Softwares To Recognize Leishmania Infection
Leishmaniases is caused by Trypanosomatid protozoan parasites which belong to the genus Leishmania. Leishmaniases is prevalent in around 98 countries where, almost 1 billion people are at a very high risk of contracting this disease. This disease can be lethal if treatment is not provided. Leishmania infection takes place when the pramastigotes inoculates into the human blood by bite of the female sandflies. The intracellular amastigote is the disease-causing stage of Leishmania.
The purpose of this study is to apply a high content analysis which uses primary macrophages as the host cells for the amastigotes of Leishmania. It also aims to use microscopy images which are obtained from any fluorescence microscope to make high output quantification of the infection assays without using expensive softwares for image analysis. It basically implements an automated image based triage assay to identify the compounds that are active against the parasite species. Usage of two softwares is done in this study to recognize Leishmania, IN Cell Investigator Developer Toolbox and CellProfiler.
According to the protocol, cultured macrophages which have been infected by L.infantum and L.amazonesis are required for analysis. Intramacrophagic Leishmania amastigotes are then identified by staining their DNA with DAPI. Images were then acquired in an IN Cell Analyzer 2000 microscope. The original images were analyzed with the IN Cell Investigator Developer Toolbox. This allowed for the identification of the host cell nuclei and the cytoplasm and also the DNA content of the parasite. Images are also analyzed by the CellProfiler, which provided the total number of macrophages and the parasites. It gave the number of the infected macrophages and the number of the parasites per infected macrophage. To compare the data taken out from IN Cell Investigator Developer Toolbox and CellProfiler a manual counting was done with the help of GraphPad Prism Software.
Amastigotes were identified by using DAPI staining. By IN Cell Investigator Developer Toolbox, it is possible to identify individual macrophages, with the association of nucleus with well defined cytoplasm and parasites within each of them. Both the softwares gave high output and reliable data of the number of the infected macrophages and parasites per infected macrophage, independently of the Leishmania species used. As IN Cell Investigator Developer Toolbox is very expensive not all institutes have this facility therefore, CellProfiler is mainly used as it is an open source software. By usage of this workflow, it is possible to obtain fast and a reliable analysis of the microscope images from Leishmania infected macrophages in any laboratory.
