Yazdanparast Ehsan, Dos Anjos Antonio, Garcia Deborah, Loeuillet Corinne, Shahbazkia Hamid Reza, Vergnes Baptiste
Universidade do Algarve, DEEI-FCT, Faro, Portugal.
MIVEGEC (UM1-CNRS 5290-IRD 224), Institut de Recherche pour le Développement (IRD), Montpellier, France.
PLoS Negl Trop Dis. 2014 May 15;8(5):e2850. doi: 10.1371/journal.pntd.0002850. eCollection 2014 May.
Intracellular protozoan parasites are causative agents of infectious diseases that constitute major health problems for developing countries. Leishmania sp., Trypanosoma cruzi or Toxoplasma gondii are all obligate intracellular protozoan parasites that reside and multiply within the host cells of mammals, including humans. Following up intracellular parasite proliferation is therefore an essential and a quotidian task for many laboratories working on primary screening of new natural and synthetic drugs, analyzing drug susceptibility or comparing virulence properties of natural and genetically modified strains. Nevertheless, laborious manual microscopic counting of intracellular parasites is still the most commonly used approach. Here, we present INsPECT (Intracellular ParasitE CounTer), an open-source and platform independent software dedicated to automate infection level measurement based on fluorescent DNA staining. It offers the possibility to choose between different types of analyses (fluorescent DNA acquisitions only or in combination with phase contrast image set to further separate intra- from extracellular parasites), and software running modes (automatic or custom). A proof-of-concept study with intracellular Leishmania infantum parasites stained with DAPI (4',6-diamidino-2-phenylindole) confirms a good correspondence between digital results and the "gold standard" microscopic counting method with Giemsa. Interestingly, this software is versatile enough to accurately detect intracellular T. gondii parasites on images acquired with High Content Screening (HCS) systems. In conclusion, INsPECT software is proposed as a new fast and simple alternative to the classical intracellular Leishmania quantification methods and can be adapted for mid to large-scale drug screening against different intracellular parasites.
细胞内原生动物寄生虫是传染病的病原体，这些传染病给发展中国家带来了重大的健康问题。利什曼原虫属、克氏锥虫或刚地弓形虫都是专性细胞内原生动物寄生虫，它们在包括人类在内的哺乳动物的宿主细胞内生存和繁殖。因此，对于许多从事新天然和合成药物初步筛选、分析药物敏感性或比较天然菌株和基因改造菌株毒力特性的实验室来说，跟踪细胞内寄生虫的增殖是一项必不可少且日常的任务。然而，费力的手动显微镜计数细胞内寄生虫仍然是最常用的方法。在这里，我们介绍INsPECT（细胞内寄生虫计数器），这是一款开源且独立于平台的软件，致力于基于荧光DNA染色实现感染水平测量的自动化。它提供了在不同类型分析（仅荧光DNA采集或与相差图像集结合以进一步区分细胞内和细胞外寄生虫）以及软件运行模式（自动或自定义）之间进行选择的可能性。一项用4',6-二脒基-2-苯基吲哚（DAPI）染色的细胞内婴儿利什曼原虫寄生虫的概念验证研究证实，数字结果与吉姆萨染色的“金标准”显微镜计数方法之间具有良好的一致性。有趣的是，该软件通用性足够强，能够在通过高内涵筛选（HCS）系统获取的图像上准确检测细胞内弓形虫寄生虫。总之，INsPECT软件被提议作为一种新的快速且简单的替代方法，用于替代传统的细胞内利什曼原虫定量方法，并且可适用于针对不同细胞内寄生虫的中大规模药物筛选。
