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.

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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) The current strategy for schistosomiasis control is based on large-scale treatments of populations aimed at reducing disease morbidity (WHO 2002). Currently, praziquantel (PZQ) is the drug of choice (Utzinger & Keiser 2004, Fenwick & Webster 2006, with the main advantages of its use being oral administration, single dose, low toxicity and low cost (Fenwick et al. 2003, Utzinger & Keiser 2004. Despite the advantages of PZQ, there is concern about the development of Schistosoma mansoni resistance to PZQ, both under laboratory and field conditions (Abdul-Ghani et al. 2009). In the laboratory, induction of resistance is based on the treatment of mice infected with S. mansoni, initially using sub-curative doses of PZQ. Afterwards, the dosage is increased for at least seven passages in mice/snails to complete the life cycle of the parasite (Ismail et al. 1994, Fallon et al. 1995.The complete mechanism of action of PZQ is still unclear (Doenhoff et al. 2008). Obtaining resistant strains is important for the evaluation of such mechanisms as well as for the development of alternative drugs for schistosomiasis treatment and control. Studies show that PZQ is effective not only in adult worms, but also in the intramolluscan phase of the parasite (Coelho et al. 1988. We report a novel meth- od for the induction (or selection) of S. mansoni worms resistant to PZQ using successive treatments of infected Biomphalaria glabrata snails. SUBJECTS, MATERIALS AND METHODSParasites and hosts -The S. mansoni (LE strain) life cycle was maintained using B. glabrata (Barreiro de Cima strain) snails as intermediate hosts and Swiss mice as definitive hosts, according to Pellegrino and Katz (1968) and Souza et al. (1995).Perfusion of adult worms from infected mice -Two methods were used. The methodology described by Pellegrino and Siqueira (1956) used a needle attached to a Brewer's automatic pipetter to inject saline solution under pressure into the descendent aorta. Afterwards, saline was injected into the hepatic hilum of mice after sectioning the portal vein, allowing the perfusion of the portal system and mesenteric veins. Worms were recovered and counted. To recover the worms using the methodology described by Smithers and Terry (1965) the portal vein of the mice was sectioned and the culture medium was gently injected into the base of the left ventricle of the infected mice's hearts. It is not possible to recover all the worms using this methodology with a lower pressure injection, but the integrity of the parasite's tegument is preserved. Therefore, this methodology is ideal for the recovery of worms when the goal is to cultivate or evaluate other parameters such as tegumental integrity and/or excretory activity.Induction of resistance to PZQ in the intramolluscan phase -Two-hundred B. glabrata were individually infected with 10 S. man...

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.

SUMMARYPraziquantel 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).

Abstract. The technique of fluorescence recovery after photobleaching was used to measure the lateral diffusion of fluorescent lipid analogues in the surface membrane of Schistosoma mansoni. Our data reveal that although some lipids could diffuse freely others exhibited restricted lateral diffusion. Quenching of lipid fluorescence by a non-permeant quencher, trypan blue, showed that there was an asymmetric distribution of lipids across the double bilayer of mature parasites. Those lipids that diffused freely were found to reside mainly in the external monolayer of the outer membrane whereas lipids with restricted lateral diffusion were located mainly in one or more of the monolayers beneath the external monolayer. Formation of surface membrane blebs allowed us to measure the lateral diffusion of lipids in the membrane without the influence of underlying cytoskeletal structures. The restricted diffusion found on the normal surface membrane of mature parasites was found to be released in membrane blebs. Quenching of fluorescent lipids on blebs indicated that all probes were present almost entirely in the external monolayer. Juvenile worms exhibited lower lateral diffusion coefficients than mature parasites: in addition, the lipids partitioned into the external monolayer. The results are discussed in terms of membrane organization, cytoskeletal contacts, and biological significance.

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