背景:丝虫是大量动物宿主的重要媒介传播病原体,包括人类,并对许多使人衰弱的被忽视的热带病负责,例如,由Wuchererriabancrofti和Brugiaspp引起的淋巴丝虫病。,以及LoaLoa引起的loiasis.此外,一些新出现或难以消除的丝虫病原体是人畜共患的,使用犬科动物作为宿主,例如Dirofilariasp.\'hongkongensis\'。通过常用方法诊断丝虫病,像显微镜一样,可能是具有挑战性的,因为微丝症可能会下降到检测限以下。相比之下,常规PCR方法更灵敏和特异,但可能显示检测合并感染以及新出现和/或新型病原体的能力有限。深度测序技术的使用消除了这些挑战,提供整个寄生虫群落的灵敏检测,同时也更适合于稀有或新型病原体的表征。因此,我们开发了一种新颖的长读元编码测定法,用于在牛津纳米孔技术公司(ONT)MinION™测序仪上对丝状线虫细胞色素c氧化酶亚基I基因进行深度测序。我们使用kappa统计量评估了我们测定的总体性能,将其与丝虫检测的常用诊断方法进行比较。如常规PCR(cPCR)与Sanger测序和基于显微镜的改良Knott检验(MKT)。
结果:我们证实了我们的代谢编码分析可以表征来自不同属的丝虫寄生虫,包括,布雷尼亚,Brugia,Cercopithifilaria,Dipetalonema,Dirofilaria,Onchocerca,Setaria,Stephanofilaria和Wuchererria.我们通过使用斯里兰卡狗的血液样本证明了该测定法的概念证明,由此我们确定了丝虫的感染Acanthocheilonemareconditum,Brugiasp.斯里兰卡基因型和人畜共患Dirofilariasp。\'hongkongensis\'。与传统诊断相比,如MKT和cPCR与Sanger测序,我们确定了一个额外的丝状物种和超过15%的单感染和共感染。
结论:我们开发的代谢编码测定法可能显示出广泛的适用性,用于代谢编码和诊断来自各种动物宿主的丝虫的全谱,包括哺乳动物和媒介,而ONT\'小型便携式MinION™的使用意味着此类方法可以部署用于现场使用。
BACKGROUND: Filarial worms are important vector-borne pathogens of a large range of animal hosts, including humans, and are responsible for numerous debilitating neglected tropical diseases such as, lymphatic filariasis caused by Wuchereria bancrofti and Brugia spp., as well as loiasis caused by Loa loa. Moreover, some emerging or difficult-to-eliminate filarioid pathogens are zoonotic using animals like canines as reservoir hosts, for example Dirofilaria sp. \'hongkongensis\'. Diagnosis of filariasis through commonly available methods, like microscopy, can be challenging as microfilaremia may wane below the limit of detection. In contrast, conventional PCR methods are more sensitive and specific but may show limited ability to detect coinfections as well as emerging and/or novel pathogens. Use of deep-sequencing technologies obviate these challenges, providing sensitive detection of entire parasite communities, whilst also being better suited for the characterisation of rare or novel pathogens. Therefore, we developed a novel long-read metabarcoding assay for deep-sequencing the filarial nematode cytochrome c oxidase subunit I gene on Oxford Nanopore Technologies\' (ONT) MinION™ sequencer. We assessed the overall performance of our assay using kappa statistics to compare it to commonly used diagnostic methods for filarial worm detection, such as conventional PCR (cPCR) with Sanger sequencing and the microscopy-based modified Knott\'s test (MKT).
RESULTS: We confirmed our metabarcoding assay can characterise filarial parasites from a diverse range of genera, including, Breinlia, Brugia, Cercopithifilaria, Dipetalonema, Dirofilaria, Onchocerca, Setaria, Stephanofilaria and Wuchereria. We demonstrated proof-of-concept for this assay by using blood samples from Sri Lankan dogs, whereby we identified infections with the filarioids Acanthocheilonema reconditum, Brugia sp. Sri Lanka genotype and zoonotic Dirofilaria sp. \'hongkongensis\'. When compared to traditionally used diagnostics, such as the MKT and cPCR with Sanger sequencing, we identified an additional filarioid species and over 15% more mono- and coinfections.
CONCLUSIONS: Our developed metabarcoding assay may show broad applicability for the metabarcoding and diagnosis of the full spectrum of filarioids from a wide range of animal hosts, including mammals and vectors, whilst the utilisation of ONT\' small and portable MinION™ means that such methods could be deployed for field use.