Praziquantel,Mefloquine-Praziquantel,and Mefloquine-Artesunate-Praziquantel against Schistosoma haematobium: A Randomized,Exploratory, Open-Label Trial

Background

Treatment and morbidity control of schistosomiasis relies on a single drug, praziquantel. Hence, there is a pressing need to develop additional therapeutics against schistosomiasis. The antimalarial drug mefloquine shows antischistosomal activity in animal models and clinical trials, which calls for further investigations.

Methodology

We comparatively assessed the efficacy and tolerability of the following treatments against Schistosoma haematobium in school-aged children in Côte d''Ivoire: (i) praziquantel (40 mg/kg; standard treatment); (ii) mefloquine (25 mg/kg) combined with praziquantel (40 mg/kg); and (iii) mefloquine-artesunate (3× (100 mg artesunate +250 mg mefloquine)) combined with praziquantel (40 mg/kg) (treatments administered on subsequent days). Two urine samples were collected before, and on days 21–22 and 78–79 after the first dosing.

Principal Findings

Sixty-one children were present on all examination time points and had complete datasets. No difference in efficacy was observed between the three treatment groups on either follow-up. On the 21–22 day posttreatment follow-up, based on available case analysis, cure rates of 33% (95% confidence interval (CI) 11–55%), 29% (95% CI 8–50%), and 26% (95% CI 5–48%) were observed for praziquantel, mefloquine-artesunate-praziquantel, and mefloquine-praziquantel, respectively. The corresponding egg reduction rates were 94% and above. On the second follow-up, observed cure rates ranged from 19% (praziquantel) to 33% (mefloquine-artesunate-praziquantel), and egg reduction rates were above 90%. Praziquantel monotherapy was the best tolerated treatment. In the mefloquine-artesunate-praziquantel group, adverse events were reported by 91% of the participants, and in the mefloquine-praziquantel group, 95% experienced adverse events. With the exception of abdominal pain at moderate severity, adverse events were mild.

Conclusions/Significance

The addition of mefloquine or mefloquine-artesunate does not increase the efficacy of praziquantel against chronic S. haematobium infection. Additional studies are necessary to elucidate the effect of the combinations against acute schistosomiasis.     

Single Versus Double Dose Praziquantel Comparison on Efficacy and Schistosoma mansoni Re-Infection in Preschool-Age Children in Uganda: A Randomized Controlled Trial

Background

Schistosoma mansoni infection is proven to be a major health problem of preschool-age children in sub-Saharan Africa, yet this age category is not part of the schistosomiasis control program. The objective of this study was to compare the impact of single and double dose praziquantel (PZQ) treatment on cure rates (CRs), egg reduction rates (ERRs) and re-infection rates 8 months later, in children aged 1-5 years living along Lake Victoria, Uganda.

Methodology/Principal Findings

Infected children (n= 1017) were randomized to receive either a single or double dose of PZQ. Initially all children were treated with a single standard oral dose 40 mg/kg body weight of PZQ. Two weeks later a second dose was administered to children in the double dose treatment arm. Side effects were monitored at 30 minutes to 24 hours after each treatment. Efficacy in terms of CRs and ERRs for the two treatments was assessed and compared 1 month after the second treatment. Re-infection with S. mansoni was assessed in the same children 8 months following the second treatment. CRs were non-significantly higher in children treated with two 40 mg/kg PZQ doses (85.5%; 290/339) compared to a single dose (83.2%; 297/357). ERRs were significantly higher in the double dose with 99.3 (95%CI: 99.2-99.5) compared with 98.9 (95%CI: 98.7-99.1) using a single dose, (P = 0.01). Side effects occurred more frequently during the first round of drug administration and were mild and short-lived; these included vomiting, abdominal pain and bloody diarrhea. Overall re-infection rate 8 months post treatment was 44.5%.

Conclusions

PZQ is efficacious and relatively safe to use in preschool-age children but there is still an unmet need to improve its formulation to suit small children. Two PZQ doses lead to significant reduction in egg excretion compared to a single dose. Re-infection rates with S. mansoni 8 months post treatment is the same among children irrespective of the treatment regimen.     

Evaluation of Oxfendazole,Praziquantel and Albendazole against Cystic Echinococcosis: A Randomized Clinical Trial in Naturally Infected Sheep

Background

Cystic Echinococosis (CE) is a zoonotic disease caused by larval stage Echinococcus granulosus. We determined the effects of high dose of Oxfendazole (OXF), combination Oxfendazole/Praziquantel (PZQ), and combination Albendazole (ABZ)/Praziquantel against CE in sheep.

Methodology/Principal Findings

A randomized placebo-controlled trial was carried out on 118 randomly selected ewes. They were randomly assigned to one of the following groups: 1) placebo; 2) OXF 60 mg/Kg of body weight (BW) weekly for four weeks; 3) ABZ 30 mg/Kg BW + PZQ 40 mg/Kg BW weekly for 6 weeks, and 4) OXF 30 mg/Kg BW+ PZQ 40 mg/Kg BW biweekly for 3 administrations (6 weeks). Percent protoscolex (PSC) viability was evaluated using a 0.1% aqueous eosin vital stain for each cyst. “Noninfective” sheep were those that had no viable PSCs; “low-medium infective” were those that had 1% to 60% PSC viability; and “high infective” were those with more than 60% PSC viability. We evaluated 92 of the 118 sheep. ABZ/PZQ led the lowest PSC viability for lung cysts (12.7%), while OXF/PZQ did so for liver cysts (13.5%). The percentage of either “noninfective” or “low-medium infective” sheep was 90%, 93.8% and 88.9% for OXF, ABZ/PZQ and OXF/PZQ group as compared to 50% “noninfective” or “low-medium infective” for placebo. After performing all necropsies, CE prevalence in the flock of sheep was 95.7% (88/92) with a total number of 1094 cysts (12.4 cysts/animal). On average, the two-drug-combination groups resulted pulmonary cysts that were 6 mm smaller and hepatic cysts that were 4.2 mm smaller than placebo (p<0.05).

Conclusions/Significance

We demonstrate that Oxfendazole at 60 mg, combination Oxfendazole/Praziquantel and combination Albendazole/Praziquantel are successful schemas that can be added to control measures in animals and merits further study for the treatment of animal CE. Further investigations on different schedules of monotherapy or combined chemotherapy are needed, as well as studies to evaluate the safety and efficacy of Oxfendazole in humans.     

Praziquantel: a new schistosomicide against Schistosoma haematobium.

The effectiveness of the new schistosomicide praziquantel was assessed in African schoolchildren infected with Schistosoma haematobium. They were stratified according to the severity of their infection and were then randomly allocated to treatment with two single-dose regimens (30 and 40 mg/kg) and a split regimen of two doses of 20 mg/kg given four hours apart. All three regimens were highly effective and produced few side effects. Children who initially had very high pretreatment egg loads showed a poorer therapeutic response at all dose levels, and further investigations are necessary to find the optimum dose. Because of its effectiveness in a single dose and lack of toxicity, praziquantel may prove to be the ideal schistosomicide.     

The zoonotic,fish-borne liver flukes Clonorchis sinensis,Opisthorchis felineus and Opisthorchis viverrini

Clonorchis sinensis, Opisthorchis felineus and Opisthorchis viverrini are the three most important liver flukes involved in human health, infecting more than 45 million people worldwide. Both C. sinensis and O. viverrini, and possibly O. felineus, can induce human cholangiocarcinoma as well as inducing other hepatobiliary pathology. Although the life cycles of all three species are similar, only that of O. felineus in Europe remains predominantly zoonotic, while O. felineus in Asia and C. sinensis have a stronger mixture of zoonotic and anthroponotic components in their life cycles. Opisthorchis viverrini from the Mekong area of southeastern Asia is predominantly anthroponotic. Here we discuss the comparative epidemiology of these three taxa comparing in detail the use of first, second and final animal hosts, and consider the potential role of humans in spreading these pathogens. In addition we discuss the genetic structure of all three species in relation to potentially cryptic species complexes.     

Praziquantel derivatives exhibit activity against both juvenile and adult Schistosoma japonicum

A praziquantel analog 10-hydroxy praziquantel and eight praziquantel/peroxide conjugates were synthesized. The biological activity of these compounds was evaluated against juvenile and adult stages of Schistosoma japonicum. Unlike praziquantel, 10-hydroxy praziquantel exhibits activity against both juvenile and adult Schistosoma japonicumin. All hybrid compounds displayed modest to significant worm killing activity. The present study has important significance for the development of hybrid antischistosomal drugs.     

Opisthorchis viverrini: ultrastructure and cytochemistry of the glycocalyx of the tegument

The ultrastructure and cytochemistry of the glycocalyx of the tegument of Opisthorchis viverrini during maturation from newly excysted juvenile to adult stages were investigated using colloidal iron, ruthenium red and lectin stainings. The results showed that the glycocalyx was intensely stained by the first two dyes, thus indicating the presence of relatively high amounts of negative charges. However, the thickness and intensity of the staining decreased during the fluke's maturation. Binding studies using lectin probes on the surface of adult parasites showed that binding sites for Canavalia ensiformis (Con A), Triticum vulgaris (WGA) and Ricinus communis I (RCA I) were present in relative large amounts on the glycocalyx of the adult tegument, whereas those for Dolichos biflorus (DBA) were relatively fewer in number, and those for Ulex europaeus I (UEA I) were absent. The binding patterns of Con A, WGA, RCA I and DBA were generally similar, and the reaction product was uniformly distributed over the dorsal and ventral surfaces of the parasite's body. These bindings, therefore, indicate the presence of D-mannose/D-glucose, N-acetyl-D-glucosamine/sialic acid, D-galactose and N-acetyl-D-galactosamine residues on the glycocalyx of the adult tegument.     

Specific serum IgG, but not IgA, antibody against purified Opisthorchis viverrini antigen associated with hepatobiliary disease and cholangiocarcinoma

Opisthorchiasis caused by Opisthorchis viverrini infection induces hepatobiliary disease (HBD)-associated cholangiocarcinoma (CCA) via a chronic inflammatory immune response. Here, we evaluated specific IgG and IgA antibodies against different fractions of O. viverrini antigen in residents from an endemic community in Northeast Thailand with varying hepatobiliary abnormalities. Crude somatic O. viverrini antigen was purified into three fractions (viz., P1, P2 and P3) by gel infiltration chromatography and these served as antigens for detection of fluke-specific IgG and IgA antibodies by enzyme-linked immunosorbent assay (ELISA). The results revealed fluke-specific IgG and IgA antibody levels—against these antigens from subjects with O. viverrini-positive HBD—higher than in subjects with O. viverrini-negative HBD. Interestingly, the rank of fluke-specific IgG (and not IgA) antibody levels against crude extract and P1 antigens was CCA > severe HBD > mild HBD > healthy individuals. Purified antigens reduced cross-reactivity with other parasites compared to the crude antigen. Multiple linear regression analysis showed that HBD status was significantly associated with the liver fluke-specific IgG antibody against purified antigens. These results suggest that purified O. viverrini-antigen improves serodiagnosis for the evaluation of opisthorchiasis-associated HBD, and may be useful in the screening of opisthorchiasis in subjects at risk of developing CCA.     

The evaluation of control measures against Schistosoma mekongi in Cambodia by a mathematical model

We constructed a mathematical model for the transmission of Schistosoma mekongi in Cambodia. The simulation of the model will be instrumental in planning schistosomiasis control measures. The model includes two definitive hosts, humans and dogs, as animal reservoirs. Dogs are recognized to play an important role in schistosomiasis transmission in Cambodia. For the purpose of dealing with age-specific prevalence and intensity of infection, the human population was classified into eight age categories in the model. To describe the seasonal fluctuation of the intermediate host population of S. mekongi, the "Post-Spate Survival" hypothesis was adopted for the population dynamics of Neotricula aperta present in the Mekong River. We carried out simulations to evaluate the effect of universal treatment (UT) and targeted mass treatment (TT) with praziquantel on the reduction in prevalence of S. mekongi. The simulations indicated that biyearly UT for 8 years or yearly TT for 5 years after three courses of yearly UT could reduce the prevalence to below 5% when a UT or TT coverage of 85% of inhabitants was achieved. The simulation suggested that the suppression of S. mekongi in Cambodia would be possible by UT or TT with a high coverage rate.     

Synergy of Omeprazole and Praziquantel In Vitro Treatment against Schistosoma mansoni Adult Worms

BackgroundTreatment and morbidity control of schistosomiasis relies on a single drug, praziquantel (PZQ), and the selection of resistant worms under repeated treatment is a concern. Therefore, there is a pressing need to understand the molecular effects of PZQ on schistosomes and to investigate alternative or synergistic drugs against schistosomiasis.MethodologyWe used a custom-designed Schistosoma mansoni expression microarray to explore the effects of sublethal doses of PZQ on large-scale gene expression of adult paired males and females and unpaired mature females. We also assessed the efficacy of PZQ, omeprazole (OMP) or their combination against S. mansoni adult worms with a survival in vitro assay.ConclusionsFunctional analysis of gene interaction networks is an important approach that can point to possible novel synergistic drug candidates. We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against S. mansoni adult worms in vitro when compared with either drug alone.     

Production and characterization of monoclonal antibodies against the excretory-secretory antigen of the liver fluke (Opisthorchis viverrini).

Monoclonal antibodies (MoAb) were produced against a major soluble metabolic product (excretory-secretory, ES) of Opisthorchis viverrini. The latter was obtained in a form of spent culture medium in which the adult flukes had been maintained in vitro. The MoAb produced were exclusively associated with either IgG or IgM isotypes. When screened against a panel of parasite antigens by indirect ELISA, these MoAb exhibited three patterns of reactivity. Approximately 50% of the MoAb were highly specific for O. viverrini and another 25% cross-reacted only with Clonorchis sinensis. The remaining 25% cross-reacted extensively with other parasites. Results from radioimmunoprecipitation and immunoblotting experiments showed all MoAb to react with the 89-kDa glycoprotein. By indirect immunofluorescence, these MoAb reacted almost exclusively with the tegumental surface, tegumental cells, cecum and developing miracidium. Different lines of evidence suggest that these MoAb reacted with different epitopes on the same 89-kDa polypeptide carrier.     

RNA interference targeting cathepsin B of the carcinogenic liver fluke, Opisthorchis viverrini

Functional genomics have not been reported for Opisthorchis viverrini or the related fish-borne fluke, Clonorchis sinensis. Here we describe the introduction by square wave electroporation of Cy3-labeled small RNA into adult O. viverrini worms. Adult flukes were subjected to square wave electroporation employing a single pulse for 20 ms of 125 V in the presence of 50 μg/ml of Cy3-siRNA. The parasites tolerated this manipulation and, at 24 and 48 h after electroporation, fluorescence from the Cy3-siRNA was evident throughout the parenchyma of the worms, with strong fluorescence evident in the guts and reproductive organs of the adult worms. Second, other worms were treated using the same electroporation settings with double stranded RNA targeting an endogenous papain-like cysteine protease, cathepsin B. This manipulation resulted in a significant reduction in specific mRNA levels encoding cathepsin B, and a significant reduction in cathepsin B activity against the diagnostic peptide, Z-Arg-Arg-AMC. This appears to be the first report of introduction of reporter genes into O. viverrini and the first report of experimental RNA interference (RNAi) in this fluke. The findings indicated the presence of an intact RNAi pathway in these parasites which, in turn, provides an opportunity to probe gene functions in this neglected tropical disease pathogen.     

Molecular Characterization of a Tetraspanin from the Human Liver Fluke, Opisthorchis viverrini

Background

The human liver fluke, Opisthorchis viverrini, is designated as a group 1 carcinogen, and is the major risk factor for cholangiocarcinoma in endemic countries throughout Southeast Asia. Proteins in the excretory-secretory products and tegumental surface membranes of the fluke have been proposed to play pivotal roles in parasite survival in the host, and subsequent pathogenesis. These macromolecules are therefore valid targets for the development of vaccines and new drugs to control the infection. Tetraspanins (TSP) are prominent components of the tegument of blood flukes where they are essential for tegument formation, are directly exposed to the immune system, and are major targets for a schistosomiasis vaccine. We propose that similar molecules in the surface membranes of O. viverrini are integral to tegument biogenesis and will be efficacious vaccine antigens.

Methodology/Principal Findings

The cDNA sequence encoding O. viverrini tetraspanin-1 (Ov-TSP-1) was identified and cloned. The Ov-tsp-1gene was isolated from a cDNA library. Ov-tsp-1 mRNA was expressed most highly in metacercariae and eggs, and to a lesser extent in juvenile and adult worms. Immunolocalization with adult flukes confirmed that Ov-TSP-1 was expressed in the tegument and eggs in utero. Western blot analysis of rOv-TSP-1 probed with sera from O. viverrini-infected humans and hamsters indicated that both hosts raise antibody responses against the native TSP. Using RNA interference we silenced the expression level of Ov-tsp-1 mRNA in adult flukes by up to 72% by 10 days after delivery of dsRNA. Ultrastructural morphology of adult worms treated with Ov-tsp-1 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls.

Conclusions/Significance

This is the first report of a tetraspanin from the tegument of a liver fluke. Our data imply that tetraspanins play important structural roles in the development of the tegument in the adult fluke. Potential uses of O. viverrini tetraspanins as novel interventions are discussed.     

Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini(Opisthorchidae,Trematoda)

The antigenic characterizations and serological reactions of human liver flukes, Clonorchis sinensis and Opisthorchis viverrini, were analyzed by immunoblot. The antigenic profiles of the crude extract of Clonorchis contained major proteins of 8, 26-28, 34-37, 43, and 70 kDa, and those of Opisthorchis 34-37, 43, 70, and 100 kDa. Of these, the 8, 26-28 and 34-37 kDa bands of Clonorchis and the 100 kDa of Opisthorchis were major components of each excretory-secretory antigen. The 8 and 26-28 kDa bands were specific to Clonorchis but the 100 kDa of Opisthorchis cross-reacted with the sera of clonorchiasis, and the 34-37, 70 and 100 kDa bands cross-reacted with sera of other helminthiases. The frequency and intensity of the immunoblot reactions were positively correlated with the intensity of the liver fluke infection.     

Schistosoma mekongi and Schistosoma japonicum: Differences in the distribution of eggs in the viscera of mice

The difference in the distribution of Schistosoma eggs in the viscera has not been clearly elucidated in the two closely related species Schistosoma japonicum and Schistosoma mekongi. In this study, we quantitatively compared the distribution of eggs in mice infected with the two species. In S. mekongi-infected mice, 56.6% to 69.4% of total eggs were found in the distal small intestine 9 to 15 weeks after infection, while in S. japonicum-infected mice, 48.8% to 71.8% of eggs were found in the proximal small intestine during the same period. There were significantly more eggs in the liver in mice infected with S. japonicum than in those infected with S. mekongi. The number of adult worms recovered did not differ between the two species during the study period. The total number of eggs laid in the tissues also did not differ between the two species at 12 to 15 weeks postinfection, but in the earlier period the total number of eggs was significantly fewer in S. mekongi-infected than in S. japonicum-infected mice, suggesting the delayed maturation of the former compared with the latter. These results clearly show that S. japonicum and S. mekongi exhibit different oviposition behavior in their hosts.     

Chromosomes and karyotype analysis of a liver fluke, Opisthorchis viverrini, by scanning electron microscopy

Opisthorchis viverrini, a human liver fluke, has been categorized as the carcinogenic organism according to the strong association with carcinogenesis of cholangiocarcinoma (CCA). The infection of this food-borne parasite is a major impact on the health of humans, especially CCA patients in the northeast of Thailand. Taxonomy, morphology, epidemiology and molecular study of O. viverrini have been publicized increasingly but the precise karyotypic study is still incomplete. In this study, the chromosomes of O. viverrini were prepared from the testes of adult worms retrieved from metacercariae infected-hamsters. The chromosomes of O. viverrini were identified in haploid (n=6) meiotic metaphase and in diploid (2n=12) mitotic metaphase by light microscopy. The chromosome number, length and nomenclature of each chromosome were determined by scanning electron microscopy. The six chromosomes consist of one large-sized metacentric, one medium-sized metacentric, two small-sized metacentric, one small-sized submetacentric and one small-sized acrocentric chromosomes with the lengths of 2.84±0.03, 2.12±0.10, 1.71±0.13, 1.44±0.04, 1.23±0.03 and 0.84±0.13 μm, respectively. This is the first karyotype analysis of O. viverrini with defined complete nomenclature.     

Spatial distribution of, and risk factors for, Opisthorchis viverrini infection in southern Lao PDR

Background

Opisthorchis viverrini is a food-borne trematode species that might give rise to biliary diseases and the fatal cholangiocarcinoma. In Lao PDR, an estimated 2.5 million individuals are infected with O. viverrini, but epidemiological studies are scarce and the spatial distribution of infection remains to be determined. Our aim was to map the distribution of O. viverrini in southern Lao PDR, identify underlying risk factors, and predict the prevalence of O. viverrini at non-surveyed locations.

Methodology

A cross-sectional parasitological and questionnaire survey was carried out in 51 villages in Champasack province in the first half of 2007. Data on demography, socioeconomic status, water supply, sanitation, and behavior were combined with remotely sensed environmental data and fed into a geographical information system. Bayesian geostatistical models were employed to identify risk factors and to investigate the spatial pattern of O. viverrini infection. Bayesian kriging was utilized to predict infection risk at non-surveyed locations.

Principal Findings

The prevalence of O. viverrini among 3,371 study participants with complete data records was 61.1%. Geostatistical models identified age, Lao Loum ethnic group, educational attainment, occupation (i.e., rice farmer, fisherman, and animal breeder), and unsafe drinking water source as risk factors for infection. History of praziquantel treatment, access to sanitation, and distance to freshwater bodies were found to be protective factors. Spatial patterns of O. viverrini were mainly governed by environmental factors with predictive modeling identifying two different risk profiles: low risk of O. viverrini in the mountains and high risk in the Mekong corridor.

Conclusions/Significance

We present the first risk map of O. viverrini infection in Champasack province, which is important for spatial targeting of control efforts. Infection with O. viverrini appears to be strongly associated with exposure to the second intermediate host fish, human behavior and culture, whereas high transmission is sustained by the lack of sanitation.     

Ultrasound-Guided Pulsed Radiofrequency for Carpal Tunnel Syndrome: A Single-Blinded Randomized Controlled Study

Objective

We assessed the therapeutic efficiency of ultrasound-guided pulsed radiofrequency (PRF) treatment of the median nerve in patients with carpal tunnel syndrome (CTS).

Methods

We conducted a prospective, randomized, controlled, single-blinded study. Forty-four patients with CTS were randomized into intervention or control groups. Patients in the intervention group were treated with PRF and night splint, and the control group was prescribed night splint alone. Primary outcome was the onset time of significant pain relief assessed using the visual analog scale (VAS), and secondary outcomes included evaluation of the Boston Carpal Tunnel Syndrome Questionnaire (BCTQ) results, cross-sectional area (CSA) of the median nerve, sensory nerve conduction velocity (SNCV) of the median nerve, and finger pinch strength. All outcome measurements were performed at 1, 4, 8, and 12 weeks after treatment.

Results

Thirty-six patients completed the study. The onset time of pain relief in the intervention group was significantly shorter (median onset time of 2 days vs. 14 days; hazard ratio = 7.37; 95% CI, 3.04–17.87) compared to the control group (p < 0.001). Significant improvement in VAS and BCTQ scores (p < 0.05) was detected in the intervention group at all follow-up periods compared to the controls (except for the severity subscale of BCTQ at week 1). Ultrasound-guided PRF treatment resulted in a lower VAS score and stronger finger pinch compared to the control group over the entire study.

Conclusions

Our study shows that ultrasound-guided PRF serves as a better approach for pain relief in patients with CTS.

Trial Registration

ClinicalTrials.gov NCT02217293