on the anticipated recruitment for a calendar year, supplies will be ordered according to 
anticipated need and in accordance with expiration dates. Bags will remain stored in the storage 
case in the locked cabinet in the pharmacy until needed on the ward. On enrollment of a 
participant a bag will be brought to the ward for initiation of therapy. Removal from the 
pharmacy will be documented on a product inventory log. The lot number and expiration date of 
the bag will be recorded on the participant CRF. Bags will not be shared between participants. 
At the end of the calendar year or when the expiry date is reached (whichever comes first), the 
remaining product will be discarded.     
6.5  Assessment of Subject Compliance with Study 
Intervention/Investigational Product  (HPS) 
HPS will be administered by dedicated, trained study nurses, with appropriate documentation.  
Any irregularities in administration will be recorded and reported as a protocol deviation.  
 
6.6   Study Product Description – Ventilatory Support    
6.6.1 Acquisition  
Age-appropriate mechanical ventilators will be used in at the bedside in the PICU; an MRI-
compatible ventilator will be used to address transport needs of study subjects from the PICU to 
the MRI suite. 
6.6.2  Formulation, Packaging, and Labeling 
NA 
6.6.3 Product Storage and Stability 
NA 
6.7    Dosage, Preparation and Administration of Study 
Intervention/Investigational Product (VENT) 
Mechanical ventilation will be initiated immediately after randomization to Study Arm 1, or in the 
event of a respiratory arrest for subjects allocated to Arms 2 and 3. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
70 
Endotracheal intubation will be performed by a pediatric intensivist after administration of IV 
anesthetic agents in doses described in the sedation section (Section 6.3). The size of the 
endotracheal tube to be used for each patient will be determined by the pediatric intensivist on-
site using standard formulas. A variety of sizes of both cuffed and uncuffed endotracheal tubes 
will be available (ranging from size 3.0 to 7.5) along with laryngoscopes and different sizes of 
blades 
At the discretion of the intensivist, initial ventilator settings may be set to deliver a Tidal Volume 
at 6-8 ml/kg, age-appropriate respiratory rate, Peak End Expiratory Pressure of 5 and Fraction 
of Inspired Oxygen at 40%. The settings will be adjusted by the pediatric intensivists to target a 
pCO2 (partial pressure of carbon dioxide) in arterial blood of 35-40 mmHg and maintain oxygen 
saturation above 90% on pulse oximetry. 
At the discretion of an intensivist, an arterial line may be placed in the ventilated subjects to 
measure arterial blood gases every 3 hours for the first 24 hours and every 6 hours 
subsequently until the participant is extubated. 
Participants will remain intubated until brain volume has decreased (brain volume score < 6) 
and the participant has improved clinically (modified Blantyre Coma Score > 3) or the patient 
has improved clinically (modified Blantyre Coma Score > 4, no repeat MRI needed) or the 
patient dies or develops severe irreversible neurologic damage or for a maximum of 7 days. 
Intubated participants will be moved from side to side every 2 hours (1 hour) to reduce 
chances of bed sores. If tolerated, they will undergo endotracheal suctioning every 6 hours (2 
hours) and more often as needed to prevent plugging of the endotracheal tube by mucoid 
secretions, using an appropriate sized suction catheter attached to wall suction.  Participants 
may be pre-medicated prior to suctioning using sedation medications described in section 6.3. 
Participants will receive chest physiotherapy as needed if they develop evidence of lung 
atelectasis. They may be premedicated with sedation medications as described in section 6.3.  
6.8     Modification of Study Intervention/Investigational Product 
for a Participant (VENT) 
In the event that a participant develops a nosocomial pneumonia, appropriate broad-spectrum 
antibiotics will be initiated after respiratory cultures are sent.  Antibiotics will be continued until 
clinical improvement is noted or for a duration determined by the pediatric intensivist.  
In the event of accidental tube dislodgement, participants will be reintubated by the pediatric 
ICU staff under the guidance of the intensivist. 
In the case of a pneumothorax, at the discretion of an intensivist a chest tube may be inserted 
and hooked to suction to aid lung re-expansion. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
71 
In the case of hypotension resulting from positive intrathoracic pressure from mechanical 
ventilation or secondary to use of sedative agents, participants may receive a 10-20 ml/kg fluid 
bolus using normal saline or Ringer’s lactate, at the discretion of the intensivist. 
Participants at risk for development of post-extubation stridor (those with a traumatic or with 
multiple intubations), will be eligible to receive a 4-dose course of dexamethasone IV prior to 
planned extubation. In case of stridor noted after extubation, nebulized racemic epinephrine will 
be administered as needed, at the discretion of the intensivist. 
 6.9  Accountability Procedures for the Study Intervention (VENT) 
NA 
6.10 Assessment of Subject Compliance with Study Intervention  
(VENT) 
NA 
6.11 Concomitant Medications/Treatments   
While on the PICU/PRW, all participants will receive standard of care anti-malarial 
treatment. This includes the following: 
x 
Anti-malarial treatment:  Artesunate will be administered according to national treatment 
guidelines upon admission, at 12- and 24-hours post-admission, and once a day 
thereafter until the child is able to take oral medication, or for a total of 5 days. After the 
child is able to take oral medications, they will be prescribed Co-Artem® (a fixed dose 
combination tablet containing artemether, 20 mg and lumefantrine, 120 mg). Dosing will 
be weight-based according to national treatment guidelines. Oral therapy is for three 
days.  
x 
Antipyretics: An aggressive fever control regimen will be undertaken in both arms – as 
pyrexia has been shown to be a major predictor of poor outcome in TBI participants 252. 
Anti-pyretic therapy will be initiated for any temperature >38°C. The two components will 
be: 
 
Paracetamol – 25mg/kg per rectum (PR) loading dose and 15 mg/kg PR 
Q6 hours thereafter 
 
Ibuprofen – 10mg/kg per naso-gastric tube (PNGT) Q6 hours 
 
Therapy will be initiated with paracetamol. If euthermia is not achieved in 3 
 
 
hours, ibuprofen will be added. The two modalities may be scheduled on   
 
 
alternating Q6 hour regimens, at the discretion of the intensivist. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
72 
x 
Anti-convulsants: Locally available anti-convulsants include paraldehyde, diazepam, 
phenobarbital, and phenytoin. Choice of a specific anti-convulsant is determined on a 
participant-to-participant basis based on the duration of seizures, available routes of 
administration, and other clinical characteristics (e.g., respiratory status) of the 
participant.  
x 
Antibiotics:   Participants initially unable to undergo lumbar puncture to rule out 
meningitis will be placed on ceftriaxone (100mg/kg IV, daily) until a lumbar puncture can 
be performed or for a maximum of seven days, whichever comes first. A blood culture 
will be collected initially on all patients, and appropriate antibiotics will be prescribed on 
the basis of a positive culture. Participants with fever refractory to anti-malarials will have 
a second venous blood sample collected for culture and the initiation of empiric antibiotic 
treatment until culture results are available.  Continued antibiotic administration will be 
dependent on blood culture results and clinical status. See Section 6.2.5 for antibiotic 
therapy for intubated patients who develop a nosocomial pneumonia. 
x 
Sedation:  Participants requiring ventilator support will be intubated after induction of 
anesthesia with ketamine IV 253 along with diazepam. Atropine will be available in case 
of bradycardia associated with direct laryngoscopy. While patients remain intubated, 
they may be pre-treated (at the discretion of the intensivist) with morphine or diazepam 
or lidocaine prior to noxious stimulation such as endotracheal tube suctioning or 
placement of gastric tubes, intravenous lines, etc. 
x 
Fluids/Nutrition: 
o At admission, all participants will be placed on maintenance fluids  
o Infusion rates will adhere to the QECH Dept. of Paediatrics protocol: 4mls/hr for 
the first 10kg, 2ml/hr for next 10kg, 1ml/hr for any weight above 20kg. 
Adjustments may be performed at the discretion of the intensivist. 
o If a participant remains comatose and is therefore unable to take oral feeds at 36 
hours post-randomization, a nasogastric tube will be placed, and a milk-based 
nutritional supplement will be administered at the same rate as peripheral IV 
fluids. IV fluids infusion rates will be decreased as NG feed volumes are 
increased.  Nasogastric feeds will be initiated when deemed safe by the PICU 
physician. 
o 50% Dextrose will be used as a rescue therapy for subjects experiencing 
hypoglycemia of < 3.0 mmol/L.   
x 
Blood transfusions will be administered as required on clinical grounds.   
x 
Oxygen therapy:  Oxygen saturation will be measured continuously in the PICU; it will be 
measured and recorded on the PRW every two hours by a dedicated study nurse using 
a portable oxygen monitor with a pediatric finger probe. From the TBI literature adequate 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
73 
oxygenation is important for treating raised intracranial pressure as injured tissue is 
highly susceptible to secondary insults including hypoxia 254-256. Supplemental oxygen 
will be provided by nasal cannula, endotracheal tube, or mask to maintain peripheral 
oxygen saturations above 90%.  
x 
Chloral hydrate:  Chloral hydrate will be used in children returning for 1-month follow-up 
visits if sedation is required for the follow-up MRI.  
x 
Omeprazole:  Omeprazole may be added at the intensivist’s discretion should any 
participants experience gastrointestinal bleeding. Study Schedule 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
74 
STUDY SCHEDULE
Screening  
Participants admitted to the PRW who meet the following initial inclusion criteria will be 
approached for consent to further screening.  
x 
Peripheral P. falciparum parasitemia of any density  
x 
Blantyre Coma Score 2 
x 
No evidence of meningitis on lumbar puncture 
x 
Does not regain consciousness after correction of hypoglycemia (if hypoglycemia was 
present)  
x 
Age between 6 months and 12 years 
x 
Oxygen saturation  85% on room air 
x 
Capillary refill  3 seconds and absence of abnormal skin tenting 
The decision to approach the parent/guardian for screening will only be made after the 
participant has been stabilized clinically and anti-malarial therapy commenced. In the context of 
obtaining informed consent, the wide spectrum of CM disease presentation will be explained 
and our desire to perform two more exams (a retinal exam and an MRI) to further characterize 
the child’s illness will be presented. The fact that neither of these exams are treatment, but that 
they might lead to further treatment, will be explained.  
If the parent/guardian consents to the screening exams, mydriatic eye drops will be added to the 
participant’s eyes and a direct and indirect ophthalmologic exam will be performed. The 
participant will be taken to MRI.  
Enrollment/Baseline 
If initial inclusion criteria for randomization are met (severely increased brain volume is 
confirmed on MRI), the parent/guardian will be approached for consent to participate in the 
clinical trial.  
Initial evaluations to be completed at the time of enrollment are detailed in the Schedule of 
Events attached as Appendix A. 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
75 
Follow-up  
The first follow up visit will be scheduled for 1 month after randomization. The participant will 
undergo detailed physical, neurological, and developmental examinations performed by a 
pediatric neurologist (or their designee), a repeat MRI, NIRS measurements of cerebral 
oxygenation and muscle reperfusion, and a repeat ONSD determination.  
Subsequent visits will be scheduled for 6- and 12-months post-randomization, at which time the 
patient will undergo detailed physical and neurological, and developmental examinations 
performed by a pediatric neurologist or their designee.  The 12-month post-randomization visit 
will be the final visit.  
All follow-up visits will be performed GD\VIURPWKHVFKHGXOHGpost-randomization visit date. 
Final Study Visit 
As described in Section 7.3, the final study visit will occur at the 12-month (14 days) post-
randomization follow-up visit. Evaluations to be completed at follow up are detailed in Appendix 
A – Schedule of Events. 
 Death 
If a death occurs in any of the study arms, transportation will be provided for the deceased and 
the family. 
Early Termination Visit 
If a participant/parent/guardian elects to terminate the study, or study participation is terminated 
early due to any of the events listed in Section 9.2.3, the parent/guardian will be asked if they 
are willing to have another MRI performed. They will also be asked if they would be willing to 
return to the PRW at 1 month (14 days) post-randomization, as this is standard-of-care for all 
participants on the PRW. 
Unscheduled Visit  
Participants will be invited to return to the PRW for any needed medical care in the interim 
between discharge from the ward and the final study visit at 12 months (14 days) post-
randomization. Any unscheduled visit will be recorded in the participant CRF and may be 
recorded as an AE or SAE if appropriate.  


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
76 
STUDY PROCEDURES/EVALUATIONS
Clinical Evaluations 
History: A brief medical history pertaining to the immediate clinical condition is obtained 
immediately on arrival to the PRW. More detailed medical history is obtained after the 
participant has been stabilized. History includes a history of the immediate illness, previous 
similar illnesses, and a brief developmental history. History is obtained by interview and 
supplemented by any records in the participant’s health passport.  
Medication history is obtained on admission to the PRW and recorded on the same enrollment 
form. This history relies heavily on the written record provided in the health passport of the 
participant. 
Physical Examination: A full physical examination is performed on arrival to the PRW and after 
immediate stabilization. Specific vital signs and organ systems evaluated can be found on the 
physical exam CRF). Twice daily after consent, a targeted physical exam is conducted by a 
study physician. The results of these daily examinations are recorded in free form on the 
continuation sheets. At minimum these exams will evaluate the neurologic status of the 
participant (as determined by BCS), examination of the heart, lungs, liver, and spleen. 
Vital Signs: Vital signs (temperature, pulse rate, blood pressure, respiratory rate, oxygen 
concentration, and BCS) will be evaluated and recorded by a dedicated study nurse every two 
hours until the participant regains consciousness (BCS 3). 
Table 6.  Blantyre Coma Score (maximum = 5): 
Motor Response 
Verbal Response 
Eye Movements 
Nil/extend                      0 
Withdraw                       1 
Localize                         2 
Nil/gasp                         0 
Abnormal cry                 1 
Normal cry or speech    2 
Unable to track or follow     0
Able to track or follow         1 
Table 7.  Modified Blantyre Coma Score (for intubated patients, maximum = 5):  
Motor Response 
Verbal Response 
Eye Movements 
Nil/extend                      0 
Withdraw                       1 
Localize                         2 
Nil/gasp                              0 
Attempt to cry, grimace      1 
Clear mouthing of words 
around ET tube                  2 
 
Unable to track or follow     0
Able to track or follow         1 
IV anti-malarial therapy: Artesunate will be administered according to national treatment 
guidelines upon admission, at 12- and 24-hours post-admission, once a day thereafter until the 
child is able to take oral medication, or for a total of 5 days. 
Oral anti-malarial therapy: After the child is able to take oral medications, they will be prescribed 
Co-Artem® (a fixed dose combination tablet containing artemether, 20 mg and lumefantrine, 
120 mg). Dosing will be weight-based, in accordance with national treatment guidelines. Oral 
therapy is for three days. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
77 
Malaria parasite (MP) and packed cell volume (PCV): An evaluation of malaria parasitemia (MP) 
and hematocrit (packed cell volume (PCV)) will be performed at initial screening and every six 
hours thereafter. These exams will continue until two consecutive examinations show an 
absence of malaria parasites. MP evaluation is performed on a thick blood smear. PCV is 
determined in a microhematocrit tube. Blood for both of these exams is obtained by fingerprick.  
Lactate and glucose check: Blood lactate and glucose levels will be measured at the bedside 
using point-of-care tests.  They will be evaluated on admission and every 6 hours thereafter until 
the participant has maintained a BCS of 3 for greater than 4 hours. 
Lumbar puncture:  Participants will be placed in the decubitus position, the L3-4 spinal 
interspace determined by palpation, and the skin cleaned three times with alcohol.  A twenty-
one-gauge needle will be used in the procedure.  Four ml (2 ml) of spinal fluid will be obtained.  
If at least 4 ml is obtained, 1 ml will be sent for each of culture and sensitivity, cell count plus 
differential, and protein and glucose determinations. 1 ml of spinal fluid will be archived for 
determination of biomarkers of increased brain volume. 
Retinal exam: Fundoscopy will be performed at admission and every 24 hours thereafter until 
the patient reaches a BCS of 3 for greater than 4 hours. Pupils of participants will be dilated with 
mydriatics (a combination of 1.0% tropicamide hydrochloride and 2.5% phenylephrine 
hydrochloride) in both eyes. A trained clinician will perform direct and indirect ophthalmoscopy 
on fully dilated eyes.   
MRI:  After consent for screening is obtained and medical stabilization has occurred, brain MRI 
images will be obtained. 
ONSD: Measurement of optic nerve sheath diameter is being investigated as a non-invasive 
measure of increased brain volume.  The procedure takes 10-15 minutes to perform. A small 
amount of ultrasound gel is placed on the eyelid and the probe gently pressed superior to the 
globe. The procedure will be repeated daily until the child maintains a BCS of 3 for four hours.   
Transcranial Doppler: TCD examination will be performed at enrollment and every 24 hours 
thereafter while the patient is hospitalized.  Middle cerebral arteries (MCAs), extracranial 
internal carotid arteries (EC-ICAs), and basilar arteries (BAs) will be insonated at 1 mm intervals 
using the transtemporal or occipital acoustic windows.  Systolic (Vs), diastolic (Vd), mean flow 
velocities (Vm) and pulsatility index (PI) will be recorded at each interval. Based on findings of 
TCD examinations, subjects will be placed into phenotypic groups.  
Neurological exam:  A focused neurological examination will be performed at admission, and at 
0800 and 1600 daily (2 hours) while the patient is comatose (BCS  2). A detailed neurological 
examination will be performed at each of the 1, 6, and 12 month ( 2 weeks) post-randomization 
follow-up visits.  While hospitalized, the examination will focus on candidate neurological exam 
markers of increased brain volume:  restriction or changes in eye movements, abnormal 
posturing (decerebrate, decorticate), changes in reflexes (corneal, gag, deep tendon).  The 
follow-up neurological history and examinations will focus on determination of hard neurological 
abnormalities:  tone, reflexes, movement, posture, interim unprovoked seizures, Neurological 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
78 
development will be determined by administration of the Malawi Developmental Assessment 
Test or Kaufman Assessment Battery for children, depending on the age of the subject at the 
time of enrollment. 
Brain swelling intervention:  After brain MRI screening, subjects with severely increased brain 
volume scores ( 6) will be eligible for randomization.  The parent/ caregiver will be approached 
for informed consent using the Randomization Consent document.  If consent for randomization 
is granted, randomization assignment will be determined online or opening the next sequentially 
numbered sealed randomization envelope (stratified by malarial retinopathy status) and the 
subject’s assignment to each of the three study arms determined.   
Subjects may be randomized to: 
x 
Arm 1:  Usual care + immediate ventilatory support for a maximum of 7 days.  These 
subjects will undergo rapid sequence intubation and be placed on a mechanical 
ventilator to maintain physiological pCO2 levels for a maximum of 7 days. Arterial blood 
gas determinations will take place every 3 hours for the first 24 hours, then every 6 
hours until the participant is extubated.  Non-scheduled blood gas determinations may 
occur at the discretion of the PICU physician.  Participants will remain in the Pediatric 
ICU during mechanical ventilation and after its discontinuance.   
x 
Arm 2:   Usual care + infusion of 3% hypertonic saline to achieve a target blood 
concentration of 150-160 mmol/L. Participants will remain in the Pediatric ICU during 
hypertonic saline infusion. Participants randomized to hypertonic saline administration 
will have electrolytes evaluated on admission and every six hours thereafter until they 
regain consciousness (BCS 3) 
x 
Arm 3:  Usual care (elevation of the head of the bed 30 degrees and intravenous 
antimalarial drugs).   
Laboratory Evaluations 
 Clinical Laboratory Evaluations 
Approximately three mls of blood will be obtained at admission, and a  fingerpick blood 
sample will be taken every 6 hours for the next 48 hours. Fingerprick samples will be 
approximately 200 ȝl in volume, resulting in an approximate total blood volume obtained 
of 3.6 mls. 
An evaluation of malaria parasitemia (MP) and hematocrit (packed cell volume (PCV)) 
will be performed at initial screening and every six hours thereafter. These exams will 
continue until two consecutive examinations show an absence of malaria parasites. MP 
evaluation is performed on a thick blood smear. PCV is determined in a microhematocrit 
tube.  


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
79 
Lumbar puncture: A lumbar puncture will be attempted at the time of admission on all 
children admitted to the PRW, and if successful, the opening pressure (mm CSF) will be 
recorded. This is performed to eliminate meningitis as an alternate etiology of coma. 
Cerebral spinal fluid (CSF) will be sent for cell count, differential, and culture. 1 ml of the 
total 4 ml volume will be archived for subsequent studies of biomarkers of increased 
brain volume. 
 
Respiratory swab:  All participants will have a nasopharyngeal swab collected at the time 
of admission.  These will be batch analyzed (after hospitalization) to detect respiratory 
viral pathogens.   
 
Red Blood Cell (RBC) Cryopreservation: RBCs will be cryopreserved and sent to an 
outside institution for bio-banking/future analysis. 
Blood for culture will be obtained on all participants admitted to the PRW. Blood will be 
obtained from either a peripheral or femoral vein and transferred to a pediatric BacTec 
bottle for subsequent analysis.  
Plasma for archive: One ml of blood will be collected in a lithium heparin anticoagulated 
tube. Plasma will be separated and archived for subsequent studies of biomarkers of 
increased brain volume. 
Urine for archive:   Urine will be collected either while inserting an indwelling urinary 
catheter or by attaching a sterile urine collection bag to the perineum, and at least one 
ml will be archived for subsequent studies of biomarkers of increased brain volume. 
Blood glucose levels will be measured using point of care testing on admission and 
every 6 hours thereafter until the participant has maintained a BCS of 3 for greater than 
4 hours. Machines will be checked for accuracy each morning with a manufacturer 
supplied test strip.  
Lactate levels will be determined on admission and every six hours thereafter until the 
participant has maintained a BCS of 3 for greater than 4 hours.  Testing is done with a 
point of care testing machine. This test uses one drop of blood obtained by finger prick. 
The monitor will be tested for accuracy on manufacturer supplied test strips every 
morning.  
HIV testing will be performed on all participants, subject to permission being granted by 
the parent/ guardian present. The test will be performed after recovery of the participant 
is anticipated by the clinical staff unless test results are thought to have relevance for 
acute clinical care. Two rapid tests will be performed on a blood sample obtained by 
finger prick. In the event of discordant results an HIV polymerase chain reaction test will 
be performed.  
Full Blood Count: Participants will have blood drawn for a full blood count (FBC) on 
admission. FBC includes hemoglobin, hematocrit, RBC indices, white blood cell (WBC) 
with 5-part differential, and platelet count.  


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
80 
Electrolytes: Participants enrolled in the study who are randomized to hypertonic saline 
administration will have electrolytes evaluated on admission and every six hours 
thereafter until they regain consciousness. As this is a crucial measurement in the 
appropriate administration of the investigational product, a back-up machine will be 
available.  
Respiratory infection screening: On admission, two nasopharyngeal swabs: one for viral 
identification and one for bacterial detection and quantification by real time PCR. For 
viral identification we will use the respiratory PCR panel (FilmArray) which includes 
simultaneous detection of 18 viruses (adenovirus, 4 endemic coronaviruses, SARS-CoV-
2, hmpv, RSV, 5 influenza viruses, rhinovirus/enterovirus and 4 parainfluenza viruses). 
Individual bacterial real time PCR analysis and quantitation will be performed 
for Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and 
Moraxella catarrhalis. 
8.3 Special Assays or Procedures 
8.3.1  Fundoscopy  
Fundoscopy will be performed at admission and every 24 hours (12 hours) thereafter until 
the patient reaches a BCS of 3 for greater than 4 hours. Pupils of participants will be dilated 
with mydriatics (a combination of 1.0% tropicamide hydrochloride and 2.5% phenylephrine 
hydrochloride) in both eyes. A trained clinician will perform direct and indirect 
ophthalmoscopy on fully dilated eyes. The elements of that will qualify a participant for being 
retinopathy positive in the study are presence one or more of the following: 
x 
Retinal hemorrhages  
x 
Whitening 
x 
Vessel changes  
Examples of each of these are included as Figure 3, Section 2.1g. 
The presence or absence of papilledema will also be noted. Presence of papilledema alone 
without any of the other three elements will not qualify the participant for classification as 
retinopathy positive. The presence or absence of papilledema will however be evaluated as 
a measure of increased brain volume.  
8.3.2  MRI 
MRI of the brain will be the “gold standard” for assessing the presence of brain volume. 
The final determination of brain volume will be made initially by one of two experienced 
radiologists.  They will use the following scale52:  
1 = severe atrophy markedly abnormal for age with diffuse prominence of the cisternae 
and sulci 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
81 
2 = mild atrophy – subtle prominence of the cisternae and sulci for age 
3 = normal brain volume 
4 = mild increased volume but with maintenance of cisternae and sulci 
5 = mild swelling – some loss of cisternae and sulci but not diffuse 
6 = moderate swelling – diffuse involvement with some loss of cisternae and sulci 
7 = severe swelling – loss of all sulci, presence of cisternal effacement, decreased 
gray/white matter delineation 
8 = severe swelling – loss of all sulci, presence of cisternal effacement, presence of 
uncal herniation, loss of gray/white matter delineation 
The presence of severely increased volume (edema score 7 or 8) identifies the subset of 
children who are most susceptible to herniation and therefore death (Figure 5).  Real-time 
interpretations during the 2018 enrollment season revealed that neuroradiologists could not 
reliably differentiate a brain volume score of 6 or 7.  In order to include all subjects who might 
benefit from the interventions, subjects with brain volume scores of 6-8 will be eligible for 
randomization (see Section 2.3.3). 
Two study radiologists will be available at all times to review MRIs and assign a brain volume 
score.  If both independently assigned brain volume scores are  6, the participant will be 
eligible for randomization.  If either independently assigned, real-time score is < 6 the participant 
will not be eligible for randomization. 
Three other study radiologists will review all screening and study scans and will arrive at a 
consensus ‘brain volume score’ for each MRI scan on a regular basis and will complete the task 
within 1-month of the end of the enrollment period each year.  If there is a discrepancy 
regarding the initial brain volume score and the consensus score in enrolled subjects any 
subjects incorrectly enrolled will be excluded from the ‘per protocol’ analyses but will remain in 
the ‘intention to treat’ analyses.  If there is a discrepancy in the serial MRIs that affects the 
decision to withdraw the experimental treatment (VENT or HPS), the discrepancy will be noted; 
any subjects incorrectly withdrawn will be excluded from the ‘per protocol’ analyses, but will 
remain in the ‘intention to treat’ analyses. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
82 
8.3.3  Optic nerve sheath diameter (ONSD) 
ONSD will be measured using a digital ultrasound machine 
with an appropriate transducer. Details of the ONSD 
determination procedure are as described in Tayal et al 196.  
 
Figure 11 Illustration of the image  
and proper area for measurement in  
ONSD determination257.   
 
 
 
 
8.3.4 Transcranial Doppler Ultrasound (TCD) 
TCD will be measured using a commercially available TCD unit with an appropriate transducer.  
Details of the TCD examination are as described in Aaslid et al72. The following diagnostic 
criteria will be used to categorize studies into phenotypes: 
 
Microvascular Obstruction  
1) Systolic flow in middle cerebral arteries was within standard deviations (SD) of normal for age and gender AND 
2) Diastolic flow in middle cerebral arteries was < 2 SD below normal for age and gender AND 
0LGGOHFHUHEUDODUWHU\*RVOLQJ¶VSXOVDWLOLW\LQGH[ZDV 
 
Pulsatility Index (PI)= (Systolic flow velocity-Diastolic flow velocity/Mean flow velocity) 
 
Hyperemia 
0HDQIORZLQPLGGOHFHUHEUDODUWHULHVZDV6'DERYHWKHDJHDQGJHQGHUQRUPDOYDOXH$1' 
2) Ratio of flow between the middle cerebral artery and extra-cranial carotid artery (Lindegaard ratio       
    (LR)) was <3 AND 
3) The dichrotic notch was absent on waveform morphology analysis of the middle cerebral artery AND  
 
Cerebral Vasospasm 
0HDQIORZLQWKHPLGGOHFHUHEUDODUWHULHVZDV6'DERYHDJHDQGJHQGHUQRUPDOYDOXH$1' 
/5ZDV$1' 
3) The dichrotic notch was present on waveform morphology analysis of the middle cerebral artery 
 
Low Flow  
6\VWROLFGLDVWROLFDQGPHDQIORZVZHUHHTXDOO\UHGXFHGLQWKHPLGGOHFHUHEUDODUWHULHV6'EHORZWKHDJHDQGJHQGHUQRrmal value AND 
2) The PI was <1.2 
 
 
Isolated Posterior Hyperemia 
0HDQIORZYHORFLW\LQWKHEDVLODUDUWHU\ZDV6'DERYHDJHDQGJHQGHUQRUPDOYDOXH$1' 
2) Mean flow velocity in both middle cerebral arteries was within 2SD of age and gender normal value  
 
Terminal Intracranial Hypertension 
1) Systolic flow in the middle cerebral artery was <2SD below age and gender normal values WITH associated systolic spikes on waveform   
    analysis AND 
2) Absence of or reversal of diastolic flow was noted 
 
 
8.3.5 Near-infrared spectroscopy 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
83 
The cerebral hemoglobin oxygenation will be assessed by near-infrared spectroscopy of 
cerebral tissue via one or two sensors placed on the skin overlying the skull. Cerebral 
oxygenation will be recorded for 30 minutes (range 15-60 minutes) on each study day per 
schedule in Appendix A.  Average saturation as well as the amplitude and frequency of 
oscillations in tissue oxygenation will be analyzed. 
Skeletal muscle reperfusion dynamics will be assessed by measuring the changes in muscle 
hemoglobin oxygenation by near-infrared spectroscopy after inflation and deflation of an 
occlusive thigh cuff on each study day per schedule in Appendix A.  The skeletal muscle 
recording will last approximately 30 minutes and includes a 3-minute inflation of a pneumatic 
cuff on the lower limb. 
 
 
8.3.6  Heart rate variability 
When patients are admitted or transferred to the PICU, they are connected to bedside 
monitoring which includes electrocardiogram (EKG), respiration waveforms, oxygen levels, and 
blood pressures.  The ICU monitors display instantaneous values for these clinical parameters 
and plots of their trends. These waveforms will be archived, compressed and uploaded for 
offline analysis of patterns of heart rate variability and integration of results with the clinical 
course.  A 60 minute reading will be taken in the supine position.  Readings will undertaken 
every six hours for the first 24 hours of admission, then daily until the patient’s Blantyre Coma 
Score is  3, and again upon discharge. The aim is to undertake the recordings at the same 
time every day; currently 0600, 1200, 1800 and 0000. Readings will be discontinued upon death 
or improvement of Blantyre coma score to  3. 
 
 
 
8.3.7 Heart and lung ultrasound 
A protocolized heart and lung ultrasound (HLUS) examination will be performed within 4 hours 
of admission. All sonographic images will be acquired with a Butterfly iQ (USA) phased array 
transducer using transverse view. Each HLUS will be performed by the same trained 
investigator. Based on the international evidence-based recommendations for POCUS of the 
lungs, a complete eight-zone examination will be used. The anterior and lateral chest wall will 
be divided and imagining obtained bilaterally in a parallel plane.  Each zone will be scored 
based on the most agreed upon scoring system for LUS which uses four ultrasound aeration 
patterns: 
  
1. Normal = the presence of lung sliding with A-lines or fewer than two isolated B-lines, scored 
as 0 
2. Moderate loss of lung aeration = multiple well-defined B-lines (B1-lines) present, scored as 1 
3. Severe loss of lung aeration = the presence of multiple coalescent B-lines (B2-lines), scored 
as 2 
4. Lung consolidation = tissue pattern characterized by dynamic air bronchograms, scored as 3. 
  


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
84 
The worst ultrasound pattern observed in each zone will be recorded and used to calculate the 
sum of the scores with a maximum total score of 24. Each child’s lung ultrasound will be 
repeated and scored daily until discharge, death, normalization, or day 4 of admission, 
whichever comes first. 
 
The cardiac function will be evaluated using a non-invasive cardiac output monitor (USCOM, 
Australia).  The ultrasound probe will be placed on the suprasternal notch and the blood flow will 
be captured.  The machine performs calculations and reports cardiac output, cardiac index, 
stroke volume, stroke volume index, systemic vascular resistance, and stroke volume 
variability.  Each child’s cardiac ultrasound will be repeated, and results recorded daily until 
discharge, death, normalization, or day 4 of admission, whichever comes first. 
8.4 Specimen Preparation, Handling, and Shipping 
8.4.1  Instructions for Specimen Preparation, Handling, and Storage 
Admission CSF, urine and plasma samples from each participant will be archived. The one ml 
blood sample dedicated for plasma archive will be aliquoted into a cryovial for long term storage 
in a -80°C freezer.  One ml of CSF will immediately be transferred to a cryovial prior to 
distributing the remainder of the sample for culture and cytology. Urine will be collected in the 
process of inserting a Foley catheter or via a sterile bag affixed to the patient and transferred to 
a cryovial.  
Cryovials for plasma, urine and CSF will be identified with a unique bar code and the location in 
the freezer recorded with dedicated sample tracking software.  Thick and thin blood smears to 
evaluate the presence of malaria parasitemia will also be stored past the conclusion of the 
study. They will be labeled with a unique bar code and stored in a slide file cabinet in a locked 
storeroom adjacent to the PRW as detailed in Section 14.7.  
Detailed protocols for both procedures are included in the study’s Manual of Operating 
Procedures (MOP).   
8.4.2 Specimen Shipment 
Samples will be shipped in accordance with IATA requirements and with any required Materials 
Transfer Agreements in place. 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
85 
ASSESSMENT OF SAFETY
Specification of Safety Parameters 
The primary safety parameter for this study is a comparison of survival of the participants in the 
three treatment arms. In addition, we will be assessing long term neurological sequelae for all 
participants.  
Methods and Timing for Assessing, Recording, and 
Analyzing Safety Parameters 
 Adverse Events
ICH E6 defines an adverse event (AE) as any untoward medical occurrence in a participant or 
clinical investigation subject exposed to an intervention or a pharmaceutical product regardless 
of its causal relationship to the study treatment.  An AE can therefore be any unfavorable and 
unintended sign (including an abnormal laboratory finding), symptom, or disease, temporally 
associated with the use of a medicinal (investigational) product.  The occurrence of an AE may 
come to the attention of study personnel during study visits and interviews of a study participant 
presenting for medical care, or upon review by a study monitor.  
Because the safety profiles of the two study interventions are fairly well-defined, and the patient 
population is critically ill, only AE considered to be possibly attributed to the study intervention 
will be collected. These will include AE known to be related to the study interventions and pre-
specified in 2.4.1.2 and any other unanticipated AE that in the opinion of the investigator was 
attributed to study interventions.  All AEs occurring while on study will be documented 
appropriately regardless of relationship.  All AEs will be followed to adequate resolution.  
Any medical condition present at the time that the participant is screened should be considered 
as baseline and not reported as an AE.  However, if the medical condition deteriorates at any 
time during the study, it will be recorded as an AE. 
All AEs will be graded for severity and relationship to study interventions.  
Since mortality is an endpoint, death will not routinely be considered an Adverse Event for study 
purposes.  Only events meeting leading to death, hospitalization or prolongation of 
hospitalization that are outlined in section 2.4.1.2, or that were unanticipated, but that are 
considered to be possibly related to the study intervention will be reported as SAE. 
9.2.1.1 Severity 
For electrolyte abnormalities, toxicity tables (Appendix B) will be used. For other, non-protocol 
specific AEs the following grading system will be used: 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
86 
x 
Mild:  events require minimal or no treatment and do not interfere with the participant’s 
daily activities. 
x 
Moderate:  events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
x 
Severe:  events interrupt a participant’s usual daily activity and may require systemic 
drug therapy or other treatment. Severe events are usually incapacitating. 
x 
Life threatening:  any adverse drug experience that places the participant or subject, in 
the view of the investigator, at immediate risk of death from the reaction as it occurred, 
i.e., it does not include a reaction that had it occurred in a more severe form, might have 
caused death. 
Changes in the severity of an AE will be documented to allow an assessment of the duration of 
the event at each level of intensity to be performed.  Onset and duration of each episode of 
intermittent adverse events will be documented. 
9.2.1.2 
Relatedness 
The relatedness of the AE will be assessed by a study clinician based on the temporal 
relationship of the AE and any interventions, as well as the likelihood of other causal etiologies 
of the specific AE. 
Related – There is a reasonable possibility that the intervention caused the adverse 
event. Reasonable possibility means that there is evidence to suggest a causal 
relationship between the study product and the adverse event. 
Not Related – There is not a reasonable possibility that the administration of the study 
product caused the event. 
9.2.1.3 
Expected Adverse Events 
 All adverse events meeting the criteria listed above will be collected. Special concern will be 
paid to the following adverse events as they are known side effect of the investigational drugs: 
x 
Potentially attributable the hypertonic saline intervention: 
 
Hypernatremia 
 
Hypokalemia 
 
Hyperkalemia 
 
Phlebitis 
x 
Potentially attributable to the intubation intervention: 
 
Pneumothorax 
 
Pneumomediastinum 
 
Subglottic stenosis 
 
Stridor 
 
Tracheostomy 
 
Bronchospasm 
 
Pulmonary hemorrhage 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
87 
 
Respiratory nosocomial infections 
 
Hypotension 
 
Endotracheal tube obstruction 
 
Inadvertent endotracheal tube dislodgment 
 
Bradycardia induced by vagal response 
 
Adverse Event grading tables (attached as Appendix B) will be used to define these conditions. 
These events will be captured in the CRF and subsequently in tabular format, listing time of 
event onset, time of event resolution, brief description of the event, clinician’s assessment of 
severity, and clinician’s assessment of relation to the intervention. They will be reported to the 
local ISM on a monthly basis, and an annual report of these events will be presented to the 
DSMB, as well as both IRBs. The events will be captured for the duration of the participants’ 
stay on the PICU/PRW, independent of whether the participant is still receiving interventional 
therapy.  
 Reactogenicity (for Vaccine Studies and Some Therapeutic Trials) 
 
NA 
 Serious Adverse Events 
An adverse event or suspected adverse reaction is considered “serious” if, in the view of either 
the investigator or sponsor, it results in any of the following outcomes:  
x 
Death, 
x 
A life-threatening adverse event,  
x 
Hospitalization or prolongation of existing hospitalization,  
x 
A persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions, or  
x 
A congenital anomaly/birth defect.   
Important medical events that may not result in death, be life-threatening, or require 
hospitalizations may be considered serious when, based upon appropriate medical judgment 
they may jeopardize the participant and may require medical or surgical intervention to prevent 
one of the outcomes listed in this definition.    Only events that are not attributable to the 
malarial illness, that are outlined in section 2.4.1.2, or that were unanticipated, but that are 
considered to be possibly related to the study intervention will be graded and reported as 
Adverse Events to the sponsor, IRBs, and independent safety monitors.   


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
88 
x 
Life-threatening adverse event: An adverse event is considered “life-threatening” if, in 
the view of either the investigator or sponsor, its occurrence places the participant or 
subject at immediate risk of death.  It does not include an adverse event, had it occurred 
in a more severe form, might have caused death. 
 
9.2.3.1 Protocol defined SAEs 
CM is a disease with a high mortality rate. Reported mortality rates range from 15 – 30%, 
with our site having a rate of 18% over the past 25 years. We anticipate the mortality rate in 
randomization arm 3 will be approximately 37%.  All deaths not attributable to the malarial 
illness will be reported to independent safety monitors, IRBs, and the sponsor as required.  
x 
Death not caused by respiratory arrest secondary to intracranial hypertension. 
x 
Prolongation of existing hospitalization for more than 7 days due to an 
intervention. 
x 
A life-threatening adverse event that is not part of the study outcomes defined in 
the study objectives, an attributable to an intervention. 
 Procedures to be Followed in the Event of Abnormal Laboratory Test 
Values or Abnormal Clinical Findings 
9.2.4.1 
Hypernatremia   
Table 3 (Section 6.1.5) categorizes the possible serum sodium levels and actions to be 
taken if they are observed. A serum sodium measurement of greater than 190 mmol/L 
will be considered an SAE and reported as such. The 3% HPS infusion will be stopped. 
Maintenance fluid infusion will continue at maintenance rate. Sodium will be rechecked 
hourly until its serum level is < 160 mmol/L and therapy will be resumed as per Table 3.  
 
9.2.4.2 
Hypokalemia  
As per the toxicity table attached as Appendix B, a serum potassium measurement of 
<2.0 mmol/L will be defined as a Grade 4 adverse event. Corrective action will be in the 
form of adding 40 mmol/L of KCl to the maintenance IV fluids (an increase from the 
17mmol/L K+ present in ½ Strength Darrow’s). IV fluid infusion rate will remain the same. 
HPS boluses will continue, and electrolytes will be monitored at 3 hour intervals until K+ 
returns to below Grade 3 disturbances.  
 
9.2.4.3 
Phlebitis  
3% hypertonic saline when administered through peripheral veins over prolonged 
periods of time may lead to phlebitis. If phlebitis or swelling of the IV catheter site is 
noted, the IV cannula will be removed and an alternate placed.  
Reporting Procedures 
 Serious Adverse Events 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
89 
The reporting period for SAEs will be between the time of randomization and the twelve month 
(2 weeks) post-randomization follow up visit. 
 
SAE reporting will be carried out as follows 
 
The study clinician will complete a Serious Adverse Event Form (Appendix D) and SAEs 
thought to be related to study related procedures will be reported within 24 hours of their 
recognition, to the independent safety monitor, the IRBs at Michigan State University and the 
University of Malawi College of Medicine, and the sponsor, unless advised otherwise by the 
DSMB. 
 
Any AE that meets a protocol-defined serious criterion will be submitted on an SAE form to the 
DMID Pharmacovigilance Group, at the following address:  
 
DMID Pharmacovigilance Group 
Clinical Research Operations and Management Support (CROMS) 
6500 Rock Spring Dr. Suite 650 
Bethesda, MD 20814, USA 
SAE Hot Line: 1-800-537-9979 (US) or 1-301-897-1709 (outside US) 
SAE FAX Phone Number: 1-800-275-7619 (US) or 1-301-897-1710 (outside US) 
SAE Email Address: PVG@dmidcroms.com
Other supporting documentation of the event may be requested by the DMID 
Pharmacovigilance Group and will be provided as soon as possible.  
 
The DMID medical monitor and clinical protocol manager will be notified of the SAE by the 
DMID Pharmacovigilance Group.  The DMID medical monitor will review and assess the SAE 
for regulatory reporting and potential impact on study subject safety and protocol conduct.  
At any time after completion of the study, if the investigator becomes aware of an SAE that is 
suspected to be related to study product, the investigator will report the event to the DMID 
Pharmacovigilance Group, the independent safety monitor, and the IRBs of the University of 
Malawi and Michigan State University.   
 Regulatory Reporting for Studies Conducted Under DMID-Sponsored 
IND 
NA 
 Regulatory Reporting for Studies Not Conducted Under 
DMID-Sponsored IND
NA 
 Other Adverse Events (if applicable) 
NA 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
90 
 Reporting of Pregnancy 
Participant population is pediatric and not prone to pregnancy. 
Type and Duration of Follow-up of Subjects after 
Adverse Events 
AEs will be followed until resolution even if this extends beyond the study-reporting period.  
Resolution of an AE is defined as the return to pretreatment status or stabilization of the 
condition with the expectation that it will remain chronic. CM is a disease with a high burden of 
long-term neurological deficits 258 . Participants will all be asked to return to clinic at 1, 3, 6 and 
12 months (2 weeks) post-randomization for evaluation of recovery; the 12-month (2 weeks) 
visit will serve as the final study visit.  
Halting Rules 
The following eventuality would trigger temporary suspension of enrollment until a safety review 
is convened and further evaluation undertaken:  
x 
Five subjects where current HPS regimen is unable to achieve the target Na+ levels of 
150-160 mmol/L. 
x 
Any SAE deemed to be related to each of the experimental medical interventions 
(ventilator support or infusion of 3% hypertonic saline solution). 
DMID retains the authority to suspend additional enrollment and study interventions during the 
entire study, as applicable. 
Safety Oversight (ISM plus SMC or DSMB) 
Safety oversight will be under the direction of a DSMB composed of a pediatrician, a pediatric 
intensivist, a statistician, and others at the discretion of NIAID. The DSMB will meet annually to 
assess safety and efficacy data on each arm of the study. The DSMB will review aggregate 
safety data for increased rate of occurrence of serious unexpected adverse reactions. If halting 
rules are initiated, more frequent meetings may be held.  The DMSB will operate under the rules 
of a DMID-approved charter that will be written at the organizational meeting of the DSMB.  At 
this time, each data element that the DSMB needs to assess will be clearly defined.  The DSMB 
will advise DMID of its findings. 
 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
91 
CLINICAL MONITORING
 Site Monitoring Plan 
Site monitors from the Research Support Centre at the University of Malawi College of Medicine 
will visit the clinical research site to review the individual subject records, including consent forms, 
CRFs, supporting data, laboratory specimen records, and medical records (physicians’ progress 
notes, nurses’ notes, participants’ hospital charts), to ensure protection of study subjects, 
compliance with the protocol, and accuracy and completeness of records. The monitors also will 
inspect sites’ regulatory files to ensure that regulatory requirements are being followed and sites’ 
pharmacies to review product storage and management. 
 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
92 
STATISTICAL CONSIDERATIONS
11.1 General Design and Objectives 
We propose a single site randomized controlled trial (RCT) of two adjunctive therapeutic 
approaches compared to usual care (UC) in treating increased brain volume in Malawian 
children with CM and MRI-supported evidence of severely increased brain volume. Children will 
be randomized to one three arms: Arm 1: UC plus immediate ventilator support (VENT), Arm 2:  
UC plus hypertonic saline (HPS), Arm 3: UC. Arm 2 and Arm 3 would receive rescue ventilation 
in the event of a respiratory arrest. 
11.2 Stratification and Randomization 
Stratification by retinopathy status (negative/positive) is necessary as the mortality rates vary 
between children of varying retinopathy status. For this study, stratification by gender or age is 
not necessary. 
 
11.3 Testing the adequacy of randomization 
Prior to analysis, the adequacy of the randomization will be evaluated by comparing all baseline 
characteristics of the three study arms. These variables include characteristics such as age, 
gender, weight, malarial retinopathy status, height and duration of symptoms prior to admission. 
Where substantive differences exist in these variables, they may be controlled for in subsequent 
analyses by regression techniques. The degree of similarity between the study arms will be 
tested using, as appropriate, ANOVA, nonparametric tests and chi-square tests. However, 
following Altman259,we will be guided by the degree of dissimilarity rather than strict statistical 
significance. 
 
11.4 Outcomes 
The primary endpoint is death within one week following randomization. In Arm 2 and Arm 3, the 
endpoint will include failure of the initial treatment allocation such that the patient requires 
rescue ventilation. Several secondary outcomes will be assessed in the trial. Table 8 gives a 
brief description and preferred analytic strategies. Additional details are provided elsewhere in 
the Protocol.  
 
11.5 Sample Size and Power 
For the primary outcome death/failure of the intervention, we conduct two parallel studies: (i) 
VENT versus control UC, (ii) HPS versus control UC. Approximately 128 children will be 
enrolled in the studies. This includes the 64 children in UC comprised of 16 randomized to UC in 
the 2018-2019 malaria seasons and augmented by 48 historical controls from previous seasons 
(2014-2017). We also randomized 16 children to VENT in the 2018-2019 malaria seasons. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
93 
 
 
 
Since the beginning of this study, we faced several logistical challenges but had success in 
recruiting 32 children in 2018-2019 (16 each in VENT and UC). Based on our current data, the 
estimated mortality in UC and VENT are respectively, 36% and 25%. Maintaining the mortality 
rate in UC, a one-sample lower test at significance level D=0.05 of a null difference has 80% 
power to detect a reduction in mortality to 18% in VENT with 33 subjects in VENT.  
 
 
11.6  Interim Analyses  
The TBS design is a 4-stage sequential plan to compare VENT to UC on the outcome of 
mortality. Based on historical controls and initial randomization to UC before 2020, mortality is 
Table 8: Summary of outcome measures and analytic plan 
Outcome measure 
Outcome type/scale 
Analytic method 
PRIMARY OUTCOME 
Success of primary intervention or 
survival in the first 7 days after 
randomization 
Binary  
Logistic regression 
SECONDARY OUTCOMES 
Brain volume determination (MRI on 
admission, then daily) 
Ordinal 
Ordinal regression 
Optic Nerve Sheath Diameter (on 
admission, then daily) 
Continuous  
Linear regression. Serial 
assessments by repeated measures 
analysis 
Transcranial Doppler ultrasound (on 
admission, then daily) 
Continuous 
Linear regression. Serial 
assessments by repeated measures 
analysis 
Opening CSF pressure (mm CSF) (on 
admission) 
Continuous 
Linear regression 
Heart rate variability patterns 
Continuous 
Linear regression.  Serial 
assessments by repeated measures 
analysis 
Neurologic exam * (on admission, then 
twice daily) 
21 binary and 5 
continuous variables  
Linear or logistic regression 
Papilledema (on admission, then  
daily) 
Binary 
Logistic regression 
Severe neurologic sequelae at 16 and 
12 months 
Binary 
Logistic regression. Serial 
assessments by repeated measures 
analysis 
Hypotension that occurs within 30 
minutes of sedation administration, 
initiation of mechanical ventilation or 
ventilator pressure adjustment. 
Binary 
Logistic regression. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
94 
assumed fixed at 
0
p =0.28. We test the hypothesis that mortality p in VENT is lower, i.e., we test 
 

0
0
0
0
:
:
H
p
p versus H
p
p
at significance level D=.05. VENT will be declared superior to 
UC, if we can demonstrate a relative risk reduction of 50% with 80% power. This needs a  
sample of 38 subjects.  
 
Boundaries for the 4-stage plan are derived from  the power family. Early stopping is to reject 
the null hypothesis. Our first interim analysis was conducted with data from the 2019-2020 
malaria seasons. Observed mortality in VENT was 12.5% (2/16). The conclusion was to 
continue to the next stage. Our current estimate, at the end of the 2021 season is 15.8% (3/19). 
In accordance with the design, we must continue to the next stage (i.e., 2022 season). The 
revised boundary information is depicted in the figure. 
 
 Figure: Boundary plot at conclusion of stage 2 
 
Legend: The scale is the maximum likelihood estimate (MLE) of the mortality difference 
(PDIFF), T  

0
p
p against the actual information level. At stage 1, observed 
T=0.1250.28=0.155, and at stage 2, T= 0.1580.28= 0.122. The rejection boundaries are not 
crossed. 
 
 
Conditional power and predictive power 
Conditional power (CP) is the power to reject the null hypothesis H0: T =0 at all future stages 
given the cumulative interim data. This calculation shows: (a) at the current estimated difference 
in mortality T=0.122, CP=76.1%, (b) at zero difference in mortality, CP=18.6%, (c) at the 
hypothesized difference, T=0.14, CP=82.9%. Predictive power (PP) is the posterior probability 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
95 
of rejecting the null at the next stage given the current data, assuming a non-informative prior on 
the mortality difference. PP=66.1% at the current stage.  
Based on these assessments it behooves that we proceed to stage 3. 
 
Hypertonic saline (HPS) vs UC 
We have our first sample of 3 subjects, one of whom died. Although hardly necessary, a formal 
analysis provides the exact 95% CI for mortality in HPS,  (0.008, 0.906).   
 
 
11.6.1  Futility Analysis  
Futility Analysis: Although VENT seems an effective treatment, does it promise at this stage 
substantial benefit  compared to UC?  To answer this question we calculated the conditional 
power to reject the null hypothesis at all future stages give the current interim data. Conditional 
power (CP) is calculated on hypothesized values of the alternative T . Our current value is –
0.122. CP at the current value is 76.1%. At the hypothesized difference,  0.140.28=0.14, 
CP=82.9%. These do suggest promise of VENT. However, if the true difference in mortality is 
actually zero (T =0)  then CP=18.6%. The literature advises that if CP is small (e.g., <20%) over 
a plausible range of T  values, then it would be futile to continue the trial. The evidence that we 
have now does not point to futility.   
 
11.7 Statistical Analysis 
For the primary outcome, the test of the null hypothesis 
T
T
 

0
1
:
0
:
0
H
vs H
 for the mortality 
difference T  

0
p
p follows from the sequential design described in 11.6. The preferred analytic 
method for secondary outcomes by type/scale is included in Table 8. The unit of analysis is the 
individual child (indexed by i) with a single outcome measure 
i
Y and covariates 
i
x which 
includes an indicator for treatment arm (VENT, HPS, UC), socio-demographic and clinical 
variables whose prognostic influence on the outcome is of interest or needs to be controlled. 
The ensuing data on the N children are 
d
d
{(
,
) :1
}
i
i
Y
i
N
x
. We expect N=128 across all three 
arms. Depending on whether the early stopping and futility rules are triggered in 11.6, sample 
size will vary by treatment arm. Minimum size in UC is 64 if no new recruitment into this arm is 
made in 2022 and 2023. Minimum size in HPS is 32, and in 21 in VENT if  futility is triggered. 
With these caveats we describe broadly our statistical analyses. 
 
Analysis of binary outcomes in Table 8 is by logistic regression. Continuous variables such as 
optic nerve sheath diameter on admission, heart rate variability patterns, and opening CSF 
pressure on admission are analyzed using linear models. To mitigate the effects of skewness in 
their distributions, if found, a logarithmic or square-root transformation g will be applied to
i
Y  
and analysis carried out on the transformed variable. To assess the effect of treatment on 
outcome we will fit the linear model 
E
c
 
( (
) |
))
i
i
i
E g Y
x
x
 and test the hypothesis 
E
E
 
 
0
1
2
:
0,
0
H
 
where E1 and E2 are the coefficients for the VENT and HPS, respectively. Point estimates and 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
96 
associated 95% confidence intervals for response mean on the original scale will be calculated 
and would involve smearing.260-262 
For assessments made serially over time, the outcome is a vector 
c
 

1
2
(
,
,
,
)
i
i
i
im
Y Y
Y
Y
of repeated 
continuous (e.g., sheath diameter on admission and then twice daily) or repeated binary 
indicators (e.g., severe neurologic sequelae at 1, 6 and 12 months). Generalized linear models 
E
c
c
 

( (
|
))
ij
i
ij
ij
i
g E Y
x
x
z b are apropos with right hand side covariates specific to the j-th time, 
ij
z
will include an intercept and terms involving time permitting assessment of the evolution of the 
mean outcome (or probability for binary outcomes) over time, both within-subject and across the 
study population (i.e., population average). The link g is the identity function for continuous 
responses, and the logit for binary responses. Correlations between components of 
i
Y are 
incorporated via the random effects. A judicious choice of covariance structure for 
i
b will be 
elicited from information criteria.263-265 Alternating logistic regression will be considered the case 
of binary responses.266 Repeated ordinal measures (e.g., brain volume) will be analyzed by 
proportional odds or partial proportional odds models.267,268 
In all analytic procedures we will examine the overall goodness-of-fit, and presence of influential 
and/or outlying data points with graphical techniques and formal residual analyses. 
 
11.7.1 Missing Data 
Although quite unlikely, mechanisms for handling missingness in our response data will be 
appropriate to the underlying analytic strategy269 but tempered by available sample size. In 
linear and nonlinear mixed models likelihood-based inference using only the non-missing 
responses is valid under the MAR, i.e., missingness is dependent only on the observed data. 
The alternative missingness mechanism--missing completely at random (MCAR) assumes 
missingness is does not depend the responses, observed or not. Other approaches such as 
inverse-probability weighting270,271 will be investigated to accommodate missing data patterns 
that are neither MCAR or MAR272,273 an approach that we used in analyzing longitudinal 
censored medical costs data.274,275 
11.7.2 Additional exploratory modeling  
Since Arm 2 (HPS) and Arm 3 (UC) are likely to have an intermediate endogenous binary 
outcome 
1
i
Y  of treatment success prior to final outcome of survival
2
iY , we will consider joint 
modeling for 
1
2
(
,
)
i
i
Y Y
 via a two-part specification: (1) conditional model for 
 
 
2
1
[
1|
1,
]
i
i
i
P Y
Y
x --
i.e., probability of survival given treatment success and (2) 
 
1
[
1|
]
i
i
P Y
x  –i.e., the probability of 
treatment success. Both two-stage and full likelihood-based estimation of the joint model are 
feasible.270,276 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
97 
SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
We will maintain appropriate medical and research records for this trial, in compliance with ICH 
E6, Section 4.9 and regulatory and institutional requirements for the protection of confidentiality 
of subjects. As part of participating in a DMID-sponsored, DMID-affiliated, or manufacturer-
sponsored study, we will permit authorized representatives of the sponsor(s), DMID, and 
regulatory agencies to examine (and when required by applicable law, to copy) clinical records 
for the purposes of quality assurance reviews, audits, and evaluation of the study safety and 
progress. 
Source data are all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial.  Examples 
of these original documents and data records include, but are not limited to, hospital records, 
clinical and office charts, laboratory notes, memoranda, subjects’ memory aid or evaluation 
checklists, pharmacy dispensing records, recorded data from automated instruments, copies or 
transcriptions certified after verification as being accurate and complete, microfiches, 
photographic negatives, microfilm or magnetic media, x-rays, and subject files and records kept 
at the pharmacy, at the laboratories, and medico-technical departments involved in the clinical 
trial. The electronic CRFs and subsequent database will be stored on a password protected 
computer. Other sources of data will include: 
x 
MRI images will be stored both at QECH and on a password protected server at 
Michigan State University.  
x 
ONSD images will be stored on an ultrasound machine at QECH. It is password 
protected and stored in a locked room adjacent to the ward.  
x 
TCD images will be stored on an ultrasound machine at QECH.  It is password 
protected and stored in a locked room adjacent to the ward.  
x 
Heart rate variability data will be stored on a Malawi-based server and securely 
transmitted to Michigan State University where it will be available for download 
and analysis by co-investigators. 
 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
98 
QUALITY CONTROL AND QUALITY ASSURANCE
A plan for local monitoring of clinical quality has been documented in a Clinical Quality 
Management Plan (CQMP) (see Manual of Operations). This document details the Standard 
Operating Procedures (SOPs) for on-site quality management including data evaluation 
methods, staff training methods, documents to be reviewed, and corrective actions to be taken 
should deficits be detected. This document will be reviewed annually and submitted to DMID.  
All clinical observations and laboratory evaluations will be recorded in accordance with the 
Manual of Procedures.   
 
Following written SOPs, the monitors will verify that the clinical trial is conducted and data are 
generated, documented (recorded), and reported in compliance with the protocol, GCP, and the 
applicable regulatory requirements.  
 
We will provide direct access to source data/documents, and reports for the purpose of monitoring 
and auditing by the sponsor, and inspection by local and regulatory authorities. 
 
The Data Manager will implement quality control procedures beginning with the data entry system 
and generate data quality control checks that will be run on the database. Any missing data or 
data anomalies will be communicated to the site for clarification/resolution. 
 
The PI is responsible for ensuring that routine quality management activities are conducted. 
 
 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
99 
ETHICS/PROTECTION OF HUMAN SUBJECTS
 Ethical Standard 
The principal investigator (PI) will ensure that this study is conducted in full conformity with the 
current revision of the Declaration of Helsinki, or with the principles set forth in The Belmont 
Report: Ethical Principles and Guidelines for the Protection of Human Subjects of Research of 
the US National Commission for the Protection of Human Subjects of Biomedical and 
Behavioral Research (April 18, 1979) and codified in 45 CFR Part 46 and/or the ICH E6; 62 
Federal Regulations 25691 (1997), whichever affords the greater protection to the subject. 
 Institutional Review Board 
Each participating institution must provide for the review and approval of this protocol and the 
associated informed consent documents and recruitment material by an appropriate 
independent ethics committee (IEC) or IRB registered with the OHRP.  Any amendments to the 
protocol or consent materials must also be approved before they are placed into use.  In the 
United States and in other countries, only institutions holding a current US Federal-Wide 
Assurance issued by OHRP may participate. All activities will be reviewed by a Michigan State 
University IRB as well as the local IRB (the University of Malawi College of Medicine Research 
and Ethics Committee [COMREC]).  
 Informed Consent Process 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in 
the study and continuing throughout the individual’s study participation.  Extensive discussion of 
risks and possible benefits of this therapy will be provided to caregivers of subjects.  Consent 
forms describing in detail the study interventions/products, study procedures, and risks are 
given to the caregiver and written documentation of informed consent is required prior to starting 
intervention/administering study product.  Consent forms will be IRB-approved and the caregiver 
will be asked to read and review the document. Upon reviewing the document, the nurse 
initiating informed consent will explain the research study to the subject and answer any 
questions that may arise.  Caregivers will sign the informed consent document prior to any 
procedures being done specifically for the study.  They will have the opportunity to discuss the 
study with their surrogates or think about it prior to agreeing to participate.  Caregivers may 
withdraw consent at any time throughout the course of the trial.  A copy of the informed consent 
document will be given to the caregivers for their records.  The rights and welfare of study 
subjects will be protected by emphasizing to caregivers that the quality of their child’s medical 
care will not be adversely affected if they decline to participate in this study. 
 Detailed Informed Consent Process 
There are two consent processes potentially involved in this study: 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
100 
1. Consent for screening.  This is the first step for all patients, and the ONLY step for 
patients who do not have increased brain volume on MRI.  These study participants will 
spend the first 24 hours on the PICU or the PRW (for continuous monitoring for HRV) 
and if in the PICU will then be moved to the PRW unless they deteriorate, clinically, and 
warrant ICU care. 
2. Consent to enroll in the interventional clinical trial.  This option is for patients with MRI 
evidence of increased brain volume on MRI  
In all cases the pertinent material will be supplied in both oral and written form. Forms will be 
available in English and the local language, Chichewa. The caregiver will be supplied with a 
copy of the consent form and given ample time to ask questions regarding specifics of the 
study. A study clinician or study nurse will review the study consent with the parents or 
guardians accompanying the child. If the parent/guardian is illiterate, the consent will be 
explained verbally. If the parent/guardian is unable to form a signature, a thumb print will be 
obtained instead.  
Although all subjects will be minors under the age of twelve, the subjects will not be capable of 
providing assent to participate because all will be, by definition, comatose. 
Exclusion of Women, Minorities, and Children (Special Populations) 
The major burden of malaria is borne by children; therefore, only children under the age of 
thirteen years will be enrolled in this study. Subjects will all be Malawian children. All children 
will be cared for on specially adapted pediatric wards. Parents/guardians are allowed to remain 
with the children throughout their hospitalization, and all meals and medications are provided 
without cost.  The licensed clinicians providing care are all trained in the management of 
children with CM, and each research unit is amply staffed with trained dedicated nursing staff.  
No participant will be excluded on the basis of sex/gender or ethnicity.  
 Subject Confidentiality 
Subject confidentiality is strictly held in trust by the participating investigators, their staff, and the 
sponsor(s) and their agents.  This confidentiality is extended to cover testing of biological 
samples in addition to the clinical information relating to participating subjects. 
The study protocol, documentation, data, and all other information generated will be held in 
strict confidence.  No information concerning the study or the data will be released to any 
unauthorized third party without prior written approval of the sponsor. 
The study monitor or other authorized representatives of the sponsor may inspect all documents 
and records required to be maintained by the investigator, including but not limited to, medical 
records (office, clinic, or hospital) and pharmacy records for the subjects in this study.  The 
clinical study site will permit access to such records. 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
101 
 Study Discontinuation 
In the event of the discontinuation of the study, any subject currently enrolled in the study will 
continue in their current study arm, unless study discontinuation was due to one arm being 
determined to be superior to the others.  In this case, study subjects will be switched to the arm 
showing highest efficacy.  
 Future Use of Stored Specimens 
Plasma, urine, and CSF samples will be stored at the study site in liquid nitrogen or at minus-
80oC, filed by study number, and de- identified.  
 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
102 
DATA HANDLING AND RECORD KEEPING
Clinical research data will be managed using the Research Electronic Data Capture 
(REDCap)277  web-based software application developed by Vanderbilt University and hosted on 
servers located at the Malaria Alert Center, Blantyre, Malawi. For the purpose of data backup, a 
copy of the data will be hosted by the Biomedical Research Informatics Core (BRIC) on servers 
at MSU. The REDCap database developed for the Study will organize patient data entered 
directly into the database by clinicians using the REDCap application deployed on tablets at the 
patient bedside, by hospital staff using paper data collection forms, and by hospital staff and 
radiologists using computers for file uploads of MRI and laboratory results. The goals of the 
Data Management Plan are to optimize workflow for the capture of high quality data, provide a 
well-documented data audit trail, deliver robust measures of data security, and facilitate easy 
data access and data sharing for the Study research team. 
 Data Management Responsibilities 
Roles for data management will engage various members of the study research team and will 
be supported by REDCap functionality. The PI will have access to all study data, and access to 
by other study personnel will be restricted as directed by the PI. REDCap user definitions and 
user permissions, which allow for role-based and form-level access permissions and 
restrictions, will be used to limit access to identifying information only to necessary individuals.  
Data exports will be configured to remove identifying information by shifting date fields and 
redacting all other fields marked as identifying, as determined by the PI. The Malawi Data 
Manager and BRIC Data Manager (DM) will be responsible for issues related to data collection 
and data quality and will train clinicians and other hospital staff on matters related to direct 
electronic data capture. The BRIC Project Supervisor and Development Supervisor will oversee 
and ensure implementation and monitoring of the data management plan.  They will work 
closely with the PI, and Study team to ensure that the informatics configuration, testing, and 
production processes are adequately documented.  They will meet regularly to review data 
quality.  
 Data Capture Methods 
Data Collection 
Clinical research data will be managed using a REDCap Study database and customized 
features to support the entry of patient data, using tablets at the patient bedside.  A majority of 
the clinical trial data will be entered directly into the database by clinicians and other hospital 
staff as the data are generated.  The appropriate patient database record will be queued for 
direct electronic data capture by the use of barcodes, which will encode the patient Study ID#.  
Biologic specimens and paper data collection forms used for data capture will be labeled with 
barcodes to identify the associated patient.  Persons entering data via the tablet will require an 
active REDCap account username and password and/or pin-authentication, and users will be 
logged out after a period of inactivity. REDCap allows tracking of form data entry through a form 
status dashboard, and each form can be signed electronically after it is moved to a ‘completed’ 
status.  In case of power failure, back up paper forms will be stored in locked file cabinets.  


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
103 
Corrections to data on paper forms will be initialed and dated, and the data error will be 
overwritten with a single strike-through line. 
 
During transmission of data, REDCap uses Secure Hypertext Transfer Protocol (HTTPS) which 
employs a standard Secure Sockets Layer (TLS 1.0, 1.1, 1.2 - 128 bit block size) encryption 
technology. SSL creates the secure connection and HTTPS transmits the data securely. All 
subject data will eventually reside in a MySQL database server. The REDCap application 
includes functionality to enable 21 CFR part 11 compliance for electronic records and 
signatures, including a comprehensive audit log that records all user activity. Audit logs are 
created automatically within the REDCap application to capture the complete user history of 
database activity.  
 
The final record for each study participant will comprise data collected over a period of   two 
years: from initial data collection during hospitalization through and 6-month periodic follow-up 
assessments to 12-months post-randomization 
 
Data Quality 
The Malawi and MSU-BRIC DMs will work together to provide ongoing management and review 
of data collection processes and data quality.  The MSU-BRIC DM will provide on-site initial 
data entry training for study personnel, and refresher trainings via annual trips.  During active 
enrollment periods, the Malawi and MSU-BRIC DMs will conference weekly to address matters 
related to data entry and data quality.  DMs will utilize standard REDCap features to ensure 
data integrity and quality.  Variables will be designed with real-time field validation rules, and 
data will be reviewed using built-in checks for missing values and outliers.  The Data Quality 
module will be used to execute quality control verification on all fields and to resolve discrepant 
data, using an audited trail of query and query resolution.  
 
Data Security 
REDCap will be hosted on a server housed in a locked and climate-controlled server room at 
the Malaria Alert Centre, Blantyre, Malawi.  The server resides on its own private segment of the 
network, protected through a firewall, which routes traffic as well as provides active intrusion 
detection and protection.  Data will be accessed through an HTTPS connection to the web 
application front end.    Daily backups of the data are made to an alternate storage location at 
an off-site location to protect data in the event of a disaster that destroys the server room.   A 
back-up copy of the Malawi REDCap study database will be maintained by MSU-BRIC, which is 
described in detail in the Project/Performance Site(s) and Resources section of the application.  
The backup of the Malawi REDCap study database will be purely file level backup and not 
generally available through a web interface.  In the event that the service in unavailable in 
Malawi, BRIC will enable access to the backup copy and it will follow the same terms of service 
as the MSU- REDCap server.  The MSU- REDCap server is housed in a facility which satisfies 
requirements for the storage of Health Insurance Portability and Accountability Act (HIPPA) 
data. The server is connected to a private segment of the BRIC network.  The segment is 
protected through a firewall, which routes traffic and provides active intrusion detection and 
protection.  Security staff patrols, key card access, and audio/video monitoring protect the 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
104 
physical location of the server.   Daily and monthly backups of the database are made and 
retained on a server for fast restore capabilities. Copies of the data are moved to tape daily and 
transported to a separate storage location.  Tapes rotate after 28 days. 
 Types of Data 
The final record for each study patient will comprise data collected during hospitalization for 
acute cerebral malaria and neurological assessment data collected at follow-up visits 1, 3, 6, 
and 12 months post-randomization. These data will include MRI images and patient clinical data 
collected during the course of care and intervention, and neurological and developmental 
assessments. 
Timing/Reports
Data will be extracted for QC/QA on an annual basis (at a minimum of every 30 subjects).  In 
addition to presenting key study outcome data, reports will consider internal data management 
of data quality, the timely use of data entry and cleaning, and the study enrollment schedule.  
The content and frequency of study quality and operational metric reports will be defined by the 
Project Manager.  Study metric reports will be interpreted to identify the need for corrective 
action such as training or re-training, modification of the protocol, or modification of data 
collection material. 
 
 Study Records Retention 
Study records in the format of paper and electronic versions are to be retained by the Malawi 
Data Management Unit (DMU) at the College of Medicine for a minimum of 2 years after the 
submission of the final report and close-out procedures. A longer retention period can be 
arranged in accordance with NIAID requirements. No records will be destroyed without the 
written consent of the sponsor, if applicable.  It is the responsibility of the NIAID to inform the 
investigator when these study records no longer need to be retained. The retention of the 
original study records shall be the responsibility of the principal investigator. 
 
REDCap is designed to export data easily in formats ready for analyses in a number of standard 
statistical software applications.  For long term storage, data will be preserved in comma-
separated-value file format. Data will be preserved in the REDCap database for approximately 
one-year following the cessation of active data collection.  The PI will be provided with metadata 
and the final de-identified dataset.   After obtaining the PI’s release signature, live data will be 
removed the server, and backup files will be phased out systematically as tapes are retired. The 
PI will serve as the primary contact for access and questions regarding the data collected for the 
Study. 
 Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, Good Clinical Practice 
(GCP), or protocol-specific Manual of Procedures (MOP) requirements.  The noncompliance 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
105 
may be either on the part of the subject, the investigator, or the study site staff.  As a result of 
deviations, corrective actions are to be developed by the site and implemented promptly.  
 
 
These practices are consistent with GCP: 
 
4.5       Compliance with protocol, sections 4.5.1, 4.5.2, and 4.5.3 
5.1 
Quality Assurance and Quality Control, section 5.1.1 
5.20 
Noncompliance, sections 5.20.1, and 5.20.2. 
 
It is the responsibility of the site PI/study staff to use continuous vigilance to identify and report 
deviations within 5 working days of identification of the protocol deviation, or within 5 working 
days of the scheduled protocol-required activity. All deviations will be promptly reported to 
DMID, via the TRI/ICON DMID-Clinical Research Operations and Management Support 
(CROMS) email (protocoldeviations@dmidcroms.com), web- (www.dmidctm.com) or fax-based 
system (1-215-699-6288).  All deviations from the protocol must be addressed in study subject 
source documents.  A completed copy of the DMID Protocol Deviation Form (TRI/ICON DMID-
CROMS or IDES form) will be maintained in the regulatory file, as well as in the subject’s source 
document.  All deviations will be reported to the local ISM quarterly, and to the IRBs at Michigan 
State University and the University of Malawi College of Medicine annually, unless advised 
otherwise by the local ISM. 
 


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
106 
PUBLICATION POLICY
Following completion of the study, the investigators will publish the results of this research in a 
scientific journal.  In accordance with the International Committee of Medical Journal Editors 
(ICMJE) member journals, the trial will be registered in a public trials registry such as 
ClinicalTrials.gov, which is sponsored by the National Library of Medicine.  It is the responsibility 
of DMID to register this trial in an acceptable registry. Financial support from NIAID will be 
acknowledged in all publications.  
The primary manuscript will be developed by the PI in conjunction with other Key Personnel. 
All publications emanating from this protocol will be submitted to the digital archive PubMed 
Central upon acceptance for publication, as required by the US National Institutes of Health 
Access Policy (Division G, Title II, Section 218 of PL 110-161 (Consolidated Appropriations Act, 
2008)).  PubMed Central reference numbers (PMCIDs) will be attached to citations of all 
manuscripts in all progress reports required for the study described with this protocol.  


V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
107 
LITERATURE REFERENCES
1. 
WHO World Malaria Report. Geneva, Switzerland: World Health Organization, 2010. 
2. 
Idro R, Ndiritu M, Ogutu B, et al. Burden, features, and outcome of neurological 
involvement in acute falciparum malaria in Kenyan children.  JAMA. United States2007: 2232-
2240. 
3. 
Bruzzone R, Dubois-Dalcq M, Grau GE, Griffin DE, Kristensson K. Infectious diseases of 
the nervous system and their impact in developing countries. PLoS Pathog 2009;5:e1000199. 
4. 
Hay SI, Okiro EA, Gething PW, et al. Estimating the global clinical burden of 
Plasmodium falciparum malaria in 2007. PLoS medicine 2010;7:e1000290. 
5. 
McGregor IA. Mechanisms of acquired immunity and epidemiological patterns of 
antibody responses in malaria in man. Bulletin of the World Health Organization 1974;50:259-
266. 
6. 
Idro R, Marsh K, John CC, Newton CR. Cerebral malaria: mechanisms of brain injury 
and strategies for improved neurocognitive outcome. Pediatric research 2010;68:267-274. 
7. 
Trape JF, Rogier C, Konate L, et al. The Dielmo project: a longitudinal study of natural 
malaria infection and the mechanisms of protective immunity in a community living in a 
holoendemic area of Senegal. Am J Trop Med Hyg 1994;51:123-137. 
8. 
Taylor T, Fu W, Carr R, et al. Differentiating the pathologies of cerebral malaria by 
postmortem parasite counts. Nat Med 2004;10:143-145. 
9. 
MacPherson GG, Warrell MJ, White NJ, Looareesuwan S, Warrell DA. Human cerebral 
malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration. The 
American journal of pathology 1985;119:385-401. 
10. 
White NJ, Warrell DA, Looareesuwan S, Chanthavanich P, Phillips RE, Pongpaew P. 
Pathophysiological and prognostic significance of cerebrospinal-fluid lactate in cerebral malaria. 
Lancet (London, England) 1985;1:776-778. 
11. 
Turner GD, Ly VC, Nguyen TH, et al. Systemic endothelial activation occurs in both mild 
and severe malaria. Correlating dermal microvascular endothelial cell phenotype and soluble 
cell adhesion molecules with disease severity. The American journal of pathology 
1998;152:1477-1487. 
12. 
Elhassan IM, Hviid L, Satti G, et al. Evidence of endothelial inflammation, T cell 
activation, and T cell reallocation in uncomplicated Plasmodium falciparum malaria. The 
American journal of tropical medicine and hygiene 1994;51:372-379. 
13. 
Brown H, Hien TT, Day N, et al. Evidence of blood-brain barrier dysfunction in human 
cerebral malaria.  Neuropathol Appl Neurobiol. England1999: 331-340. 
14. 
Newton CR, Crawley J, Sowumni A, et al. Intracranial hypertension in Africans with 
cerebral malaria. Archives of disease in childhood 1997;76:219-226. 
15. 
Clark IA, Rockett RA, Cowden WB. TNF in cerebral malaria. The Quarterly journal of 
medicine 1993;86:217-218. 
16. 
Clark IA, Cowden WB, Rockett KA. Nitric oxide in cerebral malaria. The Journal of 
infectious diseases 1995;171:1068-1069. 
17. 
Mohanty D, Ghosh K, Nandwani SK, et al. Fibrinolysis, inhibitors of blood coagulation, 
and monocyte derived coagulant activity in acute malaria.  Am J Hematol. United States1997: 
23-29. 
18. 
Hemmer CJ, Kern P, Holst FG, et al. Activation of the host response in human 
Plasmodium falciparum malaria: relation of parasitemia to tumor necrosis factor/cachectin, 
thrombin-antithrombin III, and protein C levels.  The American journal of medicine. United 
States1991: 37-44. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
108 
19. 
Clemens R, Pramoolsinsap C, Lorenz R, Pukrittayakamee S, Bock HL, White NJ. 
Activation of the coagulation cascade in severe falciparum malaria through the intrinsic 
pathway. British journal of haematology 1994;87:100-105. 
20. 
Jimmy EO, Saliu I, Ademowo O. Fibrinopeptide-A and fibrinogen interactions in acute, 
Plasmodium falciparum malaria. Annals of tropical medicine and parasitology 2003;97:879-881. 
21. 
Dondorp A, Nosten F, Stepniewska K, Day N, White N, group SEAQAMTS. Artesunate 
versus quinine for treatment of severe falciparum malaria: a randomised trial. Lancet 
2005;366:717-725. 
22. 
Dondorp AM, Fanello CI, Hendriksen IC, et al. Artesunate versus quinine in the 
treatment of severe falciparum malaria in African children (AQUAMAT): an open-label, 
randomised trial. Lancet 2010;376:1647-1657. 
23. 
ter Kuile F, White NJ, Holloway P, Pasvol G, Krishna S. Plasmodium falciparum: in vitro 
studies of the pharmacodynamic properties of drugs used for the treatment of severe malaria.  
Exp Parasitol. United States1993: 85-95. 
24. 
Enwere G. A review of the quality of randomized clinical trials of adjunctive therapy for 
the treatment of cerebral malaria.  Trop Med Int Health. England2005: 1171-1175. 
25. 
Warrell DA, Looareesuwan S, Warrell MJ, et al. Dexamethasone proves deleterious in 
cerebral malaria. A double-blind trial in 100 comatose patients. N Engl J Med 1982;306:313-
319. 
26. 
Hoffman SL, Rustama D, Punjabi NH, et al. High-dose dexamethasone in quinine-
treated patients with cerebral malaria: a double-blind, placebo-controlled trial. J Infect Dis 
1988;158:325-331. 
27. 
Taylor TE, Molyneux ME, Wirima JJ, Borgstein A, Goldring JD, Hommel M. Intravenous 
immunoglobulin in the treatment of paediatric cerebral malaria. Clin Exp Immunol 1992;90:357-
362. 
28. 
van Hensbroek MB, Palmer A, Onyiorah E, et al. The effect of a monoclonal antibody to 
tumor necrosis factor on survival from childhood cerebral malaria. The Journal of infectious 
diseases 1996;174:1091-1097. 
29. 
Di Perri G, Di Perri IG, Monteiro GB, et al. Pentoxifylline as a supportive agent in the 
treatment of cerebral malaria in children. The Journal of infectious diseases 1995;171:1317-
1322. 
30. 
Lell B, Kohler C, Wamola B, et al. Pentoxifylline as an adjunct therapy in children with 
cerebral malaria.  Malar J. England2010: 368. 
31. 
Hemmer CJ, Kern P, Holst FG, Nawroth PP, Dietrich M. Neither heparin nor 
acetylsalicylic acid influence the clinical course in human Plasmodium falciparum malaria: a 
prospective randomized study. Am J Trop Med Hyg 1991;45:608-612. 
32. 
Namutangula B, Ndeezi G, Byarugaba JS, Tumwine JK. Mannitol as adjunct therapy for 
childhood cerebral malaria in Uganda: a randomized clinical trial.  Malar J. England2007: 138. 
33. 
Mohanty S, Mishra SK, Patnaik R, et al. Brain swelling and mannitol therapy in adult 
cerebral malaria: a randomized trial.  Clin Infect Dis. United States2011: 349-355. 
34. 
Maitland K, Pamba A, English M, et al. Randomized trial of volume expansion with 
albumin or saline in children with severe malaria: preliminary evidence of albumin benefit.  Clin 
Infect Dis. United States2005: 538-545. 
35. 
Akech S, Gwer S, Idro R, et al. Volume expansion with albumin compared to gelofusine 
in children with severe malaria: results of a controlled trial. PLoS clinical trials 2006;1:e21. 
36. 
Maitland K, Kiguli S, Opoka RO, et al. Mortality after fluid bolus in African children with 
severe infection. The New England journal of medicine 2011;364:2483-2495. 
37. 
Jakobsen PH, McKay V, Morris-Jones SD, et al. Increased concentrations of interleukin-
6 and interleukin-1 receptor antagonist and decreased concentrations of beta-2-glycoprotein I in 
Gambian children with cerebral malaria. Infect Immun 1994;62:4374-4379. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
109 
38. 
Gysin J, Moisson P, Pereira da Silva L, Druilhe P. Antibodies from immune African 
donors with a protective effect in Plasmodium falciparum human infection are also able to 
control asexual blood forms of the parasite in Saimiri monkeys. Res Immunol 1996;147:397-
401. 
39. 
Hunt NH, Grau GE, Engwerda C, et al. Murine cerebral malaria: the whole story. Trends 
Parasitol 2010;26:272-274. 
40. 
Chimalizeni Y, Kawaza K, Taylor T, Molyneux M. The platelet count in cerebral malaria, 
is it useful to the clinician?  Am J Trop Med Hyg. United States2010: 48-50. 
41. 
Molyneux ME, Taylor TE, Wirima JJ, Borgstein A. Clinical features and prognostic 
indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children. Q J Med 
1989;71:441-459. 
42. 
Lewallen S, Bakker H, Taylor TE, Wills BA, Courtright P, Molyneux ME. Retinal findings 
predictive of outcome in cerebral malaria. Trans R Soc Trop Med Hyg 1996;90:144-146. 
43. 
Newton CR, Valim C, Krishna S, et al. The prognostic value of measures of acid/base 
balance in pediatric falciparum malaria, compared with other clinical and laboratory parameters.  
Clinical infectious diseases : an official publication of the Infectious Diseases Society of 
America. United States2005: 948-957. 
44. 
Haldar K, Murphy SC, Milner DA, Taylor TE. Malaria: mechanisms of erythrocytic 
infection and pathological correlates of severe disease. Annu Rev Pathol 2007;2:217-249. 
45. 
Beare NA, Taylor TE, Harding SP, Lewallen S, Molyneux ME. Malarial retinopathy: a 
newly established diagnostic sign in severe malaria. Am J Trop Med Hyg 2006;75:790-797. 
46. 
Beare NA, Lewallen S, Taylor TE, Molyneux ME. Redefining cerebral malaria by 
including malaria retinopathy. Future Microbiol 2011;6:349-355. 
47. 
Lewallen S, Taylor TE, Molyneux ME, Wills BA, Courtright P. Ocular fundus findings in 
Malawian children with cerebral malaria. Ophthalmology 1993;100:857-861. 
48. 
Lewallen S, Bronzan RN, Beare NA, Harding SP, Molyneux ME, Taylor TE. Using 
malarial retinopathy to improve the classification of children with cerebral malaria.  Trans R Soc 
Trop Med Hyg. England2008: 1089-1094. 
49. 
White VA, Lewallen S, Beare N, Kayira K, Carr RA, Taylor TE. Correlation of retinal 
haemorrhages with brain haemorrhages in children dying of cerebral malaria in Malawi. Trans R 
Soc Trop Med Hyg 2001;95:618-621. 
50. 
Beare NA, Harding SP, Taylor TE, Lewallen S, Molyneux ME. Perfusion abnormalities in 
children with cerebral malaria and malarial retinopathy. J Infect Dis 2009;199:263-271. 
51. 
Seydel KB, Fox LL, Glover SJ, et al. Plasma concentrations of parasite histidine-rich 
protein 2 distinguish between retinopathy-positive and retinopathy-negative cerebral malaria in 
Malawian children. The Journal of infectious diseases 2012;206:309-318. 
52. 
Seydel KB, Kampondeni SD, Valim C, et al. Brain swelling and death in children with 
cerebral malaria. N Engl J Med 2015;372:1126-1137. 
53. 
Wolf-Gould C, Osei L, Commey JO, Bia FJ. Pediatric cerebral malaria in Accra, Ghana. 
Journal of tropical pediatrics 1992;38:290-294. 
54. 
Edington GM. Cerebral malaria in the Gold Coast African: four autopsy reports. Annals 
of tropical medicine and parasitology 1954;48:300-306. 
55. 
Attah EB, Ejeckam GC. Clinico-pathologic correlation in fatal malaria. Tropical and 
geographical medicine 1974;26:359-362. 
56. 
Oo MM, Aikawa M, Than T, et al. Human cerebral malaria: a pathological study. Journal 
of neuropathology and experimental neurology 1987;46:223-231. 
57. 
Aikawa M. Human cerebral malaria. The American journal of tropical medicine and 
hygiene 1988;39:3-10. 
58. 
Pongponratn E, Riganti M, Punpoowong B, Aikawa M. Microvascular sequestration of 
parasitized erythrocytes in human falciparum malaria: a pathological study. Am J Trop Med Hyg 
1991;44:168-175. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
110 
59. 
Riganti M, Pongponratn E, Tegoshi T, Looareesuwan S, Punpoowong B, Aikawa M. 
Human cerebral malaria in Thailand: a clinico-pathological correlation. Immunol Lett 
1990;25:199-205. 
60. 
Boonpucknavig V, Boonpucknavig S, Udomsangpetch R, Nitiyanant P. An 
immunofluorescence study of cerebral malaria. A correlation with histopathology. Archives of 
pathology & laboratory medicine 1990;114:1028-1034. 
61. 
Dorovini-Zis K, Schmidt K, Huynh H, et al. The neuropathology of fatal cerebral malaria 
in malawian children. Am J Pathol 2011;178:2146-2158. 
62. 
Porta J, Carota A, Pizzolato GP, et al. Immunopathological changes in human cerebral 
malaria. Clin Neuropathol 1993;12:142-146. 
63. 
Walker O, Salako LA, Sowunmi A, Thomas JO, Sodeine O, Bondi FS. Prognostic risk 
factors and post mortem findings in cerebral malaria in children. Transactions of the Royal 
Society of Tropical Medicine and Hygiene 1992;86:491-493. 
64. 
Millan JM, San Millan JM, Munoz M, Navas E, Lopez-Velez R. CNS complications in 
acute malaria: MR findings. AJNR American journal of neuroradiology 1993;14:493-494. 
65. 
Cordoliani YS, Sarrazin JL, Felten D, Caumes E, Leveque C, Fisch A. MR of cerebral 
malaria. AJNR Am J Neuroradiol 1998;19:871-874. 
66. 
Looareesuwan S, Wilairatana P, Krishna S, et al. Magnetic resonance imaging of the 
brain in patients with cerebral malaria. Clin Infect Dis 1995;21:300-309. 
67. 
Rasalkar DD, Paunipagar BK, Sanghvi D, Sonawane BD, Loniker P. Magnetic 
resonance imaging in cerebral malaria: a report of four cases.  The British journal of radiology. 
England2011: 380-385. 
68. 
Potchen M, Birbeck G, Demarco J, et al. Neuroimaging findings in children with 
retinopathy-confirmed cerebral malaria. Eur J Radiol 2009. 
69. 
Potchen MJ, Kampondeni SD, Seydel KB, et al. Acute brain MRI findings in 120 
Malawian children with cerebral malaria: new insights into an ancient disease. AJNR American 
journal of neuroradiology 2012;33:1740-1746. 
70. 
Newton CR, Taylor TE, Whitten RO. Pathophysiology of fatal falciparum malaria in 
African children. Am J Trop Med Hyg 1998;58:673-683. 
71. 
Postels DG, Li C, Birbeck GL, et al. Brain MRI of children with retinopathy-negative 
cerebral malaria. Am J Trop Med Hyg 2014;91:943-949. 
72. 
Aaslid R, Markwalder TM, Nornes H. Noninvasive transcranial Doppler ultrasound 
recording of flow velocity in basal cerebral arteries. J Neurosurg 1982;57:769-774. 
73. 
LaRovere KL, O'Brien NF. Transcranial Doppler Sonography in Pediatric Neurocritical 
Care: A Review of Clinical Applications and Case Illustrations in the Pediatric Intensive Care 
Unit. Journal of ultrasound in medicine : official journal of the American Institute of Ultrasound in 
Medicine 2015;34:2121-2132. 
74. 
D'Andrea A, Conte M, Scarafile R, et al. Transcranial Doppler Ultrasound: Physical 
Principles and Principal Applications in Neurocritical Care Unit. Journal of cardiovascular 
echography 2016;26:28-41. 
75. 
Lau VI, Arntfield RT. Point-of-care transcranial Doppler by intensivists. Critical 
ultrasound journal 2017;9:21. 
76. 
O'Brien NF, Mutatshi Taty T, Moore-Clingenpeel M, et al. Transcranial Doppler 
Ultrasonography Provides Insights into Neurovascular Changes in Children with Cerebral 
Malaria. J Pediatr 2018. 
77. 
Noriyuki T, Ohdan H, Yoshioka S, Miyata Y, Asahara T, Dohi K. Near-infrared 
spectroscopic method for assessing the tissue oxygenation state of living lung. American journal 
of respiratory and critical care medicine 1997;156:1656-1661. 
78. 
Kragelj R, Jarm T, Miklavcic D. Reproducibility of parameters of postocclusive reactive 
hyperemia measured by near infrared spectroscopy and transcutaneous oximetry. Annals of 
biomedical engineering 2000;28:168-173. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
111 
79. 
Kolyva C, Kingston H, Tachtsidis I, et al. Oscillations in cerebral haemodynamics in 
patients with falciparum malaria. Advances in experimental medicine and biology 2013;765:101-
107. 
80. 
Chen Z, Ivanov P, Hu K, Stanley HE. Effect of nonstationarities on detrended fluctuation 
analysis. Physical review E, Statistical, nonlinear, and soft matter physics 2002;65:041107. 
81. 
WHO. World Malaria Report. Geneva, Switzerland: World Health Organization, 2012. 
82. 
Earle M, Martinez Natera O, Zaslavsky A, et al. Outcome of pediatric intensive care at 
six centers in Mexico and Ecuador. Critical care medicine 1997;25:1462-1467. 
83. 
Taylor T. Caring for children with cerebral malaria: insights gleaned from 20 years on a 
research ward in Malawi. Trans R Soc Trop Med Hyg 2009;103 Suppl 1:S6-10. 
84. 
Murphy S, Cserti-Gazdewich C, Dhabangi A, et al. Ultrasound findings in Plasmodium 
falciparum malaria: a pilot study. Pediatr Crit Care Med 2011;12:e58-63. 
85. 
Hinson HE, Stein D, Sheth KN. Hypertonic Saline and Mannitol Therapy in Critical Care 
Neurology.  Journal of intensive care medicine2011. 
86. 
Unterberg AW, Stover J, Kress B, Kiening KL. Edema and brain trauma. Neuroscience 
2004;129:1021-1029. 
87. 
Newton CR, Kirkham FJ, Winstanley PA, et al. Intracranial pressure in African children 
with cerebral malaria. Lancet (London, England) 1991;337:573-576. 
88. 
Prohovnik I, Pavlakis SG, Piomelli S, et al. Cerebral hyperemia, stroke, and transfusion 
in sickle cell disease. Neurology 1989;39:344-348. 
89. 
Milner DA, Jr., Whitten RO, Kamiza S, et al. The systemic pathology of cerebral malaria 
in African children. Frontiers in cellular and infection microbiology 2014;4:104. 
90. 
Ponsford MJ, Medana IM, Prapansilp P, et al. Sequestration and microvascular 
congestion are associated with coma in human cerebral malaria. The Journal of infectious 
diseases 2012;205:663-671. 
91. 
Gerardin P, Rogier C, Ka AS, Jouvencel P, Diatta B, Imbert P. Outcome of life-
threatening malaria in African children requiring endotracheal intubation. Malaria journal 
2007;6:51. 
92. 
Kenning JA, Toutant SM, Saunders RL. Upright patient positioning in the management 
of intracranial hypertension. Surg Neurol 1981;15:148-152. 
93. 
Agbeko RS, Pearson S, Peters MJ, McNames J, Goldstein B. Intracranial pressure and 
cerebral perfusion pressure responses to head elevation changes in pediatric traumatic brain 
injury. Pediatr Crit Care Med 2011. 
94. 
Durward QJ, Amacher AL, Del Maestro RF, Sibbald WJ. Cerebral and cardiovascular 
responses to changes in head elevation in patients with intracranial hypertension. Journal of 
neurosurgery 1983;59:938-944. 
95. 
Feldman Z, Kanter MJ, Robertson CS, et al. Effect of head elevation on intracranial 
pressure, cerebral perfusion pressure, and cerebral blood flow in head-injured patients. J 
Neurosurg 1992;76:207-211. 
96. 
Meixensberger J, Baunach S, Amschler J, Dings J, Roosen K. Influence of body position 
on tissue-pO2, cerebral perfusion pressure and intracranial pressure in patients with acute brain 
injury. Neurol Res 1997;19:249-253. 
97. 
Ng I, Lim J, Wong HB. Effects of head posture on cerebral hemodynamics: its influences 
on intracranial pressure, cerebral perfusion pressure, and cerebral oxygenation. Neurosurgery 
2004;54:593-597; discussion 598. 
98. 
Rosner MJ, Coley IB. Cerebral perfusion pressure, intracranial pressure, and head 
elevation. J Neurosurg 1986;65:636-641. 
99. 
Winkelman C. Effect of backrest position on intracranial and cerebral perfusion 
pressures in traumatically brain-injured adults. Am J Crit Care 2000;9:373-380; quiz 381-372. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
112 
100. 
Fan JY. Effect of backrest position on intracranial pressure and cerebral perfusion 
pressure in individuals with brain injury: a systematic review. The Journal of neuroscience 
nursing : journal of the American Association of Neuroscience Nurses 2004;36:278-288. 
101. 
Wolfe TJ, Torbey MT. Management of intracranial pressure. Curr Neurol Neurosci Rep 
2009;9:477-485. 
102. 
French LA, Galicich JH. The use of steroids for control of cerebral edema. Clinical 
neurosurgery 1964;10:212-223. 
103. 
Jelsma R, Bucy PC. The treatment of glioblastoma multiforme of the brain. J Neurosurg 
1967;27:388-400. 
104. 
Kaal EC, Vecht CJ. The management of brain edema in brain tumors.  Curr Opin Oncol. 
United States2004: 593-600. 
105. 
Vecht CJ, Hovestadt A, Verbiest HB, van Vliet JJ, van Putten WL. Dose-effect 
relationship of dexamethasone on Karnofsky performance in metastatic brain tumors: a 
randomized study of doses of 4, 8, and 16 mg per day. Neurology 1994;44:675-680. 
106. 
Ambrogio AG, Pecori Giraldi F, Cavagnini F. Drugs and HPA axis. Pituitary 2008;11:219-
229. 
107. 
van de Beek D, Farrar JJ, de Gans J, et al. Adjunctive dexamethasone in bacterial 
meningitis: a meta-analysis of individual patient data.  The Lancet Neurology. England: 2010 
Elsevier Ltd, 2010: 254-263. 
108. 
Prasad K, Garner P. Steroids for treating cerebral malaria. Cochrane Database Syst Rev 
2000:CD000972. 
109. 
Bartsch P, Swenson ER. Clinical practice: Acute high-altitude illnesses. The New 
England journal of medicine 2013;368:2294-2302. 
110. 
Roth P, Regli L, Tonder M, Weller M. Tumor-associated edema in brain cancer patients: 
pathogenesis and management. Expert review of anticancer therapy 2013;13:1319-1325. 
111. 
Sandercock PA, Soane T. Corticosteroids for acute ischaemic stroke. The Cochrane 
database of systematic reviews 2011:Cd000064. 
112. 
Rhodes JK. Actions of glucocorticoids and related molecules after traumatic brain injury. 
Current opinion in critical care 2003;9:86-91. 
113. 
Koenig MA. Brain resuscitation and prognosis after cardiac arrest. Critical care clinics 
2014;30:765-783. 
114. 
Mak CH, Lu YY, Wong GK. Review and recommendations on management of refractory 
raised intracranial pressure in aneurysmal subarachnoid hemorrhage. Vascular health and risk 
management 2013;9:353-359. 
115. 
Abraham R. Steroids in neuroinfection. Arquivos de neuro-psiquiatria 2013;71:717-721. 
116. 
Brouwer MC, McIntyre P, Prasad K, van de Beek D. Corticosteroids for acute bacterial 
meningitis. The Cochrane database of systematic reviews 2013;6:Cd004405. 
117. 
Diringer MN, Zazulia AR. Osmotic therapy: fact and fiction.  Neurocritical care. United 
States2004: 219-233. 
118. 
Ogden AT, Mayer SA, Connolly ES. Hyperosmolar agents in neurosurgical practice: the 
evolving role of hypertonic saline. Neurosurgery 2005;57:207-215; discussion 207-215. 
119. 
White H, Cook D, Venkatesh B. The use of hypertonic saline for treating intracranial 
hypertension after traumatic brain injury. Anesth Analg 2006;102:1836-1846. 
120. 
Burke AM, Quest DO, Chien S, Cerri C. The effects of mannitol on blood viscosity. J 
Neurosurg 1981;55:550-553. 
121. 
Muizelaar JP, Lutz HA, Becker DP. Effect of mannitol on ICP and CBF and correlation 
with pressure autoregulation in severely head-injured patients. J Neurosurg 1984;61:700-706. 
122. 
Warren SE, Blantz RC. Mannitol. Arch Intern Med 1981;141:493-497. 
123. 
Paczynski RP. Osmotherapy. Basic concepts and controversies. Crit Care Clin 
1997;13:105-129. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
113 
124. 
Qureshi AI, Suarez JI. Use of hypertonic saline solutions in treatment of cerebral edema 
and intracranial hypertension. Crit Care Med 2000;28:3301-3313. 
125. 
Qureshi AI, Suarez JI, Bhardwaj A, et al. Use of hypertonic (3%) saline/acetate infusion 
in the treatment of cerebral edema: Effect on intracranial pressure and lateral displacement of 
the brain. Crit Care Med 1998;26:440-446. 
126. 
Whelan TV, Bacon ME, Madden M, Patel TG, Handy R. Acute renal failure associated 
with mannitol intoxication. Report of a case. Arch Intern Med 1984;144:2053-2055. 
127. 
Dorman HR, Sondheimer JH, Cadnapaphornchai P. Mannitol-induced acute renal 
failure. Medicine (Baltimore) 1990;69:153-159. 
128. 
Rothe H. 100 cases of cerebral malaria. East African medical journal 1956;33:405-407. 
129. 
Kingston ME. Experience with urea in invert sugar for the treatment of cerebral malaria. 
J Trop Med Hyg 1971;74:249-252. 
130. 
Okoromah CA, Afolabi BB, Wall EC. Mannitol and other osmotic diuretics as adjuncts for 
treating cerebral malaria. Cochrane Database Syst Rev 2011:CD004615. 
131. 
Mahoney BD, Ruiz E. Acute resuscitation of the patient with head and spinal cord 
injuries. Emergency medicine clinics of North America 1983;1:583-594. 
132. 
Zornow MH, Prough DS. Fluid management in patients with traumatic brain injury. New 
horizons (Baltimore, Md) 1995;3:488-498. 
133. 
Ziai WC, Toung TJ, Bhardwaj A. Hypertonic saline: first-line therapy for cerebral edema? 
Journal of the neurological sciences 2007;261:157-166. 
134. 
Vassar MJ, Fischer RP, O'Brien PE, et al. A multicenter trial for resuscitation of injured 
patients with 7.5% sodium chloride. The effect of added dextran 70. The Multicenter Group for 
the Study of Hypertonic Saline in Trauma Patients. Arch Surg 1993;128:1003-1011; discussion 
1011-1003. 
135. 
Wade CE, Grady JJ, Kramer GC, Younes RN, Gehlsen K, Holcroft JW. Individual patient 
cohort analysis of the efficacy of hypertonic saline/dextran in patients with traumatic brain injury 
and hypotension. J Trauma 1997;42:S61-65. 
136. 
Prough DS, Whitley JM, Taylor CL, Deal DD, DeWitt DS. Regional cerebral blood flow 
following resuscitation from hemorrhagic shock with hypertonic saline. Influence of a subdural 
mass. Anesthesiology 1991;75:319-327. 
137. 
Schmoker JD, Zhuang J, Shackford SR. Hypertonic fluid resuscitation improves cerebral 
oxygen delivery and reduces intracranial pressure after hemorrhagic shock. J Trauma 
1991;31:1607-1613. 
138. 
Ziai WC, Toung TJ, Bhardwaj A. Hypertonic saline: first-line therapy for cerebral edema?  
Journal of the neurological sciences. Netherlands2007: 157-166. 
139. 
Koenig MA, Bryan M, Lewin JL, Mirski MA, Geocadin RG, Stevens RD. Reversal of 
transtentorial herniation with hypertonic saline. Neurology 2008;70:1023-1029. 
140. 
Badaut J, Ashwal S, Obenaus A. Aquaporins in cerebrovascular disease: a target for 
treatment of brain edema? Cerebrovascular diseases (Basel, Switzerland) 2011;31:521-531. 
141. 
Cao C, Yu X, Liao Z, et al. Hypertonic saline reduces lipopolysaccharide-induced mouse 
brain edema through inhibiting aquaporin 4 expression. Critical care (London, England) 
2012;16:R186. 
142. 
Wang WW, Xie CL, Zhou LL, Wang GS. The function of aquaporin4 in ischemic brain 
edema. Clinical neurology and neurosurgery 2014;127:5-9. 
143. 
Zelenina M. Regulation of brain aquaporins. Neurochemistry international 2010;57:468-
488. 
144. 
Nielsen S, Nagelhus EA, Amiry-Moghaddam M, Bourque C, Agre P, Ottersen OP. 
Specialized membrane domains for water transport in glial cells: high-resolution immunogold 
cytochemistry of aquaporin-4 in rat brain. The Journal of neuroscience : the official journal of the 
Society for Neuroscience 1997;17:171-180. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
114 
145. 
Verkman AS, Ratelade J, Rossi A, Zhang H, Tradtrantip L. Aquaporin-4: orthogonal 
array assembly, CNS functions, and role in neuromyelitis optica. Acta pharmacologica Sinica 
2011;32:702-710. 
146. 
Deitch EA, Shi HP, Feketeova E, Hauser CJ, Xu DZ. Hypertonic saline resuscitation 
limits neutrophil activation after trauma-hemorrhagic shock. Shock (Augusta, Ga) 2003;19:328-
333. 
147. 
Hartl R, Medary MB, Ruge M, Arfors KE, Ghahremani F, Ghajar J. 
Hypertonic/hyperoncotic saline attenuates microcirculatory disturbances after traumatic brain 
injury. The Journal of trauma 1997;42:S41-47. 
148. 
Huang LQ, Zhu GF, Deng YY, et al. Hypertonic saline alleviates cerebral edema by 
inhibiting microglia-derived TNF-alpha and IL-1beta-induced Na-K-Cl Cotransporter up-
regulation. Journal of neuroinflammation 2014;11:102. 
149. 
Pascual JL, Ferri LE, Seely AJ, et al. Hypertonic saline resuscitation of hemorrhagic 
shock diminishes neutrophil rolling and adherence to endothelium and reduces in vivo vascular 
leakage. Annals of surgery 2002;236:634-642. 
150. 
Pascual JL, Khwaja KA, Ferri LE, et al. Hypertonic saline resuscitation attenuates 
neutrophil lung sequestration and transmigration by diminishing leukocyte-endothelial 
interactions in a two-hit model of hemorrhagic shock and infection. The Journal of trauma 
2003;54:121-130; discussion 130-122. 
151. 
Howland SW, Poh CM, Gun SY, et al. Brain microvessel cross-presentation is a 
hallmark of experimental cerebral malaria. EMBO molecular medicine 2013;5:916-931. 
152. 
Nacer A, Movila A, Baer K, Mikolajczak SA, Kappe SH, Frevert U. Neuroimmunological 
blood brain barrier opening in experimental cerebral malaria. PLoS pathogens 
2012;8:e1002982. 
153. 
Nacer A, Movila A, Sohet F, et al. Experimental cerebral malaria pathogenesis--
hemodynamics at the blood brain barrier. PLoS pathogens 2014;10:e1004528. 
154. 
Pai S, Qin J, Cavanagh L, et al. Real-time imaging reveals the dynamics of leukocyte 
behaviour during experimental cerebral malaria pathogenesis. PLoS pathogens 
2014;10:e1004236. 
155. 
Khanna S, Davis D, Peterson B, et al. Use of hypertonic saline in the treatment of severe 
refractory posttraumatic intracranial hypertension in pediatric traumatic brain injury. Critical care 
medicine 2000;28:1144-1151. 
156. 
Peterson B, Khanna S, Fisher B, Marshall L. Prolonged hypernatremia controls elevated 
intracranial pressure in head-injured pediatric patients. Critical care medicine 2000;28:1136-
1143. 
157. 
Kamel H, Navi BB, Nakagawa K, Hemphill JC, Ko NU. Hypertonic saline versus mannitol 
for the treatment of elevated intracranial pressure: a meta-analysis of randomized clinical trials. 
Crit Care Med 2011;39:554-559. 
158. 
Mendelow AD, Teasdale GM, Russell T, Flood J, Patterson J, Murray GD. Effect of 
mannitol on cerebral blood flow and cerebral perfusion pressure in human head injury. J 
Neurosurg 1985;63:43-48. 
159. 
Alvarez B, Ferrer-Sueta G, Radi R. Slowing of peroxynitrite decomposition in the 
presence of mannitol and ethanol. Free Radic Biol Med 1998;24:1331-1337. 
160. 
Korenkov AI, Pahnke J, Frei K, et al. Treatment with nimodipine or mannitol reduces 
programmed cell death and infarct size following focal cerebral ischemia. Neurosurg Rev 
2000;23:145-150. 
161. 
Doyle JA, Davis DP, Hoyt DB. The use of hypertonic saline in the treatment of traumatic 
brain injury. J Trauma 2001;50:367-383. 
162. 
Vialet R, Albanese J, Thomachot L, et al. Isovolume hypertonic solutes (sodium chloride 
or mannitol) in the treatment of refractory posttraumatic intracranial hypertension: 2 mL/kg 7.5% 
saline is more effective than 2 mL/kg 20% mannitol. Critical care medicine 2003;31:1683-1687. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
115 
163. 
Cottenceau V, Masson F, Mahamid E, et al. Comparison of effects of equiosmolar doses 
of mannitol and hypertonic saline on cerebral blood flow and metabolism in traumatic brain 
injury. Journal of neurotrauma 2011;28:2003-2012. 
164. 
Schwarz S, Schwab S, Bertram M, Aschoff A, Hacke W. Effects of hypertonic saline 
hydroxyethyl starch solution and mannitol in patients with increased intracranial pressure after 
stroke. Stroke 1998;29:1550-1555. 
165. 
Battison C, Andrews PJ, Graham C, Petty T. Randomized, controlled trial on the effect of 
a 20% mannitol solution and a 7.5% saline/6% dextran solution on increased intracranial 
pressure after brain injury. Crit Care Med 2005;33:196-202; discussion 257-198. 
166. 
Ichai C, Armando G, Orban JC, et al. Sodium lactate versus mannitol in the treatment of 
intracranial hypertensive episodes in severe traumatic brain-injured patients. Intensive care 
medicine 2009;35:471-479. 
167. 
Francony G, Fauvage B, Falcon D, et al. Equimolar doses of mannitol and hypertonic 
saline in the treatment of increased intracranial pressure. Critical care medicine 2008;36:795-
800. 
168. 
Horn P, Münch E, Vajkoczy P, et al. Hypertonic saline solution for control of elevated 
intracranial pressure in patients with exhausted response to mannitol and barbiturates. Neurol 
Res 1999;21:758-764. 
169. 
Kerwin AJ, Schinco MA, Tepas JJ, Renfro WH, Vitarbo EA, Muehlberger M. The use of 
23.4% hypertonic saline for the management of elevated intracranial pressure in patients with 
severe traumatic brain injury: a pilot study. J Trauma 2009;67:277-282. 
170. 
Oddo M, Levine JM, Frangos S, et al. Effect of mannitol and hypertonic saline on 
cerebral oxygenation in patients with severe traumatic brain injury and refractory intracranial 
hypertension. J Neurol Neurosurg Psychiatry 2009;80:916-920. 
171. 
Schwarz S, Georgiadis D, Aschoff A, Schwab S. Effects of hypertonic (10%) saline in 
patients with raised intracranial pressure after stroke. Stroke 2002;33:136-140. 
172. 
Suarez JI, Qureshi AI, Bhardwaj A, et al. Treatment of refractory intracranial 
hypertension with 23.4% saline. Crit Care Med 1998;26:1118-1122. 
173. 
Schatzmann C, Heissler HE, König K, et al. Treatment of elevated intracranial pressure 
by infusions of 10% saline in severely head injured patients. Acta Neurochir Suppl 1998;71:31-
33. 
174. 
Gwer S, Gatakaa H, Mwai L, Idro R, Newton CR. The role for osmotic agents in children 
with acute encephalopathies: a systematic review.  BMC pediatrics. England2010: 23. 
175. 
Fisher B, Thomas D, Peterson B. Hypertonic saline lowers raised intracranial pressure in 
children after head trauma. J Neurosurg Anesthesiol 1992;4:4-10. 
176. 
Peltola H, Roine I, Fernández J, et al. Adjuvant glycerol and/or dexamethasone to 
improve the outcomes of childhood bacterial meningitis: a prospective, randomized, double-
blind, placebo-controlled trial. Clin Infect Dis 2007;45:1277-1286. 
177. 
Simma B, Burger R, Falk M, Sacher P, Fanconi S. A prospective, randomized, and 
controlled study of fluid management in children with severe head injury: lactated Ringer's 
solution versus hypertonic saline. Critical care medicine 1998;26:1265-1270. 
178. 
Wald SL, McLaurin RL. Oral glycerol for the treatment of traumatic intracranial 
hypertension. J Neurosurg 1982;56:323-331. 
179. 
Yildizdas D, Altunbasak S, Celik U, Herguner O. Hypertonic saline treatment in children 
with cerebral edema. Indian Pediatr 2006;43:771-779. 
180. 
Berger S, Schwarz M, Huth R. Hypertonic saline solution and decompressive 
craniectomy for treatment of intracranial hypertension in pediatric severe traumatic brain injury. 
J Trauma 2002;53:558-563. 
181. 
Newton CR, Kirkham FJ, Winstanley PA, et al. Intracranial pressure in African children 
with cerebral malaria. Lancet 1991;337:573-576. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
116 
182. 
Koenig MA, Bryan M, Lewin JL, Mirski MA, Geocadin RG, Stevens RD. Reversal of 
transtentorial herniation with hypertonic saline. Neurology 2008;70:1023-1029. 
183. 
Doyle JA, Davis DP, Hoyt DB. The use of hypertonic saline in the treatment of traumatic 
brain injury. The Journal of trauma 2001;50:367-383. 
184. 
Korenkov AI, Pahnke J, Frei K, et al. Treatment with nimodipine or mannitol reduces 
programmed cell death and infarct size following focal cerebral ischemia. Neurosurgical review 
2000;23:145-150. 
185. 
Oddo M, Levine JM, Frangos S, et al. Effect of mannitol and hypertonic saline on 
cerebral oxygenation in patients with severe traumatic brain injury and refractory intracranial 
hypertension. Journal of neurology, neurosurgery, and psychiatry 2009;80:916-920. 
186. 
Schatzmann C, Heissler HE, Konig K, et al. Treatment of elevated intracranial pressure 
by infusions of 10% saline in severely head injured patients. Acta neurochirurgica Supplement 
1998;71:31-33. 
187. 
Suarez JI, Qureshi AI, Bhardwaj A, et al. Treatment of refractory intracranial 
hypertension with 23.4% saline. Critical care medicine 1998;26:1118-1122. 
188. 
Pineda JA, Leonard JR, Mazotas IG, et al. Effect of implementation of a paediatric 
neurocritical care programme on outcomes after severe traumatic brain injury: a retrospective 
cohort study. The Lancet Neurology 2013;12:45-52. 
189. 
Maitland K, Pamba A, Fegan G, et al. Perturbations in electrolyte levels in kenyan 
children with severe malaria complicated by acidosis. Clin Infect Dis 2005;40:9-16. 
190. 
Upadhyay P, Tripathi VN, Singh RP, Sachan D. Role of hypertonic saline and mannitol 
in the management of raised intracranial pressure in children: A randomized comparative study. 
Journal of pediatric neurosciences 2010;5:18-21. 
191. 
Ayus JC, Caputo D, Bazerque F, Heguilen R, Gonzalez CD, Moritz ML. Treatment of 
hyponatremic encephalopathy with a 3% sodium chloride protocol: a case series. American 
journal of kidney diseases : the official journal of the National Kidney Foundation 2015;65:435-
442. 
192. 
Luu JL, Wendtland CL, Gross MF, et al. Three-percent saline administration during 
pediatric critical care transport. Pediatric emergency care 2011;27:1113-1117. 
193. 
Shann F. Role of intensive care in countries with a high child mortality rate. Pediatr Crit 
Care Med 2011;12:114-115. 
194. 
Helbok R, Olson DM, Le Roux PD, Vespa P. Intracranial pressure and cerebral 
perfusion pressure monitoring in non-TBI patients: special considerations. Neurocritical care 
2014;21 Suppl 2:S85-94. 
195. 
Schleien CL. Intracranial pressure: A role for a surrogate measurement? Pediatric critical 
care medicine : a journal of the Society of Critical Care Medicine and the World Federation of 
Pediatric Intensive and Critical Care Societies 2010;11:636-637. 
196. 
Tayal VS, Neulander M, Norton HJ, Foster T, Saunders T, Blaivas M. Emergency 
department sonographic measurement of optic nerve sheath diameter to detect findings of 
increased intracranial pressure in adult head injury patients.  Ann Emerg Med. United 
States2007: 508-514. 
197. 
Girisgin AS, Kalkan E, Kocak S, Cander B, Gul M, Semiz M. The role of optic nerve 
ultrasonography in the diagnosis of elevated intracranial pressure.  Emerg Med J. England2007: 
251-254. 
198. 
Girisgin AS, Kalkan E, Kocak S, Cander B, Gul M, Semiz M. The role of optic nerve 
ultrasonography in the diagnosis of elevated intracranial pressure. Emerg Med J 2007;24:251-
254. 
199. 
Karakitsos D, Soldatos T, Gouliamos A, et al. Transorbital sonographic monitoring of 
optic nerve diameter in patients with severe brain injury. Transplant Proc 2006;38:3700-3706. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
117 
200. 
Blaivas M, Theodoro D, Sierzenski PR. Elevated intracranial pressure detected by 
bedside emergency ultrasonography of the optic nerve sheath. Academic emergency medicine : 
official journal of the Society for Academic Emergency Medicine 2003;10:376-381. 
201. 
Beare NA, Kampondeni S, Glover SJ, et al. Detection of raised intracranial pressure by 
ultrasound measurement of optic nerve sheath diameter in African children. Trop Med Int Health 
2008;13:1400-1404. 
202. 
Beare NA, Glover SJ, Lewallen S, Taylor TE, Harding SP, Molyneux ME. Prevalence of 
Raised Intracranial Pressure in Cerebral Malaria Detected by Optic Nerve Sheath Ultrasound. 
Am J Trop Med Hyg 2012. 
203. 
Murphy S, Cserti-Gazdewich C, Dhabangi A, et al. Ultrasound findings in Plasmodium 
falciparum malaria: a pilot study. Pediatr Crit Care Med 2011;12:e58-63. 
204. 
Robba C, Bacigaluppi S, Cardim D, Donnelly J, Bertuccio A, Czosnyka M. Non-invasive 
assessment of intracranial pressure. Acta Neurol Scand 2016;134:4-21. 
205. 
Melo JR, Di Rocco F, Blanot S, et al. Transcranial Doppler can predict intracranial 
hypertension in children with severe traumatic brain injuries. Child's nervous system : ChNS : 
official journal of the International Society for Pediatric Neurosurgery 2011;27:979-984. 
206. 
Bellner J, Romner B, Reinstrup P, Kristiansson KA, Ryding E, Brandt L. Transcranial 
Doppler sonography pulsatility index (PI) reflects intracranial pressure (ICP). Surgical neurology 
2004;62:45-51; discussion 51. 
207. 
O'Brien NF, Maa T, Reuter-Rice K. Noninvasive screening for intracranial hypertension 
in children with acute, severe traumatic brain injury. Journal of neurosurgery Pediatrics 
2015;16:420-425. 
208. 
Cardim D, Czosnyka M, Donnelly J, et al. Assessment of non-invasive ICP during CSF 
infusion test: an approach with transcranial Doppler. Acta neurochirurgica 2016;158:279-287; 
discussion 287. 
209. 
Goh D, Minns RA, Hendry GM, Thambyayah M, Steers AJ. Cerebrovascular resistive 
index assessed by duplex Doppler sonography and its relationship to intracranial pressure in 
infantile hydrocephalus. Pediatr Radiol 1992;22:246-250. 
210. 
Goh D, Minns RA, Pye SD, Steers AJ. Cerebral blood flow velocity changes after 
ventricular taps and ventriculoperitoneal shunting. Child's nervous system : ChNS : official 
journal of the International Society for Pediatric Neurosurgery 1991;7:452-457. 
211. 
Maertzdorf WJ, Vles JS, Beuls E, Mulder AL, Blanco CE. Intracranial pressure and 
cerebral blood flow velocity in preterm infants with posthaemorrhagic ventricular dilatation. Arch 
Dis Child Fetal Neonatal Ed 2002;87:F185-188. 
212. 
Buchman TG, Stein PK, Goldstein B. Heart rate variability in critical illness and critical 
care. Current opinion in critical care 2002;8:311-315. 
213. 
Giacoppo S, Bramanti P, Barresi M, et al. Predictive biomarkers of recovery in traumatic 
brain injury. Neurocritical care 2012;16:470-477. 
214. 
Donato R. S100: a multigenic family of calcium-modulated proteins of the EF-hand type 
with intracellular and extracellular functional roles. The international journal of biochemistry & 
cell biology 2001;33:637-668. 
215. 
Marangos PJ, Schmechel DE. Neuron specific enolase, a clinically useful marker for 
neurons and neuroendocrine cells. Annual review of neuroscience 1987;10:269-295. 
216. 
Bouvier D, Fournier M, Dauphin JB, et al. Serum S100B determination in the 
management of pediatric mild traumatic brain injury. Clinical chemistry 2012;58:1116-1122. 
217. 
Bechtel K, Frasure S, Marshall C, Dziura J, Simpson C. Relationship of serum S100B 
levels and intracranial injury in children with closed head trauma. Pediatrics 2009;124:e697-704. 
218. 
Berger RP, Dulani T, Adelson PD, Leventhal JM, Richichi R, Kochanek PM. 
Identification of inflicted traumatic brain injury in well-appearing infants using serum and 
cerebrospinal markers: a possible screening tool. Pediatrics 2006;117:325-332. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
118 
219. 
Filippidis AS, Papadopoulos DC, Kapsalaki EZ, Fountas KN. Role of the S100B serum 
biomarker in the treatment of children suffering from mild traumatic brain injury. Neurosurgical 
focus 2010;29:E2. 
220. 
Day IN, Thompson RJ. UCHL1 (PGP 9.5): neuronal biomarker and ubiquitin system 
protein. Progress in neurobiology 2010;90:327-362. 
221. 
Papa L, Akinyi L, Liu MC, et al. Ubiquitin C-terminal hydrolase is a novel biomarker in 
humans for severe traumatic brain injury. Critical care medicine 2010;38:138-144. 
222. 
Papa L, Lewis LM, Silvestri S, et al. Serum levels of ubiquitin C-terminal hydrolase 
distinguish mild traumatic brain injury from trauma controls and are elevated in mild and 
moderate traumatic brain injury patients with intracranial lesions and neurosurgical intervention. 
J Trauma Acute Care Surg 2012;72:1335-1344. 
223. 
Mondello S, Linnet A, Buki A, et al. Clinical utility of serum levels of ubiquitin C-terminal 
hydrolase as a biomarker for severe traumatic brain injury. Neurosurgery 2012;70:666-675. 
224. 
Brophy GM, Mondello S, Papa L, et al. Biokinetic analysis of ubiquitin C-terminal 
hydrolase-L1 (UCH-L1) in severe traumatic brain injury patient biofluids. J Neurotrauma 
2011;28:861-870. 
225. 
Berger RP, Hayes RL, Richichi R, Beers SR, Wang KK. Serum concentrations of 
ubiquitin C-terminal hydrolase-L1 and alphaII-spectrin breakdown product 145 kDa correlate 
with outcome after pediatric TBI. J Neurotrauma 2012;29:162-167. 
226. 
Puvenna V, Brennan C, Shaw G, et al. Significance of ubiquitin carboxy-terminal 
hydrolase l1 elevations in athletes after sub-concussive head hits. PloS one 2014;9:e96296. 
227. 
Diaz-Arrastia R, Wang KK, Papa L, et al. Acute biomarkers of traumatic brain injury: 
relationship between plasma levels of ubiquitin C-terminal hydrolase-L1 and glial fibrillary acidic 
protein. J Neurotrauma 2014;31:19-25. 
228. 
Liu MC, Akinyi L, Scharf D, et al. Ubiquitin C-terminal hydrolase-L1 as a biomarker for 
ischemic and traumatic brain injury in rats. Eur J Neurosci 2010;31:722-732. 
229. 
Eng LF. Glial fibrillary acidic protein (GFAP): the major protein of glial intermediate 
filaments in differentiated astrocytes. J Neuroimmunol 1985;8:203-214. 
230. 
Pelinka LE, Kroepfl A, Leixnering M, Buchinger W, Raabe A, Redl H. GFAP versus 
S100B in serum after traumatic brain injury: relationship to brain damage and outcome. J 
Neurotrauma 2004;21:1553-1561. 
231. 
Pelinka LE, Kroepfl A, Schmidhammer R, et al. Glial fibrillary acidic protein in serum 
after traumatic brain injury and multiple trauma. J Trauma 2004;57:1006-1012. 
232. 
McMahon PJ, Panczykowski DM, Yue JK, et al. Measurement of the glial fibrillary acidic 
protein and its breakdown products GFAP-BDP biomarker for the detection of traumatic brain 
injury compared to computed tomography and magnetic resonance imaging. J Neurotrauma 
2015;32:527-533. 
233. 
Mondello S, Muller U, Jeromin A, Streeter J, Hayes RL, Wang KK. Blood-based 
diagnostics of traumatic brain injuries. Expert Rev Mol Diagn 2010;11:65-78. 
234. 
Papa L, Akinyi L, Liu MC, et al. Ubiquitin C-terminal hydrolase is a novel biomarker in 
humans for severe traumatic brain injury. Critical care medicine 2009;38:138-144. 
235. 
Papa L, Lewis LM, Falk JL, et al. Elevated levels of serum glial fibrillary acidic protein 
breakdown products in mild and moderate traumatic brain injury are associated with intracranial 
lesions and neurosurgical intervention. Ann Emerg Med 2011;59:471-483. 
236. 
Mondello S, Jeromin A, Buki A, et al. Glial neuronal ratio: a novel index for differentiating 
injury type in patients with severe traumatic brain injury. J Neurotrauma 2011;29:1096-1104. 
237. 
Czeiter E, Mondello S, Kovacs N, et al. Brain injury biomarkers may improve the 
predictive power of the IMPACT outcome calculator. J Neurotrauma 2012;29:1770-1778. 
238. 
Mannix R, Eisenberg M, Berry M, Meehan W, Hayes RL. Serum Biomarkers Predict 
Acute Symptom Burden in Children after Concussion: A Preliminary Study. J Neurotrauma 
2014. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
119 
239. 
Fraser DD, Close TE, Rose KL, et al. Severe traumatic brain injury in children elevates 
glial fibrillary acidic protein in cerebrospinal fluid and serum. Pediatric critical care medicine : a 
journal of the Society of Critical Care Medicine and the World Federation of Pediatric Intensive 
and Critical Care Societies 2011;12:319-324. 
240. 
Hayes RL, Mondello S, Wang K. Glial fibrillary acidic protein: a promising biomarker in 
pediatric brain injury. Pediatric critical care medicine : a journal of the Society of Critical Care 
Medicine and the World Federation of Pediatric Intensive and Critical Care Societies 
2011;12:603-604. 
241. 
Nylen K, Ost M, Csajbok LZ, et al. Increased serum-GFAP in patients with severe 
traumatic brain injury is related to outcome. Journal of the neurological sciences 2006;240:85-
91. 
242. 
Papa L, Lewis LM, Falk JL, et al. Elevated levels of serum glial fibrillary acidic protein 
breakdown products in mild and moderate traumatic brain injury are associated with intracranial 
lesions and neurosurgical intervention. Ann Emerg Med 2012;59:471-483. 
243. 
Honda M, Tsuruta R, Kaneko T, et al. Serum glial fibrillary acidic protein is a highly 
specific biomarker for traumatic brain injury in humans compared with S-100B and neuron-
specific enolase. J Trauma 2010;69:104-109. 
244. 
Lumpkins KM, Bochicchio GV, Keledjian K, Simard JM, McCunn M, Scalea T. Glial 
fibrillary acidic protein is highly correlated with brain injury. J Trauma 2008;65:778-782; 
discussion 782-774. 
245. 
Stein DM, Lindell AL, Murdock KR, et al. Use of serum biomarkers to predict cerebral 
hypoxia after severe traumatic brain injury. J Neurotrauma 2012;29:1140-1149. 
246. 
Bezzant TB, Mortensen JD. Risks and hazards of mechanical ventilation: a collective 
review of published literature. Disease-a-month : DM 1994;40:581-638. 
247. 
Jennett B, Bond M. Assessment of outcome after severe brain damage. Lancet (London, 
England) 1975;1:480-484. 
248. 
Bangirana P, Seggane-Musisi, Allebeck P, et al. A preliminary examination of the 
construct validity of the KABC-II in Ugandan children with a history of cerebral malaria. Afr 
Health Sci 2009;9:186-192. 
249. 
Gladstone M, Lancaster GA, Umar E, et al. The Malawi Developmental Assessment 
Tool (MDAT): the creation, validation, and reliability of a tool to assess child development in 
rural African settings. PLoS Med 2010;7:e1000273. 
250. 
Qureshi AI, Suarez JI, Bhardwaj A, et al. Use of hypertonic (3%) saline/acetate infusion 
in the treatment of cerebral edema: Effect on intracranial pressure and lateral displacement of 
the brain. Critical care medicine 1998;26:440-446. 
251. 
Qureshi AI, Suarez JI, Castro A, Bhardwaj A. Use of hypertonic saline/acetate infusion in 
treatment of cerebral edema in patients with head trauma: experience at a single center. The 
Journal of trauma 1999;47:659-665. 
252. 
Jones PA, Andrews PJ, Midgley S, et al. Measuring the burden of secondary insults in 
head-injured patients during intensive care. J Neurosurg Anesthesiol 1994;6:4-14. 
253. 
Bar-Joseph G, Guilburd Y, Tamir A, Guilburd JN. Effectiveness of ketamine in 
decreasing intracranial pressure in children with intracranial hypertension. Journal of 
neurosurgery Pediatrics 2009;4:40-46. 
254. 
Figaji AA, Zwane E, Thompson C, et al. Brain tissue oxygen tension monitoring in 
pediatric severe traumatic brain injury. Part 1: Relationship with outcome. Child's nervous 
system : ChNS : official journal of the International Society for Pediatric Neurosurgery 
2009;25:1325-1333. 
255. 
Pigula FA, Wald SL, Shackford SR, Vane DW. The effect of hypotension and hypoxia on 
children with severe head injuries.  Journal of pediatric surgery. United States1993: 310-314; 
discussion 315-316. 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
120 
256. 
Price DJ, Murray A. The influence of hypoxia and hypotension on recovery from head 
injury. Injury 1972;3:218-224. 
257. 
Tayal VS, Neulander M, Norton HJ, Foster T, Saunders T, Blaivas M. Emergency 
department sonographic measurement of optic nerve sheath diameter to detect findings of 
increased intracranial pressure in adult head injury patients. Annals of emergency medicine 
2007;49:508-514. 
258. 
Birbeck GL, Molyneux ME, Kaplan PW, et al. Blantyre Malaria Project Epilepsy Study 
(BMPES) of neurological outcomes in retinopathy-positive paediatric cerebral malaria survivors: 
a prospective cohort study. Lancet Neurol 2010;9:1173-1181. 
259. 
Altman DG. Comparability of randomised groups. The Statistician 1985;34:125-136. 
260. 
Duan N. Smearing Estimate - a Nonparametric Retransformation Method. J Am Stat 
Assoc 1983;78:605-610. 
261. 
Welsh AH, Zhou XH. Estimating the retransformed mean in a heteroscedastic two-part 
model. Journal of Statistical Planning and Inference 2006;136:860-881. 
262. 
Luo Z, Gardiner JC, Yang N. Estimation of mean response in selected samples with 
endogenous variables. Journal of Statistics & Applications 2008;3:217-238. 
263. 
Cui J. QIC program and model selection in GEE analyses. Stata Journal 2007;7:209-
220. 
264. 
Cui J, Qian G. Selection of working correlation structure and best model in GEE 
analyses of longitudinal data. Communications in Statistics-Simulation and Computation 
2007;36:987-996. 
265. 
Vaida F, Blanchard S. Conditional Akaike information for mixed-effects models. 
Biometrika 2005;92:351-370. 
266. 
Diggle PJ, Heagerty PK, Liang KY, Zeger SL. Analysis of Longitudinal Data, 2nd Edition. 
New York, NY: Oxford University Press, 2002. 
267. 
Gardiner JC, Luo Z. Logit Models in Practice: B, C, E, G, M, N, O, ...Paper 341-2011.  
SAS Global Forum, 2011; 2011; Las Vegas, NV: SAS Institute Inc. 
268. 
Peterson B, Harrell FE. PARTIAL PROPORTIONAL ODDS MODELS FOR ORDINAL 
RESPONSE VARIABLES. Applied Statistics-Journal of the Royal Statistical Society Series C 
1990;39:205-217. 
269. 
Little RJA, Rubin DB. Statistical Analysis with Missing Data, 2nd ed. New York: John 
Wiley & Sons, Inc, 2002. 
270. 
Wooldridge JM. Econometric Analysis of Cross Section and Panel Data, Second Edition. 
Cambridge, MA: MIT Press, 2010. 
271. 
Wooldridge JM. Inverse probability weighted estimation for general missing data 
problems. Journal of Econometrics 2007;141:1281-1301. 
272. 
Robins JM, Rotnitzky A, Zhao LP. Analysis of Semiparametric Regression-Models for 
Repeated Outcomes in the Presence of Missing Data. J Am Stat Assoc 1995;90:106-121. 
273. 
Dmitrienko A, Molenberghs G, Chuang-Stein C, Offen W. Analysis of Clinical Trials 
Using SAS : A Practical Guide. Cary, NC: SAS Institute Inc, 2005. 
274. 
Gardiner JC, Luo Z, Bradley CJ, Sirbu CM, Given CW. A dynamic model for estimating 
changes in health status and costs. Statistics in Medicine 2006;25:3648-3667. 
275. 
Baser O, Gardiner JC, Bradley CJ, Yuce H, Given C. Longitudinal analysis of censored 
medical cost data. Health Economics 2006;15:513-525. 
276. 
Gardiner JC. Joint modeling of mixed outcomes in health services research, Paper 435-
2013.  SAS Global Forum, 2013; 2013; San Francisco, CA: SAS Institute, Inc. 
277. 
Harris PA, Taylor R, Thielke R, Payne J, Gonzalez N, Conde JG. Research electronic 
data capture (REDCap)--a metadata-driven methodology and workflow process for providing 
translational research informatics support. J Biomed Inform 2009;42:377-381. 
 


 
 
 
 
 
                                                          V4.1 14 June 2023 
 ____________________________________________________________________________________________ 
121 
 


DMID Protocol Template
Version 3.1
01 Sept 2020
____________________________________________________________________________________________ 
____________________________________________________________________________________________ 
122 
 
PROTOCOL APPENDICES 
Appendix A  
Schedule of Events 
Appendix B  
Adverse Event Grading Table 
 


DMID Protocol Template 
Version 3.1 
 
01 Sept 2020 
 ____________________________________________________________________________________________ 
 
 ____________________________________________________________________________________________ 
123 
Appendix A 
Schedule of Events 
Clinical and Laboratory Evaluations 
      A = performed on all children on PRW/PICU [(A) = only if still unconscious] 
S = performed on children eligible to be screened for TBS study 
I = performed as part of TBS trial [(I) = only if still unconscious] 
 
Event 
Admission 
Screening 
With TBS 
consent 
Day 1 
Day 2 
Days 
3 -7 
 
Follow-
up visits 
History 
A 
 
 
 
 
 
A 
Physical Exam 
A 
 
 
A,A 
A,A 
A,A 
A 
Vital signs 
A 
 
 
A – q2 
hours 
A – q2 
hours 
A – q2 
hours 
A 
IV anti-malarial 
Rx 
A 
 
 
A,A 
A,A 
 
 
PO anti-
malarial Rx 
 
 
 
 
 
A 
(3 day 
course) 
 
MP and PCV 
A 
 
 
A A A 
A 
(A)(A) 
(A)(A) 
(A)(A) 
(A)(A) 
A 
Lactate and 
glucose check 
A 
 
 
A A A 
A 
(A)(A) 
(A)(A) 
(A)(A) 
(A)(A) 
 
Lumbar 
Puncture 
A 
 
 
 
 
 
 
Retinal exam  
 
S 
 
(I,) 
(I,) 
(I,) 
 
MRI 
 
S 
 
I 
(I) 
(I) 
I  
(1 month) 
NIRS 
 
S 
 
I 
I 
I 
I 
(1 month) 
TCD 
 
? 
I? 
I 
I 
I 
I 
(1 month) 
ONSD 
 
 
I 
I,  
(I,) 
(I,) 
I 
Expanded 
neurologic 
exam 
 
 
I 
I, I 
(I,I) 
(I,I) 
I 
Heart rate 
variability 
 
 
I 
I 
I 
I 
 
Lung 
ultrasound 
 
 
I 
I 
I 
I 
 
Brain swelling 
intervention 
 
 
I 
I 
I 
I 
 


DMID Protocol Template 
Version 3.1 
 
01 Sept 2020 
 ____________________________________________________________________________________________ 
 
 ____________________________________________________________________________________________ 
124 
Appendix B - Adverse Event Table 
Event 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Hypernatremia 
166-170 mEq/L 
171-180 mEq/L 
181-190 mEq/L 
>191 mEq/L 
Hyperkalemia 
5.4-5.9mEq/L 
6.0-6.4mEq/L 
6.5-7.0mEq/L 
> 7.0 mEq/L 
Hypokalemia 
 
 
2.0-2.4 mEq/L 
< 2.0 mEq/L 
Phlebitis 
HU\WKHPDFP
diameter at IV site 
without swelling, 
venous cord or 
fever 
erythema and 
LQGXUDWLRQFP
diameter at IV site 
without  venous cord 
or fever 
erythema and 
LQGXUDWLRQFP
diameter at IV site 
with  venous cord or 
IHYHU& 
erythema and 
LQGXUDWLRQFP
diameter at IV site 
with  venous cord 
DQGIHYHU& 
Pneumothorax 
NA 
small pneumothorax 
with no 
cardiovascular effect 
-no intervention 
required 
small pneumothorax 
requiring placement 
on 100% oxygen 
pneumothorax with 
associated 
cardiovascular 
compromise 
requiring needle 
decompression or 
placement of  chest 
tube 
Pneumomediastinum 
NA 
simple 
pneumomediastinum-
asymptomatic (No 
intervention required) 
pneumomediastinum 
with extrathoracic 
subcutaneous 
emphysema (no 
intervention 
required) 
pneumomediastinum 
with associated 
cardiovascular 
compromise 
requiring placement 
of drain or surgical 
intervention 
Subglottic stenosis 
Need for racemic 
epinephrine( X 1 
dose )use post-
extubation 
Need for use of 
multiple doses of 
racemic epinephrine 
Need for 
reintubation 
secondary to 
respiratory difficulty 
Need for surgical 
resection of 
subglottic stenosis 
and/or tracheostomy 
secondary to sub 
glottic stenosis 
Respiratory 
nosocomial infection 
Post-admit 
pneumonitis on 
CXR and phys 
exam with O2 sat 
RIRQURRP
air 
Post-admit 
pneumonitis on CXR 
and phys. Exam with 
O2 sat of < 90% on 
5$2VDWRQ
supplemental 02 
Post-admit 
pneumonitis on CXR 
and phys. exam 
after admission with 
O2 sat < 90% on 
room air, O2 sat 70-
90% with 
supplemental 02 
Post-admit 
pneumonitis on CXR 
and phys exam with 
O2 sat of < 90% on 
room air, O2 sat < 
70% with 
supplemental 02 
Hypotension 
 
Reduction of resting 
mean arterial blood 
pressure 25-49% of 
pre-intubation mean 
arterial pressure 
Reduction of resting 
mean arterial blood 
pressure 50-74% of 
pre-intubation mean 
arterial pressure 
Reduction of resting 
mean arterial blood 
pressure >75% of 
pre-intubation mean 
arterial pressure 
Endotracheal tube 
obstruction 
 
Inadvertent 
endotracheal tube 
dislodgment 
                                                                        Binary (present or absent) 
Bradycardia induced 
by vagal response 
 
Reduction of resting 
heart rate 25-49% of 
pre-intubation heart 
rate 
Reduction of resting 
heart rate 50-74% of 
pre-intubation heart 
rate 
Reduction of resting 
heart rate >75% of 
pre-intubation heart 
rate