关键词: 恶性疟原虫, 体外微量测定, 冰冻干燥, 碳酸氢钠, 抗氯喹, 液体培养, 对照井, 体外微量法, 裂殖体, 海南岛

Abstract: In order to determine the extent and degree of chloroquine resistance of falciparum malaria in our country, we have prepared both the drug-coated microplates and freeze-dried medium for the in vitro microtechnique. Each microplate has 40 wells, four horizontal rows with 10 wells each, wells 1 and 10 were untreated to serve as controls and wells 2-9 coated with 1.0, 2.0, 4.0, 5.7, 8.0, 16.0, 32.0 and 64.0 pmol of chloroquine diphosphate. The freeze-dried medium contained RPMI 1640, HEPES buffer and 15% type AB human serum. Prior to use, it was stored under refrigeration. 0.9ml of 0.21% sodium bicarbonate solution was injected into a vial with freeze-dried medium and the latter dissolved by shaking, and then 0.1ml of patient's blood added, from which an aliquot of 50μl of the mixture being placed in each well. P. falciparum kept in continuous culture for more than a year was used for laboratory assessment of reliability of the freeze-dried medium and microplates. The results obtained showed that the freeze-dried medium stored for one year at 4℃ could still support the growth of malaria parasites while those at 36℃ for 5 days and at 26℃ for 10 days failed. Field investigation was carried out in some parts of Hainan Island, South Yunnan, west Guangxi, South Guizhou and East Anhui. The results showed that the home-made plates were equally effective as those provided by WHO. The application of the freeze-dried medium in the assessment of drug-resistant, malaria furnished the test with a good readability and proved to be suitable for field use. It is clear that the home-made freeze-dried medium might be an important tool in the surveillance of chloroquine resistant malaria.(This investigation received the financial support of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, SWG on the Chemotherapy of Malaria, Project ID No:800214)