An MRI Ancillary Study of a Malaria Fever Randomized Controlled Trial (RCT)
Conditions: Brain Injuries, Malaria, MRI Imaging, Neurocognitive Deficits, Chronic Kidney Diseases, Behavior Problem
Sponsor: University of Rochester

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An MRI Ancillary Study of Malaria FEVER RCT  
NIH Grant No. R01NS111057  
Principal Investigator – Gretchen L. Birbeck 
 
Institutions:   
University of Rochester (UR), Rochester, NY, USA 
 
 
 
Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi 
 
 
 
University Teaching Hospital’s Children’s Hospital (UTH), Lusaka, Zambia 
 
 
 
Chipata Central Hospital (CCH), Chipata, Zambia 
 
 
 
Michigan State University (MSU), East Lansing, MI, USA 
 
Investigators:  
Gretchen L. Birbeck, UR & UTH 
 
 
 
Michael Potchen, UR & Lusaka Apex Medical University 
 
 
 
Sam Kampondeni, UR and Mpingwe Clinic (Blantyre) 
 
 
 
Karl Seydel, QECH & MSU 
 
 
 
Yamikani Chimalizeni, Malawi College of Medicine and QECH 
  
 
 
Manoj Mathews, UTH 
Musuku Mwenechanya, UTH 
Suzanna Mwanza, CCH 
Colleen Hammond, MSU 
John Langfitt, UR  
 
1. EXECUTIVE SUMMARY 
Despite eradication efforts, ~400,000 African children sustained brain injuries as a result of CNS malaria in 
2016. A higher maximum temperature (Tmax) during the acute malaria infection is an established risk factor for 
neurologic sequelae and a randomized controlled trial (RCT) of aggressive antipyretic therapy with 
acetaminophen and ibuprofen being conducted in Malawi and Zambia began enrollment in 2019 
(R01NS102176). In this clinical trial, the primary outcome is Tmax during the acute infection. However, the 
antipyretic therapies used in this RCT may offer neuroprotective effects without affecting Tmax--for example, 
neuroprotection through anti-inflammatory mechanisms. In An MRI Ancillary Study of Malaria FEVER RCT, 
we propose to use neuroimaging in the context of the RCT to further evaluate the potential neuroprotective 
effects of aggressive antipyretic therapy for CNS malaria and explore possible mechanisms for these effects. 
Comparing children allocated to aggressive antipyretic therapy vs. usual care on the prevalence of structural 
brain abnormalities after recovery from CNS malaria will facilitate the evaluation of non-fever pathways for 
neuroprotection. Brain MRIs will be obtained in children enrolled in the RCT at 1- and 12-months post recovery. 
Analyses will be completed comparing the odds of having any structural injury based upon RCT treatment 
allocation and based upon (Tmax) stratified by treatment allocation to assess changes specifically related to 
response to therapy in terms of fever reduction. Potential mechanisms of aggressive antipyretic-related injury 
will be evaluated including assessments for treatment-related CNS bleeds. Neuroimaging is a well-established, 
valid proxy for neurological outcomes after brain injury including in pediatric CNS malaria. Adding this MRI 
ancillary study to our fever RCT may elucidate mechanisms of treatment-associated injury and allow for early 
identification of neuroprotection from aggressive antipyretic use that would otherwise require long-term follow-
up for cognitive and behavioral assessments. This study will provide critical insights that could inform future 
neuroprotective studies of malaria that might incorporate imaging to optimize study design. 
 
2. MAIN OBJECTIVES  
This prospective observational study will seek consent from parents of children previously enrolled in the 
Aggressive Antipyretics in CNS Malaria: A Randomized Controlled Trial Assessing Antipyretic Efficacy and 
Parasite Clearance study (NIH Grant No. R01NS102176; ClinicalTrials.gov NCT03399318) to obtain 
neuroimaging and neurodevelopmental and behavioral outcomes data on their child. The imaging and 
evaluations for this observational study will occur after the child has recovered from the acute malaria 
infection and has otherwise completed the RCT intervention and safety evaluations. For some, consent will 
be sought months after the RCT-related activities for the child are completed. As such, this is an independent 
observational study. However, since the findings may offer relevant outcomes data for the RCT, data obtained 

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in this observational study will be shared with the RCT’s biostatistician for inclusion in all interim analyses and 
review by the RCT’s Data Safety Monitoring Board (DSMB). 
  
Specific Aims 
Aim 1: To compare the prevalence of  brain injury  in theaggressive antipyretic (AA)  vs. usual care (UC) groups 
of the FEVER RCT. 
 
Aim 2: To compare the prevalence of specific structural injuries  amongchildren who received AA therapy vs. UC 
with propensity score adjustment. 
 
 
 
Aim 3 Safety assessments. 
 
3. BACKGROUND AND INTRODUCTION  
CNS malaria, meaning P. falciparum parasitemia with neurologic symptoms including impaired consciousness 
and/or seizures,1-3 continues to be a major cause of neurodisabilities in African children. Brain injury with 
subsequent neurologic sequelae occur in 1/3rd of children who survive CNS malaria. Newer antimalarial 
medications rapidly clear peripheral parasitemia and improve survival, but mortality remains high at 12-25% 
with no associated decline in post-malaria neurologic injury.4 Common sequelae include epilepsy, gross motor 
deficits, deafness, blindness, cognitive impairment, and behavioral disorders.1-3, 5-8  
 
In our prior Blantyre Malaria Project Epilepsy Study (BMPES), a higher maximum temperature (Tmax) during 
the acute infection was associated with a greater risk of post-malaria neurologic sequelae. Children who 
developed epilepsy had a mean Tmax of 39.4°C vs. 38.5°C in those who did not develop epilepsy (absolute 
difference of 0.9°C; p=0.01). In addition, a higher Tmax predicted subsequent behavioral disorders (39.2°C vs. 
38.7°C; absolute difference 0.5°C; p=0.04).5 These data are biologically consistent with the substantial body of 
evidence that fevers contribute to secondary neurologic injury in the setting of stroke, meningitis, anoxia, 
hypoxia, and trauma.9-13 In the U.S., induced hypothermia is now standard-of-care for post-anoxic14 and 
neonatal hypoxic-ischemic encephalopathy15-18 and fever-reduction is an important aspect of neurocritical 
care.19-22 Malaria-induced fevers can be extremely high. Fifteen percent of the BMPES cohort experienced at 
least one temperature of ≥ 40.0°C (104.0°F). The age-related susceptibility to pediatric malaria also 
substantially overlaps with the age-related phenomena of febrile seizures. Malarial seizures are often complex, 
multifocal, and prolonged with status epilepticus being common.5, 7, 23, 24.   
 
In 2019, a randomized controlled trial (RCT) of aggressive antipyretic therapy for CNS malaria launched in 
Malawi and Zambia. This RCT compares prophylactic, scheduled acetaminophen and ibuprofen for 72 hours 
after presentation with CNS malaria to usual care with the primary outcome being Tmax as “proof-of-concept” 
for potential neuroprotective efficacy of aggressive antipyretic therapy in CNS malaria. This observational 
MRI Ancillary study will obtain neuroimaging and neuropsychiatric outcomes in children enrolled in 
the RCT.  
 
Rationale 
While a higher Tmax is associated with adverse neurologic outcomes in CNS malaria, causality is not directly 
established. CNS malaria is a highly inflammatory process25, 26 and inflammation-mediated injury is a likely 
pathway for malaria-associated brain in this population particularly for epilepsy development. The aggressive 
antipyretic regimen should offer anti-inflammatory benefits as well. Neuroimaging after recovery will facilitate 
identification of benefits associated with the aggressive antipyretic therapy that are mediated by non-fever 
pathway including reduced inflammatory factors. Our research team recently published findings from a 1-year 
follow up study of children who survived cerebral malaria compared to age-matched controls, which confirmed 
high rates of cognitive and behavioral impairments (53% at one year) in the malaria group. Importantly, 
neurodevelopmental disabilities at 12 months were associated with structural brain lesions on MRI at 1-month 
including severe atrophy and multifocal abnormalities.27 As such, utilizing neuroimaging findings captured at 1- 
and 12-months post recovery as an outcome in the RCT will also allow us to assess for potential 

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neuroprotective benefits from aggressive antipyretics in CNS malaria that are not necessarily mediated by 
fever.  
 
Neuroimaging at 1- and 12-month post recovery will also facilitate additional safety assessments. Though frank 
CNS bleeds are not associated with pediatric cerebral malaria,28 end vessel microhemorrhages are seen post 
mortem29, 30 and have been identified on brain MRIs in case reports of cerebral malaria.31, 32 Using imaging 
sequences that identify ferromagnetic substances, we will evaluate for evidence of any hemorrhages including 
parenchymal microhemorrhages and compare the presence and characteristics of any CNS bleeding between 
the two arms of the RCT. Given the antiplatelet effects of ibuprofen, increased risk of bleeding is conceivable in 
the aggressive antipyretics arm. Data from this MRI Ancillary study will be provided to the RCT’s DSMB for the 
scheduled interim analysis. 
 
4. ADMINISTRATIVE ORGANIZATION   
All patient-contact will occur in Zambia and Malawi where the RCT is ongoing. All data transmitted or conveyed 
beyond the country of enrollment will be de-identified. 
 
Study Sites: 
University Teaching Hospitals-Children’s Hospital, Lusaka, Zambia 
Chipata Central Hospital, Chipata, Zambia 
Queen Elizabeth Central Hospital, Blantyre, Malawi 
 
Study sites without patient contact or access to data with private health information include— 
University of Rochester, Rochester, NY, USA 
Michigan State University, East Lansing, MI, USA 
 
Oversight plan for non-UR affiliated US investigator: 
 
While in Malawi, the MSU PI is responsible for overseeing subject recruitment, data collection and imaging at 
Queen Elizabeth Central Hospital. As part of data cleaning, they will also have access to data with patient 
identifiers. Our office takes responsibility for the required IRB reports (the University of Rochester’s Research 
Subjects Review Board, The University of Malawi’s College of Medicine Research Ethics Committee, and the 
University of Zambia’s Biomedical Research Ethics Committee) within Malawi and Zambia. Regular 
communications between the MSU PI, Local Safety Monitors (LSM) and the PI ensure that data collected and 
contact information is up-to-date. 
 
No human subjects contact will occur at any of the US-based sites. All data exported to the US is de-identified 
prior to transfer. All RSRB determinations regarding this work will be shared with the non-affiliated 
researchers in a timely fashion.  
 
Data storage and analysis will also take place using UR servers but only de-identified data will be 
externalized. All data transmitted to UR will be de-identified. 
  
5. STUDY DESIGN 
To compare the prevalence of brain injury in the aggressive antipyretic  vs. usual care (UC) groups of the Fever 
RCT 
Children  will undergo  brain MRIs and neurodevelopmental and behavioral evaluations at 1- and 12-months  
after their acute malaria illness or as soon as possible among those children enrolled in the Fever RCT prior to 
the initiation of this MRI ancillary study. Two radiologists, blinded to treatment allocation and neurologic status, 
will independently review images and capture data using NeuroInterp.33  For children who are unable to undergo 
imaging without deep sedation, neurodevelopmental & behavioral evaluations will be used to identify those with 
brain injury. 
We hypothesize that children who receive AA therapy during CNS malaria will have lower odds of brain injury 
than those receiving UC.  

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To compare the prevalence of specific structural injuries  among children who received aggressive antipyretic 
therapy vs. usual care therapy in the Fever RCT. Specifically, we will compare 
  
a. Overall atrophy by brain volume scoring34 (atrophy being a brain volume score of 1-2 vs ≥3) 
b. Gliosis by Fazekas score35 (0, 1, 2 or 3) 
c. The presence of regional gliosis or atrophy in the following regions 
1. Cortical (present/absent) 
2. Deep grey (present/absent) 
3. Corpus callosum (present/absent) 
4. Posterior fossa (present/absent) 
 
A priori pathophysiologic attributions are as follows: 
-If focal cortical abnormalities are lower for the AA treatment group, we will conclude that the mechanism(s) 
included seizure control/prevention 
-If abnormalities are less common in the deep grey regions for the AA treatment group, we will attribute this to 
benefits of reduced sequestration in the deep vascular beds 
-If there is less diffuse atrophy or overall gliosis for the AA treatment group, we will attribute this to reductions 
in increased intracranial pressure 
 
Safety assessments 
a. In Zambia where standard imaging with GRE sequences is available, we will compare the prevalence 
of blood products in the AA vs UC groups. If present, blood products will be further characterized by 
age of blood relative to participation in the Fever RCT and blood volume based upon number and size 
of foci. 
b. The prevalence of CKD based upon blood creatinine and urine ACRs will be compared in the AA vs UC 
groups 
 
5.1. SUBJECT POPULATION 
All children enrolled in the Malaria RCT who survive to discharge are eligible for enrollment. Inclusion and 
exclusion criteria for the malaria fever study are outlined below. 
 
6. INCLUSION AND EXCLUSION CRITERIA 
All children enrolled in the malaria RCT who survive to discharge are eligible for inclusion. The malaria FEVER 
RCT inclusion and exclusion criteria are detailed below. 
 
Inclusion Criteria for RCT 
Age 2-11 years (24 to 132 months) 
Evidence of P. falciparum malaria infection by peripheral blood smear or rapid diagnostic test 
CNS symptoms associated with malaria.  
CEREBRAL MALARIA:  Impaired consciousness with a Blantyre Coma Score (BCS) ≤2 in children 
under 5 years or a Glasgow Coma score (GCS) ≤10 in children ≥5 years OR CNS MALARIA: 
Complicated seizure(s), meaning prolonged (>15 minutes), focal or multiple; or impaired consciousness 
or other evidence of impaired consciousness (confusion, delirium) without frank coma (BCS>2, GCS 
=11-14)  
 
Exclusion Criteria for RCT 
Circulatory failure (cold extremities, capillary refill > 3 seconds, sunken eyes, ↓ skin turgor) 
Vomiting in the past 2 hours 
Serum Cr > 1.2 mg/dL 
A history of liver disease 
Jaundice or a total bilirubin of >3.0mg/dL 
A history of gastric ulcers or gastrointestinal bleeding 

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A history of thrombocytopenia or other primary hematologic disorder 
Petechiae or other clinical indications of bleeding abnormalities  
A known allergy to ibuprofen, acetaminophen, aspirin or any non-steroidal medication 
Any contraindication for nasogastric tube (NGT) placement and/or delivery of enteral medications  
 
7. RECRUITMENT METHODS 
Before recruitment into this observational study will have begun, consent for future contact regarding possible 
research participation in subsequent relevant studies was sought from the parents/guardians of children enrolled 
in the Malaria FEVER RCT at the time of discharge. This process was approved by the appropriate ethical and 
institutional review boards. A list of those who consented by study ID will be cross-referenced to the contact 
details including address and cell phone numbers maintained in the study pharmacies. These parents/guardians 
will be contacted to seek consent for enrollment in this observational study.  
 
In addition, children being enrolled in the ongoing RCT will be identified as potential participants when they have 
been stabilized and regained consciousness. Study Nurses working on the ward where the children are admitted 
will then approach the parent/guardian for consent prior to discharge. 
 
8. CONSENT PROCESS 
For children newly admitted for the RCT study, nurses on the ward where the children are admitted will 
approach the parent/guardian for consent. For children recently admitted but already discharged whose 
parents consented to be contacted regarding future studies, phone calls will be placed and a brief overview of 
the study will be detailed. If the parent/guardian is willing to consider enrollment, they will be invited to come to 
the hospital for a full discussion and the consent process detailed below. 
 
Consent will take place in a private setting. Staff obtaining consent are fluent in the local languages and expert 
in conducting the informed consent discussion. All have Human Subjects Protection and Good Clinical Practice 
certifications. The consent process will be conducted in the preferred language of the parent/guardian. Only 
local staff will be involved in this process due to the geopolitical/post-colonial nature of this setting. The 
parent/guardian will be given whatever time is needed to consider whether they are willing to have their child 
enrolled including opportunities to discuss with other family members. All parents/guardians who consent will 
be provided with signed copies of the consent forms. For those parents/guardians who are unable to sign due 
to literacy limitations, a thumb print will be obtained and someone who is not associated with the study but is 
literate in the parent’s/guardian’s preferred language will be present during the consent process and will sign 
as a witness. At the time of the return visit for evaluations, for children 7 years or older, if in the 
parent’s/guardian’s assessment is that the child has the capacity to comprehend the nature of the request, 
assent will also be sought. 
 
Consent and assent forms will be developed in English and the applicable local languages. Forward- and back- 
translation will be undertaken by expert local staff with extensive experience in this regard. 
 
9. STUDY PROCEDURES 
At 1- and 12-months post admission for malaria or as soon as possible if the index illness was > 12 months 
prior to the launch of this observational study, enrolled children will undergo neuroimaging, the Neurological 
Examination for Subtle Signs (NESS) if 7 years or older, the WHO Epilepsy Screen and a single question 
behavioral assessments. In addition, depending upon age at the time of the evaluation they will undergo the 
Malawi Developmental Assessment Tool (MDAT) or the Kaufman Assessment Battery for Children (KABC). 
The MRI scan will require ~30 minutes and the remaining assessments ~1-1.5 hours. If a child is more than 1-
month post discharge are the time of consent, the first assessments will be obtained as soon as is feasible. If 
any clinically relevant findings are identified, these will be discussed with the parent/guardian and the 
appropriate referral for care will be made.  
 
MRI images will be reviewed by the technician at the time of the scanning and any findings of concern that 
might warrant gadolinium administration will be reviewed by one of the study radiologists in real time. This can 

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be done through the Baynes.net system in place for review and/or through sharing of the key images of 
concern to the technicians. This should allow for gadolinium administration when the child is initially imaged. 
However, if on review of an MRI that the technical staff had no concerns about the radiologists feels gadolinium 
is warranted for clinical care, the patient’s family will be notified and all the same financial and logistical support 
will be provided for this repeat image. This data will be used for research purposes if such images are 
recommended for clinical care. 
 
10. DISSEMINATION 
Findings from this work will initially be presented at appropriate local venues before broader dissemination. For 
example, UTH’s Neurology Research-in-Progress weekly meeting, Zambia’s Annual National Health Research 
Conference, the Wellcome Trust’s Cutting Edge weekly meeting, and/or Malawi’s College of Medicine 
Research Dissemination Day. With approvals from Zambia’s National Health Research Authority and the 
Malawi Ministry of Health, findings will then be submitted to peer reviewed journal(s) for publication and wider 
dissemination. Copies of published papers will be submitted to the associated IRBs, the College of Medicine 
Library in Malawi, and the Zambian National Health Research Authority. 
 
11. RISKS TO SUBJECTS 
Risks include loss of privacy, annoyance at the study assessments and adverse effects from sedation. MRI-
specific risks are largely associated with implanted devices which are not in use in the study populations.  
 
The study assessments including the neuroimaging are expected to take less than 2 hours. Regarding 
sedation risks--in our experience, children as young as 6 years are able to tolerate clinical MRIs without 
sedation with a parent in the scanning room to comfort and encourage them. Chloral hydrate has been 
routinely used for sedation for EEG and MRI at QECH in Malawi since both technologies became available 
over a decade ago. No serious adverse events have occurred in relation to chloral hydrate use in Malawi for 
this purpose. An estimated 6% of Malawian children given chloral hydrate develop a paradoxical hypervigilance 
sometimes with irritability and agitation lasting 4-6 hours. Nausea, vomiting and an allergic reaction are listed 
side effects of chloral hydrate though these have not been seen during their clinical use in children in Malawi 
over the last decade. Children with a history of an allergy to or adverse reactions to chloral hydrate will not undergo 
sedation with this agent. Children with a history of a paradoxical response to other sedatives will not receive chloral 
hydrate. Side effects for chloral hydrate are dose related. Although standard protocol for clinical use of chloral 
hydrate indicates repeated dosing if needed, no repeat dosing will be undertaken in this study. Due to the 
increasing concerns about tissue deposition of gadolinium, gadolinium will only be used if non-contrast imaging 
identifies a finding that clinically requires gadolinium for further assessment. By this we mean that is will not be 
administered for the purposes of this study but should an abnormality be identified that the radiologist feels 
requires gadolinium for clinical evaluation, study resources will be provided to ensure the additional imaging 
and gadolinium are provided.  
 
We have been able to harmonize sedation protocols across sites to begin with melatonin and, if needed, add 
chloral hydrate. Administration of either medication is not complex (see Addendum II of Protocol V5.0). 
Melatonin, a dietary supplement, is safe and effective for sedation in sub-Saharan Africa.36  Any interaction 
between melatonin and sedatives such as chloral hydrate are classified as minor and do not require 
adjustments in medications dosing. All children will be NPO for 4 hours prior to MRI in case any sedation is 
needed with snacks provided at the completion of the study/ 
 
In Zambia, melatonin is the standard sedative used for pediatric procedures such as EEG, but they have less 
experience with chloral hydrate. In addition, the imaging facilities in Zambia are outside of the pediatric hospital 
with limited support for managing adverse events related to sedation. As such, when chloral hydrate is used in 
Zambia, an anesthetist will administer the agent and be available until the child is fully awake.  
 
In Malawi, they are less familiar with melatonin and study staff will receive in-service training regarding 
potential side effects which include dizziness, headache and nausea. These are self-limited and likely 
uncommon--Zambian providers indicated these are not events they have had reported to them when using 

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melatonin in children for EEG. In Malawi, chloral hydrate has long been the standard management for sedation 
for EEG and MRI and imaging occurs within the pediatric hospital with ample experienced staff in attendance 
so no additional monitoring measures beyond the clinical standard will be instituted. 
 
Should the child experience any adverse effects from sedation, a study clinician will evaluate them and 
determine if further management if needed. Most anticipated side effects are self-limited and will resolve 
without intervention. If required, the clinician will provide analgesics for headache, anti-emetics for nausea, and 
oral rehydration solution for vomiting. 
 
10. POTENTIAL BENEFITS TO SUBJECTS 
Children identified as having neurologic sequelae based upon the evaluations (imaging and study instruments) 
will be referred for appropriate services that are available free of charge at both study sites. Children with 
structural lesions found on neuroimaging will be offered quarterly developmental screening assessments 
performed by our team for the duration of this study with the (MDAT/KABC, WHO Epilepsy Screening 
Questionnaire and the behavioral screen), to facilitate expedited care, if needed. Research dissemination 
meetings held with families of children who participated in our prior research conducted by this team identified 
this approach as favored by parents/guardians to help them in monitoring children at increased risk of 
neurologic sequela after recovery from cerebral malaria. 
 
When children are evaluated for research purposes and problems are identified, it is an established practice for 
both the Zambia and Malawi-based teams to provide quarterly follow-up to facilitate expedited care if children 
in the study are not seen quarterly, but these additional evaluations are not for research purposes. Full reports 
of the MDAT and KABC are provided to the clinicians and therapists caring for the patient. The findings are 
also discussed personally with the parents by the study nurses and for those children who have problems 
identified a clinician will also review the findings with the parents and explain the reason that additional care 
and monitoring is being recommended. If the child is school age, a letter is provided to the school alerting the 
teacher that the child may have neurological problems involving behavior and/or cognition related to their 
severe malaria infection, that they are under medical care for this problem, and encouraging the teacher to 
allow the child to remain in school despite their challenges.  
 
There is no precedence in this setting for special education in the public sector. Impaired children are usually 
just ejected from school and re-entry is challenging. Our goal in the communication to schools is to encourage 
the teachers to allow the children to remain in school. Providing full written reports of the MDAT and/or KABC 
would have little meaning to families or teachers in this environment. 
 
11. COSTS FOR PARTICIPATION 
Participants’ and their families will not be required to cover any of the costs associated with participation. All 
study activities and procedures will be provided free-of-charge, as well as round trip transportation for the 
children and their parents/guardians for these follow-up assessments. 
 
12. PAYMENT FOR PARTICIPATION  
• 
In Malawi, for each study visit participants and their families will be reimbursed for the roundtrip travel 
costs for the child and guardian. They will also receive remuneration consisted with published 
guidelines which is MWK4500.37 
• 
In Lusaka, for each study visit participants and their families will be reimbursed for the roundtrip travel 
costs for the child and guardian. They will also receive a total of ZMW260 for their time from work, food 
during the testing day and their child’s completion of the assessments. 
• 
In Chipata, for each study visit we will provide round trip transportation and overnight accommodation 
for the child and a parent/guardian to travel to Lusaka. They will also receive ZMW1060 for their time 
from work, food while traveling and the child’s completion of the assessments. 
 
13. SUBJECT WITHDRAWALS 
Parents/guardians may withdraw their children from the study at any time. 

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14. PRIVACY AND CONFIDENTIALITY OF SUBJECTS AND RESEARCH DATA 
To limit risk of loss of privacy, only the study ID number will identify all laboratory specimens, evaluation forms, 
reports, and other records that leave the sites. All paper records will be kept in secure study offices in locked 
filing cabinets. All computer entry and networking programs will be done using study IDs only. Study data using 
only study IDS will be store as follows: paper-based in the secure study offices at each site available only to 
study staff; neuropsychiatric outcomes Neuropsych and NeuroInterp data on the University of Rochester’s 
Research Electronic Data Capture (REDCap)RedCap server, neuroimaging files in DropBox with secured 
folders accessible only to study staff. Study staff will assure that imaging data and scanned paper-based forms 
with graphical or pictorial data containing only the study ID and are saved in secured DropBox secured (i.e. 
non-public) folders. The study offices are secured rooms with lockable file cabinets. All computers are 
password- protected. DropBox folders containing de-identified data are non-public. 
 
Clinical information will not be released without written permission of the participant’s parent or guardian, 
except as necessary for monitoring by the appropriate institutional review boards (IRBs), the NINDS, the 
OHRP, the sponsor, or the sponsor’s designee. If a family needs to be re-contacted for any reason (e.g. an 
imaging finding that is determined to warrant clinical follow-up), the study ID in this observational study will be 
cross referenced against the contact details for each child’s family that is maintained as required in the study 
pharmacy for the RCT.  
 
15. POSSIBLE CONSTRAINTS 
The RCT has commenced and those patients enrolled to date will not be able to provide 1-month imaging. 
Public health concerns related to COVID-19 is anticipated to further delay this study. Fortunately, the chronic 
structural abnormalities that are seen with cerebral malaria-associated brain injuries are not typically reversible 
and should be seen even if children are imaged later than planned. Time from malaria infection to imaging can 
be controlled for in analyses.  
 
16. DATA / SAMPLE STORAGE FOR FUTURE USE 
De-identified imaging and neuropsychiatric outcomes will be stored for future use in secured DropBox folders 
and on the UR RedCap server. No data with identifiers is obtained or stored. Study IDs are used exclusively, 
but can be linked back to the subject if needed using the contact listing maintained in the study pharmacies for 
the RCT as required. 
 
17. DATA AND SAFETY MONITORING PLAN 
This is an observational study. Potential adverse events  include breach in confidentiality and any injuries or 
inconveniences related to participation in the clinical assessments and/or imaging acquisition. Any adverse 
events identified will be reported to the appropriate institutional review boards.. Although not anticipated, 
should any Serious Adverse Events (SAE) occur during the conduct of this observational study, the Local 
Study Monitors serving the RCT will be asked to review with their assessments provided to the overseeing 
IRBs.  
 
The children in this study have participated in an ongoing blinded RCT and their allocation either to aggressive 
antipyretic therapy or routine clinical care during their acute malaria infection remains concealed. As such, the 
findings from this observational study will be provided to the RCT biostatistician for analysis and inclusion in 
safety evaluations routinely conducted by the DSMB. 
 
18. DATA ANALYSIS PLAN 
 
 
 
 
 
 
 

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The analysis of the presence of a brain injury (yes/no) will involve fitting a logistic regression model with RCT 
treatment allocation group as the factor of interest, country as a stratification factor, and a stratified propensity 
score38 to account for the fact that the treatment groups will no longer be the original randomized groups due to 
participants refusing consent and loss-to-follow-up. The propensity score will be estimated using a logistic 
regression model with treatment group as the outcome variable and baseline covariate information that is thought 
to be relevant to predicting adverse neurological outcome. These covariates include disease severity 
(presence/absence of cerebral malaria), seizures prior to admission, admission temperature, admission 
creatinine, sex, and age. The estimated treatment group odds ratio, along with its associated 95% confidence 
interval and p-value, will be derived from this model. Similar analyses will be conducted for the other outcomes 
of interest including the presence of the specific imaging outcomes outlined in Aim 2, the presence of blood 
products; and the prevalence of chronic kidney disease based upon an ACR of >1.2. For the ordinal gliosis 
outcomes in Aim 2, a proportional odds logistic regression model will be employed; if the proportional odds 
assumption appears to be seriously violated, a partial proportional odds logistic regression model or a 
multinomial logistic regression model will be used instead. 
The sample size considerations are based on the logistic regression analyses to be performed in Aim 1. 
Preliminary data indicate that 34% of pediatric CM survivors in Malawi have abnormal brain MRIs at 1 month or 
a KABC falling at least 2 standard deviations below the population norms for those over 7 years.39 A sample size 
of 184 subjects (92 per group) will provide > 85% power to detect a reduction in the percentage of subjects with 
structural injury from 35% in the UC group to 15% in the AA therapy group, using a two-tailed test and a 5% 
significance level. 
 
 
 
 
  
 
 

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Addenda 
 
Addendum I. Protocol Changes Made to Date 
 
Protocol Version Changes 
 
Version 1.0 to 2.0 
1. Removed plans for use of CT as contingency 
Version 2.0 to 2.1 
1. Reformatted for COMREC adding executive 
summary, dissemination plan and constraints 
Version 2.1 to 3.0 
1. Changed period for NPO before MRI sedation 
from 8 to 4 hours 
2. Added specific amount of payment for Malawi 
participants 
Note:  
COMREC mandated that for their review and approval the protocol be adapted to their preferred format. This 
was done using Protocol version 3.0 
  
UNZA BREC mandated that for their review and approval the protocol be adapted to their preferred format. 
This was done using Protocol version 3.0 
Version 3.0 to 4.0 
1. Added oversight plan for non-UR US investigator 
Version 4.0 to 5.0 
1. Added Addendum 1 delineating all protocol 
changes from version 1.0 onward to be updated as 
needed 
2. Revised running title in header to better reflect this 
is the MRI Ancillary and not the parent FEVER study 
protocol 
3. Sedation protocol added as addendum 2 
4. Instruments associated with the protocol were 
adjusted to add the single question behavioral 
screen and are now deemed v2.0 
5. Consents  and assents associated with protocol 
are now version 3.0 
Version 5.0 to 5.1 
1. Added further details on sedation protocols within 
both sites 
2. Added details re management of any sedation 
adverse events 
3. Added details re acute clinical review of MRI and 
use of gadolinium 
4. Added details about services provided when 
abnormalities are identified in developmental 
assessments including communications with 
teachers 
5. Added specifically that MRI will take ~30 minutes 
6. The same detail re 30 minutes for MRI was added 
to the consent form. This protocol is associated with 
Consent version 3.1 and Assent version 3.0 
 
Version 5.1 to 6.0 
1. Add BRIEF to neurodevelopmental assessment 
instruments 
2. Obtain creatinine and urine sample for dip and 
albumin: creatinine ratio to identify early signs of 
chronic kidney disease (CKD) 
3. Swab to identify co-infection at time of malaria 
infestion with SARS-Cov-2 

MRI Ancillary Study Protocol Version 7.1 19Oct23 
11 
 
 
 
Version 6.0 to 6.1 
Minor verbiage adjustments in protocol requested 
per RSRB for clarity only. 
Version 6.1 to 7.0 
 
1. Sedation clarifications 
a. In Malawi, drop melatonin and allow snack 
b. In Zambia, if imaging undertaken at Medland 
Hospital and sedation is needed, management for 
sedation is deferred to consultant anesthesiologist 
2. At Chipata Central Hospital only, option for 
participation in expanded study of autism and 
epileptogenesis. This has a separate consent. 
Version 7.0 to 7.1 
Amendments proposed 
1. Changing of analysis plan, utilizing the propensity 
score adjustment and  
2. aligning that change to slightly change the aims of 
the study 
 
Rationale ;  
bring clarity  
 
Risks 
No risks since no changes to the human subjects or 
enrolment process. 
 
 
Addendum II. Dosing schedule for sedation 
Given the potential need for sedation, children will be NPO 4 hours prior to the scheduled MRI. Snacks will be 
provided at the completion of the MRI. 
 
a. Melatonin 
Provided in 3 mg chewable tabs 
Time: 30 min – 60 min before MRI 
Dose:  
< 6 y/old: ½ tab (1.5mg) 
>6 y/old: 1 tab (3 mg) 
>10 y/old: 1 +1/2 tab (4.5mg) 
 
If at 60-minutes post dosing the child is still too active for scanning, proceed to chloral hydrate. In Zambia, 
this requires administration by an anesthetist and rescheduling the procedure may be necessary. In 
Zambia, the anesthetist will remain with the child from the administration of chloral hydrate until the child 
fully regains consciousness (BCS 5/5). 
 
b. Chloral hydrate 
Provided in liquid form. Give 50mg/kg po 30 minutes before MRI. Do not repeat dose. 
 
Addendum III.  
Protocol Amendment version 5.1 to 6.0 
Amendment changes proposed: 
1. Add BRIEF (Behavioral Rating Inventory of Executive Function) for school and pre-school age children 
to assessment instruments f 

MRI Ancillary Study Protocol Version 7.1 19Oct23 
12 
 
2. Evaluate renal function at 1- and 12 months follow-up looking for any evidence of early chronic renal 
disease 
a. Creatinine by fingerprick point-of-service test 
b. Urine sample for dipstick evaluation and albumin/Cr ratio 
3. Offer SARS-CoV-2 testing at enrollment which coincides with discharge from acute RCT admission to 
determine if the child had concurrent SARS-CoV-2 infection  
 
Rationale  
For adding BRIEF 
Behavioral abnormalities are well-documented sequelae in children surviving cerebral malaria (CM).5 Previous 
work with instruments developed for pediatric populations in the west, such as the Child Behavior Checklist, 
has shown a disappointing performance in our patient population. The BRIEF has been translated and is 
presently being used by the neuropsychological assessment team at QECH with promising early findings. This 
is an 86 question instrument with versions for both school age and preschool age children adding about 15 
minutes to the evaluations.  
 
For adding renal evaluations 
Since the clinical trial linked to this study began, a growing body of literature has begun to elucidate a 
connection between acute kidney injury (AKI), such as can occur with CM,40 and the subsequent risk of chronic 
kidney injury.41 Ibuprofen may contribute to renal injury. As such, there are stopping rules within the FEVER 
RCT protocol and data safety and monitoring plan to moderate these risks and the annual interim analysis (two 
conducted to date) specifically includes an unblinded analysis aimed at determining if there is evidence of 
excess renal injury in children allocated to the active study drug arm that includes ibuprofen. In this follow-up 
observational study of children who have completed the FEVER study, we are seeking to add renal function 
assessments to determine if there is evidence of chronic kidney disease (CKD) in CM survivors enrolled in 
FEVER and whether CKD is associated with study treatment allocation. This question is made even more 
relevant by a recent small clinical trial showing acetaminophen to be provide renal protection in malaria thought 
to be mediated by its ability to inhibit injurious plasma hemoglobin mediated oxidation.42  
For SARS-Cov-2 testing 
Cerebral malaria brain injury occurs in the setting of a proinflammatory cytokine cascade 43 8, increased 
excitotoxicity44, 45, refractory seizures mediated by excitotoxic neurotransmitters44, 45, and acceleration of free 
radical production.46, 47, 48 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) down regulates the 
angiotensin converting enzyme-2 receptor causing over activation of the renin-angiotensin system and 
producing disruption in vasodilation, neuroinflammation, oxidative stress and thrombotic response.49 
Consequently, malaria coinfections with SARS-CoV-2 could modify disease severity, worsen neurologic injuries 
and outcome and/or shift the age-susceptibility of malaria disease.50 If the inflammatory milieu of SARS-Co-V2 
further contributes to the endothelial dysfunction and blood brain barrier disruption associated with CM, this 
might contribute to prolonged parasitemia, more prolonged coma, increased cerebral edema (and thus higher 
mortality rates)51, more severe and refractory seizures, and a greater burden of neurological sequelae 
including epilepsy. A SARS-Cov-2 test is done at discharge from the acute admission when child is still not 
100% cognitively recovered. Assent is sought when the child returns for an MRI after the child is fully 
recovered and can participate in an assent process.  
 
Risk and Benefits 
 
Adding the BRIEF to the neurobehavioral outcomes assessments increases the evaluation time by about 15 
minutes and thus potentially increases annoyance and inconvenience This has been added to the consent 
form.  
 
Adding the renal assessments requires a finger prick and urine sample. Any child found to have evidence of 
CKD will be notified and an appropriate referral made to assure clinical follow-up is in place. Monitoring is 
indicated to assure timely initiation of antihypertensive treatment if needed. Careful documentation in the child’ 
medical record and in family education would also be undertaken so that renal toxic medications can be 

MRI Ancillary Study Protocol Version 7.1 19Oct23 
13 
 
avoided with any subsequent illness. Data regarding CKD outcomes will be provided to the FEVER Study for 
inclusion in interim analyses as well. 
 
We are seeking to obtain a swab for SARS-Cov-2 testing around the time of enrollment. SARS-CoV-2 
screening is not routinely conducted in the inpatient pediatric settings where FEVER is being conducted, there 
are no specific treatment for COVID and universal precautions with masking etc. are already in place. As such, 
there is little benefit to the patient to testing although notification that the child is positive may allow the family 
to make more informed decisions regarding behaviors and exposures. SARS-Cov-2 testing will be an option 
aspect of the consent. 
Addendum IV. 
Proposed Protocol Amendment version 6.1 to 7.0 
 
Amendment changes proposed 
1. Sedation changes/clarification 
a. In Malawi, melatonin will no longer be used for sedation. Children will be provided with a snack. 
b. In Zambia, if children are imaged at Medland Hospital and sedation is needed, this will be 
managed by their consultant anesthesiologist responsible for sedation 
2. In Zambia only, families will have the option of participating in an expanded study of epileptogenesis 
and autism that includes 
a. At enrollment, 10 question developmental screen and additional questions regarding past CNS 
injuries  
b. An EEG at the 1-and 12-month scheduled MRI Ancillary study follow-up.  
c. Additional follow-up at 6, 18 and 24 months’ post recovery from CM which will include an EEG, 
an epilepsy screening questionnaire, gross motor function classification system clinical 
assessment, neurological examination and EEG 
Rationale 
For sedation changes/clarifications 
IN MALAWI 
As per protocol, when sedation is needed, the Malawi team has used melatonin as the first line medication. 
They report that melatonin has been almost universally ineffective in their children especially for those children 
who have behavioral issues evident post CM. This then requires (as per protocol) administration of the second 
drug planned for sedation—chloral hydrate. Given the general failure of melatonin to provide sufficient sedation 
for MRI completion in the Malawian setting and the delays and inconvenience for the family when two drugs 
given in series are needed, melatonin will no longer be used for MRI sedation. If sedation is needed, chloral 
hydrate (the drug listed as second line in the original protocol) will be used. 
 
IN ZAMBIA 
Given technical and scheduling challenges at the Cancer Disease Hospital (CDH) and the need to assure that 
research patients do not displace imaging for clinical patients, imaging may occur at Medland Hospital. This 
contingency (i.e. imaging at private facilities) was outlined in the original approved protocol. Understandably, 
Medland Hospital policies prohibit non-Medland staff from administering sedation to their patients. As such, 
should sedation be needed for children imaged at Medland Hospital, this would be managed by the Consultant 
Anesthesiologist for Medland with the understanding that if light sedation with an oral medication is not 
sufficient, imaging will be deferred. No children will be required to have intravenous or injectable medications. 
 
AT BOTH SITES 
Children are often arriving for their follow-up and imaging evaluations having eaten nothing before an early 
departure from home. As such, we will provide food for them before their evaluations. 
 
For expanded study of epileptogenesis and autism at Chipata Central Hospital only 
After recovery from CM, ~10% of children will develop epilepsy although this will usually occur more than 12 
months after recovery and is thus an outcome that will not be captured in the MRI Ancillary Study which has 
only 1- and 12-month follow-ups.5 Within the context of this prospective cohort study, adding longer-term 

MRI Ancillary Study Protocol Version 7.1 19Oct23 
14 
 
follow-up could allow this important outcome to be identified. Additional funding through NINDS has been 
sought to support longer term follow-up with additional assessments at 6, 12, 18 and 24-months post CM 
recovery. A separate consent will be sought for this longer term study which will also address two additional 
scientific questions.  
 
Scientific query 1: Is the autism spectrum disorder (ASD) part of the neurodevelopmental problems 
that afflict children who have survived CM? It is well-established that CM places surviving children at risk of 
motor, sensory and language deficits.5 Whether they are also at risk of autism remains unknown. Autism is 
increasingly recognized as being among the contributors to childhood developmental problems globally and in 
Zambia. 52 Understanding whether CM-related brain injury is contributing to this burden would help inform 
public policy and care. To identify the development of autism and other contextual neurodevelopmental 
problems among children post CM recovery, this expanded study will include: 
 
At baseline—the 10 questions screen and additional queries re past CNS injuries 
At 6, 12, 18 and 24-months follow-up—expanded neurodevelopmental evaluation with the 23-Questions 
Developmental Assessment (23Q), the Gross Motor Function Classification System (GMFCS) clinical 
assessment, and a neurological examination. 
 
 
Scientific query 2: Can quantitative analysis of serial EEG data identify before the development of 
epilepsy those post CM children who will ultimately development epilepsy? Although 10% of children 
who survive CM can be expected to develop epilepsy, resource limited setting often do not have sufficient 
resources to provide ongoing follow-up and monitoring of all children who have recovered from severe malaria. 
Understandably, families are also often reluctant to continue regular follow-ups for a child who appears entirely 
well. Being able to recognize children who are going to develop epilepsy based upon EEG findings would allow 
for improvements in care and might inform future neuroprotective clinical trials. Identifying EEG changes that 
occur in association with epileptogenesis would also make substantial contributions to our understanding of 
this process. Professor Birbeck (C-PI for the MRI Ancillary study) in collaboration with Dr. Archana Patel (child 
neurologist and epileptologist affiliated with Zambia’s Paediatric Centre of Excellence and Boston Children’s 
Hospital), recently showed that quantitative analysis of acute EEG obtained with a standard scalp (i.e. non-
invasive) recording offers some capacity to predict which children will go on to develop epilepsy. 53 We propose 
to use these same quantitative analytic methods applied to the EEGs obtained during the acute CM admission 
and then at 1, 6, 12, 18 and 24 months while also completing a brief epilepsy screening questionnaire to 
identify incident epilepsy. Hence the expanded study will include: 
 
Epilepsy screening questionnaire and routine EEG at the 1, 6, 12, 18, and 24-month follow-up 
 
Risk and Benefits 
All evaluations completed as part of this expanded study of autism and epileptogenesis are non-invasive. No 
biological samples are required. While the data collection instruments used for this expansion include data 
such as admission quantitative parasite count during acute CM, this is data already being collected in the 
primary study and is not, in fact, novel information being collected for this expansion only. 
 
Adding the additional follow-ups at 6, 18 and 24 months requires time and effort from the family. A separate 
informed consent process will be undertaken and families may opt out of this longer term study but remain in 
the MRI Ancillary study with the shorter period of follow-up. Families will be compensated for their round trip 
travel as well as refreshments during their visit. All the same measures will be in place to protect the family’s 
privacy—all data will be entered into RedCap with only the study subject ID (no names). Paper-based records 
with identifiers will remain on site in locked study offices accessible only to research staff, all of whom have 
undergone training in the protection of human subjects.  
 
The expanded neurodevelopmental assessments and epilepsy screening tool may facilitate for a more 
expedient diagnosis of seizure disorders and/or developmental problems in children as they are recovering 
from CM. Identifying these problems earlier will allow for more rapid treatment including antiseizure 

MRI Ancillary Study Protocol Version 7.1 19Oct23 
15 
 
medications to prevent seizures, status epilepticus and seizure-related injuries, counseling of families 
regarding safety measures to prevent seizure-related injuries, communications with schools to encourage 
retention of children in school if behavioral issues limit their full engagement in school, and individualized 
therapy for children based upon their developmental challenges. Dr. Patel has substantial experience in this 
realm including at the PCOE DIC Clinic. Zambian staff have already undergone training to conduct the 
neurodevelopmental evaluations and during Dr. Patel’s time in Zambia (some months each year) she will 
further train staff in therapeutics. Essentially, care for any conditions identified over the course of the study will 
be provided through the facility where the work is being conducted. Epilepsy care services are already 
available at all study facilities and additional EEGs if needed for clinical care can be acquired.  
 
 
 
 
 
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