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Schistosoma mansoni is less susceptible to the antiparasitic drug ivermectin than other helminths. By inhibiting the P-glycoprotein or cytochrome P450 3A in mice host or parasites in a murine model, we aimed at increasing the sensitivity of S. mansoni to the drug and thus preventing infection. We assigned 124 BALB/c mice to no treatment, treatment with ivermectin only or a combination of ivermectin with either cobicistat or elacridar once daily for three days before infecting them with 150 S. mansoni cercariae each. The assignment was done by batches without an explicit randomization code. Toxicity was monitored. At eight weeks post-infection, mice were euthanized. We determined number of eggs in intestine and liver, adult worms in portal and mesenteric veins. Disease was assessed by counting granulomas/cm2 of liver and studying organ weight indices and total weight. IgG levels in serum were also considered. No difference between groups treated with ivermectin only or in combination with cobicistat or elacridar compared with untreated, infected controls. Most mice treated with ivermectin and elacridar suffered severe neurological toxicity. In conclusion, systemic treatment with ivermectin, even in the presence of pharmacological inhibition of P-glycoprotein or cytochrome P450 3A, did not result in effective prophylaxis for S. mansoni infection in an experimental murine model. [ABSTRACT FROM AUTHOR]

NGC 2548 is a ~400-500 Myr old open cluster with evidence of spatial substructures likely caused by its interaction with the Galactic disc. In this work we use precise astrometric data from the Carte du Ciel - San Fernando (CdC-SF) catalogue to study the clumpy structure in this cluster.We confirm the fragmented structure of NGC 2548 but, additionally, the relatively high precision of our kinematic data lead us to the first detection of substructures in the proper motion space of a stellar cluster. There are three spatially separated cores each of which has its own counterpart in the proper motion distribution. The two main cores lie nearly parallel to the Galactic plane whereas the third one is significantly fainter than the others and it moves towards the Galactic plane separating from the rest of the cluster.We derive core positions and proper motions, as well as the stars belonging to each core. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis is a water-borne disease afflicting over 261 million people in many areas of the developing countries with high morbidity and mortality. The control relies mainly on treatment with praziquantel. Fatty acid binding proteins (FABP) have demonstrated high levels of immune-protection against trematode infections. This study reports the immunoprotection induced by cross-reacting Fasciola hepatica FABP, native (nFh12) and recombinantly expressed using two different expression systems Escherichia coli (rFh15) and baculovirus (rFh15b) against Schistosoma mansoni infection. Methods: BALB/c mice were vaccinated with native nFh12 or recombinant rFh15 and rFh15 FABP from F. hepatica formulated in adjuvant adaptation (ADAD) system with natural or chemical synthesised immunomodulators (PAL and AA0029) and then challenged with 150 cercariae of S. mansoni. Parasite burden, hepatic lesions and antibody response were studied in vaccination trials. Furthermore differences between rFh15 and rFh15b immunological responses (cytokine production, splenocyte population and antibody levels) were studied. Results: Vaccination with nFh12 induced significant reductions in worm burden (83 %), eggs in tissues (82-92 %) and hepatic lesions (85 %) compared to infected controls using PAL. Vaccination with rFh15 showed lower total worm burden (56-64 %), eggs in the liver (21-61 %), eggs in the gut (30-77 %) and hepatic damage (67-69 %) using PAL and AA0029 as immunomodulators. In contrast, mice vaccinated with rFh15b showed only reductions in eggs trapped in the liver and intestine (53 and 60 %, respectively), and hepatic lesions (45 %). We observed a significant rise in TNFα, IL-6, IL-2, IL-4 and high antibody response (IgG, IgG1, IgG2a, IgM and IgE) in mice immunised with either rFh15 or rFh15b. Moreover, mice immunised with rFh15b showed an increase in IFNγ and a decrease in B220 cells compared to untreated mice, and less production of IgG1 and IgM than in mice immunised by rFh15. Conclusions: Higher level of protection is obtained by using Fasciola hepatica-derived FABP protein against Schistosoma mansoni infection. Native FABP is more effective than both recombinant systems. It could be due to posttranslational modifications or FABP isoform or changes in the recombinant proteins. [ABSTRACT FROM AUTHOR]

Fatty acid binding proteins (FABP) from Fasciola hepatica have demonstrated immune cross-protection against schistosomes. The present study was conducted to develop a new formulation of the recombinant FABP rFh15 with the synthetic immunomodulator AA0029 in the adjuvant adaptation (ADAD) vaccination system and to evaluate its ability to induce immune response and protection against the challenge with Schistosoma bovis cercariae. Immunization of BALB/c mice showed high levels of TNFα, IFNγ, interleukin (IL)-2, IL-6, and IL-4 in splenocyte supernatant culture and also high levels of serum specific anti-rFh15 IgG, IgG1, IgG2a IgE and IgM antibodies suggesting a mixed Th1/Th2 immune response. Using this approach, high levels of protection against experimental challenge with S. bovis cercariae were observed in the mouse and hamster models. A marked reduction up to 64% in worm burden, as well as in the number of eggs retained in liver (66%) and intestine (77%) and hepatic lesions (42%), was achieved in vaccinated BALB/c mice. Golden hamsters vaccinated and challenged in similar conditions had reductions in recovered worms (83%), liver eggs (90%), intestine eggs (96%), liver lesions (56%) and worm fecundity (48–80%). These data suggest that formulation of rFh15 in the ADAD vaccination system using the AA0029 immunomodulator could be a good option to drive an effective immunological response against schistosomiasis. [ABSTRACT FROM AUTHOR]

Several efforts have been made to identify anti-schistosomiasis vaccine candidates and new vaccination systems. The fatty acid binding protein (FAPB) has been shown to induce a high level of protection in trematode infection. The adjuvant adaptation (ADAD) vaccination system was used in this study, including recombinant FABP, a natural immunomodulator and saponins. Mice immunised with the ADAD system were able to up-regulate proinflammatory cytokines (IL-1 and IL-6) and induce high IgG2a levels. Moreover, there was a significant reduction in worm burden, egg liver and hepatic lesion in vaccinated mice in two independent experiments involving Schistosoma bovis infected mice. The foregoing data shows that ADAD system using FABP provide a good alternative for triggering an effective immune response against animal schistosomiasis. [ABSTRACT FROM AUTHOR]

Schistosomiasis is a parasitic disease that affects 143 million people in endemic countries. This work analyzed overexpressed sequences from the cercaria phase to the early schistosomulum phase using bioinformatics tools to predict host interaction and selected proteins for predicting T cell epitopes. The final peptides were chemically synthesized, and their toxicity was evaluated in vitro. Peptides were formulated in the Adjuvant Adaptation (ADAD) vaccination system and injected into BALB/c mice that were challenged with S. mansoni cercariae to assess protection and immunogenicity. A total of 39 highly expressed S.mansoni proteins were identified as being of potential interest. Three T cell peptides predicted to bind MHC mouse and human class II were synthesized and formulated for vaccination. SmGSP and SmIKE reduced the number of eggs trapped in the liver by more than 50% in challenged BALB/c mice. The liver of mice vaccinated with either SmGSP or SmTNP had a significantly reduced affected liver surface. Transcriptome-based T cell peptides elicit partial protection and could be candidates for a multiantigen vaccine. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis has been identified as a major public health problem in tropical countries. The present study aimed to investigate the schistosomicidal effects of the methanolic extract of Argemone mexicana L. and its active component, berberine against Schistosoma mansoni on in-vitro experiments. Methods: S. mansoni adults were used. Various concentrations of the methanolic extract (10 - 200 µg/ml) and berberine (2.5 - 50 µM) were tested from 24 to 72 h. The viability of S. mansoni was confirmed with an invertoscope-microscope. Furthermore, cytotoxic (Hemolysis test), and antioxidant (DPPH radical scavenging assay) capacities were determined. Results: The viability tests on S. mansoni showed that A. mexicana at 50 μg/mL is lethal at 48 h and berberine at 10 μM is lethal at 24 h. The hemolytic activity at 1,000 μg/mL was 2.9% for A. mexicana and 90.2% for berberine. The antioxidant capacities shown by A. mexicana and berberine, were EC50 156.3 and 84.1 μg/mL, respectively. Conclusion: The extract of A. mexicana and berberine demonstrated high antischistosomal activities in low concentration and short exposure time on the in-vitro model. [ABSTRACT FROM AUTHOR]

Only a small number of infected people are highly susceptible to schistosomiasis, showing high levels of infection or severe liver fibrosis. The susceptibility to schistosome infection is influenced by genetic background. To assess the genetic basis of susceptibility and identify the chromosomal regions involved, a backcross strategy was employed to generate high variation in schistosomiasis susceptibility. This strategy involved crossing the resistant C57BL/6J mouse strain with the susceptible CBA/2J strain. The resulting F1 females (C57BL/6J × CBA/2J) were then backcrossed with CBA/2J males to generate the backcross (BX) cohort. The BX mice exhibited a range of phenotypes, with disease severity varying from mild to severe disease, lacking a fully resistant group. We observed four levels of infection intensity using cluster and principal component analyses and K-means based on parasitological, pathological, and immunological trait measurements. The mice were genotyped with 961 informative SNPs, leading to the identification of 19 new quantitative trait loci (QTL) associated with parasite burden, liver lesions, white blood cell populations, and antibody responses. Two QTLs located on chromosomes 15 and 18 were linked to the number of granulomas, liver lesions, and IgM levels. The corresponding syntenic human regions are located in chromosomes 8 and 18. None of the significant QTLs had been reported previously. [ABSTRACT FROM AUTHOR]

SUMMARY We studied the cytoplasmic signalling pathways involved in the generation of nitric oxide (NO) after stimulation with adult e x cretory/secretory antigens (ESA) of Toxocara canis. The pathways of phospholipase A2 (PLA2) and phospholipase C (PLC) were considered as potentially involved in the synthesis of nitric oxide. We used inhibitors of these pathways at different levels. Several concentrations of lithium chloride, verapamil, TMB-8 and staurosporine were used to inhibit the PLC pathway. Inhibition of the PLA2 pathway was attempted with mepacrine, diethylcarbamazine or meloxicam. Lithium chloride, verapamil and TMB-8 reduced the production of NO induced by ESA in a concentration-dependent manner. Regarding the PLA2 pathway, a range of concentrations of mepacrine greatly reduced the production of NO induced by ESA. Meloxicam inhibition was always higher than 50%. Diethylcarbamazine showed a dose-dependent effect on the production of NO induced by the ESA. Our results suggest that both the PLC and the PLA2 pathways play an essential role in activating the production of macrophage NO triggered by the ESA of T. canis. This could indicate that NO production in our experimental conditions is due to both an increase of intracellular calcium and to the participation of the arachidonic acid cascade. The implications of these activations on the host–parasite relationship are discussed and compared with LPS-stimulated macrophages. [ABSTRACT FROM AUTHOR]

Schistosomiasis is considered a neglected parasitic disease. Around 280,000 people die from it annually, and more than 779 million people are at risk of getting infected. The schistosome species which infect human beings are Schistosoma mansoni, Schistosoma haematobium, Schistosoma intercalatum, Schistosoma japonicum, Schistosoma guineensis, and Schistosoma mekongi. This disease is also of veterinary significance; the most important species being Schistosoma bovis since it causes the disease in around 160 million livestock in Africa and Asia. This work was aimed at designing and developing a genus-specific loop-mediated isothermal amplification (LAMP) method for detecting the most important schistosome species affecting humans and for the species-specific detection of S. bovis. Bioinformatics tools were used for primer design, and the LAMP method was standardised for detecting the ITS-1 region from S. intercalatum, S. haematobium, S. mansoni, S. japonicum, and S. bovis DNA (generic test) and the NADH 1 gene for specifically detecting S. bovis (at different DNA concentrations). Detection limits achieved were 1 pg DNA for S. mansoni, 0.1 pg for S. haematobium, 1 pg for S. intercalatum, and 10 pg for S. bovis. No amplification for S. japonicum DNA was obtained. The LAMP designed for the amplification of S. bovis NADH-1 worked specifically for this species, and no other DNA from other schistosome species included in the study was amplified. Two highly sensitive LAMP methods for detecting different Schistosoma species important for human and veterinary health were standardised. These methods could be very useful for the diagnosis and surveillance of schistosome infections. [ABSTRACT FROM AUTHOR]

Background: Mansonella perstans infection can be considered one of the most neglected tropical infectious diseases. Very few studies have reported on the clinical picture caused by infection with this nematode. Therefore, our study was aimed to describe the clinical patterns and treatment of imported M. perstans infection by migrants from Africa. Methods: The present study evaluated a large cohort of migrants who have been diagnosed, examined and treated for imported M. perstans infection at a Spanish reference center (Hospital Carlos III Tropical Medicine Unit, Madrid, Spain) over a 19-year period. Most patients voluntarily attend the emergency unit or are referred from primary care or general hospitals in Madrid. Chi-square test was used to compare the association between categorical variables. The continuous variables were compared by Student's t-test or the Mann–Whitney test. The corresponding regression models were used for multivariate analysis. Results: Five hundred three cases of migrants from tropical and subtropical areas with M. perstans infection were identified. Two hundred sixty-four patients were female (52.5%). The mean age (± SD) was 44.6 ± 18.2 years (range: 16–93 years). The mean time (± SD) between the arrival in Spain and the first consultation was 8.6 ± 18.0 months. The major origin of the patients was Equatorial Guinea (97.6%). Regarding the clinical picture, 257 patients were asymptomatic (54.7%) and 228 were symptomatic (45.3%); 190 patients had pruritus (37.8%), 50 (9.9%) had arthralgia, 18 patients had Calabar-like swelling (3.6%), and 15 (3%) had abdominal pain. Four hundred forty-two (87.9%) migrants had hyper-IgE, and 340 (67.6%) had eosinophilia. One hundred ninety-five patients had coinfections with other filarial nematodes (38.8%), and 308 migrants had only M. perstans infection (61.2%). Four hundred thirty-seven cases (86.9%) had been treated with anti-filarial drugs; 292 cases were treated with one anti-filarial drug, and 145 cases were treated with combined anti-filarial therapy. Additionally, 20 (4%) cases received steroids and 38 (7.6%) cases received antihistamines. Conclusions: A long series of M. perstans infections is presented in sub-Saharan immigrants whose data indicate that it should be included in the differential diagnosis in patients with pruritus or analytical alterations such as eosinophilia or hyper-IgE presentation, and they also have a high number of coinfections with other microorganisms whose treatment needs to be protocolized. [ABSTRACT FROM AUTHOR]

Leishmaniasis comprises a group of neglected diseases caused by the protozoan parasite Leishmania spp. As is the case for other trypanosomatid parasites, Leishmania is auxotrophic for heme and must scavenge this essential compound from its human host. In mammals, the SLC transporter FLVCR2 mediates heme import across the plasma membrane. Herein we identify and characterize Leishmania major FLVCRb (LmFLVCRb), the first member of the FLVCR family studied in a non-metazoan organism. This protein localizes to the plasma membrane of the parasite and is able to bind heme. LmFLVCRb levels in Leishmania, which are modulated by overexpression thereof or the abrogation of an LmFLVCRb allele, correlate with the ability of the parasite to take up porphyrins. Moreover, injection of LmFLVCRb cRNA to Xenopus laevis oocytes provides these cells with the ability to take up heme. This process is temperature dependent, requires monovalent ions and is inhibited at basic pH, characteristics shared by the uptake of heme by Leishmania parasites. Interestingly, LmFLVCRb is essential as CRISPR/Cas9-mediated knockout parasites were only obtained in the presence of an episomal copy of the gene. In addition, deletion of just one of the alleles of the LmFLVCRb gene markedly impairs parasite replication as intracellular amastigotes as well as its virulence in an in vivo model of cutaneous leishmaniasis. Collectively, these results show that Leishmania parasites can rescue heme through plasma membrane transporter LFLVCRb, which could constitute a novel target for therapeutic intervention against Leishmania and probably other trypanosomatid parasites in which FLVCR genes are also present. [ABSTRACT FROM AUTHOR]

Chagas' disease is a neglected tropical disease caused by Trypanosoma cruzi which is endemic throughout Latin America and is spread by worldwide migration. Diagnosis is currently limited to serological and molecular techniques having variations regarding their sensitivity and specificity. This work was aimed at developing a new sensitive, applicable, and cost-effective molecular diagnosis technique for loop-mediated isothermal amplification-based detection of T. cruzi (Tc-LAMP). The results led to determining a highly homologous satellite repeat region (231 bp) among parasite strains as a molecular marker for diagnosing the disease. Tc-LAMP was performed correctly for detecting parasite DNA (5 fg for the CL Brener strain and 50 fg for the DM28, TcVI, and TcI strains). Assay results proved negative for DNA from 16 helminth species and 7 protozoa, including Leishmania spp. Tc-LAMP based on the highly repeated T. cruzi satellite region is thus proposed as an important alternative for diagnosing T. cruzi infection, overcoming other methods' limitations such as their analytic capability, speed, and requiring specialized equipment or highly trained personnel. Tc-LAMP could be easily adapted for point-of-care testing in areas having limited resources. [ABSTRACT FROM AUTHOR]

Background: Loiasis is an uncommon and poorly understood parasitic disease outside endemic areas of Africa. The aim of this study was to describe the clinical and biological patterns and treatment of imported loiasis by sub-Saharan migrants diagnosed in Madrid, Spain. Methods: A retrospective study was conducted with sub-Saharan immigrants seen at the Tropical Medicine Unit of the Carlos III Hospital in Madrid, Spain, a reference center, over 19 years. Categorical variables were expressed as frequency counts and percentages. Continuous variables were expressed as the mean and standard deviation (SD) or median and interquartile range (IQR: Q3–Q1). Chi-square tests were used to assess the association between categorical variables. The measured outcomes were expressed as the odds ratio (OR) with a 95% confidential interval. Continuous variables were compared by Student's t-tests or Mann-Whitney U tests. Binary logistic regression models were used. P < 0.05 was considered a statistically significant difference. Results: One hundred thirty-one migrants from tropical and subtropical areas with loiasis were identified. Forty-nine patients were male (37.4%). The migrants' mean age (±SD) was 42.3 ± 17.3 years, and 124 (94.7%) were from Equatorial Guinea. The median time (IQR) between arrival in Spain and the first consultation was 2 (1–7) months. One hundred fifteen migrants had eosinophilia, and one hundred thirteen had hyper-IgE syndrome. Fifty-seven patients had pruritus (43.5%), and thirty patients had Calabar swelling (22.9%). Seventy-three patients had coinfections with other filarial nematodes (54.2%), and 58 migrants had only Loa loa infections (45.8%). One hundred two patients (77.9%) were treated; 45.1% (46/102) patients were treated with one drug, and 54.9% (56/102) patients were treated with combined therapy. Adverse reactions were described in 14 (10.7%) migrants. Conclusions: Our patients presented early clinical manifestations and few atypical features. Thus, physicians should systematically consider loiasis in migrants with a typical presentation. However, considering that 72.5% of the patients had only positive microfilaremia without any symptoms, we suggest searching for microfilaremia in every migrant from endemic countries for loiasis presenting with eosinophilia. [ABSTRACT FROM AUTHOR]

Background: Schistosoma mansoni is the main species causing hepatic and intestinal schistosomiasis in Sub-Saharan Africa, and it is the only species in South America. Adult stages of the parasite reside in the mesenteric venous plexus of infected hosts, and eggs are shed in feces. Collecting patient stool samples for S. mansoni diagnostic purposes is difficult in large-scale field trials. Urine samples would be an alternative approach for molecular S. mansoni detection since they have several advantages over stool samples, including better handling, management and storage. Additionally, loop-mediated isothermal amplification (LAMP) technology is a powerful molecular diagnostic tool for infectious diseases, particularly under field conditions in developing countries. The present study aimed to assess the effectiveness of our previously developed LAMP assay (SmMIT-LAMP) for S. mansoni-specific detection in clinical urine samples. Methodology/Principal findings: The sensitivity of SmMIT-LAMP in urine was established in simulated fresh human urine samples artificially spiked with genomic DNA from S. mansoni. LAMP for 120 min instead of 60 min improved the sensitivity, reaching values of 0.01 fg/μL. A set of well-defined frozen stored human urine samples collected from Sub-Saharan immigrant patients was selected from a biobank to evaluate the diagnostic validity of SmMIT-LAMP. The set included urine samples from patients with microscopy-confirmed infections with S. mansoni, S. haematobium and other nonschistosome parasites, as well as urine samples from patients with microscopy-negative eosinophilia without a confirmed diagnosis. The SmMIT-LAMP was incubated for 60 and 120 min. A longer incubation time was shown to increase the LAMP-positive results in patient urine samples. We also tested urine samples from mice experimentally infected with S. mansoni, and LAMP-positive results were obtained from the third week after infection. A real-time LAMP assay was also performed with three individual urine samples. Conclusions/Significance: The SmMIT-LAMP could effectively detect S. mansoni DNA in mouse urine samples and produced promising results for human clinical samples. The detection of S. mansoni DNA in mouse urine samples from the third week after infection indicates that early diagnosis of active S. mansoni infection is possible using urine as a source of DNA. Further studies are still needed, but our method could be used as a promising molecular tool applicable to urine samples to diagnose human intestinal schistosomiasis caused by S. mansoni. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the performance of Rapid-Heat LAMPellet assay in field conditions for diagnosis of urogenital schistosomiasis in an endemic area in Cubal, Angola, and to assess the reproducibility in a reference laboratory.Methods: A total of 172 urine samples from school-age children were tested for microhaematuria, microscopic detection of Schistosoma haematobium eggs and LAMP for DNA detection. Urine samples were stored in a basic equipped laboratory. Field-LAMP tests were performed with and without prior DNA extraction from urine samples, and the results were read by turbidity and by colour change. When field procedures were finished, samples were sent to a reference laboratory to be reanalysed by LAMP.Results: A total of 83 of 172 (48.3%) were positive for microhaematuria, 87/172 (50.6%) were microscopy-positive for S. haematobium eggs detection, and 127/172 (73.8%) showed LAMP-positive results for detecting S. haematobium using purified DNA and 109/172 (63.4%) without prior DNA extraction. MacNemar's test showed a statistical significant relation between LAMP results and microscopy-detected S. haematobium infections and microhaematuria (P < 0.001 in both cases), respectively. When samples of purified DNA were reanalysed in a reference laboratory in Spain using the same LAMP methodology, the overall reproducibility achieved 72.1%.Conclusions: The ease of use, simplicity and feasibility demonstrated by LAMP assay in field conditions together with the acceptable level of reproducibility achieved in a reference laboratory support the use of LAMP assay as an effective test for molecular diagnosis of urogenital schistosomiasis in endemic remote areas. [ABSTRACT FROM AUTHOR]

Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients' stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas. [ABSTRACT FROM AUTHOR]

Background: Amphimeriasis is a fish-borne disease caused by the liver fluke Amphimerus spp. that has recently been reported as endemic in the tropical Pacific side of Ecuador with a high prevalence in humans and domestic animals. The diagnosis is based on the stool examination to identify parasite eggs, but it lacks sensitivity. Additionally, the morphology of the eggs may be confounded with other liver and intestinal flukes. No immunological or molecular methods have been developed to date. New diagnostic techniques for specific and sensitive detection of Amphimerus spp. DNA in clinical samples are needed. Methodology/Principal findings: A LAMP targeting a sequence of the Amphimerus sp. internal transcribed spacer 2 region was designed. Amphimerus sp. DNA was obtained from adult worms recovered from animals and used to optimize the molecular assays. Conventional PCR was performed using outer primers F3-B3 to verify the proper amplification of the Amphimerus sp. DNA target sequence. LAMP was optimized using different reaction mixtures and temperatures, and it was finally set up as LAMPhimerus. The specificity and sensitivity of both PCR and LAMP were evaluated. The detection limit was 1 pg of genomic DNA. Field testing was done using 44 human stool samples collected from localities where fluke is endemic. Twenty-five samples were microscopy positive for Amphimerus sp. eggs detection. In molecular testing, PCR F3-B3 was ineffective when DNA from fecal samples was used. When testing all human stool samples included in our study, the diagnostic parameters for the sensitivity and specificity were calculated for our LAMPhimerus assay, which were 76.67% and 80.77%, respectively. Conclusions/Significance: We have developed and evaluated, for the first time, a specific and sensitive LAMP assay for detecting Amphimerus sp. in human stool samples. The procedure has been named LAMPhimerus method and has the potential to be adapted for field diagnosis and disease surveillance in amphimeriasis-endemic areas. Future large-scale studies will assess the applicability of this novel LAMP assay. [ABSTRACT FROM AUTHOR]

Background: Immigrants to Spain are mainly from low- and middle-income countries, and around 20% are children. Absolute eosinophilia is defined as >0.45×109eosinophilic leucocytes/L of peripheral blood. Absolute eosinophilia in travelers and immigrants from tropical and sub-tropical areas is frequently associated with parasitic diseases. However, the significance of relative eosinophilia in immigrant children, defined as >5% eosinophilic leucocytes in those with <0.45×109eosinophils/L, is unresolved. Objectives: To describe the importance of relative eosinophilia in a cohort of immigrant children (<18 years) from sub-Saharan Africa, North Africa and Latin America. Methods: 176 immigrant children without absolute eosinophilia were prospectively evaluated. Results: 25 of them (14.2%) had relative eosinophilia. 10 patients with relative eosinophilia had no diagnosis. 15 with relative eosinophilia (60%) were diagnosed with a parasitic disease, 7 (46.7%) of whom had only one parasite, while co-infection accounted for 8 of the 15 cases (53.3%). Of the parasitic infections, the most frequent causes of relative eosinophilia were filariasis spp. (7/15, 46.7%), strongyloides spp. (5/15, 33.3%), schistosoma spp. (4/15, 26.6%) andAscaris lumbricoides(2/15, 13.3%). Conclusion: The findings suggest that relative eosinophilia is frequently associated with helminthic infection in immigrant children from tropical and sub-tropical areas, so a thorough parasitological study is highly advisable in this group of patients. [ABSTRACT FROM AUTHOR]

Background: Fasciolosis remains a significant food-borne trematode disease causing high morbidity around the world and affecting grazing animals and humans. A deeper understanding concerning the molecular mechanisms by which Fasciola hepatica infection occurs, as well as the molecular basis involved in acquiring protection is extremely important when designing and selecting new vaccine candidates. The present study provides a first report of microarray-based technology for describing changes in the splenic gene expression profile for mice immunised with a highly effective, protection-inducing, multi-epitope, subunit-based, chemically-synthesised vaccine candidate against F. hepatica.Methods: The mice were immunised with synthetic peptides containing B- and T-cell epitopes, which are derived from F. hepatica cathepsin B and amoebapore proteins, as novel vaccine candidates against F. hepatica formulated in an adjuvant adaptation vaccination system; they were experimentally challenged with F. hepatica metacercariae. Spleen RNA from mice immunised with the highest protection-inducing synthetic peptides was isolated, amplified and labelled using Affymetrix standardised protocols. Data was then background corrected, normalised and the expression signal was calculated. The Ingenuity Pathway Analysis tool was then used for analysing differentially expressed gene identifiers for annotating bio-functions and constructing and visualising molecular interaction networks.Results: Mice immunised with a combination of three peptides containing T-cell epitopes induced high protection against experimental challenge according to survival rates and hepatic damage scores. It also induced differential expression of 820 genes, 168 genes being up-regulated and 652 genes being down-regulated, p value <0.05, fold change ranging from -2.944 to 7.632. A functional study of these genes revealed changes in the pathways related to nitric oxide and reactive oxygen species production, Interleukin-12 signalling and production in macrophages and Interleukin-8 signalling with up-regulation of S100 calcium-binding protein A8, Matrix metallopeptidase 9 and CXC chemokine receptor 2 genes.Conclusion: The data obtained in the present study provided us with a more comprehensive overview concerning the possible molecular pathways implied in inducing protection against F. hepatica in a murine model, which could be useful for evaluating future vaccine candidates. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis remains one of the most common endemic parasitic diseases affecting over 230 million people worlwide. Schistosoma mansoni is the main species causing intestinal and hepatic schistosomiasis and the fresh water pulmonate snails of the genus Biomphalaria are best known for their role as intermediate hosts of the parasite. The development of new molecular monitoring assays for large-scale screening of snails from transmission sites to detect the presence of schistosomes is an important point to consider for snail control interventions related to schistosomiasis elimination. Our work was focussed on developing and evaluating a new LAMP assay combined with a simple DNA extraction method to detect S. mansoni in experimentally infected snails as a diagnostic tool for field conditions. Methodology/Principal findings: A LAMP assay using a set of six primers targeting a sequence of S. mansoni ribosomal intergenic spacer 28S-18S rRNA was designed. The detection limit of the LAMP assay was 0.1 fg of S. mansoni DNA at 63°C for 50 minutes. LAMP was evaluated by examining S. mansoni DNA in B. glabrata snails experimentally exposed to miracidia at different times post-exposure: early prepatent period (before cercarial shedding), light infections (snails exposed to a low number of miracidia) and detection of infected snails in pooled samples (within a group of uninfected snails). DNA for LAMP assays was obtained by using a commercial DNA extraction kit or a simple heat NaOH extraction method. We detected S. mansoni DNA in all groups of snails by using no complicated requirement procedure for DNA obtaining. Conclusions/Significance: Our LAMP assay, named Biompha-LAMP, is specific, sensitive, rapid and potentially adaptable as a cost-effective method for screening of intermediate hosts infected with S. mansoni in both individual snails and pooled samples. The assay could be suitable for large-scale field surveys for schistosomes control campaigns in endemic areas. [ABSTRACT FROM AUTHOR]

Background: Strongyloides stercoralis, the chief causative agent of human strongyloidiasis, is a nematode globally distributed but mainly endemic in tropical and subtropical regions. Chronic infection is often clinically asymptomatic but it can result in severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. There is a great diversity of techniques used in diagnosing the disease, but definitive diagnosis is accomplished by parasitological examination of stool samples for morphological identification of parasite. Until now, no molecular method has been tested in urine samples as an alternative to stool samples for diagnosing strongyloidiasis. This study aimed to evaluate the use of a new molecular LAMP assay in a well-established Wistar rat experimental infection model using both stool and, for the first time, urine samples. The LAMP assay was also clinically evaluated in patients´ stool samples. Methodology/Principal Findings: Stool and urine samples were obtained daily during a 28-day period from rats infected subcutaneously with different infective third-stage larvae doses of S. venezuelensis. The dynamics of parasite infection was determined by daily counting the number of eggs per gram of feces from day 1 to 28 post-infection. A set of primers for LAMP assay based on a DNA partial sequence in the 18S rRNA gene from S. venezuelensis was designed. The set up LAMP assay (namely, Strong-LAMP) allowed the sensitive detection of S. venezuelensis DNA in both stool and urine samples obtained from each infection group of rats and was also effective in S. stercoralis DNA amplification in patients´ stool samples with previously confirmed strongyloidiasis by parasitological and real-time PCR tests. Conclusions/Significance: Our Strong-LAMP assay is an useful molecular tool in research of a strongyloidiasis experimental infection model in both stool and urine samples. After further validation, the Strong-LAMP could also be potentially applied for effective diagnosis of strongyloidiasis in a clinical setting. [ABSTRACT FROM AUTHOR]

Background: Strongyloidiasis is a parasitic disease widely present in tropical and subtropical areas. Strongyloides stercoralis represents the main species that infects human beings. Ivermectin is the current drug of choice; however, issues related with treatment failure in patients with diabetes or infected with T-lymphotropic virus-1 make the identification of new molecules for alternative treatment a priority. In the present study, the activity of sphingosine-related aminoalcohol and diamine were evaluated against Strongyloides venezuelensis third-stage larva (L3) cultures and experimental infections in mice. Methods: The efficacy of each compound against L3 was assessed using both XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay and microscopic observation with concentrations ranging from 1 to 350 μM. Cytotoxicity was evaluated using J774.2 macrophage cell line and XTT assay. Lethal concentration 50 (LC50), selectivity index (SI) and structure-activity relationships were established. The activity compounds 4 (2-(ethylamino) hexadecan-1-ol), 6 (2-(butylamino) hexadecan-1-ol), 17 (tert-butyl N-(1-aminododecan-2-yl) carbamate) and 18 (tert-butyl N-(1-aminohexadecan-2-yl) carbamate) were further assessed against experimental S. venezuelensis infections in CD1 mice measuring reductions in the numbers of parthenogenetic females and egg passed in faeces. Mice were infected with 3,000 L3 and treated with 20 mg/kg/day for five days. Results: In the screening study of 15 aminoalcohols [lauryl (n = 9); palmityl (n = 13); stearyl (n = 15) and alcohol derivatives], the presence of a palmitol chain was associated with the highest efficacy against L3 (LC50 31.9-39. 1 μM). Alkylation of the 2-amino group with medium size fragments as ethyl or n-butyl showed the best larvicidal activity. The dialkylation did not improve efficacy. Aminoalcohols 4 and 6 showed the highest SI (1.5 and 1.6, respectively). With respect to diamine derivative compounds, a chain size of sixteen carbon atoms (palmitoyl chain, n = 13), and the alkylation of the 2-amino group with medium-sized fragments, were associated with the highest lethal activities. The presence of carbamoyl group in diamines 17 and 18 yielded high SI (1.7 and 1.4, respectively). Infected mice treated with aminoalcohol 6 showed reduction in parthenogenetic females (59 %) and eggs in faeces (51 %). Conclusions: These results support the potentiality of aminoalcohol and diamine sphingosine-related compounds as suitable prototypes for developing new promising drugs against strongyloidiasis. [ABSTRACT FROM AUTHOR]

Background: Fasciola hepatica infection still remains one of the helminthic neglected tropical diseases (NTDs). It has a huge worldwide distribution, affecting mainly cattle and, sometimes, human beings. In addition to data reported about the immunological response induced by helminthic infections and that induced by Fasciola hepatica, little is known about the gene expression profile in its organ target, the liver, which is where adult worms are established and live for long periods of time, causing its characteristic pathology. In the present work, we study both the early and late gene expression profiles in the livers of mice infected with F. hepatica metacercariae using a microarray-based methodology. Methodology: A total of 9 female-6-week-old BALB/c mice (Charles River Laboratories, Barcelona, Spain) weighing 20 to 35 g were used for the experiments. Two groups of BALB/c mice were orally infected with seven F. hepatica metacercariae, and the other group remained untreated and served as a control. Mice were humanely euthanized and necropsied for liver recovery, histological assessment of hepatic damage, RNA isolation, microarray design and gene expression analysis on the day of infection (t0), seven days post-infection (t7) and twenty-one days post-infection (t21). Results: We found that F. hepatica infection induces the differential expression of 128 genes in the liver in the early stage of infection and 308 genes in the late stage, and most of them are up-regulated. The Ingenuity Pathway Analysis revealed significant changes in the pathways related to metabolism, biosynthesis and signaling as well as genes implicated in inducing liver-toxicity, injury and death. Conclusion: The present study provides us insights at the molecular level about the underlying mechanisms used by F. hepatica, leading to liver damage and its subsequent pathophysiology. The expression pattern obtained here could also be used to explain the lack of association between infection with F. hepatica and cholangiocarcinoma. However, more studies should be performed to confirm this hypothesis. [ABSTRACT FROM AUTHOR]

Background: Urogenital schistosomiasis due to Schistosoma haematobium is a serious underestimated public health problem affecting 112 million people - particularly in sub-Saharan Africa. Microscopic examination of urine samples to detect parasite eggs still remains as definitive diagnosis. This work was focussed on developing a novel loop-mediated isothermal amplification (LAMP) assay for detection of S. haematobium DNA in human urine samples as a high-throughput, simple, accurate and affordable diagnostic tool to use in diagnosis of urogenital schistosomiasis. Methodology/Principal Findings: A LAMP assay targeting a species specific sequence of S. haematobium ribosomal intergenic spacer was designed. The effectiveness of our LAMP was assessed in a number of patients´ urine samples with microscopy confirmed S. haematobium infection. For potentially large-scale application in field conditions, different DNA extraction methods, including a commercial kit, a modified NaOH extraction method and a rapid heating method were tested using small volumes of urine fractions (whole urine, supernatants and pellets). The heating of pellets from clinical samples was the most efficient method to obtain good-quality DNA detectable by LAMP. The detection limit of our LAMP was 1 fg/µL of S. haematobium DNA in urine samples. When testing all patients´ urine samples included in our study, diagnostic parameters for sensitivity and specificity were calculated for LAMP assay, 100% sensitivity (95% CI: 81.32%-100%) and 86.67% specificity (95% CI: 75.40%-94.05%), and also for microscopy detection of eggs in urine samples, 69.23% sensitivity (95% CI: 48.21% -85.63%) and 100% specificity (95% CI: 93.08%-100%). Conclusions/Significance: We have developed and evaluated, for the first time, a LAMP assay for detection of S. haematobium DNA in heated pellets from patients´ urine samples using no complicated requirement procedure for DNA extraction. The procedure has been named the Rapid-Heat LAMPellet method and has the potential to be developed further as a field diagnostic tool for use in urogenital schistosomiasis-endemic areas. [ABSTRACT FROM AUTHOR]

Fasciolosis is considered the most widespread trematode disease affecting grazing animals around the world; it is currently recognised by the World Health Organisation as an emergent human pathogen. Triclabendazole is still the most effective drug against this disease; however, resistant strains have appeared and developing an effective vaccine against this disease has increasingly become a priority. Several bioinformatics tools were here used for predicting B- and T-cell epitopes according to the available data for Fasciola hepatica protein amino acid sequences. BALB/c mice were immunised with the synthetic peptides by using the ADAD vaccination system and several immune response parameters were measured (antibody titres, cytokine levels, T-cell populations) to evaluate their ability to elicit an immune response. Based on the immunogenicity results so obtained, seven peptides were selected to assess their protection-inducing ability against experimental infection with F. hepatica metacercariae. Twenty-four B- or T-epitope-containing peptides were predicted and chemically synthesised. Immunisation of mice with peptides so-called B1, B2, B5, B6, T14, T15 and T16 induced high levels of total IgG, IgG1 and IgG2a (p<0.05) and a mixed Th1/Th2/Th17/Treg immune response, according to IFN-γ, IL-4, IL-17 and IL-10 levels, accompanied by increased CD62L+ T-cell populations. A high level of protection was obtained in mice vaccinated with peptides B2, B5, B6 and T15 formulated in the ADAD vaccination system with the AA0029 immunomodulator. The bioinformatics approach used in the present study led to the identification of seven peptides as vaccine candidates against the infection caused by Fasciola hepatica (a liver-fluke trematode). However, vaccine efficacy must be evaluated in other host species, including those having veterinary importance. [ABSTRACT FROM AUTHOR]

Background: Systematic screening for antibodies against SARS-CoV-2 is a crucial tool for surveillance of the COVID-19 pandemic. The University of Salamanca (USAL) in Spain designed a project called "DIANCUSAL" (Diagnosis of New Coronavirus, COVID-19, in University of Salamanca) to measure antibodies against SARS-CoV-2 among its ~34,000 students and academic staff, as the influence of the university community in the spread of the SARS-CoV-2 pandemic in the city of Salamanca and neighboring towns hosting USAL campuses could be substantial. Objective: The aim of this study was to estimate the prevalence of SARS-CoV-2 antibodies among USAL students, professors and staff and to evaluate the demographic, academic, clinical and lifestyle and behavioral factors related to seropositivity. Methodology: The DIANCUSAL study is an ongoing university population-based cross-sectional study, with the work described herein conducted from July–October 2020. All USAL students, professors and staff were invited to complete an anonymized questionnaire. Seroprevalence of anti-SARS-CoV-2 antibodies was detected and quantified by using chemiluminescent assays for IgG and IgM. Principal findings: A total of 8197 (24.71%) participants were included. The mean age was 31.4 (14.5 SD) years, and 66.0% of the participants were female. The seroprevalence was 8.25% overall and was highest for students from the education campus (12.5%) and professors from the biomedical campus (12.6%), with significant differences among faculties (p = 0.006). Based on the questionnaire, loss of smell and fever were the symptoms most strongly associated with seropositivity, and 22.6% of seropositive participants were asymptomatic. Social distancing was the most effective hygiene measure (p = 0.0007). There were significant differences in seroprevalence between participants with and without household exposure to SARS-CoV-2 (p = 0.0000), but not between students who lived in private homes and those who lived in dormitories. IgG antibodies decreased over time in the participants with confirmed self-reported COVID-19 diagnoses. Conclusions: The analysis revealed an overall 8.25% seroprevalence at the end of October 2020, with a higher seroprevalence in students than in staff. Thus, there is no need for tailored measures for the USAL community as the official average seroprevalence in the area was similar (7.8% at 22 June and 12.4 at 15 November of 2020). Instead, USAL members should comply with public health measures. [ABSTRACT FROM AUTHOR]

Strongyloidiasis is a parasitosis that represents a public health problem, in tropical regions. The present study aimed to investigate the anthelmintic effects of several extracts of Argemone mexicana , as well as its main component berberine (Ber) against the third-stage larvae (L3) of Strongyloides venezuelensis in-vitro experiments. Also, the anti-hemolytic activity of the extract, fractions, and Ber were tested in human erythrocytes. A dose-response anthelminthic bioassay demonstrated Ber as the most effective component, followed by methanolic subfraction (Fr3) and finally the crude extract of A. mexicana (Am) showing LC 50 response values of 1.6, 19.5, and 92.1 μg/mL, at 96 h respectively. Also, Am, Fr3, and Ber did not produce significant hemolysis against human erythrocytes (p ≤ 0.05). Am and Fr3 showed erythrocyte protection effect capacity at the membrane level (p ≤ 0.05). Furthermore, Ber was found to have an antioxidant activity of 168.18 μg/mL. According to the results, the Fr3 of A. mexicana , and particularly Ber, exhibited potent in-vitro effects against L3 of S. venezuelensis , without hemolytic activity against human erythrocytes and presented good antioxidant capacity. In conclusion, the extracts of A. mexicana and the main component have activity against S. venezuelensis , nevertheless, further studies are required to elucidate the mechanism of action. Image 1 • Anthelmintic activity of A. mexicana methanolic extract, active fraction, and it 's main component. • Development of a new alternative to a conventional anthelmintics. • The capacity of the berberine alkaloid is reported, which has a response against the viability of S. venezuelensis (L3). • The safety of the methanolic extract, active fraction, and berberine, was evaluated by an in-vitro hemolytic bioassay. [ABSTRACT FROM AUTHOR]

• Elderly acute myeloblastic leukaemia patients on intensive chemotherapy included. • The Acute Leukaemia French Association model's 'benefit' group had longer survival. • Better survival identified by Haematological Oncology Clinical Studies Group model. • MD Anderson Cancer Center points model may identify patients for intensive therapy. Selection of elderly patients (aged ≥60 years) for intensive chemotherapy treatment of acute myeloblastic leukaemia (AML) remains challenging. Several cooperative groups such as Acute Leukaemia French Association (ALFA), Haematological Oncology Clinical Studies Group (HOCSG) and MD Anderson Cancer Center (MDACC) have developed predictive models to select those patients who can benefit from intensive chemotherapy. Our purpose is to validate and compare these three models in a cohort of patients treated in real-life setting. For this, a total of 1724 elderly AML patients and treated with intensive chemotherapy regimens were identified in the PETHEMA registry. Median age was 67.2 years (range, 60–84,9) and median overall survival [OS] 9 months (95 % confidence interval [CI], 8.2–9.7). Taking into account the ALFA group's model, patients likely to benefit from intensive chemotherapy had longer OS (14 months, 95 % CI 12.3–15.7) than those unlikely to benefit (5 months, 95 % CI 4.1–5.9; p < 0.001). Significant differences in OS were observed between patients with favourable risk (17 months, 95 % CI 13.2–20.7), intermediate risk (11 months, 95 % CI 9.3–12.6) and adverse risk (6 months, 95 % CI 5.1–6.4; p < 0.001) according to the HOCSG model. No significant differences in OS were observed between patients with 0, 1, 2 or ≥3 points according to the MDACC model. However, when patients with ≥1 point were compared with those with 0 points, median OS was significantly longer in the latter [15 months (95 % CI 12.1–17.8) vs 7 (95 % CI 5.7–8.5)]. This retrospective study validates predictive models proposed by the ALFA, HOCSG and MDACC groups in this real-life cohort. [ABSTRACT FROM AUTHOR]