Development of a new drug sensitivity test for Plasmodium falciparum applicable in the field

随着恶性疟原虫耐药株的流行和疟疾在世界范围内的流行，建立简便、标准化的体外药敏试验方法势在必行。目前，已通过WHO标准方法检查寄生虫的药物敏感性，该方法需要由训练有素的显微镜工作者对寄生虫进行目视分类计数。我们使用溴化乙锭进行荧光测定，以便在项目的第一年能够客观地阅读结果。在测试中获得了可靠的结果，但是，该测定需要诸如离心机和荧光分光光度计之类的设施，这些设施在现场可能无法获得。下一步，使用偶联碱性磷酸酶的合成寡核苷酸将DNA杂交技术应用于试验。染色斑点的密度清楚地区分了培养物中寄生虫的发育。在中国海南岛的田间条件下对新测定法进行了评估。试验表明，在体外药敏试验中，DNA杂交技术可获得与血涂片镜检结果平行的结果。所有操作均在海南岛一家普通医院实验室进行。因此，我们认为使用非放射性DNA探针的新药物敏感性试验适用于该领域。我们正在研制一些适当的测试工具包，预计不久将能够向疟疾流行地区提供这些工具包。

The spread of drug-resistant Plasmodium falciparum accompanied by the world-wide resurgence of malaria urges the necessity, for the establishment of simplified and standardized method for in vitro drug sensitivity test. Currently drug sensitivity of the parasites has been examined by WHO standard method which requires visual differential counting of parasites by well-trained microscopists. We applied a fluoroassay using ethidium bromide to the test in order to enable objective reading of the results in the first year of the program. Reliable results were obtained in the test, however, the assay requires facilities such as a centrifuge and a fluorescent spectrophotometer that may not be available in the field. As the next step, a DNA hybridization technique was applied to the test using a synthetic oligonucleotide coupled to alkaline phosphatase. The density of the colored spot clearly distinguished development of parasites in culture. The new assay was assessed in Hainan Island, China, under field conditions. The trial demonstrated that, in the in vitro drug sensitivity test, the DNA hybridization technique could yield results parallel to those obtained by microscopical examination of blood smears. All procedures were carried out in an ordinary hospital laboratory on Hainan Island. Thus we consider that the new drug sensitivity test using a non-radioactive DNA probe is applicable to use in the field. We are developing some appropriate kits for the test and are expecting to be able to supply them to malaria endemic areas in the near future.