Do mutations in Plasmodium falciparum dihydropteroate synthase and dihydrofolate reductase confer resistance to sulfadoxine-pyrimethamine in Iran?

The key codons of dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) genes implicated in sulfadoxine-pyrimethamine (SP) resistance were determined by allele-specific polymerase chain reaction in Plasmodium falciparum isolates collected in 1999 from 35 Iranian patients treated with SP. Seven isolates had Glu-540 dhps allele but 5 of these isolates were characterized to possess wild-type dhfr alleles. Seven additional isolates were polyclonal with mixed Lys- and Glu-540. The key dhfr mutation associated with pyrimethamine resistance, Asn-108, was found in 4 isolates. In one patient the presence of Lys- and Glu-540 in dhps and Asn-108 and Arg-59 in dhfr was associated with treatment failure. However, more studies are needed to determine whether clinical response to SP and mutations in these genes are correlated in Iran.     

Transmission of Plasmodium falciparum in urban Yaoundé, Cameroon, is seasonal and age-dependent

Data on malaria transmission intensity and prevalences of asexual parasites and of gametocytes were obtained in an urban district of Yaoundé, Cameroon. The transmission level from mosquito to human was determined by indoor night capture of mosquitoes on human volunteers, revealing a calculated entomological inoculation rate of 34 infectious bites per person per year. Only Anopheles gambiae and A. funestus contributed to malaria transmission and their distribution was seasonal. Cross-sectional surveys every 2 months from July 1999 to May 2000 (n = 965) showed average annual prevalences of 35% Plasmodium falciparum asexual parasites (range 29-38%) and 4.4% gametocytes (range 0-6.7%). Prevalence of high parasitaemia (> 400 parasites/microL) and of gametocytes was seasonal. Prevalence of asexual parasitaemias and of gametocytaemias was age-dependent. The potential infectious reservoir in this area is dominated by the age group 0-15 years, representing 75% of carriers of asexual parasites (P < 0.001), 85% of carriers of high parasitaemias (P < 0.001), and 83% of gametocyte carriers (P = 0.03). Full year logistic models developed from the available data accurately predicted parasite prevalences in subsequent analyses, thus permitting a precise determination of study samples for intervention and seroepidemiology studies, and analysis of the infectious reservoir in this area.     

Emergence of sulfadoxine–pyrimethamine resistance in Indian isolates of Plasmodium falciparum in the last two decades

Genotyping the sulfadoxine–pyrimethamine (SP) genes will help in identifying the genes under drug selection and the emergence of resistance in dhfr and dhps genes. India is an important hotspot for studying malaria due to the immense climatic diversity prevalent in the country. The central and eastern parts of the country are most vulnerable sites where malaria cases are reported throughout the year. From different regions of the country 173 field isolates were genotyped at various loci in dhfr and dhps genes collected between 1994 and 2013. This encompasses the period before antimalarial resistance emerged and the period after the use of combination therapy was made mandatory in the country. We observed the rise of resistant SP alleles from very low frequencies (in the year 1994) to steadily rising (in the year 2000) and maintaining this increasing trend subsequently (in the year 2013) as shown by the sequence analysis of dhfr and dhps genes. This study assessed the prevalence of mutations in dhfr and dhps genes associated with SP resistance in samples indicative of increase in resistance levels of Plasmodium falciparum to SP even after the change in malaria treatment policy in the country.     

In vitro demonstration of pyrimethamine resistance of “wild” Plasmodium falciparum in the Gambia

The in vitro response of wild¹ Gambian Plasmodium falciparum to pyrimethamine is described. Parasites were grown in 100 μl cultures through schizogony. The number of rings present after 48 hours in drug-treated cultures was expressed as a percentage of the controls. Neither a medium and drug change after 24 hours nor different starting parasitaemias were found to affect the outcome of the assay, although a medium and drug change did increase the multiplication rate. 60 randomly taken pure P. falciparum infections were studied. 57 were sensitive. The ID50 and ID90 of drug-sensitive infections were 1·7 × 10^?9M and 4·5 × 10^?9M pyrimethamine respectively. Three infections were resistant (5%) with individual ID50 values of > 10^?6M, 3 and 4 × 10^?9M and ID90 values of > 10^?6M, 8 and 9 × 10^?7M.     

Haplotypes associated with resistance to sulfadoxine–pyrimethamine in Plasmodium falciparum in two malaria endemic locations in Colombia

Colombia has four main malaria transmission zones. In vivo efficacy studies carried out in these areas showed big differences in the response of Plasmodium falciparum to treatment with sulphadoxine–pyrimethamine. In addition, there is still insufficient information about the genetics of P. falciparum populations. The objective of this study was to determine the haplotypes in dhfr and dhps genes of P. falciparum circulating in two distinct endemic zones. Samples from patients with non-complicated P. falciparum malaria were collected: 135 from Tumaco and 206 from Tierralta. Alleles 108 and 51 of the dhfr gene, and 437 and 540 of the dhps gene were analyzed by PCR/enzymatic restriction, while alleles 59 and 164 (dhfr), and 581(dhps) by PCR/dot blot/hybridization. Five different haplotypes were found, of which the triple mutant 51I/C59/108N/I164/437G/K540/A581 was the most frequent (54.6%). In Tumaco, the parasites with wild haplotype predominated, while mutant parasites predominated in Tierralta. Another interesting finding is the presence of the C59R mutation in the dhfr gene in two samples, a mutation rarely found in South America. These data provide information about parasite population genetics and highlight the importance of starting a long term molecular surveillance program.     

Molecular inference of sources and spreading patterns of Plasmodium falciparum malaria parasites in internally displaced persons settlements in Myanmar–China border area

In Myanmar, civil unrest and establishment of internally displaced persons (IDP) settlement along the Myanmar–China border have impacted malaria transmission. The growing IDP populations raise deep concerns about health impact on local communities. Microsatellite markers were used to examine the source and spreading patterns of Plasmodium falciparum between IDP settlement and surrounding villages in Myanmar along the China border. Genotypic structure of P. falciparum was compared over the past three years from the same area and the demographic history was inferred to determine the source of recent infections. In addition, we examined if border migration is a factor of P. falciparum infections in China by determining gene flow patterns across borders. Compared to local community, the IDP samples showed a reduced and consistently lower genetic diversity over the past three years. A strong signature of genetic bottleneck was detected in the IDP samples. P. falciparum infections from the border regions in China were genetically similar to Myanmar and parasite gene flow was not constrained by geographical distance. Reduced genetic diversity of P. falciparum suggested intense malaria control within the IDP settlement. Human movement was a key factor to the spread of malaria both locally in Myanmar and across the international border.     

Pharmacoinformatics based elucidation and designing of potential inhibitors against Plasmodium falciparum to target importin α/β mediated nuclear importation

Plasmodium falciparum, the prime causative agent of malaria, is responsible for 4, 05,000 deaths per year and fatality rates are higher among the children aged below 5 years. The emerging distribution of the multi-drug resistant P. falciparum becomes a worldwide concern, so the identification of unique targets and novel inhibitors is a prime need now. In the present study, we have employed pharmacoinformatics approaches to analyze 265 lead-like compounds from PubChem databases for virtual screening. Thereafter, 15 lead-like compounds were docked within the active side pocket of importin alpha. Comparative ligand properties and absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile were also assessed. Finally, a novel inhibitor was designed and assessed computationally for its efficacy. From the comparative analysis we have found that our screened compounds possess better results than the existing lead ivermectin; having the highest binding energy of −15.6 kcal/mol, whereas ivermectin has −12.4 kcal/mol. The novel lead compound possessed more fascinating output without deviating any of the rules of Lipinski. It also possessed higher bioavailability and the drug-likeness score of 0.55 and 0.71, respectively compared to ivermectin. Furthermore, the binding study was confirmed by molecular dynamics simulation over 25 ns by evaluating the stability of the complex. Finally, all the screened compounds and the novel compound showed promising ADMET properties likewise. To end, we hope that our proposed screened compounds, as well as the novel compound, might give some advances to treat malaria efficiently in vitro and in vivo.     

Slow Clearance of Plasmodium falciparum in Severe Pediatric Malaria,Uganda, 2011–2013

Plasmodium falciparum resistance to artemisinin derivatives is emerging in Asia. We examined molecular markers of resistance in 78 children in Uganda who had severe malaria and were treated with intravenous artesunate. We observed in the K13-propeller domain, A578S, a low-frequency (3/78), nonsynonymous, single-nucleotide polymorphism associated with prolonged parasite clearance.     

Can pretreatment screening for dhps and dhfr point mutations in Plasmodium falciparum infections be used to predict sulfadoxine-pyrimethamine treatment failure?

This study examines the relationship between malaria treatment failure after sulfadoxine-pyrimethamine (S-P) chemotherapy and presence of mutations in the Plasmodium falciparum dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) genes (associated with resistance in vitro to S and P) before treatment. In Kenya, 38 malaria patients in a holoendemic area, and 21 in an epidemic area, participated in the trial in 1997-98. In the 2 areas, drug failure occurred in 76% and 75% of cases where any mutation in dhfr was seen (positive predictive values 76% and 75%: P = 0.003 and 0.008) and an identical association was seen with dhfr Asn-108. In the holoendemic area all occurrences of > or = 2 mutations in dhfr predicted drug failure. Only 3 instances were seen in the epidemic focus, but treatment failed in all. Only in the epidemic focus, 7 (88%) of 8 occurrences of > or = 1 mutations in dhps, and all occurrences of the Gly-437 allele of dhps, predicted failure. Association between mutations in dhps and mutations in dhfr was noted in the combined sites, irrespective of outcome. Although this makes the relationship of combined dhfr and dhps mutations to failure more difficult to interpret, it nevertheless supports S-P selection acting on both genes. In the holoendemic site, treatment success increased with age. In this location, acquired immunity may mask the impact of mutations in dhps, since sulfadoxine is a less effective treatment than pyrimethamine.     

Potential determinants of deductible uptake in health insurance: How to increase uptake in The Netherlands?

In health insurance, voluntary deductibles are offered to the insured in return for a premium rebate. Previous research has shown that 11 % of the Dutch insured opted for a voluntary deductible (VD) in health insurance in 2014, while the highest VD level was financially profitable for almost 50 % of the population in retrospect. To explain this discrepancy, this paper identifies and discusses six potential determinants of the decision to opt for a VD from the behavioral economic literature: loss aversion, risk attitude, ambiguity aversion, debt aversion, omission bias, and liquidity constraints. Based on these determinants, five potential strategies are proposed to increase the number of insured opting for a VD. Presenting the VD as the default option and providing transparent information regarding the VD are the two most promising strategies. If, as a result of these strategies, more insured would opt for a VD, moral hazard would be reduced.     

High prevalence of sulphadoxine–pyrimethamine resistance-associated mutations in Plasmodium falciparum field isolates from pregnant women in Brazzaville,Republic of Congo

Intermittent preventive treatment during pregnancy with sulfadoxine–pyrimethamine (IPTp-SP) has not been evaluated in the Republic of Congo since its implementation in 2006 and there is no published data on molecular markers of SP resistance among Plasmodium falciparum isolates from pregnant women. This first study in this country aimed to describe the prevalence of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) point mutations and haplotypes in P. falciparum isolates collected from pregnant women with asymptomatic infection. From March 2012 to December 2013, pregnant women attending Madibou health centre (in Southern Brazzaville) for antenatal visits were enrolled in this study after obtaining their written informed consent. Blood samples were collected and P. falciparum infections were characterized using PCR. A total of 363 pregnant women were enrolled. P. falciparum infection was detected in 67 (18.4%) samples as their PCR amplification of dhfr and dhps genes yielded bands and all the PCR products were successfully digested. Out of these 67 isolates, 59 (88%), 57 (85%) and 53 (79.1%) carried 51I, 59R and 108N dhfr mutant alleles, respectively. The prevalence of dhps 436A, 437G and 540E mutations were 67.1% (45/67), 98.5% (66/67) and 55.2% (37/67), respectively. More than one-half of the isolates carried quintuple mutations, with highly resistant haplotype dhfr51I/59R/108N + dhps437G/540E detected in 33% (22/67) whereas 25% (17/67) were found to carry sextuple mutations. We observed significantly higher frequencies of triple dhps mutations 436A/437G/540E and quintuple mutations dhfr51I/59R/108N + dhps437G/540E in isolates from women who received IPTp-SP than those who did not. Overall, this study shows high prevalence rates of SP-associated resistance mutations in P. falciparum isolates collected from pregnant women. The presence of the dhps mutant allele 540E and the high prevalence of isolates carrying quintuple dhfr/dhps mutations are here reported for the first time in the Republic of Congo. The increasing prevalence of multiple mutant alleles observed in this study is alarming and may present a challenge for the future interventions including IPTp-SP in the country.     

Growth of protozoa in tissue culture. II.—Plasmodium relictum,exoerythrocytic forms

The exoerythrocytic forms of Plasmodium relictum have been grown in tissue culture, using the technique previously described for P. gallinaceum. Growth was terminated after 19 days. Fluid removed from the culture flasks after 5 and 9 days was infective for a canary.Growth took place in the presence of penicillin and of streptomycin.This technique is advantageous for studying the morphology of the exoerythrocytic forms, especially in the different phases of schizogony. The various stages are illustrated.     

Prevalence of 5′ insertion mutants and analysis of single nucleotide polymorphism in the erythrocyte binding-like 1 (ebl-1) gene in Kenyan Plasmodium falciparum field isolates

Plasmodium merozoites attach to and invade red blood cells (RBCs) during the erythrocytic cycle. The invasion process requires recognition of RBC surface receptors by proteins of the Plasmodium Duffy binding like erythrocyte binding like (DBL-EBP) family. Clones and isolates of Plasmodium falciparum have varying abilities to utilize different RBC receptors, and multiple distinct pathways so far identified depend on glycophorins A, B, C, and as yet unidentified receptors. At present, five members of the DBL-EBP family have been identified in the P. falciparum genome, based on gene structure and amino acid sequence homology. The cardinal features of this family consist of conserved 5′ and 3′ cysteine-rich regions (regions II and VI, respectively) whose cysteine residues are highly conserved along with the majority of aromatic amino acids. In contrast to the single DBL-EBP family member in Plasmodium vivax, in P. falciparum all DBL-EBP family members have a duplication of the conserved 5′ cysteine-rich region denoted as the F1 and F2 domains. These cysteine-rich regions are considered crucial in recognition of erythrocyte receptors and it has been shown that several bind to glycophorins on the erythrocyte surface. Several studies, on both field isolates and laboratory strains have uncovered a relatively high degree of sequence polymorphism in the DBP-EBL genes. This study is now extended to include field isolates collected from sites within Kenya. DNA isolated from blood samples of infected patients was utilized to amplify the region I sequence of ebl-1 gene in order to investigate polymorphism in the region immediately adjacent to the 5′ cysteine-rich domains, and to determine the prevalence of an insertion mutant that effectively knocks out the gene.     

The Relation of a Woman’s Impaired in Utero Growth and Association of Diabetes During Pregnancy

Small for gestational age (weight for gestational age <10th percentile, SGA) birth status and adulthood susceptibility to diabetes is well established, but the relationship to diabetes during pregnancy is incompletely understood. The authors investigated the association between women’s impaired fetal growth (as measured by SGA status) and diabetes mellitus (DM) during pregnancy. Stratified and multivariable binomial regression analyses were performed on the Illinois transgenerational dataset. Former SGA (n = 13,934) mothers had a greater prevalence of DM during pregnancy than former appropriate for gestational age (AGA) mothers (n = 116,683): 2.7 versus 1.9 %, relative prevalence (RP) equaled 1.4 [95 % confidence interval (CI)1.3, 1.6]. In a multivariable binomial regression model, the adjusted RP (95 %CI) (controlling for maternal age, education, parity, plurality, marital status, and race/ethnicity) for DM during pregnancy for former SGA (compared to AGA) mothers equaled 1.5 (1.3, 1.6). When stratified by race/ethnicity, the adjusted RP (95 % CI) of DM during pregnancy for former SGA (compared to AGA), non-Latina White, African-American, and Mexican-American mothers was 1.4 (1.3, 1.6), 1.6 (1.2, 2.1), and 2.3 (1.1, 4.7), respectively. The authors conclude that impaired fetal growth (as measured by SGA status) is a risk factor for DM during pregnancy among the leading racial/ethnic groups in the United States.