Simultaneous targeted amplicon deep sequencing and library preparation for a time and cost-effective universal parasite diagnostic sequencing approach.

We recently described a targeted amplicon deep sequencing (TADS) strategy that utilizes a nested PCR targeting the 18S rDNA gene of blood-borne parasites. The assay facilitates selective digestion of host DNA by targeting enzyme restriction sites present in vertebrates but absent in parasites. This enriching of parasite-derived amplicon drastically reduces the proportion of host-derived reads during sequencing and results in the sensitive detection of several clinically important blood parasites including Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes. Despite these promising results, high costs and the laborious nature of metagenomics sequencing are prohibitive to the routine use of this assay in most laboratories. We describe and evaluate a new metagenomic approach that utilizes a set of primers modified from our original assay that incorporates Illumina barcodes and adapters during the PCR steps. This modification makes amplicons immediately compatible with sequencing on the Illumina MiSeq platform, removing the need for a separate library preparation, which is expensive and time-consuming. We compared this modified assay to our previous nested TADS assay in terms of preparation speed, limit of detection (LOD), and cost. Our modifications reduced assay turnaround times from 7 to 5 days. The cost decreased from approximately $40 per sample to $11 per sample. The modified assay displayed comparable performance in the detection and differentiation of human-infecting Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes in clinical samples. The LOD of this modified approach was determined for malaria parasites and remained similar to that previously reported for our earlier assay (0.58 Plasmodium falciparum parasites/µL of blood). These modifications markedly reduced costs and turnaround times, making the assay more amenable to routine diagnostic applications.

Assessment of the circulation of Dirofilaria immitis in dogs from northern Portugal through combined analysis of antigens, DNA and parasite forms in blood.

Dirofilariasis is a vector-borne disease frequent in many countries. Not only infected dogs, but also cats and wild canids (including wolves and foxes), represent important sources of infection for mosquitoes, which are the pathogen vectors. The disease is endemic in Mediterranean countries with increasing incidence in Italy, France, Greece and Spain, but limited epidemiological data is available from Portugal regarding its distribution and impact. Aiming to clarify this, canine whole blood samples (n = 244) from the north of Portugal were tested for Dirofilaria spp. antigens by use of a commercial rapid immunomigration test. Polymerase chain reaction (PCR) and the modified Knott test were also used to assess the presence of microfilariae. Results were also compared to assess the performance of each test used. Of the 244 animals tested, 118 (48.4%) were positive for Dirofilaria immitis (heartworm) in the serological adult worm rapid antigen detection test, and 36 (14.8%) had circulating microfilariae, identified as D. immitis. A combined positivity of 51.6% (126/244) was found. Results indicate that the risk of exposure to D. immitis in dogs is high in this region of Portugal, and that prophylaxis against the parasite is advisable to decrease the occurrence of canine infection and disease. The present study highlights the diagnostic value of serological and molecular tests in determining the prevalence of D. immitis.

The evolution of social parasitism in Formica ants revealed by a global phylogeny.

Studying the behavioral and life history transitions from a cooperative, eusocial life history to exploitative social parasitism allows for deciphering the conditions under which changes in behavior and social organization lead to diversification. The Holarctic ant genus Formica is ideally suited for studying the evolution of social parasitism because half of its 172 species are confirmed or suspected social parasites, which includes all three major classes of social parasitism known in ants. However, the life history transitions associated with the evolution of social parasitism in this genus are largely unexplored. To test competing hypotheses regarding the origins and evolution of social parasitism, we reconstructed a global phylogeny of Formica ants. The genus originated in the Old World ∼30 Ma ago and dispersed multiple times to the New World and back. Within Formica, obligate dependent colony-founding behavior arose once from a facultatively polygynous common ancestor practicing independent and facultative dependent colony foundation. Temporary social parasitism likely preceded or arose concurrently with obligate dependent colony founding, and dulotic social parasitism evolved once within the obligate dependent colony-founding clade. Permanent social parasitism evolved twice from temporary social parasitic ancestors that rarely practiced colony budding, demonstrating that obligate social parasitism can originate from a facultative parasitic background in socially polymorphic organisms. In contrast to permanently socially parasitic ants in other genera, the high parasite diversity in Formica likely originated via allopatric speciation, highlighting the diversity of convergent evolutionary trajectories resulting in nearly identical parasitic life history syndromes.

Intestinal parasitic infections in a population of BaAka Pygmies inhabiting the Congo Basin in the Central African Republic.

INTRODUCTION: Pygmy tribes inhabit tropical environment of Central Africa. After expulsion from their original habitat by the Bantu people, they settled in a local forest ecosystem where they live with very low sanitary standards. Their actual morbidity remains unknown. OBJECTIVE: The aim of the study was to analyze the prevalence of intestinal parasitic infections in BaAka Pygmies inhabiting the Congo Basin in the Central African Republic. MATERIAL AND METHODS: The study was conducted in 2015, and involved a group of 950 Pygmies living inthe Sangha-Mbaere and Lobaye prefectures. Single stool samples were collected from study participants, fixed in 10% formalin, transported from Africa to Europe, and analyzed by light microscopy using 5 different diagnostic methods (direct smear, decantation with distilled water, Fülleborne's flotation, Kato-Miura thick smear, DiaSyS/PARASYS system sedimentation) at the Military Institute of Medicine in Warsaw, Poland. RESULTS: Microscopic examination revealed infections with 14 different species of intestinal nematodes, cestodes, trematodes and protozoa. According to the study findings, 90.5% of BaAka Pygmies were found to be infected with intestinal parasites, and 70.8% had mixed infections. Most of the pathogenic intestinal parasites were nematodes (85.0%), with Asrcaris lumbricoides (29.8%), hookworm (29.4%) and Trichuris trichiura (10.7%) being predominant. CONCLUSIONS: Poor sanitation, limited the high prevalence of intestinal parasitic infections in the community of Pygmies. The negative test results may prove the effectiveness of periodic deworming campaigns chich, implemented by non-governmental organizations, are voluntary with respect to the informed consent principle.

Parasitic infections as major cause of abattoir condemnations in cattle slaughtered at an Ethiopian abattoir: 10-year retrospective study.

Abattoirs are vital for gathering information on animal diseases and protecting the public from consuming infected or unhygienic meat. To assess the major reasons for organ and carcass condemnations and their financial implications, we reviewed 10-year abattoir records of slaughtered bovines between January 2005 and December 2014 at Kombolcha ELFORA abattoir, north-east Ethiopia. Of the 46,913 cattle slaughtered during that period, 17,963 (38.3%) had at least one disease condition. Lungs (10.67%) and liver (25%) were the most affected and condemned organs, followed by heart (1.53%), head (0.56%), tongue (0.17%) and kidney (0.32%). The major conditions responsible for condemnation were fasciolosis (49.89%), hydatid cyst (55.55%), pericarditis (78.2%), hydronephrosis (35.8%), abscess (71.7%) and abscess (43.9%), in liver, lung, heart, kidneys, head and tongue, respectively. The direct financial losses incurred from organ and carcass condemnation over the 10-year period amounted to ETB 1,219,399 (USD 61,946.9), with parasitic diseases such as fascioliasis and hydatidosis accounting for ETB 256,837.5 (USD 13,047.64) and ETB 170,827.5 (USD 8678.23) in losses, respectively. This study describes a significant loss of cheap and reliable sources of protein due to non-utilization of infected organs or carcasses, emphasizing the need to implement integrated approaches in disease surveillance and control programmes.

WaspBase: a genomic resource for the interactions among parasitic wasps, insect hosts and plants.

Insect pests reduce yield and cause economic losses, which are major problems in agriculture. Parasitic wasps are the natural enemies of many agricultural pests and thus have been widely used as biological control agents. Plants, phytophagous insects and parasitic wasps form a tritrophic food chain. Understanding the interactions in this tritrophic system should be helpful for developing parasitic wasps for pest control and deciphering the mechanisms of parasitism. However, the genomic resources for this tritrophic system are not well organized. Here, we describe the WaspBase, a new database that contains 573 transcriptomes of 35 parasitic wasps and the genomes of 12 parasitic wasps, 5 insect hosts and 8 plants. In addition, we identified long non-coding RNA, untranslated regions and 25 widely studied gene families from the genome and transcriptome data of these species. WaspBase provides conventional web services such as Basic Local Alignment Search Tool, search and download, together with several widely used tools such as profile hidden Markov model, Multiple Alignment using Fast Fourier Transform, automated alignment trimming and JBrowse. We also present a collection of active researchers in the field of parasitic wasps, which should be useful for constructing scientific networks in this field.

Parasitic infections and resource economy of Danish Iron Age settlement through ancient DNA sequencing.

In this study, we screen archaeological soil samples by microscopy and analyse the samples by next generation sequencing to obtain results with parasites at species level and untargeted findings of plant and animal DNA. Three separate sediment layers of an ancient man-made pond in Hoby, Denmark, ranging from 100 BC to 200 AD, were analysed by microscopy for presence of intestinal worm eggs and DNA analysis were performed to identify intestinal worms and dietary components. Ancient DNA of parasites, domestic animals and edible plants revealed a change in use of the pond over time reflecting the household practice in the adjacent Iron Age settlement. The most abundant parasite found belonged to the Ascaris genus, which was not possible to type at species level. For all sediment layers the presence of eggs of the human whipworm Trichuris trichiura and the beef tapeworm Taenia saginata suggests continuous disposal of human faeces in the pond. Moreover, the continuous findings of T. saginata further imply beef consumption and may suggest that cattle were living in the immediate surrounding of the site throughout the period. Findings of additional host-specific parasites suggest fluctuating presence of other domestic animals over time: Trichuris suis (pig), Parascaris univalens (horse), Taenia hydatigena (dog and sheep). Likewise, alternating occurrence of aDNA of edible plants may suggest changes in agricultural practices. Moreover, the composition of aDNA of parasites, plants and vertebrates suggests a significant change in the use of the ancient pond over a period of three centuries.

Inbreeding in stochastic subdivided mating systems: the genetic consequences of host spatial structure, aggregated transmission dynamics and life history characteristics in parasite populations.

Inbreeding in parasite populations can have important epidemiological and evolutionary implications. However, theoretical models have predominantly focussed on the evolution of parasite populations under strong selection or in epidemic situations, and our understanding of neutral gene dynamics in parasite populations at equilibrium has been limited to verbal arguments or conceptual models. This study focusses on how host-parasite population dynamics affects observed levels of inbreeding in a random sample of parasites from an infinite population of hosts by bridging traditional genetic and parasitological processes utilizing a backward-forward branching Markov process embedded within a flexible statistical framework, the logarithmic-poisson mixture model. My results indicate that levels of inbreeding in parasites are impacted by demographic and/or transmission dynamics (subdivided mating, aggregated transmission dynamics and host spatial structure), and that this inbreeding is poorly estimated by 'equilibrium' levels of inbreeding calculated assuming regular systems of mating. Specifically, the model reveals that at low levels of inbreeding (F ≤ 0.1), equilibrium levels of inbreeding are lower than those observed, while at high levels of inbreeding the opposite pattern occurs. The model also indicates that inbreeding could have important epidemiological implications (e.g., the spread of recessive drug resistance genes) by directly impacting the observed frequency of rare homozygotes in parasite populations. My results indicate that frequencies of rare homozygotes are affected by aggregated transmission dynamics and host spatial structure, and also that an increase in the frequency of rare homozygotes can be caused by a decrease in effective population size solely due to the presence of a subdivided breeding system.

Genome mining offers a new starting point for parasitology research.

Parasites including helminthes, protozoa, and medical arthropod vectors are a major cause of global infectious diseases, affecting one-sixth of the world's population, which are responsible for enormous levels of morbidity and mortality important and remain impediments to economic development especially in tropical countries. Prevalent drug resistance, lack of highly effective and practical vaccines, as well as specific and sensitive diagnostic markers are proving to be challenging problems in parasitic disease control in most parts of the world. The impressive progress recently made in genome-wide analysis of parasites of medical importance, including trematodes of Clonorchis sinensis, Opisthorchis viverrini, Schistosoma haematobium, S. japonicum, and S. mansoni; nematodes of Brugia malayi, Loa loa, Necator americanus, Trichinella spiralis, and Trichuris suis; cestodes of Echinococcus granulosus, E. multilocularis, and Taenia solium; protozoa of Babesia bovis, B. microti, Cryptosporidium hominis, Eimeria falciformis, E. histolytica, Giardia intestinalis, Leishmania braziliensis, L. donovani, L. major, Plasmodium falciparum, P. vivax, Trichomonas vaginalis, Trypanosoma brucei and T. cruzi; and medical arthropod vectors of Aedes aegypti, Anopheles darlingi, A. sinensis, and Culex quinquefasciatus, have been systematically covered in this review for a comprehensive understanding of the genetic information contained in nuclear, mitochondrial, kinetoplast, plastid, or endosymbiotic bacterial genomes of parasites, further valuable insight into parasite-host interactions and development of promising novel drug and vaccine candidates and preferable diagnostic tools, thereby underpinning the prevention and control of parasitic diseases.

Assessment of helminth biodiversity in wild rats using 18S rDNA based metagenomics.

Parasite diversity has important implications in several research fields including ecology, evolutionary biology and epidemiology. Wide-ranging analysis has been restricted because of the difficult, highly specialised and time-consuming processes involved in parasite identification. In this study, we assessed parasite diversity in wild rats using 18S rDNA-based metagenomics. 18S rDNA PCR products were sequenced using an Illumina MiSeq sequencer and the analysis of the sequences using the QIIME software successfully classified them into several parasite groups. The comparison of the results with those obtained using standard methods including microscopic observation of helminth parasites in the rat intestines and PCR amplification/sequencing of 18S rDNA from isolated single worms suggests that this new technique is reliable and useful to investigate parasite diversity.

Genome profiling of sterol synthesis shows convergent evolution in parasites and guides chemotherapeutic attack.

Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering drugs are blockbusters in human health, and inhibitors of ergosterol biosynthesis are widely used as antifungals. Inhibitors of ergosterol synthesis are also being developed for Chagas's disease, caused by Trypanosoma cruzi. Here we develop an in silico pipeline to globally evaluate sterol metabolism and perform comparative genomics. We generate a library of hidden Markov model-based profiles for 42 sterol biosynthetic enzymes, which allows expressing the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally groups eukaryote proteomes and reveals convergent evolution, in particular metabolic reduction in obligate endoparasites. We experimentally explore sterol metabolism by testing a set of sterol biosynthesis inhibitors against trypanosomatids, Plasmodium falciparum, Giardia, and mammalian cells, and by quantifying the expression levels of sterol biosynthetic genes during the different life stages of T. cruzi and Trypanosoma brucei. The phenotypic data correlate with genomic makeup for simvastatin, which showed activity against trypanosomatids. Other findings, such as the activity of terbinafine against Giardia, are not in agreement with the genotypic profile.

Molecular diagnosis in clinical parasitology: when and why?

Microscopic detection and morphological identification of parasites from clinical specimens are the gold standards for the laboratory diagnosis of parasitic infections. The limitations of such diagnostic assays include insufficient sensitivity and operator dependence. Immunoassays for parasitic antigens are not available for most parasitic infections and have not significantly improved the sensitivity of laboratory detection. Advances in molecular detection by nucleic acid amplification may improve the detection in asymptomatic infections with low parasitic burden. Rapidly accumulating genomic data on parasites allow the design of polymerase chain reaction (PCR) primers directed towards multi-copy gene targets, such as the ribosomal and mitochondrial genes, which further improve the sensitivity. Parasitic cell or its free circulating parasitic DNA can be shed from parasites into blood and excreta which may allow its detection without the whole parasite being present within the portion of clinical sample used for DNA extraction. Multiplex nucleic acid amplification technology allows the simultaneous detection of many parasitic species within a single clinical specimen. In addition to improved sensitivity, nucleic acid amplification with sequencing can help to differentiate different parasitic species at different stages with similar morphology, detect and speciate parasites from fixed histopathological sections and identify anti-parasitic drug resistance. The use of consensus primer and PCR sequencing may even help to identify novel parasitic species. The key limitation of molecular detection is the technological expertise and expense which are usually lacking in the field setting at highly endemic areas. However, such tests can be useful for screening important parasitic infections in asymptomatic patients, donors or recipients coming from endemic areas in the settings of transfusion service or tertiary institutions with transplantation service. Such tests can also be used for monitoring these recipients or highly immunosuppressed patients, so that early preemptive treatment can be given for reactivated parasitic infections while the parasitic burden is still low.

The genotypic structure of a multi-host bumblebee parasite suggests a role for ecological niche overlap.

The genotypic structure of parasite populations is an important determinant of ecological and evolutionary dynamics of host-parasite interactions with consequences for pest management and disease control. Genotypic structure is especially interesting where multiple hosts co-exist and share parasites. We here analyze the natural genotypic distribution of Crithidia bombi, a trypanosomatid parasite of bumblebees (Bombus spp.), in two ecologically different habitats over a time period of three years. Using an algorithm to reconstruct genotypes in cases of multiple infections, and combining these with directly identified genotypes from single infections, we find a striking diversity of infection for both data sets, with almost all multi-locus genotypes being unique, and are inferring that around half of the total infections are resulting from multiple strains. Our analyses further suggest a mixture of clonality and sexuality in natural populations of this parasite species. Finally, we ask whether parasite genotypes are associated with host species (the phylogenetic hypothesis) or whether ecological factors (niche overlap in flower choice) shape the distribution of parasite genotypes (the ecological hypothesis). Redundancy analysis demonstrates that in the region with relatively high parasite prevalence, both host species identity and niche overlap are equally important factors shaping the distribution of parasite strains, whereas in the region with lower parasite prevalence, niche overlap more strongly contributes to the distribution observed. Overall, our study underlines the importance of ecological factors in shaping the natural dynamics of host-parasite systems.

Evolution of eukaryotic microbial pathogens via covert sexual reproduction.

Sexual reproduction enables eukaryotic organisms to reassort genetic diversity and purge deleterious mutations, producing better-fit progeny. Sex arose early and pervades eukaryotes. Fungal and parasite pathogens once thought asexual have maintained cryptic sexual cycles, including unisexual or parasexual reproduction. As pathogens become niche and host adapted, sex appears to specialize to promote inbreeding and clonality yet maintain outcrossing potential. During self-fertile sexual modes, sex itself may generate genetic diversity de novo. Mating-type loci govern fungal sexual identity; how parasites establish sexual identity is unknown. Comparing and contrasting fungal and parasite sex promises to reveal how microbial pathogens evolved and are evolving.

Origin of the parasites of an invading species, the Australian cane toad (Bufo marinus): are the lungworms Australian or American?

Phylogeographical analyses that identify the geographical origin of parasites in invading species can clarify the parasites' potential for biological control of the invader and the risks posed by the parasite to native species. Our data on nuclear and mitochondrial genetic sequences show that the nematode lungworms (Rhabdias spp.) in invasive Australian populations of cane toads (Bufo marinus) are Rhabdias pseudosphaerocephala, a South American species. We did not find this lungworm species in any Australian frogs sympatric with cane toads, suggesting that the parasite does not attack Australian frogs and hence may offer potential as a biocontrol agent of the toad.

Evolution of pathogens in a man-made world.

Human activities have resulted in substantial, large-scale environmental modifications, especially in the past century. Ecologists and evolutionary biologists are increasingly coming to realize that parasites and pathogens, like free-living organisms, evolve as the consequence of these anthropogenic changes. Although this area now commands the attention of a variety of researchers, a broad predictive framework is lacking, mainly because the links between human activities, the environment and parasite evolution are complex. From empirical and theoretical examples chosen in the literature, we give an overview of the ways in which humans can directly or indirectly influence the evolution of different traits in parasites (e.g. specificity, virulence, polymorphism). We discuss the role of direct and indirect factors as diverse as habitat fragmentation, pollution, biodiversity loss, climate change, introduction of species, use of vaccines and antibiotics, ageing of the population, etc. We also present challenging questions for further research. Understanding the links between anthropogenic changes and parasite evolution needs to become a cornerstone of public health planning, economic development and conservation biology.

The mitochondrial genomics of parasitic nematodes of socio-economic importance: recent progress, and implications for population genetics and systematics.

Mitochondria are subcellular organelles in which oxidative phosphorylation and other important biochemical functions take place within the cell. Within these organelles is a genome, called the mitochondrial (mt) genome, which is distinct from, but cooperates closely with the nuclear genome of the cell. Investigating mt genomes has significant implications for various fundamental research areas, including mt biochemistry and physiology, and, importantly, such genomes provide a rich source of markers for population genetic and systematic studies. While approximately 250 complete mt genome sequences have been determined for a range of metazoan organisms from various phyla, few of these represent parasitic helminths. Until 1998, only two mt genome sequences had been determined for parasitic nematodes, in spite of their socio-economic importance and the need for investigations into their population genetics, taxonomy and evolution. However, since that time, there has been some progress. The main focus of the present chapter is to review the state of knowledge of the mt genomics for parasitic nematodes, to describe recent technological improvements to mt genome sequencing, to summarize applications of mt gene markers for studying the systematics and population genetics of parasitic nematodes, and to emphasize prospects and opportunities for future research in these areas.

Genomics and the biology of parasites.

Despite the advances of modern medicine, the threat of chronic illness, disfigurement, or death that can result from parasitic infection still affects the majority of the world population, retarding economic development. For most parasitic diseases, current therapeutics often leave much to be desired in terms of administration regime, toxicity, or effectiveness and potential vaccines are a long way from market. Our best prospects for identifying new targets for drug, vaccine, and diagnostics development and for dissecting the biological basis of drug resistance, antigenic diversity, infectivity and pathology lie in parasite genome analysis, and international mapping and gene discovery initiatives are under way for a variety of protozoan and helminth parasites. These are far from ideal experimental organisms, and the influence of biological and genomic characteristics on experimental approaches is discussed, progress is reviewed and future prospects are examined.