A new long-read mitochondrial-genome protocol (PacBio HiFi) for haemosporidian parasites: a tool for population and biodiversity studies.

BACKGROUND: Studies on haemosporidian diversity, including origin of human malaria parasites, malaria's zoonotic dynamic, and regional biodiversity patterns, have used target gene approaches. However, current methods have a trade-off between scalability and data quality. Here, a long-read Next-Generation Sequencing protocol using PacBio HiFi is presented. The data processing is supported by a pipeline that uses machine-learning for analysing the reads. METHODS: A set of primers was designed to target approximately 6 kb, almost the entire length of the haemosporidian mitochondrial genome. Amplicons from different samples were multiplexed in an SMRTbell® library preparation. A pipeline (HmtG-PacBio Pipeline) to process the reads is also provided; it integrates multiple sequence alignments, a machine-learning algorithm that uses modified variational autoencoders, and a clustering method to identify the mitochondrial haplotypes/species in a sample. Although 192 specimens could be studied simultaneously, a pilot experiment with 15 specimens is presented, including in silico experiments where multiple data combinations were tested. RESULTS: The primers amplified various haemosporidian parasite genomes and yielded high-quality mt genome sequences. This new protocol allowed the detection and characterization of mixed infections and co-infections in the samples. The machine-learning approach converged into reproducible haplotypes with a low error rate, averaging 0.2% per read (minimum of 0.03% and maximum of 0.46%). The minimum recommended coverage per haplotype is 30X based on the detected error rates. The pipeline facilitates inspecting the data, including a local blast against a file of provided mitochondrial sequences that the researcher can customize. CONCLUSIONS: This is not a diagnostic approach but a high-throughput method to study haemosporidian sequence assemblages and perform genotyping by targeting the mitochondrial genome. Accordingly, the methodology allowed for examining specimens with multiple infections and co-infections of different haemosporidian parasites. The pipeline enables data quality assessment and comparison of the haplotypes obtained to those from previous studies. Although a single locus approach, whole mitochondrial data provide high-quality information to characterize species pools of haemosporidian parasites.

New member of Plasmodium (Vinckeia) and Plasmodium cyclopsi discovered in bats in Sierra Leone - nuclear sequence and complete mitochondrial genome analyses.

Malaria remains the most important arthropod-borne infectious disease globally. The causative agent, Plasmodium, is a unicellular eukaryote that develops inside red blood cells. Identifying new Plasmodium parasite species that infect mammalian hosts can shed light on the complex evolution and diversity of malaria parasites. Bats feature a high diversity of microorganisms including seven separate genera of malarial parasites. Three species of Plasmodium have been reported so far, for which scarce reports exist. Here we present data from an investigation of Plasmodium infections in bats in the western Guinean lowland forest in Sierra Leone. We discovered a new Plasmodium parasite in the horseshoe bat Rhinolophus landeri. Plasmodium cyclopsi infections in a member of leaf-nosed bats, Doryrhina cyclops, exhibited a high prevalence of 100%. Phylogenetic analysis of complete mitochondrial genomes and nine nuclear markers recovered a close relationship between P. cyclopsi and the new Plasmodium parasite with the rodent species Plasmodium berghei, a widely used in vivo model to study malaria in humans. The data suggests that the "rodent/bat" Plasmodium (Vinckeia) clade represents a diverse group of malarial parasites that would likely expand with a systematic sampling of small mammals in tropical Africa. Identifying the bat Plasmodium repertoire is central to our understanding of the evolution of Plasmodium parasites in mammals.

The Genome of Plasmodium gonderi: Insights into the Evolution of Human Malaria Parasites.

Plasmodium species causing malaria in humans are not monophyletic, sharing common ancestors with nonhuman primate parasites. Plasmodium gonderi is one of the few known Plasmodium species infecting African old-world monkeys that are not found in apes. This study reports a de novo assembled P. gonderi genome with complete chromosomes. The P. gonderi genome shares codon usage, syntenic blocks, and other characteristics with the human parasites Plasmodium ovale s.l. and Plasmodium malariae, also of African origin, and the human parasite Plasmodium vivax and species found in nonhuman primates from Southeast Asia. Using phylogenetically aware methods, newly identified syntenic blocks were found enriched with conserved metabolic genes. Regions outside those blocks harbored genes encoding proteins involved in the vertebrate host-Plasmodium relationship undergoing faster evolution. Such genome architecture may have facilitated colonizing vertebrate hosts. Phylogenomic analyses estimated the common ancestor between P. vivax and an African ape parasite P. vivax-like, within the Asian nonhuman primates parasites clade. Time estimates incorporating P. gonderi placed the P. vivax and P. vivax-like common ancestor in the late Pleistocene, a time of active migration of hominids between Africa and Asia. Thus, phylogenomic and time-tree analyses are consistent with an Asian origin for P. vivax and an introduction of P. vivax-like into Africa. Unlike other studies, time estimates for the clade with Plasmodium falciparum, the most lethal human malaria parasite, coincide with their host species radiation, African hominids. Overall, the newly assembled genome presented here has the quality to support comparative genomic investigations in Plasmodium.

Assessment of Plasmodium falciparum drug resistance associated molecular markers in Mandla, Madhya Pradesh, India.

BACKGROUND: Resistance against artemisinin-based combination therapy is one of the challenges to malaria control and elimination globally. Mutations in different genes (Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1) confer resistance to artesunate and sulfadoxine-pyrimethamine (AS + SP) were analysed from Mandla district, Madhya Pradesh, to assess the effectiveness of the current treatment regimen against uncomplicated Plasmodium falciparum. METHODS: Dried blood spots were collected during the active fever survey and mass screening and treatment activities as part of the Malaria Elimination Demonstration Project (MEDP) from 2019 to 2020. Isolated DNA samples were used to amplify the Pfdhfr, Pfdhps, Pfk13 and Pfmdr1 genes using nested PCR and sequenced for mutation analysis using the Sanger sequencing method. RESULTS: A total of 393 samples were subjected to PCR amplification, sequencing and sequence analysis; 199, 215, 235, and 141 samples were successfully sequenced for Pfdhfr, Pfdhps, Pfk13, Pfmdr1, respectively. Analysis revealed that the 53.3% double mutation (C59R, S108N) in Pfdhfr, 89.3% single mutation (G437A) in Pfdhps, 13.5% single mutants (N86Y), and 51.1% synonymous mutations in Pfmdr1 in the study area. Five different non-synonymous and two synonymous point mutations found in Pfk13, which were not associated to artemisinin resistance. CONCLUSION: The study has found that mutations linked to SP resistance are increasing in frequency, which may reduce the effectiveness of this drug as a future partner in artemisinin-based combinations. No evidence of mutations linked to artemisinin resistance in Pfk13 was found, suggesting that parasites are sensitive to artemisinin derivatives in the study area. These findings are a baseline for routine molecular surveillance to proactively identify the emergence and spread of artemisinin-resistant parasites.

Leucocytozoon cariamae n. sp. and Haemoproteus pulcher coinfection in Cariama cristata (Aves: Cariamiformes): first mitochondrial genome analysis and morphological description of a leucocytozoid in Brazil.

The distribution of avian haemosporidians of the genus Leucocytozoon in the Neotropics remains poorly understood. Recent studies confirmed their presence in the region using molecular techniques alone, but evidence for gametocytes and data on putative competent hosts for Leucocytozoon are still lacking outside highland areas. We combined morphological and molecular data to characterize a new Leucocytozoon species infecting a non-migratory red-legged seriema (Cariama cristata), the first report of a competent host for Leucocytozoon in Brazil. Leucocytozoon cariamae n. sp. is distinguished from the Leucocytozoon fringillinarum group by its microgametocytes that are not strongly appressed to the host cell nucleus. The bird studied was coinfected with Haemoproteus pulcher, and we present a Bayesian phylogenetic analysis based on nearly complete mitochondrial genomes of these 2 parasites. Leucocytozoon cariamae n. sp. morphology is consistent with our phylogenetic analysis indicating that it does not share a recent common ancestor with the L. fringillinarum group. Haemoproteus pulcher and Haemoproteus catharti form a monophyletic group with Haemocystidium parasites of Reptilia, supporting the polyphyly of the genus Haemoproteus. We also discussed the hypothesis that H. pulcher and H. catharti may be avian Haemocystidium, highlighting the need to study non-passerine parasites to untangle the systematics of Haemosporida.

Origin and diversity of malaria parasites and other Haemosporida.

Symbionts, including parasites, are ubiquitous in all world ecosystems. Understanding the diversity of symbiont species addresses diverse questions, from the origin of infectious diseases to inferring processes shaping regional biotas. Here, we review the current approaches to studying Haemosporida's species diversity and evolutionary history. Despite the solid knowledge of species linked to diseases, such as the agents of human malaria, studies on haemosporidian phylogeny, diversity, ecology, and evolution are still limited. The available data, however, indicate that Haemosporida is an extraordinarily diverse and cosmopolitan clade of symbionts. Furthermore, this clade seems to have originated with their vertebrate hosts, particularly birds, as part of complex community level processes that we are still characterizing.

Morphometric and molecular characterization of an unpigmented haemosporidian parasite in the Neotropical turnip-tailed gecko (Thecadactylus rapicauda).

Morphological traits from blood stages have been the gold standard for determining haemosporidian parasite species. However, the status of some taxa and the value of such traits in parasites from reptiles remain contentious. The scarce sampling of these species worsens the situation, and several taxa lack molecular data. A survey was performed in the Magdalena Department in Colombia, where 16 species of reptiles were captured. A peculiar haemosporidian parasite was found in the Turnip-tailed gecko Thecadactylus rapicauda. This haemosporidian does not show malarial pigment in blood stages under light microscopy; thus, it fits the Garnia genus's characters belonging to the Garniidae. However, the phylogenetic analyses using a partial sequence of cytochrome b and the mitochondrial DNA placed it within the Plasmodium clade. Our findings suggest that many putative Garnia species belong to the genus Plasmodium, like the one reported here. This study either shows that visible malarial pigment in blood stages is not a diagnostic trait of the genus Plasmodium or malarial pigment might be present in an undetectable form under a light microscope. In any case, the current taxonomy of haemosporidian parasites in reptiles requires revision. This study highlights the importance of using molecular and morphological traits to address taxonomic questions at the species and genus levels in haemosporidian parasites from reptiles.

The first complete genome of the simian malaria parasite Plasmodium brasilianum.

Naturally occurring human infections by zoonotic Plasmodium species have been documented for P. knowlesi, P. cynomolgi, P. simium, P. simiovale, P. inui, P. inui-like, P. coatneyi, and P. brasilianum. Accurate detection of each species is complicated by their morphological similarities with other Plasmodium species. PCR-based assays offer a solution but require prior knowledge of adequate genomic targets that can distinguish the species. While whole genomes have been published for P. knowlesi, P. cynomolgi, P. simium, and P. inui, no complete genome for P. brasilianum has been available. Previously, we reported a draft genome for P. brasilianum, and here we report the completed genome for P. brasilianum. The genome is 31.4 Mb in size and comprises 14 chromosomes, the mitochondrial genome, the apicoplast genome, and 29 unplaced contigs. The chromosomes consist of 98.4% nucleotide sites that are identical to the P. malariae genome, the closest evolutionarily related species hypothesized to be the same species as P. brasilianum, with 41,125 non-synonymous SNPs (0.0722% of genome) identified between the two genomes. Furthermore, P. brasilianum had 4864 (82.1%) genes that share 80% or higher sequence similarity with 4970 (75.5%) P. malariae genes. This was demonstrated by the nearly identical genomic organization and multiple sequence alignments for the merozoite surface proteins msp3 and msp7. We observed a distinction in the repeat lengths of the circumsporozoite protein (CSP) gene sequences between P. brasilianum and P. malariae. Our results demonstrate a 97.3% pairwise identity between the P. brasilianum and the P. malariae genomes. These findings highlight the phylogenetic proximity of these two species, suggesting that P. malariae and P. brasilianum are strains of the same species, but this could not be fully evaluated with only a single genomic sequence for each species.

Malaria Resilience in South America: Epidemiology, Vector Biology, and Immunology Insights from the Amazonian International Center of Excellence in Malaria Research Network in Peru and Brazil.

The 1990s saw the rapid reemergence of malaria in Amazonia, where it remains an important public health priority in South America. The Amazonian International Center of Excellence in Malaria Research (ICEMR) was designed to take a multidisciplinary approach toward identifying novel malaria control and elimination strategies. Based on geographically and epidemiologically distinct sites in the Northeastern Peruvian and Western Brazilian Amazon regions, synergistic projects integrate malaria epidemiology, vector biology, and immunology. The Amazonian ICEMR's overarching goal is to understand how human behavior and other sociodemographic features of human reservoirs of transmission-predominantly asymptomatically parasitemic people-interact with the major Amazonian malaria vector, Nyssorhynchus (formerly Anopheles) darlingi, and with human immune responses to maintain malaria resilience and continued endemicity in a hypoendemic setting. Here, we will review Amazonian ICEMR's achievements on the synergies among malaria epidemiology, Plasmodium-vector interactions, and immune response, and how those provide a roadmap for further research, and, most importantly, point toward how to achieve malaria control and elimination in the Americas.

Great-tailed Grackles (Quiscalus mexicanus) as a tolerant host of avian malaria parasites.

Great-tailed Grackles (Quiscalus mexicanus) are a social, polygamous bird species whose populations have rapidly expanded their geographic range across North America over the past century. Before 1865, Great-tailed Grackles were only documented in Central America, Mexico, and southern Texas in the USA. Given the rapid northern expansion of this species, it is relevant to study its role in the dynamics of avian blood parasites. Here, 87 Great-tailed grackles in Arizona (a population in the new center of the range) were screened for haemosporidian parasites using microscopy and PCR targeting the parasite mitochondrial cytochrome b gene. Individuals were caught in the wild from January 2018 until February 2020. Haemosporidian parasite prevalence was 62.1% (54/87). A high Plasmodium prevalence was found (60.9%, 53/87), and one grackle was infected with Haemoproteus (Parahaemoproteus) sp. (lineage SIAMEX01). Twenty-one grackles were infected with P. cathemerium, sixteen with P. homopolare, four with P. relictum (strain GRW04), and eleven with three different genetic lineages of Plasmodium spp. that have not been characterized to species level (MOLATE01, PHPAT01, and ZEMAC01). Gametocytes were observed in birds infected with three different Plasmodium lineages, revealing that grackles are competent hosts for some parasite species. This study also suggests that grackles are highly susceptible and develop chronic infections consistent with parasite tolerance, making them competent to transmit some generalist haemosporidian lineages. It can be hypothesized that, as the Great-tailed Grackle expands its geographic range, it may affect local bird communities by increasing the transmission of local parasites but not introducing new species into the parasite species pool.

The evolution of primate malaria parasites: A study on the origin and diversification of Plasmodium in lemurs.

Among the primate malaria parasites, those found in lemurs have been neglected. Here, six Plasmodium lineages were detected in 169 lemurs. Nearly complete mitochondrial genomes (mtDNA, ≈6Kb) and apicoplast loci (≈6Kb) were obtained from these parasites and other Haemosporida species. Plasmodium spp. in lemurs are a diverse clade that shares a common ancestor with other primate parasites from continental Africa. Time-trees for the mtDNA were estimated under different scenarios, and the origin of the lemur clade coincides with the proposed time of their host species' most recent common ancestor (Lemuridae-Indriidae). A time tree with fewer taxa was estimated with mtDNA + Apicoplast loci. Those time estimates overlapped but were younger and had narrower credibility intervals than those from mtDNA alone. Importantly, the mtDNA + Apicoplast estimates that the clade including the most lethal malaria parasite in humans, Plasmodium falciparum, may have originated with Homininae (African apes). Finally, the phylogenetic congruence of the lemurs and their parasites was explored. A statistically significant scenario identified four cospeciation, two duplications, four transfer (host-switches), and zero loss events. Thus, the parasite species sampled in lemurs seem to be radiating with their hosts.

Why Plasmodium vivax and Plasmodium falciparum are so different? A tale of two clades and their species diversities.

The global malaria burden sometimes obscures that the genus Plasmodium comprises diverse clades with lineages that independently gave origin to the extant human parasites. Indeed, the differences between the human malaria parasites were highlighted in the classical taxonomy by dividing them into two subgenera, the subgenus Plasmodium, which included all the human parasites but Plasmodium falciparum that was placed in its separate subgenus, Laverania. Here, the evolution of Plasmodium in primates will be discussed in terms of their species diversity and some of their distinct phenotypes, putative molecular adaptations, and host-parasite biocenosis. Thus, in addition to a current phylogeny using genome-level data, some specific molecular features will be discussed as examples of how these parasites have diverged. The two subgenera of malaria parasites found in primates, Plasmodium and Laverania, reflect extant monophyletic groups that originated in Africa. However, the subgenus Plasmodium involves species in Southeast Asia that were likely the result of adaptive radiation. Such events led to the Plasmodium vivax lineage. Although the Laverania species, including P. falciparum, has been considered to share "avian characteristics," molecular traits that were likely in the common ancestor of primate and avian parasites are sometimes kept in the Plasmodium subgenus while being lost in Laverania. Assessing how molecular traits in the primate malaria clades originated is a fundamental science problem that will likely provide new targets for interventions. However, given that the genus Plasmodium is paraphyletic (some descendant groups are in other genera), understanding the evolution of malaria parasites will benefit from studying "non-Plasmodium" Haemosporida.

Larval habitats, species composition and distribution of malaria vectors in regions with autochthonous and imported malaria in Roraima state, Brazil.

BACKGROUND: Malaria control requires local action. Assessing the vector diversity and abundance provides information on the local malariogenic potential or risk of transmission. This study aimed to determine the Anopheles species composition, habitats, seasonal occurrence, and distribution in areas with autochthonous and imported malaria cases in Roraima State. METHODS: A longitudinal study was conducted from January 2017 to October 2018, sampling larvae and adult mosquitoes in three municipalities of Roraima State: Boa Vista, Pacaraima and São João da Baliza. These areas have different risks of malaria importation. Four to six mosquito larval habitats were selected for larval sampling at each municipality, along with two additional sites for adult mosquito collection. All larval habitats were surveyed every two months using a standardized larval sampling methodology and MosqTent for adult mosquitoes. RESULTS: A total of 544 Anopheles larvae and 1488 adult mosquitoes were collected from the three municipalities studied. Although the species abundance differed between municipalities, the larvae of Anopheles albitarsis s.l., Anopheles nuneztovari s.l. and Anopheles triannulatus s.l. were collected from all larval habitats studied while Anopheles darlingi were collected only from Boa Vista and São João da Baliza. Adults of 11 species of the genus Anopheles were collected, and the predominant species in Boa Vista was An. albitarsis (88.2%) followed by An. darlingi (6.9%), while in São João da Baliza, An. darlingi (85.6%) was the most predominant species followed by An. albitarsis s.l. (9.2%). In contrast, the most abundant species in Pacaraima was Anopheles braziliensis (62%), followed by Anopheles peryassui (18%). Overall, the majority of anophelines exhibited greater extradomicile than peridomicile-biting preference. Anopheles darlingi was the only species found indoors. Variability in biting times was observed among species and municipalities. CONCLUSION: This study revealed the composition of anopheline species and habitats in Boa Vista, Pacaraima and São João da Baliza. The species sampled differed in their behaviour with only An. darlingi being found indoors. Anopheles darlingi appeared to be the most important vector in São João da Baliza, an area of autochthonous malaria, and An. albitarsis s.l. and An. braziliensis in areas of low transmission, although there were increasing reports of imported malaria. Understanding the diversity of vector species and their ecology is essential for designing effective vector control strategies for these municipalities.

Evolutionary Insights into the Microneme-Secreted, Chitinase-Containing High-Molecular-Weight Protein Complexes Involved in Plasmodium Invasion of the Mosquito Midgut.

While general mechanisms by which Plasmodium ookinetes invade the mosquito midgut have been studied, details regarding the interface of the ookinete, specifically its barriers to invasion, such as the proteolytic milieu, the chitin-containing, protein cross-linked peritrophic matrix, and the midgut epithelium, remain to be understood. Here, we review our knowledge of Plasmodium chitinases and the mechanisms by which they mediate ookinetes crossing the peritrophic matrix. The integration of new genomic insights into previous findings advances our understanding of Plasmodium evolution. Recently obtained Plasmodium species genomic data enable identification of the conserved residues in the experimentally demonstrated hetero-multimeric, high-molecular-weight complex comprised of a short chitinase covalently linked to binding partners, von Willebrand factor A domain-related protein (WARP) and secreted ookinete adhesive protein (SOAP). Artificial intelligence-based high-resolution structural modeling using the DeepMind AlphaFold algorithm yielded highly informative three-dimensional structures and insights into how short chitinases, WARP, and SOAP may interact at the atomic level to form the ookinete-secreted peritrophic matrix invasion complex. Elucidating the significance of the divergence of ookinete-secreted micronemal proteins among Plasmodium species may lead to a better understanding of the ookinete invasion machinery and the coevolution of Plasmodium-mosquito interactions.

Complement component 3 mutations alter the longitudinal risk of pediatric malaria and severe malarial anemia.

Severe malarial anemia (SMA) is a leading cause of childhood morbidity and mortality in holoendemic Plasmodium falciparum transmission regions. To gain enhanced understanding of predisposing factors for SMA, we explored the relationship between complement component 3 (C3) missense mutations [rs2230199 (2307C>G, Arg>Gly102) and rs11569534 (34420G>A, Gly>Asp1224)], malaria, and SMA in a cohort of children (n = 1617 children) over 36 months of follow-up. Variants were selected based on their ability to impart amino acid substitutions that can alter the structure and function of C3. The 2307C>G mutation results in a basic to a polar residue change (Arg to Gly) at position 102 (ß-chain) in the macroglobulin-1 (MG1) domain, while 34420G>A elicits a polar to acidic residue change (Gly to Asp) at position 1224 (α-chain) in the thioester-containing domain. After adjusting for multiple comparisons, longitudinal analyses revealed that inheritance of the homozygous mutant (GG) at 2307 enhanced the risk of SMA (RR = 2.142, 95%CI: 1.229-3.735, P = 0.007). The haplotype containing both wild-type alleles (CG) decreased the incident risk ratio of both malaria (RR = 0.897, 95%CI: 0.828-0.972, P = 0.008) and SMA (RR = 0.617, 95%CI: 0.448-0.848, P = 0.003). Malaria incident risk ratio was also reduced in carriers of the GG (Gly102Gly1224) haplotype (RR = 0.941, 95%CI: 0.888-0.997, P = 0.040). Collectively, inheritance of the missense mutations in MG1 and thioester-containing domain influence the longitudinal risk of malaria and SMA in children exposed to intense Plasmodium falciparum transmission.

Negligible evidence for detrimental effects of Leucocytozoon infections among Emperor Geese (Anser canagicus) breeding on the Yukon-Kuskokwim Delta, Alaska.

Emperor Geese (Anser canagicus) are iconic waterfowl endemic to Alaska and adjacent areas of northeastern Russia that are considered to be near threatened by the International Union for Conservation. This species has been identified as harboring diverse viruses and parasites which have, at times, been associated with disease in other avian taxa. To better assess if disease represents a vulnerability for Emperor Geese breeding on the Yukon-Kuskokwim Delta, Alaska, we evaluated if haemosporidian parasites were associated with decreased mass or survival among adult female nesting birds captured during 2006-2016. Through molecular analyses, we detected genetically diverse Leucocytozoon, Haemoproteus, and Plasmodium parasites in 28%, 1%, and 1% of 607 blood samples screened in triplicate, respectively. Using regression analysis, we found evidence for a small effect of Leucocytozoon infection on the mass of incubating adult female Emperor Geese. The estimated mass of infected individuals was approximately 43 g (95% CI: 20-67 g), or approximately 2%, less than uninfected birds when captured during the second half of incubation (days 11-25). We did not, however, find support for an effect of Leucocytozoon infection on survival of adult female nesting Emperor Geese using a multi-state hidden Markov framework to analyze mark-resight and recapture data. Using parasite mitochondrial DNA cytochrome b sequences, we identified 23 haplotypes among infected Emperor Geese. Leucocytozoon haplotypes clustered into three phylogenetically supported clades designated as 'L. simondi clade A', 'L. simondi clade B', and 'other Leucocytozoon'. We did not find evidence that parasites assigned to any of these clades were associated with differential mass measures among nesting adult female Emperor Geese. Collectively, our results provide negligible evidence for Leucocytozoon parasites as causing detrimental effects to adult female Emperor Geese breeding on the Yukon-Kuskokwim Delta.

The clinical-epidemiological profile of malaria patients from Southern Venezuela, a critical hotspot in Latin America.

BACKGROUND: Venezuela accounted for 55% of the cases and 73% of the malaria deaths in the Americas in 2019. Bolivar state, in the southeast, contributes > 60% of the country's Plasmodium vivax and Plasmodium falciparum cases every year. This study describes the clinical-epidemiological characteristics of clinical malaria patients in this high-transmission area. METHODS: A prospective study was conducted on patients seeking medical attention in three medical centres in the state capital, Ciudad Bolivar, between June and October 2018. Malaria diagnosis was carried out using microscopy following national standards. Malaria-positive patients were examined for clinical symptoms, and haematological tests were performed at the time of diagnosis. Patients were followed up by telephone to evaluate malaria recurrences. RESULTS: Out of 287 patients, 200 (69.7%) were positive for P. vivax, 69 (24%) for P. falciparum, and 18 (6.3%) had mixed (P. vivax/P. falciparum) infections. Patients' median age was 33 years (IQR 20), 168 (69%) were men, and 40% practiced gold mining as the main occupation. Fever (96.5%), chills (91.3%), and headaches (90.6%) were the most frequent symptoms. At least one symptom associated with severe malaria was observed in 69 out of 161 patients with complete clinical evaluation (42.9%). Plasmodium vivax infections were found in 42 out of 69 (60.9%) severe cases; by contrast, P. falciparum and mixed malaria caused 34.8% (24/69) and 4.4% (3/69) of infections, respectively. Two patients died of cerebral malaria. Mean hemoglobin was lower in the patients infected with P. falciparum than those infected with P. vivax. Regardless of the parasite causing the infection, patients presented high levels of total bilirubin, aminotransferases (AST, ALT), and lactate dehydrogenase (LDH). Out of the 142 patients followed up by phone for three months (49.5% of the 287 patients), 35 (24.7%) reported recurrences. CONCLUSIONS: The high malaria prevalence among young male adults practicing gold mining suggests that this occupation is a significant risk factor. The unexpected high prevalence of P. vivax patients with at least one criteria of severe clinical disease is a matter of concern. Whether it is the result of a lack of timely diagnosis and effective treatment should be explored.

Plasmodium ouropretensis, n. sp., a new case of non-erythrocytic species within lizard malaria parasites.

Delimiting and describing Plasmodium species in reptiles remains a pressing problem in Haemosporida taxonomy. The few morphological characters used can overlap, and the significance of some life-history traits is not fully understood. Morphologically identical lizard Plasmodium forms have been reported infecting different cell types (red and white blood cells) in the same host and have been considered the same species. An example is Plasmodium tropiduri tropiduri, a species known to infect erythrocytes, thrombocytes and lymphocyte-like cells. Here, both forms of P. t. tropiduri were analysed using light microscope-based morphological characteristics and phylogenetic inferences based on almost complete mitochondrial genomes of parasites naturally infecting lizards in southeastern Brazil. Although morphologically similar, two distinct phylogenetic lineages infecting erythrocytes and non-erythrocytic cells were found. The lineage found in the erythrocytes forms a monophyletic group with species from Colombia. However, the non-erythrocytic lineage shares a recent common ancestor with Plasmodium leucocytica, which infects leucocytes in lizards from the Caribbean islands. Here, Plasmodium ouropretensis n. sp. is described as a species that infects thrombocytes and lymphocyte-like cells.

The apicoplast of Haemoproteus columbae: A comparative study of this organelle genome in Haemosporida.

Apicomplexa is a phylum of parasitic protozoa; among them are the order Haemosporida, vector-borne parasites that include those that cause malaria (genus Plasmodium). Most Apicomplexa species have a non-photosynthetic plastid or apicoplast. Given its unique metabolic pathways, this organelle is considered a target for malaria therapeutics. Regardless of its importance, there is a paucity of complete apicoplast genome data hindering comparative studies. Here, the Haemoproteus (Haemoproteus) columbae apicoplast genome (lineage HAECOL1) was obtained using next-generation sequencing. This genome was included in a comparative analysis with other plastids. This 29.8 kb circular genome shares the same structure found in Plasmodium parasites. It is A + T rich (87.7%), comparable but at the higher end of A + T content observed in Plasmodium species (85.5-87.2%). As expected, considering its high A + T content, the synonymous codon usage (RSCU) and the effective number of codons (ENc) showed a moderate codon bias. Several apicoplast genes have a phylogenetic signal. However, unlike mitochondrial genes, single-gene phylogenies have low support in haemosporidian clades that diverged recently. The H. columbae apicoplast genome suggests that the apicoplast function may be conserved across Haemosporida. This parasite could be a model to study this organelle in a non-mammalian system.

Association of Plasmodium falciparum kelch13 R561H genotypes with delayed parasite clearance in Rwanda: an open-label, single-arm, multicentre, therapeutic efficacy study.

BACKGROUND: Partial artemisinin resistance is suspected if delayed parasite clearance (ie, persistence of parasitaemia on day 3 after treatment initiation) is observed. Validated markers of artemisinin partial resistance in southeast Asia, Plasmodium falciparum kelch13 (Pfkelch13) R561H and P574L, have been reported in Rwanda but no association with parasite clearance has been observed. We aimed to establish the efficacy of artemether-lumefantrine and genetic characterisation of Pfkelch13 alleles and their association with treatment outcomes. METHODS: This open-label, single-arm, multicentre, therapeutic efficacy study was done in 2018 in three Rwandan sites: Masaka, Rukara, and Bugarama. Children aged 6-59 months with P falciparum monoinfection and fever were eligible and treated with a 3-day course of artemether-lumefantrine. Treatment response was monitored for 28 days using weekly microscopy screenings of blood samples for P falciparum. Mutations in Pfkelch13 and P falciparum multidrug resistance-1 (Pfmdr1) genes were characterised in parasites collected from enrolled participants. Analysis of flanking microsatellites surrounding Pfkelch13 was done to define the origins of the R561H mutations. The primary endpoint was PCR-corrected parasitological cure on day 28, as per WHO protocol. FINDINGS: 228 participants were enrolled and 224 (98·2%) reached the study endpoint. PCR-corrected efficacies were 97·0% (95% CI 88-100) in Masaka, 93·8% (85-98) in Rukara, and 97·2% (91-100) in Bugarama. Pfkelch13 R561H mutations were present in 28 (13%) of 218 pre-treatment samples and P574L mutations were present in two (1%) pre-treatment samples. 217 (90%) of the 240 Pfmdr1 haplotypes observed in the pretreatment samples, had either the NFD (N86Y, Y184F, D1246Y) or NYD haplotype. Eight (16%) of 51 participants in Masaka and 12 (15%) of 82 participants in Rukara were microscopically positive 3 days after treatment initiation, which was associated with pre-treatment presence of Pfkelch13 R561H in Masaka (p=0·0005). Genetic analysis of Pfkelch13 R561H mutations suggest their common ancestry and local origin in Rwanda. INTERPRETATION: We confirm evidence of emerging artemisinin partial resistance in Rwanda. Although artemether-lumefantrine remains efficacious, vigilance for decreasing efficacy, further characterisation of artemisinin partial resistance, and evaluation of additional antimalarials in Rwanda should be considered. FUNDING: The US President's Malaria Initiative. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.