Faculty of Science, University of Melbourne, Parkville, VIC 3052, Australia.
Institute of Parasitology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 266a, Zürich, 8057, Switzerland.
Int J Parasitol. 2024 Mar;54(3-4):185-193. doi: 10.1016/j.ijpara.2023.12.001. Epub 2023 Dec 13.
In recent years, Angiostrongylus vasorum, Crenosoma vulpis, Eucoleus aerophilus (syn. Capillaria aerophila) and Eucoleus boehmi (syn. Capillaria boehmi), commonly referred to as canine lungworms, have gained a growing interest worldwide as the result of their geographical expansion. Each of these nematode species differs considerably in its biology and pathogenicity. Despite their impact on dogs' health, these parasites are often underdiagnosed owing to diagnostic challenges. Here, we describe the development and validation of a Taq-Man-based multiplex quantitative PCR (qPCR) for the simultaneous detection of the main species of canine lungworms in faeces of infected dogs. Using 10-fold serial dilutions of synthetic gene block fragments containing individual sequence targets of each lungworm species, the analytical sensitivity of the assay ascertained was 1.84 ng/μl for A. vasorum, 3.08 ng/μl for C. vulpis and 0.79 ng/μl for Eucoleus spp. The sensitivity of the assays and their ability to detect mixed species infections were compared with microscopy-based techniques (faecal floatation and Baermann technique) applied to faecal samples submitted for lungworm testing through an accredited diagnostic laboratory at the Institute of Parasitology, University of Zurich, Switzerland, and from community dogs as part of a research project on canine endoparasites in Cambodia. The multiplex qPCR displayed high diagnostic sensitivity (42/46, 91.3%; 95% Confidence Interval (CI): 79.1-97.1%) and a diagnostic specificity of 100% (45/45, 95% CI: 90.6-100%), and was able to detect 42.9% additional mixed lungworm species infections compared with microscopy-based methods. Kappa statistics showed substantial agreement between the qPCRs and microscopy for mixed infections (κ = 0.72, 95% CI: 0.4-1) and Eucoleus spp. (κ = 0.65, 95% CI: 0.45-0.85) and almost perfect agreement for C. vulpis (κ = 0.85, 95% CI: 0.63-1) and A. vasorum (κ = 0.92, 95% CI: 0.84-1). This multiplex qPCR enables timely, accurate, and sensitive diagnosis of canine lungworm species in faecal samples and can be used to monitor the geographical distribution and emergence of these parasitic species, globally.
近年来，犬肺线虫（即血管圆线虫、犬钩口线虫、嗜气毛细线虫和犬弓首蛔虫）作为一种地理分布广泛的寄生虫，由于其地理分布范围的扩大，逐渐引起了全世界的关注。这些线虫种类在生物学和致病性方面有很大的不同。尽管这些寄生虫对狗的健康有影响，但由于诊断挑战，它们往往被漏诊。在这里，我们描述了一种 Taq-Man 多重实时定量 PCR（qPCR）的开发和验证，用于检测感染犬粪便中的主要犬肺线虫。使用包含每个肺线虫物种个体序列靶标的 10 倍系列稀释的合成基因块片段，确定该检测方法的分析灵敏度为血管圆线虫为 1.84ng/μl，犬钩口线虫为 3.08ng/μl，嗜气毛细线虫为 0.79ng/μl。比较了该检测方法的灵敏度及其检测混合物种感染的能力，与瑞士苏黎世大学寄生虫学研究所认可的诊断实验室提交的粪便样本进行的显微镜检查技术（粪便漂浮和巴氏法）以及柬埔寨社区狗进行的犬内寄生虫研究项目中的粪便样本进行了比较。多重 qPCR 显示出高诊断灵敏度（42/46，91.3%；95%置信区间（CI）：79.1-97.1%）和 100%的诊断特异性（45/45，95%CI：90.6-100%），并与显微镜检查方法相比，能够检测到 42.9%的额外混合肺线虫物种感染。Kappa 统计显示，qPCR 与显微镜检查对于混合感染（κ=0.72，95%CI：0.4-1）和嗜气毛细线虫（κ=0.65，95%CI：0.45-0.85）具有实质性一致性，对于犬钩口线虫（κ=0.85，95%CI：0.63-1）和血管圆线虫（κ=0.92，95%CI：0.84-1）具有几乎完美的一致性。这种多重 qPCR 能够及时、准确、敏感地诊断粪便中的犬肺线虫物种，并可用于监测这些寄生虫种的地理分布和出现情况，具有全球性意义。
