Evidence of Artemisinin-Resistant Malaria in Africa.

BACKGROUND: In the six Southeast Asian countries that make up the Greater Mekong Subregion, Plasmodium falciparum has developed resistance to derivatives of artemisinin, the main component of first-line treatments for malaria. Clinical resistance to artemisinin monotherapy in other global regions, including Africa, would be problematic. METHODS: In this longitudinal study conducted in Northern Uganda, we treated patients who had P. falciparum infection with intravenous artesunate (a water-soluble artemisinin derivative) and estimated the parasite clearance half-life. We evaluated ex vivo susceptibility of the parasite using a ring-stage survival assay and genotyped resistance-related genes. RESULTS: From 2017 through 2019, a total of 14 of 240 patients who received intravenous artesunate had evidence of in vivo artemisinin resistance (parasite clearance half-life, >5 hours). Of these 14 patients, 13 were infected with P. falciparum parasites with mutations in the A675V or C469Y allele in the kelch13 gene. Such mutations were associated with prolonged parasite clearance half-lives (geometric mean, 3.95 hours for A675V and 3.30 hours for C469Y, vs. 1.78 hours for wild-type allele; P<0.001 and P = 0.05, respectively). The ring-stage survival assay showed a higher frequency of parasite survival among organisms with the A675V allele than among those with the wild-type allele. The prevalence of parasites with kelch13 mutations increased significantly, from 3.9% in 2015 to 19.8% in 2019, due primarily to the increased frequency of the A675V and C469Y alleles (P<0.001 and P = 0.004, respectively). Single-nucleotide polymorphisms flanking the A675V mutation in Uganda were substantially different from those in Southeast Asia. CONCLUSIONS: The independent emergence and local spread of clinically artemisinin-resistant P. falciparum has been identified in Africa. The two kelch13 mutations may be markers for detection of these resistant parasites. (Funded by the Japan Society for the Promotion of Science and others.).

Detection of drug-resistant malaria in resource-limited settings: efficient and high-throughput surveillance of artemisinin and partner drug resistance.

OBJECTIVES: Artemisinin-resistant Plasmodium falciparum malaria is currently spreading globally, including in Africa. Artemisinin resistance also leads to resistance to partner drugs used in artemisinin-based combination therapies. Sequencing of kelch13, which is associated with artemisinin resistance, culture-based partner drug susceptibility tests, and ELISA-based growth measurement are conventionally used to monitor resistance; however, their application is challenging in resource-limited settings. METHODS: An experimental package for field studies with minimum human/material requirements was developed. RESULTS: First, qPCR-based SNP assay was applied in artemisinin resistance screening, which can detect mutations within 1 h and facilitate sample selection for subsequent processes. It had 100% sensitivity and specificity compared with DNA sequencing in the detection of the two common artemisinin resistance mutations in Uganda, C469Y and A675V. Moreover, in the partner drug susceptibility test, the cultured samples were dry-preserved on a 96-well filter paper plate and shipped to the central laboratory. Parasite growth was measured by ELISA using redissolved samples. It well reproduced the results of direct ELISA, reducing significant workload in the field (Pearson correlation coefficient: 0.984; 95% CI: 0.975-0.990). CONCLUSIONS: Large-scale and sustainable monitoring is required urgently to track rapidly spreading drug-resistant malaria. In malaria-endemic areas, where research resources are often limited, simplicity and feasibility of the procedure is especially important. Our approach combines a qPCR-based rapid test, which is also applicable to point-of-care diagnosis of artemisinin resistance and centralized analysis of ex vivo culture. The approach could improve efficiency of field experiments and accelerate global drug resistance surveillance.

The Impact of Sequestration on Artemisinin-Induced Parasite Clearance in Plasmodium falciparum Malaria in Africa.

BACKGROUND: Artemisinin-resistant Plasmodium falciparum is spreading in Southeast Asia and Africa. In vivo susceptibility to artemisinin is studied by looking at the rate of decline of peripheral parasitemia (parasite clearance half-life). However, parasites that are adhered/sequestered to the endothelium and undetectable in the peripheral blood are not considered in the estimation of parasite clearance. Here, we evaluated the influence of sequestration on in vivo artemisinin efficacy in Uganda, where artemisinin resistance is spreading. METHODS: We analyzed 133 patients with P. falciparum malaria included in an in vivo study on artemisinin efficacy in northern Uganda in 2018 and 2019. The parasite clearance half-life was estimated from peripheral parasitemia after artemisinin monotherapy. P. falciparum histidine-rich protein 2 (PfHRP2) was measured in pretreatment plasma. The number of sequestered parasites was estimated from PfHRP2 concentration and peripheral parasitemia. RESULTS: The estimated number of sequestered parasites per plasma volume ranged from 0 to 2 564 000/µL. Inflammation, thrombocytopenia, and dyslipidemia were significantly associated with sequestration independent of peripheral parasitemia. The median parasite clearance half-lives were 1.65 hours in patients infected with Pfkelch13 wild-type parasites (n = 104) and 3.95 hours in those with A675V artemisinin-resistant mutant (n = 18). In the multivariable model for the wild-type population, 1 000 000/µL of sequestered parasites were estimated to delay parasite clearance by 16.8% (95% confidence interval, 5.1%-28.5%), although it was not clear in the A675V population. CONCLUSIONS: In patients with P. falciparum malaria without artemisinin-resistant mutations, intensive sequestration delays parasite clearance after treatment, which may contribute to reduced artemisinin efficacy.

The diversity of Plasmodium falciparum isolates from asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo.

BACKGROUND: Understanding Plasmodium falciparum population diversity and transmission dynamics provides information on the intensity of malaria transmission, which is needed for assessing malaria control interventions. This study aimed to determine P. falciparum allelic diversity and multiplicity of infection (MOI) among asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo (DRC). METHODS: A total of 438 DNA samples (248 asymptomatic and 190 symptomatic) were characterized by nested PCR and genotyping the polymorphic regions of pfmsp1 block 2 and pfmsp2 block 3. RESULTS: Nine allele types were observed in pfmsp1 block2. The K1-type allele was predominant with 78% (229/293) prevalence, followed by the MAD20-type allele (52%, 152/293) and RO33-type allele (44%, 129/293). Twelve alleles were detected in pfmsp2, and the 3D7-type allele was the most frequent with 84% (256/304) prevalence, followed by the FC27-type allele (66%, 201/304). Polyclonal infections were detected in 63% (95% CI 56, 69) of the samples, and the MOI (SD) was 1.99 (0.97) in P. falciparum single-species infections. MOIs significantly increased in P. falciparum isolates from symptomatic parasite carriers compared with asymptomatic carriers (2.24 versus 1.69, adjusted b: 0.36, (95% CI 0.01, 0.72), p = 0.046) and parasitaemia > 10,000 parasites/µL compared to parasitaemia < 5000 parasites/µL (2.68 versus 1.63, adjusted b: 0.89, (95% CI 0.46, 1.25), p < 0.001). CONCLUSION: This survey showed low allelic diversity and MOI of P. falciparum, which reflects a moderate intensity of malaria transmission in the study areas. MOIs were more likely to be common in symptomatic infections and increased with the parasitaemia level. Further studies in different transmission zones are needed to understand the epidemiology and parasite complexity in the DRC.

Circulation of an Artemisinin-Resistant Malaria Lineage in a Traveler Returning from East Africa to France.

A returned traveler to Uganda presented with a Plasmodium falciparum kelch13 A675V mutant infection that exhibited delayed clearance under artesunate therapy. Parasites were genetically related to recently reported Ugandan artemisinin-resistant A675V parasites. Adequate malaria prevention measures and clinical and genotypic surveillance are important tools to avoid and track artemisinin resistance.

Emergence of artemisinin-resistant Plasmodium falciparum with kelch13 C580Y mutations on the island of New Guinea.

The rapid and aggressive spread of artemisinin-resistant Plasmodium falciparum carrying the C580Y mutation in the kelch13 gene is a growing threat to malaria elimination in Southeast Asia, but there is no evidence of their spread to other regions. We conducted cross-sectional surveys in 2016 and 2017 at two clinics in Wewak, Papua New Guinea (PNG) where we identified three infections caused by C580Y mutants among 239 genotyped clinical samples. One of these mutants exhibited the highest survival rate (6.8%) among all parasites surveyed in ring-stage survival assays (RSA) for artemisinin. Analyses of kelch13 flanking regions, and comparisons of deep sequencing data from 389 clinical samples from PNG, Indonesian Papua and Western Cambodia, suggested an independent origin of the Wewak C580Y mutation, showing that the mutants possess several distinctive genetic features. Identity by descent (IBD) showed that multiple portions of the mutants' genomes share a common origin with parasites found in Indonesian Papua, comprising several mutations within genes previously associated with drug resistance, such as mdr1, ferredoxin, atg18 and pnp. These findings suggest that a P. falciparum lineage circulating on the island of New Guinea has gradually acquired a complex ensemble of variants, including kelch13 C580Y, which have affected the parasites' drug sensitivity. This worrying development reinforces the need for increased surveillance of the evolving parasite populations on the island, to contain the spread of resistance.

Low prevalence of Plasmodium falciparum parasites lacking pfhrp2/3 genes among asymptomatic and symptomatic school-age children in Kinshasa, Democratic Republic of Congo.

BACKGROUND: Loss of efficacy of diagnostic tests may lead to untreated or mistreated malaria cases, compromising case management and control. There is an increasing reliance on rapid diagnostic tests (RDTs) for malaria diagnosis, with the most widely used of these targeting the Plasmodium falciparum histidine-rich protein 2 (PfHRP2). There are numerous reports of the deletion of this gene in P. falciparum parasites in some populations, rendering them undetectable by PfHRP2 RDTs. The aim of this study was to identify P. falciparum parasites lacking the P. falciparum histidine rich protein 2 and 3 genes (pfhrp2/3) isolated from asymptomatic and symptomatic school-age children in Kinshasa, Democratic Republic of Congo. METHODS: The performance of PfHRP2-based RDTs in comparison to microscopy and PCR was assessed using blood samples collected and spotted on Whatman 903™ filter papers between October and November 2019 from school-age children aged 6-14 years. PCR was then used to identify parasite isolates lacking pfhrp2/3 genes. RESULTS: Among asymptomatic malaria carriers (N = 266), 49%, 65%, and 70% were microscopy, PfHRP2_RDT, and pfldh-qPCR positive, respectively. The sensitivity and specificity of RDTs compared to PCR were 80% and 70% while the sensitivity and specificity of RDTs compared to microscopy were 92% and 60%, respectively. Among symptomatic malaria carriers (N = 196), 62%, 67%, and 87% were microscopy, PfHRP2-based RDT, pfldh-qPCR and positive, respectively. The sensitivity and specificity of RDTs compared to PCR were 75% and 88%, whereas the sensitivity and specificity of RDTs compared to microscopy were 93% and 77%, respectively. Of 173 samples with sufficient DNA for PCR amplification of pfhrp2/3, deletions of pfhrp2 and pfhrp3 were identified in 2% and 1%, respectively. Three (4%) of samples harboured deletions of the pfhrp2 gene in asymptomatic parasite carriers and one (1%) isolate lacked the pfhrp3 gene among symptomatic parasite carriers in the RDT positive subgroup. No parasites lacking the pfhrp2/3 genes were found in the RDT negative subgroup. CONCLUSION: Plasmodium falciparum histidine-rich protein 2/3 gene deletions are uncommon in the surveyed population, and do not result in diagnostic failure. The use of rigorous PCR methods to identify pfhrp2/3 gene deletions is encouraged in order to minimize the overestimation of their prevalence.

Increase in the proportion of Plasmodium falciparum with kelch13 C580Y mutation and decline in pfcrt and pfmdr1 mutant alleles in Papua New Guinea.

BACKGROUND: The C580Y mutation in the Plasmodium falciparum kelch13 gene is the most commonly observed variant in artemisinin-resistant isolates in the Greater Mekong Subregion (GMS). Until 2017, it had not been identified outside the GMS, except for Guyana/Amazonia. In 2017, three parasites carrying the C580Y mutation were identified in Papua New Guinea (PNG). As the C580Y allele rapidly spread in the GMS, there is concern that this mutant is now spreading in PNG. METHODS: In 2020, a cross-sectional survey was conducted at two clinics in Wewak, PNG. Symptomatic patients infected with P. falciparum were treated with artemether plus lumefantrine following a national treatment policy. Blood samples were obtained before treatment, and polymorphisms in kelch13, pfcrt, and pfmdr1 were determined. Parasite positivity was examined on day 3. The results were compared with those of previous studies conducted in 2002, 2003, and 2016-2018. RESULTS: A total of 94 patients were included in this analysis. The proportion of C580Y was significantly increased (2.2% in 2017, 5.7% in 2018, and 6.4% in 2020; p = 4.2 × 10-3). A significant upward trend was observed in the wild-type proportion for pfcrt (1.9% in 2016 to 46.7% in 2020; p = 8.9 × 10-16) and pfmdr1 (59.5% in 2016 to 91.4% in 2020; p = 2.3 × 10-6). Among 27 patients successfully followed on day 3, including three with C580Y infections, none showed positive parasitaemia. CONCLUSIONS: Under the conditions of significant increases in pfcrt K76 and pfmdr1 N86 alleles in PNG, the increase in kelch13 C580Y mutants may be a warning indicator of the emergence of parasites resistant to the currently used first-line treatment regimen of artemether plus lumefantrine. Therefore, nationwide surveillance of molecular markers for drug resistance and assessment of its therapeutic effects are important.

Malaria parasite species composition of Plasmodium infections among asymptomatic and symptomatic school-age children in rural and urban areas of Kinshasa, Democratic Republic of Congo.

BACKGROUND: Malaria remains a major public health concern in the Democratic Republic of Congo (DRC), and school-age children are relatively neglected in malaria prevalence surveys and may constitute a significant reservoir of transmission. This study aimed to understand the burden of malaria infections in school-age children in Kinshasa/DRC. METHODS: A total of 634 (427 asymptomatic and 207 symptomatic) blood samples collected from school-age children aged 6 to 14 years were analysed by microscopy, RDT and Nested-PCR. RESULTS: The overall prevalence of Plasmodium spp. by microscopy, RDT and PCR was 33%, 42% and 62% among asymptomatic children and 59%, 64% and 95% in symptomatic children, respectively. The prevalence of Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale spp. by PCR was 58%, 20% and 11% among asymptomatic and 93%, 13% and 16% in symptomatic children, respectively. Among P. ovale spp., P. ovale curtisi, P. ovale wallikeri and mixed P. ovale curtisi + P. ovale wallikeri accounted for 75%, 24% and 1% of infections, respectively. All Plasmodium species infections were significantly more prevalent in the rural area compared to the urban area in asymptomatic infections (p < 0.001). Living in a rural as opposed to an urban area was associated with a five-fold greater risk of asymptomatic malaria parasite carriage (p < 0.001). Amongst asymptomatic malaria parasite carriers, 43% and 16% of children harboured mixed Plasmodium with P. falciparum infections in the rural and the urban areas, respectively, whereas in symptomatic malaria infections, it was 22% and 26%, respectively. Few children carried single infections of P. malariae (2.2%) and P. ovale spp. (1.9%). CONCLUSION: School-age children are at significant risk from both asymptomatic and symptomatic malaria infections. Continuous systematic screening and treatment of school-age children in high-transmission settings is needed.

Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda.

BACKGROUND: Usage of chloroquine was discontinued from the treatment of Plasmodium falciparum infection in almost all endemic regions because of global spread of resistant parasites. Since the first report in Malawi, numerous epidemiological studies have demonstrated that the discontinuance led to re-emergence of chloroquine-susceptible P. falciparum, suggesting a possible role in future malaria control. However, most studies were cross-sectional, with few studies looking at the persistence of chloroquine recovery in long term. This study fills the gap by providing, for a period of at least 6 years, proof of persistent re-emergence/stable recovery of susceptible parasite populations using both molecular and phenotypic methods. METHODS: Ex vivo drug-susceptibility assays to chloroquine (n = 319) and lumefantrine (n = 335) were performed from 2013 to 2018 in Gulu, Northern Uganda, where chloroquine had been removed from the official malaria treatment regimen since 2006. Genotyping of pfcrt and pfmdr1 was also performed. RESULTS: Chloroquine resistance (≥ 100 nM) was observed in only 3 (1.3%) samples. Average IC50 values for chloroquine were persistently low throughout the study period (17.4-24.9 nM). Parasites harbouring pfcrt K76 alleles showed significantly lower IC50s to chloroquine than the parasites harbouring K76T alleles (21.4 nM vs. 43.1 nM, p-value = 3.9 × 10-8). Prevalence of K76 alleles gradually increased from 71% in 2013 to 100% in 2018. CONCLUSION: This study found evidence of stable persistence of chloroquine susceptibility with the fixation of pfcrt K76 in Northern Uganda after discontinuation of chloroquine in the region. Accumulation of similar evidence in other endemic areas in Uganda could open channels for possible future re-use of chloroquine as an option for malaria treatment or prevention.

Artemisinin-Resistant Plasmodium falciparum with High Survival Rates, Uganda, 2014-2016.

Because ≈90% of malaria cases occur in Africa, emergence of artemisinin-resistant Plasmodium falciparum in Africa poses a serious public health threat. To assess emergence of artemisinin-resistant parasites in Uganda during 2014-2016, we used the recently developed ex vivo ring-stage survival assay, which estimates ring-stage-specific P. falciparum susceptibility to artemisinin. We conducted 4 cross-sectional surveys to assess artemisinin sensitivity in Gulu, Uganda. Among 194 isolates, survival rates (ratio of viable drug-exposed parasites to drug-nonexposed controls) were high (>10%) for 4 isolates. Similar rates have been closely associated with delayed parasite clearance after drug treatment and are considered to be a proxy for the artemisinin-resistant phenotype. Of these, the PfKelch13 mutation was observed in only 1 isolate, A675V. Population genetics analysis suggested that these possibly artemisinin-resistant isolates originated in Africa. Large-scale surveillance of possibly artemisinin-resistant parasites in Africa would provide useful information about treatment outcomes and help regional malaria control.

Lack of significant recovery of chloroquine sensitivity in Plasmodium falciparum parasites following discontinuance of chloroquine use in Papua New Guinea.

BACKGROUND: Chloroquine treatment for Plasmodium falciparum has been discontinued in almost all endemic regions due to the spread of resistant isolates. Reversal of chloroquine susceptibility after chloroquine discontinuation has been reported in dozens of endemic regions. However, this phenomenon has been mostly observed in Africa and is not well documented in other malaria endemic regions. To investigate this, an ex vivo study on susceptibility to chloroquine and lumefantrine was conducted during 2016-2018 in Wewak, Papua New Guinea where chloroquine had been removed from the official malaria treatment regimen in 2010. Genotyping of pfcrt and pfmdr1 was also performed. RESULTS: In total, 368 patients were enrolled in this study. Average IC50 values for chloroquine were 106.6, 80.5, and 87.6 nM in 2016, 2017, and 2018, respectively. These values were not significantly changed from those obtained in 2002/2003 (108 nM). The majority of parasites harboured a pfcrt K76T the mutation responsible for chloroquine resistance. However, a significant upward trend was observed in the frequency of the K76 (wild) allele from 2.3% in 2016 to 11.7% in 2018 (P = 0.008; Cochran-Armitage trend test). CONCLUSIONS: Eight years of chloroquine withdrawal has not induced a significant recovery of susceptibility in Papua New Guinea. However, an increasing tendency of parasites harbouring chloroquine-susceptible K76 suggests a possibility of resurgence of chloroquine susceptibility in the future.

Absence of in vivo selection for K13 mutations after artemether-lumefantrine treatment in Uganda.

BACKGROUND: Individual drug treatment may select resistant parasites in the human body, a process termed in vivo selection. Some single nucleotide polymorphisms in Plasmodium falciparum chloroquine-resistance transporter (pfcrt) and multidrug resistance gene 1 (pfmdr1) genes have been reportedly selected after artemether-lumefantrine treatment. However, there is a paucity of data regarding in vivo selection of P. falciparum Kelch propeller domain (pfkelch13) polymorphisms, responsible for artemisinin-resistance in Asia, and six putative background mutations for artemisinin resistance; D193Y in ferredoxin, T484I in multiple resistance protein 2, V127M in apicoplast ribosomal protein S10, I356T in pfcrt, V1157L in protein phosphatase and C1484F in phosphoinositide-binding protein. METHODS: Artemether-lumefantrine efficacy study with a follow-up period of 28 days was conducted in northern Uganda in 2014. The above-mentioned genotypes were comparatively analysed before drug administration and on days; 3, 7, and 28 days after treatment. RESULTS: In 61 individuals with successful follow-up, artemether-lumefantrine treatment regimen was very effective with PCR adjusted efficacy of 95.2%. Among 146 isolates obtained before treatment, wild-type alleles were observed in 98.6% of isolates in pfkelch13 and in all isolates in the six putative background genes except I356T in pfcrt, which had 2.4% of isolates as mixed infections. In vivo selection study revealed that all isolates detected in the follow-up period harboured wild type alleles in pfkelch13 and the six background genes. CONCLUSION: Mutations in pfkelch13 and the six background genes may not play an important role in the in vivo selection after artemether-lumefantrine treatment in Uganda. Different mechanisms might rather be associated with the existence of parasites after treatment.

Little Polymorphism at the K13 Propeller Locus in Worldwide Plasmodium falciparum Populations Prior to the Introduction of Artemisinin Combination Therapies.

The emergence and spread of artemisinin-resistant Plasmodium falciparum is of huge concern for the global effort toward malaria control and elimination. Artemisinin resistance, defined as a delayed time to parasite clearance following administration of artemisinin, is associated with mutations in the Pfkelch13 gene of resistant parasites. To date, as many as 60 nonsynonymous mutations have been identified in this gene, but whether these mutations have been selected by artemisinin usage or merely reflect natural polymorphism independent of selection is currently unknown. To clarify this, we sequenced the Pfkelch13 propeller domain in 581 isolates collected before (420 isolates) and after (161 isolates) the implementation of artemisinin combination therapies (ACTs), from various regions of endemicity worldwide. Nonsynonymous mutations were observed in 1% of parasites isolated prior to the introduction of ACTs. Frequencies of mutant isolates, nucleotide diversity, and haplotype diversity were significantly higher in the parasites isolated from populations exposed to artemisinin than in those from populations that had not been exposed to the drug. In the artemisinin-exposed population, a significant excess of dN compared to dS was observed, suggesting the presence of positive selection. In contrast, pairwise comparison of dN and dS and the McDonald and Kreitman test indicate that purifying selection acts on the Pfkelch13 propeller domain in populations not exposed to ACTs. These population genetic analyses reveal a low baseline of Pfkelch13 polymorphism, probably due to purifying selection in the absence of artemisinin selection. In contrast, various Pfkelch13 mutations have been selected under artemisinin pressure.

Differential remodelling of peroxisome function underpins the environmental and metabolic adaptability of diplonemids and kinetoplastids.

The remodelling of organelle function is increasingly appreciated as a central driver of eukaryotic biodiversity and evolution. Kinetoplastids including Trypanosoma and Leishmania have evolved specialized peroxisomes, called glycosomes. Glycosomes uniquely contain a glycolytic pathway as well as other enzymes, which underpin the physiological flexibility of these major human pathogens. The sister group of kinetoplastids are the diplonemids, which are among the most abundant eukaryotes in marine plankton. Here we demonstrate the compartmentalization of gluconeogenesis, or glycolysis in reverse, in the peroxisomes of the free-living marine diplonemid, Diplonema papillatum Our results suggest that peroxisome modification was already under way in the common ancestor of kinetoplastids and diplonemids, and raise the possibility that the central importance of gluconeogenesis to carbon metabolism in the heterotrophic free-living ancestor may have been an important selective driver. Our data indicate that peroxisome modification is not confined to the kinetoplastid lineage, but has also been a factor in the success of their free-living euglenozoan relatives.

Household clustering of asymptomatic malaria infections in Xepon district, Savannakhet province, Lao PDR.

BACKGROUND: In the Lao PDR, malaria morbidity and mortality have remarkably decreased over the past decade. However, asymptomatic infections in rural villages contribute to the on-going local transmission. The primary objective of this study was to explore the characteristics of infections in a malaria-endemic district of the Lao PDR. The specific objectives were to investigate the prevalence and species of malaria parasites using molecular methods and to assess individual and household parasite levels and the characteristics associated with malaria infection. METHODS: The study population included 870 participants from 236 households in 10 villages of the Xepon district. Interviews, blood examinations and body temperature measurements were conducted between August and September 2013. A multilevel logistic regression model, with adjustment for clustering effects, was used to assess the association between predictor variables and an outcome variable (malaria infection status as principally determined by PCR). The predictive factors included individual-level factors (age, gender, past fever episode, and forest activity during night time) and household-level factors (household member size, household bed net usage/density and a household with one other malaria-infected member). RESULTS: Fifty-two participants (including 26 children) tested positive (positive rate: 6.0 %): Plasmodium falciparum mono-infection was the most common infection (n = 41, 78.8 %), followed by P. falciparum and Plasmodium vivax mixed infections (n = 9, 17.3 %). The majority of infected participants (n = 42, 80.8 %) had no fever episodes in the two previous weeks or a measurable fever (>37 °C) at the time of survey. Living in a household with one other malaria-infected member significantly increased the odds of infection (odds ratio 24.33, 95 % confidence interval 10.15-58.32). Among the 40 households that had at least one infected member, nine households were responsible for 40.4 % of the total infections. CONCLUSIONS: Plasmodium vivax was detected more frequently than it was reported from the district hospital. Most infections were asymptomatic and sub-microscopic and were highly clustered within households. To further eliminate malaria in Xepon and other similar settings in the country, the National Malaria Control Programme should consider household-based strategies, including reactive case detection targeting the household members of index cases.

A dominant negative form of inositol 1,4,5-trisphosphate receptor induces metacyclogenesis and increases mitochondrial density in Trypanosoma cruzi.

Inositol 1,4,5-trisphosphate receptor (IP3R) is a key regulator of intracellular Ca(2+) concentration that release Ca(2+) from Ca(2+) stores in response to various external stimuli. IP3R also works as a signal hub which form a platform for interacting with various proteins involved in diverse cell signaling. Previously, we have identified an IP3R homolog in the parasitic protist, Trypanosoma cruzi (TcIP3R). Parasites expressing reduced or increased levels of TcIP3R displayed defects in growth, transformation, and infectivity. In the present study, we established parasitic strains expressing a dominant negative form of TcIP3R, named DN-TcIP3R, to further investigate the physiological role(s) of TcIP3R. We found that the growth of epimastigotes expressing DN-TcIP3R was significantly slower than that of parasites with TcIP3R expression levels that were approximately 65% of wild-type levels. The expression of DN-TcIP3R in epimastigotes induced metacyclogenesis even in the normal growth medium. Furthermore, these epimastigotes showed the presence of dense mitochondria under a transmission electron microscope. Our findings confirm that TcIP3R is crucial for epimastigote growth, as previously reported. They also suggest that a strong inhibition of the IP3R-mediated signaling induces metacyclogenesis and that mitochondrial integrity is closely associated with this signaling.

Ordered accumulation of mutations conferring resistance to sulfadoxine-pyrimethamine in the Plasmodium falciparum parasite.

BACKGROUND: Monitoring the prevalence of drug resistant Plasmodium falciparum is essential for effective malaria control. Resistance to pyrimethamine and sulfadoxine increases as mutations accumulate in the parasite genes encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps), respectively. Although parasites are exposed to these antifolate drugs simultaneously, it remains virtually unknown whether dhfr and dhps mutations accumulate along interrelated paths. METHODS: We investigated the order of step-wise accumulation in dhfr and dhps by cumulative analyses using binomial tests in 575 P. falciparum isolates obtained from 7 countries in Asia and Melanesia. RESULTS: An initial step in the accumulation of mutations preferentially occurred in dhfr (2 mutations), followed by 1 mutation in dhps. In a subsequent step, mutations were estimated separately for 5 dhfr/dhps-resistant lineages identified using 12 microsatellites flanking dhfr and dhps. Among these lineages, we found 3 major mutational paths, each of which follows a unique stepwise trajectory to produce the most highly resistant form with 4 mutations in dhfr and 3 in dhps. CONCLUSIONS: The ordered accumulation of mutations in dhfr and dhps elucidated here will assist in predicting the status and progression of antifolate resistance in malaria-endemic regions where antifolate drugs are used for intermittent preventive treatment.

A longer-chain acylated derivative of Dictyostelium differentiation-inducing factor-1 enhances the antimalarial activity against Plasmodium parasites.

The spread of malarial parasites resistant to first-line treatments such as artemisinin combination therapies is a global health concern. Differentiation-inducing factor 1 (DIF-1) is a chlorinated alkylphenone (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-one) originally found in the cellular slime mould Dictyostelium discoideum. We previously showed that some derivatives of DIF-1, particularly DIF-1(+2) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) octan-1-one), exert potent antimalarial activities. In this study, we synthesised DIF-1(+3) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) nonan-1-one). We then evaluated the effects of DIF-1(+3) in vitro on Plasmodium falciparum and in vivo over 7 days (50-100 mg/kg/day) in a mouse model of Plasmodium berghei. DIF-1(+3) exhibited a half-maximal inhibitory concentration of approximately 20-30 % of DIF-1(+2) in three laboratory strains with a selectivity index > 263, including in strains resistant to chloroquine and artemisinin. Parasite growth and multiplication were almost completely suppressed by treatment with 100 mg/kg DIF-1(+3). The survival time of infected mice was significantly increased (P = 0.006) with no apparent adverse effects. In summary, addition of an acyl group to DIF-1(+2) to prepare DIF-1(+3) substantially enhanced antimalarial activity, even in drug-resistant malaria, indicating the potential of applying DIF-1(+3) for malaria treatment.