Treatment Of Plasmodium Falciparum Malaria Research Articles

Genetic characteristics of P. falciparum parasites collected from 2012 to 2016 and anti-malaria resistance along the China-Myanmar border.

The therapeutic efficacy studies of DHA-PIP for uncomplicated Plasmodium falciparum patients were implemented from 2012 to 2016 along China (Yunnan province)-Myanmar border, which verified the high efficacy of DHA-PIP. With the samples collected in these studies, the genetic characteristics of P. falciparum parasites based on in vivo parasite clearance time (PCT) was investigated to explore if these parasites had developed resistance to DHA and PIP at molecular level. The genetic characteristics were investigated based on K13 genotypes, copy numbers of genes pfpm2 and pfmdr1, and nine microsatellite loci (Short Tandem Repeats, STR) flanking the K13 gene on chromosome 13. The PCT 50s were compared based on different K13 genotypes, sites, periods and copy numbers. In the NW (North-West Yunnan province bordering with Myanmar) region, F446I was the main K13 genotype. No significant differences for PCT 50s presented among three K13 genotypes. In SW (South-West Yunnan province bordering with Myanmar) region, only wild K13 genotype was detected in all parasite isolates whose PCT 50s was significantly longer than those in NW region. For the copy numbers of genes, parasite isolates containing multiple copies of pfmdr1 gene were found in both regions, but only single copy of pfpm2 gene was detected. Though the prevalence of parasite isolates with multiple copies of pfmdr1 gene in SW region was higher than that in NW region, no difference in PCT 50s were presented between isolates with single and multiple copies of pfmdr1 gene. The median He values of F446I group and Others (Non-F446I K13 mutation) group were 0.08 and 0.41 respectively. The mean He values of ML group (Menglian County in SW) and W (wild K13 genotype in NW) group were 0 and 0.69 respectively. The mean Fst values between ML and W groups were significantly higher than the other two K13 groups. P. falciparum isolates in NW and SW regions had very different genetic characteristics. The F446I was hypothesized to have independently appeared and spread in NW region from 2012 and 2016. The high susceptibility of PIP had ensured the efficacy of DHA-PIP in vivo. Multiple copy numbers of pfmdr1 gene might be a potential cause of prolonged clearance time of ACTs drugs along China-Myanmar border. Trial registration: ISRCTN, ISRCTN 11775446. Registered 17 April 2020-Retrospectively registered, the registered name was Investigating resistance to DHA-PIP for the treatment of Plasmodium falciparum malaria and chloroquine for the treatment of Plasmodium vivax malaria in Yunnan, China. http://www.isrctn.com/ISRCTN11775446.

Screening for K13-Propeller Mutations Associated with Artemisinin Resistance in Plasmodium falciparum in Yambio County (Western Equatoria State, South Sudan).

Artemisinin-combined treatments are the recommended first-line treatment of Plasmodium falciparum malaria, but they are being threatened by emerging artemisinin resistance. Mutations in pfk13 are the principal molecular marker for artemisinin resistance. This study characterizes the presence of mutations in pfk13 in P. falciparum in Western Equatoria State, South Sudan. We analyzed 468 samples from patients with symptomatic malaria and found 15 mutations (8 nonsynonymous and 7 synonymous). Each mutation appeared only once, and none were validated or candidate markers of artemisinin resistance. However, some mutations were in the same or following position of validated and candidate resistance markers, suggesting instability of the gene that could lead to resistance. The R561L nonsynonymous mutation was found in the same position as the R561H validated mutation. Moreover, the A578S mutation, which is widespread in Africa, was also reported in this study. We found a high diversity of other pfk13 mutations in low frequency. Therefore, routine molecular surveillance of resistance markers is highly recommended to promptly detect the emergence of resistance-related mutations and to limit their spread.

SERIOUS ANTIMALARIA RESISTANCE, GENETIC MARKERS OF KELCH 13, PLASMEPSINE 2 CNV ASSOCIATED WITH DIHYDROARTEMISININE - PIPERAQUINE RESISTANCE IN Plasmodium falciparum POPULATION IN MALARIA ENDEMIC ZONE OF DAKLAK PROVINCE, 2019 - 2020

Backgrounds: Dihydroartemisinin - piperaquine is a current frontline drug recommended in global by WHO for the treatment of Plasmodium falciparum malaria (WHO, 2015), but is now failing in Vietnam provinces where border Cambodia, and has emerged and spread. The purpose of this study was to evaluate efficacy and molecular markers of dihydroartemisinin - piperaquine failures in Daklak province. 
 Methods: A study design of non - randomized controlled study design for the 42 day - course follow - up in vivo test, and the molecular markers analysis. 
 Findings: The data showed that adequate clinical and parasitological response was sharply declined of 12,1%, late clinical failure of 51.5%, late parasitological failure of 36.4%, proportion of positive parasitemia at D3 is 37%, slope half - life of 5.36 hrs, and progressive parasite clearance PC50, PC75, PC 90, PC95, and PC99 were 13.24; 19.29; 25.69; 29.97 and 39.15 hrs, respectively. Molecular markers of C580Y Kelch mutation observed 100% (50/50) in the patients, increased of Plasmepsine 2 CNV of 72% (36/50), and both K13 and Plasmepsine 2 of 72% (36/50). 
 Conclusions: The DHA - PPQ efficacy severely decreased of 12.1%, overall treatment failure of 87.9% with the prominent C580Y mutant plus increased Plasmepsine 2 copy number variation in delayed asexual P. falciparum parasite clearance. These obvious data need to urgently change antimalarial policy in DHA - PPQ resistance zones.

Integrating Pharmacokinetic-Pharmacodynamic Modeling and Physiologically Based Pharmacokinetic Modeling to Optimize Human Dose Predictions for Plasmodium falciparum Malaria: a Chloroquine Case Study.

The translation of a preclinical antimalarial drug development candidate to the clinical phases should be supported by rational human dose selection. A model-informed strategy based on preclinical data, which incorporates pharmacokinetic-pharmacodynamic (PK-PD) properties with physiologically based pharmacokinetic (PBPK) modeling, is proposed to optimally predict an efficacious human dose and dosage regimen for the treatment of Plasmodium falciparum malaria. The viability of this approach was explored using chloroquine, which has an extensive clinical history for malaria treatment. First, the PK-PD parameters and the PK-PD driver of efficacy for chloroquine were determined through a dose fractionation study in the P. falciparum-infected humanized mouse model. A PBPK model for chloroquine was then developed for predicting the drug's PK profiles in a human population, from which the human PK parameters were determined. Lastly, the PK-PD parameters estimated in the P. falciparum-infected mouse model and the human PK parameters derived from the PBPK model were integrated to simulate the human dose-response relationships against P. falciparum, which subsequently allowed the determination of an optimized treatment. The predicted efficacious human dose and dosage regimen for chloroquine were comparable to those recommended clinically for the treatment of uncomplicated, drug-sensitive malaria, which provided supportive evidence for the proposed model-based approach to antimalarial human dose predictions.

Novel Plasmodium falciparum k13 gene polymorphisms from Kisii County, Kenya during an era of artemisinin-based combination therapy deployment

BackgroundCurrently, chemotherapy stands out as the major malaria intervention strategy, however, anti-malarial resistance may hamper global elimination programs. Artemisinin-based combination therapy (ACT) stands as the drug of choice for the treatment of Plasmodium falciparum malaria. Plasmodium falciparum kelch13 gene mutations are associated with artemisinin resistance. Thus, this study was aimed at evaluating the circulation of P. falciparum k13 gene polymorphisms from Kisii County, Kenya during an era of ACT deployment.MethodsParticipants suspected to have malaria were recruited. Plasmodium falciparum was confirmed using the microscopy method. Malaria-positive patients were treated with artemether-lumefantrine (AL). Blood from participants who tested positive for parasites after day 3 was kept on filter papers. DNA was extracted using chelex-suspension method. A nested polymerase chain reaction (PCR) was conducted and the second-round products were sequenced using the Sanger method. Sequenced products were analysed using DNAsp 5.10.01 software and then blasted on the NCBI for k13 propeller gene sequence identity using the Basic Local Alignment Search Tool (BLAST). To assess the selection pressure in P. falciparum parasite population, Tajima’ D statistic and Fu & Li’s D test in DnaSP software 5.10.01 was used.ResultsOut of 275 enrolled participants, 231 completed the follow-up schedule. 13 (5.6%) had parasites on day 28 hence characterized for recrudescence. Out of the 13 samples suspected of recrudescence, 5 (38%) samples were positively amplified as P. falciparum, with polymorphisms in the k13-propeller gene detected. Polymorphisms detected in this study includes R539T, N458T, R561H, N431S and A671V, respectively. The sequences have been deposited in NCBI with bio-project number PRJNA885380 and accession numbers SAMN31087434, SAMN31087433, SAMN31087432, SAMN31087431 and SAMN31087430 respectively.ConclusionsWHO validated polymorphisms in the k13-propeller gene previously reported to be associated with ACT resistance were not detected in the P. falciparum isolates from Kisii County, Kenya. However, some previously reported un-validated k13 resistant single nucleotide polymorphisms were reported in this study but with limited occurrences. The study has also reported new SNPs. More studies need to be carried out in the entire country to understand the association of reported mutations if any, with ACT resistance.

Molecular markers of dihydroartemisinin-piperaquine resistance in northwestern Thailand

BackgroundDihydroartemisinin-piperaquine (DHA-PPQ) combination therapy is the current first-line treatment for Plasmodium falciparum malaria in Thailand. Since its introduction in 2015, resistance to this drug combination has emerged in the eastern part of the Greater Mekong Subregion including the eastern part of Thailand near Cambodia. This study aimed to assess whether the resistance genotypes have arisen the western part of country.MethodsFifty-seven P. falciparum-infected blood samples were collected in Tak province of northwestern Thailand between 2013 and 2019. Resistance to DHA was examined through the single nucleotide polymorphisms (SNPs) of kelch13. PPQ resistance was examined through the copy number plasmepsin-2 and the SNPs of Pfcrt.ResultsAmong the samples whose kelch13 were successfully sequenced, approximately half (31/55; 56%) had mutation associated with artemisinin resistance, including G533S (23/55; 42%), C580Y (6/55; 11%), and G538V (2/55; 4%). During the study period, G533S mutation appeared and increased from 20% (4/20) in 2014 to 100% (9/9) in 2019. No plasmepsin-2 gene amplification was observed, but one sample (1/54) had the Pfcrt F145I mutation previously implicated in PPQ resistance.ConclusionsKelch13 mutation was common in Tak Province in 2013–2019. A new mutation G533S emerged in 2014 and rose to dominance in 2019. PPQ resistance marker Pfcrt F145I was also detected in 2019. Continued surveillance of treatment efficacy and drug resistance markers is warranted.

Genome-wide functional screening of drug-resistance genes in Plasmodium falciparum

The global spread of drug resistance is a major obstacle to the treatment of Plasmodium falciparum malaria. The identification of drug-resistance genes is an essential step toward solving the problem of drug resistance. Here, we report functional screening as a new approach with which to identify drug-resistance genes in P. falciparum. Specifically, a high-coverage genomic library of a drug-resistant strain is directly generated in a drug-sensitive strain, and the resistance gene is then identified from this library using drug screening. In a pilot experiment using the strain Dd2, the known chloroquine-resistant gene pfcrt is identified using the developed approach, which proves our experimental concept. Furthermore, we identify multidrug-resistant transporter 7 (pfmdr7) as a novel candidate for a mefloquine-resistance gene from a field-isolated parasite; we suggest that its upregulation possibly confers the mefloquine resistance. These results show the usefulness of functional screening as means by which to identify drug-resistance genes.

Genetic variability (785A > G, 516G > T and 64C > T) in the CYP2B6 gene among individuals in Southwest Nigeria: Implications for malaria treatment

Malaria remains a disease of major public health concern globally, with endemicity more pronounced in the sub-Saharan Africa while children below the age of five alongside pregnant women are the most affected groups. The artemisinin combination therapy (ACT) is the recommended first line treatment for Plasmodium falciparum malaria in Nigeria. However, Single nucleotide polymorphisms of the cytochrome P450 2B6 gene (known for the primary induction of artemisinin) can affect the enzyme function as it either enhances, decreases, or abolishes its enzymatic activities depending on the drug substrates. This study investigated the frequency of commonly observed CYP2B6 variants (516G > T {rs3745274}, 785A > G{rs2279343}, 64C > T {rs2279345}) and the association with malaria in a Yoruba population of Nigeria. Genotyping of CYP2B6 genetic polymorphisms was carried out among 200 children using PCR-RFLP protocol. Statistical package for social sciences (SPSS) was used in analyzing demographic and clinical data. Genotypic and Allelic frequencies were calculated, conformity with Hardy-Weinberg equilibrium and associations were tested for under three different genetic models using PLINK v1.90 and Haploview 4.2. We observed a minor allelic frequency (MAF) of 0.12, 0.375, 0.423 for 785A > G, 64C > T and 516G > T allelic variants respectively. The Three SNPs were not in linkage disequilibrium, the variants 785A > G and 516G > T were in conformity with Hardy-Weinberg equilibrium. The genotype distributions of 785A > G and 64C > T differed significantly across all malaria groups. Further analysis showed a statistically significant association of 785 A > G with malaria. The 785A > G variant under the dominant and additive genetic models reduced the risk of developing uncomplicated malaria (95% CI = 0.15–0.81, OR = 0.35, p = 0.0142) and severe malaria (95% CI = 0.20–0.97, OR = 0.44, p = 0.0439) from asymptomatic malaria. This finding indicates that the cytochrome p450 gene variant, 785A > G may confer protection against malaria development in addition to its drug metabolism functions. Further large-scale studies on CYP2B6 variants are recommended to explore the impact in malaria development or susceptibility.

Assessment of the Anti-Malarial Properties of Dihydroartemisinin- Piperaquine Phosphate Solid Lipid-Based Tablets.

Artemisininbased combination therapies (ACTs) typified by dihydroartemisinin- piperaquine phosphate are first-line drugs used in the treatment of Plasmodium falciparum malaria. However, the emergence of drug resistance to ACTs shows the necessity to develop novel sustained release treatments in order to ensure maximum bioavailability. To formulate dihydroartemisinin (DHA)-piperaquine phosphate (PQ) sustained release tablets based on solidified reverse micellar solutions (SRMS). The SRMS was prepared by fusion using varying ratios of Phospholipon® 90H and Softisan® 154 and characterised. The tablets were prepared by using an in-house made and validated mould. The formulations were tested for uniformity of weight, hardness, friability, softening time, erosion time and in vitro-in vivo dissolution rate. Antimalarial properties were studied using modified Peter's 4-days suppressive test in mice. One-way analysis of variance (ANOVA) was used in the analysis of results. Smooth caplets, with average weight of 1300 ± 0.06 mg to 1312 ± 0.11 mg, drug content of 61 mg for DHA and t 450 mg for PQ. Tablet hardness ranged from 7.1 to 9.0 Kgf and softening time of 29.50 ± 1.90 min. Erosion time of 62.00 ± 2.58 to 152.00 ± 1.89 min were obtained for tablets formulated with Poloxamer 188 (Batches R2, S2 and T2) which significantly reduced the softening and erosion time (p < 0.05). In vitro release showed that the optimized formulations had a maximum release at 12 h. Formulations exhibited significantly higher parasitaemia clearance and in vivo absorption compared to marketed formulations at day 7 (p < 0.05). DHA-PQ tablets based on SRMS were much easier and relatively cheaper to produce than compressed tablets. They also showed exceptionally better treatment of malaria owing to their sustained release properties and improved bioavailability and are recommended to Pharmaceutical companies for further studies.

The influence of biological, epidemiological, and treatment factors on the establishment and spread of drug-resistant Plasmodium falciparum.

The effectiveness of artemisinin-based combination therapies (ACTs) to treat Plasmodium falciparum malaria is threatened by resistance. The complex interplay between sources of selective pressure-treatment properties, biological factors, transmission intensity, and access to treatment-obscures understanding how, when, and why resistance establishes and spreads across different locations. We developed a disease modelling approach with emulator-based global sensitivity analysis to systematically quantify which of these factors drive establishment and spread of drug resistance. Drug resistance was more likely to evolve in low transmission settings due to the lower levels of (i) immunity and (ii) within-host competition between genotypes. Spread of parasites resistant to artemisinin partner drugs depended on the period of low drug concentration (known as the selection window). Spread of partial artemisinin resistance was slowed with prolonged parasite exposure to artemisinin derivatives and accelerated when the parasite was also resistant to the partner drug. Thus, to slow the spread of partial artemisinin resistance, molecular surveillance should be supported to detect resistance to partner drugs and to change ACTs accordingly. Furthermore, implementing more sustainable artemisinin-based therapies will require extending parasite exposure to artemisinin derivatives, and mitigating the selection windows of partner drugs, which could be achieved by including an additional long-acting drug.

Effectiveness of Artemether Lumefantrine and Dihydroartemisinin Piperaquine in Clearance of Gametocytes in Uncomplicated Plasmodium falciparum Malaria in Tiwi Kenya

Background: Over 80 countries worldwide have now implemented WHO recommendations to use Artemisinin-Based Combination Therapy as a first-line treatment for Plasmodium falciparum malaria. The sexual stage of P. falciparum is responsible for the transmission of malarial parasites to infectious mosquitoes. Studies on gametocytes are generally based on microscopic detection, which is not sensitive, and there is a need for more sensitive molecular techniques that can detect and quantify gametocytes at densities as low as 0.02 to 0.1 gametocytes per micro-litre. The objective of this study was to determine the clearance rates of gametocytes after AL and DHA&amp;P in uncomplicated P. falciparum and to compare the effectiveness of microscopy and reverse transcriptase-polymerase chain reaction in gametocyte detection. Methods: In a randomised controlled clinical trial of samples collected, gametocyte densities were quantified by microscopy by counting against 500 leukocytes in the thick smear converted to numbers of parasites per micro-litre by assuming a standard count of 800 leukocytes per micro-litre of blood after staining with 10% Giemsa stain and by reverse transcriptase-polymerase chain reaction using primers specific to the pfs25 gene. Results: There was no significant difference between the drug’s gametocyte clearance (p&lt;.082). The drugs cleared gametocytes in infected patients by day 28 as detected by microscopy. There was a significant difference in the detection of gametocytes by RT-PCR and microscopy (p&lt;.001). Conclusion: This study showed that Artemether-Lumefantrine and Dihydroartemisinin piperaquine have gametocytocidal effects on P. falciparum and the study on the clearance of gametocytes using both artemether-lumefantrine and Dihydroartemisinin piperaquine may be carried out using a larger sample size for policy implementation. The reverse transcriptase-polymerase chain reaction is more effective than microscopy in detecting low levels of gametocytes and the pfs25 gene can be used in the detection of gametocytes in the field to monitor the clearance of gametocytes.

Detection of artemisinin resistance-associated Kelch 13 mutations in Plasmodium falciparum from India

Artemisinin-based combination therapies (ACT) are currently the first line drug for the treatment of Plasmodium falciparum malaria in all malaria-endemic countries including India. Recent reports on artemisinin treatment failure against P. falciparum from different parts of the world including Southeast Asia and Africa have raised a great concern. The sporadic reports of treatment failures from northeastern part of India has substantiated a need to look into the molecular markers for artemisinin resistance, especially the Kelch 13 (K13) protein. In the present study, we analyzed the PfK13 gene mutations from Bikaner, Rajasthan, which is reported as one of the endemic regions in North-West India. Further, to analyze artemisinin pressure on the Plasmodium vivax population, we studied the mutations in the orthologous gene, PvK13, from two distinct regions: Bikaner, known for occurrence of both P. falciparum and P. vivax infections; and Madurai, Tamil Nadu, predominantly exhibiting P. vivax infections. For the Pf K13 gene, we identified seven mutations, of which two were validated nonsynonymous mutations (F446I, C580Y), one was candidate mutation (P574L) and two were reported synonymous mutations (R471R and Y500Y). Along with the reported mutations, we also observed a novel nonsynonymous mutation V487L in PfK13 gene. For P. vivax, all the samples from Madurai exhibited wild type phenotype; however, an isolate from Bikaner exhibited a synonymous mutation (A418A). To the best of our knowledge, this is the first report of validated PfK13 C580Y mutation from India and indicates development of artemisinin resistance in the parasite population.

Efficacy of Artemether-Lumefantrine for the Treatment of Plasmodium falciparum Malaria in Bohicon and Kandi, Republic of Benin, 2018-2019.

In 2005, artemether-lumefantrine (AL), an artemisinin-based combination therapy, was introduced as the first-line treatment of uncomplicated Plasmodium falciparum malaria in Benin. Per World Health Organization recommendations to monitor the efficacy of antimalarial treatment, we conducted a therapeutic efficacy study with AL for uncomplicated P. falciparum malaria in Bohicon and Kandi, Benin, from 2018 to 2019. Febrile patients aged 6 to 59 months with confirmed P. falciparum monoinfection received supervised doses of AL for 3 days. We monitored patients clinically and parasitologically on days 1, 2, 3, 7, 14, 21, and 28. A molecular analysis to detect mutations in the P. falciparum Kelch propeller gene (Pfk13) gene was carried out on day 0 samples. A total of 205 patients were included in the study. In Bohicon, the uncorrected adequate clinical and parasitological response (ACPR) proportion was 91.3% (95% confidence interval [CI]: 84.6-95.8%), whereas in Kandi this proportion was 96.7% (95% CI: 90.6-99.3%). Genotype-corrected ACPR proportions were 96.3% (95% CI: 90.9-99.0%) and 96.7% (95% CI: 90.6-99.3%) in Bohicon and Kandi, respectively. On day 3, 100% of patients in Bohicon and 98.9% of patients in Kandi had undetectable parasitemia. The C580Y mutation in the Pfk13 gene was not observed. AL remains effective for P. falciparum malaria in these two sites in Benin. Monitoring antimalarial efficacy and prevalence of molecular-resistance markers in Benin should be continued to allow for early detection of antimalarial resistance and to guide treatment policies.

Decreased prevalence of the Plasmodium falciparum Pfcrt K76T and Pfmdr1 and N86Y mutations post-chloroquine treatment withdrawal in Katete District, Eastern Zambia

BackgroundIn 2002, Zambia withdrew chloroquine as first-line treatment for Plasmodium falciparum malaria due to increased treatment failure and worldwide spread of chloroquine resistance. The artemisinin combination regimen, artemether–lumefantrine, replaced chloroquine (CQ) as first choice malaria treatment. The present study determined the prevalence of CQ resistance molecular markers in the Pfcrt and Pfmdr1 genes in Eastern Zambia at 9 and 13 years after the removal of drug pressure.MethodsSamples collected from Katete District during the drug therapeutic efficacy assessments conducted in 2012 and 2016 were assayed by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) to determine the prevalence of genetic mutations, K76T on the Pfcrt gene and N86Y on the Pfmdr1 gene. A total of 204 P. falciparum-positive DBS samples collected at these two time points were further analysed.ResultsAmong the samples analysed for Pfcrt K76T and Pfmdr1 N86Y in the present study, 112 (82.4%) P. falciparum-infected samples collected in 2012 were successfully amplified for Pfcrt and 94 (69.1%) for Pfmdr1, while 69 (65.7%) and 72 (68.6%) samples from 2016 were successfully amplified for Pfcrt and Pfmdr1, respectively. In 2012, the prevalence of Pfcrt 76K (sensitive) was 97.3%, 76T (resistant) was 1.8%, and 0.8% had both 76K and 76T codons (mixed). Similarly in 2012, the prevalence of Pfmdr1 86N (sensitive) was 97.9% and 86Y (resistant) was 2.1%. In the 2016 samples, the prevalence of the respective samples was 100% Pfcrt 76K and Pfmdr1 86N.ConclusionThis study shows that there was a complete recovery of chloroquine-sensitive parasites by 2016 in Katete District, Eastern Zambia, 13 years following the withdrawal of CQ in the country. These findings add to the body of evidence for a fitness cost in CQ-resistant P. falciparum in Zambia and elsewhere. Further studies are recommended to monitor resistance countrywide and explore the feasibility of integration of the former best anti-malarial in combination therapy in the future.

Natural products for treatment of Plasmodium falciparum malaria: An integrated computational approach

Malaria is a life-threatening infectious disease with an estimated 229 million cases in the year 2019 worldwide. Plasmodium falciparum 1-deoxy-d-xylulose-5-phosphate reductoisomerase (PfDXR) is one of the key enzymes in the biosynthetic pathway of isoprenoid, (required for parasite growth and survival) and considered as an attractive target for designing anti-malarial drugs. Fosmidomycin is an effective DXR inhibitor and has been proven effective and safe against P. falciparum in clinical trials. However, due to low bioavailability and inappropriate drug attributes, it is not a preferred option. The present study was performed to identify PfDXR inhibitors with improved pharmacology/safety. For this purpose, an integrated computational framework, comprising of pharmacophore modeling, virtual screening, molecular docking, molecular dynamics (MD) simulation and MM/PBSA, was used. The binding free energy analysis was performed using a focused library of phytochemicals established from medicinal plants. The study identified four bioactive compounds namely, Myricetin 3-rhamnoside, 7-O-Galloyltricetiflavan, (25S)-5-beta-spirostan-3-beta-ol 3-O-beta-d-glucopyranosyl-(1->2)-beta-d-glucopyranoside, and Oleanolic acid 28-O-beta-d-glucopyranoside as potential inhibitors of PfDXR. The selection of these four compounds was based on pharmacophore mapping, docking score, binding stability, molecular interactions with the residues of PfDXR active site, binding stability and free energy estimation. In conclusion, medicinal plant-based scaffolds were predicted with enhanced efficacy and adequate physiochemical/pharmacokinetic profile that might be helpful in controlling malaria.

Effectiveness and Tolerability of the ASAQ &amp;lt;i&amp;gt;versus&amp;lt;/i&amp;gt; AL Association in Children 6-59 Months for the Treatment of Uncomplicated &amp;lt;I&amp;gt;P.&amp;lt;/I&amp;gt; &amp;lt;i&amp;gt;falciparum&amp;lt;/i&amp;gt; Malaria in Massakory (Chad)

Background and Objective: Artemisinin-based combination therapies are the first-line antimalarial drugs used to treat uncomplicated Plasmodium falciparum malaria in many endemic countries worldwide. This study was conducted to assess the efficacy and tolerability of two fixed-dose formulations of artesunate-amodiaquine and artemether-lumefantrine for the treatment of Plasmodium falciparum malaria in Chad. Methodology and Results: A two-arm single cohort study was conducted assessing the efficacy artesunate-amodiaquine and artemether-lumefantrine for the treatment of children with uncomplicated falciparum malaria. This study was carried out from December 14, 2019 to March 14, 2020 at the Massakory I Health Center in Chad. Primary efficacy endpoint was day 28, parasitological cure rate. Secondary endpoints were parasite and fever clearance times and tolerability. A total of 113 patients were included, including 56 in the artesunate-amodiaquine arm and 57 in the artemether-lumefantrine arm. In intention to treat these patients, the Adequate Clinical and Parasitological Response on day 28 were 100% for the two groups. No early treatment failure was observed. The drugs were well tolerated and no serious adverse events were noted. Conclusion: Both forms of Artemisinin-based combination therapy were still effective and safe in the treatment of uncomplicated P. falciparum malaria in Chad. Further studies are warranted in different regions of Chad for monitoring of drug resistance.

Serious antimalaria resistance, genetic markers of Kelch 13, plasmepsine 2 CNV associated with dihydroartemisinine-piperaquine phosphate resistance in Plasmodium falciparum population in malaria hyperendemic zone of Dak Lak Province (2019-2020)

Dihydroartemisinin-piperaquine (DHA-PPQ) is a current frontline drug recommended in global by WHO for the treatment of Plasmodium falciparum malaria (WHO, 2015), but is now failing in Vietnam’s provinces where border Cambodia, and has emerged and spread. The purpose of this study was to evaluate the efficacy and molecular markers of DHA-PPQ failures in Dak Lak province. Methods: A study design of non-randomized controlled study design for the 42 day-course follow-up in vivo test, and the molecular markers analysis. Findings: The data showed that adequate clinical and parasitological response was sharply declined to 12,1%, the proportion of late clinical failure was 51.5%, and 36.4% of patients had late parasitological. The proportion of positive parasitemia at D3 was 37%, the slope half-life was 5.36 hrs, and the progressive parasite clearance (PC) PC50, PC75, PC 90, PC95, and PC99 were 13.24; 19.29; 25.69; 29.97 and 39.15 hours, respectively. Molecular markers of C580Y Kelch mutation were observed in 100% (50/50) of the patients, the increased of Plasmepsine 2 copy number variation (CNV) was 72% (36/50), and the proportion of patients who had both K13 and increased Plasmepsine 2 CNV was 72% (36/50). Conclusions: The DHA-PPQ efficacy severely decreased to 12.1%, overall treatment failure was 87.9% with the prominent C580Y mutant plus increased Plasmepsine 2 CNV in delayed asexual P. falciparum parasite clearance. These obvious data suggest the urgency to change antimalarial policy in DHA-PPQ resistance zones, especially in Dak Lak province.

Elucidation of DNA Repair Function of PfBlm and Potentiation of Artemisinin Action by a Small-Molecule Inhibitor of RecQ Helicase.

Artemisinin (ART)-based combination therapies are recommended as first- and second-line treatments for Plasmodium falciparum malaria. Here, we investigated the impact of the RecQ inhibitor ML216 on the repair of ART-mediated damage in the genome of P. falciparumPfBLM and PfWRN were identified as members of the RecQ helicase family in P. falciparum However, the role of these RecQ helicases in DNA double-strand break (DSB) repair in this parasite has not been explored. Here, we provide several lines of evidence to establish the involvement of PfBlm in DSB repair in P. falciparum First, we demonstrate that PfBlm interacts with two well-characterized DSB repair proteins of this parasite, namely, PfRad51 and PfalMre11. Second, we found that PfBLM expression was upregulated in response to DNA-damaging agents. Third, through yeast complementation studies, we demonstrated that PfBLM could complement the DNA damage sensitivity of a Δsgs1 mutant of Saccharomyces cerevisiae, in contrast to the helicase-dead mutant PfblmK83R Finally, we observe that the overexpression of PfBLM induces resistance to DNA-damaging agents and offers a survival advantage to the parasites. Most importantly, we found that the RecQ inhibitor ML216 inhibits the repair of DSBs and thereby renders parasites more sensitive to ART. Such synergism between ART and ML216 actions was observed for both drug-sensitive and multidrug-resistant strains of P. falciparum Taken together, these findings establish the implications of PfBlm in the Plasmodium DSB repair pathway and provide insights into the antiparasitic activity of the ART-ML216 combination.IMPORTANCE Malaria continues to be a serious threat to humankind not only because of the morbidity and mortality associated with the disease but also due to the huge economic burden that it imparts. Resistance to all available drugs and the unavailability of an effective vaccine cry for an urgent discovery of newer drug targets. Here, we uncovered a role of the PfBlm helicase in Plasmodium DNA double-strand break repair and established that the parasitic DNA repair mechanism can be targeted to curb malaria. The small-molecule inhibitor of PfBlm tested in this study acts synergistically with two first-line malaria drugs, artemisinin (ART) and chloroquine, in both drug-sensitive and multidrug-resistant strains of P. falciparum, thus qualifying this chemical as a potential partner in ART-based combination therapy. Additionally, the identification of this new specific inhibitor of the Plasmodium homologous recombination (HR) mechanism will now allow us to investigate the role of HR in Plasmodium biology.

Artemether\u2013lumefantrin treatment adherence among uncomplicated plasmodium falciparum malaria patients, visiting public health facilities in AsgedeTsimbla district, Tigray, Ethiopia: a cross-sectional study

BackgroundEthiopia has set a goal to eliminate malaria by 2030; Artemether–lumefantrine (AL) is put as one of the cornerstone strategies for uncomplicated plasmodium falciparum malaria treatment. However, only focusing on prescribing of the treatment without assessing patients’ adherence could lead to the resistance of the drug. In Ethiopia, there is limited evidence about patients’ adherence to AL and its influencing factors. Therefore, this study aimed at addressing this information gap.MethodsA health facility based cross-sectional study was employed. Participants were selected using simple random sampling technique from registration books of the public health facilities in AsgedeTsimbla. Data were collected from March 24th to April 30th, 2018. We interviewed participants using a pre-tested structured questionnaire, and the blister pack was also inspected at their homes on day 4. Data were entered into Epi-Info and analyzed using SPSS 21. Odds ratios with 95% Confidence Intervals were estimated and the level of significance was declared at p-value ≤ 0.05.ResultsA total of 384 study participants were interviewed with a response rate of 95.5%. The overall AL adherence was 53.6% (95% CI 48.4–58.3%). Children aged < 5 years [AOR: 0.4, 95% CI (0.2–0.8)], and being treated in health post [AOR: 0.3, 95% CI (0.1–0.5)] were more likely to show AL adherence whereas illiteracy [AOR: 9.4, 95% CI (4.2–21.3)], didn’t know the consequence of discontinued AL [AOR: 4.0, 95% CI (2.1–7.6)], had concomitant drugs [AOR: 2.5, 95% CI (1.4–4.5)], and stopped/saved drug when improved before tablet got finished [AOR: 3.2, 95% CI (1.7–5.9)] were factors less likely to be associated with AL adherence.ConclusionAL adherence was low. Children aged < 5 years, and being treated in health post were determinants of AL adherence whereas illiteracy, didn’t know the consequence of discontinued the drug, had concomitant drugs, and stopped/saved drug when improved before tablet got finished were factors that hindered the AL adherence. Stakeholders should emphasize designing appropriate strategies including educational interventions to increase the AL adherence and prevent drug resistance. Further research should be conducted to evaluate AL resistance.

Genetic screens reveal a central role for heme metabolism in artemisinin susceptibility

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; however, resistance threatens to undermine global control efforts. To broadly explore artemisinin susceptibility in apicomplexan parasites, we employ genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncover the putative transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provide evidence that mitochondrial metabolism can modulate resistance. We show that disrupting a top candidate from the screens, the mitochondrial protease DegP2, lowers porphyrin levels and decreases DHA susceptibility, without significantly altering parasite fitness in culture. Deleting the homologous gene in P. falciparum, PfDegP, similarly lowers heme levels and DHA susceptibility. These results expose the vulnerability of heme metabolism to genetic perturbations that can lead to increased survival in the presence of DHA.