Prevalence of Polymorphisms in Plasmodium Falciparum Parasites in Pregnant and Non-pregnant Women and Potential Resistance to Antimalarial Drugs in Western Kenya

Abstract

Malaria infection in pregnancy results in maternal anaemia, placental accumulation of parasites, low birth weight infants together with maternal mortality owing to diminished maternal malaria immunity. In regions having high malaria transmission, majority of the population are presumed immune to malaria, with non-pregnant women showing quicker clearance of parasites in comparison to the pregnant. This observation suggests that the non-immune environment in pregnant women may select for parasite strains that are linked to artemisinin resistance. Our study focussed on genetic variation in Plasmodium falciparum parasites in pregnant versus non-pregnant women. Blood samples were collected at hours 0, 8, 24 and at days 7 and 28 from 75 women positive for malaria comprising of 50 pregnant women in their second or third trimesters and 25 non-pregnant women, enrolled in an ACT efficacy study at Ahero in Kenya. Malaria diagnosis was done for all samples using PCR targeting 18S rRNA gene of Plasmodium. SNP genotyping for the K13, Pfmdr1, Pfmrp1, Pfdhfr, Pfdhps and Pfcrt genes was done to determine mutations in parasites with varying clearance using Sanger sequencing and MassARRAY. For MassARRAY SNP genotyping, the calls for the genotypes were made using SpectroTyper 4.0 software (Agena). The K13 Sanger sequencing reads assembly and mapping to the reference 3D7 genome was done using CLC Main Workbench software and DNA Baser. SNPs analysis was done using multiple sequence alignment by Clustal W in Bioedit. Of the 75 women; 45 consisting pregnant 2nd trimester (n = 15), pregnant 3rd trimester (n = 12) and non-pregnant women (n = 18) had samples at all study time points merited to be included in the analysis. The 45 individuals comprised: Hour 0 = 100.0 %; Hour 8 = 84.4 %; Hour 24 = 26.7 %; Day 7 = 24.4 % and Day 28 = 4.4 % samples positive for Plasmodium. At hour 0, P. falciparum species were observed in 20.0 % of pregnant women in second trimester, 33.3 % of pregnant women in third trimester and 72.2 % of the non-pregnant women. P. malariae parasites were present in 8.3 % of pregnant women in third trimester and P. ovale wallikeri parasites in 13.3 % of pregnant women in second trimester, 58.3 % of third trimester pregnant women and 27.8 % of the non-pregnant women. At hour 8 of study, 28.6 %, 11.1 % and 62.5 % of the women in the second and third trimesters of pregnancy and the non-pregnant women respectfully had P. falciparum parasites. P. ovale wallikeri infections were observed in 28.6 % second trimester pregnant women, 22.2 % of the pregnant women in third trimester and 37.5 % of women in non-pregnant group. At hour 24 of the study, P. falciparum infections were only present in the non-pregnant women and constituted 100.0 % of the infections while P. ovale wallikeri infections were detected in 66.7 % of pregnant women in the second trimester and 100.0 % of pregnant women in third trimester. On study follow up day 7 parasites were present only in the second trimester pregnant women and they consisted of 10.0 % P. ovale wallikeri parasites. No parasites were present on follow up day 28 of the study. A total of 225 samples including all subsequent positives were carried on to MassARRAY and Sanger sequencing for SNPs genotyping. For lacking some study time points, we eliminated 30 participants samples from the study. Pregnant women showed slower clearance of non-falciparum parasites after 24 hours of treatment with ACTs. 2nd trimester pregnant women had the highest cases (40.0 %) followed by the 3rd trimester (16.7 %) and non-pregnant (5.6 %) with the lowest number of samples having parasites at hour 24. Parasites were present even on follow up day 7 in 66.7 % of the pregnant xxiii women in 2nd trimester. None of the study participants had parasites present on follow up day 28 of the study. K13 sequencing showed no nonsynonymous nor synonymous mutations in our Western Kenya samples. The mutations common in SEA like C580Y, I543T were not present in our Kenyan samples. Those common to Africa like A578S and S522C (Uganda) were also not present in our study samples. For the MassARRAY SNP genotyping, Pfmdr1 gene had low frequency of mutations at codon 86 and showed similar frequencies of mutations and wild genotypes at codon 184 in the pregnant and non-pregnant women. Pfdhfr gene had high frequency of wild genotypes at codons 16, 22 and 164 while codons 59 and 108 had high number of mutants. These frequencies were distributed similarly between the two groups of pregnant and the non-pregnant women. Pfdhps gene showed similar trends in pregnant and also non-pregnant women with codons 436, 581 and 613 having high frequency of the wild type genotypes and codon 437 being majorly mutant. Pfcrt gene had similar trends in both study arms with all codons being mostly wild type genotype. The Pfmrp1 gene was wild type in codons 191, 437 and 1390 in both study groups with codon 876 having both wild and mutant genotypes expressed similarly in both the pregnant and the non-pregnant women. The pregnant women were found to have higher cases of non-falciparum infections compared to the non-pregnant women. This could be credited to their low immunity and/or the IPTp-SP prophylaxis which targets falciparum parasites. Pregnant women did not carry parasites strains having mutations conferring resistance to artemisinin. The study did not find statistical differences between the pregnant and non-pregnant women groups to support suggestions that low immunity in pregnancy could be a source of resistant parasites strains or their reservoir. Use of ACTs for uncomplicated malaria in pregnant women was not seen to affect parasites genotypes probably since the loss of immunity is transient, relative to parasite lifecycle, hence is not a risk factor for development of resistance to ACTs. This also means IPTp-SP continues being effective as prophylaxis for malaria during pregnancy although its role in selection of species need to be considered.