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Clonorchis sinensis is a significant zoonotic food-borne parasite that causes a range of hepatobiliary diseases, which in severe cases can even lead to cholangiocarcinoma. To explore new diagnostic and treatment strategies, the dynamic RNA regulatory processes across different developmental stages of C. sinensis were analyzed by using whole-transcriptome sequencing. The chromosomal-level genome of C. sinensis was used for sequence alignment and annotation. In this study, we identified a total of 59,103 RNAs in the whole genome, including 2,384 miRNAs, 25,459 mRNAs, 27,564 lncRNAs and 3,696 circRNAs. Differential expression analysis identified 6,556 differentially expressed mRNAs, 2,231 lncRNAs, 877 miRNAs and 20 circRNAs at different developmental stages. Functional enrichment analysis highlighted the critical role of microtubule-related biological processes in the growth and development of C. sinensis. And coexpression analysis revealed 97 lncRNAs and 85 circRNAs that were coexpressed with 42 differentially expressed mRNAs that associated with microtubules at different developmental stages of C. sinensis. The expression of the microtubule-related genes dynein light chain 2 (DLC2) and dynein light chain 4 (DLC4) increased with C. sinensis development, and DLC2/4 could be inhibited by albendazole. Finally, by constructing competing endogenous RNA (ceRNA) networks, the lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA regulatory relationships were constructed, and the ceRNA networks of MSTRG.14258.5-novel_miR_2287-newGene_28215 and MSTRG.14258.5-novel_miR_2216-CSKR_109340 were verified. This study suggests, through whole transcriptome sequencing, that the context of microtubule regulation may play an essential role in the development and growth of C. sinensis. Author summary: This study is the first to comprehensively elucidate the entire transcriptome of Clonorchis sinensis by exploring the expression and regulatory dynamics of noncoding RNAs and mRNAs at different developmental stages in its life cycle. By using next-generation sequencing technology, this research demonstrated new mRNAs, miRNAs, lncRNAs and circRNAs, and established valuable resources for future parasitic research. Compared with mRNAs, lncRNAs have shorter gene lengths, lower expression levels, fewer exons and fewer isoforms. Differential expression analysis demonstrated stage-specific patterns of noncoding RNAs and mRNAs in C. sinensis during its life cycle, thus providing crucial insights into the role of noncoding RNAs in parasite growth and development. Furthermore, the construction of ceRNA regulatory networks revealed that microtubule-related lncRNAs and circRNAs in C. sinensis may play an important role in its growth and development. This study advances our understanding of the entire transcriptome of C. sinensis and introduces novel perspectives for future parasitic infection treatment strategies. In summary, this study establishes a foundation for in-depth investigations into the interplay between noncoding RNAs and parasites, as well as the associated biological processes, thus demonstrating broad research and therapeutic potential. [ABSTRACT FROM AUTHOR]

Fasciolosis caused by Fasciola hepatica is a major public health and economic problem worldwide. Due to the lack of a successful vaccine and emerging resistance to the drug triclabendazole, alternative phytotherapeutic approaches are being investigated. This study investigated the in vitro anthelmintic activity of Lavender (Lavandula angustifolia) and carob (Ceratonia siliqua L.) essential oils (EOs) against F. hepatica. The in vitro study was based on an egg hatch assay (EHA), adult motility inhibition assays, DNA damage, reactive oxygen species (ROS) level along with several oxidative stress biomarkers including glutathione peroxidase (GSH), and glutathione-S-transferase (GST), superoxide dismutase (SOD) and malondialdehyde (MDA). To this end, different concentrations of L. angustifolia and C. siliqua EOs (1, 5, 10, 25 and 50 mg/mL) were used to assess anthelmintic effects on different life stages including egg, and adults of F. hepatica for 24 hrs. The results indicated that these EOs play a significant role as anthelminthics, and the effect was dependent on time and concentration. The in vitro treatment of F. hepatica worms with both L. angustifolia and C. siliqua EOs increased DNA damage, ROS production and induction of oxidative stress (decreased SOD, GST and GSH, and increased MDA), significantly compared to control. Therefore, it can be concluded that L. angustifolia and C. siliqua EOs have the potential to be used as novel agents for the control and treatment of F. hepatica infections. Further studies are required to investigate their pharmacological potential and effectiveness in vivo for the treatment of parasitic infections. Author summary: Fasciolosis is a disease caused by a leaf-shaped trematode called Fasciola hepatica. This disease is becoming increasingly common in humans and has been reported on five continents. F. hepatica primarily infects the bile ducts of cattle, sheep and goats, causing significant economic losses to the global livestock industry. Unfortunately, this disease has been neglected and there is currently no commercially viable vaccine to prevent it. The main treatment for this disease is chemotherapy, with triclabendazole (TCBZ) being the drug of choice for controlling these parasites. However, there is growing concern about the increase in reports of drug resistance to TCBZ in parasites. Therefore, there is an urgent need to find alternative treatment methods. Natural herbal products give great hope as they contain a large reservoir of medicinal ingredients that are effectively used against various parasitic diseases. The aim of the current study is to evaluate the in vitro effect of essential oils of lavender (Lavandula angustifolia) and carob (Ceratonia siliqua L.) on F. hepatica. According to the findings of this study, L. angustifolia and C. siliqua essential oils (EOs) have significant effects as anthelmintics. The use of L. angustifolia and C. siliqua EOs in vitro significantly increased DNA damage, ROS production, and oxidative stress in F. hepatica worms. Therefore, it can be concluded that these EOs have the potential to be used as novel agents for the control and treatment of F. hepatica. [ABSTRACT FROM AUTHOR]

Leishmania, the dixenous trypanosomatid parasites, are the causative agents of leishmaniasis currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania, and the recently described Mundinia, consisting of six species distributed sporadically all over the world infecting humans and/or animals. These parasites infect various mammalian species and also cause serious human diseases, but their reservoirs are unknown. Thus, adequate laboratory models are needed to enable proper research of Mundinia parasites. In this complex study, we compared experimental infections of five Mundinia species (L. enriettii, L. macropodum, L. chancei, L. orientalis, and four strains of L. martiniquensis) in three rodent species: BALB/c mouse, Chinese hamster (Cricetulus griseus) and steppe lemming (Lagurus lagurus). Culture-derived parasites were inoculated intradermally into the ear pinnae and progress of infection was monitored for 20 weeks, when the tissues and organs of animals were screened for the presence and quantity of Leishmania. Xenodiagnoses with Phlebotomus duboscqi were performed at weeks 5, 10, 15 and 20 post-infection to test the infectiousness of the animals throughout the experiment. BALB/c mice showed no signs of infection and were not infectious to sand flies, while Chinese hamsters and steppe lemmings proved susceptible to all five species of Mundinia tested, showing a wide spectrum of disease signs ranging from asymptomatic to visceral. Mundinia induced significantly higher infection rates in steppe lemmings compared to Chinese hamsters, and consequently steppe lemmings were more infectious to sand flies: In all groups tested, they were infectious from the 5th to the 20th week post infection. In conclusion, we identified two rodent species, Chinese hamster (Cricetulus griseus) and steppe lemming (Lagurus lagurus), as candidates for laboratory models for Mundinia allowing detailed studies of these enigmatic parasites. Furthermore, the long-term survival of all Mundinia species in steppe lemmings and their infectiousness to vectors support the hypothesis that some rodents have the potential to serve as reservoir hosts for Mundinia. Author summary: Leishmania parasites of the subgenus Mundinia are an emerging health and veterinary problem that should not be ignored. Being the most recent of all Leishmania described, many aspects of Mundinia biology are enigmatic. We have very scarce data on their life cycles and biology, thus proper laboratory research must be done to enable their better understanding. One of the most crucial parts of the life cycle of Leishmania is the development in the mammalian host. In the past, we worked on establishment of other laboratory models for the subgenus, but neither Arvicanthis, Mastomys, nor guinea pigs proved to be a good choice. Other authors performed experiments with BALB/c mice using various inoculation techniques, but they also failed. Here we describe the establishment of two new potential laboratory model species, Chinese hamsters and steppe lemmings, which proved to be susceptible to Mundinia and such findings will enable other scientists to continue in research of these parasites. [ABSTRACT FROM AUTHOR]

Background: Chagas disease, affecting approximately eight million individuals in tropical regions, is primarily transmitted by vectors. Rhodnius prolixus, a triatomine vector, commonly inhabits in ecotopes with diverse palm tree species, creating optimal conditions for vector proliferation. This study aims to explore the transmission ecology of Trypanosoma cruzi, the causative parasite of Chagas disease, by investigating the feeding patterns and natural infection rates of R. prolixus specimens collected from various wild palm species in the Colombian Orinoco region. Materials and methods: To achieve this objective, we sampled 35 individuals from three palm species (Attalea butyracea, Acrocomia aculeata, and Mauritia flexuosa) in a riparian forest in the Casanare department of eastern Colombia, totaling 105 sampled palm trees. DNA was extracted and analyzed from 115 R. prolixus specimens at different developmental stages using quantitative PCR (qPCR) for T. cruzi detection and identification of discrete typing units. Feeding preferences were determined by sequencing the 12S rRNA gene amplicon through next-generation sequencing. Results: A total of 676 R. prolixus specimens were collected from the sampled palms. The study revealed variation in population densities and developmental stages of R. prolixus among palm tree species, with higher densities observed in A. butyracea and lower densities in M. flexuosa. TcI was the exclusive T. cruzi discrete typing unit (DTU) found, with infection frequency positively correlated with R. prolixus abundance. Insects captured in A. butyracea exhibited higher abundance and infection rates than those from other palm species. The feeding sources comprised 13 mammal species, showing no significant differences between palm species in terms of blood sources. However, Didelphis marsupialis and Homo sapiens were present in all examined R. prolixus, and Dasypus novemcinctus was found in 89.47% of the insects. Conclusion: This study highlights the significance of wild palms, particularly A. butyracea, as a substantial risk factor for T. cruzi transmission to humans in these environments. High population densities and infection rates of R. prolixus were observed in each examined palm tree species. Author summary: Previous studies on Chagas disease in the Orinoco region primarily focused on domestic and peri-domestic areas of rural residences. However, to fully grasp the dynamics of T. cruzi transmission from natural, preserved areas to humans, it is crucial to comprehend the habitats and food resources that kissing bugs encounter in their natural distribution zones. In this study, we delved into both the habitat conditions and the food sources available on wild palms in the eastern plains region of Colombia, with the aim of understanding the establishment of the Chagas disease vector. Notably, our findings revealed that the studied kissing bugs fed on both wild and domestic animals, as well as humans, across all stages of their development. Furthermore, this research identified differences in the structural physiognomy of wild palms directly linked to the establishment of bug colonies. This knowledge enhances our understanding of the role of wild habitats in the dynamics of T. cruzi transmission in endemic areas. [ABSTRACT FROM AUTHOR]

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a serious chronic parasitic disease, currently treated with Nifurtimox (NFX) and Benznidazole (BZ). In addition to high toxicity, these drugs have low healing efficacy, especially in the chronic phase of the disease. The existence of drug-resistant T. cruzi strains and the occurrence of cross-resistance between BZ and NFX have also been described. In this context, it is urgent to study the metabolism of these drugs in T. cruzi, to better understand the mechanisms of resistance. Prostaglandin F2α synthase (PGFS) is an enzyme that has been correlated with parasite resistance to BZ, but the mechanism by which resistance occurs is still unclear. Our results show that the genome of the CL Brener clone of T. cruzi, contains five PGFS sequences and three potential pseudogenes. Using CRISPR/Cas9 we generated knockout cell lines in which all PGFS sequences were disrupted, as shown by PCR and western blotting analyses. The PGFS deletion did not alter the growth of the parasites or their susceptibility to BZ and NFX when compared to wild-type (WT) parasites. Interestingly, NTR-1 transcripts were shown to be upregulated in ΔPGFS mutants. Furthermore, the ΔPGFS parasites were 1.6 to 1.7-fold less tolerant to oxidative stress generated by menadione, presented lower levels of lipid bodies than the control parasites during the stationary phase, and were less infective than control parasites. Author summary: Prostaglandin F2α synthase (PGFS) has been associated with T. cruzi resistance to benznidazole (BZ), but the real involvement of this enzyme in the resistance phenotype is still uncertain since different studies in the literature point in different directions. Here we demonstrated that the deletion of all copies of the PGFS gene in T. cruzi does not affect the parasite's resistance to BZ or nifurtimox (NFX), but results in reduced tolerance to oxidative stress caused by menadione. The PGFS knockout mutants are less infective, and, at the stationary phase, the parasites have fewer lipid bodies than the control parasites. Thus, our results suggest that this enzyme appears to have a regulatory role in defence against oxidative stress and parasite infectivity. [ABSTRACT FROM AUTHOR]

Background: Annual mass drug administrations (MDA) of ivermectin will strongly reduce Onchocerca volvulus microfilariae (mf) in the skin and in the onchocerciasis patients' eyes. Ivermectin treatment will also affect the expression of immunity in patients, such that activated immune defenses may help control and contribute to clearance of mf of O. volvulus. Longitudinal surveys are a prerequisite to determining the impact of ivermectin on the status of anti-parasite immunity, notably in risk zones where parasite transmission and active O. volvulus infections persist. Methodology/Principal findings: Onchocerciasis patients were treated annually with ivermectin and their Onchocerca volvulus antigen (OvAg) specific IgG and cellular responses were investigated before and at 30 years post initial ivermectin treatment (30yPT). Repeated annual ivermectin treatments eliminated persisting O. volvulus microfilariae (mf) from the skin of patients and abrogated patent infections. The OvAg-specific IgG1 and IgG4 responses were diminished at 30yPT to the levels observed in endemic controls. Prior to starting ivermectin treatment, OvAg-induced cellular productions of IL-10, IFN-γ, CCL13, CCL17 and CCL18 were low in patients, and at 30yPT, cellular cytokine and chemokine responses increased to the levels observed in endemic controls. In contrast, mitogen(PHA)- induced IL-10, IFN-γ, CCL17 and CCL18 cellular production was diminished. This divergent response profile thus revealed increased parasite antigen-specific but reduced polyclonal cellular responsiveness in patients. The transmission of O. volvulus continued at the patients' location in the Mô river basin in central Togo 2018 and 2019 when 0.58% and 0.45%, respectively, of Simulium damnosum s.l. vector blackflies carried O. volvulus infections. Conclusions/Significance: Repeated annual ivermectin treatment of onchocerciasis patients durably inhibited their patent O. volvulus infections despite ongoing low-level parasite transmission in the study area. Repeated MDA with ivermectin affects the expression of immunity in patients. O. volvulus parasite-specific antibody levels diminished to levels seen in infection-free endemic controls. With low antibody levels, antibody-dependent cellular cytotoxic responses against tissue-dwelling O. volvulus larvae will weaken. O. volvulus antigen inducible cytokine and chemokine production increased in treated mf-negative patients, while their innate responsiveness to mitogen declined. Such lower innate responsiveness in elderly patients could contribute to reduced adaptive immune responses to parasite infections and vaccines. On the other hand, increased specific cellular chemokine responses in mf-negative onchocerciasis patients could reflect effector cell activation against tissue invasive larval stages of O. volvulus. The annual Simulium damnosum s.l. biting rate observed in the Mô river basin was similar to levels prior to initiation of MDA with ivermectin, and the positive rtPCR results reported here confirm ongoing O. volvulus transmission. Author summary: Onchocerciasis is a neglected tropical disease, and a major cause of debilitating skin disease and ocular damage that can lead to irreversible blindness. Annual mass drug administrations (MDA) of ivermectin strongly reduces the load of Onchocerca volvulus microfilaria (mf) in the skin and in the patients' eyes. Evolution of onchocerciasis as a disease is prevented by MDA, but recent studies have shown that O. volvulus transmission has not been completely interrupted. Repeated MDA with ivermectin affects immune responses, such that activated immune defenses may enhance clearance of mf of O. volvulus. Longitudinal surveys are required to determine the impact of ivermectin on the status of immunity, notably in risk zones where parasite transmission and active O. volvulus infections persist. We examined the changes of O. volvulus parasite-specific antibody and cellular immune responsiveness in patients treated annually with ivermectin for 30 years. Treatment prevented patent O. volvulus infections, whilst parasite antigen-specific cytokine and chemokine responses increased but O.volvulus-specific antibody responses declined. Such decreased antibody levels could weaken antibody-dependent cellular cytotoxic responses to infective and tissue-dwelling O. volvulus larvae. Strengthened monocyte attracting and activation regulated chemokine responses could enhance effector cell migration and activation against larval stages of O.volvulus, possibly also eliciting resistance to further parasite infections. [ABSTRACT FROM AUTHOR]

Background: Chagas disease (CD) is a neglected disease that induces heart failure and arrhythmias in approximately 30% of patients during the chronic phase of the disease. Despite major efforts to understand the cellular pathophysiology of CD there are still relevant open questions to be addressed. In the present investigation we aimed to evaluate the contribution of the Na+/Ca2+ exchanger (NCX) in the electrical remodeling of isolated cardiomyocytes from an experimental murine model of chronic CD. Methodology/Principal findings: Male C57BL/6 mice were infected with Colombian strain of Trypanosoma cruzi. Experiments were conducted in isolated left ventricular cardiomyocytes from mice 180–200 days post-infection and with age-matched controls. Whole-cell patch-clamp technique was used to measure cellular excitability and Real-time PCR for parasite detection. In current-clamp experiments, we found that action potential (AP) repolarization was prolonged in cardiomyocytes from chagasic mice paced at 0.2 and 1 Hz. After-depolarizations, both subthreshold and with spontaneous APs events, were more evident in the chronic phase of experimental CD. In voltage-clamp experiments, pause-induced spontaneous activity with the presence of diastolic transient inward current was enhanced in chagasic cardiomyocytes. AP waveform disturbances and diastolic transient inward current were largely attenuated in chagasic cardiomyocytes exposed to Ni2+ or SEA0400. Conclusions/Significance: The present study is the first to describe NCX as a cellular arrhythmogenic substrate in chagasic cardiomyocytes. Our data suggest that NCX could be relevant to further understanding of arrhythmogenesis in the chronic phase of experimental CD and blocking NCX may be a new therapeutic strategy to treat arrhythmias in this condition. Author summary: Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is a neglected disease that induces heart failure and arrhythmias in approximately 30% of patients during the chronic phase of the disease. There are several substrates for arrhythmias in the heart. Some of them involve changes in the electrical properties of cardiomyocytes, the working cells of the heart. In our study we evaluate the potential involvement of Na+/Ca2+ exchanger (NCX) in the arrhythmic phenotype of cardiomyocytes isolated from mice infected with Trypanosoma cruzi, between 180- and 200- days post-infection, which is considered the chronic phase of CD in this animal model. In our study we found several arrhythmogenic membrane potential oscillations during action potential measurements, in rest and using a protocol to simulate a pause after a tachycardia. Using pharmacological approach, we determine that NCX significantly contributed to the arrhythmogenic phenomena observed. Thus, in our study we demonstrate that NCX may be relevant to the cellular arrhythmogenic profile observed in cardiomyocytes during the chronic phase of experimental CD and blocking NCX may be a new therapeutic strategy to treat arrhythmias in this condition. [ABSTRACT FROM AUTHOR]

Cystic echinococcosis is still a major concern in South America. While some regions show advances in the control of the disease, others have among the highest incidence in the world. To reverse this situation the Pan American Health Organization (PAHO) has launched a regional project on cystic echinococcosis control and surveillance. An early concern of the program was the lack of a standardized diagnostic tool to monitor infection in dogs, a key target of control programs. Under this premise, we have developed a new copro-ELISA test after extensive screening of a large panel of monoclonal antibodies (MAbs) and polyclonal sera, which performs with high standards of sensitivity (92.6%) and specificity (86.4%) as established by necropsy diagnosis of dogs. The key component of the test, MAbEg9 has a convenient IgG isotype and reacts with a periodate-resistant epitope found in high molecular weight components of the worm. Time-course analysis of experimentally infected dogs showed that even animals with a very low number of parasites could be detected as early as day 20 post infection. The test was formulated in a ready-to-use kit format with proven stability of each component for a minimum of 3 months at room temperature. This characteristic facilitates its standardized use and shipping to other laboratories, which was demonstrated by the identical results obtained by two different laboratories in Peru and our own laboratory when a large number of field samples were analyzed independently in a blind fashion.

The parasitic mite Sarcoptes scabiei is an economically highly significant parasite of the skin of humans and animals worldwide. In humans, this mite causes a neglected tropical disease (NTD), called scabies. This disease results in major morbidity, disability, stigma and poverty globally and is often associated with secondary bacterial infections. Currently, anti-scabies treatments are not sufficiently effective, resistance to them is emerging and no vaccine is available. Here, we report the first high-quality genome and transcriptomic data for S. scabiei. The genome is 56.6 Mb in size, has a a repeat content of 10.6% and codes for 9,174 proteins. We explored key molecules involved in development, reproduction, host-parasite interactions, immunity and disease. The enhanced 'omic data sets for S. scabiei represent comprehensive and critical resources for genetic, functional genomic, metabolomic, phylogenetic, ecological and/or epidemiological investigations, and will underpin the design and development of new treatments, vaccines and/or diagnostic tests. Author summary: Scabies is a highly significant parasitic disease caused by the mite S. scabiei. This NTD has a major adverse impact in disadvantaged communities around the world, particularly when associated with secondary bacterial infections and clinical complications. Here we report the first high-quality genome and transcriptomic data for S. scabiei and explore molecular aspects of S. scabiei/scabies. This genome (56.6 Mb, encoding ~ 9,200 proteins) provides a solid foundation for fundamental investigations of the molecular biology of the mite, host-parasite interactions and disease processes as well as for translational research to develop new treatments, vaccines and diagnostic tests. [ABSTRACT FROM AUTHOR]

Lymphatic filariasis is caused by filarial nematode parasites, including Brugia malayi. Adult worms live in the lymphatic system and cause a strong immune reaction that leads to the obstruction of lymph vessels and swelling of the extremities. Chronic disease leads to the painful and disfiguring condition known as elephantiasis. Current drug therapy is effective against the microfilariae (larval stage) of the parasite, but no drugs are effective against the adult worms. One of the major stumbling blocks toward developing effective macrofilaricides to kill the adult worms is the lack of a high throughput screening method for candidate drugs. Current methods utilize systems that measure one well at a time and are time consuming and often expensive. We have developed a low-cost and simple visual imaging system to automate and quantify screening entire plates based on parasite movement. This system can be applied to the study of many macroparasites as well as other macroscopic organisms.

BackgroundThe possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3) genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi.Methodology/principal findingsWe investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (ds)RNA (approximately 500 bp) designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose-dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s) within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was transcript-dependent (from 40 to >75% knockdown relative to controls) and this may have contributed to the lack of obvious phenotypes observed, even after prolonged incubations of 3 weeks. Within minutes of their mechanical preparation from cercariae, schistosomula accumulated fluorescent macromolecules in the gut indicating that the gut is an important route through which RNAi is expedited in the developing parasite.ConclusionsTransient RNAi operates gene-selectively in S. mansoni newly transformed schistosomula yet the sensitivity of individual gene targets varies. These findings and the operational parameters defined will facilitate larger RNAi screens.

Background: Schistosomiasis is a chronic parasitic disease, that affects over 207 million people and causes over 200,000 deaths annually, mainly in sub-Saharan Africa. Although many health measures have been carried out to limit parasite transmission, significant numbers of non-human primates such as Chlorocebus aethiops (Ch. aethiops) (vervet) and Papio anubis (baboon) are infected with S. mansoni, notably in Ethiopia, where they are expected to have potentially significant implications for transmission and control efforts. Objective: The objective of this study was to assess and compare the genetic diversity and population structure of S. mansoni isolates from human and non-human primates free-ranging in close proximity to villages in selected endemic areas of Ethiopia. Methods: A cross-sectional study was conducted in three transmission sites: Bochesa, Kime and Fincha. A total of 2,356 S. mansoni miracidia were directly isolated from fecal specimens of 104 hosts (i.e. 60 human hosts and 44 non-human primates). We performed DNA extraction and PCR amplification using fourteen microsatellite loci. Results: At population scale we showed strong genetic structure between the three sample sites. At the definitive host scale, we observed that host factors can shape the genetic composition of parasite infra-populations. First, in male patients, we observed a positive link between parasite genetic diversity and the age of the patients. Second, we observed a difference in genetic diversity which was high in human males, medium in human females and low in non-human primates (NHPs). Finally, whatever the transmission site no genetic structure was observed between human and non-human primates, however, there appears to be little barriers, if any, host specificity of the S. mansoni populations with cross-host infections. Conclusion: Occurrence of infection of a single host with multiple S. mansoni strains and inter- and intra-host genetic variations was observed. Substantial genetic diversity and gene flow across the S. mansoni population occurred at each site and non-human primates likely play a role in local transmission and maintenance of infection. Therefore, public health and wildlife professionals should work together to improve disease control and elimination strategies. Author summary: Schistosomiasis is a chronic disease caused by flukes (trematodes). The definitive host spectrum of schistosomes, whether human, non-human primates (NHPs) or other mammals, is highly dependent on the schistosome species concerned. Genetic diversity and population structure studies of S. mansoni have provided insights into the variation of natural populations. Understanding S. mansoni genetic diversity and population structure of isolates from human and non-human primate hosts living in close proximity showed the occurrence of infection of a single host with multiple S. mansoni strains and inter- and intra-host genetic variations. In this article, the researchers assert the fact that genetic approach reveals that parasites from the three different sites are independent. Thus, we could consider the three sites as geographical replicates showing the influence of NHPs in parasitic transmission in Ethiopia. This study provides insights into the epidemiology, genetic diversity and population structure of S. mansoni in human and non-human primates in Ethiopia, all of which are crucial for the control of schistosomiasis. [ABSTRACT FROM AUTHOR]

Genetic polymorphisms in natural Leishmania populations have been reported in endemic areas. Microsatellite typing is a useful tool to elucidate the genetic variability of parasite strains, due to its capability for high-resolution mapping of genomic targets. The present study employed multilocus microsatellite typing (MLMT) to explore the genotypic composition of Leishmania infantum in naturally infected dogs by genotyping parasites infecting different tissues with or without in vitro expansion. Eighty-six samples were collected from 46 animals in an endemic region of visceral leishmaniasis (VL). MLMT was performed for 38 spleen samples and 48 L. infantum cultures isolated from different tissues. Of the 86 samples, 23 were effectively genotyped by MLMT, identifying nine multilocus genotypes (MLG; referred to as MLG A–I). MLGs A, B and C were detected in more than one type of tissue and in more than one sample. Conversely, MLG D-I were uniquely detected in one sample each. The results showed that multiple genotype infections occur within a single host and tissue. Paired sample analysis revealed the presence of different MLMT alleles in 14 dogs, while the same MLG allele was present in 15 animals. STRUCTURE analysis demonstrated the presence of two populations; 13 samples displayed a similar admixture of both ancestral populations, and these were not assigned to any population. Only samples for which Q ≥ 0.70 after CLUMPP alignment were considered to be part of Population 1 (POP1) or Population 2 (POP2). POP2 comprised the majority of samples (n = 54) compared to POP1 (n = 19). This study presents evidence of multiple genotype infections (caused by L. infantum) in dogs in an area with high VL transmission. Further investigations must be undertaken to determine the multiple effects of infection on the host immune response and disease dynamics and treatment. Author summary: American visceral leishmaniasis (VL) is a parasitic disease caused by the protozoan Leishmania infantum. This parasite can infect humans and animals and is transmitted by sand flies. Domestic dogs are considered an important host, and like humans, they can manifest the disease or present asymptomatic infections. Studies have identified genetic variations among L. infantum parasites from different endemic regions in the American continent. For other parasitic diseases (e.g., malaria), studies have suggested that multigenetic infection predicts the development of symptoms and can lead to a high level of transmission. However, the effects of the genetic composition of Leishmania parasites on VL need to be ascertained. This study used highly variable microsatellite markers to investigate multigenotype L. infantum populations among naturally infected dogs living in an area in which VL is highly prevalent. Samples obtained from different tissues were examined to identify the occurrence of multiple genotypes in the same animal and even within the same tissue. [ABSTRACT FROM AUTHOR]

Background: Vivax malaria is an important public health problem in the Greater Mekong Subregion (GMS), including the China-Myanmar border. Previous studies have found that Plasmodium vivax has decreased sensitivity to antimalarial drugs in some area of GMS, but the sensitivity of P. vivax to antimalarial drugs is unclear in the China-Myanmar border. Here, we investigate the drug sensitivity profile and genetic variations for two drug resistance related genes, in P. vivax isolates to provide baseline information for future drug studies in the China-Myanmar border. Methodology/Principal findings: A total of 64 P. vivax clinical isolates collected from the China-Myanmar border area were assessed for ex vivo susceptibility to eight antimalarial drugs by the schizont maturation assay. The medians of IC50 (half-maximum inhibitory concentrations) for chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate, artemether, dihydroartemisinin were 84.2 nM, 34.9 nM, 4.0 nM, 22.3 nM, 41.4 nM, 2.8 nM, 2.1 nM and 2.0 nM, respectively. Twelve P. vivax clinical isolates were found over the cut-off IC50 value (220 nM) for chloroquine resistance. In addition, sequence polymorphisms in pvmdr1 (P. vivax multidrug resistance-1), pvcrt-o (P. vivax chloroquine resistance transporter-o), and difference in pvmdr1 copy number were studied. Sequencing of the pvmdr1 gene in 52 samples identified 12 amino acid substitutions, among which two (G698S and T958M) were fixed, M908L were present in 98.1% of the isolates, while Y976F and F1076L were present in 3.8% and 78.8% of the isolates. Amplification of the pvmdr1 gene was only detected in 4.8% of the samples. Sequencing of the pvcrt-o in 59 parasite isolates identified a single lysine insertion at position 10 in 32.2% of the isolates. The pvmdr1 M908L substitutions in pvmdr1 in our samples was associated with reduced sensitivity to and chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate and dihydroartemisinin. Conclusions: Our findings depict a drug sensitivity profile and genetic variations of the P. vivax isolates from the China-Myanmar border area, and suggest possible emergence of chloroquine resistant P. vivax isolates in the region, which demands further efforts for resistance monitoring and mechanism studies. Author summary: Vivax malaria is an important parasitic disease in the Greater Mekong Subregion (GMS). Plasmodium vivax, which is less sensitive to chloroquine, has been reported in the region. However, as part of the GMS, the drug sensitivity of P. vivax is unclear in the China-Myanmar border area. In view of the important role of antimalarial drugs in malaria elimination strategies, we investigate the drug sensitivity profile and genetic variations of the P. vivax isolates from the China-Myanmar border. We obtained baseline data of ex vivo sensitivities to eight antimalarial drugs and found that chloroquine-resistant P. vivax was possibly emerging this area. Studies of genetic polymorphisms in two candidate genes of drug resistance (pvmdr1 and pvcrt-o) of the P. vivax isolates from this area and found association between the M908L substitution in pvmdr1 with reduced sensitivities to and chloroquine, mefloquine, pyronaridine, piperaquine, quinine, artesunate and dihydroartemisinin. It is worth noting that the M908L substitution was almost fixed in our samples, which limits the robustness of the conclusion. Our finding suggests that more rigorous in vivo and ex vivo studies are needed to monitor the efficacy of the frontline drugs and discover drug resistance mechanisms. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis is a neglected tropical parasitic disease associated with severe pathology, mortality and economic loss worldwide. Programs for disease control may benefit from specific and sensitive diagnostic methods to detect Schistosoma trematodes in aquatic environments. Here we report the development of novel environmental DNA (eDNA) qPCR assays for the presence of the human-infecting species Schistosoma mansoni, S. haematobium and S. japonicum. Methodology/Principal findings: We first tested the specificity of the assays across the three species using genomic DNA preparations which showed successful amplification of target sequences with no cross amplification between the three focal species. In addition, we evaluated the specificity of the assays using synthetic DNA of multiple Schistosoma species, and demonstrated a high overall specificity; however, S. japonicum and S. haematobium assays showed cross-species amplification with very closely-related species. We next tested the effectiveness of the S. mansoni assay using eDNA samples from aquaria containing infected host gastropods, with the target species revealed as present in all infected aquaria. Finally, we evaluated the effectiveness of the S. mansoni and S. haematobium assays using eDNA samples from eight discrete natural freshwater sites in Tanzania, and demonstrated strong correspondence between infection status established using eDNA and conventional assays of parasite prevalence in host snails. Conclusions/Significance: Collectively, our results suggest that eDNA monitoring is able to detect schistosomes in freshwater bodies, but refinement of the field sampling, storage and assay methods are likely to optimise its performance. We anticipate that environmental DNA-based approaches will help to inform epidemiological studies and contribute to efforts to control and eliminate schistosomiasis in endemic areas. Author summary: Schistosomiasis, otherwise known as bilharzia or snail fever, is a prevalent human disease found across tropical regions of the world and is a major cause of disability. The disease is acquired from exposure to the schistosome infectious larvae released by infected host snails in freshwaters. Programs to restrict the transmission of schistosomiasis would benefit from rapid and reliable diagnostic methods to detect schistosomes. Here we report a study that has developed new diagnostic tools to identify the DNA from three human-infecting Schistosoma species within water samples. This "environmental DNA" (eDNA) approach requires the filtering and laboratory analyses of water samples, and avoids the requirements to locate, identify and individually test the infectious status of host snails. Our results showed that eDNA methods detect the presence of the parasite in freshwater bodies. However, there is need for further refinement for sampling and laboratory techniques to improve the performance of the assays. We anticipate that eDNA approaches will provide information on the distribution and abundance the water-borne parasites, and potentially contribute to the control and elimination of schistosomiasis. [ABSTRACT FROM AUTHOR]

Whole genome sequencing (WGS) is increasingly used for molecular diagnosis and epidemiology of infectious diseases. Current Leishmania genomic studies rely on DNA extracted from cultured parasites, which might introduce sampling and biological biases into the subsequent analyses. Up to now, direct analysis of Leishmania genome in clinical samples is hampered by high levels of human DNA and large variation in parasite load in clinical samples. Here, we present a method, based on target enrichment of Leishmania donovani DNA with Agilent SureSelect technology, that allows the analysis of Leishmania genomes directly in clinical samples. We validated our protocol with a set of artificially mixed samples, followed by the analysis of 63 clinical samples (bone marrow or spleen aspirates) from visceral leishmaniasis patients in Nepal. We were able to identify genotypes using a set of diagnostic SNPs in almost all of these samples (97%) and access comprehensive genome-wide information in most (83%). This allowed us to perform phylogenomic analysis, assess chromosome copy number and identify large copy number variants (CNVs). Pairwise comparisons between the parasite genomes in clinical samples and derived in vitro cultured promastigotes showed a lower aneuploidy in amastigotes as well as genomic differences, suggesting polyclonal infections in patients. Altogether our results underline the need for sequencing parasite genomes directly in the host samples Author summary: Visceral leishmaniasis (VL) is caused by parasitic protozoa of the Leishmania donovani complex and is lethal in the absence of treatment. Whole Genome Sequencing (WGS) of L. donovani clinical isolates revealed hitherto cryptic population structure in the Indian Sub-Continent and provided insights into the epidemiology and potential mechanisms of drug resistance. However, several biases are likely introduced during the culture step. We report here the development of a method that allows determination of parasite genomes directly in clinical samples, and validate it on bone marrow and splenic aspirates of VL patients in Nepal. Our study sheds a new light on the biology of Leishmania in the human host: we found that intracellular parasites of the patients had very low levels of aneuploidy, in sharp contrast to the situation in cultivated isolates. Moreover, the observed differences in genomes between intracellular amastigotes of the patient and the derived cultured parasites suggests polyclonality of infections, with different clones dominating in clinical samples and in culture, likely due to fitness differences. We believe this method is most suitable for clinical studies and for molecular tracking in the context of elimination programs. [ABSTRACT FROM AUTHOR]

Background: The full scope of the genes expressed by schistosomes during intramolluscan development has yet to be characterized. Understanding the gene products deployed by larval schistosomes in their snail hosts will provide insights into their establishment, maintenance, asexual reproduction, ability to castrate their hosts, and their prolific production of human-infective cercariae. Using the Illumina platform, the intramolluscan transcriptome of Schistosoma mansoni was investigated in field-derived specimens of the prominent vector species Biomphalaria pfeifferi at 1 and 3 days post infection (d) and from snails shedding cercariae. These S. mansoni samples were derived from the same snails used in our complementary B. pfeifferi transcriptomic study. We supplemented this view with microarray analyses of S. mansoni from B. glabrata at 2d, 4d, 8d, 16d, and 32d to highlight robust features of S. mansoni transcription, even when a different technique and vector species was used. Principal findings: Transcripts representing at least 7,740 (66%) of known S. mansoni genes were expressed during intramolluscan development, with the greatest number expressed in snails shedding cercariae. Many transcripts were constitutively expressed throughout development featuring membrane transporters, and metabolic enzymes involved in protein and nucleic acid synthesis and cell division. Several proteases and protease inhibitors were expressed at all stages, including some proteases usually associated with cercariae. Transcripts associated with G-protein coupled receptors, germ cell perpetuation, and stress responses and defense were well represented. We noted transcripts homologous to planarian anti-bacterial factors, several neural development or neuropeptide transcripts including neuropeptide Y, and receptors that may be associated with schistosome germinal cell maintenance that could also impact host reproduction. In at least one snail the presence of larvae of another digenean species (an amphistome) was associated with repressed S. mansoni transcriptional activity. Conclusions/Significance: This in vivo study, emphasizing field-derived snails and schistosomes, but supplemented with observations from a lab model, provides a distinct view from previous studies of development of cultured intramolluscan stages from lab-maintained organisms. We found many highly represented transcripts with suspected or unknown functions, with connection to intramolluscan development yet to be elucidated. [ABSTRACT FROM AUTHOR]

Based on our initial observations showing that mice consuming a probiotic product develop more severe cryptosporidiosis, we investigated the impact of other dietary interventions on the intracellular proliferation of Cryptosporidium parvum and C. tyzzeri in the mouse. Mice were orally infected with oocysts and parasite multiplication measured by quantifying fecal oocyst output. High-throughput sequencing of 16S ribosomal RNA amplicons was used to correlate oocyst output with diet and with the composition of the intestinal microbiota. On average, mice fed a diet without fiber (cellulose, pectin and inulin) developed more severe infections. As expected, a diet without fibers also significantly altered the fecal microbiota. Consistent with these observations, mice fed a prebiotic product sold for human consumption excreted significantly fewer oocysts. The fecal microbiota of mice consuming no plant polysaccharides was characterized by a lower relative abundance of Bacteroidetes bacteria. Since bacterial metabolites play an important role in the physiology of intestinal enterocytes, we hypothesize based on these observations that the impact of diet on parasite proliferation is mediated primarily by the metabolic activity of the anaerobic microbiota, specifically by the effect of certain metabolites on the host. This model is consistent with the metabolic dependence of intracellular stages of the parasite on the host cell. These observations underscore the potential of dietary interventions to alleviate the impact of cryptosporidiosis, particularly in infants at risk of recurrent enteric infections. [ABSTRACT FROM AUTHOR]

Background: The National Program for Chagas disease was implemented in Bolivia in 2006, and it greatly decreased the number of infections through vector control. Subsequently, a treatment regimen of benznidazole (BNZ) was started in seropositive school-age children living in certified vector control areas. Methods and findings: We conducted a 12-month follow-up study and seven blood samples were taken during and after the treatment. Serology, conventional diagnostic PCR (cPCR) and quantitative Real-time PCR (qPCR) were performed. Plasma Th1/Th2/Th17 cytokines levels were also determined. Approximately 73 of 103 seropositive children complied with BNZ, with three interruptions due to side effects. To evaluate each individual’s treatment efficacy, the cPCR and qPCR values during the final 6 months of the follow-up period were observed. Among 57 children who completed follow-up, 6 individuals (11%) showed both cPCR(+) and qPCR(+) (non reactive), 24 (42%) cPCR(-) but qPCR(+) (ambiguous) and 27 (47%) cPCR(-) and qPCR(-) (reactive). Within 14 Th1/Th2/Th17 cytokines, IL-17A showed significantly higher levels in seropositive children before the treatment compared to age-matched seronegative children and significantly decreased to the normal level one-year after. Moreover, throughout the follow-up study, IL-17A levels were positively co-related to parasite counts detected by qPCR. At the 12 months’ time point, IL-17A levels of non-reactive subjects were significantly higher than either those of reactive or ambiguous subjects suggesting that IL-17A might be useful to determine the reactivity to BNZ treatment. Conclusions: Plasma levels of IL-17A might be a bio-marker for detecting persistent infection of T. cruzi and its chronic inflammation. [ABSTRACT FROM AUTHOR]

Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease that affects 5–6 million people in endemic areas of the Americas. Presently, chemotherapy relies on two compounds that were proposed as trypanocidal drugs four decades ago: nifurtimox and benznidazole. Both drugs are able to eliminate parasitemia and to avoid seroconversion in infected people when used in the acute phase; however, their use in the chronic phase (the time when the majority of cases are diagnosed) is limited due to their serious side effects. Memantine is a glutamate receptor antagonist in the central nervous system of mammals that has been used for the treatment of Alzheimer’s disease. Our group previously reported memantine as a trypanocidal drug that is able to induce apoptosis-like death in T. cruzi. In the present work, we further investigated the effects of memantine on the infection of RAW 264.7 macrophages and in vivo (in BALB/c mice). Here, we showed that memantine is able to diminish NO and Ca2+ entry in both LPS-activated and non-activated cells. These results, together with the fact that memantine was also able to reduce the infection of macrophages, led us to propose that this drug is able to activate a pro-oxidant non-NO-dependent cell defense mechanism. Finally, infected mice that were treated with memantine had diminished parasitemia, cardiac parasitic load, and inflammatory infiltrates. In addition, the treated mice had an increased survival rate. Taken together, these results indicate memantine to be a candidate drug for the treatment of Chagas disease. [ABSTRACT FROM AUTHOR]

Plasmodium vivax invasion of human erythrocytes depends on the Duffy Binding Protein (PvDBP) which interacts with the Duffy antigen. PvDBP copy number has been recently shown to vary between P. vivax isolates in Sub-Saharan Africa. However, the extent of PvDBP copy number variation, the type of PvDBP multiplications, as well as its significance across broad samples are still unclear. We determined the prevalence and type of PvDBP duplications, as well as PvDBP copy number variation among 178 Ethiopian P. vivax isolates using a PCR-based diagnostic method, a novel quantitative real-time PCR assay and whole genome sequencing. For the 145 symptomatic samples, PvDBP duplications were detected in 95 isolates, of which 81 had the Cambodian and 14 Malagasy-type PvDBP duplications. PvDBP varied from 1 to >4 copies. Isolates with multiple PvDBP copies were found to be higher in symptomatic than asymptomatic infections. For the 33 asymptomatic samples, PvDBP was detected with two copies in two of the isolates, and both were the Cambodian-type PvDBP duplication. PvDBP copy number in Duffy-negative heterozygotes was not significantly different from that in Duffy-positives, providing no support for the hypothesis that increased copy number is a specific association with Duffy-negativity, although the number of Duffy-negatives was small and further sampling is required to test this association thoroughly. [ABSTRACT FROM AUTHOR]

Background: Evaluation of therapeutic response in chronic Chagas disease is a major challenge, due to prolonged persistence of Trypanosoma cruzi-specific antibodies, lack of sensitivity of parasitological tests, and need for long-term follow-up to observe negative seroconversion of conventional serological tests (CS). The objective of this study was to evaluate F2/3-ELISA serology, a promising early biomarker of therapeutic response, and T.cruzi Polymerase chain reaction (PCR) for T. cruzi Deoxyribonucleic acid (DNA), for neonatal diagnosis and evaluation of parasitemia after treatment. Methods: Prospective cohort study, with three-year clinical, serological and parasitological follow-up of pediatric Chagas disease patients treated with benznidazole. Serology was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA), Indirect hemagglutination (IHA) and F2/3-ELISA; Parasitemia by microhematocrit (MH) and PCR. Results: A cohort of 107 pediatric patients treated with benznidazole was enrolled in the study. ELISA and IHA were initially reactive in 100% of patients, F2/3-ELISA serology was reactive in 80% (86/107) and 91% (97/107) had detectable parasitemia. Seventy-six (71%) patients completed at least 36 months of serological follow up after treatment. Although a similar decreasing linear trend was observed for all serological tests, F2/3-ELISA presented earlier, age dependent, negative seroconversion compared to CS. All patients reaching undetectable CS titers had previously seroreverted by F2/3-ELISA. All patients with persistently decreasing antibody titers had negative PCRs throughout the follow up period. No new cardiological lesions were observed during the 3 years follow-up period. Conclusions: The data reported here, using CS, F2/3 ELISA and PCR provide support for the efficacy of benznidazole in congenital Chagas diseases. These results provide support for scaling up of screening, diagnosis and access to benznidazole treatment. Trial registration: ClinicalTrials.gov 0028/04 in the Research Council, Secretary of Health Buenos Aires city Goberment. [ABSTRACT FROM AUTHOR]

Background: The Plasmodium vivax Reticulocyte Binding Protein (PvRBP) family is involved in red blood cell recognition and members of this family are potential targets for antibodies that may block P. vivax invasion. To date, the acquisition of immunity against PvRBPs in low malaria transmission settings and in a broad age group of exposed individuals has not been investigated. Methodology/Principal findings: Total IgG antibody levels to six members of the PvRBP family (PvRBP1a, PvRBP1b, PvRBP2a, PvRBP2b, a non-binding fragment of PvRBP2c (PvRBP2cNB) and PvRBP2-P2) were measured in samples collected from individuals living in two regions of low P. vivax endemicity in Brazil and Thailand. In both settings, levels of total IgG to PvRBP1a, PvRBP2b, PvRBP2cNB, and PvRBP2P-2 increased significantly with age (rho = 0.17–0.49; P<0.001). IgG responses to PvRBP1a, PvRBP2b and PvRBP2cNB were significantly higher in infected individuals by using Wilcoxon’s signed-rank test (P<0.001). Of the six PvRBPs examined, only antibodies to PvRBP2b were associated with protection against clinical malaria in both settings. Conclusion/Significance: Our results indicate that PvRBP2b warrants further preclinical development as a blood-stage vaccine candidate against P. vivax. Total IgG responses to PvRBPs were also shown to be promising immunological markers of exposure to P. vivax infection. [ABSTRACT FROM AUTHOR]

Background: Amoebiasis, caused by Entamoeba histolytica infection, is a global public health problem. However, available drugs to treat amoebiasis are currently limited, and no effective vaccine exists. Therefore, development of new preventive measures against amoebiasis is urgently needed. Methodology/Principal findings: Here, to develop new drugs against amoebiasis, we focused on E. histolytica adenosine 5′-phosphosulfate kinase (EhAPSK), an essential enzyme in Entamoeba sulfolipid metabolism. Fatty alcohol disulfates and cholesteryl sulfate, sulfolipids synthesized in Entamoeba, play important roles in trophozoite proliferation and cyst formation. These processes are closely associated with clinical manifestation and severe pathogenesis of amoebiasis and with disease transmission, respectively. We validated a combination approach of in silico molecular docking analysis and an in vitro enzyme activity assay for large scale screening. Docking simulation ranked the binding free energy between a homology modeling structure of EhAPSK and 400 compounds. The 400 compounds were also screened by a 96-well plate-based in vitro APSK activity assay. Among fifteen compounds identified as EhAPSK inhibitors by the in vitro system, six were ranked by the in silico analysis as having high affinity toward EhAPSK. Furthermore, 2-(3-fluorophenoxy)-N-[4-(2-pyridyl)thiazol-2-yl]-acetamide, 3-phenyl-N-[4-(2-pyridyl)thiazol-2-yl]-imidazole-4-carboxamide, and auranofin, which were identified as EhAPSK inhibitors by both in silico and in vitro analyses, halted not only Entamoeba trophozoite proliferation but also cyst formation. These three compounds also dose-dependently impaired the synthesis of sulfolipids in E. histolytica. Conclusions/Significance: Hence, the combined approach of in silico and in vitro-based EhAPSK analyses identified compounds that can be evaluated for their effects on Entamoeba. This can provide leads for the development of new anti-amoebic and amoebiasis transmission-blocking drugs. This strategy can also be applied to identify specific APSK inhibitors, which will benefit research into sulfur metabolism and the ubiquitous pathway terminally synthesizing essential sulfur-containing biomolecules. [ABSTRACT FROM AUTHOR]

Babesiosis is considered an emerging disease because its incidence has significantly increased in the last 30 years, providing evidence of the expanding range of this rare but potentially life-threatening zoonotic disease. Babesia divergens is a causative agent of babesiosis in humans and cattle in Europe. The recently sequenced genome of B. divergens revealed over 3,741 protein coding-genes and the 10.7-Mb high-quality draft become the first reference tool to study the genome structure of B. divergens. Now, by exploiting this sequence data and using new computational tools and assembly strategies, we have significantly improved the quality of the B. divergens genome. The new assembly shows better continuity and has a higher correspondence to B. bovis chromosomes. Moreover, we present a differential expression analysis using RNA sequencing of the two different stages of the asexual lifecycle of B. divergens: the free merozoite capable of invading erythrocytes and the intraerythrocytic parasite stage that remains within the erythrocyte until egress. Comparison of mRNA levels of both stages identified 1,441 differentially expressed genes. From these, around half were upregulated and the other half downregulated in the intraerythrocytic stage. Orthogonal validation by real-time quantitative reverse transcription PCR confirmed the differential expression. A moderately increased expression level of genes, putatively involved in the invasion and egress processes, were revealed in the intraerythrocytic stage compared with the free merozoite. On the basis of these results and in the absence of molecular models of invasion and egress for B. divergens, we have proposed the identified genes as putative molecular players in the invasion and egress processes. Our results contribute to an understanding of key parasitic strategies and pathogenesis and could be a valuable genomic resource to exploit for the design of diagnostic methods, drugs and vaccines to improve the control of babesiosis. [ABSTRACT FROM AUTHOR]

Background: Babesia, a tick-borne genus of intraerythrocytic parasites, is understudied in humans outside of established high-endemic areas. There is a paucity of data on Babesia in Africa, despite evidence that it is regionally present. A pilot study suggested that Babesia was present in a rural district of Tanzania. Methodology/Principal findings: A cross-sectional study was conducted July-August 2017: residents in a case hamlet that had clustering of subjects with high signal-to-cut off (S/CO) ratios for antibodies against B. microti in the pilot study, and a control hamlet that had lacked significant signal, were evaluated for B. microti. Subjects aged ≥15yrs (n = 299) underwent clinical evaluation and household inspections; 10ml whole blood was drawn for Babesia transcription mediated amplification (TMA), B. microti indirect fluorescent antibody testing (IFA) and rapid diagnostic testing (RDT) for Plasmodium spp. Subjects aged <15yrs (n = 266) underwent a RDT for Plasmodium and assessment by ELISA for B. microti antibodies. A total of 570 subjects participated (mean age 22 [<1 to 90yrs]) of whom 50.7% were female and 145 (25.5%) subjects were Plasmodium RDT positive (+). In those <15yrs, the median ELISA S/CO was 1.11 (IQR 0.80–1.48); the median S/CO in the case (n = 120) and control (n = 146) hamlets was 1.19 (IQR 0.81–1.48) and 1.06 (IQR 0.80–1.50) respectively (p = 0.4). Children ≥5yrs old were more likely to have a higher S/CO ratio than those <5yrs old (p<0.001). One hundred (38%) subjects <15yrs were Plasmodium RDT+. The median S/CO ratio (children <15yrs) did not differ by RDT status (p = 0.15). In subjects ≥15yrs, no molecular test was positive for Babesia, but four subjects (1.4%) were IFA reactive (two each at titers of 128 and 256). Conclusions/Significance: The findings offer further support for Babesia in rural Tanzania. However, low prevalence of seroreactivity questions its clinical significance. [ABSTRACT FROM AUTHOR]

Background: Ghana started its national programme to eliminate lymphatic filariasis (LF) in 2000, with mass drug administration (MDA) with ivermectin and albendazole as main strategy. We review the progress towards elimination that was made by 2016 for all endemic districts of Ghana and analyze microfilaria (mf) prevalence from sentinel and spot-check sites in endemic districts. Methods: We reviewed district level data on the history of MDA and outcomes of transmission assessment surveys (TAS). We further collated and analyzed mf prevalence data from sentinel and spot-check sites. Results: MDA was initiated in 2001–2006 in all 98 endemic districts; by the end of 2016, 81 had stopped MDA after passing TAS and after an average of 11 rounds of treatment (range 8–14 rounds). The median reported coverage for the communities was 77–80%. Mf prevalence survey data were available for 430 communities from 78/98 endemic districts. Baseline mf prevalence data were available for 53 communities, with an average mf prevalence of 8.7% (0–45.7%). Repeated measurements were available for 78 communities, showing a steep decrease in mean mf prevalence in the first few years of MDA, followed by a gradual further decline. In the 2013 and 2014 surveys, 7 and 10 communities respectively were identified with mf prevalence still above 1% (maximum 5.6%). Fifteen of the communities above threshold are all within districts where MDA was still ongoing by 2016. Conclusions: The MDA programme of the Ghana Health Services has reduced mf prevalence in sentinel sites below the 1% threshold in 81/98 endemic districts in Ghana, yet 15 communities within 13 districts (MDA ongoing by 2016) had higher prevalence than this threshold during the surveys in 2013 and 2014. These districts may need to intensify interventions to achieve the WHO 2020 target. [ABSTRACT FROM AUTHOR]

Background: The Madagascar National Strategic Plan for Malaria Control 2018 (NSP) outlines malaria control pre-elimination strategies that include detailed goals for mosquito control. Primary surveillance protocols and mosquito control interventions focus on indoor vectors of malaria, while many potential vectors feed and rest outdoors. Here we describe the application of tools that advance our understanding of diversity, host choice, and Plasmodium infection in the Anopheline mosquitoes of the Western Highland Fringe of Madagascar. Methodology/Principal findings: We employed a modified barrier screen trap, the QUadrant Enabled Screen Trap (QUEST), in conjunction with the recently developed multiplex BLOOdmeal Detection Assay for Regional Transmission (BLOODART). We captured a total of 1252 female Anopheles mosquitoes (10 species), all of which were subjected to BLOODART analysis. QUEST collection captured a heterogenous distribution of mosquito density, diversity, host choice, and Plasmodium infection. Concordance between Anopheles morphology and BLOODART species identifications ranged from 93–99%. Mosquito feeding behavior in this collection frequently exhibited multiple blood meal hosts (single host = 53.6%, two hosts = 42.1%, three hosts = 4.3%). The overall percentage of human positive bloodmeals increased between the December 2017 and the April 2018 timepoints (27% to 44%). Plasmodium positivity was frequently observed in the abdomens of vectors considered to be of secondary importance, with an overall prevalence of 6%. Conclusions/Significance: The QUEST was an efficient tool for sampling exophilic Anopheline mosquitoes. Vectors considered to be of secondary importance were commonly found with Plasmodium DNA in their abdomens, indicating a need to account for these species in routine surveillance efforts. Mosquitoes exhibited multiple blood feeding behavior within a gonotrophic cycle, with predominantly non-human hosts in the bloodmeal. Taken together, this complex feeding behavior could enhance the role of multiple Anopheline species in malaria transmission, possibly tempered by zoophilic feeding tendencies. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax and Hepatitis B virus (HBV) are globally outspread in similar geographic regions. The concurrence of both infections and its association with some degree of protection against symptomatic and/or severe vivax malaria has been already described. Nevertheless, data on how host response to both pathogens undermines the natural progression of the malarial infection are scarce. Here, a large cohort of vivax malaria and HBV patients is retrospectively analyzed in an attempt to depict how inflammatory characteristics could be potentially related to the protection to severe malaria in coinfection. Methods: A retrospective analysis of a databank containing 601 individuals from the Brazilian Amazon, including 179 symptomatic P. vivax monoinfected patients, 145 individuals with asymptomatic P. vivax monoinfection, 28 P. vivax-HBV coinfected patients, 29 HBV monoinfected subjects and 165 healthy controls, was performed. Data on plasma levels of multiple chemokines, cytokines, acute phase proteins, hepatic enzymes, bilirubin and creatinine were analyzed to describe and compare biochemical profiles associated to each type of infection. Results: Coinfected individuals predominantly presented asymptomatic malaria, referred increased number of previous malaria episodes than symptomatic vivax-monoinfected patients, and were predominantly younger than asymptomatic vivax-monoinfected individuals. Coinfection was hallmarked by substantially elevated concentrations of interleukin (IL)-10 and heightened levels of C-C motif chemokine ligand (CCL)2. Correlation matrices showed that coinfected individuals presented a distinct biomarker profile when compared to asymptomatic or symptomatic P. vivax patients, or HBV-monoinfected individuals. Parasitemia could distinguish coinfected from symptomatic or asymptomatic P. vivax-monoinfected patients. HBV viremia was associated to distinct inflammatory profiles in HBV-monoinfected and coinfected patients. Conclusion: The findings demonstrate a distinct inflammatory profile in coinfected patients, with characteristics associated with immune responses to both pathogens. These host responses to P. vivax and HBV, in conjunction, could then be potentially associated, if not mainly responsible, for the protection against symptomatic vivax malaria. [ABSTRACT FROM AUTHOR]

Background: Soil-transmitted helminth (STH) infections remain highly endemic across the Philippines, and are believed to be important contributors to delayed cognitive development of school-aged children. Identification of communities where children are at risk of functional illiteracy is important for the attainment of Sustainable Development Goals target for literacy. We aimed to quantify the associations between the spatial variation of STH infections and functional literacy indicators adjusting for other important contributors, and identify priority areas in the Philippines in need of interventions. Methodology/Principal findings: We used data from 11,313 school-aged children on functional literacy indicators collected in 2008. Nested fixed-effects multinomial regression models were built to determine associations between STH endemicity and geographical distribution of functional literacy, adjusting for demographics, household level variables, and the prevalence of malaria. Bayesian multinomial geostatistical models were built to geographically predict the prevalence of each level of functional literacy. The number of school-aged children belonging to each of the functional literacy indicator classes was forecast for 2017. We estimated 4.20% of functional illiteracy burden among school-aged children in Mindanao might be averted by preventing T. trichiura infections. Areas predicted with the highest prevalence of functional illiteracy were observed in localised areas of the eastern region of the Visayas, and the south-eastern portion of Mindanao. Conclusions/Significance: The study demonstrates significant geographical variation in burden of functional illiteracy in school-aged children associated with STH infections suggesting that targeted helminth control could potentially promote the development of cognitive function of school-aged children in the Philippines. The benefits of a spatially targeted strategy should be tested by future studies. [ABSTRACT FROM AUTHOR]

Background: Giardia lamblia is a very common cause of gastrointestinal symptoms worldwide. There are several methods for the diagnosis of Giardia infection, however none are ideal. We aim to find a new, microRNA-based method that will improve the currently available diagnostic methods for giardiasis. Methods: Deep-sequence profiling of Giardia small-RNA revealed that miR5 and miR6 are highly expressed in Giardia. These miRNAs were tested by qRT-PCR in duodenal biopsies of patients with giardiasis who were positive by microscopic pathological evaluation. The gastric biopsies of the same patients served as negative control tissues. Additionally, these miRNAs were evaluated in stool samples of patients with proven giardiasis. Results: All histologically proven duodenal biopsies of patients with Giardia infection were positive for Giardia miR5, with a mean threshold cycle (Ct) of 23.7, as well as for Giardia DNA qPCR (16S-like gene, mean Ct 26.3). Gastric biopsies which were tested as a control all were negative. Stool evaluation of miR6 in patients with giardiasis showed 90% specificity but only 66% sensitivity, and a lower accuracy rate was obtained with miR5. Conclusion: Giardia miR5 testing in duodenal biopsies may be a new method for the diagnosis of giardiasis. It seems to be more sensitive when compared with testing for Giardia DNA by qPCR in duodenal biopsies. It will be important to investigate the contribution of routine Giardia miRNA testing in duodenal biopsies from patients with persistent abdominal symptoms [ABSTRACT FROM AUTHOR]

Background: African trypanosomosis, primarily transmitted by tsetse flies, remains a serious public health and economic challenge in sub-Saharan Africa. Interventions employing natural repellents from non-preferred hosts of tsetse flies represent a promising management approach. Although zebras have been identified as non-preferred hosts of tsetse flies, the basis for this repellency is poorly understood. We hypothesized that zebra skin odors contribute to their avoidance by tsetse flies. Methodology/Principal findings: We evaluated the effect of crude zebra skin odors on catches of wild savannah tsetse flies (Glossina pallidipes Austen, 1903) using unbaited Ngu traps compared to the traps baited with two known tsetse fly management chemicals; a repellent blend derived from waterbuck odor, WRC (comprising geranylacetone, guaiacol, pentanoic acid and δ-octalactone), and an attractant comprising cow urine and acetone, in a series of Latin square-designed experiments. Coupled gas chromatography-electroantennographic detection (GC/EAD) and GC-mass spectrometry (GC/MS) analyses of zebra skin odors identified seven electrophysiologically-active components; 6-methyl-5-hepten-2-one, acetophenone, geranylacetone, heptanal, octanal, nonanal and decanal, which were tested in blends and singly for repellency to tsetse flies when combined with Ngu traps baited with cow urine and acetone in field trials. The crude zebra skin odors and a seven-component blend of the EAD-active components, formulated in their natural ratio of occurrence in zebra skin odor, significantly reduced catches of G. pallidipesby 66.7% and 48.9% respectively, and compared favorably with the repellency of WRC (58.1%– 59.2%). Repellency of the seven-component blend was attributed to the presence of the three ketones 6-methyl-5-hepten-2-one, acetophenone and geranylacetone, which when in a blend caused a 62.7% reduction in trap catch of G. pallidipes. Conclusions/Significance: Our findings reveal fundamental insights into tsetse fly ecology and the allomonal effect of zebra skin odor, and potential integration of the three-component ketone blend into the management toolkit for tsetse and African trypanosomosis control. [ABSTRACT FROM AUTHOR]

Plasmodium vivax causes the majority of malaria outside Africa, but is poorly understood at a cellular level partly due to technical difficulties in maintaining it in in vitro culture conditions. In the past decades, drug resistant P. vivax parasites have emerged, mainly in Southeast Asia, but while some molecular markers of resistance have been identified, none have so far been confirmed experimentally, which limits interpretation of the markers, and hence our ability to monitor and control the spread of resistance. Some of these potential markers have been identified through P. vivax genome-wide population genetic analyses, which highlighted genes under recent evolutionary selection in Southeast Asia, where chloroquine resistance is most prevalent. These genes could be involved in drug resistance, but no experimental proof currently exists to support this hypothesis. In this study, we used Plasmodium knowlesi, the most closely related species to P. vivax that can be cultured in human erythrocytes, as a model system to express P. vivax genes and test for their role in drug resistance. We adopted a strategy of episomal expression, and were able to express fourteen P. vivax genes, including two allelic variants of several hypothetical resistance genes. Their expression level and localisation were assessed, confirming cellular locations conjectured from orthologous species, and suggesting locations for several previously unlocalised proteins, including an apical location for PVX_101445. These findings establish P. knowlesi as a suitable model for P. vivax protein expression. We performed chloroquine and mefloquine drug assays, finding no significant differences in drug sensitivity: these results could be due to technical issues, or could indicate that these genes are not actually involved in drug resistance, despite being under positive selection pressure in Southeast Asia. These data confirm that in vitro P. knowlesi is a useful tool for studying P. vivax biology. Its close evolutionary relationship to P. vivax, high transfection efficiency, and the availability of markers for colocalisation, all make it a powerful model system. Our study is the first of its kind using P. knowlesi to study unknown P. vivax proteins and investigate drug resistance mechanisms. [ABSTRACT FROM AUTHOR]

A reduction in the global burden of malaria over the past two decades has encouraged efforts for regional malaria elimination. Despite the need to target all Plasmodium species, current focus is mainly directed towards Plasmodium falciparum, and to a lesser extent P. vivax. There is a substantial lack of data on both global and local transmission patterns of the neglected malaria parasites P. malariae and P. ovale spp. We used a species-specific real-time PCR assay targeting the Plasmodium 18s rRNA gene to evaluate temporal trends in the prevalence of all human malaria parasites over a 22-year period in a rural village in Tanzania.We tested 2897 blood samples collected in five cross-sectional surveys conducted between 1994 and 2016. Infections with P. falciparum, P. malariae, and P. ovale spp. were detected throughout the study period, while P. vivax was not detected. Between 1994 and 2010, we found a more than 90% reduction in the odds of infection with all detected species. The odds of P. falciparum infection was further reduced in 2016, while the odds of P. malariae and P. ovale spp. infection increased 2- and 6-fold, respectively, compared to 2010. In 2016, non-falciparum species occurred more often as mono-infections. The results demonstrate the persistent transmission of P. ovale spp., and to a lesser extent P. malariae despite a continued decline in P. falciparum transmission. This illustrates that the transmission patterns of the non-falciparum species do not necessarily follow those of P. falciparum, stressing the need for attention towards non-falciparum malaria in Africa. Malaria elimination will require a better understanding of the epidemiology of P. malariae and P. ovale spp. and improved tools for monitoring the transmission of all Plasmodium species, with a particular focus towards identifying asymptomatic carriers of infection and designing appropriate interventions to enhance malaria control. [ABSTRACT FROM AUTHOR]

Background: Chemotherapy is a principle tool for the control and prevention of piroplasmosis. The search for a new chemotherapy against Babesia and Theileria parasites has become increasingly urgent due to the toxic side effects of and developed resistance to the current drugs. Chalcones have attracted much attention due to their diverse biological activities. With the aim to discover new drugs and drug targets, in vitro and in vivo antibabesial activity of trans-chalcone (TC) and chalcone 4 hydrate (CH) alone and combined with diminazene aceturate (DA), clofazimine (CF) and atovaquone (AQ) were investigated. Methodology/Principal findings: The fluorescence-based assay was used for evaluating the inhibitory effect of TC and CH on four Babesia species, including B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, the combination with DA, CF, and AQ on in vitro cultures, and on the multiplication of a B. microti–infected mouse model. The cytotoxicity of compounds was tested on Madin–Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3), and human foreskin fibroblast (HFF) cell lines. The half maximal inhibitory concentration (IC50) values of TC and CH against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 69.6 ± 2.3, 33.3 ± 1.2, 64.8 ± 2.5, 18.9 ± 1.7, and 14.3 ± 1.6 μM and 138.4 ± 4.4, 60.9 ± 1.1, 82.3 ± 2.3, 27.9 ± 1.2, and 19.2 ± 1.5 μM, respectively. In toxicity assays, TC and CH affected the viability of MDBK, NIH/3T3, and HFF cell lines the with half maximum effective concentration (EC50) values of 293.9 ± 2.9, 434.4 ± 2.7, and 498 ± 3.1 μM and 252.7 ± 1.7, 406.3 ± 9.7, and 466 ± 5.7 μM, respectively. In the mouse experiment, TC reduced the peak parasitemia of B. microti by 71.8% when administered intraperitoneally at 25 mg/kg. Combination therapies of TC–DA and TC–CF were more potent against B. microti infection in mice than their monotherapies. Conclusions/Significance: In conclusion, both TC and CH inhibited the growth of Babesia and Theileria in vitro, and TC inhibited the growth of B. microti in vivo. Therefore, TC and CH could be candidates for the treatment of piroplasmosis after further studies. [ABSTRACT FROM AUTHOR]

Background: Taenia solium is a neglected zoonotic parasite endemic throughout many low-income countries worldwide, including Zambia, where it causes human and pig diseases with high health and socioeconomic burdens. Lack of knowledge is a recognized risk factor, and consequently targeted health educational programs can decrease parasite transmission and disease occurrence in endemic areas. Preliminary assessment of the computer-based education program ‘The Vicious Worm’ in rural areas of eastern Zambia indicated that it was effective at increasing knowledge of T. solium in primary school students. The aim of this study was to evaluate the impact of ‘The Vicious Worm’ on knowledge retention by re-assessing the same primary school students one year after the initial education workshops. Methodology/Principal findings: Follow-up questionnaires were administered in the original three primary schools in eastern Zambia in 2017, 12 months after the original workshops. In total, 86 pupils participated in the follow-up sessions, representing 87% of the initial workshop respondents. Knowledge of T. solium at ‘follow-up’ was significantly higher than at the initial ‘pre’ questionnaire administered during the Vicious Worm workshop that took place one year earlier. While some specifics of the parasite’s life cycle were not completely understood, the key messages for disease prevention, such as the importance of hand washing and properly cooking pork, remained well understood by the students, even one year later. Conclusions/Significance: Results of this study indicate that ‘The Vicious Worm’ may be an effective tool for both short- and long-term T. solium education of rural primary school students in Zambia. Inclusion of educational workshops using ‘The Vicious Worm’ could be recommended for integrated cysticercosis control/elimination programs in sub-Saharan Africa, particularly if the content is simplified to focus on the key messages for prevention of disease transmission. [ABSTRACT FROM AUTHOR]

Background: Acanthamoeba castellanii, which causes keratitis and blindness in under-resourced countries, is an emerging pathogen worldwide, because of its association with contact lens use. The wall makes cysts resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. Methodology/Principal findings: Transmission electron microscopy and structured illumination microscopy (SIM) showed purified cyst walls of A. castellanii retained an outer ectocyst layer, an inner endocyst layer, and conical ostioles that connect them. Mass spectrometry showed candidate cyst wall proteins were dominated by three families of lectins (named here Jonah, Luke, and Leo), which bound well to cellulose and less well to chitin. An abundant Jonah lectin, which has one choice-of-anchor A (CAA) domain, was made early during encystation and localized to the ectocyst layer of cyst walls. An abundant Luke lectin, which has two carbohydrate-binding modules (CBM49), outlined small, flat ostioles in a single-layered primordial wall and localized to the endocyst layer and ostioles of mature walls. An abundant Leo lectin, which has two unique domains with eight Cys residues each (8-Cys), localized to the endocyst layer and ostioles. The Jonah lectin and glycopolymers, to which it binds, were accessible in the ectocyst layer. In contrast, Luke and Leo lectins and the glycopolymers, to which they bind, were mostly inaccessible in the endocyst layer and ostioles. Conclusions/Significance: The most abundant A. castellanii cyst wall proteins are three sets of lectins, which have carbohydrate-binding modules that are conserved (CBM49s of Luke), newly characterized (CAA of Jonah), or unique to Acanthamoebae (8-Cys of Leo). Cyst wall formation is a tightly choreographed event, in which lectins and glycopolymers combine to form a mature wall with a protected endocyst layer. Because of its accessibility in the ectocyst layer, an abundant Jonah lectin is an excellent diagnostic target. [ABSTRACT FROM AUTHOR]

The present study investigated the presence of endo- and ecto-parasites, and vector-borne pathogens, in dogs from four islands of Greece. A total of 200 (123 owned and 77 sheltered) dogs were examined with different microscopic, serological and molecular methods. Of the examined dogs, 130 (65%) were positive for one or more parasites and/or vector-borne pathogens. The most common zoonotic intestinal helminths recorded were Ancylostomatidae (12.5%) and Toxocara canis (3.5%). Ninety-three dogs (46.5%) seroreacted to Rickettsia conorii. Twenty-two (11%) of them were also PCR positive and 7 (3.5%) showed corpuscles suggestive of Rickettsia spp. on the blood smears. Nineteen dogs (9.5%) were seropositive for Ehrlichia canis, three of them being also PCR positive. Dogs positive for Anaplasma phagocytophilum-Anaplasma platys (1%), Dirofilaria immitis (0.5%) and Babesia canis (0.5%) were also found. Fleas and ticks were recorded in 53 (26.5%) and 50 (25%) dogs, respectively, and all specimens were identified as Ctenocephalides felis felis and Rhipicephalus sanguineus sensu latu. Binary multiple univariate Generalized Linear Models were used to investigate factors and clinical signs related to the recorded positivity, while the association of specific signs with the pathogens was evaluated using tests of independence. Knowledge of occurrence and impact of zoonotic parasites and vector-borne pathogens in dog populations is crucial to prevent the infection in animals and people, and to control the risk of spreading of these pathogens in endemic and non-endemic areas. [ABSTRACT FROM AUTHOR]

Strongyloidiasis is a much-neglected but sometimes fatal soil born helminthiasis. The causing agent, the small intestinal parasitic nematode Strongyloides stercoralis can reproduce sexually through the indirect/heterogonic life cycle, or asexually through the auto-infective or the direct/homogonic life cycles. Usually, among the progeny of the parasitic females both, parthenogenetic parasitic (females only) and sexual free-living (females and males) individuals, are present simultaneously. We isolated S. stercoralis from people living in a village with a high incidence of parasitic helminths, in particular liver flukes (Clonorchis sinensis) and hookworms, in the southern Chinese province Guangxi. We determined nuclear and mitochondrial DNA sequences of individual S. stercoralis isolated from this village and from close by hospitals and we compared these S. stercoralis among themselves and with selected published S. stercoralis from other geographic locations. For comparison, we also analyzed the hookworms present in the same location. We found that, compared to earlier studies of S. stercoralis populations in South East Asia, all S. stercoralis sampled in our study area were very closely related, suggesting a recent common source of infection for all patients. In contrast, the hookworms from the same location, while all belonging to the species Necator americanus, showed rather extensive genetic diversity even within host individuals. Different from earlier studies conducted in other geographic locations, almost all S. stercoralis in this study appeared heterozygous for different sequence variants of the 18S rDNA hypervariable regions (HVR) I and IV. In contrast to earlier investigations, except for three males, all S. stercoralis we isolated in this study were infective larvae, suggesting that the sampled population reproduces predominantly, if not exclusively through the clonal life cycles. Consistently, whole genome sequencing of individual worms revealed higher heterozygosity than reported earlier for likely sexual populations of S. stercoralis. Elevated heterozygosity is frequently associated with asexual clonal reproduction. [ABSTRACT FROM AUTHOR]

Background: Knowledge of the foci of Plasmodium species infections is critical for a country with an elimination agenda. Namibia is targeting malaria elimination by 2020. To support decision making regarding targeted intervention, we examined for the first time, the foci of Plasmodium species infections and regional prevalence in northern Namibia, using nested and quantitative polymerase chain reaction (PCR) methods. Methods: We used cross-sectional multi-staged sampling to select 952 children below 9 years old from schools and clinics in seven districts in northern Namibia, to assess the presence of Plasmodium species. Results: The median participant age was 6 years (25–75%ile 4–8 y). Participants had a median hemoglobin of 12.0 g/dL (25–75%ile 11.1–12.7 g/dL), although 21% of the cohort was anemic, with anemia being severer in the younger population (p<0.002). Most of children with Plasmodium infection were asymptomatic (63.4%), presenting a challenge for elimination. The respective parasite prevalence for Plasmodium falciparum (Pf), Plasmodium vivax (Pv) and Plasmodium ovale curtisi (Po) were (4.41%, 0.84% and 0.31%); with Kavango East and West (10.4%, 6.19%) and Ohangwena (4.5%) having the most prevalence. Pv was localized in Ohangwena, Omusati and Oshana, while Po was found in Kavango. All children with Pv/Pf coinfections in Ohangwena, had previously visited Angola, affirming that perennial migrations are risks for importation of Plasmodium species. The mean hemoglobin was lower in those with Plasmodium infection compared to those without (0.96 g/dL less, 95%CI 0.40–1.52 g/dL less, p = 0.0009) indicating that quasi-endemicity exists in the low transmission setting. Conclusions: We conclude that Pv and Po species are present in northern Namibia. Additionally, the higher number of asymptomatic infections present challenges to the efforts at elimination for the country. Careful planning, coordination with neighboring Angola and execution of targeted active intervention, will be required for a successful elimination agenda. [ABSTRACT FROM AUTHOR]

Background: Malaria remains endemic in several countries of South America with low to moderate transmission intensity. Regional human migration through underserved endemic areas may be responsible for significant parasite dispersion making the disease resilient to interventions. Thus, the genetic characterization of malarial parasites is an important tool to assess how endemic areas may connect via the movement of infected individuals. Here, four sites in geographically separated areas reporting 80% of the malaria morbidity in Colombia were studied. The sites are located on an imaginary transect line of 1,500 km from the northwest to the south Pacific Coast of Colombia with a minimal distance of 500 km between populations that display noticeable ethnic, economic, epidemiological, and ecological differences. Methodology/Principal findings: A total of 624 Plasmodium vivax samples from the four populations were genotyped by using eight microsatellite loci. Although a strong geographic structure was expected between these populations, only moderate evidence of genetic differentiation was observed using a suite of population genetic analyses. High genetic diversity, shared alleles, and low linkage disequilibrium were also found in these P. vivax populations providing no evidence for a bottleneck or clonal expansions as expected from recent reductions in the transmission that could have been the result of scaling up interventions or environmental changes. These patterns are consistent with a disease that is not only endemic in each site but also imply that there is gene flow among these populations across 1,500 km. Conclusion /Significance: The observed patterns in P. vivax are consistent with a “corridor” where connected endemic areas can sustain a high level of genetic diversity locally and can restore parasite-subdivided populations via migration of infected individuals even after local interventions achieved a substantial reduction of clinical cases. The consequences of these findings in terms of control and elimination are discussed. [ABSTRACT FROM AUTHOR]

Babesia microti is tick-borne disease that is an emerging threat to public health due to increasing prevalence and expanding geographic range. Detection and constant surveillance of babesiosis is imperative for predicting pathogen expansion. Leveraging our whole genome sequence (WGS) analyses of B. microti, we developed a single nucleotide polymorphism (SNP)-based high resolution melt (HRM) surveillance tool. We developed our HRM assay using available sequence data and identified 775 SNPs. From these candidate SNPs, we developed a 32-SNP barcode that is robust and differentiates geographically distinct populations; it contains SNPs that are putatively neutral, located in nuclear, mitochondrial, and apicoplastal regions. The assays are reproducible and robust, requiring a small quantity of DNA (limit of detection as low as 10 pg.). We analyzed the performance of our HRM assay using 26 B. microti clinical samples used in our WGS study from babesiosis endemic regions in the United States. We identified a minimal barcode consisting of 25 SNPs that differentiate geographically distinct populations across all clinical samples evaluated (average minor allele frequency > 0.22). Supporting our previous WGS findings, our 25-SNP barcode identified distinct barcode signatures that segregate B. microti into two lineages: Northeast and Midwest, with the Northeast having three subpopulations: Connecticut/Rhode Island, Nantucket, and the R1 reference group. Our 25-SNP HRM barcode provides a robust means genetic marker set that will aid in tracking the increasing incidence and expanding geographic range of B. microti infections. [ABSTRACT FROM AUTHOR]

Aurora kinases constitute a family of enzymes that play a key role during metazoan cells division, being involved in events like centrosome maturation and division, chromatin condensation, mitotic spindle assembly, control of kinetochore-microtubule attachments, and cytokinesis initiation. In this work, three Aurora kinase homologues were identified in Trypanosoma cruzi (TcAUK1, -2 and -3), a protozoan parasite of the Kinetoplastida Class. The genomic organization of these enzymes was fully analyzed, demonstrating that TcAUK1 is a single-copy gene, TcAUK2 coding sequence is present in two different forms (short and long) and TcAUK3 is a multi-copy gene. The three TcAUK genes are actively expressed in the different life cycle forms of T. cruzi (amastigotes, trypomastigotes and epimastigotes). TcAUK1 showed a changing localization along the cell cycle of the proliferating epimastigote form: at interphase it is located at the extremes of the kinetoplast while in mitosis it is detected at the cell nucleus, in close association with the mitotic spindle. Overexpression of TcAUK1 in epimastigotes leaded to a delay in the G2/M phases of the cell cycle due a retarded beginning of kinetoplast duplication. By immunofluorescence, we found that when it was overexpressed TcAUK1 lost its localization at the extremes of the kinetoplast during interphase, being observed inside the cell nucleus throughout the entire cell cycle. In summary, TcAUK1 appears to be a functional homologue of human Aurora B kinase, as it is related to mitotic spindle assembling and chromosome segregation. Moreover, TcAUK1 also seems to play a role during the initiation of kinetoplast duplication, a novel role described for this protein. [ABSTRACT FROM AUTHOR]

Cryptosporidiosis caused by the protozoan parasites Cryptosporidium hominis and C. parvum, threatens the lives of young children in developing countries. In veterinary medicine, C. parvum causes life-threatening diarrhea and dehydration in newborn dairy calves. Protocols to detect Cryptosporidium spp. oocysts using flow cytometry have been reported; however, these protocols use antibodies against the parasite and typically focus on detection of oocysts, not quantification. These techniques are not well-suited for studies that generate large variations in oocyst burdens because the amount of antibody required is proportional to the number of oocysts expected in samples. Also, oocysts are lost in washes in the staining protocol, reducing accuracy of oocyst counts. Moreover, these protocols require costly fluorochrome-conjugated monoclonal antibodies and are not optimal for studies involving large numbers of samples. Here we present an optimized protocol for purifying oocysts from mouse stool and intestine samples combined with a reliable method to quantify oocysts in a relatively pure population without the need for antibody staining. We used morphology (SSC-A vs FSC-A) and the innate characteristics of C. parvum oocysts compared to fecal and intestinal contaminants to develop a two-step gating strategy that can differentiate oocysts from debris. This method is a fast, reliable, and high-throughput technique to promote research projects on C. parvum infections in mice and potentially other animal hosts. [ABSTRACT FROM AUTHOR]

Objective: Cotrimoxazole prevents opportunistic infections including falciparum malaria in HIV-infected individuals but there are concerns of cross-resistance to other antifolate drugs such as sulphadoxine-pyrimethamine (SP). In this study, we investigated the prevalence of antifolate-resistance mutations in Plasmodium falciparum that are associated with SP resistance in HIV-infected individuals on antiretroviral treatment randomized to discontinue (STOP-CTX), or continue (CTX) cotrimoxazole in Western Kenya. Design: Samples were obtained from an unblinded, non-inferiority randomized controlled trial where participants were recruited on a rolling basis for the first six months of the study, then followed-up for 12 months with samples collected at enrollment, quarterly, and during sick visits. Method: Plasmodium DNA was extracted from blood specimens. Initial screening to determine the presence of Plasmodium spp. was performed by quantitative reverse transcriptase real-time PCR, followed by genotyping for the presence of SP-resistance associated mutations by Sanger sequencing. Results: The prevalence of mutant haplotypes associated with SP-resistant parasites in pfdhfr (51I/59R/108N) and pfdhps (437G/540E) genes were significantly higher (P = 0.0006 and P = 0.027, respectively) in STOP-CTX compared to CTX arm. The prevalence of quintuple haplotype (51I/59R/108N/437G/540E) was 51.8% in STOP-CTX vs. 6.3% (P = 0.0007) in CTX arm. There was a steady increase in mutant haplotypes in both genes in STOP-CTX arm overtime through the study period, reaching statistical significance (P < 0.0001). Conclusion: The frequencies of mutations in pfdhfr and pfdhps genes were higher in STOP-CTX arm compared to CTX arm, suggesting cotrimoxazole effectively controls and selects against SP-resistant parasites. Trial registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

Taenia solium is known to cause human cysticercosis while T. saginata does not. Comparative in vitro and in vivo studies on the oncosphere and the postoncospheral (PO) forms of T. solium and T. saginata may help to elucidate why cysticercosis can occur from one and not the other. The aim of this study was to use in vitro culture assays and in vivo models to study the differences in the development of the T. solium and T. saginata oncosphere. Furthermore, this study aimed to evaluate the expression of cytokines and metalloproteinases (MMPs) in human peripheral blood mononuclear cells (PBMCs), which were stimulated by these oncospheres and PO antigens. T. solium and T. saginata activated oncospheres (AO) were cultured in INT-407 and HCT-8 intestinal cells for 180 days. The T. solium began to die while the T. saginata grew for 180 days and developed to cysticerci in INT-407 cells. Rats were inoculated intracranially with AO and PO forms of either T. saginata or T. solium. Rats infected with T. solium AO and PO forms developed neurocysticercosis (NCC), while those infected with the T. saginata did not. Human PMBCs were stimulated with antigens of AO and PO forms of both species, and the production of cytokines and metalloproteinases (MMPs) was measured. The T. solium AO antigen stimulated a higher production of IL-4, IL-5, IL-13, IFN-γ, and IL-2 cytokines compared to T. saginata AO. In the PO form, the T. saginata PO antigen increased the production of IL-4, IL-5, IL-13, IFN-γ, IL-1β, IL-6, IL-10, TNF-α and IL-12 cytokines compared to T. solium, suggesting that this global immune response stimulated by different forms could permit survival or destruction of the parasite depending of their life-cycle stage. Regarding MMPs, T. solium AO antigen stimulated a higher production of MMP-9 compared to T. saginata AO antigen, which may be responsible for altering the permeability of intestinal cells and facilitating breakdown of the blood-brain barrier during the process of invasion of host tissue. [ABSTRACT FROM AUTHOR]

Currently, malaria rapid diagnostic tests (RDTs) are widely used for malaria diagnosis, but test performance and the factors that lead to failure of Plasmodium ovale detection are not well understood. In this study, three pLDH-based RDTs were evaluated using cases in China that originated in Africa. The sensitivity of Wondfo Pf/Pan, CareStart pLDH PAN and SD BIOLINE Pf/Pan in P. ovale detection was 70, 55 and 18%, respectively. CareStart was worse at detecting P. o. curtisi (36.5%) than at detecting P. o. wallikeri (75.0%), and SD could not detect P. o. curtisi. The overall detection ratio of all three RDTs decreased with parasite density and pLDH concentration. Wondfo, CareStart and SD detected only 75.0, 78.1 and 46.9% of the P. ovale cases, respectively, even when the parasitemia were higher than 5000 parasites/μL. Subspecies of P. ovale should be considered while to improve RDT quality for P. ovale diagnosis to achieve the goal of malaria elimination. [ABSTRACT FROM AUTHOR]

Background: Alveolar echinococcosis (AE) is a lethal zoonosis caused by the metacestode larva of the tapeworm Echinococcus multilocularis. The infection is characterized by tumour-like growth of the metacestode within the host liver, leading to extensive fibrosis and organ-failure. The molecular mechanisms of parasite organ tropism towards the liver and influences of liver cytokines and hormones on parasite development are little studied to date. Methodology/Principal findings: We show that the E. multilocularis larval stage expresses three members of the fibroblast growth factor (FGF) receptor family with homology to human FGF receptors. Using the Xenopus expression system we demonstrate that all three Echinococcus FGF receptors are activated in response to human acidic and basic FGF, which are present in the liver. In all three cases, activation could be prevented by addition of the tyrosine kinase (TK) inhibitor BIBF 1120, which is used to treat human cancer. At physiological concentrations, acidic and basic FGF significantly stimulated the formation of metacestode vesicles from parasite stem cells in vitro and supported metacestode growth. Furthermore, the parasite’s mitogen activated protein kinase signalling system was stimulated upon addition of human FGF. The survival of metacestode vesicles and parasite stem cells were drastically affected in vitro in the presence of BIBF 1120. Conclusions/Significance: Our data indicate that mammalian FGF, which is present in the liver and upregulated during fibrosis, supports the establishment of the Echinococcus metacestode during AE by acting on an evolutionarily conserved parasite FGF signalling system. These data are valuable for understanding molecular mechanisms of organ tropism and host-parasite interaction in AE. Furthermore, our data indicate that the parasite’s FGF signalling systems are promising targets for the development of novel drugs against AE. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is a disease caused by the protozoan parasite, Leishmania. The disease remains a global threat to public health requiring effective chemotherapy for control and treatment. In this study, the effect of some selected phenolic compounds on Leishmania donovani was investigated. The compounds were screened for their anti-leishmanial activities against promastigote and intracellular amastigote forms of Leishmania donovani. Methodology/Principal findings: The dose dependent effect and cytotoxicity of the compounds were determined by the MTT assay. Flow cytometry was used to determine the effect of the compounds on the cell cycle. Parasite morphological analysis was done by microscopy and growth kinetic studies were conducted by culturing cells and counting at 24 hours intervals over 120 hours. The cellular levels of iron in promastigotes treated with compounds was determined by atomic absorption spectroscopy and the effect of compounds on the expression of iron dependent enzymes was investigated using RT-qPCR. The IC50 of the compounds ranged from 16.34 μM to 124 μM compared to amphotericin B and deferoxamine controls. Rosmarinic acid and apigenin were the most effective against the promastigote and the intracellular amastigote forms. Selectivity indexes (SI) of rosmarinic acid and apigenin were 15.03 and 10.45 respectively for promastigotes while the SI of 12.78 and 5.20 respectively was obtained for intracellular amastigotes. Morphologically, 70% of rosmarinic acid treated promastigotes showed rounded morphology similar to the deferoxamine control. About 30% of cells treated with apigenin showed distorted cell membrane. Rosmarinic acid and apigenin induced cell arrest in the G0/G1 phase in promastigotes. Elevated intracellular iron levels were observed in promastigotes when parasites were treated with rosmarinic acid and this correlated with the level of expression of iron dependent genes. Conclusions/Significance: The data suggests that rosmarinic acid exerts its anti-leishmanial effect via iron chelation resulting in variable morphological changes and cell cycle arrest. [ABSTRACT FROM AUTHOR]