Adult triclabendazole-resistant Fasciola hepatica: morphological changes in the tegument and gut following in vivo treatment with artemether in the rat model

A study has been carried out to determine the morphological changes to the adult liver fluke, Fasciola hepatica after treatment in vivo with artemether. Rats were infected with the triclabendazole-resistant Sligo isolate of F. hepatica, dosed orally with artemether at a concentration of 200mg/kg and flukes recovered at 24, 48 and 72h post-treatment (p.t.). Surface changes were monitored by scanning electron microscopy and fine structural changes to the tegument and gut by transmission electron microscopy. Twenty-four hours p.t., the external surface showed minor disruption, in the form of mild swelling of the tegument. The tegumental syncytium and sub-tegumental tissues appeared relatively normal. Forty-eight and seventy-two hours p.t., disruption to the tegumental system increased, with isolated patches of surface blebbing and reduced production of secretory bodies by the tegumental cells being the main changes seen. The gastrodermal cells showed a relatively normal morphology 24h p.t. By 48h, large numbers of autophagic vacuoles and lipid droplets were present. Autophagy increased in magnitude by 72h p.t. and substantial disruption to the granular endoplasmic reticulum was observed. Results from this study show that flukes treated in vivo with artemether display progressive and time-dependent alterations to the tegument and gut. Disruption to the gut was consistently and substantially more severe than that to the tegument, suggesting that an oral route of uptake for this compound predominates. This is the first study providing ultrastructural information on the effect of an artemisinin compound against liver fluk

Electron microscopical study to assess the in vitro effects of the synthetic trioxolane OZ78 against the liver fluke, Fasciola hepatica

Adult Fasciola hepatica were incubated for 48 h in vitro in the synthetic peroxide, OZ78 at a concentration of 100 μg/ml and then prepared for scanning and transmission electron microscopy. There was limited disruption to the external fluke surface, with only slight swelling and blebbing of the interspinal tegument in the midbody and ventral tail regions. By contrast, significant disruption was observed to the ultrastructure of the tegument and subtegumental tissues. There was severe swelling of the basal infolds in the tegumental syncytium and the flooding spread internally to affect the subtegumental tissues. In the tegumental system, there was swelling of the cisternae of granular endoplasmic reticulum and of the mitochondria, with the latter showing signs of breaking down. Autophagic vacuoles and lipid droplets were present and the synthesis of tegumental secretory bodies was much reduced. The gastrodermal cells were severely affected, with swelling and degeneration of the mitochondria and the presence of autophagic vacuoles and lipid droplets. The granular endoplasmic reticulum was swollen and vesiculated and the cells contained few secretory bodies. Both the vitelline and testis follicles showed evidence of extensive cellular disruption and degeneration. This study confirms previous data indicating the potential flukicidal activity of OZ7

Activity of artemether and OZ78 against triclabendazole-resistant Fasciola hepatica

Triclabendazole is the drug of choice against Fasciola hepatica infections in humans and animals. However, parasite resistance against triclabendazole is spreading in the veterinary field, and there are no drugs of comparable activity currently available for the treatment and control of fascioliasis. We investigated the efficacy of single oral doses of artemether and OZ78 against adult triclabendazole-resistant F. hepatica harboured in rats, and compared the results with triclabendazole administered at two different doses. Single oral doses of 100 mg/kg OZ78 and 200 mg/kg artemether resulted in worm burden reductions of 100%. Whereas a single 10 mg/kg dose of triclabendazole achieved a worm burden reduction of only 4.0%, a five-fold higher dose yielded a significant worm burden reduction of 60.9%. However, the lower dose of triclabendazole administered to rats harbouring a triclabendazole-sensitive F. hepatica isolate resulted in a worm burden reduction of 95.3%. Our findings confirm that artemether and OZ78 possess good fasciocidal properties, even against a triclabendazole-resistant F. hepatica isolate, and hence these drugs might become useful in areas where triclabendazole resistance is commo

Efficacy and Safety of Artemether in the Treatment of Chronic Fascioliasis in Egypt: Exploratory Phase-2 Trials

Fasciola hepatica and F. gigantica are two liver flukes that parasitize herbivorous large size mammals (e.g., sheep and cattle), as well as humans. A single drug is available to treat infections with Fasciola flukes, namely, triclabendazole. Recently, laboratory studies and clinical trials in sheep and humans suffering from acute fascioliasis have shown that artesunate and artemether (drugs that are widely used against malaria) also show activity against fascioliasis. Hence, we were motivated to assess the efficacy and safety of oral artemether in patients with chronic Fasciola infections. The study was carried out in Egypt and artemether administered according to two different malaria treatment regimens. Cure rates observed with 6×80 mg and 3×200 mg artemether were 35% and 6%, respectively. In addition, high efficacy was observed when triclabendazole, the current drug of choice against human fascioliasis, was administered to patients remaining Fasciola positive following artemether treatment. Concluding, monotherapy with artemether does not represent an alternative to triclabendazole against fascioliasis, but its role in combination chemotherapy regimen remains to be investigated

Tenofovir in second-line ART in Zambia and South Africa: collaborative analysis of cohort studies

Tenofovir (TDF) is increasingly used in second-line antiretroviral treatment (ART) in sub-Saharan Africa. We compared outcomes of second-line ART containing and not containing TDF in cohort studies from Zambia and the Republic of South Africa (RSA)

Identification of Thioredoxin Glutathione Reductase Inhibitors That Kill Cestode and Trematode Parasites

Parasitic flatworms are responsible for serious infectious diseases that affect humans as well as livestock animals in vast regions of the world. Yet, the drug armamentarium available for treatment of these infections is limited: praziquantel is the single drug currently available for 200 million people infected with Schistosoma spp. and there is justified concern about emergence of drug resistance. Thioredoxin glutathione reductase (TGR) is an essential core enzyme for redox homeostasis in flatworm parasites. In this work, we searched for flatworm TGR inhibitors testing compounds belonging to various families known to inhibit thioredoxin reductase or TGR and also additional electrophilic compounds. Several furoxans and one thiadiazole potently inhibited TGRs from both classes of parasitic flatworms: cestoda (tapeworms) and trematoda (flukes), while several benzofuroxans and a quinoxaline moderately inhibited TGRs. Remarkably, five active compounds from diverse families possessed a phenylsulfonyl group, strongly suggesting that this moiety is a new pharmacophore. The most active inhibitors were further characterized and displayed slow and nearly irreversible binding to TGR. These compounds efficiently killed Echinococcus granulosus larval worms and Fasciola hepatica newly excysted juveniles in vitro at a 20 µM concentration. Our results support the concept that the redox metabolism of flatworm parasites is precarious and particularly susceptible to destabilization, show that furoxans can be used to target both flukes and tapeworms, and identified phenylsulfonyl as a new drug-hit moiety for both classes of flatworm parasites

Development of Functional Genomic Tools in Trematodes: RNA Interference and Luciferase Reporter Gene Activity in Fasciola hepatica

The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite–host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi) reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC). We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA) specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP), and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth parasites. These could facilitate the study of gene function and the identification of relevant targets for intervention in organisms that are by other means intractable. More specifically, these results open new perspectives for functional genomics of F. hepatica, which hopefully can lead to the development of new interventions for fascioliasis

Development and Validation of a Risk Score for Chronic Kidney Disease in HIV Infection Using Prospective Cohort Data from the D:A:D Study

Ristola M. on työryhmien DAD Study Grp ; Royal Free Hosp Clin Cohort ; INSIGHT Study Grp ; SMART Study Grp ; ESPRIT Study Grp jäsen.Background Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. Methods and Findings A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with >= 3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1: 393 chance of developing CKD in the next 5 y in the low risk group (risk score = 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. Conclusions Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.Peer reviewe