INTRODUCTION

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In recent years, several reports of apparent failures in the treatment of human schistosomes and nematodes have been published (33, 81, 116, 132). Although the interpretation and the implications of these studies are still being debated, they have led to an increased awareness of the potential problem of anthelmintic resistance (AR) in the treatment and control of human helminths.

In view of the short but worrying history of AR in livestock, such concerns are not superfluous. At present, AR is the most important disease problem of the sheep-farming industry in Australia, South Africa, and possibly South America (140, 146, 147). Twenty years ago, however, many scientists considered drug resistance in livestock helminths an unimportant phenomenon. High prevalences of AR, often exceeding 50%, have now been reported in all parts of the world for gastrointestinal helminths of sheep, goats, and horses kept in industrial livestock systems. Surprisingly, up to now very few problems with AR have been noticed in cattle helminths (58). Table 1 summarizes the helminth species and the anthelmintic classes most frequently involved.

Even multiple drug resistance is not uncommon in helminths of veterinary importance. In parts of Paraguay (95) and South Africa (140), helminths are resistant to all available broad-spectrum anthelmintics and farmers have started to give up sheep farming because of insurmountable problems with AR (138).

For purposes of discussion, AR is defined as a heritable reduction in the sensitivity of a parasite population to the action of a drug. The reduction is expressed as the decrease of the frequency of individual parasites affected by exposure to the drug, compared to the frequency observed in the same population upon initial or prior to exposure (31). Although not unequivocal but generally considered the most adequate, this definition encompasses two biologically distinct but not always distinguishable processes: (i) existing drug-tolerant parasite lines may become more frequent, particularly under drug pressure, and (ii) previously susceptible parasites may undergo genetic mutations, possibly induced by drug exposure, and be selected under drug pressure.

The term "tolerance" refers to the innate unresponsiveness of a parasite to a drug, independent of prior exposure to that drug or to others belonging to the same class.

In advancing the cause for the widespread use of drugs to control human helminths, Cerami and Warren (20) believed that "helminths are less likely to develop resistance or would do so more slowly" compared to other infectious agents because they multiply at a lower rate. This assumption has certainly not appeared valid for livestock helminths, justifying caution in the treatment of human helminths as well. AR may not be a medical problem yet, but for all we know the few reports so far may represent only the tip of an iceberg. Veterinary experiences have shown that the problems becomes apparent only when it is too late and reversion to susceptibility is no longer possible (31). Individual treatment failures may often remain unnoticed, since most helminth infections lead only to subclinical disease. Epidemiologically, there have been few efforts so far to examine or monitor the problem. The development of drug resistance, and AR in particular, usually follows a sigmoidal pattern: a long period of incubation with only a few scattered cases is followed by a sudden explosion of the problem (145). Once AR becomes apparent, it may very quickly become a major problem in both clinical and preventive medicine.

For more than a decade, veterinary researchers have drawn the attention of the medical community to the risk of AR development in human helminths, such as schistosomes and hookworms (26, 28, 62, 128). Drawing from the lessons and errors in their own field, they urged medical workers to use anthelmintics more carefully in order to avoid or at least to delay the development of AR. Nevertheless, the widespread drug use for the control of schistosomiasis, onchocerciasis, and geohelminths has been increasingly advocated by scientists and international organizations, with drug companies willing to offer assistance (1, 17, 113, 151). In light of these issues, in this paper we critically review the available evidence for drug resistance of human helminths at present and discuss the prospects for the future, taking the veterinary experiences into account.

Early reports on possible resistance to santonin in Ascaris lumbricoides (86) and diethylcarbamazine (DEC) in Onchocerca volvulus (143) were not well documented and cannot be assessed for accuracy and relevance. In this section, we concentrate on the more recent and better documented reports on AR of human nematodes (hookworms) and trematodes (schistosomes). AR of human cestodes has not yet been reported. Also, livestock cestodes do not seem to develop drug resistance easily; only a single report of drug resistance in tapeworms of sheep (Moniezia expansa) has been published (144).

Use of anthelmintics. The main drugs used to treat human nematodes nowadays are mebendazole, albendazole, pyrantel pamoate, and levamisole for intestinal nematodes, ivermectin (IVM) for onchocerciasis, and DEC alone or DEC-albendazole and IVM-albendazole combination treatments for filariasis (1, 35, 154). Depending on local epidemiology, availability, and cost, these drugs have been widely available in most health care systems for the curative treatment of clinical cases for many years. In addition, the use of anthelmintics is now being strongly advocated in a preventive, population-based way as well (1, 17, 113, 151, 155). It is estimated that some 1.3 to 2.0 billion people in the world suffer from helminth infections. Although direct mortality is low, intestinal helminth infections are believed to contribute to "general morbidity." Both intestinal helminths and schistosomiasis have been associated with anemia, stunted growth, poor nutritional status, and reduced physical and intellectual abilities (17, 18, 151); onchocerciasis has been associated with severe itching, skin diseases, poor health, and even reduced chances for marriage. By providing single-dose anthelminthics on a regular basis to entire populations or high-risk groups (such as schoolchildren and pregnant women), it is hoped to reduce both morbidity and transmission. It has even been proposed to combine albendazole, IVM, and praziquantel (PZQ) at a low dose in a single tablet and to distribute it to virtually all school-age children in the developing world (148, 149). The proponents of these strategies recognize the risk of emergence of AR but usually judge it to be insignificant. As mentioned above, veterinary experiences dictate otherwise. The recently published reports on AR in human helminths must thus be taken seriously, yet examined critically.

Problems of defining drug resistance in hookworms. It should first be noted that complete cure of hookworm infection (and most other helminth infections for that matter) is usually not achieved with any drug. Depending on the dosage and the coprological method applied (with lack of standardization and control methods being a noteworthy problem), cure rates as low as 61% (400 mg) and 67% (800 mg) for albendazole, 0% (single dose) and 23% (repeated dose) for levamisole, 30% (single) and 37% (repeated) for pyrantel pamoate, 27% for thiabendazole, 19% (single) and 45% (repeated) for mebendazole have been reported (35, 88).

Reports of drug resistance in hookworms. Two recent publications have invoked AR as the probable cause of failure of anthelmintic treatment of human hookworms. Both are community-based studies in field conditions, not clinical observations. De Clercq et al. (33) described a failure of mebendazole to treat N. americanus in Mali, whereas Reynoldson et al. (116) reported poor efficacy of pyrantel pamoate against A. duodenale in northwestern Australia. The salient features of both reports are summarized in Table 2. The authors mentioned other possible causes of reduced drug sensitivity of the hookworms such as a genetic change in the susceptibility of the local strain of hookworms (i.e., not through selection pressure by the drug) or host factors (such as local diets) which might have altered the pharmacodynamic properties of the drug. However, some features which were present in one or both localities are suggestive of possible drug resistance. Since regions in Mali and Australia are remote, relatively isolated areas with probably a rather limited influx of infected foreigners, local helminth populations may have been isolated with little dilution or replenishment by (susceptible) helminths from elsewhere. Under these circumstances, AR would develop more rapidly, because of the lack of influx of susceptible genotypes (2).

Use of antischistosomal drugs. Praziquantel (PZQ) is the most common drug for the treatment of human schistosomiasis (32, 89, 155), since it is active against all the Schistosoma species (Schistosoma mansoni, S. haematobium, S. japonicum, S. intercalatum, and S. mekongi). In the field, particularly in community treatment, the usual dosage is 40 mg/kg of body weight in a single dose; higher dosages or split regimens result in lower compliance (89). In hospitalized patients, particularly for S. japonicum and S. mekongi, and for heavy infections with the other species, the recommended dose is 30 mg/kg, up to three times daily, for two consecutive days (32, 35, 89). The drug is safe, with few or limited side effects; in heavy infections with S. mansoni, acute abdominal cramps and bloody diarrhea are frequent but always transient. CR with 40 mg/kg are usually between 70 and 90%; ERR are above 90% (32, 71, 89).

Reports on resistance to schistosomicides. As with nematodes, it should first be noted that CR and ERR in therapeutic trials with any drug for human schistosomes rarely reach 100%, even in situations where reinfection is excluded (32, 71). Moreover, reported cure rates considerably overestimate real CR. Many light infections (with EPGs below the detection limit of the coprological techniques) that persist after treatment are not detected by the usual diagnostic methods but require repeated or very sensitive examinations (37, 70). Thus, the recommended doses of schistosomicides should be considered subcurative (41). In light of these data, it is safe to assume that in schistosome populations, some individual parasites are tolerant to the drug to some degree, at least at the usual dosages.

High treatment frequency. Barton (6) and Martin et al. (98, 99) have shown in well-controlled trials that a high treatment frequency selects for resistance more strongly than do less frequent dosing regimens. There is also strong evidence that resistance develops more rapidly in regions where animals are dewormed regularly. Serious problems with AR in Haemonchus contortus were reported in some humid tropical areas where 10 to 15 treatments per year were used to control this parasite in small ruminants (42).

Single-drug regimens. Often a single drug, which is usually very effective in the first years, is continuously used until it no longer works. In a survey of sheep farmers in the United States, Reinemeyer et al. (112) found that one out of every two flocks were dosed with a single anthelmintic until it failed. Long-term use of levamisole in cattle also led to the development of resistance, although the annual treatment frequency was low and cattle helminths seem to develop resistance less easily than do worms in small ruminants (58, 61). Frequent use of IVM without alternation with other drugs has also been reported as the reason for the fast development of resistance in H. contortus in South Africa and New Zealand (127, 139). In the light of these data, the frequent and continuous use of single drugs such as albendazole for the control of intestinal helminths, IVM for onchocerciasis, or PZQ for schistosomiasis in humans may raise concern. The quickness with which AR to BZ in livestock nematodes has spread is described above; if similar strategies are to be applied in humans, there is no reason why the same problems would not arise as well.

Targeting and timing of mass treatment. Prophylactic mass treatments of domestic animals have certainly contributed to the widespread development of AR in helminths. Although no data are available from experimental studies, computer models (5) indicate that the development of resistance is delayed when 20% of the flock is left untreated. This approach would ensure that the progeny of the worms surviving treatment will not consist only of resistant worms. Given the well-known overdispersed distribution of helminths, leaving part of the group untreated, especially the members carrying the lowest worm burdens, should not necessarily reduce the overall impact of the treatment.

Underdosing. Underdosing is generally considered an important factor in the development of drug resistance, because subtherapeutic doses might allow the survival of heterozygous resistant worms (128). Several laboratory experiments have shown that underdosing indeed contributes to the selection of resistant or tolerant strains (43, 78). Some indirect field evidence further supports this assumption. Recently, it was shown that the bioavailability of BZ and levamisole is much lower in goats than in sheep and that goats should be treated with dosages 1.5 to 2 times higher than those given to sheep (77). For many years, however, sheep and goats were given the same anthelmintic doses. The fact that AR is very frequent and widespread in goats may be a direct consequence. Recent modeling exercises suggest that the field situation of AR is not always as simple (129). Depending on the initial frequency of the resistance alleles, there might be a range of dose levels where underdosing promotes resistance and a range of dose levels where it actually impedes resistance.

Benzimidazoles. The best known mechanism of resistance is the one to BZ. No information is available about the resistance mechanisms present in BZ-resistant human hookworms, but veterinary helminthologists have studied BZ resistance of H. contortus in detail. The BZ exert their anthelmintic activity by binding to -tubulin, which interferes with the polymerisation of the microtubuli. Several authors (9, 120) showed that there is an extensive polymorphism of the -tubulin gene in susceptible H. contortus populations. Roos et al. (120) proved that selection for resistance to BZ is accompanied by a loss of alleles at the locus of -tubulin isotype 1. Kwa et al. (91) nicely demonstrated that resistance to BZ is correlated with a conserved mutation at amino acid 200 in -tubulin isotype 1 (with Phe being replaced by Tyr).

Levamisole. Levamisole and the related anthelmintics pyrantel and morantel are cholinergic agonists with a selective action on nematode receptors. The mechanism of resistance to levamisole has not yet been elucidated. Sangster (122) thoroughly reviewed the pharmacology of levamisole resistance. It is thought to be caused either by a reduction of the number of nicotinic acetylcholinesterase receptors or by a decreased affinity of these receptors for the drug. Hoekstra et al. (79) were able to clone the gene Hca 1, encoding the nicotinic acetylcholinesterase receptor from H. contortus. Although polymorphism at the amino acid level could be demonstrated, these authors could not find evidence that alleles at this locus were involved in selection for resistance to levamisole. A similar gene, tar-1, was identified on the X chromosome in T. colubriformis (150). However, although statistical comparison of allele frequencies from individual male and female worms was consistent with sex linkage of tar-1, no correlation was found with levamisole resistance status.

Ivermectin. IVM and other macrocyclic lactones affect gastrointestinal nematodes by causing starvation and/or paralysis by opening chloride channels, which are thought to be associated with alfa-subunits of glutamate-gated ion channels located on muscles of the pharynx and possibly the somatic musculature (122). Rohrer et al. (117) compared IVM-resistant and -susceptible H. contortus populations and found that resistance is not due to an alteration in the binding of IVM to glutamate gated chloride channel receptors. Nevertheless, Blackhall et al. (13) did report that one allele of the putative alfa-subunit gene is associated with resistance to the drug. Recently, Blackhall et al. (12) reported considerable genetic variation of a P-gp locus in H. contortus. In several drug-selected strains of the parasite, selection for the same allele was observed. Using different approaches, Xu et al. (158) and Sangster et al. (124) came to the conclusion that P-gp might be involved in resistance to IVM in this helminth species. Other mechanisms of resistance may be present as well, as suggested by Gill et al. (64) and Gill and Lacey (65). The latter described five possible types of resistance to IVM in H. contortus based on different behavior in in vitro tests (larval development assay and L3 motility tests), different sensitivity to paraherquamide (an anthelmintic with a completely different structure and different binding sites from IVM), and different inheritance (in at least two of the five resistance types). Gill and Lacey (65) also suggested that the mechanism of resistance to IVM might be different from one species of helminth to another, because the critical events leading to expulsion have been shown to be different, e.g., when O. ostertagi is compared to H. contortus and T. colubriformis. Further research is needed to confirm these observations, to which the relevance to human O. volvulus is at present not clear.

Antischistosomal drugs (oxamniquine and praziquantel). The mechanism of action of oxamniquine is closely associated with its irreversible inhibition of nucleic acid synthesis in schistosomes (23). Based on cross-breeding experiments using susceptible and drug-selected schistosome strains exhibiting stable resistance, Cioli et al. (24) suggested that oxamniquine is not bioactivated in resistant worms, allowing them to survive the drug action. The activating enzyme, which is present in sensitive and absent in resistant schistosomes, seems to be a sulfotransferase. There is no clear understanding of the mode of action of PZQ, which also hampers the elucidation of possible mechanisms of resistance to PZQ. Redman et al. (111) have reviewed the existing knowledge and consider the PZQ-induced Ca²⁺ influx across the tegument as vital in the effect of this drug. However, the mechanisms leading to this alteration in Ca²⁺ homeostasis are not clear at all (22).

Nematodes. Nematode parasite populations are genetically heterogeneous and thus able to respond to selective pressures, i.e., anthelmintic drugs (67). Widespread drug pressure will favor and select parasite lines carrying tolerance or resistance alleles. The rate at which resistance spreads in the parasite population depends on many factors. One key factor is the proportional contribution that helminths surviving therapy will make to the next generation. This contribution is influenced by the drug pressure (frequency and timing of treatment), the drug efficacy, the gene flow (the introduction of susceptible genotypes from elsewhere), the generation time and fecundity of the worms, the frequency of resistance alleles prior to drug use, the number of genes involved, and the dominance or recessiveness of these genes. Since it is quite difficult to set up experiments to examine the influence of these different factors, several mathematical models have been developed to simulate the development of AR in gastrointestinal helminths (5, 63, 128, 129). Although these models have their limitations and must certainly be interpreted with caution (39), models such as the one of Barnes et al. (5) concerning Trichostrongylus colubriformis in grazing sheep provide interesting insights. The model allowed up to three genes for drug resistance, each with two alleles, that were combined independently under random mating. Worms of all genotypes were assumed to be equally fit in the absence of the anthelmintic. The initial frequency of resistance alleles in the worm population was assumed to be very low and was set at 0.01%. To examine the effect of using either mixtures of two drugs or rotations of a single one, two independent genes for resistance to two drugs (with different mechanisms of action) were simulated, with resistance being codominant and each drug killing 99, 50, and 10% of worms of homozygous susceptible (SS), heterozygous (RS), and homozygous resistant (RR) genotypes, respectively. The simulations were run for a period of 20 years, with treatment once a year for the ewes and three times a year for the lambs. These resulted in little development of resistance when the two drugs were used together (mixture). Substantial resistance, however, developed for all rotation strategies, 1-, 5-, and 10 yearly, with slowest development of AR in the annual rotation strategy. Assuming equal initial drug efficacy and equal resistance allele frequency, resistance developed more rapidly if it was determined by a single gene than when two or more genes were involved. Furthermore, resistance evolved fastest when it was dominant, slower when it was codominant, and slowest when it was recessive. When 20% of the flock was never treated, resistance was delayed at the expense of worm control.

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Trematodes. The genetics of resistance of schistosomes to oxamniquine are quite well known, but this is not the case for PZQ. In contrast to the development of classical drug resistance in helminths, which spreads gradually through a population as a consequence of selection of resistant phenotypes present at low frequency, resistance to hycanthone-oxamniquine appeared universally in the first filial progeny of parasites exposed to the drug (16). This strongly suggests that resistance is induced rather than selected from preexisting forms (16). The crossbreeding experiments of Cioli et al. (23, 25) and Pica-Mattocia et al. (107) have clearly shown that oxamniquine resistance is controlled by a single autosomal recessive gene. Resistance to oxamniquine does not appear to spread easily within communities but, rather, tends to remain limited to individual cases. According to Cioli et al. (25), this could be due to a selective disadvantage of resistant schistosomes in the absence of drug pressure. The fact that resistance is induced rather than selected might also contribute to this phenomenon.

Fecal egg count reduction test. The most commonly used test to detect problems of anthelmintic resistance is the fecal egg count reduction test (FECRT), which compares the egg count before and after treatment with an anthelmintic drug. A standardized protocol for the FECRT is available for the detection of anthelmintic resistance in nematodes of veterinary importance (27). In small ruminants, fecal samples are taken from two groups of at least 15, preferably young animals, which have been bred on the farm and not treated in the previous 8 to 12 weeks. Animals are randomly distributed into a treatment and a control group. Fecal samples are collected 10 to 14 days after treatment. To reduce the workload, no pretreatment samples may be taken; it has been shown that comparing treatment and control groups posttreatment is as reliable as comparing pre- and posttreatment samples. Egg counts are performed using a standardized McMaster method (27). The EPG in the feces of the control group should be higher than 150 to allow valid comparison. The following formula is used to calculate the percent reduction of the EPG: ERR = 100(1  X_t/X_c), where X is the arithmetic mean EPG and c and t indicate the control and treated groups respectively. According to the guidelines of the WAAVP, drug resistance in helminths of small ruminants is considered to be present when ERR <95% and the lower 95% confidence interval is below 90%. If only one of both criteria is met, resistance is suspected (27).

(ii) Parasitological methods. A standardized egg-counting technique should be used to determine individual egg counts. For schistosomes, Ascaris and Trichuris, the Kato technique can be used in a standard way, as described by Katz et al. (83), Peters et al. (106), or Polderman et al. (109). Slides should preferably be stored for later reference and quality control.

Laboratory tests for detection of resistance in livestock helminths. A variety of different laboratory tests have been described for the detection of AR in livestock helminths (31). Those which are most commonly used and which might be applied to detect AR in human helminths are briefly described here.

(i) Egg hatch test. The egg hatch test is an in vitro test, which is used only for the detection of BZ resistance in livestock helminths; it is based on the ovicidal activity of this group of molecules. The original test was described by Le Jambre (94); a standardized protocol was adopted by the WAAVP (27). Freshly collected fecal samples (within 3 h of being shed) are needed to obtain reliable data. If this is not possible, samples must be stored anaerobically; this storage does not influence the outcome of the test, at least for the major gastrointestinal helminths of small ruminants (80). Helminth eggs are purified and incubated with a series of concentrations of thiabendazole (TBZ). This compound was selected because it dissolves readily in dimethyl sulfoxide and because side resistance is usually present with other members of the BZ group. After 24 h, the number of hatched larvae is counted. When resistance develops, the ovicidal activity decreases, which results in a higher percentage of eggs that hatch. Based on vast experience with the test, WAAVP considers resistance to be present when the 50% effective dose is 0.1 µg/ml (27). This in vitro test has the advantage of requiring only one fecal sample. However, several authors have reported poor correlations between the results of the FECRT and the egg hatch test for helminths of livestock (14, 42).

(ii) Larval development assay. The larval development assay is more laborious and time-consuming than the egg hatch test but allows the detection of resistance to the major broad-spectrum anthelmintic classes, including the avermectins-mylbemycins. It was originally described by Coles et al. (30) and further improved by several others (66, 93) and is now commercially available (DrenchRite; Horizon Technology). In the larval development assay, nematode eggs or L1 larvae are exposed to different concentrations of anthelmintics incorporated into agar wells in a microtiter plate. The effect of the drugs on the subsequent development into L3 larvae is measured. The results correlate well with those of in vivo tests. It is claimed that this test is more sensitive than FECRT and egg hatch test and detects AR when about 10% of the worm population carries resistance genes (40), but this remains to be proven.

(iii) Larval motility or paralysis test. Several in vitro assays to detect resistance to BZ, macrocyclic lactones or levamisole-morantel have been described which are based on the motility of larvae (31). For the latter group of anthelmintics, a clear-cut distinction between susceptible and resistant strains is not always possible (60, 142). A similar motility test has been used to evaluate the sensitivity of O. volvulus microfilariae to ivermectin (135). To render the interpretation more objective, a micromotility meter has been developed (11). Folz et al. (55, 56) used this apparatus to detect drug resistance in H. contortus and T. colubriformis, but other authors have found it less reliable (142; S. Geerts, unpublished results).

(iv) PCR. The first specific primers to detect drug-resistant parasitic nematodes were developed by Kwa et al. (91). These primers discriminated between heterozygous and homozygous BZ-resistant H. contortus for the alleles in question (-tubulin isotype 1), even when these genotypes are phenotypically indistinguishable, and could also identify BZ-resistant T. colubriformis. According to Roos et al. (120), PCR detected 1% of resistant individuals within a susceptible worm population, a tremendous improvement over other in vivo and in vitro tests.

Laboratory tests for detection of resistance in human helminths. Apart from the use of the egg hatch test for hookworms in the Mali study (33), in vitro tests for AR in human nematodes have so far not been developed, adapted, or validated. A major problem is obviously the lack of reference resistant strains. If these were available, the egg hatch test and the larval development assay, as well as the promising new PCRs, could probably easily be validated for human hookworms.