Determination of in vivo parasite-clearance efficacy and prophylactic effect of SP

In vivo parasite-clearance efficacy and prophylactic effect of SP was established based on malaria microscopy [24] and merozoite surface protein-2 (MSP2) genotyping [29, 30] which differentiated cases of recrudescence from reinfection. The two blood smears collected at enrolment on day 0 and day 28 post-treatment were used to estimate the in vivo parasite-clearance efficacy and prophylactic effect. Women were classified as having parasitological-clearance failures if they had a malaria-positive blood smear both at day 0 and day 28, and genotyping of MSP2 confirmed recrudescence and ruled out new infections [29, 30]. Women who had a negative blood slide at day 0 and became slide-positive at day 28 and those who were positive at day 0 and had new infections by day 28 defined by MSP2 genotyping, were classified as prophylactic failures.

Polymorphic regions of MSP2 were amplified by nested PCR. Primary PCR primers corresponding to the conserved sequence flanking this region [31] were used, whereas the secondary PCR primers were used to amplify the IC3D7 and FC27 allelic families of MSP2 [32]. For controls, DNA from HB3 and 3D7 laboratory strains were used. Briefly, all PCR reactions were carried out in total volumes of 25 µl using Thermo Scientific^® Dream Taq PCR Master Mix (2X) and 0.5 µM of each primer and 2 µl of template. A 2 µl of the primary amplicon was used as a template in the secondary reaction.

The secondary amplicon from each sample was then analysed using electrophoresis on 2% ethidium bromide stained agarose gels. Samples from individual participants were loaded in adjacent lanes. In cases where there was no amplification, PCR was repeated using five-times the quantity of template DNA. In cases where no amplicon was detected after the second reaction, amplification was classified unsuccessful [33]. Two independent laboratory personnel compared band sizes manually; any discordant classification was settled by a third laboratory staff member. Reinfection and recrudescence were defined as described elsewhere [33]. Briefly, reinfection was defined as having completely different alleles between parasites from day 0 and from day 28; recrudescent infection was assigned if the same allele was found between parasites at day 0 and day 28.