A common polymorphism in the mechanosensitive ion channel PIEZO1 is associated with protection from severe malaria in humans.

Malaria caused by the apicomplexan parasite Plasmodium falciparum has served as a strong evolutionary force throughout human history, selecting for red blood cell polymorphisms that confer innate protection against severe disease. Recently, gain-of-function mutations in the mechanosensitive ion channel PIEZO1 were shown to ameliorate Plasmodium parasite growth, blood-brain barrier dysfunction, and mortality in a mouse model of malaria. In humans, the gain-of-function allele PIEZO1 E756del is highly prevalent and enriched in Africans, raising the possibility that it is under positive selection due to malaria. Here we used a case-control study design to test for an association between PIEZO1 E756del and malaria severity among children in Gabon. We found that the E756del variant is strongly associated with protection against severe malaria in heterozygotes. In subjects with sickle cell trait, heterozygosity for PIEZO1 E756del did not confer additive protection and homozygosity was associated with an elevated risk of severe disease, suggesting an epistatic relationship between hemoglobin S and PIEZO1 E756del. Using donor blood samples, we show that red cells heterozygous for PIEZO1 E756del are not dehydrated and can support the intracellular growth of P. falciparum similar to wild-type cells. However, surface expression of the P. falciparum virulence protein PfEMP-1 was significantly reduced in infected cells heterozygous for PIEZO1 756del, a phenomenon that has been observed with other protective polymorphisms, such as hemoglobin C. Our findings demonstrate that PIEZO1 is an important innate determinant of malaria susceptibility in humans and suggest that the mechanism of protection may be related to impaired export of P. falciparum virulence proteins.

Molecular surveillance of pfhrp2 and pfhrp3 genes deletion in Plasmodium falciparum isolates and the implications for rapid diagnostic tests in Nigeria.

Prompt diagnosis and appropriate treatment of malaria remain the hallmark for reducing malaria-related mortality in high transmission areas. Plasmodium falciparum histidine-rich protein2 (PfHRP2) based rapid diagnostic tests (RDT) play a vital role in prompt and accurate malaria diagnosis. However, pfhrp2 gene deletion threatens the RDT test sensitivity. This study reports the presence of pfhrp2 and pfhrp3 genes deletion among parasite isolates in Nigeria. Febrile children were screened using histidine-rich protein (HRP2) specific RDT (SD-Bioline RDT) and microscopy for P. falciparum infections. All RDT negative samples were re-evaluated by polymerase chain reaction (PCR). The presence of parasite in RDT false negative cases and randomly selected RDT positive cases were validated using PCRs targeting glutamate-rich protein (glurp) and merozoite surface proteins (msp-1 and msp-2). Thereafter, exon 2 of pfhrp2 and pfhrp3 were amplified, and Sanger sequenced. A total of 511 febrile children were enrolled out of which 309 (61%) were positive by RDT. The presence of pfhrp2 and pfhrp3 genes were analyzed in 66 PCR positive samples comprising of 31 RDT false negative and 35 RDT true positive randomly selected samples. The pfhrp2 and pfhrp3 genes failed to amplify in 17% (11/66) and 6% (4/66) samples, respectively. Seven of the eleven samples had only pfhrp2 deletion while four had both pfhrp2 and pfhrp3 deletions. The absence of the pfhrp2 gene may be responsible for the seven RDT false negative cases observed. Three RDT positive cases lacked pfhrp2 whereas pfhrp3 was absent in only four RDT false negative cases. The pfhrp2 and pfhrp3 amino acid repeat sequences were highly diverse. The P. falciparum isolates lacking pfhrp2 and pfhrp3 genes may be circulating and contributing to RDT false negativity in Nigeria. More studies in larger population and seasonally defined cases will be needed to determine the extent of pfhrp2/3 genes deletion in different geographical areas of Nigeria.

High prevalence of dihydrofolate reductase gene mutations in Plasmodium falciparum parasites among pregnant women in Nigeria after reported use of sulfadoxine-pyrimethamine.

This study assesses the prevalence of asymptomatic Plasmodium falciparum parasitemia positivity and P. falciparum dihydrofolate reductase (pfdhfr) mutations in parasite isolates among pregnant women in Southwest Nigeria. Plasmodium falciparum parasitemia was confirmed by microscopy and nested PCR in 200 pregnant women attending antenatal care. The prevalence of pfdhfr polymorphisms was determined by direct sequencing of the gene fragments containing the C50R, N51I, C59R, S108N, and I164L mutations. Information on the use of antimalarial drugs and methods applied to prevent malaria were obtained by a questionnaire. The prevalence of asymptomatic P. falciparum infection was 30% (60/200). The frequency of the pfdhfr triple-mutant alleles (N51I, C59R, and S108N) was 63% (38/60); none of the isolates carried the I164L mutation. Among the investigated pregnant women, 40% used un-prescribed antimalarials such as dihydroartemisinin (18%), chloroquine (14%) or pyrimethamine (9%), while only 20.5% used sulfadoxine-pyrimethamine for prevention and 39.5% did not use any drug. The prevalence of P. falciparum parasitemia (37%) was higher among pregnant women who had not taken any antimalarial drugs. A significant difference in the prevalence of the pfdhfr triple-mutant alleles was observed among women who took SP (90%) compared to those who did not take any drug (82%) and women who took dihydroartemisinin (67%) p = 0.007). Poor adherence to the World Health Organisation (WHO) strategies for malaria prevention among pregnant women was observed in addition to high prevalence of pfdhfr mutations. These findings underline the need to improve control of malaria among pregnant women in the study area.

Genetic Diversity of the Plasmodium falciparum Glutamate-Rich Protein R2 Region Before and Twelve Years after Introduction of Artemisinin Combination Therapies among Febrile Children in Nigeria.

The genetic diversity of glutamate-rich protein (GLURP) R2 region in Plasmodium falciparum isolates collected before and 12 years after the introduction of artemisinin combination treatment of malaria in Osogbo, Osun State, Nigeria, was compared in this study. Blood samples were collected on filter paper in 2004 and 2015 from febrile children from ages 1-12 years. The R2 region of the GLURP gene was genotyped using nested polymerase chain reaction and by nucleotide sequencing. In all, 12 GLURP alleles were observed in a total of 199 samples collected in the two study years. The multiplicity of infection (MOI) marginally increased over the two study years; however, the differences were statistically insignificant (2004 samples MOI = 1.23 versus 2015 samples MOI = 1.47). Some alleles were stable in their prevalence, whereas two GLURP alleles, VIII and XI, showed considerable variability between both years. This variability was replicated when GLURP sequences from other regions were compared with ours. The expected heterozygosity (He) values (He = 0.87) were identical for the two groups. High variability in the rearrangement of the amino acid repeat units in the R2 region were observed, with the amino acid repeat sequence DKNEKGQHEIVEVEEILPE more prevalent in both years, compared with the two other repeat sequences observed in the study. The parasite population characterized in this study displayed extensive genetic diversity. The detailed genetic profile of the GLURP R2 region has the potential to help guide further epidemiological studies aimed toward the rational design of novel chemotherapies that are antagonistic toward malaria.

Geographical distribution of complement receptor type 1 variants and their associated disease risk.

BACKGROUND: Pathogens exert selective pressure which may lead to substantial changes in host immune responses. The human complement receptor type 1 (CR1) is an innate immune recognition glycoprotein that regulates the activation of the complement pathway and removes opsonized immune complexes. CR1 genetic variants in exon 29 have been associated with expression levels, C1q or C3b binding and increased susceptibility to several infectious diseases. Five distinct CR1 nucleotide substitutions determine the Knops blood group phenotypes, namely Kna/b, McCa/b, Sl1/Sl2, Sl4/Sl5 and KCAM+/-. METHODS: CR1 variants were genotyped by direct sequencing in a cohort of 441 healthy individuals from Brazil, Vietnam, India, Republic of Congo and Ghana. RESULTS: The distribution of the CR1 alleles, genotypes and haplotypes differed significantly among geographical settings (p≤0.001). CR1 variants rs17047660A/G (McCa/b) and rs17047661A/G (Sl1/Sl2) were exclusively observed to be polymorphic in African populations compared to the groups from Asia and South-America, strongly suggesting that these two SNPs may be subjected to selection. This is further substantiated by a high linkage disequilibrium between the two variants in the Congolese and Ghanaian populations. A total of nine CR1 haplotypes were observed. The CR1*AGAATA haplotype was found more frequently among the Brazilian and Vietnamese study groups; the CR1*AGAATG haplotype was frequent in the Indian and Vietnamese populations, while the CR1*AGAGTG haplotype was frequent among Congolese and Ghanaian individuals. CONCLUSION: The African populations included in this study might have a selective advantage conferred to immune genes involved in pathogen recognition and signaling, possibly contributing to disease susceptibility or resistance.

Molecular markers of anti-malarial drug resistance in Central, West and East African children with severe malaria.

BACKGROUND: The Plasmodium falciparum multidrug resistance 1 (PfMDR1), P. falciparum Ca2+-ATPase (PfATP6) and Kelch-13 propeller domain (PfK13) loci are molecular markers of parasite susceptibility to anti-malarial drugs. Their frequency distributions were determined in the isolates collected from children with severe malaria originating from three African countries. METHODS: Samples from 287 children with severe malaria [(Gabon: n = 114); (Ghana: n = 89); (Kenya: n = 84)] were genotyped for pfmdr1, pfatp6 and pfk13 loci by DNA sequencing and assessing pfmdr1 copy number variation (CNV) by real-time PCR. RESULTS: Pfmdr1-N86Y mutation was detected in 48, 10 and 10% in Lambaréné, Kumasi and Kisumu, respectively. At codon 184, the prevalence of the mutation was 73% in Lambaréné, 63% in Kumasi and 49% Kisumu. The S1034C and N1042D variants were absent at all three sites, while the frequency of the D1246Y mutation was 1, 3 and 13% in Lambaréné, Kumasi and Kisumu, respectively. Isolates with two pfmdr1 gene copy number predominantly harboured the N86Y wild-type allele and were mostly found in Kumasi (10%) (P < 0.0001). Among the main pfmdr1 haplotypes (NFD, NYD and YFD), NYD was associated with highest parasitaemia (P = 0.04). At the pfatp6 locus, H243Y and A623E mutations were observed at very low frequency at all three sites. The prevalence of the pfatp6 E431K variant was 6, 18 and 17% in Lambaréné, Kumasi and Kisumu, respectively. The L263E and S769N mutations were absent in all isolates. The pfk13 variants associated with artemisinin resistance in Southeast Asia were not observed. Eleven novel substitutions in the pfk13 locus occurring at low frequency were observed. CONCLUSIONS: Artemisinins are still highly efficacious in large malaria-endemic regions though declining efficacy has occurred in Southeast Asia. The return of chloroquine-sensitive strains following the removal of drug pressure is observed. However, selection of wild-type alleles in the multidrug-resistance gene and the increased gene copy number is associated with reduced lumefantrine sensitivity. This study indicates a need to constantly monitor drug resistance to artemisinin in field isolates from malaria-endemic countries.

Molecular surveillance of Plasmodium falciparum drug resistance in the Republic of Congo: four and nine years after the introduction of artemisinin-based combination therapy.

BACKGROUND: Resistance to anti-malarial drugs hinders efforts on malaria elimination and eradication. Following the global spread of chloroquine-resistant parasites, the Republic of Congo adopted artemisinin-based combination therapy (ACT) in 2006 as a first-line treatment for uncomplicated malaria. To assess the impacts after implementation of ACT, a molecular surveillance for anti-malarial drug resistance was conducted in Congo 4 and 9 years after the introduction of ACT. METHODS: Blood samples of 431 febrile children aged 1-10 years were utilized from two previous studies conducted in 2010 (N = 311) and 2015 (N = 120). All samples were screened for malaria parasites using nested PCR. Direct sequencing was used to determine the frequency distribution of genetic variants in the anti-malarial drug-resistant Plasmodium falciparum genes (Pfcrt, Pfmdr1, Pfatp6, Pfk13) in malaria-positive isolates. RESULTS: One-hundred and nineteen (N = 70 from 2010 and N = 49 from 2015) samples were positive for P. falciparum. A relative decrease in the proportion of chloroquine-resistant haplotype (CVIET) from 100% in 2005, 1 year before the introduction and implementation of ACT in 2006, to 98% in 2010 to 71% in 2015 was observed. Regarding the multidrug transporter gene, a considerable reduction in the frequency of the mutations N86Y (from 73 to 27%) and D1246Y (from 22 to 0%) was observed. However, the prevalence of the Y184F mutation remained stable (49% in 2010 compared to 54% in 2015). Isolates carrying the Pfatp6 H243Y was 25% in 2010 and this frequency was reduced to null in 2015. None of the parasites harboured the Pfk13 mutations associated with prolonged artemisinin clearance in Southeast Asia. Nevertheless, 13 new Pfk13 variants are reported among the investigated isolates. CONCLUSION: The implementation of ACT has led to the decline in prevalence of chloroquine-resistant parasites in the Republic of Congo. However, the constant prevalence of the PfMDR1 Y184F mutation, associated with lumefantrine susceptibility, indicate a selective drug pressure still exists. Taken together, this study could serve as the basis for epidemiological studies monitoring the distribution of molecular markers of artemisinin resistance in the Republic of Congo.

Glucose-6-phosphate dehydrogenase deficiency and reduced haemoglobin levels in African children with severe malaria.

BACKGROUND: Extensive studies investigating the role of host genetic factors during malaria associate glucose-6-phosphate dehydrogenase deficiency with relative protection. G6PD deficiency had been reported to associate with anti-malarial drug induced with haemolytic anaemia. METHODS: A total of 301 Gabonese, Ghanaian, and Kenyan children aged 6-120 months with severe malaria recruited in a multicentre trial on artesunate were included in this sub-study. G6PD normal (type B), heterozygous (type A(+)) and deficient (type A(-)) genotypes were determined by direct sequencing of the common African mutations G202A and A376G. Furthermore, multivariate analyses were executed to associate possible contributions of G6PD deficiency with baseline haemoglobin levels, parasitaemia and with severe malarial anaemia. RESULTS: Two hundred and seventy-eight children (132 females and 146 males) were successfully genotyped for G6PD variants. The overall prevalence of G6PD deficiency was 13 % [36/278; 3 % (4/132) female homozygous and 22 % (32/146) male hemizygous], 14 % (40/278) children were female heterozygous while 73 % (202/278) were G6PD normal [67 % (88/132) females and 78 % (114/146) males] individuals. Multivariate regression revealed a significant association of moderately and severely deficient G6PD genotypes with haemoglobin levels according to the baseline data (p < 0.0001; G6PD heterozygous: p < 0.0001; G6PD deficient: p = 0.009), but not with severe malarial anaemia (p = 0.66). No association of G6PD genotypes with baseline parasitaemia. CONCLUSIONS: In this study, moderately (type A(+)) and severely (type A(-)) G6PD deficiency showed significant association with lower haemoglobin concentrations at baseline in African children with severe malaria without leading to severe malarial anaemia. In addition, there was no association of G6PD variant types with parasite densities on admission.