J. R. Kusel

Resistance of Schistosoma mansoni to praziquantel: is there a problem?

Evidence for resistance to praziquantel (PZQ) in Schistosoma mansoni has been sought in parasites taken from treated, but uncured human patients, and in a laboratory isolate of S. mansoni subjected to successive passages under drug pressure. Patients from villages in Egypt and Senegal have yielded isolates that can tolerate higher dosages of PZQ than other ostensible control isolates when passaged and subjected to drug treatment in mice. In vitro tests on these and the laboratory-selected isolate support the conclusion that a degree of resistance to PZQ can occur in S. mansoni, but the levels of drug resistance found so far are low. Preliminary studies have begun on these isolates to identify genetic, physiological and morphological characteristics associated with PZQ resistance and some of these may find use as markers for monitoring whether or not resistance is developing in endemic areas where the drug is used. More intensive application of PZQ can be expected in future, particularly in other parts of Africa, and vigilance will be needed to ensure that it continues to be useful as a drug for treatment of schistosomiasis. Further work is needed to elucidate the mode of action of PZQ and there is already a need for alternative drugs to treat PZQ-resistant schistosomiasis, such as already exists in northern Senegal.

Praziquantel: An urgent and exciting challenge

The anthelmintic drug praziquantel has proved useful in the treatment of schistosomiasis. The precise mechanism by which praziquantel kills the parasites has yet to be elucidated. Here, John Kusel and colleagues review the current theories on praziquantel action and suggest future avenues for research, which becomes urgent in the light of some reports of drug resistance.

Increased exposure of parasite antigens at the surface of adult male Schistosoma mansoni exposed to praziquantel in vitro.

Praziquantel is a broad-spectrum anthelmintic active against schistosome species which are major parasites of man. Two major effects on Schistosoma mansoni have been demonstrated; (i) spastic paralysis of the parasite musculature, possibly arising as a consequence of an influx of Ca2+ into the worm (Pax, Bennett & Fetterer, 1978; Coles, 1979) and (ii) vacuolation and degeneration of the worm tegument (Becker, Mehlhorn, Andrews, Thomas & Eckert, 1980). These events may contribute to the elimination of schistosomes in vivo, but this elimination may partly be dependent on the host immune response as infected T-cell-deprived mice are less able than immunologically intact animals to reduce their worm burdens following drug treatment (Doenhoff, Harrison, Sabah, Murare, Dunne & Hassounah, 1982). This latter observation raises the possibility that praziquantel may lower the ability of the parasite to evade host immunity by increasing the exposure of parasite antigens capable of acting as targets for host antibody, or antibody-armed cells at the worm surface. Consistent with this idea is the observation that adult schistosomes in praziquantel-treated mice are invaded a few hours after treatment by host granulocytes (Mehlhorn, Becker, Andrews, Thomas & Frenkel, 1981).

The effect of temperature and antimetabolites on antibody binding to the outer surface of second stage Toxocara canis larvae

In vitro maintained second stage Toxocara canis larvae do not bind antiserum raised to their excretions and secretions (ES) at 37 degrees C as detected by indirect fluorescence. However, when these larvae were incubated at 2 degrees C under the same conditions intense fluorescence on the whole outer surface was observed. This fluorescence remained as long as the larvae were maintained at 2 degrees C. When these larvae were reincubated at 37 degrees C a gradual loss of fluorescence along their outer surfaces occurred. This loss was complete after 3 h. Larvae which were preincubated in antimetabolites at 37 degrees C exhibited intense fluorescence on their outer surfaces as did those incubated at 2 degrees C with antimetabolites. It is concluded that antigens present in ES occur along the whole length of the larval outer surface and turn over at 37 degrees C. This turnover occurs along the whole outer surface and is metabolically dependent. Should this occur in vivo it could afford the parasite with a mechanism for evasion of the immune response.

Increased oviposition and growth in immature Biomphalaria glabrata after exposure to Schistosoma mansoni.

Biomphalaria glabrata snails are known to be castrated by infection with the trematode parasite Schistosoma mansoni 4-6 weeks post-infection. The pattern of oviposition in the first 35 days post-exposure (p.e.) was investigated, in snails aged 14 weeks and measuring 7-10 mm diameter which had not commenced egg-laying, by counting the numbers of eggs laid in 7-day intervals. A group of exposed snails was compared with a control non-exposed group. The exposed group included both parasitized and non-parasitized snails, and showed a significant increase in the median number of eggs laid during the periods 14-21 and 22-28 days p.e. Throughout the entire 35-day period exposed non-parasitized snails laid significantly more eggs than control snails, while parasitized snails laid significantly more eggs than controls during days 22-28 p.e. and significantly fewer during days 29-35 p.e. Parasitized snails also laid significantly more eggs/egg mass in the period 16-28 days p.e. than did control snails. Growth of the snails was measured. By day 28 p.e. the mean diameter of the exposed group was significantly greater than that of the control group. The increase in oviposition by snails soon after exposure is discussed in terms of a compensatory response for expected future suppression of egg-laying. The fact that parasitized and non-parasitized snails both show increased oviposition indicates that normal development of the parasite is not necessary to trigger the response.

Biophysical properties of the surface lipid of parasitic nematodes

The biophysical properties of the surface lipid layer (the epicuticle) of living parasitic nematodes (Trichinella spiralis and Toxocara canis) were examined using fluorescent lipid analogues. A variety of such probes were screened, and only 5-N-(octadecanoyl)-aminofluorescein was found to insert into the outer lipid layer. Fluorescence quenching experiments showed that this probe was confined to the surface, and the rate of its lateral diffusion was then measured by Fluorescence Recovery After Photobleaching. This showed that the probe was not free to diffuse within the plane of the epicuticle. This structure is, therefore, extraordinary in its selectivity to lipid probes, and in the restricted lateral mobility of inserted lipid components.

Schistosoma mansoni: a method for inducing resistance to praziquantel using infected Biomphalaria glabrata snails

To elucidate the mechanisms of antischistosoma resistance, drug-resistant Schistosoma mansoni laboratory isolates are essential. We developed a new method for inducing resistance to praziquantel (PZQ) using successive drug treatments of Biomphalaria glabrata snails infected with S. mansoni. Infected B. glabrata were treated three times with 100 mg/kg PZQ for five consecutive days with a one-week interval between them. After the treatment, the cercariae (LE-PZQ) produced from these snails and the LE strains (susceptible) were used to infect mice. Forty-five days after infection, mice were treated with 200, 400 or 800 mg/kg PZQ. Thirty days post-treatment, we observed that the mean number of worms recovered by perfusion was significantly higher in the group of mice infected with the LE-PZQ isolate treated with 200 and 400 mg/kg in comparison to the LE strain with the same treatment. Moreover, there was a significant difference between the ED50 (effective dose required to kill 50% of the worms) of the LE-PZQ isolate (362 mg/kg) and the LE strain (68 mg/kg). In the in vitro assays, the worms of the LE-PZQ isolate were also less susceptible to PZQ. Thus, the use of infected snails as an experimental model for development of resistance to S. mansoni is effective, fast, simple and cheap.

Biochemistry of irradiated parasite vaccines: Suggested models for their mode of action

Irradiated larvae of a remarkably wide range of parasite species induce potent protective immunity against subsequent challenge of experimental hosts. Ann Wales and John Kusel have studied the irradiated cercariae of Schistosoma mansoni and suggest here that the effects of irradiation on conformation and presentation of parasite antigens may be crucial in enhancing parasite immunogenicity so effectively.

The schistosome in the mammalian host: understanding the mechanisms of adaptation

In this review, we envisage the host environment, not as a hostile one, since the schistosome thrives there, but as one in which the relationship between the two organisms consists of constant communication, through signalling mechanisms involving sense organs, surface glycocalyx, surface membrane and internal organs of the parasite, with host fluids and cells. The surface and secretions of the schistosome egg have very different properties from those of other parasite stages, but adapted for the dispersal of the eggs and for the preservation of host liver function. We draw from studies of mammalian cells and other organisms to indicate how further work might be carried out on the signalling function of the surface glycocalyx, the raft structure of the surface and existence of pores in the surface membrane, the repair of the surface membrane, the role of the membrane structure in ion channel function (including recent work on the actin cytoskeleton and calcium channels) and the possible role of P-glycoproteins in the adaptation of the parasite to its environment. We are speculative in some areas, such as the suggestions that variability in surface properties of schistosomes may relate to the existence of membrane rafts and that parasite communities may exhibit quorum sensing. This speculative approach is adopted with the hope that future work on the whole organisms and their interactions will be encouraged.

Biophysical studies of the schistosome surface and their relevance to its properties under immune and drug attack

Schistosomiasis is a widespread tropical disease caused by trematode worms, which can live for many years in the bloodstream of the human or experimental host. There is evidence that immunity against reinfection can develop in human populations but such immunity is slow to develop and never complete (Butterworth et al. 1985, Hagan et al. 1987). Incomplete protection against reinfection is also seen in experimental animals immunized by a natural infection (Clegg & Smithers 1972), irradiated cercariae (Dean 1983), or by vaccination with a variety of antigen preparations (Smith & Clegg 1985, Kelly 1987, James 1985). The host’s immune response acts to damage the parasite during its migration and development, but rarely damages the adult parasite, except in abnormal hosts (Smithers & Terry 1965) or after drug treatment (Harnett 1988, Doenhoff, Modha & Lambertucci 1989, Brindley & Sher 1987). The incompleteness of the protection after immunization may be due to (a) variability in the efficacy of local (skin, lung or liver) responses in the host, or (b) variability in the surface or metabolic properties within the challenge parasite population (Jones & Kusel 1989). This variability (b) may reflect differences in the responses of each individual parasite to the effects of the host’s immune system. For example, those parasites metabolizing very rapidly might repair damaged membrane with greater efficiency. What methods are available to study the responses of living parasites to interactions with the immune system? Electrophysiology (Pax et al. 1983, Pax, Chen & Bennett 1987), pharmacology (Estey & Mansour 1988) and membrane signalling (Kalopothakis, Rumjanek & Evans 1987, Podesta et al. 1987b) are yielding interesting and valuable information about surface properties. In this article we describe studies which can be carried out on individual living parasites using a variety of fluorescent techniques. It is possible to study the lateral diffusion of macromolecules in or close to the surface membrane. Some of the results from these studies are providing new insights into the complexity of the surface structures and suggest mechanisms by which these structures may respond to attack by the immune system of the host.