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The emergence of artemisinin-resistant parasites since the late 2000s at the border of Cambodia and Thailand poses serious threats to malaria control globally, particularly in Africa which bears the highest malaria transmission burden. This study aimed to obtain reliable data on the current state of the kelch13 molecular marker for artemisinin resistance in Plasmodium falciparum in Cameroon. DNA was extracted from the dried blood spots collected from epidemiologically distinct endemic areas in the Center, Littoral and North regions of Cameroon. Nested PCR products from the Kelch13-propeller gene were sequenced and analyzed on an ABI 3730XL automatic sequencer. Of 219 dried blood spots, 175 were sequenced successfully. We identified six K13 mutations in 2.9% (5/175) of samples, including 2 non-synonymous, the V589I allele had been reported in Africa already and one new allele E612K had not been reported yet. These two non-synonymous mutations were uniquely found in parasites from the Littoral region. One sample showed two synonymous mutations within the kelch13 gene. We also observed two infected samples with mixed K13 mutant and K13 wild-type infection. Taken together, our data suggested the circulation of the non-synonymous K13 mutations in Cameroon. Albeit no mutations known to be associated with parasite clearance delays in the study population, there is need for continuous surveillance for earlier detection of resistance as long as ACTs are used and scaled up in the community. [ABSTRACT FROM AUTHOR]

Introduction: Plasmodium falciparum synthesizes phosphatidylcholine for the membrane development through serine decarboxylase–phosphoethanolamine methyltransferase pathway for growth in human host. Phosphoethanolamine-methyltransferase (PfPMT) is a crucial enzyme for the synthesis of phosphocholine which is a precursor for phosphatidylcholine synthesis and is considered as a pivotal drug target as it is absent in the host. The inhibition of PfPMT may kill malaria parasite and hence is being considered as potential target for rational antimalarial drug designing. Methods: In this study, we have used computer aided drug designing (CADD) approaches to establish potential PfPMT inhibitors from Asinex compound library virtually screened for ADMET and the docking affinity. The selected compounds were tested for in-vitro schizonticidal, gametocidal and cytotoxicity activity. Nontoxic compounds were further studied for PfPMT enzyme specificity and antimalarial efficacy for P. berghei in albino mice model. Results: Our results have identified two nontoxic PfPMT competitive inhibitors ASN.1 and ASN.3 with better schizonticidal and gametocidal activity which were found to inhibit PfPMT at IC50 1.49μM and 2.31μM respectively. The promising reduction in parasitaemia was found both in orally (50 & 10 mg/kg) and intravenous (IV) (5& 1 mg/kg) however, the better growth inhibition was found in intravenous groups. Conclusion: We report that the compounds containing Pyridinyl-Pyrimidine and Phenyl-Furan scaffolds as the potential inhibitors of PfPMT and thus may act as promising antimalarial inhibitor candidates which can be further optimized and used as leads for template based antimalarial drug development. [ABSTRACT FROM AUTHOR]

Drug-resistant strains of malaria parasites have emerged for most of antimalarial medications. A new chemotherapeutic compound is needed for malarial therapy. Antimalarial activity against both drug-sensitive and drug-resistant P. falciparum has been reported for an isocryptolepine derivative, 8-bromo-2-fluoro-5-methyl-5H-indolo[3,2-c]quinoline (ICL-M), which also showed less toxicity to human cells. ICL-M has indoloquinoline as a core structure and its mode of action remains unclear. Here, we explored the mechanisms of ICL-M in P. falciparum by assessing the stage-specific activity, time-dependent effect, a proteomic analysis and morphology. Since human topo II activity inhibition has been reported as a function of isocryptolepine derivatives, malarial topo II activity inhibition of ICL-M was also examined in this study. The ICL-M exhibited antimalarial activity against both the ring and trophozoite stages of P. falciparum. Our proteomics analysis revealed that a total of 112 P. falciparum proteins were differentially expressed after ICL-M exposure; among these, 58 and 54 proteins were upregulated and downregulated, respectively. Proteins localized in the food vacuole, nucleus, and cytoplasm showed quantitative alterations after ICL-M treatment. A bioinformatic analysis revealed that pathways associated with ribosomes, proteasomes, metabolic pathways, amino acid biosynthesis, oxidative phosphorylation, and carbon metabolism were significantly different in P. falciparum treated with ICL-M. Moreover, a loss of ribosomes was clearly observed by transmission electron microscopy in the ICL-M-treated P. falciparum. This finding is in agreement with the proteomics data, which revealed downregulated levels of ribosomal proteins following ICL-M treatment. Our results provide important information about the mechanisms by which ICL-M affects the malaria parasite, which may facilitate the drug development of isocryptolepine derivatives. [ABSTRACT FROM AUTHOR]

Introduction: Asymptomatic carriage of P. falciparum is frequent in areas endemic for malaria and individual diagnosis of clinical malaria attacks is still difficult. We investigated the impact of changes in malaria endemicity on the diagnostic criteria for malaria attacks in an area of seasonal malaria transmission. Methods: We analyzed the longitudinal data collected over 20 years from a daily survey of all inhabitants of Ndiop, a rural community in central Senegal, in a logistic regression model to investigate the relationship between the level of Plasmodium falciparum parasitemia and the risk of fever, with the aim of determining the best parasitemia thresholds for attributing to malaria a fever episode. Results: A total of 34,136 observations recorded from July 1993 to December 2013 from 850 individuals aged from 1 day to 87 years were included. P. falciparum asymptomatic carriage declined from 36% to 1% between 1993 and 2013. A total of 9,819 fever episodes were associated with a positive blood film for P. falciparum. Using age-dependent parasitemia thresholds for attributing to malaria a fever episode, we recorded 6,006 malaria attacks during the study period. Parasitemia thresholds seemed to be lower during the low-to-zero transmission season and tended to decrease with changes in control policies. The number of clinical malaria attacks was overestimated for all age groups throughout the study when all fever episodes associated with P. falciparum parasitemia were defined as malaria attacks. Conclusion: Pyrogenic thresholds are particularly sensitive to changes in malaria epidemiology and are therefore an interesting tool to accurately assess the burden of malaria in the context of declining transmission. [ABSTRACT FROM AUTHOR]

The battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum, the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase (NDH2) of P. falciparum, PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasite’s susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc1 complex. Our results suggest that PfNDH2 is not likely a good antimalarial drug target. [ABSTRACT FROM AUTHOR]

Background: In Vietnam, a rapid decline of P. falciparum malaria cases has been documented in the past years, the number of Plasmodium falciparum malaria cases has rapidly decreased passing from 19.638 in 2012 to 4.073 cases in 2016. Concomitantly, the spread of artemisinin resistance markers is raising concern on the future efficacy of the ACTs. An evaluation of the clinical impact of the artemisinin resistance markers is therefore of interest. Methods: The clinical effectiveness of dihydroartemisinin-piperaquine therapy (DHA-PPQ) has been evaluated in three districts characterized by different rates of ART resistance markers: K13(C580Y) mutation and delayed parasite clearance on day 3 (DPC3). Patients were stratified in 3 groups a) no markers, b) one marker (suspected resistance), c) co-presence of both markers (confirmed resistance). In the studied areas, the clinical effectiveness of DHA-PPQ has been estimated as malaria recrudescence within 60 days. Results: The rate of K13(C580Y) ranged from 75.8% in Krong Pa to 1.2% in Huong Hoa district. DPC3 prevalence was higher in Krong Pa than in Huong Hoa (86.2% vs 39.3%). In the two districts, the prevalence of confirmed resistance was found in 69.0% and 1.2% of patients, respectively. In Thuan Bac district, we found intermediate prevalence of confirmed resistance. Treatment failure was not evidenced in any district. PPQ resistance was not evidenced. Confirmed resistance was associated to the persistence of parasites on day 28 and to 3.4-fold higher parasite density at diagnosis. The effectiveness of malaria control strategies was very high in the studied districts. Conclusion: No treatment failure has been observed in presence of high prevalence of ART resistance and in absence of PPQ resistance. K13(C580Y) was strongly associated to higher parasitemia at admission, on days 3 and 28. Slower parasite clearance was also observed in younger patients. [ABSTRACT FROM AUTHOR]

Background: Malaria reduction and future elimination in China is made more difficult by the importation of cases from neighboring endemic countries, particularly Myanmar, Laos, and Vietnam, and increased travel to Africa by Chinese nationals. The increasing prevalence of artemisinin resistant parasites across Southeast Asia highlights the importance of monitoring the parasite importation into China. Artemisinin resistance in the Mekong region is associated with variants of genes encoding the K13 kelch domain protein (pf13k), found in specific genetic backgrounds, including certain alleles of genes encoding the chloroquine resistance transporter (pfcrt) and multidrug resistance transporter PgH1 (pfmdr1). Methods: In this study we investigated the prevalence of drug resistance markers in 72 P. falciparum samples from uncomplicated malaria infections in Tengchong and Yingjiang, counties on the Yunnan-Myanmar border. Variants of pf13k, pfcrt and pfmdr1 are described. Results: Almost all parasites harboured chloroquine-resistant alleles of pfcrt, whereas pfmdr1 was more diverse. Major mutations in the K13 propeller domain associated with artemisinin resistance in the Mekong region (C580Y, R539T and Y493H) were absent, but F446I and two previously undescribed mutations (V603E and V454I) were identified. Protein structural modelling was carried out in silico on each of these K13 variants, based on recently published crystal structures for the K13 propeller domain. Whereas F446I was predicted to elicit a moderate destabilisation of the propeller structure, the V603E substitution is likely to lead to relatively high protein instability. We plotted these stability estimates, and those for all previously described variants, against published values for in vivo parasitaemia half-life, and found that quadratic regression generates a useful predictive algorithm. Conclusion: This study provides a baseline of P. falciparum resistance-associated mutations prevalent at the China-Myanmar border. We also show that protein modelling can be used to generate testable predictions as to the impact of pfk13 mutations on in vivo (and potentially in vitro) artemisinin susceptibility. [ABSTRACT FROM AUTHOR]

Background: Malaria rapid diagnostic tests (RDT) have limitations due to the persistence of histidine-rich protein 2 (HRP2) antigen after treatment and low sensitivity of Plasmodium lactate dehydrogenase (pLDH) based RDTs. To improve the diagnosis of malaria in febrile children, two diagnostic algorithms, based on sequential interpretation of a malaria rapid diagnostic test detecting two different targets of Plasmodium species and followed by expert microscopy, were evaluated. Methods: Two diagnostic algorithms were evaluated using 407 blood samples collected between April and October 2016 from febrile children and the diagnostic accuracy of both algorithms was determined. Algorithm 1: The result of line T1-HRP2 were read first; if negative, malaria infection was considered to be absent. If positive, confirmation was done with the line T2-pLDH. If T2-pLDH test was negative, the malaria diagnosis was considered as “inconclusive” and microscopy was performed; Algorithm 2: The result of line T2-pLDH were read first; if positive, malaria infection was considered to be present. If negative, confirmation was done with the line T1-HRP2. If T1-HRP2 was positive the malaria diagnosis was considered as “inconclusive” and microscopy was performed. In absence of malaria microscopy, a malaria infection was ruled out in children with an inconclusive diagnostic test result when previous antimalarial treatment was reported. Results: For single interpretation, the sensitivity of PfHRP2 was 98.4% and the specificity was 74.2%, and for the pLDH test the sensitivity was 89.3% and the specificity was 98.8%. Malaria was accurately diagnosed using both algorithms in 84.5% children. The algorithms with the two-line malaria RDT classified the test results into two groups: conclusive and inconclusive results. The diagnostic accuracy for conclusive results was 98.3% using diagnostic algorithm 1 and 98.6% using algorithm 2. The sensitivity and specificity for the conclusive results were 98.2% and 98.4% for algorithm 1, and 98.6% and 98.4% for algorithm 2, respectively. There were 63 (15.5%) children who had an “inconclusive” result for whom expert microscopy was needed. In children with inconclusive results (PfHRP2+/pLDH- only) previous antimalarial treatment was reported in 16 children with malaria negative microscopy (16/40; 40%) and 1 child with malaria positive microscopy (1/23; 4.3%). Conclusion: The strategy of sequential interpretation of two-line malaria RDT can improve the diagnosis of malaria. However, some cases will still require confirmative testing with microscopy or additional investigations on previous antimalarial treatment. [ABSTRACT FROM AUTHOR]

Artemisinin resistance is a major threat to malaria control efforts. Resistance is characterized by an increase in the Plasmodium falciparum parasite clearance half-life following treatment with artemisinin-based combination therapies (ACTs) and an increase in the percentage of surviving parasites. The remarkably short blood half-life of artemisinin derivatives may contribute to drug-resistance, possibly through factors including sub-lethal plasma concentrations and inadequate exposure. Here we selected for a new strain of artemisinin resistant parasites, termed the artemisinin resistant strain 1 (ARS1), by treating P. falciparum Palo Alto (PA) cultures with sub-lethal concentrations of dihydroartemisinin (DHA). The resistance phenotype was maintained for over 1 year through monthly maintenance treatments with low doses of 2.5 nM DHA. There was a moderate increase in the DHA IC50 in ARS1 when compared with parental strain PA after 72 h of drug exposure (from 0.68 nM to 2 nM DHA). In addition, ARS1 survived treatment physiologically relevant DHA concentrations (700 nM) observed in patients. Furthermore, we confirmed a lack of cross-resistance against a panel of antimalarials commonly used as partner drugs in ACTs. Finally, ARS1 did not contain Pfk13 propeller domain mutations associated with ART resistance in the Greater Mekong Region. With a stable growth rate, ARS1 represents a valuable tool for the development of new antimalarial compounds and studies to further elucidate the mechanisms of ART resistance. [ABSTRACT FROM AUTHOR]

Malaria causes about half a million deaths annually, with Plasmodium falciparum being responsible for 90% of all the cases. Recent reports on artemisinin resistance in Southeast Asia warrant urgent discovery of novel drugs for the treatment of malaria. However, most bioactive compounds fail to progress to treatments due to safety concerns. Drug repositioning offers an alternative strategy where drugs that have already been approved as safe for other diseases could be used to treat malaria. This study screened approved drugs for antimalarial activity using an in silico chemogenomics approach prior to in vitro verification. All the P. falciparum proteins sequences available in NCBI RefSeq were mined and used to perform a similarity search against DrugBank, TTD and STITCH databases to identify similar putative drug targets. Druggability indices of the potential P. falciparum drug targets were obtained from TDR targets database. Functional amino acid residues of the drug targets were determined using ConSurf server which was used to fine tune the similarity search. This study predicted 133 approved drugs that could target 34 P. falciparum proteins. A literature search done at PubMed and Google Scholar showed 105 out of the 133 drugs to have been previously tested against malaria, with most showing activity. For further validation, drug susceptibility assays using SYBR Green I method were done on a representative group of 10 predicted drugs, eight of which did show activity against P. falciparum 3D7 clone. Seven had IC50 values ranging from 1 μM to 50 μM. This study also suggests drug-target association and hence possible mechanisms of action of drugs that did show antiplasmodial activity. The study results validate the use of proteome-wide target similarity approach in identifying approved drugs with activity against P. falciparum and could be adapted for other pathogens. [ABSTRACT FROM AUTHOR]

Background: While recognizing the recent remarkable achievement in the global malaria reduction, the disease remains a challenge to the malaria endemic countries in Africa. Beyond the huge health consequence of malaria, policymakers need to be informed about the economic burden of the disease to the households. However, evidence on the economic burden of malaria in Ethiopia is scanty. The aims of this study were to estimate the economic burden of malaria episode and to identify predictors of cost variability to the rural households. Methods: A prospective costing approach from a household perspective was employed. A total of 190 malaria patients were enrolled to the study from three health centers and nine health posts in Adami Tullu district in south-central Ethiopia, in 2015. Primary data were collected on expenditures due to malaria, forgone working days because of illness, socioeconomic and demographic situation, and households’ assets. Quantile regression was applied to predict factors associated with the cost variation. Socioeconomic related inequality was measured using concentration index and concentration curve. Results: The median cost of malaria per episode to the household was USD 5.06 (IQR: 2.98–8.10). The direct cost accounted for 39%, while the indirect counterpart accounted for 61%. The history of malaria in the last six months and the level of the facility visited in the health system predominantly influenced the direct cost. The indirect cost was mainly influenced by the availability of antimalarial drugs in the health facility. The concentration curve and the concentration index for direct cost indicate significant pro-rich inequality. Plasmodium falciparum is significantly more costly for households compared to Plasmodium vivax. Conclusion: The economic burden of malaria to the rural households in Ethiopia was substantial—mainly to the poor—indicating that reducing malaria burden could contribute to the poverty reduction as well. [ABSTRACT FROM AUTHOR]

Malaria continues to be a devastating disease, largely caused by Plasmodium falciparum infection. We investigated the effects of opioid and cannabinoid receptor antagonists on the growth of intraerythrocytic P. falciparum. The delta opioid receptor antagonist 7-benzylidenenaltrexone (BNTX) and the cannabinoid receptor antagonists rimonaband and SR144528 caused growth arrest of the parasite. Notably BNTX and the established antimalarial drug dihydroartemisinin induced prominent pyknosis in parasite cells after a short period of incubation. We compared genome-wide transcriptome profiles in P. falciparum with different degrees of pyknosis in response to drug treatment, and identified 11 transcripts potentially associated with the evoking of pyknosis, of which three, including glutathione reductase (PfGR), triose phosphate transporter (PfoTPT), and a conserved Plasmodium membrane protein, showed markedly different gene expression levels in accordance with the degree of pyknosis. Furthermore, the use of specific inhibitors confirmed PfGR but not PfoTPT as a possible factor contributing to the development of pyknosis. A reduction in total glutathione levels was also detected in association with increased pyknosis. These results further our understanding of the mechanisms responsible for P. falciparum development and the antimalarial activity of dihydroartemisinin, and provide useful information for the development of novel antimalarial agents. [ABSTRACT FROM AUTHOR]

Malaria is a major public health problem in the Democratic Republic of Congo. Despite progress achieved over the past decade in the fight against malaria, further efforts have to be done such as in the surveillance and the containment of Plasmodium falciparum resistant strains. We investigated resistance to artemisinin-based combination therapies currently in use in Democratic Republic of Congo by surveying molecular polymorphisms in three genes: pfcrt, pfmdr1 and pfk13 to explore possible emergence of amodiaquine, lumefantrine or artemisinin resistance in Democratic Republic of Congo. This study essentially revealed that resistance to chloroquine is still decreasing while polymorphism related to amodiaquine resistance seems to be not present in Democratic Republic of Congo, that three samples, located in the east of the country, harbor Pfmdr1 amplification and that none of the mutations found in South-East Asia correlated with artemisinine resistance have been found in Democratic Republic of Congo. But new mutations have been identified, especially the M476K, occurred in the same position that the M476I previously identified in the F32-ART strain, strongly resistant to artemisinine. Antimalarial first-line treatments currently in use in Democratic Republic of Congo are not associated with emergence of molecular markers of resistance. [ABSTRACT FROM AUTHOR]

Background: Single low dose primaquine (SLD PQ, 0.25mg/kg) is recommended in combination with artemisinin-based combination therapy (ACT) as a gametocytocide to prevent Plasmodium falciparum transmission in areas threatened by artemisinin resistance. To date, no randomized controlled trials have measured primaquine’s effect on infectiousness to Anopheline mosquitoes in Southeast Asia. Methods: Cambodian adults with uncomplicated falciparum malaria were randomized to receive a single 45mg dose of primaquine (equivalent to three SLD PQ) or no primaquine after the third dose of dihydroartemisin-piperaquine (DHP) therapy. A membrane-feeding assay measured infectiousness to Anopheles dirus on days 0, 3, 7, and 14 of blood-stage therapy. Gametocytemia was evaluated by microscopy and reverse-transcriptase PCR. Results: Prior to trial halt for poor DHP treatment efficacy, 101 participants were randomized and 50 received primaquine. Overall microscopic gametocyte prevalence was low (9%), but gametocytemic subjects given primaquine were gametocyte-free by day 14, and significantly less likely to harbor gametocytes by day 7 compared to those treated with DHP-alone, who remained gametocytemic for a median of two weeks. Only one infectious subject was randomized to the primaquine group, precluding assessment of transmission-blocking efficacy. However, he showed a two-fold reduction in oocyst density of infected mosquitoes less than 24 hours after primaquine dosing. In the DHP-alone group, four subjects remained infectious through day 14, infecting roughly the same number of mosquitoes pre and post-treatment. Overall, microscopic gametocytemia was an excellent predictor of infectiousness, and performed better than submicroscopic gametocytemia post-treatment, with none of 474 mosquitoes infected post-treatment arising from submicroscopic gametocytes. Conclusions: In a setting of established ACT resistance, a single dose of 45mg primaquine added to DHP rapidly and significantly reduced gametocytemia, while DHP-alone failed to reduce gametocytemia and prevent malaria transmission to mosquitoes. Continued efforts to make single dose primaquine widely available are needed to help achieve malaria elimination. [ABSTRACT FROM AUTHOR]

Genetic manipulation of the deadly malaria parasite Plasmodium falciparum remains challenging, but the rise of CRISPR/Cas9-based genome editing tools is increasing the feasibility of altering this parasite’s genome in order to study its biology. Of particular interest is the investigation of drug targets and drug resistance mechanisms, which have major implications for fighting malaria. We present a new method for introducing drug resistance mutations in P. falciparum without the use of plasmids or the need for cloning homologous recombination templates. We demonstrate this method by introducing edits into the sodium efflux channel PfATP4 by transfection of a purified CRISPR/Cas9-guide RNA ribonucleoprotein complex and a 200-nucleotide single-stranded oligodeoxynucleotide (ssODN) repair template. Analysis of whole genome sequencing data with the variant-finding program MinorityReport confirmed that only the intended edits were made, and growth inhibition assays confirmed that these mutations confer resistance to the antimalarial SJ733. The method described here is ideally suited for the introduction of mutations that confer a fitness advantage under selection conditions, and the novel finding that an ssODN can function as a repair template in P. falciparum could greatly simplify future editing attempts regardless of the nuclease used or the delivery method. [ABSTRACT FROM AUTHOR]

Malaria, particularly due to Plasmodium falciparum, remains a major public health threat in Ethiopia. Artemether-lumefantine (AL) has been the first-line antimalarial drug against uncomplicated P. falciparum malaria in the country since 2004. Regular monitoring of antimalarial drugs is recommended by the World Health Organization (WHO) to help early detection of drug resistant strains of the parasite and contain their rapid spread. The objective of this study was to assess the therapeutic efficacy of AL in a high-transmission setting in Ethiopia. The study site was Setit Humera, northwest Ethiopia. Single-arm prospective study of a 28-day follow-up was conducted from October 2014 to January 2015 according to the revised WHO 2009 drug efficacy study protocol. Study end-points were classified into primary end-point and secondary end-point. While the primary end-point was the day-28 adequate clinical and parasitological response the secondary end-points were clinical and parasitological evaluations (parasite, fever and gametocyte clearance rate, incidence of drug adverse events) and the relative increment in hemoglobin (Hb) level from baseline to day (D) 14 and D28. A total of 92 patients were enrolled and 79 had completed the 28-day follow-up period. The overall cure rate was 98.8% with 95% confidence interval of 0.915–0.998 without polymerase chain reaction correction. The parasite clearance rate was high with fast resolution of clinical symptoms; 100% of the study participants cleared parasitaemia and fever on D3. Gametocyte carriage was reduced from 7% on D0 to 1% on D3 and complete clearance was achieved on D14. Mean Hb concentration significantly increased on D28 compared to that on D14. There was no serious adverse event. AL was efficacious and safe in a high-transmission setting for treatment of uncomplicated falciparum malaria. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide is an important antimicrobial agent but is also crucially involved in redox signaling and pathogen-host cell interactions. As a basis for systematically investigating intracellular H2O2 dynamics and regulation in living malaria parasites, we established the genetically encoded fluorescent H2O2 sensors roGFP2-Orp1 and HyPer-3 in Plasmodium falciparum. Both ratiometric redox probes as well as the pH control SypHer were expressed in the cytosol of blood-stage parasites. Both redox sensors showed reproducible sensitivity towards H2O2 in the lower micromolar range in vitro and in the parasites. Due to the pH sensitivity of HyPer-3, we used parasites expressing roGFP2-Orp1 for evaluation of short-, medium-, and long-term effects of antimalarial drugs on H2O2 levels and detoxification in Plasmodium. None of the quinolines or artemisinins tested had detectable direct effects on the H2O2 homeostasis at pharmacologically relevant concentrations. However, pre-treatment of the cells with antimalarial drugs or heat shock led to a higher tolerance towards exogenous H2O2. The systematic evaluation and comparison of the two genetically encoded cytosolic H2O2 probes in malaria parasites provides a basis for studying parasite-host cell interactions or drug effects with spatio-temporal resolution while preserving cell integrity. [ABSTRACT FROM AUTHOR]

Chloroquine (CQ) and fansidar (sulphadoxine-pyrimethamine, SP) were widely used for treatment of Plasmodium falciparum for several decades in Malaysia prior to the introduction of Artemisinin-based Combination Therapy (ACT) in 2008. Our previous study in Kalabakan, located in south-east coast of Sabah showed a high prevalence of resistance to CQ and SP, suggesting the use of the treatment may no longer be effective in the area. This study aimed to provide a baseline data of antimalarial drug resistant markers on P. falciparum isolates in Kota Marudu located in the north-east coast of Sabah. Mutations on genes associated with CQ (pfcrt and pfmdr1) and SP (pfdhps and pfdhfr) were assessed by PCR amplification and restriction fragment length polymorphism. Mutations on the kelch13 marker (K13) associated with artemisinin resistance were determined by DNA sequencing technique. The assessment of pfmdr1 copy number variation associated with mefloquine resistant was done by real-time PCR technique. A low prevalence (6.9%) was indicated for both pfcrt K76T and pfmdr1 N86Y mutations. All P. falciparum isolates harboured the pfdhps A437G mutation. Prevalence of pfdhfr gene mutations, S108N and I164L, were 100% and 10.3%, respectively. Combining the different resistant markers, only two isolates were conferred to have CQ and SP treatment failure markers as they contained mutant alleles of pfcrt and pfmdr1 together with quintuple pfdhps/pfdhfr mutation (combination of pfdhps A437G+A581G and pfdhfr C59R+S108N+I164L). All P. falciparum isolates carried single copy number of pfmdr1 and wild type K13 marker. This study has demonstrated a low prevalence of CQ and SP resistance alleles in the study area. Continuous monitoring of antimalarial drug efficacy is warranted and the findings provide information for policy makers in ensuring a proper malaria control. [ABSTRACT FROM AUTHOR]

Background: Seasonal malaria chemoprevention (SMC) with sulphadoxine-pyrimethamine (SP) plus amodiaquine (AQ) is being scaled up in Sahelian countries of West Africa. However, the potential development of Plasmodium falciparum resistance to the respective component drugs is a major concern. Methods: Two cross-sectional surveys were conducted before (August 2012) and after (June 2014) a pilot implementation of SMC in Koutiala, Mali. Children aged 3–59 months received 7 rounds of curative doses of SP plus AQ over two malaria seasons. Genotypes of P. falciparum Pfdhfr codons 51, 59 and 108; Pfdhps codons 437 and 540, Pfcrt codon 76 and Pfmdr1codon 86 were analyzed by PCR on DNA from samples collected before and after SMC, and in non-SMC patient population as controls (November 2014). Results: In the SMC population 191/662 (28.9%) and 85/670 (12.7%) of children were P. falciparum positive by microscopy and were included in the molecular analysis before (2012) and after SMC implementation (2014), respectively. In the non-SMC patient population 220/310 (71%) were successfully PCR analyzed. In the SMC children, the prevalence of all molecular markers of SP resistance increased significantly after SMC including the Pfdhfr-dhps quintuple mutant genotype, which was 1.6% before but 7.1% after SMC (p = 0.02). The prevalence of Pfmdr1-86Y significantly decreased from 26.7% to 15.3% (p = 0.04) while no significant change was seen for Pfcrt 76T. In 2014, prevalence of all molecular markers of SP resistance were significantly higher among SMC children compared to the non-SMC population patient (p < 0.01). No Pfdhfr-164 mutation was found neither at baseline nor post SMC. Conclusion: SMC increased the prevalence of molecular markers of P. falciparum resistance to SP in the treated children. However, there was no significant increase of these markers of resistance in the general parasite population after 2 years and 7 rounds of SMC. [ABSTRACT FROM AUTHOR]

Background: Artemether-lumefantrine (AL) was introduced for treatment of uncomplicated malaria in Guinea-Bissau in 2008. Malaria then resurged and recurrent malaria after treatment with AL and stock-outs of AL were common. This study therefore aimed to assess the efficacy of AL and identify an alternative second line antimalarial. Dihydroartemisinin-piperaquine (DP) was chosen as it has been shown to be safe and efficacious and to reduce the incidence of recurrent malaria. Methods and Findings: In a multicentre randomised open-label non-inferiority clinical trial, AL or DP were given over 3 days to children aged 6 months-15 years with uncomplicated P. falciparum mono-infection. Intake was observed and AL was given with milk. Children were seen on days 0, 1, 2 and 3 and then weekly days 7–42. Recurring P. falciparum were classified as recrudescence or new infections by genotyping. Between November 2012 and July 2015, 312 children were randomised to AL (n = 155) or DP (n = 157). The day 42 PCR adjusted per protocol adequate clinical and parasitological responses were 95% and 100% in the AL and DP groups respectively, Mantel-Haenszel weighted odds ratio (OR) 0.22 (95% CI 0–0.68), p = 0.022. In a modified intention to treat analysis in which treatment failures day 0 and reinfections were also considered as treatment failures adequate clinical and parasitological responses were 94% and 97% (OR 0.42 [95% CI, 0.13–1.38], p = 0.15). Parasite clearance and symptom resolution were similar with both treatments. Conclusions: Both treatments achieved the WHO recommended efficacy for antimalarials about to be adopted as policy. DP was not inferior to AL for treatment of uncomplicated P. falciparum malaria in Guinea-Bissau. Trial Registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

The development of resistance to insecticides by the vector of malaria and the increasingly faster appearance of resistance to antimalarial drugs by the parasite can dangerously hamper efforts to control and eradicate the disease. Alternative ways to treat this disease are urgently needed. Here we evaluate the in vitro effect of direct current (DC) capacitive coupling electrical stimulation on the biology and viability of Plasmodium falciparum. We designed a system that exposes infected erythrocytes to different capacitively coupled electric fields in order to evaluate their effect on P. falciparum. The effect on growth of the parasite, replication of DNA, mitochondrial membrane potential and level of reactive oxygen species after exposure to electric fields demonstrate that the parasite is biologically able to respond to stimuli from DC electric fields involving calcium signaling pathways. [ABSTRACT FROM AUTHOR]

Background: As per the WHO recommendation, the development of resistance by P. falciparum to most artemisinin combination therapies (ACTs) triggered the need for routine monitoring of the efficacy of the drugs every two years in all malaria endemic countries. Hence, this study was carried out to assess the therapeutic efficacy of Artemether-Lumefantrine (Coartem®) in treating the uncomplicated falciparum malaria, after 9 years of its introduction in the Metehara, Eastern Ethiopia. Method: This is part of the therapeutic efficacy studies by the Federal Ministry of Health Ethiopia, which were conducted in regionally representative sentinel sites in the country from October 2014 to January 2015. Based on the study criteria set by WHO, febrile and malaria suspected outpatients in the health center were consecutively recruited to study. A standard six-dose regimen of AL was administered over three days and followed up for measuring therapeutic responses over 28 days. Data entry and analysis was done by using the WHO designed Excel spreadsheet and SPSS version 20 for Windows. Statistical significant was considered for P-value less than 0.05. Result: Of the 91 patients enrolled, the day-28 analysis showed 83 adequate clinical and parasitological responses (ACPRs). Per protocol analysis, PCR-uncorrected & corrected cure rates of Coartem® among the study participants were 97.6% (95%CI: 93.6–99.5) and 98.8% (CI: 93.5–100%), respectively. No parasite detected on day 3 and onwards. Fever clearance was above 91% on day-3. Mean hemoglobin was significantly increased (P<0.000) from 12.39 g/dl at day 0 to 13.45 g/dl on day 28. No serious adverse drug reactions were observed among the study participants. Conclusion: This study showed high efficacy of AL in the study area, which suggests the continuation of AL as first line drug for the treatment of uncomplicated P. falciparum malaria in the study area. This study recommends further studies on drug toxicity, particularly on repeated cough and oral ulceration. [ABSTRACT FROM AUTHOR]

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC50 < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible. [ABSTRACT FROM AUTHOR]

Background: The use of antimalarial drugs for prevention and treatment is a major strategy in the prevention of malaria in pregnancy. Although sulphadoxine-pyrimethamine (SP) is currently recommended for intermittent preventive treatment of malaria during pregnancy in Nigeria, previously used drugs for prophylaxis such as chloroquine (CQ) and pyrimethamine are accessible as they are purchased over the counter. This study describes the markers of absence or presence of resistance to quinoline (Pfcrt and Pfmdr 1) and type 1 antifolate antimalarial medicines (Pfdhfr). Methods: Plasmodium falciparum-positive dried blood spots from pregnant women attending antenatal clinics for the first time during current pregnancy were investigated for the presence of mutations at codons 72–76 of Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene by real time polymerase chain reaction (PCR) using haplotype-specific probes. PCR followed by sequence analysis was used to identify mutations at codons 86, 184, 1034, 1042 and 1246 of P. falciparum multi-drug resistance-1 (Pfmdr1) gene; and codons 16, 50, 51, 59, 108, 140 and 164 of Pfdhfr gene. Results: Two haplotypes of Pfcrt (n = 54) were observed: CVMNK 13(24.2%) and CVIET 41 (75.9%) of the samples. The SVMNT haplotype was absent in this population. The Pfmdr1 (n = 28) haplotypes were NYSND 15(53.6%), YYSND 5(17.9%), NFSND 6(21.4%) and YFSND 2(7.1%). The Pfdhfr (n = 15) were ACNCSVI 4(26.7%), and ACICNSVI 1(6.7%) and ACIRNVI 10 (66.7%). The rate of occurrence of Pfcrt 76T, Pfdhfr108N, Pfmdr186Yand184F were 75.9%, 73.3%, 25% and 28.1% respectively. The Pfmdr1 86Y was associated with low parasitaemia (median = 71 parasites/μl, P = 0.024) while Pfcrt 76T was associated with young maternal age (mean 24.1 ± 4.5 years; P = 0.006). The median parasitaemia were similar (P>0.05) in wild and mutant strains of Pfcrt 76, Pfmdr1 184 and Pfdhfr 108. There was no association between gravidity or gestational age of the women and presence of mutations in the Pfcrt, Pfmdr1 or Pfdhfr genes (P>0.05). Conclusion: Markers of resistance to chloroquine and pyrimethamine were high, whereas cycloguanil-resistance marker was not present in the studied population. The low level of mutations in the Pfmdr1gene indicates likely efficacy of amodiaquine against malaria in pregnancy. [ABSTRACT FROM AUTHOR]