Study Site, Population and Sample Collection
The study was conducted in Kombewa district hospital in Kisumu County, western Kenya from June 2013 through November 2014. Detailed clinical study will be reported elsewhere. Briefly, Kisumu County is a malaria holoendemic lake region with intense malaria transmission through-out the year, with annual entomological inoculation rates (EIR) of 31.1 infected bite per year [12]. This was a two-arm, randomized open-label study, where patients presenting with uncomplicated malaria at the Kombewa district hospital aged between 6 months to 65 years were recruited. Study participants were randomized to receive AL or ASMQ using block randomization schemes with varying block sizes. Venous blood samples were collected at hours 0, 4, 8, 12, 18, 24 and then every 6 hours until 2 consecutive smears became negative. Giemsa-stain films were prepared per World Health Organization (WHO) guidance and read by two independent expert microscopists. The geometric mean of the parasite count per microliter from each participant at each sampling time point was then calculated. Participants were followed for a total of 42 days. A total of 118 participants were enrolled in the study, 59 from each arm. From these, 105 blood samples from study participants (46 and 62 from individuals <5 and ≥5 years, respectively) were used in the GIA analysis.
P. falciparum negative venous blood samples were obtained from six male adults aged 18-65 years, with blood groups A, B and O and hemoglobin level of ≥ 13 g/dL living in a malaria non-endemic regions (Kericho and Nairobi) with no travel history to malaria endemic areas in the last six months of Kenya were used as non-immune controls for the GIA experiments.
For the maintenance of the parasite culture and performing the GIA assays, Red Blood Cells (RBCs) were obtained from blood group “O” donors, aged between 18 and 50 years, with hemoglobin level of 14 – 18g/dL for males and 12 – 16g/dL for females living in a malaria non-endemic area (Kericho and Nairobi) with no travel history to malaria endemic areas in the last six months. Briefly, 7 mL aliquots of the whole blood were added to 7 mL wash medium in 15mL centrifuge tubes, centrifuged at 800g for 10 minutes. (Eppendorf 5804R centrifuge, Eppendorf®) and buffy coat containing most White Blood Cells (WBCs) gently aspirated, followed by the entire supernatant, leaving the packed RBCs. The WBCs still remaining among the cells were removed/washed out by three-time centrifugation in wash media. For each subsequent wash, the buffy coat containing most WBCs was aspirated first followed by the entire supernatant. The packed RBCs were then gently re-suspended in 7 mL wash media and re-centrifuged at 800 g till clear of the WBCs. The cells were then suspended in equivalent wash medium at 50% hematocrit (packed cells /wash medium v/v), then stored at 2-8˚C.
Parasite clearance rates calculation
The statistical models used to estimate the parasite clearance measures and lag phase duration were fitted using the Parasite Clearance Estimator (PCE) tool developed by Worldwide Antimalarial Resistance Network [13]. The following parameters were estimated: parasite clearance half-life, parasite clearance rate constant (K/hour), and the estimated time to reduce parasitemia by 50% (PC50), 90% (PC90), 95% (PC95), and 99% (PC99). Log transformed parasite density was plotted against time in hours to generate slope half-life (T1/2) which is defined as the time needed for parasitemia to be reduced by half [14]. This constant is independent of starting value of parasitemia. The slope half-life was calculated as follows:
T1/2= loge (2)/K
= 0.692/K, where K is the clearance rate constant
The clearance rate K represents the rate of parasite clearance after start of drug treatment.
Culture media
P. falciparum in vitro culture medium (Life technologies, Carlsbad, CA) was prepared as previously described [15]. Briefly, basic media comprised of 10.4 g Roswell Park Memorial Institute (RPMI; Invitrogen, Inc., Carlsbad, California, USA) powder combined with 2 g of glucose (Sigma Inc., St Louis, Missouri, USA) and 5.95 g HEPES (Sigma, USA) dissolved to homogeneity in 1 litre of de-ionized water and sterilized with a 0.2 µM filter. RPMI 1640 tissue culture media (TCM), for all parasite culture and drug dilutions, consisted of RPMI 1640 basic media with 0.5% Albumax II (Gibco, Grand Island, NY), 3.2% (vol/vol) sodium bicarbonate (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) and 4 µg/mL hypoxanthine (Sigma Inc., St Louis, Missouri, USA). Complete RPMI 1640 media was stored at 2-8 ºC.
Serum free media consisted of 1.0 mL of 1.45 mM sterile hypoxanthine to 43.4 mL of RPMI basic medium, 1.6 mL of sterile sodium bicarbonate (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA), 7.5% and sterilized with 0.2 µM filter then put in 2-8˚C. 10% complete media for Control was prepared by adding 5 mL of serum to 43.4 mL of RPMI basic medium, 1.0 mL of 1.45 mM sterile hypoxanthine, 1.6 mL of sterile sodium bicarbonate, 7.5% then sterilized with 0.8 µM, 0.45 µM, 0.2 µM filters and put at 2-8˚C
P. falciparum culture
P. falciparum-3D7 strain obtained through Biodefense and Emerging Infections Research Resources Repository (BEI Resources), NIAID, NIH: falciparum, Strain 3D7, MRA-102 were maintained in continuous culture as previously described [16]. Briefly, P. falciparum-3D7 were suspended in 5 mL of CMS at 6% hematocrit in tissue culture flasks (Corning Glass Works, Corning, NY). The flasks were flushed with a gas mixture consisting of 5% oxygen, 5% carbon (IV) oxide and 90% nitrogen (Air Products Corp., Allentown, PA), sealed, and incubated at 37°C. The medium was changed daily and fresh RBCs added when required. Parasite growth was monitored microscopically by counting Giemsa-stained thin blood smears. When parasitemia exceeded 3%, ring-stage parasites were synchronized by sorbitol lysis (5 % D-sorbitol (Sigma, St. Louis, MO) as follows: Parasites were mixed and transferred into a 15 mL centrifuge tube and centrifuged at 3,000 rpm for 10 min. After removing the supernatant, 5% D-sorbitol pre-warmed to 37°C was added to the RBC pellet, incubated for 10 min at 37°C, vortexed for 1 min and re-centrifuged at 3,000 rpm for 10 min. After aspirating the supernatant, and repeating the process twice using tissue culture media (TCM) ≥ 90% ring forms was achieved.
Serum preparation
Serum was obtained from blood samples as previously described [17] with minor modifications. Briefly, whole blood collected in plain (anticoagulant free) blood bags from each study participant was stored at 2-8 °C overnight to allow settling of the serum and cells portions. Serum portion was transferred into 50 mL centrifuge tubes and centrifuged at 800 g. The supernatant was transferred into 50 mL centrifuge tube to remove all cell components. This step was repeated three times and serum was stored at -65 to -80 °C until further use. The serum from the six male adults (non-immune) were pooled together forming approximately 2.0 L for use as control for subsequent experiments. Serum from each of the immune or non-immune pooled serum was divided into two equal portions. One portion was heat inactivated at 56˚C for 30 minutes in a water bath as previously described [7].
Growth inhibition assay (GIA)
In vitro anti-plasmodial susceptibility assay was performed as previously described [15] with some modifications. Briefly, 10-fold dilution in TCM was done for immune serum, non-immune serum, and negative control wells. Parasitemia in the GIA assays of continuous culture ≥ 3% lowered to 1% were adjusted to final 1-2% hematocrit, a ratio of 1:10 serum dilution in 100 µL final volume was dispensed on Nalgene Nunc, 96 flat-bottom well cell cultures sterile with lid microtiter plates (Magna, Leicestershire, UK) and 10 µL of 3D7 parasite was added in each well then incubate at 37˚C for 72 hours in modular reservoirs, gassed with 90% nitrogen, 5% carbon dioxide and 5% oxygen. Parasite growth was evaluated using SYBR Green I dye, thus 4 µL of SYBR Green I was added to 2 mL of malaria SYBR Green 1 fluorescence (MSF) lysis buffer [20 mM Tris (pH 7.5), 5 mM EDTA, 0.16% (wt/vol) saponin, and 1.6% (vol/vol) Triton X-100], 10× _ SYBR green I dye concentration) and 100 µL of a mixture of SYBR Green 1 and MSF was transferred to each well. The plates were stored in the dark at ambient temperature for 24 hours [18]. The plates were examined for relative fluorescence units (RFUs) per well using a fluorescence plate reader (Tecan™ Morrisville, North Carolina, United States). Parasite replication was quantified for each serum sample and expressed as the mean percentage growth inhibition by
