National Reference Laboratory of Parasitic and Fungal Infections, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016, Lisbon, Portugal.
Faculty of Sciences, BioISI-Biosystems & Integrative Sciences Institute, University of Lisboa, Lisboa, Portugal.
Parasitol Res. 2020 Feb;119(2):587-599. doi: 10.1007/s00436-019-06523-3. Epub 2020 Jan 2.
Advances in molecular epidemiology of Toxoplasma gondii are hampered by technical and cost-associated hurdles underlying the acquisition of genomic data from parasites. In order to implement an enhanced genotyping approach for molecular surveillance of T. gondii, we applied a multi-locus amplicon-based sequencing strategy to samples associated with human infection. This approach, targeting genome-dispersed polymorphic loci potentially involved in adaptation and virulence, genetically discriminated almost all 68 studied strains and revealed a scenario of marked genomic mosaicism. Two-thirds (n = 43) of all strains were classified as recombinant, although recombination seemed to be linked to the classical archetypal lineage. While 92% of the Sag2 archetype I strains revealed genetic mosaicism, only 45% of Sag2 archetype II strains were identified as recombinant. Contrarily to the virulence-associated archetype I, most type II strains (regardless of their recombination background) were non-virulent in mouse. Besides Sag2, some of the newly studied loci (namely the type I/I-like alleles of Sag1, B17, PK1, and Sag3 and type III/III-like alleles of TgM-A) constitute promising candidates to rapidly infer T. gondii mouse virulence. Our successful attempt to capture microsatellite length variation launches good perspectives for the straightforward transition from the laborious intensive historical method to more informative next-generation sequencing (NGS)/bioinformatics-based methodologies. Overall, while T. gondii whole-genome sequencing will be hardly feasible in most laboratories, this study shows that a discrete loci panel has the potential to improve the molecular epidemiology of T. gondii towards a better monitoring of circulating genotypes with clinical importance.
弓形虫分子流行病学的进展受到获取寄生虫基因组数据的技术和成本相关障碍的阻碍。为了实施增强的分子监测弓形虫的基因分型方法，我们应用了一种基于多位点扩增子的测序策略来分析与人类感染相关的样本。这种方法针对可能涉及适应和毒力的基因组分散多态性位点，对几乎所有 68 株研究菌株进行了遗传区分，并揭示了明显的基因组镶嵌现象。三分之二（n=43）的所有菌株被分类为重组株，尽管重组似乎与经典原型谱系有关。虽然 92%的 Sag2 原型 I 株显示出遗传镶嵌现象，但只有 45%的 Sag2 原型 II 株被鉴定为重组株。与毒力相关的原型 I 不同，大多数 II 型株（无论其重组背景如何）在小鼠中均无毒性。除 Sag2 外，一些新研究的基因座（即 Sag1、B17、PK1 和 Sag3 的 I 型/I 样等位基因和 TgM-A 的 III 型/III 样等位基因）构成了快速推断弓形虫小鼠毒力的有希望的候选基因座。我们成功地尝试捕获微卫星长度变化，为从费力的传统方法向更具信息量的下一代测序（NGS）/基于生物信息学的方法的直接转变开辟了良好的前景。总体而言，虽然大多数实验室几乎不可能对弓形虫全基因组进行测序，但本研究表明，离散基因座面板具有改善弓形虫分子流行病学的潜力，能够更好地监测具有临床重要性的循环基因型。
