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Immunization with Plasmodium falciparum sporozoites under chemoprophylaxis can protect against controlled human malaria infection with the same strain for at least 10 weeks, and protection correlates with polyfunctional T-cell memory. The search for a malaria vaccine The best candidates for a malaria vaccine so far have been radiation-attenuated Plasmodium falciparum sporozoites (PfSPZ) inoculated by mosquitos, intravenous injection of radiation-attenuated, cryopreserved PfSPZ, and infectious PfSPZ inoculated by mosquitos in people taking chloroquine or mefloquine. Here Stephen Hoffman, Peter Kremsner and colleagues report that inoculation of volunteers taking chloroquine with direct intravenous injection of aseptic, cryopreserved, non-irradiated PfSPZ can induce protection against infection with the same strain for at least ten weeks. The authors show that protection correlates with polyfunctional T-cell memory. A highly protective malaria vaccine would greatly facilitate the prevention and elimination of malaria and containment of drug-resistant parasites 1 . A high level (more than 90%) of protection against malaria in humans has previously been achieved only by immunization with radiation-attenuated Plasmodium falciparum (Pf) sporozoites (PfSPZ) inoculated by mosquitoes 2 , 3 , 4 ; by intravenous injection of aseptic, purified, radiation-attenuated, cryopreserved PfSPZ (‘PfSPZ Vaccine’) 5 , 6 ; or by infectious PfSPZ inoculated by mosquitoes to volunteers taking chloroquine 7 , 8 , 9 , 10 or mefloquine 11 (chemoprophylaxis with sporozoites). We assessed immunization by direct venous inoculation of aseptic, purified, cryopreserved, non-irradiated PfSPZ (‘PfSPZ Challenge’ 12 , 13 ) to malaria-naive, healthy adult volunteers taking chloroquine for antimalarial chemoprophylaxis (vaccine approach denoted as PfSPZ-CVac) 14 . Three doses of 5.12 × 10 4 PfSPZ of PfSPZ Challenge 12 , 13 at 28-day intervals were well tolerated and safe, and prevented infection in 9 out of 9 (100%) volunteers who underwent controlled human malaria infection ten weeks after the last dose (group III). Protective efficacy was dependent on dose and regimen. Immunization with 3.2 × 10 3 (group I) or 1.28 × 10 4 (group II) PfSPZ protected 3 out of 9 (33%) or 6 out of 9 (67%) volunteers, respectively. Three doses of 5.12 × 10 4 PfSPZ at five-day intervals protected 5 out of 8 (63%) volunteers. The frequency of Pf-specific polyfunctional CD4 memory T cells was associated with protection. On a 7,455 peptide Pf proteome array, immune sera from at least 5 out of 9 group III vaccinees recognized each of 22 proteins. PfSPZ-CVac is a highly efficacious vaccine candidate; when we are able to optimize the immunization regimen (dose, interval between doses, and drug partner), this vaccine could be used for combination mass drug administration and a mass vaccination program approach to eliminate malaria from geographically defined areas.

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 mutations have originated locally, and that kelch13 shows a normal variation pattern relative to other genes in Africa, whereas in Southeast Asia there is a great excess of non-synonymous mutations, many of which cause radical amino-acid changes. Thus, kelch13 is not currently undergoing strong selection in Africa, despite a deep reservoir of variations that could potentially allow resistance to emerge rapidly. The practical implications are that public health surveillance for artemisinin resistance should not rely on kelch13 data alone, and interventions to prevent resistance must account for local evolutionary conditions, shown by genomic epidemiology to differ greatly between geographical regions. Malaria is an infectious disease caused by a microscopic parasite called Plasmodium, which is transferred between humans by mosquitos. One species of malaria parasite called Plasmodium falciparum can cause particularly severe and life-threatening forms of the disease. Currently, the most widely used treatment for P. falciparum infections is artemisinin combination therapy, a treatment that combines the drug artemisinin (or a closely related molecule) with another antimalarial drug. However, resistance to artemisinin has started to spread throughout Southeast Asia. Artemisinin resistance is caused by mutations in a parasite gene called kelch13, and researchers have identified over 20 different mutations in P. falciparum that confer artemisinin resistance. The diversity of mutations involved, and the fact that the same mutation can arise independently in different locations, make it difficult to track the spread of resistance using conventional molecular marker approaches. Here, Amato, Miotto et al. sequenced the entire genomes of more than 3,000 clinical samples of P. falciparum from Southeast Asia and Africa, collected as part of a global network of research groups called the MalariaGEN Plasmodium falciparum Community Project. Amato, Miotto et al. found that African parasites had independently acquired many of the same kelch13 mutations that are known to cause resistance to artemisinin in Southeast Asia. However the kelch13 mutations seen in Africa remained at low levels in the parasite population, and appeared to be under much less pressure for evolutionary selection than those found in Southeast Asia. These findings demonstrate that the emergence and spread of resistance to antimalarial drugs does not depend solely on the mutational process, but also on other factors that influence whether the mutations will spread in the population. Understanding how this is affected by different patterns of drug treatments and other environmental conditions will be important in developing more effective strategies for combating malaria.

In 2006, Senegal adopted artemisinin-based combination therapy (ACT) as first-line treatment in the management of uncomplicated malaria. This study aimed to update the status of antimalarial efficacy more than ten years after their first introduction. This was a randomized, three-arm, open-label study to evaluate the efficacy and safety of artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ) and dihydroartemisinin-piperaquine (DP) in Senegal. Malaria suspected patients were screened, enrolled, treated, and followed for 28 days for AL and ASAQ arms or 42 days for DP arm. Clinical and parasitological responses were assessed following antimalarial treatment. Genotyping ( msp1 , msp2 and 24 SNP-based barcode) were done to differentiate recrudescence from re-infection; in case of PCR-confirmed treatment failure, Pfk13 propeller and Pfcoronin genes were sequenced. Data was entered and analyzed using the WHO Excel-based application. A total of 496 patients were enrolled. In Diourbel, PCR non-corrected/corrected adequate clinical and parasitological responses (ACPR) was 100.0% in both the AL and ASAQ arms. In Kedougou, PCR corrected ACPR values were 98.8%, 100% and 97.6% in AL, ASAQ and DP arms respectively. No Pfk13 or Pfcoronin mutations associated with artemisinin resistance were found. This study showed that AL, ASAQ and DP remain efficacious and well-tolerated in the treatment of uncomplicated P. falciparum malaria in Senegal.

Following the diagnosis of a falciparum malaria case imported from Djibouti and not detected by a pfHRP2-based rapid diagnostic test (RDT), we investigated the prevalence of the pfhrp2/pfhrp3-deleted parasites in Djibouti using 378 blood samples collected between January and May 2019, from Djiboutian patients with suspected malaria. Malaria diagnosis by quantitative PCR confirmed the presence of Plasmodium falciparum for 20.9% (79/378) samples while RDTs did not detect HRP2 antigen in 83.5% (66/79) of these samples. Quantitative PCRs targeting the pfhrp2/pfhrp3 genes confirmed the absence of both genes for 86.5% of P. falciparum strains. The very large number (86.5%) of falciparum parasites lacking the pfhrp2/pfhrp3 genes observed in this study, now justifies the use of non-HRP2 alternative RDTs in Djibouti. In this area and in most countries where HRP2-based RDTs constitute the main arsenal for falciparum malaria diagnosis, it is important to implement a systematic surveillance and to inform biologists and clinicians about the risk of malaria misdiagnosis. Further investigations are needed to better understand the mechanism of selection and diffusion of the pfhrp2/pfhrp3-deleted parasites.

Background Despite widespread coverage of the emergence of artemisinin resistance, relatively little is known about the parasite populations responsible. The use of PCR genotyping around the highly polymorphic Plasmodium falciparum msp1 , msp2 and glurp genes has become well established both to describe variability in alleles within a population of parasites, as well as classify treatment outcome in cases of recurrent disease. The primary objective was to assess the emergence of minority parasite clones during seven days of artesunate (AS) treatment in a location with established artemisinin resistance. An additional objective was to investigate whether the classification of clinical outcomes remained valid when additional genotyping was performed. Methods Blood for parasite genotyping was collected from 143 adult patients presenting with uncomplicated falciparum malaria during a clinical trial of AS monotherapy in Western Cambodia. Nested allelic type-specific amplification of the genes encoding the merozoite surface proteins 1 and 2 ( msp1 and msp2 ) and the glutamate-rich protein ( glurp ) was performed at baseline, daily during seven days of treatment, and again at failure. Allelic variants were analysed with respect to the size of polymorphisms using Quantity One software to enable identification of polyclonal infections. Results Considerable variation of msp2 alleles but well-conserved msp1 and glurp were identified. At baseline, 31% of infections were polyclonal for one or more genes. Patients with recurrent malaria were significantly more likely to have polyclonal infections than patients without recurrence (seven of nine versus 36 of 127, p = 0.004). Emergence of minority alleles during treatment was detected in only one of twenty-three cases defined as being artemisinin resistant. Moreover, daily genotyping did not alter the final outcome classification in any recurrent cases. Conclusions The parasites responsible for artemisinin-resistant malaria in a clinical trial in Western Cambodia comprise the dominant clones of acute malaria infections rather than minority clones emerging during treatment. Additional genotyping during therapy was not beneficial. Disproportionately high rates of polyclonal infections in cases of recurrence suggest complex infections lead to poor treatment outcomes. Current research objectives should be broadened to include identification and follow-up of recurrent polyclonal infections so as to define their role as potential agents of emerging resistance.

Background The Republic of Congo adopted a new anti-malarial treatment policy in 2006, with artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) as the first- and second-line anti-malarial drugs, respectively. Only three clinical studies had been conducted before the policy change. A randomized study on these two artemisinin-based combinations was conducted, and the effect that sickle cell trait may have on treatment outcomes was evaluated in children under 10 years old followed during 12 months in Brazzaville in 2010–2011. Methods A cohort of 330 children under 10 years of age living in a suburban area in the south of Brazzaville were passively followed for registration of malaria episodes. Uncomplicated Plasmodium falciparum episodes were randomly treated with co-formulated ASAQ (Coarsucam ® ) or AL (Coartem ® ). Patients were followed according to the 2009 World Health Organization protocol for the evaluation of anti-malarial drug efficacy. Plasmodium falciparum recrudescent isolates were compared to pre-treatment isolates by polymerase chain reaction (PCR) to distinguish between re-infection and recrudescence. PCR-uncorrected and PCR-corrected responses to treatment were determined using per protocol analysis. Haemoglobin type (AA, AS, SS) was determined by PCR. Results Of 282 clinical malaria episodes registered during 1-year follow-up period, 262 children with uncomplicated malaria were treated with ASAQ (129 patients) or AL (133 patients). The PCR-corrected efficacy, expressed as the percentage of adequate clinical and parasitological response, was 97.0 % for ASAQ and 96.4 % for AL. Among ASAQ-treated patients, 112 (86.8 %) carried AA genotype and 17 (13.2 %) were AS carriers. The PCR-corrected efficacy was 96.4 % for AA-carriers and 100 % for AS-carriers treated with ASAQ [relative risk (RR) = 0.96; 95 % confidence interval, 0.93–1.00, p = 0.5]. Among 133 AL-treated children, 109 (82 %) carried AA, and 24 (18 %) AS genotypes. The PCR-corrected efficacy was 96.7 % among AA-carriers and 95.2 % among AS-carriers [RR = 1.01 (0.92–1.12), p = 0.6]. Nausea, jaundice, headache, dizziness, vomiting, pruritus, abdominal pain, and diarrhoea were registered as adverse events in both groups. ASAQ was associated with significantly more frequent adverse events (P < 0.05). Conclusion This first randomized study in Brazzaville confirmed the excellent efficacy of these co-formulated anti-malarial drugs in children. Sickle cell genotype did not influence the treatment efficacy of artemisinin-based combination therapy.

Background In 2005, the Democratic Republic of Congo (DRC) adopted artesunate and amodiaquine (ASAQ) as first-line anti-malarial treatment. In order to compare the efficacy of the fixed-dose formulation ASAQ versus artemether-lumefantrine (AL), a randomized, non-inferiority open-label trial was conducted in Katanga. Methods Children aged six and 59 months with uncomplicated Plasmodium falciparum malaria were enrolled and randomly allocated into one of the two regimens. The risk of recurrent parasitaemia by day 42, both unadjusted and adjusted by PCR genotyping to distinguish recrudescence from new infection, was analysed. Results Between April 2008 and March 2009, 301 children were included: 156 with ASAQ and 145 with AL. No early treatment failures were reported. Among the 256 patients followed-up at day 42, 32 patients developed late clinical or parasitological failure (9.9% (13/131) in the ASAQ group and 15.2% (19/125) in the AL group). After PCR correction, cure rates were 98.3% (95%CI, 94.1-99.8) in the ASAQ group and 99.1% (95%CI, 94.9-99.9) in the AL group (difference −0.7%, one sided 95% CI −3.1). Kaplan-Meier PCR-adjusted cure rates were similar. Both treatment regimens were generally well tolerated. Conclusion Both ASAQ and AL are highly effective and currently adequate as the first-line treatment of uncomplicated falciparum malaria in this area of Katanga, DRC. However, in a very large country, such as DRC, and because of possible emergence of resistance from other endemic regions, surveillance of efficacy of artemisinin-based combination treatments, including other evaluations of the resistance of ASAQ, need to be done in other provinces. Trial registration The protocol was registered with the clinicaltrials.gov, open clinical trial registry under the identifier number NCT01567423.

We assessed the influence that consecutive-day blood sampling, compared with single-day blood sampling, had on polymerase chain reaction (PCR)-adjusted parasitological cure after stepwise genotyping of merozoite surface proteins 2 (msp2) and 1 (msp1) in 106 children in Tanzania who had uncomplicated falciparum malaria treated with either sulfadoxine-pyrimethamine or artemether-lumefantrine; 78 of these children developed recurrent parasitemia during the 42-day follow-up period. Initial msp2 genotyping identified 27 and 33 recrudescences by use of single- and consecutive-day sampling, respectively; in subsequent msp1 genotyping, 17 and 21 of these episodes, respectively, were still classified as recrudescences; these results indicate a similar sensitivity of the standard single-day PCR protocol—that is, 82% (27/33) and 81% (17/21), in both genotyping steps. Interpretation of PCR-adjusted results will significantly depend on methodology.

Artemisinin-resistant Plasmodium falciparum has been reported in Pailin, western Cambodia, detected as a slow parasite clearance rate in vivo. Emergence of this phenotype in western Thailand and possibly elsewhere threatens to compromise the effectiveness of all artemisinin-based combination therapies. Parasite genetics is associated with parasite clearance rate but does not account for all variation. We investigated contributions of both parasite genetics and host factors to the artemisinin-resistance phenotype in Pursat, western Cambodia. Between June 19 and Nov 28, 2009, and June 26 and Dec 6, 2010, we enrolled patients aged 10 years or older with uncomplicated falciparum malaria, a density of asexual parasites of at least 10 000 per μL of whole blood, no symptoms or signs of severe malaria, no other cause of febrile illness, and no chronic illness. We gave participants 4 mg/kg artesunate at 0, 24, and 48 h, 15 mg/kg mefloquine at 72 h, and 10 mg/kg mefloquine at 96 h. We assessed parasite density on thick blood films every 6 h until undetectable. The parasite clearance half-life was calculated from the parasite clearance curve. We genotyped parasites with 18 microsatellite markers and patients for haemoglobin E, α-thalassaemia, and a mutation of G6PD, which encodes glucose-6-phosphate dehydrogenase. To account for the possible effects of acquired immunity on half-life, we used three surrogates for increased likelihood of exposure to P falciparum: age, sex, and place of residence. This study is registered with ClinicalTrials.gov, number NCT00341003. We assessed 3504 individuals from all six districts of Pursat province seeking treatment for malaria symptoms. We enrolled 168 patients with falciparum malaria who met inclusion criteria. The geometric mean half-life was 5·85 h (95% CI 5·54–6·18) in Pursat, similar to that reported in Pailin (p=0·109). We identified two genetically different parasite clone groups: parasite group 1 (PG1) and parasite group 2 (PG2). Non-significant increases in parasite clearance half-life were seen in patients with haemoglobin E (0·55 h; p=0·078), those of male sex (0·96 h; p=0·064), and in 2010 (0·68 h; p=0·068); PG1 was associated with a significant increase (0·79 h; p=0·033). The mean parasite heritability of half-life was 0·40 (SD 0·17). Heritable artemisinin resistance is established in a second Cambodian province. To accurately identify parasites that are intrinsically susceptible or resistant to artemisinins, future studies should explore the effect of erythrocyte polymorphisms and specific immune responses on half-life variation. Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Background Rodent malaria parasites (RMP) are used extensively as models of human malaria. Draft RMP genomes have been published for Plasmodium yoelii , P. berghei ANKA ( Pb A) and P. chabaudi AS ( Pc AS). Although availability of these genomes made a significant impact on recent malaria research, these genomes were highly fragmented and were annotated with little manual curation. The fragmented nature of the genomes has hampered genome wide analysis of Plasmodium gene regulation and function. Results We have greatly improved the genome assemblies of Pb A and Pc AS, newly sequenced the virulent parasite P. yoelii YM genome, sequenced additional RMP isolates/lines and have characterized genotypic diversity within RMP species. We have produced RNA-seq data and utilised it to improve gene-model prediction and to provide quantitative, genome-wide, data on gene expression. Comparison of the RMP genomes with the genome of the human malaria parasite P. falciparum and RNA-seq mapping permitted gene annotation at base-pair resolution. Full-length chromosomal annotation permitted a comprehensive classification of all subtelomeric multigene families including the ` Plasmodium interspersed repeat genes' ( pir ). Phylogenetic classification of the pir family, combined with pir expression patterns, indicates functional diversification within this family. Conclusions Complete RMP genomes, RNA-seq and genotypic diversity data are excellent and important resources for gene-function and post-genomic analyses and to better interrogate Plasmodium biology. Genotypic diversity between P. chabaudi isolates makes this species an excellent parasite to study genotype-phenotype relationships. The improved classification of multigene families will enhance studies on the role of (variant) exported proteins in virulence and immune evasion/modulation.