Sulphadoxine pyrimethamine SP is an anti

Sulphadoxine-pyrimethamine (SP) is an anti-malarial combination drug that was once commonly used, but that has been abandoned as first-line treatment due to increasing resistance in malaria parasites. At present, SP is used for intermittent preventive treatment with SP during pregnancy (IPTp-SP) in most Sub-Saharan African countries. IPTp-SP helps in preventing malaria related morbidity during pregnancy, like maternal anemia and low birth weight of the offspring . However, because of a continuous increase in SP resistant , there are now also concerns about the efficacy of IPTp-SP. Mutations in two genes, dihydropterate synthase ( and dihydrofolate reductase (, encoding the target proteins of sulphadoxine and pyrimethamine respectively, are associated with resistance against SP. Each of these (S436, A436, K540, A581, K613) and (N51, C59, S108, I164) mutations contribute to the level of clinical resistance and especially the double (A437, K540) with triple (N51, C59, S108) combination (quintuple mutant) and the more recently upcoming sextuple mutant (additional A581 mutation) are associated with SP resistance , . In East Africa, prevalence of the quintuple S63845 is high and has reached near 100% saturation in some areas, not surprisingly there are reports of IPTp-SP having lost effectiveness or even having detrimental effects in these countries , . In contrast, in West Africa S436 and A437 and the triple mutations are fairly common, but the K540 and consequently the quintuple mutation is hardly described . Efficacy of IPTp-SP is therefore still sufficient, but surveillance of possible emergent SP resistance mutations is extremely important . In an intervention trial situated around Nanoro, Burkina Faso, pregnant women were followed up from first antenatal care visit (ANC) to delivery as described by Scott Informed consent was obtained for all participants. All women received standard care at the ANC, including IPTp-SP, and additionally women from intervention villages received monthly home screening for malaria with rapid diagnostic tests. Women with positive RDTs were treated with artemether-lumefantrine. At each ANC visit, blood spots on filter papers were collected. These filter papers were used for DNA extraction. eal-time PCR positive samples from first ANC (random selection) and from delivery collected from May 2014 till September 2015 were included for mutation analyses. An additional sample set was collected from the general population (GP). For this sample set a cross-sectional survey was done in April and May 2015 in the same area as the longitudinal study in pregnant women. From randomly selected households two members (non-pregnant and aged≥6months old) were enrolled after informed consent was obtained. For underaged children informed consent was given by parents or guardians. Blood spots on filter papers were collected and processed similarly to the longitudinal study. A random selection of real-time PCR positive samples were included in mutation analyses performed in the Netherlands. One blood spot was punched out from each filter paper using skin biopsy punchers (acuderm inc, USA). DNA was extracted by first lysing the filter papers in polypropylene tubes on a roller bank for 30min using EasyMAG lysis buffer (bioMérieux). Subsequently the lysed fluid was transferred to EasyMAG vessels and incubated with magnetic bead silica (bioMérieux) according to manufacturer’s instructions. The DBS 1.0 protocol was used to extract the DNA in the NucliSENS EasyMAG. Extraction controls were included (positive filter papers of blood spots spiked with diluted FCR3 or 3D7 culture, and negative filter papers with blood spots of uninfected erythrocytes).