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目的测定日本血吸虫中国大陆主要流行区现场分离株虫卵、毛蚴和尾蚴阶段对吡喹酮的敏感性,为建立日本血吸虫吡喹酮敏感性检测技术提供基础。方法分别从中国湖南、湖北、江西、安徽、江苏和云南6省日本血吸虫病流行区病人粪便标本中分离虫卵,建立日本血吸虫现场分离虫株;采用实验室已有的3株曼氏血吸虫株为对照。将各虫株虫卵分别孵育于5×10-6、10-6、5×10-7、10-7mol/L吡喹酮溶液中24h后移至清水孵化,观察虫卵的孵化率。将各虫株毛蚴分别暴露于5×10-6、10-6、5×10-7、10-7mol/L吡喹酮溶液中,0、1、5min后观察比较毛蚴的运动及形态学变化。将各虫株尾蚴分别暴露于10-5、6×10-7、4×10-7、10-7mol/L吡喹酮溶液中,0、20、40、60、80、100min后,解剖镜下观察尾蚴的泳动、收缩和断尾率的变化。并将结果与曼氏血吸虫株进行比较。结果经10-6,5×10-7、10-7mol/L吡喹酮溶液中24h后,日本血吸虫虫卵的孵化率分别为0.52%、11.90%和49.15%,曼氏血吸虫分别为4.17%、31.37%和92.53%。当暴露于10-6mol/L吡喹酮1min后,日本血吸虫毛蚴的变形率为100.00%,曼氏血吸虫为55.73%;当分别暴露于5×10-7、10-7mol/L吡喹酮5min后,日本血吸虫毛蚴的变形率为96.82%和21.80%,曼氏血吸虫毛蚴为21.80%和0。当暴露于10-5mol/L吡喹酮中40min,日本血吸虫尾蚴的断尾率为96.75%,曼氏血吸虫为28.30%;暴露于4×10-7mol/L吡喹酮中100min,日本血吸虫尾蚴的断尾率为95.82%,曼氏血吸虫为11.40%;当暴露于10-7mol/L吡喹酮中80min,日本血吸虫尾蚴的断尾率为29.65%,曼氏血吸虫尾蚴为0。结论日本血吸虫各现场分离株间在虫卵、毛蚴和尾蚴阶段对吡喹酮的敏感性无明显差异,但均明显高于曼氏血吸虫。研究提示,将毛蚴移入5×10-7mol/L吡喹酮溶液中1min,镜下观察其变形率,可作为日本血吸虫对吡喹酮敏感性的观察指标,可用于现场判断病人化疗失败的原因是否是由于吡喹酮不敏感株产生所引起。将尾蚴移入4×10-7mol/L吡喹酮溶液中80～100min,镜下观察其断尾率,可用于螺体内虫株吡喹酮敏感性监测。
Objective To determine the susceptibility to praziquantel of schistosoma japonicum isolates from the major epidemic areas of mainland China during the stages of eggs, miracidia and cercariae, and to provide a basis for the establishment of a sensitive assay for praziquantel in Schistosoma japonicum. Methods The eggs were isolated from the stool specimens of patients with schistosomiasis japonica endemic areas in Hunan, Hubei, Jiangxi, Anhui, Jiangsu and Yunnan provinces, and the isolates of Schistosoma japonicum were isolated on the spot. Three strains of Schistosoma mansoni As a control. The worm eggs of each strain were respectively incubated in praziquantel solution of 5 × 10-6, 10-6, 5 × 10-7 and 10-7mol / L for 24 hours, and then hatched in clean water to observe the hatching rate of eggs. Each strain of miracidia were exposed to 5 × 10-6,10-6,5 × 10-7,10-7mol / L praziquantel solution, 0,1,5min after the observation of movement and morphological changes of miracidia . Each strain of cercariae were exposed to 10-5,6 × 10-7,4 × 10-7,10-7mol / L praziquantel solution, 0,20,40,60,80,100min, dissecting Under the observation of cercariae migration, contraction and tailing rate changes. The results were compared with Schistosoma mansoni. Results The hatching rates of Schistosoma japonicum eggs were 0.52%, 11.90% and 49.15% respectively after treated with 10-6, 5 × 10-7 and 10-7mol / L praziquantel for 24 hours, and the rates of schistosoma mansoni were 4.17% , 31.37% and 92.53% respectively. When exposed to 10-6mol / L praziquantel for 1min, the deformation rate of S. japonicum miracidia was 100.00% and that of S. mansoni was 55.73%. When exposed to 5 × 10-7,10-7mol / L praziquantel for 5min After that, the deformation rate of Schistosoma japonicum miracidia was 96.82% and 21.80%, and the Schistosoma mansoni miracidia was 21.80% and 0 respectively. When exposed to 10-5mol / L praziquantel for 40min, the cut-off rate of cercariae of Schistosoma japonicum was 96.75% and that of Schistosoma mansoni was 28.30%. After exposure to 4 × 10-7mol / L praziquantel for 100min, , 95.82% of them were cut off, and Schistosoma mansoni was 11.40%. When exposed to 10-7mol / L praziquantel for 80min, the cut-off rate of cercariae of Schistosoma japonicum was 29.65% and that of cercariae of Schistosoma mansoni was 0. Conclusions There is no significant difference in the sensitivity of praziquantel to the isolates of schistosoma japonicum among the isolates of Schistosoma japonicum at the stages of eggs, miracidia and cercariae, but both of them are obviously higher than that of Schistosoma mansoni. The study suggests that the miracidia transferred to 5 × 10-7mol / L praziquantel solution for 1min, microscopic observation of the deformation rate, can be used as an indicator of Schistosoma japonicum sensitivity to praziquantel can be used to determine the reasons for the failure of chemotherapy in patients Whether it is caused by the praziquantel insensitive strain. The cercariae was transferred into 4 × 10-7mol / L praziquantel solution for 80 ~ 100min, the end of the tailed rate was observed microscopically, which could be used to monitor the sensitivity of praziquantel strain.