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Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that affects about 2.5 million people worldwide. In MS, the patients' immune system starts to attack the myelin sheath, leading to demyelination, neurodegeneration, and, ultimately, loss of vital neurological functions such as walking. There is currently no cure for MS and the available treatments only slow the initial phases of the disease. The later-disease mechanisms are poorly understood and do not directly correlate with the activity of immune system cells, the main target of the available treatments. Instead, evidence suggests that disease progression and disability are better correlated with the maintenance of a persistent low-grade inflammation inside the CNS, driven by local glial cells, like astrocytes and microglia. Depending on the context, astrocytes can (a) exacerbate inflammation or (b) promote immunosuppression and tissue repair. In this review, we will address the present knowledge that exists regarding the role of astrocytes in MS and experimental animal models of the disease.

Fumonisins are naturally occurring mycotoxins that contaminate food for human and animal consumption. They have neurotoxic effects, but the mechanisms by which these toxins affect the nervous system are not fully known. In the present study, male Wistar rats were fed between 21 and 63 days of age with diets that contained fumonisins B

Exosomes are cell-derived vesicles that act as carriers for proteins and nucleic acids, with therapeutic potential and high biocompatibility. We propose a new concept of exosome-like liposomes for controlled delivery. The goal of this work was to develop a new type of liposomes with a unique mixture of phospholipids, similar to naturally occurring exosomes but overcoming their limitations of heterogeneity and low productivity, for therapeutic delivery of bioactive compounds. Curcumin was chosen as model compound, as it is a phytochemical molecule known to have antioxidant and anti-inflammatory properties, which can protect the brain against oxidative stress and reduce β-amyloid accumulation, major hallmarks of Alzheimer's disease (AD). These new liposomes can efficiently encapsulate hydrophobic curcumin, yielding particles with a size smaller than 200 nm, and a polydispersity index lower than 0.20, which make them ideal for crossing the blood-brain barrier. These particles have a long shelf life, being stable up to 6 months. The curcumin encapsulation efficiency was higher than 85% (up to approximately 94%). Curcumin-loaded liposomes were not cytotoxic (up to 20 μM curcumin, and 200 μM of exo-liposomes), and significantly reduced oxidative stress induced in SH-SY5Y neuronal cells, indicating their potential for neuroprotection. They also do not show any toxicity and are internalized in zebrafish embryos, concentrating in lipid enriched areas, as the brain and the yolk sac. Such innovative carriers are a new effective approach to deliver drugs into the brain, as these are stable, protect the cargo and are uptaken by neuronal cells. Upon internalization, liposomes release the therapeutic biomolecules, resulting in successful neuroprotection, being a positive alternative strategy for AD therapy., Strategic Programme UID/BIA/04050/2020 funded by National funds through the Fundação para a Ciência e Tecnologia I.P., and the project “FUN2CYT: Harnessing the Potential for Biomedical Applications of Pleiotropic Cytokines LIF and Oncostatin M” (POCI-01-0145-FEDER-030568), supported by Programa Operacional Competitividade e Internacionalização (FEDER) and FCT, IP. Mário Fernandes (SFRH/BD/147819/2019) and Cláudia Botelho (SFRH/BPD/111291/2015) hold scholarships from Fundação para a Ciência e Tecnologia, and Ivo Lopes is a recipient of a scholarship from the Direção Regional da Ciência e Tecnologia, Governo Regional dos Açores (M3.1.a/F/128/2015)., info:eu-repo/semantics/publishedVersion

Aging is associated with cognitive decline. Herein, we studied a large cohort of old age and young adult male rats and confirmed that, as a group, old rats display poorer spatial learning and behavioral flexibility than younger adults. Surprisingly, when animals were clustered as good and bad performers, our data revealed that while in younger animals better cognitive performance was associated with longer dendritic trees and increased levels of synaptic markers in the hippocampus and prefrontal cortex, the opposite was found in the older group, in which better performance was associated with shorter dendrites and lower levels of synaptic markers. Additionally, in old, but not young individuals, worse performance correlated with increased levels of BDNF and the autophagy substrate p62, but decreased levels of the autophagy complex protein LC3. In summary, while for younger individuals "bigger is better", "smaller is better" is a more appropriate aphorism for older subjects., Portuguese Foundation for Science and Technology (FCT) with fellowships granted to: Cristina Mota (SFRH/BD/81881/2011), Susana Monteiro (SFRH/BD/69311/2010), Sofia Pereira das Neves and Sara Monteiro-Martins (PIC/IC/83213/2007); and by the European Commission within the 7th framework program, under the grant agreement: Health-F2-2010-259772 (Switchbox). In addition, this work was co-funded by the Northern Portugal Regional Operational Programme (ON.2 SR&TD Integrated Program – NORTE-07-0124-FEDER-000021), through the European Regional Development Fund (FEDER) and by national funds granted by FCT (PEst-C/SAU/LA0026/2013), and FEDER through the COMPETE (FCOMP-01-0124-FEDER-037298).

Accepted manuscript, The process of generating new functional neurons in the adult mammalian brain occurs from the local neural stem and progenitor cells and requires tight control of the progenitor cell's activity. Several signaling pathways and intrinsic/extrinsic factors have been well studied over the last years, but recent attention has been given to the critical role of cellular metabolism in determining the functional properties of progenitor cells. Here, we review recent advances in the current understanding of when and how metabolism affects neural stem cell (NSC) behavior and subsequent neuronal differentiation and highlight the role of lipocalin-2 (LCN2), a protein involved in the control of oxidative stress, as a recently emerged regulator of NSC activity and neuronal differentiation., IF/00231/2013 of the Portuguese Foundation for Science and Technology (FCT, Portugal). This work was supported by the Foundation for Science and Technology (FCT) and COMPETE through the project EXPL/NEU-OSD/2196/2013. The work has been developed under the scope of the project NORTE-01-0145-FEDER-000013, which is supported by the Northern Portugal Regional Operational Programme (NORTE 2020) under the Portugal 2020 Partnership Agreement through the European Regional Development Fund (FEDER) and is funded by FEDER funds through the Competitiveness Factors Operational Programme (COMPETE) and by National funds through the Foundation for Science and Technology (FCT) under the scope of the project POCI-01-0145-FEDER-007038, info:eu-repo/semantics/publishedVersion

The following are available online. Scheme S1: Synthesis of peracetylated azidomannose (Ac4ManNAz); Scheme S2: Alternative synthesis of GlcNAz using chloroacetic anhydride and NaOH as a base; Scheme S3: Synthesis of peracetylated azidofucose (Ac4FucAz); Figure S1: 1H-NMR spectra of ManNAz (D2O, 300 MHz); Figure S2: 1H-NMR spectra of Ac4ManNAz (CDCl3 , 300 MHz), mixture of anomers; Figure S3: HPLC chromatogram of purified Ac4ManNAz showing the two anomers; Figure S4: 1H-NMR spectra of GlcNAz (D2O, 300MHz); Figure S5: 1H-NMR spectra of Ac4GlcNAz (CDCl3 , 300 MHz), mixture of anomers; Figure S6: HPLC chromatogram of purified Ac4GlcNAz showing the two anomers; Figure S7: 1H-NMR spectra of 6-azido-1,2,3,4-tetra-O-acetyl-6-deoxy-α,β-L-galactopyranose Ac4FucAz (CDCl3 , 300 MHz): mixture of anomers; Table S1: Primer sequences used in qRT-PCR., The expression of different glycans at the cell surface dictates cell interactions with their environment and other cells, being crucial for the cell fate. The development of the central nervous system is associated with tremendous changes in the cell glycome that is tightly regulated. Herein, we have employed biorthogonal Cu-free click chemistry to image temporal distribution of different glycans in live mouse hippocampal neurons during their maturation in vitro. We show development-dependent glycan patterns with increased fucose and decreased mannose expression at the end of the maturation process. We also demonstrate that this approach is biocompatible and does not affect glycan transport although it relies on an administration of modified glycans. The applicability of this strategy to tissue sections unlocks new opportunities to study the glycan dynamics under more complex physiological conditions., This research was funded by the European Union Framework Programme for Research and Innovation Horizon 2020 under grant agreement n.º 668983—FoReCaST, by European Union’s Horizon 2020 Research and Innovation programme, under the Grant Agreement number 739572–The Discoveries CTR, and the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER).

Background Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., is commonly found in cereals ingested by humans and animals. Its ingestion is correlated with hepatic, hematologic, renal, splenic, cardiac, gastrointestinal, and neural damages, according to dose, duration of exposure and species. In this work, the effects of the ingestion of DON-contaminated diet at concentrations considered tolerable for human and animal intake were assessed. Methods Male Wistar rats aging 21 days were allotted to five groups that were given, for 42 days, diets contaminated with different concentrations of DON (0, 0.2, 0.75, 1.75, and 2 mg kg-1 of chow). Food ingestion, bodyweight, oxidative status and morphometric analyses of gliocytes, and neurons of jejunal myenteric ganglia were recorded. Key results At these concentrations, there was no food rejection, decrease in bodyweight gain, changes in oxidative status, or loss of either neurons or gliocytes. However, DON decreased gliocyte area, general neuronal population, nitrergic, cholinergic and NADH-diaphorase positive subpopulations and, as a result, ganglion area. Conclusions & inferences It was concluded that, even in the absence of visible effect, DON exposure reduces cell body area of gliocytes and neurons of the myenteric plexus of the rat jejunum.

In the adult mammalian brain, newborn granule cells are continuously integrated into hippocampal circuits, and the fine-tuning of this process is important for hippocampal function. Thus, the identification of factors that control adult neural stem cells (NSCs) maintenance, differentiation and integration is essential. Here we show that the deletion of the iron trafficking protein lipocalin-2 (LCN2) induces deficits in NSCs proliferation and commitment, with impact on the hippocampal-dependent contextual fear discriminative task. Mice deficient in LCN2 present an increase in the NSCs population, as a consequence of a G0/G1 cell cycle arrest induced by increased endogenous oxidative stress. Of notice, supplementation with the iron-chelating agent deferoxamine rescues NSCs oxidative stress, promotes cell cycle progression and improves contextual fear conditioning. LCN2 is, therefore, a novel key modulator of neurogenesis that, through iron, controls NSCs cell cycle progression and death, self-renewal, proliferation and differentiation and, ultimately, hippocampal function., AC Ferreira is a recipient of PhD fellowship and B Sampaio-Marques is a recipient of postdoctoral fellowship from the Foundation for Science and Technology (FCT, Portugal)/FEDER. F Marques is an assistant researcher IF/00231/2013 of the Foundation for Science and Technology (FCT, Portugal). This work was supported by Foundation for Science and Technology (FCT) and COMPETE through the project EXPL/NEU-OSD/2196/2013 (to F Marques) and by the Bial Foundation through Grant 217/12 (to JC Sousa). The work at ICVS/3B's has been developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), and funded by FEDER funds through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038. The work at CICS-UBI has the support of FEDER funds through the POCI - COMPETE 2020 - Operational Programme Competitiveness and Internationalisation in Axis I - Strengthening research, technological development and innovation (Project POCI-01-0145-FEDER-007491) and National Funds by Foundation for Science and Technology (Project UID/Multi /00709/2013)., info:eu-repo/semantics/publishedVersion

Exposure to chronic stress leads to an array of anatomical, functional, and metabolic changes in the brain that play a key role in triggering psychiatric disorders such as depression. The hippocampus is particularly well known as a target of maladaptive responses to stress. To capture stress-induced changes in metabolic and functional connectivity in the hippocampus, stress-resistant (low-responders) or -susceptible (high-responders) rats exposed to a chronic unpredictable stress paradigm (categorized according to their hormonal and behavioral responses) were assessed by multimodal neuroimaging; the latter was achieved by using localized 1H MR spectroscopy and resting-state functional MRI (fMRI) at 11,7T data from stressed (n = 25) but also control (n = 15) male Wistar rats.Susceptible animals displayed increased GABA-glutamine (+19%) and glutamate-glutamine (+17%) ratios and decreased levels of macromolecules (-11%); these changes were positively correlated with plasma corticosterone levels. In addition, the neurotransmitter levels showed differential associations with functional connectivity between the hippocampus and the amygdala, the piriform cortex and thalamus between stress-resistant and -susceptible animals. Our observations are consistent with previously reported stress-induced metabolomic changes that suggest overall neurotransmitter dysfunction in the hippocampus. Their association with the fMRI data in this study reveals how local adjustments in neurochemistry relate to changes in the neurocircuitry of the hippocampus, with implications for its stress-associated dysfunctions.SIGNIFICANCE STATEMENT Chronic stress disrupts brain homeostasis, which may increase the vulnerability of susceptible individuals to neuropsychiatric disorders such as depression. Characterization of the differences between stress-resistant and -susceptible individuals on the basis of noninvasive imaging tools, such as magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI), contributes to improved understanding of the mechanisms underpinning individual differences in vulnerability and can facilitate the design of new diagnostic and intervention strategies. Using a combined functional MRI/MRS approach, our results demonstrate that susceptible- and non-susceptible subjects show differential alterations in hippocampal GABA and glutamate metabolism that, in turn, associate with changes in functional connectivity., This work is part of the Sigma project (FCT-ANR/NEU-OSD/0258/2012) cofinanced by the French ANR (Agence National pour la Recherche, APP Blanc International II 2012), the Portuguese FCT (Fundacao para a Ciencia e Tecnologia) and by the project NORTE-01-0124-FEDER-000021, supported by the Portuguese North Regional Operational Program (ON.2-O Novo Norte), under the National Strategic Reference Framework (QREN) through the European Regional Development Fund (FEDER), by the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement through the FEDER as well as the Competitiveness Factors Operational Programme (COMPETE), and the Foundation for Science and Technology (FCT) under the scope of the project POCI-01-0145-FEDER-007038, and performed on a platform of the France Life Imaging network partly funded by Grant ANR-11-INBS-0006; R.M. was supported by Grant PDE/BDE/113604/2015 from the PhD-iHES program of the School of Medicine of Universidade do Minho. We thank Douglas L. Rothman, Grame F. Mason, and Osborne Almeida for their feedback.

The continuous generation of new neurons in the adult mammalian hippocampus is a form of neural plasticity that modulates learning and memory functions, and also emotion (anxiety and depression). Among the factors known to modulate adult hippocampal neurogenesis and brain function, lipocalin-2 (LCN2) was recently described as a key regulator of neural stem cells (NSCs) proliferation and commitment, with impact on several dimensions of behaviour. Herein, we evaluated whether voluntary running, a well-known regulator of cell genesis, rescue the deficient adult hippocampal neurogenesis observed in mice lacking LCN2. We observed that running, by counteracting oxidative stress in NSCs, reverses LCN2-null mice defective hippocampal neurogenesis, as it promotes NSCs cell cycle progression and maturation, resulting in a partial reduction in anxiety and improved contextual behaviour. Together, these findings demonstrate that running is a positive modulator of adult hippocampal neurogenesis and behaviour in mice lacking LCN2, by impacting on the antioxidant kinetics of NSCs., Ana Catarina Ferreira is recipient of PhD fellowship from the Foundation for Science and Technology (FCT, Portugal)/FEDER. Fernanda Marques is an assistant researcher IF/00231/2013 of the Foundation for Science and Technology (FCT, Portugal). Tis work was supported by Foundation for Science and Technology (FCT) and COMPETE through the project EXPL/NEU-OSD/2196/2013 (to Marques F) and by the Bial Foundation through Grant 217/12 (to Sousa JC). Te work at ICVS/3B’s has been developed under the scope of the project NORTE01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), and funded by FEDER funds through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038.

Background Better knowledge of the efficacy and safety of single-dose 40 mg/kg oral praziquantel in preschool-age children is required, should preventive chemotherapy programs for schistosomiasis be expanded to include this age group. Methodology We analyzed individual participant-level data from 16 studies (13 single-arm or cohort studies and three randomized trials), amounting to 683 preschool-age children (aged, Author summary Schistosomiasis is a diseases caused by helminths (parasitic worms) which affects the intestinal and urogenital systems. In areas where schistosomiasis is endemic, the disease is controlled by the large scale distributing of praziquantel, primarily targeting school-age children. Younger children (preschool-age) too might be affected by schistosomiasis, but are currently not receiving praziquantel within treatment campaigns. Instead, preschool-age children are treated on a case-by-case basis because the current praziquantel formulation is not adapted to young children. Questions have also been raised as to whether the standard dose of 40 mg/kg given once is effective in preschool-age children. To answer this question, we collected individual-participant data from a series of studies in which 40 mg/kg of praziquantel had been given to children with intestinal or urinary schistosomiasis, and compared its efficacy and tolerability across age-groups. Since few direct comparisons had been made, we used statistical tools to make these comparisons. We found no evidence that treatment is less efficacious in preschool- than in school-age children and conclude that 40 mg/kg praziquantel may be given to preschool-age children in large-scale programs. When this happens, efficacy and tolerability will have to be closely monitored.

This study documented the population dynamics ofBiomphalariaand associated natural infections with digenetic trematodes, along the shores of Lake Albert and Lake Victoria, recording local physicochemical factors. Over a two-and-a-half-year study period with monthly sampling, physicochemical factors were measured at 12 survey sites and all freshwater snails were collected. RetainedBiomphalariawere subsequently monitored in laboratory aquaria for shedding trematode cercariae, which were classified as either human infective (Schistosoma mansoni) or nonhuman infective. The population dynamics ofBiomphalariadiffered by location and by lake and had positive relationship with pH (P<0.001) in both lakes and negative relationship with conductivity (P=0.04) in Lake Albert. Of theBiomphalariacollected in Lake AlbertN=6,183, 8.9% were infected with digenetic trematodes of which 15.8% were sheddingS. mansonicercariae and 84.2% with nonhuman infective cercariae. In Lake Victoria, 2.1% of collectedBiomphalaria N=13,172were infected with digenetic trematodes with 13.9% sheddingS. mansonicercariae, 85.7% shedding nonhuman infective cercariae, and 0.4% of infected snails shedding both types of cercariae. Upon morphological identification, species ofBiomphalariainfected includedB. sudanica,B. pfeifferi, andB. stanleyiin Lake Albert andB. sudanica,B. pfeifferi, andB. choanomphalain Lake Victoria. The study found the physicochemical factors that influencedBiomphalariapopulation and infections. The number and extent of snails sheddingS. mansonicercariae illustrate the high risk of transmission within these lake settings. For better control of this disease, greater effort should be placed on reducing environmental contamination by improvement of local water sanitation and hygiene.

Background This study was designed to determine the distribution and identity of potential intermediate snail hosts of Schistosoma spp. in Bengo, Luanda, Kwanza Norte and Malanje Provinces in north-western Angola. This is an area where infection with Schistosoma haematobium, causing urogenital schistosomiasis, is common but little is yet known about transmission of the disease. Angola has had a varied past with regard to disease control and is revitalising efforts to combat neglected tropical diseases. Methods Snails were sampled from 60 water-contact points. Specimens of the genera Bulinus, Biomphalaria or Lymnaea were screened for trematode infections by inducing cercarial shedding. Snails were initially identified using shell morphology; subsequently a cytochrome c oxidase subunit 1 (cox1) gene fragment was amplified from a subset of snails from each site, for molecular identification. Cercariae were captured onto FTA cards for molecular analysis. Specimens of Bulinus angolensis collected from the original locality of the type specimen have been characterised and comparisons made with snails collected in 1957 held at the Natural History Museum, London, UK. Results In total snails of nine genera were identified using morphological characteristics: Biomphalaria, Bulinus, Gyraulus, Lanistes, Lentorbis, Lymnaea, Melanoides, Physa and Succinea. Significant for schistosomiasis transmission, was the discovery of Bulinus globosus, B. canescens, B. angolensis, B. crystallinus and Biomphalaria salinarum in their type-localities and elsewhere. Bulinus globosus and B. angolensis occurred in two distinct geographical areas. The cox1 sequence for B. globosus differed markedly from those from specimens of this species collected from other countries. Bulinus angolensis is more closely related to B. globosus than originally documented and should be included in the B. africanus group. Schistosoma haematobium cercariae were recovered from B. globosus from two locations: Cabungo, Bengo (20 snails) and Calandula, Malanje (5 snails). Schistosoma haematobium cercariae were identified as group 1 cox1 corresponding to the type common throughout the African mainland. Conclusions Various freshwater bodies in north-western Angola harbour potential intermediate snail hosts for urogenital schistosomiasis, highlighting the need to map the rest of the country to identify areas where transmission can occur and where control efforts should be targeted. The molecular phylogeny generated from the samples confirmed that considerable variation exists in B. globosus, which is the primary snail host for S. haematobium in many regions of Africa. Electronic supplementary material The online version of this article (10.1186/s13071-017-2395-y) contains supplementary material, which is available to authorized users.

Stress is a well-established trigger for a number of neuropsychiatric disorders, as it alters both structure and function of several brain regions and its networks. Herein, we conduct a longitudinal neuroimaging study to assess how a chronic unpredictable stress protocol impacts the structure of the rat brain and its functional connectome in both high and low responders to stress. Our results reveal the changes that stress triggers in the brain, with structural atrophy affecting key regions such as the prelimbic, cingulate, insular and retrosplenial, somatosensory, motor, auditory and perirhinal/entorhinal cortices, the hippocampus, the dorsomedial striatum, nucleus accumbens, the septum, the bed nucleus of the stria terminalis, the thalamus and several brain stem nuclei. These structural changes are associated with increasing functional connectivity within a network composed by these regions. Moreover, using a clustering based on endocrine and behavioural outcomes, animals were classified as high and low responders to stress. We reveal that susceptible animals (high responders) develop local atrophy of the ventral tegmental area and an increase in functional connectivity between this area and the thalamus, further spreading to other areas that link the cognitive system with the fight-or-flight system. Through a longitudinal approach we were able to establish two distinct patterns, with functional changes occurring during the exposure to stress, but with an inflection point after the first week of stress when more prominent changes were seen. Finally, our study revealed differences in functional connectivity in a brainstem-limbic network that distinguishes resistant and susceptible responders before any exposure to stress, providing the first potential imaging-based predictive biomarkers of an individual's resilience/vulnerability to stressful conditions., This work is part of the Sigma project with the reference FCT-ANR/NEU-OSD/ 0258/2012 co-financed by the French public funding agency ANR (Agence National pour la Recherche, APP Blanc International II 2012), the Portuguese FCT (Fundação para a Ciência e Tecnologia) and by the Portuguese North Regional Operational Program (ON.2 – O Novo Norte) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER) as well as the Projecto Estratégico co-funded by FCT (PEst-C/SAU/LA0026-/2013) and the European Regional Development Fund COMPETE (FCOMP-01-0124-FEDER-037298). DAB and AN were funded by grants from FCT-ANR/NEU-OSD/0258/2012. RM is supported by the FCT fellowship grant with the reference PDE/BDE/113604/2015 from the PhDiHES program; AC was supported by a grant from the foundation NRJ. PM was funded by Fundação Calouste Gulbenkian (Portugal; ‘Better mental health during ageing based on temporal prediction of individual brain ageing trajectories (TEMPO)’), Grant Number P-139977. We thank Drs Patrício Costa and Pedro Moreira for support on the various statistical analyses., info:eu-repo/semantics/publishedVersion

This article brings the choroid plexus into the context of health and disease. It is remarkable that the choroid plexus, composed by a monolayer of epithelial cells that lie in a highly vascularized stroma, floating within the brain ventricles, gets so little attention in major physiology and medicine text books and in the scientific literature in general. Consider that it is responsible for producing most of the about 150 mL of cerebrospinal fluid that fills the brain ventricles and the subarachnoid space and surrounds the spinal cord in the adult human central nervous system, which is renewed approximately 2-3 times daily. As such, its activity influences brain metabolism and function, which will be addressed. Reflect that it contains an impressive number of receptors and transporters, both in the apical and basolateral sides of the epithelial cells, and as such is a key structure for the communication between the brain and the periphery. This will be highlighted in the context of neonatal jaundice, multiple sclerosis and Alzheimer's disease. Realize that the capillaries that irrigate the choroid plexus stroma do not possess tight junctions and that the blood flow to the choroid plexus is five times higher than that in the brain parenchyma, allowing for a rapid sensing system and delivery of molecules such as nutrients and metals as will be revised. Recognize that certain drugs reach the brain parenchyma solely through the choroid plexus epithelia, which has potential to be manipulated in diseases such as neonatal jaundice and Alzheimer's disease as will be discussed. Without further notice, it must be now clear that understanding the choroid plexus is necessary for comprehending the brain and how the brain is modulated and modulates all other systems, in health and in disease. This review article intends to address current knowledge on the choroid plexus, and to motivate the scientific community to consider it when studying normal brain physiology and diseases of the central nervous system. It will guide the reader through several aspects of the choroid plexus in normal physiology, in diseases characteristic of various periods of life (newborns-kernicterus, young adults-multiple sclerosis and the elder Alzheimer's disease), and how sex-differences may relate to disease susceptibility., The work at ICVS/3B's has the support of Portuguese North Regional Operational Program (ON.2 – O Novo Norte) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER). JCS, FM and JAP have the support of a fellowship from Bial Foundation through Grant 217/12. Fernanda Marques is a recipient of a FCT Investigator award (IF/ 00231/2013) of the Fundação para a Ciência e Tecnologia (FCT, Portugal). The work by JP has been financed by the following grants: Deutsche Forschungsgemeinschaft DFG PA930/9, DFG PA930/12; VIAA Latvia NFI/ R/2014/023; Leibniz Society SAW-2015-IPB-2; HelseSØ No: 2016062; Norsk forskningsrådet: NFR251290, NFR246392, NFR248772, JPND NeuroGEM NFR247179, JPND PROP-AD NFR260786. The work by CS has been supported by the Portuguese Foundation for Science and Technology (FCT, Portugal – hrrp://www.fct.pt) project grants (PTDC/SAU-NEU/114800/2009); and by FEDER funds through the POCI - COMPETE 2020 - Operational Programme Competitiveness and Internationalisation in Axis I - Strengthening research, technological development and innovation (Project No. 007491) and National Funds by FCT - Foundation for Science and Technology (Project UID/ Multi/00709). The work by AB at Faculty of Pharmacy, Universidade de Lisboa, was funded, in part, by iMed.ULisboa (UID/DTP/04138/2013) from Fundação para a Ciência e a Tecnologia (FCT), Portugal., info:eu-repo/semantics/publishedVersion

The production, accumulation and aggregation of amyloid beta (Aß) peptides in Alzheimer's disease (AD) are influenced by different modulators. Among these are iron and iron-related proteins, given their ability to modulate the expression of the amyloid precursor protein and to drive Aß aggregation. Herein, we describe that lipocalin 2 (LCN2), a mammalian acute-phase protein involved in iron homeostasis, is highly produced in response to Aß1-42 by choroid plexus epithelial cells and astrocytes, but not by microglia or neurons. Although Aß1-42 stimulation decreases the dehydrogenase activity and survival of wild-type astrocytes, astrocytes lacking the expression of Lcn2 are not affected. This protection results from a lower expression of the proapoptotic gene Bim and a decreased inflammatory response. Altogether, these findings show that Aß toxicity to astrocytes requires LCN2, which represents a novel mechanism to target when addressing AD.Cell Death and Differentiation advance online publication, 23 May 2014; doi:10.1038/cdd.2014.68., We thank Dr. Ioannis Sotiropoulos for reagents and comments. Sandro Da Mesquita and Ana Catarina Ferreira are recipients of PhD fellowships and Fernanda Marques is recipient of a postdoctoral fellowship by the Fundacao para a Ciencia e Tecnologia (FCT, Portugal)/FEDER. This work was supported by a grant from FCT/FEDER (EXPL/NEUOSD/2196/2013).

Following major water development schemes in the 1980s, schistosomiasis has become a serious parasitic disease of children living in the Senegal River Basin. Both urogenital (Schistosoma haematobium) and intestinal (Schistosoma mansoni) schistosomiasis can be highly prevalent in school-aged children, with many individuals infected with both parasites. In order to investigate the transmission and re-infection dynamics of both parasite species, single and mixed infection foci at three villages (Nder and Temeye; S. mansoni and S. haematobium foci and Guia; S. haematobium focus) were studied. In each focus infected children were identified and selected for a 12-month study involving two treatments with praziquantel (40mg/kg) three weeks apart at the beginning of the study and again 6 months into the study. Urine and stool samples were examined for schistosome eggs before and at 6 weeks and 6 months after chemotherapy. Prevalence and intensity of infection were recorded for each child at each time point. Before treatment, in all three villages, the prevalence and intensity of infection was extremely high for both S. mansoni (79-100%) and S. haematobium (81-97%). With the first round of chemotherapy sufficient cure rates (CRs) of both species were achieved in all villages (38-96%) with high egg reduction rates (ERRs) (97-99%). The data show that high and rapid re-infection rates occur, especially for S. mansoni, within a six-month period following treatment. Re-infection must be highly linked to ecological and seasonal factors. The persistence of S. mansoni in Nder could raise concern as levels of infection intensity remain high (geometric mean intensity at baseline 653epg changed to 705epg at 12 months) after four rounds of chemotherapy. This phenomenon could be explained by extremely rapid re-infection dynamics or a sub-optimal efficacy of praziquantel against S. mansoni in this village. High intensities in mixed infections may influence disease epidemiology and control warranting further studies. The disease situation in the SRB must be monitored closely and new treatment regimes should be designed and implemented to control schistosomiasis in the school-age population.

BACKGROUND A sensitive and reliable rapid diagnostic test (RDT) which should have comparable diagnostic performance against reference host serological methods is urgently needed for use in point-of-care (POC) diagnosis of intestinal schistosomiasis in pre school-aged children. METHODS The diagnostic accuracy of a RDT incorporating Schistosoma mansoni cercarial transformation fluid (SmCTF) for anti-schistosome antibody detection was evaluated with serum samples from a cohort of children from Uganda: 42 children aged under the age of 3 years and 40 children aged between 3 and 5 years. The infection status of these children had been previously determined by inspection of quadruplicate Kato-Katz faecal smears, a single urine circulating cathodic antigen (CCA) dipstick and antibody titres to S. mansoni soluble egg antigen (SmSEA) with a commercially available ELISA. RESULTS Upon comparison with quadruplicate Kato-Katz the sensitivity and specificity of the RDT were 75.7% and 31.1%, respectively. When using the SmSEA-ELISA as an alternate reference test, the RDT achieved 81.3% sensitivity and 61.1% specificity. Sensitivity and specificity compared to the urine-CCA test was 74.5% and 32.3% respectively. Sensitivity differed significantly according to age group. CONCLUSIONS The performance of the RDT within this study appeared favourable when compared with the currently-available SmSEA-ELISA. Looking to the future a serological POC test would be particularly promising for use in disease mapping in younger children especially in guiding administration of praziquantel treatment in selective treatment settings.

The occurrence of schistosomiasis within African infants and preschool children has been much better documented in recent years, revealing an important burden of disease previously overlooked. Despite mounting evidence showing that treatment with praziquantel is safe, beneficial, and could be delivered within ongoing public health interventions, young children still do not have satisfactory access to this drug, and a significant treatment gap exists. Progress towards resolution of this unfortunate health inequity is highlighted, including the development of an appropriate paediatric praziquantel formulation, and present blocks are identified on securing this issue within the international health agenda.

Each year, millions of African children receive praziquantel (PZQ) by mass drug administration (MDA) to treat schistosomiasis at a standard single dose of 40 mg/kg of body weight, a direct extrapolation from studies of adults. A higher dose of 60 mg/kg is also acceptable for refractory cases. We conducted the first PZQ pharmacokinetic (PK) and pharmacodynamic (PD) study in young children comparing dosing. Sixty Ugandan children aged 3 to 8 years old with egg patent Schistosoma mansoni received PZQ at either 40 mg/kg or 60 mg/kg. PK parameters of PZQ racemate and enantiomers (R and S) were quantified. PD outcomes were assessed by standard fecal egg counts and novel schistosome-specific serum (circulating anodic antigen [CAA]) and urine (circulating cathodic antigen [CCA]) antigen assays. Population PK and PD analyses were performed to estimate drug exposure in individual children, and the relationship between drug exposure and parasitological cure was estimated using logistic regression. Monte Carlo simulations were performed to identify better, future dosing regimens. There was marked PK variability between children, but the area under the concentration-time curve (AUC) of PZQ was strongly predictive of the parasitological cure rate (CR). Although no child achieved antigenic cure, which is suggestive of an important residual adult worm burden, higher AUC was associated with greater CAA antigenic decline at 24 days. To optimize the performance of PZQ, analysis of our simulations suggest that higher doses (>60 mg/kg) are needed, particularly in smaller children., IMPORTANCE Schistosomiasis is a neglected tropical disease, typically associated with chronic morbidity, and its control is a global health priority. Praziquantel (PZQ) is the only available antiparasitic drug and is often given out, as a single oral dose (40 mg/kg), to school-aged children by mass drug administration (MDA) schemes operating within preventive chemotherapy campaigns as endorsed by the World Health Organization (WHO). This current strategy has several limitations. (i) It excludes preschool children who can be patently infected. (ii) It delivers PZQ at a dose directly extrapolated from adult pharmacological studies. To address these problems, we conducted the first pharmacokinetic and pharmacodynamic study of young children within an area of Uganda where Schistosoma mansoni is hyperendemic. Our results demonstrate that a higher dose (>60 mg/kg) is required, especially in smaller children, and draw attention to the need for further optimization of PZQ treatment based on schistosome antigenic assays, which are more sensitive to pharmacodynamic markers.

Cell culture systems, either 2D or explant based, have been pivotal to better understand the pathophysiology of several central nervous system (CNS) disorders. Recently, bioengineered cell culture systems have been proposed as an alternative to the traditional setups. These innovative systems often combine different cell populations in 3D environments that more closely recapitulate the different niches that exist within the developing or adult CNS. Given the importance of such systems for the future of CNS-related research, we discuss here the most recent advances in the field, particularly those dealing with neurodegeneration, neurodevelopmental disorders, and trauma., Financial support is acknowledged from Prémios Santa Casa Neurociências – Prize Melo e Castro for Spinal Cord Injury Research; Portuguese Foundation for Science and Technology [Doctoral fellowship (SFRH/BD/103075/2014) to E.D.G.; IF Development Grant to A.J.S.; Starting Grant to F. Marques; PostDoctoral fellowship SFRH/BPD/97701/2013 to N.A.S.]; this work was co-funded by Programa Operacional Regional do Norte (ON.2 – O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER)., info:eu-repo/semantics/publishedVersion

There is a need for field-applicable markers to assess morbidity associated with intestinal schistosomiasis, especially in the context of preventive chemotherapy in young children. We investigated whether fecal occult blood (FOB) point-of-care tests could be used to assess intestinal pathology over a 12-month period in a cohort of 382 children (< 5 years of age). We found a strong association between egg-patent schistosomiasis and FOB at baseline (odds ratio [OR] = 3.1, P < 0.0001), 6 months (OR = 3.4, P < 0.0001), and 12 months (OR = 3.5, P < 0.0001), despite repeated chemotherapy. There were tendencies for prevalence of FOB to decrease in children who became egg negative and increase in those who became egg positive. Our results demonstrate overt disease in children less than five years of age. We therefore propose that FOB is useful for assessing dynamics of intestinal morbidity in young children at the community level and monitoring changes in morbidity after mass chemotherapy.

Urine-dipstick diagnostic tests that detect schistosome circulating cathodic antigen (CCA) have the potential to provide more sensitive and rapid testing for intestinal schistosomiasis in field-based surveys; this is especially so when examining preschool children, from whom it may be difficult to obtain consecutive stool samples. To assess the performance of urine dipsticks, 569 preschool children from four villages along the shore of Lake Albert, Uganda, were screened for Schistosoma mansoni by Kato-Katz (K-K) examination of a single stool sample and CCA urine dipsticks. The prevalence of infection was 32.2% by K-K and 40.0% by CCA tests. Sensitivity and specificity were influenced by whether 'trace' results from the CCA test were characterised as positive or negative for infection with S. mansoni; ambiguities around this issue need to be resolved. Nevertheless, the CCA test showed particular promise for routine epidemiological screening in this setting.

SUMMARYAnaemia is a severe public health issue among African preschool-aged children, yet little effective progress has been made towards its amelioration, in part due to difficulties in unravelling its complex, multifactorial aetiology. To determine the current anaemia situation and assess the relative contribution of malaria, intestinal schistosomiasis and infection with soil-transmitted helminths, two separate cross-sectional epidemiological surveys were carried out in Uganda including 573 and 455 preschool-aged children (⩽6 years) living along the shores of Lake Albert and on the islands in Lake Victoria, respectively. Anaemia was found to be a severe public health problem in Lake Albert, affecting 68·9% of children (ninety-five percent confidence intervals (95% CI) 64·9–72·7%), a statistically significant higher prevalence relative to the 27·3% detected in Lake Victoria (95% CI: 23·3–31·7%). After multivariate analysis (controlling for sex and age of the child), the only factor found to be significantly associated with increased odds of anaemia in both lake systems was malaria (Lake Albert, odds ratio (OR)=2·1, 95% CI: 1·4–3·2; Lake Victoria, OR=1·9, 95% CI: 1·2–2·9). Thus intervention strategies primarily focusing on very young children and combating malaria appear to represent the most appropriate use of human and financial resources for the prevention of anaemia in this age group and area. Looking to the future, these activities could be further emphasised within the National Child Health DaysPLUSagenda.

Ascariasis is of public health importance on the islands of Zanzibar (Unguja and Pemba). To shed light on the molecular epidemiology of this parasite, 68 Ascaris worms, obtained from 14 individuals in four Ungujan villages, were examined by isoenzyme analysis (ISA), DNA barcoding and microsatellite DNA profiling. ISA revealed genetic variation, which was confirmed by DNA barcoding. Nineteen worms recovered from individuals in Uganda were included for comparison. Sixteen unique DNA barcodes were identified, 15 on Unguja and three in Uganda with two shared between. These two barcodes were found in all four Ungujan villages. Worms from Tumbatu-Jongowe, an isolated village on an islet off Unguja, seemed particularly diverse. Within our barcodes, three exact matches were found with Chinese Ascaris retrieved from pigs, which is perhaps surprising given the present rarity of these animals on Unguja. Microsatellite profiling and population genetic analysis revealed further genetic diversity within our samples although population sub-structuring within Unguja was minor in comparison to that between Unguja and Uganda. As African Ascaris has not been subjected to detailed molecular scrutiny, this new diversity represents an important piece in its evolutionary jigsaw and such population markers are informative in monitoring worm dynamics during ongoing control.

The choroid plexus, being part of the blood-brain barriers and responsible for the production of cerebrospinal fluid, is ideally positioned to transmit signals into and out of the brain. This study, using microarray analysis, shows that the mouse choroid plexus displays an acute-phase response after an inflammatory stimulus induced in the periphery by lipopolysaccharide (LPS). Remarkably, the response is specific to a restricted number of genes (out of a total of 24,000 genes analyzed, 252 are up-regulated and 173 are down-regulated) and transient, as it returns to basal conditions within 72 h. The up-regulated genes cluster into families implicated in immune-mediated cascades and in extracellular matrix remodeling, whereas those down-regulated participate in maintenance of the barrier function. Importantly, several acute-phase proteins, whose blood concentrations rise in response to inflammation, may contribute to the effects observed in vivo after LPS injection, as suggested by the differential response of primary choroid plexus epithelial cell cultures to LPS alone or to serum collected from animals exposed to LPS. By modulating the composition of the cerebrospinal fluid, which will ultimately influence the brain parenchyma, the choroid plexus response to inflammation may be of relevance in brain homeostasis in health and disease., This work was supported by grant POCTI/SAU-NEU/ 56618/2004 from the Portuguese Foundation for Science and Technology (FCT) /FEDER; and from a grant from the DANA foundation; Marques F, Falcao AM, and Rodrigues AJ are recipients of fellowships from FCT/FEDER.

We characterized the molecular diversity of vanA vancomycin-resistant enterococci (VRE; 176 isolates/87 pulsed-field gel electrophoresis types) from different sources and cities in Portugal (1996 to 2004): (i) food animals (FA; n = 38 isolates out of 31 samples), hospitalized humans (HH; n = 101/101), healthy human volunteers (HV; n = 7/4), and environmental sources ( n = 30/10). Some strains were isolated from different hosts and persistently recovered for years. Twenty-four Tn 1546 variants were identified, all located on plasmids (30 to 250 kb). Some Tn 1546 variants were associated with specific sources such as FA (3 types), HH (11 types), or HV (1 type), while others were recovered from isolates of different origins (8 types). Polymorphisms in the central vanRSHA region of Tn 1546 were scarcely detected, while alterations upstream of vanR and downstream of vanA were frequently identified involving mutations ( vanS and vanX ), deletions ( vanY ), insertions (IS 1216V , IS Ef1 , and IS 19 ; sequences with or without homology with others available in GenBank databases), and different genetic rearrangements. Most Tn 1546 variants contained IS 1216V (14 types) or IS Ef1 (6 types). IS 1216V was found alone or associated with an IS 3 -like element at different orientations and positions in Tn 1546 from human, animal, and environmental samples. IS Ef1 was located within vanX - vanY region at nucleotide 9044 of Tn 1546 variants mostly associated with clinical isolates, suggesting a common genetic platform. IS 19 was observed within the vanX - vanY region in one Tn 1546 variant from poultry. Recent spread of VRE in Portugal reflects a complex epidemiology involving both clonal spread and plasmid dissemination containing a variety of Tn 1546 types. Apparent Tn 1546 heterogeneity among enterococci from human, animal, and environmental sources might reflect frequent genetic exchange events and evolution of particular widely disseminated genetic elements.

To investigate the diversity of integrons and extended-spectrum beta-lactamases (ESBLs) among Enterobacteriaceae from chickens and swine in Portugal and analyse the clonal relationships between Portuguese ESBL-producing isolates of animal and human origin.We analysed samples from faeces of healthy swine (HSF, n = 35), from uncooked chicken carcasses (CM, n = 20) and from faeces of healthy chickens (HCF, n = 20). Samples were plated on MacConkey agar with and without ceftazidime (1 mg/L) or cefotaxime (1 mg/L). ESBLs were characterized by PCR and DNA sequencing. Bacterial identification, antibiotic susceptibility and conjugation assays were performed by standard procedures. Isolate clonal relatedness was established by PFGE and by RAPD for PFGE non-typeable isolates. Escherichia coli phylogenetic groups were identified by a multiplex PCR. Integron analysis was accomplished by PCR-RFLP and sequencing.ESBL-producing Enterobacteriaceae were identified in 60% of CM, 10% of HCF and 5.7% of HSF samples, respectively, mostly corresponding to E. coli (phylogroups A, D and B1). TEM-52, SHV-2 and CTX-M-1 were detected from chicken and SHV-12 from swine samples. High clonal diversity was observed and most bla(ESBL) genes were transferable (67%). Class 1 and/or class 2 integrons were identified in 80% of CM, 10% of HCF and 63% of HSF samples, with class 1 integrons more common than class 2 integrons (36% versus 12% of the isolates recovered, respectively). Ten class 1 integron types are described, aadA1 and dfrA1-aadA1 being the most frequently found. Two class 1 integron types (aadA13-estX and dfrA14-aadA1-catB2) and one class 2 integron (aadA1) are first reported here.This study is the first report of ESBLs and integrons from chickens and swine in Portugal and highlights the antibiotic-resistant bacteria and/or resistance genes that might be acquired by humans through the food chain.

Increased levels of neuropeptide Y have been reported in transthyretin-null mice. This effect might be related to transthyretin ligands (retinol and thyroxine) since, through binding to nuclear receptors, they modulate the expression of genes that control cellular metabolism. The retinoic X receptors form obligatory heterodimers with peroxisome proliferator-activated receptors and liver X receptors - potent regulators of fat, glucose and cholesterol homeostasis. We used transthyretin-null mice to investigate whether the absence of transthyretin influences metabolism. Transthyretin-null mice do not differ from controls in body weight and white adipose tissue morphology, nor in basal or fast-induced circulating levels of glucose, lipids, and leptin. Glucose tolerance tests show that transthyretin-null mice have normal capacity to remove and metabolize energy substrates. Expression of genes encoding lipid transporters and nuclear receptors are also similar in transthyretin-null and control mice. Therefore, the absence of transthyretin does not seem to influence the regulation of lipid and glucose metabolism., POCTI / NSE / 37315 / 2001 and POCI/SAU-NEU/56618/2004, FEDER from Fundação para a Ciência e Tecnologia (Portugal)

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a marked decline in cognition and memory function. Increasing evidence highlights the essential role of neuroinflammatory and immune-related molecules, including those produced at the brain barriers, on brain immune surveillance, cellular dysfunction and amyloid beta (Aß) pathology in AD. Therefore, understanding the response at the brain barriers may unravel novel pathways of relevance for the pathophysiology of AD. Herein, we focused on the study of the choroid plexus (CP), which constitutes the blood-cerebrospinal fluid barrier, in aging and in AD. Specifically, we used the PDGFB-APPSwInd (J20) transgenic mouse model of AD, which presents early memory decline and progressive Aß accumulation, and littermate age-matched wild-type (WT) mice, to characterize the CP transcriptome at 3, 5-6 and 11-12months of age. The most striking observation was that the CP of J20 mice displayed an overall overexpression of type I interferon (IFN) response genes at all ages. Moreover, J20 mice presented a high expression of type II IFN genes in the CP at 3months, which became lower than WT at 5-6 and 11-12months. Importantly, along with a marked memory impairment and increased glial activation, J20 mice also presented a similar overexpression of type I IFN genes in the dorsal hippocampus at 3months. Altogether, these findings provide new insights on a possible interplay between type I and II IFN responses in AD and point to IFNs as targets for modulation in cognitive decline., The authors would like to acknowledge Pedro Moreira, Madalena Esteves and Dr. Patricio Costa (all from the ICVS/3B's - PT Government Associate Laboratory) for comments and scientific input regarding the statistical analysis of this study. Sandro Da Mesquita and Ana C. Ferreira are recipients of PhD fellowships by the Fundacao para a Ciencia e Tecnologia (FCT, Portugal)/FEDER and Programa Operacional Potencial Humano (POPH/FSE), references SFRH/BD/69706/2010 and SFRH/BD/51989/2012, respectively. Fernanda Marques is an assistant researcher and recipient of a FCT Investigator Grant with the reference IF/00231/2013. This work was supported by FCT and COMPETE through the project EXPL/NEU-OSD/2196/2013 and the Bial Foundation through the Grant 217/12.

Background The control of schistosomiasis emphasizes preventive chemotherapy with praziquantel, which aims at decreasing infection intensity and thus morbidity in individuals, as well as transmission in communities. Standardizing methods to assess treatment efficacy is important to compare trial outcomes across settings, and to monitor program effectiveness consistently. We compared customary methods and looked at possible complementary approaches in order to derive suggestions for standardizing outcome measures. Methodology/Principal Findings We analyzed data from 24 studies conducted at African, Asian, and Latin American sites, enrolling overall 4,740 individuals infected with Schistosoma mansoni, S. haematobium, or S. japonicum, and treated with praziquantel at doses of 40–80 mg/kg. We found that group-based arithmetic and geometric means can be used interchangeably to express egg reduction rates (ERR) only if treatment efficacy is high (>95%). For lower levels of efficacy, ERR estimates are higher with geometric than arithmetic means. Using the distribution of individual responses in egg excretion, 6.3%, 1.7% and 4.3% of the subjects treated for S. haematobium, S. japonicum and S. mansoni infection, respectively, had no reduction in their egg counts (ERR = 0). The 5th, 10th, and 25th centiles of the subjects treated for S. haematobium had individual ERRs of 0%, 49.3%, and 96.5%; the corresponding values for S. japonicum were 75%, 99%, and 99%; and for S. mansoni 18.2%, 65.3%, and 99.8%. Using a single rather than quadruplicate Kato-Katz thick smear excluded 19% of S. mansoni-infected individuals. Whilst the effect on estimating ERR was negligible by individual studies, ERR estimates by arithmetic means were 8% lower with a single measurement. Conclusions/Significance Arithmetic mean calculations of Schistosoma ERR are more sensitive and therefore more appropriate to monitor drug performance than geometric means. However, neither are satisfactory to identify poor responders. Group-based response estimated by arithmetic mean and the distribution of individual ERRs are correlated, but the latter appears to be more apt to detect the presence and to quantitate the magnitude of suboptimal responses to praziquantel., Author Summary To identify whether a person is infected with parasitic worms, stool or urine samples are examined for worm eggs. The drug praziquantel is used against the parasitic disease schistosomiasis. However, there is no definitive agreement as to how the efficacy of praziquantel is best expressed. We put together a database from various studies of the efficacy of praziquantel against schistosomiasis. Efficacy was measured using customary methods: cure rate (CR: percentage of people with eggs in their stool/urine before treatment who became egg-negative after treatment); and egg reduction rate (ERR; percentage reduction in the number of eggs in the stool/urine after treatment, where the mean number of eggs from all people treated is calculated using either geometric or arithmetic means). We found that arithmetic and geometric means can be used interchangeably only if treatment efficacy is very high; arithmetic means are more sensitive to capture drops in efficacy expressed by ERR. A valid complement for drug efficacy monitoring is to study the distribution of individual responses in egg excretion that allows identifying in a single measure both those who had an adequate response to treatment and those who respond less well; e.g., the 5% of the patients with the lowest ERRs.

Early-life stress (ELS) induces long-lasting changes in gene expression conferring an increased risk for the development of stress-related mental disorders. Glucocorticoid receptors (GR) mediate the negative feedback actions of glucocorticoids (GC) in the paraventricular nucleus (PVN) of the hypothalamus and anterior pituitary and therefore play a key role in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and the endocrine response to stress. We here show that ELS programs the expression of the GR gene (Nr3c1) by site-specific hypermethylation at the CpG island (CGI) shore in hypothalamic neurons that produce corticotropin-releasing hormone (Crh), thus preventing Crh upregulation under conditions of chronic stress. CpGs mapping to the Nr3c1 CGI shore region are dynamically regulated by ELS and underpin methylation-sensitive control of this region's insulation-like function via Ying Yang 1 (YY1) binding. Our results provide new insight into how a genomic element integrates experience-dependent epigenetic programming of the composite proximal Nr3c1 promoter, and assigns an insulating role to the CGI shore., European Union Directorate General for Research & Innovation through the CRESCENDO Consortium (O.F.X.A. and D.S) and the NINA Initial Training Program (D.S. and O.F.X.A)

Lipocalin-2 (LCN2) is an acute-phase protein that, by binding to iron-loaded siderophores, acts as a potent bacteriostatic agent in the iron-depletion strategy of the immune system to control pathogens. The recent identification of a mammalian siderophore also suggests a physiological role for LCN2 in iron homeostasis, specifically in iron delivery to cells via a transferrin-independent mechanism. LCN2 participates, as well, in a variety of cellular processes, including cell proliferation, cell differentiation and apoptosis, and has been mostly found up-regulated in various tissues and under inflammatory states, being its expression regulated by several inducers. In the central nervous system less is known about the processes involving LCN2, namely by which cells it is produced/secreted, and its impact on cell proliferation and death, or in neuronal plasticity and behaviour. Importantly, LCN2 recently emerged as a potential clinical biomarker in multiple sclerosis and in ageing-related cognitive decline. Still, there are conflicting views on the role of LCN2 in pathophysiological processes, with some studies pointing to its neurodeleterious effects, while others indicate neuroprotection. Herein, these various perspectives are reviewed and a comprehensive and cohesive view of the general function of LCN2, particularly in the brain, is provided., Ana Catarina Ferreira and Sandro Da Mesquita are recipients of PhD fellowships by the Fundação para a Ciência e Tecnologia (FCT, Portugal)/FEDER. Fernanda Marques is an assistant researcher IF/ 00231/2013 of the Fundação para a Ciência e Tecnologia (FCT, Portugal). This work was supported by Fundação para a Ciência e Tecnologia (FCT) and COMPETE through the project: EXPL/NEUOSD/2196/2013 (to Marques F). The authors thank Nadine Santos for the helpful comments on the manuscript.

We determined the presence of antibiotic-resistant enterococci (ARE) in commercialized poultry samples from Portugal and analysed their clonal diversity and the resistance genes harboured by these strains.Ninety-nine retail poultry samples of 10 widely commercialized brands were studied (1999-2001). Samples were enriched and plated on selective media with and without vancomycin, gentamicin, streptomycin or kanamycin. Antibiotic susceptibility was established following standard criteria. Identification and detection of genes coding for resistance were determined by PCR. Clonal relatedness was established by PFGE.A high percentage of samples contained vancomycin-resistant enterococci (VRE) (48%), or enterococci highly resistant (HLR) to gentamicin (34%), streptomycin (32%) or kanamycin (30%). Co-resistance to tetracycline, erythromycin, ciprofloxacin and quinupristin/dalfopristin was observed in most of these isolates. VRE were classified as VanA phenotype-vanA genotype (38% of samples), VanB phenotype-vanA (13%) or VanC phenotype-vanC1 (23%). All HLR to gentamicin isolates contained aac(6')-Ie-aph(2'')-Ia. We detected erm(B) in both erythromycin-resistant and -susceptible isolates. Some VRE and HLR to gentamicin strains were recovered from different samples and brands. Long-term persistence of particular VRE strains (2 years), exhibiting different Van phenotypes, was observed.High occurrence of ARE suggests maintenance of selective pressure by the use of antibiotics/other substances in the Portuguese poultry environment. Persistence of a number of widespread PFGE types containing different resistance genes might reflect environmental/host-adapted enterococcal strains that might contribute to the maintenance of antibiotic resistance, thus constituting a resistance reservoir that is non-sensitive to banning interventions.

The aims were to provide data on antibiotic residues in pig tissues and the trends in the occurrence of antibiotic resistance in Escherichia coli (a commensal bacteria that can colonize the intestinal tract of humans and animals) isolated from the animals studied. Muscle and kidney samples collected from freshly slaughtered pigs were tested for the presence of antibiotic residues using rapid screening tests. Ninety muscle and 90 kidney samples from the same animals, collected in the slaughterhouse of the central region of Portugal, were screened by microbial tests and only six samples did not fulfil the European Community regulations concerning maximum residue limits. When a more sensitive method was used for the detection of sulphamethazine, 36% of the animals contained residues of the antibiotic. This indication of a larger antibiotic occurrence than anticipated was confirmed by studying the susceptibility of their enteric E. coli. In all the animal faeces studied (n = 60), E. coli isolates resistant to amoxycillin, tetracyclines and sulphonamides were found. The majority of the faecal samples demonstrated a high degree of E. coli resistant to sulphonamides and tetracyclines, respectively 88% and 85%. These data raise important questions about the relevance of the detection of antibiotic residues as the only method to establish the quality and safety of animal products for human consumption.

Transthyretin is the major thyroxine-binding protein in the plasma of rodents, and the main thyroxine-binding protein in the cerebrospinal fluid of both rodents and humans. The choroid plexus synthesizes transthyretin and secretes it to the cerebrospinal fluid. Although it was suggested that transthyretin might play an important role in mediating thyroxine transfer from the blood into the brain across the choroid plexus–cerebrospinal fluid barrier, newer findings question this hypothesis. Because thyroid hormone passage across brain barriers is a precondition for its action in the CNS, and because brain is an important target of thyroid hormone action, we investigated the role of transthyretin in mediating thyroid hormone access to and distribution within the brain in a transthyretin-null mouse model system. In this report we describe the results derived from use of film autoradiography, a technique that yields definitive morphological results. Film autoradiograms were prepared at 3 and 19 h after intravenous injection of either high specific activity [125I]thyroxine or [125I]triiodothyronine. Image analyses were designed to demonstrate regional changes in hormone distribution, and to highlight alterations in iodothyronine delivery from ventricles to brain parenchyma. We find no qualitative or quantitative differences in these parameters between the transthyretin-null and the wild-type mouse brain after either [125I]thyroxine or [125I]triiodothyronine administration. The data presented here now provide definitive evidence that, under standard laboratory conditions, transthyretin is not required for thyroid hormone access to or distribution within the mouse brain. This study also provides the first map of iodothyronine distribution in the brain of the mouse.

SUMMARYWithin the World Health Organization 2012–2020 roadmap for control and elimination of schistosomiasis, the scale-up of mass drug administration with praziquantel is set to change the epidemiological landscape across Africa and Arabia. Central in measuring progress is renewed emphasis upon diagnostics which operate at individual, community and environmental levels by assessing reductions in disease, infections and parasite transmission. However, a fundamental tension is revealed between levels for present diagnostic tools, and methods applied in control settings are not necessarily adequate for application in elimination scenarios. Indeed navigating the transition from control to elimination needs careful consideration and planning. In the present context of control, we review current options for diagnosis of schistosomiasis at different levels, highlighting several strengths and weaknesses therein. Future challenges in elimination are raised and we propose that more cost-effective diagnostics and clinical staging algorithms are needed. Using the Kingdom of Saudi Arabia as a contemporary example, embedding new diagnostic methods within the primary care health system is discussed with reference to both urogenital and intestinal schistosomiasis.

SUMMARYIt is generally recommended to perform multiple stool examinations in order to improve the diagnostic accuracy when assessing the impact of mass drug administration programmes to control human intestinal worm infections and determining efficacy of the drugs administered. However, the collection and diagnostic work-up of multiple stool samples increases costs and workload. It has been hypothesized that these increased efforts provide more accurate results when infection and drug efficacy are summarized by prevalence (proportion of subjects infected) and cure rate (CR, proportion of infected subjects that become egg-negative after drug administration), respectively, but not when these indicators are expressed in terms of infection intensity and egg reduction rate (ERR). We performed a meta-analysis of six drug efficacy trials and one epidemiological survey. We compared prevalence and intensity of infection, CR and ERR based on collection of one or two stool samples that were processed with single or duplicate Kato-Katz thick smears. We found that the accuracy of prevalence estimates and CR was lowest with the minimal sampling effort, but that this was not the case for estimating infection intensity and ERR. Hence, a single Kato-Katz thick smear is sufficient for reporting infection intensity and ERR following drug treatment.

Fumonisins are mycotoxins that naturally occur as contaminants in grains that are destined for animal and human consumption. These mycotoxins cause hepatotoxic, nephrotoxic, carcinogenic, teratogenic, immunotoxic, and neurotoxic effects in different intensities based on dose, time of exposure, and animal species. In the present study, male Wistar rats were fed between postnatal days 21 and 63 with diets that contained fumonisins B1+B2 at concentrations of 1 and 3mg/kg. The objective of the present study was to evaluate the effects of fumonisins on food intake, growth, weight gain, serum activity of the alanine aminotransferase and aspartate aminotransferase enzymes, and quantitative and morphometric parameters of myenteric neurons in the jejunum that are immunoreactive to HuC/D protein and neuronal nitric oxide synthase enzyme (nNOS). Diets that contained fumonisins did not significantly alter food intake or body and blood parameters. We did not observe significant differences in the neuronal density and proportion of nitrergic neurons but found a significant reduction of cell body areas in both neuronal populations. This study is the first to report the effects of fumonisins in the enteric nervous system. The possible mechanisms by which fumonisins impair neuronal development and the use of the enteric nervous system as a tool for the study of the neurotoxic effects of fumonisins are discussed. In conclusion, fumonisin-containing food negatively affected the growth of myenteric neurons.

Specific immunoglobulin E (IgE) responses are upregulated during chronic schistosome infection and during allergy. These responses are tightly regulated during schistosomiasis. We have previously shown that IgE regulation depends on the extent and length of exposure to individual parasite allergen-like proteins. Here we compare the development of IgE and immunoglobulin G4 (IgG(4)) responses to the differentially expressed allergen-like proteins SmTAL1 and SmTAL2 among preschool-aged children from 2 villages with different levels of Schistosoma mansoni transmission. We found a lack of SmTAL1 responsiveness among all children, but evidence for IgG(4)-dependent IgE-SmTAL2 desensitization in both villages, occurring earlier among children from the village where the level of transmission was greater. Findings provide insights into the development and regulation of allergic-type immune responses.

Similarly to schistosomiasis, a single dose of PZQ eliminates the vast majority of Hymenolepis egg excretion [10–12]. However, it is likely that a PZQ gap exists in many communities being targeted by MDA in that the geographical location of Hymenolepis infections may not necessarily overlap with that of schistosomiasis. Guiding PZQ delivery solely on the basis of the distribution of schistosomiasis may miss communities endemic to Hymenolepis infection also in need of PZQ. This means that the populations at risk of hymenolepiasis may need to also be identified so that PZQ delivery can be extended to those areas. In order to test our proposition, we have analysed data from a parasitic disease survey of 2,168 children aged #15 years, including 1,098 girls and 1,070 boys in the Dande municipality in Northern Angola. Previous analysis of this dataset revealed that children were at significantly increased risk of H. nana infection (prevalence of 6.2% [95% CI: 4.9–7.8%] in preschool children and 7.3% [95% CI: 5.8–9.0%] in school-age children) compared to adults (prevalence of 1.9% [95% CI: 1.1–3.1%]) [13]. Using these data, we aimed to describe the epidemiology of H. nana infection by quantifying the role of individual and household factors and the physical environment (land surface temperature, distance to irrigation canals and rivers) in H. nana infection; quantify the role of H. nana infection on morbidity outcomes such as anaemia, diarrhoea, abdominal pain, and growth; quantify the geographical variation in H. nana infection prevalence in children aged #15 years; generate the first high-resolution H. nana infection map; and compare this map with a preexisting S. haematobium

The subependymal zone (SEZ), frequently named as adult subventricular zone (SVZ), is a niche of adult neural stem and progenitor cells that lines a large extension of the lateral ventricles of the brain. The majority of the studies do not analyze the SEZ throughout its entire extension. Instead, studies of cell populations within the SEZ typically focus their analysis on a narrow space between specific bregma coordinates that provides a perspective of only a small portion of the SEZ. We have previously proposed a standard division for the SEZ at the anterior-posterior and dorsal-ventral axes based on external brain anatomical hallmarks (Falcao et al., PLoS One 7:e38647, 2012). Herein, we describe in detail the procedure and a stereological approach that can be used to obtain an unbiased estimation of the SEZ cell proliferation under physiological and pathological conditions. This approach takes into consideration clear SEZ anatomical divisions, both on the anterior-posterior and dorsal-ventral axes, which will standardize future studies on the SEZ.

Diagnosis of schistosomiasis at the point-of-care (POC) is a growing topic in neglected tropical disease research. There is a need for diagnostic tests which are affordable, sensitive, specific, user-friendly, rapid, equipment-free and delivered to those who need it, and POC is an important tool for disease mapping and guiding mass deworming. The aim of present study was to evaluate the relative diagnostic performance of two urine-circulating cathodic antigen (CCA) cassette assays, one commercially available and the other in experimental production, against results obtained using the standard Kato-Katz faecal smear method (six thick smears from three consecutive days), as a ‘gold-standard’, for Schistosoma mansoni infection in different transmission settings in Uganda. Our study was conducted among 500 school children randomly selected across 5 schools within Bugiri district, adjacent to Lake Victoria in Uganda. Considering results from the 469 pupils who provided three stool samples for the six Kato-Katz smears, 293 (76%) children had no infection, 109 (23%) were in the light intensity category, while 42 (9%) and 25 (5%) were in the moderate and heavy intensity categories respectively. Following performance analysis of CCA tests in terms of sensitivity, specificity, negative and positive predictive values, overall performance of the commercially available CCA test was more informative than single Kato-Katz faecal smear microscopy, the current operational field standard for disease mapping. The current CCA assay is therefore a satisfactory method for surveillance of S. mansoni in an area where disease endemicity is declining due to control interventions. With the recent resolution on schistosomiasis elimination by the 65th World Health Assembly, the urine POC CCA test is an attractive tool to augment and perhaps replace the Kato-Katz sampling within ongoing control programmes.

Background Calprotectin is a calcium-binding cytoplasmic protein found in neutrophils and increasingly used as a marker of bowel inflammation. Fecal occult blood (FOB) is also a dependable indicator of bowel morbidity. The objective of our study was to determine the applicability of these tests as surrogate markers of Schistosoma mansoni intestinal morbidity before and after treatment with praziquantel (PZQ). Methods 216 children (ages 3–9 years old) from Buliisa District in Lake Albert, Uganda were examined and treated with PZQ at baseline in October 2012 with 211 of them re-examined 24 days later for S. mansoni and other soil transmitted helminths (STH). POC calprotectin and FOB assays were performed at both time points on a subset of children. Associations between the test results and infection were analysed by logistic regression. Results Fecal calprotectin concentrations of 150–300 µg/g were associated with S. mansoni egg patent infection both at baseline and follow up (OR: 12.5 P = 0.05; OR: 6.8 P = 0.02). FOB had a very strong association with baseline anemia (OR: 9.2 P = 0.03) and medium and high egg intensity schistosomiasis at follow up (OR: 6.6 P = 0.03; OR: 51.3 P = 0.003). Both tests were strongly associated with heavy intensity S. mansoni infections. There was a significant decrease in FOB and calprotectin test positivity after PZQ treatment in those children who had egg patent schistosomiasis at baseline. Conclusions Both FOB and calprotectin rapid assays were found to correlate positively and strongly with egg patent S. mansoni infection with a positive ameloriation response after PZQ treatment indicative of short term reversion of morbidity. Both tests were appropriate for use in the field with excellent operational performance and reliability. Due to its lower-cost which makes its scale-up of use affordable, FOB could be immediately adopted as a monitoring tool for PC campaigns for efficacy evaluation before and after treatment., Author Summary The severity of intestinal schistosomiasis, a disease caused by Schistosoma mansoni infection, is likely under-reported in part due to the scarcity of field-appropriate morbidity markers. Downstream potential complications of this disease include anemia, failure to thrive, and chronic multi-organ damage. Point-of-care (POC) tools to monitor intestinal schistosomiasis in low resource settings are urgently needed to better quantify the burden of disease in endemic countries and to gauge the clinical impact of scale-up of preventive PC. For the present study in rural Uganda, fecal occult blood and fecal calprotectin were identified as potential surrogate markers of intestinal morbidity. We tested both POC tests and found that they were both associated with active schistosomiasis as detected by eggs in stool with significant decrease in test positivity after PZQ treatment demonstrating short term morbidity reversion. Calprotectin was a strong indicator of intestinal inflammation, however, owing to its high per-test price makes it difficult to scale-up accordingly. Conversely, fecal occult blood was technically feasible, low-cost and had optimal performance as a morbidity marker, hence we strongly advocate for its immediate inclusion as a monitoring tool for PC programs.

testing method (version 3). Journal of Antimicrobial Chemotherapy 53, 713–28. 3. Andrews, J. M. (2001). The development of the BSAC standardized method of disc diffusion testing. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 29–42. 4. MacGowan, A. P. & Wise, R. (2001). Establishing MIC breakpoints and the interpretation of in vitro susceptibility tests. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 17–28. 5. Perez-Vazquez, M., Roman, F., Aracil, B. et al. (2004). Laboratory detection of Haemophilus influenzae with decreased susceptibility to nalidixic acid, ciprofloxacin, levofloxacin, and moxifloxacin due to gyrA and parC mutations. Journal of Clinical Microbiology 42, 1185–91.

The establishment of a national control programme (NCP) in Uganda has led to routine treatment of intestinal schistosomiasis with praziquantel in the communities along Lake Albert. However, because regular water contact remains a way of life for these populations, re-infection continues to mitigate the sustainability of the chemotherapy-based programme. A six-month longitudinal study was conducted in one Lake Albert community with the aim of characterizing water contact exposure and infection among mothers and their young preschool-aged children as the latter are not yet formally included within the NCP. At baseline the cohort of 37 mothers, 36 preschool-aged children had infection prevalences of 62% and 67%, respectively, which diminished to 20% and 29%, respectively, at the 6-month post-treatment follow-up. The subjects wore global positioning system (GPS) datalogging devices over a 3-day period shortly after baseline, allowing for the estimation of time spent at the lakeshore as an exposure metric, which was found to be associated with prevalence at follow-up (OR = 2.1, P = 0.01 for both mothers and young children and odds ratio (OR) = 4.4, P = 0.01 for young children alone). A social network of interpersonal interactions was also derived from the GPS data, and the exposures were positively associated both with the number and duration of peer interaction, suggesting the importance of socio-cultural factors associated with water contact behaviour. The findings illustrate reduction in both prevalence and intensity of infection in this community after treatment as well as remarkably high rates of water contact exposure and re-infection, particularly among younger children. We believe that this should now be formally considered within NCP, which may benefit from more in-depth ethnographic exploration of factors related to water contact as this should provide new opportunities for sustaining control.

The emerging model for the adult subependymal zone (SEZ) cell population indicates that neuronal diversity is not generated from a uniform pool of stem cells but rather from diverse and spatially confined stem cell populations. Hence, when analysing SEZ proliferation, the topography along the anterior-posterior and dorsal-ventral axes must be taken into account. However, to date, no studies have assessed SEZ proliferation according to topographical specificities and, additionally, SEZ studies in animal models of neurological/psychiatric disorders often fail to clearly specify the SEZ coordinates. This may render difficult the comparison between studies and yield contradictory results. More so, by focusing in a single spatial dimension of the SEZ, relevant findings might pass unnoticed. In this study we characterized the neural stem cell/progenitor population and its proliferation rates throughout the rat SEZ anterior-posterior and dorsal-ventral axes. We found that SEZ proliferation decreases along the anterior-posterior axis and that proliferative rates vary considerably according to the position in the dorsal-ventral axis. These were associated with relevant gradients in the neuroblasts and in the neural stem cell populations throughout the dorsal-ventral axis. In addition, we observed spatially dependent differences in BrdU/Ki67 ratios that suggest a high variability in the proliferation rate and cell cycle length throughout the SEZ; in accordance, estimation of the cell cycle length of the neuroblasts revealed shorter cell cycles at the dorsolateral SEZ. These findings highlight the need to establish standardized procedures of SEZ analysis. Herein we propose an anatomical division of the SEZ that should be considered in future studies addressing proliferation in this neural stem cell niche., Fundação para a Ciência e a Tecnologia (FCT)

SUMMARYWhere very young children come into contact with water containing schistosome cercariae, infections occur and schistosomiasis can be found. In high transmission environments, where mothers daily bathe their children with environmentally drawn water, many infants and preschool-aged children have schistosomiasis. This ‘new’ burden, inclusive of co-infections with Schistosoma haematobium and Schistosoma mansoni, is being formally explored as infected children are not presently targeted to receive praziquantel (PZQ) within current preventive chemotherapy campaigns. Thus an important PZQ treatment gap exists whereby infected children might wait up to 4–5 years before receiving first treatment in school. International treatment guidelines, set within national treatment platforms, are presently being modified to provide earlier access to medication(s). Although detailed pharmacokinetic studies are needed, to facilitate pragmatic dosing in the field, an extended ‘dose pole’ has been devised and epidemiological monitoring has shown that administration of PZQ (40 mg/kg), in either crushed tablet or liquid suspension, is both safe and effective in this younger age-class; drug efficacy, however, against S. mansoni appears to diminish after repeated rounds of treatment. Thus use of PZQ should be combined with appropriate health education/water hygiene improvements for both child and mother to bring forth a more enduring solution.

Following our previous field surveys for strongyloidiasis in western Uganda, 120 mothers and 232 children from four villages in eastern Uganda were examined, with two subsequent investigative follow-ups. As before, a variety of diagnostic methods were used: Baermann concentration, Koga agar plate and strongyloidid enzyme-linked immunosorbent assay (ELISA), as well as Kato–Katz faecal smears for detection of eggs of other helminths. At baseline, the general prevalence ofStrongyloides stercoraliswas moderate: 5.4% as estimated by Baermann and Koga agar methods combined. A much higher estimate was found by ELISA (42.3%) which, in this eastern setting, appeared to be confounded by putative cross-reaction(s) with other nematode infections. Preventive chemotherapy using praziquantel and albendazole was offered to all participants at baseline. After 21 days the first follow-up was conducted and ‘cure rates’ were calculated for all parasites encountered. Eleven months later, the second follow-up assessed longer-term trends. Initial treatments had little, if any, effect onS. stercoralis,and did not alter local prevalence, unlike hookworm infections and intestinal schistosomiasis. We propose that geographical patterns of strongyloidiasis are likely not perturbed by ongoing praziquantel/albendazole campaigns. Antibody titres increased after the first follow-up then regressed towards baseline levels upon second inspection. To better define endemic areas forS. stercoralis, careful interpretation of the ELISA is warranted, especially where diagnosis is likely being confounded by polyparasitism and/or other treatment regimens; new molecular screening tools are clearly needed.