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Objectives This study was performed to identify and characterize circulating Plasmodium species in three provinces of Mindanao approaching malaria elimination. Methods Rapid diagnostic tests (RDT), microscopic examination, and PCR were used to detect malaria parasites. PCR-positive isolates were genotyped for polymorphisms in loci of interest. Results A total of 2639 participants were surveyed in Mindanao between 2010 and 2013. Malaria prevalence by PCR was 3.8% (95% confidence interval (CI): 2.7–5.2%) in Sarangani, 10% (95% CI: 7.7–12.7%) in South Cotabato, and 4.2% (95% CI: 3.2–5.6%) in Tawi-Tawi. P. falciparum and P. vivax were identified in all three provinces, and there was one case of P. malariae in South Cotabato. RDT was inferior to PCR for detecting asymptomatic P. falciparum and P. vivax. In Tawi-Tawi, microscopy failed to identify 46 PCR-positive malaria infections. The presence of pfcrt haplotypes CVMNK, CVIET, and SMNT (codons 72–76), pfmdr1 haplotype NFSND (codons 86, 184, 1034, 1042, 1246), and pvmdr1 haplotype NFL (codons 91, 976, 1076) was confirmed in Mindanao. Conclusions Asymptomatic Plasmodium infections persisted in local communities between 2010 and 2013. PCR successfully identified subpatent malaria infections, and can better characterize malaria epidemiology in communities seeking malaria control and elimination at the local level.

Highlights • Malaria cases in UK and Ireland travellers can give false-negative HRP-RDT results. • Histidine-rich protein (HRP2/3) deletions can cause false-negative HRP-RDT results. • High parasitemia may give false-negative RDT results due to a prozone-like effect. • False-negative RDT results may also be a result of low parasite density. • Next-generation sequencing can elaborate the breakpoints of HRP2/3 deletions., Objectives Our objective was to systematically investigate false-negative histidine-rich protein 2 rapid diagnostic tests (HRP2-RDT) in imported Plasmodium falciparum malaria cases from travelers to the UK and the Republic of Ireland (RoI). Methods Five imported malaria cases in travellers returning to the UK and RoI from East Africa were reported to the PHE Malaria Reference Laboratory as negative according to histidine-rich protein (HRP2)-RDT. The cases were systematically investigated using microscopic, RDT, molecular, genomic, and in in vitro approaches. Results In each case, HRP2-RDT was negative, whereas microscopy confirmed the presence of P. falciparum. Further analysis revealed that the genes encoding HRP2 and HRP3 were deleted in three of the five cases. Whole-genome sequencing in one of these isolates confirmed deletions in P. falciparum chromosomes 8 and 13. Our study produced evidence that the fourth case, which had high parasitemia at clinical presentation, was a rare example of antigen saturation (‘prozone-like effect’), leading to a false negative in the HRP2-RDT, while the fifth case was due to low parasitemia. Conclusions False-negative HRP2-RDT results with P. falciparum are concerning. Our findings emphasise the necessity of supporting the interpretation of RDT results with microscopy, in conjunction with clinical observations, and sets out a systematic approach to identifying parasites carrying pfhrp2 and pfhrp3 deletions.

Several promising antimalarial drugs are currently being tested in human trials, such as artefenomel, cipargamin, ferroquine and ganaplacide. Many of these compounds were identified using high throughput screens against a single species of human malaria, Plasmodium falciparum, under the assumption that effectiveness against all malaria species will be similar, as has been observed for other antimalarial drugs. However, using our in vitro adapted line, we demonstrated recently that P. knowlesi is significantly less susceptible than P. falciparum to some new antimalarial drugs (e.g., cipargamin and DSM265), and more susceptible to others (e.g., ganaplacide). There is, therefore, an urgent need to determine the susceptibility profile of all human malaria species to the current generation of antimalarial compounds. We obtained ex vivo malaria samples from travellers returning to the United Kingdom and, using the [3H]hypoxanthine incorporation method, compared susceptibility to select established and experimental antimalarial agents among all major human infective Plasmodium species. We demonstrate that P. malariae and P. ovale spp. are significantly less susceptible than P. falciparum to cipargamin, DSM265 and AN13762, but are more susceptible to ganaplacide. Preliminary ex vivo data from single isolates of P. knowlesi and P. vivax demonstrate a similar profile. Our findings highlight the need to ensure cross species susceptibility profiles are determined early in the drug development pipeline. Our data can also be used to inform further drug development, and illustrate the utility of the P. knowlesi in vitro model as a scalable approach for predicting the drug susceptibility of non-falciparum malaria species in general., Graphical abstract Image 1, Highlights • P. knowlesi less susceptible than P. falciparum to some new antimalarials in vitro. • ex vivo malaria samples used to test susceptibility of other human malaria species. • P. malariae and P. ovale show similar drug susceptibility profile to P. knowlesi. • ex vivo drug screens from all malaria species can inform new drug development.

A recent randomized controlled trial, the WANECAM (West African Network for Clinical Trials of Antimalarial Drugs) trial, conducted at seven centers in West Africa, found that artemether-lumefantrine, artesunate-amodiaquine, pyronaridine-artesunate, and dihydroartemisinin-piperaquine all displayed good efficacy. However, artemether-lumefantrine was associated with a shorter interval between clinical episodes than the other regimens. In a further comparison of these therapies, we identified cases of persisting submicroscopic parasitemia by quantitative PCR (qPCR) at 72 h posttreatment among WANECAM participants from 5 sites in Mali and Burkina Faso, and we compared treatment outcomes for this group to those with complete parasite clearance by 72 h. Among 552 evaluable patients, 17.7% had qPCR-detectable parasitemia at 72 h during their first treatment episode. This proportion varied among sites, reflecting differences in malaria transmission intensity, but did not differ among pooled drug treatment groups. However, patients who received artemether-lumefantrine and were qPCR positive at 72 h were significantly more likely to have microscopically detectable recurrent Plasmodium falciparum parasitemia by day 42 than those receiving other regimens and experienced, on average, a shorter interval before the next clinical episode. Haplotypes of pfcrt and pfmdr1 were also evaluated in persisting parasites. These data identify a possible threat to the parasitological efficacy of artemether-lumefantrine in West Africa, over a decade since it was first introduced on a large scale.

Rapid diagnostic tests (RDTs) play a critical role in malaria diagnosis and control. The emergence of Plasmodium falciparum parasites that can evade detection by RDTs threatens control and elimination efforts. These parasites lack or have altered genes encoding histidine-rich proteins (HRPs) 2 and 3, the antigens recognized by HRP2-based RDTs. Surveillance of such parasites is dependent on identifying false-negative RDT results among suspected malaria cases, a task made more challenging during the current pandemic because of the overlap of symptoms between malaria and COVID-19, particularly in areas of low malaria transmission. Here, we share our perspective on the emergence of P. falciparum parasites lacking HRP2 and HRP3, and the surveillance needed to identify them amid the COVID-19 pandemic.

Ultrasensitive molecular diagnostics are lowering the limit of detection for malaria parasites in the blood and providing insights not captured by conventional tools such as microscopy and rapid antigen tests. Low-level malaria infections identified by molecular tools may influence clinical outcomes, transmission events, and elimination efforts. While many ultrasensitive molecular methods require well-equipped laboratories, technologies such as loop-mediated isothermal amplification and recombinase polymerase amplification provide more portable and analytically sensitive solutions. These tools may benefit asymptomatic patient screening, antenatal care, and elimination campaigns. We review the recent evidence, offer our perspective on the impact of these new tests, and identify future research priorities.

Background Many health facilities in malaria endemic countries are dependent on Rapid diagnostic tests (RDTs) for diagnosis and some National Health Service (NHS) hospitals without expert microscopists rely on them for diagnosis out of hours. The emergence of P. falciparum lacking the gene encoding histidine-rich protein 2 and 3 (HRP2 and HRP3) and escaping RDT detection threatens progress in malaria control and elimination. Currently, confirmation of RDT negative due to the deletion of pfhrp2 and pfhrp3, which encodes a cross-reactive protein isoform, requires a series of PCR assays. These tests have different limits of detection and many laboratories have reported difficulty in confirming the absence of the deletions with certainty. Methods We developed and validated a novel and rapid multiplex real time quantitative (qPCR) assay to detect pfhrp2, pfhrp3, confirmatory parasite and human reference genes simultaneously. We also applied the assay to detect pfhrp2 and pfhrp3 deletion in 462 field samples from different endemic countries and UK travellers. Results The qPCR assay demonstrated diagnostic sensitivity of 100% (n = 19, 95% CI= (82.3%; 100%)) and diagnostic specificity of 100% (n = 31; 95% CI= (88.8%; 100%)) in detecting pfhrp2 and pfhrp3 deletions. In addition, the assay estimates P. falciparum parasite density and accurately detects pfhrp2 and pfhrp3 deletions masked in polyclonal infections. We report pfhrp2 and pfhrp3 deletions in parasite isolates from Kenya, Tanzania and in UK travellers. Interpretation The new qPCR is easily scalable to routine surveillance studies in countries where P. falciparum parasites lacking pfhrp2 and pfhrp3 are a threat to malaria control.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Funder: Public Health England; doi: https://doi.org/10.13039/501100002141, Funder: European Developing Countries Trials Platform, Funder: Isaac Newton Trust, Funder: Alborada Fund, Funder: Global Health Innovative Technology Fund; doi: https://doi.org/10.13039/501100013996, Funder: Royal Society, New antimalarial therapeutics are needed to ensure that malaria cases continue to be driven down, as both emerging parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides threaten control programmes. Plasmodium, the apicomplexan parasite responsible for malaria, causes disease pathology through repeated cycles of invasion and replication within host erythrocytes (the asexual cycle). Antimalarial drugs primarily target this cycle, seeking to reduce parasite burden within the host as fast as possible and to supress recrudescence for as long as possible. Intense phenotypic drug screening efforts have identified a number of promising new antimalarial molecules. Particularly important is the identification of compounds with new modes of action within the parasite to combat existing drug resistance and suitable for formulation of efficacious combination therapies. Here we detail the antimalarial properties of DDD01034957—a novel antimalarial molecule which is fast-acting and potent against drug resistant strains in vitro, shows activity in vivo, and possesses a resistance mechanism linked to the membrane transporter PfABCI3. These data support further medicinal chemistry lead-optimization of DDD01034957 as a novel antimalarial chemical class and provide new insights to further reduce in vivo metabolic clearance.

Within the overlapping geographical ranges of P. knowlesi monkey hosts and vectors in Southeast Asia, an estimated 1.5 billion people are considered at risk of infection. P. knowlesi can cause severe disease and death, the latter associated with delayed treatment occurring from misdiagnosis. Although microscopy is a sufficiently sensitive first-line tool for P. knowlesi detection for most low-level symptomatic infections, misdiagnosis as other Plasmodium species is common, and the majority of asymptomatic infections remain undetected. Current point-of-care rapid diagnostic tests demonstrate insufficient sensitivity and poor specificity for differentiating P. knowlesi from other Plasmodium species. Molecular tools including nested, real-time, and single-step PCR, and loop-mediated isothermal amplification (LAMP), are sensitive for P. knowlesi detection. However, higher cost and inability to provide the timely point-of-care diagnosis needed to guide appropriate clinical management has limited their routine use in most endemic clinical settings. P. knowlesi is likely underdiagnosed across the region, and improved diagnostic and surveillance tools are required. Reference laboratory molecular testing of malaria cases for both zoonotic and non-zoonotic Plasmodium species needs to be more widely implemented by National Malaria Control Programs across Southeast Asia to accurately identify the burden of zoonotic malaria and more precisely monitor the success of human-only malaria elimination programs. The implementation of specific serological tools for P. knowlesi would assist in determining the prevalence and distribution of asymptomatic and submicroscopic infections, the absence of transmission in certain areas, and associations with underlying land use change for future spatially targeted interventions.

Background In Nigeria, indiscriminate use of antimalarial drugs may contribute to the threat of drug resistance, but this has not been evaluated among people living with human immunodeficiency virus (HIV). Methods HIV-positive adults attending a university hospital HIV clinic and HIV-negative adult volunteers from the university hospital community with a positive blood film were treated with artemether–lumefantrine. Parasite DNA from before and after treatment was polymerase chain reaction amplified to identify molecular markers of drug susceptibility. Results The pfcrt76T genotype was prevalent among both HIV-positive and HIV-negative participants (78.6% and 68.2%, respectively). Three new mutations in the pfmdr1 gene—F73S, S97L and G165R—and the uncommon pfdhps S436F variant were detected, whereas pfdhps K540E and pfdhfr I164L were absent. The A437G allele of pfdhps predominated (62/66 [94%]). The I431 V mutation was found in 19 of 66 pretreatment pfdhps sequences (28.8%). The pfmdr1 86N allele was significantly more common at day 3 post-treatment than at baseline (odds ratio 8.77 [95% confidence interval 1.21 to 380]). Conclusions We found evidence of continued chloroquine use among HIV-positive individuals. Selection for the pfmdr1 86N after artemether–lumefantrine treatment was observed, indicating a possible threat to antimalarial efficacy in the study area. The complexity of pfdhps haplotypes emphasises the need for careful monitoring of anti-folate susceptibility in Nigeria.

BackgroundRapid diagnostic tests (RDTs) that detect the malaria antigen histidine-rich protein 2 (HRP2) are widely used in endemic areas globally to confirm Plasmodium falciparum infection in febrile patients. The emergence of parasites lacking the gene encoding HRP2 and escaping RDT detection threatens progress in malaria control and elimination. Many health facilities in malaria endemic countries are dependent on RDTs for diagnosis and some National Health Service hospitals without expert microscopists rely on them for diagnosis out of hours. It is vital to study the emergence and the extent of such parasites globally to guide diagnostic policy. Currently, verification of the presence of such parasites in a blood sample requires a series of PCR assays to confirm the presence of P. falciparum and in the absence of amplicons from pfhrp2 and/or pfhrp3, which encodes a cross-reactive protein isoform. These tests have different limits of detection and many laboratories have reported difficulty in confirming the absence of pfhrp2 and pfhrp3 with certainty.MethodsWe developed and validated a novel and rapid multiplex real time quantitative (qPCR) assay to detect pfhrp2, pfhrp3, confirmatory parasite and human reference genes simultaneously. We also applied the assay to detect pfhrp2 and pfhrp3 deletion in 462 field samples from different endemic countries and UK travellers.ResultsThe qPCR assay showed limit of detection and quantification of 0.76-1.5 parasites per μl. The amplification efficiency, coefficient of determination (R2) and slope for the genes were 96-1.07%, 0.96-0.98 and −3.375 2 to −3.416 respectively. The assay demonstrated diagnostic sensitivity of 100% (n=19, 95% CI= (82.3%; 100%)) and diagnostic specificity of 100% (n=31; 95% CI= (88.8%; 100%)) in detecting pfhrp2 and pfhrp3 in. In addition, the qPCR assay estimates P. falciparum parasite density and can detect pfhrp2 and pfhrp3 deletions masked in polyclonal infections. We report pfhrp2 and pfhrp3 deletions in parasite isolates from Kenya, Tanzania and in UK travellers.ConclusionThe new qPCR assay is simple to use and offers significant advantages in speed and ease of interpretation. It is easily scalable to routine surveillance studies in countries where P. falciparum parasites lacking pfhrp2 and pfhrp3 are a threat to malaria control.

Treating malaria in HIV-coinfected individuals should consider potential drug-drug interactions. Artemether-lumefantrine is the most widely recommended treatment for uncomplicated malaria globally. Lumefantrine is metabolized by CYP3A4, an enzyme that commonly used antiretrovirals often induce or inhibit. A population pharmacokinetic meta-analysis was conducted using individual participant data from 10 studies with 6,100 lumefantrine concentrations from 793 nonpregnant adult participants (41% HIV-malaria-coinfected, 36% malaria-infected, 20% HIV-infected, and 3% healthy volunteers)., Treating malaria in HIV-coinfected individuals should consider potential drug-drug interactions. Artemether-lumefantrine is the most widely recommended treatment for uncomplicated malaria globally. Lumefantrine is metabolized by CYP3A4, an enzyme that commonly used antiretrovirals often induce or inhibit. A population pharmacokinetic meta-analysis was conducted using individual participant data from 10 studies with 6,100 lumefantrine concentrations from 793 nonpregnant adult participants (41% HIV-malaria-coinfected, 36% malaria-infected, 20% HIV-infected, and 3% healthy volunteers). Lumefantrine exposure increased 3.4-fold with coadministration of lopinavir-ritonavir-based antiretroviral therapy (ART), while it decreased by 47% with efavirenz-based ART and by 59% in the patients with rifampin-based antituberculosis treatment. Nevirapine- or dolutegravir-based ART and malaria or HIV infection were not associated with significant effects. Monte Carlo simulations showed that those on concomitant efavirenz or rifampin have 49% and 80% probability of day 7 concentrations

Background We assessed the efficacy of artemisinin-based combination therapies for treatment of uncomplicated falciparum malaria, with or without co-infecting Plasmodium spp., in Sumatera, Indonesia. Methods Febrile patients aged >6 months with uncomplicated P. falciparum were randomized to receive dihydroartemisinin-piperaquine or artemether-lumefantrine, plus single-dose primaquine, and were followed for 42 days. Mixed Plasmodium infections were included; P. vivax infections received 14 days of primaquine. We retrospectively restricted the analysis to cases with polymerase chain reaction (PCR)–confirmed parasitemia. Recurrent parasitemia in follow-up was identified by species-specific nested PCR. Results Of the 3731 participants screened, 302 were enrolled and randomized. In the dihydroartemisinin-piperaquine arm, P. falciparum infections were confirmed by PCR in 59 participants, with mixed infections in 23 (39.0%). In the artemether-lumefantrine arm, P. falciparum infections were confirmed by PCR in 55 participants, with mixed infections in 16 (29.0%). Both regimens were well tolerated, and symptoms improved rapidly in all treated participants. In the dihydroartemisinin-piperaquine arm, 1 P. falciparum recurrence (on day 7) and 6 P. malariae recurrences (1 had a mixed infection with P. falciparum) were identified during days 3–42 of follow-up. In the artemether-lumefantrine arm, 1 P. falciparum/P. malariae/P. vivax recurrence occurred on day 35. Submicroscopic persistence occurred during follow-up in 21 (37%) of 57 receiving dihydroartemisinin-piperaquine and 20 (39%) of 51 receiving artemether-lumefantrine. Conclusions In Sumatera, both regimens effectively cleared initial parasitemia, but P. falciparum and P. malariae persisted in some individuals. Molecular species detection should be deployed in antimalarial efficacy trials in Indonesia. Trial registration NCT02325180.

Although Plasmodium vivax parasites are the predominant cause of malaria outside of sub-Saharan Africa, they not always prioritised by elimination programmes. P. vivax is resilient and poses challenges through its ability to re-emerge from dormancy in the human liver. With observed growing drug-resistance and the increasing reports of life-threatening infections, new tools to inform elimination efforts are needed. In order to halt transmission, we need to better understand the dynamics of transmission, the movement of parasites, and the reservoirs of infection in order to design targeted interventions. The use of molecular genetics and epidemiology for tracking and studying malaria parasite populations has been applied successfully in P. falciparum species and here we sought to develop a molecular genetic tool for P. vivax. By assembling the largest set of P. vivax whole genome sequences (n = 433) spanning 17 countries, and applying a machine learning approach, we created a 71 SNP barcode with high predictive ability to identify geographic origin (91.4%). Further, due to the inclusion of markers for within population variability, the barcode may also distinguish local transmission networks. By using P. vivax data from a low-transmission setting in Malaysia, we demonstrate the potential ability to infer outbreak events. By characterising the barcoding SNP genotypes in P. vivax DNA sourced from UK travellers (n = 132) to ten malaria endemic countries predominantly not used in the barcode construction, we correctly predicted the geographic region of infection origin. Overall, the 71 SNP barcode outperforms previously published genotyping methods and when rolled-out within new portable platforms, is likely to be an invaluable tool for informing targeted interventions towards elimination of this resilient human malaria., Author summary Plasmodium vivax is the most widespread parasite causing human malaria, with more than one-third of the world’s population being at risk of infection. P. vivax is resilient due to its dormant liver phase, and there are increasing reports of drug-resistance and life-threatening infections. Despite this, P. vivax malaria is not always prioritised by elimination programmes. New molecular tools are needed to inform elimination efforts, including through better understanding the geographical source and outbreaks of P. vivax, thereby leading to the halting of transmission and the targeting of reservoirs of infection. Our work describes a 71 genetic marker barcode for P. vivax that has high predictive ability to identify the geographic origin, and has the potential to distinguish local transmission networks. If the 71 genetic marker barcode is implemented within new portable molecular platforms, it is likely to be an invaluable tool for informing targeted interventions towards elimination of this resilient human malaria.

Malaria infection during pregnancy, caused by the sequestering of Plasmodium falciparum parasites in the placenta, leads to high infant mortality and maternal morbidity. The parasite-placenta adherence mechanism is mediated by the VAR2CSA protein, a target for natural occurring immunity. Currently, vaccine development is based on its ID1-DBL2Xb domain however little is known about the global genetic diversity of the encoding var2csa gene, which could influence vaccine efficacy. In a comprehensive analysis of the var2csa gene in >2,000 P. falciparum field isolates across 23 countries, we found that var2csa is duplicated in high prevalence (>25%), African and Oceanian populations harbour a much higher diversity than other regions, and that insertions/deletions are abundant leading to an underestimation of the diversity of the locus. Further, ID1-DBL2Xb haplotypes associated with adverse birth outcomes are present globally, and African-specific haplotypes exist, which should be incorporated into vaccine design.

Management of uncomplicated malaria worldwide is threatened by the emergence in Asia of Plasmodium falciparum carrying variants of the pfk13 locus and exhibiting reduced susceptibility to artemisinin. Mutations in two other genes, ubp1 and ap2μ, are associated with artemisinin resistance in rodent malaria and with clinical failure of combination therapy in African malaria patients. Transgenic P. falciparum clones, each carrying orthologues of mutations in pfap2μ and pfubp1 associated with artemisinin resistance in Plasmodium chabaudi, were derived by Cas9 gene editing. Susceptibility to artemisinin and other antimalarial drugs was determined. Following exposure to 700 nM dihydroartemisinin in the ring-stage survival assay, we found strong evidence that transgenic parasites expressing the I592T variant (11% survival), but not the S160N variant (1% survival), of the AP2μ adaptin subunit were significantly less susceptible than the parental wild-type parasite population. The V3275F variant of UBP1, but not the V3306F variant, also displayed reduced susceptibility to dihydroartemisinin (8.5% survival versus 0.5% survival). AP2μ and UBP1 variants did not elicit reduced susceptibility to 48 h of exposure to artemisinin or to other antimalarial drugs. Therefore, variants of the AP2 adaptor complex μ-subunit and of the ubiquitin hydrolase UBP1 reduce in vitro artemisinin susceptibility at the early ring stage in P. falciparum These findings confirm the existence of multiple pathways to perturbation of either the mode of action of artemisinin, the parasite's adaptive mechanisms of resistance, or both. The cellular role of UBP1 and AP2μ in Plasmodium parasites should now be elucidated.

Antimalarial drug resistance is a substantial impediment to malaria control. The spread of resistance has been described using genetic markers which are important epidemiological tools. We carried out a temporal analysis of changes in allele frequencies of 12 drug resistance markers over two decades of changing antimalarial drug policy in Kenya. We did not detect any of the validated kelch 13 (k13) artemisinin resistance markers, nonetheless, a single k13 allele, K189T, was maintained at a stable high frequency (>10%) over time. There was a distinct shift from chloroquine resistant transporter (crt)-76, multi-drug resistant gene 1 (mdr1)-86 and mdr1-1246 chloroquine (CQ) resistance alleles to a 99% prevalence of CQ sensitive alleles in the population, following the withdrawal of CQ from routine use. In contrast, the dihydropteroate synthetase (dhps) double mutant (437G and 540E) associated with sulfadoxine-pyrimethamine (SP) resistance was maintained at a high frequency (>75%), after a change from SP to artemisinin combination therapies (ACTs). The novel cysteine desulfurase (nfs) K65 allele, implicated in resistance to lumefantrine in a West African study, showed a gradual significant decline in allele frequency pre- and post-ACT introduction (from 38% to 20%), suggesting evidence of directional selection in Kenya, potentially not due to lumefantrine. The high frequency of CQ-sensitive parasites circulating in the population suggests that the re-introduction of CQ in combination therapy for the treatment of malaria can be considered in the future. However, the risk of a re-emergence of CQ resistant parasites circulating below detectable levels or being reintroduced from other regions remains.

Many rapid diagnostic tests (RDT) used on suspected malaria cases are based on the detection of the protein encoded by the Plasmodium falciparum histidine-rich protein-2 (pfhrp2) gene, which shares a high sequence homology with pfhrp3 in the 3D7 reference genome. Parasite isolates showing pfhrp2 and pfhrp3 gene deletions have been emerging over the years, but a comprehensive genetic analysis of these variants is still lacking. With this purpose, genomic data from experimental P. falciparum genetic crosses between different laboratory lines (3D7, HB3, DD2, 7G8 and GB4) were first analysed (n = 98). The frequency of pfhrp2 deletions was consistent with a Mendelian prediction in HB3 × DD2 (56.7%; 95%CI = (39.5%-72.9%)). Moreover, the pfhrp2 and pfhrp3 deletions segregated independently of each other in the same genetic cross. Analysis of 3D7 × HB3 and 7G8 × GB4 estimated the probability of spontaneously generating a pfhrp2 deletion during sexual recombination to be up to 6.2%. Next, whole genome sequence data from 1970 P. falciparum isolates collected globally were analysed. Nine samples displayed depth of coverage consistent with pfhrp2 deletions (0.5%), but the corresponding split-read analysis could not confirm deletions in seven of these samples. Twenty-eight isolates had evidence of pfhrp3 deletions (1.4%), which are widespread in Southeast Asia. Finally, a meta-analysis of published data revealed a positive mean association between the frequencies of pfhrp2 and pfhrp3 deletions in Africa and South America. This result suggested a shared selective pressure acting on these genetic variants. In conclusion, evidence of genetic selection on both pfhrp2 and pfhrp3 deletions was presented, but experimental crosses do not provide evidence of a fitness cost of these variants. Further work is urgently needed to accurately determine the prevalence and the degree of association between these genetic variants, and the respective impact on diagnostic accuracy of many in-use RDT.

Plasmodium knowlesi, a common parasite of macaques, is recognised as a significant cause of human malaria in Malaysia. The P. knowlesi A1H1 line has been adapted to continuous culture in human erythrocytes, successfully providing an in vitro model to study the parasite. We have assembled a reference genome for the PkA1-H.1 line using PacBio long read combined with Illumina short read sequence data. Compared with the H-strain reference, the new reference has improved genome coverage and a novel description of methylation sites. The PkA1-H.1 reference will enhance the capabilities of the in vitro model to improve the understanding of P. knowlesi infection in humans.

Much attention has been directed toward a recent slowing of progress in malaria control worldwide, with particular emphasis on growing evidence which suggests that the twin pillars of malaria reduc...

Drug resistance is a recurrent problem in the fight against malaria. Genetic and epidemiological surveillance of antimalarial resistant parasite alleles is crucial to guide drug therapies and clinical management. New antimalarial compounds are currently at various stages of clinical trials and regulatory evaluation. Using ∼2000 Plasmodium falciparum genome sequences, we investigated the genetic diversity of eleven gene-targets of promising antimalarial compounds and assessed their potential efficiency across malaria endemic regions. We determined if the loci are under selection prior to the introduction of new drugs and established a baseline of genetic variance, including potential resistant alleles, for future surveillance programmes., Graphical abstract Image 1, Highlights • Genetic diversity of 11gene-targets of new antimalarial compounds was investigated. • Genetic data was analysed for 18 malaria endemic countries. • No signals of selective pressure was observed. • A mutation linked to resistance to a new antimalarial was found in African parasites. • Amino-acid changes close to reported antimalarial resistant mutations were detected.

Summary Indonesia is aiming for malaria elimination by 2030. A parasitological survey of 3731 individuals in North Sumatera province used polymerase chain reaction to identify 1169 with parasitemia and highly specific amplification of sicavar genes to identify Plasmodium knowlesi infection in 377., Background. As Indonesia works toward the goal of malaria elimination, information is lacking on malaria epidemiology from some western provinces. As a basis for studies of antimalarial efficacy, we set out to survey parasite carriage in 3 communities in North Sumatera Province. Methods. A combination of active and passive detection of infection was carried out among communities in Batubara, Langkat, and South Nias regencies. Finger-prick blood samples from consenting individuals of all ages provided blood films for microscopic examination and blood spots on filter paper. Plasmodium species were identified using nested polymerase chain reaction (PCR) of ribosomal RNA genes and a novel assay that amplifies a conserved sequence specific for the sicavar gene family of Plasmodium knowlesi. Results. Of 3731 participants, 614 (16.5%) were positive for malaria parasites by microscopy. PCR detected parasite DNA in samples from 1169 individuals (31.3%). In total, 377 participants (11.8%) harbored P. knowlesi. Also present were Plasmodium vivax (14.3%), Plasmodium falciparum (10.5%) and Plasmodium malariae (3.4%). Conclusions. Amplification of sicavar is a specific and sensitive test for the presence of P. knowlesi DNA in humans. Subpatent and asymptomatic multispecies parasitemia is relatively common in North Sumatera, so PCR-based surveillance is required to support control and elimination activities.

Tackling relapsing Plasmodium vivax and zoonotic Plasmodium knowlesi infections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressing P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.

Tackling relapsingPlasmodium vivaxand zoonoticPlasmodium knowlesiinfections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adaptedP. knowlesistrain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressingP. vivaxDuffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism ofP. knowlesi. Critically, antibodies raised against theP. vivaxantigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.

Summary Background Seasonal malaria chemoprevention (SMC) aims to prevent malaria in children during the high malaria transmission season. The Achieving Catalytic Expansion of SMC in the Sahel (ACCESS-SMC) project sought to remove barriers to the scale-up of SMC in seven countries in 2015 and 2016. We evaluated the project, including coverage, effectiveness of the intervention, safety, feasibility, drug resistance, and cost-effectiveness. Methods For this observational study, we collected data on the delivery, effectiveness, safety, influence on drug resistance, costs of delivery, impact on malaria incidence and mortality, and cost-effectiveness of SMC, during its administration for 4 months each year (2015 and 2016) to children younger than 5 years, in Burkina Faso, Chad, The Gambia, Guinea, Mali, Niger, and Nigeria. SMC was administered monthly by community health workers who visited door-to-door. Drug administration was monitored via tally sheets and via household cluster-sample coverage surveys. Pharmacovigilance was based on targeted spontaneous reporting and monitoring systems were strengthened. Molecular markers of resistance to sulfadoxine–pyrimethamine and amodiaquine in the general population before and 2 years after SMC introduction was assessed from community surveys. Effectiveness of monthly SMC treatments was measured in case-control studies that compared receipt of SMC between patients with confirmed malaria and neighbourhood-matched community controls eligible to receive SMC. Impact on incidence and mortality was assessed from confirmed outpatient cases, hospital admissions, and deaths associated with malaria, as reported in national health management information systems in Burkina Faso and The Gambia, and from data from selected outpatient facilities (all countries). Provider costs of SMC were estimated from financial costs, costs of health-care staff time, and volunteer opportunity costs, and cost-effectiveness ratios were calculated as the total cost of SMC in each country divided by the predicted number of cases averted. Findings 12 467 933 monthly SMC treatments were administered in 2015 to a target population of 3 650 455 children, and 25 117 480 were administered in 2016 to a target population of 7 551 491. In 2015, among eligible children, mean coverage per month was 76·4% (95% CI 74·0–78·8), and 54·5% children (95% CI 50·4–58·7) received all four treatments. Similar coverage was achieved in 2016 (74·8% [72·2–77·3] treated per month and 53·0% [48·5–57·4] treated four times). In 779 individual case safety reports over 2015–16, 36 serious adverse drug reactions were reported (one child with rash, two with fever, 31 with gastrointestinal disorders, one with extrapyramidal syndrome, and one with Quincke's oedema). No cases of severe skin reactions (Stevens-Johnson or Lyell syndrome) were reported. SMC treatment was associated with a protective effectiveness of 88·2% (95% CI 78·7–93·4) over 28 days in case-control studies (2185 cases of confirmed malaria and 4370 controls). In Burkina Faso and The Gambia, implementation of SMC was associated with reductions in the number of malaria deaths in hospital during the high transmission period, of 42·4% (95% CI 5·9 to 64·7) in Burkina Faso and 56·6% (28·9 to 73·5) in The Gambia. Over 2015–16, the estimated reduction in confirmed malaria cases at outpatient clinics during the high transmission period in the seven countries ranged from 25·5% (95% CI 6·1 to 40·9) in Nigeria to 55·2% (42·0 to 65·3) in The Gambia. Molecular markers of resistance occurred at low frequencies. In individuals aged 10–30 years without SMC, the combined mutations associated with resistance to amodiaquine (pfcrt CVIET haplotype and pfmdr1 mutations [86Tyr and 184Tyr]) had a prevalence of 0·7% (95% CI 0·4–1·2) in 2016 and 0·4% (0·1–0·8) in 2018 (prevalence ratio 0·5 [95% CI 0·2–1·2]), and the quintuple mutation associated with resistance to sulfadoxine–pyrimethamine (triple mutation in pfdhfr and pfdhps mutations [437Gly and 540Glu]) had a prevalence of 0·2% (0·1–0·5) in 2016 and 1·0% (0·6–1·6) in 2018 (prevalence ratio 4·8 [1·7–13·7]). The weighted average economic cost of administering four monthly SMC treatments was US$3·63 per child. Interpretation SMC at scale was effective in preventing morbidity and mortality from malaria. Serious adverse reactions were rarely reported. Coverage varied, with some areas consistently achieving high levels via door-to-door campaigns. Markers of resistance to sulfadoxine–pyrimethamine and amodiaquine remained uncommon, but with some selection for resistance to sulfadoxine–pyrimethamine, and the situation needs to be carefully monitored. These findings should support efforts to ensure high levels of SMC coverage in west and central Africa. Funding Unitaid.

Despite concerted efforts to eliminate malaria, it remains a major global cause of morbidity and mortality with over 200 million annual cases. Significant gains have been made, with the annual global malaria incidence and mortality halving over the past twenty years, using tools such as long-lasting insecticide-treated bed nets and artemisinin-based therapies. Malaria is also a significant cause of life-threatening imported infection in the UK. It is vital for front line clinical staff involved in the assessment of acutely ill patients to be aware of the need for early diagnostic testing, malaria epidemiology, markers of severe infection and developments in antimalarial treatments to optimise patient management. The difference between a good and poor outcome is early diagnosis and treatment. Many of the challenges faced in the quest for global eradication, such as availability of appropriate diagnostic tests, and drug and insecticide resistance could also have future implications for imported malaria.

Most malaria-endemic countries are heavily reliant upon rapid diagnostic tests (RDT) for malaria case identification and treatment. RDT previously used for malaria diagnosis can subsequently be used for molecular assays, including qualitative assessment of parasite species present or the carriage of resistance markers, because parasite DNA can be extracted from the blood inside the RDT which remains preserved on the internal components. However, the quantification of parasite density has not previously been possible from used RDT. In this study, blood samples were collected from school-age children in Western Kenya, in the form of both dried blood spots on Whatman filter paper, and the blood spot that is dropped into rapid diagnostic tests during use. Having first validated a robotic DNA extraction method, the parasite density was determined from both types of sample by duplex qPCR, and across a range of densities. The methods showed good agreement. The preservation of both parasite and human DNA on the nitrocellulose membrane inside the RDT was stable even after more than one year’s storage. This presents a useful opportunity for researchers or clinicians wishing to gain greater information about the parasite populations that are being studied, without significant investment of resources.

Background Human ovale malaria is caused by the two closely related species, Plasmodium ovale curtisi and P. ovale wallikeri. Both species are known to relapse from quiescent hepatic forms months or years after the primary infection occurred. Although some studies have succeeded in establishing mosquito transmission for ovale malaria, none have specifically described transmission and human hepatocyte infection of both sibling species. Methods Here we describe a simplified protocol for successful transmission of both P. ovale curtisi and P. ovale wallikeri to Anopheles coluzzii mosquitoes and streamlined monitoring of infection using sensitive parasite DNA detection, by loop-activated amplification, in blood-fed mosquitoes. Results In one experimental infection with P. ovale curtisi and one with P. ovale wallikeri, viable sporozoites were isolated from mosquito salivary glands and used to successfully infect cultured human hepatocytes. Conclusions This protocol provides a method for the utilisation of pretreatment clinical blood samples from ovale malaria patients, collected in EDTA, for mosquito infection studies and generation of the hepatic life cycle stages of P. ovale curtisi and P. ovale wallikeri. We also demonstrate the utility of loop-activated amplification as a rapid and sensitive alternative to dissection for estimating the prevalence of infection in Anopheles mosquitoes fed with Plasmodium-infected blood.

Malaria infections remain a serious global health problem in the world, particularly among children and pregnant women in Sub-Saharan Africa. Moreover, malaria control and elimination is hampered by rapid development of resistance by the parasite and the vector to commonly used antimalarial drugs and insecticides, respectively. Therefore, vaccine-based strategies are sorely needed, including those designed to interrupt disease transmission. However, a prerequisite for such a vaccine strategy is the understanding of both the human and vector immune responses to parasite developmental stages involved in parasite transmission in both man and mosquito. Here, we review the naturally acquired humoral and cellular responses to sexual stages of the parasite while in the human host and the Anopheles vector. In addition, updates on current anti-gametocyte, anti-gamete, and anti-mosquito transmission blocking vaccines are given. We conclude with our views on some important future directions of research into P. falciparum sexual stage immunity relevant to the search for the most appropriate transmission-blocking vaccine.

Vaccines that target Plasmodium falciparum gametocytes have the potential to reduce malaria transmission and are thus attractive targets for malaria control. However, very little is known about human immune responses to gametocytes present in human hosts. We evaluated naturally-acquired antibodies to gametocyte-infected erythrocytes (gametocyte-IEs) of different developmental stages compared to other asexual parasite stages among naturally-exposed Kenyan residents. We found that acquired antibodies strongly recognized the surface of mature asexual-IEs, but there was limited reactivity to the surface of gametocyte-IEs of different stages. We used genetically-modified P. falciparum with suppressed expression of PfEMP1, the major surface antigen of asexual-stage IEs, to demonstrate that PfEMP1 is a dominant target of antibodies to asexual-IEs, in contrast to gametocyte-IEs. Antibody reactivity to gametocyte-IEs was similar to asexual-IEs lacking PfEMP1. Significant antibody reactivity to the surface of gametocytes was observed when outside of the host erythrocyte, including recognition of the major gametocyte antigen, Pfs230. This indicates that there is a deficiency of acquired antibodies to gametocyte-IEs despite the acquisition of antibodies to gametocyte antigens and asexual IEs. Our findings suggest that the acquisition of substantial immunity to the surface of gametocyte-IEs is limited, which may facilitate immune evasion to enable malaria transmission even in the face of substantial host immunity to malaria. Further studies are needed to understand the basis for the limited acquisition of antibodies to gametocytes and whether vaccine strategies can generate substantial immunity.

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.

Background Plasmodium ovale spp. and P. malariae cause illness in endemic regions and returning travellers. Far less is known about these species than P. falciparum and P. vivax. Methods The UK national surveillance data, collected 1987 to 2015, were collated with the International Passenger Survey and climatic data to determine geographical, temporal and seasonal trends of imported P. ovale spp. and P. malariae infection. Results Of 52,242 notified cases of malaria, 6.04% (3157) were caused by P. ovale spp. and 1.61% (841) by P. malariae; mortality was 0.03% (1) and 0.12% (1), respectively. Almost all travellers acquired infection in West or East Africa. Infection rate per travel episode fell fivefold during the study period. The median latency of P. malariae and P. ovale spp. was 18 and 76 days, respectively; delayed presentation occurred with both species. The latency of P. ovale spp. infection imported from West Africa was significantly shorter in those arriving in the UK during the West African peak malarial season compared to those arriving outside it (44 days vs 94 days, p Conclusion In West Africa, where malaria transmission is highly seasonal, P. ovale spp. may have evolved to relapse during the malarial high transmission season. This has public health implications. Deaths are very rare, supporting current guidelines emphasising outpatient treatment. However, late presentations do occur.

Plasmodium falciparum parasites resistant to antimalarial treatments have hindered malaria disease control. Sulfadoxine-pyrimethamine (SP) was used globally as a first-line treatment for malaria after wide-spread resistance to chloroquine emerged and, although replaced by artemisinin combinations, is currently used as intermittent preventive treatment of malaria in pregnancy and in young children as part of seasonal malaria chemoprophylaxis in sub-Saharan Africa. The emergence of SP-resistant parasites has been predominantly driven by cumulative build-up of mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) genes, but additional amplifications in the folate pathway rate-limiting pfgch1 gene and promoter, have recently been described. However, the genetic make-up and prevalence of those amplifications is not fully understood. We analyse the whole genome sequence data of 4,134 P. falciparum isolates across 29 malaria endemic countries, and reveal that the pfgch1 gene and promoter amplifications have at least ten different forms, occurring collectively in 23% and 34% in Southeast Asian and African isolates, respectively. Amplifications are more likely to be present in isolates with a greater accumulation of pfdhfr and pfdhps substitutions (median of 1 additional mutations; P, Author summary Malaria causes approximately 435,000 deaths per year, concentrated in sub-Saharan Africa and among children under the age of five years. Global efforts to control and eliminate malaria are hampered by the emergence of Plasmodium falciparum malaria parasites resistant to currently available antimalarial drugs. Sulfadoxine-pyrimethamine (SP) was used globally as a first-line treatment for malaria and, although replaced by artemisinin combinations, is still used for the prevention of malarial disease in vulnerable groups (e.g. pregnant women). SP resistance is caused by mutations in the P. falciparum parasite genes pfdhfr and pfdhps, but recently novel structural variants in and around the pfgch1 gene have been described. By analysing genome sequence data of 4,134 P. falciparum across 29 malaria endemic countries, we establish there are at least ten different pfgch1 structural variants, existing in the presence of pfdhfr and pfdhps mutations, and occur increasingly and at high frequency in some Southeast Asian and African populations. These pfgch1 structural variants may enhance the survival of those parasites with pfdhfr and pfdhps substitutions, potentially threatening the efficacy of SP for prevention of malaria in vulnerable groups. Therefore it is important that they are monitored in molecular surveillance studies. Our work will assist epidemiological studies, laboratory and surveillance activities looking at the diversity and role of pfgch1 structural variants and their prevalence across malaria endemic regions, especially in countries using SP regimens.

This study primarily aimed to identify the causative species of cutaneous leishmaniasis (CL) in the Khyber Pakhtunkhwa Province of Pakistan and to distinguish any species-specific variation in clinical manifestation of CL. Diagnostic performance of different techniques for identifying CL was assessed. Isolates of Leishmania spp. were detected by in vitro culture, polymerase chain reaction (PCR) on DNA extracted from dried filter papers and microscopic examination of direct lesion smears from patients visiting three major primary care hospitals in Peshawar. A total of 125 CL patients were evaluated. Many acquired the disease from Peshawar and the neighboring tribal area of Khyber Agency. Military personnel acquired CL while deployed in north and south Waziristan. Leishmania tropica was identified as the predominant infecting organism in this study (89.2%) followed by Leishmania major (6.8%) and, unexpectedly, Leishmania infantum (4.1%). These were the first reported cases of CL caused by L. infantum in Pakistan. PCR diagnosis targeting kinetoplast DNA was the most sensitive diagnostic method, identifying 86.5% of all samples found positive by any other method. Other methods were as follows: ribosomal DNA PCR (78.4%), internal transcribed spacer 2 region PCR (70.3%), culture (67.1%), and microscopy (60.5%). Clinical examination reported 14 atypical forms of CL. Atypical lesions were not significantly associated with the infecting Leishmania species, nor with “dry” or “wet” appearance of lesions. Findings from this study provide a platform for species typing of CL patients in Pakistan, utilizing a combination of in vitro culture and molecular diagnostics. Moreover, the clinical diversity described herein can benefit clinicians in devising differential diagnosis of the disease.

There are few published reports of mutations in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes in P. falciparum populations in Nigeria, but one previous study has recorded a novel dhps mutation at codon 431 among infections imported to the United Kingdom from Nigeria. To assess how widespread this mutation is among parasites in different parts of the country and consequently fill the gap in sulfadoxine-pyrimethamine (SP) resistance data in Nigeria, we retrospectively analysed 1000 filter paper blood spots collected in surveys of pregnant women and children with uncomplicated falciparum malaria between 2003 and 2015 from four sites in the south and north. Genomic DNA was extracted from filter paper blood spots and placental impressions. Point mutations at codons 16, 50, 51, 59, 108, 140 and 164 of the dhfr gene and codons 431, 436, 437, 540, 581 and 613 of the dhps gene were evaluated by nested PCR amplification followed by direct sequencing. The distribution of the dhps-431V mutation was widespread throughout Nigeria with the highest prevalence in Enugu (46%). In Ibadan where we had sequential sampling, its prevalence increased from 0% to 6.5% between 2003 and 2008. Although there were various combinations of dhps mutations with 431V, the combination 431V + 436A + 437G+581G+613S was the most common. All these observations support the view that dhps-431V is on the increase. In addition, P. falciparum DHPS crystal structure modelling shows that the change from Isoleucine to Valine (dhps-431V) could alter the effects of both S436A/F and A437G, which closely follow the 2nd β-strand. Consequently, it is now a research priority to assess the implications of dhps-VAGKGS mutant haplotype on continuing use of SP in seasonal malaria chemoprevention (SMC) and intermittent preventive treatment in pregnancy (IPTp). Our data also provides surveillance data for SP resistance markers in Nigeria between 2003 and 2015., Graphical abstract Image 1, Highlights • We present data on dhps and dhfr mutations in P. falciparum populations in Nigeria. • Increased prevalence of I431V mutation was seen between 2003 and 2015 from 0 to 36%. • The 431V + 436A + 437G + 581G + 613S was the most common with dhps-431V mutation. • Crystal structure modelling of Pf DHPS shows that 431Vcould alter S436A and A437G.

Several species of the genus Leishmania are causative agents of cutaneous leishmaniasis in Pakistan. This study aimed to determine phylogenetic placement of Leishmania species causing cutaneous leishmaniasis in Khyber Pakhtunkhwa province, Pakistan (34 Leishmania tropica, 3 Leishmania infantum), in-relation to species from other geographical areas using gene sequences encoding cytochrome b (cytb) and internal transcribed spacer 2 (its2). Based on cytochrome b sequence analysis, L. tropica strains from Pakistan and other geographical regions were differentiated into two genotype groups, A and B. Within the province, five distinct L. tropica genotypes were recognized; two in group A, three in group B. Two L. infantum isolates from the province were closely associated with both Afro-Eurasian and American species of the Leishmania donovani complex, including Leishmania chagasi, L. infantum and L. donovani from Sudan and Ethiopia; while a third L. infantum isolate could not be differentiated from visceralizing Kenyan and Indian L. donovani. We observed apposite phylogenetic placement of CL-causing L. tropica and L. infantum from Khyber Pakhtunkhwa. Affinities ascribed to Leishmania spp. From the region are valuable in tracing potential importation of leishmaniasis.

Malaria case management across endemic regions of the globe relies on artemisinin (ART) combination therapies for the rapid treatment of acute Plasmodium infection and prevention of severe disease. However, the efficacy of combination therapy is threatened by reduced sensitivity of Plasmodium falciparum to ART and partner drugs, which, in turn, may compromise the progress of control and elimination campaigns. Clinically, this phenomenon is most prevalent in Southeast Asia, where it manifests in vivo as measurably slower clearance of parasitemia (1⇓–3). There are also limited reports of patients with recrudescent infections of African origin (4⇓–6); however, slow clearance, as measured by the microscopic criterion deployed in the Greater Mekong region, is not present. Much effort has gone into understanding the genetic basis of ART susceptibility. After the development of an in vitro correlate of clinical ART susceptibility by Witkowski et al. (7), it was demonstrated that variants in the pfk13 locus mediate the slow-clearance phenotype in Southeast Asia (8). The genetic diversity of this locus worldwide has since been studied extensively in vivo (9⇓⇓–12) and in vitro (7, 11). Further, reverse genetic approaches with genome editing technologies, including zinc-finger nucleases and CRISPR-Cas9, have validated the involvement of these pfkelch13 mutations in ring-stage ART resistance (12, 13). Now, in PNAS, Demas et al. (14) report and validate the role of the actin-binding protein Coronin in reducing P. falciparum ART susceptibility in vitro. Conventional approaches to evolving and studying parasite resistance to ART have been challenging. Unlike other clinically approved antimalarial drugs, ART does not seem to inhibit a single target or pathway in the cell. Instead, the drug rapidly and promiscuously oxidizes intracellular material, overwhelming the parasite cell’s damage-response machinery (15). Despite its disseminated action, ART derivates are fully metabolized … [↵][1]1To whom correspondence should be addressed. Email: colin.sutherland{at}lshtm.ac.uk. [1]: #xref-corresp-1-1

Plasmodium falciparum gametocytes develop over 9-12 days while sequestered in deep tissues. On emergence into the bloodstream, they circulate for varied amounts of time during which certain host factors might influence their further development. We aimed to evaluate the potential association of patient clinical parameters with gametocyte development and carriage via in vivo methods. Seventy-two patients were enrolled from three hospitals in the Volta region of Ghana in 2016. Clinical parameters were documented for all patients, and gametocyte prevalence by microscopy was estimated at 12.5%. By measuring RNA transcripts representing two distinct gametocyte developmental stages using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), we obtained a more precise estimate of gametocyte carriage while also inferring gametocyte maturation. Fifty-three percent of the study participants harbored parasites expressing transcripts of the immature gametocyte-specific gene (PF3D7_1477700), whereas 36% harbored PF3D7_1438800 RNA-positive parasites, which is enriched in mid and mature gametocytes, suggesting the presence of more immature stages. Linear logistic regression showed that patients older than 5 years but less than 16 years were more likely to carry gametocytes expressing both PF3D7_1477700 and PF3D7_1438800 compared with younger participants, and gametocytemia was more likely in mildly anemic individuals compared with those with severe/moderate anemia. These data provide further evidence that a greater number of malaria patients harbor gametocytes than typically estimated by microscopy and suggest a possible association between age, fever, anemia, and gametocytemia.

Summary Background Artemether–lumefantrine and artesunate–amodiaquine are used as first-line artemisinin-based combination therapies (ACTs) in west Africa. Pyronaridine–artesunate and dihydroartemisinin–piperaquine are potentially useful for diversification of ACTs in this region, but further safety and efficacy data are required on malaria retreatment. Methods We did a randomised, multicentre, open-label, longitudinal, controlled phase 3b/4 clinical trial at seven tertiary centres in Burkina Faso, Guinea, and Mali. Eligible participants for first malaria episode and all retreatment episodes were adults and children aged 6 months and older with microscopically confirmed Plasmodium spp malaria (>0 to

Malaria parasites (Plasmodium) can change the attractiveness of their vertebrate hosts to Anopheles vectors, leading to a greater number of vector-host contacts and increased transmission. Indeed, naturally Plasmodium-infected children have been shown to attract more mosquitoes than parasite-free children. Here, we demonstrate Plasmodium-induced increases in the attractiveness of skin odor in Kenyan children, and reveal quantitative differences in the production of specific odor components in infected, versus parasite-free, individuals. We found the aldehydes heptanal, octanal and nonanal to be produced in greater amounts by infected individuals, and detected by mosquito antennae. In behavioral experiments, we demonstrated that these, and other, Plasmodium-induced aldehydes enhanced the attractiveness of a synthetic odor blend mimicking ‘healthy’ human odor. Heptanal alone increased the attractiveness of ‘parasite-free’ natural human odor. Should the increased production of these aldehydes by Plasmodium-infected humans lead to increased mosquito biting in a natural setting, this would likely affect the transmission of malaria.

The WHO recommends that children living in areas of highly seasonal malaria transmission in the Sahel subregion should receive seasonal malaria chemoprevention (SMC) with sulfadoxine-pyrimethamine plus amodiaquine (SPAQ). We evaluated the use of dihydroartemisinin-piperaquine (DHAPQ) as an alternative drug that could be used if SPAQ starts to lose efficacy. A total of 1,499 children 3 to 59 months old were randomized to receive SMC with SPAQ or DHAPQ over 3 months. The primary outcome measure was the risk of clinical malaria (fever or a history of fever with a parasite density of at least 3,000/μl). A cohort of 250 children outside the trial was followed up as a control group. Molecular markers of drug resistance were assessed. The risk of a malaria attack was 0.19 in the DHAPQ group and 0.15 in the SPAQ group, an odds ratio of 1.33 (95% confidence interval [CI], 1.02 to 1.72). Efficacy of SMC compared to the control group was 77% (67% to 84%) for DHAPQ and 83% (74% to 89%) for SPAQ. pfdhfr and pfdhps mutations associated with antifolate resistance were more prevalent in parasites from children who received SPAQ than in children who received DHAPQ. Both regimens were highly efficacious and well tolerated. DHAPQ is a potential alternative drug for SMC. (This trial is registered at ClinicalTrials.gov under registration no. NCT00941785.)

In this study, in vitro anti-leishmanial activity of buparvaquone was evaluated against promastigotes and intracellular amastigotes of Pakistani Leishmania tropica isolate KWH23 in relation to the current standard chemotherapy for leishmaniasis (sodium stibogluconate, sodium stibogluconate, amphotericin B and miltefosine). For buparvaquone, mean % inhibition in intracellular amastigotes at four different concentrations (1.35 µM, 0.51 µM, 0.17 µM and 0.057 µM) was 78%, 44%, 20% and 14% respectively, whereas, against promastigotes it was 89%, 77%, 45% and 35% respectively. IC50 values calculated to estimate the anti-leishmanial activity of buparvaquone against intra-cellular amastigotes and promastigotes was 0.53 µM (95% C.I. = 0.32-0.89) and 0.15 µM (95% C.I. = 0.01-1.84) respectively. Amphotericin B was the most potent in-vitro drug tested, with an IC50 of 0.075 µM (95% C.I. = 0.006-0.907) against promastigotes, and 0.065 µM (95% C.I. = 0.048-0.089) against intra-cellular amastigotes. Amphotericin B was more cytotoxic against THP1 cells, with an IC50 of 0.15 µM (95% C.I. = 0.01-0.95) and an apparent in-vitro therapeutic index of 2.0, than was buparvaquone, with an IC50 of 12.03 µM (95% C.I. = 5.36-26.96) against THP1 cells and a therapeutic index of 80.2. The study proposes that buparvaquone may be further investigated as a candidate drug for treatment of cutaneous leishmaniasis.

The vast majority of malaria patients worldwide are currently treated with combination therapy comprising one of the artemisinin family of drugs, characterised by rapid action and short plasma half-life, co-formulated with a longer-lasting drug from the amino aryl-alcohol or quinoline families. There is now a widely perceived threat to treatment efficacy, as reduced susceptibility to rapid artemisinin clearance in vivo has become prevalent among populations of Plasmodium falciparum in the Greater Mekong subregion since 2008. In vitro and in vivo drug selection studies, heterologous cell expression experiments and genetic epidemiology have identified many candidate markers of reduced ring-stage susceptibility to artemisinin. Certain variants of the P. falciparum pfk13 gene, which encodes a kelch domain protein implicated in the unfolded protein response, are strongly associated with slow parasite clearance by artemisinin in the Mekong subregion. However, anomalies in the epidemiological association of pfk13 variants with true treatment failure in vivo and the curious cell-cycle stage specificity of this phenotype in vitro warrant exploration in some depth. Taken together, available data suggest that the emergence of P. falciparum expressing K13 variants has not yet precipitated a public health emergency. Alternative candidate markers of artemisinin susceptibility are also described, as K13-independent treatment failure has been observed in African P. falciparum and in the rodent malaria parasite Plasmodium chabaudi.

Leishmania tropica is the causative agent of cutaneous leishmaniasis in Pakistan. Here, intraspecific diversity of L. tropica from northern Pakistan was investigated using multilocus microsatellite typing. Fourteen polymorphic microsatellite markers were typed in 34 recently collected L. tropica isolates from Pakistan along with 158 archival strains of diverse Afro-Eurasian origins. Previously published profiles for 145 strains of L. tropica originating from different regions of Africa, Central Asia, Iran, and Middle East were included for comparison. Six consistently well-supported genetic groups were resolved: 1) Asia, 2) Morroco A, 3) Namibia and Kenya A, 4) Kenya B/Tunisia and Galilee, 5) Morocco B, and 6) Middle East. Strains from northern Pakistan were assigned to Asian cluster except for three that were placed in a geographically distant genetic group; Morocco A. Lesion variability among these Pakistani strains was not associated with specific L. tropica genetic profile. Pakistani strains showed little genetic differentiation from strains of Iraq, Afghanistan, and Syria (FST = 0.00–0.06); displayed evidence of modest genetic flow with India (FST = 0.14). Furthermore, genetic structuring within these isolates was not geographically defined. Pak–Afghan cluster was in significant linkage disequilibrium (IA = 1.43), had low genetic diversity, and displayed comparatively higher heterozygosity (FIS = −0.62). Patterns of genetic diversity observed suggest dominance of a minimally diverse clonal lineage within northern Pakistan. This is surprising as a wide clinical spectrum was observed in patients, suggesting the importance of host and other factors. Further genotyping studies of L. tropica isolates displaying different clinical phenotypes are required to validate this potentially important observation.

Background Although Plasmodium vivax contributes to almost half of all malaria cases outside Africa, it has been relatively neglected compared to the more deadly P. falciparum. It is known that P. vivax populations possess high genetic diversity, differing geographically potentially due to different vector species, host genetics and environmental factors. Results We analysed the high-quality genomic data for 46 P. vivax isolates spanning 10 countries across 4 continents. Using population genetic methods we identified hotspots of selection pressure, including the previously reported MRP1 and DHPS genes, both putative drug resistance loci. Extra copies and deletions in the promoter region of another drug resistance candidate, MDR1 gene, and duplications in the Duffy binding protein gene (PvDBP) potentially involved in erythrocyte invasion, were also identified. For surveillance applications, continental-informative markers were found in putative drug resistance loci, and we show that organellar polymorphisms could classify P. vivax populations across continents and differentiate between Plasmodia spp. Conclusions This study has shown that genomic diversity that lies within and between P. vivax populations can be used to elucidate potential drug resistance and invasion mechanisms, as well as facilitate the molecular barcoding of the parasite for surveillance applications.

We present case histories of four patients treated with artemether-lumefantrine for falciparum malaria in UK hospitals in 2015 to 2016. Each subsequently presented with recurrent symptoms and Plasmodium falciparum parasitemia within 6 weeks of treatment with no intervening travel to countries where malaria is endemic. Parasite isolates, all of African origin, harbored variants at some candidate resistance loci. No evidence of pfk13 -mediated artemisinin resistance was found. Vigilance for signs of unsatisfactory antimalarial efficacy among imported cases of malaria is recommended.

Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens., IMPORTANCE Malaria is a disease caused by Plasmodium parasites that caused 214 million symptomatic cases and 438,000 deaths in 2015. Plasmodium vivax is the most widely distributed species, causing the majority of malaria infections outside sub-Saharan Africa. Whole-genome sequencing (WGS) of Plasmodium parasites from clinical samples has revealed important insights into the epidemiology and mechanisms of drug resistance of malaria. However, WGS of P. vivax is challenging due to low parasite levels in humans and the lack of a routine system to culture the parasites. Selective whole-genome amplification (SWGA) preferentially amplifies the genomes of pathogens from mixtures of target and host gDNA. Here, we demonstrate that SWGA is a simple, robust method that can be used to enrich P. vivax genomic DNA (gDNA) from unprocessed human blood samples and dried blood spots for cost-effective, high-quality WGS.