Real time PCR as described previously with minor modificatio

Real-time PCR, as described previously with minor modifications, was used to detect DNA . The PCR mixture (Qiagen) was as follows: 5mM MgCl2, 2.5μl of 10x PCR Buffer, 0.125μl of HotStarTaq DNA Polymerase, 0.25mM of each dNTP, 0.4μM of each primer and 0.1μM of FAMlabelled probe (‘5-aacaattggagggcaagg-3′, Biolegio). 2.5μl of DNA was used in each reaction. A dilution series of FCR3 culture was included (104 parasites/μL–1 parasites/μL) as well as positive and negative DNA extraction controls and Milli-Q water. Reactions were run on BioRad CFX real-time PCR machine with the following settings: initial denaturation 95°C for 10min, 40 cycles of 95°C for 60s and 60°C for 20s. Results were analysed using Bio- Rad CFX manager software. Nested PCR of the and Esomeprazole Sodium sale were performed for real-time PCR positive samples. Nest 1 primers were based on Duraisingh Nest 2 primers, primer mixes and settings can be found in . Positive ( D10 genomic DNA, MRA-201G, MR4, ATCC Manassas Virginia) and negative controls (uninfected erythrocytes) were included in each PCR. After running the products on a 1% agarose gel, samples with clear bands of the correct size were sent for sequencing with the corresponding nest 2 primers (Macrogen, Amsterdam, the Netherlands). For four sequencing reactions were performed (two additional primers) targeting two parts of the region of interest, thereby circumventing a homopolymer region in the middle of the PCR product that caused polymerase slippage. Results were analysed with Geneious 8.1.8 using a 3D7 reference genome. Base pair calling was manually confirmed in both sequencing directions for each sample. Heterozygote results had to be confirmed in both sequencing directions to be qualified as a mixed infection. Mixed infections were considered mutants. For estimation of prevalence with 95% confidence intervals (CI), generalized estimating equations with village as panel variable was used in Stata 14.1. Ethical approval for both studies was obtained at Comité D’Ethique Institutionnel du Centre Muraz, Bobo-Dioulasso, Burkina Faso (ref A20-2013/CE-CM and DELIBERATION N\' 2015-01-O1O). A total of 223 delivery samples, 400 first ANC visit samples, and 400 GP samples were included in mutation analyses. Reliable sequence results were available for 186 delivery samples, 380 ANC samples and 355 GP samples. Reliable sequence results were available for 175 delivery samples, 359 ANC samples and 352 GP samples. Results of the full study are published in a separate paper . However, an important discovery was the presence of K540 mutations in 2 delivery samples (1.1%; 95% CI 0.3-4.4%) and 4 samples of the general population (1.1%; 95% CI 0.4- 2.7%). All samples with the K540 mutation were in fact quintuple mutants, except one sample from the general population that did not have reliable results for the gene sequence. The two women with quintuple mutants in their delivery samples were both multigravida (6th and 7th pregnancy) and gave birth to babies with a normal birth weight (3260g and 3710g). The participants of the GP with K540 mutations were two males of 8 and 15 years old and two females of 6 months and 51 years old. Each came from a different village. None of them had fever at the time of sample collection or in the previous month. The girl of 6 months old had a parasitemia of 480 parasites/μL, the boy of 8 had a parasitemia of 27 parasites/μL while the other two both had parasitemias <10 parasites/μL. In ANC samples no K540 mutation was found. In Burkina Faso, the K540 mutation has only been reported once before, and this particular study found a remarkably high prevalence in the north of Burkina Faso of 9–12% . Yet, none of these samples harboured the quintuple mutation. Other studies, one in the same study region as ours and one in the southeast of Burkina Faso, did not show any K540 mutations , . We could therefore argue that this is the first alarming evidence on the presence of the quintuple mutant in Burkina Faso. Of the neighbouring countries of Burkina Faso K540 mutations have been found in Mali, Ghana and Benin, although also in low numbers , . However, it is unclear how rapidly the K540 and quintuple mutants may increase and spread in these populations. For example, a study by Maiga et al. in Mali showed a rapid increase in prevalence of K540 mutations and quintuple mutations 8 months after the last dose of seasonal malaria chemoprevention (SMC) with SP compared with baseline (from 3.1% to 10.6% and 1.6% to 7.1% respectively). Nevertheless, no increase in K540 and quintuple mutation prevalence was found in children living in the same study area who were not eligible for SMC . The effect of SMC or other SP based interventions on the spread of SP resistance mutations is therefore still unclear. At population level, an estimated prevalence of >50% of quintuple mutants leads to loss of effectiveness of IPTp-SP in preventing low birth weight caused by malaria in pregnancy . The effectiveness of IPTp-SP in our study area is therefore not immediately threatened. The fact that the two pregnant women with quintuple mutations were both multigravida, who harbour more immunity against malaria in pregnancy than primi- or secundigravidae , could have contributed to the absence of malaria related morbidity. In conclusion, although the numbers of quintuple mutants are still very low, the fact that we have identified these parasites raises concerns about the efficacy of IPTp-SP in the future. This finding therefore warrants thorough follow-up of and mutation prevalence in Burkina Faso.