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.

Drug resistance within the major malaria parasites Plasmodium vivax and Plasmodium falciparum threatens malaria control and elimination in Southeast Asia. Plasmodium vivax first-line treatment drug is chloroquine together with primaquine, and the first-line treatment for P. falciparum malaria is artemisinin in combination with a partner drug. Plasmodium vivax and P. falciparum parasites resistant to their respective first-line therapies are now found within Southeast Asia. The resistance perimeters may include high transmission regions of Southern Thailand which are underrepresented in surveillance efforts.

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.

To monitor drug resistance in Plasmodium vivax, a multidrug resistance 1 (Pvmdr1) gene and a putative transporter protein (Pvcrt-o) gene were used as molecular markers for chloroquine resistance. The biomarkers, the dihydrofolate reductase (Pvdhfr) gene and the dihydropteroate synthetase (Pvdhps) gene, were also used for the detection of resistance to sulphadoxine-pyrimethamine (SP); this drug is often accidentally used to treat P. vivax infections. Clinical blood samples (n = 120) were collected from patients who had been to one of eight malaria-endemic countries and diagnosed with P. vivax infection. The chloroquine resistance marker, the Pvmdr1 gene, showed F976:L1076 mutations and L1076 mutation. A K10 insertion in the Pvcrt-o gene was also found among the samples successfully sequenced. A combination of L/I57:R58:M61:T117 mutations in the Pvdhfr gene and G383:G553 mutations in the Pvdhps gene were also observed. Mutations found in these genes indicate that drug resistance is present in these eight countries. Whether or not countries are using chloroquine to treat P. vivax, there appears to be an increase in mutation numbers in resistance gene markers. The detected changes in mutation rates of these genes do suggest that there is still a trend towards increasing P. vivax resistance to chloroquine. The presence of the mutations associated with SP resistance indicates that P. vivax has had exposure to SP and this may be a consequence of either misdiagnosis or coinfections with P. falciparum in the past.

The extensive use of insecticides for vector control has led to the development of insecticide resistance in Aedes aegypti populations on a global scale, which has significantly compromised control actions. Insecticide resistance, and its underlying mechanisms, has been investigated in several countries, mostly in South American and Asian countries. In Africa, however, studies reporting insecticide resistance are rare and data on resistance mechanisms, notably knockdown resistance (kdr) mutations, is scarce. In this study, the recently described V410L kdr mutation is reported for the first time in old world Ae. aegypti populations, namely from Angola and Madeira island. Two additional kdr mutations, V1016I and F1534C, are also reported for the first time in populations from Angola and Cape Verde. Significant associations with the resistance phenotype were found for both V410L and V1016I individually as well as for tri-locus genotypes in the Angolan population. However, no association was found in Madeira island, probably due to the presence of a complex pattern of multiple insecticide resistance mechanisms in the local Ae. aegypti population. These results suggest that populations carrying the same kdr mutations may respond differently to the same insecticide, stressing the need for complementary studies when assessing the impact of kdr resistance mechanisms in the outcome of insecticide-based control strategies.

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.

Advancements in next-generation sequencing techniques have led to a substantial increase in the genomic information available for analyses in evolutionary biology. As such, this data requires the exponential growth in bioinformatic methods and expertise required to understand such vast quantities of genomic data. Alignment-free phylogenomics offer an alternative approach for large-scale analyses that may have the potential to address these challenges. The evolutionary relationships between various species within the trypanosomatid family, specifically members belonging to the genera Leishmania and Trypanosoma have been extensively studies over the last 30 years. However, there is a need for a more exhaustive analysis of the Trypanosomatidae, summarising the evolutionary patterns amongst the entire family of these important protists. The mitochondrial DNA of the trypanosomatids, better known as the kinetoplast, represents a valuable taxonomic marker given its unique presence across all kinetoplastid protozoans. The aim of this study was to validate the reliability and robustness of alignment-free approaches for phylogenomic analyses and its applicability to reconstruct the evolutionary relationships between the trypanosomatid family. In the present study, alignment-free analyses demonstrated the strength of these methods, particularly when dealing with large datasets compared to the traditional phylogenetic approaches. We present a maxicircle genome phylogeny of 46 species spanning the trypanosomatid family, demonstrating the superiority of the maxicircle for the analysis and taxonomic resolution of the Trypanosomatidae.

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.

A significant proportion of vulnerable people in sub-Saharan Africa (SSA) remain at risk for contracting diarrhoeal diseases due to the presence of many risk factors facilitating their transmission. A systematic review of published articles from the SSA region was done to determine the prevalence and types of diarrhoeal pathogens in circulation, based on a search of databases, including EBSCO host, PubMed, Scopus, Science Direct, Google scholar and Web of Science was done between September 2009 and December 2010. Data were summarized from 27 studies, with pooled data analysed and reported. Pathogens were isolated from between 26.8-65.6% of cases, with an overall isolation rate of 55.7% (95% CI, 48.2-62.9%). Isolation rates were highest amongst adult cases followed by children, and the odds of isolating a pathogen was greater in diarrhoeal cases (Odds Ratio 4.93 (95% CI, 1.99 to 12.23), than in asymptomatic controls. Overall isolation ranged from 8% to 99%; and heterogeneity testing suggests differences between age groups (Q=5.806; df=2, P=0. 055). Mixed E. coli spp., (29.95%), Cryptosporidium (21.52%), Cyclospora (18%), Entamoeba. (13.8%), Shigella spp. (10.49%), Salmonella spp. (8.36%), and Campylobacter spp. (8.33%), were most commonly reported, and rotavirus was the most common virus isolated. This is the first review to look at the range of enteric pathogens circulating in SSA, and has confirmed high rates of isolation of pathogens from diarrhoeal cases. Public health practitioners can use this information to understanding the challenges related to diarrhoeal illness and set priorities for their prevention and control.

Energy dissipation and the transfer rate of adsorbed molecules do not only determine the rates of chemical reactions but are also a key factor that often dictates the growth of organic thin films. Here, we present a study of the surface dynamical motion of cobalt phthalocyanine (CoPc) on Ag(100) in reciprocal space based on the helium spin-echo technique in comparison with previous scanning tunnelling microscopy studies. It is found that the activation energy for lateral diffusion changes from 150 meV at 45-50 K to ≈100 meV at 250-350 K, and that the process goes from exclusively single jumps at low temperatures to predominantly long jumps at high temperatures. We thus illustrate that while the general diffusion mechanism remains similar, upon comparing the diffusion process over widely divergent time scales, indeed different jump distributions and a decrease of the effective diffusion barrier are found. Hence a precise molecular-level understanding of dynamical processes and thin film formation requires following the dynamics over the entire temperature scale relevant to the process. Furthermore, we determine the diffusion coefficient and the atomic-scale friction of CoPc and establish that the molecular motion on Ag(100) corresponds to a low friction scenario as a consequence of the additional molecular degrees of freedom.

Detection of histidine-rich protein 2 (HRP2) from the malaria parasite Plasmodium falciparum provides evidence for active or recent infection, and is utilized for both diagnostic and surveillance purposes, but current laboratory immunoassays for HRP2 are hindered by low sensitivities and high costs. Here we present a new HRP2 immunoassay based on antigen capture through a bead-based system capable of detecting HRP2 at sub-picogram levels. The assay is highly specific and cost-effective, allowing fast processing and screening of large numbers of samples. We utilized the assay to assess results of HRP2-based rapid diagnostic tests (RDTs) in different P. falciparum transmission settings, generating estimates for true performance in the field. Through this method of external validation, HRP2 RDTs were found to perform well in the high-endemic areas of Mozambique and Angola with 86.4% and 73.9% of persons with HRP2 in their blood testing positive by RDTs, respectively, and false-positive rates of 4.3% and 0.5%. However, in the low-endemic setting of Haiti, only 14.5% of persons found to be HRP2 positive by the bead assay were RDT positive. Additionally, 62.5% of Haitians showing a positive RDT test had no detectable HRP2 by the bead assay, likely indicating that these were false positive tests. In addition to RDT validation, HRP2 biomass was assessed for the populations in these different settings, and may provide an additional metric by which to estimate P. falciparum transmission intensity and measure the impact of interventions.

The broad heterogeneity of antigen-antibody interactions brings tremendous challenges to the design of a widely applicable learning algorithm to identify conformational B-cell epitopes. Besides the intrinsic heterogeneity introduced by diverse species, extra heterogeneity can also be introduced by various data sources, adding another layer of complexity and further confounding the research.

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 M2 muscarinic receptor is the prototypic model of allostery in GPCRs, yet the molecular and the supramolecular determinants of such effects are unknown. Monomers and oligomers of the M2 muscarinic receptor therefore have been compared to identify those allosteric properties that are gained in oligomers. Allosteric interactions were monitored by means of a FRET-based sensor of conformation at the allosteric site and in pharmacological assays involving mutants engineered to preclude intramolecular effects. Electrostatic, steric, and conformational determinants of allostery at the atomic level were examined in molecular dynamics simulations. Allosteric effects in monomers were exclusively negative and derived primarily from intramolecular electrostatic repulsion between the allosteric and orthosteric ligands. Allosteric effects in oligomers could be positive or negative, depending upon the allosteric-orthosteric pair, and they arose from interactions within and between the constituent protomers. The complex behavior of oligomers is characteristic of muscarinic receptors in myocardial preparations.

In this study, the BALB/c and Qs mouse responses to infection by the parasite Neospora caninum were investigated in order to identify host response mechanisms. Investigation was done using gene set (enrichment) analyses of microarray data. GSEA, MANOVA, Romer, subGSE and SAM-GS were used to study the contrasts Neospora strain type, Mouse type (BALB/c and Qs) and time post infection (6 hours post infection and 10 days post infection). The analyses show that the major signal in the core mouse response to infection is from time post infection and can be defined by gene ontology terms Protein Kinase Activity, Cell Proliferation and Transcription Initiation. Several terms linked to signaling, morphogenesis, response and fat metabolism were also identified. At 10 days post infection, genes associated with fatty acid metabolism were identified as up regulated in expression. The value of gene set (enrichment) analyses in the analysis of microarray data is discussed.

B-cell epitope is a small area on the surface of an antigen that binds to an antibody. Accurately locating epitopes is of critical importance for vaccine development. Compared with wet-lab methods, computational methods have strong potential for efficient and large-scale epitope prediction for antigen candidates at much lower cost. However, it is still not clear which features are good determinants for accurate epitope prediction, leading to the unsatisfactory performance of existing prediction methods.