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.

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.

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.

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.

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.

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.

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.

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.

Complexity of malaria transmission dynamics in the Brazilian Atlantic Forest.

Plasmodium malariae and Plasmodium vivax are protozoan parasites that can cause malaria in humans. They are genetically indistinguishable from, respectively, Plasmodium brasilianum and Plasmodium simium, i.e. parasites infecting New World non-human primates in South America. In the tropical rainforests of the Brazilian Atlantic coast, it has long been hypothesized that P. brasilianum and P. simium in platyrrhine primates originated from P. malariae and P. vivax in humans. A recent hypothesis proposed the inclusion of Plasmodium falciparum into the transmission dynamics between humans and non-human primates in the Brazilian Atlantic tropical rainforest. Herein, we assess the occurrence of human malaria in simians and sylvatic anophelines using field-collected samples in the Capivari-Monos Environmental Protection Area from 2015 to 2017. We first tested simian blood and anopheline samples. Two simian (Aloutta) blood samples (18%, n = 11) showed Plasmodium cytb DNA sequences, one for P. vivax and another for P. malariae. From a total of 9,416 anopheline females, we found 17 pools positive for Plasmodium species with a 18S qPCR assay. Only three showed P. cytb DNA sequence, one for P. vivax and the others for rodent malaria species (similar to Plasmodium chabaudi and Plasmodium berghei). Based on these results, we tested 25 rodent liver samples for the presence of Plasmodium and obtained P. falciparum cytb DNA sequence in a rodent (Oligoryzomys sp.) liver. The findings of this study indicate complex malaria transmission dynamics composed by parallel spillover-spillback of human malaria parasites, i.e. P. malariae, P. vivax, and P. falciparum, in the Brazilian Atlantic forest.

A phylogenetic study of Haemocystidium parasites and other Haemosporida using complete mitochondrial genome sequences.

Haemosporida are diverse vector-borne parasites associated with terrestrial vertebrates. Driven by the interest in species causing malaria (genus Plasmodium), the diversity of avian and mammalian haemosporidian species has been extensively studied, relying mostly on mitochondrial genes, particularly cytochrome b. However, parasites from reptiles have been neglected in biodiversity surveys. Reptilian haemosporidian parasites include Haemocystidium, a genus that shares morphological features with Plasmodium and Haemoproteus. Here, the first complete Haemocystidium mitochondrial DNA (mtDNA) genomes are studied. In particular, three mtDNA genomes from Haemocystidium spp. sampled in Africa, Oceania, and South America, are described. The Haemocystidium mtDNA genomes showed a high A + T content and a gene organization, including an extreme fragmentation of the rRNAs, found in other Haemosporida. These Haemocystidium mtDNA genomes were incorporated in phylogenetic and molecular clock analyses together with a representative sample of haemosporidian parasites from birds, mammals, and reptiles. The recovered phylogeny supported Haemocystidium as a monophyletic group apart from Plasmodium and other Haemosporida. Both the phylogenetic and molecular clock analyses yielded results consistent with a scenario in which haemosporidian parasites radiated with modern birds. Haemocystidium, like mammalian parasite clades, seems to originate from host switches by avian Haemosporida that allowed for the colonization of new vertebrate hosts. This hypothesis can be tested by investigating additional parasite species from all vertebrate hosts, particularly from reptiles. The mtDNA genomes reported here provide baseline data that can be used to scale up studies in haemosporidian parasites of reptiles using barcode approaches.

Changes in the frequencies of Plasmodium falciparum dhps and dhfr drug-resistant mutations in children from Western Kenya from 2005 to 2018: the rise of Pfdhps S436H.

BACKGROUND: Sulfadoxine-pyrimethamine (SP) is the only anti-malarial drug formulation approved for intermittent preventive treatment in pregnancy (IPTp). However, mutations in the Plasmodium falciparum dhfr (Pfdhfr) and dhps (Pfdhps) genes confer resistance to pyrimethamine and sulfadoxine, respectively. Here, the frequencies of SP resistance-associated mutations from 2005 to 2018 were compared in samples from Kenyan children with malaria residing in a holoendemic transmission region. METHODS: Partial sequences of the Pfdhfr and Pfdhps genes were amplified and sequenced from samples collected in 2005 (n = 81), 2010 (n = 95), 2017 (n = 43), and 2018 (n = 55). The frequency of known mutations conferring resistance to pyrimethamine and sulfadoxine were estimated and compared. Since artemisinin-based combination therapy (ACT) is the current first-line treatment for malaria, the presence of mutations in the propeller domain of P. falciparum kelch13 gene (Pfk13) linked to ACT-delayed parasite clearance was studied in the 2017/18 samples. RESULTS: Among other changes, the point mutation of Pfdhps S436H increased in frequency from undetectable in 2005 to 28% in 2017/18. Triple Pfdhfr mutant allele (CIRNI) increased in frequency from 84% in 2005 to 95% in 2017/18, while the frequency of Pfdhfr double mutant alleles declined (allele CICNI from 29% in 2005 to 6% in 2017/18, and CNRNI from 9% in 2005 to undetectable in 2010 and 2017/18). Thus, a multilocus Pfdhfr/Pfdhps genotype with six mutations (HGEAA/CIRNI), including Pfdhps S436H, increased in frequency from 2010 to 2017/18. Although none of the mutations associated with ACT-delayed parasite clearance was observed, the Pfk13 mutation A578S, the most widespread Pfk13 SNP found in Africa, was detected in low frequency (2.04%). CONCLUSIONS: There were changes in SP resistance mutant allele frequencies, including an increase in the Pfdhps S436H. Although these patterns seem consistent with directional selection due to drug pressure, there is a lack of information to determine the actual cause of such changes. These results suggest incorporating molecular surveillance of Pfdhfr/Pfdhps mutations in the context of SP efficacy studies for intermittent preventive treatment in pregnancy (IPTp).

The endangered California Condor (Gymnogyps californianus) population is exposed to local haemosporidian parasites.

The endangered California Condor (Gymnogyps californianus) is the largest New World Vulture in North America. Despite recovery program success in saving the species from extinction, condors remain compromised by lead poisoning and limited genetic diversity. The latter makes this species especially vulnerable to infectious diseases. Thus, taking advantage of the program of blood lead testing in Arizona, condor blood samples from 2008 to 2018 were screened for haemosporidian parasites using a nested polymerase chain reaction (PCR) protocol that targets the parasite mitochondrial cytochrome b gene. Plasmodium homopolare (Family Plasmodiidae, Order Haemosporida, Phylum Apicomplexa), was detected in condors captured in 2014 and 2017. This is the first report of a haemosporidian species infecting California Condors, and the first evidence of P. homopolare circulating in the Condor population from Arizona. Although no evidence of pathogenicity of P. homopolare in Condors was found, this study showed that the California Condors from Arizona are exposed to haemosporidian parasites that likely are spilling over from other local bird species. Thus, active surveillance should be an essential part of conservation efforts to mitigate the impact of infectious diseases, an increasingly recognized cause of global wildlife extinctions worldwide, particularly in avian populations considered vulnerable or endangered.

Malaria in Venezuela: changes in the complexity of infection reflects the increment in transmission intensity.

BACKGROUND: Malaria incidence has reached staggering numbers in Venezuela. Commonly, Bolívar State accounted for approximately 70% of the country cases every year. Most cases cluster in the Sifontes municipality, a region characterized by an extractive economy, including gold mining. An increase in migration to Sifontes, driven by gold mining, fueled a malaria spillover to the rest of the country and the region. Here samples collected in 2018 were compared with a previous study of 2003/2004 to describe changes in the parasites population structures and the frequency of point mutations linked to anti-malarial drugs. METHODS: A total of 88 Plasmodium falciparum and 94 Plasmodium vivax isolates were collected in 2018 and compared with samples from 2003/2004 (106 P. falciparum and 104 P. vivax). For P. falciparum, mutations linked to drug resistance (Pfdhfr, Pfdhps, and Pfcrt) and the Pfk13 gene associated with artemisinin delayed parasite clearance, were analysed. To estimate the multiplicity of infection (MOI), and perform P. falciparum and P. vivax population genetic analyses, the parasites were genotyped by using eight standardized microsatellite loci. RESULTS: The P. falciparum parasites are still harbouring drug-resistant mutations in Pfdhfr, Pfdhps, and Pfcrt. However, there was a decrease in the frequency of highly resistant Pfdhps alleles. Mutations associated with artemisinin delayed parasite clearance in the Pfk13 gene were not found. Consistent with the increase in transmission, polyclonal infections raised from 1.9% in 2003/2004 to 39% in 2018 in P. falciparum and from 16.3 to 68% in P. vivax. There is also a decrease in linkage disequilibrium. Bayesian clustering yields two populations linked to the time of sampling, showing that the parasite populations temporarily changed. However, the samples from 2003/2004 and 2018 have several alleles per locus in common without sharing multi-locus genotypes. CONCLUSIONS: The frequency of mutations linked with drug resistance in P. falciparum shows only changes in Pfdhps. Observations presented here are consistent with an increase in transmission from the previously circulating parasites. Following populations longitudinally, using molecular surveillance, provides valuable information in cases such as Venezuela with a fluid malaria situation that is affecting the regional goals toward elimination.

Haemocystidium spp., a species complex infecting ancient aquatic turtles of the family Podocnemididae: First report of these parasites in Podocnemis vogli from the Orinoquia.

The genus Haemocystidium was described in 1904 by Castellani and Willey. However, several studies considered it a synonym of the genera Plasmodium or Haemoproteus. Recently, molecular evidence has shown the existence of a monophyletic group that corresponds to the genus Haemocystidium. Here, we further explore the clade Haemocystidium spp. by studying parasites from Testudines. A total of 193 individuals belonging to six families of Testudines were analyzed. The samples were collected in five localities in Colombia: Casanare, Vichada, Arauca, Antioquia, and Córdoba. From each individual, a blood sample was taken for molecular analysis, and peripheral blood smears were made, which were fixed and subsequently stained with Giemsa. The prevalence of Haemocystidium spp. was 1.55% (n = 3/193); all infected individuals belonged to Podocnemis vogli (Savanna Side-necked turtle) from the department of Vichada. This is the first report of Haemocystidium spp. in Colombia and in this turtle species. The phylogenetic analysis of a mitochondrial cytb fragment revealed Haemocystidium spp. as a monophyletic group and as a sister taxon of Haemoproteus catharti and the genus Plasmodium. Haemocystidium spp. are difficult to identify by morphology only. As a result, it is possible that some of the taxa, such as Haemocystidium (Simondia) pacayae, represent a species complex. The parasite found in our study is morphologically indistinguishable from Haemocystidium (Simondia) pacayae reported in Peru. However, the new lineage found in P. vogli shows a genetic distance of 0.02 with Hae. pacayae and 0.04 with Hae. peltocephali. It is proposed that this divergent lineage might be a new species. Nevertheless, additional molecular markers and ecological features could support this hypothesis in the future.

Malaria Molecular Epidemiology: An Evolutionary Genetics Perspective.

Malaria is a vector-borne disease that involves multiple parasite species in a variety of ecological settings. However, the parasite species causing the disease, the prevalence of subclinical infections, the emergence of drug resistance, the scale-up of interventions, and the ecological factors affecting malaria transmission, among others, are aspects that vary across areas where malaria is endemic. Such complexities have propelled the study of parasite genetic diversity patterns in the context of epidemiologic investigations. Importantly, molecular studies indicate that the time and spatial distribution of malaria cases reflect epidemiologic processes that cannot be fully understood without characterizing the evolutionary forces shaping parasite population genetic patterns. Although broad in scope, this review in the Microbiology Spectrum Curated Collection: Advances in Molecular Epidemiology highlights the need for understanding population genetic concepts when interpreting parasite molecular data. First, we discuss malaria complexity in terms of the parasite species involved. Second, we describe how molecular data are changing our understanding of malaria incidence and infectiousness. Third, we compare different approaches to generate parasite genetic information in the context of epidemiologically relevant questions related to malaria control. Finally, we describe a few Plasmodium genomic studies as evidence of how these approaches will provide new insights into the malaria disease dynamics. *This article is part of a curated collection.