Comparison of different diagnostic protocols for the detection of Toxocara spp. in faecal samples of cats and dogs.

IF 3 2区 医学 Q1 PARASITOLOGY Parasites & Vectors Pub Date : 2024-10-24 DOI:10.1186/s13071-024-06524-x
Deliah Tamsyn Winterfeld, Birgit Schauer, Majda Globokar, Nikola Pantchev, Susan Mouchantat, Franz Josef Conraths, Helge Kampen, Johanna Dups-Bergmann, Gereon Schares, Pavlo Maksimov
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Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

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比较用于检测猫和狗粪便样本中毒原虫属的不同诊断方案。
背景:犬弓形虫(Toxocara canis)和猫弓形虫(Toxocara cati)是寄生在世界各地的线虫。由于环境中的Toxocara属胚胎虫卵会造成人畜共患病风险,尤其是对儿童而言,因此必须采用最佳的诊断方法来有效应对和管理疾病,包括进行监测。然而,人们对检测猫和狗粪便中毒原虫的不同诊断方案的性能知之甚少,这阻碍了最佳诊断程序的发展。本研究旨在比较检测方法,包括新开发的顺序筛分方案(SF-SSV)和基于高通量多重 qPCR 的方法,以促进流行病学研究:方法:使用物种特异性弓形虫卵悬浮液和来自野外的犬科和猫科动物粪便样本来评估方案的分析和诊断灵敏度。通过多重 qPCR(针对犬弓形虫和猫弓形虫特异性基因组序列)比较了使用酶解和机械裂解的两种自动 DNA 提取方案的性能。所有样本均通过显微镜技术、沉降浮选技术(SF)和新开发的 SF-SSV 技术进行检测、富集和纯化寄生虫卵。对所有方案所需的成本和处理时间进行了估算,并比较了单个样本和每组 100 个样本所需的成本和处理时间:结果:在检测弓形虫卵方面,SF-SSV 的分析灵敏度最高,诊断灵敏度明显高于 DNA 检测方法。在自动提取 DNA 时,机械裂解法的效果优于酶裂解法。在自动 DNA 提取中,96 孔板的性能优于 24 孔板。DNA 检测法和基于显微镜的寄生虫学方法所得出的结果基本一致。基于显微镜的技术对单个样本的成本要求最低,所需的动手时间最少。然而,当对一组 100 个样本的成本和人工进行估算时,使用 96 孔板提取 DNA 的检测方案显示出与 SF-SSV 相似的成本和最快的处理时间:结论:SF-SSV 在检测弓形虫卵的分析和诊断灵敏度方面更胜一筹。对于较大的样本集,基于多重 qPCR 的 DNA 检测是显微镜方法的一种替代方法,因为它能以与 SF-SSV 相似的成本更快地处理样本,并能提供物种特异性诊断。
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来源期刊
Parasites & Vectors
Parasites & Vectors 医学-寄生虫学
CiteScore
6.30
自引率
9.40%
发文量
433
审稿时长
1.4 months
期刊介绍: Parasites & Vectors is an open access, peer-reviewed online journal dealing with the biology of parasites, parasitic diseases, intermediate hosts, vectors and vector-borne pathogens. Manuscripts published in this journal will be available to all worldwide, with no barriers to access, immediately following acceptance. However, authors retain the copyright of their material and may use it, or distribute it, as they wish. Manuscripts on all aspects of the basic and applied biology of parasites, intermediate hosts, vectors and vector-borne pathogens will be considered. In addition to the traditional and well-established areas of science in these fields, we also aim to provide a vehicle for publication of the rapidly developing resources and technology in parasite, intermediate host and vector genomics and their impacts on biological research. We are able to publish large datasets and extensive results, frequently associated with genomic and post-genomic technologies, which are not readily accommodated in traditional journals. Manuscripts addressing broader issues, for example economics, social sciences and global climate change in relation to parasites, vectors and disease control, are also welcomed.
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