Resistance to niclosamide in Oncomelania hupensis, the intermediate host of Schistosoma japonicum: should we be worried?

As the currently only available molluscicide, niclosamide has been widely used for snail control for over 2 decades in China. There is therefore a concern about the emergence of niclosamide-resistant snail populations following repeated, extensive use of the chemical. The purpose of this study was to investigate the likelihood of niclosamide resistance in Oncomelania hupensis in China. Active adult O. hupensis snails derived from 20 counties of 10 schistosomiasis-endemic provinces of China, of 10 snails in each drug concentration, were immersed in solutions of 1, 0.5, 0.25, 0.125, 0.063, 0.032, 0.016 and 0.008 mg L-1 of a 50% wettable powder of niclosamide ethanolamine salt (WPN) for 24 and 48 h at 25 °C, and the median lethal concentration (LC50) was estimated. Then, the 24- and 48-h WPN LC50 values were compared with those determined in the same sampling sites in 2002. The results indicated that the 24- and 48-h WPN LC50 values for O. hupensis were not significantly different from those determined in 2002 (P = 0.202 and 0.796, respectively). It is concluded that the current sensitivity of O. hupensis to niclosamide has not changed after more than 2 decades of repeated, extensive application in the main endemic foci of China, and there is no evidence of resistance to niclosamide detected in O. hupensis.

Efficacy of artemether and artesunate in mice infected with praziquantel non-susceptible isolate of Schistosoma japonicum.

Praziquantel is currently the only drug of choice for the treatment of human Schistosoma japonicum infections, and praziquantel-based chemotherapy has been proved to be generally effective to control the morbidity and reduce the prevalence and intensity of S. japonicum infections. However, the potential emergence of praziquantel resistance in S. japonicum seriously threatens the elimination of this neglected tropical disease in China. The purpose of this study was designed, in mouse animals, to evaluate the in vivo efficacy of artemether and artesunate against praziquantel non-susceptible S. japonicum. Mice infected with a praziquantel non-susceptible isolate and a praziquantel-susceptible isolate of S. japonicum were treated with artemether and artesunate at a single oral dose of 300 mg/kg given once on each of days 7-8 and 35-36 post-infection to assess the efficacy against juvenile and adult worms. Administration with artemether and artesunate at a single oral dose of 300 mg/kg on each of days 7-8 post-infection resulted in total worm burden reductions of 72.8 and 73.5% in mice infected with praziquantel-susceptible S. japonicum, and 77.9 and 74.1% in mice infected with the non-susceptible isolate (both P values >0.05), while the same treatments given on days 35-36 post-infection reduced total worm burdens by 71.4 and 69.6% in mice infected with the susceptible isolate, and 75.3 and 69.6% in mice infected with the non-susceptible parasite (both P values >0.05). It is concluded that there is no evidence for reduced susceptibility of artemether and artesunate in praziquantel non-susceptible S. japonicum.

Is there a reduced sensitivity of dihydroartemisinin against praziquantel-resistant Schistosoma japonicum?

Praziquantel is currently the only drug of choice for the treatment of human schistosomiases. However, it has been proved that Schistosoma japonicum subjected to drug pressure may develop resistance to praziquantel. To evaluate the efficacy of dihydroartemisinin against praziquantel-resistant S. japonicum, mice infected with a praziquantel-resistant isolate and a praziquantel-susceptible isolate of S. japonicum were treated with dihydroartemisinin at a single oral dose of 300 mg/kg given once on each of 35-36 post-infection days, while infected but untreated mice served as controls. All mice were sacrificed 50 days post-infection, and the worm burden reductions were estimated. Administration of dihydroartemisinin at a single oral dose of 300 mg/kg on each of 35-36 post-infection days reduced total worm burdens of 69.8% and female worm burdens of 86% in mice infected with the praziquantel-susceptible isolate, and total worm burdens of 66.1% and female worm burdens of 85.1% in mice infected with the praziquantel-resistant isolate (both P values > 0.05). It is concluded that the sensitivity of artemisinin derivative dihydroartemisinin does not reduce in praziquantel-resistant S. japonicum.

In vivo activity of dihydroartemisinin against Schistosoma mansoni schistosomula in mice.

Dihydroartemisinin, an anti-malarial agent, has been shown to exhibit activity against Schistosoma japonicum and S. mansoni. The purpose of the present study was to investigate the in vivo activity of dihydroartemisinin against juvenile S. mansoni and the changes to the genital system among worms surviving drug treatment. Mice were infected with 200 S. mansoni cercariae each and randomly assigned to groups. Dihydroartemisinin at a single oral dose of 300 mg/kg was given to mice on Days 14 or 16, 18, 20, 21, 22, 24, 26 or 28 post-infection, to assess the efficacy of dihydroartemisinin against juvenile S. mansoni. Mice were treated with dihydroartemisinin using various protocols with the total drug dose of 900 mg/kg, to investigate the efficacy of dihydroartemisinin against the schistosomula of S. mansoni. In addition, changes to the genital system among worms surviving dihydroartemisinin treatment, were recorded. An oral dose of dihydroartemisinin of 300 mg/kg was given to mice on Days 14, 16, 18, 20, 21, 22, 24, 26 or 28 days post-infection; this resulted in a 65.0-82.4% reduction in total worm burden and a 70.9-83.0% female worm burden. Better results were seen when treatment was given 20-24 days post-infection. Administration of multiple-dose and low-oral-dose dihydroarteminisinin (at doses of 90, 180, 300 and 450 mg/kg) at different times, reduced total worm burdens by 88.7-99.1% and female worm burdens by 93.2-99.5%. The egg tubercles in mice livers were significantly reduced following treatment; in some mice no egg tubercles were found. These findings indicate dihydroartemisinin exhibits high in vivo activity against the schistosomula of S. mansoni. It causes damage to the genital system of worms, influences the development of of S. mansoni worms, reduces the oviposition of surviving worms and enhances the formation of granulomas around tissue-trapped eggs, thereby reducing damage to the infected mammalian host.

Effect of the in vivo activity of dihydroartemisinin against Schistosoma mansoni infection in mice.

Dihydroartemisinin, formerly known as an antimalarial drug, is the main metabolite of the mother compound artemisinins, as well as of artemether and artesunate. It has been shown that the drug exhibits antischistosomal efficacy against Schistosoma japonicum. The purpose of the current study was to assess the in vivo effect of dihydroartemisinin against Schistosoma mansoni infection in mice. Drugs at a single oral dose of 300 mg/kg were given to mice to assess the efficacy against different developmental stages of the parasite; juvenile and adult S. mansoni were treated with single doses of dihydroarteminisin with different regimens (at 200, 300, 400 or 600 mg/kg) in the stage of drug sensitivity, and the dose-response relationship was assessed; and the effect of multiple doses (at 200, 300 or 400 mg/kg) on juvenile and adult S. mansoni was also observed. The results showed that a single oral dose (300 mg/kg) of dihydroartemisinin reduced total worm burdens by 13.8-82.1% and female worm burdens by 13-82.8%, and the greatest reductions were seen when treatment was given on day 21 post-infection, with total and female worm burden reductions of 82.1% and 82.8%. Administration of a single oral dose of dihydroartemisinin on day 21 post-infection with different drug dosage (at 200, 300, 400 or 600 mg/kg) reduced total worm burdens by 70.3-87.3% and female worm burdens by 73.5-92.4%, depending on dosage. Similar treatments given on day 49 post-infection reduced total worm burdens by 48.7-68.73% and female worm burdens by 63.25-94.6%. There was obvious dose-response relationship of dihydroartemisinin against the schistosomula and adult worms of S. mansoni observed. Administration with dihydroartemisinin at oral doses of 200, 300 and 400 mg/kg, given once on each of days 20-22 post-infection of three successive days, reduced total worm burdens by 88.5-90.1% and female worm burdens by 89.2-92.1%, depending on dosage. Similar treatments given once on each of days 48-50 post-infection reduced total worm burdens by 60-70.3% and female worm burdens by 77.5-94.9%. It is concluded that dihydroartemisinin exhibits in vivo activity against various developmental stages of S. mansoni, particularly the 21-day schistosomula, and there is obvious dose-response relationship of dihydroartemisinin against the schistosomula and adult worms of S. mansoni observed.

In vivo efficiency of praziquantel treatment of single-sex Schistosoma japonicum aged three months old in mice.

Schistosomiasis is a major neglected tropical disease mainly caused by Schistosoma haematobium, S. japonicum and S. mansoni, and results in the greatest disease burden. Mass drug administration (MDA) with praziquantel (PZQ), a single drug only available for the disease, has played a vital role in schistosomiasis control. Therefore, any possibility of selection of the parasites for PZQ resistance or low sensitivity may hamper the 2030's target of global disease elimination. We had experimentally demonstrated the long-term survival and reproductive potential of single-sex (of either sex) S. japonicum infections in definitive hosts mice. What has not yet been adequately addressed is whether the long live single-sex schistosomes remain sensitive to PZQ, and what reproduction potential for those schistosomes surviving treatment may have. We therefore performed experimental mice studies to explore the treatment effectiveness of PZQ (at total doses of 200 or 400 mg/kg, corresponding to the sub-standard or standard treatment doses in humans) for single-sex S. japonicum aged three months old. The results showed that no treatment efficiency was observed on female schistosomes, whereas on male schistosomes only at PZQ 400 mg/kg a significant higher efficiency in reducing worm burdens was observed. Moreover, either schistosome males or females surviving PZQ treatment remained their reproduction potential as normal. The results indicate that long (i.e., three months) live single-sex S. japonicum can easily survive the current treatment strategy, and moreover, any schistosomes, if with PZQ resistance or low sensitivity, could be easily transmitted in nature. Therefore, in order to realize the target for the national and the global schistosomiasis elimination, there is undoubtedly a great need for refining PZQ administration and dosage, looking for alternative therapies, and/or developing vaccines against schistosome.

Effects of artemether, artesunate and dihydroartemisinin administered orally at multiple doses or combination in treatment of mice infected with Schistosoma japonicum.

Artemether and artesunate, derivatives of the antimalarial artemisinin, as well as their main metabolite, dihydroartemisinin, all exhibit antischistosomal activities. The purpose of the current study was to compare the effects of artemether, artesunate and dihydroartemisinin administered orally at multiple doses or combination in treatment of mice infected with Schistosoma japonicum. We carried out experiments with mice, infected with 40 cercariae of S. japonicum, and treated with artemether, artesunate and dihydroartemisinin (all at a single dose of 300 mg/kg, and the dose of the mixed three drugs is also 300 mg/kg) at multiple doses or combination therapy on days 6-8 or 34-36 post-infection. Administration with artemether, artesunate or dihydroartemisinin for 3 successive days reduced total worm burdens by 79.5-86% (30.86 ± 4.98 of mean total worm burden in control), female worm burdens by 79.4-86.7% (11.29 ± 2.63 of mean female worm burden in control) (all P values <0.01 vs. control), depending on different treatment protocols given on days 6-8 post-infection. However, no differences were seen between each treatment group (all P > 0.05). While the same treatment was given on days 34-36 post-infection, total worm burden reductions of 73.8-75.8% were achieved (29.44 ± 3.36 of mean total worm burden in control), which were significant when compared with the untreated control group (all P values <0.01). In all different treatment groups, female worm reductions (ranging from 88.7% to 93.1%, while the mean female worm burden in control is 10.33 ± 1.80) were consistently higher than the total worm reductions, resulting always in significantly lower female worm burdens when compared to the corresponding control (all P values < 0.01). However, there were no significant differences found between each treatment group (all P values >0.05). It is concluded that artemether, artesunate and dihydroartemisinin can be used to control schistosomiasis japonica, as a strategy to prevent S. japonicum infection. Administration with artemether, artesunate and dihydroartemisinin at multiple doses or in combined treatment damages both juvenile and adult S. japonicum, without statistically significant differences among the three drugs at the same dose.

[Cloning and expression of Schistosoma japonicum VCP gene and its mRNA expression levels in different stages].

OBJECTIVE: To prokaryotically express the valosin-containing protein (VCP) of Schistosoma japonicum, and analyze its VCP mRNA expressions in the cercaria, schistosomulum, adult worm (female and male worms) and egg. METHODS: RNA of S. japonicum eggs were extracted, and reversely transcribed into cDNA. The VCP gene of S. japonicum was amplified by using polymerase chain reaction (PCR), and subcloned into the prokaryotically expressed vector pET15b. The recombined plasmid was transformed into BL21 cells, and the expression of the target gene was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). The recombinant protein was yielded through the purification of inclusion body, and identified by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The RNA (s) of cercaria, schistosomulum, female adult worm, male adult worm, and egg of S. japonicum were extracted, digested with DNase, purified, and reversely transcribed into cDNA. The mRNA expressions of the VCP gene in various developmental stages of S. japonicum were determined by using fluorescence-based quantitative real-time PCR. RESULTS: The VCP gene of S. japonicum was yielded by PCR amplification, and the recombinant protein was obtained through recombinant plasmid expression and purification of inclusion body. The highest VCP mRNA expression in S. japonicum cercaria was detected by the fluorescence-based quantitative real-time PCR, while low expressions were found in the schistosomulum, egg, female and male adult worms. CONCLUSION: The recombinant protein encoded by the VCP gene of S. japonicum is successfully obtained, and the VCP mRNA expression is determined in various developmental stages of S. japonicum.

[Surveillance and forecast system of schistosomiasis in Jiangsu Province. VI. Detection technology of water infectivity based on enrichment of Schistosoma japonicum cercariae on water surface].

OBJECTIVE: To explore the enrichment technique of Schistosoma japonicum cercariae on the water surface, so as to establish a new method combined with the existing technology to detect the cercarial infested water body quickly and sensitively. METHODS: Soybean oil, gasoline, kerosene and isophorone were screened as expanding agents. The cercariae were enriched by the thrust of the expanding agents when diffusing on the water surface, and PE adsorption film and C-6 film were applied to seize them so as to determine the infectivity of the water quickly. The relationship between the dose of expanding agents and diffusion radius were explored. RESULTS: Gasoline, kerosene and isophorone were suitable expanding agents, and the diffusion effect of isophorone was the best. After the enrichment by the expanding agents, the detection rate of cercariae of the method seizing cercariae with the film significantly improved in the water. CONCLUSION: This new method could effectively improve the detection rate of the cercarial infested water and is suitable for the low-degree infested water.

[Preparation and molluscicidal effect of 5% niclosamide ethanolamine granules].

OBJECTIVE: To explore the preparation of 5% niclosamide ethanolamine granules and evaluate its molluscicidal effect. METHODS: The optimal formula was obtained by the selection of wetting agents, dispersants, adhesives and carriers. The molluscicidal effect of 5% niclosamide ethanolamine granules was measured by the spray methods in the laboratory and field. RESULTS: The mixed 5% niclosamide ethanolamine, 0.2% sodium lauryl sulfate, and 1% alkylphenol sulfonic polyxyethylene ether sulfonate were crushed by gas flow, and then mixed with 93.7% quartz sand and 0.1% polyvinyl alcohol water solution, drying to 5% niclosamide ethanolamine granules. The density, bulk density, and moisture of 5% niclosamide ethanolamine granules were 1.4 g/ml, 1.3 g/ml, and 2.4%, respectively, and the hot storage stability was qualified. Under the lab condition, the death rates of Oncomelania hupensis snails sprayed with 0.5 g/m2 (7 d) or 1.0 g/m2 (1 d) of 5% niclosamide ethanolamine granules were higher than 95%. In the field, the death rates of the snails sprayed with 0.5 g/m2 (7 d) or 1.0 g/m2 (1 d) were higher than 85%. CONCLUSION: There is a high molluscicidal effect of 5% niclosamide ethanolamine granules and it is suitable for field application.

[Distribution of niclosamide spreading oil on water surface and its efficacy against cercariae of Schistosoma japonicum].

OBJECTIVE: To investigate the distribution and spreading speed of niclosamide spreading oil, as well as its effect against cercariae of Schistosoma japonicum. METHODS: The foamed plastic with a diameter of 4 mm served as a buoyage, which was placed at the center of the still water surface. The niclosamide spreading oil was dropped at 0.5 cm from the buoyage, the floating distance of the buoyage was observed, and the spreading speed and area of the niclosamide spreading oil were measured. A cylindrical bucket (at a diameter of 40 cm and height of 50 cm) was filled with de-chlorinated water at a temperature of 25 +/- 1 degrees C, and then 60 microl of the spreading oil was dropped at the center of the water surface. At 10 cm and 20 cm from the center, 1 ml water was sampled at water depths of 10, 20, 30, 40 cm and 50 cm, respectively, and the niclosamide concentrations were determined by using high-performance liquid chromatography in each sample. The niclosamide spreading oil was diluted into solutions at effective concentrations of 1.25 mg/L and 0.63 mg/L with ethanol, and then 10 microl of each solution was added to 24-well plates which contained S. japonicum cercariae to yield the niclosamide concentration of 6.25 x 10(-3) mg/L and 3.13 x 10(-3) mg/L per well, respectively. The survival of the cercariae was observed at different time. RESULTS: The spreading speeds and areas were 59, 55, 71, 90, 111, 122 cm/s and 153 cm/s, and 5.31, 5.89, 7.07, 10.06, 12.56, 15.20 m2 and 16.61 m2, respectively, while dropping 20, 30, 40, 50, 60, 70 microl and 80 microl of the niclosamide spreading oil on water surface. The spreading showed an accelerating trend with the increasing dropping volume, and there was a good linear relationship observed between them. In addition, the spreading area also enlarged with the increase in the dropping volume. After dropping 60 microl of the niclosamide spreading oil on water surface, the peak concentration of niclosamide reached 1.27 mg/L on water surface, and remained more than 0.07 mg/L 2 h later. However, the concentration of niclosamide was all lower than 0.04 mg/L at 10 cm under surface or more. Following the treatment with 6.25 x 10(-3) mg/L of niclosamide spreading oil for 1 min, all the cercariae were dead, while the mortality rates of the cercariae were 0, 1.39%, 13.89%, 19.44%, 43.06%, 69.44% and 79.17% at 1, 2, 3, 5, 10, 20 min and 30 min, respectively, after the treatment with 3.13 x 10(-3) mg/L of the drug. CONCLUSIONS: The niclosamide spreading oil is fast to spread and is kept retention for a long time on water surface, and exhibits high activity against S. japonicum cercariae, and it can be used for killing the cercariae on water surface and interrupting the transmission of schistosomiasis in the endemic field.

[Studies on resistance of Schistosoma to praziquantel XIIV sensitivity of Schistosoma japonicum to praziquantel in marshland and lake endemic regions of Chinese Mainland].

OBJECTIVE: To compare the sensitivities of different isolates of Schistosoma japonicum in marshland and lake regions of Chinese Mainland to praziquantel, so as to provide experimental evidence for establishing the techniques of detecting and monitoring praziquantel sensitivity. METHODS: Mice were infected with cercariae released from the S. japonicum-infected snails collected from the marshland and lake endemic regions of Hunan, Hubei, Jiangxi, Anhui and Jiangsu provinces, grouped, and treated with praziquantel at single oral doses of 37.5, 75, 150, 300 mg/kg and 600 mg/kg, while mice infected but treated with 2.5% Cremophor EL served as controls. The worm burden reductions caused by praziquantel treatment were observed, and the 50% effective dose (ED50 value) was calculated. RESULTS: The administration with praziquantel at single oral doses of 37.5, 75, 150, 300 mg/kg and 600 mg/kg achieved worm burden reductions of 10.37%-19.81%, 23.22%-33.09%, 39.25%-49.61%, 62.87%-74.44% and 91.26%-98.09%, respectively, and no significant differences in worm burden reductions of different isolates of S. japonicum were detected following treatment with different doses of praziquantel (P > 0.05). The praziquantel ED50 values against different isolates of S. japonicum ranged from 134.1 to 186.7 mg/kg, and no significant differences of praziquantel ED50 values were found among different isolates (P > 0.05). CONCLUSIONS: There were no significant differences of praziquantel sensitivities of different isolates of S. japonicum in marshland and lake regions of Chinese Mainland. Praziquantel ED50 value against schistosomes, as a quantitative index, can truly reflect the sensitivity of schistosome populations to praziquantel, which can be used to assess the risk of emergence of praziquantel resistance in schistosomes.

[Studies on standardization of methods for screening molluscicides in laboratory IV sensitivity of Oncomelania snails from different months to niclosamide].

OBJECTIVE: To understand the sensitivity of Oncomelania snails collected from different months to niclosamide, so as to provide the scientific evidence for the standardization of methods for molluscicide screening and efficacy evaluation in laboratory. METHODS: The snails collected from the marshland of Zhenjiang City, Jiangsu Province month by month from June 2010 to May 2011. After being raised in laboratory for 24 h, the snails were randomly grouped, and then immersed in different concentrations of 50% wettable powder formulation of niclosamide ethanolamine salt at (25 +/- 1) degrees C with a humidity of 60%. The dead snails were identified and counted, and the mortality rate of snails and median lethal concentration (LC50) were calculated. RESULTS: When the snails were immersed in the solutions of niclosamide at concentrations of more than 0.5 mg/L for 24 h, all the snails collected from different months were dead, while 60%-100% of the snail mortality was achieved for 0.250 mg/L niclosamide, 3%-27% for 0.125 mg/L niclosamide, and 3%-20% for 0.062 5 mg/L niclosamide. When the concentration was lower than 0.032 mg/L, the niclosamide was not toxic to the snails within 24 h. The LC50 value was 0.140-0.209 mg/L for 24 h. When the snails were immersed in the solutions of niclosamide at concentrations of more than 0.5 mg/L for 48 h, all the snails collected from different months were dead, while 90%-100% of the snail mortality was achieved for 0.250 mg/L niclosamide, 3%-57% for 0.125 mg/L niclosamide, 3%-13% for 0.062 5 mg/L niclosamide, and 0-10% for 0.032 mg/L niclosamide. When the concentration was lower than 0.016 mg/L, the niclosamide was not toxic to the snails within 48 h. The LC50 value was 0.112-0.170 mg/L for 48 h. There were no significant differences in the mortality of snails caused by niclosamide treatment observed at 24 and 48 h (P values = 0.374 and 0.267, respectively). CONCLUSIONS: There are little changes in the sensitivity of snails collected from different months to niclosamide, with minor fluctuations in LC50 values. Such a finding indicates that the snails collected from different months have few effects on screening of molluscicides and efficacy evaluation.

A strategy for emergency treatment of Schistosoma japonicum-infested water.

BACKGROUND: Schistosomiasis japonica, caused by contact with Schistosoma japonicum cercaria-infested water when washing, bathing or production, remains a major public-health concern in China. The purpose of the present study was to investigate the effect of a suspension concentrate of niclosamide (SCN) on killing cercaria of S. japonicum that float on the water surface, and its toxicity to fish, so as to establish an emergency-treatment intervention for rapidly killing cercaria and eliminating water infectivity. RESULTS: At 30 min after spraying 100 mg/L SCN, with niclosamide dosages of 0.01, 0.02, 0.03, 0.04 g/m², the water infectivity reduced significantly and no infectivity was found at 60 min after spraying SCN. The surface of static water was sprayed with 100 mg/L SCN, the peak concentration was found at 0 min, and the solution diffused to site with a water depth of 10 cm after 10 min. 30 min later, SCN diffused to the whole water body, and distributed evenly. After spraying 100 mg/L SCN onto the surface of the water with a volume of(3.14 × 202 × 50)cm³, with niclosamide dosages of 0.02 g/m², 96 h later, no death of zebra fish was observed. CONCLUSIONS: By spraying 100 mg/L SCN, with a niclosamide dosage of 0.02 g/m² onto the surface of S. japonicum-infested water, infectivity of the water can be eliminated after 30-60 min, and there is no evident toxicity to fish. This cercaria-killing method, as an emergency-treatment intervention for infested water, can be applied in those forecasting and early warning systems for schistosomiasis.

[Study on preservation of niclosamide in water samples].

OBJECTIVE: To investigate the preservation condition of niclosamide in water samples and establish a high performance liquid chromatography (HPLC) method by solid-phase extraction (SPE) which is used for determination and preservation of niclosamide in water samples. METHODS: Under the simulated setting in laboratory, niclosamide was formulated into the solutions of 0.50, 1.00 mg/L and 2.00 mg/L with fishpond water. The solutions were preserved at 25, 4, -40 degrees C and -40 degrees C (repeated freezing and thawing), respectively, or 0.2% acetic acid and twice volume of acetonitrile were added into the water samples, and then the solutions were passed through the activated column, and stored at room temperature, 16 days later, the column was washed, dried and re-dissolved, and niclosamide was determined by using the HPLC method. RESULTS: The concentration of niclosamide in water samples decreased at 25 degrees C or 4 degrees C, and did not change at -40 degrees C, but decreased after repeated freezing and thawing. No decrease of niclosamide was found in water samples for 16 days by using SPE. The recovery rate of HPLC was 98.12%-100.06% and the relative standard deviation (RSD) was less than 5%. CONCLUSIONS: The preservation of niclosamide in water samples is frozen and protected from light without repeated freezing and thawing. The SPE method does not need to be protected from light and freezing, which can simply and effectively purify, concentrate and store niclosamide in water samples.

[Studies on resistance of Schistosoma to praziquantel XIII resistance of Schistosoma japonicum to praziquantel is experimentally induced in laboratory].

OBJECTIVE: To investigate the possibility of the emergence of praziquantel resistance in Schistosoma japonicum in Mainland China under drug pressure. METHODS: S. japonicum cercaria were released from the infected Oncomelania hupensis snails collected from the marshland in Hunan Province that was endemic for schistosomiasis japonica and raised in the laboratory of Jiangsu Institute of Parasitic Diseases, and mice were infected. O. hupensis snails were infected with miracidia hatched from the schistosome mature eggs that were isolated from the liver of the infected mice. The life cycles of a field isolate and a laboratory passage isolate of S. japonicum were established in laboratory via the cycle of mouse-snail. The mice were infected with 40 cercariae each, 35 days later post-infection, were grouped randomly into control and resistance-induced groups. All the mice in the control group were sacrificed on day 45 post-infection, and any adult S. japonicum worms in the hepatic and portomesenteric veins were recovered and counted, and the worm burdens were calculated. The mice in the resistance-induced group were administered orally with the sub-curative dose of praziquantel, and were sacrificed 22 days post-treatment. Any adult S. japonicum worms in the hepatic and portomesenteric veins were recovered and counted, and the worm burdens and reduction in the worms recovered which were obviously caused by the praziquantel treatment were calculated. The eggs in the liver of the mice in the resistance-induced group were isolated and hatched to yield miracidia, and then the snails were again infected with the newly hatched miracidia to complete the first-passage inducement. After raising in laboratory at 25 degrees C for 60-70 days post-infection, the infected snails were isolated and shed cercaria, and the mice were infected with the newly released cercaria to start a new passage of resistance-inducement. The oral dose of praziquantel for the first-passage inducement was 100 mg/kg, and an additional 100 mg/kg was given every 2-3 passages. The mice were infected with cercariae of the parasite with 8-passge resistance-inducement and the isolate that was not induced, and 35 days post-infection, were administered with praziquantel at a single oral doses of 300 mg/kg and 600 mg/kg respectively. All the mice were sacrificed 14 days post - treatment, and any adult S. japonicum worms in the hepatic and portomesenteric veins were recovered and counted, and the reductions in the worm burdens were calculated to assess the sensitivity of praziquantel in the parasites after 8-passage resistance-inducement. RESULTS: Two isolates of Jiangsu laboratory passage of Jiangsu and field isolate of Hunan were established in the laboratory, and a total 8-passage resistance -inducement was completed. For the laboratory passage isolate, the worm burden reduction was 22.3% post-treatment with 100 mg/kg praziquantel during the first-passage inducement, and 53.7% post-treatment with 300 mg/kg praziquantel during the 8-passage inducement, appearing that the worm burden reduction increased with the increasing dose of praziquantel. For the field-collected isolate, the worm burden reduction was 66.8% post-treatment with 100 mg/kg praziquantel during the first-passage inducement, and 20.6% post-treatment with 300 mg/kg praziquantel during the 8-passage inducement, indicating that the worm burden reduction markedly decreased with the increasing dose of praziquantel. The worm burden reductions were 71.5% and 97.4% respectively for the mice infected with the non-induced laboratory passage isolate, while administered with praziquantel at doses of 300 mg/kg and 600 mg/kg respectively. After 8-passage treatment with sub-curative praziquantel, the corresponding worm burden reductions decreased to 32.6% and 68.1%, respectively. For the field-collected isolate without inducement, the worm burden reductions in the mice were 70.8% and 97.5% respectively post-treatment with praziquantel at doses of 300 mg/kg and 600 mg/ kg respectively, and the corresponding worm burden reductions decreased to 45.7% and 61.9%, respectively after 8-passage treatment. COCLUSIONS: S. japonicum (strain of Mainland China) is able to develop resistance to praziquantel under continuous drug pressure. However, there are variations in the potential of the emergence of resistance due to various susceptibility of praziquantel among different isolates. The successful establishment of praziquantel-resistant strain of S. japonicum (Mainland China) will provide the basis for exploring the mechanism of praziquantel resistance in S. japonicum, and developing related techniques to detect and monitor praziquantel resistance.

[Studies on resistance of Schistosoma to praziquantel XIV experimental comparison of susceptibility to praziquantel between PZQ-resistant isolates and PZQ-susceptible isolates of Schistosoma japonicum in stages of adult worms, miracidia and cercariae].

OBJECTIVE: To investigate the changes of sensitivity to praziquantel (PZQ) about PZQ-resistant isolates of Schistosoma japonicum established in laboratory by means of the resistance-inducement method during the stages of adult worms, cercariae and miracidia, so as to provide the basis for establishing the sensitivity-detecting technique to praziquantel. METHODS: A Jiangsu laboratory-maintaining isolate and a Hunan field-collecting isolate of S. japonicum that were never treated with PZQ were as PZQ-susceptible isolates, and two PZQ-induced isolates that were established via drug-treated passage in laboratory were as PZQ-resistant isolates. Mice were infected with S. japonicum cercariae collected from above four isolates each. Thirty-five days after the infection, the mice were divided into 6 groups and administered orally with PZQ at dosages of 0, 37.5, 75, 150, 300 mg/kg and 600 mg/kg, respectively. All the mice were sacrificed two weeks after the treatment, and all the adult worms in the hepatic and portomesenteric veins were recovered and counted. The mean worm burden and reductions were calculated and input into Graphpad Prism 5.0 software, and the PZQ ED50 values of four isolates were calculated by the software. The cercariae of above four isolates were exposed to 10(-5), 5 x 10(-6), 10(-6), 5 x 10(-7), 10(-7) mol/L PZQ solutions for 20, 40, 60, 80, 100 min and the changes of tail shedding were observed under a dissecting microscope, then the tail shedding rates of cercariae were calculated. The miracidia of above four isolates were exposed to 5 x 10(-6), 10(-6), 5 x 10(-7), 10(-7) mol/L PZQ solutions for 1, 3 and 5 min and the morphological changes were observed under a dissecting microscope, then the morphological change rates of miracidia were calculated. RESULTS: The PZQ ED50 values of PZQ-susceptible and PZQ-resistant isolates of Jiangsu were 147.7 mg/kg and 565.5 mg/kg, respectively, and the PZQ ED50 values of PZQ-susceptible and PZQ-resistant isolates of Hunan were 151.8 mg/kg and 467.2 mg/kg, respectively. When the cercariae were exposed to 10(-5) mol/L PZQ solution over 20 min, the tail shedding rate of cercariae from PZQ-susceptible isolate of Jiangsu was 68.8%, and the tail shedding rate of cercariae from PZQ-resistant isolate of Jiangsu was 38.2% (P < 0.01). When the cercariae were exposed to 10(-7) mol/L PZQ solution over 100 min, the tail shedding rate of cercariae from PZQ-susceptible isolate of Jiangsu was 15.9%, and the tail shedding rate of cercariae from PZQ-resistant isolate of Jiangsu was 6.7% (P < 0.01). When the cercariae were exposed to 10(-5) mol/L PZQ solution over 20 min, the tail shedding rates of cercariae from PZQ-susceptible isolate of Hunan was 59.4%, and the tail shedding rates of cercariae from PZQ-resistant isolate of Hunan was 54.6% (P < 0.05). When the cercariae were exposed to 5 x 10(-7) mol/L PZQ solution over 40 min, the tail shedding rates of cercariae from PZQ-susceptible isolate of Jiangsu was 34.3%, and the tail shedding rates of cercariae from PZQ-resistant isolate of Jiangsu was 18.4% (P < 0.01). When the miracidia were exposed to 5 x 10(-7) mol/L and 10(-7) mol/L PZQ solutions for 1, 3 and 5 min respectively, the morphological change rates of miracidia from PZQ-susceptible isolates of Jiangsu and Hunan were significantly higher than those of PZQ-resistant isolates (P < 0.01). CONCLUSIONS: PZQ-resistant isolates of S. japonicum has been established in mice with sub-curative doses of PZQ by artificial selection in laboratory, and their sensitivities to PZQ are significantly lower than those of the isolates never treated with PZQ. The drug-resistance could exhibit in the stages of adult worms, cercariae and miracidia. The PZQ ED50 value of adult worms, the tail shedding rates of cercariae and the morphological change rates of miracidia as quantitative indicators can be used for monitoring the S. japonicum sensitivity to PZQ.

[Surveillance and forecast system of schistosomiasis in Jiangsu Province. IV. Establishment of Schistosoma japonicum cercaria-killing method by spraying niclosamide suspension on water surface].

OBJECTIVE: To investigate the effect of suspension concentrate of niclosamide (SCN) on killing cercariae of Schistosoma japonicum on water surface, optimization and impact on fish, so as to establish an emergency-treatment intervention for rapidly killing cercariae and eliminating water infectivity. METHODS: SCN was formulated into different concentrations of solutions, and then the solutions were sprayed on the surface of water containing S. japonicum cercariae. The water infectivity was determined by using mice at 0, 10, 30 min after spraying SCN. SCN was formulated into a solution of 100 mg/L and then sprayed on the surface of the water by using the spraying values of 0.01, 0.02, 0.03 g/m2 and 0.04 g/m2. At 30 min and 60 min after spraying, the water infectivity was determined by using mice. Zebra fish were transferred into the static water, then 100 mg/L SCN (s), using spraying values of 0.01, 0.02, 0.03 g/m2 and 0.04 g/m2, were sprayed on water surface. At 0, 10, 30, 60 min after spraying, the samples were collected at water depths of 0, 10, 20, 30, 40 cm, and niclosamide was determined by using high-performance liquid chromatography. The death of zebra fish was continually observed within 96 h after spraying SCN. RESULTS: At 0, 10, 30 min after spraying 1 000, 100, 10, 1, 0.1 mg/L SCN on water surface, the infectivity of water significantly decreased. At 30 min after spraying 1 000 mg/L and 100 mg/L SCN, no schistosome infectivity was detected in the water. At 30 min after spraying 100 mg/L SCN, with spraying values of 0.01, 0.02, 0.03, 0.04 g/m2, the water infectivity significantly reduced, and no infectivity was found 60 min after spraying SCN. After the surface of static water was sprayed with 100 mg/L SCN, the peak concentration was found at 0 min, and the solution diffused to site with a water depth of 10 cm after 10 min, and 30 min later, SCN diffused to the whole water body, and distributed evenly. After spraying 100 mg/L SCN on the surface of water with a volume of (3.14 x 20(2) x 50) cm3, by using the spraying value of 0.02 g/m2, 96 h later, no death of zebra fish was found. CONCLUSIONS: From 30 to 60 min after spraying 100 mg/L SCN, with the value of 0.02 g/m2, on the surface of S. japonicum-infested water, the water infectivity can be eliminated, and there is no evident toxicity to fish. This cercaria-killing method, as an emergency-treatment intervention for infested water, can be applied in those surveillance and forecast sites.