Positron emission tomography and magnetic resonance imaging in experimental human malaria to identify organ-specific changes in morphology and glucose metabolism: A prospective cohort study

Background Plasmodium vivax has been proposed to infect and replicate in the human spleen and bone marrow. Compared to Plasmodium falciparum, which is known to undergo microvascular tissue sequestration, little is known about the behavior of P. vivax outside of the circulating compartment. This may be due in part to difficulties in studying parasite location and activity in life. Methods and findings To identify organ-specific changes during the early stages of P. vivax infection, we performed 18-F fluorodeoxyglucose (FDG) positron emission tomography/magnetic resonance imaging (PET/MRI) at baseline and just prior to onset of clinical illness in P. vivax experimentally induced blood-stage malaria (IBSM) and compared findings to P. falciparum IBSM. Seven healthy, malaria-naive participants were enrolled from 3 IBSM trials: NCT02867059, ACTRN12616000174482, and ACTRN12619001085167. Imaging took place between 2016 and 2019 at the Herston Imaging Research Facility, Australia. Postinoculation imaging was performed after a median of 9 days in both species (n = 3 P. vivax; n = 4 P. falciparum). All participants were aged between 19 and 23 years, and 6/7 were male. Splenic volume (P. vivax: +28.8% [confidence interval (CI) +10.3% to +57.3%], P. falciparum: +22.9 [CI −15.3% to +61.1%]) and radiotracer uptake (P. vivax: +15.5% [CI −0.7% to +31.7%], P. falciparum: +5.5% [CI +1.4% to +9.6%]) increased following infection with each species, but more so in P. vivax infection (volume: p = 0.72, radiotracer uptake: p = 0.036). There was no change in FDG uptake in the bone marrow (P. vivax: +4.6% [CI −15.9% to +25.0%], P. falciparum: +3.2% [CI −3.2% to +9.6%]) or liver (P. vivax: +6.2% [CI −8.7% to +21.1%], P. falciparum: −1.4% [CI −4.6% to +1.8%]) following infection with either species. In participants with P. vivax, hemoglobin, hematocrit, and platelet count decreased from baseline at the time of postinoculation imaging. Decrements in hemoglobin and hematocrit were significantly greater in participants with P. vivax infection compared to P. falciparum. The main limitations of this study are the small sample size and the inability of this tracer to differentiate between host and parasite metabolic activity. Conclusions PET/MRI indicated greater splenic tropism and metabolic activity in early P. vivax infection compared to P. falciparum, supporting the hypothesis of splenic accumulation of P. vivax very early in infection. The absence of uptake in the bone marrow and liver suggests that, at least in early infection, these tissues do not harbor a large parasite biomass or do not provoke a prominent metabolic response. PET/MRI is a safe and noninvasive method to evaluate infection-associated organ changes in morphology and glucose metabolism.


Background Information
Morbidity and mortality from malaria infection remains significant despite antiparasitic treatments. Understanding the pathophysiology of disease may aid in the development of adjunctive therapies to improve survival in severe cases. Parasite sequestration in tissue microvasculature permits evasion of the reticulo-endothelial system and increased biomass [1]. Organ specific sequestration contributes to end organ dysfunction in severe malaria syndromes. Owing to the inaccessibility of sequestered parasites, estimations of total biomass and sites of sequestration have historically relied on biochemical markers [2][3][4][5], animal models [1, 6,7] and post mortem studies [8]. Determination of the biodistribution of malaria with functional nuclear medicine imaging may allow for more direct study of sequestration in human models. This may aid developing a better understanding of the organ dysfunction experienced in severe cases.
Nuclear medicine imaging techniques, particular hybrid PET/CT and PET/MRI have a central role primarily in oncology for assessing the biodistribution and activity of malignancy. The ability of these techniques to detect and locate biological and biochemical changes have more recently been applied to other medical fields including Infectious Diseases, though there are no studies in malaria.
This EXPLORATORY STUDY has been designed as a prospective pilot investigation to establish the role of 18 F FDG-PET/MRI in studying the pathophysiology of subpatent malaria following low dose Plasmodium falciparum exposure in healthy adults. Collection of baseline and post inoculation imaging will provide information about changes in host/parasite glucose metabolism that may be used to estimate parasite biomass and biodistribution. Glucose uptake is increased up to 100-fold in parasitised erythrocytes [9], suggesting that 18 F FDG may be a viable radiotracer to help evaluate the disease.
Whole body PET/MRI is a functional imaging modality that provides detailed soft tissue anatomical information with lower ionizing radiation exposure compared to equivalent PET/CT. The radiotracer 18 F FDG is a safe and well-validated biomimetic for demonstrating glucose uptake. Imaging with 18 F FDG-PET/MRI is an ideal model for exploring nuclear medicine functional imaging in a healthy human volunteer population.
Biochemical estimates of parasite biomass and parasitaemia will be compared to quantified radiotracer uptake measurements. Comparison of interval changes in host/parasite glucose metabolism will add to our understanding of the metabolic changes associated with disease [10]. Although there have been several trials using MRI imaging to study malaria, none to date have been prospective. Comparison of MRI brain data with existing literature in uncomplicated malaria may help further describe the changes seen in early disease [11]. The feasibility of 18 F FDG-PET/MRI may contribute to developing a further role for nuclear medicine imaging in malaria.
Successful application of nuclear medicine imaging would provide a greater understanding of parasite sequestration dynamics, which may aid in disease modeling and development of treatments to prevent end organ damage in severe disease.

Study Objectives
Primary: • To investigate the use of 18 F FDG-PET/MRI to estimate the biodistribution of P. falciparum in human volunteer induced blood stage malaria studies. • To investigate the use of 18 F FDG-PET/MRI to estimate the biomass of P. falciparum. Secondary: • To describe the relative burden of organ-specific tissue sequestration in P. falciparum. • To describe the impact of early malaria infection on glucose metabolism. • To describe the MRI brain findings of early malaria infection in a prospective participant cohort.

i. Research Question
The present EXPLORATORY STUDY has been designed to establish the role of 18 F FDG-PET/MRI in studying the pathophysiology of subpatent malaria following low dose Plasmodium falciparum exposure in healthy adults.

Primary
The primary objective of the study is to assess the application of whole body 18 F FDG-PET/MRI in describing the parasite biodistribution and biomass in subpatent malaria. This is a hypothesis generating pilot investigation expected to have predominately descriptive outcomes.
The primary outcome measures will be the quantified and semi-quantified 18 F FDG uptake values from specific regions of interest. Regions of interest for quantification of uptake will be spleen, bone marrow (lumbar spine and/or pelvis), muscle bulk (quadriceps) and brain. Post inoculation measurements will be compared to baseline pre inoculation uptake values. Biochemical markers of parasitaemia and total biomass from MAIN study data will be compared to 8 F FDG uptake values.
Any other regions of interested (as deemed by the investigators) identified after imaging will have semi-quantified 18 F FDG uptake values calculated for further comparison.

Secondary
The relative burden of organ specific sequestration will be determined by comparing 18 F FDG uptake values for each region of interest.
The impact of early malaria infection on glucose metabolism will be evaluated in assessing each of the above outcomes.
The MRI findings of early malaria will be presented in a descriptive manner, outlining any interval changes in high-resolution brain MRI for this prospective cohort.

iii. Study Rationale
This EXPLORATORY STUDY has been designed to establish the role of 18 F FDG-PET/MRI in studying the pathophysiology of subpatent malaria. The selection of this radiotracer and imaging modality offers a well-validated model for exploring nuclear medicine functional imaging of malaria infection in a healthy human volunteer population.

Hypotheses
• Whole body 18 F FDG-PET/MRI is a technically feasible imaging modality for the study of subpatent malaria infection. • Quantitative and semi-quantitative uptake of 18 F FDG is proportional to estimated parasite biomass and parasitaemia. • Subtle changes in the vasculature of the brain are present and identifiable on MRI imaging in subpatent malaria infection.

iv. Potential Risks and Benefits
Potential Risks There is a small risk associated with radiation exposure from the radiotracer 18 F FDG. This falls into the low risk category of the ARPANSA guidelines. This is outlined in the radiation assessment report (Appendix A). There is a small risk of perturbation in blood glucose levels following 18 F FDG administration. To minimize this risk all participants will undergo blood glucose testing at screening and further testing prior to any administration if there are clinical concerns to ensure they are suitable to receive the radiotracer. There is a very small risk of a reaction to the infusion of the radiotracer 18 F FDG. This is outlined in section 8.4. A product information sheet for 18 F FDG will be provided to each participant (Appendix G.) There is the risk of incidental abnormalities being identified on whole body PET/MRI imaging. These will be managed on an individual basis in consultation between the reporting radiologist, study doctor, principal investigator and participant.
Other risks, including those associated with IBSM inoculation and blood collection are as described in the MAIN study. EXPLORATORY STUDY involvement is not expected to alter these pre-existing risks.

Potential Benefits
There are no direct health benefits from participation in this EXPLORATORY STUDY.
Other potential benefits are as described in the MAIN study.

STUDY DESIGN
This is an EXPLORATORY STUDY comprising a population recruited from a singlecenter, IBSM model MAIN study. This EXPLORATORY STUDY is a pilot investigation to establish the role of 18 F FDG-PET/MRI in studying the pathophysiology of subpatent malaria. The population will consist of two healthy adults inoculated with P. falciparum from existing MAIN study population.
MAIN study refers to the parallel IBSM MAIN study QP15C20 SJ733 Participants will receive two whole body PET/MRI scans: the first within one week prior to MAIN study inoculation and the second one to two days prior to confinement with peak parasitaemia post BSP inoculation (estimated MAIN study InD 6 to 7). Blood samples will be collected at MAIN study timepoints.

Preparation:
At screening, participants will be consented for EXPLORATORY STUDY inclusion. At the time of consent the participant and study doctor will complete the MRI checklist (see Appendix B) and education regarding pre-imaging preparation. In the 24 hours prior to image collection, a low carbohydrate diet is to be followed, and strenuous exercise is to be avoided. These activities are to be recorded on the provided diet and activity sheet (see Appendix C).
On the day of imaging, participants are advised to wear warm clothing and arrive fasted for 6 hours prior (water is permitted, and good hydration encouraged). Participants will arrive to HIRF where the MRI checklist and diet and activity checklist will be reviewed. A peripheral intravenous cannula will be inserted for radiotracer administration. A bedside blood glucose measurement will be measured if there is clinical suspicion of a blood glucose abnormality.
Whole body imaging will be performed on the Biograph mMR PET/MRI system after the intravenous infusion of the radiotracer 18 F FDG. Dynamic tracer uptake will be recorded over a 45-60 minute period from the abdomen for quantitative measurement of FDG uptake in the spleen. Participants will be offered a short break, followed by collection of static images of the whole body and brain, over an estimated 30 minute period.
Dedicated brain imaging will take place using the MAGNETOM Prisma 3T MRI system. This includes MP2RAGE, T2FLAIR and diffusion weighted MRI sequences of the brain for assessment of the effects of very subtle oedema and inflammatory responses. The estimated time of image acquisition is 45 minutes per patient. Depending on initial results, there may be some changes to MRI sequences to improve data collection. Participant activities or exposures will not be affected by any technical imaging sequence change.
Following image acquisition participants will be allowed food and drink and encouraged to drink water. Each HIRF visit is approximately 4 hours in total duration.

Image Interpretation:
Collected images will be reviewed and reported by specialist radiologists at HIRF/RBWH. Imaging metrics (FDG uptake and kinetic parameters) will be compared between baseline and follow up scans. Quantitative 18 F FDG uptake will be calculated using Patlak model analysis from dynamic uptake imaging, yielding a Ki value ( 18 F FDG influx constant.) A nominated region of interest will have quantitative 18 F FDG uptake calculated for each scan. Semi-quantitative 18 F FDG uptake will be calculated using SUVs measured during static uptake imaging. All regions of interest will have SUVs calculated.

Other data collection:
Other blood samples will be collected as per the MAIN study.

Evaluation of data:
All data will be presented descriptively. Pre and post inoculation quantitative imaging metrics will be compared with paired T-tests or Mann-Whitney U tests. Groups will be compared with unpaired T-tests with consideration of T-value adjustment for population size and Mann-Whitney U tests. Any groups identified based on imaging results will be described with respect to demographic, clinical and biochemical data collected as part of the MAIN study.

PARTICIPANT ENROLLMENT AND WITHDRAWAL 4.1 Recruitment
Following receipt and signing the MAIN study consent forms, subjects will be fully informed of the nature of this optional EXPLORATORY STUDY, and the specific risks associated with this EXPLORATORY STUDY. A separate 'Participation Information Sheet and Consent Form' will be provided for this purpose (Appendix D).
The 'Informed Consent' will be signed and dated by the participants in the presence of an investigator. Subjects will also be given a copy of their signed 'Informed Consent'. A copy of the HIRF site map (Appendix E), diet and activity sheet (Appendix C) and 18 F FDG Product Information (FDA information, see section 12. Appendix G) will be provided for participant reference.

Eligibility Criteria 4.2.1 Inclusion Criteria
In order to be eligible to participate in this study, an individual must meet all of the following criteria: • Provide signed and dated informed consent form • Able to lie supine and still for duration of image acquisition • All other criteria as outlined in MAIN study protocol(s)

Exclusion Criteria
An individual who meets any of the following criteria will be excluded from participation in the study: • Known allergic reactions to components of the study radiotracer 18

Participant Withdrawal
Participants have the right to withdraw from the study at any time for any reason. The investigator also has the right to withdraw patients from the study in the event of any clinical adverse event (AE), laboratory abnormality, or other medical condition or situation occurs such that continued participation in the study would not be in the best interests of the participant OR the participant meets and exclusion criterion (either newly developed or not previous recognized) that precludes further study participation.
Following consent, data acquired up until withdrawal will be included in the EXPLORATORY STUDY. No further samples will be collected from the time of withdrawal notification.

Permanent Termination or Suspension of Exploratory Study
The principal investigator(s), Human Research Ethics Committee (HREC) and Regulatory Authorities independently reserve the right to discontinue the study at any time for safety or other reasons. This will be done in consultation with the MAIN study sponsor where practical. The MAIN study sponsor, in consultation with the investigators may request for suspension of the EXPLORATORY STUDY.
After such a decision, the investigator(s) will ensure that adequate consideration is given to the projection of the participants' interests. The investigator must review all participants as soon as practical and complete all required records.

STUDY INVESTIGATIONS
Whole body imaging will be performed using the Biograph mMR PET/MRI system, with concurrent PET and MRI acquisition. Dedicated Brain MRI sequences will be performed using the MAGNETOM Prisma 3T MRI system. This equipment is property of HIRF.
The standard radiotracer 18 F FDG will be used for PET imaging. This radiotracer has previously been approved by the Australian Register of Therapeutic Goods as a consumable for use in PET imaging (Austin Health) and attracts an MBS rebate for use in multiple conditions.
The radiotracer is to be purchased from the RBWH Nuclear Medicine Department, a TGA licensed manufacturer of 18 F FDG. The 18 F FDG is produced under the Good Manufacturing Practice (GMP) conditions in accordance with the British Pharmacopeia (see Appendix F.). The research will not alter the formulation of the product. The research will be using an identical product in an identical way in an identical formulation to that used for clinical diagnostic PET imaging. 18 F FDG is supplied as a clear/colourless or slightly yellow solution containing the radionuclide 18 F conjugated to the biologically active ligand glucose. It is administered as an intravenous injection prior to imaging.
All radiotracer for use will be released as per existing RBWH Nuclear Medicine department practices. Dosing of the radiotracer is at a standard weight-based dosage. This is determined based on participant radiation exposure, not pharmacological effect. The effective dose of the metabolically active ligand (FDG) is considered a micro-dose.
Due to the unique nature of radiopharmaceuticals 18 F FDG is considered an investigational product by the TGA for purposes of this research project.

Radiotracer Dosing Regimen
• Prior to administration of 18 F FDG participants are to fast for roughly 6 hours.
• Prior to administration of 18 F FDG participants are to avoid strenuous exercise and adhere to a low carbohydrate diet for 24 hours.
An estimated dose of 4.5MBq per kg (based on screening weight) 18 F FDG is to be administered by infusion (minimum dose 90MBq, maximum dose 400MBq). This is the lowest possible dose to yield reliable data. The radioactive half-life of 18 F FDG is 110 minutes. Estimated radiation exposure is included in the radiation assessment report (see Appendix A).
• Following administration of 18 F FDG participants are encouraged to drink water to promote the renal excretion of radiotracer.

Modification of Radiotracer Administration for a Participant
Any participant experiencing an AE to any part of the investigation (participation in imaging process or infusion of radiotracer) will be individually evaluated. Where it is considered unsafe to repeat the process by the investigator(s) in discussion with the medical monitor, no further imaging will take place.

Participant Compliance
Participant non-compliance with preparation for image acquisition or non-compliant with instruction during imaging itself (e.g. excessive movement) will be recorded in the participant file as this may have an impact on the quality of data collected. This will include reference to the diet and exercise activity sheet assessed prior to imaging.

Radiotracer Manufacture, Handling and Accountability
The formulation, packaging and labeling of the standard radiotracer 18 F FDG is defined by licensed manufacturer. The 18 F FDG is stored at room temperature in accordance to the conditions set by the manufacturer.
Handling of the radiopharmaceutical will follow the standard HIRF protocols for all injectable radiopharmaceuticals The 18 F FDG will be ordered on an as needed basis. The 18 F FDG is supplied on the day of each study. The product expiry time is on the same day as supply due to the short ½ life of the radioactive element. There will be no excess product. The administration of the GMP approved standard radiotracer 18 F FDG will be as per existing HIRF practices, including record keeping of 18 F FDG lot number, patient injected activity and time of administration using the Venstra system. Product accountabilty and quality control will be as per existing RBWH Nuclear Medicine Department practices under the Quality GMP Agreement for TGA licensing for the manufacture and supply of radiopharmaceuticals.

STUDY SCHEDULE 6.1 Screening
Participants consenting for participation in this EXPLORATORY STUDY must meet all inclusion and exclusion criteria for both MAIN and EXPLORATORY STUDY participation.
This includes EXPLORATORY STUDY specific screening for: • Fasted blood glucose (exclude if >6.0mmol/L) • Significant prior radiation exposure precluding participation (see exclusion criteria) • MRI checklist completion, including evaluation for claustrophobia and ability to comfortably lie supine that may preclude participation • Evaluation of patient understanding of preparation for imaging acquisition instructions: fast 6 hours prior; abstain from vigorous exercise and adherence to low carbohydrate diet 24 hours prior.

Enrollment/Baseline
Baseline fasted blood glucose and MRI checklist must be completed. Low carbohydrate diet options will be discussed.

Intermediate Visits
The first visit (within one week prior to MAIN study inoculation) will include whole body PET/MRI scanning.

Final Exploratory Study Visit
The second visit (MAIN study one to two days prior to confinement, InD 6 to 7) will include whole body PET/MRI scanning. This will also be the final study visit. Any AEs that require follow up will be reviewed at subsequent MAIN study visits.

Withdrawal Visit
A withdrawal visit coinciding with a scheduled MAIN study visit (or MAIN study withdrawal visit) will take place to review imaging results as required.

Unscheduled Visit
There are no unscheduled visits expected for this EXPLORATORY STUDY. In any case where an unscheduled visit is required, attempts will be made to ensure this occurs at a pre-existing MAIN study visit. prior recorded -insertion of a peripheral intravenous cannula -review of MRI checklist -infusion of 18 F FDG via peripheral intravenous cannula -image acquisition -participant supine on scanner for duration of image collection -image review and reporting by specialist radiologist -18 F FDG uptake quantification by Patlak model analysis by medical physicist, SUV semiquantification of remaining regions of interest • Dedicated brain MRI imaging -image acquisition -participant supine on scanner for duration of image collection -image review and reporting by specialist radiologist

CLINICAL LABORATORY EVALUATIONS
MAIN study fasted blood glucose measurements will be used as part of the EXPLORATORY STUDY screening process. Bedside capillary blood glucose measurement will take place prior to PET/MRI scanning if there are clinical concerns. MAIN study baseline and serial safety blood tests (full blood count and biochemistry) will be used for comparison of imaging results.

SPECIAL ASSAYS OR PROCEDURES
Collection of blood samples for parasitaemia and parasite biomass measurements will take place as part of the MAIN study protocols.

ASSESSMENT OF SAFETY 8.1 Specification of Safety Parameters
As per MAIN study

Definition of an Adverse Event and of a Serious Adverse Event ADVERSE EVENT
Any untoward medical occurrence in a patient or clinical investigation participant administered a pharmaceutical product and which does not necessarily have a causal relationship with this 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 product, whether or not related to the product.

SERIOUS ADVERSE EVENT
A serious AE is any untoward medical occurrence that, at any dose: a) Results in death b) Is life threatening (the term 'life-threatening' in the definition of 'serious' refers to an event in which the participant was at risk of death at the time of the event. If does not refer to an event, which hypothetically might have caused death if it were more severe. c) Required hospitalization or prolongation of an existing hospitalization (hospitalization for elective treatment of a pre-existing condition that did not worsen from baseline is not considered an AE) d) Results in disability/incapacity e) Is a congenital abnormality/birth defect.
Use medical and scientific judgment when deciding whether reporting is appropriate in other situations, such as other important medical events that may not be immediately life-threatening or result in death or hospitalization, but may jeopardize the participant or may require medical or surgical intervention to prevent one of the outcomes listed in the above definition.

SUSPECTED UNEXPECTED SERIOUS ADVERSE REACTION (SUSAR)
A suspected unexpected serious adverse reaction is an AE that is determined to be related to the radiotracer and is both serious and unexpected. The term 'unexpected' means that nature or severity of the event is not consistent with the applicable product information (Appendix G.)

Documentation and classification of study specific adverse events
For regulatory purposes to use the standard radiotracer 18 F FDG in this EXPLORATORY STUDY all AEs deemed related to radiotracer administration will be documented in an EXPLORATORY STUDY AE log including diagnosis, severity, toxicity, date of onset and resolution and any action taken. Toxicity will be determined using existing CTCAE version 4.0 criteria, consistent with those proposed for use in the MAIN studies. Severity will be determined based on the following scale: o Mild: does not interfere with participant's usual function o Moderate: interferes to some extent with participant's usual function o Severe: interferes significantly with participant's usual function All AEs will be recorded in the MAIN study participant file as per MAIN study processes. Where an AE is deemed a result of EXPLORATORY STUDY activities, this will be made clear in documentation and discussed with the MAIN study principal investigator prior to any appropriate escalation to MAIN study medical monitor and sponsor.

Relationship to Study Investigation
For the purposes of this study for an AE to be considered related to 18 F FDG it must occur within 5 half-lives, or 550 minutes of administration. For an AE to be considered related to other imaging procedures, it must occur during the procedure itself.
Any AEs deemed related to the above EXPLORATORY STUDY procedures will be documented in the EXPLORATORY STUDY AE log by one the Investigator or one of his representatives.

EXPECTEDNESS OF SAEs
There are no specific expected SAEs from participation in whole body PET/MRI or dedicated brain MRI imaging.
Incidental abnormalities detected on imaging will be discussed between the reporting radiologist, study doctor, principal investigator and participant. If required, further review will be arranged by referral to the general practitioner or appropriate specialist. Any incidental findings are expected to be asymptomatic and unpredictable, given the study population of otherwise well, healthy adult volunteers.
There are no commonly reported adverse reactions to radiopharmaceuticals and 18 F FDG in the literature (Appendix G). A multicenter review of over 80 000 administrations found no reported AEs [12]. A single case of a probable cutaneous adverse drug reaction has been reported in an individual with multiple existing drug allergies [13]. This did not preclude the patient from repeated dosing. A single case episode of possible anaphylaxis has been reported in the context of the administration of multiple other medications [14]. In both cases symptomatic treatment was effective. Based on the volume of 18 F FDG use in the clinical setting, and paucity of published adverse events, the risk of such events is considered to be extremely low.

Recording and Prompt Reporting of Events
In all cases of AEs, the reporting process used for the MAIN study from which the participant was recruited will be used. If MAIN study procedures are not available recording and reporting of AEs will be in accordance with QIMRCTSOP013 and QIMRCTSOP016.
Any AE deemed related to the administration of 18 F FDG or other imaging procedures will be discussed where possible to further clarify causality with the nominated Nuclear Medicine Independent Medical Advisor.
Any SAE deemed related to the administration of 18 F FDG or other imaging procedures will be reported to the Sponsor within 24 hours following discussion with the medical monitor and where possible with the nominated Nuclear Medicine Independent Medical Advisor. The TGA and any other relevant authorities will be notified by the Sponsor (or institutionally approved representative) within 72 hours as per existing TGA guidelines.

REPORTING OF PREGNANCY
The MAIN study from which EXPLORATORY STUDY participants will be recruited will only include males and women of non-childbearing potential.

Halting Rules
The investigator(s) reserve the right to halt the study at any point. There are no specific safety findings expected that may prompt suspension or termination of the EXPLORATORY STUDY. In a situation where the MAIN study is halted, the EXPLORATORY STUDY will also be halted as required.

QUALITY ASSURANCE
EXPLORATORY STUDY materials and quality assurance related to the imaging of participants and any involvement onsite at HIRF will take place as per existing HIRF/RBWH processes. Data quality will be considered and described in the presentation of results.
The MAIN study is a registered clinical trial employing an external study monitor responsible for quality assurance with respect to inoculum and participant safety (including pathology testing and clinical examination/investigations.)

STATISTICAL CONSIDERATIONS 10.1 Sample Size
This is a pilot investigation to assess the application of functional nuclear medicine in subpatent malaria. The population will comprise of two adults (male and female of non-childbearing potential) participants between 18 and 55 years of age, recruited from the IBSM model MAIN study population.
It is expected that this study will be hypothesis generating. There are no pre-existing PET or prospective MRI imaging trials in malaria published in the literature. Data from this pilot investigation may be used in future power calculations if required.

Statistical Analysis Plan
All data will be presented descriptively. Pre and post inoculation quantitative imaging metrics will be compared with paired T-tests or Mann-Whitney U tests (statistical significance p<0.05). Imaging results may be described with respect to demographic, clinical and biochemical data collected as part of the MAIN study.

SOURCE DOCUMENTS AND ACCESS
Upon request, the investigator(s)/institution(s) will permit direct access to source data/ documents for trial-related monitoring, audits, HREC review, and regulatory inspection(s) by the Sponsor (or their appropriately qualified delegate) and Regulatory Authorities provided this is feasible from a technical standpoint. Direct access includes examination, analysis, verification and reproduction of records and reports that are important to the evaluation of the study.

ADMINISTRATIVE PROCEDURES ETHICAL CONSIDERATIONS
The amount of blood to be sampled in the study is not considered to be excessive in healthy adult participants. This study will be carried our according to the Declaration of Helsinki, the NHMRC National Statement on Ethical Conduct in Human Research (2007) and the Notes for Guidance on Good Clinical Practice as adopted by the Australian Therapeutic Goods Administration (2000) (CPMP/CH/135/95) and the ICH GCP Guidelines.
ETHICAL REVIEW COMMITTEE This Protocol will be submitted for approval to QIMR-B HREC, Clinical Trial Protocol Committee. This protocol will then be submitted for executive approval from RBWH HREC. Following this the HIRF Scientific Advisory Committee will review the Protocol and QIMR-B HREC assessment(s) in order to facilitate expedited Site-Specific Approval from the RBWH/Metro North HREC. Written approvals will be obtained before volunteers are recruited and participants are enrolled. The Investigators will receive all the documentation needed for submitting the present Protocol to the HREC. It is the responsibility of the Investigator to report study progress to the HREC as required or at intervals not greater than one year.
An earlier version of this protocol was submitted to the QIMR Clinical Trials Protocol Committee and was referred for review by the Human Scientific Sub-Committee on the grounds that the proposed study is not a clinical trial. Subsequent discussion with the TGA and study sites have established that the HREC review process described above is required to meet the regulatory requirements for research using the standard radiotracer 18 F FDG and between the nominated sites.
REGULATORY AUTHORITIES This is an EXPLORATORY STUDY to run in parallel to an existing MAIN study clinical trial. The investigation uses the application of imaging using a standard radiotracer at standard dosages that is widely used in the clinical setting. This is administered as per existing local institution processes. Due to the unique nature of radiopharmaceuticals, facilities are able to extemporaneously produce and administer FDG onsite for their own purposes. The RBWH Nuclear Medicine department has TGA approval for manufacture of this radiotracer (Appendix F). This radiotracer is not ARTG listed for this facility, but is approved for GMP production and commercial sale from this manufacturer to other facilities within the state of Queensland. Following discussion with the TGA and study sites, this EXPLORATORY STUDY has a CTN registration for the regulatory purpose of accessing the standard radiotracer 18 F FDG in a manner that ensures prospective record keeping and transparent usage. This includes processes for AE reporting as documented below.
An independent nuclear medicine medical advisor has been nominated to ensure participant safety, to provide advice and ongoing review of any SAEs that may be related to this EXPLORATORY STUDY activities. No DSMB has been nominated given the low risk nature of this small population EXPLORATORY STUDY. Participants will receive a copy of a current FDA template for 18 F FDG product information (Appendix G), as no generic equivalent exists in Australia.

INFORMED CONSENT
Before recruitment and enrolment into the study, each prospective participant must be given a full explanation of the nature and purpose of the study, and a copy of the Participant Information Sheet and Consent Form to review. This form complies with the consent form requirements outlined in the TGA guidelines. Once the essential study information has been provided, and the Investigator is assured that each individual volunteer understands the implications of participating in the study, the participants will be asked to give consent to participate in the study by signing the informed consent form. The consent form shall be signed and dated by the appropriate parties. A notation that written informed consent has been obtained will be made on the participants' MAIN study file. The completed consent forms will be retained by the Investigator and a copy of these will be provided by the Investigator to the participant.
PARTICIPANT RE-IMBURSEMENT Participants who are found eligible and complete the study will be compensated $160 for their time. This is in addition to MAIN study compensation.
EMERGENCY CONTACT WITH INVESTIGATORS All participants will be provided with a Participant Information Sheet and Informed Consent Form with contact details of whom to contact in the case of an emergency related to the EXPLORATORY STUDY.
Participants will also be provided with a contact card as per the MAIN study.

NOTIFICATION OF PRIMARY CARE PHYSICIAN
Where it is deemed appropriate by the investigator(s), study doctor and/or reporting radiologist a letter will be sent to the physician stating the nature of the EXPLORATORY STUDY including any adverse events or incidental findings. A copy shall be retained by the study site as part of the MAIN study participant file.
PROTOCOL DEVIATIONS All protocol deviations must be reported to the principal investigator. Protocol deviations will be assessed for significance by the principal investigator. Those deviations deemed to have a potential impact on the integrity of the study results, patient safety or the ethical acceptability of the trial will be reported to the HREC prior to completion of participant enrollment. Where deviations to the protocol identify issues for protocol review, the protocol may be amended as above.

RETENTION OF OTHER STUDY SPECIFIC SAMPLES
Electronic imaging data will be retained as per HIRF guidelines. No other samples will be collected.

DATA HANDLING, RECORD KEEPING AND PUBLICATION POLICY
DATA CONFIDENTIALITY Only the investigators, designated staff and designated MAIN study staff will have access to information that identifies a study participant. All MAIN study participants will be assigned a unique identifier. Participant identifiers in this EXPLORATORY STUDY will be consistent with those used in corresponding MAIN studies.

DATA CAPTURE METHODS
All imaging data will be collected in accordance with site-specific HIRF protocols.
TYPES OF DATA Imaging data, reports and quantitative imaging metrics will be electronically stored in accordance with HIRF guidelines.

RECORD KEEPING
All study related documents and records are to be retained for a minimum of fifteen years after MAIN study completion. Written agreement from the Sponsor must precede destruction of the same. In cases where HIRF guidelines preclude the storage of source imaging data, data will be transferred to the sponsor for storage.

PUBLICATIONS POLICY
Publication and reporting of results and outcomes of this trial will be accurate and honest, undertaken with integrity and transparency and in accordance with QIMR BERGHOFER's Policy on Criteria for Authorship. Publication of results will be subjected to fair peer-review. Authorship will be given to all persons providing significant input into the conception, design, execution or reporting of the research according to QIMR BERGHOFER Policy on the Criteria for Authorship. Phase: exploratory

Background Information
Morbidity and mortality from malaria infection remains significant despite antiparasitic treatments. Understanding the pathophysiology of disease may aid in the development of adjunctive therapies to improve survival in severe cases. Parasite sequestration in tissue microvasculature permits evasion of the reticulo-endothelial system and increased biomass [1]. Organ specific sequestration contributes to end organ dysfunction in severe malaria syndromes. Owing to the inaccessibility of sequestered parasites, estimations of total biomass and sites of sequestration have historically relied on biochemical markers [2][3][4][5], animal models [1, 6,7] and post mortem studies [8]. Determination of the biodistribution of malaria with functional nuclear medicine imaging may allow for more direct study of sequestration in human models. This may aid developing a better understanding of the organ dysfunction experienced in severe cases.
Nuclear medicine imaging techniques, particular hybrid PET/CT and PET/MRI have a central role primarily in oncology for assessing the biodistribution and activity of malignancy. The ability of these techniques to detect and locate biological and biochemical changes have more recently been applied to other medical fields including Infectious Diseases, though there are no studies in malaria.
Other advanced imaging techniques including dedicated MRI imaging have been successfully applied to examine the pathophysiology of malaria, predominately in patients presenting with severe disease. There are few studies in early or uncomplicated infection, and none of these studies have been prospective. As a result there remains a paucity of imaging data in the early stages of infection.
This EXPLORATORY STUDY has been designed as a prospective pilot investigation to establish the role of 18 F FDG-PET/MRI and advanced dedicated MRI imaging techniques in studying the pathophysiology of subpatent malaria following low dose Plasmodium exposure in healthy adults. Collection of baseline and post inoculation PET/MRI imaging will provide information about changes in host/parasite glucose metabolism that may be used to estimate parasite biomass and biodistribution. Glucose uptake is increased up to 100-fold in parasitised erythrocytes [9], suggesting that 18 F FDG may be a viable radiotracer to help evaluate the disease.
Whole body PET/MRI is a functional imaging modality that provides detailed soft tissue anatomical information with lower ionizing radiation exposure compared to equivalent PET/CT. The radiotracer 18 F FDG is a safe and well-validated biomimetic for demonstrating glucose uptake. Imaging with 18 F FDG-PET/MRI is an ideal model for exploring nuclear medicine functional imaging in a healthy human volunteer population.
Biochemical estimates of parasite biomass and parasitaemia will be compared to quantified radiotracer uptake measurements. Comparison of interval changes in host/parasite glucose metabolism will add to our understanding of the metabolic changes associated with disease [10]. The feasibility of 18 F FDG-PET/MRI may contribute to developing a further role for nuclear medicine imaging in malaria.
Dedicated MRI imaging can demonstrate inflammation other subtle metabolic changes, including changes in vascular function. Comparison of interval changes in dedicated MRI imaging will add to our understanding of early disease progression and permit comparison with the existing literature in more advanced disease.
Successful application of advanced medical imaging would provide a greater understanding of parasite sequestration dynamics, which may aid in disease modeling and development of treatments to prevent end organ damage in severe disease.

Study Objectives
Primary: • To investigate the use of 18 F FDG-PET/MRI to estimate the biodistribution of Plasmodium species in human volunteer induced blood stage malaria studies. • To investigate the use of 18 F FDG-PET/MRI to estimate the biomass of Plasmodium species. • To investigate the use of advanced dedicated MRI techniques to early Plasmodium infection. Secondary: • To describe the relative burden of organ-specific tissue sequestration in Plasmodium infection. • To describe the impact of early malaria infection on glucose metabolism. • To describe the MRI brain findings of early malaria infection in a prospective participant cohort.

i. Research Question
The present EXPLORATORY STUDY has been designed to establish the role of 18 F FDG-PET/MRI and advanced dedicated MRI techniques in studying the pathophysiology of subpatent malaria following low dose Plasmodium exposure in healthy adults.

Primary
The primary objective of the study is to assess the application of whole body 18 F FDG-PET/MRI in describing the parasite biodistribution and biomass in subpatent malaria. This is a hypothesis generating pilot investigation expected to have predominately descriptive outcomes.
The primary outcome measures will be the quantified and semi-quantified 18 F FDG uptake values from specific regions of interest. Regions of interest for quantification of uptake will be spleen, bone marrow (lumbar spine and/or pelvis), muscle bulk (quadriceps) and brain. Post inoculation measurements will be compared to baseline pre inoculation uptake values. Biochemical markers of parasitaemia and total biomass from MAIN study data will be compared to 8 F FDG uptake values.
Any other regions of interested (as deemed by the investigators) identified after imaging will have semi-quantified 18 F FDG uptake values calculated for further comparison.
The other primary objective seeks to apply advanced MRI techniques to early Plasmodium infection. This will generate descriptive outcomes and inform future MRI sequence selection to optimize imaging in malaria.

Secondary
The relative burden of organ specific sequestration will be determined by comparing 18 F FDG uptake values for each region of interest.
The impact of early malaria infection on glucose metabolism will be evaluated in assessing each of the above outcomes.
The MRI findings of early malaria will be presented in a descriptive manner, outlining any interval changes in high-resolution brain MRI for this prospective cohort.

iii. Study Rationale
This EXPLORATORY STUDY has been designed to establish the role of 18 F FDG-PET/MRI and advanced MRI techniques in studying the pathophysiology of subpatent malaria. The selection of this radiotracer and imaging modality offers a well-validated model for exploring nuclear medicine functional imaging of malaria infection in a healthy human volunteer population.
Hypotheses  Whole body 18 F FDG-PET/MRI is a technically feasible imaging modality for the study of subpatent malaria infection.
 Quantitative and semi-quantitative uptake of 18 F FDG is proportional to estimated parasite biomass and parasitaemia.  Subtle changes in the vasculature of the brain are present and identifiable on MRI in subpatent malaria infection.

iv. Potential Risks and Benefits
Potential Risks There is a small risk associated with radiation exposure from the radiotracer 18 F FDG. This falls into the low risk category of the ARPANSA guidelines. This is outlined in the radiation assessment report (Appendix A). There is a small risk of perturbation in blood glucose levels following 18 F FDG administration. To minimize this risk all participants will undergo blood glucose testing at screening and further testing prior to any administration if there are clinical concerns to ensure they are suitable to receive the radiotracer. There is a very small risk of a reaction to the infusion of the radiotracer 18 F FDG. This is outlined in section 8.4. A product information sheet for 18 F FDG will be provided to each participant (Appendix G.) There is the risk of incidental abnormalities being identified on whole body PET/MRI imaging. These will be managed on an individual basis in consultation between the reporting radiologist, study doctor, principal investigator and participant.
Other risks, including those associated with IBSM inoculation and blood collection are as described in the MAIN study. EXPLORATORY STUDY involvement is not expected to alter these pre-existing risks.

Potential Benefits
There are no direct health benefits from participation in this EXPLORATORY STUDY.
Other potential benefits are as described in the MAIN study.

STUDY DESIGN
This is an EXPLORATORY STUDY comprising a population recruited from singlecenter, IBSM model MAIN studies. The population will consist of up to eight healthy adults inoculated with Plasmodium species parasite from existing MAIN study population.
MAIN study refers to the parallel IBSM MAIN study from which the participant is drawn from.
Participants will receive two scans: the first within one week prior to MAIN study inoculation and the second one to two days prior to confinement with peak parasitaemia post BSP inoculation (estimated InD 6 to 7 for P. falciparum and InD 8 to 9 for P. vivax). Blood samples will be collected at MAIN study timepoints, with no additional collections planned for the EXPLORATORY STUDY.

Preparation:
At screening, participants will be consented for EXPLORATORY STUDY inclusion. At the time of consent the participant and study doctor will complete the MRI checklist (see Appendix B) and education regarding pre-imaging preparation. In the 24 hours prior to PET/MRI image collection, a low carbohydrate diet is to be followed, and strenuous exercise is to be avoided. These activities are to be recorded on the provided diet and activity sheet (see Appendix C).
On the day of PET/MRI imaging, participants are advised to wear warm clothing and arrive fasted for 6 hours prior (water is permitted, and good hydration encouraged).
Participants will arrive to HIRF where the MRI checklist and diet and activity checklist will be reviewed. A peripheral intravenous cannula will be inserted for radiotracer administration. A bedside blood glucose measurement will be measured if there is clinical suspicion of a blood glucose abnormality.
Whole body PET/MRI imaging will be performed on the Biograph mMR PET/MRI system after the intravenous infusion of the radiotracer 18 F FDG. Dynamic tracer uptake will be recorded over a 45-60 minute period from the abdomen for quantitative measurement of FDG uptake in the spleen. Participants will be offered a short break, followed by collection of static images of the whole body and brain, over an estimated 30 minute period.
Dedicated brain imaging will take place using the MAGNETOM Prisma 3T MRI system. This includes MP2RAGE, T2FLAIR and diffusion weighted MRI sequences of the brain for assessment of the effects of very subtle oedema and inflammatory responses. The estimated time of image acquisition is 45 minutes per patient. Depending on initial results, there may be changes to MRI sequences to improve data collection. Participant activities or exposures will not be affected by any technical imaging sequence change.
Following image acquisition participants will be allowed food and drink and encouraged to drink water. A light meal will be provided prior to exit from HIRF. Each HIRF visit is approximately 4 hours in total duration.

Image Interpretation:
Collected images will be reviewed and reported by specialist radiologists at HIRF/RBWH. Imaging metrics (FDG uptake and kinetic parameters) will be compared between baseline and follow up scans. Quantitative 18 F FDG uptake will be calculated using Patlak model analysis from dynamic uptake imaging, yielding a Ki value ( 18 F FDG influx constant.) A nominated region of interest will have quantitative 18 F FDG uptake calculated for each scan. Semi-quantitative 18 F FDG uptake will be calculated using SUVs measured during static uptake imaging. All regions of interest will have SUVs calculated.

Other data collection:
Other blood samples will be collected as per the MAIN study.

Evaluation of data:
P2261 30-09-2016 Ver1.0 All data will be presented descriptively. Pre and post inoculation quantitative imaging metrics will be compared with paired T-tests or Mann-Whitney U tests. Plasmodium spp. groups will be compared with unpaired T-tests with consideration of T-value adjustment for population size and Mann-Whitney U tests. Any groups identified based on imaging results will be described with respect to demographic, clinical and biochemical data collected as part of the MAIN study.

Study Flow Chart
Screening Visit

PARTICIPANT ENROLLMENT AND WITHDRAWAL 4.1 Recruitment
Following receipt and signing the MAIN study consent forms, subjects will be fully informed of the nature of this optional EXPLORATORY STUDY, and the specific risks associated with this EXPLORATORY STUDY. A separate 'Participation Information Sheet and Consent Form' will be provided for this purpose (Appendix D).
The 'Informed Consent' will be signed and dated by the participants in the presence of an investigator. Subjects will also be given a copy of their signed 'Informed Consent'. A copy of the HIRF site map (Appendix E), diet and activity sheet (Appendix C) and 18 F FDG Product Information (FDA information, see section 12. Appendix G) will be provided for participant reference.

Eligibility Criteria 4.2.1 Inclusion Criteria
In order to be eligible to participate in this study, an individual must meet all of the following criteria:  Provide signed and dated informed consent form  Able to lie supine and still for duration of image acquisition  All other criteria as outlined in MAIN study protocol(s)

Exclusion Criteria
An individual who meets any of the following criteria will be excluded from participation in the study:  Known allergic reactions to components of the study radiotracer 18 F FDG  Fasted screening blood glucose elevated above the normal range (BSL >6.0mmol/L)  Failure to meet/provide the standard MRI checklist requirements  Claustrophobia precluding image acquisition  Significant previous radiation exposure as defined (lifetime exposure): o Any fluoroscopic imaging (e.g. coronary angiography) o Any nuclear medicine imaging (e.g. myocardial perfusion scan) o Greater than one previous CT scan o Note: previous plain film X-Rays and mammography are acceptable  All other criteria as outlined in MAIN study

Participant Withdrawal
Participants have the right to withdraw from the study at any time for any reason. The investigator also has the right to withdraw patients from the study in the event of any clinical adverse event (AE), laboratory abnormality, or other medical condition or situation occurs such that continued participation in the study would not be in the best interests of the participant OR the participant meets and exclusion criterion (either newly developed or not previous recognized) that precludes further study participation.
Following consent, data acquired up until withdrawal will be included in the EXPLORATORY STUDY. No further samples will be collected from the time of withdrawal notification.

Permanent Termination or Suspension of Exploratory Study
The principal investigator(s), Human Research Ethics Committee (HREC) and Regulatory Authorities independently reserve the right to discontinue the study at any time for safety or other reasons. This will be done in consultation with the MAIN study sponsor where practical. The MAIN study sponsor, in consultation with the investigators may request for suspension of the EXPLORATORY STUDY.
After such a decision, the investigator(s) will ensure that adequate consideration is given to the projection of the participants' interests. The investigator must review all participants as soon as practical and complete all required records.

STUDY INVESTIGATIONS
Whole body imaging will be performed using the Biograph mMR PET/MRI system, with concurrent PET and MRI acquisition. Dedicated Brain MRI sequences will be performed using the MAGNETOM Prisma 3T MRI system. This equipment is property of HIRF.
The standard radiotracer 18 F FDG will be used for PET imaging. This radiotracer has previously been approved by the Australian Register of Therapeutic Goods as a consumable for use in PET imaging (Austin Health) and attracts an MBS rebate for use in multiple conditions.) The radiotracer is to be purchased from the RBWH Nuclear Medicine Department, a TGA licensed manufacturer of 18 F FDG. The 18 F FDG is produced under the Good Manufacturing Practice (GMP) conditions (see Appendix F) in accordance with the British Pharmacopeia. The research will not alter the formulation of the product. The research will be using an identical product in an identical way in an identical formulation to that used for clinical diagnostic PET imaging. 18 F FDG is supplied as a clear/colourless or slightly yellow solution containing the radionuclide 18 F conjugated to the biologically active ligand glucose. It is administered as an intravenous injection prior to imaging.
All radiotracer for use will be released as per existing RBWH Nuclear Medicine department practices. Dosing of the radiotracer is at a standard weight-based dosage. This is determined based on participant radiation exposure, not pharmacological effect. The effective dose of the metabolically active ligand (FDG) is considered a micro-dose.
Due to the unique nature of radiopharmaceuticals 18 F FDG is considered an investigational product by the TGA for purposes of this research project.

Radiotracer Dosing Regimen
 Prior to administration of 18 F FDG participants are to fast for roughly 6 hours.  Prior to administration of 18 F FDG participants are to avoid strenuous exercise and adhere to a low carbohydrate diet for 24 hours.
An estimated dose of 4.5MBq per kg (based on screening weight) 18 F FDG is to be administered by infusion (minimum dose 90MBq, maximum dose 400MBq). This is the lowest possible dose to yield reliable data. The radioactive half-life of 18 F FDG is 110 minutes. Estimated radiation exposure is included in the radiation assessment report (see Appendix A).
 Following administration of 18 F FDG participants are encouraged to drink water to promote the renal excretion of radiotracer.

Modification of Radiotracer Administration for a Participant
Any participant experiencing an AE to any part of the investigation (participation in imaging process or infusion of radiotracer) will be individually evaluated. Where it is considered unsafe to repeat the process by the investigator(s) in discussion with the Nuclear Medicine Independent Medical Advisor, no further imaging will take place.

Participant Compliance
Participant non-compliance with preparation for image acquisition or non-compliant with instruction during imaging itself (e.g. excessive movement) will be recorded in the participant file as this may have an impact on the quality of data collected. This will include reference to the diet and exercise activity sheet assessed prior to imaging.

Radiotracer Manufacture, Handling and Accountability
The formulation, packaging and labeling of the standard radiotracer 18 F FDG is defined by licensed manufacturer. The 18 F FDG is stored at room temperature in accordance to the conditions set by the manufacturer.
Handling of the radiopharmaceutical will follow the standard HIRF protocols for all injectable radiopharmaceuticals The 18 F FDG will be ordered on an as needed basis. The 18 F FDG is supplied on the day of each study. The product expiry time is on the same day as supply due to the short half life of the radioactive element. There will be no excess product. The administration of the GMP approved standard radiotracer 18 F FDG will be as per existing HIRF practices, including record keeping of 18 F FDG lot number, patient injected activity and time of administration using the Venstra system. Product accountabilty and quality control will be as per existing RBWH Nuclear Medicine Department practices under the Quality GMP Agreement for TGA licensing for the manufacture and supply of radiopharmaceuticals.

STUDY SCHEDULE 6.1 Screening
Participants consenting for participation in this EXPLORATORY STUDY must meet all inclusion and exclusion criteria for both MAIN and EXPLORATORY STUDY participation.
This includes EXPLORATORY STUDY specific screening for:  Fasted blood glucose (exclude if >6.0mmol/L)  Significant prior radiation exposure precluding participation (see exclusion criteria)  MRI checklist completion, including evaluation for claustrophobia and ability to comfortably lie supine that may preclude participation  Evaluation of patient understanding of preparation for imaging acquisition instructions: fast 6 hours prior; abstain from vigorous exercise and adherence to low carbohydrate diet 24 hours prior.

Enrollment/Baseline
Baseline fasted blood glucose and MRI checklist must be completed. Low carbohydrate diet options will be discussed.

Intermediate Visits
The first visit (within one week prior to MAIN study inoculation) will include advanced medical imaging.

Final Exploratory Study Visit
The second visit (MAIN study one to two days prior to confinement) will include advanced medical imaging. This will also be the final study visit. Any AEs that require follow up will be reviewed at subsequent MAIN study visits.

Withdrawal Visit
A withdrawal visit coinciding with a scheduled MAIN study visit (or MAIN study withdrawal visit) will take place to review imaging results as required.

Unscheduled Visit
There are no unscheduled visits expected for this EXPLORATORY STUDY. In any case where an unscheduled visit is required, attempts will be made to ensure this occurs at a pre-existing MAIN study visit.

CLINICAL LABORATORY EVALUATIONS
MAIN study fasted blood glucose measurements will be used as part of the EXPLORATORY STUDY screening process.
Bedside capillary blood glucose measurement will take place prior to PET/MRI scanning if there are clinical concerns. Urine point of care pregnancy testing will be performed at either of QPharm or HIRF within 24 hours prior to PET/MRI imaging. MAIN study baseline and serial safety blood tests (full blood count and biochemistry) will be used for comparison of imaging results.

SPECIAL ASSAYS OR PROCEDURES
Collection of blood samples for parasitaemia and parasite biomass measurements will take place as part of the MAIN study protocols.

Definition of an Adverse Event and of a Serious Adverse Event ADVERSE EVENT
Any untoward medical occurrence in a patient or clinical investigation participant administered a pharmaceutical product and which does not necessarily have a causal relationship with this 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 product, whether or not related to the product.

SERIOUS ADVERSE EVENT
A serious AE is any untoward medical occurrence that, at any dose: a) Results in death b) Is life threatening (the term 'life-threatening' in the definition of 'serious' refers to an event in which the participant was at risk of death at the time of the event. If does not refer to an event, which hypothetically might have caused death if it were more severe. c) Required hospitalization or prolongation of an existing hospitalization (hospitalization for elective treatment of a pre-existing condition that did not worsen from baseline is not considered an AE) d) Results in disability/incapacity e) Is a congenital abnormality/birth defect.
Use medical and scientific judgment when deciding whether reporting is appropriate in other situations, such as other important medical events that may not be immediately life-threatening or result in death or hospitalization, but may jeopardize the participant or may require medical or surgical intervention to prevent one of the outcomes listed in the above definition.

SUSPECTED UNEXPECTED SERIOUS ADVERSE REACTION (SUSAR)
A suspected unexpected serious adverse reaction is an AE that is determined to be related to the radiotracer and is both serious and unexpected. The term 'unexpected' means that nature or severity of the event is not consistent with the applicable product information (Appendix G.)

Documentation and classification of study specific adverse events
For regulatory purposes to use the standard radiotracer 18 F FDG in this EXPLORATORY STUDY all AEs deemed related to radiotracer administration will be documented in an EXPLORATORY STUDY AE log including diagnosis, severity, toxicity, date of onset and resolution and any action taken. Toxicity will be determined using existing CTCAE version 4.0 criteria, consistent with those proposed for use in the MAIN studies. Severity will be determined based on the following scale: o Mild: does not interfere with participant's usual function o Moderate: interferes to some extent with participant's usual function o Severe: interferes significantly with participant's usual function All AEs will be recorded in the MAIN study participant file as per MAIN study processes. Where an AE is deemed a result of EXPLORATORY STUDY activities ( 18 F FDG radiotracer administration or other imaging procedures), this will be made clear in documentation and discussed with the MAIN study principal investigator prior to any appropriate escalation to MAIN study medical monitor and sponsor.

Relationship to Study Investigation
For the purposes of this study for an AE to be considered related to 18 F FDG it must occur within 5 half-lives, or 550 minutes of administration. For an AE to be considered related to other imaging procedures, it must occur during the procedure itself.
Any AEs deemed related to the above EXPLORATORY STUDY procedures will be documented in the EXPLORATORY STUDY AE log by one the Investigator or one of his representatives.

EXPECTEDNESS OF SAEs
There are no specific expected SAEs from participation in whole body PET/MRI or dedicated MRI imaging.
Incidental abnormalities detected on imaging will be discussed between the reporting radiologist, study doctor, principal investigator and participant. If required, further review will be arranged by referral to the general practitioner or appropriate specialist. Any incidental findings are expected to be asymptomatic and unpredictable, given the study population of otherwise well, healthy adult volunteers.
There are no commonly reported adverse reactions to radiopharmaceuticals and 18 F FDG in the literature (Appendix G). A multicenter review of radiopharmaceutical use (including 18 F FDG) administrations found an AE incidence rate of 2.1 per 100 000 administrations [12]. A single case of a probable cutaneous adverse drug reaction has been reported in an individual with multiple existing drug allergies [13]. This did not preclude the patient from repeated dosing. A single case episode of possible anaphylaxis has been reported in the context of the administration of multiple other medications [14]. In both cases symptomatic treatment was effective. Based on the volume of 18 F FDG use in the clinical setting, and paucity of published adverse events, the risk of such events is considered to be extremely low.

Recording and Prompt Reporting of Events
In all cases of AEs, the reporting process used for the MAIN study from which the participant was recruited will be used. If MAIN study procedures are not available recording and reporting of AEs will be in accordance with QIMRCTSOP013 and QIMRCTSOP016.
Any AE deemed related to the administration of 18 F FDG or other imaging procedures will be discussed where possible to further clarify causality with the nominated Nuclear Medicine Independent Medical Advisor.
Any SAE deemed related to the administration of 18 F FDG or other imaging procedures will be reported to the Sponsor within 24 hours following discussion with the medical monitor and where possible with the nominated Nuclear Medicine Independent Medical Advisor. The TGA and any other relevant authorities will be notified by the Sponsor (or institutionally approved representative) within 72 hours as per existing TGA guidelines.

REPORTING OF PREGNANCY
Female participants of child bearing potential will undergo pregnancy testing as outlined and as per MAIN study protocol. All pregnancy-related policy and procedures will be managed as per MAIN study protocol in addition to exclusion from further EXPLORATORY STUDY participation.
If the MAIN study from which EXPLORATORY STUDY participants only recruits women of non-childbearing potential, no point of care testing or pregnancy monitoring will take place.

Halting Rules
The investigator(s) reserve the right to halt the study at any point. There are no specific safety findings expected that may prompt suspension or termination of the EXPLORATORY STUDY. In a situation where the MAIN study is halted, the EXPLORATORY STUDY will also be halted as required.

QUALITY ASSURANCE
EXPLORATORY STUDY materials and quality assurance related to the imaging of participants and any involvement onsite at HIRF will take place as per existing HIRF/RBWH processes. Data quality will be considered and described in the presentation of results.
The MAIN study is a registered clinical trial employing an external study monitor responsible for quality assurance with respect to inoculum and participant safety (including pathology testing and clinical examination/investigations.)

STATISTICAL CONSIDERATIONS 10.1 Sample Size
This is a pilot investigation to assess the application of advanced medical imaging in subpatent malaria. The population will comprise of eight adults (male and nonpregnant, non-lactating female participants between 18 and 55 years of age, recruited from the IBSM model MAIN study population.
It is expected that this study will be hypothesis generating. There are no pre-existing PET or prospective MRI imaging trials in malaria published in the literature. Data from this pilot investigation may be used in future power calculations if required.

Statistical Analysis Plan
All data will be presented descriptively. Pre and post inoculation quantitative imaging metrics will be compared with paired T-tests or Mann-Whitney U tests (statistical significance p<0.05). Imaging results may be described with respect to demographic, clinical and biochemical data collected as part of the MAIN study.

SOURCE DOCUMENTS AND ACCESS
Upon request, the investigator(s)/institution(s) will permit direct access to source data/ documents for trial-related monitoring, audits, HREC review, and regulatory inspection(s) by the Sponsor (or their appropriately qualified delegate) and Regulatory Authorities provided this is feasible from a technical standpoint. Direct access includes examination, analysis, verification and reproduction of records and reports that are important to the evaluation of the study.

ADMINISTRATIVE PROCEDURES ETHICAL CONSIDERATIONS
The amount of blood to be sampled in the study is not considered to be excessive in healthy adult participants. This study will be carried our according to the Declaration of Helsinki, the NHMRC National Statement on Ethical Conduct in Human Research (2007) and the Notes for Guidance on Good Clinical Practice as adopted by the Australian Therapeutic Goods Administration (2000) (CPMP/CH/135/95) and the ICH GCP Guidelines.
ETHICAL REVIEW COMMITTEE This Protocol will be submitted for approval to QIMR-B HREC, Clinical Trial Protocol Committee. This protocol will then be submitted for executive approval from RBWH HREC. Following this the HIRF Scientific Advisory Committee will review the Protocol and QIMR-B HREC assessment(s) in order to facilitate expedited Site-Specific Approval from the RBWH/Metro North HREC. Written approvals will be obtained before volunteers are recruited and participants are enrolled. The Investigators will receive all the documentation needed for submitting the present Protocol to the HREC. It is the responsibility of the Investigator to report study progress to the HREC as required or at intervals not greater than one year.
REGULATORY AUTHORITIES This is an EXPLORATORY STUDY to run in parallel to an existing MAIN study clinical trial. The investigation uses the application of imaging using a standard radiotracer at standard dosages that is widely used in the clinical setting. This is administered as per existing local institution processes. Due to the unique nature of radiopharmaceuticals, facilities are able to extemporaneously produce and administer FDG onsite for their own purposes. The RBWH Nuclear Medicine department has TGA approval for manufacture of this radiotracer (Appendix F). This radiotracer is not ARTG listed for this facility, but is approved for GMP production and commercial sale from this manufacturer to other facilities within the state of Queensland. Following discussion with the TGA and study sites, this EXPLORATORY STUDY has a CTN registration for the regulatory purpose of accessing the standard radiotracer 18 F FDG in a manner that ensures prospective record keeping and transparent usage. This includes processes for AE reporting as documented below.
A Nuclear Medicine Independent Medical Advisor has been nominated to ensure participant safety, to provide advice and ongoing review of any SAEs that may be related to EXPLORATORY STUDY activities. No DSMB has been nominated given the low risk nature of this small population EXPLORATORY STUDY. Participants will receive a copy of a current FDA template for 18 F FDG product information (Appendix G), as no generic equivalent exists in Australia.

INFORMED CONSENT
Before recruitment and enrolment into the study, each prospective participant must be given a full explanation of the nature and purpose of the study, and a copy of the Participant Information Sheet and Consent Form to review. This form complies with the consent form requirements outlined in the TGA guidelines. Once the essential study information has been provided, and the Investigator is assured that each individual volunteer understands the implications of participating in the study, the participants will be asked to give consent to participate in the study by signing the informed consent form. The consent form shall be signed and dated by the appropriate parties. A notation that written informed consent has been obtained will be made on the participants' MAIN study file. The completed consent forms will be retained by the Investigator and a copy of these will be provided by the Investigator to the participant.
PARTICIPANT RE-IMBURSEMENT Participants who are found eligible and complete the study will be compensated $160 for their time. This is in addition to MAIN study compensation.

EMERGENCY CONTACT WITH INVESTIGATORS
P2261 30-09-2016 Ver1.0 All participants will be provided with a Participant Information Sheet and Informed Consent Form with contact details of whom to contact in the case of an emergency related to the EXPLORATORY STUDY.
Participants will also be provided with a contact card as per the MAIN study.

NOTIFICATION OF PRIMARY CARE PHYSICIAN
Where it is deemed appropriate by the investigator(s), study doctor and/or reporting radiologist a letter will be sent to the physician stating the nature of the EXPLORATORY STUDY including any adverse events or incidental findings. A copy shall be retained by the study site as part of the MAIN study participant file.

PROTOCOL AMENDMENTS
The Investigator must not modify the Protocol without first obtaining the agreement of QIMR Berghofer in writing. No changes (amendments) to the Protocol may be implemented without prior approval of QIMR Berghofer and the appropriate HREC. If a Protocol amendment requires changes to the Informed Consent Form, the revised Informed Consent Form, prepared by the Investigator, must be approved by the appropriate HREC.
PROTOCOL DEVIATIONS All protocol deviations must be reported to the principal investigator. Protocol deviations will be assessed for significance by the principal investigator. Those deviations deemed to have a potential impact on the integrity of the study results, patient safety or the ethical acceptability of the trial will be reported to the HREC prior to completion of participant enrollment. Where deviations to the protocol identify issues for protocol review, the protocol may be amended as above.

RETENTION OF OTHER STUDY SPECIFIC SAMPLES
Electronic imaging data will be retained as per HIRF guidelines. No other samples will be collected.

INSURANCE AND INDEMNITY
This EXPLORATORY STUDY is an observational study to run parallel with existing MAIN study clinical trials. There are no specific expected SAEs from participation requiring indemnity, however in the event of an AE that is deemed related to EXPLORATORY STUDY participation (following Investigator and medical monitor review) the sponsor holds No-Fault Compensation for Clinical Trials insurance policy for this study. The site HIRF is part of the RBWH campus.
The MAIN study holds a No-Fault Compensation for Clinical Trials insurance policy. Only the investigators, designated staff and designated MAIN study staff will have access to information that identifies a study participant. All MAIN study participants will be assigned a unique identifier. Participant identifiers in this EXPLORATORY STUDY will be consistent with those used in corresponding MAIN studies.
DATA CAPTURE METHODS All imaging data will be collected in accordance with site-specific HIRF protocols.
TYPES OF DATA Imaging data, reports and quantitative imaging metrics will be electronically stored in accordance with HIRF guidelines.
RECORD KEEPING All study related documents and records are to be retained for a minimum of fifteen years after MAIN study completion. Written agreement from the Sponsor must precede destruction of the same. In cases where HIRF guidelines preclude the storage of source imaging data, data will be transferred to the sponsor for storage.

PUBLICATIONS POLICY
Publication and reporting of results and outcomes of this trial will be accurate and honest, undertaken with integrity and transparency and in accordance with QIMR BERGHOFER's Policy on Criteria for Authorship. Publication of results will be subjected to fair peer-review. Authorship will be given to all persons providing significant input into the conception, design, execution or reporting of the research according to QIMR BERGHOFER Policy on the Criteria for Authorship.