Comparison of multi-parallel quantitative real-time PCRs targeting different DNA regions and detecting soil-transmitted helminths in stool.

BACKGROUND: Soil-transmitted helminths infect an estimated 18% of the world's population, causing a significant health burden. Microscopy has been the primary tool for diagnosing eggs from fecal samples, but its sensitivity drops in low-prevalence settings. Quantitative real-time polymerase chain reaction (qPCR) is slowly increasing in research and clinical settings. However, there is still no consensus on preferred qPCR targets. METHODS: We aimed to compare soil-transmitted helminth (STH) DNA detection methods by testing naïve stool samples spiked with known quantities of STH eggs and larvae. DNA extracts from spiked samples were tested using independent quantitative realtime PCR (qPCR) assays targeting ribosomal or putative non-protein coding satellite sequences. RESULTS: For Trichuris trichiura, there was a strong correlation between egg/larvae counts and qPCR results using either qPCR method (0.86 and 0.87, respectively). Strong correlations also existed for A. lumbricoides (0.60 and 0.63, respectively), but weaker correlations were found for Ancylostoma duodenale (0.41 for both assays) and Strongyloides stercoralis (0.48 and 0.65, respectively). No correlation for Necator americanus was observed when testing with either qPCR assay. Both assays had fair-to-moderate agreement across targets when using field-collected stool samples (0.28-0.45, for all STHs), except for S. stercoralis (0.12) with slight agreement. CONCLUSIONS: There is a strong correlation between qPCR results and egg/larvae counts. Our study confirms that qPCR is an effective diagnostic tool, even with low-intensity infections, regardless of the DNA-based diagnostic marker used. However, the moderate agreement between the two different qPCR assays when testing field samples highlights the need to understand the role of these targets in the genome so that the parasite burden can be quantified more accurately and consistently by qPCR.

Fatal Hymenolepis nana-associated visceral larva migrans in captive juvenile white-tailed antsangies (Brachytarsomysalbicauda).

White-tailed antsangies (Brachytarsomys albicauda) are Madagascan rodents uncommonly kept in captivity. Hymenolepis nana is a cestode with an unusual life cycle, incorporating direct, indirect and autoinfective stages. This case series represents the first reported outbreak of H. nana cestodiasis in white-tailed antsangies, summarizing macroscopic and histological findings in four cases. On post-mortem examination (PME), numerous cysticerci were detected consistently throughout the intestinal serosa, liver, mesenteric lymphatic vasculature and mesenteric lymph nodes. Pancreatic cysticerci were observed in one case. Adult tapeworms, larvae and eggs were found only in the small intestine, and faecal egg shedding was a feature. Histopathological examination identified adult, larval and encysted cestodes within the respective gross lesions, with pulmonary, pancreatic and splenic involvement detected in a single case. The cestodes sampled on PME were identified by polymerase chain reaction and DNA sequencing, with H. nana confirmed in all cases. Visceral larva migrans was consistent throughout all specimens, in contrast with the natural infections of standard rodent hosts, and may be considered a likely pathological feature of H. nana infection in white-tailed antsangies.

Evidence that a lineage of teleost-infecting blood flukes (Aporocotylidae) infects bivalves as intermediate hosts.

The family Aporocotylidae is recognized as having the widest intermediate host usage in the Digenea. Currently, intermediate host groups are clearly correlated with definitive host groups; all known life cycles of marine teleost-infecting aporocotylids involve polychaetes, those of freshwater teleost-infecting aporocotylids involve gastropods, and those of chondrichthyan-infecting aporocotylids involve bivalves. Here we report the life cycle for a marine elopomorph-infecting species, Elopicola bristowi Orélis-Ribeiro & Bullard in Orélis-Ribeiro, Halanych, Dang, Bakenhaster, Arias & Bullard, 2017, as infecting a bivalve, Anadara trapezia (Deshayes) (Arcidae), as the intermediate host in Moreton Bay, Queensland, Australia. The cercaria of E. bristowi has a prominent finfold, distinct anterior and posterior widenings of the oesophagus, a tail with symmetrical furcae with finfolds, and develops in elongate to oval sporocysts. We also report molecular data for an unmatched aporocotylid cercaria from another bivalve, Megapitaria squalida (G. B. Sowerby I) (Veneridae), from the Gulf of California, Mexico, and six unmatched cercariae from a gastropod, Posticobia brazieri (E. A. Smith) (Tateidae), from freshwater systems of south-east Queensland, Australia. Phylogenetic analyses demonstrate the presence of six strongly-supported lineages within the Aporocotylidae, including one of elopomorph-infecting genera, Elopicola Bullard, 2014 and Paracardicoloides Martin, 1974, now shown to use both gastropods and bivalves as intermediate hosts. Of a likely 14 aporocotylid species reported from bivalves, six are now genetically characterised. The cercarial morphology of these six species demonstrates a clear distinction between those that infect chondrichthyans and those that infect elopomorphs; chondrichthyan-infecting aporocotylids have cercariae with asymmetrical furcae that lack finfolds and develop in spherical sporocysts whereas those of elopomorph-infecting aporocotylids have symmetrical furcae with finfolds and develop in elongate sporocysts. This morphological correlation allows predictions of the host-based lineage to which the unsequenced species belong. The Aporocotylidae is proving exceptional in is propensity for major switches in intermediate host use, with the most parsimonious interpretation of intermediate host distribution implying a minimum of three host switches within the family.

Exploring gene sequences and phylogenetic relationships of four terrestrial planarian species (Platyhelminthes; Tricladida; Geoplanidae) in Europe.

Partial mitochondrial cox1 gene sequences from four recently recognised European species of terrestrial planarians, and ribosomal ITS1 sequences for two of them, are presented: Marionfyfea adventor, Artioposthia exulans (both introduced from New Zealand), Australopacifica atrata (from Australia) and specimens putatively identified as Microplana edwardsi, presumed to be native to the UK. The sequences are compared with those from other terrestrial planarian species and analysed phylogenetically. Results indicate that the sister group of M. adventor comprises a clade constituted by at least the genus Arthurdendyus. The phylogenetic position of Ar. exulans remains less certain, Australopacifica atrata might be closely related to the species Parakontikia ventrolineata and Endeavouria septemlineata. The specimens of M. cf. edwardsi are distinct from all other Microplana species for which sequences are available.

Worms and bugs of the gut: the search for diagnostic signatures using barcoding, and metagenomics-metabolomics.

Gastrointestinal (GI) helminth infections cause significant morbidity in both humans and animals worldwide. Specific and sensitive diagnosis is central to the surveillance of such infections and to determine the effectiveness of treatment strategies used to control them. In this article, we: (i) assess the strengths and limitations of existing methods applied to the diagnosis of GI helminth infections of humans and livestock; (ii) examine high-throughput sequencing approaches, such as targeted molecular barcoding and shotgun sequencing, as tools to define the taxonomic composition of helminth infections; and (iii) discuss the current understanding of the interactions between helminths and microbiota in the host gut. Stool-based diagnostics are likely to serve as an important tool well into the future; improved diagnostics of helminths and their environment in the gut may assist the identification of biomarkers with the potential to define the health/disease status of individuals and populations, and to identify existing or emerging anthelmintic resistance.

Expansion of Cyclophyllidea Biodiversity in Rodents of Qinghai-Tibet Plateau and the "Out of Qinghai-Tibet Plateau" Hypothesis of Cyclophyllideans.

The Cyclophyllidea comprises the most species-rich order of tapeworms (Platyhelminthes, Cestoda) and includes species with some of the most severe health impact on wildlife, livestock, and humans. We collected seven Cyclophyllidea specimens from rodents in Qinghai-Tibet Plateau (QTP) and its surrounding mountain systems, of which four specimens in QTP were unsequenced, representing "putative new species." Their complete mitochondrial (mt) genomes were sequenced and annotated. Phylogenetic reconstruction of partial 28S rDNA, cox1 and nad1 datasets provided high bootstrap frequency support for the categorization of three "putative new species," assigning each, respectively, to the genera Mesocestoides, Paranoplocephala, and Mosgovoyia, and revealing that some species and families in these three datasets, which contain 291 species from nine families, may require taxonomic revision. The partial 18S rDNA phylogeny of 29 species from Taeniidae provided high bootstrap frequency support for the categorization of the "putative new species" in the genus Hydatigera. Combined with the current investigation, the other three known Taeniidae species found in this study were Taenia caixuepengi, T. crassiceps, and Versteria mustelae and may be widely distributed in western China. Estimates of divergence time based on cox1 + nad1 fragment and mt protein-coding genes (PCGs) showed that the differentiation rate of Cyclophyllidea species was strongly associated with the rate of change in the biogeographic scenarios, likely caused by the uplift of the QTP; i.e., species differentiation of Cyclophyllidea might be driven by host-parasite co-evolution caused by the uplift of QTP. We propose an "out of QTP" hypothesis for the radiation of these cyclophyllidean tapeworms.

Factors associated with soil-transmitted helminths infection in Benin: Findings from the DeWorm3 study.

BACKGROUND: Despite several years of school-based MDA implementation, STH infections remain an important public health problem in Benin, with a country-wide prevalence of 20% in 2015. The DeWorm3 study is designed to assess the feasibility of using community-based MDA with albendazole to interrupt the transmission of STH, through a series of cluster-randomized trials in Benin, India and Malawi. We used the pre-treatment baseline survey data to describe and analyze the factors associated with STH infection in Comé, the study site of the DeWorm3 project in Benin. These data will improve understanding of the challenges that need to be addressed in order to eliminate STH as a public health problem in Benin. METHODS: Between March and April 2018, the prevalence of STH (hookworm spp., Ascaris and Trichuris trichiura) was assessed by Kato-Katz in stool samples collected from 6,153 residents in the community of Comé, Benin using a stratified random sampling procedure. A standardized survey questionnaire was used to collect information from individual households concerning factors potentially associated with the presence and intensity of STH infections in pre-school (PSAC, aged 1-4), school-aged children (SAC, aged 5-14) and adults (aged 15 and above). Multilevel mixed-effects models were used to assess associations between these factors and STH infection. RESULTS: The overall prevalence of STH infection was 5.3%; 3.2% hookworm spp., 2.1% Ascaris lumbricoides and 0.1% Trichuris. Hookworm spp. were more prevalent in adults than in SAC (4.4% versus 2.0%, respectively; p = 0.0001) and PSAC (4.4% versus 1.0%, respectively; p<0.0001), whilst Ascaris lumbricoides was more prevalent in SAC than in adults (3.0% versus 1.7%, respectively; p = 0.004). Being PSAC (adjusted Odds Ratio (aOR) = 0.2, p< 0.001; adjusted Infection Intensity Ratio (aIIR) = 0.1, p<0.001) or SAC (aOR = 0.5, p = 0.008; aIIR = 0.3, p = 0.01), being a female (aOR = 0.6, p = 0.004; aIIR = 0.3, p = 0.001), and having received deworming treatment the previous year (aOR = 0.4, p< 0.002; aIIR = 0.2, p<0.001) were associated with a lower prevalence and intensity of hookworm infection. Lower income (lowest quintile: aOR = 5.0, p<0.001, 2nd quintile aOR = 3.6, p = 0.001 and 3rd quintile aOR = 2.5, p = 0.02), being a farmer (aOR = 1.8, p = 0.02), medium population density (aOR = 2.6, p = 0.01), and open defecation (aOR = 0.5, p = 0.04) were associated with a higher prevalence of hookworm infection. Lower education-no education, primary or secondary school- (aIIR = 40.1, p = 0.01; aIIR = 30.9, p = 0.02; aIIR = 19.3, p = 0.04, respectively), farming (aIIR = 3.9, p = 0.002), natural flooring (aIIR = 0.2, p = 0.06), peri-urban settings (aIIR = 6.2, 95%CI 1.82-20.90, p = 0.003), and unimproved water source more than 30 minutes from the household (aIIR = 13.5, p = 0.02) were associated with a higher intensity of hookworm infection. Improved and unshared toilet was associated with lower intensity of hookworm infections (aIIR = 0.2, p = 0.01). SAC had a higher odds of Ascaris lumbricoides infection than adults (aOR = 2.0, p = 0.01) and females had a lower odds of infection (aOR = 0.5, p = 0.02). CONCLUSION: Hookworm spp. are the most prevalent STH in Comé, with a persistent reservoir in adults that is not addressed by current control measures based on school MDA. Expanding MDA to target adults and PSAC is necessary to substantially impact population prevalence, particularly for hookworm. TRIAL REGISTRATION: ClinicalTrials.gov NCT03014167.

Evolutionary transitions in broad tapeworms (Cestoda: Diphyllobothriidea) revealed by mitogenome and nuclear ribosomal operon phylogenetics.

Broad tapeworms (Diphyllobothriidea) are parasites whose adults are capable of infecting a wide range of freshwater, marine and terrestrial tetrapods including humans. Previous works examining the evolution of habitat and host use in this group have been hampered by the lack of a well-resolved phylogeny. In order to produce a robust phylogenetic framework for diphyllobothriideans, we sequenced the complete mitochondrial genome of 13 representatives, carefully chosen to cover the major clades, and two outgroup species representing the Spathebothriidea and Haplobothriidea. In addition, complementary data from the nuclear ribosomal operon was sequenced for 10 representative taxa. Mitogenomes and ssrDNA and lsrDNA were used towards elucidating the phylogenetic framework for the Diphyllobothriidea. The Cephalochlamydidae is confirmed as the earliest diverging diphyllobothriidean lineage, and Solenophoridae and Diphyllobothriidae are sister groups. We infer a probable freshwater origin of the diphyllobothriideans. The ancestral condition for life cycle complexity could not be unambiguously resolved. However, we infer exclusive use of a three-host life cycle following the origin of the Solenophoridae + Diphyllobothriidae. Regarding definitive host use, although we infer reptiles as the most likely ancestral condition, this result should be revisited with a more densely sampled phylogeny in future studies. Freshwater habitat is used by the early diverging lineages within the Solenophoridae + Diphyllobothriidae clade. For the latter, habitat use shifts between freshwater and marine environments, and definitive host use includes marine and terrestrial mammals and birds. We use mitochondrial genomes to distinguish Schistocephalus species occurring in different species of sticklebacks and demonstrate conspecificity of Ligula cf. intestinalis specimens collected from two Fennoscandian ringed seal subspecies.

How qPCR complements the WHO roadmap (2021-2030) for soil-transmitted helminths.

Complementing the launch of the World Health Organization (WHO) roadmap (2021-2030) we explore key elements needing attention before recruitment of qPCR as the main diagnostics tool to confirm reduction or elimination of soil-transmitted helminth (STH) transmission in both control and elimination programmes. Given the performance limitations of conventional methods, a proposed harmonised qPCR will provide a diagnostic tool, with the sensitivity and specificity required to monitor low-intensity infections, following mass drug administration (MDA). Technical and logistical challenges associated with introducing qPCR as a stand-alone tool are highlighted, and a decision-making scheme on how qPCR can support surveillance, resistance detection, and elimination is presented. An accurate point-of-care (POC) diagnostic test needs to be developed to support STH control in the field, and STH biorepositories need to be established and maintained to ensure that reference materials are available for research and validation.

Nanopore Sequencing Resolves Elusive Long Tandem-Repeat Regions in Mitochondrial Genomes.

Long non-coding, tandem-repetitive regions in mitochondrial (mt) genomes of many metazoans have been notoriously difficult to characterise accurately using conventional sequencing methods. Here, we show how the use of a third-generation (long-read) sequencing and informatic approach can overcome this problem. We employed Oxford Nanopore technology to sequence genomic DNAs from a pool of adult worms of the carcinogenic parasite, Schistosoma haematobium, and used an informatic workflow to define the complete mt non-coding region(s). Using long-read data of high coverage, we defined six dominant mt genomes of 33.4 kb to 22.6 kb. Although no variation was detected in the order or lengths of the protein-coding genes, there was marked length (18.5 kb to 7.6 kb) and structural variation in the non-coding region, raising questions about the evolution and function of what might be a control region that regulates mt transcription and/or replication. The discovery here of the largest tandem-repetitive, non-coding region (18.5 kb) in a metazoan organism also raises a question about the completeness of some of the mt genomes of animals reported to date, and stimulates further explorations using a Nanopore-informatic workflow.

Phylogenetic reconstruction of early diverging tapeworms (Cestoda: Caryophyllidea) reveals ancient radiations in vertebrate hosts and biogeographic regions.

Tapeworms of the order Caryophyllidea are the earliest diverging 'true' tapeworms (Eucestoda) and parasitise cypriniform and siluriform fishes almost exclusively. They are typified by a monozoic (non-proglottised) body plan, which is a characteristic shared with early diverging 'cestodarians' Gyrocotylidea and Amphilinidea. Here we present the most comprehensive multi-gene molecular phylogeny of this group, to date. Specimens of 63 species from 32 genera (~50% and ~75% of known species and genus diversity, respectively) were gathered during an intense and targeted 15-year collecting effort. Phylogenetic reconstructions provide high nodal support for three major lineages, which only partly correspond to currently recognised families. The three well-supported clades were as follows: Clade A was in an unsupported position at the base of the tree and was almost exclusively comprised of parasites of catfishes (Siluriformes) from the Afrotropical and Indomalayan regions, including the type genus of the Lytocestidae (Lytocestus). Clade B formed the sister group to the remaining taxa (Clade C) and was composed of species that parasitise cyprinids and loaches (Cypriniformes: Cyprinoidei and Cobitoidei) from the Palaearctic Region. This clade included the type genus of the Caryophyllaeidae (Caryophyllaeus). Clade C comprised Nearctic species from suckers and minnows (Cypriniformes: Catostomidae and Cyprinoidei), which were previously accommodated in two families, i.e. Capingentidae and Caryophyllaeidae. This clade included the type genus of the Capingentidae (Capingens). In addition to Clades A-C, Balanotaenia bancrofti from the monotypic Balanotaeniidae, which parasitises plotosid catfishes in Australia, and Lytocestoides tanganyikae, which parasitises African cichlids, formed a poorly supported clade at the base of the tree. Whereas morphological characteristics traditionally used to differentiate caryophyllidean families do not characterise molecular lineages, host association and biogeographical distribution play a key role in the circumscription of the three well-supported clades revealed by molecular data. Thus, the taxonomic rearrangement proposed herein was guided by the molecular clades. The names of all four extant families were preserved and family affinity was determined by topological clustering with the type genera of the families. The family diagnoses of the Lytocestidae, Caryophyllaeidae and Capingentidae are amended. Biogeographic patterns are indicative of separate Gondwanan and Laurasian radiations having taken place. Regarding the Gondwanan radiation in the Siluriformes, the topology in Clade A indicates an Asian origin with a subsequent African colonisation. Concerning Laurasia, separate radiations appear to have taken place in the Cypriniformes in the temperate zones of North America and Eurasia. Complete absence of caryophyllideans in the Neotropical Region, where numerous catfishes occur, may be due to the Gondwanan radiation having taken place after the continental separation of Africa and South America.

Molecular circumscription of new species of Gyrocotyle Diesing, 1850 (Cestoda) from deep-sea chimaeriform holocephalans in the North Atlantic.

Chimaeras, or ratfishes, are the only extant group of holocephalan fishes and are the sole host group of gyrocotylidean cestodes, which represent a sister group of the true tapeworms (Eucestoda). These unique, non-segmented cestodes have been known since the 1850s and multiple species and genera have been erected despite a general agreement that the delineation of species on the basis of morphology is effectively impossible. Thus, in the absence of molecular studies, the validity of gyrocotylid taxa and their specific host associations has remained highly speculative. Here we report the presence of Gyrocotyle spp. from rarely-caught deep-sea chimaeras collected in the North-East Atlantic, and describe two new species: G. haffii n. sp. from the bent-nose chimaera, Harriota raleighana Goode & Bean, and G. discoveryi n. sp. from the large-eyed rabbit fish, Hydrolagus mirabilis (Collett). Nuclear ribosomal sequence data were generated for individual parasites taken from different host species collected on different dates and from different localities and were combined with previously published sequences. Phylogenetic analyses supported the recognition of independent lineages and clusters, indicative of species, but were indecisive in recovering the root of the tree in analyses that included non-gyrocotylid outgroup taxa. The molecular data reveal variation not reflected in morphology and point to a complex picture of genetic divergence shaped by both isolation and migration in the deep-sea environment.

Pooling as a strategy for the timely diagnosis of soil-transmitted helminths in stool: value and reproducibility.

BACKGROUND: The strategy of pooling stool specimens has been extensively used in the field of parasitology in order to facilitate the screening of large numbers of samples whilst minimizing the prohibitive cost of single sample analysis. The aim of this study was to develop a standardized reproducible pooling protocol for stool samples, validated between two different laboratories, without jeopardizing the sensitivity of the quantitative polymerase chain reaction (qPCR) assays employed for the detection of soil-transmitted helminths (STHs). Two distinct experimental phases were recruited. First, the sensitivity and specificity of the established protocol was assessed by real-time PCR for each one of the STHs. Secondly, agreement and reproducibility of the protocol between the two different laboratories were tested. The need for multiple stool sampling to avoid false negative results was also assessed. Finally, a cost exercise was conducted which included labour cost in low- and high-wage settings, consumable cost, prevalence of a single STH species, and a simple distribution pattern of the positive samples in pools to estimate time and money savings suggested by the strategy. RESULTS: The sensitivity of the pooling method was variable among the STH species but consistent between the two laboratories. Estimates of specificity indicate a 'pooling approach' can yield a low frequency of 'missed' infections. There were no significant differences regarding the execution of the protocol and the subsequent STH detection between the two laboratories, which suggests in most cases the protocol is reproducible by adequately trained staff. Finally, given the high degree of agreement, there appears to be little or no need for multiple sampling of either individuals or pools. CONCLUSIONS: Our results suggest that the pooling protocol developed herein is a robust and efficient strategy for the detection of STHs in 'pools-of-five'. There is notable complexity of the pool preparation to ensure even distribution of helminth DNA throughout. Therefore, at a given setting, cost of labour among other logistical and epidemiological factors, is the more concerning and determining factor when choosing pooling strategies, rather than losing sensitivity and/or specificity of the molecular assay or the method.

Integrated Approach Reveals Role of Mitochondrial Germ-Line Mutation F18L in Respiratory Chain, Oxidative Alterations, Drug Sensitivity, and Patient Prognosis in Glioblastoma.

Glioblastoma is the most common and malignant primary brain tumour in adults, with a dismal prognosis. This is partly due to considerable inter- and intra-tumour heterogeneity. Changes in the cellular energy-producing mitochondrial respiratory chain complex (MRC) activities are a hallmark of glioblastoma relative to the normal brain, and associate with differential survival outcomes. Targeting MRC complexes with drugs can also facilitate anti-glioblastoma activity. Whether mutations in the mitochondrial DNA (mtDNA) that encode several components of the MRC contribute to these phenomena remains underexplored. We identified a germ-line mtDNA mutation (m. 14798T > C), enriched in glioblastoma relative to healthy controls, that causes an amino acid substitution F18L within the core mtDNA-encoded cytochrome b subunit of MRC complex III. F18L is predicted to alter corresponding complex III activity, and sensitivity to complex III-targeting drugs. This could in turn alter reactive oxygen species (ROS) production, cell behaviour and, consequently, patient outcomes. Here we show that, despite a heterogeneous mitochondrial background in adult glioblastoma patient biopsy-derived cell cultures, the F18L substitution associates with alterations in individual MRC complex activities, in particular a 75% increase in MRC complex II_III activity, and a 34% reduction in CoQ10, the natural substrate for MRC complex III, levels. Downstream characterisation of an F18L-carrier revealed an 87% increase in intra-cellular ROS, an altered cellular distribution of mitochondrial-specific ROS, and a 64% increased sensitivity to clomipramine, a repurposed MRC complex III-targeting drug. In patients, F18L-carriers that received the current standard of care treatment had a poorer prognosis than non-carriers (373 days vs. 415 days, respectively). Single germ-line mitochondrial mutations could predispose individuals to differential prognoses, and sensitivity to mitochondrial targeted drugs. Thus, F18L, which is present in blood could serve as a useful non-invasive biomarker for the stratification of patients into prognostically relevant groups, one of which requires a lower dose of clomipramine to achieve clinical effect, thus minimising side-effects.

Calculating the prevalence of soil-transmitted helminth infection through pooling of stool samples: Choosing and optimizing the pooling strategy.

Prevalence is a common epidemiological measure for assessing soil-transmitted helminth burden and forms the basis for much public-health decision-making. Standard diagnostic techniques are based on egg detection in stool samples through microscopy and these techniques are known to have poor sensitivity for individuals with low infection intensity, leading to poor sensitivity in low prevalence populations. PCR diagnostic techniques offer very high sensitivities even at low prevalence, but at a greater cost for each diagnostic test in terms of equipment needed and technician time and training. Pooling of samples can allow prevalence to be estimated while minimizing the number of tests performed. We develop a model of the relative cost of pooling to estimate prevalence, compared to the direct approach of testing all samples individually. Analysis shows how expected relative cost depends on both the underlying prevalence in the population and the size of the pools constructed. A critical prevalence level (approx. 31%) above which pooling is never cost effective, independent of pool size. When no prevalence information is available, there is no basis on which to choose between pooling and testing all samples individually. We recast our model of relative cost in a Bayesian framework in order to investigate how prior information about prevalence in a given population can be used to inform the decision to choose either pooling or full testing. Results suggest that if prevalence is below 10%, a relatively small exploratory prevalence survey (10-15 samples) can be sufficient to give a high degree of certainty that pooling may be relatively cost effective.

Phylogeny of hymenolepidid cestodes (Cestoda: Cyclophyllidea) from mammalian hosts based on partial 28S rDNA, with focus on parasites from shrews.

The aims of the study are to enrich the partial 28S rDNA dataset for hymenolepidids by adding new sequences for species parasitic in the genera Sorex, Neomys and Crocidura (Soricidae) and to propose a new hypothesis for the relationships among mammalian hymenolepidids. New sequences were obtained for Coronacanthus integrus, C. magnihamatus, C. omissus, C. vassilevi, Ditestolepis diaphana, Lineolepis scutigera, Spasskylepis ovaluteri, Staphylocystis tiara, S. furcata, S. uncinata, Vaucherilepis trichophorus and Neoskrjabinolepis sp. The phylogenetic analysis (based on 56 taxa) confirmed the major clades identified by Haukisalmi et al. (Zool Scr 39:631-641, 2010) based on analysis of 31 species: Ditestolepis clade, Hymenolepis clade, Rodentolepis clade and Arostrilepis clade; however, the support was weak for the early divergent lineages of the tree and for the Arostrilepis clade. Novelties revealed include the molecular evidence for the monophyly of Coronacanthus, the non-monophyletic status of Staphylocystis and the polyphyly of Staphylocystoides. The analysis has confirmed the monophyly of Hymenolepis, the monophyly of hymenolepidids from glirids, the position of Pararodentolepis and Nomadolepis as sister taxa, the polyphyly of Rodentolepis, the position of Neoskrjabinolepis and Lineolepis as sister taxa, and the close relationship among the genera with the entire reduction of rostellar apparatus. Resolved monophyletic groups are supported by the structure of the rostellar apparatus. The diversification of the Ditestolepis clade is associated with soricids. The composition of the other major clades suggests multiple evolutionary events of host switching, including between different host orders. The life cycles of Coronacanthus and Vaucherilepis are recognised as secondarily aquatic as these taxa are nested in terrestrial groups.

Assessing the feasibility of interrupting the transmission of soil-transmitted helminths through mass drug administration: The DeWorm3 cluster randomized trial protocol.

Current control strategies for soil-transmitted helminths (STH) emphasize morbidity control through mass drug administration (MDA) targeting preschool- and school-age children, women of childbearing age and adults in certain high-risk occupations such as agricultural laborers or miners. This strategy is effective at reducing morbidity in those treated but, without massive economic development, it is unlikely it will interrupt transmission. MDA will therefore need to continue indefinitely to maintain benefit. Mathematical models suggest that transmission interruption may be achievable through MDA alone, provided that all age groups are targeted with high coverage. The DeWorm3 Project will test the feasibility of interrupting STH transmission using biannual MDA targeting all age groups. Study sites (population ≥80,000) have been identified in Benin, Malawi and India. Each site will be divided into 40 clusters, to be randomized 1:1 to three years of twice-annual community-wide MDA or standard-of-care MDA, typically annual school-based deworming. Community-wide MDA will be delivered door-to-door, while standard-of-care MDA will be delivered according to national guidelines. The primary outcome is transmission interruption of the STH species present at each site, defined as weighted cluster-level prevalence ≤2% by quantitative polymerase chain reaction (qPCR), 24 months after the final round of MDA. Secondary outcomes include the endline prevalence of STH, overall and by species, and the endline prevalence of STH among children under five as an indicator of incident infections. Secondary analyses will identify cluster-level factors associated with transmission interruption. Prevalence will be assessed using qPCR of stool samples collected from a random sample of cluster residents at baseline, six months after the final round of MDA and 24 months post-MDA. A smaller number of individuals in each cluster will be followed with annual sampling to monitor trends in prevalence and reinfection throughout the trial. TRIAL REGISTRATION: ClinicalTrials.gov NCT03014167.

Deep sequencing reveals the mitochondrial DNA variation landscapes of breast-to-brain metastasis blood samples.

Breast-to-brain metastasis (BBM) often represents a terminal event, due to the inability of many systemic treatments to cross the blood-brain barrier (BBB), rendering the brain a sanctuary site for tumour cells. Identifying genetic variations that can predict the patients who will develop BBM would allow targeting of adjuvant treatments to reduce risk while disease bulk is minimal. Germ-line genetic variations may contribute to whether a BBM forms by influencing the primary tumour subtype that presents, or by influencing the host response to the tumour or treatment regimen, or by facilitating transition of tumour cells across the BBB and establish a viable brain metastasis. The role of mitochondrial DNA (mtDNA) variants specifically in BBM is underexplored. Consequently, using a sensitive deep sequencing approach, we characterized the mtDNA variation landscapes of blood samples derived from 13 females who were diagnosed with early-onset breast cancer and later went on to develop BBM. We also predicted the potential pathogenic significance of variations identified in all mtDNA-encoded oxidative phosphorylation (OXPHOS) proteins using 3D protein structural mapping and analysis, to identify variations worthy of follow-up. From the 70 variations found in protein coding regions, we reveal novel links between three specific mtDNA variations and altered OXPHOS structure and function in 23% of the BBM samples. Further studies are required to confirm the origin of mtDNA variations, and whether they correlate with (1) the predicted alterations in mitochondrial function and (2) increased risk of developing breast-to-brain metastasis using a much larger cohort of samples.

The catholic taste of broad tapeworms - multiple routes to human infection.

Broad tapeworms (Cestoda: Diphyllobothriidea) are the principal agents of widespread food-borne cestodosis. Diphyllobothriosis and diplogonoporosis, caused by members of the genera Diphyllobothrium, Diplogonoporus and Adenocephalus, are the most common fish cestodoses with an estimated 20million people infected worldwide, and has seen recent (re)emergences in Europe due to the increasing popularity of eating raw or undercooked fish. Sparganosis is a debilitating and potentially lethal disease caused by the larvae of the genus Spirometra, which occurs throughout much of the (sub)tropics and is caused by the consumption of raw snakes and frogs, and drinking water contaminated by infected copepods. Both diseases are caused by several species, but the frequency by which the transition to humans has occurred has never been studied. Using a phylogenetic framework of 30 species based on large and small nuclear ribosomal RNA subunits (ssrDNA, lsrDNA), large subunit mitochondrial ribosomal RNA (rrnL) and cytochrome c oxidase subunit I (cox1), we hypothesize that humans have been acquired asaccidental hosts four times across the tree of life of diphyllobothriideans. However, polytomies prevent an unambiguous reconstruction of the evolution of intermediate and definitive host use. The broad host spectrum and the frequency with which switching between major host groups appears to have occurred, may hold the answer as to why accidental human infection occurred multiple times across the phylogeny of diphyllobothriideans. In this study Diplogonoporus is determined to be the junior synonym of Diphyllobothrium. Furthermore, we divide the latter polyphyletic genus into (i) the resurrected genus Dibothriocephalus to include freshwater and terrestrial species including Dibothriocephalus dendriticus, Dibothriocephalus latus and Dibothriocephalus nihonkaiensis as the most common parasites of humans, and (ii) the genus Diphyllobothrium to accommodate parasites from cetaceans including the type species Diphyllobothrium stemmacephalum and Diphyllobothrium balaenopterae n. comb. known also from humans. The non-monophyletic aggregate of marine species from seals is provisionally considered as incertae sedis.

Animal Evolution: Last Word on Sponges-First?

A major problem in understanding animal evolution is where early branching phyla, especially sponges and comb jellies (sea gooseberries), sit in the tree of life. A new study seeks to overcome this problem by sampling more species and data cleansing.