Introduction
Fasciolosis, caused by the liver fluke, Fasciola hepatica, is an extremely important disease of livestock in temperate areas of the world. In recent years, the disease has undergone a sharp rise, which has been attributed to climate change. The human form of the disease has also become a major public health problem in several parts of the world. Triclabendazole (TCBZ) (marketed as Fasinex®) has become established as the main drug used to treat fluke infections in ruminants since its introduction in the early 1980s, due to its high efficacy against all stages of infection in the mammalian host. It is now the drug of choice for human fascioliasis as well, marketed as Egaten®. This apparently rosy scenario is being threatened by the development of resistance among fluke populations, first reported in Australia, but now present in several countries in western Europe. The situation is one of concern, given the over-reliance on a single drug and the zoonotic potential of the disease. There is a need to learn more about the activity of TCBZ and the epidemiology of the disease, to counter the threat of resistance.
Following the Second Ken Mott Symposium at EMOP IX in Valencia, 2004, I wrote a review on TCBZ (Fairweather, Reference Fairweather2005). The current review, stemming from last year's Symposium (the Third Ken Mott Symposium) held at EMOP X in Paris, 2008, is intended primarily to provide an update in our understanding of several aspects of TCBZ activity, based largely on research published since 2004. For earlier studies, the reader is referred to my previous review (Fairweather, Reference Fairweather2005) and the review by Keiser et al. (Reference Keiser, Engels, Buscher and Utzinger2005). The main topics that will be discussed below are: TCBZ pharmacokinetics; the mechanisms of action and resistance; biological differences between fluke isolates of known susceptibility to TCBZ; the development of alternative drugs and control strategies; and the use of TCBZ for the treatment of human fascioliasis. Information on the unusual chemical structure and narrow spectrum of activity of TCBZ was covered previously and the information will not be repeated here.
TCBZ pharmacokinetics
The basic pattern of biotransformation of TCBZ in the ruminant host was established by Hennessy et al. (Reference Hennessy, Lacey, Steel and Prichard1987). Briefly, TCBZ is completely removed from the portal blood by the liver and cannot be detected in the plasma. It is oxidized to the sulphoxide (TCBZ.SO) and sulphone (TCBZ.SO2) metabolites, which are the main metabolites present in the plasma. Hydroxylation of TCBZ and its two metabolites takes place in the liver, too, giving rise to the corresponding hydroxy metabolites, which are excreted in the bile (Hennessy et al., Reference Hennessy, Lacey, Steel and Prichard1987). The flavin monooxygenase (FMO) pathway is the main pathway involved in the conversion of TCBZ to TCBZ.SO, while it contributes equally with the cytochrome P450 (P450) enzyme system to the sulphonation of TCBZ.SO to TCBZ.SO2 (Mottier et al., Reference Mottier, Virkel, Solana, Alvarez, Salles and Lanusse2004; Virkel et al., Reference Virkel, Lifschitz, Sallovitz, Pis and Lanusse2006). It has been shown that the rumen microflora are capable of carrying out the sulphoreduction of TCBZ.SO and OH-TCBZ.SO to TCBZ and OH-TCBZ, respectively, suggesting that the rumen can act as a reservoir of TCBZ compounds. This could serve as a slow-release system for the further availability of TCBZ in the digestive tract, from where it could be absorbed and passed to the liver (Virkel et al., Reference Virkel, Lifschitz, Sallovitz, Pis and Lanusse2006). TCBZ can also be oxidized to TCBZ.SO by digestive microflora prior to its absorption or by the intestinal wall during absorption (Mestorino et al., Reference Mestorino, Formentini, Lucas, Fernandez, Modamio, Mariño Hernández and Errecalde2008). It is evident, then, that the mechanisms of TCBZ metabolism are complex, but serve (together with the strong binding to plasma proteins) to maintain active concentrations of TCBZ compounds in the host for considerable periods of time, and this undoubtedly enhances drug efficacy.
A recent study compared the pharmacokinetics of TCBZ in sheep and cattle (Mestorino et al., Reference Mestorino, Formentini, Lucas, Fernandez, Modamio, Mariño Hernández and Errecalde2008). Parameters for TCBZ.SO were similar in the two species, although maximum blood levels were reached later in cattle (30 h as against 22 h in sheep). However, the data were very different for TCBZ.SO2, which reached a higher peak concentration and persisted at higher levels for longer in cattle (Mestorino et al., Reference Mestorino, Formentini, Lucas, Fernandez, Modamio, Mariño Hernández and Errecalde2008).
The studies on enzyme pathways cited above (Mottier et al., Reference Mottier, Virkel, Solana, Alvarez, Salles and Lanusse2004; Virkel et al., Reference Virkel, Lifschitz, Sallovitz, Pis and Lanusse2006) were carried out in vitro with liver microsomes. A follow-up experiment has been conducted by the same group, to determine whether co-administration of TCBZ with metabolic inhibitors would alter the systemic availability of TCBZ metabolites in a natural host (the sheep). Methimazole (MTZ, an FMO inhibitor) did not affect TCBZ disposition kinetics in vivo, although it inhibited both TCBZ.SO and TCBZ.SO2 formation in vitro (Virkel et al., 2009). This may have been due to its rapid elimination from the body after the intravenous administration route used in the study, rather than the more typical oral route. In contrast, co-administration with the P450 inhibitors, piperonyl butoxide (PB) and ketoconazole (KTZ) lead to an increase in the maximum blood level of TCBZ.SO and greater plasma availability of this metabolite. The maximum plasma concentration and bioavailability of TCBZ.SO2 were enhanced following co-administration of TCBZ with PB, but not with MTZ or KTZ (Virkel et al., 2009). So, the experiment showed that it is possible to enhance the availability of TCBZ metabolites, which would extend the exposure of the fluke to the drugs and lead to an improvement in the efficacy of TCBZ.
Triclabendazole is marketed in combination with other anthelmintics and there may be interactions between the drugs that affect its pharmacokinetics. One such combination is TCBZ plus ivermectin. Ivermectin itself has no activity against trematodes such as Fasciola (Shoop et al., Reference Shoop, Ostlind, Rohrer, Mickle, Haines, Michael, Mrozik and Fisher1995), but a recent study has shown how it can affect the disposition of TCBZ and its metabolites (Lifschitz et al., Reference Lifschitz, Virkel, Ballent, Sallovitz and Lanusse2009). Thus, the systemic availability of TCBZ was reduced, but the maximum plasma levels of TCBZ.SO and TCBZ.SO2 were enhanced and the plasma availability of the two metabolites was increased for the first 12 and 24 h, respectively (Lifschitz et al., Reference Lifschitz, Virkel, Ballent, Sallovitz and Lanusse2009). The relevance of this observation to dealing with the problem of drug resistance will be dealt with later in this review.
Impairment of drug metabolism in heavily fluke-infected animals has been advanced as a possible explanation for product failure and mis-diagnosis of TCBZ resistance. This idea has not been tested in livestock, but a study on patients in Egypt has shown that fluke infection did not affect either TCBZ pharmacokinetics or drug efficacy (El-Tantawy et al., Reference El-Tantawy, Salem and Mohammed Safwat2007). The level of infection was not determined, so it is not possible to know how much liver damage can be tolerated before drug metabolism is compromised.
Pharmacodynamics and drug action
The extensive metabolism of TCBZ by the host means that (potentially) the adult fluke is exposed to a number of different forms of TCBZ. Moreover, each of the compounds is capable of entering the fluke via diffusion, entry being closely related to their lipophilicity. TCBZ, TCBZ.SO and TCBZ.SO2 demonstrate a similar ability to diffuse into the fluke and their level of diffusion is higher than that for the corresponding hydroxyl compounds (Mottier et al., Reference Mottier, Virkel, Solana, Alvarez, Salles and Lanusse2004, Reference Mottier, Alvarez, Ceballos and Lanusse2006a).
Entry of TCBZ compounds into the fluke has been shown to take place principally by means of diffusion across the tegument, rather than by oral ingestion, a result that is surprising, perhaps, given the strong binding of the metabolites to plasma proteins. Two approaches have been used to confirm this idea, one pharmacological, one morphological. Both made use of flukes that had been ligatured to prevent oral entry of drug. Following incubation in TCBZ.SO, its concentration in the fluke was similar, irrespective of whether the fluke had been ligatured or not. When an excess of bovine serum albumin (BSA) was added to the incubation medium, in order to allow most of the drug to bind to it, the concentration of TCBZ.SO was reduced (by 85%) in both ligatured and non-ligatured flukes (Mottier et al., Reference Mottier, Alvarez, Ceballos and Lanusse2006a). A parallel morphological study has been carried out, to compare drug-induced changes to the tegument and gut following incubation with TCBZ.SO. Disruption to the tegument, as assessed by scanning electron microscopy (SEM), was similar in ligatured and non-ligatured flukes, indicating that restricting the oral uptake of drug does not affect the ability of TCBZ.SO to enter the fluke and exert its effect (Toner et al., Reference Toner, McConvery, Brennan, Meaney and Fairweather2009). Incubation with TCBZ.SO in the presence of an excess of BSA led to a reduction in the level of tegumental disruption. In all experiments, the gut remained unaffected by TCBZ.SO action, suggesting that the oral uptake of drug plays only a (very) minor role in drug entry (Toner et al., Reference Toner, McConvery, Brennan, Meaney and Fairweather2009). The results of the two studies complement each other.
In terms of drug action, the fluke is known to play a more active role than simply being subject to the passive uptake of drug and its diffusion to the site of action, as it has been shown to be capable of metabolizing TCBZ to TCBZ.SO andTCBZ.SO to TCBZ.SO2 (Mottier et al., Reference Mottier, Virkel, Solana, Alvarez, Salles and Lanusse2004; Robinson et al., Reference Robinson, Lawson, Trudgett, Hoey and Fairweather2004a).
It is clear from what has been described above, that the fluke is exposed to a number of different forms of TCBZ. Since TCBZ.SO is the main metabolite present in both blood plasma and bile, it has been assumed to be the active form of TCBZ and even the only active metabolite. However, after the initial (24–30 h) exposure to TCBZ.SO, the fluke will be exposed to TCBZ.SO2 for a prolonged period of time (Hennessy et al., Reference Hennessy, Lacey, Steel and Prichard1987). Moreover, TCBZ.SO2 has been shown to have some activity in its own right in vivo: it caused a 41% reduction in worm burden against a juvenile fluke infection in sheep (Büscher et al., Reference Büscher, Bowen, Strong and Crowfoot1999). In addition, TCBZ.SO2 has been shown to be capable of binding to fluke tubulin, in a colchicine binding assay (Fetterer, Reference Fetterer1986). A recent study has been carried out in vitro to compare the action of TCBZ, TCBZ.SO and TCBZ.SO2 against F. hepatica. It involved the use of SEM (for surface changes) and transmission electron microscopy (TEM, for internal changes) to determine the relative disruption to the tegument caused by the three compounds (Halferty et al., Reference Halferty, Brennan, Trudgett, Hoey and Fairweather2009). The level of surface disruption induced by the three compounds varied from region to region, and overall was similar, but that caused by TCBZ was slightly greater than that produced by the two metabolites. Internal changes observed were greatest following treatment with TCBZ.SO2 and, while TCBZ.SO was also disruptive, TCBZ was far less disruptive. Combining the results for surface and internal changes, the order of severity of disruption was TCBZ.SO2 >TCBZ.SO>>TCBZ (Halferty et al., Reference Halferty, Brennan, Trudgett, Hoey and Fairweather2009). So, TCBZ.SO2 may well contribute to drug action in vivo and is not the inactive metabolite that it was previously thought to be. It may further disrupt flukes already affected by TCBZ.SO. The hydroxy forms of TCBZ.SO and TCBZ.SO2 have also been shown to be capable of disrupting the tegument of F. hepatica (unpublished observations), so drug action may be the combined effect of several metabolites, rather than being due to a single compound.
Some idea of the time-scale of drug action has been provided by recent studies in sheep. Following treatment of a juvenile (4-week) infection with TCBZ (10 mg kg− 1), flukes were still active at 48 h post-treatment (p.t.) and displayed limited surface disruption, as observed by SEM (Halferty et al., Reference Halferty, Brennan, Hanna, Edgar, Meaney, McConville, Trudgett, Hoey and Fairweather2008). By 72 h p.t., all but one of the recovered flukes were dead and they displayed a range of disruption. In most, there was severe swelling over all the body surface, with areas of tegumental sloughing in the tail region. Other flukes were more severely affected, with more widespread loss of the tegument and exposure of the underlying parenchyma. At 96 h p.t., all the flukes were dead and they were grossly disrupted. The tegument had been totally removed and lesions were present in the basal lamina, exposing the internal tissues (Halferty et al., Reference Halferty, Brennan, Hanna, Edgar, Meaney, McConville, Trudgett, Hoey and Fairweather2008). In adult infections, the posterior end of the fluke's body becomes elongated and exhibits a green discolouration after 72 h p.t.. This phenomenon coincides with the movement of flukes into the gall bladder and their subsequent expulsion from the sheep (personal observations). So, drug action is relatively slow and this would be compatible with a microtubule-based action, rather than one based on energy disruption, for example.
Most of the studies on the mechanism of action of TCBZ have been carried out with TCBZ.SO. The precise mechanism remains to be fully elucidated, but there is more evidence for an action against microtubules and microtubule-based processes than for other possibilities, such as against energy metabolism or neuromuscular co-ordination, for example (for a more complete discussion of the evidence, see Fairweather, Reference Fairweather2005).
Mechanism of resistance
Since the previous review in 2005, another report of TCBZ resistance has been published, from north-west Spain (Alvarez-Sánchez et al., Reference Alvarez-Sánchez, Mainar-Jaime, Pérez-García and Rojo-Vázquez2006). This means that resistance has now been reported in several countries in western Europe, in addition to the original report in Australia in the mid-1990s (for associated references, see Fairweather, Reference Fairweather2005). It should be noted that not all reports, anecdotal or otherwise, have been confirmed by rigorous trials. There is convincing evidence for a number of isolates used in TCBZ studies: the Dutch, Oberon and Sligo isolates (previous references are in Fairweather, Reference Fairweather2005; see also, Keiser et al., Reference Keiser, Utzinger, Vennerstrom, Dong, Brennan and Fairweather2007a; McConville et al., Reference McConville, Brennan, Flanagan, Edgar, Hanna, McCoy, Gordon, Castillo, Hernández-Campos and Fairweather2009a). It is essential that such supporting data are obtained; otherwise, the purported cases could be explained by incorrect (under-) dosing, product failure, reduced metabolism as a result of liver damage, even inadequate and incorrect diagnostic tests. Interestingly, in the Spanish report, the flukes (which we have designated the Leon isolate) were described as being resistant to albendazole and clorsulon (in combination with ivermectin) as well (Alvarez-Sánchez et al., Reference Alvarez-Sánchez, Mainar-Jaime, Pérez-García and Rojo-Vázquez2006). If validated, this would be the first instance of multiple drug resistance in the liver fluke. The Sligo isolate has now been shown to be resistant at three stages of development in the mammalian host: 3 days, 4 weeks and 12 weeks post-infection (Coles et al., 2000; Coles & Stafford, 2001; McCoy et al., Reference McCoy, Fairweather, Brennan, Kenny and Forbes2005; McConville et al., Reference McConville, Brennan, Flanagan, Edgar, Hanna, McCoy, Gordon, Castillo, Hernández-Campos and Fairweather2009a).
Since TCBZ is a benzimidazole compound, there is the assumption that its target is tubulin. Immunocytochemical studies using an anti-tubulin antibody have demonstrated that tubulin immunoreactivity in the tegument of TCBZ-susceptible (TCBZ-S) Cullompton flukes is abolished by treatment with TCBZ.SO, whereas that in TCBZ-resistant (TCBZ-R) Sligo flukes is unaffected (Robinson et al., Reference Robinson, Trudgett, Hoey and Fairweather2002; McConville et al., 2006). Another assumption following on from this has been that mutations in the β-tubulin molecule have led to the development of resistance against TCBZ, as is known to be the case for other benzimidazoles in nematode parasites. In the latter, there are three principal substitutions associated with the presumed drug-binding site: the phenylalanine–tyrosine substitution at position 200, the phenylalanine–tyrosine or histidine substitution at position 167 and the glutamic acid–alanine substitution at position 198 (Wolstenholme et al., Reference Wolstenholme, Fairweather, Prichard, von Samson-Himmelstjerna and Sangster2004; Ghisi et al., Reference Ghisi, Kaminsky and Mäser2007). Six β-tubulin isotypes have been sequenced in the TCBZ-S Cullompton isolate and in the TCBZ-R Sligo and Oberon isolates (Ryan et al., Reference Ryan, Hoey, Trudgett, Fairweather, Fuchs, Robinson, Chambers, Timson, Ryan, Fetwell, Ivens, Bentley and Johnston2008). However, no differences have been detected between the isotypes in the three isolates. Phenylalanine is present at position 167 and glutamic acid at position 198 in all six isotypes; at position 200, tyrosine is present in three, phenylalanine in two and leucine in one of the isotypes (Ryan et al., Reference Ryan, Hoey, Trudgett, Fairweather, Fuchs, Robinson, Chambers, Timson, Ryan, Fetwell, Ivens, Bentley and Johnston2008). The presence of tyrosine at position 200 in TCBZ-S flukes would render the binding site inaccessible to classical benzimidazoles such as albendazole, and this would go some way towards explaining why F. hepatica is refractory to many benzimidazole anthelmintics. In benzimidazole-resistant nematodes, the drug-binding cleft is closed off by the presence of tyrosine at position 200 and this forms the basis of the resistance mechanism (Robinson et al., Reference Robinson, McFerran, Trudgett, Hoey and Fairweather2004b). If TCBZ does target tubulin, its binding site may be in a different position on the tubulin molecule, but this remains to be determined.
While, to date, there is no convincing evidence of a role for tubulin mutations in resistance to TCBZ, there is evidence to indicate that altered uptake and metabolism of TCBZ may be involved. Comparison between the Cullompton (TCBZ-S) and Sligo (TCBZ-R) isolates of F. hepatica has shown that the diffusion of both TCBZ and TCBZ.SO is significantly lower in TCBZ-R than in TCBZ-S flukes (Alvarez et al., Reference Alvarez, Solana, Mottier, Virkel, Fairweather and Lanusse2005; Mottier et al., Reference Mottier, Alvarez, Fairweather and Lanusse2006b). Interestingly, this was not true for the related benzimidazole, albendazole, whose uptake was similar in both isolates (Mottier et al., Reference Mottier, Alvarez, Fairweather and Lanusse2006b). The results suggest that the mechanism is specific to TCBZ and that P-glycoprotein (Pgp)-linked drug efflux pumps may be involved in the resistance mechanism. Overexpression of Pgp has been linked to resistance in nematodes against different classes of anthelmintics (Kerboeuf et al., Reference Kerboeuf, Blackhall, Kaminsky and von Samson-Himmelstjerna2003; Wolstenholme et al., Reference Wolstenholme, Fairweather, Prichard, von Samson-Himmelstjerna and Sangster2004). Experiments with Pgp inhibitors have shown that it is possible to ‘reverse’ the condition of the flukes, from resistant to susceptible. For example, co-incubation with ivermectin increased the uptake of TCBZ and TCBZ.SO in TCBZ-R Sligo flukes to levels comparable to those in TCBZ-S Cullompton flukes (Mottier et al., Reference Mottier, Alvarez, Fairweather and Lanusse2006b). In contrast, ivermectin had no impact on the uptake of albendazole in either TCBZ-S or -R flukes (Mottier et al., Reference Mottier, Alvarez, Fairweather and Lanusse2006b). The consequence of Pgp inhibition to the condition of TCBZ-R flukes has been demonstrated in a separate morphological (SEM) study with another Pgp inhibitor, R(+)-verapamil. Co-incubation of R(+)-verapamil with TCBZ.SO led to severe disruption of the tegument of TCBZ-R (Oberon) flukes, whereas treatment with TCBZ.SO on its own (even at a high concentration) caused minimal changes to the tegumental surface (Fairweather et al., Reference Fairweather, Meaney, Savage, Brennan, Hoey and Trudgett2008). The disruption to the TCBZ-R flukes, which took the form of widespread tegumental sloughing, was greater than that seen in the TCBZ-S Cullompton fluke following treatment with TCBZ.SO. While a change in efflux pump activity may simply represent a non-specific mechanism (although the albendazole result suggests that this is not the case), nevertheless it is likely to play a significant role in the development of resistance.
There is a marked difference in the ability of TCBZ-S and TCBZ-R isolates to metabolize TCBZ. Thus, TCBZ-R (Sligo) flukes have been shown to carry out the metabolism of TCBZ to TCBZ.SO, and TCBZ.SO to TCBZ.SO2, at a significantly higher rate than that achieved by TCBZ-S (Cullompton) flukes (Robinson et al., Reference Robinson, Lawson, Trudgett, Hoey and Fairweather2004a; Alvarez et al., Reference Alvarez, Solana, Mottier, Virkel, Fairweather and Lanusse2005). Methimazole, an FMO inhibitor, had a significantly greater inhibitory effect on TCBZ sulphoxidation in TCBZ-R than -S flukes, reducing it to a level comparable to that in TCBZ-S flukes (Alvarez et al., Reference Alvarez, Solana, Mottier, Virkel, Fairweather and Lanusse2005). By comparison, the cytochrome P450 inhibitor, PB had a lesser effect on TCBZ.SO formation and the effect was similar in the two isolates (Alvarez et al., Reference Alvarez, Solana, Mottier, Virkel, Fairweather and Lanusse2005). These experiments were carried out on microsomal fractions of flukes. A subsequent study on intact flukes in vitro has shown that it is possible to reverse the TCBZ-R condition of a fluke (in this case, the Oberon isolate) by co-incubation of TCBZ with MTZ (Devine et al., Reference Devine, Brennan, Lanusse, Alvarez, Trudgett, Hoey and Fairweather2009). Treatment with either TCBZ or TCBZ.SO on their own resulted in more severe disruption to the TCBZ-S Cullompton isolate than the TCBZ-R Oberon isolate, as visualized by surface changes to the tegument (Devine et al., Reference Devine, Brennan, Lanusse, Alvarez, Trudgett, Hoey and Fairweather2009). Methimazole alone had no effect on either isolate, but when it was included alongside TCBZ or TCBZ.SO, disruption to the Oberon isolate was greater than that to the Cullompton isolate, and greater than that in both isolates after either drug on its own. Severe swelling and blebbing of the tegument occurred all over the body and stripping of the apical plasma membrane was observed in the oral cone and midbody regions (Devine et al., Reference Devine, Brennan, Lanusse, Alvarez, Trudgett, Hoey and Fairweather2009). The study showed the morphological manifestation of what the inhibition of drug metabolism by the fluke can lead to in terms of the whole fluke.
Biological differences between isolates
Studies on the various isolates of F. hepatica have revealed interesting differences between them, in relation to their fitness, which have implications for the spread of resistance in the field. For example, in a snail and rat study on the Oberon (TCBZ-resistant) and Fairhurst (TCBZ-susceptible) isolates, the Oberon isolate was shown to be faster to egg hatch (by 2 days: 12 days as against 14 days); faster to produce cercariae (by 4 days: 49 days as against 53 days); and it produced more cercariae (>4 times as many). Moreover, the metacercariae were more infectious to the rat hosts and the flukes reached patency more quickly (by 11 days: 59 days as against 70 days) (Walker et al., Reference Walker, Hoey, Fletcher, Brennan, Fairweather and Trudgett2006). Across the life cycle, the Oberon isolate could be gaining an approximately 2.5 week advantage over the Fairhurst isolate if the isolates were competing with each other. From egg hatch, it would be infecting the mammalian host ~1 week before the Fairhurst isolate and it would be releasing eggs ~1.5 weeks earlier. This would give it a considerable advantage. The results also indicated that the development of drug resistance by the Oberon isolate has not led to a reduction of fitness by comparison with the Fairhurst isolate. In fact, the data showed that the Oberon isolate maintained a higher level of fitness throughout the life cycle. This goes against the general rule that resistance to benzimidazoles results in reduced fecundity (Maingi et al., Reference Maingi, Scott and Prichard1990), although this rule is not absolute (Kelly et al., Reference Kelly, Whitlock, Thompson, Hall, Campbell, Martin and Lejambre1978; Elard et al., Reference Elard, Sauve and Humbert1998). This is important, because if resistant isolates can maintain fecundity, there will be no reversion to a drug-susceptible status; this idea is supported by field data from The Netherlands, which showed no reversion of TCBZ resistance after a 3-year period when TCBZ was not used for treatment (Borgsteede et al., Reference Borgsteede, Moll, Vellema and Gaasenbeek2005).
A separate comparison has been made between infections of the Cullompton (TCBZ-susceptible) and Sligo (TCBZ-resistant) isolates in sheep. Sligo flukes were smaller than their Cullompton counterparts, but migrated more quickly, reaching the bile ducts 1 week earlier (week 7 post-infection, as against week 8), and they produced eggs more quickly (60 days as against 75 days). On the other hand, Sligo flukes produced relatively fewer eggs (approximately one-third as many as the Cullompton flukes) and they were less infectious to sheep (24% of the metacercarial dose reached maturity, as against 57%) (McConville et al., Reference McConville, Brennan, Flanagan, Edgar, Hanna, McCoy, Gordon, Castillo, Hernández-Campos and Fairweather2009a). The more rapid egg production would be an advantageous quality, but the Sligo isolate appears to have sacrificed a number of physiological attributes in order to survive TCBZ treatment. The Cullompton flukes are known to be aspermic and triploid, so perhaps they can devote more energy to growth, resulting in their larger size (Hanna et al., Reference Hanna, Edgar, Moffett, McConnell, Fairweather, Brennan, Trudgett, Hoey, Cromie, Taylor and Daniel2008). Spermatogenesis in Cullompton flukes does not proceed beyond the primary spermatocyte stage, presumably due to a failure of meiosis. Despite this, Cullompton flukes produce normal-looking eggs which are capable of hatching and undergoing parthenogenic development (Hanna et al., Reference Hanna, Edgar, Moffett, McConnell, Fairweather, Brennan, Trudgett, Hoey, Cromie, Taylor and Daniel2008). Sligo flukes show two different phenotypes: in one, the testis contains fully developed sperm, whereas in the other, spermatogenesis is halted at the spermatid stage, due to the failure of nuclear elongation that leads to sperm formation. The two phenotypes are present in the same animal and cross-fertilization between the two takes place (Hanna et al., Reference Hanna, Edgar, Moffett, McConnell, Fairweather, Brennan, Trudgett, Hoey, Cromie, Taylor and Daniel2008). Other fluke isolates (including the Oberon isolate) undergo full sperm development, are diploid and produce normal eggs (Hanna et al., Reference Hanna, Edgar, Moffett, McConnell, Fairweather, Brennan, Trudgett, Hoey, Cromie, Taylor and Daniel2008). The Cullompton result shows that, in the field, it would be possible for there to be a rapid evolution of clonal populations following selection for resistance. So, the limited data show the variation between fluke populations and this needs to be taken into account when understanding fluke population dynamics and the epidemiology of fascioliasis.
As well as fluke isolates having differing sensitivities to TCBZ, studies have shown that they differ in their response to other anthelmintics. For example, the activity of nitroxynil (a fasciolicide) has been compared against four isolates of F. hepatica: the TCBZ-S Cullompton and Fairhurst isolates and the TCBZ-R Oberon and Sligo isolates. The impact of nitroxynil action was assessed by fine structural changes to the tegument and gut. In terms of the severity of disruption observed, the isolates were ranked in the following order: Cullompton >Sligo >Oberon >Fairhurst (McKinstry et al., Reference McKinstry, Brennan, Halferty, Forbes and Fairweather2007, Reference McKinstry, Halferty, Brennan and Fairweather2009). Interestingly, this ranking does not coincide with their susceptibility to TCBZ, which was: Cullompton >Fairhurst >Oberon >Sligo. The Sligo isolate appears to be particularly susceptible to nitroxynil, whereas the Fairhurst isolate is more refractory. Such variations may need to be taken into account in the field when designing control strategies, although the data on nitroxynil will only apply to adult flukes as it is not active against juveniles.
Dealing with resistance
This topic was discussed in the previous review (Fairweather, Reference Fairweather2005). Strategies include the better use of existing fasciolicides, the use of drug combinations and the development of new drugs.
Use of current drugs
A number of current drugs have been shown to be active against the Sligo TCBZ-R isolate of F. hepatica: albendazole, clorsulon (in combination with ivermectin), closantel, nitroxynil and oxyclozanide (Coles et al., Reference Coles, Rhodes and Stafford2000; Moll et al., Reference Moll, Gaasenbeek, Vellema and Borgsteede2000; Coles & Stafford, Reference Coles and Stafford2001). In terms of the response to albendazole and clorsulon, the data for Sligo is at odds with that for the Leon isolate, which indicated that the isolate was resistant to these compounds. Perhaps the drug status of fluke populations from different geographical regions varies, a point that needs to be taken into account when considering alternative therapies. The value of using existing flukicides would be restricted to treatment of adult fluke infections, as they are not effective against the juvenile stages. However, there is evidence that, because of the perceived (but not necessarily proven) problem of TCBZ resistance, farmers are turning away from the use of TCBZ to older compounds, such as closantel and nitroxynil (Hanna, personal communication).
One way to enhance the efficacy of TCBZ would be to modulate its pharmacokinetics. As described above, this can be achieved by co-administration of TCBZ with metabolic and Pgp inhibitors (Lifschitz et al., Reference Lifschitz, Virkel, Ballent, Sallovitz and Lanusse2009; Virkel et al., Reference Virkel, Lifschitz, Sallovitz, Ballent, Scarcella and Lanusse2009). The feasibility of adopting this approach has been demonstrated in studies on a number of anthelmintics: e.g. albendazole, ivermectin and oxfendazole (Lanusse & Prichard, Reference Lanusse and Prichard1991, Reference Lanusse and Prichard1992; López-Garcia et al., Reference López-Garcia, Torrado, Torrado, Martinez and Bolás1998; Alvinerie et al., Reference Alvinerie, Dupuy, Eeckhoutte and Sutra1999; Sánchez et al., Reference Sánchez, Small, Jones and McKellar2002; Merino et al., Reference Merino, Molina, Garcia, Pulido, Prieto and Alvarez2003; Ballent et al., Reference Ballent, Lifschitz, Virkel, Sallovitz and Lanusse2006, Reference Ballent, Lifschitz, Virkel, Sallovitz and Lanusse2007; see also reviews by Alvarez et al., Reference Alvarez, Merino, Molina, Pulido, McKellar and Prieto2006; Lespine et al., Reference Lespine, Alvinerie, Vercruysse, Prichard and Geldhof2008). More significantly, co-administration of anthelmintic-plus-inhibitor has been shown to lead to greater efficacy against drug-resistant nematodes (Benchaoui & McKellar, Reference Benchaoui and McKellar1996; Molento & Prichard, Reference Molento and Prichard1999). TCBZ is marketed in combination with ivermectin and this combination needs to be examined further, to determine whether it possesses activity against TCBZ-R fluke infections.
Use of drug combinations
Drug combinations are a routine part of parasite control in livestock, often being used to treat mixed infections. For example, TCBZ is marketed with levamisole, oxfendazole, ivermectin and abamectin, to provide fluke and nematode control. Drug combinations are also considered to be the most effective way of slowing down the development of resistance and extending the life span of the drugs (Barnes et al., Reference Barnes, Dobson and Barger1995; Sangster, Reference Sangster2001). This is particularly true if the drugs are from different chemical groupings and possess different mechanisms of action, because this opens up the possibility of producing additive or synergistic effects. Moreover, the latter would permit the use of lower quantities of drugs, with the added advantage of reducing drug residues in host tissues and in the environment. There are reports of synergistic interactions between drugs used for schistosome and soil-transmitted helminth infections in humans (see reviews by Albonico, Reference Albonico2003; Utzinger & Keiser, 2004). Synergistic combinations have also been described for veterinary infections (e.g. Bennet et al., Reference Bennet, Behm, Bryant and Chevis1980; Hopkins & Gyr, Reference Hopkins and Gyr1991). Synergism between TCBZ and clorsulon or luxabendazole (at greatly reduced dose rates) has been demonstrated for F. hepatica (data in Fairweather & Boray, Reference Fairweather and Boray1999). Two recent studies have examined the morphological effects of a TCBZ+clorsulon combination against adult flukes (Meaney et al., Reference Meaney, Allister, McKinstry, McLaughlin, Brennan, Forbes and Fairweather2006, Reference Meaney, Allister, McKinstry, McLauglin, Brennan, Forbes and Fairweather2007). The two drugs have different mechanisms of action, with clorsulon targeting energy metabolism and TCBZ (presumably) microtubules; also, they have different routes of entry into the fluke – clorsulon oral and TCBZ trans-tegumental. The combination of the two drugs at half-normal dose rates induced greater disruption than either drug on its own (at reduced and normal levels). Surface changes observed with the combination treatment were: stripping of the apical plasma membrane in the anterior half of the fluke, spine loss, blebbing and swelling (Meaney et al., Reference Meaney, Allister, McKinstry, McLaughlin, Brennan, Forbes and Fairweather2006). Among the internal changes seen with the combination were a reduction in the production of secretory bodies in the tegumental cells, swelling of the basal infolds and autophagy in the syncytium, flooding of the internal tissues and disruption to the spines (Meaney et al., Reference Meaney, Allister, McKinstry, McLauglin, Brennan, Forbes and Fairweather2007). Such changes are likely to lead to the surface changes just described. The results pointed to additive or synergistic effects of the two drugs when used together and support the concept of employing drug combinations against fluke infections. The studies were carried out on the Cullompton (TCBZ-S) isolate of F. hepatica; it remains to be seen whether the phenomenon can be replicated in TCBZ-R flukes. In a separate study in sheep, no synergism was demonstrated between TCBZ and nitroxynil (at normal dose rates) against juvenile (4-week-old) TCBZ-R (Sligo) flukes (McCoy et al., Reference McCoy, Fairweather, Brennan, Kenny and Forbes2005). The study showed that the Sligo isolate was resistant to TCBZ at a juvenile stage; all other studies have been concerned with the adult fluke.
Development of new drugs
In relation to new compounds, information on the TCBZ derivative, compound alpha, was presented in the previous review. It showed promise as an alternative to TCBZ, since it possesses a spectrum of activity similar to that of TCBZ itself. A number of studies on compound alpha have been carried out since 2005. Treatment of both adult and juvenile TCBZ-S Cullompton flukes in vivo (in sheep) led to progressive disruption over time: the disruption took the form of tegumental loss, degeneration of the sub-tegumental tissues, internal flooding and disruption of the muscle bundles (McConville et al., Reference McConville, Brennan, Flanagan, Edgar, McCoy, Castillo, Hernández-Campos and Fairweather2008, Reference McConville, Brennan, Flanagan, Edgar, Castillo, Hernández-Campos and Fairweather2009b). The most significant changes occurred between 48 and 72 h p.t., indicating a slow action, even though maximum blood levels are reached quite quickly – after only 10–14 h p.t. (Rivero et al., Reference Rivero, Jung, Castillo and Hernández-Campos1998; Ramírez et al., Reference Ramírez, Mayet, Del Rivero, Ibarra-Velarde, Castillo, Hernández-Campos and Jung-Cook2009). The effect was more rapid with juvenile than adult flukes: after 72 h treatment, almost 90% of juvenile flukes were dead, whereas only 23% of adult flukes were dead at this time. However, ~50% of the flukes displayed a discolouration in the midbody region, which coincided with the loss of the tegument (McConville, unpublished observations). Compound alpha causes a significant reduction in tubulin immunostaining in TCBZ-S flukes (McConville et al., 2006), suggesting that it may share a target and mode of action similar to those of TCBZ. Experiments carried out in vitro with adult and juvenile stages of the Sligo TCBZ-R isolate showed that compound alpha affects tegumental structure, and more severely than that induced by TCBZ.SO, although the changes were not accompanied by any loss of tubulin immunoreactivity (McConville et al., 2006, Reference McConville, Brennan, McCoy, Castillo, Hernández-Campos, Ibarra and Fairweather2007). Unfortunately, when tested in vivo (in sheep) against the Sligo isolate, compound alpha treatment did not result in a reduction of fluke burden at 3 days, 4 weeks and 12 weeks post-infection (McConville et al., Reference McConville, Brennan, Flanagan, Edgar, Hanna, McCoy, Gordon, Castillo, Hernández-Campos and Fairweather2009a). So, the in vitro data did not translate into in vivo efficacy. It is possible that the flukes can survive any initial impact of drug action and recover: they are known to be able to mount a stress response to drug action (McConville et al., Reference McConville, Brennan, Flanagan, Edgar, McCoy, Castillo, Hernández-Campos and Fairweather2008; Halferty et al., Reference Halferty, Brennan, Hanna, Edgar, Meaney, McConville, Trudgett, Hoey and Fairweather2008). As a consequence of this result, the potential of compound alpha to replace TCBZ for the treatment of TCBZ-R fluke infections may be limited. A carbamate derivative of compound alpha has been synthesized and shown to possess a high level of efficacy against the gut paramphistome, Calicophoron calicophorum (Reyes et al., Reference Reyes, Ibarra, Vera, Cantó, Hernández, Hernández, Castillo and Villa2008); unfortunately, its activity against flukes has not been tested. Tribendimidine is an anthelmintic that is effective against soil-transmitted helminths and the intestinal trematode, Echinostoma caproni. When tested against the Cullompton (TCBZ-S) isolate of F. hepatica, it had no impact on fluke burdens in rats. Nor was it effective against Schistosoma mansoni, although it showed activity against Clonorchis sinensis and Opisthorchis viverrini (Keiser et al., Reference Keiser, Shu-Hua, Chollet, Tanner and Utzinger2007b).
Recently, there has been an upsurge of interest in making use of natural plant products that have been used as traditional medicines in developing countries (Hammond et al., Reference Hammond, Fielding and Bishop1997; Iqbal et al., Reference Iqbal, Akhtar, Sindhu, Khan and Jabbar2003; Kayser et al., Reference Kayser, Kiderlen and Croft2003; Anthony et al., Reference Anthony, Fyfe and Smith2005; Crump, Reference Crump2006; Stepek et al., Reference Stepek, Lowe, Buttle, Duce and Behnke2007). The marketing of ‘Mirazid’, derived from the myrrh tree Commiphora molmol was discussed in the previous review and the reader is referred to that review for a discussion of the controversy surrounding its use as an anti-schistosomal drug (Fairweather, Reference Fairweather2005). Other natural products with reported efficacy against F. hepatica include extracts of the fern Matteuccia orientalis (Shiramizu et al., Reference Shiramizu, Tuchida and Anu1993); the black-fruited galangal Alpinia nigra (Roy & Tandon, Reference Roy and Tandon1999); the fineleaf fumitory Fumaria parviflora, the nickernut Caesalpinia crista and the black cumin Nigella sativa (Akhtar et al., Reference Akhtar, Iqbal, Khan and Lateef2000); the silk tree Albizia anthelmintica and the soapberry tree Balanites aegyptiaca (Koko et al., Reference Koko, Galal and Khalid2000); the toothache tree Zanthoxylum alatum (Tagboto & Townson, Reference Tagboto and Townson2001); persimmon Albizia anthelmintica, the coral tree Diospyrus, henna Erythrina, Lawsonia and katigua pyta Trichilla (Iqbal et al., Reference Iqbal, Akhtar, Sindhu, Khan and Jabbar2003).
Another natural product is genistein. It is an isoflavone derivative of Flemingia vestita, known as Soh-Phlang in north-east India, and extracts of the plant are eaten raw as a cure for various helminth infections, including that caused by the trematode, Fasciolopsis buski (Rao, Reference Rao1981). The activity of genistein has been tested in vitro against F. hepatica. Incubation of intact flukes at a concentration of 0.27 mg ml− 1 ( = 1 mm) led to a rapid loss of movement (in less than 3 h), while exposure of fluke muscle strips led to significant increases in the frequency and/or amplitude of muscle contractions at concentrations of 10 μm to 10 mm (Toner et al., Reference Toner, Brennan, Wells, McGeown and Fairweather2008). Genistein is believed to affect Ca2+ homeostasis as a result of modulating nitric oxide activity, via changes to cyclic guanosine monophosphate (cGMP) levels (Das et al., Reference Das, Tandon and Saha2007, Reference Das, Tandon, Lyndem, Gray and Ferro2009). Within the short time-frame of 3 h, genistein caused marked surface changes to the tegument of F. hepatica: the changes included widespread blebbing and swelling of the tegument, and spine loss (Toner et al., Reference Toner, Brennan, Wells, McGeown and Fairweather2008). The internal tissues were severely affected, too: there was reduced secretory activity together with autophagy in the tegumental and gastrodermal cells and inhibition of cell development and differentiation in the testis and vitelline follicles (Toner et al., Reference Toner, Brennan, Wells, McGeown and Fairweather2008). In other organisms, genistein is known to inhibit mitosis, induce apoptosis and interfere with signalling pathways, as it is an inhibitor of tyrosine-specific protein kinases. The pathways are present in Schistosoma and Echinococcus and the changes observed in F. hepatica could have a similar basis (for the relevant references on genistein action and signalling in helminths, see Toner et al., Reference Toner, Brennan, Wells, McGeown and Fairweather2008).
Propolis, or bee glue, is a resinous hive product that has been used for a number of medicinal purposes and has anti-protozoal activity (Higashi & de Castro, Reference Higashi and de Castro1994). Incubation of Fasciola gigantica in propolis in vitro led to severe disruption of the surface tegument: there was swelling, blebbing, loss of spines, formation of lesions and (in extreme cases) loss of the tegument (Hegazi et al., Reference Hegazi, Abd El Hady and Shalaby2007a). It was described as being relatively more disruptive than TCBZ itself. A separate study showed that propolis inhibits the development and hatching of fluke eggs (Hegazi et al., Reference Hegazi, Abd El Hady and Shalaby2007b). It is a very complex mixture of components, so it may be extremely difficult to identify the active constituent(s).
Although the results of studies on natural products as discussed above are interesting, it remains to be seen whether their use will make a significant contribution to fluke therapy in the future. Much further evaluation and testing will be required before their true usefulness will be known. It is relatively easy to find compounds that are active in vitro, but less so to translate that to efficacy in vivo.
One group of drugs derived from natural products that has attracted considerable attention in recent years is the artemisinins. Artemisinin itself was originally isolated from the wormwood plant Artemisia annua; extracts of the plant have been used in China for more than 2 millennia as traditional herbal remedies for the treatment of various illnesses (Li & Wu, Reference Li and Wu2003; Woodrow et al., Reference Woodrow, Haynes and Krishna2005). Semi-synthetic derivatives of artemisinin were isolated in the 1970s and are well-established anti-malarial drugs: they include artemether, artesunate, arteether and their principal metabolite, dihydroartemisinin (Borstnik et al., Reference Borstnik, Paik, Shapiro and Posner2002; Woodrow et al., Reference Woodrow, Haynes and Krishna2005). Further modification of the compounds led to the development of the synthetic 1,2,4-trioxolanes, which retain the peroxide moiety essential for antiparasitic activity, yet are simpler molecules, easier to synthesize and have improved pharmacokinetic properties (e.g. greater stability, better absorption, longer half-life). One of the compounds, OZ277, has gone to clinical trials as part of the Medicines for Malaria Venture (Vennerstrom et al., Reference Vennerstrom, Arbe-Barnes, Brun, Charman, Chiu, Chollet, Dong, Dorn, Hunziker, Matile, McIntosh, Padmanilayam, Tomas, Scheurer, Scorneaux, Tang, Urwyler, Wittlin and Charman2004). In addition to their use as anti-malarials, artemisinins are used for the treatment of schistosome infections, especially in combination with praziquantel. The combination is valuable as the artemisinins have activity against juvenile stages, whereas praziquantel targets the adult worms (Xiao, Reference Xiao2005; Utzinger et al., Reference Utzinger, Xiao, Tanner and Keiser2007). Recent studies have shown that artemisinin compounds are active against other trematode parasites, such as C. sinensis, O. viverrini and E. caproni, both in vitro and in vivo in rodent models (Keiser et al., Reference Keiser, Xiao, Jian, Chang, Odematt, Tesana, Tanner and Utzinger2006a, Reference Keiser, Brun, Fried and Utzingerb, Reference Keiser, Utzinger, Tanner, Dong and Vennerstromc, Reference Keiser, Xiao, Dong, Utzinger and Vennerstrom2007c; Shu-Hua et al., Reference Shu-Hua, Jian, Tanner, Yong-Nian, Keiser, Utzinger and Hui-Qiang2008; see also the reviews by Keiser & Utzinger, Reference Keiser and Utzinger2007a, Reference Keiser and Utzingerb). In contrast, artemether (and tribendimidine) lacks activity against Paragonimus westermani (Xue et al., Reference Xue, Utzinger, Zhang, Tanner, Keiser and Xiao2008). Little is known about the activity of artemisinins against tapeworm parasites. In one study, artesunate and dihydroartemisinin (but not artemether) were effective against the protoscoleces of Echinococcus granulosus in vitro, but were ineffective against Echinococcus multilocularis metacestodes in an in vivo mouse model (Spicher et al., Reference Spicher, Roethlisberger, Lany, Stadelmann, Keiser, Ortega-Mora, Gottstein and Hemphill2008).
A number of artemisinin compounds have been tested against F. hepatica, both in vitro and in the rat model: artemether, artesunate and the synthetic trixolane, OZ78 (Keiser et al., Reference Keiser, Utzinger, Tanner, Dong and Vennerstrom2006c, Reference Keiser, Shu-Hua, Tanner and Utzingerd, Reference Keiser, Utzinger, Vennerstrom, Dong, Brennan and Fairweather2007a; Keiser & Morson, Reference Keiser and Morson2008a, Reference Keiser and Morsonb; O'Neill et al., Reference O'Neill, Johnston, Halferty, Brennan, Keiser and Fairweather2009). They displayed high levels of efficacy against both adult and juvenile flukes, with relatively greater activity against the adult stage. The compounds were also shown to be capable of inducing marked changes to the surface tegument. The disruption became progressively more severe over time following treatment in vivo, leading to the death and expulsion of flukes after approximately 72–96 h (Keiser & Morson, Reference Keiser and Morson2008a, Reference Keiser and Morsonb; Keiser et al., Reference Keiser, Utzinger, Tanner, Dong and Vennerstrom2006c, Reference Keiser, Shu-Hua, Tanner and Utzingerd). Of particular interest is that artemether and OZ78 showed an extremely high level of efficacy against the TCBZ-R Oberon isolate in a rodent model (Keiser et al., Reference Keiser, Utzinger, Vennerstrom, Dong, Brennan and Fairweather2007a). If that activity could be repeated in a ruminant animal, this would be a promising breakthrough and the result warrants further investigation.
Greater disruption to the tegument was observed when haemin was incorporated in the culture medium. This result ties in with the idea that activation of artemisinin-type compounds depends on the presence of an iron-containing compound (as would be derived from haemoglobin in vivo). Activation leads to cleavage of the peroxide bridge in the drug and the generation of free radicals. These free radicals are damaging to flukes (Xiao et al., Reference Xiao, Wu, Tanner, Wu, Utzinger, Mei, Scorneaux, Chollet and Zhai2003). It has been suggested that artemisinins need to be ingested by the blood fluke, Schistosoma in order for activation to occur (Xiao et al., Reference Xiao, Wu, Tanner, Wu, Utzinger, Mei, Scorneaux, Chollet and Zhai2003), and this may be true for F. hepatica as well, since it is an haematophagous feeder. In a recent study on the action of artemether against F. hepatica, the gut was seen to be more severely affected than the tegument following treatment in vivo, and this result supports the idea that oral ingestion is the main route of entry into the fluke (O'Neill et al., Reference O'Neill, Johnston, Halferty, Brennan, Keiser and Fairweather2009).
A pilot study in human patients has been carried out in Vietnam, to compare the impact of artesunate and TCBZ on relieving the symptoms (abdominal pain) of fascioliasis. The initial response in the artesunate-treated group was better than in the TCBZ-treated group, but 3 months after treatment the response was lower (Hien et al., Reference Hien, Truong, Minh, Dat, Dung, Hue, Dung, Tuan, Campbell, Farrar and Day2008). The study needs to be followed up by a more rigorous assessment of the efficacy of artemisinin compounds, before any conclusion can be reached as to what role they might play in controlling fluke infections in humans. The extremely high value placed on these compounds in malarial areas may limit their use against other parasites, due to the risk of promoting drug resistance.
Use of TCBZ for the treatment of human fascioliasis
In recent years, fascioliasis has emerged as a major zoonotic disease, with an increase in the number of human cases, and it is a serious health problem in a number of countries (Mas-Coma et al., Reference Mas-Coma, Bargues and Valero2005; WHO, 2007). TCBZ is the drug of choice for treating human fascioliasis: a summary of its use was given in my previous review (Fairweather, Reference Fairweather2005; see also Keiser et al., Reference Keiser, Engels, Buscher and Utzinger2005; WHO, 2007). The success of a selective treatment programme targeted to schoolchildren in Egypt has been presented by Curtale et al. (Reference Curtale, Hassanein and Savioli2005). Despite this success, there was a real concern that Novartis would stop production of Egaten®, the human formulation of TCBZ (Curtale, Reference Curtale2006). Fortunately, that decision was rescinded and Novartis resumed production. Moreover, the company decided to donate 600,000 tablets to WHO, to ensure that the drug was available in endemic countries (Curtale, Reference Curtale2008). Egypt, Iran, Bolivia, Peru, Vietnam, Georgia and the Yemen have benefited from this scheme, made possible by the generosity of Novartis.
TCBZ is also effective against lung infections of human paragonimiasis (Calvopiña et al., Reference Calvopiña, Guderian, Paredes and Cooper2003; Keiser et al., Reference Keiser, Engels, Buscher and Utzinger2005), but showed no activity against the human blood fluke, Schistosoma mansoni in patients co-infected with Fasciola spp. (Barduagni et al., Reference Barduagni, Hassanein, Mohamed, El Wakeel, El Sayed, Hallaj and Curtale2008).
Conclusions
It is fair to say that a substantial amount of work has been carried out since the previous review. We have learned a lot about the pharmacokinetics and pharmacodynamics of TCBZ, although identification of its target molecule remains tantalizingly elusive. Some progress has been made on clarifying the mechanism(s) of resistance and that information may be of use in designing new strategies to deal with resistance to TCBZ. But has the knowledge gained actually led to a greater understanding of these topics? Probably not entirely, not yet, but the results have opened up new lines of enquiry to pursue, to edge us closer to a more complete understanding of TCBZ action in all its facets. In terms of addressing the problem of resistance, evaluation of novel compounds and drug combinations has attracted a lot of interest, but it is uncertain whether this will translate into marketable therapies. However, one area highlighted in the previous review remains neglected and that concerns the development of reliable tests, not just for diagnosis of fluke infection, but for detection of resistance. Until tests are standardized, apparent cases of resistance may continue to be reported that turn out not to be the case at all and the true extent of resistance will remain confused.
Acknowledgements
The work carried out at Queen's University Belfast and by Professor Carlos Lanusse's group in Argentina has been supported by the DELIVER grant (Design of Effective and sustainable control strategies for LIVER fluke in Europe) from the European Union (FOOD-CT-200X-023025). This financial support is gratefully acknowledged.
