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The quinazolines CBR417 and CBR490 were previously shown to be potent anti-wolbachials that deplete Wolbachia endosymbionts of filarial nematodes and present promising pre-clinical candidates for human filarial diseases such as onchocerciasis. In the present study we tested both candidates in two models of chronic filarial infection, namely the Litomosoides sigmodontis and Brugia pahangi jird model and assessed their long-term effect on Wolbachia depletion, microfilariae counts and filarial embryogenesis 16−18 weeks after treatment initiation (wpt). Once per day (QD) oral treatment with CBR417 (50 mg/kg) for 4 days or twice per day (BID) with CBR490 (25 mg/kg) for 7 days during patent L. sigmodontis infection reduced the Wolbachia load by >99% and completely cleared peripheral microfilaremia from 10–14 wpt. Similarly, 7 days of QD treatments (40 mg/kg) with CBR417 or CBR490 cleared >99% of Wolbachia from B. pahangi and reduced peritoneal microfilariae counts by 93% in the case of CBR417 treatment. Transmission electron microscopy analysis indicated intensive damage to the B. pahangi ovaries following CBR417 treatment and in accordance filarial embryogenesis was inhibited in both models after CBR417 or CBR490 treatment. Suboptimal treatment regimens of CBR417 or CBR490 did not lead to a maintained reduction of the microfilariae and Wolbachia load. In conclusion, CBR417 or CBR490 are pre-clinical candidates for filarial diseases, which achieve long-term clearance of Wolbachia endosymbionts of filarial nematodes, inhibit filarial embryogenesis and clear microfilaremia with treatments as short as 7 days., Graphical abstract Image 1, Highlights • CBR417 and CBR490 provide long-term effects in 2 chronic filaria jird models. • CBR417 and CBR490 deplete >99% Wolbachia in B. pahangi and L. sigmodontis filariae. • CBR417 and CBR490 clear L. sigmodontis microfilariae after 10–14 weeks. • CBR417 and CBR490 inhibit filarial embryogenesis in both models. • Suboptimal doses do not maintain reduction of microfilariae and Wolbachia.

Treatment of schistosomiasis relies precariously on just one drug, praziquantel (PZQ). In the search for alternatives, 15 S-[2-(alkylamino)alkane] thiosulfuric acids were obtained from a previous research program and profiled in mice for efficacy against both mature (>42-day-old) and juvenile (21-day-old) Schistosoma mansoni using a screening dose of 100 mg/kg PO QDx4. One compound, S-[2-(tert-butylamino)-1-phenylethane] thiosulfuric acid (TPT sulfonate), was the most effective by decreasing female and male worm burdens by ≥ 90% and ≥46% (mature), and ≥89% and ≥79% (juvenile), respectively. In contrast, PZQ decreased mature female and male worm burdens by 95% and 94%, respectively, but was ineffective against juvenile stages. Against 7-day-old lung-stage worms, TPT sulfonate was only effective at twice the dose decreasing female and male burdens by 95 and 80%, respectively. Single oral doses at 400 and/or 600 mg/kg across various developmental time-points (1-, 7-, 15-, 21- and/or 42 day-old) were consistent with the QD x4 data; efficacy was strongest once the parasites had completed lung migration, and female and male burdens were decreased by at least 90% and 80%, respectively. In vitro, TPT sulfonate is inactive against the parasite suggesting a pro-drug mechanism of action. In mice, TPT sulfonate is fully absorbed and subject to rapid, non-CYP-mediated, first-pass metabolism that is initiated by desulfation and yields a series of metabolites. The initially-formed free thiol-containing metabolite, termed TP thiol, was chemically synthesized; it dose-dependently decreased S. mansoni and Schistosoma haematobium motility in vitro. Also, when administered as a single 50 mg/kg IP dose, TP thiol decreased 33-day-old S. mansoni female and male burdens by 35% and 44%, with less severe organomegaly. Overall, TPT sulfonate's efficacy profile is competitive with that of PZQ. Also, the characterization of a parasiticidal metabolite facilitates an understanding and improvement of the chemistry, and identification of the mechanism of action and/or target., Graphical abstract Image 1, Highlights • TPT sulfonate provides single dose efficacy that is competitive with the current drug, praziquantel. • TPT sulfonate must be biotransformed to be active. • TPT sulfonate is fully absorbed and subject to rapid, non-CYP-mediated, first-pass metabolism. • One of the key metabolites, TP thiol, is anti-schistosomal in vitro and in vivo.

The rapid global spread of the coronavirus disease (COVID-19) has inflicted significant health and socioeconomic burden on affected countries. As positive cases continued to rise in Malaysia, public health laboratories experienced an overwhelming demand for COVID-19 screening. The confirmation of positive cases of COVID-19 has solely been based on the detection of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) using real-time reverse transcription polymerase chain reaction (qRT-PCR). In efforts to increase the cost-effectiveness and efficiency of COVID-19 screening, we evaluated the feasibility of pooling clinical Nasopharyngeal/Oropharyngeal (NP/OP) swab specimens during nucleic acid extraction without a reduction in sensitivity of qRT-PCR. Pools of 10 specimens were extracted and subsequently tested by qRT-PCR according to the WHO-Charite protocol. We demonstrated that the sample pooling method showed no loss of sensitivity. The effectiveness of the pooled testing strategy was evaluated on both retrospective and prospective samples, and the results showed a similar detection sensitivity compared to testing individual sample alone. This study demonstrates the feasibility of using a pooled testing strategy to increase testing capacity and conserve resources, especially when there is a high demand for disease testing.

Background: The giant amoebal viruses of Mimivirus and Marseillevirus are large DNA viruses and have been documented in water, soil, and sewage samples. The trend of discovering these giant amoebal viruses has been increasing throughout Asia with Japan, India, and Saudi Arabia being the latest countries to document the presence of these viruses. To date, there have been no reports of large amoebal viruses being isolated in South East Asia. Objective: In this study, we aim to discover these viruses from soil samples in an aboriginal village (Serendah village) in Peninsular ­Malaysia. Method and Results: We successfully detected and isolated both Mimivirus-like and Marseillevirus-like viruses using Acanthamoeba castellanii. Phylogeny analysis identified them as Mimivirus and Marseillevirus, respectively. Conclusion: The ubiquitous nature of both Mimivirus and Marseillevirus is further confirmed in our study as they are detected in higher quantity in soil that is near to water vicinities in an aboriginal village in Peninsular Malaysia. However, this study is limited by our inability to investigate the impact of Mimivirus and Marseillevirus on the aboriginal villagers. More studies on the potential impact of these viruses on human health, especially on the aborigines, are warranted.

River blindness and lymphatic filariasis are two filarial diseases that globally affect millions of people mostly in impoverished countries. Current mass drug administration programs rely on drugs that primarily target the microfilariae, which are released from adult female worms. The female worms can live for several years, releasing millions of microfilariae throughout the course of infection. Thus, to stop transmission of infection and shorten the time to elimination of these diseases, a safe and effective drug that kills the adult stage is needed. The benzimidazole anthelmintic flubendazole (FBZ) is 100% efficacious as a macrofilaricide in experimental filarial rodent models but it must be administered subcutaneously (SC) due to its low oral bioavailability. Studies were undertaken to assess the efficacy of a new oral amorphous solid dispersion (ASD) formulation of FBZ on Brugia pahangi infected jirds (Meriones unguiculatus) and compare it to a single or multiple doses of FBZ given subcutaneously. Results showed that worm burden was not significantly decreased in animals given oral doses of ASD FBZ (0.2–15 mg/kg). Regardless, doses as low as 1.5 mg/kg caused extensive ultrastructural damage to developing embryos and microfilariae (mf). SC injections of FBZ in suspension (10 mg/kg) given for 5 days however, eliminated all worms in all animals, and a single SC injection reduced worm burden by 63% compared to the control group. In summary, oral doses of ASD formulated FBZ did not significantly reduce total worm burden but longer treatments, extended takedown times or a second dosing regimen, may decrease female fecundity and the number of mf shed by female worms., Author summary Safe and effective macrofilaricidal drugs are critically needed to treat onchocerciasis and lymphatic filariasis, which affect over 54 million people worldwide. Flubendazole (FBZ) in its current commercial formulations is an effective anthelminthic for intestinal soil transmitted helminth (STH) infections but not for filarial infections due to its low bioavailability. The purpose of this study was to assess the efficacy of a new amorphous solid dispersion (ASD) formulation of FBZ given orally to jirds (Meriones unguiculatus) infected with the filarial nematode Brugia pahangi and compare it to FBZ (in suspension) given subcutaneously as a single or multiple dose. Our results indicated that treatment with ASD FBZ did not significantly reduce the total number of worms. However, doses as low as 1.5 mg/kg caused ultrastructural damage to the early stages of developing embryos. No worms were recovered from jirds given 10 mg/kg SC injections of FBZ (suspension) for 5 days when necropsied 68 days after the first-dose, and the number of mf at necropsy was significantly decreased compared to the control group. Animals given only a single 10 mg/kg SC injection had a 63% decrease in the number of adult worms. These animals also had fewer female worms and mf compared to the control group suggesting that even a single SC injection of FBZ had an effect on female survival and fecundity. TEM of worms recovered from jirds given low doses of the ASD formulated FBZ (1.5 mg/kg) suggested that female fecundity and mf production were reduced, but longer treatments, longer takedown times or a second dosing regimen of ASD FBZ may be needed to significantly decrease the total worm burden.

Human toxocariasis which is caused mainly by the larvae of Toxocara canis and Toxocara cati, is a worldwide zoonotic disease that can be a potentially serious human infection. The enzyme-linked immunosorbent assay (ELISA) using T. canis excretory-secretory (TES) antigens harvested from T. canis larvae is currently the serological test for confirming toxocariasis. An alternative to producing large amounts of Toxocara TES and improved diagnosis for toxocariasis is through the development of highly specific recombinant antigens such as the T. canis second stage larva excretory-secretory 30 kDa protein (recTES-30). The aim of this study was to evaluate the sensitivity and specificity of a rapid diagnostic kit (RDT, named as iToxocara kit) in comparison to recTES-30 ELISA in Serendah Orang Asli village in Selangor, Malaysia. A total of 133 subjects were included in the study. The overall prevalence rates by ELISA and RDT were 29.3% and 33.1%, respectively, with more positive cases detected in males than females. However, no association was found between toxocariasis and gender or age. The percentage sensitivity, specificity, positive predictive value and negative predictive value of RDT were 85.7%, 90.1%, 80% and 93.2%, respectively. The prevalence for toxocariasis in this population using both ELISA and RDT was 27.1% (36/133) and the K-concordance test suggested good agreement of the two tests with a Cohen's kappa of 0.722, P

Dengue fever is endemic in Malaysia, contributing to significant economic and health burden in the country. Aedes aegypti and Ae. albopictus are the main vectors of the dengue virus (DENV), which circulates in sylvatic and human transmission cycles and has been present in Malaysia for decades. The study investigated the presence and distribution of DENV in urban localities in the Klang Valley, Peninsular Malaysia. A total of 364 Ae. aegypti and 1,025 Ae. albopictus larvae, and 10 Ae. aegypti and 42 Ae. albopictus adult mosquitoes were screened for the presence of DENV. In total, 31 (2.2%) samples were positive, of which 2 Ae. albopictus larvae were co-infected with two serotypes, one with DENV-2 and DENV-3 and the other with DENV-3 and DENV-4. Phylogenetic analysis determined that the isolates belonged to DENV-1 genotype I (1 Ae. aegypti adult), DENV-2 (1 Ae. albopictus larva), DENV-3 genotype V (3 Ae. aegypti larvae and 10 Ae. albopictus larvae) and DENV-4 genotype IV (6 Ae. aegypti larvae and 12 Ae. albopictus larvae), a sylvatic strain of DENV-4 which was most closely related with sylvatic strains isolated from arboreal mosquitoes and sentinel monkeys in Peninsular Malaysia in the 1970s. All four DENV serotypes were co-circulating throughout the study period. The detection of a sylvatic strain of DENV-4 in Ae. aegypti and Ae. albopictus mosquitoes in urban areas in Peninsular Malaysia highlights the susceptibility of these vectors to infection with sylvatic DENV. The infectivity and vector competence of these urban mosquitoes to this strain of the virus needs further investigation, as well as the possibility of the emergence of sylvatic virus into the human transmission cycle., Author summary Dengue is a mosquito-borne disease that has re-emerged as a major public health problem in the 21st Century. The four serotypes of dengue virus are capable of infecting humans with a range of symptoms and clinical outcomes, with the change in predominating serotypes often responsible for new dengue outbreaks. In this study, we surveyed urban sites in the Klang Valley, Malaysia, for Aedes aegypti and Aedes albopictus mosquito vectors and confirmed the presence of all four serotypes of the virus. We detected the presence of genotype V of dengue serotype 3, which has previously not been reported in the country, as well as a sylvatic strain of dengue serotype 4 that was closely related to strains obtained from forest-dwelling mosquito vectors and sentinel monkeys in Malaysia. The detection of a sylvatic strain of dengue virus in urban vectors raises questions over the risk of virus transmission to humans, especially with recent reports of severe clinical cases as a result of infection with sylvatic dengue virus serotypes. Further studies will be needed to determine the source and origins of these strains as well as the severity of dengue disease upon infection, especially with the development of new dengue vaccines.

Using a sensitive transgenic reporter mouse system and in vivo biophotonic imaging techniques, we present a dynamic analysis of eosinophil responses to schistosome infection. Use of this methodology provided previously unattainable detail on the spatial and temporal distribution of tissue eosinophilia and eosinopoietic responses to schistosome worms and eggs. Dramatic hepatic and intestinal eosinophilia in response to the deposition of schistosome eggs, with accompanying eosinopoiesis in the bone marrow, was observed between weeks 8 and 10 p.i., with subsequent downregulation evident by week 11. Contrary to expectations, we also demonstrate that schistosome parasites themselves induce significant intestinal eosinophilia and eosinopoiesis in the bone marrow at very early stages during prepatent infection.

Background Cercarial elastase is the major invasive larval protease in Schistosoma mansoni, a parasitic blood fluke, and is essential for host skin invasion. Genome sequence analysis reveals a greatly expanded family of cercarial elastase gene isoforms in Schistosoma mansoni. This expansion appears to be unique to S. mansoni, and it is unknown whether gene duplication has led to divergent protease function. Methods Profiling of transcript and protein expression patterns reveals that cercarial elastase isoforms are similarly expressed throughout the S. mansoni life cycle. Computational modeling predicts key differences in the substrate-binding pockets of various cercarial elastase isoforms, suggesting a diversification of substrate preferences compared with the ancestral gene of the family. In addition, active site labeling of SmCE reveals that it is activated prior to exit of the parasite from its intermediate snail host. Conclusions The expansion of the cercarial gene family in S. mansoni is likely to be an example of gene dosage. In addition to its critical role in human skin penetration, data presented here suggests a novel role for the protease in egress from the intermediate snail host. This study demonstrates how enzyme activity-based analysis complements genomic and proteomic studies, and is key in elucidating proteolytic function., Author Summary Schistosome parasites are a major cause of disease in the developing world. The larval stage of the parasite transitions between an intermediate snail host and a definitive human host in a dramatic fashion, burrowing out of the snail and subsequently penetrating human skin. This process is facilitated by secreted proteases. In Schistosoma mansoni, cercarial elastase is the predominant secreted protease and essential for host skin invasion. Genomic analysis reveals a greatly expanded cercarial elastase gene family in S. mansoni. Despite sequence divergence, SmCE isoforms show similar expression profiles throughout the S. mansoni life cycle and have largely similar substrate specificities, suggesting that the majority of protease isoforms are functionally redundant and therefore their expansion is an example of gene dosage. However, activity-based profiling also indicates that a subset of SmCE isoforms are activated prior to the parasite's exit from its intermediate snail host, suggesting that the protease may also have a role in this process.

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., Author Summary The World Health Organization estimates that there are approximately 37 million people who are afflicted by Onchocerca volvulus (the parasitic worm that causes river blindness) and over 120 million people afflicted by the filarial worms Wuchereria and Brugia spp. (causative agents of lymphatic filariasis or elephantiasis). Current mass drug administration includes albendazole and either diethylcarbamazine or ivermectin. These drugs, however, are effective at killing the early larval stage (microfilariae) released from adult female worms but they do not kill the adult worms. Adult worms can live up to 10 or more years, releasing thousands of microfilariae per day. It is essential therefore to treat infected individuals with macrofilaricides in order to prevent the adult parasites from producing microfiliariae for the duration of the infection and to treat the disease. In order to screen candidate drugs for use as macrofilaricides, we have developed an inexpensive system and simple method for quantifying the effectiveness of drugs on parasite movement. The apparatus uses a commodity video camera, a computer and a newly developed free and open source software application to provide automated and quantitative measurements of parasite motility on each plate of worms. This system is not only useful for high throughput screening of macroparasites but can also be applied to the study of other macroscopic organisms as well.

Background Eggs deposited in the liver of the mammalian host by the blood fluke parasite, Schistosoma mansoni, normally drive a T-helper-2 (Th2)-mediated granulomatous response in immune-competent mice. By contrast, in mice deprived of T-cells and incapable of producing granulomata, egg-secreted proteins (ESP) induce acute hepatic injury and death. Previous work has shown that one such ESP, the T2 ribonuclease known as omega-1, is hepatotoxic in vivo in that specific antisera to omega-1 prevent hepatocyte damage. Methodology/Principal Findings Using an in vitro culture system employing mouse primary hepatocytes and alanine transaminase (ALT) activity as a marker of heptocyte injury, we demonstrated that S. mansoni eggs, egg-secreted proteins (ESP), soluble-egg antigen (SEA), and omega-1 are directly hepatotoxic and in a dose-dependent manner. Depletion of omega-1 using a monoclonal antibody abolished the toxicity of pure omega-1 and diminished the toxicity in ESP and SEA by 47 and 33%, respectively. Anion exchange chromatography of ESP yielded one predominant hepatotoxic fraction. Proteomics of that fraction identified the presence of IPSE/alpha-1 (IL-4 inducing principle from S. mansoni eggs), a known activator of basophils and inducer of Th2-type responses. Pure recombinant IPSE/alpha-1 also displayed a dose-dependent hepatotoxicity in vitro. Monoclonal antibody depletion of IPSE/alpha-1 abolished the latter's toxicity and diminished the total toxicity of ESP and SEA by 32 and 35%, respectively. Combined depletion of omega-1 and IPSE/alpha-1 diminished hepatotoxicity of ESP and SEA by 60 and 58% respectively. Conclusions We identified IPSE/alpha-1 as a novel hepatotoxin and conclude that both IPSE/alpha-1 and omega-1 account for the majority of the hepatotoxicity secreted by S. mansoni eggs., Author Summary The flatworm disease, schistosomiasis, is a major public health problem in sub-Saharan Africa, South America and East Asia. A hallmark of infection with Schistosoma mansoni is the immune response to parasite eggs trapped in the liver and other organs. This response involves an infiltration of cells that surround the parasite egg forming a “granuloma.” In mice deprived of T-cells, this granulomatous response is lacking, and toxic products released by eggs quickly cause liver damage and death. Thus the granulomata protect the host from toxic egg products. Only one hepatotoxic molecule, omega-1, has been described to date. We set out to identify other S. mansoni egg hepatotoxins using liver cells grown in culture. We first showed that live eggs, their secretions, and pure omega-1 are toxic. Using a physical separation technique to prepare fractions from whole egg secretions, we identified the presence of IPSE/alpha-1, a protein that is known to strongly influence the immune system. We showed that IPSE/alpha-1 is also hepatotoxic, and that toxicity of both omega-1 and IPSE/alpha-1 can be prevented by first mixing the proteins with specific neutralizing antibodies. Both proteins constitute the majority of hepatotoxicity released by eggs.

Background The 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 Findings We 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. Conclusions Transient 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., Author Summary RNA interference (RNAi) is a technique to selectively suppress mRNA of individual genes and, consequently, their cognate proteins. RNAi using double-stranded (ds) RNA has been used to interrogate the function of mainly single genes in the flatworm, Schistosoma mansoni, one of a number of schistosome species causing schistosomiasis. In consideration of large-scale screens to identify candidate drug targets, we examined the selectivity and sensitivity (the degree of suppression) of RNAi for 11 genes produced in different tissues of the parasite: the gut, tegument (surface) and otherwise. We used the schistosomulum stage prepared from infective cercariae larvae which are accessible in large numbers and adaptable to automated screening platforms. We found that RNAi suppresses transcripts selectively, however, the sensitivity of suppression varies (40%–>75%). No obvious changes in the parasite occurred post-RNAi, including after targeting the mRNA of genes that had been computationally predicted to be essential for survival. Additionally, we defined operational parameters to facilitate large-scale RNAi, including choice of culture medium, transfection strategy to deliver dsRNA, dose- and time-dependency, and dosing limits. Finally, using fluorescent probes, we show that the developing gut allows rapid entrance of dsRNA into the parasite to initiate RNAi.

Two major human diseases caused by filariid nematodes are onchocerciasis, or river blindness, and lymphatic filariasis, which can lead to elephantiasis. The drugs ivermectin, diethylcarbamazine (DEC), and albendazole are used in control programs for these diseases, but are mainly effective against the microfilarial stage and have minimal or no effect on adult worms. Adult Onchocerca volvulus and Brugia malayi worms (macrofilariae) can live for up to 15 years, reproducing and allowing the infection to persist in a population. Therefore, to support control or elimination of these two diseases, effective macrofilaricidal drugs are necessary, in addition to current drugs. In an effort to identify macrofilaricidal drugs, we screened an FDA-approved library with adult worms of Brugia spp. and Onchocerca ochengi, third-stage larvae (L3s) of Onchocerca volvulus, and the microfilariae of both O. ochengi and Loa loa. We found that auranofin, a gold-containing drug used for rheumatoid arthritis, was effective in vitro in killing both Brugia spp. and O. ochengi adult worms and in inhibiting the molting of L3s of O. volvulus with IC50 values in the low micromolar to nanomolar range. Auranofin had an approximately 43-fold higher IC50 against the microfilariae of L. loa compared with the IC50 for adult female O. ochengi, which may be beneficial if used in areas where Onchocerca and Brugia are co-endemic with L. loa, to prevent severe adverse reactions to the drug-induced death of L. loa microfilariae. Further testing indicated that auranofin is also effective in reducing Brugia adult worm burden in infected gerbils and that auranofin may be targeting the thioredoxin reductase in this nematode., Author Summary Onchocerciasis or river blindness, and lymphatic filariasis, which can lead to disfiguring elephantiasis, are two neglected tropical diseases that affect millions of people, primarily in developing countries. Both diseases are caused by filariid nematodes; onchocerciasis is caused by Onchocerca volvulus and lymphatic filariasis is caused by Brugia malayi, B. timori, and Wuchereria bancrofti. Currently, there are no drugs available that are highly efficacious against adult worms; existing drugs mainly kill the first-stage larvae (microfilariae). While these drugs can reduce the transmission of infections in a population, the adult filariids (macrofilariae) can continue to produce microfilariae and perpetuate the cycle of infection. Finding a drug that could kill the adult worms would be an important tool in eliminating onchocerciasis and lymphatic filariasis. To identify potential macrofilaricidal drugs, we developed a high throughput screening method to test FDA-approved drugs on adult Brugia spp., which serves as a model for O. volvulus. Using this screening method, we identified a drug called auranofin that kills adult Onchocerca and adult Brugia spp. in vitro, inhibits the molting of O. volvulus L3s, and reduces the worm burden in an in vivo gerbil-B. pahangi model system. Auranofin is known to inhibit a critical enzyme called thioredoxin reductase in some parasite species, and subsequent testing of the effects of auranofin on the thioredoxin reductase of Brugia indicates that this may be auranofin’s mode of action in this nematode as well.

Using a genomic microarray, gene expression at three different developmental stages of the schistosome parasite were analyzed, resulting in the identification of 1154 developmentally enriched transcripts., Background Schistosome bloodflukes are complex trematodes responsible for 200 million cases of schistosomiasis worldwide. Their life cycle is characterized by a series of remarkable morphological and biochemical transitions between an invertebrate host, an aquatic environment, and a mammalian host. We report a global transcriptional analysis of how this parasite alters gene regulation to adapt to three distinct environments. Results Utilizing a genomic microarray made of 12,000 45-50-mer oligonucleotides based on expressed sequence tags, three different developmental stages of the schistosome parasite were analyzed by pair-wise comparisons of transcript hybridization signals. This analysis resulted in the identification of 1,154 developmentally enriched transcripts. Conclusion This study expands the repertoire of schistosome genes analyzed for stage-specific expression to over 70% of the predicted genome. Among the new associations identified are the roles of robust protein synthesis and programmed cell death in development of cercariae in the sporocyst stages, the relative paucity of cercarial gene expression outside of energy production, and the remarkable diversity of adult gene expression programs that reflect adaptation to the host bloodstream and an average lifespan that may approach 10 years.

Schistosoma blood flukes are trematode parasites with a cosmopolitan distribution that infect over 200 million people globally. We previously showed that Schistosoma mansoni growth and development in the mammalian host is dependent on signals from host CD4+ T cells. To gain insight into the mechanisms that underlie this dependence, we sought to determine the evolutionary origins and limits of this aspect of the host-pathogen relationship. By infecting RAG-1−/− mice with a range of different schistosome species and strains, we tested several hypotheses concerning the time during Schistosoma evolution at which this dependence arose, and whether this dependence is specific to Schistosoma or is also found in other blood flukes. Our data indicate that the developmental dependence on CD4+ T cells previously described for S. mansoni is conserved in the evolutionarily basal species Schistosoma japonicum, suggesting this developmental adaptation arose early in Schistosoma evolution. We also demonstrate that the development of the more evolutionarily-derived species Schistosoma haematobium and Schistosoma intercalatum are dependent on adaptive immune signals. Together, these data suggest that the blood fluke parasites of humans utilize common mechanisms to infect their hosts and to co-opt immune signals in the coordination of parasite development. Thus, exploitation of host-schistosome interactions to impair or prevent parasite development may represent a novel approach to combating of all the schistosome pathogens of humans.

The blood fluke Schistosoma mansoni is a causative agent of schistosomiasis and a major global health problem. After penetrating the skin of the host, schistosomula migrate via the bloodstream to the portal circulation, arriving during the second week of infection [1]. On reaching the liver, migration ceases, and rapid growth and development ensues [2], culminating in the sexual maturation of the parasites, pairing, and the onset of oviposition at ~5 weeks after infection. Eggs are deposited in and transit across the wall of the intestine, emerging into the lumen and passing from the body in feces. Deposition of eggs in host tissues and the resulting inflammatory and immune responses are the primary cause of the pathological and clinical manifestations associated with schistosome infection. Deposition of schistosome eggs in the bowel wall and other tissues induces formation of granulomas around the eggs, a process that is mediated by CD4+ Th2 responses to egg antigens [3]. Formation of granulomas around schistosome eggs appears to facilitate the transit of eggs across the intestinal wall and their egress from the body [4]. Interestingly, CD4+ T cells also facilitate S. mansoni development during the prepatent period, because parasite development is severely impaired in immunodeficient mice and normal development is restored by adoptive transfer of CD4+ T cells into immunodeficient mice [5]. Taken together, these findings may explain why patients with schistosomiasis who are coinfected with HIV excrete fewer eggs in their feces than patients who are HIV negative [6]. Observations from patients with schistosome-HIV coinfection highlight a general recurring theme in helminth-HIV coinfections—that, in contrast to bacteria, fungi, and protozoa, immunosuppression by HIV does not lead to fulminant helminth infections [7]. Rather, HIV coinfection either has little effect on helminth infections, as is the case with most intestinal helminths [7], or may create conditions that are unfavorable to helminth development in the host, as is the case for Strongyloides stercoralis infection [8]. The latter finding correlates well with the notable lack of association between HIV infection and disseminated strongyloidiasis [7]. Attenuated schistosome development was also reported in IL-7–deficient (IL-7−/−) mice [9], suggesting that this host factor may play a direct role in promoting parasite development. Because IL-7 is not expressed by T lymphocytes but is produced by endothelial and epithelial cells such as thymic stromal cells, it is possible that IL-7 represents a distinct mechanism by which host factors modulate schistosome development. However, IL-7 is critical for the thymic development of T lymphocytes and their subsequent survival in the periphery, as demonstrated by the severely lymphopenic phenotype of IL-7−/− [10] and IL-7 receptor (IL-7R)−/− [11] mice. We, therefore, hypothesized that the defective schistosome development reported in IL-7−/− mice was the indirect result of impaired T cell development and T lymphopenia in the absence of IL-7 signaling rather than the result of a direct effect that IL-7 has on schistosomes. To test this hypothesis, we examined schistosome infections in mice that lack components of the IL-7R complex (IL-7Rα and common γ [γc] chains) because IL-7 signaling is abrogated in these mice [11, 12] but IL-7 synthesis is intact, as demonstrated by their ability to sustain homeostatic proliferation of adoptively transferred IL-7R–expressing T cells [13]. Our data support the conclusion that the effects that IL-7 has on schistosome development are indeed mediated indirectly via the effects that IL-7 has on T cell homeostasis. Furthermore, our analyses identify an indirect role that another γc cytokine, IL-2, plays in schistosome development. Thus, although expression of Th1 or Th2 phenotypes and their associated effector cytokines by CD4+ T cells does not play a role in schistosome development [5, 14], autocrine production of the T cell growth factor IL-2 by CD4+ T cells is essential to the mechanism by which these cells modulate blood fluke development.

Background Schistosomiasis is a chronic, debilitating parasitic disease infecting more than 200 million people and is second only to malaria in terms of public health importance. Due to the lack of a vaccine, patient therapy is heavily reliant on chemotherapy with praziquantel as the World Health Organization–recommended drug, but concerns over drug resistance encourage the search for new drug leads. Methods and Findings The efficacy of the vinyl sulfone cysteine protease inhibitor K11777 was tested in the murine model of schistosomiasis mansoni. Disease parameters measured were worm and egg burdens, and organ pathology including hepato- and splenomegaly, presence of parasite egg–induced granulomas in the liver, and levels of circulating alanine aminotransferase activity as a marker of hepatocellular function. K11777 (25 mg/kg twice daily [BID]), administered intraperitoneally at the time of parasite migration through the skin and lungs (days 1–14 postinfection [p.i.]), resulted in parasitologic cure (elimination of parasite eggs) in five of seven cases and a resolution of other disease parameters. K11777 (50 mg/kg BID), administered at the commencement of egg-laying by mature parasites (days 30–37 p.i.), reduced worm and egg burdens, and ameliorated organ pathology. Using protease class-specific substrates and active-site labeling, one molecular target of K11777 was identified as the gut-associated cathepsin B1 cysteine protease, although other cysteine protease targets are not excluded. In rodents, dogs, and primates, K11777 is nonmutagenic with satisfactory safety and pharmacokinetic profiles. Conclusions The significant reduction in parasite burden and pathology by this vinyl sulfone cysteine protease inhibitor validates schistosome cysteine proteases as drug targets and offers the potential of a new direction for chemotherapy of human schistosomiasis., A significant reduction in parasite burden and pathology by a vinyl sulfone cysteine protease inhibitor suggests a new direction for chemotherapy of human schistosomiasis., Editors' Summary Background. Schistosomiasis, a disease caused by a type of parasitic flatworm that lives in the blood, infects around 200 million people worldwide. The disease is a serious problem in sub-Saharan Africa, South America, China, and southeast Asia. Although this disease can kill, it is better known as a lifelong chronic infection with debilitating symptoms mainly due to an immune reaction raised against parasite eggs trapped in the liver, spleen, and gut. The worm's life cycle is complicated and involves a free-swimming form that emerges from certain types of snails that live in lakes and ponds. This can penetrate the skin of people in contact with the water. After a period spent in the skin and around the lungs, the parasites move to veins around the gut, and develop into adult worms that mate and lay eggs. These eggs eventually return to the water through the person's feces or urine. A particular group of proteins called cysteine proteases are thought to be very important in the biology of these worms, especially in their function as digestive enzymes in the parasite's gut. These proteases could represent an exciting opportunity for development of new drugs to treat schistosomiasis. The researchers are looking at whether it is possible to block the activity of cysteine proteases and, as a result, kill the worms or prevent them from developing and thriving. Why Was This Study Done? At the moment there is only one drug, praziquantel, in common use for treatment of schistosomiasis; it is cheap and effective. However many organizations are worried about relying on a single drug to treat a serious disease which affects so many people worldwide. The research group here has been looking at molecules that block cysteine protease activity, to see if any of these could be good drug candidates for schistosomiasis. One molecule they have been looking at goes by the name of K11777, which is under evaluation as a drug candidate for another parasitic infection (Chagas' disease). Here, the researchers wanted to find out whether K11777 had any activity against schistosome worms. What Did the Researchers Do and Find? In this study, the researchers deliberately infected laboratory mice with the schistosome parasite. These mice were then either injected with K11777 solution twice daily, or with equivalent volumes of water as a comparison. The researchers examined the effects of injecting K11777 either “early” in infection (using a 14 day course, starting 1 day after infection with the parasite) or “late” in the worms' development (using an 8 day treatment course starting 30 days after infection). The outcomes used as measures of success of treatment with K11777 included the number of worms recovered from mice after euthanasia, the number of worm eggs counted in the liver; the extent of the damage to the liver; and finally, the researchers also looked at activity levels of cysteine proteases in the worms themselves, in particular, those proteases associated with the parasite gut. The results of the early-treatment experiment showed a substantial decrease in worm numbers and egg production. In five of the seven mice treated, eggs were eliminated entirely. Also, there was little measurable liver damage. For the late-treatment experiment, decreased burdens of worms and eggs in the livers of K11777 treated mice were also found, and there was less damage to the livers. Those worms surviving treatment and removed from mice also had much less activity of gut cysteine proteases suggesting that K11777 exerts its effects by targeting worm cysteine proteases. What Do These Findings Mean? These experiments show that K11777 is a potent antischistosomal agent in mice. It might therefore be a good ‘candidate' molecule for developing future treatments for human schistosomiasis. However, before that stage can be reached, it would be important to carry out clinical trials to test whether K11777 is both safe and effective in schistosomiasis patients. Full details as to which worm cysteine protease(s) is the critical target of K11777 would also need to be worked out, and more information would be needed as to whether the dosing plan used in this study (twice-daily injections for a week to 14 days) can be decreased. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040014. World Health Organization pages about schistosomiasis including links to details on further research into the disease Information from the US Centers for Disease Control for patients and health professionals about schistosomiasis Wikipedia pages on schistosomiasis (Wikipedia is an internet encyclopedia anyone can edit) PLoS Neglected Tropical Diseases is a new journal from the Public Library of Science that is devoted to publishing research on the world's most neglected tropical diseases, including schistosomiasis

CD4(+) T cell responses and macrophage activation are essential components of schistosome egg-induced granuloma formation. Previous studies implicated tumour necrosis factor (TNF) as a potential mediator of macrophage recruitment and activation during schistosome infection. Here we demonstrate that signalling by TNF and its receptors can influence granuloma formation, but is ultimately dispensable for granuloma formation in this system. However, we identify a previously unrecognised role for TNF in limiting hepatocellular damage in response to schistosome eggs. Further, we show that this activity of TNF is independent of TNF receptors (TNFR1 and TNFR2). Taken together, these data suggest that additional, as yet unrecognised receptors exist for TNF and that these receptors are capable of mediating important pathological effects in the liver. Finally, we provide evidence that TNF plays an unexpected role in maintaining adult schistosome viability in the portal system.

Development of female schistosomes from infectious cercariae to mature egg-producing adults requires both male schistosomes and an intact adaptive immune system. By examining single sex infections in immunodeficient mice, we provide evidence that female schistosome development is not directly influenced by the adaptive immune system, whereas male development is. Our data are consistent with a sequential model of schistosome development, where the adaptive immune system signals development of mature males, which subsequently stimulate development of mature females. The male schistosome therefore appears to play a central role both in transducing signals from the adaptive immune system and in facilitating female development.

Invasion of skin by schistosome cercariae is facilitated by a serine protease secreted from the acetabular cells of cercariae in response to skin lipid. Specific inhibitors of the protease, when applied to human skin in formulations designed to retain the inhibitor on and in the upper stratum corneum layers, block cercarial invasion of human skin. Both peptide-based, irreversible inhibitors and non-peptide, reversible inhibitors block cercarial invasion when applied in a propylene glycol:isopropyl alcohol (3:1) formulation in vitro. Arrest of cercarial invasion could be achieved even after immersion of treated skin in water for 2 hr. Peptide-based irreversible inhibitors in the presence of three different Topicare Delivery Compounds optimized arrest of cercarial invasion. The three Topicare Delivery Compounds applied alone prevented 80-100% of cercarial invasion. With inclusion of the inhibitor, there was 97-100% inhibition in vitro. The optimal formulation with inhibitor was then applied to the tails of BALB/c mice, and the mice were exposed to 120 cercariae by tail immersion. With the carrier lotion alone, there was a 50% reduction in worm burden and a 70% reduction in egg burden. When inhibitor was included, an 80% reduction in worm burden and a 92% reduction in egg burden was observed.

Background Skin invasion is the initial step in infection of the human host by schistosome blood flukes. Schistosome larvae have the remarkable ability to overcome the physical and biochemical barriers present in skin in the absence of any mechanical trauma. While a serine peptidase with activity against insoluble elastin appears to be essential for this process in one species of schistosomes, Schistosoma mansoni, it is unknown whether other schistosome species use the same peptidase to facilitate entry into their hosts. Methods Recent genome sequencing projects, together with a number of biochemical studies, identified alternative peptidases that Schistosoma japonicum or Trichobilharzia regenti could use to facilitate migration through skin. In this study, we used comparative proteomic analysis of human skin treated with purified cercarial elastase, the known invasive peptidase of S. mansoni, or S. mansoni cathespin B2, a close homolog of the putative invasive peptidase of S. japonicum, to identify substrates of either peptidase. Select skin proteins were then confirmed as substrates by in vitro digestion assays. Conclusions This study demonstrates that an S. mansoni ortholog of the candidate invasive peptidase of S. japonicum and T. regenti, cathepsin B2, is capable of efficiently cleaving many of the same host skin substrates as the invasive serine peptidase of S. mansoni, cercarial elastase. At the same time, identification of unique substrates and the broader species specificity of cathepsin B2 suggest that the cercarial elastase gene family amplified as an adaptation of schistosomes to human hosts., Author Summary Schistosome parasites are a major cause of disease in the developing world, but the mechanism by which these parasites first infect their host has been studied at the molecular level only for S. mansoni. In this paper, we have mined recent genome annotations of S. mansoni and S. japonicum, a zoonotic schistosome species, to identify differential expansion of peptidase gene families that may be involved in parasite invasion and subsequent migration through skin. Having identified a serine peptidase gene family in S. mansoni and a cysteine peptidase gene family in S. japonicum, we then used a comparative proteomic approach to identify potential substrates of representative members of both classes of enzymes from S. mansoni in human skin. The results of this study suggest that while these species evolved to use different classes of peptidases in host invasion, both are capable of cleaving components of the epidermis and dermal extracellular matrix, as well as proteins involved in the host immune response against the migrating parasite.

In this third of a series of studies on experimental dirofilariasis in primate hosts, 3 Macaca cynomolgus, 6 M. speciosa, and 2 M. mulatta were inoculated with infective larvae of Dirofilaria tenuis, a parasite of the raccoon, and were killed at periods ranging from 2 weeks to 13 months of infection. One M. mulatta and one M. speciosa were treated daily with prednisolone beginning 2 months after inoculation; worms were recovered from these and 5 other monkeys (7 of 11). Microfilaremia was detected during the 28th week of infection in a monkey under prednisolone treatment. Microfilaremia was found to be aperiodic in that monkey and in a raccoon. Eosinophilia and antibodies to the microfilaria reached appreciable levels in three monkeys harboring gravid worms. Development of D. tenuis in M. speciosa appeared to be comparable to that observed in man.

The chemoprophylactic use of diethylcarbamazine citrate at total oral doses of 15--180 mg/kg body weight was tested against subperiodic Brugia malayi infection in the leaf monkey (Presbytis melalophos). A total dose of 45 mg/kg body weight given over 9 days killed all developing infective larvae. Similarly, a total dose of 35 mg/kg body weight given over 7 days killed all fourth stage larvae. The minimum effective dose that prevents infection would be 5 mg/kg body weight daily for 7 days every month.