Garcia H, Garcia M-E, Perez H, Mendoza-Leon A
Laboratorio de Investigación, Cátedra de Parasitología, Departamento de Patología Veterinaria, Facultad de Ciencias Veterinarias, Universidad Central de Venezuela.
Ann Trop Med Parasitol. 2005 Jun;99(4):359-70. doi: 10.1179/136485905X36271.
The usefulness of PCR-based assays for detecting trypanosomiasis in water buffaloes and other livestock was explored, under field conditions, in Venezuela. The sensitivity and specificity of the assays, which were based on established primer pairs (21-mer/22-mer and ILO1264/ILO1265), were evaluated, partly by comparison with the results of parasitological tests (stained bloodsmears and microhaematocrit centrifugation) and immunological assays (IFAT) run in parallel. The optimised PCR-based assays showed a sensitivity of 10 pg DNA. The use of the 21-mer/22-mer primer pair gave a test that was specific for species in the subgenus Trypanozoon (including Trypanosoma evansi), whereas use of ILO1264/ILO1265 produced a test that was specific for T. vivax. The results of a hybridization assay using T. evansi-DNA and T. vivax-DNA probes indicated no cross-hybridization between the T. evansi and T. vivax PCR products.The results of the bloodsmear examinations, microhaematocrit centrifugations (MHC) and IFAT indicated that 23 (6.7%), 39 (11.4%) and 135 (39.5%) of the 342 blood samples investigated (including 316 from water buffaloes) contained trypanosomes, respectively. The results of the PCR-based assays indicated that 68 (19.9%) of the same blood samples contained T. vivax (or at least T. vivax DNA), and that none contained T. evansi or any other member of the subgenus Trypanozoon. For the detection of trypanosomes, the assay therefore appeared almost twice as sensitive as the MHC. These results are the first on the molecular characterization of the trypanosomes infecting water buffaloes in Venezuela. When the results of the MHC (which is the most practical, and frequently used, alternative detection method) were used as the gold standard, the PCR-based assay for T. vivax was found to have 100% sensitivity, 90.4% specificity, a positive predictive value of 0.57, a positive likelihood ratio of 10.45, and a negative likelihood ratio of 0.00. The assay therefore appears a reasonable choice for detecting T. vivax in the mammalian livestock of Venezuela and elsewhere.
在委内瑞拉的田间条件下，对基于聚合酶链反应（PCR）的检测法用于检测水牛及其他家畜锥虫病的效用进行了探索。对基于既定引物对（21聚体/22聚体以及ILO1264/ILO1265）的检测法的敏感性和特异性进行了评估，部分是通过与同时进行的寄生虫学检测（染色血涂片和微量血细胞比容离心法）及免疫学检测（间接荧光抗体试验，IFAT）的结果作比较。优化后的基于PCR的检测法显示出10皮克DNA的敏感性。使用21聚体/22聚体引物对得到的检测法对布氏锥虫亚属（包括伊氏锥虫）的物种具有特异性，而使用ILO1264/ILO1265得到的检测法对活跃锥虫具有特异性。使用伊氏锥虫DNA和活跃锥虫DNA探针进行的杂交试验结果表明，伊氏锥虫和活跃锥虫的PCR产物之间不存在交叉杂交。血涂片检查、微量血细胞比容离心法（MHC）及IFAT的结果表明，在所调查的342份血样（包括316份来自水牛的血样）中，分别有23份（6.7%）、39份（11.4%）和135份（39.5%）含有锥虫。基于PCR的检测法结果表明，相同的血样中有68份（19.9%）含有活跃锥虫（或至少含有活跃锥虫DNA），且没有一份含有伊氏锥虫或布氏锥虫亚属的任何其他成员。因此，对于锥虫的检测，该检测法的敏感性几乎是MHC的两倍。这些结果是关于委内瑞拉感染水牛的锥虫分子特征的首批结果。当将MHC（这是最实用且常用的替代检测方法）的结果用作金标准时，发现基于PCR的活跃锥虫检测法具有100% 的敏感性、90.4% 的特异性、0.57的阳性预测值、10.45的阳性似然比以及0.00的阴性似然比。因此，该检测法似乎是检测委内瑞拉及其他地方哺乳动物家畜中活跃锥虫的合理选择。
