http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.970289
ORIGINAL ARTICLE
Evaluation of a method for induction of praziquantel resistance in Schistosoma mansoni Wael M. Lotfy1, Mohamed G. Hishmat1, Ahmed S. El Nashar1, and Hanaa M. Abu El Einin2 Department of Parasitology, Medical Research Institute, Alexandria University, Alexandria, Egypt and 2Department of Environmental Researches and Medical Malacology, Theodor Bilharz Research Institute, Imbaba, Giza, Egypt
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Abstract
Keywords
Context: Praziquantel (PZQ) is a highly efficacious anthelmintic against many flatworms including schistosomes. PZQ has been in use for more than 25 years, and concern is increasing that resistance has emerged in human schistosomes in Egypt and other endemic countries. Objective: The current study was designed to evaluate a recently described method for induction of PZQ resistance in Schistosoma mansoni. Materials and methods: Successive subcurative drug treatments of Biomphalaria alexandrina snails infected with an Egyptian strain of S. mansoni were undertaken. Cercariae shed from snails exposed and unexposed to PZQ were used to infect mice. Forty-five days after infection, mice were treated with a single oral dose of PZQ in 2% aqueous solution of Cremophor-ELÕ . The concentration of PZQ was 0, 200, 400, or 800 mg/kg. Thirty-three days after treatment, all groups of mice were dissected to collect the S. mansoni worms by the perfusion technique. In addition, the oogram pattern was examined to study the production, maturity, and death of S. mansoni eggs in the different groups of mice. Results: The present study has shown that the sublethal dose for induction of PZQ resistance in the intra-molluscan S. mansoni stages was 500 mg/kg. The worm count and the percentage of immature eggs in different groups of mice were significantly affected by the intra-molluscan exposure to PZQ and the drug concentration used to treat infected mice. Discussion and conclusion: The results obtained herein confirm the possibility of using successive drug treatments of infected B. alexandrina to induce PZO resistance in S. mansoni.
Antischistosomal, Biomphalaria alexandrina, drug, intra-molluscan, sub-curative
Introduction Schistosoma is one of the most important parasites infecting humans. It is estimated that about 41 000 people die from the disease each year, and approximately 90% of all cases occur in Africa (WHO, 2010). In Egypt, it is the most important parasitic disease (WHO, 2010). It has plagued its people throughout recorded history (Nunn & Tapp, 2000). A new era in the control of the disease in the country has started in 1988 with the distribution of praziquantel (PZQ), free of charge, to all diagnosed cases through the government health facilities. From October 1997, after a ministerial decree that authorized the distribution of PZQ without prior diagnosis, annual treatment of primary school children was implemented in all governorates of the country, and for the entire population in high-prevalence villages (Curtale et al., 2003). Until now, PZQ is the drug of choice for treatment of the disease, with the main advantages of its use being oral administration, single dose, low toxicity, and low cost (Botros et al., 2004). PZQ has been in use for more than 25 years (King & Correspondence: Prof. Wael M. Lotfy, Department of Parasitology, Medical Research Institute, 165 El-Horreya Avenue, P.O. Box 21561, Alexandria, Egypt. Tel: +20 100 815 4959. E-mail: waelotfy@ alexu.edu.eg
History Received 1 June 2014 Accepted 23 September 2014 Published online 22 January 2015
Mahmoud, 1989), and concern is increasing that resistance has emerged in human schistosomes. PZQ-resistant strains of Schistosoma mansoni have been reported in Egypt (Ismail et al., 1994a; 1996; 1999), Senegal (Gryseels et al., 1994; Stelma et al., 1995), and even in Brazil where it is rarely used (Araujo et al., 1996; Bonesso-Sabadini & Dias, 2002; Gomes et al., 1993). Under laboratory conditions, induction of resistance is based on the treatment of mice infected with S. mansoni, initially using sub-curative doses of PZQ. Then, the drug dose is increased for at least seven passages in mice/snails to complete the life cycle of the parasite (Fallon et al., 1995; Ismail et al., 1994b). Yue et al. (1990) failed to induce drug resistance in Schistosoma japonicum after exposure of infected mice to PZQ for several generations. Fallon and Doenhoff (1994) succeeded to induce PZQ resistance in S. mansoni and demonstrated that S. mansoni subjected to drug pressure may develop resistance to the drug over the course of relatively few passages. Liang et al. (2002) confirmed these results. Ismail et al. (2002) used isolates of S. mansoni originally showing marked diminished susceptibility to PZQ, passaged them in mice, and treated them with sub-curative doses of PZQ. The results showed that repeated passage of S. mansoni isolates in the laboratory did not render
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them more susceptible to PZQ. Indeed, these resistant isolates showed less susceptibility to the drug than before, or at least they retained their original level of insusceptibility to PZQ. William et al. (2001) tested the stability of six S. mansoni isolates derived from human infections not cured by three successive doses of PZQ and produced infections in mice that were significantly more difficult to cure than infections with control worms. They reported that only three of the six isolates retained their decreased response to PZQ after multiple passages through the life-cycle in the absence of therapeutic pressure. They concluded that the stability of some of the isolates and the reversion of others indicate that the biological or genetic factors conferring decreased PZQ response vary among the isolates. Couto et al. (2011) described a novel method for induction of PZQ resistance in a South American strain of S. mansoni. This was achieved by using successive exposures of the intramolluscan phase of the parasite in B. glabrata to PZQ. To the best of our knowledge, this method was never applied before on the Egyptian B. alexandrina snails infected with S. mansoni. The aim of the present work is to evaluate induction of S. mansoni resistance to PZQ by using successive drug treatments of B. alexandrina snails infected with S. mansoni.
Materials and methods Infection of snails with S. mansoni Laboratory-reared B. alexandrina snails (6–8 mm shell diameter) were supplied by the Department of Medical Malacology at Theodor Bilharz Research Institute (TBRI), Giza, Egypt. Miracidia of S. mansoni were purchased from the Schistosome Biological Supply Program at TBRI, Giza, Egypt. For infection, a total of 350 B. alexandrina snails were individually exposed to 10 freshly collected S. mansoni miracidia for 3 h in 1–2 ml water. Starting from the 30th day of post-infection, exposed snails were examined daily for cercarial shedding by exposing them to fluorescent light for 2 h according to the method described by Lewis et al. (1993). Shedding snails were selected and kept in separate aquaria. Treatment of infected snails with PZQ For treatment of infected snails, the drug was incorporated into the snail food which was prepared from Purina mouse chow ground up with 10% of calcium carbonate and TetraminÕ . Water was also added in order to obtain a smooth paste. The snails were individually weighed using an analytical balance and the snails’ mean weight was used to calculate the drug dosage to be administered. The drug utilized was mixed into the chow and 100 mg from this mixture (chow and drug) were weighed and allowed to consume everyday by each snail. In the present experiment, the mean weight of the group of snails was 600 mg and if the dose of PZQ to be administered was 1000 mg/kg, the amount of drug incorporated into the 100 mg food would be calculated accordingly as follows: 1000:1 000 000 ¼ X:600 mg, i.e., 0.600 mg of PZQ. Throughout the experiment, the snails were individually maintained in plates for cell culture with dechlorinated water. The food with PZQ and water was changed the second day after exposure. Exposure
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of snails to PZQ was repeated three times, with a 1-week interval between the treatments (Couto et al., 2011; Mattos et al., 2007). Standardization of the PZQ dose used for S. mansoni intra-molluscan exposure Two groups of snails, each composed of 12 snails shedding S. mansoni cercariae, were treated with PZQ after 30 d of the infection with the parasite. The first group was exposed to PZQ in a dose of 1000 mg/kg; while for the second group, the dose was 500 mg/kg. A control group composed of 12 snails was kept under the same conditions but without any exposure to the drug. Assessment of the impact of intra-molluscan S. mansoni exposure to PZQ on cercarial shedding To quantify the number of cercariae shed per snail, 12 positive snails from each group were isolated and kept in separate aquaria under complete darkness. Dead snails were removed daily and their number was recorded. After exposure of snails to light, emerged cercariae were counted, the length of cercarial incubation and the periodic cercarial production were determined individually, and this was carried out weekly (Mahmoud, 2011). Infection of mice with S. mansoni and treatment of infected mice A total of 88 mice were infected with approximately 40 S. mansoni cercariae per mouse by subcutaneous injection (Pellegrino & Katz, 1968). According to the intra-molluscan exposure to PZQ, mice were divided into two main large groups: 44 mice were infected with cercariae derived from snails treated with PZQ and 44 mice were infected with cercariae developed from untreated snails. Forty-five days after infection, mice were treated with a single oral dose of PZQ in 2% aqueous solution of Cremophor-ELÕ . The concentration of PZQ was 0, 200, 400, or 800 mg/kg, thus mice were finally divided into eight groups: (1) Sm-Un: Mice infected with S. mansoni unexposed to PZQ. (2) Sm-Un200: Mice infected with S. mansoni unexposed to PZQ during the intra-molluscan phase and treated with PZQ at a dose of 200 mg/kg. (3) Sm-Un400: Mice infected with S. mansoni unexposed to PZQ during the intra-molluscan phase and treated with PZQ at a dose of 400 mg/kg. (4) Sm-Un800: Mice infected with S. mansoni unexposed to PZQ during the intra-molluscan phase and treated with PZQ at a dose of 800 mg/kg. (5) Sm-PZQ: Mice infected with S. mansoni exposed to PZQ during the intra-molluscan phase. (6) Sm-PZQ200: Mice infected with S. mansoni exposed to PZQ during the intra-molluscan phase and treated with PZQ at a dose of 200 mg/kg. (7) Sm-PZQ400: Mice infected with S. mansoni exposed to PZQ during the intra-molluscan phase and treated with PZQ at a dose of 400 mg/kg.
Induction of praziquantel resistance in S. mansoni
DOI: 10.3109/13880209.2014.970289
Worm recovery Thirty-three days after treatment, all groups of mice were killed by cervical dislocation and dissected. Schistosomes were then removed from the hepatic portal system by the perfusion technique (Duvall & DeWitt, 1967; Smithers & Terry, 1965).
14 12 No. of snails
(8) Sm-PZQ800: Mice infected with S. mansoni exposed to PZQ during the intra-molluscan phase and treated with PZQ at a dose of 800 mg/kg.
3
Exposed to PZQ
Unexposed to PZQ
10 8 6 4 2 0 0
1
2
3
4
5
6
7
8
Weeks
Figure 1. Survival of infected snails.
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Production, maturity, and death of S. mansoni eggs in different groups of mice The oogram pattern was examined to show the different developmental stages of S. mansoni eggs in the small intestines of mice to study the effect of different concentrations of PZQ on egg production, maturity, and death of eggs. Also, this effect was studied in relation to the intra-molluscan exposure of the parasite to the drug. After the perfusion, a fragment from the median part of the small intestine (10 mm) was cut-off and processed for oogram. Eggs were then counted and classified according to the different stages of development (Pellegrino et al., 1962). The eggs were classified into immature, mature, or dead.
Table 1. Numbers of cercariae shed by individual snails exposed and unexposed to PZQ. Number of cercariae/ snail/stimulation Groups of snails
Mean ± SD (range)
Exposed to 500 mg/kg (no. of examinations ¼ 24) Unexposed to PZQ (no. of examinations ¼ 29)
81.7 ± 45.8 (6–200)
Independent samples t-test (p value) 0.0001
192.7 ± 106.6 (0–412)
The number of examinations: total number of snail examinations for shedding during the examination period.
Statistical analyses Statistical analyses were run on an IBM compatible PC using SPSS for windows statistical package (SPSS Statistics Base 17.0; SPSS Inc., Chicago, IL). All parameters were treated as parametric data. They are expressed as mean ± SD (min– max). Comparison between two means was done using independent samples’ t-test. The survival distributions of the two groups of snails were compared by using the log-rank (Mantel–Cox) test. A multiple linear regression model was used to predict S. mansoni worm count based on the intramolluscan exposure to PZQ and the drug dose used to treat mice. The p value below 0.05 was considered significant.
Results Standardization of the PZQ dose used for S. mansoni intra-molluscan phase The results of the present study showed that snails treated with a dose of 1000 mg/kg PZQ stopped shedding cercariae after treatment until they died, but snails exposed to 500 mg/ kg shed cercariae. Effect of PZQ on survival of infected B. alexandrina snails The snails exposed to the drug survived 6 weeks after treatment, while the unexposed snails survived up to 7 weeks (Figure 1). The survival distributions of the two groups were compared using the log-rank (Mantel–Cox) test, and the difference was insignificant (p ¼ 0.396). Effect of PZQ on cercarial production by B. alexandrina snails Cercariae from snails exposed and unexposed to PZQ were counted weekly starting from the 37th day after infection until
Table 2. Schistosoma mansoni worm recovery from the different groups of mice.
Mice groups Sm-PZQ (no. ¼ 8) Sm-Un (no. ¼ 7) Sm-PZQ200 (no. ¼ 8) Sm-Un200 (no. ¼ 8) Sm-PZQ400 (no. ¼ 9) Sm-Un400 (no. ¼ 7) Sm-PZQ800 (no. ¼ 7) Sm-Un800 (no. ¼ 9)
Mean ± SD 15.00 ± 2.39 11.71 ± 1.11 9.00 ± 2.56 6.63 ± 1.69 7.78 ± 1.99 1.14 ± 1.07 1.00 ± 0.82 0.22 ± 0.44
(11–18) (10–13) (6–14) (5–10) (6–12) (0–3) (0–2) (0–1)
t-Test (p value) 0.005 0.046 0.000 0.028
Percentages of worm reduction – – 40 43 48 90 93 98
No.: the number of surviving mice.
all the infected snails died after 7 weeks. Snails exposed to PZQ shed significantly (p ¼ 0.0001) lower numbers of cercariae than those unexposed to the drug (Table 1). Schistosoma mansoni worm recovery from different groups of mice In the two untreated mouse groups, the numbers of worms recovered from mice infected with S. mansoni cercariae exposed to PZQ during the intra-molluscan phase (Sm-PZQ) were significantly higher than those recovered from mice infected with S. mansoni cercariae unexposed to the drug (Sm-Un) (Table 2). In the three groups of mice infected with S. mansoni exposed to PZQ during the intra-molluscan phase (Sm-PZQ200, Sm-PZQ400, and Sm-PZQ800), the increase of the PZQ dose from 200 mg/kg to 400 mg/kg was used to treat mice, and then 800 mg/kg resulted in a gradual decrease in the
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Table 3. Percentages of the different stages of S. mansoni eggs recovered from the groups of mice.
Mice groups
Immature, mean ± SD (min–max)
Mature, mean ± SD (min–max)
Dead, mean ± SD (min–max)
Sm-PZQ (no. 8) Sm-Un (no. 7) t-Test (p value) Sm-PZQ200 (no. 8) Sm-Un200 (no. 8) t-Test (p value) Sm-PZQ400 (no. 9) Sm-Un400 (no. 7) t-Test (p value) Sm-PZQ800 (no. 7) Sm-Un800 (no. 9)
50.5 ± 3.0 (47.6–54.7) 49.2 ± 4.2 (44.0–54.4) 0.529 30.8 ± 7.3 (24.3–47.3) 9.6 ± 2.0 (7.1–12.0) 0.000 5.6 ± 0.1 (5.5–5.8) 0.0 0.000 0.0 0.0
42.0 ± 2.9 (37.2–44.7) 44.7 ± 3.9 (39.6–50.0) 0.148 42.4 ± 9.0 (23.7–52.6) 36.1 ± 1.8 (33.3–39.3) 0.070 20.8 ± 4.2 (15.7–27.7) 55.6 ± 18.5(33.3–83.3) 0.002 0.0 0.0
7.4 ± 1.3 (5.6–9.5) 5.8 ± 0.6 (5.1–6.5) 0.010 26.4 ± 4.9 (18.4–33.3) 54.3 ± 2.3 (51.8–59.2) 0.000 73.6 ± 4.2 (66.6–78.9) 44.3 ± 18.5 (16.6–66.6) 0.005 100 100
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No.: the number of surviving mice.
Table 4. Multiple linear regression models to predict S. mansoni worm count based on the intra-molluscan exposure to PZQ and the drug dose used to treat mice. Unstandardized coefficients Model Constant Intra-molluscan exposure to PZQ Drug concentration used to treat mice
B
Standard error
Significance (p value)
10.477 2.885
0.548 0.555
0.000 0.000
0.015
0.001
0.000
number of worms recovered from them (Table 2). The same condition was observed in the other three groups of mice that were infected with S. mansoni unexposed to PZQ during the intra-molluscan phase: Sm-Un200, Sm-Un400, and SmUn800 (Table 2). Significant differences were found between different corresponding groups, i.e., Sm-PZQ200 with SmUn200, Sm-PZQ400 with Sm-Un400, and Sm-PZQ800 with Sm-Un800. Production, maturity, and death of S. mansoni eggs in different groups of mice In the two untreated mice groups (Table 3), the differences in the percentages of immature and mature eggs recovered from the two groups were insignificant. However, the percentage of dead eggs was significantly higher in the mice infected with S. mansoni exposed to the drug during the intra-molluscan phase (Sm-PZQ) than the other group (Sm-Un). In the two groups of mice treated with PZQ at a dose of 200 mg/kg, the percentages of the immature eggs were significantly higher in mice infected with S. mansoni exposed to PZQ during the intramolluscan phase (Table 3); in contrast, the percentages of the dead eggs were significantly lower. In the two groups of mice treated with PZQ at a dose of 400 mg/kg, immature eggs could not be detected in mice infected with S. mansoni previously unexposed to PZQ and significantly higher percentages of mature eggs were found in mice infected with S. mansoni exposed to PZQ during the intra-molluscan phase (Tables 3). In the two groups of mice treated with PZQ at a dose of 800 mg/kg, all the detected eggs were dead (Tables 3).
Prediction of worm count based on the intra-molluscan exposure to PZQ and the drug concentration used to treat mice The results of the multiple linear regression analysis showed that the overall model was significant, p50.001, R2 ¼ 81.4% (Table 4).
Discussion PZQ is a broad-spectrum, highly efficacious, and safe anthelmintic against trematode and cestode infections in humans and animals, respectively (Chai, 2013). Emerging problems include the appearance of PZQ-resistant strains or isolates in S. mansoni and S. japonicum in the laboratory (Liang et al., 2002, 2010) or in the field (Wang et al., 2012). According to the present results, based on cercarial shedding, the recommended sublethal dose of PZQ for exposure of S. mansoni intra-molluscan phase in B. alexandrina was 500 mg/kg. The present results showed that the snails exposed to the drug survived 6 weeks after treatment with subcurative doses of 500 mg/kg, while the unexposed snails survived up to 7 weeks (Figure 1). The difference between survival of the two groups of snails was insignificant (p ¼ 0.396). This result indicated that exposure of B. alexandrina snails to the drug did not affect their survival. Despite the differences in survival rates between B. alexandrina and B. glabrata, a similar result was reported by Mattos et al. (2007), after exposure of B. glabrata to PZQ. Snails unexposed to PZQ shed higher numbers of cercariae than those exposed to the drug, with significant differences between the two groups (p ¼ 0.000). This finding is in accordance with the results reported by other authors in different studies after the intra-molluscan exposure of S. mansoni to PZQ in B. glabrata snails (Mattos et al., 2007; Riley & Chappell, 1990). Results of the current study showed that the numbers of worms recovered from mice infected with S. mansoni unexposed to PZQ were significantly lower than those recovered from the mice infected with S. mansoni exposed to the drug during the intra-molluscan phase (Table 2). This may indicate that stress posed by previous exposure of S. mansoni intra-molluscan stages to PZQ had positive effect on success of S. mansoni to colonize mice and achieve
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DOI: 10.3109/13880209.2014.970289
maturity. In the mouse groups treated with PZQ, the increase of the drug dose from 200 mg/kg to 400 mg/kg, and then 800 mg/kg resulted in a gradual decrease in the number of recovered worms (Table 2). Comparable results were reported by Couto et al. (2011). By comparing the number of worms recovered from different groups of mice infected with parasites previously unexposed to the drug during the intramolluscan phase with their corresponding exposed groups, the numbers were significantly higher in the groups infected with schistosomes previously exposed to the drug; significant differences were found between them (Table 2). These results were in agreement with the findings reported by Couto et al. (2011), which may indicate that intra-molluscan exposure of S. mansoni has induced resistance to the drug in the adult stage. In mice infected with S. mansoni, oviposition begins about day 30 of post-infection. Immature eggs require about 6 d for the miracidium to develop (Pellegrino et al., 1962). Studying the oogram pattern permits a direct assessment of the production and development of S. mansoni eggs, and their passage from mesenteric vessels into the intestinal lumen in mice (Mati & Melo, 2013). A drug that shows a chemotherapeutic effect causes a progressive change in the percentages of different developmental stages with a decrease in immature stages and an increase in the percentages of mature and/or dead eggs (Delgado et al., 1992). These changes are due to the interruption of oviposition in the intestinal wall and the maturation of viable eggs already there (Delgado et al., 1992; Pellegrino et al., 1962). Results of the present work indicate that without mice treatment the differences between the two mice groups as regards percentages of immature or mature S. mansoni eggs were insignificant (Table 3). This may indicate that the intra-molluscan exposure to PZQ had no effect on the success of S. mansoni eggs to achieve maturity in the murine host. After treatment, the immature eggs showed significantly higher percentages in the mice group infected with S. mansoni exposed to PZQ during the intra-molluscan phase compared with the unexposed group denoting oviposition by a greater number of surviving worms (Table 3). Thus, the results of oogram pattern confirm that the intra-molluscan exposure of S. mansoni has induced drug resistance in the adult stage. To confirm the present results, a multiple linear regression model was carried out (Table 4). The model indicated that the worm count in different groups of mice was significantly affected by the intra-molluscan exposure to PZQ and the drug concentration used to treat infected mice. The present study has important implications for the use of B. alexandrina to induce PZO resistance in S. mansoni. Also, it is the first report of laboratory induction of PZQ resistance in an Egyptian S. mansoni strain in B. alexandrina.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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Stelma FF, Talla A, Sow S, et al. (1995). Efficacy and side-effects of praziquantel in an epidemic focus of Schistosoma mansoni. Am J Trop Med Hyg 53:167–70. Wang W, Wang L, Liang YS. (2012). Susceptibility or resistance of praziquantel in human schistosomiasis: A review. Parasitol Res 111: 1871–7. WHO. (2010). First report on neglected tropical diseases. Working to overcome the global impact of neglected tropical diseases. Geneva, Switzerland: World Health Organization. William S, Sabra A, Ramzy F, et al. (2001). Stability and reproductive fitness of Schistosoma mansoni isolates with decreased sensitivity to praziquantel. Int J Parasitol 31:1093–100. Yue WJ, Yu SH, Xu XJ. (1990). Failure to induce resistance of Schistosoma japonicum to praziquantel. Southeast Asian J Trop Med Public Health 21:85–9.
ORIGINAL ARTICLE
Evaluation of a method for induction of praziquantel resistance in Schistosoma mansoni Wael M. Lotfy1, Mohamed G. Hishmat1, Ahmed S. El Nashar1, and Hanaa M. Abu El Einin2 Department of Parasitology, Medical Research Institute, Alexandria University, Alexandria, Egypt and 2Department of Environmental Researches and Medical Malacology, Theodor Bilharz Research Institute, Imbaba, Giza, Egypt
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Abstract
Keywords
Context: Praziquantel (PZQ) is a highly efficacious anthelmintic against many flatworms including schistosomes. PZQ has been in use for more than 25 years, and concern is increasing that resistance has emerged in human schistosomes in Egypt and other endemic countries. Objective: The current study was designed to evaluate a recently described method for induction of PZQ resistance in Schistosoma mansoni. Materials and methods: Successive subcurative drug treatments of Biomphalaria alexandrina snails infected with an Egyptian strain of S. mansoni were undertaken. Cercariae shed from snails exposed and unexposed to PZQ were used to infect mice. Forty-five days after infection, mice were treated with a single oral dose of PZQ in 2% aqueous solution of Cremophor-ELÕ . The concentration of PZQ was 0, 200, 400, or 800 mg/kg. Thirty-three days after treatment, all groups of mice were dissected to collect the S. mansoni worms by the perfusion technique. In addition, the oogram pattern was examined to study the production, maturity, and death of S. mansoni eggs in the different groups of mice. Results: The present study has shown that the sublethal dose for induction of PZQ resistance in the intra-molluscan S. mansoni stages was 500 mg/kg. The worm count and the percentage of immature eggs in different groups of mice were significantly affected by the intra-molluscan exposure to PZQ and the drug concentration used to treat infected mice. Discussion and conclusion: The results obtained herein confirm the possibility of using successive drug treatments of infected B. alexandrina to induce PZO resistance in S. mansoni.
Antischistosomal, Biomphalaria alexandrina, drug, intra-molluscan, sub-curative
Introduction Schistosoma is one of the most important parasites infecting humans. It is estimated that about 41 000 people die from the disease each year, and approximately 90% of all cases occur in Africa (WHO, 2010). In Egypt, it is the most important parasitic disease (WHO, 2010). It has plagued its people throughout recorded history (Nunn & Tapp, 2000). A new era in the control of the disease in the country has started in 1988 with the distribution of praziquantel (PZQ), free of charge, to all diagnosed cases through the government health facilities. From October 1997, after a ministerial decree that authorized the distribution of PZQ without prior diagnosis, annual treatment of primary school children was implemented in all governorates of the country, and for the entire population in high-prevalence villages (Curtale et al., 2003). Until now, PZQ is the drug of choice for treatment of the disease, with the main advantages of its use being oral administration, single dose, low toxicity, and low cost (Botros et al., 2004). PZQ has been in use for more than 25 years (King & Correspondence: Prof. Wael M. Lotfy, Department of Parasitology, Medical Research Institute, 165 El-Horreya Avenue, P.O. Box 21561, Alexandria, Egypt. Tel: +20 100 815 4959. E-mail: waelotfy@ alexu.edu.eg
History Received 1 June 2014 Accepted 23 September 2014 Published online 22 January 2015
Mahmoud, 1989), and concern is increasing that resistance has emerged in human schistosomes. PZQ-resistant strains of Schistosoma mansoni have been reported in Egypt (Ismail et al., 1994a; 1996; 1999), Senegal (Gryseels et al., 1994; Stelma et al., 1995), and even in Brazil where it is rarely used (Araujo et al., 1996; Bonesso-Sabadini & Dias, 2002; Gomes et al., 1993). Under laboratory conditions, induction of resistance is based on the treatment of mice infected with S. mansoni, initially using sub-curative doses of PZQ. Then, the drug dose is increased for at least seven passages in mice/snails to complete the life cycle of the parasite (Fallon et al., 1995; Ismail et al., 1994b). Yue et al. (1990) failed to induce drug resistance in Schistosoma japonicum after exposure of infected mice to PZQ for several generations. Fallon and Doenhoff (1994) succeeded to induce PZQ resistance in S. mansoni and demonstrated that S. mansoni subjected to drug pressure may develop resistance to the drug over the course of relatively few passages. Liang et al. (2002) confirmed these results. Ismail et al. (2002) used isolates of S. mansoni originally showing marked diminished susceptibility to PZQ, passaged them in mice, and treated them with sub-curative doses of PZQ. The results showed that repeated passage of S. mansoni isolates in the laboratory did not render
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them more susceptible to PZQ. Indeed, these resistant isolates showed less susceptibility to the drug than before, or at least they retained their original level of insusceptibility to PZQ. William et al. (2001) tested the stability of six S. mansoni isolates derived from human infections not cured by three successive doses of PZQ and produced infections in mice that were significantly more difficult to cure than infections with control worms. They reported that only three of the six isolates retained their decreased response to PZQ after multiple passages through the life-cycle in the absence of therapeutic pressure. They concluded that the stability of some of the isolates and the reversion of others indicate that the biological or genetic factors conferring decreased PZQ response vary among the isolates. Couto et al. (2011) described a novel method for induction of PZQ resistance in a South American strain of S. mansoni. This was achieved by using successive exposures of the intramolluscan phase of the parasite in B. glabrata to PZQ. To the best of our knowledge, this method was never applied before on the Egyptian B. alexandrina snails infected with S. mansoni. The aim of the present work is to evaluate induction of S. mansoni resistance to PZQ by using successive drug treatments of B. alexandrina snails infected with S. mansoni.
Materials and methods Infection of snails with S. mansoni Laboratory-reared B. alexandrina snails (6–8 mm shell diameter) were supplied by the Department of Medical Malacology at Theodor Bilharz Research Institute (TBRI), Giza, Egypt. Miracidia of S. mansoni were purchased from the Schistosome Biological Supply Program at TBRI, Giza, Egypt. For infection, a total of 350 B. alexandrina snails were individually exposed to 10 freshly collected S. mansoni miracidia for 3 h in 1–2 ml water. Starting from the 30th day of post-infection, exposed snails were examined daily for cercarial shedding by exposing them to fluorescent light for 2 h according to the method described by Lewis et al. (1993). Shedding snails were selected and kept in separate aquaria. Treatment of infected snails with PZQ For treatment of infected snails, the drug was incorporated into the snail food which was prepared from Purina mouse chow ground up with 10% of calcium carbonate and TetraminÕ . Water was also added in order to obtain a smooth paste. The snails were individually weighed using an analytical balance and the snails’ mean weight was used to calculate the drug dosage to be administered. The drug utilized was mixed into the chow and 100 mg from this mixture (chow and drug) were weighed and allowed to consume everyday by each snail. In the present experiment, the mean weight of the group of snails was 600 mg and if the dose of PZQ to be administered was 1000 mg/kg, the amount of drug incorporated into the 100 mg food would be calculated accordingly as follows: 1000:1 000 000 ¼ X:600 mg, i.e., 0.600 mg of PZQ. Throughout the experiment, the snails were individually maintained in plates for cell culture with dechlorinated water. The food with PZQ and water was changed the second day after exposure. Exposure
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of snails to PZQ was repeated three times, with a 1-week interval between the treatments (Couto et al., 2011; Mattos et al., 2007). Standardization of the PZQ dose used for S. mansoni intra-molluscan exposure Two groups of snails, each composed of 12 snails shedding S. mansoni cercariae, were treated with PZQ after 30 d of the infection with the parasite. The first group was exposed to PZQ in a dose of 1000 mg/kg; while for the second group, the dose was 500 mg/kg. A control group composed of 12 snails was kept under the same conditions but without any exposure to the drug. Assessment of the impact of intra-molluscan S. mansoni exposure to PZQ on cercarial shedding To quantify the number of cercariae shed per snail, 12 positive snails from each group were isolated and kept in separate aquaria under complete darkness. Dead snails were removed daily and their number was recorded. After exposure of snails to light, emerged cercariae were counted, the length of cercarial incubation and the periodic cercarial production were determined individually, and this was carried out weekly (Mahmoud, 2011). Infection of mice with S. mansoni and treatment of infected mice A total of 88 mice were infected with approximately 40 S. mansoni cercariae per mouse by subcutaneous injection (Pellegrino & Katz, 1968). According to the intra-molluscan exposure to PZQ, mice were divided into two main large groups: 44 mice were infected with cercariae derived from snails treated with PZQ and 44 mice were infected with cercariae developed from untreated snails. Forty-five days after infection, mice were treated with a single oral dose of PZQ in 2% aqueous solution of Cremophor-ELÕ . The concentration of PZQ was 0, 200, 400, or 800 mg/kg, thus mice were finally divided into eight groups: (1) Sm-Un: Mice infected with S. mansoni unexposed to PZQ. (2) Sm-Un200: Mice infected with S. mansoni unexposed to PZQ during the intra-molluscan phase and treated with PZQ at a dose of 200 mg/kg. (3) Sm-Un400: Mice infected with S. mansoni unexposed to PZQ during the intra-molluscan phase and treated with PZQ at a dose of 400 mg/kg. (4) Sm-Un800: Mice infected with S. mansoni unexposed to PZQ during the intra-molluscan phase and treated with PZQ at a dose of 800 mg/kg. (5) Sm-PZQ: Mice infected with S. mansoni exposed to PZQ during the intra-molluscan phase. (6) Sm-PZQ200: Mice infected with S. mansoni exposed to PZQ during the intra-molluscan phase and treated with PZQ at a dose of 200 mg/kg. (7) Sm-PZQ400: Mice infected with S. mansoni exposed to PZQ during the intra-molluscan phase and treated with PZQ at a dose of 400 mg/kg.
Induction of praziquantel resistance in S. mansoni
DOI: 10.3109/13880209.2014.970289
Worm recovery Thirty-three days after treatment, all groups of mice were killed by cervical dislocation and dissected. Schistosomes were then removed from the hepatic portal system by the perfusion technique (Duvall & DeWitt, 1967; Smithers & Terry, 1965).
14 12 No. of snails
(8) Sm-PZQ800: Mice infected with S. mansoni exposed to PZQ during the intra-molluscan phase and treated with PZQ at a dose of 800 mg/kg.
3
Exposed to PZQ
Unexposed to PZQ
10 8 6 4 2 0 0
1
2
3
4
5
6
7
8
Weeks
Figure 1. Survival of infected snails.
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Production, maturity, and death of S. mansoni eggs in different groups of mice The oogram pattern was examined to show the different developmental stages of S. mansoni eggs in the small intestines of mice to study the effect of different concentrations of PZQ on egg production, maturity, and death of eggs. Also, this effect was studied in relation to the intra-molluscan exposure of the parasite to the drug. After the perfusion, a fragment from the median part of the small intestine (10 mm) was cut-off and processed for oogram. Eggs were then counted and classified according to the different stages of development (Pellegrino et al., 1962). The eggs were classified into immature, mature, or dead.
Table 1. Numbers of cercariae shed by individual snails exposed and unexposed to PZQ. Number of cercariae/ snail/stimulation Groups of snails
Mean ± SD (range)
Exposed to 500 mg/kg (no. of examinations ¼ 24) Unexposed to PZQ (no. of examinations ¼ 29)
81.7 ± 45.8 (6–200)
Independent samples t-test (p value) 0.0001
192.7 ± 106.6 (0–412)
The number of examinations: total number of snail examinations for shedding during the examination period.
Statistical analyses Statistical analyses were run on an IBM compatible PC using SPSS for windows statistical package (SPSS Statistics Base 17.0; SPSS Inc., Chicago, IL). All parameters were treated as parametric data. They are expressed as mean ± SD (min– max). Comparison between two means was done using independent samples’ t-test. The survival distributions of the two groups of snails were compared by using the log-rank (Mantel–Cox) test. A multiple linear regression model was used to predict S. mansoni worm count based on the intramolluscan exposure to PZQ and the drug dose used to treat mice. The p value below 0.05 was considered significant.
Results Standardization of the PZQ dose used for S. mansoni intra-molluscan phase The results of the present study showed that snails treated with a dose of 1000 mg/kg PZQ stopped shedding cercariae after treatment until they died, but snails exposed to 500 mg/ kg shed cercariae. Effect of PZQ on survival of infected B. alexandrina snails The snails exposed to the drug survived 6 weeks after treatment, while the unexposed snails survived up to 7 weeks (Figure 1). The survival distributions of the two groups were compared using the log-rank (Mantel–Cox) test, and the difference was insignificant (p ¼ 0.396). Effect of PZQ on cercarial production by B. alexandrina snails Cercariae from snails exposed and unexposed to PZQ were counted weekly starting from the 37th day after infection until
Table 2. Schistosoma mansoni worm recovery from the different groups of mice.
Mice groups Sm-PZQ (no. ¼ 8) Sm-Un (no. ¼ 7) Sm-PZQ200 (no. ¼ 8) Sm-Un200 (no. ¼ 8) Sm-PZQ400 (no. ¼ 9) Sm-Un400 (no. ¼ 7) Sm-PZQ800 (no. ¼ 7) Sm-Un800 (no. ¼ 9)
Mean ± SD 15.00 ± 2.39 11.71 ± 1.11 9.00 ± 2.56 6.63 ± 1.69 7.78 ± 1.99 1.14 ± 1.07 1.00 ± 0.82 0.22 ± 0.44
(11–18) (10–13) (6–14) (5–10) (6–12) (0–3) (0–2) (0–1)
t-Test (p value) 0.005 0.046 0.000 0.028
Percentages of worm reduction – – 40 43 48 90 93 98
No.: the number of surviving mice.
all the infected snails died after 7 weeks. Snails exposed to PZQ shed significantly (p ¼ 0.0001) lower numbers of cercariae than those unexposed to the drug (Table 1). Schistosoma mansoni worm recovery from different groups of mice In the two untreated mouse groups, the numbers of worms recovered from mice infected with S. mansoni cercariae exposed to PZQ during the intra-molluscan phase (Sm-PZQ) were significantly higher than those recovered from mice infected with S. mansoni cercariae unexposed to the drug (Sm-Un) (Table 2). In the three groups of mice infected with S. mansoni exposed to PZQ during the intra-molluscan phase (Sm-PZQ200, Sm-PZQ400, and Sm-PZQ800), the increase of the PZQ dose from 200 mg/kg to 400 mg/kg was used to treat mice, and then 800 mg/kg resulted in a gradual decrease in the
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Table 3. Percentages of the different stages of S. mansoni eggs recovered from the groups of mice.
Mice groups
Immature, mean ± SD (min–max)
Mature, mean ± SD (min–max)
Dead, mean ± SD (min–max)
Sm-PZQ (no. 8) Sm-Un (no. 7) t-Test (p value) Sm-PZQ200 (no. 8) Sm-Un200 (no. 8) t-Test (p value) Sm-PZQ400 (no. 9) Sm-Un400 (no. 7) t-Test (p value) Sm-PZQ800 (no. 7) Sm-Un800 (no. 9)
50.5 ± 3.0 (47.6–54.7) 49.2 ± 4.2 (44.0–54.4) 0.529 30.8 ± 7.3 (24.3–47.3) 9.6 ± 2.0 (7.1–12.0) 0.000 5.6 ± 0.1 (5.5–5.8) 0.0 0.000 0.0 0.0
42.0 ± 2.9 (37.2–44.7) 44.7 ± 3.9 (39.6–50.0) 0.148 42.4 ± 9.0 (23.7–52.6) 36.1 ± 1.8 (33.3–39.3) 0.070 20.8 ± 4.2 (15.7–27.7) 55.6 ± 18.5(33.3–83.3) 0.002 0.0 0.0
7.4 ± 1.3 (5.6–9.5) 5.8 ± 0.6 (5.1–6.5) 0.010 26.4 ± 4.9 (18.4–33.3) 54.3 ± 2.3 (51.8–59.2) 0.000 73.6 ± 4.2 (66.6–78.9) 44.3 ± 18.5 (16.6–66.6) 0.005 100 100
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No.: the number of surviving mice.
Table 4. Multiple linear regression models to predict S. mansoni worm count based on the intra-molluscan exposure to PZQ and the drug dose used to treat mice. Unstandardized coefficients Model Constant Intra-molluscan exposure to PZQ Drug concentration used to treat mice
B
Standard error
Significance (p value)
10.477 2.885
0.548 0.555
0.000 0.000
0.015
0.001
0.000
number of worms recovered from them (Table 2). The same condition was observed in the other three groups of mice that were infected with S. mansoni unexposed to PZQ during the intra-molluscan phase: Sm-Un200, Sm-Un400, and SmUn800 (Table 2). Significant differences were found between different corresponding groups, i.e., Sm-PZQ200 with SmUn200, Sm-PZQ400 with Sm-Un400, and Sm-PZQ800 with Sm-Un800. Production, maturity, and death of S. mansoni eggs in different groups of mice In the two untreated mice groups (Table 3), the differences in the percentages of immature and mature eggs recovered from the two groups were insignificant. However, the percentage of dead eggs was significantly higher in the mice infected with S. mansoni exposed to the drug during the intra-molluscan phase (Sm-PZQ) than the other group (Sm-Un). In the two groups of mice treated with PZQ at a dose of 200 mg/kg, the percentages of the immature eggs were significantly higher in mice infected with S. mansoni exposed to PZQ during the intramolluscan phase (Table 3); in contrast, the percentages of the dead eggs were significantly lower. In the two groups of mice treated with PZQ at a dose of 400 mg/kg, immature eggs could not be detected in mice infected with S. mansoni previously unexposed to PZQ and significantly higher percentages of mature eggs were found in mice infected with S. mansoni exposed to PZQ during the intra-molluscan phase (Tables 3). In the two groups of mice treated with PZQ at a dose of 800 mg/kg, all the detected eggs were dead (Tables 3).
Prediction of worm count based on the intra-molluscan exposure to PZQ and the drug concentration used to treat mice The results of the multiple linear regression analysis showed that the overall model was significant, p50.001, R2 ¼ 81.4% (Table 4).
Discussion PZQ is a broad-spectrum, highly efficacious, and safe anthelmintic against trematode and cestode infections in humans and animals, respectively (Chai, 2013). Emerging problems include the appearance of PZQ-resistant strains or isolates in S. mansoni and S. japonicum in the laboratory (Liang et al., 2002, 2010) or in the field (Wang et al., 2012). According to the present results, based on cercarial shedding, the recommended sublethal dose of PZQ for exposure of S. mansoni intra-molluscan phase in B. alexandrina was 500 mg/kg. The present results showed that the snails exposed to the drug survived 6 weeks after treatment with subcurative doses of 500 mg/kg, while the unexposed snails survived up to 7 weeks (Figure 1). The difference between survival of the two groups of snails was insignificant (p ¼ 0.396). This result indicated that exposure of B. alexandrina snails to the drug did not affect their survival. Despite the differences in survival rates between B. alexandrina and B. glabrata, a similar result was reported by Mattos et al. (2007), after exposure of B. glabrata to PZQ. Snails unexposed to PZQ shed higher numbers of cercariae than those exposed to the drug, with significant differences between the two groups (p ¼ 0.000). This finding is in accordance with the results reported by other authors in different studies after the intra-molluscan exposure of S. mansoni to PZQ in B. glabrata snails (Mattos et al., 2007; Riley & Chappell, 1990). Results of the current study showed that the numbers of worms recovered from mice infected with S. mansoni unexposed to PZQ were significantly lower than those recovered from the mice infected with S. mansoni exposed to the drug during the intra-molluscan phase (Table 2). This may indicate that stress posed by previous exposure of S. mansoni intra-molluscan stages to PZQ had positive effect on success of S. mansoni to colonize mice and achieve
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DOI: 10.3109/13880209.2014.970289
maturity. In the mouse groups treated with PZQ, the increase of the drug dose from 200 mg/kg to 400 mg/kg, and then 800 mg/kg resulted in a gradual decrease in the number of recovered worms (Table 2). Comparable results were reported by Couto et al. (2011). By comparing the number of worms recovered from different groups of mice infected with parasites previously unexposed to the drug during the intramolluscan phase with their corresponding exposed groups, the numbers were significantly higher in the groups infected with schistosomes previously exposed to the drug; significant differences were found between them (Table 2). These results were in agreement with the findings reported by Couto et al. (2011), which may indicate that intra-molluscan exposure of S. mansoni has induced resistance to the drug in the adult stage. In mice infected with S. mansoni, oviposition begins about day 30 of post-infection. Immature eggs require about 6 d for the miracidium to develop (Pellegrino et al., 1962). Studying the oogram pattern permits a direct assessment of the production and development of S. mansoni eggs, and their passage from mesenteric vessels into the intestinal lumen in mice (Mati & Melo, 2013). A drug that shows a chemotherapeutic effect causes a progressive change in the percentages of different developmental stages with a decrease in immature stages and an increase in the percentages of mature and/or dead eggs (Delgado et al., 1992). These changes are due to the interruption of oviposition in the intestinal wall and the maturation of viable eggs already there (Delgado et al., 1992; Pellegrino et al., 1962). Results of the present work indicate that without mice treatment the differences between the two mice groups as regards percentages of immature or mature S. mansoni eggs were insignificant (Table 3). This may indicate that the intra-molluscan exposure to PZQ had no effect on the success of S. mansoni eggs to achieve maturity in the murine host. After treatment, the immature eggs showed significantly higher percentages in the mice group infected with S. mansoni exposed to PZQ during the intra-molluscan phase compared with the unexposed group denoting oviposition by a greater number of surviving worms (Table 3). Thus, the results of oogram pattern confirm that the intra-molluscan exposure of S. mansoni has induced drug resistance in the adult stage. To confirm the present results, a multiple linear regression model was carried out (Table 4). The model indicated that the worm count in different groups of mice was significantly affected by the intra-molluscan exposure to PZQ and the drug concentration used to treat infected mice. The present study has important implications for the use of B. alexandrina to induce PZO resistance in S. mansoni. Also, it is the first report of laboratory induction of PZQ resistance in an Egyptian S. mansoni strain in B. alexandrina.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References Araujo N, de Souza CP, Passos LK, et al. (1996). Susceptibility to chemotherapeutic agents of Schistosoma mansoni isolates from patients treated with oxamniquine and praziquantel and not cured. Rev Soc Bras Med Trop 29:467–76.
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