The Development of Resistance to Anthelmintics: A Perspective with an Emphasis on the Antischistosomal Drug Praziquantel

EXPERIMENTAL PARASITOLOGY ARTICLE NO. PR974229

87, 260–267 (1997)

The Development of Resistance to Anthelmintics: A Perspective with an Emphasis on the Antischistosomal Drug Praziquantel James L. Bennett,*,1 Tim Day,* Liang Feng-Tao,† Magdi Ismail,‡ and Adel Farghaly‡ *Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan, U.S.A.; †Center for Tropical Diseases and Biomedicine, University of Massachusetts at Lowell, Lowell, Massachusetts, U.S.A.; and ‡Department of Parasitology, University of Zagazig, Zagazig, Egypt Bennett, J. L., Day, T., Feng-Tao, L., Ismail, M., and Farghaly, A. 1997. The development of resistance to anthelmintics: A perspective with an emphasis on the antischistosomal drug praziquantel. Experimental Parasitology 87, 260–267. © 1997 Academic Press

GENERAL CONCEPTS REGARDING THE DEVELOPMENT OF RESISTANCE TO ANTHELMINTICS There have been several major reviews describing various aspects of why and how helminths develop resistance to antiparasitics (Warwick 1994; Jackson 1993; Roos et al. 1993; Waller 1993, 1994). In general, studies on the genetics of resistance suggest that resistance is normally polygenic and arises from within the normal phenotypic range and that there are three phases in the selection process. An initial susceptible phase is followed by an intermediate one in which heterozygous resistant individuals are common within the population and finally homozygous resistant individuals predominate within the population. Selection models from anthelmintic resistance studies, where treatment is usually of short persistence and discriminatory and where there is high but not absolute efficacy, suggest (again) that resistance develops from the upper phenotypic range of susceptibility. Resistance selected in this manner will generally fall under control of more than one gene (LeJambre 1992). These general concepts have been validated, for the most part, by studies (Waller 1994) examining the molecular basis for the action of the benzimidazoles, a group of very active antinem1 To whom correspondence should be addressed at Keystone Symposia, Silverthorne, CO 80498. Fax: (970)2621525. E-mail: [email protected].

260 0014-4894/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.

atodal agents. Here it was shown that selection for resistance removes the susceptible alleles from the population and only resistant alleles remain. Thus, it was shown that a susceptible population is genetically and phenotypically heterogeneous and, importantly, does not require that resistance arises as a result of a new mutational event. In this particular case there exists four different genes expressing variants of the tubulin protein which is the target of the drug. Although knowing the molecular nature of the receptor which initiates the drug’s therapeutic action helps in the development of methods for detecting resistance to a drug, a number of powerful molecular biological tools exist which can be used to research the genetics of resistance (LeJambre 1992) and actually help in elucidating the molecular mechanism of the drug’s action. A critical aspect to the evolution of drug resistance among helminths is that the methods used to detect the developing resistance do not exist or are too insensitive. This ensures that most cases of resistance will not be detected at an early stage which, in turn, reduces the likelihood of reversion occurring. For example, if resistance was caused by one gene that is recessive then this means that the gene frequency of the resistant gene is already 0.5 and reversion at that time is very difficult. To avoid increasing levels of resistance, conventional wisdom suggests prolonged or total withdrawal of the se-

SCHISTOSOMAL RESISTANCE TO PZQ

lecting drug. In the case of drugs like praziquantel (PZQ) this is difficult to do since it is the only drug (at least for African and Oriental schistosomiasis) that can be seriously considered for controlling the parasite. At this point it is important to provide what most consider to be a definition of the word ‘‘resistance’’ when used to describe a drug that is less effective than predicted by earlier clinical trials (Cioli et al. 1993). Resistance is usually defined as a genetically transmitted loss of sensitivity in a parasite population that was previously sensitive to a given drug. The lack of sensitivity in a previously unexposed population is often termed tolerance. The boundary between the two situations is rather blurred when viewed in molecular terms, since in either case one has to deal with one or more mutations differentiating a given population from another one. There is strong evidence that schistosomes have the potential to evade the therapeutic action of drugs. Work by Bueding and his colleagues clearly demonstrated that schistosomes can develop resistance to antischistosomals (Rodger and Bueding 1971). Specifically, the drug hycanthone when administered in low doses to infected mice produced progeny that were highly resistant to hycanthone and oxaminquine a structural analog). The resistance was associated with a particular laboratory strain of the parasite because these results could not be reproduced in other strains of the parasite. In 1980 a Brazilian strain was isolated from a patient that turned out to be resistant to hycanthone (Katz 1980). Later a resistant isolate was identified in Kenya (Katz 1980). In Egypt, the Zagazig University Department of Parasitology laboratory (personal communication from Dr. Magdi Ismail, Zagazig University, Zagazig, Egypt) have identified over 15 isolates that are resistant to hycanthone. Bueding’s initial observations were extended by a series of eloquent experiments by Cioli and colleagues (Crop 1992; Cioli et al. 1993). Work from this group demonstrated that hycanthone stopped, irreversibly, the synthesis of RNA and DNA in schistosomes. This was predicted by Cioli because it

261

was earlier observed that this drug was a frameshift mutagen capable of intercalating between DNA base pairs. Using a resistant isolate it was demonstrated that the transmission of this phenotype followed a genetic pattern characteristic of a single, autosomal, recessive gene. During this work it was observed that the back crosses produced numbers lower than expected. This was due to a reduced fitness of the resistant phenotype. This appears to be a very critical observation and may explain why resistance to hycanthone has not spread more rapidly than predicted. This work ultimately directed these investigators to a critical schistosome enzyme that was necessary for the conversion of hycanthone to the active antischistosomal. In schistosomes that were resistant to the effects of hycanthone, the activity of this enzyme could not be detected. Finally, in a study performed on seven strains of Schistosoma mansoni that were isolated from the feces of Brazilian patients, three of which were known to harbor worms that would not respond to hycanthone, it was reported that after treating each strain with one of three different antischistosomals (hycanthone/oxaminquine and niridazole) that significant differences were observed in the efficacy of each of these drugs. Most importantly, such differences were dependent on the strain’s specific characters rather than their known responsiveness to the antischistosomal drugs. This unexpected finding seems to reinforce the idea that there is a great deal of genetic variation among schistosomes even in a specific region of the world (Katz 1980; Minchella et al. 1994; Gasser et al. 1996). PROBLEMS LIMITING THE THERAPEUTIC IMPACT OF PRAZIQUANTEL ON SCHISTOSOMIASIS During the early 1980s the cost and availability of PZQ limited its distribution in areas such as Egypt and Sudan. In the middle and late 1980s PZQ was being manufactured and more importantly produced in the endemic area. The cost of the drug was lowered and its availability was increased which resulted in a dramatic increase in its distribution along the Nile Valley.

262

BENNETT ET AL.

As would be predicted reports began to appear in the late 1980s and early 1990s that the problem of schistosomiasis (Michelson et al. 1993), especially in Egypt, was detectably less and that most of the observed decrease in the prevalence of the infection was due to the increased availability of PZQ. As with any drug the problems associated with its extensive use often results in the development of resistance to the drug. Recognizing this the Schistosomiasis Research Project, funded by the United States Agency for International Development and the Egyptian Ministry of Health, provided funding to determine if resistance to PZQ was a problem in villages where the drug had been extensively used over the past 10 years. These villages were located in the delta region north of Cairo. The author along with several Egyptian colleagues, most notably Dr. Magdi Ismail (Zagazig University) and Dr. Aiesha Metwally (Theodor Bilharz Research Institute, Cairo, Egypt), have been actively investigating whether resistance to PZQ is a problem in Egypt. Concurrently studies were also being conducted in Senegal on the development of resistance to PZQ at a site where a dam has been recently constructed (Fallon et al. 1995). Work conducted in Egypt was initially focused on identifying patients that were not successfully cured after receiving three doses of PZQ (Ismail 1996). Specifically, we treated 1607 infected villagers, located in eight villages, with two successive doses of PZQ. The drug was given orally at 40 mg/kg and then given again (6–8 weeks after the first dose) if the villager was still infected. If the villager was still excreting viable eggs after the second dose they were treated with a third dose of 60 mg/kg. Eggs from stools were isolated from villagers that were cured after the first dose. These eggs were used to establish an infection, a PZQsensitive isolate, in mice. Eggs were also retrieved from patients that were not cured after the second or third infection (PZQ-insensitive isolates) and used to establish an infection in mice, i.e., the eggs were hatched to infect snails and then cercariae from the infected snails were

used to infect outbred mice. Since the concentration of PZQ in the blood is critical to the drug’s efficacy, blood was taken from all patients not cured after the third dose and from a sample of those not cured after the second dose and from a sample of those cured after first dose. Blood samples were taken at various intervals after dosing to ensure that a proper pharmacokinetic profile could be generated so that parameters like the ‘‘area under the curve’’ (AUC) could be determined. The percentage of patients who could not be cured after the three doses of PZQ is presented in Table I. We observed that about 2.3% of the patients could not be successfully cured but that there was a great deal of variation from village to village (range 0.3 to 6.0%). Failure to respond to treatment was not due to an abnormally low concentration of PZQ in those not successfully cured since analysis of the AUC values (total amount of drug available over time) in those cured versus those not cured was not significantly different (Metwally et al. 1995; Ismail 1996). As was mentioned above, eggs from villagers cured and not cured were isolated and used to establish an infection in mice. Our rationale for this study was to remove as many of the confounding factors associated with PZQ’s action in humans (Ismail 1996) by isolating the infection and placing it in experimental animals housed in a controlled environ-

TABLE I Percentage of Infected Villagers Not Responding to Three Doses of Praziquantel

Village number

Number initially infected

Number not cured after three doses of PZQ

Percentage of failure

1 2 3 4 5 6 7 8 Total

33 272 283 391 329 139 382 158 1987

2 1 4 3 13 2 12 9 46

6 0.3 1.4 0.7 3.9 1.4 3.1 5.6 Mean, 2.3

263

SCHISTOSOMAL RESISTANCE TO PZQ

ment. Once the infection was established in mice we treated the infected animals with four different doses of the drug to determine the dose that would kill 50% (ED50 value) of the worms. Two independent labs were involved in this phase of the study. One lab (Zagazig University) was located close to the site of the collection of the eggs while the other lab was the NIH schistosome reference lab located at the University of Massachusetts—Lowell. From this work we reported (Ismail et al. 1996) some initial observations on 21 isolates. Six of the isolates came from patients that were cured after one dose, 8 came from patients cured after two doses, 3 came from patients cured after three doses, while 4 isolates came from patients that were not cured after three doses. Five of these isolates were significantly more difficult to treat compared to our control isolates which came from humans successfully cured after a single dose. Two of the five isolates came from patients who required three doses of PZQ to cure them of their infection, while the other three came from patients who could not be cured with three doses of PZQ. Isolates which had the highest ED50 values (the most difficult to treat) came from patients who could not be cured. The ED50 value for these isolates was three to five times higher (288–534 mg/kg) than the ED50 value (104 mg/kg) for isolates retrieved from patients successfully treated with PZQ. It is important to note that two isolates, which came from patients who required three doses of PZQ, were found to be susceptible to PZQ when placed in mice. Finally, the NIH schistosome reference lab was able to confirm significantly higher ED50 values for the isolates that came from humans we could not cure (Ismail et al. 1996). In a reexamination of our data, we have just completed an analysis of 73 isolates obtained from infected Egyptian villagers. Results (Table II) clearly indicate that isolates from patients successfully cured after a single dose of PZQ differ significantly from isolates obtained from patients requiring three or more doses of the drug. Although the percentage of worm reduction values vary by only 21% between isolates obtained from patients not cured after three

TABLE II Efficacy of Praziquantel on Mice Infected with Schistosome Isolates Obtained by Isolation of Eggs from Egyptian Villagers who Varied in Their Response (Clinical Group) to the Drug

Clinical group Cured after one dose Cured after two doses Cured after three doses Not cured after three doses

Number in group

Percentage of worm reduction after treating isolate with 5× 100 mg/kg PZQa

14 3 9

85 ± 9 80 ± 7 69 ± 8a

31

64 ± 13a

a 10 mice were infected with cercariae from a particular isolate and then treated; i.e., 570 mice were utilized in this study. P < 0.01 as determine by Dunnet’s t test when compared to group cured after a single dose.

doses and isolates obtained from patients cured after one dose, when we performed a complete analysis of the ED50 values they ranged between 80 and 104 mg/kg for the group cured after a single dose versus 123–680 mg/kg for the group that could not be cured after three doses. Thus, in some of the more difficult to treat human patients we identified schistosomes that were six times less sensitive to PZQ. IMPLICATIONS OF WORK DONE ON SCHISTOSOMES ISOLATED FROM HUMANS NOT RESPONDING TO PZQ Although the data we have obtained confirm the clinical efficacy of PZQ in Egyptian villagers, the information we have gathered is suggestive of a process that, as we would predict, is slow but showing signs of a possible break in the ability of a selected population of schistosomes to escape the therapeutic consequences of the drug. Most notable are the poor cure rates we observed among the 46 people located in various villages around the city of Zagazig. If a population of schistosomes is being selected out then we will have to determine if there are significant genetic variants of the parasite within the Delta region of the Nile and then prove that one or more of these variants is found much more frequently in schistosomes less suscep-

264

BENNETT ET AL.

tible to PZQ. The need to continue with a carefully designed program to monitor the response of ‘‘Egyptian schistosomes’’ to this drug is critical. Results describing the effects of PZQ on infected mice harboring isolates from humans who did and did not respond to PZQ treatment are important for a number of reasons. First, the antischistosomal action of PZQ (at least in mice) is contingent upon the ability of the host’s immune system to recognize key epitopes on the worm’s surface (Brindley et al. 1989; Tanaka et al. 1995). Since exposure of schistosomes to PZQ disrupts their surface it is thought that this action of PZQ allows the host’s immune system to bind to these epitopes. If the same relationship between the drug and the immune system is operable in humans then infected villagers who have an immune system that does not recognize these key epitopes may not be able to clear their infection when given PZQ. Second, PZQ’s antischistosomal action is not expressed on developing or immature worms. Thus, it is possible that some of our incurable patients had immature schistosomes when they were treated. Finally, the concentration of PZQ varies dramatically from patient to patient (thus it is possible that a patient may not be cured because of inadequate levels of PZQ in their blood (Metwally et al. 1995). This last confounding factor in PZQ’s effectiveness was controlled for in our study, but not the first two; thus the need to examine the effects of PZQ on schistosomes derived from our villagers was critical to determine whether the isolate was truly less susceptible to the drug. As we stated above our work in mice infected with these isolates produced the following results: We identified isolates that were difficult to treat from patients who were likewise difficult to treat. We identified isolates that were not difficult to treat in mice but were difficult to treat in their human host. For example, among the 31 patients who we could not cure with PZQ, 6 generated isolates that were susceptible to the drug in mice, while 25 produced isolates that were difficult to cure in mice. These results suggest that some patients are not responding to PZQ and

that the reason for this nonresponse is not due to a reduction in the susceptibility of their schistosomes to PZQ nor the lack of drug in their blood stream. It is as yet another unknown factor. Although our results clearly demonstrate the presence of schistosomes in humans who can tolerate high doses of PZQ several important points need to be made. First, of the 1987 infected patients less than 2.3% carry schistosomes that are less responsive to PZQ. Based upon the fact that of every five incurable patients we produced one isolate that could be cured in mice it is possible that the 2.3% value should be reduced to 1.8%. More importantly, the significance of our observation concerning the percentage of patients not responding to PZQ is difficult to interpret since no other report has described this kind of research on PZQ. In brief, it is very possible that if our studies had been done 10 years ago we would have obtained the same results. Thus two important questions concerning this data are, first, has this subpopulation of schistosomes always been present (as we suspect) and, second, is the prevalence of this subpopulation growing as PZQ therapy is applied? It should be noted that early research (Andrews and Thomas 1983) performed on various geographic isolates of S. mansoni showed no variation in their response to the drug while wide variation in efficacy was reported for oxaminiquine and hycanthone (Cioli et al. 1993). At present it is difficult to determine whether schistosomes that have been isolated from humans who do not respond to the drug are truly resistant to PZQ. For example, one possibility is that PZQ may simply be selecting for schistosomes that are slow in their development and thus unresponsive to PZQ, an important point since the drug is less effective against the immature schistosome (Andrew and Thomas 1983). Furthermore, PZQ at nanomolar concentrations can induce a rapid, quantifiable contraction of the worm’s muscles (Pax et al. 1979). Thus we need to determine if the receptor (yet to be identified) that induces this response, and associated events like tegumental damage and

SCHISTOSOMAL RESISTANCE TO PZQ

an increase in calcium permeability, varies from PZQ-susceptible to less susceptible schistosomes. OTHER EXPERIMENTAL WORK RELATED TO THE DEVELOPMENT OF PZQ RESISTANCE In a recent report (Fallon and Doenhoff 1994) it was clearly demonstrated that treating infected mice, which harbored a heterogenetic pool of schistosomes, with low doses of PZQ and then isolating the eggs from the surviving schistosomes and repeating the life cycle seven times resulted in the production of schistosomes that were ‘‘resistant’’ to the effects of the drug. Specifically, mice harboring the wild-type population of schistosomes and treated with 300 mg/kg of PZQ for 3 days lost 95% of their worms, while those selected for PZQ resistance lost only 13% of their schistosomes. A factor which could have biased the results of these investigators was that the doses were given on days 28 through 37 following the infection; during this time the schistosomes are known to be less, if at all, susceptible to PZQ (Andrews and Thomas 1983). The question of whether PZQ is selecting for schistosomes that are delayed in developing their response to PZQ was not answered nor was there any genetic evidence that the PZQ-resistant strain was different from the various geographic isolates used to generate the pool of schistosomes that were used in the first passage. It should be noted that schistosomes do not develop synchronously. If there is a genetic basis for this then it is possible that PZQ maybe selecting for worms that develop slowly. Regardless, this report was the first to demonstrate that ‘‘resistance’’ to PZQ can be induced under experimental conditions and it may have implications for what we are observing in the field. OTHER FIELD WORK RELATED TO THE DEVELOPMENT OF PZQ RESISTANCE Two reports (Kuman and Gryseels 1994; Gryseels 1994) from investigators working in Senegal clearly demonstrated that PZQ efficacy is surprisingly low in this population. Senegal is unique in that this community has only recently

265

been exposed to S. mansoni and thus to PZQ. Thus unlike Egypt, there has been very little therapeutic pressure placed upon the S. mansoni population in Senegal. Following standard treatment only 18% of the patients stopped excreting eggs. A number of factors have been proposed to account for the low PZQ cures in this population (Fallon et al. 1996), including rapid reinfection after treatment; presence of immature parasites at the time of drug treatment; low levels of anti-schistosome immunity that may be required to enhance the efficacy of PZQ; and the possible resistance of tolerance of the Senegal parasite to PZQ. Regardless, the Senegalese strain of S. mansoni was isolated and placed in mice and treated with PZQ along with mice harboring a Puerto Rican and Kenyan strain. Mice were treated 35 and 37 days postinfection. At one dose the authors reported a 2% reduction in the worm burden of mice harboring the Senegalese strain while mice infected with the Puerto Rican and Kenyan strains experienced a 43 and 44% reduction, respectively. Using a F3 isolate (third laboratory generation) of the Senegalese stain they reported a 19% reduction in the worm burden of the infected mice while observing a 72% reduction in mice harboring the Puerto Rican strain. They stated that ‘‘this experimental evidence may be cause for considerable concern.’’ CONCLUSION The ability of various infectious agents to develop resistance to a particular drug is now a familiar story. Going back to the development of the first antibiotics (sulfa drugs) it was soon recognized that bacteria had developed the ability to evade the action of these drugs and to pass those evasive strategies on to their progeny. Although the ‘‘genetic flexibility’’ of bacteria is probably much greater than what we observe in helminths, the evidence is clear (although sometimes slow in appearing) that resistance of helminths to drugs is a reality which must be dealt with. Recently several solutions have been employed to overcome problems associated ‘‘drug resistance’’ and they involve (1) changing the pattern of drug usage, (2) changing patient atti-

266

BENNETT ET AL.

tudes, (3) increasing surveillance of drugresistant organisms, (4) improving the techniques associated with testing for drug susceptibility, and (5) developing new drugs (Waller 1993). With regard to option 1 it is possible to switch to the drug oxamniquine but for African nations that would be very expensive and cumbersome. Since there are no new drugs being developed for schistosomiasis we are left with options 2, 3, and 4. Changing patient attitudes, from the perspective of not allowing the drug to be dispensed freely on the open market and within government health clinics (as we find in present Egypt), could help to lower the therapeutic pressure and thus the rate at which resistance develops, but to enforce this in a free market economy is very difficult. Options 3 and 4 are extremely important given the fact that the other options are not available. In essence, if we cannot do anything else we should at least monitor for the development of resistance to PZQ. If resistance becomes a problem then its early detection could bring about an effective strategy to slow or stop the further development of the problem—in essence the international community may find the resources to adopt some of the above-mentioned options that are presently not being pursued. ACKNOWLEDGMENTS This work was sponsored by the Schistosomiasis Research Project administered by the Egyptian Ministry of Health and the United States Agency for International Development under Contract 263-0140-C-9081-00 to the Medical Service Corporation.

REFERENCES Andrews, P., and Thomas, H. 1983. Praziquantel. Medicinal Research Review 3, 147–200. Brindley, P. 1994. Drug resistance to schistosomicides and other anthelmintics of medical significance. Acta Tropical 56, 213–221. Brindley, P. J., Stand, M., Norden, A., and Sher, A. 1989. Role of host antibody in the chemotherapeutic action of praziquantel against S. mansoni. Molecular and Biochemical Parasitology 34, 99–108. Cioli, D., Pica-Mattoccia, L., and Moroni, R. 1992. S. mansoni: Hycanthone/oxamiquine resistance is controlled by a single autosomal recessive gene. Experimental Parasitology 75, 425–432. Cioli, D., Pica-Mattoccia, L., and Archer, S. 1993. Drug

resistance in schistosomes. Parasitology Today 9, 162– 168. Day, T., Bennett, J. L., and Pax, R. A. 1992. Praziquantel: The enigmatic antiparasitic. Parasitology Today 8, 343– 344. Fallon, P., and Doenhoff, M. F. 1994. Drug-resistance schistosomiasis: resistance to praziquantel and oxaminquine induced in S. mansoni in mice is drug specific. American Journal Tropical Medicine and Hygiene 51, 83–88. Fallon, P. G., Sturrock, R. F., Capron, A., Niang, M., and Doenhoff, M. J. 1995. Short report: Diminished susceptibility to praziquantel in a Sengal isolate of S. mansoni. American Journal Tropical Medicine Hygiene 53, 61–62. Fallon, P., Tao, F-F., Ismail, M. M., and Bennett, J. L. 1996. Schistosome resistance to praziquantel: Fact or artifact. Parasitology Today 12, 316–320. Gasser, R. B., Baozhen, Q., Nansen, P., Johansen, M. V., and Bogh, H. 1996. Use of RAPD for the detection of genetic variation in the human blood fluke, Schistosoma japonicum, from mainland China. Molecular and Cellular Probes 10, 353–358. Grant, W. 1994. Genetic variation in parasitic nematodes and its implications. International Journal Parasitology 24, 821–830. Gryseels, B. 1994. Epidemiology, immunology and chemotherapy of S. mansoni infections in a recently exposed community in Senegal. Tropical and Geographic Medicine 46, 209–219. Ismail, M., Metwally, A., Farghly, A., Bruce, J., Tao, L. F., and Bennett, J. L. 1996. Characterization of isolates of S. mansoni from Egyptian villagers that tolerate high doses of praziquantel. American Journal Tropical Medicine and Hygiene 55, 214–218. Jackson, F. 1993. Anthelmintic resistance: The state of play. British Veterinary Journal 149, 123–138. Katz, N. 1980. Susceptibility to chemotherapeutic agents of strains of S. mansoni isolated from treated and untreated patients. American Journal Tropical Medicine 29, 890– 894. Kuman, V., and Gryseels, B. 1994. Use of praziquantel against schistosomiasis: A review of current status. Int. J. Antimicrobiol Agents 4(4), 313–321. LeJambre, L. F. 1992. Molecular biology and anthelmintic resistance in parasitic nematodes. In ‘‘Resistance of Parasites to Antiparasitic Drugs’’ (J. C. Boray, P. J. Martin, and R. T. Roush, Eds.), pp. 155–164. SMD Agvet, Rahway, NJ. Minchella, D. J., Lewis, F. A., Sollenberger, K. M., and Williams, J. A. 1994. Genetic diversity of Schistosoma mansoni: quantifying strain heterogeneity using a polymorphic DNA element. Metwally, A., Bennett, J. L., Botros, S., Ebeid, F., and ElAttar, G. 1995. Impact of drug dosage and brand on bioavailability and efficacy of proziquantel. Pharmacology Research 31, 53–59. Michelson, M. K., Axxis, F. A., Gamil, F. M., and Spencer,

SCHISTOSOMAL RESISTANCE TO PZQ H. C. 1993. Recent trends in the prevalence and distribution of schistosomiasis in the Nile delta region. American Journal Tropical Medicine and Hygiene 49(1), 76–87. Pax, R., Bennett, J. L., and Fetterer, R. 1979. A benzodiazepine derivative and praziquantel: Effects on musculature of S. mansoni and S. japonicum. Naunyn-Schmiedeberg’s Archives Pharmacology 304, 309–314. Rodger, S. H., and Bueding, E. 1971. Resistance to hycanthone: Development in Schistosoma mansoni. Science 172, 1057–1058. Roos, M. H., Kwa, M. S. G., Veenstra, J. G., Kooyman,

267

F. N. J., and Boersema, J. 1993. Molecular aspects of drug resistance in parasitic helminths. Pharmacology and Therapeutics 60, 331–345. Waller, P. J. 1993. Control strategies to prevent resistance. Veterinary Parasitology 46, 133–142. Waller, P. J. 1994. The development of anthelmintic resistance in ruminant livestock. Acta Tropical 56, 223–243. Received 5 June 1997; accepted with revision 27 August 1997