Experimental infection and protection against ... - TI Pharma

134 Chapter 7
insectary of the Radboud University Nijmegen Medical Centre. Feeding sessions
were repeated when necessary, with a smaller number of mosquitoes until each
volunteer had been exposed to exactly five mosquitoes that took a blood meal
and had Pf sporozoites in their salivary glands. One feeding session was
sufficient for one and four volunteers in the NF135.C10 and NF54 groups
respectively, two and one volunteers required two sessions and three feeding
sessions were needed in three volunteers of the NF135.C10 group
(Supplementary Table 1). Starting from day five post-infection, volunteers were
subjected to intensive follow-up with up to thrice daily visits to the LUMC outpatient
clinical research department. All signs and symptoms (solicited and
unsolicited) were recorded and graded by the attending physician as follows:
mild (easily tolerated), moderate (interferes with normal activity), or severe
(prevents normal activity), or in case of fever grade 1 (>37.5°C – 38.0°C), grade 2
(>38.0°C – 39.0°C) or grade 3 (>39.0°C). Haematological and biochemical
parameters were monitored daily. Because of a previously reported serious
cardiac adverse event after a malaria challenge infection in a separate study
[23], particular attention was paid to markers of coagulation or cardiac damage
with daily follow-up of highly sensitive troponin, platelets, d-dimer and lactate
dehydrogenase during the period of expected blood stage parasitemia.
Whenever abnormal, blood samples were checked for the presence of
fragmentocytes and von Willebrand cleaving protease activity. Promptly after
identification of a positive blood smear, volunteers were treated with a curative
regimen of four tablets of 250/100mg atovaquone/proguanil once daily for three
days. Volunteers whose blood smears remained free of parasites until day 21
after challenge presumptively received the same curative treatment with followup
to the end of the study at day 28. Complete cure was always confirmed by
two consecutive parasite-negative blood smears. The trial was performed in
accordance with Good Clinical Practice and approved by the Central Committee
for Research Involving Human Subjects of The Netherlands (CCMO
NL30350.058.09). Clinicaltrials.gov identifier: NCT01002833.
Parasitological Outcomes
Thick blood smears were examined by microscopy twice daily on days five and
six post-challenge, thrice daily on days seven to eleven, twice daily on days 12-
15 and once daily on days 16-21 post-challenge. 15µl of EDTA-anti-coagulated
blood was spread over the standardised surface of one well of a 3-well glass
slide (CEL-LINE Diagnostic Microscope Slides, 30-12A-black-CE24). After drying,
wells were stained with Giemsa for 30 minutes. Slides were read at 1000x