Development of an automated image analysis protocol for quantification of intracellular forms of Leishmania spp

Leishmania parasites cause a set of neglected tropical diseases with considerable public health impact, the leishmaniases, which are often fatal if left untreated. Since current treatments for the leishmaniases exhibit high toxicity, low efficacy and prohibitive prices, many laboratories throughout the world are engaged in research for the discovery of novel chemotherapeutics. This entails the necessity of screening large numbers of compounds against the clinically relevant form of the parasite, the obligatory intracellular amastigote, a procedure that in many laboratories is still carried out by manual inspection. To overcome this well-known bottleneck in Leishmania drug development, several studies have recently attempted to automate this process. Here we implemented an image-based high content triage assay for Leishmania which has the added advantages of using primary macrophages instead of macrophage cell lines and of enabling identification of active compounds against parasite species developing both in small individual phagolysosomes (such as L. infantum) and in large communal vacuoles (such as L. amazonensis). The automated image analysis protocol is made available for IN Cell Analyzer systems, and, importantly, also for the opensource CellProfiler software, in this way extending its implementation to any laboratory involved in drug development as well as in other aspects of Leishmania research requiring analysis of in vitro infected macrophages.Project Norte-01-0145-FEDER-000012-Structured program on bioengineered therapies for infectious diseases and tissue regeneration, supported by Norte Portugal Regional 17 Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). AGGA and TC were supported by contracts SFRH/BD/93766/2013, financed by POPHÐQREN, and subsidized by Fundo Social Europeu and Ministério da Ciência, Tecnologia e Ensino Superior, and PTDC/QEQ-MED/7097/2014, funded by National Funds through FCT, respectively. HC is funded by FCT under the "Investigador FCT" Programme