Matovu Enock, Katiti Dianah, Kitibwa Annah, Kazibwe AnneJ, Kato Charles Drago, Biéler Sylvain, Ndung'u Joseph Mathu
College of Veterinary Medicine, Animal Resources and Biosecurity (COVAB), Makerere University, P. O. Box 7062, Kampala, Uganda.
College of Veterinary Medicine, Animal Resources and Biosecurity (COVAB), Makerere University, P. O. Box 7062, Kampala, Uganda.
Exp Parasitol. 2020 Feb 29;211:107844. doi: 10.1016/j.exppara.2020.107844.
The Loopamp™ Trypanosoma brucei Detection Kit is the latest addition of molecular techniques for amplification of parasite DNA in biological materials. We have evaluated the kit on a number of preparations of crude templates from the blood of experimentally infected rodents, to provide the best option that can be extrapolated to resource-poor healthcare settings where human African trypanosomiasis (HAT) is endemic. We used rodent blood spiked with T. b. brucei at various serial dilutions to test whole blood, that was concentrated by differential lysis of red blood cells (RBCs) followed by centrifugation, or buffy coat samples recovered from whole blood after centrifugation. We also tested crude templates produced after lysis of blood with sodium dodecyl sulphate (SDS) or Triton X, and storage for up to 28 days at room temperature after spotting on filter paper or glass slides. Concentration by RBC lysis provided the highest analytical sensitivity (0.04 trypanosomes/ml), closely followed by the much cheaper SDS at 0.1 trypanosomes/ml sensitivity. We also monitored the persistence of DNA in lysed blood dried onto filter papers by testing them weekly with the LAMP kit and by PCR for the 177bp repeats characteristic of the T. brucei subspecies. At a concentration of 100 trypanosomes/ml, signals indicating presence of parasite DNA could be detected up to week 10, while at 10 trypanosomes/ml detection of signals was limited to week 4. Thus, an ordinary filter paper provides a convenient medium for preservation of trypanosome DNA at ambient conditions for use with the LAMP kit in the short run. Lysis of samples with SDS enhanced sensitivity by facilitating parasite DNA availability. This opens the avenue to incorporate LAMP in routine algorithms for HAT diagnosis and surveillance, as well as for monitoring elimination programs.
Loopamp™ 布氏锥虫检测试剂盒是用于扩增生物材料中寄生虫DNA的最新分子技术产品。我们已在多种来自实验感染啮齿动物血液的粗模板制剂上对该试剂盒进行了评估，以提供可外推至非洲人类锥虫病（HAT）流行的资源匮乏医疗环境的最佳选择。我们使用添加了不同系列稀释度布氏布氏锥虫的啮齿动物血液来检测全血，全血先通过红细胞（RBC）的差异裂解然后离心进行浓缩，或者检测离心后从全血中回收的血沉棕黄层样本。我们还测试了用十二烷基硫酸钠（SDS）或 Triton X裂解血液后产生的粗模板，以及点样到滤纸或载玻片上后在室温下保存长达28天的样本。通过RBC裂解进行浓缩提供了最高的分析灵敏度（0.04个锥虫/毫升），紧随其后的是便宜得多的SDS，灵敏度为0.1个锥虫/毫升。我们还通过每周用LAMP试剂盒和PCR检测布氏锥虫亚种特有的177bp重复序列，监测了干燥在滤纸上的裂解血液中DNA的持久性。在浓度为100个锥虫/毫升时，直到第10周都能检测到表明存在寄生虫DNA的信号，而在10个锥虫/毫升时，信号检测仅限于第4周。因此，普通滤纸为在环境条件下保存锥虫DNA提供了一种方便的介质，以便短期内与LAMP试剂盒一起使用。用SDS裂解样本通过促进寄生虫DNA的可利用性提高了灵敏度。这为将LAMP纳入HAT诊断和监测的常规算法以及监测消除计划开辟了道路。
