Plasmodium falciparum malaria remains one of the world's foremost health problems, primarily in highly endemic regions such as Sub-Saharan Africa, where it is responsible for substantial morbidity, mortality and economic losses. Malaria is a significant cause of severe disease and death in pregnant women and newborns, with pathogenesis being associated with expression of a unique variant of the multidomain Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) called VAR2CSA. Here, we characterize the polymorphism of the DBL3X domain of VAR2CSA and identify regions under selective pressure among placental parasites from women living in endemic western Kenya. In addition to significant levels of polymorphism, our analysis reveals evidence for diversification through intra-segmental recombination and novel mutations that likely contributed to the high number of unique VAR2CSA sequence types identified in this study. Interestingly, we also identified a number of critical residues that may be implicated in immune evasion through switching (or toggling) to alternative amino acids, including an arginine residue within the predicted binding pocket in subdomain III, which was previously implicated in binding to placental CSA. Overall, these findings are important for understanding parasite diversity in pregnant women and will be useful for identifying epitopes and variants of DBL3X to be included in a vaccine against placental malaria.

The genus Leishmania includes approximately 53 species, 20 of which cause human leishmaniais; a significant albeit neglected tropical disease. Leishmaniasis has afflicted humans for millennia, but how ancient is Leishmania and where did it arise? These questions have been hotly debated for decades and several theories have been proposed. One theory suggests Leishmania originated in the Palearctic, and dispersed to the New World via the Bering land bridge. Others propose that Leishmania evolved in the Neotropics. The Multiple Origins theory suggests that separation of certain Old World and New World species occurred due to the opening of the Atlantic Ocean. Some suggest that the ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia evolved on Gondwana between 90 and 140 million years ago. In the present study a detailed molecular and morphological characterisation was performed on a novel Australian trypanosomatid following its isolation in Australia's tropics from the native black fly, Simulium (Morops) dycei Colbo, 1976. Phylogenetic analyses were conducted and confirmed this parasite as a sibling to Zelonia costaricensis, a close relative of Leishmania previously isolated from a reduviid bug in Costa Rica. Consequently, this parasite was assigned the name Zelonia australiensis sp. nov. Assuming Z. costaricensis and Z. australiensis diverged when Australia and South America became completely separated, their divergence occurred between 36 and 41 million years ago at least. Using this vicariance event as a calibration point for a phylogenetic time tree, the common ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia appeared in Gondwana approximately 91 million years ago. Ultimately, this study contributes to our understanding of trypanosomatid diversity, and of Leishmania origins by providing support for a Gondwanan origin of dixenous parasitism in the Leishmaniinae.

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

The spread of drug resistance to antimalarial treatments poses a serious public health risk globally. To combat this risk, molecular surveillance of drug resistance is imperative. We report the prevalence of mutations in the Plasmodium falciparum kelch 13 propeller domain associated with partial artemisinin resistance, which we determined by using Sanger sequencing samples from patients enrolled in therapeutic efficacy studies from 9 sub-Saharan countries during 2014-2018. Of the 2,865 samples successfully sequenced before treatment (day of enrollment) and on the day of treatment failure, 29 (1.0%) samples contained 11 unique nonsynonymous mutations and 83 (2.9%) samples contained 27 unique synonymous mutations. Two samples from Kenya contained the S522C mutation, which has been associated with delayed parasite clearance; however, no samples contained validated or candidate artemisinin-resistance mutations.

Hierarchical clustering of pathogen genotypes is widely used to complement epidemiologic investigations of outbreaks. Investigators must dissect trees to obtain genetic partitions that provide epidemiologists with meaningful information. Statistical approaches to tree dissection often require a user-defined parameter to predict the optimal partition number and augmenting this parameter can drastically impact resultant partition memberships. Here, we demonstrate how to optimize a given tree dissection parameter to maximize accuracy irrespective of the tree dissection method used. We hierarchically clustered 1,873 genotypes of the foodborne pathogen Cyclospora spp., including 587 possessing links to historic outbreaks. We dissected the resulting tree using a statistical method requiring users to select the value of a 'stringency parameter' (s), with a recommended value of 95% to 99.5%. We dissected this hierarchical tree across s-values from 94% to 99.5% (at increments of 0.25%), to identify a value that maximized partitioning accuracy, defined as the degree to which genetic partitions conform to known epidemiologic groupings. We show that s-values of 96.5% and 96.75% yield the highest accuracy (> 99.9%) when clustering Cyclospora sp. isolates with known epidemiologic linkages. In practice, the optimized s-value will generate robust genetic partitions comprising isolates likely derived from a common food source, even when the epidemiologic grouping is not known prior to genetic clustering. While the s-value is specific to the tree dissection method used here, the optimization approach described could be applied to any parameter/method used to dissect hierarchical trees.

The recent emergence of artemisinin resistance in the Greater Mekong Subregion poses a major threat to the global effort to control malaria. Tracking the spread and evolution of artemisinin-resistant parasites is critical in aiding efforts to contain the spread of resistance. A total of 417 patient samples from the year 2007, collected during malaria surveillance studies across ten provinces in Thailand, were genotyped for the candidate Plasmodium falciparum molecular marker of artemisinin resistance K13. Parasite genotypes were examined for K13 propeller mutations associated with artemisinin resistance, signatures of positive selection, and for evidence of whether artemisinin-resistant alleles arose independently across Thailand. A total of seven K13 mutant alleles were found (N458Y, R539T, E556D, P574L, R575K, C580Y, S621F). Notably, the R575K and S621F mutations have previously not been reported in Thailand. The most prevalent artemisinin resistance-associated K13 mutation, C580Y, carried two distinct haplotype profiles that were separated based on geography, along the Thai-Cambodia and Thai-Myanmar borders. It appears these two haplotypes may have independent evolutionary origins. In summary, parasites with K13 propeller mutations associated with artemisinin resistance were widely present along the Thai-Cambodia and Thai-Myanmar borders prior to the implementation of the artemisinin resistance containment project in the region.

Plasmodium falciparum resistance to artemisinin has emerged in the Greater Mekong Subregion and now poses a threat to malaria control and prevention. Recent work has identified mutations in the kelch propeller domain of the P. falciparum K13 gene to be associated artemisinin resistance as defined by delayed parasite clearance and ex vivo ring stage survival assays. Species specific primers for the two most prevalent human malaria species, P. falciparum and P. vivax, were designed and tested on multiple parasite isolates including human, rodent, and non- humans primate Plasmodium species. The new protocol described here using the species specific primers only amplified their respective species, P. falciparum and P. vivax, and did not cross react with any of the other human malaria Plasmodium species. We provide an improved species specific PCR and sequencing protocol that could be effectively used in areas where both P. falciparum and P. vivax are circulating. To design this improved protocol, the kelch gene was analyzed and compared among different species of Plasmodium. The kelch propeller domain was found to be highly conserved across the mammalian Plasmodium species.

Cyclospora cayetanensis is an intestinal parasite responsible for the diarrheal illness, cyclosporiasis. Molecular genotyping, using targeted amplicon sequencing, provides a complementary tool for outbreak investigations, especially when epidemiological data are insufficient for linking cases and identifying clusters. The goal of this study was to identify candidate genotyping markers using a novel workflow for detection of segregating single nucleotide polymorphisms (SNPs) in C. cayetanensis genomes. Four whole C. cayetanensis genomes were compared using this workflow and four candidate markers were selected for evaluation of their genotyping utility by PCR and Sanger sequencing. These four markers covered 13 SNPs and resolved parasites from 57 stool specimens, differentiating C. cayetanensis into 19 new unique genotypes.

Previously, it was suggested that haemadipsid leeches represent an important vector of trypanosomes amongst native animals in Australia. Consequently, Chtonobdella bilineata leeches were investigated for the presence of trypanosome species by polymerase chain reaction (PCR), DNA sequencing and in vitro isolation. Phylogenetic analysis ensued to further define the populations present. PCR targeting the 28S rDNA demonstrated that over 95% of C. bilineata contained trypanosomes; diversity profiling by deep amplicon sequencing of 18S rDNA indicated the presence of four different clusters related to the Trypanosoma (Megatrypanum) theileri. Novy–MacNeal–Nicolle slopes with liquid overlay were used to isolate trypanosomes into culture that proved similar in morphology to Trypanosoma cyclops in that they contained a large numbers of acidocalcisomes. Phylogeny of 18S rDNA/GAPDH/ND5 DNA sequences from primary cultures and subclones showed the trypanosomes were monophyletic, with T. cyclops as a sister group. Blood-meal analysis of leeches showed that leeches primarily contained blood from swamp wallaby (Wallabia bicolour), human (Homo sapiens) or horse (Equus sp.). The leech C. bilineata is a host for at least five lineages of Trypanosoma sp. and these are monophyletic with T. cyclops; we propose Trypanosoma cyclops australiensis as a subspecies of T. cyclops based on genetic similarity and biogeography considerations.

Recent anti-malarial resistance monitoring in Angola has shown efficacy of artemether-lumefantrine (AL) in certain sites approaching the key 90% lower limit of efficacy recommended for artemisinin-based combination therapy. In addition, a controversial case of malaria unresponsive to artemisinins was reported in a patient infected in Lunda Sul Province in 2013.

The ability to identify mixed-species infections and track the origin of Plasmodium parasites can further enhance the development of treatment and prevention recommendations as well as outbreak investigations. Here, we explore the utility of using the full Plasmodium mitochondrial genome to classify Plasmodium species, detect mixed infections, and infer the geographical origin of imported P. falciparum parasites to the United States (U.S.). Using the recently developed standardized, high-throughput Malaria Resistance Surveillance (MaRS) protocol, the full Plasmodium mitochondrial genomes of 265 malaria cases imported to the U.S. from 2014-2017 were sequenced and analyzed. P. falciparum infections were found in 94.7% (251/265) of samples. Five percent (14/265) of samples were identified as mixed- Plasmodium species or non-P. falciparum, including P. vivax, P. malariae, P. ovale curtisi, and P. ovale wallikeri. P. falciparum mitochondrial haplotypes analysis revealed greater than eighteen percent of samples to have at least two P. falciparum mitochondrial genome haplotypes, indicating either heteroplasmy or multi-clonal infections. Maximum-likelihood phylogenies of 912 P. falciparum mitochondrial genomes with known country origin were used to infer the geographical origin of thirteen samples from persons with unknown travel histories as: Africa (country unspecified) (n = 10), Ghana (n = 1), Southeast Asia (n = 1), and the Philippines (n = 1). We demonstrate the utility and current limitations of using the Plasmodium mitochondrial genome to classify samples with mixed-infections and infer the geographical origin of imported P. falciparum malaria cases to the U.S. with unknown travel history.

The World Health Organization recommends regular therapeutic efficacy studies (TES) to monitor the performance of first and second-line anti-malarials. In 2016, efficacy and safety of artemether-lumefantrine (AL) for the treatment of uncomplicated falciparum malaria were assessed through a TES conducted between April and October 2016 at four sentinel sites of Kibaha, Mkuzi, Mlimba, and Ujiji in Tanzania. The study also assessed molecular markers of artemisinin and lumefantrine (partner drug) resistance.

Sexually reproducing pathogens such as Cyclospora cayetanensis often produce genetically heterogeneous infections where the number of unique sequence types detected at any given locus varies depending on which locus is sequenced. The genotypes assigned to these infections quickly become complex when additional loci are analysed. This genetic heterogeneity confounds the utility of traditional sequence-typing and phylogenetic approaches for aiding epidemiological trace-back, and requires new methods to address this complexity. Here, we describe an ensemble of two similarity-based classification algorithms, including a Bayesian and heuristic component that infer the relatedness of C. cayetanensis infections. The ensemble requires a set of haplotypes as input and assigns arbitrary distances to specimen pairs reflecting their most likely relationships. The approach was applied to data generated from a test cohort of 88 human fecal specimens containing C. cayetanensis, including 30 from patients whose infections were associated with epidemiologically defined outbreak clusters of cyclosporiasis. The ensemble assigned specimens to plausible clusters of genetically related infections despite their complex haplotype composition. These relationships were corroborated by a significant number of epidemiological linkages (P < 0.0001) suggesting the ensemble's utility for aiding epidemiological trace-back investigations of cyclosporiasis.

The current first-line treatments for uncomplicated malaria recommended by the National Malaria Control Programme in Mali are artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ). From 2015 to 2016, an in vivo study was carried out to assess the clinical and parasitological responses to AL and ASAQ in Sélingué, Mali.

Targeted amplicon deep sequencing (TADS) of the 16S rRNA gene is commonly used to explore and characterize bacterial microbiomes. Meanwhile, attempts to apply TADS to the detection and characterization of entire parasitic communities have been hampered since conserved regions of many conserved parasite genes, such as the 18S rRNA gene, are also conserved in their eukaryotic hosts. As a result, targeted amplification of 18S rRNA from clinical samples using universal primers frequently results in competitive priming and preferential amplification of host DNA. Here, we describe a novel method that employs a single pair of universal primers to capture all blood-borne parasites while reducing host 18S rRNA template and enhancing the amplification of parasite 18S rRNA for TADS. This was achieved using restriction enzymes to digest the 18S rRNA gene at cut sites present only in the host sequence prior to PCR amplification.

Neospora caninum is an apicomplexan parasite responsible for neosporosis, a disease causing hind limb paralysis in dogs and abortion in cattle, resulting in substantial economic losses to beef and dairy industries. Marked differences in pathogenicity exist between N. caninum strains suggesting that intrinsic genetic differences exist between them. These differences likely exist in genes expressed during the tachyzoite lifecycle stage which is responsible for the pathogenesis of neosporosis. An improved understanding of these genetic differences is essential to understanding N. caninum virulence, though such knowledge is scarce. Using a variant detection workflow we compared the tachyzoite transcriptomes of two N. caninum strains with different virulence properties: NC-Liverpool (virulent) and NC-Nowra (avirulent). This workflow identified 3130 SNPs and 6123 indels between the strains, and nine markers capturing 30 variants were Sanger sequenced for both strains. Sequencing of these loci was extended to an additional eight strains and subsequent phylogenetic analysis supported a genetic population structure comprised of two major clades with no geographical segregation. Sequence polymorphisms within coding regions of tachyzoite-associated genes were concentrated on chromosomes XI and XII, with 19 distinct tachyzoite-associated SNP hotspot regions identified within coding regions of the N. caninum nuclear genome. The variants were predominantly located in loci associated with protein binding, protein-protein interactions, transcription, and translation. Furthermore, 468 nonsynonymous SNPs identified within protein-coding genes were associated with protein kinase activity, protein binding, protein phosphorylation, and proteolysis. This work may implicate these processes and the specific proteins involved as novel effectors of N. caninum tachyzoite virulence.

Accurate diagnosis of malaria infections continues to be challenging and elusive, especially in the detection of submicroscopic infections. Developing new malaria diagnostic tools that are sensitive enough to detect low-level infections, user friendly, cost effective and capable of performing large scale diagnosis, remains critical. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. ovale by real-time PCR. In our study, a total of 173 clinical samples, consisting of different malaria species, were utilized to test this novel PET-PCR primer. The sensitivity and specificity were calculated using nested-PCR as the reference test. The novel primer set demonstrated a sensitivity of 97.5% and a specificity of 99.2% (95% CI 85.2-99.8% and 95.2-99.9% respectively). Furthermore, the limit of detection for P. ovale was found to be 1 parasite/μl. The PET-PCR assay is a new molecular diagnostic tool with comparable performance to other commonly used PCR methods. It is relatively easy to perform, and amiable to large scale malaria surveillance studies and malaria control and elimination programs. Further field validation of this novel primer will be helpful to ascertain the utility for large scale malaria screening programs.

Cyclosporiasis is an infection caused by Cyclospora cayetanensis, which is acquired by consumption of contaminated fresh food or water. In the United States, cases of cyclosporiasis are often associated with foodborne outbreaks linked to imported fresh produce or travel to disease-endemic countries. Epidemiologic investigation has been the primary method for linking outbreak cases. A molecular typing marker that can identify genetically related samples would be helpful in tracking outbreaks. We evaluated the mitochondrial junction region as a potential genotyping marker. We tested stool samples from 134 laboratory-confirmed cases in the United States by using PCR and Sanger sequencing. All but 2 samples were successfully typed and divided into 14 sequence types. Typing results were identical among samples within each epidemiologically defined case cluster for 7 of 10 clusters. These findings suggest that this marker can distinguish between distinct case clusters and might be helpful during cyclosporiasis outbreak investigations.

Anti-malarial resistance is a threat to recent gains in malaria control. This study aimed to assess the efficacy and safety of artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) in the management of uncomplicated malaria and to measure the prevalence of molecular markers of resistance of Plasmodium falciparum in sentinel sites in Maferinyah and Labé Health Districts in Guinea in 2016.

Targeted amplicon deep sequencing (TADS) has enabled characterization of diverse bacterial communities, yet the application of TADS to communities of parasites has been relatively slow to advance. The greatest obstacle to this has been the genetic diversity of parasitic agents, which include helminths, protozoa, arthropods, and some acanthocephalans. Meanwhile, universal amplification of conserved loci from all parasites without amplifying host DNA has proven challenging. Pan-eukaryotic PCRs preferentially amplify the more abundant host DNA, obscuring parasite-derived reads following TADS. Flaherty et al. (2018) described a pan-parasitic TADS method involving amplification of eukaryotic 18S rDNA regions possessing restriction sites only in vertebrates. Using this method, host DNA in total DNA extracts could be selectively digested prior to PCR using restriction enzymes, thereby increasing the number of parasite-derived reads obtained following NGS. This approach showed promise though was only as sensitive as conventional PCR.