Institute of Epidemiology, Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany.
Institute for Community Medicine, Section SHIP-KEF, University Medicine Greifswald, Greifswald, Germany.
Parasit Vectors. 2024 Oct 24;17(1):436. doi: 10.1186/s13071-024-06524-x.
Toxocara canis and Toxocara cati are parasitic nematodes that occur worldwide. As embryonated Toxocara spp. eggs in the environment pose a zoonotic risk, especially for children, optimal diagnostic approaches are necessary for effective disease response and management, including surveillance. However, little is known about the performance of different diagnostic protocols for detecting Toxocara spp. in the faeces of cats and dogs, hampering movement towards an optimal diagnostic process. This study aimed to compare detection methods, including a newly developed sequential sieving protocol (SF-SSV) and a high-throughput multiplex qPCR-based method to facilitate epidemiological studies.
Species-specific Toxocara spp. egg suspensions and canine and feline faecal samples from the field were used to estimate analytical and diagnostic sensitivity of the protocols. The performance of two automated DNA extraction protocols using enzymatic and mechanical lysis were compared by multiplex qPCR, targeting both T. canis and T. cati-specific genomic sequences. All samples were examined by microscopy-based techniques, the sedimentation flotation technique (SF) and a newly developed SF-SSV for the detection, enrichment and purification of parasite eggs. The costs and processing times necessary for all protocols were estimated and compared for both single samples and sets of 100 samples.
To detect Toxocara spp. eggs, SF-SSV showed the highest analytical sensitivity and a significantly higher diagnostic sensitivity than the DNA detection methods. Mechanical lysis performed better than enzymatic lysis for automated DNA extraction. In automated DNA extraction, 96-well plates performed better than 24-well plates. DNA detection and microscopy-based parasitological methods showed substantial agreement between the results generated by each method. Microscopy-based techniques required the lowest costs and least hands-on time for a single sample. However, when costs and labour were estimated for a set of 100 samples, the DNA detection protocol using 96-well plates for extraction revealed costs similar to SF-SSV and the fastest processing times.
SF-SSV was superior in terms of analytical and diagnostic sensitivity for the detection of Toxocara spp. eggs. For larger sets of samples, multiplex qPCR-based DNA detection represents an alternative to microscopy-based methods, based on the possibility of faster sample processing at similar costs to SF-SSV, and the ability to provide species-specific diagnoses.
犬弓首蛔虫和猫弓首蛔虫是寄生线虫，在全球范围内存在。由于环境中含有胚胎的犬弓首蛔虫和猫弓首蛔虫卵具有动物传染病风险，特别是对儿童而言，因此需要优化诊断方法，以有效应对和管理疾病，包括进行监测。然而，对于检测猫和犬粪便中犬弓首蛔虫和猫弓首蛔虫的不同诊断方案的性能，人们知之甚少，这阻碍了向最佳诊断过程的发展。本研究旨在比较不同的检测方法，包括新开发的序贯筛检方案（SF-SSV）和高通量多重 qPCR 检测方法，以促进流行病学研究。
使用犬弓首蛔虫和猫弓首蛔虫特异性卵悬浮液以及现场采集的犬和猫粪便样本，来估计这些方案的分析和诊断灵敏度。通过多重 qPCR 比较了两种基于酶和机械裂解的自动化 DNA 提取方案的性能，该多重 qPCR 针对犬弓首蛔虫和猫弓首蛔虫的特异性基因组序列。所有样本均通过显微镜检查、沉淀浮聚技术（SF）和新开发的 SF-SSV 进行检查，以检测、富集和纯化寄生虫卵。估计了所有方案的成本和处理时间，并对单个样本和 100 个样本组进行了比较。
为了检测犬弓首蛔虫和猫弓首蛔虫卵，SF-SSV 的分析灵敏度和诊断灵敏度均最高，明显高于 DNA 检测方法。与酶裂解相比，机械裂解更有利于自动化 DNA 提取。在自动化 DNA 提取中，96 孔板的性能优于 24 孔板。DNA 检测和基于显微镜的寄生虫学方法在每种方法产生的结果之间具有显著的一致性。基于显微镜的技术对于单个样本而言，所需的成本最低，人工操作时间最少。但是，当估计 100 个样本组的成本和劳动时间时，使用 96 孔板进行提取的 DNA 检测方案显示出与 SF-SSV 相似的成本和最快的处理时间。
SF-SSV 在检测犬弓首蛔虫和猫弓首蛔虫卵的分析和诊断灵敏度方面具有优势。对于更大的样本组，基于多重 qPCR 的 DNA 检测是基于显微镜的方法的替代方法，基于更快的样本处理速度，成本与 SF-SSV 相似，并且能够提供种特异性诊断。
