Following intravenous injection of P. vivax (day 0), subjects were monitored by daily telephone calls until day 4, when subjects visited the clinical unit daily until the day of apheresis. Subjects were monitored for adverse events (AEs), signs and symptoms of malaria infection, and blood was collected for 18S qPCR measurement of parasitaemia. The severity of AEs were determined by the common terminology of clinical trial adverse events (CTCAE) v. 4.03 [17].

The threshold for commencement of apheresis and treatment with artemether–lumefantrine was within 24 h of a parasitaemia > 20,000 parasites/mL, or the Malaria Clinical Score reaching > 6 [10], or at the Investigator’s discretion. The morning that this threshold was reached (anticipated based on previous studies to be Day 10 or 11 [9], subjects were admitted to the clinical unit (Q-Pharm) for initial safety assessments before being escorted to the Apheresis Unit at RBWH by Q-Pharm staff. The Apheresis Unit is located in the Haematology Department at RBWH where patients are subject to donor or therapeutic apheresis. At the Apheresis Unit the subjects underwent the apheresis procedure as per the Standard Operating Procedure (Additional files 2, 3, 4 and 5) whilst being supervised by the apheresis specialist nurse and under the supervision of the responsible clinical haematologist (GK). The same apheresis nurse performed the apheresis procedure for all four subjects. The apheresis procedure lasted 1–4 h. Subjects were then escorted back to the clinical unit and began treatment with artemether–lumefantrine (Riamet®, Novartis Pharmaceuticals Australia Pty Ltd). Treatment consisted of six doses of 4 tablets at 12 hourly intervals (each tablet contains 20 mg artemether and 120 mg of lumefantrine). Subjects remained confined within the clinical unit for 48–72 h for safety monitoring. Following release from confinement, subjects attended protocol specified visits until three months post-treatment to monitor for signs of recrudescent parasitaemia and to assess late safety signals. Relapse is not a concern in the P. vivax IBSM studies as liver infection is bypassed and hypnozoites are not produced. A schematic of the study design is shown in Additional file 6: Fig. S1.

This study used an iterative adaptive design approach where subject safety and outcome data were analysed after each subject and modifications made to improve the chances of meeting the exploratory objectives in the subsequent subject. A summary of the changes instituted is shown in Table Table11.

Summary of main study design differences between subjects

From the primary apheresis (HCT): Intermediate (64%)

From the secondary apheresis (HCT): Final (3%) Spare (5%) Waste (42%)

CMNC; continuous mononuclear cell collection, HCT; haematocrit. Protocols for subjects 1 to 4 and all experiments can be found in Additional files 7, 8, 9 and 10. *Biological duplicates involved repeat 18S qPCR testing from two separate blood samples from each HCT layer collected using apheresis.

When a HCT range is included the sample was taken from multiple HCT layers e.g. 5–7% = 5%, 6% and 7% HCT. +Originally aimed to sample 8% HCT layer but actual sample consisted of 11% HCT. #During cohort 4 a red cell depletion was carried out, producing an intermediate bag sample, followed by a second apheresis procedure on the red cell depletion product. The second apheresis procedure involved sampling of ~ 100 ml of the lowest HCT layers of the sample (final bag) followed by ~ 100mls of the subsequent lowest HCT layers (spare bag) and then the remainder ~ 100mls (waste bag).

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