背景:锥虫是对公共卫生最关键的寄生虫之一,因为它们对人类的影响,动物,植物健康。与这些病原体相关的疾病主要表现在贫困和脆弱人群中,社会,环境,和生物因素调节病例发病率和地理分布。
方法:我们在来自不同哺乳动物的样本中使用基于Sanger和扩增子的下一代测序(NGS)来鉴定哥伦比亚几个部门的锥虫虫感染。总共174个DNA样本(18个人,83只狗和73种野生哺乳动物)通过使用热休克蛋白70(Hsp70)基因片段的常规PCR进行分析,并对阳性样品进行Sanger测序。使用相同的基因片段将27个样品用于基于扩增子的NGS。获得的数据用于进行多样性分析。
结果:通过Hsp70片段进行PCR检测,一百一十三个样本呈阳性;这些对应于22.1%的利什曼原虫。,18.6%亚马逊乳杆菌,9.7%巴西乳杆菌,14.2%L.infantum,8%的巴拿马乳杆菌,和27.4%克氏锥虫。通过使用的两种测序技术对鉴定的物种的比较导致97%的一致性。α和β多样性指数显著,主要针对狗;在分析的样本中有一个有趣的共感染事件指数:不同的利什曼原虫物种以及在分析的样本之一中同时存在T.cruzi甚至T.rangeli。此外,在野生哺乳动物的样品中观察到巴西乳杆菌的含量较低。有趣的是,根据我们的知识,这是哥伦比亚Hydrochaerishydrochaeris(水仙花)中检测到利什曼原虫的第一份报告。
结论:本研究中使用的Hsp70片段是在许多宿主中鉴定锥虫的最佳分子标记,并且当使用基于扩增子的测序时,可以鉴定同一样品中的不同物种。然而,应该仔细解释该片段通过常规PCR进行分子诊断的用途,因为它具有相同的鉴定几种寄生虫的能力。由于锥虫科不同属的共同流通,这一点在南美高度流行的国家中至关重要。研究结果表明,可以研究共同进化和载体-宿主相互作用的一种健康方法的有趣起点。
BACKGROUND: Trypanosomatids are among the most critical parasites for public health due to their impact on human, animal, and plant health. Diseases associated with these pathogens manifest mainly in poor and vulnerable populations, where social, environmental, and biological factors modulate the case incidence and geographical distribution.
METHODS: We used Sanger and amplicon-based next-generation sequencing (NGS) in samples from different mammals to identify trypanosomatid infections in several departments in Colombia. A total of 174 DNA samples (18 humans, 83 dogs, and 73 wild mammals) were analyzed by conventional PCR using a fragment of the heat shock protein 70 (Hsp70) gene and Sanger sequenced the positive samples. Twenty-seven samples were sent for amplicon-based NGS using the same gene fragment. Data obtained were used to perform diversity analyses.
RESULTS: One hundred and thirteen samples were positive for PCR by Hsp70 fragment; these corresponded to 22.1% Leishmania spp., 18.6% L. amazonensis, 9.7% L. braziliensis, 14.2% L. infantum, 8% L. panamensis, and 27.4% Trypanosoma cruzi. Comparison of the identified species by the two sequencing technologies used resulted in 97% concordance. Alpha and beta diversity indices were significant, mainly for dogs; there was an interesting index of coinfection events in the analyzed samples: different Leishmania species and the simultaneous presence of T. cruzi and even T. rangeli in one of the samples analyzed. Moreover, a low presence of L. braziliensis was observed in samples from wild mammals. Interestingly, to our knowledge, this is the first report of Leishmania detection in Hydrochaeris hydrochaeris (capybara) in Colombia.
CONCLUSIONS: The Hsp70 fragment used in this study is an optimal molecular marker for trypanosomatid identification in many hosts and allows the identification of different species in the same sample when amplicon-based sequencing is used. However, the use of this fragment for molecular diagnosis through conventional PCR should be carefully interpreted because of this same capacity to identify several parasites. This point is of pivotal importance in highly endemic countries across South America because of the co-circulation of different genera from the Trypanosomatidae family. The findings show an interesting starting point for One Health approaches in which coevolution and vector-host interactions can be studied.