Tsetse & Trypanosomiasis Research Institute, P.O. Box 1026, Tanga, Tanzania.
Vet Parasitol. 2013 Nov 8;197(3-4):549-56. doi: 10.1016/j.vetpar.2013.05.022. Epub 2013 Jun 6.
Detection of trypanosomes that cause disease in human beings and livestock within their tsetse fly hosts is an essential component of vector and disease control programmes. Several molecular-based diagnostic tests have been developed for this purpose. Many of these tests, while sensitive, require analysis of trypanosome DNA extracted from single flies, or from pooled tsetse fly heads and amplified trypanosome DNA. In this study, we evaluated the relative analytical and diagnostic sensitivities of two PCR-based tests (ITS and TBR) and a Trypanozoon specific LAMP assay using pooled whole tsetse flies and midguts spiked with serially diluted procyclics of a laboratory strain of Trypanosoma brucei brucei (KETRI 3386). Test sensitivity was also evaluated using experimentally infected tsetse flies. The aim was to determine the most appropriate pooling strategy for whole tsetse and midguts. RIME-LAMP had the highest diagnostic sensitivity (100%) followed by TBR-PCR (95%) and ITS-PCR (50%) in detecting trypanosome DNA from pooled tsetse midguts. RIME-LAMP also had the best diagnostic specificity (75%) followed by ITS-PCR (68%) and TBR-PCR (50%). The relative detection limit determined by serial dilution of procyclics was below 10(-6) (equivalent to 1parasite/ml). Using TBR-PCR, ITS-PCR and RIME-LAMP, it was possible to detect trypanosome DNA in single flies or in pools of 2, 3, 4, 5, 10, or 15 flies/midguts. The proportion of positive pools declined by up to 60% when testing pools of 15 whole flies as opposed to testing pools of 5-10 flies. Additionally, it was possible to detect DNA in a single infected tsetse fly in the background of 4, 9, or 14 uninfected tsetse flies. Averaged across pool sizes and tsetse species, RIME-LAMP detected the highest proportion of positive pools in spiked whole tsetse and midguts (86.6% and 87.2%) followed by TBR-PCR (78. 6% and 79.2%) and ITS-PCR (34.3% and 40.2%). There were no significant differences between the proportions of positive pools detected in whole flies and midguts. We conclude that pooling of whole tsetse/midguts is an effective strategy to reduce hands-on-time and hence has potential application in large scale xenomonitoring to generate epidemiological data for decision making. RIME-LAMP offers the best diagnostic sensitivity and specificity on pooled tsetse midguts, thus demonstrating its superior diagnostic performance when compared with TBR-PCR and ITS-PCR. Using pools of whole tsetse or midguts as source of DNA does not have any significant effect on test results and is more representative of the field conditions where the proportion of flies with infected midguts tends to be higher than flies with infected salivary glands. Therefore to save time and minimize costs, pooling of whole tsetse flies is recommended.
在传播媒介和疾病控制计划中，检测在采采蝇宿主体内引起人类和家畜疾病的锥虫是必不可少的。为此目的已经开发了几种基于分子的诊断测试。这些测试中的许多虽然灵敏，但需要分析从单个苍蝇或从混合的采采蝇头部中提取的锥虫 DNA，并扩增锥虫 DNA。在这项研究中，我们评估了两种基于 PCR 的测试（ITS 和 TBR）和一种锥虫特异性 LAMP 检测方法的相对分析和诊断灵敏度，使用混合的整个采采蝇和中肠，并用实验室培养的布氏锥虫布鲁斯氏锥虫（KETRI 3386）的连续稀释的前鞭毛体进行了接种。还使用实验感染的采采蝇评估了测试的敏感性。目的是确定最适合整个采采蝇和中肠的混合策略。RIME-LAMP 在检测混合的采采蝇中肠的锥虫 DNA 时具有最高的诊断灵敏度（100％），其次是 TBR-PCR（95％）和 ITS-PCR（50％）。RIME-LAMP 还具有最佳的诊断特异性（75％），其次是 ITS-PCR（68％）和 TBR-PCR（50％）。通过前鞭毛体的连续稀释确定的相对检测限低于 10（相当于 1 个寄生虫/ml）。使用 TBR-PCR、ITS-PCR 和 RIME-LAMP，可以在单个苍蝇或 2、3、4、5、10 或 15 只苍蝇/中肠的混合池中检测到锥虫 DNA。当测试 15 只全蝇混合池时，与测试 5-10 只苍蝇混合池相比，阳性混合池的比例下降了多达 60％。此外，还可以在 4、9 或 14 只未感染的采采蝇的背景下检测到单个感染的采采蝇中的 DNA。平均而言，在整个池大小和采采蝇物种中，RIME-LAMP 在接种的整个采采蝇和中肠中检测到最高比例的阳性池（86.6％和 87.2％），其次是 TBR-PCR（78.6％和 79.2％）和 ITS-PCR（34.3％和 40.2％）。在全蝇和中肠中检测到的阳性池比例之间没有显着差异。我们得出的结论是，混合整个采采蝇/中肠是一种有效的减少动手时间的策略，因此具有在大规模 Xenomonitoring 中应用的潜力，以生成用于决策的流行病学数据。RIME-LAMP 在混合的采采蝇中肠上提供了最佳的诊断灵敏度和特异性，因此与 TBR-PCR 和 ITS-PCR 相比，显示出卓越的诊断性能。使用整个采采蝇或中肠作为 DNA 来源对测试结果没有任何重大影响，并且更能代表现场条件，其中感染中肠的苍蝇比例往往高于感染唾液腺的苍蝇比例。因此，为了节省时间和最小化成本，建议混合整个采采蝇。
