Markers for Benzimidazole Resistance in Human Parasitic Nematodes?

1087 Markers for benzimidazole resistance in human parasitic nematodes?

ROGER K. PRICHARD* Institute of Parasitology, McGill University, 21111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, Canada, H9X 3V9

SUMMARY

Benzimidazole (BZ) resistance is widespread and appears to be readily selected in a variety of nematode parasites of animals. There have been reports of a lack of efficacy of BZ anthelmintics against soil transmitted nematode parasites of humans. However, resistance to BZs in nematodes of humans has not been confirmed. It is difficult to perform tests to confirm anthelmintic resistance in humans for a variety of technical and ethical reasons. The use of anthelmintic drugs for the control of helminth parasites in people is increasing massively as a result of numerous programmes to control gastrointestinal nematode parasites in children, the Global Program for the Elimination of Lymphatic Filariasis and other programmes. Many of these programmes are dependent on BZ anthelmintics and this will increase the pressure for resistance development to BZ anthelmintics in nematode parasites of people. We need to perform monitoring for anthelmintic resistance in these programmes and we need new tools to make that monitoring sensitive, inexpensive and practical. There is a real need for DNA-based markers for BZ resistance in nematode parasites of humans. We have a reasonable understanding of the molecular mechanisms and genetics of BZ resistance in some nematode parasites of animals and similar mechanisms are likely to prevail in nematodes of humans. Based on the likelihood that similar single nucleotide polymorphisms (SNPs) will be involved in BZ resistance in human, as in animal nematode parasites, rapid SNP assays have been developed for possible BZ resistance development in Wuchereria bancrofti.

Key words: Benzimidazole anthelmintics, drug resistance, human parasitic nematodes, albendazole, mebendazole, resistance markers.

INTRODUCTION some human nematode parasites, in our understanding of the mechanisms of, and markers for, Benzimidazole (BZ) resistance appeared in veterin- anthelmintic resistance in nematodes and, perhaps ary nematode parasites soon after the introduction of most important, in the adoption of several large the ﬁrst BZ, thiabendazole (Conway, 1964). Much programmes for the mass administration of anthel- work has been done to understand the mechanism mintic drugs to control and/or eliminate a number of of BZ resistance and to develop SNP markers for parasitic diseases. this resistance in veterinary parasites; this has been The BZs albendazole (ABZ) and mebendazole reviewed by Samson-Himmelstjerna et al. (in this (MBZ) are used extensively for the control of soil special issue) and will not be repeated in this dis- transmitted nematodes in people, such as Ascaris cussion. However, the experience in veterinary lumbricoides, the hookworms Ancylostoma duodenale, parasitology suggests that the development of BZ A. ceylanicum and Necator americanus, the whip- resistance in human nematode parasites could occur. worm Trichuris trichiura, strongyloidiasis caused by Markers for BZ resistance, based on similar single Strongyloides stercoralis and the pinworm Enterobius nucleotide polymorphisms (SNPs) as seen in veter- vermicularis. In addition, ABZ is being used against inary parasites, should be developed so that the Trichinella spiralis, the cestodes Taenia spp., possible development and spread of BZ resistance in Hymenolepis nana (Horton, 2000) and hydatid human parasites can be monitored before parasite cysts, the intestinal protozoa Giardia intestinalis control becomes problematic. The status of anthel- and microsporidia such as Enterocytozoon bieneusi mintic resistance in helminths of humans was re- and Encephalitozoon spp. (Farthing, 2006). More viewed by Geerts and Gryseels (2001). Since then, recently, ABZ is being administered widely in com- there have been substantial changes in the extent bination with either diethylcarbamazine (DEC) or of anthelmintic resistance in helminth parasites of ivermectin (IVM) as part of the Global Program animals, in reports of ineﬃcacy to anthelmintics in to Eliminate Lymphatic Filariasis (GPELF). Many hundreds of millions of doses of ABZ have been administered to humans since its development. * Corresponding author: Institute of Parasitology, McGill University, 21111 Lakeshore road, Ste-Anne-de-Bellevue, Given this heavy and increasing use of BZs against Quebec, Canada, H9X 3V9. Tel: +1-514-398-7729. Fax: human parasites in many parts of the world, what +1-514-398-7857. E-mail: [email protected] evidence is there that BZ resistance may have

Parasitology (2007), 134, 1087–1092. f 2007 Cambridge University Press doi:10.1017/S003118200700008X Printed in the United Kingdom R. K. Prichard 1088 developed in some human parasites and what should enable, for example, larval development assays to be be the concerns of public health authorities that it performed. Nor can the parasite that is the principal might develop in the near or medium term future? cause of LF, W. bancrofti, be maintained in any experimental animal host in which anthelmintic response assays could be conducted. Therefore, EVIDENCE FOR BZ RESISTANCE IN HUMAN parasitological evidence of resistance can only be PARASITES obtained by following the response of this parasite to Reduced efficacy of BZ anthelmintics against soil anthelmintics, in humans, over a long period of time. transmitted nematodes has been reported, but not The most important effect of ABZ on W. bancrofti so far confirmed, by biological tests and genomic is the prolonged suppression of reproduction (for analysis (Albonico et al. 2004a). De Clercq et al. approximately 9 months) by the adult parasites (1997) reported a failure of MBZ to eliminate human (Michael et al. 2004). To assess shortening of the hookworm infection in Mali, but did not provide duration of suppression of reproduction, it would be follow up confirmation that the failure was due to necessary to determine blood microfilarial densities resistance. Similarly, Albonico et al. (2003) reported at intervals over this 9 month period. Given that the inefficacy of MBZ against intestinal nematode microfilaraemia is usually periodic (requiring blood infections in people in Zanzibar who had been re- samples to be taken in the night), it would not be peatedly treated. ABZ resistance has been reported trivial to evaluate the parasitological responses to in the intestinal protozoa, Giardia (Upcroft et al. ABZ if resistance is suspected. Furthermore, mod- 1996; Reynoldson et al. 1998). These authors did elling of anthelmintic resistance development and not find that ABZ resistance was associated with a spread in W. bancrofti (Schwab et al. 2006, 2007) codon 200 SNP in b-tubulin, as is commonly seen indicates that frank parasitological evidence of an- in BZ resistance in trichostrongylid nematodes of thelmintic resistance will not be obvious while the livestock. However, in the human filarial nematode, GPELF is active in a region, until treatment stops Wuchereria bancrofti, the Phe200Tyr SNP has been and microfilarial levels bounce back but are refrac- found in b-tubulin and increased dramatically in tory to the existing drug combinations. For these microfilariae obtained from patients in Burkina Faso, reasons, there is a need for molecular markers for after combination treatment with ABZ and IVM anthelmintic resistance in W. bancrofti and for (Schwab et al. 2005). Nevertheless, parasitological monitoring to be conducted as part of the GPELF confirmation of BZ resistance in W. bancrofti is throughout the period of mass drug administration still lacking, although the treated people still had (MDA) as well as, for a period, after MDA has microfilariae in their blood seven days after the stopped. chemotherapy.

SHOULD WE BE CONCERNED ABOUT THE DIFFICULTY OF ANALYZING FOR BZ ANTHELMINTIC RESISTANCE DEVELOPING RESISTANCE IN NEMATODE PARASITES IN NEMATODE PARASITES OF PEOPLE? OF PEOPLE It has been argued that anthelmintic resistance is It is difficult to confirm unequivocally that anthel- unlikely to develop in lymphatic filariae for several mintic resistance occurs in human helminth para- reasons: (a) ABZ and MBZ have been used against sites, particularly in filarial parasites, such as soil transmitted nematodes of humans for many years W. bancrofti, which have no free-living stages and and unequivocal proof of BZ-resistance is still lack- cannot be cultured in model hosts. In veterinary ing in any of them; (b) anthelmintic resistance has medicine, unequivocal evidence of anthelmintic not arisen in animal filarial parasites, such as resistance has usually come from collection of the Dirofilaria immitis, which are biologically closely egg or larval stages in animal faeces, culture to related to the human filarial nematodes; resistance the infective stage and experimental infection of problems in livestock have been in trichostrongylid parasite-naı¨ve hosts with the infective stage, followed nematodes, which are biologically different from by anthelmintic treatment and post treatment nec- the filariae; (c) filarial nematodes have a long life ropsy of the experimentally infected and treated span and a generation interval of about one year, animals. Such strategies for confirmation of anthel- which would slow selection for resistance; (d) for mintic resistance are impossible for several obvious LF, combination MDA with drugs with different reasons with parasites of humans unless the human modes of action are being used (ABZ+IVM, or parasite can be cultured and used to infect model ABZ+DEC) rather than monotherapy; and (e) in animal hosts. In general, this is not possible. contrast to the frequent use of anthelmintics at short It is also difficult to undertake in vitro biological intervals in livestock, the MDA for LF is only once assays for anthelmintic susceptibility for many hu- per year. man helminth parasites. The nematodes that cause It is important to examine these points and lymphatic filariasis (LF) have no free living stages to others which might influence the rate of resistance Benzimidazole resistance in human parasitic nematodes 1089 development in the human filarial nematodes. With While resistance remains to be confirmed in respect to point (a), there are some reports which D. immitis, we cannot take comfort from the recent suggest that resistance may have developed in a few experience in this animal filarial infection. In ad- instances in human soil transmitted nematodes (see dition to these considerations, there is evidence for above). Furthermore, investigators have not been non-random mating in filarial nematodes such as looking for anthelmintic resistance in human nema- W. bancrofti and O. volvulus (Schwab et al. 2007; tode parasites, so we do not really know the extent Bourguinat, C., Pion, S. D. S., Kamgno, J., Gardon, to which it exists, if any. In addition, there has J., Duke, B. O. L., Boussinesq, M. and Prichard, recently been a marked intensification of MDA for R. K., personal communication). BZ resistance in human helminth parasites as seen with the GPELF, parasitic nematodes is usually recessive (Prichard, the FRESH (Focusing Resources on Effective School 2001) and if non-random mating typifies these in- Health, Hygiene & Nutrition) programme launched fections, ABZ resistance will more rapidly develop by WHO, UNICEF, UNESCO, World Bank and than if mating was random, because non-random others in 2000 (see www.schoolsandhealth.org), mating will increase the proportion of the population APOC (African Program for Onchocerciasis Con- that is homozygous (see Schwab et al. 2007 for fur- trol) and many other large programmes. These large- ther discussion). scale MDA programmes in which a high degree of With respect to point (c), it is true that filarial community coverage is sought could be putting nematodes of people have a longer life span (esti- strong pressure on the parasites to develop resist- mates range from 5–8 years for W. bancrofti; 12–15 ance. However, monitoring for drug resistance years for O. volvulus), in the absence of antiparasite has not been a component of these programmes. In chemotherapy, than most soil transmitted nematodes addition, it must be remembered that in the case of of animals or humans. Filariae typically require the human filarial parasites such as W. bancrofti, about 1 year between microfilarial production and there are no free living stages which could provide achieving sexual maturity, compared with a minimal refugia at the time of MDA. Almost all of the parasite prepatent period for, e.g., trichostrongylid parasites population is in the people being treated, with only a of livestock (18 days). However, in reality, tricho- very small portion of the total parasite population in strongylid nematodes of livestock will often only the vector. Thus, almost the whole of the parasite have 1 or 2 generations in a year. The longer life span population will be under drug selection pressure. and longer generation time for the human filariae will This is in contrast to the soil transmitted nematodes tend to slow the selection for anthelmintic resistance. of both humans and animals, where normally much However, it must also be recognized that existing of the total parasitic nematode population, at the time anthelmintics used in MDA programmes are only of treatment, will be on soil as the free-living egg or slightly/moderately macrofilaricidal (see e.g. Duke larval stages. Therefore, the selection pressures for et al. 1990; Michael et al. 2004). The most important resistance are likely to be much greater in the case of effect of MDA against the human filarial parasites is the human filarial nematodes than for soil trans- the prolonged suppression of reproduction by sur- mitted nematodes of humans or animals. viving adult worms. If some adult worms are more Two important considerations are relevant to likely to survive repeated treatment and can resume point (b). First, it is true that there are some bio- reproduction more rapidly than other adult worms, logical differences between the trichostrongylid these more tolerant (resistant?) worms will be pro- nematode parasites of livestock in which anthelmin- ducing progeny for a long time while other more tic resistance has, in many cases, become a crisis, and susceptible adult worms will either be removed or the human filarial and gastrointestinal nematodes. have their reproduction almost totally suppressed However, we use the same anthelmintics and the throughout their life. This could result in a huge available evidence strongly indicates that the re- reproductive advantage for any resistant parasites ceptors for them are similar in the human and live- and very strong selection pressure for resistance, and stock parasites. Furthermore, there are no a priori possibly a much stronger selective effect than in the reasons why similar resistance mechanisms should case of parasites with a short prepatent period and a not be expected in the different parasitic nematodes. shorter life span than the human filariae. Secondly, the belief that no resistance is occurring in In general, the use of combination chemotherapy, the canine filarial nematode D. immitis is unsub- if the different drugs do not share the same mech- stantiated by the lack of surveys designed to detect it. anisms of resistance, should delay the development Despite the fact that IVM is used at a much lower of drug resistance (point (d)). If drug resistance is dose rate as a prophylaxis against canine heartworm entirely associated with changes in the drug receptor than in human filariases, the Food and Drug and the component drugs target different receptors, Administration in the USA is concerned about then their use in combination may be expected to microfilarial breakthroughs in dogs that have been delay the development of resistance to both. on ML prophylaxis (Hampshire, 2005) as possible However, if the mechanism of resistance is in part indicator of anthelmintic resistance development. due to non-receptor related factors, such as changes R. K. Prichard 1090 in the levels of ABC transporters, then the benefits of for IVM resistance in lymphatic filariae could be combination chemotherapy may not be realized, if developed based on understanding IVM resistance, similar changes can affect both drug classes. IVM is and markers for resistance, in other nematodes an excellent substrate for some P-glycoproteins and (see McCavera et al. and Prichard and Roulet, in ABC transporters appear to be involved in IVM this special issue). The possibility of developing resistance (see Prichard and Roulet, in this special markers for DEC resistance is virtually non-existent issue). BZ drugs such as ABZ can also be substrates at this time, as we do not even know how the drug for ABC transporters (Nare et al. 1994; Kerboeuf works. et al. 2003), so it is possible that combination MDA ABZ and MBZ are the main anthelmintics used to with ABZ+IVM could result in enhanced selection control soil transmitted nematodes in humans. As on ABC transporters involved with resistance to both noted above, reports of treatment failures with BZs classes of anthelmintics. In addition, it has recently in these parasites remain to be confirmed. There is a been found that IVM selects on b-tubulin in both critical need to develop assays to monitor for an- O. volvulus and H. contortus (Eng and Prichard, thelmintic resistance in soil transmitted nematodes 2005; Eng et al. 2006; Bourguinat, C., Pion, S. D. S., (Albonico et al. 2004a). An in vitro test based on an Kamgno, J., Gardon, J., Duke, B. O. L., Boussinesq, assessment of drug effects on the mobility of human M. and Prichard, R. K., personal communication), hookworm (Ancylostoma spp. and N. americanus) and which is known to be the receptor for ABZ. Strongyloides spp. infective-stage larvae was devel- Therefore, it is possible that combination chemo- oped to allow an estimate to be made of anthel- therapy with ABZ+IVM may accelerate anthelmintic responsiveness (Kotze et al. 2004). However, mintic resistance selection rather than delay it. While such an assay would not readily work with parasites this is speculative at this stage, it should be noted that such as A. lumbricoides and T. trichiura, for which the the frequency of the Phe200Tyr SNP in b-tubulin in infective stage is an egg. Recently, the hamster has W. bancrofti microfilariae increased dramatically been proposed as a laboratory animal model to with one or two rounds of this combination treatment examine anthelmintic resistance in human hook- in patients in Burkina Faso (Schwab et al. 2005). worms. Hamsters were infected with N. americanus Finally, annual MDA for LF should impose less and dosed with ABZ, MBZ and other anthelmintic selection pressure for anthelmintic resistance than drugs (Xue et al. 2005). These biological tests would the more frequent treatment regimens which for anthelmintic resistance in some soil transmitted are often used in livestock (point (e)). It should be nematodes of people will be useful. Nevertheless, noted that anthelmintic resistance has occurred molecular markers for BZ resistance in soil trans- rapidly in parasites of sheep even in cases in which mitted nematodes are desirable and would enable only two treatments per year were used (Besier and monitoring to be more easily undertaken. In an at- Love, 2003). This occurred in a situation of a low tempt to determine whether the SNPs in b-tubulin level of refugia, the situation that prevails in LF which cause BZ resistance in nematodes also occur when MDA is applied. Furthermore, as noted above, in T. trichiura and hookworms, this gene was the anthelmintics used for LF MDA exert a pro- sequenced from several isolates (Bennett et al. 2002; longed effect over several months, whereas with Albonico et al. 2004b). The resistance associated livestock parasites the main effect is the rapid killing SNPs was not detected in the relatively small of all of the parasitic stages. The prolonged and number of worms sequenced. However, a more continuous effect of ABZ, IVM (and DEC) against expansive investigation of these SNPs, coupled to W. bancrofti could be expected to exert a very high field egg count monitoring and laboratory biological level of selection pressure for resistance, possible assays for resistance, is needed. Molecular assays exceeding that experienced when anthelmintics are similar to those developed for livestock parasites (see used several times a year against soil transmitted Samson-Himmelstjerna et al. in this special issue) or nematodes. for W. bancrofti (Schwab et al. 2005) are needed for For all of these compelling reasons, it would soil transmitted nematode parasites of humans. appear to be folly not to be concerned about anthelmintic resistance development in lymphatic filariae CONCLUSION and not to monitor for resistance as part of the GPELF. However, as discussed above, it is not There is a serious risk that BZ resistance will develop easy to monitor, parasitologically, for anthelmintic and spread in parasitic nematodes of humans as a resistance in lymphatic filariae. For these reasons, result of intensification of BZ anthelmintic use as- DNA-based assays for anthelmintic resistance are sociated with larg-scale parasite control programmes urgently needed. Schwab et al. (2005) have reported such as the GPELF, FRESH and others. Virtually Phe200Tyr and Phe167Tyr SNP assays, using either no monitoring for anthelmintic resistance is being Real Time PCR or Pyrosequencing, to monitor for undertaken, and it has been predicted that a resist- the genetic changes most commonly associated with ance problem in lymphatic filariae, for example, ABZ resistance in many nematodes. Monitoring would not become obvious, without appropriate Benzimidazole resistance in human parasitic nematodes 1091 monitoring, until MDA stopped. 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