Antiplasmodial action of 4-nitrobenzenesulfonamide chalcones: Design, synthesis, characterisation, in vitro and in silico evaluation against blood stages of Plasmodium falciparum 3D7.

Malaria is an intracellular protozoan parasitic disease caused by Plasmodium species with significant morbidity and mortality in endemic regions. The complex lifecycle of the parasite and the emergence of drug-resistant Plasmodium falciparum have hampered the efficacy of current anti-malarial agents. To circumvent this situation, the present study attempts to demonstrate the blood-stage anti-plasmodial action of 26 hybrid compounds containing the three privileged bioactive scaffolds (sulfonamide, chalcone, and nitro group) with synergistic and multitarget action. These three parent scaffolds exhibit divergent activities, such as antibacterial, anti-malarial, anti-fungal, anti-inflammatory, and anticancer. All the synthesised compounds were characterised using various spectroscopic techniques. The in vitro blood-stage inhibitory activity of 26 hybrid compounds was evaluated against mixed-stage culture (asynchronize) of human malarial parasite P. falciparum, Pf 3D7 at different concentrations ranging from 25.0 µg/mL to 0.78 µg/mL using SYBR 1 green assay, with IC50 values determined after 48 h of treatment based on the drug-response curves. Two potent compounds (11 and 10), with 2-Br and 2,6-diCl substitutions, showed pronounced activity with IC50 values of 5.4 µg/mL and 5.6 µg/mL, whereas others displayed varied activity with IC50 values ranging from 7.0 µg/mL to 22.0 µg/mL. Both 11 and 10 showed greater susceptibility towards mature-stage trophozoites than ring-stage parasites. The hemolytic and in vitro cytotoxicity assays revealed that compounds 11 and 10 did not cause any toxic effects on host red blood cells (uninfected), human-derived Mo7e cells, and murine-derived BA/F3 cells. The in vitro observations are consistent with the in silico studies using P. falciparum-dihydrofolate reductase, where 11 and 10 showed a binding affinity of -10.4 Kcal/mol. This is the first report of the hybrid scaffold, 4-nitrobenzenesulfonamide chalcones, demonstrating its potential as an anti-plasmodial agent.

Varied Prevalence of Antimalarial Drug Resistance Markers in Different Populations of Newly Arrived Refugees in Uganda.

Newly arrived refugees offer insights into malaria epidemiology in their countries of origin. We evaluated asymptomatic refugee children within 7 days of arrival in Uganda from South Sudan and the Democratic Republic of Congo (DRC) in 2022 for parasitemia, parasite species, and Plasmodium falciparum drug resistance markers. Asymptomatic P. falciparum infections were common in both populations. Coinfection with P. malariae was more common in DRC refugees. Prevalences of markers of aminoquinoline resistance (PfCRT K76T, PfMDR1 N86Y) were much higher in South Sudan refugees, of antifolate resistance (PfDHFR C59R and I164L, PfDHPS A437G, K540E, and A581G) much higher in DRC refugees, and of artemisinin partial resistance (ART-R; PfK13 C469Y and A675V) moderate in both populations. Prevalences of most mutations differed from those seen in Ugandans attending health centers near the refugee centers. Refugee evaluations yielded insights into varied malaria epidemiology and identified markers of ART-R in 2 previously little-studied countries.

Assessment of Plasmodium falciparum drug resistance associated molecular markers in Mandla, Madhya Pradesh, India.

BACKGROUND: Resistance against artemisinin-based combination therapy is one of the challenges to malaria control and elimination globally. Mutations in different genes (Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1) confer resistance to artesunate and sulfadoxine-pyrimethamine (AS + SP) were analysed from Mandla district, Madhya Pradesh, to assess the effectiveness of the current treatment regimen against uncomplicated Plasmodium falciparum. METHODS: Dried blood spots were collected during the active fever survey and mass screening and treatment activities as part of the Malaria Elimination Demonstration Project (MEDP) from 2019 to 2020. Isolated DNA samples were used to amplify the Pfdhfr, Pfdhps, Pfk13 and Pfmdr1 genes using nested PCR and sequenced for mutation analysis using the Sanger sequencing method. RESULTS: A total of 393 samples were subjected to PCR amplification, sequencing and sequence analysis; 199, 215, 235, and 141 samples were successfully sequenced for Pfdhfr, Pfdhps, Pfk13, Pfmdr1, respectively. Analysis revealed that the 53.3% double mutation (C59R, S108N) in Pfdhfr, 89.3% single mutation (G437A) in Pfdhps, 13.5% single mutants (N86Y), and 51.1% synonymous mutations in Pfmdr1 in the study area. Five different non-synonymous and two synonymous point mutations found in Pfk13, which were not associated to artemisinin resistance. CONCLUSION: The study has found that mutations linked to SP resistance are increasing in frequency, which may reduce the effectiveness of this drug as a future partner in artemisinin-based combinations. No evidence of mutations linked to artemisinin resistance in Pfk13 was found, suggesting that parasites are sensitive to artemisinin derivatives in the study area. These findings are a baseline for routine molecular surveillance to proactively identify the emergence and spread of artemisinin-resistant parasites.

Hybrid PABA-glutamic acid conjugated 1,3,5-triazine derivatives: Design, synthesis, and antimalarial activity screening targeting Plasmodium falciparum dihydro folate reductase enzyme.

Despite the successful reduction in the malaria health burden in recent years, it continues to remain a significant global health problem mainly because of the emerging resistance to first-line treatments. Also because of the disruption in malaria prevention services during the COVID-19 pandemic, there was an increase in malaria cases in 2021 compared to 2020. Hence, the present study outlined the in silico study, synthesis, and antimalarial evaluation of 1,3,5-triazine hybrids conjugated with PABA-glutamic acid. Docking study revealed higher binding energy compared to the originally bound ligand WR99210, predominant hydrogen bond interaction, and involvement of key amino acid residues, like Arg122, Ser120, and Arg59. Fourteen compounds were synthesized using traditional and microwave synthesis. The in vitro antimalarial evaluation against chloroquine-sensitive 3D7 and resistant Dd2 strain of Plasmodium falciparum showed a high to moderate activity range. Compounds C1 and B4 showed high efficacy against both strains and a further study revealed that compound C1 is non-cytotoxic against the HEK293 cell line with no acute oral toxicity. In vivo, study was performed for the most potent antimalarial compound C1 to optimize the research work and found to be effectively suppressing parasitemia of Plasmodium berghei strain in the Swiss albino mice model.

Therapeutic efficacy of artesunate-amodiaquine and artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in Chad: clinical and genetic surveillance.

BACKGROUND: Artesunate-amodiaquine (AS-AQ) and artemether-lumefantrine (AL) are the currently recommended first-and second-line therapies for uncomplicated Plasmodium falciparum infections in Chad. This study assessed the efficacy of these artemisinin-based combinations, proportion of day 3 positive patients, proportions of molecular markers associated with P. falciparum resistance to anti-malarial drugs and variable performance of HRP2-based malaria rapid diagnostic tests (RDTs). METHODS: A single-arm prospective study assessing the efficacy of AS-AQ and AL at three sites (Doba, Kelo and Koyom) was conducted between November 2020 to January 2021. Febrile children aged 6 to 59 months with confirmed uncomplicated P. falciparum infection were enrolled sequentially first to AS-AQ and then AL at each site and followed up for 28 days. The primary endpoint was PCR-adjusted adequate clinical and parasitological response (ACPR). Samples collected on day 0 were analysed for mutations in pfkelch13, pfcrt, pfmdr-1, pfdhfr, pfdhps genes and deletions in pfhrp2/pfhrp3 genes. RESULTS: By the end of 28-day follow-up, per-protocol PCR corrected ACPR of 97.8% (CI 95% 88.2-100) in Kelo and 100% in Doba and Kayoma were observed among AL treated patients. For ASAQ, 100% ACPR was found in all sites. All, but one patient, did not have parasites detected on day 3. Out of the 215 day 0 samples, 96.7% showed pfkelch13 wild type allele. Seven isolates carried nonsynonymous mutations not known to be associated artemisinin partial resistance (ART-R). Most of samples had a pfcrt wild type allele (79% to 89%). The most prevalent pfmdr-1 allele detected was the single mutant 184F (51.2%). For pfdhfr and pfdhps mutations, the quintuple mutant allele N51I/C59R/S108N + G437A/540E responsible for SP treatment failures in adults and children was not detected. Single deletion in the pfhrp2 and pfhrp3 gene were detected in 10/215 (4.7%) and 2/215 (0.9%), respectively. Dual pfhrp2/pfhrp3 deletions, potentially threatening the efficacy of HRP2-based RDTs, were observed in 5/215 (2.3%) isolates. CONCLUSION: The results of this study confirm that AS-AQ and AL treatments are highly efficacious in study areas in Chad. The absence of known pfkelch13 mutations in the study sites and the high parasite clearance rate at day 3 suggest the absence of ART-R. The absence of pfdhfr/pfdhps quintuple or sextuple (quintuple + 581G) mutant supports the continued use of SP for IPTp during pregnancy. The presence of parasites with dual pfhrp2/pfhrp3 deletions, potentially threatening the efficacy of HRP2-based RDTs, warrants the continued surveillance. Trial registration ACTRN12622001476729.

Genomic characterization of Plasmodium falciparum genes associated with anti-folate drug resistance and treatment outcomes in eastern India: A molecular surveillance study from 2008 to 2017.

Introduction: After being used vigorously for the previous two decades to treat P. falciparum, chloroquine and sulfadoxine-pyrimethamine were replaced in 2009 with an artemisinin-based combination therapy (artesunate-sulfadoxine-pyrimethamine) in an effort to combat multidrug-resistant parasites. Methods: We set out to assess the genetic variants of sulfadoxine-pyrimethamine resistance and the effectiveness of its treatment in eastern India prior to, during, and 6 to 8 years following the introduction of the new pharmacological regime. In 2008-2009, 318 P. falciparum-positive patients got the recommended doses of sulfadoxine-pyrimethamine. We used 379 additional isolates from 2015 to 2017 in addition to the 106 isolates from 2010. All 803 isolates from two study sites underwent in vitro sulfadoxine-pyrimethamine sensitivity testing and genomic characterisation of sulfadoxine-pyrimethamine resistance (pfdhfr and pfdhps). Results: In Kolkata and Purulia, we observed early treatment failure in 30.7 and 14.4% of patients, respectively, whereas recrudescence was found in 8.1 and 13.4% of patients, respectively, in 2008-2009. In 2017, the proportion of in vitro pyrimethamine and sulfadoxine resistance steadily grew in Kolkata and Purulia despite a single use of sulfadoxine-pyrimethamine. Treatment failures with sulfadoxine-pyrimethamine were linked to quintuple or quadruple pfdhfr- pfdhps mutations (AICII-AGKAT, AICII-AGKAA, AICII-SGKGT, AICII-AGKAA, AICNI-AGKAA) in 2008-2009 (p < 0.001). The subsequent spread of mutant-haplotypes with higher in vitro sulfadoxine-pyrimethamine resistance (p < 0.001), such as the sextuple (dhfr-AIRNI+dhps-AGEAA, dhfr-ANRNL+dhps-AGEAA) and septuple (dhfr-AIRNI+dhps-AGEAT), mutations were observed in 2015-2017. Discussion: This successive spread of mutations with high in vitro sulfadoxine-pyrimethamine resistance confirmed the progressive increase in antifolate resistance even after an 8-year withdrawal of sulfadoxine-pyrimethamine.

Prevalence of Mutations in the Pfdhfr, Pfdhps, and Pfmdr1 Genes of Malarial Parasites Isolated from Symptomatic Patients in Dogondoutchi, Niger.

The effectiveness of artemisinin-based combination therapies (ACTs) depends not only on that of artemisinin but also on that of partner molecules. This study aims to evaluate the prevalence of mutations in the Pfdhfr, Pfdhps, and Pfmdr1 genes from isolates collected during a clinical study. Plasmodium genomic DNA samples extracted from symptomatic malaria patients from Dogondoutchi, Niger, were sequenced by the Sanger method to determine mutations in the Pfdhfr (codons 51, 59, 108, and 164), Pfdhps (codons 436, 437, 540, 581, and 613), and Pfmdr1 (codons 86, 184, 1034, and 1246) genes. One hundred fifty-five (155) pre-treatment samples were sequenced for the Pfdhfr, Pfdhps, and Pfmdr1 genes. A high prevalence of mutations in the Pfdhfr gene was observed at the level of the N51I (84.97%), C59R (92.62%), and S108N (97.39%) codons. The key K540E mutation in the Pfdhps gene was not observed. Only one isolate was found to harbor a mutation at codon I431V. The most common mutation on the Pfmdr1 gene was Y184F in 71.43% of the mutations found, followed by N86Y in 10.20%. The triple-mutant haplotype N51I/C59R/S108N (IRN) was detected in 97% of the samples. Single-mutant (ICS and NCN) and double-mutant (IRS, NRN, and ICN) haplotypes were prevalent at 97% and 95%, respectively. Double-mutant haplotypes of the Pfdhps (581 and 613) and Pfmdr (86 and 184) were found in 3% and 25.45% of the isolates studied, respectively. The study focused on the molecular analysis of the sequencing of the Pfdhfr, Pfdhps, and Pfmdr1 genes. Although a high prevalence of mutations in the Pfdhfr gene have been observed, there is a lack of sulfadoxine pyrimethamine resistance. There is a high prevalence of mutation in the Pfmdr184 codon associated with resistance to amodiaquine. These data will be used by Niger's National Malaria Control Program to better monitor the resistance of Plasmodium to partner molecules in artemisinin-based combination therapies.

Microwave synthesis and antimalarial screening of novel 4-amino benzoic acid (PABA)-substituted pyrimidine derivatives as Plasmodium falciparum dihydrofolate reductase inhibitors.

Antimalarial drug resistance is a major threat due to the emerging resistance to all the available drugs in the market. In an approach to develop alternative drugs, a novel class of Pf-DHFR inhibitors was developed using pyrimidine as the core nucleus and substituting the 4- and 6- positions with amines and 4-amino benzoic acid (PABA) to avoid the problem of drug resistance. The resultant compounds 3(a-j) after primary in silico screening and filtering were synthesized using microwave efficiently in high yield and reduced time period compared to conventional synthesis. The antimalarial assay was performed in vitro, against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strains of Plasmodium falciparum using chloroquine as a reference standard. The IC50 values were in the range of 5.26-106.76 µg/ml for 3D7 and in Dd2 the value ranges from 4.71 to 112.98 µg/ml. Compounds 3d, 3e, 3f and 3h showed significant antimalarial activity against both the strains of P. falciparum with no cytotoxicity against fibroblast cell line and 3f was found to be the most potent among them. The hemolysis assay of all the compounds in fresh erythrocytes showed insignificant hemolysis below 5% at a higher dose level. Hence, the present study suggests the possible utility of PABA-substituted pyrimidine scaffold for further development of new Pf-DHFR inhibitors. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03236-w.

Decreased Susceptibility to Dihydrofolate Reductase Inhibitors Associated With Genetic Polymorphisms in Ugandan Plasmodium falciparum Isolates.

BACKGROUND: The Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors pyrimethamine and cycloguanil (the active metabolite of proguanil) have important roles in malaria chemoprevention, but drug resistance challenges their efficacies. A new compound, P218, was designed to overcome resistance, but drug-susceptibility data for P falciparum field isolates are limited. METHODS: We studied ex vivo PfDHFR inhibitor susceptibilities of 559 isolates from Tororo and Busia districts, Uganda, from 2016 to 2020, sequenced 383 isolates, and assessed associations between genotypes and drug-susceptibility phenotypes. RESULTS: Median half-maximal inhibitory concentrations (IC50s) were 42 100 nM for pyrimethamine, 1200 nM for cycloguanil, 13000 nM for proguanil, and 0.6 nM for P218. Among sequenced isolates, 3 PfDHFR mutations, 51I (100%), 59R (93.7%), and 108N (100%), were very common, as previously seen in Uganda, and another mutation, 164L (12.8%), had moderate prevalence. Increasing numbers of mutations were associated with decreasing susceptibility to pyrimethamine, cycloguanil, and P218, but not proguanil, which does not act directly against PfDHFR. Differences in P218 susceptibilities were modest, with median IC50s of 1.4 nM for parasites with mixed genotype at position 164 and 5.7 nM for pure quadruple mutant (51I/59R/108N/164L) parasites. CONCLUSIONS: Resistance-mediating PfDHFR mutations were common in Ugandan isolates, but P218 retained excellent activity against mutant parasites.

Microwave-assisted synthesis of hybrid PABA-1,3,5-triazine derivatives as an antimalarial agent.

The present manuscript deals with the development of novel p-aminobenzoic acid (PABA) associated 1,3,5-triazine derivatives as antimalarial agents. The molecules were developed via microwave-assisted synthesis and structures of compounds were ascertained via numerous analytical and spectroscopic techniques. The synthesized compounds were also subjected to ADMET analysis. In a docking analysis, the title compounds showed high and diverse binding affinities towards wild (-162.45 to -369.38 kcal/mol) and quadruple mutant (-165.36 to -209.47 kcal/mol) Pf-DHFR-TS via interacting with Phe58, Arg59, Ser111, Ile112, Phe116. The in vitro antimalarial activity suggested that compounds 4e, 4b, and 4h showed IC50 ranging from 4.18 to 8.66 µg/ml against the chloroquine-sensitive (3D7) strain of Plasmodium falciparum. Moreover, compounds 4g, 4b, 4e, and 4c showed IC50 ranging from 8.12 to 12.09 µg/ml against chloroquine-resistant (Dd2) strain. In conclusion, our study demonstrated the development of hybrid PABA substituted 1,3,5-triazines as a novel class of Pf-DHFR inhibitor for antimalarial drug discovery.

Surveillance of Antimalarial Drug-Resistance Genes in Imported Plasmodium falciparum Isolates From Nigeria in Henan, China, 2012-2019.

Malaria remains a major public health issue in Nigeria, and Nigeria is one of the main sources of imported malaria in China. Antimalarial drug resistance is a significant obstacle to the control and prevention of malaria globally. The molecular markers associated with antimalarial drug resistance can provide early warnings about the emergence of resistance. The prevalence of antimalarial drug resistant genes and mutants, including PfK13, Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps, was evaluated among the imported Plasmodium falciparum isolates from Nigeria in Henan, China, from 2012 to 2019. Among the 167 imported P. falciparum isolates, the wild-type frequency of PfK13, Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps was 98.7, 63.9, 34.8, 3.1, and 3.1%, respectively. The mutation of PfK13 was rare, with just two nonsynonymous (S693F and Q613H) and two synonymous mutations (C469C and G496G) identified from four isolates. The prevalence of Pfcrt mutation at codon 74-76 decreased year-by-year, while the prevalence of pfmdr1 86Y also decreased significantly with time. The prevalence of Pfdhfr and Pfdhps mutants was high. Combined mutations of Pfdhfr and Pfdhps had a high prevalence of the quadruple mutant I51R59N108-G437 (39.0%), followed by the octal mutant I51R59N108-V431A436G437G581S613 (17.0%). These molecular findings update the known data on antimalarial drug-resistance genes and provide supplemental information for Nigeria.

Reduced Birth Weight Caused by Sextuple Drug-Resistant Plasmodium falciparum Infection in Early Second Trimester.

Mutations in the Plasmodium falciparum genes Pfdhfr and Pfdhps, particularly the sextuple mutant haplotype threatens the antimalarial effectiveness of sulfadoxine-pyrimethamine (SP) as intermittent preventive treatment during pregnancy (IPTp). To explore the impact of sextuple mutant haplotype infections on outcome measures after provision of IPTp with SP, we monitored birth outcomes in women followed up from before conception or from the first trimester until delivery. Women infected with sextuple haplotypes, in the early second trimester specifically, delivered newborns with a lower birth weight compared with women who did not have malaria during pregnancy (difference, -267 g; 95% confidence interval, -454 to -59; P = .01) and women infected with less SP-resistant haplotypes (-461 g; -877 to -44; P = .03). Thus, sextuple haplotype infections seem to affect the effectiveness of SP for IPTp and directly affect birth outcome by lowering birth weight. Close monitoring and targeted malaria control during early pregnancy is therefore crucial to improving birth outcomes.

Temporal evolution of sulfadoxine-pyrimethamine resistance genotypes and genetic diversity in response to a decade of increased interventions against Plasmodium falciparum in northern Ghana.

BACKGROUND: Anti-malarial drug resistance remains a key concern for the global fight against malaria. In Ghana sulfadoxine-pyrimethamine (SP) is used for intermittent preventive treatment of malaria in pregnancy and combined with amodiaquine for Seasonal Malaria Chemoprevention (SMC) during the high malaria season. Thus, surveillance of molecular markers of SP resistance is important to guide decision-making for these interventions in Ghana. METHODS: A total of 4469 samples from uncomplicated malaria patients collected from 2009 to 2018 was submitted to the Wellcome Trust Sanger Institute, UK for DNA sequencing using MiSeq. Genotypes were successfully translated into haplotypes in 2694 and 846 mono infections respectively for pfdhfr and pfdhps genes and the combined pfhdfr/pfdhps genes across all years. RESULTS: At the pfdhfr locus, a consistently high (> 60%) prevalence of parasites carrying triple mutants (IRNI) were detected from 2009 to 2018. Two double mutant haplotypes (NRNI and ICNI) were found, with haplotype NRNI having a much higher prevalence (average 13.8%) than ICNI (average 3.2%) across all years. Six pfdhps haplotypes were detected. Of these, prevalence of five fluctuated in a downward trend over time from 2009 to 2018, except a pfdhps double mutant (AGKAA), which increased consistently from 2.5% in 2009 to 78.2% in 2018. Across both genes, pfdhfr/pfdhps combined triple (NRNI + AAKAA) mutants were only detected in 2009, 2014, 2015 and 2018, prevalence of which fluctuated between 3.5 and 5.5%. The combined quadruple (IRNI + AAKAA) genotype increased in prevalence from 19.3% in 2009 to 87.5% in 2011 before fluctuating downwards to 19.6% in 2018 with an average prevalence of 37.4% within the nine years. Prevalence of parasites carrying the quintuple (IRNI + AGKAA or SGEAA) mutant haplotypes, which are highly refractory to SP increased over time from 14.0% in 2009 to 89.0% in 2016 before decreasing to 78.9 and 76.6% in 2017 and 2018 respectively. Though quintuple mutants are rising in prevalence in both malaria seasons, together these combined genotypes vary significantly within season but not between seasons. CONCLUSIONS: Despite high prevalence of pfdhfr triple mutants and combined pfdhfr/pfdhps quadruple and quintuple mutants in this setting SP may still be efficacious. These findings are significant as they highlight the need to continuously monitor SP resistance, particularly using deep targeted sequencing to ascertain changing resistance patterns.

Low prevalence of highly sulfadoxine-resistant dihydropteroate synthase alleles in Plasmodium falciparum isolates in Benin.

BACKGROUND: In 2004, in response to high levels of treatment failure associated with sulfadoxine-pyrimethamine (SP) resistance, Benin changed its first-line malaria treatment from SP to artemisinin-based combination therapy for treatment of uncomplicated Plasmodium falciparum malaria. Resistance to SP is conferred by accumulation of single nucleotide polymorphisms (SNPs) in P. falciparum genes involved in folate metabolism, dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps), targeted by pyrimethamine and sulfadoxine, respectively. Because SP is still used for intermittent preventive treatment in pregnant women (IPTp) and seasonal malaria chemoprevention (SMCP) in Benin, the prevalence of Pfdhfr and Pfdhps SNPs in P. falciparum isolates collected in 2017 were investigated. METHODS: This study was carried out in two sites where the transmission of P. falciparum malaria is hyper-endemic: Klouékanmey and Djougou. Blood samples were collected from 178 febrile children 6-59 months old with confirmed uncomplicated P. falciparum malaria and were genotyped for SNPs associated with SP resistance. RESULTS: The Pfdhfr triple mutant IRN (N51I, C59R, and S108N) was the most prevalent (84.6%) haplotype and was commonly found with the Pfdhps single mutant A437G (50.5%) or with the Pfdhps double mutant S436A and A437G (33.7%). The quintuple mutant, Pfdhfr IRN/Pfdhps GE (A437G and K540E), was rarely observed (0.8%). The A581G and A613S mutant alleles were found in 2.6 and 3.9% of isolates, respectively. Six isolates (3.9%) were shown to harbour a mutation at codon I431V, recently identified in West African parasites. CONCLUSIONS: This study showed that Pfdhfr triple IRN mutants are near fixation in this population and that the highly sulfadoxine-resistant Pfdhps alleles are not widespread in Benin. These data support the continued use of SP for chemoprevention in these study sites, which should be complemented by periodic nationwide molecular surveillance to detect emergence of resistant genotypes.

In silico analysis, synthesis and biological evaluation of DHFR inhibitors.

INTRODUCTION: Malaria is one of the varieties of fatal diseases caused by a protozoan parasite that is now considered to be the greatest global health challenge. A parasite of Plasmodium species triggers it transmitting the disease to humans by the bites of female Anopheles mosquitoes. AIM: To screen out designed molecules by molecular docking analysis and assess their pharmacokinetic properties using SwissADME. To synthesize the designed compounds. To characterize the synthesized compounds by TLC, melting point, IR spectroscopy, mass spectrometry, 1H NMR, and 13C NMR. To evaluate the synthesized compounds for antimalarial activity. MATERIALS AND METHODS: In silico analysis was performed with SWISSADME, and molecular docking was performed by AutoDock Vina version 4.2. In vitro antimalarial activity study was performed. RESULTS: In-vitro studies of synthesized molecules showed that compounds C2 (IC50 1.23), C6 (IC50 0.48), C10 (IC50 0.79), and C14 (IC50 0.19) possess good antimalarial activity. CONCLUSIONS: 7-chloroquinoline-piperazine derivatives exhibited potential antimalarial compounds for pf-DHFR inhibitors.

Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda.

BACKGROUND: In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies. METHODS: Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays. RESULTS: Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0-46% in 2018; 0-23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively). CONCLUSIONS: We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

Efficacy of artemether-lumefantrine for treating uncomplicated Plasmodium falciparum cases and molecular surveillance of drug resistance genes in Western Myanmar.

BACKGROUND: Currently, artemisinin-based combination therapy (ACT) is the first-line anti-malarial treatment in malaria-endemic areas. However, resistance in Plasmodium falciparum to artemisinin-based combinations emerging in the Greater Mekong Sub-region is a major problem hindering malaria elimination. To continuously monitor the potential spread of ACT-resistant parasites, this study assessed the efficacy of artemether-lumefantrine (AL) for falciparum malaria in western Myanmar. METHODS: Ninety-five patients with malaria symptoms from Paletwa Township, Chin State, Myanmar were screened for P. falciparum infections in 2015. After excluding six patients with a parasite density below 100 or over 150,000/µL, 41 P. falciparum patients were treated with AL and followed for 28 days. Molecular markers associated with resistance to 4-amino-quinoline drugs (pfcrt and pfmdr1), antifolate drugs (pfdhps and pfdhfr) and artemisinin (pfk13) were genotyped to determine the prevalence of mutations associated with anti-malarial drug resistance. RESULTS: For the 41 P. falciparum patients (27 children and 14 adults), the 28-day AL therapeutic efficacy was 100%, but five cases (12.2%) were parasite positive on day 3 by microscopy. For the pfk13 gene, the frequency of NN insert after the position 136 was 100% in the day-3 parasite-positive group as compared to 50.0% in the day-3 parasite-negative group, albeit the difference was not statistically significant (P = 0.113). The pfk13 K189T mutation (10.0%) was found in Myanmar for the first time. The pfcrt K76T and A220S mutations were all fixed in the parasite population. In pfmdr1, the Y184F mutation was present in 23.3% of the parasite population, and found in both day-3 parasite-positive and -negative parasites. The G968A mutation of pfmdr1 gene was first reported in Myanmar. Prevalence of all the mutations in pfdhfr and pfdhps genes assessed was over 70%, with the exception of the pfdhps A581G mutation, which was 3.3%. CONCLUSIONS: AL remained highly efficacious in western Myanmar. Pfk13 mutations associated with artemisinin resistance were not found. The high prevalence of mutations in pfcrt, pfdhfr and pfdhps suggests high-degree resistance to chloroquine and antifolate drugs. The pfmdr1 N86/184F/D1246 haplotype associated with selection by AL in Africa reached > 20% in this study. The detection of > 10% patients who were day-3 parasite-positive after AL treatment emphasizes the necessity of continuously monitoring ACT efficacy in western Myanmar.

Analysis of sulphadoxine-pyrimethamine resistance-associated mutations in Plasmodium falciparum isolates obtained from asymptomatic pregnant women in Ogun State, Southwest Nigeria.

Intermittent preventive treatment in pregnancy with sulphadoxine-pyrimethamine (IPTp-SP) is one of the main strategies for protecting pregnant women, fetus, and their new-born against adverse effects of P. falciparum infection. The development of the drug resistance linked to mutations in P. falciparum dihydrofolate reductase gene (pfdhfr) and P. falciparum dihydropteroate synthase gene (pfdhps), is currently threatening the IPTp-SP approach. This study determined the prevalence of pfdhfr and pfdhps mutations in isolates obtained from pregnant women with asymptomatic P. falciparum infection in Nigerian. Additionally, P. falciparum genetic diversity and multiplicity of infection (MOI) was assessed by genotyping the P. falciparum merozoite surface Protein 1 and 2 (pfmsp-1 and pfmsp-2) genes. The pfdhfr and pfdhps were genotyped by direct sequencing, and the pfmsp-1 and pfmsp-2 fragment analysis by polymerase chain reaction was used to determine P. falciparum genetic diversity. Of the 406 pregnant women recruited, 123 had P. falciparum infection by PCR, and of these, 52 were successfully genotyped for pfdhfr and 42 for pfdhps genes. The pfdhfr triple-mutant parasites (N51I, C59R, and S108N) or the IRN haplotype were predominant (98%), whereas pfdhfr mutations C50R and I164L did not occur. For pfdhps gene, the prevalence of A437G, A581G, A436A, and A613S mutations were 98, 71, 55, and 36%, respectively. Nineteen (44%) isolates with quintuple mutations (CIRNI- SGKGA) had the highest combined pfdhfr-pfdhps haplotype. Isolates with sextuple mutants; CIRNI- AGKAS and CIRNI- AGKGA had a prevalence of 29 and 14%, respectively. High genetic diversity (7 pfmsp-1 alleles and 10 pfmsp-2 alleles) and monoclonal infection rate (76%) was observed. This study demonstrated a continuous high prevalence of pfdhfr mutation and an increase in pfdhps mutations associated with SP-resistance in southwest Nigeria. Continuous surveillance of IPTp-SP effectiveness and consideration of alternative IPTp strategies is recommended.

Stable high frequencies of sulfadoxine-pyrimethamine resistance associated mutations and absence of K13 mutations in Plasmodium falciparum 3 and 4 years after the introduction of artesunate plus sulfadoxine-pyrimethamine in Ujjain, Madhya Pradesh, India.

BACKGROUND: Artesunate plus sulfadoxine-pyrimethamine (ASP) is first-line treatment for uncomplicated Plasmodium falciparum malaria in most of India, except for six North-eastern provinces where treatment failure rates were high. In Ujjain, central India, the frequency of mutations associated with increased drug tolerance, but not overt resistance to sulfadoxine and pyrimethamine were 9% and > 80%, respectively, in 2009 and 2010, just prior to the introduction of ASP. The frequency of drug resistance associated mutations in Ujjain in 2015-2016 after 3-4 years of ASP use, are reported. METHODS: Blood samples from patients with P. falciparum mono-infection verified by microscopy were collected on filter-paper at all nine major pathology laboratories in Ujjain city. Codons pfdhfr 16-185, pfdhps 436-632 and K13 407-689 were identified by sequencing. Pfcrt K76T and pfmdr1 N86Y were identified by restriction fragment length polymorphism. RESULTS: Sulfadoxine-pyrimethamine resistance-associated pfdhfr 108 N and 59R alleles were found in 100/104 (96%) and 87/91 (96%) samples, respectively. Pfdhps 437G was found in 10/105 (10%) samples. Double mutant pfdhfr 59R + 108 N were found in 75/81 (93%) samples. Triple mutant pfdhfr 59R + 108 N and pfdhps 437G were found in 6/78 (8%) samples. Chloroquine-resistance-associated pfcrt 76T was found in 102/102 (100%). Pfmdr1 N86 and 86Y were identified in 83/115 (72%) and 32/115 (28%) samples, respectively. CONCLUSION: The frequency of P. falciparum with reduced susceptibility to sulfadoxine-pyrimethamine remained high, but did not appear to have increased significantly since the introduction of ASP. No polymorphisms in K13 associated with decreased artemisinin susceptibility were found. ASP probably remained effective, supporting continued ASP use.

Occurrence of septuple and elevated Pfdhfr-Pfdhps quintuple mutations in a general population threatens the use of sulfadoxine-pyrimethamine for malaria prevention during pregnancy in eastern-coast of Tanzania.

BACKGROUND: Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) mutations compromise the effectiveness of sulfadoxine-pyrimethamine (SP) for treatment of uncomplicated malaria, and are likely to impair the efficiency of intermittent preventive treatment during pregnancy (IPTp). This study was conducted to determine the level of Pfdhfr-Pfdhps mutations, a decade since SP was limited for IPTp use in pregnant women in Tanzania. METHODS: P. falciparum genomic DNA was extracted from dried blood spots prepared from a finger prick. Extracted DNA were sequenced using a single MiSeq lane by combining all PCR products. Genotyping of Pfdhfr and Pfdhps mutations were done using bcftools whereas custom scripts were used to filter and translate genotypes into SP resistance haplotypes. RESULTS: The Pfdhfr was analyzed from 445 samples, the wild type (WT) Pfdhfr haplotype NCSI was detected in 6 (1.3%) samples. Triple PfdhfrIRNI (mutations are bolded and underlined) haplotype was dominant, contributing to 84% (number [n] = 374) of haplotypes while 446 samples were studied for Pfdhps, WT for Pfdhps (SAKAA) was found in 6.7% (n = 30) in samples. Double Pfdhps haplotype (SGEAA) accounted for 83% of all mutations at Pfdhps gene. Of 447 Pfdhfr-Pfdhps combined genotypes, only 0.9% (n = 4) samples contained WT gene (SAKAA-NCSI). Quintuple (five) mutations, SGEAA-IRNI accounted for 71.4% (n = 319) whereas 0.2% (n = 1) had septuple (seven) mutations (AGKGS-IRNI). The overall prevalence of Pfdhfr K540E was 90.4% (n = 396) while Pfdhps A581G was 1.1% (n = 5). CONCLUSIONS: This study found high prevalence of Pfdhfr-Pfdhps quintuple and presence of septuple mutations. Mutations at Pfdhfr K540E and Pfdhps A581G, major predictors for IPTp-SP failure were within the recommended WHO range. Abandonment of IPTp-SP is recommended in settings where the Pfdhfr K540E prevalence is > 95% and Pfdhps A581G is > 10% as SP is likely to be not effective. Nonetheless, saturation in Pfdhfr and Pfdhps haplotypes is alarming, a search for alternative antimalarial drug for IPTp in the study area is recommended.