Resistant Falciparum Research Articles

Synergistic interaction of two antimalarial drugs, artemisinin and concanamycin A

Artemisinin is a powerful drug that has been combined with other antimalarial drugs to combat malaria and it has been crucial to recent achievements in reducing malaria cases. However, the emergence of Plasmodium falciparum resistance against artemisinin has become a serious problem in malaria treatment. Our previous studies reported that artemisinin alkalinised the digestive vacuole of P. falciparum similarly to concanamycin A. Concanamycin A is a specific inhibitor of V-type H+-ATPase, a proton pump located on the membrane of the digestive vacuole. A study also showed that a low concentration of concanamycin A is required to kill 50% of the parasites. Therefore, this study aimed to determine the interaction of artemisinin with concanamycin A by using the isobologram analysis of effects on parasite growth. The antimalarial activity (IC50) of artemisinin and concanamycin A was evaluated by using a malarial SBYR Green I fluorescence-based (MSF) assay prior to isobologram analysis. Based on their IC50 values, six different combination solutions of the drugs were assigned and used in the isobologram analysis. The IC50 of artemisinin and concanamycin A was 13 ± 2.52 nM and 7 ± 1.15 nM, respectively. The interaction of artemisinin and concanamycin A was found to be synergistic, indicating that the combination of these drugs could kill the parasites more effectively. This study suggests that artemisinin and concanamycin A combination can be a new candidate in artemisinin-based combination therapies.

Chemogenomic Profiling of a Plasmodium falciparum Transposon Mutant Library Reveals Shared Effects of Dihydroartemisinin and Bortezomib on Lipid Metabolism and Exported Proteins.

The antimalarial activity of the frontline drug artemisinin involves generation of reactive oxygen species (ROS) leading to oxidative damage of parasite proteins. To achieve homeostasis and maintain protein quality control in the overwhelmed parasite, the ubiquitin-proteasome system kicks in. Even though molecular markers for artemisinin resistance like pfkelch13 have been identified, the intricate network of mechanisms driving resistance remains to be elucidated. Here, we report a forward genetic screening strategy that enables a broader identification of genetic factors responsible for altering sensitivity to dihydroartemisinin (DHA) and a proteasome inhibitor, bortezomib (BTZ). Using a library of isogenic piggyBac mutants in P. falciparum, we defined phenotype-genotype associations influencing drug responses and highlighted shared mechanisms between the two processes, which mainly included proteasome-mediated degradation and the lipid metabolism genes. Additional transcriptomic analysis of a DHA/BTZ-sensitive piggyBac mutant showed it is possible to find differences between the two response mechanisms on the specific components for regulation of the exportome. Our results provide further insight into the molecular mechanisms of antimalarial drug resistance. IMPORTANCE Malaria control is seriously threatened by the emergence and spread of Plasmodium falciparum resistance to the leading antimalarial, artemisinin. The potent killing activity of artemisinin results from oxidative damage unleashed by free heme activation released by hemoglobin digestion. Although the ubiquitin-proteasome system is considered critical for parasite survival of this toxicity, the diverse genetic changes linked to artemisinin resistance are complex and, so far, have not included the ubiquitin-proteasome system. In this study, we use a systematic forward genetic approach by screening a library of P. falciparum random piggyBac mutants to decipher the genetic factors driving malaria parasite responses to the oxidative stress caused by antimalarial drugs. This study compares phenotype-genotype associations influencing dihydroartemisinin responses with the proteasome inhibitor bortezomib to delineate the role of ubiquitin-proteasome system. Our study highlights shared and unique pathways from the complex array of molecular processes critical for P. falciparum survival resulting from the oxidative damage of artemisinin.

Assessment of Plasmodium falciparum Resistance to Artemisinin Among Attendees of Selected Health Facilities in Rivers State, Nigeria

Across-sectional research, aimed at evaluating artemisinin drug resistance in falciparum malaria patients attending selected health facilities in Rivers State, Nigeria, was done between July 2019 and March 2020. The selected hospitals were Rivers State University Teaching Hospital (RSUTH), University of Port Harcourt Teaching Hospital (UPTH) and Model Primary Health Centre (MPHC), Iriebe, all in Rivers State, Nigeria. The study population was 1000 subjects with an overall malaria prevalence of 25.5%, the number of enrolled participants from RSUTH was 494 with the highest infection rate of 32.5%, 300 from UPTH which had the least (11%) prevalence, while 206 were from MPHC, Iriebe with 29.6% prevalence accordingly. Blood samples were collected from these subjects by phlebotomy method. Plasmodium falciparum parasite was determined by microscopy using thick and thin blood films stained with Giemsa staining technique. Artemisinin resistance markers were assessed using the nested polymerase chain reaction method. The study population consisted of children, pregnant women, and other adults of both sexes. Out of the forty-seven isolates assayed for K13 and ATPase 6 genes, who carried the artemisinin-sensitive wild-type allele, none of these isolates exhibited both ATPase 6 and Kelch -13 resistance genes/mutations hence no single nucleotide polymorphism was observed. This suggests that this part of the country is not under the threat of artemisinin resistance. However, this should not undermine the need for continuous monitoring and surveillance of the response of P. falciparum to Artemisinin and Artemisinin combination therapy (ACTs) in River State.

Population Genomics of Plasmodium falciparum and Malaria Control Implications in Abidjan (Cote d’ivoire)

Introduction: The onset of Plasmodium falciparum (P. falciparum) resistance to antimalarial drugs requires the careful surveillance of African parasite populations. Genomic tools are implemented to detect evolutionary changes that could impact malaria control and elimination strategies. Here, we evaluate the genome-wide pattern of selection and sequence variation in P. falciparum populations in Abidjan, Côte d’Ivoire. Methods: The study was conducted in three localities of Abidjan from 2013 to 2014. We collected 70 blood samples after receiving written informed consent from patients above two years of age. After extracting P. falciparum and human DNA from isolates, we performed whole-genome sequencing and used population genomics approaches to investigate the genetic diversity and complexity of infections and identify loci under positive directional selection. Results: We observed an excess of rare variants in the population, showing a clear mutation process in the isolates. Moderate Fst estimates (0.3) was detected for surfin, an immune invasion gene family. Seven iHS regions that had at least two SNPs with a score > 3.2 were identified. These regions code for genes that have been under strong directional selection. Two of these genes were the chloroquine resistance transporter (crt) on chromosome 7 and the dihydropteroate reductase (dhps) on chromosome 8. Our analyses showed that a recent selective sweep occurred for the erythrocyte membrane protein (Pfemp1). Conclusion: Our analyses identified genes under selective drug pressure and balancing selection on protective immune-specific genes. These findings demonstrate the effectiveness of genomics analyses to follow the evolution of malaria parasites and adopt appropriate strategies to eliminate malaria in Côte d’Ivoire.

Sub-microscopic Plasmodium falciparum parasitaemia, dihydropteroate synthase (dhps) resistance mutations to sulfadoxine–pyrimethamine, transmission intensity and risk of malaria infection in pregnancy in Mount Cameroon Region

BackgroundPlasmodium falciparum resistance to intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) continues to spread throughout sub-Saharan Africa. This study assessed the occurrence of microscopic and sub-microscopic P. falciparum parasitaemia, dihydropteroate synthase mutations associated with resistance to SP and maternal anaemia in the Mount Cameroon area.MethodsConsenting pregnant women living in semi-rural and semi-urban/urbanized settings were enrolled in this cross-sectional study. Socio-demographic, antenatal and clinical data were documented. Microscopic and sub-microscopic parasitaemia were diagnosed using peripheral blood microscopy and nested polymerase chain reaction (PCR) respectively. The dhps mutations were genotyped by restriction fragment length polymorphism analysis. The presence of A437G, K540E, and A581G was considered a marker for high-level resistance. Haemoglobin levels and anaemia status were determined.ResultsAmong the women, the prevalence of microscopic and sub-microscopic P. falciparum infection were 7.7% (67/874) and 18.6% (93/500) respectively. Predictors of microscopic infection were younger age (< 21 years) (AOR = 2.89; 95% CI 1.29–6.46) and semi-rural settings (AOR = 2.27; 95% CI 1.31–3.96). Determinants of sub-microscopic infection were the rainy season (AOR, 3.01; 95% CI 1.77–5.13), primigravidity (AOR = 0.45; 95% CI 0.21–0.94) and regular ITN usage (AOR = 0.49; 95% CI 0.27–0.90). Of the145 P. falciparum isolates genotyped, 66.9% (97) carried mutations associated with resistance to SP; 33.8% (49), 0%, 52.4% (76) and 19.3% (28) for A437G, K540E, A581G and A437G + A581G respectively. The A581G mutation was associated with ≥ 3 SP doses evident only among sub-microscopic parasitaemia (P = 0.027) and multigravidae (P = 0.009). Women with microscopic infection were more likely from semi-rural settings (AOR = 7.09; 95% CI 2.59–19.42), to report history of fever (AOR = 2.6; 95% CI 1.07–6.31), to harbour parasites with double resistant mutations (AOR = 6.65; 95% CI 1.85–23.96) and were less likely to have received 2 SP doses (AOR = 0.29; 95% CI 1.07–6.31). Microscopic infection decreased Hb levels more than sub-microscopic infection.ConclusionThe occurrence of sub-microscopic P. falciparum parasites resistant to SP and intense malaria transmission poses persistent risk of malaria infection during pregnancy in the area. ITN usage and monitoring spread of resistance are critical.

Design and synthesis of novel glycyrrhetinic acid-triazole derivatives that exert anti-plasmodial activity inducing mitochondrial-dependent apoptosis in Plasmodium falciparum

Malaria is an enormous threat to public health due to the emergence of Plasmodium falciparum resistance to widely used anti-malarials, such as chloroquine.

Fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum clinical isolates collected from unrest tribal agencies of Pakistan.

Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.

4-Aminoquinolines as Antimalarial Agents: Review of A Medicinal Chemistry Perspective

Abstract: Malaria is a potentially fatal parasitic disease brought on by five Plasmodium species, including Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. The fast emergence of P. falciparum resistance to currently available treatments has increased health concerns in developing countries. The growth in resistance emphasizes the need for a novel, secure, and cost-effective antimalarial drug that can treat malaria resistant to multiple antimalarial drugs. Therefore, it has become crucial to create new therapeutic approaches to deal with the rise of parasites resistant to artemisinin. For the treatment of P. falciparum malaria in children living in areas with moderate to high transmission, as established by WHO, the malaria vaccine RTS, S has been authorized. The WHO recommends that governments consider this immunization against a human parasite when deciding the optimum subnational combination of measures for maximum impact. The current research synthesizes numerous 4-aminoquinoline derivatives successfully treating malaria and its diverse etiological species. Additionally crucial to the management and prevention of malaria are antibiotics. Effective and well-tolerated antimalarial medications include tetracycline and chloramphenicol, chloroquine, primaquine, pamaquine, and artemisinins are some of the drugs used to treat malaria; however, numerous new compounds have proven to be even more efficient. This review, based on literature reports, will give medicinal chemists ideas for new malaria drugs to develop. In addition, this review will help search for new antimalarial drug leads in the future.

Antimalarials: A Patents Landscape Study (2015-Present)

Abstract: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and P. knowlesi are five Plasmodium species that cause malaria, a life-threatening parasitic disease. In the developing world, the rapid development of Plasmodium falciparum resistance to currently available treatments has become a serious health concern. This work reports a patent landscape analysis of patent documents related to antimalarial. The patent search was conducted using the commercially available CAS SciFinder database and the open-source patent database, The Lens. Seven hundred ninety-seven patents from The Lens and 1172 patent Sci-finder were exported using the antimalarial drug as a keyword. After the initial screening, 58 patent documents were shortlisted for in-depth analysis. After analysis, it was found that most of the top applicants come from the United States and Switzerland, which shows that market protection is more important in these two countries. The top applicants come from private companies, universities, and public-private partnerships. The United States, Europe, China, Canada, and the Republic of Korea lead the patent race in this area. The most-recorded IPC code is A61P33/06, related to the chemical substances or pharmaceutical formulations showing antimalarials activity. Most of the time, antimalarial drugs were made from quinine, artemisinin, trioxolane, naphthoquinones, and isoquinoline derivatives. Quinine and artemisinin are well-established classes of antimalarials with the maximum number of antimalarial drugs in the market. The research and innovations disclosed in most patents were focused on exploring or evaluating the new scaffolds and their mechanism of action against the normal and resistant malarial parasite. In conclusion, it has been found that various scaffolds are needed to be explored further in search of new antimalarial compounds.

Targeting Artemisinin-Resistant Malaria by Repurposing the Anti-Hepatitis C Virus Drug Alisporivir.

The emergence of Plasmodium falciparum resistance raises an urgent need to find new antimalarial drugs. Here, we report the rational repurposing of the anti-hepatitis C virus drug, alisporivir, a nonimmunosuppressive analog of cyclosporin A, against artemisinin-resistant strains of P. falciparum. In silico docking studies and molecular dynamic simulation predicted strong interaction of alisporivir with PfCyclophilin 19B, confirmed through biophysical assays with a Kd value of 354.3 nM. Alisporivir showed potent antimalarial activity against chloroquine-resistant (PfRKL-9 with resistance index [Ri] 2.14 ± 0.23) and artemisinin-resistant (PfKelch13R539T with Ri 1.15 ± 0.04) parasites. The Ri is defined as the ratio between the IC50 values of the resistant line to that of the sensitive line. To further investigate the mechanism involved, we analyzed the expression level of PfCyclophilin 19B in artemisinin-resistant P. falciparum (PfKelch13R539T). Semiquantitative real-time transcript, Western blot, and immunofluorescence analyses confirmed the overexpression of PfCyclophilin 19B in PfKelch13R539T. A 50% inhibitory concentration in the nanomolar range, together with the targeting of PfCyclophilin 19B, suggests that alisporivir can be used in combination with artemisinin. Since artemisinin resistance slows the clearance of ring-stage parasites, we performed a ring survival assay on artemisinin-resistant strain PfKelch13R539T and found significant decrease in parasite survival with alisporivir. Alisporivir was found to act synergistically with dihydroartemisinin and increase its efficacy. Furthermore, alisporivir exhibited antimalarial activity in vivo. Altogether, with the rational target-based Repurposing of alisporivir against malaria, our results support the hypothesis that targeting resistance mechanisms is a viable approach toward dealing with drug-resistant parasite.

Molecular surveillance for anti-malarial drug resistance and genetic diversity of Plasmodium falciparum after chloroquine and sulfadoxine-pyrimethamine withdrawal in Quibdo, Colombia, 2018

BackgroundResistance to anti-malarial drugs is associated with polymorphisms in target genes and surveillance for these molecular markers is important to detect the emergence of mutations associated with drug resistance and signal recovering sensitivity to anti-malarials previously used.MethodsThe presence of polymorphisms in genes associated with Plasmodium falciparum resistance to chloroquine and sulfadoxine-pyrimethamine was evaluated by Sanger sequencing, in 85 P. falciparum day of enrollment samples from a therapeutic efficacy study of artemether–lumefantrine conducted in 2018–2019 in Quibdo, Colombia. Samples were genotyped to assess mutations in pfcrt (codons 72–76), pfdhfr (codons 51, 59, 108, and 164), and pfdhps genes (codons 436, 437, 540, and 581). Further, the genetic diversity of infections using seven neutral microsatellites (NMSs) (C2M34, C3M69, Poly α, TA1, TA109, 2490, and PfPK2) was assessed.ResultsAll isolates carried mutant alleles for pfcrt (K76T and N75E), and for pfdhfr (N51I and S108N), while for pfdhps, mutations were observed only for codon A437G (32/73, 43.8%). Fifty samples (58.8%) showed a complete neutral microsatellites (NMS) profile. The low mean number of alleles (2 ± 0.57) per locus and mean expected heterozygosity (0.17 ± 0.03) showed a reduced genetic diversity. NMS multilocus genotypes (MMG) were built and nine MMG were identified.ConclusionsOverall, these findings confirm the fixation of chloroquine and pyrimethamine-resistant alleles already described in the literature, implying that these drugs are not currently appropriate for use in Colombia. In contrast, mutations in the pfdhps gene were only observed at codon 437, an indication that full resistance to sulfadoxine has not been achieved in Choco. MMGs found matched the clonal lineage E variant 1 previously reported in northwestern Colombia.

The protein aggregation inhibitor YAT2150 has potent antimalarial activity in Plasmodium falciparum in vitro cultures

BackgroundBy 2016, signs of emergence of Plasmodium falciparum resistance to artemisinin and partner drugs were detected in the Greater Mekong Subregion. Recently, the independent evolution of artemisinin resistance has also been reported in Africa and South America. This alarming scenario calls for the urgent development of new antimalarials with novel modes of action. We investigated the interference with protein aggregation, which is potentially toxic for the cell and occurs abundantly in all Plasmodium stages, as a hitherto unexplored drug target in the pathogen.ResultsAttempts to exacerbate the P. falciparum proteome’s propensity to aggregation by delivering endogenous aggregative peptides to in vitro cultures of this parasite did not significantly affect their growth. In contrast, protein aggregation inhibitors clearly reduced the pathogen’s viability. One such compound, the bis(styrylpyridinium) salt YAT2150, exhibited potent antiplasmodial activity with an in vitro IC50 of 90 nM for chloroquine- and artemisinin-resistant lines, arresting asexual blood parasites at the trophozoite stage, as well as interfering with the development of both sexual and hepatic forms of Plasmodium. At its IC50, this compound is a powerful inhibitor of the aggregation of the model amyloid β peptide fragment 1-40, and it reduces the amount of aggregated proteins in P. falciparum cultures, suggesting that the underlying antimalarial mechanism consists in a generalized impairment of proteostasis in the pathogen. YAT2150 has an easy, rapid, and inexpensive synthesis, and because it fluoresces when it accumulates in its main localization in the Plasmodium cytosol, it is a theranostic agent.ConclusionsInhibiting protein aggregation in Plasmodium significantly reduces the parasite’s viability in vitro. Since YAT2150 belongs to a novel structural class of antiplasmodials with a mode of action that potentially targets multiple gene products, rapid evolution of resistance to this drug is unlikely to occur, making it a promising compound for the post-artemisinin era.

High prevalence of Pfcrt 76T and Pfmdr1 N86 genotypes in malaria infected patients attending health facilities in East Shewa zone, Oromia Regional State, Ethiopia

BackgroundPlasmodium falciparum resistance to series of anti-malarial drugs is a major challenge in efforts to control and/or eliminate malaria globally. In 1998, following the widespread of chloroquine (CQ) resistant P. falciparum, Ethiopia switched from CQ to sulfadoxine–pyrimethamine (SP) and subsequently in 2004 from SP to artemether–lumefantrine (AL) for the treatment of uncomplicated falciparum malaria. Data on the prevalence of CQ resistance markers after more than two decades of its removal is important to map the selection pressure behind the targets codons of interest. The present study was conducted to determine the prevalence of mutations in Pfcrt K76T and Pfmdr1 N86Y codons among malaria-infected patients from Adama, Olenchiti and Metehara sites of East Shewa zone, Oromia Regional State, Ethiopia.MethodsFinger-prick whole blood samples were collected on 3MM Whatman ® filter papers from a total of 121 microscopically confirmed P. falciparum infected patients. Extraction of parasite DNA was done by Chelex-100 method from dried blood spot (DBS). Genomic DNA template was used to amplify Pfcrt K76T and Pfmdr1 N86Y codons by nested PCR. Nested PCR products were subjected to Artherobacter protophormiae-I (APoI) restriction enzyme digestion to determine mutations at codons 76 and 86 of Pfcrt and Pfmdr1 genes, respectively.ResultsOf 83 P. falciparum isolates successfully genotyped for Pfcrt K76T, 91.6% carried the mutant genotypes (76T). The prevalence of Pfcrt 76T was 95.7%, 92.5% and 84.5% in Adama, Metehara and Olenchiti, respectively. The prevalence of Pfcrt 76T mutations in three of the study sites showed no statistical significance difference (χ2 = 1.895; P = 0.388). On the other hand, of the 80 P. falciparum samples successfully amplified for Pfmdr1, all carried the wild-type genotypes (Pfmdr1 N86).ConclusionAlthough CQ officially has been ceased for the treatment of falciparum malaria for more than two decades in Ethiopia, greater proportions of P. falciparum clinical isolates circulating in the study areas carry the mutant 76T genotypes indicating the presence of indirect CQ pressure in the country. However, the return of Pfmdr1 N86 wild-type allele may be favoured by the use of AL for the treatment of uncomplicated falciparum malaria.

Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysis.

CRISPR editing has enabled the rapid creation of fluorescent Plasmodium transgenic lines, facilitating a deeper understanding of parasite biology. The impact of genetic perturbations such as gene disruption or the introduction of drug resistance alleles on parasite fitness is typically quantified in competitive growth assays between the query line and a wild type reference. Although fluorescent reporter lines offer a facile and frequently used method to measure relative growth, this approach is limited by the strain background of the existing reporter, which may not match the growth characteristics of the query strains, particularly if these are slower-growing field isolates. Here, we demonstrate an efficient CRISPR-based approach to generate fluorescently labelled parasite lines using mNeonGreen derived from the LanYFP protein in Branchiostoma lanceolatum, which is one of the brightest monomeric green fluorescent proteins identified. Using a positive-selection approach by insertion of an in-frame blasticidin S deaminase marker, we generated a Dd2 reporter line expressing mNeonGreen under the control of the pfpare (P. falciparum Prodrug Activation and Resistance Esterase) locus. We selected the pfpare locus as an integration site because it is highly conserved across P. falciparum strains, expressed throughout the intraerythrocytic cycle, not essential, and offers the potential for negative selection to further enrich for integrants. The mNeonGreen@pare line demonstrates strong fluorescence with a negligible fitness defect. In addition, the construct developed can serve as a tool to fluorescently tag other P. falciparum strains for in vitro experimentation.

Anti-folate quintuple mutations in Plasmodium falciparum asymptomatic infections in Yaoundé, Cameroon

A major challenge in the fight to effectively control malaria is the emergence of resistant parasite to drugs used in therapy as well as for chemoprevention. In this study, single nucleotide polymorphisms (SNPs) associated with Plasmodium falciparum resistance to sulfadoxine-pyrimethamine (SP), one of the partner drugs in artemisinin-based therapies (ACTs) were studied in asymptomatic P. falciparum isolates from Cameroon. Dried Blood spots were collected from children with asymptomatic malaria enrolled during a household survey. The P. falciparum dihydrofolate reductase (Pfdhfr), dihydropteroate synthase (Pfdhps) and Kelch 13 genes were amplified and point mutations in these gene sequences were analyzed by sequencing. Among a total of 234 samples collected, 51 showed parasitaemia after microscopic examination of which 47 were P. falciparum mono-infections. Molecular analysis revealed 97.3% of mutant alleles at codons 51I, 59R and 108 N in Pfdhfr gene. In Pfdhps gene the most common mutation was 437G (83.3%); followed by 436A (47.6%) and 436F (28.6%). The association of mutations in the two genes (dhfr + dhps) showed 11 different haplotypes including three sextuple mutants (IRNI + AGKGA, IRNI + AAKGS, IRNI + AGKAS) and one septuple mutant (IRNI + AGKGS). For K13 gene no SNPs were seen in the studied asymptomatic malaria samples. The findings revealed presence of SP-resistant alleles in asymptomatic infected individuals with presence of sextuples and septuple SNPs. This emphasizes that regular profiling of antimalarial drugs resistance markers in such population is essential for malaria control and elimination programmes.

Spatiotemporal spread of Plasmodium falciparum mutations for resistance to sulfadoxine-pyrimethamine across Africa, 1990-2020.

BackgroundSulfadoxine-pyrimethamine (SP) is recommended in Africa in several antimalarial preventive regimens including Intermittent Preventive Treatment in pregnant women (IPTp), Intermittent Preventive Treatment in infants (IPTi) and Seasonal Malaria Chemoprevention (SMC). The effectiveness of SP-based preventive treatments are threatened in areas where Plasmodium falciparum resistance to SP is high. The prevalence of mutations in the dihydropteroate synthase gene (pfdhps) can be used to monitor SP effectiveness. IPTi-SP is recommended only in areas where the prevalence of the pfdhps540E mutation is below 50%. It has also been suggested that IPTp-SP does not have a protective effect in areas where the pfdhps581G mutation, exceeds 10%. However, pfdhps mutation prevalence data in Africa are extremely heterogenous and scattered, with data completely missing from many areas.Methods and findingsThe WWARN SP Molecular Surveyor database was designed to summarize dihydrofolate reductase (pfdhfr) and pfdhps gene mutation prevalence data. In this paper, pfdhps mutation prevalence data was used to generate continuous spatiotemporal surface maps of the estimated prevalence of the SP resistance markers pfdhps437G, pfdhps540E, and pfdhps581G in Africa from 1990 to 2020 using a geostatistical model, with a Bayesian inference framework to estimate uncertainty. The maps of estimated prevalence show an expansion of the pfdhps437G mutations across the entire continent over the last three decades. The pfdhps540E mutation emerged from limited foci in East Africa to currently exceeding 50% estimated prevalence in most of East and South East Africa. pfdhps540E distribution is expanding at low or moderate prevalence in central Africa and a predicted focus in West Africa. Although the pfdhps581G mutation spread from one focus in East Africa in 2000, to exceeding 10% estimated prevalence in several foci in 2010, the predicted distribution of the marker did not expand in 2020, however our analysis indicated high uncertainty in areas where pfdhps581G is present. Uncertainty was higher in spatial regions where the prevalence of a marker is intermediate or where prevalence is changing over time.ConclusionsThe WWARN SP Molecular Surveyor database and a set of continuous spatiotemporal surface maps were built to provide users with standardized, current information on resistance marker distribution and prevalence estimates. According to the maps, the high prevalence of pfdhps540E mutation was to date restricted to East and South East Africa, which is reassuring for continued use of IPTi and SMC in West Africa, but continuous monitoring is needed as the pfdhps540E distribution is expanding. Several foci where pfdhps581G prevalence exceeded 10% were identified. More data on the pfdhps581G distribution in these areas needs to be collected to guide IPTp-SP recommendations. Prevalence and uncertainty maps can be utilized together to strategically identify sites where increased surveillance can be most informative. This study combines a molecular marker database and predictive modelling to highlight areas of concern, which can be used to support decisions in public health, highlight knowledge gaps in certain regions, and guide future research.

Keystone Malaria Symposium 2022: a vibrant discussion of progress made and challenges ahead from drug discovery to treatment

Keystone Malaria Symposium 2022: a vibrant discussion of progress made and challenges ahead from drug discovery to treatment

Low Complexity of Infection Is Associated With Molecular Persistence of Plasmodium falciparum in Kenya and Tanzania.

Plasmodium falciparum resistance to artemisinin-based combination therapies (ACTs) is a threat to malaria elimination. ACT-resistance in Asia raises concerns for emergence of resistance in Africa. While most data show high efficacy of ACT regimens in Africa, there have been reports describing declining efficacy, as measured by both clinical failure and prolonged parasite clearance times. Three hundred children aged 2-10 years with uncomplicated P. falciparum infection were enrolled in Kenya and Tanzania after receiving treatment with artemether-lumefantrine. Blood samples were taken at 0, 24, 48, and 72 h, and weekly thereafter until 28 days post-treatment. Parasite and host genetics were assessed, as well as clinical, behavioral, and environmental characteristics, and host anti-malarial serologic response. While there was a broad range of clearance rates at both sites, 85% and 96% of Kenyan and Tanzanian samples, respectively, were qPCR-positive but microscopy-negative at 72 h post-treatment. A greater complexity of infection (COI) was negatively associated with qPCR-detectable parasitemia at 72 h (OR: 0.70, 95% CI: 0.53-0.94), and a greater baseline parasitemia was marginally associated with qPCR-detectable parasitemia (1,000 parasites/uL change, OR: 1.02, 95% CI: 1.01-1.03). Demographic, serological, and host genotyping characteristics showed no association with qPCR-detectable parasitemia at 72 h. Parasite haplotype-specific clearance slopes were grouped around the mean with no association detected between specific haplotypes and slower clearance rates. Identifying risk factors for slow clearing P. falciparum infections, such as COI, are essential for ongoing surveillance of ACT treatment failure in Kenya, Tanzania, and more broadly in sub-Saharan Africa.

Novel pfk13 polymorphisms in Plasmodium falciparum population in Ghana

The molecular determinants of Plasmodium falciparum artemisinin resistance are the single nucleotide polymorphisms in the parasite’s kelch propeller domain, pfk13. Validated and candidate markers are under surveillance in malaria endemic countries using artemisinin-based combination therapy. However, pfk13 mutations which may confer parasite artemisinin resistance in Africa remains elusive. It has therefore become imperative to report all observed pfk13 gene polymorphisms in malaria therapeutic efficacy studies for functional characterization. We herein report all novel pfk13 mutations observed only in the Ghanaian parasite population. In all, 977 archived samples from children aged 12 years and below with uncomplicated malaria from 2007 to 2017 were used. PCR/Sanger sequencing analysis revealed 78% (763/977) of the samples analyzed were wild type (WT) for pfk13 gene. Of the 214 (22%) mutants, 78 were novel mutations observed only in Ghana. The novel SNPs include R404G, P413H, N458D/H/I, C473W/S, R529I, M579T/Y, C580R/V, D584L, N585H/I, Q661G/L. Some of the mutations were sites and ecological zones specific. There was low nucleotide diversity and purifying selection at the pfk13 locus in Ghanaian parasite population. With increasing drug pressure and its consequent parasite resistance, documenting these mutations as baseline data is crucial for future molecular surveillance of P. falciparum resistance to artemisinin in Ghana.

Pfkelch13 Plasmodium falciparum Mutations in Huambo, Angola.

Artemisinin (ART) is recommended as the first-line drug for P. falciparum infections combined with a long-acting partner drug. The emergence of P. falciparum resistance to ART (ARTR) is a concern for malaria. The most feared threat remains the spread of ARTR from Southeast Asia to Africa or the independent emergence of ARTR in Africa, where malaria accounts for 93% of all malaria cases and 94% of deaths. To avoid this worst-case scenario, surveillance of Pfkelch13 mutations is essential. We investigated mutations of Pfkelch13 in 78 P. falciparum samples from Huambo, Angola. Most of the parasites had a wild-type Pfkelch13 allele. We identified one synonymous mutation (R471R) in 10 isolates and one non-synonymous mutation (A578S) in two samples. No Pfkelch13 validated or candidate ARTR mutants were identified. The finding suggests that there is little polymorphism in Pfkelch13 in Huambo. Since cases of late response to ART in Africa and the emergence of ARTR mutations in Rwanda and Uganda have been reported, efforts should be made toward continuous molecular surveillance of ARTR. Our study has some limitations. Since we analyzed P. falciparum parasites from a single health facility, the study may not be representative of all Angolan endemic areas.