Llewellyn Martin S, Messenger Louisa A, Luquetti Alejandro O, Garcia Lineth, Torrico Faustino, Tavares Suelene B N, Cheaib Bachar, Derome Nicolas, Delepine Marc, Baulard Céline, Deleuze Jean-Francois, Sauer Sascha, Miles Michael A
The London School of Hygiene and Tropical Medicine, London, United Kingdom; Molecular Ecology and Fisheries Genetics Laboratory, School of Biological Sciences, University of Wales, Bangor, Bangor, Gwynedd, United Kingdom.
The London School of Hygiene and Tropical Medicine, London, United Kingdom.
PLoS Negl Trop Dis. 2015 Apr 7;9(4):e0003458. doi: 10.1371/journal.pntd.0003458. eCollection 2015 Apr.
Chagas disease results from infection with the diploid protozoan parasite Trypanosoma cruzi. T. cruzi is highly genetically diverse, and multiclonal infections in individual hosts are common, but little studied. In this study, we explore T. cruzi infection multiclonality in the context of age, sex and clinical profile among a cohort of chronic patients, as well as paired congenital cases from Cochabamba, Bolivia and Goias, Brazil using amplicon deep sequencing technology.
METHODOLOGY/ PRINCIPAL FINDINGS: A 450bp fragment of the trypomastigote TcGP63I surface protease gene was amplified and sequenced across 70 chronic and 22 congenital cases on the Illumina MiSeq platform. In addition, a second, mitochondrial target--ND5--was sequenced across the same cohort of cases. Several million reads were generated, and sequencing read depths were normalized within patient cohorts (Goias chronic, n = 43, Goias congenital n = 2, Bolivia chronic, n = 27; Bolivia congenital, n = 20), Among chronic cases, analyses of variance indicated no clear correlation between intra-host sequence diversity and age, sex or symptoms, while principal coordinate analyses showed no clustering by symptoms between patients. Between congenital pairs, we found evidence for the transmission of multiple sequence types from mother to infant, as well as widespread instances of novel genotypes in infants. Finally, non-synonymous to synonymous (dn:ds) nucleotide substitution ratios among sequences of TcGP63Ia and TcGP63Ib subfamilies within each cohort provided powerful evidence of strong diversifying selection at this locus.
CONCLUSIONS/SIGNIFICANCE: Our results shed light on the diversity of parasite DTUs within each patient, as well as the extent to which parasite strains pass between mother and foetus in congenital cases. Although we were unable to find any evidence that parasite diversity accumulates with age in our study cohorts, putative diversifying selection within members of the TcGP63I gene family suggests a link between genetic diversity within this gene family and survival in the mammalian host.
恰加斯病由二倍体原生动物寄生虫克氏锥虫感染引起。克氏锥虫具有高度的遗传多样性，个体宿主中的多克隆感染很常见，但研究较少。在本研究中，我们使用扩增子深度测序技术，在一组慢性患者以及来自玻利维亚科恰班巴和巴西戈亚斯的配对先天性病例中，探讨年龄、性别和临床特征背景下的克氏锥虫感染多克隆性。
方法/主要发现：在Illumina MiSeq平台上，对70例慢性病例和22例先天性病例的锥鞭毛体TcGP63I表面蛋白酶基因的450bp片段进行扩增和测序。此外，对同一病例队列中的第二个线粒体靶点——ND5进行测序。产生了数百万条读数，并在患者队列（戈亚斯慢性病例，n = 43；戈亚斯先天性病例，n = 2；玻利维亚慢性病例，n = 27；玻利维亚先天性病例，n = 20）中对测序读深度进行了标准化。在慢性病例中，方差分析表明宿主内序列多样性与年龄、性别或症状之间没有明显相关性，而主坐标分析表明患者之间没有按症状聚类。在先天性病例对之间，我们发现了多种序列类型从母亲传播到婴儿的证据，以及婴儿中广泛存在新基因型的情况。最后，每个队列中TcGP63Ia和TcGP63Ib亚家族序列之间的非同义与同义（dn:ds）核苷酸替换率提供了该位点强烈多样化选择的有力证据。
结论/意义：我们的结果揭示了每个患者体内寄生虫离散型单元的多样性，以及先天性病例中寄生虫菌株在母婴之间传播的程度。虽然在我们的研究队列中未能找到任何证据表明寄生虫多样性随年龄积累，但TcGP63I基因家族成员内的假定多样化选择表明该基因家族内的遗传多样性与在哺乳动物宿主中的生存之间存在联系。
