The hatchability of Schistosoma haematobium eggs excreted by patients treated with praziquantel (PZQ) was examined. The rate of hatching of the eggs excreted by the patients before treatment was 90.1%. The rates of hatching of the eggs excreted by the patients 3 hr, 24 hr and 48 hr after treatment were 25.5%, 19.2% and 29.9% respectively. In vivo PZQ inhibits the hatching of S. haematobium eggs. The result of in vivo experiment was strongly supported by the results of the in vitro experiments. The hatching of the eggs exposed to PZQ at a concentration of 0.1μg/ml for one hour in vitro was inhibited

Aoki Yoshiki

Katsumata Tatsuya

Kimura Eisaku

Migwi D. K.

Muhoho N. D.

Shimada Masaaki

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Trop. Med.; 35 (1), 17-21, March, 1993 17Effect of Praziquantel on Hatching ofSchistosoma haematobium EggsTatsuya KATSUMATA1, D. K. MIGWI2, N. D. MUHOHO2,Eisaku KIMURA1, Masaaki SHIMADA1 and Yoshiki AOKI1department of Parasitology, Institute of Tropical Medicine, Nagasaki University,1-12-4 Sakamoto, Nagasaki 852, Japan2Centre for Microbiology Research, Kenya Medical Research Institute, P. O. Box54840, Nairobi, KenyaAbstract: The hatchability of Schistosoma haematobium eggs excreted by patients treatedwith praziquantel (PZQ) was examined. The rate of hatching of the eggs excreted by the pa-tients before treatment was 90.1%. The rates of hatching of the eggs excreted by the pa-tients 3 hr, 24 hr and 48 hr after treatment were 25.5%, 19.2% and 29.9% respectively. Invivo PZQ inhibits the hatching of S. haematobium eggs. The result of in vivo experimentwas strongly supported by the results of the in vitro experiments. The hatching of the eggsexposed to PZQ at a concentration of 0.1ƒÊg/ml for one hour in vitro was inhibited.Key words: Schistosoma haematobium, Egg, Hatching, Inhibition, PraziquantelINTRODUCTIONPraziquantel (PZQ) is a potent antischistosomal drug. The widespread use of PZQreduces contamination of the environment with schistosome eggs, thus reducing transmis-sion; in addition it reduces and prevents morbidity (WHO, 1977). The egg production ofadult female wormsceased immediately after treatment with PZQ (Andrew, 1981). However,the eggs were excreted for months after successful treatment (McMahon and Kolstrup, 1979;Doehring et al., 1985). It is, therefore, obligatory subject to measure ovicidal effect of PZQ.Since 1981, a research programme on urinary schistosomiasis has been carried out atMwachinga village, Kwale, Kenya, under the joint sponsership of the Kenya Government andthe Japan International Cooperation Agency. The selective mass-chemotherapy with PZQ wasattempted on July 1986. The present study was designed to examine the hatchability of S.haematobium eggs which were excreted by the patients treated with PZQ and those of eggsexposed to PZQ in vitro.Received for Publication January ll, 1993.Contribution No. 2719 from the Institute of Tropical Medicine, Nagasaki University_I18MATERIALS AND METHODSHatching of eggs excreted by patients treated with PZQ:The subjects enrolled in this study were 20 schoolchildren of Mwachinga village bet-ween 10 and 13 years of age whose intensity of infection was moderate to severe. Theschoolchildren received a single oral dose of 40mg/kg body weight of PZQ at noon. Theurine was collected one hour before treatment, 3hr, 24 hr and 48hr after treatment. The hat-chability of the eggs was examined immediately after collection of urine. Urine was filteredthrough a nuclepore membranewith 12 fim porosity and 25 mmin diameter so as to leave200 and more eggs on the membrane. Volume of urine filtered was then recorded. The mem-brane was then soaked in a small bottle filled with 4 ml of distilled water and left in daylight for one hour. The hatching of the eggs was stopped by the addition of 1 ml of 10% for-maline solution. The number of miracidium and unhatched eggs were then counted. Thenumber of eggs excreted by a patient was expressed by egg counts/ 10 ml of urine.Hatching of eggs exposed to PZQ in vitro:S. haematobium eggs were obtained from the schoolchildren at Mtsangatam PrimarySchool. Urine was filtered through a series of the meshes with different pore size (Nos. 5.5,16 and 282). The eggs thus collected were washed repeatedly with Dulbecco's (-)phosphate-buffered saline (PBS), and incubated in PBS containing various concentrations ofPZQ for different periods of time. The eggs then exposed to water after complete removal ofPZQ solution. The hatching of the eggs was stopped by the addition of 10% formaline solu-tion. The number of micracidia and unhatched eggs were then counted.RESULTSHatching of eggs excreted by patients treated with PZQ:The geometric mean of the number of eggs excreted by 20 schoolchildren before treat-ment was 537±4 eggs/10 ml of urine. Those of the eggs excreted 3 hr, 24 hr, and 48 hrafter treatment were 759+7, 246±3 and 363±3 eggs/10 ml of urine respectively. There wasno significant difference in the egg output during the initial 48 hr after treatment (Scheffe'stest).When placed in water, the eggs of S. haematobium easily hatch. The rates of the hat-ching of eggs excreted by each of 20 schoolchildren before treatment varied from 76.3% to97.6%, with an average of 90.1%. The hatching rates of eggs excreted 3 hr, 24 hr and 48 hrafter treatment were 25.5%, 19.2% and 29.9% respectively. Fig 1. represents the change inthe percentages of hatching of the eggs excreted by each of 20 schoolchildren during the in-itial 48 hr after treatment. Althogh individual variations exist, a definite pattern emergedfrom the results is that hatching tends to decrease during the initial 24 hr after treatmentand increase slightly 48 hr after PZQ treatment.Hatching of eggs exposd to PZQ in vitro:19Table 1 shows the hatchability of the eggs exposed to various concentrations of PZQfor different periods of time. The hatching rates of the eggs exposed to a high concentrationof PZQ (10 fjig/ml) for 10 minutes or more were lower than that of control eggs (chi square,p<0.01). PZQ-inhibited hatching was depend on the concentration of PZQ and period of ex-posure to PZQ. PZQ, at a concentration of 0.01 ^g/ml, did not affect the hatchability of theeggs.(%)100,JD£50uTOI0à"*à"\ -VV /s0 3 24 48Time in hours after the administration of praziquantel(40mg/kg)Fig. 1. Hatching of eggs excreted by patients during initial 48 hr after ad-ministration of praziquantel.Table 1. Hatching of eggs exposed to various concentrations of praziquantel fordifferent periods of time in vitroI ncubation Periodin PBS containingPraziquantel (min)C oncentration of Praziquantel (//g/ml)0 0.01 0.1 1 10M/M+UE M/M+ UE M/M+ UE M/M+UE M/M+UE10306012036096/238 (40.3) 130/304 (42.8)225/473 (47.6) 127/294 (43.2)80/214 (37.4) 76/218 (34.9)116/300 (38.7) 130/331 (39.3)93/206 (45.1) 136/272 (50.0)129/289 (44.6) 124/354 (35.0) 90/359 (25.1)*108/261 (41.4) 151/449 (33.6)* 41/145 (28.3)*34/188 (18.1)* 62/260 (23.8)* 36/226 (15.9)*70/388 (18.0)* 48/306 (15.7)* 19/253 ( 7.5)*56/335 (16.7)* 57/307 (18.6)* 25/274 ( 9.1)*Eggs were pre-incubated in PZQ solution. After complete removal of PZQ by repeated washing, eggs wereallowed to hatch in water.M: miracidium, UE: unhatched eggs, ( ): % hatched* Differences were statistically significant (chi square, p < 0. 01).20DISCUSSIONSince ova of S. haematobium take several days to pass through the bladder wall intourine (Belding, 1965), the eggs examined in this study were evidently those which hadalready been deposited in the venules of the bladder at the time of treatment. The presentstudy, therefore, was successfully designed to examine the effect of PZQ on the eggs of S.haematobium in vivo. The present study revealed that the hatching of eggs excreted aftertreatment was inhibited. It is a growing belief that active miracidia is responsible forbreakage of the eggshell (Samuelson et aL, 1984; Katsumata et al., 1988; Katsumata et al.,1989). It would appear that PZQ has injurious effects upon the miracidia enclosed within theeggshell in vivo.When a single oral dose of 40 mg/kg body weight of PZQ was given the healthyvolunteers, the maximumplasma concentration of PZQ reached 0.7 //g/ml 3 hr later, and thehigh plasma concentration of PZQ (over 0.1 jwg/ml) was observed for 24 hr after administra-tion of PZQ (Mandour efaL, 1990). The eggs examined in our study, therefore, were pro-bably exposed to the high concentration of PZQ (over 0.1 /*g/ml) for several hours. Theresult of in vivo study was strongly supported by the results of in vitro experiments. Whenthe eggs were incubated in PBS containing PZQ at a concentration of 0.1 ^g/ml for onehour, the hatching of the eggs was inhibited.Andrew (1978) reported the fact that in vivo PZQ inhibited hatching of S. mansonimiracidia for 24 hr after administration of 500 mg/kg to infected mice. Interestingly enough,the eggs passed after 48 hr hatched normally and were infective to Biomphalaria glabratasnails. In our study, the hatchability of the eggs excreted by patients 48 hr after treatmentwith PZQ was likely to recover partially. PZQ-inhibited hatching of the eggs of S.haematobium may be temporary in man as in a case of S. mansoni-mouse model, the in-hibitory action of PZQ may be longer in man than in mice though.It has been well known that the patients with urinary schistosomiasis excretes eggs forseveral months after treatment (McMahon and Kolstrup, 1979). Doehring et al. (1985)reported that many viable eggs were excreted by the patients during the initial 5 days aftertreatment with PZQ, the hatchability of the eggs was not examined though. The presentstudy revealed that the hatchability of the eggs excreted by the patients treated with PZQwas inhibited, even if the inhibitory action of PZQ would be temporary. The majority of thepatients treated with PZQ can be expected to contribute little to transmission of disease im-mediately after treatment with PZQ.ACKNOWLED GMENTSWeare indebted to Prof. M. Mugambi, the ex-Director of the Kenya Medical ResearchInstitute for his permission and encouragement in conducting this study. This study was sup-ported by the Kenya Medical Research Institute and the Japan International CooperationAgency. Japan.21REFERENCE S1) Andrew, P. (1978): Effect of praziquantel on the free-living stages of Schistosoma mansoni. Z.Parasitol., 56, 99-106.2) Andrew, P. (1981): A summary of the efficacy of praziquantel against schistosomes in animal ex-periments and notes on its mode of action. Arzneimittelforsh, 31, 538-541.3) Belding, D.L. (1965): The blood flukes of man. pp. 751-753. In Textbook of Parasitology Third Edi-tion. Appleton-Century-Crofts, New York.4) Doehring, E., Reider, F., Schmidt-Ehry, G. & Ehrich, J.H.H. (1985): Reduction of pathological fin-dings in urine and bladder lesions in infection with Schistosoma haematobium after treatment withpraziquantel. J. Inf. Dis. 152, 807-810.5) Katsumata, T., Shimada, M., Sato, K. & Aoki, Y. (1988): Possible involvement of calcium ions inthe hatching of Schistosoma mansoni eggs in water. J. Parasitol., 74, 1040-1041.6) Katsumata, T., Kohno, S., Yamaguchi, K., Kara, K. & Aoki, Y. (1989): Hatching of Schistosoma man-soni eggs is a Ca2+/ calmodulin-dependent process. Parasitol. Res., 76, 90-91.7) Mandour, M.M., Turabi, H., Homeida, M.M.A., Sadig, T., Ali, H.M., Bennett, J.L., Leahey, WJ. &Harron, D.W.G. (1990): Pharmacokinetics of praziquantel in healthy volunteers and patients withschistosomiasis. Trans. Roy. Trop. Med. Hyg., 84, 389-393.8) McMahon, J.E. & Kolstrup, N. (1979): Praziquantel: a new schistosomicide against Schistosomahaematobium. British Med. J., 2, 1396-1399.9) Samuelson, J.C., Quinn, JJ. & Caulfield, J.P. (1984): Hatching, chemokinesis, and transformation ofmiracidia of Schistosoma mansoni. J. Parasitol., 70, 321-331.10) WHO scientific working group on schistosomiasis (1977): Chemotherapy in control, pp. 7-8. Reportof the first meeting. TDR/MPD/SWG-SCH(l)/77.3.

Effect of Praziquantel on Hatching of Schistosoma haematobium Eggs

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Effect of Praziquantel on Hatching of Schistosoma haematobium Eggs

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