Treatment Failure after Multiple Courses of Triclabendazole among Patients with Fascioliasis in Cusco,Peru: A Case Series

Triclabendazole is reported to be highly effective in treatment of human fascioliasis. We present 7 of 19 selected cases of human fascioliasis referred to our center in the Cusco region of Peru that failed to respond to triclabendazole. These were mostly symptomatic adults of both sexes that continued passing Fasciola eggs in the stool despite multiple treatments with 2 doses of triclabendazole at 10 mg/kg per dose. We documented the presence of eggs by rapid sedimentation and Kato Katz tests after each treatment course. We found that repeated triclabendazole courses were not effective against fascioliasis in this group of people. These findings suggest that resistance to triclabendazole may be an emerging problem in the Andes.     

Advances in Fasciola hepatica research using ‘omics’ technologies

The liver fluke Fasciola hepatica is an economically important pathogen of livestock worldwide, as well as being an important neglected zoonosis. Parasite control is reliant on the use of drugs, particularly triclabendazole, which is effective against multiple parasite stages. However, the spread of parasites resistant to triclabendazole has intensified the pursuit for novel control strategies. Emerging 'omics' technologies are helping advance our understanding of liver fluke biology, specifically the molecules that act at the host-parasite interface and are central to infection, virulence and long-term survival within the definitive host. This review discusses the technological sequencing advances that have facilitated the unbiased analysis of liver fluke biology, resulting in an extensive range of ‘omics’ datasets. In addition, we highlight the ‘omics’ studies of host responses to F. hepatica infection that, when combined with the parasite datasets, provide the opportunity for integrated analyses of host-parasite interactions. These extensive datasets will form the foundation for future in-depth analysis of F. hepatica biology and development, and the search for new drug or vaccine interventions.     

Insights into the Interactions of Fasciola hepatica Cathepsin L3 with a Substrate and Potential Novel Inhibitors through In Silico Approaches

Background Fasciola hepatica is the causative agent of fascioliasis, a disease affecting grazing animals, causing economic losses in global agriculture and currently being an important human zoonosis. Overuse of chemotherapeutics against fascioliasis has increased the populations of drug resistant parasites. F. hepatica cathepsin L3 is a protease that plays important roles during the life cycle of fluke. Due to its particular collagenolytic activity it is considered an attractive target against the infective phase of F. hepatica.Conclusions/SignificanceThe present study is the first computer-aided drug design approach against F. hepatica cathepsins. Here we predict the principal determinants of binding of FhCL3 in complex with a natural substrate by detailed energetic characterization of protease interaction surface. We also propose novel compounds as FhCL3 inhibitors. Overall, these results will foster the future rational design of new inhibitors against FhCL3, as well as other F. hepatica cathepsins.     

Liver fluke vaccines in ruminants: strategies,progress and future opportunities

The development of a vaccine for Fasciola spp. in livestock is a challenge and would be advanced by harnessing our knowledge of acquired immune mechanisms expressed by resistant livestock against fluke infection. Antibody-dependent cell-mediated cytotoxicity directed to the surface tegument of juvenile/immature flukes is a host immune effector mechanism, suggesting that antigens on the surface of young flukes may represent prime candidates for a fluke vaccine. A Type 1 immune response shortly after fluke infection is associated with resistance to infection in resistant sheep, indicating that vaccine formulations should attempt to induce Type 1 responses to enhance vaccine efficacy. In cattle or sheep, an optimal fluke vaccine would need to reduce mean fluke burdens in a herd below the threshold of 30–54 flukes to ensure sustainable production benefits. Fluke infection intensity data suggest that vaccine efficacy of approximately 80% is required to reduce fluke burdens below this threshold in most countries. With the increased global prevalence of triclabendazole-resistant Fasciolahepatica, it may be commercially feasible in the short term to introduce a fluke vaccine of reasonable efficacy that will provide economic benefits for producers in regions where chemical control of new drug-resistant fluke infections is not viable. Commercial partnerships will be needed to fast-track new candidate vaccines using acceptable adjuvants in relevant production animals, obviating the need to evaluate vaccine antigens in rodent models.     

The Diterpenoid 7-Keto-Sempervirol,Derived from Lycium chinense,Displays Anthelmintic Activity against both Schistosoma mansoni and Fasciola hepatica

BackgroundTwo platyhelminths of biomedical and commercial significance are Schistosoma mansoni (blood fluke) and Fasciola hepatica (liver fluke). These related trematodes are responsible for the chronic neglected tropical diseases schistosomiasis and fascioliasis, respectively. As no vaccine is currently available for anti-flukicidal immunoprophylaxis, current treatment is mediated by mono-chemical chemotherapy in the form of mass drug administration (MDA) (praziquantel for schistosomiasis) or drenching (triclabendazole for fascioliasis) programmes. This overreliance on single chemotherapeutic classes has dramatically limited the number of novel chemical entities entering anthelmintic drug discovery pipelines, raising significant concerns for the future of sustainable blood and liver fluke control.

Methodology/ Principle Findings

Here we demonstrate that 7-keto-sempervirol, a diterpenoid isolated from Lycium chinense, has dual anthelmintic activity against related S. mansoni and F. hepatica trematodes. Using a microtiter plate-based helminth fluorescent bioassay (HFB), this activity is specific (Therapeutic index = 4.2, when compared to HepG2 cell lines) and moderately potent (LD₅₀ = 19.1 μM) against S. mansoni schistosomula cultured in vitro. This anti-schistosomula effect translates into activity against both adult male and female schistosomes cultured in vitro where 7-keto-sempervirol negatively affects motility/behaviour, surface architecture (inducing tegumental holes, tubercle swelling and spine loss/shortening), oviposition rates and egg morphology. As assessed by the HFB and microscopic phenotypic scoring matrices, 7-keto-sempervirol also effectively kills in vitro cultured F. hepatica newly excysted juveniles (NEJs, LD₅₀ = 17.7 μM). Scanning electron microscopy (SEM) evaluation of adult F. hepatica liver flukes co-cultured in vitro with 7-keto-sempervirol additionally demonstrates phenotypic abnormalities including breaches in tegumental integrity and spine loss.

Conclusions/ Significance

7-keto-sempervirol negatively affects the viability and phenotype of two related pathogenic trematodes responsible for significant human and animal infectious diseases. This plant-derived, natural product is also active against both larval and adult developmental forms. As such, the data collectively indicate that 7-keto-sempervirol is an important starting point for anthelmintic drug development. Medicinal chemistry optimisation of more potent 7-keto-sempervirol analogues could lead to the identification of novel chemical entities useful for future combinatorial or replacement anthelmintic control.     

Resistance-induced changes in triclabendazole transport in Fasciola hepatica: ivermectin reversal effect

Triclabendazole (TCBZ) and albendazole (ABZ) are flukicidal benzimidazole compounds extensively used in veterinary medicine. Although TCBZ has excellent activity against mature and immature stages of the liver fluke, Fasciola hepatica, ABZ action is restricted to flukes older than 12 wk. The intensive use of TCBZ has resulted in the development of resistance. To gain insight into the mechanisms of resistance to TCBZ, the ex vivo diffusion of TCBZ, TCBZ sulfoxide (TCBZSO, the active metabolite of TCBZ), and ABZ into TCBZ-susceptible and -resistant adult flukes was compared. TCBZ-susceptible (Cullompton) and -resistant (Sligo) flukes were incubated in Krebs-Ringer Tris buffer with either TCBZ, TCBZSO, or ABZ (5 nmol/ ml) for 90 min. Drug/metabolite concentrations were quantified by high-performance liquid chromatography. All the assayed molecules penetrated through the tegument of both susceptible and resistant flukes. However, significantly lower concentrations of TCBZ and TCBZSO were recovered within the TCBZ-resistant flukes. In contrast, ABZ entrance into the susceptible and resistant flukes was equivalent. The influx/efflux balance for TCBZ, TCBZSO, and ABZ in susceptible and resistant flukes in the presence or absence of a substrate (ivermectin) of the drug transporter P-glycoprotein was assessed. The ivermectin-induced modulation of P-glycoprotein activity decreased TCBZ efflux from the resistant flukes. Higher concentrations of TCBZ and TCBZSO were recovered from the resistant liver flukes in the presence of ivermectin. Thus, an altered influx/efflux mechanism may account for the development of resistance to TCBZ in F. hepatica.     

Anthelmintics for ruminants

There is now a wide range of potent broad-spectrum anthelmintics for cattle and small ruminants, including the benzimidazoles, the avermectins, tetrahydropyrimidines and imidothiazoles which are highly efficacious against parasite gastroenteritis and bronchitis. Fascioliasis can be controlled with the salicy-lanilide and related phenolic compounds, the benzimidazoles, albendazole and triclabendazole the latter being the first flukicide with excellent activity against all stages of liver fluke infestation.The major developments for anthelmintic therapy have been and will be in the use of intraruminal boluses with either sustained release or pulse release of anthelmintic and other methods for group medication. The implications of such strategies for helminth resistance and individual immunity require further evaluation.     

Triclabendazole Sulfoxide Causes Stage-Dependent Embryolethality in Zebrafish and Mouse In Vitro

Background

Fascioliasis and paragonimiasis are widespread foodborne trematode diseases, affecting millions of people in more than 75 countries. The treatment of choice for these parasitic diseases is based on triclabendazole, a benzimidazole derivative which has been suggested as a promising drug to treat pregnant women and children. However, at the moment, this drug is not approved for human use in most countries. Its potential adverse effects on embryonic development have been scarcely studied, and it has not been assigned a pregnancy category by the FDA. Thus, to help in the process of risk-benefit decision making upon triclabendazole treatment during pregnancy, a better characterization of its risks during gestation is needed.

Methodology

The zebrafish embryo test, a preimplantation and a postimplantation rodent whole embryo culture were used to investigate the potential embryotoxicity/teratogenicity of triclabendazole and its first metabolite triclabendazole sulfoxide. Albendazole and albendazole sulfoxide were included as positive controls.

Principal Findings

Triclabendazole was between 10 and 250 times less potent than albendazole in inducing dysmorphogenic effects in zebrafish or postimplantation rodent embryos, respectively. However, during the preimplantation period, both compounds, triclabendazole and triclabendazole sulfoxide, induced a dose-dependent embryolethal effect after only 24 h of exposure in rodent embryos and zebrafish (lowest observed adverse effect concentrations = 10 μM).

Conclusions/Significance

In humans, after ingestion of the recommended doses of triclabendazole to treat fascioliasis and paragonimiasis (10 mg/kg), the main compound found in plasma is triclabendazole sulfoxide (maximum concentration 38.6 μM), while triclabendazole concentrations are approximately 30 times lower (1.16 μM). From our results it can be concluded that triclabendazole, at concentrations of the same order of magnitude as the clinically relevant ones, does not entail teratogenic potential in vitro during the organogenesis period, but its first metabolite triclabendazole sulfoxide has a high embryotoxic capacity in vitro during the preimplantation stage.     

Piperonyl butoxide enhances triclabendazole action against triclabendazole-resistant Fasciola hepatica

A study has been carried out to determine whether the action of triclabendazole (TCBZ) against the liver fluke, Fasciola hepatica is altered by inhibition of the cytochrome P450 (CYP 450)-mediated drug metabolism pathway. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for these experiments, the basic design of which is given in the paper by Devine et al. (2010a). Piperonyl butoxide (PB) was the CYP P450 inhibitor used. Morphological changes resulting from drug treatment and following metabolic inhibition were assessed by means of transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with PB+TCBZ, but more particularly PB+TCBZ.SO, led to greater changes to the TCBZ-resistant isolate than with each drug on its own, with blebbing of the apical plasma membrane, severe swelling of the basal infolds and their associated mucopolysaccharide masses in the syncytium and flooding in the internal tissues. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis and production of secretory bodies were badly disrupted. The mitochondria were swollen throughout the tegumental system and the somatic muscle blocks were disrupted. With the TCBZ-susceptible Cullompton isolate, there was a limited increase in drug action following co-incubation with PB. The results provide evidence that the condition of a TCBZ-resistant fluke can be altered by inhibition of drug metabolism. Moreover, they support the concept that altered drug metabolism contributes to the mechanism of resistance to TCBZ.     

Fas2-ELISA in the detection of human infection by Fasciola hepatica

Fasciola hepatica has recently emerged as a major pathogen of humans from reports on areas of endemicity and hyper-endemicity for fascioliasis. This situation is aggravated by the lack of standard assays for the screen diagnosis of F. hepatica infection in humans living in endemic areas. Our laboratory has developed an enzyme-linked immunosorbent assay (Fas2-ELISA) based on the capture of IgG antibody by a purified protein Fas2, which is an adult fluke cysteine proteinase. Fas2-ELISA exhibited 95% sensitivity and 100% specificity in 38 individuals infected with F. hepatica diagnosed by finding eggs in stools and 46 serum samples from healthy volunteers. No cross-reaction was observed with 54 serum samples from patients with ten different parasitic infections including the trematodes Paragonimus westermani and Schistosoma mansoni. The high antigenicity of Fas2 is suggested by the fact that antibodies to Fas2 rise rapidly by 1-2 weeks of infection and rise until patency at 8 weeks of infection in experimentally infected alpacas. Field screening for human fascioliasis using Fas2-ELISA and coprology in three endemic locations of the Peruvian Andes resulted in 95.5% sensitivity, 86.6% specificity in a population of 664 children in an age range of 1 to 16 years old. These results provide evidence of the clinical potential of Fas2-ELISA to diagnose fascioliasis in humans exposed to liver fluke infection in endemic areas for this parasite. Fas2-ELISA is currently developed as a standard assay for both field screening for fascioliasis in people living in endemic areas and detecting occasionally F. hepatica infected patients in clinical laboratories.     

Characterisation of a beta-tubulin gene from the liver fluke, Fasciola hepatica

This study represents the first beta-tubulin sequence from a trematode parasite, namely, the liver fluke, Fasciola hepatica. PCR of genomic DNA showed that at least one beta-tubulin gene from F. hepatica contains no introns. A number of amino acids in the primary sequence of fluke tubulin are different from those described previously in various nematode species and the cestode, Echinococcus multilocularis. beta-Tubulin is an important target for benzimidazole anthelmintics, although (with the exception of triclabendazole) they show limited activity against F. hepatica. The amino acid differences in fluke beta-tubulin are discussed in relation to the selective toxicity of benzimidazoles against helminths and the mechanism of drug resistance.     

头孢地尔:新型铁载体头孢菌素抗多重耐药革兰阴性杆菌感染北大核心CSCD

抗菌药物耐药是目前全世界面临的重要公共卫生问题之一,亟需开发有效的广谱抗菌药物以应对多重耐药革兰阴性杆菌的感染。头孢地尔是日本Shionogi公司开发的新型铁载体头孢菌素类抗菌药物。该药物对碳青霉烯耐药的肠杆菌目细菌(carbapenem resistant Enterobacterales,CRE)、铜绿假单胞菌、鲍曼不动杆菌和嗜麦芽窄食单胞菌等具有广谱强效的抗菌活性。该药物克服了革兰阴性杆菌因外膜孔道蛋白表达量下调和主动外排泵表达量上调产生的耐药性,并对丝氨酸酶和金属碳青霉烯酶具有较好的稳定性。该药可用于治疗由革兰阴性杆菌引起的复杂性尿路感染(包括肾盂肾炎)、院内获得性肺炎和呼吸机相关性肺炎。文中通过汇总头孢地尔的化学结构、抗菌作用机制、体外抗菌活性、药代动力学、药效学和临床治疗等信息,展现头孢地尔作为新型铁载体头孢菌素在治疗多重耐药革兰阴性杆菌感染中的应用前景。     

IAP家族分子与肿瘤靶向治疗

凋亡抑制因子(inhibitor of apoptosis proteins,IAPs)是一类高度保守的内源性抗细胞凋亡因子家族,主要通过抑制Caspase活性和参与调节核因子NF-κB的作用而抑制细胞凋亡。细胞抗凋亡机制在肿瘤发生、发展以及肿瘤耐药性形成中发挥重要作用。肿瘤细胞高表达IAPs是导致肿瘤细胞抵抗凋亡的关键。细胞凋亡调控异常与肿瘤细胞耐药密切相关,增强肿瘤细胞对化疗药物的敏感性成为近年来肿瘤治疗的重要策略之一。该文综述了IAP家族蛋白的结构、生物学特性及其作为肿瘤治疗靶点的研究进展。     

Drug targeting in Leishmania donovani infections using tuftsin-bearing liposomes as drug vehicles

The efficacy of sodium stibogluconate against Leishmania donovani infections was markedly enhanced by encapsulating this drug in tuftsin-bearing liposomes. Also, pretreatment of the animals with these liposomes (free of drug) rendered them resistant to this infection, possibly by activating the host's macrophages. These results demonstrate that tuftsin-bearing liposomes besides delivering the drug to the target cells could also enhance the nonspecific resistance against infections, thus offering an additional advantage over the use of tuftsin-free liposomes as drug carriers in leishmania therapy.     

Administration of triclabendazole is safe and effective in controlling fascioliasis in an endemic community of the bolivian altiplano

Background

The Bolivian northern Altiplano is characterized by a high prevalence of Fasciola hepatica infection. In order to assess the feasibility, safety and efficacy of large-scale administration of triclabendazole as an appropriate public health measure to control morbidity associated with fascioliasis, a pilot intervention was implemented in 2008.

Materials and Methods

Schoolchildren from an endemic community were screened for fascioliasis and treated with a single administration of triclabendazole (10 mg/kg). Interviews to assess the occurrence of adverse events were conducted on treatment day, one week later, and one month after treatment. Further parasitological screenings were performed three months after treatment and again two months later (following a further treatment) in order to evaluate the efficacy of the intervention.

Results

Ninety infected children were administered triclabendazole. Adverse events were infrequent and mild. No serious adverse events were reported. Observed cure rates were 77.8% after one treatment and 97.8% after two treatments, while egg reduction rates ranged between 74% and 90.3% after one treatment, and between 84.2% and 99.9% after two treatments. The proportion of high-intensity infections (≥400 epg) decreased from 7.8% to 1.1% after one treatment and to 0% after two treatments.

Conclusion

Administration of triclabendazole is a feasible, safe and efficacious public health intervention in an endemic community in the Bolivian Altiplano, suggesting that preventive chemotherapy can be applied to control of fascioliasis. Further investigations are needed to define the most appropriate frequency of treatment.     

抗菌肽的作用机制、生物活性及应用研究进展

抗菌肽广泛存在于生物界,是辅助生物机体抵抗外来病原体入侵的重要防御分子。抗菌肽不仅能抑制、杀灭多种细菌,而且具有抗真菌、抗寄生虫、抗病毒、抗肿瘤和免疫调节等生物学活性。抗菌肽的作用机制与传统抗生素不同,不仅具有广谱抗微生物作用,而且不易诱导机体产生耐药性,因此,在治疗临床耐药菌株方面具有极大的开发潜力。     

Fascioliasis and Intestinal Parasitoses Affecting Schoolchildren in Atlixco,Puebla State,Mexico: Epidemiology and Treatment with Nitazoxanide

Background

The Atlixco municipality, Puebla State, at a mean altitude of 1840 m, was selected for a study of Fasciola hepatica infection in schoolchildren in Mexico. This area presents permanent water collections continuously receiving thaw water from Popocatepetl volcano (5426 m altitude) through the community supply channels, conforming an epidemiological scenario similar to those known in hyperendemic areas of Andean countries.

Methodology and Findings

A total of 865 6–14 year-old schoolchildren were analyzed with FasciDIG coproantigen test and Lumbreras rapid sedimentation technique, and quantitatively assessed with Kato-Katz. Fascioliasis prevalences ranged 2.94–13.33% according to localities (mean 5.78%). Intensities were however low (24–384 epg). The association between fascioliasis and the habit of eating raw vegetables was identified, including watercress and radish with pronouncedly higher relative risk than lettuce, corncob, spinach, alfalfa juice, and broccoli. Many F. hepatica-infected children were coinfected by other parasites. Entamoeba histolytica/dispar, Giardia intestinalis, Blastocystis hominis, Hymenolepis nana and Ascaris lumbricoides infection resulted in risk factors for F. hepatica infection. Nitazoxanide efficacy against fascioliasis was 94.0% and 100% after first and second treatment courses, respectively. The few children, for whom a second treatment course was needed, were concomitantly infected by moderate ascariasis burdens. Its efficacy was also very high in the treatment of E. histolytica/E. dispar, G. intestinalis, B. hominis, H. nana, A. lumbricoides, Trichuris trichiura, and Enterobius vermicularis. A second treatment course was needed for all children affected by ancylostomatids.

Conclusions

Fascioliasis prevalences indicate this area to be mesoendemic, with isolated hyperendemic foci. This is the first time that a human fascioliasis endemic area is described in North America. Nitazoxanide appears as an appropriate alternative to triclabendazole, the present drug of choice for chronic fascioliasis. Its wide spectrum efficacy against intestinal protozooses and helminthiasis, usually coinfecting liver fluke infected subjects in human endemic areas, represents an important added value.     

Atypical multidrug resistance may be associated with catalytically active mutants of human DNA topoisomerase II alpha

In human cells, atypical drug resistance was previously identified with reduced catalytic activity or nuclear localization efficiency of DNA topoisomerase II alpha (TOP2 alpha). We have shown two etoposide resistant hTOP2 alpha mutants, K798L and K798P confer resistance to etoposide. In this work, we showed these mutants are also resistant against doxorubicin and mAMSA in vivo in the yeast strain ISE2, rad52, top2-4 at the non-permissive temperature. We purified these mutants to characterize the drug resistant mechanism. Purified recombinant proteins were 8- to 12-fold more resistant to etoposide and doxorubicin than wild type TOP2 alpha, and 2-fold more resistant to amsacrine, as measured by accumulation of cleavable DNA. These data show that K798L and K798P may be intrinsically resistant against these drugs in vitro and that this character may confer atypical multidrug resistant phenotype in vivo in yeast.     

In vitro activity of cefamandole, cefoxitin, cefuroxime, and carbenicillin, alone and in combination with aminoglycosides against Serratia marcescens.

Synergistic antibiotic studies were undertaken to compare the effectiveness of two new beta-lactamase resistant cephalosporins, cefamandole, and carbenicillin, with four aminoglycosides against clinical strains of Serratia marcescens. The strains demonstrated various combinations of resistance and/or susceptibility to the antibiotics tested. Tobramycin was the most effective aminoglycoside when used in combination with beta-lactam antibiotics. Carbenicillin and cefamandole demonstrated similar activity with aminoglycosides in synergy experiments. Tobramycin-carbenicillin was found to be the superior pairs as indicated by the total number of strains inhibited. This combination was the only one effective against certain high drug resistant strains and the strain resistant to all four aminoglycosides. Carbenicillin or cefamandole with tobramycin exhibited comparable activity against multiple drug resistant organisms. However, mutants significantly more resistant to cefamandole developed during susceptibility testing. The findings of this study have clinical relevance for treating infections by this formidable pathogen.