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.

Evaluation of genome skimming to detect and characterise human and livestock helminths.

The identification of gastrointestinal helminth infections of humans and livestock almost exclusively relies on the detection of eggs or larvae in faeces, followed by manual counting and morphological characterisation to differentiate species using microscopy-based techniques. However, molecular approaches based on the detection and quantification of parasite DNA are becoming more prevalent, increasing the sensitivity, specificity and throughput of diagnostic assays. High-throughput sequencing, from single PCR targets through to the analysis of whole genomes, offers significant promise towards providing information-rich data that may add value beyond traditional and conventional molecular approaches; however, thus far, its utility has not been fully explored to detect helminths in faecal samples. In this study, low-depth whole genome sequencing, i.e. genome skimming, has been applied to detect and characterise helminth diversity in a set of helminth-infected human and livestock faecal material. The strengths and limitations of this approach are evaluated using three methods to characterise and differentiate metagenomic sequencing data based on (i) mapping to whole mitochondrial genomes, (ii) whole genome assemblies, and (iii) a comprehensive internal transcribed spacer 2 (ITS2) database, together with validation using quantitative PCR (qPCR). Our analyses suggest that genome skimming can successfully identify most single and multi-species infections reported by qPCR and can provide sufficient coverage within some samples to resolve consensus mitochondrial genomes, thus facilitating phylogenetic analyses of selected genera, e.g. Ascaris spp. Key to this approach is both the availability and integrity of helminth reference genomes, some of which are currently contaminated with bacterial and host sequences. The success of genome skimming of faecal DNA is dependent on the availability of vouchered sequences of helminths spanning both taxonomic and geographic diversity, together with methods to detect or amplify minute quantities of parasite nucleic acids in mixed samples.

Can avian flyways reflect dispersal barriers of clinostomid parasites? First evidence from the mitogenome of Clinostomum complanatum.

Clinostomum complanatum (Rudolphi, 1814) is an economically important parasitic flatworm (Trematoda, Digenea), yet little is known on the population structure of these animals. We characterise a new mitochondrial genome for C. complanatum, derived from an Iranian specimen. The newly obtained sequence is used to position the species in the digenean tree of life. The first-ever intraspecific comparison at mitogenome scale within C. complanatum revealed a high degree of similarity to the previously sequenced mitogenome of a distant (Italian) population. Avian migratory routes mirror phylogenetic clustering, and hence we suggest that infection of a flying host enables genetic exchange between parasites across large geographic distances. Comparative mitogenomic work in Clinostomum spp. at both the intra- (C. complanatum) and interspecific (C. complanatum-C. sinensis) level further shows that usage of new and/or additional mitochondrial markers is preferred over single-gene methods for high-resolution diagnostics and population biology.

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.

Phylogeny of hymenolepidids (Cestoda: Cyclophyllidea) from mammals: sequences of 18S rRNA and COI genes confirm major clades revealed by the 28S rRNA analyses.

The aim of the study is to test a hypothesis for the phylogenetic relationships among mammalian hymenolepidid tapeworms, based on partial (D1-D3) nuclear 28S ribosomal RNA (rRNA) genes, by estimating new molecular phylogenies for the group based on partial mitochondrial cytochrome c oxidase I (COI) and nuclear 18S rRNA genes, as well as a combined analysis using all three genes. New sequences of COI and 18S rRNA genes 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 analyses confirmed the major clades identified by Haukisalmi et al. (Zoologica Scripta 39: 631-641, 2010): Ditestolepis clade, Hymenolepis clade, Rodentolepis clade and Arostrilepis clade. While the Ditestolepis clade is associated with soricids, the structure of the other three clades suggests multiple evolutionary events of host switching between shrews and rodents. Two of the present analyses (18S rRNA and COI genes) show that the basal relationships of the four mammalian clades are branching at the same polytomy with several hymenolepidids from birds (both terrestrial and aquatic). This may indicate a rapid radiation of the group, with multiple events of colonizations of mammalian hosts by avian parasites.

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.

The first mitochondrial genomes of endosymbiotic rhabdocoels illustrate evolutionary relaxation of atp8 and genome plasticity in flatworms.

The first three mitochondrial (mt) genomes of endosymbiotic turbellarian flatworms are characterised for the rhabdocoels Graffilla buccinicola, Syndesmis echinorum and S. kurakaikina. Interspecific comparison of the three newly obtained sequences and the only previously characterised rhabdocoel, the free-living species Bothromesostoma personatum, reveals high mt genomic variability, including numerous rearrangements. The first intrageneric comparison within rhabdocoels shows that gene order is not fully conserved even between congeneric species. Atp8, until recently assumed absent in flatworms, was putatively annotated in two sequences. Selection pressure was tested in a phylogenetic framework and is shown to be significantly relaxed in this and another protein-coding gene: cox1. If present, atp8 appears highly derived in platyhelminths and its functionality needs to be addressed in future research. Our findings for the first time allude to a large degree of undiscovered (mt) genomic plasticity in rhabdocoels. It merits further attention whether this variation is correlated with a symbiotic lifestyle. Our results illustrate that this phenomenon is widespread in flatworms as a whole and not exclusive to the better-studied neodermatans.

The increased sensitivity of qPCR in comparison to Kato-Katz is required for the accurate assessment of the prevalence of soil-transmitted helminth infection in settings that have received multiple rounds of mass drug administration.

BACKGROUND: The most commonly used diagnostic tool for soil-transmitted helminths (STH) is the Kato-Katz (KK) thick smear technique. However, numerous studies have suggested that the sensitivity of KK can be problematic, especially in low prevalence and low intensity settings. An emerging alternative is quantitative polymerase chain reaction (qPCR). METHODS: In this study, both KK and qPCR were conducted on stool samples from 648 participants in an STH epidemiology study conducted in the delta region of Myanmar in June 2016. RESULTS: Prevalence of any STH was 20.68% by KK and 45.06% by qPCR. Prevalence of each individual STH was also higher by qPCR than KK, the biggest difference was for hookworm with an approximately 4-fold increase between the two diagnostic techniques. Prevalence of Ancylostoma ceylanicum, a parasite predominately found in dogs, was 4.63%, indicating that there is the possibility of zoonotic transmission in the study setting. In individuals with moderate to high intensity infections there is evidence for a linear relationship between eggs per gram (EPG) of faeces, derived from KK, and DNA copy number, derived from qPCR which is particularly strong for Ascaris lumbricoides. CONCLUSIONS: The use of qPCR in low prevalence settings is important to accurately assess the epidemiological situation and plan control strategies for the 'end game'. However, more work is required to accurately assess STH intensity from qPCR results and to reduce the cost of qPCR so that is widely accessible in STH endemic countries.

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.

Quantitative PCR-Based Diagnosis of Soil-Transmitted Helminth Infections: Faecal or Fickle?

Treatment and control programmes tackling soil-transmitted helminth (STH) infections require sensitive, reliable, and accurate diagnostic tools. There is a growing need for measures of infection intensity as programmes approach STH control. Quantitative real-time PCR (qPCR) is well suited to the detection of DNA targets present in stool, even in low-prevalence settings. Detecting low levels of infection becomes increasingly important when the breakpoint of transmission is approached, and is vital when monitoring for recrudescence once control, or possibly 'elimination', is achieved. We address key challenges and questions that remain as barriers to incorporating qPCR as a cornerstone diagnostic tool for STH infections.